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)

Antagonism is the ability of a modified antigenic peptide (altered peptide ligand, APL) to prevent CD4 T cell activation by the original peptide. Here we show that antagonistic activity can be conferred to peptides of HIV envelope glycoprotein gp120 and reverse transcriptase p66 by adding flanking polypeptide sequences at the C or at the N terminus by genetic engineering, rather than by introducing substitutions by synthesis. The glutathione S-transferase (GST)-peptide system has been used to produce molecules that display the peptide at the appropriate end of the GST carrier. When the gp120 peptide 191-205 (pep24) was expressed at the C terminus of GST (GST-24), antigenicity of specific human CD4 T cells was maintained. In contrast, when the peptide was expressed at the N terminus of GST (24-GST), antigenicity was abolished and antagonistic activity was introduced. Similar results were obtained with a p66-derived peptide at the C terminus of the GST carrier. Antagonism was (1) specific; proliferation of a CD4 T cell line from the same donor responding to the envelope glycoprotein of another retrovirus, HTLV-1, was not affected; (2) reversible; proliferative response was rescued in T cells exposed to antigen-presenting cells (APC) pulsed with the antagonist; (3) dominant; T cells cultured with APC pulsed with the agonist and with APC pulsed with the antagonist did not proliferate. The carrier could be cleaved by proteolysis while the antagonistic activity was preserved. Thus a minimal sequence that confers antagonistic activity can be engineered or synthesized with peptides to antagonize undesired CD4 responses as an alternative to the use of APL.
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PMID:Antagonistic activity of HIV-1 T helper peptides flanked by an unrelated carrier protein. 1035 98

Glycolipid transfer protein (GLTP) is a small (23-24 kDa), basic protein (pI congruent with 9.0) that accelerates the intermembrane transfer of various glycolipids. Here, we report the first cloning of cDNAs that encode the bovine and porcine GLTPs. The cDNA open reading frame for bovine GLTP was constructed by bridge-overlapping extension polymerase chain reaction (PCR) after obtaining partial coding cDNA clones by hot start, seminested, and rapid amplification of cDNA ends-PCR. The cDNA open reading frame for porcine GLTP was constructed by reverse transcriptase-PCR. The encoded amino acid sequences in the full-length bovine and porcine cDNAs were identical, consisting of 209 amino acid residues, and were nearly the same as the published sequence determined by Edman degradation. The cDNA encoded one additional amino acid at the N terminus (methionine), arginine at positions 10 and 200 instead of lysine, and threonine at position 65 instead of alanine. Expression of GLTP-cDNA in Escherichia coli using pGEX-6P-1 vector resulted in glutathione S-transferase (GST)-GLTP fusion protein. Regulation of growth and induction conditions led to approximately 50% of expressed fusion protein being soluble and active. Proteolytic cleavage of GST-GLTP fusion protein (bound to GST-Sepharose) and affinity purification resulted in fully active GLTP. Northern blot analyses of bovine tissues showed a single transcript of approximately 2.2 kilobases and the following hierarchy of mRNA levels: cerebrum > kidney > spleen congruent with lung congruent with cerebellum > liver > heart muscle. Reverse transcriptase-PCR analyses of mRNA levels supported the Northern blot results.
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PMID:Cloning and expression of glycolipid transfer protein from bovine and porcine brain. 1067 54

Quercetin is one of the most abundant of the naturally occurring flavonoids. It has been estimated that about 25-50 mg of quercetin are consumed from the daily diet. The chemopreventive effect of quercetin on dietary carcinogen has been intensely studied in animal models; however, knowledge regarding the molecular mechanism is still limited. In this study, the human hepatoma Hep G2 cell line was used to investigate how quercetin prevents benzo[a]pyrene (B[a]P)-induced DNA adducts. The Hep G2 cells were treated with 10 microM B[a]P for 18 hours in the presence or absence of quercetin. The DNA adduct levels, evaluated by 32P postlabeling, decreased in a dose-dependent manner after treatment with quercetin. Cytochrome P-450 1A1 (CYP1A1) and glutathione S-transferase involvement have been well demonstrated in the modulation of B[a]P-induced DNA damage. From the assays of both enzyme activities, quercetin inhibits CYP1A1-linked ethoxyresorufin O-dealkylase activity more effectively than glutathione S-transferase activity. To elucidate the molecular mechanisms, reverse transcriptase-polymerase chain reaction and Western blot were used to evaluate whether the decrease in CYP1A1 enzyme activity by quercetin is mediated because of alterations of CYP1A1 transcription or mRNA stability. The results indicated that quercetin significantly inhibits B[a]P-induced CYP1A1 mRNA and protein expression. From these findings, we conclude that quercetin suppresses B[a]P-induced DNA damage in human Hep G2 cells by altering CYP1A1 gene expression. Thus we suggest that dietary quercetin may have a long-term preventive effect on chemical carcinogenesis, especially in people who eat a diet rich in fruits and vegetables.
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PMID:Quercetin inhibits benzo[a]pyrene-induced DNA adducts in human Hep G2 cells by altering cytochrome P-450 1A1 gene expression. 1069 72

