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 alpha6 subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) has been implicated in mediating the intoxicating effects of ethanol and the motor ataxic effects of general anesthetics. To test this hypothesis, we used gene targeting in embryonic stem cells to create mice lacking a functional alpha6 gene. Homozygous mice are viable and fertile and have grossly normal cerebellar cytoarchitecture. Northern blot and reverse transcriptase-polymerase chain reaction analyses demonstrated that the targeting event disrupted production of functional alpha6 mRNA. Autoradiography of histological sections of adult brains demonstrated that diazepam-insensitive binding of [3H]Ro15-4513 to the cerebellar granule cell layer of wild-type mice was completely absent in homozygous mice. Cerebellar GABA(A)-R density was unchanged in the mutant mice; however, the apparent affinity for muscimol was markedly reduced. Sleep time response to injection of ethanol after pretreatment with vehicle or Ro15-4513 did not differ between genotypes. Sleep time response to injection of pentobarbital and loss of righting reflex and response to tail clamp stimulus in mice anesthetized with volatile anesthetics also did not differ between genotypes. Thus, the alpha6 subunit of the GABA(A)-R is not required for normal development, viability, and fertility and does not seem to be a critical or unique component of the neuronal pathway mediating the hypnotic effect of ethanol and its antagonism by Ro15-4513 in mice. Similarly, the alpha6 subunit does not seem to be involved in the behavioral responses to general anesthetics or pentobarbital.
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PMID:Gene knockout of the alpha6 subunit of the gamma-aminobutyric acid type A receptor: lack of effect on responses to ethanol, pentobarbital, and general anesthetics. 910 23

IL-1 beta converting enzyme (ICE), a proteolytic enzyme that converts the inactive precursor of interleukin-1 beta (IL-1 beta) to its mature active form, has been abundantly detected in the IL-1 beta producing cells in the spleen. Since IL-1 beta is a potent neuro-endocrine-immuno modulator, alterations in the production of IL-1 beta by an exogenous factor, such as morphine or ethanol, may have deleterious effects on the system as a whole. In this study, we examined the expression of ICE in the spleens of rats given chronic treatment with morphine versus ethanol using reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of ICE in the spleen of rats given chronic morphine was clearly less than that of the animals given placebo, however, it was similar for both rats on ethanol and control diets. These data suggest that chronic use of morphine, but not ethanol, attenuates the expression of ICE in the spleen.
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PMID:Chronic exposure to morphine, but not ethanol, attenuates the expression of interleukin-1 beta converting enzyme in rat spleen. 929 96

A protocol for the in situ polymerase chain reaction (IS-PCR) detection of viral nucleic acid in the heart tissue of four-to-five-week-old CD1 mice infected with coxsackievirus B3 (CBV3) Nancy strain is described. To compare the effects of formalin concentration on the IS-PCR process, two different concentrations (10 and 37%) were employed. Using 37% formalin, 25 PCR cycles were sufficient and a permeabilization step could be omitted. However, postfixation of tissues with 4% paraformaldehyde and 100% ethanol after the deparaffinization, reverse transcriptase and amplification steps was required in order to minimize artefacts. When the tissues were fixed in 10% formalin, postfixation with 4% paraformaldehyde was not required, but a permeabilization step had to be employed and 40 cycles of PCR amplification were needed. To detect the PCR product in the 10% formalin-fixed samples, incubation with 0.3 U/ml of an anti-digoxigenin antibody conjugated to alkaline phosphatase was performed for 90 min. When 37% formalin-fixed samples were used, the concentration of the antibody conjugate had to be increased to 3 U/ml and the exposure time was decreased to 30 min. Enterovirus (EV) nucleic acid was detected in the cytoplasm of myocytes. Thus, IS-PCR was successful in localizing EV nucleic acid in the cytoplasm of myocytes in mice infected with a cardiotropic strain of CBV3. Using this technique, 10% formalin-fixed tissues gave better results than 37% formalin-fixed tissues.
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PMID:Comparison of procedures for the detection of enteroviruses in murine heart samples by in situ polymerase chain reaction. 949 12

