Regulatory proteins of vertebrate eye tissues

In our work the new proteins likely belonged to the microenvironment of pigmented epithelium cells and retinal neurons in mammalian eye were studied. We attempted to understand the role of these proteins in the maintenance of normal morphological and functional state of these eye tissues. Earlier for the first time we identified the adhesion molecules with physico-chemical and biological properties much different from other known cell adhesion molecules of bovine eye. Probably, they represent one family of low molecular weigh, highly glicosylated proteins, that express biological activity in extremely low doses--10(-10) mg/ml. The homogeneity of studying proteins is confirmed by HPLC and SDS-electrophoresis in PAAG. It is shown also that these proteins are N-glycosylated, because they contain mannose and N-acetilglucosamine residues. They demonstrate as well a high calcium-binding activity, with Kd corresponded to 10(-4)-10(-6) mg/ml. For a study of the biological effect of these glycoproteins in extremely low doses, a new experimental model was proposed and developed. It was the cultivation in vitro of the posterior part of the eye obtained from the newt Pleurodeles waltl. In short-time culture system it was demonstrated that the studied glycoproteins could stabilize pigment epithelium cell differentiation and cellular interactions in the neural retina in vitro. In addition, glycoproteins, obtained from the pigmented epithelium of bovine eye could decrease the rate of bipolar cell apoptosis in the neural retina. Therefore, the novel adhesion glycoproteins, expressing their biological activity in extremely low doses, pretend to be the regulatory molecules with vivid gomeostatic effects necessary for the delicate adjustment of cell behavior action and function in sensory tissues.     

The activity of xylose reductase and xylitol dehydrogenase in yeasts

The activity and the cofactor specificity of xylose reductase and xylitol dehydrogenase were studied in extracts of yeasts from the genera Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis grown under microaerobic conditions. It was found that xylitol dehydrogenase in all of the yeast species studied is specific for NAD+; xylose reductase in the xylitol-producing species C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, Kl. fragilis, Kl. marxianus, P. guillermondii, and T. molishiama is specific for NADPH; and xylose reductase in the ethanol-producing species P. stipitis, C. shehatae, and Pa. tannophilus is specific for both NADPH and NADH.     

The accumulation and degradation dynamics of cyanophycin in cyanobacteria grown in symbiotic associations with plant tissues and cells

Five different artificial associations of cyanobacterial cells with the cells or tissues of nightshade and rauwolfia were studied. The associations grown on nitrogen-containing media produced heterocysts. Cyanobacterial cells in the associations retained their ability to take up bound nitrogen from the medium, to store it in the form of cyanophycin granules, and to use them in the process of symbiotic growth. The synthesis and degradation of cyanophycin granules in cyanobacterial cells were more active in the associations than in monocultures. In the symbiotic associations of Chlorogloeopsis fritschii ATCC 27193 with Solanum laciniatum cells and of Nostoc muscorum CALU 304 with the Rauwolfia serpentina callus, heterocysts were produced at 3- to 30-fold higher cyanophycin contents than in cyanobacterial monocultures. In contrast, in the association of N. muscorum CALU 304 with the Solanum dulcamara callus, heterocysts were produced at lower cyanophycin contents than in the N. muscorum CALU 304 monoculture. The degradation of cyanophycin granules in N. muscorum CALU 304 cells grown in associations with plant tissues or cells was subjected to mathematical analysis. The activation of cyanophycin degradation and heterocyst production in the associations N. muscorum CALU 304-R. serpentina and C. fritschii-S. laciniatum was accompanied by an enhanced synthesis of the nitrogen-containing alkaloids in plant cells. The data obtained suggest that an integrated system of nitrogen homeostasis can be formed in symbiotic associations. Depending on the growth stage of an association, its plant member can either stimulate the accumulation of bound nitrogen in vegetative cyanobacterial cells in the form of cyanophycin granules, or activate their degradation, or initiate the formation of heterocysts independently of the cyanobacterial sensory-signalling system.     

