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Biomedical subjects

F Oesch

Publications and source records attributed to F Oesch.

At least 91 records · Page 5Linked to original sources
PubMed1996

Inverse relationship of melanocyte differentiation antigen expression in melanoma tissues and CD8+ cytotoxic-T-cell responses: evidence for immunoselection of antigen-loss variants in vivo.

Antigenic peptides derived from differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for MHC-restricted cytotoxic T lymphocytes (CTL). CTL directed against peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens can be detected in melanoma patients and in healthy controls. The presence of defined antigenic peptides and corresponding precursor CTL in patients with metastatic melanoma opens perspectives for the development of antigen-specific tumor vaccines. In this study, we examined the expression of Melan A/MART-1, tyrosinase and gp100lPmel17 in fresh melanoma tissues of HLA-A2+ patients and the spontaneous CTL reactivity against antigenic peptides derived from these antigens. Our results demonstrate an inverse correlation of antigen expression and CTL response to Melan A/MART-1 and tyrosinase in patients with metastatic melanoma. In 2 patients with advanced disease, CTL responses against Melan A/MART-1 and tyrosinase were induced by intradermal immunization with synthetic nona- or deca-peptides derived from these antigens. Metastases increasing in size over time showed a loss of Melan A/MART-1 expression in the presence of CTL in one patient. The regression of a metastasis with persistent tyrosinase expression was observed in the other patient after the induction of CTL, reactive against tyrosinase. We conclude that CTL responses against melanocyte differentiation antigens may mediate regression of antigen-positive tumors and select for antigen-loss variants in vivo.

Amino Acid Sequence↗
PubMed1996

Asp333, Asp495, and His523 form the catalytic triad of rat soluble epoxide hydrolase.

On the basis of the sequence similarity between mammalian epoxide hydrolases and bacterial haloalkane dehalogenase reported earlier (Arand, M., Grant, D. F., Beetham, J. K., Friedberg, T., Oesch, F., and Hammock, B. D. (1994) FEBS Lett. 338, 251-256; Beetham, J. K., Grant, D., Arand, M., Garbarino, J., Kiyosue, T., Pinot, F., Oesch, F., Belknap, W. R., Shinozaki, K., and hammock, B. D. (1995) DNA Cell. Biol. 14, 61-71) we selected candidate amino acid residues for the putative catalytic triad of the rat soluble epoxide hydrolase. The predicted amino acid residues were exchanged by site-directed mutagenesis of the epoxide hydrolase cDNA, followed by the expression of the respective mutant enzymes in Escherichia coli. A total of 25 different mutants were analyzed for their epoxide hydrolase activity toward the model substrate trans-stilbene oxide. In case of impaired catalytic activity of a given mutant, the structural integrity of the recombinant enzyme protein was assessed either by its ability to covalently bind the substrate trans-stilbene oxide or by affinity purification on benzyl thio-Sepharose, using the soluble epoxide hydrolase-specific competitive inhibitor 4-fluorochalcone oxide to release the bound enzyme from the affinity matrix. Of the mutants under investigation, only those with changes in the positions Asp333, Asp495, and His523 were completely inactive toward the model substrate trans-stilbene oxide while retaining the proper protein fold. These amino acids were exactly those previously predicted by sequence alignment. Exchange of the amino acid residues flanking the catalytic nucleophile Asp333 significantly changed the kinetic properties of the enzyme. Mutation of His332 to Gln had no apparent effect on the Km but led to a heavily reduced Vmax (5% that of the wild type) of the mutant enzyme, while the exchange of Trp334 against Phe strongly increased the Km (7-fold) and also moderately enhanced the Vmax (2-fold) of the corresponding mutant. Mutation of Trp540 apparently had a strong effect on the protein conformation.

Amino Acid Sequence↗
PubMed1996

Prognostic significance of c-erB-2 mRNA in ovarian carcinoma.

