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)

Although 2% glutaraldehyde is often the first-line agent for endoscopic disinfection, its adverse reactions are common among staff and it is less effective against certain mycobacteria and spore-bearing bacteria. Chlorine dioxide is a possible alternative and an automated washer-disinfector fitted with this agent is currently available. This study was conducted to evaluate the effectiveness of chlorine dioxide in endoscopic disinfection after upper gastrointestinal examination. In vitro microbicidal properties of chlorine dioxide solutions were examined at high (600 ppm) and low (30 ppm) concentrations against various microbes including Pseudomonas aeruginosa, Helicobacter pylori, Mycobacterium avium-intracellulare and Bacillus subtilis in the presence or absence of bovine serum albumin (BSA). Immediately following endoscopic procedures and after application to the automated reprocessor incorporating chlorine dioxide at 30 ppm for 5 min, endoscopic contamination with infectious agents, blood, H. pylori ureA gene DNA and HCV-RNA was assessed by cultivation, sensitive test tape, polymerase chain reaction (PCR) and reverse transcriptase-PCR analysis, respectively. Chlorine dioxide at 30 ppm has equivalent microbicidal activity against most microbes and faster antimicrobial effects on M. avium-intracellulare and B. subtilis compared with 2% glutaraldehyde, but contamination with BSA affected the microbicidal properties of chlorine dioxide. Endoscopic contamination with microbes, blood and bacterial DNA was eliminated after application of the automated reprocessor/chlorine dioxide system. Thus, chlorine dioxide is a potential alternative to glutaraldehyde. The use of automated reprocessors with compatibility to chlorine dioxide, coupled with thorough pre-cleaning, can offer effective, faster and less problematic endoscopic disinfection.
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PMID:Endoscope disinfection using chlorine dioxide in an automated washer-disinfector. 1665 May 6

When grown in divalent cation-limited medium, Pseudomonas aeruginosa becomes resistant to cationic antimicrobial peptides and polymyxin B. This resistance is regulated by the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems. To further characterize Mg(2+) regulation in P. aeruginosa, microarray transcriptional profiling was conducted to compare wild-type P. aeruginosa grown under Mg(2+)-limited and Mg(2+)-replete conditions to isogenic phoP and pmrA mutants grown under Mg(2+)-limited conditions. Under Mg(2+)-limited conditions (0.02 mM Mg(2+)), approximately 3% of the P. aeruginosa genes were differentially expressed compared to the expression in bacteria grown under Mg(2+)-replete conditions (2 mM Mg(2+)). Only a modest subset of the Mg(2+)-regulated genes were regulated through either PhoP or PmrA. To determine which genes were directly regulated, a bioinformatic search for conserved binding motifs was combined with confirmatory reverse transcriptase PCR and gel shift promoter binding assays, and the results indicated that very few genes were directly regulated by these response regulators. It was found that in addition to the previously known oprH-phoP-phoQ operon and the pmrHFIJKLM-ugd operon, the PA0921 and PA1343 genes, encoding small basic proteins, were regulated by Mg(2+) in a PhoP-dependent manner. The number of known PmrA-regulated genes was expanded to include the PA1559-PA1560, PA4782-PA4781, and feoAB operons, in addition to the previously known PA4773-PA4775-pmrAB and pmrHFIJKLM-ugd operons.
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PMID:Contribution of the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems to Mg2+-induced gene regulation in Pseudomonas aeruginosa. 1670 91

Through chemical contamination of natural environments, microbial communities are exposed to many different types of chemical stressors; however, research on whole-genome responses to this contaminant stress is limited. This study examined the transcriptome response of a common soil bacterium, Pseudomonas aeruginosa, to the common environmental contaminant pentachlorophenol (PCP). Cells were grown in chemostats at a low growth rate to obtain substrate-limited, steady-state, balanced-growth conditions. The PCP stress was administered as a continuous increase in concentration, and samples taken over time were examined for physiological function changes with whole-cell acetate uptake rates (WAURs) and cell viability and for gene expression changes by Affymetrix GeneChip technology and real-time reverse transcriptase PCR. Cell viability, measured by heterotrophic plate counts, showed a moderately steady decrease after exposure to the stressor, but WAURs did not change in response to PCP. In contrast to the physiological data, the microarray data showed significant changes in the expression of several genes. In particular, genes coding for multidrug efflux pumps, including MexAB-OprM, were strongly upregulated. The upregulation of these efflux pumps protected the cells from the potentially toxic effects of PCP, allowing the physiological whole-cell function to remain constant.
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PMID:Transcriptome analysis reveals that multidrug efflux genes are upregulated to protect Pseudomonas aeruginosa from pentachlorophenol stress. 1752 77

