Stories of Success, Volume 45: Personal Recollections. X (Comprehensive Biochemistry) [10 ed.] 0444522468, 9780444522467, 9780080553573

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COMPREHENSIVE BIOCHEMISTRY

COMPREHENSIVE BIOCHEMISTRY SECTION I (VOLUMES 1–4) PHYSICO-CHEMICAL AND ORGANIC ASPECTS OF BIOCHEMISTRY SECTION II (VOLUMES 5–11) CHEMISTRY OF BIOLOGICAL COMPOUNDS SECTION III (VOLUMES 12–16) BIOCHEMICAL REACTION MECHANISMS SECTION IV (VOLUMES 17–21) METABOLISM SECTION V (VOLUMES 22–29) CHEMICAL BIOLOGY SECTION VI, A (VOLUMES 30–34 and 39) SELECTED TOPICS IN THE HISTORY OF BIOCHEMISTRY SECTION VI, B (VOLUMES 35–38 and 40–45) STORIES OF SUCCESS – PERSONAL RECOLLECTIONS, I–X

COMPREHENSIVE BIOCHEMISTRY Series Editor: GIORGIO SEMENZA Swiss Federal Institute of Technology, Department of Biochemistry, ETH-Zentrum, CH-8092 Zurich (Switzerland) and University of Milan, Department of Chemistry, Biochemistry, and Biotechnologies for Medicine, I-20133 Milan (Italy)

VOLUME 45 STORIES OF SUCCESS – PERSONAL RECOLLECTIONS. X Volume Editor: GIORGIO SEMENZA Swiss Federal Institute of Technology, Department of Biochemistry, ETH-Zentrum, CH-8092 Zurich (Switzerland) and University of Milan, Department of Chemistry, Biochemistry, and Biotechnologies for Medicine, I-20133 Milan (Italy)

AMSTERDAM
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First edition 2007 Copyright r 2007 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://www.elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloguing-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-444-52246-7 ISSN: 0069-8032 For information on all Elsevier publications visit our website at books.elsevier.com Printed and bound in Italy 07 08 09 10 11 10 9 8 7 6 5 4 3 2 1

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PREFACE TO VOLUME 45 Stories of Success – Personal Recollections. X

O brave new world, That has such people in it! Shakespeare, The Tempest, Act V

‘‘Luck’’ does not come to a scientist the way it does to a winner of a lottery. Luck in science is man-made, mostly scientist-made, through his or her own ingenuity, hard work, endurance (or, stubbornness, as others might say). Fruitful ideas come to the ‘‘prepared mind’’ only, as Pasteur [1] and my mentor Tiselius used to say [2]. You, dear reader, will find these components of scientific success in the lives of the (auto)biographic chapters collected in this volume and in previous volumes of this subseries. You will read how a few grams of tryptophan (despite the lack of any additional financial support) triggered the beginning of more than 60 years of extremely fruitful scientific productivity for a young Japanese MD, returning after World War II to a Japan which then consisted only of ruins. In his chapter, the youngest contributor tells of the joys and frustrations experienced during a difficult and challenging ‘‘work in progress’’. Again, ingenuity, novel ideas, hard work and endurance were needed to overcome the immense obstacles on the way to obtain 3D e.m. pictures of individual proteins; or to discover the nonorthodox use of a stop codon in the biosynthetic incorporation of a non-canonic amino acid in a polypeptide chain; or to demonstrate unequivocally the liquid crystal nature of the lipid bilayer in biological membranes; or to demonstrate convincingly the nature

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PREFACE

and function of a peculiar blood clotting factor; or to discover the alpha-feto-protein, which initiated cancer immunodiagnostics (and in particular the carcino-embryonic research). The same ‘‘ingredients’’, as it were, were needed in the initial period of metabolic biochemistry also, for the discovery of, e.g., the Embden– Meyerhof–Parnas pathway of glucose degradation. ‘‘Luck’’ can be a helpful ingredient among the components, which lead to a discovery. However, the unprecedented explosion of molecular biological sciences began during the ‘‘age of extremes’’ of the so-called short 20th century [3]. The scientists, their families and friends, like other citizens, were hit by an avalanche of horrors which swept through Europe and much of the rest of the world before, during and after World War II. ‘‘Luck in science’’ was often nullified by these catastrophic events. Many were drafted and sent to the front; those who returned would find their countries in ruins and shambles. Even after the aftermath of World War II, emigration (which is always traumatic even in the most favorable of circumstances) was often a required condition to be scientifically active at all. The Jews – always prominent in the molecular biological scientific community – were discriminated against and persecuted well before World War II began, and even more so during the war, not only in Nazi-occupied Europe; but often in Soviet-block countries too. Emigration had become for many a disguised sort of good luck. But also non-Jews could be silenced for years – in some cases forever – for whatever whimsical political [4] or pseudo-scientific [5] reasons (to name but two examples). Young molecular bioscientists, in particular, are invited to read the scientific and tragic private life of J. Parnas. They may master the intricacies of mutual interactions of protein kinases; but they need not know that radioactive phosphate was introduced in biochemistry by him, or that 1,6-phosphofructokinase and the degradation of glycogen to eventually glucose 6-phosphate were discovered in his laboratory. Parnas had created an excellent department of biochemistry in Lemberg (this name was later changed to Lviv and Lvov), which was also a flourishing center of Jewish intelligentsia and activity. The Nazis destroyed it utterly. Parnas escaped just in time; he was taken to central Asia and then to Moscow, where he resumed his biochemical research – naturally by cultivating also his contacts with the West. In so doing he tempted his own fate as he was arrested and eventually taken to

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the Lubyanka where he died, not even having been formally accused, let alone tried. This takes me to the second, very important goal of this volume and of this subseries. The ghosts of the past, which my generation thought had been killed forever, are lurking and, occasionally, spring back to life. Beware. Those who forget their past are prone to repeat past errors – and suffer past horrors anew. Our children and children’s children must not be tempted to repeat the mistakes, which our fathers and fathers’ fathers tragically made. Not just molecular biosciences, but indeed any intellectual and decent human initiative can thrive only in freedom and peace. We hope that these volumes will convey this message to our present and – importantly – to our future colleagues. ACKNOWLEDGMENTS

The editor wishes to thank the authors who have prepared such excellent (auto) biographic chapters. He thanks also Dr. Ned Mantei of the ETH in Zurich, and the staff of Elsevier in Amsterdam, in particular Anne Russum, for their help and co-operation. REFERENCES [1] See, e.g., in Dubos, R. (1960) Pasteur and Modern Science, republished by the Amer. Soc. for Microbiol., in 1998. [2] Tiselius, A. (l968) Annu. Rev. Biochem. 37, 1–24. [3] Hobsbawm, E. (1995) Age of Extremes: The Short Twentieth Century, 1914– 1991. London, UK, Abacus (Little, Brown and Company). [4] Bayev, A.A. (1995) The paths of my life. In Comprehensive Biochemistry (IV of the ‘‘Personal Recollections’’ subseries) (Slater, E.C., Jaenicke, R. and Semenza, G., eds.), Vol. 38, pp. 439–479. Amsterdam, Elsevier. [5] Levina, E.S., Yesakov, V.D. and Kisselev, L.L. (2005) Nikolai Vavilov: Life in the cause of science or science at the cost of life. In Comprehensive Biochemistry (IX of the ‘‘Personal Recollections’’ subseries) (Semenza, G. and Turner, A.J., eds.), Vol. 44, pp. 345–410. Amsterdam, Elsevier.

Swiss Institute of Technology, Zurich, Switzerland, and University of Milan, Italy, 2007

Giorgio Semenza

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CONTRIBUTORS TO THIS VOLUME G. I. ABELEV Member of the Russian Academy of Sciences, Russian Cancer Research Center, Moscow, Russia

ADRIANO AGUZZI The Institute of Neuropathology, University Hospital of Zurich, Schmelzbergstrasse 12, CH 8090 Zurich, Switzerland

´ SKA JOLANTA BARAN Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Pasteura 3, PL 02-093 Warsaw, Poland

WOLFGANG BAUMEISTER Department of Structural Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

¨ CK BIRGER BLOMBA Karolinska Institutet, Nanna Svartz va¨g 2, 17177 Solna, Sweden

¨ CK AUGUST BO Department of Biology I, University of Munich, LindenstraX e 10, D-82269 Geltendorf, Germany

ANDRZEJ DZUGAJ Department of Animal Physiology, Wrocław University, Cybulskiego 30, PL 50-205 Wrocław, Poland

OSAMU HAYAISHI Osaka Bioscience Institute, Suita, Osaka, Japan

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CONTRIBUTORS TO THIS VOLUME

JANINA KWIATKOWSKA-KORCZAK Department of Medical Biochemistry, Medical University, Chalubinskiego 10, PL 50-368 Wrocław, Poland

PETER J. QUINN Department of Biochemistry, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom

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CONTENTS VOLUME 45

COMPREHENSIVE BIOCHEMISTRY Stories of Success – Personal Recollections. X Preface to Volume 45 . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors to this Volume . . . . . . . . . . . . . . . . . . . . ix Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Chapter 1. Odyssey of a Biochemist by OSAMU HAYAISHI Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . Growing Up in Three Different Countries. Diamonds in the Desert . . . . . . . . . . . . . . Back to U.S.A. . . . . . . . . . . . . . . . . . . . . . Discovery of Oxygenases . . . . . . . . . . . . . . Kyoto University, My Second Alma Mater. The Mystery of Sleep . . . . . . . . . . . . . . . . Osaka Bioscience Institute . . . . . . . . . . . . Nature is the Guide . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 2. A Voyage to the Inner Space of Cells by WOLFGANG BAUMEISTER Abstract . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . Apprenticeship with Great Freedom . Heading into New Directions . . . . . .

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The First Decade in Martinsried: Studying Protein Architecture on Prokaryotic Cell Surfaces. . . . . . . . . . . . . . The Next Decade: Proteasomes, Thermosomes and Other Elements of Intracellular Protein Quality Control. . . The Latest Frontier: Charting Molecular Landscapes Inside Cells by Cryoelectron Tomography . . . . . . . . . . . . . . Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 3. Fixed to Translation: A Recollection by AUGUST BO¨CK Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Early Years. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . From Aminoacyl-tRNA Synthetases to Ribosomes . An Excursion to Archaea . . . . . . . . . . . . . . . . . . . Regulation of Fermentation . . . . . . . . . . . . . . . . .

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The Formate Regulon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The Biosynthesis and Insertion of the Metal Centre of [NiFe]–Hydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selenium Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Specific Incorporation of Selenium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Non-Specific Selenium Incorporation . . . . . . . . . . . . . . . . . . . . . . . . . 112 Selenium Toxicity and Resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Relations to Industry Resume and Outlook. Remarks . . . . . . . . . . References . . . . . . . .

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Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dennis Chapman: The Scientist . . . . . . . . . . . . . . . . . . Gonville and Caius, Cambridge . . . . . . . . . . . . . . . . . . . The Frythe Laboratories, Welwyn . . . . . . . . . . . . . . . . . Reckitt & Coleman, Sheffield University . . . . . . . . . . . . London University, Chelsea College . . . . . . . . . . . . . . . London University, Royal Free Hospital Medical School London University, Interdisciplinary Research Centre .

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Chapter 4. Dennis Chapman: Oiling the Path to Biomembrane Structure by PETER J. QUINN

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Dennis Chapman, The Man . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 5. Embden–Meyerhof–Parnas, the First Metabolic Pathway: The Fate of Prominent Polish Biochemist Jakub Karol Parnas by JOLANTA BARAN´SKA, ANDRZEJ DZUGAJ and JANINA KWIATKOWSKA-KORCZAK Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biography: Life, Work and Tragic Fate of Jakub Karol Parnas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1902–1913, Learning and Studying in Different Cities of Europe: Beginning of scientific activity . . . . . . . . . . . . . . . . . . . . . . . . . . . 1914–1920, the First World War: Decision to Work and Live in the Independent Poland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1920–1939, The Best, Successful Years in Lviv . . . . . . . . . . . . . . . Parnas as a Teacher and Master . . . . . . . . . . . . . . . . . . . . . . . Parnas’ Scientific Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . Nominations, Honors, Awards . . . . . . . . . . . . . . . . . . . . . . . . . Parnas’ Personality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1939–1941, the Second World War in Lviv: Soviet Occupation . . . June 1941, Leaving Lviv to Ufa . . . . . . . . . . . . . . . . . . . . . . . . . . 1943–1949, Stay in Moscow . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Parnas’ and Collaborators’ Contribution to Discovery of Glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discovery of Reactions of Glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . .

The Parnas’ School of Biochemistry . . . . . . . . . . The Polish Biochemical Society and Its Activities Undertaken to Honor Jakub Karol Parnas . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biographic Data . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . And Then There Was a New Disease . . . . . . . . . . . . . . . . Bleeding Time Factor – Really?. . . . . . . . . . . . . . . . . . . . .

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Chapter 6. A Journey with Bleeding Time Factor by BIRGER BLOMBA¨CK

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Factor Aims at Platelets . . . . . . . . . . . . . . . . . . . . Factor Dimmed and Brought to Light . . . . . . . . . . Factor, A Series of Huge Polymers . . . . . . . . . . . . Factor, Polymers of a Single-Chain Protein. . . . . . Factor, We See You! . . . . . . . . . . . . . . . . . . . . . . . Factor Loves Factor VIII. . . . . . . . . . . . . . . . . . . . Factor in Dialogue with Platelets and Vessel Wall. Factor Missing or Malformed . . . . . . . . . . . . . . . . Factor Checked by Guardian. . . . . . . . . . . . . . . . . Has Factor Other Guardians? . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 7. A Neuropathologist’s Diary by ADRIANO AGUZZI Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . Zurich, Part I: Brains of Humans and Mice The Vienna Experience. . . . . . . . . . . . . . . . Zurich, Part II: Tracking Prions . . . . . . . . . A Function for the Prion Protein . . . . . . . . The Future of Prion Therapeutics . . . . . . . Immunotherapy against Prions? . . . . . . . . . Soluble Prion Antagonists. . . . . . . . . . . . . . Inflammation: A License to Replicate? . . . . Homework for the Next Few Years . . . . . . . Life Science and Animal Experiments . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 8. An Autobiographical Sketch: 50 Years in Cancer Immunochemistry by G.I. ABELEV Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . University. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In the Laboratory: Searches and Findings. . . . . . . . . . . . . In Zilber’s Laboratory . . . . . . . . . . . . . . . . . . . . Immunodiffusion . . . . . . . . . . . . . . . . . . . . . . . . Individual Antigens and Monospecific Antibodies Alpha-fetoprotein (AFP) . . . . . . . . . . . . . . . . . . .

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Resonance and Subsequent Studies. Concluding Remarks. . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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G. Semenza (Ed.) Stories of Success – Personal Recollections. X (Comprehensive Biochemistry Vol. 45) r 2007 Elsevier B.V. All rights reserved. DOI: 10.1016/S0069-8032(07)45001-X

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Chapter 1

Odyssey of a Biochemist OSAMU HAYAISHI Osaka Bioscience Institute, Suita, Osaka, Japan

Abstract I was born in Stockton, California, U.S.A. in 1920 and soon went to Germany and lived in Berlin for about 2 years. Thereafter, I came back to Japan and graduated from Osaka University School of Medicine in 1942. After serving in the Japanese Navy as a medical officer, I returned to Osaka University to start my career as microbiologist. By the use of enrichment technique, I was able to isolate from the scorched soil, a strain of soil microorganism Pseudomonas that was able to grow on tryptophan as a sole source of carbon and nitrogen. Unlike in animals in which tryptophan is degraded to anthranilate and other intermediate metabolites and secreted in the urine, in this pseudomonas anthranilate was degraded to catechol and muconic acid, ultimately to CO2, NH3 and H2O. Furthermore, I was able to isolate a novel enzyme that catalyzed the oxidative cleavage of the aromatic structure of catechol yielding cis, cis-muconic acid as the reaction product. Molecular oxygen was obsolutely required for the reaction and could not be replaced by the known electron acceptor dyes or coenzymes. This finding together with various other properties of this enzyme indicated strongly the possibility that it could be an oxygen transferase rather than an oxidase. Therefore, it was named as ‘‘pyrocatechase’’ rather than catechol oxidase. However, the enzymatic incorporation of molecular oxygen into substrates had been completely ruled out in biological systems by the famous

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‘‘dehydrogenation theory’’ by Heinrich Wieland, and my interpretation was not immediately accepted. In 1949, I went to U.S.A. as a postdoctoral fellow and then in 1954 became Chief of Section on Toxicology, NIH, Bethesda. In 1955, I was able to show by the use of 18O, a stable isotope of oxygen, that the oxygen atoms incorporated into the product, muconic acid, was completely derived from molecular oxygen and not from H2O. Thus, I proposed to name such an enzyme as ‘‘oxygenases.’’ In 1958, I came back from U.S.A. to become professor and chairman of the Department to Medical Chemistry, Kyoto University School of Medicine. Thereafter, I extended my work on tryptophan metabolism and oxygenases for almost 25 years. After retirement from Kyoto University, I switched my field of interest to the elucidation of the molecular mechanisms of sleep wake regulation and found that prostaglandin D2 is a sleep hormone which is involved in the regulation of both circadian and homeostatic regulation of non-REM sleep. Keywords: tryptophan; pyrochatechase; oxygenases; sleep

Growing Up in Three Different Countries I was born in Stockton, California, U.S.A., on January 8, 1920. This city is not too far from Modest, which lies about 40 km to the southeast and is the birthplace of A. A. Benson, a noted plant biochemist [1]. It was soon after the end of the World War I, and there was a large population of immigrants of Japanese origin on the west coast of America mainly working as farmers, laborers and menial workers. My parents were not exactly typical immigrants. My father, Jitsuzo Hayaishi, was born on February 2, 1882, in a small village, Ejiri, now a part of the city of Miyazu, which is situated about 100 km north of Kyoto. Ejiri was located near ‘‘Amano Hashidate,’’ translated literarily as ‘‘a bridge in the heavens,’’ one of the three most beautiful scenic sites in Japan (Fig. 1). His father passed away at a relatively young age, and so he was raised by his mother, Tei, who was a silk merchant. He graduated from Osaka Jikei Hospital Medical School in 1900 at the age of 18, passed the national medical examination in the

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Fig. 1. Amano Hashidate (A bridge in the heavens). One of the three most beautiful scenic sites in Japan.

same year and was granted a license to practice medicine (license number 15170) in the next year. Thus, he must have been a very bright person to become an exceptionally young medical doctor in the Japan of 100 years ago. He then spent a year at the Department of Pathology, Kyoto University School of Medicine in Kyoto, and studied pathology under the guidance of Professor Kan Fujinami, a famous oncologist who was one of the pioneering researchers in the field of the viral origin of cancer. He returned to Miyazu, the new name of his hometown, and practiced medicine for almost eight years. Even though he was quite young, he was very successful, well-respected and popular in the Miyazu area. During this period, he became acquainted with Father Louis Relave, a catholic priest who was the founder of the Catholic Miyazu Church (St. John the Baptist church) and who taught English and French to my father. It may have been partly due to his influence that my father decided to close his clinic and go abroad to start his medical training all over again. I must say that my father must have been a very courageous as well as adventurous person to have not been content to be a country physician in a small town and to have given up his successful medical practice to go overseas for advanced training, especially in the first decade of the last century when air transportation was not

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available and presumably he had no friends or relatives in the United States. In 1909, he took a boat to California, safely arrived in America, but was told soon that it was not so easy to live and study medicine on the west coast due to racial discrimination against Japanese. He then took a train, crossed a big country, and ended up in Baltimore, Maryland, because he was told that the Johns Hopkins University had one of the best medical schools in U.S.A. However, owing to the high cost of tuition and insufficient qualifications on his part, his application was turned down; but he was advised to try the University of Baltimore, now the University of Maryland. Eventually, he was admitted, received his M.D., passed the State Board Examination and returned to California to practice medicine. Soon he met and married my mother, Mitsu Uchida, who came to see her brother Takashi, a deputy manager of Mitsui Co. Ltd. in San Francisco. My father was a conscientious and hardworking physician, always kind and devoted to his patients. Furthermore, at that time there were not many legally licensed Japanese doctors on the west coast. Consequently, he had a large number of patients, not only those coming from the vicinity of Stockton and the state of California but also others from distant places, such as Oregon, Arizona and other states. In addition, he now had a son, Osamu. Although he was quite successful and respected both socially and professionally, my father decided to close his clinic after almost ten years to obtain further advanced training in clinical and basic medicine. So I left Stockton shortly after my first birthday and started my pilgrimage with my parents. We first went to Rochester, Minnesota, where my father joined the Mayo Clinic to brush up on his clinical experience for about six months. Then the opportunity arose for my father to carry out research in immunology at the Robert Koch Institute, under the guidance of Professor A. Schnabel; and so we crossed the Atlantic Ocean and ended up in Berlin, Germany. During his nearly two-year stay at the institute, he published three papers all in German [2], which, upon his return to Japan later, were submitted to Kyushu Imperial University Medical School in Fukuoka as part of his Ph.D. thesis. I have dim memories of playing almost every day with a German boy, Hans, who lived in the next door in our apartment building in Berlin. Besides Japanese as my first language, German rather than American English was my second language. Although I later

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5

forgot both English and German as well as French, which my father spoke a little bit due to his association with Father L. Relave in Miyazu, my childhood experiences with other languages may have helped me to have an aptitude for foreign languages to some extent in later years. About 60 years later, on October 5, 1986, I visited the Robert Koch Institute with my friend Dr. Hans-Joachim Glotz of Schering A. G., and was very happy to see my father’s publications, which had been filed and stored in the library of the institute. My father passed away on August 12, 1977, at the age of 95. He had devoted his life to clinical practice. He was always respected and trusted by his colleagues and patients, because he was highly studious and always tried hard to keep up with new developments in the medical sciences. Even in his late years after officially retired from medical practice, his main hobby was reading new medical journals and books published in Japan as well as in the United States. His sincere attitude toward medical science and the scholarly atmosphere at home were what led my two brothers and myself to a career in medicine and even in basic research in my case. In September 1923, while sailing from Hamburg, Germany, to return to U.S.A., we received shockingly sad news from Japan that a horrendous earthquake had struck Tokyo, almost completely destroying the entire city. We went back to Japan via United States and safely returned to Japan in November (Fig. 2). After having spent about two years in Fukuoka, where my father received his Ph.D. from the Kyushu Imperial University, we finally settled in Osaka, the second largest city in Japan and the center of commerce in western Japan. There my father started a new clinic in Semba, a district almost in the center of the city.

Diamonds in the Desert I went to Naniwa Elementary School and Kitano High School, both of which were the best schools in Osaka, and had a wonderful education. I did not experience any bullying, even though some children born or raised in foreign countries were having a hard time by being bullied in some schools in Japan, which problem exists even at the present time. I graduated from the Osaka University School of Medicine in 1942 at the age of 22. It was in the middle of World War II, and I was almost immediately

6

Fig. 2.

OSAMU HAYAISHI

Leaving Europe with my father and mother in Berlin in September 1923.

enlisted in the Japanese Navy and served as a medical officer until the end of the war in 1945. Fortunately, I was stationed in Hokkaido and very soon was able to come back to Osaka only to find that the entire city had been demolished by air raids. I was devastated to see that my parents’ house had been almost

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completely destroyed and was discouraged by the realization that all of the memorable sites of my boyhood and youth during the past 25 years had also been wiped out. Food was scarce, and supplies of commodities were limited. I thought of joining my parents, who had taken refuge in Miyazu, and helping my father with his medical practice. However, before leaving Osaka, I went to see my former mentor, Professor Tenji Taniguchi, Department of Bacteriology of Osaka University School of Medicine, to tell him my plans. Contrary to my expectation, he was in roaring spirits and told me ‘‘Japan lost the war but did not disappear. You are young and are lucky to have survived. You should try to rebuild the country, and in order to do that one should try to start by rebuilding the foundation. To rebuild Japanese medical science, one should start from the basic research.’’ However, I was reluctant to agree with him under the sad social and financial circumstances that prevailed almost immediately after the war. Finally, he said, ‘‘Do you know the old Japanese saying: ‘One should take a seed of persimmon rather than a ball of rice’?’’ For a moment, I was puzzled and speechless but soon began to realize what my old professor was trying to tell me. Food was scarce in those days and rice was rationed. A ball of white rice was a big feast. But then, if one should choose persimmon seeds instead, the seeds will grow into many trees that will bear delicious fruits and beautiful flowers in 10 or 20 years. I was standing at a cross road of my life and was pondering over the question ‘‘Should I be a clinician like my father or should I be a research scientist in basic medicine like Professor Taniguchi?’’ Finally, I asked my father who, as I said, had settled in his hometown and was already 64 years old. To my surprise, he almost immediately responded in agreement with Professor Taniguch, and encouraged me to become a research scientist. He also told me that if I would remain in Osaka, he would come back there and open a clinic to help as many patients as possible, as adequate medical facilities were not yet plentiful and satisfactory in the city. I was so glad that my father was still full of adventurous spirit, and so I decided to return to my alma mater, where I was appointed as a junior faculty member and started my career as a microbiologist. However, the living conditions were miserable. My starting salary was 60 yen per month, approximately 17 cents at the exchange rate in those days. If I had chosen the practice of medicine, as had most of my classmates, I would have

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been economically much more comfortable even in those difficult times. Furthermore, laboratory conditions were almost hopeless; even the supply of electricity, gas and water was inadequate, and research money was extremely limited. There were no sources of experimental animals, much less the food to feed them. It seemed very difficult to even survive, let alone to carry out any experimental research in basic science. However, I was very fortunate to have been surrounded by a number of excellent and ambitious senior colleagues, such as Teishiro Seki, Masami Suda, Tsunehisa Amano and others. Despite the disastrous events during and after World War II, every one in the department was enthusiastic and devoted to basic science and held Professor Taniguchi in high esteem. Furthermore, the morale was generally high. Soon we learned that the Rockefeller Foundation had contributed a huge number of back issues of major scientific journals, as well as books, to the library of Tokyo University. So we saved money from our meager salaries, took trips to Tokyo to scan through the multitude of major scientific publications and brought back the essence of the latest information to Osaka, where we had seminars almost every day. It was Teishiro Seki who kindly showed and encouraged me to read Bacterial Metabolism by Majory Stephenson and Dynamic Aspects of Biochemistry by Ernest Baldwin. Reading these two books opened my eyes and shifted my interest from infectious diseases to metabolism. Even so, I still had to be content with seminars and discussions every day due to lack of money and inadequate facilities and supplies in the laboratory. One day in the early spring of 1946, I was quite unexpectedly visited by Dr. Yashiro Kotake, Emeritus Professor of Osaka University. Osaka University was a center of biochemical research in prewar Japan, because the Department of Biochemistry at this university had been created in 1905 by Kotake, where he elucidated major metabolic pathways of important amino acids such as tryptophan, histidine, and so forth and discovered and characterized kynurenine as the key intermediate in tryptophan metabolism. In 1934 and 1935, he wrote two solicited review articles on these accomplishments in the Annu. Rev. Biochem., vols. 3 and 4, and received many awards and honors for his work. He kindly provided us with several grams of tryptophan, kynurenine and other related chemicals and told us that he had

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already retired some time ago and had no use for these valuable chemicals and would be very happy if I could use them in my research. I was very happy to have met him again and find him in excellent health after all these years, because I had attended his course on biochemistry as a first-year medical student in 1939 and vividly remembered his eloquent lectures presented in a mixture of Japanese and fluent German, which presentations were very impressive but extremely difficult for me to understand. I thought I would never be able to become a biochemist. In 1925, Kotake and his collaborators elucidated the major metabolic pathways of tryptophan in mammals and, as I mentioned above, had discovered and identified kynurenine as the key intermediate, which was further metabolized to kynurenic acid, anthranilic acid or xanthurenic acid. These metabolites were then excreted in the urine (Fig. 3). Needless to say, I was grateful to him for his valuable gifts, but frankly speaking did not know what to do with them, partly because I did not know much about tryptophan metabolism and partly because Kotake and his colleagues had devoted almost their entire lives on this subject. Thus I felt that there was very little left for a rookie scientist like me to make exciting new discoveries in this field of research. One day, I came across a paper by G. S. Mirick, while looking through back issues of journals in OH N

COOH

Kynurenic acid CH2CHCOOH N H

NH2

Tryptophan

O CCH2CHCOOH

COOH

OH

NH2

NH2

OH

NH2

Kynurenine

Anthranilic acid

Catechol

COOH COOH

OH

Muconic acid N

COOH

OH

Xanthurenic acid CO2, NH3, H2O

Fig. 3. Metabolic pathways of tryptophan in 1949. Solid arrows ( ) indicate reactions in animals and white arrows ( ) indicate newly discovered reactions in Pseudomonas.

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the Tokyo University library. This article described soil microorganisms that could be selectively fished out from soil by the socalled enrichment culture technique and that produced adaptive enzymes capable of metabolizing various aromatic compounds such as o- and p-aminobenzoate, tryptophan and so forth [3]. I was intrigued by this new approach and upon returning to Osaka, immediately went out to the backyard of the laboratory, obtained several soil samples from the scorched ground, mixed them with a trace amount of tryptophan and tap water in test tubes, went home, and waited to see what would happen. After a few days, some cloudiness appeared in the supernatant solution indicating the growth of soil bacteria that could utilize tryptophan as their sole source of carbon and nitrogen for growth; but nothing happened in test tubes without tryptophan. By transferring the supernatant into the tryptophan solution, and repeating the subculture process several times, I was able to isolate in pure form a soil microorganism that could degrade tryptophan, which was later identified as Pseudomonas sp. It was such a simple and undoubtedly the least expensive experiment I ever performed throughout my entire career. But I did not realize at the time that I had found ‘‘a diamond in the desert’’ that played a decisive and valuable role in my research career. In these bacteria, I discovered that tryptophan was degraded to anthranilate via kynurenine, exactly the same sequence of reactions Professor Kotake had shown to occur in mammals many years ago. However, I was startled to find out that in this soil bacterium, anthranilate was further metabolized to catechol and ultimately completely degraded to CO2, NH3, and H2O (Fig. 3). To elucidate the metabolic steps involved in this newly discovered pathway, I then attempted to extract and purify the enzymes involved in it. In those days, enzymes were usually extracted from animal tissues and organs by the use of homogenizers such as the Waring blender, but these methods could not be applied to bacterial cells. However, after trying several rather primitive methods of extraction, I was able to solubilize only one enzyme in a cell-free form, namely, the enzyme that catalyzed the conversion of catechol, a newly found metabolite of tryptophan in this soil microorganism, to muconic acid (Fig. 3). Using a partially purified enzyme preparation, I measured oxygen consumption during the enzymic reaction with a Warburg manometer (respirometer), the only modern scientific instrument in our laboratory at that time. My colleagues

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and I were able to demonstrate clearly a stoichiometric relation, that is, one mole of atmospheric oxygen was consumed, while one mole of catechol was converted to one mole of muconic acid. The product of the reaction was extracted with ether from the acidified reaction mixture, and identified to be in the cis, cis-form. However, one crucial question as to the role of molecular oxygen in this catalytic mechanism remained unanswered at that time. The fundamental mechanisms underlying the metabolism of molecular oxygen in tissue respiration have long been one of the most important and challenging subjects in the field of bioenergetics, ever since Lavoisier initiated the study of biological oxidation processes in the 18th century. Lavoisier defined the term ‘‘oxidation’’ as the addition of oxygen atoms to a substrate, S, and referred to the opposite process, that of reduction, as the removal of oxygen atoms from an oxide, as shown here. oxidation S + O2

SO2 reduction

This role of oxygen molecules in biological oxidation was, however, vigorously challenged upon the discovery by Schardinger of an enzyme that catalyzed the conversion of aldehydes to acids in the presence of methylene blue but in the complete absence of atmospheric oxygen. In this case, the oxidation of aldehydes was accomplished by the concomitant reduction of methylene blue under anaerobic conditions. This finding prompted Wieland to propose a generalized mechanism of biological oxidation referred to as ‘‘the dehydrogenation theory’’ [4]. According to this theory, the essential characteristic of biological oxidation processes is the removal or transfer of electrons or hydrogen atoms from the substrate molecule (SH2) to an appropriate acceptor (A). oxidation SH2 + A

S + AH2 reduction

These enzymes were termed as ‘‘dehydrogenases’’; and in cases in which molecular oxygen served as the ultimate hydrogen

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acceptor producing H2O or H2O2, the enzymes were referred to as ‘‘oxidases.’’ However, according to the Wieland’s theory, which became the central dogma in the biological oxidation studies, the direct incorporation of atmospheric oxygen to a substrate was completely ruled out as a part of biological oxidation mechanisms [4] (for the more detail account of historical documentation, see Ref. [5]). It soon became apparent to me that this enzyme that I had discovered and partially purified from this soil microorganism appeared to be clearly different from all hitherto known oxidases and dehydrogenases described in the literature at that time. First, molecular oxygen was absolutely and specifically required for the reaction and no other oxidant such as methylene blue and any other dyes or coenzymes could substitute for molecular oxygen. Second, the partially purified enzyme preparation did not contain nor did it react with heme, flavins or pyridine nucleotides. Third, o-benzoquinone appeared not to be involved as a dissociable intermediate. Furthermore, the enzyme activity was not inhibited by KCN, NaN3 or CO but was inhibited completely by 10 3 M AgNO3. These and other lines of experimental evidence indicated strongly that this enzyme was not a typical oxidase or dehydrogenase and that molecular oxygen may have been directly incorporated into the substrate, catechol. These properties of the enzyme and the unique oxidative cleavage of the aromatic ring structure clearly showed this enzyme to be a novel one different from phenolase and other similar enzymes involved in the metabolism of numerous phenolic compounds previously reported in plants, mushrooms, insects and so forth. We therefore proposed to name it ‘‘pyrocatechase’’ [6]. Subsequent studies revealed that it was a trivalent non-heme iron protein and had an MW of approximately 90,000 Da. I then presented this work at the first Annual Meeting of the Japanese Biochemical Society after World War II in April 1949 at Kyoto University. Professor Kotake, who was sitting in the front seat, stood up and applauded our work, and mentioned that when he was young and studied biochemistry at the University of Ko¨nigsberg, Professor Max Yaffe, his mentor, had fed benzene to dogs, isolated muconic acid from their urine, and proposed that an enzyme like our pyrocatechase might be present in mammals. Comments from most other people in the audience were very encouraging and supportive, and many congratulated me for the

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discovery of pyrocatechase and a new metabolic pathway of tryptophan found in this soil microorganism. Even so, most people were skeptical about my hypothesis that this enzyme is an oxygen transferase rather than an oxidase, simply because the dehydrogenation theory of H. Wieland was an undisputable central dogma in the textbooks of biochemistry and enzymology at that time. In fact, in many textbooks published later, for example, in the Textbook of Enzymology authored by Hoffmann-Ostenhof [7], pyrocatechase was classified in the ‘‘miscellaneous’’ or ‘‘unclassified’’ category of oxidoreductases. Thus I did not dare propose the additive oxygenation mechanism in my first publication [6], and simply stated the experimental observations as mentioned above; because I was afraid that my paper would have been rejected if I had clearly stated my interpretation of the data. In the mean time, I married Takiko Satani in 1946, whose father, Yukichi, had studied urology and dermatology at the Johns Hopkins University in Baltimore, was a close friend of my father for a long time, and was the dean of the Osaka University School of Medicine when I received my M.D. degree. We then welcomed the arrival of our daughter Mariko in 1947.

Back to U.S.A. While I was preparing the manuscript of the pyrocatechase paper for publication, I wrote a letter to Professor David E. Green and asked him to make comments about my hypothesis; because in Japan, most people including my mentors and friends were skeptical about my interpretation, while some other people were enthusiastic and encouraged me to propose the additive oxygenation mechanism for ‘‘pyrocatechase.’’ Although I had never met Dr. Green before, I was aware that he had spent many years in Cambridge, England, where Sir Frederick Hopkins had discovered tryptophan and studied its metabolism and Dr. Green had been recently appointed as the first director of the newly created Enzyme Institute at the University of Wisconsin in Madison. He did not respond to my letter for some weeks, and so I decided to send the manuscript to the Japanese Journal of Biochemistry, the official journal of the Japanese Biochemical Society and one of the few journals that published in English in Japan at that time. Soon the manuscript was accepted for publication without any

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criticisms or revisions. After a few days, I received a letter from Dr. Green. When I opened it, I was startled to find that he did not say much about my paper except for several enthusiastic and polite comments but that he wanted me to come to Madison to join his group and was offering me a William Waterman Fellowship in Enzyme Chemistry. I was of course delighted but hesitated to accept his offer immediately, because Japan was still an occupied country and I thought there might be some legal problems as well as some hostile feelings toward me as a Japanese in light of the recent war. In fact, when I mentioned it to my mentors and colleagues at Osaka University, some people advised me to wait several years until the situation had improved. However, research facilities and working conditions were presumably much better in the United States, and there was the financial aspect to be considered. My starting salary as an instructor at Osaka University was 60 yen per month, approximately 17 US cents, not dollars! while the fellowship Green had arranged for me was $250 per month, an almost 1,500-fold difference! Finally, I decided to take a chance. I went to General McArthur’s head quarters in Tokyo to obtain a visa instead of the American embassy. To my surprise, I was interviewed by Surgeon General Sams and was encouraged and treated politely. In November 1949, I received an airplane ticket from Dr. Green and boarded a double-decker four-engine Boeing B-377 from Tokyo airport, which was a luxury in those days. It took about 36 h to fly from Tokyo to San Francisco with three stops for refueling. In San Francisco, I met my mother’s elder brother, Takashi Uchida, and his family and spent a few days recuperating and listening to the sad story about their experiences during World War II. Yoshi Uchida, the younger daughter, was a writer and had published more than 30 books and received several awards. Among her books, the best known was ‘‘Desert Exile,’’ in which she described the forced relocation of Japanese and Japanese–Americans during the war. At that time, there were about 120,000 people of Japanese origin living in the United States and about two-thirds of them had American citizenship. In May 1942, the Uchidas were evacuated from their home and were moved to Tanforan, a horserace track north of San Francisco, where they were housed in a stable. In September, they were moved to a concentration camp in Toparz, Utah, a desert town and released in 1943. Their story was scary and

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discouraging, so much so that I almost considered going back to Japan. Nevertheless, I decided to continue my venture and thanked my uncle and his family for their advice and took a plane to Chicago, and another small one to Madison. It was midNovember 1949 and snowing as I landed at the Madison airport. There I met two young Americans, Bernard Katchman and Ephraim Kaplan, both postdoctoral fellows of Green’s group in the Enzyme Institute. I soon got to know both Bernie and Eph, and found them to be the kindest and most considerate human beings I have ever met. In spite of the language barrier and the ethnic and religious differences, they treated me like a real brother, instructing me in various matters of daily life, especially American idioms (even some Yiddish jokes!), as well as acquainting me with procedures and jargon used in the lab and giving me considerable other advice. One day, they asked me what my name, ‘‘Hayaishi,’’ meant in Japanese; and so I replied, ‘‘quick stone.’’ They said ‘‘Schnellstein’’ might be a good translation into German, referring to ‘‘Einstein;’’ and so I was christened ‘‘Samuel Schnellstein’’ as a joke, the Samuel probably came from ‘‘Osamu.’’ Without their help, I would probably have been lost, lonesome and homesick. Thanks to their kindness and friendship, it did not take too long to get adjusted to and enjoy the American way of life in this beautiful university town. Soon I made many friends, among them Philip Feigelson, Philip Cohen, Henry Lardy, Takeru Higuchi, a Japanese-American who later became a famous pharmacology professor, and many others. At the Enzyme Institute, however, my work under the guidance of Dr. Green was not very successful, because my experimental results often contradicted his cyclophorase hypothesis, which turned out to be very controversial. In April 1950, I attended the Federation Meetings at Atlantic City and was greatly impressed by the presentation of a young anonymous scientist, who gave a 10-minute talk on the enzyme ‘‘isocitrate dehydrogenase.’’ I spoke to Bernie Katchman, who was sitting next to me, about this young man and he told me that the speaker’s name was Arthur Kornberg, head of the enzyme section of NIAMD, NIH in Bethesda and a new star in biochemistry in America. He also suggested that I should try to be mentored by such a young, promising biochemist rather than work with a famous, already well-established, but busy, big-name scientist. Soon afterwards Arthur came to Madison for a lecture; and, when

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I approached him, he kindly suggested that I apply for an NIH fellowship. I immediately sent in an application; but, in the mean time, Professor Roger Stanier, at the University of California at Berkeley, invited me to come to his laboratory to work for a collaborative project on the tryptophan metabolism of pseudomonas. He had independently studied tryptophan metabolism by a soil bacterium but had found another pathway, which he named the ‘‘quinolinic’’ pathway. He proposed to name my pathway the ‘‘aromatic’’ one, and wanted to clarify the difference between these two pathways by extracting all the enzymes involved to elucidate their detailed mechanism. Since it was a very challenging problem and closely related to my previous experience, I accepted his offer and, with Dr. Green’s consent, resigned from my post at the Enzyme Institute at the end of August 1950. Roger’s lab was located in the Life Science Building at Berkeley, and I was fortunate to meet there a number of outstanding scientists, such as C. B. van Niel, H. A. Barker, Michael Doudoroff, William Hassid and others, all of whom were very helpful, kind and welcoming. During my stay in Roger’s lab, we both worked very hard and were able to publish six papers during my fourmonth stay, which appeared in Science, Journal of Biochemistry and Journal of Bacteriology [8–13]. It was undoubtedly one of the most productive periods of my career, at least quantitatively. As the end of December approached, I received good news from Arthur that my fellowship application had been approved. So I asked my wife, Takiko, and my daughter, Mariko, to join me; and we moved to Bethesda, Maryland. Before, we left Berkeley, we met my uncle and his family and told them that I had had a wonderful time during the past year, one of the most enjoyable and profitable experiences of my life; and we thanked them for their help and advice. They were back in Oakland and had settled into a new cozy house. We congratulated each other on the upturn in our lives. The Enzyme Section at the NIH was a utopia for me. Arthur was doing his own experiments almost all the time, with the help of Bill Price, a senior technician, and a co-worker. I was the only postdoctoral fellow when I came to the lab, but later some new fellows joined us. In addition, Bernard Horecker and Leon Heppel were the senior and independent members. This was quite a contrast to Green’s lab, where he had more than a dozen postdoctoral fellows and few senior members. Every day, we had a

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luncheon seminar, at which every one brought his own lunch and took turns in alphabetical order to lead the seminar. One important paper was selected for critical discussion of its strategy, logic and methodology; unlike in the case of the journal clubs in other laboratories, where only the abstracts or conclusions were introduced without debating the experimental evidence, let alone having more detailed meaningful discussions. This seminar was often attended by people from other sections and departments, such as Herb Tabor, Alan Mehler, Bruce Ames, Jesse Rabionowitz, Terry and Earl Stadtman, Hans Klenow and others. Such a seminar became a legend in many other laboratories including my own; and when I moved to Kyoto University in 1958, the luncheon seminar became a tradition there and was nicknamed as the ‘‘Hayaishi training school.’’ During the two years at the NIH, I worked with Arthur on bacterial degradation of uracil and found malonyl CoA and acetyl CoA as intermediates. These findings prompted Feodor Lynen to come and to meet me at NIH. This was the beginning of a long association with ‘‘Fitzy,’’ which was not only scientifically rewarding, but was also cemented by a most heart-warming friendship. Fitzy was a son-in-law of Professor H. Wieland, who, as I mentioned earlier, had denied the existence of oxygentransferring enzymes and had proposed the dehydrogenation theory, and so Fitzy was always appreciative of my work on oxygenases. Two years passed since we had settled in Bethesda, and all of us were having a wonderful time; because Bethesda was a pleasant residential suburb of Washington, DC, and was socially and culturally very active and attractive. Even our daughter, Mariko, enjoyed a wonderful time as she quickly started to learn American English, to adjust to the American way of living and to make many good friends in the kinder-garten she attended. One day quite unexpectedly, Arthur invited me to his office and told me that he was going to move to St. Louis to accept the position as Chair and Professor of the Department of Microbiology at the Washington University School of Medicine. To my surprise, he then asked me if I was interested to come along with him to the Washington University as an assistant professor. I was not able to answer immediately, as it was only my third year in the United States and I did not think I would be capable of handling teaching and administrative duties in English. After a

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long talk with Arthur, I finally accepted the offer; and so my family and I moved to St. Louis in late 1952. Contrary to our expectation, life in St. Louis was quite pleasant, despite the fact that I had to spend so much time in preparing lectures, seminars and laboratory exercises for medical students, as well as being involved in the renovation of the old research building, the purchasing of new equipment and chemicals, the preparation of grant applications and so forth. I met a number of outstanding and friendly scientists who were kind and helpful in both teaching and research. Carl and Gerty Cori were especially understanding, as they were from abroad, too, originally from Prague. Stanley Cohen was living in the same faculty apartment building as the Hayaishi’s, and we became very good friends. Other notable scientists who became life long-friends included Martin Kamen, Oliver Lowry, Robert Furchgot and others. Time passed quickly and in the autumn of 1954, I received a phone call from Dr. S. Rosenthal of NIAMD, NIH, who asked me if I would be interested in coming back to Bethesda to take the position of Chief of the Section of Toxicology at NIAMD. I felt very much flattered but honored and thanked Dr. Rosenthal for thinking of me. I decided to accept his offer, and so we moved back to Bethesda in December 1954. This acceptance meant the fourth move of our family during five years since we came to the United States. I personally enjoyed having lived in different parts of the U.S.A. and also having made so many new excellent friends, but it must have been a lot of work for my family, especially for our daughter, who had to change schools and become accustomed to new neighbors and a different environment after each move. However, both Takiko, my wife, and Mariko, my daughter, had no problem at all getting adjusted to new neighbors and they, too, met so many kind and friendly people wherever they went (Fig. 4).

Discovery of Oxygenases Dr. Rosenthal was well aware that I was originally a microbiologist who then became a full-fledged biochemist-enzymologist, and so he wanted me to change the classical toxicology laboratory to a more modern and biochemical toxicology. In reorganizing the research program of the Section on Toxicology, I immediately

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Fig. 4. With Takiko and Mariko in St. Louis, March 1954.

thought of ‘‘pyrocatechase,’’ because I considered that the reaction catalyzed by this type of enzyme might be involved in detoxification mechanisms and that the molecular mechanisms underlying such an enzymatic reaction might be relevant to modern toxicology. Therefore, in a lecture I immediately presented my idea to the members of the Section and also several friends who had been invited from other departments (Fig. 5). As expected, most people were rather skeptical, because, after all, the ‘‘Dehydrogenation theory’’ of Professor H. Wieland was still the dominant theory in the textbooks of biochemistry and direct addition of molecular oxygen was generally believed to be nonexistent in living systems. The only persons who were very

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Fig. 5. Members of the section on toxicology, NIAMD, NIH, January 1956, the man on the right of the front row is Dr. Masayuki Katagiri, a co-author of my first paper on oxygenases in 1955.

enthusiastic about the adventurous project were Dr. Masayuki Katagiri, a postdoctoral fellow from Kanazawa University, Japan and Mr. Simon Rothberg, an expert in mass spectrometry. So, the three of us decided to join forces and were determined to carry out a crucial experiment. At that time, O18 2 , a stable isotope of molecular oxygen, was not commercially available; but some one told me that O18 2 was being produced as a by-product during distillation of salt water from the Dead Sea in Israel. So I immediately wrote a letter to the Weizmann Institute; and Dr. David Samuel, head of the Isotope Department, promptly responded and provided me with a highly concentrated preparation of H2O18. We 18 and then carried out generated O18 2 by the electrolysis of H2O the pyrocatechase reaction by using a highly purified preparation of the enzyme and the substrate catechol in the presence of O18 2 18 and H2O16 in one flask and of O16 in the other. The 2 and H2O product, muconic acid, was isolated, purified and analyzed by mass spectrometry for its O18 content. If pyrocatechase were a dehydrogenase or an oxidase, the incorporated oxygen should

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have been derived from H2O18 according to the Wieland’s theory. However, our results clearly demonstrated that the incorporated oxygen atoms were derived almost exclusively from O18 2 , not from H2O18. A small amount of O18 was lost, presumably due to the exchange reaction during the process of isolation and purification of the product, muconic acid. These data unequivocally demonstrated that enzymatic fixation of molecular oxygen did occur, contrary to the ‘‘dehydrogenation theory’’ [14]. I then proposed to name these oxygen fixation enzymes as ‘‘oxygenases.’’ Concurrently and independently, H. S. Mason and co-workers reported that a mushroom phenolase complex incorporated one atom of molecular oxygen into the substrate 3,4-dimethylphenol to form 4,5-dimethylcatechol while the other atom was reduced to water [15]. Mason proposed to name such an enzyme a ‘‘mixed function oxidase.’’ In 1956, I was asked to organize the first symposium on oxygenases at the American Chemical Society meeting, held that year in Atlantic City; and so I invited H. S. Mason, K. Bloch, M. Hayano and other speakers. I proposed the terms dioxygenase and monooxygenase for the oxygenases incorporating two and one oxygen atoms, respectively, per substrate. In 1957, Professor Otto HoffmanOsternhof of the University of Vienna came to the NIH for a visit and invited me to organize and chair a colloquium on oxygenases at the 4th Congress of the International Union of Biochemistry (IUB), which would be held in Vienna in 1958. In late 1957, Kyoto University School of Medicine decided to appoint me as Chairman and Professor of the Department of Medical Chemistry. This department, one of the oldest departments of biochemistry in Japan, had been founded in 1899 by Torasaburo Araki, the last student of the famous Hoppe-Seyler. It was considered the most prestigious center for biochemistry in Japan. Furthermore, Yashiro Kotake was Araki’s prote´ge´ and had spent some time in this department, although Kotake was a graduate of Osaka University. In February 1958, I accepted the offer from Kyoto and returned to Japan. In the summer of that year, I went to Vienna to attend the 4th IUB Congress and chaired the Colloquium on Oxygenases. It was the first international meeting on oxygenases and was very successful. Furthermore, after the Congress, I was invited to a number of universities and institutions and spent almost a month touring in Europe, lecturing and meeting famous biochemists, whose names were known to me only from their

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publications and correspondence. When I visited Karolinska ¨m greeted me warmly and Institute in Stockholm, Sune Bergstro showed me around the institute. He knew about our work on oxygenases and told me about his discovery of prostaglandins and we discussed the possibility that prostaglandin synthase might be a unique dioxygenase. This was my first exposure to prostaglan¨m, dins and the beginning of the long association with Bergstro Bengt Samuelsson, Sten Orrenius, Peter Reichard and their colleagues at the Karolinska Institute and Lars Ernster of Stockholm University. I also had a very pleasant and most memorable meeting with Otto Warburg, the ‘‘Pope’’ of biochemistry and famous for his oxygen activation theory. I told him that tryptophan dioxygenase is a hemoprotein and that we were able to demonstrate the Enzyme:Substrate:Oxygen complex spectrophotometrically as an obligatory intermediate. He appeared to be very pleased. In 1962, I edited and Academic Press published a monograph entitled Oxygenases, the first comprehensive treatise on this subject [5]. In 1964, I had the pleasure of presenting a plenary lecture entitled ‘‘Oxygenases’’ at the Sixth IUB Congress in New York. The session was chaired by Professor John T. Edsall. I had never met him before but will never forget his kind and encouraging remarks.

Kyoto University, My Second Alma Mater My first job as a newly appointed professor at the prestigious but ancient Department of Medical Chemistry was to renovate the old building and purchase numerous modern equipment and apparatus so that I could continue my research as actively as in the United States. The financial conditions in Japan were still unsatisfactory; and my salary as the youngest professor in Kyoto University was about one-thirteenth (7.7%) of my salary at the NIH. Fortunately, the Japanese Government had made a special effort to provide me with a large amount of grants; and many foundations and pharmaceutical companies offered their generous support in the form of starting grants. In addition, the NIH provided a substantial amount of research grant; and the Jane Coffin Memorial Fund, the Rockfeller Foundation, China Medical Board and several pharmaceutical companies in the United States contributed significant amounts of money, not only for research

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but also to rebuild and refurnish the old buildings and even to build a new building for radioactive experiments and a library. When I arrived in Kyoto in February 1958, the first graduate student who applied to join me was Yasutomi Nishizuka, who later became one of the most famous biochemists in Japan as the discoverer of protein kinase C. Soon the department became filled with young and ambitious postdoctoral fellows, graduate students and visiting scientists from all over Japan as well as from abroad. All these people were bright, highly motivated and hard working; above all they were all eager to learn new dynamic biochemistry and enzymology, which I had inherited and learned from my experience during the past 10 years in the United States from my mentors and friends, especially Arthur Kornberg. The time spent with Arthur, the first two years as a post-doctoral fellow in Bethesda and subsequent two years as assistant professor in St. Louis, provided valuable, indispensable and unforgettable experiences for me; and our friendship will last the rest of our lives (Fig. 6). At first, oxygenase-catalyzed reactions were generally thought to be rather unusual and might be limited to primitive forms of life, such as soil bacteria and mushrooms. Subsequent work in my own Kyoto laboratory and experiments by others all over the world revealed that oxygenases are ubiquitously found in animals, plants and microorganisms and play important roles not

Fig. 6. Arthur and Carolyn Kornberg, Takiko and Osamu Hayaishi on February 21, 1999, at Kornberg’s home in Portola Valley, CA.

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only in the biosynthesis and degradation of natural compounds but also in the degradation of synthetic compounds such as drugs, insecticides, chemicals, toxins and so forth. During my term in Kyoto, I was fortunate to collaborate with more than 500 individuals, including staff members, postdoctoral fellows, graduate students and visiting scientists. Among them more than 150 have been appointed as professors of biochemistry in various universities all over Japan and abroad. By collaborating with these able and outstanding scientists, whose names are too numerous to mention here, my project on oxygenases has proceeded into two major directions: (i) basic studies concerning the properties of oxygenases and characterization of the basic mechanism involved in their catalysis and (ii) elucidation of new metabolic pathways of physiological importance in which various oxygenases are involved, such as prostaglandins, tryptophan and other amino acids, poly- and mono-ADP-ribosylations, indoleamine dioxygenase and so forth. The year 2005 marked the 50th anniversary of the discovery of oxygenase. In commemoration of this event, two journals, J. Biol. Inorg. Chem. and Biochem. Biophys. Res. Commun., published special issues [16,17]. On April 10–12, Takeda Science Foundation Symposium on Oxygenases was held in Kyoto and a commemorative symposium was held as part of the 20th IUBMB Congress on June 19 also in Kyoto. It would be safe to say that the discovery of oxygenases has created a new vista in the field of bioenergetics and triggered a research explosion that has had an enormous impact on nearly all fields of medical, biological and physicochemical sciences as well as on environmental health science (see Refs. [5, 18–24] for general review articles).

The Mystery of Sleep Sleep is perhaps one of the most important and yet least understood of the physiological functions of the brain. Although we repeat sleep-wake cycles every day and night and spend almost one-third of our precious life time sleeping in bed or somewhere else, yet we are not able to answer even the most simple questions about sleep, such as ‘‘what is sleep?’’, ‘‘why do we need to sleep?’’ and most importantly, ‘‘where and how are sleep and arousal regulated and controlled?’’. Thus, sleep remains as one of the

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greatest mysteries in medical science in this new century. In the mean time, the number of patients suffering from sleep disorders has recently been increasing exponentially and now exceeds more than 30% of the total population in most countries. More than 88 different sleep disorders or sleep-related diseases have been described and classified in textbooks on sleep; but in most instances, the aetiologies are not clearly understood, simply because basic sleep science is still in its infancy. The humoral theory of sleep regulation, the concept that sleep is regulated by a hormone-like chemical substance rather than by a neural network, was initially proposed by a French neuroscientist, Henri Pie´ron, in Paris, and independently and concurrently by Kuniomi Ishimori in Nagoya, Japan, in the first decade of the last century. They took samples of cerebrospinal fluid from sleep-deprived dogs and infused them into the brains of normal dogs. The recipient dogs soon fell asleep. Thus these researchers became the first to demonstrate the existence of endogenous sleep substances. However, the chemical nature of their sleep substance was not identified. During the next 90 years or so, more than 30 so-called endogenous sleep substances were reported by numerous investigators to be present in the brain, CSF, and other organs and tissue of mammals. However, their physiological relevance has remained uncertain in most instances (see Ref. [25] for a review). In 1984, Professor Alexander Borbe´ly of the University of Zu ¨ rich in Switzerland proposed his famous two-process model of sleep regulation in his book Das Geheimnis des Schlafs or in English, Secrets of Sleep [26]. As is well known, the need to sleep grows stronger, the longer we stay awake. This ‘‘homeostatic’’ process is controlled by the sleep pressure that accumulates during the course of wakefulness and dissipates during sleep. In contrast, the ‘‘circadian’’ process, namely, the sleep-wake cycle operating during the day and night, is controlled by an internal pacemaker and is independent of prior sleep and waking. However, the molecular mechanisms underlying the sleep-wake regulation in these processes have so far remained a complete mystery. Prostaglandins (PGs) are sometimes referred to as tissue or local hormones, because nearly 30 different kinds of PGs are widely distributed in virtually all tissues and organs; and they exert numerous and diverse biological effects on a wide variety of

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physiological and pathological activities. However, relatively little had been known about prostaglandins in the brain until recently. In the late 1970s, we showed prostaglandin D2 (PGD2) to be the most abundant prostanoid in the brains of rats and other mammals including humans. Since PGD2 had long been considered as a minor and biologically inactive prostanoid, our findings suggested that it might be a unique constituent molecule of the brain and might have some specific and important function in this organ. Sure enough, we soon found out that PGD2 induced sleep when microinjected into the brains of rats. This serendipitous discovery occurred in 1981, shortly before I retired from Kyoto University at the mandatory retirement age of 63, and was published in 1982. The Ministry of Education almost immediately provided me a large research grant; and subsequently the Exploratory Research for Advanced Technology (ERATO), a subsidiary of the Ministry of Science and Technology, gave me a grant-in-aid of approximately 1.5 billion yen (15 million US dollars) for this project in 1983–1988, after I retired from Kyoto University and became the President of Osaka Medical College in Takatsuki City, about 50 km west of Kyoto. I still remember the remark made by Mr. Genya Chiba, who supported and initiated this project, ‘‘Dr. Hayaishi, do not be afraid to get a lot of strikeouts but try to hit just one big out-of-the-park homer.’’ He knew that sleep is such a formidable problem and that I would have to overcome so many difficult problems. He was quite right. It has been more than 20 years since then, but I feel myself having hit a big home run by my recently published article [27]. Sleep is a very complex phenomenon; wakefulness and the two major types of sleep, namely, non-REM sleep or slow-wave sleep and rapid eye movement sleep or REM sleep, which is sometimes referred to as paradoxical sleep, can be judged from the animal’s behavior. However, a more precise qualitative and quantitative assessment of the sleep-wake pattern is made by use of the EEG, electroencephalogram, the recording of brain waves, the EOG or electrooculogram, the recording of eye movement; and the EMG or electromyogram, that is, the recording of muscle tension. By means of a microinjection pump, the desired substance, such as PGD2, is infused continuously and slowly through a cannula chronically implanted into the third ventricle of a freely moving rat. The sleep stages are then determined on the basis of polygraphic recordings of EEG and EMG. Brain temperature as

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well as food and water intake is also monitored, and the behavior of the rat is recorded by use of a video-recording system under infrared light. When PGD2 was infused continuously into the third ventricle of a freely moving rat for 10 h during the night, when rats are normally active, both SWS and REMS increased significantly during the time of infusion. The effect was specific to PGD2 and dose-dependent. As little as picomolar amounts of PGD2 delivered per minute were effective in inducing excess sleep. Most importantly, however, the sleep induced by PGD2 was indistinguishable from physiological sleep as judged by several behavioral and electrophysiological criteria, including power spectral data, thus indicating the possibility that PGD2 might be a sleep hormone. In the brain of mammals, PGD2 is produced from the substrate PGH2 by the action of prostaglandin D synthase abbreviated here as PGDS. Two types of PGDS have been purified from the brain and crystallized in my laboratory. Lipocalin type, or L-PGDS, found in the leptomeninges, is mainly involved in sleep regulation; whereas the hematopoietic type or H-PGDS, localized in microglia and mast cells, is mainly involved in inflammatory processes. These enzymes are structurally different proteins but catalyze essentially the same reaction. L-PGD is a monomeric glycoprotein with a molecular weight of approximately 25,000 Da. After a long and extensive search for a specific inhibitor of this enzyme, we discovered inorganic tetravalent (4+) selenium compounds to be potent, relatively specific and reversible inhibitors when tested in vitro. They act upon both L- and H-type PGD synthases having essentially similar IC50 values of about 40 and 90 mM, respectively. These inhibitors seem to interact with the free sulfhydryl group in the active site of the enzyme, because the inhibition can be reversed by the addition of excess amounts of SH compounds such as glutathione or DTT. We then administered selenium tetrachloride into the third ventricle of a sleeping rat to see if it would inhibit sleep. When selenium chloride was infused into the third ventricle of a sleeping rat during the daytime, sleep was inhibited promptly and effectively. After about 2 h from the start of the infusion, sleep was almost completely inhibited. The effect was reversible, for when the infusion was interrupted, sleep was restored. Furthermore, the inhibition was reversed by the simultaneous

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infusion of SH compounds such as DTT and reduced GSH, as in the case of the in vitro enzyme activity. More recently, we infused a PGD receptor (DPR) antagonist, ONO-4127, into the subarachnoid space in the rostral basal forebrain of a sleeping rat, where receptors are densely localized. Sleep was inhibited dose- and time-dependently and reversibly during the infusion [27]. These results taken together clearly showed that PGD synthase and the PGD receptor play a crucial role in the maintenance of natural sleep and therefore, that PGD2 is involved in sleep under physiological conditions in rodents. In the mean time, in 1985 Roberts and coworkers in the U.S.A. reported that endogenous production of PGD2 increased up to 150-fold in systemic mastocytosis patients during deep sleep episodes. Then, in 1990, Pentreath and coworkers in the U.K. reported that the PGD2 concentration was elevated progressively and selectively up to 1000-fold in the CSF of patients with African sleeping sickness caused by Trypanosoma. These clinical observations are consistent with the notion that excessive endogenous production of PGD2 induces sleep in man under certain pathological conditions. In 1991, about 10 years after we started our project on sleep and prostaglandins, a re´sume´ of our experimental results appeared on the cover of the FASEB Journal [28]. PGD2 induces sleep in the preoptic area; whereas PGE2, its positional isomer, promotes wakefulness in the posterior hypothalamus. The molecular mechanisms involved in sleep regulation by PGD2 have been worked out during the past 10 years or so in my own and other laboratories in Japan, the U.S.A., especially by Dr. Saper’s group at Harvard and in Europe, especially by Dr. Luppi’s group in Lyon. L-PGD synthase, the key enzyme in sleep induction is present mainly, if not exclusively, in the membrane system surrounding the brain, namely, the arachnoid membrane, and the choroid plexus; although some enzyme protein is also detectable in oligodendrocytes in the brain parenchyma. Once produced, PGD2 is secreted into the CSF and circulates in the ventricular and subarachnoidal space. In contrast, PGD receptors, abbreviated as DPR, are exclusively localized in a small area on the ventro rostral surface of the basal forebrain. PGD2 circulating in the CSF then binds to this receptor, where a signal for sleep is generated. The signal initiated by the binding of PGD2 to its specific receptor is transmitted across

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the pia membrane and through the brain parenchyma to the VLPO or ventrolateral preoptic area, a known sleep center, where the so-called sleep neurons are activated. This transduction is mediated via adenosine by way of the A2A adenosine receptor. The VLPO projects to the TMN or tuberomammilary nucleus, a wake center located in the posterior hypothalamus, through GABAergic and galaninergic neurons and sends inhibitory signals to downregulate the wake neurons. Thus sleep is induced by upregulation of the sleep neurons and at the same time downregulation of the wake neurons by a flip–flop mechanism. To substantiate our conclusion, and to further elucidate the exact role of the PGD2 system in the whole live animal, we then generated knock-out mice for L-prostaglandin D synthase. These mice are supposedly unable to produce PGD2 and therefore might be predicted to be unable to sleep. Contrary to our expectation, however, the KO mice were viable and appeared to be quite healthy and to grow, breed and even sleep normally. The circadian profiles of NREM and REM sleep of wild-type mice, and L-PGDS KO mice, appear to be essentially identical under macroscopic examination and there is no major difference between them in the daily amounts of sleep and wakefulness. The same results were also obtained for PGD receptor KO mice. These findings apparently contradict the previous pharmacological experimental results I mentioned above, namely, that the PGDS inhibitor and the PGDR antagonist can promptly and effectively block sleep by i.c.v. infusion. However, such a lack of effect on phenotypes is not uncommon among KO mice for neurotransmitters, peptides and so forth that are reportedly involved in sleep or arousal regulation. For example, Ohtsu and Watanabe and coworkers reported that mice lacking histidine decarboxylase or histamine H1 receptors exhibited a circadian rhythm almost identical to that of the wild-type mice, even though each of these abolished systems is thought to regulate wakefulness [29]. All these experimental results from my own and other laboratories taken together were interpreted to mean that, because sleep is essential for life, the sleep-regulatory system is composed of a complicated network with built-in redundancies; Therefore, a deficiency caused by removing a gene in one system may be effectively compensated by other systems during early ontogenic development. To obtain more convincing evidence that would fully justify such an interpretation, I wanted to demonstrate

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conclusively that the inhibitory effect of SeCl4 on sleep was due exclusively and specifically to the inhibition of PGD synthase activity and not to some non-specific or general toxic effect of selenium. As mentioned earlier, selenium-induced inhibition of sleep in rats was initially reported by Matsumura in my laboratory in 1991 [30]. We have now confirmed these results in mice by administering an intraperitoneal bolus injection of SeCl4 to wildtype mice at eleven o’clock in the morning. In good agreement with the previous experiments on rats, both non-REM and REM sleep were inhibited promptly and effectively and almost completely after about 1 hr, and this inhibitory effect lasted about 5–6 h under these conditions [27]. When L-PGDS KO mice were used instead of the wild-type mice, no inhibition was observed at all, clearly indicating that the sleep inhibition by SeCl4 was due to its specific inhibitory effect on L-PGDS and not to some other non-specific toxic effect. When SeCl4 was administered to L- and H-PGDS double KO mice under the same experimental conditions, neither the inhibition of sleep nor the increase in sleep during the night was observed. SeCl4 had no effect at all on the sleep of L- and H-double KO mice. These results clearly show that SeCl4 is not toxic to sleep per se but inhibits sleep by inhibiting the endogenous production of PGD2 by PGDS. The sleep of DPR KO mice was also not inhibited significantly by selenium, indicating that in these mutant mice their PGD2 system was not functioning and had probably been replaced by some other system during embryonic development. We then subjected these mice to sleep deprivation to find out if PGD2 is also involved in the homeostatic regulation of sleep. As shown previously, the circadian profiles of the sleep-wake patterns of the wild-type (WT) and PGDS KO mice appeared to be essentially identical under macroscopic examination. However, when the WT mice were subjected to sleep deprivation for 6 h immediately before the onset of their wake period, a pronounced rebound was observed in NREMS; whereas little, if any, rebound occurred in NREMS in the PGDS KO mice. The total amount of NREM sleep rebound exceeded more than 60 min in the WT mice as well as in the histamine H1 receptor KO mice and prostaglandin EP1 receptor KO mice, which were used as controls. However, the sleep rebound was almost non-existent in the KO mice for

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PGD synthase as well as in those for the PGD receptor and or adenosine A2A receptor. These results clearly show that the PGD2 system, including the adenosine A2AR, plays a crucial role in the homeostatic regulation of NREM sleep [31]. Thus, it may not be too far-fetched to say that prostaglandin D2 is most likely the endogenous sleep substance that was described by Pie´ron and Ishimori almost 100 years ago. These results altogether showed: (1) Prostaglandin D2 is essential for the maintenance of the sleep state in normal or wild-type animals. (2) The adenosine and A2A receptor system is a link between the humoral and neural mechanisms of sleep-wake regulation. (3) Prostaglandin D2 plays a crucial role in the circadian [27] and homeostatic [31] regulation of NREM sleep (see reviews [32] and [33]). During the term of my professorship at Kyoto University School of Medicine, which lasted over 25 years, I also served concurrently as an adjunct professor of Kyoto University Institute of Chemical Research from 1959 to 1976, of Osaka University School of Medicine from 1963 to 1965, of Tokyo University School of Medicine from 1970 to 1974 and of several other universities in Japan as well as abroad, in addition to serving as the dean of the Kyoto University School of Medicine from 1979 to 1981. I also held the office of President of International Union of Biochemistry (IUB) from 1973 to 1976. I was extremely fortunate to have had a number of colleagues assisting me in the administrative duties related to the IUB, such as Professor William Whelan, general secretary of IUB during my term of the President. I also count myself lucky to be able to mention numerous scientific colleagues who have collaborated with me all these years, including Yasutomi Nishizuka, Tasuku Honjo, Mitsuhiro Nozaki, Shozo Yamamoto, Yuzuru Ishimura, Yoshihiro Urade and many others. It has been only through such collaborative efforts that I have been able to keep up my research activities all these years.

Osaka Bioscience Institute Shortly after I retired from Kyoto University, Mr. Yasushi Ohshima, then the mayor of Osaka, the second largest city in Japan, and Professor Yuichi Yamamura, President of Osaka University, a renowned biochemist and a long-time friend of

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mine, approached me and invited me to become the director of a new research institute focusing on bioscience and biotechnology. This undertaking had been planned to commemorate the 100th anniversary of the foundation of the Osaka Municipal Government. The Kansai region, where Osaka, Kyoto and Kobe are situated, has long been involved in various fields of bioscience, such as fermentation, brewing, drug manufacturing, chemical industry and so forth. It was Mr. Ohshima’s idea to take advantage of this tradition and to create a research institute with the aim of conducting world-class research on more advanced basic science. He also encouraged me to build a second and modern ‘‘Tekijuku’’ to train, educate and nurture future leaders in bioscience. Tekijuku was an exclusive private school in Osaka during the Edo period in the 18th century, the only source of Western scientific knowledge in Japan during the period of national isolation. Tekijuku attracted many ambitious and gifted young people from all over Japan. I was very much attracted by his idealism and accepted his offer under the conditions that this new institute would be operated by an entirely new system, one that has never existed in Japan in the past. For example, there would be no tenure positions: A review system by outside specialists, complete academic freedom of research subjects, and so forth. My conditions were accepted, and so I became the director of this new and innovative institute, known by the acronym OBI. The institute consisted of four departments to start with, each department having about ten employees, including head, vice head, two members plus 5 to 6 other positions. This is quite in contrast to the traditional university system, whereby each ‘‘Koza’’ or department usually has only 3 to 4 members including professor, assistant professo, and 1 or 2 assistants. Salaries at OBI are higher than those at other comparable institutions and financial support for research is much more abundant. Once in a year, all scientists are required to present their accomplishments in English at the meeting of the advisory board, which consists of several distinguished scientists from Japan and abroad. The head of each department has complete freedom in their research but also is completely responsible for the outcome. Unlike many Japanese traditional institutions, employment is judged solely on the basis of scientific merit; and no consideration is paid to nationality, school, degree, age, sex, etc. of the candidates. Maybe this description of OBI sounds like

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Fig. 7. With the statue of Don Quixote around 1987 at OBI.

too idealistic a view. In fact, a friend of mine nicknamed me as the ‘‘Japanese Don Quixote’’ and gave me a wooden statue of Don Quixote and Sancho Panza to congratulate the creation of OBI (Fig. 7). It has been 19 years since then and the OBI has published many scientific achievements during the relatively short period of its existence. Some of the best-known examples are those regarding the basic mechanisms of apoptosis by Shigekazu Nagata, control mechanisms of sleep-wake regulation by my own group, regulatory mechanisms of neural networks by Shigetada

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Nakanishi, and molecular mechanisms of cancer by Hidesaburo Hanafusa and others. A 2002 report from ISI Thompson ranked OBI as #1 internationally in terms of the impact of molecular biology and genetics papers published during 1991–2001. OBI was thus reported as a world leader in scientific research in a recent article in ASBMB Today [34]. Dr. Samuel Coleman remarked, ‘‘Japan needs far more OBI-like institutions to promote career development’’ in his book Japanese Science – From the Inside [35]. In fact, many of the leading universities and scientific institutions in Japan are now adapting their programs along the lines of the OBI system. Nature is the Guide Professor Yashiro Kotake once gave me a handwritten prose that read as follows: ‘‘You must read books. You must also think hard. But an ordinary person must work hard. To work hard means to become intimate with Nature. You must observe Nature. Then you will be able to find Nature.’’ I shall be 87 years old on January 8, 2007. I feel extremely fortunate to have been able to enjoy a good life and to continue to do research almost all my life. Over the years, I have been very lucky to have met and become associated with so many outstanding, kind and helpful people, both scientists and non-scientists. Their names are too numerous to mention here, but to all of them I express my deepest gratitude. As to a small accomplishments I have made, most of them have been due to my association with my teachers, mentors, collaborators and friends. However, I honestly feel that the most important teacher for me has always been the great Nature, and I am grateful to have been a bench scientist all my life and to have been close to Nature rather than to have been a teacher, a statesman or an administrator. ACKNOWLEDGMENTS

I dedicate this article to Jitsuzo and Mitsu Hayaishi who gave me good DNA. They both passed away at the age of 95 and did not have any major diseases. I would like to thank my wife, Takiko, for her many years of faithful support and devotion and both her and our daughter Mariko and her family for providing one big

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happy family. I thank Professor G. Semenza for inviting me to write this article and his constant encouragement to this lazy author, Dr. L. Frye and Mrs. Mariko Akizuki for their help in the preparation of this manuscript, and Miss Naoko Ueda for her secretarial assistance.

REFERENCES [1] Benson, A.A. (2002) Annu. Rev. Plant. Biol. 53, 1–25. [2] Hayaishi, J. (1924) Z. Hyg. Infekt.-Kr. 102, 87–93; 102, 201–205; 103, 59–77. [3] Mirick, G.S. (1943) J. Exp. Med. 78, 255–272. [4] Wieland, H. (1932) On the Mechanism of Oxidation. Yale University Press, New Haven. [5] Hayaishi, O. (1962) In Oxygenases (Hayaishi, O., ed.), p. 1. Academic Press, New York. [6] Hayaishi, O. and Hashimoto, Z. (1950) J. Biochem. 37, 371–374. [7] Hoffman-Ostenhof, O. (1954) Enzymologie. Springer-Verlag, Vienna. [8] Stanier, R.Y. and Hayaishi, O. (1951) Science 114, 326–330. [9] Stanier, R.Y., Hayaishi, O. and Tsuchida, M. (1951) J. Bacteriol. 62, 355–366. [10] Stanier, R.Y. and Hayaishi, O. (1951) J. Bacteriol. 62, 367–375. [11] Hayaishi, O. and Stanier, R.Y. (1951) J. Bacteriol. 62, 691–701. [12] Tsuchida, M., Hayaishi, O. and Stanier, R.Y. (1952) J. Bacteriol. 64, 49–54. [13] Hayaishi, O. and Stanier, R.Y. (1952) J. Biol. Chem. 195, 735–740. [14] Hayaishi, O., Katagiri, M. and Rothberg, S. (1955) J. Am. Chem. Soc. 77, 5450–5451. [15] Mason, H.S., Fowlks, W.L. and Peterson, E. (1955) J. Am. Chem. Soc. 77, 2914–2915. [16] Hayaishi, O. (2005) J. Biol. Inorg. Chem. 10, 1–2. [17] Hayaishi, O. (2005) Biochem. Biophys. Res. Commun. 338, 2–6. [18] Bloch, K. and Hayaishi, O. (1966) Biological and Chemical Aspects of Oxygenases. Maruzen Co., Ltd., Tokyo. [19] Hayaishi, O. (1974) Molecular Mechanisms of Oxygen Activation. Academic Press, New York. [20] Hayaishi, O. (1974) Molecular Oxygen in Biology. North-Holland, Amsterdam. [21] Hayaishi, O. and Asada, K. (1977) Biochemical and Medical Aspects of Active Oxygen. University of Tokyo Press, Tokyo. [22] Nozaki, M., Yamamoto, S., Ishimura, Y., Coon, M.J., Ernster, L. and Estabrook, R.W. (1982) Oxygenases and Oxygen Metabolism. Academic Press, New York. [23] Yamamoto, S., Nozaki, M. and Ishimura, Y. (1991) International Symposium on Oxygenases and Oxygen Activation. Yamada Science Foundation, Osaka.

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[24] Ishimura, Y., Nozaki, M., Yamamoto, S., Shimizu, T., Narumiya, S. and Mitani, F. (2002) Oxygen and Life – Oxygenases, Oxidases and Lipid Mediators. Elsevier, Amsterdam. [25] Inoue´, S. (1989) Biology of Sleep Substances. CRC Press, Boca Raton, FL. [26] Borbe´ly, A. (1986) Secrets of Sleep. Basic Books, New York. [27] Qu, W.-M., Huang, Z.-L., Xu, X.-H., Aritake, K., Eguchi, N., Nambu, F., Narumiya, S., Urade, Y. and Hayaishi, O. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 17949–17954. [28] Hayaishi, O. (1991) FASEB J. 5, 2575–2581. [29] Ohtsu, H. and Watanabe, T. (2003) Biochem. Biophys. Res. Commun. 305, 443–447. [30] Matsumura, H., Takahata, R. and Hayaishi, O. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9046–9050. [31] Hayaishi, O., Urade, Y., Eguchi, N. and Huang, Z.-L. (2004) Arch. Ital. Biol. 142, 533–539. [32] Hayaishi, O. (2002) J. Appl. Physiol. 92, 863–868. [33] Hayaishi, O. (2005) In Sleep: Circuits and Functions (Luppi, P.-H., ed.), pp. 65–82. CRC Press, Boca Raton, FL. [34] ASBMB Today (2006) October, 5 pp. 8–10. [35] Coleman, S. (1999) In Japanese Science –From the Inside (Coleman, S., ed.), pp. 81–127. Routledge, London.

