Cholesterol is not crucial for the existence of microdomains in kidney brush-border membrane models.

The external membrane leaflet plays a key role in the organization of the cell plasma membrane as a mosaic of ordered microdomains enriched in sphingolipids and cholesterol and of fluid domains. In this study, the thermotropic behavior and the topology of bilayers made of a phosphatidylcholine/sphingomyelin mixture, which mimicks the lipid composition of the external leaflet of renal brush-border membranes, were examined by differential scanning calorimetry and atomic force microscopy. In the absence of cholesterol, a broad phase separation process occurred where ordered gel phase domains of size varying from the mesoscopic to the microscopic scale, enriched in sphingomyelin, occupied half of the bilayer surface at room temperature. Increasing amounts of cholesterol progressively decreased the enthalpy of the transition and modified the topology of membranes domains up to a concentration of 33 mol % for which no membrane domains were detected. These results strongly suggest that, in membranes highly enriched in sphingolipids like renal and intestinal brush borders, there is a threshold close to the physiological concentration above which cholesterol acts as a suppressor rather than as a promoter of membrane domains. They also suggest that cholesterol depletion does not abolish the lateral heterogenity in brush-border membranes.     

A low background high-throughput screening (HTS) fluorescence assay for lipases and esterases using acyloxymethylethers of umbelliferone

Esters and acyloxymethyl ethers of umbelliferone were evaluated as fluorogenic substrates for lipases and esterases with respect to stability and resistance to non-specific hydrolysis. Isobutyryloxymethyl ether 1c and pivaloxymethyl ether 1d were found to be optimal substrates for enzyme assays, particularly with respect to HTS applications.     

Discovery of biaryl inhibitors of H+/K+ ATPase

We report the identification of a novel biaryl template for H⁺/K⁺ ATPase inhibition. Evaluation of critical SAR features within the biaryl imidazole framework and the use of pharmacophore modelling against known imidazopyridine and azaindole templates suggested that the geometry of the molecule is key to achieving activity. Herein we present our work optimising the potency of the molecule through modifications and substitutions to each of the ring systems. In particular sub-micromolar potency is achieved with (4b) presumably through a proposed intramolecular hydrogen bond that ensures the required imidazole basic centre is appropriately located.     

An H5N1 highly pathogenic avian influenza virus isolated from a local tree sparrow in Indonesia

The isolation of an H5N1 influenza A virus from a tree sparrow (Passer montanus) captured in East Java, Indonesia in 2010 is reported here. Its hemagglutinin and neuraminidase were genetically similar to those of human isolates from 2006-2007 in Indonesia. The finding of a tree sparrow H5N1 virus that possesses genetically similar surface molecules to those of human viruses highlights the importance of monitoring resident wild birds, as well as migratory birds, for pandemic preparedness.     

PP2A-mediated dephosphorylation of p107 plays a critical role in chondrocyte cell cycle arrest by FGF

FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations. This phenomenon requires the function of the p107 and p130 members of the Rb protein family, and p107 dephosphorylation is one of the earliest distinguishing events in FGF-induced growth arrest. To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex. CyclinD/cdk4-expressing RCS cells became resistant to FGF-induced p107 dephosphorylation and growth arrest, and maintained significantly high levels of cyclin E/cdk2 activity and of phosphorylated p130 at later times of FGF treatment. We explored the involvement of a phosphatase in p107 dephosphorylation. Expression of the SV40 small T-Ag, which inhibits the activity of the PP2A phosphatase, or knockdown of the expression of the PP2A catalytic subunit by RNA interference prevented p107 dephosphorylation and FGF-induced growth arrest of RCS cells. Furthermore, an association between p107 and PP2A was induced by FGF treatment. Our data show that p107 dephosphorylation is a key event in FGF-induced cell cycle arrest and indicate that in chondrocytes FGF activates the PP2A phosphatase to promote p107 dephosphorylation.     

