A convenient lactic dehydrogenase-coupled assay for determining pyridoxal 5'-phosphate in plasma

An assay for determining the concentration of pyridoxal 5'-phosphate in plasma from 0.4 ml whole blood is reported. The assay consists of incubating deproteinized plasma with -serine apodehydratase from Escherichia coli in 0.5 N-2-hydroxyethyl-piperazine-N'-3-propanesulfonic acid, pH 7.8, at 37[deg]C for 15 min, and then determining the -serine dehydratase activity of an aliquot of the incubation mixture. A lactic dehydrogenase-coupled assay (at 25[deg]C) was used to measure the rate of enzymically catalyzed conversion of D-serine to pyruvate, wherein depletion of NADH was followed continuously at 340 nm. The concentration of pyridoxal 5'-phosphate in the plasma sample was estimated from the enzymic activity which is a linear function of the amount of pyridoxal 5'-phosphate present in the assay.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23279/1/0000216.pd

Biomarkers differentiate drug-induced liver injury from other liver injury: PONDER study

Background and Aim: Drug-induced liver injury (DILI) is a known complication of volatile anesthetic (VA) agents, and, despite being rare, DILI can be serious. One mechanism of VA-DILI occurs via interleukin 4 (IL-4)driven upregulation of cytochrome P450-2E1, leading to the formation of drug metabolites (haptens) that trigger IL-4-driven antigen-specific T cells and autoantibodies. Our group has developed biomarkers for liver injury and have examined this in patients before and after VA exposure. The aim of this prospective study was to determine the early markers of VA-DILI. Methods: We prospectively followed patients having a VA general anesthetic (sevoflurane and/or desflurane) and compared them with those who received regional or total intravenous anesthesia. Exclusion criteria were known liver disease or any episode of significant hypotension. Baseline data on patient demographics and comorbidities were collected, and blood was analyzed for liver biochemistry, macrophage activation markers (CD206, CD163), and IgG1 and IgG4 antibodies to JHDN5 (the CYP2E1 epitope) and trifluoroacetyl (TFA), the VA drug hapten. Follow-up blood samples were taken 48 h postoperatively and compared with baseline results. DILI was defined as an alanine aminotransferase (ALT) level greater than two times the upper limit of normal (ULN) and post-review agreement by an expert panel, taking into account the pattern of liver function test result derangement and intraoperative events. Results: Of 229 patients recruited, 16 developed an ALT level > 2 × ULN. Twelve were considered likely to have VA-DILI, including four with an ALT rise >3 × ULN. There was a trend to associate VA-DILI with obesity (RR, 2.98; P = 0.063); however, the association with dyslipidemia (RR, 1.47; P = 0.72), male sex (RR, 1.18; P = 0.76), history of atopy (RR, 1.16; P = 0.79), and heavy ethanol consumption (RR, 1.09; P = 0.89) was not statistically significant. Prior VA exposure was not a risk factor (RR, 0.89; P = 0.83). There was a rise in CD206 and decline in CD163 from baseline in all patients. However, in the patients with VA-DILI, the levels were significantly different from all other groups. TFA IgG1 and IgG4 antibodies were elevated in the VA-DILI group when compared with controls. Conclusion: Recognizing that our results may be skewed by our cohort, this work suggests the known immunological pathway mediated by IL-4 in response to an injury: rise in CD206 to stimulate an inflammatory response, and decrease in CD163 to modulate the response. The increase in TFA IgG1 and IgG4 antibodies in the VA-DILI group is consistent with metabolism and the heightened immune response in those who develop DILI. At this early juncture, JHDN5 IgG4 autoantibodies were not detected. Ongoing work is looking at other DILI, and how these markers can be used in DILI

Copy Number Variation Affecting the Photoperiod-B1 and Vernalization-A1 Genes Is Associated with Altered Flowering Time in Wheat (Triticum aestivum)

The timing of flowering during the year is an important adaptive character affecting reproductive success in plants and is critical to crop yield. Flowering time has been extensively manipulated in crops such as wheat (Triticum aestivum L.) during domestication, and this enables them to grow productively in a wide range of environments. Several major genes controlling flowering time have been identified in wheat with mutant alleles having sequence changes such as insertions, deletions or point mutations. We investigated genetic variants in commercial varieties of wheat that regulate flowering by altering photoperiod response (Ppd-B1 alleles) or vernalization requirement (Vrn-A1 alleles) and for which no candidate mutation was found within the gene sequence. Genetic and genomic approaches showed that in both cases alleles conferring altered flowering time had an increased copy number of the gene and altered gene expression. Alleles with an increased copy number of Ppd-B1 confer an early flowering day neutral phenotype and have arisen independently at least twice. Plants with an increased copy number of Vrn-A1 have an increased requirement for vernalization so that longer periods of cold are required to potentiate flowering. The results suggest that copy number variation (CNV) plays a significant role in wheat adaptation

Flavopiridol Pharmacogenetics: Clinical and Functional Evidence for the Role of SLCO1B1/OATP1B1 in Flavopiridol Disposition

