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81.
Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes
with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental
niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic
activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based
microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied.
Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches.
As culture-dependent methods have thus far resulted in the isolation of only a tiny percentage of the marine microbiota the
application of metagenomic strategies holds great potential to study and exploit the enormous microbial biodiversity which
is present within these marine environments. 相似文献
82.
Clapp C Thebault S Arnold E García C Rivera JC de la Escalera GM 《American journal of physiology. Endocrinology and metabolism》2008,295(4):E772-E778
Disruption of the quiescent state of blood vessels in the retina leads to aberrant vasopermeability and angiogenesis, the major causes of vision loss in diabetic retinopathy. Prolactin is expressed throughout the retina, where it is proteolytically cleaved to vasoinhibins, a family of peptides (including the 16-kDa fragment of prolactin) with potent antiangiogenic, vasoconstrictive, and antivasopermeability actions. Ocular vasoinhibins act directly on endothelial cells to block blood vessel growth and dilation and to promote apoptosis-mediated vascular regression. Also, vasoinhibins prevent retinal angiogenesis and vasopermeability associated with diabetic retinopathy, and inactivation of endothelial nitric oxide synthase via protein phosphatase 2A is among the various mechanisms mediating their actions. Here, we discuss the potential role of vasoinhibins both in the maintenance of normal retinal vasculature and in the cause and prevention of diabetic retinopathy and other vasoproliferative retinopathies. 相似文献
83.
Objectives: The aim of this study was to assess persistence and tissue invasion of Candida albicans strains isolated from a 65 year‐old patient with chronic hyperplastic candidosis (CHC), that subsequently developed into squamous cell carcinoma (SCC). Materials and Methods: C. albicans (n=7) were recovered from the oral cavity of the patient over seven years. Confirmation of CHC and SCC in this patient was achieved by histopathological examination of incisional biopsy tissue. DNA fingerprinting was performed on the seven isolates from the CHC patient together with a further eight isolates from patients with normal oral mucosa (n=2), chronic atrophic candidosis (n=1), SCC (n=1) and CHC (n=4). Genotyping involved the use of inter‐repeat PCR using the eukaryotic repeat primer 1251. Characterisation of the tissue invasive abilities of the isolates was achieved by infecting a commercially available reconstituted human oral epithelium (RHE; SkinEthic, Nice, France). After 24 h. C. albicans tissue invasion was assessed by histopathological examination. Results: DNA fingerprinting demonstrated strain persistence of C. albicans in the CHC patient over a seven year period despite provision of systemic antifungal therapy. The strain of C. albicans isolated from this patient was categorised as a high invader within the RHE compared to other isolates. Conclusions: Candidal strain persistence was evident in a patient with CHC over seven years. This persistence may be due to incomplete eradication from the oral cavity following antifungal therapy or subsequent recolonisation from other body sites or separate exogenous sources. The demonstration of enhanced in vitro tissue invasion by this particular strain may, in part, explain the progression to carcinoma. 相似文献
84.
Neurofibromin GTPase-activating protein-related domains restore normal growth in Nf1-/- cells 总被引:2,自引:0,他引:2
Hiatt KK Ingram DA Zhang Y Bollag G Clapp DW 《The Journal of biological chemistry》2001,276(10):7240-7245
Members of the Ras superfamily of signaling proteins modulate fundamental cellular processes by cycling between an active GTP-bound conformation and an inactive GDP-bound form. Neurofibromin, the protein product of the NF1 tumor suppressor gene, and p120GAP are GTPase-activating proteins (GAPs) for p21(Ras) (Ras) and negatively regulate output by accelerating GTP hydrolysis on Ras. Neurofibromin and p120GAP differ markedly outside of their conserved GAP-related domains (GRDs), and it is therefore unknown if the respective GRDs contribute functional specificity. To address this question, we expressed the GRDs of neurofibromin and p120GAP in primary cells from Nf1 mutant mice in vitro and in vivo. Here we show that expression of neurofibromin GRD, but not the p120GAP GRD, restores normal growth and cytokine signaling in three lineages of primary Nf1-deficient cells that have been implicated in the pathogenesis of neurofibromatosis type 1 (NF1). Furthermore, utilizing a GAP-inactive mutant NF1 GRD identified in a family with NF1, we demonstrate that growth restoration is a function of NF1 GRD GAP activity on p21(Ras). Thus, the GRDs of neurofibromin and p120GAP specify nonoverlapping functions in multiple primary cell types. 相似文献
85.
Acacia mangium and Paraserianthes falcataria are leguminous tree species widely grown for timber in Indonesia and other tropical countries, yet little is known about the identity of their rhizobial symbionts. Polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS) analysis of the 16S rRNA gene was used along with sequencing to assess the diversity of 57 rhizobia isolated from nodules of A. mangium and P. falctaria in Indonesia. In total, 26 rhizobia isolated from A. mangium were analysed by PRS and sequencing. The PRS patterns indicated that 12 (46%) clustered with Bradyrhizobium elkanii , 13 (50%) with B. lianoningense / japonicum and one (4%) with Mesorhizobium loti . Thirty-one isolates were analysed from P. falcataria : five (16%) clustered with B. elkanii and 26 (84%) with B. lianoningense / japonicum. These results were confirmed by phylogenetic analysis of sequences. Intraspecific diversity of the 16S rRNA genes from rhizobia nodulating A. mangium and P. falcataria revealed by PRS was low, only one genotype was found within the isolates that clustered with B. elkanii and two within the B. liaoningense / japonicum group. These Bradyrhizobium species are apparently ubiquitous throughout the Indonesian archipelago and it is clear why the two tree species are able to successfully establish outside their native range without the need for inoculation with indigenous rhizobia. 相似文献
86.
