Biological effects of individually synthesized TNF-binding domain of variola virus CrmB protein.

The biological characteristics of a 17-kDa protein synthesized in bacterial cells, a TNF-binding domain (VARV-TNF-BP) of a 47-kDa variola virus CrmB protein (VARV-CrmB) consisting of TNF-binding and chemokine-binding domains, were studied. Removal of the C-terminal chemokine-binding domain from VARV-CrmB protein was inessential for the efficiency of its inhibition of TNF cytotoxicity towards L929 mouse fibroblast culture and for TNF-induced oxidative metabolic activity of mouse blood leukocytes. The results of this study could form the basis for further studies of VARV-TNF-BP mechanisms of activity for prospective use in practical medicine.

Selection of naturally occurring autoantibodies to interleukin-18 from phage display library.

Four unique phage single-chain variable fragments (scFvs) to recombinant human interleukin 18 (IL-18) has been selected from a naïve combinatorial library of human scFvs. Binding of unique phage antibodies with IL-18 was tested by ELISA and Western-blotting. No cross reactivity with tumor necrosis factor α, interferons α and γ was shown for the selected antibodies. The gene segments encoding V(D)J regions of selected antibodies exhibited a high degree of homology to germline genes, therefore we suggest that the selected scFv's belong to repertoire of naïve autoantibodies.

[Modeling spatial structures of variola and cowpox virus TNF-binding CrmB proteins bound to murine or human TNF].

Orthopoxviruses bear in their genomes several genes coding for homologous secreted proteins able to bind tumor necrosis factor. Different species of the genus possess different sets of these tumor necrosis factor-binding proteins. Viriola virus encodes the only one of them named CrmB. Despite sharing high sequence identity, CrmB proteins belonging to distinct orthopoxviral species were shown to significantly differ by their physico-chemical and biological properties. We modeled spatial structures of tumor necrosis factor receptor domains of variola and cowpox virus CrmB proteins bound to either murine, or human or mutated human tumor necrosis factor. In the sequence of last the arginine residue at position 31 is substituted with glutamine that is characteristic for murine tumor necrosis factor. Theoretical analysis of modeled ligand-receptor complexes revealed that the least stable should be the complex of cowpox virus CrmB with human tumor necrosis factor, and that arginine to glutamine substitution at position 31 should significantly stabilize binding of corresponding human tumor necrosis factor mutant to cowpox virus CrmB. Experimental evaluation of recombinant variola and cowpox virus CrmB efficiencies in inhibiting cytotoxic effect of all these tumor necrosis factors have approved our predictions.

Recombinant TNF-binding protein from variola virus as a novel potential TNF antagonist.

Gel-filtration chromatographic separation of the lysate of Sf21 insect cells infected with recombinant baculovirus BVi67 containing the gene for TNF-binding protein (CrmB) of variola virus (VARV) revealed that hTNF-cytotoxicity neutralization activity is associated with a fraction corresponding mainly to high molecular weight proteins (above 500 kDa) and less with fractions corresponding to proteins of 270 or 90 kDa. The recombinant VARV-CrmB protein has been purified by affinity chromatography. Difference in the experimentally determined and estimated (according to amino acid composition) VARV-CrmB molecular weight is due to glycosylation of the recombinant protein expressed in the insect cells. VARV-CrmB neutralizes in vitro the cytotoxic effect of hTNF and hLTalpha, and its TNF-neutralizing activity is two to three orders of magnitude higher compared to the analogous effects of type I and II soluble TNF receptors, comparable with the activity of mAb MAK195, and somewhat lower than the effect of the commercial drug Remicade.

[Characteristics of A-interferon-generated dendritic cells in patients with pulmonary tuberculosis].

The phenotype of the dendritic cells (DC) generated from the adhesion fraction of mononuclear cells in the presence of GM-CSF and alpha-interferon was studied in patients with pulmonary tuberculosis. Despite the absence of significant differences in the count of mature CD83+DCs in the groups of patients (n = 38) and healthy donors (n = 30), elevated CD14(+)-monocyte levels and few activated CD25(+)-DCs were indicative of the impaired process of DC maturation/generation in patients with pulmonary tuberculosis, particularly in a subgroup of patients with a low T-cell proliferative response against PPD (PPD-anergy, n = 10). The patients with tuberculosis showed the lower relative levels of CD11c(-)-CD123(+)-DC and the normal levels of myeloid CD11c(+)D123(-)DCs. However, in patients with PPD-anergy, the content of myeloid CD11c(+)CD123(-)-DCs was significantly higher than that in PPD-reactive patients. Moreover, the patients with PPD-anergy were characterized by the elevated peripheral blood levels of CD14+CD16(+)-monocytes, which was associated with the high suppressive activity of monocytes (r(s) = 0.53; p < 0.05). The impaired process of DC generation/maturation in patients with pulmonary tuberculosis is believed to be associated with the changes in the phenotypic and functional properties of monocytes and to be a cause of an inadequate antigen-specific response in tuberculous infection.

