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

---

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

The development of riboprobe expression cassettes for phosphorimager-based quantitation of steady-state transcripts for three different genes using solution hybridization, RNase protection assays is described. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin genes are widely used as reporter genes to estimate the amount and integrity of RNA as well as for comparing gene expression among different tissues. To directly compare expression of these two genes in lymphoid tissue and liver, cDNA fragments of beta-actin and GAPDH from both mice and rats were generated by RT-PCR and cloned together into pGEM1 under control of the T7 RNA polymerase promoter. Antisense transcripts from this fusion construct protected the appropriate-sized fragments of beta-actin (115 nt) and GAPDH (214 nt) in RNA isolated from rat spleen, thymus and liver. Expression of GAPDH transcripts was less variable across tissues because this mRNA was only two-fold lower in liver as compared to either thymus or spleen, whereas expression of beta-actin transcripts was eight-fold lower in liver than in these tissues. Two other riboprobe expression cassettes (IGF-I/actin) were constructed by ligating a cDNA fragment of mouse or rat beta-actin that would protect 115 nt to either a mouse or rat IGF-I genomic DNA fragment containing 182 bp of exon 4. These mouse and rat IGF-I/actin riboprobes were used to conclusively demonstrate that rat CSF-1-derived bone marrow macrophages, mouse elicited peritoneal macrophages and the murine PU5-1R macrophage cell line synthesize abundant transcripts for both IGF-I and beta-actin. However, the mouse M1 progenitor myeloid cell line does not express RNA for IGF-I, as demonstrated by the absence of protected transcripts for IGF-I in the presence of abundant protected transcripts for beta-actin. Phosphorimager scanning of the gels revealed that macrophages of both mice and rats express IGF-I transcripts at a level of 60-100% of those found in liver. These data show that a single riboprobe can be developed to generate multigene antisense RNAs that can then be used to quantitatively compare IGF-I transcripts in macrophages and other tissues to an internal standard, with GAPDH transcripts being less variable among tissues than those for beta-actin. This approach should be broadly applicable for measuring a variety of markers of cellular activation.
...
PMID:Riboprobe expression cassettes for measuring IGF-I, beta-actin and glyceraldehyde 3-phosphate dehydrogenase transcripts. 830 98

We determined during photoreceptor development if there is a retina-specific hypomethylation of the mouse gene encoding interphotoreceptor retinoid-binding protein (IRBP) that is associated with its activation. Second, the role of IRBP gene and protein expression in development was assessed by determining if their expression occurs before that of opsin. Retina-specific hypomethylation of the IRBP promoter region started on Embryonic (E) Day 11, at the time of cone formation, increased from E12 to E14, at the time of rod formation, and reached a peak on Postnatal (P) Day 4, which was followed thereafter by a slow decrease. Starting on E11, IRBP and opsin mRNA levels were quantitated relative to that of the beta-actin gene with RNase protection analysis. beta-Actin and IRBP transcripts were readily detected on E11. beta-actin levels remained constant during embryonic and early postnatal stages and decreased slightly afterward. On the other hand, beginning on E13, when the rods are formed, the IRBP level markedly increased. In contrast, the opsin transcript first appeared later on P0 and then increased from P3 onward. After P6, the opsin and IRBP transcript levels became comparable and by P20 their levels reached constancy. The timing of the onset of protein expression for the IRBP and opsin genes was determined during the last proliferative cycle of the rod precursor cells before their differentiation. Mice at P2 or P3 were injected with bromodeoxyuridine (BrdU) and their retinal cells were dissociated and then double-labeled with antibodies against BrdU and either IRBP or opsin. Cells positive for both IRBP and BrdU were always observed as soon as 2 hr after injection but it took at least 40 hr before they became positive for both opsin and BrdU. Taken together, these results indicate that IRBP gene activation is associated with hypomethylation during the last mitosis before photoreceptor cell differentiation.
...
PMID:Timing of interphotoreceptor retinoid-binding protein (IRBP) gene expression and hypomethylation in developing mouse retina. 831 88

