Cell Electrophoresis: Proceedings of the International Meeting Rostock, German Democratic Republic, September 24–28, 1984 [Reprint 2019 ed.] 9783110860559, 9783110101775

Citation preview

Cell Electrophoresis

Takeo Mori, 27. 9.1984, Rostock

Cell Electrophoresis Proceedings of the International Meeting Rostock, German Democratic Republic September 24-28,1984 Editors W Schüft • H. Klinkmann

W G DE

Walter de Gruyter • Berlin • New York 1985

Editors Wolfgang Schürt, Dr. Horst Klinkmann, Prof. Dr. sc. Wilhelm-Pieck-Universität Rostock Klinik für Innere Medizin DDR-2500 Rostock German Democratic Republic

Library of Congress Cataloging in Publication Data

Cell electrophoresis. „International Meeting on Cell Electrophoresis"--Pref. Includes bibliographies and indexes. 1. Electrophoresis-Congresses. 2. Cytology-Methodology-Congresses. 3. Cell separation-Congresses. I. Schiitt, W (Wolfgang), 1945. II. Klinkmann, Horst. III. International Meeting on Cell Electrophoresis (1984 : Rostock, Germany) [DNLM: 1. Cytologycongresses. 2. Electrophoresis-congresses. QU 25C393 1984] QH585.5.E46C45 1985 574.87 85-10378 ISBN 0-89925-067-X (U.S.)

CIP-Kurztitelaufnahme der Deutschen

Bibliothek

Cell Electrophoresis : Cell Electrophoresis 1984 : proceedings of the internat, meeting, Rostock, German Democrat. Republic, September 24-28,1984 / ed. W Schiitt ; H. Klinkmann. - Berlin ; New York : de Gruyter, 1985. ISBN 3-11-010177-7 (Berlin) ISBN 0-89925-067-X (New York) NE: Schiitt, Wolfgang [Hrsg.]; Cell Electrophoresis nineteen hundred and eighty-four

ISBN 311010177 7 Walter de Gruyter • Berlin • New York ISBN 0-89925-067-X Walter de Gruyter, Inc., New York Copyright © 1985 by Walter de Gruyter & Co., Berlin 30 All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. Binding: Dieter Mikolai, Berlin. Printed in Germany.

PREFACE

When, during the "Electrophoresis

'83" in Tokyo, we announced that the

International Meeting on Cell Electrophoresis was going to take place in Rostock, we did not expect quite such an overwhelming response. But what pleased us most was that, for the first time, the majority of teams working on this topic in East and West came together at this joint meeting concerning cell electrophoresis. 130 participants from 18 countries

accepted

the invitation extended by the Wilhelm Pieck University, Rostock, to meet at the oldest university in Northern Europe.

After the initial contacts established at the symposia in Munich Bristol

(1979, 1981) and Tokyo (1983), in the course of mutual

(1979),

study visits

and our PARMOQUANT workshops held in Pushchino, Moscow and Kasan, we and the staff of the Applications Laboratory for Special Medical

Techniques

of the Department of Internal Medicine considered it a great honour to offer our hospitality and provide people engaged in the application

of

cell electrophoresis with the opportunity for an intensive exchange of experience.

The scientific programme of the meeting ranged from a summary review of "Electrophoresis

'83" and a report on recent experiments

concerning

electrophoresis in space given by Prof. Hashimoto (Japan) and Prof. Todd (USA), respectively, to interesting applications of cell in biophysical, biochemical, pharmacological

electrophoresis

and immunological

for clinical, hematological, neurological, neonatological

research,

and pulmonolo-

gical practice and for tumor research. These problems were described and discussed in 75 papers and on 32 posters.

The development of automatic measuring systems such as the analytic free flow electrophoresis equipment, Laser Doppler electrophoresis

equipment,

the Pen Kem and the PARMOQUANT will make a major contribution to the further popularisation of cell electrophoresis. The fact that it has now become possible to measure objectively changes of less than 1 % in mobility

VI

makes i t possible to check past r e s u l t s arid, simultaneously, to elaborate on new a p p l i c a t i o n s . The high r e s o l u t i o n of histograms based on a representative number of c e l l s yielded by modern cell electrophoresis systems reduces the danger of misinterpretation and thus enables the method to be used for routine work. We wish to thank the authors for presenting their valuable

scientific

contributions at the meeting and also those additional teams who were unable to attend for sending their c o n t r i b u t i o n s . We believe that they have enabled us to present in t h i s volume an almost complete review of the most recent r e s u l t s obtained in the f i e l d of cell

electrophoresis.

The editors express their profound gratitude at t h i s point to the Wilhelm Pieck U n i v e r s i t y Rostock, and p a r t i c u l a r l y to i t s rector magnificus, Professor W. Brauer, and i t s director of medical research, Professor W. Finck, for the encouragement received during the preparations and during the meeting i t s e l f . We are indebted to the s t a f f of our Applications Laboratory, Drs. U. Thomaneck, E. Knippel, J. Rychly and Mrs. Ch. Grewe and B. Linke for valuable collaboration. Grateful acknowledgement i s also due to Mr. B. Petchett and Mrs. K. Laxon for their technical assistance in r e v i s i n g several manuscripts and to Mrs. S i b y l l e Lange for help with the layout of the typescript. In p a r t i c u l a r we wish to express our thanks to the s t a f f of Walter de Gruyter Publishers for i t s excellent cooperation. Horst Klinkmann

Wolfgang Schutt Rostock, February 1985

LIST OF CHAIRMEN

Opening Speech

W. Brauer (GDR), Rector magnificus

Plenary Lecture N. Hashimoto (Japan), P.W. Todd (USA)

Cell Electrophoresis in Basic Studies: Theoretical and Methodical Aspects, Topochemistry J.N. Mehrishi (England), M. Kantcheva

(Bulgaria)

Subcellular Particles, Bacteria, Vesicles, Liposomes I.

L. Wojtczak (Poland), N. Crawford

II.

M. De Cuyper (Belgium), W. Schutt (GDR)

(England)

Characterization of Lymphocytes in Cancer, Neurological Diseases, Pregnancy,

Transplantation

I.

P.W. Todd (USA), H. Hashimoto (Japan), Sun Ling (China)

II.

D. Sabolovic (France), E. Hansen (FRG)

Malignant Transformation - Cell Characterization in Leukaemia and Lymphomas I.

J. Holowiecki (Poland), 0. Babusikova U. Thomaneck

II.

(Czechoslovakia),

(GDR)

J. Bubenik (Czechoslovakia), T. Iwaguchi J. Rychly (GDR)

(Japan),

VIII Influence of Exogenic Factors: I.

Radiation K.A. Chaubal (India), A.I. Miroshnikov

II.

(USSR)

Interaction with Antibodies N. Kraskina (USSR), W. Hoffmann (FRG), E. Knippel (GDR)

III. Interaction with Drugs and Mitogens H. Hayashi (Japan), G. Hennighausen (GDR)

Detection of Multiple Sclerosis E.J. Field (England), H. Meyer-Rienecker

(GDR)

Cell Electrophoresis Techniques P.W. Todd (USA), A. W. Preece (England), W. Schütt (GDR)

Dielectrophoresis, Magnetic Immuno-Microspheres Z.M. Agadshanyan (USSR), B. Bohn (FRG)

Detection of Lymphokines: Methodical Aspects, Cancer,. Renal Transplantation, Drugs I.

E.J. Field (England), M. Müller (GDR)

II.

B. Shenton (England), B. von Broen (GDR)

Closing Session W. Finck (GDR), H. Klinkmann (GDR)

CONTENTS

OPENING SPEECH

Microgravity cell electrophoresis experiments on the Space shuttle: A 1984 overview P.W.Todd

3

CELL ELECTROPHORESIS TECHNIQUES

Free zone electrophoresis of animal cells: Experiments on cell-cell interactions P.W.Todd, St.Hjerten

23

Video image correlation electrophoresis: An intermediate stage of automation J. 0. T. Deeley, A.J.Bater ...-.

33

System 3000 Automated Electrokinetics Analyzer for biomedical applications Ph.J.Goetz

41

Automated single cell electrophoresis realized in PARMOQUANT 2 and PARMOQUANT L: Test Results W.Schiitt, U.Thomaneck, E.Knippel, J.Rychly, H.Klinkmann, H.Hayashi, N.Toyoama, M.Fujii, F.Hirose, Ch.Yoshikumi, Ch. Onodera, K.Kotagawa, K.Endo, Y.Nishimura, Y.Kawai

DIELECTROPHORESIS, MAGNETIC

55

IMMUNO-MICROSPHERES

Dielectrophoretic characterisation and separation of biological cells I.Lamprecht, M.Mischel

75

X Cell separation: Comparison between magnetic immunomicrospheres (MIMS) and FACS J.Kandzia, W.Haas, G.Leyhausen, W. Miiller-Ruchholtz ....

87

CELL ELECTROPHORESIS IN BASIC STUDIES: THEORETICAL AND METHODICAL ASPECTS; TOPOCHEMISTRY

Electrokinetic evidence for blood cell membrane - drug interactions: Usefulness and applications in monitoring treatment of mental and neuroendocrine disorders J.N.Mehrishi

97

Is the method of electrophoresis in determination of electric surface properties of particles a problem for erythrocytes ? V.L.Sigal, I.N.Simonov

113

Influence of surface structure on cell electrophoresis E.Donath, A.Voigt

123

The influence of particle concentration on microelectrophoretic mobility and on the electrooptical effect I.B.Petkanchin, T.T.Suong, G.S.Todorov

137

Phytohemagglutinin-induced effects on the passive electric and electrophoretic properties of erythrocytes F.Pliquett, V.Lap, M.Kantcheva

145

Electrophoretic mobility, surface carbohydrates and cell interactions - Studies on erythrocytes and melanoma cell lines St.J.Luner

147

XI

SUBCELLULAR PARTICLES, BACTERIA, VESICLES, LIPOSOMES

Effect of changes of surface potential of cellular organelles on their enzymic activities L.Wojtczak

159

Surface charge density of purple membrane fragments N.Popdimitrova, M.Kantcheva, Zs.Dancshazy, S.Stoylov

167

The electric surface charge of photosynthetic membranes M.Kantcheva, V.Goltzev, S.Todorov

173

Electrophoretic behaviour of Escherichia coli strains in dependence of different surface antigens G.Zingler, W.Nimmich, U.Thomaneck

183

A method for checking the fermentation processes of Escherichia coli strains with fimbrial antigens P.Gallien, U.Kludas, K.Kruger

191

Influence of Escherichia coli bacteria on the surface charge of lymphocytes J.Rychly, G.Zingler

197

Free flow electrophoresis study of the non-protein supported transfer of phosphatidic acid and polyglycerophospholipids between artificial membranes M.De Cuyper, M.Joniau

203

Free flow electrophoresis used to study the interaction of pig brain microtubular proteins with phospholipid model membranes - Role of phospholipid composition M.Joniau, M.De Cuyper

211

Influence of cations on the surface potential of negatively charged vesicles 0.Zschbrnig, K.Arnold, H.-P.Kertscher, R.Krahl

219

XII

The J - and "V - p o t e n t i a l 0 TMMA-salts A.Hattenbach,

of a r t i f i c i a l

membranes w i t h

D.Haroske

223

Use of f r e e flow e l e c t r o p h o r e s i s i n s t u d i e s of s u r f a c e and i n t r a c e l l u l a r membranes and f u n c t i o n a l l y i m p o r t a n t domains i n blood p l a t e l e t s and l e u c o c y t e s N.Crawford

225

I n v e s t i g a t i o n o f body f l u i d s by p a r t i c l e e l e c t r o p h o r e s i s I . I n f l u e n c e of c e r e b r o s p i n a l f l u i d ( C S F ) on t h e e l e c t r o p h o r e t i c m o b i l i t y (EPM) o f a r t i f i c i a l p a r t i c l e s - A u s e ful t o o l f o r the e v a l u a t i o n of m e n i n g i t i s ? D.Hobusch, E . K n i p p e l , H. M e y e r - R i e n e c k e r

E.Peters,

I n v e s t i g a t i o n o f body f l u i d s II. Particle electrophoresis pulmonary d i s e a s e s E.Knippel,

H.Wendel,

W.Schiitt,

H. ¥ .

Meyer,

247

by p a r t i c l e e l e c t r o p h o r e s i s f o r d i f f e r e n t i a t i o n between

W.Schiitt,

K.Diwok

261

I n v e s t i g a t i o n of body f l u i d s by p a r t i c l e e l e c t r o p h o r e s i s I I I . R e s p i r a t o r y d i s t r e s s syndrome (RDS) i n newborn i n f a n t s and c y s t i c f i b r o s i s as d e t e r m i n e d by i n v e s t i g a t i o n o f p h a r y n g e a l a s p i r a t e s and serum E.Knippel,

M.Müller,

W.Schiitt,

J.Hein

271

CHARACTERIZATION OF LYMPHOCYTES: METHODOLOGY AND CLINICAL APPLICATIONS

Cell electrophoresis

- P a s t and

future

D.Sabolovic Preparative A review E.Hansen

283 free

flow e l e c t r o p h o r e s i s

o f lymphoid c e l l s : 287

XIII

Analytical and preparative free flow cell electrophoresis : an alternative and complementary method to flow cytometry and sorting B.Bohn, C.T.Nebe

305

Suitability of automated single cell electrophoresis (ASCE) for biomedical and clinical applications: General remarks W.SchUtt, U.Thomaneck, E.Knippel, J.Rychly, H.Klinkmann

313

Electrophoretic mobility and monoclonal antibody combined in the study of human peripheral blood lymphocytes D.Sabolovic, E.Knippel, U.Thomaneck, J.Rychly, W.Schutt

333

Lymphocyte electrophoresis in tumor-bearing mice and its application to drug evaluation T.Iwaguchi, M.Shimizu

345

The changes of lymphocyte electrophoretic mobility in cancer patients T.Mori, G.Kohsaki

355

Cell electrophoretic investigation of peripheral blood lymphocytes from patients with bronchial carcinoma before, during and after radiation therapy U.Thomaneck, W.Schutt, D.Hamann, E.Hamann

367

Influence of BCG immunotherapy on circulating lymphoid populations of cancer patients I. Results in melanoma patients treated for residual disease M.Legros, J.Chassagne, D.Bernard, J.-P.Perrière, F.Gachon, Ph.Chollet, G.Gaillard, R.Plagne 373 Influence of BCG immunotherapy on circulating lymphoid populations of cancer patients II. Results in breast cancer patients treated for residual disease J.-P.Ferriére, M.Legros, D.Bernard, J.Chassagne, G.Besse, Ch.Gardon-Mollard, Ph.Chollet, G.Gaillard, R.Plagne, G. Bétail

385

XIV Histogram of regional lymph nodes in tumors of the ear, nose and throat (ENT) B.Kramp, H.L.Jenssen, E. Mix, W. Schtitt

397

Cell electrophoretic characterization of lymphocytes from patients with treated and untreated sarcoidosis G.Bulow, E.Behm, K.Diwok

403

Cell electrophoretic studies on lymphocytes after transplantation D.Mucke, M. Lehmann, H.Hi la cher, J.Rychly

405

Further studies on the influence of the method of isolation of human peripheral blood lymphocytes on the distribution of their electrophoretic mobilities J.N.Mehrishi, M.Wioland, E.Gomariz-Zilber

411

Characterization of electrophoretically separated lymphocytes subpopulations in human peripheral blood R.V.Petrov, I.M.Dozmorov, T.V.Lutsenko, I.S.Nikolayeva, A. M. Saposzhnikov

421

Electrophoretic characterization of lymphocyte separated on Percoll density gradients J.Rychly, W.SchUtt, D.Sabolovic

429

Enrichment of chicken peripheral blood B lymphocytes by preparative free flow electrophoresis E.Burkhardt, W.Scheffel

435

A method of analysing a polymodal histogram of electrophoretic mobility M.Kracht, K.H.Hildebrandt, G.Kundt, J.Towe, W.SchUtt, E. Knippel

441

XV MALIGNANT TRANSFORMATION - CELL CHARACTERIZATION

IN LEUKAEMIA

Differences in the electrophoretic mobilities of pigmented and non-pigmented melanoma cells K.Hyrc, M. Kapiszewska, K.Cieszka

451

Electrophoresis of mouse leukocytes and leukemia cells J.Bubenik, D.Bubenikova

459

Application of the cell electrophoresis for characterisation of leukemic cells J.Holowiecki, K.Jarczok, K.Jagoda

467

Electrophoretic mobility distribution of cells in leukemia J.Rychly, O.Anders, G.Eggers, M.Schulz

477

Electrophoretic mobility distributions and membrane phenotypes of some hemopoietic cell lines and fresh leukemic cells 0.Babusikova, E.Konikova, B.Chorvath, P.Ujhazy

485

Electrophoresis of leukemic white blood cells with Lazypher R.Steiner, O.Ottmann, R.Kaufmann, W.Hoffmann

493

Cell electrophoretic and size distribution investigations of thrombocytes of patients with acute leukemia and hemorrhagic diathesis U.Thomaneck, O.Anders, E.Knippel, H.Konrad, H.Klug

503

INFLUENCE OF EXOGENIC FACTORS: RADIATION, STRESS, ANTIBODIES, DRUGS, MITOGENS

Cell electrophoretic mobility as an aid to study biological systems K. A. Chaubal

515

XVI

The biochemical and morphologic blood analysis and electrophoretic behaviour of rat erythrocytes after gamma irradiation St.Ivanov, M.Kantcheva, B.Galutzov, D.Kovatchev

527

Electrophoretic mobility of erythrocyt es from gamma— or alpha-irradiated rats protected by adeturone T.Vranska, T.Pantev, K.Kuzova, N.Ryzhov, B.Fedorenko ..

529

The use of the electrophoretic microscope PARMOQUANT 2 for the investigation of erythrocytes in dynamics in the therapy of patients with psoriasis Ju.K.Khudensky, Ju.M.Bochkarev, P.Y.Lawin, V.S.Polkanov, N.B.Olkhovikova, V.V.Tkachev

537

Effect of ultraviolet irradiation on electrophoretic behaviour of red blood cells K.Redmann

541

Stress induced alterations in electrophoretic and morphological properties of thymus cells Zh.M.Agadshanyan, A.V.Temnov

549

Erythrocyte electrophoretic mobility and its membrane state in conditions of adrenalin stress V.A.Baronenko, V.G.Shamratova, M.G.Beljaeva

557

Some aspects of cell electrophoresis application in immunological investigations M. Kraskina, M.Bliacher, I.Pedorova, E.Knippel, W.Schiitt

565

Application of antibodies to cell electrophoresis H.Hayashi, N.Toyama, N.Takahashi, Y.Oguchi, K.Matsunaga, M.Fujii, F.Hirose, Ch.Yoshikumi, T.Hotta, M.Yanagisawa

581

In vitro differential effects of chloropromazine on the surface charge of murine T cell subsets M.Donner, S.Droesch, J.F.Stoltz

589

XVII

Purification of intestinal intraepithelial lymphocytes by preparative electrophoresis H.Cohly, C.van Oss, M.Weiser, B.Albini

603

Microelectrophoresis of epithelial cells and lymphocytes H.Cohly, B.Albini, M.Weiser, K.Green, C.van Oss

611

Monitoring of ATG production using cell electrophoresis E.Knippel, S.Preussner, E.Dirks

617

Cell electrophoretic detection of pregnancy factors E.Knippel, W.Straube, V.Briese, R.Sudik, R.Fliess

621

Isolation of cationic proteins from bovine allantoic fluid - A preliminary study of their biological properties J.J.Sennesael, M.lammens-Verslijpe, P.P.Lambert

625

Preliminary investigation of the effect of Chinese herbs on lymphocyte stimulation by cell electrophoresis Sun Ling, Chen Min, Shen Xiao-Hua, Xu Ying-Hui

635

The variation in the electrophoretic mobility of erythrocytes after massive blood replacement by "Perftoran" A.I.Miroshnikov, E.V.Grishina, B.I.Islamov

645

The application of cell electrophoresis to the production of erythrocyte diagnostic preparations A.I.Miroshnikov, A.V.Temnov, A.Yu.Ivanov, N.V.Dulatova, 0.V.Savitskaya

651

Electrophoretic mobility, ecto-ATPase activity and mitotic rate of thymocytes after treatment of mice with immunosuppressive drugs G. Hennighausen, R. Claus , J.Rychly, W.Schiitt

657

On the sensitivity of several indicators of cytotoxic drug effects of thymocytes in vitro G.Hennighausen, J.Rychly, S.Szymaniec

663

XVIII

Discrimination between functional and non-functional ConAreceptors by means of cell electrophoresis Ch.Huckel, J.Brock, J.Rychly, U.Schulz, P. Kusnierczyk, E. Paitasz

669

Mechanism of cell charge alteration of T lymphocytes after ConA stimulation Ch.Huckel, J.Brock, J.Rychly, H.Werner

677

The effect of peanut agglutinin on the electrophoretic mobility of lymphocyte populations J.Rychly, P.Ziska

681

Phytohaemagglutinin-induced changes of electrophoretic mobility and functional activity of PEA chloroplasts V.Goltzev, M.Kantcheva, V.Doltchinkova, D.Kovatchev

691

Cell electrophoretic detection of the influence of transfer factor on trypsinated T lymphocytes I.Schroder, W.Schutt, J.Rychly

697

DETECTION OF MULTIPLE SCLEROSIS

Family studies in multiple sclerosis: Frequency amongst relatives of a proband E.J.Field, G.Joyce

703

Multiple sclerosis: Diagnosis by He-Ne laser irradiation of erythrocytes E.J.Field, G.Joyce, D.Field

721

Laser Cytopherometry in multiple sclerosis A.W.Preece, N.P.Luckman, R.J.Jones

733

XIX

DETECTION OF L Y M P H O K I N E S : RENAL T R A N S P L A N T A T I O N ,

METHODICAL A S P E C T S ,

CANCER,

DRUGS

The macrophage e l e c t r o p h o r e t i c a p p l i c a t i o n s : A guide

mobility t e s t :

Clinical

E.J.Field

747

Enrichment and c h a r a c t e r i z a t i o n of the macrophage s l o w i n g f a c t o r r e l e a s e d by mononuclear b l o o d c e l l s upon c o n t a c t w i t h CEA or t e r a t o m a - d e r i v e d p r o t e i n s H. Wagner,

H.Grossmann,

J.Irmscher,

M.Kotzsch,

M.Miiller

.

757

C h a r a c t e r i z a t i o n of the p h y t o h a e m a g g l u t i n i n - i n d u c e d macrophage s l o w i n g f a c t o r (PHA-MSF) G.Metzner,

D.Haroske,

Failure

the MEM t e s t

of

B.Fahlbusch for

cancer

765 diagnosis

D. Zemanova, E . N i n g e r , J . K o v a r i k , L. P o p e l i n s k y , M.Jira, I.Malbohan, J.Andrlikova, R.Bischof

J. S t r e ¡jcek ,

777

Long-time s t u d y i n melanoma p a t i e n t s w i t h TAA-melanoma and PPD 9 y e a r s a f t e r primary o p e r a t i o n by MEM-test H.Sochor,

M.Gehre,

H.Werner

783

I n v e s t i g a t i o n of o b l i t e r a t i v e the PARMOQUANT 2 R.lambrecht,

K.Plate,

vascular

H.Kosowski,

diseases

with

P.Heinrich

785

The t r a n s f e r of c e l l u l a r immunity i n the i n v i t r o MEM t e s t by immune RNA i s o l a t e d from g u i n e a - p i g s immunized w i t h b a s i c p r o t e i n of human g l i o m a Shi Yongde, Che Y u f a n

Tang Zhenshen,

Xiao

Baoguo,

Ye

A l t e r a t i o n of e l e c t r o p h o r e t i c m o b i l i t y of p e r i t o n e a l macrophages by c a r r a g e e n a n H. Schaf f n e r

Qingwei,

guinea

789

pig 801

XX

The tanned erythrocyte electrophoretic mobility (TEEM) test B.K.Shenton, A.Alomran, P.K.Donnelly, T.W.J.Lennard, G.Proud, R.M.R.Taylor

807

Effect of supernatant from stimulated lymphocyte subpopulation on sheep erythrocyte electrophoretic mobility N.Hashimoto, S.Nose

819

Influence of autacoids and some drugs on lymphoid cells in vitro: Secretion of charge changing lymphokines and modulation of the ConA response H.Werner, I.Paegelow

829

Assessment of transfer factor activity using the tanned erythrocyte electrophoretic mobility test H.Werner, I.Schroder

847

Basic mechanisms of EMT: Investigations of some effects U.D.Koenig, B.Kozan

851

Histospecific tumor - associated antigens from normal and malignant tissues treated with viral neuraminidase: Their application in the electrophoretic mobility test (EM-test) 861

N.L.Novichenko Cancer diagnosis and other applications by using the electrophoretic mobility-test (EM-test in the last ten years (1974-1984): A critical review K.B.Mross

ABSTRACTS

AUTHOR

SUBJECT

871

OF

INDEX

INDEX

PAPERS

NOT R E C E I V E D

AS

MANUSCRIPTS

895

903

907

OPENING SPEECH

MICROGRAVITY CELL ELECTROPHORESIS EXPERIMENTS ON THE SPACE SHUTTLE:

A 198 4 OVERVIEW

Paul Todd Molecular and Cell Biology Program, The Pennsylvania State University, 403 Althouse Laboratory. University

Park,

Pennsylvania 16802

Summary Gravity affects the preparative electrophoresis of particles and cells in three ways:

it causes particle sedimentation

during exposure of particles to the electric field, it causes zone sedimentation during density

instabilities caused by

diffusion boundaries, and it causes convection during heating of the suspending fluid by the electric current.

Cell and

particle electrophoresis experiments were performed on three flights of the U.S. Space Transportation System (Shuttle, or STS) to test the effects of the absence of these three influences of gravity.

Test particles consisted of fixed

erythrocytes (human and rabbit) having different electrophoretic mobilities and polystyrene latex spheres having three different mobilities and three different colors. Living cell samples consisted of cultured human embryonic kidney cells, suspensions of rat anterior pituitary cells, and suspenions of canine pancreatic islet cells.

Test methods

were static column electrophoresis and free-flow electrophoresis.

The effect of particle sedimentation on the

preparative purification of pituitary cells, which are

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

4 heterogeneous with respect to size and density, was evident when microgravity density

free-flow electrophoresis was compared with

gradient electrophoresis at 1 g.

The effect of zone

sedimentation was evident when static column

electrophoresis

of erythrocytes, which can be studied at very concentrations, was compared with density

high

gradient

electrophoresis at 1 g. The effect of convection was evident in both static and free-flow electrophoresis when chambers too large to be cooled effectively successfully

separation

on earth were used

in microgravity.

Introduction Detailed descriptions of the biological studies performed on cells retrieved been presented the 3rd

from U.S. Space Shuttle

(STS) flights have

in articles on cell experiments conducted on

(1-3) and 8th (4,5) flights.

Fixed erythrocytes were

separated by static column electrophoresis, and no substantial effect of cell concentration at extremely was seen.

Polystyrene

high

concentrations

latex spheres having three different

electrophoretic mobilities were separated by continuous flow electrophoresis on the 7th flight, and the effect of conductance gaps was observed unpublished observations). human embryonic kidney

(6, R. S. Snyder et al.,

Canine pancreatic

islet cells,

cultured cells, and rat anterior

pituitary

cells were returned alive from the 8th flight, and

separated

fractions were analysed

produced.

for the products they

In summary, electrophoretic

in insulin-producing urokinase-producing

subtractions

enriched

cells, growth hormone cells, and cells, respectively were

expected on the basis of ground-control

found, as

experiments.

5 Cell and particle electrophoresis studies in microgravity on earlier U.S. space flights have been reported in considerable detail (7-11), so the summary that follows is limited to STS flight results that relate directly to the effects of gravity on preparative electrophoretic processes. Gravity affects the preparative electrophoresis of particles and cells in three ways:

it causes particle sedimentation

during exposure of particles to the electric field, it causes zone sedimentation during density

instabilities caused by

difussion boundaries, and it causes convection during heating of the suspending fluid by the electric current.

One of the

promises of microgravity electrophoresis is the elimination of these gravity-dependent effects, which, in some cases, limit sample size and compromise electrophoretic resolution.

This

summary of recent electrophoresis research conducted in microgravity

on the U.S. Space Transportation System (STS, or

"Space Shuttle") has as its purpose the comparison of 1-g and microgravity

experiments in which each of these three effects

is evident.

Materials The cells and particles used in STS experiments are summarized in Table 1.

Methods Two cell electrophoresis methods were used on STS flights: static column electrophoresis in the Electrophoresis Equipment Verification Test (EEVT) and the McDonnell-Douglas Continuous Flow Electrophoretic Separator (CFES). Schematically

in Figures 1 and 2.

These are described

The electrophoresis chamber

6 Table 1.

Cells and particles subjected to microgravity electrophoresis on Space Shuttle flights

STS-3

APR 82

Human RBC Rabbit RBC

Test effect of cell concentration

STS-7

AUG 83

Polystyrene Latex Spheres

Test stability and effect of conductance

STS-8

SEP 83

Canine Pancreas

Purify

Human embryonic k idney

Purify urokinaseproducing cells

Rat pituitary

Purify individual hormone-producing cells

buffers used were designated and "CFES Buffer" given

"D-l Buffer"

in the case of CFES.

Beta cells

in the case of EEVT

Their compositions are

in the captions of Figures 1 and 2, respectively.

two electrophoresis methods were semi-automated, with insertion and experiment

The

sample

initiation being performed by

astronauts or payload specialists, while the apparatus operated

unattended during electrophoretic

separations.

Periodic photographs were taken during separations with astronaut

participation.

Experimental

Results.

Specific effects of particle sedimentation, zone sedimentation, and convection on static column

electrophoresis

and

qualitatively,

free-flow electrophoresis can be predicted

and these effects have now been demonstrated by direct comparison of experimental results in lg and microgravity environment.

7

© 120cm

® ¡—+

1 CD !

4-I5CI*-» Figure 1.

Schemetic diagram of static column zero-g electrophoresis device. (1) Electrophoresis column in which cells move left to right in buffer "D-1" (6.42 mM NaCl, 0.367 mM K H 2 P 0 4 , 1.76 mM N a 2 H P 0 4 , 222 mM glucose, 0.336 mM Na 2 EDTA, 5% dimethylsulfoxide, pH 7.2, 0.015 g-ions/1 ionic strength, conductivity = 0.9 mmho/cm) under an applied field of 13.6 v/cm at 14°C. (2) Meters for monitoring column voltage and temperature, toggle switches, and clock in the viewing field of the camera (4), which took photographs of the meters every minute and of the columns, when their thermal cover was removed briefly, every 10 min. (3) Power supply and electrode buffer circulating system. (5) Illuminator. (6) Accelerometer for detecting small non-zero-g accelerations during cell electrophoresis.

Figure

Schematic diagram of continuous flow zero-g electrophoresis device. (1) Continuous sample input at 0.05 ml/min. (2) Computer-controlled pumping system for continuous buffer input at 40 ml/min into the separation chamber (3) where cells migrate from left to right in a 40 V/cm field while flowing upward. The system pumps separate buffer into the electrode compartments (4). (5) Sample collection system which divides the end of the column into 198 outlets and delivers electrophoretic subfractions to 198 receptacles (6). A modification of triethanolamine-acetate buffer of Hannig et al^. was used in the chamber.

2.

8 1.

Particle

sedimentation

Particle sedimentation

(or buoyancy) is a significant

phenomenon only when the sedimentation velocity exceeds the Brownian velocity of the suspended particles.

In the case of

macromolecules, particle sedimentation at 1 g is obviously not a problem, and, even in an ultracentrifuge, diffusion and sedimentation of very large molecules are study simultaneously. The velocity of a spherical particle of radius a, density po, and mobility p undergoing electrophoresis in a density gradient with density and viscosity p D (x) and r\a(x) , respectively, is v = v E + V s = u(x)E(x) + 2(p-p n (x))a 2 9 uri ( x )

(1)

The sedimentation term (second term) is very small for bacteria and most subcellular particles.

The sedimentation

term could be 10% or more of the electrophoretic velocity

term

(first term) in the case of whole cells, owing to their large radius (12).

The effect of particle sedimentation on the

electrophoretic migration of cells in a density gradient has been demonstrated theoretically

and experimentally

for cells

of different sizes and densities being electrophoresed in a Ficoll gradient

(13).

upward

One procedure used to prevent

particle sedimentation during electrophoresis is to perform separations in an horizontal tube and to rotate the tube at approximately

70 RPM (14).

In continuous flow electrophresis the electrophoretic velocity vector is orthogonal to the sedimentation vector in most configurations.

However, migration distance is established

by time spent in the horizontal electric field.

Figure 3

illustrates what may happen in different configurations of

9

ft

r*

VF±-|a2(A>-?0)g

EQUAL fj,

B

DIFFERENT SEDIMENTATION

EQUAL fi DIFFERENT BUOYANCY

111111 Figure

3.

continous

E f f e c t s of particle s e d i m e n t a t i o n on c o n t i n u o u s flow e l e c t r o p h o r e s i s of cells. In A and B the c i r c l e s show the path of m o r e rapidly s e d i m e n t i n g cells. In C and D the c i r c l e s show the path of m o r e b u o y a n t cells. All cells are c o n s i d e r e d to have the same e l e c t r o p h o r e t i c m o b i l i t y . flow e l e c t r o p h o r e s i s

sedimentation. downward while horizontal

as a c o n s e q u e n c e

For e x a m p l e , a particle flowing

electric

that

upward spends m o r e

of

sediments

time

in the

field and t h e r e f o r e m o v e s farther

cells of lower s e d i m e n t a t i o n v e l o c i t y .

A hint of the

of s e d i m e n t a t i o n

4, in w h i c h

is indicated

in Figure

m i g r a t i o n d i s t a n c e s of rat pituitary different

c o n d i t i o n s are c o m p a r e d .

cells

gradient

the

three

In the case of

e l e c t r o p h o r e t i c m o t i o n of rat a n t e r i o r in a Ficoll density

under

than effect

pituitary

cells

(upper p a n e l ) , g r o w t h

upward

hormone

10

secreting

cells migrated

flow e l e c t r o p h o r e s i s

the least d i s t a n c e .

(center panel),

In c o n t i n u o u s

in w h i c h

flow w a s

the same p o p u l a t i o n of cells m i g r a t e d the g r e a t e s t In m i c r o g r a v i t y

(lower panel),

hormone

secreting

Zone

is i r r e l e v a n t ,

growth

the g r e a t e s t

distance.

gradient electrophoresis

electrophoresis

are limited

by z o n e , or d r o p l e t , concentration

achievable

and c o n t i n u o u s

in their cell processing

sedimentation.

The m a x i m u m

d e p e n d s on cell size,

v i s c o s i t y , cell d e n s i t y ,

and m e d i u m d e n s i t y ,

l i m i t s have bee n work ed out by Ma son

(15).

have r e v e a l e d gradient, than about

that stable

are d i f f i c u l t

suspensions,

medium

and

corresponding

even

(16,17)

in a density

to achieve at higher cell

densities

10 m i l l i o n / m l .

Shuttle

at high cell density

of zone s e d i m e n t a t i o n . electrophoresis of 0.5 x 109

This experiment

simulated

of high cell c o n c e n t r a t i o n s by

e a c h of fixed human and rabbit limit at unit gravity

absence

the

using a m i x t u r e

erythrocytes.

to exceed the zone

(16,17), and

m o b i l i t i e s of the cells had been d e t e r m i n e d 2 compares

the

in the b u f f e r

the e l e c t r o p h o r e t i c m o b i l i t i e s of the

test p a r t i c l e s m e a s u r e d

in the laboratory

electrophoresis

by

D-l.

two

microscopic

(2) w i t h the m o b i l i t i e s of

and trailing e d g e s of m i g r a t i n g

on

of

in the p r e s u m e d

This c o n c e n t r a t i o n had been d e m o n s t r a t e d sedimentation

experiments

flight STS-3 w a s the e v a l u a t i o n

electrophoresis

leading

capacity

Deta i1ed

One of the o b j e c t i v e s of the e l e c t r o p h o r e s i s

analytical

flow

cell

e x a m i n a t i o n s of the b e h a v i o r of fixed e r y t h r o c y t e s

Table

cells

sedimentation

B o t h density

Space

is

cells m i g r a t e d w i t h the high mobility

but did not m i g r a t e

2.

distance.

in w h i c h s e d i m e n t a t i o n

a b s e n t and d i r e c t i o n of m i g r a t i o n

upward,

the

c e l l s d e t e r m i n e d by

11

o o o

o N LlJ z o z a: o x

to o z < CO 0 1

UJ z o s CE

o X

IIO FRACTION

Figure

4.

130

:—inn

0 1

NUMBER

Top panel: Density-gradient electrophoresis of growth-hormone-cell enriched rat anterior pituitary cell suspension. Electrophoretic migration was from bottom to top of Ficoll gradient, right to left; v = migration vector, g = gravity vector. Center panel: Electrophoretic profiles of rat anterior pituitary cells and their growth hormone concent in separations performed using the McDonnell-Douglas continuous flow electrophoretic separator with upward buffer flow. Electrophoretic migration was from left to right, and gravity was orthogonal but affected the cells' rate of transport in the moving buffer stream. Bottom panel: Electrophoretic profiles of rat anterior pituitary cells and the free hormone content of corresponding fractions. Migraton was from left to right on the plot, microgravity conditions. (Redrawn from refs. 4 and 13).

12 dividing every

their v e l o c i t i e s , m e a s u r e d

11 m i n u t e s by the STS-3 a s t r o n a u t s

field s t r e n g t h , also mobilities 10

9

from p h o t o g r a p h s (1), by the

in the p h o t o g r a p h s ,

are the same

in m i c r o g r a v i t y

c e l l s / m l as unit gravity

taken

of

monitored

13.5 v / c m .

The

at a c o n c e n t r a t i o n

at a c o n c e n t r a t i o n of

10

of

6

cells/ml. Table 2. E l e c t r o p h o r e t i c m o b i l i t i e s of fixed human and rabbit e r y t h r o c y t e s in "D-l" buffer in the laboratory and in space m e a s u r e d at 12°C, in units of u m / s e c per V / c m .

CONDITION

CONCENTRATON (cells/ml)

METHOD

Space

10 9

Static column

Laboratory

RBC M O B I L I T I E S HUMAN RABBIT

Microscopic

10

-1.32

6

- 1 . 42±0.18

It w a s pointed out in p r e v i o u s d i s c u s s i o n s sedimentation, because causes

(16) that

it is a s t o c h a s t i c p h e n o m e n o n

intolerable broadening

density

-0.83

c o e f f i c i e n t s of v a r i a t i o n

droplet (15),

of cell z o n e s , especially

gradient electrophoresis.

droplet sedimentation were

-0.94+0.12

T w o m e t h o d s of

tested, and the

resulting

(relative zone w i d t h , d e f i n e d as the

ratio of s t a n d a r d d e v i a t i o n to m e a n m i g r a t i o n d i s t a n c e ) c o m p a r e d w i t h those previously gradient experiments. results,

Table

gradient),

vector

orthogonal

to m i g r a t i o n

free-zone electrophoresis),

or z e r o

the to

in a density

(horizontal m i g r a t i o n (in the

were

density

is a n t i p a r a l l e l

(upward e l e c t r o p h o r e t i c m i g r a t i o n

experiments). width

found in v e r t i c a l

3 is a list of some of

in w h i c h the gravity

migration

in

eliminating

in

STS-3

The e f f e c t of d r o p l e t s e d i m e n t a t i o n on zone

is d e m o n s t r a t e d

in this

comparison.

13

T a b l e 3. E f f e c t of d r o p l e t s e d i m e n t a t i o n on the of v a r i a t i o n of the e l e c t r o p h o r e t i c m i g r a t i o n of ery throcy tes. CELLS PER ML 1

COEFFICIENT OF VARIATION

GRAVITY

10 7

X

5. 0

Antiparallel

7 5 x 10

13.7

8 1 x 10

20. 4

8 5 x 10

29. 3

1 x 109

>90

2 x 108 2 x IO

Orthogonal

5.5

9

6.8

2 x 10®

Zero

4.5

1 x 109

16.9

In c o n t i n u o u s - f l o w

electrophoresis droplet

the same e f f e c t as particle e f f e c t of

increasing

flow

upward

too b u o y a n t . the

field

long e n o u g h flow

depicted

in Figure

not normally

s e d i m e n t a t i o n , w i t h the sample

is too b u o y a n t

for cell d e f l e c t i o n

if it is too d e n s e .

in the laboratory

observations).

the

if it is

it w i l l not remain in upward

flow or

four c o n d i t i o n s

using c e l l s , they

using high c o n c e n t r a t i o n s of p r o t e i n s

have been shown to be a b s e n t unpublished

In

5, and, a l t h o u g h these e x t r e m e cases

studied

been demonstrated

These

has

additional

stream w i l l not enter

if it is too dense or d o w n w a r d

If a sample

downward

sedimentation

cell band w i d t h s at the o u t l e t .

e x t r e m e cases, a too-dense chamber

coefficient fixed

in m i c r o g r a v i t y

(J. W.

in in

are are have and

Lanham,

14

II I I I I I I I

n* TOO DENSE

TOO BUOYANT

Figure 5.

3.

Sketch of the consequences of sample zones that are too dense or too buoyant in continuous flow electrophoresis.

Convection

The non-uniform heating of buffers by the current in electrophoresis chambers causes convection, and nearly all chambers are designed to minimize convection through a system of uniform, and sometimes extensive, cooling. All electrophoresis chambers have at least one very

samll

dimension, to allow optimum cooling, and this small dimension usually

limits sample size and/or throughput rate.

microgravity

Successful

electrophoresis experiments with chambers having

15

dimensions

that

convection

indicate

avoided

not be a d e q u a t e l y

that

have been used

have not y e t

been t e s t e d

increase

increased

size

chamber

Laboratory

All

Shuttle-based

and f o l l o w e d Examples of 4,

alone

electrophoresis

obtained

(4).

Migration

similar.

on STS-3 w i t h after

resulted

The s t a t i c 109

experiments migration

laboratory of

(1,4)

produced an tapering

free-zone,

rotating

profile

of of

pancreas

experiments experiments

unexpected c e l l s was human

(1-3).

seen

(leading)

Laboratory

electrophoresis However, when ony present

electroosmosic

the m e t h y l c e l l u l o s e the c e l l

of

e x p e r i m e n t s were

as human c e l l s were amount o f

through e r o s i o n

a migration

tube

two sharp bands.

in

microgravity

and c a n i n e

one of

using

them.

shown

profiles and in

band of

2 sharp bands,

erythrocytes

cells

are

column e l e c t r o p h o r e s i s

(trailing)

and when a small

preceded

simulated

in the two t y p e s of

One l a r g e than

that

combinations

erythrocytes

in the e x p e c t e d

introduced

the

al.,

e x p e r i m e n t s were

rabbit

1/2 as many r a b b i t sample, (14),

profiles

profile.

simulations

a 4-8-

(as

on the b a s i s of

experiments

in the

Similar

60 min r a t h e r

and one of

of sizes

chambers,

( J . W. Lanham, e t

such " g r o u n d - c o n t r o l "

cells

migration

sample

is achievable

p e r f o r m e d on human embryonic kidney were

flow

6 mm

3.0 mm

evidence

cell

are

simulations

by l a b o r a t o r y

a r e compared cells.

without

solutions)

in which e l e c t r o p h o r e t i c

pituitary

convection

observations).

4.

Fig.

prevent

columns more than

in c o n t i n u o u s

in sample thoughput protein

unpublished

thermal

convection-limited

demonstrated w i t h

to

f l o w chambers more than

in m i c r o g r a v i t y

Although

fold

Thus s t a t i c

and c o n t i n u o u s

convection.

cooled

the e f f e c t s of

in m i c r o g r a v i t y .

in d i a m e t e r thick

could

mixture

in

the

was coating

resembling

that

16 o b s e r v e d on flight STS-3 w a s o b t a i n e d . obtained Figure

in both cases by o p t i c a l

These

profiles,

s c a n n i n g , are c o m p a r e d

in

6.

CO

Figure

6.

Bottom: O p t i c a l scan of negative of photo taken 51 m i n after the i n i t i a t i o n of e l e c t r o p h o r e s i s of " C o l u m n 019" on STS-3 m i s s i o n (Sarnoff et al., Strip-chart 1983; M o r r i s o n and Lewis, 1983). Top: r e c o r d of e l e c t r o p h o r e t i c s e p a r a t i o n of 1 x 1 0 9 / m l h u m a n R B C ' s in free-zone e l e c t r o p h o r e s i s tube w i t h some e l e c t r o s m o s i s , 60 m i n after the b e g i n n i n g of electrophoresis.

Discussion Three g r a v i t y - d e p e n d e n t electrophoresis: sedimentation, demonstrated

free-fluid

s e d i m e n t a t i o n , zone

and c o n v e c t i o n .

in laboratory

continuous-flow demonstrated

p h e n o m e n a affect

particle

in m i c r o g r a v i t y

droplet)

All three of these are

experiments

electrophoresis,

(or

readily

in d e n s i t y - g r a d i e n t

and their a b s e n c e has

experiments.

been

or

17

Particle

s e d i m e n t a t i o n at unit gravity

p o s i t i o n of macroraolecules as cells, have a very subject they

they

in s o l u t i o n .

if they

are s u s p e n d e d .

are d e n s e r

They

desireable.

W h e n rat pituitary

cells a c c o r d i n g

cells are s u b j e c t e d

Ambiguities

due to s e d i m e n t a t i o n are a b s e n t

Zone, or d r o p l e t ,

have the

and cells have

into their zone, m a k i n g The r e s u l t

w h e n the d e n s e r zone buoyancy density

occurs. gradient

electrophoresis flow

lies above the resulting

When

molecules surrounding

sedimentation less-dense

for e l e c t r o p h o r e s i s is injected

or a

zone.

zone

fixed e r y t h r o c y t e s are s u s p e n d e d

in a

several-percent

into a c o n t i n u o u s - f l o w

c h a m b e r , zone s e d i m e n t a t i o n

(or

in the m i l d e s t cases and

in e x t r e m e cases.

Space Shuttle

diffusivity

than the

is "rainfall" or d r o p l e t

c a u s e s zone b r o a d e n i n g zone

it d e n s e r

is m u c h

low

is made d e n s e r than the p a r t i c l e z o n e ,

protein solution

rate

concentration

they do not d i f f u s e out of their z o n e , but small

If the m e d i u m

same

in m i c r o g r a v i t y .

in w h i c h their c o n c e n t r a t i o n

Because macromolecules

solution.

smaller

s e d i m e n t a t i o n o c c u r s w h e n there e x i s t s a

s u r r o u n d e d by a zone

diffuse

the

in cell m i g r a t i o n

z o n e of large m o l e c u l e s or cells at high lower.

to

from their

less dense c o u n t e r p a r t s even w h e n they

electrophoretic mobility.

to

not be

electrophoresis,

larger a n d / o r d e n s e r cells are s e p a r a t e d

if

Sedimentation

this s e p a r a t i o n may or may

d e n s i t y - g r a d i e n t or c o n t i n u o u s - f l o w and/or

fluid

to buoyancy

fluid.

are useful w a y s of separating

size and d e n s i t y ; h o w e v e r ;

such

and are

than the

are s u b j e c t

are less dense that the suspending

and buoyancy

the

Large p a r t i c l e s ,

low d i f f u s i o n c o e f f i c i e n t

to s e d i m e n t a t i o n

in w h i c h

does not a f f e c t

buoyancy) unmanageable

E x p e r i m e n t s c o n d u c t e d on the

have shown that m i c r o g r a v i t y

eliminates

zone

sedimentation. Convection

is a b u o y a n t

surroundings. gradient.

The

flow of fluid less dense than

usual case

is a t h e r m a l l y - i n d u c e d

A s o l u t i o n that passes a c u r r e n t

is h e a t e d

its density in

18 p r o p o r t i o n of the c u r r e n t d e n s i t y . density-gradient upward

electrophoresis

In a w a t e r - j a c k e t e d

column convective

in the center and d o w n w a r d at the w a l l s .

typically

induced by e l e c t r i c c u r r e n t s

migration

rates >1 cm/hr

electrophoresis,

using

thick

cylindrical

Space

convection

flow c h a m b e r s

c o o l e d at the w a l l s . demonstrated

In

cell

continuous-flow

that can only

less than

rates

be

1 m m in d i a m e t e r ,

The absence of c o n v e c t i v e d i s t u r b a n c e

and c o n t i n u o u s - f l o w

in two m i c r o g r a v i t y

Shuttle

that produce

(rather slow).

is

c u r r e n t s r e q u i r e d to achieve m i g r a t i o n

of several m m / m i n produce p r e v e n t e d by

flow

It is

c h a m b e r s has

in

been

electrophoresis devices

on

flights.