[Construction of combinatorial immune library of single chain human antibodies to orthopoxviruses and selection from this library antibodies to recombinant protein prA30L of variola virus].

A combinatorial immune library of human single-chain antibody fragments (scFv) was constructed on the base of genes encoding variable domains of heavy and light chains of immunoglobulins cloned from the lymphocytes of four vaccinia virus (VACV) vaccinated donors. The size of the library was 3 x 10(7) independent clones. After the library was enriched with the clones producing scFv against recombinant analogue of variola virus surface protein prA30L, a panel of unique antibodies specific to both prA30L and VACV was selected from the library. A plaque reduction neutralization test was performed for all selected antibodies and two antibodies were shown to be able to neutralize plaque formation of VACV in Vero E6 cells monolayer. Binding specificities of these antibodies were confirmed using ELISA and Western blot analysis. To determine the amino acid sequences of neutralizing antibodies their genes were sequenced.

[Fusion proteins encoded by orf 129L of ectromelia and orf A30L of smallpox viruses cross-react with neutralizing monoclonal antibodies].

Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.

[Expression of genes for orthopoxviral TNF-binding proteins and study resulted recombinant proteins].

Genes for TNF-binding proteins (CrmBs) of variola virus (VARV), monkeypox virus (MPXV) or cowpox (CPXV) were isolated with PCR from viral genomes and expressed within baculovirus DNAs in Sf21 insect cell line. Properties of resulted recombinant proteins were studied with physical-chemical and immunological methods. It was shown with solid phase enzyme-linked immunoassay that viral proteins inhibited hTNF binding with polyclonal hTNF-antibodies. The strongest inhibitor was VARV-CrmB, the less one was MPXV-CrmB. Biological activity of recombinant protein preparations was studied in the test of neutralization of TNF cytotoxicity for L929 murine fibroblast cells. It was shown that recombinant CrmBs neutralized cytotoxicity of hTNF, mTNF or rTNF in species-specific manner. It was shown also that effectiveness of hTNF cytotoxicity inhibition in vitro with VARV-CrmB exceeded the same effect of polyclonal hTNF-antibody. A possibility of the elaboration of new therapeutics for anti-TNF therapy on the base of CrmB-like proteins is discussed.

Cytotoxic effects of dynorphins through nonopioid intracellular mechanisms.

Dynorphin A, a prodynorphin-derived peptide, is able to induce neurological dysfunction and neuronal death. To study dynorphin cytotoxicity in vitro, prodynorphin-derived peptides were added into the culture medium of nonneuronal and neuronal cells or delivered into these cells by lipofection or electroporation. Cells were unaffected by extracellular exposure when peptides were added to the medium. In contrast, the number of viable cells was significantly reduced when dynorphin A or "big dynorphin," consisting of dynorphins A and B, was transfected into cells. Big dynorphin was more potent than dynorphin A, whereas dynorphin B; dynorphin B-29; [Arg(11,13)]-dynorphin A(-13)-Gly-NH-(CH(2))(5)-NH(2), a selective kappa-opioid receptor agonist; and poly-l-lysine, a basic peptide more positively charged than big dynorphin, failed to affect cell viability. The opioid antagonist naloxone did not prevent big dynorphin cytotoxicity. Thus, the toxic effects were structure selective but not mediated through opioid receptors. When big dynorphin was delivered into cells by lipofection, it became localized predominantly in the cytoplasm and not in the nuclei. Big dynorphin appeared to induce toxicity through an apoptotic mechanism that may involve synergistic interactions with the p53 tumor-suppressor protein. It is proposed that big dynorphin induces cell death by virtue of its net positive charge and clusters of basic amino acids that mimic (and thereby perhaps interfere with) basic domains involved in protein-protein interactions. These effects may be relevant for a pathophysiological role of dynorphins in the brain and spinal cord and for control of death of tumor cells, which express prodynorphin at high levels.

p53 Latency. C-terminal domain prevents binding of p53 core to target but not to nonspecific DNA sequences.

The p53 transcription factor is either latent or activated through multi-site phosphorylation and acetylation of the negative regulatory region in its C-terminal domain (CTD). How CTD modifications activate p53 binding to target DNA sequences via its core domain is still unknown. It has been proposed that nonmodified CTD interacts either with the core domain or with DNA preventing binding of the core domain to DNA and that the fragments of the CTD regulatory region activate p53 by interfering with these interactions. We here characterized the sequence and target specificity of p53 activation by CTD fragments, interaction of activating peptides with p53 and target DNA, and interactions of "latent" p53 with DNA by a band shift assay and by fluorescence correlation spectroscopy. In addition to CTD fragments, several long basic peptides activated p53 and also transcription factor YY1. These peptides and CTD aggregated target DNA but apparently did not interact with p53. The potency to aggregate DNA correlated with the ability to activate p53, suggesting that p53 binds to target sequences upon interactions with tightly packed DNA in aggregates. Latent full-length p53 dissociated DNA aggregates via its core and CTD, and this effect was potentiated by GTP. Latent p53 also formed complexes via both its core and CTD with long nontarget DNA molecules. Such p53-DNA interactions may occur if latent p53 binding to DNA via CTD prevents the interaction of the core domain with target DNA sites but not with nonspecific DNA sequences.

