![CHAPTER XII
BENEATH THE SURFACE
The terrible story I had been listening to for two weeks broke over
me like a storm. Was this the Revolution I had believed in all my life,
yearned for, and strove to interest others in, or was it a caricature—a
hideous monster that had come to jeer and mock me? The
Communists I had met daily during six months—self-sacrificing,
hard-working men and women imbued with a high ideal—were such
people capable of the treachery and horrors charged against them?
Zinoviev, Radek, Zorin, Ravitch, and many others I had learned to
know—could they in the name of an ideal lie, defame, torture, kill?
But, then—had not Zorin told me that capital punishment had been
abolished in Russia? Yet I learned shortly after my arrival that
hundreds of people had been shot on the very eve of the day when
the new decree went into effect, and that as a matter of fact
shooting by the Tcheka had never ceased.
That my friends were not exaggerating when they spoke of tortures
by the Tcheka, I also learned from other sources. Complaints about
the fearful conditions in Petrograd prisons had become so numerous
that Moscow was apprised of the situation. A Tcheka inspector came
to investigate. The prisoners being afraid to speak, immunity was
promised them. But no sooner had the inspector left than one of the
inmates, a young boy, who had been very outspoken about the
brutalities practised by the Tcheka, was dragged out of his cell and
cruelly beaten.
Why did Zorin resort to lies? Surely he must have known that I
would not remain in the dark very long. And then, was not Lenin
also guilty of the same methods? Anarchists of ideas [ideyni] are
not in our prisons, he had assured me. Yet at that very moment
numerous Anarchists filled the jails of Moscow and Petrograd and of](https://image.slidesharecdn.com/24155-250417075732-f9d9871d/85/HIV-Protocols-2nd-Edition-Kimdar-Sherefa-Kemal-Phd-27-320.jpg)
![But knowing her as I did, it was not difficult for me to understand
how bitter was her grief over the hapless and formless thing that
had come to Russia. But now her beloved Italians were coming!
They would bring with them the warmth and colour of Italy.
The Italians came and with them new festivities, demonstrations,
meetings, and speeches. How different it all appeared to me from
my memorable first days on Belo-Ostrov. No doubt the Italians now
felt as awed as I did then, as inspired by the seeming wonder of
Russia. Six months and the close proximity with the reality of things
quite changed the picture for me. The spontaneity, the enthusiasm,
the vitality had all gone out of it. Only a pale shadow remained, a
grinning phantom that clutched at my heart.
On the Uritski Square the masses were growing weary with long
waiting. They had been kept there for hours before the Italian
Mission arrived from the Tauride Palace. The ceremonies were just
beginning when a woman leaning against the platform, wan and
pale, began to weep. I stood close by. It is easy for them to talk,
she moaned, but we've had no food all day. We received orders to
march directly from our work on pain of losing our bread rations.
Since five this morning I am on my feet. We were not permitted to
go home after work to our bit of dinner. We had to come here.
Seventeen hours on a piece of bread and some kipyatok [boiled
water]. Do the visitors know anything about us? The speeches went
on, the Internationale was being repeated for the tenth time, the
sailors performed their fancy exercises and the claqueurs on the
reviewing stand were shouting hurrahs. I rushed away. I, too, was
weeping, though my eyes remained dry.
The Italian, like the English, Mission was quartered in the Narishkin
Palace. One day, on visiting Angelica there, I found her in a
perturbed state of mind. Through one of the servants she had
learned that the ex-princess Narishkin, former owner of the palace,
had come to beg for the silver ikon which had been in the family for
generations. Just that ikon, she had implored. But the ikon was
now state property, and Balabanova could do nothing about it. Just](https://image.slidesharecdn.com/24155-250417075732-f9d9871d/85/HIV-Protocols-2nd-Edition-Kimdar-Sherefa-Kemal-Phd-35-320.jpg)
![escape: he landed in the Tcheka. The next morning the Moscow
Pravda and the Izvestia denounced the action of the Printers' Union
as counter-revolutionary, and raged about Tchernov having been
permitted to speak. The papers called for exemplary punishment of
the printers who dared defy the Soviet Government.
The Bakers' Union, a very militant organization, had also been
suppressed, and its management replaced by Communists. Several
months before, in March, I had attended a convention of the bakers.
The delegates impressed me as a courageous group who did not
fear to criticise the Bolshevik régime and present the demands of the
workers. I wondered then that they were permitted to continue the
conference, for they were outspoken in their opposition to the
Communists. The bakers are 'Shkurniki' [skinners], I was told;
they always instigate strikes, and only counter-revolutionists can
wish to strike in the workers' Republic. But it seemed to me that the
workers could not follow such reasoning. They did strike. They even
committed a more heinous crime: they refused to vote for the
Communist candidate, electing instead a man of their own choice.
This action of the bakers was followed by the arrest of several of
their more active members. Naturally the workers resented the
arbitrary methods of the Government.
Later I met some of the bakers and found them much embittered
against the Communist Party and the Government. I inquired about
the condition of their union, telling them that I had been informed
that the Russian unions were very powerful and had practical control
of the industrial life of the country. The bakers laughed. The trade
unions are the lackeys of the Government, they said; they have no
independent function, and the workers have no say in them. The
trade unions are doing mere police duty for the Government. That
sounded quite different from the story told by Melnichansky, the
chairman of the Moscow Trade Union Soviet, whom I had met on my
first visit to Moscow.
On that occasion he had shown me about the trade union
headquarters known as the Dom Soyusov, and explained how the](https://image.slidesharecdn.com/24155-250417075732-f9d9871d/85/HIV-Protocols-2nd-Edition-Kimdar-Sherefa-Kemal-Phd-44-320.jpg)
![six compartments, two of which were converted into a dining room
and kitchen. They were of diminutive size, but we managed to make
a presentable dining room of one, and the kitchen might have made
many a housekeeper envy us. A large Russian samovar and all
necessary copper and zinc pots and kettles were there, making a
very effective appearance. We were especially proud of the
decorative curtains on our car windows. The other compartments
were used for office and sleeping quarters. I shared mine with our
secretary, Miss A. T. Shakol.
Besides Alexander Berkman, appointed by the Museum as chairman
and general manager, Shakol as secretary, and myself as treasurer
and housekeeper, the Expedition consisted of three other members,
including a young Communist, a student of the Petrograd University.
En route we mapped out our plan of work, each member of the
Expedition being assigned some particular branch of it. I was to
gather data in the Departments of Education and Health, the
Bureaus of Social Welfare and Labour Distribution, as well as in the
organization known as Workers' and Peasants' Inspection. After the
day's work all the members were to meet in the car to consider and
classify the material collected during the day.
Our first stop was Kursk. Nothing of importance was collected there
except a pair of kandai [iron handcuffs] which had been worn by a
revolutionist in Schlüsselburg. It was donated to us by a chance
passer-by who, noticing the inscription on our car, Extraordinary
Commission of the Museum of the Revolution, became interested
and called to pay us a visit. He proved to be an intellectual, a
Tolstoian, the manager of a children's colony. He succeeded in
maintaining the latter by giving the Soviet Government a certain
amount of labour required of him: three days a week he taught in
the Soviet schools of Kursk. The rest of his time he devoted to his
little colony, or the Children's Commune, as he affectionately called
it. With the help of the children and some adults they raised the
vegetables necessary for the support of the colony and made all the
repairs of the place. He stated that he had not been directly](https://image.slidesharecdn.com/24155-250417075732-f9d9871d/85/HIV-Protocols-2nd-Edition-Kimdar-Sherefa-Kemal-Phd-60-320.jpg)
HIV Protocols 2nd Edition Kimdar Sherefa Kemal Phd
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- 8. M E T H O D S I N M O L E C U L A R B I O L O G YTM John M. Walker, SERIES EDITOR 489. Dynamic Brain Imaging: Methods and Protocols, edited by Fahmeed Hyder, 2009 485. HIV Protocols: Methods and Protocols, edited by Vinayaka R. Prasad and Ganjam V. Kalpana, 2009 484. Functional Proteomics: Methods and Protocols, edited by Julie D. Thompson, Christine Schaeffer-Reiss, and Marius Ueffing, 2008 483. Recombinant Proteins From Plants: Methods and Protocols, edited by Loı̈c Faye and Veronique Gomord, 2008 482. Stem Cells in Regenerative Medicine: Methods and Protocols, edited by Julie Audet and William L. Stanford, 2008 481. Hepatocyte Transplantation: Methods and Protocols, edited by Anil Dhawan and Robin D. Hughes, 2008 480. Macromolecular Drug Delivery: Methods and Protocols, edited by Mattias Belting, 2008 479. Plant Signal Transduction: Methods and Protocols, edited by Thomas Pfannschmidt, 2008 478. Transgenic Wheat, Barley and Oats: Production and Characterization Protocols, edited by Huw D. Jones and Peter R. Shewry, 2008 477. Advanced Protocols in Oxidative Stress I, edited by Donald Armstrong, 2008 476. Redox-Mediated Signal Transduction: Methods and Protocols, edited by John T. Hancock, 2008 475. Cell Fusion: Overviews and Methods, edited by Elizabeth H. Chen, 2008 474. Nanostructure Design: Methods and Protocols, edited by Ehud Gazit and Ruth Nussinov, 2008 473. Clinical Epidemiology: Practice and Methods, edited by Patrick Parfrey and Brendon Barrett, 2008 472. Cancer Epidemiology, Volume 2: Modifiable Factors, edited by Mukesh Verma, 2008 471. Cancer Epidemiology, Volume 1: Host Susceptibility Factors, edited by Mukesh Verma, 2008 470. Host-Pathogen Interactions: Methods and Protocols, edited by Steffen Rupp and Kai Sohn, 2008 469. Wnt Signaling, Volume 2: Pathway Models, edited by Elizabeth Vincan, 2008 468. Wnt Signaling, Volume 1: Pathway Methods and Mammalian Models, edited by Elizabeth Vincan, 2008 467. Angiogenesis Protocols: Second Edition, edited by Stewart Martin and Cliff Murray, 2008 466. Kidney Research: Experimental Protocols, edited by Tim D. Hewitson and Gavin J. Becker, 2008. 465. Mycobacteria, Second Edition, edited by Tanya Parish and Amanda Claire Brown, 2008 464. The Nucleus, Volume 2: Physical Properties and Imaging Methods, edited by Ronald Hancock, 2008 463. The Nucleus, Volume 1: Nuclei and Subnuclear Components, edited by Ronald Hancock, 2008 462. Lipid Signaling Protocols, edited by Banafshe Larijani, Rudiger Woscholski, and Colin A. Rosser, 2008 461. Molecular Embryology: Methods and Protocols, Second Edition, edited by Paul Sharpe and Ivor Mason, 2008 460. Essential Concepts in Toxicogenomics, edited by Donna L. Mendrick and William B. Mattes, 2008 459. Prion Protein Protocols, edited by Andrew F. Hill, 2008 458. Artificial Neural Networks: Methods and Applications, edited by David S. Livingstone, 2008 457. Membrane Trafficking, edited by Ales Vancura, 2008 456. Adipose Tissue Protocols, Second Edition, edited by Kaiping Yang, 2008 455. Osteoporosis, edited by Jennifer J. Westendorf, 2008 454. SARS- and Other Coronaviruses: Laboratory Protocols, edited by Dave Cavanagh, 2008 453. Bioinformatics, Volume 2: Structure, Function, and Applications, edited by Jonathan M. Keith, 2008 452. Bioinformatics, Volume 1: Data, Sequence Analysis, and Evolution, edited by Jonathan M. Keith, 2008 451. Plant Virology Protocols: From Viral Sequence to Protein Function, edited by Gary Foster, Elisabeth Johansen, Yiguo Hong, and Peter Nagy, 2008 450. Germline Stem Cells, edited by Steven X. Hou and Shree Ram Singh, 2008 449. Mesenchymal Stem Cells: Methods and Protocols, edited by Darwin J. Prockop, Douglas G. Phinney, and Bruce A. Brunnell, 2008 448. Pharmacogenomics in Drug Discovery and Development, edited by Qing Yan, 2008. 447. Alcohol: Methods and Protocols, edited by Laura E. Nagy, 2008 446. Post-translational Modifications of Proteins: Tools for Functional Proteomics, Second Edition, edited by Christoph Kannicht, 2008. 445. Autophagosome and Phagosome, edited by Vojo Deretic, 2008 444. Prenatal Diagnosis, edited by Sinhue Hahn and Laird G. Jackson, 2008. 443. Molecular Modeling of Proteins, edited by Andreas Kukol, 2008. 442. RNAi: Design and Application, edited by Sailen Barik, 2008. 441. Tissue Proteomics: Pathways, Biomarkers, and Drug Discovery, edited by Brian Liu, 2008 440. Exocytosis and Endocytosis, edited by Andrei I. Ivanov, 2008 439. Genomics Protocols, Second Edition, edited by Mike Starkey and Ramnanth Elaswarapu, 2008 438. Neural Stem Cells: Methods and Protocols, Second Edition, edited by Leslie P. Weiner, 2008 437. Drug Delivery Systems, edited by Kewal K. Jain, 2008 436. Avian Influenza Virus, edited by Erica Spackman, 2008 435. Chromosomal Mutagenesis, edited by Greg Davis and Kevin J. Kayser, 2008 434. Gene Therapy Protocols: Volume 2: Design and Characterization of Gene Transfer Vectors, edited by Joseph M. Le Doux, 2008 433. Gene Therapy Protocols: Volume 1: Production and In Vivo Applications of Gene Transfer Vectors, edited by Joseph M. Le Doux, 2008 432. Organelle Proteomics, edited by Delphine Pflieger and Jean Rossier, 2008 431. Bacterial Pathogenesis: Methods and Protocols, edited by Frank DeLeo and Michael Otto, 2008 430. Hematopoietic Stem Cell Protocols, edited by Kevin D. Bunting, 2008 429. Molecular Beacons: Signalling Nucleic Acid Probes, Methods and Protocols, edited by Andreas Marx and Oliver Seitz, 2008 428. Clinical Proteomics: Methods and Protocols, edited by Antonia Vlahou, 2008 427. Plant Embryogenesis, edited by Maria Fernanda Suarez and Peter Bozhkov, 2008 426. Structural Proteomics: High-Throughput Methods, edited by Bostjan Kobe, Mitchell Guss, and Thomas Huber, 2008 425. 2D PAGE: Sample Preparation and Fractionation, Volume 2, edited by Anton Posch, 2008