Clinical studies suggest that moderate alcohol consumption may decrease the risk for coronary artery disease and myocardial infarction. This effect may be attributed, in part, to the alcohol-mediated increase in endothelial cell (EC)-mediated fibrinolytic activity mediated by the increase in synthesis and/or activity of tissue-type plasminogen activators (t-PAs) and/or urokinase-type PA (u-PAs). To determine whether low alcohol levels (0.01 to 0.1%, v/v) induced the expression of these proteins, cultured human saphenous vein ECs (HSVECs) were preincubated in the absence/presence of ethanol for 5 to 120 min at 37 degrees C, washed, refed, and further incubated for 8 and 24 hr without alcohol. PA mRNA (reverse transcriptase-polymerase chain reaction) and secreted antigen (ELISA) levels were analyzed after incubation for 8 and 24 hr and the net expression of (sustained) endogenous PA-mediated surface-localized HSVEC fibrinolytic activity (plasmin generation) quantitated by activation of 125I-Glu-plasminogen after incubation for 24 hr. A brief 5 to 30 min preincubation (induction) of both t-PA and u-PA antigen increased approximately 3-fold (t-PA control, 14.2 +/- 1.7, plus alcohol, 25.4 +/- 5 ng/ml; u-PA control, 15 +/- 0.8, plus alcohol, 46.4 +/- 1.3 ng/ml) and mRNA levels approximately 2-fold, as compared with controls. Increased PA expression was associated with a significant concomitant approximately 2-fold increase in surface-localized fibrinolytic activity (control, 96 +/- 2.8, plus alcohol, 255 +/- 42 fmol/ well). These combined results indicate that a brief exposure (<30 min) to low levels of alcohol can induce synthesis of EC-produced t-PA and u-PA resulting in an increased expression of HSVEC surface-localized fibrinolytic activity and may account, in part, for the apparent cardioprotective benefit associated with moderate alcohol consumption.
Alcohol Clin Exp Res 1998 Apr
PMID:Alcohol-induced upregulation of plasminogen activators and fibrinolytic activity in cultured human endothelial cells. 958 43

A rapid, efficient and inexpensive method was developed to concentrate poliovirus type 1 (PV1), rotavirus (RV) and hepatitis A virus (HAV) from artificially spiked samples of tap and surface water. The method consists of adsorbing the viruses to silicon dioxide (SiO2) in the presence of 0.5 mM AlCl3 and adjustment of the pH to 3.5. The silica-adsorbed virus was collected by low speed centrifugation. Viral RNA was then extracted with guanidium thiocyanate (GT), and environmental nucleases and inhibitors of reverse transcriptase and Taq polymerase were further eliminated from concentrates by sequential treatment with GT, ethanol and acetone. Subsequent RT-PCR allowed the detection of as few as 1 to 10 TCID50 of PV1, RV, and HAV in seeded 1 liter samples of tap water. The same protocol was then used with effluents from two local sewage treatment plants. These samples, found to be free of HAV, were most commonly contaminated with enteroviruses and rotaviruses. Addition of 1000 TCID50 of HAV, PV1 or RV to a second 1 liter sample, taken at the same time from the corresponding surface waters allowed detection of the input virus without discernible inhibition by amplification inhibitors. The newly established method seems amenable to scaling up and promising for virus monitoring in different water types. The method is rapid and results can be obtained within 24 to 36 hours.
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PMID:Use of silica as a carrier to recover and prepare waterborne enteric viruses for detection by RT-PCR. 963 82

