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)

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

Immunocytochemical distribution of the fetal protein fetuin in the neocortex of developing rat brain and the presence of its mRNA, as detected by using reverse transcriptase-polymerase chain reaction analysis, was studied in fetuses at embryonic day 15 (E15) through E22, in neonates at postnatal day 0 (P0) through P20, and in adults. Quantitative estimates of fetuin in cerebrospinal fluid (CSF) and plasma were obtained over the same period. Exogenous (bovine) fetuin injected intraperitoneally into fetal and postnatal rats was used to study the uptake of fetuin into CSF and brain and its distribution compared with endogenous fetuin; bovine albumin was used as a control. Fetuin was identified immunocytochemically in the cortical plate and subplate cells of the developing neocortex. In the rat fetus, fetuin first was apparent at E17, mainly in cell processes, but a few subplate cells also were positive. By E18, there was strong staining in subplate neurons and in inner cells of the cortical plate. At E21, these inner cells of the cortical plate were beginning to differentiate into layer VI neurons, many of which were positive for fetuin. By P0-P1, more layer VI neurons and some layer V neurons had become positive for fetuin. Fetuin immunoreactivity generally was weaker at P1, and, by P2-P3, it had disappeared from all of the layers of the developing neocortex. Bovine fetuin (but not albumin), probably taken up through CSF over the neocortical dorsal surface, had a cytoplasmic distribution; endogenous rat fetuin was both cytoplasmic and membrane bound. Thus, much of this fetuin can be accounted for by uptake, although the presence of fetuin mRNA indicates that in situ synthesis may also contribute.
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PMID:Fetuin in the developing neocortex of the rat: distribution and origin. 1087 79

Early detection of recurrence is valuable for monitoring hepatocellular carcinoma (HCC) progression. By quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we derived calibration curves for alpha-fetoprotein (afp) and albumin (alb) mRNAs using 40 matched tumors and non-tumor liver tissues from HCC/adenoma patients. We prospectively quantified tumor cells and non-tumor liver cells in 62 patients' blood samples before, during and after surgery. Expression of both mRNAs was heterogeneous (1-10(5)-fold) between tumors and HepG2 cell line. The alb-mRNA levels in non-tumor liver cells were 2-10-fold higher than in tumor cells. The afp-mRNA levels in HCC cells were 30-1000-fold higher than in non-tumor cells. The alb-mRNA level in blood may reflect the number of liver cells, whereas the afp-mRNA level may represent mostly the number of HCC cells. We found different ratios of circulating HCC cells to non-tumor liver cells during the clinical course of patients, in association with the subsequent development of recurrence/metastasis. This approach may prove useful for detecting and monitoring HCC progression.
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PMID:Quantitative comparison of alpha-fetoprotein and albumin mRNA levels in hepatocellular carcinoma/adenoma, non-tumor liver and blood: implications in cancer detection and monitoring. 1088 Jul 63

Primary cultures of intrahepatic bile duct epithelial (IBDE) cells isolated from duckling livers were successfully grown for studies of duck hepatitis B virus (DHBV). The primary IBDE cells were characterized by immunohistochemistry using CAM 5.2, a cytokeratin marker which was shown to react specifically to IBDE cells in duck liver tissue sections and in primary cultures of total duck liver cells. Immunofluorescence assay using anti-duck albumin, a marker for hepatocytes, revealed that these IBDE cultures did not appear to contain hepatocytes. A striking feature of these cultures was the duct-like structures present within each cell colony of multilayered IBDE cells. Normal duck serum in the growth medium was found to be essential for the development of these cells into duct-like structures. When the primary cultures of duck IBDE cells were acutely infected with DHBV, dual-labeled confocal microscopy using a combination of anti-DHBV core proteins and CAM 5.2 or a combination of anti-pre-S1 proteins and CAM 5.2 revealed that the IBDE cell colonies contained DHBV proteins. Immunoblot analysis of these cells showed that the DHBV pre-S1 and core proteins were similar to their counterparts in infected primary duck hepatocyte cultures. Southern blot analysis of infected IBDE preparations using a digoxigenin-labeled positive-sense DHBV riboprobe revealed the presence of hepadnavirus covalently closed circular (CCC) DNA, minus-sense single-stranded (SS) DNA, double-stranded linear DNA, and relaxed circular DNA. The presence of minus-sense SS DNA in the acutely infected IBDE cultures is indicative of DHBV reverse transcriptase activity, while the establishment of a pool of viral CCC DNA reveals the ability of these cells to maintain persistent infection. Taken collectively, the results from this study demonstrated that primary duck IBDE cells supported hepadnavirus replication as shown by the de novo synthesis of DHBV proteins and DNA replicative intermediates.
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PMID:Duck hepatitis B virus replication in primary bile duct epithelial cells. 1146 37

