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R Gebhardt

Publications and source records attributed to R Gebhardt.

At least 73 records · Page 4Linked to original sources
PubMed1996

Glial fibrillary acidic protein as a marker of perisinusoidal stellate cells that can distinguish between the normal and myofibroblast-like phenotypes.

Glial fibrillary acidic protein (GFAP) has recently been shown to provide a marker for normal perisinusoidal stellate cells (PSC) in the liver. However, nothing was known so far about the changes in the intercellular abundancy of GFAP during transformation of PSC into myofibroblasts, a process characterized by marked changes in the expression of elements of the cytoskeleton. In order to address this question, we have used double-labelling immunofluorescence techniques for detecting smooth muscle alpha-actin (SMAA) and the intermediate filament proteins, GFAP, desmin and vimentin, taking advantage of the fact that PSC present in primary cultures of rat hepatocytes proliferate and transform. GFAP and vimentin were expressed in PSC throughout cultivation, while desmin which stained only about half of the PSC in early cultures was expressed in all GFAP-positive cells later on. The intensity of staining for GFAP in PSC transiently increased till the second day of cultivation followed by a decrease. At the 6th day of cultivation, staining for GFAP was seen only in the perinuclear region and as a faint rim at the contours of the cells. In contrast, SMAA, an established marker for transformed PSC, started to be expressed at the third day. Thereafter, immunoreactivity for SMAA increased continuously, indicating an inverse expression of GFAP and SMAA during transformation. These results indicate that GFAP, due to the plasticity of its expression, can discriminate between the normal untransformed and transformed phenotypes of PSC. Furthermore, they suggest a role of GFAP not only as a reliable marker, but also in the maintenance and/or function of the normal differentiated phenotype of liver PSC.

Actins↗
PubMed1995

Different proliferative responses of periportal and pericentral rat hepatocytes to hepatocyte growth factor.

Stimulation of DNA-synthesis by HGF was compared in cultured periportal and pericentral hepatocyte populations. Periportal hepatocytes showed a higher maximal stimulation of the BrdU labeling index than pericentral hepatocytes (7-fold vs 4-fold), while the stimulation for total hepatocytes was in between (5-fold). The glutamine synthetase-positive hepatocytes adjacent to the central veins responded much less to HGF than did the pericentral cells in general. These inherent differences in the growth potential of hepatocytes from different lobular localizations may contribute to the enhanced growth of hepatocytes in the periportal zone in normal and regenerating liver.

Animals↗
PubMed1995

Significant amounts of glycogen are synthesized from 3-carbon compounds in astroglial primary cultures from mice with participation of the mitochondrial phosphoenolpyruvate carboxykinase isoenzyme.

The incorporation was studied of the gluconeogenic substrates lactate, alanine, aspartate and glutamate into glycogen of astroglial primary cultures derived from mouse brain. The incorporation was inhibited by 3-mercaptopicolinate, an inhibitor of one of the characteristic gluconeogenic enzymes, phosphoenolpyruvate carboxykinase. Only the mitochondrial isoenzyme of phosphoenolpyruvate carboxykinase was detectable in the astroglial primary cultures. After the incubation of glucose-starved cells with medium containing a mixture of [6-3H]glucose and [U-14C]glucose, the newly synthesized glycogen showed a 3H/14C ratio which was approximately 15% less than the isotope ratio for the medium. The decrease of the isotope ratio was not significantly inhibited by 3-mercaptopicolinate, indicating a cycling of approximately 15% of the glucose to the level of the triose phosphates before its incorporation into astroglial glycogen. During the initial phase of glycogen resynthesis, the contribution of the gluconeogenic substrates appeared to be higher. This was in agreement with the accumulation of fructose 2,6-bisphosphate during refeeding. A participation of gluconeogenic substrates in glycogen metabolism was also detectable when the glycogen content was not changing significantly.

Animals↗
PubMed1995

Heterogeneous expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase genes in the rat liver lobulus.

