[Experience of anticipatory therapy with IL-6 receptor inhibitors and perspectives for its use in the evolution of COVID-19].

The Advisory Board chaired by the chief specialist in infectious diseases of the Ministry of Health of Russian Federation, Professor V.P. Chulanov was held on June 18, 2022 in Saint Petersburg. Aim. The main purpose of the Board was following discussion: the analysis of the real-world data of levilimab as an anticipatory therapy for COVID-19 in hospitalized patients; the review of the experience and perspectives of levilimab as an anticipatory anti-inflammatory option for outpatient patients who meet defined clinical and laboratory criteria. Results. The analyzed data on clinical efficacy and safety formed the basis of recommendations proposed by experts for the use of levilimab in the inpatient and outpatient medical care for COVID-19.

[The value of bone mineral density assessment by dual-energy x-ray absorptiometry for prediction of cardiovascular mortality in patients treated with hemodialysis].

AIM: Determine whether bone mineral density (BMD) assessed by dual-energy x-ray absorptiometry can be used as predictor of increased risk of death in hemodialysis patients. MATERIALS AND METHODS: A prospective study was performed of 516 patients with chronic kidney disease treated with hemodialysis (men 265, women 251, mean age 44.811.4 years) who were observed for 5.73.2 years. Before inclusion in the study, in all patients was analyzed bone mineral density using dual-energy X-ray absorptiometry in three standard departments: lumbar vertebrae, proximal femur and distal forearm. The probability analysis of the outcome was carried out using the KaplanMeier method and Cox. RESULTS: During follow-up period 111 (21.5%) patients died, 50.5% from cardiovascular events. Survival analysis by KaplanMeier method allowed to prove the increased risk of death from cardiovascular pathology in hemodailysis patients with low bone mineral density of all evaluated areas. Step-by-step multivariate Cox regression analysis showed that the T score of the femur, showing the difference of BMD of the patient with normal value of BMD for young adult, had the greatest prognostic significance. CONCLUSION: Reduced bone mineral density in patients receiving hemodialysis is associated with an increased risk of death from cardiovascular disease. Dual energy x-ray absorptiometry can be used for assessment of this risk.

Probing the uniformity of hydrogen intercalation in quasi-free-standing epitaxial graphene on SiC by micro-Raman mapping and conductive atomic force microscopy.

In this paper, micro-Raman mapping and conductive atomic force microscopy (C-AFM) were jointly applied to investigate the structural and electrical homogeneity of quasi-free-standing monolayer graphene (QFMLG), obtained by high temperature decomposition of 4H-SiC(0001) followed by hydrogen intercalation at 900 °C. Strain and doping maps, obtained by Raman data, showed the presence of sub-micron patches with reduced hole density correlated to regions with higher compressive strain, probably associated with a locally reduced hydrogen intercalation. Nanoscale resolution electrical maps by C-AFM also revealed the presence of patches with enhanced current injection through the QFMLG/SiC interface, indicating a locally reduced Schottky barrier height (ΦB). The ΦB values evaluated from local I-V curves by the thermionic emission model were in good agreement with the values calculated for the QFMLG/SiC interface using the Schottky-Mott rule and the graphene holes density from Raman maps. The demonstrated approach revealed a useful and non-invasive method to probe the structural and electrical homogeneity of QFMLG for future nano-electronics applications.

[Structural changes of eye chorioretinal complex after total cerebral ischemia and their correction].

Structural changes of eye chorioretinal complex were investigated in 40 adult male outbred albino rats after total transient cerebral ischemia using electron microscopy and morphometric analysis. Furthermore, the influence of a new sterically hindered phenolic antioxidant dibornol on these processes was estimated. Our studies demonstrated that total transient cerebral ischemia in rats resulted in the capillary thrombosis of the choriocapillary lamina of the uvea, structural disturbances of the blood-retinal barrier, degeneration of the retinal neurons and radial glia. Course administration of dibornol was shown to improve the microcirculation and to protect the retinal neuronal structures, pigment epithelium, and radial glia.

Thermal impurity reactions and structural changes in slightly carbonated hydroxyapatite.

