Nucleotide pyrophosphatase of rat liver. A comparative study on the enzymes solubilized and purified from plasma membrane and endoplasmic reticulum.

Studies on the subcellular distribution of rat liver nucleotide pyrophosphatase activity revealed its presence in the plasma membrane and the endoplasmic reticulum only. The enzymes from either source were solubilized specifically with trypsin without an apparent change of their catalytic properties. A 200-fold and 1600-fold purification, respectively, was achieved by a procedure including DEAE-cellulose and affinity-chromatography with AMP as ligand, gel filtration on Sephadex G-200 and gel electrophoresis. Both nucleotide pyrophosphatases were isolated as electrophoretically homogeneous soluble proteins. They were shown to contain carbohydrate moieties. The electrophoretic mobility of both enzymes in polyacrylamide gels was identical at three pH values. Dodecylsulfate gel electrophoresis indicated a molecular weight of 137 000 for both glycoproteins. The enzymes hydrolyze a variety of purine and pyrimidine nucleotides yielding a 5'-nucleoside monophosphate. Adenosine 3':5'-monophosphate, nucleic acids and phosphate monoesters are not cleaved, but p-nitrophenyl-thymidine5'-monophosphate is readily hydrolyzed. In view of their substrate and inhibitor specificities the enzymes are considered nucleotide pyrophosphatases rather than phosphodiesterases.     

The recycling of a secretory granule membrane protein

We have used N-hydroxysuccinimido-d-biotin as a reagent for labeling proteins exposed at the surface of cultured bovine adrenal chromaffin cells during Ba2+-stimulated secretion. A specific secretory granule membrane constituent, dopamine-beta-hydroxylase (DBH), has been investigated using immunoprecipitation followed by electrophoresis. Within 30 min of stimulation, exposed DBH had been cleared from the cell surface. Nevertheless, quantitation of labeled DBH using [125I] streptavidin suggested that it remained undegraded over a period of 24 h, a time during which secretory granule stores of catecholamines were being replenished. Subcellular fractionation of the cultured cells suggested that, after 3 or 4 h, the biotinylated DBH, which was still membrane-bound, was located in particulate material that also contained cytochrome b561, another major secretory granule membrane component.     

Presence of a small plasmid in clinical isolates of Streptococcus pneumoniae

Twenty-four of 63 enteric Gram-negative organisms (38.1%) which were isolated from 35 apparently healthy Nigerian students were found to have low trimethoprim resistance (MIC less than 1000 mg/l). These isolates were also found to be resistant to several other antibiotics and trimethoprim resistance was found to be transferable from 15 (62.5%) of the trimethoprim resistant organisms into E. coli EC 1005. It is likely that the high percentage of trimethoprim resistance encountered in this study is related to the high rate of resistance transfer which was observed.     

Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6–7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2’-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.     

Functional Effects of Adiponectin on Endothelial Progenitor Cells

Adiponectin is an adipokine whose plasma levels are inversely correlated to metabolic syndrome components. Adiponectin protects against atherosclerosis and decreases risks in myocardial infarction. Endothelial progenitor cells (EPCs) are a heterogeneous population of circulating cells involved in vascular repair and neovascularization. EPCs number is reduced in patients with cardiovascular disease. We hypothesize that the positive effects of adiponectin against atherosclerosis are explained in part by its interactions with EPCs. Cells were obtained from healthy volunteers' blood by mononuclear cell isolation and plating on collagen‐coated dishes. Three sub‐populations of EPCs were identified and characterized using flow cytometry. EPCs' expression of adiponectin receptors, AdipoR1, and AdipoR2 was evaluated by quantitative PCR. The effects of recombinant adiponectin on EPCs' susceptibility to apoptosis were assessed. Finally, expression of neutrophil elastase by EPCs and activity of this enzyme on adiponectin processing were assessed. Quantitative PCR analysis of EPCs mRNAs showed that AdipoR1 mRNA is expressed at higher levels than AdipoR2. Expression of AdipoR1 protein was confirmed by western blot. Adiponectin significantly increased survival of two sub‐populations of EPCs in conditions of serum deprivation. Such effect could not be demonstrated in the third EPCs sub‐population. We also demonstrated that EPCs, particularly one sub‐population, express neutrophil elastase. Neutrophil elastase activity was confirmed in EPCs' conditioned media. Adiponectin protects some EPCs sub‐populations against apoptosis and therefore could modulate EPCs ability to induce repair of vascular damage. Neutrophil elastase activity of EPCs could locally modulate adiponectin activity by its involvement in the generation of the globular form of adiponectin.     

