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)

Protein kinase C (PKC) inhibitors decrease alpha1(I) collagen mRNA in stellate cells exposed to 200 micromol/liter of acetaldehyde. The purpose of these studies was to determine whether PKC activation plays a role in transcriptional activation of the alpha2(I) collagen gene. Cultured stellate cells were exposed to 200 micromol/liter of acetaldehyde. PKC, inositol triphosphate, diacylglycerol (DAG), and intracellular free calcium (Ca2+i) were measured. Alpha1(I) and alpha2(I) collagen messages were determined by reverse transcriptase-polymerase chain reaction. Activation of the alpha2(I) collagen promoter was determined in transiently transfected stellate cells. Acetaldehyde exposure enhanced PKC activity translocation to the particulate fraction at 20 min. Acetaldehyde did not increase Ca2+i, or inositol triphosphate but increased DAG levels at 20 min and 3 hr. Acetaldehyde increased both the alpha1(I) and alpha2(I) collagen messages in stellate cells. Calphostin C, a specific PKC inhibitor, which blocks DAG binding, eliminated both activation of the alpha2(I) collagen promoter by acetaldehyde and mRNA production by reverse transcriptase-polymerase chain reaction analysis. Similarly, D609, an inhibitor of DAG production, also inhibited alpha2(I) collagen gene expression. This study shows that collagen production by acetaldehyde is mediated by a calcium-independent PKC mechanism.
Alcohol Clin Exp Res 1999 Feb
PMID:Acetaldehyde enhances murine alpha2(I) collagen promoter activity by Ca2+-independent protein kinase C activation in cultured rat hepatic stellate cells. 1006 57

Biotransformation of all-trans-retinol (t-ROH) and all-trans-retinal (t-RAL) to all-trans-retinoic acid (t-RA) in human prenatal hepatic tissues (53-84 gestational days) was investigated with HPLC using human adult hepatic tissues as positive controls. Catalysis of the biotransformation of t-ROH by prenatal human cytosolic fractions resulted in accumulation of t-RAL with minimal t-RA. Oxidations of t-ROH catalyzed by prenatal cytosol were supported by both NAD+ and NADP+, although NAD+ was a much better cofactor. In contrast, catalysis of the oxidation of t-RAL to t-RA appeared to be solely NAD+ dependent. Substrate Km values for conversions of t-ROH to t-RAL and of t-RAL to t-RA were 82.4 and 65.8 microM, respectively. At concentrations of 10 and 90 mM, ethanol inhibited the conversion of t-ROH to t-RAL by 25 and 43%, respectively, but did not inhibit the conversion of t-RAL to t-RA significantly. In contrast, acetaldehyde reduced the conversion of t-RAL to t-RA by 25 and 87% at 0.1 and 10 mM respective concentrations. Several alcohols and aldehydes known to be generated from lipid peroxides also exhibited significant inhibition of t-RA biosynthesis in human prenatal hepatic tissues. Among the compounds tested, 4-hydroxy-2-nonenal (4-HNE) was highly effective in inhibiting the conversion of t-RAL to t-RA. A 20% inhibition was observed at a concentration of only 0.001 mM, and nearly complete inhibition was produced at 0.1 mM. Human fetal and embryonic hepatic tissues each exhibited significant CYP2E1 expression as assessed with chlorzoxazone 6-hydroxylation, a highly sensitive western blotting technique, and reverse transcriptase-polymerase chain reaction (PCR) (RT-PCR), suggesting that lipid peroxidation can be initiated via CYP2E1-catalyzed ethanol oxidation in human embryonic hepatic tissues. In summary, these studies suggest that ethanol may affect the biosynthesis of t-RA in human prenatal hepatic tissues directly and indirectly. Ethanol and its major oxidative metabolite, acetaldehyde, both inhibit the generation of t-RA. Concurrently, the CYP2E1-catalyzed oxidation of ethanol can initiate lipid peroxidation via generation of a variety of free radicals. The lipid peroxides thereby generated could then be further converted via CYP2E1-catalyzed reactions to alcohols and aldehydes, including 4-HNE, that act as potent inhibitors of t-RA synthesis.
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PMID:Inhibition of human prenatal biosynthesis of all-trans-retinoic acid by ethanol, ethanol metabolites, and products of lipid peroxidation reactions: a possible role for CYP2E1. 1007 87

