Possibility of Application of Extracellular Protease of the Micromycetе <Emphasis Type="Italic">Aspergillus ochraceus</Emphasis> VKM F-4104D for Determination of the Protein C Content in Human Blood Plasma

The protein C activator activity, determined in normal plasma by using A. ochraceus protease is comparable with the activity of a commercial protease analogue from the South American copperhead venom (Protac®). As in the case of Protac®, the A. ochraceus protease can be used for protein C determination in plasma with its reduced content. Comparison of the activator protein C activity of A. ochraceus protease and the commercial analogue showed some excess of the activator activity of the fungal preparation, which may be a promising substitute for the snake activator in diagnostical kits for determining the protein C content in clinical laboratories.     

Preparations for serodiagnosis of diseases due to causative agents of ixode tick-borne Borreliosis (Lyme disease). Communication 2. Comparative study of recombinant enzyme immunoassay test-systems (rELISA) for serological diagnosis of ixode tick-borne Borreliosis]

One foreign and two Russian recombinant enzyme immunoassay test-systems were comparatively investigated under conditions of an encoded experiment. Sensitivity in the experiment with the Russian test-systems Borreliosis-ELISA-IgG and Lyme Best was 63.8 and 68.8% respectively. As for the test-system Borrelia IgG Recombinant, it was 47.5%. All the test-systems were highly specific (94.4 to 99.5%). The test-systems Lyme Best and Borrelia IgG Recombinant revealed partial cross reactions with sera from patients with systemic lupus erythematosus and leptospirosis.     

Adsorption of the antibiotic, nisin, to Streptococcus lactis cells

Nizin is produced by Str. lactis, strain MSU. During biosynthesis it is excreted into the fermentation broth and gradually adsorbed on the organism cells. This was confirmed by experiments with an inactive variant of Str. lactis IIa. The cells of this culture adsorbed nizin from "active" fermentation broth. Adsorption of nizin depended on pH of the medium; at pH 2,3 the cells did not adsorbe the antibiotic and at pH 6.6 the amount of the antibiotic adsorbed by the cells was maximum.     

Class B scavenger receptor types I and II and CD36 mediate bacterial recognition and proinflammatory signaling induced by Escherichia coli, lipopolysaccharide, and cytosolic chaperonin 60

Class B scavenger receptors (SR-B) are lipoprotein receptors that also mediate pathogen recognition, phagocytosis, and clearance as well as pathogen-induced signaling. In this study we report that three members of the SR-B family, namely, CLA-1, CLA-2, and CD36, mediate recognition of bacteria not only through interaction with cell wall LPS but also with cytosolic chaperonin 60. HeLa cells stably transfected with any of these SR-Bs demonstrated markedly (3- to 5-fold) increased binding and endocytosis of Escherichia coli, LPS, and chaperonin 60 (GroEL) as revealed by both FACS analysis and confocal microscopy imaging. Increased pathogen (E. coli, LPS, and GroEL) binding to SR-Bs was also associated with the dose-dependent stimulation of cytokine secretion in the order of CD36 > CLA-2 > CLA-1 in HEK293 cells. Pathogen-induced IL-6-secretion was reduced in macrophages from CD36- and SR-BI/II-null mice by 40-50 and 30-40%, respectively. Intravenous GroEL administration increased plasma IL-6 and CXCL1 levels in mice. The cytokine responses were 40-60% lower in CD36(-/-) relative to wild-type mice, whereas increased cytokine responses were found in SR-BI/II(-/-) mice. While investigating the discrepancy of in vitro versus in vivo data in SR-BI/II deficiency, SR-BI/II(-/-) mice were found to respond to GroEL administration without increases in either plasma corticosterone or aldosterone as normally seen in wild-type mice. SR-BI/II(-/-) mice with mineralocorticoid replacement demonstrated an ～40-50% reduction in CXCL1 and IL-6 responses. These results demonstrate that, by recognizing and mediating inflammatory signaling of both bacterial cell wall LPS and cytosolic GroEL, all three SR-B family members play important roles in innate immunity and host defense.     

Therapeutic siRNAs and nonviral systems for their delivery

Gene-directed therapy with small interfering RNA (siRNA) has a tremendous potential and will undoubtedly occupy one of the leading positions among other therapeutic methods in the future. The lack of efficient and targeted delivery vectors delays a successful implementation of this method in medicine. To develop such systems, one needs a comprehensive insight into the interactions of siRNAs, its delivery systems, and an organism. The review covers the properties of therapeutic siRNAs and nonviral systems for their delivery.     

