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Low-temperature stress is one of the main factors limiting the distribution and reducing the yield of many subtropical crops, including the tea crop. Efficient breeding to develop frost-tolerant cultivars requires a reliable set of genetic markers for identifying resistance donors, and that is why it is necessary to reveal the specific genetic response in frost-tolerant genotypes in comparison with frost- susceptible ones. In this work, we performed a comparative analysis of the expression of 18 tea genes (ICE1, CBF1, DHN1, DHN2, DHN3, NAC17, NAC26, NAC30, bHLH7, bHLH43, P5CS, WRKY2, LOX1, LOX6, LOX7, SnRK1.1, SnRK1.2, SnRK1.3) under cold and frost conditions in two tea genotypes, tolerant and susceptible. Low-temperature stress was induced by placing the potted plants in cold chambers and lowering the temperature to 0…+2 °С for 7 days (cold stress), followed by a decrease in temperature to –4…–6 °С for 5 days (frost stress). Relative electrical conductivity of leaf was measured in response to the stress treatments, and a significant difference in the frost tolerance of the two tea genotypes was confirmed. Cold exposure did not lead to a change in the electrical conductivity of leaf tissue. On the other hand, frost treatment resulted in increased REC in both genotypes and to a greater extent in the susceptible genotype. Increased expression of all the genes was shown during cold and frost. The genes that were strongly expressed in the tolerant tea genotype were revealed: ICE1, CBF1, DHN2, NAC17, NAC26, bHLH43, WRKY2, P5CS, LOX6, SnRK1.1, SnRK1.3. These genes can be proposed as markers for the selection of frost-tolerance donors in tea germplasm collections. Additionally, it was shown that the tolerant genotype is characterized by an earlier response to stress at the stage of cold acclimation. The study of the expression of the identified genes in different organs of tea plants and in different exposures to low temperature is relevant for further investigations.

Molecular chaperon Hsp60 and protein kinase p70S6K play an important functional role in the regulation of cardiomyocytes vital function or apoptosis. Aim. To study a possibility of in vivo complex formation between Hsp60 and p70S6K in cardiomyocytes. Methods. Co-immunoprecipitation, Western-blot analysis. Results. We have identified in vivo interaction between molecular chaperone Hsp60 and two isoforms of proteinkinase p70S6K in human myocardium, normal and affected by cardiomyopathy. Conclusions. The results obtained suggest a possible participation of molecular chaperon Hsp60 in regulation of p70S6K activity in stressinduced apoptotic signaling pathway in cardiomyocytes. [ABSTRACT FROM AUTHOR]

Protein kinase FGFR1 plays a key role in oncogenic transformation and oncovasculogenesis. Inhibitors of this kinase can be used as anticancer drugs. Protein kinase FGFR1 inhibitors searching were performed among oxindole derivatives. 9 compounds from 920 oxindole derivatives have been chosen using virtual screening. The most active compound was 2-Hydroxybenzoic acid (2-oxo-1,2-dihydroindol-3-ylidene)-hydrazide (??50 1.25 μM). Structure-activity relationship of tested compounds was investigated and optimization strategies were proposed. [ABSTRACT FROM AUTHOR]

Inhibition activity against protein kinase CK2 of thieno[2,3-d]pyrimidinone derivatives was discovered. In vitro testing of 86 compounds allowed finding of 15 compounds with IC50 less than 20 μM. The most active compound is 4-{[5-(4-ethylphenyl)-4-oxothieno[2,3-d]pyrimidine-3-(4H)-yl]methyl} benzoic acid (IC50=2.5 μM). The structure- actvity relationship of thieno[2,3-d]pyrimidinones was investigated and the binding modes of their interaction with protein kinase CK2 were proposed. [ABSTRACT FROM AUTHOR]

Identifying amino acid positions that determine the specific interaction of proteins with small molecule ligands, is required for search of pharmaceutical targets, drug design, and solution of other biotechnology problems. We studied applicability of an original method SPrOS (specificity projection on sequence) developed to recognize functionally significant positions in amino acid sequences. The method allows residues specific to functional subgroups to be determined within the protein family based on their local surroundings in amino acid sequences. The efficiency of the method has been estimated on the protein kinase family. The residues associated with the protein specificity to inhibitors have been predicted. The results have been verified using 3D structures of protein-ligand complexes. Three small molecule inhibitors have been tested. Residues predicted with SPrOS either in contacted the inhibitor or influenced the conformation of the ligand-binding area. Excluding close homologues from the studied set makes it possible to decrease the number of difficult to interpret positions. The expediency of this procedure was determined by the relationship between an inhibitory spectrum and phylogenic partition. Thus, the method efficiency has been confirmed by matching the prediction results with the protein 3D structures.

A basically new system has been developed to screen carcinogens in Drosophilidae, which is based on somatic mutagenesis and recombination. The system may induce tumors in Drosophilidae, which are recorded in adult insects. The test uses recessive mutation in the suppressor gene of growth of warts (wts) tumors. The new system is sensitive to a wide spectrum of mutagenic carcinogens that are naturally encountered. The sensitivity of the system to polycyclic aromatic hydrocarbons and aromatic amides is higher than that of classical tests. Dominant p53 gene mutation that ceases mutagen-induced apoptosis has been shown to increase the incidence of wts tumors by many times. The magnitude of the effect depends on the rate of mutant p53 expression. The increasing effect of p53 mutation extends to both somatic recombination and point mutations and deletions at the wts locus.