Rats and mice are frequently used in studies of the regulation of lipoprotein metabolism. Although the species are closely related, they differ dramatically in the responses of their lipoproteins to estrogen administration. In rats, estrogens produce profound decreases in the levels of all plasma lipoproteins and this is attributed largely to estrogen-induced increases of hepatic low-density lipoprotein receptor (LDL-receptor) activity. Estrogens affect mouse plasma lipoproteins to a much lesser extent. Therefore, one of our aims was to compare the regulation of LDL-receptor gene expression in rats and mice at several potential loci of regulation. To assess the specificity of the estrogen effect, we also compared the responses of apolipoprotein AI (apoAI), apolipoprotein B (apoB), and beta-actin to the response of the LDL-receptor. In male Sprague Dawley rats given 17 beta-estradiol or 17 alpha-ethinyl estradiol at supraphysiological doses of 5 micrograms/g body mass/day, plasma total cholesterol and triacylglycerols fell to approximately 5% and approximately 50%, and, plasma apoAI and apoB fell to approximately 12% and approximately 16% of controls, respectively. By contrast, in male C3H/HeJ mice the above parameters dropped only to approximately 65% of controls and apoB concentrations rose to approximately 200% of controls. In rats, relative rates of LDL-receptor mRNA transcription (nuclear 'run-off' assay) and total hepatic, nuclear and polysomal LDL-receptor mRNA levels (RNase protection assay) increased by 1.5-2-fold, while synthesis of LDL-receptor protein on hepatic polysomes (in a wheat-germ translation system) increased 8-fold and LDL-receptor protein mass in hepatic plasma membranes increased 10-fold (by immunoblotting). In mouse liver, too, LDL-receptor mRNA levels increased 1.5-fold and the LDL-receptor mRNA transcription start sites in rat and mouse were found to be the same, but mouse LDL-receptor protein mass did not change, i.e. LDL-receptors of mice were similar to rat with respect to transcriptional regulation, but differed in their post-transcriptional control mechanisms. In rats, estrogen administration increased apoAI mRNA transcription rates 1.6-fold and also apoAI mRNA levels in total liver homogenates, nuclei and polysomes, (2-fold for each) consistent with transcriptional regulation. However, apoAI synthesis on total RNA increased less than apoAI mRNA, indicating that apoAI translational control mechanisms, at least in part, also regulate hepatic rates of apoAI production. ApoB mRNA transcription rates and levels showed small increases following estrogen administration. Hepatic beta-actin mRNA transcription and levels did not change.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:In vivo regulation of low-density lipoprotein receptors by estrogen differs at the post-transcriptional level in rat and mouse. 837 91

During the menstrual cycle, the endometrium undergoes characteristic changes in response to circulating sex steroids. Intense mitotic activity of glands and stroma occurs in the proliferative (estradiol-dominant) phase, and glandular secretion and stromal differentiation in the secretory (progesterone-dominant) phase. The insulin-like growth factors (IGF-I and IGF-II) promote cellular growth and differentiation and have been proposed to participate in these cyclic endometrial events, acting as mediators of steroid hormones. The objective of this study was to determine whether the messenger RNAs (mRNAs) encoding the IGF peptides and the type I and type II IGF receptors are differentially expressed in human endometrium during the menstrual cycle and in early pregnancy. A solution hybridization ribonuclease protection assay, using 32P-labeled riboprobes for IGF-I, IGF-II, and beta-actin (control), revealed IGF-I gene expression primarily in proliferative and early secretory endometrium and abundant IGF-II gene expression in mid-late secretory endometrium and early pregnancy decidua. Northern analysis, using IGF-I and IGF-II complementary DNA probes, revealed multiple IGF-I mRNAs [2-7.6 kilobase (kb)], expressed primarily in proliferative and early secretory endometrium, and IGF-II mRNAs (1.4-6.0 kb), expressed primarily in secretory endometrium and in early pregnancy decidua. The 7.6-kb IGF-I mRNA and the 6.0-kb IGF-II mRNA were most abundantly expressed. IGF-IEa and IGF-IEb mRNA splicing variants were present in a ratio of about 9:1, respectively. Type I and type II IGF receptor gene expression in endometrium was investigated using specific riboprobes and the ribonuclease protection assay. Messenger RNAs encoding both receptors were more abundantly expressed in the secretory phase and during early pregnancy, compared to the proliferative phase. These results show that mRNAs encoding the IGF peptides and their receptors are differentially expressed in human endometrium, depending on the steroid hormone milieu. The preferential expression of IGF-I mRNA in the proliferative phase supports the hypothesis that IGF-I is an estromedin in human endometrium. The expression of endometrial IGF-II mRNA in the mid to late secretory phase and in early pregnancy supports a role for IGF-II in differentiative functions of the endometrium, perhaps including endometrial tissue shedding in the menstrual cycle or remodeling during early pregnancy.
...
PMID:Differential expression of messenger ribonucleic acids encoding insulin-like growth factors and their receptors in human uterine endometrium and decidua. 849

