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Biomedical subjects

M D Burke

Publications and source records attributed to M D Burke.

At least 19 recordsLinked to original sources
PubMed1992

Cytochrome P450IA expression in adult and fetal human liver.

A monoclonal antibody has been produced that recognizes the cytochrome P450 form, cytochrome P450IA1, but not cytochrome P450IA2 in rats and recognizes a single protein band of similar mol. wt on immunoblots of human liver microsomes. Immunohistochemical studies have been carried out with this antibody to investigate the localization and distribution of cytochrome(s) P450 of the P450IA family in human liver. Cytochrome P450IA was identified in both adult and fetal liver and in each case it was localized predominantly to hepatocytes. In adult liver there was a heterogeneous distribution of cytochrome P450IA immunoreactivity with cytochrome P450IA mainly present in zone 3 hepatocytes of the liver acinus. Within fetal liver there was a uniform distribution of cytochrome P450IA immunoreactivity with no apparent zonal distribution. Bile duct epithelium did not show definite immunostaining for cytochrome P450IA in either adult or fetal liver.

Adult↗
PubMed1990

7-Alkoxyquinolines: new fluorescent substrates for cytochrome P450 monooxygenases.

A series of 7-alkoxyquinolines was synthesized and tested as substrates with hepatic microsomes prepared from male Wistar rats. Microsomal O-dealkylation rates and kinetic constants were determined for the 7-alkoxyquinolines with microsomes from control, 3-methylcholanthrene (MC)-pretreated, and phenobarbitone (PB)-pretreated rats. Structure-activity relationship studies indicated that the 7-benzyloxyquinoline was the most rapidly metabolized substrate for control microsomes and those from PB-pretreated rats, whereas the 7-ethoxy- and 7-propoxyquinolines were O-dealkylated more rapidly by microsomes of MC-pretreated animals. Differences in activities occurred in Vmax and apparent Km values; however, there does not appear to be a correlation between these two values for the different quinoline substrates. Apparent Km and Vmax values for the 7-alkoxyquinolines were: control microsomes, Km = 71-773 microM, Vmax = 0.37-8.4 nmol 7-quinolinol/min/mg protein; MC microsomes, Km = 0.5-14 microM, Vmax = 0.29-2.7 nmol 7-quinolinol/min/mg protein; PB microsomes, Km = 2.8-46 microM, Vmax = 0.9-12 nmol 7-quinolinol/min/mg protein. All of the quinoline substrates gave Type I binding spectra with control and MC microsomes. With PB microsomes, Type I. Reverse Type I, and a mixture of the two types of binding spectra were observed. Comparisons of the structure-activity relationships, levels of induction, and kinetic constants were made with 7-alkoxycoumarin and 7-alkoxyphenoxazone analogs. In addition, three new coumarin substrates (7-pentoxy-, 7-hexoxy-, and 7-benzyloxycoumarin) are described.

Animals↗
PubMed1990

Variation in drug-metabolizing enzyme activities during the growth of human Hep G2 hepatoma cells.

1. The activities of several drug-metabolizing enzymes change during the growth cycle (exponential growth to confluence) of Hep G2 cells in culture. As the rate of cell growth slowed down (days 7 to 10 after passage) the activities of ethoxy- and methoxy-resorufin O-dealkylase and of NADPH cytochrome c- and NADH cytochrome b5-reductase increased. In contrast, the O-dealkylations of pentoxy- and benzyloxy-resorufin did not change significantly during culture. 2. UDP-glucuronyltransferase activities also showed substrate-dependent alterations with time in culture. In contrast, glutathione-S-transferase activity remained constant despite a decline in the intracellular reduced glutathione content. 3. Epoxide hydrolase activity altered throughout time in culture, with an initial decrease in activity followed by a marked increase between days 7 and 10 after passage. 4. These results indicate the importance of standardizing the protocol with regard to the timing of experiments within the growth period of the cells when using hepatoma cell lines for assessing the metabolism and cytotoxicity of chemicals.

Carcinoma, Hepatocellular↗
PubMed1990

Diagnosis of ectopic pregnancy by the rate of increase of choriogonadotropin in serum: diagnostic criteria compared.

We compare three methods for using the rate of change of human choriogonadotropin (hCG) concentration in serum to diagnose ectopic pregnancy. With Method I, the lower limit for the rate of increase of serum hCG in normal pregnancy is 66% per 48 h. With Method II, a different lower limit of normal is specified for each of four discrete sampling intervals of hCG. With Method III, the lower limit of normal is determined by a continuous discriminant function of the initial hCG concentration. If the initial hCG concentration is less than or equal to 2000 int. units/L (Second International Standard), all three methods have acceptable diagnostic efficiencies, and there are no statistically significant differences among conclusions from the methods. None of the three methods performs satisfactorily if the initial hCG concentration is greater than 2000 int. units/L. We recommend Method I because it is simpler than the other two.

Chorionic Gonadotropin↗
PubMed1989

Differences in adult and foetal human cytochrome P-450 forms recognized by monoclonal antibodies with specificity for the P450III family.

Six murine monoclonal antibodies raised against a major human adult liver cytochrome P-450 (P-450) of the PCN family (P450III) detected a protein in human foetal liver microsomes (microsomal fractions) which had an approx. 1 kDa higher molecular mass on SDS/polyacrylamide-gel electrophoresis than the protein recognized in human adult liver microsomes. Although each of the antibodies recognized both the adult and the foetal forms, antibody HL4 showed higher affinity for the foetal form. Recognition by the monoclonal antibodies of peptides generated by proteolytic cleavage of microsomal proteins showed different patterns for the adult and foetal forms. It is concluded that the foetal P-450 form recognized by antibodies to the major human adult liver form P450hA7, although structurally similar, is either a distinct P-450 isoenzyme or that the adult and foetal proteins have different covalent modification. Immunoquantification experiments showed comparable levels of the P-450 forms in adult and foetal liver, although there appeared to be less inter-individual variation in foetal livers.

Adult↗
PubMed1989

Enzyme histochemistry on freeze-dried, resin-embedded tissue.

We have developed a method for histochemical demonstration of a wide range of enzymes in freeze-dried, resin-embedded tissue. Freeze-dried tissue specimens were embedded without fixation at low temperature (4 degrees C or -20 degrees C) in glycol methacrylate resin or LR Gold resin. Enzyme activity was optimally preserved by embedding the freeze-dried tissue in glycol methacrylate resin. All enzymes studied (oxidoreductases, esterases, peptidases, and phosphatases), except for glucose-6-phosphatase, were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-drying combined with low-temperature resin embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, high enzyme activity, and excellent tissue morphology.

Animals↗