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1.
The Hep G2 human hepatoma cell line has been recognized as an excellent in vitro human model system. For this reason, this line was used to study the effect of ethanol on HMG-CoA reductase activity concerning cell growth and cholesterol metabolism. Cells were incubated in ethanol-containing medium (0-400 mmol/L) for up to 102 h. Ethanol caused an inhibition in the growth rate and in HMG-CoA reductase activity that could be reverted by the removal of ethanol from the culture medium, indicating no cellular damage. These changes cannot be ascribed to the regulatory effect of cholesterol levels, since its content was not modified either in the cells or in the medium. The addition of mevalonate to the culture medium could not revert the growth rate inhibition evoked by ethanol. Moreover, ethanol produced an increment in the cholesterol efflux in [3H]cholesterol-prelabeled cells. We conclude that the decrease in HMG-CoA reductase activity evoked by ethanol treatment on Hep G2 cells would not be the cause but the consequence of the impairment in cellular growth, since this impairment could not be reverted by the addition of mevalonate to the culture medium.  相似文献   

2.
We have localized p55, a thyroid hormone-binding protein found in the endoplasmic reticulum in cultured cells, in samples of normal human and monkey tissues, using a monoclonal antibody with cryostat sections and immunoperoxidase histochemistry. Large amounts of p55 were found in many tissues, generally corresponding to the amount of endoplasmic reticulum contained in each cell type. Intense localization of p55 was found in cells of the anterior and intermediate pituitary lobes, in epithelial cells of thyroid follicles, in the glandular epithelium of mammary gland, in hepatocytes, in Paneth cells and Brunner's glands in duodenum, in acinar cells of pancreas, in adrenal cortical cells, and in scattered interstitial fibroblastic cells in many tissues. These results suggest a potential role for thyroid hormone and p55 in regulating protein synthesis or secretion in multiple organs.  相似文献   

3.
Our purpose was to examine the roles of natural (estradiol (E2) and estrone (E1)) and synthetic estrogens (ethinyl estradiol (EE), moxestrol (MOX), and tamoxifene (TAM)) in regulating production of sex hormone-binding globulin (SHBG) by human hepatoma G2 (Hep G2) cells, the rationale being that synthetic estrogens are less rapidly metabolized than natural estrogens and, thus, may alter SHBG levels more readily. In Hep G2 cells, E2, E1, and EE at 10(-7) M did not result in significantly greater SHBG secretion compared to control cells. The synthetic estrogens, MOX and TAM, caused significant, P < 0.001, increases of 30% and 51% in SHBG secretion at 10(-7) M compared to controls. However, when TAM and E2 were added together, each at 10(-7) M, no increase in SHBG secretion was noted. We conclude that natural estrogens at physiologic concentrations do not increase SHBG secretion by Hep G2 cells, but the increase of SHBG secretion caused by MOX and TAM suggests that the lack of effect of E2 and E1 may, in part, be due to their rapid metabolism. In addition, TAM stimulates SHBG secretion by interaction with the genome that is different, in certain respects, from that of E2.  相似文献   

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The hepatic IL-6-receptor is composed of an 80 kDa IL-6-binding protein and a 130 kDa polypeptide (gp130) believed to be involved in signal transduction. Previous experiments have shown that the 80 kDa IL-6-receptor is up-regulated by glucocorticoids, but not by IL-6. Here we demonstrate that IL-6 together with the synthetic glucocorticoid dexamethasone induces the expression of mRNA for gp130 approximately 5-fold in HepG2 cells. The induction was dose- and time-dependent. Dexamethasone alone, interferon-gamma, IL-1 alpha and IL-1 beta had no effect. A possible role for the regulation of the IL-6-signal transducing protein gp130 in various inflammatory states is proposed.  相似文献   

