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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ribonuclease (RNase) protection assay (RPA) was evaluated as a method to estimate genetic distances among sequence variants of RNA viruses. The patterns of fragments generated, under different RPA conditions, by three sets of RNA sequence variants of known nucleotide sequence, were analyzed. Both the effectiveness of cleavage (i.e. the probability of cleavage in a certain heteroduplex) and its degree (i.e. in all the molecules in the assay or in a part of them) varied largely according to the nature of the mismatch. Probability and degree of cleavage were also dependent on distant sequence context effects. No correlation could be established between context and cleavage, so that the pattern of fragments in RPA cannot be unequivocally predicted from sequence information. Accordingly, nucleotide sequence differences between two sequence variants cannot be directly derived from RPA data. For all three sequence sets linear relationships were found between the number of non-shared fragments in the RPAs of two variants and their nucleotide sequence differences. Nevertheless, both linearity and the linear regression parameters varied largely according to the sequence set and according to RPA conditions, in a non-predictable way. Thus, under experimental conditions, RPA may not be as appropriate a method to estimate genetic distances between RNA sequences as simulation under an ideal model suggested. Possible ways to diminish the gap between the ideal model and the experimental procedure are proposed.
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PMID:Experimental evaluation of the ribonuclease protection assay method for the assessment of genetic heterogeneity in populations of RNA viruses. 766 91

Two patients with histologically proven mycosis fungoides, a malignancy of phenotypically mature T cells, received a topical challenge with mechlorethamine to areas of clinically uninvolved skin to exclude possible hypersensitivity reactions to this chemotherapeutic agent. In both patients, allergic contact dermatitis (ACD) developed at the sites of the application and resolved completely after withdrawal of mechlorethamine. The lesions were biopsied and analyzed for the presence of clonal T-cell receptor (TCR)-gamma gene rearrangements using two polymerase chain reaction (PCR)-based assays involving denaturing gradient gel electrophoresis (PCR/DGGE) and ribonuclease protection analysis (PCR/RPA). The former method has a clonal detection threshold of 10(-3)-10(-2), while the latter has a sensitivity of 10(-5). In both cases, the ACD lesions were polyclonal by PCR/DGGE. In contrast, PCR/RPA detected tumor-specific TCR-gamma gene rearrangements in these same lesions. This indicates that the ACD lesions contained tumor cells at a density within the 10(-5)-10(-2) range. Analysis of peripheral blood mononuclear cells from both patients failed to detect the malignant clone and showed the same result as blood from four normal individuals. The normal skin from one skin patient also lacked detectable TCR-gamma gene rearrangements. These results indicate that mycosis fungoides tumor are present within ACD lesions induced in mycosis fungoides patients and that this phenomenon does not appear to be due to the ubiquitous presence of detectable levels of these tumor cells in the blood or skin. These findings might be explained by nonspecific recruitment of malignant T cells to sites of local inflammation mediated by non-neoplastic antigen-specific T cells. Alternatively, they might be due to the local proliferation of very rare tumor cells in apparently normal skin in response to cytokines generated during the ACD reaction. In either case, the present study offers evidence that the malignant cells in myosis fungoides retain at least some capability of responding in vivo to physiologic stimuli.
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PMID:Detection of low-level tumor cells in allergic contact dermatitis induced by mechlorethamine in patients with mycosis fungoides. 861 5

Hormones can regulate the expression of their own receptor. We have examined whether adrenalectomy (ADX) and hormone replacement by physiological doses of aldosterone or dexamethasone could modulate the expression of glucocorticoid receptor (GR) or mineralocorticoid receptor (MR) at the mRNA level in the rat kidney, distal colon, and heart. Adult rats were adrenalectomized and received or did not receive an infusion of aldosterone (5 micrograms.100 g-1.day-1) or dexamethasone (10 micrograms.100 g-1.day-1). No significant change in steady-state levels of both MR and GR mRNA was detectable by using ribonuclease (RNase) protection assay (RPA) after either ADX or hormone replacement. Because the kidney is heterogeneous with regard to MR expression, RPA was adapted for measurements on microdissected nephron segments. GR mRNA is expressed at comparable levels all along the nephron, whereas MR mRNA is restricted to the distal nephron. No effect of ADX or GR and MR mRNA levels was detected in any nephron segment that was either aldosterone sensitive or insensitive. In situ hybridization confirmed the absence of corticosteroid-dependent modulation of MR mRNA in all kidney cell types. We conclude that variations of corticosteroid status do not affect MR and GR mRNA steady-state levels in heart, colon, and kidney and thus do not participate to the functional adaptations that are known to depend on hormonal status.
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PMID:Corticosteroid receptor mRNA expression is unaffected by corticosteroids in rat kidney, heart, and colon. 896 34

