The role of the tryptophyl residue in the hypotensive peptide xenopsin

The role of the Trp⁶ residue in the biological activity of the hypotensive peptide xenopsin (<Glu-Gly-Lys-Arg-Pro-Trp-Ile-Leu-OH) was investigated. This residue was satisfactorily reduced to 2,3-dihydro-Trp on treatment with excess pyridine-borane in trifluoroacetic acid without any detectable change in other parts of the molecule. The analogous peptide, (Lys², Gly³) xenopsin, was also reduced in a similar manner. Both reduction products were purified by gel filtration and characterized by UV absorption, amino acid composition, and structural analysis.The reduced peptides were assayed on the fundus strip of isolated rat stomach and were found to possess less than 1 percent of the activity of the original peptides. Although each of the reduced analogs had an indoline substituted for an indole in the tryptophyl residue, their biological activity was virtually lost. This suggests that the tryptophyl residue of xenopsin is crucial for its biological activity.     

Purification of hepatitis B virus gene X product synthesized in Escherichia coli and its detection in a human hepatoblastoma cell line producing hepatitis B virus

A fused gene containing 94% of the hepatitis B virus (HBV) open reading frame X was expressed in Escherichia coli, and its 17-kDa product was purified by ion-exchange chromatography. Antibody elicited against the X-gene product reacted with materials proximal to the nuclear membrane of a human hepatoblastoma cell line producing HBV particles. No such reaction was observed with the same cell line that did not produce HBV particles.     

Reduced Visual Cortex Gray Matter Volume and Thickness in Young Adults Who Witnessed Domestic Violence during Childhood

Exposure to interparental violence is associated with negative outcomes, such as depression, post-traumatic stress disorder and reduced cognitive abilities. However, little is known about the potential effects of witnessing domestic violence during childhood on gray matter volume (GMV) or cortical thickness. High-resolution 3.0 T volumetric scans (Siemens Trio Scanner) were obtained on 52 subjects (18–25 years) including 22 (6 males/16 females) with a history of visually witnessing episodes of domestic violence, and 30 (8 males/22 females) unexposed control subjects, with neither a current nor past DSM-IV Axis I or II disorder. Potential confounding effects of age, gender, level of parental verbal aggression, parental education, financial stress, full scale IQ, and total GMV, or average thickness were modeled using voxel based morphometry and FreeSurfer. Witnessing domestic violence subjects had a 6.1% GMV reduction in the right lingual gyrus (BA18) (P = 0.029, False Discovery Rate corrected peak level). Thickness in this region was also reduced, as was thickness in V2 bilaterally and left occipital pole. Theses regions were maximally sensitive to exposure to witnessing domestic violence between 11–13 years of age. Regional reductions in GMV and thickness were observed in both susceptible and resilient witnessing domestic violence subjects. Results in subjects witnessing domestic violence were similar to previously reported results in subjects with childhood sexual abuse, as the primary region affected was visual cortex. Brain regions that process and convey the adverse sensory input of the abuse may be specifically modified by this experience, particularly in subjects exposed to a single type of maltreatment. Exposure to multiple types of maltreatment is more commonly associated with morphological alterations in corticolimbic regions. These findings fit with preclinical studies showing that visual cortex is a highly plastic structure.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae.

The hepatitis B virus genome carries the surface antigen (SAg) gene and an open reading frame that encodes two SAg-related polypeptides: SAg with a 55-amino-acid N-terminal extension polypeptide and SAg with a 174-amino-acid N-terminal extension polypeptide. These are termed middle S and large S, respectively. These polypeptides or their glycosylated derivatives have been detected in Dane particles, but their chemical and biological properties have remained largely unknown because of their limited availability. We attempted to produce these proteins in Saccharomyces cerevisiae by placing the coding regions under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Yeast cells carrying middle S and large S coding sequences produced 33,000- and 42,000-dalton products, respectively, each of which reacted with anti-S antibody and bound to polymerized human serum albumin, in accordance with the known properties of pre-S proteins from particles in human sera (K. H. Heermann, U. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten, and W. H. Gerlich, J. Virol. 52:396-402, 1984; A. Machida, S. Kishimoto, H. Ohnuma, K. Baba, Y. Ito, H. Miyamoto, G. Funatsu, K. Oda, S. Usuda, S. Togami, T. Nakamura, Y. Miyakawa, and M. Mayumi, Gastroenterology 86:910-918, 1984). The middle S polypeptide is glycosylated and can be assembled into particles whose size and density are similar to those of SAg. However, this polypeptide was highly susceptible to proteolytic degradation into 29,000- and 26,000-dalton polypeptides, of which only the former retained the binding activity to polymerized albumin. The large S polypeptides are nonglycosylated, relatively stable, and do not seem to assemble into particles by themselves.     

