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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA replication and repair require a specific mechanism to join the 3'- and 5'-ends of two strands to maintain DNA continuity. In order to understand the details of this process, we studied the activity of the 5' nucleases with substrates containing an RNA template strand. By comparing the eubacterial and archaeal 5' nucleases, we show that the polymerase domain of the eubacterial enzymes is critical for the activity of the 5' nuclease domain on RNA containing substrates. Analysis of the activity of chimeric enzymes between the DNA polymerases from Thermus aquaticus (TaqPol) and Thermus thermophilus (TthPol) reveals two regions, in the "thumb" and in the "palm" subdomains, critical for RNA-dependent 5' nuclease activity. There are two critical amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates. Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA-dependent 5' nuclease activity 4-10-fold. Furthermore, the RNA-dependent DNA polymerase activity is controlled by a completely different region of TaqPol and TthPol, and mutations in this region do not affect the 5' nuclease activity. The results presented here suggest a novel substrate binding mode of the eubacterial DNA polymerase enzymes, called a 5' nuclease mode, that is distinct from the polymerizing and editing modes described previously. The application of the enzymes with improved RNA-dependent 5' nuclease activity for RNA detection using the invasive signal amplification assay is discussed.
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PMID:RNA template-dependent 5' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases. 1082 84

PEPCK is a key enzyme of gluconeogenesis in liver and kidney. Recently, we have shown that small intestine also contributes to the endogenous glucose production in insulinopenia in rats and that glutamine is the main precursor of glucose synthesized in this tissue. The expression of the PEPCK gene in rat and human small intestine and the effect of streptozotocin-induced diabetes and fasting have been studied using reverse transcriptase-polymerase chain reaction, Northern blot analysis, and determination of enzyme activity. The PEPCK gene is expressed along the whole small intestine in adult rat and human. The abundance of PEPCK mRNA was increased approximately 30 times in the duodenum, 15 times in the jejunum, and only 3 times in the ileum from diabetic rats. PEPCK mRNA was downregulated in all parts of the tissue upon insulin treatment for 10 h. In 48-h fasted rats, the PEPCK mRNA abundance was increased 17 times in the duodenum and the jejunum and 3 times in the ileum, and it was normalized upon refeeding for 7 h. PEPCK activity was also increased 2-5 times in diabetic and fasted rats in the duodenum and jejunum but not in the ileum. We conclude that PEPCK is a crucial enzyme contributing to the induction of gluconeogenesis in small intestine, just as it is well known to be in the liver and kidney.
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PMID:Induction of PEPCK gene expression in insulinopenia in rat small intestine. 1090 74

The role of antioxidants in preventing apoptosis and viral activation in HIV is well documented. N-acetylcysteine, glutathione, and alpha-lipoic acid have been shown to interrupt the process of viral activation and CD4 cell death. L-glutamine has been shown to improve glutathione levels and significantly increase lean body mass in HIV infection. The literature on the use of L-carnitine and acetyl-L-carnitine in treating mitochondrial toxicity, both in muscle and nerve pathologies is relevant in nutritional treatment of HIV, given the mitochondrial toxicity of nucleoside analog reverse transcriptase inhibitor therapy. The current use of highly active antiviral therapies, their toxicity, and significant failure rates have created the need for a more conservative reassessment of HIV treatment. The adjunctive use of nutrient therapy in the treatment of HIV is reviewed here.
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PMID:Nutrients and HIV: part three - N-acetylcysteine, alpha-lipoic acid, L-glutamine, and L-carnitine. 1095 77

Total parenteral nutrition (TPN) decreases intestinal IgA and levels of Th2 cytokines, interleukin (IL)-4, and IL-10 within the supernatants of intestinal homogenates. These cytokines are known to stimulate IgA production in vitro by cells of the gut-associated lymphoid tissue (GALT). Glutamine (GLN) supplementation of TPN normalizes GALT mass and cytokine levels. Because intestinal homogenates contain mucosa which itself is a source of cytokines, it was unclear whether cytokines change within the GALT itself. This study investigates dietary effects on IL-4 and IL-10 cytokine mRNA expression within isolated GALT lamina propria cells after lipopolysaccharide (LPS) stimulation. Prospective randomized experimental trials were used in this study. Fifty-nine mice were randomized to chow, intravenous TPN (IV-TPN), intragastric TPN (IG-TPN), complex enteral diet (CED), or 2% GLN-supplemented TPN (GLN-TPN). In experiment 1, animals were fed chow, IV-TPN, IG-TPN, or CED for 5 days and received intraperitoneal LPS (100 microg/kg BW), and then were sacrificed 1 h later. Intestine was harvested for GALT lamina propria. Total RNA was extracted from lamina propria cells and cytokine mRNA for IL-4, and IL-10 was measured by reverse transcriptase polymerase chain reaction. IgA levels of intestinal washing were also measured with ELISA. In experiment 2, mRNA for IL-4 and IL-10, and intestinal IgA levels were measured in mice fed chow, IV-TPN, or GLN-TPN as in experiment 1. Both IL-4 and IL-10 mRNA expression decreased significantly in IV-TPN mice compared to chow or CED feeding. IG-TPN resulted in IL-10 mRNA expression significantly lower than chow or CED but significantly better than IV-TPN. GLN preserved IL-4 and IL-10 mRNA levels, which correlated with intestinal IgA levels. Route and type of nutrition as well as GLN influence message for the Th2 type IgA-stimulating cytokines, IL-4 and IL-10, within the primary site of GALT IgA production, the lamina propria.
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PMID:TPN decreases IL-4 and IL-10 mRNA expression in lipopolysaccharide stimulated intestinal lamina propria cells but glutamine supplementation preserves the expression. 1130 33