The absence of direct relationship between the ability of yeasts to grow at elevated temperatures and their survival after lethal heat shock

The study of the growth of the yeasts Rhodotorula rubra, Saccharomyces cerevisiae, and Debaryomyces vanriji at elevated temperatures and their survival after transient lethal heat shock showed that the ability of these yeasts to grow at supraoptimal temperatures (i.e., their thermoresistance) and their ability to tolerate lethal heat shocks (i.e., their thermotolerance) are determined by different mechanisms. The thermotolerance of the yeasts is suggested to be mainly determined by the division rate of cells before their exposure to heat shock.     

Influence of the degree of aeration on halotolerance of yeasts of the genera Candida,Rhodotorula, and Malassezia

The biochemical mechanisms were studied that determine different reactions of yeasts of different genera to two simultaneously imposed stressors, hypoxia and osmotic shock. For Candida lipolytica, these two stressors were antagonistic, which resulted in stimulation of yeast growth by NaCl (in a wide range of concentrations) under microaerobic conditions. The reaction of Malassezia sp. was different: the degree of halotolerance of this microorganism was lower under microaerobic conditions. An intervening reaction pattern was characteristic of Rhodotorula aurantiaca. These differences were found to be determined, above all, by the induction of a salt-resistant respiratory system (oxidase) in Candida lipolytica, which could not be induced in Malassezia sp. In addition, the synthesis of catalase was enhanced in Candida lipolytica, which provided for neutralization of the active forms of oxygen accumulating as a result of inhibition of other protective enzymes by salt.     

Effect of cytochrome oxidase inhibitors on the yeast thermotolerance

The investigation of the effect of the cytochrome oxidase inhibitors sodium cyanide and sodium azide on the thermotolerance of the yeasts Rhodotorula rubra, Debaryomyces vanriji, and Saccharomyces cerevisiae showed that these inhibitors diminish the thermotolerance of R. rubra and D. vanriji, but do not affect the thermotolerance of S. cerevisiae. Taking into account the fact that, unlike the latter yeast, R. rubra and D. vanriji are nonfermentative yeasts, the difference in the effects of the inhibitors on the yeast thermotolerance can be readily explained by the different types of glucose utilization (either oxidative or fermentative) in these yeasts. The data obtained also provide evidence that there is a correlation between the functional activity of mitochondria and the thermotolerance of yeast cells.     

Quantitative isolation of microbial DNA from the different types of soils of natural and agricultural ecosystems

A novel procedure was developed for direct quantitative isolation of microbial DNA from soil. This technique was used to evaluate microbial DNA pools in soils of contrasting types (chernozems and brown forest soils) under different anthropogenic loads. A strong correlation was found between microbial biomass and DNA contents in soils of different types (R2 = 0.799). The ratio of soil CO2 emission rate to the amount of extractable DNA in the soil was shown to reflect physiological state of the soil microbial community; this ratio can be used as an ecophysiological parameter similarly to the metabolic quotient qCO2.     

Construction of the synthetic genes for protein analogs of spider silk carcass spidroin 1 and their expression in tobacco plants

To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f9 coding for spidroin 1 analogs were 3'-fused in-frame with the reporter lichenase gene. The Tr2' weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements. The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks. On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one. Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants. The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.     

Determination of glycyrrhizic acid binding sites by a phage display method

Phages that expose peptides specifically interacting with glycyrrhizic acid (GA) were selected from a phage peptide library by affinity selection and ELISA. Amino acid sequence analysis of the selected peptides and human proteins with the SIM program revealed homology to tyrosine protein kinases, serine/threonine protein kinases, tyrosine phosphatases, and some receptors. Analysis of the peptide and virus protein sequences with the BLAST program showed that GA has affinity for various surface proteins of several human viruses such as HIV-1, hepatitis C virus, and herpesviruses.     

Signaling pathways that control the growth and survival of prostate tumor cells in the absence of androgens

Androgen-dependent human prostate adenocarcinoma cell line LNCaP was used to study the effect of androgen deprivation on the cell response to TNF-related cytokines. Several signaling pathways were implicated in cell survival in the absence of androgens. In androgen-deprived LNCaP cells, TNF-alpha and TRAIL stimulated the cell growth and activated the mitogenic and antiapoptotic signaling pathways involving NF-kappa B, STAT3, PI3K, and beta-catenin. The results suggested a role of cytokines in the survival of prostate adenocarcinoma cells deprived of androgens in vitro.