Stem cell regulation and engraftment
 
RAINER STORB    Leukemia Vol 7, Suppl 2
Fred Hutchinson Cancer Research Center, and the Department of Medicine, University of Washington School of Medicine, Seattle, Washington, USA Transplantation of hematopoietic stem cells, usually in the form of marrow grafts, has been increasingly used over the last 21/2 decades to treat patients with various malignant and nonmalignant hematological diseases. The number of transplants is steadily increasing worldwide. Marrow for transplantation is usually obtained by aspiration from the iliac crests. It is placed into tissue culture medium and screened before infusion into the recipient. The marrow is composed of a mixture of cells including large numbers of differentiated and mature cells, many committed progenitors with defined function and limited proliferation potential, and rare pluripotent stem cells that have broad developmental and proliferative potential. The ability of the pluripotent stem cells not only to self-renew, but also to differentiate into the various committed hematopoietic precursors, has made marrow transplantation possible. After the marrow harvest and screening, the marrow is placed into a blood administration bag and infused intravenously into the recipient whose underlying disease has been eradicated and whose immune system suppressed by high doses of chemoradiotherapy .Infused stem cells circulate through the blood stream and, presumably with the help of homing receptors, enter the marrow cavity , attach to the stroma bed, and begin to divide under the influence of humoral and cellular stimuli. In most recipients of HLA-identical sibling transplants, the cell inoculum settles in uneventfully. With extraordinary speed, the progenitor cells produce progeny 0 The first mature cells begin to appear in the circulation within 2-3 weeks of transplantation, normal granulocyte and platelet counts may be reached as early as day 40 to 50 after transplant, and normal hematocrit values are seen between the second and third months after transplantation. In most patients, hematopoiesis is entirely of marrow donor origin, although a minority may show a mixture of host and donor cells. Hematopoiesis is stable. The longest survivors after marrow transplantation have passed the 25-year mark, and they display a completely normal hematopoietic and immune system of donor type. Recent studies using molecular probes for methylation site polymorphisms on the inactivated X-chromosome have shown that hematopoietic repopulation after transplantation is of polyclonal origin, although one group of investigators reported clonal origin in a small minority of patients (1,2). Factors governing the fate of the marrow transplant include the immunosuppressive effects of the conditioning programs, the degree of histoincompatibility between donor and recipient, the composition of the cells in the graft, and the quality of the marrow bed. Inadequacies with any of these factors may lead to a problem, graft failure. With HLA-identical sibling donors, most grafts are successful. Graft failure is seen in less than 2% of recipients. In contrast with HLA-haploidentical family members who are mismatched for 1, 2 or 3 HLA-Ioci on the nonshared haplotype, graft rejection is seen in 7-20% of cases (3,4). In almost 80% of patients with graft failure, residual host lymphocytes could be seen in the peripheral blood, which is suggestive of host-mediated immune rejection. Graft failure is accompanied by a case fatality rate of 97%. Studies in a canine model of marrow transplantation had predicted that engraftment problems might arise in the HLA-nonidentical transplant setting (5). While grafts with marrow from DLA-identical littermates were successful in 95% of the cases, transplants from DLA-haploidentical littermates or DLA-nonidentical unrelated dogs had graft failure rates of 92% and 95 % 0 , respectively. At the time of rejection, dogs showed an increase in peripheral blood large granular lymphocytes of host type which functioned as natural killer cells (NK cells) (6). In coculture experiments, these recipient cells suppressed marrow colony-forming unit granulocytemacrophage production, not only from donor marrow but also from marrow of unrelated dogs that were either phenotypically DLA-identical or DLA-nonidentical with the marrow