Synthesis and biological activities of glycosphingolipid analogues from marine sponge Aplysinella rhax

A novel glycosphingolipid, beta-D-GalNAcp(1-->4)[alpha-D- Fucp(1-->3)]-beta-D-GlcNAcp(1-->)Cer (1), isolated from the marine sponge Aplysinella rhax, has a unique structure, with D-fucose and N-acetyl-D-galactosamine attached to a reducing-end N-acetyl-D-glucosamine through an alpha1-->3 and beta1-->4 linkage, respectively. We synthesized glycolipid analogues carrying a 2-branched fatty alkyl residue or a 2-trimethylsilyl ethyl residue in place of ceramide (2 and 3), non-natural type trisaccharide analogue containing an L-fucose residue (4), and other analogues (5 and 6). Among these prepared compounds, 2 showed the most potent nitric oxide (NO) production inhibitory activity against LPS-activated J774.1 cells. In addition, their structure-activity relationships were established.     

Purification and characterization of rhodobactin: a mixed ligand siderophore from Rhodococcus rhodochrous strain OFS

The siderophore produced by Rhodococcus rhodochrous strain OFS, rhodobactin, was isolated from iron-deficient cultures and purified by a combination of XAD-7 absorptive/partition resin column and semi-preparative HPLC. The siderophore structure was characterized using 1D and 2D ¹H, ¹³C and ¹⁵N NMR techniques (DQFCOSY, TOCSY, NOESY, HSQC and LR-HSQC) and was confirmed using ESI-MS and MS/MS experiments. The structural characterization revealed that the siderophore, rhodobactin, is a mixed ligand hexadentate siderophore with two catecholate and one hydroxamate moieties for iron chelation. We further investigated the effects of Fe concentrations on siderophore production and found that Fe limiting conditions (Fe concentrations from 0.1 μM to 2.0 μM) facilitated siderophore excretion. Our interests lie in the role that siderophores may have in binding metals at mixed contamination sites (containing metals/radionuclides and organics). Given the broad metabolic capacity of this microbe and its Fe scavenging ability, R. rhodochrous OFS may have a competitive advantage over other organisms employed in bioremediation. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

2,5-Dimethyl-4-hydroxy-3(2H)-furanone as an Anti-biofilm Agent Against Non-Candida albicans Candida Species

Mycopathologia - The predominance of non-Candida albicans Candida (NCAC) species causing healthcare-associated infections has increased over the last decade pertaining to their ability to form...     

Seasonal variation in HSP70 expression and oxidative stress in skin of zebu (Tharparkar) and crossbred (Karan Fries) cattle under tropical climate

The study aimed to evaluate inducible HSP70 (HSP70.1 and HSP70.2) gene expression and oxidative stress status in skin of cattle during different seasons. Ten each of Tharparkar (zebu) and Karan Fries (crossbred) heifers were selected from NDRI herd, Karnal. Animals were maintained under standard managemental practices followed at the farm. Skin biopsies were aseptically collected from each animal during winter, spring, and summer. Real time PCR was performed to examine HSP70 expression. Reactive oxygen species (ROS) and antioxidant enzymes (SOD and CAT) were determined by ELISA. In both the breeds, significantly higher (p < 0.05) levels of HSP70 expression, ROS, caspases, and antioxidant enzymes were observed during summer followed by winter and spring. Breeds showed no significant difference during winter and spring. During summer, HSP70 expression, ROS, and antioxidant enzymes were higher (p < 0.05) in Karan Fries than Tharparkar, whereas caspases levels were higher in Tharparker than Karan Fries. The study concludes that levels of HSP70 expression, ROS, caspases, and antioxidant enzymes in skin of cattle were strongly affected by seasonal change in temperature. Differences exist in skin tissue thermotolerance of Tharparkar and Karan Fries cattle. This might be an efficient and centrally important mechanism for better adaptability of zebu cattle to heat stress.     

O-fucosylation stabilizes the TSR3 motif in thrombospondin-1 by interacting with nearby amino acids and protecting a disulfide bond

Thrombospondin type-1 repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by β3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization are poorly understood. Here, we conducted molecular dynamics (MD) simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER.     

Characterization of lipid efflux particles generated by seminal phospholipid-binding proteins.