Ack n o w l e d g e m e n t s The r e s e a r c h s u m m a r i z e d G. C. M a r s h a l l Center,

in this report w a s c a r r i e d out at the

Space Flight C e n t e r , the L.B. J o h n s o n

Lehigh U n i v e r s i t y ,

University,

the O r e g o n H e a l t h

the M i c h a e l Rees R e s e a r c h F o u n d a t i o n ,

Pennsylvania

State U n i v e r s i t y , M c D o n n e l l - D o u g l a s

Corporation,

and W a s h i n g t o n U n i v e r s i t y .

a u t h o r s of

(refs.

1-9).

U.S. N a t i o n a l A e r o n a u t i c s McDonnell-Douglas

Space

Sciences The Astronautics

P a r t i c i p a n t s are

The r e s e a r c h w a s s u p p o r t e d by and Space A d m i n i s t r a t i o n

Astronautics

the

the

and

Corp.

References 1.

M o r r i s o n , D. R. and Lewis, M. L.: E l e c t r o p h o r e s i s tests on STS-3 and g r o u n d control e x p e r i m e n t s : A basis for future b i o l o g i c a l sample s e l e c t i o n s . In Proc. 33rd I n t e r n a t i o n l A s t r o n a u t i c a l F e d e r a t i o n C o n g r e s s , Paper No. 85-152, (1983).

2.

S n y d e r , R. S., R h o d e s , P. H., H e r r e n , B. J., M i l l e r , T. S e a m a n , G. V. F., T o d d , P., K u n z e , M. E., and S a r n o f f , B. E.: E l e c t r o p h o r e s i s (submitted 1984).

3.

S a r n o f f , B. E., Kunze, M. E., and Todd, P.: A s t r o n a u t . Sci. 53, 139-148 (1983).

Adv.

Y.,

19 4.

M o r r i s o n , D. R. , B a r l o w , G. H., C l e v e l a n d , C., F a r r i n g t o n , M. A., G r i n d e l a n d , R., H a t f i e l d , J. M., H y m e r , W. C., K u n z e , M. E., L a n h a m , J. W., Lewis, M. L., Adv. Space N a c h t w e y , D. S., T o d d , P., and W i l f i n g e r , W.: Res. (in press, 1984).

5.

M o r r i s o n , E. R., Lewis, M. L. , B a r l o w , G. H., T o d d , P., K u n z e , M. E., S a r n o f f , B. E., and Li, Z. K.: A d v . Space Res. (in press, 1984).

6.

R h o d e s , P. H. and S n y d e r , R. S.: In M a t e r i a l s P r o c e s s i n g in the R e d u c e d Gravity of Space, Ed. G. E. R i n d o n e , N o r t h H o l l a n d , New York, 1982, pp. 225-232.

7.

A l l e n , R. E., Rhodes, P. H., S n y d e r , R. S., B a r l o w , G. H., B i e r , M., B i g a z z i , P. E., van Oss, C. J., Knox, R. J., S e a m a n , G. V. F., M i c a l e , F. J., and V a n d e r h o f f , J. W.: Sep. Purif. Meth. _6, 1-59 (1977).

8.

A l l e n , R. E. et al^. E l e c t r o p h o r e s i s t e c h n o l o g y . In A p o l l o - S o y u z T e s t P r o j e c t Summary P r o g r e s s R e p o r t V o l . 412. N a t i o n a l A e r o n a u t i c s and Space A d m i n i s t r a t i o n Report NASA SP-412 (1977).

9.

M c K a n n a n , E. C., K r u p n i c k , A. C., G r i f f i n , R. N. and M c C r e i g h t , L. R.: E l e c t r o p h o r e t i c s e p a r a t i o n in s p a c e — A p o l l o 14. N a t i o n a l A e r o n a u t i c s and Space A d m i n i s t r a t i o n R e p o r t NASA TMX-64 611 (1971).

10.

S n y d e r , R. S.: E l e c t r o p h o r e s i s d e m o n s t r a t i o n on A p o l l o 16. N a t i o n a l A e r o n a u t i c s and Space A d m i n i s t r a t i o n R e p o r t NASA T M X - 6 4 7 2 4 (1972).

11.

S n y d e r , R. S., B i e r , M., G r i f f i n , R. N., J o h n s o n , A. J., L e i d h e i s e r , H., M i c a l e , E. J., Ross, S., and van Oss, C. J.: Sep. Purif. Meth. 2, 258-282 (1973).

12.

B o l t z , R. C., Jr., Todd, P., S t r e i b e l , M. J., and Louie, K.: P r e p a r a t i v e B i o c h e m . 3, 383-401 (1973).

13.

P l a n k , L. D. , H y m e r , W. C. , Kunze, M. E. , Marks, G. M. , L a n h a m , J. W., and Todd, P.: J. B i o c h e m . B i o p h y s . Meth. 8, 275-289 (1983).

14.

H j e r t e n , S.: Free Zone E l e c t r o p h o r e s i s . Wik s e l l s Bktr., U p p s a l a , 1967.

15.

M a s o n , D. W. :

16.

B o l t z , R. C., Jr., and T o d d , P.: In E l e c t r o k i n e t i c S e p a r a t i o n M e t h o d s (Eds. P. G. R i g h e t t i , C. J. van Oss, and J. V a n d e r h o f f ) E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press, A m s t e r d a m , 1978, pp. 229-250.

17.

O m e n y i , S. N., S n y d e r , R. S., A b s o l o m , D. T., N e u m a n n , A. W . , and van Oss, C. J.: J. C o l l o i d Interface Sci. 81, 4 2 0 - 4 0 9 (1981).

B i o p h y s . J. JL6, 407

Almqvist

M.

and

( 1976).

CELL ELECTROPHORESIS TECHNIQUES

FREE

ZONE

ELECTROPHORESIS

CELL-CELL Paul

Todd

and Stellan

Institute Uppsala,

OF A N I M A L C E L L S

I. E X P E R I M E N T S

ON

INTERACTIONS Hjerten

of B i o c h e m i s t r y , Box

576, S - 7 5 1

Biomedical

23 U p p s a l a ,

Center,

University

of

Sweden

Summary Free zone

electrophoresis

of m a t e r i a l s zones.

The

introduced progress

is m o n i t o r e d

by

photometer.

The

migration

is the h o r i z o n t a l

into a r o t a t i n g

absence

the of

velocities

of any m a t e r i a l

particles,

cells).

interaction free zone

by

of erythrocytes 2 x 10

9

velocity

increasing

cells/ml were

human glial

permitted

to

patterns

interact

electrophoresis, upon cell

during

or by

interactions

human The

the

not mixing

Concentrations

by

up

containing cells

molecule-

free

appear

were

altered

in the c a s e of studied

by

from

erythrocytes was

electrophoresis, As

to

high

buffers.

When zones

previously

cell-cell

at

of c u l t u r e d

and n e u r o b l a s t o m a

occurred.

interactions

can be s t u d i e d

erythrocytes

species.

used.

cells

macromolecules,

is a l s o o r t h o g o n a l

strength

fixed

of

electrophoretic

concentration

from d i f f e r e n t

migration molecule

of

zones

ultraviolet

fixed e r y t h r o c y t e s

ionic

cell

migrating

dense

particle-particle

and m i x t u r e s

in low

tube as

in the d i r e c t i o n

of

mobility

Fixed

of

species,

studied

electrophoretic

to

flows

sedimentation e f f e c t s of

mixtures

an

(small m o l e c u l e s ,

on e l e c t r o p h o r e t i c

animal

cells were

cultured

tube w i t h fluid

electrophoresis.

different

altered

As

motion,

concentrations,

quartz

of e l e c t r o p h o r e t i c a l l y

scanning

permits direct measurement

electrokinetic

electrophoresis

to

type.

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

zone depend

24 I n t r o d u c t ion Free zone e l e c t r o p h o r e s i s

(FZE)

been applied

of p r o t e i n s

to the study

b u f f e r s of widely a p p l i e d also

varying

in a rotating

of the

(1-4).

attributable

Any

The m e t h o d has collide

s u b s e q u e n t mobility

to the o c c u r r e n c e

(3) in been

i n t e r a c t i o n of samples

two z o n e s w h e n the zones e l e c t r o p h o r e t i c a l l y one a n o t h e r

(1) has

(2) and cells

composition.

to the study

tube

changes

of a c h e m i c a l

in

with are

reaction

during

collision. The free

interaction fluid

of animal

is d i f f i c u l t

sedimentation and Snyder

cells during e l e c t r o p h o r e s i s

at high cell c o n c e n t r a t i o n s

e n v i r o n m e n t , where zone of c e l l - c e l l

unambiguous;

the optical

two clearly using

high

not

human (7).

consistent

firm and

for a n a l y s i s

(7-9).

tested; however,

did

Previous

of a g g r e g a t i o n ionic

of

strength

microorganisms

of buffer c o n d i t i o n s at high

field s t r e n g t h .

after p r o l o n g e d

This

exposure

aggregation

(ca. 30 m i n . )

to

fields.

B e c a u s e zone s e d i m e n t a t i o n migration

does not affect

in FZE, and because

interactions

electrophoretic

this m e t h o d also

reveals

that affect e l e c t r o p h o r e t i c m i g r a t i o n ,

c h o s e n as a m e a n s of e x p l o r i n g cell

peaks

under all c o n d i t i o n s of

and high

abruptly

although

images a v a i l a b l e

under a w i d e variety

concentration

might

concerning

microgravity

experiments,

FZE (3) indicated a lack

fresh e r y t h r o c y t e s

occurred

Omenyi

in e x p e r i m e n t s w i t h in the

interaction, were

separated

and buffer c o m p o s i t i o n aggregated

But

s e d i m e n t a t i o n does not occur

The r e s u l t s of m i c r o g r a v i t y w i t h a lack

studies

and e v i d e n c e

i n t e r a c t i o n w a s sought

and rabbit e r y t h r o c y t e m i x t u r e s

not show

(5).

(6) have p r e s e n t e d data that e r y t h r o c y t e s

i n t e r a c t at high c o n c e n t r a t i o n , cell-cell

in

to e v a l u a t e due to d r o p l e t , or zone,

interactions

possible

on e l e c t r o p h o r e t i c

e f f e c t s of

mobility.

FZE was animal

25 Materials and Methods Cells.

Human erythrocytes were obtained

healthy

male donor fcy venipuncture and

from a 47-year-old

immediately

into isotonic phosphate buffer containing anticoagulant. were suspended

0.01% EDTA as

After three rinses in this buffer the cells in 2.5% paraformaldehyde

buffer at ambient temperature erythrocytes were obtained

in isotonic

(19-24 °C) for 2 weeks.

3 times in electrophoresis buffer

prior to experiments. were used:

Two strains of cultured

cells "SKNSH-5YSY", kindly

Pclhlmann of the Wallenberg

The

"D-l"

human cells

supplied by Dr. S.

Laboratory, Uppsala.

from monolayer culture by rinsing

EDTA in phos-phate-buffered incubating

(7).

Normal human cells of glial origin, "787CG" and

neuroblastoma suspended

Rabbit New

treated

in the same manner as the human RBC's

cells were washed

phosphate

from a 6-month-old healthy

Zealand white rabbit by ear venipuncture and subsequently

diluted

isotonic saline

5 min at 37°C in 0.25% trypsin

They were

5 min in 0.02% (PBS) and

(1-300) in PBS.

The action of trypsin was stopped by the addition of complete culture medium

(Eagle's Minimum Essential Medium) containing

10% fetal bovine serum. centrifugation

Cells were removed

and suspended

(6.2% sucrose) to discourage

Buffers.

in buffer C-l with sedimentation and

The blood-collection

buffer are described

buffer and the

in Table 1, which

tions of the ingredients. "C-l" used

3.4% Ficoll aggregation.

cell-fixation

lists the concentra-

The electrophoresis buffer used

for RBC electrophoresis, designated described

from medium by

"D-l", and the buffer

for cultured human cell electrophoresis are also in Table 1.

Electrophoresis.

The free zone electrophoresis

(1), consists of a thermostated coated with methy lcellulose

apparatus

rotating horizontal

to prevent

tube

electroosmotic

26 back flow. with

the

rotation

The RBC e x p e r i m e n t s following speed,

of buffer,

parameters:

of 2 x 10 9

concentrations

experiments were temperature

conditions were

time,

introduced

in two

2 cm apart

following

of buffer,

4.0 m A ; p o t e n t i a l , usually

starting

strip-chart

human

cell

conditions:

1.5 m m h o / c m ;

1450 V.

at 5 m i n

(with

at

The c u l t u r e d

under the

column

1 min

Other

the same as for RBC e x p e r i m e n t s .

recorded,

w i t h an analog 1.

performed

optical

290/320 nm ratio;

cells/ml.

3°C; conductivity

applied current,

Table

Cells were

"origin" of the tube

tube

conductivity

0.80 m A ;

5 m i n ; column scanning

field turned off). b a n d s at the

320 nm or

performed

14°C;

3.0 m m ;

0.90 m m h o / c m ; applied c u r r e n t ,

interval,

s c a n s were

here were

temperature,

40 rpm; tube d i a m e t e r ,

monitoring wavelength, scanning

reported

Optical

intervals, on

paper

recorder.

C o m p o s i t i o n of buffers

used

erythrocyte

and fixation and in the

collection

free zone e l e c t r o p h o r e s i s (concentrations COMPONENT Na 2HPO4

BLOOD COLLECTION

NaH2P04

in human and

tube and

rabbit

electrodes

are mM or %) RBC FIX 53. 7

TUBE D-l 1.76

ELECTRODE D-l 1.76

C-l 8.10 1.47

13.5

KH2PO4

0. 367

0. 367

NaCl

6. 42

6. 42

0.889

0. 889

145.0

150.0

KC1 MgCl 2 Na2EDTA

0.336

4.0

222.0

Glucose

55. 5 199. 0

Sucrose 5%

DimethyISO Paraformaldehyde Conductivity

2. 65 0.48

1.5%

(mmho/cm)

Ionic S t r e n g t h

(g-ions/1itre)

0.9

1.5

0. 015

0.030

27

F r a c t i o n s of e l e c t r o p h o r e t i c a l l y - s e p a r a t e d collected used

from the tube w i t h

for inserting

corresponded collected

the long, thin needle

samples z o n e s .

Each

to a 2 cm interval along

fraction was added directly

culture medium of each cell

type per m i c r o s c o p e

types w e r e d i s t i n g u i s h a b l e

total m a g n i f i c a t i o n . multiple

sample zone

Figure

the

the tube.

Each

to 5 ml of dish.

complete The

field w a s c o u n t e d 28 hours

1 indicates

number

in ten

later.

in phase c o n t r a s t

at

The

two

160x

the p r i n c i p l e

insertion, and figure

of two e r y t h r o c y t e z o n e s

normally

fraction

in a 60 mm plastic culture

fields by phase c o n t r a s t microscopy cell

human cells w e r e

of

2 is a p h o t o g r a p h

inserted at d i f f e r e n t

locations

in

tube.

Figure

1.

The insertion of m u l t i p l e z o n e s into the free zone e l e c t r o p h o r e s i s tube.

rotating

Figure

2.

P h o t o g r a p h of 2 zones of e r y t h r o c y t e s inserted into the rotating free zone e l e c t r o p h o r e s i s tube.

28

Results Cell-cell factor

i n t e r a c t i o n w a s not found to be a s i g n i f i c a n t

in the e l e c t r o p h o r e s i s

of fixed e r y t h r o c y t e s .

3 is a series of o p t i c a l

scans of the

phoresis

intervals

tube at various

free zone

after

the

e l e c t r o p h o r e t i c m i g r a t i o n of two cell z o n e s apart. rabbit

The human cell zone cell zone

velocity

Figure

or zone

3.

Cell-cell

("H") m i g r a t e d

initiation initially

of

2 cm

through

the

("R") w i t h o u t any m o d i f i c a t i o n of

its

shape.

O p t i c a l scans of m i g r a t i n g zones of fixed human ("H") e r y t h r o c y t e s and fixed rabbit ("R") erythrocytes. Each zone c o n t a i n e d 2 x 10 9 cells. interaction was

erythroid cells.

Figure

found to occur

in living,

4, lower panel,

t h r o u g h a lower-mobility These cells

the a g g r e g a t e s

non-

is a graph of

m i g r a t i o n of a rapidly m o v i n g zone of c u l t u r e d glial cells.

Figure

electro-

zone of c u l t u r e d

neuroblastoma

interacted w h e n the z o n e s c o l l i d e d ,

that formed w e r e v i s i b l e

the

cells

so their m o t i o n

and in

29

0

Figure

4.

50

100

M I G R A T I O N T I M E , MIN Electrophoretic migration profile (top) and distance migrated vs time (bottom) of human neuroblastoma (circles) and glial (dots) cells inserted as zones initially separated by 4 cm. The glial cell zone (1.4 x 103 cells) with a higher mobility, collided with the neuroblastoma cell zone (1.3 x 10 4 cells) after 30-50 min of migration, during which aggregates formed and migrated more slowly (indicated by x's). The two individual cell types continued to migrate with their original mobilities. The aggregates that formed during collision contained both cell types as indicated by their simultaneous presence in fraction 6 in the top figure, where the glial cell count has been multiplied by 5.

30 the field could be followed.

Their mobility was lower than

that of the individual cell types.

After the field was

switched off, and the cells were collected

from the tube in

2 cm increments, most of the cells of both types appeared the fraction that contained

in

the aggregates.

Discussion This study

has confirmed

previous findings(3) that

erythrocytes do not interact with one another affects electrophoretic mobility findings to very

in a way

and has extended

these

high cell concentrations, mixtures of cells

from different species, and conditions encountered preparative cell electrophoresis.

in

On the other hand, living

animal cells do interact, consistent with observations

that

earlier

involving microbial cells and leukocytes

(3,4).

Ack nowledgements We thank

Irja Johansson and Karin Elenbring

technical assistance.

for superb

This research was supported by the

International Union Against Cancer

(UICC) through a

Yamagiwa-Yoshida Visiting Cancer Research Fellowship and by the Universities Space Research Association Visiting

Scientist

Grant.

(USRA) through a

31 References 1.

H j e r t e n , S.: Free Zone E l e c t r o p h o r e s i s . Wik s e l l s Bktr., U p p s a l a , 1967.

2.

K a r l s s o n , E., Eaker, D. L., and P o r a t h , J.: B i o p h y s . A c t a 127, 505 (1966).

3.

H j e r t e n , S.: In Cell S e p a r a t i o n M e t h o d s (Ed. H. B l o e m e n d a l ) E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press, A m s t e r d a m , 1977, pp. 119-128.

4.

A g a r w a l , K. N. and H j e r t e n , S.: S u p p l . 93, 53 (1964).

5.

Boltz, R. C., Jr., and Todd, P.: Electrokinetic S e p a r a t i o n M e t h o d s (Eds. P. G. R i g h e t t i , C. J. van Oss, and J. V a n d e r h o f f ) E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l P r e s s , A m s t e r d a m , 1978, pp. 229-250.

6.

O m e n y i , S. N., S n y d e r , R. S., A b s o l o m , A. W. , and van Oss, C. J.: J. C o l l o i d 81, 420-409 (1981).

7.

Sriyder, R. S. , Rhodes, P. H. , H e r r e n , B. J., Miller, T. Y., Seaman, G. V. F., T o d d , P., K u n z e , M. E., and S a r n o f f , B. E.: E l e c t r o p h o r e s i s (submitted 1984).

8.

S a r n o f f , B. E., Kunze, M. E., and Todd, P.: A s t r o n a u t . Sei. _53, 139-148 (1983).

9.

T o d d , P.: In: C e l l E l e c t r o p h o r e s i s '84 (Eds. W. Schiitt, H. K l i n k m a n n ) . W a l t e r de G r u y t e r , B e r l i n , pp. 3-19 (1985).

Acta

Almqvist

and

Biochim.

Endocrinol.,

D. T., N e u m a n n , Interface Sei.

Adv.

VIDEO of

IMAGE C O R R E L A T I O N

ELECTROPHORESIS:

An

Intermediate

Stage

South Wales

Radiotherapy

and

Automation.

J . O . T . Deeley and A.J. Radiation Oncology United

Science

Bater

Laboratories,

Service,

Velindre H o s p i t a l ,

Cardiff,

CF4 7XL

Wales,

Kingdom.

Analytical

particle micro-electrophoresis

study surface charge c h a r a c t e r i s t i c s . requires

a small

d i l u t e sample of

strictly

electrophoretic measurements

has

long been used to

The t e c h n i q u e

particles.

only

Although

indicate the

electronic

charge d e n s i t y of a shell of ions a small d i s t a n c e from p a r t i c l e surface estimation increased

such m e a s u r e m e n t s

of the actual

have been used to p r o v i d e

s u r f a c e charge d e n s i t y .

membrane

biophysical

CO

measurements re-evaluation

in clinical

research

(see £ 3 ]

of a v a i l a b l e e q u i p m e n t .

of our e q u i p m e n t was s t i m u l a t e d by the announcements

the [2 !J

electrophoretic

) t h e r e has been a

Indeed, the Field and

development

Caspary

of the m a c r o p h a g e e l e c t r o p h o r e t i c m o b i l i t y

t e s t for c a n c e r C 4 > 5 U 1

use of

The e q u i p m e n t a v a i l a b l e

in the

s w o r k e d a d e q u a t e l y for short e x p e r i m e n t a l

f o u n d to be i n a p p r o p r i a t e

of an improved

(MEM)

early

runs but was

for the rapid t h r o u g h p u t of

day after day r e q u i r e d of a routine clinical detailed description

an

in

and e l e c t r o c h e m i c a l

p r o p e r t i e s t o g e t h e r with the potential

1970

With

a w a r e n e s s of the role of s u r f a c e charge

determining

the

samples

procedure.

A

micro-electrophoresis

a p p a r a t u s d e v e l o p e d at V e l i n d r e H o s p i t a l ( t h e m o d i f i e d

Rank

has been p r e s e n t e d

Briefly

by S u t h e r l a n d and P r i t c h a r d C6l] .

the a p p a r a t u s c o n s i s t s c h a m b e r of internal

of a r e c t a n g u l a r glass

d i m e n s i o n s 4.2mm high,

long.

Glass e l e c t r o d e

tubing

s l e e v e s to each end of the c h a m b e r .

electrophoresis

1mm d e e p and

vessels are c o n n e c t e d

mk3)

via

50mm

silicone

The e l e c t r o l y t e

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

in

34

the

electrode

chamber

vessels

by a s e m i - p e r m e a b l e

insert with a positive isolate

'0-rings' line

the

chamber

bath

circulating

with

vertically

up o v e r

(i) s t a b l e

substained for more small

use,

of

present

samples,

and

determining

television

(v)

adaptable,

The m o d i f i e d

for

during

last d e c a d e C 7 - 1 0 J

the

tool.

development

electrophoretic

of d e t e c t i n g

Rank .

mk3

In p a r t i c u l a r ,

of an a u t o m a t e d

used

Our

has

cancer

for

automatic

satisfactorily

micro-electrophoresis

means

reassemble

a

mobility.

to

of

suitable

facility,

laboratory

in t h e

and

(iii)

the manual

been

water-

designed

periods

has

in p a r t i c l e

research

involved

was

long

with and

and

directed

Cambridge)

u s e as a p o s s i b l e

a general

viewing

providing

insert

temperature

to d i s m a n t l e

(iv) e q u i p p e d of

taps

being

repairs,

the

plastic

PTFE

The a p p a r a t u s easy

of

The c h a m b e r

input flow

or for

affecting

of w o r k e r s

the

'drift' during

for ease

Bros.,

interest

its

from

electophoretic

(Rank

by a n u m b e r

the

on a

Rotaflow

t i p of

in a c o n s t a n t

chamber.

cleaning

without

assessment

been

the

and m o n i t o r

apparatus

water,

contents

supported

measurements.

(ii) q u i c k

of

at t h e

immersed

free

elaborate

preferably

from

and

volumes

camera

are

the

as s e a l s .

seal

during

vessels

from

membrane

acting

contact

electrode

be

is s e p a r a t e d

moved

to o n e we

of

have

means

of

mobility C 1 1 , 1 2 , 1 3 H .

Automat i on. The

standard

involves

a

method

glass

chamber across

is u s u a l l y

balanced

flow

any

the

return

location possible

the

of

(the

The

of

electric

The

camera

in a movement

and

in t h e

monitor

chamber

at a l o c a t i o n

chamber

the m i d d l e .

so-called

effect

particles

of t h e

down

of an

apart.

of f o c u s )

mobility

a particle

a television

depth

walls

flow

of

influence

distance

system.

a narrow

along

by t h e

under

a known

by m e a n s

optical

(with

at t h i s

To r e d u c e

it m o v e s points

observed

are f o c u s e d

flow

as two

to t h e

the fluid

electrophoretic

'time of f l i g h t ' m e a s u r e m e n t

field

coupled

for d e t e r m i n i n g

where

(endo-osmosis) The n e t

stationary

unidirectional

layer) fluid

is

fluid is

zero.

drift

it

35 is u s u a l

to

reverse

measurement distance

arid t i m e

electric

significance

it

a large

number

of c e l l s .

errors

that

from

conducting

The a p p r o a c h e s fall

into

spectroscopy light

to

three

tangential

D O

velocity

velocity

of t h e

of

and

television

image

individual

particles

C16,173

These

.

greatly

computer equipment system

which

measurements expense

necessary

The

the

Video

Figure

form

in

for

those

1 shows

the modified

which to

be

Correlation a general

Rank m k 3

determinations.

is a n e e d

The

with

and

and an

(iii)

general

scan that

to

stage

An

and

'add-on'

where

cannot

the manual

be

facility

operator

the

wishes

is to

measured.

Electrophoresis of t h e

apparatus

(VICE)

VICE system

usually

consists

of

used

for

a cell

of

existing

electrophoresis

techniques

in

they

extensive

intermediate

studies

the

of advance

albeit

operation.

where

the

field

require

the

to a

on

a considerable

low c o s t ,

where

which

the m o v e m e n t

mobility

for

at

retains

view

system

field

laser of

respect

is d e p e n d a n t

its m a n u a l

of m o r e

techniques

s h i f t of

a television

equipment

situations

particles

Image

image

examines

sophisticated

A system

Doppler

represent

and

many

Doppler field

laboratories

part

tedium

inhibited

Cl5jin

across

be f i t t e d ,

affecting

value

of t h e m o r e

justified. select

There can

without

is of

which

c o s t of

the

movements

transduction

of e l e c t r o p h o r e t i c

to t h e

laser

signal

same

studies.

in t h e e l e c t r i c

moving

the

in an e l e c t r i c

a particle

techniques

support.

automation

(i) the

image

reference

analysis

the d e t e r m i n a t i o n add

moving

particle

to t i m e

involved,

over the adequate

micro-electrophoresis

which measures (ii)

first

particle

probably

electrophoresis

by c e l l s

grating

has

categories:

polarity,

rotating

The t i m e occur

the

To e s t a b l i s h

is n e c e s s a r y

automating

broad

scattered

alternating

can

after

of t h e

direction.

associated workers

field

the m o v e m e n t

in t h e o p p o s i t e

statistical of

the

System.

connected manual

detector

and

to

36

Figure 1. A g e n e r a l v i e w of t h e v i d e o i m a g e c o r r e l a t i o n electrophoresis apparatus. (1) m i c r o e l e c t r o p h o r e s i s apparatus a n d t e l v i s i o n c a m e r a , (2) c e l l d e t e c t o r a n d c o r r e l a t i o n u n i t , (3) m i c r o c o m p u t e r a n d (4) t e l e v i s i o n m o n i t o r .

correlator (labelled

(Malvern 2

television Commodore The

in

Figure

camera

and

CBM-4032

presence

registered

of

as

Instruments)

inserted,

monitor.

in

raster

signal.

line

in a s h i f t

stored

interval,

the

appear

different

the

on

number

detected

of

The

scan

line,

system

which

crosses

detections 256

moved

lines.

Their

in

scan

each

of

register

are

cross-correlated

shift

register

and

the

cross-correlation

The

process

with

buffer.

continously

the

existing

coefficient The

position

channels.

updated buffer

is d i s p l a y e d (in

terms

of

as

The

is

TV

the

The

television

line

and

functions

C131.

offset)

in

a

an now

the are

and

second

in t h e

second

first stored

in

output integrated

cross-correlation

monitor of

and

in t h e

coefficient

repeated

fresh

contents on

those

scan

After

is d e t e c t e d

stored

detections

with

data.

a

in e a c h

distance

presence line

the

a

is

channels.

some

by

producing

of

register

output

line

cell

of

have

the

processes

number

shift

buffer

between

scan

the

unit

is c o n t r o l l e d

also

shift

an

128

a single

register

cells

detections

as

television

scan

characteristic is

any

in

The

microcomputer

a cell

the

1)

fitted

(Figure

the major

peak

2). of

37

Figure 2. D e t a i l of the t e l e v i s i o n m o n i t o r s h o w i n g the d i s p l a y of t h e c o r r e l a t i o n c o e f f i c i e n t . The b o l d v e r t i c a l l i n e is t h e reference television line. Detected cells are t a g g e d with a w h i t e m a r k e r f o r t h e i n f o r m a t i o n of t h e o p e r a t o r .

the

function

across line,

the the

represents

screen. mean

determined. most

arrangement field

are

consists

x40

adjusted

the

determinations direction.

satisfactory. direction

pH

run

the

fresh 50

focus

In o u r

on

the

electrode

vessels

25

the

of

are We

of

be

immediately

biological

optical

and

a

a

bright

long

working

of

good

contrast,

is

It

is

important

across has

the

been

cells (about

washed

and

have

found

steps The to

to

the

is

any

in

the

applied pH

and

refilled an

determine

using

is

each 20ml

interval for

function the

of

field

after

suitable

correlation

in

of

influence

electric

that

ensure

mobility

field

in t h e

25s)

be

to

lamp

quite

field

changes

To m i n i m i s e

a run

of

in

and

electric

the

cells.

after

similar

electrophoretic

runs

correlation

is

condenser

this

time

television

be

The

version)

moving

The

move

occur.

a short

examination analysed

when

electrolyte.

between

since

cells

the

can

system

condenser

image,

the

of

cells

objective.

prolonged

behaviour for

width

manual

cells

of

system.

monitor.

as

can

the

VICE

experience

However,

applied

buffered

frames

long

is d r i f t - f r e e

electrolyte

changes

generally

the

in t h e

are made

shift

detected

(as

television

during

line

manual

a suitable

system

electrode

of

the

until

that

one

of

immersion

on

the

the

water

obtained

one

that

water-immersed

distance

of

operation

to

mean

calibrating

mobility

The

respects

By

the

mean

of

of the can

38

KEY smoothed input data derived triangle —

fitted curve residual signal

MOBILITY V) F

o ~

a 2

lLotex porticiT] I 28* 4 0 00* (C V • 066 %) 1.0

IHotm RBC| U4ri 0.01* (C.V • I 4%)

i floe

i 0-8' |Sh«6p RBC 1 099° i O.OO" (C.V. • 0.53 % I J

b

06

Eagle's MEM

¡sa oJ 9 (*) h (**) d*

(9)

J

!

e *

s

/ P

(*)e'Z*dx

(*)

v s

'c* /f tx>cosh

(*x)dix

(AO)

For the special case of constant fixed charge density the electroosmotic velocity profile v

is:

j-cosh[(x~S)z]+ cosh(zx)e'

es

i dJ

x^S

with

0 « a.y% s r rz-1 clsh(„S)

7

2 2 -rrrr cosh (at) * * /« '

'

+ «/***

tmkM)[tmmhM*

1, ,r Tt corh(*J) a/x]J

v

e + e~*S['C«hk*)+c*il,Mt L - ta»h (cuf) sihh ( , l/|s](mM)-'

•

Fig 1 Effect of ionic surfactants on the kinetics of glycerol-3-phosphate dehydrogenase in mitochondria from insect muscles (a) and monoamine oxidase in mitochondria from rat liver (B). Lineweaver-Burk plots. Oleate and cetyltrimethyl-ammonium bromide (CTAB) were added at concentrations of 68 jiM and 36-39 jiM, respectively. From (3,5). ned unchanged.

Such situation

is compatible with the

assump-

tion that the effect is due to changes of the local concentration of the substrate at the locus of the enzyme. Similar

results

microsomes somes,

were

obtained with

arylsulphatase

(Table I), acetylcholinesterase

dimethylaniline

oxidase

(3)

from liver

from brain synapto-

and glucose-6-phasphatase

(5) from liver microsomes. The effects of ionic amphiphiles and metal cations disappeared when

the

gent, the

membranes

e.g.

Lubrol

enzymes

again

into

indicates

became WX,

and

solubilized re-appeared

phospholipid

that

the

in

a non-ionic

upon

membranes

effect

was

due

deter-

incorporation of (liposomes).

to

the

This

action

on

the embrane and not on the enzyme protein. Assuming that the change of apparent K^ results from a change of

the

local

surface, the

concentration change

of

the

substrate

at the membrane

of the surface

potential

( A"Y

calculated from the following equation (1-3)

) can be

162

II

K m

AY

(2)

K m

it K and K i n d i c a t e the apparent M i c h a e l i s constants m m b e f o r e and a f t e r the t r e a t m e n t , r e s p e c t i v e l y . Such c a l c u l a -

where tions

made f o r

of A V

liver

microsomes show a f a i r l y

good agreement

values obtained in t h i s way w i t h those c a l c u l a t e d from

the binding of anilinonaphthalene sulphonate

(Table

II).

Table I . E f f e c t of i o n i c s u r f a c t a n t s on the apparent K value of a r y l s u l p h a t a s e in r a t l i v e r microsomes. From Ionic character

Surfactant

K m

None

(mM)

2,0

Palmitoyl-CoA,

40 jiM

Dodecylsulphate, Oleate,

(3).

100 jiM

100 jiM

Cetyltrimethylammonium, Cetylpyridinium,

150 yM

150 jiM

anionic

2,2

anionic

3,0

anionic

9,9

cationic

1,5 1,2

cationic

Table I I . E f f e c t of i o n i c s u r f a c t a n t s on the s u r f a c e p o t e n t i a l of r a t l i v e r microsomes. Changes of the s u r f a c e p o t e n t i a l were c a l c u l a t e d from the d i s s o c i a t i o n constant of a n i linonaphthalene sulphonate ( K ^ ) and from the change of appar e n t K m values of a r y l s u l p h a t a s e . C a l c u l a t e d from data in Table I . From ( 3 )

calculated from K,

Added s u r f a c t a n t

Oleate,

100 jiM

12

Cetyltrimethylammonium, Cetylpyridinium,

150 jiM

150 nM

AY

(mV) calculated from K m 18

10

8

14

14

163

E f f e c t of

phosphorylation

Phosphorylation surface ried

of

charge o f

out

by

fragments w i t h

o f membrane

membrane membranes

(6,

7).

cellular

ATP i n

presence

curred. due t o t h e p r e s e n c e could

proteins

incubating the

be p o t e n t i a t e d

proteins

of

increases

negative

P h o s p h o r y l a t i o n was

car-

organelles or membrane 2+ o f Mg . The p r o c e s s o c -

endogenous

by a d d i t i o n

the

of

protein kinases

soluble

cytoplasmic

but ki-

nases . As

shown

proteins with

in

Fig.

anionic

thylaniline again

activity

substrate)

manifested

little

change Similar were

phosphorylation

the

oxidase

fects. teins

2,

decreased

of

also

by

^max-

o f microsomal

arylsulphatase

and i n c r e a s e d

(cationic a

of

change

the

activity

substrate). of

apparent

Dephosphorylation for

monoamine

K , of

oxidase

of

dime-

effect with

reversed

changes upon p h o s p h o r y l a t i o n observed

The

membrane (reacting

no

these

membrane (7)

and

was or efprogly-

Fig. 2 E f f e c t o f p h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n o f membrane p r o t e i n s on t h e k i n e t i c s o f a r y l s u l p h a t a s e ( A ) and d - i m e t h y l a n i l i n e o x i d a s e ( B ) i n r a t l i v e r microsomes. L i n e w e a v e r - B u r k p l o t s . D e p h o s p h o r y l a t i o n was c a r r i e d out by p o s t - i n c u b a t i o n f o r 15 min i n t h e p r e s e n c e o f EDTA. I n d i c a tions: •, control microsomes; o, after phosphorylation; A , a f t e r d e p h o s p h o r y l a t i o n . From ( 6 ) .

164

cerol-3-phosphate

dehydrogenase

published) in mitochondria.

(Famulski

These effects

and Wojtczak,

un-

disappeared after

solubilization of the membranes in non-ionic detergents, indicating that they were not of the enzyme

protein

due to a specific modification

but, rather,

to an unspecific action

on the membrane itself. A comparison of phorylation

of

values upon phosphorylation and dephosliver

microsomes,

calculated

from a change

of apparent K m values of two enzymes, with those measured by the binding of anilinonaphthalene

sulphonate

shows again an

excellent agreement (Table III).

Effect of changing phospholipid This

was

studied

solubilized corporated

in

reconstituted

in cholate into

composition systems.

Membranes were

and their integral proteins were in-

liposomes

of known

by dialyzing off the detergent

phospholipid composition

(8). In this way kinetic

pa-

rameters of several membrane enzymes could be related to the phospholipid

composition

of

the

membrane.

made from either phosphatidylcholine •phosphatidylserine phosphatidylcholine

(acidic with

were

(neutral phospholipid),

phospholipid),

either

Liposomes

anionic

or

a

mixture

of

(dicetylphosphate,

Table III. Effect of phosphorylation and dephosphorylation of membrane proteins on the surface potential of rat liver microsomes. A"^ was calculated from the binding of anilinonaphthalene sulphonate (K,) and from apparent K values of arylsulphatase and dimethylaniline oxidase. From f6). (mV) calculated from: K of arylsulpRatase

K of dimethylaniline oxidase

Treatment

K

Phosphorylation

-10

-11

-10

0

+5

0

Dephosphorylation

d

165

Table IV. E f f e c t of p h o s p h o l i p i d composition of liposomes on apparent K values of i n c o r p o r a t e d membrane enzymes. l i posomes were prepared from the f o l l o w i n g p h o s p h o l i p i d s : phosphatidylcholine (PC), phosphatidylserine (PS), phosphatid y l c h o l i n e c o n t a i n i n g 20 mol % d i c e t y l p h o s p h a t e (PC + DCP), p h o s p h a t i d y l c h o l i n e c o n t a i n i n g 30-40 mol % phosphatidic a c i d (PC + PA) and p h o s p h a t i d y l c h o l i n e c o n t a i n i n g 30-40 mol % t r i d e c y l a m i n e (PC + TDA). Prom ( 9 ) . Phospholipid

Apparent K^ values

composition

arylsulphatase

PC

(mM)

monoamine oxidase

3.2

0.17

PS

0.14

PC + DCP

5.6

PC + PA

7.1

0.12

PC + TDA

2.0

0.24

phosphatidic

a c i d ) or c a t i o n i c

I t was observed depending the

were

the

those

containing

same enzyme

For

incorporated

increased

phospholipids

tridecylamine,

alone.

K values d i f f e r e d m of liposomes to which

enzymes

K^ was

(anionic)

additions.

apparent

composition

apparent

acidic

phatidylcholine

that

incorporated.

substrates

containing in

IV)

on phospholipid

enzymes

anionic

(Table

(tridecylamine)

into

as

reacting

with

in

liposomes

and

decreased

compared

with

Km of

liposomes made from phos-

A reversed

r e l a t i o n s h i p was

f o r enzymes r e a c t i n g w i t h c a t i o n i c

observed

substrates.

Conclusion Studies

presented

ties

enzymes

of

enced changes are

by of

the

surface

the

produced

here

located

by

have

revealed

in

biological

potential.

surface factors

potential likely

We

that

kinetic

membranes have

also

of i n t r a c e l l u l a r to

occur

proper-

are

shown t h a t membranes

in a l i v i n g

as f o r example changes of the l e v e l of n a t u r a l

influ-

cell,

amphiphiles

166

and divalent metal cations, phosphorylation of membrane teins should and

and alterations also

be

pathogens

taken may

of their phospholipid into have

consideration a

similar

pro-

composition. It

that certain drugs

effect.

Therefore, the

surface potential of cellular membranes should be considered as one of the

controlling

factors

of membrane-bound enzymes

in vivo.

References 1. Katchalski, E., Silman, I., Goldman, R.: Adv. Enzymol. 34. 445-536 (1971). 2. Goldstein, L.. Levin, Y., Katchalski, E.: Biochemistry 3, 1913-1919 (1964). 3. Wojtczak, L., Nalecz, M.J.: Eur. J. Biochem. 94, 99-107 (1979). 4. Haynes, D.H.: J. Membrane Biol. 17, 341-366 (1974). 5. Nalecz, M.J., Wojtczak, L.: Postepy Biochem. 28, 191-225 (1982) /in Polish/-

(Warsaw)

6. Famulski, K.S. Nalecz, M.J., Wojtczak, L.: FEBS Lett. 103, 260-264 (1979). 7. Famulski, K.S., Nalecz. M.J., Wojtczak, L.: FEBS Lett. 157, 124-128 (1983). 8. Ragan, C.I., Racker, E.: J. Biol. Chem. 248. 6876-6884 (1973). 9- Nalecz, M.J., Zborowski, J., Famulski, K.S., Wojtczak, L.: Eur, J. Biochem. 11_2, 75-80 (1980).

SURFACE CHARGE DENSITY OF PURPLE MEMBRANE

Nezabravka

FRAGMENTS

Popdimitrova

D e p a r t m e n t of P h y s i c s a n d B i o p h y s i c s , M e d i c a l 1431 S o f i a , B u l g a r i a Maria

Academy

Kantcheva

D e p a r t m e n t of B i o p h y s i c s , B i o l o g i c a l F a c u l t y , S o f i a U n i v e r s i t y 1421 S o f i a , B u l g a r i a Zs.

Dancshazy

I n s t i t u t e of B i o p h y c i c s , B i o l o g i c a l R e s e a r c h H u n g a r i a n A c a d e m y of S c i e n c e s Szeged, Hungary Stoyl

Center,

Stoylov

I n s t i t u t e of P h y s i c a l C h e m i s t r y , B u l g a r i a n A c a d e m y of 1040 Sofia, B u l g a r i a

Sciences

Introduction Purple membranes m a membrane

are f o u n d as i r r e g u l a r r e g i o n s

of H a l o b a c t e r i u m ,

in the

a n d c o n t a i n only one

c o m p o n e n t , b a c t e r i o r h o d o p s i n , w h i c h acts as a

plas-

protein

light-driven

proton pump

(1) (Fig. 1). P r o t e i n m o l e c u l e s

meric units

form a h e x a g o n a l l a t t i c e in the p u r p l e

o r g a n i z e d in

E a c h p r o t e i n m o l e c u l e is f o l d e d into s e v e n closely

tri-

membrane. packed

o C - h e l i c e s that pass t r a n s v e r s e l y t h r o u g h the l i p i d

bilayer.

B a c t e r i o r h o d o p s i n has its N - t e r m i n a l r e g i o n e x p o s e d to the c y t o p l a s m i c s i d e . The t r a n s v e r s a l m o l e c u l a r a s y m m e t r y

obser-

v e d for p r o t e i n a n d l i p i d m e m b r a n e c o m p o n e n t s gives rise

to

a d i f f e r e n t s u r f a c e c h a r g e on e a c h side of the m e m b r a n e .

The

n e g a t i v e s u r f a c e charge d e n s i t y on the t w o s u r f a c e s p u r p l e m e m b r a n e d e m o n s t r a t e d q u a l i t a t i v e l y by c a t i o n

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

of the ferritin

168

HALOBACTERIUM

* oYJJhi

&

....

PURPLE

MEMBRANE

OUT

J

BACTERIORHODOPSINE C0 0 H

OUT P i g . 1 . Schematic drawings o f : a / Halobacterium redrawn from ( 2 ) ; b/ t h r e e - d i m e n s i o n a l s t r u c t u r e of the purple membrane derived from image p r o c e s s e d p i c t u r e s ( 3 ) ; c / s i n g l e b a c t e r i o r h o d o p s i n molecule.

169 binding and electron microscopy trophoresis

(4). M o v i n g b o u n d a r y

elec-

i n d i c a t e d a l i g h t - i n d u c e d i n c r e a s e in the n e t

n e g a t i v e charge of p u r p l e m e m b r a n e f r a g m e n t s the r e s t r i c t e d d i m e n s i o n s

(5). B e c a u s e

of the p u r p l e m e m b r a n e

of

fragments

- the d i a m e t e r is a b o u t 500 n m and they are 5 n m t h i c k

-

s i n g l e f r a g m e n t s are n o t d e t e c t a b l e by l i g h t

microscopy.

P u r p l e m e m b r a n e s are s t a b l e at v e r y low s a l t

concentrations

of the s u s p e n d i n g m e d i u m . The p H value a n d i o n i c s t r e n g t h of the e l e c t r o l y t i c s o l u t i o n g o v e r n purple m e m b r a n e c l u s t e r m a t i o n . S u c h an a s s o c i a t i o n of purple m e m b r a n e c o u l d be m o n i t o r e d m i c r o s c o p i c a l l y

fragments

for c a l c u l a t i o n the

elec-

t r o p h o r e t i c m o b i l i t y in an e l e c t r i c f i e l d . The m o b i l i t y of p u r p l e m e m b r a n e depends

for-

value

clusters w i t h a d i a m e t e r of a b o u t 5 n m

on the e l e c t r i c double layer p r o p e r t i e s

(6). The

re-

l a t i v e l y l o w i s o e l e c t r i c p o i n t e s t a b l i s h e d at a b o u t p H 1.7 c o u l d be r e l a t e d to the l i p i d c o n t e n t of the m e m b r a n e

surface

(7).

Results and

Discussion

In order to o b t a i n a b e t t e r i n s i g h t into the p u r p l e s t r u c t u r e a n d light e n e r g y c o n v e r s i o n , the properties various

membrane

electrokinetic

of m e m b r a n e c l u s t e r s were i n v e s t i g a t e d r e l a t i v e

ionic e n v i r o n m e n t s . A b r i e f outline

cal c o n s i d e r a t i o n s

and assumptions

of the

follows:

1. P u r p l e m e m b r a n e c l u s t e r s are c o n s i d e r e d as

approximately

e l l i p t i c a l p a r t i c l e s h a v i n g a d i a m e t e r of 5 V1111) m u c h t h a n the t h i c k n e s s

of the i o n i c d o u b l e l a y e r . The

larger

relation

b e t w e e n e l e c t r o p h o r e t i c m o b i l i t y and z e t a - p o t e n t l a l

at the

h y d r o d y n a m i c plane of s h e a r c o u l d be c a l c u l a t e d a c c o r d i n g the H e l m h o l t z - S m o l u c h o w s k i

to

theoreti-

equation: (1)

w h e r e V) and D are a s s u m e d to be the b u l k v i s c o s i t y a n d the

to

170 dielectric constant

of the s u s p e n d i n g m e d i u m

respectively.

2. The s u r f a c e charge d e n s i t y , (5 , is c a l c u l a t e d w i t h a m o d i f i e d f o r m of the G o u y - C h a p m a n e q u a t i o n , as p r e s e n t e d Mehrishi

(9), at a t e m p e r a t u r e

of 25° C a n d an i o n i c

by

strength

of 0.14 N a C l . The s u r f a c e charge d e n s i t y is g i v e n by the following S = where

equation:

[ 1 + (1-oc)1/2

]

13.410-sinh

( £ /51.3)

(2)

is t h a t f r a c t i o n of the t o t a l s p a c e on the

surface

w h i c h is n o t a v a i l a b l e to c o u n t e r ions. The value of ) (jiC/cni )

Source

Particle electrophoresis pH = 7.0

Thylakoids

- 1 .00

(12

)

Parti cle electrophoresis pH = 8.0

Thylakoids

- 1 .20

(13

)

Laser Doppler velocimetry pH = 7.0

Broken chloroplasts

- 0 .54

(14

)

Particle electrophores is pH ^ 7.0

Broken chloroplasts

- 1 .00

(15

)

Particle electrophoresis pH = 6.4

Broken dark chloroplasts light

- 0 .86 - 1 .10

(16

)

177 some t h y l a k o i d m e m b r a n e g a l a c t o l i p i d s membrane surface

sis are s u p p o r t e d by r e s u l t s methods

are e x p o s e d at the

(18). The d a t a from p a r t i c l e

electrophore-

o b t a i n e d w i t h a n u m b e r of

of e x a m i n i n g s u f a c e c h a r g e d e n s i t y s u c h as

fluorescence

induction, fluorescence

fluorescence quenching

change,

other

EPR,

absorption,

etc.