Because bacterial translocation from the gut is one of the important sources of bacterial infection in acute necrotizing pancreatitis (ANP) and growth hormone (GH) has the ability to promote the intestinal epithelial proliferation, we investigated the effects of GH on bacterial translocation in a rat ANP model. ANP was induced in rats by injection of 5% sodium taurocholate into the biliopancreatic duct. The rats with ANP were treated with either human recombinant GH or placebo. Laparotomized animals without induction of ANP (sham operation [SO]) served as controls. At 24 hours after operation, blood was drawn for bacterial culture and determination of amylase, lipase, and endotoxin. Peritoneal fluid and specimens of mesenteric lymph nodes (MLN), liver, pancreas, and spleen were taken for bacterial culture by standard techniques. Intestinal mucosal permeability was assessed by measuring the movement of 125I-labeled albumin from blood to intestinal lumen. Insulin-like growth factor-1 (IGF-1) mRNA was detected in the liver and ileum by reverse transcriptase-polymerase chain reaction (RT-PCR). Morphologic changes of pancreas and ileum were also analyzed. Administration of GH significantly decreased the serum amylase, lipase activities, plasma endotoxin level, and incidence of bacterial translocation. Moreover, the survival rate of ANP rats was improved. The severity of inflammation in pancreas and ileum was alleviated by GH treatment. Ileal mucosal thickness, villus height, and crypt depth in GH treatment rats were obviously increased compared with those of ANP rats. The intestinal permeability was markedly improved in the GH group versus the ANP group. GH treatment resulted in up-regulation of IGF-1 mRNA expression in ileum, but not in liver. These results suggested that exogenous GH had beneficial effects in maintaining the integrity of intestinal mucosal barrier and reducing the incidence of bacterial translocation in rats with ANP. One of the mechanisms might be the up-regulation of IGF-1 mRNA in intestine by GH treatment.
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PMID:Beneficial effects of growth hormone on bacterial translocation during the course of acute necrotizing pancreatitis in rats. 1148 17