The oncogene specific mRNA of c-erbB-2 was detected by the S1 nuclease protection assay in 95 ovarian cancer specimens. In 79 primary carcinomas, we found 16 (20%) with strong expression, 13 (17%) with weak expression, 4 (5%) with very weak expression, and 46 (58%) with no expression. In 3 of 16 recurrencies (19%) a strong expression of c-erbB-2 mRNA was detected, in 2 (12%) weak expression was detected, and in 11 (69%) no expression of c-erbB-2 mRNA was detected. Kaplan-Meier analysis revealed no significant association between strong expression of c-erbB-2 mRNA and survival of the 79 patients with primary cancer. However, in the subgroup of patients with FIGO (International Federation of Gynecology and Obstetrics) stages III and IV (n = 60) a shorter median survival time (12 months) was obtained for patients with strong c-erbB-2 mRNA expression compared to patients with no, very weak, and weak c-erbB-2 mRNA expression (25 months, P = 0.04). In addition, strong expression of c-erbB-2 mRNA did not depend on histologic grade, histologic type, and FIGO stage. Adverse prognostic factors include histologic type (serous carcinoma), high grade, high stage (FIGO stages III and IV), and residual of tumor after surgery. From our results we conclude that for all patients (FIGO stages I-IV) strong expression of c-erbB-2 mRNA is not a prognostic parameter, but in the subpopulation of patients with FIGO stage III and IV only, an association between strong c-erbB-2 mRNA expression and shorter median survival time was found, although statistical significance was weak (P = 0.04).

Female↗
PubMed1996

Primary DNA damage in peripheral mononuclear blood cells of workers exposed to bitumen-based products.

The genotoxic effect of occupational exposure to bitumen-based products was determined by the extent of DNA strand breaks and alkali-labile sites of the DNA of peripheral mononuclear blood cells from seven roofers, 18 road paving workers, and nine bitumen painters. In order to evaluate short-term genotoxic effect the workers were investigated on Fridays and on Mondays after a weekend free of occupational exposure. The roofers (all cigarette smokers) showed a significantly (P < 0.002) 43% higher mean level of alkaline DNA strand breaks on Friday than did the ten smoking controls included in this study. Also, comparison of the individual levels of alkaline strand breaks on Mondays and on Fridays revealed a significant increase (P < 0.05, Wilcoxon test) during the work week. In the road paving workers and the bitumen painters no statistically significant difference in the mean levels of alkaline strand breaks could be found compared to controls either for the measurement on Mondays or for that on Fridays. However, interesting tendencies were observed. As in the group of roofers, the mean level of alkaline DNA strand breaks as well as the majority of the individual levels of alkaline strand breaks of road paving workers was higher on Fridays than on Mondays. In contrast, bitumen painters exhibited a relatively high level of alkaline DNA strand breaks on Mondays and a decreased mean level of strand breaks on Fridays. DNA adducts could be detected at a low level (up to 2.9 adducts per 10(9) bases) in 10 of 14 road paving workers and bitumen painters using the 32P-postlabelling assay. The number of DNA adducts correlated with the years spend in the present job. Road paving workers and bitumen painters showed only suggestive evidence for a possible genotoxic effect due to their occupational exposure. Because we cannot exclude the formation of DNA cross-links in these workers, a more detailed investigation of the hazard is urgently needed. For roofers, substantial genotoxic damage in peripheral mononuclear blood cells was observed in this study.

Adult↗
PubMed1996

Distribution and induction of aflatoxin B1-9a-hydroxylase activity in rat liver parenchymal and non-parenchymal cells.

Chronic administration of aflatoxin B1 (AFB1) to rats gives rise to hepatocellular and cholangiocellular carcinomas without affecting Kupffer and endothelial cells. The enzymatic conversion of AFB1 to AFB1-8,9-epoxide is the critical step in the activation of the myocotoxin, while the conversion of AFB1 to aflatoxin M1 (AFM1), catalyzed by the AFB1-9a-hydroxylase, is considered to be a detoxication route for the toxin. In the present study the distribution and inducibility of AFB1-9a-hydroxylase were analyzed in microsomes derived from freshly isolated liver parenchymal (PC) and nonparenchymal cells (i.e. Kupffer + endothelial cells, NPC). AFB1-9a-hydroxylase activity was clearly measurable in NPC and similar to that of PC. In NPC the rate of formation of AFM1 was higher (when incubating with 16 microM AFB1) than or similar (with 128 microM AFB1) to that of AFB1-8,9-epoxide, while in PC it was significantly lower. Taken together, these results suggest that the AFB1-9a-hydroxylase activity might be particularly important in NPC to protect these cells from AFB1 by converting it to a significantly less mutagenic metabolite and by reducing the amount of AFB1 available for epoxidation. Furthermore, it is shown that AFB1-9a-hydroxylase activity is inducible by phenobarbital (only in PC), 3-methylcholanthrene, isosafrole and Aroclor 1254, thus indicating that in rat liver the conversion of AFB1 to AFM1 is catalyzed by members of the cytochrome 1A and 2B families.

