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)

Reaction conditions for Rous sarcoma virus ribonucleic acid (RNA)-instructed deoxyribonucleic acid (DNA) polymerase activity are described whereby the viral RNA is relatively protected from endogenous or added nuclease activity. Three analyses of reaction product nucleic acids ((3)H-RNA, (32)P-DNA) were compared, namely, gel electrophoresis, Cs(2)SO(4) gradient centrifugation, and hydroxyapatite column chromatography. It was found that hydroxyapatite analysis could be misleading unless the state of the template RNA was monitored concomitantly with the DNA analysis. Gel electrophoresis and Cs(2)SO(4) gradient centrifugation gave comparable results. It was concluded that analyses of the product of reverse transcriptase reactions should not only refer to the template RNA and product DNA species, but also be performed with virus or viral RNA which do not have or obtain nicks in the 60S RNA. Otherwise, interpretation of the results would have the ambiguity of potential artifacts caused by those degraded RNA molecules.
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PMID:Deoxyribonucleic acid polymerase of Rous sarcoma virus: reaction conditions and analysis of the reaction product nucleic acids. 433 43

Drastic inhibition of the human immunodeficiency virus (HIV) reverse transcriptase (RT) by mycoplasma has been noted in many laboratories causing confusion in data interpretation. The mycoplasma-related inhibitor of HIV-1 RT was identified as a soluble protein in the particle-free supernatant of a contaminated culture. Gel filtration studies revealed the molecular mass of this protein to be about 70 kDa. This RT-inhibitor contained a DNase with strong activity on both linear and circular DNAs. Addition of this inhibitor after completion of reverse transcription still reduced the final outcome of the RT assay significantly, implying that the inhibitory mechanism occurred mainly by its DNase activity. Treatment of the culture with an antimycoplasma drug cured the mycoplasma contamination, removed the RT-inhibitor and abolished the DNase activity.
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PMID:The mycoplasma-related inhibitor of HIV-1 reverse transcriptase has a DNase activity and is present in the particle-free supernatants of contaminated cultures. 751 27

Cholic acid (sodium cholate) exhibits a strong spermicidal and antiviral [anti-human immunodeficiency virus (HIV)-1] activity. The same effects are observed for F-5 Gel, the active mixture of a new contraceptive sponge (Protectaid), which contains sodium cholate together with low concentrations (0.5%) of nonoxynol-9 and benzalkonium chloride. Both cholic acid and the F-5 Gel exerted a dose-dependent, in-vitro inhibitory effect 1) on the activity of HIV-1 associated reverse transcriptase in an acellular system (their 50% inhibitory dose was 7.2 mM and 0.8 x 10 -3 v/v, respectively, and 2) on the potential of HIV-1 to infect human lymphocytes efficiently. In the 3 semen samples examined, sperm motility was instantaneously inhibited by the addition of a 6 mM solution of sodium cholate or of a 1:10 dilution of F-5 Gel. Both cholic acid and F-5 Gel affected in a dose-dependent manner the viability of normal peripheral blood lymphocytes (NPBL) and CEM cells. The Protectaid contraceptive sponge impregnated with F-5 Gel was given to 20 young women aged 19-25 years for a period of 1 year who had chosen this method for both contraception and against sexually transmitted diseases. All women were instructed to insert the sponge within the 12 hours preceding each sexual intercourse and to remove it 4-6 hours afterwards. During 12 months of use with at least 3 intercourse per week, the contraceptive efficacy of the Protectaid vaginal sponge was 100%. Cervical cultures at 6-month intervals showed the presence of Mycoplasma hominis and Candida albicans in 1 and 2 cases, respectively. The combined spermicidal and anti-HIV properties of cholic acid reported and used in the Protectaid sponge offer a new and modern protective method of contraception. At the end of the study, cervical cultures revealed the presence of Escherichia coli and Candida albicans in 1 case each. No slide effects were recorded, and only 1 woman complained of discomfort.
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PMID:Spermicidal and antiviral properties of cholic acid: contraceptive efficacy of a new vaginal sponge (Protectaid) containing sodium cholate. 768 80