Making certain assumptions, Barber

(5) a p p l i e d the

Poisson-

B o l t z m a n n a p p r o a c h a d o p t e d in the G o u y - C h a p m a n t h e o r y to the t h y l a k o i d m e m b r a n e s u r f a c e a n d p r e d i c t e d in terms the d i f f e r e n t s c r e e n i n g effects

qualitative

o b t a i n e d by the

addi-

t i o n of m o n o - and d i v a l e n t c a t i o n s . C o m p u t e r a n a l y s i s d a t a c o n c e r n i n g changes in the s p a c e s u r f a c e charge c o u l d be a membrane

u s e f u l i n d i c a t i o n of the b e h a v i o u r

components

of

of

density

charged

in the i n t r i n s i c e l e c t r i c f i e l d of the

m e m b r a n e . The p r e s e n t a t i o n of the e l e c t r i c f i e l d of the membrane components

in the i n t r i n s i c e l e c t r i c f i e l d of the

m e m b r a n e . The p r e s e n t a t i o n

of the e l e c t r i c p o t e n t i a l

profi-

le of the t h y l a k o i d m e m b r a n e p r o v i d e s some i n t e r e s t i n g f o r m a t i o n a b o u t the s p e c i f i c b e h a v i o u r of c h a r g e d complexes

in the p r o c e s s of m e m b r a n e

Our i n v e s t i g a t i o n s

f o c u s e d on two

functional

in-

membrane

activity.

points:

1. Is the e n e r g i z a t i o n of the t h y l a k o i d m e m b r a n e by p h o t o n a d s o r p t i o n r e l a t e d to changes in the s u r f a c e charge s i t y w h i c h can be d e t e c t e d by p a r t i c l e

den-

electrophoresis

measurements? 2. W h a t are the o p t i m a l e x p e r i m e n t a l

conditions

for

investi-

g a t i n g the s t a b i l i z i n g e f f e c t of the s u r f a c e charge s i t y on the f u n c t i o n of the

den-

chloroplast?

There are c o n f l i c t i n g r e p o r t s a b o u t the e x i s t e n c e a n d t e n t of changes in s u r f a c e charge d e n s i t y r e s u l t i n g

ex-

from

the a b s o r p t i o n of p h o t o n s . N o b e l and Mel found a s m a l l crease in the e l e c t r o p h o r e t i c m o b i l i t y branes

(7). N a k a t a n i et al.

of p e a t h y l a k o i d

(8) f a i l e d to d e t e c t a n y

et al.

(9) w h o r e p o r t e d that

mem-

signi-

f i c a n t e f f e c t of l i g h t on the e l e c t r o p h o r e t i c m o b i l i t y , c o n t r a s t to S c h a p e n d o u k

in-

in

illu-

178 m i n a t i o n h a d a g r e a t e f f e c t on the e l e c t r o p h o r e t i c of

mobility

chloroplasts.

In our e x p e r i m e n t s s t i m u l a t i o n of the e l e c t r o p h o r e t i c by i l l u m i n a t i o n is a b o u t 20 to 30 % a n d d e p e n d s the s u s p e n d i n g m e d i u m

mobility

on the p H of

(16). By r e c o r d i n g the s t i m u l a t i o n

by

l i g h t of the s u r f a c e charge d e n s i t y of the m e m b r a n e a pH v a l u e of 6.5 was f o u n d to be o p t i m u m . The s p e c i f i c e l e c t r o n p o r t i n h i b i t o r DMCU as well as the u n c o u p l e r s

of

trans-

photophospho-

r i l a t i o n CCCP and g r a m i c i d i n e l i m i n a t e d the p h o t o - i n d u c e d m u l a t i o n of the s u r f a c e charge d e n s i t y

sti-

(unpublished data).

is s u g g e s t e d that these p h o t o - i n d u c e d e f f e c t s are s t r o n g l y l a t e d to p r o t o n m o v e m e n t across the m e m b r a n e , r e s u l t i n g

It re-

in

changes in the s u r f a c e and t r a n s m e m b r a n e p o t e n t i a l . Fig. 1 shows the p o s s i b l e r e a r r a n g e m e n t s

of t h y l a k o i d m e m b r a n e

n e n t s , due to the p h o t o - i n d u c e d e l e c t r o c h e m i c a l p r o t o n The c o n f o r m a t i o n a l r e a r r a n g e m e n t tein-lipid interactions

i n c l u d e d changes in the

and in the s u b s e q u e n t t r a n s v e r s a l

m e n t of the i n t r i n s i c p r o t e i n s . The pH d e p e n d e n c e cular rearrangement

compogradient. promove-

of s u c h m o l e -

is p r o b a b l y due to the b a l a n c e of

ionogenic

F i g . 1 A m o d e l for the p o s s i b l e t r a n s v e r s e m o v e m e n t of t h y l a k o i d m e m b r a n e p r o t e i n c o m p o n e n t s a n d changes in the s u r f a c e charge d e n s i t y i n d u c e d by the p h o t o s y n t h e t i c r e a c t i o n

179

groups determining the net negative charge of the chloroplast membrane surface. If photo-induced acidification of the intrathylakoid phase occurs, the isoelectric point for inner membrane proteins is attained due to neutralization of the surface ionogenic groups. An increase in the hydrophobic properties of the surface leads to conformational changes, and protein components escape from the inner surface deeper into the lipid matrix. The transversal motion of the intrinsic protein molecules is associated with the increased exposure of the external surface charge groups and subsequently increase the surface charge density. The mechanism suggested does not exclude the possibility of other light induced conformational changes related to surface charge density effects . These could include the lateral diffusion of negatively charged molecules like the phosphoryla-

A - stacked membrane B - unstacked membrane

Fig. 2 A model of the lateral diffusion of proteins and membrane unstacking resulting in the modification of surface charge density • - PS II pigment protein; Q, D -LHC- light harvesting complex; 0-PS I pigment protein; g -coupling factor; P-phosphorylated light harvesting complex.

180 t e d light h a r v e s t i n g c o m p l e x of P h o t o s y s t e m II

(LHCP-PSII)

(Fig. 2). The light d r i v e n p r o t o n g r a d i e n t o b s e r v e d , w h i c h is due to e l e c t r o n t r a n s p o r t and t h y l a k o i d m e m b r a n e

swelling,

causes a s i g n i f i c a n t d e c r e a s e in the a p p r e s s e d a r e a of the m e m b r a n e . As s u g g e s t e d by B a r b e r t i o n of L H C P - P S I I - c o m p l e x e s

(5) this leads to

, lateral diffusion, and

z a t i o n of LHCP in the n o n a p p r e s s e d and s t r o m a l Such rearrangements

dissociarandomi-

thylakoids.

c o u l d be r e l a t e d to the a d d i t i o n a l

ve s u r f a c e charge d e t e c t e d in the e l e c t r o p h o r e t i c a l l y s i b l e c h l o r o p l a s t r e g i o n . I n v e s t i g a t i n g the time

negatiacces-

dependence

of the light i n d u c e d e l e c t r o p h o r e t i c m o b i l i t y s t i m u l a t i o n two d i f f e r e n t t e m p e r a t u r e s , we f o u n d t h a t a low temperature

of 4° C is m o r e c o n v e n i e n t

at

incubation

for m a i n t a i n i n g

e f f e c t . The d a t a were in line w i t h the fact that low tures p r e s e r v e the f u n c t i o n a l a c t i v i t y of i s o l a t e d

this

tempera-

chloro-

plasts. In our e x p e r i m e n t s

c h l o r o p l a s t f u n c t i o n a l a c t i v i t y was

red using delayed fluorescence measurements

monito-

owing to the

back

r e a c t i o n b e t w e e n the p r i m a r y e l e c t r o n d o n o r in p h o t o s y s t e m and one of the p r i m a r y e l e c t r o n a c c e p t o r s . An e x p o n e n t i a l p e n d e n c e was o b s e r v e d b e t w e e n d e l a y e d f l u o r e s c e n c e and the e l e c t r o n p o t e n t i a l d i f f e r e n c e

de-

intensity

(18,19). The t h e o r y

the e l e c t r i c a l double layer a p p l i e d to t h y l a k o i d

phyll fluorescence, thylakoid stacking and electron to be pH d e p e n d e n t w h e n the d e g r e e of d e l a y e d

of

membranes

e x p l a i n s the role of the s u r f a c e charge in c o n t r o l l i n g (4). The in v i t r o a g e i n g of i s o l a t e d c h l o r o p l a s t s

chloro-

transport

was

found

fluorescence

i n t e n s i t y was t a k e n i n t o a c c o u n t . W h e n the d e n s i t y of the face charge is m o d i f i e d w i t h OA a n d CTAB, the d e g r e e d e n c e is g r e a t l y a f f e c t e d , but a pH value

of

sur-

depen-

of a b o u t 6.0 was

f o u n d once m o r e to be b e s t for the c h l o r o p l a s t activity

II

functional

(17,20).

T h e r e is some e v i d e n c e s u g g e s t i n g changes in the d e n s i t y the s u r f a c e

charge in v i v o d u r i n g n o r m a l p l a s t i d

(11). S t o k i n g a n d F r a n c e s c h i r e p o r t e d t h a t the

of

development

electrophore-

181

tic mobility increases during plastid maturation.

Summarizing

our i n f o r m a t i o n a b o u t the role p l a y e d by the d e n s i t y of the c h l o r o p l a s t s u r f a c e charge, it can be s t a t e d t h a t the

inter-

p l a y b e t w e e n s u r f a c e c h a r g e , s u r f a c e p o t e n t i a l a n d the p e r t i e s of the s u s p e n d i n g m e d i u m - pH v a l u e , s a l t tion, temperature

and dielectric properties- responsible

the rate of m e t a b o l i c a n d f u n c t i o n a l a c t i v i t y in membranes

is s i g n i f i c a n t .

f o r w a r d by B a r b e r

pro-

concentrathylakoid

In v i e w of the basic c o n c e p t s

(4,5), the r e l a t i o n b e t w e e n s u r f a c e

t i a l a n d the rate of r e a c t i o n s t a k i n g p l a c e at the s u r f a c e s u g g e s t e d by I t o h et al.

put

poten-

membrane

(20) and this s h o r t

presenta-

t i o n of our data, we c o n f i r m t h a t the s u r f a c e p o t e n t i a l forms a r e g u l a t o r y

f u n c t i o n in the c o n v e r s i o n

of s o l a r

C o n t r o l a n d r e g u l a t i o n of the f u n c t i o n a l a c t i v i t y roplast will become increasingly

perenergy.

of the

c o m m o n in the n e a r

m a i n l y in a p p l i e d biote'chnological

for

chlo-

future,

investigations.

References 1. D o u c e , R., J o y a r d , J.: In "Methods in c h l o r p l a s t m o l e c u l a r b i o l o g y , E l s e v i e r B i o m e d i c a l p r e s s , 2 3 9 - 2 5 6 (1983) 2. B o g o r a d , L.: The J. of cell biol. 91., 3, 2 5 6 - 2 7 0

(1981).

3. A l l e n , C.F., T r o s p e r , T.Z., Park, R.B.: B i o c h e m . R e s . C o m m u n . 48, 9 0 7 - 1 0 0 5 (1972).

Biophys.

4. B a r b e r , J.: Ann. Rev. P l a n t P h y s i o l .

(1982).

33, 2 6 1 - 2 9 5

5. B a r b e r , J.: B i o c h i m . B i o p h y s . A c t a 594, 2 5 3 - 3 0 8 6. M e r c e r , F . V . , H o d g e , A . J . , H o p e , A . B . , M c L e a n , A u s t . J. Biol. Sci. 8, 1 - 1 8 (1955). 7. Nobel, P . S . , M e l , H.C.: A r c h . B i o c h e m . B i o p h y s .

695-702

(1966).

8. N a k a t a n i , H . Y . , B a r b e r , J. F o r r e s t e r , J.A.: B i o p h y s . A c t a 504, 2 1 5 - 2 2 5 (1978).

(1980). J.D.: 113,

Biochim.

9. S c h a p e n d o n k , A d . H . , H e m r i k a - W a g n e r , A . D . , T h e u v e n e t , R., W o n g F o n g Sang, A., V r e d e n b e r g , W . J . , K r a a y e n h o f , R.: Biochemistry

19, 1 9 2 2 - 1 9 2 7

(1980).

182 10. S t o c k i n g , R . C . , F r a n c e s c h i , V . R . : P l a n t P h y s i o l . 1 2 5 5 - 1 2 5 9 (1982).

70,

11. M a n s f i e l d , R.W., N a k a t a n i , H . Y . , B a r b e r , J. M a u r o , S. L o n n o y e , L.: FEBS L e t t . 1 3 7 , 1, 1 3 3 - 1 3 6 (1982). 12. Barber, J., C h o w , W . S . : F E B S Lett. 1 0 5 , 5 - 1 0 13. B a r b e r , J., M i l l s , J.: F E B S Lett. 68, 2 8 8 - 2 9 2

(1979). (1976).

14. V e r n o t t e , C., S o l i s , C., M o y a , I., Mais on, B., B r i a n t a i s , J., A r r i o , B., J o h a n n i n , G.: B i o c h i m . B i o p h y s . A c t a 725, 3 7 6 - 3 8 3 (1983). 15. M a s a m o t o , K., Itoh, S., N i s h i m u r a , M.: B i o c h i m . A c t a 591, 1 4 2 - 1 5 2 (1980).

Biophys.

16. G o l t z e v , V., K a n t c h e v a , M.: B i o e l e c t r o c h e m i s t r y B i o e n e r g e t i c s (1984) in p r e s s .

and

17. G o l t z e v , V., D o l t c h i n k o v a , V., K a n t c h e v a , M.: b i o p h y s . 94, 5 7 - 5 8 (1983).

Studia

18. F l e i s c h m a n n , (1971).

277-286

D. E.: P h o t o c h e m . P h o t o b i o l . 1 4 ,

19. V e n e d i c t o v , P . S . , G o l t z e v , V., S h i n k a r e v , V.: B i o p h y s . A c t a 593, 1 2 5 - 1 3 2 (1980).

Biochim.

20. V e n e d i c t o v , P.S., K r i v o s h e j e v a , A . A . : P l a n t a 160, (1984).

200-203

21. Itoh, S.N., T a m u r a , K., H a s h i m o t o , M., N i s h i m u r a , M.: The o x y g e n e v o l v i n g s y s t e m of p h o t o s y n t h e s i s , A c a d e m i c press J a p a n (1983).

ELECTROPHORETIC

BEHAVIOUR

OF

ESCHERICHIA

D E P E N D E N C E OF D I F F E R E N T S U R F A C E Gerhard Zingler, Wolfgang

COLI

STRAINS

IN

ANTIGENS

Nimmich

I n s t i t u t für M e d i z i n i s c h e M i k r o b i o l o g i e u n d E p i d e m i o l o g i e , Wilhelm-Pieck-Universität Rostock, DDR-2500 Rostock Uwe

Thomaneck

K l i n i k für Innere M e d i z i n , W i l h e l m - P i e c k - U n i v e r s i t ä t DDR-2500 Rostock

Rostock,

Introduction The

virulence

governed

of

uropathogenic

by a v a r i e t y

0-and K-antigens

(4,

5) a n d f i m b r i a e

7)

considered

serum

to

activity,

bactericidal

be

virulence

Colicin activity.

Many

Fimbriae than

(c.f. 8, 9 for +

carrying

(fim )

nonfimbriated

infections stant

(UTI)

(10,

E.coli

11).

addition

to to

have

been

made

(EPM) as a m a r k e r for (Fig.

1a)

isolated

Fimbriae

bac-

of h u m a n

are

from

induce

a

more

urinary

common tract

mannose-resiand

exhicells

(Fig.

(HA)

(6,

bit the a b i l i t y to a d h e r e s p e c i f i c a l l y t o u r o e p i t h e l i a l that

haemagglutination

(F)

and r e s i s t a n c e

attempts

in

is

antigens

review).

strains

among

factors,

V production,

to use the e l e c t r o p h o r e t i c m o b i l i t y terial virulence

strains

2, 3). S u r f a c e

certain

are

coli

(1,

s u c h as

haemolytic

Escherichia

of factors

erythrocytes

1b) (12). E a r l i e r studies by B R I N T O N et al. fim+

cells

The

purpose

EPM

can

thogenic

be

of

of our

used

to

E. coli

of

a

identify

(13) s h o w e d

l o w e r EPM t h a n fim

investigation was

E.coli strains,

the degree

have

to

cells.

find out w h e t h e r

two virulence

factors

in u r o p a -

viz. the f o r m a t i o n of K - a n t i g e n s

fimbriation.

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

the and

184

Fig. Fig. 1a

1a

Fig.

E l e c t r o n m i c r o g r a p h of a c e l l . (magn. x 39 000)

1b

fimbriate

strains :

All

from p a t i e n t s w i t h UTI. 37°C

on

C1212-77,

and were

kindly

Copenhagen, serotyping:

performed techniques

as

agar

C 1214-77,

Escherichia

stitut

strains

E . c o l i bacinterference

(14).

for

the

and

24

h.

isolated

The s t r a i n s

reference

supplied

were

s t r a i n s w e r e grown at

by

Klebsiella

strains

Dr.

18°C

C1254-79,

for

fimbrial

I d a j3rskov,

Interna-

Centre,

Statens

Serumin-

Denmark. Serotyping

described

counter-current

investigated

The E. c o l i

nutrient

antigens, tional

E.coli

and Methods

Bacterial

0:K:H

coated

Squamous e p i t h e l i a l c e l l with attached teria (enlargement x 800, Nomarski m i c r o s c o p e , l e i t z ) from ( 1 2 ) .

Material

and

gold

1b

For

of

the

previously, K

0-

and

using

identification

Immunoelectrophoresis

tube

H-antigens

agglutination

specific

were

was

phages

applied

(15,

and 16,

17). Haemagglutination : and were

guinea

pig

performed

phate-buffered mannose

(18).

Human

erythrocytes

erythrocytes with

dense

saline

(pH

were

used.

bacterial 7,2)

of

with

blood

group

Agglutination suspensions

the

addition

in of

0P^ tests phos1 %

185

Crossed

Immunoelectrophoresis :

E.coli

strains

crossed (19).

with

HA were

0RSKOV e t a l .

fimbrial

(F)

serologically

immunoelectrophoresis

The CIE t e s t

The (CIE)

of WEEKE (20,

as

antigens

analysed

described

of

using

priviously

21) was used as m o d i f i e d by

(22).

Cell

electrophoresis:

made

on c e l l s

three

times

which

electrophoretic

finally

treated

measurements

with

resuspended

were

f o r m a l i n , washed

phosphate-buffered 7 8 (pH 7 , 2 ) t o a f i n a l c o n c e n t r a t i o n of 10 -10 organisms

saline

and

The

had been

in

per ml. EPM measurements were made w i t h the Parmoquant 2 (VEB Carl

Zeiss

Jena,

GDR) in

measured 30-50 p a r t i c l e s

the at

manual

mode.

25°C and at

In

all

a current

The EPM values are r e l a t e d t o human red blood

cases we of

10 mA.

cells

(1 ,08 x 1 0 " 8 m 2 s _ 1 V " 1 ) .

Results and Discussion Urinary

isolates

of

E.coli

serotypes

were

examined

and 37°C,

respectively.

belonging

for

to

EPM a f t e r

different

0:K:H:F

cultivation

at

HA a b i l i t y was used t o i n d i c a t e presence of f i m b r i a e . tion bility

of

capsular

of s t r a i n s

The r e s u l t s in

Table

tions

antigens

concluded

from

Produc-

inagglutina-

in homologous a n t i - 0 serum.

obtained w i t h

1 .

was

18°C

Fig

2 shows

13 s e l e c t e d s t r a i n s the

are

presented

d i f f e r e n t frequency

distribu-

obtained a f t e r incubation at 18 and 37°C using,

in t h i s

case, the E. c o l i s t r a i n 11195 as an example. It

is

evident

that,

haemagglutination JAMES e t

al.

as

takes

(23),

reported place

in

in

the

the

we were unable t o

literature 18°C

(22),

cultures.

Like

f i n d any d i f f e r e n c e

EPM t h a t was r e l a t e d t o f i m b r i a t i o n ( T a b l e 1 ) .

These

no in

results

are c o n t r a d i c t o r y t o those presented by BRINT0N ( 1 3 ) . 0:K:H

serotypes

incubation

at

can 18°C.

be

agglutinated

Apparently

the

in

anti-0

capsular

serum

after

antigens

dealt

186

Table 1 E.coli 0:K:H serotypes and the effect of different growth temperatures on the haemagglutination (HA), the agglutination with anti-0 sera, and the electrophoretic mobility (EPM) S t r a i n

0:K:H

Growth

d e s i g n a t i o n

Serotype

temperature

HA

F i m b r i a -

A g g l u t i n a t i o n

type

w i t h

a n t i - 0

aera

BP*

+

SD

, 4 n - f l 2^-1 -1) I Xl U HI Y 0

C C )

075:K"

C125^-79

FZ

S

64

075:K

018: K

170

0

11195

0

C1214-77

0

C1212-77

N

103

FZ

N

5

12/1

162

2 : K

6:K

6 :K

5:H"

5: H

+

8

-

-

7

+

FS

8

-

-

7

-

-

8

5:H1

2:H1

2 : H1

7

-

-

8

-

-

7

•

8

-

7

>

8

-

-

7

-f

F7

a )

5521

1736

VP.

done:

a) b)

with

in

•

4 . 6

*

•

10,1

*

F1

1.83

•

2.9

*

0.89

•

8 . 0

S

1.83

*

4.9

+

0.13

•

5.6

*

1.84

•

4.1

t

+

0.52

•

7.2

*

1 .92 •

4.9

t

-

0

not

1.56

0.15

-

0

1 :K

1 ;H~

1 :K51:H~

mannoae-aena1 spontaneous

our

study

t

ND

-

7

1:H~

*

4.8

F?

8

1:K

10,0

*

1:H4

0

•

.54 +

1

1

2:K

+

+

F11

8

-

-

7

+

F9

8

-

-

7

6131

0.15

8.2

0

1:H~

X

7.4

-

1:K

*

9.5

+

-

+

2.8

1 .57 •

8

7

•

t

0.72

2 : H1

0

+

0.16 0.16

+

2: K

1:H~

+ +

-

0

OR:K

370

:H5

+

b)

.04

1

.88 •

+

F11

i

1

*

5 - 9 3.8

*

ND 1.89

•

4.4

t

0.54

1

•

6.4

1 .90 •

3.8

*

8

0.56

•

6.9

*

7

1.90

•

4.0

*

0.42

•

11.4

t

2.24

I

3.3

*

8

-

-

7

•

F9

8

-

-

7

•

F?

Ive

*

•

h a e m a g g l u t i n a t i o n

a g l u t l n a t l o n

were

in

s a l i n e

produced

s o l u t i o n

temperature

dependently.

This has already been suggested previously by other authors (24, 25, 26).

187

Pig. 2 The electrophoretic mobility distributions of E.coli cultures (strain 11195) grown at 18°C (A) and at 37°C (B). Further

evidence

noncapsular values

at

cultured

for

strain 18 and

at

37°C

our

conclusion

(C1254-79) 37°C, whereas is

2 -

is

(Table

the

1) has

fact

that the

identical EPM

the EPM of capsular strains

10 times

higher

than

the EPM of

those cultured at 18°C. We conclude that the EPM of capsular 37°C cultures is governed by the nature of their K-antigens, i.e.

by the kind

and number

of the charged groups of mole-

cules (Table 2). Table 2

The average EPM of E.coli strains in relation to the composition of their K-antigens K Antigen

Components

1,56

K

5

N-acetyl-glucosamine, Glucuronic acid

1 : 1

K

2

Galactose, Glycerolphosphate

1

: 1

1 ,90

K

1

N-acetyl-neuraminic Acid

2, 24

K 51

1

: 2

en co

EPM

Molar ratio

N-acetyl-glucosamine-phosphate, 0-acetyl

Composition of E.coli K-antigens from (27).

188

Conclusions 1. 2.

The

EPM of

dent

of

The

EPM

their of

than t h a t 3.

All

state

failed

after

Escherichia Further

of

cells to

cells

for

to

be

indepen-

was

higher

(2

to

10 t i m e s )

produce with

fimbrial

and

capsular

capsular

antigens

18°C. different

EPM v a l u e s .

investigations

be u s e f u l

found

ones.

i n c u b a t i o n at

coli

was

fimbriation.

capsular

have d i f f e r e n t 5.

organisms

of n o n - c a p s u l a r

strains

antigens 4.

capsular

will

detection

show i f

EPM d e t e r m i n a t i o n may-

of K - a n t i g e n s

(except

for

acidic

0-antigens) . 6.

From t h e a g g l u t i n a b i l i t y in for

anti-0

serum i t

of

0:K:H s t r a i n s

grown a t

may be c o n c l u d e d t o use

18°C

18°C-cultures

0-serotyping.

References 1.

M o l l b y , R. , K a l l e n i u s , G . , Korhonen, T . K . , Svenson, S.B. : I n f e c t i o n H , 68-72 ( 1 9 8 3 ) .

2.

Tschape, (1984).

3.

Block, L . H . , Georgopoulos , 43-48 ( 1 9 8 4 ) .

4.

0rskov,

5.

Jann, K . : ACS Symposium S e r i e s , No. 231, B a c t e r i a l L i p o polysaccharides: Structure, Synthesis, and B i o l o g i c a l Activities, Anderson, L . , Unger, F . M . ( e d s - ) , Copyright 1983 by t h e American Chemical S o c i e t y .

6.

Nimmich, W. , 258, 104-111

7.

0rskov, I . , 0 r s k o v , F. , B i r c h - A n d e r s e n , A . , Klemm, P . , Svanborg-Eden, C. . ' P r o t e i n a t t a c h m e n t factors (fimbriae in adhering E s c h e r i c h i a c o l i s t r a i n s ) . I n : Robbins, J . B . , H i l l , J . C . , S a d o f f , J . C . ( e d s . ) : Seminars i n i n f e c t i o u s D i s e a s e , V o l . I V , 97-103. Thieme, New Y o r k - S t u t t g a r t 1982

8.

Vransky,

H. ,

F.:

Prager,

J.

Infect.

Zingler, (1984).

V.K.:

R. :

Z.

Urol. A.:

D i s . 137,

G. , 0 r s k o v ,

Winberg,

Nephrol.

Miinch.

Die Z e l l e l e k t r o p h o r e s e ,

407-413

med. Wschr.

630-633

I. :

78,

J.,

126,

(1978).

Zbl.

In:

Bakt.

Beier,

Hyg.

W.

A

(ed.)

189

F o r t s c h r i t t e d e r e x p e r i m e n t e l l e n und t h e o r e t i s c h e n p h y s i k , Bd. 18, 81-84. Georg Thieme, L e i p z i g 1974. 9.

Bio-

S h e r b e t , G . V . : The b i o p h y s i c a l c h a r a c t e r i s a t i o n of t h e c e l l s u r f a c e , pp 78-86, Academic P r e s s , London-New Y o r k San F r a n c i s c o 1978

10. 0 r s k o v , I., 0rskov, F., Birch-Andersen, A., Kanamori, M., Svanborg-Eden, C . : Scand. J. i n f e c t . D i s . Suppl. 33, 18-25 ( 1 9 8 2 ) . 11.

Väisänen-Rhen, V. , E l o , J . , V ä i s ä n e n , E. , S i i t o n e n , A . , 0rskov, I., 0rskov, F., Svenson, S.B., Mäkelä, P . H . , Korhonen, T . K . : I n f e c t . Immun. 43, 149-155 ( 1 9 8 4 ) .

12.

Svanborg-Eden, S4-S69 ( 1 9 7 8 ) .

13.

Brinton jr., C.C., Buzzell, A., B i o p h y s . A c t a 15. 533-542 ( 1 9 5 4 ) .

14. 0 r s k o v , F . , 83, 595-600

C.:

Scand.

0rskov, (1975).

I.;

15. G r o s s , R . J . , C h e a s t y , 548-550 ( 1 9 7 7 ) . 16. K a i j s e r , (1984).

B. ,

Jodal,

Acta

T., U. :

J.

B.:

Clin.

Suppl.

Microbiol.

B. ,

18.

Evans, D . G . , Evans 18, 330-337 ( 1 9 7 7 ) .

D.J.,

Tjoa,

19.

0rskov, I., 0rskov, F., Immun. 27, 657-666 ( 1 9 8 0 ) .

Jann,

Scand. B

Microbiol.

K. :

Bacteriol.

W. : I n f e c t .

B i r c h - A n d e r s en,

6,

19_. 264-266

Microbiol.

Jann,

15,

M. A. : B i o c h i m .

J.Clin.

17. 0 r s k o v , I . , 0rskov, F., Rev. 41_, 667-710 ( 1 9 7 7 ) . jr.,

Dis.

Laufer,

Pathol.

Rowe, J.

infect.

A.:

Immun. Infect.

20. Weeke,

B.:

Scand.

J.

Immunol.

2,

Suppl.

1,

15-35

(1973).

21. Weeke,

B.:

Scand.

J.

Immunol.

2,

Suppl.

1,

47-56

(1973).

22.

0 r s k o v , I . , 0 r s k o v , F . : S e r o l o g y of E s c h e r i c h i a c o l i f i m b r i a e . I n : Hanson, L . A . , K a i l o s , P . , W e s t p h a l , 0 . ( e d s . ) : P r o g r e s s i n A l l e r g y , V o l . 33, 80-105. S. K a r g e r , B a s e l 1983.

23. James, A.M., List, 307-317 ( 1 9 6 6 ) . 24

C.F.:

Biochim.

Biophys.

0rskov, I . , J0rskov, F . , S o j k a , W . J . , Leach, P a t h o l . M i c r o b i o l . Scand. 53, 404-422 ( 1 9 6 1 ) .

25. Glynn,

A.A.,

Howard, H.U.:

C.J.:

Path.

Acta

J.M. : A c t a

Immunology 1_8 331-346 Microbiol.

¿6,

112,

215-222

(1970).

26.

Bertschinger,

(1970).

27.

Jann, K. , Jann, B. : The K a n t i g e n s of E s c h e r i c h i a c o l i . I n : Hanson, L . A . , K a i l o s , P . , Westphal, 0. ( e d s ) : P r o g r e s s i n A l l e r g y , V o l . 33, 53-79. S. K a r g e r , B a s e l 1983.

A METHOD FOR CHECKING THE FERMENTATION PROCESSES OF ESCHERICHIA COLI STRAINS WITH FIMBRIAL ANTIGENS

Peter Gallien, Uwe Kludas, Kurt Krüger Institut für Impfstoffe Dessau, Kombinat Veterinärimpfstoffe Dessau, DDR-4500 Dessau

Introduction The investigation of the electrophoretic properties of bacteria cells has gained in significance recently. As bacteria can cause diseases in human beings and animals, it is important to study the relationships between

^-potential and bacteria

toxicity and surface structures. For several years numerous authors have been engaged in morphological and genetic studies of nonflagellate bacterial parts, the fimbriae.

(1,2,3)

Fimbriae are consisting nearly completely of proteins. They include small quantities of polysaccharides and phosphorus. Classifications of these thread-shaped structures have been carried out mainly by Duguid (4) and Ottow (5). The settlement of a host by fimbriae-bearing microorganism takes place in by the lectines within the fimbriae tips host organism (6,7). This ability to adhere makes the fimbriae an important virulence factor (8-11). The formation of the fimbriae depends to a high degree on the conditions of cultivation (12-17). Optimal pH-values as well as amino acid concentrations of the nutrient medium cause among other things a satisfactory formation of fimbriae. The purpose of our investigations was to propose both an objectively qualitative and a numerically quantitative method of determination for fimbriae antigens in Escherichia coli strains with the aid of cell electrophoresis

(18).

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

192

Material and methods Escherichia coli strains with the fimbriae antigens F 41 , K 99, K 88 and 987 P have been gained from strain keeping cultures of the Institute for vaccines Dessau by fermentation. pure cultures samples were cell electrophoretically

Besides

examined

too, which were contaminated by strange germs (species of bacilli and cocci as well as grampositive little rods with formation of spores). Before each measurement the cell electrophoresis

equipment

"PARMOQUANT 2" (Carl Zeiss Jena, GDR) was calibrated by a standard sample.

Results and Discussions Samples from about 600 cultures of Escherichia coli strains were examined and measured by cell electrophoresis. Table 1 Characteristic EPM-values of the Escherichia coli strains with fimbriae antigens F 41, K 99, K 88 and 987 p Escherichia coli strain

EPM (10

01 01

K

(A)~

K 99 : F 41

0, 40

0101

k

(Ar

K 99

0, 60

0147

K 89 (B)

K 88

0,90

OS 2011

K (A)"

987 P

1,35

8

m2v

1

s

1

Table 2 Dependence of the fimbriae-bearing and the EPM-values of the cells on the stirring speed (model: OS 2011 : K (A) : 987 p) RPM

2000

2000 6000 10000 14000

,. . time in s 3 x 30 120 3 x 20 120 120

/ r,-8 2 - 1 EPM ( A1 0 m v s 1 ,30 1 ,25 1 . 20 1,10 1 , 04

— 1 \ standard ; , ... deviation

m

Pig.

1

E l e c t r o n m i c r o s c o p i c p i c t u r e s o f a a ) w e l l and b ) w o r s e f i m b r i a e b e a r i n g c e l l (OS 2011 : K ( A ) : 987 P )

Fig. fHufcrm tHh OH

It

i s known a t a s e r i e s

Escherichia the

coli

f o r m a t i o n of

- that

interest

fimbriae

(1,19).

determination

f o r the production

formation i t

- in p a r t i c u l a r

are immunogens. which

antibodies

quantitative

and f i m b r i a e v a c c i n e s ) . of

b a c t e r i a kinds

fimbriae

protective

f o r m a t i o n and t h e i r special

of

2

Scheme o f a ) w e l l and b ) worse f i m b r i a e bearing c e l l

The

at

induce

fimbriae

therefore

are

of

vaccines

(live

On t h e base o f

an e x a c t

determination

is

possible,

to

of

vaccines

f i x t h e end o f

the

194

»I

Fig.

so-

C e l l e l e c t r o p h o r e t i c a l l y checking o f group f e r m e n t a t i o n o f E s c h e richia coli strains with fimb r i a e a n t i g e n s F 41, K 99, K 88 and 987 p ( v a l u e s s e e t a b l e 1 )

30

il

n 10

3

II «.3

».t

Li

e.» EPM-1Ó*m'Y*t~ 4

EPM

Fig.

tilfrT

4

F e r m e n t a t i o n of E s c h e r i c h i a c o l i 0147: K 8 9 ( B ) : K 88. The e l e c t r o c y t o g r a m m shows s t r a n g e germ c o n t a m i n a t i o n s by ( 1 , 8 - 2 , 4 ) . 1 0 ~ 8 m 2 . v ~ 1 . s _ 1 f e r m e n t a t i o n i n t h a t way, priate

microorganisms

production This

of

of

of v a c c i n e s ,

fimbriae

containing

electron

optical

values

i s kept

vaccines

possibility

duality

that the a n t i g e n constant

from charge t o

standardisation

content

i n the

of

course

approof

the

charge.

determines

decisirely

which have been produced on t h e base bacteria.

It

investigations,

downwards s i g n i f y a w o r s e

c o u l d be p o i n t e d out that

deviations

of

f i m b r i a e bearing of

the of

by

t h e EPMthe

cells.

195

(Fig. cells (for

1a,

1b,

2 ) The g r a d u a t e d EPM v a l u e s

c o u l d be r e l a t e d instance

to t h e i r

0-or K-antigen

Owing t o t h e s i g n i f i c a n t w i t h the f i m b r i a e of

the s u r f a c e

Escherichia

surface

F 41,

EPM o f

K 88,

Escherichia

f e r m e n t a t i o n w i t h aid of

an e l e c t r o c y t o g r a m m

moreover o f f e r s s t i l l

strange

of

cultivation

a first

germ c o n t a m i n a t i o n

(Fig.

coli

cells

K 99 and 987 P t h e

The c e l l processes

structures

f o r m a t i o n can be watched a l s o

electrophoresis

coli

(20))

different

antigens

antigen

different

of

(Fig.

possibility

at

3)

during

for

growth

group

the

checking a

4).

Conclusions -

Application

of

f o r instance antigens

F 41,

available

t h e method f o r

for Escherichia K 88,

after

- Utilisation

of

c h e c k i n g group coli

Examination o f

- Utilisation r i n g the

of

fimbriae

K 99 and 987 P : measured d a t a

10 min and h i s t o g r a m s t h e method f o r the g e n e t i c

after

are

30 min.

checking t r e n d e s t i m a t i o n

fermentation processes with strange -

fermentations,

c e l l s w i t h the

germ

stability

t h e method f o r

of

of

contamination s t r a i n keeping

checking f i m b r i a e

cultures

formation

du-

fermentation

Re f e r e n c e s 1.

Gaastra, (1982)

W.,

de G r a a f ,

2.

Rhen, M. , Wahlström, E. , Korhonen, l e t t e r s 18, 227-232 ( 1 9 8 3 )

3.

To, S . C . - M . , 1-5 (1984)

4.

Duguid,

Moon,

J.P.:

Microbiol.

H.W. , Runnels,

Nature 215,

Evans, D . J . , Evans, 336-346, ( 1 9 7 9 ) J.C.G.:

F.K.:

D.G.,

Annal.

rev.

93-94

Rev.

46,

129-161

T . K . : FEMS m i c r o b i o l . P.L.:

Infec.

Immun.

43

(1967)

Du P o n t ,

H.L.:

microbiol.

29,

Infec.

5.

Ottow,

6.

M o r r i s , J . A . , Thorns, C . , S c o t t , A . C . , S o j k a , G . A . : I n f e c . Immun. 36, 1146-1153 ( 1 9 8 2 )

79-108

Immun.

23,

(1975)

W.J.,

Wells,

196

7. S e i l w o o d , R.: Biochim.

biophys.

a c t a 632,

326-335

8. Jones, G.W., Rutter, J.M.: J. gen. m i c r o b i o l . (1974) 9. I s a a c s o n ,

R.E., R i c h t e r ,

P.: J. Bact. 1 4 6 ,

10. I s a a c s o n , R.E., C o l m e n e r o , J., R i c h t e r , biol. letters 1_2, 2 2 9 - 2 3 2 (1981)

(1980)

84,

135-144

784-789

(1981)

P.: F E M S m i c r o -

11. K u s e c e k , B., W l o c h , H., M e r c e r , A., V a i s ä n e n , V. , P l u s c h k e , G., K o r h o n e n , T., A c h t m a n , M . : Infec. Immun. 43, 3 6 8 - 3 7 9 (1984) 12. F r a n c i s , D.H., R e m m e r s , G . A . , de Zeeuw, P . S . : J. m i c r o b i o l . 1_5, 1 8 1 - 1 8 3 (1982)

clin,

13. G i r a r d e a u , J.P., D u b o u r g u i e r , H.C., G o u e t , Ph.: J. m i c r o b i o l . 1 2 8 , 4 6 3 - 4 7 0 and 2 2 4 3 - 2 2 4 9 (1982) 14. de Graaf, F.K., K l a a s e n - P o o t , Immun. 30, 1 2 5 - 1 2 8 (1980) 15. de G r a a f , F.K., Klemm, 8 7 7 - 8 8 3 (1980)

P., v a n Hess, J . E . :

P., G a a s t r a , W.: Infec.

16. de G r a a f , F.K., R o o r d a , J.: Infec.

Immun.

gen. Infec.

Immun.

36, 7 5 1 - 7 5 8

17. M a s a l m e h , M . A . , Moon, H.W., R u n n e l s , P.L., S c h n e i d e r , Infec. Immun. 35, 3 0 5 - 3 1 3 (1982) 18. G a l l i e n , (1984)

P., K l u d a s , U., K r ü g e r , K.: J e n a Rev. 4,

19. Ketyi, J.: a c t a m i c r o b i o l . hung. 20. James, A.M., List, C . F . : Biochem. 317 (1966)

31, 1 - 2 5 biophys.

33, (1982) R.A.:

190-191

(1984) a c t a 112,

307-

I N F L U E N C E OF E S C H E R I C H I A C H A R G E OF

Joachim

COLI B A C T E R I A ON THE

LYMPHOCYTES

Rychly

K l i n i k für Innere M e d i z i n , DDR-2500 Rostock Gerd

SURFACE

Wilhelm-Pieck-Universität

Zingler

I n s t i t u t für M i k r o b i o l o g i e u n d E p i d e m i o l o g i e , Pieck-Universität DDR-2500 Rostock

Wilhelm-

Introduction The i n t e r a c t i o n of b a c t e r i a w i t h cells is of i n t e r e s t several reasons.

Adherence

of b a c t e r i a ,

for

e s p e c i a l l y to

lial cells, has b e e n i n v e s t i g a t e d w i t h r e g a r d to the

epithepathoge-

n i c i t y of b a c t e r i a (1,2). The fact t h a t b a c t e r i a can a l s o a d h e r e s p e c i f i c a l l y to l y m p h o c y t e s immunologists racterize

a r o u s e d the i n t e r e s t

in u t i l i z i n g this e f f e c t to i d e n t i f y a n d

lymphocyte subpopulations.

different strains lymphocytes,

or s p e c i e s

but m u t a n t s

It has b e e n s h o w n t h a t

of b a c t e r i a b i n d to

distinct

of the same s t r a i n of E . c o l i

a d h e r e to B cells only or to T cell s u b p o p u l a t i o n s We s t u d i e d the i n t e r a c t i o n of s t r a i n s

also

(3).

of E . c o l i c u l t u r e d at

different temperatures w i t h murine lymphocytes retic mobility

of cha-

by

electropho-

measurements.

M a t e r i a l and M e t h o d s Five s t r a i n s and serotypes

of E s c h e r i c h i a coli w e r e used. The

designation

of the b a c t e r i a are l i s t e d in Table 1. All

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

198

Table 1 Serotype

Strain designation 6131

01

K1

H°

F9

5521

01

K1

H°

F°

8278

01

K1

H7

F11

S370

01

K1

H"

F11

C1214-77

06

K2

H1

F1

strains were cultured both at 18° C and 37° C before the expeg riments. A suspension of 8 x 10 bacteria/ml phosphate buffered saline (PBS) was prepared for incubation with lymphocytes. Cell preparation

Spleen cells and thymocytes were taken from

CBA mice (3 month old), the organs were removed, and minced in Eagle medium, and the cells were filtered through gauze. Erythrocytes were removed by hemolysis with cold distilled water. The cells were then washed twice in phosphate buffered saline (PBS) and adjusted to a concentration of 8 x 10^ cells/ml. Preparation of lipopolysaccharide

lipopolysaccharide

(LPS)

was extracted from the surface of bacteria with phenol/water and further purified according to Gmeiner (4). Incubation and electrophoresis

1 ml of the splenocyte or

thymocyte suspension was centrifuged and the supernatant decanted. 1 ml of bacterial suspension was added to the pellet of spleen cells or thymocytes, and the cells were resuspended. This suspension was incubated at room .temperature for 30, 60, 90 and 120 min. Splenocytes (1 ml) were incubated with LPS for 45 min at room temperature. After incubation, the electrophoretic mobility (EPM) of the spleen cells and thymocytes was measured in an automatic cell electrophoresis equipment PARM0QUANT (YEB Carl Zeiss JENA). The EPM of 400 cells in each sample was measured automatically, and histograms were printed out.

199

Results and Discussion The electrophoretic distributions of mouse spleen cells reveal two populations representing B (low mobility cells) and T lymphocytes (high mobility cells) respectively (5). Bacteria cultured at 18" C increased the electrophoretic mobility of the fast cell population (T lymphocytes), but had no effect on B cells (Pig. 1). The histograms also indicate that charge was altered only in subpopulations of these cells. The effect observed was nearly identical with all strains of bacteria at 18° C. The maximal increase in EPM was reached when the cells were incubated for 90 min. In contrast, none of the five strains of bacteria influenced the EPM of spleen cells when cultured at 37° C. E. coli bacteria cultured at 18" C had no effect on the electrophoretic mobility of thymocytes, which suggests that changes in the lymphocyte surface during the maturation of T cells are important for the effect we detected in T splenocytes. Direct contact with the bacteria Fig. 1 Histograms of spleen cells after incubation with E. coli bacteria ( control, • with bacteria)

a) with 37° C bacteria (summary histogram with all 5 strains tested) 0.6 O.B 1.0 1.2 EPM

ir.

b) with 18° C bacteria (summary histogram, 30 min incubation) Q8

1.0

1.2 EPM

c) with 18' C bacteria (strain 8278 90 min incubation) 06

Of

(0

U EPM

200

seems t o be a p r e r e q u i s i t e charge

f o r the a l t e r a t i o n

of the s u r f a c e

because incubation of s p l e e n c e l l s w i t h supernatants

of the b a c t e r i a showed no e f f e c t . The i n c r e a s e in the EPM of T c e l l s must be caused by changes i n the c e l l s u r f a c e i t s e l f

and not by the s u r f a c e charge

of

p o s s i b l y adherent b a c t e r i a because the EPM of the 18° C b a c t e r i a was low ( 6 ) . responsible

It

i s not y e t c l e a r what the mechanisms are

f o r changing the EPM of c e l l s

w i t h the b a c t e r i a .

In g e n e r a l i t

interaction

i s suggested t h a t

adhere t o lymphocytes by carbohydrates f a c e which bind t o l e c t i n s

after

bacteria

of the b a c t e r i a l

on the lymphocyte ( 7 ) .

It

sur-

has been

shown t h a t the b a c t e r i a l o s e t h e i r capsule when c u l t u r e d at 18° C ( 6 ) .

T h e r e f o r e these b a c t e r i a possess a c c e s s i b l e

saccharide

chains which could contact the c e l l s ,

in

poly-

contrast

t o the b a c t e r i a c u l t u r e d at 37° C. However in our experiments

lipopolysaccharide

the b a c t e r i a l s u r f a c e did not i n c r e a s e , t r o p h o r e t i c m o b i l i t y of trations

(Fig.

B splenocytes

prepared from

but reduced, the at higher

concen-

2).

The r e s u l t s showed that E . c o l i b a c t e r i a under s p e c i a l conditions

elec-

(18° C) s e l e c t i v e l y

m o b i l i t y of T lymphocytes

i n f l u e n c e the

of mouse spleen

culture

electrophoretic

cells.

EPM

Fig.

2

EPM of T and B s p l e n o c y t e s i n dependence on the c o n c e n t r a t i o n of added LPS

1.0

0.9.

0 2

5

10

20

50

100

200 jug/ml LPS

201

Re ferences 1. Ellen, R.P., G i b b o n s , (1972).

R.J.: Infect.

Immun.

5,

826-830

2. S e i i n g e r , D.S., Julie, N., Reed, W.P., W i l l i a m s R.C.: S c i e n c e 201, 4 5 5 - 4 5 7 (1978). 3. B r a t e s c u , A., M a y e r , E.P., T e o d o r e s c u , M.: J. 131, 1 1 8 9 - 1 1 9 4 (1983). 4. G m e i n e r , J . : Europ. J. B i o c h e m . 5. S a b o l o v i c , (1972).

58, 6 2 1 - 6 2 6

Jr.,

Immunol.

(1975).

D., S a b o l o v i c , N., D u m o n t , F.: L a n c e t 2,

6. Z i n g l e r , G., T h o m a n e c k , U., N i m m i c h , W.: this

volume

7. L e h m a n n , V. , S t r e c k , H., M i n n e r , I., K r a m m e r , P.H., R u s c h m a n n , E. : Eur. J. Immunol. 1_0, 685 (1980).