- 9. ME T H O D S I N MO L E C U L A R BI O L O G Y TM HIV Protocols SecondEdition Edited By Vinayaka R. Prasad, PhD and Ganjam V. Kalpana, PhD AlbertEinsteinCollegeofMedicine,Bronx,NY,USA
- 10. Editor Vinayaka R. Prasad Ganjam V. Kalpana Albert Einstein College of Medicine Albert Einstein College of Medicine Bronx, NY Bronx, NY USA USA prasad@aecom.yu.edu kalpana@aecom.yu.edu Series Editor John M. Walker University of Hertfordshire Hatfield Herts UK ISBN: 978-1-58829-859-1 e-ISBN: 978-1-59745-170-3 ISSN: 1064-3745 e-ISSN: 1940-6029 DOI 10.1007/978-1-59745-170-3 Library of Congress Control Number: 2008938341 c Humana Press, a part of Springer Science+Business Media, LLC 2009 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper springer.com
- 11. Preface Why another book of HIV protocols? The question is sure to arise in the minds of the readers. The AIDS epidemic continues unabated despite strong advances in thera- peutics and an unprecedented level of efforts in vaccine development. Although some of the reasons for the failure in AIDS control can be attributed to poor prevention measures or paucity of antiretrovirals, a major drawback is the absence of efficacious, potent antiretrovirals that can both suppress viral load completely and that do not have toxic side effects. Toxicity can lead to nonadherence, which in turn results in poor virus control, emergence of drug resistance and the eventual clinical drug fail- ure. Development of novel drugs and vaccines require definition of new targets, bet- ter definition of already known viral targets and understanding the interplay between viral and host factors. Similarly, in order to develop an AIDS vaccine, we need to be equipped with effective methods to measure immune response. More importantly, these studies require the development of efficient and powerful in vitro and in vivo systems to study viral replication and pathogenesis. Therefore, HIV researchers have a real need for access to well-described, state-of-the-art methods to study HIV. Approaches in HIV/AIDS investigation have continuously advanced in tune with the evolution of modern experimental science. Development of new technologies in investigating familiar aspects of HIV replication or immune response to it have led to new insights that have improved our understanding of the biology of HIV. In compiling this collection, our objective is threefold. First, we aim to document up-to- date protocols available for select aspects of HIV biology. Second, we bring together both virological and immunological approaches in a single volume. Third, we provide a comprehensive account of techniques that are not already part of an existing HIV protocol book. HIV Protocols, Second Edition, is organized into five sections. Section I delin- eates the methods to isolate full-length DNA clones of HIV-1 from patient samples, isolation of HIV-1 particles free of contaminating cellular proteins and a method to titer these virus particles. Section II delineates the study of early and late events. Early events include entry, reverse transcription, nuclear transport, integration as well as recombination, a process that occurs during reverse transcription. A thorough study of early events would be incomplete without the analysis of complexes formed during reverse transcription and prior to integration as well as the interaction between viral and host proteins within these complexes. In the subsection on late events, we take you through methods to study assembly and particle production within the producer cells and the use of cell free systems to study the interaction of viral proteins and nucleic acids including the cognate tRNALys,3. No HIV-1 investigation is complete without the analysis of the dynamics of host- virus interactions. HIV-1, being an intra-cellular parasite, not only invades the host, but also subverts cellular antiviral mechanisms and hijacks host proteins for its own purposes. Section III explores approaches to investigate the interplay between the v
- 12. vi Preface host and the virus by employing genetic, molecular and cellular techniques including novel small animal models. Methods to investigate specific, immunological techniques to understanding host-HIV-1 interplay are discussed in Section IV. Chapters in this section delineate methods to study mucosal immunity, T-cell responses and antiviral responses in cell culture and in Rhesus monkey models. The last section of the book delves into the intense battle between the host and the HIV-1. The virus continues to evade antiretrovirals owing to its ability to develop drug resistance and its baffling ability to evolve and escape the immune system. The chapters included should facilitate investigations of drug resistant viruses and virus evolution. We would like to draw the readers’ attention to the Notes sections in each chapter. These notes come from the experts who have used these methods successfully many times and contain many ‘tricks’ and little details that are rarely mentioned in standard protocols. We find them to be a unique aspect of the Methods in Molecular Biology series. We would like to thank Humana Press for the opportunity to edit this book, the series editor, Dr. John Walker, for his continuous support and guidance and David Casey of Humana Press for his patience and support. We extend our sincerest gratitude to all contributors for their submission of critical contributions to this collection. The advice of Dr. Barbara Shacklett of the University of California, Davis in the selection of immunological topics is specially acknowledged. The administrative assistance of Ms. Emilia Ortiz in the production of the book went beyond the call of duty and we are thankful to her. We also wish to express our gratitude to our graduate students, post-doctoral fellows and other close colleagues at Einstein who helped with editing the book for scientific content (Dhivya Ramalingam, Elizabeth Hanna Luke, Sonald Duclair and Vasudev Rao) or for style (Andrea Provost, Aviva Joseph) and in the beta testing and improving of the subject index (Chisanga Lwatula, James Gaudette, Jennifer Cano, Melissa Smith, SeungJae Lee, Sheeba Mathew, Sohrab Khan, Supratik Das). We would like to thank Humana Press for the opportunity to edit this book as well as the series editor, Dr. John Walker, for his continuous support and guidance. We acknowledge the advice of Dr. Barbara Shacklett of the University of California, Davis in the selection of immunological topics. The administrative assistance of Ms. Emilia Ortiz often went beyond the call of duty. We also wish to express our grati- tude to colleagues at Einstein who helped with editing for scientific content (Dhivya Ramalingam, Elizabeth Hanna Luke, Sonald Duclair and Vasudev Rao) or for style (Andrea Provost). Finally, we extend our sincerest gratitude to all contributors for their submission of critical contributions to this collection. Vinayaka R. Prasad, PhD Ganjam V. Kalpana, PhD
- 13. Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi SECTION I. PREPARATION OF VIRUS PARTICLES AND THEIR ANALYSIS 1 Methods for Viral RNA Isolation and PCR Amplification for Sequencing of Near Full-Length HIV-1 Genomes Kimdar Sherefa Kemal, Milan Reinis, Barbara Weiser, and Harold Burger . . . . . . . . 3 2 Purification of HIV-1 Virions by Subtilisin Digestion or CD45 Immunoaffinity Depletion for Biochemical Studies David E. Ott . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 3 Calculating HIV-1 Infectious Titre Using a Virtual TCID50 Method Yong Gao, Immaculate Nankya, Awet Abraha, Ryan M. Troyer, Kenneth N. Nelson, Andrea Rubio, and Eric J. Arts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 SECTION II. METHODS TO STUDY HIV-1 REPLICATION SUBSECTION A. EARLY EVENTS 4 Cell-Free Assays for HIV-1 Uncoating Christopher Aiken. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 5 Real-Time PCR Analysis of HIV-1 Replication Postentry Events Jean L. Mbisa∗, Krista A. Delviks-Frankenberry∗, James A. Thomas, Robert J. Gorelick, Vinay K. Pathak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 6 Analysis of 2-LTR Circle Junctions of Viral DNA in Infected Cells Dibyakanti Mandal and Vinayaka R. Prasad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 7 HIV-1 Recombination: An Experimental Assay and a Phylogenetic Approach Michael D. Moore, Mario P.S. Chin, and Wei-Shau Hu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 8 Methods of Preparation and Analysis of Intracellular Reverse Transcription Complexes Ariberto Fassati. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107 9 Analysis of Viral and Cellular Proteins in HIV-1 Reverse Transcription Complexes by Co-immunoprecipitation Sergey N. Iordanskiy and Michael I. Bukrinsky . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121 10 Isolation and Analysis of HIV-1 Preintegration Complexes Alan Engelman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .135 11 Bisarsenical Labeling of HIV-1 for Real-Time Fluorescence Microscopy Nathalie J. Arhel and Pierre Charneau . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151 SUBSECTION B. LATE EVENTS 12 Methods for the Study of HIV-1 Assembly Abdul A. Waheed, Akira Ono, and Eric O. Freed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .163 vii
- 14. viii Contents 13 Assembly of Immature HIV-1 Capsids Using a Cell-Free System Jaisri R. Lingappa and Beth K. Thielen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .185 14 Preparation of Recombinant Hiv-1 Gag Protein and Assembly of Virus-Like Particles In Vitro Siddhartha A.K. Datta and Alan Rein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .197 15 Methods for the Analysis of HIV-1 Nucleocapsid Protein Interactions with Oligonucleotides Andrew G. Stephen and Robert J. Fisher. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .209 16 Methods for Analysis of Incorporation and Annealing of tRNALys in HIV-1 Shan Cen, Fei Guo, and Lawrence Kleiman. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .223 SECTION III. SPECIALIZED APPROACHES TO STUDY HIV-1 BIOLOGY AND PATHOGENESIS 17 Somatic Cell Genetic Analyses to Identify HIV-1 Host Restriction Factors Susana T. Valente and Stephen P. Goff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .235 18 Rapid, Controlled and Intensive Lentiviral Vector-Based RNAi Manuel Llano, Natassia Gaznick, and Eric M. Poeschla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .257 19 Reverse Two-Hybrid Screening to Analyze Protein–Protein Interaction of HIV-1 Viral and Cellular Proteins Supratik Das and Ganjam V. Kalpana. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .271 20 Methods to Study Monocyte Migration Induced by HIV-Infected Cells Vasudev R. Rao, Eliseo A. Eugenin, Joan W. Berman, and Vinayaka R. Prasad . . . .295 21 Novel Mouse Models for Understanding HIV-1 Pathogenesis Aviva Joseph, Kaori Sango, and Harris Goldstein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .311 SECTION IV. IMMUNOLOGICAL STUDIES OF HIV SUBSECTION A. MUCOSAL IMMUNOLOGY 22 Mucosal Antibody Responses to HIV Zina Moldoveanu and Jiri Mestecky . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .333 23 Isolating Mucosal Lymphocytes from Biopsy Tissue for Cellular Immunology Assays Barbara L. Shacklett, J. William Critchfield, Donna Lemongello . . . . . . . . . . . . . . . . . . . . .347 SUBSECTIOM B. MEASURING T CELL RESPONSES VIA FLOW CYTOMETRY 24 Quantifying HIV-1-Specific CD8+ T-Cell Responses Using ELISPOT and Cytokine Flow Cytometry Barbara L. Shacklett, J. William Critchfield, and Donna Lemongello . . . . . . . . . . . . . . . .359 25 Multiparameter Flow Cytometry Monitoring of T Cell Responses Holden T. Maecker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .375 SUBSECTION C. ANTIVIRAL RESPONSES 26 Measuring HIV Neutralization in a Luciferase Reporter Gene Assay David C. Montefiori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .395 27 Assessing the Antiviral Activity of HIV-1-Specific Cytotoxic T Lymphocytes Otto O. Yang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .407
