[The family 28 carbohydrate-binding module of the thermostable endo-1,4-beta-glucanase CelD Caldicellulosiruptor bescii maximizes the enzyme's activity and binds irreversibly to amorphous cellulose].

The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second one of 749 aa encoding a multimodular endo-1,4-beta-glucanase CelD (85019 Da). N-terminal region of the protein includes the signal peptide and the catalytic module of glycoside hydrolase family 5 (GH5), followed by the substrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic one and CBM28, were produced in E. coli cells and purified to homogeneity. Analysis of the catalytic properties showed CelD to be endo-1,4-beta-glucanase whose maximum activity was exhibited on beta-glucan of barley at pH 6.2 and 70 degrees C. The enzyme was stable at 50 degrees C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 decreased on cellulose substrates, and its thermostability was dropped. Binding of CBM28 to amorphous cellulose was almost irreversible as it could not be removed from this substrate in a range of pH 4-11, temperatures--of 0-75 degrees C, and NaCl concentration--of 0-5 M. Only 100% formamide or 1% SDS were able to remove the protein.

[Thermotoga neopolitina gene cluster, participating in degradation of starch and maltodextrins: expression of aglB and aglA gene in Escherichia coli, properties of recombinant enzymes]. / Klaster genov Thermotoga neapolitana, uchastvuiushchikh v degradatsii krakhmala i mal'todekstrinov: éksptressiia genov aglB i aglA v Escherichia coli, svoistva rekombinantykh fermentov.

The aglB and aglA genes from the starch/maltodextrin utilization gene cluster of Thermotoga neapolitana were subcloned into pQE vectors for expression in Escherichia coli. The recombinant proteins AglB and AglA were purified to homogeneity and characterized. Both enzymes are hyperthermostable, the highest activity was observed at 85 degrees C. AglB is an oligomer of identical 55-kDa subunits capable of aggregation. This protein hydrolyses cyclodextrins and linear maltodextrins to glucose and maltose by liberating glucose from the reducing end of the molecules, and it is a cyclodextrinase with alpha-glucosidase activity. The pseudo-tetrasaccharide acarbose, a potent alpha-amylase and alpha-glucosidase inhibitor, does not inhibit AglB but, on the contrary, acarbose is degraded quantitatively by AglB. Recombinant AglB is activated in the presence of CaCl2, KCl, and EDTA, as well as after heating of the enzyme. AglA is a dimer of two identical 54-kDa subunits, and it hydrolyses the alpha-glycoside bonds of disaccharides and short maltooligosaccharides, acting on the substrate from the non-reducing end of the chain. It is a cofactor-dependent alpha-glucosidase with a wide action range, hydrolysing both oligoglucosides and galactosides with alpha-link. Thereby, the enzyme is not specific with respect to the configuration at the C4 position of its substrate. For the enzyme to be active, the presence of NAD+, DTT, and Mn2+ is required. Enzymes AglB and AglA supplement one another in substrate specificity and ensure complete hydrolysis to glucose for the intermediate products of starch degradation.

[Effect of gamma-plant preparation on production of TNF-alpha, IL-1 beta, and IL-6 in human peripheral blood mononuclear cells in vitro]. / Vliianie preparata gamma-plant na produktsiiu FNO-al'pha, IL-1 beta i IL-6 mononuklearami perifericheskoi krovi cheloveka in vitro.

The effect of a new medicine gamma-plant has antiinflammatory activity on human peripheral blood mononuclear cells obtained from healthy donors was studied. Its ability to influence TNF, IL-1 and IL-6 production via mononuclear cells was determined. Lymphokines spontaneous production and lymphokines productions by cells stimulated by LPS was studied. IL-1 content was defined with IL-1 sensitive cell line D10G4.1 while IL-6 content with IL-6 dependent heterohybridoma D6C8. TNF activity in supernatants was determined as lysis grade of TNF-sensitive cells of mouse fibrosarcoma L-929. It was shown that gamma-plant acted as stimulator when IL-1 production was low and as inhibitor when it was high. Stimulation of IL-6 production induced with LPS was achieved by low gamma-plant doses while for stimulation of spontaneous production higher doses were required. After nonstimulated LPS peripheral blood cells were treated with gamma-plant statistically sensible stimulated TNF production was observed apart from TNF synthesis level in a control group. The TNF production inhibition could never be demonstrated in subjects that showed in control cultures stimulated with LPS a level of TNF production lower than 100 pg/ml. In four of the five high TNF producers the cytokine release inhibition in LPS stimulated cultures has been observed. It is likely that the mild stimulation of the proinflammatory cytokine system has taken place. For example, simultaneously with stimulation of cytokine production the circulating IL-1 receptor antagonist and/or soluble TNF receptor may be induced.