927

FREE-FLOW

ELECTROPHORESIS

STUDY

OF THE

NON-PROTEIN

SUPPORTED

TRANSFER OF PHOSPHATIDE ACID AND POLYGLYCEROPHOSPHOLIPIDS

BETWEEN

ARTIFICIAL MEMBRANES

M a r c e l De Cuyper and M a r c e l

Joniau

K a t h o l i e k e U n i v e r s i t e i t L e u v e n , Campus K o r t r i j k , I n t e r d i s c i p l i n a i r Research Centrum, B-8500 K o r t r i j k ,

Belgium.

Introduction In

their

cytosolic

pioneering

proteins

between

natural

proved

that,

able

partition

to

low

transfer biological

in

of

this

1976,

absence

the

Martin of

and

found of

MacDonald

proteins,

Since

(1)

transfer

membranes

media.

lipids (2)

(commonly

related

(fig.

1).

unilamellar

As

spite the

of

: PA,

their

operational

authors

also

in

cardiolipin)

most

and the

which

mammalian

charged C

PA,

chains

14:0 acceptor in

transfer

event,

discretely

tissues PG a n d

(6).

The

bis(diacyl-

were i n v e s t i g a t e d

particles their

is

transfer

we

use

electrical are

as

small surface

monitored

by

electrophoresis.

phosphatidic

phosphatidylglycerol ; TES,

be

are

spontaneous

the non-protein-mediated

Alterations

during

continuous f r e e - f l o w

Abbreviations

in

carrying

donor

vesicles.

occurring

named

negatively all

might

also

phospholipids in

then,

that

5).

we c h e c k

mitochondria

glycero) phosphate,

myristoyl

In

Zilversmit

catalyze

mechanism

( 3 , 4,

paper,

DPG to

structurally

PG ,

to

and

artificial

aqueous

that present

localized

charge,

the

between

membranes

the

capacity

well

able

membranes.

in

Wirtz

phenomenon h a s become a f a s c i n a t i n g t o p i c and many

suggested In

are

even

solubility

work

; DPG,

acid

; PC,

phosphatidylcholine;

diphosphatidylglycerol

, 14:0 2-[ [ t r i s ( h y d r o x y m e t h y l ) m e t h y l ] a m i n o ] - e t h a n e s u l f o n i c

acid.

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

; C

204 0 R —C —0 —CH2 0 II R —C —0 —CH

phoshatidic

acid

0 II H£ — 0 — P — OH I 0" 0 R —C —0 —CH2

HJCOH

0 II R —C —0 —CH

H —C —OH

phosphatidylglycerol

0 II H£ — 0 — P — 0 — CH2 0"

R —C —0 —CH2

H£ — 0 — C — R

0 II R —C —0 —CH

0 ll HC —0 —C —R

b i s ( d i a c ylglycero)phosphate

Hf— 0 — P — 0 — CH2 0" 0 R —C —0 —CH2 0 » R —C —0 —CH

Hi —0 —P —CH2 I 0"

0 II diphoshatidylglycerol H —C —OH H —C —0—C —R 0

Hf —0 —P —0 —CH;

Fig.

Hi —0 —C —R

1 : S t r u c t u r a l f o r m u l a e of p h o s p h a t i d i c a c i d and p o l y g l y c e r o p h o s p h o l i p i d s u s e d in t h i s s t u d y . R r e p r e s e n t s CJ^.Q r e s i d u e s , e x c e p t in t h e c a s e of DPG where R r e p r e s e n t s an h e t e r o g e n e o u s m i x t u r e of f a t t y a c y l c h a i n s (see "Materials").

205 Experimental

Procedures

Lipids DiC As

PC and bovine h e a r t DPG were o b t a i n e d f r o m S i g m a ( U . S . A . ) . 14:0 shown by g a s - l i q u i d c h r o m a t o g r a p h y , t h e f a t t y a c i d d i s t r i b u t i o n

of

DPG was a s

, 1% ; C , 4% ; C , 2% ; C 16:0 16:1 18:0 18:1 6% ; C , 71% ; C , 6% ; C ,5%. S a t u r a t e d DPG w a s 18:2 18:3 20:0 o b t a i n e d by q u a n t i t a t i v e h y d r o g e n a t i o n of t h e u n s a t u r a t e d c h a i n s of DPG ( 7 ) .

follows:

C

DiC

and

PA and diC PG were s y n t h e t i z e d a c c o r d i n g 14:0 14:0 b i s ( d i C ^ ^ g l y c e r o l ) p h o s p h a t e f o l l o w i n g t h e Dang and

(9)

procedure.

Vesicle Small

unilamellar

sonication

The

vesicles

Results

vesicles,

procedure which a c t

and

Sonication presence

were

the

concentration) e t h a n e s u l f onic glycine

as

were as

:

(pH

(pH 6 . 0 ) ,

Stoffel

electrophoresis Heidelberg,

chamber

contained

except

for

the from

FRG)

t h e same

transfer a

10 mM p o t a s s i u m components 4.5,

(pH 7 . 0 , pH's

of

(,see

[

marker chloride

(each

in

C] (11).

in

a

the

5 mM

4- m or ph o1 i n e -

5.1), 7.2,

event

trace

by

(10).

7.4),

Tris

15 mM h y d r o g e n

(pH

chloride

8.4) or

used.

was as

described as

incubation

The r a t e the

performed

buffers

unbuffered

t h e TES b u f f e r .

calculated

with

described

non-exchangeable

4.1,

At e x t r e m e

the

were p r e p a r e d

as

electrophoresis

Free-flow (Desaga,

a

TES

15 mM p o t a s s i u m h y d r o x i d e were Free-flow

during

buffer

acetate

(pH 9 . 0 ) .

diameter

labeled

made up of

following

acid

30 nm in

acceptors

(Amersham)

media of

about

and were c h a r a c t e r i z e d

Discussion)

cholesteryloleate

in

(8)

preparation

the

and

to

constant

time-dependent

d i s t a n c e of t h e v e s i c l e s

(10).

on

(10).

used

the

changes

FF48

The

for

mixtures of

a

electrophoresis

vesicle which

preparation, were

transfer in

apparatus

the

injected

process

was

migration

206 R e s u l t s and

Discussion

T r a n s f e r of It

is

well

phenomena charge

a n i o n i c p h o s p h o l i p i d s a t n e u t r a l pH. established

are influenced

(13)

of

the

that

spontaneous

by the

physical

bilayers.

To

phospholipid

state

unify

transfer

( 1 2 ) and t h e

these

surface

conditions

in

the

d i f f e r e n t s e t - u p s of t h i s work, our e x p e r i m e n t s were c o n d u c t e d with diC PC v e s i c l e s a s r e c i p i e n t p a r t i c l e s , w h e r e a s t h e d o n o r s were 14:0 composed of the same m a t r i x l i p i d c o n t a i n i n g one of t h e n e g a t i v e l y charged group the

lipid

ratio

of

donors

9/1.

These

(fig.l)

between values

the

electric

field,

whereas

equimolar

23

are

PC v e s i c l e s ( 2 3 ° C ) . 14:0 At pH 7 . 0 (5mM T E S ) ,

chamber,

of

particles

reaction

kinetics.

be

visualized

donors,

convects

acceptors

(10,

practically

kinetic

process

4 times

faster

resulting

in

other

in

that

this

through

the

For

156 min,

higher

the

for

aqueous

14:0 whereas

We found

the

electric

peculiar

polyunsaturated

fatty

that

chains,

type and

first-order process

escapes and

can

from

absorbs

halftime-value

the

to

the

for

the

T h i s d i f f e r e n c e in t r a n s f e r

speed

-OH g r o u p s of

the

behaviour acyl

this

lipid donor

about

the

only

For

transfers

donors

field

side.

14:0 to true

t h e PG a n a l o g u e

degree DPG

phase

in

electrophoresis

PG,

lipid

PA t h e

immobile

on t h e a c i d i c

responsible

diC

pure

electrophoretograms

diC

anionic

glycero) phosphate natural

the

of

anionic

for

the anodic

14:0 each o t h e r a c c o r d i n g

:

a

near

PA and

by t h e p r e s e n c e of

With

complicated.

diC

found

free-flow

way, depending

With

( h a l f t i m e = 4 1 min).

with bis( diC ^ unchanged.

the elute

d o n o r s and a c c e p t o r s

follows

is

can be e x p l a i n e d PG,

in

head-

transition

are

donors

polar

on t h e

the a c c e p t o r s

approach

14).

phase

depending value

The mechanism

as

crystal

28°C

close

generated

donors.

acceptor

and

the

t h e mixed

mixtures

in t h e

a zwitterionic/acidic

to

may v a r y in a t i m e - d e p e n d e n t present

at

The g e l - t o - l i q u i d

varied

component. diC

species

in t h e

solvation

(15). is

and a c c e p t o r s

is

a certain due

as

In

part

to

remain

somewhat

more

approached

extent. by

the

each

We a s s u m e

peroxidation

detected

of

contrast,

electrophoretograms situation

to

polar

of

method

the of

207 Klein

(16).

rupture

of

This

the

reaction

chains

indeed

resulting

can

ultimately

in an enhanced

transfer

therefore preferred

to work with the hydrogenated

matter

Chapman

of

fact,

unsaturation its

water

of

a lipid

solubility

transfer

model

molecule and

thus

on

its

-

bis(diC^ ^glycerophosphate remains

completely

explanation

for

fixed this

the

The 33°C

effect

was

14:0 of

also

-

have

in

the

that

much

view

that

big

As a

degree

the

upon

aqueous

As

with

An

of

acceptable

both

acidic

hydrophobic

lipid-bilayer

DPG

moiety

dissociation.

pH

on

the

transfer

(fig. 2).

potency

Below

; between

pH 4.5

pH 4.5 and

of the

diC

14:0

PA

In this pH

rate

7.0 a constant

value

( - 160 min) is found, and above pH 7.2 an

region

the

halftime

three distinct zones in the transfer with

the

pure

PA

state

of

vesicles

respectively

ionization in

100-200

(18)].

The

is less

than

10

accurate

min.

curve can be roughly

of

the

phosphate

mM

sodium

relatively

[pK

for a These

correlated

and

chloride, are fast

at

transfer

m e a s u r e m e n t of the time course is limited by the time required run.

of

effect

of

bilayer.

their

We

hydrogenated

character

to

the

potency.

found

donor

rate.

a

PA as a function of pH.

decreases

for the halftimes

not

to

form of DPG.

that

transfer

barrier

also examined

progressively

should

also

in

is,

energy

Transfer of diC

we

supposes

immobile

tetraacylphospholipids increases

(17)

lead

pK

in

3,5 and

8-9

transfer

speed

of

form

originate

2-

d i C ^ ^PA from (ii)

as c o m p a r e d

: (i) larger an

diC^

^PA

by a lower group

hydration

H-bonds

(19).

transfer

upon

degree

(15).

progressively

the

electrostatic

enhanced

stabilizing

with

Also

of

(15)

decrease.

; and

(iii)

the

gradual

Similarly, decreasing

impact

may

repulsions at the m e m b r a n e

solvatation

the

monovalent

of

the

of

pH

the

below

of

retardation

undissociated

the low

paucity

4.5 may

electrostatic

Possibly, at

the

surface ;

be

of

caused

phosphate

repulsions

pH-values, only

will the

208 undissociated

form

molecules

the d o n o r s

phosphate diC

14:0

Fig.

2

of of

will

PC i s

2-3

transfer. may a d a p t (18)]

so

Indeed,

donors

and

of

diC

PC m a t r i x 14:0 c h a r a c t e r [pK of t h e

a cationic that

PA m o l e c u l e s w i l l become f i x e d ( s e e

: Transfer

the

the

diC

remaining

monovalent

13).

PA b e t w e e n diC PC/diC PA ( 9 : 1 ) 14:0 14:0 14:0 diC PC a c c e p t o r s a s a f u n c t i o n o f pH. 14:0

209 Physiological Significance From a biological standpoint, an interesting question is whether or not spontaneous transfer of phospholipids can be contributory in membrane biogenesis and maintenance. For instance, in the case of DPG biosynthesis, w h i c h e x c l u s i v e l y o c c u r s in the i n n e r mitochondrial membrane (6), the PA precursor has to move from the outer to the inner membrane. Since no transfer catalyzing proteins are found in the inter membrane space, Baranska and Wojtczak (5) claim that spontaneous transfer must occur. Our results suggest that pH-conditions may exert here an important role. Furthermore, our observations may also provide interesting background information concerning the distinct localization of D P G in the i n n e r mitochondrial m e m b r a n e . Most probably the presence of four hydrophobic chains in DPG make the molecule uncapable to transfer to other membrane entities. Possibilities

and

Limits

of

Free-Flow

Electrophoresis

Although the free-flow electrophoresis technique was originally developed for the separation of proteins, the many applications in the past ten years prove that it has become more important for the fractionation of biological particles (20). A few years ago, we also i n t r o d u c e d t h e t e c h n i q u e in the field of artificial phospholipid vesicles, which we could separate in a reproducible and non-destructive way according to their membrane charge (11). Since electrophoresis occurred in the absence of stabilizing carriers, we found that all vesicle types w e r e completely recovered. This contrasts to ion-exchange chromatography where satisfactory recovery of only one vesicle population is obtained on the condition that presaturation of the supporting matrix with lipid molecules has occurred (21). The applicability of free-flow electrophoresis may be limited, h o w e v e r , in the case of relatively fast kinetic processes where the separation time may be too long to allow accurate m e a s u r e m e n t s . Nevertheless, we s h o w e d here that electrokinetic measurements in the area of basic research can be successfully done by free-flow electrophoresis. Acknowledgments We thank Wim Noppe for technical assistance, Steven Van Cauwenberghe for preparing the manuscript and the Belgian F.G.W.O for financial support.

210 References 1.

Wirtz, K.W.A., 3596-3602 (1968).

2.

Martin, (1976).

F.J.,

MacDonald,

3. Stuhne-Sekalec, 407-413 (1978). 4. Stuhne-Sekalec, 137-143 (1982). 5. Baranska, (1984).

Zilversmit,

L.,

D.B.

R.C.

Stanacev,

L., Stanacev,

: J.

Biol.

: Biochemistry N.Z.

N.Z.

Chen. ¡5

: Can.

J.

6. Hostetler, K.J. : Phospholipids (Hawthorne, Eds.) Elsevier Biomedical Press, Amsterdam P.J.

: Proc. Natl.

8. Eibl, H., Kovatchev, S. : Methods J.M., Ed.) Vol.72, 632 639 (1981). 9. Dang, Q.Q., (1983).

Stoffel,

W.

10. De Cuyper, M., Joniau, 415-420 (1983).

in

: Chem.

M.,

Biochem.

A.M.,

73,

(Lowenstein,

Lipids

H.

go,

773, 23-31

Sei. U.S.A.

Enzymology

Phys.

Dangreau,

5g i

J.N., Ansell.G.B., p. 215-261 (1982).

Acad.

33,

33-40

: Biochemistry

22,

H. : Biochem. Biophys.

11. De Cuyper, M., Joniau, M., Dangreau, Res. Commun. 95, 1224-1230 (1980). 12. Massey, J.B , Gotto, 3630-3636 (1982).

321-327

: Can. J. Biochem.

J., Wojtczak, L. : Biochim. Biophys. Acta

7. Chapman, D., Quinn, 3971-3975 (1976).

243,

Pownall, H.J.

: Biochemistry

21,

13.

De Cuyper, M., Joniau, M., Engberts, J.B.F.N., Sudhölter, E.J.R. : Colloids and Surfaces 10, 313-319 (1984).

14.

Nichols, (1982).

J.W.,

Pagano,

R.E.

: Biochemistry

21,

1720-1726

15. Cevc, G. : Studia Biophysica 91, 45-52 (1982). 16. Klein, R.A.

: Biochim.

Biophys.

17. C h a p m a n , D. : Form and Function G.B., H a w t h o r n e , J.N., D a w s o n , Amsterdam, p. 140 (1973). 18.

Boggs,

J.M.

19. Elamrani, (1984).

: Can.

J.

Acta,

of Phospholipids R.M.C., E d s . )

Biochem.

K., Blume, A. : Biochim.

210, 486-489

58,

(Ansell, Elsevier,

755-770

Biophys. Acta

(1970).

(1980).

769, 578-584

20. Hannig, K. : Electrophoresis 3,235-243 (1982). 21.

McLean, (1981).

L.R.,

Phillips,

M.C.

: Biochemistry

20,

2893-2900

FREE FLOW ELECTROPHORESIS USED TO STUDY THE INTERACTION OF PIG BRAIN MICROTUBULAR

PROTEINS

WITH

PHOSPHOLIPID

MODEL

MEMBRANES

ROLE OF PHOSPHOLIPID COMPOSITION

M a r c e l J o n i a u and M a r c e l De C u y p e r Interdisciplinary Research I n s t i t u t e , Katholieke L e u v e n , Campus K o r t r i j k , B - 8 5 0 0 - K o r t r i j k , Belgium

Universiteit

Introduction Tubulin,

the

main c o n s t i t u e n t

h a s been r e p o r t e d & Berlin

(2)

and

Klausner

of

composed

pu're

of

the

tubulin

et

(DPPC)

only

(T

Subsequently,

t

at

al.

their

the

introduced

interaction

free

of

pig

sonicated

unilamellar

or

mixtures

their

(e.g.

DMPC) c o u l d

(DMPC)

amounts

of

glycerol

(DMPG), w e r e

further

report

of

an

(5,

different

phospholipid

on t h e

been

8). to

species,

Neutral, bind

interaction in

of

of

analogue

temperature

shown

to

occur

study

(MTP)

of

with

phospholipids phospholipids

Only when

sufficient

dimyristoylphosphatidy1occurred.

MTP w i t h

the

pure

the

amphionic

MTP.

e.g.

interaction

be

of

(A).

(SUV) composed

Also,

that

vesicles

(FFE) i n

will

membranes

dipalmitoyl

microtubular

7,

systematical

transition has

Caron

reported

proteins

added

membrane f l u i d i t y w i l l be

have

sonicated its

phase

a

cells,

(1).

model

brain 6,

charge. species

started with

flow e l e c t r o p h o r e s i s

be shown

anionic

and

interaction

vesicles

not

with

eukaryotic

membranes

These a u t h o r s

respective this

have

tubulin

interacts

o v e r a wide r a n g e of t e m p e r a t u r e s We h a v e

(3)

of

phospholipids.

strongly

in

a l s o in p l a s m a

interaction

dimy r i s t o y l p h o s p h a t i d y l c h o l i n e ).

microtubuli

t o be p r e s e n t

investigation brain

of

anionic

compared.

Here,

phospholipid series,

Finally,

discussed.

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

the

we

will

vesicles

different role

of

212 M a t e r i a l s and

1)

Methoas

Phospholipids.

DMPC

and

Most

DPPC

were

phosphatidylserine choline

phospholipids

(DOPC)

synthetic

(DMPS) and

-

was

were

were

prepared

from Amersham.

cardiolipin

Vesicle

prepared single, and

described

or

mM p h o s p h a t e

pH 6 . 4 ) .

interior

Excess

AcA 54 3)

pH 6 . 4 ,

phospholipids

vesicle (5).

were

of

two, 10

as

[14C]

liver,

[3H] DOPC

Avanti)

natural

and

products.

vesicles (MSE

(SUV)

150

W)

mg

of

(except

for

CL

:

15

in

sonication with

buffer

1 mM EGTA and

done

was removed

0.5

mM MgS0 4 o

by i n c o r p o r a t i n g mixture.

fluorescent

were

30

ml e l e c t r o p h o r e s i s

was

loaded

well

and

phospholipids

Labeling

was

DPPC

Serdary.

Biochemicals.

M sucrose,

the

from

(bovine

sonicating

min in

0.25

fluorochrome

[ H]—

Mostly,

pyranine

by g e l f i l t r a t i o n

(6 on

or the

mg/ml) Ultrogel

(LKB).

Microtubular

were

30

Dimyristoyl-

DPPG, a s

unilamellar

by

mixtures

for

were

[14C]

Sigma)

Small

(7),

equimolar

(SP-buffer

(9).

origin:

dioleoylphosphatidy1-

DMPG and

was a p r o d u c t o f P - L

stearylamine)

[l^C ]

heart,

;

(DOPG)

(DM P A ) ,

commercial

(Sigma).

Avanti

described

preparation.

as

from

Phosphatidylinositol

(bovine

Stearylamine 2)

as

were o f

products

glycerol

Dimyristoylphosphatidate DMPC,

used

prepared

described solution

proteins. by

(10)

in

two

and

Pig

cycles

stored

polymerization

brain of

in

microtubular

reversible

liquid

buffer

(MTP)

polymerization

nitrogen

(0.1

proteins

as

M phosphate

a ca.

as

10

mg/ml

pH 6 . 4 ,

1 mM

EGTA, 0 . 5 mM MgS0 4 and 1 mM GTP). 4)

Protein-vesicle

short 100

: freshly 000

buffer mixture

g),

interaction.

Was

cold-depolymerized

and 0 . 2 5

was added

to

was i n c u b a t e d

ml of 0.50 for

a

ml o f

1.5

done

MTP were mg/ml

as

described

ultracentrifuged

solution

a 3 mg/ml v e s i c l e

3 0 min. a t

in

(7). (lh

In at

electrophoresis

preparation.

23°C and a n a l y z e d

by f r e e

The flow

213 electrophoresis

on a

Electrophoresis

was

effluent

collected

recovered

DESAGA done

for

at

about

and a n a l y z e d

FF48

for

apparatus

80V/cm 30 min.

: right

(ca.

as

200

described

mA)

Forty-eight

angle light

and

23°C

fractions

scattering

R e s u l t s and

C]

rag/ml,

conc.

Discussion

interaction

pure

release

MTP, of

nearly

a final

position

1C),

(Fig.

concentration

interaction

was o b s e r v e d

(Fig

protein

the

shift

of

1:9 w e i g h t IF)

cathodic

with

of

towards

(c)

a

7,

using

mixture

(Fig

that

pronounced

SUV c o m p o s e d

indicated

(6,

1A)

the

ratio

SUV p o s i t i o n

before

experiment

components

(final

H]-radioactivity

light,

In a s i m i l a r

a

[

of

and run on FFE,

reported

t h e SUV ( F i g

Finally,

in

very

a

23°C,

as

in s c a t t e r e d

ID).

stearylamine

at

IB)

of

both

containing

to

(a)

pyranine.

(Fig.

unaffected

30 min. (Fig

:

an i n c r e a s e

entrapped

DMPC v e s i c l e s

by

anodic

(b)

for

occurred

was e v i d e n c e d

from t h e o r i g i n a l of

When MTP, a t

2.0 mg/ml), incubated

This

nra),

(405/512

were mixed with SUV composed of DMPG/DMPC ( 5 0 / 5 0 )

extensive 8).

were

radioactivity.

1 . E f f e c t of v e s i c l e c h a r g e . 0.5

and

(325

t r y p t o p h a n f l u o r e s c e n c e ( 2 8 0 / 3 3 6 nra), p y r a n i n e f l u o r e s c e n c e nra ) and [ 3 H ] - or [

(9).

remain

of

IE),

a

by a c o m p l e t e and a l a r g e

pure

DMPC strong

shift

of

increase

in

opacity. Thus,

the

slightly

with n e u t r a l (2,

3).

probably

phospholipids,

Their of

negatively

a

interaction purely

charged in

contrast

with

electrostatic

(because

physiologically

negatively

charged

phospholipids

play

a role as

evidenced

with

positively

interesting

effects

MTP o b v i o u s l y

origin.

other

puzzling.

by ( a )

vesicles

However,

the

of

is

more

interaction

Either,

the l e a k a g e

interact

publications

charged

more r e l e v a n t ) is

do not

with

hydrophobic the

vesicle

214

FRACTION NUMBER Fig.

1 : E l e c t r o p h e r o g r a m s o f SUV only ( A , C , E ) o r t h e i r mixtures ( B , D , F ) w i t h MTP i n a 1/4 w e i g h t r a t i o (MTP/SUV) a f t e r i n c u b a t i o n f o r 30 min a t 2 3 ° C . They were a n a l y z e d for l i g h t s c a t t e r i n g ( s o l i d l i n e ) , pyranine content ( d a s h e d ) , phospholipid radioactivity ( d o t t e d ) and tryptophan f l u o r e s c e n c e ( d o t , d a s h ) a s d e s c r i b e d i n t h e t e x t . The t h r e e SUV p r e p a r a t i o n s were : ( A , B ) DMPG/DMPC ( 5 0 / 5 0 ) , (C,D) DM PC, and ( E , F ) s t e a r y 1 a m i n e / D M P C (10/90 ; w/w). The p o s i t i o n o f f r e e MTP ( T ) i s i n d i c a t e d in e a c h c a s e .

215 interior,

and

(b)

changes

observations). mixture more

present The

as

-

(b)

interaction (7,

Effect

effort

linkers

of

the

the

cationic

: (a)

small

components amounts

a g r o u p of

of

latter

with

cationic

anionic

(unpublished present

free

microtubule-associated

in MTP and c o n t a i n i n g

reported 2)

Alternatively,

may a c t

likely

in membrane m i c r o v i s c o s i t y

in

Ca^+

or

proteins

(11).

has

been

8).

of

type.

In

an

t o s t u d y t h e i n f l u e n c e of t h e p o l a r head g r o u p q u a l i t y

on

the

interaction compared.

anionic

with

phospholipid

MTP,

a

series

polar

of

head

group

anionic

phospholipids

were

They were mixed in a 5 0 / 5 0 molar r a t i o with DMPC ( e x c e p t

f o r CL where e q u a l w e i g h t s were m i x e d ) , s o n i c a t e d and c o n f r o n t e d , a 2 mg/ml d i s p e r s i o n , occurred degree

to

varying

of

(a)

radioactive most

marker

pronounced

DMPG ( F i g

interpreted

SUV peak i s

of

substantiated, 2C)

and

aforementioned population content. partial,

MTP with

(these)

(MAP)

resp. PI

by g e l (Fig

systems

by

the

of

in

the

differ

with

(PI)

MTP.

2k), which

interaction

:

scatter

indicate labile,

Both

that

or i s

hypotheses 7).

without

the (CL)

either

its

weak

(triphasic

to are

Cardiolipin

original

of

the

due

amount

curves

and This

from

is

the

leakage

considerably

or

2B)

fluorescence.

and FFE ( 6 ,

interaction biphasic

is

The

DMPS ( F i g

light

may

SUV e i t h e r

of

DMPC).

and p y r a n i n e

with t r y p t o p h a n

: a substantial

the

for

strong

and

different shift

DMPA ( F i g

obtained

peak

filtration

2D)

either

In both c a s e s shown

present

with

t o the c a t h o d e ,

DMPG t o o ,

by t h e

[ H]

as

interaction

cathodic o

cases

indicate

resultant

for

remains,

as

the

observed

minor c o m p o n e n t s

(Fig

shifted

(b)

all

The p i c t u r e

The

evidenced

occurred

similarly

before.

and

(in

d o e s not c o r r e l a t e

occasionally

interaction

to

However,

radioactivity

2) a s

release,

IB).

completely

complete.

(Fig

interaction

can

is

extents

phospholipid

as

be

with 0 . 5 mg MTP a s

pyranine

behaved

is

-

(MAP)

components

vesicles

the

for

the

3 SUV

pyranine or CL)

only for

216

A

D

J' X

V

1 '

b.

*

\

C

V

1

\ \

y

B

V

V

J

1

/

A

A1 / /

a! v \ 1 \ T

r\ \iSTAr4c£

—

Fig. 2. The charge characteristic on w h i c h electrophoretic mobilities depend is not the true surface charge of a particle, expressed as a summation of all the surface ions, but the zeta potential. In an aqueous ionic medium bioparticles have a surrounding hydrated zone into which ions from the m e d i u m can enter [Stern layer]. These ions alter the true surface charge on the particle through electrostatic interactions. The zeta potential is the electrokinetic potential expressed at the outer perimeter of the Stern layer, the plane of shear between the particle and the ionic environment around.

Use of F F E in the study of platelet surface and intracellular membranes As most of y o u will know, the circulating blood platelet is discoid shaped rPLATE l] but w h e n damage occurs in a blood vessel wall it has the capacity to respond to haemostatic stimuli at its surface w h i c h "trigger" a wide range of morphological and metabolic changes.

One feature of this

morphological change is the rapid conversion from a disc to a sphere and following this the appearance of surface protuberances which develop into long thin spiny processes arising from the surface membrane ["PLATE 2]. These processes thicken and extend into long pseudopodia.

At the same

time the platelet releases a whole range of small and high molecular weight substances from intracellular granule stores b y a membrane

fusion

event [exocytosis] which ensures that constituents of the cytosol are not

229

Plate 1. Transmission electron micrograph of unactivated platelets discoid shape with many internal granules and membrane complexes.

showing

Plate 2. Scanning electron micrograph of activated platelets showing spiny processes and pseudopodia arising from their surfaces.

simultaneously lost.

Some of these specifically released components are

procoagulants which serve to attenuate the clotting cascade whilst others, such as the vasoactive amine [5 hydroxytryptamine] and mitogenic proteins [platelet derived growth factor and smooth muscle proliferating factor] act locally in blood vessel repair and restoration processes. Concomitantly w i t h these platelet secretory events the activated cell changes its "social habits", becomes sticky and adheres to vessel wall constituents exposed through damage and to other platelets to form a consolidated platelet mass w h i c h seals off the site of injury and prevents blood loss. Interest in the platelet surface membrane of course arises from our n e e d to k n o w more about the surface receptor sites at w h i c h haemostatic agents exert their effects of how the resulting signals at the surface are transduced into the interior of the platelet to initiate the various metabolic and morphological changes.

As w i t h many other cells, Ca

2+

is an

230

important second messenger and the platelet contains a wide spectrum of

2+

Ca

dependent systems, some of w h i c h are listed b e l o w : -

1. Force-generating changes in the platelet's cytoskeletal

equipment

[microfilaments and microtubules]. 2. Other polymeric changes affecting cytosol sol/gel

transitions.

3. Prostanoid synthesis and release of proaggregatory compounds

[e.g.

TXA 2 ] 4. Surface membrane reorganisations at the cytoplasmic face affecting revelation of surface oriented receptors and other ligand binding sites. 5. Protein kinases involved in phosphorylation mechanisms w h i c h alter the properties of structural and metabolic proteins. 6. cAMP 2+ levels, through phosphodiesterase and perhaps cyclase by Ca

regulation

/calmodulin.

7. F u s i o n events between plasma membrane domains and secretory granules for exocytotic release of procoagulants and other stored constituents. 2+ Exactly how the quiescent platelet maintains Ca hemeostasis is not known in detail but, like the red cell, the platelet surface membrane m a y 2+ 2+ 2+ have a Ca extrusion pump associated w i t h a Ca Mg -ATPase w h i c h is

2+ responsible for the efflux of any Ca

w h i c h leaks into the cell from the

high millimolar concentrations in plasma.

Also within the cytoplasmic

matrix are many intracellular membrane complexes [see Plate ll whose functions are believed to be analogous to the2+ endoplasmic reticulum of other cells but w h i c h also appear to have Ca transporting properties similar to the sarcoplasmic reticulum of muscle tissues. ular Ca

2+

pump, w h i c h is also associated w i t h a Ca

2+

Mg

2+

This

intracell-

-ATPase activity,

presumably operates collaboratively w i t h the surface membrane extrusion of C a ^ + to maintain cytosolic [ C a ^ + ] low and the platelet in an inactivated

2+

state.

To date our understanding of these important Ca

mobilising

systems has been frustrated by the lack of good isolation procedures capable of providing discrete membrane fractions 2+ representing these surface and intracellular membranes.

This Ca

mobilising problem was

one of our interests w h i c h provided the initial stimulus for us to investigate the value of F F E for platelet differential membrane

isolation.

Using a combination of conventional density gradient sedimentation and h i g h voltage F F E we have b e e n able to isolate from fresh platelet

231 sonicates discrete fractions representing surface and intracellular membrane elements.

The details of these procedures have b e e n documented

in a number of papers from this laboratory [l, 2, 3, 4] and will not be repeated here, but briefly, the procedure depends upon reducing the electronegativity of the surface membrane by pre-treatment w i t h neuraminidase at the whole cell level.

Sufficient negatively charged

sialic acid moeities are removed so that w h e n a m i x e d membrane vesicle population harvested from the low density region of the sorbitol density gradient is applied to the free flow electrophoresis chamber the surface and intracellular membranes separate into distinct peaks w i t h the intracellular membrane fraction always on the anodal side of the electrophoresis profile.

A flow diagram of this isolation and

fractionation procedure is shown in Fig. 3-

The profiles in Fig, k also

show the advantages of pre-treatment at the whole cell level w i t h neuraminidase and that by a simple change in the density gradient [presence or absence of EDTA] the surface membrane separates as a single peak or shows two components

[N^J

and

N J J J ] *

The neuraminidase

treatment

appears to be one of the most innocuous covalent modifications one can perform at the whole cell level to a membrane and by measurement of a variety of analytical and enzymatic parameters on the density gradient m i x e d membrane fractions prepared from neuraminidase-treated and untreated platelets we have been unable to detect any significant differences other than in sialoglycoprotein heterogeneity. and intracellular membrane fractions have been extensively

These surface characterised

by polypeptide, lipid, phospholipid and enzyme profiling and the disparity between them is in m a n y respects quite striking.

In particular the

surface membrane is rich in cytoskeletal proteins [actin, myosin, actin binding protein, a actinin, etc.] whereas the intracellular membrane is essentially devoid of these.

Similarly the well known platelet

glycoproteins GPIb, lib, Ilia, Illb and IV are all well represented in the surface membrane fraction but absent from the intracellular membrane. Figs. 5a and b show two dimensional isoelectric

focussing/polyacrylamide

gel separations of the two membrane fractions run under reducing gel conditions.

One can readily see that the surface membrane polypeptides

occupy a wide range of isoelectric points and molecular size, whereas

232

COMBINED SUPERNATANT [ P A V L O A D T O SORBITOL G R A D I E N T ] , 1 0 m M HEPES pH 7 . 2 | 1 5 0 m M Nacl

WASHED PLATELETS. 2x

4 m M Kcl

SORBITOL

3 m M EDTA

INCUBATED 3 7 ° C

FOR

MIXED -

GRADIENT IM - 3

R E S U S P E N D E D IN S A M E BUFFER pH A D J U S T E D T O 6 . 2

I*

5M

MEMBRANE

[LINEAR]

10mln.

4 2 0 0 0 xg 90 mm I N C U B A T E D 2 0 mln 3 7 C W I T H N E U R A M I N I D A S E [ 0 . 0 3 - 0 . 0 5 ( j / m l ]

W A S H E D 3 « S S U S P E N D E D IN 1 0 m M HEPES pH 7 . 2

FREE FLOW

C O N T A I N I N G 0 . 3 M S O R B I T O L . [ E D T A O M I T T E D , 5 0 u M LEUPEPTIN A D D E D ]

ELECTROPHORESIS

S O N I C A T E D 2 x 1 0 s e c l D A W E S O N I C A T O R 4 m m PROBE L E V E L 6] AT 4 ° C

FRACTION

C E N T R I F U G E D 2 0 0 0 xg 2 0 m l n [ P E L L E T R E S U S P E N D E D AND

N°

SONICATED AGAIN]

Fig. 3* Flow diagram of platelet isolation, sonication and density gradient fractionation preparatory to free flow electrophoresis

Table 1. 10

Identity* Microtubule-associated ABP P235 MHC GPIat GPIba GPIIat GPIIbat GPIIIat a-Actinin GPIIIb (IV) Gelsolin GPV Actin Tropomyosin GPIIbß GPlbß GP17

('MAPs')

_3

x M range r 1 ^approx.)\ 300 260 235 200 150-165 135-150 125-135 115-130 90-95 95 90-95 85 80-85 43 29 25-28 21-23 17-20

separation

PI range (approx.) 6.0-5.2 6.0-7.0 6.4-7.0 5.7-6.7 5.3-6.0 5.0-5.8 5.5-5.7 5.3-6.1 5.2-5.8 5.9-6.2 5.2-5.4 6 . 2 , 6 . 3 , 6.4 6.0-6.5 5.6-5.8 5.4-5-6 5.1-5.6 6.4-7.0 6.4-6.7

233

WITHOUT

NEURAMINIOASE

FRACTION b)

PRETREATMENT

N°

WITH NEURAMINIDASE

PRETREATMENT SURFACE MEMBRANES

FRACTION

N°

Fig. 4. Free flow electrophoresis profiles derived from fractions taken from the chamber,

a, b & c illustrate experiments with and without

neuraminidase pre-treatment and with and without EDTA in the sorbitol density gradient [see text].

234

Figs. 5a & b. Two dimensional SDS polyacrylamide isoelectric focusing gels of a) surface and b) intracellular membrane polypeptides. Conventional nomenclature has been used for the glycoproteins and where appropriate a and 3 subunits identified. Tm = tropomyosin. CBP = Con. binding protein not present in the surface membrane.

235 those in the intracellular membrane span a m u c h narrower range [pi 4.7 to 6.5 and Mr.

30,000 to 90,000].

W e are only in an early cataloguing

stage w i t h these membrane components and Table 1 shows the range of polypeptides definitively identified in the surface membrane. Table 2 shows the analytical data accumulated to date about the membranes and Table 3 shows some of the enzyme activities w h i c h have been investigated.

F r o m this latter table it can be seen that in addition to

the enrichment of the E R enzyme N A D H cytochrome-c-reductase the p h o s p h o lipid modifying enzymes Phospholipase A ^ and diacylglycerol lipase are predominant features of the intracellular membrane.

These findings

together w i t h the almost exclusive localisation of cyclooxygenase

and

thromboxane synthase in the intracellular membrane suggest that the complete enzymatic machinery for the liberation of arachidonic acid and its conversion to thromboxanes is structurally localised at an intracellular site a n d not associated w i t h the surface membrane as formerly believed.

A n additional property of the intracellular membrane vesicles

is their capacity to sequester Ca

2+

.

This is not a feature of the surface

membrane vesicles since they are oriented outside out and cannot display their C a Ca

2+

2+

extrusion properties.

The intracellular membrane uptake of

is ATP-dependent, saturates at an external level of Ca

10 lomolar and reaches steady state levels in about 12 mins.

2+

around The Ca

2+

ionophore A23187 releases sequestered C a ^ + rapidly [tj_ 1-2 m i n ] but 2

extensive investigations using phosphatidic acid, various prostaglandin endoperoxides and also generating thromboxanes within the vesicles have 2+ failed to show any Ca -ionophoric properties by any of these compounds.

2+

H o w Ca known.

is transported out of or released from these vesicles is not yet These isolation procedures w h i c h include the use of free flow

electrophoresis have allowed us to probe some of the properties of human platelet surface and intracellular membranes using purified fractions that have hitherto not b e e n available.

Our understanding at the molecular

level of the platelet's remarkable range of metabolic and functional activities is still rather limited but knowledge of the platelet's role in normal haemostatic behaviour and in the pathological

events

associated w i t h thrombosis, atherosclerosis transplant rejection etc. depends u p o n intensifying our efforts at the subcellular and molecular

236

Table 2. ANALYTICAL AND ENZYMATIC DATA FOR HUMAN PLATELET SURFACE MEMBRANES ISOLATED BY FREE FLOW ELECTROPHORESIS [Values as Mean ± SD for at least 3 determinations] SURFACE MEMBRANE

CONSTITUENT CHOLESTEROL [|u mol/mg] PHOSPHOLIPIDS [|i mol/mg] CHOL/P-LIPID MOLAR RATIO SPHINGOMYELIN a) ,. PHOSPHATIDYL SERINE1' ' PHOSPHATIDYL ETHANOLAMINEv PHOSPHATIDYL CHOLINE(^) PHOSPHATIDYL INOSITOL^) MICROVISCOSITY [POISE] 25° (37°) a) °/o TOTAL LIPIDS

0.69 0.93 0.74 24.8

2.8

41.9 5O.7 4.4 4.7

INTRACELLULAR MEMBRANE

0.06 0.06 0.05 1.8 0.06 1.5 2.5 1.5 (2.5)

O.O3 0.05 0.01

0.27 O.92 O.29

2.6

2.6

3.0 28.5 60.3 8.7 3.1

0.07 1.4

2.0

0.7 (2.0)

b) °/c OF SUM OF ALL GLYCEROPHOSPHOLIPIDS

Table 3ENZYME ADENYLATE CYCLASE (a) NADH cyt-c REDUCTASE ( b ) LEUCYL AMINO PEPTIDASE 5'NUCLEOTIDASE 2+

(c)

^

2+

Ca Mg ATPase ^ PHOSPHOLIPASE-An (d) DIGLYCERIDE LIPASE ( d ) CYCLOOXYGENASE ^ THROMBOXANE SYNTHASE (a) LIPOXYGENASE ^ FATTY ACYL-CoA TRANSFERASE ( b )

(a) p mol min * mg ^

(b) n mol min ^ mg ^

(d) n mol h ^ mg ^

N.D. = not detected.

(c) Ji mol h

-1

mg

-1

237 levels.

A more rational approach to drug design to prevent these

conditions may then become possible. I

I

Separation of different functionally active surface membrane domains in the phagocytosing polymorphonuclear TPMN] leucocyte The response of PMN-leucocytes to chemotactic and phagocytic stimuli are surface membrane mediated events which involve not only topographical changes in integral membrane proteins but also changes in the character and disposition of the cell's cytoskeletal equipment, some components of which [e.g. actin] are associated in polymeric form with surface membrane constituents on the cytoplasmic face of the surface membrane. For detailed studies of these membrane phenomena a highly purified surface membrane fraction is required which is free from contaminating intracellular components and of course essentially unaltered in associated constituents by the isolation and purification procedures. Most of the more conventional techniques for isolating leucocyte plasma membranes result in a rather heterogenous population of membrane vesicles containing elements of both surface and intracellular membrane origin. Additionally, if care is not taken to maintain the integrity of the leucocyte's granular organelles, fragments of their boundary membranes may also be included in mixed membràne fractions.

For our research interests

in leucocyte motile activités as expressed in phagocytic and chemotactic activities we have combined conventional density gradient sedimentation procedures with high voltage free flow electrophoresis.

These have

produced surface membrane subfractions from resting and phagocytosing cells which have allowed direct comparison of analytical, enzymatic and other properties of the membranes at rest and during phagocytosis.

Additionally

using a phagocytic model system it is possible to compare phagocytic vesicle membranes with surface domains uninvolved in the internalisation process and aspects of membrane reorganisation during the phagocytic event can then be studied.

The full experimental protocol for separating and

purifying these surface membrane domains has been reported in detail elsewhere [Stewart & Crawford, 1983], but briefly resting or phagocytosing cells are homogenised with a tight fitting pestle homogeniser and the homogenate is applied to sorbitol density gradients [iy/c w/v to 55°/» v/v

238 sorbitol] which are linear over the density range 1.08 to 1.23.

After

centrifugation at 70,000 x g for 50 min. a mixed membrane fraction which locates in the lower density region of the gradient is removed, washed, concentrated and then applied to the chamber of the free flow electrophoresis apparatus. [Bender Hobein Elphor VAP5].

The chamber buffer

consisting of 0.275M sorbitol, 10 mM triethanolamine and acetic acid [pH 7*3 300 ideal milliosmols] is run at a flow rate of 3*8-4.0 ml/hour per fraction and the membrane sample is injected into this chamber at a flow rate of 1.5-2.0 ml/h.

The potential difference applied across the

chamber is of the order 100 v/cm at a current o f ' ^ 1 5 0 mA. For all these studies peritoneal PMN-leucocytes have been used. These were taken from the abdominal cavity of rabbits 8-10 h. after an injection of sterile saline containing 0.1^ w/v glycogen. leucocytes collected by abdominal lavage are preparation contains ^95°/o leucocytes with a few lymphocytes.

The PMN

96°/o viable and the final 2^] contaminating

The phagocytic model is based upon that described by

Stossel and his colleagues [Stossel et al. 1972] using opsonised paraffin oil as the stimulus and containing "Oil Red 0".

In our experience the

leucocytes take up 3-4 large droplets of the oil and 1 or 2 smaller ones after 20 min. phagocytosis.

For quantitative purposes aliquots of the

cells containing the phagocytosed oil are dried down and the oil/dye mixture extracted with dioxane for colorimetric measurements at 524 nm. Figs. 6a and b show a time course for phagocytosis by rabbit PMNleucocyte and a typical cell containing internalised oil.

It can be seen

that oil entry ceases after 10-12 min. when presumably the cell will not further compromise its surface area by enclosing oil particles. Fig. 7 shows the appearance of the sorbitol gradients after separating homogenates prepared from resting and phagocytosing cells. The resting leucocyte produces a well defined zone of mixed membranes [surface and intracellular] in the density range 1.13 to 1.18.

There is

a similar well defined membrane zone in the homogenates from cells taken after 12-15 min. phagocytosis.

However, in the low density microsome

region of the gradient from these phagocytosing cells another discrete membrane zone at the air/microsome interface is seen and this contains all the Oil Red 0 consumed during the phagocytic event.

This fraction,

239

0.4 -

0.2

T

10

20 TIME (minutes)

Fig. 6a. Time curve for the phagocytosis of opsonised paraffin oil droplets in which the dye oil red '0' has been incorporated.

Fig. 6b. A blood leucocyte with ingested paraffin-oil droplets (O) x 9000 [Castle 198^].

240

we believe, contains the phagocytic vesicles and since the whole internalisation process is really a frustrated phagocytic event and does not proceed to lysosome fusion these can be isolated independently and

Phagocytic Vesicles(PV) 13%«i

Sx

Resting Cell Homogenate

d. 1.08

Sorbitol Gradient

ÉÉ

1.11

Phagocytic Cell Homogenate

S, PELLET

55%-

ninvolved surface Membrane

Fig. 7. Appearance of the sorbitol density gradients after homogenates from resting and phagocytosing leucocytes.

separating

The m i x e d membrane

vesicles [Sg] locate in the density range 1.08-1.11 and the phagocytic vesicles containing the oil float to the meniscus. cell cytosol and S^ the nuclei and granules.

S^ and S^ contain

S^. is a faint zone of

unknown particles. after washing and hypotonic lysis to free the boundary membrane

from

around the oil droplet regarded as pure phagocytic vesicle membranes. The membrane fraction locating at higher density in this sorbitol gradient contains surface membrane domains not involved in the phagocytic process. The mixed membrane fractions are then applied to the

electrophoresis

chamber using a buffer and electrical conditions similar to those used for platelet membrane isolation.

Fig. 8. is a composite diagram of the

electrophoretic profiles obtained w i t h resting and phagocytic cell membranes.

It will be seen that the surface membrane marker probe applied

at the whole cell level

Lens culinaris] locates almost exactly in

the same fractions as the surface m a r k e r enzyme

5'nucleotidase.

The phagocytic vesicle membranes which are normally electrophoresed

241 P

©

Plasma

Membrane

Resting

Cells

^ ^

•lo

lio-

100-

Ili

I-Lectin,

15

«0-

—r»0

40 Plasma

60

Membrane

^yphagocytosing

Cells

O

-?000

ISO5 Nucleo-

tldase —

n ;. ^,

,» >* i

100-

.1000

«h

"

l-

—I—

XO

FRACTION

—I—

AC

60

No.

Fig. 8. Composite diagram of the free flow-electrophoresis profiles from resting (a) and phagocytosing (b) cells. The F^ and F^l fractions represent surface membranes as indicated by almost exclusive localisation of the 125 I-labelled Lens cul inaris and 5'-nucleotidase activity. Their electrophoretic mobilities are identical. The peak 'Pv' in the lower profile represents phagocytic vesicle membranes. These are normally electrophoresed separately but the profile is superimposed here for comparison.

242

separately have a higher electrophoretic mobility than the surface membrane from w h i c h they were derived. 125 Analysis of the distribution of

I labelled Lens culinaris and

the activity of the enzyme 5'nucleotidase in the various membrane fractions [Table ¿t~] shows that there is no significant difference in bound lectin between the surface membranes from resting and phagocytic cells and the phagocytic vesicle membrane. to homogenate between 8 and 9 fold.

All are enriched w i t h respect

However, w i t h respect to the activity

of 5'nucleotidases, this is 11 fold enriched in the surface membrane from resting cells but 35 fold enriched in the uninvolved surface domain of the phagocytic cells.

