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)

Expression of the Cyp2b-9 and Cyp2b-10 genes was investigated in primary cultured adult C57BL/6NCrj mouse hepatocytes in monolayers or during the formation of spheroids (multicellular aggregates). Both the constitutive and phenobarbital-inducible expression of Cyp2b-9 and Cyp2b-10 mRNA decreased rapidly after transferring the hepatocytes to monolayer culture. The decrease was dependent on cell-density, and became more rapid in more densely seeded cells. However, in spheroid culture, the Cyp2b-9 and Cyp2b-10 mRNAs were induced by phenobarbital at high levels for at least 4 days either with continuous exposure from the start of cultivation or for 24 h before harvesting. The expression of both Cyp2b-9 and Cyp2b-10 species was confirmed by either reverse transcriptase-polymerase chain reaction or Western blotting. Although more Cyp2b-9 than Cyp2b-10 was expressed in the liver of control mice, the amounts of the latter became relatively overwhelming in untreated hepatocytes because of a faster decline of Cyp2b-9 species in culture. The level of mRNA induced by phenobarbital was concentration-dependent, the highest being at 2 or 4 mM, which was equivalent to in vivo treatment levels. There was more Cyp2b-10 species than those of Cyp2b-9 after exposure to phenobarbital both in vivo and in vitro. Phenobarbital also induced CYP1A2 mRNA, which was again peculiar to spheroid culture. Although the expression levels of both Cyp2b-9 and Cyp2b-10 species was very low in hepatocytes cultured without dexamethasone even in the presence of phenobarbital, the addition of 10(-7) or 10(-6) M dexamethasone caused an increase in the mRNA. When given concomitantly with phenobarbital, the expression was greatly enhanced. Nicotinamide or isonicotinamide similarly enhanced the expression of the two mRNA species, but the levels in monolayer cultures were still far lower than those in vivo. In contrast to the findings for mRNA expression, the protein levels in the presence of nicotinamide were similar to those in vivo under both monolayer and spheroid conditions. Since previous efforts to maintain expression of the CYP2B1 and CYP2B2 genes in primary cultured rat hepatocytes, orthologous to mouse Cyp2b-9 and Cyp2b-10 genes, required special cell attachment factors, our spheroid culture system, in which mouse hepatocytes are simply seeded onto noncoated dishes, has advantages for mechanistic studies.
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PMID:Maintenance of phenobarbital-inducible Cyp2b gene expression in C57BL/6 mouse hepatocytes in primary culture as spheroids. 784 Jun 37

Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in the glycolytic pathway. Since its transcript levels do not vary in most experimental conditions, it has been often used as a control in northern blot or reverse transcriptase-polymerase chain reaction analysis. We have cloned and sequenced a gene encoding glyceraldehyde-3-phosphate dehydrogenase (Tthgapdh) from Tetrahymena thermophila cDNA library and determined whether the Tthgapdh mRNA is a loading control in gene expression studies of T. thermophila cell. The open reading frame encoded a protein of 341 amino acid residues (36.8 kDa) containing a nicotinamide adenine dinucleotide-binding domain and a catalytic domain, which was highly similar to those of other organisms. Its mRNA levels at different growth stages were examined by northern blot analysis. The fragment of the isolated cDNA was hybridized to a 1.3-kb mRNA transcript. There was a marked increase in Tthgapdh mRNA level at the mid-exponential phase, followed by a gradual decrease. Therefore, much caution should be made to use Tthgapdh mRNA as an internal standard for northern blot analysis in Tetrahymena.
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PMID:Cloning and sequencing of a cDNA encoding glyceraldehyde-3-phosphate dehydrogenase from Tetrahymena thermophila: growth-associated changes in its mRNA expression. 930 12

N-(trifluoromethylphenyl)-2-cyano-3-hydroxy-crotonic acid amide (A77 1726), the physiologically active metabolite of leflunomide, has been described to exert antiproliferative effects in vitro and anti-inflammatory actions in several animal models. Currently, its use is being evaluated in clinical trials in psoriasis, which is characterized by epidermal hyperproliferation and infiltration of inflammatory cells. We studied the effects of A77 1726 on growth and gene expression in cultured epidermal cells by 5-bromo-2'-deoxy-uridine (BrdU) incorporation, reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot hybridizations and flow cytometry. A77 1726 inhibited epidermal proliferation at concentrations above 5 microM after 24 hr. However, the cells were still fully viable at a concentration of 100 microM. The drug caused a dose-dependent reduction in the mRNA level of the type A receptor for the proinflammatory cytokine interleukin-8 (IL-8-RA) and, in contrast, induced gene expression of the receptor for the anti-inflammatory cytokine IL-10 (IL-10R) at the mRNA and protein levels. In addition, the mRNA and protein levels of the p53 gene, which is a negative cell cycle regulator, were up-regulated by A77 1726. These data suggest that A77 1726 exerts its anti-inflammatory action via the modulation of epidermal gene expression.
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PMID:Differential modulation of pro- and anti-inflammatory cytokine receptors by N-(4-trifluoromethylphenyl)-2-cyano-3-hydroxy-crotonic acid amide (A77 1726), the physiologically active metabolite of the novel immunomodulator leflunomide. 1007 46

