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F Oesch

Publications and source records attributed to F Oesch.

At least 181 records · Page 10Linked to original sources
PubMed1992

Carbohydrate metabolism in human renal clear cell carcinomas.

BACKGROUND: Renal cell carcinomas can be subclassified into clear cell carcinomas, chromophobe cell carcinomas, chromophilic cell carcinomas, and oncocytomas. Previous studies, in which no distinction among the different types of renal cell tumors and their grades of malignancy was performed, showed that these tumors had high glycolytic rates. EXPERIMENTAL DESIGN: The carbohydrate metabolism of control human kidney samples and renal clear cell carcinomas with different degrees of cytologic malignancy (G I, G II, and G III) was studied by determining the glycogen and glucose-6-phosphate levels and the activities of key enzymes involved in glycolysis (hexokinase, glucokinase, pyruvate kinase), gluconeogenesis (glucose-6-phosphatase, fructose-1,6-diphosphatase), and the pentose phosphate pathway (glucose-6-phosphate dehydrogenase) in these tissues and compared with those of a limited number of chromophilic cell carcinomas, chromophobe cell carcinomas, and oncocytomas. RESULTS: The glycogen and glucose-6-phosphate levels were significantly higher in G I, G II, and G III clear cut carcinomas than in control kidneys; glucokinase, hexokinase, and glucose-6-phosphate dehydrogenase activities remained unchanged, pyruvate kinase activity was enhanced, and glucose-6-phosphatase as well as fructose-1,6-diphosphatase activities were strongly reduced when compared with control kidney values. In chromophilic cell carcinomas glycogen content, glucose-6-phosphate dehydrogenase, and pyruvate kinase activities were elevated, while fructose-1,6-diphosphatase activity was reduced. In chromophobe cell carcinomas glycogen content was elevated and gluconeogenesis was reduced, whereas glycolysis was not activated. In oncocytomas glycogen was not detected and glucose-6-phosphate dehydrogenase, pyruvate kinase, and fructose-1,6-diphosphatase activities remained unchanged. CONCLUSIONS: It has been demonstrated that a series of characteristic changes occur in the carbohydrate metabolism of renal clear cell carcinomas: glycogen and glucose-6-phosphate levels increase, glycolysis is activated, and gluconeogenesis is reduced. Furthermore, the alterations of the carbohydrate metabolism within clear cell carcinomas are clearly distinct from those observed in chromophilic cell carcinomas, chromophobe cell carcinomas, and oncocytomas.

Adenocarcinoma↗
PubMed1991

An impaired peroxisomal targeting sequence leading to an unusual bicompartmental distribution of cytosolic epoxide hydrolase.

To gain an understanding of the mechanism by which the subcellular distribution of cytosolic epoxide hydrolase (cEH) is directed, we have analyzed the carboxy terminal region of rat liver cEH by means of cDNA cloning to define the structure of its possible peroxisomal targeting sequence (PTS). Purified cEH was subjected to peptide analysis following endoproteinase Glu-C digestion and HPLC-separation of the fragments. The obtained sequence information was used to perform PCR experiments resulting in the isolation of a 680 bp cDNA clone encoding the carboxy terminus of cEH. The deduced amino acid sequence displays a terminal tripeptide Ser-Lys-Ile which is highly homologous to the PTS (Ser-Lys-Leu) found in other peroxisomal enzymes. This slight difference appears to be sufficient to convert the signal sequence into an impaired and therefore ambivalent PTS, directing the enzyme partly to the peroxisomes and allowing part to reside in the cytosol.

Amino Acid Sequence↗
PubMed1991

Influence of the level of cytosolic epoxide hydrolase on the induction of sister chromatid exchanges by trans-beta-ethylstyrene 7,8-oxide in human lymphocytes.