[Gamma-plant as a new nontoxic interferon inducer]. / Gamma-plant kak novyi netoksichnyi induktor interferona.

The interferon-inducing activity of gamma plant was studied in vivo in mice and in vitro in human whole blood cells. The maximum serum interferon (IF) production after intraperitoneal injection of gamma plant was as high as 256 IU/ml. The titers of IF produced in response to gamma plant were 16 to 32 times higher than the level of IF induced by gossipol. The capacity of human whole blood cells to produce IF under the effects of various doses of different inducers (Newcastle disease virus, staphylococcal enterotoxin A, gossipol, and gamma plant) was studied in vitro. In contrast to gossipol and staphylococcal enterotoxin A, gamma plant stimulated the cell production of IF with a high antiviral effect in culture medium. The findings permit a conclusion on a high potency of gamma plant as an IF inducer.

[Antiviral activity of the natural immunostimulant "Vegetan" in murine herpetic meningoencephalitis, guinea pig genital herpes, and canine parvoviral enteritis]. / Antivirusnaia aktivnost' prirodnogo immunostimuliatora "Vegetan" pri gerpeticheskom meningoéntsefalite myshei, genital'nom gerpes morskikh svinok i parvovirusnom énterite sobak.

Antiviral activity of vegetan, a new natural immunostimulator, in herpetic meningoencephalitis of mice, genital herpes of guinea pigs, and parvoviral enteritis of dogs was compared to that of acylguanosine and phosphonoformic acid (PFA). Vegetan incorporated in liposomes or pure was the most active if used for prevention 5 days before intracerebral infection of mice with herpes simplex type 1 virus, whatever the dose of infective agent (10 to 100 LD50). Protective efficacy of vegetan was 57-63%, whereas for ara-A, acylguanosine, and PFA these values were, respectively, 20, 25, and 33%. Use of liposomal vegetan preparation ensured a 80% protective effect. Vegetan had a manifest antiviral effect in guinea pigs with genital herpes and in dogs with parvoviral enteritis.

[Formation of DNA-protein links upon interaction between sodium bisulfite and phage DNP]. / K voprosu ob obrazovanii DNK-belkovykh sshivok pri deístvii bisul'fita natriia na fagovyi DNP.

The conditions for linkage formation in phages Sd and P22 DNP under effects of sodium bisulfite were studied. Th linkage was estimated by the fall in phage infectiosity. After removal of the reagent excess prior to inoculation the modified phage was transferred to various buffers (12 salt systems). The correlation between infectiosity of preparations and changes in their CD spectra in corresponding calt solutions was established.

[Bacteriophage P22 DNA structure in situ]. / Issledovanie struktury DNK in situ Bakteriofaga.

The structure of phage P22 DNA in situ was investigated by optical methods and by chemical modification with sodium bisulfite. On disruption of the phage particles by heating at 45 degrees a drop in absorbance at the 250 nm to 290 nm region was observed. At 260 nm this hypochromism was about 12%. CD spectra of intraphage DNA differed from that of free P22 DNA in the intensity as well as in the position of the positive band (lambda max 280 nm, delta epsilon max=1.3). In the intraphage DNA 21 per cent of cytosines reacted with sodium bisulfite. Cytosyl-amino acid products were found in the HClO4 and HCl hydrolysates of the modified phage. The main amino acid component of the product was identified as lysine. It was shown by means of gradient centrifugation and electron microscopy that the cytosyl-amino acid products result in the crosslinking of DNA to protein in the phage particles.

[Identification of the amino acids participating in interaction with DNA in the makeup of a viral nucleoprotein]. / Ob identifikatsii aminokislot, uchastvuiushchikh vo vzaimodeistvii s DNK v sostave virusnogo nukleoproteida.

A method is described for identification of amino acids participating in covalent DNA-protein cross-links formed in the course of modification of native CD phage by soldium bisulfite. The method is based on estimation of increased content of free amino acids, formed after treatment of the isolated cross-link products using equimolar mixture of O-methyl hydroxylamine and sodium bisulfite. Thin layer chromatography on the plates Fixion 50 x 8 was used for comparative semi-quantitative analysis of amino acids.