- 15. Contents ix 28 Methods for Quantitating Antigen-Specific T Cell Responses Using Functional Assays in Rhesus Macaques Rama Rao Amara. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .417 SECTION V. DRUG RESISTANT VIRUSES AND VIRAL EVOLUTION 29 Isolation of Drug-Resistant Mutant HIV Variants Using Tissue Culture Drug Selection Maureen Oliveira, Bluma G. Brenner, and Mark A. Wainberg . . . . . . . . . . . . . . . . . . . . . .427 30 Virus Evolution as a Tool to Study HIV-1 Biology Ben Berkhout and Atze T. Das. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .435 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .453
- 16. Contributors AWET ABRAHA, BA • Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA CHRISTOPHER AIKEN, PHD • Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA RAMA RAO AMARA, PHD • Department of Microbiology and Immunology, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA NATHALIE J. ARHEL, PHD • Institut Pasteur, Molecular Virology and Vectorology Group, Virology Department, Paris, France ERIC J ARTS, PHD • Division of Infectious Diseases, Department of Medicine; Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH, USA BEN BERKHOUT, PHD • Laboratory of Experimental Virology, Academic Medical Center, Amsterdam, The Netherlands JOAN W. BERMAN, PHD • Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA BLUMA G. BRENNER, PHD • McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada MICHAEL I. BUKRINSKY, MD, PHD • The George Washington University, Washington, DC, USA HAROLD BURGER, MD, PHD • Wadsworth Center, New York State Department of Health, Albany, New York Albany Medical College, Albany, NY, USA SHAN CEN, PHD • Department of Medicine, McGill University, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada PIERRE CHARNEAU, PHD • Institut Pasteur, Molecular Virology and Vectorology Group, Virology Department, Paris, France MARIO P.S. CHIN, PHD • HIV Drug Resistance Program, National Cancer Institute, Frederick, MD, USA J. WILLIAM CRITCHFIELD, PHD • Department of Medical Microbiology and Immunology and School of Medicine, University of California, Davis, CA, USA ATZE T. DAS, PHD • Laboratory of Experimental Virology, Academic Medical Center, Amsterdam, The Netherlands SUPRATIK DAS, PHD • Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY, USA SIDDHARTHA A.K. DATTA, PHD • HIV Drug Resistance Program, National Cancer Institute, Frederick, MD, USA KRISTA A. DELVIKS-FRANKENBERRY, PHD • HIV-Drug Resistance Program, NCI-Frederick, Frederick, MD, USA xi
- 17. xii Contributors ALAN ENGELMAN, PHD • Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Division of AIDS, Harvard Medical School, Boston, MA, USA ELISEO A. EUGENIN, PHD • Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA ARIBERTO FASSATI, MD, PHD • Wohl Virion Centre and MRC Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, London, UK ROBERT J. FISHER, PHD • Protein Chemistry Laboratory, Research Technology Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA ERIC O. FREED, PHD • Virus-Cell Interaction Section, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, MD, USA YONG GAO, MD, PHD • Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA NATASSIA GAZNICK, BS • Molecular Medicine Program, Mayo Clinic College of Medicine, Rochester, MN, USA STEPHEN P. GOFF, PHD • Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, College of Physicians and Surgeons, New York, NY, USA HARRIS GOLDSTEIN, MD • Departments of Microbiology and Immunology and Pediatrics, Albert Einstein College of Medicine, Bronx, NY, USA ROBERT J. GORELICK, PHD • AIDS Vaccine Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA FEI GUO, PHD • Lady Davis Institute for Medical Research, Jewish General Hospital Montreal, QC, Canada WEI-SHAU HU, PHD • HIV Drug Resistance Program, National Cancer Institute, Frederick, MD, USA SERGEY N. IORDANSKIY, PHD • The D.I. Ivanovsky Institute of Virology, Moscow, Russia AVIVA JOSEPH, PHD • Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA GANJAM V. KALPANA, PHD • Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY, USA KIMDAR SHEREFA KEMAL, PHD • Wadsworth Center, New York State Department of Health, Albany, NY, USA LAWRENCE KLEIMAN, PHD • Department of Medicine, McGill University, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada DONNA LEMONGELLO, BS • Department of Medical Microbiology and Immunology and School of Medicine, University of California, Davis, CA, USA JAISRI R. LINGAPPA, MD, PHD • Departments of Pathobiology and Medicine, University of Washington, Seattle, WA, USA MANUEL LLANO, MD, PHD • Molecular Medicine Program, Mayo Clinic College of Medicine, Rochester, MN, USA HOLDEN T. MAECKER, PHD • Palo Alto, CA, USA
- 18. Contributors xiii DIBYAKANTI MANDAL, PHD • Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA JEAN L. MBISA, PHD • HIV-Drug Resistance Program, NCI-Frederick, Frederick, MD, USA JIRI MESTECKY, MD, PHD • University of Alabama, Birmingham, AL, USA ZINA MOLDOVEANU, PHD • University of Alabama, Birmingham, AL, USA DAVID C. MONTEFIORI, PHD • Department of Surgery, Laboratory for AIDS Vaccine Research and Development, Duke University Medical Center, Durham, NC, USA MICHAEL D. MOORE, PHD • HIV Drug Resistance Program, National Cancer Institute, Frederick, MD, USA IMMACULATE NANKYA, MBCHB, PHD • Division of Infectious Diseases, Department of Medicine, Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH, USA KENNETH N. NELSON, BA • Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA MAUREEN OLIVEIRA, BSC • McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada AKIRA ONO, PHD • Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA DAVID E. OTT, PHD • AIDS Vaccine Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA VINAY K. PATHAK, PHD • HIV-Drug Resistance Program and AIDS Vaccine Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA ERIC M. POESCHLA, MD • Molecular Medicine Program, Mayo Clinic College of Medicine, Rochester, MN, USA VINAYAKA R. PRASAD, PHD • Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA VASUDEV R. RAO, MBBS, MS • Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA ALAN REIN, PHD • HIV Drug Resistance Program, National Cancer Institute, Frederick, MD, USA MILAN REINIS, PHD • Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague ANDREA RUBIO, BSC • National Reference Center for AIDS, Department of Microbiology, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina KAORI SANGO, MS • Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA BARBARA L. SHACKLETT, PHD • Department of Medical Microbiology and Immunology and Division of Infectious Diseases, Department of Internal Medicine, School of Medicine, University of California, Davis, CA, USA ANDREW G. STEPHEN, PHD • Protein Chemistry Laboratory, Research Technology Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA BETH K. THIELEN, BS • Departments of Pathobiology and Medicine, University of Washington, Seattle, WA, USA
- 19. xiv Contributors JAMES A. THOMAS, PHD • AIDS Vaccine Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA RYAN M. TROYER, PHD • Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA SUSANA T. VALENTE, PHD • Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, College of Physicians and Surgeons, New York, NY, USA ABDUL A. WAHEED, PHD • Virus-Cell Interaction Section, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, MD, USA MARK A. WAINBERG, PHD • McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada BARBARA WEISER, MD • Wadsworth Center, New York State Department of Health, and Albany Medical College, Albany, NY, USA OTTO O. YANG, MD • Division of Infectious Diseases, Department of Medicine, Department of Microbiology, Immunology, and Molecular Genetics and AIDS Institute, Geffen School of Medicine, UCLA Medical Center, Los Angeles, CA, USA
- 20. Section I Preparation of Virus Particles and Their Analysis
- 21. Chapter 1 Methods for Viral RNA Isolation and PCR Amplification for Sequencing of Near Full-Length HIV-1 Genomes Kimdar Sherefa Kemal, Milan Reinis, Barbara Weiser, and Harold Burger Abstract HIV-1 in plasma represents the viral quasispecies replicating in the patient at any given time. Studies of HIV-1 viral RNA from plasma or other body fluids therefore reflect the virus present in real time. To obtain near full-length genomic sequences derived from virion RNA it is first necessary to carefully isolate and amplify the RNA. The procedure described below, involves viral RNA extraction, reverse transcription (RT) of the extracted RNA to produce cDNA copies, and PCR amplification of long HIV-1 gene fragments using site-specific, overlapping primers. The primers are based on subtype B HIV-1 strains, and plasma speci- mens are used in the procedures. However, the protocol can easily be adapted to other HIV-1 subtypes by modifying the primers to match the subtype of interest. Key words: HIV-1, HIV-1 viral RNA, HIV-1 primers, Long RT-PCR amplification of HIV-1. 1. Introduction HIV-1 infection is characterized by continuous replication of ∼ 9 kb RNA genomes resulting in a viral swarm of closely related molecules called quasispecies (1, 2). Sequence variation is a hall- mark of lentivirus infection; surviving viral species reflect replica- tion and selection (3). When studying the relationship between HIV-1 sequences and pathogenesis, it is highly desirable to ana- lyze the complete HIV-1 genome because variability in multiple regions of the genome may play a role in pathogenesis and virus– host interactions (4–6). Full-length HIV-1 sequencing provides essential data needed to address vaccine design and molecular epidemiology (7, 8). Full-length sequence analysis also helps to identify the presence of dual infections and recombination in vivo Vinayaka R. Prasad, Ganjam V. Kalpana (eds.), HIV Protocols: Second Edition, vol. 485 C 2009 Humana Press, a part of Springer Science+Business Media DOI 10.1007/978-1-59745-170-3 1 Springerprotocols.com 3
- 22. 4 Kemal et al. (9–11). Furthermore, knowledge of the complete genomic HIV- 1 RNA sequence and HLA type of the infected individual makes it possible, with the use of an immunologic database (http://hiv- web.lanl.gov/content/immunology/index), to predict cytotoxic T-cell epitopes encoded by the virus (4,5). HIV-1 in plasma represents the replicating virus population at any given time; by contrast, proviral DNA, integrated in cel- lular genomes, represents a repository of older sequences, the majority of which have been shown to lack replication compe- tence (12,13). Amplification of a full-length HIV-1 genome requires careful step-by-step procedures involving viral RNA extraction, synthesis of cDNA copies of the viral RNA (RT-PCR), PCR amplification of target gene fragments using specific primers, and the purifica- tion of the amplified PCR products for further analyses. Successful amplification of a near full-length HIV-1 viral RNA depends on several factors, including viral RNA load in the specimen, quality of specimen, viral RNA isolation methods used, cDNA synthesis from RNA using RT-PCR, primer selection, and PCR amplifica- tion conditions (6, 9, 14–17). The quality of the specimen, such as plasma, is directly affected by factors such as storage temper- ature, repeated freezing and thawing, and general handling of the specimen from initial processing to the RNA extraction steps. Although we cannot rule out the possibility of amplifying full- length HIV-1 genomes from specimens with viral loads as low as 5,000 copies/mL, we recommend using samples with viral loads of at least 10,000 copies/mL. Specimens should be aliquoted into 1 mL volume and stored at temperatures below −80 ◦C. Repeated freeze and thaw steps need to be avoided (see Note 1 ). 2. Materials 2.1. HIV-1 Viral RNA Isolation 1. RNAgentsR Total RNA Isolation System kit (Promega). The kit includes combined guanidine thiocyanate crystals and the Citrate/Sarcosine/β-Mercaptoethanol (CSB) buffer in a single bottle of denaturing solution. Phenol/chloroform/ isoamyl alcohol solution, isopropanol, 2 M sodium acetate (pH 4.0), and nuclease free water are also included. 2. Yeast t-RNA (Sigma). 3. Glycogen (Sigma). 4. Siliconized Flat Top microfuge tubes (Fisher Scientific). 2.2. Reverse Transcription and cDNA Synthesis SuperScript TM First-strand Synthesis System for RT-PCR kit (Invitrogen).