The interaction between lymphoma cells and vascular endothelial cells (EC) is the first critical step in the invasion of lymphoma cells. We found that invasive human CCRF-CEM T lymphoma cells (CEM) released a factor that upregulates the expression of adhesion molecules on vascular EC. The supernatant of CEM (CEM-SUP) increased the expression of both ICAM-1 and ELAM-1 in time- and dose-dependent manners as shown by cell enzyme-linked immunoabsorbent assay (ELISA). In contrast, the induction of VCAM-1 on EC with CEM-SUP was relatively weak. No activity for interleukin-1 alpha (IL-1 alpha), IL-1 beta, interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha), which are known to augment ICAM-1 expression, was detected in CEM-SUP by ELISA. In reverse transcriptase polymerase chain reaction (RT-PCR) assay, CEM expressed a minimum amount of TNF-alpha mRNA, but absolutely no IL-1 beta or IFN-gamma mRNA. In addition, antibodies for cytokines did not inhibit the upregulatory effect of CEM-SUP. Semipurified CEM-SUP further increased the cellular binding between CEM cells and EC in vitro. This factor was stable to heat (65 degrees C, 30 minutes) and labile to acid (pH 2.0). Gel filtration and chromatofocusing estimated its molecular weight at 50 kd, with an isoelectric point of pH 7.2. Production of this factor might contribute to the invasive character of CEM through upregulation of adhesion molecules on EC.
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PMID:Invasive human T lymphoma cells produce a novel factor that upregulates expression of adhesion molecules on endothelial cells. 769 38

Proteoglycans are mediators of cellular adhesion and regulate growth factor activities. Proteoglycans of B lymphocytes undergo structural changes during B cell ontogeny which may correspond to the specific requirements of the respective microenvironment of the maturing cell. We analyzed three human B cell lines representing pre-B cells (Nalm-6), activated B cells (Jok-1) and plasma cells (U266) for their cellular proteoglycans. Gel filtration of the 35S-labeled macromolecules of the three cell lines revealed an increase in size in the order Nalm-6 < Jok-1 < U266. In Jok-1 and U266 cells the major pool of proteoglycans consisted of proteochondroitin sulfates of 50 to 90 kDa. These proteolglycans carried a protein core of approx. 30 kDa to which 1 to 3 glycosaminoglycan chains in the range of 28 to 32 kDa were attached. In Nalm-6 cells only free chondroitin sulfate chains of 23 kDa, but no intact proteoglycans, were detected. Chondroitin sulfate chains were predominantly composed of chondroitin-4-sulfate, those of Nalm-6 and U266 cells additionally contained 10-20% of unsulfated disaccharides. In U266 cells 30% of glycosaminoglycans consisted of heparan sulfate either bound to pure proteoheparan sulfate or to chondroitin sulfate/heparan sulfate hybrid-proteoglycans. Earlier, syndecan-1 was described as a hybrid proteoglycan containing heparan sulfate/chondroitin sulfate chains which is transcribed by murine B cells at early and late maturation stages. In order to see whether syndecan is transcribed by the human B cell lines used here, we measured expression of syndecan mRNA by the reverse transcriptase polymerase chain reaction. Similar to murine lymphocytes, syndecan-specific mRNA was detected in Nalm-6 and U266 cells, equivalent to early and late B cells, but not in lymphoblastoid Jok-1 cells. However, Nalm-6 cells do not produce proteoheparan sulfate. In these cells, syndecan synthesis may be blocked at the translational level. Also, the proteoglycans of U266 are different from syndecan-1 in their composition of glycosaminoglycans and in size of protein cores. Together, these results indicate that the major pool of proteoglycans produced by human B cells consists of proteochondroitin sulfate and additionally in later stages of a smaller proportion of proteoheparan sulfate which is not identical to syndecan-1. During distinct phases of B cell differentiation, modulations in the glycosaminoglycan moiety concerning size and sulfation of glycosaminoglycan chains were also found.
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PMID:Modulated glycosylation of proteoglycans during differentiation of human B lymphocytes. 777 69