trans-beta-Ethylstyrene 7,8-oxide, a substrate of cytosolic epoxide hydrolase, and 4-fluorochalcone oxide, an inhibitor of this enzyme, were investigated on induction of sister chromatid exchanges (SCE) in human lymphocytes. Both epoxides enhanced the frequency of SCE. 4-Fluorochalcone oxide at low concentration (2.5 microM) inhibited cytosolic epoxide hydrolase activity towards trans-beta-ethylstyrene 7,8-oxide in lymphocytes by 74% and had no effect on glutathione transferase activity using this substrate. At this concentration it did not induce SCE itself, but it potentiated the effect of trans-beta-ethylstyrene 7,8-oxide several fold. In lymphocytes from different subjects, the number of SCE induced by a low concentration of trans-beta-ethylstyrene 7,8-oxide correlated negatively with the individual cytosolic epoxide hydrolase activity (r = -0.72; -0.73 in two series of experiments). The number of SCE induced by a high concentration of trans-beta-ethylstyrene 7,8-oxide did not correlate with cytosolic epoxide hydrolase activity (r = 0.004; -0.24), but a negative correlation was found with glutathione transferase activity (r = -0.50). This finding is consistent with the results of biochemical studies in lymphocytes in which we determined the relative contribution of cytosolic epoxide hydrolase and glutathione transferase to the metabolism of trans-beta-ethylstyrene 7,8-oxide at varying substrate concentrations. The study demonstrates that the level of genotoxic effects induced in human lymphocytes is influenced by the individual level of detoxifying enzymes. At low concentrations, cytosolic epoxide hydrolase was more important than glutathione transferase activity.

Cells, Cultured↗
PubMed1991

The in vitro metabolic activation of dibenz[a,h]anthracene, catalyzed by by rat liver microsomes and examined by 32P-postlabelling.

DNA has been incubated in vitro with dibenz[a,h]anthracene (DB[a,H]A) and the related 5,6-diol and 3,4-diol in the presence of 3-methylcholanthrene- or Aroclor 1254-induced rat liver microsomes. After incubation, the DNA was extracted and examined for the presence of aromatic adducts using the nuclease P1 modification of the 32P-postlabelling technique. The maps of PEI-cellulose plates and autoradiography showed that 92% of the radioactivity contained in DB[a,h]A-DNA adduct spots is derived from the related 3,4-diol and that about 50% of the adducts may be formed following the conversion of this diol to the bay-region anti- and syn-3,4-diol 1,2-oxides.

Animals↗
PubMed1991

Fjord- and bay-region diol-epoxides investigated for stability, SOS induction in Escherichia coli, and mutagenicity in Salmonella typhimurium and mammalian cells.

The fjord-region diol-epoxides of benzo(c)phenanthrene combine high mutagenic and carcinogenic activity with low chemical reactivity. To study whether this is a unique property of these compounds or a more general characteristic of fjord-region diol-epoxides, we have synthesized the anti- and syn-diastereomers of r-9,t-10-dihydroxy-11,12-oxy-9,10,11,12-tetrahydrobenzo(c)chrysene and r-11-t-12-dihydroxy-13,14-oxy-11,12,13,14-tetrahydrobenzo(g)chrysene. These compounds as well as the anti- and syn-diastereomers of the fjord-region diol-epoxides of benzo(c)phenanthrene and of the bay-region diol-epoxides of phenanthrene, chrysene, and benzo(a)pyrene were investigated for their half-lives in a physiological buffer, for their mutagenicity in Salmonella typhimurium (reversion of the his- strains TA97, TA98, TA100, and TA104), for induction of SOS response in Escherichia coli (SOS chromotest in strain PQ37) and for their mutagenicity in V79 Chinese hamster cells (acquisition of resistance to 6-thioguanine). All six of the investigated fjord-region diol-epoxides were more stable in physiological buffer at 37 degrees C (t1/2 greater than 2 h) than the six bay-region diol-epoxides (t1/2 = 0.011 to 1.2 h). The half-lives correlated negatively with the calculated delta Edeloc values for the formation of the benzylic carbocations, and were consistently shorter for the syn- than for the corresponding anti-diastereomer. All fjord-region diol-epoxides showed extraordinarily high activity in all six genotoxicity assays used. In mammalian cells, the anti-diol-epoxide of benzo(c)chrysene was 8.6 and 12 times more active than the anti-diol-epoxides of benzo(c)phenanthrene and benzo(a)pyrene, respectively, which were the most potent mutagens among the reference compounds. The other three newly available fjord-region diol-epoxides were also markedly more mutagenic in mammalian cells than the reference compounds. Whereas the syn-diastereomers of the simple bay-region diolepoxides were clearly less mutagenic in mammalian cells than the corresponding anti-diastereomers, the differences in potency between diastereomers were small for the fjord-region diol-epoxides. In conclusion, the diol-epoxides of benzo(c)phenanthrene are not unique in their high biological activities. The two newly available diastereomeric pairs of fjord-region diol-epoxides of benzo(g)- and benzo(c)chrysene proved to be even more active. For one of them, the diol-epoxides of benzo(g)chrysene, the delta Edeloc value for the formation of the benzylic carbocation is lower than for the benzo(c)phenanthrene diol-epoxides, for the other it is higher.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

DNA binding, adduct characterisation and metabolic activation of aflatoxin B1 catalysed by isolated rat liver parenchymal, Kupffer and endothelial cells.

