New delta8(14)-ketosterols with an oxygenated side chain

New analogues of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) with modified 17-chains [(22S,23S,24S)- and (22R,23R,24S)-3beta-hydroxy-24-methyl-22,23-oxido-5alpha-cholest-8(14)-en-15-ones and (22RS,23xi,24S)-24-methyl-5alpha-cholesta-3beta,22,23-triol-15-one] were synthesized from (22E,24S)-3beta-acetoxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one. The chiralities of their 22 and 23 centers were determined by NMR spectroscopy. The isomeric 22,23-epoxides effectively inhibited cholesterol biosynthesis in hepatoma Hep G2 cells (IC50 0.9 +/- 0.2 and 0.7 +/- 0.2 microM, respectively), and their activities significantly exceeded those of 15-ketosterol (IC50 4.0 +/- 0.5 microM), (22E,24S)-3beta-hydroxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one (IC50 3.1 +/- 0.4 microM), and the 3beta,22,23-triol synthesized (IC50 6.0 +/- 1.0 microM). The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.     

The in vitro motility assay to study the calcium-mechanical relationship in skeletal and cardiac muscles

In a series of experiments on regulated contractile systems (i.e., in vitro mobile systems with reconstructed thin filaments), the velocities of the movement of a thin filament on the surface covered by either rabbit skeletal or rat cardiac myosin at various concentrations of calcium ions in solution (in the pCa range from 4 to 8) were assessed. The corresponding "pCa-velocity" relationships were plotted, which proved to be of the sigmoid form. It was found that, at a saturating calcium concentration (pCa 4), the velocity of regulated thin filaments was 65% higher than for unregulated ones in the case of skeletal myosin and 87% higher than for unregulated thin filaments in the case of cardiac myosin. It was also found that the Hill coefficient was 1.95 and 2.5 for skeletal and cardiac myosins, respectively. The difference in the Hill coefficients for skeletal and cardiac myosins is discussed in terms of the difference in contribution of cooperativity mechanisms of contractile and regulatory proteins in the regulation of contraction in these types of muscles.     

Genetic identification of rickettsiae of the tick-borne spotted fever group, isolated in the foci of tick-borne rickettsiosis

A total of 25 rickettsial cultures of the tick-borne spotted fever (TBSF) group from the collection of the Research Institute of Infections in Omsk, isolated from different sources in the territory of the Russian Federation (from the Urals to the Far East) during the period of 1954-2001) were studied by the methods of genetic analysis. The fragments of the gene coding the outer-membrane protein of 190 kD (ompA) and synthetase citrate (gltA) of the rickettsiae under the study were sequenced. 23 isolates were identified as R. sibirica, among them 3 isolates obtained from patients, 16 isolates obtained from Dermacentor ticks, 2 isolates from Haemaphysalis concinna and 2--from Ixodes persulcatus. The strain Primorye 32/84, isolated from D. silvarum ticks in the Far East and earlier identified as S. sibirica by the results of the PCR-RFLP analysis proved to be a genovariant Rickettsia spBJ-90, i.e. close to this species. Strain Karpunino 19/69, isolated in the Kurgan region, was identified as R. slovaca. The results obtained extended our notions of the spectrum of rickettsiae group TBSF in Russia as well as their vectors.     

Hydrodynamic parameters of native C-reactive protein molecule in a solution

The hydrodynamic properties of the C-reactive protein in solution (pH 6.8) were studied using quasi-elastic light scattering and size-exclusion liquid chromatography. It was shown that the solution containing the C-reactive protein represents a polydisperse system. The values of the translation diffusion coefficient and the apparent molecular weight of the C-reactive protein in solution at pH 6.8 were determined. The values of the translation diffusion coefficient, molecular weight and the hydration radius obtained suggest that the native pentameric C-reactive protein is the major form of the protein in solution at pH 6.8.     

