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Biomedical subjects

B Burchell

Publications and source records attributed to B Burchell.

At least 127 records · Page 7Linked to original sources
PubMed1990

Chromosomal mapping of a human phenol UDP-glucuronosyltransferase, GNT1.

A 5' fragment of a full-length cDNA clone encoding a human phenol UDP-glucuronosyltransferase was used to produce a specific probe for this gene. DNA isolated from a panel of 18 human-rodent somatic cell hybrids was analysed by Southern-blot hybridization. The results indicate that this UDP-glucuronosyltransferase is encoded by a single gene, designated GNT1, located on human chromosome 2.

Blotting, Southern↗
PubMed1990

The role of xenobiotic glucuronidating enzymes in drug resistance of tumour tissues and cells.

Xenobiotic and endobiotic glucuronidation is regulated by many cellular features such as (a) access of substrates to a family of microsomal enzymes, the UDP-glucuronosyltransferases (UDPGTs) and (b) mechanisms of transport and excretion of glucuronides from the cell. We have isolated molecular biological probes identifying human UDPGTs to facilitate the examination of mechanisms regulating the functional expression of UDPGTs during natural development, in genetic diseases and in cancer cells. UDPGTs are encoded by a multigene family. Seven rat and four human UDPGT cDNAs have been cloned and expressed in cell culture to determine their substrate specificity and ability to glucuronidate xenobiotics. Analysis of the amino acid sequences of cloned UDPGTs has provided information about their method of synthesis and topological orientation with the lumen of the endoplasmic reticulum (ER). The location of hepatic UDPGT raises interesting questions about the role of possible transporters essential for functional glucuronidation. Measurement of the UDPGT activities and UDPGT mRNA in carcinoma tissue and in hepatic nodules from rats suggest that increases in the level of a phenol-UDPGT may contribute to drug resistance in cancer. Human UDPGTs stably expressed in cell lines have been used to study the glucuronidation of carcinogens and anticancer drugs. Human phenol UDPGT will catalyse the glucuronidation of carcinogens. The high levels of expression of human phenol UDPGT in tissue culture cells could also lead to an increased resistance to xenobiotic toxicity caused by mitoxantrone, mitomycin C, and adriamycin. This molecular cell biology approach is being used to further assess the glucuronidation and effective toxicity of anticancer drugs.

Animals↗
PubMed1989

The enantioselective glucuronidation of morphine in rats and humans. Evidence for the involvement of more than one UDP-glucuronosyltransferase isoenzyme.

The formation of morphine glucuronides is enantio- and regioselective in rats and humans. In rat liver microsomes, natural (-)-morphine formed only the 3-O-glucuronide, whereas the unnatural (+)-morphine formed glucuronides at both the 3-OH and 6-OH positions, with the 6-O-glucuronide being the principal product. In human liver microsomes, both the 3-OH-and 6-OH positions were glucuronidated with each of the enantiomers, with the 3-O-glucuronide being the major product with (-)-morphine, and the 6-OH position preferred with the (+)-enantiomer. By using a series of biochemical and biological situations such as induction by xenobiotics, ontogeny, selective inhibition and genetic deficiencies, which are considered to be diagnostic of UDP-glucuronosyltransferase heterogeneity, we determined that two UDP-glucuronosyltransferase isoenzymes were responsible for the glucuronidation of morphine in rat liver. One isoenzyme (the so-called "morphine UDP-glucuronosyltransferase") was responsible for the glucoronidation at the (-)-3-OH and (+)-6-OH positions of morphine, whereas the other formed only the (+)-morphine-3-glucuronide. Evidence from enzyme induction and the genetically deficient deficient Gunn rat suggested that bilirubin UDPGT may be responsible for the (+)-morphine-3-UDP-glucuronosyltransferase activity. In human kidney, glucuronidation of both (-)- and (+)-enantiomers at the 6-OH position was deficient, whereas the activity at the 3-OH positions was still present, which indicated the involvement of two UDP-glucuronosyltransferases in the glucuronidation of morphine in man, as well as rats.

Age Factors↗
PubMed1989

Novel inhibitors and substrates of bilirubin: UDP-glucuronosyltransferase. Arylalkylcarboxylic acids.