G. Semenza (Ed.) Stories of Success – Personal Recollections. X (Comprehensive Biochemistry Vol. 45) r 2007 Elsevier B.V. All rights reserved. DOI: 10.1016/S0069-8032(07)45002-1

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Chapter 2

A Voyage to the Inner Space of Cells$ WOLFGANG BAUMEISTER Department of Structural Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany E-mail: [email protected]

$

This is an updated version of the Stein and Moore Award Address, which was originally published in Protein Science (2005) 14, 257–269 and appears here with permission.

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Abstract This article is a personal account of the development of biomolecular electron microscopy over a period of almost four decades. The nineteen seventies were marked by the advent of electron crystallography and, later, we have seen the emergence of single particle methods as a powerful tool for investigating the structure and function of macromolecular complexes. Recently, the development of electron tomography has opened the door for studying macromolecular structures in their functional environment – the cell. Keywords: Structural biology; Electron microscopy; Protein degradation; Protein quality control; Proteomics

Introduction The title I have chosen for my personal recollections describes, in a nutshell, the direction my scientific endeavors from the time of my PhD thesis, which I began in 1970, to the present day. Over the years, I have changed fields a number of times. There were periods when I was preoccupied with the development of methods; at other times, the focus was on biological problems. Science can be advanced by new hypotheses about how things work, which can be tested and proven right or wrong, and by new methods which enable us to tackle questions that we were unable to address with the existing methods. Or, as Richard Feynman put it: ‘Science means, sometimes, a special method of finding things out. Sometimes it means the body of knowledge arising from the things found out. It may also mean the new things you can do when you have found something out, or the actual doing of new things. This last field is usually called technology y’ (R. P. Feynman in the John Danz Lectures, 1963). Apprenticeship with Great Freedom After graduating from the University of Bonn in late 1969, I joined the Institute of Biophysics and Electron Microscopy at the University of Du ¨ sseldorf in January 1970. The director of the Institute at the time was Helmut Ruska, who became my PhD supervisor. Helmut Ruska, a medical doctor, was the younger brother of

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Ernst Ruska, the electrical engineer who in 1932, at the age of 26, had published his calculations on the theoretical resolving power of an electron microscope and, in the face of strong scepticism, had completed the development of a commercial instrument by 1939 [1]. Rarely has a scientific instrument had such an impact on so many branches of science, and yet it took more than 50 years before Ernst Ruska was rewarded with the Nobel Prize in Physics in 1986 for his fundamental work in electron optics and his design of the first electron microscope. Helmut Ruska, who was very close to his brother, realized immediately the potential of such an instrument for the biomedical sciences, in particular the visualization of hitherto invisible infectious agents and for ultrastructural studies of cells (Figure 1). Helmut Ruska played a very important role in the early days of electron microscopy, not only by raising awareness and support – his clinical mentor at the Charite´ in Berlin, Richard Siebeck, became a decisive advocate at a critical time – but also by his achievements in the visualization of viruses, bacteria and blood cells (for a review see [2]; for relevant references see also [1]). At the time I joined Helmut Ruska’s laboratory, the performance of transmission electron microscopes had reached a level that allowed the imaging of single heavy atoms. Several groups in Europe, the United States and in Japan tried to take advantage of

Fig. 1. Pioneers of electron microscopy. Left: Ernst Ruska (1906–1988) Right: Helmut Ruska (1908–1973).

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this capability and to use heavy atoms as site-specific labels, e.g. for mapping the bases in strands of DNA. In the same vein, Helmut Ruska gave me the task of exploring the use of heavy atom labels to study membrane topology. I decided to begin with well-defined model membranes before tackling membranes of biological relevance. I never got that far! Using Langmuir-Blodgett techniques, I prepared monomolecular layers at the water–air interface and transferred them under precisely controlled conditions to specimen supports, but when I exposed my carefully designed lipid layers to the electron beam, they faded away before I was able to take a picture. Occasionally, I obtained images of remnants of them with the heavy atoms coalesced into clusters. Eventually, with an unusually radiation-resistant organometallic compound of no relevance to biology, thorium-hexafluoracetylacetonate, I succeeded in obtaining images showing a heavy atom pattern that was consistent with my design plan [3]. Helmut Ruska was preoccupied with administrative duties during my time as a graduate student in his laboratory and, as a consequence, his supervision of me was very casual. Nevertheless, he was very supportive and he gave me all the resources I needed for my work. In late August 1973, only a few months after receiving my PhD, Helmut Ruska died after a short illness. I had offers from other places, but decided to stay in Du ¨ sseldorf, and since it took several years until a successor for Helmut Ruska was found, I enjoyed complete freedom during my postdoctoral years. Thanks to benevolent reviewers, I obtained my first grant in 1974 and I began to work on radiation damage – the electron microscopist’s greatest foe. I used a variety of methods for a quantitative assessment of radiation damage in lipids and proteins under the conditions encountered in electron microscopy [4,5]. My hope was that a better understanding of the underlying radiation chemistry might enable us to find a remedy – a vain hope as it turned out [6].

Heading into New Directions Having realized that I was on an unproductive path, I had to change direction. While the electron microscopy community in Germany with its strong tradition in electron optics was preoccupied with ‘high resolution’, others, driven more strongly by their

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desire to obtain insights into biomolecular architectures, took more pragmatic approaches. Already in 1968, De Rosier and Klug had formulated the principles for the three-dimensional reconstruction of objects from projection images and applied them to the tail of bacteriophage T4, taking advantage of its helical symmetry [7]. When working with periodic or repetitive structures, one can minimize radiation damage by underexposing the samples; the information is retrieved from the statistically noisy images by averaging over many identical structures. Averaging can be performed by direct superposition (see, for example, [8]) or by Fourier-transform filtering (see, for example, [9]) using both optical and digital methods [10]. Using the aforementioned stratagem and applying it to unstained, glucose-embedded purple mem˚ brane, Henderson and Unwin succeeded in 1975 in obtaining a 7 A structure of bacteriorhodopsin [11], which became the paradigm of a membrane protein structure. Having read about a bacterium of legendary radiation resistance, Micrococcus radiodurans, (now Deinococcus radiodurans), and knowing from the literature that a regular protein layer was a component of its cell wall, I focussed my work on this structure. Before long, I obtained decent micrographs of this structure (Figure 2a), which I called the hexagonally packed-intermediate (HPI) layer, but I ran into a dilemma with the image processing. After having done some initial experiments with optical filtration, I became convinced that computer methods were the future, however, with the notable exception of Walter Hoppe in Munich, optical methods were preferred to computer methods in Germany at the time. The arguments in favor of optical image processing (averaging and correction of contrast transfer function) were the size of the images that could be processed and the speed. The downside was lack of flexibility, and the fabrication of suitable masks became a serious bottleneck. Since I had neither access to the necessary infrastructure nor the know-how for computer-based processing, I started a collaboration with Olaf Ku ¨ bler at the ETH in Zu ¨ rich who was in an inverse position: he had the software and hardware that was needed but was short of data. In the following years, we made substantial progress in elucidating the molecular architecture of the D. radiodurans cell envelope [12–15]. In 1979, I organized a meeting entitled ‘Electron Microscopy at Molecular Dimensions’ held at Burg Gemen near Mu ¨ nster, Germany, which in retrospect became quite influential (see, e.g., [16]).

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Fig. 2. (a) Electron micrograph of a metal-shadowed HPI-layer as obtained by detergent extraction of the Deinococcus radiodurans cell envelope. Areas marked R show the rough inner surface; areas marked S show the smoother outer surface (for details see [15]). (b) 8 A˚ projection map of the HPI-layer embedded in aurothioglucose (for details see [86]).

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Besides state-of-the art applications, it covered many developments in technology from image recording and processing to low-temperature EM, or strategies for making regular 2-D arrays [17]. The intrinsic disorder in the 2-D protein arrays limits the resolution one can attain by Fourier filtering, and during the Gemen meeting, I became convinced that there are better ways of dealing with imperfect 2-D crystals. The emerging methods for averaging of single molecules offered an alternative, and I decided to join forces with Joachim Frank. A few months later when I visited him in Albany, we explored the application of correlationbased averaging to the micrographs of the HPI-layer, but to our disappointment we failed to obtain meaningful results during this rather short period of time. A year later, when I spent several months at the Cavendish Laboratory in Cambridge, England, working with Owen Saxton, we were able to overcome the problems and obtained the first correlation-averaged images of the HPI-layer with a significantly improved resolution. In trying to get this work published, we faced unusual problems; it took several rounds of reviewing and several steps down the ladder of (journal) prestige, until our manuscript was finally published [18]. In retrospect, however, it is gratifying to see that more than 20 years later, this paper is still cited frequently and, in the guise of ‘lattice unbending’ ([19]) our stratagem for overcoming the limitations due to lattice disorder became part of the standard repertoire used for processing images of 2-D crystals. Shortly thereafter, we applied correlation averaging to STEM images of unstained preparations of the HPI-layer and obtained the first quantitative mass maps [20], the beginning of a long-standing and successful collaboration with Andreas Engel at the Biocenter in Basel, Switzerland.

The First Decade in Martinsried: Studying Protein Architecture on Prokaryotic Cell Surfaces At the beginning of 1982, I moved to the Max-Planck-Institute of Biochemistry where, after a short overlap period, I was appointed successor of Walter Hoppe. Hoppe was a microscopist-turned X-ray crystallographer of remarkable originality and theoretical ability but with limited interest in the practical aspects of structural biology. No humble man, he insisted that his ‘nonconventional’ approach to the structural analysis of individual macromolecules,

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as he liked to call it, was superior to other strategies (for a review, see [21]). I remained unconvinced, and felt that the clever tactics of ‘single particle analysis’ as pioneered by Joachim Frank, a former student of Hoppe, and Marin van Heel at the Fritz-Haber-Institute in Berlin held greater promise. Not only did their approach greatly simplify data acquisition, the combination of intelligent image classification procedures and extensive averaging had the great advantage of yielding significant and interpretable structural data (for a recent review see [22]). In all fairness, I must add that in spite of a fierce public dispute I had with Walter Hoppe a few years earlier [23,24]) and divergent views on the course to take, he was, in general, supportive when I arrived in Martinsried and began to set up my laboratory. We continued our work with the HPI-layer; a 3-D model was generated in due course and, using cryomicroscopy, ˚ projection map was also obtained [25,26] (Figure 2b). an 8 A With the plentiful resources now at our disposal, we not only extended our structural studies to several other bacterial surface layers, we also widened our repertoire of methods. Our comparative structural studies revealed some common architectural principles [25,27]) and sequence analyses led to the identification of new motifs [28–30]) such as the S-layer homology domain (for a recent review see [31]) but the biological function of S-layers remained an enigma. Intuitively, I still feel that there must be some function beyond mediating adhesion to animate or inanimate surfaces or protecting underlying components of the cell envelope, but this remains pure speculation. Colleagues in Martinsried (Wolfram Zillig) and in Regensburg (Karl-Otto Stetter) introduced me to the exciting world of extremophiles. Most hyperthermophiles belong to the archaeal domain of life where (glyco)protein surface layers are common. They represent the main macromolecular component of the cell envelope and are intimately associated with the plasma membrane. Some show a high degree of order and have a role in maintaining and possibly determining cell shape [32,33] while others form poorly ordered and flexible surface networks on pleomorphic cells [34,35]. In spite of their apparent diversity, archaeal surface layers have some common structural principles: a stalk of variable length (10–70 nm) emanates from a membrane-anchoring domain and connects to a highly variable (filiform or bulky) domain that forms a canopy-like layer by means of end-to-end contacts enclosing a

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quasi-periplasmic space [36]. A periplasmic space of unusual width and maintained by a rod-shaped spacer protein (Omp a) is also found in the hyperthermophilic ancestral bacterium Thermotoga maritima [37,38]. The structural principles of archaeal surface layer proteins is exemplified particularly clearly by tetrabrachion, the giant glycoprotein found on the surface of Staphylothermus marinus, where it forms a poorly ordered, branched network [35]. This filiform molecule is anchored in the cell membrane at the C-terminal end of a 70-nm long stalk and branches at the other end into four arms, each of 24 nm length, which form the canopy-like meshwork. A hybrid approach, which used EM and biochemical data as well as molecular biology and bioinformatics, led to a very detailed model structure (Figure 3) [39], the salient features of which, in the meantime, have been confirmed by X-ray crystallography [40]. The C-terminal part is formed by a right-handed, coiled coil of four a-helices; the almost flawless pattern of aliphatic residues, mainly leucine and isoleucine, throughout the hydrophobic core of the stalk provides an explanation for its exceptional stability. At a proline residue, the stalk switches from a righthanded supercoil to a left-handed one. At a flexible glycine-rich hinge region, the stalk branches into four arms, each formed by a ‘heavy chain’ and a ‘light chain’ which in turn are each derived from the translated 1524 residue polypeptide by internal proteolytic cleavage. The most likely topology of the arms is a threestranded coil of antiparallel b-sheets. There is a patch of negative charges on the outer face of the coiled coil near the middle of the stalk, which serves as an anchoring device for a large, hyperthermostable protease of the subtilisin family; in the stalk-bound form the protease is resistant to heat inactivation up to a temperature of 1251C [41], while the stalk withstands heating up to 1301C. Obviously, one function of the Staphylothermus surface layer is to provide an extracellular holding compartment for a protease that could otherwise cause havoc.

The Next Decade: Proteasomes, Thermosomes and Other Elements of Intracellular Protein Quality Control In 1989, my laboratory became interested in studying the structure and function of a large (20S) protein complex, at the time known as

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Fig. 3. (a) The Staphylothermus marinus surface layer as revealed by freezeetching (left). In the absence of MgCl2 the detergent extracted surface layer dissociates into micelles formed by the tetrabrachion–protease complexes (right). (b) The tetrabrachion–protease complex. Left: Model showing the mode of interaction of the tetrabrachion–protein complexes in the layer structure. Center: Electron micrograph of the negatively stained complex released from the surface layer meshwork by SDS-heat treatment (for details see [77]). Right: Folding topology of tetrabrachion. The location of N-terminal residues, cysteine residues and the unique proline residue separating the left- and right-handed supercoiled domains are marked by circles. Putative disulfide bridges are indicated. The flexible hinge segment, the protease-binding region and the membrane anchor are marked by rectangles (for details see [76]) (see Colour Plate Section at the end of this volume).

the multicatalytic proteinase [42]. Already in 1980, a large, multisubunit protease had been isolated and characterized [43–45]. Initially, the multicatalytic proteinase was believed to be composed of 3–5 subunits, ranging from 24 kDa to 28 kDa in size; it displayed three distinct proteolytic activities (trypsin-like, chymotrypsin-like and peptidylglutamylpeptide-hydrolyzing) when assays were performed with small synthetic peptides, and it was noted that the integrity of the 20S complex was essential for all proteolytic activities. Attempts were made to assign specific activities to distinct subunits, but in spite of the efforts of many groups, the nature of the active sites remained enigmatic. Along a different line, a particle named ‘prosome’ was under intensive investigation in the

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mid-1980s (for a review see [46]). Reminiscent in size and subunit composition of the multicatalytic protease complex, it appeared to be associated with RNA and it was suggested to have a role in the regulation of gene expression. In 1988, it was established beyond doubt that the prosome and the multicatalytic proteinase complex were one and the same particle [47,48] and the name ‘proteasome’ was coined by Alfred L. Goldberg (Harvard Medical School) to highlight its only established function, the proteolytic one, and its complex structure. In the following years, evidence began to accumulate that the 20S proteasome was part of an even larger complex, the 26S proteasome, which was implicated in the ATP-dependent degradation of ubiquitin-conjugated proteins [49–51]. By 1990, the 20S proteasome was structurally rather featureless and its subunit composition and stoichiometry were ill-defined. Reports that proteasomes could undergo changes in subunit composition during development [52] made its structural analysis a daunting challenge, since structural methods rely, in one guise or another, on averaging and, therefore, on homogeneous preparations of molecules. This led us to search for proteasomes of hopefully simpler subunit composition in prokaryotic cells. While our initial attempts to find proteasomes in bacteria were unsuccessful, we found them in the archaeon Thermoplasma acidophilum [42]. The Thermoplasma proteasome turned out to be very similar in size and shape to proteasomes from eukaryotic cells, but much simpler in subunit composition; it comprises only two subunits, a (25.8 kDa) and b (22.3 kDa). The two subunits have significant sequence similarity, suggesting that they arose from a common ancestor via gene duplication [53,54]. Owing to its relative simplicity, the ensuing years saw the Thermoplasma proteasome play a pivotal role in elucidating the structure and enzymatic mechanism of this intriguing protein degradation machine. In 1991, a first, three-dimensional structure of the Thermoplasma proteasome was obtained by EM single particle analysis, showing with remarkable clarity the organization of the barrelshaped complex with its tripartite inner compartment [55]. Immunoelectron microscopy studies allowed us to assign the a-subunits to the two outer rings of the barrel and the b-subunits to the inner rings [56]. Mass measurements by STEM (scanning transmission electron microscopy) helped us to establish the stoichiometry (a7b7b7a7) and metal decoration studies of proteasome crystals (not yet good enough for high-resolution X-ray

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crystallography) clearly revealed the symmetry of the 20S complex. The structural model we put forward on the basis of these data stood the test of time and it recurred in all proteasomes, eukaryotic and prokaryotic [57]. Another important advance was the expression of fully assembled and functional 20S proteasomes in E. coli [58] (Figure 4a). It not only allowed us to perform systematic mutagenesis studies aimed at identifying the active site, it also greatly facilitated the growth of crystals diffracting to high resolution [59]. In 1995, the crystal structure analysis was completed in a collaboration with the group of Robert Huber [60] (Figure 4b). The long-sought catalytic nucleophile of the 20S proteasome, the N-terminal threonine of the mature b-subunit was identified independently and almost simultaneously by site-directed mutagenesis and crystal structure analysis [6,61]. As anticipated from their sequence similarity the a- (non-catalytic) and the b-type (catalytic) subunits showed the same fold: a four-layer a+b structure with two antiparallel fivestranded b sheets, flanked on one side by two, and on the other side by three a-helices. In the b-type subunits, the b-sheet sandwich is closed at one end by four hairpin loops and opens at the opposite end to form the active-site cleft; the cleft is oriented toward the inner surface of the central cavity. In the a-type subunits, an additional helix formed by an N-terminal extension crosses the top of the b-sheet sandwich and fills this cleft. Initially, the proteasome fold was believed to be unique; however, it turned out to be common to a new superfamily of proteins referred to as Ntn (N-terminal nucleophile) hydrolases [62]. Beyond the common fold, members of this family share the mechanisms of the nucleophilic attack and self-processing (for reviews see [63–66]). The crystal structure revealed that access to the inner cavity that harbors the active sites is controlled by four constrictions. The constrictions in the a-rings which give access to the two ‘antechambers’ are narrow and partially obstructed, while the constrictions which regulate access to the central cavity are wider. We were able to show with NanogoldTM-labeled substrates, visible in electron micrographs, that polypeptides indeed enter the proteasome via the orifice at the center of the a-rings. Bulky additions to the polypeptide chain, such as a gold cluster, prevent passage into the interior, suggesting that the discrimination between folded and unfolded substrates is based on a size-exclusion mechanism [67]. Thus, the 20S proteasome is a molecular nano-compartment that

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Fig. 4. The 20S proteasome from Thermoplasma acidophilum. (a) Electron micrograph of recombinant 20S proteasomes in vitreous ice. (b) Top left: Structure of the 20S proteasome in surface representation, low-pass filtered to 1 nm resolution. The a- and b-subunits are located in the outer and inner rings, respectively. Top right: The same structure cut open along the 7-fold axis to display the inner compartments with the active sites of the b-subunits in the central chamber marked in red. Bottom left and right: Similar fold of the a- (left) and b- subunits (right). Both subunits contain a sandwich of two, five-stranded antiparallel b sheets flanked by helices (for details see [64,113]) (see Colour Plate Section at the end of this volume).

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confines the proteolytic reaction to its interior and sequesters it from the crowded environment of the cell. Interestingly, formation of the active sites by the post-translational removal of the propeptides of the b-subunits [68] is coupled to the assembly of the 20S proteasome in such a manner that activation is delayed until the assembly is complete (for review see [65]). This led us to propose the concept of self-compartmentalization as a regulatory principle [63,69]. As mentioned earlier, it began to transpire in the early 1990s that the 20S proteasome of eukaryotes associates with regulatory complexes, in an ATP-dependent manner, to form the 26S proteasome. Now it is firmly established that this 2.5 MDa complex altogether comprising more than 30 different subunits acts downstream in the ubiquitin–proteasome pathway and is the central player in intracellular proteolysis. Proteins destined for degradation are marked by covalent attachment of Ub chains, which mediate recognition by the 26S proteasome (for recent reviews see [70,71]). In 1993, we were able to provide the first detailed description of the 26S complex, based on electron microscopy and image analysis [72]. The averages showing the regulatory (19S) particles attached to one or both ends of the 20S proteasome core particle (the ‘dragon-head’ or ‘double dragon-head’ motif) became the classical textbook images of the 26S proteasome. Since then, however, progress has been embarrassingly slow; the notorious instability of the complex and its dynamics have made it a daunting challenge to establish a detailed structural model [73–76]. While it is clear that the role of the 19S regulatory complexes (Figure 5) is the preparation of substrates for degradation in the 20S core particle – involving the recognition of ubiquitinated substrates, the removal of the polyubiquitin chains, the unfolding of substrates and assistance in translocation across the gates of the 20S complex – the precise topology and role of the 19S subunits is hitherto only dimly understood [77]. In 1991, we found, in a serendipitous manner, a novel ATPase complex. During the lysis of accidentally heat-shocked Pyridictium cells on electron microscopy grids, a massive release of toroidal particles composed of the stacked octameric rings was observed [78]. Not only the shape, but also the heat-shock induction of this complex was reminiscent of the GroEL/Hsp60 family, and therefore raised the possibility that it represented an archaeal chaperonin. Subsequently, we named it ‘thermosome’ to highlight its

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Fig. 5. Structure of the 26S proteasome from Drosophila melanogaster as determined by cryoelectron tomography and displayed in surface (left) and cut-open views (right) (for details see [73]) (see Colour Plate Section at the end of this volume).

heat induction and extreme thermostability [79]. Independently, a closely related complex (TF55) was discovered in the laboratory of Art Horwich in Yale [80]. The thermosome or TF55 were the first representatives of the Group II chaperonins found in archaea and in the eukaryotic cytosol. The main structural feature distinguishing the Group II from the Group I chaperonins is, in the absence of a co-chaperonin, a built-in lid provided by the protrusions of the apical domains which can seal the folding chamber by an iris-type closure mechanism [81,82]. In 1996, in our quest for a more comprehensive understanding of the protein quality control machinery in Thermoplasma we found a fascinating, large proteolytic complex that works in conjunction with an array of aminopeptidases [83]. In view of the shape of the hexamer, we named it ‘tricorn protease’; soon thereafter we were able to show that tricorn protease exists in the cell as a giant icosahedral complex of approximately 15 MDa, which in addition to its peptide-cleaving activity, appears to serve as an organizing center for the more downstream elements of the protein degradation pathway [84]. Tricorn protease converts the oligo-peptides (typically about 8 aa-residues) released by the proteasome into smaller (2–4 residues) peptides which are degraded further by aminopeptidases [85]. These findings stimulated the search for ‘functional homologs’ of tricorn protease in eukaryotic cells; one of

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Fig. 6. Giant complexes involved in intracellular protein degradation. Left: Tricorn protease capsids from Thermoplasma (15 MDa). Right: Tripeptidylpeptidase II from Drosophila (6 MDa) (for details see [89,106]) (see Colour Plate Section at the end of this volume).

the candidates is tripeptidylpeptidase (TPP) II, another giant protein complex with an intriguing structure [86–89] (Figure 6). In 2000, we completed the sequencing of the genome of Thermoplasma acidophilum, an endeavor we had undertaken with modest resources [90]. It not only served to further establish Thermoplasma as a model system for studying cellular protein quality control, it also provided the platform for a very ambitious project, namely the mapping of its cellular proteome by cryoelectron tomography; this, in turn, can be expected to shed new light on the pathways of intracellular protein quality control (Figure 7). The Latest Frontier: Charting Molecular Landscapes Inside Cells by Cryoelectron Tomography The foundations of electron tomography were laid already in the late 1960s. In their landmark paper, De Rosier and Klug outlined very clearly and in general terms the principles of 3-D reconstruction from electron micrographs [7]. Being aware of the practical problems in recording 3-D datasets, they took advantage of the helical symmetry of the bacteriophage T4 tail in a very pragmatic

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Fig. 7. The protein quality control system in Thermoplasma acidophilum. Components of the proteolytic pathway are shown in yellow, chaperones in green. The numbers refer to the ORF code (for details see [90]) (see Colour Plate Section at the end of this volume).

manner. Walter Hoppe, guided by his background in X-ray crystallography, also realized the potential of 3-D electron microscopy. Diverging from the approaches taken by most others, he focused on the development of methods suitable for studying individual structures (‘Crystallography of crystals consisting of a single unit cell’) [91]. In fact, his group presented as early as 1974 a 3-D

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reconstruction of single fatty acid synthetase molecules obtained by tomography [92]. As mentioned earlier, the ‘brute force’ approach they used provoked some criticism. Besides doubts that negative staining can portray details of the underlying structure to the resolution they claimed, the main concern was the enormous electron dose to which the specimen was exposed during recording of the data. There was much discussion in the following years as to whether it might ultimately be possible to do electron tomography with acceptable electron doses. Also in 1968, R. G. Hart at the Lawrence Livermore Laboratory published a paper entitled ‘Electron microscopy of unstained biological material: The polytropic montage’ [113]. Despite its vision, the Hart paper had negligible impact. For a vision to materialize, timing is a crucial element; if it is too early, the necessary technologies might not yet exist. The key problem in electron tomography, which for many years was a formidable obstacle and a deterrent, is to reconcile two requirements that are in conflict with each other: to obtain a reconstruction that is detailed and largely undistorted, one has to collect data over as wide a tilt range as possible with increments as small as possible (for a review see [93]). At the same time, the electron dose must be minimized. Above a critical dose, the specimen undergoes structural degradation that, in the worst case, can render a reconstruction meaningless. In principle, one could fractionate the dose over as many projections as an optimized tilt geometry might require. However, there is a practical limitation; the signal-to-noise ratio of the 2-D images has to be sufficient to permit their accurate alignment by cross-correlation. This problem is further aggravated by the far-from-perfect mechanical accuracy of the tilting devices that causes image shifts and changes of focus. Therefore, following each change of tilt angle, the specimen (or its image) have to be realigned and refocused. Doing this manually and with minimal exposure to the electron beam is utterly impossible. In the late 1980s, when computer-controlled electron microscopes and large-area CCD cameras became available, we saw an opportunity to automate tomographic data acquisition [94–96, 114]. This made the recording of datasets not only less cumbersome, but first and foremost it allowed the cumulative electron dose to be kept within tolerable limits. The fraction of the dose that is spent on overhead (search, re-centering, (auto-)focusing) can be kept as low as 3% of the total dose; in other words, almost

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all electrons are used for gaining information [97]. As is evident from the recent resurgence this has changed the perspectives of electron tomography in a most profound manner; electron tomography had been used from time to time for ultrastructural studies, mostly of plastic embedded biological material, but it has gathered momentum only recently. As demonstrated originally with ‘phantom cells’, i.e. lipid vesicles encapsulating specific sets of macromolecules, automated tomography in a ‘low-dose mode’ has enabled us to combine the potential of 3-D imaging with the best possible preservation of biological samples, i.e. embedded in vitreous ice [96,98]. Vitrification by rapid freezing ensures not only a close-to-life preservation of molecular and cellular structures, but it also allows the capture of dynamic events [99]. It avoids the risks of artifacts traditionally associated with chemical fixation and staining or with the dehydration of cellular structures. Equally important, tomograms of frozen-hydrated structures represent their natural density distribution whereas staining reactions tend to produce intricate mixtures of positive and negative staining. As a consequence, the interpretation of such tomograms in molecular terms may be very problematic if not impossible [100]. With the use of automated procedures and user-friendly software meanwhile the recording of tilt series and their processing has become routine. It is in fact now less cumbersome and less time-consuming to obtain a cryotomogram than going through the conventional procedures of plastic embedding and sectioning the material. With smaller structures (e.g. bacteriophages docked onto proteoliposomes) a resolution of 2.5 nm has been obtained [101]. With whole prokaryotic cells or thin eukaryotic cells grown directly on EM grids, resolution is usually in the range of 4–5 nm, but prospects for further improvements are good [102]. Better detectors, in particular, will allow a finer 3-D sampling, which, in turn, will improve resolution (see above) and allow tomography to enter the realm of molecular resolution (2–3 nm). Even at the present practical level of resolution, cryotomograms of organelles or cells contain an imposing amount of information. They are, essentially, 3-D images of entire proteomes, and they should ultimately enable us to map the spatial relationships of the full complement of macromolecules in an unperturbed cellular context; however, new strategies and innovative image analysis techniques are needed for ‘mining’ this information. Retrieving it is

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confronted with two major problems: Cryotomograms are ‘contaminated’ by residual noise, and they are distorted by missing data – in spite of optimized image acquisition schemes. Moreover, the cytoplasm of most cells is densely packed (‘crowded’) with molecules literally touching each other. It is therefore often impossible to perform a segmentation and to extract features, based on visual inspection of the tomograms. Denoising procedures [103] can facilitate the visualization of features, but advanced pattern recognition techniques are needed for detecting and identifying specific macromolecules by their respective structural signatures. The most powerful method for improving the signal-to-noise ratio is averaging. Although averaging can obviously not be applied to tomograms of pleomorphic structures in a first instance, such tomograms may nevertheless contain repetitive elements which can be extracted in silico, and the subtomograms containing them can be subjected to classification and averaging. These averages can be used subsequently for replacing the original data in the tomograms, resulting in ‘synthetic’ tomograms with a locally improved signal-to-noise ratio. This strategy was used, for example, to obtain a density map of whole Herpes simplex virions [104]. In spite of the low signal-to-noise ratio of tomograms, continuous structures such as membranes of cytoskeletal filaments are easy to recognize. Cryotomograms of Dictyostelium discoideum cells grown directly on carbon support films have provided unprecedented insights into the organization of actin filaments in an unperturbed cellular environment [105,106]. The tomograms show, on the level of individual filaments, their modes of interaction (isotropic networks, bundles, etc.), they allow to determine the branching angles precisely (in 3-D), and they reveal the structure of membrane attachment sites. For the quantitative analysis of large datasets, as is needed for extracting statistically significant quantitative data, it will be necessary to develop algorithms for automated segmentation, to establish connectivity of filaments in noisy datasets – a notoriously difficult problem – and measure structural parameters of filaments (Figure 8a). Cryoelectron tomography enables us to obtain images of single macromolecules inside intact cells as is exemplified by Figure 8b, which shows a single 26S proteasome within the cytoplasm of a Dictyostelium cell. Although in this case the detection and identification was facilitated by the large size (2.5 MDa) and the peculiar shape of this complex, it indicates that a molecular

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Fig. 8. Cryoelectron tomography of Dictyostelium discoideum cell. (a) Visualization of the actin network and cytoplasmic complexes in a Dictyostelium cell grown directly on an EM grid and embedded in vitreous ice (for details see [71]). (b) Visualization of a 26S proteasome within an intact Dictyostelium cell. Left: Slice from a tomogram. Dominant features are ribosomes, some of them attached to the endoplasmic reticulum (lower left corner), and actin filaments. The encircled particle is a 26S proteasome. Right: Enlarged contour plot of the single (unaveraged) 26S proteasome (projection of a stack of slices from tomogram) (see Colour Plate Section at the end of this volume).

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signature-based approach to mapping cellular proteomes should become feasible. Alternatively, one could envisage strategies for introducing electron-dense labels marking the spatial distribution of the molecules of interest. Such an approach, however, would no longer be noninvasive – unless it is based entirely on genetic manipulations – and it would be difficult, if not impossible, to achieve quantitative detection. Moreover, it is hard to imagine how this approach could be parallelized such that it becomes a high-throughput technology capable of mapping entire proteomes. For every molecule of interest it would be necessary to repeat the whole procedure, the labeling of cells, the recording of tilt series and the tomographic reconstruction. Even if this could be accomplished, it would be a daunting challenge to interrelate the individual maps and to reveal the structure of molecular networks, owing to the stochastic nature of cellular supramolecular architecture. Therefore, there is a strong incentive to exploit the information content of cryotomograms by means of intelligent pattern recognition algorithms. With this approach, a tomogram needs to be produced only once, and it is then interpreted in a sequential manner in terms of its molecular architecture. The strategy we are pursuing is ‘template matching’ [107,108]. Provided that high- or medium-resolution structures of the macromolecules of interest are available, they can be used for a systematic interrogation of the tomograms (Figure 9). Image simulations have shown that template matching is indeed a feasible approach for identifying macromolecules in ‘noisy’ tomograms. Experimental studies with ‘phantom cells’, i.e. lipid vesicles encapsulating known sets of proteins provide a means of validating the results of the template matching. At the present resolution of 4–5 nm only very large complexes (ribosomes, 26S proteasomes) can be mapped with high fidelity (495%). An improvement in resolution to approximately 2 nm will be required for the mapping of medium-sized complexes (200–400 kDa, depending on shape). While tomograms with a resolution of 2 nm are a realistic prospect, major technical innovations would be required to go beyond [109,110]. Once the challenges of obtaining a sufficiently good resolution are met, the next challenge will be to create comprehensive libraries of templates. A whole array of methods can be used to this end. Worldwide structural genomics efforts will increase the pace with which high-resolution structures of domains, subunits

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Fig. 9. Strategy for the detection and identification of macromolecules in cellular volumes. Because of the crowded nature of cells and the high-noise levels in tomograms (left), an interactive segmentation and feature extraction is, in most cases, not feasible. It requires automated pattern recognition techniques to exploit the rich information content of such tomograms. An approach that has been demonstrated to work is based on the recognition of the structural signature (size, shape) of molecules by template matching. Templates of the macromolecules under scrutiny are obtained by high- or medium-resolution techniques. These templates are then used to search the volume of the tomograms (Vin) systematically for matching structures by cross-correlation. The tomogram has to be scanned for all possible Eulerian angles around three different axes, with templates of all the different protein structures in which one is interested. The search is computationally demanding, but can be parallelized efficiently. The output information (Vout) is a set of coordinates that describes the positions and orientation of all the molecules found in the tomogram (for details see [38]) (see Colour Plate Section at the end of this volume).

and larger molecular entities become available and eventually provide a comprehensive structural dictionary. Integrative hybrid approaches, combining information gathered by a variety of techniques, will play a crucial role (for a recent review see [111]). EM-based ‘single’ particle analysis will undoubtedly become a major player in furnishing medium-resolution (1 nm) structures of complexes. Currently, this technique is slow and cumbersome, but great strides have been made in recent years toward improving throughput by automating data acquisition and analysis [112]. With cryoelectron tomography providing 3-D images at molecular resolution of cells in a close-to-life state, and with the availability of image analysis tools for interpreting the tomograms, we are poised now to integrate structural information gathered at multiple levels – from atoms to cells – into pseudoatomic maps of organelles or cells. The move from proteomics parts lists to precise

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Fig. 10. Protein atlas of a cellular (Thermoplasma) volume obtained by cryoelectron tomography. The positions and orientations of several molecular species (displayed in different colors) were determined by ‘template matching’ (for details see [72]) (see Colour Plate Section at the end of this volume).

maps of supramolecular landscapes will provide unprecedented insights into the network structures that underlie higher cellular functions and the global and local principles that orchestrate such complex networks of interactions (Figure 10). Epilogue An epilogue is the place for reflections and also for acknowledgments. I have deliberately changed fields a few times. In doing so, the decision to leave a field was usually more difficult than the decision to embark on a new one. After working on a problem for a significant period of time, one becomes emotionally attached to it or even obsessed by it, but there is also an element of convenience: one knows the field with all of its ramifications, and one becomes established and is recognized by his/her peers. However, a change of fields can be rejuvenating. One is less inhibited by the knowledge of problems or obstacles, and more willing to take new approaches. By looking at a problem from a different angle, new

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opportunities arise and it is often the interface between fields and disciplines where the sparks fly. I believe, for example, that our work in cryoelectron tomography will eventually enable us to address problems in intracellular protein quality control in a new manner. I was privileged to witness the development of biomolecular electron microscopy from humble beginnings to its maturation and to partake in it. Electron microscopy now has a firm place in the repertoire of methods structural biologists have at their disposal and it has unique potential to integrate structural data gathered at different levels, bridging the gap between molecular and cellular studies. The work I have described in this article would not have been accomplished without the support and the great efforts of many coworkers and colleagues. I have been fortunate to work with generations of talented and motivated students and postdoctoral fellows and I greatly enjoyed the collaboration with many fine colleagues and friends, with several of them over long periods of time to this day. I mentioned a few of them in the main text, but for the sake of the space it was impossible to acknowledge them all; the names of most of them appear as coauthors in the list of references. I wish to thank them all. Finally, I had the privilege to work in environments that were very supportive and allowed me to undertake the projects I liked to do, irrespective of the chances of success.

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[71] Voges, D., Zwickl, P. and Baumeister, W. (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Ann. Rev. Biochem. 68, 1015–1068. [72] Peters, Jan-M., Cejka, Z., Harris, J.R., Kleinschmidt, J.A. and Baumeister, W. (1993) Structural features of the 26S proteasome complex. J. Mol. Biol. 234, 932–937. [73] Walz, J., Erdmann, A., Kania, M., Typke, D., Koster, A.J. and Baumeister, W. (1998) 26S proteasome structure revealed by threedimensional electron microscopy. J. Struct. Biol. 121, 19–29. [74] Glickman, M.H., Rubin, D.M., Coux, O., Wefes, I., Pfeifer, G., Cejka, Z., Baumeister, W., Fried, V.A. and Finley, D. (1998) A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and elF3. Cell 94, 615–623. ¨lzl, H., Kapelari, B., Kellermann, J., Seemu [75] Ho ¨ ller, E., Su ¨ megi, M., Udvardy, A., Medalia, O., Sperling, J., Mu ¨ ller, J.A., Engel, A. and Baumeister, W. (2000) The regulatory complex of Drosophila melangolaster 26S proteasomes: subunit composition and localization of a deubiquitylating enzyme. J. Cell Biol. 150, 119–129. [76] Nickell, S., Mihalache, O., Beck, F., Hegerl, R., Korinek, A. and Baumeister, W. (2007) Structural analysis of the 26S proteasome by cryoelectron tomography. Biochem. Biophys. Res. Commun. 353, 115–120. [77] Zwickl, P., Voges, D. and Baumeister, W. (1999) The proteasome: a macromolecular assembly designed for controlled proteolysis. Philos. T. Roy. Soc. B. 354, 1501–1511. [78] Phipps, B.M., Hoffmann, A., Stetter, K.O. and Baumeister, W. (1991) A novel ATPase complex selectively accumulated upon heat shock is a major cellular component of thermophilic archaebacteria. EMBO J. 10, 1711–1722. [79] Phipps, B.M., Typke, D., Hegerl, R., Volker, S., Hoffmann, A., Stetter, K.O. and Baumeister, W. (1993) Structure of a molecular chaperone from a thermophilic archaebacterium. Nature 361, 475–477. [80] Trent, J.D., Nimmesgern, E., Wall, J.S., Hartl, F.U. and Horwich, A.L. (1991) A molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1. Nature 354, 490–493. [81] Klumpp, M., Baumeister, W. and Essen, L.-O. (1997) Structure of the substrate binding domain of the thermosome, an archaeal group II chaperonin. Cell 91, 263–270. [82] Gutsche, I., Essen, L.-O. and Baumeister, W. (1999) Group II chaperonins: new TRiC(k)s and turns of a protein folding machine. J. Mol. Biol. 293, 295–312. [83] Tamura, T., Tamura, N., Cejka, Z., Hegerl, R., Lottspeich, F. and Baumeister, W. (1996) Tricorn protease – the core of a modular proteolytic system. Science 274, 1385–1389. [84] Walz, J., Tamura, T., Tamura, N., Grimm, R., Baumeister, W. and Koster, A.J. (1997) Tricorn protease exists as an icosahedral supermolecule in vivo. Mol. Cell 1, 59–65. [85] Tamura, N., Lottspeich, F., Baumeister, W. and Tamura, T. (1998) The role of tricorn protease in its aminopeptidase-interacting factors in cellular protein degradation. Cell 95, 637–648.

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[86] Geier, E., Pfeifer, G., Wilm, M., Lucchiari-Hartz, M., Baumeister, W., Eichmann, K. and Niedermann, G. (1999) A giant protease with potential to substitute for some functions of the proteasome. Science 283, 978–981. [87] Rockel, B., Peters, J., Ku ¨ hlmorgen, B., Glaeser, R.M. and Baumeister, W. (2002) A giant protease with a twist: the TPP II complex from Drosophila studied by electron microscopy. EMBO J. 21, 5979–5984. [88] Rockel, B., Peters, J., Mu ¨ ller, S.A., Seyit, G., Ringler, P., Hegerl, R., Glaeser, R.M. and Baumeister, W. (2005) Molecular architecture and assembly mechanism of Drosophila tripeptidyl peptidase II. Proc. Natl. Acad. Sci. U.S.A. 102, 29: 10135–10140. [89] Seyit, G., Rockel, B., Baumeister, W. and Peters, J. (2006) Size matters for the tripeptidylpeptidase II complex from Drosophila. The 6-MDa spindle form stabilizes the activated state. J. Biol. Chem. 281, 35: 25723–25733. [90] Ruepp, A., Graml, W., Santos-Martinez, M.-L., Koretke, K.K., Volker, C., Mewes, H.W., Frishman, D., Stocker, S., Lupas, A.N. and Baumeister, W. (2000) The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum. Nature 407, 508–513. [91] Hoppe, W. (1978) Three-dimensional electron microscopy of individual structures: crystallography of ‘crystals’ consisting of a single unit cell. Chem. Scr. 79, 227–243. [92] Hoppe, W., Gassmann, J., Hunsmann, N., Schramm, H.J. and Sturm, M. (1974) Three-dimensional reconstruction of individual negatively stained yeast fatty-acid synthetase molecules from tilt series in the electron microscope. Hoppe-Seyler’s Z. Physiol. Chem. 355, 1483–1487. [93] Baumeister, W., Grimm, R. and Walz, J. (1999) Electron tomography of molecules and cells. Trends Cell Biol. 9, 81–85. [94] Dierksen, K., Typke, D., Hegerl, R., Koster, A.J. and Baumeister, W. (1992) Towards automatic electron tomography. Ultramicroscopy 40, 71–87. [95] Koster, A.J., Chen, H., Sedat, J.W. and Agard, D.A. (1992) Automated microscopy for electron tomography. Ultramicroscopy 46, 207–227. [96] Dierksen, K., Typke, D., Hegerl, R., Walz, J., Sackmann, E. and Baumeister, W. (1995) Three-dimensional structure of lipid vesicles embedded in vitreous ice and investigated by automated electron tomography. Biophys. J. 68, 1416–1422. [97] Koster, A.J., Grimm, R., Typke, D., Hegerl, R., Stoschek, A., Walz, J. and Baumeister, W. (1997) Perspectives of molecular and cellular electron tomography. J. Struct. Biol. 120, 276–308. ¨rmann, M., Ha ¨ckl, W., Typke, D., Sackmann, E. and [98] Grimm, R., Ba Baumeister, W. (1997) Energy filtered electron tomography of iceembedded actin and vesicles. Biophys. J. 72, 482–489. [99] Dubochet, J., Adrian, M., Chang, J.J., Homo, J.C., Lepault, J., McDowall, A.W. and Schulz, P. (1988) Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, 129–228. [100] Baumeister, W. (2002) Electron tomography: towards visualizing the molecular organization of the cytoplasm. Curr. Opin. Struct. Biol. 12, 679–684. [101] Bo¨hm, J., Lambert, O., Frangakis, A., Letellier, L., Baumeister, W. and Rigaud, J.L. (2001) FhuA-mediated phage genome transfer into liposomes: a cryo-electron tomography study. Curr. Biol. 11, 1168–1175.

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¨rster, F., Gross, A. and [102] Plitzko, J., Frangakis, A.S., Nickell, S., Fo Baumeister, W. (2002) In vivo veritas: electron cryotomography of cells. Trends Biotechnol. 20, S40–S44. [103] Frangakis, A. and Hegerl, R. (2001) Noise reduction in electron tomographic reconstructions using nonlinear anisotropic diffusion. J. Struct. Biol. 135, 239–250. [104] Gru ¨ newald, K., Desai, P., Winkler, D.C., Heymann, J.B., Belnap, D.M., Baumeister, W. and Steven, A.C. (2003) Three-dimensional structure of Herpes simplex virus from cryo-electron tomography. Science 302, 1396–1398. [105] Medalia, O., Weber, I., Frangakis, A., Nicastro, D., Gerisch, G. and Baumeister, W. (2002) Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography. Science 298, 1209–1213. [106] Medalia, O., Beck, M., Ecke, M., Weber, I., Neujahr, R., Baumeister, W. and Gerisch, G. (2007) Organization of actin networks in intact filopodia. Curr. Biol. 17, 79–84. [107] Bo¨hm, J., Frangakis, A., Hegerl, R., Nickell, S., Typke, D. and Baumeister, W. (2000) Toward detecting and identifying macromolecules in a cellular context: template matching applied to electron tomograms. Proc. Natl. Acad. Sci. U.S.A. 97, 14245–14250. ¨rster, F., Nickell, S., Nicastro, D., Typke, D., [108] Frangakis, A., Bo¨hm, J., Fo Hegerl, R. and Baumeister, W. (2002) Identification of macromolecular complexes in cryoelectron tomograms of phantom cells. Proc. Natl. Acad. Sci. U.S.A. 99, 14153–14158. [109] Nickell, S., Kofler, C., Leis, L. and Baumeister, W. (2006) A visual approach to proteomics. Nat. Rev. Mol. Cell Biol. 7, 225–230. [110] Ortiz, J., Fo¨rster, F., Ku ¨ rner, J., Linaroudis, A. and Baumeister, W. (2006) Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition. J. Struct. Biol. 156, 334–341. [111] Sali, A., Glaeser, R., Earnest, T. and Baumeister, W. (2003) From words to literature in structural proteomics. Nature 422, 216–225. [112] Carragher, B., Fellmann, D., Guerra, F., Milligan, R.A., Mouche, F., Pulokas, J., Sheehan, B., Quispe, J., Suloway, C., Zhu, Y. and Potter, C.S. (2004) Rapid routine structure determination of macromolecular assemblies using electron microscopy: current progress and further challenges. J. Synchrotron Radiat. 11, 83–85. [113] Hart, R.G. (1968) Electron microscopy of unstained biological material: the polytropic montage. Science 159, 1464–1467. [114] Typke, D., Dierksen, K. and Baumeister, W. (1991) Automatic electron tomography. In Proceedings of the 49th Annual Meeting on Electr. Micr. Soc. Am. (Bailey, W., ed.), pp. 544–545. San Francisco Press, San Francisco.

G. Semenza (Ed.) Stories of Success – Personal Recollections. X (Comprehensive Biochemistry Vol. 45) r 2007 Elsevier B.V. All rights reserved. DOI: 10.1016/S0069-8032(07)45003-3

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Chapter 3

Fixed to Translation: A Recollection$ ¨ CK AUGUST BO Department of Biology I, University of Munich, LindenstraX e 10, D-82269 Geltendorf, Germany E-mail: [email protected]

$ Dedicated to Irmi, our children Evi and Uli, our grandchildren Claire and Isabelle, and to my friends.

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Abstract I had the great opportunity to start my scientific career at the time of the onset of molecular biology and my initial work laid the foundation for later contributions made in the fields of archaeal and bacterial protein synthesis and its regulation. I recount my academic life in two German universities and the role, which mentors played in the development of my career and how the excellence of my students contributed to the achievements. Keywords: Prokaryotic protein synthesis; anaerobic gene expression; fermentation; metalloprotein synthesis; selenoprotein synthesis.

During my scientific career I had the chance to experience an unbelievable revolution of life sciences from a more or less descriptive to a predominantly chemical and physical discipline. Moreover, I spent my entire career in German universities, I was educated in one of the established historical institutions, had the chance to participate in the foundation of a biology faculty in one of the new universities and finished my career again in the established one. Dramatic changes have been implemented during these 50 years and I hope that I manage to bring over some of the flavour of these times of changes.

Early Years Two years before the onset of Second World War, I was born as the third of four children in Kaltenberg, a small village of less than 300 people but with a long history dating back to times of the foundation of Munich in 1180. The village is topped by a medieval castle owned by direct descendents of our Bavarian kings, but more known by the premium beer they are brewing there. Our father was a carpenter, but because of the lack of appropriate work in his profession he earned his money in this local brewery. Between 1943 and 1947, I attended Primary School in Geltendorf and experienced a very fragmentary basic education because of the difficult living situation in the last years of the war and the disastrous economic situation in the immediate years after. Four age groups were crowded in one classroom and were supervised by a single teacher. Fortunately, this was compensated by the extreme

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dedication of this teacher who also convinced my parents that I should continue my studies in the so-called ‘‘Oberschule’’, which, at least in the country side, was considered the privilege of children of physicians or of those who were supposed to become priests. I was fortunate that my parents chose the Oberrealschule in Augsburg, which had an emphasis in science with strong mathematics, physics and chemistry. I finished the studies in 1957 and subsequently enrolled in the Faculty of Biology at the University of Munich. Studying biology in Germany at that time opened just two main perspectives for entering a profession, namely teaching biology in schools or starting a scientific career. The major reason was that the discipline was dichotomously divided into botany and zoology with a high emphasis in morphology, cytology and taxonomy. A main occupation of students in the laboratory courses, for example, consisted of performing hand drawings of images from macroscopic or microscopic samples for almost two years. Since there were no professorships in biochemistry, microbiology or genetics in most of the faculties, entering a job in industry was not obvious, also aggravated by the lack of an appropriate graduation examination, like the diploma was in chemistry. Looking back I am convinced that this maintenance of the historical view of the bipartite structure of biology contributed to the very sluggish introduction of quantitative and molecular aspects into teaching and research in biology faculties of many universities in Germany. In the recruitment of new staff by the established universities there was and still is a tendency to maintain this ‘‘balance’’ which limits the rapid adjustment to scientific developments. To be on the safer side, I chose the curriculum for the education of schoolteachers which besides biology obligatorily involved also chemistry and geography which included the fundamentals of geology. The curriculum in chemistry for teachers was very basic and I suffered from this weakness during my entire career. On a personal level, however, I benefited very much from geography and geology since it endowed me with the ability to not only enjoy the beauty but also to understand the fascinating world of landscapes during our many family hikes and travels. Since there was no official requirement for an examination or graduation, I started already working on a doctoral thesis in the group of Werner Rau parallel to my studies. The topic was to resolve the still nowadays timely question of the interaction of two microorganisms: one of them was the mould Penicillium funiculosum

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which produced a deep-red pigment at the contact interface with the mycelium of other moulds. The results lead to the conclusion that P. funiculosum secretes a fungistatic naphthoquinone which chemically interacts with amino groups from compounds produced and secreted by the second organism. Because of the lack of causal experimental approaches like mutant isolation and analysis the outcome was rather phenomenological. During my entire career I benefited from the mentoring and the advice of a number of eminent scientists. The first one to mention was Meinhart Zenk who was assistant professor during my studies in botany, having just arrived from Purdue University where he had conducted his doctoral work. He took great care in tutoring his students and to introducing them into modern fields of plant physiology and biochemistry outside the regular courses. It was also Meinhart Zenk who advised me to take a postdoctoral stay in the United States after I had worked for two years as a postdoctoral research assistant of Leo Brauner on projects dealing with the perception of gravity by plants and with their geotropic growth response. After intensive reading, my decision was to apply for a fellowship to work in the group of Frederick Neidhardt in the Life Science Department of Purdue University. I was happy to be offered a position as research associate. I was also a recipient of a travel grant from the Fulbright Foundation, and in September 1964, I boarded the ship and crossed the Atlantic with several hundred fellow students; it was an exciting journey and I am still wondering that we did not sink the vessel. Neidhardt had just developed the concept of the analysis of temperature-sensitive mutants of bacteria in order to identify genes and reactions that are indispensable for growth and cannot be supplemented by any addition to the medium. I became involved in this exciting development and attempted to obtain strains with a temperature-sensitive RNA polymerase, one of the most wanted enzymes at that time, to characterise the gene(s) and later on the enzyme. This actually ended up with a mutant with a blockade in the carbon flux through glycolysis because of a temperature-sensitive fructose-1,6 bisphosphate aldolase [1]. It displayed the phenotype and the biochemical properties of an RNA polymerase mutant which pointed to some regulatory link between energy metabolism and RNA synthesis. In addition to providing interesting information on the effect of the accumulation of fructose 1,6-bisphosphate on carbon metabolism it was important

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for my training, especially in firmly believing in the power of mutant analysis as the only means to correlate biochemical results with the physiology of a cell and to obtain causal answers. Another project in which I was involved during my postdoctoral stay at Purdue was the mapping of genes coding for aminoacyl-tRNA synthetases by means of conditional mutants from Escherichia coli. Such mapping projects were very time consuming, they took months, because of the scarcity of chromosomal markers and the odd phenotypes that had to be employed. For example, mapping of the locus for glycyl-tRNA synthetase had to be achieved by exploiting affinity differences of the enzymes from two strains for the substrate glycine which necessitated Km assays of all recombinants obtained from the crosses. The overall information gained from much work was that these genes are not linked to the biosynthetic operons for the corresponding amino acid and that they also are not organised in clusters [2]. It provided genetic evidence that the genes coding for enzymes involved in the biosynthesis of the amino acids and their attachment to the cognate tRNA are differentially regulated and may have evolved independently. The opportunity to work in Fred Neidhardt’s group had great influence on my entire further scientific career. It was the time of the beginning of microbial molecular biology and I learnt very much from his integrated view on the cell biology of E. coli and his perspective thinking. He also was a great teacher and the weekly lab seminars served an important function for our ability to give clear and logical presentations. I still remember the rather painful trials to write my first paper in English which was returned and returned until he finally accepted it as appropriate.

From Aminoacyl-tRNA Synthetases to Ribosomes Amongst various options of institutions to which I had the opportunity to return to Germany I accepted an offer from Otto Kandler for an assistant professorship position in the Botany Institute of the University of Munich, the place where I had conducted my doctoral work. When I returned in 1966, he had accepted the chair but not yet moved with his group which meant that I came into an interregnum situation without any institutional support apart from a refrigerator and glass Petri dishes. So we were doing cheap science, namely genetics, and my first two co-workers, Herrmann

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Weber and Heinz Kosakowski, enthusiastically joined me in the isolation of new temperature-sensitive mutants and in characterising their lessions. Since the greatest enthusiasm cannot compensate for the lack of money, I discussed the matter with Peter Starlinger on the occasion of one of the Genetics Meetings in Cologne to which I was invited and he told me to immediately write an application to the Deutsche Forschungsgemeinschaft and ask for everything I needed. I did as he said and I got everything within a very short time. He must have played God. I owe Otto Kandler very much for the opportunity to work in his institute. He not only offered perfect physical and personnel infrastructure after his arrival, but also, and this was and still is not obvious for German universities, complete independence of the scientific development of the young staff. He took continuous interest in our work and, although not in the immediate field, contributed to our strategies by his admirably broad knowledge over all biological fields and by his wide perspectives. I decided to continue working on aminoacyl-tRNA synthetases in a biochemical direction, especially with the aim to unravel the structural basis of temperature-sensitivity of the mutant enzymes. It was found via structure–function analyses of wild-type and mutant alanyl-tRNA synthetase and phenylalanyl-tRNA synthetase that the temperature sensitivity of the enzyme variants resided in a change of the quaternary structure at elevated temperature [3]. In 1969, I went through the procedure of ‘‘Habilitation’’ in which one acquires the qualification for independent teaching. It encompassed the writing of a thesis on the research performed as an assistant professor, a lecture on one of three choice topics and the defence of 10 theses one had to propose and which were announced to the public. I remember that one of my general theses (one had to include three of them in addition to the seven scientific ones) was that within universities students should be encouraged to refrain from addressing their teachers with their academic titles in order to create more openness for discussions. It was 1969! Almost all the questions from the attendees addressed this thesis, an indication that I may have hit a sensitive issue. The topic of my lecture dealt with the metabolism and physiology of polyphosphates, still a timely subject. Fortunately, I passed the procedure and was appointed to ‘‘Privatdozent’’ with the venia legendi in botany because the type of research I was following did not fit well into the heart of contemporary microbiology which was

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predominantly classical at that time in Germany. Also fortunately, however, I was obliged to teach the introductory microbiology course to diploma students, apart from teaching botany. I have been teaching this course and the connected laboratory course in introductory microbiology for altogether 32 years, and I must confess that I liked it very much since it is their first occasion when students get in contact with micro-organisms and it is easy to raise their enthusiasm for the discipline. Over the last decade, the abolition of the Habilitation was cherished as one of the most important issues of the never-ending ¨tsreform’’. I never understood all the emotions con‘‘Universita nected with it in view of the real structural and management problems of German University science, since one can see that the procedure serves two purposes: first, it controls that new lecturers have the experience and practice required to meet the challenge of handling a large crowd of students, and second, it presents a time slot when the faculty has to decide whether the candidate should enter a academic career or not. In 1971, I accepted the offer for a full professorship in microbiology at the University of Regensburg. This university was one of a whole series of new foundations in that time period, so chances for young scientists like me to enter an academic career were excellent and much better than nowadays when an exceedingly high number of young researchers are competing for a very narrow tip of top positions. The task of contributing to the establishment of teaching and research from scratch in a congenial collaboration with Widmar Tanner, Rainer Jaenicke and Herrmann Eggerer was pioneering in any respect. I was fortunate that my entire working group joined me in moving to Regensburg so that we could set up a microbiology teaching curriculum within a very short time and in parallel continue our projects without delay. Thus, a detailed structure– function analysis was performed by Hauke Hennecke of the phenylalanyl–tRNA synthetase from E. coli and he also characterised the threonine specific enzyme and the respective genes [4,5]. In a cooperation with Gisela Nass the mode of resistance of E. coli to the antibiotic borrelidin could be attributed to a mutation of the enzyme either reducing the affinity towards its substrate threonine and thereby also to borrelidin, or to promoter-up mutations causing an overproduction of the enzyme and consequently a titration of the antibiotic [5].

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A serendipitous discovery led the way into the field of the genetics and biosynthesis of ribosomes. It was found that suppressors of the temperature-sensitivity of mutants with an altered aminoacyl-tRNA synthetase isolated at semi-permissive temperature possessed mutations in genes coding for ribosomal proteins. In a co-operation between Peter Buckel and H. G. Wittmann, the mutant ribosomal proteins were identified; most of them bind directly to ribosomal RNA, like S5, S8 or S20. The biochemical basis for the suppression of the aminoacyl-tRNA synthetase mutation by ribosomal mutations was attributed to the fact that all these mutants exhibited an assembly defect in the biosynthesis of ribosomes, thus lowering the actual cellular concentration of active ribosomes. In this way the reduced capacity for aminoacylation of the respective tRNA species was adjusted to its need in protein synthesis and the toxic effect of an unoccupied A site at the ribosome is avoided. Such an empty A site is known to cause a permanent stringent response with overshooting synthesis of guanosine tetra- and pentaphosphates and the consequent mal-adaptation of numerous regulatory circuits [6]. Other projects followed in parallel concentrated on the protein chemical characterisation of the ribosomal mutations to which Wolfgang Piepersberg, now at the University of Wuppertal contributed prominently [7]. The question of whether ribosomal proteins or rRNA are the catalytically active components in protein synthesis, especially in the peptidyl-transfer reaction stimulated the mutational analysis of ribosomes considerably in this time period. Different approaches were followed and eventually variants of most of the ribosomal proteins could be identified and their genes mapped. However, apart from influencing the path of assembly or in some cases the binding of antibiotics no further clues on a role of ribosomal proteins beyond maintaining the structure of the ribosome were gathered [7]. We turned our attention then to organisms producing antibiotics interfered with functions of the ribosome in translation since during their evolution they should have developed mechanisms conferring resistance to their own products. Our choice was Micromonospora species since they are able to produce a multitude of such inhibitors that interfere with different partial reactions of the translation process. It was found that the producers were resistant to their own product because they possessed intrinsically resistant ribosomes and in criss–cross subunit reconstitution

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experiments done by Wolfgang Piendl in collaboration with Eric Cundliffe from the University of Leicester it turned out that the component conferring resistance was rRNA [8]. As Micromonospora species produce a variety of aminocyclitols/ aminoglycosides that interfere with initiation, elongation and the fidelity of protein synthesis, the information that it was rRNA which confers resistance provided one of the first pieces of evidence for an important catalytic role in translation. Wolfgang Piendl continued this project after his change to the University of Innsbruck. The development of a superior method for the separation of all ribosomal proteins in a single two-dimensional gel by Dieter Geyl allowed us to approach the analysis of how their synthesis is regulated [9]. Gene dose experiments employing merodiploid strains carrying plasmids with genes for ribosomal proteins showed that an increase of the number of a gene did not result in an increased cellular level of the protein, indicating a regulatory feedback which couples the rate of synthesis of the individual proteins to the amount needed in ribosome synthesis [10]. To test this contention in vitro a l phage specifically transducing the gene for ribosomal protein S20 was isolated and a transcription/ translation system was set up and employed by Reinhard Wirth, now at the University of Regensburg, to study S20 formation in vitro. The major finding was that 16s rRNA which binds S20 protein stimulated synthesis dramatically allowing the conclusion that S20 represses its own synthesis and that the repression is relieved by the consumption of the gene product in ribosome assembly [11]. It added an early stone to the mosaic of impressive work from Masayasu Nomura’s group in setting up a model for the feedback regulation of ribosomal protein synthesis, determined by their relative affinities to their rRNA-binding sites and the binding motif in the 50 region of the mRNA [12].

An Excursion to Archaea In 1978, I accepted a full professorship position at my original alma mater, the University of Munich. Intriguingly, this was the last but one German university which established microbiology as a discipline in a faculty of biology. The decision to leave Regensburg was difficult and was mainly determined by the fact that the curriculum there almost entirely aimed at the education of teachers

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and that it was hard to recruit students to enter diploma and doctoral work in microbiology which was not considered as relevant by students since it was not significantly included in the official catalogue of subjects covered by examinations. The faculty of biology in Munich, however, had meanwhile established a curriculum aimed at educating students also for professions outside of an academic career and we were supposed to structure the section of microbiology. The mixture of basic and applied topics which we offered attracted a high number of gifted students who were a rich source for very dedicated and engaged scientific co-workers over all the years. A more aesthetic aspect of the move also was that our laboratories were housed in one of the wings of the summer palace of the Bavarian kings in Nymphenburg. The palace was constructed in the first half of the 18th century as a replica of Versailles and offered a beautiful ambience. A disadvantage was that it was far off from other science institutions in Munich, although the remoteness to the university administration was sometimes considered as an advantage. Also in 1978, initiated by Otto Kandler and Rolf Thauer, a priority programme of the Deutsche Forschungsgemeinschaft was established with the aim to foster research in Germany on the biology of archaea in general and on methanogens in particular. We joined the programme with the aim of characterising the structure and function of the translational machinery as an evolutionary decisive cellular system. Coming from E. coli microbiology, a major difficulty was encountered when we implemented facilities and techniques for handling the obligate anaerobes. As an anecdote for such a genuine microbiological problem, I remember that we added supernatant of fermented sewage sludge to the cultures as a source for at that time unknown micro-nutrients. So I sent one of the students to the municipal sewage plant to get some sludge and he returned with a 25 l vessel filled to the top with it. He stoppered it very tightly and put it into the 371C incubator room. What must have happened, happened and the worst-case scenario took place. The stopper was blown out and the whole content was exploded into the room covering walls and equipment with a black layer of awfully smelling sludge. This was the only time when none of the other co-workers showed solidarity in helping with the cleaning task. The differential susceptibility of eukaryal 80s ribosomes and bacterial 70s ribosomes is traditionally considered as one of the

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distinguishing features of the two lines of descent. Accordingly, we started out with the sensitivity testing of halophilic and methanogenic organisms for antibiotics with different spectra of activity. In parallel, we also developed in vitro polynucleotidedirected polypeptide synthesis systems to exclude misinterpretations of the in vivo susceptibility results which could be caused by some problem in the cellular uptake of the compounds. It was found that archaea display a mosaic pattern of sensitivity to translational inhibitors. All archaeal ribosomes were refractory to inhibition by compounds like streptomycin or chloramphenicol whereas they were sensitive to the anti-70s directed compound thiostrepton. Most of them were found to be susceptible to some of the normally anti-80s active compounds like anisomycin and they were insusceptible to many other anti-70s and also anti-80s acting antibiotics, like erythromycin or cycloheximide, respectively [13]. Studies in the group of Piero Cammarano in Rome complemented this survey with the analysis of ribosomes from extreme thermophiles which showed traits even more different from those of the bacterial and eukaryal systems [14]. Although being phenotypic and non-quantitative markers, this pattern of antibiotic susceptibility pointed to the existence of a third class of translational machinery bearing functional traits of both 80s and 70s ribosomes [15]. To search for a structural basis for this mixed pattern of antibiotic sensitivity we initiated a sequencing project for ribosomal RNA and ribosomal proteins of methanogenic archaea. Methanococcus vannielii was chosen as model organism. Sequencing in the beginning of the eighties was conducted solely by hand since sequenators, gel readers or drawing software had not been developed yet. The co-workers involved in the sequencing project ran some kind of an Olympic competition for the longest readable sequence ladder and, as an anecdote, I remember that I hired the girl friend of one of my doctoral students to manually draw the secondary structure of the 23s RNA with the use of letter sets for publication. The saleswoman at the stationers was curious why we only bought sheets with Gs, As, Cs and Us. The product looked beautiful but it took her two months to finish the structure. The effort, however, was worth in two directions. First, the 16s RNA sequence of the methanogen determined by Michael Jarsch showed the overall primary and secondary structure architecture as the previously sequenced homologues from halophilic organisms,

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supporting the conclusions on their close relationship derived from the oligonucleotide pattern determined before. More importanly, when he continued his work in the determination of the 23s RNA sequence of this organism many of the antibiotic sensitivity/ resistance traits could be correlated with specific single base or signature motif differences of the ribosomal RNAs from the different lineages [16]. It thus supported our conclusion derived from studies on ribosomes from antibiotic producer organisms, namely that a major target of many antibiotics is the RNA part of the ribosome rather than the ribosomal proteins. The vast majority of bacteria contain multiple copies of the operon coding for ribosomal RNAs, so genetic proof for the correlation between antibiotic sensitivity and a sequence feature of the rRNA was not possible since sensitivity is dominant over resistance. The finding that many archaeal species contain only a single copy prompted us to prove the correlation experimentally using archaeal species. Heidi Hummel isolated thiostrepton resistant strains from a halophilic organism, and after sequencing the rDNA she found that they had replacements of the A-1067 base of 23s RNA which is essential for inhibition by the antibiotic in bacteria. A-1067 was replaced by a G (as characteristic of eukaryal 25s RNA) or a U. The same phenotype is generated in the thiostrepton producer organism by 2-O-methylation at the ribose moiety of A-1067 [7]. In another approach, mutants of Methanobacterium formicicum were isolated resistant against anisomycin, a classical inhibitor of cytoplasmic ribosomes from eukarya, analogous in its inhibitory action to the anti-70s compound chloramphenicol. Several mutational sites of the anisomycin-resistant derivatives were positioned within the so-called peptidyl-transferase loop of 23s RNA and one of them was identical to the exchange in murine mitochondrial 23s RNA from chloramphenicol resistant ribosomes. Altogether, the conclusion was that the antibiotic sensitivity pattern of ribosomes is a core phylogenetic feature and can be correlated with specific sequence motifs of the ribosomal RNA providing another line of evidence for the now recognised important role of ribosomal RNA in protein synthesis [15]. In the wave of excitement over the recognition of a third evolutionary lineage the genes for a great number of ribosomal proteins, for the two translation elongation factors and for a series of tRNAs were sequenced by Johannes Auer, Konrad Lechner and Gu ¨ nter Wich. An intriguing finding, besides the discovery of novel

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introns in tRNA genes [17], was that archaeal genes for ribosomal proteins were clustered within the different operons in the same organisation as they are on bacterial chromosomes. The sequences, however, were disperse in their conservation, some of them close to those from bacterial 70s or from eukaryotic 80s ribosomes and others were without any apparent relationship at all [18]. Another puzzling finding was that the latter ‘‘new’’ genes were intercalated into operons with an otherwise apparent bacterial-type organisation of genes. We worried considerably about the fact that the dendrograms for the individual protein sequences indicated very different relationships and did not follow the pattern of 16s rRNA sequence relationships. This inexplicability – much later dealt with in an elegant critical consideration by Ford Doolittle [19] – was eventually the reason why we left the field of comparative sequence analysis. A very rewarding spin-off result, however, still came from the availability of the sequences and upstream regions of many genes from the translational system. Since their expression should be coordinately activated we thought that they must contain identical transcription initiation motifs. Therefore, they offered themselves for the identification of transcription initiation signals in archaea, eagerly hunted for already in various groups but with inconclusive information because upstream regions of genes whose expression is governed by different physiological regimes were compared. Alignments of the upstream regions of genes coding for tRNA, rRNA and ribosomal proteins by Gu ¨ nter Wich highlighted two conserved motifs, designated box A and box B by us [20], with box A being an A/T rich signature later re-designated as archaeal TATA box and shown to constitute the equivalent of the eukaryal TATA motif in transcription initiation [21].

Regulation of Fermentation The move of our group to the University of Munich induced discussions on the directions the scientific projects should take in the future. Work with ribosomes became increasingly technologyoriented which required infrastructure and method platforms for which the manpower of a small team was limiting. Also, ribosome research was not in the heart of the expectations and requirements for the education of microbiology students for a professional

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career in industry or in science, apart from the observation that many students did not consider comparative sequence analysis as a creative activity after a while. This was one of the reasons to enter a new field, namely anaerobic metabolism of E. coli and its regulation. The choice was greatly influenced by the sabbatical stay of Frederick Neidhardt in our group at the University of Regensburg. With admirable intensity he worked at the lab bench and adapted the O’Farrell technique for the separation of proteins [22] to the analysis of the E. coli protein pattern, especially of cells subjected to some physical or chemical stress. I remember the excitement with which he showed me two-dimensional gels in which he had separated proteins from UV stressed cells and which just displayed a few spots of predominantly synthesised proteins, later on identified as components of the bacterial SOS response. This work clearly marks the onset of the era of proteomics [23]. Apart from Fred’s experimental achievements, our group took great ambition to train him and his wife Geri to become skilled mountaineers climbing peaks in the Bavarian, Austrian and North Italian Alps. Additional input to study anaerobic gene expression came from Hauke Hennecke, now at the ETH Zu ¨ rich, who had just returned from a postdoctoral stay at UC Davis and established his own group as Assistant Professor in the faculty. His elegant work on the NifA-mediated oxygen regulation of nitrogenase synthesis by Bradyrhizobium japonicum [24] raised our interest in the control of fermentation in E. coli. As a strategy, phage Mudlac was randomly inserted into the chromosome with the expectation that insertion of the phage into genes coding for enzymes with a function in fermentation should yield derivatives forming b-galactosidase solely under anoxic conditions. The experiments were conducted by Chaiwat Jatisatienre, a Thai doctoral student from Chiang Mai who had joined us to expand his experience in bacterial genetics and molecular biology. During a very broad and diligent screening project, he discovered two recombinants harbouring insertions that should keep almost my entire laboratory busy for the next 20 years [25]: One of the strains, mutant M9s has an insertion in the gene (fdhF) for the selenopolypeptide of formate dehydrogenase H and another one, mutant M17s in one of the genes (hycB) for the formate hydrogenlyase (FHL) system. M9s guided us towards unravelling the basic biochemistry of selenocysteine biosynthesis and insertion into proteins and M17s

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towards the elucidation of the regulatory network of the formate regulon and into the field of the synthesis and insertion of a complex metal cofactor into proteins, namely the [NiFe]-centre of hydrogenases. At that time, I was hardly aware that selenium is an element with biological function nor was there much information on the chemistry of metal centres, on their biosynthesis and on integration into the apoproteins. I still feel guilty for my university that Chaiwat could not continue his work in our group. The reason was the requirement for knowledge of the German language. From the training in a Goethe Institute he spoke it decently but in the language examination by the university he had dealt with complicated articles from a Munich newspaper. After failing several times, he quit and changed to the Technical University which did not demand such an examination. He performed excellent work there and teaches microbiology now in the University of Chiang Mai. In a second experimental approach, Anita Pecher separated cellular proteins from aerobically and fermentatively grown cells on two-dimensional gels in the search for proteins that are present only in anaerobically grown cells. Her work resulted in the discovery of the pyruvate formatelyase gene (pflA) which catalyses the non-oxidative cleavage of pyruvate, the reaction in the heart of the anaerobic metabolism of enteric bacteria (see Figure 1) [26]. The plasmid carrying the gene was handed over to J. Knappe, who had characterised PFL as a SAM radical enzyme; the gene rendered the system amenable for molecular analysis of the reaction mechanism. The work around pflA regulation, however, was followed by Gary Sawers, first as a postdoctoral associate and later as Assistant Professor and independent group leader. The Formate Regulon Pyruvate formatelyase and formate hydrogenlyase catalyse the key reactions within the mixed acid fermentation system of enterobacteria like E. coli (see Figure 1). Although it was known that the formate hydrogenlyase system consists of a formate dehydrogenase and a hydrogenase component [31], a genetic analysis had not been performed. The phenotype of strain M17s which Chaiwat Jatisatienre had isolated was a tool to identify putative structural genes since the integration abolished formate hydrogenlyase activity.

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Fig. 1. Regulation of carbon flux in the formate regulon of E. coli. Regulatory acting proteins are shaded and their interactions are given by broken arrows (for abbreviations see text).

Sequencing the chromosomal region around the insertion site of the Mudlac phage in strain M17s by graduate student Robert Bo¨hm revealed that the phage had inserted into a gene (hycB) belonging to an operon comprising altogether nine genes [27]; the introduction of an in-frame deletion into each of them by Martin Sauter showed that mutations in eight of them blocked the generation of formate hydrogenlyase activity [28]. An operational model was set up by Martin Sauter implicating that the system catalysing this seemingly simple reaction of formate disproportionation discovered by Stephenson and Stickland [29] consists of the seleno-molybdo-iron-polypeptide of formate dehydrogenase H, two FeS-cluster containing redox carriers, two very hydrophobic membrane-integral proteins plus the small and large subunits of a hydrogenase, designated hydrogenase 3. Five of the gene products displayed significant sequence similarity with components of the NADH-ubiquinone oxidoreductase (complex I) of the respiratory chain, a finding whose evolutionary relevance was not fully appreciated by us at that time. Subsequent detailed

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sequence comparisons by other groups showed later on that indeed many genes of the components of the mitochondrial complex I have their evolutionary origin in genes coding for components of hydrogenases [30]. A lucid although phenomenological description of the factors regulating formate hydrogenalyase formation (oxygen, formate, pH, nitrate) had been given already in 1957 by Peck and Gest [31] and with the DNA sequence available we were able to characterise this very complex regulatory circuit at the molecular level (for review see Ref. [32]). Over the years, this was unravelled by the doctoral works of a group of five superb graduate students. The first one was Angelika Birkmann who showed that expression of the fdhF gene and the hyc operon required upstream activating sequences and together with Scarlett Lutz it was worked out that sigma-54 (RpoN) is also required. Until then sigma-54 was assumed to specifically act in the activation of the expression of genes involved in nitrogen metabolism. A screen for mutations affecting expression in trans by Verena Schlensog lead to the discovery of the master switch protein FhlA. She showed that it is a member of the 2-component regulatory system family of proteins, but like XylR from Pseudomonas [33] FhlA is not activated by the activity of a sensory kinase via phosphorylation but by binding of a ligand that was identified by her as formate. Accordingly, instead of a phosphorylation input site, FhlA possesses an N-terminal extra domain for ligand coordination. Finally, Sylvia Hopper and Ingrid Korsa characterised the regulatory activity of FhlA by in vitro assays. Like XylR, FhlA is very prone to inclusion body formation when overproduced from a plasmid and only a fortuitous discovery facilitated the purification of sufficient protein to set up the in vitro system in which its regulatory activity could be analysed. It was found that although inclusion bodies were immediately formed during induction of the fhlA gene expression they resolved partially into soluble protein when the culture was continuously incubated for further 24 h. An intriguing aspect of the regulatory activity is that binding of the ligand formate modulates the overall ATPase activity of FhlA required for open complex formation at the promoter, mainly by increasing ATP-binding affinity of FhlA. The results were integrated into a model of sophisticated control of carbon flux which is depicted in Figure 1. When cells are shifted from respiratory to fermentative conditions, cleavage of pyruvate by pyruvate dehydrogenase becomes displaced by the

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non-oxidative cleavage by pyruvate formatelyase. The switch is facilitated by a basal level of pyruvate formatelyase present already in aerobic cells, which is inactive but rapidly activated when oxygen becomes limiting [34]. Work conducted by Gary Sawers showed that this activation is also accompanied by a massive increase of expression rendering pyruvate formatelyase the most abundant protein in the fermentatively grown cell. The induction of expression appears to involve a multi-component transcription activation complex at the 50 -upstream region of the pflA gene [34]. The activation of pre-existing inactive enzyme plus enzyme formation results in a massive production of formate, the primary signal molecule of fermentation. The concentration of formate is sensed by the FhlA regulator and binding of the ligand not only activates transcription of the fhlA gene in a forward feedback reaction (auto-activation) but also the expression of the formate hydrogenlyase genes. The resulting increase of the formate hydrogenlyase activity decreases the pool of formate and counterbalances the activation of gene expression by FhlA. The system is further poised by the activity of the first gene of the formate hydrogenlyase operon, hycA. Its product acts as an anti-activator of FhlA. The question of how external electron acceptors like nitrate suppress fermentation was addressed by Reinhild Rossmann in her diploma thesis. She studied the expression of transcriptional fusions of hycB with lacZ in response to the presence of oxygen, formate and nitrate and dependent on the pH of the medium. All these effectors act via the concentration of a single compound, namely formate [32]. Under aerobiosis, PFL is inactive and all formate generated via other routes is consumed by the aerobic formate dehydrogenase FDH-O. Under fermentative conditions, the formate produced by pyruvate formatelyase is exported at neutral pH, presumably via the formate transporter FocA [35]. At more acidic conditions an internal pool of formate builds up which can be several mM in size and converts FhlA into the active transcription factor inducing the formation of the formate hydrogenlyase system. Inclusion of nitrate in the medium represses fermentative enzyme formation indirectly by decreasing the pool of formate below levels required for binding to FhlA, which is around 5 mM. This is because PFL, the sole pyruvate cleavage enzyme, is partially replaced by pyruvate dehydrogenase, and secondly, formate dehydrogenase N is induced which due to its

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high affinity for formate and overall high activity keeps formate levels low. To my opinion, control of the carbon flux through pyruvate during the transition from aerobic to anaerobic growth of E. coli presents one of the most complex and refined adjustments of metabolism known. It integrates the availability of external electron acceptors like oxygen and nitrate, the pH of the medium, the availability of metals like Mo, Fe, Ni and of the half-metal Se and the concentration of the end product of fermentation, namely formate. Elegantly, formate is converted into hydrogen under acidic growth conditions as a means of pH homoeostasis whereby the energy content is maintained. The system would be a challenging topic for the computational analysis of carbon flux distribution at a metabolic branch point.