The RNase protection assay was applied to quantify mRNA expression of five principal mammalian water channels in 18 different rat tissues, and to determine the influence of dehydration on renal water channel expression. Probes consisted of labeled cRNAs transcribed from cDNA fragments of rat CHIP28 (AQP-1, bp 238-575 of coding sequence), AQP-CD (AQP2, bp 53-606), MIWC (AQP4, bp 235-572), GLIP (AQP3, bp 219-604), and AQP5 (bp 56-612). Results were normalized to expression of rat beta-actin by quantitative densitometry of autoradiograms. CHIP28 mRNA was expressed strongly in heart, kidney > placenta, skeletal muscle, and urinary bladder and detected weakly in eye, lung, trachea, spleen, liver, colon, prostate, and skin. AQP-CD was detected only in kidney. MIWC mRNA expression was highest in brain, followed by eye, trachea, lung, stomach, kidney, and skeletal muscle. GLIP was found in eye, trachea, kidney, urinary bladder, skin, prostate, placenta, and skeletal muscle. AQP5 was detected in salivary gland, eye, lung, and trachea. An alternatively spliced form of MIWC (sMIWC) was also identified in lung and kidney by RNase protection assay, corresponding to deletion of exon 2 of MIWC. In response to dehydration (3 days, -15 % body weight), renal expression of CHIP28 and MIWC were unchanged, whereas expression of AQP-CD and GLIP were increased significantly by 2.18 +/- 0.04 and 1.36 +/- 0.11 fold (SE, n = 5), respectively. These results establish quantitative values for aquaporin transcript expression in multiple mammalian tissues. The sensitive RNase protection assay revealed the expression of water channels in several tissues not studied previously or in which mRNA levels were too low to detect by Northern blot analysis. The observation of GLIP up-regulation in kidney by dehydration suggests a role in the urinary concentrating mechanism.
...
PMID:Quantitative analysis of aquaporin mRNA expression in rat tissues by RNase protection assay. 867 43

We have examined the influence of nutrition on plasma IGF-I, IGF-II and IGF-binding protein (IGFBP) levels and on hepatic IGF-I gene expression in young meat-type chickens. Plasma IGF concentrations were measured by using RIA with recombinant chicken IGFs as standards. In chickens fed the control diet containing 200 g/kg dietary protein ad libitum for 7 days, plasma IGF-I concentrations increased significantly from those found in the initial control group. Food restriction for either 4 or 7 days decreased plasma IGF-I by 30% from the initial control. When chickens were refed ad libitum for 3 days after 4 days of restricted feeding, plasma IGF-I levels recovered to those of the control birds fed ad libitum. In chickens eating a low protein diet (100 g/kg protein), the plasma IGF-I tended to be lowered but the decrease was not significant. Although the intensity of IGF-I and beta-actin mRNA bands protected in the RNase protection assay was changed by nutrition, no statistical effect of nutrition on the ratio of IGF-I to beta-actin was observed. The nutritional treatments had no effect on plasma IGF-II concentrations. Western ligand blot and chromatographic analyses were used to investigate the influence of nutrition on IGFBP profiles. Both IGF-I and IGF-II ligands in the Western ligand blot revealed the most intense binding at 30 kDa for plasma obtained from chickens with restricted food intake. The 30 kDa band also appeared at a lower intensity in the group fed a low protein diet but not in any other groups. These observations were confirmed by neutral gel chromatography. The chicken IGF-II ligand revealed an intensely labelled band corresponding to 75 kDa and this was not affected by nutrition. IGF-I and IGFBP concentrations in the plasma of young broiler chickens were influenced by nutritional state but IGF-II concentrations were not. The lack of a response in circulating IGF-II levels may have been due to the presence of high concentrations of a 75 kDa specific binding protein which did not respond to nutrition in this experiment.
...
PMID:Influence of nutrition on hepatic IGF-I mRNA levels and plasma concentrations of IGF-I and IGF-II in meat-type chickens. 867 50