donor, consistent with the notion of cytotoxicity which was not restricted by the major histocompatibility complex (MHC) (Raff et al., unpublished). In agreement with the notion that NK-Iike cells were involved in graft rejection was the finding that agents directed against macrophages and T lymphocytes were ineffective in enhancing marrow engraftmentacross MHC barriers in the dog. Accordingly, we developed monoclonal antibodies (MAb) that recognized those host cells which mediate rejection. To this purpose we harvested canine marrow cells 6 days after TBI and immunized mice. Among the antibodies produced, one, S5, had a broad hematopoietic distribution, binding to normal marrow cells, peripheral blood mononuclear cells, granulocytes and lymph node lymphocytes. The antibody was used in a controlled in vivo experiment in which dogs were given 9.2 Gy of TBI followed by DLA-nonidentical unrelated marrow grafts (7). In this setting, graft failure was seen in 33 of 36 control dogs not given additional therapy, in 6 of 7 dogs given the isotype matched irrelevant MAb 6.4 before TBI, and in only 4 of 19 dogs given antibody S5 at a dose of 0.2 mg/kg/day from day -7 to day -2 before TBI. Thus, antibody S5 resulted in a significant enhancement of engraftment over that seen in control dogs given either no antibody or an isotype matched irrelevant antibody. Subsequent work using techniques of sequential immunoprecipitation, tryptic digestion, and N-glyconase digestion revealed antibody 55 to detect the CD44 antigen (8). CD44 functions in cell-cell and extracellular matrix adhesions and has been implicated in lymphocyte traffic. It is also involved in T cell activation via CD2 and CD3. Binding of anti-CD44 monoclonal antibodies causes secretion of IL-l and TNF-alfa. Further studies using positive selection have shown CD44 to be expressed on hematopoietic stem cells that can repopulate dog marrow after a supralethal dose of TBI. The mechanism by which an antibody to CD44 enhances engraftment across a major histocompatibility barrier is as yet unexplained. Alternative modes of action include that MAb 55 affects traffic of host immune cells, thereby permitting marrow to home, or increases the radiosensitivity of host NK cells thought to be involved in graft resistance. Elucidation of these mechanisms is important for clinical application of antibody-mediated enhancement of marrow grafts. Previous studies in dogs indicated that hematopoietic stem cells express MHC class II molecules as recognized by the anti-class II MAb 7.2 (9,10). Treatment of canine marrow in vitro with antibody 7.2 and rabbit complement prevents autologous marrow engraftment after lethal TBI, whereas selected 7.2-positive marrow cells are capable of permanently repopulating the hematopoietic system of irradiated dogs. Class II molecules are differentially expressed on hematopoietic progenitor cells of man. Committed progenitor cells assayed as colony-forming unit granulocyte-macrophage, mixed colonies, and burst-forming unit erythroid express HLA-DR and DP antigens whereas colony-forming units erythroid are predominantly HLA-DR negative. Low HLA-DR expression has been seen on murine and human long-term marrow culture initiating cells. It is not clear whether truly pluripotent stem cells express class II MHC antigens. If present, the class II molecules on stem cells could have significant clinical implications, because investigators have described a possible role of HLA-D restricted cell interactions in hematopoiesis. Such restrictions could be important for interactions between transplantable cells and as yet undetected accessory cells in the hematopoietic microenvironment. We have established a model of canine marrow autografts after 9.2 ay TBI to study the role of class II antigens in hematopoietic stem cell growth and differentiation (11). Twenty dogs were given 9.2 ay TBI marrow and intravenous murine anti-class II MAb. Infusion of 0.6 mg/kg/day of antibody H81.98.21, an Iga2a MAb reactive with HLA-DR, on days 0 to 4 after TBI did not prevent initial engraftment, but all dogs died with late graft failure. The critical time for effect of the antibody is during the first 4 days after transplant. Results of extensive studies argued against several pathogenetic mechanisms, including removal of antibody coated stem cells by the