We investigated the lobular localization and molecular level of expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene transcription of cholesterol 7 alpha-hydroxylase were predominant in pericentral hepatocytes of control rats, being 7.9-, 9.9-, and 4.4-fold higher than in periportal hepatocytes, respectively. Similar localization was found for sterol 27-hydroxylase: 2.9-, 2.5-, and 1.7-fold higher enzyme activity, mRNA, and gene transcription, respectively, was found in pericentral hepatocytes. Interruption of the enterohepatic circulation with colestid resulted in upregulation of these parameters for both enzymes, as a consequence of stimulated gene expression mainly in the periportal zone. In contrast, mRNA levels and gene transcription of 3-hydroxy-3-methylglutaryl CoA reductase showed opposite lobular distribution. Selective periportal expression for the latter was enhanced, but remained local, after colestid treatment. In situ hybridization showed unambiguously that cholesterol 7 alpha-hydroxylase mRNA is localized exclusively in the pericentral zone and that sterol 27-hydroxylase mRNA is expressed preferentially in the pericentral region, though less pronounced. Administration of colestid led to expression of both genes within a larger area of the liver lobulus. In conclusion, we suggest that cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase are coordinately regulated by the bile acid gradient over the lobulus, resulting in predominant expression in the pericentral zone. Opposite lobular localization of cholesterol and bile acid synthesis provides an alternative view to interregulation of these metabolic pathways.

Animals↗
PubMed1995

Heterogeneous hepatocellular expression of glutamine synthetase in developing mouse liver and in testicular transplants of fetal liver.

BACKGROUND: Glutamine synthetase is exclusively expressed in pericentral hepatocytes in mammalian liver, but its regulation mechanism is still largely unknown. EXPERIMENTAL DESIGN: Heterogeneous expression of glutamine synthetase was examined in detail during mouse liver development by immunohistochemistry and by in situ hybridization. Heterogeneous expression of this enzyme was also analyzed in immature liver fragments transplanted to an ectopic site where no portal blood flow exists. RESULTS: At 18.5 days of gestation, a random, spotty distribution of low levels of glutamine synthetase mRNA was observed all over the liver parenchyma, but the enzyme protein was not detectable immunohistochemically in the liver at any fetal stage. Glutamine synthetase and its mRNA began to be heterogeneously expressed in pericentral hepatocytes 2 to 3 days after birth, when glycogen accumulation in the liver parenchyma was rather homogeneous. In the early postnatal development, a mosaic distribution of positive and negative hepatocytes with respect to glutamine synthetase protein and mRNA was noted around the central veins. Subsequently, mRNA distribution gradually became continuous, although some hepatocytes still lacked protein, indicating partial regulation at the translational level. When fetal liver fragments that had not yet heterogeneously expressed glutamine synthetase were cultured under the testis capsule of male mice, only pericentral hepatocytes expressed this enzyme after 2 months. However, the distribution of glutamine synthetase protein- and mRNA-positive hepatocytes around the central veins was patchy rather than continuous, as in perinatal livers. CONCLUSIONS: These results support the importance of local interactions of hepatocytes with intrahepatic cell populations and/or structural elements. Furthermore, they demonstrate that the capacity for the positional expression of glutamine synthetase is already established at a fetal age before expression of glutamine synthetase can be detected.

Animals↗
PubMed1994

Isolation of rat pgp3 cDNA: evidence for gender and zonal regulation of expression in the liver.

Distinct differences exist in the function and regulation of the individual p-glycoprotein (pgp) members in many species. In order to study regulation of individual pgp mRNA isoforms it is, therefore, necessary to have probes that can distinguish between the various pgp isoforms. However, to date few studies examining hepatic gene expression in rat liver have used pgp gene specific probes. Towards this end we screened a cDNA library constructed from a normal rat liver with a human pgp1 cDNA and isolated a partial cDNA for class III pgp, rat pgp3. By comparison of the sequence of this new rat pgp3 cDNA with genomic and cDNA sequences for rat pgp1 and rat pgp2 we selected oligonucleotide probe sequences that would allow us to differentiate between the highly homologous rat pgp2 and pgp3 genes on Northern blots and by polymerase chain reaction (PCR). We found that pgp3, for both male and female rats, was the predominant form of pgp expressed in normal rat liver with males consistently expressing several-fold lower levels of pgp3 than females. Because many genes are zonally expressed in the hepatic acinus we examined the possibility that pgp3 might show heterogeneous distribution as well. We found, by in situ hybridization of paraformaldehyde-fixed rat liver sections that pgp3 was distributed non-uniformly across the hepatic acinus with a gradient that showed the highest expression toward the terminal hepatic venule. We confirmed this finding by selectively isolating hepatocytes from either the terminal hepatic venular or periportal zones using a digitonin/collagenase perfusion procedure. Application of specific pgp3 PCR primers to RNA isolated from hepatocytes from these areas confirmed that pgp3 mRNA was the predominant form in the hepatocytes surrounding the terminal hepatic venule. Finally, we examined pgp3 expression in a variety of tissues by Northern blot analysis and found that pgp3 was most highly expressed in the liver and gastrointestinal tract (with a gradient of expression from small to large intestine), while low levels were found in the kidney, heart and brain. Pgp3 mRNA was undetectable in the adrenal gland and in skeletal muscle. In summary, using rat pgp gene specific oligonucleotide probes we found that pgp3 gene expression is regulated by anatomic location with the highest mRNA expression in organs that are involved in drug detoxification. Our results also demonstrate heterogeneity of hepatic rat pgp3 gene expression, which is influenced by both gender and by acinar location.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1994

