On Immunological Studies at Sirius University of Science and Technology.

This short report summarizes the results of recent immunological studies performed at new Sirius University of Science and Technology. The report focuses on studying the features of the immune response to vaccination and revaccination against SARS-CoV-2, as well as on a search of potential agents to prevent infection with this virus.

[On Immunological Studies at Sirius University of Science and Technology].

This short report summarizes the results of recent immunological studies performed at the new Sirius University of Science and Technology. The report focuses on studying the features of the immune response to vaccination and revaccination against SARS-CoV-2, as well as on a search of potential agents to prevent infection with this virus.

Antibodies to the N-Terminal Domain of Angiotensin-Converting Enzyme (ACE2) That Block Its Interaction with SARS-CoV-2 S Protein.

SARS-CoV-2 is a new coronavirus that is the cause of COVID-19 pandemic. To enter the cell, the virus interacts via its surface S protein with angiotensin-converting enzyme 2 (ACE2), the main entry receptor on the cell membrane. Most of protective antibodies, including those induced by vaccinations, target the S protein, preventing its interaction with the ACE2 receptor. We have evaluated an alternative strategy for blocking the S-ACE2 interaction using new antipeptide antibodies to the N-terminus of the ACE2 molecule. These antibodies allow detection of human ACE2 in vitro and ex vivo.

Generation and Evaluation of Bispecific Anti-TNF Antibodies Based on Single-Chain VHH Domains.

Systemic cytokine inhibition may be an effective therapeutic strategy for several autoimmune diseases. However, recent studies suggest that tissue or cell type-specific targeting of certain cytokines, including TNF, may have distinct advantages and show fewer side effects. Here we describe protocols for generating and testing bispecific cytokine inhibitors using variable domain of single-chain antibodies from Camelidae (VHH) with a focus on cell-specific TNF inhibitors.

[The action of the tumor necrosis factor (TNF) on the recovery processes of hemopoietic colony-forming cells in irradiated mice]. / Deistvie tumornekroticheskogo faktora (TNF) na protsessy vosstanovleniia gemopoéticheskikh kolonieobrazuiushchikh kletok u obluchennykh myshei.

The influence of a tumor-necrotic factor (TNF) on the CFUs population has been studied normally and after irradiation. An inhibitory effect on the pool of the seven-day and doubling of the yield of the eleven-day colonies have been observed in mice received TNF 20 h before bone marrow removal as compared with the controls. The kinetics of restoration of bone marrow cellularity and CFUs number in mouse donors treated with TNF 20 h before irradiation (5.0 Gy) has demonstrated the stimulatory effect of the agent on both indices.

[Model polycistron operon for studying conjugated translation in E. coli cells. The role of a stream of ribosomes]. / Model'nyi politsistronnyi operon dlia izucheniia sopriazhennoi transliatsii v kletkakh E. coli. Rol' potoka ribosom.

The role of "stream" of ribosomes upon translation of polycistronic mRNAs has been studied using an artificial polycistron. It has been found that the levels of activation of cistron Ci + 1 out of two adjacent cistrons (Ci and Ci + 1) depends, in addition to earlier described effects of mutual arrangement of initiation and termination signals, also on efficiency of translation of the foregoing cistron Ci. The results obtained lead to the conclusion that in polycistronic systems the levels of translation of cistron Ci + 1 can be regulated by "stream" of ribosomes resulted from translation of the proximal cistron Ci.

Enhancement of tumour necrosis factor production and sensitivity to interleukin-2 of human peripheral blood mononuclear cells stimulated by lipopolysaccharide and muramyl dipeptide in vitro.

The effect of lipopolysaccharide, muramyl dipeptide, and a combination of these agents in vitro on the production of tumour necrosis factor (TNF) by human peripheral blood mononuclear cells (PBMC; monocytes and lymphocytes) and on the sensitivity of these cells to recombinant interleukin-2 (IL-2) was studied. Both lipopolysaccharide and muramyl dipeptide, alone and combined, induced the production of TNF alpha by blood monocytes and lymphocytes, although the T-cells made only a very small contribution to the TNF produced by the lymphocyte fraction, most being produced by the B-cells. The B-lymphocytes and monocytes also spontaneously produced TNF alpha. Pretreatment of the mononuclear cells with a combination of lipopolysaccharide and muramyl dipeptide, but not with each preparation separately, led to enhancement of the proliferative response of these cells to recombinant IL-2. Incubation of mononuclear cells with muramyl dipeptide, muramyl dipeptide plus lipopolysaccharide, and to a lesser extent with lipopolysaccharide alone led to the generation of more-active lymphokine-activated killer cells in the presence of IL-2 than when IL-2 was used alone. The resultant lymphokine-activated killer cells lysed both human and murine tumour target cells.

