Neutralization of hepatitis B virus surface antigen with immunoglobulin preparations

The in vitro experiments with immunoglobulin and blood plasma containing hepatitis B virus (HBV) markers, revealed that the control of immunoglobulin preparations for the presence of hepatitis B virus surface antigen (HBsAg), mandatory for Russia, was not sufficiently informative. The neutralization of HBsAg with specific antibodies to the level, not determined by the EIA method, reached not less than 24 ng/ ml in 2 hours of incubation and not less than 49 ng/ml in 24 hours of incubation, which, when evaluated in 1 lU of anti-HBs, was 34.6 +/- 0.9 ng and 70.7 +/- 1.8 ng of HBsAg respectively. The process of the formation of immune complexes depended mainly on the time of incubation of experimental samples and on the antibody--antigen proportion in the system. The neutralization of viruses by antibodies had no influence on the capacity of the polymerase chain reaction to detect HBV DNA.     

The store-operated calcium entry pathways in human carcinoma A431 cells: functional properties and activation mechanisms

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca2+ from intracellular Ca2+ stores and activation of Ca2+ influx across the plasma membrane. Two types of Ca2+ channels, highly Ca2+-selective ICRAC and moderately Ca2+-selective ISOC, support store-operated Ca2+ entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated Imin/ICRACL plasma membrane Ca2+ influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca2+ influx pathways in A431 cells. We discovered that (a) ICRAC and ISOC are present in A431 cells; (b) ICRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca2+ store depletion; (c) ISOC currents are moderately selective for divalent cations (PBa/PCs = 14.5) and require at least 300 s for full activation; (d) ICRAC and ISOC currents are activated by PLC-coupled receptor agonists; (e) ISOC currents are supported by Imin/ICRACL channels that display 8.5-10 pS conductance for sodium; (f) ICRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) Imin/ICRACL channels are activated in excised patches by an amino-terminal fragment of InsP3R1 (InsP3R1N); and (h) InsP3 binding to InsP3R1N is necessary for activation of Imin/ICRACL channels. Our findings provide novel information about store-operated Ca2+ influx pathways in A431 cells.     

Synthesis of polymeric neoglycoconjugates based onN-substituted polyacrylamides

Several types of polymeric glycoconjugates,N-substituted polyacrylamides, have been synthesized by the reaction of activated polymers with -aminoalkylglycosides: (i) (carbohydrate-spacer) _n -polyacrylamide, pseudopolysaccharides; (ii) (carbohydrate-spacer) _n -phosphatidylethanolamine _m -polyacrylamide, neoglycolipids, derivatives of phosphatidylethanolamine; (iii) (carbohydrate-spacer) _n -biotin _m -polyacrylamide, biotinylated probes; (iv) (carbohydrate-spacer) _n -polyacrylamide-(macroporous glass), affinity sorbents based on macroporous glass, covalently coated with polyacrylamide. An almost quantitative yield in the conjugation reaction makes it possible to insert in the conjugate a predetermined quantity of the ligand(s).Pseudopolysaccharides proved to be a suitable form of antigen for activation of polystyrene and poly(vinyl chloride) plates (ELISA) and nitrocellulose membranes (dot blot), being advantageous over traditional neoglycoproteins. Polyvalent glycolipids insert well in biological membranes: their physical properties, particularly solubility, can be changed in a desired direction. Biotinylated derivatives were used as probes for detection and analysis of lectins.Abbreviations sp spacer arm - PAA polyacrylamide - PE phosphatidylethanolamine - Biot biotin - MPGlass macroporous glass 200 Å - ELISA enzyme-linked immunosorbent assay - HPLC high-performance liquid chromatography - AIBN azodiisobutyronitrile - BSA bovine serum albumin - DMG 3,6-di-O-methyl-d-glucose - TLC thin-layer chromatography - DMF dimethylformamide - DMSO dimethylsulfoxide - RI refractive index - PBS phosphate buffered saline (0.14m NaCl, 0.01m sodium phosphate, pH 7.3)     

Immobilization of proteases in composite hydrogel based on poly(N-vinyl caprolactam)

Summary A novel one-step method was developed for entrapment of proteolytic enzymes, namely carboxypeptidase B and trypsin, in polymer composite gel based on poly(N-vinyl caprolactam). Native proteases and enzymes previously stabilized by covalent attachment to poly(vinylpyrrolidone-co-acrolein) were immobilized in gels. After immobilization, about 90% and 75% of original trypsin and CPB activities, respectively, were retained. The immobilized enzymes were active within a wide pH range. The optimum temperature of the entrapped enzymes was approximately 25°C higher than that of the soluble enzymes. The entrapped enzymes were successfully used to obtain human insulin from recombinant proinsulin.     

