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)

mRNA was isolated and purified from human liver, and it was also used as templet for cDNA synthesis under the existence of reverse transcriptase. Two primers were designed and synthesized according to GST gene sequence which has been reported, GST gene was obtained using cDNA as templet and PCR technique. The sequencing result indicated that the GST gene is reliable, it was subcloned into NdeI and Bg1 II sites of plasmid pIJ6021, and then introduced into Streptomyces lividans TK54. Proteins were isolated from transformants (TK54/pIJ4486 and TK54/pIJ6021) respectively, SDS-PAGE result showed that the GST over-expressed and its yield is about 15% in soluble proteins in Streptomyces.
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PMID:[Over-expression of glutathione S-transferase in Streptomyces]. 763 3

Leukophysin (LKP) is a 28-kDa protein of CTL and U937 monocytic cells that is located in the membrane of high density granules as well as lighter cytoplasmic granules or vesicles. mAbs to KLP were used to clone a full length cDNA clone with an open reading frame coding for a 235-amino acid polypeptide with a molecular mass of 24.3 kDa and two potential transmembrane regions. The nucleotide sequence was highly homologous to the 3' end of human RNA helicase A. Expression of the LKP was confirmed as a reverse transcriptase-PCR product that may be an alternately spliced product of RNA helicase A. The cDNA contained a repetitive motif that was similar to synaptophysin 1, a protein that is important for synaptic vesicle exocytosis. A polyclonal Ab directed against the 17 carboxyl-terminal amino acids of LKP detected the same 28-kDa granule membrane protein as the D545, one of the mAbs used to clone the cDNA. In addition, the D545 mAb reacted strongly with the GST fusion protein of the bacterially expressed LKP cDNA. In confocal immunofluorescence studies, the anti-LKP peptide Ab reacted with granzyme A-negative granules and vesicles in CD8+ CTL lymphocytes from normal and Chediak-Higashi patients. Thus, based on the expression of the C-terminal LKP epitope, vesicular structures an granules have been detected in CTL that are distinct from classical granzyme-containing cytolytic granules.
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PMID:Leukophysin: an RNA helicase A-related molecule identified in cytotoxic T cell granules and vesicles. 869 Aug 89

Immune responses in the Australian common brushtail possum (Trichosurus vulpecula) and in particular the role of cytokines are poorly understood. We have undertaken to isolate cytokine genes using reverse transcriptase-polymerase chain reaction (RT-PCR) and in this study describe the molecular cloning of TNF-alpha. Primers were designed from consensus sequences at the N-terminus end of eutherian mammalian TNF-alpha and the possum cDNA, derived from spleen RNA, identified by RT-PCR. The complete cDNA encoding possum TNF-alpha was amplified from lymphocyte RNA by 5' and 3' rapid amplification of cDNA ends (RACE). The nucleotide sequence of the protein coding region of this cDNA shared 66-69% identity with other mammalian TNF-alpha genes. The predicted protein of 233 amino acids shared 56-58% identity with eutherian mammalian TNF-alpha was expressed in both Saccharomyces cerevisiae and Escherichia coli by constructing expression plasmid derivatives of the vectors pYES2 and pGEX-2T respectively. Cell extracts prepared from transformants and the purified GST/TNF-alpha fusion protein exhibited cytotoxic activity on the TNF-alpha-sensitive murine fibroblast L929 cells and stimulated proliferation of possum thymocyte cells. The induction of possum TNF-alpha mRNA in alveolar macrophages was analysed by RT-PCR using possum-specific TNF-alpha primers. Macrophages cultured in the presence of LPS showed enhanced transcription of TNF-alpha mRNA. This is the first report of the cloning and sequence analysis of the cDNA encoding a marsupial cytokine gene.
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PMID:Molecular cloning and characterization of tumor necrosis factor alpha (TNF-alpha) from the Australian common brushtail possum, Trichosurus vulpecula. 872 2

Human bronchial epithelial cells (BEC), a primary defense against inhaled materials, are the progenitor cells for bronchogenic carcinomas and have important metabolic capabilities. We used reverse transcriptase-polymerase chain reaction (RT-PCR) to identify xenobiotic metabolism enzymes expressed in primary BEC and alveolar macrophages (AM) of non-smoking volunteers. Cytochromes P450 (CYP) 1A1, 1B1, 2B7, 2E1, and 4B1 and microsomal epoxide hydrolase (mEH) were expressed in BEC but not AM. CYP2F1 was expressed in BEC, but it was expressed at barely detectable levels or not at all in AM. NADPH oxidoreductase (NADPH OR), microsomal glutathione transferase (GST 12), glutathione transferase mu, phenol sulfotransferase (PST), thermolabile phenol sulfotransferase (TL PST), and the clara cell-specific gene, CC10 were expressed in both BEC and AM. CYP3A4 and glucuronosyl transferases-1 and 2 were not expressed in either BEC or AM. In contrast to primary BEC, of the genes evaluated, the immortalized human bronchial epithelial cell line BEP2D constitutively expressed only CYP1A1, CYP2E1, NADPH OR, glucuronosyl transferase 1, GST 12, GST mu, PST, TL PST, and CC10. The loss of xenobiotic metabolism enzyme gene expression in the BEP2D cell line may result from either reduced exposure to inducing agents, or loss of differentiative characteristics in culture. It is clear from the data comparing BEC and AM that there are important intertissue differences in expression of xenobiotic metabolism enzymes.
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PMID:Xenobiotic metabolism enzyme gene expression in human bronchial epithelial and alveolar macrophage cells. 884 77