CD4+ T cells play an important role in the aetiology of inflammatory bowel disease (IBD), but it is not clear which factor(s) cause activation of these cells. The aim of this study was to examine the effects of adoptive transfer of splenic (CD4+) T cells from TNBS/ethanol-sensitized donor rats to naive recipients and the migration pattern of transferred T cells. For the transfer experiments, colitis was induced in rats by colonic administration of TNBS/ethanol. Seventeen days later, either total splenic T cells or CD4+, or CD8+ T cells were transferred to naive recipients. At days 1, 2 and 3 after transfer, the recipients were killed and the migration pattern of the transferred T cells was studied, as well as inflammatory cells in several organs, including the colon. To determine cytokine profiles of the T cells, colitis was induced in mice. Therefore, different combinations of 2,4-dinitrobenzene sulfonic acid (DNBS) in ethanol or saline, or ethanol alone were intrarectally administered. At day 9 after induction of colitis, mice were killed and cytokine profiles in the colon were studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The results show that CD4+ T cells from donor rats with TNBS/ethanol-induced colitis migrate in particular to the colon upon transfer to naive recipients, and that this process is down-regulated by CD8+ T cells. This migration is probably caused by T cell recognition of the colonic bacterial flora and initiates an inflammatory reaction in the recipient's colon, characterized by an increase of the recipient's own T cells, macrophages, and neutrophils. In the mice experiments we showed that a second administration of DNBS/ethanol or ethanol alone, which presumably causes bacterial translocation, results in increased numbers of T cells into the colon, accompanied by an increase in Th1 cytokines. These data suggest that Th1 cells recognize the colonic bacterial flora.
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PMID:CD4+ T cells from 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis rodents migrate to the recipient's colon upon transfer; down-regulation by CD8+ T cells. 964 83

We have established a novel injury model in the central nervous system by a stereotaxic injection of ethanol into rat striatum to induce necrosis. With this model, we clarify a function of inducible nitric oxide synthase (iNOS) in a healing mechanism around a necrotic lesion. A semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that the iNOS mRNA arose at 6 h, peaked at 24 h, and declined to a lower level 48 h after an intrastriatal 5-microL ethanol injection. From in situ hybridization, this iNOS mRNA was expressed in the area surrounding the injury. By immunohistochemistry, mononuclear cells at this boundary area of necrosis were stained with anti-iNOS antibody on the first day after the injury. These cells turned out to be reactive microglia from the positive staining of GSA-I-B4, ED-1 and OX-42. Haematoxylin-eosin (HE) staining showed that neurons in this boundary area gradually disappear up to 5 days after the injury with an increment of microglial cells, and this area became cavernous. Nuclei of neurons in this area were stained positive by the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end-labelling (TUNEL) assay on the first day after the injury. These TUNEL-positive neurons gradually disappeared toward the third day, while microglial cells increased. L-Ng-nitro-arginine methylester (L-NAME), a competitive NOS inhibitor, administration diminished the elimination of neurons by microglia in this boundary area surrounding necrosis. Microglial NO may act as a neurotoxic agent to eliminate damaged neurons near the necrosis in the form of delayed neuronal death, and may reintegrate the neuronal circuits with functionally intact neurons.
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PMID:Microglial NO induces delayed neuronal death following acute injury in the striatum. 975 Nov 34

In this study, cytochrome P450 (CYP; EC 1.14.14.1)-dependent activities and P450 isoenzyme patterns were determined in human monocytes and macrophages, which play a major role in antigen processing including small molecular weight compounds which cause contact dermatitis or drug-allergic reactions. Using reverse transcriptase-polymerase chain reaction (RT-PCR) we determined the mRNA expression of eight CYPs (1A1, 1A2, 1B1, 2B6/7, 2E1, 3A3/4, 3A7 and 4B1) in human blood monocytes and macrophage subsets 27E10 and RM3/1. To study the influence of known P450 inducers, monocytes were incubated in vitro with ethanol, dexamethasone, cyclosporin A (CSA), benzanthracene (BA), phenobarbital (PB), lipopolysaccharide (LPS) and 12-O-tetradecanoyl-phorbol-13-acetat (TPA) for 24 hr. Percoll density gradient isolated monocytes as well as the pro-inflammatory macrophage subtype 27E10 expressed 1B1, 2E1 and 2B6/7. On the other hand, in the anti-inflammatory macrophage subtype RM3/1, predominantly 1B1 and to some extent 2B6/7 were found. Treatment with cyclosporin A, phenobarbital, benzanthracene or ethanol resulted in induction of the expression of 3A3/4. CYP1B1 was the predominant isoenzyme in all monocytes and macrophages. In monocytes purified by adherence or induced by benzanthracene, lipopolysaccharide or 12-O-tetradecanoyl-phorbol-13-acetat, 1A1 was also expressed. Northern blot analysis confirmed the presence of CYP1B1 in monocytes and macrophages, a presence which was also demonstrated on the protein level by immunoblot and by immunohistochemical staining of the cells. The expression of several CYPs in monocytes/macrophages suggests that these cells may be important in the metabolism of small molecular weight compounds, which play a role in allergic contact dermatitis and drug reactions. Of particular interest is the remarkably strong expression of the recently identified dioxin inducible CYP1B1, known to be present in a wide range of malignant tumors.
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PMID:Cytochrome P450 1B1: a major P450 isoenzyme in human blood monocytes and macrophage subsets. 980 19

