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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

T3 inhibits transcription of the rat TSH beta gene, and two T3 response elements have been identified that bind T3 receptors and that share sequence homology with the consensus sequence that is also recognized by retinoic acid receptors (RARs). We, therefore, asked whether RA was a regulator of TSH beta gene expression in vivo. Using RNase protection analysis, we found that vitamin A deficiency led to a 2-fold increase in rat pituitary TSH beta messenger RNA (mRNA) levels, which returned to normal 18 h after treatment with RA (20 micrograms/rat). Vitamin A deficiency had no effect on TSH beta mRNA levels in hypothyroid rats. Coadministration of RA and T3 (10 micrograms/100g body wt) to either vitamin A-deficient or vitamin A-deficient, hypothyroid animals caused decreases in TSH beta mRNA content that were indistinguishable from those seen with T3 alone. Surprisingly, vitamin A deficiency had no significant effect on GH mRNA levels in euthyroid or hypothyroid rats. Furthermore, treatment of vitamin A-deficient, hypothyroid animals with RA for either 18 or 72 h had no effect on GH mRNA levels, whereas T3 caused 11-fold and 18-fold increases in GH mRNA, respectively, at these times. We also used transient transfection to test for direct, retinoid receptor-mediated regulation of TSH beta gene transcription by RA. A plasmid TSH beta luciferase, containing 0.8 kilobases of rat TSH beta gene 5'-flanking sequences, exon 1, and 150 base pairs of intron 1, was transfected into CV-1 cells. Cotransfection with RAR alpha and retinoid X receptor-beta induced TSH beta expression by 3.5-fold, and treatment with RA suppressed this induction by 46%. These results show that vitamin A levels play a significant role in regulating the expression of the TSH beta gene, but not the GH gene, in vivo and suggest that RA may suppress TSH beta gene transcription directly by an RAR-retinoid X receptor heterodimer-mediated mechanism.
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PMID:Regulation of thyroid-stimulating hormone beta-subunit and growth hormone messenger ribonucleic acid levels in the rat: effect of vitamin A status. 783 86

Neuropeptide-Y (NPY), a hypothalamic peptide, is involved in stimulation of LHRH and LH surges during proestrus and those induced by ovarian steroids in ovariectomized (ovx) rats. The NPY neurons that reside in the arcuate nucleus of the medial basal hypothalamus (MBH) and accumulate 17 beta-estradiol participate in the initiation of LHRH and LH surges. To determine whether NPY synthesis is altered in conjunction with the LH surge, we studied the dynamic changes in prepro-NPY mRNA levels in the MBH in association with the LH surge elicited by estradiol benzoate (EB) alone or by progesterone (P) in EB-primed ovx rats. Five days after ovariectomy, rats received oil or EB (30 micrograms/rat) at 1000 h on day 0. On day 2, these rats were injected with either oil or P (2 mg/rat) at 1000 h. Rats were killed before (1000 h) and at 2-h intervals after oil or P injection. The MBHs were dissected out and processed for determination of prepro-NPY mRNA levels by solution hybridization/RNase protection assay using a cRNA probe. Although in control ovx rats, prepro-NPY mRNA levels remained unchanged between 1000-1600 h, prepro-NPY mRNA levels showed dynamic changes in steroid-primed rats. In the EB-primed rats, prepro-NPY mRNA levels rose significantly (100%) at 1200 and 1400 h before the LH rise at 1600 h, and the levels remained elevated up to 1800 h. After P injection to the EB-primed rats, this response was further augmented, with a slightly different temporal pattern. Prepro-NPY mRNA levels rose at 1400 h (600%) before the onset of the LH rise at 1600 h and declined steadily to significantly lower values at 1800 h, coincident with the highest rate of LH secretion. These studies demonstrate dynamic shifts in hypothalamic NPY gene expression in association with the LH (LHRH) surge, and that maximal increases occur before the onset of the LH rise, but thereafter, NPY gene expression diverged in the two ovarian-steroid treatment models. These findings along with previous evidence of similar antecedent increases in NPY content in the median eminence, followed by release, suggest that augmented NPY synthesis and release are two temporally dissociable neural events for the LHRH and LH surges.
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PMID:Hypothalamic neuropeptide-Y gene expression increases before the onset of the ovarian steroid-induced luteinizing hormone surge. 811 37

