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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exposure for 20 min of stationary phase cells of Salmonella typhimurium to a combined triple stress system (TSS) treatment comprising hypochlorite derived 5 ppm free available chlorine in solution acidified with 1% succinate (pH 2.5) and at a chill shock temperature of 5 degrees C resulted in symptoms of injury. Cells became sensitive to 40 micrograms/ml lysozyme, 50 micrograms/ml actinomycin D and 100 micrograms/ml ribonuclease B, to which control cells were resistant. Metabolic injury was indicated by reduction in colony forming ability of stressed cells on minimal salts glucose agar M9 medium. There was no detectable leakage loss of 260-280 nm-absorbing materials. This was also confirmed by assay of the cellular RNA material components. Loss of alkaline phosphatase activity was observed in the stressed cells. The intensity of induced cellular damage as measured by lysozyme sensitivity was greatest in the cells exposed to the complete TSS, followed by those stressed in 1% succinate at 5 degrees C, then 5 ppm chlorine at 5 degrees C and the singular chill shock stress at 5 degrees C, respectively. The magnitudes of cellular damage, however, were suggestive of synergistic interactions among the component stress factors of the TSS. The findings obtained indicated impairment of the structural integrity and functional capabilities of the permeability barriers and the inactivation of certain periplasmic enzymes. The resultant cumulative cellular damage from the TSS exposure may therefore enhance greater sensitivity of treated cells to subsequent stress factors.
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PMID:Mechanisms of triple stress-mediated damage in stationary phase cells of Salmonella typhimurium exposed to succinate-acidified hypochlorite system at 5 degrees C. 242

Chloride channels supply critical functions in epithelial cells throughout the body. Although function of the volume- and voltage-gated C1C-2 is uncertain, its wide tissue distribution of mRNA suggests C1C-2 has important housekeeping functions. This study's objective was to identify the extent of not only C1C-2 mRNA expression but also protein expression as a measure of the capacity for C1C-2 chloride secretion in epithelial tissues. Using quantitative ribonuclease protection assay, we found that C1C-2 mRNA transcripts were abundant in fetal and postnatal brain, fetal kidney, liver, intestine, and lung. In contrast to brain, C1C-2 mRNA transcripts were downregulated during late gestation in lung, kidney, and intestine. The lung expressed the least C1C-2 mRNA. Immunoblotting demonstrated similar tissue- and gestation-dependent variations in C1C-2 protein expression. To determine if there is a correlation between the sites of C1C-2 protein expression and cystic fibrosis transmembrane conductance regulator (CFTR), another epithelial chloride channel, a polyclonal COOH-terminal C1C-2 antibody and an anti-R domain CFTR anti-body were used. C1C-2 and CFTR were expressed in different sites in lung and kidney.
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PMID:Gestational and tissue-specific regulation of C1C-2 chloride channel expression. 894 27

Chloride transport is critical to many functions of the lung. Molecular defects in the best-known chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), lead to impaired function of airway defensins, hydration of airway surface fluid, and mucociliary clearance leading to chronic lung disease, and premature death, but do not cause defects in lung development. We examined the expression of one member of the ClC family of volume- and voltage-regulated channels using the ribonuclease protection assay and Western blot analysis in rats. ClC-5 mRNA and protein are most strongly expressed in the fetal lung, and expression is maintained although downregulated postnatally. In addition, using immunocytochemistry, we find that ClC-5 is predominantly expressed along the luminal surface of the airway epithelium, suggesting that ClC-5 may participate in lung chloride secretion. Identifying candidate genes for critical ion transport functions is essential for understanding normal lung morphogenesis and the pathophysiology of several lung diseases. In addition, the manipulation of non-CFTR chloride channels may provide a viable approach for treating cystic fibrosis lung disease.
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PMID:ClC-5: ontogeny of an alternative chloride channel in respiratory epithelia. 1183 44

We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay. The mRNA of Ad2 was detected as early as 6 h after infection at 10(6) infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as 24 h after infection at 10(6) IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation. Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner. These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples.
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PMID:Detection of infectious adenovirus in cell culture by mRNA reverse transcription-PCR. 1466 Mar 88