Many bacteria living in soil have developed the ability to use a wide variety of organosulfur compounds. Pseudomonas putida strain DS1 is able to utilize dimethyl sulfide as a sulfur source via a series of oxidation reactions that sequentially produce dimethyl sulfoxide, dimethyl sulfone (DMSO2), methanesulfonate, and sulfite. To isolate novel genes involved in DMSO2 utilization, a transposon-based mutagenesis of DS1 was performed. Of c. 10,000 strains containing mini-Tn5 inserts, 11 mutants lacked the ability to utilize DMSO2, and their insertion sites were determined. In addition to the cysNC, cysH, and cysM genes involved in sulfate assimilation, the ptsP gene encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) family protein EI(Ntr) was identified, which is necessary for DMSO2 utilization. Using quantitative reverse transcriptase-polymerase chain reaction analysis, it was demonstrated that the expression of the sfn genes, necessary for DMSO2 utilization, was impaired in the ptsP disruptant. To the authors' knowledge, this is the first report of a PTS protein that is involved in bacterial assimilation of organosulfur compounds.
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PMID:The ptsP gene encoding the PTS family protein EI(Ntr) is essential for dimethyl sulfone utilization by Pseudomonas putida. 1771 52

Gene expression in biofilms is dependent on bacterial responses to the local environmental conditions. Most techniques for studying bacterial gene expression in biofilms characterize average values across the entire population. Here, we describe the use of laser capture microdissection microscopy (LCMM) combined with multiplex quantitative real-time reverse transcriptase PCR (qRT-PCR) to isolate and quantify RNA transcripts from small groups of cells at spatially resolved sites within biofilms. The approach was first tested and analytical parameters were determined for Pseudomonas aeruginosa containing an isopropyl-beta-D-thiogalactopyranoside-inducible gene for the green fluorescent protein (gfp). The results show that the amounts of gfp mRNA were greatest in the top zones of the biofilms, and that gfp mRNA levels correlated with the zone of active green fluorescent protein fluorescence. The method then was used to quantify transcripts from wild-type P. aeruginosa biofilms for a housekeeping gene, acpP; the 16S rRNA; and two genes regulated by quorum sensing, phzA1 and aprA. The results demonstrated that the amount of acpP mRNA was greatest in the top 30 microm of the biofilm, with little or no mRNA for this gene at the base of the biofilms. In contrast, 16S rRNA amounts were relatively uniform throughout biofilm strata. Using this strategy, the RNA amounts of individual genes were determined, and therefore the results are dependent on both gene expression and the half-life of the transcripts. Therefore, the uniform amount of rRNA throughout the biofilms likely is due to the stability of the rRNA within ribosomes. The levels of aprA mRNA showed stratification, with the largest amounts in the upper 30-microm zone of these biofilms. The results demonstrate that mRNA levels for individual genes are not uniformly distributed throughout biofilms but may vary by orders of magnitude over small distances. The LCMM/qRT-PCR technique can be used to resolve and quantify this RNA variability at high spatial resolution.
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PMID:Localized gene expression in Pseudomonas aeruginosa biofilms. 1848 1

In the present study we tested the responsiveness of human corneal epithelial cells (HCECs) and corneal fibroblasts to lipopolysaccharide (LPS), a Toll-like receptor (TLR) 4 ligand. Purified Pseudomonas aeruginosa LPS was used to stimulate telomerase-immortalized HCECs (HUCL) and stromal fibroblast (THK) cell lines. Exposure of cells to LPS induced a time-dependent activation of NF-kappaB in THK but not in HUCL cells, as assessed by an increase in IkappaB-alpha phosphorylation and degradation. Concomitant with NF-kappaB activation, LPS-treated THK cells, but not HUCL cells, produced a significantly larger number of cytokines than control untreated cells. A cell surface biotinylation assay revealed that HUCL cells express TLR4 intracellularly, whereas TLR5 is expressed on the cell surface. Furthermore, reverse transcriptase-PCR analysis revealed that HUCL and primary HCECs, in contrast to THK cells, do not express myeloid differentiation (MD)-2. Thus, our results demonstrate that the LPS unresponsiveness of HCECs might be due to deficient expression of MD-2, an essential component for LPS-TLR4 signaling.
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PMID:Lack of MD-2 expression in human corneal epithelial cells is an underlying mechanism of lipopolysaccharide (LPS) unresponsiveness. 1893 73

In this study, Pseudomonas savastanoi pv. savastanoi isolates were demonstrated to contain two iaaL paralogs, which are both chromosomally located in most strains. Comparative analysis of iaaL nucleotide sequences amplified from these two paralogs revealed that one paralog, iaaL(Psn), is 100% identical to iaaL from P. savastanoi pv. nerii, while the other paralog, iaaL(Psv), exhibited 93% identity to iaaL from Pseudomonas syringae pv. tomato (iaaL(Pto)). A 3-nucleotide motif (TAC) comprised of 3 to 15 repeats, which remained stable after propagation of the strains in olive plants, was found in iaaL(Psv). Based on the observed nucleotide sequence variations, a restriction fragment length polymorphism assay was developed that allowed differentiation among iaaL(Psn), iaaL(Psv), and iaaL(Pto)(.) In addition, reverse transcriptase PCR on total RNA from P. savastanoi pv. savastanoi strains demonstrated that both iaaL(Psv) and iaaL(Psn) containing 14 or fewer TAC repeats are transcribed. Capillary electrophoresis analysis of PCR-amplified DNA fragments containing the TAC repeats from iaaL(Psv) allowed the differentiation of P. savastanoi pv. savastanoi isolates.
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PMID:Pseudomonas savastanoi pv. savastanoi contains two iaaL paralogs, one of which exhibits a variable number of a trinucleotide (TAC) tandem repeat. 1909 22