Cell envelope fluidity modification for an effective glutamate excretion in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> 2262

1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was used to assess the cell envelope fluidity of Corynebacterium glutamicum 2262 during a temperature-triggered glutamate producing process. Because the fluorescence lifetime of TMA-DPH was shown to be constant all over the process, fluorescence anisotropy can be considered as a good index of cell envelope fluidity. When the temperature of the fed-batch culture was increased from 33 to 39°C to induce glutamate excretion, the fluorescence anisotropy values decreased from 0.212 ± 0.002 to 0.186 ± 0.002 (corresponding to an increase in the cell fluidity), while the specific glutamate production rate reached its maximal value. The increase in fluidity of the C. glutamicum cell envelope was not due to a physical effect related to the temperature elevation, but rather to an alteration of the composition of the cell envelope. Using a mutant devoid of corynomycolates, significant differences in fluorescence anisotropy values were obtained compared to the wild-type strain, suggesting that TMA-DPH is mainly anchored into the corynomycomembrane. Differences in fluorescence anisotropy were also observed when the bacteria were cultivated at 33, 36, 38, and 39°C in batch cultures, and a linear relationship was obtained between the maximum specific glutamate production rate and the measured fluidity. When using the glutamate non-producing variant of C. glutamicum 2262, the fluorescence anisotropy remained constant at 0.207 ± 0.003 whatever the applied temperature shift. This suggests that the fluidity of the Corynebacteria mycomembrane plays an important role in glutamate excretion during the temperature-triggered process.     

Melatonin does not prevent the protection of ischemic preconditioning in vivo despite its antioxidant effect against oxidative stress

Free radicals are involved in the protective mechanism of preconditioning (PC), whereas antioxidant compounds abolish this benefit. Melatonin is a hormone with antioxidant properties. The aim of our study was to evaluate the effect of melatonin on infarct size in ischemic preconditioning in vivo. We randomly divided 33 male rabbits into four groups and subjected them to 30 min of myocardial ischemia and 3 h of reperfusion with the following prior interventions: (i) no intervention, (ii) iv melatonin at a total dose of 50 mg/kg, (iii) PC with two cycles of 5 min ischemia and 10 min reperfusion, and (iv) combined melatonin and PC. In a second series of experiments, another antioxidant agent N-acetylcysteine (NAC) was used in a control and in a PC group. Myocardial infarct size was determined and blood samples were drawn at different time points for the determination of lipid peroxidation products, total superoxide dismutase (SOD) activity, and (1)H-NMR spectra to evaluate the changes in the metabolic profile. Melatonin showed no effect on myocardial infarct size in the group of sustained ischemia (42.9 +/- 3.6% vs 47.4 +/- 4.9%) and it did not attenuate the reduction of myocardial infarct size in the PC group (13.6 +/- 2.4% vs 14.0 +/- 1.7%). A similar effect was found in NAC-treated groups (44.8 +/- 3.4% vs 14.3 +/- 1.3%). Lipid peroxidation product levels were significantly elevated in the control and PC groups, whereas melatonin decreased them in both groups. The SOD activity was enhanced in the PC group compared to controls; melatonin kept SOD activity unchanged during ischemia/reperfusion and enhanced its activity when it was combined with PC. Melatonin did not change the metabolic profile of the control and PC groups. Melatonin does not prevent the beneficial effect of ischemic PC on infarct size despite its antioxidant properties.     

Adaptation of aequorin functional assay to high throughput screening

AequoScreen, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal:background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z' values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.     

Changes in cardiac substrate transporters and metabolic proteins mirror the metabolic shift in patients with aortic stenosis

In the hypertrophied human heart, fatty acid metabolism is decreased and glucose utilisation is increased. We hypothesized that the sarcolemmal and mitochondrial proteins involved in these key metabolic pathways would mirror these changes, providing a mechanism to account for the modified metabolic flux measured in the human heart. Echocardiography was performed to assess in vivo hypertrophy and aortic valve impairment in patients with aortic stenosis (n = 18). Cardiac biopsies were obtained during valve replacement surgery, and used for western blotting to measure metabolic protein levels. Protein levels of the predominant fatty acid transporter, fatty acid translocase (FAT/CD36) correlated negatively with levels of the glucose transporters, GLUT1 and GLUT4. The decrease in FAT/CD36 was accompanied by decreases in the fatty acid binding proteins, FABPpm and H-FABP, the β-oxidation protein medium chain acyl-coenzyme A dehydrogenase, the Krebs cycle protein α-ketoglutarate dehydrogenase and the oxidative phosphorylation protein ATP synthase. FAT/CD36 and complex I of the electron transport chain were downregulated, whereas the glucose transporter GLUT4 was upregulated with increasing left ventricular mass index, a measure of cardiac hypertrophy. In conclusion, coordinated downregulation of sequential steps involved in fatty acid and oxidative metabolism occur in the human heart, accompanied by upregulation of the glucose transporters. The profile of the substrate transporters and metabolic proteins mirror the metabolic shift from fatty acid to glucose utilisation that occurs in vivo in the human heart.