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51.
Functional analysis of the cag pathogenicity island in Helicobacter pylori isolates from patients with gastritis, peptic ulcer, and gastric cancer 总被引:9,自引:0,他引:9 下载免费PDF全文
Backert S Schwarz T Miehlke S Kirsch C Sommer C Kwok T Gerhard M Goebel UB Lehn N Koenig W Meyer TF 《Infection and immunity》2004,72(2):1043-1056
Helicobacter pylori is the causative agent of a variety of gastric diseases, but the clinical relevance of bacterial virulence factors is still controversial. Virulent strains carrying the cag pathogenicity island (cagPAI) are thought to be key players in disease development. Here, we have compared cagPAI-dependent in vitro responses in H. pylori isolates obtained from 75 patients with gastritis, peptic ulcer, and gastric cancer (n = 25 in each group). AGS gastric epithelial cells were infected with each strain and assayed for (i) CagA expression, (ii) translocation and tyrosine phosphorylation of CagA, (iii) c-Src inactivation, (iv) cortactin dephosphorylation, (v) induction of actin cytoskeletal rearrangements associated with cell elongation, (vi) induction of cellular motility, and (vii) secretion of interleukin-8. Interestingly, we found high but similar prevalences of all of these cagPAI-dependent host cell responses (ranging from 56 to 80%) among the various groups of patients. This study revealed CagA proteins with unique features, CagA subspecies of various sizes, and new functional properties for the phenotypic outcomes. We further showed that induction of AGS cell motility and elongation are two independent processes. Our data corroborate epidemiological studies, which indicate a significant association of cagPAI presence and functionality with histopathological findings in gastritis, peptic ulcer, and gastric cancer patients, thus emphasizing the importance of the cagPAI for the pathogenicity of H. pylori. Nevertheless, we found no significant association of the specific H. pylori-induced responses with any particular patient group. This may indicate that the determination of disease development is highly complex and involves multiple bacterial and/or host factors. 相似文献
52.
The L RNA of three Lassa virus strains originating from Nigeria, Ghana/Ivory Coast, and Sierra Leone was sequenced and the data subjected to structure predictions and phylogenetic analyses. The L gene products had 2218-2221 residues, diverged by 18% at the amino acid level, and contained several conserved regions. Only one region of 504 residues (positions 1043-1546) could be assigned a function, namely that of an RNA polymerase. Secondary structure predictions suggest that this domain is very similar to RNA-dependent RNA polymerases of known structure encoded by plus-strand RNA viruses, permitting a model to be built. Outside the polymerase region, there is little structural data, except for regions of strong alpha-helical content and probably a coiled-coil domain at the N terminus. No evidence for reassortment or recombination during Lassa virus evolution was found. The secondary structure-assisted alignment of the RNA polymerase region permitted a reliable reconstruction of the phylogeny of all negative-strand RNA viruses, indicating that Arenaviridae are most closely related to Nairoviruses. In conclusion, the data provide a basis for structural and functional characterization of the Lassa virus L protein and reveal new insights into the phylogeny of negative-strand RNA viruses. 相似文献
53.
When Marburg virus (MBGV) nucleoprotein (NP) is expressed in insect cells, it binds to cellular RNA and forms NP-RNA complexes such as insect cell-expressed nucleoproteins from other nonsegmented negative-strand RNA viruses. Recombinant MBGV NP-RNA forms loose coils that resemble rabies virus N-RNA. MBGV NP monomers are rods that are spaced along the coil similar to the nucleoprotein monomers of the rabies virus N-RNA. High salt treatment induces tight coiling of the MBGV NP-RNA, again a characteristic observed for other nonsegmented negative-strand virus N-RNAs. Electron microscopy of fixed Marburg virus particles shows that the viral nucleocapsid has a smaller diameter than the free, recombinant NP-RNA. This difference in helical parameters could be caused by the interaction of other viral proteins with the NP-RNA. A similar but opposite phenomenon is observed for rhabdovirus nucleocapsids that are condensed by the viral matrix protein upon which they acquire a larger diameter. Finally, there appears to be an extensive and regular protein scaffold between the viral nucleocapsid and the membrane that seems not to exist in the other negative-strand RNA viruses. 相似文献
54.
Zipfel S Schneider A Wild B Löwe B Jünger J Haass M Sack FU Bergmann G Herzog W 《Psychosomatic medicine》2002,64(5):740-747
OBJECTIVE: This study explored the value of preoperative self-reported assessment for depression and anxiety in patients who had undergone heart transplantation (HTx). The initial sample was divided into subgroups of patients with ischemic (ICMP) and dilated cardiomyopathy (DCMP). Patient depression and anxiety scores were measured in both subgroups and their impact on pre- and postoperative mortality investigated. METHOD: An initial sample of 152 patients with either ICMP (N = 57) or DCMP (N = 95) and end-stage heart disease awaiting heart transplantation were assessed in a multidimensional longitudinal study, including psychological and somatic variables. One hundred and three patients received a HTx and were followed up for a mean of 4.4 years. Proportional hazard models were computed to test for the influence of psychosocial and somatic factors on outcome. RESULTS: Preoperative depression and state anxiety scores were significantly higher in the ICMP group. In addition to donor and recipient age, ICMP patients in the preoperative high depression group also showed a significantly higher mortality after HTx. This result remained significant even after controlling for sociodemographic and somatic variables. CONCLUSIONS: Patient self-reported depression, but not anxiety, can contribute to the identification of subgroups of patients with an unfavorable outcome after HTx. It therefore may be helpful to screen for depression, particularly in patients with an ischemic cause of their end-stage heart disease. Specific intervention programs should be further developed and evaluated. 相似文献
55.