Animals↗
PubMed1996

Ha-rasVal12 but not p53Ser247 leads to a significant neoplastic transformation rate of the putative rat liver stem cells (oval cell).

In order to test the controversially discussed hypothesis that oval cells are part of a liver stem cell compartment and can give rise to cholangiocellular as well as hepatocellular carcinomas in the course of liver carcinogenesis, we transfected an oval cell line established in our laboratory with an oncogenically activated genomic Ha-ras clone (pUC EJ 6.6), carrying a valine at position 12 instead of the wild-type glycine, or a rat p53 cDNA mutated by site-directed mutagenesis at codon 247, which corresponds to codon 249 in the human p53. This codon is of particular interest since it represents a mutation hotspot observed in hepatocellular carcinoma especially in regions with high aflatoxin B1 exposure. Independent Ha-rasVal12 and p53Ser247 recombinant clones were subcutaneously injected into syngeneic newborn rats and the resulting tumours were analysed histopathologically. Each of two p53Ser247 clones gave negligible tumour yields (one tumour out of 13 injected animals), whereas each of two Ha-rasVal12 clones gave marked tumour yields (four tumours out of 13 and seven out of 12 treated animals, respectively). In addition, the p53Ser247-induced tumours appeared only after 11 months and were small, whereas the Ha-rasVal12-induced tumours appeared already after 6-8 weeks and grew rapidly. Histopathological analysis of the tumours revealed only undifferentiated carcinomas. Interestingly, one tumour that arose upon injection of Ha-rasVal12-transfected cells stained positive for albumin, showing at least a partial hepatocytic differentiation.

Animals↗
PubMed1996

Inducing properties of rifampicin and rifabutin for selected enzyme activities of the cytochrome P-450 and UDP-glucuronosyltransferase superfamilies in female rat liver.

Important species differences have been reported concerning the induction properties of rifampicin towards enzymes of the P-450 superfamily. Mice, rabbits and humans are far more responsive than rats and guinea pigs. In the present study a strong induction of cytochrome P-450 3A-dependent enzyme activities was observed in female rat liver microsomes after high dose treatment (> or = 250 mg/kg/day for 9 days) with rifampicin, resulting in an up to 30-fold enhanced hydroxylation rate of testosterone in the 2 beta-, 6 beta- and 15 beta-position in vitro. Other cytochrome P-450 isozyme-selective reactions were not, or only marginally, affected. A steep increase in cytochrome P-450 3A activity on a moderate elevation of the dose administered, together with the previously observed lack of efficient induction with doses below 200 mg/kg/day demonstrated that there is a threshold in enzyme induction by rifampicin. For rifabutin such a threshold was not apparent. Induction by rifabutin showed an isoenzyme-selectivity profile similar to that produced by rifampicin, but the maximally achievable induction of cytochrome P-450 3A by rifabutin was about two-fold lower compared with rifampicin. Rifampicin and rifabutin enhanced the glucuronidation of 1-naphthol, 4-hydroxybiphenyl and beta-estradiol by a factor of two to three. The potential implications of the enzyme induction by rifampicin derivatives in terms of possible drug-drug interactions are discussed.

Animals↗
PubMed1996

Cytolytic T cell reactivity against melanoma-associated differentiation antigens in peripheral blood of melanoma patients and healthy individuals.