In vitro studies with rat liver parenchymal, Kupffer and endothelial cells isolated from male Sprague-Dawley rats were undertaken to investigate cell-specific bioactivation of aflatoxin B1, DNA binding and adduct formation. In the mutagenicity studies, using homogenates of all three separated liver cell populations (co-incubated with NADP+ and glucose-6-phosphate as cofactors for the cytochrome P-450 monooxygenase system) parenchymal, Kupffer and endothelial cells were able to activate aflatoxin B1 to a metabolite mutagenic to Salmonella typhimurium TA 98. In the case of nonparenchymal cells (i.e. Kupffer and endothelial cells) 10-fold higher concentrations of aflatoxin B1 had to be used to obtain a similar number of revertants to that observed with parenchymal cells. Induction studies with Aroclor 1254 led to a striking decrease in the activation of aflatoxin B1 in parenchymal cells, whereas nonparenchymal cells had a slightly enhanced metabolic activation capacity for aflatoxin B1. Metabolism studies with microsomes from induced and noninduced cells using testosterone as substrate revealed comparable results: after induction with Aroclor 1254, parenchymal cells showed a 60% decrease in the formation rate of 2 alpha-hydroxytestosterone, whereas the formation rate of this metabolite remained unchanged in nonparenchymal cells; 2 alpha-hydroxytestosterone is specifically formed by cytochrome P-450 IIC11, which also catalyses the activation of aflatoxin B1 to its epoxide. When freshly isolated, intact cells were incubated with tritiated aflatoxin B1, a dose-dependent aflatoxin B1 binding to DNA in parenchymal and nonparenchymal cells was observed. HPLC analysis of DNA acid hydrolysates of all three cell types showed the major adduct to be 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1.

Aflatoxin B1↗
PubMed1991

Differential stabilization of cytochrome P-450 isoenzymes in primary cultures of adult rat liver parenchymal cells.

Cytochrome P-450 dependent hydroxylation of testosterone was measured in 7-day-old cultures of primary rat liver parenchymal cells. Determinations were carried out in monocultures of parenchymal cells and co-cultures of parenchymal cells with rat liver nonparenchymal epithelial cells, or mouse embryo fibroblasts. In the monoculture system, testosterone metabolism was drastically reduced and hardly measurable after 7 days in culture. In the co-culture systems, individual P-450 isoenzymes were stabilized on different levels. P-450s p and presumably c were well preserved, P-450 a was reduced but clearly measurable, P-450 h was totally lost whereas P-450s b and e were not measurable after 7 days (the activities of these isoenzymes however were already low in freshly isolated parenchymal cells). The results were independent of the cell line used for co-cultivation and of the method of parenchymal cell isolation, that is whether collagenase or EDTA was used as the agent for dissociating the cells from the liver. The results showed that the co-cultivation of liver parenchymal cells with other nonparenchymal cells significantly improved the differentiated status of the former. In this cell culture system however, not every parameter was equally well stabilized.

Animals↗
PubMed1991

Tissue-specific expression and differential inducibility of several microsomal epoxide hydrolase mRNAs which are formed by alternative splicing.

mRNA was isolated from several rat tissues and subjected to either the nuclease S1 or the RNAseA protection assay with probes covering the 5' end, the middle part, and the 3' end of the microsomal epoxide hydrolase (mEHb) cDNA. Whereas probes directed against the latter two regions yielded a single protected fragment, a probe which covered base pairs -148 to +453 (+1 defines the start of protein biosynthesis) yielded two protected fragments. The degree of protection of the two fragments was strongly dependent on the tissue from which the mRNA had been isolated. Thus at least two mEHb mRNAs which differ at their 5' ends are differentially expressed in various tissues. In addition the mRNAs corresponding to the two protected fragments were clearly differentially inducible by Aroclor 1254 treatment of the animals. Primer extension analysis with hepatic RNA from untreated animals yielded three primer-extended products corresponding to three mRNAs which differ at their 5' ends. As already seen in the nuclease S1 protection assay, one of the mRNAs was induced by Aroclor 1254 treatment. The expression of the two other mRNAs was either repressed or stable. Thus besides the mRNA already characterized for mEHb, there are at least two other mEHb mRNAs. This result was confirmed by the isolation of a mEHb cDNA which is completely distinct in its sequence in a region just preceding the initiation codon for protein biosynthesis. From that point on, the sequence of our cDNA becomes identical to the published mEHb cDNA. This point corresponds exactly to the start of exon 2 as determined from the genomic sequence. Thus the region where both mEHb cDNAs differ is encoded by two different exons 1, which are joined to exon 2 by alternative splicing. The tissue-specific expression and the different inducibility of the various mEHb mRNAs might indicate that their expression is governed by different promoters.