reticuloendothelial system, canine complement-mediated cytotoxic effects on stem cells, antibody-dependent cellular cytotoxicity , and inactivation of antibody coated cells by dog anti-mouse antibody. To distinguish between antibody-induced damage to microenvironment/accessory cells and late graft failure from lack of pluripotent stem cells, three dogs were given TBI, a marrow autograft, and antibody H81.98.21 on days 0 to 4; one given thoracic duct lymphocytes on day 6 developed graft failure; the other two given marrow depleted of accessory cells by L-Ieucil L-Ieucine O-methyl ester had sustained grafts. Findings support the notion that originally transplanted pluripotent stem cells are no longer present on day 6 and that the marrow microenvironment is functional and able to support newly injected stem cells. These initial observations on the development of graft failure after injection of MAb to class II during the early period after transplant suggest a role for class II molecules in the engraftment and/or regulation of pluripotent stem cells required for sustained marrow function. The mechanism by which MAb to class II induce late marrow graft failure is currently under investigation (12). Hematopoietic engraftment of donor type is "permanent" in human patients as indicated by blood genetic marker studies in patients, many of whom have now been followed for 10-25 years after transplant. Given the permanence of the engraftment and the fact that recovery in most patients is polyclonal, gene transfer into hematopoietic stem cells has become an attractive possibility .Gene transfer might be used to inhibit or alter genes causing disease, for example, in patients with hemoglobinopathies or inborn errors of metabolism, to confer drug resistance to marrow cells in patients undergoing high-dose chemotherapy, and to transfer histocompatibility genes with the aim of inducing tolerance to MHC gene products. For gene therapy to be accomplished, a sample of the patient's marrow would be obtained, placed into a long-term culture system, and exposed to a retroviral vector containing the genes of interest. In the interim, the patient would be treated to eradicate the underlying disease. That accomplished, the transduced marrow would be injected in hopes that genes would be expressed in the progeny of hematopoietic progenitor cells. Our work was conducted in a preclinical canine model using two Moloney leukemia virus based vectors, LNCA and LASN, which contain both the neomycin phosphotransferase gene and a human adenosine deaminase gene (13,14). The system was helper virus free. Two approaches were taken. In one, prospective recipient dogs were treated with canine recombinant granulocyte colony-stimulating factor for 7 days. Then, marrow was harvested and placed into long-term culture which was regularly fed with supernatant from a virusproducing feeder layer. After the marrow was in culture for 4 days, the dog was given 9.2 ay of TBI and the transduced marrow infused. The dog was then treated with granulocyte colony-stimulating factor until hematopoietic engraftment. The other approach involved pretreatment of the recipient dog with a dose of 40 mg of cyclophosphamide per kg intravenously, a maneuver which led to profound pancytopenia. At the time of greatest pancytopenia, marrow was harvested and cocultivated for 48 hours with a viral-producing feeder layer. Then the dog was given 9.2 Gy of TBI and the marrow was infused. The dog then received granulocyte colony-stimulating factor until recovery of peripheral blood granulocyte counts. Six dogs were treated with long-term cultured marrow. Of these six, two died with intercurrent infection, on the day of TBI and marrow transplantation and on day 15, respectively, while four became long-term survivors. The four are surviving between 2 and more than 4 years after transplant. Of the four animals treated with cyclophosphamide, two died with pneumonia on days 1 and 12 after TBI, respectively, and two are surviving between 2 and more than 4 years after transplant. At regular intervals, marrow has been harvested from these dogs and placed into colony-forming unit granulocytemacrophage cultures with and without the chemical G418 to test for neomycin resistance. For now up to more than 4 years after transplant, between 1% and 10% G418-resistant colony-forming