DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation

Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive inherited PD (Bonifati et al. 2003). In a companion article (Martinat et al. 2004), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to alpha-synuclein, a protein implicated in PD pathogenesis.     

Two distinct Gb3/CD77 signaling pathways leading to apoptosis are triggered by anti-Gb3/CD77 mAb and verotoxin-1

Globotriasosylceramide (Gb3), a neutral glycosphingolipid, is the B-cell differentiation antigen CD77 and acts as the receptor for most Shiga toxins, including verotoxin-1 (VT-1). We have shown that both anti-Gb3/CD77 mAb and VT-1 induce apoptosis in Burkitt's lymphoma cells. We compared the apoptotic pathways induced by these two molecules by selecting cell lines sensitive to only one of these inducers or to both. In all these cell lines (including the apoptosis-resistant line), VT-1 was transported to the endoplasmic reticulum and inhibited protein synthesis similarly, suggesting that VT-1-induced apoptosis is dissociated from these processes. VT-1 triggered a caspase- and mitochondria-dependent pathway (rapid activation of caspases 8 and 3 associated with a loss of mitochondrial membrane potential (Deltapsim) and the release of cytochrome c from mitochondria). In contrast, the anti-Gb3/CD77 mAb-induced pathway was caspase-independent and only involved partial depolarization of mitochondria. Antioxidant compounds had only marginal effects on VT-1-induced apoptosis but strongly protected cells from anti-Gb3/CD77 mAb-induced apoptosis. VT-1- and anti-Gb3/CD77 mAb-treated cells displayed very different features on electron microscopy. These results clearly indicate that the binding of different ligands to Gb3/CD77 triggers completely different apoptotic pathways.     

Preparative enantiomeric separation of new aromatase inhibitors by HPLC on polysaccharide-based chiral stationary phases: Determination of enantiomeric purity and assignment of absolute stereochemistry by X-ray structure analysis

The development of high-performance liquid chromatography methods on polysaccharide-based stationary phases (cellulose or amylose derivatives) has permitted preparative enantioseparations of various 6-[1(imidazol-1-yl)-1-phenylmethyl]-3-methyl-1,3-benzoxazol-2(3H)-one and 6-[1(imidazol-1-yl)-1-phenylmethyl]-3-methyl-1,3-benzothiazol-2(3H)-one, aromatase inhibitors, with satisfactory yields. Analytical enantioseparation methods using both UV and evaporative light-scattering detection (ELSD) were validated to determine the enantiomeric purity of these compounds. Using UV detection, linear calibration curves in the range from 4 x 10(-6) to 4.8 x 10(-4) M range were obtained; repeatability, limits of detection (LD), and quantification (LQ) were determined: LD varied, for the various solutes, from 1 to 80 microg/l and from 2.05 to 10.05 mg/l with UV detection and ELSD, respectively. Single-crystal X-ray analysis was successful in determining the absolute configuration of the individual enantiomers. The relationship between retention order and absolute configuration of the enantiomers was established.     

Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid- and solution-phase immunochemical assays, employing anti-ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin ( ca . 8 × 10⁸ molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13-fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later-stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus . The results of pulse-labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12-fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin-encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.     

The Genomic Basis of Color Pattern Polymorphism in the Harlequin Ladybird

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High-Resolution Denitrification Kinetics in Pasture Soils Link N2O Emissions to pH,and Denitrification to C Mineralization