The phagocytic vesicle domain concomitantly is

depleted in this enzyme activity to avoid the levels found in homogenate. This finding suggests that the 5'nucleotidase w h i c h is the major sialoglycoprotein of the rabbit PMN-leucocytes selectively segregates moving out of the vesicle domain and into the uninvolved surface membrane during phagocytosis. evenly distributed. Fig, 9«

O n the other hand Lens culinaris receptors remain This phenomena is illustrated diagrammatically in

Further studies [not included here] have shown that during the

phagocytic event there is a kW/o increase above endogenous levels in filamentous actin associated w i t h the uninvolved surface membrane of the phagocytic cell w i t h a significant shift in the equilibrium state between monomeric

'G' actin and filamentous

'F' actin in the leucocyte

No actin can be detected in the phagocytic vesicle membrane

cytosol.

suggesting

that the endogenous actin network associated w i t h the cytoplasmic face of the resting cell surface membrane is dissociated from the vesicle preparatory to a fusion event.

We believe that this phagocytic model

system and the membrane isolation procedure described here are highly suitable for the study of other surface membrane constituents

and

receptors which m a y selectively segregate during a phagocytic event and further studies are being carried out to explore other ligand binding processes. In conclusion I have attempted, using two examples from our own research interests, to illustrate the value of high voltage free flow electrophoresis in membrane

research.

243

Tabi* 4

REDISTRIBUTION OF [ 1 2 5 I ] LECTIN 3 dpm x 10 mg protein ^

HOMOGENATE SURFACE MEMBRANE

LECTIN AND 5 'NUCLEOTIDASE ACTIVITY

RESTING

PHAGOCYTOSING

CELLS

CELLS

244 ( l . O )

222 (l.O)

2070 (8.5)

1980 (8.9)

PHAGOCYTIC VESICLE MEMBRANE

5'NUCLEOTIDASE ACTIVITY [n mol AMP mg ^ protein h ^

HOMOGENATE SURFACE MEMBRANE PHAGOCYTIC VESICLE MEMBRANE

1830 (8.3)

RESTING

PHAGOCYTOSING

CELLS

CELLS

1.2 (l.O)

1.3 (1.0)

13.0 (10.8)

45.0 (34.6) 1.4 (1.0)

Figures in parenthesis are enrichments of the specific activities with respect to homogenates [= 1.0]

244

• -5 NUCLEOTIDASE V RECEPTORS FOR

LECTIN

Fig. 9.

SELECTIVE SEGREGATION OF SURFACE MEMBRANE CONSTITUENTS DURING PHAGOCYTOSIS

The ecto-enzyme 5'-nucleotidase ( 90K sialoglycoprotein) migrates out of forming phagocytic vesicle into uninvolved membrane domain. Lectin receptors remain evenly distributed throughout, but now face interior of internalised phagosome.

245 These applications have facilitated the separation of conventionally produced m i x e d membrane fractions from blood platelets and blood granulocytes into discrete subfractions containing highly purified surface and intracellular membrane elements.

Analytical, enzymatic and

functional parameters for these two membrane fractions can now be established w i t h a degree of confidence not possible hitherto. Additionally, free flow electrophoresis procedures can be carried out preparatively to produce high yields of fractions suitable for the isolation of physiologically important intrinsic membrane such as enzymes, and receptors for molecular

constituents

characterisation.

The author w o u l d like to thank the organisers of this Symposium for the opportunity of presenting this work and also his colleagues [Drs,. K. S. Authi, N . Hack and D. I. H . Stewart] for kindly allowing h i m to draw freely upon their own research data.

The Medical Research Council

and the British Heart Foundation are also acknowledged for providing financial support for some of these studies.

KEY REFERENCES A.

F R E E F L O W ELECTROPHORETIC PROCEDURES F O R MEMBRANES

1.

Menashi, S., Weintroub, H. and Crawford, N . (l98l).

Characterisation

of human platelet surface and intracellular membranes isolated by free flow electrophoresis. 2.

J . Biol. Chem. 256. 4095-4101.

Lagarde, M., Guichardant, M., Menashi, S. and Crawford, N.

(1982).

The phospholipid and fatty acid composition of human platelet surface and intracellular membranes isolated by high voltage free flow electrophoresis. 3.

J. Biol. Chem. 256. 3100-3104.

Menashi, S., Authi, K . S., Carey, F . and Crawford, N .

(1984).

Characterisation of the calcium sequestering process associated w i t h human platelet intracellular membranes isolated by free flow electrophoresis. 4.

Biochem. J. 222, 413-417.

Carey, F., Menashi, S. and Crawford, N . (l982).

Localisation of

246

4.

Carey, F., Menashi, S. and Crawford, N . (1982).

Localisation of

cyclooxygenase and thromboxane synthetase in human platelet intracellular membranes. 5.

Biochem. J. 204, 847-851.

Hack, N . and Crawford, N . (1984).

Two-dimensional polyacrylamide gel

electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes.

Biochem. J . 222,

235-246. Carey, F. 6.

Hack, N . / a n d Crawford, N . (1984).

The inhibition of platelet

cyclooxygenase is associated with the acetylation of a 72 kDa polypeptide of the intracellular membranes. 7.

Stewart, D. I. H . and Crawford, N . (1983).

Biochem. J . 223, 105-111. Preparation of a highly

purified surface membrane fraction from rabbit PMN-leucocytes by free flow electrophoresis.

Biochim. Biophys. Acta, 733, 154-162.

B.

PHAGOCYTOSIS A N D MEASUREMENTS O F PHAGOCYTIC ACTIVITY

1.

Castle, A . G. (1984) Phagocytosis. Biologist, 31. 9-14.

2.

Stossel, T. P., Mason, R. J., Hartwig, J. and Vaughan, M . J . Clin. Invest.

3.

(1972).

615-624.

Stewart, D. I. H . and Crawford, N . (1983). Redistribution of membrane 5' nucleotidase in rabbit PMN-leucocytes during phagocytosis. FEBS Letters. 156, 329-334.

I N V E S T I G A T I O N OF BODY F L U I D S BY P A R T I C L E I. I n f l u e n c e of

ELECTROPHORESIS

c e r e b r o s p i n a l f l u i d (CSF) on the

phoretic mobility

electro-

(EPM) of a r t i f i c i a l p a r t i c l e s - a

useful

tool for the e v a l u a t i o n of m e n i n g i t i s ?

Dirk H o b u s c h , E v a P e t e r s Wilhelm-Pieck-Universitat Kinderklinik DDR-2500 Rostock Eberhard Knippel, Wolfgang

Rostock

Schütt

Wilhelm-Pieck-Universität Rostock K l i n i k für I n n e r e M e d i z i n DDR-2500 Rostock

Helmut Walter

Meyer

Friedrich-Schiller-Universität Jena A b t e i l u n g für E l e k t r o n e n m i k r o s k o p i e des B e r e i c h e s DDR-6900 Jena Hans

Medizin

Meyer-Rienecker

Wilhelm-Pieck-Universitat Nervenklinik DDR-2500 Rostock

Rostock

Introduction Electrophoresis electrical

is a simple m e t h o d for m e a s u r i n g the n e t

s u r f a c e c h a r g e of a p a r t i c l e - a q u a n t i t y

which

y i e l d s i n f o r m a t i o n r e g a r d i n g the s t r u c t u r e a n d q u a l i t y of the p a r t i c l e s u r f a c e a n d h o w it is a f f e c t e d by its

environment.

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

248 As a rule the particles investigated by biological and medical electrophoretic studies are cells; electrophoretic

studies

dealing with non-cellular particles are the exception rather than the rule, particularly in connection with clinical problems. The starting point for the work described here was the use of paticle electrophoresis to differentiate between non-cellular components (liposomes) in amniotic fluid as a means of gaining information regarding prenatal lung maturation (1). It seemed possible that this method, which is simpler and less time-consuming than conventional techniques, could also be used to examine other body fluids to obtain additional parameters for the diagnosis of pathological states (2, 3). The method has not so far been used to examine cerebrospinal fluid (CSF). Since it produces results rapidly, however, its use for the diangosis of meningitis could be important because it is still necessary to differentiate between the different forms of meningitis, i.e. purulent, viral and initially treated, before appropriate treatment can be instituted. Since none of the methods currently in use has proved to be completely reliable, we wanted to find out whether electrophoretic examination of the CSF could be considered a useful tool for differentiating between purulent and viral meningitis . In contrast to amniotic fluid, most samples of CSF contain too few small particles (- 1 um) to permit electrophoretic measurements. The results presented here are therefore based mainly on investigations into the effects of substances dissolved in the CSF, such as proteins, lipides, phospholipides, sugars and enzymes, on the EPM of artificial particles (droplets of silicone oil) produced in siliconized centrifuge tubes. Particles in the CSF were also included to a very slight extent in the studies. In contrast to procedures for measuring a single component, electrophoresis is used to measure the joint action of several different quantities simultaneously, and

249

t h i s , it is h o p e d , m a y h e l p d i f f e r e n t i a t e b e t w e e n

patholo-

g i c a l c h a n g e s in the C S F .

Material and Methods C o l l e c t i o n a n d i n v e s t i g a t i o n of C S F -

B a s e l i n e d a t a for

our s t u d i e s w e r e o b t a i n e d by m e a s u r i n g the EPM of C S F

sam-

differen-

p l e s t a k e n by l u m b a r p u n c t u r e f r o m 95 c h i l d r e n for

tial d i a g n o s t i c p u r p o s e s or to e l i m i n a t e m e n i n g i t i s as a p o s s i b l e d i a g n o s i s . N o n e of the c h i l d r e n s u f f e r e d f r o m z u r e s , a n d the l i q u o r s a m p l e s w e r e t a k e n b e f o r e the

sei-

children

h a d b r e a k f a s t e d . All of t h e s e l i q u o r s a m p l e s h a d an a p p r o p r i a t e cell c o u n t (4) for the age of the s u b j e c t , a n o r m a l protein content, a normal blood sugar-to-liquor sugar a n d a n o r m a l l a c t a t e v a l u e . T h e y c o n t a i n e d no

erythrocytes

a n d h a d a n o r m a l cell d i s t r i b u t i o n in the l i q u o r after SAYK

ratio,

sediment

(5).

The v a l i d i t y of the m e t h o d as d i a g n o s t i c i n s t r u m e n t m e n i n g i t i s was i n v e s t i g a t e d by e x a m i n i n g

for

cerebrospinal

f l u i d f r o m 25 c h i l d r e n a g e d 4 m o n t h s to 15 y e a r s w i t h p u r u l e n t m e n i n g i t i s a n d 42 c h i l d r e n a g e d 10 m o n t h s to 15 y e a r s w i t h v i r a l m e n i n g i t i s . In the f o r m e r g r o u p the n e s s was c a u s e d by N. m e n i n g i t i d i s in n i n e cases,

ill-

Haemo-

p h i l u s i n f l u e n z a e in t h r e e cases, D i p l o c o c c u s p n e u m o n a e t h r e e c a s e s a n d E. coli in one case. E f f o r t s to the g e r m w e r e u n s u c c e s s f u l

in

culture

in s e v e n cases, but N.

menin-

g i t i d e s was s u s p e c t e d in s e v e n of t h e m on a c c o u n t of the c l i n i c a l p i c t u r e . A m o n g the 42 c h i l d r e n a g e d 10 m o n t h s 15 y e a r s w i t h v i r a l m e n i n g i t i s ,

a v i r u s or a

to

corresponding

t i t r e i n c r e a s e in the c o m p l e m e n t b i n d i n g r e a c t i o n was t e c t e d i n 15 c a s e s . T h e g e r m s w e r e i d e n t i f i e d as

de-

Coxsackie

v i r u s in five c a s e s , ECHO in five, m u m p s v i r u s in four

and

i n f l u e n z a v i r u s in one. P a r a m e t e r s m e a s u r e d in the l a b o r a t o r y in a d d i t i o n to the

250 EPM i n c l u d e d cell c o u n t s u s i n g the F u c h s - R o s e n t h a l

chamber,

cell s e d i m e n t a t i o n a f t e r SAYK, p r o t e i n c o n t e n t by

turbidity

m e a s u r e m e n t a f t e r a d d i t i o n of E x t o n r e a g e n t , a l b u m i n a n d IgG by M a n c i n i ' s R I D m e t h o d , b l o o d a n d C S F g l u c o s e

concentra-

tions with O-toluidine and glacial acetic acid, and c o n c e n t r a t i o n by an e n z y m a t i c m e t h o d a f t e r The C S F was e x a m i n e d b a c t e r i o l o g i c a l l y beacteriological

by G r a m ' s s t a i n a n d

culturing experiments, and diagnostic

e x a m i n a t i o n w a s p e r f o r m e d on b l o o d s a m p l e s , t h r o a t and

lactate

deproteinization. viral

swabs

stool.

Electrophoretic measurements -

The C S F s a m p l e s w e r e

stored

at - 2 5 °C u n t i l the e l e c t r o p h o r e t i c m e a s u r e m e n t s c o u l d be p e r f o r m e d . B e f o r e the m e a s u r e m e n t s the s a m p l e s were

centrifuged

at 250 g for 10 m i n . , w h e r e u p o n 100 ;ul of s u p e r n a t a n t

were

t h o r o u g h l y s h a k e n w i t h 2 ml p h o s p h a t e b u f f e r e d s a l i n e

(PBS)

for 3 m i n . in s i l i c o n i z e d c e n t r i f u g e t u b e s ( s i l i c o n e s i o n NE 4 / N u n c h r i t z

oil emul-

) to o b t a i n a s u f f i c i e n t n u m b e r of

cone oil d r o p l e t s . The t i m e r e q u i r e d for s a m p l e

sili-

preparation

f r o m d e f r e e z i n g to EPM m e a s u r e m e n t was a b o u t 20 m i n .

T h e EPM of the c h a r g e d s i l i c o n e oil d r o p l e t s a n d of the o t h e r p a r t i c l e s in the C S F s a m p l e s was m e a s u r e d in a P A R M 0 Q U A N T a u t o m a t i c cell e l e c t r o p h o r e s i s

apparatus

Z e i s s J e n a , 6), w h i c h p e r m i t s the EPM of d i s c r e t e

(Carl

particles

to be m e a s u r e d q u i c k l y a n d a c c u r a t e l y . It is able to p r o d u c e the EPM of s t a n d a r d p a r t i c l e s s u c h as h u m a n t h r o c y t e s w i t h an a c c u r a c y b e t t e r t h a n + 1

%. In

s a m p l e , 25 to 50 of the s m a l l e s t p a r t i c l e s

(^ 1 u m )

reery-

each were

s e l e c t e d s u b j e c t i v e l y a n d t h e i r m e a n EPM w a s m e a s u r e d constant conditions

(pH, c o n d u c t i v i t y ) ,

5 m i n . per

samples

b i n g r e q u i r e d for the m e a s u r e m e n t s . The a p p a r a t u s w a s b r a t e d w i t h h u m a n e r y t h r o c y t e s w i t h a m e a n EMP of

under cali-

251

1 . 0 8 x 1 0 " 8 m 2 s ~ 1 V ~ 1 . The t - t e s t ( S t u d e n t ) a n d W E L C H w e r e u s e d for the s t a t i s t i c a l a n a l y s i s of the

Electron microscopy -

test

results.

The s a m p l e s for e l e c t r o n

microscopic

e x a m i n a t i o n w e r e a l s o c e n t r i f u g e d for 10 m i n u t e s at 250 g and then frozen (Cryo-JET freezer, Messrs. Balzers),

which

e n s u r e s e x t r e m e l y r a p i d f r e e z i n g w i t h o u t the f o r m a t i o n of t r o u b l e s o m ice c r y s t a l s . The f r o z e n s a m p l e s w e r e t h e n

cle-

aned with sodium hypochlorite, flushed with distilled

water

a n d e x a m i n e d in a TESLA BS 500 e l e c t r o n

Infra red spectroscopy -

microscope.

The i n f r a r e d s p e c t r a w e r e

d e d on K B r p h o t o g r a p h i c p l a t e s w i t h an I R - 1 2

m e t e r ( M e s s r s . B e c k m a n ) . The a c c u r a c y of the w a v e d a t a is + 0.5 cm

-1

to + 1.4 cm

-1

recor-

spectrophotonumber

, d e p e n d i n g on the

range.

Results The e l e c t r o n m i c r o s c o p i c , e x a m i n a t i o n

of v a r i o u s

samples

( b a s e l i n e s a m p l e s a n d s a m p l e s from p a t i e n t s w i t h

purulent

a n d v i r a l m e n i n g i t i s ) s h o w e d , t h a t the few p a r t i c l e s

con-

t a i n e d i n the C S F were m e m b r a n e v e s i c l e s r a n g i n g

from

150 to 700 n m in d i a m e t e r , w h i c h e x h i b i t e d m a j o r

particle

a g g r e g a t i o n s as a s i g n of d a m a g e in some c a s e s , a n d u n i d e n t i f i a b l e p a r t i c l e s w h i c h m a y h a v e c o n s i s t e d of tinated membrane material

agglu-

(Fig.1).

M o s t of the p a r t i c l e s m e a s u r e d e l e c t r o p h o r e t i c a l l y

produced

the same IR s p e c t r u m as the p u r e s i l i c o n e oil w i t h the l o w i n g b a n d s : 2962 c m 676 cm

-1

,

1260 c m

-1

,

1094 c m

-1

,

800 c m

. The E P M of the pure s i l i c o n e oil d r o p l e t s

2,15 e l e c t r o p h o r e t i c u n i t s

(e.u.) but t h a t of the

p a r t i c l e s i n f l u e n c e d by the C S P w a s l o w e r . The d e v i a t i o n of the C S F - i n f l u e n c e d s i l i c o n e oil

-1

foland

was

silicone

standard

particles

252

Pig. 1 Electron microscopic p h o t o g r a p h of m e m b r a n e v e s i c l e s in the c e r e b r o spinal fluid

r a n g e d f r o m 3 % to 8 %; it was c o m m o n l y a r o u n d 5 %. The m e a n EPM of our 95 b a s e l i n e s a m p l e s was 1.33 + 0.34

e.u.

As T a b l e 1 s h o w s , the EPM v a r i e s w i t h age. D i f f e r e n c e s w e r e s i g n i f i c a n t at the 1 % level w e r e o b s e r v e d

that

between

g r o u p s 2 a n d 4, 2 a n d 5, 3 a n d 4, 3 a n d 5 a n d b e t w e e n g r o u p 1 a n d all o t h e r

groups.

The m e a n EPM of the d r o p l e t s in C S F s a m p l e s f r o m

patients

w i t h p u r u l e n t a n d v i r a l m e n i n g i t i s w e r e 0.87 + 0.10 a n d 0.99 + 0.12 e.u. r e s p e c t i v e l y

on the day of

e.u.

hospital-

i z a t i o n . The i n d i v i d u a l EPM v a l u e s are s h o w n in Fig. 2. The two m e a n s d i f f e r from e a c h o t h e r a n d from the b a s e l i n e

Table 1

E P M - v a l u e s (10 u p o n the age

group

age

8

m2

s

1

V

n u m b e r of subj ects

1

) in C S F in

mean

1

0 -

2 months

15

1 ,05

2

3 -

6 months

3

7 - 1 2

EPM

dependency

SD

+0,11

13

1 ,22

+0,17

nonths

13

1,20

+0,15

4

1 - 6

years

31

1,42

+0,25

5

7 - 1 5

years

23

1,53

+ 0,49

253 EPM

(10"8m2s-1V-1)

1.3 1.2

1.1 i.o0,95 0,9' Fig. 2 The i n d i v i d u a l E P M v a l u e s of patients with b a c t e r i a l (b) and non-bacterial (ab) meningitis

0.8' 0,7' b m

significantly

(p 0 , 0 8

056 i0.CH

046 0,9510,11 ±0,08

0 , 9 6 ! 0,11

0S0

•OXK

1.40 1,30 1.20

!

1,10

>

• 8"

i 1,00

• It Ll

0,90

i

1. I

0.80

i l ii1

!• :•

I

1

1.

M e a n v a l u e s of e l e c t r o phoretic mobilities ( 1 0 ~ 8 m 2 s ~ 1 V _ 1 ) of s i l i cone oil d r o p l e t s i n c u b a ted with bronchoalveolar lavage fluid from 8 normal c o n t r o l s and 61 p a t i e n t s with pneumonia, lung carcinoma, bronchitis and sarcoidosis

I •

8 8.

•

0,70 0.60 0.50 n = 9 control

n=10 pneumonia

n = 20 carcinoma

n=15 bronchitis

n = 15 sarcoidosis

Fig. 3 shows the r e g i o n s of the EPM m e a n s . E x c e p t for the g r o u p s h e a l t h y l u n g / p n e u m o n i a and bror.chitis/sarcoidosis, g r o u p s d i f f e r f r o m e a c h other s i g n i f i c a n t l y at the p < level or, for the g r o u p s t u m o u r / b r o n c h i t i s

and

all

0.01

tumour/sarcoi-

d o s i s , at the p ^ 0.05 l e v e l . The d i f f e r e n c e b e t w e e n

pneumonia

and t u m o u r is p a r t i c u l a r l y d i s t i n c t : 90 % of all EPM

values

for p n e u m o n i a c a s e s are s i t u a t e d above a n a r b i t r a r y

threshold

of 1.02 e.u. a n d 95 % of all EPM for t u m o u r s are b e l o w threshold,

this

so t h a t in this case it m a y e v e n be p o s s i b l e

d i s t i n g u i s h b e t w e e n the d i s e a s e s in i n d i v i d u a l c a s e s . t u m o u r s i n v o l v e d can be d i v i d e d into two g r o u p s on the of r a d i o l o g i c a l a n d h i s t o l o g i c a l

to

The basis

f i n d i n g s a n d the d e g r e e

metastazatior., a n d t h e s e g r o u p s also y i e l d d i f f e r e n t EPM a small s t a n d a r d d e v i a t i o n

(4-5 %) / T a b l e

1).

of with

267 Table 1

Relationship between clinical assessment c a r c i n o m a a n d EPM v a l u e s

n = 7 evaluation

EPM

Roentgen

Histological findings Metastazation

less

lung

n = 11

>0.90

focus

findings

of

e.u.

small

mature

no

I 1 to m f O o. E o o

in

+1 S g ~

< w M

e

c

I £

•S

g

00 H tO •O'"

O CS

-h t^

00

o

CM

a

—

I M

V Ol

S

o>

in

+1

+1

S E

tO

f-'

o o -o

CS CO

o

^

r-l

CS r—1

sO o»

l/> %o

+1

+1

o to

o CO

to

+1

+1

CS O ò in

o*

^ £ . 0 )

with

p.

•

r e g a r d e d as a f u n c t i o n

it

that

come

) = x

k V M

and

observations

of

n J T S(X. ,2) 1 i =1

L is

o f X)

now i s L,

density

=

0im s " s l o w m o v i n g " c e l l s (EPM = 0.82 vim s

-1

V

_1

V -1

V

cm) -1

cm)

conand

cm).

T h e r e is n o s i g n i f i c a n t d i f f e r e n c e b e t w e e n the m e a n

values

of the EPM a n d e l e c t r o p h o r e t i c p r o f i l e s of cells f r o m

tu-

m o u r s a n d cell c u l t u r e s . T h e d u r a t i o n of in v i t r o

culturing

(72 h o u r s ) is c o n s i d e r e d to be s u f f i c i e n t for the

regeneration

of the cell s u r f a c e a f t e r e n z y m e t r e a t m e n t (10). F o r r e a s o n it seems t h a t t r y p s i n d i g e s t i o n h a s no e f f e c t on the EPM of m e l a n o m a c e l l s . S i m i l a r

this

significant conclusions

h a v e b e e n d r a w n f r o m e x p e r i m e n t s c a r r i e d out on v a r i o u s

li-

n e s of m e l a n o m a c e l l s (11). On the o t h e r h a n d , it is k n o w n t h a t t r y p s i n d i g e s t i o n l i b e r a t e s some g l y c o p r o t e i n s

from

the s u r f a c e of B o m i r s k i h a m s t e r m e l a n o m a c e l l s ( 1 2 , 1 3 ) . The e l e c t r o p h o r e t i c p r o f i l e s of c e l l s f r o m n o n - p i g m e n t e d t u m o u r s a n d f r o m cell c u l t u r e s are p r e s e n t e d in F i g .

2.

The cell p o p u l a t i o n t a k e n d i r e c t l y f r o m t u m o u r s (EPM = —1 —1 1 . 1 2 vim s V cm) c o n s i s t s of two s u b p o p u l a t i o n s , the —1 —1 m a i n g r o u p (EPM = 1 .08 Jims

V

cm) c o n t a i n i n g a b o u t 85 %

of the c e l l s . The h i s t o g r a m of the EPM of cells f r o m ry cell c u l t u r e s is v e r y s i m i l a r to t h a t of c e l l s

prima-

from

t u m o u r s a l t h o u g h the m e a n v a l u e of the EPM of c u l t u r e (EPM = 0.97 u m s ~ 1 V ~ 1

cm) is s m a l l e r t h a n t h a t of

c e l l s . The e l e c t r o p h o r e t i c p r o f i l e s of n o n - p i g m e n t e d m a i n t a i n e d in v i t r o for a l o n g e r time h a v e shapes.

cells

tumour cells

different

T h e p o p u l a t i o n c o n s i s t s of t h r e e cell g r o u p s

with

d i f f e r e n t E P M . The d i s c r e p a n c i e s b e t w e e n t h e m e a n

values

of the E P M a n d e l e c t r o p h o r e t i c p r o f i l e s c a n n o t be

attri-

b u t e d e x c l u s i v e l y to the a c t i o n of t r y p s i n on the

cell

surface

( 1 0 , 1 1 , 1 2 , 1 3 ) . S o m e c h a n g e s m a y be c a u s e d by t h e

454

12-

J

8-

0.4

Fig. 1

0.8

1.2

1.6

2.0

1.6

2.0

Histograms of the electrophoretic mobility (EPM) of pigmented Bomirski hamster melanoma cells derived from tumours by trypsin digestion (A) and from cell cultures without using enzymes (B)

20-J J1

0.4

0.8

1.2

EPM

n

2016-

128-

\s\

4-

—I-1 0.4 20-

1

1

0.8

1.2

1-1

Fig. 2

1—

1.6

2.0

1.6

2.0

Histograms of the electrophoretic mobility (EPM) of non-pigmented Bomirski hamster melanoma cells derived from tumours by trypsin digestion (A) and from cell cultures without using enzymes (B)

n

16" 128-

un

0.4

-i

0.8

r

1.2

EPM

455 s p e c i f i c c o n d i t i o n s of cell d e v e l o p m e n t i n cell

cultures.

U n f o r t u n a t e l y , no s u c h c o m p a r i s o n can be m a d e for

pigmen-

t e d B o m i r s k i h a m s t e r m e l a n o m a b e c a u s e t h e s e c e l l s can be c u l t u r e d i n v i t r o for only a few days w i t h o u t l o s i n g

pig-

m e n t . The s e p a r a t i o n of p i g m e n t e d B o m i r s k i h a m s t e r

melanoma

c e l l s in a s u c r o s e d e n s i t y g r a d i e n t i n d i c a t e s t h a t

the

p o p u l a t i o n is c o m p o s e d of two cell g r o u p s

(Pig.

3).

Fig. 3 D i s t r i b u t i o n of r e l a t i v e cell c o n c e n t r a t i o n s for a p o p u l a t i o n of p i g m e n t e d B o m i r s k i h a m s t e r m e l a n o m a c e l l s s e p a r a t e d by s e d i m e n t a t i o n in a s u c r o s e d e n s i t y g r a d i e n t T h e c e l l s f r o m f r a c t i o n 11 are large a n d w e l l

pigmented.

The c e l l s f r o m f r a c t i o n 6, in c o n t r a s t are r a t h e r small do n o t c o n t a i n w e l l - d e f i n e d m e l a n o s o m e s . The

and

elec-

t r o p h o r e t i c a n a l y s i s of c e l l s f r o m f r a c t i o n s 6 a n d 11 (Fig. 4 ) s h o w s t h a t f r a c t i o n 11 c o n t a i n i n g

well-de-

v e l o p e d c e l l s is c o n s i d e r a b l y e n r i c h e d w i t h "fast

moving"

cells, whereas fraction 6 consists almost exclusively

of

" s l o w m o v i n g " c e l l s . It a p p e a r s t h a t w e l l - d i f f e r e n t i a -ted pigmented cells have a greater electrophoretic

mobility

456 t h a n p o o r l y p i g m e n t e d c e l l s . T h i s is an u n u s u a l

biological

f e a t u r e b e c a u s e h i g h e l e c t r o p h o r e t i c m o b i l i t y is to be a s s o c i a t e d w i t h f a s t g r o w i n g , p o o r l y c e l l s r a t h e r t h a n w i t h d e v e l o p e d ones

(14,15).

To c h a r a c t e r i z e the b a s i c f e a t u r e s of t h e s e cells from both fractions

subpopulations,

(6 a n d 11) w e r e i n j e c t e d

g o l d e n h a m s t e r s . The fact t h a t p i g m e n t e d t u m o u r s in h a m s t e r s

considered

differentiated

into

developed

injected w i t h cells from either fraction

shows

t h a t c e l l s f r o m b o t h f r a c t i o n s are able to s y n t h e t i z e

mela-

nin. Selected characteristics

con-

of t h e s e c e l l s ( m e l a n i n

t e n t , rate of o x y g e n c o n s u m p t i o n ) a n d d e r i v e d

tumours

(rate of g r o w t h , d i s t r i b u t i o n s of e l e c t r o p h o r e t i c t i e s ) are b e i n g

mobili-

investigated.

Fig. 4

0 4

0.8

1.2

1.6

2.0

0.4

0.8

1.2

1.6

2.0

H i s t o g r a m s of the e l e c trophoretic mobility (EPM) of c e l l s f r o m f r a c t i o n s 6 (A) a n d 11 (B) s e p a r a t e d f r o m a s u s p e n s i o n of B o m i r s ki h a m s t e r m e l a n o m a c e l l s by s e d i m e n t a t i o n in a sucrose density gradient

EPM

457

References 1. B o m i r s k i , A.: B i o l o g i c a l P r o p e r t i e s of T r a n s p l a n t a b l e M e l a n o m a s in S y r i a n H a m s t e r s D u r i n g 16 Y e a r s of M a i n t e n ance by S e r i a l P a s s a g a s , M e d i c a l A c a d e m y G d a n s k 1977. 2. P a j a k , S., S u b c z y n s k i , ¥ . , P a n z , T., i u k i e w i c z , F o l i a H i s t o c h e m . C y t o c h e m . 1 8 , 3 3 - 4 0 (1980).

S.:

3. S l o m i n s k i , A., S c i s l o w s k i , P., B o m i r s k i , A.: Int. J. B i o c h e m . 1 6 , 3 2 3 - 3 2 6 (1984). 4. S c i s l o w s k i , W . , S l o m i n s k i , A., B o m i r s k i , A.: Int. J. B i o c h e m . 1 6 , 327-331 (1984). 5. M i y a m o t o , K . , T e r a s a k i , T.: C a n e . R e s . 40, (1980).

4751-4757

6. P a r k , B.H., F i k e , R . M . , K i m , U.: I R C S M e d . B i o c h e m . 1 0 , 9 6 - 9 7 (1982).

Science

7. R o m a n i , N., S c h ü l e r , 6 . , P r i t s c h , P.: A r c h . R e s . 275, 3 9 7 - 4 0 2 (1983).

Dermatol.

8. N i c o l s o n , G . L . : B i o c h i m . B i o p h y s . A c t a 695.

113-176

(1982).

9. W e i s s , L.: in F u n d a m e n t a l A s p e c t s of M e t a s t a s i s , N o r t h H o l l a n d P u b l i s h i n g C o m p . A m s t e r d a m 1976. 10. S i m o n - R e u s s , J . , C o o k , G . M . V . : C a n e . R e s . 24, (1963).

51-70,

2038-2045

11. B o s m a n n , H . B . , B i e b e r , G . F . , B r o w n , A . E . , C a s e , K . R . , G e r s t e n , D . M . , K i m m e r e r , T . W . , L i o n e , A.: N a t u r e 246, 4 8 7 - 4 8 9 (1973). 12. K o z l o w s k a , K., B o m i r s k i , A . , Z u r a w s k a - C z u p a , A r c h . D e r m . R e s . 256, 1 9 7 - 2 0 3 (1976).

B.:

13. K o z l o w s k a , K., B o m i r s k i , A., Z u r a w s k a - C z u p y , B.: A r c h . I m m u n o l . T h e r a p i a e E x p t l . 25, 1 0 7 - 1 1 0 (1977). 14. E i s e n b e r g , S., B e n - O r , S., D o l j a n s k i , F.: E x p . Cell R e s . 26, 451-461 (1962). 15. Elul, R., B r o n s , J.: N a t u r e 258, 6 1 6 - 6 1 7

(1975).

ELECTROPHORESIS OF MOUSE LEUKOCYTES AND LEUKEMIA CELLS

Oan Bubenik Institute of Molecular Genetics, Czechoslovak Academy of Sciences, 166 37 Prague Dana Bubenikovi Institute of Hygiene and Epidemiology, 100 42 Prague, Czechoslovakia

Int roduction The kinetics of the cell populations involved in leukemias and related neoplasms is one of the crucial factors influencing progression, remissions and therapy of these diseases. In order to study the cell population kinetics, reliable markers of the cell populations are needed. It has been previously shown that mean electrophoretic mobility (EPM) represents one of the relatively stabile characteristics of various populations and subsets of cells, such as macrophages (1-4), lymphocytes, thymus cells, erythrocytes (5) and sarcoma cells (6). Therefore, the method of cell electrophoresis has been used in this study to investigate and compare the electrokinetic properties of the leukemic blast populations present in various histological types of leukemias. It is difficult to obtain pure populations of leukemic blasts, and various procedures used to purify leukemic cells may alter the EPM of the blasts. An alternative approach has been proposed (7), which is based on simultaneous assessment of EPM and diameter of individual cells in suspensions prepared from enlarged and heavily infiltrated leukemic lymph nodes. The mean EPM is calculated and considered separately

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

460 for two subpopulations differing in cell diameter. One subpopulation corresponds in size to normal lymph node cells (LNC, 6-9yum) and the other to large leukemic blasts

(LLB,

10-16yum). Using this approach, it is not possible to discriminate between small (6-9yum) leukemic blasts and normal LNC remaining in the infiltrated lymph nodes. However, it is possible to distinguish and study the characteristics of LLB in various histological leukemia types and cell

compartments.

Some of our findings have been previously reported

(7-9).

Material and methods Animals. Females from a closed colony of random-bred

ICR

Swiss mice (VELAZ, Prague, Czechoslovakia) with high incidence of spontaneous neoplastic diseases of and lymphoid tissues (5,7) were

hematopoietic

used.

Leukemias. Twenty-one primary leukemias, which

originated

in 194-694-day-old ICR Swiss mice, were examined. The

classi-

fication of leukemias was based on autopsy and histology pei— formed independently by two pathologists (7). The

original

structure was obliterated and replaced by neoplastic cells in all lymph nodes used for EPM examination (7). Lymph node samples from 6 mice with generalized thymic lymphoma or lymphosarcoma, one mouse with stem-cell leukemia, 9 mice with reticulum cell sarcoma and 5 mice w i t h myeloid were

leukemia

examined.

In addition, four long-transplanted thymic

lymphomas,

EL-4, EL-4R, BW5147 and BH2 (8,9), and normal ICR lymph node cell suspensions (5) were

examined.

Cell electrophoresis was performed as described in detail elsewhere

(5-8). Briefly, the automated analytical cell

electrophoresis apparatus, Parmoquant

(C. Zeiss, O e n a , G D R ) ,

equipped with a microcomputer and a data-printing

system,

was used to examine v a r i o u s reference cell populations

461

(erythrocytes, LNC, thymus cells). For detailed analysis of normal and leukemic lymph node cell populations, an Opton cytopherometer (C. Zeiss, Oberkochen, FRG) was utilized (52 3 8). A total of 2 x 10 - 2 x 10 cells from each cell population were examined. Cell diameter was measured during the EPM determination using an eyepiece

grid.

Nylon wool-adherent and nonadherent LNC were separated in plastic columns as described earlier (5). T h y 1,2* cells were Ig+

determined with monoclonal HO-13-4 a n t i b o d y and surface cells with fluorescein-labelled anti-immunoglobulin

(5).

Results and Discussion It has been previously reported (5) that normal ICR LNC show a distinctly bimodal pattern of electrophoretic

distri-

bution with the lowest point of the electrophoretogram at an EPM of - 0 . 9 5 . As can be seen in Table 1, grouping of LNC into faster and slower than this value gave a group of

fast-moving

LNC with the mean EPM value of - 1 . 1 8 and a group of slow-moving LNC w i t h the mean EPM value of - 0 . 7 6 . The majority of fast-moving LNC were nonadherent, T h y 1.2 + cells, whereas the majority of slow-moving LNC were nylon w o o l - a d h e r e n t ,

slg+

cells (Table 1). Both the expression of cell membrane

markers

and the percentage of the cells in ICR lymph nodes (Table 1) indicate that most of the fast-moving LNC have the

characte-

ristics of T lymphocytes and most of the slow-moving LNC have the characteristics of B lymphocytes. The typical

bimodal

pattern of the electrophoretic distribution is changed into a unimodal pattern due to the infiltration with leukemic (7). The analysis of lymph node electrophoretograms mice with primary leukemias reveals remarkable

blasts

from 21

heterogeneity

of the mean EPM values of individual leukemic cell populations (Table 2 - 4 ) . Despite this heterogeneity, some

general

conclusions can be made. The mean EPM of cells from primary thymic lymphomas ( - 1 . 1 3 ,

462 Table 1 Electrophoretic mobility s e p a r a t e d i n n y l o n w o o l columns Lymph node c e l l s

of

Mean anodic EPM in vim. s Slow c e l l s

Fast c e l l s

0.76+0.01

1.18+0.01

(25.5) Nylon wool-adherent

0.75+0.01

Nylon wool-nonadherent

0.75+0.01

1 ,,V 1 .cm

+ SE

Thy 1.2 + s l g +

Total

(%)

(«

1.09+0.02

72.0

23.6

0.82+0.02

8.7

91.3

1.13+0. 01

90.2

9.7

(%)

(%) Unfractionated

ICR LNC s u b p o p u l a t i o n s

(74.5) 1.16+0.01 (10.8)

(89.2)

1.16+0.01 (88.5)

(11.5)

Table 2 Thymic lymphomas. E l e c t r o p h o r e t i c s u b p o p u l a t i o n s from l e u k e m i c lymph nodes

mobility

of

cell

—1 —1 Leukemia

Mean anodic EPM (jjm.s

.V

. cm + SE) of c e l l s d i f f e r i n g in diameter

6 - 9 pm Slow c e l l s

(%)

10 - 16 vim

(%)

Total

Past c e l l s (%)

T1

0.70

(3)

1.20

(77)

1.13

(20)

1.17+0.03

T2

0.85

(1)

1.17

(33)

1.12

(66)

1.13+0.03

T3

0.90

(1)

1.20

(45)

1.09

(54)

1.14+0.03

T5

-

-

1.05

(14)

1.06

(86)

1.06+0.01

T6

-

-

1.19

(31)

1.05

(69)

1.09+0.04

T7

—

_

1.19

(68)

1.12

(32)

1.17+0.03

(D

1.18+0.03

(45)

(54)

1.13+0.04

Mean

0.75

1.09+0.03

463

Table 3 Reticulum c e l l sarcomas. Electrophoretic of c e l l subpopulations from leukemic lymph nodes

mobility

—1 —1 Leukemia Mean anodic EPM (nm.s . V . cm + SE) of cells differing in diameter 6-91® 10 - 16 nm 00 Slow cells (%) Fast cells (%) Total R 1

0.70

(1)

1..23

(57)

1.15

(42)

1..19+0.02

R3 R4

0.75 0.90

(1) (2)

1,.19 1..19

( 9) (80)

1.05 1.08

(90) (18)

1,.06+0.03 1,,16+0.03

1..22 1.23

(79)

1.24

(21)

1.,22+0.02

(91)

1.22

(53) (77)

1.08

( 9) 1,.23+0.02 (44) 1..12+0.04

R6 R7 R8 R10 R11 R12 Mean

-

-

-

-

0.85 0.80

(3)

1,.16

(3)

0.90

(1)

1,.19 1,.20

-

0.83

-

(1)

1..18

( 8) (62)

1.20+0.05

(57)

1.09 1.06

(20)

1.08

(91) (38)

1.12+0.07

(42)

1,.16+0.03 1,.07+0.03 1,.14+0.03 1,.15+0.06

Table 4 Myeloid and s t e m - c e l l leukemias. Electrophoretic m o b i l i t y of c e l l subpopulations from leukemic lymph nodes Leukemia Mean anodic EPM (pm.s (Myeloid) diameter 6 - 9 vim Slow cells

(50

Fast cells

-

1 . 22

0..75

(2)

M4 M5 M6

—1 —1 . V .cm + SE) of cells differing in 10 - 16 pm 00 00

Total 1. 27

1.16

(47) (52) (53) (86)

1.17 1. 23 1. 23

(53) 1,,25+0. 03 (46) 1,.16+0. 02

M7 M8

-

1.19 1. 23

0..75

(4)

1.18

(48)

1.15

(47) 1..21+0. 02 (14) 1,.23+0. 03 (48) 1.,15+0. 02

Mean

0.,75

(1)

1.20+0.03

(57)

1.21+0.05

(42) 1,.20+0. 04

Stem-cell S1 0.,80

(3)

1. 20

(28)

1. 06

(69) 1,.09+0. 03

-

464 Table 2) was similar to that of primary reticulum cell sarcomas (-1.15, Table 3) but significantly ( P < 0.025) lower than that of myeloid leukemias (-1.20, Table 4). Similar and even more pronounced differences among the three groups of leukemias were noted when the LLB cell populations were

conside-

red. The mean EPM of LLB from thymic lymphomas ( - 1 . 0 9 , Table 2) and reticulum cell sarcomas ( - 1 . 1 2 , Table 3) were

similar

and significantly ( P < 0.005 and P < 0.025, respectively)

lower

than that of LLB from myeloid leukemias ( - 1 . 2 1 , Table 4). Interestingly, the mean EPM ( - 0 . 8 5 + 0.06yum.s""*".V'^.cm) of the group of four long-transplanted

thymic lymphoma cell

lines,

EL-4 (-0.91 + 0.01), EL-4R (-0.96 + 0.05), BV75147 (-0 . 69 + 0.02. and BH2 ( - 0 . 8 3 + 0.05), was significantly ( P < 0.001) lower than that of primary thymic lymphomas ( - 1 . 1 3 , Table 2). In the 6-9yum cell subpopulations

from leukemic lymph nodes

(Table 2-4) electrophoretically slow cells corresponding EPM to B lymphocytes were almost completely depleted

in

(1%),

irrespective of the histopathological classification of the leukemia. The decrease in the number of T LNC could not be assessed, since the approach used did not allow us to discriminate between normal LNC and small leukemic blasts in the 6-9yum cell

subpopulation.

References 1. Bubenik, 3., Indrová, M., Némecková, §., Malkovsky, M., Vo n Broen, B» , Palek, A n d r l í k o v á , 0 • : In t • 3 • Csncer 2 1 , 348-355 (1978). 2. M a l k o v s k y , M., Bubenik, D., Holán, V. , Hasek, M.: Folia biol. (Prague) 25, 17S-187 (1579). 3. Halkovsky, M., Bubenik, 3., Boubelík, M., Hausner, P.: T r a n s p l a n t a t i o n 28, 121-124 (1579). 4. Bubenik, 3.. Cochran, A . , T o d d , G. , Malkovsky, M., Oandlová, T . , Suhajová, E. , Boubelik, M.: Neoplasma 28, 185-193 (1581). 5. Bubenik, 0., Bubeniková, D., Lastovicka, 0., Dandlová, T.: Neoplasma 28, 517-525 (1581).

465

5. Bubenik, 3. , 3andlovä, T. , Suhajovä, E. , Malkovsky, H. : Neoplasma 26, 499-501 (1579). 7. Bubenikovä, D., Bubenik, 3.: Neoplasma 30, 691-700

(1983).

8. Bubenik, 3., Perlmann, P . , 3onsdöttir, I., Kypenovä, H. , Bubenikovä, D. , 5imova, 3.: Brit. 3. Cancer 44, 692-699 (19 01). 9. Bubenik, 3., Bubenikovä, D., 3andlovä, T . , Perlmann, P., Biberfeld, P.: Tumour Progression and Markers, Kugler Publ. , Amsterdam, p. 361-370 (1582).

A P P L I C A T I O N OF THE C E L L E L E C T R O P H O R E S I S OF L E U K E M I C

FOR

CHARACTERISATION

CELLS

Jerzy Holowiecki, Krzysztof

Jarczok, Krystyna

D e p a r t m e n t of H e m a t o l o g y , S i l e s i a n M e d i c a l 40-029 Katowice, Poland

Jagoda

Academy

Introduction The m a i n a i m of our i n v e s t i g a t i o n was to find out cell e l e c t r o p h o r e s i s

whether

(CE) can be c o n s i d e r e d a u s e f u l

for e x a m i n i n g l e u k e m i c a n d l y m p h o m a c e l l s . In

particular

we w a n t e d to k n o w if C E is an e f f i c i e n t m e t h o d of out w h e t h e r the g r o w t h p r o c e s s

originates

tool

finding

from the B or T

cell line, but at the same time we w e r e i n t e r e s t e d in the q u e s t i o n of a p o s s i b l e c o r r e l a t i o n b e t w e e n CE r e s u l t s the F A B c l a s s i f i c a t i o n .

and

Our f i r s t e x p e r i e n c e w i t h C E in

n e c t i o n w i t h l e u k e m i c p a t i e n t s s h o w e d us that these g a t i o n s w o u l d i n v o l v e p a r a l l e l t r i a l s . For t e c h n i c a l

con-

investirea-

sons we i n v e s t i g a t e d w h e t h e r cells s u b m i t t e d to C E m a y be s t o r e d at l o w t e m p e r a t u r e s

b e f o r e m e a s u r e m e n t . It

appeared

a l s o i m p o r t a n t to look for a g e n t s w h i c h m i g h t i n f l u e n c e m e m b r a n e p o t e n t i a l , s u c h as n e u r a m i n i d a s e , or m o n o c l o n a l a n t i b o d i e s

electric

the

field

(Mo-Ab).

Materials and Methods We e x a m i n e d 24 h e a l t h y b l o o d donors a n d 44 p a t i e n t s

with

l e u k e m i a a n d l y m p h o m a . M o n o n u c l e a r cells were i s o l a t e d by density gradient centrifugation. PARMOQUANT-2

CE was p e r f o r m e d w i t h the

(VEB Carl Zeiss J E N A ) a p p a r a t u s

(1,2). The

cu-

m u l a t i v e m e t h o d of e x a m i n a t i o n was u s e d - e a c h a n a l y s i s

was

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

468 b a s e d on c o u n t i n g 900 cells in t o t a l . B e f o r e e a c h m e a s u r e m e n t , the m o b i l i t y of r e d cells f r o m h e a l t h y i n d i v i d u a l s was —1 —1 a n d was f o u n d to range f r o m 0.96 jam sec V cm to 1.1 - 1

sec

checked

- 1

V

cm. The a p p a r a t u s was r e c a l i b r a t e d w i t h the s a m e

red

cells a f t e r e a c h m e a s u r e m e n t , and, if this r e s u l t d i d n o t fer f r o m the o r i g i n a l c a l i b r a t i o n by more t h a n + 0.02 V ly

sec

cm, the t e s t was r e g a r d e d as h a v i n g b e e n p e r f o r m e d . To i d e n t i f y any i n f l u e n c e

of l o w t e m p e r a t u r e

correct»

storage,

some of l y m p h o c y t e s were e x a m i n e d i m m e d i a t e l y a f t e r

isolation

a n d the r e m a i n d e r were s u s p e n d e d in a f r e e z i n g m i x t u r e ml P a r k e r m e d i u m ,

0.1 ml DMS0 a n d 0.2 ml s e r u m . The

analysis

(1), d e f r o z e n a n d then t e s t e d by CE. c o n s i s t e d of S t u d e n t ' s

of 0.7

latter

f r a c t i o n was f r o z e n a n d c r y o p r e s e r v e d for 10 days as elsewhere

described

Statistical

"t" t e s t for c o r r e l a t e d

a b l e s . To d e t e r m i n e the i n f l u e n c e

dif-

of n e u r a m i n i d a s e the

variery-

t h r o c y t e s f r o m 14 h e a l t h y donors were i n c u b a t e d w i t h n e u r a m i -4 -4 nidase at c o n c e n t r a t i o n s of 3.25 x 10 U / m l to 65 x 10 U/ml a n d h i g h e r for one h o u r . To d e t e r m i n e the i n f l u e n c e of m o n o clonal antibodies

lymphocytes

chronic

from 11 p a t i e n t s w i t h

l y m p h o c y t i c l e u k e m i a (CLL) were i n c u b a t e d w i t h V I B - C 5 or E3 M o - A b ( a g a i n s t B c e l l s ) , w i t h c o m p l e m e n t , a n d w i t h m e n t + M o - A b . The s u s p e n s i o n s

of m o n o n u c l e a r

cells

(10 / m l )

were w a s h e d t w i c e w i t h P B S before i n c u b a t i o n w i t h the substances

VIB-

compleabove

(mo-Ab at a c o n c e n t r a t i o n of 5 n g / m l ) at 4" C for

30 m i n , t h e n w a s h e d twice and u s e d for

testing.