Mice are resistant to the carcinogenic effects of the mycotoxin aflatoxin B(1) (AFB(1)) because they constitutively express an alpha-class glutathione S-transferase (mGSTA3-3) that has high (approximately 200,000 pmol/min/mg) activity toward aflatoxin B(1)-8, 9-epoxide (AFBO). Rats do not constitutively express a GST with high AFBO-conjugating activity and are sensitive to AFB(1)-induced hepatocarcinogenesis. Constitutively expressed human hepatic alpha-class GSTs (hGSTA1-1 and hGSTA2-2) possess little or no AFBO-detoxifying activity (<2 pmol/min/mg). Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), exhibits significant (approximately 300 pmol/min/mg) constitutive hepatic GST activity towards AFBO. To determine which specific GST isoenzyme(s) is (are) responsible for this activity, MF: GSTs were purified from liver tissue and characterized and, Mf mu-class GST cDNAs were cloned by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Purification by glutathione agarose (GSHA) affinity chromatography yielded a protein, GSHA-GST, that exhibited relatively high AFBO-conjugating activity (239 pmol/min/mg) compared to other GST-containing peaks. Western blotting and enzymatic activity analyses revealed that GSHA-GST belongs to the mu class. Two distinct mu-class GST cDNAs, mfaGSTM1 (GenBank accession # AF200709) and mfaGSTM2 (GenBank accession # AF200710), were generated by RT-PCR. CDNA-derived amino acid sequence analysis revealed that mfaGSTM1 and mfaGSTM2 share 97% and 96% homology with the human mu-class GSTs hGSTM4 and hGSTM2, respectively. In contrast to recombinant mfaGSTM1-1, which had no detectable AFBO-conjugating activity, mfaGSTM2-2 exhibited this activity at 333 pmol/min/mg. Activity profiles for the stereoisomers exo- and endo-AFBO, and of 1-chloro-2,4-dinitrobenzene of the purified protein GSHA-GST and recombinant mfaGSTM2-2, suggested that they are two distinct enzymes. Our results indicate that, in contrast to rodents, mu-class GSTs are responsible for the majority of AFBO-conjugating activity in the liver of Macaca fascicularis.
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PMID:Mu-class GSTs are responsible for aflatoxin B(1)-8, 9-epoxide-conjugating activity in the nonhuman primate macaca fascicularis liver. 1086 51

We have shown previously that integrin-linked kinase (ILK) is upregulated in human HT-144 melanoma cells following TGF-beta1 stimulation. Using mRNA from TGF-beta1 stimulated HT-144 cells and reverse transcriptase polymerase chain reaction, we have isolated a cDNA encoding a protein highly homologous to ILK. Sequencing of the full-length 1359 base pair cDNA and polypeptide translation revealed that this protein, designated ILK-2, differs from the known ILK (hereafter called ILK-1) by only four amino acids, while the cDNA sequence diverges by 102 nucleotides, thus excluding that ILK-2 is an allelic variant of ILK-1. Expression of ILK-2 mRNA was observed in metastatic human HT-144 melanoma and HT-1080 fibrosarcoma cell lines, but not in normal human tissues. Moreover, stimulation of HT-144 cells with TGF-beta1, but not with EGF, PDGF-AB or insulin, induced a selective overexpression of ILK-2 mRNA as compared to ILK-1 mRNA. Bacterially-expressed GST/ILK-2 autophosphorylated and labeled myelin basic protein as well as a recombinant GST/beta3 integrin cytoplasmic tail peptide. Transfection of either ILK-2 or ILK-1 cDNA into the non-metastatic melanoma cell line SK-Mel-2, expressing exclusively ILK-1, induced anchorage independent cell growth and cell proliferation, as demonstrated by growth in soft agar. Our data provide evidence that ILK-2 is a new isoform of ILK-1 that is expressed in some highly invasive tumor cell lines but not in normal adult human tissues and whose expression is regulated by TGF-beta1.
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PMID:Cloning of an isoform of integrin-linked kinase (ILK) that is upregulated in HT-144 melanoma cells following TGF-beta1 stimulation. 1087 59

G protein betagamma (Gbetagamma) complexes are considered to play an important role in second messenger signaling of phospholipase C (PLC). Monitoring the inositol 1,4,5-trisphosphate (IP(3)) response in circumvallate tissue homogenates upon stimulation with denatonium benzoate, it was demonstrated that a glutathione S-transferase-GRK3ct fusion protein-a Gbetagamma scavenger-attenuates the bitter tastant-induced second messenger reaction. Towards an identification of the Gbetagamma complex involved in rat bitter taste transduction, it was found that the G protein beta(3) subtype is specifically expressed in taste receptor cells of circumvallate papillae. Gbeta(3)-specific antibodies blocked the denatonium benzoate-induced IP(3) formation in a dose-dependent manner; the inhibitory effect was reversed by preincubation with the antigenic peptide. A less pronounced inhibition was observed using Gbeta(1)-specific antibodies. Analyzing individual taste cells by single cell reverse transcriptase-polymerase chain reaction approaches, overlapping expression patterns for PLCbeta(2), Galpha(gust), Gbeta(3) and Ggamma(3) could be demonstrated. Furthermore, the co-expression of all profiled signal transduction components in individual taste receptor cells could be detected. These data support the concept that the denatonium benzoate-induced IP(3) response is mediated by an activation of PLCbeta(2) via a Gbetagamma complex, possibly composed of Gbeta(3) as the predominant beta subunit and Ggamma(3), and imply that multiple second messenger pathways may exist in individual taste receptor cells.
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PMID:G protein betagamma complexes in circumvallate taste cells involved in bitter transduction. 1094 5

