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81.
82.
Chiu LL Perng DW Yu CH Su SN Chow LP 《Journal of immunology (Baltimore, Md. : 1950)》2007,178(8):5237-5244
Allergenic serine proteases are important in the pathogenesis of asthma. One of these, Pen c 13, is the immunodominant allergen produced by Penicillium citrinum. Many serine proteases induce cytokine expression, but whether Pen c 13 does so in human respiratory epithelial cells is not known. In this study, we investigated whether Pen c 13 caused IL-8 release and activated protease-activated receptors (PARs) in airway epithelial cells. In airway-derived A549 cells and normal human airway epithelial cells, Pen c 13 induced IL-8 release in a dose-dependent manner. Pen c 13 also increased IL-8 release in a time-dependent manner in A549 cells. Pen c 13 cleaved PAR-1 and PAR-2 at their activation sites. Treatment with Pen c 13 induced intracellular Ca(2+) mobilization and desensitized the cells to the action of other proteases and PAR-1 and PAR-2 agonists. Moreover, Pen c 13-mediated IL-8 release was significantly decreased in Ca(2+)-free medium and was abolished by the protease inhibitors, PMSF and 4-(2-aminoethyl) benzenesulfonyl fluoride. Blocking Abs against the cleavage sites of PAR-1 and PAR-2, but not of PAR-4, inhibited Pen c 13-induced IL-8 production, as did inhibition of phospholipase C. Pen c 13 induced IL-8 expression via activation of ERK 1/2, and not of p38 and JNK. In addition, treatment of A549 cells or normal human airway epithelial cells with Pen c 13 increased phosphorylation of ERK 1/2 by a Ca(2+)-dependent pathway. These finding show that Pen c 13 induces IL-8 release in airway epithelial cells and that this is dependent on PAR-1 and PAR-2 activation and intracellular calcium. 相似文献
83.
The speed of the human genome project (Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C. et al., Nature 2001, 409, 860-921) was made possible, in part, by developments in automation of sequencing technologies. Before these technologies, sequencing was a laborious, expensive, and personnel-intensive task. Similarly, automation and robotics are changing the field of proteomics today. Proteomics is defined as the effort to understand and characterize proteins in the categories of structure, function and interaction (Englbrecht, C. C., Facius, A., Comb. Chem. High Throughput Screen. 2005, 8, 705-715). As such, this field nicely lends itself to automation technologies since these methods often require large economies of scale in order to achieve cost and time-saving benefits. This article describes some of the technologies and methods being applied in proteomics in order to facilitate automation within the field as well as in linking proteomics-based information with other related research areas. 相似文献
84.
Chew WM Xu MJ Cordova CA Chow HH 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2006,844(2):235-239
A sensitive HPLC-tandem mass spectrometry method was developed for determination of buspirone levels in human plasma. After solid phase extraction and reversed phase HPLC separation, detection of buspirone and the internal standard (prazosin) was performed using eletrospray ionization and selected reaction monitoring in the positive ion mode. Linear calibration curves were established over a concentration range of 0.025-2.5 ng/ml when 0.5 ml aliquots of plasma were used. Satisfactory results of within-day precision (RSD of 1.9-7.7%) and accuracy (% difference of 0.5-6.6%) and between-day precision (RSD of 3.7-11.1%) and accuracy (% difference of 2.2-6.8%) were obtained. The assay has been successfully applied to the analysis of buspirone levels in more than 500 human plasma samples collected from a drug interaction study. 相似文献
85.
Ye L Zhu W Backx PH Cortez MA Wu J Chow YH McKerlie C Wang A Tsui LC Gross GJ Hu J 《Journal of cellular and molecular medicine》2011,15(11):2307-2316
The identification and analysis of several cationic ion channels and their associated genes have greatly improved our understanding of the molecular and cellular mechanisms of cardiac arrhythmia. Our objective in this study was to examine the involvement of anionic ion channels in cardiac arrhythmia. We used a transgenic mouse model to overexpress the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-regulated chloride channel. We used RNase protection and in situ hybridization assays to determine the level of CFTR expression, and radiotelemetry and in vivo electrophysiological study in combination with pharmacological intervention to analyse the cardiac function. Cardiac CFTR overexpression leads to stress-related sudden death in this model. In vivo intracardiac electrophysiological studies performed in anaesthetized mice showed no significant differences in baseline conduction parameters including atrial-His bundle (AH) or His bundle-ventricular (HV) conduction intervals, atrioventricular (AV) Wenckebach or 2:1 AV block cycle length and AV nodal functional refractory period. However, following isoproterenol administration, there was marked slowing of conduction parameters, including high-grade AV block in transgenic mice, with non-sustained ventricular tachycardia easily inducible using programmed stimulation or burst pacing. Our sudden death mouse model can be a valuable tool for investigation of the role of chloride channels in arrhythmogenesis and, potentially, for future evaluation of novel anti-arrhythmic therapeutic strategies and pharmacological agents. 相似文献
86.
