Ceramide-induced intracellular oxidant formation, iron signaling, and apoptosis in endothelial cells: protective role of endogenous nitric oxide

Sphingolipid ceramide (N-acetylsphingosine), a bioactive second messenger lipid, was shown to activate reactive oxygen species (ROS), mitochondrial oxidative damage, and apoptosis in neuronal and vascular cells. The proapoptotic effects of tumor necrosis factor-alpha, hypoxia, and chemotherapeutic drugs were attributed to increased ceramide formation. Here we investigated the protective role of nitric oxide (.NO) during hydrogen peroxide (H(2)O(2))-mediated transferrin receptor (TfR)-dependent iron signaling and apoptosis in C(2)-ceramide (C(2)-cer)-treated bovine aortic endothelial cells (BAECs). Addition of C(2)-cer (5-20 microm) to BAECs enhanced .NO generation. However, at higher concentrations of C(2)-cer (> or =20 microm), .NO generation did not increase proportionately. C(2)-cer (20-50 microm) also resulted in H(2)O(2)-mediated dichlorodihydrofluorescein oxidation, reduced glutathione depletion, aconitase inactivation, TfR overexpression, TfR-dependent uptake of (55)Fe, release of cytochrome c from mitochondria into cytosol, caspase-3 activation, and DNA fragmentation. N(w)-Nitro-l-arginine methyl ester (l-NAME), a nonspecific inhibitor of nitricoxide synthases, augmented these effects in BAECs at much lower (i.e. nonapoptotic) concentrations of C(2)-cer. The 26 S proteasomal activity in BAECs was slightly elevated at lower concentrations of C(2)-cer (< or =10 microm) but was greatly suppressed at higher concentrations (>10 microm). Intracellular scavengers of H(2)O(2), cell-permeable iron chelators, anti-TfR receptor antibody, or mitochondria-targeted antioxidant greatly abrogated C(2)-cer- and/or l-NAME-induced oxidative damage, iron signaling, and apoptosis. We conclude that C(2)-cer-induced H(2)O(2) and TfR-dependent iron signaling are responsible for its prooxidant and proapoptotic effects and that .NO exerts an antioxidative and cytoprotective role.     

Comparative FISH mapping of 32 loci reveals new homologous regions between donkey and horse karyotypes

A total of 32 loci comprising specific genes, microsatellites and anonymous BAC clones from horse and cattle were mapped on donkey chromosomes. Of these, 13 markers were also mapped for the first time in the horse. This information, together with that previously available in donkey and horse updates the comparative status of the karyotypes of the two species. The findings of the present study for the first time show correlation between eleven equine acrocentric autosomes and the donkey chromosomes and in part enable detection of rearrangements between them. There are still 7-8 pairs of chromosomes/arms for which no correspondence is known. At least 20 chromosome rearrangements (inversions, fusions and fissions) are already identified that differentiate the two karyotypes. More will be known once complete correspondence is deduced between them. These observations match similar differences observed between human-gibbon and mouse-rat karyotypes that show considerable rearrangements in relation to each other. How donkey and horse karyotypes gathered these differences within a short period of 5-10 Myr since divergence from a common ancestor will be known only after an ancestral equid karyotype is deduced, and the direction of change leading to chromosome rearrangements is clearly understood.     

Glycolipids and other constituents from Desmodium gangeticum with antileishmanial and immunomodulatory activities

Nineteen compounds of various classes, such as flavonoid glycosides, pterocarpanoids, lipids, glycolipids, and alkaloids, were isolated and identified from the Desmodium gangeticum whole plant. Aminoglucosyl glycerolipid (8) is reported here for the first time. Its structure has been elucidated by spectroscopic and degradation studies. This novel compound exhibited in vitro antileishmanial and immunomodulatory activities, as it enhanced nitric oxide (NO) production and provided resistance against infection established in peritoneal macrophages by the protozoan parasite Leishmania donovani. Another known compound, glycosphingolipid (cerebroside) (7) was found to possess significant in vitro antileishmanial and immunomodulatory activities against the same parasite. Other compounds were found to be inactive.     

Diversity of ‘benzenetriol dioxygenase’ involved in p-nitrophenol degradation in soil bacteria

Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featuring in the microbial metabolism of several xenobiotic aromatic compounds. One such RHDO is benzenetriol dioxygenase (BtD) which constitutes the metabolic machinery of microbial degradation of several mono- phenolic and biphenolic compounds including nitrophenols. Assessment of the natural diversity of benzenetriol dioxygenase (btd) gene sequence is of great significance from basic as well as applied study point of view. In the present study we have evaluated the gene sequence variations amongst the partial btd genes that were retrieved from microorganisms enriched for PNP degradation from pesticide contaminated agriculture soils. The gene sequence analysis was also supplemented with an in silico restriction digestion analysis. Furthermore, a phylogenetic analysis based on the deduced amino acid sequence(s) was performed wherein the evolutionary relatedness of BtD enzyme with similar aromatic dioxygenases was determined. The results obtained in this study indicated that this enzyme has probably undergone evolutionary divergence which largely corroborated with the taxonomic ranks of the host microorganisms.     