Galfione M Luo W Kim J Hawke D Kobayashi R Clapp C Yu-Lee LY Lin SH 《Protein expression and purification》2003,28(2):252-258
The 16-kDa fragment of prolactin (16-kDa PRL), derived from proteolytic cleavage of 23-kDa PRL, was shown to have antiangiogenic activity. Previous studies have shown that recombinant 16-kDa PRL produced from bacteria often contained endotoxins, which are cytotoxic to endothelial cells, and varied in its biological activity due to changes in its refolding from inclusion bodies. These problems limited the use of recombinant 16-kDa PRL. To improve the generation of recombinant 16-kDa PRL, we expressed 16-kDa PRL in Sf9 insect cells using a baculoviral expression system. The signal sequence of the human PRL gene and codons for seven histidines were added to the N- and C-termini, respectively, of the 16-kDa PRL cDNA construct. Recombinant 16-kDa PRL was detected in both the cell pellet and the medium. About 0.28 mg purified protein was isolated from the cell pellet of 4 x 10(7) infected cells using nickel affinity chromatography. Sixteen kilodalton PRL was posttranslationally modified with apparent molecular weights of 16 and 18 kDa on SDS-PAGE. The level of 18-kDa protein was significantly reduced after digestion with peptidyl-N-glycosidase, suggesting that the heterogeneity was due to glycosylation of 16-kDa PRL. N-terminal sequence analysis confirmed the fact that both proteins were human 16-kDa PRL and the signal sequences were cleaved at the same position as that of human PRL. Consistent with its role as an angiogenesis inhibitor, purified recombinant 16-kDa PRL inhibits the proliferation of endothelial cells with a potency similar to that previously reported for the protein generated in Escherichia coli. This 16-kDa PRL expressed in Sf9 cells is a useful reagent for functional studies and for the purification and identification of its receptor. 相似文献
87.
Mycolic acid-containing actinomycetes capable of metabolizing nitriles were recovered from deep-sea sediments and terrestrial soils by enrichment culture on acetonitrile, benzonitrile, succinonitrile or bromoxynil. A total of 43 nitrile-degrading strains were isolated and, together with previously recovered nitrile-degrading rhodococci, were identified by a polyphasic taxonomic approach, which included mycolic acid profiles, pyrolysis mass spectrometry (PyMS), genomic fingerprinting based on sequence variability of the 16S ribosomal RNA gene using polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism, and 16S rRNA gene sequence comparison. Isolates phylogenetically related to Rhodococcus erythropolis dominated the culturable microorganisms from most marine and terrestrial samples. These isolates clustered together in a major pyrogroup that showed high congruence with PRS profiles of the 16S rRNA gene. Such high congruence also was obtained for other recovered isolates that were assigned to species of Rhodococcus and Gordonia. Sequencing data validated the results obtained by PRS analysis and enabled phylogenetic relationships to be established. Some of the recovered bacteria probably represent novel microbial species. The fact that nitrile-metabolizing microorganisms were recovered from a wide range of habitat types suggests that nitrile transforming enzymatic activity is geographically widely distributed in nature. 相似文献
88.
ATP-sensitive potassium channels (K(ATP) channels) are formed from an octameric complex of an inwardly rectifying K(+) channel (Kir6.1, Kir6.2) and a sulfonylurea receptor (SUR1, SUR2A, and SUR2B). In this study we have attempted to address the question of whether SUR heteromultimers can form using a combination of biochemical and electrophysiological approaches. We have constructed monoclonal stable lines in HEK293 cells co-expressing Kir6.2 with SUR1 and SUR2A. Using coimmunoprecipitation analysis with SUR isotype-specific antibodies two biochemical populations are distinguished, one containing SUR1 and the other SUR2A. It is not possible to detect immune complexes containing both SUR1 and SUR2A. Functional studies were undertaken and whole cell membrane currents were studied using the patch clamp. Concentrations of sulfonylureas and potassium channel openers were determined that selectively inhibited or activated SUR1/Kir6.2 and SUR2A/Kir6.2. In the cell line expressing SUR1/SUR2AKir6.2 we were unable to demonstrate a population of channels with unique pharmacological properties. Thus we conclude from these studies that heteromultimeric channel complexes containing both SUR1 and SUR2A are not formed, suggesting an incompatibility between different SUR subtypes. This incompatibility limits the pharmacological complexity of K(ATP) channels that may be observed in native tissues. 相似文献
89.
Juan Pablo Robles Magdalena Zamora Elva Adan-Castro Lourdes Siqueiros-Marquez Gonzalo Martinez de la Escalera Carmen Clapp 《The Journal of biological chemistry》2022,298(3)
Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5β1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1β, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5β1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1β, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5β1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5β1 as a promising target for treating vascular inflammation in COVID-19. 相似文献
90.
William L. Clapp Barry S. Fagg Carr J. Smith 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》1999,446(2):27
The mutagenic activity of cigarette smoke condensates (CSC) made from tobacco before and after removal of protein was assessed by the Ames Salmonella assay in bacterial strains TA98 and TA100. Removal of protein and peptides from flue-cured tobacco via water extraction followed by protease digestion reduced the mutagenicity of the resultant CSC by 80% in the TA98 strain and 50% in the TA100 strain. Similarly, reductions of 81% in TA98 and 54% in TA100 were seen following water extraction and protease digestion of burley tobacco. The significant reductions in Ames mutagenicity following protein removal suggest that protein pyrolysis products are a principal contributor to the genotoxicity of CSC as measured in this assay. 相似文献