Denitrification in pasture soils is mediated by microbial and physicochemical processes leading to nitrogen loss through the emission of N₂O and N₂. It is known that N₂O reduction to N₂ is impaired by low soil pH yet controversy remains as inconsistent use of soil pH measurement methods by researchers, and differences in analytical methods between studies, undermine direct comparison of results. In addition, the link between denitrification and N₂O emissions in response to carbon (C) mineralization and pH in different pasture soils is still not well described. We hypothesized that potential denitrification rate and aerobic respiration rate would be positively associated with soils. This relationship was predicted to be more robust when a high resolution analysis is performed as opposed to a single time point comparison. We tested this by characterizing 13 different temperate pasture soils from northern and southern hemispheres sites (Ireland and New Zealand) using a fully automated-high-resolution GC detection system that allowed us to detect a wide range of gas emissions simultaneously. We also compared the impact of using different extractants for determining pH on our conclusions. In all pH measurements, soil pH was strongly and negatively associated with both N₂O production index (IN₂O) and N₂O/(N₂O+N₂) product ratio. Furthermore, emission kinetics across all soils revealed that the denitrification rates under anoxic conditions (NO+N₂O+N₂ μmol N/h/vial) were significantly associated with C mineralization (CO₂ μmol/h/vial) measured both under oxic (r² = 0.62, p = 0.0015) and anoxic (r² = 0.89, p<0.0001) conditions.     

Quantitative Autoradiography of Tyrosine Hydroxylase Immunoreactivity in the Rat Brain

Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S-labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high-sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer-assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S-labelled antibody, the detection of TH could be performed at the cellular level.     

The Beneficial Effect of Organic Solvents on the Enzymatic Synthesis of Nucleoside Analogues Using N-Deoxyribosyltransferases from Lactobacillus leichmannii (E.C. 2.4.2.6)

Addition of ethylene glycol (10% v/v) has a beneficial effect on the synthesis of 2'-deoxynucleosides catalysed by crude preparations of N-deoxyribosyltransferases from Lactobacillus leichmannii. In the absence of added organic solvent decomposition of products and starting materials by deamination or hydrolysis occurs giving rise to poor yields of products if the transfer reaction is slow. The glycosyl transfer reaction is unaffected by addition of organic solvent but decomposition of products and starting materials is largely suppressed. The organic solvent appears to inhibit selectively contaminating enzymes in the crude N-deoxyribosyltransferase preparation as the purified transferase does not possess hydrolytic or deaminating activity. Different concentrations of ethylene glycol and other organic solvents have been examined as inhibitors of the side reactions but 10% (v/v) appears to be the most effective. Using the N-deoxyribosyltransferase in the presence of ethylene glycol, a number of 2'-deoxynucleosides of 6-substituted nucleosides have been obtained in high yield on a preparative scale.     

Small,dense HDL 3 particles attenuate apoptosis in endothelial cells: pivotal role of apolipoprotein A‐I

Plasma high‐density lipoproteins (HDLs) protect endothelial cells against apoptosis induced by oxidized low‐density lipoprotein (oxLDL). The specific component(s) of HDLs implicated in such cytoprotection remain(s) to be identified. Human microvascular endothelial cells (HMEC‐1) were incubated with mildly oxLDL in the presence or absence of each of five physicochemically distinct HDL subpopulations fractionated from normolipidemic human plasma (n= 7) by isopycnic density gradient ultracentrifugation. All HDL subfractions protected HMEC‐1 against oxLDL‐induced primary apoptosis as revealed by nucleic acid staining, annexin V binding, quantitative DNA fragmentation, inhibition of caspase‐3 activity and reduction of cytoplasmic release of cytochrome c and apoptosis‐inducing factor. Small, dense HDL 3c displayed twofold superior intrinsic cytoprotective activity (as determined by mitochondrial dehydrogenase activity) relative to large, light HDL 2b on a per particle basis (P < 0.05). Equally, all HDL subfractions attenuated intracellular generation of reactive oxygen species (ROS); such anti‐oxidative activity diminished from HDL 3c to HDL 2b. The HDL protein moiety, in which apolipoprotein A‐I (apoA‐I) predominated, accounted for ～70% of HDL anti‐apoptotic activity. Furthermore, HDL reconstituted with apoA‐I, cholesterol and phospholipid potently protected HMEC‐1 from apoptosis. By contrast, modification of the content of sphingosine‐1‐phosphate in HDL did not significantly alter cytoprotection. We conclude that small, dense, lipid‐poor HDL 3 potently protects endothelial cells from primary apoptosis and intracellular ROS generation induced by mildly oxLDL, and that apoA‐I is pivotal to such protection.