Mutant alpha-synuclein-induced degeneration is reduced by parkin in a fly model of Parkinson's disease.

Parkinson's disease (PD) patients show a characteristic loss of motor control caused by the degeneration of dopaminergic neurons. Mutations in the genes that encode alpha-synuclein and parkin have been linked to inherited forms of this disease. The parkin protein functions as a ubiquitin ligase that targets proteins for degradation. Expression of isoforms of human alpha-synuclein in the Drosophila melanogaster nervous system forms the basis of an excellent genetic model that recapitulates phenotypic and behavioural features of PD. Using this model, we analysed the effect of parkin co-expression on the climbing ability of aging flies, their life span, and their retinal degeneration. We have determined that co-expression of parkin can suppress phenotypes caused by expression of mutant alpha-synuclein. In the developing eye, parkin reduces retinal degeneration. When co-expressed in the dopaminergic neurons, the ability to climb is extended over time. If conserved in humans, we suggest that upregulation of parkin may prove a method of suppression for PD induced by mutant forms of alpha-synuclein.     

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.     

Single-stranded circular DNA generated from broad host range plasmid R1162 and its derivatives

Extraction of R1162 plasmid DNA with the alkaline lysis method yields considerable amounts of single-stranded circular plasmid DNA. Destabilization of plasmid DNA is stimulated by the R1162 mob region in cis. The formation of single-stranded circular DNA is initiated at a specific site on the plasmid, presumably the origin of transfer (oriT).     

Lysosomes and pancreatic islet function

Summary Ultrastructural studies of pancreatic islets have suggested that crinophagy provides a possible mechanism for intracellular degradation of insulin in the insulin-producing B-cells. In the present study, a quantitative estimation of crinophagy in mouse pancreatic islets was attempted by morphometric analysis of lysosomes containing immunoreactive insulin. Isolated islets were incubated in tissue culture for one week in 3.3, 5.5 or 28 mmol/l glucose. The lysosomes of the pancreatic B-cells were identified by morphological and enzyme-cytochemical criteria and divided into three subpopulations comprising primary lysosomes and insulin-positive or insulin-negative secondary lysosomes. Both the volume and numerical density of the primary lysosomes increased with increasing glucose concentration. The proportion of insulin-containing secondary lysosomes was highest at 5.5 and lowest at 3.3 mmol/l glucose. Insulin-negative secondary lysosomes predominated at 3.3 mmol/l glucose. Studies of the dose-response relationships of glucose-stimulated insulin biosynthesis and insulin secretion of the pancreatic islets showed that biosynthesis had an apparent K_m-value for glucose of 7.0 mmol/l, whereas it was 14.5 mmol/l for secretion. The pronounced crinophagic activity at 5.5 mmol/l glucose may thus be explained by the difference in glucose sensitivity between insulin biosynthesis and secretion resulting in an intracellular accumulation of insulin-containing secretory granules. The predominance of insulin-negative secondary lysosomes at 3.3 mmol/l glucose may reflect an increased autophagy, whereas the predominance of primary lysosomes at 28 mmol/l glucose may reflect a generally low activity of intracellular degradative processes.     

First reliable record of a fossil species of Anthomyiidae (Diptera), with comments on the definition of recent and fossil clades in phylogenetic classification

All previous records of fossil Anthomyiidae are shown to be unsubstantiated. A female anthomyiid of a new genus and species is hereby described from a piece of Dominican amber (Upper Eocene-Oligocene). Character analysis suggests that the fossil, Coenosopsites poinari gen. & sp. nov. , belongs to a Neotropical clade with two recent genera, Phaonantho Albuquerque and Coenosopsia Malloch. Evidence for a sister-group relationship between Coenosopsites poinari and the genus Coenosopsia is provided. Clades are the only acceptable units of phylogenetic classification. Combining fossil and recent clades in phylogenetic classification requires them to be temporally delimited. Proper application of phylogenetic definitions is essential for this purpose. It is proposed that the units of phylogenetic classification should be taxa for recent clades and plesia for fossil clades. A taxon is defined as node-based with reference to its recent species, while a plesion is defined as apomorphy-based. The term lineage is proposed for a recent clade defined as stem-based with reference to its recent sister group. Individual recent species represent clades that can be incorporated into phylogenetic classification as minimal taxon units. Individual fossil species may not represent clades and thus do not count as proper units of phylogenetic classification. However, the names of fossil species are readily construed also to signify plesia with the fossil species as their only known component. As such, they are proper units of phylogenetic classification.     

Excess capacity of H(+)-ATPase and inverse respiratory control in Escherichia coli.

With succinate as free-energy source, Escherichia coli generating virtually all ATP by oxidative phosphorylation might be expected heavily to tax its ATP generating capacity. To examine this the H(+)-ATPase (ATP synthase) was modulated over a 30-fold range. Decreasing the amount of H(+)-ATPase reduced the growth rate much less than proportionally; the H(+)-ATPase controlled growth rate by < 10%. This lack of control reflected excess capacity: the rate of ATP synthesis per H(+)-ATPase (the turnover number) increased by 60% when the number of enzymes was decreased by 40%. At 15% H(+)-ATPase, the enzyme became limiting and its turnover was increased even further, due to an increased driving force caused by a reduction in the total flux through the enzymes. At smaller reductions of [H(+)-ATPase] the total flux was not reduced, revealing a second cause for increased turnover number through increased membrane potential: respiration was increased, showing that in E.coli, respiration and ATP synthesis are, in part, inversely coupled. Indeed, growth yield per O2 decreased, suggesting significant leakage or slip at the high respiration rates and membrane potential found at low H(+)-ATPase concentrations, and explaining that growth yield may be increased by activating the H(+)-ATPase.     

Reaction intensification for biocatalytic production of polyphenolic natural product di-C-β-glucosides

Polyphenolic aglycones featuring two sugars individually attached via C-glycosidic linkage (di-C-glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di-C-glycosides limits their use-inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction-intensified production of the di-C-β-glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl-trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two-fold C-glycosylation of the respective aglycone, supplied as inclusion complex with 2-hydroxypropyl β-cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di-C-glycosyltransferase (di-CGT), and it uses UDP-Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C-glycosylation as rate-limiting (0.4–0.5 mmol/L/min) for the di-C-glucoside production. With internal supply from sucrose keeping the UDP-Glc at a constant steady-state concentration (≥50% of the UDP added) during the reaction, the di-C-glycosylation is driven to completion (≥95% yield). Contrary to the mono-C-glucoside intermediate which is stable, the di-C-glucoside requires the addition of reducing agent (10 mmol/L 2-mercaptoethanol) to prevent its decomposition during the synthesis. Both di-C-glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di-C-β-glucoside synthesis from expedient substrates.