Metallothioneins (MT) are low-molecular-weight, heat-stable, cysteine-rich proteins with four isoforms. MT-I and MT-II are ubiquitous and are induced by oxidative, physical, and chemical stress. MT-I is an efficient scavenger of superoxide (*O2) and hydroxyl ion (OH(-)). We have demonstrated that *O2 and hypohalous acid can cause an increase in glomerular albumin permeability (P(alb)) in vitro. The purpose of this study was to document the protective effect of MT gene product on the *O2-mediated increase in P(alb). Glomeruli from Sprague-Dawley rats in 4% BSA medium were incubated for 4 hr at 37 degrees C in duplicate tubes. Each set contained glomeruli alone or with 5 microM Cd(++), 0.3 mM Spermine-NONOate (NO donor), 0.3 mM Sulfo-NONOate (nitrous oxide donor), 0.6 mM SNP (nonspecific NO donor) and SNP + carboxy-PTIO (10 mg/ml). After incubation, one set of tubes was used to isolate total RNA for the measurement of the mRNA levels of MT-I by reverse transcriptase polymerase chain reaction (RT-PCR). Duplicate tubes were incubated for an additional 10 min with 10 nM of *O2, and P(alb) was measured using video microscopy. RT-PCR of total RNA from Cd(++) and Spermine-NONOate treated glomeruli revealed a 2-fold induction of MT-I expression at the mRNA level. *O2 caused a significant increase in P(alb) (0.8 +/- 0.06 vs. control 0.0 +/- 0.12, P < 0.05) and induction of MT-I in glomeruli by Cd(++) or by Spermine-NONOate blocked this effect (0.21 +/- 0.12 and 0.24 +/- 0.19, respectively, P < 0.05 vs. *O2). In contrast, Sulfo-NONOate and SNP did not induce mRNA for MT-I in glomeruli and did not provide protection against *O2-mediated increase in P(alb.) We conclude that MT-I gene products may play an important role in protecting the glomerular filtration barrier from the injury induced by reactive oxygen species in immune and/or nonimmune renal diseases.
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PMID:Induction of metallothionein-I protects glomeruli from superoxide-mediated increase in albumin permeability. 1178 80

AIM:To investigate the characteristics of newly established four hepatocellular carcinoma cell lines (SNU-739, SNU-761, SNU-878 and SNU-886) from Korean hepatocellular cancer patients.METHODS:Morphologic and genetic studies were done.RESULTS:All four lines grew as a monolayer with an adherent pattern, and their doubling times ranged from 20 to 29 hours. The viability rate was relatively high (88%-94%).Neither mycoplasmal nor bacterial contamination was present.The lines showed different patterns in fingerprinting analysis. The hepatitis B virus (HBV) DNA was integrated in the genomes of all four lines, and in all of them HBx,HBc and HBs transcripts were detected by reverse transcriptase-PCR methods. Among the three cell lines used as control (Hep 3B, SK Hep1 and Hep G2),only Hep 3B showed HBx expression, and this line was used as a HBV integrated control.The RNA of albumin was detected in three lines (SNU-761, SNU-878 and SNU-886), that of transferrin in two lines (SNU-878, SNU-886), and that of IGF-II was detected in none of the cell lines.CONCLUSION:These well characterized cell lines may be very useful for studying the biology of hepatocellular carcinoma in association with the hepatitis Bvirus.
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PMID:Establishment and characterization of four human hepatocellular carcinoma cell lines containing hepatitis B virus DNA. 1181 50

Therapies for liver diseases with stem and progenitor cells will require a detailed knowledge of the molecular mechanisms driving the in vivo differentiation process toward adult hepatic tissue. We applied quantitative gene expression methods to analyze the differentiation process of fetal liver progenitor cells after transplantation into an animal model of liver regeneration. Enhanced green fluorescent protein (EGFP)-transgenic liver progenitor cells were isolated from fetal mouse liver at stage embryonic day 13.5 and transplanted into uPA/RAG-2 mice. Two, 4, and 6 weeks after cell transplantation cryosections of liver tissue were analyzed for EGFP-positive regeneration nodules. RNA from laser-microdissected EGFP-positive tissue was isolated and used as template for quantitative real-time reverse transcriptase-polymerase chain reaction. Phenotypic differentiation was analyzed by staining of the canalicular marker enzyme dipeptidyl-peptidase IV. Proliferation in regenerative nodules and surrounding tissue was monitored with the BrdU incorporation assay. Alpha fetoprotein gene expression had already decreased 2 weeks after transplantation in EGFP-positive regeneration nodules compared to pretransplantation values and was not detectable after 4 and 6 weeks, whereas albumin slightly increased in transplanted cells indicating differentiation into a mature phenotype. The dipeptidyl-peptidase IV antigen was associated with some liver progenitor cells 2 weeks after transplantation and in virtually all cells after 4 and 6 weeks. Cell proliferation index in transplanted cells was maximally increased (4.8% BrdU-positive cells) after 2 weeks and decreased (0.4%) after 6 weeks to normal levels. Our results demonstrate that gene expression in liver progenitor cells changes from fetal to adult phenotype within 4 to 6 weeks after transplantation despite ongoing proliferation of the transplanted cells in a mouse model of liver regeneration. Quantitative gene expression profiles as shown here will have important implications in our understanding of the in vivo differentiation process of stem cells.
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PMID:Quantitative gene expression analysis reveals transition of fetal liver progenitor cells to mature hepatocytes after transplantation in uPA/RAG-2 mice. 1250 88