The subcellular distribution of hepatic alanine:glyoxylate aminotransferase 1 (AGT) has changed, under the influence of dietary selection pressure, on several o occasions during the evolution of mammals. In some species (e.g. human and rabbit) AGT is entirely peroxisomal; in other species (e.g. marmoset and rat) this enzyme is found in similar amounts in peroxisomes and mitochondria; in yet other species (e.g. cat) it is mainly mitochondrial. The molecular basis of the species-specific dual intracellular targeting of AGT has been partially elucidated in the human and rabbit (as examples of the first group), and in the rat and marmoset (as examples of the second group). As part of a wider study on the molecular evolution of AGT intracellular targeting, we report in the present paper the results of an investigation into the molecular basis of the subcellular distribution of AGT in the cat (as an example of the third group). Cat liver AGT cDNA has been cloned and sequenced, and shown to have a high degree of similarity to AGT from human, rabbit, marmoset and rat. Southern-blotting analysis showed that AGT in the cat is probably encoded by a single gene, as it is in other species. Transcript analysis by RNase protection indicated that almost all of the AGT mRNA would possess an open reading frame encoding a polypeptide of 414 amino acids and a molecular mass of 45,508 Da. The N-terminal 22 amino acids comprised the putative mitochondrial-targeting sequence (by analogy with the equivalent sequence in marmoset and rat pre-mitochondrial AGT). The very low level of peroxisomal AGT in cat liver is compatible with the absence of any RNase-protected transcripts initiating downstream of the first putative translation initiation codon (i.e. absence of any transcripts in which the mitochondrial-targeting sequence is excluded from the open reading frame). In vitro studies showed that the 45 kDa polypeptide was imported into rat liver mitochondria and processed to a mature protein of approximately 43 kDa, compatible with the cleavage of the N-terminal 22 amino acids, as is also the case in rat and marmoset. A polypeptide in which the N-terminal 22 amino acids was absent could not be imported into mitochondria in vitro.
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PMID:Molecular evolution of alanine/glyoxylate aminotransferase 1 intracellular targeting. Analysis of the feline gene. 816 41

Perfusion of liver of rats toxicated with galactosamine or thioacetamide with a 0.02% solution of picroliv (glycoside fraction of Picrorhiza kurroa) for 30 min (1 ml/min; 6 mg/rat), significantly reversed toxicant-induced changes in the activities of several enzymes. Galactosamine induced increases in the activities of alkaline phosphatase, gamma-glutamyl transpeptidase, acid ribonuclease, acid phosphatase, succinate dehydrogenase and decreases in the activities of Na(+)-K(+)-adenosine triphosphatase (ATPase) and glucose-6-phosphatase (reversed by 40-87%). Similarly, thioacetamide-induced inhibitions of the activities of Na(+)-K(+)-ATPase, Ca(++)-ATPase, Mg(++)-ATPase, succinate dehydrogenase and elevations in the activities of alkaline phosphatase, gamma-glutamyl transpeptidase, and acid ribonuclease were also significantly reversed. A significant reversal of the toxicants-induced decrease in [14C]-leucine incorporation was also observed. These results indicate that picroliv can also reverse D-galactosamine- or thioacetamide-induced hepatic damage in rats.
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PMID:Perfusion with picroliv reverses biochemical changes induced in livers of rats toxicated with galactosamine or thioacetamide. 825 34

Glycoprotein IX is a relatively small (M(r) 20,000) surface glycoprotein of human platelets; one of three (Ib alpha, Ib beta, IX) polypeptide chains present in the glycoprotein Ib-IX complex that functions as the von Willebrand factor receptor and mediates platelet adhesion in the arterial circulation. Using a cDNA for human glycoprotein IX as the probe, clones were isolated from a human genomic library, and the genomic sequence for glycoprotein IX (3.2 kilobases) was determined. The transcriptional start site was located by RNase protection and primer extension experiments. The gene includes three exons and two introns within 1.6 kilobases of DNA, and the entire open reading frame for glycoprotein IX is included within the third exon. The genes for glycoproteins IX and Ib alpha share similar exon sequences on the 5' side of their ATG start codons, and both genes possess introns in this region. The glycoprotein IX gene contains two consensus regulatory sequences (GATA and ACTTCCT [ets]) in its promoter region (5' flank, within 67 bases of the start site) that are also present in similar sites in the previously described "megakaryocyte-platelet" genes (glycoprotein IIb, platelet factor 4, beta-thromboglobulin: human and rat). Thus, the glycoprotein IX gene shares structural features with other megakaryocyte-platelet genes and contains at least two consensus cis-acting regulatory elements that may govern gene expression.
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PMID:Characterization of the gene encoding human platelet glycoprotein IX. 842 20

