Soluble and particulate methane monooxygenase gene clusters in the marine methanotroph Methylomicrobium sp. strain NI

Soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO) gene clusters in the marine methanotroph Methylomicrobium sp. strain NI were completely sequenced and analysed. Degenerated primers were newly designed and used to amplify the gene fragments containing intergenic mmoX-Y and mmoD-C regions and a partial pmoC region. Phylogenetic analysis of amino acid sequences deduced from mmoX and pmoA, as well as of 16S rRNA gene sequences, indicated that this strain was most closely related to the halotolerant methanotroph Methylomicrobium buryatense. There were putative sigma(54)- and sigma(70)-dependent promoter sequences upstream of the sMMO and pMMO genes, respectively, and mmoG, which is known to be related to the expression and assembly of sMMO, existed downstream of the sMMO genes. These findings suggest that the major components and regulation of MMOs in this marine methanotroph are quite similar to those in freshwater methane oxidizers, despite the difference in their habitats.     

The coexistence of Ser84 in renin and His13 in angiotensinogen brings a pH profile of two separate peaks to the reaction of human renin and sheep angiotensinogen

The pH dependence and kinetics parameters of renin-angiotensinogen reactions were determined using wild-type and S84G mutant human renins and wild-type and H13Y mutant sheep angiotensinogens. It is explained in this report that (i) renin catalyzes acidic and basic reactions of which the optimum pHs are 5.5 and 7.5-8.2 respectively, both of which produce angiotensin I; (ii) Ser84 specific to human renin accelerates the acidic reaction by 75-110% through elevation of V(max), and shifts the optimum pH of the basic reaction from 7.5 to 8.0-8.2; and (iii) His13 specific to sheep angiotensinogen accelerates the acidic and basic reactions by 25-42% through reduction of K(m). It is concluded from these results that the coexistence of Ser84 in renin and His13 in angiotensinogen brings a pH profile of two separate peaks at pHs 5.5 and 8.2 to the reaction of human renin and sheep angiotensinogen.     

Association of single-nucleotide polymorphisms in RHOB and TXNDC3 with knee osteoarthritis susceptibility: two case-control studies in East Asian populations and a meta-analysis

Introduction

Conflicting findings on the association of single nucleotide polymorphisms (SNPs) in RHOB and TXNDC3 with susceptibility to knee osteoarthritis (OA) have been reported in European Caucasians. To examine the associations of these SNPs with OA in East Asian populations and to evaluate their global significance, we conducted two case-control studies in 955 Chinese and 750 Japanese patients.

Methods

We genotyped the previously implicated SNPs rs585017 (in RHOB) and rs4720262 (in TXNDC3) in patients with primary symptomatic knee OA with radiographic confirmation and in matched control individuals, and analyzed their associations. We further conducted a meta-analysis of the study findings together with those of previously reported European studies using the DerSimonian-Laird procedure.

Results

A significant association of RHOB with knee OA was observed in male Chinese patients (P = 0.02). No significant associations were found for RHOB in any other comparisons in the East Asian populations. The association of TXNDC3 was replicated in Chinese female (P = 0.04) and Japanese (P = 0.03) patients, although none of these associations persisted after Bonferroni correction. Significant association (P = 0.02 for the allelic frequency) with nonsignificant heterogeneity was found in the East Asian replication study. No significant association was found in any comparison in the meta-analysis for all studies.

Conclusion

Our study replicates the association, previously reported in European Caucasians, of TXNDC3 with knee OA susceptibility in an East Asian population.     

Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both α and β crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded β-sheets, was composed of a typical β-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.     

Molecular mapping of QTLs for resistance to Gibberella ear rot, in corn, caused by Fusarium graminearum.