Lattice and surface impurity reactions and structural changes induced by them in slightly carbonated hydroxyapatite (SCHA) treated at 25-1100 degrees C were comprehensively studied. The SCHA was processed by a conventional wet synthesis at a high possible temperature(96 degrees C) using ammonium containing parent reagents. IR-spectroscopy, XRD, TG-DTA technique and mass spectrometric thermal analysis (MSTA) were employed for characterization of the samples. NH4+ with H3O+ in cationic-and CO3(2-) (A- and B-positions) with HPO4(2-) in anionic sites, and H2O, CO3(2-)(HCO3(-)) NO3(-), NxHy on the surface of particles were found and considered as impurity groups. Complicated changes in lattice constants of theSCHA stepwise annealed in air (for 2 h) were revealed; the changes were associated with reactions of the impurity groups. Filling the hexed sites with hydroxyl ions above 500 degrees C was shown to happen partly due to lattice reactions but was mainly owing to hydrolysis of the SCHA by water molecules in air. Decomposition of CO3(2-) groups proceeded through both thermal destruction and reactions with some of the impurity ions. The decarbonation in A-sites occurred at much lower temperatures (450-600 degrees C) than in B-sites (700-950 degrees C) and was first revealed to happen in two stages: due to an impurity reaction around 500 degrees C, and then through thermal destruction at 570 degrees C. A redistribution of CO3(2-) ions, decreasing in amount on the whole, was observed upon annealing above 500 degrees C. To avoid possible erroneous conclusions from TG-data, a sensitive method was shown to be required for monitoring gaseous decomposition products (such as the MSTA in this study), in case several impurity groups were present in a SCHA.

RNA - DNA hybridization on membrane filters with fragmented mammalian DNA.

The possibilities of using fragmented mammalian DNA for hybridization on membrane filters were investigated. The adsorption and release of fragmented DNA were studied as influenced by various factors of the hybridization procedure. It was found that working with preparations sufficiently homogeneous in molecular weight with fragment size 4.8-6.5 S, dissolved in 6 times SSC at neutral pH, the adsorption on filters was almost 100%. After incubation of filters for 18 h in 2 times SSC at 65 degrees C about 50% of the fragmented DNA and 20% of the high molecular weight DNA were released. The degree of release differed for the different families of repeated DNA sequences. Lowest release was obtained with the highly repeated DNA (20%) and highest with the unique DNA (63%), i.e. the release was inversely proportional to the renaturation rate of DNA. In the course of release of fragmented total DNA the material remaining on the filters became enriched in highly repeated sequences, due to selective release of the slowly reassociating fractions. As a result, the percentage of fragmented DNA which hybridized with heterogeneous nuclear RNA was higher than that of high molecular weight DNA. The thermal stabilities of the hybrids with fragmented and high molecular weight DNA were identical. The conditions are defined which permit application of the membrane filter hybridization technique to fragmented mammalian DNA.

Radioiodination of naturally occurring phospholipids.

A simple and rapid nonenzymatic method for radioiodination of phospholipids is described. It involves oxidation of Na125I with TlCl3 (or chloramine-T) in an aqueous medium, with subsequent exposure of the phospholipids, dissolved in chloroform/methanol, to the action of the oxidizing mixture. Purification of the radiolabelled phospholipids was effected by washing with sodium thiosulphate followed by thin-layer chromatography on silica gel. Specific radioactivity of 125I-labelled phosphatidylcholine was estimated to be about 10 muCi/mg phospholipid. The method is designed for radioiodination of various naturally occurring phospholipids.

Unusual effect of clusters of rare arginine (AGG) codons on the expression of human interferon alpha 1 gene in Escherichia coli.

The human interferon (hIFN alpha 1) gene contains 11 arginine (Arg) codons AGG or AGA, which are extremely rare for bacteria, four of which are organized in tandems. The two AGG tandems (corresponding to Arg12 Arg13 and Arg163 Arg164) are known to inhibit the translation of hIFN alpha 1 mRNA and therefore they are considered to be responsible for the poor expression of hIFN alpha 1 gene in bacterial cells. To study the effect of these two tandems on the expression of hIFN alpha 1 in E. coli, four new gene variants were designed to contain preferential Arg codons (CGT) substituted for the rare AGG codons in either the first, the second or both AGG tandems. We found that, whereas the yield of hIFN alpha 1 protein per cell remained unchanged, the level of hIFN alpha 1 mRNA decreased gradually (by a factor of two) with the consecutive substitution of the first, second and both AGG tandems. These results indicated, first, that the AGG clusters might have a stabilizing effect on the mRNA, and second, that mRNAs devoid of such clusters were translated at a higher rate in vivo. The protein products of the four genes (having the same amino acid sequence) showed different specific antiviral activity. The most active was the product of gene hIFN alpha 1(c) in which the second AGG tandem (corresponding to Arg163, Arg164) was preserved while the least active was the protein of gene hIFN alpha 1(d) (devoid of both AGG clusters). The role of the AGG tandems in folding of the gene product is discussed.

