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 level of the terminal complement components secreted by human umbilical vein endothelial cells (HUVEC) was measured by a sensitive ELISA which allows the detection of 30-50 pg/ml of these components. C7 was the only terminal component detected in measurable amounts in the cell supernatant. The mean value was 11 ng/106 cells at 96 h and was slightly higher than that of C3 (9 ng/106 cells). HUVEC and serum C7 analysed by SDS-PAGE and immunoblot exhibited the same electrophoretic mobility. A proportion of C7 secreted by HUVEC was incorporated into the terminal complement complex (TCC) assembled spontaneously in the supernatant of cells cultured in C7-deficient human serum, and was not detected by the standard ELISA for C7 measurement. By adding the amount of C7 present in the TCC to that of free C7, the total amount of the component released by HUVEC was calculated to be approximately 35 ng/106 cells. Further TCC was produced following complement activation of the cell supernatant through the alternative pathway. Synthesis of C7 by HUVEC was confirmed by inhibition experiments in the presence of cycloheximide and by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of C7 mRNA expression. Addition of IL-1alpha and tumour necrosis factor-alpha to the cell culture stimulated the secretion of C3, but had no effect on the synthesis of C7. By contrast, interferon-gamma had only a marginal effect on the production of C3, but markedly down-regulated the synthesis of C7 as assessed both by ELISA and RT-PCR.
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PMID:The endothelium is an extrahepatic site of synthesis of the seventh component of the complement system. 1088 32

Astrocytes contain transport systems that are capable of removing various neurotransmitters from the synaptic cleft by transporters present in the plasma membrane. Glial serotonin transporter (SERT) plays an important role in the re-uptake of 5-hydroxytryptamine (5-HT). We examined the pharmacological characterization of 5-HT uptake into rat cortical synaptosomes and cultured rat astrocytes, and the immunodetection of glial SERT proteins using specific site-directed monoclonal antibodies (MoAb). Furthermore, using a reverse transcriptase-polymerase chain reaction (RT-PCR) method, we addressed the expression of SERT mRNA in cultured rat astrocytes. We investigated the inhibitory effects of various monoamine uptake inhibitors on the uptake of [3H]5-HT into cultured astrocytes and cortical synaptosomes. Tricyclic antidepressants (clomipramine and imipramine) as well as selective serotonin re-uptake inhibitors (fluvoxamine, fluoxetine and zimelidine) were very potent inhibitors of [3H]5-HT uptake in both preparations. In contrast, the inhibitory effects of NE uptake inhibitors (nisoxetine and desipramine) and cocaine were weaker than those of 5-HT uptake inhibitors. In addition, dopamine (DA) uptake inhibitors (nomifensine and GBR-12935) exhibited a Ki value in the low micromolar range. The inhibitory potencies were in the order 5-HT uptake inhibitors (clomipramine, fluvoxamine, fluoxetine, imipramine and zimelidine) > NE uptake inhibitors (nisoxetine and desipramine) = cocaine > DA uptake inhibitors (nomifensine and GBR-12935). There was no difference in the order of the inhibitory effects of various monoamine uptake inhibitors between the two preparations. A correlation analysis of the potencies of various monoamine uptake inhibitors in the inhibition of [3H]5-HT into cultured astrocytes and cortical synaptosomes produced a highly significant correlation coefficient of 0.9893 (P < 0.0001). Immunocytochemical staining using anti-SERT MoAb in cultured astrocytes revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or golgi membranes, showed a considerable level of immunoreactivity. Extracts of astrocytes and synaptosomes from the cortex were immunoblotted with anti-SERT MoAb. SDS-PAGE/Western blots indicate that anti-SERT MoAb recognized two bands of 120 and 73 kDa in both preparations. RT-PCR demonstrated that astrocytes in cultured expressed mRNA for the cloned SERT protein, which has been characterized as the neuronal SERT. These pharmacological experiments indicate that this uptake process takes place through glial SERT that is very similar to neuronal SERT. Furthermore, the present data also indicate that the presence of the mRNA and protein for the neuronal SERT were established in cultured rat astrocytes, and the polypeptide portion of SERT in astrocytes and frontal cortex could be the same gene product.
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PMID:Pharmacological characterization and visualization of the glial serotonin transporter. 1131 48