It is well known that hypothalamic endogenous opioid peptides (EOP) exert an inhibitory influence on LH release, and that a restraint on this inhibitory tone triggers preovulatory LHRH and LH hypersecretion. Recent evidence suggests that hypothalamic neuropeptide Y (NPY) is an important component of the neural circuitry that participates in induction of the LH surge. We have reported previously that blockade of EOP influence by the opioid receptor antagonist, naloxone (NAL), stimulated NPY accumulation in the median eminence (ME) in association with increased LH release in estradiol-17 beta-primed ovariectomized rats. To evaluate whether a restraint on the EOP system will result in an increase in NPY synthesis, the effects of NAL infusion on preproNPY messenger RNA (mRNA) levels in the medial basal hypothalamus were studied by solution hybridization/RNase protection assay and by in situ hybridization. NAL (2 mg/0.6 ml.h) or saline (SAL, 0.6 ml/h) was infused for 3 h (1100-1400 h) via an intrajugular cannula in estradiol-17 beta-primed ovariectomized rats. In accord with previous studies, NAL infusion significantly increased plasma LH levels at 1400 h. concomitant with this activation of LH release, NPY gene expression was also augmented. As compared with initial control levels at 1100 h, preproNPY mRNA levels in the medial basal hypothalamus increased at 1400 h in SAL (106%)- and NAL (202%)-infused rats, and at this time, preproNPY mRNA levels were significantly higher in NAL-infused rats than in SAL-infused rats. In situ hybridization studies showed that NAL infusion significantly increased the preproNPY mRNA signal at 1400 h mainly in the rostral and middle regions of the arcuate nucleus (ARC) as compared with that seen at 1100 and 1400 h in SAL-infused rats. To examine further the relationship between the NAL-induced increase in LH release and increase in NPY gene expression, the effects of NPY mRNA antisense oligonucleotides (oligos) on NPY levels in the ME-ARC and on plasma LH levels were studied in NAL-infused rats. In NAL-infused rats, intracerebroventricular administration of a NPY antisense oligo (25 micrograms/rat) at 1000, 1200, and 1300 h decreased NPY levels in the ME-ARC and blocked the increase in plasma LH levels at 1500 h, whereas control missense oligos had no effect. Collectively, these results show that a decrease in opioid influence rapidly augments NPY gene expression in a subpopulation of neurons in the ARC, and support the hypothesis that a disinhibition from opioid influence acutely promotes NPY synthesis and release, which is necessary for phasic LH discharge in rats.
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PMID:Disinhibition from opioid influence augments hypothalamic neuropeptide Y (NPY) gene expression and pituitary luteinizing hormone release: effects of NPY messenger ribonucleic acid antisense oligodeoxynucleotides. 853 45

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non-selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32P or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).
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PMID:In situ hybridization: identification of rare mRNAs in human tissues. 938 82

Although ciliary neurotropic factor (CNTF) is a tropic factor in nervous system development and maintenance, peripheral administration of this cytokine also causes severe anorexia and weight loss. The neural mechanism(s) mediating the loss of appetite is not known. As hypothalamic neuropeptide Y (NPY) is a potent orexigenic signal, we tested the hypothesis that CNTF may adversely affect NPYergic signaling in the hypothalamus. Intraperitoneal administration of CNTF (250 microg/kg) daily for 4 days significantly suppressed 24-h food intake in a time-dependent manner and decreased body weight. The loss in body weight was similar to that which occurred in pair-fed (PF) rats. As expected, hypothalamic NPY gene expression, determined by measurement of steady state prepro-NPY messenger RNA by ribonuclease protection assay, significantly increased in PF rats in response to energy imbalance. However, despite a similar loss in body weight, there was no increase in NPY gene expression in CNTF-treated rats. Daily administration of CNTF intracerebroventricularly (0.5 or 5.0 microg/rat) also produced anorexia and body weight loss. In this experiment, negative energy balance produced by both PF and food deprivation augmented hypothalamic NPY gene expression. However, despite reduced intake and loss of body weight, no similar increment in hypothalamic NPY gene expression was observed in CNTF-treated rats. In fact, in rats treated with higher doses of CNTF (5.0 microg/rat), NPY gene expression was reduced below the levels seen in control, freely fed rats. Furthermore, CNTF treatment also markedly decreased NPY-induced feeding. These results suggested that anorexia in CNTF-treated rats may be due to a deficit in NPY supply and possibly in the release and suppression of NPY-induced feeding. The possibility that CNTF-induced anorexia may be caused by increased leptin was next examined. Daily intracerebroventricular injections of leptin (7 microg/rat) decreased food intake, body weight, and hypothalamic NPY gene expression in a manner similar to that seen after CNTF treatment. Leptin administration also suppressed NPY-induced feeding. However, peripheral and central CNTF injections markedly decreased leptin messenger RNA in lipocytes, indicating a deficiency of leptin in these rats; thus, leptin was unlikely to be involved in appetite suppression. Thus, these results show that a two-pronged central action of CNTF, causing diminution in both NPY availability and the NPY-induced feeding response, may underlie the severe anorexia. Further, unlike other members of the cytokine family, suppression of NPYergic signaling in the hypothalamus by CNTF does not involve up-regulation of leptin, but may involve a direct action on hypothalamic NPY neurons or on neural circuits that regulate NPY signaling in the hypothalamus.
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PMID:Anorectic effects of the cytokine, ciliary neurotropic factor, are mediated by hypothalamic neuropeptide Y: comparison with leptin. 944 12