Flavopiridol is a cyclin-dependent kinase inhibitor in phase II clinical development for treatment of various forms of cancer. When administered with a pharmacokinetically (PK)-directed dosing schedule, flavopiridol exhibited striking activity in patients with refractory chronic lymphocytic leukemia. This study aimed to evaluate pharmacogenetic factors associated with inter-individual variability in pharmacokinetics and outcomes associated with flavopiridol therapy.Thirty-five patients who received single-agent flavopiridol via the PK-directed schedule were genotyped for 189 polymorphisms in genes encoding 56 drug metabolizing enzymes and transporters. Genotypes were evaluated in univariate and multivariate analyses as covariates in a population PK model. Transport of flavopiridol and its glucuronide metabolite was evaluated in uptake assays in HEK-293 and MDCK-II cells transiently transfected with SLCO1B1. Polymorphisms in ABCC2, ABCG2, UGT1A1, UGT1A9, and SLCO1B1 were found to significantly correlate with flavopiridol PK in univariate analysis. Transport assay results indicated both flavopiridol and flavopiridol-glucuronide are substrates of the SLCO1B1/OATP1B1 transporter. Covariates incorporated into the final population PK model included bilirubin, SLCO1B1 rs11045819 and ABCC2 rs8187710. Associations were also observed between genotype and response. To validate these findings, a second set of data with 51 patients was evaluated, and overall trends for associations between PK and PGx were found to be consistent.Polymorphisms in transport genes were found to be associated with flavopiridol disposition and outcomes. Observed clinical associations with SLCO1B1 were functionally validated indicating for the first time its relevance as a transporter of flavopiridol and its glucuronide metabolite. A second 51-patient dataset indicated similar trends between genotype in the SLCO1B1 and other candidate genes, thus providing support for these findings. Further study in larger patient populations will be necessary to fully characterize and validate the clinical impact of polymorphisms in SLCO1B1 and other transporter and metabolizing enzyme genes on outcomes from flavopiridol therapy

Topoisomerase IIα Binding Domains of Adenomatous Polyposis Coli Influence Cell Cycle Progression and Aneuploidy

Truncating mutations in the tumor suppressor gene APC (Adenomatous Polyposis Coli) are thought to initiate the majority of colorectal cancers. The 15- and 20-amino acid repeat regions of APC bind beta-catenin and have been widely studied for their role in the negative regulation of canonical Wnt signaling. However, functions of APC in other important cellular processes, such as cell cycle control or aneuploidy, are only beginning to be studied. Our previous investigation implicated the 15-amino acid repeat region of APC (M2-APC) in the regulation of the G2/M cell cycle transition through interaction with topoisomerase IIalpha (topo IIalpha).We now demonstrate that the 20-amino acid repeat region of APC (M3-APC) also interacts with topo IIalpha in colonic epithelial cells. Expression of M3-APC in cells with full-length endogenous APC causes cell accumulation in G2. However, cells with a mutated topo IIalpha isoform and lacking topo IIbeta did not arrest, suggesting that the cellular consequence of M2- or M3-APC expression depends on functional topoisomerase II. Both purified recombinant M2- and M3-APC significantly enhanced the activity of topo IIalpha. Of note, although M3-APC can bind beta-catenin, the G2 arrest did not correlate with beta-catenin expression or activity, similar to what was seen with M2-APC. More importantly, expression of either M2- or M3-APC also led to increased aneuploidy in cells with full-length endogenous APC but not in cells with truncated endogenous APC that includes the M2-APC region.Together, our data establish that the 20-amino acid repeat region of APC interacts with topo IIalpha to enhance its activity in vitro, and leads to G2 cell cycle accumulation and aneuploidy when expressed in cells containing full-length APC. These findings provide an additional explanation for the aneuploidy associated with many colon cancers that possess truncated APC

Advanced backcross QTL mapping of resistance to Fusarium head blight and plant morphological traits in a Triticum macha × T. aestivum population

While many reports on genetic analysis of Fusarium head blight (FHB) resistance in bread wheat have been published during the past decade, only limited information is available on FHB resistance derived from wheat relatives. In this contribution, we report on the genetic analysis of FHB resistance derived from Triticum macha (Georgian spelt wheat). As the origin of T. macha is in the Caucasian region, it is supposed that its FHB resistance differs from other well-investigated resistance sources. To introduce valuable alleles from the landrace T. macha into a modern genetic background, we adopted an advanced backcross QTL mapping scheme. A backcross-derived recombinant-inbred line population of 321 BC2F3 lines was developed from a cross of T. macha with the Austrian winter wheat cultivar Furore. The population was evaluated for Fusarium resistance in seven field experiments during four seasons using artificial inoculations. A total of 300 lines of the population were genetically fingerprinted using SSR and AFLP markers. The resulting linkage map covered 33 linkage groups with 560 markers. Five novel FHB-resistance QTL, all descending from T. macha, were found on four chromosomes (2A, 2B, 5A, 5B). Several QTL for morphological and developmental traits were mapped in the same population, which partly overlapped with FHB-resistance QTL. Only the 2BL FHB-resistance QTL co-located with a plant height QTL. The largest-effect FHB-resistance QTL in this population mapped at the spelt-type locus on chromosome 5A and was associated with the wild-type allele q, but it is unclear whether q has a pleiotropic effect on FHB resistance or is closely linked to a nearby resistance QTL