Different drug metabolizing capacities in cultured periportal and pericentral hepatocytes.

Isolated and cultured periportal (PP) and pericentral (PC) hepatocytes were used for studying the acinar distribution of several phase I and phase II drug metabolizing reactions and their induction by phenobarbital (PB) and 3-methylcholanthrene (MC) or a combination thereof. Ethoxycoumarin-o-deethylase (EC 1.14.14.1) (ECOD) activity was found to predominate in PC hepatocytes even after induction with MC and PB. Metabolism of biphenyl to a monosulfated product also predominated in PC hepatocytes as did the conversion of harmine to harmine glucuronide and sulfate. In contrast, metabolism of lonazolac was not zonated. The metabolism of all three substrates declined during cultivation for 24 hr and was differentially induced (biphenyl and harmine) or not affected (lonazolac) by MC or PB. Metabolic heterogeneity was best maintained by the combination of MC and PB indicating that the zonal differences are due to a specific balance of phase I and phase II reactions. Glutathione-S-transferase (GST) activities against 1-chloro-2, 4-dinitrobenzene (CDNB) were higher in PC hepatocytes and remained so after induction with PB. In contrast, GST activities against 1,2-dichloro-4-nitrobenzene (DCNB) were almost twice as high in PP cells but equilibrated due to a spontaneous increase during cultivation, particularly in PC hepatocytes. In the presence of PB or both, MC and PB, induction of the activity against DCNB occurred exclusively in the PP hepatocytes. The distribution of the GST subunits Ya and Yb1 roughly corresponded to the pattern of GST activities against CDNB and DCNB, respectively. These results agree with earlier reports demonstrating that PP and PC hepatocytes show different patterns of phase I and phase II drug metabolizing enzymes which are maintained during short-term cultivation. In vitro induction of these activities does not result in equilibration but rather maintenance or even pronounciation of these zonal differences.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1994

Inhibition of cholesterol biosynthesis by allicin and ajoene in rat hepatocytes and HepG2 cells.

Exposure of primary rat hepatocytes and human HepG2 cells to allicin and ajoene resulted in the concentration-dependent inhibition of cholesterol biosynthesis at different steps of this metabolic pathway. At low concentrations of ajoene sterol biosynthesis from [14C]acetate in rat hepatocytes was decreased by 18% with an IC50-value of 15 microM, while allicin was almost uneffective. In HepG2 cells, both compounds significantly inhibited sterol biosynthesis by 14% and 19% with IC50-values of 7 and 9 microM for allicin and ajoene, respectively. This inhibition was exerted at the level of HMG-CoA-reductase as revealed by the absence of inhibition, if [14C]acetate was replaced by [14C]mevalonate as a precursor, and by direct determination of enzyme activity. At somewhat higher concentrations inhibition of cholesterol biosynthesis by both, allicin and ajoene, was also observed at late steps resulting in the accumulation of the precursor lanosterol. Alliin instead was completely inactive. In the case of allicin, small amounts of dihydrolanosterol and 7-dehydrocholesterol were formed at intermediate concentrations of 5-10 microM. From these results it is concluded that a major point of inhibition at the late steps occurs at the level of lanosterol 14 alpha-demethylase.

Acetates↗
PubMed1994

Overexpression of glutamine synthetase in human primary liver cancer.

