Mechanisms of maternal aneuploidy: FISH analysis of oocytes and polar bodies in patients undergoing assisted conception.

We have examined unfertilised oocytes and their first polar bodies (PBs) to determine the way in which the frequency of whole chromosome imbalance compares with that involving single chromatids and whether the precocious separation of chromatids prior to anaphase I affects all pairs of chromosomes. We have applied the technique of fluorescent in situ hybridisation in a three-stage method by using locus-specific probes for chromosomes 13 and 21 and alpha-satellite probes for chromosomes 1, 9, 16, 18 and X to determine the chromosome status of oocytes and their PBs. We obtained analysable results from 127 oocytes and 57 PBs from 72 patients of average age 33 years. Six oocytes and three PBs had extra signals but, of these, three were derived from a single patient, aged 26. Anomalies were seen in chromosomes 13, 16, 18, X and, notably, 21 but none were observed in chromosomes 1 and 9. Half of the anomalies involved additional chromatids rather than whole chromosomes. Since particular chromatids were found to be prematurely separated in the metaphase II oocyte, this may provide further evidence for an additional mechanism of maternal aneuploidy that operates at anaphase II. Detailed analyses of both oocytes and PBs have elucidated possible mechanisms leading to aneuploid gametes in this group of patients with fertility problems.

Introduction of heteroplasmic mitochondrial DNA (mtDNA) from a patient with NARP into two human rho degrees cell lines is associated either with selection and maintenance of NARP mutant mtDNA or failure to maintain mtDNA.

Mitochondria from a patient heteroplasmic at nucleo-tide position 8993 of mitochondrial DNA (mtDNA) were introduced into two human tumour cell lines lacking mtDNA. The donor mitochondria contained between 85 and 95% 8993G:C mtDNA. All detectable mtDNA in the mitochondrially transformed cells contained the pathological 8993G:C mutation 3 months after transformation. These results suggest that 8993G:C mtDNA had a selective advantage over 8993T:A mtDNA in both lung carcinoma and osteo-sarcoma cell backgrounds. In contrast, two other presumed pathological mtDNA variants were lost in favour of 'wild-type' mtDNA molecules in the same lung carcinoma cell background. Taken together, these findings suggest that the transmission bias of mtDNA variants is dependent upon a combination of nuclear background and mtDNA genotype. A second phenomenon observed was a marked decrease in the growth rate of many putative transformed cell lines after 6 weeks of culturing in selective medium, and in these cell lines mtDNA was not readily detectable by Southern blotting. Restriction endonuclease analysis and sequencing of amplified mtDNA demonstrated that the slow growing cells contained little or no mtDNA. It is concluded that these cells represented transient mitochondrial transformants.

Drug glucuronidation by human renal UDP-glucuronosyltransferases.

The UDP-glucuronosyltransferases catalyse the conjugation of glucuronic acid to a wide variety of endobiotics and xenobiotics, representing one of the major conjugation reactions in the conversion of both exogenous (e.g. drugs and pesticides) and endogenous compounds (e.g. bilirubin and steroid hormones). The liver is the major site of glucuronidation, however a number of extrahepatic tissues exhibit particular UDP-glucuronosyltransferase activities. The present study was undertaken to assess the human renal UDP-glucuronosyltransferase system. Enzymatic analysis of human kidney showed that a limited number of UDP-glucuronosyltransferase isoforms were expressed in this tissue. However the level of renal UGT activity towards the anaesthetic propofol was higher compared with human liver. The glucuronidation of propofol is catalysed by UGT1A8/9 suggesting higher levels of this isoform in the kidney. Immunoblot analysis revealed two major UDP-glucuronosyltransferase immunopositive bands to be present in human kidney as compared to four major bands in human liver. The human kidney was capable of conjugating various structurally diverse drugs and xenobiotics.

Regulation of the human bilirubin UDP-glucuronosyltransferase gene.

The human UGT1 gene is a single copy gene consisting of four common exons and more than 13 variable exons which span more than 200 kb of the human genome. A single variable exon is spliced to the four common exons to form the mRNA for synthesis of a single UDP-glucuronosyltransferase (UGT) isoenzyme. Treatment of humans or hepatoma cell lines with drugs such as phenobarbital causes the induction of hepatic bilirubin UGT by increased transcription from the UGT1 gene. The upstream region of UGT1*1 (bilirubin UGT) was sequenced and found to contain consensus sequences for several transcriptional regulatory elements including a 'BARBIE box'. An unusual 'TATA' promoter sequence A(TA)6TAA was also observed. The 5' region flanking the UGT1*1 exon when cloned into reporter constructs and transfected into four cells lines was capable of promoting reporter gene expression, but not when transfected into monkey kidney cell fibroblasts (COS-7 cells) indicating a cell specific expression. Sequential deletion of the 5' flanking region in the plasmid constructs did not cause any significant reduction in reporter expression. Treatment of cells transfected with these plasmid constructs with drugs did not cause a significant increase in reporter expression except with retinoic acid plus WY 14643. Introduction of an additional two base pairs (TA) into the 'TATA' box of the 5' gene sequence (as observed in Gilbert's patients) did not significantly change reporter expression levels. The regulation of the biliruibin UGT gene by drugs is not yet understood and it will be important to identify additional genetic elements possibly further than -2kb upstream of the UGT1*1 coding region, which regulate the expression of this gene.

