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1.
Nobukazu Suganuma Satoru Ito Hiromichi Aso Masashi Kondo Mitsuo Sato Masahiro Sokabe Yoshinori Hasegawa 《PloS one》2012,7(9)
It is suggested that migration of airway smooth muscle (ASM) cells plays an important role in the pathogenesis of airway remodeling in asthma. Increases in intracellular Ca2+ concentrations ([Ca2+]i) regulate most ASM cell functions related to asthma, such as contraction and proliferation. Recently, STIM1 was identified as a sarcoplasmic reticulum (SR) Ca2+ sensor that activates Orai1, the Ca2+ channel responsible for store-operated Ca2+ entry (SOCE). We investigated the role of STIM1 in [Ca2+]i and cell migration induced by platelet-derived growth factor (PDGF)-BB in human ASM cells. Cell migration was assessed by a chemotaxis chamber assay. Human ASM cells express STIM1, STIM2, and Orai1 mRNAs. SOCE activated by thapsigargin, an inhibitor of SR Ca2+-ATPase, was significantly blocked by STIM1 siRNA and Orai1 siRNA but not by STIM2 siRNA. PDGF-BB induced a transient increase in [Ca2+]i followed by sustained [Ca2+]i elevation. Sustained increases in [Ca2+]i due to PDGF-BB were significantly inhibited by a Ca2+ chelating agent EGTA or by siRNA for STIM1 or Orai1. The numbers of migrating cells were significantly increased by PDGF-BB treatment for 6 h. Knockdown of STIM1 and Orai1 by siRNA transfection inhibited PDGF-induced cell migration. Similarly, EGTA significantly inhibited PDGF-induced cell migration. In contrast, transfection with siRNA for STIM2 did not inhibit the sustained elevation of [Ca2+]i or cell migration induced by PDGF-BB. These results demonstrate that STIM1 and Orai1 are essential for PDGF-induced cell migration and Ca2+ influx in human ASM cells. STIM1 could be an important molecule responsible for airway remodeling. 相似文献
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A convenient method using commercial aqueous concentrated HCl (conc. HCl; 35%, w/w) as an acid catalyst was developed for preparation of fatty acid methyl esters (FAMEs) from sterol esters, triacylglycerols, phospholipids, and FFAs for gas-liquid chromatography (GC). An 8% (w/v) solution of HCl in methanol/water (85:15, v/v) was prepared by diluting 9.7 ml of conc. HCl with 41.5 ml of methanol. Toluene (0.2 ml), methanol (1.5 ml), and the 8% HCl solution (0.3 ml) were added sequentially to the lipid sample. The final HCl concentration was 1.2% (w/v). This solution (2 ml) was incubated at 45°C overnight or heated at 100°C for 1–1.5 h. The amount of FFA formed in the presence of water derived from conc. HCl was estimated to be <1.4%. The yields of FAMEs were >96% for the above lipid classes and were the same as or better than those obtained by saponification/methylation or by acid-catalyzed methanolysis/methylation using commercial anhydrous HCl/methanol. The method developed here could be successfully applied to fatty acid analysis of various lipid samples, including fish oils, vegetable oils, and blood lipids by GC. 相似文献
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Intraperitoneal administration of leupeptin to rats induced a hemoglobin-hydrolyzing protease which was most active at pH 3.5 and was insensitive to pepstatin in various tissues such as the liver, kidney, and muscle, as observed previously in adult rat hepatocytes in primary culture (Tanaka, K., Ikegaki, N., and Ichihara, A. (1979) Biochem. Biophys. Res. Commun. 91, 102-107). The induced acidic protease was purified about 600-fold in 30% yield from rat liver by conventional chromatographic techniques. The purified enzyme appeared homogeneous by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate and was a monomeric protein of Mr = 20,000. The enzyme appeared to be a glycoprotein because its induction was blocked by the addition of tunicamycin to cultures of hepatocytes and because the induced protease was absorbed on concanavalin A-Sepharose and eluted with methylglucoside. It seemed to be present in lysosomes and was fairly stable at various pH values and temperatures. It showed endopeptidase activity on various protein substrates, but scarcely hydrolyzed N-substituted derivatives of arginine. It did not hydrolyze esters, showed no aminopeptidase or carboxypeptidase activity, and did not inactivate glucose-6-phosphate dehydrogenase or aldolase. The enzyme appeared to be a thiol protease, since it was strongly inhibited by sulfhydryl-reactive compounds and N-( [N-(1-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine and was not inhibited by reagents specific for carboxyl-, serine-, or metalloproteases. This induced protease could be separated from cathepsins B, D, and H by chromatography. The enzyme was similar to cathepsin L in chromatographic behavior, Mr and pI, but differed from the latter in stability and in its inability to inactivate some enzymes. These results suggest that it differs from any known proteases found previously in rat liver. 相似文献