Gibberella ear rot, caused by the fungus Fusarium graminearum Schwabe, is a serious disease of corn (Zea mays) grown in northern climates. Infected corn is lower yielding and contains toxins that are dangerous to livestock and humans. Resistance to ear rot in corn is quantitative, specific to the mode of fungal entry (silk channels or kernel wounds), and highly influenced by the environment. Evaluations of ear rot resistance are complex and subjective; and they need to be repeated over several years. All of these factors have hampered attempts to develop F. graminearum resistant corn varieties. The aim of this study was to identify molecular markers linked to the genes for resistance to Gibberella ear rot. A recombinant inbred (RI) population, produced from a cross between a Gibberella ear rot resistant line (CO387) and a susceptible line (CG62), was field-inoculated and scored for Gibberella ear rot symptoms in the F4, F6, and F7 generations. The distributions of disease scores were continuous, indicating that resistance is probably conditioned by multiple loci. A molecular linkage map, based on segregation in the F5 RI population, contained 162 markers distributed over 10 linkage groups and had a total length of 2237 cM with an average distance between markers of 13.8 cM. Composite interval mapping identified 11 quantitative trait loci (QTLs) for Gibberella ear rot resistance following silk inoculation and 18 QTLs following kernel inoculation in 4 environments that accounted for 6.7%-35% of the total phenotypic variation. Only 2 QTLs (on linkage group 7) were detected in more than 1 test for silk resistance, and only 1 QTL (on linkage group 5) was detected in more than 1 test for kernel resistance, confirming the strong influence of the environment on these traits. The majority of the favorable alleles were derived from the resistant parent (CO387). The germplasm and markers for QTLs with significant phenotypic effects may be useful for marker-assisted selection to incorporate Gibberella ear rot resistance into commercial corn cultivars.     

4E10-Resistant HIV-1 Isolated from Four Subjects with Rare Membrane-Proximal External Region Polymorphisms

Human antibody 4E10 targets the highly conserved membrane-proximal external region (MPER) of the HIV-1 transmembrane glycoprotein, gp41, and has extraordinarily broad neutralizing activity. It is considered by many to be a prototype for vaccine development. In this study, we describe four subjects infected with viruses carrying rare MPER polymorphisms associated with resistance to 4E10 neutralization. In one case resistant virus carrying a W680G substitution was transmitted from mother to infant. We used site-directed mutagenesis to demonstrate that the W680G substitution is necessary for conferring the 4E10-resistant phenotype, but that it is not sufficient to transfer the phenotype to a 4E10-sensitive Env. Our third subject carried Envs with a W680R substitution causing variable resistance to 4E10, indicating that residues outside the MPER are required to confer the phenotype. A fourth subject possessed a F673L substitution previously associated with 4E10 resistance. For all three subjects with W680 polymorphisms, we observed additional residues in the MPER that co-varied with position 680 and preserved charged distributions across this region. Our data provide important caveats for vaccine development targeting the MPER. Naturally occurring Env variants described in our study also represent unique tools for probing the structure-function of HIV-1 envelope.     

Transcriptional Regulation of Cannabinoid Receptor-1 Expression in the Liver by Retinoic Acid Acting via Retinoic Acid Receptor-γ

Alcoholism can result in fatty liver that can progress to steatohepatitis, cirrhosis, and liver cancer. Mice fed alcohol develop fatty liver through endocannabinoid activation of hepatic CB₁ cannabinoid receptors (CB₁R), which increases lipogenesis and decreases fatty acid oxidation. Chronic alcohol feeding also up-regulates CB₁R in hepatocytes in vivo, which could be replicated in vitro by co-culturing control hepatocytes with hepatic stellate cells (HSC) isolated from ethanol-fed mice, implicating HSC-derived mediator(s) in the regulation of hepatic CB₁R (Jeong, W. I., Osei-Hyiaman, D., Park, O., Liu, J., Bátkai, S., Mukhopadhyay, P., Horiguchi, N., Harvey-White, J., Marsicano, G., Lutz, B., Gao, B., and Kunos, G. (2008) Cell Metab. 7, 227–235). HSC being a rich source of retinoic acid (RA), we tested whether RA and its receptors may regulate CB₁R expression in cultured mouse hepatocytes. Incubation of hepatocytes with RA or RA receptor (RAR) agonists increased CB₁R mRNA and protein, the most efficacious being the RARγ agonist CD437 and the pan-RAR agonist TTNPB. The endocannabinoid 2-arachidonoylglycerol (2-AG) also increased hepatic CB₁R expression, which was mediated indirectly via RA, because it was absent in hepatocytes from mice lacking retinaldehyde dehydrogenase 1, the enzyme catalyzing the generation of RA from retinaldehyde. The binding of RARγ to the CB₁R gene 5′ upstream domain in hepatocytes treated with RAR agonists or 2-AG was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift and antibody supershift assays. Finally, TTNPB-induced CB₁R expression was attenuated by small interfering RNA knockdown of RARγ in hepatocytes. We conclude that RARγ regulates CB₁R expression and is thus involved in the control of hepatic fat metabolism by endocannabinoids.     