units granulocytemacrophage have been found in each of the six surviving animals, indicating successful gene transfer . Furthermore, peripheral blood granulocytes, peripheral blood lymphocytes, lymph node lymphocytes, and marrow cells have all been tested for both the presence of the neomycin phosphotransferase gene and the adenosine deaminase gene using a polymerase chain reaction based test. The genes were found to be present for up to more than 4 years. Dogs have remained healthy. Tests for helper virus have remained negative. These data indicate for the first time successful and persistent transduction of long-term repopulating marrow cells in a large random bred species. It is clear that much work still needs to be done to increase the relatively low levels of gene expression in vivo through improvements in the development of both retroviral vectors and of packaging cell lines so that the highest possible virus titers can be generated in the absence of any detectable helper virus. Perhaps results can be improved through the use of various hematopoietic growth factors. Much work needs to be done in regards to developing safe conditioning programs for the recipient which do not cause life threatening pancytopenias. As our understanding of gene transfer techniques increases and our knowledge of gene regulation in the course of differentiation improves, gene transfer for the treatment of hematopoietic disorders may one day become clinical reality . ACKNOWLEDGMENTS This work was supported by grants HL36444, CA18221, CA31787, CA18029, and CA15704 from the National Institutes of Health, DHHS. REFERENCES 1. Nash R, Storb R, Neiman P. Polyclonal reconstitution of human marrow after allogeneic bone marrow transplantation. Blood 1988;72:2031-2037. 2. Turhan AG, Humphries RK, Phillips GL, Eaves AC, Eaves CJ. Clonal hematopoiesis demonstrated by X-Iinked DNA polymorphisms after allogeneic bone marrow transplantation. N Engl J Med 1989;320: 1655-1661. 3. Beatty PG, Clift RA, Mickelson EM et al. Marrow transplantation from related donors other than HLA-identical siblings. N Engl J Med 1985;313: 765-771. 4. Anasetti C, Amos D, Beatty PG et al. Effect ofHLA compatibility on engraftment of bone marrow transplants in patients with leukemia or lymphoma. N Engl J Med 1989;320: 197-204. 5. Storb R, Deeg HJ. Failure of allogeneic canine marrow grafts after total body irradiation: Allogeneic "resistance" vs transfusion induced sensitization. Transplantation 1986;42:571-580. 6. Raff RF, Deeg HJ, Loughran TP,Jr. et al. Characterization of host cells involved in resistance to marrow grafts in dogs transplanted from unrelated DLA-nonidentical donors. Blood 1986;68:861-868. 7. Schuening F, Storb R, Goehle Set al. Facilitation of engraftment ofDLA-nonidentical marrow by treatment of recipients with monoclonal antibody directed against marrow cells surviving radiation. Transplantation 1987;44:607-613. 8. Sandmaier BM, Storb R, Appelbaum FR et al. An antibody that facilitates hematopoietic engraftment recognizes CD44. Blood 1990;76:630-635. 9. Szer J, Deeg HJ, Appelbaum FR, Storb R. Failure of autologous marrow reconstitution after cytolytic treatment of marrow with anti-Ia monoclonal antibody. Blood 1985;65:819-822. 10. Schuening F, Storb R, Goehle Set al. Canine pluripotent hemopoietic stem cells and cFu-GM express la-Iike antigens as recognized by two different class-1I specific monoclonal antibodies. Blood 1987;69:165-172. 11. areinix HT, Ladiges WC, Graham TC et al. Late failure of autologous marrow grafts in lethally irradiated dogs given anti-class-1I monoclonal antibody. Blood 1991;78:2131-2138. 12. areinix HT, Storb R, Bartelmez SH. Specific growth inhibition of primitive hematopoietic progenitor cells mediated through monoclonal antibody binding to major histocompatibility class II molecules. Blood 1992;80:1950-1956. 13. Schuening Fa, Storb R, Stead RB et al. Improved retroviral transfer of genes into canine hematopoietic progenitor cells kept in long-term marrow culture. Blood 1989;74:152-155. 14. Schuening Fa, Kawahara K, Miller DA et al. Retrovirus-mediated gene transduction into long-term repopulating marrow cells of dogs. Blood 1991;78:2568-2576.
Prof. Yuri Ovchninnikov
 
V.T. Ivanov and V. A. Nesmeyanov     Haematol. Bluttransf. Vol 35

Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow, USSR.