Thrombin is one of the first regulatory molecules present at sites of CNS trauma or injury. Exposure of neuronal and glial cells to thrombin produces potent morphological as well as cytoprotective and cytotoxic effects, but little is known about how this important modulator affects neurotransmitter signaling. In astrocyte cultures that have been morphologically differentiated by exposure to transforming growth factor-alpha, addition of thrombin induced a retraction of astrocytic processes and suppressed the stimulation of phosphoinositide hydrolysis by the selective metabotropic glutamate receptor (mGluR) agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid. In addition to the suppression of phosphoinositide hydrolysis, thrombin treatment produced a corresponding reduction in level of mGluR5 mRNA as demonstrated with ribonuclease protection assay and reduced content of mGluR5 receptor protein as seen with western blotting. In contrast, thrombin exposure up-regulated astrocyte beta-actin mRNA levels. A synthetic hexapeptide with a sequence corresponding to the amino-terminus of the thrombin receptor's tethered ligand also mimicked the ability of thrombin to suppress mGluR5 levels and to increase beta-actin mRNA content, suggesting that these effects of thrombin are mediated by proteolytically activated cell surface thrombin receptors. Thrombin's suppressive effect on mGluR5 was resistant to pretreatment with pertussis toxin or various protein kinase and protein phosphatase inhibitors. However, the serine/threonine protein kinase inhibitor H-7 did prevent thrombin-induced reversal of astrocyte stellation and induction of beta-actin mRNA levels, indicating that these effects of thrombin involve a signaling pathway distinct from the one that mediates the suppressive effects of thrombin on mGluR5.
...
PMID:Exposure of astrocytes to thrombin reduces levels of the metabotropic glutamate receptor mGluR5. 885 25

Particulate and other pollutant exposures are associated with lung injury and inflammation. The purpose of this study was to develop an approach by which intact RNA could be obtained from inflamed lung tissue from particulate-exposed animals in order to correlate injury with specific gene expression. Male Sprague Dawley (SD) and Fischer-344 (F-344) rats were intratracheally instilled with saline or residual oil fly ash (ROFA) particles, 8.3 mg/kg body weight in saline. At various time points following ROFA instillation, lungs were either lavaged or used for RNA isolation. ROFA exposure produced an increase in bronchoalveolar lavage fluid (BALF) neutrophils in both SD and F-344 rats. A time-dependent increase in eosinophils occurred only in SD rats but not in F-344 rats. Extraction of inflamed pulmonary tissue having a high influx of eosinophils for RNA using the conventional acid guanidinium thiocyanate phenol-chloroform (AGPC) procedure failed to provide undegraded RNA suitable for RT-PCR and Northern blot analysis of beta-actin mRNA expression. Mixing intact total RNA from saline control rat lungs with degraded RNA samples from inflamed lung yielded a gel profile of degraded RNA, indicating the presence of ribonuclease-like activity in the RNA extracted from lung tissues having eosinophil influx. Evidently, the conventional AGPC procedure failed to completely remove ribonuclease activity associated with ROFA-induced pulmonary eosinophil influx. This study reports a single-step modification to the AGPC extraction method that does not require additional reagents or additional precipitation steps for extracting undegraded RNA from nuclease-rich inflamed lung tissue. The aqueous layer resulting from mixing homogenate and chloroform is extracted a second time using an equal volume of AGPC buffer followed by addition of chloroform and centrifugation. The second aqueous phase is then treated as described in the conventional RNA extraction protocol. This simple and convenient modification does not require multiple precipitations of RNA and yields undegraded RNA from inflamed lung tissue with a slightly higher A260/A280 ratio without affecting overall RNA recovery. The results indicate that undegraded RNA could not be isolated using the routine AGPC-based isolation technique from lung tissue containing eosinophils following ROFA exposure. The degraded RNA preparations were unsuitable for gene expression studies. However, undegraded RNA can be isolated from these tissues by modifying the original AGPC RNA extraction procedure, which is suitable for gene expression analysis using northern blot and RT-PCR techniques.
...
PMID:Eosinophilic lung inflammation in particulate-induced lung injury: technical consideration in isolating RNA for gene expression studies. 888 58

