Competitive binding of various beta-lactam compounds to penicillin-binding proteins of Escherichia coli

Competing interaction of two novel N-acyl derivatives of ampicillin i.e. N'-benzylchlorbenzimidazole (No. 48) and N-pyrazolytiazole (No. 72) derivatives and 14C-benzylpenicillin with penicillin-binding proteins (PBP) of E. coli was studied. It was shown that ampicillin and its derivative No. 48 markedly differed in their affinity to various PBPs. Derivative No. 72 did not prevent binding of the labeled benzylpenicillin to any PBP which corresponded to its low antimicrobial activity. Analogous experiments with new cephalosporin structures i.e. active and inactive N-acyl derivatives of cephalosporin showed that the active derivative No. 94 i.e. N-methyltiobenzimidazole derivative had the highest affinity to PBP-2 and PBP-5. The inactive derivative No. 68 i.e. N-chlorbenzimidazole derivative also had high affinity to PBP-1b, PBP-2 and PBP-3 essential for the cell. No activity of the latter compound against intact cells of E. coli was probably due to its low penetration through the outer membrane of the bacterial cell. Estimation of affinity of the beta-lactam structures to various PBPs not only provided data on the mechanism of their action but also made it possible to explain in some cases the peculiarities of their antimicrobial spectrum.     

Molecular evolution of the tick-borne encephalitis and Powassan viruses

Issues associated with newly emerging viruses, their genetic diversity, and viral evolution in modern environments are currently attracting growing attention. In this study, a phylogenetic analysis was performed and the evolution rate was evaluated for such pathogenic flaviviruses endemic to Russia as tick-borne encephalitis virus (TBEV) and Powassan virus (PV). The analysis involved 47 nucleotide sequences of the TBEV genome region encoding protein E and 17 sequences of the PV NS5-encoding region. The nucleotide substitution rate was estimated as 1.4 × 10⁻⁴ and 5.4 × 10⁻⁵ substitutions per site per year for the E protein-encoding region of the TBEV genome and for the NS5 genome region of PV, respectively. The ratio of non-synonymous to synonymous nucleotide substitutions (dN/dS) in viral sequences was calculated as 0.049 for TBEV and 0.098 for PV. The highest dN/dS values of 0.201–0.220 were found in the subcluster of Russian and Canadian PV strains, and the lowest value of 0.024 was observed in the cluster of Russian and Chinese strains of the Far Eastern TBEV genotype. Evaluation of time intervals between the events of viral evolution showed that the European subtype of TBEV diverged from the common TBEV ancestor approximately 2750 years ago, while the Siberian and Far Eastern subtypes emerged approximately 2250 years ago. The PV was introduced into its natural foci of the Russian Primorskii krai only approximately 70 years ago; these strains were very close to Canadian PV strains. The pattern of PV evolution in North America was similar to the evolution of the Siberian and Far Eastern TBEV subtypes in Asia. The moments of divergence between major genetic groups of TBEV and PV coincide with historical periods of climate warming and cooling, suggesting that climate change was a key factor in the evolution of flaviviruses in past millennia.     

Possible use of a concentrated vaccine against tick-borne encephalitis for immunizing donors to obtain specific immunoglobulin

The immunization of donors with dried, purified, inactivated and concentrated cell-culture tick-borne encephalitis vaccine, produced at the Institute of Poliomyelitis and Viral Encephalitides in Moscow, has been carried out with the aim of obtaining immune raw material for the production of specific donor immunoglobulin. The high immunogenic potency and low reactogenicity of the vaccine, introduced in 2 injections at an interval of 6 months, have been confirmed. Immunization in 2 injections has proved to be as effective as that achieved by 3 injections. The expediency of the immunization of regularly bled donors has been established: hemagglutinin titers in these donors have been found to be significantly higher than in a group of persons never bled before. The optimum time for taking blood has been determined: 3 weeks for regularly bled donors and 2 weeks for donors bled for the first time. Blood may be taken again 2 months after the first bleeding without booster immunization and 2 weeks after the third injection of the vaccine.     

Ribosomal dysentery vaccine. I. Method of production, physical and chemical properties]

Studies of A. and G. Youmans on the experimental tuberculosis led to discovery of a fundamentally new type of vaccines (ribosomal vaccines) which proved to be highly effective in the prophylaxis of many experimental infections. Therefore it seems reasonable to prepare analogous vaccine from Shigellae for the study of its efficiency in experimental shigellosis. Ribosomal preparations from Shigella sonnei were prepared by sonic disruption of microbial cells followed by differential ultracentrifugation according to A. and G. Youmans' method with slight modifications. The yeild of ribosomal fraction was about 2 per cent by weight; all the series had an UV adsorption maximum at 260 nm, the ratio OD260:OD280 being approximately 2. They contained about 55% of RNA, 35% of protein and no more than 8% of saccharides. As shown by centrifugation in sucrose gradient and by analytical ultracentrifugation the preparations were homogeneous. The presence of undissociated ribosomes was confirmed by electron microscopy. Thus, the ribosomal preparations obtained proved to be sufficiently purified for carrying out experimental investigations of their biological activity.     