Yuri A. Ovchinnikov started his carrier within the precincts of Moscow University at the Chemical Department under Professor Yu. A. Arbuzov. The project of his masters' degree (1957) provided material for the first publication on a new technique for the synthesis of pyrrolidine and thiophan derivatives. By that time, the gifted student had already shown a disposition toward synthetic organic chemistry. It was at this period that his belief took shape that the chemistry of living organisms was by far the most attractive area for an organic chemist to enter. Therefore, having begun his postgraduate course at the Chemical Department, Y. A. Ovchinnikov readily accepted an invitation to participate in the project on the complete synthesis of an important group of antibiotics, the tetracyclines. While working toward his doctorate, Yuri Ovchinnikov met M. M. Shemyakin, the leader of the project. The joint work led to a long-lasting collaboration between the two scientists, whose contribution to the foundation and advancement of physicochemical biology in the USSR was outstanding. After finished his postgraduate course, Ovchinnikov joined the Institute for Chemistry of Natural Products of the USSR Academy of Sciences, set up not long ago. Here, Professor Shemyakin proposed that he go into peptide chemistry. The subject under study depsipeptide antibiotics, atypical peptides containing hydroxy and amino acid residues. The problems of synthesis of the optically active N-methylated amino acids, reversible protection of the hydroxyl function of hydroxy acids, and cyclization of linear depsipeptides were rapidly solved and compounds with the structures proposed in the 1940s by Swiss researchers for antibiotics enniatins A and B were prepared. However, the samples obtained were devoid of antimicrobial activity and their physicochemical properties differed much from those of the naturally reliably confirmed, it remained to conclude that the formulae proposed for enniatins A and B were incorrect. Several alternative structures differing in ring size were suggested and accordingly synthesized. Two of them were indistinguishable from the natural enniatins A and B, which meant a solution to structural problems. Later (1964-1970), Ovchinnikov and his colleagues performed a series of elegant syntheses of some other naturally occurring depsipeptides (sporidesmolides I-IV, angolidc, serratamolide, esperin, beauvericin). he was awarded a D.Sc. in 1966 for the synthesis of natural depsipeptides and their analogs. In 1967, Shemyakin, Ovchinnikov, and their team formulated the original (socalled topochemical) principle of transformation of biologically active peptides: novel molecules can be designed by such deep structural modifications as reversal of the acylation direction and the configuration of asymmetric centers, replacement of ester bonds by amide bonds and vica versa, cyclization of linear molecules, etc. The conditions favorable for retaining the original stereo electronic parameters and, consequently, biological properties of the molecule were found. Ideas from this pioneer research were taken up by many laboratories and served to create novel highly active peptides (hormones, antibiotics, neuropeptides, enzyme substrates, and inhibitors). The experience accumulated during this synthetic work served as a basis for the next and the culiminative step in studying the depsipeptide antibiotics. Bearing in mind the recently discovered ability of valinomycin and enniatins to induce permeability of lipid membranes to alkali metals ions, Y. A. Ovchinnikov and his colleagues undertook a study of the physicochemical basis of the phenomenon. It appeared that valinomycin binds potassium ions in solution, yielding stable complexes, and shows a unique K/Na-selectivily of complex formation unsurpassed in nature. Enniatins bind virtually all alkali and alkali-earth cations, though with a lower selectivity. These complexes are the ion-transporting species, and selectivity of ion binding is the origin of the selectivity of transmembrane ion transport. Further, the threedimensional structures of the free antibiotics and their complexes were established. It was shown for the first time that such sophisticated structures can be resolved not only by X-ray analysis but also in solution by spectral methods. The bound ion appeared to reside always in the center of the depsipeptide molecular cavity and be kept in place by ion-dipole interactions with the carbonyl oxygens. The size of the valinomycin cavity is limited by a bracelet-like system of six intramolecular hydrogen bonds that accounts for its inability to adapt to smaller sized ions such as sodium or lithium. Enniatin structures are more flexible, which enables adjustment of the cavity to the size of the bound ion. The molecular periphery of both valinomycin and enniatin complexes is fully hydrophobic, which allows them to migrate freely across lipid zones of the membrane. Several laboratories outside the USSR were about to get similar results, but "the train had already gone." Step-by-step protein compounds, the major working bodies of any living system, began to occupy the prominent place in Ovchinnikov's research activity. The 1970s witnessed a series of studies on the primary structures of porcine aspartate aminotransferase, and toxins from the venoms of cobra, bee, scorpion, etc. As a result, more than 20 structures were added to international data banks and atlases of protein structures. Inspired by these advances, Ovchinnikov and his group tackled the deciphering of the primary structure of E. coli DNA dependent RNA polymerase, a key transcription enzyme investigated in many laboratories. Ovchinnikov had a very strong team, but even for them the problem seemed extremely difficult, since RNA polymerase is built of several subunits, among them two very large - and '-subunits (each over 1300 amino acid residues). 1ndeed, after rapid sequencing of the alfa-subunit (over 300 amino acids) it became clear that analysis of the - and '-subunits exclusively by conventional methods of protein chemistry could take many years. A decision was made to utilize the methods of genetic engineering and to analyze the sequences of genes coding for the subunits. In those days, such an approach was new for this country, and elsewhere it was at the early stages of development. Genes for large subunits of DNAdependent RNA polymerase form the socalled operon rpo BC and contain about 10000 base pairs. They were isolated, inserted into plasmids, and sequenced. Structures of pep tides of large subunits were detected in parallel and independently. That was of use: when the structural analysis of genes was completed and the structures of corresponding proteins were derived according to the genetic code, they appeared to coincide with the peptide structures and, consequently, were determined correctly. Soon after that, other laboratories reported the gene fragments but not the complete gene. It is worthwhile noting that the structures of these fragments contained errors. only the combined use of the methods of protein and nucleotide chemistry provided reliable results. The structural analysis of RNA polymerase served as a basis for a thorough investigation of the mechanism of action of the enzyme, for numerous genetic and biochemical studies. That was in the late 1970s. More and more laboratories outside the USSR were successfully applying genetic engineering methods to microbiological synthesis of practically important proteins. Yuri Ovchinnikov was the first in the USSR to assess the prospects. He united enthusiasts and headed the work on improving the methods of chemical synthesis and directed mutagenesis of DNA to create microorganisms producing alien peptides and proteins. As a result, strains producing an opioid neuropeptide, leucine-enkephalin (1979), the antiviral and antitumour human protein interferon-alfa2 (1981), and the precursor of human insulin, proinsulin (1983), were obtained. Despite these advances of Yuri Ovchinnikov in genetic engineering and biotechnology, the bioorganic chemistry of peptides and proteins was always his major interest and devotion. In the mid-1970s, he, N. Abdulaev, and a group of colleagues focused their interest on the molecular mechanisms of photoreception. By that time, a series of substantial discoveries had been made that paved the way for solving the problem of how light energy is transformed into the electric energy of the nerve impulse by rhodopsin, a well-known light-sensitive protein from the animal retina. Soon after wards, there appeared data on the membrane protein -bacteriorhodopsin- found in microorganisms living in salt lakes. The protein was given that name because of its similarity to the visual rhodopsin (the presence of the bound retinal, light-sensitivity, etc.). Though bacteriorhodopsin functioned as a light-dependent proton pump, from the viewpoint of the primary photochemical properties it was very similar to rhodopsin. At the same time, bacteriorhodopsin is more readily available in large amounts and has a simpler structure than the visual rhodopsin, the main effort was initially directed to that protein. It was also considered that bacteriorhodopsin was (and still is) an ideal model for structure-functional analysis of membrane proteins. Simaltaneously with Prof. G. Khorana of the USA, the Nobel prize winner, Ovchinnikov succeeded in determining the amino acid sequence of bacteriorhodopsin, is was the first time that the chemical structure of the membrane protein had been deciphered (1987). Ovchinnikov and his team were then pioneers in solving the structure of rhodopsin from bovine retina (1981). Research into the topography of polypeptide chains of these proteins in native membranes and elucidation of the structure of their active sites and disposition of functionally important groups were the next steps in this project. Using a variety of approaches including chemical modification, enzymatic treatment, and immunochemical methods, Yuri Ovchinnikov and his colleagues