To examine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thymic gene expression in vitro, freshly isolated rat thymocytes were incubated with 10 nM TCDD, and reverse transcriptase polymerase chain reaction experiments were performed using primers specific for prostaglandin G/H synthase (PGHS) and glyceraldehyde 3-phosphate dehydrogenase. TCDD selectively repressed PGHS gene expression, with maximal inhibition occurring within 60 min. Gel retardation assays demonstrated that dioxin transiently induced binding of the ubiquitous transcription factor NF kappa B to its cognate response element at early time points. However, TCDD had little ability to induce transformation of the Ah receptor to the xenobiotic responsive element in thymic cytosol. These results indicate that TCDD exerts changes in thymocyte gene expression prior to inducing toxicity.
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PMID:2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated gene expression in the immature rat thymus. 782 58

HES-5 is a mammalian basic helix-loop-helix factor that has a distant sequence homology to the product of the Drosophila pair-rule gene hairy. HES-5 mRNA is present exclusively in the developing nervous system, but its level decreases as neural differentiation proceeds. In this study, to characterize the molecular mechanism of the neural-specific expression of HES-5 we isolated the mouse HES-5 gene. This gene consists of three exons, and Southern blot analysis shows that it is a single copy gene. The transcription initiation site, determined by primer extension and reverse transcriptase-mediated polymerase chain reaction, is located 26 nucleotides downstream of a TATAbox. Transient transfection analysis shows that the upstream region of the HES-5 gene can direct efficient expression in neural precursor cells and moderate expression in undifferentiated NCB20 neuroblastoma-brain hybrid cells but not in glioma or fibroblast cells. The moderate level of expression in NCB20 cells decreases when differentiation into neuron-like cells is induced. Further promoter analysis shows that this undifferentiated neural-specific expression is mediated by the multiple GC stretches present in the HES-5 promoter. Gel mobility shift analysis suggests the presence of a neural precursor cell-specific protein that binds to the GC stretches. These results raise the possibility that HES-5 expression in the developing nervous system is regulated by the GC stretch-binding protein.
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PMID:Structure and promoter analysis of the gene encoding the mouse helix-loop-helix factor HES-5. Identification of the neural precursor cell-specific promoter element. 783 1

Human immunodeficiency virus type 1 (HIV-1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2',3'-dideoxy-3'-thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventual outgrowth of viruses encoding valine (Val). Similar replication kinetics were measured for wild-type and 3TC-resistant HIV-1 viruses in tissue culture infections of a T cell line, but we measured reduced polymerase activity for the two mutant RT enzymes compared with the wild-type enzyme (Ile = 43% and Val = 67%). Gel analysis of the reverse transcription products revealed that both 3TC-resistant RT mutants produce significantly shorter cDNA molecules than the wild-type enzyme [Met (wt)>Val>Ile], indicating that 3TC-resistant RT polymerases are less processive enzymes. Interestingly, these enzyme defects were more pronounced under limiting dNTP concentrations and we therefore assayed virus replication in primary cells that contain relatively low dNTP levels. Under these conditions, we measured significantly reduced replication kinetics for the 3TC-resistant HIV-1 variants [Met (wt)>Val>Ile]. If the level of virus replication can be similarly reduced in 3TC-treated patients that develop drug-resistant HIV-1 variants, this may be of considerable clinical benefit.
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PMID:Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme. 867 Sep 8