Human immunodeficiency virus type-1 Tat has been proposed to play a role in the regulation of reverse transcription. We previously demonstrated that wild-type Tat can augment viral infectivity by suppressing the reverse transcriptase (RT) reaction at late stages of the viral life cycle in order to prevent the premature synthesis of potentially deleterious viral DNA products. Here we have performed a detailed analysis of the cell-free reverse transcription reaction to elucidate the mechanism(s) whereby Tat can affect this process. Our results show that Tat can suppress nonspecific DNA elongation while moderately affecting the specific initiation stage of reverse transcription. In addition, Tat has an RNA-annealing activity and can promote the placement of tRNA onto viral RNA. This points to a functional homology between Tat and the viral nucleocapsid (NC) protein that is known to be directly involved in this process. Experiments using a series of mutant Tat proteins revealed that the cysteine-rich and core domains of Tat are responsible for suppression of DNA elongation, while each of the cysteine-rich, core, and basic domains, as well as a glutamine-rich region in the C-terminal domain, are important for the placement of tRNA onto the viral RNA genome. These results suggest that Tat can play at least two different roles in the RT reaction, i.e., suppression of DNA polymerization and placement of tRNA onto viral RNA. We believe that the first of these activities of Tat may contribute to the overall efficiency of reverse transcription of the viral genome during a new round of infection as well as to enhanced production of infectious viral particles. We hypothesize that the second activity, illustrating functional homology between Tat and NC, suggests a potential role for NC in the displacement of Tat during viral maturation.
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PMID:The Tat protein of human immunodeficiency virus type 1 (HIV-1) can promote placement of tRNA primer onto viral RNA and suppress later DNA polymerization in HIV-1 reverse transcription. 1190 3

Three fish retroviruses infecting walleyes constitute the recently recognized genus called epsilonretrovirus. The founding member of this group, walleye dermal sarcoma virus (WDSV), induces benign skin tumors in the infected fish and replicates near 4 degrees C. While the viral genomic sequence is known, biochemical characterization of the virus has been limited to the identification of the mature structural and envelope proteins present in virions. We undertook this study to determine the cleavage sites in the WDSV Pro and Pol proteins and to characterize the viral protease (PR) in vitro. A recombinant PR was expressed in and purified from Escherichia coli as a larger fusion with additional nucleocapsid and reverse transcriptase residues flanking the PR domain. Autocleavage produced a functional, mature PR. Autocleavage as well as cleavage of peptides and of Gag protein by the mature PR occurred at a pH optimum of 7.0, higher than that of other retroviral proteases. Analysis of the cleavage sites identified a glutamine residue in the P2 position of all WDSV sites, both in Gag and in Pol. Amino acid sequence alignments of Gag-Pro-Pol from WDSV, walleye epidermal hyperplasia virus type 1, and walleye epidermal hyperplasia virus type 2 showed the P2 glutamine to be conserved in all cleavage sites in these three viruses. Such conservation is unprecedented in other retroviruses.
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PMID:Characterization of the protease of a fish retrovirus, walleye dermal sarcoma virus. 1193

N-methyl-D-aspartate receptor (NMDA-R) is an amino acid receptor and membrane calcium channel. NMDA-R is activated by binding of coagonists, L-glutamine and L-glycine. In the brain, calcium entry via NMDA-R activates type I nitric oxide synthase (NOS I). The kidney also contains NOS I and vasodilates in response to L-glycine. In this study, NMDA-R mRNA was demonstrated in rat kidney cortex by reverse transcriptase-PCR and cDNA sequencing. NMDA-R protein was demonstrated in kidney cortex by immunoblotting. To study the functional role of renal NMDA-R, renal hemodynamic effects of NMDA-R inhibition were assessed in rats using a blocker of the NMDA calcium channel (75 mg/kg MK-801 intraperitoneally) or an inhibitor of glycine binding to NMDA-R (30 mg/kg 5,7-dichlorokynurenic acid intraperitoneally). Renal blood flow was measured by perivascular pulse Doppler. GFR was measured by 3H-inulin clearance. Measurements were made before and during glycine infusion. Both NMDA-R antagonists caused renal vasoconstriction and attenuated the renal vasodilatory response to glycine infusion. These effects were not mediated by the renal nerves. The glycine response was not inhibited by aortic snare used to mimic the effects of NMDA-R inhibitors on basal renal blood flow. NMDA-R are expressed in kidney cortex, where they exert a tonic vasodilatory influence and may account for the vasodilatory response to glycine infusion.
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PMID:Vasodilatory N-methyl-D-aspartate receptors are constitutively expressed in rat kidney. 1196 Oct 27