We showed that transposon P(GUS.p53.259H), mapped to chromosome 3 and carrying a dominant mutation p53(259)H.GUS, has a positive effect on the frequency of spontaneous and carcinogen-induced tumor mosaic clones warts- in Drosopila melanogaster heterozygotes for the tumor suppressor gene warts located in the same chromosome. The transposon effect could be explained either by the arrest of apoptosis in the cells expressing mutant p53(259)H.GUS gene and containing carcinogen-induced pre-mutations, and/or by genetic instability introduced into chromosome 3 by the P(GUS.p53.259H) transposon itself. The effect of the P(GUS.p53.259H) appeared to be carcinogen-specific. It substantially increased the frequency of tumors induced by supermutagenic platinum complex, oxoplatin, and did not increase the frequencies of tumors induced by polycyclic aromatic hydrocarbons, benzo(alpha)pyrene and pyrene. In the spectrum of mutations induced by all carcinogens tested, somatic recombination events prevailed over somatic mutations. Hence, carcinogen-specificity of the P(GUS.p53.259H) effect cannot be explained by preferential induction of somatic mutations or somatic recombination by one of the carcinogens. Organ-specificity of the increased frequency of mosaic warts- clones induced by P(GUS.p53.259H) was established.

Recently we identified a new class of protein kinases with a novel type of catalytic domain structurally and evolutionarily unrelated to the conventional eukaryotic protein kinases. This new class, which we named alpha-kinases, is represented by eukaryotic elongation factor-2 kinase and the Dictyostelium myosin heavy chain kinases. Here we cloned, sequenced and analyzed the tissue distribution of five new putative mammalian alpha-kinases: melanoma alpha-kinase, kidney alpha-kinase, heart alpha-kinase, skeletal muscle alpha-kinase, and lymphocyte alpha-kinase. All five are large proteins of more than 1000 amino acids with an alpha-kinase catalytic domain located at the very carboxyl-terminus. We expressed the catalytic domain of melanoma alpha-kinase in Escherichia coli, and found that it autophosphorylates on threonine residues, demonstrating that it is a genuine protein kinase. Unexpectedly, we found that the long amino-terminal portions of melanoma and kidney alpha-kinases represent new members of the transient receptor potential (TRP) ion channel family, which are implicated in the mediation of capacitative Ca2+ entry in nonexcitable mammalian cells. This suggests that melanoma and kidney alpha-kinases, which represent a novel type of signaling molecule, are involved in the regulation of Ca2+ influx in mammalian cells.

An earlier investigation of the superoxide-neutralizing activity of blood serum sampled from patients with primary multiple malignancies detected for the first time RF-proteins as well as their tropism to the target organ from which tumor was removed 2-30 years before. A possible explanation may be that a general reaction of the body presented itself. The present study has led to a conclusion that, in cases of polyneoplasia, successful complex treatment of primary malignancy, carried out however for back, might be masked by RF-protein function: the latter "indicates" the presence of foci of malignancy which are not there any longer.

It is shown that the uptake of K+ ions by anaerobically grown E. coli bacteria, which perform glycolysis with the production of H2 in exchange for H+ ions, which are extruded from the cells, occurs with a fixed stoichiometry of N,N'-dicyclohexylcarbodiimide-sensitive ion fluxes (2H+/K+). This process is observed in the precursor strain and arcB mutant, and is destroyed in arcA mutant. The K(+)-uptake by the latter mutant, which proceeds with a moderate affinity (KM 2.0 mM) and is triggered by a positive shock, is sensitive to external osmotonicity. The K(+)-uptake by the arcA mutant is also inhibited by N,N'-dicyclohexylcarbodiimide and has a variable stoichiometry of N,N'-dicyclohexylcarbodiimide-sensitive ion fluxes and does not significantly change in the presence of valinomycin and with varying temperature, whereas the intracellular activity of K+ ions is lower. The Arc-system for the control of synthesis of respiratory enzymes in E. coli participates in the regulation of the K(+)-transporting TrkA system, which directly interacts with F0F1 H-ATPasa; this system in the arcA mutant operates independent of F0F1 and interacts with the latter by mediation of the proton gradient.

Mutants without pseudo-HPr activity intrinsic to the H domain of the FruB protein were obtained in Escherichia coli K12 through insertional mutagenesis by means of the MudIlac phage and TnPhoA transposon. For isolating these mutants, double mutants of enteric bacterium (ptsH fruR of ptsH fruS) were used as original strains. These double mutants were inactive with respect to the total HPr protein of the phosphoenolpyruvate-carbohydrate phosphotransferase system and could not provide constitutive synthesis of fructose-specific proteins of this system. The mutants obtained were designated fruH, mapped, and genetically characterized. Insertions were shown to be located in exactly the portion of the fruB gene that specifies synthesis of the H domain of the FruB protein. The fruH mutations decreased motility of bacterial both in a medium free of carbohydrates and in a medium with glucose, fructose, or maltose. In addition, fruH mutants with the MudIlac genome insertion had sharply decreased activity of beta-galactosidase.

