Full, persistent blockade of central neurokinin-1 (NK1) receptors may be a potential antidepressant mechanism. The selective NK1 antagonist orvepitant (GW823296) was used to test this hypothesis. A preliminary positron emission tomography study in eight male volunteers drove dose selection for two randomized six week studies in patients with major depressive disorder (MDD). Displacement of central [(11)C]GR205171 binding indicated that oral orvepitant doses of 30-60 mg/day provided >99% receptor occupancy for ≥24 h. Studies 733 and 833 randomized patients with MDD and 17-item Hamilton Depression Rating Scale (HAM-D)≥22 to double-blind treatment with orvepitant 30 mg/day, orvepitant 60 mg/day or placebo (1:1:1). Primary outcome measure was change from baseline in 17-item HAM-D total score at Week 6 analyzed using mixed models repeated measures. Study 733 (n=328) demonstrated efficacy on the primary endpoint (estimated drug-placebo differences of 30 mg: -2.41, 95% confidence interval (CI) (-4.50 to -0.31) p=0.0245; 60 mg: -2.86, 95% CI (-4.97 to -0.75) p=0.0082). Study 833 (n=345) did not show significance (estimated drug-placebo differences of 30 mg: -1.67, 95% CI (-3.73 to 0.39) p=0.1122; 60 mg: -0.76, 95% CI (-2.85 to 1.32) p=0.4713). The results support the hypothesis that full, long lasting blockade of central NK1 receptors may be an efficacious mechanism for the treatment of MDD.
A soybean flour-induced, soluble cytochrome P-450 (P-450,,.Y) was purified 130-fold to homogeneity from Streptomyces gyiseus. Native cytochrome P-450,,. is a single polypeptide, with a molecular weight of 47,500, in association with one ferriprotoporphyrin IX prosthetic group. Oxidized P-450S exhibited visible absorption maxima at 394, 514, and 646 nm, characteristic of a high-spin cytochrome P-450. The CO-reduced difference spectrum of P-450O had a Soret maximum at 448 nm. When reconstituted with spinach ferredoxin and spinach ferredoxin;NADP+ oxidoreductase, purified cytochrome P-450O, catalyzed the NADPH-dependent oxidation of the xenobiotic substrates precocene II and 7-ethoxycoumarin. In vitro proteolysis of cytochrome P-450soy generated a stable and catalytically active cytochrome P-450, designated P-450SOYA.Streptomyces griseus (ATCC 13273) has the remarkable ability to catalyze the stereo-and regiospecific oxidation of a diverse array of xenobiotics. The types of reactions performed by S. griseus include aromatic, cyclic, and aliphatic hydroxylations, 0-, S-, and N-oxidations, C-C fission, epoxidation, and 0-and N-dealkylations. These transformations, which occur with compounds such as alkaloids (17,27), coumarins (25), rotenoids (28), chromenes (30), and other complex xenobiotics (7,26), parallel those performed by mammalian cytochromes P-450 (34, 36). However, the nature of the enzymatic system(s) employed by S. griseus for such diverse reactions has remained unresolved (26). The biotransformations catalyzed by S. griseus occur primarily following growth of the organism on a complex medium enriched with soybean flour (26). Recently, Sariaslani and Kunz (29) demonstrated that soybean flour and one of its isoflavonoid constituents, genistein, induce the synthesis of cytochrome P-450 in S. griseus and inferred a role for this enzyme in the biotransformation reactions performed by this organism. Here we report the purification and characterization of a soybean flour-induced cytochrome P-450 and demonstrate the transformation of two xenobiotic substrates, precocene II and 7-ethoxycoumarin, by purified preparations of this enzyme.( was determined by the method of Omura and Sato (23). Heme was analyzed as its reduced pyridine hemochrome derivative (10). High-pressure liquid chromatography (HPLC) was performed on a system consisting of an LKB 2152 controller, LKI3 2150 pump, and a Hewlett-Packard 1040A diode array detector.Cytochrome P-450.,y purification protocol. All procedures except steps 4 and 5 were carried out at 4°C. Glycerol (20% [vol/vol]) was included in all buffers following the salt precipitation step to prevent degradation of the P-450 to its inactive P-420 species. Protease inhibitors used in steps 1 to 3 to inhibit a wide range of protease activities were pepstatin (an acid protease inhibitor), leupeptin (a broad-range protease inhibitor), and phenylmethylsulfonyl fluoride (PMSF; a serine protease inhibitor). Fractions were stored between purification steps at -80°C with no discernable loss o...
The genome of the pufferfish (Fugu rubripes) (400 Mb) is -7.5 times smaller than the human genome, but it has a similar gene repertoire to that of man. If regions of the two genomes exhibited conservation of gene order (i.e., were syntenic), it should be possible to reduce dramatically the effort required for identification of candidate genes in human disease loci by sequencing syntenic regions of the compact Fugu genome. We have demonstrated that three genes (dihydrolipoamide succinyltransferase, S31iii125, and S20il5), which are linked to FOS in the familial Alzheimer disease locus (AD3) on human chromosome 14, have homologues in the Fugu genome adjacent to Fugu cFOS. The relative gene order of cFOS, S31iii125, and S20i15 was the same in both genomes, but in Fugu these three genes lay within a 12.4-kb region, compared to >600 kb in the human AD3 locus. These results demonstrate the conservation of synteny between the genomes of Fugu and man and highlight the utility of this approach for sequence-based identification of genes in human disease loci.
An unusual Xanthobacter sp., capable of independent growth on cyclohexane as the sole source of carbon and energy, has been isolated from soil by using classical enrichment techniques. The mean generation time for growth on cyclohexane was 6 h. The microorganism showed a limited ability to utilize hydrocarbons, with only alicyclic hydrocarbons closely related to cyclohexane supporting growth. Ultrastructural studies indicated the presence of electron-transparent vesicles in the cyclohexane-grown Xanthobacter sp., but the presence of complex intracytoplasmic membranes could not be identified. A soluble inducible enzyme capable of oxidizing cyclohexane was identified in cell extracts. This enzyme had a pH optimum of 6.5, an absolute specificity for NADPH, and a stoichiometric requirement for molecular 02 which was consistent with the formation of cyclohexanol. The enzyme showed no activity towards straight chain alkanes and only a limited activity towards unsaturated ring compounds. Enzymatic studies with cell extracts have indicated the main route of metabolism of cyclohexane by this Xanthobacter sp. to proceed via cyclohexane-* cyclohexanol-* cyclohexanone-* 1-oxa-2-oxocycloheptane (r-caprolactone)-* 6-hydroxyhexanoate (6-hydroxycaproate)-*-* adipic acid. Alternative routes involving initial double hydroxylation of the cyclohexane ring may operate fortuituously but are unlikely to represent major pathways for the dissimilation of cyclohexane by this microorganism.
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