The aim of this study was to develop a method for investigating the stability of the human NoV capsid in response to disinfectants and sanitisers (virucides) as an indirect method for determining virus infectivity. Capsid destruction or "virolysis" was measured using the reverse transcribed quantitative PCR (RT-QPCR) reaction in conjunction with RNase treatment (in order to destroy any exposed RNA). Two commercially available alcohol based handwashes, alcohols (75% (v/v) ethanol or isopropanol), quaternary ammonium compounds (0.14% BAC or 0.07% DIDAC), and chlorine dioxide (200 ppm) were all ineffective at promoting virolysis of human norovirus present in dilute clinical samples at the concentrations tested. GII.4 NoVs were sensitive to a combination of heat and alkali. These data show that NoVs present in dilute stool samples are resistant to virolysis using virucides that are used commonly.
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PMID:Measurement of the virolysis of human GII.4 norovirus in response to disinfectants and sanitisers. 2141 62

The relationship between the infectivity of the feline calicivirus (FCV) vaccine strain F-9 and capsid destruction (virolysis) in response to available chlorine was investigated under standardized light soil disinfection conditions. Virolysis was measured using RNase pretreatment (in order to destroy exposed RNA following chlorine treatment) and quantitative reverse transcription PCR. A comparison between the results of plaque assays and virolysis following exposure of FCV F-9 grown in tissue culture to different concentrations of available chlorine showed a similar log-linear relationship, with >4-log reductions occurring at 48 and 66 ppm, respectively. Three non-epidemiologically linked human GII.4 noroviruses (NoVs) present in dilute clinical samples showed behavior similar to each other and were 10 times more resistant to virolysis than cultured FCV F-9. FCV F-9 when present in dilute human GII.4 samples acquired increased resistance to virolysis approaching that of human NoVs. This study represents a direct comparison between the virolysis of a surrogate virus (FCV F-9) and that of human GII.4 NoVs within the same matrix in response to available chlorine. The results support the view that matrix effects have a significant effect on virus survival.
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PMID:Virolysis of feline calicivirus and human GII.4 norovirus following chlorine exposure under standardized light soil disinfection conditions. 2218 52

Viruses excreted by humans affect the commercial and recreational use of coastal water. Shellfish produced in contaminated waters have been linked to many episodes and outbreaks of viral gastroenteritis, as well as other food-borne diseases worldwide. The risk can be reduced by appropriate treatment following harvesting and by depuration. The kinetics of inactivation of murine norovirus 1 and human adenovirus 2 in natural and artificial seawater by free available chlorine was studied by quantifying genomic copies (GC) using quantitative PCR and infectious viral particles (PFU). Human JC polyomavirus Mad4 kinetics were evaluated by quantitative PCR. DNase or RNase were used to eliminate free genomes and assess potential viral infectivity when molecular detection was performed. At 30 min of assay, human adenovirus 2 showed 2.6- and 2.7-log(10) GC reductions and a 2.3- and 2.4-log(10) PFU reductions in natural and artificial seawater, respectively, and infectious viral particles were still observed at the end of the assay. When DNase was used prior to the nucleic acid extraction the kinetic of inactivation obtained by quantitative PCR was statistically equivalent to the one observed by infectivity assays. For murine norovirus 1, 2.5, and 3.5-log(10) GC reductions were observed in natural and artificial seawater, respectively, while no viruses remained infectious after 30 min of contact with chlorine. Regarding JC polyomavirus Mad4, 1.5- and 1.1-log(10) GC reductions were observed after 30 min of contact time. No infectivity assays were conducted for this virus. The results obtained provide data that might be applicable to seawater used in shellfish depuration.
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PMID:Comparative inactivation of murine norovirus, human adenovirus, and human JC polyomavirus by chlorine in seawater. 2277 37

Chemical modification of proteins is gaining importance due to the improvement in properties and the broader range of applications that these protein conjugates have. Once modified, several purification strategies need to be applied to isolate the conjugates of interest. Aqueous two-phase systems (ATPS) are an attractive alternative for the primary recovery of proteins and their conjugates. However, to better understand which biochemical parameters affect in greater degree the partition behavior of these modified proteins in ATPS, it becomes necessary to characterize the partition behavior of different species. In this work, ribonuclease A (RNase A) was selected as a model protein to address the partition behavior of chemically modified proteins in ATPS. Native, mono-PEGylated, Uniblue A, Dabsyl Chloride, and Direct Red 83 chemically modified RNase A's were partitioned in 16 different polyethylene glycol (PEG)-potassium phosphate ATPS. Results suggest that while the effects of system design parameters govern the partition of native RNase A, the behavior of the chemically modified species is more influenced by the physicochemical characteristics of the modifying molecules, that in most cases promote partition toward the top polymer-rich phase with recovery percentages as high as 86%. It has been found that both, the hydrophobicity and molecular weight of the modifying species play a preponderant role in conjugate partition behavior since as hydrophobicity increases partition is promoted towards the PEG-rich phase balancing the effect of the molecular weight of the modifying molecules that tends to shift partition towards the salt rich phase.
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PMID:Effects of chemical modifications in the partition behavior of proteins in aqueous two-phase systems: a case study with RNase A. 2329 81