- 23. PCR Amplification of Full-Length HIV-1 5 2.3. PCR Amplification 1. GeneAmp XL PCR Kit (Applied Biosystems). 2. AmpliWax, PCR Gem 50 (Applied Biosystems). 3. 10 mM dNTP Mix with dTTP (Applied Biosystems). 4. TE buffer (pH 8.0). 2.4. PCR Primers All the PCR primers and PCR amplification programs specific for each fragment are listed in Table 1.1. 2.5. Agarose Gel Analysis 1. Agarose (Fisher Scientific). 2. 6X gel-loading buffer: 0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol, water up to desired volume. A stock solution can be prepared and aliquots can be made in 1.5 mL microfuge tubes and stored in a refrigerator. Once a tube is taken out of the refrigerator, it can be stored at room temper- ature and used. 3. Tris-Borate-EDTA (TBE) buffer: 0.45 M Tris-borate, 0.01 M EDTA, pH 8.3. 4. Ethidium bromide tablets, 100 mg/tablet (Sigma). To have a 10 mg/mL stock solution, dissolve one tablet in 10 mL deion- ized water and keep the solution at room temperature away from direct light (see Note 2 ). 5. DNA ladder, 1 kb plus (Invitrogen). 6. Disposable scalpels. 7. QIAquick Gel Extraction Kit (Qiagen). 3. Methods HIV-1 viral RNA isolation is one of the critical steps for the success of the rest of the procedures. RNA should always be han- dled with care; gloves should be worn at all times to help elim- inate the introduction of endonucleases. It is important to work in an RNase-free environment and use RNase-free reagents. Work should be done in a Bio-safety level 2 (BSL-2) laminar flow hood; RNA isolation areas should be separate from DNA or PCR ampli- fication areas in the lab. It is also important to have dedicated pipettes for RNA isolation. If this is not possible, always clean the hood and the pipettes with RNase and DNA contaminant remov- ing reagents and turn on the UV light, in the laminar flow hood, for 20–30 min after use. To increase viral recovery, we suggest pel- leting the plasma virions in siliconized microfuge tubes. We also recommend using 0.5–1.0 mL sample volumes, particularly if the viral load is low ( 10, 000 copies/mL). When available, 1 mL is preferred. Using this method, RNA for full-length amplification can be recovered from plasma samples with viral loads as little as 5,000 RNA copies/mL (see Note 3 ).
- 24. 6 Kemal et al. Table 1.1 PCR primers and amplification conditions used to amplify a 9-kb HIV-1 subtype B genome as four overlapping fragments Primer Primer sequences (5 − 3) HXB2 location Fragment 1/partial 5 LTR and gag 517FA CTT-AAG-CCT-CAA-TAA-AGC-TTG-CCT-TGA 517–543B 2348RC TAC-TGT-ATC-ATC-TGC-TCC-TGT-ATC 2348–2325 548F TCA-AGT-AGT-GTG-TGC-CCG-TCT-G 549–570 2314R TCC-TTT-AGT-TGC-CCC-CCT-ATC 2314–2294 PCR conditions: 1X 94 ◦C: 5 min 5X 94 ◦C: 15 s, 45 ◦C: 45 s, 72 ◦C: 3 min 30X 94 ◦C: 15 s, 55 ◦C: 45 s, 72 ◦C: 3 min 1X 72 ◦C: 10 min hold at 4 ◦C: until next step Fragment 2/pol 1999F AAT-TGC-AGG-GCC-CCT-AGA-AAA-AAG-GGC-TGT 1999–2028 5304R TTC-TAT-GGA-GAC-TCC-CTG-ACC-CAA-ATG-CCA 5304–5275 2138F AGA-GCA-GAC-CAG-AGC-CAA-CAG 2138–2158 5202R TCC-CCT-AGT-GGG-ATG-TGT-ACT 5222–5202 PCR conditions: 1X 94 ◦C: 5 min 5X 94 ◦C: 15 s, 45 ◦C: 45 s, 72 ◦C: 5 min; 30X 94 ◦C: 15 s, 55 ◦C: 60 s, 72 ◦C: 4 min; 1X 72 ◦C: 10 min hold at 4 ◦C: until next step Fragment 3/env and accessory genes 4650F ATT-CCC-TAC-AAT-CCC-CAA-AGT-CAA-G 4650–4674 9626R CTT-GAA-GCA-CTC-AAG-GCA-AGC-TTT-ATT-G 9626–9599 4956F TGG-AAA-GGT-GAA-GGG-GCA-GTA-GTA-ATA-CAA-G 4956–5014 9173R TGG-TGT-GTA-GTT-CTG-CCA-ATC-AGG-GAA-G 9173–9146 PCR conditions: 1X 94 ◦C: 5 min 5X 94 ◦C: 15 s, 55 ◦C: 45 s, 72 ◦C: 5 min; 30X 94 ◦C: 15 s, 60 ◦C: 5 min 1X 72 ◦C: 10 min hold at 4 ◦C: until next step (continued)
- 25. PCR Amplification of Full-Length HIV-1 7 Table 1.1 (continued) Fragment 4/nef and LTR 8081F TGG-AAT-AAC-ATG-ACC-TGG-AGG-G 8091–8112 9626R CTT-GAA-GCA-CTC-AAG-GCA-AGC-TTT-ATT-G 9626–9599 8296F CAT-AAT-GAT-AGT-AGG-AGG-CTT-GG 8279–8301 9608R TGA-AGC-ACT-CAA-GGC-AAG-C 9608–9590 PCR conditions: 1X 94 ◦C: 5 min 35X 94 ◦C: 15 s; 55 ◦C: 45 s; 72 ◦C: 3 min 1X 72 ◦C: 10 min hold at 4 ◦C: until next step AF: stands for forward primers, the numbers indicate the nucleotide positions of the for- ward or reverse genome fragment. BIndicates nucleotide positions in HIV-1 HXB2 strain. CR: stands for reverse primers. 3.1. RNA Extractions 3.1.1. Beginning 1. Clarify plasma by spinning at 400 × g, at room temperature, for 10 min (see Note 4 ). 2. Transfer the plasma to sterile, siliconized, 1.5 mL microfuge tubes. 3. To pellet virions, centrifuge the plasma 18, 500 × g, at 10 ◦C, for 90 min. 4. Remove most of the now virion-free plasma without disturb- ing the pellet (see Note 5 ). 5. To extract virion-associated RNA, re-suspend the pellet in 300 μL pre-chilled denaturing solution. 6. Vortex the pellet until it is completely dissolved. This usually takes 5–10 min (see Note 6 ). 7. Mix in order: 1 μL yeast tRNA (1 μg/μL), 1 μL glycogen (2 μg/μL) and 30 μL of 2 M sodium acetate, pH 4.0 (see Note 7 ). 8. Add 32 μL of the above mixture into each tube, and then add 0.5 mL phenol/chloroform/isoamyl alcohol solution, being careful to remove only from the lower organic phase (see Note 8 ). 9. Vortex for 10 s, then chill on ice for 20 min. 10. Centrifuge at 18, 500 × g for 20 min, at 4 ◦C. 11. Transfer the top phase, which contains the RNA, to fresh microfuge tubes (see Note 9 ). 12. To precipitate the RNA, add an equal volume of isopropanol (0.3–0.5 mL) and keep the tubes at −20 ◦C for at least 2 h (see Note 10 ).
- 26. Discovering Diverse Content Through Random Scribd Documents
- 27. CHAPTER XII BENEATH THE SURFACE The terrible story I had been listening to for two weeks broke over me like a storm. Was this the Revolution I had believed in all my life, yearned for, and strove to interest others in, or was it a caricature—a hideous monster that had come to jeer and mock me? The Communists I had met daily during six months—self-sacrificing, hard-working men and women imbued with a high ideal—were such people capable of the treachery and horrors charged against them? Zinoviev, Radek, Zorin, Ravitch, and many others I had learned to know—could they in the name of an ideal lie, defame, torture, kill? But, then—had not Zorin told me that capital punishment had been abolished in Russia? Yet I learned shortly after my arrival that hundreds of people had been shot on the very eve of the day when the new decree went into effect, and that as a matter of fact shooting by the Tcheka had never ceased. That my friends were not exaggerating when they spoke of tortures by the Tcheka, I also learned from other sources. Complaints about the fearful conditions in Petrograd prisons had become so numerous that Moscow was apprised of the situation. A Tcheka inspector came to investigate. The prisoners being afraid to speak, immunity was promised them. But no sooner had the inspector left than one of the inmates, a young boy, who had been very outspoken about the brutalities practised by the Tcheka, was dragged out of his cell and cruelly beaten. Why did Zorin resort to lies? Surely he must have known that I would not remain in the dark very long. And then, was not Lenin also guilty of the same methods? Anarchists of ideas [ideyni] are not in our prisons, he had assured me. Yet at that very moment numerous Anarchists filled the jails of Moscow and Petrograd and of
- 28. many other cities in Russia. In May, 1920, scores of them had been arrested in Petrograd, among them two girls of seventeen and nineteen years of age. None of the prisoners were charged with counter-revolutionary activities: they were Anarchists of ideas, to use Lenin's expression. Several of them had issued a manifesto for the First of May, calling attention to the appalling conditions in the factories of the Socialist Republic. The two young girls who had circulated a handbill against the labour book, which had then just gone into effect, were also arrested. The labour book was heralded by the Bolsheviki as one of the great Communist achievements. It would establish equality and abolish parasitism, it was claimed. As a matter of fact, the labour book was somewhat of the character of the yellow ticket issued to prostitutes under the Tsarist régime. It was a record of every step one made, and without it no step could be made. It bound its holder to his job, to the city he lived in, and to the room he occupied. It recorded one's political faith and party adherence, and the number of times he was arrested. In short, a yellow ticket. Even some Communists resented the degrading innovation. The Anarchists who protested against it were arrested by the Tcheka. When certain leading Communists were approached in the matter they repeated what Lenin had said: No Anarchists of ideas are in our prisons. The aureole was falling from the Communists. All of them seemed to believe that the end justified the means. I recalled the statements of Radek at the first anniversary of the Third International, when he related to his audience the marvellous spread of Communism in America. Fifty thousand Communists are in American prisons, he exclaimed. Molly Stimer, a girl of eighteen, and her male companions, all Communists, had been deported from America for their Communist activities. I thought at the time that Radek was misinformed. Yet it seemed strange that he did not make sure of his facts before making such assertions. They were dishonest and an insult to Molly Stimer and her Anarchist comrades, added to the injustice they had suffered at the hands of the American plutocracy.