Isolation of flagellin antigens of salmonellae by the method of ultracentrifugation and their use for preparation of erythrocytic H-diagnostic agents]

Flagellar preparations were obtained from 6 salmonellae strains by differential ultracentrifugation; they were characterized by morphological, immunochemical, and serological tests. The results of investigations showed that the preparations possessed high serological activity; somatic antigen admixture was insignificant. Flagellins extracted from the flagellae were used to prepare erythrocytic H-diagnostic agents. The results of their use in the examination of sera of healthy persons, and of those suffering from typhoid fever and salmonellosis indicated the expediency of using passive H-hemagglutination for diagnostic purposes.     

Metabolism of 3-hydroxybenzoate and gentisate by strain <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> 1CP

The ability of strain Rhodococcus opacus 1CP to utilize 3-hydroxybenzoate (3-HBA) and gentisate in concentrations up to 600 and 700 mg/L, respectively, as sole carbon and energy sources in liquid mineral media was demonstrated. Using high-performance liquid chromatography (HPLC) and thin-layer chromatography, 2,5-dihydroxybenzoate (gentisate) was identified as the key intermediate of 3-hydroxybenzoate transformation. In the cell-free extracts of the strain grown on 3-HBA or gentisate, the activities of 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase, and maleylpyruvate isomerase were detected. During growth on 3-HBA, low activity of catechol 1,2-dioxygenase was detected. Based on the data obtained, the pathway of 3-HBA metabolism by strain R. opacus 1CP was proposed.     

Reduction in number of sarcolemmal KATP channels slows cardiac action potential duration shortening under hypoxia

The cardiovascular system operates under demands ranging from conditions of rest to extreme stress. One mechanism of cardiac stress tolerance is action potential duration shortening driven by ATP-sensitive potassium (K_ATP) channels. K_ATP channel expression has a significant physiologic impact on action potential duration shortening and myocardial energy consumption in response to physiologic heart rate acceleration. However, the effect of reduced channel expression on action potential duration shortening in response to severe metabolic stress is yet to be established. Here, transgenic mice with myocardium-specific expression of a dominant negative K_ATP channel subunit were compared with littermate controls. Evaluation of K_ATP channel whole cell current and channel number/patch was assessed by patch clamp in isolated ventricular cardiomyocytes. Monophasic action potentials were monitored in retrogradely perfused, isolated hearts during the transition to hypoxic perfusate. An 80–85% reduction in cardiac K_ATP channel current density results in a similar magnitude, but significantly slower rate, of shortening of the ventricular action potential duration in response to severe hypoxia, despite no significant difference in coronary flow. Therefore, the number of functional cardiac sarcolemmal K_ATP channels is a critical determinant of the rate of adaptation of myocardial membrane excitability, with implications for optimization of cardiac energy consumption and consequent cardioprotection under conditions of severe metabolic stress.     

Comparative characteristics of penicillin-binding proteins of representatives of the genus Streptomyces

Penicillin-binding proteins (PBPs) in Strepomyces strains producing clavulanic acid and beta-lactamase and in Streptomyces strains not producing these compounds were studied comparatively. In S. clavuligerus, the organism producing clavulanic acid, there were detected 3 PBPs in the membrane fraction. S. griseus, the organism producing beta-lactamase, contained 6 PBP. In S. cacaoi and S. olivaceus, organisms producing neither beta-lactams nor beta-lactamase, there were detected 5 and 4 PBP, respectively. The set of the PBP in the organism producing clavulanic acid varied during fermentation. In a variant of S. clavuligerus isolated after protoplasting of the mycelial cells and their regeneration the content of the electrophoretically most mobile PBP lowered. The PBP of S. clavuligerus did no show any high affinity to other beta-lactams such as methicillin and ampicillin tested as competing agents of 14C-benzylpenicillin.     

Primary pathological changes in cells infected with tick-borne encephalitis virus]

Synthesis and the content of RNA and protein in cells of the pig embryo kidneys infected with tick--borne encephalitis virus were studied by means of autoradiography and cytophotometry. Acceleration of synthesis and accumulation of RNA (in cellular structures) were estimated as over 125--200% of the initial level followed by a decrease in 12 hr when the virus release from the cells occurred. By the 2nd and 6--8th hr of infection (in the period of synthesis of early and late virus material), two peaks of protein contents over 123--131% were observed. The RNA and protein shifts are well correlated with the virus reproduction stages.