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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cDNA fragment (HSD-1) coding for part of a human sperm membrane protein (hSMP-1) was previously isolated from a human testis cDNA expression library, with the serum from an infertile patient used as a probe. By rescreening human testis cDNA libraries with the HSD-1 insert and using rapid amplification of cDNA ends, the complete cDNA of 2482 bp was identified and sequenced. An open reading frame of 1572 bp encodes 523 amino acid residues with a computed molecular mass of 55.08 kDa. This protein sequence does not match any other sequence in the databases, indicating that it represents a novel sperm antigen. Northern blot analysis of human and rat testis poly(A) mRNA detected a band of approximately 2.5 kb in both species. Reverse transcriptase polymerase chain reaction analysis showed that hSMP-1 mRNA was present in human testis but was not in either kidney or liver. When the cDNA was expressed in Escherichia coli under the control of the T7 promoter, the expressed protein accumulated to a level of about 50% of the total cellular protein. The expressed protein, which contained an N-terminal poly(his) sequence tag, was purified by chromatography on an nitrilo-tri-acetic acid affinity resin. Approximately 10 mg of pure protein was obtained from a 500-ml culture, purified, and used as antigen to generate a polyclonal antiserum in rabbits. Western blot analysis of human sperm extracts showed a single specific band at 55.5 kDa. Immunofluorescence data showed that hSMP-1 was localized to the head of human sperm. The fluorescent staining formed a cap-shaped pattern that was similar in morphology to the human sperm acrosome. The availability of large amounts of recombinant hSMP-1 and its antiserum will facilitate studies on the function and expression of the protein during spermatogenesis and the assessment of its potential value as a contraceptive immunogen.
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PMID:Expression and characterization of a novel human sperm membrane protein. 878 82

A full-length cDNA sequence was isolated from kidney total RNA of di(2-ethylhexyl) phthalate-treated sea bass by reverse-transcriptase polymerase chain reaction and then rapid amplification of cDNA ends. The deduced amino acid sequence, which has been named CYP4T2, shared 69 and 54.4% amino acid identity with rainbow trout CYP4T1 and rat CYP4B1, respectively. RNA blot analysis using the CYP4T2 cDNA as a probe indicated that the mRNA was rather abundant in kidney, and less so in liver, small intestine, and brain. Treatment of sea bass with peroxisome proliferators showed marked tissue-specific induction. CYP4 inducers clofibrate, di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxy acetic acid (2,4-D) were administered by intraperitoneal injection. The strongest induction was found in kidney after a DEHP treatment (6.5-fold) or a 2,4-D treatment (9-fold), while no induction was observed in liver.
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PMID:Molecular cloning of a phthalate-inducible CYP4 gene (CYP4T2) in kidney from the sea bass, Dicentrarchus labrax. 979 Sep 33

Leptin was shown to exhibit similar to cholecystokinin (CCK) cytoprotective activity against acute gastric lesions, but its role in ulcer healing has not been examined. The aims of this study were: (1) to compare the effects of exogenous leptin to those of CCK on the course of healing of chronic gastric ulcers; (2) to study the gene and protein expression of leptin at the ulcer margin during ulcer healing; and (3) to assess the effects of leptin administration on the mucosal gene expression of main growth factor such as transforming growth factor alpha (TGFalpha). Gastric ulcers were produced in rats by the acetic acid method. Rats with ulcers were divided in following treatment groups: (1) vehicle; (2) leptin (10 microg/kg i.p.); (3) CCK (10 microg/kg s.c.); and (4) leptin or CCK with or without tyrphostin A46 (200 microg/kg i.p.), an inhibitor of epidermal growth factor (EGF)-receptor tyrosine kinase or NG-nitro-L-arginine (20 mg/kg i.g.), a blocker of nitric oxide synthase. Animals were euthanized 9 days after ulcer induction. The area of gastric ulcers and the gastric blood flow at the ulcer area were determined. In addition, mucosal biopsy samples were taken from the ulcer area for histological evaluation as well as for the determination of mRNA and protein expression for leptin and constitutive nitric oxide synthase (cNOS) and inducibile nitric oxide synthase (iNOS) by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot, respectively. In addition, the gene expression for TGFalpha was analyzed by RT-PCR. Both leptin and CCK reduced significantly the ulcer area as compared to vehicle-treated group by approximately 50%. The treatment with tyrphostin or N(G)-nitro-L-arginine reversed in part the acceleration of ulcer healing by leptin and CCK. The expression of leptin mRNA and protein was significantly increased at the ulcer edge. The leptin-induced acceleration of ulcer healing was associated with increased expression of transcripts for TGFalpha as well as increased mRNA and protein expression for cNOS and iNOS at the ulcer margin. We conclude that leptin accelerates ulcer healing by mechanisms involving the up-regulation of TGFalpha and increased production of nitric oxide due to up-regulation of cNOS and iNOS in the ulcer area.
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PMID:Role of leptin in ulcer healing. 1123 Sep 99