Swiatek M Greiner S Kemp S Drescher A Koop HU Herrmann RG Maier RM 《Current genetics》2003,43(1):45-53
The genetic transformation of plastids of higher plants has developed into a powerful approach for both basic research and biotechnology. Due to the high copy number of the plastid genome per plastid and per cell, repeated cycles of shoot regeneration under conditions selective for the modified plastid chromosome are required to obtain transformants entirely lacking wild-type plastid genomes. The presence of promiscuous plastid DNA in nuclear and/or mitochondrial genomes that generally contaminate even gradient-purified plastid fractions reduces the applicability of the highly sensitive PCR approach to monitor the absence of residual wild-type plastid chromosomes in transformed lines. It is therefore difficult, or even impossible, to assess reliably the hetero- or homoplastomic state of plastid transformants in this manner. By analysing wild-type and transplastomic mutants of tobacco, we demonstrate that separation of plastid chromosomes isolated from gradient-purified plastid fractions by pulsed-field gel electrophoresis can overcome the problem of (co)amplification of interfering promiscuous plastid DNA. PCR analyses with primers specific for plastid, mitochondrial and nuclear genes reveal an impressive purity of such plastid DNA fractions at a detection limit of less than one wild-type plastid chromosome copy per ten transplastomic cells. 相似文献
56.
Tommasi S Fedele V Crapolicchio A Bellizzi A Paradiso A Reshkin SJ 《International journal of molecular medicine》2003,12(1):131-134
The erbB2 gene is often found amplified and/or overexpressed in breast cancer in which it has clinical relevance as prognostic and predictive factor. It is involved in growth regulation and has a role in the initial phases of cell proliferation, while in vivo and in vitro studies have suggested an involvement also in cell invasion and metastases. It is not clear if these two roles are mutually exclusive and little is known about the mechanisms by which erbB2 may be involved in the control of these processes. Our previous data on patient series suggested that erbB2 might be regulated in different ways depending on the neoplastic status of the cells and that it might be involved in different regulatory pathways. To test this hypothesis we have measured the serum-dependent regulation of erbB2 as a function of the expression of the antimetastatic gene, nm23, in a panel of breast cancer cell lines. The experimental model consisted of three cell lines having different proliferative and invasive potentials: a non-metastatic estrogen receptor (ER) positive cell line, MCF-7; a highly metastatic ER negative cell line, MDA-MB435; and the MDA-MB435 cell line transfected with the nm23-H1 antimetastatic gene (clone H1-177) which has lost the ability to invade and metastasize. We first analysed the serum concentration dependence of invasion and proliferation after 3-4 days of serum deprivation confirming the proliferative and invasive potential of the three cell lines. Modulation of erbB2 expression by different concentrations of serum was then studied. ErbB2 expression in MCF-7 cells showed a complex pattern due to serum modulation, whereas, it was not longer regulated by serum in the MDA-MB435 cell line. In H1-177 cells the erbB2 response to serum was restored and it was very similar to that observed in MCF-7. These data showed a tight association between nm23 and the regulation of erbB2 expression by serum factors suggesting that the role of erbB2 in invasion might be dependent on nm23 expression. 相似文献
57.
Kemp S Valianpour F Denis S Ofman R Sanders RJ Mooyer P Barth PG Wanders RJ 《Molecular genetics and metabolism》2005,84(2):144-151
X-linked adrenoleukodystrophy (X-ALD) is a progressive neurodegenerative disorder characterized by the accumulation of saturated and mono-unsaturated very long-chain fatty acids (VLCFA) and reduced peroxisomal VLCFA beta-oxidation activity. In this study, we investigated the role of VLCFA biosynthesis in X-ALD fibroblasts. Our data demonstrate that elongation of both saturated and mono-unsaturated VLCFAs is enhanced in fibroblasts from patients with peroxisomal beta-oxidation defects including X-ALD, and peroxisome biogenesis disorders. These data indicate that enhanced VLCFA elongation is a general phenomenon associated with an impairment in peroxisomal beta-oxidation, and not specific for X-ALD alone. Analysis of plasma samples from patients with X-ALD and different peroxisomal beta-oxidation deficiencies revealed increased concentrations of VLCFAs up to 32 carbons. We infer that enhanced elongation does not result from impaired peroxisomal beta-oxidation alone, but is due to the additional effect of unchecked chain elongation. We demonstrate that elongated VLCFAs are incorporated into complex lipids. The role of chain elongation was also studied retrospectively in samples from patients with X-ALD previously treated with "Lorenzo's oil." We found that the decrease in plasma C26:0 previously found is offset by the increase of mono-unsaturated VLCFAs, not measured previously during the trial. We conclude that evaluation of treatment protocols for disorders of peroxisomal beta-oxidation making use of plasma samples should include the measurement of saturated and unsaturated VLCFAs of chain lengths above 26 carbon atoms. We also conclude that chain elongation offers an interesting target to be studied as a possible mode of treatment for X-ALD and other peroxisomal beta-oxidation disorders. 相似文献
58.