The in vitro inhibitory potency of 20 structurally related alkanoic and arylalkanoic acids has been investigated on rat liver UDP-glucuronosyltransferase. These compounds were tested on the microsomal and purified enzyme, and a cloned cDNA expressed in COS 7 cell cultures. Among all the acids tested, 7,7,7-triphenylheptanoic acid was the most powerful inhibitor of bilirubin:UDP-glucuronosyltransferase with a lower effect on 1-naphtol, androsterone and testosterone glucuronidation. The inhibition was competitive towards the microsomal and purified bilirubin:UDP-glucuronosyltransferases with Kiapp values of 12.0 microM and 1.6 microM, respectively. Twenty analogues were examined, and the results showed that their inhibitory potency on bilirubin:UDP-glucuronosyltransferase activity was a function of at least three structural features (a) the presence of a hydrophobic triphenyl moiety; (b) the length of the aliphatic chain and (c) the presence of a carboxylic group. These inhibitors were also tested as possible substrates of UDP-glucuronosyltransferases. The strongest inhibitors were poor substrates of rat liver microsomal UDP-glucuronosyltransferases. However, 7,7,7-triphenylheptanoic acid was actively glucuronidated by purified bilirubin:UDP-glucuronosyltransferase, in contrast to its analogues with decreasing alkyl chain length. In addition, glucuronidation of this molecule was enhanced by clofibrate treatment but could not be detected in Gunn rats, which are deficient in bilirubin:UDP-glucuronosyltransferase, further indicating that the glucuronidation of this compound was catalysed by bilirubin:UDP-glucuronosyltransferase. The results suggest that 7,7,7-triphenylheptanoic acid may be a useful structural probe to investigate the molecular basis of glucuronidation of bilirubin and carboxylic acids.

Animals↗
PubMed1989

An investigation of the transverse topology of bilirubin UDP-glucuronosyltransferase in rat hepatic endoplasmic reticulum.

Bilirubin UDP-glucuronosyltransferase (UDPGT) activity in sealed hepatic microsomes from clofibrate-treated rats was highly latent and was fully expressed by disruption of vesicles with detergents. Antibodies raised against purified bilirubin UDPGT were used to study the transmembrane orientation of the protein to provide a molecular understanding of the UDPGT latency. Immunoblot analysis of sealed microsomes, and microsomes after treatment with proteinases, showed that only a small portion of the protein resides on the cytoplasmic side of the microsomal vesicles. Treatment of microsomes with sodium deoxycholate allowed subtilisin and proteinase K to cleave the transferase, causing loss of activity and the release of smaller immunodetectable peptides. Treatment of the purified bilirubin UDPGT with peptide N-glycosidase F indicated that the enzyme was a glycoprotein. A working model of the transmembrane topology of bilirubin UDPGT is described.

Animals↗
PubMed1989

Expression of a human liver cDNA encoding a UDP-glucuronosyltransferase catalysing the glucuronidation of hyodeoxycholic acid in cell culture.

A cDNA encoding a human liver UDPGT (HLUG 25) transcribed and translated in vitro showed that the encoded protein was synthesized as a precursor and was cleaved and glycosylated when dog pancreatic microsomes were present during translation. The UDPGT cDNA was transiently expressed in mammalian cell culture (COS-7 cells) resulting in the biosynthesis of a polypeptide of 52 kDa. This expressed UDPGT glycoprotein catalysed the glucuronidation of hyodeoxycholic acid forming an ether glucuronide. These results suggest that this UDPGT isoenzyme may be responsible for the glucuronidation of 6 alpha-hydroxy bile acids in human liver.

Animals↗
PubMed1989

Development of human liver UDP-glucuronosyltransferases.

The development of multiple UDPGT activities towards eight substrates has been studied in fetal term and adult post-mortem (less than 5 h after death) liver samples. Most fetal and term liver activities were less than 14% of adult values, except that towards 5-hydroxytryptamine which was present in fetal and term liver at adult levels. The majority of UDPGT activities develop to adult levels within 10-20 weeks postnatally, and even premature (30 weeks) which survive for up to 10 weeks will develop these enzyme activities. Immunoblot analysis of human liver microsomes and cDNA cloning of human UDPGT shows the existence of the family of isoenzymes in man, and it is important to determine the developmental pattern of individual drug glucuronidating enzymes in liver. Immunoblot analysis of developing liver shows the presence of two major UDPGT polypeptides in fetal liver, whereas more than five are observed in adult liver. The investigation of substrate specificity of individual UDPGTs by expression of cloned genes in COS-7 cells and the use of antibodies will facilitate the identification of enzymes present in perinatal liver.

Cloning, Molecular↗
PubMed1988

Expression of human liver epoxide hydrolase in Saccharomyces pombe.

Human liver microsomal epoxide hydrolase cDNA was inserted into the yeast expression vector pEVP11. The resulting recombinant plasmid was introduced into Saccharomyces pombe. The epoxide hydrolase protein and enzymic activity was subsequently expressed and identified in the 105,000 g pellet after centrifugal fractionation of homogenized yeast cells. This method will provide a useful source of human liver epoxide hydrolase, avoiding the problems of obtaining human tissue.

Cloning, Molecular↗
PubMed1988

A new microtechnique for the analysis of the human hepatic microsomal glucose-6-phosphatase system.