Results EM of b l o o d l y m p h o c y t e s

s t o r e d at - 1 9 6 ° C.

The EM of

lympho-

cytes before f r e e z i n g r a n g e d from 1.0 to 1.09 (mean 1 . 0 4 ) after defreezing,

f r o m 0.96 to 1 . 0 8 (mean 1.03). The

lity of the l y m p h o c y t e s

and

viabi-

as e s t i m a t e d by the t r y p a n blue

s i o n t e c h n i q u e was 92 % b e f o r e f r e e z i n g a n d 86 % a f t e r

excludefree-

z i n g . F i g . 1 p r e s e n t s the h i s t o g r a m s y i e l d e d by our t e s t s

be-

469 fore (A) and after (B) storage in liquid nitrogen.

Fig. 1 EM of cryopreserved lymphocytes (A before, B after)

Influence of neuraminidase on EM. The results are presented in Pig. 2. The EM of untreated erythrocytes from healthy donors was 1.06, but treatment with neuraminidase reduced the EM distinctly to an extent depending on the neuraminidase concentration: even very low concentrations of 3.25 x -4 10

U/ml slowed the EM down by about 20 %, and a concentra-

tion of 65 x 10 ^ U/ml reduced the EM to about one third of the untreated value. Higher neuraminidase concentrations induced no further reduction plateau at this level.

in the EM, the curve reaching its

470

Fig. 2 EM of e r y t h r o cytes t r e a t e d with neuraminidase

3.25x10""

26x10~*

Influence

52*10~A65*10^

130

of m o n o c l o n a l a n t i b o d i e s

xlO'4-

on EM.

The m e a n EM of

u n t r e a t e d l y m p h o c y t e s was 0.97 + 0.04; it was 0.97 +

0.04

a f t e r t r e a t m e n t w i t h m o u s e c o m p l e m e n t , 0 . 9 8 + 0.04

after

t r e a t m e n t w i t h V I B - C 5 M o - A b , a n d 0.95 + 0.04 a f t e r

treatment

with mouse complement and VIB-C5 Mo-Ab The r e s u l t s d e m o n s t r a t e ,

(A

t h a t t h e r e is n o

f e r e n c e b e t w e e n the EM of C L L l y m p h o c y t e s

not

significant).

significant incubated

dif-

with

V I B - C 5 M o - A b , V I B - C 5 + c o m p l e m e n t a n d c o m p l e m e n t only. r e c e n t l y p e r f o r m e d p i l o t tests w i t h V I B - E 3 a n d V I M - D 5 Ab s u g g e s t h o w e v e r , t h a t a l t h o u g h the m e a n EM is n o t

Our Mo-

affected,

the i s t o g r a m s m a y c h a n g e , a n d this i m p l i e s t h a t only a p a r t of the l e u k e m i c cells are i n f l u e n c e d by M o - A b . In other

words

471

the proportion of cells reacting with Mo-Ab may be relevant. Our investigations into the influence of VIE-G4 Mo-Ab (against glycophorin A) on the EM of red cells met difficulties owing to clumping even when very low concentrations of VIE-G4 Mo-Ab were used. EM of CLL lymphocytes•

Lymphocytes isolated from the blood

of healthy individuals showed a mean EM 1.00 + 0.06. The histogram is an assymetric curve (Fig. 1 A). In 15 patients with CLI (Pig. 3) the mean EM was 1.01 + 0.04, all patients having a similar value. A slower fraction with an EM of 0.93 + 0.05 was observed in one case only. The final diagnosis in this patient was prolymphocytic leukemia, which took a rather severe clinical course. EM of mononuclear cells of ALL.

The EM values ranged from

0.91 to 1.09, and the histograms revealed a predominantly uniform population (Pig. 4). Comparison with the histograms of normal lymphocytes revealed a striking difference is the lack of a distinct low mobility fraction. In this preliminary

060 Fig. 3

OX

QtO

090

100

W

IX

IX

M0 EM

EM of CLL lymphocytes

an 0» 0.95 e.u. Fig. 5 shows the PVD-curves in both normal subjects and patients with AL, which were performed parallel with the EPM measurements. The relative PVD-curves for patients suffering from AL w.r. displayed a shift towards smaller thrombocytes (p < 0.01). In cases with a remission the relative PVD-curves 1.1

0.89i0.01

0.89*0.02

N = 36

0.91*0.03 N = 7

0.94*0.05 N =

38

0.91 *0.02

0.99*1104

N = 23

N =

15

Fig. 1 1.0.

0.9-

:I

II

0.8. complete [ e m E s m port id rwrossKn withajt remission

acute

. . leukemia

without bleeding with H e e d i n g

acute leukemia without remission

Mean EPM of platelets of patients with acute leukemia in different stages and comparison between patients with and without bleeding

507

Fig. 2 Cytopherograms of all measured thrombocytes of patients in acute leukemia (AL), a.: normal subjects (N = 1088), b.: AL without remission (N = 1288), c.: AL in complete remission (N= 220), d.: AL in partial remission (N = 243), N represents the number of measured cells

EPM

resembled that of normal subjects more closely (Fig. 5). When the thrombocytes of patients suffering from AL w.r. were examined under an electron microscope they displayed a slightly more spherical form (Fig. 6), whereas the granulomers in comparison to normal thrombocytes are showing distinct changes in form of vacuolic-like structures (Fig. 7). Furthermore, in such a case no well-defined particles of glycogen can be observed and the cell membrane appears also partly altered.

Fig. 3

1.1

1.2

1.3

1.4

EPM MO'Wsec'V '3

Cytopherograms of all measured thrombocytes of patients in acute leukemia without remission a.: without bleeding (N=796) and b. : with bleeding (N=513), N represents the number of measured cells

508

pat. C.S. j

~

1 -1

5

1.0

JE

pat. U.H. 0.95 e . u . )

i n d i c a t e t h a t a hemorrhagic

t h e s i s can be expected ( F i g .

4).

The c o n t r a r y ,

i.e.

dia-

a reduction

in the tendency t o b l e e d may be assumed in the case of a n o r mal EPM. For i n s t a n c e , case of

t h e r e was no b l e e d i n g tendency in one

chronic myeloid leukemia w i t h a normal EPM of

predominantly small p l a t e l e t s

the

although t h e i r number was only

1 Gpt/1. Our i n v e s t i g a t i o n s

are unable t o e x p l a i n the e l e v a t e d number

of p l a t e l e t s w i t h an i n c r e a s e d n e g a t i v e charge in cases of AL w . r . w i t h hemorrhaging.

Possible

causes may be a r e d u c t i o n

the numbers of the amino groups or a change in the content of the thrombocyte s u r f a c e as a r e s u l t

in

phospholipid

of t h e i r

activa-

tion. The parameters

obtained by conductance measurements,

i n the u l t r a s t r u c t u r e t r o n microscopy,

by changes

of the granulomers e s t a b l i s h e d by e l e c -

and by the c e l l e l e c t r o p h o r e t i c

measurements

of the net s u r f a c e charge suggest that AL w . r . w i t h hemorrhag i n g i s probably a s s o c i a t e d w i t h p a t h o l o g i c a l thrombocytes.

changes in the

The measurement of the volumes and EPM of

bocytes can enhance the i n f o r m a t i o n content of the

throm-

thrombocyte

p r o f i l e in cases of AL and may be u s e f u l as an i n d i c a n t thrombocyte

for

substitution.

References 1. Eldor, A . , A v i t z o u r , R., Or, R., Hanna, R., B r i t . Med. J. 285, 397-400 (1982).

Penchas,

S.:

2. Hamberg, M., Svenson, J . , Wakabayashi, T . , Samuelsson, Proc. Nat. Acad. S c i . ( U S A ) 71, 345-349 (1974). 3. D i e d e r i c h , G. , Kriiger, U. : Z. med. Labor-Diagn. 155-162 ( 1 9 8 1 ) .

22,

4. Schutt, W. : Laborpraxis in der Medizin 3,

(1981).

16-26

5. Anders, 0. , Thomaneck, U. , K e l l e r , D. , Konrad, H. , Giinther, I . , Lakner, V . : Wiss. Z. d. W i l h . - P i e c k - U n i v . 32 ( 7 ) , 84-88 (1983).

B.:

Rost.

512

6. Thorn, R., Hampe, A . , Sauerbrey, 151, 331-349 (1969).

G.: Z. g e s .

exper.

Med.

7. Kachel, V. , Metzger, H. , Ruhenstroth-Bauer, exper. Med. 153, 331-347 (1970).

G. : Z.

ges.

8. F r o j m o v i c , M.M., M i l t o n , 261 (1982).

J.G.:

Physiol.

9. Bessman, J . D . , W i l l i a m s , L . J . , Gilmer, c l i n . Path. 78, 150-153 (1982). 10. G i l e s , 11. Nelson,

C.: B r i t .

J. Haematol.

R . B . , Kehl,

Rev. 62,

185-

P . R . : Amer.

48, 31-37

J.

(1981).

D.: Cancer 48, 954-956

(1981).

12. Dumoulin-Lagrange, M., Tirmache, M., Cousten, B., Hotchen, M., Samama, M.: Acta haemat. 71, 25-31 (1984). 13. Nouvel, C., Caranobe, C., S i e , P . , C a p d e v i l l e , J . , P r i s , J . , Boneu, B . : Scand. J. Haematol. 2±, 421-426 (1978). 14. C a g l i e r o , E., P o r t a , M., Cousins, S . , Kohner, E.M.: Haemostasis 12, 293 (1982). 15. Seaman, G . V . F . : Thrombosis Research 8 (Suppl. I I ) , 235246 (1976). 16. Sherbet, G . V . : "The B i o p h y s i c a l C h a r a c t e r i s a t i o n of the C e l l S u r f a c e " , pp 68-78, Acad. Press London - New York San F r a n c i s c o , 1978 17. Mehrishi,

J . N . : Prog.

Biophys. Mol. B i o l .

18. Mehrishi,

J . N . : Eur. J. Cancer 6, 127-137

25, 1-70 ( 1 9 7 0 ) . (1970).

19. Gröttum, K . A . , Solum, N.O.: B r i t . J. Haematol. 1_6, 277290 (1969). 20. Gröttum, K . A . : Scand. J. Haematol. H , 166-176 (1973).

INFLUENCE OF EXOGENIC FACTORS: RADIATION/ STRESS/ DRUGS/

MITOGENS

ANTIBODIES/

CELL ELECTROPHORETIC

MOBILITY

A S A N A I D TO S T U D Y

BIOLOGICAL

SYSTEMS

K . A.

Chaubal

Biophysics Division, Cancer Research Institute, Centre, Parel, Bombay 400 012, India

Tata

A n a r e a of i n t e r e s t t h a t c o n t i n u e s

a

revolves

a r o u n d the m e m b r a n e

to challange

of a l i v e a n i m a l

difference

of t h e o r d e r

across

it r e v e a l s

holes

ebonite.

A n o t h e r i l l u s t r a t i o n w o u l d be of t h e t o t a l

other membranes

of a n i n d i v i d u a l .

of m a t e r i a l

electric

N a t u r e has

created such conditions

lamp illuminated

with a purpose

activity

of

poten-

would charge

it w o u l d

for several

years.

of u n b e l i e v a b l e

a n d it is h e n c e

u n d e r s t a n d the purpose and to r e l a t e electrical

has

as t o u g h as

If it is c i r c u l a t e d ,

an average

in living systems

a

of 1 0 0 , 0 0 0 V o l t s / c m w h i c h

puncture the cells

into

ulti-

The Nature

some amazing properties. An estimate

its w i d t h a n d p o t e n t i a l tial gradient

researcher

c e l l - its

m a t e m o d e l a n d its a l m o s t b a f f l i n g p r o p e r t i e s . e n d o w e d the m e m b r a n e

Memorial

on keep

The

magnitude

essential

to

i t , if p o s s i b l e , w i t h

associated w i t h the membranes

of

the

live

cells . The membrane

of a c e l l b e a r i n g e l e c t r i c a l

charges

p a r e d to a c h a r g e d c o n d e n s e r w h e r e the a d d i t i o n ge n e c e s s i t a t e s

performance

d e d is s t o r e d i n t h e cell perhaps

has

of e v e r y

of w o r k a n d t h e e n e r g y t h u s

condenser.

Likewise,

s t o r e d i n a c e l l m e m b r a n e by v i r t u e the

could be

its o w n w a y s

of its

charexpen-

the energy may charge

and

of t a p p i n g it as p e r

com-

be

the its

needs. The presence

of c h a r g e

the comparative sition,

on a c e l l m e m b r a n e

properties

temperature

etc.

seems

t o be d u e

s u c h as p H , i o n i c s t r e n g t h , of t h e m e d i u m b a t h i n g t h e

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

to

compo-

cell.

The

516

ultimate result of the interaction between a cell and the surrounding medium could be an equilibrium between the two which culminates, amongst other things, into the development of an appropriate electrical charge on the membrane. Its magnitude may vary with the physiological status of the cells and also with the position of the cell in the 'cell cycle*. The association of electric charge with the cell membrane is realised when a live cell moves in an electric field (1,2) and also when a microelectrode in close contact with the membrane registers an electric potential with reference to another electrode kept in the surrounding medium. These two parameters are on the decline when the death of a cell approaches (3,4-) • Hence, in order to translate the biological functions and properties of a cell membrane into appropriate physical principles , dependent on its charge, it is essential to measure, directly or indirectly, the magnitude of its charge. A number of procedures, some of these very elaborate, have been designed to measure the cell electrophoretic mobility (EPM) and therefrom, by calculations, the cell surface charge or potential. We have contributed to a procedure where the cell EPM is measured by observing the motion of a cell in a cylindrical capillary, under

the action of vertically directed electrical field (5).

The electrophoresis chamber is easy to fabricate, sturdy, simple to operate and reliable (Fig. 1). It permits repeated observations on the same cell, without causing its displacement from the stationary layer (Table 1). The effect of gravity, which causes such as displacement in procedures where the cell under electric field moves in»a horizontal direction, is either eliminated or minimized by raising the density of buffer medium. Glycerol, which is non-toxic to the cells and which is routinely used during freeze-preservation of cells, is suitable for this purpose. The other suitable material is Percoll. The EPM of a cell is obtained by measuring the velocity of the cell twice, once when the electric field assists residual

517

Fig. 1 A schematic diagram of electrophoresis chamber with cylindrical capillary, platinum electrodes in the cups A and B and a teflon tubing connecting cups B and C

ELECTRODE

gravity, if any, and the other when it opposes gravity. In EPM measurements, the distance between the divisions of a scale in the eyepiece serves to measure the linear movement of a cell. The magnification of the scale by the eyepiece remaining same, it is often debated whether the total magnification of the object should be high or low. At higher magnification, the cell appears enlarged and the observations on its movement made with reference to a region of its periphery may become vitiated if the cell rotates about itself during its motion. At lower magnification, the cell will appear almost as point object and its motion could be observed with reference to the whole cell-periphery. Hence, lower magnification, xIO, of the objective is preferable. It has another advantage that it permits simultaneous observation of cells in both the stationary layers, thereby increasing the scope of work (Fig. 2).

518 Table 1 Electrophoretic mobility measurements human erythrocyte Velocity N° of Observation

Lower electrode +ve (V T )

on a s i n g l e

()*m/s)

lower Electrode -ve (V„)

Electrophoretic Mobility V

1

+ V

2

2

/

/

1

3..63

2 .46

0..94

2

3..72

2,.67

0..99

3

3..30

3..06

0..98

4

3..53

3..55

1 .09 .

5

3,.52

3,.50

1 .08 .

6

3..28

3..08

0..98

7

3..28

3 .00

0..97

8

3..28

2..82

0..94

I

D

D i s t a n c e b e t w e e n e l e c t r o d e s (D), 1 5 - 5 cm; d i s t a n c e t r a v e l l e d by the cell, 69 n m > v o l t a g e (V), 50 V; c u r r e n t , 0.29 mA; e l e c t r o p h o r e s i s m e d i u m , G M 5 ; r o o m t e m p e r a t u r e 27°C, A v e r a g e e l e c t r o p h o r e t i c m o b i l i t y (EPM), 0.99 + 0.02 ]im/s/V/cm. G M 5 : P h y s i o l o g i c a l s a l i n e w i t h 5 % G l y c e r o l , pH 7 . 2 A care w h i c h n e e d s to be s t r i c t l y t a k e n in EPM w o r k is t h a t of meticulous

cleaning, with scrubbing,

of the i n n e r s u r f a c e

of

the c a p i l l a r y in w h i c h the cells m o v e . This is b e c a u s e , no t e r w h a t s o a p or d e t e r g e n t is u s e d , the i n n e r s u r f a c e c o a t e d w i t h c e l l u l a r m a t e r i a l a f t e r a few sets S u c h cell e l e c t r o p h o r e s i s

of

gets

observations.

a p p a r a t u s have b e e n e x t e n s i v e l y

p l o y e d to m e a s u r e the EPM of a v a r i e t y of c e l l s . The EPM f i l e s , thus o b t a i n e d , m a y be of l i m i t e d i n t e r e s t b e c a u s e d i f f e r e n t cell types

mat-

c a n n o t be a t t r i b u t e d by d i s t i n c t i v e

empro-

the EPM

v a l u e s . H o w e v e r , more p r a g m a t i c a p p l i c a t i o n of EPM m e a s u r e m e n t is s e e n in s t u d i e s w h e r e the cells of a g i v e n type s u b j e c t e d to v a r y i n g e x p e r i m e n t a l c o n d i t i o n s , s u c h as

are

irra-

519

Fig. 2 S c h e m a t i c d i a g r a m of v i e w t h r o u g h the e y e p i e c e s h o w i n g the s c a l e a n d edges of the c a p i l l a r y . The curves of u p p e r p a r t are EPM Vs d i s t a n c e a l o n g the s c a l e at t w o d i f f e r e n t p H of the b u f f e r a n d s h o w p o s i t i o n s of s t a t i o n a r y layers

EYE-PIECE

VIEW

( Mognificotion- 1mm of «ye piece s c a l e > 0 109 mm at object plane )

d i a t i o n , h e a t i n g , d r u g t r e a t m e n t , t u m o r i n d u c t i o n etc. In s u c h studies, conclusions

are d r a w n by m e a s u r i n g the a v e r a g e

cell

E P M b e f o r e a n d a f t e r a p p l y i n g the s t r e s s e s . A f e w t y p i c a l

stu-

dies of b i o l o g i c a l s y s t e m s u s i n g cell EPM as a m e a s u r a b l e

para-

m e t e r are d i s c u s s e d

below.

E l e c t r o p h o r e t i c m o b i l i t y of cells in r a d i o b i o l o g i c a l

studies

A n i m p o r t a n t a s p e c t of r a d i o b i o l o g i c a l w o r k is to s e a r c h for c h e m i c a l s w h i c h w i l l act as r a d i o - s e n s i t i z e r s tissues

and r a d i o - p r o t e c t o r s

of

cancerous

of n o r m a l t i s s u e s . T h e r e is n o

u n i v e r s a l t e s t s y s t e m for this p u r p o s e . The i n f o r m a t i o n the s u r v i v a l curves

from

of E. coli B/r or the c o l o n y g r o w t h of

cells in t i s s u e c u l t u r e or the life s p a n a n d w e i g h t s

of

organs

of e x p e r i m e n t a l a n i m a l s , s u b j e c t e d to i r r a d i a t i o n , m a y or m a y n o t agree w i t h e a c h other. The e x p l o r a t i o n of this

information

is f i n a l l y for the b e n e f i t of the h u m a n b e i n g a n d h e n c e ,

quite

520

Fig. 3 EPM d a t a of h u m a n a m n i o n cells t r e a t e d w i t h barbiturates

TIME IN HOURS A F T E R IRRADIATION ( D 0 9 E - 5 0 0 rod )

often, i n f e r e n c e s are d r a w n f r o m a c o m m o n p a t t e r n n o t i c e d the m a j o r i t y of s u c h o b s e r v a t i o n s . One can a d d to t h e s e

in

crite-

r i a the a v e r a g e EPM of cells, m e a s u r e d at s e v e r a l

intervals,

a f t e r i r r a d i a t i o n w i t h a s u b l e t h a l dose. S u c h EPM

patterns

l e a d to m e a n i n g f u l

conclusions(6.7.8),

out in the f o l l o w i n g F i g u r e s In this w o r k

a n d t h i s has b e e n

brought

3 and 4.

(7,8) the cells were i r r a d i a t e d in the p r e s e n c e

a b s e n c e of c h e m i c a l s s u c h as b a r b i t u r a t e s

a n d h e p a r i n . It is

s e e n t h a t the r e c o v e r y of EPM a f t e r i r r a d i a t i o n is m o r e

rapid

Fig. 4 EPM d a t a of h u m a n a m n i o n cells t r e a t e d w i t h h e p a r i n

or

521 Table 2

E l e c t r o p h o r e t i c m o b i l i t y of

Cell Types

cells

Electrophoretic Mobility jim/sec/V/cm

Mouse

Control

- 0 .85 + 0..05

Fibrosarcoma

Heparin treated

- 1 .42 + 0..08

Dextran sulfate treated

- 1 .20 + 0,.16

Ascites Cells

Control

- 1 .09 + 0..17

Heparin treated

- 1 .64 + 0..21

Dextran sulfate treated

- 1 .36 4- 0..12 4

C o n e , of H e p a r i n = 0.2 m g / m l / 2 x 10

cells

C o n e , of D e x t r a n s u l f a t e = 1 m g / m l / 2 x 10^

cells

for cells t r e a t e d w i t h these c h e m i c a l s . It is a l s o

noticed

t h a t the s l o p e of the above curves in Fig. 4 over a s e l e c t e d r e g i o n , g i v e s the rate of r e c o v e r y of EPM a n d shows a h i g h e r rate for h e p a r i n t r e a t e d cells . Since the i n f o r m a t i o n

from

Fig. 3 and 4 is in a g r e e m e n t w i t h that f r o m the other

tests,

the EPM d a t a of the a b o v e n a t u r e m a y be u s e f u l in ding radio-protectivity/sensitivity studies

understan-

of c h e m i c a l s . The r e c e n t

(not r e p o r t e d ) on I n s u l i n p o i n t out t h a t it is

radio-

protective .

E l e c t r o p h o r e t i c m o b i l i t y of cells in the s t u d y of in

metastasis

cancer

The only l a b o r a t o r y p r o c e d u r e

of s t u d y i n g e x p e r i m e n t a l

meta-

s t a s i s is to i n j e c t r a d i o a c t i v e l y l a b e l l e d t u m o r cells, t h e i r s u r f a c e charge m o d i f i e d c h e m i c a l l y ,

if r e q u i r e d ,

with

through

the tail v e i n of l a b o r a t o r y a n i m a l s a n d to e n q u i r e on the l o d g e m e n t of cells on d i f f e r e n t o r g a n s . The a n o d i c EPM of a s c i t e s a n d M F S t u m o r cells t r e a t e d w i t h h e p a r i n a n d

dextran

522

Table 3 O r g a n d i s t r i b u t i o n s t u d i e s on m o u s e f i b r o s a r c o m a (MFS) and a s c i t e s cells in s w i s s m i c e at v a r i o u s i n t e r v a l s Treatment

Mouse

Time after Inoculation

Cells

(%) l o d g e d in v a r i o u s Kidney

organs

Lungs

Liver

Spleen

24 hr

0,.12

0,.64

2,.26

0..20

72 hr

0..10

0 .76

1 .94 ,

0..18

24 hr

0..12

1 .00 ,

2..35

0..16

72 hr

0..10*

1 .46 ,

2..70

0,.15

24 hr

0..16

1 .85 ,

3..07

0..50

72 hr

0,.10*

1 .61 .

2..90

0,.10

24 hr

1 .59 .

2..37

4..11

1 .27 ,

72 hr

1 .45 .

4 .15

8..12

1 .16 ,

24 hr

2,.07

3 .66

7..78

1 .80 ,

72 hr

1 .76 .

4 .55

8..35

1 .33 ,

24 hr

2 .71

4 .06

8..91

2 .77

72 hr

1 .98 ,

5 .64

12,.47

2..07

fibrosarcoma

Control

Dextran Sulfate treated Heparin treated

A s c i t e s t u m o r cells Control

Dextran Sulfate treated Heparin treated * not

significant

s u l f a t e are s h o w n in Table 2 and f o l l o w the p a t t e r n : EPM of h e p a r i n t r e a t e d cells > EPM of d e x t r a n s u l f a t e t r e a t e d >

EPM of u n t r e a t e d cells

(9). F r o m the v a l u e s

cells

of Table 3, the

p r o p o r t i o n of h e p a r i n t r e a t e d M F S c e l l s , l o d g e d on the

organs,

is h i g h e s t a n d is f o l l o w e d by d e x t r a n s u l f a t e t r e a t e d cells u n t r e a t e d c e l l s . F u r t h e r , the l o d g e m e n t of u n t r e a t e d

and

ascites

cells w i t h h i g h e r a n o d i c EPM is m o r e t h a n t h a t of u n t r e a t e d M F S c e l l s . These r e s u l t s s u g g e s t t h a t the cells w i t h h i g h e r a n o d i c EPM or h i g h e r s u r f a c e n e g a t i v e c h a r g e m a y be m o r e

prone

523 to cause metastasis in cancer. Electrophoretic mobility of cells in the study of cell-to-cell interaction The forces responsible for the development of cell-to-cell contact may be many; but the more dominant of these appear to be dependent on the electrical activity associated with the surface of the two contacting cells . It has been hypothesized that one of the two contacting cells should possess a lower surface negative charge (10) i.e. lower anodic EPM. This becomes evident in the attempts made to understand the mechanism of cell-mediated cytolysis, where the interaction between nonadherent splenic cells of Swiss mice and syngeneic MFS tumor cells was investigated (11). The primary step in such an interaction is the development of contact between the killer cell and the target cell. The Table 4 depicts the EPM profiles of splenic cells from the normal and tumor (MFS) bearing Swiss mice after separating the cells into lower density ( < 1.057 to just < 1.069 gm/ml) and higher density (1.069 to just

1.087 gm/ml) groups on Percoll

step gradient. The % distribution values are with respect to Table 4 Percentage distribution of splenic cells with respect to an EPM of 0.85 Normal Mice EPM*

Splenic Cells < Lower density Higher density

Total

Tumor bearing Mice

0.85 21

EPM* >0.85

0.85

29

38

12

8

42

18

32

29

71

56

44

* EPM , electrophoretic mobility in )im/sec/Volt/cm

524

Table 5 Cytotoxicity of higher (H) and lower (L) density splenic cells from normal (N) and MFS tumor (T) bearing swiss mice towards MFS cells Cytotoxicity Exp. N°

T

L

T

N

H

Ratio of % Cytotoxicity L

N

H

T

L/TH

N

L/NH

T

L/NL

V

N

H

MFS cells (primary cultures) 1

57 ±

2

32 + 3

39 + 2

25 + 2

1 .8

1 .6

1 .5

1 .3

2

48 ±

2

32 + 2

38 + 2

22 + 1

1 .5

1 .6

1 .3

1 .3

3

28 t

2

19 + 1

22 + 1

16 + 1

1 .5

1 .4

1 .3

1 .2

4

18 + 2

15 + 1

16 + 1

12 + 1

1 .2

1.3

1 .2

1 .3

The percentage cytotoxicity values with SEM given above are from 4 independent observations. the average EPM of 0.85 jim/sec/Volts/cm

0f

the target MFS cells.

This information can be compared with the % cytotoxicity values of Table 5 from an independent experiment on similar cells 51 where the lysis of Cr labelled MFS cells by the splenic cells from normal and MFS tumor bearing mice was investigated. Of the sixteen ratios of % cytotoxicity in Table 5, thirteen are significant and three, of value 1.2, could not be taken with a degree of confidence. A comparison of the corresponding values of Tables 4 and 5 reveals, apart from other things, that the lower density splenic cells of tumor bearing mice are more cytotoxic than the lower density splenic cells of normal mice. This may be because, amongst the splenic cells of lower density, in tumor bearing mice, the cells with lower anodic EPM (
J antiserum :o tetanus antigen

572

tions and particularly in the structure of the surface membrane. These differences in surface composition my be reflected in electrophoretic mobility. Differences in the mobility of human (18,19,20,21,22) and animal (11,15) T and B lymphocytes are well known, but it may be supposed that further deep investigation of EPM will yield more detailed knowledge of lymphocyte subpopulation composition. It has been shown that cortison-susceptible and cortison-resistant thymocytes differ in terms of EPM, the former having a much lower value (23,24). Age-dependent changes in lymphocyte subset composition are accompanied by corresponding changes in the EPM of the cells (15,25-27). Ageing in mice is accompanied by an increase in the number of high mobility cells and a lower number of low mobility cells in the thymus. Various factors influencing the immune system induce changes in lymphocyte EPM. Cortison injection leads to the accumulation of fast moving cells in the mouse thymus (12). Injection of anti-T serum reduces the EPM of fast moving T cells in the mouse spleen (15). Thymectomy or chronic camrulation of the thoracic duct lead to a reduction in the percentage of high mobility cells (23,28). %

Titre

inhibition % of EPM inhibition - a PHA ( 1 9 S * 7 S ) "

Fig. 7

A /

\

PHA ( 7 S ) * *

1=10240 1:2560 1:640 1160 140 110

1

2 3 A 5 days after immunization

6

Antibodies in blood sera from mice immunized with diphtheria toxoid (*passive haemagglutination test, ** PHA test with sera treated with mercaptoethanol)

573 It should be noted that data concerning lymphocyte EPM in the course of an immune response is scant. Changes in the histograms have been reported for lymph node cells of mice immunized with tumor cells (29) and rabbits immunized with sheep erythrocytes (30). The principal effect was the appearance in the histogram of a "fast" peak which was absent in the control. An attempt was made to evaluate various influences on the lymphocyte subset composition by using electrophoretic and immunologic methods in parallel. Immunologic methods included the cytotoxic test with antisera to various subpopulations, the detection of cells with receptors to Fc-fragment of IgGp and to the third component of complement, the detection of antibody producing cells and the evaluation of T suppressor activity.

It was demonstrated that defini-

te changes in spleen subset composition correspond to changes in the spleen histogram pattern. An example is furnished by a comparison of normal histograms and histograms obtained from "B-mice" (irradiated and protected by bone marrow

rel. frequency // /

10

\

Fig. 8

\

\

Electrophoretic mobility pattern of splenocytes from normal mice ( ) and Bmice ( )

LL

0.50

2 EPM-101-8Jcm

1.00

1.50

574 transplantation

(Fig. 8). The s p l e e n of the " B - m i c e "

p r a c t i c a l l y no m a t u r e T c e l l s , a n d the h i s t o g r a m s s p l e e n s lack the peak t y p i c a l

such

of T c e l l s . A n a n a l o g o u s

re was r e p o r t e d for " n u d e " m o u s e s p l e e n s In our e x p e r i m e n t s

of

have pictu-

(11,12).

the s u b p o p u l a t i o n s w e r e i n v e s t i g a t e d

ly by i m m u n i z i n g m i c e w i t h v a r i o u s b a c t e r i a l a n t i g e n s .

mainTheir

s p l e e n s w e r e e x a m i n e d by e l e c t r o p h o r e t i c a n d i m m u n o l o g i c in p a r a l l e l on v a r i o u s days a f t e r i m m u n i z a t i o n . E a c h

tests

animal

was e x a m i n e d s e p a r a t e l y . To s t a n d a r d i z e the e x p e r i m e n t a l

con-

d i t i o n s m i c e w e r e i m m u n i z e d , k i l l e d and e x a m i n e d on same

day.

Our e a r l i e r e x p e r i m e n t s

of this type s h o w e d that

of m e n and m i c e w i t h v a r i o u s b a c t e r i a l

antigens

immunization (vaccines)

i n d u c e s p r o n o u n c e d c h a n g e s in the s u b s e t c o m p o s i t i o n of p h o i d cells. The n a t u r e a n d i n t e n s i t y on the p r o p e r t i e s

of t h e s e c h a n g e s

of the a n t i g e n (31). S t r i k i n g c y c l i c

t i o n s w e r e seen in the n u m b e r and f u n c t i o n a l ferent lymphocyte

lymdepends varia-

a c t i v i t y of

dif-

subpopulations.

E x p e r i m e n t s u s i n g cell e l e c t r o p h o r e s i s

revealed

strong

shifts in the s p l e e n h i s t o g r a m s a f t e r i m m u n i z a t i o n . Fig. 9 i l l u s t r a t e s the c h a n g e s on the 6

day a f t e r the

injection

of s o l u b l e p e r t u s s i s a n t i g e n . A m o r e d e t a i l e d a n a l y s i s c h a n g e s in the r e l a t i v e f r e q u e n c y EPM of these s u b p o p u l a t i o n s

showed

of T a n d B cells a n d of the

(Fig. 10). The p e r c e n t a g e

of T

Fig. 9 Changes in h i s t o g r a m p a t t e r n of mouse splenocytes after immunization w i t h pertussis antigen ( before immunization, ^ o n the 6 day a f t e r i m m u n i zation) 0.70

1.00

130

EPM 1 0 "

575

cells increased significantly on days

6 and 12 after immuni-

zation and the percentage of B cells decreased correspondingly. Cytophoretic analysis showed that not only the numbers but also the properties of the T and B cells change, depending on their according differentiation and functions. This is evident from the change in the EPM, particularly that of the T lymphocytes. The EPM of T cells was significantly lower for many days after immunization. The greatest EPM inhibition was registered on the days 6 and 12, when their numbers increased. It may be supposed that the increase in the quantity of T cells is due to the appearance of a particular subpopulation with a low EPM. The results of electrophoretic analysis are consistent with data obtained from immunologic tests. Fig. 11 illustrates the relative frequencies of T cells as measured by cytotoxic and electrophoretic tests at various times after the application of pertussis antigen. It can be seen that the results yielded by these methods are basically the same although the electrophoretic values are higher at all times. It is not yet clear why this is. Immunologic determination of the quantity and activity of nonspecific T suppressors by method (31) has shown that their number was greatly increased on days 6 and 12. It can be assumed that T suppressors have lower EPM than other T cell supbopulations (32). EPM-10 8

% cells

1.00

%

B

3 4 6 10 11 days after immunization EPM

0.90

Fig. 10 Changes in cell number and EPM of different subsets of splenocytes after immunization with pertussis antigen (the points which significantly differ from day 0 are marked © a n d V )

576 The third line cell

of i n v e s t i g a t i o n

electrophoresis

ficulty

is c o n n e c t e d w i t h the

to d e t e c t a n t i g e n - f i x i n g

of d e t e r m i n i n g

the number and avidity

cells. The of t h e s e

is w e l l k n o w n . W e s u p p o s e d t h a t c o n t a c t w i t h a n t i g e n cells possessing

specific receptors

l e a d to a c h a n g e i n t h e i r

to t h i s a n t i g e n

o b t a i n e d by S u n d a r a m e t a l .

of would

supposition

(24): t r e a t m e n t w i t h the

t h e E P M of l y m p h o c y t e s

were homo-

n o t of n o r m a l r a t s . C o n t a c t b e t w e e n P P D a n d l y m p h o c y t e s of a p e c u l i a r

"fast" subpopulation of T c e l l s

tact with macrophages that the effect further

but of

appearance

( 3 3 ) . T h i s is c o n t r a r y

of s e n s i t i z e d m e n a f t e r t h e

treated with PPD

( 3 4 ) . I t is

to

con-

clear

of a n a n t i g e n on t h e E P M of a c l o n e

needs

investigation.

We investigated phi

a

of i m m u n e

m e n w h o a r e s e n s i t i z e d to t h i s a n t i g e n i n d u c e s t h e EPM inhibition

dif-

cells

s u r f a c e m e m b r a n e a n d h e n c e to

change in their EPM. D a t a confirming this logous antigen inhibits

use

the effect

of t r e a t m e n t w i t h O - a n t i g e n S

o n t h e E P M of s p l e e n c e l l s of n o r m a l m i c e

mice primed with this antigen. ted that a single injection

It h a s a l r e a d y

ty-

(control) and been

of O - a n t i g e n i n m i c e d o e s n o t

d u c e a n t i b o d y p r o d u c t i o n b u t i n s t e a d l e a d s to the

in-

formation

% T-cells -

cell electrophoresis cytotoxic test with ATS

p. n g >

. . 11

C h a n g e s i n the r e l a t i v e f r e q u e n c y of T c e l l s in splenocytes after immunization with pertussis antigen

70

20 0

3 6 days a f t e r

9 12 immunization

of

demonstra-

of an immunologic memory-intensive antibody production comes in response to the second injection of the antigen (35). The data in Table 3 and Fig. 12 show the principal

effect:

EPM of spleen cells is reduced by contact with 0-antigen, and the effect is much more pronounced with the cells of primed animals. So low doses of antigen (1 mkg) affect only primed cells. Interesting results were obtained after analysing the effect of 0-antigen on separate T and B subpopulations. 0-antigen slightly inhibits T cell EPM but does not influence B cells from normal spleen. It seems possible that non-immunized animals have few cells with receptors to 0-ant gen, and that these cells are T lymphocytes. On the other hand, the incubation of primed mice cells with 0-antigen has only a slight effect on the frequencies of the subpopulations but it strongly affects their EPM. In the presence of 10 mkg of 0-antigen, the mean EPM of T cells is reduced by 11 %, and that of B cells by 17 %. This suggests that the result of priming with 0-antigen is a great increase in the number of cells with receptors to this antigen. The immunologic memory is formed by both subpopulations, i.e. T and B cells. % inhibition

Fig. 12

22 20

Influence of 0-antigen on the EPM of mouse spleen cells

18

16

%

12 10 8 6 U 2

0 1 10 102103 0 1 10 102103 0 1 10 102103 concentration of 0-antigen (mkg)

578

43

«H *H O 43 C SÄ -H

O o o o

S 0 . 0 5 ) .

of a l l measurements

( F - t e s t ) r e v e a l e d no s i g n i f i c a n t

from

diffe-

This means t h a t no group worked more or

l e s s a c c u r a t e l y than another. Table 1 presents the p o s i t i v e r e s u l t s

of the MEM t e s t

in t h r e e l a b o r a t o r i e s w i t h t h r e e antigen e x t r a c t s . t a g e of p o s i t i v e r e s u l t s

obtained

The p e r c e n -

f o r tumour p a t i e n t s was v e r y low

( o n l y up t o 31 %). Comparison of the p o s i t i v e r e s u l t s

f o r the

cancer p a t i e n t s and f o r the c o n t r o l s showed no s i g n i f i c a n t differences

(Fischer's

test,

p > 0.05).

In f a c t no l a b o r a t o r y

s u c c e s s f u l l y i d e n t i f i e d any i n d i v i d u a l a n t i g e n .

The summari-

zed r e s u l t s are a l s o shown in Table 1. In conclusion we can say t h a t t h e r e was no s i g n i f i c a n t d i f f e r e n c e between the mour and c o n t r o l groups as regards the percentage of

tu-

positive

results. No d i s t i n c t i o n

could be made between p a t i e n t s

according t o EF, nor did the e x t r a c t s

and c o n t r o l s

from b r e a s t and lung

cancer p r o v i d e any i n f o r m a t i o n about the d i a g n o s i s s a t i o n of the tumour.

or

locali-

780 Table 1 Positive results of the MEM test in three laboratories using three coded antigenic extracts, antigens: human basic myelinprotein (EF), breast cancer extract (BC), lung cancer extract (IC). N = number of people examined, n = number of positive results Laboratory

Diagnosis

N

n EF

Brno

Praha

Pribram

summarized results

BC

IC

breast Ca

16

3

3

5

controls

12

1

2

1

breast Ca

5

1

1

_

lung Ca

8

1

-

-

controls

11

—

—

—

lung Ca

14

—

2

2

controls

8

-

-

-

breast Ca

21

4 (19

lung Ca

22

controls

31

1 ( 4 1 ( 3

%) %) %)

4 (19 2 ( 9 2 ( 6

%) %) %)

5 (24 %) 2 ( 9 5) 1 ( 3 %)

Dis cuss ion Previous studies performed on patients with tumours showed controversial results as shown by the literature reviewed by Hoffmann and Berthold (6,7). It is difficult to compare results from different laboratories due to the patients themselves, the procedures and the type of cytopherometer used for the MEM test. Therefore we considered a joint study by three laboratories to be inevitable. We wanted to standardise the technical

781

c o n d i t i o n s as fg.r as p o s s i b l e . l a b o r a t o r y r a t e of v a r i a t i o n s

Besides e s t a b l i s h i n g the

inter-

in the r e s u l t s we expected t o

obtain more i n f o r m a t i o n about the c l i n i c a l u s e f u l n e s s of

the

t e s t in p a r t i c u l a r w i t h r e s p e c t t o i t s tumour s p e c i f i c i t y . According t o our e x p e r i e n c e the major c o m p l i c a t i o n of the i s not only the number of v a r i a b l e s - a n t i g e n s , which makes i t

practically

macrophages

i m p o s s i b l e t o reach standard

t i o n s and r e p r o d u c i b l e r e s u l t s ,

test -

condi-

but a l s o the measuring system

itself. The cause of the u n s a t i s f a c t o r y r e s u l t s

r e p o r t e d by some

authors may be the i m p e r f e c t i o n and d e f e c t i v e n e s s

of the appa-

ratuses used. This a p p l i e s i n our case t o Brno, where f r e q u e n t t e c h n i c a l breakdowns arose in the course of the study,

namely

w i t h the t h e r m o r e g u l a t i o n system and v a l v e s . We are not able t o g i v e our opinion on the p r i n c i p l e s test

of

the

or on the hypotheses concerning i t s mechanisms as they

are not c l e a r as y e t .

We have only shown t h a t our performance

of the t e s t demonstrated no tumour s p e c i f i c i t y and s e n s i t i v i t y so t h a t i t s

c l i n i c a l usefulness is highly

doubtful.

References 1. Kovarik, J . , Zemanova, D., Ninger, E . : Short communication at the Symposium on c e l l e l e c t r o p h o r e s i s in Rostock, 15.-17.4.1982. 2. S t r e j c e k , J . , J i r a , M. , Suhajova, E . , I . : C a s . l e k . c e s . 123, 511 (1984).

Pisa,

P.,

Malbohan,

3. E y l a r , E.H., Thompson, M.: Arch. Biochem. Biophys. 129, 468 (1969). 4. M i i l l e r , M. , Irmscher, J . , F i s c h e r , R. , Grossmann, H. : Dtsch. Gesundh. Wesen ¿ 0 , 1836 (1975). 5. P r i t c h a r d , J . A . V . , Moore, J . L . , Sutherland, W.H., J o s l i n , B r i t . J. Cancer27, 1 (1973). 6. B e r t h o l d , F . , B e r t h o l d , R . , Brockmeier, D., Engel, R., Lampert, F . : Tumordiagnostik 2, 91 (1981). 7. Hoffmann, W., Kaufmann, R., S t e i n e r , J. Cancer 43, 598 (1981).

R., Werner, W.:

Brit.

C.:

LONG-TIME STUDY IN MELANOMA PATIENTS WITH TAA-MELANOMA AND PPD 8 YEARS AFTER PRIMARY OPERATION BY MEM-TEST

Heinz Sochor, Marina Gehre, Heinz Werner Institut für Dermatologie, Medizinische Akademie DDR-Magdeburg

Introduction For years it has been strongly suggested that the host is capable of establishing a defense mechanism against his own tumour. The host environment interaction is regarded as the immunologic basis of host resistance to malignant melanoma. The important role of lymphocytes and macrophages in tumor transplantation reactions has been recognised. If we want to employ this pnehomenon for immunodlagnosis or immunotherapy, we must take a few factors into consideration when interpreting our results, e.g. in the host: immunologic status, age and sex, localisation of the tumour, other diseases and effects of tumor cells, level of invasion, inflammatory cells. This study was aimed to identify potential applications of the macrophage electrophoretic mobility test (MEM-test, Field) for investigating the relationship between host and tumor and to determine the value of this method when performed with the PARMOQUANT (VEB Carl Zeiss JENA, GDR). 55 patients who survived treatment for pigmented skin tumors and metastatic nodes in 1974 to 1976 were followed up for 8 to 10 years. 40 patients died after 10 years. TAA were prepared from melanoma metastic nodes by the 3-M-KCl-technique after Meltzer as modified by Werner (Rostock) and were characterized as TAA 6/P 2 and TAA 5. PPD was prepared by the Institut fur Impfstoffe, Dessau. A positive result means more than 10 %

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

784

reduction of mobility. Antigen concentrations were 100 and 200 ug/ml.

Results and conlusions A significant difference in TAA 6/P 2 was detected between volunteers and melanoma patients treated 8 years earlier. No significant differences were found in TAA 5 and PPD: Antigen

volunteers

melanoma patients

Cell type alveolar spindle/mixed

TAA 6/P 2

1/16

12/29*

11/18*

1/11

TAA 5

0/10

6/20

5/14

1/7

PPD

4/16

6/26

4/16

1/10

(Number of positive results/number of patients; * significant) No significant difference was found between tumor melanoma types (nodular or superficial spreading, lentigo maligna melanoma and non classified melanoma) and between levels of invasion by tumor cells. Using TAA 6/P 2 a significant difference was detected between tumor cell types (alveolar or spindle/mixed type) This analysis suggests that the lymphokine assay performed as a MIM-test can be used for the diagnosis of malgnant melanoma and as a control, especially with TAA 6/P 2. The cell type of the tumor significantly affected the chance of immunologic diagnosis of malignant melanoma in a long time study. The MEM-test is sensitive, but antigen preparation for tumor type of tumor tissue is at present not specific. Monoclonal antibodies may offer new possibilities. The individual genetic influences on the immunsystem of the host were included in our interpretation of immunologic tumor diagnosis data and limited the individual immunologic diagnosis alone.

I N V E S T I G A T I O N OF O B L I T E R A T I V E V A S C U L A R D I S E A S E S W I T H

THE

PARMOQUANT 2

R e i n h a r d L a m b r e c h t , K a r i n Plate, H e i n r i c h Peter

Heinrich

C h i r u r g i s c h e Klinik d e r M e d i z i n i s c h e n DDR-3090 Magdeburg Jochen

Kosowski,

Akademie

Morenz

I n s t i t u t für M e d i z i n i s c h e M i k r o b i o l o g i e u n d DDR-3090 Magdeburg

Epidemiologie

Introduction A c c o r d i n g to V o l l m a r ' s include degenerative, emboloid diseases.