We reported recently that the choline phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa) of bovine seminal plasma (BSP) stimulate cholesterol and choline phospholipid efflux from fibroblasts. In this study, we characterized the lipid efflux particles generated by BSP proteins. The density gradient ultracentrifugation of the efflux medium from radiolabeled fibroblasts incubated with BSP proteins showed a single peak of [3H]cholesterol between density (d) 1.12 and 1.14 g/ml, which is in the range of high-density lipoproteins. Size-exclusion chromatographic and immunoblot analysis revealed that the efflux particles have a large size equal to or bigger than very low-density lipoproteins and contained BSP proteins. Lipid analysis of density gradient and gel filtration fractions from efflux medium of simultaneously labeled fibroblasts ([3H]cholesterol and [3H]choline) incubated with BSP proteins showed that the efflux particles were homogeneous and composed of cholesterol and choline phospholipids. The lipid particles contained BSP proteins, cholesterol and choline phospholipids in molar ratio of 0.05:1.21:1, respectively. Agarose gel electrophoresis showed that the BSP-generated lipid particles had a gamma migration pattern which is slower than low-density lipoproteins. The sonication of cholesterol and BSP proteins followed by gel filtration chromatographic analysis indicated no direct binding of cholesterol to BSP proteins. These results taken together indicate that BSP proteins induce a concomitant cholesterol and choline phospholipid efflux and generate large protein-lipid particles.     

Paternal imprinting of the SLC22A1LS gene located in the human chromosome segment 11p15.5

Prostate cancer is the most commonly diagnosed cancer in men and one of the leading causes of cancer deaths. There is strong genetic evidence indicating that a large proportion of prostate cancers are caused by heritable factors but the search for prostate cancer susceptibility genes has thus far remained elusive. TGFBR1*6A, a common hypomorphic variant of the type I Transforming Growth Factor Beta receptor, is emerging as a tumor susceptibility allele that predisposes to the development of breast, colon and ovarian cancer. The association with prostate cancer has not yet been explored. A total of 907 cases and controls from New York City were genotyped to test the hypothesis that TGFBR1*6A may contribute to the development of prostate cancer. TGFBR1*6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1*6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67). Our data suggest that TGFBR1*6A does not contribute to the development of prostate cancer.     

Effect of fungicides on mycorrhizal colonization and growth of onion

Summary Effect of five fungicidesviz Agrosan. Benlate, Captan, Ceresan and Plantavax on VA mycorrhizal symbiosis in onion was studied in a phosphorus deficient, sandy loam soil. Two levels of fungicides (2.5 g and 25 g/g soil) were applied to the soil. Agrosan, Benlate and Plantavax applied at 25 g/g soil reduced the mycorrhizal colonization significantly. All the fungicides, except Captan, applied at lower concentration reduced plant growth and P uptake. Captan applied at the lower concentration had no effect on mycorrhizal colonization, plant growth and P uptake, and when applied at higher concentration had least effect on plant growth compared to other fungicides.Contribution of U.A.S. Research project DR/AMB-1.     

Estimation of helix-helix association free energy from partial unfolding of bacterioopsin

To obtain thermodynamic information about interactions between transmembrane helices in integral membrane proteins, partial unfolding of bacterioopsin in ethanol/water mixtures was studied by F?rster-type resonance energy transfer (FRET) from tryptophan to a dansyl group on Lys 41. Tryptophan to dansyl FRET was detected by measuring sensitized emission at 490-500 nm from 285 nm excitation. FRET was observed in dansylbacterioopsin in apomembranes and in detergent micelles but not in 90% ethanol/water or in the chymotrypsin fragment C2 (residues 1-71). The main fluorescence donors are Trp 86 and Trp 182. Increase of FRET from C2 with added chymotrypsin fragment C1 (residues 72-248) provides an estimate of the C1-C2 association constant as 7.7 x 10(6) M(-1). With increasing ethanol concentration, the FRET signal from dansylbacterioopsin in detergent micelles disappeared with a sharp transition above 60% ethanol. No transition occurred in Trp fluorescence from bacterioopsin lacking the dansyl acceptor, nor did dansyl model compounds undergo a similar transition. Light scattering measurements show that the detergent micelles dissipate below 50% ethanol. Thus the observed transition is likely to be a partial unfolding of bacterioopsin. Assuming a two-state unfolding model, the free energy of unfolding was obtained by extrapolation as 9.0 kcal/mol. The slope of the transition (m-value) was -0.8 kcal mol(-1) M(-1). The unfolding process probably involves dissociation of several helices. The rate of association was measured by stopped-flow fluorometry. Two first-order kinetic processes were observed, having approximately equal weights, with rate constants of 2.32 s (-1) and 0.185 s(-1).