We investigated the ability of a genetically altered embryonic stem (ES) cell line to promote endodermal differentiation toward hepatocytes in vitro by transfecting the hepatocyte nuclear factor-3beta (HNF-3beta) gene. Parental and HNF-3beta-transfected ES cells were initiated toward differentiation in embryoid bodies (EBs) for 5 days and the resulting EBs were transferred to an attached culture system. Albumin production was observed using an immuno-cytochemical method 7 days after induction of differentiation in almost all differentiating HNF-3beta-transfected ES cells, whereas scant immuno-reactivity against albumin was found on the same day in the cultures of differentiating parental ES cells. An analysis using a reverse transcriptase polymerase chain reaction revealed the HNF-4alpha expression in the HNF-3beta-transfected ES cells and also demonstrated that the expression of endodermal and hepatocyte-related markers, such as transthyretin, alpha-fetoprotein, albumin, alpha-1 antitrypsin, tryptophan-2,3-dioxygenase and phosphoenol-pyruvate carboxykinase, could be observed at an early stage in the outgrowths of HNF-3beta-transfected ES cells compared to the parental ES cells. These results suggest that HNF-3beta-transfected ES cells may be useful for the efficient induction of hepatocytes in vivo.
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PMID:In vitro differentiation of hepatocyte-like cells from embryonic stem cells promoted by gene transfer of hepatocyte nuclear factor 3 beta. 1285 Jun 95

Chemicals known as peroxisome proliferators (PPs) are the subject of intense study because of their ability to cause hepatocellular carcinoma in laboratory rodents. These chemicals act through a family of proteins termed the peroxisome proliferator-activated receptors (PPARs), in particular PPARalpha. It has become increasingly apparent that the role of the PPs in the development of cancer encompasses many different aspects of cell growth regulation. Immortalized hepatocytes from wild-type (PPARalpha(+/+)) and PPARalpha(-/-) mice were generated using a temperature-sensitive SV40 virus. Characterization of the murine SV40 hepatocytes (MuSH) generated from both genotypes (MuSHalpha(+/+), MuSHalpha(-/-)) show markers of differentiation such as albumin expression, but is devoid of Kupffer cell contamination. Hallmark PPARalpha-mediated responses such as induction of acyl-CoA oxidase mRNA by PPs are present in the MuSHalpha(+/+) but are absent in MuSHalpha(-/-) cells. In contrast to most cell culture systems, the wild-type MuSH hepatocytes retain the mitogenic activity of PPs, whereas the MuSHalpha(-/-) does not respond in this manner, thus making this cell culture system an ideal tool to examine growth regulatory gene expression affected by PPs. Microarray experiments performed on both cell types identified many genes in which regulation is dependent on the presence of PPARalpha, and these changes were verified with reverse transcriptase-PCR. Genes involved in carcinogenesis and control of the cell cycle that are regulated by PPs in a PPARalpha-dependent manner include ubiquitin COOH-terminal hydrolase 37 (also known as UCT-L5) and cyclin T1. These results show that MuSH cells reflect the biological properties of both the wild-type and PPARalpha-null animals and can be used to identify novel PPARalpha-regulated genes that could be involved in regulation of the cell cycle and carcinogenesis.
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PMID:Comprehensive gene expression analysis of peroxisome proliferator-treated immortalized hepatocytes: identification of peroxisome proliferator-activated receptor alpha-dependent growth regulatory genes. 1452 98


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