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81.
The way that UL42, the processivity subunit of the herpes simplex virus DNA polymerase, interacts with DNA and promotes processivity remains unclear. A positively charged face of UL42 has been proposed to participate in electrostatic interactions with DNA that would tether the polymerase to a template without preventing its translocation via DNA sliding. An alternative model proposes that DNA binding by UL42 is not important for processivity. To investigate these issues, we substituted alanine for each of four conserved arginine residues on the positively charged surface. Each single substitution decreased the DNA binding affinity of UL42, with 14- to 30-fold increases in apparent dissociation constants. The mutant proteins exhibited no meaningful change in affinity for binding to the C terminus of the catalytic subunit of the polymerase, indicating that the substitutions exert a specific effect on DNA binding. The substitutions decreased UL42-mediated long-chain DNA synthesis by the polymerase in the same rank order in which they affected DNA binding, consistent with a role for DNA binding in polymerase processivity. Combining these substitutions decreased DNA binding further and impaired the complementation of a UL42 null virus in transfected cells. Additionally, using a revised mathematical model to analyze rates of dissociation of UL42 from DNAs of various lengths, we found that dissociation from internal sites, which would be the most important for tethering the polymerase, was relatively slow, even at ionic strengths that permit processive DNA synthesis by the holoenzyme. These data provide evidence that the basic surface of UL42 interacts with DNA and support a model in which DNA binding by UL42 is important for processive DNA synthesis.  相似文献   
82.
Liu J  Brown CR  Chiang HL 《Autophagy》2005,1(3):146-156
The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced during glucose starvation. After the addition of glucose, inactivated FBPase is selectively targeted to Vid (vacuolar import and degradation) vesicles and then to the vacuole for degradation. To identify proteins involved in this pathway, we screened various libraries for mutants that failed to degrade FBPase. Via these approaches, subunits of the vacuolar- H+ -ATPase (V-ATPase) have been identified repeatedly. The V-ATPase has established roles in endocytosis, sorting of carboxypeptidase Y and homotypic vacuole fusion. Here, we show that mutants lacking Stv1p, Vph1p, and other subunits of the V-ATPase are defective for FBPase degradation. FBPase was detected in Vid vesicles. However, most FBPase was resistant to proteinase K digestion in the Deltavph1 or vma mutants, whereas the majority of FBPase was sensitive to proteinase K digestion in the Deltastv1 mutant. Therefore, STV1 and VPH1 have distinct functions in FBPase degradation. In cells lacking V0 genes, Vma2p and Vma5p were still detected on Vid vesicles and vacuoles, suggesting that the distribution of V1 proteins is independent of V0 genes. The V0 and V1 domains are assembled following a glucose shift and the assembly is not regulated by protein kinase A and RAV genes. Assembly of the V0 complex is necessary for FBPase trafficking, since mutants that block the assembly and transport of V0 out of the ER were defective in FBPase degradation.  相似文献   
83.
Cultures of JU56 cells were irradiated with 2.5 Gy X-rays and 16 h later the cultures were exposed to a moderately inhibitory dose of 1-beta-D-arabinofuranosylcytosine (ara-C) or aphidicolin (APC) and to colcemid, for 2 h. The c-metaphases collected for examination had therefore been exposed to X-rays in G1 or early S, and to the repair inhibitors APC and ara-C during the latter half of G2. It was found that treatment of cells irradiated early in cell cycle, that is, in G1 and early S, with APC or ara-C in G2, (1) reduced the frequency of chromatid and chromosome exchanges below that of cells treated with X-rays alone, (2) produced no more chromatid breaks and gaps than were seen in unirradiated cells, (3) increased the number of chromosome fragments and gaps in a more than additive fashion, and (4) produced only an additive effect, by comparison with the effect of X-rays and drug given separately, on the total number of chromosomal aberrations.  相似文献   
84.
The mycotoxin ochratoxin A (OA) consists of 5-chloro-3-methyl-3,4-dihydro-8-hydroxyisocoumarin moiety linked by an amide bond to β-L-phenylalanine. When added to washed rat platelets in vitro, OA caused a dose-dependent inhibition of aggregation induced by agonists such as adenosine diphosphate (ADP) or thrombin. The aggregatory response induced by prior addition of an agonist was also reversed in a dose-dependent manner by OA. Inhibition of aggregation appeared to be irreversible since exposure of platelets to OA followed by several washings removed most of the mycotoxin associated with the platelets but did not diminish the inhibitory response. Serotonin secretion from dense granules and arachidonic acid release from membrane phospholipid (especially phosphatidylcholine) as well as its further metabolism were also inhibited by OA. These results suggest that a disruption of the platelet plasma membrane structure by OA is probably responsible for inhibition of the primary and secondary phases of aggregation.  相似文献   
85.