[Construction of original artificial immunogens--candidate vaccines against HIV-1]. / Konstruirovanie original'nykh iskusstvennykh immunogenov--kandidatov v vaktsiny protiv HIV-1.

An original method for making effective artificial vaccines has been developed. Immunogens are virus-like particles containing a DNA molecule covered with recombinant proteins carrying the pathogen epitopes. The recombinant proteins are exposed on the surface of the particle and are attached to DNA via spermidine-polygluquine-glutathione or galactopyranoside conjugates (depending on the hybrid protein composition). Two variants of artificial immunogens-candidates for antiHIV vaccine have been prepared.

Clustering of apoptotic cells via bystander killing by peroxides.

Clustering of apoptotic cells is a characteristic of many developing or renewing systems, suggesting that apoptotic cells kill bystanders. Bystander killing can be triggered experimentally by inducing apoptosis in single cells and may be based on the exchange of as yet unidentified chemical cell death signals between nearby cells without the need for cell-to-cell communication via gap junctions. Here we demonstrate that apoptotic cell clusters occurred spontaneously, after serum deprivation or p53 transfection in cell monolayers in vitro. Clustering was apparently induced through bystander killing by primary apoptotic cells. Catalase, a peroxide scavenger, suppressed bystander killing, suggesting that hydrogen peroxide generated by apoptotic cells is the death signal. Although p53 expression increased the number of apoptoses, clustering was found to be similar around apoptotic cells whether or not p53 was expressed, indicating that there is no specific p53 contribution to bystander killing. Bystander killing through peroxides emitted by apoptotic cells may propagate tissue injury in different pathological situations and be relevant in chemo-, gamma-ray, and gene therapy of cancer.

[A model system for the assessment of the stability of eukaryotic genes and expressed proteins in extended space flight]. / Model'naia sistema dlia izucheniia stabil'nosti éukarioticheskikh genov i ékspressiruemykh belkov v usloviiakh dlitel'nogo kosmicheskogo poleta.

Evidence was obtained that the changed gravity, tension profiles of the magnetic fields inside the orbital station and other spaceflight factors (SFF) substantially influence the cell genome and synthesis of recombinant proteins. The authors proposed a technique of fixation of possible alterations in genes and expressed recombinant proteins in strains-producers of human alpha-interferon: HuIFN-alpha 2b, HuIFN-alpha 8a, HuIFN-alpha 10a, and HuIFN-alpha 14a. Spaceflight factors were simulated by way of gamma-irradiation at 10 Gy and 50 Gy by a cobalt unit, and centrifugation of samples at 10 g and 50 g. Cultivation of the strains-producers during the SFF simulation yielded HuIFN-alpha proteins with altered functional characteristics. Following exposure to simulated SFF, strains-producers expressed HuIFN-alpha that preserved a high titre of antiviral activity (AVA) but suppressed the antiproliferative activity (APA) inferring some structural/functional shifts in the HuIFN-alpha molecule due to, presumably, mutations in the active center responsible for APA. Determination of species specificity of the HuIFN-alpha recombinant proteins following exposure to SFF revealed dissociation of cross-AVA in homologous and heterologous cell cultures which can be also attributed to the structural/functional shifts in the HuIFN-alpha molecule. These changes can be localized in the receptor cluster of molecule or consequent to modification of the center defining HuIFN-alpha species specificity. The proposed simulation system allows fixation of shifts in the HuIFN-alpha structural/functional characteristics and investigations of the stability of eucaryotic genes in long-term space flights.

[Development of approaches to studying the structural and functional organization of protein molecules aboard the International space station]. / Razrabotka podkhodov dlia izucheniia strukturno-funktsional'noi organizatsii belkovykh molekul na Mezhdunarodnoi kosmicheskoi stantsii.

