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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tight junctions (TJs) provide a barrier function, inhibiting solute and water flow through the paracellular space. There had been no analysis until now as to how tight junction molecules could be involved in the pathology of patients with chronic venous insufficiency. The aim of the study was to analyse the expression pattern of TJ-molecules occludin (OCLN), claudin-1 (CLDN-1), claudin-3 (CLDN-3) and claudin-5 (CLDN-5) on mRNA and protein level in patients with edema, venous leg ulcers and healthy controls. Biopsy specimens were taken in healthy individuals and in patients before, and four weeks after compression therapy. mRNA-expression was determined by using reverse-transcriptase and polymerase chain reaction (RT-PCR) and the protein-expression was determined by Western blotting from tissue specimens. Quantification performed determining the expression for TJ-molecules displayed diminished expression for CLDN-1 (p<0.01) and CLDN-5 (p<0.01) in patients with chronic venous insufficiency in comparison with healthy controls on mRNA as well as protein level. No statistical differences were detected for OCLN and CLDN-3 between the edema group and healthy controls. There was a significantly elevated expression (p<0.01) on mRNA and protein level between the leg ulcer group and healthy controls for OCLN and CLDN-3. Densitometric evaluation revealed a more significantly elevated expression (p<0.01) for CLDN-1 and CLDN-5 on mRNA and protein level after four weeks of compression therapy in comparison with prior to treatment for the edema as well as the leg ulcer group. Compression therapy tightens the paracellular barrier via elevated expression of specific TJs and prevents thereby the progression of chronic venous insufficiency due to inhibited permeability of fluid into the perivascular tissue.
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PMID:Tight junctions and compression therapy in chronic venous insufficiency. 1678 76

Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performance of nucleic acid sequence-based amplification (NASBA), we developed a real-time norovirus NASBA targeting part of the RNA-dependent RNA polymerase (RdRp) gene. Specificity of the assay was studied with 33 divergent clones that contained part of the targeted RdRp gene of noroviruses from 15 different genogroups. Viral RNA originated from commercial oysters, surface waters, and sewage treatment plants in The Netherlands. Ninety-seven percent of the clones derived from human noroviruses were detected by real-time NASBA. Two clones containing animal noroviruses were not detected by NASBA. We compared the norovirus detection by real-time NASBA with that by conventional reverse transcriptase PCR (RT-PCR) with large-volume river water samples and found that inhibitory factors of RT-PCR had little or no effect on the performance of the norovirus NASBA. This consequently resulted in a higher sensitivity of the NASBA assay than of the RT-PCR. We show that by combining an efficient RNA extraction method with real-time NASBA the sensitivity of norovirus detection in water samples increased at least 100 times, which consequently has implications for the outcome of the infectious risk assessment.
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PMID:Real-time detection of noroviruses in surface water by use of a broadly reactive nucleic acid sequence-based amplification assay. 1688 86

This study focuses on the effect of electroacupuncture (EA) on the gastric mucosal histology and ITF (intestinal trefoil factor) mRNA in stress-related rat, and the relationship between the gastric protective mechanism of EA at acupoints of Stomach Meridian of Foot-Yangming (SMFY) group and Gallbladder Meridian of Foot-Shaoyang (GMFS) group. Forty rats were randomly divided into 4 groups: blank control group (BCG), model control group (MCG), SMFY group (EA at acupoints of SMFY for 7 days before model inducing), and GMFS group (EA at acupoints of GMFS for 7 days before model inducing). All rats (except normal group) were made model by water immersion and restriction (WRS) on day 7, then the gastric mucosal lesion index (GUI) was accessed, ITF mRNA expression of the tissue was detected by reverse- transcriptase-polymerase chain reaction (RT-PCR) method, and the histological change under light microscope was observed. As a result, the GUI value in SMFY/GMFS groups decreased significantly (p < 0.05 or 0.01). The level of ITF mRNA expression in SMFY group was significantly higher than that in MCG (p < 0.01), while that in GMFS group was higher than MCG but there was no statistical difference (p < 0.05). This result may be due to the intrinsic mechanism of EA's gastric mucosal protection by the upregulation of ITF mRNA expression in gastric mucosal tissue, and the expression variance indicated the classical traditional Chinese medicine (TCM) theory "Relative Particularity between SMFY and Stomach.".
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PMID:The effect of EA on the gastric mucosal histology and ITF mRNA expression in stress-induced rats. 1716 89