6.
There is a growing body of evidence to show that that C-reactive protein (CRP), an acute phase reactant, is one of the most valuable predictors of future cardiovascular events. Since CRP proteins directly contribute to the development and progression of atherosclerosis as well, reduction of CRP levels may be a novel therapeutic target for the treatment of cardiovascular disease. In this study, we examined whether pigment epithelium-derived factor (PEDF) could block the interleukin-6-induced CRP expression in cultured human hepatoma cells and the way that it might achieve this effect. PEDF inhibited the IL-6-induced CRP expression in Hep3B cells at both mRNA and proteins levels. PEDF suppressed the intracellular reactive oxygen species generation in IL-6-exposed Hep3B cells. Anti-oxidants mimicked the effects of PEDF. PEDF was also found to inhibit the IL-6-elicited Rac-1 activation, whereas dominant-negative Rac-1 dose-dependently decreased the CRP mRNA levels. PEDF blocked the IL-6-induced STAT3 phosphorylations and NF-kappaB p65 activity in Hep3B cells. Our present study suggests that PEDF could be one of the potent suppressors of CRP production by the liver and may play a protective role against atherosclerosis.  相似文献   

7.
The purpose of the present study was the development of a protocol for detecting chemically-induced DNA damage, using the alkaline single-cell gel electrophoresis (SCGE) assay with human-derived, metabolically competent hepatoma (Hep G2) cells. Previous studies indicated that Hep G2 cells have retained the activities of certain phase I and phase II enzymes and reflect the metabolism of genotoxins in mammals better than other in vitro models which require addition of exogenous activation mixtures. The optimal trypsin concentration for the removal of the cells from the plates were found to be 0.1%. Dimethylsulfoxide, at concentrations up to 2%, was an appropriate solvent for water-insoluble compounds. To determine the optimal exposure periods for mutagen treatment, the time kinetics of comet formation was investigated with genotoxic chemicals representing various classes of promutagens namely benzo[a]pyrene (B[a]P), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and N-nitrosodimethylamine (NDMA) and with N-nitrosomethylurea (NMU). All compounds caused a statistically significant induction in DNA damage. With the promutagens, comet formation increased gradually as a function of the exposure duration, and reached maximum values between 20-24 h. With NMU, comet induction maximized already after a short exposure (1 h) and remained at a constant level for up to 24 h. Based on these results, the Hep G2/SCGE assay appears to be a suitable approach for investigating DNA damaging potential of chemicals. Further experiments with IQ and B[a]P showed that the assays are highly reproducible. Comparisons of the present results with those from earlier experiments in which other endpoints (induction of sister chromatid exchanges, micronuclei and chromosomal aberrations) were measured in Hep G2 cells, indicated that the sensitivity of the SCGE assays is more or less identical. Since the SCGE assay is less time consuming than other genotoxicity assays we anticipate that it might be a suitable approach to investigate DNA damaging effects of chemicals in the human-derived, metabolically competent cell line.  相似文献   

8.
A cDNA for the gene that encodes for a human cellular thyroid hormone-binding protein (p55) has recently been isolated and sequenced. The sequence of p55 indicates that it is identical to the protein disulfide isomerase and the beta-subunit of prolyl 4-hydroxylase. By in situ hybridization, the gene for p55 was localized on chromosome 17 at band q25. This localization shows that the p55 gene is not linked to either erbA1 or erbA2; two other thyroid hormone-binding protein genes are located at 17q 11-21 and 3p21-pter, respectively. The localization of p55 gene will permit the evaluation of the possible effects of chromosome changes on the structure and activity of the p55 gene in chromosome syndromes or neoplasms.  相似文献   

9.
Many secretory proteins contain an amino-terminal propeptide extension which is removed prior to secretion. The point of cleavage is usually marked by a basic pair of amino acids containing arginine. Canavanine, an analogue of arginine, is incorporated into protein and has been shown to inhibit the proteolytic processing of several of these prosecretory proteins. The addition of 3 mM canavanine to Hep G2 cells incubated with L-[35S]methionine inhibited the secretion of 11 plasma proteins studied. Of the secretory proteins studied only albumin is thought to contain a propeptide, which is marked by a pair of arginine residues at its point of proteolytic processing. Canavanine had varying effects on the secretion of plasma proteins; ranging from a 43-53% inhibition of secretion of alpha 1 antitrypsin and alpha 1 anti-chrymotrypsin to nearly abolishing (93% inhibition) secretion of transferrin. Canavanine also caused most of the proteins studied to migrate slower on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two of the canavanine-treated proteins (albumin and transferrin) which underwent marked changes in electrophoretic mobility were more sensitive than untreated proteins to proteolysis by Staphylococcus Aureus V8 proteinase. The slower electrophoretic migration and the greater sensitivity to proteolysis of these proteins may be attributed to marked structural changes caused by the incorporation of canavanine. This suggests that the inhibition of plasma protein secretion by canavanine is not only due to an inhibition of the processing of proteins but may be caused by structural distortions of the secretory proteins.  相似文献   