A total of 264 nonduplicate strains of imipenem (IPM)-nonsusceptible Pseudomonas aeruginosa were isolated from hospitals in 16 different regions throughout China. These 264 IPM-nonsusceptible clinical isolates of P. aeruginosa were examined by PCR, a metallo-beta-lactamase (MBL) Etest, a double-disk synergy test (DDST), and a test using combined IPM disks supplemented with various amounts of EDTA. A total of 24 strains positive for MBLs were confirmed by PCR and DNA sequence analysis: 10 strains positive for the bla(VIM-2) gene, 13 strains positive for the bla(IMP-9) gene, and 1 strain positive for the bla(IMP-1) gene. Real-time reverse transcriptase PCR (RT-PCR) was used to verify whether the isolates harboring MBL genes produced the enzyme and was considered the standard for evaluation of the methodology in this study. Of these 24 MBL-positive stains, 21 were confirmed as MBL-producing strains by real time RT-PCR for MBL expression and the other 3 had no expression of MBLs. The sensitivities, specificities, and positive and negative predictive values for the MBL Etest, the DDST, and the combined disk (CD) assay were evaluated. The best method for screening for MBL production in P. aeruginosa strains from China was the CD assay (IMP-EDTA) using 750 microg of EDTA/disk with a breakpoint of >or=6 mm. In the CD assay (IPM-EDTA) with 290 microg and 750 microg EDTA, the zone diameter increases for VIM-2-producing P. aeruginosa isolates were greater than those for IMP-9-producing P. aeruginosa isolates (P = 0.00).
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PMID:Evaluation of phenotypic tests for detection of metallo-beta-lactamase-producing Pseudomonas aeruginosa strains in China. 1921 96

To analyze the genetic mechanisms of carbapenem and ciprofloxacin resistance in clinical isolates of Pseudomonas putida, 27 clinical isolates (comprising 11 carbapenem- and ciprofloxacin-resistant strains, 13 carbapenem-resistant and ciprofloxacin-susceptible strains, and 3 carbapenem- and ciprofloxacin-susceptible strains) were collected from different patients. Carbapenem resistance was examined by polymerase chain reaction (PCR) and DNA sequencing for metallo-beta-lactamase (MBL) and integrase genes (IntI-1 and IntI-3), and by reverse transcriptase-PCR (RT-PCR) for expression of the porin gene (oprD). Ciprofloxacin resistance was characterized by PCR and DNA sequencing for mutations in the quinoloneresistance determining regions of the gyrA and parC genes. The blaIMP-1 MBL and intI-1 and/or intI-3 genes were detected in all carbapenem-resistant strains, and decreased expression of the oprD gene as compared to carbapenemsusceptible strains was observed in several strains. All the 11 strains with ciprofloxacin minimal inhibitory concentrations (MICs) of > or =64 mg/l had substitution in GyrA (Thr83Ile), and one (ciprofloxacin MIC of 512 mg/l) of these strains also had substitution in ParC (Ser87Leu). Overproduction of the efflux pump was observed in 10 of the 11 ciprofloxacin-resistant strains. We concluded that the production of IMP-1 type MBL was the most critical factor in developing high-level resistance to carbapenems, and mutations in the target proteins and overproduction of the efflux pump synergistically contribute to the acquisition of high-level resistance to ciprofloxacin in clinical isolates of P. putida.
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PMID:Molecular characterizations of carbapenem and ciprofloxacin resistance in clinical isolates of Pseudomonas putida. 1928 Feb 93

Abstract A diverse assemblage of diazotrophic bacteria exists in the rhizosphere of the smooth cordgrass, Spartina alterniflora, but the taxa actively involved in nitrogen fixation have not been determined. In order to identify the diazotrophs that were actively expressing nifH, the gene encoding the nitrogenase iron protein, mRNA was extracted from Spartina rhizosphere samples and nifH-specific seminested reverse transcriptase-PCR performed. Expressed nifH sequences were recovered from organisms affiliated with the (gamma-+beta-) Proteobacteria and the anaerobes. Most of the expressed nifH sequences were highly similar (>/=95% similarity) to sequences previously recovered from Spartina rhizosphere DNA using conventional nifH-specific PCR. These sequences were also similar, although not identical to the nifH sequences of Pseudomonas stutzeri, Vibrio diazotrophicus, Desulfovibrio africanus, and Desulfovibrio gigas.
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PMID:Expression of nifH genes by diazotrophic bacteria in the rhizosphere of short form Spartina alterniflora. 1971 72


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