Bacterial lipopolysaccharide (LPS) or endotoxin induces neurological manifestations including anorexia. It is proposed that LPS-induced cytokine production is involved in the generation of neurological manifestations and in neuroinflammatory/immunological responses during gram-negative infections. For example, LPS-induced effects can be blocked or ameliorated by the interleukin-1 receptor antagonist (IL-1Ra). Here, sensitive and specific RNase protection assays were used to investigate the effects of the intracerebroventricular (i.c.v.) administration of LPS on mRNA levels of interleukin-1beta (IL-1beta) system components, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, and neuropeptide Y (NPY) in the cerebellum, hippocampus, and hypothalamus. The same brain region sample was analyzed with all of the antisense probes. The data show simultaneous local induction of multiple cytokine components messenger ribonucleic acids (mRNAs) within specific brain regions in anorectic rats responding to i.c.v. administered LPS (500 ng/rat). Interleukin-1beta and IL-1Ra had a similar mRNA induction profile (hypothalamus > cerebellum > hippocampus). Interleukin-1 receptor type I (IL-1RI) mRNA also increased in all three brain regions examined, and the soluble form of IL-1 receptor accessory protein (IL-1R AcP II) mRNA was induced in the hypothalamus. Tumor necrosis factor-alpha mRNA levels increased in the hypothalamus > hippocampus > cerebellum. Levels of membrane bound IL-1R AcP, TGF-beta1, and NPY mRNAs did not change significantly in any brain region. The results suggest that: (1) endogenous up-regulation of IL-1beta and TNF-alpha in the hypothalamus contribute to LPS-induced anorexia; and (2) the ratio IL-1Ra/IL-1beta, and IL-1beta <--> TNF-alpha interactions may have implications for gram-negative infections associated with high levels of LPS in the brain-cerebrospinal fluid.
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PMID:Interleukin-1beta system (ligand, receptor type I, receptor accessory protein and receptor antagonist), TNF-alpha, TGF-beta1 and neuropeptide Y mRNAs in specific brain regions during bacterial LPS-induced anorexia. 957 Jul 21

A series of contiguous deletions were made in a cDNA encoding the ribonuclease restrictocin with the purpose of identifying the amino acids that are essential for the cleavage of the phosphodiester bond on the 3' side of G4325 in the alpha-sarcin/ricin domain of mammalian (rat) 28S rRNA. In all 93 of 149 amino acids, 62% of the residues in restrictocin, were not essential for the action of the toxin. Of the five residues that have been proposed to constitute the active site, three could be deleted without loss of activity if they were part of a deletion of three or five amino acids but not if they were removed singly. It is likely that the loss of these three residues is compensated for by a neighboring residue that occupies the structural space created by the larger amino acid deletions. This was demonstrated to be the case for the active site residue Glu95 which in the deletion mutant Delta91-95 is replaced by Asp90. Systematic deletion of amino acids is a rapid, cost effective method for identifying the residues in a protein likely to contribute directly to function and, hence, deserving of closer scrutiny. Moreover, a semiquantitative estimate of the contribution of the residue to function can be made. For this reason the method may be useful for functional proteomics.
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PMID:Analysis by systematic deletion of amino acids of the action of the ribotoxin restrictocin. 1182 14


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