Expression of human beta-defensins is correlated with differentiation in the oral epithelium, consistent with their function as part of the epithelial antimicrobial barrier. Because calcium is a known regulator of epithelial differentiation, we tested the hypothesis that calcium concentration mediates beta-defensin expression. Gingival epithelial cells were cultured in medium containing low calcium concentration (0.03 mM), then either changed to high extracellular calcium concentrations or stimulated with thapsigargin to release intracellular calcium stores in the presence or absence of BAPTA-AM, a calcium chelator. Human beta-defensin-2 (hBD-2) mRNA expression was rapidly induced by thapsigargin, and more slowly induced by high extracellular calcium. Induction of hBD-2 peptide was confirmed by immunofluorescence. BAPTA-AM inhibited hBD-2 induction by both thapsigargin and calcium in a dose-dependent fashion. In addition, BAPTA-AM inhibited hBD-2 induction by a bacterial stimulant. Collectively, these findings demonstrate that intracellular calcium is a critical mediator of hBD-2 expression. Abbreviations used in this study are: BAPTA-AM, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid tetrakis (acetoxymethyl ester); DMSO, dimethylsulfoxide; F. nucleatum, Fusobacterium nucleatum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBDs, human beta-defensins; HGECs, human gingival epithelial cells; MAP, mitogen-activated protein; and RT-PCR, reverse-transcriptase/polymerase chain-reaction.
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PMID:Intracellular calcium in signaling human beta-defensin-2 expression in oral epithelial cells. 1457 98

Growth hormone secretion is under the control of a pair of hypothalamic factors, growth hormone releasing hormone and somatostatin. The growth hormone secretagogue receptor (GHSR) and its endogenous ligand represent a novel third method regulating the release of growth hormone. Early chicken embryonic development has been proposed to be independent of GH. However, recent evidence shows that peripheral GH secretion has paracrine/autocrine functions during embryonic development. In the current study, we used the reverse-transcriptase polymerase chain reaction to determine the expression pattern of the GHSR during embryonic development and the effects of in ovo recombinant human (rh) IGF-I administration on its expression pattern. Eggs were injected once with 100 ng rhIGF-I in 10 mM acetic acid, and 0.1% BSA per embryo on embryonic day 3. Total RNA was isolated from whole embryos on embryonic day (E) 0-6 (n=6 per day), thoracic/abdominal halves of the embryos on E7- E8 (n= 6 per day) and Pectoralis muscle on E9-E20 (n= 4 per day). We found that GHSR expression was low during E0-E4, followed by an increase on E5 and remained constant through E17. GHSR expression then increased on E18 before reducing on E20. A similar pattern was found in the rhIGF-I treated embryos with the exception of a significant increase in GHSR expression on E8. These data indicate that the GHSR may be active in regulating GH secretion during early embryonic development, and upregulation of the GHSR gene following IGF-I administration may have an important role in the determination of postnatal muscle growth.
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PMID:The effects of in ovo rhIGF-I administration on expression of the growth hormone secretagogue receptor (GHSR) during chicken embryonic development. 1530 60