Antigenic peptides derived from several differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs). To examine their potential role in tumour-directed immune responses in vivo, we determined CTL reactivity against seven antigenic peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens in the peripheral blood of 10 HLA-A2+ healthy controls and 26 HLA-A2+ melanoma patients. The influenza matrix peptide (GILGFVFTL) presented by HLA-A2.1 was used as a control peptide. CTL reactivity was assessed in a mixed lymphocyte 'peptide' culture assay. Reactivity against Melan A/MART-1-derived peptide antigens was readily detectable in both melanoma patients and controls. Reactivity directed against tyrosinase-derived peptide antigens was also detected in both melanoma patients and healthy individuals, but less frequently. A measurable response against gp100/Pmel17-derived antigens was found in 1/10 controls and in 1/26 of the melanoma patients. Reactivity against the influenza matrix peptide was common in both melanoma patients and controls. Our findings show that precursor CTLs against melanocyte differentiation antigens can be detected in peripheral blood of melanoma patients and healthy individuals. The pattern of CTL reactivity directed against melanoma-associated antigens does not seem to be altered in melanoma patients. Despite antigen-specific CTL reactivity, tumour growth was not prevented in melanoma patients and autoimmune phenomena were not detected in healthy individuals. It remains to be determined whether precursor CTLs recognizing melanocyte differentiation antigens can be activated by immunization and lead to effective tumour rejection in vivo.

Amino Acid Sequence↗
PubMed1996

Transient increase in DNA strand breaks in car refinishing spray painters.

OBJECTIVES: Genotoxic risk was evaluated for spray painters possibly exposed to polyester resins and acrylic enamel-based paints in automotive body repair shops. METHODS: DNA (deoxyribonucleic acid) strand breaks and alkali-labile sites were measured in peripheral mononuclear blood cells ex vivo using the alkaline elution method. Samples of venous blood were taken on Monday after a free weekend and again on Friday from 38 male and 1 female spray painters and compared with the blood samples from 36 male and 3 female referents. The elution rate of each DNA sample was standardized by dividing it by the elution rate obtained from simultaneously sampled untreated Chinese hamster V79 cells. RESULTS: The spray painters showed a significantly (P < 0.001) higher mean level of strand breaks and alkali-labile sites in the Friday samples [2.05 (SE 0.17)] compared with the Monday samples [1.38 (SE 0.07)]. The Monday results of the spray painters were not distinguishable from the referents' [1.41 (SE 0.10)]. The increase in DNA damage was numerically higher, but only weakly significant (use of masks, P < 0.05) or nonsignificant (use of spray booths), when fewer safety provisions were taken. CONCLUSIONS: A significant increase in DNA strand breaks and alkali-labile sites was found in spray painters after a week's work. However, DNA damage seems to be reversible. The use of modern safety equipment seems to affect DNA damage only marginally. There is an urgent need to identify the genotoxic chemicals in the occupational environment of spray painters and to develop corresponding satisfactory safety measures.

Acrylic Resins↗
PubMed1995

Relationship between the microsomal epoxide hydrolase and the hepatocellular transport of bile acids and xenobiotics.

Recently two different bile-acid carriers for the hepatocellular sodium-dependent uptake of taurocholate have been described. The first transport system was isolated and characterized by functional expression cloning in Xenopus laevis oocytes. The corresponding cDNA clone, named Ntcp for Na+/taurocholate co-transporting polypeptide, codes for a protein of 362 amino acids and shows no similarity to previously known sequences. The transport function of this carrier system is well documented by expression in Xenopus laevis oocytes and by transient and stably transfected cell lines. In addition, several lines of evidence implied that the well-known xenobiotic-metabolizing enzyme microsomal epoxide hydrolase (mEH, EC 3.3.2.3) is also able to mediate sinusoidal uptake of taurocholate. Furthermore, it was claimed that the same enzyme also mediates the uptake of the conjugated bile acid into the smooth endoplasmic reticulum (ER). No direct proof of the transport function of mEH by its heterologous expression has yet been published. In the present work we used a stable transfected cell line that expressed high levels of heterologous mEH for uptake studies of various bile acids and the loop diuretic bumetanide. The uptake of the conjugated bile acid taurocholate, of the non-conjugated bile acid cholate and of the organic anion bumetanide was measured in the transfected as well as in the non-transfected parental cell line. These organic anions represent the main substrates of the known transport systems for organic anions in the rat liver. The results show that the microsomal epoxide hydrolase is unable to transport taurocholate, cholate or bumetanide. Furthermore, Western-blot analysis revealed the expression of mEH in hepatoma tumor cell lines, which show no transport activity for these organic anions. These results show that it is unlikely that mEH can mediate the transport of these substrates.