The Biosynthesis and Insertion of the Metal Centre of [NiFe]– Hydrogenases E. coli has the capacity to synthesise three active hydrogenases, two of them, isoenzymes 1 and 2, had been purified and characterised biochemically before we initiated the project on hydrogenase synthesis and maturation [36]. Isoenzyme 3, the component of the formate hydrogenlyase complex, was identified genetically in our group during our work on regulation of fermentation. Whereas mutations in the genes of each of the three operons for these hydrogenases blocked the formation of the respective isoenzyme only, there are mutational loci outside these structural gene operons which pleiotropically affect the build-up of activity of more than one of these enzymes. Most of these genes mapped at the 50 -side of the operon for hydrogenase 3 (hyc, see Figure 1) on the E. coli chromosome. Intriguingly, the effect of mutations in one of these genes could be rescued by high nickel content of the medium which suggested a direct role of the gene product in incorporation of this metal [37]. In the beginning of this project we wanted to address the question on how nickel is inserted into the hydrogenase apoprotein and we assumed to obtain easy experimental access to answering it by sequencing the chromosomal region in which the gene is located whose lesion can be complemented by an increased supply of the metal in the medium. As the complex structure of the metal centre was not known this assumption appears somewhat naive now in

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retrospect as we now know the machinery involved, comprising biosynthetic enzymes for CO and CN, a nickel chaperone, a GTPase, a redox-active 4Fe-4S cluster containing protein, a small chaperone for the large subunit and an endopeptidase. Scarlett Lutz and Alexander Jacobi who cooperated in the sequencing project discovered a divergently oriented operon comprising six genes; the promoter-distal one of them had been already known as fhlA, the regulator of the formate regulon (see above). The introduction of in-frame deletions into the five genes located upstream of fhlA showed that their products were involved in hydrogenase maturation (see Figure 1) [38]. A further gene (hypF) was discovered at the downstream side of the hyc operon and was also identified as maturation gene by Thomas Maier in our group [39]. We suggested a designation for these genes as ‘‘hyp’’ because mutant alleles pleiotropically abolished the capacity to form all three hydrogenases in their active state. During the Hydrogenase Congress in Albertville in 1997 organised by Paulette Vignais there was a lively discussion on the appropriateness of the designation because it does not apply to organisms possessing only a single hydrogenase; however, the recent information on the biochemical functions of the gene products as components of a hydrogenase maturation machinery favoured the acceptance of the nomenclature. With the support of the ‘‘Wasserstoffprogramm des Bundesministeriums fu ¨ r Forschung und Technologie’’ initiated and coordinated by Rolf Thauer and the two COST programmes of the European Union organised by Kornel Kovacs we set out to unravel the biochemical roles of these maturation proteins in the biosynthesis of [NiFe]–hydrogenases. A prerequisite and indispensable tool was the introduction of in-frame deletions into each hyc and hyp gene located at their genuine chromosomal site. As much as possible of each coding region was removed to preclude any interference of a truncated gene product with the maturation process. Each of the genes was also cloned and its gene product purified; antibodies were elicited in rabbits as a tool for qualitative and quantitative assessment of the antigen. Use of the hexa-his tag in the purification and characterisation protocols was avoided to exclude any false interaction with metal containing proteins during metal chelate affinity chromatography. Instead, the Strep tagII system was adapted to the purification of metallo-proteins in collaboration with the ¨ttingen [40]. Institut fu ¨ r Bioanalytik GmbH, Go

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A characteristic phenotype of these hyp mutants was that the large subunit of the heterodimeric hydrogenases from E. coli lacked nickel and that it existed in a more slowly migrating form [41] which later was identified as a precursor containing a C-terminal extension. Working with the Azotobacter hydrogenase, Gollin and coworkers had observed this extension and described its proteolytic removal already in 1992 [42], several years before the crystal structure directly proved that the mature large subunit is shorter than expected from the gene sequence [43]. The pending question we asked was on the role of this proteolytic processing and Reinhild Rossmann addressed it by assessing the substrate specificity of the cleavage enzyme. She purified the nickelcontaining precursor from a mutant devoid of the processing activity. Cleavage of the precursor took place when nickel was present but not when it had been removed by incubation with 2-mercaptoethanol. The cogent conclusion was that the presence of nickel is required for cleavage to take place [44]. Reinhild Rossmann furthermore succeeded in purifying the endopeptidase involved and by reverse genetics she identified the structural gene (hycI) as the promoter-distal one of the hyc operon (see Figure 1). The endopeptidase is a monomeric protein of about 17 kDa molecular mass; the purified protein does not contain any metal [45]. A joint project between Erich Fritsche from Robert Huber’s group and Athanasios Paschos was initiated to crystallise the HycI maturation endopeptidase but all attempts to obtain crystals failed. Surprisingly, they immediately succeeded, however, in the crystallisation of HybD, the maturation endopeptidase for hydrogenase 2 from E. coli and to resolve its three-dimensional structure. The crystallised protein contains a cadmium ion from the crystallisation buffer which is coordinated by the carboxyl oxygens of a glutamic and an aspartic acid and the imidazole nitrogen of a histidine residue in a unique binding motif [46]. Since replacements of these residues by chemically dissimilar ones were detrimental for activity and since no other essential residue could be detected in a mutational screen we assumed that the cadmium-binding site reflects the binding motif for nickel. Our present model for catalysis is that the enzyme belongs to the class of metallo-proteases which use the metal ion in the substrate for activity [47]. The proof for this assumption requires the resolution of the crystal structure of a substrate complex which is not yet available.

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What is the rationale for the removal of the C-terminal extension of the large subunit by endoproteolysis? As long as we do not have the three-dimensional structures of maturation intermediates of the large subunit there is no convincing conclusion. What we observed, however, is that the proteolytic processing causes a large shift in the electrophoretic mobility indicative of an intensive conformational change triggered by the cleavage. We take this as circumstantial evidence that for the insertion of the metals the protein needs to be in an ‘‘open’’ conformation and the conformational change brings the metal centre into the interior. So the extension may act as an intra-molecular chaperone; such a function also is in accord with the observation that expression of the large subunit from a gene with a deletion of the fragment coding for the C-terminal extension renders a product amenable to rapid degradation. The substrate specificity of the maturation endopeptidase is high explaining the necessity that the maturation of each hydrogenase affords its specific protease; E. coli thus possesses three maturation endopeptidases for the formation of hydrogenases 1, 2 and 3. The requirement of proteolysis as a final step in metal incorporation into an apoprotein has another example in a very different biological system: The incorporation of manganese into the Mn–Ca cluster of the D2 protein of the photosystem II from chloroplasts also involves the proteolytic cleavage of the protein after metal incorporation [48]. Before the chemical structure of the [NiFe] metal centre was published in 1996 (see Figure 2) [49,50], the biochemical function of another member from this zoo of maturation proteins was

Fig. 2. Established and postulated reactions involved in the synthesis and insertion of the metal centre of [NiFe]–hydrogenases as worked out for the model system of hydrogenase 3 from E. coli. (a) Synthesis of the cyanide ligand by maturation proteins HypF and HypE. (b) Scheme for the cyanation of Fe, the transfer to the precursor of the large subunit by ligand exchange followed by the insertion of nickel and proteolytic processing. The scheme shows the transfer of one CN moiety to the HypC  HypD complex and from there to the large subunit. This ligand transfer cycle must be repeated three times for full coordination. The following abbreviations are not mentioned in the text: CP, carbamoylphosphate; pre-L-SU, precursor of the large subunit of the [NiFe]-L-SU, mature large subunit of the [NiFe]–hydrogenases. Fig. 2b is a reprinted version of Fig. 10 from Advances in Microbial Physiology, Vol. 51, pp. 1–71 (2006), with permission from Elsevier.

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identified, namely HypB. Its sequence features the function of an NTPase and Thomas Maier demonstrated GTP binding and low intrinsic GTP hydrolysis activity for the purified protein. The exchange of one of the essential amino acids involved in binding or hydrolysis of GTP blocked hydrogenase maturation indicating

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that GTP hydrolysis rather than just binding is necessary. HypB was thus the first member of the GTPase family for which a function in metalloprotein synthesis was described [51]. Its characterisation was followed by the findings that urease formation, CO dehydrogenase synthesis and [FeFe]–hydrogenase maturation also require functionally homologous enzymes (see Ref. [52]). The identification of the active site metal centre as NiFe (CO)(CN)2 [49,50] opened experimental approaches for the elucidation of the function of the other maturation proteins. Accordingly, we expected activities for the anoxic synthesis of CO and CN which had not been described yet for E. coli. Furthermore, mechanisms ought to exist for the coordination of these ligands by the Fe atom and for the maintenance of the 1:2 stoichiometry between CO and CN, for bridging the iron and the nickel sites and for internalisation of the cluster into the body of the protein. Athanasios Paschos searched in the sequences of all Hyp proteins for signatures indicating some function in organic synthesis and he discovered relevant motifs within the HypF sequence. Apart from the already known acylphosphatase motif in the N-terminal part of HypF he found a short sequence stretch with a faint similarity to motifs characteristic of O- and N-carbamoyltransferases. The substrate of these enzymes, carbamoylphosphate, appeared to us as a compelling precursor for the formation of CO or CN or of both and, indeed, mutations in one of the carbamoylphosphate synthetase genes (carAB) abolished the capacity of E. coli to form active hydrogenases [53]. What we had missed at that time was a report from Erika Barrett’s laboratory – years before the chemical structure of the metal centre had been elucidated that a screen in Salmonella for hydrogenase negative mutants had delivered strains with a block in carAB [54]. A regulatory role had been assumed by these authors but the credits for an essential role of carbomylphosphate in the generation of active enzyme go to these authors. It was very fortunate that Richard Glass from the Chemistry Department of the University of Arizona spent a sabbatical in our group in Munich at that time. He had joined us with the intention to work on the selenium biochemistry project but his expertise and advice were indispensable for us in making progress in the elucidation of the path for synthesis of the CN ligand. As a chemist, he immediately pointed out to us that chemical reactions are known which are able to convert an iron carbamoyl complex into a

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carbonyl or cyanyl complex [53]. Accordingly, we assumed that biosynthesis of CO and CN takes place from carbamoylphosphate by ligand chemistry at the iron and we were very surprised when we found that CN formation takes place by sulphur chemistry instead and, later on, that CO must be formed via a different path. Thus, CN synthesis involves an ATP-dependent carbamoylation of the thiolate of the C-terminal cysteine in maturation protein HypE yielding HypE-thiocarboxamide (Figure 2a). In a second step, the carboxamide moiety is dehydrated via ATP-dependent phosphorylation of the enolic form and the subsequent dephosphorylation into HypE-thiocyanate. These steps could be carried out by in vitro assays employing purified components and 14C-labelled carbamoylphosphate and the products were identified via mass spectrometry by Stefanie Reissmann in cooperation with Friedrich Lottspeich from the Max-Planck-Institute of Biochemistry [55]. The unprecedented biochemical question how HypE-S-CN donates the cyano group to the iron was addressed by Melanie Blokesch in her doctoral work. She starved cells for carbamoylphosphate and searched for changes in the electrophoretic migration pattern of Hyp maturation proteins. Indeed, under this condition cellular extracts contained a tight complex between two maturation proteins, namely proteins HypC and HypD (Figure 2b). Members of the HypC protein family contain an essential Cys residue at their N-terminus (the methionine is posttranslationally removed), and members of the HypD family possess an EPR silent and reducible 4Fe-4S cluster [52]. This complex disappeared when citrulline was donated to the carAB mutant as a source of carbamoylphosphate. The resolution was not observed when the large subunit of the hydrogenase was not present because of a deletion of the gene. Under this condition, the HypC  HypD complex changed to a slower migration position indicative of some modification like one observes in precursor product relations [56]. With this information at hand, Melanie Blokesch succeeded to reproduce the in vivo results with purified components in vitro. She isolated the HypC  HypD complex by affinity chromatography using a HypC variant with a Strep tag at the C-terminus. When isolated from anaerobically grown cells and under anoxic conditions, the HypC  HypD complex could be cyanated with HypE-S-CN as a CN donor (Figure 2b, step A). Oxidation of the cluster destroyed the acceptor activity meeting our expectation that coordination of one CN group would require two electrons [57].

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Haofan Wang in the group of Richard Glass studied the transfer of the cyano group to iron in elegant chemical model reactions. He achieved efficient cyanation of Fe in model compounds both by nucleophilic and electrophilic substitution that demonstrates the feasibility of the biochemical reaction. Conversion of a thiocarboxamide into a thiocyanate could be demonstrated in these model reactions as well [55]. A major worry we still have concerns the chemical nature of the bond between CN and the HypC  HypD complex. Its stability characteristics indicate that it cannot be linked to a thiolate like in HypE-S-CN and it is rapidly lost during mild denaturation of the HypC  HypD complex. As a working model we assume presently that CN is coordinated by one of the irons of the 4Fe-4S cluster from HypD which is also coordinated to protein HypC. Support for this assumption comes from the facts that exchange of the N-terminal Cys residue of HypC which is strictly conserved in the entire HypC family and of a Cys motif within HypD abolishes complex formation and consequently CN acceptor activity. Such a mechanism would also be attractive since the 4Fe-4S cluster could be re-reduced after each cycle of ligand coordination and, after transfer of the HypC-Fe(CO(CN)2 to the precursor of the large subunit (Figure 2b, step B) Fe could be replenished like in the case of the 3Fe-4S centre in the iron cycle of aconitase [58] (Figure 2b, steps E and F). The transfer of the fully coordinated Fe atom to the large subunit of the hydrogenase could take place by ligand exchange which liberates HypD and now leaves HypC bound to the large subunit (Figure 2b, step B). Such a complex between HypC and the precursor of the large subunit has been detected very early in the course of our studies and characterised intensively by Nicola Drapal [59]. It is accumulated to particularly large levels in strains which are blocked in nickel insertion or in proteolytic processing. The biosynthesis and coordination of the CO ligand has not yet been addressed thoroughly. 13CO2-labelling studies performed by the group of Siem Albracht with Allochromatium vinosum had indicated some time ago that CN and CO in the enzyme of this organism come from different sources [60]. The result was surprising since CO2 was the only carbon source offered in the medium of this photosynthetic organism. Also, in our group we were very convinced that the chemistry of carbamoylphosphate

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suggests that it is a very compelling precursor also for the formation of the CO ligand. A very neat story then is that the EDTA used for chelating Fe in the trace element solution for the cultivation of A. vinosum was found to liberate some acetate which was used by the organism for the synthesis of CO. The C1 carbon of acetate was incorporated preferentially over the C2 carbon (560). Melanie Blokesch followed this lead in the E. coli system and analysed whether HypE-thiocarboxamide can donate the carbamoyl group to the HypC  HypD complex as a putative precursor for the formation of CO. The experiment was performed with a mutant HypE protein unable to bind ATP and therefore unable to dehydrate the HypE-S-carboxamide into the HypE-thiocyanate: She found that this HypE variant could still be carbamoylated but neither dehydration to the thiocyanate nor transfer to the HypC  HypD complex did take place. This can be taken as convincing evidence that as in A. vinosum carbamoylphosphate is not the educt for CO biosynthesis in E. coli [61]. Thus, the two ligands are derived from different sources, once again showing that plausible chemistry is not necessarily followed in biological systems. When we initiated the project on hydrogen maturation our main aim was to study nickel insertion into the protein. Now we know that nickel insertion (Figure 2b, step C) follows insertion of the fully coordinated iron into the precursor of the large subunit. Which arguments do we have? The major one is that nickel incorporation and further maturation into an active hydrogenase by purified endopeptidase can be demonstrated in in vitro assays, even in the absence of the HypB GTPase [62]. Equally, in vivo a hypB mutant synthesises active hydrogenase when grown in the presence of an excess of nickel, although with a lower efficiency. The situation became even more complicated by the finding of Michaela Hube that maturation protein HypA also plays a role [63]. Both HypA and HypB bind nickel but it is still open which one is the physiological nickel donor. The resolation of the crystal structure for HypB from an archaeon in Alfred Wittinghofer’s group revealed the existence of a potential metal-binding site at the interface of the subunits of the dimeric enzyme [64]. A role for GTP hydrolysis in the release of the metal seems plausible although other mechanism like donation of nickel by the HypA protein with GTP hydrolysis by HypB as a pure switch cannot be excluded yet. An in vitro system mimicking the dynamics of the process is required.

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As already pointed out, insertion of nickel converts the precursor of the large subunit into a substrate for the endopeptidase. Removal of the extension triggers a conformational switch in which the newly generated C-terminus with cysteine 4 (Figure 2b, step C) bridges the two metals and ‘‘closes’’ the centre. The conformational change also triggers the internalisation of the centre into the body of the large subunit close to the interface followed by the docking to the small subunit (Figure 2b, step D). Although we can outline a certain sequence of events in the maturation process there are still many gaps to be filled. Several of the steps illustrated in Figure 2b are still highly speculative, e.g. concerning the source of CO, the site of coordination of the Fe atom with the ligands or the functions of the HypA and HypB proteins in nickel insertion. Despite these gaps in our information it is clear that the path of maturation of [NiFe]–hydrogenases is fundamentally different from those of the [FeFe]–hydrogenases [51] and possibly also from that of the FeS-cluster free hydrogenases. The minimal property which the three enzyme classes share is the existence of an iron-carbonyl which may have been the first primitive catalyst for hydrogen oxidation/proton reduction. As outlined by Rees and Howard [65], the optimisation of primordial inorganic catalysts for higher efficiency necessitated the development of complex machineries rendering the metal centre more sophisticated and also providing the necessary chemical environment by insertion of the cluster into a protein. In the case of the three classes of hydrogenases the optimisation must have followed three different directions both concerning the catalyst and consequently also the apoprotein. In my opinion this is one of the most spectacular cases of convergent evolution in biochemical systems.

Selenium Biochemistry Specific Incorporation of Selenium As already mentioned, a serendipitous spin-off from our project on the control of expression of genes coding for fermentative enzymes lead us into the field of selenium biochemistry, in particular into the analysis of selenocysteine biosynthesis and its incorporation into polypeptides. This was because one of the mutants with a

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Mudlac insertion in such an anaerobically expressed gene showed a deficiency in the oxidation of formate paralleled by a blockade of fermentative hydrogen formation. It was concluded that the phage had inserted into the gene for the formate dehydrogenase H (FDH-H) [31]. The gene was mapped in a hitherto unknown chromosomal site and was designated fdhF [66]. Since according to the work of Jane Pinsent formate dehydrogenase formation by E. coli occurs only when the medium contained selenium and molybdate (which actually was the first proof for a biological role of this element) [67], and because John DeMoss and colleagues had just a few years before devised a protocol for the incorporation of radio-active selenium into polypeptides of E. coli [68], Anita Pecher had the tools at hand to demonstrate that the Mu insertion was located in the gene for the selenopolypeptide of FDH-H [66]. She also found that the strain is still able to form the isoenzyme FDH-N whose activity is coupled to nitrate reductase, the conclusion therefore was that fdhF is the structural gene of FDH-H and not a gene involved in some general mechanism for the incorporation of selenium. The gene was handed over to Franz Zinoni to initiate studies on the mechanisms of selenium incorporation and on the nature of the selenium compound. He detected the presence of an in-frame TGA stop codon in the sequence of the open reading frame which, of course, let us first assume that it was the consequence of some sequencing or reading error. After ruling out such trivial error possibilities by re-cloning and re-sequencing we were forced to get acquainted with the idea that there may be a connection with selenium incorporation [69]. We had considerable worries on how to prove the validity of this provocative idea. As the chemical nature of the selenium-containing compound in FDH-H was not known, I discussed the matter with Thressa Stadtman during a Gordon Research Conference on ‘‘Methanogenesis’’ in 1984 and we agreed to cooperate. In 1976, Thressa Stadtman had resolved that the selenium compound in selenoprotein A of the glycine reductase from Clostridium sticklandii was selenocysteine [70]. It took her several years to confirm by peptide sequencing employing fusions between the TGA-containing fragment of the fdhF gene and lacZ that selenocysteine is also the selenium-compound in the FdhF polypeptide [71]. The reason for the delay was that classical peptide sequencing strategies could not be employed because of the instability of selenocysteine in most of the sequencing

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reagents and the conditions employed. Intriguingly, the presence and sequence position of selenocysteine in the glutathione peroxydase protein from rat had been known long before the determination of the nucleotide sequence revealed that the mRNA contained a UGA stop codon at the respective position. The latter finding was published one month before the appearance of our manuscript on the fdhF gene sequence [72]. A major inherent problem we encountered was that the co-linearity between the TGA codon in the gene and the selenocysteine in the polypeptide chain principally is not sufficient to prove that the codon directs the insertion; consequently, it is not possible to discriminate between co-translational insertion and post-translational modification. Mechanisms alternative to the cotranslational incorporation could have been the conversion of a classical amino acid inserted by some UGA suppressor tRNA which is converted into selenocysteine either before or after incorporation. Definite proof for the co-translational incorporation model, however, came from genetic experiments in which different 50 -portions of the fdhF gene containing or lacking the TGA codon site were fused to lacZ: Read-through over the UGA was only observed when selenium was present in the medium. Most satisfying was that the alteration of the UGA into a classical codon obviated the requirement of selenium for b-galactosidase formation [73]. It took us 3 years from the detection of the TGA codon and the definite proof that it is decoded with a selenium-containing amino acid and 5 years until we knew that this is identical with selenocysteine. I have to add that as being newcomers to the selenium club we had to learn considerably from the many faces of this element, particularly about its ubiquitous presence as a contaminant in chemicals. We were surprised over the difficulty to devise seleniumpoor medium and even more, when we found that minimal medium contained much more selenium than rich medium. The rich medium compounds had of course already been subjected to a biological discrimination process which decreases the ratio of selenium relative to the sulphur compounds, so the best ‘‘low-selenium’’ source of sulphur was found to be cysteine because it is solely produced by the hydrolysis of biological material like hair. Because of all this experience we are worried sometimes by the unhesitant assignment of UGA as selenocysteine codon in gene annotations without biochemical controls, like labelling the gene product with radioactive selenium.

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Having established that selenocysteine incorporation into polypeptides takes place co-translationally at the ribosome we were confronted with the questions how the amino acid is synthesised, how it is attached to some (unknown) tRNA and how the UGASec codon is discriminated at the ribosome from a classical UGA stop codon. Genetics again was extremely helpful in unravelling the components involved in these steps. Mutations in the genes whose products are involved should lead to pleiotropic deficiency in the synthesis of all three formate dehydrogenase isoenzymes from E. coli. Strains carrying such mutations (fdhA, fdhB and fdhC) had been described in the literature and they were kindly provided by Marie-Andre´e Mandrand-Berthelot from Lyon [74]. Walfred Leinfelder in our group also initiated such a mutant search and demonstrated that the fdhA locus previously described actually contains two genes and he cloned and sequenced all these fdh genes. They were shown to code for components of the selenoprotein synthesis machinery because mutants with lesions in these genes were unable to synthesise all of the three formate dehydrogenase isoenzymes. Consequently, we re-designated the genes as selA, selB (previously fdhA), selC (previously fdhC) and selD (previously fdhB) [75]. The elucidation of the biochemical function of their products went very quick, owing to the experience present in our group of techniques connected with work with aminoacyl-tRNA synthetases and ribosomes (Figure 3b). The sequence of selC showed that it does not code for a protein but its derived RNA product could be folded into a tRNA-like structure. The putative tRNA differs, however, greatly from that of canonical elongator tRNA species both in size, primary and secondary structure features. Its putative anticodon ACU matches the UGA codon which was very surprising for us in view of the canonical use of UGA as stop signal. Despite these differences this tRNA could be charged, albeit sluggishly, with L-serine by the canonical seryl-tRNA synthetase which also aminoacylates the serine family of tRNAs (SerS, Figures 3b and 5) [76]. Information on the functions of the other three gene products was collected by Walfred Leinfelder in a heroic approach in which he isolated a great batch of tRNA from cells grown in the presence of 75 Se-selenite of high specific radioactivity and after alkaline hydrolysis analysed the aminoacyl group by thin layer chromatography. E. coli wild type contained selenocysteyl-tRNA in small amounts, selA and selD mutants were devoid of it whereas selB

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Fig. 3. Scheme of the incorporation of the 20 canonical amino acids (a) and of selenocysteine incorporation (b) into polypeptides. Note that elongation factor Tu (EF-Tu) acts in the form of a ternary complex with one of the standard 20 aminoacyl-tRNAs and GTP from solution, whereas SelB forms a quaternary complex with selenocysteyl-tRNA, GTP and the SECIS element of the selenoprotein mRNA. Release factor 2 (RF2) recognises cognate UGA stop codons but not the UGA in the context with the SECIS element (courtesy of Martin Thanbichler) (see Colour Plate Section at the end of this volume).

strains accumulated it at a grossly increased level [77]. The cogent conclusion was that SelA and SelD are biosynthetic enzymes with a function in converting the seryl-moiety of tRNASec into the selenocysteyl group and that SelB must act ‘‘downstream’’, i.e. in the decoding process. This also confirmed that the carbon skeleton of serine is used as an entity for selenocysteine biosynthesis as published by Sunde and collaborators before [78] and showed that the biosynthesis takes place in the tRNA-bound state. A tRNA species with similar structural properties as tRNASec had been reported by Dolph Hatfield to be present in many eukaryotic organisms and to carry the O-phosphoseryl group; it had been assumed that it inserts O-phosphoserine into proteins. Since we were intrigued by this similarity of tRNASec with this tRNA concerning both sequence and secondary structure features we communicated our results via Thressa Stadtman to Dolph

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Hatfield. Subsequently, his group could identify that this tRNA actually inserts selenocysteine into eukaryal proteins [79]. The side-by-side publications of our two groups reported the first evidence for a structural and functional conservation of selenoprotein formation in bacteria and eukaryotes [77,79]. A functional difference between protein SelA and protein SelD was that mutations in selA blocked only selenoprotein formation whereas those in selD lead to a blockade of both selenoprotein synthesis and the conversion of mnm5S2U to mnm5Se2U which is a modified base in tRNALys and tRNAGlu (see Figure 5). The function of SelA was unravelled by Karl Forchhammer, now at the University of Tu ¨ bingen. He purified the protein and discovered that it contains a pyridoxal phosphate prosthetic group and it reacts with seryl-tRNASec. In the absence of a selenium donor in the assay SelA synthesised a product determined as dehydroalanyltRNASec [80] because of its reducibility with borohydride to alanyltRNASec. The reaction thus follows a classical 2,3-elimination of water from the serine moiety. Full conversion into selenocysteyltRNASec required the presence of a source for reduced selenium plus ATP and protein SelD. As a source for the reduced selenium we used selenite plus dithiothreitol; the problem is that during the time course of the assay the selenite is reduced through all oxidation states of selenium ending eventually in the elemental form. The structure of SelA, which was designated as selenocysteine synthase, was addressed by Harald Engelhard from the Max-Planck-Institute of Biochemistry in Martinsried. His impressive electron microscopy studies confirmed Karl Forchhammer’s biochemical results that the enzyme from E. coli is a homodecamer and showed that it is built of two pentameric rings stacked on top of each other. Two juxtaposed subunits, one from each ring, act as a functional unit in binding one serylated tRNASec [81]. The fully loaded protein thus carries five molecules of charged tRNAs, indeed an unprecedented substrate complex of the molecular mass comparable a 30 s ribosomal subunit. Barbara Veprek and Armin Ehrenreich purified the SelD protein from E. coli in the functional state and they showed that the ATP required to activate selenium is cleaved into AMP and inorganic phosphate [82]. Elegant enzymological analysis of the unique reaction carried out in Thressa Stadtman’s group unequivocally characterised the activated selenium species as monoselenophosphate, the first direct selenium–phosphate bond in

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a natural compound [83]. SelD accordingly was designated as selenophosphate synthetase. The biological origin of the reduced selenium species which is used as a substrate by SelD is not fully clarified, mainly because of the outlined difficulty to maintain a defined redox state of selenium ions in solution. Some choices are selenide or a selenium species liberated from selenocysteine by the action of one of the selenocysteine lyases from E. coli [84]. The SelD protein of most organisms differs from that of E. coli by containing a selenocysteine residue instead of the essential cysteine residue in the amino acid position 17 of the E. coli protein. Its role in the catalytic cycle is not clear. An unsolved puzzle also is that most of the eukarya contain a second type of SelD protein, SelD-like, in which this cysteine (selenocysteine) at position 17 is replaced by some other amino acid. Despite an extensive sequence similarity the purified variant from Drosophila was unable to catalyse the formation of monoselenophosphate from reduced selenium and ATP [85]. What biological function does it serve and, above all, what is the solution of the dilemma that a component of the selenocysteine insertion machinery is a selenoprotein itself? How can a pool of active enzyme be built up when an organism recovers from selenium starvation? The existence of an in-frame UGA codon violates the rule that within one genetic system no codon must have more than one meaning. Our two-fold dilemma therefore was to delineate mechanisms which explain (i) why the in-frame UGA is not recognised as a translation termination signal and (ii) why ordinary UGA stop codons do not serve as selenocysteine codons. Clearly, codon– anticodon recognition and interaction cannot explain the difference between the two readout meanings, some outside structure which differentiates between sense and nonsense must be involved. Alternatives which we discussed were either some specificity component of the ribosome or an inherent property of the mRNA. The answer to the question came again from genetic experiments in which Franz Zinoni fused fdhF with the lacZ gene at different distances to the TGA codon at the 30 -side. He discovered that a hairpin structure of the mRNA is required following the UGA at the 30 -side and that the nucleotides in the loop region of this hairpin were of paramount importance (Figures 3 and 4a). We nominated this phenomenon as ‘‘coding from a distance’’ since the change of one of the essential parameters resulted in alteration of the function of the UGA: it was converted into a termination

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Fig. 4. Recognition of the SECIS element by elongation factor SelB. (a) Stemloop structure of the fdhF mRNA from E. coli and the 17 nucleotides minihelix (shaded). Nucleotides protected by SelB against chemical modification are indicated by triangles. (b) and (c) Interaction of the SelB protein and the SECIS element from Moorella thermoacetica. In (b), the C-terminal two winged-helixmotifs are shown from which the ultimate one interacts with the SECIS RNA via its base G23 and a string of charged residues. In (c), a summary of the functional groups of G23 and of the phosphates of the RNA backbone interacting with groups from SelB are given [101] (permission granted by Nature Publishing Group). (d) Image of the 50s ribosomal subunit (crown view) into which the Methanococcus maripaludis SelB-selenocysteyl-tRNA complex has been modelled [106] (courtesy of Nenad Ban and Marc Leibundgut) (see Colour Plate Section at the end of this volume).

codon [86]. Following a suggestion of Marla Berry who worked on selenoprotein formation by eukaryal organisms, the stem-loop structure was designated as SECIS (selenocysteine insertion sequence) element [87]. This context dependence of UGA decoding brought an explanation for the aforementioned dilemma number (ii): ordinary stop codons are not decoded by selenocysteine since they lack such a SECIS recognition structure in the appropriate location. For addressing dilemma number (i) we had to postulate that the recognition sequence must interact with some partner, either the ribosome or another molecule. We proposed SelB as a critical interaction partner since our experiments

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had shown that it acts ‘‘downstream’’ of selenocysteyl-tRNASec formation [77]. Accordingly, Karl Forchhammer became interested in the characterisation of the functon of SelB. The derived amino acid sequence again gave the key information since it predicted that the N-terminal two-thirds of the 69 kDa protein bear similarity with the sequence of guanine nucleotide-binding proteins, in particular with that of elongation factor Tu (EF-Tu) and somewhat less with that of initiation factor 2 [88]. He purified SelB from an overproducing recombinant host and confirmed that it possesses a low intrinsic GTPase activity. Intriguingly, different from the binding characteristics of EF-Tu, the apparent affinities for GTP and GDP were in the same order of magnitude, suggesting that the two substrates might exchange chemically and without the involvement of a guanine nucleotide release protein. The potential functional homology between SelB and EF-Tu was then further substantiated by the demonstration that SelB binds charged tRNASec, most importantly, however, only in the selenocysteylated but not the serylated form. SelB thus discriminates the aminoacyl residue of tRNASec whereas EF-Tu on the average indiscriminately binds the charged forms of all tRNAs that insert the 20 standard amino acids. The finding from Matthias Sprinzl’s group that EF-Tu conversely does not use tRNASec as a ligand under physiological conditions [89] left us with the conclusion that SelB fulfils the function of EF-Tu in the insertion of selenocysteine into polypeptides (Figure 3). It was Johann Heider, who moved to the University of Darmstadt recently, who came up with a both challenging and provocative idea about a possible function of SelB, namely, which was that the protein also might bind to the mRNA coding for selenoproteins, in particular to the 40-bases long stem-loop structure located downstream of the UGASec codon and thereby ‘‘guide’’ the selenocysteyl-tRNA to the decoding site of the ribosome. In a congenial collaboration with a second graduate student, Christian Baron who is now at McMaster University, they proved the validity of this assumption via mobility shift assays: presence of both the charged tRNA and of the SECIS element caused an extensive supershift of the electrophoretic position of SelB. In addition, by using mutated versions of the stem-loop structure they highlighted nucleotides essential for complex formation [90]. Supporting information for this unique situation that an elongation

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factor binds the charged tRNA species and also a motif of the mRNA to be decoded was brought about by several independent experimental strategies. Probing of the nucleotides of the SECIS which are protected against chemical modification when it is bound to SelB showed that a GU doublet at the tip of the hairpin was essential [91] (Figure 4a). In a cooperative project with Michael Famulok intriguing information was obtained via the in vitro selection of aptamers binding to the protein [92]. About half of them possessed the sequence and putative secondary structure of the wild-type element. Amongst the residual aptamers there were binders which interacted with even a higher affinity with SelB but they did not display biological activity. This was taken as evidence that the geometry or dynamics of the interaction is of paramount importance. Finally, the isolation of intergenic suppressor mutations in which the detrimental effect of mutations in the SECIS stem-loop structure were compensated by amino acid exchanges of SelB highlighted possible segments of SelB taking part in the interaction [93]. During a follow-up doctoral work performed by Matthias Kromayer, the domains of SelB participating in this interaction with the mRNA were characterised. We had long been intrigued by the fact that SelB possesses a C-terminal extension which bears no similarity at all with EF-Tu on the one hand or with such extensions of SelB species from distantly related organisms on the other hand. Since the SECIS elements from mRNAs coding for functionally different enzymes also differ, a plausible assumption was that the SelB extension is the site for binding of the SECIS and that its heterogeneity reflects the evolutionary adaptation of SelB to this ligand. Matthias Kromayer approached the question by truncating the extension of SelB and also the mRNA containing the SECIS to the minimal sizes still functioning in this interaction. The result was that the minimal domain of SelB was the 17 kDa ultimate C-terminus and that the SECIS element could be reduced to a 17 base mini-helix (shaded in Figure 4a) that constitutes the top part of the SECIS element without loss of complex formation [94]. SelB thus binds two RNA ligands, the selenocysteyl-tRNA by the EF-Tu-like N-terminal part and the 17 base secondary structure of the selenoprotein mRNA by the C-terminal extension. Together with GTP they constitute a quaternary complex which is preformed at the mRNA and which contains the crucial components of a decoding complex (see Figure 3b). In the group we wildly

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speculated whether this may reflect some archetypal ‘‘miniribosome’’ because SelB may provide the codon in juxtaposition to the anticodon of a charged tRNA. Shortly after our discovery of the context-dependence of UGA decoding, Marla Berry reported that in eukaryal selenoprotein mRNAs the SECIS element is located in the 30 -nontranslated region (see Ref. 87). In view of this fundamental difference Reinhard Wilting got interested in the analysis of the archaeal selenoprotein synthesis system. He followed his interest when I was away for a meeting and upon my arrival presented an astounding wealth of information. Not only did he identify all genes coding for selenoproteins (some of them already wrongly annotated) in the database of Methanococcus jannaschii but he also highlighted that the archaeal SECIS element (aSECIS) is also positioned in the 30 -nontranslated region of the respective mRNAs. He achieved everything ‘‘by hand’’ analysis of the sequence since the application of the RNA fold programs available at that time was not successful. aSECIS differed completely from the structure of the eukaryotic (eSECIS) counterpart suggesting an independent evolution [95]. Since such major discoveries were made several times during my absence my graduate students recommended further-on that I should go travelling if they had a particularly tough problem to solve. The archaeal selenoprotein formation system should also prove to be instrumental for gaining insight into the mechanism of action of translation factor SelB. We decided to initiate a search for the archaeal counterpart (aSelB) of the bacterial protein since we assumed that the common features which the system shares with the eukaryal one and the inherently less complex genetic composition might facilitate both the search and the biochemical analysis of the components involved. The identification of aSelB via its similarity with elongation factors amongst the annotated genes from M. jannaschii was a relatively easy task [96] but provided important clues that facilitated the successful search for eukaryal SelB (eSelB) by two groups [97,98]. A common feature which aSelB and eSelB share was that the C-terminal extension is much shorter than that of bSelB, prompting the question whether they are able to bind to their 30 -SECIS sequences like bSelB does to the in-frame SECIS element. Indeed, like in the eukaryal system no complex formation between aSelB and aSECIS could be demonstrated. However, since this is a negative result this issue

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requires additional experiments. In the case of the eukaryal system, the interaction between eSelB and eSECIS is mediated by a second protein [99]. Despite several attempts we were unable to demonstrate the existence of such a ‘‘communicator protein’’ in archaea. Ever since we had characterised bSelB as a selenocysteinespecific translation factor we had attempted to obtain its threedimensional structure by entering co-operations with X-ray crystallography groups but all trials to obtain crystals from the entire protein failed. This was also experienced for the protein of a moderately thermophilic organism, Moorella thermoacetica, whose bSelB gene had been cloned by Reinhard Wilting and handed over to Maria Selmers from Lund. Happily, however, she detected crystals in a preparation stored for several months in the refrigerator and even more enjoyably they consisted of a proteolytically generated fragment containing the entire C-terminal extension. The resolution of the structure yielded information on the existence of four similar structural sub-domains with a hinge region between sub-domains 2 and 3 [100]. Successful resolution of the three-dimensional structure of a complex between the C-terminal extension of bSelB from the same organism by Yoshizawa and collaborators [101] shed light on many interesting features of this RNA–protein complex. First, within the complex the stem-loop RNA is paired to the very top, leaving only two nucleotides unpaired which are ‘‘extruded’’ from the double helix, namely the GU doublet highlighted by our chemical probing experiments. Second, the sub-domains of the extension are winged-helix motifs from which only motif 4 (the immediate C-terminal one) interacts with the SECIS RNA. Finally, specific interaction of the RNA with this winged helix is mediated by a single base, the G of the GU doublet, which talks with the protein side chains via all its functional groups. The interaction is further stabilised by a string of positively charged side chain groups of the protein with the phosphates of the RNA backbone (Figures 4b and 4c) [101]. The structure thus supports the genetic and biochemical results collected by us over many years and describes the mechanisms with which SelB scans cellular mRNAs for the presence of SECIS elements at the molecular level. As a soluble protein, EF-Tu donates the charged tRNA species to the ribosomal A-site from solution (Figure 3a), whereas SelB only acts so in the pre-bound state at the mRNA which carries the

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UGASec codon. For a considerable time we thought that the mechanistic rationale for the difference is that in this way the selenocysteyl-tRNA is tethered to the A-site in a stereochemically controlled manner. Meanwhile, although such tethering may be involved we became convinced by a number of different results that the predominant reason rather is that SelB needs the SECIS element as a ligand for being converted into a functionally competent conformation. What was the evidence? First, it was observed by Matthias Kromayer in mobility shift experiments that presence of selenocysteyl-tRNA causes SelB to bind much tighter to the SECIS mini-helix than in its absence [89]. Second, in collaboration with the group of Roger Goody, Martin Thanbichler succeeded in highlighting the biochemical basis of this apparent cooperativity. In his doctoral work he devised rapid methods for large-scale purification of all components of the selenocysteine insertion machinery and he attached fluorescent labels to GTP, GDP and to the mini-helix RNA which represents the SECIS ligand. Using the stopped-flow method, the real-time association and dissociation constants of the interaction of SelB with the ligands were quantitatively assessed. It was found that the mini-helix binds to SelB with a very high interaction affinity (about 1 nM) which is further maximised by the presence of selenocysteyl-tRNA [102]. This suggests that the release of the charged tRNASec at the A-site of the ribosome decreases the interaction of SelB with the SECIS element and facilitates to free the mRNA for the translation of codons downstream of the UGA. Presence of the guanine nucleotides does not influence the kinetics of interaction with the RNA ligands. The experiments also supported our previous results that the exchange of GDP by GTP occurs chemically and does not require the activity of a release factor. The role of the SECIS element as a ligand that confers a conformational switch to SelB was directly proven by measuring of the stimulation of its intrinsic GTPase activity. When incubated with 70s ribosomes, this low intrinsic activity of SelB was not augmented significantly in the absence of SECIS RNA. Addition of a synthetic mRNA containing the motif greatly stimulated GTP hydrolysis and nearly the same increase was observed by addition of the mini-helix alone [103]. Finally, a beautiful piece of work which demonstrated that this ligand role of the bSECIS element also is decisive for the in vivo

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function of SelB was conducted by Elias Arne´r. He joined our group in 1997 as a guest scientist from the Karolinska Institute and he arrived with the idea to express the mammalian thioredoxin reductase (trxR) gene in E. coli. As detected by Thressa Stadtman, TrxR is a selenoprotein in which selenocysteine is the penultimate amino acid residue at the C-terminus [104]. Selenocysteine insertion in the mammalian system is, of course, directed by an eSECIS element located in the 30 -nontranslated region and the mRNA consequently should not be translated in E. coli since it lacks the in-frame bSECIS. Elias Arne´r constructed a series of trxR alleles and could show that a large amount of correct gene product is formed when the bSECIS motif is fused into the nontranslated region separated from the UGASec codon by one or two genuine stop codons [105]. Thus, the SECIS sequence does not need to be translated to direct selenocysteine insertion. These results again emphasised the role of the SECIS as a ligand for activating SelB into the productive conformation and they explained that the inability of ordinary UGA stop codons to direct selenocysteine insertion is based on the lack of the SECIS motif. After all the failure to crystallise the intact SelB protein we were extremely pleased when Nenad Ban from the ETH Zu ¨ rich approached us und suggested to cooperate in the X-ray structural analysis of an archaeal SelB species. We provided the aSelB gene from Methanococcus maripaludis and Marc Leibundgut from Ban’s group succeeded in obtaining diffracting crystals and he resolved the structure within an astoundingly short time [106]. The structure shed light on many important features of aSelB in the binding of the charged tRNASec and they nicely complemented genetic data on the specificity of selenocysteyl-tRNA binding worked out by Martin Thanbichler in parallel and the results on the kinetics of interaction between SelB and its ligands discussed above. aSelB has the overall structure of a chalice in which the three EF-Tu-like domains are connected to domain 4 (the C-terminal extension) via two b strands in a very flexible connection. It constitutes a structural chimaera between elongation factor Tu and initiation factor 2 which is reminiscent of the similar interaction kinetics with GTP/GDP and also with the fact that both initiation factor 2 and elongation factor SelB discriminate the aminoacyl-moiety of their cognate charged tRNA species, namely formyl-methionyltRNAfmet and selenocysteyl-tRNASec, respectively.

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An earlier comparison of the structural models of the aminoacyl binding pockets of EF-Tu and SelB had revealed that whereas that of EF-Tu is open and suited to accept the side chains of all standard amino acids, two arginyl side chains (E. coli situation) protrude into the pocket of the bSelB counterpart [107]. In aSelB, one of the arginyl moieties is replaced by a histidyl residue. Exchange of one of the two positive charges by mutation did not abolish the UGA readout activity but replacement of both was detrimental. Intriguingly, modelling the selenocysteyl-tRNA into the aSelB structure according to the coordinates of the resolved cysteyl-tRNA  EFTu complex places the selenolate in the immediate proximity of the two positive charges. It is thus assumed that the deprotonated selenolate is sandwiched between these two charges and this interaction may serve as one of the recognition features [106]. The X-ray structure also shed light on one of the most intriguing features of tRNASec, namely its exclusive binding to SelB accompanied by the rejection of all the standard tRNAs. Sequence comparisons of tRNASec species from all three phylogenetic lineages and our previous extensive mutagenic analysis had indicated that the elongated aminoacyl-T-stem helix may be crucial. Indeed, modelling selenocysteyl-tRNA into the tRNA binding site of aSelB suggests that the protein possesses an extra loop not present in EF-Tu which intrudes into the groove of the RNA helix and provides contact with the backbone of the RNA molecule. Ordinary tRNAs are rejected because of their one base pair-shorter helix. SelB thus acts as a ruler that measures the length of the A/T-stem [106]. Finally, a model of the aSelB molecule into the EF-Tu binding site of the ribosome provides the view that domain 4, i.e. the C-terminal extension, is positioned in the mRNA entry cleft of the 30s ribosomal subunit (Figure 4d). The consequence of this positioning is still open; possibilities are the prevention of termination or the correct exposure of the anticodon to the A-site UGA codon. Thus, the resolution of the structure was at the end of a long road from detection of the UGA codon in 1984, of the identification of the specific tRNASec in 1988 and of the alternate elongation factor SelB in 1989. Since selenocysteine insertion shows all properties of a DNA directed process (own codon, specific tRNA, involvement of a translation factor) we proposed it to be the 21st amino acid [108] and suggested ‘‘U’’ for its nomenclature in the amino acid code which is now accepted.

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An inevitable question which I routinely have been asked at the occasion of presentations in seminars or conferences concerns the evolution of the process. In view of the oxygen sensitivity of the selenol group and without the wealth of information available at present we initially had speculated that it is a relic of an early form of the genetic code, carried over into special biochemical niches from times when the atmosphere was lacking oxygen. Such an early property of the genetic code would be in excellent agreement with the hypothesis by Wong on the co-evolution between amino acid biosynthesis and the development of the genetic code [109] since UGA belongs to the serine family of codons and selenocystene is synthesised from serine as a precursor. An early evolutionary origin is indeed suggested for some of the selenoproteins by bioinformatics analysis and also by the existence of a pseudo-SECIS element in the 30 -nontranslated region of homologous variants of selenoproteins which contain cysteine instead of selenocysteine [110]. However, in other cases there is convincing evidence that selenoproteins developed from sulphur-containing precursors [111]. A discussion of the evolutionary origin must consider the selective advantage for an enzyme of having a selenol instead of a thiol in the active site and also the efficiency of the selenocysteine insertion step. The answer to the first question is clear-cut and came from the construction of a cysteine containing variant of the formate dehydrogenase H from E. coli by Franz Zinoni, the purification of the S and Se variants and the comparison of their kinetic constants by Milton Axley in Thressa Stadtman’s group. It was found that the Se form is superior because of an about 100-fold increased reaction velocity [112]. Similar results were later on obtained for other systems. The analysis of the efficiency of the selenocysteine insertion process was assessed by ligating the TGA plus bSECIS segment in between the coding regions for two proteins whose quantities are easy to determine. In such a construct, appearance of the enzyme activity derived from the downstream located reading frame can be taken as a measure for readthrough over the UGASec codon. Our own measurements conducted by Britt Persson, who had joined us as a postdoctoral fellow, together with Sabine Suppmann showed that the process is kinetically inefficient [113]. The step time for the insertion amounted to 8s and its extraordinary length was found to be due predominantly to the binding of SelB to the

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mRNA and to the melting of the complex after release of the tRNA. Follow-up measurements by Warren Tate’s group indicated that our results might be at the lower edge and that the insertion might be more efficient [114]. The kinetic advantage of having a selenocysteine is therefore somewhat balanced by the costs for the reduced efficiency in formation of the selenoprotein. However, much lower cellular levels of selenoenzymes are apparently required as a consequence of the improved kinetics to satisfy the physiological needs. In summary, there is definitely an advantage for a cell to possess an enzyme which has selenocysteine as a catalytically active residue instead of cysteine. As already stated, many of my graduate students made it their ambition to follow side projects on their own and to surprise me with some intriguing result. So, Martin Thanbichler one day came to me and told me that he found an apparent SECIS element in the 50 -nontranslated region of the selAB operon from E. coli. The two genes are translationally coupled where selA codes for the biosynthetic enzyme selenocysteine synthase and selB for the translation factor. We decided to analyse whether this is of physiological consequence. SECIS-like, as we termed it, indeed bound SelB together with selenocysteyl-tRNA and in this form repressed expression of the selAB operon at the translational level [115]. In the in vivo situation, SECIS-like, therefore, competes with the in-frame bSECIS of selenoprotein mRNAs for SelB and the charged tRNA. When both are abundant, the formation of the selenocysteine synthase and SelB are tuned down, when one or both are limiting selAB expression is maximised to provide optimal levels of the charged tRNA and the translation factor for selenocysteine formation and insertion. In this way, the availability of selenium in the medium is also tied in since as a precursor for selenocysteine-tRNA it participates in this regulatory cascade. We also believe that in order to act as a regulator selenium must be specifically incorporated into some macromolecule since in the low molecular state it is prone to be confused with sulphur-containing homologues. Non-Specific Selenium Incorporation The close chemical similarity between selenium and sulphur poses a particular challenge for biological systems to discriminate the two elements. Many anecdotal stories have travelled around

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narrating cases of excessive uptake of selenium resulting in intoxication. The historically first and amongst the most wellknown ones is the description by Marco Polo that his package animals lost hairs and hooves because they fed on poisonous plants on the highlands of Tibet, whereas the domestic animals had learnt to despise these plants, today recognised as certain species of the genus Astragalus which accumulate high amounts of organo-selenium compounds. Georges Cohen was the first biochemist who reported precise results on the unspecific incorporation of selenomethionine into protein [116] and this finding was developed into an invaluable tool for crystallographers to solve the phasing problem [117]. Relatively less, however, is known on the path of selenium incorporation into macromolecules, especially proteins, when cells are grown on selenite or selenate. Sabine Mu ¨ ller addressed this question by using mutants of E. coli with structural and regulatory gene mutations in the pathways for cysteine and methionine biosynthesis and analysing them for the incorporation of radioactive selenium (Figure 5, pathways in bold letters). All genes involved in cysteine biosynthesis were required for the non-specific incorporation of selenium except those for sulfite reduction indicating that Se incorporation takes place via the cysteine pathway and that intracellular selenite, in contrast to sulphite, is reduced non-enzymatically [118]. Blockade of the methionine synthesis pathway or feeding with excess methionine was without influence which suggested that in this organism Se is incorporated in the form of selenocysteine and not methionine. In other organisms, however, seleno-methionine is the preferentially selenated compound found in proteins of organisms grown on selenite-containing media [119] One can conclude that the ability to discriminate the two elements in biological systems rests on the differential affinities of cysteyl-tRNA synthetase and cystathionine-g-synthase or cystathionine -b-lyase for the substrates cysteine versus selenocysteine (see Figure 5): A cysteyl-tRNA synthetase equally activating cysteine and selenocysteine allows the incorporation of selenocysteine; if the enzyme discriminates the two substrates selenocysteine is rejected but it is still available for the synthesis of selenomethionine via Se-cystathionine if the enzymes of the transsulfuration pathway are promiscuous concerning these substrates (Figure 5). With this information as a basis, Sabine Mu ¨ ller in cooperation with Hans Senn from the Roche company devised a method for

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the preferential labelling of proteins with selenocysteine [120]. With thioredoxin as a model protein they showed that both activesite cysteine groups of the protein together could be replaced by selenocysteine with an efficiency of 75 to 80% and one of the two cysteines was exchanged in 5–10% of the gene product. The Se-Se, Se–S and S–S forms could be separated and the di-selenide form exhibited remarkably different redox properties and it was found by NMR analysis that it is isomorphous with the di-sulphide form. The expression system and the replacement strategy prove to be a valuable tool for X-ray crystallographers for solving the phasing problem by multiwavelength anomalous dispersion (MAD), in addition to the well-established technique of selenomethionine incorporation. By our collaborators Marie-Paule Strub and Andre Aumelas from Montpellier this expression system was optimised for sideby-side co-incorporation of selenocysteine plus selenomethionine. With two model proteins they demonstrated the isomorphous replacement by both seleno amino acids and they showed that the cognate di-sulphide bridges in one of the proteins were converted into unstrained di-selenide bridges [121]. The approach improves the efficiency of the MAD method and is especially valuable for proteins with a low content of one of these amino acids. Selenium Toxicity and Resistance Selenium was considered a toxic element until the mid-fifties of the last century when Jane Pinsent [67] and Karl Schwarz [122] described its role as a nutrient for the growth of E. coli and rats, respectively. As already mentioned, selenium toxicity is caused mainly by the so-called selenium accumulator plants which when Fig. 5. Schematic representation of specific and non-specific incorporation of selenium into macromolecules. Components of the non-specific route are written in bold letters. Abbreviations: mnm5S2U, 5-methyl-aminomethyl-2-thiouridine; mnm5Se2U, 5-methyl-aminomethyl-2-seleno-uridine; SMT, selenocysteine methyl transferase; HMT, homocysteine methyl transferase; AdoMet, S-adenosylmethionine; CH3-Met, methyl-methionine; CH3-Se-Cys, Se-methyl-selenocysteine; O-Ac-Ser, O-acetyl-serine; EF-Tu, protein synthesis elongation factor Tu; [HSe-], reduced selenium species acting as a substrate for the SelD protein; Selenylated proteins, polypeptides with a random replacement of S against Se amino acids; Selenoproteins, proteins with a specifically incorporated selenocysteine residue; R-SH, reducing agent like glutathion.

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fed by animals lead to intoxication symptoms. A considerable amount of work has been conducted accordingly in agricultural departments and the pioneering work mainly of Alexander Shrift revealed that these plants accumulate low-molecular weight selenium compounds which constitute derivatives of proteinogenic selenium amino acids, like selenocysteine, which are converted into forms which can no longer be incorporated into proteins, like Se-methyl-selenocysteine [123]. Since the enzymes involved in this conversion and their evolutionary origin were largely unknown, Bernhard Neuhierl in our group addressed this question by means of plant cell cultures from a selenium accumulator plant, Astragalus bisulcatus, and a closely related non-accumulator species, A. cicer. The cell cultures were provided by Meinhart Zenk and Bernhardt Neuhierl showed that they maintained their accumulator and non-accumulator phenotypes in culture. He succeeded in cloning the gene and purifying the gene product that converts selenocysteine into Se-methyl-selenocysteine with S-adenosyl-methionine as a methyl group donor [124]. A database search showed that the enzyme is representative of a whole family of proteins with hitherto unassigned functions. A member is also formed by wild-type E. coli strains and its purification and characterisation identified it as an enzyme transferring a methyl group from S-methyl-methionine to homocysteine, selenohomocysteine or selenocysteine. Via mutant analysis performed by Martin Thanbichler its role in E. coli was found as that of a (third) methionine biosynthetic enzyme synthesising two methionines from a homocysteine plus S-methyl-methionine [125] (Figure 5); S-methyl-methionine is a compound accumulated by many plants. A closer investigation of the methyltransferase from A. bisulcatus showed that S-methyl-methionine is also the preferred physiological methyl group donor in plants. It appears therefore that the Se-selenocysteine methyltransferase of the selenium accumulator plant has developed from an S-methyl-methionine-dependent thiol/selenol methyltransferase under selective pressure. Accordingly, selenium resistance or tolerance can be visualised as the scanning of the cysteine pool for the intrusion of non-specifically formed selenocysteine and its removal into a non-proteinogenic amino acid by methylation. Overexpression of the gene in E. coli indeed provided resistance to high selenate concentrations in the medium. Also, with the aid of the methyltransferase gene from A. bisulcatus, transgenic plants were constructed; they exhibited

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increased selenium tolerance and are discussed to be of value for selenium remediation processes [126].

Relations to Industry Since its establishment as an own biological discipline, microbiology always had close relations to application and thereby also to industry. My personal interest in such contacts dates back to the days as a doctoral student in the Botanical Institute at the University of Munich when I served as a technical hand in the determination of the degradation of chemical compounds in sewage plants. This test was performed in special Warburg vessels and one had to read oxygen consumption continuously for several days. Fortunately (or unfortunately), equipment was not automatic in these days so one had to sleep side-by-side with the machine in the lab and read and reset the manometers at regular intervals dictated by an alarm clock during the nights. These relations and contacts with companies continued parallel to the scientific projects followed in the group after I had been appointed to assistant professor and resulted in publications in non-main-stream journals like ‘‘Tenside, Detergents’’ [127]. One of the goals, for example, attained in such experiments was the development of chemically modified starch derivatives for the use as biologically degradable ingredients of washing powder. Later on, after the move from Regensburg to Munich, we analysed the maturation pathway of penicillin acylase, one of the top ten technical enzymes, which is used for the formation of 6-amino-penicillanic acid needed as a precursor for the chemical synthesis of semisynthetic penicillins. Following the expression of a plasmid-encoded gene provided by a company it was found that in contrast to previous work the active enzyme is a heterodimer and is synthesised as a polyprotein precursor [128]. The precursor is secreted into the periplasm guided by a classical signal peptide and it is processed there into the a- and b-subunits by endoproteolytic removal of a short peptide [129]. The results aided in the establishment of an overproduction process for the protein. Particularly rewarding was the cooperation with Frank Mu ¨ ller, director of research of a major Munich-based chemical company. He decided to enter R&D in biotechnology and to enforce the already existing fine chemicals department, now headed by my

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former student Gerhard Schmid, in the direction of biological products. In addition to two other colleagues I was asked to provide guidance. The development of fermentation processes for the amino acids tryptophan and cysteine by E. coli which we accompanied was a major achievement of this group. This work also induced a paradigmatic shift in the belief of applied microbiologist that the superior organism for mass production of amino acids by fermentation must be a Brevibacterium or Corynebacterium species. Our results provided a new look on the regulation of amino acid biosynthesis in general and cysteine biosynthesis specifically since two exporters for cysteine and its precursor O-acetylserine were identified. One of them was so efficient that, when overproduced, generated a deficiency in reduced sulphur in the cell leading to growth cessation [130,131]. So, in my opinion E. coli is the ideal organism for the design of pathways to achieve production of commodity compounds. Scientifically equally attractive was the establishment of a procedure for the production of cyclodextrins (CD). Cyclodextrins are ring-shaped and therefore cylindrical molecules of 6 (a-CD), 7 (b-CD) or 8 (g-CD) glucose units. They possess a hydrophilic surface and a hydrophobic cavity into which non-polar compounds can be included. Depending on their guest molecules, cyclodextrins are of wide use in pharmacy, nutrition or plant protection formulations. The first gene for the enzyme producing CD from starch, CD glycosyl-transferase from Klebsiella oxytoca was cloned in our group by Florian Binder [132] and a periplasmically leaky strain of E. coli was developed which releases the protein into the medium. Meanwhile a production plant is operating in Iowa that utilises the knowledge and is based on further R&D work in the company. Equally interesting was the elucidation of the path of CD degradation. An operon (cym) was characterised also from K. oxytoca by Gabriele Fiedler harbouring eight genes whose products are all involved in the metabolism of CDs [133]. A monomeric, unusual porin characterised by Markus Pajatsch in cooperation with Roland Benz from the University of Wu ¨ rzburg allows the entrance of a-CD and b-CD into the periplasm [134] and a CD-specific ABC transport system catalyses the active transport of these bulky molecules with the significant diameter of 1.37 nm for a-CD and 1.53 nm for b-CD into the cytoplasm. There, a CD-specific cylodextrinase opens the incoming molecules and feeds the linearised oligosaccharides into the maltose pathway. The

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pathway is evolutionary related to the maltose degradation route but specifically induced only in the presence of CDs. On a personal rather than economic basis, all these projects are topped, however, by the outreach of a scientific excursion which I had the chance to participate in and which was guided by Wolfram Zillig. We intended to isolate new, extremely thermophilic organisms in hot springs at Iceland. We started out in Munich in a DFG-owned archetype of an SUV, drove to the Northern tip of Denmark with a maximal speed of 80 km per hour and boarded a ferry to Iceland with a 2-days stop at the Faeroer Islands to do some training. Our aims on Iceland were hot springs that break ¨kull Glacier creating ice-walled funnels with a through the Vatnajo hot spring at the bottom. The scientific outcome was the description of an archaeon which I had the honour to co-author and which is the only taxonomic paper on my publication list [135]. The practical result was a strain which produced a heat-stable a-galactosidase which we handed over to a company on exchange for a 2-years salary for a postdoctoral researcher. The enzyme was developed by the company to remove raffinose from sugar beat melasses because raffinose inhibits crystallisation of sucrose. The pursuit of such joint projects with industry was not only personally enjoyable but also highly profitable for the entire group. Their discussion during the research seminars introduced the fundamentals of economic thinking to the students and it also melted into the subjects of teaching. I experienced that applied aspects immediately caught the attention of students. Not the least, the generous support of the work by our industrial partners provided the whole group with flexible money to support basic projects and also saved many hours for grant writing to agencies or the always tedious argumentations for more money within the university. Many students after finishing their Ph.D. also found a job in one of these cooperating partner institutions and strengthened the relationships. It is also remarkable to see how the general view on such co-operations has changed during the last 30 years. At the end of the sixties and during the seventies of the last century, I experienced once that the dean of my faculty did not dare to put my application for permission of such a co-operation on the agenda of a faculty meeting. Relations with industry were considered unethical since science was argued to become dominated by economic principles, to loose its identity. What a change to nowadays

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when we are inundated with requests from the technology transfer department of the university offering courses in patent writing and advising us that we should clone money rather than genes.

Resume and Outlook Looking back at all these years, I appreciated most the scientific freedom which I enjoyed in following my ideas, although sometimes there may have been too many of them. I never was constrained or even restricted to adjust or fit our plans to constructions like ‘‘clusters’’ or ‘‘networks’’, somehow it was all curiosity-driven sports to us. As an aficionado of university research I still strongly believe in the power of small and creative groups which in no way can be replaced by politically pushed and a priori formulated scientific goals. Most of the really novel findings were cases of serendipity anyway and such top-down approaches leave less room for serendipitous discoveries and for their pursuit. Of course, we received numerous co-operations and I enjoyed them greatly but they always followed the initiation of a project and never were the prerequisite for it. However, although we got much technical support from colleagues from the chemistry department or from the Max Planck Institute of Biochemistry, the progress of our work was retarded or even hampered by the lack of a sufficiently equipped technical platform maintained by experts as an independent unit within the faculty. The establishment of such platforms and also the unlimited access to scientific journals would have been very beneficial for our work. Finally, our independence rested firmly on the support by our fabulous granting agency DFG which always provided individual grants and still backs up basic research without asking for practical spin-off. Thanks for the fact that in handling such individual grants they never demanded a detailed account on whether we spent grant money in exactly the same way for which we had applied it, provided some interesting result came out! It was a great pleasure to educate and to cooperate with doctoral students and watch them develop into mature scientists and find their career in academia or industry. A culmination of such pleasure was that when I received the honorary degree of the ETH Zu ¨ rich in 2005, my former student Hauke Hennecke in his

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function as Chairman of the Department of Biology presented the laudation. Most of the work rested on the shoulders of my co-workers whether research or teaching and they followed their work independently and with great enthusiasm and diligence. They all delivered a comprehensive scientific story in their theses and I hope that they have forgiven me already for brutally correcting their stochastic use of commas or their overly epic way of writing. Remarks Forty-five years is a long-time period and when writing this article, I had to make a compromise which was dictated by space limitation and comprehensiveness. Regrettably, many contributions and co-workers had to remain unaddressed as a consequence and I apologise to those concerned. It is in no way a matter of relevance or quality. I like to thank also all persons providing the physical conditions to follow the research projects, whether from granting agencies or the industry. Finally, I appreciated very much working in two Bavarian universities where I always experienced a very positive attitude towards basic science. REFERENCES [1] [2] [3] [4] [5] [6] [7]

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G. Semenza (Ed.) Stories of Success – Personal Recollections. X (Comprehensive Biochemistry Vol. 45) r 2007 Elsevier B.V. All rights reserved. DOI: 10.1016/S0069-8032(07)45004-5

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Chapter 4

Dennis Chapman: Oiling the Path to Biomembrane Structure PETER J. QUINN Department of Biochemistry, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom E-mail: [email protected]

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Abstract Research on the structural changes in glycerides associated with temperature-dependent phase changes in these lipids led Dennis Chapman onto the path of defining the dynamic behaviour of cell membranes. He developed the concept of membrane fluidity in which the lipid matrix of the membrane, represented by a bilayer of polar lipids, allowed diffusion of proteins and other membrane components in the plane of the membrane. It was also recognized that membrane fluidity could be modulated by cholesterol leading to the contemporary views of lateral phase separations and membrane rafts. He invented methods of mimicking membranes to coat prosthetic devices so as to render them haemocompatible. Dennis Chapman’s career spanned both industrial and academic service but with a singular aim of transforming his scientific discoveries to the benefit of mankind. Keywords: Biological membranes; membrane fluidity; lipid structure; secondary protein structure; protein rotation.

Dennis Chapman: The Scientist Dennis Chapman’s contribution to the field of biological membranes, particularly to our understanding of membrane lipid dynamics was that of a giant upon whose shoulders the remainder stood. He made a number of key discoveries that turned out to be of fundamental importance as to how membranes perform their tasks. Of equal importance were his personal qualities that acted to inspire generations of those who followed his footsteps. Chapman’s scientific career began as a graduate technical assistant at the Port Sunlight laboratory of Unilever. His early promise was recognized by an award of a maintenance grant from the Department of Scientific and Industrial Research to enable him to study on secondment for a research degree at the nearby University of Liverpool. The topic of his research appears to have been undefined at the outset and not of obvious relevance to the interests of the company. Chapman relished the freedom of these circumstances provided and set about experiments in Professor Meek’s Department of Electronics under the guidance of Mr Hopwood who suggested the subject he examine. The

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Fig. 1. Correlation between stability of complex formation and electronegativity of metals. Adapted from Ref. [1].

originality of his graduate studies concerned the experimental determination of electron affinities of negative ions created when electrons collide with molecules. From this work he was able to correlate the electronegativity of metal ions with the stability of complexes they form with different ligands in terms of the strength of metal–ligand bonds. This correlation is illustrated in Figure 1 that shows the relationship he established between electronegativity of different metals and the stability constant of complexes formed with them. The results were presented in a thesis entitled ‘‘A Spectroscopic Study of a Spark Discharge in Oxygen’’ for which he was awarded the Degree of PhD in 1952. The work was published two years after he graduated from his private address in Wharfedale Avenue, Birkenhead [1]. He was able to conclude that the observed order of stability of metal complexes with a variety of ligands, such as organic acids, reported by H. Irving and R.J.P. Williams [2], was governed by (a) the tendency towards or ease of formation of the complex dictated, for example, by the second ionization potential of the metal ion and (b) the strength of the metal–ligand bonds after complex formation

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predicated by the magnitude of the difference in electronegativity between metal and ligand. He recognized that these effects could be modulated, in turn, by steric factors, hybridisation, inductive effects, etc. which could influence the difference in electronegativity between interacting species and thus complex stability. Chapman resumed full time post-doctoral work at Port Sunlight where his attention was diverted to the study of materials in the product range marketed by Unilever, namely, glycerides such as cocoa butter which are primary ingredients of chocolate and other foods. His initial studies were performed under the direction of R.J. Taylor and addressed the assignment of CQC vibrational modes in vitamin A palmitate. He showed that CQC stretching vibrations in the conjugated configuration in the molecule could be readily distinguished from bands originating from isolated double bonds [3]. These initial studies of unsaturated lipids stimulated Chapman to contemplate the use of infrared spectroscopy to characterize the thermotropic polymorphism of lipids, notably, the glycerides that was central to understanding the changes that are associated with food processing. The standard method of characterizing the complex mesomorphism of lipids at that time was X-ray diffraction which was essentially a static method requiring relative long exposures to accumulate sufficient scattered X-rays for analysis. It was therefore impractical to use the method to explore transitions between different structures. Chapman constructed a thermostatically controlled (711) sample cell so that spectra could be recorded over the range 650–3500 cm 1 during rapid heating and cooling scans. The lipid samples were examined in the form of films between rock-salt windows so that inhomogeneities in structure due to the relatively low thermal conductivity of glycerides could be avoided. He was able to identify structural transitions from changes in band position and intensity of different vibrational modes in this group of molecules. Furthermore, the method he was able to identify hitherto unidentified polymorphic forms of glycerides. This is exemplified by the spectra of monosterin presented in Figure 2. This shows infrared spectra of the monoglyceride in liquid state and its different polymorphic forms each recorded at characteristic temperatures showing subtle changes in vibrational modes of particular groups that characterize each of the structures. A particular feature of the spectra in the region about 719–720 cm 1 assigned to a methylene rocking mode of the

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Fig. 2. (A) Infrared absorption spectra of polymorphic forms of monostearin. (B) Conversion of the b0 -form of tristearin to the b-form via an intermediate liquid. Adapted from Ref. [5].

acyl chains was found to be critical in discriminating between two closely related forms of orthorhombic packing of the acyl chains. The overall conclusions from these early studies were that infrared spectral features of glycerides were consistent with the classification of subcell symmetry of chain packing deduced from wide-angle X-ray powder diffraction but that the versatility of the infrared absorption method had the potential to reveal lattice distortions that occur during transitions from one structure to another. Evidence for this is shown in Panel B of Figure 2 that shows detail of the C–H vibrational mode of tristerin during the transition from the b0 -form to the b-structure of the lipid. It is

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clear that a transient disordering of the b0 -structure to a liquidlike state occurs before reordering into the b-structure at 651C. The results of these detailed infrared studies were published in the Journal of the Chemical Society in a series of five, single author papers, during 1956-1958 [4–8]. In addition to these studies, he examined the infrared spectra of long-chain anhydrous soaps such as sodium stearate and sodium palmitate. A detailed examination of the temperature-dependence of absorption bands in anhydrous soaps, such as sodium palmitate, led to the conclusion that the hydrocarbon chains undergo a partial melting process some 2001 below the capillary melting point [9]. Thus, in the spectral region of 1250 cm 1 as the temperature approaches 1001C the distribution of bands becomes less well resolved. He interpreted this to mean that the hydrocarbon chains begin to twist and flex so that a range of rotational isomerizations are generated each with its own frequency in this region thereby serving to smear out the sharp spectral bands. This was consistent with a slight decrease in intensity of the band at 719 cm 1, which he had already assigned as due to in-phase motion of all CH2 groups, so that twisting of parts of the chain reduces the intensity of the absorption. At temperatures of 120–1301C the spectra were found to be entirely liquid-like in character. It is not until the capillary melting point is reached at about 3201C that the strong bond between the highly polar sodium ions and the carboxylate groups are ruptured. This notion of partial melting of the fatty acid chains represented a major step in the development of the concept of fluidity in which the polar part of the molecule retains a relatively ordered structure while temperature-dependent changes between different configurations of the hydrocarbon chains take place. These ideas were to be subsequently extended from simple soap systems to more complicated phospholipid mixtures. As an appendix to these infrared spectroscopic studies, Chapman borrowed a differential scanning calorimeter from his colleagues and undertook one of the first thermal analysis of glycerides in which transitions between different polymorphic forms could be demonstrated [7]. The results of this study are presented in Figure 3 that shows enthalpic transitions in four molecular species of triglyceride. The method clearly identified the temperature ranges over which the particular polymorphic phases existed and the endo- or exothermic processes that were associated with transitions between the phase states. Despite the fact that his work at

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Fig. 3. Differential scanning calorimetric curves of (a) 1-stearodimyristin, (b) 2-myristodistearin, (c) 1-palmitodimyristin and (d) 2-myristodipalmitin. Adapted from Ref. [7].

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the Port Sunlight laboratory of Unilever was to be the foundation upon which his later research career would be based he showed a remarkable reticence to acknowledge how influential this early phase of his working life had been.

Gonville and Caius, Cambridge In 1960 Chapman was given leave of absence from Unilever to take up a Berkeley Bye-Fellowship at Gonville and Caius College, Cambridge to continue his work on lipid structure. From his own account he received little encouragement. ‘‘I was keen to learn about molecular orbital theory and I had many discussions with the theoretical chemists. On one occasion I was asked by one of the young scientists about my own research interests and confessed to a fascination with lipids. ‘Lipids?’, he said; ‘how many atoms are there in lipids?’ I replied ‘quite a few’. ‘More than four?’ he asked. ‘Well yes’ I replied. ‘More than eight?’ he challenged with some surprise in his voice. ‘Well,’ I said somewhat apologetically, ‘yes, I’m afraid so’. ‘Oh dear,’ he replied; Dennis, you are wasting your time working on those molecules’!’’ He was gainfully distracted in his first year at Cambridge by starting work on a comparatively simple molecule, S4N4, and a relative, S2N2. This proved prophetic in the sense that he was introduced to a variety of physical techniques that he would subsequently employ to great effect. He used electron spin resonance spectroscopy to demonstrate, for the first time, that electron delocalization could take place in an inorganic ring molecule [10] and this led to other studies of [SN]x polymer, charge alternation and molecular orbital calculations [11,12]. Not content to abandon his interest in lipids he returned to the subject in the second half of his fellowship at Cambridge. Infrared spectroscopy at that time was not able to deal with hydrated specimens because of the dominating O–H stretch vibrations. He therefore initiated proton magnetic resonance spectroscopic studies of partially hydrated lipids. He foresaw the importance of characterizing the structure of polar lipids, particularly phospholipids, that were being shown to act reliable models of biological membranes in pioneering studies of R.M.C. Dawson and A.D. Bangham at the then AFRC Institute of Animal Physiology in nearby Babraham. His interest in these lipids was also heightened

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by a chance meeting with L.L.M. van Deenen working in Utrecht who had recorded an infrared spectrum of phospholipids but was puzzled by the fact that spectra of related isomers such as 1-oleoyl-2-stearoyl- and 1-stearoly-2-oleoyl- derivatives of phosphatidylethanolamine were relatively broad and, unexpectedly, indistinguishable from each other [13]. Similar broad spectra were recorded from a range of other phospholipids. Chapman instantly recognized these features from his earlier work on soaps and glycerides as arising from a liquid-crystalline phase. Thus isomers of glycerides with closely related structures like 1-oleoyldistearin and 2-oleoyldistearin he had shown give identical spectra when examined as liquids but markedly different spectra when recorded in the crystalline state [14]. He quickly applied infrared spectroscopy to reexamine the phospholipids using his temperature controlled sample cell. The results of these studies are summarized in Figure 4 which clearly demonstrated the liquid crystalline nature of phospholipids [15]. He drew some important conclusions from these studies. He observed that phospholipids of biological origin contain unsaturated fatty acid residues that have a lower melting point than saturated fatty acids so that phospholipids with mixed chains, such as oleoyl/stearoyl phospholipid derivatives, would be in a liquid state at body temperature despite the fact that the capillary melting point was in the region of 2001C. He also recognized that the presence of cis double bonds in lipids of biological origin would cause a much greater reduction in melting point than fatty acids with trans double bonds. Furthermore, the possibility that the presence of water would have a profound effect on lipid properties and, in turn, membrane structure and permeability was considered. He next embarked on studies employing a range of biophysical methods to determine the extent of molecular motion in the molecules above the critical temperature and then to relate this to thermotropic phase behaviour of the phospholipid to its behaviour in solids, lipid-water systems and monomolecular films at the air–water interface.