Molecular variants of human papillomavirus type 16 from four continents suggest ancient pandemic spread of the virus and its coevolution with humankind. 总被引:7,自引:0,他引:7 下载免费PDF全文
S Y Chan L Ho C K Ong V Chow B Drescher M Dürst J ter Meulen L Villa J Luande H N Mgaya et al. 《Journal of virology》1992,66(4):2057-2066
We have amplified by the polymerase chain reaction, cloned, and sequenced genomic segments of 118 human papillomavirus type 16 (HPV-16) isolates from 76 cervical biopsy, 14 cervical smear, 3 vulval biopsy, 2 penile biopsy, 2 anal biopsy, and 1 vaginal biopsy sample and two cell lines. The specimens were taken from patients in four countries--Singapore, Brazil, Tanzania, and Germany. The sequence of a 364-bp fragment of the long control region of the virus revealed 38 variants, most of which differed by one or several point mutations. Phylogenetic trees were constructed by distance matrix methods and a transformation series approach. The trees based on the long control region were supported by another set based on the complete E5 protein-coding region. Both sets had two main branches. Nearly all of the variants from Tanzania were assigned to one (African) branch, and all of the German and most of the Singaporean variants were assigned to the other (Eurasian) branch. While some German and Singaporean variants were identical, each group also contained variants that formed unique branches. In contrast to the group-internal homogeneity of the Singaporean, German, and Tanzanian variants, the Brazilian variants were clearly divided between the two branches. Exceptions to this were the seven Singaporean isolates with mutational patterns typical of the Tanzanian isolates. The data suggest that HPV-16 evolved separately for a long period in Africa and Eurasia. Representatives of both branches may have been transferred to Brazil via past colonial immigration. The comparable efficiencies of transfer of the African and the Eurasian variants to the New World suggest pandemic spread of HPV-16 in past centuries. Representatives of the African branch were possibly transferred to the Far East along old Arab and Indonesian sailing routes. Our data also support the view that HPV-16 is a well-defined virus type, since the variants show only a maximal genomic divergence of about 5%. The small amount of divergence in any one geographic location and the lack of marked divergence between the Tanzanian and Brazilian African genome variants two centuries after their likely introduction into the New World suggest a very slow rate of viral evolution. The phylogenetic tree therefore probably represents a minimum of several centuries of evolution, if not an age equal to that of the respective human races. 相似文献
87.
Upon binding to the poliovirus receptor (PVR), the poliovirus 160S particles undergo a conformational transition to generate 135S particles, which are believed to be intermediates in the virus entry process. The 135S particles interact with host cell membranes through exposure of the N termini of VP1 and the myristylated VP4 protein, and successful cytoplasmic delivery of the genomic RNA requires the interaction of these domains with cellular membranes whose identity is unknown. Because detergent-insoluble microdomains (DIMs) in the plasma membrane have been shown to be important in the entry of other picornaviruses, it was of interest to determine if poliovirus similarly required DIMs during virus entry. We show here that methyl-beta-cyclodextrin (MbetaCD), which disrupts DIMs by depleting cells of cholesterol, inhibits virus infection and that this inhibition was partially reversed by partially restoring cholesterol levels in cells, suggesting that MbetaCD inhibition of virus infection was mediated by removal of cellular cholesterol. However, fractionation of cellular membranes into DIMs and detergent-soluble membrane fractions showed that both PVR and poliovirus capsid proteins localize not to DIMs but to detergent-soluble membrane fractions during entry into the cells, and their localization was unaffected by treatment with MbetaCD. We further demonstrate that treatment with MbetaCD inhibits RNA delivery after formation of the 135S particles. These data indicate that the cholesterol status of the cell is important during the process of genome delivery and that these entry pathways are distinct from those requiring DIM integrity. 相似文献
88.
A mutation in VP4 defines a new step in the late stages of cell entry by poliovirus. 总被引:2,自引:16,他引:2 下载免费PDF全文
During the entry of poliovirus into cells, a conformational transition occurs within the virion that is dependent upon its binding to the cell surface receptor. This conformational rearrangement generates an altered particle of 135S, results in the extrusion of capsid protein VP4 and the amino terminus of VP1 from the virion interior, and leads to the acquisition of membrane-binding properties by the 135S particle. Although the subsequent fate of VP4 is unknown, its apparent absence from purified 135S particles has long suggested that VP4 is not directly involved during virus entry. We report here the construction by site-specific mutagenesis of a nonviable VP4 mutant that upon transfection of the cDNA appears to form mature virus particles. These particles, upon interaction with the cellular receptor, undergo the 135S conformational transition but are defective at a subsequent stage in virus entry. The results demonstrate that the participation of VP4 is required during cell entry of poliovirus. In addition, these data indicate the existence of additional stages in the cell entry process beyond receptor binding and the transition to 135S particles. These post-135S stages must include the poorly understood processes by which nonenveloped viruses cross the cell membrane, uncoat, and deliver their genomes into the cytoplasm. 相似文献
89.
90.
Birgit Eisenhaber Stephan Eisenhaber Toh Yew Kwang Gerhard Grüber 《Cell cycle (Georgetown, Tex.)》2014,13(12):1912-1917
The transamidase subunit GAA1/GPAA1 is predicted to be the enzyme that catalyzes the attachment of the glycosylphosphatidyl (GPI) lipid anchor to the carbonyl intermediate of the substrate protein at the ω-site. Its ~300-amino acid residue lumenal domain is a M28 family metallo-peptide-synthetase with an α/β hydrolase fold, including a central 8-strand β-sheet and a single metal (most likely zinc) ion coordinated by 3 conserved polar residues. Phosphoethanolamine is used as an adaptor to make the non-peptide GPI lipid anchor look chemically similar to the N terminus of a peptide. 相似文献