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Biomedical subjects

G F Weber

Publications and source records attributed to G F Weber.

At least 37 records · Page 2Linked to original sources
PubMed1994

The pathophysiology of reactive oxygen intermediates in the central nervous system.

Current evidence for the participation of free radicals in diseases of the central nervous system is reviewed. We conclude that the pathogenesis is based on a uniform mechanism: free radicals preferentially attack myelin, which contains easily peroxidizable phospholipids. The basal ganglia seem to be a brain area that is especially susceptible to radical damage which is possibly related to the synthesis of neurotransmitters. The clinical picture of the resulting cell death is dominated by convulsions and retardation in childhood and by psychomotoric disability and dementia in adulthood.

Cell Death↗
PubMed1993

Differential activation of cyclophosphamide and ifosphamide by cytochromes P-450 2B and 3A in human liver microsomes.

The present study identifies the specific human cytochrome P-450 (CYP) enzymes involved in hydroxylation leading to activation of the anticancer drug cyclophosphamide and its isomeric analogue, ifosphamide. Substantial interindividual variation (4-9-fold) was observed in the hydroxylation of these oxazaphosphorines by a panel of 12 human liver microsomes, and a significant correlation was obtained between these 2 activities (r = 0.85, P < 0.001). Enzyme kinetic analyses revealed that human liver microsomal cyclophosphamide 4-hydroxylation and ifosphamide 4-hydroxylation are best described by a 2-component Michaelis-Menten model composed of both low Km and high Km P-450 4-hydroxylases. To ascertain whether one or more human P-450 enzymes are catalytically competent in activating these oxazaphosphorines, microsomal fractions prepared from a panel of human B-lymphoblastoid cell lines stably transformed with individual P-450 complementary DNAs were assayed in vitro for oxazaphosphorine activation. Expressed CYP2A6, -2B6, -2C8, -2C9, and -3A4 were catalytically competent in hydroxylating cyclophosphamide and ifosphamide. Whereas CYP2C8 and CYP2C9 have the characteristics of low Km oxazaphosphorine 4-hydroxylases, CYP2A6, -2B6, and -3A4 are high Km forms. In contrast, CYP1A1, -1A2, -2D6, and -2E1 did not produce detectable activities. Furthermore, growth of cultured CYP2A6- and CYP2B6-expressing B-lymphoblastoid cells, but not of CYP-negative control cells, was inhibited by cyclophosphamide and ifosphamide as a consequence of prodrug activation to cytotoxic metabolites. Experiments with P-450 form-selective chemical inhibitors and inhibitory anti-P-450 antibodies were then performed to determine the contributions of individual P-450s to the activation of these drugs in human liver microsomes. Orphenadrine (a CYP2B6 inhibitor) and anti-CYP2B IgG inhibited microsomal cyclophosphamide hydroxylation to a greater extent than ifosphamide hydroxylation, consistent with the 8-fold higher activity of complementary DNA-expressed CYP2B6 with cyclophosphamide. In contrast, troleandomycin, a selective inhibitor of CYP3A3 and -3A4, and anti-CYP3A IgG substantially inhibited microsomal ifosphamide hydroxylation but had little or no effect on microsomal cyclophosphamide hydroxylation. By contrast, the CYP2D6-selective inhibitor quinidine did not affect either microsomal activity, while anti-CYP2A antibodies had only a modest inhibitory effect. Overall, the present study establishes that liver microsomal CYP2B and CYP3A preferentially catalyze cyclophosphamide and ifosphamide 4-hydroxylation, respectively, suggesting that liver P-450-inducing agents targeted at these enzymes might be used in cancer patients to enhance drug activation and therapeutic efficacy.

Antibodies↗
PubMed1993

Activation of the anti-cancer drug ifosphamide by rat liver microsomal P450 enzymes.

