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)

The cDNA for a membrane-associated cGMP-dependent protein kinase (cGK II) was cloned from rat intestine using reverse transcriptase PCR and oligonucleotide primers encoding two conserved motifs of known cGMP-dependent protein kinases and subsequently by screening a rat intestine cDNA library. A full-length clone encodes a protein of 761 amino acids with an estimated size of 87 kDa. Sequences of eight peptides from purified pig intestinal mucosa cGK II were found in the derived amino acid sequence of this clone, identifying it as rat intestinal cGK II. Phylogenetic analysis showed that rat intestinal cGK II is less related to mammalian cGK I than to the Drosophila DG1 gene product and most closely related to a recently cloned mouse brain CGKII isoform. Like several other cGK sequences, that of cGK II contained a leucine/isoleucine heptad repeat motif that has been implicated in dimer formation in cGK I. Expression of cGK II cDNA in HEK 293 cells followed by subcellular fractionation revealed cGK II localization in the cell particulate fraction, consistent with the membrane association of endogenous rat cGK II. On Northern blots, the major cGK II poly(A) RNA form was 4.8 kb, with minor forms of 6.2 and 3.1 kb. The cGK II RNA was highly expressed in rat intestinal mucosa and was 20 times less abundant in rat brain and kidney. The localization of endogenous cGK II RNA in rat small intestine was shown by in situ hybridization to be in villous epithelial cells and to some extent in crypt cells.
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PMID:Cloning, expression, and in situ localization of rat intestinal cGMP-dependent protein kinase II. 793 83

Dendritic cells are potent antigen-presenting cells involved in the initiation of immune responses. The trafficking of these cells to tissues and lymph nodes is mediated by members of the chemokine family. Recently, a novel CC chemokine known as MIP-3alpha or liver and activation-regulated chemokine has been identified from the EMBL/GenBank/DDBJ expressed sequence tag database. In the present study, we have shown that the messenger RNA for MIP-3alpha is expressed predominantly in inflamed and mucosal tissues. MIP-3alpha produced either synthetically or by human embryonic kidney 293 cells is chemotactic for CD34(+)-derived dendritic cells and T cells, but is inactive on monocytes and neutrophils. MIP-3alpha was unable to displace the binding of specific CC or CXC chemokines to stable cell lines expressing their respective high affinity receptors, namely CCR1-5 and CXCR1 and CXCR2, suggesting that MIP-3alpha acts through a novel CC chemokine receptor. Therefore, we used degenerate oligonucleotide-based reverse transcriptase PCR to identify candidate MIP-3alpha receptors in lung dendritic cells. Our results show that the orphan receptor known as GCY-4, CKRL-3, or STRL-22 is a specific receptor for MIP-3alpha, and that its activation leads to pertussis toxin-sensitive and phospholipase C-dependent intracellular Ca2+ mobilization when it is expressed in HEK 293 cells.
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PMID:Cloning and characterization of a specific receptor for the novel CC chemokine MIP-3alpha from lung dendritic cells. 929 37

Hepatitis B virus (HBV) variant strains may develop during therapy for chronic infection with the nucleoside analog 2',3'-dideoxy-3'-thiacytidine (3TC). HBV mutants result from isoleucine (I) or valine (V) substitutions in the methionine (M) of the YMDD motif in the viral reverse-transcriptase catalytic domain. In addition, other mutations in the reverse-transcriptase "B domain" involving either a phenylalanine (F)-to-leucine (L) at amino acid 501 (F501L) or an L-to-M substitution at amino acid 515 (L515M) have been observed during 3TC and Famciclovir therapy as well. To determine the biologic consequences of these mutations on viral replication, variant viral genomes were constructed and transiently transfected into hepatocellular carcinoma (HCC) and HEK 293 human embryo kidney-derived cell lines. In transiently transfected HCC cells, the viruses bearing the YI/VDD or F501L mutations had greatly impaired replication as compared to wild-type virus, whereas the virus carrying the L515M substitution showed the least defect. Double mutants with the L515M substitution showed intermediate defect between the YI/VDD or F501L and the L515M single-mutant strains. In contrast, when transfected into HEK 293 cells, the viruses bearing the YI/VDD or L515M mutation replicated as wild-type. However, under conditions of deoxynucleotide depletion produced by hydroxyurea treatment of HEK 293 cells, all mutants but not the wild-type virus exhibited a reduced replication phenotype similar to that observed in HCC cells. In both HCC and HEK 293 cells, the mutant viruses carrying the F501L substitution showed a decreased pregenomic RNA encapsidation level, suggesting that the defect in HBV DNA synthesis occurs at the RNA packaging level. These findings show that 3TC and Famciclovir selected mutations alter the properties of the HBV reverse transcriptase, resulting in impaired viral replication within the cell.
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PMID:Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective. 946 67