Hepatocytes entrapped in collagen gel and cultured in serum-free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c-fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin-stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 +/- 0.37 and 9 +/- 2.7 nmol glucose/h/microg DNA). Collagen-cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 +/- 4 nmol urea/h/microg DNA). The rate of albumin synthesis in collagen-entrapped cells was maintained above the day-1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 +/- 152 pmol/microg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen-cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and glutathione-transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione-transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP-alpha and -beta, and HNF-1 and -4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate-synthetase I).
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PMID:Long-term expression of differentiated functions in hepatocytes cultured in three-dimensional collagen matrix. 1009 8

The concept of chronic hepatitis induced by alcohol (AL-CH) has not been widely accepted, because AL-CH may be due to non-A-E hepatitis virus in heavy drinkers. Recently, hepatitis G virus (HGV) was identified as a positive-strand RNA virus related to members of the Flaviviridae family. In this study, we determined serum HGV in patients with AL-CH and analyzed the clinicopathological changes after abstinence to evaluate whether AL-CH is caused by alcohol or not. Serum samples were obtained from 16 patients with AL-CH who had neither hepatitis B nor C virus. The diagnosis was confirmed histologically. In eight patients, liver biopsy was performed twice, within 3 days and 4 to 8 weeks after abstinence. The NS3 region of the HGV genome was detected using an reverse transcriptase-polymerase chain reaction method. Serum levels of AST, ALT and gamma-glutamyltranspeptidase were measured once a week sequentially after admission. Serum HGV-RNA was detected in only one patient with AL-CH (6.3%). In all patients, including one patient with HGV, serum levels of AST, ALT and gamma-glutamyltranspeptidase clearly decreased to normal levels after abstinence. Inflammatory activity in the periportal area of patients with actively drinking decreased or disappeared after abstinence for 4 to 8 weeks. These results suggest that HGV may not play an important role for development of AL-CH, and that AL-CH may be caused by alcohol itself, although a more larger number of patients with AL-CH are needed to obtain definitive conclusions.
Alcohol Clin Exp Res 1999 Apr
PMID:Clinicopathological study of chronic hepatitis induced by alcohol with or without hepatitis G virus. 1023 75

The expression of the ethanol-metabolizing cytochrome P450 (CYP2E1) in human monocyte-derived macrophages was studied at the mRNA and protein levels. The presence of mRNA was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) and protein by immunocytochemistry. The data show that CYP2E1 is expressed in human monocyte-derived macrophages at a level similar to that demonstrated in other extrahepatic tissues. Although there is circumstantial evidence for the presence of CYP2E1 in human macrophages, it has not previously been demonstrated directly. Its presence in macrophages underlines the potential importance of these cells in initiating alcohol-induced cytotoxicity.
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PMID:Expression of CYP2E1 by human monocyte-derived macrophages. 1039 64

Gut-derived endotoxins (lipopolysaccharide, LPS) complexed to LPS-binding protein (LBP) activate liver Kupffer cells via their CD14 receptor. Pro-inflammatory cytokines are released and this is postulated to promote liver injury. We previously demonstrated enhanced expression of CD14 endotoxin receptor after 2 weeks of alcohol administration. A similar result, based on 6 weeks of ethanol treatment, was recently reported and suggested to correlate with alcohol-induced liver injury. To establish whether this occurs prior to or after the initiation of damage, we investigated the temporal effect of continuous ethanol exposure on the expression of CD14 and the associated LBP. In addition, we studied the effect of treatment with gadolinium chloride (GdCl3) that inactivates Kupffer cells and alleviates alcohol-induced liver damage. The amount of CD14 and LBP mRNA, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR), was unchanged 4-8 h after intragastric ethanol administration. However, after 24-48 h of repeated ethanol administration, CD14 and LBP mRNA both increased significantly and reached a level similar to that observed after 6 weeks of ethanol exposure by liquid diet. Immunostaining experiments with ED2 antibody demonstrated that GdCl3 efficiently inactivated Kupffer cells. However, there was no concomitant reduction in the expression of CD14 mRNA, suggesting that compensatory infiltration by ED2-negative, but CD14-positive, macrophages had occurred. Our results demonstrate that soon after the initiation of ethanol exposure, i.e. within 24-48 h, the hepatic expression of both the CD14 receptor and LBP is increased. This suggests that these increases could contribute to the initiation of alcoholic damage rather than being a consequence of the injury.
Alcohol Alcohol
PMID:Short-term ethanol exposure increases the expression of Kupffer cell CD14 receptor and lipopolysaccharide binding protein in rat liver. 1041 5

