The registration of nitric oxide production in mammals using a water-soluble complex of N-methyl-D,L-glucamine dithiocarbamate with Fe3+

The level of nitric oxide production in the intact rabbit organism was studied using the water-soluble complex of Fe3+ with MGD as a selective spin trap for nitric oxide. The Fe(3+)-MGD3 complex was injected intravenously. It was shown by the EPR method that this injection resulted in the formation of paramagnetic complexes in the urine, as Cu(2+)-MGD2, and nitric oxide spin adducts: nitric oxide-Fe(2+)-MGD2 and nitric oxide-Fe(3+)-MGD2. The level of nitric oxide production was estimated by the ratio of the total amount of these adducts to the nitric oxide-Fe(2+)-MGD2 level, formed after the addition of excessive S-nitrosoglutathione. This value for intact animals was 1.33 +/- 0.13%.     

Functional sulfurtransferase is associated with mitochondrial complex I from Yarrowia lipolytica, but is not required for assembly of its iron-sulfur clusters

Here, we report that in the obligate aerobic yeast Yarrowia lipolytica, a protein exhibiting rhodanese (thiosulfate:cyanide sulfurtransferase) activity is associated with proton pumping NADH:ubiquinone oxidoreductase (complex I). Complex I is a key enzyme of the mitochondrial respiratory chain that contains eight iron-sulfur clusters. From a rhodanese deletion strain, we purified functional complex I that lacked the additional protein but was fully assembled and displayed no functional defects or changes in EPR signature. In contrast to previous suggestions, this indicated that the sulfurtransferase associated with Y. lipolytica complex I is not required for assembly of its iron-sulfur clusters.     

Modulation of macrophage phenotype by soluble product(s) released from neutrophils

The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-alpha, 168% more IL-6, and 61% less TGF-beta1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-alpha staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-alpha and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-alpha and IL-6 release from LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-alpha release from LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-alpha and superoxide release from murine peritoneal macrophages. The TNF-alpha inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.     

Recognition and separation of single particles with size variation by statistical analysis of their images

Macromolecules may occupy conformations with structural differences that cannot be resolved biochemically. The separation of mixed molecular populations is a pressing problem in single-particle analysis. Until recently, the task of distinguishing small structural variations was intractable, but developments in cryo-electron microscopy hardware and software now make it possible to address this problem. We have developed a general strategy for recognizing and separating structures of variable size from cryo-electron micrographs of single particles. The method uses a combination of statistical analysis and projection matching to multiple models. Identification of size variations by multivariate statistical analysis was used to do an initial separation of the data and generate starting models by angular reconstitution. Refinement was performed using alternate projection matching to models and angular reconstitution of the separated subsets. The approach has been successful at intermediate resolution, taking it within range of resolving secondary structure elements of proteins. Analysis of simulated and real data sets is used to illustrate the problems encountered and possible solutions. The strategy developed was used to resolve the structures of two forms of a small heat shock protein (Hsp26) that vary slightly in diameter and subunit packing.     

Nitrogen availability and TOR regulate the Snf1 protein kinase in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the Snf1 protein kinase of the Snf1/AMP-activated protein kinase (AMPK) family regulates a wide range of responses to stress caused by glucose deprivation. The stress signal is relayed via upregulation of Snf1, which depends on phosphorylation of its activation loop Thr210 residue by upstream kinases. Although Snf1 is also required for coping with various stresses unrelated to glucose deprivation, some evidence suggests a role for low-level basal activity of unphosphorylated Snf1, rather than a specific signaling function. We previously found that Snf1 is required for diploid pseudohyphal differentiation, a developmental response to nitrogen limitation. Here, we present evidence that Snf1 is directly involved in nitrogen signaling. First, genetic analyses suggest that pseudohyphal differentiation depends on the stimulatory phosphorylation of Snf1 at Thr210. Second, immunochemical data indicate that nitrogen limitation improves Thr210 phosphorylation. Analyses of pseudohyphal differentiation in cells with catalytically inactive and hyperactive Snf1 support the role of Snf1 activity. Finally, we show that Snf1 is negatively regulated by the rapamycin-sensitive TOR kinase which plays essential roles in signaling nitrogen and amino acid availability. This and other evidence implicate Snf1 in the integration of signals regarding nitrogen and carbon stress. TOR and Snf1/AMPK are highly conserved in evolution, and their novel functional interaction in yeast suggests similar mechanisms in other eukaryotes.     

Comparative analysis of the flea fauna and its epizootic importance in the deserts of Southern Kazakhstan

Common features and interregional differences in the structure of the flea fauna from Moiynkum (Chu-Talas watershed), Eastern Kyzyl Kum and Betpak Dala are described. The main role of the fleas Xenopsylla gerbilli minax (Moiynkum and Betpak Dala) and X. gerbilli caspica (Eastern Kyzyl Kum) in the transmission of plague microbes is demonstrated. The long-term dynamics of these species population in different desert regions were studied. The main factors responsible for this dynamics are demonstrated.     

Somatic mutagenesis at T-cell receptor locus in inhabitants of radiation polluted regions as a result of the Chernobyl disaster

In the period of 2001-2004, frequency of cells bearing mutations at T-cell receptor (TCR) locus was assessed in 553 inhabitants of radiation polluted regions of the Russian Federation and 154 unexposed control persons. The inhabitants were divided into three groups according to age at the moment of the Chernobyl disaster and 137Cs pollution density: 1) in utero, 37-555 kBq/m2; 2) 0-14 years old, 20-555 kBq/m2; 3) 18 and more years old, highest 137Cs density (185 more than 555 kBq/m2). The most intense changes of the TCR-mutant cell frequency were observed in the group of persons exposed to ionizing radiation in utero. The mean frequency of the mutant cells was higher in the first group than in age-matched control group by about 1.5-fold: 4.0 x 10(-4) vs 2.7 x 10(-4) accordingly (p < 0.0001). Elevation in the mean TCR-mutant cell frequency was less expressed in group of inhabitants aged 0-14 years at the moment of irradiation start: 1.3-fold increase in comparison to age-matched control (3.8 x 10(-4) vs 2.9 x 10(-4), p = 0.0002). It was not found significant differences in mutant cell frequencies between control group and adults consisting in the third group (18 and more years old at the moment of the Chernobyl accident). The changes of the TCR-mutant cell frequency in persons exposed in pre- and postnatal periods differ not only quantitatively, but qualitatively. In the fist case all persons react to irradiation by increasing number of the TCR-mutant cells in some degree. In the second case - only a part of population. Proportion of reacting persons depends on age at the start of irradiation and, perhaps, on dose absorbed. The TCR-mutant frequency was significantly higher in persons with benign tumors of different localizations and nodules in thyroid gland than in persons without this pathology.     

Diatoms of the genus Attheya west, 1860 from the Sea of Japan

This paper documents the species diversity of the diatom genus Attheya from the Sea of Japan. Two species (A. arenicola and A. longicornis) were studied in field samples and in culture. Their morphology is described using light and electron microscopy; the ecology and the distribution data are provided. A. arenicola is a new record for the seas of Russia.