[The vector containing a signal for specific degradation of chimeric proteins. Synthesis of [Leu5]enkephalin using enteropeptidase]. / Vektor, soderzhashchii signal dlia spetsificheskogo rasshchepleniia khimernykh belkov. Poluchenie [Leu5]énkefalina s pomoshch'iu énteropeptidazy.

A plasmid vector (pEK1) coding, in framework of beta-galactosidase gene, for the amino acid sequence (Asp)4Lys which is recognized by bovine enteropeptidase has been constructed. Using this vector and chemically synthesized DNA coding for the [Leu5]-enkephalin, a plasmid (pEK-ENK) has been obtained in which the beta-galactosidase gene is fused, through the enteropeptidase linker, with the gene for [Leu5]enkephalin. The chimeric protein produced by expression of this plasmid has been isolated and then cleaved by the enteropeptidase to give [Leu5]enkephalin with the yield 74%.

A simple and rapid method for sequencing DNA.

A simplified technique for DNA sequence analysis has been developed, based on modification of a previous method [(1980) Methods Enzymol. 65, 499-560]. It employs an adsorptive immobilization of terminally labelled DNA on DEAE paper followed by G, A+G, C+T and C specific modification and cleavage reactions. This solid-phase technique is faster and more convenient than the original method. The efficiency is comparable. The total processing time taken to produce cleaved fragments loaded on a gel is less than 2 h.

DNA rearrangements generating artificial promoters.

The promoter-cloning plasmid pBRH4 (a derivative of pBR322 with a partially deleted promoter of the tet gene) is shown to contain a sequence which is located near the EcoRI site and can operate as an effective Pribnow box, but is not the remainder of the deletion-inactivated tet promoter of pBR322. If there is a sequence homologous to the '-35' promoter region at the border of the DNA fragment inserted at the EcoRI site, then a compound promoter arises and activates the tet gene. Point mutations in the nonfunctional--35 region of pBRH4 also activate the cryptic Pribnow box. Several compound promoters were obtained through deleting small portions of DNA around the HindIII site of pBR322; the deletions moved various sequences that could operate as Pribnow boxes towards the -35 region of the tet promoter.

[A solid phase method of determining DNA sequence]. / Tverdofaznyi metod opredeleniia nukleotidnoi posledovatel'nosti DNK.

A solid-phase method for DNA sequencing has been developed which involves immobilization of the terminally labeled DNA fragment on the DEAE-paper followed by chemical modification and cleavage at G, A + G, C + T, and C sites. As compared to the Maxam and Gilbert method, the new technique is more rapid and less laborious, being of the same efficiency.

[Analysis and properties of a bacterial clone containing a gene fragment of an immunoglobulin L chain]. / Analiz i svoistva bakterial'nogo klona, soderzhashchego fragment gena L-tsepi immunoglobulina.

Restriction analysis of hybrid plasmids from a number of clones allowed to screen out the plasmid p8-1. A detailed analysis of this plasmid and that of the inserted DNA was made using HhaII, AluI, Sau96I, Sau3A, MspI and BspI restriction endonucleases. The results demonstrated that the inserted DNA corresponds to the 3'-nontranslated region and to a portion of the C fragment of the light-chain immunoglobulin gene. Establishing the partial structure of the recombinant plasmid exhibited a complete coincidence of the inserted DNA 3'-terminus and the primary structure of the light-chain mRNA synthesized by MOPC-21 myeloma cells. This sequence corresponds to the amino acid sequence in the light-chain constant region (207-214 residues). The analysis of p8-1 plasmid showed that nucleotides of pBR322 plasmid at positions from 376 to 618 were deleted. The deletion might be predetermined by the plasmid property to be amplified more readily than all the other hybrid plasmids which were not deleted.

Synthesis of a model promoter for gene expression in Escherichia coli.

We have synthesized a 67-membered double-stranded DNA containing putative initiation sites for transcription and translation in E. coli. Among the potential applications of this DNA are construction of artificial genes, cloning of foreign genes in E. coli, and study of structure - activity relations in promoter and ribosome binding sites.