Factors encoded by and affecting the holding stability of yeast plasmid pSR1

Summary A 2 m DNA-like plasmid, pSR1, isolated from a strain of Zygosaccharomyces rouxii has three coding frames, P, S and R. Insertional inactivation of R completely abolished the intramolecular recombination, and the defect was complemented by an intact R frame on a coexistent plasmid molecule. The P and S regions were also transactive and important, but not essential, for the stable maintenance of the plasmid molecules. Insertional disruption of the P frame suggested that it produces a protein factor. Similar insertional disruption of the S frame affected the plasmid stability in Z. rouxii and Saccharomyces cerevisiae hosts differently, depending on whether the inserted DNA fragment was a short 8 bp SalI linker or a long (2.2 kb) DNA fragment. Results strongly suggested that the S region encodes two factors, one RNA and the other a protein, and that the S protein is compatible with a sprecific hostfactor in Z. rouxii, but not in S. cerevisiae. In addition, a cis-acting locus, Z, was found at a site in the plasmid molecule where no distinct open reading frames were located. No long direct repeats or inverted repeats were observed in the Z region, such as are found in the REP3 locus of 2 m DNA.     

Isolation and characterization of a novel 2-aminoethylphosphonyl-glycosphingolipid from the sea hare, Aplysia kurodai

A novel phosphonoglycosphingolipid named SGL-I' containing 1 mol of 2-aminoethylphosphonate residue was isolated from the skin of Aplysia kurodai using two silicic acid chromatography systems. Data obtained on methanolysis, permethylation, mild acid hydrolysis, and hydrogen fluoride treatment combined with thin-layer chromatography, gas liquid chromatography, gas chromatography-mass spectrometry, and proton magnetic resonance spectrometry showed that this glycolipid was 3-O-MeGal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2]Gal beta 1----4Glc beta 1----1Ceramide. Palmitic acid, octadeca-4-sphingenine and anteiso-nonadeca-4-sphingenine are its major aliphatic components. The new glycolipid has essentially the same structure as another major phosphonoglycosphingolipid in the skin of Aplysia, SGL-II, that contains 2 mol of 2-aminoethylphosphonate residue, suggesting a metabolic relationship between the two.     

Characterization of a polysaccharide component of lipopolysaccharide from Pseudomonas aeruginosa IID 1008 (ATCC 27584) as D-rhamnan

Structural studies were carried out on a rhamnose-rich polysaccharide isolated from the O-polysaccharide fraction of lipopolysaccharide in Pseudomonas aeruginosa IID 1008 (ATCC 27584) after destruction of the major O-specific chain by alkaline treatment. The isolated polysaccharide contained rhamnose, 3-O-methyl-6-deoxyhexose, glucose, xylose, alanine, galactosamine and phosphorus in a molar ratio of 67:6.9:4.3:2.1:1.1:1.0:4.1. Data from analysis involving Smith degradation, methylation, 1H-NMR spectroscopy and optical rotation measurement showed that the polysaccharide was built up of three moieties, a rhamnan chain composed of about 70 D-rhamnose residues, the core chain and an oligosaccharide chain comprising 3-O-methyl-6-deoxyhexose, xylose, rhamnose and probably glucose. The repeating unit of the rhamnan chain was indicated to have the following structure:----3)D-Rha(alpha 1----3)D-Rha(alpha 1----2)D-Rha(alpha 1----. This structure is identical with that proposed previously for the repeating unit of the side chain of lipopolysaccharide from plant pathogenic bacteria Pseudomonas syringae pv. morsprunorum C28 [Smith, A.R.W., Zamze, S.E., Munro, S.M., Carter, K. J. and Hignett, R.C. (1985) Eur. J. Biochem. 149, 73-78].