A second putative mRNA binding site on the Escherichia coli ribosome.

Translation in bacteria is initiated by a base-pairing interaction between the extreme 3'-end of the small-subunit rRNA and a purine-rich domain (Shine-Dalgarno (SD) sequence) preceding the initiation codon at the 5'-end of most bacterial mRNAs. Here, we describe the identification of a second functional and alternative site on the Escherichia coli ribosome which is capable of interacting with mRNA devoid of SD sequences and initiate the translation. This site is localized between nt 1340 and 1360 of the 16S rRNA in E. coli and is complementary to the untranslated region at the 5'-end of tobacco mosaic virus RNA (omega sequence).

Interaction of homo-aza-steroidal ester of [p-[bis-(2-chloroethyl) amino]phenyl]acetic acid (ASE) with DNA of Ehrlich ascites tumor cells.

A cytostatic, homo-aza-steroidal ester of [p-[bis-(2-chloroethyl) amino]phenyl]acetic acid (ASE) was reduced with NaB3H4 and [3H]ASE-treated DNA prepared in vitro. We found that: (1) ASE reacts preferentially with purines; (2) ASE decreases the thermal stability of the double helix upon binding to DNA; (3) [3H]ASE binding sites are clustered along the DNA molecules; (4) ASE binding sites probably represent oligo- or polypurine sequences.

Fluorescence spectral characteristics of novel asymmetric monomethine cyanine dyes in nucleic acid solutions.

Six new asymmetric monomethine cyanine dyes have been synthesized and their fluorescence characteristics in the presence of nucleic acids studied. The new dyes have no fluorescence of their own in water solutions upon excitation at 480 nm but they become strongly fluorescent in the presence of nucleic acids. The fluorescence maxima of the investigated dyes are found at 525-545 nm when bound to dsDNA and around 600 nm upon binding to RNA and ssDNA. Fluorescence quenching studies with increasing concentrations of NaCl indicate that the cyanine dyes have a mixed (intercalating and groove binding) type of interaction with dsDNA.

Comparative study on the effect of signal peptide codons and arginine codons on the expression of human interferon-alpha 1 gene in Escherichia coli.

Human interferon-alpha 1 (HuIFN-alpha 1) gene containing signal peptide codons is poorly expressed in bacteria, and this is explained by the presence of clusters of rare (AGG) arginine codons in its structure. In this study, we have constructed a series of modified HuIFN-alpha 1 genes to study the effect of both residual signal peptide codons and clusters of AGG codons on gene expression in Escherichia coli cells. Our results showed that substitution of preferential for rare arginine codons in two clusters did not affect the yield, whereas deletion of the signal peptide codons led to a 10-fold increase in the yield of recombinant protein. To understand the mechanism of interference of gene structure on the expression of the HuIFN-alpha 1 gene in vivo, both the level and stability of HuIFN-alpha 1 mRNA were measured. The amount of HuIFN mRNA increased almost five times on deletion of the signal peptide codons from HuIFN-alpha 1 gene constructs (containing AGG clusters or not). The stability of mRNA obtained from all gene constructs was shown to be the same (half-life of 60 +/- 5 secs), indicating that the signal peptide codons interfere with both the efficiency of transcription of the HuIFN-alpha 1 gene and translation of its mRNA.

Effect of N-terminal deletions on the activity of pokeweed antiviral protein expressed in E. coli.

