An affinity matrix for the purification of poly(ADP-ribose) glycohydrolase.

The preparation of quantities of poly(ADP-ribose) glycohydrolase sufficient for detailed structural and enzymatic characterizations has been difficult due to the very low tissue content of the enzyme and its lability in late stages of purification. To date, the only purification of this enzyme to apparent homogeneity has involved a procedure requiring 6 column chromatographic steps. Described here is the preparation of an affinity matrix which consists of ADP-ribose polymers bound to dihydroxyboronyl sepharose. An application is described for the purification of poly(ADP-ribose) glycohydrolase from calf thymus in which a single rapid affinity step was used to replace 3 column chromatographic steps yielding enzyme of greater than 90% purity with a 3 fold increase in yield. This matrix should also prove useful for other studies of ADP-ribose polymer metabolism and related clinical conditions.     

Effect of the myorelaxant clenbuterol on the oxytocin-induced release of prostaglandin F2 alpha in ovariectomized heifers

Clenbuterol, as other sympathomimetic drugs, relaxes the myometrium, thus causing a short-term inhibition of labor and the delay of parturition. This study has examined the influence of clenbuterol on the release of prostaglandin F2 alpha (PGF2 alpha) induced by oxytocin alone or with estradiol-17 beta. Five bilaterally ovariectomized heifers, primed with progesterone for 14 days, were used in two experiments. In the first they received two i.v. injections of oxytocin 6h apart, with and without an i.v. injection of clenbuterol before the second oxytocin injection; the second experiment was similar to the first except that the animals were given estradiol-17 beta 30 min after the first oxytocin injection. Frequent blood samples were taken for the measurement of 13,14-dihydro-15-keto-PGF2 alpha by radioimmunoassay. The data show that clenbuterol does not influence PGF2 alpha release in response to oxytocin alone or with estradiol-17 beta, and it does not inhibit the basal release of PGF2 alpha. This suggests that clenbuterol does not act on the endometrium to alter the secretion of PGF2 alpha in the non-pregnant cow.     

The tet(K) gene from Staphylococcus aureus mediates the transport of potassium in Escherichia coli.

The tet(K) gene, encoding the tetracycline efflux protein from Staphylococcus aureus, mediates the transport of potassium in an Escherichia coli mutant defective in potassium uptake. Deletion mapping indicates that the first third of the tet(K) gene is sufficient to mediate potassium transport.     

Genetic analysis suggests functional interactions between the N- and C-terminal domains of the TetA(C) efflux pump encoded by pBR322.

Genetic analysis of the tetA(C) gene of pBR322 indicates the importance of two-cytoplasmic loops in the TetA(C) protein (P. McNicholas, I. Chopra, and D. M. Rothstein, J. Bacteriol. 174:7926-7933, 1992). In this study, we characterized second-site suppressor mutations that suggest a functional interaction between these two cytoplasmic regions of the protein.     

Origin and developmental patterns of lactase and other glycosidases in sheep amniotic and allantoic fluid.

Intestinal lactase activity (with its associated cellobiase, 4-methylumbelliferyl-beta-galactosidase and -beta-glucosidase activities) was used as a specific intestinal marker enzyme to study the release of protein and enzymes of intestinal origin in sheep amniotic fluid during gestation. In amniotic fluid, intestinal lactase activity peaked at 66--85 days of gestation and then decreased with gestation. This enzyme activity was very low or absent in allantoic fluid throughout gestation suggesting that there is no important transfer of amniotic fluid lactase towards the allantoic cavity. Maltase and 4-methylumbelliferyl-alpha-glucosidase showed no statistically significant variation with gestation in both amniotic and allantoic fluid whereas alpha-galactosidase and N-acetyl-beta-hexosaminidase which were first higher in allantoic than in amniotic fluid increased in amniotic fluid to reach allantoic fluid levels near term. Such patterns are consistent with the suggestion that the fetal urine is a source of alpha-galactosidase and N-acety-beta-hexosaminidase activities and that sheep urine is first accumulated in the allantoic sac via the urachus up to 86--90 days of gestation and thereafter passes more and more into the amniotic sac.     

