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Biomedical subjects

B Burchell

Publications and source records attributed to B Burchell.

At least 55 records · Page 3Linked to original sources
PubMed1997

ATP-dependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocyte canalicular isoform MRP2.

Bilirubin is secreted from the liver into bile mainly as monoglucuronosyl and bisglucuronosyl conjugates. We demonstrate for the first time that ATP-dependent transport of both bilirubin glucuronides is mediated by the multidrug resistance protein (MRP1) as well as by the distinct canalicular (apical) isoform MRP2, also termed cMRP or cMOAT (canalicular multispecific organic anion transporter). In membrane vesicles from MRP1-transfected HeLa cells mono[3H]glucuronosylbilirubin and bis[3H]glucuronosylbilirubin (each at 0.5 microM) were transported with rates of 5.3 and 3.1 pmol/min per mg of protein respectively. Rat hepatocyte canalicular membrane vesicles, which contain Mrp2 (the rat equivalent of MRP2), transported mono[3H]glucuronosylbilirubin and bis[3H]glucuronosylbilirubin at rates of 8.9 and 8.5 pmol/min per mg of protein, whereas membrane vesicles from mutant liver lacking Mrp2 showed no transport of the conjugates. In membrane vesicles from human hepatoma Hep G2 cells, which predominantly expressed MRP2, transport rates were 8.3 and 4.4 pmol/min per mg of protein for monoglucuronosylbilirubin and bisglucuronosylbilirubin respectively. ATP-dependent transport of the glutathione S-conjugate -3H-leukotriene C4, an established high-affinity substrate for MRP1 and MRP2, was inhibited by both bilirubin glucuronides with IC50 values between 0.10 and 0.75 microM. The ratios of leukotriene C4 transport and bilirubin glucuronide transport, determined in the same membrane vesicle preparation, indicated substrate specificity differences between MRP1 and MRP2 with a preference of MRP2 for the glucuronides.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1997

A general strategy for the expression of recombinant human cytochrome P450s in Escherichia coli using bacterial signal peptides: expression of CYP3A4, CYP2A6, and CYP2E1.

Heterologous expression of unmodified recombinant human cytochrome P450 enzymes (P450s) in Escherichia coli has proved to be extremely difficult. To date, high-level expression has only been achieved after altering the 5'-end of the native cDNA, resulting in amino acid changes within the P450 protein chain. We have devised a strategy whereby unmodified P450s can be expressed to high levels in E. coli, by making NH2-terminal translational fusions to bacterial leader sequences. Using this approach, we initially tested two leader sequences, pelB and ompA, fused to CYP3A4. These were compared with an expression construct producing a conventional NH2-terminally modified CYP3A4 (17alpha-3A4). Both leader constructs produced spectrally active, functional protein. Furthermore, the ompA-3A4 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with 17alpha-3A4. We then tested the ompA leader with CYP2A6 and CYP2E1, again comparing with the conventional (17alpha-) approach. As before, the leader construct produced active enzyme, and, for CYP2E1 at least, gave a higher level of expression than the 17alpha-construct. The ompA fusion strategy thus appears to represent a significant advance for the expression of P450s in E. coli, circumventing the previous need for individual optimization of P450 sequences for expression.

Aminolevulinic Acid↗
PubMed1997

Preclinical prediction of factors influencing the elimination of 5,6-dimethylxanthenone-4-acetic acid, a new anticancer drug.

The glucuronidation of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a newly developed anticancer drug, was investigated in vitro to determine factors likely to affect the elimination of this compound in patients. Human liver microsomal DMXAA glucuronidation followed Michaelis-Menten kinetics, with a mean apparent Km of approximately 100 microM. Two cDNA-expressed UGT isoforms, UGT1*02 and UGT2B7, had the capacity to glucuronidate DMXAA, although comparative kinetic and inhibitor studies were more consistent with a greater contribution of UGT2B7 to the human hepatic reaction. Microsomal DMXAA glucuronide formation was screened for inhibition by drugs known to be eliminated by glucuronidation. Of the drugs screened, significant inhibition was observed with diclofenac, epirubicin, indomethacin, R,S-ketoprofen, lorazepam, S-naproxen, oxazepam, and temazepam; apparent Ki values ranged from 9.5-318 microM. These values are substantially above unbound concentrations of the individual drugs achieved in vivo. DMXAA glucuronide was found to be unstable at physiological pH values, and the rate of degradation was marginally increased in the presence of albumin. Taken together, these data indicate that the kinetics of DMXAA glucuronidation in vivo are likely to be linear and unaffected by the coadministration of most glucuronidated drugs, but plasma DMXAA clearance may be decreased in patients with renal dysfunction. This study illustrates the utility of in vitro techniques for the prediction of potential drug interactions and other dispositional characteristics of newly developed anticancer drugs before their administration to patients.

