High-throughput scintillation proximity assay for stearoyl-CoA desaturase-1

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of monounsaturated fatty acids and has been implicated in a number of disease states, including obesity and diabetes. To find small-molecule inhibitor leads, a high-throughput scintillation proximity assay (SPA) was developed using the hydrophobic binding characteristics of a glass microsphere scintillant bead to capture SCD1 from a crude lysate of recombinant SCD1 in Sf9 lysate coupled with the strong binding characteristics of an azetidine compound ([(3)H]AZE). The SPA assay was stable over 24 h and could detect compounds with micromolar to nanomolar potencies. A robust 1536-well high-throughput screening assay was developed with good signal-to-noise ratio (10:1) and excellent Z' factor (0.8). A screening collection of 1.6 million compounds was screened at 11 μM, and approximately 7700 compounds were identified as initial hits, exhibiting at least 35% inhibition of [(3)H]AZE binding. Further screening and confirmation with an SCD enzyme activity assay led to a number of new structural leads for inhibition of the enzyme. The SPA assay complements the enzyme activity assay for SCD1 as a tool for the discovery of novel leads in drug discovery.     

Group composition in wild and commensal hamadryas baboons: A comparative study in Saudi Arabia

Papio hamadryas was surveyed throughout its range in Saudi Arabia and was observed at altitudes ranging from 0 to 2300 m. Wild populations occur along the whole range of altitude, while commensal populations are only found above 850 m altitude. No variation in group size was found with altitude. Comparison of wild and commensal populations showed the following. (1) Their composition in terms of age and sex classes, overall adult sex ratios, and group size does not significantly differ. (2) Groups of both populations include, in similar proportions, three types of parties: one-male units (>70%), two-male units (>13%), and a few other units of variable composition. (3) The mean size of commensal parties is significantly larger than in the wild population; specifically one-male units are larger in the commensal population due to a larger number of females per male. Thus, female distribution in commensal groups is more inequitable than that in wild groups. (4) Finally, the number of females included in two-male units increases with altitude. These differences are discussed in terms of food availability and predator pressure and are compared with results obtained on other Arabian and Ethiopian populations.     

Microbial transformation of ethylpyridines

Pyridine and its derivatives have been found as pollutants in the environment. Although alkylpyridines constitute the largest class of pyridines contaminating the environment, little information is available concerning the fate and transformation of these compounds. In this investigation ethylpyridines have been used as model compounds for investigating the biodegradability of alkylpyridines. A mixed culture of ethylpyridine-degrading microorganisms was obtained from a soil that had been exposed to a variety of pyridine derivatives for several decades. The enrichment culture was able to degrade 2-, 3-, and 4-ethylpyridine (100 mg/L) at 28° C and pH 7 within two weeks under aerobic conditions. The degradation rate was greatest for 2-ethylpyridine and least for 3-ethylpyridine. Transformation of ethylpyridines was dependent on substrate concentration, pH, and incubation temperature. Studies on the metabolic pathway of 4-ethylpyridine revealed two products; these chemicals were identified by MS and NMR analyses as 4-ethyl-2(1H)-pyridone and 4-ethyl-2-piperidone. 6-Ethyl-2(1H)-pyridone was determined to be a product of 2-ethylpyridine degradation. These results indicate that the transformation mechanism of ethylpyridines involves hydroxylation and reduction of the aromatic ring before ring cleavage.     

sst2 Somatostatin receptor inhibits cell proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 activation

The G protein-coupled sst2 somatostatin receptor is a critical negative regulator of cell proliferation. sstII prevents growth factor-induced cell proliferation through activation of the tyrosine phosphatase SHP-1 leading to induction of the cyclin-dependent kinase inhibitor p27Kip1. Here, we investigate the signaling molecules linking sst2 to p27Kip1. In Chinese hamster ovary-DG-44 cells stably expressing sst2 (CHO/sst2), the somatostatin analogue RC-160 transiently stimulates ERK2 activity and potentiates insulin-stimulated ERK2 activity. RC-160 also stimulates ERK2 activity in pancreatic acini isolated from normal mice, which endogenously express sst2, but has no effect in pancreatic acini derived from sst2 knock-out mice. RC-160-induced p27Kip1 up-regulation and inhibition of insulin-dependent cell proliferation are both prevented by pretreatment of CHO/sst2 cells with the MEK1/2 inhibitor PD98059. In addition, using dominant negative mutants, we show that sst2-mediated ERK2 stimulation is dependent on the pertussis toxin-sensitive Gi/o protein, the tyrosine kinase Src, both small G proteins Ras and Rap1, and the MEK kinase B-Raf but is independent of Raf-1. Phosphatidylinositol 3-kinase (PI3K) and both tyrosine phosphatases, SHP-1 and SHP-2, are required upstream of Ras and Rap1. Taken together, our results identify a novel mechanism whereby a Gi/o protein-coupled receptor inhibits cell proliferation by stimulating ERK signaling via a SHP-1-SHP-2-PI3K/Ras-Rap1/B-Raf/MEK pathway.     

Metabolism and Solubilization of Cellulose by Clostridium cellulolyticum H10

When Clostridium cellulolyticum was grown with cellulose MN300 as the substrate, the rates of growth and metabolite production were found to be lower than those observed with soluble sugars as the substrate. At low cellulose concentrations, the growth yields were equal to those obtained with cellobiose. The main fermentation products from cellulose and soluble sugars were the same. Up to 15 mM of consumed hexose, a change in the metabolic pathway favoring lactate production similar to that observed with soluble sugars was found to occur concomitantly with a decrease in molar growth yield. With cellulose concentrations above 5 g/liter, accumulation of soluble sugars occurred once growth had ceased. Glucose accounted for 30% of these sugars. A kinetic analysis of cellulose solubilization revealed that cellulolysis by C. cellulolyticum involved three stages whatever cellulose concentration was used. Analysis of these kinetics showed three consecutive enzymatic activity levels having the same K_m (0.8 g of cellulose per liter, i.e., 5 mM hexose equivalent) but decreasing values of V_max. The hypothesis is suggested that each step corresponds to differences in cellulose structure.     

Preparation and crystal structure of bis(acetato)(trans-1,2-diaminocyclohexane)platinum(II)

The structure of the complex [Pt(trans-1,2-di- aminocyclohexane) (acetate)₂]·H₂O has been determined by X-ray diffraction. This racemic compound is orthorhombic, space group Aba2, a = 20.813(9), b = 7.926(5), c = 17.296(8) Å, Z = 8. The structure was refined on 1214 nonzero Cu Kα reflections to R = 0.028. The square planar environment of Pt includes the amino groups of the diamine in cis positions and oxygens from two monodentate acetates. The PtN and PtO distances average 2.00(3) and 2.02(3) Å, respectively. The bite of the diamine ligand imposes a NPtN angle of 85(1)°, whereas the small OPtO angle of 85(1)° probably results from packing effects. The average plane through the puckered cyclohexyl ring makes an angle of 19° with the PtN₂O₂ plane. The molecules are stacked by pairs along the b axis. The two molecules of each pair are 180° apart about the stacking axis, and form altogether four NH···O hydrogen bonds.