- 29. During the past several months I had seen and heard enough to become somewhat conversant with the Communist psychology, as well as with the theories and methods of the Bolsheviki. I was no longer surprised at the story of their double-dealing with Makhno, the brutalities practised by the Tcheka, the lies of Zorin. I had come to realize that the Communists believed implicitly in the Jesuitic formula that the end justifies all means. In fact, they gloried in that formula. Any suggestion of the value of human life, quality of character, the importance of revolutionary integrity as the basis of a new social order, was repudiated as bourgeois sentimentality, which had no place in the revolutionary scheme of things. For the Bolsheviki the end to be achieved was the Communist State, or the so-called Dictatorship of the Proletariat. Everything which advanced that end was justifiable and revolutionary. The Lenins, Radeks, and Zorins were therefore quite consistent. Obsessed by the infallibility of their creed, giving of themselves to the fullest, they could be both heroic and despicable at the same time. They could work twenty hours a day, live on herring and tea, and order the slaughter of innocent men and women. Occasionally they sought to mask their killings by pretending a misunderstanding, for doesn't the end justify all means? They could employ torture and deny the inquisition, they could lie and defame, and call themselves idealists. In short, they could make themselves and others believe that everything was legitimate and right from the revolutionary viewpoint; any other policy was weak, sentimental, or a betrayal of the Revolution. On a certain occasion, when I passed criticism on the brutal way delicate women were driven into the streets to shovel snow, insisting that even if they had belonged to the bourgeoisie they were human, and that physical fitness should be taken into consideration, a Communist said to me: You should be ashamed of yourself; you, an old revolutionist, and yet so sentimental. It was the same attitude that some Communists assumed toward Angelica Balabanova, because she was always solicitous and eager to help wherever possible. In short, I had come to see that the Bolsheviki were social
- 30. puritans who sincerely believed that they alone were ordained to save mankind. My relations with the Bolsheviki became more strained, my attitude toward the Revolution as I found it more critical. One thing grew quite clear to me: I could not affiliate myself with the Soviet Government; I could not accept any work which would place me under the control of the Communist machine. The Commissariat of Education was so thoroughly dominated by that machine that it was hopeless to expect anything but routine work. In fact, unless one was a Communist one could accomplish almost nothing. I had been eager to join Lunacharsky, whom I considered one of the most cultivated and least dogmatic of the Communists in high position. But I became convinced that Lunacharsky himself was a helpless cog in the machine, his best efforts constantly curtailed and checked. I had also learned a great deal about the system of favouritism and graft that prevailed in the management of the schools and the treatment of children. Some schools were in splendid condition, the children well fed and well clad, enjoying concerts, theatricals, dances, and other amusements. But the majority of the schools and children's homes were squalid, dirty, and neglected. Those in charge of the preferred schools had little difficulty in procuring everything needed for their charges, often having an over-supply. But the caretakers of the common schools would waste their time and energies by the week going about from one department to another, discouraged and faint with endless waiting before they could obtain the merest necessities. At first I ascribed this condition of affairs to the scarcity of food and materials. I heard it said often enough that the blockade and intervention were responsible. To a large extent that was true. Had Russia not been so starved, mismanagement and graft would not have had such fatal results. But added to the prevalent scarcity of things was the dominant notion of Communist propaganda. Even the children had to serve that end. The well-kept schools were for show, for the foreign missions and delegates who were visiting Russia.
- 31. Everything was lavished on these show schools at the cost of the others. I remembered how everybody was startled in Petrograd by an article in the Petrograd Pravda of May, disclosing appalling conditions in the schools. A committee of the Young Communist organizations investigated some of the institutions. They found the children dirty, full of vermin, sleeping on filthy mattresses, fed on miserable food, punished by being locked in dark rooms for the night, forced to go without their suppers, and even beaten. The number of officials and employees in the schools was nothing less than criminal. In one school, for instance, there were 138 of them to 125 children. In another, 40 to 25 children. All these parasites were taking the bread from the very mouths of the unfortunate children. The Zorins had spoken to me repeatedly of Lillina, the woman in charge of the Petrograd Educational Department. She was a wonderful worker, they said, devoted and able. I had heard her speak on several occasions, but was not impressed: she looked prim and self-satisfied, a typical Puritan schoolma'am. But I would not form an opinion until I had talked with her. At the publication of the school disclosures I decided to see Lillina. We conversed over an hour about the schools in her charge, about education in general, the problem of defective children and their treatment. She made light of the abuses in her schools, claiming that the young comrades had exaggerated the defects. At any rate, she added, the guilty had already been removed from the schools. Similarly to many other responsible Communists Lillina was consecrated to her work and gave all her time and energies to it. Naturally, she could not personally oversee everything; the show schools being the most important in her estimation, she devoted most of her time to them. The other schools were left in the care of her numerous assistants, whose fitness for the work was judged largely according to their political usefulness. Our talk strengthened my conviction that I could have no part in the work of the Bolshevik Board of Education.
- 32. The Board of Health offered as little opportunity for real service— service that should not discriminate in favour of show hospitals or the political views of the patients. This principle of discrimination prevailed, unfortunately, even in the sick rooms. Like all Communist institutions, the Board of Health was headed by a political Commissar, Doctor Pervukhin. He was anxious to secure my assistance, proposing to put me in charge of factory, dispensary, or district nursing—a very flattering and tempting offer, and one that appealed to me strongly. I had several conferences with Doctor Pervukhin, but they led to no practical result. Whenever I visited his department I found groups of men and women waiting, endlessly waiting. They were doctors and nurses, members of the intelligentsia—none of them Communists—who were employed in various medical branches, but their time and energies were being wasted in the waiting rooms of Doctor Pervukhin, the political Commissar. They were a sorry lot, dispirited and dejected, those men and women, once the flower of Russia. Was I to join this tragic procession, submit to the political yoke? Not until I should become convinced that the yoke was indispensable to the revolutionary process would I consent to it. I felt that I must first secure work of a non-partisan character, work that would enable me to study conditions in Russia and get into direct touch with the people, the workers and peasants. Only then should I be able to find my way out of the chaos of doubt and mental anguish that I had fallen prey to.
- 33. CHAPTER XIII JOINING THE MUSEUM OF THE REVOLUTION The Museum of the Revolution is housed in the Winter Palace, in the suite once used as the nursery of the Tsar's children. The entrance to that part of the palace is known as detsky podyezd. From the windows of the palace the Tsar must have often looked across the Neva at the Peter-and-Paul Fortress, the living tomb of his political enemies. How different things were now! The thought of it kindled my imagination. I was full of the wonder and the magic of the great change when I paid my first visit to the Museum. I found groups of men and women at work in the various rooms, huddled up in their wraps and shivering with cold. Their faces were bloated and bluish, their hands frost-bitten, their whole appearance shadow-like. What must be the devotion of these people, I thought, when they can continue to work under such conditions. The secretary of the Museum, M. B. Kaplan, received me very cordially and expressed the hope that I would join in the work of the Museum. He and another member of the staff spent considerable time with me on several occasions, explaining the plans and purposes of the Museum. They asked me to join the expedition which the Museum was then organizing, and which was to go south to the Ukraina and the Caucasus. Valuable material of the revolutionary period was to be gathered there, they explained. The idea attracted me. Aside from my general interest in the Museum and its efforts, it meant non-partisan work, free from Commissars, and an exceptional opportunity to see and study Russia. In the course of our acquaintance I learned that neither Mr. Kaplan nor his friend was a Communist. But while Mr. Kaplan was strongly pro-Bolshevik and tried to defend and explain away everything, the other man was critical though by no means antagonistic. During my
- 34. stay in Petrograd I saw much of both men, and I learned from them a great deal about the Revolution and the methods of the Bolsheviki. Kaplan's friend, whose name for obvious reasons I cannot mention, often spoke of the utter impossibility of doing creative work within the Communist machine. The Bolsheviki, he would say, always complain about lack of able help, yet no one—unless a Communist— has much of a chance. The Museum was among the least interfered with institutions, and work there had been progressing well. Then a group of twenty youths were sent over, young and inexperienced boys unfamiliar with the work. Being Communists they were placed in positions of authority, and friction and confusion resulted. Everyone felt himself watched and spied upon. The Bolsheviki care not about merit, he said; their chief concern is a membership card. He was not enthusiastic about the future of the Museum, yet believed that the coöperation of the Americans would aid its proper development. Finally I decided on the Museum as offering the most suitable work for me, mainly because that institution was non-partisan. I had hoped for a more vital share in Russia's life than the collecting of historical material; still I considered it valuable and necessary work. When I had definitely consented to become a member of the expedition, I visited the Museum daily to help with the preparations for the long journey. There was much work. It was no easy matter to obtain a car, equip it for the arduous trip, and secure the documents which would give us access to the material we set out to collect. While I was busy aiding in these preparations Angelica Balabanova arrived in Petrograd to meet the Italian Mission. She seemed transformed. She had longed for her Italian comrades: they would bring her a breath of her beloved Italy, of her former life and work there. Though Russian by birth, training, and revolutionary traditions, Angelica had become rooted in the soil of Italy. Well I understood her and her sense of strangeness in the country, the hard soil of which was to bear a new and radiant life. Angelica would not admit even to herself that the much hoped-for life was stillborn.
- 35. But knowing her as I did, it was not difficult for me to understand how bitter was her grief over the hapless and formless thing that had come to Russia. But now her beloved Italians were coming! They would bring with them the warmth and colour of Italy. The Italians came and with them new festivities, demonstrations, meetings, and speeches. How different it all appeared to me from my memorable first days on Belo-Ostrov. No doubt the Italians now felt as awed as I did then, as inspired by the seeming wonder of Russia. Six months and the close proximity with the reality of things quite changed the picture for me. The spontaneity, the enthusiasm, the vitality had all gone out of it. Only a pale shadow remained, a grinning phantom that clutched at my heart. On the Uritski Square the masses were growing weary with long waiting. They had been kept there for hours before the Italian Mission arrived from the Tauride Palace. The ceremonies were just beginning when a woman leaning against the platform, wan and pale, began to weep. I stood close by. It is easy for them to talk, she moaned, but we've had no food all day. We received orders to march directly from our work on pain of losing our bread rations. Since five this morning I am on my feet. We were not permitted to go home after work to our bit of dinner. We had to come here. Seventeen hours on a piece of bread and some kipyatok [boiled water]. Do the visitors know anything about us? The speeches went on, the Internationale was being repeated for the tenth time, the sailors performed their fancy exercises and the claqueurs on the reviewing stand were shouting hurrahs. I rushed away. I, too, was weeping, though my eyes remained dry. The Italian, like the English, Mission was quartered in the Narishkin Palace. One day, on visiting Angelica there, I found her in a perturbed state of mind. Through one of the servants she had learned that the ex-princess Narishkin, former owner of the palace, had come to beg for the silver ikon which had been in the family for generations. Just that ikon, she had implored. But the ikon was now state property, and Balabanova could do nothing about it. Just
- 36. think, Angelica said, Narishkin, old and desolate, now stands on the street corner begging, and I live in this palace. How dreadful is life! I am no good for it; I must get away. But Angelica was bound by party discipline; she stayed on in the palace until she returned to Moscow. I know she did not feel much happier than the ragged and starving ex-princess begging on the street corner. Balabanova, anxious that I should find suitable work, informed me one day that Petrovsky, known in America as Doctor Goldfarb, had arrived in Petrograd. He was Chief of the Central Military Education Department, which included Nurses' Training Schools. I had never met the man in the States, but I had heard of him as the labour editor of the New York Forward, the Jewish Socialist daily. He offered me the position of head instructress in the military Nurses' Training School, with a view to introducing American methods of nursing, or to send me with a medical train to the Polish front. I had proffered my services at the first news of the Polish attack on Russia: I felt the Revolution in danger, and I hastened to Zorin to ask to be assigned as a nurse. He promised to bring the matter before the proper authorities, but I heard nothing further about it. I was, therefore, somewhat surprised at the proposition of Petrovsky. However, it came too late. What I had since learned about the situation in the Ukraina, the Bolshevik methods toward Makhno and the povstantsi movement, the persecution of Anarchists, and the Tcheka activities, had completely shaken my faith in the Bolsheviki as revolutionists. The offer came too late. But Moscow perhaps thought it unwise to let me see behind the scenes at the front; Petrovsky failed to inform me of the Moscow decision. I felt relieved. At last we received the glad tidings that the greatest difficulty had been overcome: a car for the Museum Expedition had been secured. It consisted of six compartments and was newly painted and cleaned. Now began the work of equipment. Ordinarily it would have taken another two months, but we had the coöperation of the man at the head of the Museum, Chairman Yatmanov, a Communist. He
- 37. was also in charge of all the properties of the Winter Palace where the Museum is housed. The largest part of the linen, silver, and glassware from the Tsar's storerooms had been removed, but there was still much left. Supplied with an order of the chairman I was shown over what was once guarded as sacred precincts by Romanov flunkeys. I found rooms stacked to the ceiling with rare and beautiful china and compartments filled with the finest linen. The basement, running the whole length of the Winter Palace, was stocked with kitchen utensils of every size and variety. Tin plates and pots would have been more appropriate for the Expedition, but owing to the ruling that no institution may draw upon another for anything it has in its own possession, there was nothing to do but to choose the simplest obtainable at the Winter Palace. I went home reflecting upon the strangeness of life: revolutionists eating out of the crested service of the Romanovs. But I felt no elation over it.
