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81.
Setting up standards and a reference map for the alkaline proteome of the Gram-positive bacterium Lactococcus lactis 总被引:2,自引:0,他引:2
Despite the fact that almost 39% of the theoretical expressed proteins of Lactococcus lactis have a predicted isoelectric point above 7, these proteins have not been studied in previous proteome analyses. In the present study, we set up a reference map of alkaline lactococcal proteins by using immobilized pH gradients (IPG) spanning pH 6 to 12 and 9 to 12, and protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Different electrophoresis systems for isoelectric focusing were evaluated to optimize the first dimension. Best results were obtained by sample application using cup-loading at the anodic side and increasing the final voltage up to 8000 V for IPGs, using N,N-dimethylacrylamide as monomer. After two-dimensional gel electrophoresis of extracts obtained from exponentially growing cells, about 200 protein spots were selected for identification by peptide mass fingerprinting. With MALDI-TOF MS, 153 proteins were identified that were the products of 85 different genes. Their predicted isoelectric points range from as high as 11.31 to as low as 6.34. Ribosomal proteins, hypothetical proteins and proteins with unknown function represent the largest groups of identified proteins. For further classification, the codon adaptation index (CAI) and grand average of hydropathicity (GRAVY) for each lactococcal protein were calculated. The protein with the lowest CAI identified in this study is the manganese ABC transporter ATP-binding protein. Less than 10% of the alkaline lactococcal proteins have a smaller CAI. The highest GRAVY for an identified protein is 0.26. The complete in silico data of Lactococcus lactis as well as clickable reference maps are available at www.wzw.tum.de/proteomik/lactis. 相似文献
82.
Background
E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated. 相似文献83.
Wendt T Karabay A Krebs A Gross H Walker R Hoenger A 《Journal of molecular biology》2003,333(3):541-552
ncd is a minus-end directed, kinesin-like motor, which binds to microtubules with its motor domain and its cargo domain as well. Typical of retrograde motors, the motor domain of ncd locates to the C-terminal end of the polypeptide chain, and hence, the cargo domain constitutes the N-terminal region. To date, several studies have investigated the interaction properties of the motor domain with microtubules, but very few structural data are available about the tail itself or its interaction with microtubules as cargo. Here, we applied cryo-electron microscopy and helical 3D image reconstruction to 15 protofilament microtubules decorated with an ncd tail fragment (N-terminal residues 83-187, named NT6). In our study, the ncd tail shows a behaviour resembling filamentous MAPs such as tau protein, exhibiting a highly flexible structure with no large globular domains. NT6 binds to four different sites on the outer side of microtubules within the proximity of the kinesin motor-binding site. Two of these sites locate within the groove between two neighbouring protofilaments, and appear as strong binding sites, while the other two sites, located at the outer rim, appear to play a secondary role. In addition, the ncd tail fragment induces the formation of large protofilament sheets, suggesting a tail-induced modification of lateral protofilament contacts. 相似文献
84.
Veldman C Stauber A Wassmuth R Uter W Schuler G Hertl M 《Journal of immunology (Baltimore, Md. : 1950)》2003,170(1):635-642
Pemphigus vulgaris (PV) is the most severe autoimmune bullous skin disorder and is primarily associated with circulating autoantibodies (autoAb) against desmoglein 3 (Dsg3). In light of recent evidence that autoreactive T cells are critical for the induction and regulation of Ab production, the goal of this study was to characterize and quantitate autoreactive T cells in patients with PV and healthy controls. Peripheral Dsg3-reactive Th cells from 28 patients with acute-onset, chronic active, and remittent PV were quantitated by MACS secretion assay. Dsg3-reactive Th2 cells were detected at similar frequencies in all studied PV patients, while the number of autoreactive Th1 cells exceeded those of Th2 cells in chronic active PV. In contrast, healthy carriers of the PV-associated HLA class II alleles, DRB1*0402 and DQB1*0503, exhibited exclusively Dsg3-reactive Th1 cell responses, while healthy carriers of other HLA class II alleles did not. Moreover, the presence of IgG1 and IgG4 against Dsg3 was directly related to the ratio of Dsg3-reactive Th1/Th2 cells. T cell recognition of Dsg3 was restricted by HLA-DRB1*0402 and DQB1*0503 in PV patients and Dsg3-responsive healthy donors. These observations strongly suggest 1) that the appearance of Dsg3-reactive Th2 cells is restricted to patients with PV; 2) that specific HLA class II alleles that are prevalent in PV are critical for T cell recognition of Dsg3 in PV patients and Dsg3-responsive healthy donors; and 3) that autoAb production is associated with both Th1 and Th2 cells. 相似文献
85.
86.