Animals↗
PubMed1991

Selective induction of bilirubin UDP-glucuronosyl-transferase by perfluorodecanoic acid.

Differential effects of perfluorodecanoic acid (PFDA) on rat liver UDP-glucuronosyltransferase isoenzymes have been observed after a single i.p. administration of the compound to young male Sprague-Dawley rats. (1) Bilirubin glucuronidation was induced 2-fold. The induced state was stable for at least 3 weeks. (2) Glucuronidation of 1-naphthol, morphine and testosterone was decreased to half of the control values. These decreases were maximal after 12 days but all three activities returned to normal levels after 3 weeks. (3) Immunoblotting experiments indicated that the differential effects of PFDA on UDP-glucuronosyltransferase activities were due to modulation of enzyme protein concentrations rather than activation/inactivation mechanisms. With respect to its influence on UDP-glucuronosyltransferase isoenzymes, PFDA may be classified as a clofibrate-type inducer. The persistence of the induction after a single application however is unique among peroxisome proliferators and therefore PFDA may be a useful, elective inducer of bilirubin glucuronidation.

Animals↗
PubMed1991

Relationship between mutagenicity and DNA adduct formation in mammalian cells for fjord- and bay-region diol-epoxides of polycyclic aromatic hydrocarbons.

Chinese hamster V79 cells were treated with the anti- and syn-diastereomers of the bay- or fjord-region diol-epoxides of four polycyclic aromatic hydrocarbons, namely benzo[a]pyrene (BP), benzo[c]chrysene (BcC), benzo[g]chrysene (BgC) and benzo[c]phenanthrene (BcPh). The frequency of induction of 6-thioguanine-resistant mutations was determined, and the extent of formation of DNA adducts was measured by 32P-postlabelling. When expressed as mutation frequency per nanomoles compound per millilitre incubation medium, this group of chemicals expressed a 160-fold range in potency. In agreement with previous experimental studies, the anti-diol-epoxide of BcC was highly mutagenic, inducing in excess of 3 x 10(4) mutations/10(6) cells per nmol compound/ml. The mutagenic activities of the anti- and syn-diol-epoxides of BP were 10- and 100-fold lower, respectively. Both diol-epoxides of BgC, the syn-BcC and the anti-BcPh derivatives were also highly mutagenic, and only the syn-BcPh diol-epoxide was less mutagenic than the anti-diol-epoxide of BP. Determination of the levels of DNA adducts formed by the diol-epoxides indicated that the most mutagenic compounds were the most DNA reactive, although the fjord-region diol-epoxides gave rise to more complex patterns of adducts than those of the BP diol-epoxides. When the mutagenicity results were expressed as mutations per femtomoles total adducts formed, all compounds showed similar activities. Thus the potent mutagenicity of the fjord region diol-epoxides appears to be due to the high frequency with which they form DNA adducts in V79 cells, rather than to formation of adducts with greater mutagenic potential.

Animals↗
PubMed1991

Regiospecific oxidation of polycyclic aromatic dihydrodiols by rat liver dihydrodiol dehydrogenase.