- 38. CHAPTER XIV PETROPAVLOVSK AND SCHLÜSSELBURG As some time was to pass before we could depart, I took advantage of the opportunity which presented itself to visit the historic prisons, the Peter-and-Paul Fortress and Schlüsselburg. I recollected the dread and awe the very names of these places filled me with when I first came to Petrograd as a child of thirteen. In fact, my dread of the Petropavlovsk Fortress dated back to a much earlier time. I think I must have been six years old when a great shock had come to our family: we learned that my mother's oldest brother, Yegor, a student at the University of Petersburg, had been arrested and was held in the Fortress. My mother at once set out for the capital. We children remained at home in fear and trepidation lest Mother should not find our uncle among the living. We spent anxious weeks and months till finally Mother returned. Great was our rejoicing to hear that she had rescued her brother from the living dead. But the memory of the shock remained with me for a long time. Seven years later, my family then living in Petersburg, I happened to be sent on an errand which took me past the Peter-and-Paul Fortress. The shock I had received many years before revived within me with paralyzing force. There stood the heavy mass of stone, dark and sinister. I was terrified. The great prison was still to me a haunted house, causing my heart to palpitate with fear whenever I had to pass it. Years later, when I had begun to draw sustenance from the lives and heroism of the great Russian revolutionists, the Peter-and-Paul Fortress became still more hateful. And now I was about to enter its mysterious walls and see with my own eyes the place which had been the living grave of so many of the best sons and daughters of Russia.
- 39. The guide assigned to take us through the different ravelins had been in the prison for ten years. He knew every stone in the place. But the silence told me more than all the information of the guide. The martyrs who had beaten their wings against the cold stone, striving upward toward the light and air, came to life for me. The Dekabristi, Tchernishevsky, Dostoyevsky, Bakunin, Kropotkin, and scores of others spoke in a thousand-throated voice of their social idealism and their personal suffering—of their high hopes and fervent faith in the ultimate liberation of Russia. Now the fluttering spirits of the heroic dead may rest in peace: their dream has come true. But what is this strange writing on the wall? To-night I am to be shot because I had once acquired an education. I had almost lost consciousness of the reality. The inscription roused me to it. What is this? I asked the guard. Those are the last words of an intelligent, he replied. After the October Revolution the intelligentsia filled this prison. From here they were taken out and shot, or were loaded on barges never to return. Those were dreadful days and still more dreadful nights. So the dream of those who had given their lives for the liberation of Russia had not come true, after all. Is there any change in the world? Or is it all an eternal recurrence of man's inhumanity to man? We reached the strip of enclosure where the prisoners used to be permitted a half-hour's recreation. One by one they had to walk up and down the narrow lane in dead silence, with the sentries on the wall ready to shoot for the slightest infraction of the rules. And while the caged and fettered ones treaded the treeless walk, the all- powerful Romanovs looked out of the Winter Palace toward the golden spire topping the Fortress to reassure themselves that their hated enemies would never again threaten their safety. But not even Petropavlovsk could save the Tsars from the slaying hand of Time and Revolution. Indeed, there is change; slow and painful, but come it does. In the enclosure we met Angelica Balabanova and the Italians. We walked about the huge prison, each absorbed in his own thoughts
- 40. set in motion by what he saw. Would Angelica notice the writing on the wall, I wondered. To-night I am to be shot because I had once acquired an education. Some time later several of our group made a trip to Schlüsselburg, the even more dreadful tomb of the political enemies of Tsarism. It is a journey of several hours by boat up the beautiful River Neva. The day was chilly and gray, as was our mood; just the right state of mind to visit Schlüsselburg. The fortress was strongly guarded, but our Museum permit secured for us immediate admission. Schlüsselburg is a compact mass of stone perched upon a high rock in the open sea. For many decades only the victims of court intrigues and royal disfavour were immured within its impenetrable walls, but later it became the Golgotha of the political enemies of the Tsarist régime. I had heard of Schlüsselburg when my parents first came to Petersburg; but unlike my feeling toward the Peter-and-Paul Fortress, I had no personal reaction to the place. It was Russian revolutionary literature which brought the meaning of Schlüsselburg home to me. Especially the story of Volkenstein, one of the two women who had spent long years in the dreaded place, left an indelible impression on my mind. Yet nothing I had read made the place quite so real and terrifying as when I climbed up the stone steps and stood before the forbidding gates. As far as any effect upon the physical condition of the Peter-and-Paul Fortress was concerned, the Revolution might never have taken place. The prison remained intact, ready for immediate use by the new régime. Not so Schlüsselburg. The wrath of the proletariat struck that house of the dead almost to the ground. How cruel and perverse the human mind which could create a Schlüsselburg! Verily, no savage could be guilty of the fiendish spirit that conceived this appalling tomb. Cells built like a bag, without doors or windows and with only a small opening through which the victims were lowered into their living grave. Other cells were stone cages to drive the mind to madness and lacerate the heart of the
- 41. unfortunates. Yet men and women endured twenty years in this terrible place. What fortitude, what power of endurance, what sublime faith one must have had to hold out, to emerge from it alive! Here Netchaev, Lopatin, Morosov, Volkenstein, Figner, and others of the splendid band spent their tortured lives. Here is the common grave of Ulianov, Mishkin, Kalayev, Balmashev, and many more. The black tablet inscribed with their names speaks louder than the voices silenced for ever. Not even the roaring waves dashing against the rock of Schlüsselburg can drown that accusing voice. Petropavlovsk and Schlüsselburg stand as the living proof of how futile is the hope of the mighty to escape the Frankensteins of their own making.
- 42. CHAPTER XV THE TRADE UNIONS It was the month of June and the time of our departure was approaching. Petrograd seemed more beautiful than ever; the white nights had come—almost broad daylight without its glare, the mysterious soothing white nights of Petrograd. There were rumours of counter-revolutionary danger and the city was guarded against attack. Martial law prevailing, it was forbidden to be out on the streets after 1 A. M., even though it was almost daylight. Occasionally special permits were obtained by friends and then we would walk through the deserted streets or along the banks of the dark Neva, discussing in whispers the perplexing situation. I sought for some outstanding feature in the blurred picture—the Russian Revolution, a huge flame shooting across the world illuminating the black horizon of the disinherited and oppressed—the Revolution, the new hope, the great spiritual awakening. And here I was in the midst of it, yet nowhere could I see the promise and fulfilment of the great event. Had I misunderstood the meaning and nature of revolution? Perhaps the wrong and the evil I have seen during those five months were inseparable from a revolution. Or was it the political machine which the Bolsheviki have created—is that the force which is crushing the Revolution? If I had witnessed the birth of the latter I should now be better able to judge. But apparently I arrived at the end—the agonizing end of a people. It is all so complex, so impenetrable, a tupik, a blind alley, as the Russians call it. Only time and earnest study, aided by sympathetic understanding, will show me the way out. Meanwhile, I must keep up my courage and—away from Petrograd, out among the people. Presently the long-awaited moment arrived. On June 30, 1920, our car was coupled to a slow train called Maxim Gorki, and we pulled
- 43. out of the Nikolayevski station, bound for Moscow. In Moscow there were many formalities to go through with. We thought a few days would suffice, but we remained two weeks. However, our stay was interesting. The city was alive with delegates to the Second Congress of the Third International; from all parts of the world the workers had sent their comrades to the promised land, revolutionary Russia, the first republic of the workers. Among the delegates there were also Anarchists and syndicalists who believed as firmly as I did six months previously that the Bolsheviki were the symbol of the Revolution. They had responded to the Moscow call with enthusiasm. Some of them I had met in Petrograd and now they were eager to hear of my experiences and learn my opinions. But what was I to tell them, and would they believe me if I did? Would I have believed any adverse criticism before I came to Russia? Besides, I felt that my views regarding the Bolsheviki were still too unformed, too vague, a conglomeration of mere impressions. My old values had been shattered and so far I have been unable to replace them. I could therefore not speak on the fundamental questions, but I did inform my friends that the Moscow and Petrograd prisons were crowded with Anarchists and other revolutionists, and I advised them not to content themselves with the official explanations but to investigate for themselves. I warned them that they would be surrounded by guides and interpreters, most of them men of the Tcheka, and that they would not be able to learn the facts unless they made a determined, independent effort. There was considerable excitement in Moscow at the time. The Printers' Union had been suppressed and its entire managing board sent to prison. The Union had called a public meeting to which members of the British Labour Mission were invited. There the famous Socialist Revolutionist Tchernov had unexpectedly made his appearance. He severely criticised the Bolshevik régime, received an ovation from the huge audience of workers, and then vanished as mysteriously as he had come. The Menshevik Dan was less successful. He also addressed the meeting, but he failed to make his
- 44. escape: he landed in the Tcheka. The next morning the Moscow Pravda and the Izvestia denounced the action of the Printers' Union as counter-revolutionary, and raged about Tchernov having been permitted to speak. The papers called for exemplary punishment of the printers who dared defy the Soviet Government. The Bakers' Union, a very militant organization, had also been suppressed, and its management replaced by Communists. Several months before, in March, I had attended a convention of the bakers. The delegates impressed me as a courageous group who did not fear to criticise the Bolshevik régime and present the demands of the workers. I wondered then that they were permitted to continue the conference, for they were outspoken in their opposition to the Communists. The bakers are 'Shkurniki' [skinners], I was told; they always instigate strikes, and only counter-revolutionists can wish to strike in the workers' Republic. But it seemed to me that the workers could not follow such reasoning. They did strike. They even committed a more heinous crime: they refused to vote for the Communist candidate, electing instead a man of their own choice. This action of the bakers was followed by the arrest of several of their more active members. Naturally the workers resented the arbitrary methods of the Government. Later I met some of the bakers and found them much embittered against the Communist Party and the Government. I inquired about the condition of their union, telling them that I had been informed that the Russian unions were very powerful and had practical control of the industrial life of the country. The bakers laughed. The trade unions are the lackeys of the Government, they said; they have no independent function, and the workers have no say in them. The trade unions are doing mere police duty for the Government. That sounded quite different from the story told by Melnichansky, the chairman of the Moscow Trade Union Soviet, whom I had met on my first visit to Moscow. On that occasion he had shown me about the trade union headquarters known as the Dom Soyusov, and explained how the
- 45. organization worked. Seven million workers were in the trade unions, he said; all trades and professions belonged to it. The workers themselves managed the industries and owned them. The building you are in now is also owned by the unions, he remarked with pride; formerly it was the House of the Nobility. The room we were in had been used for festive assemblies and the great nobles sat in crested chairs around the table in the centre. Melnichansky showed me the secret underground passage hidden by a little turntable, through which the nobles could escape in case of danger. They never dreamed that the workers would some day gather around the same table and sit in the beautiful hall of marble columns. The educational and cultural work done by the trade unions, the chairman further explained, was of the greatest scope. We have our workers' colleges and other cultural institutions giving courses and lectures on various subjects. They are all managed by the workers. The unions own their own means of recreation, and we have access to all the theatres. It was apparent from his explanation that the trade unions of Russia had reached a point far beyond anything known by labour organizations in Europe and America. A similar account I had heard from Tsiperovitch, the chairman of the Petrograd trade unions, with whom I had made my first trip to Moscow. He had also shown me about the Petrograd Labour Temple, a beautiful and spacious building where the Petrograd unions had their offices. His recital also made it clear that the workers of Russia had at last come into their own. But gradually I began to see the other side of the medal. I found that like most things in Russia the trade union picture had a double facet: one paraded before foreign visitors and investigators, the other known by the masses. The bakers and the printers had recently been shown the other side. It was a lesson of the benefits that accrued to the trade unions in the Socialist Republic. In March I had attended an election meeting arranged by the workers of one of the large Moscow factories. It was the most
- 46. exciting gathering I had witnessed in Russia—the dimly lit hall in the factory club rooms, the faces of the men and women worn with privation and suffering, the intense feeling over the wrong done them, all impressed me very strongly. Their chosen representative, an Anarchist, had been refused his mandate by the Soviet authorities. It was the third time the workers gathered to re-elect their delegate to the Moscow Soviet, and every time they elected the same man. The Communist candidate opposing him was Semashko, the Commissar of the Department of Health. I had expected to find an educated and cultured man. But the behaviour and language of the Commissar at that election meeting would have put a hod-carrier to shame. He raved against the workers for choosing a non- Communist, called anathema upon their heads, and threatened them with the Tcheka and the curtailment of their rations. But he had no effect upon the audience except to emphasize their opposition to him, and to arouse antagonism against the party he represented. The final victory, however, was with Semashko. The workers' choice was repudiated by the authorities and later even arrested and imprisoned. That was in March. In May, during the visit of the British Labour Mission, the factory candidate together with other political prisoners declared a hunger strike, which resulted in their liberation. The story told me by the bakers of their election experiences had the quality of our own Wild West during its pioneer days. Tchekists with loaded guns were in the habit of attending gatherings of the unions and they made it clear what would happen if the workers should fail to elect a Communist. But the bakers, a strong and militant organization, would not be intimidated. They declared that no bread would be baked in Moscow unless they were permitted to elect their own candidate. That had the desired effect. After the meeting the Tchekists tried to arrest the candidate-elect, but the bakers surrounded him and saw him safely home. The next day they sent their ultimatum to the authorities, demanding recognition of their choice and threatening to strike in case of refusal. Thus the bakers triumphed and gained an advantage over their less courageous
- 47. brothers in the other labour organizations of minor importance. In starving Russia the work of the bakers was as vital as life itself.