Three members of the water channel (aquaporin) family are expressed in adult rat lung: CHIP28 (AQP-1), MIWC (AQP-4), and AQP-5. Because water channels may be important in the clearance of fluid from the newborn lung, the expression of water channels just before and after birth was investigated using the ribonuclease (RNAse) protection assay. RNA was isolated from lungs, brain, and heart of prenatal rats (fetal days F19, F20, and F21) and postnatal rats (days +1, +2, +5, +7, +21, and adult). Transcript expression was measured relative to a beta-actin control by quantitative densitometry. Whereas beta-actin mRNA expression was nearly constant over time, distinct expression patterns were observed for the three water channels. CHIP28 mRNA expression rose slowly from days F19 to +1, then strongly at day +2, and remained elevated over the first week. MIWC mRNA was weakly expressed prenatally, but strongly increased just after birth. AQP-5 mRNA increased slowly and monotonically between days F20 and +7. These patterns contrasted sharply with the developmental expression of CHIP28 in heart, which decreased over time, and MIWC in brain. Immunocytochemistry showed CHIP28 protein expression in capillary endothelia and MIWC in airway epithelia by day +1; quantitative immunoblot analysis showed increased CHIP28 protein expression over time. These findings are consistent with a role of lung water channels in perinatal fluid clearance; however, proof of physiologic significance will require functional measurements of air space-capillary water permeability.
...
PMID:Sharp increase in rat lung water channel expression in the perinatal period. 891 74

Apolipoprotein (apo) E is associated with several classes of lipoproteins and serves as a ligand for the receptor mediated uptake of cholesterol-rich particles by hepatocytes and peripheral tissues. Variant forms of apo E is also associated with dyslipidemia and late-onset of Alzheimer's Disease (AD). We report here expression of apoE in various mouse tissues, and regulation of apoE in liver, kidney, brain and testes by supraphysiological doses of estrogen. ApoE mRNA was quantified by RNase protection assay and translatable apoE mRNA by in vitro translation. As an internal control the levels of beta-actin mRNA were also quantified. Highest levels of apoE were expressed in liver (220-280 pg/mu g RNA) with negligible levels in small intestine. Brain expressed highest levels of total (35-40 pg/mu g RNA) and translatable apoE mRNA next only to liver. Other tissues that expressed relatively higher levels of apoE were adrenals, testes and ovary. ApoE was also found to be expressed in heart, lung, kidney and spleen. Regulation of apoE gene expression by estrogen (3 mu g 17beta-estradiol/ g body weight/ day for 5 consecutive days) was studied in liver, kidney, brain and testes of 4 mouse strains. Hepatic apoE mRNA did not change significantly in any of the mouse strains following estradiol administration. Of note was significant increases in the levels of brain apoE mRNA in the strain C3H. These studies demonstrate that estrogen regulates apoE gene expression in a tissue-specific manner in mice, and increases in apoE mRNA in the brain by estrogen may have implications in late-onset of Alzheimer's Disease.
...
PMID:Apolipoprotein E gene expression in various tissues of mouse and regulation by estrogen. 893 23


<< Previous 1 2 3 4 5 6 7 Next >>

---