Antineoplastic Agents↗
PubMed1997

Influence of the angiotensin converting enzyme I/D gene polymorphisms on left ventricular diastolic filling in patients with essential hypertension.

BACKGROUND: An insertion/deletion (I/D) polymorphism in the angiotensin converting enzyme (ACE) gene accounts for 50% of the variance in serum ACE activity. ACE is responsible for the generation of angiotensin II, which not only has pressor and mitogenic activities but also exerts effects on left ventricular diastolic performance. OBJECTIVE: To investigate the contribution of genetic polymorphisms at the ACE gene to the development of diastolic functional abnormalities in 100 patients with essential hypertension. METHODS AND RESULTS: The left ventricular mass (LVMI) of each patient was assessed echocardiographically. We calculated peak and integral early:late left ventricular diastolic filling ratios (E:AP, and E:AI, respectively) and determined the ACE genotype from leukocyte DNA. There was no significant difference in age, sex, blood pressure and LVMI among genotype groups. Analysis of covariance modelled for indices of diastolic function, adjusted for age, sex, heart rate and LVMI, demonstrated that the E:AP interacted with age (P < 0.0001), heart rate (P < 0.001) and ACE genotype (P = 0.018). Similarly, the E:AI interacted with age (P < 0.001), heart rate (P = 0.025) and ACE genotype (P = 0.047). There was a strong correlation between the E:AP and the LVMI for the DD group (r = -0.81, P < 0.0001) but not for the ID (r = -0.03, P = 0.83) and II (r = -0.23, P = 0.23) groups. CONCLUSIONS: These findings suggest that that the I/D polymorphism of the ACE gene influences the relationship between left ventricular mass and echocardiographic left ventricular diastolic filling abnormalities in patients with essential hypertension.

Adult↗
PubMed1997

The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence.

This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution. Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably. At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily. Comparison of a relatedness tree of proteins leads to the definition of 33 families. It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution. For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g. 'human UGT2B4' and 'mouse Ugt2b5'. We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown. Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g. 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g. UGT1A1, UGT1A2, ... UGT1A12). When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized. We suggest that the Human Gene Nomenclature Guidelines (http://www.gene.acl.ac.uk/nomenclature/guidelines.html++ +) be used for all species other than the mouse and Drosophila. Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g. UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines. It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.

Amino Acid Sequence↗
PubMed1997

Genetic and environmental factors associated with variation of human xenobiotic glucuronidation and sulfation.

Glucuronidation and sulfation are phase 2 metabolic reactions catalyzed by large families of different isoenzymes in man. The textbook view that glucuronidation and sulfation lead to the production of harmless conjugates for simple excretion is not valid. Biologically active and toxic sulfates and glucuronides are produced and leed to adverse drug reactions, including immune hypersensitivity. Considerable variation in xenobiotic conjugation is observed as a result of altered expression of UDP-glucuronosyltransferases (UGTs) and sulfotransferases (STs). Recent cloning and expression of human cDNA encoding UGTs and STs has facilitated characterization of isoform substrate specificity, which has been further validated using specific antibodies and human tissue fractions. The availability of cloned/expressed human enzymes and specific antibodies has enabled the investigation of xenobiotic induction and metabolic disruption leeding to adverse responses. Genetic polymorphisms of glucuronidation and sulfation are known to exist although the characterization and assessment of the importance of these variations are hampered by appropriate ethical studies in men with suitable safe model compounds. Genetic analysis has allowed molecular identification of defects in well-known hyperbilirubinemias. However, full characterization of the specific functional roles of human UGTs and STs requires rigorous kinetic and molecular analyses of the role of each enzyme in vivo through the use of specific antibodies and inhibitors. This will leed to the better prediction of variation of xenobiotic glucuronidation and sulfation in man.