- 48. CHAPTER XVI MARIA SPIRIDONOVA The Commissariat of Education also included the Department of Museums. The Petrograd Museum of the Revolution had two chairmen; Lunacharsky being one of them, it was necessary to secure his signature to our credentials which had already been signed by Zinoviev, the second chairman of the Museum. I was commissioned to see Lunacharsky. I felt rather guilty before him. I left Moscow in March promising to return within a week to join him in his work. Now, four months later, I came to ask his coöperation in an entirely different field. I went to the Kremlin determined to tell Lunacharsky how I felt about the situation in Russia. But I was relieved of the necessity by the presence of a number of people in his office; there was no time to take the matter up. I could merely inform Lunacharsky of the purpose of the expedition and request his aid in the work. It met with his approval. He signed our credentials and also supplied me with letters of introduction and recommendation to facilitate our efforts in behalf of the Museum. While our Commission was making the necessary preparations for the trip to the Ukraine, I found time to visit various institutions in Moscow and to meet some interesting people. Among them were certain well-known Left Social Revolutionists whom I had met on my previous visit. I had told them then that I was eager to visit Maria Spiridonova, of whose condition I had heard many conflicting stories. But at that time no meeting could be arranged: it might have exposed Spiridonova to danger, for she was living illegally, as a peasant woman. History indeed repeats itself. Under the Tsar Spiridonova, also disguised as a country girl, had shadowed Lukhanovsky, the Governor of Tamboy, of peasant-flogging fame.
- 49. Having shot him, she was arrested, tortured, and later sentenced to death. The western world became aroused, and it was due to its protests that the sentence of Spiridonova was changed to Siberian exile for life. She spent eleven years there; the February Revolution brought her freedom and back to Russia. Maria Spiridonova immediately threw herself into revolutionary activity. Now, in the Socialist Republic, Maria was again living in disguise after having escaped from the prison in the Kremlin. Arrangements were finally made to enable me to visit Spiridonova, and I was cautioned to make sure that I was not followed by Tcheka men. We agreed with Maria's friends upon a meeting place and from there we zigzagged a number of streets till we at last reached the top floor of a house in the back of a yard. I was led into a small room containing a bed, small desk, bookcase, and several chairs. Before the desk, piled high with letters and papers, sat a frail little woman, Maria Spiridonova. This, then, was one of Russia's great martyrs, this woman who had so unflinchingly suffered the tortures inflicted upon her by the Tsar's henchmen. I had been told by Zorin and Jack Reed that Spiridonova had suffered a breakdown, and was kept in a sanatorium. Her malady, they said, was acute neurasthenia and hysteria. When I came face to face with Maria, I immediately realized that both men had deceived me. I was no longer surprised at Zorin: much of what he had told me I gradually discovered to be utterly false. As to Reed, unfamiliar with the language and completely under the sway of the new faith, he took too much for granted. Thus, on his return from Moscow he came to inform me that the story of the shooting of prisoners en masse on the eve of the abolition of capital punishment was really true; but, he assured me, it was all the fault of a certain official of the Tcheka who had already paid with his life for it. I had opportunity to investigate the matter. I found that Jack had again been misled. It was not that a certain man was responsible for the wholesale killing on that occasion. The act was conditioned in the whole system and character of the Tcheka.
- 50. I spent two days with Maria Spiridonova, listening to her recital of events since October, 1917. She spoke at length about the enthusiasm and zeal of the masses and the hopes held out by the Bolsheviki; of their ascendancy to power and gradual turn to the right. She explained the Brest-Litovsk peace which she considered as the first link in the chain that has since fettered the Revolution. She dwelt on the razverstka, the system of forcible requisition, which was devastating Russia and discrediting everything the Revolution had been fought for; she referred to the terrorism practised by the Bolsheviki against every revolutionary criticism, to the new Communist bureaucracy and inefficiency, and the hopelessness of the whole situation. It was a crushing indictment against the Bolsheviki, their theories and methods. If Spiridonova had really suffered a breakdown, as I had been assured, and was hysterical and mentally unbalanced, she must have had extraordinary control of herself. She was calm, self- contained, and clear on every point. She had the fullest command of her material and information. On several occasions during her narrative, when she detected doubt in my face, she remarked: I fear you don't quite believe me. Well, here is what some of the peasants write me, and she would reach over to a pile of letters on her desk and read to me passages heart-rending with misery and bitter against the Bolsheviki. In stilted handwriting, sometimes almost illegible, the peasants of the Ukraine and Siberia wrote of the horrors of the razverstka and what it had done to them and their land. They have taken away everything, even the last seeds for the next sowing. The Commissars have robbed us of everything. Thus ran the letters. Frequently peasants wanted to know whether Spiridonova had gone over to the Bolsheviki. If you also forsake us, matushka, we have no one to turn to, one peasant wrote. The enormity of her accusations challenged credence. After all, the Bolsheviki were revolutionists. How could they be guilty of the terrible things charged against them? Perhaps they were not responsible for the situation as it had developed; they had the whole
- 51. world against them. There was the Brest peace, for instance. When the news of it first reached America I happened to be in prison. I reflected long and carefully whether Soviet Russia was justified in negotiating with German imperialism. But I could see no way out of the situation. I was in favour of the Brest peace. Since I came to Russia I heard conflicting versions of it. Nearly everyone, excepting the Communists, considered the Brest agreement as much a betrayal of the Revolution as the rôle of the German Socialists in the war—a betrayal of the spirit of internationalism. The Communists, on the other hand, were unanimous in defending the peace and denouncing as counter-revolutionist everybody who questioned the wisdom and the revolutionary justification of that agreement. We could do nothing else, argued the Communists. Germany had a mighty army, while we had none. Had we refused to sign the Brest treaty we should have sealed the fate of the Revolution. We realized that Brest meant a compromise, but we knew that the workers of Russia and the rest of the world would understand that we had been forced to it. Our compromise was similar to that of workers when they are forced to accept the conditions of their masters after an unsuccessful strike. But Spiridonova was not convinced. There is not one word of truth in the argument advanced by the Bolsheviki, she said. It is true that Russia had no disciplined army to meet the German advance, but it had something infinitely more effective: it had a conscious revolutionary people who would have fought back the invaders to the last drop of blood. As a matter of fact, it was the people who had checked all the counter-revolutionary military attempts against Russia. Who else but the people, the peasants and the workers, made it impossible for the German and Austrian army to remain in the Ukraine? Who defeated Denikin and the other counter- revolutionary generals? Who triumphed over Koltchak and Yudenitch? Lenin and Trotsky claim that it was the Red Army. But the historic truth was that the voluntary military units of the workers and peasants—the povstantsi—in Siberia as well as in the south of Russia —had borne the brunt of the fighting on every front, the Red Army
- 52. usually only completing the victories of the former. Trotsky would have it now that the Brest treaty had to be accepted, but he himself had at one time refused to sign the treaty and Radek, Joffe, and other leading Communists had also been opposed to it. It is claimed now that they submitted to the shameful terms because they realized the hopelessness of their expectation that the German workers would prevent the Junkers from marching against revolutionary Russia. But that was not the true reason. It was the whip of the party discipline which lashed Trotsky and others into submission. The trouble with the Bolsheviki, continued Spiridonova, is that they have no faith in the masses. They proclaimed themselves a proletarian party, but they refused to trust the workers. It was this lack of faith, Maria emphasized, which made the Communists bow to German imperialism. And as concerns the Revolution itself, it was precisely the Brest peace which struck it a fatal blow. Aside from the betrayal of Finland, White Russia, Latvia, and the Ukraine—which were turned over to the mercy of the German Junkers by the Brest peace—the peasants saw thousands of their brothers slain, and had to submit to being robbed and plundered. The simple peasant mind could not understand the complete reversal of the former Bolshevik slogans of no indemnity and no annexations. But even the simplest peasant could understand that his toil and his blood were to pay the indemnities imposed by the Brest conditions. The peasants grew bitter and antagonistic to the Soviet régime. Disheartened and discouraged they turned from the Revolution. As to the effect of the Brest peace upon the German workers, how could they continue in their faith in the Russian Revolution in view of the fact that the Bolsheviki negotiated and accepted the peace terms with the German masters over the heads of the German proletariat? The historic fact remains that the Brest peace was the beginning of the end of the Russian Revolution. No doubt other factors contributed to the debacle, but Brest was the most fatal of them.