BACKGROUND/AIMS: We have identified several clones specifically expressed during malignant cell proliferation by screening a complementary DNA library constructed from a human primary liver cancer with subtractive probes. One clone was identified as the glutamine synthetase (GS) transcript. Its expression is tightly regulated during development, especially in the hepatic lobule. Because this enzyme is involved in nitrogen homeostasis, it might contribute to tumor development/progression in primary liver cancer. METHODS: We compared the expression of GS messenger RNA (mRNA) and protein in tumorous and nontumorous liver from 34 patients with primary liver cancers, using a combination of Northern blot, dot blot, western blot, and determination of GS enzyme activity. RESULTS: GS mRNA was higher in tumors versus nontumors in 23 of 34 primary liver cancer samples. GS activity was higher in 6 of 8 selected primary liver cancer samples with high RNA levels. GS protein levels were proportional to enzyme activity. A major GS transcript of 2.8 kilobase was detected by Northern blotting and sequencing. This comprised the minor 1.8-kb transcript and a long 3' untranslated region; the latter contained an AT-rich zone, fully conserved in the chicken, mouse, and rat, which might be important for stability. CONCLUSIONS: Our results show an overexpression of GS in human primary liver cancers and, thus, point to its potential involvement in hepatocyte transformation.

Amino Acid Sequence↗
PubMed1994

Heterogeneous (positional) expression of hepatic glutamine synthetase: features, regulation and implications for hepatocarcinogenesis.

Glutamine synthetase expression in liver parenchyma is restricted to a small population of pericentral hepatocytes surrounding the central veins. Studies on the development of this heterogeneous (positional) gene expression and of the changes observed in response to experimental alterations of liver physiology or manipulations of hepatocytes in culture have revealed that it is dependent on cell-cell and cell-matrix interactions rather than on the levels of hormones and other modulating factors. The considerable stability of GS expression may point to further events leading to a defined differentiated GS+ phenotype. Observations during experimental hepatocarcinogenesis indicate that strong GS expression may be used for tracing hepatocellular lineages during preneoplastic and early neoplastic stages. Furthermore, these studies suggest a relationship between the GS+ phenotype and enhanced growth of these lesions. Future studies should help to define the diagnostic value of GS and its significance for new chemotherapeutic strategies.

Adipose Tissue↗
PubMed1994

Implications of the concepts "coping" and "quality of life" for criteria of course and outcome.

The Berlin Coping Study is a prospective longitudinal project with schizophrenic patients investigating the relationship between coping strategies and course and outcome. We find that some psychological issues relevant for both clinical practice and psychiatric research, and especially for pharmacological therapy, have been rather neglected up to now. The investigation of coping strategies leads to accentuating the patients' subjective individual views concerning their demands, goals, and coping activities in daily living and their schizophrenic illness. The concept of quality of life is increasingly receiving attention in various domains of professional care and it is very relevant to the issues discussed here. Consequences of this approach for therapy and research are outlined.

Adaptation, Psychological↗
PubMed1994

Promotion of preneoplastic foci in rat liver with 2,3,7,8-tetrachlorodibenzo-p-dioxin, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin and a defined mixture of 49 polychlorinated dibenzo-p-dioxins.

In a two-stage initiation-promotion experiment the hypothesis was investigated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TE), calculated from data of CYP1A induction in hepatocytes in primary culture, or international TCDD equivalents (ITE) are useful for evaluating the tumor-promoting potency of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) and of a defined mixture (M2) of 49 polychlorinated dibenzo-p-dioxins (PCDDs) in comparison with TCDD. Therefore, female Wistar rats were treated with an initiating dose of N-nitrosomorpholine, and subsequently received daily doses of 2, 20 and 200 ng TCDD/kg or equivalent doses of HpCDD or M2, based on TE values. After a promotion phase of 13 weeks, hepatic PCDD levels, CYP1A activity and the relative hepatic volume of adenosinetriphosphatase-negative or glutathione S-transferase P-positive preneoplastic foci were determined. After logarithmic transformation, linear PCDD level-response relationships were obtained for induction of CYP1A activity with TCDD, HpCDD or M2. Based on TE values, inducing potencies of both HpCDD and M2 were over-estimated at higher doses, whereas induction was approximately equivalent at the lowest dose. The best fit of PCDD level-response relationships of relative hepatic volumes of preneoplastic lesions was achieved using a four-parameter logistic model. Significantly different functions were calculated for promotion with TCDD or HpCDD. It is concluded that (i) different PCDD level-response relationships exist for the induction of hepatic CYP1A activity and the promotion of preneoplastic liver foci, and (ii) that TE or ITE factors provide only a rough estimate of the tumor-promoting potency of a PCDD mixture but may overestimate the risk from exposure to higher-chlorinated 2,3,7,8-substituted congeners such as HpCDD.