Glucuronates↗
PubMed1997

Arginine 52 and histidine 54 located in a conserved amino-terminal hydrophobic region (LX2-R52-G-H54-X3-V-L) are important amino acids for the functional and structural integrity of the human liver UDP-glucuronosyltransferase UGT1*6.

The hepatic UDP-glucuronosyltransferase UGT1*6 is actively involved in the glucuronidation of short and planar phenols in humans. Based on the irreversible inhibition of the enzyme on chemical modification by 2,3-butanedione and diethyl pyrocarbonate, the roles of His54 and Arg52 were investigated by oligonucleotide site-directed mutagenesis. These amino acids belong to a consensus sequence LX2-R52-G-H54-X3-V-L located in a conserved hydrophobic region of the variable amino-terminal domain of UGT. Arg52 was replaced by alanine (mutant R52A), and His54 was replaced by alanine or glutamine (mutants H54A and H54Q). The immunological and catalytic properties of UGT1*6 and mutants were examined after stable expression in V79 cell lines. Immunoblots and immunoprecipitation studies revealed that the mutant and UGT1*6 proteins were expressed in the microsomal membranes in similar amounts. However, replacement of His54 by glutamine led to a complete loss of activity toward 4-methylumbelliferone, and the Vmax value was decreased 4-5-fold in the mutants R52A and H54A compared with the wild-type enzyme. The dissociation constants that characterize the binding of 4-methylumbelliferone and UDP-glucuronic acid to UGT1*6 were not greatly affected by the mutations. Interestingly, H54Q was not recognized by specific antibodies to the amino-terminal portion of UGT1*6, thereby indicating that this amino acid was critical to antibody recognition. In contrast, the mutants R52A and H54A could not be differentiated from the wild-type protein by pH optimum or thermal denaturation. Furthermore, these mutants were still sensitive to irreversible inhibition by diethyl pyrocarbonate and 2,3-butanedione, with second-order inactivation constant values similar to those obtained for UGT1*6. Altogether, the strict conservation of His54 and Arg52 and the mutational analysis of these residues suggest that these amino acids in the hydrophobic amino-terminal consensus sequence LX2-R52-G-H54-X3-V-L are important for the function and the structure required for optimal catalytic efficiency of UGT1*6.

Animals↗
PubMed1997

Heterologous expression of human drug-metabolizing enzymes.

This article is a report on a symposium held at the March 1997 meeting of the American Society for Pharmacology and Experimental Therapeutics in San Diego. Current developments in the heterologous expression of cytochrome P450, NADPH-cytochrome P450 reductase, glutathione transferase, and UDP-glucuronosyltransferase enzymes are described. Systems include bacteria, insect cells, and transient and stable mammalian cells. Uses of the products are described for discernment of which enzymes are involved in metabolism of drugs, genotoxicity assays, mutagenesis (for structure-activity relationships), large scale production of enzyme products, antibody production, and production of proteins for biophysical studies.

Enzymes↗
PubMed1996

Coexpression of a human P450 (CYP3A4) and P450 reductase generates a highly functional monooxygenase system in Escherichia coli.

The catalytic activities of recombinant cytochrome P450s expressed in E. coli have been impeded by the absence of endogenous P450 reductase. To solve this problem, we coexpressed P450 reductase with CYP3A4. Membranes from this strain contained 215 pmol P450/mg protein and a reductase activity of 1315 nmol cytochrome c reduced/min per mg. We detected 6beta-hydroxylation of testosterone and oxidation of nifedipine in vivo with turnover numbers of 15.2 and 17.3 min(-1), respectively. These values compare favourably with those obtained using an optimally reconstituted system. Our data demonstrate that a catalytically efficient human P450 system can be generated in E. coli.

Base Sequence↗
PubMed1996

Genetic variation in bilirubin UPD-glucuronosyltransferase gene promoter and Gilbert's syndrome.