Although ethanol has long been recognized as an immunosuppressant, the effects of ethanol on immune functions in the central nervous system (CNS) have not been well characterized. Glial cells function as immune effector cells within the CNS. Nitric oxide (NO), generated by inducible NO synthase (iNOS) of activated glial cells, appears to participate in the immune defense and the pathogenesis of brain injury and several neurologic diseases. The goal of the present study was to examine the effects of ethanol on NO production and mRNA expression of iNOS following its induction by bacterial endotoxin lipopolysaccharide (LPS) in cultured glial cells. After incubation of mixed glia with LPS for 24 hr, the levels of nitrite in the culture medium were assayed by Griess reaction. We found that LPS (10-500 ng/ml) induced a concentration-dependent increase in the production of NO which was abolished by the selective iNOS inhibitor aminoguanidine. While ethanol treatment (25 to 400 mM, 24 hr exposure) had no direct effect on basal NO production, it significantly suppressed the LPS-induced increase of nitrite levels in a concentration-dependent manner. Using a semiquantitative reverse transcriptase polymerase chain reaction, we found that while ethanol by itself was unable to induce iNOS mRNA, it nevertheless suppressed LPS-induced iNOS mRNA expression. Our results that ethanol had no direct effect on NO production but inhibited LPS-induced NO, indicated an immunomodulatory role by ethanol. These findings suggest that ethanol may ameliorate the consequences of overwhelming NO generation through iNOS induction in glial cells following infection, inflammation or CNS injuries.
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PMID:Ethanol modulates induction of nitric oxide synthase in glial cells by endotoxin. 980 68

Distal symmetrical peripheral neuropathy is a common adverse experience in persons with HIV infection. This condition, which presents as a pain, numbness. burning and/or dysaethesia initially in the feet, is often multi-factorial in its origin. Nucleoside analogue reverse transcriptase inhibitors represent an important contributor to peripheral neuropathy. Specifically, around 10% of patients receiving stavudine or zalcitabine and 1 to 2% of didanosine recipients may have to discontinue therapy with these agents due to neuropathy. Prompt withdrawal of these therapies enables gradual resolution of signs and symptoms in most patients, although a period of symptom intensification may occur shortly after withdrawal. Risk factors for developing peripheral neuropathy during nucleoside analogue therapy include low CD4+ cell count (<100 cells/mm3), a prior history of an AIDS defining illness or neoplasm, a history of peripheral neuropathy, use of other neurotoxic agents including high alcohol (ethanol) consumption and nutritional deficiencies such as low serum hydroxocobalamin levels. Thus, patients at increased risk of peripheral neuropathy should potentially avoid the use of the neurotoxic nucleoside analogues or be more carefully monitored during therapy. Management of this problem includes patient education. prompt withdrawal of the likely causative agent (giving consideration not to leave the patient on a sub-optimal therapy regimen) and simple analgesia. with augmentation with tricyclic antidepressants or anticonvulsant agents when pain is severe. New agents that may assist in managing this condition include levacecarnine (acetyl-L-carnitine) and nerve growth factors such as recombinant human nerve growth factor.
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PMID:Peripheral neuropathy with nucleoside antiretrovirals: risk factors, incidence and management. 988 91


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