The Frythe Laboratories, Welwyn He returned to the industrial fold by joining the scientific staff of the Unilever Research Laboratories at the Frythe in Welwyn,

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Fig. 4. Infrared absorption spectra of dipalmitoylphosphatidylethanolamine recorded at designated temperatures. Adapted from Ref. [15].

Hertfordshire. He set about the synthesis of pure molecular species of a range of phospholipids that he then proceeded to characterize by calorimetry, paramagnetic resonance spectroscopy, infrared spectroscopy, X-ray diffraction and monomolecular film techniques.

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One of the most obvious advantages of all these methods was that phospholipids could be examined in aqueous dispersions and he had already recognized that the properties of anhydrous phospholipids could only go part way to explain the function of these lipids in cell membranes. His establishment at the Frythe Laboratories turned out to be one of the most productive and exciting times in his research career. He rose quickly through the ranks to become Director of the Molecular Biophysics Group in which he assembled a team of talented young scientists who set about transforming the notion that living cell membranes were static structures into our present view of fluid and dynamic molecular assemblies. The concept of membrane fluidity originated from the discovery that phospholipids, like he had shown earlier in soaps, exist as liquid crystals at temperatures well below their capillary melting temperatures [16]. Its original formulation was as follows: ‘‘We envisage that one of the functions of the distribution of fatty acid residues observed with these phospholipids is to provide the correct fluidity at the particular environmental temperature so as to match the required diffusion or rate of metabolic process for the tissue. Thus in membranes where metabolic and diffusion processes are required to be of a rapid nature, such as in the mitochondria, the average transition temperature of the phospholipids present will probably be low compared with the biological environmental temperature, while in membranes where these processes are slow, e.g. in myelin of the central nervous system, the average transition temperature of the phospholipids will be higher and may be close to that of the biological environmental temperature.’’ It was also noted that the fatty acyl composition of the membrane phospholipids of poikilothermic organisms varied with the growth temperature in such a way as to preserve the fluidity within relatively narrow limits. Sterols were known to be prominent constituents of biological membranes and in animal cells the sterol is cholesterol. Chapman turned his attention to how cholesterol modulated the dynamic motion of the acyl chains of the membrane phospholipids. The condensing effect of cholesterol on phospholipids was well known from the Croonian Lectures of J.B. Leathes published in 1925 [17] who noted from studies of mixed monolayers at the air–water interface that ‘‘it (the condensing effect) is observed with fatty acids even more than lecithine, though still observable with this,

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temps one to suppose that the action is between cholesterol and the paraffin chains rather than the complex glyceryl cholyl phosphoric acid, and therefore is again a change in physical behaviour, an alteration in the force of cohesion between these chains, that depends upon chemical characteristics not capable of resulting in actual chemical union in the ordinary sense.’’ The physical consequences of the interaction of cholesterol with phospholipids was examined in a landmark publication [16] in which he reported that cholesterol ‘‘condenses’’ both saturated and unsaturated phospholipid monolayers and in subsequent calorimetric studies it was shown that cholesterol broadens the thermotropic phase transition and reduces the transition enthalpy. The first high-resolution proton magnetic resonance spectrum (60 MHz) of a model membrane was published at this time and the method was used to show the condensing effect of cholesterol at the molecular level [18]. The spectra of egg-yolk lecithin and lecithin codispersed with an equal molar amount of cholesterol are presented in Figure 5. The spectral assignments are indicated and show that the presence of cholesterol causes an almost complete loss of resolution of resonances arising from the acyl chains of the phospholipid. At the same time, the resonances from the choline methyl protons are only reduced in intensity which may have been due to the effect of cholesterol on the macroscopic size of the sonicated dispersion. The evidence suggested to him that there was a direct interaction of a portion of the phospholipid, and particularly the hydrocarbon chains, with the cholesterol molecule and the formation of a molecular complex between phospholipid and cholesterol. The formation of the complex resulted in a reduction in molecular motion of the acyl chains. While Chapman initially espoused the idea that phospholipid formed a complex with cholesterol he subsequently held to the belief that there were no specific interactions between cholesterol and phospholipids (or peptides) and considerable effort was devoted to molecular dynamic studies of arrays of acyl chains (or peptides) and cholesterol on the assumption that they obeyed a hard sphere model [19]. He was wrong on this score and remained unconvinced to the end. Nevertheless, he can be credited with highlighting the role of cholesterol in the function of biological membranes. His time at the Frythe was marked by an extraordinary list of influential publications reporting on the work undertaken by his

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Fig. 5. Proton nuclear magnetic resonance spectra of egg-yolk lecithin and an equimolar mixture of egg-yolk lecithin and cholesterol dispersed in D2O. Adapted from Ref. [18].

team. Literally thousands of scientific papers from scientists throughout the world were generated directly from the lead taken by the group. Indeed, Chapman’s work was judged by the Institute of Scientific Information as being amongst the most cited studies ever carried out in the United Kingdom.

Reckitt & Coleman, Sheffield University The era came to an end in 1969 when Unilever took the decision to close the Frythe laboratory and to amalgamate it with the laboratories at Coleworth House in Bedfordshire. Chapman was not able to negotiate a future for his research with the company and was recruited by Reckitt and Coleman as Director of Research and joined the University of Sheffield as a Visiting Professor. His team at Sheffield retained much of the talent built-up in the

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Frythe but expanded to some 25 young researchers and distinguished visitors as his reputation grew. It was at Sheffield that he became interested in the proteins in membranes and particularly the effect of lipid fluidity on the motion of the proteins. In a seminal paper published in 1970 by L.D. Frye and M. Edidin [20] it was found that using fluorescence probe methods that proteins are able to diffuse laterally in the plane of cell membranes establishing that the lipid bilayer matrix possessed a fluid character. Shortly after R.A. Cone [21] demonstrated, using the 11-cis-retinal chromophore of rhodopsin, that the protein rotated about an axis perpendicular to the plane of the retinal rod disk membrane with a relaxation time of about 20 ms. This was orders of magnitude slower than the rotational motion of soluble proteins like bovine serum albumin, for example, which exhibit relaxation rates in the timescale of ns which is appropriate to the decay of fluorescence of probes attached to the molecule. To address the question of the rotational motion of proteins a flash photolysis instrument was constructed under his direction. He used the instrument to detect photoexcitation products in the purple membrane of Halobacterium halobium that exhibited polarized transient absorbance spectra extending to about 10 ms at a wavelength maximum of 410 nm. Although it was not possible to identify the source of the intrinsic chromophores the timescale of the decay indicated that the protein was much less mobile in the purple membrane than observed for rhodopsin in the retinal rod outer segment disk membrane. It was concluded that bacteriorhodopsin, which is a major integral protein of the purple membrane of Halobacterium halobium, was severely constrained in its rotational diffusion motion showing that not all membranes were fluid. In an imaginative step Chapman’s group developed the use of triplet probes that could be covalently bound to proteins devoid of an intrinsic chromophore, like rhodopsin, so as to enable measurements of their rotational diffusion [22]. Accordingly, eosin was covalently attached to bovine serum albumin and dichroism of the eosin triplet–triplet absorption band in the visible region (610 nm) was recorded. No dichroism could be detected at room temperature from labeled protein suspended in 80:20 glycerol:water solution because rotational relaxation time predicted to be about 6 ms was shorter than the limits detectable by their instrument (about 20 ms). However, as can be seen in Figure 6 when the suspension was cooled to 101C a transient dichroism is clearly observed from

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Fig. 6. Transient dichroism of triplet–triplet absorbance at 610 nm of eosin covalently bound to bovine serum albumin at 101C. Adapted from Ref. [22].

which they were able to calculate a relaxation time of about 0.7 ms. The principle established in this work was subsequently extended to investigate the motion and interactions of a wide variety of membrane and soluble proteins [23]. Also at Sheffield he began a systematic examination of interactions between lipids and proteins in membranes. He used a model initiated earlier at the Frythe Laboratory to study how the presence of intrinsic peptides like the cyclic antibiotics affects the motion of the acyl chains of the lipid. One approach to this problem was to use deuterium substitution for protons in the system and to follow the relaxation of the deuterium atoms by deuterium nuclear magnetic resonance spectroscopy. This proved to be an incisive method subsequently widely exploited to build up a detailed picture of the motion of lipid hydrocarbon chains in membranes and hydration of the membrane–water interface. Chapman was also instrumental in overcoming problems in needing to prepare highly dispersed phospholipid systems in order to resolve high-resolution nuclear magnetic resonance spectra. He instigated experiments using the so-called magic-angle sample spinning in which multibilayer dispersions were rotated at several kHz in a gas turbine and the dipolar interactions between protons, responsible for broadening the spectrum, were averaged out by the sample motion [24]. The improvement of the broadline spectrum

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by spinning at the magic angle (ca. 54.71) is seen in Figure 7. This shows a 60 MHz 1H-NMR spectrum of a multilamellar dispersion of egg-yolk phosphatidylcholine at a temperature of 251C at which the phospholipid is in the liquid-crystal phase when rotated at 3.5 kHz perpendicular (spectrum a) and at the magic angle with respect to the main magnetic field (spectrum b). Spinning at the magic angle is seen to resolve a peak at 6.8 t which is assigned to the –+N(CH3)3 protons of the choline head group and a prominent signal at 9.1 t assigned to the terminal methyl groups of the acyl chains from the very broad (500 Hz) absorption seen in spectrum a. From the narrowing linewidths of the resonances achieved by magic angle spinning he was able to deduce that the range of microscopic motions of the choline head group of the phospholipid in liquid-crystal phase contains modes with correlation times of less than 100 ms. In contrast, when compared to linewidths of the choline methyl protons of phospholipid in gel phase the correlation time increases to about 1 ms indicating an order of magnitude decrease in motion. Although modest achievements in resolution could be obtained at the field strength (about 1.4 Tesla) then in use it took improvements in instrumentation to produce truly high-resolution spectra from solid-state samples [25]. The principles established by Chapman at the outset, however, paved the way for these later developments. Chapman’s ease in both industrial and academic environments was reflected in the authority carried by his inaugural speech delivered for the Chair at Sheffield in 1969. The title of the address was ‘‘Industry and the Universities; Collision or Collaboration.’’ In this he argued cogently that exchange of personnel and ideas between research laboratories in academia and industry were essential to exploit fundamental discoveries in development of products that could be marketed profitably. Indeed, his ethos expounded in this address and in talks presented on programmes broadcast by the BBC was to apply his discoveries to serve the health and wealth of his fellow man.

London University, Chelsea College After leaving Sheffield he took up a Senior Wellcome Trust Fellowship in 1975 at Chelsea College, University of London with the intention of setting up a new Department when the College

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Fig. 7. Proton nuclear magnetic resonance spectra (60 MHz) of egg-yolk lecithin dispersed in D2O recorded at 251C rotating at 3.5 kHz perpendicular or at the magic angle with respect to H0. Adapted from Ref. [24].

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merged with St George’s Hospital Medical School in the formation of what was to have been a multifaculty University in South London. The plan failed to materialize but he set about new research designed to modulate the fluidity of membranes by manipulating the number of double bonds of fatty acids acylated to membrane phospholipids. It was known that the length of these fatty acids and the extent of unsaturation were the two main factors governing the fluidity and attempts had been made to alter the fatty acids of cells by nutritional, genetic and environmental manipulation [26,27]. The project initiated by Chapman arose as an attempt to reconstitute cytochrome oxidase into a defined lipid environment in order to examine how membrane fluidity modulated enzyme function. Methods for removal of all lipids without damaging the protein had not yet been developed so the idea of saturating the double bonds of the residual lipids by catalytic hydrogenation was considered. Initial attempts using Adams’ catalyst, a conventional heterogeneous catalyst, were unsuccessful although complete hydrogenation could be achieved with the phospholipids dissolved in solvent. In a brilliant stroke he tried Wilkinson’s catalyst, a homogeneous catalyst, and achieved rapid and complete hydrogenation of phospholipid dispersions and respectable hydrogenation of suspensions of biological membranes including rat liver mitochondria and microsomes as well as rabbit muscle sarcoplasmic reticulum [28]. This is illustrated in Table 1 which shows the fatty acid composition of phospholipids of sarcoplasmic reticulum before and after a brief incubation with Wilkinson’s catalyst, Rh(PPh3)3Cl. Wilkinsons’ catalyst is insoluble in water and had to be added in a small amount of solvent such as tetrahydrofuran whence it TABLE 1 Fatty acid composition of the phospholipids of rabbit muscle sarcoplasmic reticulum before and after partial hydrogenation in the presence of Wilkinson’s catalyst

Fatty Acid

16:0 18:0 18:1 18:2

Composition (mole%) Initial

Hydrogenated

34.5 15.2 20.8 29.6

36.7 20.5 21.8 21.0

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Fig. 8. Changes in C18 fatty acid composition of soya lecithin dispersions during hydrogenation in the presence of a water-soluble homogeneous catalyst. Adapted from Ref. [30].

partitioned into the membrane and catalysed the reaction. Chapman was concerned that the method of addition of the catalyst or its presence in the membrane may have detrimental effects and sought to solve this problem by persuading Wilkinson to synthesize a water-soluble form of his catalyst. Accordingly Wilkinson obliged by substituting diphenylphosphinobenzenem-sulphonate for triphenylphosphine to produce a water-soluble catalyst [29]. Figure 8 shows the C18 fatty acid composition of a sonicated soya lecithin dispersion incubated in the presence of the water-soluble catalyst. It can be seen that there is progressive hydrogenation of C18:3-C18:2-C18:1-C18:0 and that complete hydrogenation of the phospholipid is achieved after about 3 h incubation [30]. Another advantage of the water-soluble catalyst for biological applications apart from avoiding the need for solvent to deliver the catalyst to the membrane was that it could be removed at the completion of the reaction. From a commercial viewpoint development of the water-soluble catalysts overcome the

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major problem of removing the catalyst from the products at the end of the reaction by performing the reaction at an oil (substrate)–water interface. Furthermore, substrates can be presented at oil–water interfaces that allow the high specificity of homogeneous catalysts to be fully exploited [31]. His insight into the utility of the method was apparent from the exchange with A.D. Bangham recorded after a lecture to the New York Academy of Sciences in 1978 where he presented the work described in Figure 8: BANGHAM: What worries me a little about this is that there is a good deal of evidence that suggests that the animals, as it were, can adapt membranes for a change. For example, if you put goldfish into a warm or cold environment, their membranes change. If you are going to hydrogenate their membranes and they don’t like it, so to speak, they are going to modify other parts of the membrane components to adapt to that. CHAPMAN: We know that poikilothermic organisms do vary their fatty acids to match their environmental temperature. When the environmental temperature is raised, the fatty acids become more saturated; when the environmental temperature is lowered, the membrane will become more unsaturated in the cell. But what happens when we hydrogenate at constant temperatures? What are the biochemical processes that control membrane fluidity? That is what we are asking. BANGHAM: Quite so; but then there is going to be a possibility of an adaptation to the modified membranes, at least this is what happens in nutritional states. CHAPMAN: That’s true, except that in the nutritional state you have a considerable length of time in which adaptation can occur. When you’ve changed the membrane fluidity within 45 minutes, what are the cells going to do about it? With regard to nutritional variation, if we are interested to know the role of the highly unsaturated fatty acids in excitable tissues, such as the squid axon, then the possibility of getting major changes in the fatty acids as a result of nutrition is very limited indeed. In principle it is possible to take an axon and hydrogenate it selectively, step by step, and see how some of the electrical functions are changed. But that’s speculative at present!

The method was indeed widely applied to investigate the role of unsaturated lipids in membrane function and biochemical homeostatic mechanisms long before the advent of genetic engineering to address these questions. He also showed prescience of the industrial applications of such catalysts and obtained a patent for their use [32].

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London University, Royal Free Hospital Medical School Chapman accepted a Personal Chair of Biophysical Chemistry of London University and moved to the Department of Biochemistry and Chemistry at the Hunter Street site of the Royal Free Hospital Medical School in 1978. He continued to apply the catalytic hydrogenation method in new ways including using hydrogenation as a probe of membrane and lipoprotein structure [33]. While his initial attempts to polymerize phospholipid dispersions at Chelsea had been unsuccessful he followed the strategy of G. Wegner [34] and succeeded in forming polymeric membranes from photoactivable derivatives of phospholipids. The method differed from that of H.G. Khorana who had developed fatty acid derivatives containing carbine precursors [35] that were employed to investigate the interactions between phospholipids and phospholipids and proteins in membranes. Chapman chose to use diacetylenic fatty acyl residues which he synthesized and incorporated into phosphatidycholine [36]. Irradiation of the phospholipid derivatives with UV light of around 254 nm when presented as monomolecular films at an air–water interface, in multilayers deposited on a teflon support, as dispersions of multilamellar liposomes or mixed with KBr and compressed into transparent discs was examined to test whether polymerization into covalently linked structures could be achieved. It was demonstrated by changes in spectral properties of the irradiated samples that polymerization occurred provided the phospholipid derivatives were in a condensed phase, i.e. below the fluid phase transition temperature. The formation of polyconjugated phospholipid polymer from diacetylenicphosphatidylcholine as envisaged by the reaction is illustrated in Figure 9. To demonstrate the biological utility of the method he found that diacetylenic fatty acids could be biosynthetically incorporated into the membranes of Acholeplasma laidlawii when the cells were cultured in the presence of the fatty acid derivatives. Furthermore, irradiation of the treated cells resulted in spectral changes consistent with polymerization of the phospholipids in the membranes of the cells [36]. Chapman’s group moved to the Royal Free Hospital Medical School’s Hampstead campus in the early 1980s where he founded the Department of Protein and Molecular Biology. Many of his colleagues were intrigued by the title of his Department that made no reference to his by now well-established reputation as a

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Fig. 9. The mechanism of polymerization of diacetylenic fatty acid derivatives of phospholipids after irradiation by UV light.

lipidologist. He justified this by embarking on a new line of investigation of the structure of membrane proteins using techniques he had applied so effectively in the past to the study of lipid polymorphism. Infrared spectroscopy had by this time been adapted to deal with hydrated specimens and he undertook to characterize the amide bands of membrane polypeptides and proteins by Fourier transform infrared spectroscopy [37]. The conformation of a variety of receptors, channels and membrane pumps was examined qualitatively using the method. This is illustrated in Figure 10 that shows difference and secondderivative spectra of rabbit muscle sarcoplasmic reticulum. The spectral assignments to secondary structure were performed by comparing these spectra with those obtained from membranes suspended in D2O. In his detailed study of bacteriorhodopsin from Halobacterium halobium and Ca2+-ATPase from rabbit muscle sarcoplasmic reticulum it was concluded that performing secondderivative spectral calculations greatly assisted resolution of minor amide I and amide II components from the broad band envelopes and in assigning these to particular conformational features. Thus, from these second-derivative spectra he was able to demonstrate that both Ca2+-ATPase and bacteriorhodopsin contain antiparallel b-sheet structure in addition to a-helicle and random coil structure. The secondary structure was shown to be

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Fig. 10. Difference (a) and second-derivative (b) infrared spectra in the amides I and II region of sarcoplasmic reticulum. Adapted from Ref. [37].

unaffected by whether the surrounding lipids were in gel or in fluid phase. Second-derivative analysis of the carbonyl stretching bands from the phospholipids showed that the presence of the proteins perturb the static order of the bilayer interface when the phospholipids were in the gel phase but no detectable effects were observed in fluid phase bilayers. In further refinements, methods based on deconvolution and derivative spectra were developed to identify the number and position of components of amide I and amide II bands so as to provide a quantitative analysis of protein secondary structure [38]. These studies occupied much of the last 10 years of his active research career. With his characteristically astute commercial sense he recognized the potential of stabilized phospholipid structures as vectors for drug targeting and delivery or for the production of haemocompatible surfaces. It was long known that failure of prosthetic devices implanted in the cardiovascular system was caused by a tendency of such devices to induce thrombosis. It was also known that haemo-compatibility within the circulatory system was

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achieved by preserving choline phosphatides (namely, phosphatidylcholine and sphingomyelin) on the surface of cells. Chapman conceived of how polymerized films of choline phosphatides and later phosphoryl choline, could be used to form stable haemocompatible interfaces to prevent prosthetic devices from causing blood clotting. The idea was formulated in a paper published in 1982 [39]: We have shown that it is possible to coat many materials (glass, quartz, Perspex, Teflon and steel) with ordered layers of diacetylenic-containing phosphatidylcholine molecules and that upon irradiation these molecules polymerize. Furthermore, layers can be produced in such a way that the polar groups of the lipid form the outer coated surface. The layers after polymerization are quite stable in aggressive media and can also, with some precautions, be handled without damage. It is expected that the same technique can be used to deposit other phospholipids and glycolipids so that particular types of charged and zwitterionic phospholipid polar groups form the outer surfaces y In this way, stable polymerized surfaces consisting of the charged polar groups which make up the inner surface of erythrocytes can be modeled. Stable polymeric surfaces consisting of carbohydrate groups of certain cell membranes may be modeled by the biosynthesis of various glycolipid molecules containing these diacetylene groups. These various surfaces may be useful for studies of blood coagulation processes, the adsorption of various types of protein such as fibrinogen, and also for cell-cell contact investigations. The ability to coat a surface such as glass or metal to produce a stable surface having the polar characteristics akin to that of the outer layer of erythrocyte membranes may make such surfaces useful in certain biomedical applications such as the production of biocompatible surfaces.

He was granted a patent for the production of biocompatible surfaces [40] that was to reestablish his credentials in the industrial sector. To exploit this discovery he set up the UK firm ‘Biocompatibles International’ in 1984; the company was successfully floated on the London Stock Exchange in 1995 and was headquartered in Farnham in Surrey. The basic hypothesis of biocompatibility was rapidly expanded to embrace the polymerization of lipid molecules with other species to form a new type of plastic material the so called friendly plastics because they do not provoke adverse haematological or immunological reactions in the body. Biocompatibles exploited these plastics to produce eye contact lenses and other ophthalmic surgical devices through their Eye Care Division, urological stents and catheters manufactured by Biocompatibles’ wholly owned subsidiary, Urotech,

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GmbH, and lipid-coated coronary stents. Coronary stents manufactured by Biocompatibles are currently the first choice amongst many cardiologists. At the time of his death, Biocompatibles International PLC maintained manufacturing facilities in the US, Ireland and Germany making it a truly international company with more than 300 employees and a budget of US$95 m for product development. London University, Interdisciplinary Research Centre Dennis Chapman was a founding co-director of the University of London Interdisciplinary Research Centre (IRC) in Biomedical Materials based at Queen Mary University of London, which included collaborating groups at St Bartholomew’s and the Royal London School of Medicine and Dentistry, with associated laboratories at Royal Free Hospital School of Medicine, Institute of Orthopaedics and Imperial College of Science, Technology and Medicine. The IRC was founded in 1991, with a core programme grant from the Science and Engineering Research Council (SERC) and expanded to a complement of over 100 staff and research students 10 years later. The IRC research activity is based on a multidisciplinary approach, with relevant aspects of cell biology, biochemistry, basic medical science, materials science, bioengineering, clinical dentistry and medicine. Chapman’s experience and temperament ideally suited him to these ideals and his talents as a scientific facilitator came to the fore. This was of particular value in setting up the Industrial Affiliates Club of the IRC and an associated company, Abonetics Ltd, to facilitate technology transfer in the development of medical implants and prostheses. Dennis Chapman, The Man Dennis Chapman was born in May 6, 1927 in Sunderland, County Durham the only son of George and Katherine Chapman. His formative years were dominated by the hardships visited on working class families living in the industrial North of England during the depression of the 1930s and the attentions paid to a major shipbuilding port by the Luftwaffe during the early phase of Second World War. He attended Sunderland Junior Technical

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School from 1938 to 1943 where his parents expected him to learn a trade and make his own way in the world. He left at the age of 16 without any qualifications. The shipyards and coalmines apparently held little attraction for him and he was determined to improve his prospects. To this end he worked in unskilled jobs by day and studied during the evenings at Sunderland Polytechnic gaining an external BSc from London University in 1948. He courted one of the star pupils of the local grammar school, Margaret Stephenson, during this period. His suit was somewhat hampered, however, by an insistence on being chaperoned by Margaret’s sister, Mary. Despite this encumbrance they married in Hampstead in 1949; he then aged 22 years. Their marriage represented a considerable sacrifice for Margaret as she was obliged to decline an offer of a place to read for a degree at Cambridge and move across the Pennines to set up house with her husband in Birkenhead. The couple were also indebted to Margaret’s parents who provided considerable assistance to set up their residence at 29 Wharfedale Avenue. He had a particular affection for his mother-in-law, Elsie, who was a reliable supporter of his family throughout her life. The move from Tyne & Wear to Mersyside provided new opportunities and challenges for the ambitious graduate from Sunderland Polytechnic. He relied on his mentor, Dr J. Toping, who recommended him for the award that supported his attendance at Liverpool University after securing a position at Unilever in Birkenhead. He had the luxury of directing his interests to whatever he chose and exploited this to manifestly productive ends during his post-graduate studies on metal ions. Upon his return to the commercial laboratory at Port Sunlight his research interests become constrained but he set about the task of infrared characterization of lipids with flare and dedication. When again faced with a free hand in choosing his research topic at Cambridge, he set off in the direction he had taken during his post-graduate studies at Liverpool but finally saw a connection between his spectroscopic studies of glycerides in foodstuffs and the challenges offered by the properties of the much more complex lipid mixtures found in biological membranes. His prescience and skill in persuading his employers of the need to support his studies of these polar lipids enabled him to establish a pre-eminence in the field. At an early stage of his career Dennis Chapman became well known on the International Conference circuit as an enthusiastic

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and entertaining speaker. Although his lectures were delivered with apparent ease and confidence, he often suffered considerable anxiety beforehand. His public persona also masked a more private and domestic side of his character. It was reliably told that he carried his scientific training into other fields of endeavour whether this be swimming, golfing or growing tomatoes. The strategy was to read up on the theory, make careful observations of the practice then perform the task. With one exception success was assured; his failure to coordinate arm and leg movements efficiently meant that swimming and sinking were finely balanced! He also harboured a rather curious aversion to burnt toast; he maintained that the cinders contained free radicals that would harm his health. It did not seem to occur to him that all the unpaired electrons generated in the toaster would have found stable partners by the time the dressings had been applied; perhaps he just did not like the taste. In another side of his character, he managed to write a radio play that was broadcast by the BBC. When making scientific visits, even to the remoter parts of the world, the visitor was often regaled by the host on how much they had gained from Professor Chapman’s last visit. He was an inveterate traveler and, when all the children left the family home, his wife who shared his appetite for the international set usually accompanied him. He suffered the greatest adversity of his life on the tragic death of his first wife, Margaret, who provided him with a happy and stable family life during his often-turbulent scientific career. His marriage to his second wife, Franc- oise, managed to rekindle his spirits and get his scientific interests back on track. The academic community bestowed many accolades on him. He received honorary doctorates from the University of Utrecht, Memorial University of Newfoundland, Universidad del Pais Vasco, University of Cluj Napoca and the University of Ancona. He delivered the Langmuir Lecture to the American Chemical Society in 1992 and received the Harden Medal from the Biochemical Society in 1997. His appointment as a Fellow of the Royal Society he regarded as the pinnacle of his achievements because it vindicated what he believed to be his maverick approach to science. In his later years he undertook a more titular role in the university serving as the Head of the Division of Basic Medical Sciences during 1988–1989 and Vice Dean of the School during 1990–1993. He published many books and research papers but his editorship of the influential Volumes I–V of Biological Membranes

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published by Academic Press during 1968–1984 represents a lasting testament to his standing in the field. Dennis Chapman’s presence will live on with the many colleagues and students whose careers he influenced. In his valedictory address, he strongly urged young scientists to respond to the needs of society, as he had endeavoured to do with great vigour and success over a lifetime devoted to this cause.

ACKNOWLEDGEMENTS

We wish to thank the Royal Society for graciously consenting to reproduce, in part, the memoirs of Dennis Chapman published under their imprint: P.J. Quinn (2001) Dennis Chapman, 6 May 1927 to 28 October 1999, Biog. Mems. Fell. Roy. Soc. London, 47, 55–66. The photograph of Dennis Chapman was taken in December 1997 by Downing Street Studios, Farnham, Surrey, UK, and is reproduced with permission. REFERENCES [1] Chapman, D. (1954) Electronegativity and stability of metal complexes. Nature 174, 887–888. [2] H. Irving and Williams, R.J.P. (1953) The stability of transition metal complexes. J. Chem. Soc. 3192–3210. [3] Chapman, D. and Taylor, R.J. (1954) Infra-red assignments of unsaturation in the region 900–1,000 cm 1. Nature 174, 1011–1012. [4] Chapman, D. (1956) Infrared spectra and the polmorphism of glycerides. Part I. J. Chem. Soc. 12, 55–60. [5] Chapman, D. (1956) Infrared spectra and the polymorphism of glycerides. Part II: 1:3-diglycerides and saturated triglycerides. J. Chem. Soc. 487. [6] Chapman, D. (1957) Infrared spectra and the polymorphism of glycerides. Part III: Palmitodistearins dipalmitostearins. J. Chem. Soc. 523, 2715–2720. [7] Chapman, D. (1958) Infrared spectra and the polymorphism of glycerides. Part IV: Myristopalmitins and myristostearins. J. Chem. Soc. 646. [8] Chapman, D. (1958) Infrared spectra and polymorphism of glycerides. Part V: 1:2-diglycerides. J. Chem. Soc. 942, 4680–4682. [9] Chapman, D. (1958) An infrared spectroscopic examination of some anhydrous sodium soaps. J. Chem. Soc. 152, 784–789. [10] Chapman, D. (1962) Spectroscopic studies of sulphur nitride ions. Trans. Faraday Soc. 58, 1291–1298. [11] Chapman, D. and McLachlan, A.D. (1963) Bond alternation in [AB] inorganic polymers. Trans. Faraday Soc. 59, 2671–2679.

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[12] Chapman, D. and Waddington, T.C. (1962) Free-electron molecularorbital calculations for inorganic systems. 1. An electron on a sphere model for tetrasulpur tetranitride and FTR tetra-arsenic tetrasulphide. Trans. Faraday Soc. 58, 1679–1681. [13] de Haas, G.H. and van Deenen, L.L.M. (1961) Metabolism and functions of phosphatides. 19. Synthesis of enantiomeric mixed-acid phosphatides. J. Royal Netherlands Chem. Soc. 80, 951–970. [14] Chapman, D. (1960) Infrared spectroscopic characterisation of glycerides. J. Am. Oil Chem. Soc. 37, 73–77. [15] Byrne, P. and Chapman, D. (1964) Liquid crystalline nature of phospholipids. Nature 202, 987–988. [16] Chapman, D., Owens, N.F. and Walker, D.A. (1966) Physical studies of phospholipids. II. Monolayer studies of some synthetic 2,3-diacyl-DLphosphatidylethanolamines and phosphatidylcholines containing trans double bonds. Biochim. Biophys. Acta 120, 148–155. [17] Leathes, J.B. (1925) Role of fats in vital phenomena. The Lancet 205, 853–856. [18] Chapman, D. and Penkett, S.A. (1966) Nuclear magnetic resonance spectroscopic studies of the interaction of phospholipids with cholesterol. Nature 211, 1304–1305. [19] Cornell, B.A., Chapman, D. and Peel, W.E. (1979) Random close-packed arrays of membrane components. Chem. Phys. Lipids 23, 223–237. [20] Frye, L.D. and Edidin, M. (1970) The rapid intermixing of cell surface antigens after formation of mouse–human heterokaryons. J. Cell Sci. 7, 319–335. [21] Cone, R.A. (1972) Rotational diffusion of rhodopsin in the visual receptor membrane. Nature-New Biol. 236, 39–43. [22] Razi Naqvi, K., Gonzalez-Rodriguez, J., Cherry, R.J. and Chapman, D. (1973) Spectroscopic technique for studying protein rotation in membranes. Nature-New Biol. 245, 249–251. [23] Harrison, J.P., Morrison, I.E. and Cherry, R.J. (1990) Rotational diffusion studies of the lipoyl domain of 2-oxoacid dehydrogenase multienzyme complexes. Biochemistry 29, 5596–5604. [24] Chapman, D., Oldfield, E., Doskocilova, D. and Schneider, B. (1972) NMR of gel and liquid crystalline phospholipids spinning at the ‘magic angle’. FEBS Lett. 25, 261–264. [25] Adebodun, F., Chung, J., Montez, B., Oldfield, E. and Shan, X. (1992) Spectroscopic studies of lipids and biological membranes: carbon-13 and proton magic-angle sample-spinning nuclear magnetic resonance study of glycolipid–water systems. Biochemistry 31, 4502–4509. [26] Raison, J.K. (1973) The influence of temperature-induced phase changes on the kinetics of respiratory and other membrane-associated enzyme systems. J. Bioenerg. 4, 285–309. [27] Seiler, D. and Hasselbach, W. (1971) Essential fatty acid deficiency and the activity of the sarcoplasmic calcium pump. Eur. J. Biochem./FEBS 21, 385–387. [28] Chapman, D. and Quinn, P.J. (1976) A method for the modulation of membrane fluidity: homogeneous catalytic hydrogenation of phospholipids and phospholipids and phospholipid–water model biomembranes. Proc. Natl. Acad. Sci. U.S.A. 73, 3971–3975.

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[29] Borowski, A.F., Cole-Hamilton, D.J. and Wilkinson, G. (1978) Water-soluble transition-metal phosphene complexes and their use in two-phase catalytic reactions of olefins. Nouv. J. Chim. 2, 137–144. [30] Chapman, D., Peel, W.E. and Quinn, P.J. (1978) The modulation of bilayer fluidity by polypeptides and homegeneous catalysts. Ann. N.Y. Acad. Sci. 308, 67–84. [31] Quinn, P.J. and Taylor, C.E. (1981) Selective homogeneous catalysis in heterogeneous reaction systems: phospholipid bilayers in water. J. Mol. Catal. 13, 389–396. [32] Quinn, P.J., Chapman, D. and Wilkinson, G. (1976) In Hydrogenation (Office, B.P., ed.), Vol. 29997/76. [33] Katagiri, C., Owen, J.S., Quinn, P.J. and Chapman, D. (1981) Hydrogenation of plasma lipoproteins by a water-soluble catalyst: its use as a structural probe. Eur. J. Biochem./FEBS 118, 335–338. [34] Kaiser, J., Wegner, G. and Fischer, E.W. (1972) Topochemical reactions of monomers with congugated triple-bonds. 7. Mechanism of transition from monomer to polymer phase during solid-state polymerisation. Israel J. Chem. 10, 157–165. [35] Gupta, C.M., Costello, C.E. and Khorana, H.G. (1979) Sites of intermolecular crosslinking of fatty acyl chains in phospholipids carrying a photoactivable carbene precursor. Proc. Natl. Acad. Sci. U.S.A. 76, 3139–3143. [36] Johnston, D.S., Sanghera, S., Pons, M. and Chapman, D. (1980) Phospholipid polymers – synthesis and spectral characteristics. Biochim. Biophys. Acta 602, 57–69. [37] Lee, D.C., Hayward, J.A., Restall, C.J. and Chapman, D. (1985) Secondderivative infrared spectroscopic studies of the secondary structures of bacteriorhodopsin and Ca2+-ATPase. Biochemistry 24, 4364–4373. [38] Lee, D.C., Haris, P.I., Chapman, D. and Mitchell, R.C. (1990) Determination of protein secondary structure using factor analysis of infrared spectra. Biochemistry 29, 9185–9193. [39] Albrecht, O., Johnston, D.S., Villaverde, C. and Chapman, D. (1982) Stable biomembrane surfaces formed by phospholipid polymers. Biochim. Biophys. Acta 687, 165–169. [40] Chapman, D. and Durrani, A. (1980) Improvements relating to biocompatible surfaces. In (Office, E.P., ed.), 853003564.

G. Semenza (Ed.) Stories of Success – Personal Recollections. X (Comprehensive Biochemistry Vol. 45) r 2007 Elsevier B.V. All rights reserved. DOI: 10.1016/S0069-8032(07)45005-7

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Chapter 5

Embden–Meyerhof–Parnas, the First Metabolic Pathway: The Fate of Prominent Polish Biochemist Jakub Karol Parnas ´ SKAa, ANDRZEJ DZUGAJb AND JOLANTA BARAN JANINA KWIATKOWSKA-KORCZAKc a

Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Pasteura 3, PL 02-093 Warsaw, Poland b Department of Animal Physiology, Wrocław University, Cybulskiego 30, PL 50-205 Wrocław, Poland c Department of Medical Biochemistry, Medical University, Chalubinskiego 10, PL 50-368 Wrocław, Poland

Abstract The contribution of Jakub Karol Parnas (1884–1949), a prominent Polish scientist, to the understanding of muscle biochemistry, including ammonia and carbohydrates sources and nucleotide metabolism, is described. Among his main achievements was the discovery of glycogen phosphorolysis, the first use of radioactive phosphorus in biological studies, and formulation and proof of phosphate transfer between glycolytic intermediates and ATP. The rewarding and successful life led by this man of great scientific and intellectual abilities up to the beginning of the Second World War, his dramatic fate during the war, and his tragic death in a Soviet prison reflects the turbulent history of the region for the 20th century. Keywords: glycogen; glycolysis; phosphate transfer; ATP; ammonia; muscle biochemistry; Parnas’ school of biochemistry

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Introduction Prof. Jakub Karol Parnas (1884–1949) was a world-renowned scientist, the founder of the Polish School of Biochemistry (Fig. 1). After his studies in Berlin, Zurich and Munich, he was appointed to his first faculty position at the University of Strasbourg in 1907. In 1913, he has been transferred to Cambridge, but his career there was brought to an abrupt halt. In 1914, the First World War broke out and Parnas returned to Poland, which at this time started its successful fight for independence. In 1916, he lectured in physiological chemistry at the Warsaw University and in 1921, moved to Lviv where he headed the Department of Medical Chemistry at the Jan Kazimierz (Joannes Casimirus) University for 20 years. This period marked the beginning of his most fruitful years of research. In a short time he gathered a team of talented coworkers and generated a very special, stimulating

Fig. 1. Jakub Karol Parnas. Photograph made in the twenties.

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atmosphere in the department. His main field of research was muscle metabolism, particularly the pathway of ammonia formation, glycogenolysis and glycolysis. This study made important contributions to the explanation of the anaerobic glucose metabolism later called the Embden–Meyerhof–Parnas pathway. In this chapter, we will discuss the important scientific activities and trends initiated by Prof. Parnas. We will also explore some of the more interesting aspects of his personality, as well as the circumstances surrounding his tragic death in 1949 in Lubyanka, the infamous Moscow prison. The chapter will also demonstrate the depth of his impact on Polish biochemistry. After the Second World War, one of every fourth professor in biochemistry was from the Parnas’ school. Hence, we will briefly characterize some of his coworkers. Finally, we will describe the Polish Biochemical Society activity conducted to honor the memory of Jakub Karol Parnas and the cooperation in this respect with the Ukrainian Biochemical Society. In June 1941, the German eastern offensive started and Prof. Parnas and his family were forced to flee Lviv for remote Ufa, deep into the Soviet Union. In 1942, his 19-year-old son, Jan Oskar Parnas, joined the newly organized Polish Army with the full support of his father. With this Army, Jan marched out of the Soviet Union. He took part in the famous Montecassino battle in Italy, as well as many others. Jan, who survived the war, always kept a copy of a Kipling poem, translated by Prof. Parnas into Polish and given to him on the eve of his departure. It was one of the most important messages he ever received from his father. Therefore, we present this poem here, as the motto of this chapter (Fig. 2). IF If you can keep your head when all about you Are losing theirs and blaming it on you, If you can trust yourself when all men doubt you But make allowance for their doubting too, If you can wait and not be tired by waiting, Or being lied about, don’t deal in lies, Or being hated, don’t give way to hating, And yet don’t look too good, nor talk too wise: If you can dream – and not make dreams your master, If you can think – and not make thoughts your aim; If you can meet with Triumph and Disaster And treat those two impostors just the same; If you can bear to hear the truth you’ve spoken

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Fig. 2. Jakub Karol Parnas with his son, Jan Oskar on the bank of Morskie Oko Lake in High Tatras, Poland, 1937. Twisted by knaves to make a trap for fools, Or watch the things you gave your life to, broken, And stoop and build ‘em up with worn-out tools: If you can make one heap of all your winnings And risk it all on one turn of pitch-and-toss, And lose, and start again at your beginnings And never breath a word about your loss; If you can force your heart and nerve and sinew To serve your turn long after they are gone, And so hold on when there is nothing in you Except the Will which says to them: ‘‘Hold on!’’ If you can talk with crowds and keep your virtue, Or walk with kings – nor lose the common touch, If neither foes nor loving friends can hurt you; If all men count with you, but none too much,

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If you can fill the unforgiving minute With sixty seconds’ worth of distance run, Yours is the Earth and everything that’s in it, And – which is more – you’ll be a Man, my son! – Rudyard Kipling

Belonging to the third generation of Parnas’ pupils, we do believe that Jakub Karol Parnas was not only one of the most prominent biochemists of the first half of the 20th century, but also, simply, a Man. Biography: Life, Work and Tragic Fate of Jakub Karol Parnas Jakub Karol Parnas, the son of Oskar, a landowner, and Gabriela, ne´e Bernstein, was born on 16 January 1884 in Mokrzany near Tarnopol, a small town situated in the district Galicia. This area reflects the complicated history of Central and Eastern Europe. Until the end of the 18th century it had been a part of Poland. Then, up to the First World War it belonged to Austro–Hungarian monarchy. Between 1918 and 1939, the region returned to Poland again. Following the Soviet–German treaty in 1939, the land was turned over to the Soviet Union. From 1941 to 1944, it was occupied by Germany. Now, it is a part of Ukraine. The name of Lviv, the main city of this land where Parnas worked for 20 years and he made his greatest achievements experienced similar cultural changes. In the course of one century, this city was known as Lviv in English and Russian, Lwo´w in Polish (pronounced Lvoov), Lemberg in German and Lviv in Ukrainian. 1902– 1913, Learning and Studying in Different Cities of Europe: Beginning of scientific activity Parnas attended primary school in Tarnopol and then gymnasium in Lviv. After graduation in 1902, he studied chemistry at the Institute of Technology (Technische Hochschule) in BerlinCharlottenburg (1902–1904) and then organic and physiological chemistry at the University of Strasbourg (1904–1905). After completed graduation in 1906 he worked for a year at the Zurich Polytechnikum (Eidgeno¨ssische Technische Hochschule Zu ¨ rich,

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¨tter, a prominent ETH), under the guidance of Richard Willsta scientist and Nobel Prize winner, and one of the founders of modern chemistry. Parnas’ brilliance emerged early during the research in organic ¨tter’s laboratory, the staff was chemistry in his career. In Willsta searching for the third isomer of naphthoquinone that was believed to exist in addition to the two known isomers. At the age of 22, Parnas succeeded in the isolation, crystallization and characterization of 2,3-naphthoquinone (amfi-naphthoquinone). ¨tter were published [1,2], and in Two papers by Parnas and Willsta 1907, after submitted his dissertation at the University in Munich, Jakub Karol Parnas received the PhD degree in Chemistry [3]. The same year he was appointed as an assistant to Franz Hofmeister’s laboratory at the Institute of Physiological Chemistry in Strasbourg. The atmosphere in the laboratory was very stimulating. Innovative ideas were enthusiastically discussed, and efforts were made to prove them in bold experiments. In this inspiring atmosphere, many talented outstanding biochemists like Gustav Embden, Franz Knoop and others were working together with Hofmeister. Prof. Hofmeister exerted a profound influence on his pupils and, as a result, young Dr. Parnas solidified his commitment to scientific endeavors. His initiation in biochemistry took place in the Strasbourg laboratory. Here, he started to ask relevant questions that he would eventually devote his career in unraveling. According to coworkers, Prof. Parnas frequently looked back fondly upon those days spent in Strasbourg, and expressed his admiration and gratitude to Hofmeister. At that time, the development of biochemistry was extensive and rapid. Many constituents of the living cell were identified, and their metabolism was proposed. But the role of enzymes and their protein nature remained largely a mystery. The connection between glycogen or glucose breakdown and lactic acid formation was hypothetical and naively seen as a one-step reaction. The glycolytic pathway was viewed in a similar reductivist tendency, although certain reactions were already known. Jakub Karol Parnas started experiments on tissue homogenates and on perfused organs. The catalytic activity in extracts of animal tissues was at that time merely a hypothesis yet to be proven. In 1897, Edward Buchner showed that the cell-free yeast juice was capable of fermentation. In 1906, Arthur Harden and William Y. Young separated yeast juice into filtrate and residue demonstrating

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that fermentation was possible only through their mixture. Although it took many years, the common pathways of metabolism in all living organisms was eventually conceptualized. Parnas investigated porcine as well as bovine liver and lung homogenates, and their water extracts on the ability to produce acid and alcohol from aldehyde in Cannizzaro reaction. Other scientists had conducted research on acetaldehyde in the past, but with limited success. Using benzaldehyde and several aliphatic aldehydes as substrates, Parnas demonstrated that all of them were metabolized by liver extracts. Parnas’ conclusion was that liver contains a soluble ferment, able to catalyze the Cannizzaro reaction, and named it as ‘‘aldehyde mutase’’ [4]. Each of Parnas’ experiments confirmed his and Hofmeister’s conviction that enzymes catalyze reactions in living cells. Parnas had felt that he wanted to explore his physiological knowledge. This prompted him to spend a year at the Zoological Station in Naples, Italy. There he learned new experimental techniques. His main research interest was in energetics of mollusc smooth muscles. He found that the steady, maximal contraction of these muscles was not accompanied by an expenditure of energy. The total energy of this contraction does not exceed 10–4–10–5 of the values for striated muscles [5]. An excellent review on smooth muscle energetics is given in Parnas’ work in 1910 [6]. Later, back to Strasbourg, Parnas returned to studies on metabolism of animal tissues. The production of lactic acid by bacteria, yeast and vertebrate muscles was already known, but the stereochemistry of this has been studied only in microorganisms. Parnas injected solutions of L(+) and D(–) lactic acids to rabbits and analyzed its content in particular organs, muscles and urine. From this experiment he proved that L and D forms of lactate are differently metabolized. L(+) lactic acid is quickly removed from tissues, whereas D(–) lactate is extremely slowly metabolized and is toxic [7]. Results of the experiment were of great value. It was the very first observation of the stereospecificity of enzymes. In 1912, Parnas demonstrated that glycogen in liver can be synthesized from glyceraldehyde [8]. Experiments were performed by Julius Baer showed that in muscle glucose might be formed from lactate and glycerate by way of two oxidative reactions and condensation. The authors stated that three molecules of glucose are converted to six molecules of lactate in exothermic reaction, and from these

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two molecules of glucose, six of CO2 and six of H2O may be synthesized [9]. In 1913, Parnas was promoted Docent (Associate Professor) at the University of Strasbourg and was listed in its staff up to 1918, although that time he was working in Poland. His collaboration with Wagner who was located in Strasbourg would last five years more. The correlation between glycogen breakdown and lactic acid formation had been postulated in these years. Fletcher and Hopkins, Embden, Meyerhof and others did many valuable experiments, but no clear, convincing image was formed and the hypothesis was yet to be verified. Parnas and Wagner performed highly sophisticated experiments on resting and working isolated frog muscles in oxidative and anaerobic conditions. The content of glycogen, glucose, phosphate and lactate was estimated at different time of experiments. Their findings unquestionably concluded that the decrease of carbohydrate content in working muscles is accompanied by lactate formation. In aerobic conditions much less lactate is present – less is formed, or it is metabolized further [10,11]. Gustaw Embden and Otto Meyerhof later confirmed Parnas’ data and showed the equivalence between glucose decrease and lactate increase. Parnas’ early works are an important part in the history of biochemistry. Each observation represents a significant contribution to the understanding of tissue metabolism, its pathways and the role of enzymes. 1914– 1920, the First World War: Decision to Work and Live in the Independent Poland When Parnas visited Cambridge in 1913, he started a productive collaboration with Frederich Gowland Hopkins, the known authority in the research on muscle metabolism. He continued his cutting-edge investigation on the lactic acid production in working muscle. After the beginning of the First World War in 1914, Parnas unfortunately has found himself in an uncomfortable position. Despite being an Austrian citizen, he was allowed to stay in Great Britain and worked in Hopkins’ laboratory, but he was unable to leave Cambridge and was restricted to travel freely. Nevertheless, he was allowed to go to his native land. There, he was called soon to serve in the Austrian army. As a chemist and with the help of the known bacteriologist, Prof. Odo Bujwid, he

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joined the medical service to work in laboratory and stay in Galicia. During the war it soon became apparent that Poland would again become an independent state. This political change significantly influenced the remainder of Dr. Parnas’ career. Thus, in 1916, he decided to live and work in Poland, even though he could easily find a position in any of the best laboratories in Europe. He accepted a position at the University of Warsaw where he organized the Department of Physiological Chemistry. His lectures quickly gained notoriety and became popular among scientific circles and were attended by enthusiastic medical students, eager to know about the newest achievements in science. Knowing their interest, Parnas dedicated time and effort in writing the first Polish textbook on physiological chemistry. He continued studies on the metabolism of isolated frog muscles [12–15]. Soon he turned his attention to the recent discovery of pathogenesis of diabetes. This was encouraged him to investigate animal muscles in experimental diabetes [16]. In collaboration with Richard Wagner from Strasbourg, Parnas contributed to the knowledge of carbohydrate metabolism alterations in diabetic patients and in other diseases [17–19]. He became interested in the study of problems of clinical chemistry [20–22]. At that time, local research studies at the Warsaw University and their significance were often overestimated and the research of foreign investigations neglected. Parnas openly criticized this tendency as well as other irregularities. Consequently many colleagues from the University opposed him. This experience was frustrated him. These troubles caused much discouragement and consternation but he taught his coworkers to be open to world science, honest and critical of their achievements, as he always had been himself. 1920– 1939, The Best, Successful Years in Lviv The year 1920 was very fortunate for Parnas’ personal life and scientific activity. He married Renata Taubenhaus who became his devoted wife staying with him till his death. Also in 1920, Parnas decided to move to Lviv. He was given a position at the Medical Faculty of the Jan Kazimierz University as a full professor and the Head of the Department of Medical Chemistry. On 16 June 1921, in a solemn ceremony he swore an oath. The text (in Polish) signed by him is still held in the archives of

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the Faculty of Medicine (now Medical Institute) in Lviv (now Ukraine). Here is its translation: I swear to the God Almighty, that in the academic position, given to me, I will contribute with all my forces to strengthen freedom, independence and power of the Republic of Poland, which I will always serve; I will equally respect all the citizen of the country; observe all the regulation of the law, fulfill all the duties of my position ardently and conscientiously, execute orders of my superiors strictly and keep the official secrets. So help me Lord.

Then, the best and most fruitful 20 years of Jakub Karol Parnas’ life and work have begun. His life stabilized and soon two children were born, daughter Justyna in 1921 and son Jan Oskar in 1923.1 In those years the Jan Kazimierz University was a renowned scientific and intellectual center. Many outstanding scientists were working in chemistry, physics, anthropology, literature, medical sciences and other disciplines. The famous Polish school of mathematics was developed with the world-known mathematicians Hugo Steinhaus and Stefan Banach at the lead. Rudolf Weigl invented and produced antityphoid vaccine which saved so many lives during the Second World War. The name of Jakub Karol Parnas was well known at the University, and he was greeted with respect and reverence. He was given all the necessary resources to enable him to maximize his scientific, intellectual and creative potential. Parnas as a Teacher and Master In Lviv, Parnas fulfilled his pedagogical responsibilities with great enthusiasm. He always considered the students education to be a very important part of scientific work and he devoted much effort to these activities. This turned out to be mutually beneficial – students received a very good education and it was among them that Parnas found his most talented future collaborators. Parnas had all the abilities to do teaching: broad knowledge of the discipline, familiarity with relevant international research activities, ability of effective communication and an exact understanding what learners required for integrating new ideas. 1

His children from the previous marriage with Raissa, Gustav Theodore (1906–1979) and Claire Walter (1909–1972) lived their whole life in France and died without children. Renata was Jakub Karol Parnas’ second wife; however, we are unaware of the circumstances of his short first marriage and divorce.

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Parnas was an excellent lecturer. His lectures for medical students on general and physiological chemistry were vivid, brilliant, supported by experiments, and showed his competence and depth of knowledge as well as his great sense of humor. His class exemplified not only scientific knowledge, but also demonstrated his familiarity with classic and contemporary literature and art. He was also not reluctant to show his well-rounded intellectual interests, and would regularly integrate several modern languages as well as Greek and Latin into his lectures. He also quoted Homer, Virgil, Shakespeare, Rilke and Elliot as readily as the newest results in physiology or chemistry. Chemical and biochemical problems were shown within a historical perspective, from their discovery up to the present achievements. These were consistently precise and clearly formulated. He particularly emphasized the correlation between biochemical problems and practical medicine. Crowds of students attended his lectures not only from the medical but also from other faculties. The most enthusiastic listeners, sitting in the front row of the auditorium, asked a lot of questions during the time of the lectures and Parnas eagerly answered them and engaged in lively dialogue with students. As early as 1922, Prof. Parnas published his textbook Physiological Chemistry with Special Reference to Animal Physiology [23], the first one written in Polish. It was extremely important and helpful in teaching physicians and biologists. The second edition of the improved textbook of Physiological Chemistry was published in Warsaw in 1937 [24]. Many of Parnas’ students volunteered to work in his laboratory. Those selected remained there after graduation. Affectionate reminiscences by his former students and coworkers are found throughout Polish biochemical and historical journals. Parnas is shown to be a master, who profoundly influenced the scientific career paths they chose. Even clinical practitioners, pediatricians, surgeons and internists emphasized in their recollections, how invaluable the biochemical training they had received in his laboratory. Many words of respect and admiration were chronicled in all their reminiscences. Parnas’ Scientific Activities At this time, ammonia formation had been a new and poorly understood problem in biochemistry, which became the object of

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intense focus of the Department of Medical Chemistry. This led to studies on purine metabolism. To begin experiments on ammonia formation in living organism, ´zef Heller invented a new precise and reliable Parnas and Jo method, involving microdestillation, and modified the previously known Parnas–Wagner apparatus. Using this method, Parnas’ research team found that, in isolated blood, the concentration of ammonia increased with time. They concluded that ammonia was not derived from proteins or urea, but was formed enzymatically from some low-molecular weight substances present in blood cells [25–27]. Next, the ammonia level was determined in circulating human and animal blood. Several factors influencing ammonia concentration were investigated, such as starvation, acidosis, anoxia and others. It was found that under narcosis, only negligible amounts of ammonia were formed. Much higher ammonia content in intestinal venous blood was correctly attributed to an exogenic origin [28–30]. Once they completed the main objectives of the study to find the source of endogenic ammonia in blood, Parnas and Włodzimierz Mozołowski analyzed resting, injured and working muscles of various species from fish to man. They concluded that an increased level of ammonia concentration was strictly related to muscle contraction. Their unprecedented results were published and disseminated at biological and medical meetings in Innsbruck in 1924, Warsaw in 1925 and at the 12th International Physiological Congress in Stockholm in 1926 [31–37]. Parnas believed that nitrogen turnover is of real importance for muscle metabolism and function [38]. In 1927, Gustav Embden and Margarete Zimmermann discovered AMP in muscles. This turned Parnas’ attention to purine metabolism as a possible source of ammonia in muscle and blood. Parnas introduced a precise new method of free bases and nucleotide determination in small amounts of tissue. It was found that no free bases were present in muscles, and among nucleotides AMP amounted to 82–89% and IMP to 11–18% [39]. In anaerobic conditions, in injured and working muscle, AMP was converted to IMP. The decrease in adenylic acid content was found to be equivalent to ammonia formation. When oxygen was supplied, AMP was recovered from IMP, but not with utilization of ammonia, once split off. This was proved by direct experiments. Papers on these results [40–42] and presentation at the 20th Congress in

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Boston in 1929 [43] were met with great interest among biochemists and physiologists. These findings contradicted those of Embden, who considered that ammonia splitting is reversible. In a series of unique experiments, Prof. Parnas proved that he was right [44]. Perhaps today this knowledge seems obvious, but at that time true scientific imagination was necessary as well as many arduous experiments to provide irrefutable evidence for it. Ammonia metabolism was not the only focus of studies in Parnas’ laboratory. Heller effectively studied metamorphosis of insects, Dadlez the metabolism of uric acid, and Mozołowski and Hilarowicz antipepsin activity of blood serum. Parnas was in close contact with other biochemists around the world. They exchanged information about their latest experiments. Wictor Henry, the professor of physical chemistry from Zurich, Pierre Thomas from the Pasteur Institute in Paris and many others visited Parnas’ laboratory. In 1928, the University of Strasbourg donated the newest equipment for carbon determination. Prof. Parnas also successfully solicited funding from the Elli Sachs-Ploetz Foundation in New York. In the 1929/1930 academic years, Parnas was appointed Dean of the Faculty of Medicine at Lviv University. At the beginning of 1930, he utilized the facilities at the Universities of Leipzig and Strasbourg performing microchemical analyses which he was unable to do in his own laboratory. Suddenly, his record of accomplishments gave to an abrupt halt. In 1930, a tragic blow hit the Parnas family. The nine-year old Justyna died of tuberculotic encephalomyelitis. Parnas was in deep emotional despair. His loss was so profound that even staying in the apartment where Justyna lived so happily was too painful for him. He started spending more and more time withdrawing in his laboratory, and in 1931 he left with wife and son for Zurich where he spent a year as a visiting professor. Many friends, ¨tter, Naegely, Strohl and others visited him professors Willsta there. As his son, Jan Oscar Parnas later recalled that his father liked to hike with him for hours in mountains, or to ski in winter, saying ‘‘Mountains make people better’’. Back to Lviv, Parnas has shifted to faculty housing, the so-called ‘‘Professor’s housing’’ available through his institution. In this way he was in closer proximity with prominent scientists from the Jan Kazimierz University. A peculiar atmosphere reigned in the house. When Parnas was offended by literature professor Kleiner,

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for example, they did not speak to each other directly and instead exchanged letters – in Latin! In 1931, Parnas was elected a member of the Polish Academy of Arts and Sciences in Cracow. The same year he was also nominated as Head of the Division of Pharmacy at the Faculty of Medicine and the Director of the committee supervising Premedical Departments of the Faculty. Even though Parnas had multiple simultaneous administrative appointments, his research activities did not suffer. In fact, the thirties of the 20th century were his most successful and prolific years of research, full of innovative achievements that persist to be of fundamental value in biochemistry today. Studies on nucleotide deamination in muscle and heart of several animal species were continued, with modified and improved methods of AMP and ATP determination [45]. Ostern and Mann found that a very active enzyme deaminates AMP and some other compounds, probably adenosine, whereas ATP was not deaminated. This apparently simple observation was of great importance for the elucidation of nucleotide role in energy metabolism. The discovery of the inhibitory effect of phosphate on ammonia formation led to the brilliant conclusion that nucleotide metabolism is linked with glycogenolysis. According to this, in the presence of phosphate ATP is recovered from AMP, and the deamination cannot proceed. Phosphocreatine (‘‘phosphagen’’, previously described by Parnas) was considered as a phosphate donor. This paper published by Parnas, Ostern and Mann in Biochem. Z. in 1934 [46] was translated from German into English by H.M. Kalckar, and re-published in 1969 in Kalckar’s book entitled Biological Phosphorylations – Development of Concepts [47]. Later on, it was shown that iodoacetate and fluoride stimulated AMP deamination. Their role in inhibiting two glycolytic reactions was already known, so phosphoglycerate and phosphopyruvate were found to be responsible as a source of phosphate, which could be directly or via phosphagen transferred to AMP to restitute ATP [48]. Experiments with glycolytic intermediates added to the homogenates confirmed this assumption. Schematic representation of AMP formation from ATP in working muscle, and of phosphate transfer from glycolytic intermediates or phosphocreatine to ATP in resting muscle was given by Parnas and his coworkers Ostern, Lutwak-Mann, Mann and ´ ski [46–52]. Lewin

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Thus, it has been clearly shown for the first time that ATP may be regenerated at the fexpense of the energy derived from glycolysis in a process known as the ‘‘Parnas reaction’’. These results were first presented at the 4th Congress of Biochemistry in Paris in 1933 and then, in 1934, in two papers were published in Nature [53,54]. After these discoveries, research in Parnas’ laboratory was mainly concentrated on studies on glycogen and glucose metabolism. His entire staff was engaged in these experiments. Teams worked on their individual projects but in concert with each other. In short, their most important achievements were: 1. 2.

3.

The discovery of the reaction of glycogen phosphorolysis occurrs in the presence of inorganic phosphate [55–60]. The discovery that ATP synthesis, which occurs during glycolysis, involves the transfer of phosphate residues from molecule to molecule [46,47]. The application of radioactively labeled phosphorus in biological experiments.

In collaboration with G. Hevesy from the Institute of Theoretical Physics in Copenhagen, Parnas, using 32P, confirmed the reaction of phosphorolytic breakdown of glycogen [61–66]. Also with 32P application, Parnas and Tadeusz Korzybski showed in vivo a rapid turnover of b and g groups of ATP and a slow one of the phosphate moiety of AMP [67–69]. 32P-labeled adenine nucleotides and sugar esters were synthesized ‘‘Laokoon’’, a pharmaceutical company in Lviv, according to the procedure worked out in Parnas’ laboratory. Samples of these chemicals were sent abroad to colleagues, C.F. and G.T. Cori, among others. Many of Parnas’ talented coworkers were engaged in these works: Z. Augustin, T. Baranowski, K. Gibayło, J.A. Guthke, S. Hubl, T. Korzybski, C. Lutwak-Mann, T. Mann, W. Mejbaum, I. Mochnacka, W. Mozołowski, P. Ostern, J. Reis, W. Słobodzian, B. Sobczuk, J. Terszakowec´ and B. Umszweif. Parnas insisted that they should often publish their results not with him as a coauthor. C. Lutwak-Mann, T. Mann, K. Gibayło and B. Umschleif were comparing reactions of alcoholic fermentation in yeast and glycolysis in muscle, and P. Ostern, J. Guthke and W. Szankowski studied differences in aerobic and anaerobic glycolysis. P. Ostern,

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J. Terszakowec´ and J. Reis investigated ATP synthesis from adenosine and adenylic acid in yeast. Nominations, Honors, Awards Throughout his career, Jakub Karol Parnas maintained close contacts with prominent European and American scientists. He solidified these relationships during his many visits to diverse European laboratories. The Ministry of Education promoted his visits to Great Britain, Austria, France and Germany as a representative of Polish science. His on-going contacts were welcomed by the international research community, and he was invited to many laboratories and attended Congresses in Belgium and Moscow. In 1938 Prof. Parnas was invited to inaugurate the Conference on Application of Radioactive Isotopes in Biological Sciences held in Niels Bohr’s laboratory in Copenhagen. A year later he was invited as visiting professor at Gandava University, Belgium. Perhaps he would have avoided his tragic fate going there. But he did not want to interrupt his son’s education, who was attending his final year of gymnasium in Lviv. While he visited prestigious labs abroad, Prof. Parnas encouraged and helped facilitate visits from other scientists to his Department in Lviv. As it is noted in the archives of the Faculty of Medicine in Lviv, many biochemists from Belgium, Denmark, England, France, Germany and Sweden visited the Department of Medical Chemistry under his leadership. It was not only in his home institute that Parnas was recognized for his impressive list of achievements. As a world-renowned scientist, he was elected an Honorary Member of the University in Athens, Greece, and a Member of the German Leopoldina Academy of Sciences in Halle, Germany. To strengthen international scientific literature, he was invited to contribute articles on the chemistry of muscle in the first and second volumes of the prestigious Annual Review of Biochemistry in 1932 and 1933 [70,71]. Along with his research work, Parnas had been engaged in other activities. As one of the founders of the Polish Physiological Society, he was asked to inaugurate the First Plenary Meeting in 1937. In this Meeting, he gave a lecture on the mechanisms of metabolic transformations in animal tissues. He appreciated the importance of scientific writing in education and in popularization of science [72,73]. Throughout the period between

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1922 and 1926, he contributed several chapters to Textbook of Physiology, edited by A. Beck [74]; Textbook of Biological Methods, edited by E. Abderhalden [75] and Textbook of Normal and Pathological Physiology, edited by A. Bethe, G. Bergmann, G. Embden and A. Ellinger [76]. Interested in problems of nutrition, Parnas published with W. Mozołowski a monograph on the physiological and chemical aspects of dietetics [77]. An entirely new version of his Physiological Chemistry appeared in 1937 [24]. Parnas was the editor of this two-volume textbook, and contributed to important chapters himself. Several articles written on the metabolism of carbohydrates, lipids and vitamins popularized these complex processes for the general public [78]. In addition to these subjects, Parnas was also interested in challenges associated with Polish biochemical nomenclature [79,80], and biographies of prominent scientists such as O.H. Warburg, G. Embden and others [81–84]. Parnas’ Personality In his memoirs, Jan Oskar Parnas described his father’s habits of working and spending his free time. Many details can be also found in articles written by Jakub Karol Parnas’ students, coworkers and colleagues. His typical working day indicated that he was highly devoted to his work. From Monday to Friday, he spent almost the entire day in the laboratory. He used to leave home at 9 am, go by hackney carriage to the Department and stay till 8 pm. He actively performed many experiments, and closely supervised the work of all his staff. Parnas’ authority was warm and stimulating. He created a very special atmosphere in the laboratory. Everyone worked hard with enthusiasm and joy, in a spirit of collaboration and (in the good sense of the word) competition. With his jovial sense of humor, Jakub Parnas’ inclination toward non-vicious mockery only added to the friendly and informal atmosphere. Many of his staff members nostalgically recalled that Prof. Parnas’ personality had a profound influence on them lasting for all of their life. After a typical period of closely supervised work, Parnas subsequently fostered autonomous research and independent dissemination and publishing activities. He initially observed these studies cautiously, but as finding became more promising, Parnas entered actively in research. He often engaged in heated debate with authors, discussed what he considered to be potential problems, and formidably challenged the results. Once a study

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could withstand his strict internal criteria, he offered significant assistance and support to bring the results to international scientific audience. This unique process simultaneously encouraged young scientists to learn to work effectively in collaboration with diverse specialists and to develop their own fully independent critical thinking. When his absent in the laboratory by illness or travel, Parnas contacted the laboratory inquiring about results of everyone’s work. Respecting schedules of examinations, Parnas frequently invited students home. Prof. Parnas’ son Jan recalled that his mother routinely served food and sweets to overworked young students expecting exams, to calm and relax them. Weekends, however, were devoted to family. Hours were spent with Jan in bookshops, especially antiquarian. Parnas was a connoisseur of literature. His library at home held many treasures of classic and contemporary literature. He collected rare books and first editions, contacted libraries and often ordered abroad. In his free time, Parnas participated in the rich cultural life of Lviv by attending concerts, theaters and parties given by the University or City Council. Although tall and rather heavy, he was remembered by ladies as a graceful dancer. In good weather, Parnas hiked with Jan in the hills and forests surrounding Lviv. The family often spent vacations in the Carpathian or Tatra mountains. 1939– 1941, the Second World War in Lviv: Soviet Occupation This happy world came to end when the Second World War began in 1939. On 17 September, as stipulated the Ribbentrop-Molotow treaty, the Red Army entered Lviv. Soon massive arrests began. Members of the army, police, judges and other government representatives, as well as artists and other independent thinkers considered a threat to the new regime were taken into custody without trial and disappeared. Many never came back. Their families were taken at night to trucks without their belongings and sent to Siberia or further east to the Asian Republics with virtually no food or water. Under these conditions of deprivation associated with forced genocidal winter expulsion, the long, difficult journey in overcrowded cattle cars on industrial train routes, led to disease and death. When

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they arrived, many did not survive the unbearable conditions and hard work. At the end of September 1939, it was decided that the Jan Kazimierz University would be renamed Ivan Franko, and that the university would be functioning according to the Soviet model. However, on 5 October 1939 when the new academic year started, activities were carried out in the usual ways. Unfortunately, it was only the calm before the storm. In the middle of January 1940, the university status was changed and reconstructed according to the Soviet pattern; the Faculty of Medicine was separated from the University and a new National Medical Institute was formed. Parnas became the Head of the Chemical Department at this Institute (Fig. 3). In spite of that, everyday life in the city had to go on. The intensive teaching and research work was carried out as usual.

Fig. 3.

Jakub Karol Parnas. Photograph made in the forties.

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At the beginning of the war, many helpless refugees from the western and central parts of Poland occupied by Germans came to Lviv. Many of them received help from Parnas and possibility to work. Some were engaged as assistants, among them chemists from Warsaw, Heppner and Lindenfeld and young, enthusiastic ´ ska-Blauth and people, happy to work with Parnas, such as Opien others. Along with continuing studies on glycolysis, Parnas’ interest in naphthoquinones, the object of his doctoral thesis was rekindled. With Baranowski he synthesized methylnaphthoquinone, a vitamin K substitute and applied it to medical practice, in surgery and hemorrhages. Parnas showed the results of this work in the article on vitamins in 1943 [85]. Soviet authorities had a full knowledge of Parnas’ position in world science and made great efforts to enlist him as a Soviet scientist. He was promised resources to enlarge the Department and to buy new equipment, reagents and experimental animals. To make his name known to the public, several articles were published in the national journal ‘‘Izvyestya’’ in Moscow. Some were competent, some naive. They generally stated that the people, especially soldiers of the Red Army, were highly concerned with Parnas and his laboratory. At the end of 1940 Parnas was elected a member of the Regional Council of People. Parnas had been truly respected by Russian and Ukrainian scientists. Many prominent biochemists met him in 1940: A.A. Bohomolec, A.E. Braunstein, A.W. Palladin, S.S. Medviediev, B.J. Zbarski, W.A. Engelhardt and others. Parnas and Baranowski were invited to Moscow to present the results of their work and were met with appreciation. Nevertheless, most promises made by authorities remained on paper. In his letters to Mozołowski and Mann, Parnas complained about the lack of international scientific journals. He became aware that contacts with foreign colleagues would be almost impossible. Reagents and equipment were lacking.

June 1941, Leaving Lviv to Ufa On 22 June 1941 German troupes attacked the Soviet Union. A few days after the beginning of the war, an aggressive anti-German article appeared in the local journal, signed with the name of Parnas, but in fact, it was not written by him. Prof. B. Halikowski

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who visited Parnas that day saw him shocked and frustrated. It became clear that Parnas had no other choice but to leave Lviv before the Nazis would enter the city. As Jan Oskar Parnas recalled, on 26 or 27 June a car was sent by Soviets’ authorities to evacuate Parnas and his family. Only a short time had been left to take some belongings and to allow Jan, a student of medicine, to join his parents. A few days later, 23 prominent Polish professors, many of them from the Faculty of Medicine, were executed by Nazis at the suburban hills of Lviv. Although Prof. Parnas was not among them, his fate would be not much better due to his Jewish origin. Jan Oskar Parnas described the long journey to the east of the Soviet Union – several days by car to Kiev, and then, with other refugees from the Ukrainian Academy of Sciences, weeks by train to Ufa in the Bashkirian Republic, deep in the Asian part of USSR. The family got a small room in a hotel. Parnas, who tried in vain to work in the laboratory of the local University, was depressed. He changed his mind after a short visit to the Polish Embassy in Moscow. He got a good news that a Polish Army has to be formed in USSR. Then, from all parts of the Soviet Union Poles were coming to the recruitment offices; they came from labor camps and regions of displacement, often on foot. Jan Oskar Parnas recalled how one day an emaciated man in rags, almost barefoot, entered their room, bowed, and said politely: ‘‘Good morning, professor’’. Jakub Karol stood up and greeted the newcomer calmly: ‘‘How are you, mister President?’’ The man was the President of Lviv Prof. Stanisław Ostrowski (future President of the Polish Republic in exile in London, 1972–1979) who was arrested and sent to Siberia in 1939. Prof. Ostrowski described this event in his memoirs – a moving contrast of harsh circumstances and good manners. At the end of 1941, a Polish officer visited Parnas. Prof. Parnas has introduced Jan to him ‘‘Here is your first volunteer to the Army’’. Jan participated in many battles involving the Polish Army, including those in Italy. He was able to contact parents only through friends in London. After the battle at Montecassino, Jan spent several months in hospital and his mother came to see him. In 1946, Jan returned to Poland, completed his medical studies at the Wrocław Medical University and later on worked at the Clinic of Surgery.

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1943– 1949, Stay in Moscow In 1943, refugees residing in Ufa were moved to Moscow. Parnas was appointed the Director of the Chemical Department of the National Institute of Experimental Medicine. He reorganized it soon into a National Institute of Biological and Medical Sciences of Academy of Medical Sciences. He also organized a new Laboratory of Carbohydrate Metabolism. The same year he was elected as a Member of the USSR Academy of Science and in 1944 Member of the USSR Academy of Medical Sciences. During the war, no experimental work had been possible. Parnas used that time to write excellent reviews on enzymes, coenzymes and hormones, published in the biochemical journals in USSR and in Nature (London) [86–90]. He organized conferences known at the Department as ‘‘Parnas’ Thursdays’’, famous for interesting lectures and vivid discussions. Parnas insisted on the active participation of young people, making them prepare lectures on various topics. These seminars were attended not only by the staff, but also by renowned scientists, such as A.E. Braunstein, W. Bielew, M. Lyubimova, M. Shelagin all from Moscow; A. Szent-Gyo+ rgy from Hungary and B. Hastings from USA. When the war ended the experimental work began. However, many reagents were still not available, so glycogen, myosin, nucleotides and other organic compounds were isolated from rabbit muscle. Supervised by Parnas, Borys Stepanienko studied the reaction of polysaccharides with iodide. Anna Pietrova, and later on Eugenia Rosenfeld isolated phosphorylase, glycogen 1,6-glucosidase and myokinase from muscles and other tissues, and studied glycogen interactions with proteins. AMP, ADP and ATP were the other objects of research. For the first time the difference between the ‘‘muscle AMP’’ (adenosine 50 -phosphate) and ‘‘yeast’’ adenosine-30 -phosphate was established. Warm recollections of the work with Parnas were published in 1997 by professors E. Rozenfeld, A. Kotielnikova and E. Afanasjeva. All his coworkers were impressed by his personality; the style of management, his erudition and kindness. They warmly recalled that the professor and his wife invited them to home several times. The elegant dinners in these hungry days were remembered well (Fig. 4). In summer and fall of 1944 Parnas was allowed to visit Lviv. He was very interested in studies performed at his former

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Jakub Karol Parnas with wife Renata, Uzkoye, Soviet Union, 1948.

Department. He attended meetings with professors of the Medical Institute and openly discussed the way in which incompetent Communist party members criticized some professors. Talking with Dr. Zygmunt Albert, Parnas expressed his involvement in the problems of people deported to Siberia. He naively believed what he was informed by Soviet authorities that after the war those people would be transported to Western Ukraine and later on allowed to go to Poland. Nevertheless, Parnas was aware of the dramatic fate of his compatriots in the Soviet Union and even joined the Committee of Polish Patriots with the intention of helping Polish people. In many cases he truly succeeded. Many years later the testimonies of his help in liberating students and workers of the Faculty of Medicine from labor camps were disclosed by the family of one of these students, Prof. Anna Rutkowska-Brzecka, and by the Association of People Deported to Siberia. As it is evident from the correspondence with T. Korzybski and I. Mochnacka (1945–1947), Parnas wanted to return to his native country. At the turn of 1946–1947 he was allowed to visit Poland. He went to Cracow and Wrocław and was invited to accept the Chair of the Department of Physiological Chemistry in either of those academic centers. Parnas was ready to accept the position

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offered at the Jagiellonian University in Cracow. Unfortunately, his plans could not be realized. Scientists were considered to be the property of the Soviet Union. We have to remember that in 1939, the Soviet Union occupied and incorporated a large part of the eastern Poland. All inhabitants of this territory automatically were considered Soviet citizens. Communist authorities from one hand have been taking care of prominent scientists offering them slightly better standard of living or awarding them medals, on the other hand they have not been allowed to go abroad. Because of that, Parnas could not attend the First International Congress of Biochemistry in London in 1948 and was not allowed to return to Poland for good, despite his Polish nationality. His friends from the USSR Academy of Sciences advised him not to even think about leaving Moscow. Thus, the decision to remain in the Soviet Union up to the tragic end was not his choice. After the Second World War he as well as many others was not allowed to return to their own homelands. All the time, up to his last days, Parnas has been in touch with Korzybski in Warsaw. They exchanged letters, often by the courtesy of people traveling to and from Moscow. Parnas asked details about his former coworkers, their life, work and results of research. He was proud of them, and several people quoted his words: ‘‘My best successes are my students’’. Parnas maintained contact with scientists from Western Europe and from the USA. In 1945–1947, he was elected a member of the French Academy of Science, a Honorary Member of the of Sorbonne University in Paris, corresponding member of Biological Societies in Paris and Vienna, member of Chemical Societies in Paris and London, and in 1948 an active member of the Polish Academy of Arts and Sciences. In Moscow, he was a frequent guest in many embassies of Western countries. Parnas, who all his life had been in continous contact with scientists from the whole world, could not understand how extremely dangerous for him this was under a communistic regime. It was also unusual to openly present and discuss one’s own opinion in this regime. Parnas publicly criticized the pseudo-scientific theories of Lysenko, and several other projects supported by communistic authorities. Jan knew that his mother had been informed of the situation in the Soviet Union, especially after imprisonment in 1947 of W. Parin, the Secretary of the Academy of Medical Sciences and

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many prominent physicians and scientists. Over time his father also perhaps became nervous. In 1947, Parnas’ health has been impaired; he suffered from diabetes and heart disorders. He resigned from the position of the Director of the Institute and in May 1948 his resignation was accepted. That year he was invited as a Vice-President to the First International Congress of Biochemistry in London, but as already mentioned, Soviet authorities refused his permission to leave Moscow. On 29 January 1949, Parnas was expected to attend the seminar at the Institute. He did not appear, so nervous coworkers decided to visit him at home. As E. Rozenfeld recalled, they found his wife, Renata Parnas sitting in tears at the stairs before the apartment sealed. That day, after midnight, MGB (later known as KGB) officers had entered the apartment and arrested Jakub Karol Parnas, they ordered him to dress up and come with them. His last words to his wife were: ‘‘You were right’’. For months and years Renata tried to find her husband in Moscow prisons of Lubyanka, Lefortovo and Butyrki, each time hearing the same: ‘‘Nothing is still known about such person’’. Her letters to authorities were not answered. The apartment, all belongings and savings were confiscated. Renata got a small room in the collective apartment at a Moscow suburb, making a living from embroidery. Four years later, in summer of 1953 she was invited to the office of the General Prosecutor of the Army in Moscow, where R.A. Rudenko told her that her husband was arrested on accusation of espionage and died the same day in the Lubyanka prison. The testimony of death, registered on 11 July 1953, stated only that ‘‘Jakub Karol Parnas died on the 29th of January 1949, at the age of 65 years, in the city of the republic RSFSR’’ (Russian Soviet Federated Socialist Republic). Renata and his closest associates knew that the charges against him were false. Since January 1949 Jan Oscar Parnas, living in Poland, had not been able to contact his parents. When calling, the operator answered him: ‘‘There is no such number in Moscow’’. His letters were returned. He has been refused passport, and soon he lost the job in the clinic from two reasons: the arrest of his father and his past in the allied Army. Only in 1954, when working as a surgeon in the hospital of Bytom (Silesia, Poland) he received a phone call from Moscow – his mother asked him to come and see her.