Rat liver dihydrodiol dehydrogenase (DDH, E.C. 1.3.1.20) has recently been shown to oxidize the highly carcinogenic benz[a]anthracene-3,4- dihydrodiol in an NADP(+)-dependent reaction to its corresponding catechol. The present study is a systematic investigation of the substrate specificity of the purified enzyme towards synthetic trans-dihydrodiol metabolites of phenanthrene, benz[a]anthracene, chrysene, dibenz[a, h]anthracene and benzo[a]pyrene. DDH exhibited a remarkable regiospecificity of enzymatic catalysis with regard to the site of the dihydrodiol moiety of the parent hydrocarbon. M-region- and, with lower efficiency, bay-region dihydrodiols were found to be good substrates of the enzyme with maximal velocities between 20-80 nmol/min per mg enzyme and Km values in the micromolar range. K-region dihydrodiols were not accepted as substrates. Dihydrodiols situated at the terminal ring of an anthracene-type structure such as benz[a]anthracene-8,9-dihydrodiol as well as the corresponding dihydrodiol epoxides were also not oxidized by DDH at measurable rates. The results provide evidence for a detoxifying role of DDH in the metabolism of the chemical carcinogens benz[a]anthracene, chrysene and dibenz[a, h]anthracene.

Alcohol Oxidoreductases↗
PubMed1991

Mapping of phenytoin-inducible cytochrome P450 immunoreactivity in the mouse central nervous system.

The distribution of phenytoin-inducible cytochrome P450 in non-treated mouse brain and spinal cord was analysed immunohistochemically using polyclonal antibodies against phenytoin-induced mouse cerebral microsomal P450. This P450 protein was proved in Ouchterlony [Volk B. et al. (1988) Neurosci. Lett. 84, 219-224], Western blot, and immunohistochemical analyses to be reactive to the specific antibodies and an IgG fraction raised against phenobarbital-induced rat liver microsomal P450IIB1. The phenytoin-induced P450 is designated P450IIB1* because immunologically it is comparable with P450IIB1; however, it has not yet been analysed for other characteristics of this enzyme. Immunocytochemistry was performed on acetone-fixed serial cryosections of the whole brain using the avidin-biotin-peroxidase detection system. Negative controls included incubations with preimmune serum of the immunized animal instead of the primary antibody and preabsorption of the antibody with the corresponding immunogen. The pattern of immunoreactive sites indicates that P450IIB1* is not distributed evenly throughout the CNS. It was found to be restricted to only some cellular populations. The most striking aspect of immunostaining was a predominant reactivity in the evolutionary old brain parts. Neuropil and neuronal staining was found in the spinal cord (motor neurons of the ventral horn), medulla oblongata (hypoglossal nuclei, magnocellular part of the lateral reticular nuclei), pons (trigeminal, facial, cochlear and pontine nuclei), cerebellum (granule cells), midbrain (dorsal raphe nucleus) and limbic lobe (hippocampal pyramidal cells). Neuropil reactivity alone appeared in cerebellar nuclei, midbrain, thalamus, basal ganglia, neopallium and olfactory brain. Generally, pia mater/arachnoid, ependyma, choroid plexus, vascular system and some astrocytic populations were found to be strongly P450IIB1* immunoreactive. In comparison with astroglia, which is characterized by glial fibrillary acidic protein-positiveness, the astrocytes, which are also P450IIB1* reactive, occurred only in subpial and subependymal layers, and in large fiber tracts of the spinal cord and brainstem, where they were attached to the vascular system. Otherwise, the glial fibrillary acidic protein-positive astrocytes were not P450IIB1* immunoreactive in the cerebellar molecular layer (fibers of Bergmann glia), in remaining neuropils and in white matter areas.

Animals↗
PubMed1991

Enzyme histochemical and immunohistochemical characterization of oval and parenchymal cells proliferating in livers of rats fed a choline-deficient/DL-ethionine-supplemented diet.