The cold-water-fish pathogen Vibrio salmonicida expresses a functional bacterial luciferase but produces insufficient levels of its aliphatic-aldehyde substrate to be detectably luminous in culture. Our goals were to (i) better explain this cryptic bioluminescence phenotype through molecular characterization of the lux operon and (ii) test whether the bioluminescence gene cluster is associated with virulence. Cloning and sequencing of the V. salmonicida lux operon revealed that homologs of all of the genes required for luminescence are present: luxAB (luciferase) and luxCDE (aliphatic-aldehyde synthesis). The arrangement and sequence of these structural lux genes are conserved compared to those in related species of luminous bacteria. However, V. salmonicida strains have a novel arrangement and number of homologs of the luxR and luxI quorum-sensing regulatory genes. Reverse transcriptase PCR analysis suggests that this novel arrangement of quorum-sensing genes generates antisense transcripts that may be responsible for the reduced production of bioluminescence. In addition, infection with a strain in which the luxA gene was mutated resulted in a marked delay in mortality among Atlantic salmon relative to infection with the wild-type parent in single-strain challenge experiments. In mixed-strain competition between the luxA mutant and the wild type, the mutant was attenuated up to 50-fold. It remains unclear whether the attenuation results from a direct loss of luciferase or a polar disturbance elsewhere in the lux operon. Nevertheless, these findings document for the first time an association between a mutation in a structural lux gene and virulence, as well as provide a new molecular system to study Vibrio pathogenesis in a natural host.
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PMID:A novel lux operon in the cryptically bioluminescent fish pathogen Vibrio salmonicida is associated with virulence. 1727 25

The rate-limiting step for nucleotide incorporation in the pre-steady state for most nucleic acid polymerases is thought to be a conformational change. As a result, very little information is available on the role of active-site residues in the chemistry of nucleotidyl transfer. For the poliovirus RNA-dependent RNA polymerase (3D(pol)), chemistry is partially (Mg(2+)) or completely (Mn(2+)) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pK(a) values of 7.0 or 8.2 and 10.5, depending upon the divalent cation used in the reaction. A solvent deuterium isotope effect of three to seven was observed on the rate constant for nucleotide incorporation in the pre-steady state; none was observed in the steady state. Proton-inventory experiments were consistent with two protons being transferred during the rate-limiting transition state of the reaction, suggesting that both deprotonation of the 3'-hydroxyl nucleophile and protonation of the pyrophosphate leaving group occur in the transition state for phosphodiester bond formation. Importantly, two proton transfers occur in the transition state for nucleotidyl-transfer reactions catalyzed by RB69 DNA-dependent DNA polymerase, T7 DNA-dependent RNA polymerase and HIV reverse transcriptase. Interpretation of these data in the context of known polymerase structures suggests the existence of a general base for deprotonation of the 3'-OH nucleophile, although use of a water molecule cannot be ruled out conclusively, and a general acid for protonation of the pyrophosphate leaving group in all nucleic acid polymerases. These data imply an associative-like transition-state structure.
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PMID:Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases. 1736 May 13

Noroviruses have received increased attention in recent years because their role as etiologic agents in acute gastroenteritis outbreaks is now clearly established. Our inability to grow them in cell culture and the lack of an animal model hinder the characterization of these viruses. More recently, molecular approaches have been used to study the genetic relationships that exist among them. In the present study, environmental samples from seawater, estuarine water, and effluents of sewage treatment plants were analyzed in order to evaluate the role of environmental surface contamination as a possible vehicle for transmission of norovirus genogroups I and II. Novel broad-range reverse transcription-PCR/nested assays targeting the region coding for the RNA-dependent RNA polymerase were developed, amplifying fragments of 516 bp and 687 bp in the nested reactions for genogroups II and I, respectively. The assays were evaluated and compared against widely used published assays. The newly designed assays provide long regions for high-confidence BLAST searches in public databases and therefore are useful diagnostic tools for molecular diagnosis and typing of human noroviruses in clinical and environmental samples, as well as for the study of molecular epidemiology and the evolution of these viruses.
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PMID:Molecular identification and genetic analysis of Norovirus genogroups I and II in water environments: comparative analysis of different reverse transcription-PCR assays. 1748 65

Endocrine-disrupting chemicals (EDC) are linked to human health and diseases as they mimic or block the normal functioning of endogenous hormones. The present work dealt with a comparative study of the androgenic potential of wastewater treatment plant (WWTP) influents and effluents in Northern region of India, well known for its polluted water. Water samples were screened for their androgenic potential using the Hershberger assay and when they were found positive for androgenicity, we studied their mode of action in intact rats. The data showed a significant change in the weight and structure of sex accessory tissues (SATs) of castrated and intact rats. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis demonstrated a significant change in the expression patterns of the major steroidogenic enzymes in adrenal and testis: cytochrome P450(SCC), cytochrome P450(C17), 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase. This was further supported by increased enzymatic activities measured in vitro spectrophotometrically. Serum hormone profile showed a decreased level of gonadotrophic hormones and increased testosterone level. Further, increase in the serum level of alkaline phosphatase, SGPT and SGOT and histopathological changes in kidney and liver of treated animals, confirmed the toxic effects of contaminating chemicals. Analysis of water samples using HPLC and GC-MS showed the presence of various compounds and from them, four prominent aromatic compounds viz. nonylphenol, hexachlorobenzene and two testosterone equivalents, were identified. Our data suggest that despite rigorous treatment, the final treated effluent from WWTP still has enough androgenic and toxic compounds to affect general health.
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PMID:Androgenic endocrine disruptors in wastewater treatment plant effluents in India: their influence on reproductive processes and systemic toxicity in male rats. 1800 9