Pokeweed antiviral protein (PAP) from Phytolacca americana is a highly specific N-glycosidase removing adenine residues (A4324 in 28S rRNA and A2660 in 23S rRNA) from intact ribosomes of both eukaryotes and prokaryotes. Due to the ribosome impairing activity the gene coding for mature PAP has not been expressed so far in bacteria whereas the full-length gene (coding for the mature 262 amino acids plus two signal peptides of 22 and 29 amino acids at both N- and C-termini, respectively) has been expressed in Escherichia coli. In order to determine: 1) the size of the N-terminal region of PAP which is required for toxicity to E. coli; and 2) the location of the putative enzymatic active site of PAP, 5'-terminal progressive deletion of the PAP full-length gene was carried out and the truncated forms of the gene were cloned in a vector containing a strong constitutive promoter and a consensus Shine-Dalgarno ribosome binding site. The ribosome inactivation or toxicity of the PAP is used as a phenotype characterized by the absence of E. coli colonies, while the mutation of PAP open reading frames in the small number of survived clones is used as an indicator of the toxicity to E. coli cells. Results showed that the native full-length PAP gene was highly expressed and was not toxic to E. coli cells although in vitro ribosome inactivating activity assay indicated it was active. However, all of the N-terminal truncated forms (removal of seven to 107 codons) of the PAP gene were toxic to E. coli cells and were mutated into either out of frame, early termination codon or inactive form of PAP (i.e., clone PAP delta107). Deletion of more than 123 codons restored the correct gene sequence but resulted in the loss of the antiviral and ribosome inactivating activities and by the formation of a large number of clones. These results suggest that full-length PAP (with N- and C-terminal extensions) might be an inactive form of the enzyme in vivo presumably by inclusion body formation or other unknown mechanisms and is not toxic to E. coli cells. However, it is activated by at least seven codon deletions at the N-terminus. Deletions from seven through to 107 amino acids were lethal to the cells and only mutated forms (inactive) of the gene were obtained. But deletion of more than 123 amino acids resulted in the loss of enzymatic activity and made it possible to express the correct PAP gene in E. coli. Because deletion of Tyr94 and Val95, which are involved in the binding of the target adenine base, did not abolish the activity of PAP, it is concluded that the location previously proposed for PAP enzymatic active site should be reassessed.

EPR study on the interaction of spin-labelled hydrazine mustard derivatives with DNA.

The interaction of three spin-labelled compounds, derivatives of bis-(2-chloroethyl)-hydrazine (HMSL), N-methyl,N-chloroethyl-hydrazine (MCEHSL) and bis-(2-bromoethyl)-hydrazine (BEHSL) with DNA was studied by the method of electron paramagnetic resonance (EPR). It was found that HMSL (containing two chloroethyl groups) and MCEHSL (containing one chloroethyl group) gave spin-labelled dsDNA with identical strongly immobilized EPR spectra. The conclusion was drawn that only one of the alkylating groups of HMSL reacted with DNA. In contrast, the EPR spectrum of DNA spin-labelled with BEHSL was non-immobilized due to the strong destabilizing effect of this compound on the double helix. The extent of alkylation of DNA with the three hydrazine mustard derivatives was one and the same. It was found, however, that chloroethyl-containing derivatives (HMSL and MCEHSL) had an expressed base specificity and alkylated preferably the guanilic residues, and their bromo-analogue (BEHSL) did not show any base specificity and alkylated the bases of DNA at random.

Expression of human alpha 1 interferon genes in vectors containing tandemly located promoters recognized by two different RNA polymerases (Escherichia coli and T7).

An expression vector containing two tandemly located promoters (T7 and P1) and two transcription terminators recognized by two different RNA polymerases (T7 RNA polymerase and Escherichia coli RNA polymerase) was constructed. Human alpha 1 interferon gene variants were cloned in this vector and their expression was studied in E. coli strains containing [E. coli BL2I (DE3)] or devoid (E. coli BL21) of the gene for the T7 RNA polymerase. We report that simultaneous activity of the two promoters reduces the level of gene expression when compared with the levels of expression corresponding to either P1 or T7 promoter alone.

Porcine granulosa cell conditioned media as autocrine regulator of progesterone secretion.

The ability of porcine granulosa cells to release a progesterone inhibiting substance(s) was examined in vitro. Granulosa cells (SGCs, MGCs and LGCs) were harvested from small, medium or large antral follicles respectively. The effect of granulosa cell conditioned media obtained from small follicles (SGCCM) was studied in the culture of LGCs by estimation of progesterone secretion; the conditioned media evoked the inhibition of progesterone secretion by the LGCs. SGCCM produced by various numbers of cultured granulosa cells showed a dose-related inhibition of progesterone production. A maximum inhibitory effect was noted when a 5-fold concentration of SGCCM was used. The addition of SGCCM had no effect on the growth of the cultured cells. The factor(s) inhibiting progesterone secretion appeared to be a nonsteroidal substance of molecular mass greater than 10 kDa and was heat-stable and trypsin-sensitive. The data presented support the suggestion that the conditioned media generated by primary cultures of SGCs contain nonsteroidal regulators capable of inhibiting progesterone secretion by cultured LGCs; this inhibitory activity can play an important autocrine regulatory role in the process of follicular differentiation.