A novel antifungal protein, designated chrysancorin, was isolated from seeds of Chrysanthemum coronarium var. spatiosum with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue resin, ion exchange chromatography on SP-Sepharose and FPLC-gel filtration on Superdex 75. The N-terminus of chrysancorin displays sequence similarity to the genomic sequence of chromosome 1 from Arabidopsis thaliana BAC T19E23. Chrysancorin exhibits a molecular mass of 13.4 kDa in gel filtration and SDS-polyacrylamide gel electrophoresis. It stimulates the proliferation of mouse splenocytes and inhibits the activity of human immunodeficiency virus-1 reverse transcriptase. The protein possesses antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola and Physalospora piricola, but not against Rhizoctonia solani, Fusarium oxysporum and Coprinus comatus. However, we could not detect antibacterial activity against a variety of bacteria.
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PMID:Purification of chrysancorin, a novel antifungal protein with mitogenic activity from garland chrysanthemum seeds. 1150 60

Previous in vitro data on type I collagen self-assembly into fibrils suggested that the amino acid 776-796 region of the alpha1(I) chain is crucial for fibril formation because it serves as the recognition site for the telopeptide of a docking collagen monomer. We used a natural collagen mutation with a deletion of amino acids 766-801 to confirm the importance of this region for collagen fibril formation. The proband has type III osteogenesis imperfecta and is heterozygous for a COL1A1 IVS 41 A(+4) --> C substitution. The intronic mutation causes splicing of exon 41, confirmed by sequencing of normal and shorter reverse transcriptase-PCR products. Reverse transcriptase-PCR using RNA from proband dermal fibroblasts and clonal cell lines showed the mutant cDNA was about 15% of total alpha1(I) cDNA. The mutant transcript is translated; structurally abnormal alpha chains are demonstrated in the cell layer of proband fibroblasts by SDS-urea-PAGE. The proportion of mutant chains in the secreted procollagen was determined to be 10% by resistance to digestion with MMP-1, since chains lacking exon 41 are missing the vertebral collagenase cleavage site. Secreted proband collagen was used for analysis of kinetics of binding of alpha1(I) C-telopeptide using an optical biosensor. Telopeptide had slower association and faster dissociation from proband than from normal collagen. Purified proband pC-collagen was used to study fibril formation. The presence of the mutant molecules decreases the rate of fibril formation. The fibrils formed in the presence of 10-15% mutant molecules have strikingly increased length compared with normal collagen, but are well organized, as demonstrated by D-periodicity. These results suggest that some collagen molecules containing the mutant chain are incorporated into fibrils and that the absence of the telopeptide binding region from even a small portion of the monomers interferes with fibril growth. Both abnormal fibrils and slower remodeling may contribute to the severe phenotype.in
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PMID:Procollagen with skipping of alpha 1(I) exon 41 has lower binding affinity for alpha 1(I) C-telopeptide, impaired in vitro fibrillogenesis, and altered fibril morphology. 1170 4

Feline peripheral blood mononuclear cells (PBMC)-derived chemotactic factor induced by egg white derivatives (EWD) treatment was analyzed at the protein and messenger ribonucleic acid (mRNA) level. EWD itself was not active chemotactic for feline peripheral blood polymorphonuclear cells (PMN). But chemotaxis of PMN was enhanced by either culture supernatant from PBMC treated with EWD or human recombinant (hr) interleukin (IL)-8. Both hr IL-8 and the culture supernatant from PBMC treated with EWD yielded a distinct band, molecular weight of 6-8kDa, in sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 15% loading gel. Therefore, to identify this chemotactic factor, culture supernatant from PBMC treated with EWD was partially purified by anion exchange chromatography on diethylaminoethyl (DEAE)-Sepharose CL-6B and concentrated by ultrafiltration. Only the fraction, which was eluted with 0.3M NaCl, showed a high concentration of total protein and also enhanced the chemotactic activity of PMN. This activity was thereafter designated as eluate. The chemotactic activity of eluate was inhibited by anti-hr IL-8 polyclonal antibody (pAb). A single protein band with 6-8kDa was shown in both the eluate and hr IL-8 when analyzed by SDS-PAGE and Western blotting using anti-hr IL-8 pAb, suggesting that the chemotactic factor for feline PMN is IL-8, 6-8kDa, produced by PBMC treated with EWD. The physicochemical characteristics of eluate were stable in heated (60-100 degrees C), acid (pH 3.0), and alkaline (pH 9.0) conditions. The eluate under these conditions also showed a distinct band in molecular weight of 6-8kDa in SDS-PAGE and Western blotting and was very active in chemotactic activity of PMN.IL-8 mRNA gene expression on feline PBMC was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) assay using a series of oligonucleotides, each 22 mer, derived from feline IL-8. Feline IL-8 mRNA showed low level in 3-h incubation without EWD, but it was increased in a dose-dependent manner by addition of EWD. Following EWD (10 microg/ml) treatment, IL-8 mRNA expression was rapidly increased up to 6h and decreased by 12h although it was not expressed in freshly prepared PBMC. This study strongly suggested that immunoenhancing effect of EWD on chemotactic response of PMN is mediated by feline IL-8, 6-8kDa, produced by PBMC stimulated with EWD. In addition, the expression of feline IL-8 mRNA on PBMC is increased when stimulated with EWD.
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PMID:Feline interleukin-8 expression in peripheral blood mononuclear cells induced by egg white derivatives. 1194 29