Gγ1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration

The isoprenoid biosynthetic pathway leading from the production of mevalonate by HMGCoA reductase (Hmgcr) to the geranylation of the G protein subunit, Gγ1, plays an important role in cardiac development in the fly. Hmgcr has also been implicated in the release of the signaling molecule Hedgehog (Hh) from hh expressing cells and in the production of an attractant that directs primordial germ cells to migrate to the somatic gonadal precursor cells (SGPs). The studies reported here indicate that this same hmgcr→Gγ1 pathway provides a novel post-translational mechanism for modulating the range and activity of the Hh signal produced by hh expressing cells. We show that, like hmgcr, gγ1 and quemao (which encodes the enzyme, geranylgeranyl diphosphate synthetase, that produces the substrate for geranylation of Gγ1) are components of the hh signaling pathway and are required for the efficient release of the Hh ligand from hh expressing cells. We also show that the hmgcr→Gγ1 pathway is linked to production of the germ cell attractant by the SGPs through its ability to enhance the potency of the Hh signal. We show that germ cell migration is disrupted by the loss or gain of gγ1 activity, by trans-heterozygous combinations between gγ1 and either hmgcr or hh mutations, and by ectopic expression of dominant negative Gγ1 proteins that cannot be geranylated.     

Development and characterization of microsatellite markers in Indian sesame (Sesamum indicum L.)

The genetic characterization of Indian sesame cultivars and related wild species was analysed using 102 simple sequence repeat (SSR; microsatellite) markers. Of these, 62 were novel sesame-specific microsatellites isolated in the course of the present investigation by constructing genomic libraries. Characterization of the 68 sesame accessions and three related wild species using 72 polymorphic SSR primers resulted in the detection of 170 alleles. The number of alleles ranged from two to four with an average of 2.5 alleles per locus. Polymorphic information content of the markers ranged from 0.43 to 0.88 with an average of 0.66. UPGMA cluster analysis grouped all the accessions into two major clusters with a genetic similarity ranging from 0.40 to 0.91. A moderate to high level of genetic variability was observed. The three wild accessions used in the study formed separate clades and distant genetic relationships were observed between the cultivar lines and wild species. Differentiation of genotypes according to geographical region was not observed. Analysis of molecular variance (AMOVA) analysis revealed that a high percentage of variation was within populations (87.1 %). An overall F _st of 0.11 among the populations indicated low population differentiation. The SSR markers developed will be useful for further genetic analysis, linkage mapping and selection of parents in future breeding programmes.     

Partial Purification and Characterization of Chromate Reductase of a Novel Ochrobactrum sp. Strain Cr-B4

Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (E_a) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (E_d) was found to be 79.6 kJ/mol over a temperature range of 50–80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s^?1, and for deactivation of chromate reductase it was found to be 265.66 × 10³ s^?1. The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN₃, Pb²⁺, Ni²⁺, Zn²⁺, and Cd²⁺) inhibited the enzyme activity, while metals like Cu²⁺ and Fe³⁺ significantly enhanced the reductase activity. The enzyme followed Michaelis–Menten kinetics with K_m of 104.29 µM and a V_max of 4.64 µM/min/mg.     

IL-10- and TGFβ-mediated Th9 Responses in a Human Helminth Infection

Background

Th9 cells are a subset of CD4⁺ T cells that express the protoypical cytokine, IL-9. Th9 cells are known to effect protective immunity in animal models of intestinal helminth infections. However, the role of Th9 cells in human intestinal helminth infections has never been examined.

Methodology

To examine the role of Th9 cells in Strongyloidis stercoralis (Ss), a common intestinal helminth infection, we compared the frequency of Th9 expressing IL-9 either singly (mono-functional) or co-expressing IL-4 or IL-10 (dual-functional) in Ss-infected individuals (INF) to frequencies in uninfected (UN) individuals.

Principal Findings

INF individuals exhibited a significant increase in the spontaneously expressed and/or antigen specific frequencies of both mono- and dual-functional Th9 cells as well as Th2 cells expressing IL-9 compared to UN. The differences in Th9 induction between INF and UN individuals was predominantly antigen-specific as the differences were no longer seen following control antigen or mitogen stimulation. In addition, the increased frequency of Th9 cells in response to parasite antigens was dependent on IL-10 and TGFx since neutralization of either of these cytokines resulted in diminished Th9 frequencies. Finally, following successful treatment of Ss infection, the frequencies of antigen-specific Th9 cells diminished in INF individuals, suggesting a role for the Th9 response in active Ss infection. Moreover, IL-9 levels in whole blood culture supernatants following Ss antigen stimulation were higher in INF compared to UN individuals.

Conclusion

Thus, Ss infection is characterized by an IL-10- and TGFβ dependent expansion of Th9 cells, an expansion found to reversible by anti-helmintic treatment.     

Hoe 140 abolishes the blood pressure lowering effect of taurine in high fructose-fed rats

Summary. High fructose feeding induces moderate increases in blood pressure of normal rats, associated with hyperinsulinemia, insulin resistance and impaired glucose tolerance. Increased vascular resistance, and sodium retention have been proposed to contribute to the blood pressure elevation in this model. Taurine, a sulphur-containing amino acid has been reported to have antihypertensive and antinatriuretic actions. In addition, taurine is shown to increase the excretion of nitrite and kinin availability and hence would be expected to improve the vascular tone. In the present study, the involvement of kinins in the blood pressure lowering effect of taurine was investigated by coadministration of Hoe 140, a kinin B₂ receptor antagonist along with taurine. The effects of taurine on plasma and urinary concentrations of sodium and tissue kallikrein activity were studied in high fructose-fed rats. Fructose-fed rats had elevated blood pressure and decreased levels of sodium in urine. Treatment with 2% taurine in drinking water prevented the blood pressure elevation and coadministration of Hoe 140 abolished this effect of taurine in high fructose-fed rats. The findings confirm the antinatriuretic action of taurine and also suggest a role for the kinins in the mechanism of taurine action in diet-induced hypertension.