Animals↗
PubMed1995

Density-dependent regulation of cell growth by contactinhibin and the contactinhibin receptor.

BACKGROUND: The number of cells within mammalian tissues is maintained by growth-stimulating and growth-inhibiting mechanisms, with inhibitory signals being superimposed over growth stimuli. This is reflected, in the culture of normal adherent cells, by the phenomenon of density-dependent inhibition of growth: cells cease proliferation after becoming a confluent monolayer. We have shown previously that a plasma membrane glycoprotein, contactinhibin, is a major effector of negative growth regulation. Although transformed cells express contactinhibin in a functionally active form, they are not growth-inhibited, suggesting that the defects that lead to their aberrant growth are located 'downstream' of contactinhibin. RESULTS: Here, we provide evidence that a 92 kD plasma membrane protein, which we call CiR, binds specifically to contactinhibin and acts as a receptor mediating the contact-dependent inhibition of growth of cultured human fibroblasts. When polyclonal antibodies against CiR were introduced into cells using liposomes, confluent cells were released from density-dependent growth control. By contrast, cross-linking CiR that is localized to the plasma membrane, using anti-CiR antibodies, led to growth inhibition, suggesting that CiR is a signalling molecule and implicating CiR oligomerization in signal generation. This conclusion is supported by the finding that binding of contactinhibin by CiR is strongly dependent on the local concentration of both molecules and has a sharp threshold. When CiR was isolated by immuno-precipitation under conditions favouring phosphorylation, it was hyperphosphorylated on serine and threonine residues and had reduced contactinhibin-binding capacity; the binding capacity of CiR was restored after treatment with potato acid phosphatase. Fibroblasts transformed with simian virus 40 had reduced CiR expression, higher CiR phosphorylation levels, and a strongly reduced capacity of CiR to bind to contactinhibin. Phosphatase treatment of the CiR isolated from transformed cells only partially restored its contactinhibin-binding capacity. CONCLUSIONS: Homeostasis is the net result of a highly balanced network of growth-stimulating and growth-inhibitory signals. We have shown that density-dependent inhibition of growth in vitro is mediated by the interaction of contactinhibin with a 92 kD plasma membrane glycoprotein, CiR, the contactinhibin-binding capacity of which is regulated by phosphorylation.

Cell Count↗
PubMed1995

Conjugation of anti-dihydrodiol epoxides of benzo[a]pyrene, chrysene, benzo[c]phenanthrene and dibenz[a,h]anthracene with glutathione catalyzed by cytosol and by the Mu-class glutathione transferase HTP II from rat liver.

The (+/-)-anti-dihydrodiol epoxides (DE) of benzo[a]pyrene (BP), chrysene (Chr), benzo[c]phenanthrene (BcPh) and dibenz[a,h]anthracene (DBA) were incubated in the presence of glutathione (GSH) with hepatic cytosol from untreated and Aroclor 1254 pretreated rats and with the Mu-class glutathione transferase (GST) HTP II from rat liver. The diastereoisomeric GSH conjugates formed were separated, identified and quantified by HPLC employing synthetic reference compounds. All (+/-)-anti-dihydrodiol epoxides investigated in this study were proven to be substrates of the cytosolic GSTs. The highly mutagenic and carcinogenic (+)-anti-DE with R,S,S,R absolute configuration was preferentially conjugated in the case of BP and Chr. Aroclor 1254 pretreatment increased the turnover 2-3-fold and changed the enantioselectivity. The previously purified GST HTP II exhibited a high degree of enantioselectivity (> or = 95%) towards the R,S,S,R-configurated enantiomer in the case of the bay-region (+/-)-anti-BPDE, (+/-)-anti-ChrDE and (+/-)-anti-DBADE, whereas in the case of fjord-region (+/-)-anti-BcPhDE both enantiomers were good substrates. The contribution of HTP II to the enzymatic activity of the cytosolic GST pool was estimated to be in the range of 11-32%. In agreement with previous results, the observed enantioselectivity of the purified enzyme seems to be of minor significance considering the total GST pool in the liver.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

Tumor cell--endothelium adhesion in an artificial venule.