We here presented evidence that a 94-kDa glucose-regulated protein (GRP94) was associated with hepatitis B viral (HBV) polymerase in the human liver cell HepG2 and this association could be applied even in Escherichia coli. We investigated the role of GRP94 in the expression and stabilization of HBV polymerase in Escherichia coli by coexpression of the two proteins. The affinity column-purified glutathione S-transferase-tagged HBV polymerase (GST-P, 130 kDa) showed a proper molecular size and reverse transcriptase activity on several exogenous templates and was sensitive to specific inhibitors. The GST-P was associated with the maltose-binding protein-tagged GRP94 (MBP-GRP94, 130 kDa) using analyses by an affinity chromatography, native gel electrophoresis and glycerol gradient centrifugation. However, nondenaturing and partially denaturing activity gel analyses showed two active bands of approximately 260 kDa and approximately 130 kDa, respectively. Furthermore, in the presence of the encapsidation signal RNA template (HBV epsilon RNA), the approximately 260-kDa active band was gradually converted to approximately 130 kDa, which implies that HBV polymerase was dissociated from the chaperone GRP94 and bound preferentially to the HBV epsilon RNA. These results suggested that the chaperone GRP94 was necessary for the stabilization and production of HBV polymerase as an active form.
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PMID:Expression of stable hepatitis B viral polymerase associated with GRP94 in E. coli. 1096 39

Suppression subtractive hybridization analysis in our laboratory recently revealed that transferrin mRNA may be elevated in Sedeficient rat liver. In this work, we compared expression in rat liver of genes for transferrin, transferrin receptor, ferritin light and heavy chains, and iron-regulatory proteins 1 and 2 in Se adequacy and deficiency. Weanling male Sprague-Dawley rats were fed Torula yeast diets supplemented with 0 or 0.15 microg Se/kg diet as sodium selenite for 15 wk. Activity of cellular glutathione peroxidase was virtually abolished in Se-deficient rat liver, whereas activity of glutathione S-transferase was 43% higher than in Se-adequate liver. There were no differences in hematocrit, hemoglobin, or liver iron content. To examine differential gene expression, we used a multiplex relative reverse transcriptase-polymerase chain reaction method. Three of the six genes examined showed modest but consistent upregulation in Se deficiency. Transferrin mRNA was 30% more abundant in Se-deficient than in Se-adequate liver. For the transferrin receptor, the difference was 32%, and for iron regulatory protein 1, it was 63%. No consistent differences were observed for iron regulatory protein 2 or for ferritin light or heavy chain. These findings suggest a possible role for dietary Se in moderating iron metabolism.
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PMID:Selenium regulates expression in rat liver of genes for proteins involved in iron metabolism. 1104

Schistosoma mansoni p14 gene encodes an eggshell precursor that is expressed only in vitelline cells of mature female worms in response to a male stimulus. The upstream region of the p14 gene contains several potential cis-acting regulatory sequences. We used the upstream region of the p14 gene as bait in a yeast-one-hybrid screen of a S. mansoni cDNA library to identify interacting proteins. We report the identification and characterization of a cDNA (S. mansoni PUR-alpha (SmPUR-alpha)) encoding a protein homologous to single-stranded DNA transcription activator PUR-alpha, that binds to the p14 upstream region and activates transcription of the HIS3 reporter gene in yeast. SmPUR-alpha has a predicted molecular mass of 30 kDa and shares an overall homology of 63% with mammalian PUR-alpha. The DNA binding domain of SmPUR-alpha is highly conserved. We show by gel shift assays that GST-SmPUR-alpha binds to oligonucleotides comprising the p14 upstream region. SmPUR-alpha binds preferentially to single-stranded DNA and also binds RNA. Unlike the mammalian homologue, SmPUR-alpha exhibits little specificity for the PUR element GGn, but shows strong preference for a sequence containing alternating pyrimidines. Our data support that SmPUR-alpha is a single-copy gene and through reverse transcriptase-polymerase chain reaction and in situ hybridization, we show that SmPUR-alpha is constitutively transcribed in many cell types and thus likely plays a role as a general transcription activator in schistosomes.
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PMID:Identification and functional characterization of a member of the PUR-alpha family from Schistosoma mansoni. 1107 Dec 90

Smad proteins are essential intracellular signal transducers of the transforming growth factor-beta (TGF-beta) superfamily. The TGF-beta superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70- and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptase-polymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmTbetaR-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmTbetaR-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-beta(1). These data indicate that the SmSmad2 responds to the TGF-beta signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-beta effect.
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PMID:Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer. 1115 51


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