Comparative investigation of DNA from mouse and Ehrlich ascites tumor cells.

Mouse DNA and DNA of Ehrlich ascites tumor have been comparatively studied in the search for changes in DNA during tumor progression. No differences were found in kinetics of homologous (mouse X mouse) and heterologous (mouse X tumor) DNA reassociation; in thermal stability of homologous and heterologous duplexes of repeated, unique and satellite DNA; in percentage of hybridization with mouse liver heterogenous nuclear RNA; in thermal stability of the RNA.DNA hybrids. The negative results suggest that the considerable evolution of transplantable tumors (both in biological properties and in karyotype) has not been accompanied by alterations of the genome which could be detected by the now available methods of molecular hybridization for studying DNA divergence. In the light of the results obtained the functions ascribed to the mouse satellite DNA are discussed.

Efficiency of a novel non-Shine-Dalgarno and a Shine-Dalgarno consensus sequence to initiate translation in Escherichia coli of genes with different downstream box composition.

The efficiency of a novel non-Shine-Dalgarno translational initiator (ACCUACUCGAGUUAG, denoted PL) to promote translation in Escherichia coli was compared with that of the Shine-Dalgarno (SD) consensus sequence (AAGGAGGU) using four reporter genes. The obtained results showed that the genes of pokeweed antiviral protein (PAP I) and human calcitonin (CT) were poorly expressed under the conventional SD and were better expressed under the PL sequence. On the contrary, the genes of human interferon gamma (hIFN gamma) and chloramphenicol acetyltransferase (CAT) were highly expressed under SD and poorly expressed under the PL sequence. Computer search revealed a great diversity between the four reporter genes in respect to their complementarity to E. coli 16S rRNA. PAP I and CT genes were rich in nucleotides matching 16S rRNA (called downstream boxes) whereas the complementary domains in the other two (hIFN-gamma and CAT) genes were much shorter. The different behavior of the four reporter genes when placed under the translational control of SD and PL sequences was explained by the different binding energy of their mRNAs to the 30S ribosomal subunit.

Non-enzymatic RNA hydrolysis promoted by the combined catalytic activity of buffers and magnesium ions.

Although Mg2+ is an important cofactor for the specific degradation of RNA by ribozymes, it is not considered as a typical chemical nuclease. In this study we show that in combination with common buffers such as tris(hydroxymethyl)aminomethane and sodium borate, Mg2+ is a powerful catalyst for the degradation of RNA. pH and temperature are found to be the principal factors for the efficient degradation of RNA. Whereas in Tris-HCl/Mg2+ the efficient cleavage starts at pH values higher than 7.5 and temperatures higher than 37 degrees C, in sodium borate RNA degradation begins at pH 7.0 and at 37 degrees C. RNA hydrolysis promoted under the combined catalytic activity of buffer/Mg2+ results in partially degraded RNA and negligible amounts of acid-soluble material. Reaction is insensitive to the concentration of monovalent cations but is completely prevented by chelating agents (EDTA and citrate) at concentrations exceeding that of Mg2+. Borate-magnesium reaction is inhibited also by some polyvalent alcohols (glycerol) and sugars.

[Fractionation of chromatin of liver cell nuclei after mild micrococcal nuclease digestion]. / Fraktsionirovanie khromatina iader kletok pecheni posle ogranichennogo rasshchepleniia mikrokokkovoi nukleazoi.

Mild micrococcal nuclease treatment of rat and mouse nuclei and fractionation were based on the method of Tata and Baker. Three chromatin fractions, S, P1, P2, were separated, and for each of these fractions the sensitivity to the DNase 1 action was determined. The relative content in these fractions of non-transcribed DNA sequences was established by hydridization with a mouse satellite DNA, and the relative content of transcribed DNA sequences--by hydridization with DNA synthesised on the total poly (A) mRNA. None of the fractions displayed the properties characteristic of active chromatin.