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His passport had been returned. At the Moscow airport the mother told him the tragic truth. Renata Parnas returned to Poland in 1958, when the second action of repatriation of Polish citizen was organized. She lived in Warsaw and died in 1967.2 In 1992, Jan asked the General Prosecutor Office in Moscow for more details of the reasons and circumstances of his father’s imprisonment. He received a document stating that J.K. Parnas has been arrested on the accusation of the ‘‘intelligence work against USSR for a foreign Western country’’. After his arrest on 29 January 1949, he had been interrogated from 3.15 am till 5.30 pm. According to accounts after the prosecutor left, Parnas became ill and in spite of the medical help died on heart attack. On 3 April 1954 his case had been discontinued because of the ‘‘lack of evidence of the crime’’. Thus, nobody knows, in fact, when and how Jakub Karol Parnas actually died. From 1949 to 1960, speaking the name Parnas had been forbidden in the USSR. ‘‘No such Academician exists or has existed’’ – a Polish scientist was answered, when asking about Parnas. In 1960, his record was finally cleared of all accusations. A.E. Braunstein, A. Kotelnikowa, S.E. Severin and W.A. Engelhardt edited ‘‘Collected papers’’ of Jakub Karol Parnas in Moscow. In the sixties and seventies, Russian scientific literature and textbooks have cited Parnas according to the Russian style addressed by his father’s name ‘‘Yakub Oskarovitch’’. Despite his Polish nationality, he was consistently portrayed as a Soviet scientist. In the textbook Biochimja, edited by N.N. Jakovlev, Moscow, 1969, he has been described as a Soviet researcher, whose work was important for the development of Russian biochemistry [91]. A similar biographic note has been written for the 1971 edition of ‘‘Who was Who in the USSR’’, Menten, New Jersey [92]. Fortunately, this kind of misinformation currently appears with less frequency. An example of a contemporary view of Parnas from a progressive Russian perspective is a book written by Simon Sznol entitled, Heroes, Gangsters and Conformists, published in Moscow in 2000 (translated into Polish, 2005), in which the author presents a more accurate portrayal of Parnas’ experiences in Moscow [93].

2

´w, a small town in the western Jan Oskar Parnas died in 1995. The hospital in Człucho part of Poland where he was the Head of the Surgery, is now named after him. Jan’s wife, ´w. Jan’s son died young in an accident, but his Barbara Parnas, is still living in Człucho grandson, Tomasz Parnas, is living in Warsaw.

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Such was the tragic end of the brilliant scientific career of a man, who could be awarded a Nobel Prize as many biochemists presume; one of the most prominent biochemists of the first half of the 20th century; an excellent teacher and educator, founder of the Polish school of biochemistry; a noble, good and friendly person. What can be learned from Parnas’ fate? The questioning of freedom of science, freedom of opinions, freedom of mind, restrictions on free interpersonal and international communication eventually leads to tragic consequences. Parnas’ and Collaborators’ Contribution to Discovery of Glycolysis In the 19th century, an idea had been formulated that glucose oxidation is the main source of energy for living organisms. It was reported that glucose oxidation results in lactic acid production in animal muscle and alcohol in yeasts. It had even been calculated that from one molecule of glucose two molecules of lactic acid are generated. Nevertheless, the discovery of glycolysis took place only in thirties of the 20th century (for excellent reviews, see [94,95]). A number of investigators participated in discovery of it, but the main contributors who revealed this pathway are considered to be Meyerhof, Embden and Parnas. As a matter of fact, glycolysis was the first of the metabolic pathways to be unraveled. Investigation revealed six types of the reaction: 1. 2. 3. 4.

5. 6.

Phosphorylation – transfer of phosphate from ATP to sugar or from intermediates to ADP catalyzed by kinases, Isomerization of aldose to ketose and vice-versa catalyzed by hexose or triose isomerase, Mutation – transfer of phosphate from one oxygen atom to another, catalyzed by hexose or triose mutase, Cleavage of carbon bond catalyzed by aldolase as well as the reverse reaction of 3-phosphoglyceraldehyde and phosphodihydroxyaceton aldol condensation, Oxidation of aldehyde catalyzed by dehydrogenase in the presence of NAD, Dehydration of the intermediate, 2-phosphoglyceric acid, catalyzed by enolase.

Below is the list of particular glycolytic reactions and researchers who discovered them.

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Discovery of Reactions of Glycolysis Hexokinase

GLUCOSE +ATP

GLU-6-P +ADP (Meyerhof, 1927) [96]

Hexoseisomerase

GLU-6-P

FRU-6-P (Lohmann, 1933) [97]

Phosphofructokinase

FRU-6-P + ATP

FRU-1,6-P2 +ADP

(Ostern, Guthke, Terszakowec , 1936, Parnas collaborators) [98]

Aldolase

FRU-1,6-P2

GAPDH + DHAP (Meyerhof, Lohmann, Schuster, 1936) [99]

Triose-3-Phosphate Isomerase

GAPDH

DHAP (Meyerhof, Kiessling, 1935) [100]

GAPDH + NAD

+

3-Phosphoglyceraldehyde Dehydrogenase

D-1,3-BPG + NADH

(Warburg, Christian 1939) [101]

1,3-Phosphoglycerate Kinase

1,3-BPG +ADP

3-PG + ATP (Bücher, 1947) [102]

3-PG

3-Phosphoglycerate Mutase

2-PG

(Meyerhof, Kiesling 1935) [103]

2-PG

Enolase

PEP

(Lohmann, Meyerhof, 1934) [104]

Pyruvate Kinase

PEP + ADP

Pyruvate + ATP (Parnas 1934) [46]

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Parnas, during the 5th Congress of Biological Chemistry, Brussels, 1935, presented the report, subsequently published in 1936 [105]. In this report he stated he and his coworkers’ significant discovery in 1934, in which they are the first scientists revealed that ATP might be synthesized by the transfer of phosphate residues from glycolytic intermediates to ADP, and that in one of these reactions simultaneously with ATP pyruvate was formed [46]. Parnas’ data were proved by Lehmann. He showed that in this reaction phosphate was transferred from phosphoenolpyruvate (PEP) [106]. The investigation included the discovery of enzymes catalyzing particular steps of glycolysis as well as intermediates of this pathway (Table 1). TABLE 1 List of abbreviations of intermediates and researchers and data of discovery

Abbreviation

Intermediate

Glu-6-P

Glucose-6-phosphate

Fru-6-P Fru-1,6-P2

Fructose-6-phosphate Fructose-1,6-bisphosphate

GAPD DHAP 1,3-BPG

D-glyceraldehyde 3phosphate Phosphodihydroxyacetone 1,3-biphosphoglyceric acid

3-PG 2-PG

3-phosphoglyceric acid 2-phosphoglyceric acid

PEP

Phosphoenolpyruvic acid

ATP

Adenosine triphosphate Pyruvic acid

Researchers and Data of Discovery Harden and Robison, 1914 [107]; Embden and Zimmermann, 1927 [108]; Robison and King, 1931 [109] Robison, 1932 [110] Harden and Young, 1908 [111]; Embden and Zimmermann, 1924 [112]; Levene and Raymond, 1928 [113] Embden et al., 1933 [114] Embden et al., 1933 [114] Negelein and Bro¨mel, 1939 [115] Embden et al., 1933 [116] Meyerhof and Kiessling, 1935 [103] Meyerhof and Kiessling, 1935 [117] Fiske and Subbarow, 1929 [118] Neubauer and Fromherz, 1911 [119]

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At Jan Kazimierz University in Lviv, Parnas’ laboratory was well equipped in the 1930s. His collaborators were well-educated young people, mostly medical doctors with a great knowledge of chemistry, who could synthesize sugar phosphoric esters, presumed glycolytic intermediates, as well as adenylic acid and adenosine triphosphate and to identify the intermediates in muscle. Adenylic acid produced in Parnas’ laboratory was of the highest quality, probably the best in the world (Lutwak-Mann and Mann, [120]). They had investigated skeletal as well as cardiac muscle mostly from vertebrates like frogs, rabbits, dogs and horse, and, additionally, investigations had been performed on brewer yeasts. It was really advantageous to work on the broad spectrum of subjects. Identification of the same metabolite from different species indicated the universal character of glucose metabolism. Working on muscle metabolism Parnas had initially been mostly interested in ammonia generated during muscle exercise, particularly by wounded muscle [38]. Later on he focused his attention on the process of phosphorylation [62]. Either investigating the fate of ammonia or glycolytic intermediates, Parnas and his team used the thick muscle dispersion that was obtained by mixing freshly ground muscle with an equal volume of water [32]. Ammonia released in working muscle had been determined with Kjeldahl instrument improved by Parnas and Heller [25]. Ammonia was a product of nucleotides not glucose metabolism. Nevertheless, the important finding was that ammonia is released from adenylic acid (AMP) not from adenosinetriphosphoric acid (ATP) [39]. To determine ATP, the barium salt of ATP was precipitated from trichloroacetic acid extract and, in the next, dispersed in sulfuric acid and subjected to enzymatic deamination. The enzymatically released ammonia was subsequently determined. ATP and ADP take part in glycolysis and in this pathway are not further metabolized. In skeletal muscle all three nucleotides ATP, ADP and AMP are in equilibrium owing adenylate kinase catalyzing the following reaction [45]: ATP þ AMP$2ADP

Additionally in maintaining this equilibrium, creatine kinase has a role in catalyzing the transfer of phosphate from phosphocreatine to ADP [46]. It is always of great importance to choose the right subject of investigation. Many times it depends on scientist’s intuition.

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Following Parnas’ work one may conclude that he had a special gift to select a proper subject of investigation. Apparently release of ammonia could not help to reveal glycolysis, but later on it turned out how important was Parnas’ work on adenosine metabolites. The other example of the proper choice of the subject of investigation was Parnas’ and his team’s work on phosphorylation. At present, biological phosphorylation is considered as one of the most abundant and important reactions recognized not only in metabolic pathways but also in a number of physiological phenomena, like signal transduction, or posttranslational protein modification. It was also of great importance to choose a proper tool to investigate phosphorylation. In those times phosphorus could be relatively easily quantitatively determined with the use of the Fiske-Subbarow method [118]. But to follow the fate of phosphate residue in glucose metabolism, it was necessary to employ the radioactive phosphorus. This element had been successfully used by Parnas and his collaborators [61,62,67–69]. They were among the first researchers who employed radioactive phosphorus to investigate phosphorylation and used it in biochemical studies. Collaborating with Hevesy, they had been getting radioactive phosphorus [32P] from Niels Bohr laboratory in Copenhagen. Because of relatively short half-life of this element (only 14 days) it was necessary to send radioactive sodium phosphate by airplane to Parnas’ laboratory, where this salt was used in the synthesis of sugar derivatives, intermediates of glycolysis, which subsequently were used for further experiments. The substrates and products of reactions catalyzed by enzymes had been burned, in all cases the final residue was phosphoric acid. Phosphoric acid was used to obtain the mixed ammonium magnesium phosphate. This salt had been sent back to Copenhagen where its radioactivity was measured. Employing radioactive sugar it was possible to follow the fate of phosphate residue in glycolysis. In 1943, Hevesy obtained the Nobel Prize as the first one who introduced radioactive isotopes to mark biological material, while Parnas’ and his collaborators’ input into this technical advance remained unrecognized. It was always an intriguing question that neither glucose nor pyruvate, the final product of glycolysis, was phosphorylated, but all other intermediates were phosphoric esters. Why to phosphorylate compounds only to dephosphorylate them later on? Initially, the role of ATP was not clear, and nobody knew why at the first

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stage sugars are phosphorylated with the use of ATP, and later on intermediates are dephosphorylated and ATP is recovered. Parnas and his collaborators were really pioneers investigating biological phosphorylation. The first reaction of phosphorylation discovered by Parnas and his collaborators was glycogen breakdown to glucose 6-phosphate (Embden ester) occurring in the presence of inorganic phosphate [57]. This observation was confirmed employing 32P and showing that the radioactive phosphate was quantitatively transferred to glucose-6-phosphate (Hevesy, Baranowski, Guthke, Ostern, Parnas [62]). Further investigation on glycogen had been performed by Cori and Cori who found that breakdown of glycogen results in glucose-1-phosphate formation (Cori ester) [121], which subsequently was mutated into glucose-6-phosphate, one of the glycolytic intermediates (for their discovery Cori and Cori had been awarded with Nobel Prize in 1947). One of the methods used by researchers to investigate glycolysis was employment of inhibitors like iodoacetic acid or fluorides. Iodoactic acid is an inhibitor of 3-phosphoglyceraldehyde dehydrogenase, fluoride, inhibitor of enolase. Employing the inhibitors, Parnas was able to show that ATP has not been generated when iodoacetic acid was present, but it appeared in the presence of fluorides [48]. This enabled Parnas to determine the stages of ATP production. Based on their work on ammonia and phosphorylation in muscle tissue, Parnas, with his collaborators, made a very important observation that production of lactate in muscle is accompanied by ATP synthesis [46,47]. In a paper published in 1934 in Nature (London) entitled ‘‘Chemistry of anaerobic recovery in muscle’’, Parnas and Ostern stated [53]: The three series of major chemical changes which are associated with muscular activity, namely: disintegration and resynthesis of adenosinetriphosphoric acid, disintegration and resynthesis of phosphocreatine, and glycogenolysis, or transformation of glycogen into lactic acid, have been considered, until recently, as independent reactions, but as in some way linked – energetically and chemically. It was known, that the presence of adenosinetriphosphoric acid is the condition of glycolysis in muscle, and that glycolysis is a condition of the resynthesis of adenosinetriphosphoric acid from adenylic acid and phosphates. Recently it has been made clear by Lohmann, that the resynthesis of adenosinetriphosphoric acid from adenylic acid is brought about by splitting phosphate groups from phosphocreatine, at the same time, we have demonstrated the linkage between glycogenolysis and the resynthesis of adenosinetriphosphoric acid, depending probably on the intermediate resynthesis of phosphocreatine, and this is linked to a definite intermediate step of glycogenolysis.

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Thus, the quintessence of glycolysis is the breakdown of glucose to lactic acid, which results in ATP production [46,47]. Since in the thirties, glycolysis has been extensively investigated. This metabolic pathway has been found in almost all living cells and recognized as the only source of energy in anaerobic conditions. Depending on the cell and species, different isozymes of glycolysis have been found; nevertheless, different isozymes catalyze the same reaction. Investigation revealed that glycolysis is precisely regulated. Three proteins have been recognized as regulatory enzymes: hexokinase, 1,6-phosphofructokinase and pyruvate kinase. Hexokinase has been discovered by Meyerhof [96], two other regulatory enzymes of glycolysis have been found by Parnas and his collaborators. Ostern, Guthke and Terszakowec´ [98] discovered phosphofructokinase, whereas in the discovery of pyruvate kinase Ostern, Mann and Parnas themselves took part [46]. Based upon their own discoveries as well as taking into account the reports of other researchers, Parnas proposed in 1938, ¨ ber die in a paper published in Enzymologia [122], entitled ‘‘U ¨rung enzymatischen Phosphorylierungen in der alkoholischen Ga und in der Muskelglykogenolyse’’ the pathway of glycolysis is commonly accepted by biochemists [122]. As a result of that, Parnas has been considered among Embden and Meyerhof as one of the main contributors in glycolysis discovery. In Biochemists’ Songbook [123], published by Pergamon Press in 1982, Prof. Harold Baum, from the Chelsea College University of London, presented a song entitled: ‘‘In praised of E.M.P’’. One of the phrases of this song was: Of all nature’s pathways, We sing the praise today Of Parnas, Embden, Meyerhof – The glycolytic way.

The Parnas’ School of Biochemistry Parnas educated a large number of biochemists. His scientific imagination and brilliant personality attracted a group of gifted students and scholars who gathered around him and joined his staff (Fig. 5). Most of them became very well-known biochemists. Among them were Paweł Ostern, Tadeusz (Thaddeus) Mann and

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Fig. 5. Staff of Physiological Chemistry Department, Jan Kazimierz University, Lviv, Poland, 1929. Standing from left: J. Nuckowski, J. Jaworska, T. de Tesseyre, W. Chrza ˛szczewski, W. Lewin´ski, P. Ostern, C. Lutwak-Mann, technician. Sitting from left: W. Mozołowski, A. Klisiecki, J.K. Parnas, J. Heller, U. Mroczkiewicz, and J. Sieniawski, T. Mann, K. Wajda.

his wife Cecylia (Cecilia) Lutwak-Mann, Tadeusz Baranowski, Irena Mochnacka, Wanda Mejbaum-Katzenelenbogen, Leszek ´zef Heller, Włodzimierz Mozołowski, Janina Tomaszewski, Jo ´ ska-Blauth, Tadeusz Korzybski, Bohdan Sobczuk and Jurij Opien Terszakowec´. Some others became well-known physicians as Bogusław Halikowski, pediatrician; Andrzej Klisiecki, physiologist; Tadeusz Krwawicz and Julian L. Reis, ophthalmologists; Włodzimierz Antyporowicz, laryngologist; Stanisław Hubl, surgeon. After the Second World War, nearly all of those who had survived the repressive regimes of Hitler and Stalin returned to Poland, with the exception of Tadeusz and Cecylia Mann and Julian L. Reis who stayed in Cambridge and London, respectively. Among Ukrainians, Jurij Terszakowec´ moved to USA and Bohdan Sobczuk remained in Lviv, which since the Second World War belonged to the Soviet Union. Sobczuk, being a Parnas’ successor, headed the Medical Chemistry Department at the Lviv National Medical Institute. One of the most talented Parnas’ scholars was Paweł Ostern who died tragically in 1941. In 1944, Parnas wrote an obituary

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devoted to him, published in Nature [124]. He wrote, ‘‘Dr. Paul Ostern was killed by the Nazis in Lvov at the beginning of July 1941, during a pogrom in which several men of science, scholars, physicians and others died’’. However, this was only a half of the truth. The truth was even more tragic. Ostern, knowing what would happen, in the face of implacable fate, decided to commit a suicide, taking a poison. Indeed, in the night from 3 to 4 July 1941, the Nazis detained 23 professors and executed them the very same night, often together with their relatives. Among them were 13 medical doctors, as well as a well-known writer and poet, ˙ elen ´ ski-Boy, who was then employed as a professor Tadeusz Z at the Lviv University. At this moment, the crime was directed against Polish intellectuals, not yet specifically against the Jewish community. Similar action took place in Cracow (‘‘Sonderkation Krakau’’), where on the 6 November 1939, professors of the Jagiellonian University were detained and interned in Sachsenhausen and Dachau concentration camps by the Nazis. For more, Ostern, who was a Polish scientist, was also of Jewish origin, so for him the danger was double. Paweł Ostern was born in 1902, studied biochemistry in Lviv and joined Parnas’ staff in 1927. Parnas, in his obituary [124], wrote: ‘‘Although young he made brilliant contribution to biochemistry’’ y and further: ‘‘Ostern spent some time abroad, where his work with Krebs in Freiburg was interrupted by the Nazi seizure of power and Krebs leaving the country; with Verzar in Basel and then with Krebs again, in Cambridge. In Lvov he participated in the team work, with myself and Dr. T. Mann, now in Cambridge, which led to the discovery of direct enzymatic transfer of the phosphate group from phosphoglyceric to adenylic acid, with the formation of adenosintriphosphoric acid, and to the chart of the linkage of chemical transformation in glycogenolysis, as now generally accepted. With T. Baranowski and J. Reis (now in the British Eighth Army), Ostern discovered (1935) the direct transfer of phosphate from adenosintriphosphoric acid to creatine, and the role of the phosphocreatine-creatine system as an alternating acceptor and donor of phosphate’’ y ‘‘In 1937 he made his brilliant discovery of enzymatic synthesis of adenylic acid from adenosin and phosphate’’ y ‘‘His last important discovery was made with E. Holmes and D. Herbert, in 1939, during a short stay in Cambridge- that glucose is formed in liver by way of phosphorolysis of glycogen and subsequent hydrolysis of the phosphoric ester. The formation of glycogen from the Cori ester by liver enzymes was then published by these workers, simultaneously with the St. Louis group y’’.

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Paweł Ostern was not only fully respected by Parnas as the scientist; he was also his favorite pupil. There was a special kind of communication between them. Ostern was the only one who was able to read easily Prof. Parnas’ handwriting. According to Parnas’ son, Jan Oskar, the mother often called Ostern when the professor was out of Lviv and sent a postcard to home, saying: ‘‘Pawełku, please come to us and read what Kubus´ wrote’’ (in Polish: Pawełek, a nick name of Paweł; Kubus´, a nick name of Jakub). Prof. Parnas finished his obituary with the words: ‘‘Unfortunately, he did not leave the city when the Germans approached’’. This fate was spared for the Parnas’ family who left Lviv on 27 June 1941, and Tadeusz Mann, who together with wife, left Lviv in 1935, being invited to Cambridge (England) as a scholar of the Rockefeller Foundation. Tadeusz (Thaddeus) Mann (1908–1993) studied medicine at the Jan Kazimierz University in Lviv and already as a student started to work with Parnas. In 1935, he got PhD degree. His PhD thesis concerned the origin of ammonia in skeletal muscle. The same year he left Lviv to Cambridge, where for nearly a decade he cooperated with David Keilin in the ‘‘Molteno’’ Institute. At the end of the Second World War, the British Government invited him to start investigating on biochemistry of semen of domestic animals. Shortly, he became a creator of this field of biochemistry and became professor of physiology of reproduction at the Cambridge University. He published about 300 research articles and among them books Biochemistry of Semen (1954), Male Reproductive Function of Semen (together with his wife, 1981) and Spermatophores – Development, Structure, Biochemical Attributes and Role in Transfer of Spermatozoa (1984). Tadeusz Mann and Cecylia Mann had a strong relation with Poland and warmly recollected the time spent in Parnas’ laboratory. In 1955, when only the sad news of Parnas’ death reached England, Tadeusz Mann wrote an obituary devoted to his professor [125] and in 1981 together with his wife he published an article ‘‘50 years ago – The Parnas School’’ [120]. Mann was always very proud that Parnas invited him to write a few chapters to the second edition of Parnas’ two-volume textbook Physiological Chemistry, which was published in 1937 in Warsaw [24]. Another important scientist belonging to the Parnas’ school was Tadeusz Baranowski (1910–1993). After graduation at the Medical Faculty of the Jan Kazimierz University in Lviv, he joined

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the Parnas’ group in 1934. In the Parnas’ laboratory he studied the phosphate group transfer and glycolytic enzymes. When the Soviet Army entered Lviv, the Faculty, and later on, the Medical Institute, continued their function, despite the difficult situation and the shortage of chemicals. Tadeusz Barnowski was that time employed as an assistant professor and had the second position in this group. In 1940, Parnas and Barnowski were invited to visit Kiev and Moscow where Soviet scientists were particularly interested in the crystallization of some muscle proteins, the work done by Baranowski. As he had proven later on, muscle myogen A crystallized in Parnas’ laboratory had, in fact, aldolase activity. The investigation was stopped when the Nazis entered Lviv. During German occupation, all such scientific works were brought to an abrupt halt. When the Second World War was finished, Tadeusz Baranowski arrived in 1945 in Wrocław. He actively participated in the organization of the Medical Faculty at the University in the completely destroyed town. Two colleagues from the laboratory of Parnas, Irena Mochnacka and Wanda Mejbaum-Katzenellenbogen supported him. Wanda Dobryszycka, Janina KwiatkowskaKorczak and Elwira Lisowska, all of the pupils and coworkers of Baranowski, wrote in the article ‘‘Post-war biochemistry in Wrocław’’ [126]: ‘‘How different everything was in that time, no funds, no plans and reports, only some basic reagents and a lot of enthusiasm and a strong will to overcome all obstacles.’’ y ‘‘When a home-made fraction collector stopped working during the several-day experiment, the fractions were manually collected day and night. When the synthesis of glutathione and its analogs was planned, only glycine was available. Therefore, glutamic acid was obtained from wheat flour bought in a food shop, and cysteine was prepared from hair collected by helpful hairdressers’’. Such problems were common to all biochemical laboratories in Poland at those times. Despite such basic problems, Tadeusz Baranowski immediately organized classes for medical students and laboratory research in Wrocław. Glycolytic enzymes became the main subject of his research. The achievements of the team headed by Baranowski included isolation, crystallization and characterization of muscle and erythrocyte glycolytic enzymes, many of them obtained for the first time from human tissues. Kinetic and regulatory properties of phosphofructokinase, aldolase, triosephosphate isomerase,

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glyceraldehyde dehydrogenase, enolase and pyruvate kinase were described. The crystallization of muscle phosphoglycerol dehydrogenase (known as ‘‘Baranowski’s enzyme’’) was performed during his stay in Cori’s laboratory [127]. Tadeusz Baranowski took from Parnas’ laboratory not only the scientific problems but also the specific style of the work. His great merit was the education of young biochemists. One of the authors of the current work, Andrzej Dzugaj,3 was his pupil. As already mentioned, Irena Mochnacka and Wanda Mejbaum-Katzenellenbogen helped Tadeusz Baranowski in the reconstruction and organization of the scientific life in Wrocław. Both of them joined the Parnas’ staff during their studies at the Medical Faculty of Jan Kazimierz University in Lviv. Wanda Mejbaum-Katzenellenbogen (1914–1986) received her doctor’s degree in 1939 and in the same year published the results of her investigations on ribose determination done in the Parnas’ laboratory [128]. This method, used for RNA quantitative determination, was the most popular in the fifties and sixties of the last century. Current Contents distinguished this article in 1970 and 1986 (ISI Press) for ‘‘the post-war record breaking number of citation’’. In Wrocław, she was the Head of the Department of Biochemistry at the University. Irena Mochnacka (1905–1979) in the early fifties moved from Wrocław to Warsaw, where she headed the Department of Biochemistry at the Warsaw Medical University. The education of young students was her passion. Leszek Tomaszewski (1919–1997) joined Parnas’ staff in 1938 as a 19-year-old medical student. Inexperienced young men, such as he, were called ‘‘lambs’’ in the laboratory vernacular which means that they could quietly graze on green grasses of the Parnas’ lab and help in simple laboratory work. The war stopped this happy period. Tomaszewski, after the Second World War, similarly as Irena Mochnacka, organized scientific life at the Warsaw Medical University. At the end of the twenties, Prof. Parnas had among his staff two ´ zef Heller and Włodzimierz Mozołowski. senior biochemists: Jo 3

Andrzej Dzugaj was the PhD student of Prof. Baranowski and prepared his PhD dissertation under his supervision. Dzugaj’s study on muscle glycolytic enzymes is a continuation of Baranowski’s and Parnas’ research. Owing to that he used to say that he is a Parnas’ ‘‘grandson’’ and his coworkers and PhD students are ‘‘great-grandsons’’ of Parnas.

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Both of them were engaged in research on several aspects of muscle biochemistry, mainly concerned with the process of ammonio´zef Heller (1896–1982) moved to the genesis. In the thirties, Jo Hygiene Department at the Lviv University, where he continued his research in Parnas’ laboratory, his life long investigation on insect physiology and biochemistry. After the Second World War he was a founder of the Institute of Biochemistry and Biophysics of the Polish Academy of Sciences in Warsaw, which subsequently became very important for the development of scientific life in Poland. Włodzimierz Mozołowski (1895–1975) was a close coworker of Parnas. Their collaboration started in 1922 when Mozołowski finished the medical study at the Lviv University. In the Parnas’ group, his main subject of research was ammonia production in muscle and in blood. After the Second World War, Mozołowski ´ sk, organized the scientific life in destroyed and burned Gdan teaching and doing research. Studies on adenylic acid and adenine deaminases carried out at the Department of Biochemistry of the ´ sk were a continuation of the nucleoMedical University in Gdan tide metabolism investigation performed in Parnas’ laboratory. Mozołowski and his coworkers, step by step, created a strong ´ sk. biochemical center in Gdan ´ ska-Blauth (1895–1987) came to Lviv in 1939 Janina Opien and collaborated with Parnas until his departure in 1941. She was one of many refugees from Central Poland eagerly accepted into his staff. As early as in September 1944, before the end of the war, and in cooperation with other Polish scientists, she started to organize the University of Lublin. Full of enthusiasm, from 1944 she had been the Head of the Department of Physiological Chemistry at this newly formed university. Recalling that Poland had a big Ukrainian minority before the Second World War, Bohdan Sobczuk (1910–1974) and Jurij ´ (1914–1987) were its representatives. As students Terszakowec of the Medical Faculty at the Jan Kazimierz University, they joined the Parnas’ team. Sobczuk worked directly with Parnas [59], whereas Terszakowec´ belonged to the small research group directed by Ostern [98]. The Terszakowec´’ family was involved in the movement for free Ukraine, therefore in 1944 they left Lviv and moved to the United States where Terszakowec´ (George Tershakovec) became a professor of biochemistry at the University

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of Miami School of Medicine in Florida. He was a distant relative of one of the authors of the current chapter, Andrzej Dzugaj, whose Polish family living in Lviv before the Second World War had also mixed Ukrainian ancestry. In those days, families with different roots were quite common among the progressive multinational inhabitants of this city. In contrast to Terszakowec´, Sobczuk stayed in Lviv, a part of the Ukrainian Soviet Socialistic Republic till 1991. In 1944, Bohdan Sobczuk was appointed the Head of the Department of Biochemistry at the State Medical Institute, as a successor of Parnas. One of the authors of the current chapter, Janina Kwiatkowska-Korczak,4 worked in the department under supervision of Sobczuk. Here are her personal recollections: ‘‘I started to work at the Department of Biochemistry in 1953. At that time even the name of Parnas was formally forbidden. But not in our laboratory – professor Sobczuk often told us about his teacher with admiration, cited his publications, introduced the laboratory habits of old times, even in details. Textbook and papers of Parnas were to be confiscated from the Department’s library, but our boss hide them at his home. Prof. Sobczuk created a special atmosphere at the Department and inspired his young coworkers, often invoking the authority of Jakub Parnas. He insisted on knowledge of contemporary scientific literature, what was not easy, since it needed a special permission of the Rector. Nevertheless, he got it for us. He made a great effort to continue the tradition of Parnas’ laboratory, despite of very hard times and many difficulties, also personal’’. Another important member of the Parnas’ school was Tadeusz Korzybski (1906–2002). In Parnas’ laboratory he dealt with the nucleotide turnover and ATP and ITP formation. Together with Parnas they were the first who used radioactive phosphorus 32P to biochemical studies and demonstrated the turnover of phosphorus compounds in intact muscle. Their paper, published in 1939 [68], was translated from French into English by H.M. Kalckar and published again in the full length in 1969 in Kalckars’s book 4

Janina Kwiatkowska-Korczak prepared her PhD thesis under Sobczuk supervision and got PhD title in Lviv, Ukraine. When in 1959 she and her family was allowed to return to Poland, she has been accepted and started the work in Baranowski’s group in Wrocław, moving from one laboratory of Parnas’ student to another one. Prof. KwiatkowskaKorczak after Baranowski death became, as his successor, the Head of the Department of Medical Biochemistry at the Medical University in Wrocław, Poland. Now, she is Professor emeritus at the department.

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entitled Biological Phosphorylations – Development of Concepts [129]. Tadeusz Korzybski spent the Second World War in Lviv and after correction of boundaries he moved to Poland in 1945. In 1946, he went to Toronto, where the technology of penicillin production was bestowed to him as a gift of UNRRA to Poland. For this reason, he is called ‘‘the father of Polish penicillin’’. All his life he preserved the remembrance of professor Parnas, and kept old letters, photographs and documents concerning his professor. He very often motivated the Polish Biochemical Society to honor Jakub Karol Parnas’ memory through a variety of actions and recommendations. When writing about the Parnas’ school of biochemistry and his influence on people, it is necessary to mention Prof. Zofia ´ ska (1915). Because she obtained her training at the Zielin Warsaw University and after the Second World War worked at the University of Ło´dz´ and later at the Nencki Institute of Experimental Biology in Warsaw, she was never able to work directly with Parnas. She has, however, been fascinated by his research and personality. She has made a deep friendship with Parnas’ son, Jan Oskar, and his family. As long time Editor-in-Chief of Advances in Biochemistry (‘‘Post˛epy Biochemii’’, edited in Polish), she made a huge effort to popularize the figure of Jakub Karol Parnas to ´ ska, among others, the Polish biochemical community. Zofia Zielin ‘‘infected’’ one of the authors of the current work, Jolanta ´ ska,5 with this same fascination. Baran Despite the fact that professor Parnas has left us half a century ago and all his pupils have also passed away, his influence on Polish biochemistry still exists. As described in the aforementioned biographies, Parnas’ pupils after the Second World War were very active in the organization of the scientific life in devastated Poland. Working at universities and research institutes throughout Poland, they educated young generations of students with great passion. Parnas’ ideas remain a tremendous source of inspiration, instructing our studies, informing our methodology and guiding us to effectively mentor young people, who will take our place one day.

5

´ ska has received PhD title at the Nencki Institute of Experimental Biology, Jolanta Baran Warsaw, Poland. During 1998–2005 she was the President of the Polish Biochemical Society and in 2006, was appointed the Chairperson of the Federation of European Biochemical Societies (FEBS).

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The Polish Biochemical Society and Its Activities Undertaken to Honor Jakub Karol Parnas The Polish Biochemical Society from the time of its foundation in 1958 undertook numerous forms of activity to commemorate Prof. Jakub Karol Parnas. Thus, since 1963, the Society has awarded every year the best biochemical research carried out in a Polish laboratory and published in one of the best international journals. This highest and most prestigious award has been named ‘‘Parnas Award’’. In 1985, in commemoration of 100 years of Prof. Jakub Karol Parnas birth, the Polish Biochemical Society organized a Special Session dedicated to Parnas during the 21st Annual Meeting held in Cracow. In this Session, Prof. Wanda MejbaumKatzenellenbogen had a lecture entitled ‘‘Recollection of Professor Parnas’’ and Prof. Włodzimierz S. Ostrowski, ‘‘Jakub Parnas – The founder of the Polish school of biochemistry’’. Jan Oskar Parnas also took part in this celebration, recollecting his father. In 1958, 1986, 1992 and 1997, special issues of the Society journal Advances in Biochemistry (‘‘Post˛epy Biochemii’’) were devoted to Parnas [130–133]. The role of Parnas’ coworkers in all of these initiatives was essential. Contacts between Polish and Ukrainian Biochemical Societies were sporadic. These personal, direct contacts began more intensive when the political systems in both countries became transformed. Owing to that, in 1996, it became possible to organize in Lviv the 1st Polish–Ukrainian Conference to honor Jakub Karol Parnas. During this Conference, not only the lectures and recollections were held but also a plaque commemorating Jakub Karol Parnas was placed on the wall of the building where the Department of Medical Chemistry headed by Parnas was located. The Conference was a great success. Attendees passed a resolution to organize the Parnas Conference every two years, rotating between Poland and Ukraine. According to this resolution, the 2nd ´ sk (Poland), the 3nd Parnas Conference was held in 1998 in Gdan in 2000, again in Lviv (Ukraine), the 4th in 2002 in Wrocław (Poland) and the 5th in Kiev (Ukraine) in 2005. The next Parnas Conference will be held in 2007 in Cracow (Poland). The leading topic of all of these Conferences concerns molecular mechanisms of cellular signalling. 2005 Parnas Conference, organized by Prof. Sergiy Komisarenko and Prof. Jolanta

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´ ska (Presidents of Ukrainian and Polish Biochemical Baran Societies, respectively) gathered 264 researchers. Among them were representatives of Ukraine and Poland and also of Belarus, Belgium, France, Germany, Iran, Italy, Kazakhstan, Russia, Sweden, United Kingdom and United States. This provides some indication of the increasing popularity that the Parnas Conference has among the international scientific community. At the end of the chapter, we would like to remember the words of Prof. Rostislav Stoika [133], Co-Chairman from the Ukrainian side of the 1st Parnas Conference. During the Opening Ceremony he said: ‘‘As we know, relations between Poland and Ukraine varied throughout history, with periods of close cooperation alternated with times of hostility. During the period when Ukraine was part of a totalitarian communist state, it was the antagonism which was emphasized. Now, however, we can acknowledge that our two countries are joined by hundreds, if not thousands, of various links. The life of Professor Parnas is one of those threads, which bond our histories and our nations’’. These words are full of optimism. With such optimistic accent we wish to finish this story. ACKNOWLEDGMENTS

The authors are greatly indebted to their Polish colleagues, Stefan ´ ski, Jacek Kuz´nicki, Angielski, Tadeusz Chojnacki, Jerzy Duszyn Andrzej Morawiecki, Włodzimierz S. Ostrowski, Paweł Pomorski, Lech Wojtczak and Włodzimierz Zago´rski-Ostoja for their critical reading of the manuscript. We would also like to express our gratitude to Maria Korzybska (Poland), Rostislav Stoika, Ivan Holovatsky (Ukraine) and Alexander Wlodawer (USA) for their help in obtaining biographical data. Finally, we would especially ´ ska for her enormous assistance and sharing thank Zofia Zielin historical materials with us from her private archive. REFERENCES

Note: Parnas has published 130 scientifc papers and 46 reviews and textbooks. Twenty of them may be found in D.M. Needham (1971), Machina Carnis, p. 706, Cambridge. A complete list of

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Parnas’ and his coworkers’ papers (1907–1949) has been published in Post. Biochem. (Advances in Biochemistry) (1986) 32, 265–285. ¨ ber 2,6-Naphthochinon. Ber. Chem. ¨tter, R. and Parnas, J. (1907) U [1] Willsta Ges. 40, 1406–1415. ¨ ber amphi-Naphthochinone. II. ¨tter, R. and Parnas, J. (1907) U [2] Willsta Ber. Chem. Ges. 40, 3971–3978. + + [3] Parnas, J. (1908) Uber Naphtochinone. Dissertation. Munchen, pp. 9–95. + [4] Parnas, J. (1910) Uber fermentative Beschleunigung der Cannizaroschen ¨fte. I. Biochem. Z. 28, 274–294. Aldehydumlagerung durch Gewebssa [5] Parnas, J., (1909) Energetic glatter Muskeln. VIII Intern. Physiol. Kongress, Wien, 27–30, September. + [6] Parnas, J. (1910) Energetic glatter Muskeln. Pflugers Archiv. Ges. Physiol. 134, 441–495. + ¨uren im [7] Parnas, J. (1912) Uber das Schicksal der stereoismeren. Milchsa Organismus des normalen Kaninchens. Biochem. Z. 38, 53–64. + [8] Parnas, J. (1912) Uber Bildung von Glykogen aus Glycerinaldehyd in der Leber. Zbl. Physiol. 26, 671–672. + [9] Parnas, J. and Baer, J. (1912) Uber Zuckerabbau und Zuckeraufbau im Tierischen Organismus. Biochem. Z. 41, 386–418. + [10] Parnas, J. and Wagner, R. (1914) Uber den Kohlenhydratumsatz isolierter Amphibienmusklen und u ¨ ber die Beziehungen zwischen Kohlenhydratsch¨urebildung im Muskel. I. Biochem. Z. 61, 387–427. wund und Milchsa + [11] Parnas, J.K. (1915) Uber das Wesen der Muskelerholung. Zbl. Physiol. 30, 1–18. [12] Parnas, J.K. and Laska-Mintz, E. (1921) Beeinflussen subminimale Reize den Ablauf chemischer Umsetzungen in isolierten Muskel? Biochem. Z. 116, 59–70. + [13] Parnas, J.K. (1921) Uber den Kohlenhydratstoffwechsel der isolierten Amphibienmuskeln. II. Biochem. Z. 116, 71–88. + [14] Parnas, J.K. (1921) Uber den mechanischen Wirkungsgrad der in isolierten Amphibienmuskeln stattfindenden Verbrennungsprozesse. Biochem. Z. 116, 102–107. + ´ ska, Z. (1921) Uber [15] Parnas, J.K. and Krasin den Stoffwechsel der Amphibienlarven. Biochem. Z. 116, 108–137. + [16] Parnas, J.K. (1921) Uber den Kohlenhydratstoffwechsel der isolierten Amphibienmuskeln. III. Der Umsatz in Muskeln, pankreasdiabetischer. Tiere. Biochem. Z. 116, 89–101. + ¨rung des [17] Wagner, R. and Parnas, J.K. (1921) Uber die eigenartige Sto Kohlenhydratstoffwechsels und ihre Beziehungen zum Diabetes mellitus. II. Eine klinish-experimentelle Studie. Z. Gesamte. Exp. Med. 25, 361–384. [18] Parnas, J.K. and Wagner, R. (1922) Beobachtungen u ¨ ber Zuckerneubildung. I. Nach Versuchen, die an einem Falle besonderer Kohlenhydratstoffwechselstoru ¨ ng angestellt wurden. Biochem. Z. 127, 55–65. + + [19] Parnas, J.K., Rosenbluth, A. and Wagner, R. (1923) Uber den Einfluss der Kohlenhydrate auf den Grundumsatz. Nach Versuchen, die an einem Falle besonderer Kohlenhydratstoffwechselstoru ¨ ng angestellt wurden. IV. Z. Gesamte Exp. Med. 38, 445–457. [20] Wagner, R. and Parnas, J.K. (1922) Zur Korrelation der Blutdru ¨ sen. Med. Klin. 18, 135–138.

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´w krwi ´ ski, W. (1922) Rozmieszczenie składniko [21] Parnas, J.K. and Jasin niekoloidowych pomi˛edzy osocze i krwinki na podstawie analiz krwi rodzimej. Spraw. Tow. Nauk. Lwo´w 2, 163–164. + ´ ski, W. (1922) Uber [22] Parnas, J.K. and Jasin die Verteilung von Zucker, Reststickstoff und Calcium im Blute. Klin. Wochenschr. 1, 2029–2030. [23] Parnas, J.K. (1922) Chemia fizjologiczna ze szczego´lnym uwzgl˛ednieniem fizjologii zwierz˛ecej (textbook), pp. XIII–559. Warszawa-Lwo´w, Wende and Altenberg. [24] Parnas, J.K. (1937) Chemia Fizjologiczna (textbook), Vol. I, pp. XLIV–575; Vol. II, pp. XXVII–682. Warszawa, Delta. + [25] Parnas, J.K. and Heller, J. (1924) Uber den Ammoniakgehalt und u ¨ ber die Ammoniakbildung im Blute. I. Biochem. Z. 152, 1–28. + [26] Parnas, J.K. (1925) Uber den Ammoniakgehalt und u ¨ ber die Ammoniakbildung im Blute. II. Biochem. Z. 155, 247–255. + [27] Parnas, J.K. and Taubenhaus, M. (1925) Uber den Ammoniakgehalt und die Ammoniakbildung im Blute. III. Die Entstehung des Bluttammoniaks. Biochem. Z. 159, 298–310. + [28] Parnas, J.K. and Klisiecki, A. (1926) Uber den Ammoniakgehalt und die Ammoniakbildung im Blute IV. Ist im kreisenden Blute Ammoniak vorhanden? Biochem. Z. 169, 255–265. [29] Parnas, J.K. (1926) O amoniaku krwi, jego pochodzeniu i warunkach ´w. 6, 156–158. powstawania. Spraw. Tow. Nauk. Lwo [30] Parnas, J.K. (1927) Existe-t-il des sels ammoniacaux dans le sang circulant? Bull. Soc. Chim. Biol. 9, 76–90. [31] Parnas, J.K. (1926) On ammonia in blood, its formation and its physiological behaviour. XII-th International Physiological Congress held at Stockholm. Skandinav. Archiv. 49, 199–200. + [32] Parnas, J.K. and Mozołowski, W. (1927) Uber den Ammoniakgehalt und die Ammoniakbildung im Muskel und deren Zusammenhang mit Funktion ¨nderung. I. Biochem. Z. 184, 399–441. und Zustandsa ´ ski, W. (1927) Powstawanie [33] Parnas, J.K., Mozołowski, W. and Lewin amoniaku w mi˛e´sniach a praca. Spraw. Tow. Nauk. Lwo´w. 7, 167–168. ´ ski, W. (1927) Praca mi˛e´sniowa i [34] Parnas, J.K., Mozołowski, W. and Lewin amoniak krwi. Spraw. Tow. Nauk. Lwo´w. 7, 168. + ´ ski, W. (1927) Uber [35] Parnas, J.K., Mozołowski, W. and Lewin die Ammoniakbildung im isolierten Muskel und ihren Zusammenhang mit der Muskelarbeit. Klin. Wochenschr. 6, 1710–1711. + ´ ski, W. (1927) Uber [36] Parnas, J.K., Mozołowski, W. and Lewin den Ammoniakgehalt und Ammoniakbildung im Blute. IX. Der Zusammenhang des + Blutammoniaks mit der Muskelarbeit. III. Uber die Ammoniakbildung im ¨nderung. Muskel und deren Zusammenhang mit Funktion und Zustandsa Biochem. Z. 188, 15–23. [37] Parnas, J.K. (1928) Badania nad powstawaniem amoniaku i zalez˙nos´cia ˛ tej sprawy od czynnos´ci i stanu mi˛e´sni. Acta Biol. Exp. 1, 1–83. + [38] Parnas, J.K. and Mozołowski, W. (1927) Uber die Ammoniakbildung im ¨ ¨nderung. Muskel und ihren Zusammenhang mit Tatigkeit und Zustandsa Klin. Wochenschr. 6, 998–999. + [39] Parnas, J.K. (1929) Uber die Ammoniakbildung im Muskel und ihren ¨nderung. VI. Der ZusammenZusammenhang mit Function und Zustandsa hang der Ammoniakbildung mit der Umwandlung des Adeninnucleotids zu ¨ure. Biochem. Z. 206, 16–38. Inosinsa

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´ ski, W. and Mozołowski, W. (1928) O [40] Parnas, J.K., Jaworska, J., Lewin sprz˛ez˙eniu dezaminacji beztlenowych z tlenowymi i o przypuszczalnej funkcji chemicznej zasad aminopurynowych zawartych w kwasach nukleinowych. Spraw. Tow. Nauk. Lwo´w. 8, 212–215. ¨ ber den Purinstoffwechsel des Muskels und u [41] Parnas, J.K. (1928) U ¨ ber die Muttersubstanz des im Muskel entstehenden Ammoniaks. I. Klin. Wochenschr. 7, 1423–1424. ¨ ber den Purinstoffwechsel des Muskels und u [42] Parnas, J.K. (1928) U ¨ ber die Muttersubstanz des im Muskel entstehenden Ammoniaks II. Klin. Wochenschr. 7, 2011–2012. [43] Parnas, J.K. (1929) Ammonia formation in muscle and its source. Am. J. Physiol. 90, 467. + ´ ski, W., Jaworska, J. and Umschweif, B. (1930) Uber [44] Parnas, J.K., Lewin den Ammoniakgehalt und die Ammoniakbildung im Froschmuskel. VII. Biochem. Z. 228, 366–400. ¨ ber die Auswertung von Aden[45] Ostern, P. and Parnas, J.K. (1932) U osinderivaten am u ¨ berlebenden Froschherz. Biochem. Z. 248, 389–397. ¨ ber die Verkettung der [46] Parnas, J.K., Ostern, P. and Mann, T. (1934) U ¨nge im Muskel. Biochem. Z. 272, 64–70. chemischen Vorga [47] Parnas, J.K., Ostern, P. and Mann, T. (1969) Coupling of chemical processes in muscle. In Biological Phosphorylations – Development of Concepts (Kalckar, H.M., ed.), pp. 73–79. Prentice Hall, Englewood Cliffs, NJ. ¨ ber die Verkettung der [48] Parnas, J.K., Ostern, P. and Mann, T. (1935) U ¨nge im Muskel. II. Biochem. Z. 275, 74–86. chemischen Vorga ¨ ber die Verkettung der [49] Parnas, J.K., Ostern, P. and Mann, T. (1935) U ¨nge im Muskel. III. Die Phosphatu chemischen Vorga ¨ bertragung durch ¨ure. Biochem. Z. 275, 163–166. Brenztraubensa + ´ ski, W. (1935) Uber [50] Parnas, J.K. and Lewin den Ammoniakgehalt und die + Ammoniakbildung im Muskel. XXII. Uber den Zusammenhang zwischen ¨tigkeit unter aeroben Bedingungen. Ammoniakbildung und Muskelta Biochem. Z. 276, 398–407. + [51] Parnas, J.K. and Lutwak-Mann, C. (1935) Uber den Ammoniakgehalt und + Ammoniakbildung im Muskel. XXII. 1. Uber die Methode zur Best+ ¨ure. 2. Uber immung der Adenosintriphophorsa die zweite ammoniakbildende Substanz des Muskelgewebes. Biochem. Z. 278, 11–12. ¨ ber die Verkettung der chemischen [52] Parnas, J.K. and Ostern, P. (1935) U ¨nge im Muskel. IX. Die Rolle der Phosphagene. Biochem. Z. 279, Vorga 94–98. [53] Parnas, J.K. and Ostern, P. (1934) Chemistry of anaerobic recovery in muscle. Nature (London) 134, 627. [54] Parnas, J.K., Ostern, P. and Mann, T. (1934) Linkage of chemical changes in muscle. Nature (London) 134, 1007. [55] Parnas, J.K. (1935) El encadenaniente de los procesos quinicos en el musculo. Res. Circulo Med. Argent 35, 410: 819–843. [56] Parnas, J.K. and Baranowski, T. (1935) Reakcja pocza ˛tkowa glikogenolizy mi˛e´sniowej. Spraw. Tow. Nauk. Lwo´w. 15, 216–217. [57] Parnas, J.K. and Baranowski, T. (1935) Sur les phosphorylations initiales du glycogene. C. R. Soc. Biol. 120, 307–310. [58] Parnas, J.K. and Ostern, P. (1936) Le me´chanisme de la glycoge´nolyse. Bull. Soc. Chim. Biol. 18, 1471–1492.

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[59] Parnas, J.K., Mejbaum, W. and Sobczuk, B. (1936) Le me´chanisme de l’action de la phlorhizine sur la glycoge´nolyse musculaire. C. R. Soc. Chim. Biol. 122, 1148–1152. [60] Parnas, J.K. (1937) Der Mechanismus der Glykogenolyse im Muskel. Ergebn. Enzymf. 6, 57–110. [61] Hevesy, G., Baranowski, T., Guthke, J.A., Ostern, P. and Parnas, J.K. (1938) Badania nad glikoliza ˛. Nowa metoda z zastosowaniem fosforu promieniotwo´rczego. Spraw. Tow. Nauk. Lwo´w. 18, 88–95. [62] Hevesy, G., Baranowski, T., Guthke, J.A., Ostern, P. and Parnas, J.K. (1938) Untersuchungen u ¨ ber die Phosphoru ¨ bertragungen in der Glykolyse und Glycogenolyse. Acta Biol. Exp. 12, 34–39. [63] Parnas, J.K. (1938) O mechanizme myshechnego glikogenoliza. Fiziol. Zh. SSSR 24, 277–293. [64] Parnas, J.K. (1939) L’application des isotopes radioactives pour l’exploration des e´changes et des transformations biochimiques. Bull. Soc. Chim. Biol. 21, 1059–1093. [65] Parnas, J.K. (1940) Glycogenolyse. In Handbuch der Enzymologie (Nord, F.F. and Weidenhagen, R., eds.), pp. 902–967. New York/Berlin, Akad. Verlagsg, Leipzig. [66] Parnas, J.K. (1940) Primenenie radioaktiwnykh izotopow dlya issledowaniya obmena weschestw. Fiziol. Zh. SSSR 28, 571. ¨ ber Abbau und Wiederaufbau der [67] Korzybski, T. and Parnas, J.K. (1938) U ¨ure im Warmblu Adenylsa ¨ termuskel. Z. Physiol. Chem. 255, 195–204. [68] Korzybski, T. and Parnas, J.K. (1939) Observation sur les e´changes des atomes du phosphore renferme´s dans l’acide ade´nosinetriphosphorique, ´ l’aide du phosphore marque´ par du radiophosphdans l’animal vivant, a ore 32P. Bull. Soc. Chim. Biol. 21, 713–716. ¨ ber der Umsatz der Aden[69] Korzybski, T. and Parnas, J.K. (1939) U ¨ure im lebenden Tier. Acta Biol. Exp. 13, 157–166. osintriphosphorsa [70] Parnas, J.K. (1932) The chemistry of muscle. Ann. Rev. Biochem. 1, 431–456. [71] Parnas, J.K. (1933) The chemistry of muscle. Ann. Rev. Biochem. 2, 317–336. [72] Parnas, J.K. (1936) Uzupełniony schemat glikogenolizy mi˛e´sniowej. Spraw. Tow. Nauk. Lwo´w. 16, 84–87. [73] Parnas, J.K., Mochnacka, I. and Augustin, Z. (1937) O koenzymach glikogenolizy mi˛e´sniowej. Spraw. Tow. Nauk. Lwo´w. 17, 164–165. [74] Parnas, J.K. (1924) Chemizm oddychania. Czynnos´´c wa ˛troby. Mocz. Skład i włas´ciwos´ci mleka. In Podr˛ecznik fizjologii (Beck, A., ed.), Vol. II, pp. 123–157, 261–282, 283–301, 335–340. Lwo´w – Warszawa – Krako´w, Gubrynowicz. + [75] Parnas, J.K. (1923) Methoden zur Beeinflussung der thierischen Entwicklung durch Gase und der Bestimmung des respiratorischen Gaswechsels ´hrend der Entwicklungsvorga ¨nge. In Handbuch der biologischen wa Arbeitsmethoden (Abderhalden, E., ed.), Abt. V, Teil 3.A, pp. 651–670. [76] Parnas, J.K. (1926) Allgemeines und Verleichendes des Wasserhaushaltes. In Handbuch der normalen und patologischen Physiologie (Bethe, A., Bergmann, G., Embden, G. and Ellinger, A., eds.), Vol. 17, Correlationen III, J.XVII, pp. 137–160. Berlin, Springer. [77] Parnas, J.K. and Mozołowski W. (1934) Podstawy chemiczne i fizjologiczne dietetyki. In Dietetyka, pp. 3–64. Warszawa, Delta.

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[78] Parnas, J.K. (1936) Przemiana materii. Tłuszcze. Trawienie. W˛eglowodany. ´ wiat i z˙ycie., Vol. IV, pp. 441–456, 1108–1116, 1131–1144, Witaminy. In S ´w-Warszawa, Ksia 1256–1272, 1378–1386. Lwo ˛˙znica Atlas. [79] Parnas, J.K. (1937) O sprawie polskiej terminologii fizjologiczno-chemicznej. Acta Biol. Exp. 11, 357–368. [80] Parnas, J.K. (1938) W sprawie nomenklatury ciał rujop˛ednych. Endokrynol. Lek. 3, 31–34. [81] Parnas, J.K. (1927) Ernst Josef Leser. Biochem. Z. 196, 1–2. [82] Parnas, J.K. (1931) Otto Warburg. Pol. Gaz. Lek. 10, 997–998. [83] Parnas, J.K. (1933) Prof. G. Embden. Nature (London) 132, 994–995. [84] Parnas, J.K. (1936) Laureaci Nobla: H. Dale i O. Loewi. Gaz. Pol. 17, XI. [85] Parnas, Ya.O. (1943) O niekotorykh uspekhakh i itogakh izucheniya vitaminow. Izd. AN USSR, Ufa, 1–32. [86] Parnas, Ya.O. (1943) Enzimy i koenzimy. Usp. Sowrem. Biol. 16, 225. [87] Parnas, J.K. (1943) Coenzymatic reactions. Nature (London) 151, 577. [88] Parnas, J.K. (1944) Enzymes and coenzymes. Amer. Rev. Sov. Med. 1, 485–517. [89] Parnas, Ya.O. (1943) Khimiya i gormony. Sow. Med. 9, 1. [90] Parnas, Ya.O. (1945) Nowyje witaminy. Byull. Eksp. Biol. Med. 20, 3–15. ´ ska, Z. (1997) Taking over of Parnas fate and fame-documents. Post. [91] Zielin Biochem. 43, 370–383. [92] Schulz, H.E., Urban, P.K. and Lebed, A.J. (1972) Who was Who in the USSR, pp. 434–435. Metuchen, NJ, Scarecrow Press. [93] Sznol, S. (2004) Akademik Jakub Karol Parnas. In Herosi, gangsterzy, konformis´ci, pp. 296–312, Warszawa, Bellona Press. (Translation from Russian: S. Sznol (2000) In Gieroi, Zlodiei, Konformisty rossijskoj nauki. Moscow, Kron-Press.) [94] Barnett, J.A. (2005) Glucose catabolism in Yeast and Muscle. Selected Topics in the History of Biochemistry: Personal Recollections IX. In Comprehensive Biochemistry (Semenza, G., and Turner, A.J., eds), Vol. 44, pp. 1–128. Elsevier, Amsterdam. [95] Florkin, M. (1975) A history of biochemistry. Part III. History of identification of the sources of free energy in organisms. In Comprehensive Biochemistry (Florkin, M., and Stotz, E.H., eds), Vol. 31, pp. 1–473. Elsevier, Amsterdam. ¨ ber die enzymatische Milchsa ¨urebildung im Muskel[96] Meyerhof, O. (1927) U ¨urebildung aus den ga ¨rfa ¨higen extrakt. III. Mitteilung die Milchsa Hexosen. Biochem. Z. 183, 176–215. ¨ ber Phosphorylierung und Dephosphorylierung. [97] Lohmann, K. (1983) U ¨ure aus ihren KomponenBildung der natu ¨ rlichen Hexosemonophosphorsa ten. Biochem. Z. 262, 137–151. ¨ ber die Bildung des [98] Ostern, P., Guthke, J.A. and Terszakowec´, J. (1936) U ¨ure Esters und dessen umwandlung in FructoseHexose-monophosphorsa ¨ure-ester in Muskel. Z. Physiol. Chem. 243, 9–37. disphosphorsa ¨ ber die Aldolase, ein [99] Meyerhof, O., Lohmann, K. and Schuster, P. (1936) U kohlenstoff-verknu ¨ pfendes Ferment. I. Mitteilung: Aldolkondensation von Dioxyacetonphosphorsa¨ure mit Acetaldehyde. Biochem. Z. 286, 301–318.

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¨ ber die enzymatische Umwand[100] Meyerhof, O. and Kiessling, W. (1935) U ¨ure in Dioxyacetonphosphorsa ¨ure. lung von Glycerinaldehydephosphorsa Biochem. Z. 279, 40–48. [101] Warburg, O. and Christian, W. (1939) Isolierung und Kristallisation des ¨rungsferments. Biochem. Z. 303, 40–68. Proteins des oxydierenden Ga ¨ ber ein Phosphatu ¨rungsferment. [102] Bu ¨ cher, T. (1947) U ¨ bertragendes Ga Biochim. Biophys. Acta 1, 292–314. ¨ ber die Isolierung der Isomeren [103] Meyerhof, O. and Kiessling, W. (1935) U ¨ure (Glycerinsa ¨ure-2-phosphorsa ¨ure und GlycerPhosphoglycerinsa ¨ure-3-phosphorsa ¨ure) aus Ga ¨ransa ¨tzen und ihr enzymatisches insa Gleichgewicht. Biochem. Z. 276, 239–253. ¨ ber die enzymatische Umwand[104] Lohmann, K. and Meyerhof, O. (1934) U lung von Phosphoglycerinsau ¨ re in Brenztraubeensau ¨ re und Phosphorsau ¨ re. Biochem. Z. 273, 60–72. [105] Parnas, J.K. (1936) L’enchainement des processus enzymatiques dans le tissue musculaire. V Congress de Chimie Biologique, Bruxelles, 23–25 Octobre 1935. Bull. Soc. Chim. Biol. France 18, 53–95. ¨ ber die enzymatische Synthese der Kreatinpho[106] Lehmann, H. (1935) U ¨ure durch Umesterung der Phosphobrentraubensa¨ure. Biochem. Z. sorsa 281, 271–293. [107] Harden, A. and Robison, R. (1914) A new phosphoric ester obtained by the aid of yeast-juice. Proc. Chem. Soc. 30, 16–17. ¨ ber die Chemie des Lacta[108] Embden, G. and Zimmermann, M. (1927) U cidogens, 5 Mitteilung. Z. Physiol. Chem. 167, 114–136. [109] Robison, R. and King, E.J. (1931) Hexosemonophophoric Esters. Biochem. J. 25, 323–338. [110] Robison, R. (1932) Hexosemonophophoric Esters: Mannosemonophosphate. Biochem. J. 26, 2191–2202. [111] Harden, A. and Young, W.J. (1908) The alcoholic frerment of yeast-juice. Part III. The function of phosphates in the fermentation of glucose by yeast-juice. Proc. R. Soc. Lond. Ser. B 80, 299–311. ¨ ber die Chemie des Lactaci[112] Embden, G. and Zimmermann, M. (1924) U dogens, IV. Mitteilung. Z. Physiol. Chem. 167, 225–232. [113] Levene, P.A. and Raymond, A.L. (1928) Hexosediphosphate. J. Biol. Chem. 80, 633–638. ¨ ber das Vorkommen [114] Embden, G., Deuticke, H.j. and Kraft, G. (1933) U ¨ure bei der Glykolyse in der einer optisch aktiven Phosphoglycerinsa Muskulatur. Z. Physiol. Chem. 230, 12–28. [115] Negelein, E. and Bro¨mel, H. (1939) R-Diphosphoglycerinsau ¨ re, ihre Isolierung und Eigenschaften. Biochem. Z. 303, 132–144. ¨ ber das intermedia ¨ren [116] Embden, G., Deuticke, H.j. and Kraft, G. (1933) U ¨nge bei der Glycolyse in der Muskulatur. Klin. Wochenschr. 12, Vorga 213–215. ¨ ber den enzymatischen Umsatz [117] Meyerhof, O. and Kiessling, W. (1935) U ¨ure (Enolbrentraubensa ¨ureder synthetischen Phosphobrenztraubensa ¨ure). Biochem. Z. 280, 99–109. phosphorsa [118] Fiske, C.H. and Subbarow, Y. (1929) Phosphorus compounds of muscle and liver. Science 70, 381–382. ¨ ber den Abbau der Aminosa ¨uren [119] Neubauer, O. and Fromherz, K. (1911) U ¨rung. Z. Physiol. Chem. 70, 326–350. bei der Hefega

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[120] Lutwak-Mann, C. and Mann, T. (1981) 50 years ago. The Parnas school. Trends Biochem. Sci. 6, 309–310. [121] Cori, C.F. and Cori, G.T. (1936) Mechanism of formation of hexose monophosphate in muscle and isolation of a new phosphate ester. Proc. Soc. Exper. Biol. Med. 43, 702–705. ¨ ber die enzymatischen Phosphorylierungen in der [122] Parnas, J.K. (1938) U ¨rung und in der Muskelglykogenolyse. Enzymologia 5, alkoholischen Ga 166–184. [123] Baum, H. (1982) In praised of E.M.P. In The Biochemist’s Songbook, pp. 3–5. Oxford/New York, Pergamon Press. [124] Parnas, J.K. (1944) Dr. Paul Ostern. Nature (London) 154, 695–696. [125] Mann, T. (1955) Prof. J.K. Parnas. Nature (London) 175, 532–534. [126] Dobryszycka, W., Kwiatkowska-Korczak, J. and Lisowska, E. (2003) Postwar biochemistry in Wrocław. Acta Biochim. Polon. 50, IX–X. [127] Baranowski, T. (1949) Crystalline glycerophosphate dehydrogenase from rabbit muscle. J. Biol. Chem. 180, 535–541. ¨ ber die Bestimmung kleiner Pentosemenge insbe[128] Mejbaum, W. (1939) U ¨ure. Z. Physiol. Chem. 258, 117–120. sondere in Derivaten der Adenylsa [129] Korzybski, T. and Parnas, J.K. (1969) Some observations on the turnover of the phosphorous atoms of adenosine triphosphoric acid, in the living animal, by the use of radioactive 32P-labeled phosphorus. In Biological Phosphorylations – Development of Concepts (Kalckar, H.M., ed.), pp. 377–380. Englewood Cliffs, NJ, Prentice Hall. [130] Post. Biochem. (Advances in Biochemistry) (1958) Special issue devoted to Jakub Karol Parnas (Heller, J., ed.), Vol. 4, pp. 1–65. [131] Post. Biochem. (1986) Special issue devoted to Jakub Karol Parnas ´ ska, Z., ed.), Vol. 32, pp. 243–285. (Zielin [132] Post. Biochem. (1992) Special issue devoted to Jakub Karol Parnas ´ ska, Z., ed.), Vol. 38, pp. 138–150. (Zielin [133] Post. Biochem. (1997) Special issue devoted to Jakub Karol Parnas ´ ska, Z., ed.), Vol. 43, pp. 311–388. (Zielin

BIOGRAPHIC DATA Private correspondence: Tadeusz Korzybski and Irena Mochnacka with Jakub ´ ska with Jan Oskar Parnas (1990–1995); Karol Parnas (1945–1948); Zofia Zielin ´ ska with Barbara Parnas Janina Kwiatkowska-Korczak and Jolanta Baran (2005). Recollections, testimonies, documents. In Post. Biochem. (1992) Vol. 38, pp. 138–150; and Post. Biochem. (1997), Vol. 43, pp. 311–378. Polski Słownik Biograficzny (1980) Vol. 25, pp. 218–221, Wrocław, Zakład ´ skich. Narodowy im. Ossolin Lwowskie ´srodowisko naukowe w latach 1939–1945. O Jakubie Karolu Parnasie (1993) (Stasiewicz-Jasiukowska, I., ed.), Warszawa, PAN, KHNT. Heller, J. and Mozołowski, W. (1958) Jakub Karol Parnas. Działalnos´´c nauczycielska w latach 1916–1939 (Jakub Karol Parnas. Teaching activity in 1916–1939). Post. Biochem., Vol. 4, pp. 5–8.

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Korzybski, T. (1974) Parnas Jakub Karol. In Dictionary of Scientific Biography (Gillespie, C.S., ed.), Vol. 10, pp. 326–327. New York, Scribner. Mann, T. (1955) Prof. J.K. Parnas. Nature (London), Vol. 175, pp. 532–534. Mejbaum, W. (1986) Profesor Jakub Karol Parnas (Professor Jakub Karol Parnas, recollections). Post. Biochem., Vol. 32, pp. 261–264. Ostrowski, W. (1986) Jakub Karol Parnas, z˙ycie i two´rczos´´c (Jakub Karol Parnas, life and creativity). Post. Biochem., Vol. 32, pp. 247–260. ´ ska, Z. (1987) Jakub Karol Parnas. Acta Physiol. Pol., Vol. 38, pp. 91–99 Zielin (together with re-reprinted [129]).

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G. Semenza (Ed.) Stories of Success – Personal Recollections. X (Comprehensive Biochemistry Vol. 45) r 2007 Elsevier B.V. All rights reserved. DOI: 10.1016/S0069-8032(07)45006-9

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Chapter 6

A Journey with Bleeding Time Factor ¨ CK BIRGER BLOMBA Karolinska Institutet, Nanna Svartz va¨g 2, 17177 Solna, Sweden E-mail: [email protected]

Abstract The primary focus of this review is the research that resulted in the discovery of a new factor (von Willebrand factor, VWF) in the coagulation of blood; the prime motor in initial haemostasis. Further research that lead to the identification of VWF as a multimeric protein that interacts with vessel wall and platelets is also considered, as is the regulation of this factor’s activity in blood, an important aspect of both haemostasis and thrombosis. Keywords: Von Willebrand factor; VWF; VWD; multimer; platelets; factorVIII; ADAMTS-13.

The factor now known as von Willebrand factor (VWF) is a protein that is missing in severe von Willebrand’s disease (VWD). In the 1950s Swedish researchers1 presented evidence for the occurrence of a previously unknown factor in the initial phase of haemostasis. I have described the ideas and painstaking work that lead to that claim in great detail. The factor was elusive, its main signum,

Abbreviations: ADAMTS 13: a mettalloprotease. AHG and AHF: antihaemophilic factor; factor VIII. CSL: Commonwealth Serum Laboratories. c-DNA: complementary DNA. NADPH: nicotine amide adenine dinucleotide phosphate; reduced form. TTP: thrombotic thrombocytopenic purpura. 1 ¨ck, Margareta Blomba ¨ck and Birgit Hessel The members of the group were: Birger Blomba of Karolinska Institute in Stockholm, Irene von Franken of Stockholm University and Inga-Marie Nilsson of University of Lund

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shortening of bleeding time,2 no doubt gave it a spooky quality. Anyway, few in the scientific community in those days believed in its existence as a unique factor in haemostasis. VWD was rather thought of as being affected by vascular factors, clotting factors, platelets and the interactions between them. I have tried to trail VWF on its voyage from anonymity to what we now know as a gigantic multimeric protein that assists in initial haemostasis by catching platelets as a prelude to the interactions between clotting factors that eventually leads to the haemostatically secure clot at bleeding sites. Furthermore, now that the gene for VWF has been elucidated, it has been fascinating to learn how defects in the genetic message explain various forms of VWD with their great variety of phenotypic expressions. The ingenious discovery of a specific metalloprotease (ADAMTS-13) that degrades VWF, then inspired me to follow the trail of research that led to the realization that VWF is a player not only in bleeding disorders but also in thrombotic conditions. My story gives the discovery of VWF ample space because reviews of the factor seldom cover this early work. Instead, they tend date the discovery to the point in time when the antigenic expression for VWF was found.3 Although physically unknown to us, the factor and its unique actions obviously existed. There are similarities here to the way in which compounds like antibiotics and vitamins were discovered, long before they were obtained in pure chemical form. I have also given ample space in footnotes to personal recollections of events that occurred during the time I participated in research on the bleeding time factor. And Then There Was a New Disease Descriptions of bleeding disorders with prolongation of bleeding time as a common marker goes back to the turn of the 19th century 2

Piercing cuticle capillaries and measuring the time until bleeding stops (usually by piercing an earlobe and measuring how long blood continues to drip). The test measures adhesion of platelets to wound surfaces, which together with platelet aggregation and vessel contraction leads to the cessation of blood flow. 3 For instance in 1987 the history was described as follows: ‘‘In 1926, Erik von Willebrand, a German professor, described the congenital bleeding disorder that bears his name after ˚ land archipelago in the ¨, an island of the A studying several members of a family from Fo¨glo Gulf of Bothnia. After 45 years, in 1971, the protein known today as von Willebrand factor ( VWF) was first detected immunologically and named, at the time, factor VIII-related antigen’’ (Z.M. Ruggeri and T.S. Zimmerman (1987) von Willebrand factor and von Willebrand disease. Blood 70, 895–904).

A JOURNEY WITH BLEEDING TIME FACTOR

Fig. 1.

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Photograph of Dr Erik von Willebrand.

(Figure 1). The diathesis described by Dr Erik von Willebrand4 in 1926 was nevertheless a newcomer [1]. The story began with ˚ land islands in the ¨rdis S, a five-year-old girl5 from one of the A Hjo Baltic Sea, who had a severe hemorrhagic diathesis. She was admitted to the medical ward at the Deacon Hospital in Helsinki in April 1924. Her first bleeding episode had occurred when she was one-year old and, at the age of 3, she had had a more severe episode after a fall in which she hurt her lip; this was followed by severe bleedings for 3 days and confinement to bed for another 10 weeks. 4

Erik von Willebrand (1870–1949), Finnish medical doctor in Helsinki. He learned about ˚ land ¨glo ¨, an island in the A the bleeding history of Hjo¨rdis’ family through a teacher on Fo archipelago in the Baltic Sea. He apparently never visited the island. 5 Her mother, born in 1882, had bleeders among her ancestors and so had her father, born in 1875. Their eldest daughter had died of an intestinal hemorrhage at the age of 2. The second daughter died of a hemorrhage following injury at the age of 4. The third daughter ¨rdis S was to die of profuse likewise died of an intestinal hemorrhage at the age of 2. Hjo menstrual bleedings at the age of 13. A fine description of members of the family and their ¨ck’s review in 1999. ‘‘Scientific visits bleeding histories is to be found in Margareta Blomba ˚ land islands’’, Haemophilia, 5(Suppl. 2), 12–18. to the A

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More recently she had frequently bled from the nose or gums, and had also had skin bruises and bleeding in a twisted ankle. Physical investigation at the time of admission showed a more or less healthy status although her skin was pale and there were bruises in several locations. Blood cells, including platelet number, were normal and so was clot retraction, but bleeding time was much prolonged. Moreover, capillaries of nail folds appeared more twisted and their diameters were more uneven than in normal individuals. In further studies, von Willebrand found two large families related to Hjo¨rdis S and one unrelated in which bleeding symptoms similar to those in Hjo¨rdis were common [2]. He concluded that he was dealing with a hereditary disease that occurs predominantly in women and that the bleeders were of two different types: severe and mild. Von Willebrand considered the inheritance was possibly Mendelian dominant but was unable to decide whether it was sex-linked or autosomal.6 The disease was named pseudohemophilia. He further concluded that the condition differed from other, somewhat similar, bleeding disorders known at that time: classical haemophilia, essential thrombocytopenia (Morbus Werlhofi)7 and Glanzmann’s thrombasthenia. In view of his earlier work it is somewhat surprising that in a later publication von Willebrand suggested that the pathology of pseudohaemophilia included functional deficiency of platelets, acting together with components in the vessel wall [3]. The condition was now named constitutional thrombopathy8 but von Willebrand did not dismiss the notion that factors in blood plasma might also be important in its pathogenesis. 6

In his first two publications the pattern of inheritance indicated a sex-linked dominant disease. In the 1934 publication he noted that a sick father and an apparently healthy mother had two sons who had the disease. This, he concluded, contradicted sex-linked inheritance. However, the apparently healthy mother was distantly related to the husband’s family. 7 In addition to essential thrombocytopeni, there was at that time a disease that presented itself with a low platelet count but ironically led, not to bleeding, but to microthrombotic sequelae. The disease, subsequently known as thrombotic thrombocytopenic purpura, was discovered by E. Moschcowitz in 1924. About 50 years later it was shown to have a bearing on the factor missing in von Willebrand’s disease. More about this later. 8 The change in name was primarily based on a report by Morawitz and Ju ¨ rgens, who had investigated a patient with a pathophysiology similar to that of von Willebrand’s cases (case description in Ref. [3]) but arrived at the diagnosis essential thrombopathy. Using an apparatus known as capillary thrombometer, they found that their patient had a prolonged ‘‘thrombosing time’’ when whole blood was forced in alternate directions through a paraffinated capillary tube. When a few of von Willebrand’s patients were tested with this method, they were found to have greatly prolonged values and, in addition, defective platelet agglutination in a ‘‘hanging drop of platelet rich plasma’’.