Adhesion of tumor cells to endothelial cells is a crucial step in the complex sequence of metastasis. In addition to type and local density of adhesion molecules on both cell types, shear forces exerted by the blood flow have been described to be of major importance in governing cell adhesion. Most of the experiments on the molecular basis of tumor-endothelial cell adhesion have been performed as static assays which lack shear forces. We have developed an artificial venule which shares the following in vivo characteristics. A confluent layer of endothelial cells lines the luminal surface of a glass capillary of 1 mm i.d. with pores of 30 nm diameter to allow diffusion of molecules from outside the capillary. Physiological pressure of 16 mbar, flow rate of 2 cm/s, and shear forces of 2 dynes/cm2 are maintained. This device allowed us to show that under dynamic conditions adhesion of B16 mouse melanoma cells to EA.hy926 endothelial cells is mediated most likely by a lectin-like structure on B16 cells and oligosaccharide(s) on endothelial cells. In addition, endothelial activation-independent adhesion was found to be restricted to only a fraction of endothelial cells, as the number of B16 cells that adhered was independent of the number of B16 cells applied.

Animals↗
PubMed1995

Xenobiotic metabolizing enzyme activities and viability are well preserved in EDTA-isolated rat liver parenchymal cells after cryopreservation.

Rat liver parenchymal cells (PC) were isolated by EDTA perfusion and were purified by a subsequent Percoll centrifugation. The isolated PC had a viability of 95%, as judged by trypan blue exclusion. Freshly isolated PC were cryopreserved with an optimized protocol in a computer-controlled freezer. After thawing, the PC still retained a viability of 89%. The activities of representative xenobiotic metabolizing enzymes were compared between freshly isolated and cryopreserved PC after thawing. The cytochrome P450 content and the cytochrome P450 2C11 isoenzyme activity, determined by hydroxylation of testosterone in intact cells, were not affected by the cryopreservation. The following phase II enzyme activities were also well maintained after cryopreservation: Phenol sulfotransferase (92%), 1-naphthol UDP-glucuronosyl transferase (95%), soluble epoxide hydrolase (87%), and glutathione S-transferase (88%), determined with broad spectrum substrate 1-chloro-2,4-dinitrobenzene. However, there was a significant decrease in plating efficiency between freshly isolated (86%) and cryopreserved (57%) PC when they were cultured. The initial quality of the freshly isolated PC is decisive for the success of cryopreservation. These results support the use of cryopreserved PC in pharmacology and toxicology with the aim to reduce the number of experimental animals used.

Animal Testing Alternatives↗
PubMed1995

DNA single strand break analysis in mononuclear blood cells of petrol pump attendants.

DNA single strand breaks, including DNA adducts that lead to alkali-labile sites, were measured in peripheral mononuclear blood cells of 35 petrol pump attendants by alkaline filter elution. Blood samples from petrol pump attendants were taken on Monday and Friday. Additionally, DNA single strand breaks of smoking and non-smoking control persons were examined. For the smoking (n = 12) and the non-smoking controls (n = 20) a mean normalized elution rate of 1.49 +/- 0.52 (mean value +/- 95% confidence interval) and 1.32 +/- 0.28, respectively, was obtained. The difference between smoking and non-smoking controls was not statistically significant (U test). An increase in DNA single strand breaks from Monday to Friday was detected for non-smoking petrol pump attendants with a daily working time of more than 4 h at the pump station. Their mean normalized elution rate increased from 1.08 on Monday to 1.89 on Friday. This difference was statistically significant (P < 0.05; Wilcoxon test for paired data), although the 95% confidence interval was large on Friday (0.43 on Monday; 1.23 on Friday). However, no significant increase was found for non-smoking petrol pump attendants who were on duty for less than 4 h per day at the pump station. No statistically significant increase in DNA single strand breaks could be detected for smoking petrol pump attendants whether they were pumping gasoline for more or for less than 4 h per day.

Adult↗