Animals↗
PubMed1994

Changes in distribution and activity of glutamine synthetase in carbon tetrachloride-induced cirrhosis in the rat: potential role in hyperammonemia.

Cirrhosis induced in rats by carbon tetrachloride was used to study alterations in the activities and lobular distribution of carbamoylphosphate synthetase and glutamine synthetase. Specific activity of carbamoylphosphate synthetase in cirrhotic subjects was decreased to 70% of controls. Staining was homogeneous within micronodular areas, but varied from area to area and generally showed a decreased intensity. Specific activity of glutamine synthetase and the size of the glutamine synthetase-positive area were decreased to 20% and less of controls. Glutamine synthetase-positive hepatocytes were rare and scattered at the periphery of nodular areas and within fibrous septa, the normal association with the central veins being widely lost. Rarely, complete micronodules showed a slight homogeneous staining for glutamine synthetase. Arginase activity was not affected, whereas glutaminase activity was enhanced by 50%. Serum levels of ammonia were elevated more than 2-fold and those of glutamine by 30%. In contrast, urea levels tended to be slightly diminished. Serum ammonia levels showed a clear negative correlation with the specific activity of glutamine synthetase and the size of the glutamine synthetase-positive area. Furthermore, blood urea levels correlated with the sum of ammonia and glutamine concentrations, but not with each of these substrate concentrations alone. These data suggest that the changes in activity and distribution of glutamine synthetase contribute to hyperammonemia in cirrhosis. Despite a reduced activity of the initial enzyme of the urea cycle, urea synthesis is not diminished accordingly. This may be due to an enhanced flux caused by the elevated blood level of ammonia and an increased hydrolysis of glutamine, because of higher levels of glutaminase.

Ammonia↗
PubMed1993

Cytochrome P-450-induced cytotoxicity of mitoxantrone by formation of electrophilic intermediates.

Recent studies of our group have shown that the oxidation of the substituted anthraquinone skeleton is involved in the biotransformation of mitoxantrone. In this report the importance of this process with regard to the mode of action of the drug is investigated. This communication describes a new high performance liquid chromatography separation for mitoxantrone and its metabolites allowing the direct coupling of high performance liquid chromatography to mass spectrometry. Application of this technique to bile of mitoxantrone-treated pigs reveals the formation of several metabolites in addition to the drug-derived compounds found in urine. Seven biliary metabolites are identified as thioether derivatives of mitoxantrone and its side chain oxidation products. Independent synthesis and structural elucidation of 3 thioether conjugates of the drug provides unequivocal evidence that the hydroquinone moiety of mitoxantrone is the site of reaction with glutathione. Furthermore, the formation of the thioether conjugates in HepG2 hepatoma cells and in rat hepatocytes during cell incubations is demonstrated. Inhibition of cytochrome P-450 with metyrapone prevents the formation of the thioether conjugates and leads to a complete loss of the cytotoxicity of mitoxantrone in HepG2 cells and rat hepatocytes up to concentrations of 200 to 400 microM thereby indicating that mitoxantrone has a negligible effect by itself. Rat hepatocytes were found to be more susceptible for the oxidation-induced cytotoxicity than HepG2 cells. These results demonstrate that the acute cytotoxicity of mitoxantrone depends on prior oxidation of its 1,4-dihydroxy-5,8-diaminoanthraquinone moiety.

Animals↗
PubMed1993

Glycogen in astrocytes: possible function as lactate supply for neighboring cells.

In order to contribute to the elucidation of the function of astrocyte glycogen in brain, studies on the fate of the glucosyl residues of glycogen were carried out on astroglia-rich primary cultures derived from the brains of newborn rats. On glucose deprivation astroglial cells rapidly deplete their glycogen. In contrast to the situation with hepatocytes, only lactate, but not glucose, is detectable in the medium surrounding the astroglial cells. Besides glucose, astroglial cultures can also use mannose as a substrate for the synthesis of glycogen and the generation of lactate. Although mannose-fed astroglial cells contain glucose-6-phosphate, they do not release a measurable amount of glucose into the culture medium. Instead of glucose the astroglial cells release high amounts of lactate into the culture medium. Gluconolactone or 2-deoxyglucose which prevent glycogen breakdown in astroglial cells after glucose deprivation, allow to discriminate between lactate generated from glycogen and lactate from other sources. The amount of lactate found in the medium in the absence of gluconolactone (or 2-deoxyglucose) exceeds the amount found in the presence of either compound by the lactate equivalents calculated to be contained in the cellular glycogen. In conclusion, glycogen in astrocytes can be considered as a store for lactate rather than for glucose.