To elucidate the mutation in the nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) gene from a Chinese patient with hereditary methemoglobinemia type I, we analyzed the coding sequences of b5R cDNA from the patient and from normal subjects by direct sequencing the reverse transcriptase-polymerase chain reaction (RT-PCR) products. The PCR-amplified genomic DNA fragments of the b5R gene from the patient, his mother, and normal controls were analyzed by restriction enzymes MspI and RsaI. A compound heterozygote Arg57Gln (CGG-->CAG)/Cys203Tyr (TGC-->TAC) was found in the b5R gene from the patient, and a CGG-->CAG mutant allele occurred in a chromosome inherited from his mother, while TGC-->TAC occurred in a chromosome inherited from his father. In this report, we discuss a compound heterozygote first observed in the b5R gene from a patient with hereditary methemoglobinemia type I.
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PMID:A compound heterozygote in the NADH-cytochrome b5 reductase gene from a Chinese patient with hereditary methemoglobinemia type I. 1097 6

We studied the expression of Substance P (SP) and its receptor in an established human stem cell line (TF-1) and primary stem cells derived from human placental cord blood (HPCB). Using reverse transcriptase polymerase chain reaction (RT-PCR) assay, SP mRNA is detected in both TF-1 cells and HPCB stem cells. Among the alpha, beta, gamma, and delta transcripts of the SP gene, only the beta, gamma, and delta transcripts are detectable in these cells. These RT-PCR-amplified transcripts are confirmed by Southern blot assay using a specific SP probe. Sequence analysis of the RT-PCR-amplified products transcribed from mRNA extracted from the HPCB stem cells also confirmed that these transcripts are identical to those found in human neurons. At the protein level, TF-1 cells produced endogenous SP as determined by an enzyme-linked immunosorbent assay (EIA). Capsaicin, a vanillyl fatty acid amide (ingredient of hot pepper), released SP from TF-1 cells. In addition, using RT nested-PCR analysis, we identified the presence of mRNA for neurokinin-1 receptor (NK-1R, the receptor for SP) in both TF-1 cells and HPCB stem cells, which was confirmed by Southern blot and DNA sequencing analysis. The demonstration that human stem cells express SP and its receptor support the notion that SP is biologically involved in the hematopoietic regulating network.
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PMID:Human stem cells express substance P gene and its receptor. 1098 42

Evidence is rapidly accumulating that low-activity-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases homologous to that in phagocytic cells generate reactive oxygen species as signaling intermediates in both endothelium and vascular smooth muscle. We therefore explored the possibility of such an oxidase regulating growth of airway smooth muscle (AWSM). Proliferation of human AWSM cells in culture was inhibited by the antioxidants catalase and N-acetylcysteine, and by the flavoprotein inhibitor diphenylene iodonium (DPI). Membranes prepared from human AWSM cells generated superoxide anion (O) measured by superoxide dismutase-inhibitable lucigenin chemiluminescence, with a distinct preference for NADPH instead of reduced nicotinamide adenine dinucleotide as substrate. Chemiluminescence was also inhibited by DPI, suggesting the presence of a flavoprotein containing oxidase generating O as a signaling molecule for cell growth. Examination of human AWSM cells by reverse transcriptase-polymerase chain reaction consistently demonstrated transcripts with sequences identical to those reported for p22(phox). Transfection with p22(phox) antisense oligonucleotides reduced human AWSM proliferation. Inhibition of NADPH oxidase activity with DPI prevented serum-induced activation of nuclear factor-kappaB (NF-kappaB), and overexpression of a superrepressor form of the NF-kappaB inhibitor IkappaBalpha significantly reduced human AWSM growth. These findings suggest that an NADPH oxidase containing p22(phox) regulates growth-factor responsive human AWSM proliferation, and that the oxidase signals in part through activation of the prototypical redox-regulated transcription factor NF-kappaB.
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PMID:NADPH oxidase promotes NF-kappaB activation and proliferation in human airway smooth muscle. 1188 Mar 5

We previously demonstrated that cyclic ADP-ribose (cADPR) elicits Ca2+ release in airway smooth muscle (ASM) cells through ryanodine receptor channels. CD38 is a cell surface protein that catalyzes the synthesis and degradation of cADPR. In inflammatory diseases such as asthma, augmented Ca2+ responses and Ca2+ sensitivity contribute to increased ASM contractility in response to agonists. In this study, we investigated the regulation of CD38 expression and the role of cADPR-mediated Ca2+ release in airway inflammation. Human ASM cells in culture between the second and fifth passages were exposed to tumor necrosis factor alpha (TNF-alpha), interleukin 1beta, or interferon gamma, or bovine serum albumin (controls). CD38 expression was measured by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, and Western blot analysis, and ADP-ribosyl cyclase activity was assayed with nicotinamide guanine dinucleotide as the substrate. Ca2+ responses to acetylcholine, bradykinin, and thrombin were measured in fura-2AM-loaded cells by fluorescence microscopy. Cytokines caused significant augmentation of CD38 expression, ADP-ribosyl cyclase activity, and Ca2+ responses to the agonists, compared with the control. TNF-alpha effects were greater than those of the other two cytokines. The cADPR antagonist 8-bromo-cADPR attenuated the Ca2+ responses to the agonists in control and cytokine-treated cells, with the magnitude of inhibition correlating with the level of CD38. This study provides the first demonstration of a role for CD38-cADPR signaling in a model of inflammatory airway disease.
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PMID:CD38/cyclic ADP-ribose-mediated Ca2+ signaling contributes to airway smooth muscle hyper-responsiveness. 1251 17

