The role of protein binding with single-stranded DNA (SSB-protein) in DNA replication in Ehrlich ascites carcinoma cells

Possible involvement of the single-strand DNA-binding protein (SSB-protein) in DNA replication in Ehrlich ascite tumour (EAT) cells was studied. There was a direct correlation between the content of SSB-protein in chromatin and the intensity of replicative synthesis of DNA in various preparations of EAT in vitro and in vivo (the computed value of the correlation coefficient was equal to 0.9). It was shown that the addition of exogenous SSB-protein to permeable EAT cells increased the replicative synthesis. It was concluded that although eukaryotic SSB-proteins are not complete analogs of prokaryotic ones, they may participate in DNA replication in eukaryotic cells and, possibly, are intracellular regulators of proliferation.     

Optimized flow cytometry protocol for analysis of surface expression of interleukin-1 receptor types I and II

The biological effects of interleukin (IL)-1 are realized through binding to specific membrane-bound receptors. The efficiency of IL-1 action depends on the number of receptors on the cell. We determined the percentage of cells that express IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII) by flow cytometry using phycoerythrin (PE)-labelled antibodies to the IL-1Rs, and the mean absolute number of membrane-bound IL-1Rs per cell using QuantiBRITE PE calibration beads. We showed that different subpopulations of immunocompetent cells expressed different numbers of molecules of membrane-bound IL-1RI and IL-1RII. We also established that when cells were stimulated with bacterial lipopolysaccharide, there was a significant increase in the number of IL-1RI expressed, and a significant decrease in the mean number of IL-1RII molecules per cell. Determination of the mean number of membrane-bound IL-1R molecules using this protocol enables us to obtain precise and reproducible data that are necessary for full evaluation of expression levels.     

The different effects of carbon dioxide on the toxicity of silver ions for prokaryotic and eukaryotic microorganisms

The effect of carbon dioxide on survivability of bacteria Escherichia coli and the germination ability of conidia of the fungus Neurospora crassa in the presence of silver nitrate was studied. It was shown that carbon dioxide increased the toxic effect of silver ions on prokaryotic cells of E. coli but did not change the survivability of spores of the eukaryote N. Crassa.     

DNA fingerprints of Bombyx mori L. Testing of genotypic variability of parthenogenetic strains.

A comparative analysis of the total cellular DNA in certain parthenogenetic specimens of the silkworm (Bombyx mori), produced from the females of the parthenogenetic strains by two types of parthenogenesis, has been performed through the application of the DNA fingerprinting method based on M13 phage DNA as a hybridization probe. It has been shown that parental specimens and their genetically identical off-springs produced through ameiotic parthenogenesis have identical patterns of hybridization with the hypervariable DNA fragments. The off-springs produced through the meiotic type of parthenogenesis have individual-specific patterns of hybridization, revealing a high level of polymorphism of individual genotypes. The results obtained testify to the effectiveness and reliability of this promising method for identification of genotypic variability, marking and genomic characterization of parthenogenetic clones in the silkworm.     

A study of the complex of proteolytic enzymes and their protein inhibitors in embryogenesis of the silkworm

We studied changes in the activities of serin, thiol, and aspartyl proteinases and their protein inhibitors during embryogenesis of the silkworm. The dynamics of activities of the protein inhibitors and specific proteinases were interrelated, thus providing for coordination and fine regulation of functioning of the proteolytic complex of enzymes during embryogenesis. Possible functions of peptidohydrolases and their protein inhibitors in the silkworm are discussed.     

Aminotransferases in early development of salmonid fish

The activities of alanine and aspartate aminotransferases were assessed in the embryos and juveniles of the rainbow trout Parasalmo mukiss L. and chum salmon Oncorchyncus keta W. Changes in subcellular localization of these enzymes and their activities were found at different pH optima in each subcellular fraction of the rainbow trout during ontogenesis.     

Comparative analysis of prothrombin activators from the venom of Australian elapids

A key component of the venom of many Australian snakes belonging to the elapid family is a toxin that is structurally and functionally similar to that of the mammalian prothrombinase complex. In mammals, this complex is responsible for the cleavage of prothrombin to thrombin and is composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va. The snake prothrombin activators have been classified on the basis of their requirement for cofactors for activity. The two major subgroups described in Australian elapid snakes, groups C and D, are differentiated by their requirement for mammalian coagulation factor Va. In this study, we describe the cloning, characterization, and comparative analysis of the factor X- and factor V-like components of the prothrombin activators from the venom glands of snakes possessing either group C or D prothrombin activators. The overall domain arrangement in these proteins was highly conserved between all elapids and with the corresponding mammalian clotting factors. The deduced protein sequence for the factor X-like protease precursor, identified in elapids containing either group C or D prothrombin activators, demonstrated a remarkable degree of relatedness to each other (80%-97%). The factor V-like component of the prothrombin activator, present only in snakes containing group C complexes, also showed a very high degree of homology (96%-98%). Expression of both the factor X- and factor V-like proteins determined by immunoblotting provided an additional means of separating these two groups at the molecular level. The molecular phylogenetic analysis described here represents a new approach for distinguishing group C and D snake prothrombin activators and correlates well with previous classifications.     

Effect of inhibitors of enzymatic DNA methylation on the formation of reproductive structures and carotenoid production in Neurospora crassa]

The effect of inhibitors of DNA methylation on light-sensitive developmental stages of the filamentous fungus Neurospora crassa was studied. Under conditions of nitrogen starvation, when blue light induced protoperithecia development and inhibited conidia formation, 5-azacytidine (3-300 microM) inhibited protoperithecia formation and stimulated conidia formation (a 700-fold increase after light induction). After treatment of the mycelium with 5-azacytidine, the protoperithecia formation was accompanied by inversely proportional changes in the formation of conidia, both in the dark and after illumination. In the mycelium cultivated on the Vogel's medium, 5-azacytidine (up to 30 microM) and methotrexate (up to 3 microM) stimulated the light-induced carotenoid synthesis by 30%, whereas higher concentrations of these agents were toxic to carotenoid synthesis and growth.