Abacavir (ZIAGEN) is a reverse transcriptase inhibitor marketed for the treatment of HIV-1 infection. A small percentage of patients experience a hypersensitivity reaction indicating immune system involvement and bioactivation. A major route of metabolism for abacavir is oxidation of a primary betagamma unsaturated alcohol to a carboxylic acid via an aldehyde intermediate. This process was shown to be mediated in vitro by human cytosol and NAD, and subsequently the alphaalpha and gamma2gamma2 human isoforms of alcohol dehydrogenase (ADH). The alphaalpha isoform effected two sequential oxidation steps to form the acid metabolite and two isomers, qualitatively reflective of in vitro cytosolic profiles. The gamma2gamma2 isozyme generated primarily an isomer of abacavir, which was minor in the alphaalpha profiles. The aldehyde intermediate could be trapped in incubations with both isozymes as an oxime derivative. These metabolites can be rationalized as arising via the aldehyde which undergoes isomerization and further oxidation by the alphaalpha enzyme or reduction by the gamma2gamma2 isozyme. Non-extractable abacavir protein residues were generated in cytosol, and with alphaalpha and gamma2gamma2 incubations in the presence of human serum albumin (HSA). Metabolism and residue formation were blocked by the ADH inhibitor 4-methyl pyrazole (4-MP). The residues generated by the alphaalpha and gamma2gamma2 incubations were analyzed by SDS-PAGE with immunochemical detection. The binding of rabbit anti-abacavir antibody to abacavir-HSA was shown to be dependent on metabolism (i.e. NAD-dependent and 4-MP sensitive). The mechanism of covalent binding remains to be established, but significantly less abacavir-protein residue was detected with an analog of abacavir in which the double bond was removed, suggestive of a double bond migration and 1,4 addition process.
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PMID:The metabolic activation of abacavir by human liver cytosol and expressed human alcohol dehydrogenase isozymes. 1239 60

Of the HIV proteins, reverse transcriptase(RT) has been probably the most useful target protein for screening and designing of its specific inhibitors. Because retroviral replication is absolutely dependent on both the RNase H and the polymerase function of RT and, so far as is now known, RT does not play a direct role in the life cycle of a normal cell. Under suitable fermentation conditions in our experiments, HIV-1 RT was highly expressed in E. coli JM109(pKRT-2)* by inducing the trc promoter with isopropyl-beta-Dthiogalactopyranoside(IPTG). 1. 1 mg of purified RT was obtained from one liter culture of bacteria by DEAE-cellulose and phosphaellulose chromatography. SDS-PAGE analysis of the purified RT showed two major protein bands of 66 kD and 51 kD, indicating that the purified RT was a heterodimer composed of two subunits. Results of enzyme assay showed that the purified RT had high activity(1.4 x 10(4) umit/mg). We also improved the reaction system of enzyme assay. The effect of PFA on HIV-1 RT was determined with the improved enzyme assay and the mechanism of inhibition was non-competitive with respect to substrate consistent with the reports of Dr. Bo Oberg. This suggests that the purified HIV-1 RT by this simple method can be applied to the anti HIV-1-drug screening. (*E. coli JM109(pKRT2) was obtained from NIAID, NIH; pKRT2 from Dr. Richard D'Aquila and Dr. William C. Summers.)
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PMID:[The study on purification and characterization of HIV-1 reverse transcriptase from a recombinant strain of E. coli]. 1251 92