A novel nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), [(1R)-5-cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyano[3,4-b]indol-1-yl] acetic acid (HCV-371), was discovered through high-throughput screening followed by chemical optimization. HCV-371 displayed broad inhibitory activities against the NS5B RdRp enzyme, with 50% inhibitory concentrations ranging from 0.3 to 1.8 microM for 90% of the isolates derived from HCV genotypes 1a, 1b, and 3a. HCV-371 showed no inhibitory activity against a panel of human polymerases, including mitochondrial DNA polymerase gamma, and other unrelated viral polymerases, demonstrating its specificity for the HCV polymerase. A single administration of HCV-371 to cells containing the HCV subgenomic replicon for 3 days resulted in a dose-dependent reduction of the steady-state levels of viral RNA and protein. Multiple treatments with HCV-371 for 16 days led to a >3-log10 reduction in the HCV RNA level. In comparison, multiple treatments with a similar inhibitory dose of alpha interferon resulted in a 2-log10 reduction of the viral RNA level. In addition, treatment of cells with a combination of HCV-371 and pegylated alpha interferon resulted in an additive antiviral activity. Within the effective antiviral concentrations of HCV-371, there was no effect on cell viability and metabolism. The intracellular antiviral specificity of HCV-371 was demonstrated by its lack of activity in cells infected with several DNA or RNA viruses. Fluorescence binding studies show that HCV-371 binds the NS5B with an apparent dissociation constant of 150 nM, leading to high selectivity and lack of cytotoxicity in the antiviral assays.
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PMID:Novel nonnucleoside inhibitor of hepatitis C virus RNA-dependent RNA polymerase. 1556 61

The peptide transporter PEPT2 mediates transmembrane uptake of small peptides. So far, its expression has not been evidenced in the gastrointestinal tract. We have investigated peptide transport activity in the neuromuscular layers of the gastrointestinal tract by using the fluorescent tracer-dipeptide beta-Ala-Lys-Nepsilon-7-amino-4-methyl-coumarin-3-acetic acid (Ala-Lys-AMCA). Whole-mount preparations from mouse, rat, and guinea pig stomach and small and large intestine were incubated with Ala-Lys-AMCA in the presence or absence of the uptake-inhibitors L-histidine, D-phenylalanyl-L-alanine (D-Phe-Ala), glycyl-L-sarcosine (Gly-Sar), glycyl-L-glutamine (Gly-Gln), benzylpenicillin, and cefadroxil. Fluorescence microscopy revealed that Ala-Lys-AMCA specifically accumulated in both ganglionic layers of the enteric nervous system (ENS) in all regions and species studied. This could be inhibited by Gly-Sar, D-Phe-Ala, Gly-Gln, and cefadroxil, but not by free histidine and benzylpenicillin, indicating uptake via PEPT2. Accordingly, dipeptide uptake was completely abolished in PEPT2-deficient mice. Reverse transcriptase-polymerase chain reaction analysis detected a PEPT2-specific transcript in extracts from the ganglionic ENS layers of mouse small and large intestine, further proving that enteric dipeptide transport activity is specifically mediated via PEPT2. The cellular site of dipeptide uptake was immunohistochemically localized to enteric glial cells and tissue-resident macrophages. In addition, dipeptide uptake occurred in a neurochemically defined subset of neurons in the guinea pig ENS. Our results constitute the first functional evidence for dipeptide transport activity in the ENS. PEPT2-mediated dipeptide transport in enteric glia could contribute to the clearance of neuropeptides in the ENS. In addition, the fluorophore-coupled dipeptide uptake via PEPT2 is a novel vital marker for glial cells in the ENS.
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PMID:Functional expression of the peptide transporter PEPT2 in the mammalian enteric nervous system. 1604 13

Aberrant regulation of innate immune responses and uncontrolled cytokine bursts are hallmarks of sepsis and endotoxemia. Activation of the nuclear liver X receptor (LXR) was recently demonstrated to suppress inflammatory genes. Our aim was to investigate the expression of LXR in human monocytes under normal and endotoxemic conditions and to study the influence of LXR activation on endotoxin-induced cytokine synthesis and release. Adherent human monocytes or whole blood were pretreated with a synthetic LXR agonist (3-{3-[(2-chloro-3-trifluoromethyl-benzyl)-(2,2-diphenyl-ethyl)-amino]-propoxy}-phenyl)-acetic acid) and subsequently challenged with LPS (from Escherichia coli) or peptidoglycan (from Staphylococcus aureus). Cytokine release was assessed by a Multiplex antibody bead kit, and cytokine mRNA levels were measured by real-time reverse-transcriptase-polymerase chain reaction. We found that LXRalpha mRNA was up-regulated in CD14+ monocytes in LPS-challenged blood, whereas LXRbeta mRNA was not altered. Addition of 3-{3-[(2-chloro-3-trifluoromethyl-benzyl)-(2,2-diphenyl-ethyl)-amino]-propoxy}-phenyl)-acetic acid to monocytes suppressed the LPS-induced release of IL-1beta, IL-6, IL-8, IL-10, IL-12p40, TMF-alpha, macrophage inflammatory protein 1alpha, macrophage inflammatory protein 1beta, and monocyte chemoattractant protein 1 in a concentration-dependent manner. Surprisingly, an accompanying decrease in cytokine mRNA accumulation was not observed. The suppressed cytokine release could not be explained by a diminished transport of mRNA out of the nucleus or a decreased secretion of cytokines. We propose that LXR is a key regulator of cytokine release in LPS-challenged human monocytes, possibly by interfering with translational events.
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PMID:Liver X receptor is a key regulator of cytokine release in human monocytes. 1772 34