Granulomas form in the liver and intestines of mice infected with the parasite Schistosoma mansoni. Vasoactive intestinal peptide (VIP) is a neurokine that can modulate aspects of the immune response by acting through receptors within the granuloma. Cloned are two novel VIP receptor (VIPR) mRNAs (VIPR1 and VIPR2) that also bind a second neurokine called pituitary adenylated cyclase-activating polypeptide (PACAP). The objective of this study was to determine if granulomas express either VIPR1 or VIPR2. Using a radioligand-binding assay, it was established that PACAP is as effective as VIP at displacing radiolabeled VIP from splenocytes and granuloma cells, and that most if not all VIPRs in the spleen and granulomas bind PACAP. PCR amplification of reverse transcribed RNA determined that granulomas express both VIPR1 and VIPR2 mRNAs. Gel electrophoresis and nucleotide sequencing confirmed the authenticity of the PCR products. Also, both receptor subtypes were amplified from several granuloma CD4+ T cell lines; yet reverse transcribed RNA from T cell-depleted, dispersed granuloma cells had only VIPR1 RNA. It is notable that reverse transcriptase-PCR detected only VIPR1 in the thymus and spleen, which are organs rich in T lymphocytes. Thus, the granulomas and spleens from mice with schistosomiasis contain cells that display authentic VIP/PACAP receptors. Moreover, these data suggest that T cells in different compartments vary in VIPR subtype expression. VIPR1 and VIPR2 may have different physiologic roles in inflammation.
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PMID:T cell vasoactive intestinal peptide receptor subtype expression differs between granulomas and spleen of schistosome-infected mice. 868 24

Using an antibody that recognizes the products of all known members of the fos family of immediate early genes, it was demonstrated that destruction of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle produces a prolonged (>3 months) elevation of Fos-like immunoreactivity in the striatum. Using retrograde tract tracing techniques, we have previously shown that this increase in Fos-like immunoreactivity is located predominantly in striatal neurons that project to the globus pallidus. In the present study, Western blots were performed on nuclear extracts from the intact and denervated striatum of 6-OHDA-lesioned rats to determine the nature of Fos-immunoreactive protein(s) responsible for this increase. Approximately 6 weeks after the 6-OHDA lesion, expression of two Fos-related antigens with apparent molecular masses of 43 and 45 kDa was enhanced in the denervated striatum. Chronic haloperidol administration also selectively elevated expression of these Fos-related antigens, suggesting that their induction after dopaminergic denervation is mediated by reduced activation of D2-like dopamine receptors. Western blot immunostaining using an antibody which recognizes the N-terminus of FosB indicated that the 43 and 45 kDa Fos-related antigens induced by dopaminergic denervation and chronic haloperidol administration may be related to a truncated form of FosB known as deltaFosB. Consistent with this proposal, retrograde tracing experiments confirmed that deltaFosB-like immunoreactivity in the deafferented striatum was located predominantly in striatopallidal neurons. Gel shift experiments demonstrated that elevated AP-1 binding activity in denervated striata contained FosB-like protein(s), suggesting that enhanced deltaFosB levels may mediate some of the effects of prolonged dopamine depletion on AP-1-regulated genes in striatopallidal neurons. In contrast, chronic administration of the D1-like receptor agonist CY 208243 to 6-OHDA-lesioned rats dramatically enhanced deltaFosB-like immunoreactivity in striatal neurons projecting to the substantia nigra. Western blot immunostaining revealed that deltaFosB and, to a lesser extent, FosB are elevated by chronic D1-like agonist administration. Both the quantitative reverse transcriptase-polymerase chain reaction and the ribonuclease protection assay demonstrated that deltafosB mRNA levels were substantially enhanced in the denervated striatum by chronic D1-like agonist administration. Lastly, we examined the effects of chronic administration ofD1-like and D2-like dopamine receptor agonists on striatal deltaFosB expression in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) primate model of Parkinson's disease. In monkeys rendered Parkinsonian by MPTP, there was a modest increase in deltaFosB-like protein(s), while the development of dyskinesia produced by chronic D1-like agonist administration was accompanied by large increases in DeltaFosB-like protein(s). In contrast, administration of the long-acting D2-like agonist cabergoline, which alleviated Parkinsonian symptoms without producing dyskinesia reduced deltaFosB levels to near normal. Taken together, these results demonstrate that chronic alterations in dopaminergic neurotransmission produce a persistent elevation of deltaFosB-like protein(s) in both the rodent and primate striatum.
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PMID:Chronic alterations in dopaminergic neurotransmission produce a persistent elevation of deltaFosB-like protein(s) in both the rodent and primate striatum. 871 7


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