(1) d e f i n i t i o n , arteriosclerotic

obliterative and

diseases

inflammatory

The r e s u l t s of c e r t a i n s t u d i e s make it

p r o b a b l e t h a t o b l i t e r a t i v e v a s c u l a r d i s e a s e s are of

seem

immunolo-

g i c a l o r i g i n , s u c h as - the c i r c u l a t i o n of a n t i b o d i e s

and

immunocomplexes,

- the d e t e c t i o n of c o m p l e m e n t in v a s c u l a r b i o p t i c

material,

- d e p o s i t i o n of i m m u n o g l o b u l i n in the e n d o t h e l i u m of

vessels,

- p l a s m a - l y m p h o c y t i c i n f i l t r a t e in v e s s e l w a l l s , - HLA a s s o c i a t i o n s ,

and

- the a n t i g e n c h a r a c t e r of s t r u c t u r a l g l y c o p r o t e i n s w a l l s . H o r s c h , B o l l i n g e r a n d L e u (2, 3, 4), for

in v e s s e l

example,

i d e n t i f i e d a n t i e l a s t i n a n t i b o d y in p a t i e n t s w i t h

endangitis

o b l i t e r a n s . M i s h i m a (5) and S h i o n o y a (6) f o u n d a r t e r i a b o d i e s , a n d Heine

(7) o b s e r v e d the p r e s e n c e of

d e p o s i t s in the i n t i m a of e n d a n g i t i c v e s s e l s . teams w o r k i n g w i t h K i s h o v

(8) a n d Z u b z h i t z k y

H u n g a r i a n t e a m w o r k i n g w i t h Gero

anti-

immunoglobulin The

Soviet

(9) a n d the

(10) are c o n c e n t r a t i n g

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

on

786

immunological

aspects

of

We used the MEM t e c h n i q u e Carl

Zeiss

JENA, GDR) t o

arteriosclerosis. ( 1 1 ) and t h e PARMOQUANT 2 (VEB detect

associated with o b l i t e r a t i v e vessel tes

antigens.

vascular

The e l e c t r o p h o r e t i c

from c o n t r o l

with manifest

c e l l r n e d i a t e d immune diseases with mobilities

of

subjects with healthy v e s s e l s ,

arteriosclerosis

confirmed endangitis

reactions prepared thrombocy-

from

and p a t i e n t s w i t h

o b l i t e r a n s were measured and

patients

clinically compared.

Methods Peripheral gradient

mononuclear

c e l l s were i s o l a t e d

centrifugation with Ficoll-Paque

u s i n g Boyum's with a cell

(12) technique.

concentration

of

2 ml o f

by

density

(Uppsala,

lymphocyte

10^ c e l l s / m l w e r e

w i t h 84 jig o f v e s s e l

a n t i g e n p e r ml lymphocyte

and 42 jag o f

for

antigen

90 min a t

37°C.

The

Sweden)

suspension

incubated suspension

supernatants

were subsequently a concentration The EPM of

i n c u b a t e d w i t h g u i n e a p i g macrophages 7 of 10 c e l l s / m l f o r 90 min.

20 macrophages w i t h at l e a s t

two p a r a f f i n

s i o n s were measured manually i n t h e PARMOQUANT 2 a t using a current

of

inclu25°C

10 mA. The t h r o m b o c y t e m o b i l i t i e s

a l s o measured manually a t

25°C w i t h

a current

of

at

were

10 mA.

Results 1 ) Pour s u b j e c t s

among a c o n t r o l

healthy vessels

subjects

group c o m p r i s i n g

responded w i t h an i n h i b i t i o n

t h e p a r a f f i n - s t i m u l a t e d macrophages 84 jag o f

antigen

protein.

a f t e r incubation with

12 w i t h

a f t e r incubation

No i n h i b i t i o n was

42 jig o f

with

observed

antigen.

The t e s t was p e r f o r m e d under t h e same c o n d i t i o n s w i t h patients

of

s u f f e r i n g from manifest

arteriosclerosis.

18

787 Pour of these, too, responded to incubation with 84 jig of vessel antigen by the liberation of lymphokin, but none of them responded to incubation with 42 ]xg. The MEM test was also performed with 17 clinically

confirmed

endangetic patients. A response in terms of macrophage mobility was observed in 13 of the patients to incubation with 84 jig of antigen and in 10 patients to 42 jig of the antigen (Table 1). Lymphokin liberation after incubation n

84 jig antigen/ml

42 ng antigen/ml

Controls

12

4

0

arteriosclerosis

18

4

0

endangitis obliterans 17

13

10

Table 1: Lymphokin liberation after incubation with different antigen concentrations 2) The mean mobility of thrombocytes in the control group (healthy vessels, n = 12) was 0.96 * 0.12. In the group with arteriosclerosis

(n = 14) it was 0,96 * 0.08, and in

the group with endangitis obliterans 1

(n = 13) it was

1

0.92 + 0.09 jum cm V ~ s ~ .

Dis cussion The MEM test showed that peripheral lymphocytes

from most of

the patients with endangitis obliterans were sensitized

against

vessel antigen. The liberation of lymphokin after incubation with vessel antigen permits the conclusion to

be drawn that

cell-mediated immune mechanisms are also implicated in endangitis obliterans. According to Gotz (13), the

cellular

immune system plays an important role in the genesis of vasculitis, but it is not considered to be the primary

788 pathogenetic factor. The disease may be a manifestation of an immunoregulatory disturbance. Measurement of the mean thrombocyte mobility revealed no appreciable differences between the control subjects with healthy vessels and patients with blood flow problems in the peripheral arteries.

Références 1.

Vollmar, J.: Rekonstruktive Chirurgie der Arterien Georg Thieme, Stuttgart (1975)

2.

Bollinger, A., Hollmann, B., Schneider, E., Fontana, A.: Schweiz.med.Wschr. 109, 537-543 (1979)

3.

Horsch, A.K., Brechemier, D., Robert, L.. Horsch, S.: Verh.dt.Ges.inn.Med. 83, 1758-1761 (1977)

4.

Leu, H.J.: Dt.med.Wsehr. 101, 113-114 (1976)

5.

Mishima, Y. : J.cardiovasc.Surg. 11, 67-72 (1970)

6.

Shionoya, S.: Vasa 9, 270-276 (1980)

7.

Heine, H., Heinemann, 1., Thiel, Ch., Falck, P., Schmidt, H.. Philipp, H., Blauärmel, 0.. Barthelmes, H., Schneider, W., Marchlowitz, E.: Dt.Gesundh.-Wesen 34, 2174-2177 (1979)

8.

Kishov, M.G.: Patol.Fiziol.i.eksper.Ter.3,

9.

Zubzhitsky,Y.N., Nagornev, W.A.: Vestnik Akad.med.Mauk 27, 88-94 (1972) Gero, S., Szondy, E., Füst, G., Horvath, M., Szekely, J.: Progr.biochem.Pharmacol. 14, 283-286 (1977)

10.

30-36 (1974)

11.

Field, E.J., Caspary, E.A.: Lancet II, 1337-1338 (1970)

12.

Böyum, A.: Scand.J.clin.Lab.Invest. 1, 9-109 (1968)

13.

Götz, H.C.: Therapiewoche 29, 2674-2681

(1979)

THE TRANSFER OF CELLULAR IMMUNITY IN THE IN VITRO MEM TEST BY IMMUNE RNA ISOLATED FROM GUINEA-PIGS IMMUNIZED WITH BASIC PROTEIN OF HUMAN GLIOMA

Shi Yongde, Tang Zhenshen, Xiao Baoguo Department of Biophysics and Department of Neurology Shanghai First Medical College Shanghai, China Ye Qingwei, Che Yufan Shanghai Institute of Cell Biology, Academia Sinica Shanghai, China

Introduction It was reported that i-RNA extracted from the lymphoid tissues of immunized animals could transfer specific immune reactivity to transform normal nonsensitized lymphocytes into specific sensitized ones. Paque and Dray (1972) immunized monkeys with keyhole limped hemocyanin (KLH), purified protein derivate (PPD) and coccidiodin respectively and the extracted i-RNA was able to release macrophage inhibiting factors (MIF) on contact with the corresponding antigen. That was the first attempt to transfer cellular immune reactivity from animal to human using i-RNA. Vettman et al. (1974) pointed out that i-RNA from guineapigs or sheep immunized with cancer cells could transform normal human lymphocytes into "killer cells" which could effect immune cytolysis of human cancer target cells. Several experiments showed that i-RNA against cancer could help evoke a host's own specific immunity to cancer and eventually result in the inhibition or disappearance of cancer (Ramming et al., 1971; Pilch et al., 1973, Deckers et al., 1973; Ye et al., 1974). The macrophage electrophoretic mobility test (MEM) is a modern

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

790 technique for investigating cellular immunity. Using such a technique Field et al. (1970, 1973) and Pritchard et al. (1972, 1973) found that lymphocytes isolated from patients with malignant diseases released a lymphokine-like substance - a macrophage electrophoretic slowing factor (MFS) - which could make the macrophages from guinea-pigs induce an electrophoretic slowing effect when the patients' lymphocytes encountered myelin basic proteins (MBP) isolated from human brain tissues. Later they also found that when cancer basic proteins were used instead of MBP, this slowing action was emphasised for malignant patients but was almost undetectable in the lymphocytes from normal persons. We have now isolated human glioma basic proteins (GBP) for use in MEM test. It was found that such proteins could also be detected in the lymphocytes from patients with malignant diseases and it was at first used in clinics for diagnosing brain tumors and other malignancies (Shi et al.). Our paper reports that such proteins caused cellular immunity in guinea-pigs and, furthermore, that the i-RNA isolated from the lymphoid tissues of the immunized animals displayed the ability to transfer immunological information to normal human lymphocytes, which were given specific cellular immunological reactivity.

Procedures and Methods Isolation of GBP

According to the methods published, the

main steps taken were: 1. Collection of glioma: The glioma was collected from the operating table, rinsed with saline to remove the blood, stripped of the surrounding tissues, chopped and stored at -40° C or used immediately. 2. Homogenization: The chopped tissue was homogenized in a waring blender with 10 volumes of distilled water and cen-

791

trifuged at 15.000 g for 30 minutes. After the supernatant had been discarraded, the sediment was added to 10 volumes of distilled water, chopped and centrifuged twice, and then dried to powder by lyophilization. 3. Defatting: The dried powder was suspended in 5 volumes of a mixture composed of chloroform and methanol with a volume ratio of 2 : 1, chopped, shaken and then filtered by suction to remove the organic solvent. 4. Acid extraction: The defatted powder was resuspended in 5 volumes of 5 % NaCl solution and centrifuged at 15.000 g for 30 minutes. The supernatant was discarded and the pellet twice resuspended in 5 volumes of distilled water. The suspension was adjusted to pH 2.6, kept at 4° C for 24 hours and centrifuged to collect the first supernatant. The sediment was once again suspended in volumes of distilled water, and the second suspension collected according to the methods mentioned above. The first and second supernatants were combined to determine the ultra-violet absorption spectrum, which showed a maximum at 275 - 280 nm. The protein concentration of the combined supernatant was put into ampoules, frozen and dried in vacuum, and, after sealing, stored at 4° C for use. The GBP yield was about 10 mg/g of dried powder. Animals and immunization

Animals: The guinea-pig weighed

about 400 g, male and female. Each guinea-pig was first injected with 500 mg of GBP: 250 mg was dissolved in saline and injected into the animals peritoneum and a further 250 mg was made up into an emulsion by mixing and grinding with 1 ml saline and 1 ml FCA (Freund's completed adjuvant). This emulsion was then injected in 0.25 ml doses into each of 8 spots - 6 on either side of the spine and 2 on the sites of the rear feet. Secondly, two weeks later, each animal was injected with 300 mg of GBP. 150 mg was dissolved in 2 ml of saline and injected into the peritoneum and 150 mg was dissolved in 1.2 ml of

792 s a l i n e and i n j e c t e d i n t o 6 spots l o c a t e d on e i t h e r s i d e the spine i n 2 ml doses. T h i r d l y a week l a t e r ,

of

200 mg of GBP

was d i s s o l v e d in s a l i n e and i n j e c t e d i n t o the peritoneum. A week a f t e r the l a s t immunization,

one of two procedures was

c a r r i e d out: in one case blood was c o l l e c t e d from the

heart,

the lymphocytes were i s o l a t e d from the b l o o d , and the MEM t e s t f o r GBP was performed, killed,

the s p l e e n s ,

in the other case, the animals were

and lymph nodes were c o l l e c t e d and s t o -

red on d r y - i c e immediately b e f o r e i s o l a t i o n

of the RNA. This

RNA was c a l l e d i-RNA. The c o n t r o l animals were not immunized but were kept under the same c o n d i t i o n s .

Both t h e i r blood and t h e i r spleens and

lymph nodes were c o l l e c t e d :

the blood f o r the MEM t e s t

f o r GBP

and the spleens and lymphonodes t o i s o l a t e RNA. The RNA from the c o n t r o l animals was c a l l e d n-RNA. The MEM t e s t

on lymphocytes from immunized g u i n e a - p i g s

1. I s o l a t i o n

of the lymphocytes: Blood (approx 8 ml/ g u i n e a -

p i g ) was taken from the hearts of both immunized and cont r o l animals,

heparinized,

and the lymphocytes wer

isola-

t e d by c e n t r i f u g i n g the blood w i t h F i c o l l s o l u t i o n w i t h a d e n s i t y of 1.070 which was prepared as d e s c r i b e d i n an e a r l i e r paper ( S h i ,

1977).

The average number of

lympho-

cytes was 5.7 + 1.57 x 10^ (X + SD) f o r each g u i n e a - p i g . These were suspended i n t o a medium of

199.

2. Incubation of the lymphocytes w i t h GBP: The lymphocytes were suspended in 199 medium and e q u a l l y d i v i d e d i n t o two tubes. The "Antigen t u b e " a l s o contained 30 jig of GBP, had a t o t a l volume of 1.5 ml. The " c o n t r o l t u b e " contained no GBP but contained other m a t e r i a l s t o make i t up the same volume.

Both tubes were incubated at 23

0

C f o r 2 hours

then c e n t r i f u g e d t o c o l l e c t the supernatant. t i o n the lymphocytes

During incuba-

of the immunized group r e l e a s e d MSF

upon contact w i t h GBP. 3. I s o l a t i o n of macrophages from the g u i n e a - p i g s and incuba-

793 tion with the supernatant: Healthy, adult and nonpregnant guinea-pigs weighing about 400 g were used. 6 - 1 2 days before the experiment, 20 ml of sterile liquid paraffin was injected into the peritoneum of each guinea—pig. The guineapigs were killed by a blow to the head, the peritoneum was washed with 100 ml of Hank's solution, and the exudate was removed for centrifugation. The sediment obtained which consisted chiefly of macrophages, was washed twice in 5 ml of Hank's solution and finally diluted in medium 199 to make 7 up a macrophage suspension with a concentration of 7 x 10 / ml, which could be used in the test after it was X-rayed with a dose of 200 rad. The supernatant obtained by incubation with GBP and lymphocytes from both tubes (antigen and control) was added to 0.1 ml of macrophage suspension and incubated at 37 ° C for 2 hours. Finally the macrophage electrophoretic measurement was taken by a doubleblind test. 4. Macrophage electrophoretic measurement: A labmade cytopherometer (Lian et al. 1979) with on observation chamber consi2 sting of a square capillary tube 50 mm long and 1 x 1 mm in internal cross section was used. Only 0.05 ml of cell suspension was needed when changing the liquid. The cells in focus in the stationary layer were selected for measuring the electrophoretic time. Ten cells from each macrophage sample tube, were measured over a distance of 33 H® in both direction with an electrophoretic timer (Lian, 1980). The slowing rate was (t ag -t o/t o ) x 100, where t ag was the electrophoretic speed for the antigen tube and t Q the speed for the control. Isolation of the RNA

The RNA was isolated by the hot-phenol

method (Ye, 1974). Tissue which had been, stored on dry ice was placed in a waring blender with 10 volumes of tris-buffer and 88 % phenol/g of tissue and homogenized for 1 minute at 10.000 rpm: 0.1 M tris solution at pH 5.0, containing 0.5 SDS and 5 % naphthalene-1.5 disulphonic acid sodium, was added to

794 the liquid before it was again homogenized for 1 minute. The liquid was then placed in water at 60° C until it reached 55°C, and then cooled with ethanol-dry-ice. The homogenate was centrifuged at 3.000 rpm for 30 minutes to collect the water-phase and the phenol-phase was mixed with an equal volume of trispuffer and shaken for 10 minutes. The mixture was collected as above. The first and second water-phases were combined and mixed with 88 % phenol, water-phase: 88 % phenol was 2:1 and centrifuged. The water-phase was collected again and the above extraction was repeated once or twice until no protein appeared on the boundary between the phenol and the water-phase. NaCl crystals were added to the final water-phase to make the concentration up to 0.1 M, then 3 volumes of cold ethanol (-20°C) were added and the substance was stored at -20°C for 24 hours. The precipitate was collected by centrifugation, dissolved in bidistilled water and mixed with an equal volume of 4M KAc,then 3 volumes of cold ethanol were added and

this

was repeated twice. The final precipitate was dissolved into bidistilled water and centrifuged for 30 minutes at 100.000 g, which meant that the RNA contained in the supernatant was reprecipated. We found the i-RNA and n-RNA to have an E2 60/280 above and less than 0.5 % protein. Diphenolamine was not used to test for DNA. The i-RNA and n-RNA, were put into ampoules, dried by lyophilization and stored at -20® C until use. The MEM test for the transfer of immune information to human lymphocytes 1. Isolation of the human lymphocytes: 8 ml of human venous blood was collected from healthy and defibrinated with glass beads. The lymphocytes were isolated using a Ficoll solution with a density of 1.078. They were suspended in 1.5 ml of medium 199 and the suspension was divided into 3 tubes, each tube containing 1.4 x 10^ lymphocytes. 2. Incubation of the lymphocytes, i-RNA, GBP and macrophages: First the lymphocytes were incubated with i-RNA or n-RNA

795 d i s s o l v e d in medium 199 at a c o n c e n t r a t i o n of 1 mg/ml. ml of i-RNA was added t o one of the tubes of

0.5

lymphocytes

and t h i s was c a l l e d the i-RNA t u b e : n-RNA was added t o another and t h i s was c a l l e d in n-RNA tube; the t h i r d tube contained medium 199 only and was the c o n t r o l . all

They were

incubated at 37° C f o r 30 minutes and c e n t r i f u g e d , w h e r e -

upon the supernatant was d i s c a r d e d and the lymphocytes lected.

col-

Secondly the lymphocytes were incubated w i t h GBP.

30 iig of GBP d i s s o l v e d i n 1.5 ml of medium 199 was added t o the tubes.

They were incubated at 23° C f o r two hours and

then c e n t r i f u g e d t o c o l l e c t the supernatant. T h i r d l y the supernatant was incubated w i t h the

guinea-pig

macrophages a c c o r d i n g t o the aforementioned procedure. A f t e r incubation at 37° C f o r 2 hours, the

electrophoretic

m o b i l i t y was measured. 3. Macrophage e l e c t r o p h o r e t i c measurement. This was taken by the same method as the aforementioned MEM t e s t p i g lymphocytes.

of

guinea-

The r e d u c t i o n in EPM was c a l c u l a t e d as

below: f o r the i-RNA tube ( t i - t o / t o ) x 100; f o r the n-RNA tube ( t n - t o / t o ) x 100, were t i ,

tn and t o are the

electro-

p h o r e t i c times f o r the i-RNA, n-RNA and c o n t r o l tubes

re-

spectively.

Results The MEM t e s t

f o r s e n s i t i z e d g u i n e a - p i g s w i t h GBP 61 g u i n e a -

p i g s were d i v i d e d i n t o two batches, the f i r s t batch

consisting

of 35 (11 c o n t r o l and 2k immunized w i t h GBP), the second batch c o n s i s t i n g of 26 (12 c o n t r o l s and 14 immunized).

The r e s u l t s

are shown in Table 1. It

can be seen from Table 1 and P i g .

1 t h a t the r e d u c t i o n

in

EPM (%) was near z e r o in the c o n t r o l 0.96 + 0.90 % (X + S . E . ) on average and 6.13 + 1.06 % f o r the immunized groups.

These

796

Table 1

MEM t e s t f o r GBP s e n s i t i z e d g u i n e a - p i g s w i t h GBP Control Number Reduction in EPM (%) Mean S. D.

Batch 1 Batch 2 Total

11 12 23

1.,66 0.,67 0.,96

1.,44 1.,19 0.,90

Immunized Number Reduction in EPM (%) Mean S. D. 24 14 38

6. 13 6. 13 6. 13

1 .39 1 .39 1 .06

P < 0 . 0 1 f o r comparison of means r e s u l t s show a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e . They show t h a t the lymphocytes of the immunized groups had been s e n s i t i zed by GBP: they could r e l e a s e MSF when upon c o n t a c t w i t h GBP in v i t r o and produce a s i g n i f i c a n t r e d u c t i o n in EPM in the MEM t e s t . The lymphocytes from the c o n t r o l groups had not been s e n s i t i z e d by t h i s p r o t e i n , thus they had not produced any r e d u c t i o n , and a c l e a r d i f f e r e n c e can be seen between the two r e sults . The MEM t e s t f o r the t r a n s f e r of immuno i n f o r m a t i o n by i-RNA: A t o t a l of 83 g u i n e a - p i g s were u s e d , in b a t c h e s of 11, 12 and 12 as c o n t r o l groups and 24, 14 and 10 as immunized g r o u p s . The y i e l d of RNA i s o l a t e d from the s p l e e n and lymphnodes was 4 . 2 5 mg/g and 3 . 1 4 mg/g of f r e s h t i s s u e r e s p e c t i v e l y f o r the c o n t r o l and immunized groups (Table 2 ) . 30, 20,

10,

I0 iV-

0

-10 N,

N2

I,

h

P i g . 1 MEM t e s t f o r immunized and c o n t r o l groups ( N = c o n t r o l , I=immunized)

N,

I,

N;

I,

N,

I3

Fig. 2 MEM t e s t f o r i-RNA t r a n s f e r r i n g immune informat i o n t o human lymphocytes (N=n-RNA; I=i-RNA)

797

Table 2

RNA y i e l d f o r the c o n t r o l and immunized groups

Batch Groups N°

Guinea g.of fresh total yield pigs tissue per of RNA (mg) N" animal

Per an animal

Yield

Per g fresh tissues

1

N

11

1.13

65

5. 90

1

I

24

2. 09

176

7. 35

3.20

2

N

12

1.31

2.63

2

I

14

2.35

90

3. 46 6. 42

2.73

3

N

12

1.37

84

7. 02

5.10

3

I

10

1. 50

60

6. 06

4. 00

Total

N

35

1.27

190.5

5. 45

4.25

I

48

2.17

326

6. 80

3.14

N= C o n t r o l ;

41.5

5.20

1= Immunized

The MEM t e s t f o r the t r a n s f e r of immune i n f o r m a t i o n t o normal human lymphocytes was c a r r i e d out w i t h i-RNA and n-RNA on 3 batches of 21, 14 and 14 animals r e s p e c t i v e l y . are shown in Table 3 and F i g . It

The r e s u l t s

2.

can be seen from Table 3 and F i g .

2 t h a t the i-RNA could

t r a n s f e r immune r e a c t i o n i n f o r m a t i o n t o the normal

lymphocytes

because t h e i r EPM was reduced t o between 6.25 and 10.30 % w i t h an average of 7.73 %• The n-RNA produced no such e f f e c t :

the

r e d u c t i o n i n EPM i n t h i s case ranged from 0.03 t o 2.47 %, w i t h an average of 1.29

The reductions i n the EPM d i f f e r e d from

5.28 t o 7.89 %, and the average was 6.44 %, which i s cally significant.

statisti-

The r e s u l t s were s i m i l a r on the same and

the next day f o r 15 cases, showing t h a t ,

though the

values of the r e d u c t i o n might vary f o r d i f f e r e n t

absolute

subjects,

t h e r e was a high degree of agreement between in EPM produced by i-RNA and n-RNA. We a l s o incubated macrophages from 4 g u i n e a p i g s w i t h i-RNA and n-RNA only at 37° C f o r 2 hours. There was no d i f f e r e n c e between the r e s u l t s

from both i-RNA and n-RNA.

798 Table 3 MEM t e s t f o r the t r a n s f e r of immuno i n f o r m a t i o n t o human lymphocytes (X + S . E . ) by i-RNA Batch

Human

Reduction in EPM (%) in MEM test *

1

21

0.03 + 0.86

7.00 + 1.39

6.94 + 2.48

0.05

2

14

1.53 + 0.71

6.25 + 0.90

5.28 + 1.70

0.05

3

14

2.47 + 2.39

10.30 + 0.63

7.89 + 2.81

0.05

Total

49

1.29 + 0.81

7.73 + 0.97

6.44 + 1.31

0.01

* 1= i-RNA; N= n-RNA; I-N= d i f f e r e n c e between them

Discussion F i e l d et a l .

and P r i t c h a r d e t a l .

found t h a t GBP from human

brain t i s s u e s and CaBP from cancer t i s s u e s could, test,

in the MEM

produce a s i g n i f i c a n t r e d u c t i o n i n the EPM of

t e s from cancer

lymphocy-

patients.

They r e c o g n i z e d t h a t a common antigen determinant might

exist

i n GBP and CaBP and p o s t u l a t e d the e x i s t e n c e of a common cancer antigen. Shaw e t a l .

Goldstone e t a l .

(1973),

Preece and L i g h t

(1974),

(1976) have s i n c e supported t h i s p o s t u l a t i o n ,

Coates and Carnegie (1975) pointed out t h a t the

and

lymphocytes

from animals which were s e n s i t i z e d w i t h CaBP could produce s i g n i f i c a n t t r a n s f o r m a t i o n s in lymphocytes when they came i n t o 3 contact w i t h CaBP in v i t r o by HdT i n c o r p o r a t i o n i n t o lymphoc y t e s . McDermott e t a l .

(1974) r e i n f o r e e d the view of CaBP as

a s o l i d - s u p p o r t e r and used a f f i n i t y chromatography t o t h a t a common determinant s t r u c t u r e e x i s t e d .

prove

Our GBP was

also

proved t o be an a s s o c i a t e antigen which could be r e c o g n i z e d by lymphocytes from brain tumor p a t i e n t s and other body cancer patients.

This paper has proved t h a t lymphocytes from g u i n e a -

p i g s immunized w i t h GBP can r e l e a s e lymphokinase-like

substan-

799 ces and reduce the EPM of macrophages. from normal animals had no such e f f e c t .

However, the The r e s u l t s

lymphocytes indicate

t h a t GBP can s t i m u l a t e s p e c i f i c c e l l u l a r immune r e a c t i v i t y , which agree w i t h the r e s u l t s

produced by Coates e t a l .

(1975).

Our paper a l s o p o i n t s out t h a t normal human lymphocytes which were t r e a t e d w i t h i-RNA, in the MEM t e s t ,

can produce a marked r e d u c t i o n in EPM

but those t r e a t e d w i t h n-RNA had no such e f -

f e c t . However, when the macrophages were d i r e c t l y t r e a t e d w i t h i-RNA or n-RNA n e i t h e r produced an e f f e c t .

The r e s u l t s

t h a t c e l l u l a r immune r e a c t i v i t y was not caused by the e f f e c t of RNA on the macrophages,

indicate direct

but t h a t the RNA causes the

production of s e n s i t i z e d lymphocytes because,

on contact w i t h

GBP, they r e l e a s e d lymphokinases t o reduce the EPM of the macrophages. Our paper proves t h a t i-RNA can t r a n s f e r s p e c i f i c

cellu-

l a r immune i n f o r m a t i o n from g u i n e a - p i g s t o human lymphocytes i.e.

beyond s p e c i e s

Until recently,

boundaries.

only a few p r e l i m i n a r y s t u d i e s had been made on

the use of i-RNA i s o l a t e d from animals which were immunized w i t h cancer c e l l s Wu e t a l .

1977).

in c l i n i c a l

therapy ( P i l c h e t a l .

Our r e s u l t s t h e r e f o r e suggest t h a t

use should be made of

1975,

clinical

our experimental data r e g a r d i n g the use of

RNA prepared from animals immunized w i t h a s s o c i a t e a n t i g e n l a t e d from cancer c e l l s t o improve and i n c r e a s e c e l l u l a r immunological

1976;

function.

the

iso-

specific

ALTERATION OF ELECTROPHORETIC MOBILITY OF GUINEA PIG PERITONEAL MACROPHAGES BY CARRAGEENAN

Horst

Schaffner

Sektion Biowissenschaften, Karl-Marx-Universität DDR-7010 Leipzig

Leipzig

Introduction Carrageenan, a high molecular weight sulphated

polygalactose

of some Rhodophycea, has been used repeatedly for the manipulation of humoral immune response. Like others

(1,2,3,4) we

could show, that animals treated with this polysaccharide strongly immunosuppressed

are

(to be published). There are some

evidences, that the carrageenan acts on macrophages

(5,6).

The true mechanism of the effect is not yet well understood, however.

By use of the analytical cell electrophoresis we stu-

died some aspects of the in vitro interaction of carrageenan and guinea pig peritoneal macrophages. We could show, that the drug alters the surface charge of these cells.

Material and Methods Macrophages.

We harvested guinea pig peritoneal cells by

washing the peritoneal cavity with Eagle's medium Immunpräparate und Nährmedien Berlin) 5 to 10 days paraffin-oil Carrageenan• Lymphokine

(Inst, für after

stimulation. Iota-carrageenan

(Sigma) has been applied.

(MSF)-containing supernatants: a ) 1,5 x 10^ mono-

nuclear cells from human blood were incubated with 200 iig PPD in a total volume of 3 ml Eagle's medium. After 2 h incuba-

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

802 tion

at

37°C we o b t a i n e d t h e s u p e r n a t a n t s

As c o n t r o l s antigen

served supernatants

(0-controls)

supplemented w i t h a n t i g e n stituted target

controls)

cells

for

protein

HEP f o r the s t i m u l a t i o n

trophoretic

of

4,

of

described

test

case o f

supernatants

mean v a l u e o f

of

of

an

We used 200 )ig cells. of

the

elec-

Opton-cytopherome-

We measured t h e the

the

supernatants

cells

over

time,

a distan-

the measuring was done i n both

MSF (macrophage s l o w i n g

the slowing of

s u p e r n a t a n t s was c a l c u l a t e d

tical

The

above.

c e l l s we a p p l i e d

15 um. For each c e l l

taining

(recon-

later

For d e t e r m i n a t i o n

which was needed f o r t h e m i g r a t i o n In the

cells

7 and 10 days

supplemented w i t h a TV-equipment.

ce of

the

2 x 10^ lymph node

measurement.

mobility

without

(HEP) - 20 jig and CFA per mouse

lymph node c e l l s were p r e p a r e d .

were o b t a i n e d by the p r o c e d u r e

rections.

incubated

o f t h e same k i n d ,

b ) AJ-mice were immunized w i t h

footpads.

Electrophoretic

centrifugation.

c h e c k i n g an immediate i n t e r a c t i o n

and a n t i g e n ,

by i n j e c t i o n i n t o t h e

ter,

cells

a f t e r removal o f

human e n c e p h a l i t o g e n i c popliteal

of

and s u p e r n a t a n t s

by

the macrophages

in r e l a t i o n

t h e two c o n t r o l

factor)

to the

dicon-

by t h e arithme-

supernatants.

Results A f t e r incubation nan at

of

guinea pig p e r i t o n e a l

37°C the e l e c t r o p h o r e t i c

creases.

This e f f e c t

blue e x c l u s i o n

is

i t was shown,

ence on the v i a b i l i t y min i s

sufficient.

causes

a decrease

higher mobility and not the

of

the

that of

the m o b i l i t y .

depends on s u r f a c e

on a change o f

the

increased mobility.

these

is

1).

carragee-

cells

in-

By t r y p a n

no markable

influ-

An i n c u b a t i o n t i m e o f

30

the i n c u b a t i o n time

rather

It

the

is

evident,

charge

conductivity

c e l l s with carrageenan-free

of

(Fig.

there

cells.

Prolongation of

mobility

dose dependent

c e l l s with

of

changes

that of

the

t h e medium.

medium does n o t reduce

So we commonly used washed

cells.

cells

Washing their

-

803

Fig. 1 Increase of r e l a t i v e m o b i l i t y of guinea p i g p e r i t o n e a l macrophages a f t e r 30 min i n c u b a t i o n w i t h c a r r a g e e n a n and washing i n c a r r a g e e n a n - f r e e medium; 2 i n d e p e n d e n t e x p e r i m e n t s ; mean v a l u e s o f 20 c e l l s p e r p o i n t . Erythrocytes

of

the p e r i t o n e a l

several

mammalian s p e c i e s

macrophages.

show an i n c r e a s e d m o b i l i t y Mouse p e r i t o n e a l

do not r e a c t

Only e r y t h r o c y t e s

of

a f t e r treatment with

macrophages r e a c t

like

like

guinea

pigs

carrageenan.

macrophages

of

guinea

pigs . Because we a r e i n t e r e s t e d p r o o f e d the s e n s i t i v i t y

i n some a s p e c t s

of

t h e MSF. I n two i n d e p e n d e n t show,

that

these

MSF-activity

in

cells

of

t h e MEM-test, we

carrageenan-treated experimental

are w e l l

suitable

macrophages

systems we

could

f o r the d e t e c t i o n HEP-immunized

we found a s l o w i n g

macrophages

samples.

found,

that

positive

of

In t h e

carrageenan-treated case o f

The m i g r a t i o n t i m e s tests

PPD i n e a r l i e r

o n l y 50 p e r cent o f

i n t h e MEM-test.

ges we found a p o s i t i v i t y -

of

supernatants.

Both i n t h e PPD-model and i n t h e model o f test

to

of

all

and t h e two kinds

the

checked v o l u n t e e r s

i n more than 75 p e r c e n t samples w i t h t r e a t e d controls

w i t h t h e samples w i t h normal o n e s ,

by t h e

investigations

Using c a r r a g e e n a n - t r e a t e d

of

mice was

are

macropha(Fig.

2).

macrophages

- were s h o r t e r

compared

however t h e t r e a t e d

macro-

804

Fig. 2 PPD-MEM-test; comparison of untreated (left) and carrageenan-treated target cells (right); open circles: slowing of the untreated ones (identical supernatants), full circles: slowing of the treated cells is smaller; vertical bars connect double measurements of identical supernatants; each point represents 15 to 20 cells

phages were more susceptible for the slowing effect of the lymphokine. Similar results we got in the case of the HEP-induced MSF from mouse lymph node cells

(Fig. 3).

We assume, that the alterations of the surface charge are involved in some other alterations of the in vitro properties

HEP-MEM-Test, AJ-Mouse

Ca Ma

NM0 i Ca Ma

I 4d

7d

NMfl

10.d

Ca Mi

of

Fig. 3 HEP-MEM-test; slowing of normal (NM) and carrageenan treated (CaM) peritoneal macrophages by identical supernatants of the 4th, 7th and 10th day after immunization, • a n d « : two samples of supernatants, vertical bars connect the results of double estimations, each point - 20 to 25 cells

805

carrageenan-treated

macrophages.

phagocytic

of

capacity

I n our o p i n i o n indicator

cells

We c o u l d show,

carrageenan-macrophages

carrageenan-macrophages f o r MSF i n some

that is

are w e l l

the

suppressed.

suitable

as

circumstances.

Re f e r e n c e s 1.

Bice, E.C.:

E.D., Brunwell, D . I . G . , S a l v a g g i o , Immun. Commun. 1_, 615-627 ( 1 9 7 2 ) .

2. Aschheim, L . A . E . , 262 ( 1 9 7 2 ) .

Raffel,

3.

Bash, J . A . , (1980).

4.

Lukic,

5.

A l l i s o n , A.C., Harrington, Med. 1_24, 141-154 ( 1 9 6 6 ) .

M.L.,

Cochran,

F.R.:

Leskowitz,

6. Neveu, P . J . , T h i e r r y , 179 ( 1 9 8 1 ) .

S.:

S.:

J.

J.

Retic.

Retic.

D. : I n t .

J.

Hoffman,

Soc. JJ_,

Soc.

Nature 252, J.C.,

U.E.,

28,

203-211

605-606

Birbeck,

M.:

253-

J.

Immunopharmac.

(1974). exp. 175-

THE TANNED ERYTHROCYTE ELECTROPHORETIC MOBILITY (TEEM) TEST

BK S h e n t o n , A A l o m r a n , PK D o n n e l l y , TWJ L e n n a r d , G P r o u d , RMR T a y l o r D e p a r t m e n t of S u r g e r y , M e d i c a l S c h o o l , F r a m l i n g t o n P l a c e , N e w c a s t l e u p o n Tyne

Introduction

Over t h e p a s t

f e w y e a r s a number of c y t o p h e r o m e t r i c t e s t s

for

evaluating

lymphocyte f u n c t i o n have been p r o p o s e d .

These have used guinea

p e r i t o n e a l macrophages

(2) and tanned sheep red

cells

(1),

granulocytes

(TSRBC) ( 3 ) a s t h e i n d i c a t o r p a r t i c l e s

interaction.

of

T h e r e a r e many a d v a n t a g e s i n t h e u s e of a ' s t a n d a r d '

Unfortunately,

whilst

considerable

e f f o r t h a s b e e n p l a c e d on

d e v e l o p m e n t of c o m m e r c i a l m a c h i n e s b o t h t h e b i o l o g i c a l systems used and t h e i r a p p l i c a t i o n poorly investigated.

Thus,

to the c l i n i c a l

t h e a i m s of t h e p r e s e n t

t h e m e c h a n i s m of t h e TEEM t e s t

of i t s c l i n i c a l

particle

machines.

the

b a s i s of t h e

test

s i t u a t i o n have been s t u d y were

to

system and t o i l l u s t r a t e

some

applications.

B a c k g r o u n d t o t h e TEEM T e s t S y s t e m a n d

I n t h e p e r f o r m a n c e of t h e TEEM t e s t described

blood

lymphocyte-antigen

s u c h a s t h e TSRBC p a r t i c u l a r l y i n t h e d e v e l o p m e n t of a u t o m a t e d

investigate

pig

(3,4).Fresh

Methodology

the methodology has a l r e a d y

been

s h e e p b l o o d i n A l s e v e r ' s s o l u t i o n was u s e d a s

the

s o u r c e of t h e s h e e p r e d b l o o d c e l l s a n d l y m p h o c y t e s w e r e p r e p a r e d b y t h e c o n v e n t i o n a l Boyum m e t h o d .

I n c u b a t i o n of b o t h t h e f i r s t a n d s e c o n d

stages

h a v e b e e n d e s c r i b e d a n d t h e m e a s u r e m e n t of TSRBC m o b i l i t i e s was

performed

b y both t h e manual and a u t o m a t i c method.

studies

Blind r e p r o d u c i b i l i t y

have been c a r r i e d out both w i t h i n and between a s s a y s and c o r r e l a t i o n l y m p h o c y t e t r a n s f o r m a t i o n was h i g h

(5).

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

with

808 The p r e c i s e mechanism of

the TEEM t e s t i s unknown and our i n i t i a l

have attempted t o examine t h i s mechanism.

P r e v i o u s l y the k i n e t i c s of

two s t a g e TEEM t e s t have been d e s c r i b e d and s i m i l a r i t i e s MEM t e s t have been shown ( 3 , 6 ) . T h e importance of ( p o s s i b l y as a n t i g e n p r e s e n t i n g c e l l s )

studies

to those of

the the

the presence of monocytes

i n the t e s t l e u c o c y t e

suspension

have been shown by a number of workers and our s t u d i e s have shown that

the

slowing of the i n d i c a t o r TSRBC i s produced by p u r i f i e d i n t e r l e u k i n - 2 and not by i n t e r l e u k i n - 1 .

I n the dose t i t r a t i o n s t u d i e s ,

50Ug/3ml was the

optimum dose of lymphokine ( F i g l a ) . The slowing f a c t o r has a molecular weight of

15,000 d a l t o n s , and a p i point of 6.5 ( F i g . l b ) .

I t is stable

to

60°C and has an optimum pH range of 6-8 ( F i g l a ) . Our s t u d i e s have shown the slowing f a c t o r to be a product of T lymphocytes and i t s production from c e l l s c o r r e l a t e s with m i t o g e n i c f a c t o r s i n lymphocyte t r a n s f o r m a t i o n studies ( 5 ) . carried out,

Further c h a r a c t e r i s a t i o n of

the slowing f a c t o r i s

being

t o g e t h e r with the p o s s i b i l i t y of r a i s i n g an a n t i b o d y t o

Fig.la

it.

Fig.lb

T E M P E R A T U R E A N D pH S T A B I L I T Y O F T H E E F F E C T O R L Y M P H O K I N E IN T H E T E E M T E S T

U

A

D E T E R M I N A T I O N OF THE ISOELECTRIC POINT OF THE EFFECTOR L Y M P H O K I N E ON A CHROMATOFOCUSING COLUMN

20

200

o o

c = £ 8

20

30

40

Fraction number

Dose titration effect of the effector lymphokine from PPD stimulated P B L on the indicator cells ( T S R B C ) in the cytopherometer. Effect of temperature and pH change on the biological effect of effector lymphokine (75 ^ig/3 ml test) isolated from PPD

1.0 ml (0.4 mg/ml) effector lymphokine sample was run on a 30 cm long chromatofocusing column, equilibrated with start buffer (0.025 M Tris-CH, C O O H , pH 8.2). The sample was eluted with 30 : 7 0 % polybuffer 96 : 74 pH 5. Fractions collected from the column were tested for their slowing effect on indicator T S R B C .

809

U s e of the TEEM test

for cellular

sensitization

Whilst the M E M test has b e e n used in m a n y clinical s i t u a t i o n s test has not

yet been used

so w i d e l y .

T h e TEEM test has been used to

l y m p h o c y t e r e s p o n s e to both m i t o g e n s and v a r i e t y of a c u t e and chronic diseases

(6) the TEEM

(Tables 1,2.

Table

Figs.2,3).

1

L Y M P H O C Y T E R E S P O N S E T O PPD A N D M I T O G E N S IN VARIOUS PATIENT GROUPS Lymphocyte response (%)

Subject group PPD

CON A

PWM

PHA

19.5 ± 2.3 (65)

24.7 ± 3.5 (37)

31.5 ± 3 . 2 (36)

41.3 ± 11.5 (36)

Chronic renal failure (non-transfused)

14.2 ± 4 . 6 (37)

14.4 ± 4 . 2 (30)

13.9 ± 2 . 6 (27)

19.8 + 5.5 (31)

Chronic renal failure (transfused)

12.9 ± 3 . 3 (19)

12.2 ± 3 . 0 114)

11.6 ± 2 . 8 (12)

17.8 ±5.2 (14)

Normal subjects Chronic disease

Haemophilia

11.7 ± 4 . 1 (22)

11.9 ± 3 . 6 (11)

12.9 ± 5 . 0 (11)

18.0 ± 5.2 (11)

Liver disease

13.4 ± 3 . 0 (10)

19.9 ± 5 . 2 (10)

18.2 ± 7 . 1 (10)

26.9 ± 7 . 2 (101

Rheumatoid arthritis

12.9 ± 3 . 9 (12)

12.9 ± 3 . 2 (12)

12.4 ±3.7 (12)

14.9 ± 3 . 1 (12)

Diabetes

15.2 ± 2 . 5 ( 6)

18.1 ± 1.8 1 6)

18.4 ±2.7 ( 6)

18.7 ±2.7 ( 6)

12.0 ± 3 . 3 (135)

11.2 ± 3.6 (136)

16.7 ± 5 . 6 (137)

21.9 ± 4.9 (48)

21.3 ± 5 . 2 (48)

33.9 ± 10.5 (48)

Cancer Benign disease

9.7 ± 4 . 1 (137) 17.6 ± 3.1 (48)

Table

2

L Y M P H O C Y T E R E S P O N S E T O PPD A N D M I T O G E N S IN V A R I O U S PATIENT GROUPS Lymphocyte response (%)

Subject group PPD

CON A

PWM

PHA

Acute disease Bums

10.2 ± 3.3 (11)

12.0 ± 2 . 6 (11)

10.2 ± 2 . 6 (11)

15.4 ± 3.2 (11)

Acute pancreatitis

10.5 ± 2 . 2 ( 5)

11.5 ± 1.1 ( 5)

12.8 ± 1.6 (15)

15.1 ± 3.8 ( 5)

Post-operative trauma dayO

18.1 ± 2 . 1 (11)

23.8 ± 2.7 (11)

23.1 ± 3 . 6 (12)

30.0 ± 5.6 (11)

9.9 ± 2.6 (11) 6.9 ± 1 . 6 ( 6)

13.3 ± 2 . 9 (11)

11.8 ± 3.5 (11) 9.3 ± 2 . 2 ( 6)

18.5 ± 3 . 0 (11) 13.3 ±3.7 ( 6)

day 1 day 3

study

recall a n t i g e n s in patients with a

9.7 ± 2.4 ( 6)

day 5

16.2 ± 1.9 (10)

19.8 ± 4 . 1 (10)

18.4 ± 4.0 (10)

24.4 ± 9.6 (10)

day 7 day 14

19.6 ± 0 . 5 ( 9) 19.7 ± 1.1 ( 9)

25.0 ± 2.8 ( 9) 24.3 ± 3.0 ( 9)

24.1 ±3.0 ( 9) 24.3 ± 3.9 ( 9)

day 21

20.0 ± 0.7 (12)

24.7 ± 2 . 2 (12)

26.2 ± 3.4 (12)

31.4 ± 7 . 2 ( 9) 32.8 ± 5.9 ( 9) 31.6 ± 7 . 4 (12)

' day 28

19.8 ± 1.6 (11)

25.8 ± 1.2 (11)

26.5 ± 1.5 (111

37.8 ± 6.0 (11)

810

L Y M P H O C Y T E R E A C T I V I T Y T O MITOGENS A N D PPD IN PATIENTS W I T H C H R O N I C DISEASES

F i g . 2

N

N C'O' C'T' H

C C H L R 'O' 'T'

— Normals — Chronic renal failure — Chronic renal failure transfused — Haemophilia

L

—

R D C B

— — —

Liver

Rheumatoid arthritis Diabetes Cancer Benign

L Y M P H O C Y T E R E A C T I V I T Y T O M I T O G E N S A N D PPD IN PATIENTS W I T H ACUTE DISEASE

PPD

50 40 • 30 20 10 • F i g . 3

0 • 30 -

CON A

J: I. I

20 -

I

T

i Ì = I

3 5

7 H 21 28

10 0 -

N B P 0

1

3 5

7 H 21 28

Post-operative trauma N — Normals B - Burns P — Pancreatitis

0

1

Post-operative trauma

811

I n the groups of both chronic and and acute d i s e a s e p a t i e n t s studied an impairment of lymphocyte f u n c t i o n was f o u n d . I n the post o p e r a t i v e group t h i s was seen o n l y on days 1,3 and 5 a f t e r o p e r a t i o n .

Whilst a d e g r e e o f

a n e r g y has been d e s c r i b e d i n t h e s e p a t i e n t groups,

clinical

the

s i g n i f i c a n c e of t h i s i n terms of p r o g n o s t i c v a l u e i s being e v a l u a t e d . the human r e n a l t r a n s p l a n t s i t u a t i o n a good p r o g n o s t i c index of success has been shown by t e s t i n g p r e g r a f t r e c i p i e n t

In

graft

lymphocyte r e a c t i o n

to

the r e c a l l a n t i g e n PPD ( 7 ) .

Animal s t u d i e s have shown a good c o r r e l a t i o n between tumour growth and o r t h o t o p i c heart t r a n s p l a n t a t i o n r e j e c t i o n and the TEEM t e s t r e s u l t lymphocyte

sensitization.

The use of

the TEEM t e s t f o r s u p p r e s s i v e

of

activity

Many c e n t r e s have r e p o r t e d the presence and p r o g n o s t i c s i g n i f i c a n c e of n a t u r a l l y o c c u r r i n g immunosuppressive f a c t o r s , p a r t i c u l a r l y i n g l o b u l i n s and low molecular weight p e p t i d e s i n the plasma of s u f f e r i n g from a c u t e ( o p e r a t i v e or a c c i d e n t a l ) (organ f a i l u r e and malignancy)

the

patients

trauma and chronic

illness,

(8).

Good c o r r e l a t i o n between drug s e n s i t i v i t y r e a c t i o n s as measured i n the TEEM t e s t and lymphocyte t r a n s f o r m a t i o n has been shown ( 5 ) and the TEEM t e s t i s thus p a r t i c u l a r l y u s e f u l i n screening the e f f e c t s of

potential

immunosuppressive a g e n t s . The method of q u a n t i f y i n g PSA has a l r e a d y been described ( 4 ) .

We have c a r r i e d out a f i v e - y e a r study t o e v a l u a t e the

e f f e c t of n a t u r a l l y plasma s u p p r e s s i v e substances as p r o g n o s t i c i n d i c e s renal g r a f t s u r v i v a l .