Activation of protein C by thrombin bound to thrombomodulin is enhanced by endothelial protein C receptor. This pathway may inhibit inflammation. We investigated effects of protein C and activated protein C on neutrophils as well as whether an endothelial protein C receptor is involved in mediating protein C effects. Neutrophils were from venous blood of healthy donors. Cell migration, respiratory burst, phagocytic activity, and apoptosis were studied by micropore filter assays and fluorometry. Receptor expression was investigated by reverse transcriptase-polymerase chain reaction (PCR) for mRNA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of immunoprecipitated receptor protein, and fluorescence-activated cell-sorter scanner (FACS) analysis using the anti-endothelial protein C receptor antibody RCR-252. Neither protein C nor activated protein C induced migration, yet both of them inhibited neutrophil chemotaxis triggered by interleukin-8, formyl-Met-Leu-Phe, antithrombin, or C5a. A protein C activation-blocking antibody against endothelial protein C receptor diminished inhibitory effects of protein C or activated protein C on migration. No effect of either protein C preparation was seen in neutrophil's respiratory burst, bacterial phagocytosis, or apoptosis assays. Endothelial protein C receptor immunoreactivity was confirmed on neutrophils by FACS. De novo synthesis is suggested by endothelial protein C receptor mRNA expression as demonstrated by reverse transcriptase PCR and immunoprecipitation SDS-PAGE analyses. Data suggest that an endothelial protein C receptor is expressed by human neutrophils whose active site ligation with either protein C or activated protein C arrests directed cell migration. Inhibitory effects of these components of the protein C pathway on neutrophil function may play a role in the protein C-based treatment of severe sepsis.
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PMID:Expression and function of the endothelial protein C receptor in human neutrophils. 1271 92

A protein with an N-terminal sequence displaying similarities to N-terminal sequences of human calcyon and barley endo-1,4-glucanase, and to C-terminal sequences of human translation initiation factor 4 gamma and yeast superkiller viralicidic activity, was isolated from the broad bean Vicia faba. The protein, termed fabin, has a molecular mass of 34 kDa in SDS-polyacrylamide gel electrophoresis. Antifungal activity of the protein was observed against several fungal species including Rhizoctonia solani, Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. Fabin inhibits HIV-1 reverse transcriptase with an IC50 of 34 microM and translation in a rabbit reticulocyte lysate with an IC50 of 2.4 microM. At a concentration of about 1.5 microM fabin is able to elicit a 9-fold increase in the mitogenic response of murine splenocytes.
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PMID:Fabin, a novel calcyon-like and glucanase-like protein with mitogenic, antifungal and translation-inhibitory activities from broad beans. 1281 78

The allograft inflammatory factor-1 (AIF-1) is a 17-kDa IFN-gamma inducible Ca(2+)-binding EF-hand protein that is encoded within the HLA class III genomic region and is involved in immune dysfunction and smooth muscle cell activation. We used immunohistochemistry double labelling experiments to analyse the spatial distribution and cell-type-specific localization of AIF-1 in the brains of patients who died as a result of sporadic Creutzfeldt-Jakob disease (CJD) and neuropathologically unaltered controls. Significantly more AIF-1 immunoreactive macrophages/microglial cells and, interestingly, neurones were observed in CJD patients compared to controls. Western blotting confirmed more prominent AIF-1 immunoreactive bands of approximately 50 kDa in four CJD patients compared to three controls. Chaotropic SDS-PAGE of the recombinant AIF-1 resulted in almost complete reduction of the 50 kDa band and mass spectrometry revealed only AIF-1-specific tryptic protein fragments suggesting that trimerized AIF-1 is the predominant form in vivo. Finally, we analysed mechanisms of neuronal AIF-1 induction. Following H2O2 challenge, a model of general cell stress, we observed the gradual induction of AIF-1 and, more interestingly, release to the supernatant of SKNSH neurones. Parallel reverse transcriptase polymerase chain reaction and sequencing was used to confirm AIF-1 mRNA expression.
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PMID:The allograft inflammatory factor-1 in Creutzfeldt-Jakob disease brains. 1288 99


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