Induction of endo-1,4-beta-xylanase and beta-galactosidase in the original and recombinant strains of the fungus Penicillium canescens

The induction of the synthesis of secreted enzymes endo-1,4-beta-xylanase (EC 3.2.1.8) and beta-galactosidase (EC 3.2.1.23) in the original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for the induction: the synthesis of beta-galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4-beta-xylanase was observed at 5 to 10 mM. An increase in the number of endo-1,4-beta-xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of beta-galactosidase; the synthesis of endo-1,4-beta-xylanase in the high-copy-number recombinant producing beta-galactosidase was affected to a lesser extent. The amount of the enzymes synthesized did not depend on the saccharide used as a sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.     

Autoimmune disorders in liqvidators 11 years after the Chernobyl accident

We studied long-lasting consequences of the low-doses irradiation on the immune system of 71 clean-up workers who participated in the emergency work after the Chernobyl Plant accident in 1986 and 25 healthy donors from Belarus. In sera of the workers the level of autoantibodies to thyroid gland antigens (thyroglobulin and microsomal fraction of thyroid gland) was increased in 48% of cases, the level of autoantibodies to lens oculis antigen was increased in 44% of cases; the level of circulating immune complexes was elevated in 55%, and the serum level of thyroglobulin in 60% of people. Immunological disorders were found without any definite clinical evidences of diseases and this allows us to consider the examined contingent as a group of risk for the development of autoimmune pathology in the future.     

Estimating of changes in the amyloid and prion content of yeast cells

An attempt was made at estimating the overall amyloid content of yeast cells by treating crude cellular lysates with thioflavin T, the agent specifically staining amyloid fibrils. We demonstrated that overproduction of the yeast chaperone Hsp104p, as well as GuHCI treatment of the [PSI+] cells led both to elimination of the [PSI+] factor and to a stable decrease of the overall amyloid content estimated by intensity of fluorescence (IF) of the thioflavin T. At the same time, overexpression of gene SUP35, coding the protein prionizable to [PSI+], led to generation of [PSI+] clones with higher IF of thioflavin T. Cytoduction in the crosses involving PSI factor leads to considerable enhancement of IF; cytoductants with the nucleus of the recipient [psi-] strain not only got [PSI+] factor from the donor strain but also increased their amyloid content. In these model experiments all treatments modifying one of the yeast prions, [PSI+] factor, led to a predictable shift of IF of thioflavin T that behaved like a cytoplasmic hereditary determinant. The data obtained show that IF of thioflavin T staining gives reliable estimates of cellular amyloid content and that mitotically stable shift of IF after a battery of treatments modifying cellular prion set provides quantitative estimate of the input of prionizable protein molecules to the amyloid pool. The combination of thioflavin staining and prionotropic treatments applied here can be possibly used for future attempts of checking yeast strains for cryptic prions.     

Noradrenaline dynamics in prolonged ischemia after ischemic preconditioning

AIM OF THE STUDY: To determine the effects of two-staged ischemic preconditioning on myocardial noradrenaline in prolonged ischemia and reperfusion. METHODS: Thirty-two male Wistar rats anesthetised with urethane randomly divided into 2 groups: group 1 (ischemic preconditioning group, n = 16), and group 2 (control, n = 16). Myocardial interstitial noradrenaline levels were measured using a microdialysis technique. Ischemic preconditioning was elicited by two episodes: 5 min of ischemia and 10 min of reperfusion. The intermittent occlusions were followed by prolonged occlusion (60 min) and reperfusion (60 min). RESULTS: An increase in interstitial noradrenaline was observed in 10 min of prolonged ischemia in group 2, and in 20 min in group 1. After 20 min of myocardial ischemia there was a significant difference between groups (p < 0.05) in interstitial noradrenaline levels. In control group, it was 60% higher. In reperfusion, noradrenaline levels decreased markedly in group 1. CONCLUSION: We suggest that ischemic preconditioning by two episodes: 5-min ischemia and 10-min reperfusion prevents excessive noradrenaline interstitial accumulation, perhaps, through protection of physiological uptake I carrier.     