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Intramembrane metalloproteases are nearly ubiquitous in living organisms and they function in diverse processes ranging from cholesterol homeostasis and the unfolded protein response in humans to sporulation, stress responses, and virulence of bacteria. Understanding how these enzymes function in membranes is a challenge of fundamental interest with potential applications if modulators can be devised. Progress is described toward a mechanistic understanding, based primarily on molecular genetic and biochemical studies of human S2P and bacterial SpoIVFB and RseP, and on the structure of the membrane domain of an archaeal enzyme. Conserved features of the enzymes appear to include transmembrane helices and loops around the active site zinc ion, which may be near the membrane surface. Extramembrane domains such as PDZ (PSD-95, DLG, ZO-1) or CBS (cystathionine-β-synthase) domains govern substrate access to the active site, but several different mechanisms of access and cleavage site selection can be envisioned, which might differ depending on the substrate and the enzyme. More work is needed to distinguish between these mechanisms, both for enzymes that have been relatively well-studied, and for enzymes lacking PDZ and CBS domains, which have not been studied. This article is part of a Special Issue entitled: Intramembrane Proteases. 相似文献
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We attempted to identify the genes involved in cellularsenescence, telomere maintenance and telomerase regulationthrough subtractive screening of cDNA libraries prepared froma human lung adenocarcinoma cell line A549 and its sublinesnamed A5DC7, CK and AST-9. Cell phenotypes of A5DC7, CK andAST-9 are normal cell-like, cancer cell-like and intermediate,respectively. These cell lines have different phenotypes interms of telomerase activity and telomere maintenance, andthus are thought to be useful for identifying the genesinvolved in cellular senescence and telomerase regulation. In this study, we identified 86 independent cDNA clones bysubtractive screening. Among these cDNA clones, subtractingA5DC7 cDNAs from A549 cDNAs and CK cDNAs gave 7 and 3 cDNAclones which highly and specifically expressed in tester celllines. Genes corresponding to these 10 cDNA clones mightparticipate in maintaining cancer-cell phenotypes. As aresult of database searching, each four of A549 specific cDNAclones are found to correspond to known cDNAs. Each two ofA549 specific and two of CK specific cDNA clones have highhomology to independent ESTs. Sequences having homology toeach one of A549 specific and one of CK specific cDNA cloneshave not been deposited in the Genbank database, indicatingthat these two cDNA clones are part of novel genes. Weanticipate that their involvement in telomerase regulationand/or senescence program can be clarified by functionalanalysis using each full-length cDNA. 相似文献
9.
Requirement of three proteins for hydrocarbon oxidation 总被引:1,自引:0,他引:1
10.
Mutations in the profilin 1 (PFN1) gene have been identified as a cause of familial amyotrophic lateral sclerosis (ALS), and neuropathological studies indicate that TDP-43 is accumulated in brains of patients with PFN1 mutation. Here, we investigated the role of PFN1 mutations in the formation of prion-like abnormal TDP-43. Expression of PFN1 with pathogenic mutations resulted in the formation of cytoplasmic aggregates positive for p62 and ubiquitin, and these aggregates sequestered endogenous TDP-43. TDP-43 accumulation was facilitated in the presence of proteasome or lysosome inhibitor. Co-expression of mutant PFN1 and TDP-43 increased the levels of detergent-insoluble and phosphorylated TDP-43, and this increase required the C-terminal region of TDP-43. Moreover, detergent-insoluble fractions prepared from cells expressing ALS-linked mutant PFN1 induced seed-dependent accumulation of TDP-43. These findings indicate that expression of PFN1 mutants induces accumulation of TDP-43, and promotes conversion of normal TDP-43 into an abnormal form. These results provide new insight into the mechanisms of TDP-43 proteinopathies and other diseases associated with amyloid-like protein deposition. 相似文献