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)

Mycobacterium tuberculosis is known to reside latently in a significant fraction of the human population. Although the bacterium possesses an aerobic mode of metabolism, it adapts to persistence under hypoxic conditions such as those encountered in granulomas. While in mammalian systems hypoxia is a recognized DNA-damaging stress, aspects of DNA repair in mycobacteria under such conditions have not been studied. We subjected Mycobacterium smegmatis, a model organism, to the Wayne's protocol of hypoxia. Analysis of the mRNA of a key DNA repair enzyme, uracil DNA glycosylase (Ung), by real-time reverse transcriptase PCR (RT-PCR) revealed its downregulation during hypoxia. However, within an hour of recovery of the culture under normal oxygen levels, the Ung mRNA was restored. Analysis of Ung by immunoblotting and enzyme assays supported the RNA analysis results. To understand its physiological significance, we misexpressed Ung in M. smegmatis by using a hypoxia-responsive promoter of narK2 from M. tuberculosis. Although the misexpression of Ung during hypoxia decreased C-to-T mutations, it compromised bacterial survival upon recovery at normal oxygen levels. RT-PCR analysis of other base excision repair gene transcripts (UdgB and Fpg) suggested that these DNA repair functions also share with Ung the phenomenon of downregulation during hypoxia and recovery with return to normal oxygen conditions. We discuss the potential utility of this phenomenon in developing attenuated strains of mycobacteria.
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PMID:Detrimental effects of hypoxia-specific expression of uracil DNA glycosylase (Ung) in Mycobacterium smegmatis. 2097 17

Tuberculosis is the most common disease inaugural of AIDS in France and HIV serology should be offered routinely when a tuberculosis case is diagnosed. Similarly, tuberculosis should also be sought before starting antiretroviral treatment. The case of pleural tuberculosis revealing AIDS presented here illustrates the difficulties of management of this co-infection due to polychemotherapy used to treat each of these pathologies causing drug interactions requiring dose adjustments and changes in treatment protocol and an increase in side effects. This is especially true when combining rifampicin and protease inhibitors and non-nucleoside reverse transcriptase inhibitors. On the other hand, resistance of Mycobacterium tuberculosis is possible in these patients coinfected by HIV particularly among migrants and in the case history of tuberculosis treatment.
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PMID:[Tuberculosis -HIV, a common and sensitive care in connection with a case of pleural tuberculosis revealing AIDS]. 2165 54

The aim of this study was to establish and evaluate a simultaneous amplification and testing method for detection of the Mycobacterium tuberculosis complex (SAT-TB assay) in clinical specimens by using isothermal RNA amplification and real-time fluorescence detection. In the SAT-TB assay, a 170-bp M. tuberculosis 16S rRNA fragment is reverse transcribed to DNA by use of Moloney murine leukemia virus (M-MLV) reverse transcriptase, using specific primers incorporating the T7 promoter sequence, and undergoes successive cycles of amplification using T7 RNA polymerase. Using a real-time PCR instrument, hybridization of an internal 6-carboxyfluorescein-4-[4-(dimethylamino)phenylazo] benzoic acid N-succinimidyl ester (FAM-DABCYL)-labeled fluorescent probe can be used to detect RNA amplification. The SAT-TB assay takes less than 1.5 h to perform, and the sensitivity of the assay for detection of M. tuberculosis H37Rv is 100 CFU/ml. The TB probe has no cross-reactivity with nontuberculous mycobacteria or other common respiratory tract pathogens. For 253 pulmonary tuberculosis (PTB) specimens and 134 non-TB specimens, the SAT-TB results correlated with 95.6% (370/387 specimens) of the Bactec MGIT 960 culture assay results. The sensitivity, specificity, and positive and negative predictive values of the SAT-TB test for the diagnosis of PTB were 67.6%, 100%, 100%, and 62.0%, respectively, compared to 61.7%, 100%, 100%, and 58.0% for Bactec MGIT 960 culture. For PTB diagnosis, the sensitivities of the SAT-TB and Bactec MGIT 960 culture methods were 97.6% and 95.9%, respectively, for smear-positive specimens and 39.2% and 30.2%, respectively, for smear-negative specimens. In conclusion, the SAT-TB assay is a novel, simple test with a high specificity which may enhance the detection rate of TB. It is therefore a promising tool for rapid diagnosis of M. tuberculosis infection in clinical microbiology laboratories.
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PMID:Novel real-time simultaneous amplification and testing method to accurately and rapidly detect Mycobacterium tuberculosis complex. 2220 4

