A nonradiometric, high-throughput assay for poly(ADP-ribose) glycohydrolase (PARG): application to inhibitor identification and evaluation

The enzyme poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of glycosidic bonds of ADP-ribose polymers, producing monomeric ADP-ribose units. Thus, in conjunction with poly(ADP-ribose) polymerase (PARP), PARG activity regulates the extent of in vivo poly(ADP-ribosyl)ation. Small molecule inhibitors of PARP and PARG have shown considerable promise in cellular models of ischemia-reperfusion injury and oxidative neuronal cell death. However, currently available PARG inhibitors are not ideal due to cell permeability, size, and/or toxicity concerns; therefore, new small molecule inhibitors of this important enzyme are sorely needed. Existing methodologies for in vitro assessment of PARG enzymatic activity do not lend themselves to high-throughput screening applications, as they typically use a radiolabeled substrate and determine product quantities through TLC analysis. This article describes a method whereby the ADP-ribose product of the PARG-catalyzed reaction is converted into a fluorescent dye. This highly sensitive and reproducible method is demonstrated by identifying two known PARG inhibitors in a 384-well plate assay and by subsequently determining IC(50) values for these compounds. Thus, this high-throughput, nonradioactive PARG assay should find widespread use in experiments directed toward identification of novel PARG inhibitors.     

Enrichment and functional reconstitution of glutathione transport activity from rabbit kidney mitochondria: further evidence for the role of the dicarboxylate and 2-oxoglutarate carriers in mitochondrial glutathione transport

In previous studies, we provided evidence for uptake of glutathione (GSH) by the dicarboxylate and the 2-oxoglutarate carriers in rat kidney mitochondria. To investigate further the role of these two carriers, GSH transport activity was enriched from rabbit kidney mitochondria and functionally reconstituted into phospholipid vesicles. Starting with 200 mg of mitoplast protein, 2 mg of partially enriched proteins were obtained after Triton X-114 solubilization and hydroxyapatite chromatography. The reconstituted proteoliposomes catalyzed butylmalonate-sensitive uptake of [(14)C]malonate, phenylsuccinate-sensitive uptake of [(14)C]2-oxoglutarate, and transport activity with [(3)H]GSH. The initial rate of uptake of 5 mM GSH was approximately 170 nmol/min per mg protein, with a first-order rate constant of 0.3 min(-1), which is very close to that previously determined in freshly isolated rat kidney mitochondria. The enrichment procedure resulted in an approximately 60-fold increase in the specific activity of GSH transport. Substrates and inhibitors for the dicarboxylate and the 2-oxoglutarate carriers (i.e., malate, malonate, 2-oxoglutarate, butylmalonate, phenylsuccinate) significantly inhibited the uptake of [(3)H]GSH, whereas most substrates for the tricarboxylate and monocarboxylate carriers had no effect. GSH uptake exhibited an apparent K(m) of 2.8 mM and a V(max) of 260 nmol/min per mg protein. Analysis of mutual inhibition between GSH and the dicarboxylates suggested that the dicarboxylate carrier contributes a somewhat higher proportion to overall GSH uptake and that both carriers account for 70 to 80% of total GSH uptake. These results provide further evidence for the function of the dicarboxylate and 2-oxoglutarate carriers in the mitochondrial transport of GSH.     

HETEROTROPHY AND PHOTOHETEROTROPHY BY ANTARCTIC MICROALGAE: LIGHT-DEPENDENT INCORPORATION OF AMINO ACIDS AND GLUCOSE 1

Diatoms isolated from the benthic, planktonic and sea ice microbial communities in Mc Murdo Sound, Antarctica assimilated ambient concentrations of dissolved amino acids and glucose in both the light and dark. Uptake of amino acids but not glucose was influenced by the iucubation irradiance and amino acid uptake rates were up to 250 times greater than those of glucose. Amino acids were incorporated into proteins and other complex polymers and the rates of assimilation and patterns of polymer synthesis were similar to those of the light-saturated photosynthetic incorporation of inorganic carbon. This suggests that these diatoms can use exogenous amino acids to synthesize the essential macromolecules for heterotrophic growth. The assimilation of dissolved organic substrates could supplement light-limited growth during the austral spring and summer as well as potentially support the heterotrophic growth of these diatoms throughout the aphotic polar winter.     