Specificity of human UDP-glucuronosyltransferases and xenobiotic glucuronidation.

Several human liver UDP-Glucuronosyltransferases (UGTs) have been cloned and the cDNAs expressed in heterologous cell lines. This technological advance has allowed the assessment of the functional substrate specificity of these UGTs. The problems which may be encountered with the latency and assay of UGTs are briefly described. The data accumulated to date indicate that the Km, and possibly the Vmax/Km, for individual substrates are the best parameters to assess the specificity of the enzymes towards xenobiotic molecules. The substrate specificity of seven UGTs has been summarised from the currently available information. Of these, UGT1*02 and UGT2B8 appear to be key isoforms in the glucuronidation of a wide range of xenobiotic substrates. Additional UGTs have yet to be identified and characterised and their future inclusion may provide further insights. Finally, the functional role of each UGT in vivo has to be determined.

Human UDP-glucuronosyl transferases: chemical defence, jaundice and gene therapy.

Human UDP-glucuronosyltransferases (UDPGTs) are a family of enzymes which detoxify many hundreds of compounds by their conjugation to glucuronic acid, rendering them both harmless and more water soluble, hence, excretable. The level of expression of each UDPGT isoform in the body is the result of interplay between temporal, tissue-specific and environmental regulators. This complexity contributes to the difficulty in predicting the metabolic fate of compounds. Genetic defects and polymorphisms affecting individual isoform activities have deleterious and potentially lethal effects, as exemplified by the severe hyperbilirubinaemia observed in Crigler-Najjar Syndrome. Such severe genetic defects in bilirubin glucuronidation are obvious candidates for antenatal screening and gene therapy.

The protein phosphatases involved in cellular regulation. Evidence that dephosphorylation of glycogen phosphorylase and glycogen synthase in the glycogen and microsomal fractions of rat liver are catalysed by the same enzyme: protein phosphatase-1.

Glycogen synthase (labelled in sites-3) and glycogen phosphorylase from rabbit skeletal muscle were used as substrates to investigate the nature of the protein phosphatases that act on these proteins in the glycogen and microsomal fractions of rat liver. Under the assay conditions employed, glycogen synthase phosphatase and phosphorylase phosphatase activities in both subcellular fractions could be inhibited 80-90% by inhibitor-1 or inhibitor-2, and the concentrations required for half-maximal inhibition were similar. Glycogen synthase phosphatase and phosphorylase phosphatase activities coeluted from Sephadex G-100 as broad peaks, stretching from the void volume to an apparent molecular mass of about 50 kDa. Incubation with trypsin decreased the apparent molecular mass of both activities to about 35 kDa, and decreased their I50 for inhibitors-1 and -2 in an identical manner. After tryptic digestion, the I50 values for inhibitors-1 and -2 were very similar to those of the catalytic subunit of protein phosphatase-1 from rabbit skeletal muscle. The glycogen and microsomal fractions of rat liver dephosphorylated the beta-subunit of phosphorylase kinase much faster than the alpha-subunit and dephosphorylation of the beta-subunit was prevented by the same concentrations of inhibitor-1 and inhibitor-2 that were required to inhibit the dephosphorylation of phosphorylase. The same experiments performed with the glycogen plus microsomal fraction from rabbit skeletal muscle revealed that the properties of glycogen synthase phosphatase and phosphorylase phosphatase were very similar to the corresponding activities in the hepatic glycogen fraction, except that the two activities coeluted as sharp peaks near the void volume of Sephadex G-100 (before tryptic digestion). Tryptic digestion of the hepatic glycogen and microsomal fractions increased phosphorylase phosphatase about threefold, but decreased glycogen synthase phosphatase activity. Similar results were obtained with the glycogen plus microsomal fraction from rabbit skeletal muscle or the glycogen-bound form of protein phosphatase-1 purified to homogeneity from the same tissue. Therefore the divergent effects of trypsin on glycogen synthase phosphatase and phosphorylase phosphatase activities are an intrinsic property of protein phosphatase-1. It is concluded that the major protein phosphatase in both the glycogen and microsomal fractions of rat liver is a form of protein phosphatase-1, and that this enzyme accounts for virtually all the glycogen synthase phosphatase and phosphorylase phosphatase activity associated with these subcellular fractions.