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131.
Michael Habeck Haim Haviv Adriana Katz Einat Kapri-Pardes Sophie Ayciriex Andrej Shevchenko Haruo Ogawa Chikashi Toyoshima Steven J. D. Karlish 《The Journal of biological chemistry》2015,290(8):4829-4842
The activity of membrane proteins such as Na,K-ATPase depends strongly on the surrounding lipid environment. Interactions can be annular, depending on the physical properties of the membrane, or specific with lipids bound in pockets between transmembrane domains. This paper describes three specific lipid-protein interactions using purified recombinant Na,K-ATPase. (a) Thermal stability of the Na,K-ATPase depends crucially on a specific interaction with 18:0/18:1 phosphatidylserine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-l-serine; SOPS) and cholesterol, which strongly amplifies stabilization. We show here that cholesterol associates with SOPS, FXYD1, and the α subunit between trans-membrane segments αTM8 and -10 to stabilize the protein. (b) Polyunsaturated neutral lipids stimulate Na,K-ATPase turnover by >60%. A screen of the lipid specificity showed that 18:0/20:4 and 18:0/22:6 phosphatidylethanolamine (PE) are the optimal phospholipids for this effect. (c) Saturated phosphatidylcholine and sphingomyelin, but not saturated phosphatidylserine or PE, inhibit Na,K-ATPase activity by 70–80%. This effect depends strongly on the presence of cholesterol. Analysis of the Na,K-ATPase activity and E1-E2 conformational transitions reveals the kinetic mechanisms of these effects. Both stimulatory and inhibitory lipids poise the conformational equilibrium toward E2, but their detailed mechanisms of action are different. PE accelerates the rate of E1 → E2P but does not affect E2(2K)ATP → E13NaATP, whereas sphingomyelin inhibits the rate of E2(2K)ATP → E13NaATP, with very little effect on E1 → E2P. We discuss these lipid effects in relation to recent crystal structures of Na,K-ATPase and propose that there are three separate sites for the specific lipid interactions, with potential physiological roles to regulate activity and stability of the pump. 相似文献
132.
Joy Scaria Haruo Suzuki Christopher P. Ptak Jenn-Wei Chen Yongzhang Zhu Xiao-Kui Guo Yung-Fu Chang 《BMC genomics》2015,16(1)
Background
Clostridium difficile and C. sordellii are two anaerobic, spore forming, gram positive pathogens with a broad host range and the ability to cause lethal infections. Despite strong similarities between the two Clostridial strains, differences in their host tissue preference place C. difficile infections in the gastrointestinal tract and C. sordellii infections in soft tissues.Results
In this study, to improve our understanding of C. sordellii and C. difficile virulence and pathogenesis, we have performed a comparative genomic and phenomic analysis of the two. The global phenomes of C. difficile and C. sordellii were compared using Biolog Phenotype microarrays. When compared to C. difficile, C. sordellii was found to better utilize more complex sources of carbon and nitrogen, including peptides. Phenotype microarray comparison also revealed that C. sordellii was better able to grow in acidic pH conditions. Using next generation sequencing technology, we determined the draft genome of C. sordellii strain 8483 and performed comparative genome analysis with C. difficile and other Clostridial genomes. Comparative genome analysis revealed the presence of several enzymes, including the urease gene cluster, specific to the C. sordellii genome that confer the ability of expanded peptide utilization and survival in acidic pH.Conclusions
The identified phenotypes of C. sordellii might be important in causing wound and vaginal infections respectively. Proteins involved in the metabolic differences between C. sordellii and C. difficile should be targets for further studies aimed at understanding C. difficile and C. sordellii infection site specificity and pathogenesis.Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1663-5) contains supplementary material, which is available to authorized users. 相似文献133.