The possibility of substitution of the PHO2 activator protein by GCN4 protein in transcriptional regulation of acid phosphatase genes PHO5 and PHO11 was demonstrated in the yeast Saccharomyces cerevisiae. We observed the increase in acid phosphatase synthesis in the case of gcn4-delta 1 pho2-delta 1 mutant transformed with YCp88 (GCN4-wt) plasmid in comparison with that in nontransformed cells. The mode of repression of acid phosphatase synthesis in two types of transformants gcn4-delta 1 pho2-delta 1 (GCN4-wt) and gcn4-delta 1 pho2-delta 1 (PHO2-wt) was studied. It wat demonstrated that repression of acid phosphatase synthesis in the first type of transformants took place at lower concentration of Pi than was necessary for the second type of transformants. The model of interactions between the GCN4 activator protein and PHO regulatory factors is proposed.

A number of unknown ATP analogues is isolated when studying the structure of the active site of catalytic histonekinase subunit. Adenosine-5'-chloromethanepyrophosphonate adenosine-5'-(beta-bromoethanepyrophosphonate) and adenosine-5'-(p-fluorosulphonylphenylphosphate) were isolated under the reaction of chloromethanephosphonic acid, beta-bromoethanephosphonic acid and n-phenolsulphofluoride respectively with AMP imidazolide. Adenosine-5'-(beta-chloroethylphosphate) was obtained from AMP morpholide and ethylenechorohydrine. Adenosine-5'-chloracetylaminomethanephosphonate and adenosine-5'-(p-fluorosulphonylbenzoylaminomethanephosphonate) were obtained in the reaction of chloroacetyc anhydride and n-fluorosulphonylbenzoylchloride. Adenosine-5'-(p-aminophenylphosphate) is synthesized under the reduction of AMP mononitrophenyl ester. The treatment of the former with chloroacetyc anhydride produced adenosine-5'-(p-chloroacetylaminophenylphosphate. Interaction of ATP analogues obtained and also of early synthesized adenosine-5'-chloromethanephosphonate and adenosine-5'-(beta-bromoethanephosphonate) with homogenous catalytic histonekinase subunit is studied. The decrease in the reaction rate of Hi histone phosphorylation is found to take place. pH optimum of the enzyme inactivation with adenosine-5'-chloromethanepyrophosphonate and adenosine-5'-(beta-chloroethylphosphate) and the protective effect of the substrate (ATP) indicate covalent blocking imidazole ring in the active site. The date obtained suggest that the functional group of the active site of catalytic histonekinase subunit is histidine imidazole ring located close to terminal ATP phosphate.

Interaction of adenosine-3',5'-cyclosulphate (cAMS) cAMP analogue, having sulphur atom instead of phosphorus in a six-term cyclic system with pig brain proteinkinase and rabbit skeletal muscle phosphodiesterase is studied. The affinity of proteinkinase to cAMS was found to be in 25000 times lower than the affinity of cAMP, the affinity of cAMS to the active site of phosphodiesterase being high enough. It is suggested that in the regulatory subunit of proteinkinase positive kationic group participates in nucleotide binding by interacting with negative oxygen atom of six-term cyclophosphate system. There is no such a group in the active site of phospodiesterase, because the absence of negative charge in case of cAMS only slightly affects the constant of cAMS binding by phosphodiesterase.

The circular dichroism spectra of phosphorylated and non-phosphorylated forms of cAMP-dependent protein kinase from pig brain and those of the catalytical and regulatory subunits of the enzyme were studied. The percentage of the secondary structure components of the subunits was calculated. cAMP was shown to cause conformational changes of the enzyme. The conformation of the cyclic nucleotide within the cAMP--regulatory subunit complex was established. It was assumed that the conformation of the cAMP molecule during enzyme activation is subjected to inversion.

The phosphorylation of lysine-rich histones F1, F2a2 and F2b of calf thymus has been investigated using homogeneous histone kinase from pig brain. 32P-labelled phosphopeptides from tryptic digests of corresponding histones were obtained. According to N-terminal analysis and the quantitative determination of amino acid composition of the obtained radioactive peptides the sites of phosphorylation were identified in the primary structure of lysine-rich histones, namely, Ser-38 for the polypeptide chain of histone F1, Ser-19 or 18 for histone F2a2 and Ser-14 and 36 for histone F2b. Thus, the high specificity of brain histone kinase in vitro was demonstrated.

By X-ray analysis a collagen-like structure was observed in the muscle protein connectin . Hydration of species gives a clearer diffraction pattern, which is characteristic of the collagen-like structures. The functional role of connectin should consist in the fixation of maximum length of muscular cells.

The paper deals with the mechanism of the phosphotransferase reaction catalyzed by G-kinase purified from the shrimp tissue by affinity chromatography. The ATP functional groups are stated to be significant for interaction with the enzyme. A mathematical model of the reaction is suggested assuming two active centres in a G-kinase molecule.