An acyl-CoA hydrolase, referred to as hBACH, was purified from human brain cytosol. The enzyme had a molecular mass of 100 kDa and 43-kDa subunits, and was highly active with long-chain acyl-CoAs, e.g. a maximal velocity of 295 micromol/min/mg and K(m) of 6.4 microM for palmitoyl-CoA. Acyl-CoAs with carbon chain lengths of C(8-18) were also good substrates. In human brain cytosol, 85% of palmitoyl-CoA hydrolase activity was titrated by an anti-BACH antibody, which accounted for over 75% of the enzyme activity found in the brain tissue. The cDNA isolated for hBACH, when expressed in Escherichia coli, directed the expression of palmitoyl-CoA hydrolase activity and a 44-kDa protein immunoreactive to the anti-BACH antibody, which in turn neutralized the hydrolase activity. The hBACH cDNA encoded a 338-amino acid sequence which was 95% identical to that of a rat homolog. The hBACH gene spanned about 130 kb and comprised 9 exons, and was mapped to 1p36.2 on the cytogenetic ideogram. These findings indicate that the long-chain acyl-CoA hydrolase present in the brain is well conserved between man and the rat, suggesting a conserved role for this enzyme in the mammalian brain, and enabling genetic studies on the functional analysis of acyl-CoA hydrolase.

For efficient drug discovery and screening, it is necessary to simplify P-glycoprotein (P-gp) substrate assays and to provide in silico models that predict the transport potential of P-gp. In this study, we developed a simplified in vitro screening method to evaluate P-gp substrates by unidirectional membrane transport in P-gp-overexpressing cells. The unidirectional flux ratio positively correlated with parameters of the conventional bidirectional P-gp substrate assay (R 2 = 0.941) and in vivo K p,brain ratio (mdr1a/1b KO/WT) in mice (R 2 = 0.800). Our in vitro P-gp substrate assay had high reproducibility and required approximately half the labor of the conventional method. We also constructed regression models to predict the value of P-gp-mediated flux and three-class classification models to predict P-gp substrate potential (low-, medium-, and high-potential) using 2397 data entries with the largest data set collected under the same experimental conditions. Most compounds in the test set fell within two- and three-fold errors in the random forest regression model (71.3 and 88.5%, respectively). Furthermore, the random forest three-class classification model showed a high balanced accuracy of 0.821 and precision of 0.761 for the low-potential classes in the test set. We concluded that the simplified in vitro P-gp substrate assay was suitable for compound screening in the early stages of drug discovery and that the in silico regression model and three-class classification model using only chemical structure information could identify the transport potential of compounds including P-gp-mediated flux ratios. Our proposed method is expected to be a practical tool to optimize effective central nervous system (CNS) drugs, to avoid CNS side effects, and to improve intestinal absorption.

To understand the comprehensive mechanisms of gene expression and processing for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the gene organization, promoter and transcriptional initiation sites, alternative splicing and polyadenylating sites, and the cDNA structures of this gene in the Japanese flounder, Paralichthys olivaceus. The flounder IGF-I gene was found to be composed of five exons and four introns spanning 17.5 kb. By Northern blot analysis, two major mRNA classes of 4.7 kb and 2.9 kb were found in the liver. cDNA cloning and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that these two mRNA classes result from two different-sized 3'-noncoding regions generated by alternative usage of two polyadenylating signals. Further analysis by RT-PCR and sequencing revealed that these mRNA classes both contain two subclasses of mRNA encoding two forms of IGF-I prepropeptide, preproIGF-I-1 and preproIGF-I-2. The two forms of preproIGF-I share the identical signal peptide and mature IGF-I domain but contain different E domains as a result of alternative splicing in exon 3. The mature form of flounder IGF-I was found to comprise 68 amino acid residues, showing a small molecular weight, 7486. In the 5'-flanking region, one major and four minor transcription start sites have been identified by ribonuclease protection assay between -230 and -130 from the translation initiation codon, but no canonical TATA box or GC box was detected in their upstream regions up to -724. The results suggest that some unknown transcription initiation factors are functioning in the promotion of IGF-I gene expression.

A Gram-stain-positive, non-motile, mesophilic, aerobic, coccus-shaped bacterium, designated strain Y7R2T, was isolated from the brain of a Chiroteuthis picteti squid living in mesopelagic water near Muroto, Kochi, Japan. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain was most closely related to the genus Hoyosella (96.1 % similarity to the type strain of the type species Hoyosella altamirensis ) and formed a separate distinct cluster in a stable, deep-branching lineage with the type strains of Hoyosella suaedae and Hoyosella lacisalsi (98.7–99.5% similarities). The major fatty acids (>10 %) of strain Y7R2T were C17 : 1 ω8c, C15 : 0, C16 : 1 ω6c/C16 : 1 ω7c and C16 : 0, and the isoprenoid quinones were menaquinone-7 (57.8 %) and menaquinone-8 (42.2 %). The principal polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylinositol, and the DNA G+C content was 68.0 %. These chemotaxonomic features, with the exception of the fatty acid composition, were similar to those of the phylogenetically clustered species ( H. suaedae and H. lacisalsi ) but different from those of core Hoyosella species (including H. altamirensis ). These results suggested that Y7R2T, H. suaedae and H. lacisalsi strains should be assigned to a novel genus. Furthermore, strain Y7R2T showed low average nucleotide identity values (88.0–88.2 %) and low digital DNA–DNA hybridization values (34.3–34.7 %) to the type strains of H. suaedae and H. lacisalsi . These data indicated that strain Y7R2T should be assigned to a novel genus and species, for which the name Lolliginicoccus levis gen. nov., sp. nov. is proposed. The type strain is Y7R2T (=NBRC 114883T=KCTC 49749T). Accordingly, reclassification of H. suaedae and H. lacisalsi as Lolliginicoccus suaedae comb. nov. (type species) and Lolliginicoccus lacisalsi comb. nov. is also proposed.

No abstract

No abstract