Does variation in host plant association and symbiont infection of pea aphid populations induce genetic and behaviour differentiation of its main parasitoid, Aphidius ervi?

The host-associated differentiation (HAD) hypothesis states that higher trophic levels in parasitic associations should exhibit similar divergence in case of host sympatric speciation. We tested HAD on populations of Aphidius ervi the main parasitoid of the pea aphid Acyrthosiphon pisum, emerging from host populations specialized on either alfalfa or red clover. Host and parasitoid populations were assessed for genetic variation and structure, while considering geography, host plant and host aphid protective symbionts Regiella insecticola and Hamiltonella defensa as potential covariables. Cluster and hierarchical analyses were used to assess the contribution of these variables to population structure, based on genotyping pea aphids and associated A. ervi with microsatellites, and host aphid facultative symbionts with 16S rDNA markers. Pea aphid genotypes were clearly distributed in two groups closely corresponding with their plant origins, confirming strong plant associated differentiation of this aphid in North America. Overall parasitism by A. ervi averaged 21.5 % across samples, and many parasitized aphids producing a wasp hosted defensive bacteria, indicating partial or ineffective protective efficacy of these symbionts in the field. The A. ervi population genetic data failed to support differentiation according to the host plant association of their pea aphid host. Potential for parasitoid specialization was also explored in experiments where wasps from alfalfa and clover aphids were reciprocally transplanted on alternate hosts, the hypothesis being that wasp behaviour and parasitic stages should be most adapted to their host of origin. Results revealed higher probability of oviposition on the alfalfa aphids, but higher adult emergence success on red clover aphids, with no interaction as expected under HAD. We conclude that our study provides no support for the HAD in this system. We discuss factors that might impair A. ervi specialization on its divergent aphid hosts on alfalfa and clover.     

A role for ATP-citrate lyase, malic enzyme, and pyruvate/citrate cycling in glucose-induced insulin secretion

In pancreatic beta-cells, metabolic coupling factors generated during glucose metabolism and pyruvate cycling through anaplerosis/cataplerosis processes contribute to the regulation of insulin secretion. Pyruvate/citrate cycling across the mitochondrial membrane leads to the production of malonyl-CoA and NADPH, two candidate coupling factors. To examine the implication of pyruvate/citrate cycling in glucose-induced insulin secretion (GIIS), different steps of the cycle were inhibited in INS 832/13 cells by pharmacological inhibitors and/or RNA interference (RNAi) technology: mitochondrial citrate export, ATP-citrate lyase (ACL), and cytosolic malic enzyme (ME1). The inhibitors of the di- and tri-carboxylate carriers, n-butylmalonate and 1,2,3-benzenetricarboxylate, respectively, reduced GIIS, indicating the importance of transmitochondrial transport of tri- and dicarboxylates in the action of glucose. To directly test the role of ACL and ME1 in GIIS, small hairpin RNA (shRNA) were used to selectively decrease ACL or ME1 expression in transfected INS 832/13 cells. shRNA-ACL reduced ACL protein levels by 67%, and this was accompanied by a reduction in GIIS. The amplification/K(ATP)-independent pathway of GIIS was affected by RNAi knockdown of ACL. The ACL inhibitor radicicol also curtailed GIIS. shRNA-ME1 reduced ME1 activity by 62% and decreased GIIS. RNAi suppression of either ACL or ME1 did not affect glucose oxidation. However, because ACL is required for malonyl-CoA formation, inhibition of ACL expression by shRNA-ACL decreased glucose incorporation into palmitate and increased fatty acid oxidation in INS 832/13 cells. Taken together, the results underscore the importance of pyruvate/citrate cycling in pancreatic beta-cell metabolic signaling and the regulation of GIIS.