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G protein-coupled receptors. We have used homology genomic library screening and polymerase chain reaction (PCR) techniques to isolate both genomic and cDNA clones encoding the human homolog of the recently cloned rat GALR2 galanin receptor. By fluorescence in situ hybridization, the gene encoding human GALR2 (GALNR2) has been localized to chromosome 17q25.3. The two coding exons of the human GALNR2 gene, interrupted by an intron positioned at the end of transmembrane domain III, encode a 387 amino acid G protein-coupled receptor with 87% overall amino acid identity with rat GALR2. In HEK-293 cells stably expressing human GALR2, binding of [125I]porcine galanin is saturable and can be displaced by galanin, amino-terminal galanin fragments and chimeric galanin peptides but not by carboxy-terminal galanin fragments. In HEK-293 cells, human GALR2 couples both to Galphaq/11 to stimulate phospholipase C and increase intracellular calcium levels and to Galphai/o to inhibit forskolin-stimulated intracellular cAMP accumulation. A wide tissue distribution is observed by reverse transcriptase (RT)-PCR analysis, with human GALR2 mRNA being detected in many areas of the human central nervous system as well as in peripheral tissues.
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PMID:Molecular characterization, pharmacological properties and chromosomal localization of the human GALR2 galanin receptor. 968 25

Mammalian cells are able to sense oxygen and regulate a number of genes in response to hypoxia. The transcription factor Hypoxia Inducible Factor-1 (HIF-1) was identified as an important key component of the hypoxia signaling pathway. HIF-1 is a heterodimer composed of two members of the basic helix-loop-helix transcription factor superfamily containing a PAS (PER-ARNT-SIM) domain: HIF-1alpha and HIF-1beta/ARNT. During the cloning by reverse transcriptase-polymerase chain reaction of the human HIF-1alpha subunit, we isolated two cDNA clones which corresponded to alternative splicing of the HIF-1alpha gene. Polymerase chain reaction analysis and sequencing revealed that both clones possessed three additional base pairs between exons 1 and 2. Also, one of them lacked 127 base pairs corresponding to exon 14. We demonstrate that the mRNA of this truncated form is expressed in several human cells lines and human skin but apparently not in rodents. When transfected in HEK 293 cells, the corresponding 736 amino acid protein (HIF-1alpha(736)) is regulated by hypoxia in a similar manner as the full-length HIF-1alpha (HIF-1alpha(FL)). In luciferase transfection assays, both recombinant proteins HIF-1alpha(736) and HIF-1alpha(FL) dimerize with HIF-1beta/ARNT and activate the VEGF promoter upon hypoxia. However, the shorter HIF-1alpha isoform is 3-fold less active than HIF-1alpha(FL), a result consistent with the lack of the C-terminal transactivation domain. As expected, this small isoform can compete with the endogenous and transfected full-length HIF-1alpha. Altogether, these results suggest that the HIF-1alpha(736) isoform modulates gene expression upon hypoxia.
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PMID:Identification of alternative spliced variants of human hypoxia-inducible factor-1alpha. 1070 53

Neuromedins are a family of peptides best known for their contractile activity on smooth muscle preparations. The biological mechanism of action of neuromedin U remains unknown, despite the fact that the peptide was first isolated in 1985. Here we show that neuromedin U potently activates the orphan G protein-coupled receptor FM3, with subnanomolar potency, when FM3 is transiently expressed in human HEK-293 cells. Neuromedins B, C, K, and N are all inactive at this receptor. Quantitative reverse transcriptase-polymerase chain reaction analysis of neuromedin U expression in a range of human tissues showed that the peptide is highly expressed in the intestine, pituitary, and bone marrow, with lower levels of expression seen in stomach, adipose tissue, lymphocytes, spleen, and the cortex. Similar analysis of FM3 expression showed that the receptor is widely expressed in human tissue with highest levels seen in adipose tissue, intestine, spleen, and lymphocytes, suggesting that neuromedin U may have a wide range of presently undetermined physiological effects. The discovery that neuromedin U is an endogenous agonist for FM3 will significantly aid the study of the full physiological role of this peptide.
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PMID:Neuromedin U is a potent agonist at the orphan G protein-coupled receptor FM3. 1081 30

The expression of the sole alpha-subunit of muscle or brain sodium channels in frog oocytes mediates currents with a bimodal inactivation with an abnormal slow mode that is strongly depressed only by co-expression of the beta1-subunit. In contrast, in the expression of the alpha-subunit in the human embryonic kidney cell line, HEK-293, the slow mode is almost absent, suggesting an endogenous expression of the beta1-subunit. We have tested this hypothesis by reverse transcriptase-polymerase chain reaction. We found an abundant expression of mRNA encoding the beta1A splicing of the putative regulatory sodium channel subunit but no mRNA encoding the beta1-subunit in HEK cells. This finding is consistent with the idea that the endogenous beta1A-subunit is sufficient for suppressing the slowly inactivating mode of sodium currents by co-assembly with alpha-subunits, and calls attention to the reliability of effects attributed in HEK cells to alpha-beta1 co-expression.
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PMID:Endogenous expression of the beta1A sodium channel subunit in HEK-293 cells. 1081 59