We studied the effect of ageing on the mRNA levels of mitochondria-encoded polypeptides in human platelets. We used quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate the expression of selected cytochrome c oxidase (COX) genes (subunits I and III) and Complex I genes (subunits reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ND)1 and (ND)5 in platelets from young and aged healthy subjects. Northern blot analysis confirmed the PCR results. COX I expression is higher than that of COX III in both young and aged platelets. A significant increase of transcripts for Complex I was found during ageing. On the contrary, the mRNA levels of the two COX subunits did not significantly vary during ageing.
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PMID:Increased transcription of mitochondrial genes for Complex I in human platelets during ageing. 1475 9

In avian species, the ultimobranchial anlage is populated with neuronal cells derived from the distal vagal ganglion. We found that ultimobranchial C cells of chick embryos cultured in the presence of nicotinamide continued to grow for at least 60 days and exhibited profound morphological changes, resulting in the formation of dense networks of neuronal fibers. Nicotinamide, thus, facilitated the manifestation of neuronal features in C cells. The neuronal phenotypes of cultured C cells were analyzed in detail by both scanning and transmission electron microscopy. Their neural nature was also positively established by immunostaining with monoclonal antibodies to the neuronal markers neuron-specific class III beta-tubulin (TuJ1), microtubule-associated protein (MAP) 2, and synaptophysin. Confocal laser scanning microscopy confirmed that these neuron-specific proteins are colocalized with calcitonin in both the somata and the neuronal processes of C cells. Furthermore, reverse transcriptase-polymerase chain reaction analyses, performed at various times up to 30 days in culture, indicated that the C cells have persistent gene expression of calcitonin, the catecholamine-synthesizing enzyme tyrosine hydroxylase, proenkephalin, proopiomelanocortin, neuron-specific beta-tubulin (cbeta4), SCG10, and Bcl-2. The morphological responses of C cells to nicotinamide treatment were analyzed quantitatively over a period of 60 days. The area of C-cell colonies, number of processes per colony, and length of processes continued to increase until culture day 45. In conclusion, nicotinamide stimulates long-term survival and neuronal differentiation of chick embryo C cells, and this culture system may provide a useful model for studying neuronal differentiation mechanisms.
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PMID:Nicotinamide promotes long-term survival and extensive neurite outgrowth in ultimobranchial C cells cultured from chick embryos. 1621 94

SPD754 (AVX754) is a deoxycytidine analogue nucleotide reverse transcriptase inhibitor (NRTI) in clinical development. These studies characterized the in vitro activity of SPD754 against NRTI-resistant human immunodeficiency virus type 1 (HIV-1) and non-clade B HIV-1 isolates, its activity in combination with other antiretrovirals, and its potential myelotoxicity and mitochondrial toxicity. SPD754 was tested against 50 clinical HIV-1 isolates (5 wild-type isolates and 45 NRTI-resistant isolates) in MT-4 cells using the Antivirogram assay. SPD754 susceptibility was reduced 1.2- to 2.2-fold against isolates resistant to zidovudine (M41L, T215Y/F, plus a median of three additional nucleoside analogue mutations [NAMs]) and/or lamivudine (M184V) and was reduced 1.3- to 2.8-fold against isolates resistant to abacavir (L74V, Y115F, and M184V plus one other NAM) or stavudine (V75T/M, M41L, T215F/Y, and four other NAMs). Insertions at amino acid position 69 and Q151M mutations (with or without M184V) reduced SPD754 susceptibility 5.2-fold and 14- to 16-fold, respectively (these changes gave values comparable to or less than the corresponding values for zidovudine, lamivudine, abacavir, and didanosine). SPD754 showed similar activity against isolates of group M HIV-1 clades, including A/G, B, C, D, A(E), D/F, F, and H. SPD754 showed additive effects in combination with other NRTIs, tenofovir, nevirapine, or saquinavir. SPD754 had no significant effects on cell viability or mitochondrial DNA in HepG2 or MT-4 cells during 28-day exposure at concentrations up to 200 microM. SPD754 showed a low potential for myelotoxicity against human bone marrow. In vitro, SPD754 retained activity against most NRTI-resistant HIV-1 clinical isolates and showed a low propensity to cause myelotoxicity and mitochondrial toxicity.
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PMID:In vitro antiretroviral activity and in vitro toxicity profile of SPD754, a new deoxycytidine nucleoside reverse transcriptase inhibitor for treatment of human immunodeficiency virus infection. 1643 19


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