Male outbred Sprague-Dawley rats were fed a choline-deficient diet containing 0.10% DL-ethionine for up to 30 weeks. Liver slices from rats killed 4, 6, 10, 14, 22 and 30 weeks after starting the treatment were histochemically analyzed for the following parameters: basophilia, expression of cytokeratin 19 (which in the liver is bile duct epithelial cell-specific), glycogen content and activities of glycogen synthetase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6PASE), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerin-3-phosphate dehydrogenase (G3PDH), 'malic enzyme' (MDH), alkaline phosphatase (ALKPASE) and gamma-glutamyltranspeptidase (GGT). The diet induced necrosis of single parenchymal cells and a massive proliferation of oval cells within 4-6 weeks; thereafter cholangiofibroses, cystic cholangiomas and some cholangiofibromas, but no cholangiocarcinomas, were observed. Oval cells, cholangiofibroses, cystic cholangiomas and cholangiofibromas expressed cytokeratin 19, whereas parenchymal cells, foci of altered hepatocytes and hepatocellular adenomas did not; this observation does not support a precursor-product relationship between oval and parenchymal cells. SYN, PHO, G6PASE, G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT activities were detected in oval cells; cholangiofibrotic lesions, cystic cholangiomas and cholangiofibromas stained strongly for GAPDH, G3PDH and MDH. In livers from rats fed the diet for 10 weeks, single hepatocytes storing high amounts of glycogen appeared in the parenchyma. There was no indication of a transition from the oval cell population to hepatocytes storing glycogen in excess. Foci of glycogen-storing cells were scattered all over the lobes after 14 and 22 weeks; they had increased G6PASE, G6PDH, ALKPASE and GGT activities. Mixed cell foci and hepatocellular adenomas developed within 22-30 weeks and exhibited a remarkable decrease of G6PASE activity, a strong increase of G6PDH, GAPDH, G3PDH and MDH activities as well as extremely high ALKPASE and GGT activities. The data support the concept that during hepatocarcinogenesis, a number of sequential changes in the activities of various enzymes involved in carbohydrate metabolism occur and that a correlation between morphology and enzyme pattern in the focal lesions does in fact exist. Furthermore, our results suggest that two different cell lineages are involved in the development of cholangiocellular tumors from oval cells and hepatocellular tumors from hepatocytes.

Alkaline Phosphatase↗
PubMed1991

The metabolic activation of dibenz[a,h]anthracene in mouse skin examined by 32P-postlabelling: minor contribution of the 3,4-diol 1,2-oxides to DNA binding.

Dibenz[a,h]anthracene (DB[a,h]A) and the related 3,4-diol and anti- and syn-3,4-diol 1,2-oxides were applied to the shaved dorsal skin of groups of four C57Bl/CB1 mice. Twenty-four hours later the mice were killed, DNA isolated from the treated skin, hydrolysed and examined for the presence of aromatic adducts using the nuclease P1 modification of the 32P-postlabelling technique. Autoradiography of the maps obtained by chromatography on polyethyleneimine-cellulose plates showed that six DNA adduct spots that were derived from DB[a,h]A were also present in the DNA of skin treated with the DBA 3,4-diol and that, whilst four of these adduct spots were also seen in maps prepared from the DNA of skin treated with the anti-3,4-diol-1,2-oxide, they were not present in DNA from skin to which the syn-isomer had been applied. The identity of these adduct spots was confirmed by their coincidence when mixtures of different DNA hydrolysates were chromatographed together. Quantitatively, the highest levels of mouse skin modification were obtained with the diol-epoxides and the lowest with DB[a,h]A. The results suggest that most of the DNA adducts formed in DB[a,h]A-treated mouse skin arise through metabolism of the hydrocarbon to the related 3,4-diol and that some may be formed following the conversion of this diol to the bay-region anti-3,4-diol-1,2-oxide.

Animals↗
PubMed1990

Selective detection of mRNA forms encoding the major phenobarbital inducible cytochromes P450 and other members of the P450IIB family by the RNAse A protection assay.

The identification of P450 mRNAs in a tissue poses the problem that members of the same P450 gene family share a high sequence homology. Studies based on oligomer probes rely on a probe covering only a few base pairs. In contrast in our study on the expression of the P450IIB gene family we used in vitro-generated antisense transcripts, covering several hundred base pairs, of the hypervariable and constant regions of the P450IIB1 and P450IIB2 cDNA, in the RNAse A protection assay of mRNA isolated from various tissues. RNAse A concentrations were adjusted to a level where this enzyme still yielded distinct fragments for a defined P450IIB1 antisense/P450IIB2 sense heteroduplex, which contained 24 scattered mismatches within a stretch of 285 nucleotides. In contrast nuclease S1 was not useful for the detection of mismatches within this heteroduplex. With this highly sensitive RNAse A protection assay we were able to distinguish between the expression of P450IIB1 and the expression of P450IIB2 in several organs. Our results strongly support earlier studies on the tissue specific expression of these enzymes, which had used oligomer probes (Omiecinski, C. J., 1986, Nucleic Acids Res. 14, 1525-1539). Moreover we detected the constitutive hepatic expression of a P450IIB gene which was distinct from P450IIB1 and IIB2. In addition we identified a P450IIB mRNA which was expressed at high levels in the preputial gland but not in the liver or any other organ tested.

Animals↗