10.
To evaluate the impact of taurine on hepatic cholesterol catabolism low density lipoprotein (LDL) binding, internalization and degradation were measured in cultured Hep G2 cells. Preincubation of cells with 0.1-10 mM taurine for 24 h stimulated LDL receptor activity by as much as 100%. Only the high affinity LDL receptor activity (specific) was increased by taurine preincubation, whereas the low affinity receptor activity (nonspecific) remained unchanged. Scatchard analysis of the binding data revealed that taurine doubled the number of LDL receptors without affecting receptor affinity. Taurine-enhanced LDL receptor activity was most pronounced when LDL concentrations exceeded 100 micrograms/ml, but was noted at taurine concentrations as low as 0.1 mM (plasma level). Interestingly, taurine had no effect on LDL receptor activity when it was added simultaneously with 125I-LDL to Hep G2 cells, or when non-bile acid-producing human skin fibroblasts were tested. Stimulation of LDL receptor activity was also obtained with 10 mM cysteine, a taurine precursor, but not with glycine. Increased cellular concentrations of taurine and cysteine were associated with an elevated rate of bile acid synthesis and a reduced cellular free cholesterol concentration. The data suggest that taurine enhanced LDL receptor activity by sparing cysteine, a known sulfhydryl group donor and stimulator of 7 alpha-hydroxylase activity, and that the latter stimulated bile acid production leading to increased utilization of cellular free cholesterol and enhanced LDL uptake.  相似文献   

11.
The Jurkat T cell line was stably transfected with an Epstein-Barr virus-based episomal replicon designed to express high levels of the HIV-1 Tat protein. After selection in hygromycin B, high-level Tat activity was detected in 3 of 18 transfected cell lines. After stimulation with phytohemagglutinin (PHA) and phorbol myristate acetate (PMA), Tat transfectants with high Tat expression showed diminished expression of interleukin-2 (IL-2) and the interleukin-2 receptor alpha chain (IL-2R) when compared to untransfected Jurkat cells or Jurkat cell lines transfected with the parent control plasmid. Sublines derived from the high-level Tat transfectants with reduced Tat activity showed normalization of PHA/PMA-induced IL-2 expression. Northern analysis showed diminished expression of IL-2 and IL-2R mRNA in the stimulated Tat transfectants. Inhibition of IL-2 and IL-2R expression by the HIV-1 Tat protein may contribute to the immune suppression that characterizes HIV-1 infection.  相似文献   

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13.
We have studied the influence of thyroid hormone status in vivo on expression of the genes encoding guanine nucleotide-binding regulatory protein (G protein) alpha-subunits Gs alpha, Gi alpha(2), Gi alpha(3), and both the 36-kDa form (beta 1) and the 35-kDa form (beta 2) of the beta-subunit in rat ventricle. The relative amounts of immunoactive Gi alpha(2) and Gi alpha(3) were greater in ventricular membranes from hypothyroid animals than from euthyroid animals (1.9- and 2.6-fold, respectively). A corresponding 2.3-fold increase in Gi alpha(2) mRNA was observed as well as a 1.5-fold increase in Gi alpha(3) mRNA. The relative amounts of immunoactive beta 1 and beta 2 polypeptides were also increased (2.8- and 1.8-fold, respectively) in the hypothyroid state and corresponded with comparable increases in the relative levels of beta 1 and beta 2 mRNAs. No difference was seen between the amounts of Gi alpha(2), Gi alpha(3), beta 1, and beta 2 in the euthyroid state and the hyperthyroid state. In contrast to these effects of thyroid hormone status on Gi alpha and beta, the steady-state amounts of Gs alpha protein and mRNA were not altered by thyroid hormone status. Thyroid hormone status did not alter sensitivity of adenylyl cyclase to stimulation by sodium fluoride or guanyl-5'-yl imidodiphosphate (GppNHp), nor did it influence GppNHp-induced inhibition of forskolin-stimulated enzyme activity. These results demonstrate that thyroid hormone status in vivo can regulate expression of specific G protein subunits in rat myocardium. However, the physiological consequences of these changes remain unclear.  相似文献   