The NADPH-dependent metabolism of ifosphamide catalyzed by rat liver microsomes was investigated in order to identify individual P450 enzymes that activate this anti-cancer drug and to ascertain their relationship to the P450 enzymes that activate the isomeric drug cyclophosphamide. Pretreatment of rats with phenobarbital or clofibrate increased by up to 8-fold the activation of both ifosphamide and cyclophosphamide catalyzed by isolated liver microsomes. Studies using P450 form-selective inhibitory antibodies demonstrated that constitutively expressed P450s belonging to subfamily 2C (forms 2C11/2C6) make significant contributions to the activation of both oxazaphosphorines in uninduced male rat liver microsomes, while the phenobarbital-inducible P450 2B1 was shown to be a major catalyst of these activations in phenobarbital-induced microsomes. Pretreatment of rats with dexamethasone increased liver microsomal activation of ifosphamide approximately 6-fold without a corresponding effect on cyclophosphamide activation rates. Ifosphamide activation catalyzed by dexamethasone-induced liver microsomes was minimally inhibited by anti-P450 2B or anti-P450 2C antibodies, but was selectively inhibited by anti-P450 3A antibodies. Selective inhibition of liver microsomal ifosphamide activation was also effected by the macrolide antibiotic triacetyloleandomycin, an inhibitor of several dexamethasone-inducible 3A P450s. These studies establish that a dexamethasone-inducible family 3A P450 can make an important contribution to rat liver microsomal ifosphamide activation, and suggest that dexamethasone pretreatment might provide a useful approach for modulation of ifosphamide metabolism in order to improve its therapeutic efficacy in cancer patients.

Animals↗
PubMed1993

Denitrosation of the anti-cancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea catalyzed by microsomal glutathione S-transferase and cytochrome P450 monooxygenases.

The alkylating agent BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] can be inactivated through denitrosation reactions catalyzed by both cytosolic and microsomal enzymes. While previous studies have identified a class mu glutathione S-transferase [rat transferase 4-4 (Yb2)] as a major catalyst of the cytosolic denitrosation reaction, the enzymatic catalysts of BCNU denitrosation in microsomal membranes have not been identified. In the present study, both NADPH and glutathione (GSH) were found to support BCNU denitrosation catalyzed by isolated rat liver microsomes. Treatment of rats with the microsomal enzyme inducers phenobarbital and dexamethasone increased NADPH-dependent liver microsomal BCNU denitrosation up to fivefold without major effect on the GSH-dependent denitrosation activity. Although the NADPH-dependent activity was fully inhibited by antibody to NADPH-P450 reductase, purified NADPH-P450 reductase catalyzed BCNU denitrosation at rates that could only account for approximately 2-3% of the microsomal activity. Other experiments, including selective inhibition of NADPH-dependent microsomal BCNU denitrosation by chemical and antibody inhibitors of cytochrome P450, competitive inhibition of P450-catalyzed cyclophosphamide and ifosfamide activation by BCNU, and reconstitution of the denitrosation reaction by purified P450 enzyme 2B1 (major phenobarbital-inducible P450 form), established an important role for cytochrome P450 in BCNU denitrosation. By contrast, GSH-dependent microsomal BCNU denitrosation was unaffected by cytochrome P450 inhibitors, but was inhibited, with varying degrees of selectivity, by the microsomal glutathione S-transferase inhibitors ethacrynic acid, bromosulfophthalein, and indomethacin. These studies establish that BCNU inactivation can be catalyzed by two independent microsomal enzyme systems and suggest that therapeutically useful improvements in BCNU antitumor activity might be achieved through differential inhibition of these enzyme systems in tumor as compared to extratumoral sites.

Animals↗
PubMed1993

HIV glycoprotein as a superantigen. A mechanism of autoimmunity and implications for a vaccination strategy.

The pathogenic effects of HIV may reflect mimicry of several key immunological molecules. The surface glycoprotein of HIV has superantigenic properties responsible for the sequential deletion of T-cell clones. In addition, the glycoprotein has several regions sharing homology with class II MHC products. It can elicit cross-reactive antibodies which block replenishment of these T-cells in the thymus. The usefulness of conventional vaccination strategies in the fight against AIDS has been subject to debate. Based on these considerations, we argue that effective vaccines should avoid viral sequences homologous to class II MHC proteins which might exacerbate this process.

AIDS Vaccines↗
PubMed1992

Platinum anticancer drugs modulate P-450 mRNA levels and differentially alter hepatic drug and steroid hormone metabolism in male and female rats.