Replisome pausing in mutagenesis

E. coli cells containing a temperature-sensitivednaE mutation, in the α-subunit of holoenzyme DNA polymerase III, do not survive at the restrictive temperature. Such cells may survive in the presence of thepcbA1 mutation, an allele of thegyrB gene. Such survival is dependent on an active DNA polymerase I. Evidence indicates that DNA polymerase I interacts directly in the replisome (REP·A). Despite normal survival for cells using thepcbA replication pathway after some type of DNA damage, we have noted a failure of damage-induced mutagenesis. Here we present evidence supporting a model of replisome pausing in cells dependent upon thepcbA replication pathway. The model argues that the (REP·A) complex pauses longer at the site of the lesion, allowing excision repair to occur completely. In the normal replication pathway (REP·E) bypass of the lesion occurs, fixing the mutation.     

Linking micromorphism,brooding, and hermaphroditism in brachiopods: Insights from Caribbean Argyrotheca (Brachiopoda)

In extant brachiopods, parental brooding of the larvae occurs exclusively within Rhynchonelliformea. Methods of larval protection range from simple retention of the larvae within the mantle cavity, to sophisticated brood care within highly specialized brood pouches found in Argyrotheca and Joania (Terebratulida, Megathyridoidea), Gwynia (Terebratulida, Gwynioidea), and all Thecideoidea (Thecideida). Previous studies on the reproductive biology of Argyrotheca yielded contrasting results on the epithelial origin of the brood pouches in this genus. Here, representatives of different species of Argyrotheca from the Belize Barrier Reef were examined using histological section series. Brood pouches of four species, A. cf. schrammi and Argyrotheca sp. 1–3, are of the same basic structure, formed by invaginations of the anterior body wall and connected to the visceral cavity via the metanephridia. The same four species are simultaneously hermaphroditic, suggesting that fertilization is achieved, at least partly, through selfing. One species, Argyrotheca rubrocostata, differs significantly from all others as it has no brood pouch and gonochoric gonads. Thus, the presence of brood pouches and simultaneous hermaphroditism are concluded to be correlated within Megathyridoidea and proposed to be homologous traits of Joania and several but not all species of Argyrotheca, questioning the monophyletic status of both genera. In contrast to the brood pouches of Thecideoidea, lophophoral epithelium is not involved in the formation of the pouches of Argyrotheca and Joania. Therefore, megathyridoid and thecideoid brood pouches are not homologous but evolved independently within rhynchonelliform brachiopods. All brachiopods with brood pouches share a micromorphic form and a short life span, limiting the space and time available for gamete and larval development. We suggest that the brood pouches and the hermaphroditic gonads of Argyrotheca spp. and Joania compensate these limitations by minimizing the loss of gametes and larvae, and by maximizing the chances of successful fertilization. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Identification and design of a novel series of MGAT2 inhibitors

[Acyl CoA]monoacylglycerol acyltransferase 2 (MGAT2) is of interest as a target for therapeutic treatment of diabetes, obesity and other diseases which together constitute the metabolic syndrome. In this Letter we report our discovery and optimisation of a novel series of MGAT2 inhibitors. The development of the SAR of the series and a detailed discussion around some key parameters monitored and addressed during the lead generation phase will be given. The in vivo results from an oral lipid tolerance test (OLTT) using the MGAT2 inhibitor (S)-10, shows a significant reduction (68% inhibition relative to na?ve, p <0.01) in plasma triacylglycerol (TAG) concentration.