demonstrated that the two rhodopsins are arranged in the membrane in a similar way as seven extended protein segments spanning the membrane's width and connected with each other on the two sides of the membrane by short peptide links. In the mid-1980s, Y. Ovchinnikov and v. Lipkin focused their attention on the studies of other proteins involved in transmission and amplification of the visual cascade transducin and cyclic GMD phosphodiesterase. In 1985, the primary structures of the y- and alfa-subunits of transducin from bovine retinal rods were sequenced. Interestingly, the y-subunit is characterized by the two adjoining cysteine residues also connected by a disulfide bridge. The residues are apparently involved in the formation of the transducin-photoactivated rhodopsin complex. An exciting page in the scientific biography of Yuri Ovchinnikov was his last project, devoted to studies of the system of active ion transport, i.e., Na,Ktransporting adenosine triphosphatase and related proteins. In the late 1970s, Ovchinnikov initiated research into the structure of Na,K-ATPase. At the beginning, oligomeric organization of the functionally active complex in the native membrane was unraveled and the asymmetric arrangement of the subunits described. Further progress depended upon determination of the amino acid sequence of the subunits. Around 19851986, Ovchinnikov's team completed studies of the nucleotide sequences of genes for subunits and amino acid sequences of their polypeptide chains, which led to the complete primary structure of Na,K-A TPase from pig kidney outer medulla. Some research centers outside the USSR were also working intensively in these areas. The teams of S. Numa (Japan) and A. Schwartz (USA) simultaneously reported amino acid sequences of similar enzymes from other sources. However, the approach chosen by Ovchinnikov extended far beyond the primary structure determination. Complemented by spectroscopic and molecular modelling studies, it resulted in the first detailed model of the Na,K-ATPase spatial structure. Here, the alfa-subunit (1016 amino acid residues) forms seven transmembrane segments and the major portion of its hydrophilic region accommodating the catalytic site is located inside the cell. The -subunit (302 residues) spans the membrane once and the main part of its polypeptide chain forms an extracellular glycosylated domain. As for the Na,K-ATPase active site, Ovchinnikov and his team employing affinity modification by A TP analog succeeded in identifying an unknown component of the catalytic site, thus experimentally confirming its dynamic changes during enzyme functioning. Yuri Ovchinnikov, together with Eugene Sverdlov and their groups of researchers, obtained novel data on the regions of the human genome encoding the systems of active ion transport that seem to be of general biological significance. A family of at least five genes was defined in the human genome coding for several isoforms of the Na,K-A TPase catalytic subunit as well as other structurally similar ion-transporting A TPases. The discovery of the multigene family gave rise to new concepts on regulation of the active ion transport through changes in the activity of the appropriate genes. This was supported by experiments on the expression level of various genes for Na,K-ATPase in healthy and pathological human tissues. Thus, ideas on the mechanisms of genetic regulation of iontransporting enzymes received a solid foundation. Lately, the problems of immunology and hematology attracted the attention of Yuri Ovchinnikov, who believed that chemistry and biology should do more to help solving medical problems in the USSR.Intense investigations of naturally occurring regulators of immunity and hemopoiesis have been started at the Shemyakin Institute. Some presentations at this symposium deal with these problems. Above, we have outlined the scientific interests of Yuri Ovchinnikov, who was also in the driving seat in leading the chemical and biological scientific communities of his country. Ovchinnikov could not imagine how the science could evolve without intensive international cooperation. He excellently presented the advances of the Institute, and promoted scientific contacts, giving impetus to a series of bilateral symposia such as USSR-FRG, USSR-USA, France-USSR, Sweden- USSR, and Italy-USSR in various fields of physicochemical biology, many of which have now became a tradition. The remarkable symposia on Frontiers in Bioorganic Chemistry and Molecular Biology in Tashkent (1980) and Moscow-Alma-Ata (1984) were also organized and presided over by him. Of Yuri Ovchinnikov occupies a prominent place in the world's scientific heritage.
We can only guess at what his further endeavors would have been, if he were still alive. It is our hope that this numerous works will inspire many generations of bioorganic chemists to come, providing the key to solving a diversity of problems and demonstrating again and again the beauty and the attractive power of the world of science.