Peptide Modulators of ASIC Channels of the Sea Anemone Urticina aff. coriacea (Cuvier, 1798) from the Sea of Okhotsk

Russian Journal of Marine Biology - A search for biologically active peptides has been performed in an aqueous extract of the sea anemone Urticina aff. coriacea (Cuvier, 1798) (Actiniidae) from the...     

In silico screening for tumour-specific expressed sequences in human genome.

A computer-based differential display tool named HsAnalyst has been developed and successfully used for the comparison of expression patterns in a set of tumours versus a set of normal tissues. A list of EST clusters highly represented in tumours and rarely observed in normal tissues has been developed as a resulting output file of the program. These differentially expressed EST clusters (genes) can be useful for developing new tumour markers and prognostic indicators for a wide set of human malignancies. Tumour-specific protein-coding genes may be considered a manifestation of tumour-specific gene expression.     

Construction of peptide mimetics of an epitope of the human immunodeficiency virus (HIV-1) gp41 protein, recognized by virus-neutralizing antibodies 2F5

A phase peptide library was screened with virus-neutralizing monoclonal antibodies (MCA) 2F5 which recognize a conserved epitope of HIV-1 gp41. Phages that expose peptides specifically binding with MCA 2F5 were selected by ELISA. Amino acid sequence analysis revealed a homology to region 662-671 of HIV-1 HB10 gp160 for most peptides. The major role in recognition was ascribed to Asp-664, Lys-665, and Trp-666. The epitope-mimicking peptides were tested for immunogenicity. Antibodies to gp41 were detected in serum of immunized rabbits.     

Application of cellulose-based self-assembled tri-enzyme system in a pseudo-reagent-less biosensor for biogenic catecholamine detection

Amorphous cellulose was used as a specific carrier for the deposition of self-assembled multienzyme complexes capable of catalyzing coupled reactions. Naturally glycosylated fungal cellobiohydrolases (CBHs) of glycosyl hydrolase families 6 and 7 were specifically deposited onto the cellulose surface through their family I cellulose-binding modules (CBM). Naturally glycosylated fungal laccase was then deposited onto the preformed glycoprotein layer pretreated by ConA, through the interaction of mannosyl moieties of fungal glycoproteins with the multivalent lectin. The formation of a cellulase-ConA-laccase composite was proven by direct and indirect determination of activity of immobilized laccase. In the absence of cellulases and ConA, no laccase deposition onto the cellulose surface was observed. Finally, basidiomycetous cellobiose dehydrogenase (CDH) was deposited onto the cellulose surface through the specific interaction of its FAD domain with cellulose. The obtained paste was applied onto the surface of a Clark-type oxygen electrode and covered with a dialysis membrane. In the presence of traces of catechol or dopamine as mediators, the obtained immobilized multienzyme composite was capable of the coupled oxidation of cellulose by dissolved oxygen, thus providing the basis for a sensitive assay of the mediator. Swollen amorphous cellulose plays three different roles in the obtained biosensor as: (i) a gelforming matrix that captures the analyte and its oxidized intermediate, (ii) a specific carrier for protein self-assembly, and (iii) a source of excess substrate for a pseudo-reagent-less assay with signal amplification. The detection limit of such a tri-enzyme biosensor is 50-100 nM dopamine.     

Etiology of purulent septic diseases and the antibiotic resistance of the isolated causative agents

Among the causative agents of purulent septic diseases in the surgical hospital, 25 microbial species were isolated; of these, the prevailing species were Staphylococcus aureus (19.86 +/- 1.07%), Escherichia coli (16.5 +/- 0.99%) and Pseudomonas aeruginosa (10.06 +/- 0.8%). From environmental objects in the hospital 14 microbial species were isolated, among them bacteria of the genus Enterobacter (27 +/- 1.7%), E. coli (19.07 +/- 1.48%), S. aureus (14.7 +/- 1.31%), Klebsiella pneumoniae (13.73 +/- 1.31%), P. aeruginosa (7.33 +/- 0.98%). During 3 years of observation the isolation rate of K. pneumoniae from different environmental objects was found to increase threefold to 24.7 +/- 2.7%. The results of the study of the microbial picture in surgical hospitals, as well as the antibiotic resistance of circulating causative agents, should be borne in mind while taking epidemic control measures.     

The Mechanism of Inhibitory Effect of Polyanions and Polycations on the Activation of the Complement First Component

Polyethyleneimine (PEI, 50 kDa) and polymethacrylic acid (PMA, 200 kDa) were shown to inhibit the lysis of sheep erythrocytes induced by the guinea pig complement. They twofold suppress the hemolysis at the concentrations of 0.47 and 0.89 g/ml, respectively. The inhibitory effect on the binding of the C1q subunit of human complement to the sensitized sheep erythrocytes (EA) was found to depend on the component of the reaction with which the inhibitors were preliminarily incubated. When an inhibitor, C1q, and EA were simultaneously incubated, the inhibition constants for PEI and PMA were 17 ± 6 and 8.1 ± 0.1 g/ml, respectively. The preincubation of EA with PEI and the subsequent washing out of the inhibitor resulted in the inhibition constant of 22 ± 3 g/ml. No inhibitory effect was observed after a similar preincubation of EA with PMA. No inhibition was also detected when the inhibitors were added after the formation of the C1q complex with antibodies. These observations suggest that the binding of antibodies to cationic PEI prevents the C1q–antibody complex formation, while the binding of anionic PMA to the active site of C1q impedes the interaction of this subunit with immunoglobulins. Moreover, within the range of concentrations studied, the studied inhibitors did not affect the subsequent C1q binding to the C1r and C1s enzymes.     