We have previously identified a point mutation (intervening sequence (IVS) 4: T --> C) in the branchpoint consensus sequence of intron 4 of the lecithin:cholesterol acyltransferase (LCAT) gene in patients with fish-eye disease. To investigate the possible mechanisms responsible for the defective splicing, we made a series of mutations in the branchpoint sequence and expressed these mutants in HEK-293 cells followed by the analysis of pre-mRNA splicing using reverse transcriptase-polymerase chain reaction as well as LCAT activity assay. The results reveal that 1) the mutation of the branchpoint adenosine to any other nucleotide completely abolishes splicing; 2) the insertion of a normal branch site into the intronic sequence of the natural (IVS4-22c) or the branchpoint (IVS4-20t) mutant completely restores splicing; 3) the natural mutation can be partially rescued by making a single nucleotide change (G --> A) within the branchpoint consensus sequence; and 4) other single base changes, particularly around the branchpoint adenosine residue, significantly decrease the efficiency of splicing and thus enzyme activity. Surprisingly, the nucleotide transversion at the last position of the branchpoint sequence (i.e. IVS4-25a or -25g) results in a 2.7-fold increase in splicing efficiency. Therefore, these observations clearly establish the functional significance of the branchpoint sequence of intron 4 for the splicing of the human LCAT mRNA precursors.
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PMID:Characterization of the effects of mutations in the putative branchpoint sequence of intron 4 on the splicing within the human lecithin:cholesterol acyltransferase gene. 1084 35

Natural resistance-associated macrophage protein 2 (Nramp2) has been suggested to be involved in transferrin-independent iron uptake. Two isoforms of the Nramp2 gene generated by alternative splicing of the 3' exons were identified in mouse, rat and human, but it is unclear if they perform distinct functions. To rationalize our previous work, which indicated an increase in iron deposition in a Parkinsonian monkey brain, two monkey Nramp2 isoforms were isolated for a comparative study to assess their relative iron-uptake abilities, tissue distribution and subcellular localization. The monkey Nramp2 isoforms, 2a and 2b, exhibit approx. 98% identity at the amino acid level when compared with the human homologues. The Nramp2a transcript contains a canonical iron-responsive element (IRE), whereas that of Nramp2b lacks the IRE motif in the 3' untranslated region. By reverse transcriptase (RT)-PCR, the mRNAs of both isoforms were detected in all tissues examined. The amino acid differences at the C-terminus neither affected the protein expression levels in HEK-293T and COS-7 cells nor altered the subcellular localization and tissue distribution of the isoforms. Similar levels of iron uptake were detected in the HEK-293T cells transfected with either the Nramp2a or 2b gene, and a reduction of iron from the ferric (Fe(3+)) to the ferrous (Fe(2+)) state is necessary before transport can take place. However, this transferrin-independent uptake of iron into the cells is not a Ca(2+)-dependent process.
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PMID:Heterologous expression, functional characterization and localization of two isoforms of the monkey iron transporter Nramp2. 1086 Dec 41

Although accumulating data show that placenta is able to synthesize 1,25-dihydroxyvitamin D3, the presence of cytochrome P(450) enzyme capable of converting 25-hydroxyvitamin D3 (250HD(3)) to the biologically active form of vitamin D in this tissue, has not been yet clearly established. In this study, we have investigated the presence of 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-(OH)ase) gene expression products in cultured human syncytiotrophoblast. Total RNA was isolated from cultured placental cells and subjected to Northern blots or RT-PCR by using 1alpha-(OH)ase-specific primers. The amplified complementary DNA fragments were analyzed by gel electrophoresis and nucleotide sequencing. Total RNA from kidney HEK 293 cells was subjected to reverse transcriptase reaction, and a 298-bp complementary DNA 1alpha-(OH)ase probe was generated by PCR. Primary cultures of human syncytiotrophoblasts exhibited 1alpha-(OH)ase activity, and a transcript for this gene could be demonstrated in these cells. Northern blot analysis revealed the presence of a 2.5-kb product, similar in size to that previously reported in kidney. RT-PCR analysis demonstrated the presence of a single transcript with nucleotide sequence identical to that previously reported for human 1alpha-(OH)ase complementary DNA clones. In addition, data are presented which suggest that differentiation of cytotrophoblast to the syncytial state was not necessary for this gene to be expressed, which may indicate a role of this enzyme all through pregnancy. The overall results of this study provide evidence for the presence of 1alpha-(OH)ase in the human placenta, suggesting that conversion of 25OHD(3) to 1,25-dihydroxyvitamin D3 in the trophoblast is most probably attributed to an enzymatic 1alpha-hydroxylation reaction.
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PMID:Identification of a 25-hydroxyvitamin D3 1alpha-hydroxylase gene transcription product in cultures of human syncytiotrophoblast cells. 1090 6


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