To solve the problems of biological safety of the cosmonaut on long-duration space mission and prediction of changes in macroorganism as a whole and constituting protein molecules under these conditions, it is important to study the influence of spaceflight factors (SFF) on microorganisms-carriers of modeled structures, protein molecules and pro- and eukaryotic genes in particular. Within the framework of scientific cooperation NAUKA-NASA, the authors proposed a model system of prokaryotic producers of pro- and eukaryotic proteins--staphylococcus alpha-toxin (SAT)--as a key protein in pathogenesis of staphylococcal infections, and human leukocytic interferon (HuIFN-alpha) as one of the homeostasis regulating proteins with well-studied structural and functional properties. Recombinant strains of E. coli with either a single or duplicated HuIFN-alpha 2b gene or other genes of the HuIFN-alpha family: HuIFN-alpha 8a, HuIFN-alpha 10a and HuIFN-alpha 14a were selected as producers of SAT and HuIFN-alpha. This biotechnologic system allows imitation and assessment of the SFF mutagenic effect both at the levels of genome of strain-carrier and gene-insertion and expressed CAT and HuIFN-alpha molecules including transcription, translation, assembly and post-translatory modifications of the target-protein. The developed methodology allows determination of highly mutable and conservative regions in the primary structure of a hypothetical protein associated with its functional activity, prediction of specific amino acid substitutes in these regions, and comparison of test calculations with a pool of natural mutations in the family of proteins under study. The structural/functional analysis of proteins and HuIFN-alpha genes made it possible to isolate and systematize functionally significant areas in the structure of hypothetical protein HuUFN-alpha, on the basis of which the most probable amino acid substitutions were prognosticated. This will present a possibility to identify expectable mutation events in HuIFN-alpha proteins, which so far have not been found in natural genes of the human interferon. Comparison of results of SFF modeling and space experiments aboard the International Space Station with monitoring of HuIFN-alpha mutant forms will help estimation of the extent of influence of the spaceflight factors on evolution of protein molecules.

[Regulation of translation of the distal lacZ gene in polycistronic mRNA by the ribosome stream from the proximal gene]. / Reguliatsiia transliatsii distal'nogo gena lacZ politsistronnoi mRNK potokom ribosom s proksimal'nogo gena.

Four series of plasmids (pNSI, pNSII, pNLI, and pNLII) with artificial polycistrons containing the lacZ test gene were constructed. These plasmids coded for polycistronic mRNAs with two different types of cistron (orfZ and lacZ) coupling: in pNSI and pNLI, the orfZ termination codon and the lacZ initiation codon overlapped (type I); in pNSII and pNLII, the orfZ termination codon, was located upstream of the lacZ SD sequence. The length of the orfZ cistron was 60 bp in pNSI and pNSII or 300 bp in pNLI and pNLII. Plasmids with the same type of cistron coupling contained the same lacZ translation initiation region, whereas the structure of the orfZ translation initiation region varied, thereby providing varying efficiency of the orfZ gene translation. The effect of these variations on the efficiency of the lacZ gene translation was evaluated by direct measurement of the beta-galactosidase activity in Escherichia coli cells transformed with the corresponding plasmids. We found that the level of translation of the distal lacZ gene depended on the ribosome stream from the proximal gene and was maximal at the optimal ribosome stream level, which, in turn, depended on the type of cistron coupling.

[Expression of the gene for vaccinia virus 36K nonstructural protein in Escherichia coli and study of the protective properties of expression products]. / Ekspressiia gena nestrukturnogo belka 36K virusa ospovaktsiny v Escherichia coli i izuchenie protektivnykh svoistv produktov ékspressii.

The nonstructural 36K protein of vaccinia virus (VV) mapped in HindIII-P and HindIII-J fragments of VV strain L-IVP has been expressed in E. coli as a fusion protein. The products of 36K gene preserved the antigen homology with the native protein 36K and the capacity to bind specific immunoglobulins of rabbit antiVV serum. The protective properties of 36K gene products and their joint effect with the immunodominant protein 35K were investigated. The non-structural 36K gene products showed no protective activity, but increased the production of specific antibodies in mice immunized with a mixture of both protein preparations, this increase being compatible with that observed after immunization with the inactivated virus preparations.

Design of four-helix bundle protein as a candidate for HIV vaccine.

To be efficient, a synthetic vaccine should contain different T and B cell epitopes of human immunodeficiency virus (HIV) antigens, and the B epitope regions in the vaccine and in the HIV should be conformationally similar. We have suggested previously the construction of vaccines in the form of a protein with a predetermined tertiary structure, namely a four-alpha-helix bundle. Antigenic determinants of cellular and humoral immunity are blocks for the vaccine design. From experimentally studied HIV-1 T and B cell epitopes, we constructed a sequence of a four-helix protein TBI (T and B cell epitopes containing immunogen). The gene of the protein was synthesized and the protein was produced in C600 Escherichia coli cells under recA promoter from Proteus mirabelis. CD spectroscopy of the protein demonstrated that 30% of amino acid residues adopt an alpha-helical conformation. Mice immunized with TBI have shown both humoral and cellular immune responses to HIV-1. The obtained data show that the design of TBI was successful. The synthesized gene structure makes possible further reconstruction and improvement of the protein vaccine structure.