Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Infections caused by Chlamydia spp are an important cause of human disease, and the accuracy and reproducibility of antimicrobial susceptibility tests for these bacteria could have considerable clinical implications. We have developed a reverse transcriptase PCR (RT-PCR) based method to determine the antibiotic susceptibility of Chlamydia spp., and compared this with conventional tests using immunofluoresence (IF) staining. The MICs of antimicrobial agents for a test strain of Chlamydia pneumoniae were higher by RT-PCR as compared with IF staining, indicating the greater stringency of the former method. Using RT-PCR, doxycycline and tetracycline were the most active agents (MIC 1 mg/L), followed by erythromycin (1.6 mg/L), and ciprofloxacin (16 mg/L). Neither trimethoprim nor sulphamethoxazole (400 mg/L) inhibited growth as assessed by both techniques. The RT-PCR based method may thus represent an improved and less time consuming assay for in-vitro determination of the antibiotic susceptibility of Chlamydia spp.
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PMID:A reverse transcriptase-PCR based assay for in-vitro antibiotic susceptibility testing of Chlamydia pneumoniae. 872 33

Increased stimulation of Th2 cytokines may contribute to the development of persistent ocular chlamydial infection, resulting in the blinding pathological changes of trachoma. Proliferation and cytokine production profiles of PBMC in response to stimulation with antigens of Chlamydia trachomatis were compared in 30 patients with severe conjunctival scarring due to trachoma and in 30 age-, sex- and location-matched controls. Interferon-gamma (IFN-gamma) and IL-4 were detected at the single-cell level by ELISPOT assay. Transcription of the genes encoding IFN-gamma, IL-4 and IL-10 was detected in mRNA isolated from parallel cultures of PBMC using reverse transcriptase-polymerase chain reaction (RT-PCR). Incubation with the chlamydial heat shock protein (hsp)60 resulted in increased numbers of IL-4-producing cells in PBMC isolated from patients with scarring disease and increased secretion of IFN-gamma from PBMC of control subjects. Incubation with the chlamydial major outer membrane protein (MOMP) increased the number of IFN-gamma-producing cells in the control group only. Messenger RNA encoding IL-4 was only detected in PBMC of patients with scarring disease after in vitro stimulation with chlamydial antigens, but IFN-gamma mRNA and IL-10 mRNA were also more frequently detected in this group. Thirty-eight subjects were HLA-DRB1 and -DQB1 typed. Associations were observed between certain HLA class II alleles and cellular immune responses to chlamydial antigens. No HLA associations were found with clinical status, and overall we found no evidence of strong associations and the type of immune response. These data are consistent with a role for Th2 cells and cytokines in the pathogenesis of trachomatous scarring.
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PMID:T helper type-1 (Th1)/Th2 profiles of peripheral blood mononuclear cells (PBMC); responses to antigens of Chlamydia trachomatis in subjects with severe trachomatous scarring. 880 30

The presence of Chlamydia pneumoniae in atheromas has been demonstrated in several studies. Culture of the organism from arterial tissue has been difficult. We report the use of a reverse transcriptase polymerase chain reaction to detect viable Chlamydia pneumoniae in carotid atheromas. We analyzed 30 patients (14 females, mean age 69.6 +/- 8.8 years) who underwent surgery for the removal of atherosclerotic plaques from carotid arteries. During surgery, samples of lingual vein and superior thyroideal artery were also taken. We applied two molecular biology techniques to the carotid plaques on lingual vein or thyroideal artery samples: 1) polymerase chain reaction (PCR) and 2) reverse transcriptase-PCR (RT-PCR) for the detection of bacterial mRNA, employing PCR primers designed to detect a fragment of the 16S rRNA gene. Blood samples were obtained from the patients for determination of Chlamydia pneumoniae IgG, IgA, and IgM antibody titers by a microimmunofluorescence technique. The results of the present study confirmed the presence of viable Chlamydia pneumoniae in atheromas and support the hypothesis that the organism may be an active factor in the pathogenesis of atherosclerosis.
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PMID:Demonstration of viable Chlamydia pneumoniae in atherosclerotic plaques of carotid arteries by reverse transcriptase polymerase chain reaction. 1039 39

Chlamydia trachomatis is an obligate intracellular eubacteria that is dependent on a eukaryotic host cell for a variety of metabolites. For years, it has been speculated that chlamydiae are energy parasites, totally dependent on their host cell for ATP and other high-energy intermediates. To determine whether C. trachomatis contains functional enzymes that produce energy or reducing power, four enzymes involved in glycolysis or the pentose phosphate pathway, specifically pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase, were cloned, sequenced and expressed as recombinant proteins in Escherichia coli. The deduced amino acid sequences obtained show high homology to other pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase enzymes. In contrast to numerous other bacterial species, chlamydial glycolytic genes are not arranged in an operon, but are dispersed throughout the genome. Results from reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicate that all four genes are maximally expressed in the middle of the chlamydial developmental cycle. The chlamydial genes are capable of complementing mutant E. coli strains lacking the respective enzyme activities. In vitro enzyme analysis indicates that recombinant chlamydial enzymes expressed in E. coli are active and, interestingly, recombinant chlamydial pyruvate kinase is not regulated allosterically by fructose 1,6 bisphosphate or AMP, as found with other bacterial pyruvate kinases. In summary, identification and characterization of these glucose-catabolizing enzymes indicate that chlamydia contains the functional capacity to produce its own ATP and reducing power.
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PMID:Glucose metabolism in Chlamydia trachomatis: the 'energy parasite' hypothesis revisited. 1041 34

The conventional method for antimicrobial susceptibility testing of Chlamydia trachomatis is subjective and potentially misleading. We have developed a reverse transcriptase PCR (RT-PCR)-based method which is more sensitive and less subjective than the conventional method. Using 16 strains of C. trachomatis in triplicate assays, we found the RT-PCR method consistently more sensitive than the conventional technique for all eight antimicrobials tested, with resultant MICs determined by RT-PCR ranging from 1.6-fold higher (erythromycin) to >/=195-fold higher (amoxicillin).
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PMID:Antimicrobial susceptibility testing of Chlamydia trachomatis using a reverse transcriptase PCR-based method. 1047 87

The medically significant, obligate intracellular pathogen Chlamydia trachomatis replicates within vacuoles termed inclusions. A developmental cycle is initiated after entry into a host cell and is manifested by the transformation of infectious elementary bodies (EBs) to larger, non-infectious reticulate bodies (RBs). Analysis of the C. trachomatis genome has revealed that chlamydiae possess genes that may encode a type III secretion apparatus. In other Gram-negative pathogens, the type III secretion mechanism is used to target virulence factors directly to the host cell cytoplasm and is essential for full virulence. To evaluate the possibility of a functional type III secretion mechanism in C. trachomatis, we initially focused on a locus containing genes encoding products with similarity to chaperones (Scc1), secretion pore components (Cds1 and Cds2) and secreted proteins (CopN) from other type III systems. Gene expression was tested by reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNA extracted from infected HeLa cell monolayers at 2, 6, 12 and 20 h after infection and normalized for the number of C. trachomatis genomes present. Message was detected for Scc1 at all times, whereas message for all other tested genes was detected in significant amounts at 12 h and 20 h. Immunoblot analysis with Scc1- and CopN-specific antibodies revealed that CopN and Scc1 were present in EBs, RBs and whole-culture extracts harvested 20 h after infection. CopN is homologous to the secreted protein YopN of Yersinia sp., and analysis of monolayers 20 h after infection via indirect immunofluorescence showed specific labelling of inclusion membranes when probed with CopN-specific antibodies but not with Scc1-specific antibodies. His-tagged CopN and a chlamydial cytoplasmic control protein (NrdB) were expressed in Yersinia enterocolitica containing or lacking the virulence plasmid pYV. CopN, but not NrdB, was secreted by Y. enterocolitica in a Ca2+- and pYV-dependent fashion. These data indicate that components of the putative type III apparatus of C. trachomatis are expressed and that at least one of these products is secreted by chlamydiae to the inclusion membrane. The observation that CopN is also secreted by the Yersinia type III apparatus provides support for the notion that chlamydiae secrete proteins via a type III mechanism.
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PMID:Evidence for the secretion of Chlamydia trachomatis CopN by a type III secretion mechanism. 1112 78

Samples of atherosclerotic tissue from 58 patients undergoing carotid surgery were analysed by tissue culture and PCR for Chlamydia pneumoniae; PCR was performed to detect Omp1, 16S rRNA and HSP-70 genes. To understand the active pathogenic role of C. pneumoniae, a reverse transcriptase-PCR (RT-PCR) assay was applied to detect the specific RNAs expressed either in the replicative form, or in the cryptic form found in chronic infection. The C. pneumoniae omp1 gene, encoding the major outer-membrane protein (MOMP), was detected in 13 of 58 samples. Among these, the result was confirmed in 11 samples after amplification of a further target, the 16S rRNA, and the presence of the HSP-70 gene, encoding heat-shock protein 70, was revealed in only five cases. All the samples were negative for evidence of specific RNAs by RT-PCR. The presence of genomic DNA and absence of specific RNAs in atherosclerotic tissue samples suggests a lack of an active metabolic or persistent infective role for C. pneumoniae. Thus, traces of C. pneumoniae DNA in these samples could be due to a degradative pathway of the host defensive cellular and biochemical mechanisms.
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PMID:Search for Chlamydia pneumoniae genes and their expression in atherosclerotic plaques of carotid arteries. 1123 67

Chlamydia pneumoniae is associated with several chronic human diseases, including chronic obstructive pulmonary disease and atherosclerotic cardiovascular disease. During chronic disease, organisms are believed to exist in a persistent phase that is not well understood at the genetic level. Long-term in vitro continuous infections are spontaneously persistent and are less susceptible than in vitro acute infections to treatment with antibiotics, and are therefore particularly relevant as an in vitro model of in vivo chronic disease. Real-time reverse transcriptase-PCR (r-t RT-PCR) was used to quantitate transcript copy numbers of 13 genes in continuous and acute infections with C. pneumoniae. The set of genes studied encodes proteins with known or predicted functions in the cell membrane, the inclusion membrane, cell division, metabolism, and immunopathology. Significant upregulation was seen for five genes (CPn0483, nlpD, ompA, pmp1 and porB) in continuous cultures. The genes omcB, pmp1, and porB, all of which encode membrane proteins, shared similar patterns of expression over both acute and continuous profiles. These results show that Chlamydia in the long-term continuous model of persistence have a unique transcription profile, adding to our knowledge of regulation of this important stage of chlamydial growth.
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PMID:Differential expression of genes encoding membrane proteins between acute and continuous Chlamydia pneumoniae infections. 1262 Mar 80

Chlamydia pneumoniae has been shown to possess at least 13 genes that are homologous with other known type III secretion (TTS) systems. Upon infection of HEp-2 cells with C. pneumoniae, the expression of these genes was followed by reverse transcriptase PCR throughout the developmental cycle of this obligate intracellular pathogen. In addition, expression was analyzed when C. pneumoniae was grown in the presence of human gamma interferon (IFN-gamma). The groEL-1, ompA, and omcB genes were used as markers for the early, middle, and late stages of the developmental cycle, respectively, and the inhibition of expression of the fstK gene was used as a marker for the effect of IFN-gamma on the maturation of C. pneumoniae. In the absence of IFN-gamma, the TTS genes were expressed as follows: early stage (1.5 to 8 h), yscC, yscS, yscL, yscJ and lcrH-2; middle stage (by 12 to 18 h), lcrD, yscN, and yscR; and late stage (by 24 h), lcrE, sycE, lcrH-1, and yscT. Of the genes expressed early, the lcrH-2 gene was detected the earliest, at 1.5 h. Expression of the yscU gene was not detected at any of the time points examined. Under the influence of IFN-gamma, the cluster of TTS genes that were normally not expressed until the middle to late stages of the developmental cycle, namely, lcrD, lcrE, and sycE, as well as lcrH-1, were down-regulated, and expression could not be detected up to 48 h. In contrast, the expression of the other TTS genes appeared to be unchanged in the presence of IFN-gamma. The lcrH-1 and lcrH-2 genes differed from one another in both their temporal expression and response to IFN-gamma. In other TTS systems, these genes code for proteins that function in regulation of effector protein synthesis as well as serve as chaperones for proteins that provide for the translocation of the effector proteins into the host cell. In summary, the expression pattern of the TTS genes of C. pneumoniae examined suggests that they are temporally regulated throughout the developmental cycle. Furthermore, paralleling the inhibition of the maturation of the reticulate body to the elementary body, TTS genes expressed in the later stages of the cycle appear to be down-regulated when the organism is grown in the presence of IFN-gamma.
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PMID:Temporal expression of type III secretion genes of Chlamydia pneumoniae. 1270 28

Genome sequencing of C. trachomatis serovar D revealed the presence of three putative open reading frames (ORFs), CT145 (Pkn1), CT673 (Pkn5), and CT301 (PknD), encoding eukaryote-like serine/threonine kinases (Ser/Thr kinases). Two of these putative kinase genes, CT145 and CT301, were PCR amplified from serovar L2, cloned, and sequenced. Predicted translation products of the ORFs showed the presence of conserved kinase motifs at the N terminus of the proteins. CT145 and CT301 (encoding Pkn1 and PknD, respectively) were expressed in Escherichia coli as GST fusion proteins. In vitro kinase assays with Escherichia coli-derived glutathione S-transferase fusion proteins showed autophosphorylation of Pkn1 and PknD, indicating that they are functional kinases. Gene expression analysis of these kinase genes in Chlamydia by reverse transcriptase PCR indicated expression of these kinases at the early mid phase of the developmental cycle. Immunoprecipitated native chlamydial Pkn1 and PknD proteins also showed autophosphorylation in an in vitro kinase assay. Phosphoamino acid analysis by thin-layer chromatography confirmed that Pkn1 and PknD are phosphorylated on both serine and threonine residues. Interaction of Pkn1 and PknD with each other as well as interaction of Pkn1 with inclusion membrane protein G (IncG) was demonstrated by using a bacterial two-hybrid system. These interactions were further suggested by phosphorylation of the proteins in in vitro kinase assays. This report is the first description of the existence of functional Ser/Thr kinases in Chlamydia. The results of these findings should lead to a better understanding of how Chlamydia interact and interfere with host signaling pathways, since kinases represent potential mediators of the intimate host-pathogen interactions that are essential to the intracellular life cycle of Chlamydia.
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PMID:Identification of two eukaryote-like serine/threonine kinases encoded by Chlamydia trachomatis serovar L2 and characterization of interacting partners of Pkn1. 1450 Apr 99


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