Antioxidant enzymes from S. mansoni, cytosolic Cu-Zn superoxide dismutase (CT-SOD), signal-peptide-containing SOD (SP-SOD), glutathione peroxidase (GPX), and glutathione transferase (GST) were compared for their relative levels of transcript expression throughout development in a semiquantitative reverse transcriptase-polymerase chain reaction assay. All of the antioxidant enzymes exhibited a similar pattern of developmental regulation. Adult worms have the highest level of specific mRNA compared with larval stages. GST shows the highest level of expression, being approximately 10-fold more abundant than CT-SOD and SP-SOD and 100-fold more abundant than GPX. This order of expression was nearly consistent for all the developmental stages studied. To localize the antioxidant enzymes, immunofluorescence staining was performed on 3-hr schistosomula and adult worms. GPX, SP-SOD, and CT-SOD were all found to be associated with the adult tegument and gut epithelium. SP-SOD was also associated with organelle and cell membranes of parenchymal cells and interestingly with the spines of adult worms. Schistosomula, on the other hand, showed little immunofluorescence. These studies further demonstrate the developmental regulation of antioxidant enzymes and localize them to the host-parasite interface, supporting the notion that they have a role in allowing adult worms to evade immune attack.
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PMID:Schistosoma mansoni: the developmental regulation and immunolocalization of antioxidant enzymes. 914 42

Variants of the human ovarian carcinoma cell line, OAW42, exhibiting low-level intrinsic resistance (OAW42-SR) and drug-induced higher-level resistance (OAW42-A1 & OAW42-A), were studied along with a sensitive clonal population (OAW42-S) which was isolated from OAW42-SR. Expression of the MDR-associated protein P-170, the more recently discovered LRP (lung resistance-related protein) and MRP (multidrug resistance-associated protein), topoisomerase II alpha and beta, GST pi and the cytoskeletal proteins, cytokeratin 8 and vimentin, were studied (using immunocytochemistry and Western blotting techniques) in conjunction with drug (doxorubicin) accumulation and subcellular distribution. Expression of mRNA for P-170, MRP, topoisomerase 11 alpha and beta and GST pi was studied using RT-PCR (reverse transcriptase polymerase chain reaction). Results indicate differential co-expression of four MDR-associated parameters (P-170, MRP, LRP and reduced topoisomerase II alpha and beta) in the OAW42-SR and OAW42-A1 variants, whereas resistance in the OAW42-A variant appeared to be mainly P-170 mediated. Comparable amounts of MRP and greater amounts of LRP were detected in the OAW42-S cells compared to the OAW42-SR variant (which showed increased resistance compared to the OAW42-S cells), but all cell lines expressed similar low-level amounts of MRP mRNA (by RT-PCR). GST pi levels did not differ markedly between variants. Increased levels of the cytoskeletal proteins were observed with increasing levels of resistance. The relative resistance of the variants, OAW42-SR and OAW42-A1, compared with OAW42-S was seen to change during increased serial passaging of the cells. There was greater drug accumulation by the sensitive OAW42-S cell line compared with that of the resistant variants, particularly the most highly resistant OAW42-A cells. Both verapamil and cyclosporin A effectively restored the accumulation defects seen in the resistant variants, cyclosporin A being the more effective of the two. Sub-cellular location of drug was predominantly in the nucleus with maximum levels seen in the sensitive OAW42-S variant and minimum levels in the most resistant OAW42-A clone.
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PMID:Co-expression of MDR-associated markers, including P-170, MRP and LRP and cytoskeletal proteins, in three resistant variants of the human ovarian carcinoma cell line, OAW42. 927 50

Severe Plasmodium falciparum malaria is characterized by excessive sequestration of infected and uninfected erythrocytes in the microvasculature of the affected organ. Rosetting, the adhesion of P. falciparum-infected erythrocytes to uninfected erythrocytes is a virulent parasite phenotype associated with the occurrence of severe malaria. Here we report on the identification by single-cell reverse transcriptase PCR and cDNA cloning of the adhesive ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs. A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix. The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding. PfEMP1 is suggested to be the rosetting ligand and heparan sulfate, or a heparan sulfate-like molecule, the receptor both for PfEMP1 binding and naturally formed erythrocyte rosettes.
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PMID:Identification of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as the rosetting ligand of the malaria parasite P. falciparum. 941 7

Hepatocytes entrapped in collagen gel and cultured in serum-free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c-fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin-stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 +/- 0.37 and 9 +/- 2.7 nmol glucose/h/microg DNA). Collagen-cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 +/- 4 nmol urea/h/microg DNA). The rate of albumin synthesis in collagen-entrapped cells was maintained above the day-1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 +/- 152 pmol/microg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen-cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and glutathione-transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione-transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP-alpha and -beta, and HNF-1 and -4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate-synthetase I).
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PMID:Long-term expression of differentiated functions in hepatocytes cultured in three-dimensional collagen matrix. 1009 8

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


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