Teprenone, but not H2-receptor blocker or sucralfate, suppresses corpus Helicobacter pylori colonization and gastritis in humans: teprenone inhibition of H. pylori-induced interleukin-8 in MKN28 gastric epithelial cell lines

Background. The role of teprenone in Helicobacter pylori‐associated gastritis has yet to be determined. To investigate the effect of teprenone on inflammatory cell infiltration, and on H. pylori colonization of the gastric mucosa in H. pylori‐infected patients, we first compared the effect of teprenone with that of both histamine H2 receptor antagonists (H2‐RA) and sucralfate on the histological scores of H. pylori gastritis. We then examined its in vitro effect on H. pylori‐induced interleukin (IL)‐8 production in MKN28 gastric epithelial cells. Materials and Methods. A total of 68 patients were divided into three groups, each group undergoing a 3‐month treatment with either teprenone (150 mg/day), H2‐RA (nizatidine, 300 mg/day), or sucralfate (3 g/day). All subjects underwent endoscopic examination of the stomach before and after treatment. IL‐8 production in MKN28 gastric epithelial cells was measured by enzyme‐linked immunosorbent assay (ELISA). Results. Following treatment, the teprenone group showed a significant decrease in both neutrophil infiltration and H. pylori density of the corpus (before vs. after: 2.49 ± 0.22 vs. 2.15 ± 0.23, p = .009; 2.36 ± 0.25 vs. 2.00 ± 0.24, p = .035, respectively), with no significant differences seen in either the sucralfate or H2‐RA groups. Teprenone inhibited H. pylori‐enhanced IL‐8 production in MKN28 gastric epithelial cells in vitro, in a dose‐dependent manner. Conclusions. Teprenone may modify corpus H. pylori‐associated gastritis through its effect on neutrophil infiltration and H. pylori density, in part by its inhibition of IL‐8 production in the gastric mucosa.     

Activation of Notch1 signaling in cardiogenic mesoderm induces abnormal heart morphogenesis in mouse

Notch signaling is implicated in many developmental processes. In our current study, we have employed a transgenic strategy to investigate the role of Notch signaling during cardiac development in the mouse. Cre recombinase-mediated Notch1 (NICD1) activation in the mesodermal cell lineage leads to abnormal heart morphogenesis, which is characterized by deformities of the ventricles and atrioventricular (AV) canal. The major defects observed include impaired ventricular myocardial differentiation, the ectopic appearance of cell masses in the AV cushion, the right-shifted interventricular septum (IVS) and impaired myocardium of the AV canal. However, the fates of the endocardium and myocardium were not disrupted in NICD1-activated hearts. One of the Notch target genes, Hesr1, was found to be strongly induced in both the ventricle and the AV canal of NICD1-activated hearts. However, a knockout of the Hesr1 gene from NICD-activated hearts rescues only the abnormality of the AV myocardium. We searched for additional possible targets of NICD1 activation by GeneChip analysis and found that Wnt2, Bmp6, jagged 1 and Tnni2 are strongly upregulated in NICD1-activated hearts, and that the activation of these genes was also observed in the absence of Hesr1. Our present study thus indicates that the Notch1 signaling pathway plays a suppressive role both in AV myocardial differentiation and the maturation of the ventricular myocardium.