Diabetic neuropathies. Metabolism of sorbitol in sciatic nerve tissue in streptozotocin diabetes

The increase of sorbitol and fructose levels caused by aldose reductase activation and sorbitol dehydrogenase inhibition were observed in sciatic nerve of streptozotocin-diabetic rats. Elevated polyol pathway activity has been implicated in the development of diabetic complications such as neuropathy. The regulation of polyol pathway enzymes is based on the changes of redox state of free nicotinamide nucleotides. The decrease of the NADP+/NADPH ratio in cytosolic compartment of sciatic nerve cells activated aldose reductase and the decrease of the NAD+/NADH ratio inhibited sorbitol dehydrogenase. Nicotinamide as a precursor of NAD+ biosynthesis increased the free NADP+/NADPH and NAD+/NADH ratios and inhibited the activity of polyol pathway. The sorbitol level decreased in sciatic nerve of nicotinamide-treated streptozotocin-diabetic rats as compared to non-treated ones. Thus, the data provide evidence for important role of nicotinamide, as an antidiabetic drug, in prevention or correction of diabetic neuropathy.     

Habitat selection and metapopulation structure: a multi-year study of distribution of the Hodgson's pipit, Anthus Hodgsoni Richm. (Aves, Passeriformes)

Numbers and distribution of the Hodgson's pipit (Anthus hodgson Richm.: Aves, Passeriformes) were studied in the Yenissei middle taiga region over an area of about 450 sq km. Distribution of breeding pairs was mapped on fixed study plots (up to 450 ha in total) annually during 15 years. Habitat properties were described and measured in detail on 53 4-ha homogeneous plots within the area. It is shown by means of multiple regression, an average bird abundance over these plots depends on five habitat features (R2 = 0.74) including development and accessibility of moss cover and absence of a potential competitor, the tree pipit (A. trivialis L.). Correlation with these factors ruterated every year, therefore the average bird abundance was used as an index of habitat favourability. Density deviations from the mean in years of high and low numbers appeared to be closely related to habitat favourability; in particular, the relationship can alter the sign depending on the spatial scale of population structures. Changes in abundance reversely related to favourability in adjacent habitats, according to the prediction of despotic distribution hypothesis (Fretwell, Lucas, 1970), strictly indicating dominance behaviour during selection of a breeding territory. The numbers in larger population groups occupying a patchy habitat complex changed synchronously and proportionally to their average habitat quality. This assumes another mechanism governing the distribution of individuals, requiring no local knowledge and no dominance relationships. Dynamics and distribution of individuals among population groups of different hierarchic ranks agree with investigations on establishing of individual site fixation in birds and allow splitting the process into four consecutive steps. 1. During the juvenile dispersal, birds spread around quite evenly, disregarding of habitat quality. This maintains entirety of the metapopulation and occupation of isolated habitat spots. 2. The juvenile dispersal ends with switching to a search for a nearest site containing potential breeding territories. Such a site gets imprinted as the site for next breeding. According to our data, it covers an area of a few square kilometers. 3. During the pre-breeding period, an individual chooses a territory regarding to the favourability, but dominance of old residents forces it to search around for an empty patch within the imprinted area. As a result, dominance relationships affect both breeding density in best habitats and availability of empty patches. 4. The next years, an individual keeps connection with the breeding site fixed due to site tenacity. Rising of the social status allows the individual to occupy a better territory in future. Thus, a population group proportionate to individually imprinted area, with a concentration of favourable patches in the central part, comprises an elementary structural and functional unit of a metapopulation. Its individual members share the same well-known imprinted area and the social structure in common. Its numbers are regulated by density-dependent dominance relationships. Individuals spread over such groups in proportion to their carrying capacity in density-independent manner. Groups with best habitats can be more profitable at population lows, and less densely populated ones can offer more profit at population tops. Despite this, lack of information restricts profitable movements between them. Hence each group offers the same average fitness to its members.