In cerebellum, 13 different GABA(A) receptor subunits are expressed. The number of different receptor subtypes formed in this tissue, their subunit composition and their quantitative importance so far has not been determined. In the present study, immunodepletion by immunoaffinity chromatography, as well as immunoprecipitation and western blot analysis was performed using 13 different subunit-specific antibodies to provide an overview on the subunit composition and abundance of GABA(A) receptor subtypes in mouse and rat cerebellum. Results obtained indicate that alpha1betaxgamma2, alpha1alpha6betaxgamma2, alpha6betaxgamma2, alpha6betaxdelta and alpha1alpha6betaxdelta are the major GABA(A) receptor subtypes present in the cerebellum. In addition, small amounts of alpha1betaxdelta receptors and a series of minor receptor subtypes containing alpha2, alpha3, alpha4, alpha5, gamma1 or gamma3 subunits are also present in the cerebellum. Whereas the abundance of alpha1alpha6betaxgamma2, alpha6betaxdelta and alpha1alpha6betaxdelta receptors is different in mouse and rat cerebellum, that of other receptors is quite similar in these tissues. Data obtained for the first time provide an overview on the GABA(A) receptor subtypes present in the cerebellum and represent the basis for further studies investigating changes in receptor expression and composition under pathological conditions. 相似文献
87.
Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells 总被引:5,自引:0,他引:5
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In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread. We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread. 相似文献
88.
Coronin 3 is a ubiquitously expressed member of the coronin protein family in mammals. In fibroblasts and HEK 293 cells, it is localized both in the cytosol and in the submembranous cytoskeleton, especially at lamellipodia and membrane ruffles. The carboxyl terminus of all coronins contains a coiled coil suggested to mediate dimerization. We show here that in contrast to other coronin homologues, the recombinant human coronin 3 carboxyl terminus forms oligomers rather than dimers, and that this part is sufficient to bind to and cross-link F-actin in vitro. The carboxyl terminus alone also conferred membrane association in vivo, and removal of the coiled coil abolished membrane localization but not in vitro F-actin binding. Coronin 3 is exclusively extracted as an oligomer from both the cytosol and the membrane fraction. Because oligomerization was not reported for other coronins, it might be a key feature governing coronin 3-specific functions. Cytosolic coronin 3 showed a high degree of phosphorylation, which is likely to regulate the subcellular localization of the protein. 相似文献
89.
Bouazoune K Mitterweger A Längst G Imhof A Akhtar A Becker PB Brehm A 《The EMBO journal》2002,21(10):2430-2440
Drosophila Mi-2 (dMi-2) is the ATPase subunit of a complex combining ATP-dependent nucleosome remodelling and histone deacetylase activities. dMi-2 contains an HMG box-like region, two PHD fingers, two chromodomains and a SNF2-type ATPase domain. It is not known which of these domains contribute to nucleosome remodelling. We have tested a panel of dMi-2 deletion mutants in ATPase, nucleosome mobilization and nucleosome binding assays. Deletion of the chromodomains impairs all three activities. A dMi-2 mutant lacking the chromodomains is incorporated into a functional histone deacetylase complex in vivo but has lost nucleosome-stimulated ATPase activity. In contrast to dHP1, dMi-2 does not bind methylated histone H3 tails and does not require histone tails for nucleosome binding. Instead, the dMi-2 chromodomains display DNA binding activity that is not shared by other chromodomains. Our results suggest that the chromodomains act at an early step of the remodelling process to bind the nucleosome substrate predominantly via protein-DNA interactions. Furthermore, we identify DNA binding as a novel chromodomain-associated activity. 相似文献
90.
Piekorz RP Hoffmeyer A Duntsch CD McKay C Nakajima H Sexl V Snyder L Rehg J Ihle JN 《The EMBO journal》2002,21(4):653-664
TACC3 is a centrosomal/mitotic spindle-associated protein that is highly expressed in a cell cycle-dependent manner in hematopoietic lineage cells. During embryonic development, TACC3 is expressed in a variety of tissues in addition to the hematopoietic lineages. TACC3 deficiency causes an embryonic lethality at mid- to late gestation involving several lineages of cells. Hematopoietic stem cells, while capable of terminal differentiation, are unable to be expanded in vitro or in vivo in reconstitution approaches. Although gross alterations in centrosome numbers and chromosomal segregation are not observed, TACC3 deficiency is associated with a high rate of apoptosis and expression of the p53 target gene, p21(Waf1/Cip1). Hematopoietic stem cell functions, as well as deficiencies in other cell lineages, can be rescued by combining the TACC3 deficiency with p53 deficiency. The results support the concept that TACC3 is a critical component of the centrosome/mitotic spindle apparatus and its absence triggers p53-mediated apoptosis. 相似文献