Given the urgent need for designing a new antituberculosis vaccine conferring total protection on patients of all ages, following the line of research adopted by our institute, this work has identified Mycobacterium tuberculosis (Mtb) Rv3166c protein high-activity binding peptides (HABPs) which are able to inhibit bacterial invasion of U937 (monocyte-derived macrophages) and A549 (type II alveolar epithelial cells) cell lines. The presence and transcription of the rv3166c gene in the Mtb species complex was confirmed by polymerase chain reaction (PCR) and reverse transcriptase-PCR; Rv3166c expression was evaluated by western blot and cellular localisation confirmed by immunoelectron microscopy. Its presence was mainly determined on cell surface. Sixteen peptides covering its entire length were chemically synthesised and tested for their ability to bind to U937 and A549 cells. Two U937 HABPs were identified and three for A549, one of them being shared by both cell lines. The four HABPs found inhibited Mtb entry by 15.07-94.06%. These results led us to including Rv3166c HABPs as candidates for further studies contributing towards the search for a multiepitope, chemically synthesised, subunit-based antituberculosis vaccine.
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PMID:The role of Mycobacterium tuberculosis Rv3166c protein-derived high-activity binding peptides in inhibiting invasion of human cell lines. 2242 70

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population(1), and almost two million people are killed by TB each year. Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy. The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities. Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in > 95% of the MDR-TB isolates. The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community. Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene. It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results. It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated. Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.
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PMID:Diagnosing pulmonary tuberculosis with the Xpert MTB/RIF test. 2250 85

Expressions of recombinant proteins for different applications are important objectives in molecular biotechnology; however, expression of some recombinant proteins is difficult. Several methods have been designed for expression of these proteins. The aim of this study was to construct a vector containing Mtb32C fragment of Mycobacterium tuberculosis (M.tuberculosis) as a fusion partner in order to improve the expression of fused recombinant proteins. Mtb32C was amplified by polymerase chain reaction (PCR). The amplified fragment was ligated into pET21b+ vector. Colony-PCR, enzyme digestion and DNA sequencing methods were used to confirm the recombinant vector. Colony-PCR showed a 420 bp fragment in size corresponding to the correct size of our fragment. In addition the recombinant plasmids sequencing showed the accuracy of the cloned fragment. For confirming the expression, reverse transcriptase (RT)-PCR analysis was performed showing a 420 bp fragment in agarose gel electrophoresis using specific primers. The construction of a vector containing Mtb32C fragment is promising as a fusion partner for future studies as it affected the expression of the fused proteins and increased immune responses against the partner.
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PMID:Construction and Evaluation of an Expression Vector Containing Mtb32C (Rv0125) of Mycobacterium tuberculosis. 2340 10

We have determined the crystal structure of porcine quinolinate phosphoribosyltransferase (QAPRTase) in complex with nicotinate mononucleotide (NAMN), which is the first crystal structure of a mammalian QAPRTase with its reaction product. The structure was determined from protein obtained from the porcine kidney. Because the full protein sequence of porcine QAPRTase was not available in either protein or nucleotide databases, cDNA was synthesized using reverse transcriptase-polymerase chain reaction to determine the porcine QAPRTase amino acid sequence. The crystal structure revealed that porcine QAPRTases have a hexameric structure that is similar to other eukaryotic QAPRTases, such as the human and yeast enzymes. However, the interaction between NAMN and porcine QAPRTase was different from the interaction found in prokaryotic enzymes, such as those of Helicobacter pylori and Mycobacterium tuberculosis. The crystal structure of porcine QAPRTase in complex with NAMN provides a structural framework for understanding the unique properties of the mammalian QAPRTase active site and designing new antibiotics that are selective for the QAPRTases of pathogenic bacteria, such as H. pylori and M. tuberculosis.
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PMID:Crystal structure of Sus scrofa quinolinate phosphoribosyltransferase in complex with nicotinate mononucleotide. 2362 66