The electronic circular dichroism (CD) spectra of donor-acceptor binaphthyls were investigated experimentally and theoretically. The enantiomerically pure forms of 1-(2-methoxy-1-naphthyl)- and 1-(2,3-dimethoxy-1-naphthyl)-2-methylisoquinolinium tetrafluoroborates (DA and D'A) were prepared, and their UV-vis and CD spectra were compared. The donor-acceptor interaction was apparent from the absorption at longer wavelengths, whereas its strength was not very different from each other. In addition, very similar structures were obtained for the two aromatic planes in DA and D'A when the geometry was optimized by the density functional theory. The additional methoxy group in the latter spices scarcely disturbed the UV-vis spectrum but significantly affected the CD spectrum. Thus, the observed CD spectra were considerably different from each other, especially in the (1) B(b) band couplet, where the amplitude was reduced to almost one-fourth in D'A. The theoretical investigations led to the following conclusions: (1) The potential curve associated with the central C-C dihedral angle of 1,1'-binaphthyl is fairly flat at the bottom for both DA and D'A and freely rotating at an ambient temperature. The potential curve of D'A is, however, significantly different from that of DA, in which the curve is much steeper and biased to the s-cis side. As the observed CD spectrum is an ensemble of conformers of various dihedral angles, such difference in potential certainly affects the overall spectrum; (2) The additional methoxy group introduced at the 3-position effectively altered the CD spectral pattern, which was theoretically supported by the calculation at the RI-CC2/TZVPP level; (3) Consequently, the classical coupled oscillator theory, in which the angle between the transition dipole moments of two aromatic planes is solely considered, is not applicable to the quantitative evaluation of the chiroptical properties of 1,1'-binaphthyls; rather, the quantum chemical approach is preferred, permitting a direct comparison with the experiment. 相似文献
134.
Fujihara J Ueki M Yasuda T Iida R Soejima M Koda Y Kimura-Kataoka K Kato H Panduro A Tongu M Takeshita H 《DNA and cell biology》2011,30(4):205-217
The single-nucleotide polymorphisms (SNPs) in the human DNase I gene (DNASE1) might be involved in susceptibility to some common diseases; however, only limited population data are available. Further, the effects of these SNPs on in vivo DNase I activity remain unknown. The genotype and haplotype of all the SNPs in DNASE1 were determined in 3 ethnic groups including 14 populations using newly developed methods. Together with our previous data on the nonsynonymous SNPs, two major haplotypes based on the five exonic SNPs were identified; genetic diversity in the Asian population was low. Among 10 SNPs, other than exonic SNPs in the gene, only 3 were polymorphic among all the populations. Haplotype distribution, based on all the polymorphic SNPs, was clarified to be generally varied in an ethnic-dependent manner. Thus, the genetic aspects of DNASE1 with regard to all the SNPs in wide-ranging ethnic groups could be first demonstrated. Further, there was no correlation of all the polymorphic SNPs other than nonsynonymous ones with serum DNase I activity levels. Polymorphic SNPs other than the exonic SNPs might not be directly related to common diseases through alterations in in vivo levels of the activity. 相似文献
135.
Takahashi S Toyoda A Sekiyama Y Takagi H Nogawa T Uramoto M Suzuki R Koshino H Kumano T Panthee S Dairi T Ishikawa J Ikeda H Sakaki Y Osada H 《Nature chemical biology》2011,7(7):461-468
Spiroacetal compounds are ubiquitous in nature, and their stereospecific structures are responsible for diverse pharmaceutical activities. Elucidation of the biosynthetic mechanisms that are involved in spiroacetal formation will open the door to efficient generation of stereospecific structures that are otherwise hard to synthesize chemically. However, the biosynthesis of these compounds is poorly understood, owing to difficulties in identifying the responsible enzymes and analyzing unstable intermediates. Here we comprehensively describe the spiroacetal formation involved in the biosynthesis of reveromycin A, which inhibits bone resorption and bone metastases of tumor cells by inducing apoptosis in osteoclasts. We performed gene disruption, systematic metabolite analysis, feeding of labeled precursors and conversion studies with recombinant enzymes. We identified two key enzymes, dihydroxy ketone synthase and spiroacetal synthase, and showed in vitro reconstruction of the stereospecific spiroacetal structure from a stable acyclic precursor. Our findings provide insights into the creation of a variety of biologically active spiroacetal compounds for drug leads. 相似文献
136.