Expression of ergotope-associated markers of T lymphocytes in atopic dermatitis after in vitro polyclonal activation

The antiergotypic response leads to the formation of effector T cells able to eliminate activated lymphocytes independently of their antigenic specificity, since the targets of these cells are molecules produced during cell activation (ergotopes). In this paper, we describe the level of expression of the ergotope-associated markers CD25, HSP60, and HLA-DR by the T lymphocytes isolated from the blood of atopic dermatitis patients immediately after isolation and after cultivation. After 10-day cultivation in the presence of anti-CD3 antibodies and IL-2, the expression levels of early and late activation markers in T cells have changed: the shares of CD25-positive CD4⁺ and CD8⁺ lymphocytes increase to 68 and 47%, respectively, and the share of HLA-DR-positive cells increases to 26 and 33%. The density of HLA-DR molecules on the surface of activated T cells increases more than fivefold. Almost all T cells before and after cultivation express 60 kDa heatshock protein (HSP60); however, the CD4⁺ cells activated in vitro contain more HSP60 molecules than do the in vitro-activated CD8⁺ cells and the CD4⁺ cells of peripheral blood. Thus, the T cells of atopic-dermatitis patients have the status of activated cells because they express sufficient amounts of early and late activation markers; presumably, they can enhance the induction of antiergotypic response when administered to patients. Taking into account that antiergotypic regulation acts on activated T cells independently of their antigenic specificity, immunotherapy utilizing autologous activated T lymphocytes can be of interest as a method for targeted action on pathogenetic components of atopic dermatitis.     

Tightly bound adenosine diphosphate, which inhibits the activity of mitochondrial F1-ATPase, is located at the catalytic site of the enzyme

The binding of one ADP molecule at the catalytic site of the nucleotide depleted F1-ATPase results in a decrease in the initial rate of ATP hydrolysis. The addition of an equimolar amount of ATP to the nucleotide depleted F1-ATPase leads to the same effect, but, in this case, inhibition is time dependent. The half-time of this process is about 30 s, and the inhibition is correlated with Pi dissociation from the F1-ATPase catalytic site (uni-site catalysis). The F1-ATPase-ADP complex formed under uni-site catalysis conditions can be reactivated in two ways: (i) slow ATP-dependent ADP release from the catalytic site (tau 1/2 20 s) or (ii) binding of Pi in addition to MgADP and the formation of the triple F1-ATPase-MgADP-Pi complex. GTP and GDP are also capable of binding to the catalytic site, however, without changes in the kinetic properties of the F1-ATPase. It is proposed that ATP-dependent dissociation of the F1-ATPase-GDP complex occurs more rapidly, than that of the F1-ATPase-ADP complex.     

Mitochondria produce reactive nitrogen species via an arginine-independent pathway

We measured the contribution of mitochondrial nitric oxide synthase (mtNOS) and respiratory chain enzymes to reactive nitrogen species (RNS) production. Diaminofluorescein (DAF) was applied for the assessment of RNS production in isolated mouse brain, heart and liver mitochondria and also in a cultured neuroblastoma cell line by confocal microscopy and flow cytometry. Mitochondria produced RNS, which was inhibited by catalysts of peroxynitrite decomposition but not by nitric oxide (NO) synthase inhibitors. Disrupting the organelles or withdrawing respiratory substrates markedly reduced RNS production. Inhibition of complex I abolished the DAF signal, which was restored by complex II substrates. Inhibition of the respiratory complexes downstream from the ubiquinone/ubiquinol cycle or dissipating the proton gradient had no effect on DAF fluorescence. We conclude that mitochondria from brain, heart and liver are capable of significant RNS production via the respiratory chain rather than through an arginine-dependent mtNOS.     

ATP activates ataxia-telangiectasia mutated (ATM) in vitro. Importance of autophosphorylation

Ataxia-telangiectasia Mutated (ATM), mutated in the human disorder ataxia-telangiectasia, is rapidly activated by DNA double strand breaks. The mechanism of activation remains unresolved, and it is uncertain whether autophosphorylation contributes to activation. We describe an in vitro immunoprecipitation system demonstrating activation of ATM kinase from unirradiated extracts by preincubation with ATP. Activation is both time- and ATP concentration-dependent, other nucleotides fail to activate ATM, and DNA is not required. ATP activation is specific for ATM since it is not observed with kinase-dead ATM, it requires Mn2+, and it is inhibited by wortmannin. Exposure of activated ATM to phosphatase abrogates activity, and repeat cycles of ATP and phosphatase treatment reveal a requirement for autophosphorylation in the activation process. Phosphopeptide mapping revealed similarities between the patterns of autophosphorylation for irradiated and ATP-treated ATM. Caffeine inhibited ATM kinase activity for substrates but did not interfere with ATM autophosphorylation. ATP failed to activate either A-T and rad3-related protein (ATR) or DNA-dependent protein kinase under these conditions, supporting the specificity for ATM. These data demonstrate that ATP can specifically induce activation of ATM by a mechanism involving autophosphorylation. The relationship of this activation to DNA damage activation remains unclear but represents a useful model for understanding in vivo activation.