The Caenorhabditis elegans spe-38 gene encodes a novel four-pass integral membrane protein required for sperm function at fertilization

A mutation in the Caenorhabditis elegans spe-38 gene results in a sperm-specific fertility defect. spe-38 sperm are indistinguishable from wild-type sperm with regards to their morphology, motility and migratory behavior. spe-38 sperm make close contact with oocytes but fail to fertilize them. spe-38 sperm can also stimulate ovulation and engage in sperm competition. The spe-38 gene is predicted to encode a novel four-pass (tetraspan) integral membrane protein. Structurally similar tetraspan molecules have been implicated in processes such as gamete adhesion/fusion in mammals, membrane adhesion/fusion during yeast mating, and the formation/function of tight-junctions in metazoa. In antibody localization experiments, SPE-38 was found to concentrate on the pseudopod of mature sperm, consistent with it playing a direct role in gamete interactions.     

Study of the occurrence of Vibrio vulnificus in oysters in India by polymerase chain reaction (PCR) and heterogeneity among V. vulnificus by randomly amplified polymorphic DNA PCR and gyrB sequence analysis

The pathogenic bacterium Vibrio vulnificus is widely distributed in estuarine waters throughout the world. In this study, the presence of V. vulnificus in oysters was studied both by conventional culture and DNA-based molecular technique. Following enrichment in alkaline peptone water (APW), the bacteria were lysed and a nested polymerase chain reaction (PCR) for vvhA gene was performed. The effect of duration of enrichment on the sensitivity of detection by PCR was evaluated. The organism was isolated from 43% of samples after 18 h enrichment in APW by conventional culture method. Nested PCR amplifying a fragment of vvhA gene detected the organism in 11%, 60% and 81% of samples following 0, 6 and 18 h of enrichment. All the biochemically identified V. vulnificus strains possessed vvhA gene and belonged to biotype 1. The genetic relatedness among the strains was studied by randomly amplified polymorphic DNA (RAPD) PCR and gyrB sequence analysis. The results suggest the presence of two distinct clonal groups of V. vulnificus in oysters in India. The study demonstrates, for the first time that gyrB sequence analysis could be used to study the genetic diversity of V. vulnificus.     

Characterisation of Shiga toxin-producing Escherichia coli (STEC) isolated from seafood and beef

Shiga toxin-producing Escherichia coli (STEC) strains isolated in Mangalore, India, were characterised by bead-enzyme-linked immunosorbent assay (bead-ELISA), Vero cell cytotoxicity assay, PCR and colony hybridisation for the detection of stx1 and stx2 genes. Four strains from seafood, six from beef and one from a clinical case of bloody diarrhoea were positive for Shiga toxins Stx1 and Stx2 and also for stx1and stx2 genes. The seafood isolates produced either Stx2 alone or both Stx1 and Stx2, while the beef isolates produced Stx1 alone. The stx1 gene of all the beef STEC was found to be of recently reported stx1c type. All STEC strains and one non-STEC strain isolated from clam harboured EHEC-hlyA. Interestingly, though all STEC strains were negative for eae gene, two STEC strains isolated from seafood and one from a patient with bloody diarrhoea possessed STEC autoagglutinating adhesion (saa) gene, recently identified as a gene encoding a novel autoagglutinating adhesion.     

Metal Cations Defibrillize the Amyloid Beta-Protein Fibrils

Amyloid beta-protein (A) is the major constituent of amyloid fibrils composing -amyloid plaques and cerebrovascular amyloid in Alzheimer's disease (AD). We studied the effect of metal cations on preformed fibrils of synthetic A by Thioflavin T (ThT) fluorescence spectroscopy and electronmicroscopy (EM) in negative staining. The amount of cross beta-pleated sheet structure of A 1–40 fibrils was found to decrease by metal cations in a concentration-dependent manner as measured by ThT fluorescence spectroscopy. The order of defibrillization of A 1–40 fibrils by metal cations was: Ca²⁺ and Zn²⁺ (IC₅₀ = 100 M) > Mg²⁺ (IC₅₀ = 300 M) > Al³⁺ (IC₅₀ =1.1 mM). EM analysis in negative staining showed that A 1–40 fibrils in the absence of cations were organized in a fine network with a little or no amorphous material. The addition of Ca²⁺, Mg²⁺, and Zn²⁺ to preformed A 1–40 fibrils defibrillized the fibrils or converted them into short rods or to amorphous material. Al³⁺ was less effective, and reduced the fibril network by about 80 % of that in the absence of any metal cation. Studies with A 1–42 showed that this peptide forms more dense network of fibrils as compared to A 1–40. Both ThT fluorescence spectroscopy and EM showed that similar to A 1–40, A 1–42 fibrils are also defibrillized in the presence of millimolar concentrations of Ca²⁺. These studies suggest that metal cations can defibrillize the fibrils of synthetic A.     

Structural Events in a Bacterial Uniporter Leading to Translocation of Glucose to the Cytosol

Among classes of sugar transporters, there exists a comparatively new family of transporters named SWEET transporters (semi-SWEETS in bacteria) that are uniport transmembrane proteins. It is hypothesized that sugar is transported from the extracellular side (via outward-open state) to intracellular side (inward-open state) through intermediate occluded state (both extracellular and intracellular gates closed). In our study, extensive unbiased all-atom molecular dynamics simulations were carried out with the outward-open and inward-open conformations to study this transition mechanism. We find that after 100?ns, the outward-open structure without sugar bound starts changing to the occluded form leading to closure of extracellular gates stabilized by electrostatic and hydrophobic interactions. Further simulations (up to 7?μs) have led to a transition toward the inward-open form and suggest that there exists more than one intermediate occluded conformation. We have also performed 5-μs simulations on the glucose-docked structure to identify the putative substrate-bound translocation pathway. Glucose binds to semi-SWEET with strong hydrogen bonds to Asn66 and Trp50. Comparative simulations of substrate bound, and unbound forms suggested that glucose, the putative substrate, facilitates relatively rapid conformational transitions. For the first time, we captured the release of glucose to the cytosol, in this family of transporters. We find that prior to release of glucose, the glucose forms interactions with polar residues near the intracellular gate which may facilitate its release. The distance between the residues Asn31 and Gly34 of the other protomer was found to play a decisive role in the transport of glucose to the cytoplasmic side.     

Molecular Determinants of GS-9620-Dependent TLR7 Activation

GS-9620 is an orally administered agonist of Toll-like receptor (TLR)7 currently being evaluated in clinical studies for the treatment of chronic HBV and HIV patients. GS-9620 has shown antiviral efficacy in preclinical models of chronic hepadnavirus infection in woodchuck as well as chimpanzee. However, the molecular determinants of GS-9620-dependent activation of TLR7 are not well defined. The studies presented here elucidate GS-9620 subcellular distribution and characterize its molecular interactions with human TLR7 using structure-guided mutational analysis. Based on our results we present a molecular model of TLR7 bound to GS-9620. We also determine that several coding SNPs had no effect on GS-9620-dependent TLR7 activation. In addition, our studies provide evidence that TLR7 exists in a ligand-independent oligomeric state and that, TLR7 activation by GS-9620 is likely associated with compound-induced conformational changes. Finally, we demonstrate that activation of NF-κB and Akt pathways in primary plasmacytoid dendritic cells occur as immediate downstream cellular responses to GS-9620 stimulation. The data presented here further our understanding of the molecular parameters governing TLR7 activation by GS-9620, and more generally by nucleos/tide-related ligands.