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).     

Hypothermic perfusion of rabbit livers: effect of perfusate composition (Ca and lactobionate) on enzyme release and tissue swelling

Rabbit livers were preserved by continuous hypothermic (5 degrees C) perfusion at a flow rate of 1 ml/min-1 g-1 for as long as 72 hr. Cell swelling (total tissue water, TTW) and the rate at which intracellular enzymes were released into the perfusate were measured. Livers perfused with a simple NaCl-based solution containing hydroxyethyl starch as a colloid released relatively large amounts of aspartate aminotransferase (AST, 442 +/- 224 u/liter-1 100 g-1) and lactic dehydrogenase (LDH, 1580 +/- 688 u/liter-1 100 g-1) into the perfusate during 72 hr of perfusion. The addition of Ca (0.5 mmol/liter) to the perfusate reduced the leakage of enzymes into the perfusate (AST, 70 +/- 30 u; LDH, 450 +/- 50 u) and reduced cell swelling (TTW, 3.1 kg/kg dry mass vs 4.4 kg/kg dry mass without added Ca). But the use of a higher concentration of Ca (1.5 mmol/liter) caused membrane damage (AST, 4000 +/- 1500 u; LDH, 10,000 +/- 2222 u) and increased cell swelling (TTW, 3.7 kg/kg dry mass). The release of intracellular enzymes caused by continuous perfusion with a chloride-based perfusate also could be reduced by replacing the chloride with lactobionate (AST, 100 +/- 30 u; LDH, 400 +/- 100 u, at 72 hr). In the lactobionate-containing perfusate, the addition of Ca (0.5 or 1.5 mmol/liter) did not alter the rate at which intracellular enzymes were released. There was no tissue swelling after 72 hr of preservation with the lactobionate-containing perfusate, and the TTW (2.1 kg/kg dry mass) was similar to the TTW of freshly harvested rabbit livers.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Comparison of the effect of temperature on kidney cortex mitochondria from rabbit,dog, pig,and human: Arrhenius plots of ADP-stimulated respiration

The effect of temperature on the rate of ADP-stimulated respiration of mitochondria from dog, rabbit, pig, and human kidney cortex mitochondria was plotted according to the Arrhenius relationship. The temperature at which the plot demonstrated a break was at 15 °C for mitochondria from dog, pig, and human kidneys. The discontinuity occurred at 10 °C or less for mitochondria from rabbit kidneys. This difference suggests that mitochondria from rabbit kidneys undergo a lipid-phase transition at lower temperatures than for other species commonly used in experimental renal preservation. The implications of this difference suggest caution in using results obtained with rabbit kidneys for comparison to results obtained from hypothermic renal preservation of other species kidneys. Apparent fluidization of dog kidney mitochondrial membranes with adamantine abolished the discontinuity in the Arrhenius plot.     

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.     

Characterization of the activity of fatty-acid epoxide hydrolase in seeds of castor bean (Ricinus communis L.)

Epoxide hydrolase (EC 3.3.2.3) activity was measured with [1-¹⁴C]cis-9,10-epoxystearic acid as the substrate. Homogenates were prepared from the endosperm tissue of germinating seeds of castor bean (Ricinus communis L. zanzibariensis). The activity of fatty-acid epoxide hydrolase was characterized with respect to dependence on time, amount of protein, pH and temperature. Analyses of enzyme distribution in endosperm, cotyledons, root and hypocotyl showed the highest total activity in the endosperm, less in the cotyledons and low activity in the root and hypocotyl. The specific activity was similar for cotyledons and endosperm. Analysis of the temporal expression of the enzyme in the endosperm during germination revealed high activity already in the imbibed seed. Activity was maximal between days four to six and then decreased at the end of one week. Subcellular fractionation of endosperm revealed a dual distribution of activity between the glyoxysomal and the cytosolic fractions.