Serum antibodies to EpCAM in healthy donors but not ulcerative colitis patients

Purpose: The gastrointestinal carcinoma-associated antigen epithelial cell adhesion molecule (EpCAM) has been a target for passive and active immunotherapy of gastrointestinal carcinoma patients. The antigen is expressed by both tumor and normal tissues. The immunogenicity of EpCAM in colorectal cancer patients has been described previously. The purpose of this study was to evaluate humoral and cellular immune responses of healthy individuals and ulcerative colitis patients to EpCAM and to relate immune responses to colonic tissue expression of EpCAM. Methods: An inhibition radioimmunoassay was used to detect anti-EpCAM serum antibodies. Anti-EpCAM antibodies of a healthy donor were expressed by phages and sequenced. ³H-thymidine incorporation assay was used for detection of lymphoproliferative responses to stimulation with EpCAM. EpCAM tissue expression was determined by immunohistochemistry. Results: We detected anti-EpCAM serum antibodies in 4 of 10, and EpCAM-specific lymphoproliferation responses in 1 of 10 healthy volunteers. The majority of anti-EpCAM antibodies derived from a healthy donor were germline-encoded. In contrast, none of the 23 patients with ulcerative colitis showed serum antibodies to EpCAM (P=0.005). Antigen expression was greatly reduced and altered in ulcerative colitis patients, whereas colon from healthy individuals and uninvolved colon of colorectal cancer patients expressed high levels of EpCAM. Conclusion: The results of these studies suggest an association between EpCAM antibody production and colonic EpCAM expression in healthy individuals and patients with ulcerative colitis. Decreased and altered colonic EpCAM expression in ulcerative colitis patients may be related to the disease induction, based on the previously demonstrated adhesion function of this molecule. Healthy individuals with anti-EpCAM immune responses and high risk for developing colorectal carcinoma are prime candidates for prophylactic immunization against EpCAM.Emma E. Furth and Jian Li contributed equally     

An investigation of the products of 53 gene loci in three species of wild Canidae: Canis lupus,Canis latrans,and Canis familiaris

This article describes an investigation of inter- and intraspecific variation in three small populations of wild Canidae-wolf, coyote, and dingo. The products of 53 gene loci were examined. Very little interspecies variation was observed, but the level of intraspecific variation was compatible with that found in man.     

A study of short utrophin isoforms in mice deficient for full-length utrophin

Utrophin can functionally replace dystrophin in dystrophin-deficient muscle and may have a role in a therapeutic strategy for Duchenne muscular dystrophy. This has resulted in many investigations of the full-length muscle form of utrophin; however, the short utrophins and non-muscle forms have been relatively neglected, partly because they are difficult to analyze in the presence of the full-length form. Our study circumvents this problem by using mice deficient for the full-length form (UKOex6 mice) to study the translation and distribution of short utrophins. Four tissues were examined—kidney, testis, fetal hands/feet, and brain—and three novel short isoforms were identified, including Up120, which appears to be specific to kidney glomeruli, and Up 109, expressed in the fetal dermis. A third form, Up103, was found in testis but at extremely low levels. A cDNA for Up109 has been isolated and shown to have a unique NH2-terminal sequence. In addition, the first exons of Up109 and another short form, G-utrophin, have both been located within intron 55, 56 kb apart. Our immunological studies show that G-utrophin protein accumulates only in neural tissue, in line with its similarly restricted RNA distribution. Our study of testis expression shows, for the first time, that full-length utrophin is expressed at high levels in Leydig cells, raising the possibility that this protein is involved in testosterone secretion. We note that translation of the short utrophins, especially Up140 and Up71, is relatively inefficient and discuss the significance of this observation.     

An investigation of seven enzymes as possible genetic markers in horse leucocytes

In this paper we describe seven enzymes, NP, GOT_M, PGM₂, αFUC, PEP A, ADA and MPI which are found in the white cells of horses, including 39 British crossbred ponies and 16 crossbred horses, 30 Mongolian ponies and 10 Icelandic ponies. Two of these enzymes - αFUC and MPI - were polymorphic in all the populations of horses studied and could prove useful as additional markers in the paternity testing of horses. PEP A and GOT_M were also polymorphic in two of the populations studied and could be used as further markers in these populations.     

Genetics of four plasma protein loci in Equus przewalskii: new alleles at the prealbumin, postalbumin and transferrin loci

This paper reports genetic variation at the prealbumin ( Pr ), postalbumin ( Pa ) and transferrin ( Tf ) loci in Equus przewalskii found using thin layer isoelectric focusing and an amphoteric separator. The method resolves all three loci plus serum esterase ( Es ) on a single gel, and typing of all four loci is readily achieved. In addition to the esterase alleles previously reported by Fisher & Scott (1979), five alleles were found at the Pr locus. three at the Pa locus and six at the Tf locus. Analysis of several mating types confirms inheritance is autosomal and codominant for all four loci.     

An investigation of the products of 23 gene loci in the domestic cat, Felis catus

The products of 23 enzyme and protein loci were examined in the domestic cat, Felis catus. Seven of these loci showed electrophoretic variation (six with two alleles and one with four alleles). Based on evidence from a family group the variation at five of these loci can be attributed to genetic causes.