Culturable animal caliciviruses are widely-used as surrogates for human norovirus (HuNoV). The infectivity of a culturable virus was traditionally determined by plaque assay and/or 50% tissue culture infectious dose (TCID50) assay, both of which are time-consuming and labor-intensive. Molecular approaches, such as quantitative real time RT-PCR (qRT-PCR) and RT-PCR, could be used for detection of the viral genome but yet fail to determine the infectivity of a virus. In this study, we evaluated different assays for determination of infectivity of Tulane virus (TV), a surrogate for HuNoV. The infectivity of TV was measured by RNase exposure assay, RT-PCR assays, cellular-receptor-mediated capture qRT-PCR assay, receptor-mediated in situ capture qRT-PCR assay, cell-culture-mediated amplification qRT-PCR, and confirmed by TCID50 assay. RNase exposure assay was only useful for measuring TV inactivation caused by heat. Short template RT-PCR assay did not reflect inactivation status of TV. Partial reduction in viral RNA signal could be measured by long-template RT-PCR only when TV was inactivated by thermal or chlorine treatments at full-inactivation levels. Cellular-receptor-mediated capture qRT-PCR exhibited low sensitivity and specificity for the evaluation of virus infectivity. The in situ capture qRT-PCR assay could be used to evaluate virus inactivation deriving from damage to viral capsid caused by heat and chlorine. The cell-culture-mediated amplification qRT-PCR could be used as an alternative method to rapidly determine the infectivity of TV.
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PMID:Alternative methods to determine infectivity of Tulane virus: a surrogate for human nororvirus. 2579 Sep 87

Human norovirus (NoV) is the leading cause of acute gastroenteritis worldwide. Persistence on surfaces and resistance to many conventional disinfectants contribute to widespread transmission of norovirus. We examined the efficacy of neutral electrolyzed water (NEW; pH 7) for inactivation of human NoV GII.4 Sydney in suspension (ASTM method 1052-11) and on stainless steel surfaces (ASTM method 1053-11) with and without an additional soil load. The impact of the disinfectant on viral capsid was assessed using reverse transcriptase quantitative PCR (RT-qPCR; with an RNase pretreatment), SDS-PAGE, transmission electron microscopy, and a histo-blood group antigen (HBGA) receptor-binding assay. These studies were done in parallel with those using Tulane virus (TuV), a cultivable human NoV surrogate. Neutral electrolyzed water at 250 ppm free available chlorine produced a 4.8- and 0.4-log₁₀ reduction in NoV genome copy number after 1 min in suspension and on stainless steel, respectively. Increasing the contact time on surfaces to 5, 10, 15, and 30 min reduced human NoV genomic copies by 0.5, 1.6, 2.4, and 5.0 log₁₀ and TuV infectious titers by 2.4, 3.0, 3.8, and 4.1 log₁₀ PFU, respectively. Increased soil load effectively eliminated antiviral efficacy regardless of testing method and virus. Exposure to NEW induced a near complete loss of receptor binding (5 ppm, 30 s), degradation of VP1 major capsid protein (250 ppm, 5 min), and increased virus particle aggregation (150 ppm, 30 min). Neutral electrolyzed water at 250 ppm shows promise as an antinoroviral disinfectant when used on precleaned stainless steel surfaces.IMPORTANCE Norovirus is the leading cause of acute viral gastroenteritis worldwide. Transmission occurs by fecal-oral or vomitus-oral routes. The persistence of norovirus on contaminated environmental surfaces exacerbates its spread, as does its resistance to many conventional disinfectants. The purpose of this research was to evaluate the antinoroviral efficacy of neutral electrolyzed water (NEW), a novel chlorine-based disinfectant that can be used at reduced concentrations, making it more environmentally friendly and less corrosive than bleach. An industrial-scale electrochemical activation device capable of producing relatively stable electrolyzed water at a wide pH range was used in this study. Experiments showed that 250 ppm NEW effectively eliminated (defined as a 5-log₁₀ reduction) human norovirus GII.4 Sydney (epidemic strain) on clean stainless steel surfaces after a 30-min exposure. Supporting studies showed that, like bleach, NEW causes inactivation by disrupting the virus capsid. This product shows promise as a bleach alternative with antinoroviral efficacy.
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PMID:Efficacy of Neutral Electrolyzed Water for Inactivation of Human Norovirus. 2860 Mar 17


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