As shown i n F i g . 4, a high d e g r e e of

significance

was found between plasma s u p p r e s s i v e a c t i v i t y (PSA) and renal s u r v i v a l i n the f i r s t

t h r e e months.

graft

The importance of second locus

i d e n t i t y and PSA has a l s o been shown ( F i g . 5 ) .

The p o t e n t i a l importance of

PSA i n r e n a l t r a n s p l a n t a t i o n has encouraged us t o l o o k a t many acute and chronic d i s e a s e

conditions.

of

812 Fig. 4

Fig. 5

THE GRAFT SURVIVAL OF 121 CONSECUTIVE RENAL TRANSPLANTS (NON-I IMMUNOLOGICAL FAILURES EXCLUDED) WITH HIGH AND LOW PSA

THE CONTRIBUTION TO RENAL ALLOGRAFT SURVIVAL OF H L A - B LOCUS IDENTITY AND HIGH PSA IN 121 CONSECUTIVE TRANSPLANTS INON-IMMUNOLOGICAL FAILURES EXCLUDED)

Antlg.n Milch*.

A+B

DR

100

2.7

1.2

90

HLA2B + PSA < 5 ill

B0 2.5

1.1

70 f t

t

60 50

• p < 0.05 •• p < 0.01

411 30 20

Months poit transplant

10

• p < 0.05

0

T a b l e 3 shows the r e s u l t s d i f f e r e n t patient

groups.

of

12 18 24 Months post transplant

plasma s u p p r e s s i v e a c t i v i t y w i t h i n

As may be seen, a l l

an e l e v a t e d plasma s u p p r e s s i v e

the

groups of p a t i e n t s

showed

activity. Table 3

a2 M concentration (mg%)

Group (16) Normals Chronic disease. Benign (12) Chronic renal failure 'O' (10) Chronic renal failure 'T' (14) (12) Chronic liver failure Diabetes type 1 (10) (10) Diabetes type 2 Haemophilia Rheumatoid arthritis Cancers Acute pancreatitis Acute Burns

(16) (12) (47) ( 7) (11)

PSA Allogeneic Autologous

169 ± 4 2

34.3

+

0.5

34.8 ± 1.0

163 ± 10 187.7 ±60.8

24.0 4.93

+

206 ± 18 258 ± 17 257 ±39.2 266 ±82.1 253.3 ± 70.54 208.9 ± 74.1 178.8 ± 5 6 147 ± 21 125 ± 15

11.2

+

24.2 8.39 5.4

+

+

2.5 0.38

+

0.7

19.1

+

12.3 13.33 3.37 2.34

+

1.5 4.9 1.19 1.41

1.5 0.3

4.61 + 2.22 9.33 ± 4.48 1.12 + 0.7 3.2 + 1.5 6.1 ± 1.5

+

+ + +

7.54

+

1.1 1.1 2.5

+ + +

2.4 1.4

5.60 0.7 0.1 0.4

813 In a l l of t h e s e groups most of the s u p p r e s s i v e a c t i v i t y was a s s o c i a t e d with both the plasma p r o t e i n a 2

macroglobulin and low molecular weight p e p t i d e s

(< 10,000) of plasma i n a c u t e and c h r o n i c a l l y i l l p a t i e n t s ( 8 ) .

Only by

using a r a p i d a s s a y system such a s the TEEM t e s t was i t p o s s i b l e to screen l a r g e numbers of a n a l y t i c a l f r a c t i o n s .

Many of t h e s e c o n d i t i o n s

have been d e s c r i b e d to be a s s o c i a t e d with some d e g r e e of immunological anergy ( 8 ) . However, a s may be seen from the t a b l e , i n a c u t e d i s e a s e high s u p p r e s s i v e a c t i v i t y was a s s o c i a t e d with lower l e v e l s of plasma d i s e a s e t h e r e was no c l e a r r e l a t i o n s h i p between I t was s u g g e s t e d t h e r e f o r e that the groups of p a t i e n t s s t u d i e d .

^M.

In chronic

a2M l e v e l and PSA.

°c2M may be q u a l i t a t i v e l y d i f f e r e n t i n =2M i- s

a

potent p r o t e a s e i n h i b i t o r which

a c t s a s the f i n a l common pathway f o r p r o t e a s e c l e a r a n c e from the c i r c u l a t i o n by the r e t i c u l o e n d o t h e l i a l system.

The e f f e c t of p r o t e a s e

with pure ocv M and normal plasma on s u p p r e s s i v e a c t i v i t y was a s s a y e d i n the TEEM t e s t .

P r o t e a s e binding was checked by the BAPNA a s s a y ( 9 ) .

As

can be s e e n , the a d d i t i o n of p r o t e a s e to both normal plasma and pure ®»»M was accompanied by a r i s e i n s u p p r e s s i v e a c t i v i t y ( F i g s . I t thus seemed that binding of p r o t e a s e to

6,7).

2M was r e f l e c t e d i n i t s

0C

suppressive capacity. The s p e c i f i c

2M binding c a p a c i t y was c a l c u l a t e d f o r the p a t i e n t groups

0C

and i s shown below: Group

Number

MBC mg

Normal h e a l t h y

(16)

6.0+0.3

Benign preop

(12)

5.9+0.6

Liver f a i l u r e

(12)

4.8+0.4

Renal f a i l u r e

(14)

4.4+0.5

Acute p a n c r e a t i t i s ( 7)

4.3+0.4

Acute burns

5.0+0.3

(11)

814

815

These r e s u l t s suggest that p a t i e n t s with acute and chronic i l l n e s s may have s u p p r e s s i v e plasma due to inadequate handling of p r o t e a s e i n the p e r s i s t e n c e of "^M complexes w i t h i n the c i r c u l a t i o n .

resulting Thus, «^M

appears to have a c e n t r a l r o l e i n immunoregulation by v i r t u e of i t s p r o p e r t i e s of p r o t e a s e binding and suppression of lymphocytes. h y p o t h e t i c a l mechanism of i t s f u n c t i o n i s shown i n

dual

A

Fig.8.

Fig. 8 A HYPOTHETICAL MECHANISM OF IMMUNOREGULATION PRODUCED BY a 2 M—PROTEASE INTERACTION

Proteases released from: (a) the cells of the immune system ' (b) damaged cells or (c) rapidly proliferating tissue

t

Other protease inhibitors

t

a2 M a 1 Pl-enzyme complex

a 2 M protease interaction

a 2 M-enzyme complex "polyclonal activation"

Lymphokines LA F MAF RES

polymorphonuclear leukocyte

Lymphocyte activation factor Macrophage activation factor Reticuloendothelial system

Studies of immunosuppressive drugs and p e p t i d e s

We have a l r e a d y shown that the TEEM t e s t can be used to measure the immunosuppressive e f f e c t of drugs, and no d i f f e r e n c e between the of

t r a n s f o r m a t i o n were found ( F i g . 9 ) .

was studied i t

When

CC2M

protease

results

interaction

was found that a low molecular weight p e p t i d e was l i b e r a t e d

( s e e F i g 1 0 ) . This low molecular weight p e p t i d e was found to have a molecular weight of 3,000, a p i of

5 . 6 , and t o b l o c k MLR, i n t e r f e r o n

production, NK c e l l a c t i v i t y and lymphocyte response. Furthermore,

it

shared physicochemical p r o p e r t i e s with p e p t i d e s i d e n t i f i e d i n the plasma of the p a t i e n t groups studied

(Fig.11).

816 Fig.9 A C O M P A R I S O N OF THE I N H I B I T O R Y E F F E C T OF H Y D R O C O R T I S O N E ON IN V I T R O R E S P O N S E S T O PPD O F L Y M P H O C Y T E S F R O M N O R M A L S U B J E C T S (n - 61

100 Lymphocytt % inhibition in Teem Teit

90 80 70 60 Mean I D a u = 15.7 (|19/100>1I>

50 40 30 20

to 0 0.1

1

10

100

Hydrocortisone concentration (po/100 /il)

Lymphocyte * inhibition in Tri njf or mit ion allay = 19.4 (f9/100*lll

0.1

1

10 ID50 100

1000

Hydrocortiione concentretion (pfl/100 fiO

Fig.10 100

E E

c c o 3 . 4 (%) i s s i g n , P < 0 . 0 5 ) 25

30 35 FRACT. NO.

850

Dis c u s s i o n Our i n v e s t i g a t i o n s antigens

is

TEEM-test.

from normal mice s p l e e n The s e c r e t i n g

cell

s t a n d a r d of

(6).

TF-activity

?) to

hypothesis

TP.

or n o t

be a c t i v e

of

supposed t o findings

system.

conditions

may a m p l i f y a p r e - e x i s t i n g

secretion

of

antigens)

the

(6,7).

of

t h e donor

according

by t h e

IL-2

of

TF and t h e k i n d o f

But i t

(8).

the

(ubiquitous TF-extract

low-level

The b e l i e v e d ability

of

t h e machanism o f

needs more i n f o r m a t i o n involved

TF a c t s fact

This supports

antigen

in to

adjuvants

inducing

the

associated

about t h e

structures

anthe

sensiti-

13 kDa lymphokine, which c o u l d be

to

TP p r e -

i n a manner u n r e l a t e d

TP seems t o be due t o i t s of

which may h e l p

antigen structures

i n the t e s t

ubiquitous

of

the

laboratory

The open q u e s t i o n w h e t h e r the

the r e c i p i e n t

effect

in

TF t h e r a p y .

vity

the s e n s i t i v i t i e s

changing

be a T h e l p e r / a

can be answered p a r t l y

t h a t under i n v i v o

(against

charge

detectable

concerning to the procedure

TF needs some s p e c i a l

tigens

of

cells,

convenient

can be drawn from our r e s u l t s

t o the e f f i c i e n c y of antigen s p e c i f i c

is

of

can be e s t a b l i s h e d ,

and s t a n d a r d i z a t i o n

Another conclusion

that

cell

Based upon t h e s e

t o s o l v e some problems paration

TP i n p r e s e n c e

a b l e t o induce t h e s e c r e t i o n

lymphokines inducer

show t h a t

to

nature

explain

TF e f f i c i e n c y .

References 1.

Burnet,

F.M.:

2. S h i f r i n ,

M.,

J.

Allergy

clin.

Scibienski,

54,

Oncology 32,

Lawrence,

S.:

Allergol.

et

Immunopathol.

5. S c h r ö d e r , I . , Rovensky, 1_0, 171-176 ( 1 9 8 2 ) .

J.:

Allergol.

et

Immunopathol.

7.

Shenton,

B.K.

8.

Salaman,

M.R.:

et

al.:

I.: this

this

239-350

(1975).

S c h r ö d e r , I . , Lüneburg, 12, 1-5 (1984).

Paegelow,

68,

269-274

(1974).

3.

H.,

The Harvey L e c t u r e s ,

1-13

4.

6. Werner,

H.S.:

R.:

Immunol.

issue

issue

Immunology Today 3,

4-6

(1982).

(1974).

BASIC MECHANISMS OF EMT: INVESTIGATIONS OF SOME EFFECTS Ulf D. Koenig und Burak Kozan Arbeitsgruppe fu'r Reproduktions- und Tumorimmunologie Universitats-Frauenklinik Bonn Introduction The principle of MEM-(Macrophage-Electrophoretic-Mobility-) test i s generally attributed to the liberation of cytokines that reduce the electrophoretic mobility (e.m.) of peritoneal macrophages. The Electrophoretic-Mobility-Test (EMT) with s u l p h o s a l i c y l i c

acid s t a b i l i z e d and

tanned sheep erythrocytes (ETS: erythrocyte, tanned, s t a b i l i z e d ) as indicator c e l l s i s considered as an equivalent and even identical system by many authors (1). This implies the existence of mononuclear cell products, which are produced during immunological responses and reduce the e.m. of ETS. One of the main concerns of our group was a c r i t i c a l evaluation of t h i s assumption. We worked with an analytical

free-flow-cell

electrophoresis instrument after Hannig (2) and with defined animal models. Rat spleen c e l l s , immunized with antigens such as myelin basic protein (MBP) or PPD were tested by EMT. In addition we studied the mitogen stimulation capacity of these c e l l s .

Methods and r e s u l t s Table 1 shows a representative experiment with PPD and tuberculin in EMT. We tested Lewis r a t s , either immunized only with Freund's adjuvant, completed with 50 pg

mycobacteria, or with adjuvant plus 60 gg MBP.

Shown are the percentage i n h i b i t i o n s of e.m. with respect to untreated ETS. There was no s i g n i f i c a n t difference between immunized and control animals. In contrast, highly s i g n i f i c a n t differences could be observed with another, established lymphokine system, namely an migrationi n h i b i t i o n - a s s a y , which demonstrated the e x i s t i n g immunity (data not shown). Thus there i s a discrepancy between the two test systems, that supposedly both detect c e l l u l a r immune reactions. On the other hand, two other effects o r i g i n a l l y not considered in the investigations became apparent as reproducible.

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Ftinted in Germany

852 As shown, MBP inhibitis maximally at 90 minutes and decreases thereafter. The increase in e.m., as caused by Con A treatment, become significant only after 18 hours.

PPD 50

Tuberkulin 100

200

200

500

Kontrolle

0,0

-1,6

0,0

0,0

-0,6

KFA

0,0

-3,0

1,0

-0,9

3,3

KFA + MBP

0,0

-2,0

0,0

-1,0

0,0

Mg/ml

Table 1: Percentual e.m.-reductions caused by antigen stimulated rat mononuclear cell supernatants.

Figure 1: MBP reactivity of immunized and nonimmunized rat splenocytes.

853 The mobility-decreasing effect of MBP -we prefer to call this

"self-

i n h i b i t i o n " - is a well-known fact (3). Additionally a reduction of this s e l f - i n h i b i t i o n has been reported by several groups and generally attributed to the presence of mononuclear cells (4). Figure 1 shows results with EMT done with animals in three collectives: non-immunized (dotted l i n e s ) , immunized with complete adjuvant (dashed lines) and immunized with adjuvant plus MBP ( s o l i d l i n e s ) . The evaluation of mobility changes were done with two different controls: the circled symbols depict the percentual e.m.-change calculated with respect to untreated ETS. They all show a marked reduction of the ETS-mobility. In contrast, the asterix symbols refer to e.m.-changes calculated with controls consiiing of ETS plus given amounts of MBP, and thus consider the s e l f - i n h i b i t i o n of MBP. The results show a reduction of the s e l f inhibition, i.e. a relative increase of e.m. The differences between untreated and immunized animals are essentially unsignificant, suggesting a non-immunological mechanism for the MBP-self-inhibition-reduction.

In

the following this reduction i s tentatively called "MBP-degradation". The mitogenic reactivity with Concanavalin A (Con A) i s represented in figure 2. The preparation used showed no self-effects on ETS, in other words, in this case the controls of the previous experiment were identical. The meaning of the various line types of f i g . 1 are retained. These data demonstrate, that Con A-induced supernatants show an increase in e.m. of ETS. This unexpected reaction compares well to other reports, which describe similar accelerations (e.g. 5). These effects were generally interpreted as negative test results or as technical a r t i f a c t s . Our r e s u l t s , however, show this increase in ETS-mobility to be reproducible and concentration dependent. These two effects, MBP-degradation and Con A induced acceleration, were analyzed further.

Fig. 3

shows the time dependence of these reactions by

varying the time of incubation with MBP/Con A and mononuclear c e l l s . The negative values on the y-axis represent percentual reductions in e.m. of ETS, whereas the positive ones represent an increase. The MBP experiments ( s o l i d l i n e s , 100 ng/ml MBP) are compared with Con A stimulations (dashed l i n e s , 20 Mg/ml Con A). This format of representation is used for the following figures, too.

854

Figure 3: Time dependency of the incubation of mononuclear cells with MB or Con A. Fig. 4 shows the time-dependent effects of cell-free supernatants after 18 hour incubation. The mobility-decreasing effect is established within the shortest measurable time. In contrast the Con A-stimulated supernatants used 12 minutes to develop the accelerating effect.

855 10 -

zr>

S L HI "T> C S3 L 01

5

S 0

4

0'

8

12

IB

20

24

28 Minuten

-5 0 •

-10

Figure 4: Time dependency of the ETS-induced supernatant interaction. As demonstrated on fig. 5, the slowing effect of MBP-supernatants is not temperature dependent. The Con A-induced increase, however, was significantly lowered at 4°C and 60°C and showed a putative maximum at 37°C. As a further characterization of the supernatant-ETS-interaction, we tried to wash the mobility-changing effectsoff (Fig. 6), by repeatedly spinning down the ETS and resuspending them. With MBP-supernatant treated cells three wash cycles caused a slight but significant reduction of the slowing effect. Under the given experimental conditions, the Con A-induced acceleration seemed not to be removable by washing. In order to study the importance of adherent mononuclear cells for the MBP-degradation, they were depleted by plate adherence technique. Depleted and undepleted cells were incubated for different periods of time with 100 pg/ml MBP and the supernatant were added to ETS cells (Fig. 7). The column diagram shows the inhibitory effects of depleted (population B) as compared to undepleted cells (population A). After 90 minutes the differences become significant, demonstrating a possibly important role of the adherent cells, mainly spleen macrophages, on the MBP-degradation reaction. In order to assess the importance of proteolytic enzymes on the test system we used several inhibitors: EDTA, preferentially inhibiting metalloproteases (1 mM) and soja bean trypsin inhibitor (STI, 100 (jg/ml), epsilon-aminocaproic acid (-ACA, 25 mM), t-4-aminomethylcyclohexacarboic

856 acid (AMCHA, 10 mM) as inhibitors of proteases of serine-type. The i n h i b i tors were added to the f i r s t incubation step, i . e . with mononuclear c e l l s , or to the second incubation, i . e . with cell-free

Figure 6: Washing of the supernatant-treated ETS.

supernatants and ETS.

857

Population

SCN" > Br" > CI-;

increase

membranes

phase

times

liposomes.

> I0~ > C 1 0 ^ > s a l i c y l a t e 3+

10

the

effective

decreasing be

and N a

+

and

N a + = NH^ = K + .

presumably ions

pK ^ a value

ionic

strength

connected

for a c i d i c

branes .

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

sites

with

for of a

of m e m -

899

ELECTROPHORETIC STUDY ON ION BINDING TO LIPOSOMES S.A. T a t u l i a n ,

A.A.

Lev

I n s t i t u t e of C y t o l o g y , Academy of Science of USSR, Leningrad, 194064, USSR The dependence of

z e t a p o t e n t i a l of m u l t i l a m e l l a r

prepared from a z w i t t e r i o n i c choline

(DMPC),

and an a c i d i c

lipid

liposomes

dimyristoylphosphatidyl-

from a n e u t r a l l i p i d g l y c e r o l m o n o o l e a t e

(GMO)

l i p i d c o n t a i n i n g f r a c t i o n from soy-bean

( A z o l e c t i n ) on v a r y i n g c o n c e n t r a t i o n s o f d i f f e r e n t

ionic

s p e c i e s i n aqueous s o l u t i o n s was measured w i t h the

automatic

apparatus PARMOQUANT-2 ( C a r l Zeiss JENA, GDR). I t was e s t a b lished that s e l e c t i v e

a d s o r p t i o n of monovalent anions

DMPC liposomes induced c o n s i d e r a b l e i n c r e a s e i n z e t a

to poten2 -

t i a l which was not the case f o r the d i v a l e n t anion S0^ . On 2+

the o t h e r and the binding of Ca

t o DMPC turned out t o be

much more s t r o n g e r compared w i t h t h a t of Na + or K + . t i n appeared to bind s e l e c t i v e l y

K + and L i +

Azolec-

(the l a t t e r

to

be bound s t r o n g e r ) . We found only small d i f f e r e n c e s i n the binding o f I , Br , N0~, and CI , as w e l l as L i + , Na + , and + J K t o GMO liposomes. Values of i n s t r i n s i c

binding constants and numbers of

ding s i t e s

per u n i t area were determined w i t h the use

Gouy-Stern

approximation.

A sharp change i n the z e t a p o t e n t i a l

at the t r a n s i t i o n

DMPC from the g e l to l i q u i d c r y s t a l l i n e

phase was

binof of

detected.

Experimental f i n d i n g s presented p r o v i d e evidence i n favour of the e l e c t r o s t a t i c nature of ion binding t o l i p i d membranes . A schematic model f o r the molecular o r g a n i s a t i o n of terionic

l i p i d i n b i l a y e r membranes i s suggested

a zwit-

conside-

r i n g the d e f e c t s i n c r y s t a l l i n e s t r u c t u r e of the membranes as i o n - b i n d i n g

sites.

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

900 RELATIONSHIP BETWEEN THE SURFACE CHARGE AND VOLUME OF MITOCHONDRIA AT DIFFERENT ENERGETIC STATE

I. Tsoneva, T. Tomov Central Laboratory of Biophysics, Bulgarian Academy of Sciences Sofia-1113, Bulgaria The relationship between energetic state, volume and electrophoretic mobility of rat liver mitochondria has been experimentally investigated. It is shown, that in the medium with comparatively low ionic strength, the energization of mitochondria leads to a rise in the electrophoretic mobility and an increase in electrophoretic mobility are related with changes of the size of outer surface in shrinking or swelling and on the other hand with changes in the surface charge density of the outermost surface of mitochondria.

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

901

REACTIVITY OF LYMPHOCYTES TO FETAL TISSUE EXTRACTS AS MEASURED BY THE MACROPHAGE ELECTROPHORETIC MOBILITY TEST (MEM TEST) A COMPILATION OF DATA AND THEIR POSSIBLE INTERPRETATION

B. von Broen, S. Albrecht, G. Gryschek, B. Schlott, G. Pasternak Forschungszentrum für Molekularbiologie und Medizin, Akademie der Wissenschaften der DDR DDR-1115 Berlin-Buch

By use of soluble KC1 extracts of different fetal tissues it was formerly shown that lymphocytes of a high percentage of tumor-bearing men and animals react to antigenic structures of embryonic origin (cf. Abstracts of 4th PARMOQUANT-workshop, Rostock 1982). The reactivity seems to reflect a state of cellular sensitization to tumor-associated molecules of fetal type. Extendend studies revealed: 1. there is an extensive cross-reactivity of lymphocytes to fetal tissue extracts of different species origin, including even tissue extracts of lower vertebrates as frog and carp. These observations led to the conclusion that the sensitization is triggered by phylogenetically highly conserved structures unknown as yet. 2. the reactivity is not limited to neoplasia but may occur in pregnancy and several other non-malignant

conditions

too. The common feature of the phenomena under study could be a state of cellular hyperproliferation that leads to an excessive expression of fetal-type antigens. With respect to the biological significance of the immunological reactivity to fetal-type cell structures arising in adult life a hypothetical explanation is offered for discussion.

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

902 ELPHOR VAP 21 - A NEW MULTIUSER FREE PLOW ELECTROPHORESIS INSTRUMENT

Gerhard Weber Bender & Hobein GmbH, 8000 München 15

New f o c u s i n g t e c h n i q u e s w e r e i n t r o d u c e d i n t o

free

trophoresis

years.

by Wagner e t

These methods a r e : flow i s o e l e c t r i c Based upon t h e

free

al. flow

during the

free

f o c u s i n g and f r e e

flow

flow electrophoresis techniques

application.

f l u e n c i n g the discussed in

o gf

more than 10 Using f r e e

free

an i n s t r u m e n t

flow

zone

electro-

done

regarding their

data of

in

field

the i n s t r u m e n t ,

and t h e q u a l i t y

cells of

of

of

the i n s t r u m e n t ,

flow electrophoresis

in-

separation

per

proteins

the

are

preparative

can be enhanced

to

hour.

f i e l d step electrophoresis

the

preparative

c o u l d be e x t e n d e d t o more than 5 grams

hour.

Using the a p p r o p r i a t e suitable for

are d i s c u s s e d ,

potential free

flow

through-put per

al.,

detail.

Using the w h o l e through-put

free

d u r i n g 20 y e a r s .

Some t e c h n i c a l

flexibility

elec-

isotachophoresis.

c o o p e r a t i o n w i t h Wagner e t

a t e c h n i q u e which was n e a r l y e x c l u s i v e l y

The v a r i o u s of

flow

f i e l d step electrophoresis,

was d e v e l o p e d which c o u l d p e r f o r m a l l phoresis,

last

for

large-scale

analytical

With the

technique,

purifications,

is

but can a l s o

not

the d e t e c t i o n unit

be used

Elphor Scan and t h e

Elphor VAP 21, sample volumes i n the ^ 1 - r a n g e

be s e p a r a t e d

and d e t e c t e d e i t h e r

ge,

scattering

by l i g h t

only

purposes.

combination of

instrument

the i n s t r u m e n t

o r by

by absorbance

can

i n t h e UV r a n -

fluorescence.

Cell Electrophoresis © 1985 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

AUTHOR INDEX

Agadshanyan, A l b i n i , B. A l b r e c h t , S. Alomran, A. Anders, 0. Arnold, K.

Th.M. 549 603,611 897 807 477,503 219

Babusikova, 0. 485 Baronenko, V.A. 557 Bater, A . J . 33 Behm, E. 403 B e i j a e v a , M.G. 557 Bernard, D. 373,32 385 Besse, G. B e t a i l , G. 385 B i s c h o f , R. 777 B l i a c h e r , R. 565 Bochharev, J.M. 537 Bohn, B. 405 B r i e s e , V. 621 Brock, J. 669,677 Broen, B. von 897 Bubenik, J. 459 Bubenikova, D. 459 Bülow, G. 403 Burkhardt, E. 435 Chassagne, J. 373,385 Chaubal, K.A. 515 Chen Min 635 Che Yufan 789 C h o l l e t , Ph. 373,385 Chorvath, B. 485 Cieszka, K. 451 Cohly, H. 589,603 Crawford , N. 225 Dancshazy, Z. 167 De Cuyper, M. 203,211 Deeley, J . O . T . 33 Dirks, E. 617 Diwok, K. 261,403 Doltchinkova, V. 691 Donath, E. 123 D o n e l l y , P.K. 807 Donner, M. 589 Dozmorov, J.M. 421 Droesch, S. 589

Dulatova, M.V. 651 D u v i v i e r , C. 897 Eggers, G. 477 Endo, K. 55 Fahlbusch, B. 765 Fedorenko, B. 529 565 Fedorova, J . Ferriere, J . P . 373,385 F i e l d , D. 721 Field, E.J. 703,721,747 F u j i , M. 55,581 Gachon, F. 373 G a i l l a r d , G. 373,385 G a l l i e n , P. 191 G a l u t z o v , B. 527 Gardon-Mollard, Ch. 385 Gehre, M. 783 Goetz, Ph. 41 G o l z e v , V. 173,691 G o m a r i z - Z i l b e r , E. 411 Green, K. 611 G r i s h i n a , E.V. 645 Grossmann, H. 757 Gryschek, G. 897 Haas, W. 87 Hamann, D. 367 Hamann, E. 367 Hansen, E. 287 Haroske, D. 223,765 Hashimoto, N. 819 Hattenbach, A. 223 Hayashi, H. 55,581 Hein, J . 271 H e i n r i c h , P. 785 Hennighausen, G. 657,663 H i l d e b r a n d t , K.K. 441 H i l s c h e r , H. 405 H i r o s e , F. 55,581 H j e r t e n , St. 23 Hobusch, D. 247 Hoffmann, W. 493 Holowiecki, J . 467 Hotta, T. 581 Hückel, Ch. 669,677

904 Hyrc, K.

451

Lap, V. 145 Lawin, P.Y. 537 Legros, M. 373,385 Lehmann, M. 405 Lennard, T.W.J. 807 Lev, A.A. 897 Leyhausen, G. 87 Luckmann, N.P. 733 Luner, St.J. 147 Lutsenko, T.V. 421

Irmscher, J. 757 Islamov, B. I. 645 Ivanov, A.Yu. 651 Ivanov, St. 527 Iwaguchi, T. 345 Jagoda, K. 467 Janot, C. 897 Jarczok, K. 467 Jenssen, H.L. 397 777 Jira, M. 733 Jones, R.J. Joniau, M. 203,211 Joyce, G. 703,721 Kandzia, J. 87 Kantcheva, M. 145,167,173, 527,691 Kapiszewska,M. 451 Kaufmann, R. 493 Kawai , Y. 55 Kertscher, H.P. 219 Khudensky, J.K. 537 Kitagawa, K. 55 Klinkmann, H. 55,313 Kludas, U. 183 Klug, H. 503 Knlppel, E. 55,261,271,313, 333,565,617,621 Koenig, U.D. 851 Ko hs ak i , G. 355 Konikova, E. 485 Konrad, H. 503 Kosowski, H. 785 Kotsch, M. 757 Kovarik, J. 777 Kovatchev, D. 527,691 Kozan, B. 851 Kracht, M. 441 Krahl, R. 219 Kramp, B. 397 Kraskina, N. 565 Krüger, K. 191 Kundt, G. 441 Kusnirczyk , P. 669 Kuzova, K. 529 Lambert, P.P. 625 Lambrecht, R. 785 Lammens-Vers Iijpe, M. Lamprecht, I. 75

625

Malbohan, I. 777 Malher, E. 897 Matsunaga, K. 581 Mehrishi, J.N. 97, 411 Metzner, G. 765 Meyer, H.W. 247 Meyer-Rienecker, H. 247 Miroshnikov, A.J. 645,651 Mischel, M. 75 Mix, E. 397 Mori, T. 355 Mross, K.B. 871 Mücke, D. 405 Müller, M. 271 Müller, M. 757 Müller-Ruchholtz, W. 87 Nebe, Th. 305 Nikolayeva, I.S. 421 Nimmich, W. 183 Ninger, E. 777 Nishimura, Y. 55 Nose, S. 819 Novichenko, N.L. 861 Oguchi, Y. 581 Olkhovikova, N.B. Onodera, Ch. 55 Ottmann, 0. 493

537

Paegelow, I. 829 Paitasz, E. 669 Pantev, T. 529 Pasternak, G. 897 Peters, E. 247 Petkanchin, I.B. 137 Petrov, R.V. 421 Plagne, R. 373,385 Plate, K. 785 Pliquett, F. 145 Polkanov, V.S. 537 Popdimitrova, N. 167 Popelinsky, L. 777

905

Preece, A.W. 733 Preussner, S. 617 Proud, G. 807

55,581 Toyama, N. Tsoneva, I. 897 Tulupov, A.N 897

Redmann, K. Ruzhov, N. Rychly, J.

Ujhazy, P.

541 529 55,197,313,429, 333,477,663,681 Sabolovic, D. 283,333,429 Sapozhnikov, A.M. 421 Säur, F. 897 Schaffner, H. 801 Scheffel, U. 435 Schlott, B. 897 Schröder, I. 697,847 Schulz, M. 477 669 Schulz, U. Schutt, W. 55,247,261,313 333,441,429,697 Sennesael, J.J. 625 Shamratova, V.G. 557 Shen Xiao-Hua 635 Shenton, B.K. 807 Shimizu, M. 345 Shi Yongde 789 Sigal, V.L. 113 Simonov, J.N. 113 Sochor, H. 783 493 Steiner, R. Stoltz, Y.F. 589,897 Stoylov, St. 167 Straube, W. 621 777 Strejcek, J. Sudik, R. 621 Sun Ling 635 Suong, T.T. 137 Szymaniec, S. 663 581 Takahashi, N. Tatulian, S.A. 897 Tang Zhenshen 789 Taylor, R.M.R. 807 Temnov, A. V. 549,651 Thomaneck, U. 55,183,313,333, 367,503 Tkachev, V.V. 537 Todd, P.W. 3,23 Todorov, G.S. 137,173 Tomov, T. 897 Töwe, J. 441

485

603,611 van Oss, C. Voigt, A. 123 Vranska, T. 529 Wagner, Weiser, Wendel, Werner,

H. M. H. H.

757 603,611 261 677,783,829 847 411 Wioland, M. Wojtczak, L. 159 Xiao Baogno Xu Ying-Hui

789 635

581 Yanagis awa, M. Ye Qingwei 789 Yoshihumi, Ch. 55,581 Zemanova, D. 777 Zingler, G. 183,197 681 Ziska, P. Zschörnig, 0 219

SUBJECT

INDEX

ACE-710

851,871

-, carcino-embryonic

adeturone adherent cells adrenalin

agglutination aggregation

411 147,651

rachidonic acid

541,651

aging

147,747

algae

75

autocoids

alpha-L-fucose

851,747,757

amniotic fluid

41,313

75

663 75,897

antibody -, anti-lymphocyte globulin 287,313,373,565,581,747 -, anti-thymocyte globulin 589,617 -, cationised double 287 -, detection 565 -, monoclonal 287,313,467 485,581,847 — , BLT 765 — , Coulter Clone T,B 829 — , HLA-BW 4, BW 6 87 — , Leu 313,829 — , Lyt 287,589,829 — , OKT 287,385,765 — , Thy 287,829 -, sandwich technique 287 -, production 287 -, production, monitoring 617 antigen -,- antibody reaction 191 191

-, ACE-710 851,871 -, laser Doppler 733 — , LAZYPHER 493 -, PARMOQUANT - 2 55,313 - L 55,581 -, Pen Kem 3000 41 -, Video Image Correlation System 33 bacteria -, Escherichia coli 183, 191, 197,897 -, Staphylococcus aureus 897 -, Klebsiella pneumoniae 897 -, holobacterium 167 bacteriorhodopsin

167

BCG immunotherapy

373,385

beam splitter bentonite

733

137

binding constants blast cells

antideprès ants 663

-, bacteria -, c aps u1ar

69,70,71

83

automated cell electrophoresis apparatusses

alternating electric field

antibiotics

-, -, -, -,

557

affinity surface

anaesthetics

-,

287,305,313 589

733, 757 cancer basic protein 871 common tumor 747 density 565 detection 565 fixing cells 565

biological spread 581,651, 565

brain neurons

219

459 55

211

body fluids -, amniotic 41,313 -, cerebrospinal 247

908 -, lung lavage 261 -, pharyngeal aspirates -, serum 271 bone marrow

271

589

coergistic drug effects collagen disease

287,477,485

bronchoalveolar lavage

chloropromazine

261

buffers for electrophoresis 3,603,611 burst counter

733

CAM-apparatus

97,411,451,625

colloids

819

41,733

common tumor antigen

capillary

capsular antigen n 2+ pump 225 Ca carbonyl iron

203

carrageenan

801

33 467

cyclophosphamide

657

cystic fibrosis

271,313

cytopherometer 55,137,147, 159,173,459,529,541,691,703, 721,851,871 -, semi-automatic 313,807

733

cardiolipin

continous flow electrophoresis 3 convection 3 cryopreservation

191

cytostatics

cell-cell interaction

23,515

467

cytotoxic drug effects

cell separation -, antigen specific 287 -, artefacts 411 -, differentiation 225 -, free flow electrophoresis 3,203,211,287,305,421,435, 603,611 -, free zone electrophoresis 23 -, magnetic immune 87 -, velocity sedimentation 421 cycle 26,287

diet

cellular spin resonance

diffusion coefficient

central nervous system cerebrospinal fluid chemotaxis Chinese herbs chloroplast

847

75 557

247

Debye-Huckel length defibrination

635

663 123

411

delayed fluorescence density gradient dielectrophoresis

691

429 75

di-n-octyl-tin-di chloride 657 733

DLV0 theory D 2 0 gradient drugs

41

113 603,611

droplet sedimentation

173,691

55

851,871

cross-correlation

733

747

computer-controlled video system Concanavalin A

cancer 261,313,861,871 -, basic protein 871 breast 385,871 bronchial 261,313,367 ENT 397 mamma 851

663

345,807,829

3

909

-,

ecto-ATPase a c t i v i t y 645, 657,663 e l e c t r i c d i p o l e moment electric

light

137

free radicals

137

frequency spectrum

scattering

electrodes electronic

41,55,733 d i s c r i m i n a t i o n 55 e l e c t r o n microscopy 191,435, 503 electroosmosis

123,733

electrostatic repulsive

forces 541

enzymatic a c t i v i t i e s epithelial

cells

erythrocytes -, -, -, -, -,

159,225 611

3 , 3 31, 45 55 ,, 11 4173,,713233 ,

human

23,313,537,645,651, 703,721 mixture 3,41,55 rabbit 23 rat 527,529 sheep 801,819,847

fast

applications 203,211,225, 287,421,435,871

Fourier-transformation

f r e e zone e l e c t r o p h o r e s i s

gangliosides glial

cells

147

glioma b a s i c protein glutaraldehyde glycopeptide glycophorin glycoproteine

733 147 123

Goy-Chapman e q u a t i o n Goy-Chapman-Stern

Ficoll fimbria

human

75

411

grating velocimeter

flow c y t o m e t r y

87,305

f l u i d d r o p l e t model

113

f l u o r e s c e n t membrane probe 159 free-flow electrophoresis - , technique — , ACE-710 851,897 — , E l p h o r VAP 21 897 — , FF 48 203, 211

41

3 557

haemopoietic c e l l l i n e s haemorrhagic d i a t h e s i s

haemostasis

503

405

reaction 287 429,733,871

haemoglobin l e v e l

fibroblasts,

theory 219,897

graft-versus-host

fermentation

897

167

grafting - , a l l o g e n i c , syngenic - , sham 405 -, skin 405,747

gravity

fetal tissue extract

789

225

FACS, f l u o r e s c e n c e a c t i v a t e d cell sorter 87,287,305 191

23

75

23

granulocytes 41

541

225

handling - , blood 411 - , c e l l suspension heparin

485 503

411

515

histamin H 2 receptor histocompatibility

283

antigens 287,747

910

insulin

515

interference

filter

immobilization immune RNA

733

549

789

immuno e l e c t r o n

microscopy 435

immunofluorescence

287

immunosuppressive -, factor 807 - , substance 581,657,807 immunotherapy

373

i o n e x c h a n g e r model

113

ionic

159

amphiphiles

ionic

environment

ionic

strength

iron oxide

897 123,897

pigment

41

leucocytes 225,287,411 - , migration i n h i b i t i o n test 871 leukemia 313,493,503 - , acute lymphoblastic 467 acute myeloid 459,467, 493,503 B-cell 485 cell line 485 chronic g r a n u l o c y t i c chronic lymphocytic chronic myeloid stem c e l l 459 T - c e l l 485 Levamisole linoleic

703,721,733

lipids 225,557 - , exchange 203 -, fluidity 589 lipopolysaccharide

-, -, -, -,

liposomes

KCl-tumor e x t r a c t s kidney

cells

871

3

laser 41,733 - , Doppler v e l o c i m e t r y 493,721,733,897 latex

particles

LAZYPHER lectin - , PHA - , ConA -, -, LDH

LPS PWM

225,313 691,765,819,871 669,677,819,847, 851,871 819 819

355

Lentinan

41,581

493

819

493

819

acid

irradiation alpha 515,529 follow-up studies 367 gamma 527,529 ultra-violett 537,541

467 467

197

41,159,203,211, 223,313,897

lymph node c e l l s - , human 397 - , mice 287, 459 lymphocytes -, B cells 197,287,313 - , chicken 435 - , enrichment 287,411 - , helper 283,287,333,441, 589 - , human p e r i p h e r a l b l o o d 33, 2 2 5 , 2 8 3 , 2 8 7 , 3 1 3 , 3 4 5 , 3 7 3 , 385,403,421,589,635,747, 789 - , intermediate mobility 283 intraepithelial 603,611 isolation 411,421,429 mouse 287,611 null 383 stem c e l l 287 s t i m u l a t i n g agent 819 subpopulation 283,287, 313,411,421,429,565,733,819 - , suppressor c e l l s 283,287, 333,441

911

T cells

197,261,283,287, 313,493,589,669, 667,697 low m o b i l i t y 261,367 - , transformation 373 - , transformation t e s t (LTT) 807,851 lymphokine, charge changing 313,733-896 fractionation 757,765,807, 829,847,871 - , heat l a b i l i t y 757 specific activity 757 lymphoma - , thymic Lymphoprep

485 459 733

macrophage 635,721.806 - , s l o w i n g f a c t o r (MSF) 757, 765,871 magnetic immune microspheres (MIMS) 87 melanoma - , hamster 451 human 147,373,783 (non)-pigmented 451 membrane artificial 203,211,223 - , brush border 41 - , charges 225 - , drug i n t e r a c t i o n 97 -, intracellular 225 - , ion binding 897 - , neural 211 - , surface 123,203,225 MEM-test 733-805,829,871,897 - , aging 747 - , anti-lymphocytic sera 747 - , cancer d e t e c t i o n 313, 733,747,777,783,897 - , cross r e a c t i v i t y 897 - , immune RNA 789 - , mixed lymphocyte r e a c t i o n (MLR) 747 - , multiple sclerosis 733, 747

-,

non-malignant

-,

obliterative disease 785 pregnancy 747,897 skin g r a f t i n g 747 t r a n s p l a n t a t i o n 747

-, -, -,

meningitis

conditions 747,897 vascular

247

mental d i s o r d e r s metal c a t i o n s

97 159

metal s a l t s

663

metastasis

147,451,515

methyl

819

methyl c e l l u l o s e

41,411, 733 myelin basic p r o t e i n (MBP) 851,871 microcirculation microsomes

541

159

microblogulin,

Bg-

microgravity

3

microspheres

87,225

microtubules

211

mitochondria

159,897

mitotic rate

657,663

373

mixed lymphocyte c u l t u r e 225,669,747,871 MMC

345

modulation - , ConA 847 - , immuno847 monitoring - , antisera -,

production 617 r a d i a t i o n treatment 367

monoclonal antibody (see antibody) monocytes

287

mononuclear blood c e l l s 611,757,765

912

monosaccharide

—, —, —,

757

morphology o f

cells

MORPHOQUANT

549

549

multilamellar vesicles

219

313,703, 721,733 diagnosis 703,721 f a m i l i y studies 703

—,

multiple sclerosis

—,

-,

—,

muscular dystrophy

313 —,

neuraminidase

123,147,225, 467,681,861

neuroblastoma c e l l s

23

neuroendocrine d i s o r d e r s 5'-nucleotidase nystatin

97

—, —, —, —,

225

liposomes 313,225 lymph node c e l l s 397 macrophages 313,757, 765,777,783,785,807 o i l droplets 247,261, 271 p e r i p h e r a l blood lymphocytes 313,367,403,421 p e r i p h e r a l blood lymphoc y t e subpopulations 313,333,345,355,429,565, 589,617,621,677,681,697 splenocytes 405,565, 635,669 tanned sheep red blood cells 635,861 thrombocytes 503 thymocytes 313,549, 657,663 tumor c e l l s 397

particle size

75

particle

41

concentration

o b l i t e r a t i v e vascular diseases 785

passive e l e c t r i c

occlusive a r t e r i a l

path c u v e t t e

oligopeptides oil

847

droplets

optical

247,261,271

fibre

organelles

diseases 541

733 159

organo m e t a l i c

compounds 663

oxygen - , capacity 541 consumption 451 cells

properties 145

55

pea c h l o r o p l a s t s Pen Kem 3000 pentobarbitol

691

41,313 515

Perco11 429,515,603 p e r f l u o r o c a r b o n , blood substitutes 60,645 663 pesticides phagocytosis

225

pharyngeal a s p i r a t e phenothiazine

pancreatic i s l e t

3

PARMOQUANT - , t e c h n i c a l dates 55 - , measuring o b j e c t s — , bacteria 183,191,197 — , erythrocytes 55,113, 537,557,589,645,651 , mixture of 55 — , leukemic c e l l s 459, 467,477,485

137

271

589

phospholipid -, -, -,

203,211,219, 225 composition 159 transfer 203 vesicles 203

phosphorylation photodestruction photodiode

733

159 541

913

p h o t o s y n t h e t i c membranes

173, 691

phytohemagglutinin 145,527, 691,819,871 pH value

41,691

pigment content

451

pituitary cells

3

platelets 225,503,733 - , acute leukemia 503 - , e l e c t r o n micrographs - , volume d i s t r i b u t i o n polypeptide pregnancy -, factor

225 503

261

analysis

serotonin

847 147

skin -, grafting 747 -, testing 373 - , tumor 451 Smoluchowski-Hiickel 733

Space S h u t t l e

313,747,897 621

s p e c i f i c conductance splenocytes

stationary layer stress

pulmonary diseases

261

purified protein derivate (PPD) 721,783,871 733

purple membrane

137,167

r a d i o b i o l o g i c a l studies radio-protectors,

515

sensitizers 515

Rank m i c r o - e l e c t r o p h o r e s i s apparatus 33,373,385 renal transplantation rosette

formation

respiratory distress

807

313,403, 477 syndrome 271

sample m i c r o s h i f t e r sarcoidosis

403

55

75

41,55,493,733

405,549,557

surface - , antigens 287 - , bound I g 435 carbohydrates 147 - , charge 123,167,173,515, 541,581,589,663 - , potential 123,159 - , structure 123 surfactants 159 TEEM-test 807-893 cancer 313,861,871 - , drugs 829 -, lectine 819,829 -, transfer factor 847 teratom d e r i v e d p r o t e i n theophylline thrombin thylakoid

Sacchoromyces c e r e v i s i a e

451

287,405,669,667

psoriasis

537

123

3

protease 807,871 -, inhibitor 765

PVPNO

theory 113

space charge d e n s i t y

657

733 651

271 acid

smoking

225

prednisolone

serological

sialic 287

33,451

semipermeable membranes

serum

plaque-forming c e l l s

pneumonia

sedimentation

819

411 173

thymocytes 405,635 - , human 313,549 - , mice 287,459,657,663 - , rat 663

757

914

transfer factor

697,847,851

transplantation - , skin 405,747 - , renal 807 t r i a n g l e deconvolution trypsin

147,313,697

tubulin

211

33

tumor a s s o c i a t e d antigens (TAA) 313,777,783,861 ultrasound

75

velocimetry 733 - , sedimentation vesicles

287

123,225

video-image c o r r e l a t i o n viscosity,

blood

541

volume d i s t r i b u t i o n , cytes 503 yeast

33

thrombo-

75

zeta potential

113,223,897

zone sedimentation

3

NO CHARG no problem for Zetasizer II In studies of flocculation and colloid stability, the region close to the zero point of charge is the most critical. It is in precisely this region of low mobility that other electrophoresis systems are least accurate and most difficult to use. With its high frequency optical modulator, the Malvern Zetasizer II solves this problem, allowing you to measure mobility and zeta potential with uncompromising accuracy right across the range.

| Measurements are simple, fast and operator-independent and Zetasizer II also gives you built-in submicron particle size capability. Learn more about the potential of Zetasizer II in your laboratory — contact MALVERN today.

—

m§

Malvern Instruments Ltd Spring Lane South Malvern Worcestershire WR14 1AQ England Telephone: (06845) 68415 Telex: 339679

Malvern Instruments Inc 187 Oaks Road Framingham MA 01701 USA Telephone: (617)626 0200 Telex: 311397

w DE

G H. Hirai (Editor)

Walter de Gruyter Berlin-New York Electrophoresis '83

Advanced Methods Biochemical and Clinical Applications Proceedings of the International Conference on Electrophoresis • Tokyo, Japan, May 9-12,1983 1984.17cm x 24cm. XX, 787 pages. Numerous illustrations. Hardcover. DM 280,-; approx. US$93.30 ISBN 311009788 5

D. Stathakos (Editor)

Electrophoresis '82

Advanced Methods Biochemical and Clinical Applications Proceedings of the International Conference on Electrophoresis • Athens, Greece, April 21-24,1982 1983.17cm x 24cm. XVI, 867 pages. Numerous illustrations. Hardcover. DM 260,-; approx. US $86.70 ISBN 311 008791X

R. C. Allen (Editor)

Electrophoresis '81

Advanced Methods Biochemical and Clinical Applications Proceedings of the Third International Conference on Electrophoresis • Charleston, South Carolina, USA, April 1981 1981.17cm x 24cm. XVIII, 1041 pages. Numerous illustrations. Hardcover. DM 245,-; approx. US$81.70 ISBN 311 008155 5

C. J. Hoiloway

(Editor)

Analytical and Preparative Isotachophoresis Proceedings • 3rd International Symposium on Isotachophoresis • Goslar, Germany, June 1-4,1982 1984.17cm x 24 cm. XIII, 404 pages. With numerous illustrations. Hardcover DM 170,-; approx. US $56.70 ISBN 311 010178 5

Prices are subject to change without notice