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In the following years, numerous cases similar to those described by von Willebrand were described in the literature, usually under the name of pseudohaemophilia. New puzzling findings came to light in a report by Alexander and Goldstein in 1953 [4]. They confirmed the earlier findings of prolonged bleeding time, normal platelet count and function and abnormal nail bed capillaries. They also observed a prolonged coagulation time9 that was normalized by normal plasma: interestingly, the prolonged bleeding time was not affected by plasma infusion (for an explanation see pages 216–217). Their measurements of clotting factors showed that antihaemophilic factor (factor VIII) activity was low. They suggested that the deficient factor VIII activity led to deficient thrombin generation and this in turn, together with malfunctioning components of the vessel wall, led to a prolonged bleeding time. Larrieu and Soulier [5] also found low factor VIII activity and a prolonged bleeding time in pseudohaemophilia, but otherwise normal clotting factors and platelet parameters. They proposed the name von Willebrand’s syndrome for the condition but otherwise considered that the disease scenario predicted by Alexander and Goldstein was likely. Bleeding Time Factor – Really? Bleeding time – a test that for some years in the late 1950s was woven into our efforts to identify a factor in blood.10 How did we get into this bleeding time business? Actually through serendipity. ¨ck It all started with fibrinogen. At that time Margareta Blomba and I were working on purification of the fibrinogen from plasma and early in 1956, out of Cohn’s fraction I, we had obtained one fraction, I-O, that we first believed was pure fibrinogen but then found to be heavily contaminated with antihaemophilic factor (factor VIII) [6]. We had already used this fraction in treatment of 9

The time it takes for a venous blood sample to form a gel (clot), usually during discreet rocking. In contrast to bleeding time, this test measures activation of intrinsic coagulation factors in blood. 10 I remember my biochemical colleagues at Karolinska Institutet in Stockholm scolding us for using such an imprecise, unscientific method for measuring a factor. Think of it. Puncturing an earlobe and measuring how long blood continues to drip. But at that time it was the only test we had to identify what we believed to be a new factor in blood. In September 2003, I attended a meeting on haemophilia and von Willebrand’s disease. A participant told me that one of his colleagues no longer used bleeding time test, as being too vagrant, and put more trust in other diagnostic tests. I thought to myself, ‘‘Fortunately we trusted bleeding time in 1956’’.

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fibrinogen deficiencies11 and now we realized that it was also a good candidate for treatment of haemophilia A12. It so happened that Dr Inga-Marie Nilsson, who had performed the testing of factor VIII activity on our fibrinogen preparations, had at that time a patient, a 15-year-old girl, Birgitta, on her ward at the University Hospital ¨, Sweden. This girl had had a severe haemorrhagic diain Malmo thesis since childhood. At the age when her menstrual bleedings started, the disease took a grave turn and large quantities of blood were transfused to stop the bleedings. However, after a while she suddenly developed severe side-reactions when transfused, and the blood transfusions had to be abandoned. The coagulation status had shown: much prolonged bleeding time, delayed prothrombin consumption, somewhat prolonged clotting time, but normal platelet count and function. Furthermore, factor VIII activity was low. Since fraction I-O had a high concentration of factor VIII it was decided to test its effect in this patient. To our surprise, not only did plasma factor VIII activity increase as expected but bleeding time was also normalized [7]. Subsequently, under cover of fraction I-O, hysterectomy was successfully performed (Figure 2). Birgitta’s case launched our bleeding time factor story. We presented the patient initially as a case of female haemophilia, which to say the least was a misnomer. At that time we were not aware of the haemorrhagic disease which von Willebrand had first described in the 1920s. However, we were aware that the clinical and laboratory picture, especially the prolonged bleeding time, was at odds with classical haemophilia and more in line with cases of ‘‘pseudohaemophilia’’ reported previously by Alexander and Goldstein [4] and other authors. A follow-up study of patients 11

Such fractions were prepared under non-sterile conditions and sterile filtered before use. I remember the first patient with prostatic cancer, intense fibrinolysis and severe bleedings. He responded as expected to the treatment, bleedings stopped but after a few hours he developed a high fever that turned out to be due to pyrogens in the water used in the fractionation. I was contrite and had to inform Professor Erik Jorpes, head of our department. He was understanding and said he had had similar mishaps during his early work on heparin. Anyway, we learned a lesson and after that, none of the sterile products (there were more than thousand of them) we used for treatment of VWD or haemophilia A were contaminated with pyrogens. 12 In this context I remember that afterwards there was a widely spread rumor, causing ¨ck was mightily dismuch merriment in some quarters, to the effect that Birger Blomba appointed to learn that fraction I-O, instead of being pure fibrinogen, also contained the antihaemophilic factor. From our description of the use of fraction I-0 [6] it is obvious that the rumor was false. However, it may have influenced people’s conception of my interest in the factor. Nevertheless, antihaemophilic activity was not present in the highly purified fibrinogen we eventually obtained and that was also fine. How lucky we were to have Bleeding Time Factor and Factor VIII caught in a subfraction, I-O.

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Fig. 2. Effects of fraction I-O on patient Birgitta, before, during and after surgery. The Swedish text on ordinate translates from top to bottom as follows: Factor VIII activity, bleeding time, coagulation time, treatment and bleeding episodes. Arrows indicate infusions of fraction I-O (AHG) and operation for surgery. Abscissa shows time in days. From Nordisk Medicine, 56, 1654–1662 (1956).

from various families in Sweden, presenting bleeding disorders similar to our proposita, showed the disease to be inherited and the defective gene autosomal, dominant and with varying expressivity [8]. It was now that the von Willebrand’s early studies started to attract our attention. The similarities between our cases ˚ land Islands and his cases were striking. An expedition to the A was called for to study the descendants of the families that von Willebrand had studied in the1920s.13 In fact some of the original members were still alive. Platelet function, other laboratory ˚ land families were indisparameters and clinical features of the A tinguishable from those in the Swedish patients [9]. Following the proposal by Larrieu and Soulier [5] we named the condition von Willebrand’s disease (VWD). 13 ˚ land island, who prompted I suspect it was Professor Erik Jorpes, born and raised on an A us to investigate von Willebrand’s original cases.

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Birgitta’s combination of factor VIII deficiency and prolonged bleeding time was puzzling. Like Alexander and Goldstein [4], our first hypothesis was that low factor VIII level combined with capillary abnormality was responsible for prolongation of both clotting time and bleeding time. However, when we found that transfusion of fraction I-O, that had lost most of its factor VIII activity by adsorption, brought about immediate correction of bleeding time in a VWD patient,14 we started to question this explanation. It was obvious that the effect on bleeding time was independent of factor VIII activity in fraction I-O. Furthermore, our cases appeared not to have the vascular or platelet abnormalities proposed by several authors. We dismissed disturbance of the capillary wall as an etiologic factor, since ophthalmologic examination of our patients had not revealed any vessel pathology and since platelet factors (1, 3 and 4) were normal, constitutional thrombopathy [3] was considered less likely. An idea was born: plasma contains a factor that controls bleeding time and a deficiency of this factor causes VWD. But were we really dealing with a new ‘‘bleeding time factor’’ ? Further experiments were required to prove the notion [9–13]. As bleeding time is normal in patients with severe haemophilia A, we prepared fraction I-O from blood of individuals with this disease and injected it into patients with VWD. The effect on bleeding time was the 14

The story behind this finding is as follows. Treatment of haemophilia A and VWD requires a sterile and pyrogen-free preparation of fraction I-O. Early on the fraction was prepared under non-sterile conditions and the process ended with filtration through Hyflo Super Cel and sintered glass filters to obtain sterile preparations. It turned out that factor VIII activity was often lost through this filtration [6]. So we set to work on a fractionation procedure that would be aseptic all the way from drawing of blood to the final product. The resultant recovery of factor VIII activity and bleeding time effect was good, if not excellent. Filtered preparations without or with low factor VIII activity had been kept in storage and were now used as controls in transfusion experiments in VWD, i.e. we expected that these fractions would have little or no effect on bleeding time since factor VIII activity was low. To our surprise, bleeding time was normalized after injection of such preparations. The proposition that we were dealing with a new bleeding time factor was reported for the first time at the Conference on Blood Cells and Plasma Proteins in Albany, NY, 1957 [10]. In his discussion of the talk, Dr Anderson, Michigan Department of Health, Mich, said: ‘‘His method has also provided important clues for separation of other components (than fibrinogen, author’s addition) of fraction I; namely antihemophilic globulin and properdin. His observation that ‘fraction I-O’ deprived of AHG (factor VIII, author’s note) by adsorption, still controlled bleeding time and capillary bleedings, is welcomed by our group’’. He somehow missed our point when he subsequently said: ‘‘His observation that ‘purified fibrinogen (fraction I-2) from stored blood had no’ effect on bleeding time and capillary bleeding prompts me to suggest that highly purified fibrinogen may not be the best product for the treatment of hypofibrinogenemia’’ He obviously thought that the bleeding time correction was a property inherent in fibrinogen present in fraction I-O and not due to a unique plasmatic factor, as I had pointed out in my talk.

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same as (or even better than) with the fraction from normal blood. Then obviously we would expect fraction I-O originating from VWD patients to have no effect and that proved to be the case. Fraction I-O consists to about 90% of fibrinogen. To rule out the possibility that fibrinogen was responsible for the bleeding time effect, we administered a large excess of purified fibrinogen and found no effect on bleeding time. Still there was the possibility that the shortening of bleeding time was due to platelets or platelet factors, contaminating our fraction I-O. This turned out to be unlikely since the effect on bleeding time was the same whether the fraction had been prepared from platelet rich or platelet poor plasma. Furthermore, infusion of a platelet suspension from a normal donor to a VWD patient had no effect on either bleeding time or bleeding tendency. Neither did injection of fraction I-O into a patient with thrombocytopenia. We concluded that platelets or platelet factors were not identical with the bleeding time factor in fraction I-O. There was still another problem. Alexander and Goldstein [4] had reported that plasma did not affect bleeding time in their patient. This and frequent reports that infusion of blood or plasma had little effect on bleeding time in cases of ‘‘pseudohemophilia’’ turned out to be mainly a logistic problem. Although transfusion of 500 ml of blood did not effect bleeding time, transfusion of 1350 ml produced a shortening. This should be compared with the 3200 ml of blood from which a normal dose of fraction I-O was prepared. An entirely different puzzle was the deficiency of factor VIII in VWD and how it might be related to the bleeding time factor. In all the cases studied, the level of factor VIII was somehow correlated with the bleeding time: the lower the factor VIII level, the longer the bleeding time. More important was our early observation that after administration of fraction I-O to patients with VWD, the level of factor VIII was elevated more than one would expect from the concentration of factor VIII in fraction I-O. Furthermore, the concentration of factor VIII several hours or even days after the injection was even higher than immediately after. Here are a few examples: ‘‘the AHF level in the sample drawn 2 hours after injection had increased from 5 to 28% of normal; on the next day it was higher, i.e. 73% and on the third day it was still as high as 67%’’ [13]. The slow turn-over of factor VIII activity in VWD contrasted sharply with the fast disappearance of factor VIII after administration of normal fraction I-O to patients with

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haemophilia A. Administration of fraction I-O from haemophilia A plasma, with no factor VIII activity, to VWD patients produced unexpected results [12]. After 2 h the level of factor VIII began to rise and, after 10 h, it had increased from about 6% to 30% and after 24 h it was still at 20% of normal.15 No such elevation of factor VIII occurred after infusion of fraction I-O obtained from plasma of individuals with severe VWD. Our conclusion from the above studies was that we had evidence of the occurrence in normal plasma of a unique factor, missing in VWD, that controls bleeding time by acting on the capillary wall and/or the platelets [11]. Furthermore, we suggested that this factor directs the production or activation of factor VIII. Deficiency of this factor would then explain ‘‘why prolonged bleeding time and factor VIII deficiency occur in association with this disease’’. The name we had used, bleeding time factor, was now changed to von Willebrand Factor (VWF)16 to honor the discoverer of the disease. The claim that a previously unknown factor in plasma had been discovered was communicated at the Congress of the International Society of Haematology in Rome 1958. In the following years efforts were made to purify the elusive VWF, which very likely was of protein nature since it occurred in what appeared to be an high-molecular-weight protein complex. A major problem was the factor’s lability. This called for the use of fresh blood, collected in silicon-treated bottles and a swift preparation procedure. In those days, bleeding time was the factor’s only reliable marker. It would take several years until in vitro assays for VWF became available. An illustration of our difficulties is the fact that all preparations had to be tested in vivo by administration to individuals with VWD.17 Further attempts to purify the factor at that time were therefore considered neither realistic nor ethically defensible. However, the success with fraction I-O in our clinical studies had shouldered us with the responsibility for producing this fraction for regular or intermittent treatment of haemophilia A and VWD in Sweden. Clinical experience of the treatment of these conditions accumulated over these years [12]. 15 At about that time Cornu et al. [16,17] had also shown that injection of plasma from haemophilia A patients to patients with VWD elicited a gradual increase in factor VIII level. 16 Looking back, I recall that it was the head of the chemistry department at Karolinska Insttutet, Professor Erik Jorpes, who insisted that we call it von Willebrand factor. 17 Today, the constrains on in vivo experiments, in order to contain viral infections like hepatitis and HIV, would simply have ruled out some of the work we did in the Fifties.

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At first the fraction was administered to patients during bleeding episodes and surgical interventions. However, a foundation was also laid for long-term treatment of haemophilia A and VWD by intermittent administration of the fraction. The above undertaking required a good supply of absolutely fresh blood and a laboratory where fractionation under aseptic conditions could be performed and to the latter end a new laboratory was inaugurated in 196018 (Figure 3). The main steps in the fractionation procedure was precipitation of fraction I from plasma and subsequent extraction of contaminants with an aqueous glycine–citrate–ethanol solution, at –3 to –41C, to yield fraction I-O. The concentration of glycine was 1 M and ethanol about 6% (V/V). Surprisingly, Cohn’s fraction I, the starting material in our fractionation procedure, was in other laboratories found to have little or no effect on bleeding time, although it did affect the level of factor 18

Before that time we had used localities housing also other investigators engaged in a variety of projects (e.g. extraction of secretin from pig intestines). A growing problem now was to find a steady supply of fresh blood. For an interim solution we approached Professor Clarence Crafoord, head of the Thoracic Surgery Clinic at Sabbatsberg’s Hospital in Stockholm. He had occasionally used our fraction I-O in treatment of bleeding episodes in his patients. Professor Crafoord gladly endorsed our project and told us to inform the Director of the Blood Donor Center, Dr Olof Ramgren, that we need the blood for Professor Crafoords research projects. This solved our blood problem until the County Comptrollers started to ask disturbing questions, since we were using a considerable part (as I remember about 10%) of the city’s total blood supply. However, by that time fraction I-O had proved its place in treatment of haemophilia A and VWD and the supply of blood could be arranged in a way that was more acceptable to the authorities. To sustain fractionation under aseptic conditions was a problem and called for new laboratory space. Fortunately, Alice and Marcus Wallenberg’s Research Foundation decided to donate a large sum of money for the construction of facility that would house our and other research groups at Karolinska Institutet. This enabled us to establish a laboratory for fractionation of blood under sterile conditions for production of fraction I-O for treatment of patients with haemophilia A and VWD in Sweden. At that time no pharmaceutical company was interested in providing this material. Now a third problem arose, this time of a financial nature. We needed money to employ staff to do the work and could not use our research grants for that purpose. Instead a price tag could be attached to the bottles we delivered to the hospitals. Professor Jorpes, head of Chemistry Department II, suggested that we contact the director of the Swedish Association of County Councils to explain the situation. We told the director that, in addition to our research work, we produced fraction I-O for the medical care of patients. He agreed that this was something the hospitals should pay for and to that end sent a recommendation to all the County Councils. That solved the money problem. The price of the product included a percentage for funding our innovative research. Fraction I-O was also sent for treatment of patients in other countries (England, France, USA). In 1966, the production of fraction I-O was taken over by Kabi AB, an arrangement that lasted for more than two decades. Moreover, a fractionation unit similar to ours was established at the Commonwealth Serum Laboratories in Melbourne, Australia. Dr Percival Bazley, head of C S L, whom I had met at ¨ck and me to come to Australia the conference in Albany in 1957, invited Margareta Blomba to do the ground work, which we did in 1961–1962. We had a wonderful time in Australia, not the least because Dr Pehr Edman was also there. He taught us the secrets of amino acid sequencing, knowledge that became extremely useful in our future scientific work. I have described this fortunate happening at some length in a biography on Pehr [140].

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Fig. 3. Inauguration of new laboratory in 1960. The laboratory was inaugurated in presence of the late King Gustaf VI Adolf and Queen Louise. Here we see the king and queen watch laboratory nurse, Gunilla, demonstrate the transfer of separated plasma from sediment of red cells after centrifugation.

VIII. A likely explanation is a lack of attention to the conditions for preparation, such as storage-time of blood and contact surfaces during fractionation. The importance of such conditions for preservation of bleeding time activity was discussed in a study by Weiss [14]. He also found that the Swedish fraction I-O was superior to other currently available preparations of factor VIII complex19 (Table 1).

19 Dr Weiss tested three different preparations on a patient with VWD (see Table 1). At one occasion, the patient ‘‘was given 4 g of the AHG-rich fibrinogen product of Merck, Sharp & Dohme’’. As may be seen (line 1, Table 1) ‘‘there was no improvement in her bleeding time. Although the above results suggested that the patient differed from the cases reported from ¨ck (Margareta Blomba ¨ck, author’s Sweden, we nevertheless enlisted the help of Dr Blomba note). The patient was given three grams of Swedish fraction I-O, obtained from about 1400 ml of plasma. As may be seen on line 2, the bleeding time from both the arm and the ear was shortened to 5 minutes following the transfusion, and 16 hours later a bleeding time of 23 minutes showed that the corrective effect still had not been completely lost. Results obtained at a later date with the Swedish fractions are shown on line 5 and 6’’. The result obtained with the Boston preparations (from Protein Foundation, Boston, MA; authors note) were ‘‘only slightly active in shortening the bleeding time’’. At another occasion, the patient had ‘‘developed unremitting bleeding from an undiagnosed gastrointestinal site’’. Laparotomy showed a bleeding point in the proximal jejunum. Surgical intervention followed’’. During the 5-hours procedure, the patient received 3 g of the Swedish fraction (line 9). Operative bleeding was minimal and the bleeding jejunal lesion was successfully plicated’’.

Infusion no.

Date Given

Fraction

Grams of Protein Transfused

Bleeding Time (min.) Following Infusion Arm (Ivy)

200 1

7–30–60

2 3 4 5 6 7 8 9 10

1–30–61 2–2–61 2–15–61 2–23–61 2–23–61 10–25–61 10–27–61 11–3–61 11–6–61

Merk AHG-rich fibrinogen Swedish 1–0 Boston 1–4 Boston 1 E–5 Swedish 1–0 Swedish 1–0 Boston 1–6 Boston 1 E–2 Swedish 1–0 Merk AHG-rich fibrinogen

4

445

3 4 4 1.5 3 4 4 3 6

5 9 9 5 5 25,6 16 7 460

2 hr

4 hr

Effect Ear (Duke)

6 hr

16 hr

445

200 445

23 460 445

5 12 7

10 8 460 460 12

8

10

20

460

460 460 2

None Excellent Slight Slight Excellent Excellent Slight Slight Very good None

A JOURNEY WITH BLEEDING TIME FACTOR

TABLE 1 Effects of various factor VIII concentrates on bleeding time in a patient with von Willebrand’s disease

Note: Fractions denoted Merck were from the company of Merck, Sharp & Dohme. Those denoted Boston were from Protein Foundation, Boston, MA. Ivy and Duke refer to the two methods used for determination of bleeding time. For more details, see footnote 19. From Vox. Sang. 7, 267–280 (1962).

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222

Factor Aims at Platelets Our idea that a plasmatic factor controls bleeding time and production or activation of factor VIII had received a mixed response. However, several reports did support the notion of a plasma factor missing in VWD [15–17]. Biggs and Macfarlane [18], who had successfully used fraction I-O to treat post-operative bleedings in a case of VWD, were not averse to the possibility of a specific plasma factor missing in VWD, but warned: ‘‘Since the nature of the haemostatic defect is still obscure it would be premature to speculate on ways in which a plasma factor may correct it’’. Nevertheless, the prolonged bleeding time in VWD invited studies of platelet adhesion to different substrata. Borchgrevink in Norway [19] found deficient adhesion of platelets to wound surfaces in VWD, a deficiency that nicely correlated with the prolonged bleeding time. Later Salzman and Zucker [20,21] found in VWD defective adhesion of platelets to glass, when blood flowed through columns of glass beads. An understanding of why some of the glass-bead tests had shown normal results in VWD was gained when O’Brien and Heywood [22] showed that the flow rate of blood was also important. Adhesion of platelets was normal in VWD at a slow flow rate but abnormal at a fast rate. Although the adhesion studies provided support for a plasma factor missing in VWD, how it worked was not clear. The studies by Zucker [21] had indicated that after primary passage of normal plasma through a glass-bead column platelet adhesiveness in VWD was normalized. Further studies by Meyer and Larrieu [23] also hinted at how the plasma factor took part in the adhesion process. They showed that platelets from VWD patients adhered to glass beads that had previously been coated with plasma from normal or haemophilia A individuals, but that adhesion was greatly deficient when coating was done with VWD plasma. Normalization of both platelet adhesion and bleeding time was observed after patients with VWD had been treated with factor VIII/VWF concentrates. The platelets apparently adhered to the mysterious VWF being adsorbed on glass during flow. Reviewing the state of the art in VWF research in the late 1960s, Stormorken [24] proposed that a ‘‘unifying concept would require that the basic defect is the plasma factor and that this factor secondarily affects both the morphology and function of platelets and the vascular wall. Hemostatically, its influence on the vessel wall should be most important since the transfusion of platelets has no

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effect on the bleeding in these patients. The factor must be able to leave its site of origin, since it is present in blood, as well as be able to enter the factor-VIII producing cell since it promotes the production of this factor. Before being used for this purpose, it could act on the process of adhesion between the platelets and the vascular wall’’. This concept comes close to our present understanding of VWF’s function. Although the interrelation of VWF and platelets continued to attract attention, the idea of capillary dysfunction as a factor in VWD, suggested by some authors (e.g. 1, 4), had somehow faded. In ultra structural studies on platelet plug formation, Hovig and Stormorken [25] showed that the defect in VWD is unlikely to be related to disturbed vessel contraction or pathological alteration of the endothelial cells but rather to disturbance of platelet function caused by the lack of a plasma factor.

Factor Dimmed and Brought to Light But what was the physical nature of this bleeding time or adhesion factor? The factor was apparently present in a protein complex of high-molecular weight, having factor VIII activity. The rest was an enigma. An important contribution toward identifying the physical factor was published by Zimmerman et al. [26]. They showed that a rabbit antibody to a Factor VIII/VWF concentrate reacted with plasma from normal and haemophilia A individuals, but not from those with VWD. The antibody also quenched factor VIII activity. The latter finding lead them to propose that the antigenic determinant represented a protein with factor VIII activity and that consequently a true deficiency of this factor existed in VWD, whereas a non-functional form of factor VIII was produced in haemophilia A. An alternative explanation was that in haemophilia A, the factor VIII-like molecule was inactive due to the presence of an inhibitor. For some time this ‘‘one-molecule’’ hypothesis dimmed the concept of a separate, unique factor missing in VWD. With hindsight, it is obvious that in attempts at this time to purify VWF or factor VIII, the strong binding between them led to enrichment of both factors and thus supported the one-molecule concept, i.e. a molecule with both factor VIII and VWF activity. The one-molecule concept certainly did not agree with our early findings and was soon questioned by other investigators. One

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concern was the marked disparity between factor VIII activity and the factor VIII/VWF-related antigen in plasma of patients with VWD, after treatment with plasma or plasma concentrates. In such a study Bloom et al. [27] presented the hypothesis that the factor VIII/VWF complex consists of a high-molecular-weight unit, the factor VIII-related antigen being identical to VWF, controlling adhesion and bleeding time and a lower-molecular-weight unit responsible for factor VIII activity. The high-molecular-weight unit was considered to stimulate the formation or release of factor VIII from cellular stores, as we had previously predicted [11], but also to act as a carrier of factor VIII. The fact that the transfused antigen disappeared quickly was explained by its clearance to hitherto empty stores or deposition on vascular endothelium. Furthermore, Zimmerman and Edgington [28] found the one-molecule concept unlikely on the ground that a human iso-antibody to factor VIII activity in haemophilia A patients and a rabbit antibody to the factor VIII/VWF-complex differentially bound and removed factor VIII activity and the VWF-related antigen from plasma. This indicated that factor VIII resides on a molecule distinct from the VWF antigen. Owen and Wagner [29] had also suggested that a high-molecular-weight protein acted as a carrier for factor VIII activity, since the latter could be dissociated from its carrier in various saline or detergent solutions. Moreover, no factor VIII activity was present in highly purified VWF preparations from human and porcine plasma, and antibodies to VWF did not inhibit factor VIII activity, nor did the material neutralize factor VIII inhibitor in haemophilia A blood [30]. It was also shown that during synthesis of the VWF antigen in endothelial cells, there was no concomitant synthesis of factor VIII in that location [31]. VWF was also shown to be synthesized by megakaryocytes, thereby explaining its presence in platelet granules [32]. By the late 1970s and early 1980s, the consensus was that factor VIII and VWF were separate molecular entities. The final eye-opener came with the isolation of factor VIII free of VWF antigen. Using immunosorbent techniques, factor VIII in a stable form was separated from human factor VIII/VWF complex [33,34]. Soon afterwards the molecular structure of a highly purified factor VIII was elucidated [35,36]. Later work by several groups showed factor VIII to be a single-chain protein of molecular weight about 300 kDa, which was processed to a series of lower molecular weight entities.

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Despite the initial misinterpretation, the finding by Zimmerman et al. [26] of an antigenic expression of the VWF complex became important in the further identification of VWF as a unique protein in blood. Equally important was the finding by Howard and Firkin [37] that the factor had a unique activity, distinct from the adhesion and bleeding time activities. They observed that the antibiotic Ristocetin20 induced platelet aggregation in normal plasma but not in plasma of patients with VWD and this was correlated to perturbed adhesion of platelets to glass beads, whereas platelet aggregation induced by collagen, ADP and thrombin was the same in VWD as in normal plasma. They suggested that Ristocetin interacted with a plasma factor and proposed for it the name: ristocetin co-factor. Factor, A Series of Huge Polymers A new concept of the fine details of VWF structure came with the demonstration by Counts et al. [38] that the high-molecular-weight compound, with the antigenic and activity profile of VWF, was composed of not one but a series of homologous multimers. To disrupt non-covalent protein–protein interactions, they subjected the purified high-molecular-weight factor VIII/VWF complex to electrophoresis on very porous acrylamide-agarose gels,21 at physiological ionic strength, and in the presence of sodium dodecyl sulphate (SDS). Under these conditions, they found that VWF turned into a series of multimers with molecular weights from the hundreds up to several millions. The same pattern was observed when plasma was analyzed. When the multimeric complex was reduced with excess of mercaptoethanol a subunit of about 240 kDa was formed, having no ristocetin co-factor activity but the factor VIII activity of the original complex appeared to be unchanged.22 At low concentrations of reducing agent a dimer was formed. The authors suggested that even numbers of such dimers (protomers) formed 20

The potent antibiotic Ristocetin, discovered in 1956, is obtained from the actinomycete, Nocardia lurida and is active against gram positive bacteria and mycobacteria. Later it was largely withdrawn from the market because of side-reactions, most notably thrombocytopenia. Howard and Firkin observed that Ristocetin at high doses caused precipitation of fibrinogen. At low doses there was no precipitation but platelet aggregation in normal plasma was not perturbed. 21 Instead of conventional electrophoresis on 5% polyacrylamide-SDS gels, gels containing 2% acrylamide and 0.5% agarose were used. 22 This led them to suggest that factor VIII is covalently attached to the subunit.

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the different members in the oligomeric series observed before reduction. A possible interpretation of this study was that reductive processes in vivo gave rise to the oligomeric series. Although such processes cannot be ruled out, we now have evidence that proteolysis, rather than reduction, is a major process in the generation of the multimeric series (see below Factor checked by guardian). Later studies on VWD patients confirmed that it was primarily the presence of high-molecular-weight multimers of VWF that influenced ristocetin co-factor activity [39]. We still do not know for certain why only high-molecular-weight multimers are active.23 Multimerization takes place intracellularly during biosynthesis of VWF in two cellular compartments: rough endoplasmic reticulum and Golgi apparatus [40]. The formation of glycosylated dimers (protomers) occurs in the former and multimerization in the latter or later compartments (e.g. Weibel-Palade storage bodies), most likely by disulfide bond formation between protomers, and in this process a pro-peptide is cleaved off from the subunits of the protomers. Factor, Polymers of a Single-Chain Protein The perception of VWF as a series of homologous multimers built up by a unique protein subunit was instrumental in the further clarification of its structure and function. Using the new techniques for assaying VWF, we started on a project to obtain factor VIII/VWF complex in a highly purified form [41]. What we had in mind was a complex that would exhibit high factor VIII activity as well as antigenic and other activities of VWF, including shortening of bleeding time in VWD.24 Only then, we thought, was the complex likely to contain the native VWF molecule. When the purified 23

It has been suggested that larger VWF multimers are more active in inducing ristocetin co-factor activity since they contain more binding sites for platelets than the smaller ones (see Ref. [135]). 24 Since further purification of VWF out of fraction I-O had not been successful, we devised a new fractionation procedure. As starting material we used a solution of cryoprecipitate, rather than Cohn’s fraction I. From this solution, fibrinogen and other contaminating proteins were precipitated by adding glycine to 2 M concentration, at 261C; the factor VIII/ VWF complex was then precipitated simply by adding sodium chloride (91 g/l) at 20–231C. Final gel-filtration resulted in a highly purified complex displaying VWF antigen, ristocetin co-factor and factor VIII activity in good yields. Since we knew that sterile filtration might lead to loss of factor VIII activity [6], we decided to use an aseptic technique in the procedure prior to gel filtration.

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complex was injected into individuals with severe VWD [41,42], bleeding time, ristocetin co-factor activity and VWF antigen level were normalized, while in haemophilia A individuals, factor VIII activity was restored.25 The effect of the complex on the adhesion of platelets to subendothelium in human arteries was also tested in vitro using an annular perfusion chamber [44]. The complex normalized platelet adherence in reconstituted blood. Platelets in plasma of persons with severe VWD showed no adhesion before but normal adhesion after they had been transfused with the purified complex. The normalization was accompanied by decrease in bleeding time and increased ristocetin co-factor activity. Hessel et al. [45] now turned their attention to the biochemical characteristics of the purified factor VIII/VWF complex. Before reduction, like in normal plasma, multimers of molecular weight between 1.5  106 and 22  106 Da were observed (Figure 4). In VWD plasma no multimers were visible. After complete reduction of the purified complex one subunit (about 260 kDa) was identified (Figure 5). Most important, amino acid sequence analysis demonstrated a single-chain protein. This chain clearly represented VWF since the same sequence was recorded after removal of factor VIII from the complex. The reason why factor VIII did not show up in gel electrophoresis or in the sequence analysis was simply a matter of quantity. In terms of the specific activity of factor VIII [35,36], the factor VIII protein constituted only a few percent of the total protein and this was below the sensitivity of the methods we used for detection. The VWF protein was rich in half-cystine residues and, since no free thiol-groups were present, these were all engaged in disulfide bridge formation. Limited reduction of disulfide bonds by thioredoxin was accompanied by decrease in multimer size, loss of ristocetin co-factor activity and early dissociation of factor VIII from VWF protein. Disulfide bridges most likely linked 25

The clinical studies were performed on the complex before gel filtration, which is the final step in the purification procedure. I believe that the product after gel filtration also has preserved bleeding time activity. First, the gel filtered complex had a high ristocetin cofactor activity, for which there is a correlation with platelet adherence to substrata [37,44], which correlates in turn with shortening of bleeding time [19,23,44]. Second, the presence of high-molecular-weight multimers points to the perseverance of bleeding time activity. It is of interest that recombinant dog VWF, which did not shorten bleeding time from cuticle wounds in VWF deficient dogs (although severe nose bleeding ceased), had lower molecular weight than VWF in normal dog plasma [43]. The molecular weight of VWF multimers may therefore be of major importance for its effect on bleeding time. I surmise that unfolding to the active state occurs at a lower shear stress, or concentration than with lowermolecular-weight species.

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Fig. 4. Gel electrophoresis of factor VIII/VWF multimers in plasma and purified complex. The gels were stained with labeled antibodies to the VWF complex. The figures to the right show the position of: 1. IgM, Mr 900000, 2. Its dimer and 3. Its trimer. D. Von Willebrand disease plasma, E. Normal plasma and F. Purified factor VIII/VWF complex. Adapted from Thromb. Res. 35, 431–450 (1984).

all the individual components of VWF multimers, since extended reduction with thioredoxin yielded the subunits. We aimed to establish the complete primary structure of VWF in the way we had used successfully in our work on fibrinogen. However, other events lead the way. In 1985, based on the known partial N-terminal and C-terminal amino acid sequences of VWF, Sadler et al. [46] were able to isolate two c-DNAs that covered both the N-terminal and the C-terminal sequences of the mature VWF, as well as part of a pro-peptide sequence. Later, Verveij et al. [47] described a full-length c-DNA that encoded signal peptide, pro-peptide and mature protein. The pro-peptide was in all likelihood identical with the VWF-antigen II that had previously been detected in plasma [48]. The translated protein appeared to represent a protein of 2813 amino acids of which 2050 belonged to the mature VWF. The protein consisted to about 90% of duplicated, triplicated and quadruplicated domains in the following

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Fig. 5. Electrophoretic pattern of purified factor VIII/VWF complex. E. Before gelfiltration, F. Before gelfiltration; reduced sample. The three adjacent bands, shown after reduction, represent fibrinogen chains. G. After gelfiltration, H. After gelfiltration; reduced sample. Adapted from Thromb. Res. 35, 431–450 (1984).

order from the N-terminal end: H-D1-D2-D0 -D3-A1-A2-A3-D4-B1B2-C1-C2-OH. Both prosequence and mature VWF appeared to harbor an Arg–Gly–Asp sequence, implicating binding sites for platelets. Confirmation of the c-DNA sequence was soon obtained by direct determination of the amino acid sequence of the purified VWF protein. For a start, in addition to a partial N-terminal sequence, Chopek et al. [49] clarified a short C-terminal sequence of the VWF subunit.26 Important was their detection of smaller fragments of the subunit, attached to it by disulfide bonds. The amounts of these fragments (120 and 140 kDa27) increased as the multimers decreased in size. They suggested that the fragments were the end result of in vivo proteolysis of the multimers observed in plasma. 26

By using cyanogen bromide for cleavage of methionyl bonds, they were able to find a small fragment that on sequence analysis showed lysine at the C-terminal end instead of a methionin derivative; obligatory if cyanogen bromide had caused cleavage of the peptide bond. Thus, lysine was considered to be C-terminal in the VWF subunit. 27 In subsequent studies, usually referred to as 140 and 176 kDa fragments.

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VWF was found to bind to platelets and this binding decreased with decreasing multimer size. In another report, Girma et al. [50] used Staphylococcus aureus V-8 protease for digestion of VWF. This turned out to be of the outmost importance for solving primary and higher order structures of VWF and for obtaining functional insights into this molecule. The protease primarily splits a single bond (Glu–Glu) in the VWF subunit, resulting in stepwise degradation of the multimers. After prolonged cleavage two dimeric fragments remained, one from the N-terminal and the other from the C-terminal end of the subunits. Like the original multimer, the N-terminal fragment did bind to platelets whereas the C-terminal fragment did not. The analysis led to the conclusion that in multimers, subunits are linked by disulfide bonds but these alternate between the C-terminal and the N-terminal regions of the subunits. Thus subunits will first be linked at their C-terminal ends and the dimers so formed are then linked at the N-terminal ends to form the multimers, or, as they put it, first ‘‘foot-to-foot’’ and then ‘‘head-to-head’’ junctions. Finally, as the climax of this tour de force in elegant biochemistry, Titani et al. [51] announced the complete amino acid sequence of VWF. Most of the structure was obtained by direct sequencing of the whole protein and of fragments obtained after limited proteolysis. However, minor parts were deduced from the nucleotide sequence of cDNA clones [46]. This study confirmed that mature VWF is a glycoprotein with 2050 amino acid residues, including 169 half-cystine residues, and with several oligosaccharide chains linked to Asn or Thr/Ser residues. Confirming the interpretation of the cDNA sequence [47], an Arg–Gly–Asp–Ser (RGDS) sequence at the C-terminal end of the subunit indicated, as in fibrinogen, a possible ligand for GPIIb+IIIa on activated platelets. The carbohydrate chains in VWF may play a role for proper function of VWF [52]. Thus, in the human complex, carbohydrate chains terminate in sialic acid, with galactose being penultimate. In bovine factor VIII/VWF complex, which supports platelet aggregation in the absence of ristocetin, the carbohydrate chains end with galactose. It was shown that removal of sialic acid from human factor VIII/VWF complex produced a protein that, like the bovine complex, aggregated platelets in the absence of ristocetin. Oxidation of penultimate galactose abated this activity. Attempts to pair the 169 half-cystine residues in the VWF protein are still in progress [53]. Intersubunit disulfide bonds

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were shown to be present in C-terminal regions. Elucidation of the crystal structure of VWF, or its domains, which has been done for the A1 domain [54], may in due course reveal the complex arrangements of all disulfides in the molecule. The elucidation of the amino acid sequence and genomic structure of VWF paved the way for new experimental approaches that underpin our present understanding of the role of VWF in haemostasis. Recombinant proteins, expressed by a number of authors, made it now possible to identify functional domains interacting with, e.g., platelet receptors, collagen, factor VIII and glycosaminoglycans such as heparin. Fragments of VWF, resulting from proteolysis, and shown to interfere with VWF function could now be located in the overall sequence. The site of binding of monoclonal antibodies, shown to interfere with particular functions of VWF, could likewise be explored (for review, see Ref. [55]).

Factor, We See You! Visual features of VWF multimers were now brought to light. Electron-microscopic pictures, by Fowler et al. [56], of purified VWF showed elongated flexible strands up to 2 mm in length, made up of dimeric units (protomers) linked to each other in an end-toend fashion. Each protomer consists of large globular end domains connected to a small central node by two flexible rod-like domains (Figure 6). Visualization of N- and C-terminal fragments of VWF [57], obtained by digestion with Staphylococcus V8 protease, confirmed the predictions of Girma et al. [50], which was also supported in a study on biosynthesis of VWF [58]. The N-terminal fragment contained a binding site for heparin and for the platelet GPIb receptor and the C-terminal fragment for the GPIIb+IIIa receptor [57]. Instead of elongated strands, the structure revealed by atomic force microscopy of VWF multimers is more compact [59]. However, on exposure to shear stress the structure unfolds into an elongated shape. The compact structure may represent the conformation of VWF in blood at low-shear rates and requires high-shear stress for unfolding. Ristocetin or botrocetin28 attachment to VWF most likely brings about another type of 28

Botrocetin is a venom protein isolated from the venom of the snake Bothrops Jararaca.

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Fig. 6. Electron micrograph of human VWF showing alternating rod and globular domains. Below the micrograph is a model showing the protomer and protomers joined together at N-terminal ends. From J. Clin. Invest.76, 1491–1500 (1985).

conformational changes that unfold sites binding to GP1b receptor on platelets [60]. It had been shown that proper multimerization depended on the release of pro-peptide, VWF antigen II, from the precursor protein [40]. This was further substantiated by studies of recombinant VWF with and without pro-peptide [61,62]. Polymerization of VWF appears, in a sense, to be similar to that of fibrinogen after its activation with thrombin. Fibrinogen is a dimer formed by two subunits that are held together by disulfide bonds; thus a construct similar to that of a VWF dimer (protomer). Dimerization of the two proteins occurs in endoplasmatic reticulum, although in fibrinogen this happens at the N-terminal ends of subunits, in VWF at the C-terminal ends. After activation, fibrinogen protomers align non-covalently in half-staggered fashion, due to interaction between sites in N- and C-terminal parts of the subunits, thus forming two identical fibrin strands (Figure 7). In fact, VWF protomers (without pro-peptide) also appears to assemble into functional polymers by non-covalent interactions in a concentration-dependent manner, as shown by Loscalzo et al. [63]. If sites of interaction similar to those in fibrinogen existed in VWF, a polymer like that shown in Figure 6 could be formed after removal of pro-peptide. In this case, disulfide bonds between

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Fig. 7. Polymerization pattern of fibrinogen dimers. N stands for aminoterminal end and C for carboxyterminal end of fibrinogen monomers.

protomers of VWF are likely to be the result of secondary processes, as in fibrin factor XIII-induced cross-linking. Polymerization in the case of fibrinogen is followed by gel formation, i.e. a polymer network is formed. Whether this occurs with VWF multimers is not known, although VWF antigen was observed being deposited in a filamentous subcellular or intercellular network matrix [64]. Factor Loves Factor VIII Our studies in the 1950s missed an important aspect of VWF function, i.e. as a carrier and protector of factor VIII. The suggestion by Bloom et al. [27] that VWF acts as a carrier for factor VIII became generally accepted and could be approached in experiments showing that purified factor VIII added to plasma at physiological calcium ion concentration swiftly recombined with VWF-related antigen [65]. Furthermore, VWF was shown to stabilize factor VIII in whole blood in vitro [66]. This was verified in vivo [67] by showing that after infusion of highly purified factor VIII into patients with heamophilia A, the half-life was about 11 h, whereas the half-life in a patient with VWD was much shorter, about 2 h. However, infusion of material also containing VWF to the latter patient led to the typical secondary rise in factor VIII and longer preservation of factor VIII in circulation. Experiments with highly purified factor VIII in haemophilic dogs and dogs with VWD gave similar results [68]. Infusion in VWD dogs did not affect haemostasis, despite high levels of factor VIII in blood. In haemophilia A dogs, however, haemostasis was restored. In our in vivo experiments [42] with highly purified VWF complex we found, as we had observed in our early experiments [11], that after injection, factor VIII activity in blood continued to increase for many hours, followed by a slow decline. The factor VIII activity remained in circulation for longer time than VWF antigen

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and ristocetin co-factor activity: the latter activities decayed as bleeding time was prolonged. Interestingly, when the VWF complex was transfused to patients with haemophilia A, the half-life of factor VIII activity is about the same as for VWF antigen or ristocetin-cofactor activity in VWD29 (Figure 8). This supported the notion that factor VIII is strongly bound to VWF in both conditions and decays at the same rate. However, the longevity of factor VIII activity in VWD is still a mystery, though it is most likely explained by VWF exerting a long-lasting effect on the synthesis or secretion of factor VIII. The VWF antigen binds to vascular endothelium [93–95] and it is possible that this binding affects the synthesis of factor VIII, as suggested by Bloom et al. [27]. Thus, the carrier function may, more or less, go hand in hand with stimulation of synthesis or secretion of factor VIII. Release of pro-peptide may be necessary for the mature VWF to bind and stabilize factor VIII [62]. The binding of factor VIII occurs in the N-terminal part of VWF [69]. It is therefore of some interest that we found release of factor VIII from VWF in conjunction with reduction of disulfides (at positions 4 and 13) in the N-terminal part of VWF [45]. In vitro studies have indicated that complex formation between factor VIII and VWF is extremely rapid in plasma [70]. Thus, the time needed to bind 50% of factor VIII in plasma is about 2 s. Plasma contains a molar excess of VWF, with the result that only 1 out of 50 sites is occupied by factor VIII.30 The authors conjecture that complex formation most likely occurs in sinusoids of the liver, where factor VIII and VWF first meet. In line with this, it was shown that VWF could promote reconstitution of factor VIII activity from dissociated heavy and light chains of factor VIII [62], which supports the notion that VWF may promote assembly of factor VIII right at the site of the latter’s secretion. Nesheim et al. [71] demonstrated that factor VIII bound to VWF is still available for proteolysis by thrombin, which in turn leads to release of activated factor VIII, which can then participate in the amplification of thrombin formation in the milieu of aggregating platelets. Interestingly, when bound to VWF, factor VIII is protected from activation by factor Xa and from inactivation by activated protein C. 29

Not discussed in Ref. [42]. This is in accordance with the finding that in a highly purified factor VIII-VWF complex, factor VIII constitutes only a few percent of the complex [45].

30

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Fig. 8. Fate of factor VIII activity and factor VIII-related antigen (VWF) in two individuals with von Willebrand’s disease and haemophilia A after administration of the factor VIII/VWF complex. Factor VIII activity: factor VIII:C. VWF: factor VIIIR:Ag. A. Factor VIII activity (factor VIII:C) in von Willebrand’s disease, B. VWF (factor VIIIR:Ag) in von Willebrand’s disease and C. Factor VIII activity (factor VIII:C) in haemophilia A. Adapted from Thromb. Res. 31, 375–385 (1983).

Factor in Dialogue with Platelets and Vessel Wall Right from the beginning it was suspected that in exerting its haemostatic effect, VWF somehow interacts with platelets and/or vessel wall [19–21]. This was not simply an interaction under static conditions. Flow came into the picture with O’Brien and

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Heywood’s findings on the adhesion of platelets to glass [22]. Weiss et al. [72] subsequently demonstrated that, in native blood, the adhesion of platelets to subendothelium is shear-rate dependent and that the same applies to the adhesion of platelets to glass beads. At a low wall shear rate there was no difference in adhesion between normal blood and blood from patients with VWD. At shear rates similar to those in microvasculature, however, adhesion was greatly impaired in VWD but also in Bernard-Soulier’s syndrome. This raised the issue of to which compound in subendothelium the platelets bound and what was VWF’s role in that process. Meyer and Larrieu [23] had shown that adsorption of factor VIII/VWF complex onto glass precedes platelet adhesion to that matrix. How this could apply to subendothelium was an open question. Studies indicated that microfibrils or collagen might be important in platelet adhesion to the basement membrane in the presence of VWF complex [73,74]. The fact that VWF complex binds to collagen prompted the proposal that VWF adhered to collagen present in subendothelium [75].31 Sakariassen et al. [76] clearly showed that platelets do adhere to VWF attached to the subendothelium. The amount of bound VWF correlated with the quantity of platelets adhering to the surface and adhesion was deficient in VWD. VWF is deposited in subendothelium during biosynthesis [64,77]. Plasma VWF appears to be in a dynamic equilibrium with the VWF present on subendothelium [78]. Thus, it was projected that at high-shear rates platelet adherence is mediated by VWF present in both compartments. Furthermore, it was shown that the N-terminal fragment of VWF substituted functionally for VWF in supporting platelet adhesion at a high-shear rate [79].32 This part of the molecule contained a site for binding to collagen. The C-terminal part also bound to collagen but did not support platelet adhesion. The dynamic equilibrium between soluble VWF and surface bound VWF was explored in another study [80]. When the A1 domain of recombinant VWF, i.e. the N-terminal domain that binds to platelets, was missing both in immobilized VWF and in VWF in flowing 31

On the other hand, VWF binding to collagen in the fluid phase in the presence of platelets led to platelet aggregation. Surprisingly, these latter results indicated that a multimeric structure of VWF was not required for platelet adhesion. Cautiously, the authors end by saying: ‘‘our results do not stipulate that in vivo the large multimers are unimportant for normal hemostasis but only indicate that in vitro a specific fragment may substitute for the fully polymerized VWF in promoting platelet adhesion to collagen’’. 32

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blood, no adhesion occurred. The addition to the fluid phase of VWF with functional A1 domain led to restoration of the binding function. Apparently the latter bound to the surface and, in doing so, displayed the binding site on A1 domain that mediated platelet adhesion. Unfolding of VWF by shear forces might be important for exposing this binding site [59]. The platelet membrane has specific receptors for VWF. It was suggested that VWF binds to a specific glycoprotein that is present on normal platelets and on platelets in VWD but absent on platelets in Bernard-Soulier’s syndrome [81]. Glycocalicin [82], a part of a larger hydrophobic membrane glycoprotein, appeared to be responsible for binding to VWF [83]. Thus, platelet response to VWF was reduced at the same time as the glycocalicin was released from the platelets as a result of proteolysis. Glycocalicin was shown to be derived from the membrane glycoprotein component, GPIb, missing in Bernard-Soulier’s syndrome [84]. GPIb consists of two glycoprotein chains, a and b. Glycocalicin is part of the larger a-chain. The high specificity of the interaction between VWF and GP1ba is exemplified by the finding that a monoclonal antibody to GPIba that inhibited binding of VWF to the platelet membrane, did not inhibit aggregation induced by ADP, collagen, thrombin or arachidonic acid [85]. A major axis of platelet adhesion in the presence of VWF was revealed: platelet GPIba-VWF-subendothelium; the active component in subendothelium being one of its matrix components, possibly collagen, microfibrils or other components, possibly acting in concert. Interaction between VWF and the GP1ba receptor appears to activate platelets and expose the GPIIb+IIIa receptor [86–88], which in turn can interact with VWF, fibrinogen or fibronectin as a preliminary to platelet aggregation. A real-time analysis of thrombus formation on collagen surfaces [89] showed adhesion, at high-shear rates, to be the result of adsorption of VWF multimers onto collagen followed by binding of platelet GPIba to the insolubilized VWF. Aggregation occurred subsequently and required the binding of ligands, including VWF via its RGD (i.e. the Arg–Gly–Asp sequence) binding domain, to the GPIIb+IIIa receptor on platelets. Another real-time study analyzed platelet adhesion and thrombus development after flowing blood had been exposed at a high-shear rate to a surface of collagen fibrils or subendothelial matrix [90]. VWF was rapidly adsorbed onto both

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substrates, and platelets became initially tethered by interaction between the GP1b receptor and VWF. However, irreversible adhesion and thrombus growth required additional interactions involving other receptors, among them IIb+IIIa. The role of VWF multimers versus that of fibrinogen in haemostatic processes should be considered, since both bind to activated platelets through interaction with GPIIb+IIIa. However, fibrinogen seems to inhibit the binding of VWF to the latter receptor [91]. Since the concentration of fibrinogen in blood is much higher than that of VWF, this may mean that following platelet activation fibrinogen will be one of the main denominators of platelet aggregation and release of procoagulant factors. The generation of thrombin is followed by fibrin formation at the site of injury. However, an afibrinogenemic patient with a normal level of VWF in blood was shown to have surprisingly few major bleeding events [92]. The authors suggested that in this case VWF acts as a standin ligand in platelet aggregation and that this together with effective platelet adhesion in small vessels, where shear rate is high, upholds haemostasis without fibrin clot formation. VWF binds not only to subendothelial structures but also to components on the endothelial cell surface. It was shown that a rabbit antibody to VIII/VWF complex bound to vascular endothelium [93]. Holmberg et al. [94] further demonstrated that such an antibody bound to the intimal layer of veins in normal subjects and in haemophilia A but not in severe VWD. Recent experiments proved that stimulated endothelial cells (mice) in vivo rapidly secret VWF from Weibel-Palade bodies [95]. VWF bound to the cell membrane within seconds and subsequently induced, even at a low-shear rate, platelet adhesion mediated by GP1ba. Translocation occurred but no aggregation. The fact that VWF harbors heparin-binding sites in the A1 domain [96,97] has led to the proposition that VWF binds to glycosaminoglycans, e.g. heparansulfate on endothelium, and this might rapidly recruit platelets to a site of injury or inflammation [95].

Factor Missing or Malformed Von Willebrand’s disease was presented to us in the 1950s in two forms Erik von Willebrand had described [1,2]. There were the mild and the severe forms, now named VWD Type 1 and VWD

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Type 3, respectively. Type 3 has no VWF protein in plasma, low or absent ristocetin co-factor activity, much prolonged bleeding time and severe bleedings. The mutations in the VWD Type 3 gene in cohorts of families, originally investigated by von Willebrand, were ¨ck’s group [98]. A homozygous clarified by Margareta Blomba frameshift in exon 18 was found. This would lead to premature termination of the chain within the pro-peptide region, explaining why no mature VWF is present in the blood of these patients. The multimeric pattern in plasma of VWD Type 1 is qualitatively similar to that in normal plasma, but less VWF protein is present and less VWF activity. Bleeding time is variously prolonged and bleeding tendency is usually minor. VWD Type 1 was found in individuals with heterozygous mutations in the VWF gene, i.e. a null allele as in VWD Type 3 may combine with a wild type allele, or with an allele bearing a missense mutation [99]. The picture of VWD has become much more complicated in recent years. It turns out that the disease harbors a multitude of forms – in addition to those mentioned above – with different pathogenetic mechanisms, as described in recent reviews [55,100,101]. Let me dwell on some of these forms. In a variant form of the disease, known as Type 2A, there is bleeding tendency and ristocetin co-factor activity in plasma is decreased [102]. In a subgroup of this type, the larger forms of VWF multimers in blood are missing due to proteolysis of multimers [103,104]. The mutations are clustered in the A2 domain of VWF but also occur in the A1 domain, containing the binding site for platelet GP1ba, as well as in the pro-peptide region [see 101]. In another interesting variant with bleeding diathesis, named 2B, binding of VWF to platelets occurs at a lower ristocetin concentration than in normal plasma [105]. Thrombocytopenia may occur. In these patients the larger forms of VWF are sometimes missing in blood but not in platelets. In this type, mutations are recorded in the A1 domain and the mutation has, in contrast to the situation in 2A, apparently facilitated binding of VWF to GP1ba [106,33107]. One explanation for the pathology would be that, in vivo, VWF interacts with platelets spontaneously, leading to consumption both of larger multimers and of platelets. This might favor thrombotic sequelae but since, as in Type 2A,

33

¨. Designated type 1 New York or type 1 Malmo

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increased proteolysis also occurs, the balance is tipped toward bleeding diathesis. Finally, there is an interesting VWD variant that mimics haemophilia A. In this, Type 2N, binding of factor VIII to VWF is hampered [108]. Since VWF stabilizes factor VIII in circulation, factor VIII infused into these patients has a short half-life, as in VWD Type III, where VWF is missing (see Ref. [67]). Mutations in Type IIN are found in the N-terminal part of mature VWF, the binding region for factor VIII. The mutations are essentially located in the D0 region [109].

Factor Checked by Guardian In 1924, two years before Erik von Willebrand discovered the disease bearing his name, Dr Eli Moschcowitz in New York [110] described a pathological condition that later became known under the name thrombotic thrombocytopenic purpura (TTP) and was connected with VWF half a century later. The proposita was a young girl with the clinical picture of sudden onset of fever, anemia, renal dysfunction, impairment of central nervous system and cardiac failure; five cardinal symptoms of this new syndrome. She died in coma and of particular interest at autopsy was the occurrence of hyaline thrombi in arterioles and capillaries of several organs, especially the heart. Several investigators subsequently described the same condition, which turned out to be associated with thrombocytopenia. The hyaline thrombi of Moschcowitz were in fact aggregates of platelets with little fibrin [111,112]. Still, the pathophysiology of TTP was left in conundrum. A link to VWF started to emerge. Studies of autopsy cases revealed the presence of intracapillary thrombi and endothelial deposits that stained weakly for fibrinogen/fibrin but strongly for factor VIII/VWF complex [113]. This was in stark contrast to autopsy findings in cases with disseminated intravascular coagulation (DIC) and periarteritis nodosa, in whom thrombi stained strongly for fibrinogen/fibrin and weakly or not at all for VWF complex. The observation by Moake and associates that unusually large VWF multimers were present during remission in TTP and that they became smaller during relapse established another important link [114,115]. They suggested that ‘‘a previously unrecognized regulatory process in normal plasma may prevent the

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Fig. 9. Von Willebrand factor synthesized by endothelial cells compared to von Willebrand factor as present in plasma. EC, Endothelial cell-derived VWF; NP, VWF multimers in plasma. Adapted from Blood 73, 2074–2076 (1989).

circulation of unusually large VIII:VWF multimers derived from endothelial cells’’. Later, animal models in dogs and pigs supported the view that VWF multimers, in activated34 form, are crucial in eliciting TTP [116]. Other studies now showed that endothelial cells in vitro produced not a series of multimers but what appeared to be a single ultra-large multimer (Figure 9); more efficient than the plasma forms in inducing platelet aggregation [117,118]. The series of multimers observed in plasma were then probably the products of proteolysis, or other rearrangements, and must have been formed at or after secretion into blood. In that case, a possible explanation for the presence of ultra-large multimers in TTP might be deficient proteolysis. Other studies [49,103] led to the suggestion that the small fragments of VWF (140 and 176 kDa), observed in normal plasma and in plasma of VWD Type 2A and 2B (after reduction), were the end result of proteolytic cleavages of the VWF multimers. Strong evidence for proteolysis was presented by Dent et al. [119], who showed that cleavage of the peptide bond 34

In this case activated with the snake venom enzyme Botrocetin.

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between residues Tyr-842 and Met-843 in a VWF protomer would generate the fragments of 140 and 176 kDa observed in plasma. Furthermore, cleavage of this single bond could explain the creation of multimeric series. Proteolysis would result in the multimers being degraded to smaller and smaller sizes and eventual cleavage of the VWF protomer would, on reduction, give rise to the above fragments [120,121]. The question now arose: which agent was involved in the degradation? Since Tyr–Met bonds were split, the likely culprit was a proteolytic enzyme. Plasmin was first considered but was ruled out, because it did not split Tyr–Met bonds, and so were several other proteolytic enzymes. Another suggestion was that a disulfide reductase might be involved. An interesting observation was provided by Frangos et al. [122] who found that the accumulation of ultralarge VWF polymers, secreted from endothelial cells exposed to shear stress, was prevented by cryosupernatants of normal or VWDType 3 plasmas. Interestingly, the ultra-large multimers that were deposited in retrograde direction into subendothelial space were not affected. Finally, Tsai et al. [123] showed a shear-stress dependent loss of the largest multimers in normal plasma. Most important, this loss was accompanied by the emergence of the specific split products (140 and 176 kDa) normally seen in plasma. It seemed most likely that the shear stress unfolded the multimers and made certain peptide bonds susceptible to cleavage by a plasma protease. Tsai [124] further showed that VWF multimers could be unfolded not just by shear stress but also by chaotropic agents, such as guanidine hydrochloride; in both cases with ensuing proteolysis. The enigmatic enzyme required calcium ions and was inhibited by EDTA, but not by a panel of synthetic and natural serine protease inhibitors. Partial purification indicated an enzyme belonging to the metallo-proteinase family. It was further shown that the enzyme spontaneously cleaved recombinant VWF Type 2A (group 2), in contrast to wild type VWF, without exposure to shear force [104]. The mutation had apparently changed the conformation of VWF in such a way that unfolding was not needed to induce proteolysis. Independently of Tsai’s group, Furlan et al. [125] analyzed fractions obtained by gelfiltration of VWF multimers in low ionic strength buffers35 containing urea. A fraction that separated from 35

A low ionic strength buffer was used because a previous study had shown that exposure of factor VIII-VWF complex to this milieu yielded two components, which at the time was taken to be evidence of a physical dissociation of the complex [126].

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the main multimers swiftly degraded VWF multimers into the fragments present in normal plasma (i.e. 140 and 170 kDa fragments). The enzyme required divalent cat ions and was inhibited by EDTA and EGTA. It had no effect on plasma proteins apart from VWF and collagen, suggesting high specificity for VWF. Further purification and sequence analysis led to the enzyme being finally identified as belonging to the ADAMTS family of metalloproteases [127]. In a subsequent study, the gene that was affected in four families with TTP, was sequenced and a new member of the ADAMTS family (a disintergrin and metalloprotease with thrombospondin motif) of metalloproteinases, ADAMTS 13, was identified [128]. Genome-wide linkage analysis placed the genetic locus in chromosome 9q34. The gene was expressed, resulting in an enzyme with the same specificity as that isolated from plasma [129]. The discovery of ADAMTS-13 was a major contribution to our understanding of the delicate balance that governs the action of VWF in haemostasis. The enzyme controls the size of VWF multimers, right at their loci of release from vascular wall, and thus diminishes their remarkable tendency to interact with platelets. In fact, deficient activity of the enzyme was found both in familial and in acquired cases of TTP [130,131]. In the latter cases inhibitors (IgG) to the enzyme were demonstrated. Deficiency of ADAMTS-13 may be part of the pathology in other diseases, e.g. in vascular diseases caused by artherosclerotic changes in the vasculature. In fact, ADAMTS-13 deficiency was recently suggested to play a role in coronary heart disease [132]. In other cases, a mutation of VWF may make multimers more susceptible to digestion by the enzyme and cause bleeding complications, as we see in VWD Type 2A. Likewise, it is possible that in VWD Type 2B, where the mutation has rendered the VWF multimers more reactive toward platelets, it has also made them more susceptible to proteolysis, which would explain both the absence of thrombotic manifestations and the presence of bleedings in this disease. One problem with assays of ADAMTS 13 is the difficulty in obtaining concordant results from different laboratories. This probably has to do with the indirect methods used for determining enzyme activity. The recently described synthetic substrate for ADAMTS 13 may clear the way for specific assays of the enzyme [133]. In a construct covering the sequence D1596 to R1668 of the A2 domain, the enzyme rapidly cleaved the Tyr1605–Met1506

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bond (corresponding to 842Tyr–843Met in the plasma form of VWF). Cleavage occurred under static conditions and in the absence of denaturants. The substrate was not cleaved by plasma of patients with TTP but was cleaved by normal plasma and by plasma of a patient with haemolytic uremic syndrom (HUS). VWF bound to subendothelial components may be the substrate favored by ADAMTS-13. However, as mentioned before, VWF multimers also bind to stimulated endothelium [93–95], and binding in this case is likely to be to glycosaminoglycans in the endothelium [95]. Such multimers are also good substrates for ADAMTS-13. It was shown that ultra-large multimers produced by stimulated endothelial cells, under static36 as well as under shear stress conditions, formed long (up to 3 mm) strings, decorated with platelets in the direction of flow [134,135]. As they were formed, these strings were anchored to endothelium and then rapidly (within minutes) cleaved by plasma ADAMTS-13. The digested multimers were similar to those normally present in plasma, which do not bind GP1b spontaneously but require modulators like ristocetin or botrocetin for that purpose. It is likely that the interaction between GP1b on platelets and its binding site in the A1 domain of VWF makes the multimers susceptible to digestion [136]. Surprisingly, heparin also stimulated proteolysis, possibly by a similar mechanism since studies have indicated that heparin, like ristocetin, is able to induce interaction between VWF and GPIb on platelets [137]. Glycosaminoglycans, like heparan sulfate, which is widely distributed on endothelium, may therefore have an in vivo role as a regulator of haemostasis by both binding the VWF multimers as well as stimulating their proteolysis.

Has Factor Other Guardians? As regards regulation of multimer size in blood, it is possible that processes other than proteolysis are important. Take, for example, the plasma of patients with TTP during remission. There is a multimeric ladder containing not only the ultra-large VWF multimers but also a multimeric series similar to that seen in normal blood. The latter may be the result of partial reduction of 36

It was suggested that the ultralarge VWF multimers did bind under these conditions, not only because of a larger number of binding sites, but also due to a change in the conformation of the A1 domain that has made this more adhesive [135].

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disulfide bonds and such multimers would be difficult to distinguish from those obtained by proteolysis by ADAMTS-13. The thioredoxin system (NADPH, thioredoxin and thioredoxin reductase) causes swift changes in the oligomeric structure and function of the factor VIII complex in vitro [45]. In VWF, a limited reduction of disulfide bonds with thioredoxin brought about a loss of ristocetin co-factor activity and this was accompanied by dissociation of factor VIII from the multimer. Multimers of lower molecular weight appeared and after prolonged reduction, dimers and monomers appeared. Thioredoxin is present in platelets and in vascular endothelium [138]. It was suggested that at the site of a lesion in a vessel, thioredoxin, released from platelets or present in subendothelium, may participate in decreasing the size of VWF multimers [45]. This would lead to decreased platelet adhesion but also to increased coagulation prowess since factor VIII at the same time dissociates from the multimers. However, reduction of disulfide bonds attenuates rapidly as average multimer size decreases. This is because reduction is dependent on NADPH and oxidation of this compound would lead to an increase in NADP+, which would favor reoxidation and formation of disulfide bonds. When, in one experiment, reduction was carried out in an O2 atmosphere, no intermediary polymers appeared and loss of ristocetin cofactor activity was negligible. This supports the notion that the reduction process is reversible. Reoxidation would restore the size of VWF multimers and their function in platelet adhesion, but at the same time the capacity to reduce disulfide bonds would also be restored; so the cycle starts all over again. Other reductases may also participate in the regulation of VWF activity since recent studies have implicated thrombospondin-1 in disulfide bond exchange with VWF, thereby reducing its size and activity [139]. Thrombospondin-1 appeared not to act on VWF multimers in plasma, but VWF multimers secreted from stimulated platelets rapidly decreased in size in the presence of Thrombospondin-1. Possibly it is in the latter location that thioredoxin and/or thrombospondin-1 have the best chances of acting.

ACKNOWLEDGMENT

This review covers mainly biochemical and biophysical events that occurred between 1950 and 2005. I am most grateful for the advice

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[80] Savage, B., Sixma, J.J. and Ruggeri, Z.M. (2002) Functional self-association of von Willebrand factor during platelet adhesion under flow. Proc. Natl. Acad. Sci. U.S.A. 99, 425–430. [81] Jenkins, C.S.P., Phillips, D.R., Clemetson, K.J., Meyer, D., Larrieu, M.-J. and Lu ¨ scher, E.F. (1976) Platelet membrane glycoproteins implicated in ristocetin-induced aggregation: studies of the proteins on platelets from patients with Bernard-Soulier syndrome and von Willebrand’s disease. J. Clin. Invest. 57, 112–124. [82] Okumura, T., Lombart, C. and Jamieson, G.A. (1976) Platelet glycocalicin. II. Purification and characterization. J. Biol. Chem. 251, 5950–5955. [83] Solum, N.O., Hagen, I., Filion-Myklebust, C. and Stabaek, T. (1980) Platelet glycocalicin: its membrane association and solubilization in aqueous media. Biochim. Biophys. Acta 597, 235–246. [84] Clemetson, K.J., Naim, H.Y. and Lu ¨ scher, E.F. (1981) Relationship between glycocalicin and glycoprotein Ib of human platelets. Proc. Natl. Acad. Sci. U.S.A. 78, 2712–2716. [85] Ruan, C., Tobelem, G., McMichael, A.J., Drouet, L., Legrand, Y., Degos, L., Kieffer, N., Lee, H. and Caen, J.P. (1981) Monoclonal antibody to human platelet glycoprotein I. II. Effects on human platelet function. Br. J. Haematol. 49, 511–519. [86] Sakariassen, K.S., Nievelstein, P.F.E.M., Coller, B.S. and Sixma, J.J. (1986) The role of platelet membrane glycoproteins Ib and IIb+IIIa in platelet adherence to human artery subendothelium. Br. J. Haematol. 63, 681–691. [87] Turitto, V.T., Weiss, H.J. and Baumgartner, H.R. (1984) Platelet interaction with rabbit subendothelium in von Willebrand’s disease: altered thrombus formation distinct from defective platelet adhesion. J. Clin. Invest. 74, 1730–1740. [88] Ikeda, Y., Handa, M., Kawano, K., Kamata, T., Murata, M., Araki, Y., Anbo, H., Kawai, Y., Watanabe, K., Itagaki, I., Sakai, K. and Ruggeri, Z.M. (1991) The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J. Clin. Invest. 87, 1234–1240. [89] Alevriadou, B.R., Moake, J.L., Turner, N.A., Ruggeri, Z.M., Folie, B.J., Phillips, M.D., Schreiber, A.B., Hrinda, M.E. and McIntire, L.V. (1993) Real-time analysis of shear-dependent thrombus formation and its blockade by inhibitors of von Willebrand factor binding to platelets. Blood 81, 1263–1276. [90] Savage, B., Almus-Jacobs, F. and Ruggeri, Z.M. (1998) Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell 94, 657–666. [91] Pie´tu, G., Cherel, G., Margurie, G. and Meyer, D. (1984) Inhibition of von Willebrand factor-platelet interaction by fibrinogen. Nature 308, 648–649. [92] Fellowes, A.P., Brennan, S.O., Holme, R., Stormorken, H., Brosstad, F.R. and George, P.M. (2000) Homozygous truncation of the fibrinogen Aa chain within the coiled coil causes congenital afibrinogenemia. Blood 96, 773–775. [93] Bloom, A.L., Giddings, J.C. and Wilks, C.J. (1973) Factor VIII on the vascular intima: possible importance in haemostasis and thrombosis. Nature New Biol. 241, 217–219.

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[94] Holmberg, L., Mannucci, P.M., Turesson, I., Ruggeri, Z.M. and Nilsson, I.M. (1974) Factor VIII antigen in the vessel walls in von Willebrand’s disease and haemophilia A. Scand. J. Haematol. 13, 33–38. [95] Andre´, P., Denis, C.V., Ware, J., Saffaripour, S., Hynes, R.O., Ruggeri, Z.M. and Wagner, D.D. (2000) Platelets adhere to and translocate on von Willebrand factor presented by endothelium in stimulated veins. Blood 96, 3322–3328. [96] Sobel, M., Soler, D.F., Kermode, J.C. and Harris, R.B. (1992) Localization and characterization of a heparin binding domain peptide of human von Willebrand factor. J. Biol. Chem. 267, 8857–8862. [97] Rastegar-Lari, G., Villoutreix, B.O., Ribba, A.-S., Legendre, P., Meyer, D. and Baruch, D. (2002) Two clusters of charged residues located in the electropositive face of Von Willebrand factor A1 domain are essential for heparin binding. Biochemistry 41, 6668–6678. ¨ck, M., Nyman, D. and Anvret, M. (1993) Mutations [98] Zhang, Z.P., Blomba of von Willebrand factor gene in families with von Willebrand disease in ˚ land Islands. Proc. Natl. Acad. Sci. U.S.A. 90, 7937–7940. the A ¨ck, M. and Anvret, M. (1995) Effects of [99] Zhang, Z., Lindstedt, M., Blomba the mutant von Willebrand factor gene in von Willebrand disease. Hum. Genet. 96, 388–394. [100] Schneppenheim, R., Budde, U. and Ruggeri, Z.M. (2001) A molecular approach to the classification of von Willebrand disease. Best Pract. Res. Clin. Haematol. 14, 281–298. [101] Sadler, J.E. (2005) New concepts in von Willwbrand disease. Annu. Rev. Med. 56, 173–191. [102] Peake, I.R., Bloom, A.L. and Giddings, J.C. (1974) Inherited variants of factor VIII-related protein in von Willebrand’s disease. N. Engl. J. Med. 291, 113–117. [103] Zimmerman, T.S., Dent, J.A., Ruggeri, Z.M. and Nannini, L.H. (1986) Subunit composition of plasma von Willebrand factor. Cleavage is present in nomal individuals, increased in IIA and IIB von Willebrand disease, but minimal in variants with aberrant structure of individual oligomers (Types IIC, IID and IIE). J. Clin Invest. 77, 947–951. [104] Tsai, H.-M., Sussman, I.I., Ginsburg, D., Lankhof, H., Sixma, J.J. and Nagel, R.L. (1997) Proteolytic cleavage of recombinant Type 2A von Willebrand factor mutants R834W and R834Q: inhibition by doxycycline and by monoclonal antibody VP-I. Blood 89, 1954–1962. [105] Ruggeri, Z.M., Pareti, F.I., Mannucci, P.M., Ciavarella, N. and Zimmerman, T.S. (1980) Heightened interaction between platelets and factor VIII/ von Willebrand factor in a new subtype of von Willebrand’s disease. N. Engl. J. Med. 302, 1047–1051. [106] Holmberg, L., Dent, J.A., Schneppenheim, R., Budde, U., Ware, J. and Ruggeri, Z.M. (1993) von Willebrand factor mutation enhancing interaction with platelets in patients with normal multimeric structure. J. Clin. Invest. 91, 2169–2177. [107] Ribba, A.S., Christophe, O., Derlon, A., Cherel, G., Siguret, V., Lavergne, J.M., Girma, J.P., Meyer, D. and Pietu, G. (1994) Discrepancy between IIA phenotype and IIB genotype in a patient with a variant of von Willebrand disease. Blood 83, 833–841.

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[108] Nishino, M., Girma, J.-P., Rothschild, C., Fressinaud, E. and Meyer, D. (1989) New variant of von Willebrand disease with defective binding to factor VIII. Blood 74, 1591–1599. [109] Hilbert, L., Jorieux, S., Proulle, V., Favier, R., Goudemand, J., Parquet, A., Meyer, D., Fressinaud, E., Mazurier, C. and the INSERM network on molecular abnormalities in von Willebrand disease. (2003) Two novel mutations, Q1053 H and C1060R, located in the D3 domain of von Willebrand factor, are responsible for decreased F VIII-binding capacity. Br. J. Haematol. 120, 627–632. [110] Moschcowitz, E. (1924) Hyaline thrombosis of the terminal arterioles and capillaries: a hitherto undescribed disease. Proc. New York Pathol. Soc. 24, 21–24. [111] Baehr, G., Klemperer, P. and Schifrin, A. (1936) An acute febrile anemia and thrombocytopenic purpura with diffuse platelet thromboses of capillaries and arterioles. Trans. Assoc. Am. Physician 65, 43–58. [112] Neame, P.B., Lechago, J., Ling, E.T. and Koval, A. (1973) Thrombotic thrombocytopenic purpura: report of a case with disseminated intravascular platelet aggregation. Blood 42, 805–814. [113] Asada, Y., Sumiyoshi, A., Hayashi, T., Suzumiya, J. and Kaketani, K. (1985) Immunohistochemistry of vascular lesion in thrombotic thrombocytopenic purpura, with special reference to factor VIII related antigen. Thromb. Res. 38, 469–479. [114] Moake, J.L., Rudy, C.K., Troll, J.H., Weinstein, M.J., Colannino, N.M., Azocar, J., Seder, R.H., Hong, S.L. and Deykin, D. (1982) Unusually large plasma factor VIII: von Willebrand factor multimers in chronic relapsing thrombotic thrombcytopenic purpura. N. Engl. J. Med. 307, 1432–1435. [115] Moake, J.L. and McPherson, P.D. (1989) Abnormalities of von Willebrand factor multimers in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome. Am. J. Med. 87, 9N–15N. [116] Sanders, W.E. Jr., Reddick, R.L., Nichols, T.C., Brinkhous, K.M. and Read, M.S. (1995) Thrombotic thrombocytopenia induced in dogs and pigs: the role of plasma and platelet vWF in animal models of thrombotic thrombocytopenic purpura. Arterioscl. Thromb. Vas. Biol. 15, 793–800. [117] Moake, J.L., Turner, N.A., Stathopoulos, N.A., Nolasco, L.H. and Hellums, J.D. (1986) Involvement of large plasma von Willebrand factor (vWF) multimers and unusually large vWF forms derived from endothelial cells in shear stress-induced platelet aggregation. J. Clin. Invest. 78, 1456–1461. [118] Tsai, H.-M., Nagel, R.L., Hatcher, V.B. and Sussman, I.I. (1989) Multimeric composition of endothelial cell-derived von Willebrand factor. Blood 73, 2074–2076. [119] Dent, J.A., Berkowitz, S.D., Ware, J., Kasper, C.K. and Ruggeri, Z.M. (1990) Identification of a cleavage site directing the immunochemical detection of molecular abnormalities in Type IIA von Willebrand factor. Proc. Natl. Acad. Sci. U.S.A. 87, 6306–6310. [120] Dent, J.A., Galbusera, M. and Ruggeri, Z.M. (1991) Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit. J. Clin. Invest. 88, 774–782. ¨mmle, B. [121] Furlan, M., Robles, R., Affolter, D., Meyer, D., Baillod, P. and La (1993) Triplet structure of von Willebrand factor reflects proteolytic

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G. Semenza (Ed.) Stories of Success – Personal Recollections. X (Comprehensive Biochemistry Vol. 45) r 2007 Elsevier B.V. All rights reserved. DOI: 10.1016/S0069-8032(07)45007-0

Chapter 7

A Neuropathologist’s Diary ADRIANO AGUZZI The Institute of Neuropathology, University Hospital of Zurich, Schmelzbergstrasse 12, CH 8090 Zurich, Switzerland

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Abstract Besides directing the MD-PhD program at the University of Zurich and chairing the Department of Pathology, Adriano Aguzzi runs a laboratory which is entirely dedicated to the study of prion diseases. In this autobiographical essay, he traces the motifs behind his tortuous trajectory which started in Italy and led him to Switzerland after two decades of stints in Germany, Austria, and the United States. Keywords: prions; neurodegeneration; transgenic mice; knockout mice

Autobiographical essays are the most difficult of all writings – particularly for scientists. When I was invited by Giorgio Semenza to write such a piece, therefore, I was left with mixed feelings. At the age of 45, I am pretty much in the middle of my scientific activity and I am still hopeful that there will still be more scientific thrills ahead than behind. But then, is there a reasonable approach to a personal look-back? Certainly, I would not be interested in writing a selfcelebration – and most importantly, I would wish that my writing be useful at least to somebody. The latter point has motivated me to tackle this task despite all the above caveats. My scientific activities in the past two decades have been reasonably successful: it may be interesting to some younger scientists – in addition to representing a useful introspection for me – to analyze their framework, and to identify pivotal decisional points that have contributed to the success of specific projects. Therefore, I will try and trace my scientific motives and achievements, and also describe the impact of those scientists who taught me how to research the molecular bases of diseases. In 1979, the Italian undergraduate curriculum in the medical sciences was utterly uninviting. Therefore, although my hometown Pavia (Italy) sports a distinguished university, after no more than one semester I gave up my enrollment at the medical faculty and decided to continue my studies in Germany. Three years into medical school in Freiburg im Breisgau, I felt the urge to learn the methods of basic science. Thanks to the mediation of an old friend, Gianpaolo Merlini (thence postdoc in New York), I was given the opportunity to do an elective in Soldano Ferrone’s lab at Columbia University, where Patrizio Giacomini taught me how to

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make monoclonal antibodies, and introduced to the thenomnipresent methods of immunochemistry: sandwich assays, radioimmunoprecipitation, immunofluorescence, and so on. As naı¨ve as this may seem with nowadays’ hindsight, in 1983 immunotherapy of cancer seemed to lie just around the corner. A wealth of tumor antigens was being discovered, and the booming technology of monoclonal antibodies made it seem likely that one could soon use them as magic bullets against cancer [1]. Several of the genes encoding interferons had been cloned [2,3], and since these substances seemed to hold some promise in the therapy of malignant melanoma, Patrizio and I deemed it reasonable to investigate the effects of interferons on the expression of melanoma-associated tumor antigens [4,5] and of histocompatibility antigens [6,7]. At that time I would not have imagined that, some ten years later, I was to make the acquaintance of some of the most prominent scientist in the interferon field, Jean Lindenmann who discovered interferon [8], and Charles Weissmann who cloned the first interferon gene [2], and to initiate an extraordinarily fruitful scientific collaboration with the latter scientist – albeit in an area which, to the best of my and Charles’ knowledge, is totally unrelated to interferons. As we all now know, tumor immunotherapy – with or without interferons – in the years to come was not to hold much of what it had been promising. While the last word may not have been written yet on that subject, by the end of my medical training I decided that I had had enough of tumor immunology, and opted for moving upwards, i.e. into the central nervous system. In July 1986 I took on a residency in neuropathology with Paul Kleihues, at the time Director of the Institute of Neuropathology at the University of Zurich. Ever since the time of Alois Alzheimer, neuropathology has always been an exciting field, right at the interface between clinical medicine and basic science. When compared to our colleagues in general histopathology, we neuropathologists are a privileged lot: we do not need to spend much of our time diagnosing warts and inflamed gall bladders under the microscope. On top of that, since surgeons are in general more inclined to removing gall bladders than brains, the total diagnostic workload of neuropathologists is often smaller than that of general pathology. But such privileges bear also duties, and therefore one should insist that respectable neuropathologists in academic institutions

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be engaged in scientific activities – whose excellence ought to be measured against the same standards by which our colleagues in the natural sciences are evaluated.