Treatment of male rats with the anticancer drug cisplatin leads to feminization of the profile of cytochrome P-450 and other microsomal enzymes involved in steroid hormone and drug metabolism (G.A. LeBlanc, and D.J. Waxman, J. Biol. Chem., 263: 15732-15739, 1988). The present study uses the rat model to evaluate the differential effects of cisplatin treatment on liver microsomal enzymes between genders, and also examines whether the modulation of enzyme activities by cisplatin and its analogues involves changes in P-450 gene expression. While cisplatin treatment of male rats caused a severalfold increase in female-predominant hepatic enzymes, including testosterone 5 alpha-reductase and testosterone 7 alpha-hydroxylase (P-450 form 2A1), it partially decreased the expression of these enzymes in females. The reduced expression of these estrogen-dependent enzymes in females may derive from the loss of circulating estradiol that was shown to occur in response to cisplatin treatment. Analysis of mRNA levels of individual P-450 forms revealed that the effects of cisplatin on P-450-catalyzed steroid hydroxylase activities in both male and female rats are primarily operative through the drug's effects on P-450 mRNA expression. P-450-dependent cyclophosphamide activation was significantly compromised in male rats after cisplatin administration; however, this activity was not altered in cisplatin-treated females. This sex-dependent effect of cisplatin was due to its suppression of P-450 form 2C11, a male-specific P-450 that is a major contributor to microsomal cyclophosphamide bioactivation in male rat liver. The clinically active cisplatin analogue iproplatin elicited effects very similar to those of cisplatin, while carboplatin and transplatin did not have significant effects on hepatic P-450 expression. Together, these findings demonstrate that the response of rat liver to cisplatin-induced changes in hepatic P-450 enzyme profiles and cyclophosphamide bioactivation capacity differs between the sexes, and in addition, these effects can be minimized by use of carboplatin in place of cisplatin.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1992

Inhibition of the neutrophil NADPH oxidase by folic acid and antagonists of the folic acid metabolism.

The influence of folic acid and several antagonists of the folic acid metabolism on neutrophil superoxide generation was investigated with the cytochrome c reduction assay. The compounds were found to be partial competitive inhibitors of the NADPH oxidase, their activity apparently increasing with larger substituents at the 10 position. There is evidence that compounds with a 4-oxo substituent are taken up more slowly by neutrophils than those with a 4-amino functionality. Scavenging properties could be excluded from control measurements with the xanthine/xanthine oxidase assay.

Adult↗
PubMed1992

Current methods in histopathology.

The past decade has presented many new methods to histopathology. However, there are important reasons for establishing these methods in every institute for pathology. It is not enough just to exchange old equipment into new systems. It takes a careful judgement what will be the benefits from a new system for the institute, the hospital and the patient. Important factors like institute management, quality assurance, quality control, speed, competitiveness, profitability and environmental safety aspects are high ranking tasks in the modern institute of pathology, next to the specialization in new diagnostic areas like immunohistochemistry, bone-pathology (bone-morphometry/undecalcifield iliac crest biopsy histology), flow cytometry, image analyzing or PCR-techniques etc. The main target of each institute of pathology, weather of private nature or a governmental/state institution, is the establishment of a high quality, safe and reliable diagnosis in the fastest time and for a price based on a standardized, accepted and economical system. Institutions will be more controlled by certain economical departments which have to watch the cost-/profit situation of the hospital and it's individual institutions in comparison to the average country standard and the average European standard. The introduced methods are mostly standard in "Western" institutes. However, it is of utmost importance that the institutes in "Eastern Europe" establish these standards as soon as possible in order to be competitive and to be able to join research-projects with "Western" universities - a necessary source of income.

Europe, Eastern↗
PubMed1991

Glutathione peroxidase deficiency and childhood seizures.

4 children with intractable seizures, repeated infections, and intolerance to anticonvulsants had evidence of glutathione peroxidase deficiency. 2 had low intracellular enzyme activity but normal blood selenium and high plasma glutathione peroxidase concentrations. The other 2 had low intracellular glutathione peroxidase activity with low circulating glutathione peroxidase and selenium concentrations. The clinical state of the children improved after discontinuation of anticonvulsant medication and selenium substitution.

Anticonvulsants↗
PubMed1991

The antifolate 10-deazaaminopterin inhibits neutrophil chemotaxis and superoxide generation.