A novel method to measure mRNA levels has been developed by combining the detection capabilities of RNase protection (RPA) with the quantification advantages of scintillation proximity assay (SPA) technology. Sample processing is reduced to the addition of a single reagent post RNase digestion. As a model system, the inducible expression of rat apolipoprotein-A1 mRNA has been measured by both traditional gel-based RPAs and the SPA-based RPA assay. Results demonstrate that the ribonuclease protection proximity assay (RiPPA) faithfully reproduces the gel-based results and is at least as sensitive as many existing methods.
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PMID:A homogeneous method to quantify mRNA levels: a hybridization of RNase protection and scintillation proximity assay technologies. 920 50

Neuropeptide Y (NPY) is highly expressed in hypothalami of undernourished and genetically obese animals, and is a potent regulator of food intake and reproduction. Leptin, a protein expressed by adipocytes, has been reported to reduce hypothalamic NPY expression. We recently detected (by ribonuclease protection assay [RPA]) expression of the NPY receptor subtype Y1 (but not Y2) mRNA in adipose tissue. Based on these observations we hypothesized that NPY-Y1 receptors in adipose may represent a peripheral mechanism by which NPY can regulate leptin expression in a direct and rapid manner. To test this hypothesis, adipose samples were biopsied from the tailhead region of 48 +/- 3 kg wether lambs immediately before and 30 min after a single intravenous injection of 50 micrograms porcine NPY ("treated" animals, n = 5), or vehicle ("control" animals, n = 4). Injection of NPY resulted in an increase in expression (P = 0.013; as measured by RPA) of both leptin and NPY-Y1 mRNA. In treated animals, negative correlations were found between response in leptin expression and body weight (r = -0.82, P = 0.092), and between leptin response and initial leptin mRNA levels (r = -0.81, P = 0.097). These data provide evidence of a peripheral mechanism by which NPY may regulate adipocyte expression of both leptin and NPY-Y1 receptor mRNA.
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PMID:Effects of an intravenous injection of NPY on leptin and NPY-Y1 receptor mRNA expression in ovine adipose tissue. 934 53

A ribonuclease (RNase) protection assay (RPA) has been used to detect nucleotide sequence variation within the nucleoprotein gene of 39 viral haemorrhagic septicaemia virus (VHSV) isolates of European marine origin. The classification of VHSV isolates based on RPA cleavage patterns permitted the identification of ten distinct groups of viruses based on differences at the molecular level. The nucleotide sequence of representatives of each of these groupings was determined and subjected to phylogenetic analysis. This revealed grouping of the European marine isolates of VHSV into three genotypes circulating within distinct geographic areas. A fourth genotype was identified comprising isolates originating from North America. Phylogenetic analyses indicated that VHSV isolates recovered from wild caught fish around the British Isles were genetically related to isolates responsible for losses in farmed turbot. Furthermore, a relationship between naturally occurring marine isolates and VHSV isolates causing mortality among rainbow trout in continental Europe was demonstrated.
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PMID:Analysis of the nucleoprotein gene identifies distinct lineages of viral haemorrhagic septicaemia virus within the European marine environment. 1050 14

The localization of the multidrug resistance gene (mdr-1b) messenger ribonucleic acid (mRNA) along the rat nephron and its regulation was investigated under two different experimental situations: dehydration and high-Na+ diet. The mdr-1b mRNA was detected in glomeruli, proximal tubule segments, cortical and medullary thick ascending limbs, inner medullary collecting ducts and thin limbs of Henle's loop. Using the ribonuclease (RNase) protection assay (RPA), the abundance of mdr-1b mRNA was shown to be 35% less in renal cortex than in medulla. The mdr-1b mRNA expression in dehydrated rats in cortex or medulla did not differ from control. However, after 5 or 14 days on a high-Na+ diet, mdr-1b expression had decreased significantly in both cortex and medulla. There was no change in protein expression in dehydrated rats but a significant decrease occurred in rats fed the high-salt diet, confirming the results obtained with RPA. Our results suggest that the mdr-1b product is involved in extracellular volume regulation in rats.
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PMID:Modulation of the mdr-1b gene in the kidney of rats subjected to dehydration or a high-salt diet. 1065 Sep 88