Valianpour F Selhorst JJ van Lint LE van Gennip AH Wanders RJ Kemp S 《Molecular genetics and metabolism》2003,79(3):189-196
Elevated levels of very long-chain fatty acids (VLCFA) in plasma and tissues are the biochemical hallmark for patients with X-linked adrenoleukodystrophy (X-ALD). Current methods for the determination of VLCFA levels are laborious and time-consuming. We describe a rapid and easy method using electrospray ionization mass spectrometry (ESI-MS) with deuterated internal standards. VLCFA are hydrolyzed, extracted, and quantified in less than 4h. This includes 2h of hydrolysis and 4min of quantification. We validated the method by analyzing 60 plasma samples from controls and patients with X-ALD or Zellweger syndrome using both the ESI-MS protocol and an established method for VLCFA analysis using gas chromatography (GC). The C26:0 concentrations determined with ESI-MS in plasma and fibroblasts of X-ALD patients are in good agreement with those reported previously for GC and GC-MS. Besides saturated straight chain VLCFA, we also determined the concentrations of the mono-unsaturated VLCFA C24:1 and C26:1 and established that while C24:1 levels are not elevated, C26:1 levels are elevated in both plasma and fibroblasts from X-ALD patients. 相似文献
59.
The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation 总被引:8,自引:0,他引:8
Géza Szücs Stephan Heinke Christine De Greef Luc Raeymaekers Jan Eggermont Guy Droogmans Bernd Nilius 《Pflügers Archiv : European journal of physiology》1996,431(4):540-548
We employed the patch-clamp technique to investigate the effects of various phosphorylation pathways on activation and modulation of volume-activated Cl- currents (I
Cl,vol) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation ofI
Cl,vol occurred at a hypotonicity of 27.5 ± 1.2%. Run-down of the current upon repetitive activation was less than 15% within 60 min. Stimulation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) or by (–)-indolactam did not affectI
Cl,vol. Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 mol/l PMA, or its inhibition by loading the cells with the specific inhibitory 19–31 pseudosubstrate peptide, did not influenceI
Cl,vol. Trifluoperazine and tamoxifen fully blockedI
cCl,vol with concentrations required for half-maximal inhibition of 3.0 and 2.4 mol/1 respectively. This inhibitory effect is probably not mediated by the calmodulin-antagonistic action of these compounds, because it occurs at free intracellular [Ca2+] of 50 nmol/l, which are below the threshold for calmodulin activation. The tyrosine kinase inhibitor herbimycin A (1 ol/1) and genistein (100 ol/1) did not affectI
Cl,vol Exposing CPAE cells to lysophosphatidic acid (1mol/1), an activator of p42 MAPkinase and the focal adhesion kinase p125FAK in endothelial cells, neither evoked a Cl– current nor affectedI
Cl,vol Neither wortmannin (10 mol/1), an inhibitor of MAP kinases and of PI-3 kinase, nor rapamycin (0.1 mmol/1), which interferes with the p70S6 kinase pathway, affectedI
Cl,vol Exposure of CPAE cells to heat or Na-arsenite, both activators of a recently discovered stress-activated tyrosine phosphorylation pathway, neither activated a current nor affected the hypotonic solution-induced Cl– current. We conclude that none of the studied phosphorylation pathways is essential for the activation of the Cl– current induced by hypotonicity. 相似文献
60.
Stephan A. Wacker Franz Oswald Jörg Wiedenmann Walter Knöchel 《Developmental dynamics》2007,236(2):473-480
Lineage labeling is one of the most important techniques in developmental biology. Most recently, a set of photoactivatable fluorescent proteins originating from marine cnidarians became available. Here, we introduce the application of the green to red photoconvertible protein EosFP as a novel technique to analyze early vertebrate development. Both injection of EosFP mRNA and purified, recombinant EosFP followed by a light-driven green to red conversion allow lineage labeling in virtually any temporal and spatial dimension during embryonic development for at least 2 weeks. Specific staining of cells from nonsurface layers is greatly facilitated by light-driven conversion of EosFP compared with traditional methods. Therefore, green to red photoactivatable proteins promise to be a powerful tool with the potential to satisfy the increasing demand for methods enabling detailed phenotypical analyses after manipulations of morphogenetic events, gene expression, or signal transduction. 相似文献