Zurich, Part I: Brains of Humans and Mice The bread and butter of neuropathology is the diagnosis of neuroectodermal tumors resected by neurosurgeons, and that of neurodegenerative diseases – the latter being, for obvious reasons, mainly a postmortem exercise. I found each one of these fields exciting enough to dedicate the next 12 years of my scientific existence to both. Besides being an eminent morphologist, and the driving force behind the current World Health Organization classification of brain tumors, Paul Kleihues was obsessed by the problem of carcinogenesis in the nervous system, and by the events driving progression of benign gliomas to states of higher malignancy. I was assigned the project of investigating whether expression of tyrosine kinase oncogenes of the src family might initiate glioma tumorigenesis in mice. Since the activity of c-src is tightly regulated by tyrosine phosphorylation of the SRC protein, I feared that overexpression of c-src in transgenic mice might not be helpful to reach the above goal, and thought it more promising to use constitutively active SRC variants. Unavoidably, I came across the work of Erwin Wagner who had pioneered the field of in vivo analysis of oncogenes with transgenic mice. Another few days, and I was driving to Heidelberg (with Otmar Wiestler and Roy Weller, a British neuropathologist on sabbatical in Zurich) to meet Erwin at EMBL and to discuss a possible collaboration. I thought it intriguing to express the viral v-src oncogene, or the middle T antigen of polyoma virus which binds and activates SRC along with a bunch of other enzymes [9], in astrocytes of mice – but I soon learnt from Lindsay Williams and Catherine Boulter that constitutive expression of either gene would kill mice during embryogenesis [10,11]. Therefore, generation of transgenic or chimaeric mice with any of the systems available at that time might not prove useful to investigate whether these proteins were tumorigenic for neural tissue, and which kind of tumors they would induce. We therefore transduced primary embryonic neuroectodermal cells of rats with v-src, middle T, and c-src527Tyr-Phe using replication-defective

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retroviral vectors, and resorted to neural transplantation into wild-type hosts to analyze the consequences. While middle T induced cavernous hemangiomas in the brain of recipient animals, v-src led to the development of astrocytomas [12], yet when middle T was introduced into the germ line of mice using an extremely similar construct, neuroblastomas developed [13]. Several years later, I (with the help of my co-workers Gerhard Malin, Jakob Weissenberger, and Joachim Steinbach) ended up hooking v-src to the glial fibrillary acidic protein (GFAP) promoter, and producing what still seems to be the only available transgenic model for astrocytoma development [14]. Only few GFAP-v-src transgenic mice develop full-blown astrocytomas, but all of them display preneoplastic lesions: this renders them potentially useful to identify the genetic lesions responsible for progression of gliomas to malignancy. We have pursued the latter idea using proviral tagging and grafting of GFAP-v-src neuroectodermal tissue, an approach similar to the classic studies of Anton Berns on lymphoma development in mice infected with Moloney murine leukemia virus [15].

The Vienna Experience The application of in vivo gene transfer methodologies to neuropathological questions fascinated me: therefore it seemed logical to join Erwin Wagner who had just been recruited to the Institute of Molecular Pathology in Vienna. With Erwin’s and Max Birnstiel’s support, I had the opportunity to set up a tiny unit of ‘‘molecular morphology’’ (dealing with techniques like in situ hybridizations, etc.), and to analyze genotype–phenotype correlations in a variety of transgenic models of disease. It was Ivan Horak and Axel Rethwilm who sparked my interest in transgenic models of neurodegenerative diseases, and together with them I spent the next several years in the attempt to understand the neural damage occurring in transgenic mice expressing genes of the human foamy virus (HFV), a relatively obscure retrovirus which had been claimed to be prevalent in certain regions of Africa. The detailed analysis of HFV transgenic mice led to the identification of viral genes responsible for the various aspects of their complex neurodegenerative phenotype [16–24]. The system has not lost any of its fascination: since HFV is extremely

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promiscuous in its choice of host cells, there is some hope that the elimination of the determinants of neurotoxicity may lead to its exploitation as a versatile vector for gene therapy [25,26]. After three years of intimacy with transgenic mice, I felt like moving back into clinical neuropathology. I therefore returned to Zurich, and took up a position as registrar in neuropathology in July 1992. A few months before my departure from Vienna, Erwin had taken me along to a meeting of the Schering Foundation in Berlin on ‘‘transgenic models of disease’’, and it was there that I had the occasion to spend a memorable evening with Charles Weissmann. By then, Weissmann had been working on prion diseases for almost a decade, and his accomplishment in this field had been instrumental in shifting prion research from rather esoteric grounds onto the domain of solid molecular biology. Among other things, Charles had isolated Prnp, the gene which encodes the normal prion protein PrPC, thereby providing crucial evidence in favor of Stan Prusiner’s ‘‘protein-only’’ hypothesis of prion replication, and suggesting the possibility that infectious prions might replicate by recruitment and conversion of normal prion protein [27–29]. He had also produced a remarkable amount of theoretical work on the protein-only theory [30–32]. With his colleagues Michel Aguet and Hansru ¨ edi Bu ¨ eler, Charles had gone on to create Prnp knockout mice [33].

Zurich, Part II: Tracking Prions Charles Weissmann was not entirely happy with the histopathological analysis of these mice, and when he learnt that I intended to move to Zurich he immediately proposed that we collaborate on the attempt of infecting Prnpo/o mice with a mouse-adapted strain of the sheep scrapie agent. Sure enough, knockout mice stayed healthy and displayed no neuropathological changes in their brains [34]; moreover they did not support replication of the infectious agent [35]. My involvement in these now-famous studies was essentially restricted to histopathological analyses: it is a typical sign of Charles Weissmann’s generosity that he let me participate in this project, and in fact also in all other prionrelated experiments going on in his laboratory. Ever since, Charles Weissmann unconditionally and enthusiastically supported my wish to establish an own prion research program.

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My initial desire was to elucidate the pathogenetic events which are initiated by the introduction of prions into the brain, and which eventually result in the tremendous cerebral damage typical of patients who develop Creutzfeldt-Jakob disease. Almost nothing was known about the molecular basis of this process. In theory, CNS damage could come along because of PrPSc toxicity, or because the conversion of PrPC into PrPSc may abruptly reduce its availability to cellular processes for which it may be needed. That Prnp knockout mice do not suffer from spontaneous scrapie does not necessarily rule out the second hypothesis, since absence of PrPC from early development on may arguably be compensated for more easily than withdrawal during adult life. By then, we had learnt many facts about the biology of neural grafts [36–38], and I had convinced myself that the neurografting technology could be useful for addressing a variety of questions not necessarily related to carcinogenesis. Therefore, it was only logical to try and figure whether neurografting could represent a possible approach to the question of prion neurotoxicity. My original idea was that a piece of brain highly overexpressing PrPC grafted in the middle of a Prnpo/o brain might, once infected, serve as a continuous source of PrPSc. At this point, I had some luck: Sebastian Brandner had started as intern in neuropathology, and did not feel enough challenged with the clinical tasks which Paul Kleihues had assigned to him. Sebastian volunteered to devote his spare time to the grafting experiment detailed above, and this was the beginning of a collaboration which has fruitfully evolved during the last 6 years. Predictably, prion-infected neurografts derived from PrP-overexpressing transgenic donors designated tga20 [39] and infected with prions underwent severe degeneration, yet PrP-deficient host mice did not develop clinical symptoms of disease, and brain tissue surrounding the grafts was histologically unaffected despite significant leaks of PrPSc from the graft [40,41]. Therefore, expression of PrPC is important not only for replication of the pathological agent, but also for initiation of pathogenesis: PrPC-deficient neurons failed to succumb to scrapie despite chronic exposure to the agent and to PrPSc – these two terms, given the current state of knowledge, do not necessarily need to indicate the same physical entity [42]. The apparent lack of clinical symptoms in mice harboring a scrapiesick graft allowed us to determine what would happen if disease was allowed to progress for a period of time much longer than the

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usual life span of mice. Under such circumstances, we found neurons to be virtually wiped out while other cell types turned out to be essentially resistant to the disease [43]. In March 1996, the Edinburgh neurology group of Robert Will described the occurrence of a new variant of Creutzfeldt-Jakob disease (nvCJD) which occurred exclusively in very young individuals [44], and which much circumstantial evidence is incriminating as being caused by the infection of humans with the agent of bovine spongiform encephalopathy (BSE) [45–47]. The impact of the BSE crisis on the prion research community was already becoming enormous, and was precipitated by this dramatic announcement. From its status of a ‘‘non-existent’’ disease (as one colleague neurologist in Zurich described it), CreutzfeldtJakob disease had become literally overnight a household byword. It became compelling to wonder whether the research we were doing might bear any practical relevance to the question of whether, and how, the BSE agent can get into the brain of humans. After all, intracerebral inoculation with prions, though convenient for eliciting spongiform encephalopathy in experimental animals with high efficiency and with minimal latency, is not really the commonest way by which infection occurs in real life. How do prions, when entering the body through extracerebral sites, manage to reach the central nervous system, which is the only place in the body where they are capable of wreaking havoc? Within the framework of the protein-only hypothesis, at least in its most simplified incarnation, it might be appealing to speculate that single molecules of the scrapie agent might just randomly diffuse through the body, and with some bad luck (from the viewpoint of the patient) hit a nerve cell and start the chain reaction which ultimately leads to spongiform encephalopathy. One argument, however, deposes strongly against this hypothesis: the latency period from inoculation into the peritoneum cavity to onset of clinical symptoms and to death of experimental animals, is extremely precise. Although such latency typically extends over 200–250 days depending on the prion and mouse strains used, its standard deviation when inoculating a defined number of infectious units of scrapie infectivity into the peritoneum of mice, is typically in the range of a few percent. Such a precise process bears resemblance to the functioning of a Swiss clockwork, and one is reluctant to believe that it owes to a random

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diffusion event. Besides, a neuropathologist’s clinical experience teaches that most neurotropic viruses have evolved extremely sophisticated mechanisms for invading the brain once inoculated into distant sites. Think of herpes viruses, or of rabies virus, both of which exploit retrograde axonal transport and have been conveniently used as tracer to identify networks of neuronal connectivity. In the case of scrapie prions, a considerable body of older evidence indicated that the peripheral nervous system plays an important role in prion neuroinvasion. Already in the 1970s the scientists at the Neuropathogenesis Unit of Edinburgh demonstrated that neural projections, both in the central and in the peripheral nervous system, were pivotal to prion spread: when injected into the footpad or the peritoneal cavity, prions were shown to replicate first in those segments of the spinal cord where the corresponding spinal nerves projected. And intraocular injection led to targeting of the contralateral colliculus superior as expected by the neuroanatomy of the retinotectal projection pathway [48]. The other relevant player for neuroinvasion, which had been already identified when we started our work, was the immune system. Elegant studies by Tetsuyuki Kitamoto [49] and others [50,51] had shown that the lack of an intact immune system would prevent neuroinvasion of prions administered to the peritoneal cavity of mice. If prions utilize these organs systems to spread throughout the body, which are of the molecules necessary for this process? Once identified, such molecules could be used to interfere with the spread of prions. A straightforward assumption was that PrPC itself might represent a crucial element. PrP is not only expressed in the central nervous system, but in many other cells of the body, most notably lymphocytes. But how to detect spread of infectivity to the nervous system if one uses PrP-deficient mice? Such mice are, as repeatedly shown by several groups [34,52,53], totally resistant to scrapie and cannot replicate the agent. We resorted to introducing a neural graft, which in this case would act as an indicator of neuroinvasion. If the graft developed histopathologically recognizable disease, and accumulated PrPSc and infectivity, this would mean that neuroinvasion had taken place. It turned out that peripheral inoculation at all sites we tried never sufficed to achieve neuroinvasion in this system. This was true for the eye, for the peritoneal cavity, and for the footpad.

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At first blush, these results would seem to indicate that the absence of normal prion protein totally prevents neuroinvasion, in addition to preventing replication of the agent and neurotoxicity. The initial enthusiasm we had developed for this theory became somewhat dampened though, when Charles Weissmann critically reviewed the data and suggested that Prnp knockout mice, which have never seen normal prion protein before in their life, might develop a humoral immune response against PrP when grafted with neuroectodermal tissue heavily overexpressing this protein. Upon his suggestion, we looked for evidence of such an immune response, and to our consternation we promptly found it: Western blots of recombinant and of mammalian PrP probed with serum of grafted PrP-deficient mice, indicated high titers of anti-PrP antibodies [54]. Therefore, it was not clear whether spread of prion infectivity was prevented by the presence of antibodies, or by the absence of PrP. Thanks to the helpfulness of Andreas Sailer, Alex Raeber and Charles Weissmann, we could use a line of mice called tg33, which they had developed for other purposes and which expresses high levels of PrP in T-lymphocytes. Presumably owing to clonal deletion during thymic development, these mice are incapable of mounting an anti-PrP immune response. When we repeated the experiment with tg33 mice, we found that intraocular (but not intraperitoneal) inoculation still failed to produce disease in the graft. Therefore, at least for the intraocular route, we conclude that absence of PrP effectively prevents the spread of prions [54]. This does not necessarily mean, however, that anti-PrP antibodies will not prevent prion spread, and in fact a wealth of evidence now indicates that prions can be neutralized immunologically [55]. Given the large amount of literature supporting an important role of the immune system in prion spread, we embarked onto a bone marrow grafting enterprise using PrP-expressing (wild-type or tga20 transgenic) mice as donors, and Prnp knockout mice with a PrP-expressing graft in their brain as recipients. Thomas ¨ttler, a student in my laboratory who in the meantime has Bla gone on to become a neurologist, has been performing these studies with significant help from the crew of Rolf Zinkernagel. Thomas found that this manipulation sufficed to restore accumulation of prions in spleen, and we were certainly predicting that it would suffice to restore neuroinvasion. Instead, we were

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almost shocked to learn that the contrary was the case. We detected scrapie histopathology in only one of twenty-eight grafts of bone marrow-reconstituted mice. This led us to propose a model in which the immune system is important for moving prions from the site of injection to lymphatic organs; however, PrP expression in peripheral nerves might be crucial for actual neuroinvasion [56]. Unfortunately, the latter hypothesis was to prove rather resilient to all attempts at positive experimental verification. Ideally, one would wish to reconstitute expression of PrPC on peripheral nerves selectively, and show this manipulation to be sufficient for restoration of neuroinvasion. We are attempting to achieve this with the help of PrP-transducing adenoviral vectors which were generated by Eckhard Flechsig and Charles Weissmann. Conversely, Weissmann and Bea Navarro are attempting a similar experiment by the transgenic route using promoters specific to the peripheral nervous system. Since manipulation of the immune system seemed easier to accomplish, we sought to identify specific cell types in lymphoid organs which might be relevant to neuroinvasion. At this point, I was extremely lucky to elicit in Rolf Zinkernagel some interest for this project. Zinkernagel was (and probably still is) not exactly a fervent supporter of the protein-only hypothesis: he was intrigued by the possibility that prion diseases may be intimately connected to dysfunctions of the immune system – although he never yielded to pleas to explain such thoughts in more detail. Rolf Zinkernagel and Hans Hengartner, who are conveniently located one floor above us at the Institute of Experimental Immunology in Zurich, had assembled a collection of various immunodeficient mice gathered from laboratories all over the world with specific defects of the immune system, and made them available to us for inoculation into brain and peripheral sites. Michael Klein, who had joined my laboratory after a postdoctoral period in experimental neuropathology in Munich, took on the task of assessing the sensitivity to scrapie of all these mutant strains. Contrary to Zinkernagel’s guess (which he underlined by betting some champagne with us on its correctness), all mice suffering from immune deficiencies developed scrapie within the normal latency when inoculated intracerebrally. After intraperitoneal inoculation, however, all mice that lacked terminally differentiated B-lymphocytes did not develop disease, suggesting

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a crucial role for these cells in transport of prions [57]. Even mMT mice carrying a targeted disruption of the transmembrane domain of the immunoglobulin m chain, whose only defect consists in lack of terminal maturation of B-cells [58], were protected.

A Function for the Prion Protein The original Prnpo/o mice did not display any severe abnormalities. However, some of the knockout lines generated later develop progressive cerebellar Purkinje cell degeneration with ataxia in advanced age. This phenotype was originally attributed to the lack of PrPC and ran counter to the two PrP knockout mouse lines produced earlier: the ZH-I Prnpo/o [33] and the Edbg Prnp / mice [59]. The characterization of Ngsk Prnp / mice was particularly conscientious: the authors reintroduced Prnp as a transgene by genetic crosses, and showed that this manipulation rescued the Purkinje cell degeneration. It seemed entirely reasonable, hence, to conclude that PrPC is necessary for cerebellar homeostasis. Yet this interpretation could not be easily reconciled with the lack of phenotype in the remaining knockout lines, and eventually was proven to be incorrect. The inconsistency was eventually resolved by David Westaway’s discovery of a novel gene located just 16 kilobases downstream of Prnp and encoding a 179-residue protein that has sequence similarities to the C-terminus of PrP and was thus termed Doppel or Dpl [60]. It then emerged that the gene targeting strategy in all ataxic PrP-deficient mice was associated with deletion of a splice acceptor site located on the coding exon of Prnp. This modification effectively places Dpl under transcriptional control of the Prnp promoter. As a consequence, brain expression of Dpl, which is normally very low, skyrockets in Nsgk, ZH-II and Rcm0 mice [61]. This is clearly neurotoxic, as ablation of the Dpl reading frame from ZH-II mice abolishes the Purkinje cell degeneration phenotype [62,63]. Most intriguingly, Dpl-dependent neurodegeneration is abolished by cell-autonomous co-expression of full-length PrP [64]. Formally, this indicates that Dpl and PrPC act antagonistically, maybe because they bind to a hitherto conjectural common ligand which was provisionally termed LPrP [65]. Alternatively, PrPC and Dpl might engage in heterooligomeric complexes, whose

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function could depend on their stoichiometric composition [66]. The same mechanism may be operative in transgenic mice produced by Doron Shmerling and Charles Weissmann [65], in an attempt to specify the domain of PrPC required for prion replication. Expression of a PrP variant that lacks a large part of the N-terminus of PrP in Prnpo/o mice induces spontaneous cerebellar degeneration, which however affects granule cells rather than Purkinje cells (the promoter used was inactive in Purkinje cells), and can also be prevented by the co-expression of a single endogenous Prnp allele. Structural studies have shown that human Dpl contains a relatively short, flexibly disordered ‘‘tail’’ comprising residues 24–51, and a globular domain extending from residues 52 to 149 for which a detailed structure was obtained [67]. Despite their highly divergent primary sequence, Dpl is largely superimposable to the carboxy proximal half of PrPC. The molecular pathways by which Dpl and amino proximally truncated PrP damage the cerebellum are unknown. However, the suppressibility of both phenotypes by full-length PrPC is indicative of a high degree of specificity. Therefore, I am still convinced that this model represents the best-validated window of entry to determine the function of PrPC in vivo.

The Future of Prion Therapeutics An impressive wealth of molecules was touted as potential antiprion lead compounds. However, none of these therapeutical leads have proven their usefulness yet in clinical settings, and some have conspicuously failed. One of the possible problems derives from the fact that most antiprion compounds were identified in cell culture assays, where chronically prion-infected neuroblastoma cells are ‘‘cured’’ of their PrPSc and prion burden. A startling variety of substances appears to possess such prioncuring properties: a non-exhaustive list includes compounds as diverse as Congo red [68], amphotericin B, anthracyclins [69], sulfated polyanions [70], porphyrins [71], branched polyamines [72], ‘‘beta-sheet breakers’’ [73], and the spice curcumin [74]. Disappointingly, none of these compounds proved very effective for actual therapy of sick animals – let alone patients. We therefore believe that it is premature to treat patients with alleged antiprion drugs on the sole basis of antiprion efficacy in neuroblastoma cells.

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This shortcut was taken in the case of quinacrine, which cures scrapie-infected cultured cells with impressive efficacy [75], yet appears to be utterly ineffective in scrapie-infected mice [76] and in CJD patients [77], besides being severely hepatotoxic [78]. Why do scrapie-infected cells fare so poorly as model system for prion therapy? In our experience, infection rarely hits all cells in any given culture, and the prion-infected state can be quite unstable. Therefore, one could speculate that a variety of stressors may masquerade as antiprion cures by conferring a selective advantage to non-infected cells. This interpretation would explain the puzzling observation that antiprion ‘‘cure’’ is brought about by compounds with no structural or biological similarities. Cytidyl-guanyl oligodeoxynucleotides (CpG-ODN), which bind Toll-like receptor 9 (TLR9) and stimulate innate immune responses, were reported to delay disease upon chronic administration to scrapie-infected mice [79]. The contention that immune stimulation might protect against prions is extraordinary, and is difficult to reconcile with the observation that immune deficiencies of all kinds inhibit prion spread [57,80–83]. Besides, MyD88 / mice undergo normal prion pathogenesis despite abrogation of TLR9 signaling [84], and we could not evidence any major effects of TLR9 stimulation on the course of disease – in a paradigm identical ¨lder and AA, to that described originally (MP, Mathias Heikenwa unpublished results). Hence, more detailed studies will be needed to understand the basis of the antiprion effect of CpG-ODN. On a more positive note, the tremendous interest in this field has attracted researchers from various neighboring disciplines, including immunology, genetics, and pharmacology, and therefore it is to hope that rational and efficient methods for managing prion infections will be developed in the future.

Immunotherapy against Prions? Prions are sturdy and their resistance against sterilization is proverbial, yet exposure in vitro to anti-PrP antisera can reduce the titer of infectious hamster brain homogenates [85]. Anti-PrP antibodies were found to inhibit formation of protease-resistant PrP in a cell-free system [86]. Also, antibodies [82] and F(ab) fragments to PrP [87,88] can suppress prion replication in cultured cells.

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While these data suggest the feasibility of antiprion immunoprophylaxis, the mammalian immune system is essentially tolerant to PrPC; this is hardly a surprise, given that PrPC is expressed on T- and B-cells. Ablation of Prnp [33] renders mice highly susceptible to immunization with prions [54], and indeed some of the best monoclonal antibodies to PrPC were generated in Prnpo/o mice [89]. Tolerance was circumvented by transgenic expression of an immunoglobulin m chain containing the epitope-interacting region of 6H4, a high-affinity anti-PrP monoclonal antibody [90]. The transgenic m chain associated with endogenous k and l chains: some pairings lead to reactive moieties and, consequently, to high anti-PrPC titers in Prnpo/o and Prnp+/+ mice. The buildup of anti-PrPC titers, however, was more sluggish in the presence of endogenous PrPC, suggesting that clonal deletion is actually occurring. B cell clones with the highest affinity to PrPC are probably eliminated by tolerance, while clones with medium affinity are retained. The latter sufficed to block prion pathogenesis upon intraperitoneal prion inoculation [55]. Hence, B cells are not intrinsically tolerant to PrPC, and can – in principle – mount a protective humoral response against prions. It was then found, in a follow-up study, that passive transfer of anti-PrP monoclonal antibodies (in admittedly heroic amounts) can delay the onset of scrapie in mice infected with prions intraperitoneally, albeit not such infected intracerebrally [91]. The challenges to a practical antiprion immunization, however, are enormous. While providing an encouraging proof of principle, transgenic immunization cannot easily be reduced to practice. Further, no protection was observed if treatment was started after the onset of clinical symptoms, suggesting that passive immunization might be a good candidate for prophylaxis rather than therapy of TSEs. Active immunization, like in most antiviral vaccines, may be more effective, but is rendered exceedingly difficult by the stringent tolerance to PrPC.

Soluble Prion Antagonists In several paradigms, expression of two PrPC moieties subtly different from each other antagonizes prion replication. For example, humans heterozygous for a common Prnp polymorphism at

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codon 129 are largely protected from CJD: this effect is so important that it may have acted as selective evolutionary pressure [92]. Similarly, transgenic expression of hamster PrPC renders Prnpo/o mice highly susceptible to hamster prions, whereas co-expression of mouse PrPC diminishes this effect. Transdominant single nucleotide mutations of Prnp have also been described [93]. The molecular basis for these effects is unknown; perhaps the subtly modified PrPC acts as a decoy by binding incoming PrPSc (or protein X), and sequestering it into a complex incapable of further replication. We tested the latter hypothesis by fusing an immunoglobulin Fcg domain to PrPC. The Fcg tail served multiple purposes: (i) ligand dimerization, which may enhance its avidity for interacting partners, (ii) provision of a convenient tag for affinity purification, (iii) expression of the protein as a soluble moiety, which allows for testing cell-autonomous effects, and (iv) increased stability in body fluids. Excitingly, the PrP-Fc2 fusion protein was found to compete with PrPC for PrPSc, and to prolongs the latency period of prion infection upon expression in transgenic mice [94]. It will be exciting to determine whether PrP-Fc2 can act cell-autonomously when delivered as a drug. If that proves true, soluble prion protein mutants may represent useful prionostatic compounds.

Inflammation: A License to Replicate? As lymphoid infectivity occurs in most examples of prion disease, and proinflammatory cytokines and immune cells are involved in lymphoid prion replication [49,95–99], we assessed whether chronic inflammatory conditions within non-lymphoid organs could affect the dynamics of prion distribution. Indeed, inclusion body myositis, which is an inflammatory disease of muscle, was reported to lead to the presence of large PrPSc deposits in muscle [100]. Indeed, many chronic inflammatory conditions, some of which are very common and include rheumatoid arthritis, type-I diabetes, Crohn’s disease, Hashimoto’s disease, and chronic obstructive pulmonary disease, result in organized inflammatory foci of B-lymphocytes, FDCs, DCs, macrophages and other immune cells associated with germinal centers [101–104]. In

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addition, extranodal metastases of Hodgkin’s disease and nonHodgkin’s lymphomas may contain neoplastic follicles with FDCs [105,106]. Furthermore, the meninges can also develop ectopic, lymphoid follicles under conditions of chronic inflammation [107]. Most importantly, tertiary follicles can be induced in non-lymphoid organs by naturally occurring infections in ruminants, and are surprisingly prevalent in non-lymphoid organs [104,108]. To investigate whether inflammatory diseases influence prion pathogenesis, mice with a variety of five inflammatory diseases of the kidney, pancreas or liver were inoculated with the RML prion strain. In all cases and in all organs tested, chronic lymphocytic inflammation resulted in prion accumulation in otherwise prionfree organs. The presence of inflammatory foci consistently correlated with upregulation of LT and the ectopic induction of PrPC-expressing FDC cells. Inflamed organs of mice lacking LTa or LTbR did not accumulate either PrPSc or infectivity upon prion inoculation. Hence scrapie infection of mice suffering from nephritis, hepatitis, or pancreatitis induces unexpected prion deposits at the sites of inflammation [109]. These data raised concerns that analogous phenomena might occur in farm animals, since these are very commonly in contact with inflammogenic pathogens. Indeed, we found that sheep with natural scrapie infections and also mastitis, contained PrPSc in their mammary glands [110]. These observations indicate that inflammatory conditions induce accumulation and replication of prions in organs previously believed to be prion-free. These findings could have an impact on the risk assessment of biologicals (e.g. milk) and could lead to a readjustment of current rules. In addition, it was hypothesized that inflammatory conditions could result in the shedding of prions via excretory organs (e.g. kidney). To investigate this hypothesis, various transgenic and spontaneous mouse models of nephritis were analyzed to ascertain whether prions could be excreted via urine [111]. Indeed, prion infectivity was observed in the urine of mice with both subclinical and terminal scrapie, and with inflammatory conditions of the kidney [111]. The factors that enable horizontal prion spread between hosts have been discussed by veterinarians without resolution for over 100 years. It is possible that the horizontal spread of prions is mediated by secreted body fluids (e.g. urine; milk) that are derived from potentially infectious secretory organs (e.g. mammary

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gland, kidney). Placenta of infected ewes could provide a source of prion infectivity for horizontal transmission [112] – yet the set of data supporting the latter hypothesis is scant at best. Public health considerations mandate that we should increase our understanding of the altered prion tropism observed in ruminants (e.g. sheep, cattle, goat, elk and deer) and the underlying mechanisms. Future experiments should include an analysis of the effect of other common chronic inflammatory disorders (e.g. granulomatous diseases) in prion-infected rodents and farm animals. Moreover, it remains unclear whether prion infectivity is actively or passively transported into tertiary follicles. As LT is up-regulated in almost all states of inflammation and LTb / or LTbR / mice do not support prion replication in organs with lymphocytic inflammation (e.g. liver, kidney), it is reasonable to suggest that LT plays a crucial role in the induction of ectopic prion replication [113]. Indeed, it is plausible that LT itself induces prion replication competence on various cell types (e.g. stromal or mesenchymal cells). Future experiments will test the hypothesis that LT, induced by various exogenous stimuli (viral, bacterial or parasitic infection), promotes a microenvironment capable of prion replication. It is possible that different physiological states, such as the number of PPs [80] or the presence of prion infectivity in blood [114], alter the molecular and cellular preconditions required for ectopic prion accumulation and replication.

Homework for the Next Few Years Does any of the above bear any relevance to the BSE crisis? Undoubtedly, it would be highly desirable to develop some sort of post-exposure prophylaxis for prion diseases – especially when one considers that a large fraction of the population may have been exposed to the BSE agent in Great Britain and in continental Europe. But this will only be possible if prionotropic molecules and cells can be identified, which may offer a handle to interfere with the spread of prions. It is unclear how long prions persist in the body when their replication is prevented [115], but under certain circumstances prion clearance may be rather fast. Yet, total immune depletion does not represent a feasible practical solution for humans – except perhaps after massive exposure at a

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known and well-defined time point [116]. Even selective killing of B-lymphocytes may be problematic. I deem it realistic to hope that in the next few years the refinement of our understanding of the mechanisms of prion neuroinvasion will lead to the development of effective strategies of post-exposure prophylaxis, and perhaps even of therapy. Finally, I do not want to leave unmentioned the unusual events which have kept me busy in the first half of the year in 1998. Swiss scientists have just escaped a terrible threat. The population had been asked to vote on an amendment of the Swiss Constitution that would ban all genetic manipulation of animals, including worms and flies. Needless to say, this would have throttled Swiss biomedical research. At the beginning of the year 1998 the polls where thoroughly negative and provoked a lot of gloom within the Swiss research community. The neurografting experiments described above seemed to be just the things that would enrage the vociferous coalition of animal righters and ‘‘gene protectionists’’ which supported the initiative. So my colleagues and I undertook to go public and explain, in podium discussions, interviews on radio, on TV, and in the newspapers, what we were doing and why. These activities demanded a large share of our time and certainly affected our scientific productivity but, as a result, reason eventually prevailed and the ill-conceived ‘‘gene protection’’ initiative was defeated by a 2:1 ratio. The lesson I personally learned from these events is that education of the population with respect to the methods and the goals of science belongs to our job just as much as experimental work. Take both tasks seriously, and we will retain the support of the public which we depend on.

Life Science and Animal Experiments Like any other natural scientist, I love Nature and I am fascinated by living beings of all kinds. Can you think of any better way than life science for safeguarding the diversity of biological species, for preventing deadly epidemics of emergent infectious diseases, for installing early-warning systems, and for preserving the wellbeing of animals in their habitats? All these aspects of animal protection rely on understanding of animal physiology and pathophysiology. Hence life scientists are a highly effective group of animal protectionists.

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As documented by innumerable examples, animal research is a good thing also for the sake of animals. However, we scientists have to put up with a cross fire of physical, ideological, moral, and legal harassment by belligerent groups of self-proclaimed animal protectors! Clearly, biologists need to become more vocal about their own contribution to animal welfare. That should not be too difficult, since every democratic test of the case has confirmed that a majority is enthusiastic about biomedical research – including research on animals. In Switzerland, the pro-science community achieved two landslide victories in popular votes on genetic modification of animals (1998) and human embryonic stem cells (2004), and a series of national referendums against animal experimentation over the last 20 years were invariably rejected. Bewilderingly, some societies appear to condone the fanaticism of ‘‘animal liberators’’. For fear of attacks, the United Kingdom has refrained from all primate research on prion transmission. This is particularly shameful in the face of the victims of variant Creutzfeldt-Jakob-Disease. There is no sensitive blood testing method, no validated strategy of postexposure prophylaxis, and no therapy for those who fall prey of the disease. If primates had been inoculated with prions, they would now provide an opportunity for testing prion-removing blood filters, novel screening methodologies, and original experimental therapies. But no such studies were initiated – thanks to the Animal Liberation Front and to complacent authorities. Swiss animal protectionists may be less physically violent than the animal terrorists in the UK – but their methods are equally disruptive. Having failed at gaining control democratically, they have quietly infiltrated regulatory offices within cantonal administrations. These offices regulate all animal experimentation here, and are entrusted with far-reaching powers. Besides hampering the advancement of science, they also act as ‘‘mouse police’’, raiding scientific institutions and instilling a climate of harassment and intimidation among life scientists. As detailed above, we found that inflammatory states modulate the organ tropism of prions. Lack of IL-10 causes an inflammatory bowel disease, and we deemed it important to ask whether this may affect susceptibility to prions. If so, people with gastrointestinal disorders may bear a heightened risk of contracting Mad Cow Disease. However, a particularly militant local bureaucrat vetoed our experiment arguing that the severity of the IL-10 /

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phenotype (which is comparable to Crohn’s disease) could not possibly justify any advance in knowledge. Graciously, she volunteered to propose, and approve, an ersatz experiment: inoculation of hemizygous IL-10+/ mice. There is just one problem with this bureaucrat-driven pseudoscience: IL-10, like the overwhelming majority of mammalian genes, is not haploinsufficient. Hence hemizygous IL-10+/ mice are ‘‘severely normal’’ and could at best serve as negative control! All of this may sound hilarious – if it were not for the fact that these people have the authority of closing down your lab if you upset them.

ACKNOWLEDGMENTS

I am indebted to those scientists who have taught me the craftsmanship of neuropathology and of molecular biology, most notably Charles Weissmann. The current crew at the Institute of Neuropathology in Zurich consists of extremely motivated, clever, and hard-working colleagues from many different countries: it is an immense pleasure to devise experiments and discuss results with them. Special thanks also to Norbert Wey for help with artwork and digital imaging through several years. The work described in these pages was supported by numerous funding agencies, most notably the Kanton of Zurich, the Swiss National ¨mter fu Science Foundation, the Bundesa ¨ r Gesundheit, ¨rwesen, und Erziehung, the Migros Foundation, the Veterina Ernst-Jung foundation, and the European Union. REFERENCES [1] Natali, P.G., Aguzzi, A., Veglia, F., Imai, K., Burlage, R.S., Giacomini, P. and Ferrone, S. (1983) J. Cutan. Pathol. 10, 6: 514–528. [2] Nagata, S., Taira, H., Hall, A., Johnsrud, L., Streuli, M., Ecsodi, J., Boll, W., Cantell, K. and Weissmann, C. (1980) Nature 284, 5754: 316–320. [3] Gray, P.W., Leung, D.W., Pennica, D., Yelverton, E., Najarian, R., Simonsen, C.C., Derynck, R., Sherwood, P.J., Wallace, D.M., Berger, S.L., Levinson, A.D. and Goeddel, D.V. (1982) Nature 295, 5849: 503–508. [4] Giacomini, P., Aguzzi, A., Pestka, S., Fisher, P.B. and Ferrone, S. (1984) J. Immunol. 133, 3: 1649–1655. [5] Giacomini, P., Imberti, L., Aguzzi, A., Fisher, P.B., Trinchieri, G. and Ferrone, S. (1985) J. Immunol. 135, 4: 2887–2894. [6] Giacomini, P., Aguzzi, A., Tecce, R., Fisher, P.B. and Ferrone, S. (1985) Eur. J. Immunol. 15, 9: 946–951.

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G. Semenza (Ed.) Stories of Success – Personal Recollections. X (Comprehensive Biochemistry Vol. 45) r 2007 Elsevier B.V. All rights reserved. DOI: 10.1016/S0069-8032(07)45008-2

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Chapter 8

An Autobiographical Sketch: 50 Years in Cancer Immunochemistry$ G.I. ABELEV Member of the Russian Academy of Sciences, Russian Cancer Research Center, Moscow, Russia

Abstract Brief description of scientific life of Russian biochemist G.I. Abelev in tumor immunochemistry. Main attention is devoted to develop the system of analysis of complex system on the level of individual antigens. The discovery of alpha-fetoprotein and its use as tumor marker is described. $

Based on an interview given to G.Yu. Moshkova, Research Officer of the Institute of natural sciences and Technique history of the Academy of Sciences of the USSR in 1986–1987, with additions.

284 Keywords: immunodiffusion; immunofiltration; alpha-fetoprotein in tumors; tumor markers.

G.I. ABELEV alpha-fetoprotein

in

ontogenesis;

In my childhood and youth I knew nothing about the existence of research institutes and researchers. No one among the members of my family or their friends was involved in research. My interest in science arose in the time of World War II when I was a schoolboy. In the very beginning it was mainly of philosophical type. I wanted to understand the thinking process and how everything is organized. Perhaps initially I was interested in astronomy, particularly due to the popular books by Flammarion. My school years – from the 6th to 10th classes – proceeded in the wartime. Naturally, life at that time was very hard for everybody, and the harder it was, the more attractive seemed to me the life in science – bright, interesting, and noble. Life within the walls of university became the goal of all my dreams. After the 8th class I acquired more definite interest in psychological problems. I tried to read Ivan Sechenov, Vladimir Bekhterev, Ivan Pavlov, and I even organized a home ‘‘seminar’’ where we discussed their studies and problems related to psychology. Graduating from school in 1945, I was at the ‘‘crossroad’’: what institute should I try to enter? At that time the Chair of psychology had just been opened at the Department of Philosophy, Moscow State University. I had no interest in biology in itself, but I was interested in physiology of higher nervous activity and of the work of the brain. At that time I used to listen to public lectures in the Moscow Polytechnic Museum. The distinguished physiologist Professor P.K. Anokhin had given a lecture on higher nervous activity. Although I could not dare to ask him for advice, I felt that he was the person who may really help me to choose my own way. At last I became brave enough to ask his advice, saying that I am particularly interested in what are the brain and the thoughts? Anokhin gave me a clear answer. He said that if you enter the Chair of Psychology it will be interesting for you to learn and read literature, but nothing would be done by yourself. Studying physiology you cannot do much, but all you will have done would be your own work. His advice was consistent with my own ideas and desires, and so I went in for the entrance examinations at the Department of Biology, Moscow State University. Although I prepared for these exams very seriously, I nearly failed.

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The reason was that my entrance to the university meant too much to me and I probably overworked during preparation for the examinations. The competition for entrance was quite strong, about six competitors per university seat. In spite of my so-called semiacceptable score, I was accepted as a student of the Biological Department, possibly due to my gender. At that time the majority of boys preferred to enter Moscow Aviation Institute, Moscow Energetic Institute, Moscow Technical High School, or at least the Departments of Physics or Mathematics of Moscow State University.

University Soon after my entrance to the Biological Faculty, I became a member of the Students’ Scientific Society at the Chair of Physiology of Higher Nervous Activity. It was directed by Professor Kh.S. Kashtoyants and Associate Professor M.V. Kirzon. Possibly due to my activity (I was interested in this subject) Kashtoyants said to his students that he would like to talk with me. However, I was very shy and did not see him. Soon after this he suddenly died. With my friend Alexander Zotin, who later became Professor and Head of the Laboratory at the Kol’tsov Institute of Developmental Biology, we organized our own student scientific society. We called it ‘‘student scientific society group for biophysics’’, but in reality we were interested in mechanisms determining physiological processes. Now this field of science is known as molecular biology. We invited Professor S.S. Vassiliev, who gave lectures in physical chemistry at our Biological Faculty, to become the head of this group. The personality of this enthusiastic charming man substantial influenced my life and those of my friends. Zotin worked on energetics of development all his life, and his research interests originated from problems that we analyzed with Professor Vassiliev in our student scientific society group. During the first and second years I, with 3–4 other students, began to work with Professor Alexander Gurvich, an outstanding world-renowned cell biologist, who proposed the theory of biological field. (He used to be the Director of the Institute of Experimental Biology of the All-Union Institute of Experimental

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medicine.) We visited his laboratory after lectures and seminars (and sometimes instead of them!) and worked there until very late in the evening. This was our first involvement in real research work. It was neither simple nor easy. Thus, from the very beginning of my studentship, I met and worked with outstanding people in remarkable laboratories and institutes. I think that Moscow State University of the postwar period (up to the sadly known August 1948, when Lyssenko came to power in biology) had major influence on my formation as a scientist. I should say that even the university walls, painted by famous Russian artist Vasnetsov, ‘‘emitted’’ special aura and charm. Romantics, moral purity, democratic principles, nice people, and real scientists open to communication with students created my image of science, type of relations and communications, and the norm of scientific activity which remain with me all my life. Among professors who influenced my choice of scientific interests and my way in science, I should again mention S.S. Vassiliev, the head of our scientific student group. He worked with us, his students, regretting neither his time nor effort. Especially for us, a small group of students, he prepared wonderful courses of lectures on mathematics, physical chemistry, chemical kinetics, and thermodynamics. This experience, together with knowledge I gained from his lessons, still remains with me, and I am very grateful to Professor Vassiliev. Professor Alexander Gurvich and his daughter, Anna Gurvich, with whom I worked during the first and second years of my studentship, had no direct relation to Moscow State University. Nevertheless, they significantly influenced my viewpoints on science and scientific human relations. I accepted their type of human relations, scientific spirit, and democracy. We ‘‘inhaled’’ their aura, which still remains inside us as some scientific norm, requested type, and mode of scientific relations. In Gurvich’s laboratory I was working on the problem of the nature of nerve excitation, which attracted my interest and ‘‘drove’’ me to this particular laboratory. I thought that I could get inside elemental steps of the nerve process. In agreement with Gurvich’s theory, I thought that using mitogenetic emission1 1

Mitogenetic radiation – a weak ultraviolet emission accompanying different biochemical processes.

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accompanying the process of nerve excitation propagating along a nerve fiber it would be possible to get deep inside the nature of this process. I cannot say that the work in Gurvich’s laboratory was quite successful for me in spite of his kind attention to my friends and me. The thing is that the method employed in his laboratory for registration of the mitogenetic emission was based on stimulation of cell division in yeasts. So each experiment required estimation of the number of budding yeast cells and comparison of this parameter in normal cells. This was a rather subjective and laborconsuming procedure. In some cases such calculation was successful, whereas in other cases it was not. Ms. Gurvich specially taught us, but the results of estimation were nevertheless very unreliable. In spite of enormous efforts and time spent, there was little progress in that work. As to me, I was not sure that my results would be reproducible and that they did represent ‘‘objective reality’’. I was not sure when the experiment was quite reliable and when not. This stimulated my search for methodological tools that would give me confidence in reliability of both methods used and results obtained in my experiments. I think that this search together with some other reasons (which I did not realize at that time or did not want to realize at the subconscious level) turned me toward biochemistry. Biochemistry, the so-called test-tube biology, employing a chemical approach for analysis of biological phenomena, looked more reliable to me. It provided a firm methodological background required for my sense of satisfaction. This was the major factor that influenced my decision to change the area of my scientific interests. I should also say that in the second half of the 1940s it became clear that the nature of biological processes would be understood on the chemical level rather than on the energetic or physical levels. Intention to understand the nature of biological processes (including nerve processes) would definitely be directed toward investigation of chemistry of these processes. At that time I did not understand this point consciously, but I definitely felt it subconsciously. I also had some problems with experimental work employing such biological objects as a neuromuscular preparation or experimental animals. Possibly such work requires a ‘‘special personal

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arrangement’’ of a scientist. I think that a good physiologist should be a skillful surgeon strong enough to perform vivisections. My personality did not meet such criterion. I was more interested in the field of the ‘‘test-tube biology’’, elemental chemical processes, and in chemical rather than surgical or physiological methods. And, of course, I wanted to get reliable and reproducible results from my experimentation. Thus, in the third year of my university studentship I desperately wanted to specialize in biochemistry. So, I made an application to the Chair of Animal Biochemistry and would have definitely continued my education at this chair, but suddenly (I cannot explain why!) the Chair of Plant Biochemistry attracted my attention. The reason was that my deeper study of biochemistry resulted in understanding of the fact that I became more and more interested in this science as such rather than a useful tool for investigation of nerve processes. Biochemistry as the basis for understanding the nature of life became my major interest. In this respect plants were much more interesting objects than animals, because plant biochemistry was much more ‘‘diverse’’, and it started from the very beginning of life, photosynthesis, the synthesis of organic matter. Elemental biochemical processes in plants are also much more diverse and variable than in animals. (At that time biochemistry of microorganisms was also referred to as plant biochemistry.) I was deeply interested in these processes. There was another reason by which I chose the Chair of Plant Biochemistry. I mean styles of learning biochemistry at the Chair of Animal Biochemistry and the Chair of Plant Biochemistry. In animal biochemistry a ‘‘ladies’’ style dominated. Its chairman, the famous scientist Professor Sergey Severin, was a brilliant lecturer, and we all attended his university course of lectures in biochemistry. However, his associates and various assistants involved in the education at this chair almost deified him. They taught their students very meticulously and scrupulously. They accepted a word from the professor as unquestioned dogma and his viewpoint as ‘‘the truth of the final instance’’. At the Chair of Plant Biochemistry, directed by Academician Alexander Oparin, there was a completely different style because of the relatively young Professor Andrey Belozersky, who was in charge of the educational and pedagogical processes.

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Soon after the beginning of my specialization in animal biochemistry, I realized that I desperately wanted to continue my education at the Chair of Plant Biochemistry. So I made an application to this chair to accept me for subsequent specialization. It was a rather difficult procedure, which was not well understood by my friends, teachers (and to be honest, by myself). However, I felt that I needed to change chair while it was not too late. In the very beginning Professor Belozersky was rather skeptical of my intention to change chair after the beginning of an academic year. He tried to persuade me to leave the situation just as it was. He explained that research is not as romantic as I had suggested and hard-working ability was the major factor, while philosophy and biophysics were good of course. Real research requires solid reproducible observations. The most important in our work is the ‘‘coincidence of duplicate determinations’’, he said. Professor Belozersky was a wonderful person and influence on my scientific fate and my scientific development. I feel that my scientific viewpoints, style of work and human relations, criteria of evaluation of results, reference group on which I oriented and began to orient later, as well as personal self-evaluation were initially formed under the strong influence of Belozersky’s personality. (I am not sure that he realized such influence of his own personality.) Belozersky was a very talented, charming, and benevolent person (Figure 1). He was a real scientist. He was real in everything. Being a genuine researcher, he belonged to a cohort of investigators who ‘‘extracted’’ primary facts hidden in nature. He had surprisingly simple viewpoints on many problems, common sense in evaluation of scientific events, results, and people. He demonstrated extraordinary respect to students, colleagues, and other scientists. Belozersky was characterized by such a wonderful feature as ‘‘presumption’’ of respect to everyone and everything. He was very delicate. His intention not to offend persons (especially students), complete lack of haughtiness and snobbism, and simultaneously his accessibility but without familiarity were very typical of him. These characteristics of Belozersky were wonderful and absolutely natural. In the very beginning of my studentship at the Chair of Plant Biochemistry I was not very successful, especially in learning the ‘‘art’’ of experimental work. (At that time it was definitely art

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Fig. 1. Professor A.N. Belozersky (center) and docent N.I. Proscuryakov (right) with students of plant biochemistry chair. In the center of the second row G.I. Abelev (1950).

rather than ordinary skill.) Experimental work was very empirical and strongly depended on ‘‘good hands’’, scientific ‘‘flair’’, and the sixth sense with respect to the material used. I found perfect understanding of my problems, some indulgence, healthy humor, i.e. normal human relations. I think that Belozersky with his large experience well understood how touchy a young student is under these circumstances. So all that I accepted from Belozersky was ‘‘grown in a well prepared and very sensitive background’’. He never taught us ‘‘do this or that’’. He never taught what is right or wrong. He just commented on our activities, discussed our problems with us, gave his advice and recommendations, and that was all. All features of his personality influenced me (and others as well): his direct evaluations of my work, his comments, lectures, talks, short remarks during conversations, etc. One of the main understandings which appeared there and which basically predetermined my scientific carrier was the sense that I should work as hard as I can and as much as possible. I also began to realize that real experimentation requires knowledge of a research object, possession of the art of experimental work, reliability, and reproducibility of results obtained and methods employed. Evaluation of results obtained by yourself and your

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colleagues also required some proportion of healthy significance and simplicity. I think that I came to such understanding mainly due to communication with Belozersky, and I am confident that this understanding is still with me. Professor Belozersky treated me kindly, maybe too kindly, but it was typical of him: there were just a few male students at his chair. Such kind attention to his male students was maintained during the whole studentship and even afterwards up to the end of his life. He always helped me and was involved in my fate and my work. He was always benevolent, ready to help and advise. Later he involved me in work at the university, where I still teach students at the Chair of Virology, which he founded. I am very grateful to him for his kind attention to me. My diploma project was about nuclear proteins. I thought it would be a very interesting project, and indeed it was. It was the first continuation of initial observation by American researchers, Mirsky and Pollister, that nuclei of plant cells as well as nuclei of animal cells contained histone, the basic protein of chromosome structure. At that time this fact was not known; moreover, it was believed that the presence of histone was restricted to animal cells. However, using some remarks made by Mirsky and Pollister and employing their method, I demonstrated that plants also contained histone. In my diploma project I isolated large amounts (900 mg!) of this protein, analyzed its amino acid composition, and thus provided convincing evidence for the existence of histone in plants. Belozersky was deeply interested in this work. However, it was performed in 1949–1950, right after events accompanying the sadly known session of the Academy of Agricultural Sciences headed by Lyssenko. These events significantly disturbed development of research related to nucleic acids, chromosomes, and nuclear proteins in the USSR. This was a time when real normal scientists, classical biochemists, tried to avoid research projects related to such areas. Belozersky was not an exception: he had many problems because of his works related to nuclear proteins, DNA, and his contacts with classical genetics at the university. So he did not want my project to be done at his chair, and he suggested projects on bacterial antigens. However, I had no choice and finally we succeeded to obtain approval for the project thanks to A.I. Oparin. He had just obtained a large project for the whole

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chair on oak as the basic tree for the so-called field-protecting forest strips; this problem was quite popular and ‘‘fashionable’’. (Such field-protecting forest strips dominated in the ‘‘Stalin plan for reorganization of nature’’.) Oparin suggested that the nucleoprotein problem could be investigated using oak as the research object. Of course, it was difficult and inconvenient to employ oak as the research object, but the major thing was that the nucleoprotein project could be started. I began to work on this project (with oak as the research object), but several months later I continued my work using real research material – wheat germ – and obtained interesting results on histones. I was totally inside the problem of nuclear proteins and their role in cell differentiation and mitosis. Although Professor Belozersky worked for the N.F. Gamaleya Microbiology Institute2 as a consultant and worked on bacterial antigens, I had no interest in immunology. Diploma projects of my friends dealt with bacterial antigens, but those problems were absolutely out of my interest. Moreover, I did not accept Belozersky’s proposal to work on bacterial antigens because I was keenly interested in nuclear proteins, nucleoproteins, and global problems of cell division and differentiation. I was confident that this was real biochemistry. I graduated from Moscow State University in 1950. It was a difficult time and I had to join Professor I.B. Zbarsky, who was Head of the Biochemical Laboratory at P.A. Gertsen Research Institute of Oncology. He was interested in tumor cell nucleoproteins, particularly in the so-called tumoroproteins, putative tumorspecific proteins. (A hypothesis on tumor-specific proteins and their role in tumor cells was proposed by his father, B.I. Zbarsky, an outstanding Soviet biochemist, Chairman of Biochemistry at the First Moscow Medical Institute and also Director of the Lenin Mausoleum Laboratory.) My relations with I.B. Zbarsky had a good start: being a student, I gave a talk at his laboratory seminar, and he wanted to see me in his laboratory. I also wanted to continue my work on nuclear proteins even if from different object(s). For me it was important to continue research work within the same problem irrespectively to research objects. 2

Institute of Epidemiology and Microbiology named after famous microbiologist N.F. Gamaleya and belonging to the USSR Academy of Medical Sciences.

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To exclude all possible complications during my postgraduate employment, Dr. I.B. Zbarsky asked his father to make an application to the University Postgraduate Employment Commission to send me to the Lenin Mausoleum Laboratory, which was organized to look after Lenin’s body for prevention of any signs of its decay. (Here I should explain that due to free of charge education in the USSR, the graduates had to work for at least three years for organizations that sent their applications to the University Postgraduate Employment Commission. This commission ‘‘distributed’’ the graduates between applicants.) I.B. Zbarsky promised that in spite of my position at the Mausoleum Laboratory, I would be working at the Institute for Oncology. I accepted his offer because I relied on his experience. So such application was made, but it played a bad role in my fate. Entering the office of the University Graduate Employment Commission, I felt that things had gone a way I did not want. Members of the commission explained that they were not satisfied with the application from the Mausoleum Laboratory because employment at this laboratory requires special selection. I was told that I could be sent to any other laboratory. Here I should say that an anti-Semitic tendency was just beginning in science in contrast to art, literature, or theater, but together with my low social (political) activity it was sufficient reason to prevent my entrance into the Mausoleum Laboratory. I thought that all my ‘‘rosy’’ perspectives collapsed in one moment, but Belozersky immediately began to investigate possibilities of my postgraduate employment. He quickly found that Dr. V.A. Blagoveschensky, a biochemist and bacteriologist who was organizing the large Biochemical Department at the Gamaleya Microbiology Institute, needed junior staff members. They rapidly made the appropriate application offering me a position of junior technician. Dr. Blagoveschensky treated me very kindly. As I was a student of Professor Belozersky, he permitted me to continue my university project on nucleoproteins, but on bacterial objects.

In the Laboratory: Searches and Findings Thus, in summer of 1950 I began my work at the department of Biochemistry, Gamaleya Institute for Epidemiology and Microbiology, Academy of Medical Sciences of the USSR.

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Dr. Blagoveschensky was interested in problems of vaccination and bacterial antigens, but I was indifferent to them. On the third floor of that institute there was a department directed by Professor L.A. Zilber, an outstanding scientist who was successfully working on cancer immunology. He was basically one of the founders of this discipline. Although I had no interest in immunology there was one important point that attracted my attention. Zilber not only developed immunology of cancer, but actively investigated nucleoproteins and nuclear proteins of tumor cells. Based on his theoretical considerations originating from the viral theory of cancer, he believed that specific tumor antigens ought to be found mainly among nucleoproteins. (This is because viral proteins should be associated with the nucleoprotein fraction.) This explains why biochemists of his laboratory were working on tumor nucleoproteins. A.N. Belozersky was a consultant in these studies carried out by Z.A. Avenirova and V.A. Artamonova. In that year the University Graduate Employment Commission sent four graduates to the Gamaleya Institute. Dr. Blagoveschensky could not accept all of them. I still remember the episode when we, four young specialists, were in the office of the Staff Department. The head of this department phoned to Zilber and asked him to come to her office and to choose some of us for work in his laboratory. Zilber came. He was a big, tall, confident, and very impressive man (a man from another world!). He briefly interviewed each of us, asking about our diploma projects and our supervisors. He was going to accept me, because I was a pupil of Belozersky, who was a consultant of some projects in his laboratory and whom Zilber highly evaluated and respected. He also appreciated that I had good background in the field of nucleoprotein research, and his laboratory used the techniques developed by Belozersky. However, Blagoveschensky refused to send me to Zilber’s laboratory. They had a hot conversation, and Zilber was offended by his decision and refused to accept anybody. Thus, starting my work with Blagoveschensky, I already wanted to work with Zilber because nucleoprotein research had top priority in his laboratory. It was a reasonable intention of a young researcher to join a laboratory in which scientific problems of his interest represented ‘‘the main stream’’ of its research as well. In Blagoveschensky’s laboratory the nucleoprotein

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project was given to me just due to good relations between Blagoveschensky and Belozersky. Anyway, I started my work in Blagoveschensky’s laboratory. However, one year later Blagoveschensky was invited to work in another bacteriology institute out of Moscow, and he left the Gamaleya Institute. The next head of that Department, Professor V.S. Gostev, was a completely different person. To begin with, he was a disciple of Zhukov-Verezhnikov, a powerful microbiologist supporting Lyssenko’s views. He was interested in completely different studies, whereas I desperately wanted to continue my work in Zilber’ department.

In Zilber’s Laboratory Immunological methods developed by Zilber for detection of specific tumor antigens (anaphylaxis with desensitization) looked very attractive to me because it opened new horizons in studies of specificity of nucleoproteins, their functioning, and isolation of tumor-specific nuclear proteins. All these studies were carried out in Zilber’s laboratory. The other reason was Zilber’s personality. He was an outstanding scientist involved in solution of very interesting problems. It was clear for me that my work with him would find resonance and support and would attract interest to my research, which I was limited in at that time. It is very natural for a young researcher to have a very competent, very distinguished supervisor who would be interested in your work, who would encourage you to overcome difficulties and problems in your research. Simple benevolence is good but it is not enough. These were my reasons, which drove me to move to Zilber’s laboratory or at least to have a joint project with him. I established contacts with his biochemists, and we discussed possibilities of a joint research project. This was right after Blagoveschensky left and Gostev appeared. Gostev sharply objected to any contacts with Zilber. He was also interested in tumor immunology but had ‘‘cold relations’’ with Zilber. I do not think that this was due to competition in science, because Zilber’s personality was much higher in all respects. Gostev’s ideals were very far from the ideals of my university teachers, Belozersky, or Gurvich. He was a completely different person.

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Although it was clear for me that I would do my best to join Zilber and his team, I discussed this problem with my friends, my wife, and of course with Belozersky. He was the only person who strongly recommended me to go to Zilber. He said it would not be easy to work with Zilber; many people believed that he was very autocratic and even a despotic person, but I would be happy to work with him. Belozersky said that Zilber was a very enthusiastic man actively working in science and we would easily establish our relations. This was the only definitive viewpoint. Most of my friends were rather skeptical of my intention to change laboratory. They argued that I was a professional biochemist, working just at the Biochemical Department. Moving to Zilber I would be surrounded by specialists in medicine, immunologists (basically non-biochemists). They felt that I would do all biochemical studies by myself, and nobody would help me in solving biochemical problems. But I ignored these skeptical warnings. Thus, I directly applied to Zilber asking him to accept me as a member of his department (Figure 2). Zilber was keenly interested to accept a student of Belozersky into his department, but it was very difficult. There were long-term negotiations (for several months) with the institute director, Professor V.D. Timakov, but

Fig. 2.

L.A. Zilber (1937).

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finally this lucky event happened, and I became a member of Zilber’s department. This change in laboratory was also accompanied by a better position: I became a senior technician with a higher salary. (This was also important because I had family.) However, the final result was that I had come to a laboratory that needed my work and was keenly interested in it. Thus, in the very beginning of 1952 I began my work in Zilber’s department. This was a very important and decisive event in my life. I started my work from fractionation of tumor nucleoproteins. Fractionation was very important for isolation of tumor-specific antigens under control of immunological tests developed in that laboratory. In the very beginning of my work I was not familiar with these tests, and so I worked in close contacts with medical immunologists Ms. Zinaida Baidakova and Dr. Nikolay Narcissov. (They were the staff members closest to Professor Zilber.) The anaphylactic reaction with desensitization (A-D) was the major test for tumor antigens. Guinea pigs were initially sensitized with tumor nucleoprotein; three weeks after sensitization the guinea pigs were desensitized with gradually increasing doses of nucleoproteins isolated from normal tissues. When the guinea pigs were totally desensitized to normal antigens, they received a single dose of initial tumor nucleoprotein. Usually such administrations were accompanied by anaphylactic shocks of various intensities. This suggested the presence of some tumor antigens specific for a particular tumor and absent from normal tissues. This rather simple but elegant and convincing method developed by Zilber and his team brought major success in that direction of tumor immunology. This method represented the important tool employed in Zilber’s research. So I fractionated tumor nucleoproteins on the basis of this reaction. I was especially interested in nucleoprotein fractionation based on differential centrifugation. Several years earlier such approach was proposed by the Belgian biologist Albert Claude. Employing this method it was possible to isolate ribonucleoprotein-enriched cytoplasmic granules, nuclei, and non-structural cytoplasm. For this purpose G.S. Bezverkhyi, an engineer who used to work with Blagoveschensky, and I adapted milk separators for isolation of cytoplasmic granules (mitochondria and microsomes), and then we applied our method to nucleoprotein fractionation. Pilot results were clear and promising. They suggested special activity and the presence of specific antigen in the cytoplasmic

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granule fraction. However, subsequent results were less stable and reliable; their reproduction depended on anaphylactoid reactions (i.e. false anaphylactic reactions induced by protein aggregates or cell debris), which were obtained using these corpuscular, poorly soluble fractions. I was upset and depressed about not being able to reproduce some results and the conclusions, or lack thereof, we had to draw from that fact. I worked very hard and tried to use different variants of the method. I also started parallel work with Dr. Narcissov. Using complement-fixation reaction with sera of tumor-bearing rats, the results became more stable, reliable, and reproducible, and the reaction itself was much simpler. So, under control of complement-fixation reaction Narcissov and I provided convincing evidence for the localization in the mitochondrial/microsomal fraction of an antigen responsible for reaction with sera obtained from tumor-bearing rats. That was the time (1952) when immunogenetics was formed as a science. It became clear that such studies would employ a so-called syngenic system (i.e. a system of genetic identity of tumor donor and recipient, although this term appeared later.) We started to think about various controls and tried to work on primary tumors and sera of the same animals. Generally we confirmed the major conclusion that during growth of sarcomas induced by carcinogens (primary or transplanted sarcomas), the sera obtained from the tumor-bearing animals exhibit complement binding reaction with extracts from the same tumors, mainly with the cytoplasmic granule fraction isolated from these tumors [1]. In parallel work, together with a group of engineers from the Research Institute of Chemical Machinery, we tried to improve the separators. This work culminated in several models of reasonably good separators applicable for isolation of cytoplasmic granules. Furthermore, we developed a new type of separator, the chamber separator, which was able to separate particles from the flow of tissue homogenate by their size and density. Thus, we were able to use both differential centrifugation and density gradient separation in one procedure. (I should say that using our device it was possible to work with large volumes of starting material.) This was reasonably good work of 1955. The problem was that there were just one or maximally two copies of our separators, and our work could not be further developed [2].

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Thus, in the 1950s I was involved in all studies carried out by Zilber’s department. I worked on separators, fractionation, and analysis of antigenic properties of various intracellular components, and also I learned cancer virology and immunology. In parallel, I set up the method of free electrophoresis after Tiselius and employed it for characterization of proteins obtained during fractionation of cell components. Together with Narcissov, we studied antigenic properties of these fractions using sera obtained from tumor-bearing rats. In 1955 I received the Ph.D. degree. However, I should say that the title of my thesis, ‘‘Study of Some Fractions from Tumor Tissue’’, was a bit boring. At that time I was interested in immunology mainly as a tool for studies of cell structures, tissue-specific proteins, and a tool for recognition of proteins specific for tumor tissue. Immunology attracted me as a method that would help solve biological and oncologic problems related to cell differentiation. I was poorly familiar with immunology and spent much time in attempts to understand it. There were a few handbooks of immunology, but they had a strong emphasis on medicine and microbiology. So it was difficult for me to understand the main notions and principles of immunology. (Literally speaking, all immunology of that period ‘‘sank’’ in abundant complex medical terminology, which often substituted real understanding of actual scientific links by numerous unrelated phenomena.) Zilber’s seminar organized in the Gamaleya Institute for medical aspirants (which was also attended by senior technicians) was very helpful for understanding immunology. Basically, Zilber clarified many problems of immunology for me. I still remember that I started to understand the unitary theory of immunity: the reaction between antigen and antibody represents the basis for all multiplicity of various phenomena joined by immunology, and secondary immunological manifestations can have various phenomenological features. The Russian translation of the book by William Boyd ‘‘Principles of Immunity’’ (or possibly ‘‘Principles of Immunology’’) published in 1947 (if I remember correctly) also helped me in learning immunology. This very good textbook with emphasis on immunochemistry clearly described the principles of immunity. Only reading this book and participating in Zilber’s seminars cleared my brain in terms of immunology, and I began to understand the basic principles of this science.

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In 1956 William Boyd visited Moscow, and he had many discussions with us, members of Zilber’s department. Looking after him in Moscow, I asked him how he managed to write such a wonderful book, describing basic principles of immunity in a simpler way compared with other textbooks. He said that it was difficult for him (a chemist interested in immunology) to get inside the immunologic literature, very complicated with phenomenology and immunological notions and terms. So he started to learn principles of immunity from the chemical viewpoint and later, when he became well familiar with them, he wrote that book. Perhaps a reader lacking medical and immunological background needed such logics of a ‘‘xenobiotic’’ expert for understanding this science. The second edition of this textbook was also translated into Russian. However, it was larger and more complex and was not as clear as the first edition both in construction and style of presentation. Immunodiffusion In the middle of the 1950s I became familiar with the method of antigen precipitation in gel. In laboratory seminar we discussed the method of Ouchterlony applicable for analysis of tissue proteins. In two papers by B. and V. Bjo¨rklund published in 1952, this method was used for studies of some tissue proteins. We actively discussed applicability of this method for detection of specific tumor antigens. We understood the great possibility and facilities of this method, its resolution and analytical power. We started to realize that this method might be used for studies of individual antigens, individual proteins (without their purification from a complex mixture), for comparison of these proteins, for elucidation of not only species but also tissue (including tumor) specificity. Nevertheless, working with anaphylaxis we believed that the method of gel precipitation was rather crude and insensitive for detection of fine differences, which do exist between the antigenic structure of normal and tumor cells. This was our strong belief. However, I suggested that this method was very valuable for us anyway, because it could be used to compare antigenic structure of different cytoplasmic structures and to identify proteins characterizing nuclei, cytoplasm, and various tissues. I was confident

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that this method opened ‘‘colossal’’ facilities for identification and characterization of complex protein systems. There was very attractive promise for use of this approach as such (or at least as an intermediate stage) for recognition and identification of specific tumor antigens that were the basis of our studies. However, until 1955 I was working hard on the abovementioned projects and analysis of the data obtained and preparation of the Ph.D. thesis. Afterwards I continued my work on the chamber separator and electrophoresis and analysis of sera from tumor-bearing animals. I did not have time to use the gel precipitation method in my research. In 1956 Dr. Vera Parnes, a senior researcher of Zilber’s laboratory (who was also interested in the gel precipitation reaction), and I decided to set this method together. I had to work on the immunochemical part of our common project, manipulations with gels, fractions, antigen preparations. Her part of that work included selection of corresponding antiserum, potent precipitating sera, and system of normal and tumor tissues that we would compare. A very important thing was that we had an excellent precipitating serum, obtained by Z.L. Baidakova for a completely different purpose: for preparation of an antileukemic vaccine. She obtained very potent donkey antiserum against human leukemic spleen, which we also used with Parnes in our manipulations during reproduction of the gel precipitation method. Finally, we were able to distinguish the first precipitation band in gel and first reaction of identity. Everything was new for us: agar preparation (starting from alga), preparation of agar wells, and protection of this system against bacterial growth. In other words, everything was difficult, but the major result was that this method worked and its use yielded clear bands. This was perfectly wonderful! Besides the experimentations with leucosis, Avenirova and I began to compare the antigenic structure of normal liver and hepatoma. Using separators, we isolated several subcellular fractions (mitochondria, microsomes, nuclei) and compared them with each other and also with normal liver and hepatoma. We obtained very good precipitation bands with antisera against liver mitochondria and microsomes, against homogenates of normal and tumor hepatic tissues. In 1957 we had two systems in which the precipitation in gel gave results: the leukemic system used in

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studies with Parnes and the hepatic system employed for joint studies with Avenirova. Parnes continued the work with leukemia spleen, while we together with Avenirova focused our attention on the hepatic system. The hepatic project ran rather well and a question arose: is this method applicable for detection of differences between normal and tumor tissue, or is such precipitation too crude for detection of such fine differences? In other words, is this wonderful method applicable for detection of tumor tissue specificity? This issue became the major goal of our studies. I was confident that even if the sensitivity of this method would be insufficient, it might give us purer and better characterized tumor tissue fractions that would be further used in the anaphylactic reaction. We began to compare cytoplasmic granules from normal hepatic tissues and hepatoma. All this work was carried out on mice. Hepatoma-22a employed in our experiments was obtained by V.I. Gelstein. She spent some time working with Zilber and was well familiar with his studies. We had very good contacts. We started comparing the antigenic structure of normal and tumor hepatic tissues and obtained promising results almost immediately. During direct comparison of mitochondria and microsomes from normal liver and hepatoma, we found lack of at least one or even a few antigens present in the normal tissue. In 1956 E. Weiler, from West Germany, described the phenomenon of ‘‘antigenic simplification’’. Comparing antigens of normal liver and hepatoma using the complement binding reaction, he demonstrated loss of the tissue-specific liver antigens in hepatomas. Weiler defined this phenomenon as ‘‘antigenic simplification’’. This antigenic simplification was immediately detected in our experiments with precipitation in gel. We found that several tissue-specific antigens typical for normal hepatic tissue disappeared in hepatoma. This phenomenon was potent, clear, readily reproducible, reliable, and very promising. It was possible to isolate these antigens under control of reliable test and investigate their behavior during chemical carcinogenesis in various tumors. Basically this opened wide prospects for studies of this phenomenon. We were very lucky, and in collaboration with Vladimir Tsvetkov we developed a simple method for purification of one

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of the tissue-specific hepatic antigens, which we name AO-antigen, the antigen of organ specificity. This was a rather simple and good method based on ultracentrifugation and electrophoresis, which we set up in Zilber’s laboratory. This method yielded immunologically pure antigen with high activity and very low-protein content. Isolation of an individual protein was a very difficult task, but we had such an antigen. We were able to investigate semi-quantitatively the fate of this antigen in various tumors during chemical carcinogenesis. This antigen demonstrated a direction for subsequent studies employing other tissue-specific antigens (see Ref. [3], pp. 308–313). This work was quite nice even by modern criteria, but at that time when individual antigens were not available, its continuation seemed to show promise. I thought that we might spend time for working on this project, but Zilber had another viewpoint. He often said to us that it is good interesting work, which definitely opens a new chapter in immunology, but specific antigens are much more important. He ‘‘pushed’’ us toward tumor-specific antigens, which could be recognized using the same technique. He used to say that we could continue our research on the organ antigens at another time. The work on specific antigens was stimulated by another point, which was also rather important for Zilber. These events took place in 1957 and in the next year he would have to present results of his department at the 7th World Cancer Congress in London. This was his first international presentation after a 30-year forced interval, and he definitely wanted to demonstrate tumor-specific antigens to the international audience. So Zilber stimulated us to work on these specific antigens. This was a much more difficult project compared with our work on the organ-specific antigens, because such tumor-specific antigens were hard to detect. However, there were some hints in our experiments: one band (which we considered as the band of specific antigen) appeared in some experiments and disappeared in others. So we postponed this work for experiments with more reliable antigens. However, Zilber insisted, and finally we started our ‘‘pursuit’’ of this antigen ‘‘weakly twinkling’’ in the spectrum of antigens common for normal and tumor tissues. One of the major difficulties in the detection of specific antigens was the fact that specific differences could be detected on gel

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plates within a peripheral zone of antibody–antigen interaction, where this reaction is minimal, looking like a weak spur. So I was thinking of an experimental protocol in which specificity could be detected in the zone of optimal interaction between antigen and antibody, where the precipitation zone would be most pronounced. Finally we succeeded in developing a system now known in the literature as the ‘‘quadrant scheme’’. Using this scheme it was possible to compare two complete antigen–antibody systems and displace specific reaction to the most potent reaction zone [4]. This approach was very successful; it is still employed now, and since 1957 commercial firms issuing matrices for precipitation in gel include the quadrant scheme into their kits. I was very pleased that this work was appreciated and gave an expected result. We obtained clearer detection of the hepatomaspecific antigen and reliably detected its line in the complex spectrum of bands. In the summer of 1958 at the World Cancer Congress in London, Zilber presented the results of studies on tumor and normal antigens obtained by means of precipitation in gel. He demonstrated the phenomenon of antigenic simplification, and in the spectrum of precipitation bands he also showed the band corresponding to specific hepatoma antigen [5]. We continued our successful studies and a very good period in our research appeared. As I wrote above, we developed a method for purification of one of the most potent organ-specific liver antigens (AO) and demonstrated association of this antigen to particulate fraction and its easy solubilization. Now I am confident that properties of this antigen and the method for its purification well fit cytokeratins. Here I should say that the phenomenon of antigenic simplification was not as universal and abundant as Weiler believed in the very beginning, and as we suggested in the period of the first experiments. Our subsequent experiments on the behavior of the organspecific antigens in primary tumors and at various stages of carcinogenesis revealed that tumors had certain features of their organ origin and contained organ-specific antigens [6]. However, hepatoma-22a was the most non-differentiated marginal form and so all changes were highly expressed. (We were quite lucky to use this research object.) So it was reasonable to continue this work using various tumors.

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Individual Antigens and Monospecific Antibodies Our major achievements of that period (besides organ-specific antigen purification and the quadrant scheme) consisted of two more important steps in characterization of our antigens. These were preparation of monospecific antibodies to individual organand tumor-specific antigens and the development of the method of immunofiltration for immunochemical purification of these antigens. We obtained monospecific antibodies by dissociation of specific precipitates. Specific precipitate containing one antigen and corresponding antibody was obtained using a multicomponent antigen system and polyspecific antiserum. Using successive absorption of this serum by unrelated antigens and partial purification of the investigated antigen, we finally obtained the precipitate formed by just one antigen–antibody pair. Subsequent incubation of this precipitate in weakly acidic medium resulted in elution of 15% of the antibodies. This procedure was rather complex and time-consuming, whereas antibody yield was low. Such system exhibited different behavior with different antigens, but to our surprise (and satisfaction!) we obtained a whole spectrum of antibodies against various antigens. This work was carried out in collaboration with N.I. Khramkova (Kuprina). The initial work on eluates was carried out with Z.A. Avenirova (see Ref. [3], pp. 308–323). We easily obtained monospecific antibodies to AO and to our hepatoma-specific antigen. This was perfectly wonderful because using such antibodies it would be possible to detect an antigen of interest in the whole tumor extract or in extract of tumor subcellular fractions: precipitation in gel gave clear and well-detected band of the antigen, which could not be missed among other bands (Figure 3). This provided a firm and reliable background for our subsequent studies. Using such a reliable test, we continued our work. In 1957 N.V. Engelhardt joined our group after graduating from Moscow State University. At that time V.S. Tsvetkov also joined our group, and we developed the method of organ-specific antigen purification with him (see above). A.I. Gusev worked in close contact with our group. Using the same methodology and objectives, he started to work with Rous chicken sarcoma as the model. Gusev was ‘‘a master’’ of experimental work, and he also obtained very clear results with gel precipitation and antibody

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Fig. 3. Characteristics of the eluate of antibodies to the specific hepatoma antigen. EH, eluate of antibodies to hepatoma antigen; AH, anti-hepatoma serum; eN, eluate to the organ-specific liver antigen, AO; gH, gamma-globulin, isolated from mono-specific anti-hepatoma serum; AN, anti-liver serum; AS, antisplen serum; AK, anti-kidney serum; H, hepatoma extract; N, liver extract; S, spleen extract; K, kidney extract. From Ref. [3].

eluates. We had very good and very open interaction, and in 1958 our group of five researchers continued very intensive studies on tumor antigens. Together with Tsvetkov, we started to develop the method of immunofiltration. We considered the following hypothesis: our antigen (which we called H-antigen, antigen of hepatoma) was characterized by rather high-electrophoretic mobility, similar to that of serum alpha-globulin, i.e., larger than that of gammaglobulin (to which antibodies belonged). So using electrophoresis in agar it was tempting to create electroosmotic movements of antibodies to meet antigen, which would move in opposite directions: antibodies to cathode and antigen to anode. In this case the

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antigen had to pass through the antibodies. If the antigen contained ‘‘irremovable’’ electrophoretically similar contaminants common for normal and tumor tissues and gamma-globulin fraction contained antibodies against common components of normal and tumor tissue, such antigen–antibody movement in opposite directions would represent antigen ‘‘filtration’’ through antibodies. In this case all contaminants would have to bind to corresponding antibodies and precipitate or form antigen–antibody complex characterized by lower electrophoretic mobility than the antigen. Consequently, only pure antigen was able to penetrate through the ‘‘antibody filter’’, whereas all contaminations would be retained on it. Initially we developed an analytical version of such system, then we made a preparative version, and finally within one year (or even a few months) we developed several variants of the immunofiltration method. Using this method we obtained immunologically pure antigen of hepatoma. It exhibited one precipitation band with polyspecific serum against hepatoma and did not react with antiserum against liver, and it was not detected in other organs. Thus, it met all our criteria [7]. Using this method applicable for most various antigens, we obtained specific hepatoma antigen (Figure 4). We were very proud to have in hand the first pure tumorspecific antigen. However, we faced other problems: what is the nature of our antigen, its origin, and putative role (if any) in antitumor immunity?