- 53. Spiridonova asserted that the Left Socialist Revolutionary elements had warned the Bolsheviki against that peace and fought it desperately. They refused to accept it even after it had been signed. The presence of Mirbach in Revolutionary Russia they considered an outrage against the Revolution, a crying injustice to the heroic Russian people who had sacrificed and suffered so much in their struggle against imperialism and capitalism. Spiridonova's party decided that Mirbach could not be tolerated in Russia: Mirbach had to go. Wholesale arrests and persecutions followed upon the execution of Mirbach, the Bolsheviki rendering service to the German Kaiser. They filled the prisons with the Russian revolutionists. In the course of our conversation I suggested that the method of razverstka was probably forced upon the Bolsheviki by the refusal of the peasants to feed the city. In the beginning of the revolutionary period, Spiridonova explained, so long as the peasant Soviets existed, the peasants gave willingly and generously. But when the Bolshevik Government began to dissolve these Soviets and arrested 500 peasant delegates, the peasantry became antagonistic. Moreover, they daily witnessed the inefficiency of the Communist régime: they saw their products lying at side stations and rotting away, or in possession of speculators on the market. Naturally under such conditions they would not continue to give. The fact that the peasants had never refused to contribute supplies to the Red Army proved that other methods than those used by the Bolsheviki could have been employed. The razverstka served only to widen the breach between the village and the city. The Bolsheviki resorted to punitive expeditions which became the terror of the country. They left death and ruin wherever they came. The peasants, at last driven to desperation, began to rebel against the Communist régime. In various parts of Russia, in the south, on the Ural, and in Siberia, peasants' insurrections have taken place, and everywhere they were being put down by force of arms and with an iron hand. Spiridonova did not speak of her own sufferings since she had parted ways with the Bolsheviki. But I learned from others that she
- 54. had been arrested twice and imprisoned for a considerable length of time. Even when free she was kept under surveillance, as she had been in the time of the Tsar. On several occasions she was tortured by being taken out at night and informed that she was to be shot—a favoured Tcheka method. I mentioned the subject to Spiridonova. She did not deny the facts, though she was loath to speak of herself. She was entirely absorbed in the fate of the Revolution and of her beloved peasantry. She gave no thought to herself, but she was eager to have the world and the international proletariat learn the true condition of affairs in Bolshevik Russia. Of all the opponents of the Bolsheviki I had met Maria Spiridonova impressed me as one of the most sincere, well-poised, and convincing. Her heroic past and her refusal to compromise her revolutionary ideas under Tsarism as well as under Bolshevism were sufficient guarantee of her revolutionary integrity.
- 55. CHAPTER XVII ANOTHER VISIT TO PETER KROPOTKIN A few days before our Expedition started for the Ukraine the opportunity presented itself to pay another visit to Peter Kropotkin. I was delighted at the chance to see the dear old man under more favourable conditions than I had seen him in March. I expected at least that we would not be handicapped by the presence of newspaper men as we were on the previous occasion. On my first visit, in snow-clad March, I arrived at the Kropotkin cottage late in the evening. The place looked deserted and desolate. But now it was summer time. The country was fresh and fragrant; the garden at the back of the house, clad in green, smiled cheerfully, the golden rays of the sun spreading warmth and light. Peter, who was having his afternoon nap, could not be seen, but Sofya Grigorievna, his wife, was there to greet us. We had brought some provisions given to Sasha Kropotkin for her father, and several baskets of things sent by an Anarchist group. While we were unpacking those treasures Peter Alekseyevitch surprised us. He seemed a changed man: the summer had wrought a miracle in him. He appeared healthier, stronger, more alive than when I had last seen him. He immediately took us to the vegetable garden which was almost entirely Sofya's own work and served as the main support of the family. Peter was very proud of it. What do you say to this! he exclaimed; all Sofya's labour. And see this new species of lettuce—pointing at a huge head. He looked young; he was almost gay, his conversation sparkling. His power of observation, his keen sense of humour and generous humanity were so refreshing, he made one forget the misery of Russia, one's own conflicts and doubts, and the cruel reality of life.
- 56. After dinner we gathered in Peter's study—a small room containing an ordinary table for a desk, a narrow cot, a wash-stand, and shelves of books. I could not help making a mental comparison between this simple, cramped study of Kropotkin and the gorgeous quarters of Radek and Zinoviev. Peter was interested to know my impressions since he saw me last. I related to him how confused and harassed I was, how everything seemed to crumble beneath my feet. I told him that I had come to doubt almost everything, even the Revolution itself. I could not reconcile the ghastly reality with what the Revolution had meant to me when I came to Russia. Were the conditions I found inevitable—the callous indifference to human life, the terrorism, the waste and agony of it all? Of course, I knew revolutions could not be made with kid gloves. It is a stern necessity involving violence and destruction, a difficult and terrible process. But what I had found in Russia was utterly unlike revolutionary conditions, so fundamentally unlike as to be a caricature. Peter listened attentively; then he said: There is no reason whatever to lose faith. I consider the Russian Revolution even greater than the French, for it has struck deeper into the soul of Russia, into the hearts and minds of the Russian people. Time alone can demonstrate its full scope and depth. What you see to-day is only the surface, conditions artificially created by a governing class. You see a small political party which by its false theories, blunders, and inefficiency has demonstrated how revolutions must not be made. It was unfortunate—Kropotkin continued—that so many of the Anarchists in Russia and the masses outside of Russia had been carried away by the ultra-revolutionary pretenses of the Bolsheviki. In the great upheaval it was forgotten that the Communists are a political party firmly adhering to the idea of a centralized State, and that as such they were bound to misdirect the course of the Revolution. The Bolsheviki were the Jesuits of the Socialist Church: they believed in the Jesuitic motto that the end justifies the means. Their end being political power, they hesitate at nothing. The means, however, have paralysed the energies of the masses and have terrorized the people. Yet without the people, without the direct
- 57. participation of the masses in the reconstruction of the country, nothing essential could be accomplished. The Bolsheviki had been carried to the top by the high tide of the Revolution. Once in power they began to stem the tide. They have been trying to eliminate and suppress the cultural forces of the country not entirely in agreement with their ideas and methods. They destroyed the coöperatives which were of utmost importance to the life of Russia, the great link between the country and the city. They created a bureaucracy and officialdom which surpasses even that of the old régime. In the village where he lived, in little Dmitrov, there were more Bolshevik officials than ever existed there during the reign of the Romanovs. All those people were living off the masses. They were parasites on the social body, and Dmitrov was only a small example of what was going on throughout Russia. It was not the fault of any particular individuals: rather was it the State they had created, which discredits every revolutionary ideal, stifles all initiative, and sets a premium on incompetence and waste. It should also not be forgotten, Kropotkin emphasized, that the blockade and the continuous attacks on the Revolution by the interventionists had helped to strengthen the power of the Communist régime. Intervention and blockade were bleeding Russia to death, and were preventing the people from understanding the real nature of the Bolshevik régime. Discussing the activities and rôle of the Anarchists in the Revolution, Kropotkin said: We Anarchists have talked much of revolutions, but few of us have been prepared for the actual work to be done during the process. I have indicated some things in this relation in my 'Conquest of Bread.' Pouget and Pataud have also sketched a line of action in their work on 'How to Accomplish the Social Revolution.' Kropotkin thought that the Anarchists had not given sufficient consideration to the fundamental elements of the social revolution. The real facts in a revolutionary process do not consist so much in the actual fighting—that is, merely the destructive phase necessary to clear the way for constructive effort. The basic factor in a revolution is the organization of the economic life of the country. The Russian Revolution had proved conclusively that we must prepare
- 58. thoroughly for that. Everything else is of minor importance. He had come to think that syndicalism was likely to furnish what Russia most lacked: the channel through which the industrial and economic reconstruction of the country may flow. He referred to Anarcho- syndicalism. That and the coöperatives would save other countries some of the blunders and suffering Russia was going through. I left Dmitrov much comforted by the warmth and light which the beautiful personality of Peter Kropotkin radiated; and I was much encouraged by what I had heard from him. I returned to Moscow to help with the completion of the preparations for our journey. At last, on July 15, 1920, our car was coupled to a train bound for the Ukraine.
- 59. CHAPTER XVIII EN ROUTE Our train was about to leave Moscow when we were surprised by an interesting visitor—Krasnoschekov, the president of the Far Eastern Republic, who had recently arrived in the capital from Siberia. He had heard of our presence in the city, but for some reason he could not locate us. Finally he met Alexander Berkman who invited him to the Museum car. In appearance Krasnoschekov had changed tremendously since his Chicago days, when, known as Tobinson, he was superintendent of the Workers' Institute in that city. Then he was one of the many Russian emigrants on the West Side, active as organizer and lecturer in the Socialist movement. Now he looked a different man; his expression stern, the stamp of authority on him, he seemed even to have grown taller. But at heart he remained the same—simple and kind, the Tobinson we had known in Chicago. We had only a short time at our disposal and our visitor employed it to give us an insight into the conditions in the Far East and the local form of government. It consisted of representatives of various political factions and even Anarchists are with us, said Krasnoschekov; thus, for instance, Shatov is Minister of Railways. We are independent in the East and there is free speech. Come over and try us, you will find a field for your work. He invited Alexander Berkman and myself to visit him in Chita and we assured him that we hoped to avail ourselves of the invitation at some future time. He seemed to have brought a different atmosphere and we were sorry to part so soon. On the way from Petrograd to Moscow the Expedition had been busy putting its house in order. As already mentioned, the car consisted of
- 60. six compartments, two of which were converted into a dining room and kitchen. They were of diminutive size, but we managed to make a presentable dining room of one, and the kitchen might have made many a housekeeper envy us. A large Russian samovar and all necessary copper and zinc pots and kettles were there, making a very effective appearance. We were especially proud of the decorative curtains on our car windows. The other compartments were used for office and sleeping quarters. I shared mine with our secretary, Miss A. T. Shakol. Besides Alexander Berkman, appointed by the Museum as chairman and general manager, Shakol as secretary, and myself as treasurer and housekeeper, the Expedition consisted of three other members, including a young Communist, a student of the Petrograd University. En route we mapped out our plan of work, each member of the Expedition being assigned some particular branch of it. I was to gather data in the Departments of Education and Health, the Bureaus of Social Welfare and Labour Distribution, as well as in the organization known as Workers' and Peasants' Inspection. After the day's work all the members were to meet in the car to consider and classify the material collected during the day. Our first stop was Kursk. Nothing of importance was collected there except a pair of kandai [iron handcuffs] which had been worn by a revolutionist in Schlüsselburg. It was donated to us by a chance passer-by who, noticing the inscription on our car, Extraordinary Commission of the Museum of the Revolution, became interested and called to pay us a visit. He proved to be an intellectual, a Tolstoian, the manager of a children's colony. He succeeded in maintaining the latter by giving the Soviet Government a certain amount of labour required of him: three days a week he taught in the Soviet schools of Kursk. The rest of his time he devoted to his little colony, or the Children's Commune, as he affectionately called it. With the help of the children and some adults they raised the vegetables necessary for the support of the colony and made all the repairs of the place. He stated that he had not been directly
- 61. interfered with by the Government, but that his work was considerably handicapped by discrimination against him as a pacifist and Tolstoian. He feared that because of it his place could not be continued much longer. There was no trading of any sort in Kursk at the time, and one had to depend for supplies on the local authorities. But discrimination and antagonism manifested themselves against independent initiative and effort. The Tolstoian, however, was determined to make a fight, spiritually speaking, for the life of his colony. He was planning to go to the centre, to Moscow, where he hoped to get support in favour of his commune. The personality of the man, his eagerness to make himself useful, did not correspond with the information I had received from Communists about the intelligentsia, their indifference and unwillingness to help revolutionary Russia. I broached the subject to our visitor. He could only speak of the professional men and women of Kursk, his native city, but he assured us that he found most of them, and especially the teachers, eager to coöperate and even self- sacrificing. But they were the most neglected class, living in semi- starvation all the time. Like himself, they were exposed to general antagonism, even on the part of the children whose minds had been poisoned by agitation against the intelligentsia. Kursk is a large industrial centre and I was interested in the fate of the workers there. We learned from our visitor that there had been repeated skirmishes between the workers and the Soviet authorities. A short time before our arrival a strike had broken out and soldiers were sent to quell it. The usual arrests followed and many workers were still in the Tcheka. This state of affairs, the Tolstoian thought, was due to general Communist incompetence rather than to any other cause. People were placed in responsible positions not because of their fitness but owing to their party membership. Political usefulness was the first consideration and it naturally resulted in general abuse of power and confusion. The Communist dogma that the end justifies all means was also doing much harm. It had thrown the door wide open to the worst human passions, and discredited
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