The antiulcer effect of bisdemethoxycurcumin, a yellow pigment found mainly in rhizomes of Curcuma longa, was compared with curcumin in gastric ulcer model systems to validate its clinical application as a remedy for peptic ulcer. Western blot analysis of mouse macrophage cell line RAW 264.7 activated with lipopolysaccharide showed that bisdemethoxycurcumin inhibited inducible nitric oxide synthase (iNOS) production significantly but had no effect on tumor necrosis factor-alpha (TNF-alpha) production, whereas curcumin showed stronger suppression of iNOS protein production and inhibited TNF-alpha protein production significantly. However, bisdemethoxycurcumin and curcumin possessed similar potency in scavenging nitric oxide generated from mouse macrophage cell line RAW 264.7. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that both curcuminoids inhibited the induction of iNOS dose-dependently at the transcriptional level and curcumin also appeared to inhibit the induction of TNF-alpha at post-transcriptional level. In an animal model, intraduodenal administration of bisdemethoxycurcumin (5-80 mg/kg body wt.) showed a strong inhibitory effect on gastric acid secretion in pylorus-ligated rats whereas curcumin (5-20 mg/kg body wt.) showed a less inhibitory effect, with maximum potency at a dose of 20mg/kg body wt. Moreover, oral administration of bisdemethoxycurcumin at doses of 20-80 mg/kg body wt. twice daily for 10 days showed a significant curative efficacy in accelerating the healing of acetic acid-induced chronic gastric ulcer and promotion of mucosal regeneration in the ulcerated portion in a dose-related manner with potency equal to curcumin. In contrast, the curative potency of curcumin tended to decrease at doses over 160 mg/kg body wt./day. Western blot analysis in ulcerated gastric mucosa showed that bisdemethoxycurcumin dose-dependently reduced the increased protein expression level of iNOS but not TNF-alpha. These results indicated that bisdemethoxycurcumin directly accelerates gastric ulcer healing with potency equal to curcumin. Its antiulcer effect might be due to its properties of decreasing gastric acid secretion and enhancing the mucosal defensive mechanism through suppression of iNOS-mediated inflammation.
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PMID:Comparative antiulcer effect of bisdemethoxycurcumin and curcumin in a gastric ulcer model system. 1918 55

Gall formation by Pantoea agglomerans pv. gypsophilae is controlled by hrp/hrc genes, phytohormones, and the quorum-sensing (QS) regulatory system. The interactions between these three components were investigated. Disruption of the QS genes pagI and pagR and deletion of both substantially reduced the transcription levels of the hrp regulatory genes hrpXY, hrpS, and hrpL, as determined by quantitative reverse-transcriptase polymerase chain reaction. Expression of hrpL in planta was inhibited by addition of 20 microM or higher concentrations of the QS signal C(4)-HSL. The pagR and hrpL mutants caused an equivalent reduction of 1.3 orders in bacterial multiplication on bean leaves, suggesting possible mediation of the QS effect on epiphytic fitness of P. agglomerans pv. gypsophilae by the hrp regulatory system. indole-3-acetic acid (IAA) and cytokinin significantly affected the expression of the QS and hrp regulatory genes. Transcription of pagI, pagR, hrpL, and hrpS in planta was substantially reduced in iaaH mutant (disrupted in IAA biosynthesis via the indole-3-acetamide pathway) and etz mutant (disrupted in cytokinin biosynthesis). In contrast, the ipdC mutant (disrupted in IAA biosynthesis via the indole-3-pyruvate pathway) substantially increased expression of pagI, pagR, hrpL, and hrpS. Results presented suggest the involvement of IAA and cytokinins in regulation of the QS system and hrp regulatory genes.
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PMID:Regulatory interactions between quorum-sensing, auxin, cytokinin, and the Hrp regulon in relation to gall formation and epiphytic fitness of Pantoea agglomerans pv. gypsophilae. 1952 67


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