The ribonuclease (RNase) protection assay (RPA) is an extremely sensitive technique used to determine specific mRNAs from cell and tissue extracts. The present protocol presents detailed procedures for a conventional RPA using antisense RNA probes purified with a Fullengther apparatus. The Fullengther has the advantage of being a relatively quick and safe procedure compared to more conventional methods for purification of full-length RNA probes. Using this protocol, we sought to simultaneously determine multiple mRNA species, including splice variants of the type I receptor (PAC(1)) of pituitary adenylate cyclase-activating polypeptide (PACAP), an important mediator in the regulation of luteinizing hormone-releasing hormone (LHRH) synthesis by ovarian steroids such as progesterone [7]. PAC(1) has more than eight splice variants. We have been able to discriminate the hop1 variant from other splice variants. To improve our understanding of the regulation mechanism of genes that are related to each other, such as LHRH and PACAP, it is most important to simultaneously determine genes that are involved in the same physiological areas of regulation. Using only 5 microg of total RNA sample from a single rat preoptic area, we simultaneously determined five different transcripts, including four rare mRNA species such as LHRH, PACAP, and hop1 variant and other splice variants of PAC(1), as well as the internal control of cyclophilin mRNA. This protocol provides a method for the simultaneous determination of multiple transcripts using the RPA.
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PMID:Simultaneous determination of multiple transcripts and splice variants of a primary transcript using ribonuclease protection assays. 1143 Nov 30

The Bcl-2 family of apoptotic regulators comprises both pro- and anti-apoptotic proteins. Pro-apoptotic BH3-only members of the Bcl-2 family are subject to stringent control in normal proliferating cells, but can be activated by a variety of mechanisms in response to diverse apoptotic signals. To facilitate the simultaneous identification of BH3-only targets that are regulated at the RNA level, we have designed and assembled multi-probe ribonuclease protection assay templates and demonstrated their ability to detect multiple BH3-only transcripts in a variety of human cell lines and primary tissues. Following this, we have determined the modulation of BH3-only transcripts to a variety of apoptotic stimuli, in selected primary and transformed cell lines. Early experiments, showing that a given cell type upregulates a unique subset of BH3-only RNAs in response to different death-inducing stimuli, indicate that the multi-probe BH3-only RPA template sets could serve as useful diagnostic and analytical tools in cancer research.
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PMID:Multi-probe RPA template sets to study RNA modulation and transcriptional control of BH3-only members of the Bcl-2 family. 1582 80

The latency-associated transcripts (LATs) of herpes simplex virus type-1 (HSV-1) are the only viral RNAs accumulating during latent infections in the sensory ganglia of the peripheral nervous system. The major form of LAT that accumulates in latently infected neurons is a 2 kb intron, spliced from a much less abundant 8.3 primary transcript. The spliced exon mRNA has been hard to detect. However, in this study, we have examined the spliced exon RNA in productively infected cells using ribonuclease protection (RPA), and quantitative RT-PCR (q-PCR) assays. We were able to detect the LAT exon RNA in productively infected SY5Y cells (a human neuronal cell line). The level of the LAT exon RNA was found to be approximately 5% that of the 2 kb intron RNA and thus is likely to be relatively unstable. Quantitative RT-PCR (q-PCR) assays were used to examine the LAT exon RNA and its properties. They confirmed that the LAT exon mRNA is present at a very low level in productively infected cells, compared to the levels of other viral transcripts. Furthermore, experiments showed that the LAT exon mRNA is expressed as a true late gene, and appears to be polyadenylated. In SY5Y cells, in contrast to most late viral transcripts, the LAT exon RNA was found to be mainly nuclear localized during the late stage of a productive infection. Interestingly, more LAT exon RNA was found in the cytoplasm in differentiated compared to undifferentiated SY5Y cells, suggesting the nucleocytoplasmic distribution of the LAT exon RNA and its related function may be influenced by the differentiation state of cells.
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PMID:Characterization of a spliced exon product of herpes simplex type-1 latency-associated transcript in productively infected cells. 1693 24


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