Aconitine modification of cell membrane sodium channels of a neuroblastoma

Currents through normal and aconitine-modified sodium channels in the perfused neuroblastoma cell are measured under voltage clamp conditions. Aconitine shifts the voltage range of activation of the sodium channels towards more negative potentials by about 20 mV, and changes the selectivity, so that channels become more permeable to NH4+ than to Na+ ions. The currents through aconitine--modified channels are inactivated almost completely like those through the normal ones. Aconitine is effective when applied to both sides of the cell membrane. Steady-state characteristics of gating are discussed in terms of the model assuming three main states of the gate machinery: closed, open and inactivated.     

A study of moss (Marchantia polymorpha) thylakoid membrane lipids in monolayers.

Monolayers of seven fractions of natural lipids (phosphatidyl inositol, sulfoquinovosyl diacylglycerol, phosphatidylcholine, digalactosyl diacylglycerol, phosphatidyl glycerol, phosphatidylethanolamine, monogalactosyl diacylglycerol), isolated from the photoautotrophic cell culture of the moss Marchantia polymorpha grown under normal and light-stress conditions, have been prepared for the first time. We have shown that the high-intensity light affects the area occupied by the lipid molecule. In the case of digalactosyl diacylglycerol and phosphatidyl glycerol fractions, after the light stress the area significantly increased from 0.50 to 0.80 nm2 and from 0.47 to 0.63 nm2, respectively, and in the case of the sulfoquinovosyl diacylglycerol fraction, the area decreased from 0.40 to 0.32 nm2. These results are in agreement with our previous data on the redistribution of the double bonds in the aliphatic chains of these lipids and can be used to characterize the state of the lipid bilayer of the thylakoid membranes.     

Studies of Electron Transport Dynamics in Photosynthetic Reaction Centers Using Fast Temperature Changes

Rates of thermoinduced conformational transitions of reaction center (RC) complexes providing effective electron transport were studied in chromatophores and isolated RC preparations of various photosynthesizing purple bacteria using methods of fast freezing and laser-induced temperature jump. Reactions of electron transfer from the primary to secondary quinone acceptors and from the multiheme cytochrome c subunit to photoactive bacteriochlorophyll dimer were used as probes of electron transport efficiency. The thermoinduced transition of the acceptor complex to the conformational state facilitating electron transfer to the secondary quinone acceptor was studied. It was shown that neither the characteristic time of the thermoinduced transition within the temperature range 233-253 K nor the characteristic time of spontaneous decay of this state at 253 K exceeded several tens of milliseconds. In contrast to the quinone complex, the thermoinduced transition of the macromolecular RC complex to the state providing effective electron transport from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer within the temperature range 220-280 K accounts for tens of seconds. This transition is thought to be mediated by large-scale conformational dynamics of the macromolecular RC complex.     

Role of polypeptide growth factors in the regulation of epidermal keratinocyte proliferation

The growth factors, cytokines, adhesive molecules and extracellular matrix components play the leading role in the processes of intercellular interactions. Literary data on structure and mechanisms of functioning of the growth factors and their receptors are summarised in the present review. Some aspects of regulatory functions of such growth factors as EGF, TGFalpha, TGFbeta, FGF, KGF, AR, and HGF in these processes in epidermis keratinocytes both in vivo and in vitro as example were also considered.     

The OF characteristics of streptococci group A and immunity to streptococcal infection based on the results of OF and anti-OF tests

The influence of the OF activity of group A streptococci on their specific pathogenic properties has been shown, which is manifested by increased virulence of these streptococci for children of younger age groups and by a two times higher isolation rate of OF+ strains in tonsillitis than in scarlet fever. The possibility of the indirect evaluation of the content of anti-M-antibodies by the results of the anti-OF test has been revealed, which permits using this test instead of the bactericidal test, more complicated, in the study of immunity to infection induced by group A OF+ streptococci. Among the main methods of laboratory support of epidemiological surveillance on streptococcal infection, the introduction of the highly discriminating OF typing and the anti-OF test into practical use is recommended.     

Modern Approaches to Differentiation of Live and Dead Bacteria Using Selective Amplification of Nucleic Acids

Microbiology - Specific amplification of nucleic acids is a convenient and quick alternative to the culture-based method of detecting bacterial cells. However, conventional PCR and other...