The effect of the dihydrofolate reductase inhibitor 10-deazaaminopterin on several neutrophil functions was tested in vitro. At 100 uM it inhibited chemotaxis by 50% and reduced the generation of superoxide by 30%. It had no influence on phagocytosis and did not significantly change the secretion of beta-D-glucuronidase, a marker enzyme of degranulation. After preincubation of white cells with various concentrations of 10-deazaaminopterin, followed by resuspension in drug-free medium, no inhibition of chemotaxis or superoxide generation was seen. Therefore, the effects on chemotaxis and NADPH oxidase appear to be reversible and not due to metabolic transformation of the dihydrofolate reductase inhibitor.

Adult↗
PubMed1990

The measurement of oxygen-derived free radicals and related substances in medicine.

Due to the high reactivity of the chemical species and the presence of multiple potentially interfering substances, the measurement of oxygen-derived free radicals in biological material requires highly developed techniques. The currently employed methods are reviewed according to the reactions upon which they are based, the assays, possible interferences and their use in medical research. Detection of the emission of light is a very popular method. Although it is in principle unspecific, there are modifications to measure individual radical species. The only direct way to detect radicals is electron spin resonance spectroscopy. Among the specific assays for O2- the reductions of nitroblue tetrazolium or cytochrome c are predominant. For the detection of H2O2 different techniques are employed for either intracellular or extracellular determination. An array of substances has been used for the measurement of OH. Which of them is the most useful depends on the question to be answered. There are also indirect methods that determine free radicals based on chemical modifications caused by them; the most important assays of this kind quantify lipid peroxides. In addition, assays for thiobarbituric acid-reactive substances and DNA strand breaks and interstrand crosslinks are used.

Animals↗
PubMed1982

Galactosyltransferase variant in pleural effusion.

In measuring total galactosyltransferase activity in the pleural effusions from patients with benign or malignant diseases, we found no significant difference between the two groups (p greater than 0.05). However, a small amount of a galactosyltransferase variant, GT(l), could be separated from other galactosyltransferase enzymes in malignant pleural effusions by anion-exchange chromatography (DEAE-cellulose) with a buffer of low ionic strength. Other galactosyltransferases were eluted from the column with buffer of higher ionic strength. Using a mini-column method, we detected GT(l) enzyme in 19 of 26 specimens fro cancer patients, as compared with eight of 25 specimens from patients with benign disorders. The appearance of GT(l) enzyme in pleural effusion may be a tumor-associated phenomenon.

Chromatography, DEAE-Cellulose↗
PubMed1979

Nature of human serum blood group T antibodies.

Antibodies against blood group substance T in normal human sera consist of two types, depending upon whether or not the molecules remain immunochemically active at 37 degrees C. The two agglutinins were isolated by means of T antigen-coupled Sepharose 48 affinity chromatography. Temperature-sensitive agglutinin was eluted from the affinity column at 37 degrees C, while temperature-independent agglutinin remained bound to the antigen. Subsequently, the latter was dissociated from the column in the presence of 2.5 M MgCl2. Examination of six normal sera revealed that the levels of temperature-independent agglutinin was about twice that of temperature-sensitive agglutinin. More than 90% of anti-T agglutinins in normal sera were of the IgM class, in 8 of 11 samples studied. No direct relationship appeared to exist between anti-T titers and blood type. Thermal effects on hemagglutination, at least for anti-T agglutinin interacting with T antigen bearing red cells, is primarily due to the molecular characteristics of the agglutinin and not to the conformational change of the red cell membranes.

Adsorption↗
PubMed1979

Extraction of human plasma or sera by heat treatment for a solid-phase radioimmunoassay of carcinoembryonic antigen.

Heat treatment and a solid-phase radioimmunoassay are combined to give a relatively simple and rapid procedure for assay of carcinoembryonic antigen in plasma or serum. The new way we describe to extract this antigen is an alternative to the conventional method of extraction with perchloric acid. Heating plasma or serum samples in acetate buffer (0.16 mol/L, pH 5.0) at 70 degrees C for 15 min precipitates out most of the heat-labile, nonspecific plasma proteins, but leaves most of the antigen in solution, with its immunochemical properties apparently unaffected. Comparison between the heat treatment and the perchloric acid extraction yielded comparable values when tested either by solid-phase radioimmunoassay or by the zirconyl phosphate precipitation method. An added advantage of our method is that it gives the same assay values for both plasma and serum. Results for a group of pathological plasma samples, assayed by both our method and the perchloric acid-zirconyl phosphate precipitation method, gave a correlation coefficient of 0.90.

Carcinoembryonic Antigen↗