Pancreatitis is a well-described complication of human immunodeficiency virus (HIV) itself and its combination antiretroviral therapy. Historically, this has been predominantly associated with the usage of nucleoside reverse transcriptase inhibitors such as didanosine and stavudine, but only rarely with the usage of protease inhibitors via the induction of hypertriglyceridemia. Pancreatitis rates in HIV/AIDS population may have been exceedingly high because of the comorbid conditions prevalent in HIV/AIDS patients (e.g. ethanol use and biliary disease), and the use of non-combination antiretroviral therapy medications such as pentamidine, corticosteroids, ketoconazole, sulphonamides, metronidazole, isoniazid and opportunistic infections (e.g. cytomegalovirus, cryptosporidiosis, mycobacterial disease). In resource limited settings, where didanosine and stavudine are widely available in cheaper generic fixed dose combinations it is likely that their usage will remain in the first line HIV treatment in common. In such settings management or estimation of a patient's risk of pancreatitis still remains an issue of concern.
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PMID:Acute pancreatitis in HIV/AIDS patients: an issue of concern. 2373 May 53

This study was carried out to standardize reverse transcriptase PCR (RT-PCR) targeting 85B gene for the rapid detection of viable Mycobacterium tuberculosis (M tuberculosis) from sputum specimens. 100 sputum samples from clinically suspected tuberculosis patients were included in the study. The sputum samples were collected in sterile diethyl pyrocarbonate (DEPC) treated containers and transported in ice to the laboratory within 2 hours to prevent degradation of RNA. The following microbiological analysis was performed on the sputum specimens: Ziehl Neelsen staining, Mycobacterial culture by BACTEC TB 460 reader and RT-PCR targeting 85B gene. Out of the 100 sputum samples, 40 sputum samples were Ziehl Neelsen stain positive, 58 sputum samples were culture and RT-PCR positive and 42 were culture and RT-PCR were negative. Among direct smear positive specimens, 3 specimens did not grow in culture and was RT-PCR negative indicating the non-viability of the acid-fast bacilli seen in the direct smear. The results of RT-PCR and culture against control group and clinically diagnosed tuberculosis patients were statistically significant by Chi square test (P < 0.001). RT-PCR targeting 85B gene of M tuberculosis standardized in our laboratory for is a rapid, reliable and sensitive technique to detect the viable M. tuberculosis directly from sputum specimens.
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PMID:Rapid detection of viable M. tuberculosis directly from sputum by reverse transcriptase PCR targeting 85B gene. 2378 65

The highly successful pathogen Mycobacterium tuberculosis (Mtb) has evolved strategies to adapt to various stress conditions, thus promoting survival within the infected host. The two-component regulatory system (2CRS) senX3-regX3, which has been implicated in the Mtb response to inorganic phosphate depletion, is believed to behave as an auto-regulatory bicistronic operon. Unlike other 2CRS, Mtb senX3-regX3 features an intergenic region (IR) containing several mycobacterium interspersed repetitive units (MIRU) of unknown function. In this study, we used a lacZ reporter system to study the promoter activity of the 5' untranslated region of senX3, and that of various numbers of MIRUs in the senX3-regX3 IR, during axenic Mtb growth in nutrient-rich broth, and upon exposure to growth-restricting conditions. Activity of the senX3 promoter was induced during phosphate depletion and nutrient starvation, and IR promoter activity under these conditions was directly proportional to the number of MIRUs present. Quantitative reverse transcriptase (qRT)-PCR analysis of exponentially growing Mtb revealed monocistronic transcription of senX3 and regX3, and, to a lesser degree, bicistronic transcription of the operon. In addition, we observed primarily monocistronic upregulation of regX3 during phosphate depletion of Mtb, which was confirmed by Northern analysis in wild-type Mtb and by RT-PCR in a senX3-disrupted mutant, while upregulation of regX3 in nutrient-starved Mtb was chiefly bicistronic. Our findings of differential regulation of senX3-regX3 highlight the potential regulatory role of MIRUs in the Mtb genome and provide insight into the regulatory mechanisms underlying Mtb adaptation to physiologically relevant conditions.
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PMID:Differential regulation of the two-component regulatory system senX3-regX3 in Mycobacterium tuberculosis. 2472 8


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