BACKGROUND: The genetic basis of Gilbert's syndrome is ill-defined. This common mild hyperbilirubinaemia sometimes presents as an intermittent jaundice. A reduced hepatic bilirubin UPD- glucuronosyltransferase (UGT) is associated with this syndrome. We have examined variation in the gene encoding the UGT1*1 enzyme and serum bilirubin levels in a Scottish population. METHODS: Blood was collected from 12 patients with confirmed or suspected Gilbert's syndrome, from 6 members of a family with 4 Gilbert members, and from 77 non-smoking, alcohol-free, drug-free volunteers recruited from the staff of a teaching hospital in Dundee. Polymerase chain reaction amplification was used to examine sequence variation of the promoter upstream of the UGT1*1 exon I. Genotypes were assigned as follows: 6/6 (homozygous for a common allele bearing the sequence [TA](6)TAA), 7/7 (homozygous for a rarer allele with the sequence [TA](7)TAA), and 6/7 (heterozygous with one of each allele). FINDINGS: Individuals in the population with the 7/7 genotype had significantly higher bilirubin concentrations than those who had the 6/7 or 6/6 genotype. 14 volunteers underwent a 24 h fasting test to see if they had Gilbert's syndrome, and all four positives had the 7/7 genotype. One confirmed Gilbert's patient, two recurrent jaundice patients (with suspected Gilbert's syndrome), and nine clinically diagnosed cases had the 7/7 genotype. Segregation of the 7/7 genotype with the Gilbert phenotype was also demonstrated in the family with four affected members. The frequency of the 7/7 genotype in this eastern Scottish population was 10-13%. INTERPRETATION: In a healthy population there was an association between variation in bilirubin concentration and a mutation within the gene encoding the enzyme bilirubin UGT. This and other findings suggest the existence of a mild and a more severe form of Gilbert's syndrome, depending on whether the gene defect lies in the promoter sequence upstream of UGT1*I exon I, as here (mild), or in the coding sequence (severe) of the gene.

Adult↗
PubMed1996

Heterogeneous expression of sulphotransferases in periportal and perivenous hepatocytes prepared from male and female rat liver.

Sulphotransferase (ST) is a family of enzymes responsible for metabolism and detoxication of endobiotics and xenobiotics. We investigated the hepatic acinar distribution of three sulphotransferases: phenol sulphotransferase (PST), oestrogen sulphotransferase (EST), and hydroxysteroid sulphotransferase (HST) in male and female rat livers by measurement of enzyme activities in isolated periportal and perivenous hepatocytes. The distribution was confirmed by immunohistochemistry. EST activity was located predominantly in the perivenous hepatocytes in male rats but not in female rats, where residual activity is catalysed by another ST. HST activity was not significantly different in periportal and perivenous hepatocytes in either male or female rats. For PST, a more widespread distribution was observed, with slight predominance in the periportal regions. The results indicate heterogeneous distribution of ST isoenzymes in the periportal and perivenous hepatocytes isolated from male and female rat livers.

Animals↗
PubMed1996

The ontogeny of key endoplasmic reticulum proteins in human embryonic and fetal red blood cells.

Recently, using immunohistochemical methods, we surprisingly found that endoplasmic reticulum glucose-6-phosphatase is present in human embryonic and fetal red blood cells (RBCs) but not in adult RBCs. The fact that an endoplasmic reticulum enzyme, whose major site of expression in adults is the liver, is present in human embryonic and fetal RBCs, particularly nucleated cells, indicated that it would be sensible to determine whether these cells also contain other endoplasmic reticulum enzyme systems normally found in adult liver. Therefore, we have studied the expression of other endoplasmic reticulum proteins and found that human embryonic and fetal RBC precursors contain other protein components of the glucose-6-phosphatase system, ie, the phosphate and glucose transport proteins as well as other enzymes (eg, uridine diphosphate-glucuronosyltransferases, cytochrome P450 isozymes, nicotinamide adenine dinucleotide phosphate cytochrome P450 oxidoreductase, and prostaglandin H synthase). In addition, we also found the predominantly cytosolic markers 15-hydroxyprostaglandin dehydrogenase, prostaglandins PGE2 and 13,14-dihydro-15-keto-PGE2. The expression of key enzymes that control glucose production, detoxification of endobiotics and xenobiotics, and the regulation of prostaglandin levels in embryonic and early fetal RBCs means that these cells may have an important role in protecting the developing conceptus before it establishes an efficient circulation and before all tissues fully express their normal complement of these enzymes.

Adult↗