Animals↗
PubMed1993

Dose response effects of 2-acetylaminofluorene on DNA damage, cytotoxicity, cell proliferation and neoplastic conversion in rat liver.

This study measured the effect of precise doses of 2-acetylaminofluorene (AAF) in inducing DNA damage, functional changes and neoplastic conversion in rat liver. Groups of male F344 rats at 9 weeks of age were exposed to cumulative doses of 0.5 or 2.0 mmol AAF per kg body weight given by gavage daily 5 days per week over an 8-week period and maintained with no further exposure for up to 8 weeks. Administration of AAF resulted in the formation of N-deoxyguanosin-(8-yl)-2-aminofluorene in liver DNA in relationship to dose. In centrilobular hepatocytes the zone of glutamine synthetase-expressing cells was reduced by exposure. By 8 weeks, but not at 4 weeks, the higher of the two doses of AAF provoked an increase in cell proliferation measured by immunohistochemical incorporation of bromodeoxyuridine. Altered hepatocellular foci expressing the placental form of glutathione transferase were induced by the high dose of AAF at 4 weeks, but not at the low dose. At 8 weeks the incidence of foci at the high dose was 79 times that induced by the low dose. These foci were highly proliferative. In animals exposed to AAF for 8 weeks and maintained for 4 weeks with no exposure, DNA adducts decreased by 80% and cell proliferation subsided by 80%, although the glutamine synthetase zone remained diminished. After discontinuation of AAF, the number of foci diminished by 50% and their proliferation subsided by 80% at 4 weeks, indicating a phenotypic reversion of many foci. With this protocol of administration of precise doses of AAF, we have established non-linearity of effects and a lack of correlation between DNA adduct formation and induction of cellular lesions. We suggest that doses in the range of those reported can be used to study the contribution of epigenetic and genotoxic effects in carcinogenesis and to study threshold events.

2-Acetylaminofluorene↗
PubMed1993

Identification and functional characterization of regulatory elements of the glutamine synthetase gene from rat liver.

Hepatic glutamine synthetase (GS) shows a unique expression pattern limited to a few hepatocytes surrounding the terminal hepatic veins. Starting from the genomic clone of the rat GS gene, lambda GS1 [Van de Zande, L. P. G. W., Labruyère, W. T., Arnberg, A. C., Wilson, R. H., Van den Bogaert, A. J. W., Das, A. T., Frijters, C., Charles, R., Moorman, A. F. M. & Lamers, W. H. (1990) Gene (Amst.) 87, 225-232] additional genomic clones containing up to 9 kb of 5'flanking region were isolated in order to characterize cis-acting elements involved in the regulation of GS expression. Sequence analysis of the 5'flanking region up to -2520 bp revealed a putative AP2-binding site at -223 bp and a second GC box at -2343 bp in addition to the canonical TATA, CCAAT and GC boxes found proximal to the transcription-start site. A possible negative glucocorticoid-responsive element (GRE) and regions with very weak similarity to a GRE and to a known silencer element were noted at -506 bp, -406 bp and at -798 bp, respectively. Within the sequenced part of the 5'flanking region no known regulatory elements associated with liver-specific gene expression were found except for a putative HNF3-binding site at -896 bp. Functional analysis by transient transfection assays using constructs with the pSSCAT or the pXP1 vector revealed that the elements present within the first 153 bp and particularly the first 368 bp of upstream sequence constitute an active promoter the activity of which is decreased by additional sequences up to -2148 bp. The presence of dexamethasone led to a 2-4-fold increase in the promoter activity of all these constructs. Using the heterologous truncated thymidine-kinase-gene promoter of the plasmid pT81-luc a strong enhancer element was located between -2520 bp and -2148 bp. Its activity was not affected by dexamethasone but was negatively influenced by flanking sequences in both directions. This enhancer was also effective with the homologous GS promoter (-153 to +59 bp) and the heterologous full thymidine-kinase-gene promoter (pT109luc). No further enhancers were found up to -6200 bp. Using the same approach, a second enhancer was found between +259 bp and +950 bp within the first intron. Deoxyribonuclease-I hypersensitivity studies confirmed the presence of a hypersensitive site between +350 bp and +550 bp and suggested a second site between +850 bp and +1200 bp.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