By combining a Drosophila genome data base search and reverse transcriptase-PCR-based cDNA isolation, two G-protein-coupled receptors were cloned, which are the closest known invertebrate homologs of the mammalian opioid/somatostatin receptors. However, when functionally expressed in Xenopus oocytes by injection of Drosophila orphan receptor RNAs together with a coexpressed potassium channel, neither receptor was activated by known mammalian agonists. By applying a reverse pharmacological approach, the physiological ligands were isolated from peptide extracts from adult flies and larvae. Edman sequencing and mass spectrometry of the purified ligands revealed two decapentapeptides, which differ only by an N-terminal pyroglutamate/glutamine. The peptides align to a hormone precursor sequence of the Drosophila genome data base and are almost identical to allatostatin C from Manduca sexta. Both receptors were activated by the synthetic peptides irrespective of the N-terminal modification. Site-directed mutagenesis of a residue in transmembrane region 3 and the loop between transmembrane regions 6 and 7 affect ligand binding, as previously described for somatostatin receptors. The two receptor genes each containing three exons and transcribed in opposite directions are separated by 80 kb with no other genes predicted between. Localization of receptor transcripts identifies a role of the new transmitter system in visual information processing as well as endocrine regulation.
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PMID:Functional annotation of two orphan G-protein-coupled receptors, Drostar1 and -2, from Drosophila melanogaster and their ligands by reverse pharmacology. 1216 55

Glutamine is taken up into the rat hepatoma cell line H4-IIE-C3 by a Na+-dependent transport system which is specific for glutamine, alanine, serine, cysteine and asparagine and does not tolerate substitution of Na+ by Li+. Glutamine transport was relatively weakly inhibited by a 50-fold excess of leucine and was not inhibited by phenylalanine or N -methyl aminoisobutyrate. These general properties are characteristic of the recently identified ASCT/B0 family of transporters. Using a reverse transcriptase PCR-based homology cloning approach, we have characterized a cDNA for a novel member of this transporter family (H4-ASCT2) from H4-IIE-C3 cells. The cDNA encodes a 551-amino acid protein which exhibits similarities of between 75 and 85% with ASCT/B0 transporters previously cloned from other sources. When expressed in Xenopus oocytes, this transporter catalyses Na+-dependent glutamine uptake with characteristics very similar to those of glutamine uptake into the H4-IIE-C3 cells. This newly characterized transporter possesses a number of amino acid sequence differences from ASCT2 clones recently isolated from rat astroglial cells and from normal rat liver. In particular, the loop region between transmembrane helices 3 and 4 from H4-ASCT2 shares less than 60% sequence similarity with ASCT2 from rat liver; furthermore, there are some 25 single amino acid substitutions elsewhere in the H4-ASCT2 sequence compared with that from rat liver. Thus enhanced glutamine uptake in rat hepatoma cells is mediated by the expression of a novel ASCT/B0 transporter isoform rather than by increased expression of the ASCT2 mRNA found in normal rat liver.
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PMID:Identification of a plasma membrane glutamine transporter from the rat hepatoma cell line H4-IIE-C3. 1217 99

The heat shock protein, heme oxygenase-1 (HO-1), contributes to the protection of the intestine. Some experimental models suggest that induction of HO-1 by glutamine may contribute to the preservation of intestinal mucosa. The effect of an enteral infusion of glutamine for 6 h on HO-1 expression in duodenal mucosa was studied in healthy men and women and compared with an isonitrogenous mixture of amino acids. After enteral infusion, endoscopic duodenal biopsies were performed and either fixed in formalin for immunohistochemistry or frozen for HO-1 mRNA analysis by reverse transcriptase-polymerase chain reaction. Histologic examination revealed that HO-1 was constitutively expressed in intestinal epithelial cells (IEC), and that glutamine increased the grade of HO-1 immunostaining (P </= 0.01). Glutamine also increased the percentage of HO-1 immunoreactive lamina propria cells (LPC, 10.5 vs. 7.5%, P </= 0.05). Glutamine significantly increased HO-1 mRNA expression compared with control amino acids: median (range) 156 (102-182) vs. 100 (68-179)%, P </= 0.05. The mRNA level for HO-1 was correlated with the percentage of immunoreactive LPC (r = 0.55, P = 0.017) and the grade of immunostaining in IEC (r = 0.51, P = 0.030). In conclusion, glutamine enhanced HO-1 mRNA and protein expression in human duodenal mucosa. These data support further evaluation of the effects of glutamine on intestinal HO-1 during states of intestinal inflammation.
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PMID:Acute enteral glutamine infusion enhances heme oxygenase-1 expression in human duodenal mucosa. 1222 Dec 10


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