Evaluation of reasons of the MDR M. tuberculosis strains dissemination by analysis of the rifampicin and/or isoniazid resistant isolates

During the last years in Novosibirsk region of Russia the rate of TB patients infected by MDR strains of M. tuberculosis has been constantly increasing. This increase may occur as a result of the spontaneously mutated mycobacterium selection during treatment of patients or as a result of primary infection by the resistant M. tuberculosis, or also, as a result of both reasons in combination. If the main reason of MDR strain dissemination is selection of resistant bacterium during patient treatment, the equal apportionment of the dominated mutation into the mycobacterium genotypes would be observed. If the main reason is the primary infection by resistant M. tuberculosis, the unequal apportionment would be revealed. For deeper understanding of the main reasons of the fast MDR strains spreading in the region, the distribution of the main mutations over genotypes of strains in Novosibirsk (170 isolates) and Tomsk prison (51 isolates) was investigated. Mutations in rpoB gene associated with the rifampicin resistance and in katG (isoniazid resistance) were detected by biochips. M. tuberculosis genotypings were carried out by IS6110 PCR typing or MIRU typing, in the last method the twelve loci (MIRU 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40) have been used. The most frequent mutation in the rpoB gene was Ser531-->Leu (60-70% of the rifampicin resistant strains) and Ser315-->Thr in gene katG (80% of the isoniazid resistant M. tuberculosis). Both in Novosibirsk and in Tomsk prison the rates of clustered cases transmissions were high (69 and 63% respectively). Analysis of the distribution of the dominated mutations Ser531-->Leu (rpoB) and Ser315-->Thr (katG) revealed that all of them were detected in each clusters, but in Novosibirsk there were only two clusters, in which the percentage of strains, containing mutation Ser531-->Leu (rpoB) were higher (85.7% and 77.7% respectively, P < 0.05), then in others. Among the Tomsk prison's clusters it was revealed one in which the proportion of the Ser3 15-->Thr mutation in katGwas higher (96.4%, P < 0.05). The nonuniform distribution of the dominated mutations highlighted that the epidemic spread of drug-resistant strains of M. tuberculosis in region resulted from the selection of them during patient treatment and the subsequent transmission by TB patients.     

Human RFP2 gene promoter: unique structure and unusual strength

Human gene RFP2 is a candidate tumor suppressor located at 13q14.3 and deleted in multiple tumor types. To explore regulation of RFP2, we determined structure of the 5'-untranslated region of RFP2 gene and its promoter. RFP2 promoter area is TATA-less, highly enriched in G and C nucleotides, and contains multiple quadruplex forming GGGGA-repeats. Deletion analysis of 5'-flanking sequences demonstrated that repeat containing fragment possesses activity seven times exceeding that of the combined SV40 promoter/enhancer. Other unusual features of the RFP2 promoter include anomalously high electrostatic fields induced by sequence-dependent dipoles and very low nucleosome forming potential. A "minimized" version of the RFP2 promoter could be used for overexpression of the various transgenes in the mammalian cells.     

Last‐century changes of alpine grassland water‐use efficiency: a reconstruction through carbon isotope analysis of a time‐series of Capra ibex horns

The ecophysiological response of an alpine grassland to recent climate change and increasing atmospheric CO₂ concentration was investigated with a new strategy to go back in time: using a time‐series of Capra ibex horns as archives of the alpine grasslands' carbon isotope discrimination (¹³Δ). From the collection of the Natural History Museum of Bern, horns of 24 males from the population of the Augstmatthorn–Brienzer Rothorn mountains, Switzerland, were sampled covering the period from 1938 to 2006. Samples were taken from the beginning of each year‐ring of the horns, representing the beginning of the horn growth period, the spring. The horns' carbon ¹³C content (Δ¹³C) declined together with that of atmospheric CO₂ over the 69‐year period, but ¹³Δ increased slightly (+0.4‰), though significantly (P<0.05), over the observation period. Estimated intercellular CO₂ concentration increased (+56 μmol mol^?1) less than the atmospheric CO₂ concentration (+81 μmol mol^?1), so that intrinsic water‐use efficiency increased by 17.8% during the 69‐year period. However, the atmospheric evaporative demand at the site increased by approximately 0.1 kPa between 1955 and 2006, thus counteracting the improvement of intrinsic water‐use efficiency. As a result, instantaneous water‐use efficiency did not change. The observed changes in intrinsic water‐use efficiency were in the same range as those of trees (as reported by others), indicating that leaf‐level control of water‐use efficiency of grassland and forests followed the same principles. This is the first reconstruction of the water‐use efficiency response of a natural grassland ecosystem to last century CO₂ and climatic changes. The results indicate that the alpine grassland community has responded to climate change by improving the physiological control of carbon gain to water loss, following the increases in atmospheric CO₂ and evaporative demand. But, effective leaf‐level water‐use efficiency has remained unchanged.     

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

Background  

Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals.