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)

Recently it has been shown that infection with Mycobacterium tuberculosis increases the replication of HIV in mononuclear cells. The objective of this study was to investigate the mechanism(s) of up-regulation of HIV in primary human monocytes. Monocytes from healthy subjects were infected with HIV in vitro and then cultured with purified protein derivative (PPD) of M. tuberculosis. Culture supernatants were assessed for HIV p24 and cytokines. HIV expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). PPD induced HIV-infected monocytes to increased expression of HIV RNA and production of HIV p24. This effect correlated with production of tumour necrosis factor-alpha (TNF-alpha) in monocyte cultures. However, neutralizing antibody to TNF-alpha only partly abrogated the PPD-induced HIV p24 in these cultures. Also, PPD and culture filtrate of M. tuberculosis induced HIV mRNA expression. Further, using an adenovirus infection system containing an HIV long-terminal repeat (LTR) reporter plasmid, we showed that M. tuberculosis and its PPD induced HIV LTR. Therefore, the effect of M. tuberculosis and its PPD on HIV replication in monocytes is primarily one of transcriptional activation.
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PMID:Transcriptional activation of HIV by Mycobacterium tuberculosis in human monocytes. 1044 65

Mechanisms of protective immunity to mycobacterial infection in the lung remain poorly defined. In this study, T-cell subset expansion and cytokine expression in bronchoalveolar spaces, lung parenchyma, and mediastinal lymph nodes of mice infected intratracheally with Mycobacterium bovis-Calmette-Guerin bacillus (BCG) were analyzed in parallel with histopathology and bacterial burden. M. bovis-BCG was cleared rapidly from bronchoalveolar spaces without evidence for persistence. In lung parenchyma bacteria grew during the first 4 wk followed by gradual clearance with less than 0.1% of the original inoculum persisting for more than 8 mo. Clearance of M. bovis-BCG from bronchoalveolar lavage was associated with recruitment of both neutrophils and lymphocytes. Lung CD4(+), CD8(+), and gammadelta T-cell receptor-positive T cells expanded maximally by Week 4, and declined by Week 8 to control values despite bacterial persistence. Both CD4(+) and CD8(+) lung T cells produced interferon (IFN)-gamma in response to M. bovis-BCG. Four distinct pathologic states of lung parenchymal infection were noted. Early focal sub-bronchial inflammation with transmigration of cells into airways was followed by diffuse peribronchitis, perivasculitis, and alveolitis with activated macrophages, lymphoblasts, and occasional giant cells. The latter stage corresponded to maximal M. bovis-BCG growth. Resolving infection consisted of small lymphocytes and foamy macrophages, which coincided with decreasing M. bovis-BCG colony-forming units, T-cell infiltration, and IFN-gamma expression. A final quiescent phase consisted of residual lymphoid aggregates and perivasculitis associated with persistent spontaneous IFN-gamma production. Bacterial dissemination to lymph node and spleen occurred by Week 4 and declined in parallel to lung. In contrast to lung, IFN-gamma secretion was detected only late despite early expansion of CD4(+) and CD8(+) T cells. By reverse transcriptase/polymerase chain reaction, IFN-gamma and interleukin (IL)-12 p40 messenger RNA (mRNA) in lung paralleled IFN-gamma protein production. Tumor necrosis factor-alpha, IL-4 and IL-10 mRNA expression was not increased during M. bovis-BCG lung infection. Thus, protective immunity to M. bovis-BCG in the lung evolved differently in air space, lung, and lymph node.
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PMID:Pulmonary immune responses during primary mycobacterium bovis- Calmette-Guerin bacillus infection in C57Bl/6 mice. 1069 70

The ability of mRNA to direct synthesis of cDNA in the presence of oligo(dT) was analysed using a novel application of fluorescein-11-dUTP incorporation into cDNA by reverse transcriptase. Evidence is provided for the first time that a majority of the mycobacterial mRNA pool is polyadenylylated. mRNA transcripts of hsp65 were also amplified with specific primers from the oligo(dT)-primed cDNA preparation in Mycobacterium bovis BCG, M. smegmatis and M. vaccae. Furthermore, PCR amplication of cDNAs for genes entD, entC and trpE2 from M. bovis BCG yielded the expected products when reverse transcription was primed with oligo(dT), suggesting that polyadenylylation is a general phenomenon in mycobacteria.
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PMID:Polyadenylylation in mycobacteria: evidence for oligo(dT)-primed cDNA synthesis. 1074 66

Mycobacterium tuberculosis (MTB) the causative organism of tuberculosis can remain dormant as a non-culturable organism, reactivate and cause disease in man and animals. There is a need for proof of viability of such organisms in order to understand the process of reactivation. PCR for bacterial DNA cannot distinguish between viable and non-viable bacilli. We have tested a previously described two tube directed reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of mRNA of antigen 85B (Ag85B) of MTB that can distinguish between viable and non-viable organisms. Using a set of external and internal primers for Ag85B, a cDNA amplified product (216 bp) was seen among simulated samples containing only viable cfus at a sensitivity of >10 and <100 cfu/ml. Eucaryotic DNA rich normal mouse lung homogenate did not interfere among these samples. The method amplified the 216 bp product also among cfu positive tissues of naturally infected mice. Finally, in a mouse model of dormancy, direct RT-PCR detected a signal among multiple tissues that were negative for cfus and hence non-culturable. Ag85B is abundantly secreted by MTB and hyper-expressed under stress conditions. Thus the method to identify its mRNA message may be useful to detect viable but dormant bacteria.
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PMID:Identification of viable and non-viable Mycobacterium tuberculosis in mouse organs by directed RT-PCR for antigen 85B mRNA. 1083 70

Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene by a partially reductive pathway in which the intermediate hydroxylaminobenzene is enzymatically rearranged to 2-aminophenol by hydroxylaminobenzene mutase (HAB mutase). The properties of the enzyme, the reaction mechanism, and the evolutionary origin of the gene(s) encoding the enzyme are unknown. In this study, two open reading frames (habA and habB), each encoding an HAB mutase enzyme, were cloned from a P. pseudoalcaligenes JS45 genomic library and sequenced. The open reading frames encoding HabA and HabB are separated by 2.5 kb and are divergently transcribed. The deduced amino acid sequences of HabA and HabB are 44% identical. The HAB mutase specific activities in crude extracts of Escherichia coli clones synthesizing either HabA or HabB were similar to the specific activities of extracts of strain JS45 grown on nitrobenzene. HAB mutase activity in E. coli extracts containing HabB withstood heating at 85 degrees C for 10 min, but extracts containing HabA were inactivated when they were heated at temperatures above 60 degrees C. HAB mutase activity in extracts of P. pseudoalcaligenes JS45 grown on nitrobenzene exhibited intermediate temperature stability. Although both the habA gene and the habB gene conferred HAB mutase activity when they were separately cloned and expressed in E. coli, reverse transcriptase PCR analysis indicated that only habA is transcribed in P. pseudoalcaligenes JS45. A mutant strain derived from strain JS45 in which the habA gene was disrupted was unable to grow on nitrobenzene, which provided physiological evidence that HabA is involved in the degradation of nitrobenzene. A strain in which habB was disrupted grew on nitrobenzene. Gene Rv3078 of Mycobacterium tuberculosis H37Rv encodes a protein whose deduced amino acid sequence is 52% identical to the HabB amino acid sequence. E. coli containing M. tuberculosis gene Rv3078 cloned into pUC18 exhibited low levels of HAB mutase activity. Sequences that exhibit similarity to transposable element sequences are present between habA and habB, as well as downstream of habB, which suggests that horizontal gene transfer resulted in acquisition of one or both of the hab genes.
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PMID:Sequence analysis and initial characterization of two isozymes of hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45. 1087 93

IL-17 is a T cell cytokine with a complex and important role in the immune system. It has been detected in rheumatoid arthritis (RA) synovial membrane and found to stimulate the production of the proinflammatory cytokines IL-6, IL-8, tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. To date, there are few data available on the agents that stimulate IL-17 production. We therefore investigated the in vitro IL-17 response to a variety of mitogens and antigens, and compared the IL-17 response to interferon-gamma (IFN-gamma), IL-4, IL-10 and TNF-alpha. In this study we used a type-0 antigen, tetanus toxoid (TT), a type-1 antigen, PPD from Mycobacterium tuberculosis, a potential type-2 rye grass (RG) antigen (Lol I) and an autoantigen SS.B (La), to stimulate PBMC from healthy controls. Cytokine mRNA was measured using semiquantitative reverse transcriptase-polymerase chain reaction and cytokine protein measured using specific ELISA techniques, while the frequency of IL-17-producing T cells was determined by flow cytometry. The mitogens concanavalin A, phytohaemagglutinin and phorbol myristate acetate/ionomycin induced a significant increase in IL-17, with the highest levels being produced by anti-CD3/anti-CD28 stimulation. The antigens TT and PPD significantly increased IL-17 mRNA expression over time, but failed to have such an effect at the protein level. IL-17 protein was also detectable in both antigen-specific (TT, SS. B) and non-specific T cell clones, but at levels lower than IFN-gamma. IL-17 production did not correlate with either the type-1 cytokine IFN-gamma or TNF-alpha or the type-2 cytokine IL-4 or IL-10 at either the mRNA or protein level.
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PMID:Antigen-induced IL-17 response in the peripheral blood mononuclear cells (PBMC) of healthy controls. 1101 16

Infection with mycobacterial species, including Mycobacterium tuberculosis, has long been implicated in the etiopathology of rheumatoid arthritis (RA) on the basis of clinical and pathological similarities between tuberculosis and RA. Despite evidence of immune responses to mycobacterial antigens in RA patient synovial fluid, cross-reactivity between these and host joint antigens, and the presence of M. tuberculosis protein antigen in RA synovial fluid, a definite causal association with RA has not been shown. Previous studies from our laboratory using reverse transcriptase PCR (RT-PCR) of bacterial rRNAs have shown RA synovium to be colonized by a diverse range of bacteria, most of commensal origin. However, M. tuberculosis group organism (MTG) RNA sequences were found in one RA patient tissue. Since this was considered of sufficient interest to warrant further investigation, we devised a M. tuberculosis-specific nested RT-PCR test which could be used for detection of MTG in a mixed pool of bacterial crDNAs. This test was used to investigate the distribution of MTG in RA synovial tissue and also non-RA arthritis and healthy control tissues and was also used to examine the tissue distribution of MTG in an acute and chronic model of M. tuberculosis infection in the BALB/c mouse. MTG sequences were found in a high proportion of RA patient synovial tissues but also in non-RA arthritis control tissues at lower frequency. This likely reflects trafficking of persistent M. bovis BCG to inflamed joint tissue, irrespective of cause. MTG were not found in healthy synovial tissue or the tissue of patients with undifferentiated arthritis. In both the acute and chronic models of infection in BALB/c mice, M. tuberculosis was also found to have trafficked to joint tissues, however, no signs of inflammation, arthritis, or pathology associated with M. tuberculosis infection was seen. These combined results would argue against a specific causal role of MTG in RA-like arthritis; however, their role as adjuvant in immune dysfunction in an innately susceptible host cannot be excluded.
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PMID:Detection of Mycobacterium tuberculosis group organisms in human and mouse joint tissue by reverse transcriptase PCR: prevalence in diseased synovial tissue suggests lack of specific association with rheumatoid arthritis. 1117 60

Linear plasmids were unknown in mycobacteria until recently. Here, we report the complete nucleotide sequence of 23-kb linear plasmid pCLP from Mycobacterium celatum, an opportunistic pathogen. The sequence of pCLP revealed at least 19 putative open reading frames (ORFs). Expression of pCLP genes in exponential-phase cultures was determined by reverse transcriptase PCR (RT-PCR). Twelve ORFs were expressed, whereas no transcription of the 7 other ORFs of pCLP was detected. Five of the 12 transcribed ORFs detected by RT-PCR are of unknown function. Sequence analysis revealed similar loci in both M. celatum pCLP and the Mycobacterium tuberculosis chromosome, including transposase-related sequences. This result suggests horizontal transfer between these two organisms. pCLP also contains ORFs that are similar to genes of bacterial circular plasmids involved in partition (par operon) and postsegregational (pem operon) mechanisms. Functional analysis of these ORFs suggests that they probably carry out similar maintenance roles in pCLP.
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PMID:Genomic sequence and transcriptional analysis of a 23-kilobase mycobacterial linear plasmid: evidence for horizontal transfer and identification of plasmid maintenance systems. 1124 52

A simple ligand-protein structural optimization and binding evaluation procedure has been routinely used in high-speed ligand-protein docking studies. In this work, we examine whether such an optimization/scoring procedure is useful in indicating possible drug-resistant mutations in proteins. Crystal structures of three wild-type enzymes (HIV-1 protease, HIV-1 reverse transcriptase, and Mycobacterium tuberculosis H37Rv enoyl-ACP reductase) complexed to a variety of inhibitors are studied. Mutations are introduced into these structures by using the molecular modeling software, SYBYL. Structural optimization and scoring of a mutant complex is conducted by a procedure similar to that used in a recent docking study (Wang et al., 1999). The computed results are compared with observed drug resistance data and the profile of nonresistant mutations. Most mutations studied show an energy change in the same direction as those indicated by observed resistance data. 50% of the polar to polar or nonpolar to nonpolar mutations are found to correlate qualitatively with observed drug resistance data. Van der Waals interactions account for most of these changes, which is in agreement with conclusions from structural studies. Substantially larger deviations are found between computed results and observed data for most polar to nonpolar or nonpolar to polar mutations, which result from deficiency in modelling and scoring ligand-protein interactions in our procedure. Our results suggest that an optimization/docking scoring procedure is useful for qualitatively probing polar to polar or nonpolar to nonpolar resistant mutations in addition to its application in screening active compounds. More accurate description of ligand-protein interactions and the use of methods such as free energy perturbation and Poisson-Boltzmann may be needed to further improve the quality of prediction.
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PMID:Can an optimization/scoring procedure in ligand-protein docking be employed to probe drug-resistant mutations in proteins? 1155 85

The DNA region upstream of katG in Mycobacterium smegmatis was cloned and sequenced. The furA gene, highly homologous to Mycobacterium tuberculosis furA, mapped in this region. The furA-katG organization appears to be conserved among several mycobacteria. The transcription pattern of furA and katG in M. smegmatis upon oxidative stress was analyzed by Northern blotting and primer extension. Although transcription of both furA and katG was induced upon oxidative stress, transcripts covering both genes could not be identified either by Northern blotting or by reverse transcriptase PCR. Specific transcripts and 5' ends were identified for furA and katG, respectively. By cloning M. smegmatis and M. tuberculosis DNA regions upstream of a reporter gene, we demonstrated the presence of two promoters, pfurA, located immediately upstream of the furA gene, and pkatG, located within the terminal part of the furA coding sequence. Transcription from pfurA was induced upon oxidative stress. A 23-bp sequence overlapping the pfurA -35 region is highly conserved among mycobacteria and streptomycetes and might be involved in controlling pfurA activity. Transcription from a cloned pkatG, lacking the upstream pfurA region, was not induced upon oxidative stress, suggesting a cis-acting regulatory role of this region.
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PMID:Transcriptional regulation of furA and katG upon oxidative stress in Mycobacterium smegmatis. 1169 68


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