Alcoholics have increased susceptibility to infections including tuberculosis. Chronic alcohol treatment impairs host response to bovine mycobacterium infection from BCG. This study assesses the role of four cytokines (TNFalpha, IFNgamma, IL-4, and IL-10) in this impaired response. Twenty male C57BL/6 mice were pair-fed on the Lieber DiCarli control (LCD) or ethanol (LED) diets for 28 days. The LED treated subjects ate ad lib and consumed a mean of 13 g/kg/d of ethanol. After 14 days, based on body weight, subjects were randomly divided into four treatment groups of five each. Ten infected with 2x10(6) colony-forming units (CFU) of BCG by tail-vein. On day 28, the mice were sacrificed. Liver was cultured to determine the mycobacteria CFU/g tissue. Spleens were assayed for the levels of TNFalpha, IFNgamma, IL-4, and IL-10 mRNA relative to mRNA levels for a housekeeping gene using a quantitative reverse transcriptase PCR. Without BCG infection, only the mRNA for IFNgamma was increased by LED treatment, 51% (p = 0.0001). BCG infection significantly increased TNFalpha, IFNgamma, and IL-10 mRNA (p<0.0001). IL-4 mRNA decreased (p = 0.0006). Chronic LED plus BCG infection further increased TNFalpha (p = 0.002) and IFN-gamma (p = 0.04); IL-10 was unchanged, whereas IL-4 was marginally further decreased (p = 0.06). CFU/liver increased with LED (mean +/- SD, 72+/-33x10(5) vs. 39+/-17x10(5); p = 0.004). A significant direct correlation was observed between CFU and TNFalpha, r = 0.70, p = 0.03. In conclusion, BCG infection increases TNFalpha, IFNgamma, & IL-10 and decreases IL-4. CFU numbers correlate with mRNA for TNFalpha, and LED inhibits host containment of BCG infection as measured by liver CFU. This study could not identify cytokine alterations in either Th1- or Th2-type immune responses that might contribute to the impaired host response to the BCG infection.
Alcohol 1999 Aug
PMID:Cytokine response to BCG infection in alcohol-fed mice. 1048 89

Cytokines induce apoptosis in pancreatic beta cells, but the exact mechanisms and sequence of events are not clear. Here, we investigate a role for tumor necrosis factor alpha (TNF-alpha) in the apoptosis of beta cells. Using the ribonuclease (RNase) protection assay and the reverse transcriptase-polymerase chain reaction (RT-PCR) method, we confirmed that TNF receptor 1 (TNFR1), TNFR1-associated death domain protein (TRADD), Fas receptor-associated intracellular protein with death domain (FADD), and FADD-like interleukin-1beta-converting enzyme (FLICE) were expressed in the pancreatic beta cell line, MIN6 cells. Fluorescent microscopic examination using Hoechst 33342 dye (Sigma, St Louis, MO) demonstrated that TNF-alpha induced time- and dose-dependent apoptotic nuclear changes in these beta cells. In situ end-labeling (ISEL) DNA analysis revealed that 10 nmol/L TNF-alpha generated new 3'-OH DNA strand breaks. Moreover, qualitative assessment of the induced DNA damage on agarose gels showed that 10 nmol/L TNF-alpha produced characteristic apoptotic patterns of DNA fragments formed by internucleosomal hydrolysis of static chromatin. In addition, C2-ceramides and natural ceramides dispersed in a solvent mixture of ethanol and dodecane induced characteristic features of apoptosis in MIN6 cells, mimicking TNF-induced DNA damage. We also determined endosomal ceramide production after TNF-alpha (10 nmol/L) treatment in MIN6 cells using the diacylglycerol kinase assay. These results suggest that TNF-alpha can cause apoptosis in pancreatic beta cells through TNFR1-linked apoptotic factors, TRADD, FADD, and FLICE, and TNF-induced ceramide production may be involved in the pathways.
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PMID:Tumor necrosis factor alpha signaling pathway and apoptosis in pancreatic beta cells. 1059 77

