Wei-Jun Liang

Vitamin C transport systems of mammalian cells.

Vitamin C is essential for many enzymatic reactions and also acts as a free radical scavenger. Specific non-overlapping transport proteins mediate the transport of the oxidized form of vitamin C, dehydroascorbic acid, and the reduced form, Lascorbic acid, across biological membranes. Dehydroascorbic acid uptake is via the facilitated-diffusion glucose transporters, GLUT 1, 3 and 4, but under physiological conditions these transporters are unlikely to play a major role in the uptake of vitamin C due to the high concentrations of glucose that will effectively block influx. L-ascorbic acid enters cells via Na+-dependent systems, and two isoforms of these transporters (SVCT1 and SVCT2) have recently been cloned from humans and rats. Transport by both isoforms is stereospecific, with a pH optimum of ~ 7.5 and a Na+: ascorbic acid stoichiometry of 2 : 1. SVCT2 may exhibit a higher affinity for ascorbic acid than SVCT1 but with a lower maximum velocity. SVCT1 and SVCT2 are predicted to have 12 transmembrane domains, but they share no structural homology with other Na+ co-transporters. Potential sites for phosphorylation by protein kinase C exist on the cytoplasmic surface of both proteins, with an additional protein kinase A site in SVCT1. The two isoforms also differ in their tissue distribution: SVCT1 is present in epithelial tissues, whereas SVCT2 is present in most tissues with the exception of lung and skeletal muscle.

OPRM1 and CYP2B6 Gene Variants as Risk Factors in Methadone‐Related Deaths

Methadone is a medication valued for its effectiveness in the treatment of heroin addiction; however, many fatal poisonings associated with its use have been reported over the years. We have examined the association between CYP2B6 and µ‐opioid receptor (OPRM1) gene variations and apparent susceptibility to methadone poisoning. Genomic DNA was extracted from postmortem whole blood of 40 individuals whose deaths were attributed to methadone poisoning. The presence of CYP2B6*4,*9, and *6 alleles and the OPRM1 A118G variant was determined by SNP genotyping. CYP2B6 *4, *9, and *6 alleles were found to be associated with higher postmortem methadone concentrations in blood (P ≤ 0.05). OPRM1 A118G was also associated with higher postmortem methadone concentrations in blood but not to a level of statistical significance (P = 0.39). In these methadone‐related deaths, OPRM1 118GA was associated with higher postmortem benzodiazepine concentrations (P = 0.04), a finding not associated with morphine‐related deaths. The risk of a methadone‐related fatality during treatment may be evaluated in part by screening for CYP2B6*6 and A118G.

CYP2B6 and OPRM1 gene variations predict methadone-related deaths

The largest proportion of methadone‐associated deaths occurs during the drug induction phase. We analysed methadone‐related fatalities for gene variations linked with methadone action. A significant association between high methadone concentrations and the CYP2B6*6 allele characteristic of the slow metabolizer phenotype was identified. We suggest that the risk of methadone fatality may be predetermined in part by the CYP2B6*6 allele. A significant correlation was also observed between post‐mortem benzodiazepine concentrations and the OPRM1 A118G allele GA in methadone‐related fatalities. Screening for these susceptibility variations prior to methadone prescription could assist in reducing the potential for serious adverse effects.

The gusBC genes of Escherichia coli encode a glucuronide transport system

Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of beta-glucuronides with synthetic [(14)C]phenyl-1-thio-beta-d-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of beta-d-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respiration-generated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed.

Increased DNA Methylation of ABCB1, CYP2D6, and OPRM1 Genes in Newborn Infants of Methadone-Maintained Opioid-Dependent Mothers.

Objective To investigate whether in utero opioid exposure, which has been linked to adverse neurodevelopmental and social outcomes, is associated with altered DNA methylation of opioid‐related genes at birth. Study design Observational cohort study of 21 healthy methadone‐maintained opioid‐dependent mother‐infant dyads consecutively delivered at >36 weeks of gestation, and 2 comparator groups: smoking, “deprived” opioid‐naïve mother‐infant dyads (n = 17) and nonsmoking, “affluent” opioid‐naïve mother‐infant dyads (n = 15). DNA methylation of ABCB1, CYP2D6, and OPRM1 genes for mothers and babies was determined from buccal swabs. Plasma methadone concentrations were additionally measured for methadone‐maintained opioid‐dependent mothers. Results DNA methylation for ABCB1 and CYP2D6 was similar in opioid‐naïve infants compared with their mothers, but was less for OPRM1 (3 ± 1.6% vs 8 ± 1%, P < .0005). Opioid‐exposed newborns had similar DNA methylation to their mothers for all genes studied and greater methylation of ABCB1 (18 ± 4.8% vs 3 ± 0.5%), CYP2D6 (92 ± 1.2% vs 89 ± 2.4%), and OPRM1 (8 ± 0.3% vs 3 ± 1.6%) compared with opioid‐naïve newborns (P < .0005 for all 3 genes). Infant DNA methylation was not related to birth weight, length of hospital stay, maternal smoking, dose or plasma concentration of methadone at delivery, or postcode of residence. Conclusions In utero exposure to opioids is associated with increased methylation of opioid‐related genes in the newborn infant. It is not clear whether these findings are due to opioid exposure per se or other associated lifestyle factors.

Laboratory and field validation of a simple method for detecting four species of non-native freshwater fish using eDNA

This paper presents the first phase in the development and validation of a simple and reliable environmental (e)DNA method using conventional PCR to detect four species of non-native freshwater fish: pumpkinseed Lepomis gibbosus, sunbleak Leucaspius delineatus, fathead minnow Pimephales promelas and topmouth gudgeon Pseudorasbora parva. The efficacy of the approach was demonstrated in indoor tank (44 l) trials in which all four species were detected within 24 h. Validation was through two field trials, in which L. gibbosus was detected 6-12 h after its introduction into outdoor experimental ponds and P. parva was successfully detected in disused fish rearing ponds where the species was known to exist. Thus, the filtration of small (30 ml) volumes of pond water was sufficient to capture fish eDNA and the approach emphasised the importance of taking multiple water samples of sufficient spatial coverage for detecting species of random or patchy distribution.