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Solubilized membrane proteins of Hep G2 cells were electrophoretically separated on polyacrylamide gels and electrotransferred onto nitrocellulose paper. Overlaying the nitrocellulose with human high density lipoproteins conjugated to colloidal gold revealed the presence of a single protein band with an apparent molecular mass of 80 kDa. Binding of the conjugates to this protein was specific for high density lipoproteins in as much as it was effectively displaced by an excess of unlabelled high density lipoproteins but not by a similar excess of unlabelled low density lipoproteins. Binding was not dependent on Ca2+ as 10 mM EDTA had no effect. The binding activity of the solubilized membranes was increased by incubating the cells with non-lipoprotein cholesterol. This was detected on electroblots and quantified with a new dot blot assay using the colloidal gold-high density lipoprotein conjugates.  相似文献   

19.
Enhancing effect of taurine on CYP7A1 mRNA expression in Hep G2 cells   总被引:1,自引:0,他引:1  
Summary. Taurine has been reported to enhance cholesterol 7α-hydroxylase (CYP7A1) mRNA expression in animal models. However, no in vitro studies of this effect have been reported. The Hep G2 human hepatoma cell line has been recognized as a good model for studying the regulation of human CYP7A1. This work characterizes the effects of taurine on CYP7A1 mRNA levels of Hep G2 cells in a dose- and time-dependent manner. In the dose-dependent experiment, Hep G2 cells were treated with 0, 2, 10 or 20 mM taurine in the presence or absence of cholesterol 0.2 mM for 48 h. In the time-dependent experiment, Hep G2 cells were treated with 0 or 20 mM taurine for 4, 24 and 48 h with and without cholesterol 0.2 mM. Our data revealed that taurine showed time- and dose-response effects on CYP7A1 mRNA levels in Hep G2 cells. However, glycine – a structural analogue of taurine – did not have an effect on CYP7A1 gene expression. These results show that, in agreement to previous studies on animal models, taurine induces the mRNA levels of CYP7A1 in Hep G2 cells, which could enhance cholesterol conversion into bile acids. Also, Hep G2 cell line may be an appropriate model to study the effects of taurine on human cholesterol metabolism.  相似文献   

20.
4-Aminobiphenyl (4-ABP) and its analogue, 2-aminobiphenyl (2-ABP), were examined for their ability to induce oxidative DNA damage in Hep G2 cells. Using the alkaline comet assay, we showed that 2-ABP and 4-ABP (25-200 microM) were able to induce the DNA damage in Hep G2 cells. With both compounds, formation of intracellular reactive oxygen species (ROS) was detected using flow cytometry analysis. Post-treatment of 2-ABP and 4-ABP-treated cells by endonuclease III (Endo III) or formamidopyrimidine-DNA glycosylase (Fpg) to determine the formation of oxidized pyrimidines or oxidized purines showed a significant increase of the extent of DNA migration. This indicated that oxidative DNA damage occurs in Hep G2 cells after exposure to 2-ABP and 4-ABP. This assumption was further substantiated by the fact that the spin traps, 5,5-dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN), decreased DNA damage significantly. Furthermore, addition of the catalase (100 U/ml) caused a decrease in the DNA damage induced by 2-ABP or 4-ABP, indicating that H(2)O(2) is involved in ABP-induced DNA damage. Pre-incubation of the cells with the iron chelator desferrioxamine (DFO) (1mM) and with the copper chelator neocupronine (NC) (100 microM) also decreased DNA damage in cells treated with 200 microM 2-ABP or 200 microM 4-ABP, while the calcium chelator {1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester}(BAPTA/AM) (10 microM) decreased only DNA strand breaks in cells exposed to 4-ABP. This suggested that ions are involved in the formation of DNA strand breaks. Using RT-PCR and Western blotting, lower inhibition of the expression of the OGG1 gene and of the OGG1 protein was observed in cells treated with 4-ABP, and 2-ABP-treated cells showed a marked reduction in the expression of OGG1 gene and OGG1 protein. Taken together, our finding indicated the mechanisms of induced oxidative DNA damage in Hep G2 cell by 2-ABP and 4-ABP are different, although both tested compounds are isomers.  相似文献   

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