A microtechnique has been developed which enables a complete kinetic analysis of the human hepatic microsomal glucose-6-phosphatase system to be carried out in microsomes isolated from very small liver samples. Complete or partial deficiencies of any of the proteins of the glucose-6-phosphatase system resulting in Type 1a, 1b, 1c or 1d glycogen storage disease can be therefore be diagnosed using hepatic needle biopsy samples, whereas previous methods of diagnosis needed large wedge biopsy samples requiring laparotomy.

Female↗
PubMed1988

Induction of microsomal epoxide hydrolase by nitrosamines in rat liver. Effect on messenger ribonucleic acids.

Nitrosomethylethylamine and nitrosomethylpropylamine were found to be more potent inducers of rat liver microsomal epoxide hydrolase (styrene oxide hydrolase) than nitrosodiethylamine or nitrosodimethylamine. The time course of induction following a single administration of nitrosodimethylethylamine, nitrosomethylpropylamine or nitrosodiethylamine each showed a delay of 24 hr during which enzyme activity was unaltered. After that time activity increased and reached a maximum at between 72 and 120 hr. Increased enzyme activity following NDEA was paralleled by changes in the content of epoxide hydrolase in microsomes as measured by Western blots. Nitrosamines caused an increase of mRNA for epoxide hydrolase which was detected by probing Northern blots with a [32]-P labelled epoxide hydrolase cDNA and by in vitro translation of polyadenylated mRNA. Both methods showed a maximal increase at 72 hr after nitrosodiethylamine treatment but a significant increase was also observed at 24 hr although at this time no increase in enzyme activity was apparent.

Animals↗
PubMed1988

Cloning and substrate specificity of a human phenol UDP-glucuronosyltransferase expressed in COS-7 cells.

A rat kidney phenol UDP-glucuronosyltransferase cDNA was used to isolate a human liver phenol UDP-glucuronosyltransferase cDNA by screening of a human liver cDNA library in the expression vector lambda gt11. The 2.4-kilobase cDNA contained an open reading frame of 1593 base pairs coding for a protein of 531 residues. The human liver cDNA was subcloned into the vector pKCRH2. Transfection of this recombinant plasmid into COS-7 cells allowed the expression of a protein of approximately 55 kDa. The enzyme synthesized was a glycoprotein, as indicated by a reduction in molecular mass of approximately 3 kDa after biosynthesis in the presence of tunicamycin. The expressed enzyme rapidly catalyzed the glucuronidation of 1-naphthol, 4-methylumbelliferone, and 4-nitrophenol. The use of a related series of simple phenols provided an outline description of the substituent restrictions imposed upon the phenolic structures accepted as substrates. The glucuronidation of testosterone, androsterone, and estrone was not catalyzed by this cloned UDP-glucuronosyltransferase.

Amino Acid Sequence↗
PubMed1988

Examination of the substrate specificity of cloned rat kidney phenol UDP-glucuronyltransferase expressed in COS-7 cells.

A cDNA encoding a rat kidney UDP-glucuronyltransferase (UDPGT) was subcloned into the vector pKCRH2. Expression driven by the SV40 promoter produced enzymatically active UDPGT in COS-7 cells cultured in vitro. The appearance of enzyme activity was associated with an immunodetectable glycosylated UDPGT protein (Mr 53 kDa) in the cells. The expressed enzyme rapidly catalyzed the glucuronidation of 1-naphthol, 4-methylumbelliferone, and 4-nitrophenol. Studies using more than 20 compounds showed that the cloned UDPGT exhibited a restricted specificity towards planar phenols. A crude description of the molecular conformation of 4-alkylphenols accepted within the active site of the protein was obtained. The glucuronidation of morphine, thymol, menthol, testosterone, androsterone, or estrone was not catalyzed by this enzyme.

Animals↗
PubMed1988

The inadequacy of perinatal glucuronidation: immunoblot analysis of the developmental expression of individual UDP-glucuronosyltransferase isoenzymes in rat and human liver microsomes.

Two anti-rat UDP-glucuronosyltransferase (UDPGT) antibody preparations, exhibiting different specificity of recognition of UDPGT isoenzymes on immunoblot analysis, were used to investigate the molecular basis of the perinatal inadequacy of glucuronidation in rats and humans. Immunoblot analysis of microsomes from developing rat liver demonstrated that the deficiency in bilirubin and testosterone glucuronidation in the fetus was due to the absence of the UDPGT isoenzyme proteins responsible for these conjugations. In contrast, phenol UDPGT enzyme activity and protein was detectable in significant amounts in fetal rat liver (greater than 30% of adult levels). In human liver, only one major immunoreactive polypeptide was observed in fetal microsomes. The remaining UDPGTs present in adult human liver developed postnatally, in parallel with the appearance of enzyme activities. Therefore, there was a correlation between the development of enzyme activity and enzyme protein. The possible consequences of developmental inadequacy of conjugation reactions for the fetus is discussed.

Adult↗