To determine whether overexpression of the human MnSOD transgene protected 32D cl 3 hematopoietic progenitor cells from ionizing irradiation, 32D cl 3 cells were co-electroporated with the pRK5 plasmid containing the human MnSOD transgene and SV2-neo plasmid with G418-resistant colonies selected. Two clones (1F2 and 2C6) were identified to overexpress the human MnSOD transgene by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and increased biochemical activity. Measurement of irradiation-induced damage was determined in cells removed from G418 for 1 week before irradiation. Irradiation survival curves, apoptosis tunnel assay, and Comet assay was performed. Cell cycle distribution was determined for each line at 0, 1, 3, 6, 24, and 48 hr after 500 cGy by fixing the cells in 70% ethanol, staining with propidium iodide, and analysis by flow cytometer. Biochemical MnSOD activity in U/mg protein was 2.6 for 32D cl 3 and significantly elevated to 8.4 and 6.6 (P < 0.001) U/mg protein for subclones 1F2 and 2C6, respectively. Irradiation survival curves demonstrated an increased shoulder on the irradiation survival curve for 1F2 and 2C6 cells with an n of 4.95 +/- 0.48 (P = 0.042) and 4.95 +/- 0.13 (P = 0.011), compared with 2.77 +/- 0.20 for 32D cl 3. A higher percent of 32D cl 3 cells demonstrated apoptosis at 24 and 48 hr after 1,000 cGy irradiation, compared with 1F2 and 2C6 cells (at 24 hr, 29.37% +/- 2.01% of 32D cl 3 cells were apoptotic compared with 5.21 +/- 2.61 (P = 0.018) and 5.27 +/- 2.58 (P = 0.004) for 1F2 and 2C6, respectively). Significantly more DNA strand breaks were detected by Comet assay in 32D cl 3 cells (Comet length at 600 cGy of 103.4 +/- 50.3 units, compared with 69.7 +/- 36.3 (P < 0.001) and 48.9 +/- 27.5 (P < 0.001) for 1F2 and 2C6, respectively). In contrast, irradiation-induced cell cycle arrest was similar between the cell lines with a G2/M phase arrest at 6 hr and a G1/S phase arrest at 24 and 48 hr after irradiation. While overexpression of MnSOD increases the shoulder on the irradiation survival curve of 32D cl 3 cells, decreases irradiation-induced apoptosis, and DNA strand breaks by Comet assay, irradiation-induced alterations in cell cycle distribution were not significantly altered. These 32D cl 3 subclonal lines overexpressing MnSOD provide a potentially valuable system with which to study the mechanism of irradiation-induced cell cycle arrest separate from irradiation-induced apoptosis.
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PMID:Overexpression of the human manganese superoxide dismutase (MnSOD) transgene in subclones of murine hematopoietic progenitor cell line 32D cl 3 decreases irradiation-induced apoptosis but does not alter G2/M or G1/S phase cell cycle arrest. 1064 56

The instability of RNA in solutions during storage and travel is an impediment to its utilization in routine diagnostics. A robust and simple approach to the problem of RNA protection and processing is offered by storage of RNA desiccated with processing procedures that do not solublize the RNA until the beginning of reverse transcription. The feasibility of this general approach was tested with coxsackievirus B4 (CVB-4) from blood or culture fluid held on a storage and transport medium (FTA(R)) and analysed by reverse transcriptase PCR (RT-PCR) without removing the RNA from the FTA(R) until reverse transcription. Phase-trapping techniques based on water-miscible solvents such as ethanol or phenol were compared with simple buffers and concentrated lithium chloride solutions. RT-PCR detection of viral RNA reached a sensitivity of approximately 0.1 fg, which is comparable with other non-nested PCR techniques. Whole blood as a virus vehicle significantly interfered with CVB-4 detection, but to an acceptable degree. Desiccation-storage of the RNA of CVB-4 appears to be unaffected by weeks on the storage medium under ambient conditions. These characteristics indicate that this approach forms a credible developmental base for RNA-based pathogen diagnostics with particular application to the problem of transporting potentially infectious body fluids to a centralized laboratory for analysis.
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PMID:Reverse transcription of an RNA genome from databasing paper (FTA(R)). 1081 92


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