This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain +/- bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media approximately 90% of the total Rb influx occurred through the Na-K pump and NKCC and approximately 10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased K(c) by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 approximately 25 microM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.
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PMID:Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells. 1818 76

We wished to discover if we could gain greater insights into how biological treatment plants function by contrasting the presence and activity of the most abundant Bacteria in plug flow and completely mixed activated sludge plants. Presence was assessed by amplifying 16S rRNA gene fragments (using PCR) and activity by amplifying native 16S rRNA, using reverse-transcriptase PCR (RT-PCR), using Bacteria-specific primers. The amplified sequences were compared using denaturing gradient gel electrophoresis (DGGE). The plug flow plant exhibited a strong physico-chemical gradient with an initial anoxic zone, whilst the two completely mixed reactors did not. Similarities were observed between the profile of the banding pattern for presence and activity. However, in the plug flow reactor one prominent band was detected in the active population (16S rRNA) but was absent from the corresponding profile of the 16S rRNA gene. Sequencing of this band revealed its identity as a Nitrosomonas-like sequence. The intensity of the 16S rRNA sequenced varied along the physico-chemical gradient of the plug-flow reactor in a manner that coincided with the growth of ammonia-oxidising bacteria (AOB) and the loss of ammonia. This band was also absent from the completely mixed reactors, although significant numbers of AOB were detected in all systems ( approximately 10(6)-10(8)cells ml(-1)) by fluorescence in situ hybridisation (FISH). An abundant and highly active AOB population was present in the anoxic zone of the plug-flow reactor where up to 60% of the total ammonia was removed. An examination of nitrogen removal/production rates, together with the above data, reveal that complex nitrogen removal processes occur in this system. These data also enabled the calculation of a specific in situ growth rate for the AOB as 0.12h(-1).
Water Res 2008 Jun
PMID:Presence and activity of ammonia-oxidising bacteria detected amongst the overall bacterial diversity along a physico-chemical gradient of a nitrifying wastewater treatment plant. 1842 99

To verify previous conclusions on the use of bacterial indicators suggested in regulations and to investigate virological quality of groundwater, a 1-year study was undertaken on groundwater used as a source of drinking water in 3 provinces in Canada. Raw water from 25 municipal wells was sampled during a 1-year period for a total of 167 samples. Twenty-three sites were selected on the basis of their excellent historical bacteriological water quality data, and 2 sites with known bacteriological contamination were selected as positive controls. Water samples were analyzed for general water quality indicators (aerobic endospores, total coliforms), fecal indicators (Escherichia coli, enterococci, somatic and male-specific coliphages), total culturable human enteric viruses (determined by cell culture and immunoperoxidase), noroviruses (analyzed by reverse-transcriptase -- polymerase chain reaction (RT-PCR)), adenovirus types 40 and 41 (analyzed by integrated cell culture (ICC) - PCR), and enteroviruses and reoviruses types 1, 2, and 3 (analyzed by ICC-RT-PCR). General water quality indicators were found very occasionally at the clean sites but were frequently present at the 2 contaminated sites. Only one of 129 samples from the 23 clean sites was positive for enterococci. These results confirm the value of raw water quality historical data to detect source water contamination affecting wells that are vulnerable. Samples from the 2 contaminated sites confirmed the frequent presence of fecal indicators: E. coli was found in 20/38 samples and enterococci in 12/38 samples. Human enteric viruses were not detected by cell culture on MA-104 cells nor by immunoperoxidase detection in any sample from the clean sites but were found at one contaminated site. By ICC-RT-PCR and ICC-PCR, viruses were found by cytopathic effect in one sample from a clean site and they were found in 3 samples from contaminated sites. The viruses were not detected by the molecular methods but were confirmed as picornaviruses by electron microscopy. Noroviruses were not detected in any samples. The results obtained reinforce the value of frequent sampling of raw water using simple parameters: sampling for total coliforms and E. coli remains the best approach to detect contamination of source water by fecal pollutants and accompanying pathogens. The absence of total coliforms at a site appears to be a good indication of the absence of human enteric viruses.
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PMID:Groundwater microbiological quality in Canadian drinking water municipal wells. 1853 33


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