Arisa Miyagawa‐Yamaguchi Takuma Okami Nozomu Kira Haruo Yamaguchi Kouhei Ohnishi Masao Adachi 《Phycological Research》2011,59(2):113-119
A nuclear transformation system for the centric diatom Chaetoceros sp. has been established using two plasmids pTpfcp/nat and pTpNR/green fluorescent protein (GFP) that had been used for Thalassiosira pseudonana transformation. These contain the nourseothricin resistance gene (nat) with the fucoxanthin chlorophyll a/c binding protein (fcp) promoter/terminator from T. pseudonana and the enhanced green fluorescent protein gene (egfp), with the nitrate reductase (NR) promoter/terminator from T. pseudonana, respectively. Transformants were recovered in the presence of the antibiotic nourseothricin. One to four copies of both nat and egfp genes were integrated into genomic DNA of the transformants. Transformation efficiency was 1.5–6.0 transformants per 108 cells. This work is the first report of stable genetic transformation of Chaetoceros, which is important as not only a constituent member of marine ecosystem but also feed for aquaculture. 相似文献
137.
Minami M Koyama T Wakayama Y Fukuhara S Mochizuki N 《Molecular biology of the cell》2011,22(18):3508-3519
Insulin-like growth factor-I (IGF-I) activates not only the phosphatidylinositol 3-kinase (PI3K)-AKT cascade that is essential for myogenic differentiation but also the extracellular signal-regulated kinase (ERK) 1/2 cascade that inhibits myogenesis. We hypothesized that there must be a signal that inhibits ERK1/2 upon cell-cell contact required for skeletal myogenesis. Cell-cell contact-induced engagement of ephrin ligands and Eph receptors leads to downregulation of the Ras-ERK1/2 pathway through p120 Ras GTPase-activating protein (p120RasGAP). We therefore investigated the significance of the ephrin/Eph signal in IGF-I-induced myogenesis. EphrinA1-Fc suppressed IGF-I-induced activation of Ras and ERK1/2, but not that of AKT, in C2C12 myoblasts, whereas ephrinB1-Fc affected neither ERK1/2 nor AKT activated by IGF-I. IGF-I-dependent myogenic differentiation of C2C12 myoblasts was potentiated by ephrinA1-Fc. In p120RasGAP-depleted cells, ephrinA1-Fc failed to suppress the Ras-ERK1/2 cascade by IGF-I and to promote IGF-I-mediated myogenesis. EphrinA1-Fc did not promote IGF-I-dependent myogenesis when the ERK1/2 was constitutively activated. Furthermore, a dominant-negative EphA receptor blunted IGF-I-induced myogenesis in C2C12 and L6 myoblasts. However, the inhibition of IGF-I-mediated myogenesis by down-regulation of ephrinA/EphA signal was canceled by inactivation of the ERK1/2 pathway. Collectively, these findings demonstrate that the ephrinA/EphA signal facilitates IGF-I-induced myogenesis by suppressing the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines. 相似文献
138.
139.
140.
Nakao R Hasegawa H Ochiai K Takashiba S Ainai A Ohnishi M Watanabe H Senpuku H 《PloS one》2011,6(10):e26163
We previously reported that mutation of galE in Porphyromonas gingivalis has pleiotropic effects, including a truncated lipopolysaccharide (LPS) O-antigen and deglycosylation of the outer membrane protein OMP85 homolog. In the present study, further analysis of the galE mutant revealed that it produced little or no outer membrane vesicles (OMVs). Using three mouse antisera raised against whole cells of the P. gingivalis wild type strain, we performed ELISAs to examine the reactivity of these antisera with whole cells of the wild type or the galE mutant. All three antisera had significantly lower reactivity against the galE mutant compared to wild type. OMVs, but not LPS, retained the immunodominant determinant of P. gingivalis, as determined by ELISAs (with wild type LPS or OMVs as antigen) and absorption assays. In addition, we assessed the capacity of OMVs as a vaccine antigen by intranasal immunization to BALB/c mice. Synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [Poly (I∶C)], an agonist of Toll-like receptor 3 (TLR3), was used as the mucosal adjuvant. Vaccination with OMV elicited dramatically high levels of P. gingivalis-specific IgA in nasal washes and saliva, as well as serum IgG and IgA. In conclusion, the OMVs of P. gingivalis have an important role in mucosal immunogenicity as well as in antigenicity. We propose that P. gingivalis OMV is an intriguing immunogen for development of a periodontal disease vaccine. 相似文献