We describe a method for creating differentiated equine bronchial epithelial cell cultures that can be used for in vitro studies including airway disease mechanisms and pathogen–host interactions. Our method is based on the culturing of human tracheobronchial epithelial cells at an air–liquid interface (ALI) in specific serum-free, hormone-supplemented medium. Bronchial epithelial cells are isolated and grown on T-Clear® insert membranes. Within 2 to 3 wk, cells differentiate into ciliated and mucus producing cells as demonstrated by confocal and electron microscopy. Furthermore, the demonstration of the two major gel-forming mucin species, Muc5ac and Muc5b, in our bronchial epithelial cell culture system validates this method for studies of respiratory tract disease of the horse.  相似文献   
86.
Recipient airway epithelial cells are found in human sex-mismatched lung transplants, implying that circulating progenitor epithelial cells contribute to the repair of the airway epithelium. Markers of circulating progenitor epithelial cells and mechanisms for their trafficking remain to be elucidated. We demonstrate that a population of progenitor epithelial cells exists in the bone marrow and the circulation of mice that is positive for the early epithelial marker cytokeratin 5 (CK5) and the chemokine receptor CXCR4. We used a mouse model of sex-mismatched tracheal transplantation and found that CK5+ circulating progenitor epithelial cells contribute to re-epithelialization of the airway and re-establishment of the pseudostratified epithelium. The presence of CXCL12 in tracheal transplants provided a mechanism for CXCR4+ circulating progenitor epithelial cell recruitment to the airway. Depletion of CXCL12 resulted in the epithelium defaulting to squamous metaplasia, which was derived solely from the resident tissue progenitor epithelial cells. Our findings demonstrate that CK5+CXCR4+ cells are markers of circulating progenitor epithelial cells in the bone marrow and circulation and that CXCR4/CXCL12-mediated recruitment of circulating progenitor epithelial cells is necessary for the re-establishment of a normal pseudostratified epithelium after airway injury. These findings support a novel paradigm for the development of squamous metaplasia of the airway epithelium and for developing therapeutic strategies for circulating progenitor epithelial cells in airway diseases.  相似文献   
87.
The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.  相似文献   
88.
BackgroundGreen tea catechins have been hypothesized to increase energy expenditure and fat oxidation by inhibiting catechol-O-methyltransferase (COMT) and thus promoting more sustained adrenergic stimulation. Metabolomics may help to clarify the mechanisms underlying their putative physiological effects.ObjectiveThe study investigated the effects of 7-day ingestion of green tea extract (GTE) on the plasma metabolite profile at rest and during exercise.MethodsIn a placebo-controlled, double-blind, randomized, parallel study, 27 healthy physically active males consumed either GTE (n=13, 1200 mg catechins, 240 mg caffeine/day) or placebo (n=14, PLA) drinks for 7 days. After consuming a final drink (day 8), they rested for 2 h and then completed 60 min of moderate-intensity cycling exercise (56%±4% VO2max). Blood samples were collected before and during exercise. Plasma was analyzed using untargeted four-phase metabolite profiling and targeted profiling of catecholamines.ResultsUsing the metabolomic approach, we observed that GTE did not enhance adrenergic stimulation (adrenaline and noradrenaline) during rest or exercise. At rest, GTE led to changes in metabolite concentrations related to fat metabolism (3-β-hydroxybutyrate), lipolysis (glycerol) and tricarboxylic acid cycle (TCA) cycle intermediates (citrate) when compared to PLA. GTE during exercise caused reductions in 3-β-hydroxybutyrate concentrations as well as increases in pyruvate, lactate and alanine concentrations when compared to PLA.ConclusionsGTE supplementation resulted in marked metabolic differences during rest and exercise. Yet these metabolic differences were not related to the adrenergic system, which questions the in vivo relevance of the COMT inhibition mechanism of action for GTE.  相似文献   
89.
90.
Sialylated glycans serve as cell surface attachment factors for a broad range of pathogens. We report an atypical example, where desialylation increases cell surface binding and infectivity of adeno-associated virus (AAV) serotype 9, a human parvovirus isolate. Enzymatic removal of sialic acid, but not heparan sulfate or chondroitin sulfate, increased AAV9 transduction regardless of cell type. Viral binding and transduction assays on mutant Chinese hamster ovary (CHO) cell lines defective in various stages of glycan chain synthesis revealed a potential role for core glycan residues under sialic acid in AAV9 transduction. Treatment with chemical inhibitors of glycosylation and competitive inhibition studies with different lectins suggest that N-linked glycans with terminal galactosyl residues facilitate cell surface binding and transduction by AAV9. In corollary, resialylation of galactosylated glycans on the sialic acid-deficient CHO Lec2 cell line with different sialyltransferases partially blocked AAV9 transduction. Quantitative analysis of AAV9 binding to parental, sialidase-treated or sialic acid-deficient mutant CHO cells revealed a 3-15-fold increase in relative binding potential of AAV9 particles upon desialylation. Finally, pretreatment of well differentiated human airway epithelial cultures and intranasal instillation of recombinant sialidase in murine airways enhanced transduction efficiency of AAV9 by >1 order of magnitude. Taken together, the studies described herein provide a molecular basis for low infectivity of AAV9 in vitro and a biochemical strategy to enhance gene transfer by AAV9 vectors in general.  相似文献   
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