Fig. 4. Immunoelectrophoresis of specific hepatoma antigen, isolated by immunofiltration. From Ref. [3].

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So far we had been solving methodological problems: would precipitation in gel be applicable for detection of specific antigens, would it be possible to develop methods for isolation and identification of this antigen? When antigen isolation was the major goal of our studies, we did not think about the nature of the antigen. We thought that when this antigen would be available our experimental system would give us a hint. However, in the process of antigen isolation we became more and more concerned about its nature and putative role. First, this antigen might be an antigen of a ‘‘passenger’’ virus, which simply ‘‘settled’’ in the tumor. In such case it is natural that this antigen is detected in tumor but not in normal tissues. This might be the case especially if we took into consideration that this antigen was found mainly in our transplantable hepatoma and rather rarely in primary hepatomas induced by carcinogen. This increased the possibility that this antigen would be a secondary antigen indirectly linked to tumor transformation. Furthermore, we were not confident that our hepatoma actually represented hepatocellular cancer (i.e. the tumor originating from liver parenchymal cells representing the major population of liver cells). It might be possible that the tumor originated from bile duct cells, which are much less abundant in liver. If our antigen represented the organ-specific antigen of bile epithelium, we would not detect it in the liver due to low sensitivity of the gel precipitation method, and in tumor its content would be much higher. In such case this antigen would be just bile epithelium organ-specific antigen rather than specific tumor antigen, which we were looking for. (This possibility was quite real and our colleague Dr. A.Ya. Fridenstein suggested it as a plausible alternative explanation of our findings.) Finally, it was also possible that small amounts of this antigen present in normal liver were below the detection limits of our method, whereas in the tumor the antigen content was much higher. In the end of 1960 we started analysis of all these possibilities. We were very skeptical and expected that one of these versions would be experimentally confirmed. We developed a highly sensitive gel precipitation method, which were also based on immunofiltration. This method was 10–20 times more sensitive and could readily detect possible traces of this antigen in normal liver.

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N.V. Engelhardt started to adapt an immunofluorescence method for investigation of localization of our antigen. This was a very difficult methodological task, but we cherished hopes for use of the immunomorphological method, which would detect possible localization of our antigen in bile capillaries. However, this very difficult task was solved only in 1969 (see below). Simultaneously, we started to analyze various tumor strains for the presence of our antigen. This would give us a clear answer whether this antigen was linked to some ‘‘passenger’’ virus, which might be recognized in other tumors. These were our major goals of experiments of 1960–1961, which gave us variable results. At that moment one event sharply changed direction of our studies. Alpha-fetoprotein (AFP) In parallel with investigation of hepatoma-specific antigen, we also continued our work on ‘‘antigenic simplification’’. Our results indicated that antigenic simplification was more complex than suggested by Weiler and by us (studying hepatoma-22a only). In other hepatoma strains organ-specific antigens persisted, and later we also detected traces of one organ-specific antigen in our 22a hepatoma. Tumor progression is characterized by changes in the pattern of organ-specific antigens; each tumor has its own set of organ-specific antigens. We identified at least seven various antigens. This was the ‘‘zone of responsibility’’ for Mrs. N.I. Khramkova (Kuprina). We observed very interesting phenomenon of individual antigenic structure of various hepatomas (see Ref. [6]). We decided to investigate this individuality in more details, whether it reflected the possible existence of discrete periods in the development of liver characterized by certain sets of organ-specific antigens. It might be possible that antigenic ‘‘maturation’’ of the liver has several discrete stages, and structures of different hepatomas might correspond to different stages of liver differentiation. We had a collection of antibodies against seven organ-specific antigens (i.e. test systems for these antigens), and Khramkova and I decided to check this hypothesis. Since we also had a test system to the hepatoma antigen, we also employed that system as well. We had no hopes – we just wanted to try.

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In our laboratory there was a young student from the medical institute. She wanted to become familiar with immunology and to get some experience in laboratory work. She learned to perform precipitation in gel, and Khramkova gave her all our test systems. The student used embryonic liver for analysis of organ-specific antigens. We had never analyzed them in embryonic liver. She collected embryos at various stages of development and performed experiments with all the test systems. However, that student disappeared from our laboratory for some unknown reason, and we had to analyze her precipitation results ourselves. We were shocked! In the extract from the embryonic livers there were huge amounts of our ‘‘hepatoma antigen’’, which we never saw in the hepatoma preparations. Our first feeling was that this was experimental error of our student. However, we did not have any plausible explanation for mistakes she could make! We immediately repeated that experiment and it demonstrated exceptionally high content of our antigen again. We repeated this experiment using the liver and other embryonic organs and results clearly demonstrated very high levels of this antigen in them! These levels were much higher than in hepatoma extract! At first glance this was a dead-end. We did not have any plausible ideas on the origin of this antigen in the embryonic liver. Why were its significant amounts detected in other embryonic organs? Suddenly we realized that in reality this liver antigen might be blood antigen (serum antigen). This would explain its presence in various organs because we did not wash these organs free from blood! We immediately investigated embryonic blood serum and obtained very high titer of this antigen; we never detected such high titer in any other biological material used for analysis! It became clear that the former hepatoma antigen, (in reality the so-called hepatoma antigen) represents blood serum antigen. This conclusion was like a cold shower for us. Indeed, our antigen was a serum protein. It could be synthesized in liver and/or some other organs in response to tumor growth; it could be one of the acute phase proteins, which were already known at that time. These proteins were synthesized in liver and were released into the blood stream in response to various pathological conditions. For example, a similar protein was detected in blood of rats in response to growth of various tumors. Appearance of such protein

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would represent a nonspecific response of an organism to various pathological processes. After appearance in blood such protein could be transported to various organs and accumulated in tumors and necrotic tissues. Although we washed tumors free from blood, its traces would surely remain anyway, so that this protein might just represent contamination unrelated to the tumor. We analyzed blood of mice bearing hepatoma-22a and found very high levels of this antigen. Interestingly, blood levels were much higher than that of tumor; this antigen was also detected in organs of hepatoma-bearing animals. In previous experiments we used only normal organs of healthy animals, and this explained why we did not find our antigen there. It became clear that we were dealing with acute phase protein, which we considered as a tumor-specific antigen. If this suggestion were correct, this antigen would be formed in organisms in response to any tumor growth, say sarcoma, which is unrelated to hepatoma. So we went to Olga Lezhneva, a member of Zilber’s laboratory who was working with mice transplanted with carcinogenic sarcomas. We took blood from these animals and analyzed these samples for precipitation. The next day we detected the presence of our ‘‘hepatoma antigen’’ in one of four investigated samples. The worst suggestion was almost confirmed. The only hope was that one of the blood samples was taken from a pregnant mouse. (Since embryo contains huge amounts of this antigen, it could be also detected in blood of the pregnant mouse.) We immediately went to the animal house and found that there were newborn mice in the cage with our mice! But it was unclear which mouse gave birth to these pups! So, we took blood from male mice transplanted with sarcomas and other tumors and analyzed blood samples for the presence of the hepatoma antigen. We did not find it, and this fact gave slight hope that our antigen was really associated with liver tumors. We did not find this antigen in the blood of animals with other tumors. However, it might be possible that this antigen appears in blood only in response to hepatoma growth (or preferential hepatoma growth). To exclude all other possibilities, we had to demonstrate in direct experiments the synthesis of this antigen by our hepatoma. If we were to demonstrate this phenomenon, it would become clear that we were dealing with some embryonic antigen, which was reexpressed in liver cancer. This was very

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interesting. However, I should remind again that we had to demonstrate antigen synthesis by tumor. It was a soluble (but difficult) task. We started to solve this problem using several approaches. These included heterotransplantation of hepatoma to hamster cheek pouch and to cortisone-pretreated rats. Our suggestion was that if mouse tumor synthesizes our antigen, we would find it in hamster blood and in rats. In parallel we started our joint experiments on the hepatoma antigen in tissue culture with Dr. I.S. Irlin and Ms. S.D. Perova, who joined our group when Mrs. Khramkova went for maternity leave. We tried to get hepatoma primary cultures. If the antigen was synthesized in cell cultures, we would get the best and the most valuable evidence for its synthesis by tumor. We were quite lucky in these experiments. First, we found some quantities of our antigen in the hamster cheek pouch. We did not get large tumors in the hamster pouch, there were just small nodules. However, we were able to detect the hepatoma antigen in these nodules and their extracts. It was possible that this antigen was synthesized by tumor. However, it was also possible that we had introduced it during transplantation. In rats we got clearer results. In rats the mouse tumor had rapidly grown in two weeks and then it started to resolve. Thus, in parallel to the tumor development and resolution, we were able to detect our hepatoma antigen in blood, which disappeared during tumor resolution. However, some problems still remained: since mouse and rat are closely related species, do cross-reacting antigens exist? Using rat embryonic serum we found a crossreacting antigen, exhibiting potent suppression of our test system for hepatoma antigen. (This suggested the existence of a related antigen.) Thus, we felt that results of these experiments suggested the existence of mouse antigen analog in rats, so we were still looking for additional evidence. Two months passed and we finally obtained hepatoma primary cultures in vitro. In the culture medium we found the hepatoma antigen! We washed the cell culture to exclude possible blood contamination, we changed cultivation medium, but the antigen was detected. Thus it became clear that the hepatoma antigen had been synthesized in the tumor and that we caught an interesting

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phenomenon. In embryos this protein was probably synthesized in the liver as some serum protein, which we called af -globulin or fetal a-globulin. Embryonic concentrations of this af -globulin significantly exceeded the concentrations of serum albumin; however, during subsequent development synthesis of this antigen stopped and appeared again in primary liver cancer (hepatocellular carcinoma). Although this phenomenon was not observed in all the cases, it was definitely noted in rapidly grown anaplastic hepatomas rather than in highly differentiated hepatomas. Anyway, in most hepatomas synthesis of this antigen was resumed. Hepatoma-22a used in our crucial experiments was the most potent producer of this a-globulin. So we were very lucky to use it in the very beginning of our studies! Thus, it became clear that liver tumor might reexpress the embryonic antigen, which is almost totally suppressed in cells of adult liver. This meant that embryonic antigens specific for particular tissue might appear again in tumors derived from this particular tissue. Moreover, these antigens might appear not only in the tumor but also they could be secreted into blood (provided that this is a secretory protein). Consequently, an embryonic antigen, which is absent in blood of healthy adult mice, may be a tumor marker detectable in tumor as well as in blood of tumorbearing animals. Later we found that this phenomenon is also typical in rats. Thus it became clear that this is a common phenomenon at least in the cases of mouse and rat hepatomas. This story on the discovery of hepatoma antigen in embryonic liver began in January 1962, and in July 1962 the 8th World Cancer Congress would be held in Moscow. Our lecture was scheduled into its program. Here I should say that Professor Peter Grabar, a distinguished French immunochemist, the author of the method of immunoelectrophoresis, was familiar with results of our previous studies on the hepatoma antigen. Being interested in this problem, he invited me to report our results at a so-called panel discussion of the congress because this was the first tumor antigen isolated using immunochemical methods. We sent an abstract of our presentation before our discovery that this is the embryonic antigen: reexpression of the embryonic antigen described above became clear only in May 1962. The Gamaleya Institute was being refurbished: the administration was preparing the institute building to meet guests of the

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congress. We were constantly moved from our laboratory rooms, and I still do not understand how we were able to clarify the situation with our antigen within four months. Anyway, in July 1962 I reported our results at the 8th Cancer Congress. The audience appreciated our results, however, discussion went in several directions. Most questions were focused on the problem of the existence of specific hepatoma antigen (besides the embryonic one). Shortly after the congress our results became available for the Soviet and international scientific audience [8,9]. A very important continuation of the above data was obtained by Yu.S. Tatarinov, a biochemist from Astrakhan who was interested in human serum proteins in hepatic pathologies. We had known each other for about two years, and he already started to characterize the proteins in embryonic serum. Although it was clear that such system might have diagnostic value, we did not push forward such a project. However, some results indicated that our antigen would be a marker of hepatocyte proliferation, because its temporary synthesis was observed in mouse regenerating liver. We regard this antigen as the marker of hepatocyte proliferation rather than the immunodiagnostic marker of hepatic cancer. So, we were not inspired with the idea of clinical studies in humans. Tatarinov obtained corresponding anti-embryonic serum, absorbed it with adult serum, and then analyzed the serum obtained from a hepatoma patient. In the very first experiments he obtained convincing evidence for the presence of embryonic a-globulin in it. Thus it became clear that the described phenomenon also exists in humans. In the beginning of 1964 Tatarinov reported his data at the First All-Union Biochemical Congress in Leningrad and published a paper in the journal ‘‘Voprosy Meditsinskoi Khimii’’. Thus, he demonstrated an important diagnostic aspect of this problem. I always appreciated (and appreciate now) Tatarinov’s priority in the diagnostic aspect. His work in Astrakhan was much more difficult than ours in Moscow, in Zilber’s laboratory. So I ‘‘restrained’’ activity of my coworkers trying to get into liver cancer diagnostics and reserved this area for Tatarinov. However, in 1965 or 1966, Dr. N.I. Perevodchikova, a clinician from the Institute of Experimental and Clinical Oncology, asked me to study embryonic a-globulin in hepatic cancer for evaluation

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of its possible diagnostic value. I advised her to contact Tatarinov, but she desperately wanted to work with us. Tatarinov had already described six clinical cases and his priority in this problem was unquestioned anyway. In my papers I always cited his works. However, I was a bit skeptical about the diagnostic value of the embryonic a-globulin, and so we started to work with Dr. Perevodchikova. Quite soon we confirmed Tatarinov’s observation on appearance of af -globulin in blood of patients with primary hepatic cancer. However, we found its appearance in only 70% of cases. Furthermore, we also demonstrated appearance of af -globulin in blood of patients with testicular teratoblastomas. This was very important because it became clear that this protein appeared in tumors originating from embryonic cells. The reason for appearance of af -globulin in the embryonic cellderived tumors was unclear. Subsequent studies revealed that this was as important as the diagnostic aspect for primary liver cancer. In 1966–1967 we obtained a more or less clear picture using representative clinical material obtained from 55 patients with primary liver cancer and many patients with testicular teratoblastoma. We published results of our observations in the International Journal of Cancer [10], and this paper was appreciated by the international scientific community, especially by tumor immunologists. Together with Tatarinov’s papers, it basically opened a diagnostic aspect of this problem [11]. It became clear that embryonic antigens may appear in a tumor, be released into blood, and they may serve as diagnostic markers of tumor(s). Thus, embryonic antigens may be used for specific serologic diagnostics of cancer, for its differential diagnostics [12,13] (Figure 5). Soon after our first publications on hepatoma, Gold and Freedman (1965) referring to our works and using our approaches (including the method of precipitation in agar and the quadrant method) detected specific antigens in colorectal tumors, and then they also demonstrated the embryonic nature of those antigens. In 1969 these authors also demonstrated that low concentrations of the carcino-embryonic antigen (CEA) could be detected in blood of patients with colorectal cancer and might serve as a diagnostic marker. In the second half of the 1960s it became clear that embryonic antigens might appear in various tumors. These antigens may be

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Fig. 5. Schematic representation of alpha-fetoprotein (AFP) synthesis in normal development and pathologic states. Solid line, serum AFP level in arbitrary units; broken line, expected AFP level; ?, not known. From Ref. [12].

secreted into blood or appear in blood via other routes. Their detection in blood is a diagnostic sign of a particular form of cancer. Subsequent works by our laboratory and by Czech scientists revealed that the time-course of these antigens in blood well corresponded to the time-course of development of corresponding tumors. In the end of the 1960s, there were three tumor types that could be diagnosed by embryonic antigens: primary liver cancer, testicular teratoblastoma, and colorectal cancer.

Resonance and Subsequent Studies We started to publish results of our studies in 1959; our key paper on embryonic antigen in hepatomas appeared in 1963 and attracted much attention from the scientific community. However, this interest enormously increased after elucidation of diagnostic aspects of this problem (i.e. from 1967). This international interest in our work was also stimulated by Professor Grabar, who was very familiar with our results. His own laboratory in the Villejuif Institute for Scientific Research on Cancer in France was involved in studies of related problems, and starting in 1965 they obtained results that were consistent with our results obtained in rats. Grabar and his colleagues also started to develop the diagnostic aspect of this problem. They established good contacts with Professor Rene Masseyeff, who worked in Dakar University (Senegal) at that time. Owing to high frequency of primary liver cancer in West Africa, they collected a large amount of material supporting our first observations. Thus, interest in the problem increased.

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My contacts with Howard Goodman, Head of the Immunology Unit at the World Health Organization and a very nice and intelligent man, resulted in arrangement of an international experiment in African countries on validation of a diagnostic value of af -globulin. This experiment started in 1968 with our trip with Tatarinov to research centers of African countries involved in studies of primary liver cancer. This was a very impressive trip with adventures, demonstration, and propaganda for our results, with establishment of new, close, and open scientific contacts. Finally, our activity culminated in establishment of international experiments, which gave very clear positive results in 1969; they were published in 1970 in ‘‘Cancer’’ [14]. All these events resulted in rapid international recognition of the alpha-fetoprotein (AFP) test and its wide distribution in all countries. The AFP test was applied also in cases of testicular and ovarian teratoblastomas, where it could evaluate effectiveness of medical treatment. In contrast to primary liver cancer, these tumors can be effectively treated by surgery and chemotherapy and therefore evaluation of effectiveness of such treatment by a simple serologic test is very important. In the end of the 1970s, the total number of publications on AFP exceeded two thousand, and this problem smoothly entered both science and clinical medicine without hot discussions or skeptical resistance. Of course, there were some disputes with Jose Uriel, Grabar’s pupil, but they were devoted to some ‘‘local aspects’’, and we soon came to a common viewpoint and maintained friendly relations. In 1975 this work received one of the First Prizes on Cancer Immunology awarded by the New York Cancer Research Institute. In the very beginning of the 1970s, Japanese Professor Hidematsu Hirai organized an international group for studies of cancer embryonic proteins. In 1980 this group transformed into the International Society for Oncodevelopmental Biology and Medicine (ISOBM). In 1976 I received a prize awarded by this group for discovery of AFP in hepatoma. Earlier (in 1973) I was elected as an Honorary Member of the American Association of Immunologists. I was very pleased with these awards. (I should say that I even did not know that I had been nominated for these awards.) In 1978, together with Tatarinov, we received the USSR State Prize for the discovery and practical application of AFP in cancer. We received this prize on the ‘‘third attempt’’. In 1971,

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the Gamaleya Institute nominated us together with a group of colleagues for the State Prize, but our work was rejected. The same situation was repeated in 1976. Finally, working in the USSR Cancer Research Center, we were nominated for the State Prize and received it in 1978. In 1990 I received the Abbot Prize awarded by the International Society for Oncodevelopmental Biology and Medicine. Although the series of our studies were internationally recognized and received prestigious prizes and awards, this period of international recognition coincided with a very hard time in my life. In 1966, L.A. Zilber suddenly passed away, and all members of his department suggested me as the new Head. The administration of Gamaleya Institute and its Director, Academician O.V. Baroyan of the USSR Academy of Medical Sciences, had to accept me as the new head of Zilber’s department. For three years I worked as Executive Head of this department, and in 1969 I was elected to this position. Everything looked fine: studies successfully went in the directions we wanted. In 1971 we developed a diagnostic kit for AFP and gave it to the production department at the Gamaleya Institute. This was the first kit for immunodiagnostics of cancer in the world. During that period I was in frequent contacts with the World Health Organization, Institute for Scientific Research on Cancer in France, and often traveled abroad. However, in 1971 everything changed. In 1971 the first people who desperately wanted legal emigration to Israel appeared. They were blamed by formal Soviet public opinion and treated as ‘‘traitors to the fatherland’’. Scientists lost their jobs, and they could not get necessary documents required for emigration visas. Laboratories where such people worked had serious problems as well. Waiting for official permission for emigration varied from several months up to many years without any reasonable explanation. The presence of such persons among staff members of the institute and especially the laboratory represented basically ‘‘nightmares’’ for administrators. Usually dismissal of such potential emigrants occurred after public discussion of their personalities, which always ended by very rough blaming of ‘‘such antisocial persons’’. People with Jewish background who remained loyal to the Soviet regime had to participate actively in such a ‘‘farce’’, irrespectively to their own viewpoints. In the end of 1971 one microbiologist, a junior researcher of the institute, decided to emigrate to Israel. At the forthcoming

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meeting of the Scientific Board at Gamaleya Institute, this person would be reelected to his position. (This is a common practice in all research institutes.) However, in reality he could not be reelected for a new period. The real goal of that meeting was to blame that potential emigrant. All heads of laboratories with Jewish background had to participate in that meeting. Besides blaming the ‘‘renegade’’, the administration expected that Jews of our institute would demonstrate their own loyalty. I was not going to emigrate, but I did not want to play a role in such a shameful farce either. Although I was a member of the Scientific Board, I ignored that meeting. My ‘‘demarche’’ caused almost immediate and hard reaction from a director of the Gamaleya Institute. He slightly changed the names of two laboratories of Zilber’s department, but this caused serious consequences. My laboratory, the Laboratory of Immunochemistry, was renamed as Laboratory of Immunochemistry and Diagnostics of Cancer. The other one, the Laboratory of Cancer Virology, was renamed as Laboratory of Viral Etiology of Cancer. This formal ‘‘reorganization’’ resulted in dismissal of all coworkers, and we had to apply for a position in these ‘‘new’’ laboratories, as all scientists who should be elected anew. At the same time, the scientific community of Moscow supported us very actively. Academicians Vladimir Engelhardt and Boris Astaurov, directors of the Molecular Biology (V.E.) and Developmental Biology (B.A.) Institutes, respectively, called a conference in the academy devoted to cancer virology and immunology and asked me to present the results of our work. Our director appointed a special meeting of the institute scientific board with my account on the work of my laboratory just on the same day and at the same time, so that I could not do my presentation in the academic meeting called by Engelhardt and Astaurov. The only way to join this meeting was to retire from the work in my institute. I did so, made the presentation at the academic meeting, lost the work in the institute, got positive support from the scientific community of Moscow, and started the struggle for the transfer of my laboratory to the Oncology Institute. I asked the president of the Academy of Medical Sciences to transfer our laboratory into the Oncology Institute. My colleagues and I protested against this despotism and the director (an energetic, hard, and severe man) initiated rough

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‘‘strikebreaking’’. Echo of our ‘‘war’’ with him reached the President and the Presidium of the Academy of Medical Sciences. The Presidium formally took our side and liquidated the ‘‘fire of this war’’. However, this victory left ‘‘painful scars’’ in my soul. I should say that I have never had enemies and I do not have them now (both in scientific and human terms). Nevertheless, from time to time I am involved in conflicts with state and institute administrations. I think that such conflicts originate from my subconscious resistance to humiliations. Anyway, that ‘‘war of 1971’’ ‘‘burned’’ us. Of course we continued our work, but the whole team of our laboratory was disturbed. We felt that we were ‘‘under the gun-sight’’ of the administration of the Gamaleya Institute, and nobody inside the institute could help us (although many people did sympathize with us silently). This was a very difficult period of my life. My scientific visits abroad immediately ceased. I could not attend even conferences on AFP or cancer immunodiagnostics. Our work was completely ignored by the administration. Literally speaking, we had to keep ‘‘circle defense’’. This situation coincided with serious changes in directions in analytical and applied studies on AFP. At that moment I started my fight for transfer of the department to Blokhin’s Oncology Institute.3 I thought that this would give us the possibility for normal and productive research. Finally, in 1977 this fight with the administration of the Gamaleya Institute finished by our movement to the Oncology Institute. During these six years we worked very intensively (as intensively as before), but we were almost fully isolated from the international scientific community. Nevertheless, this was a very fruitful period, which was also appreciated by our colleagues. I published a review, one of the first reviews on alpha-fetoprotein [12], which was recognized as a Citation Classic by ‘‘Current Contents’’ (800 references during 1971–1980). The other work (by N.V. Engelhardt, A.K. Yazova, and V.S. Poltoranina) underlined reasons for synthesis of AFP by experimental testicular and ovarian teratoblastomas [15]. This work demonstrated that this protein was synthesized in germinal tumors because of development of the element of embryonic yolk sac. (Besides liver

3

Now N.N. Blokhin Russian Oncological Scientific Center of the Russian Academy of Medical Sciences.

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embryonic yolk sac, entoderm also synthesizes AFP in normal ontogenesis as well.) This was a very nice and exact work. The third work of that period was related to the development of a highly sensitive method for determination of not only AFP, but other antigens as well; this method was based on isotachophoresis in polyacrylamide gel. This method demonstrated the presence of traces of AFP in sera of normal animals and revealed the basic level of this protein in serum of healthy people. This study was subsequently confirmed by others. In collaboration with S.D. Perova, we developed a very nice, precise, and elegant variant of the method for determination of AFP synthesis by single cells of hepatoma or hepatocytes in a cell culture or in micro-colonies of these cells [16]. During that period, we (A.I. Gusev, A.K. Yazova, and S.D. Perova) also developed a commercial version of an immunodiagnostic kit for simple, convenient, and inexpensive AFP assay, which was produced by the Gamaleya Institute for about 15 years. Finally, there was one polemic paper, which I prepared together with my first (late) wife as a response (or maybe as our reaction) to all these events in our lives. Besides my resistance to the above-mentioned administrative pressure, we were writing that paper about ethics, about the role of ethics in science. This paper was ‘‘our manifesto’’ about normal human relations in science and our personal viewpoint about organization and administration in science. Literally speaking, that paper was written with our blood. We sent the manuscript to the Soviet journal ‘‘Nature’’ (‘‘Priroda’’), where it had been positively reviewed by Academician V.A. Engelhardt, but in spite of this it was not published. Quite unexpectedly, it appeared in another journal, Chemistry and Life, in the beginning of 1985 [17]. Thus, 13 years later this paper was published in slightly a shortened variant, but our major ideas are preserved there. (I should say that after publication of our paper, this journal had serious problems.) In spite of all attendant circumstances, we continued our research. We were involved in studies of a new interesting erythroblast antigen, which we intensively studied and which had clinical application as well [18]. We also studied the problem of expression of endogenous viral antigens [19] during normal mouse development and in tumors. Our investigation of morphological aspects of AFP during liver regeneration represented a basis for subsequent studies.

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Our main problem was that of AFP regulation: why does AFP cease to be produced in liver maturation, and why it is reexpressed in hepatocellular carcinomas? The main observation important for understanding of this problem was the role of cell interaction in the suppression of AFP synthesis. It was shown that AFP synthesis is resumed after poisoning of the liver by hepatotoxins in the hepatocytes bordering the necrotic area. These cells lost their contacts with neighbors and seemed to be isolated from the liver plate [20]. The isolation of hepatocytes from the adult liver by treatment with collagenase in Ca2+-free medium led to AFP production in the overwhelming majority of cells. Restitution of intercellular contacts suppressed AFP synthesis, while inclusion of hepatocytes in a three-dimensional extracellular matrix resulted in formation of liver-like islands with reestablishment of cell polarity, bile canaliculi, and full suppression of AFP [21]. The cell–extracellular matrix interaction in three-dimensions became the major factor in induction and maintenance of hepatocytes differentiation, including suppression of AFP [22]. This could be the reason for AFP reexpression in liver tumors. This is the problem of today’s and tomorrow’s research.

Concluding Remarks My opinion on the significance of our work. I should say that our studies leading to discovery of AFP in hepatomas were our most important works. On the one hand, they represented a basis for the development of the ‘‘carcino-embryonic’’ direction in oncology, and on the other they started cancer immunodiagnostics. Very important for us is the study of regulation of AFP synthesis in liver development and regeneration. However, in my very personal viewpoint, our studies of wide expression of the endogenous retroviral antigen are also very attractive and important. They basically represented the first immunologic evidence for the ubiquitous distribution of endogenous retroviruses in the mouse genome. We investigated the expression of this viral genome in mouse ontogenesis. I still like this work of 1970, and do believe that it is a very important study. This work was also appreciated by the scientific community, but to a much lesser extent compared with our studies on AFP.

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I like my early series of papers on separators, especially on the chamber separator. This was an original device of new construction in pair with a fully original method of particle separation both by dimension and density. Although these works were appreciated and recognized, there was no further development of this approach in other laboratories because of lack of laboratory separator production and commercial realization of these devices. This is very annoying and disappointing. I like my work on the method of immunofiltration that ensures isolation and purification of antigens specific for a certain particular system. I do not say that this work did not have scientific resonance. It did. And from time to time similar methods appear in various modifications. Finally, I like my work of 1979. It gave a push to a subsequent series of studies on counterflow isotachophoresis in porous membranes. I feel that in that work we found a new principle of electrophoresis, its new version: flow isoelectric focusing based on automatic isotachophoresis, rather than the specific electrophoretic method. This work required intellectual efforts and accurate and long-term analysis of very simple and unclear phenomena, which take place in hydrophilic porous membranes during electrophoresis. Suddenly, I understood the reasons for abnormal behavior of liquid in the membrane during electrophoresis. This understanding resulted in the development of a flexible method with wide capacities. Its use allows simultaneous concentrating and effective separation of proteins from a solution, which is in flow and contains trace amounts of these proteins. The lesser the concentration of protein the more effective the method is (although the method itself is rather simple and theoretically clear) [23]. This method was employed for diagnostics of malignant B-lymphomas using urinary proteins. I think this method has promise in nephrology, in obstetrics, basically everywhere that analysis of protein composition in biological liquids with low-protein content is required. Using this method we developed a completely new principle of automated multistage immunochemical reactions and a new arsenal of methods for work with monoclonal antibodies (from screening of monoclonal antibodies and including epitope analysis) [24]. Thus, certain manipulations involving monoclonal antibodies can be transformed in automated mode of analyses based on counterflow isotachophoresis in porous membranes.

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Although we employed this method for several years and have published it in Russian and international journals, its resonance is rather small. We know advantages of this method and its large capabilities, but it is still in a latent period. It is possible that this situation occurs due to popularity of methods that require commercially available reagents produced by special companies. Possibly this happens because of rather bulky description of this method; the method itself is very simple, and I do not understand why other laboratories do not use it. At least we would like to see it being employed by others. However, time goes fast and each method is good for its particular time. It is really sad that during many years this method has been used mainly in our laboratory; it would be very useful in many other studies. Some formal information. My first scientific paper was published in 1953. It dealt with the method of sample preparation for electron microscopy from saline solutions. I still like this paper; it is very short and simple. I was involved in electron microscopy in Zilber’s laboratory; he wanted me to use electron microscopy, but I did not like it. Anyway, I developed a method for electron microscopy of samples avoiding distilled water, a stage required for salt removal [25]. However, three years earlier A.N. Belozersky and N.V. Proskuryakov published their textbook Practical Works in Plant Biochemistry (1950) with some modifications of the methods, which I developed in my diploma project. They described these methods and acknowledged my authorship, and I was very proud. I obtained my Ph.D. degree in 1955 and my life changed. First of all my salary increased, and this was very important because I had a family with two children. Right after defense of my Ph.D. thesis I finished all my extra works, which I needed to get money. (I used to write abstracts, worked as a teacher, and even as a loader.) After public defense of my Ph.D. thesis I worked only as a researcher. In 1963 I defended my D.Sci. thesis devoted to identification of individual tumor antigens. It included all materials on gel precipitation, all methodical materials related to immunofiltration, isolation, and purification of tumor-specific antigen; and initial results of studies of the embryonic nature of this antigen. I should say that I did not write my thesis; my defense of the D.Sci. degree was based on a monograph written together with Zilber [3]. It was an interesting story. Zilber had a contract with

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Medgiz (State Publishing House for Medical Literature). According to this contract he had to write a book on cancer virology and immunology by 1961. Zilber was very busy and he could not finish that book by the time indicated in the contract. It was especially difficult for him to write chapters devoted to analysis of recent immunogenetic studies. During that period he was especially interested in cancer virology, and he did not have enough time for chapters on immunogenetics and histocompatibility antigens. This particularly important direction was just begun to develop very intensively. Zilber asked me to analyze the literature on these subjects and to write a chapter to his book. I readily started this work, entered the problem, and analyzed unusual and complicated literature. I wrote a chapter on transplantation antigens, tissue compatibility, and its genetic control and also on the role of the transplantation antigens and their importance for studies of cancer immunology. Zilber liked this chapter and he invited me to write one more chapter, and then other chapters y Basically I wrote almost the whole section of that book devoted to cancer immunology, which represented more than half of that book. This section contained results obtained by Zilber’s and our team. (Our laboratory was just being organized.) At that time we did not know that the hepatoma antigen is the embryonic antigen. This book was finished in 1961 just before the discovery of AFP, and it also included all methods that we developed. This explains why I did not want to ‘‘copy’’ the same material for a thesis. It was a nice liberal time in Soviet science when it was believed that scientists should do good research, whereas the form of its presentation is less important. In 1963 I defended my D.Sci. degree using the immunological section of that book, which included all data before AFP and the very beginning of alpha-fetoprotein research. My defense of the D.sci. degree was quite successful in the Academy of Medical Sciences of the USSR; however, problems began when all documents were sent to the High Attestation Committee. (This is usual practice of Soviet and Russian science: results of public defense of Ph.D. and D.Sci. theses have to be approved by the High Attestation Committee.) One referee of that committee concluded that I had to write a whole text and repeat the whole public defense again. Fortunately, the other referee wrote a very positive report about ‘‘my part’’ of the book and in 1963 the High Attestation Committee approved the results of my public defense, and thus I got my D.Sci. degree.

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The book by Zilber and me, published in 1962, was translated and edited by Pergamon Press in 1968. All my younger colleagues (N.V. Engelhardt, N.I. Khramkova, A.I. Gusev, V.S. Tsvetkov, and T.D. Beloshapkina) defended their Ph.D. theses in 1964–1967, and in 1967 I received the title of Professor. In 1964 a new important period began in my scientific life: I started to give lectures on immunochemistry at the Chair of Virology of Moscow State University organized by Professor A.N. Belozersky. I was among the first invited pedagogues at this chair. For this cycle of lectures, I had to ‘‘translate’’ medical problems of immunology into common ‘‘biological’’ language. I wanted to demonstrate to my students that immunology is a biological science, which examines problems of cell differentiation, genetic control of protein synthesis, employing unique biological models. Under certain circumstances these models underline basic biological processes even better than classic developmental biology models. I wanted to introduce immunology for university students in a way that would be interesting for them from the biological viewpoint. However, I also wanted to demonstrate to them that biological problems of immunology represent a basis that may be used for understanding of pathogenesis of many diseases and for understanding of common medical problems such as inflammation or allergy. This task, the introduction of this medical science for ‘‘purely biological students’’, was very interesting, and I surrendered wholly to this work. I think that my course in immunology was interesting for the university students. I still give a cycle of lectures in immunology (2005) using the same principles, but of course using modern scientific level and material. I think that my personal self-evaluation as a professional expert in a particular field of immunochemistry appeared after mastering the method of immunodiffusion (and related subjects). We became very familiar with all the delicate details of this method, and my confidence in this particular field extrapolated to other things. This was very typical for me. I always extended frameworks of my profession or alternatively in attempts to solve a problem I entered in a new field, which required completely new competence in this new field. Basically, the race of problem solution constantly exceeds limits of my competence. In the very beginning of my life in science I left biochemistry for immunochemistry, then from analytical immunochemistry I moved to

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immunomorphology and cell biology, but then I came back to electrophoresis. Thus, my search for a solution to particular scientific problems is accompanied by partial loss of previous competence. I think that a specialization of a scientist trying to solve a scientific problem is the specialization on resolution of non-standard problems. This requires a certain kind of confidence, which should be combined with a strong diffidence. Such combination of self-confidence and diffidence accompanies all my life in science. I do not know whether this is my personal characteristic feature or a professional feature of real scientists. I prefer to think that this is a professional feature as well. One cannot be a real researcher if one pre-limits one’s own professional competence and professional confidence. Real scientific problem always overcomes these limits. Thus, the major question is should one follow the problem, when it leaves predetermined professional limits, or not? For me there was not a question: I always left areas of my own competence. Conflicts of ideas or viewpoints. I do not remember actual conflicts as such. There were some conflicts of ideas, especially when Zilber was alive. I respected Zilber very much. He was an extraordinary, outstanding man. Zilber was ‘‘a large scale’’ man of principles, very active, very impatient. He believed that a scientist should work only on the resolution of important principal problems. He used to say that it is absolutely right, because work on principal and ‘‘second-best’’ problems is equally hard, or at least they require comparable efforts and time. Consequently, it is better to be involved in works on principal problems. He always followed this rule. Works on ‘‘second-best’’ problems were inconsistent with his nature. Zilber often said that it is impossible to ‘‘sew the last button on a work’’; basically he did not like to lead research up to a refined state. He preferred to resolve problems in general and to leave details for others. He always appreciated ‘‘large and wide’’ problems. He had a wonderful feature: he never had inertia of previous success, and he was able to change scientific problems and research areas. This was a great feature, which helped Zilber to make significant changes in his life and important independent discoveries. When we had demonstrated the presence of embryonic antigen in tumor he said: ‘‘Enough. You have made a good job and now apply your experience and your systems and approaches for

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recognition of specific tumor antigens. Embryonic antigen is interesting for a narrow group of researchers, whereas specific antigens are a really hot spot, which would be interesting for all. It will definitely change the whole of oncology’’. In his intention to persuade us to do most important things, Zilber refused to sign our requests for laboratory mice, which we needed for this work. However, for my colleagues and me, it was impossible to leave this work unfinished. I thought that if study was not clarified all crucial points, it should be continued. Really completed work, its results begin ‘‘its life’’ after elucidation of precise mechanisms of phenomenon and putative consequences. I continued my work in spite of completely different viewpoint of Zilber and would not leave it unfinished. Zilber respected me, I deeply respected him, and therefore conflicts of our ideas cannot be classified as struggle. However, I should say that such conflicts of ideas were quite frequent events. Now I should say that we both were not right at that time. We did not think about diagnostic aspects of this problem. However, subsequent development of liver cancer and germ cell tumor diagnostics originated from the discoveries associated with embryonic antigens. Anyway, this was one of the first results of basic studies that were introduced into clinical practice and gave clear clinical results. This perfectly fitted Zilber’s concept of introducing basic results into practice. I did not have real scientific opponents. Results of our work and our viewpoints were readily accepted and reproduced by scientists in different laboratories without resistance or scientific struggle. I think that there is an overestimated notion about our laboratory. This may be due to our position: we do not develop eccentric super-original concepts; we always set experiments before theoretical conclusions, which are always based on results of our experiments. Anyway, I should say that I have not met scientific opponents either in the literature or in discussions. From time to time I wage war with administrators. However, my wars are related to organization of scientific process, but not to science itself. The only things that I strongly resist since the beginning of the 1970s are hierarchical principle of organization of science, monopoly, and administration in science. I struggled for these principles in the past and I struggle for them now. I think that this is my primary scientific duty.

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I am interested in a wide spectrum of studies in my field and related areas. These include research on general immunology, T-cell receptors, and intercellular interactions. I am interested in these studies from both scientific and pedagogical viewpoints. (I always renew my lecture material for my students.) My current professional interest includes methods in immunochemistry, problems related to differentiation of tumors, immunodiagnostics of cancer and leukemias, problems in tumor biology, regulation of AFP synthesis, etc. Among studies by Russian scientists, I like to mention interesting works by the late A.Ya. Fridenstein on the microenvironment and differentiation of stem cells. He used original approaches and ideas on the differentiation of stem cells, which influenced me very much. I am interested in studies by Yu.M. Vasiliev on the role of extracellular matrix in regulation of specific biosyntheses. These works are directly related to our studies. Interesting results on immunology of metastasizing have been carried out by G.I. Deichman. I should also mention papers by A.D. Altstein on construction of antiviral vaccines that may serve as antitumor drugs. It is difficult to say which contemporary scientists–immunologists are authoritative for me now. Twenty years ago I might mention M. Burnett, G. Nossal, P. Medavar, and M. Hasek. Now it is difficult for me to select outstanding contemporary scientists. There are many ‘‘first class’’ studies and brilliant scientists, and so I am not confident that I can choose outstanding scientists determining current progress in immunology. Nevertheless, I want to mention some very distinguished experts in my field. These are George Klein (virologist, immuno¨hler (they devellogist, oncologist), Cesar Milstein and Georg Ko oped hybridoma technology), Lloyd Old and Edward Boys (among founders of cancer immunology, making important contribution to development of this field), and Susumu Tonegawa, who demonstrated genetic recombination in the production of antibodies for the first time. I have already written about my teachers, especially about my Moscow State University teachers. My reader clearly understands the particular role Zilber had in my life. I hope that everyone who reads this paper realizes that Zilber played a decisive role in my choice of research field where I am still working, on selection of

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scientific problems, and research work as well. His strongest influence on me consists in the following: he never let us (his coworkers) work on local problems. Sometimes he even forced us to leave them and to focus our attention on global problems. I lost this inestimable influence on my research with his death. Zilber was characterized by a wonderful feature, special width in his approaches to scientific and human problems. This width was ‘‘his intrinsic property’’. He could cover the whole particular problem starting from its background, monitor its development, and analyze its relation to adjacent areas. I always liked this feature of Zilber’s and I tried to inherit it from him. There are several members in my team, who jointed our laboratory for a diploma project or as graduates from the university. Now they are mature independent scientists with their own names in science. I do not know whether they consider me as their teacher. However, I taught them to elaborate and follow some criteria of their scientific work: tolerance, reliability, and reproducibility. I do not say that there were special lessons. Things came naturally during experiments and in the process of discussion of their results, when we elaborated some general criteria in evaluation of our work, its merit, originality, and mainly reliability. It is possible that this is the inherent style of my students. Generally I tended to develop their own individuality in my students and junior colleagues. In some cases I achieved my goals. I treat my colleagues very attentively and possibly I know their strongest sides better than they themselves. I always wanted that my younger colleagues should develop their best sides themselves. In people I especially like those features that I do not have myself, and I never suppress individuality of my younger colleagues. Moreover, working together I always try to identify ‘‘genes underlying their personalities’’, their inclinations, capacities, and interests. I do believe that the learning process is the process of ‘‘purification’’ of one’s own interests and talent. All my students are very different and each of them has their own individual ‘‘face’’. A.S. Gleiberman can be readily provoked on a new project; he has a perfect flair on novelty. A.I. Gusev is a brilliant bench worker, S.D. Perova is an expert of biological experimentation, whereas N.V. Engelhardt is a perfect immunomorphologist. Each of my students is unique, but they all share one common feature, absolute intolerance to negligence, approximation, and

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peacockery. They completely differ from me, we are often interested in different things, we are different personalities, but we easily come to terms. Twenty years ago scientists were subdivided into several ‘‘schools’’. There was Zilber’s school, Shabad’s school. However, this subdivision was typical for the ‘‘pre-paradigm period’’. Now, after formation of paradigms in cancer virology, cancer immunology, general immunology, I do not even know whether certain scientific schools joining groups of like-minded persons actually exist. I think that in our field number of such schools gradually decreases because of structuring of our knowledge. In my viewpoint the schools now reflect some structural scientific organizations: laboratories, departments maintaining steady groups of scientists working together for many years. Such groups share similarity in viewpoints on the investigated problems, criteria of success of their research. Perhaps this is the modern meaning of what we call the ‘‘scientific school’’ now. In my viewpoint borders between various schools of immunologists and virologists are spread out and schools themselves are ‘‘dissolved’’ into the general structure of current knowledge of a particular field. I myself was a student of Belozersky’s school, belonged to Zilber’s school. These schools influenced my subsequent activity. However, now I do not belong to any school. I think that a young person, who wants to become a scientist, should have natural and potent interest in science; this interest should be one’s own interest rather than reflection of fashion or ambitions. Real scientist should believe in their own interest in science, should follow it, and keep eyes and ears open. I think that a young scientist needs success to obtain confidence required for subsequent studies. Real interest in science together with keenness on research results in such ‘‘fusion’’ with an investigated problem, which helps better understanding of hot spots of these particular problems and creates a good background for intuition. The latter is ultimately required for success of any investigation. Such preconditions are very important for a sketch of an internal image of the research object and when logics ends the scientist will intuitively find the route, allowing to see a ‘‘light at the end of the tunnel’’ and thus solve problem and understand truth. Secondary stimuli, such as ambitions and profit, cannot develop intuition in scientists and deep understanding of research object or problem

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and if these are the only (or leading) stimuli it is impossible to get real success in science. The other important thing consists in analysis of particular problems. It is not right to pre-classify problems into primary and secondary ones. Scientists should solve problems, which they selected by themselves, and then ‘‘God decides’’ which positions of these solved problems will occupy the common tree of cognition. Anyway, I do believe that artificial selection of global or ‘‘nodal’ problems, which bode success is a wrong way leading to a dead-end. The thing is that the problems which may be formulated as promising ones may be defined as the pre-resolved problems because they have been already formulated and pathways for their solution are more or less clear. In my viewpoint scientists should work on unresolvable tasks, originating from the own particular interest of these scientists. I should say that I am happy with my life in science. Some results of our work played a certain role in immunology and oncology, some results still influence these fields. This gives me a sense of great satisfaction, because normally the net efficiency coefficient in science is very low. The major proportion of studies finishes at a dead-end without public resonance and subsequent development in other laboratories. Thus, I am pleased that at least some results of our studies have been recognized by the scientific community and received further development both in theory and practice. All my life I have been working only in accordance with my own research interest. Although my way in science has not been straight, I have always followed my interest, the way that it has driven. I almost physically could not work on uninteresting things. However, I always worked with pleasant people, with which I felt quite comfortable. It is my constant belief that one should maintain only those scientific contacts that are quite nice and pleasant and also to work with those people who do not ‘‘spoil’’ comfortable, creative, and productive atmosphere in the lab. The most successful and creative periods of my scientific carrier were in the middle of the 1950s (work of new construction of separators); the end of the 1950s and the beginning of the 1960s (works on immunodiffussion, immunofiltration, isolation of hepatoma antigen, and elucidation of its nature); the beginning and middle of the 1970s (the development of highly sensitive methods,

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study of endogenous viral antigens, the development of the method of counterflow isotachophoresis on porous membranes); the end of the 1970s and beginning of the 1980s (a burst of my engineering activity), and finally – the study of cell–extracellular matrix interaction as a critical step in liver cell differentiation. In conclusion I want to say that in spite of successful and creative life in science I am not a perfectly satisfied scientist. This lack of ‘‘complete satisfaction’’ accompanies me during all my life. It is still with me. However, I feel that this is a normal state of a real researcher.

ACKNOWLEDGMENTS

I would like to express my sincere thanks to my wife, Galina Deichman, for constant assistance in the preparation of this chapter. The help of professional translators Alexey Medvedev and Richard Lozier is greatly acknowledged. My special thanks to Olga Salnikova for all technical work connected with the preparation and corrections of the manuscript. REFERENCES [1] Narcissov, N.V. and Abelev, G.I. (1959) Antibody formation in primary rat sarcoma induced with carcinogen. Neoplasma VI(4), 353–360. [2] Novikova, E.S., Abelev, G.I., Dzincharadze, V.M. and Gussev, A.I. (1956) Biochemistry (Russian) 21, 5: 569–572. [3] Zilber, L.A. and Abelev, G.I. (1968) The Virology and Immunology of Cancer. , 2nd edn. Pergamon Press, Oxford, pp. 308–323. [4] Abelev, G.I. (1960) Modification of the agar precipitation method for comparing two antigen-antibody systems. Folia Biol. VI(1), 56–58. [5] Zilber, L.A. (1959) A study of tumor antigens. Acta Union Int. Contr. XV, 933–935. [6] Abelev, G.I. (1965) Antigenic structure of chemically induced hepatomas. Prog. Exp. Tumor Res. 7, 104–157. [7] Abelev, G.I. and Tsvetkov, V.S. (1962) The method of isolation of specific antigens of tumor and normal tissue. Acta Union Int. Contr. 18, N1-2: 91–93. [8] Abelev, G.I. (1963) Study of the antigenic structure of tumors. Acta Union Int. Contr. 19, 1/2: 80–82. [9] Abelev, G.I., Perova, S.D., Khramkova, N.I., Postnikova, Z.A. and Irlin, I.S. (1963) Production of embryonal a-globulin by transplantable mouse hepatomas. Transplantation I, N2: 174–180.

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[10] Abelev, G.I., Assekritova, I.V., Kraevsky, I.A., Perova, S.D. and Perevodchicova, N.I. (1967) Embryonal serum a-globulin in cancer patients: diagnostic value. Int. J. Cancer 2, 551–558. [11] Abelev, G.I. (1968) Production of embryonal serum a-globulin by hepatomas: review of experimental and clinical data. Cancer Res. 28, 1344–1350. [12] Abelev, G.I. (1971) Alpha-fetoprotein in ontogenesis and its association with malignant tumors. Adv. Cancer Res. 14, 295–358. [13] Abelev, G.I. and Elgort, D.A. (1982) Alpha-Fetoprotein. In Cancer Medicine (Holland, J., and Frei, E., eds), pp. 1089–1099. Lee & Febiger, New York. [14] O’Conor, G., Tatarinov, Yu.S., Abelev, G.I. and Uriel, J. (1970) A collaborative study for the evaluation of a serologic test for primary liver cancer. Cancer 25, 1091–1098. [15] Engelgardt, N.V., Poltoranina, V.S. and Yazova, A.K. (1973) Localization of alpha-fetoprotein in transplantable murine teratocarcinomas. Int. J. Cancer 11, 448–459. [16] Abelev, G.I., Engelhardt, N.V. and Elgort, D.A. (1979) Immunochemical and immunohistochemical micromethods in the study of tumor-associatid embryonic antigens (a-fetoprotein). In Methods in Cancer Research (Fishman, W.F. and Bush, H., eds.), Vol. XVIII, pp. 1–37. New York, Academic Press. [17] Abeleva, E.A. and Abelev, G.I. (1985) Ethic as a cement of science. Chemistry and Life (Khimia and Jizn) No. 2, 3–8 (in Russian). [18] Ievleva, E.S., Engelhardt, N.V. and Abelev, G.I. (1976) Specific antigen of murine erythroblasts. Int. J. Cancer 17, 798–805. [19] Abelev, G.I. and Elgort, D.A. (1970) Group-specific antigen of murine leukemia viruses in mice of low-leukemic strains. Int. J. Cancer 6, 145–152. [20] Gleiberman, A.S. and Abelev, G.I. (1985) Cell position and cell interactions in expression of fetal phenotype of hepatocyte. Int. Rev. Cytol. 95, 229–266. [21] Gleiberman, A.S., Kudrjavtseva, E.I., Yu Sharovskaya, Yu. and Abelev, G.I. (1989) The synthesis of alpha-fetoprotein in hepatocytes is coordinately regulated with cell-cell and cell-matrix interactions. Mol. Biol. Med. 6, 95–107. [22] Abelev, G.I. and Eraizer, T.L. (1999) Cellular aspects of alpha-fetoprotein reexpression in tumors. Sem. Cancer Biol. (Tumor Markers) 9, 2: 95–104. [23] Abelev, G.I. and Karamova, E.R. (1984) Counterflow immunoisotachophoresis on the cellulose acetate membranes. Anal. Biochem. 142, 437–444. [24] Abelev, G.I. and Karamova, E.R. (1997). Counterflow immunoisotachophoresis and immunoaffinity electrochromatography on porous membranes. In Immunology Methods Manual (Lefkovits, I., ed.), Ch. 8.3, pp. 499–513. London, Academic Press. [25] Abelev, G.I. and Solovjev, N.N. (1953) Method for making electronmicroscopy preparation from salt solution. Microbiology (Russian) 22, 6: 707–708.

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Index Abderhalden, E. 173 Abelev, G.I. 283–334 Afanasjeva, E. 178 Aguet, M. 262 Aguzzi, A. 257–282 Akizuki, M. 35 Albert, Z. 179 Albracht, S.P. 94 Alevriadou, B.R. 237 Alexander, B. 213 Altstein, A.D. 329 Alzheimer, A. 259 Amano, T. 8 Ames, B. 17 Andre´, P. 238 Angielski, S. 199 Anokhin, P.K. 284 Antyporowicz, W. 190 Araki, T. 21 Arne´r, E.S.J. 109 Artamonova, V.A. 294 Asada, Y. 240 Astaurov, B.L. 319 Auer, J. 80 Augustin, Z. 171 Aumelas, A. 115 Avenirova, Z.A. 294, 301–302, 305 Axley, M.J. 111 Ban, N. 103, 109 Baehr, G. 240

Baer, J. 163 Baidakova, Z.L. 297, 301 Baldwin, E. 8 Banach, S. 166 Bangham, A.D. 134, 146 Baranowski, T. 171, 176, 188, 190–194, 196 ´ ska, J. 157–207 Baran Barker, H.A. 16 Baron, C. 104 Baroyan, O.V. 318 Barrett, E.L. 92 Baum, H. 189 Baumeister, W.P. 37–68 Baumgartner, H.R. 236 Beck, A. 173 Beloshapkina, T.D. 326 Belozersky, A.N. 288–296, 324, 326, 331 Benson, A.A. 2 Benz, R. 118 Bergmann, G. 173 Bergstro¨m, S. 22 Berns, A. 261 Bernstein, G. 161 Berry, M.J. 106 Bethe, A. 173 Bezverkhyi, G.S. 297 Bielew, W. 178 Biggs, R. 222 Binder, F. 118 Birkmann, A. 85 Birnstiel, M. 261

336

Bjo¨rklund, B.H. 300 Bjo¨rklund, V. 300 Blagoveschensky, V.A. 293–295, 297 ¨ttler, T. 266 Bla Bloch, K. 21 Blokesch, M. 93, 95 Blokhin, N.N. 320 ¨ck, B. 209–255 Blomba ¨ck, M. 213, 219, 220, Blomba 239 Bloom, A.L. 224, 238 Bo¨ck, A. 69–126 Bo¨hm, R. 84 Bohomolec, A.A. 176 Bohr, N. 187 Borbe´ly, A. 25 Borchgrevink, C.F. 222 Boulter, C.A. 260 Boyd, W. 299 Boys, E. 329 Brandner, S. 263 Brauner, L. 72 Braunstein, A.E. 176, 178, 182 Brinkhous, K.M. 233 ¨mel, H. 185 Bro Buchner, E. 162 Bu ¨ chner, T. 184 Buckel, P. 76 Bu ¨ eler, H.R. 262 Bujwid, O. 164 Burnett, M. 329 Cammarano, P. 79 Chapman, D. 127–156 Chiba, G. 26 Chojnacki, T. 199 Chopek, M.W. 229 Christian, W. 184 Chrza ˛szczewski, W. 190 Claude, A. 297

INDEX

Clemetson, K.J. 237 Cohen, G.N. 113 Cohen, P. 15 Cohen, S. 18 Coleman, S. 34 Cone, R.A. 140 Cori, C.F. 18, 171, 188 Cori, G.T. 18, 188 Counts, R.B. 225 Cundliffe, E. 77 Dadlez, J. 169 Davi, E.A. 224 Dawson, R.M.C. 134 Dent, J.A. 241–242 De Rosier, D.J. 41, 52 De Tesseyre, T. 190 Dobryszycka, W. 193 Dong, J.-F. 244 Doolittle, W.F. 81 Doudoroff, M. 16 Drapal, N. 94 ´ ski, J. 199 Duszyn Dzugaj, A. 157–207 Edgington, T.S. 224 Edidin, M. 140 Edsall, J.T. 22 Edward Boys, E. 329 Eggerer, H. 75 Eguchi, N. 31 Ehrenreich, A. 101 Ellinger, A. 173 Embden, G. 157, 159, 162, 164, 168–169, 173, 183, 185, 188–189 Emsley, J. 231 Engel, A. 43 Engelhard, H. 101 Engelhardt, N.V. 305, 309, 320, 326, 330

INDEX

Engelhardt, W.A. (or V.) 176, 182, 319, 321 Ernster, L. 22 Famulok, M. 105 Fay, P.J. 224 Feigelson, P. 15 Fellowes, A.P. 238 Ferrone, S. 258 Fiedler, G. 118 Firkin, B.G. 225 Fiske, C.H. 185, 187 Flechsig, E. 267 Fletcher, W.M. 164 Forchhammer, K. 101, 104 Foster, P.A. 234 Fowler, W.E. 231 Frangos, J.A. 242 Frank, J. 43–44 Fraser, H. 265 Fridenstein, A.Ya. 308, 329 Fritsche, E. 89 Fromherz, K. 185 Frye, L.D. 35, 140 Fujikawa, K. 243 Fujinami, K. 3 Furchgot, R. 18 Furlan, M. 242–243 Gelstein, V.I. 302 Gest, H. 85 Geyl, D. 77 Giacomini, P. 258–259 Gibayło, K. 171 Girma, J.-P. 230 Glass, R.S. 92, 94 Gleiberman, A.S. 330 Glotz, H.-J. 5 Goldberg, A.L. 47 Goldstein, R. 213 Gollin, D.J. 89

337

Goody, R.S. 108 Gostev, V.S. 295 Grabar, P.N. 313, 316–317 Green, D.E. 13–16 Gurvich, A.A. 286–287 Gurvich, A.G. 285–287, 295 Gusev, A.I. 305, 321, 326, 330 Guthke, J.A. 171, 184, 188–189 Halikowski, B. 176, 190 Hanafusa, H. 34 Harden, A. 162, 185 Hart, R.G. 54 Hasek, M. 329 Hassid, W. 16 Hastings, B. 178 Hatfield, D.L. 100–101 Hayaishi, J. 2, 4, 34 Hayaishi, M. 13, 16–18, 34 Hayaishi, O. 1–36 Hayaishi, T. 2, 16, 18, 34 Hayano, M. 21 Heider, J. 104 ¨lder, M. 270 Heikenwa Heller, J. 168–169, 186, 190, 194 Heller, Y. 194–195 Henderson, R. 41 Hengartner, H. 267 Hennecke, H. 75, 82, 120 Henry, W. 169 Heppel, L. 16 Heppner, F.L. 176 Herbert, D. 191 Hessel, B. 227 Hevesy, G. 171, 187–188 Heywood, J.B. 222 Hidematsu, H. 317 Higuchi, T. 15 Hilarowicz, H. 169

338

Hilbert, L. 240 Hoffmann-Ostenhof, O. 13, 21 Hofmeister, F. 162 Holmberg, L. 224, 238–239 Holmes, E. 191 Holovatsky, I. 199 Honjo, T. 31 Hopkins, F.G. 13, 164 Hoppe, W. 41, 43–44, 53 Hopper, S. 85 Hoppe-Seyler, F. 21 Hopwood, J. 128 Horak, I. 261 Horecker, B. 16 Horwich, A. 51 Hovig, T. 223 Howard, J.B. 96 Howard, M.A. 225 Huang, Z.-L. 26 Hube, M. 95 Huber, R. 48, 89 Hubl, S. 171, 190 Hummel, H. 80 Ikeda, Y. 237 Inoue´, S. 25 Irlin, I.S. 312 Irving, H. 129 Ishimori, K. 25 Ishimura, Y. 24, 31 Jacobi, A. 88 Jaenicke, R. 75 Jaffe, E.A. 224 Jakovlev, N.N. 182 Jan, J.O. 174 Jarsch, M. 79 Jatisatienre, C. 82–83 Jaworska, J. 190 Jenkins, C.S.P. 237 Kalckar, H.M. 170, 196

INDEX

Kamen, M. 18 Kandler, O. 73–74, 78 Kaplan, E. 15 Kashtoyants, Kh.S. 285 Katagiri, M. 20 Katchman, B. 15 Keilin, D. 192 Khorana, H.G. 147 Khramkova, N.I. 305, 309–310, 312, 326 Kiessling, W. 184–185 King, E.J. 185 Kipling, R. 159, 161 Kirzon, M.V. 285 Kitamoto, T. 265 Kleihues, P. 259–260, 263 Klein, G. 329 Klein, M.A. 267 Kleiner, J. 169 Klisiecki, A. 190 Klug, A. 41, 52 Knappe, J. 83 Knoop, F. 162 Koch, R. 5 Ko¨hler, G. 329 Kokame, K. 243 Komisarenko, S. 198 Kornberg, A. 15–16, 23 Kornberg, C. 23 Korsa, I. 85 Korzybska, M. 199 Korzybski, T. 171, 179–180, 190, 196–197 Kosakowski, H. 74 Kotake, Y. 8–10, 12, 21, 34 Kotelnikowa, A. 178, 182 Kovacs, K. 88 Krebs, H.A. 191 Kromayer, M. 105, 108 Krwawicz, T. 190 Ku ¨ bler, O. 41

INDEX

Kuz´nicki, J. 199 Kwiatkowska-Korczak, J. 157–207 Lardy, H. 15 Larrieu, M.-J. 213, 222 Lavoisier, A.L. 11 Leathes, J.C. 137 Lechner, K. 80 Lehmann, H. 185 Leibundgut, M. 103, 109 Leinfelder, W. 99 Lenin, V.I. 293 Levene, P.A. 185 Levy, G.G. 243 ´ ski, W. 190 Lewin Lezhneva, O. 311 Lindenfeld, 176 Lindenmann, J. 259 Lisowska, E. 193 Lloyd Old, L. 329 Lohmann, K. 184, 188 Lo˜pes, J.A. 244 Loscalzo, J. 232 Lottspeich, F. 93 Lowry, O. 18 Lozier, R. 333 Luppi, P.-H. 28 Lutwak-Mann, C. 170–171, 186, 190 Lutz, S. 85, 88 Lynen, F. 17 Lyssenko, T.D. 180, 286, 291, 295 Lyubimova, M. 178 Macfarlane, R.G. 222 Maier, T. 88, 91 Malin, G. 261 Mandrand-Berthelot, M.-A. 99

339

Mann, T. 170–171, 176, 186, 189–192 Marder, V.J. 226 Mason, H.S. 21 Masseyeff, R. 316 Matsumura, H. 30 Medavar, P. 329 Medvedev, A. 333 Medviediev, S.S. 176 Meek, K. 128 Mehler, A. 17 Mejbaum, W. 171 Mejbaum-Katzenellenbogen, W. 190, 193–194, 198 Merlini, G. 258 Meyer, D. 222, 226, 232 Meyerhof, O. 157, 159, 164, 183, 184–185, 189 Milstein, C. 329 Mirick, G.S. 9 Mirsky, A. 291 Moake, J.L. 240–241 Mochnacka, I. 171, 179, 190, 193–194 Morawiecki, A. 199 Moschcowitz, E. 240 Moshkova, G.Yu 283 Mozołowski, W. 168–169, 171, 173, 176, 190, 194–195 Mroczkiewicz, U. 190 Mu ¨ ller, I. 117 Mu ¨ ller, S. 113 Naegely, O. 169 Nagata, S. 33 Nakanishi, S. 34 Narcissov, N.V 297–299 Nass, G. 75 Navarro, B. 267 Nazaki, M. 24 Neame, P.B. 240

340

Needham, D.M. 199 Negelein, E. 185 Neidhardt, F.C. 72–73 Nesheim, M. 234 Neubauer, O. 185 Neuhierl, B. 116 Nilsson, I.M. 214–215, 218 Nishino, M. 240 Nishio, K. 244 Nishizuka, Y. 23, 31 Nomura, M. 77 Nossal, G. 329 Nozaki, M. 24, 31 Nuckowski, J. 190 Nyman, D. 236 O’Brien, J.R. 222 O’Farrell, P.H. 82 Ohshima, Y. 31 Ohtsu, H. 29 Okumura, T. 237 Old, L. 329 Oparin, A.I. 288, 291–292 ´ ska-Blauth, J. 176, 190, Opien 195 Orrenius, S. 22 Ostern, J.K. 188 Ostern, P. 170–171, 184, 188–192, 195 Ostrowski, S. 177 Ostrowski, W.S. 198–199 Owen, W.G. 224 Pajatsch, M. 118 Palladin, A.W. 176 Parin, W. 180 Paris, H. 25 Parnas, B. 182 Parnas, J.K. 157–207 Parnas, J.O. 159, 169, 174, 177, 180–182, 192, 198

INDEX

Parnas, R. 169, 181–182, 192 Parnas, T. 182 Parnas, Y.K. 181 Parnes, V.A. 301–302 Paschos, A. 89, 92 Peake, I.R. 239 Pecher, A. 83, 97 Peck, H.D. 85 Pentreath, V.W. 28 Perevodchikova, N.I. 314–315 Perova, S.D. 312, 321, 330 Perrault, C. 244 Persson, B.C. 111 Piendl, W. 77 Pie´ron, H. 25 Piepersberg, W. 76 Pietrova, B. 178 Pie´tu, G. 238 Pimanda, J.E. 245 Pinsent, J. 97, 115 Plaimauer, B. 243 Pollister, 291 Polo, M. 113 Poltoranina, V.S. 320 Pomorski, P. 199 Price, B. 16 Prinz, M. 270 Proscuryakov, N.I. 290 Prusiner, S.B. 262 Qu, W.-M. 30 Quinn, P.J. 127–156 Quixote, D. 33 Rabionowitz, J. 17 Raeber, A. 266 Rand, J.H. 236 Rastegar-Lari, G. 238 Ratnoff, O.D. 223 Rau, W. 71 Raymond, A.L. 185

INDEX

Rees, D.L. 96 Reichard, P. 22 Reissmann, S. 93 Relave, L. 3, 5 Rethwilm, A. 261 Ribba, A.S. 239 Roberts, J.L. 28 Robison, R. 185 Rosenthal, S. 18 Rossmann, R. 86, 89 Rothberg, S. 20 Rozenfeld, E. 178, 181 Rudenko, R.A. 181 Ruggeri, Z.M. 239 Ruska, E.A.F. 39 Ruska, H.P.G. 38–40 Rutkowska-Brzecka, A. 179 Sachs-Ploetz, E. 169 Sadler, J.E. 228, 231 Sailer, A. 266 Sakariassen, K.S. 236–237 Salnikova, O. 333 Salzman, E.W. 222 Samuel, D. 20 Samuelsson, B. 22 Sanders, W.E. 241 Saper, C.B. 28 Satani, T. 13 Sauter, M. 84 Savage, B. 236–237 Sawers, G. 83, 86 Saxton, W.O. 43 Schardinger, F. 11 Schlensog, V. 85 Schmid, G. 118 Schnabel, A. 4 Schuster, P. 184 Schwarz, H.P. 227 Schwarz, K. 115 Seki, T. 8

341

Selmers, M. 107 Semenza, G. 35 Senn, H. 113 Severin, S.E. 182, 288 Shelagin, M. 178 Shmerling, D. 269 Shrift, A. 116 Siebeck, R. 39 Siedlecki, C.A. 231 Sieniawski, J. 190 Sixma, J.J. 227 Słobodzian, W. 171 Sobczuk, B. 171, 190, 195–196 Sobel, M. 238 Solum, N.O. 237 Soulier, J.P. 213 Sprinzl, M. 104 Stadtman, A. 101 Stadtman, E. 17 Stadtman, T.K. 97, 100, 109, 111 Stalin, I.V. 292 Stanier, R.Y. 16 Starlinger, P. 74 Steinbach, J.P. 261 Steinhaus, H. 166 Stel, H.V. 236 Stepanienko, B. 178 Stephenson, M. 8, 84 Stetter, K.O. 44 Stickland, L.H. 84 Stoika, R. 199 Stormorken, H. 222–223 Strohl, 169 Strub, M.P. 115 Subbarow, Y. 185, 187 Suda, M. 8 Sunde, R.A. 100 Suppmann, S. 111 Szankowski, W. 171

342

INDEX

Szent-Gyo+ rgy, A. 178 Sznol, S. 182

Urade, Y. 26, 31 Uriel, J. 317

Tabor, H. 17 Taniguchi, T. 7–8 Tanner, W. 75 Tatarinov, Yu.S. 314–315, 317 Tate, W.P. 112 Taubenhaus, R. 165 Taylor, R.J. 130 Tershakovec, G. 195 Terszakowec´, J. 171–172, 184, 189–190, 195–196 Tesseyre, 190 Thanbichler, M. 100, 108–109, 112, 116 Thauer, R.K. 78, 88 Thomas, P. 169 Thorell, L. 226 Timakov, V.D. 296 Tiselius, A. 299 Titani, K. 230 Tobelem, G. 237 Tomaszewski, L. 190, 194 Tonegawa, S. 329 Toping, J. 152 Tsai, H.-M. 239, 241–243 Tsuchida, M. 16 Tsvetkov, V.S. 302, 305–306, 326 Tuddenham, E.G.D. 224 Turitto, V.T. 237

van Deenen, L.L.M. 135 van Heel, M. 44 van Mourik, J.A. 242 van Niel, C.B. 16 von Willebrand, E.A. 211–214 Vasiliev, Yu.M. 329 Vassiliev, S.S. 285–286 Veprek, B. 101 Vermylen, J. 230 Verveij, C.L. 228, 232 Verzar, F. 191 Vignais, P. 88 Vlot, A.J. 234

Uchida, M. 4 Uchida, T. 14 Uchida, Y. 14 Ueda, N. 35 Umschleif, B. 171 Umszweif, B. 171 Unwin, P.N.T 41

Wagner, D.D. 226, 236 Wagner, E.F. 260–262 Wagner, R. 164–165, 168 Wagner, R.H. 224 Wajda, K. 190 Wang, H. 94 Warburg, O.H. 22, 173, 184 Watanabe, T. 29 Weber, H. 74 Wegner, G. 147 Weigl, R. 166 Weiler, E. 302, 309 Weiss, H.J. 233–234, 236 Weissenberger, J. 261 Weissmann, C. 259, 262, 266–267, 269 Weller, R. 260 Westaway, D. 268 Whelan, W.J. 31 Wich, G. 80–81 Wieland, H. 2, 11–13, 17, 19, 21 Wiestler, O.D. 260 Will, R.G. 264

INDEX

¨tter, R. 162, 169 Willsta Wilting, R. 106–107 Wirth, R. 77 Wittinghofer, A. 95 Wittmann, H.G. 76 Wise, R.J. 232 Wlodawer, A. 199 Wojtczak, L. 199 Wong, J.T.F. 111 Yaffe, M. 12 Yamamoto, S. 24, 31 Yamamura, Y. 31 Yazova, A.K. 320–321 Yoo, G. 243 Yoshizawa, S. 107 Young, W.Y. 162, 185 Zago´rski-Ostoja, W. 199 Zbarski, B.J. 176

343

Zbarsky, B.I. 292 Zbarsky, I.B. 292–293 ˙ elen ´ ski-Boy, T. 191 Z Zenk, M. 72, 116 Zhang, Z. 239 Zhukov-Verezhnikov, N.N. 295 ´ ska, Z. 197, 199 Zielin Zilber, L.A. 294–295, 297, 299–303, 311, 314, 318–320, 324–331 Zillig, W. 44, 119 Zimmerman, T.S. 223, 228, 239 Zimmermann, M. 168, 185 Zinkernagel, R.M. 266–267 Zinoni, F. 97, 102, 111 Zotin, A.I. 285 Zucker, M.B. 222, 233

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Colour Plate Section

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Plate 1. Ch. 2. Photo of Baumeister (for Black and White version see page 37).

Plate 2. Ch. 2-Fig. 3. (a) The Staphylothermus marinus surface layer as revealed by freeze-etching (left). In the absence of MgCl2 the detergent extracted surface layer dissociates into micelles formed by the tetrabrachion–protease complexes (right). (b) The tetrabrachion–protease complex. Left: Model showing the mode of interaction of the tetrabrachion–protein complexes in the layer structure. Center: Electron micrograph of the negatively stained complex released from the surface layer meshwork by SDS-heat treatment (for details see [77]). Right: Folding topology of tetrabrachion. The location of N-terminal residues, cysteine residues and the unique proline residue separating the left- and right-handed supercoiled domains are marked by circles. Putative disulfide bridges are indicated. The flexible hinge segment, the protease-binding region and the membrane anchor are marked by rectangles (for details see [76]) (for Black and White version see page 46).

Plate 3. Ch. 2-Fig. 4. The 20S proteasome from Thermoplasma acidophilum. (a) Electron micrograph of recombinant 20S proteasomes in vitreous ice. (b) Top left: Structure of the 20S proteasome in surface representation, low-pass filtered to 1 nm resolution. The a- and b-subunits are located in the outer and inner rings, respectively. Top right: The same structure cut open along the 7-fold axis to display the inner compartments with the active sites of the b-subunits in the central chamber marked in red. Bottom left and right: Similar fold of the a- (left) and b- subunits (right). Both subunits contain a sandwich of two, five-stranded antiparallel b sheets flanked by helices (for details see [64,113]) (for Black and White version see page 49).

Plate 4. Ch. 2-Fig. 5. Structure of the 26S proteasome from Drosophila melanogaster as determined by cryoelectron tomography and displayed in surface (left) and cut-open views (right) (for details see [73]) (for Black and White version see page 51).

Plate 5. Ch. 2-Fig. 6. Giant complexes involved in intracellular protein degradation. Left: Tricorn protease capsids from Thermoplasma (15 MDa). Right: Tripeptidylpeptidase II from Drosophila (6 MDa) (for details see [89,106]) (for Black and White version see page 52).

Plate 6. Ch. 2-Fig. 7. The protein quality control system in Thermoplasma acidophilum. Components of the proteolytic pathway are shown in yellow, chaperones in green. The numbers refer to the ORF code (for details see [90]) (for Black and White version see page 53).

Plate 7. Ch. 2-Fig. 8. Cryoelectron tomography of Dictyostelium discoideum cell. (a) Visualization of the actin network and cytoplasmic complexes in a Dictyostelium cell grown directly on an EM grid and embedded in vitreous ice (for details see [71]). (b) Visualization of a 26S proteasome within an intact Dictyostelium cell. Left: Slice from a tomogram. Dominant features are ribosomes, some of them attached to the endoplasmic reticulum (lower left corner), and actin filaments. The encircled particle is a 26S proteasome. Right: Enlarged contour plot of the single (unaveraged) 26S proteasome (projection of a stack of slices from tomogram) (for Black and White version see page 57).

Plate 8. Ch. 2-Fig. 9. Strategy for the detection and identification of macromolecules in cellular volumes. Because of the crowded nature of cells and the high-noise levels in tomograms (left), an interactive segmentation and feature extraction is, in most cases, not feasible. It requires automated pattern recognition techniques to exploit the rich information content of such tomograms. An approach that has been demonstrated to work is based on the recognition of the structural signature (size, shape) of molecules by template matching. Templates of the macromolecules under scrutiny are obtained by high- or medium-resolution techniques. These templates are then used to search the volume of the tomograms (Vin) systematically for matching structures by cross-correlation. The tomogram has to be scanned for all possible Eulerian angles around three different axes, with templates of all the different protein structures in which one is interested. The search is computationally demanding, but can be parallelized efficiently. The output information (Vout) is a set of coordinates that describes the positions and orientation of all the molecules found in the tomogram (for details see [38]) (for Black and White version see page 59).

Plate 9. Ch. 2-Fig. 10. Protein atlas of a cellular (Thermoplasma) volume obtained by cryoelectron tomography. The positions and orientations of several molecular species (displayed in different colors) were determined by ‘template matching’ (for details see [72]) (for Black and White version see page 60).

Plate 10.

Ch. 3. Photo of Bo ¨ ck (for Black and White version see page 69).

Plate 11. Ch. 3-Fig. 3. Scheme of the incorporation of the 20 canonical amino acids (a) and of selenocysteine incorporation (b) into polypeptides. Note that elongation factor Tu (EF-Tu) acts in the form of a ternary complex with one of the standard 20 aminoacyl-tRNAs and GTP from solution, whereas SelB forms a quaternary complex with selenocysteyl-tRNA, GTP and the SECIS element of the selenoprotein mRNA. Release factor 2 (RF2) recognises cognate UGA stop codons but not the UGA in the context with the SECIS element (courtesy of Martin Thanbichler) (for Black and White version see page 100).

Plate 12. Ch. 3-Fig. 4. Recognition of the SECIS element by elongation factor SelB. (a) Stem-loop structure of the fdhF mRNA from E. coli and the 17 nucleotides minihelix (shaded). Nucleotides protected by SelB against chemical modification are indicated by triangles. (b) and (c) Interaction of the SelB protein and the SECIS element from Moorella thermoacetica. In (b), the C-terminal two winged-helix-motifs are shown from which the ultimate one interacts with the SECIS RNA via its base G23 and a string of charged residues. In (c), a summary of the functional groups of G23 and of the phosphates of the RNA backbone interacting with groups from SelB are given [101] (permission granted by Nature Publishing Group). (d) Image of the 50s ribosomal subunit (crown view) into which the Methanococcus maripaludis SelB-selenocysteyl-tRNA complex has been modelled [106] (courtesy of Nenad Ban and Marc Leibundgut) (for Black and White version see page 103).

Plate 13.

Ch. 7. Photo of Aguzzi (for Black and White version see page 257).

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