G protein-coupled receptor kinase 2 mediates endothelin-1-induced insulin resistance via the inhibition of both Galphaq/11 and insulin receptor substrate-1 pathways in 3T3-L1 adipocytes

G protein-coupled receptor kinases (GRKs) regulate seven-transmembrane receptors (7TMRs) by phosphorylating agonist-activated 7TMRs. Recently, we have reported that GRK2 can function as a negative regulator of insulin action by interfering with G protein-q/11 alpha-subunit (Galphaq/11) signaling, causing decreased glucose transporter 4 (GLUT4) translocation. We have also reported that chronic endothelin-1 (ET-1) treatment leads to heterologous desensitization of insulin signaling with decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and Galphaq/11, and decreased insulin-stimulated glucose transport in 3T3-L1 adipocytes. In the current study, we have investigated the role of GRK2 in chronic ET-1-induced insulin resistance. Insulin-induced GLUT4 translocation was inhibited by pretreatment with ET-1 for 24 h, and we found that this inhibitory effect was rescued by microinjection of anti-GRK2 antibody or GRK2 short interfering RNA. We further found that GRK2 mediates the inhibitory effects of ET-1 by two distinct mechanisms. Firstly, adenovirus-mediated overexpression of either wild-type (WT)- or kinase-deficient (KD)-GRK2 inhibited Galphaq/11 signaling, including tyrosine phosphorylation of Galphaq/11 and cdc42-associated phosphatidylinositol 3-kinase activity. Secondly, ET-1 treatment caused Ser/Thr phosphorylation of IRS-1 and IRS-1 protein degradation. Overexpression of KD-GRK2, but not WT-GRK2, inhibited ET-1-induced serine 612 phosphorylation of IRS-1 and restored activation of this pathway. Taken together, these results suggest that GRK2 mediates ET-1-induced insulin resistance by 1) inhibition of Galphaq/11 activation, and this effect is independent of GRK2 kinase activity, and 2) GRK2 kinase activity-mediated IRS-1 serine phosphorylation and degradation.     

Purification and characterization of a novel aminoacylase from Streptomyces mobaraensis

A novel aminoacylase was purified to homogeneity from culture broth of Streptomyces mobaraensis, as evidenced by SDS-polyacrylamide gel electrophoresis (PAGE). The enzyme was a monomer with an approximate molecular mass of 100 kDa. The purified enzyme was inhibited by the presence of 1,10-phenanthroline and activated by the addition of Co2+. It was stable at temperatures of up to 60 degrees C for 1 h at pH 7.2. It showed broad substrate specificity to N-acetylated L-amino acids. It catalyzed the hydrolysis of the amide bonds of various N-acetylated L-amino acids, except for Nepsilon-acetyl-L-lysine and N-acetyl-L-proline. Hydrolysis of N-acetyl-L-methionine and N-acetyl-L-histidine followed Michaelis-Menten kinetics with K(m) values of 1.3+/-0.1 mM and 2.7+/-0.1 mM respectively. The enzyme also catalyzed the deacetylation of 7-aminocephalosporanic acid (7-ACA) and cephalosporin C. Moreover, feruloylamino acids and L-lysine derivatives of ferulic acid derivatives were synthesized in an aqueous buffer using the enzyme.     

Genomewide high-density SNP linkage analysis of 236 Japanese families supports the existence of schizophrenia susceptibility loci on chromosomes 1p, 14q, and 20p

The Japanese Schizophrenia Sib-Pair Linkage Group (JSSLG) is a multisite collaborative study group that was organized to create a national resource for affected sib pair (ASP) studies of schizophrenia in Japan. We used a high-density single-nucleotide–polymorphism (SNP) genotyping assay, the Illumina BeadArray linkage mapping panel (version 4) comprising 5,861 SNPs, to perform a genomewide linkage analysis of JSSLG samples comprising 236 Japanese families with 268 nonindependent ASPs with schizophrenia. All subjects were Japanese. Among these families, 122 families comprised the same subjects analyzed with short tandem repeat markers. All the probands and their siblings, with the exception of seven siblings with schizoaffective disorder, had schizophrenia. After excluding SNPs with high linkage disequilibrium, we found significant evidence of linkage of schizophrenia to chromosome 1p21.2-1p13.2 (LOD=3.39) and suggestive evidence of linkage to 14q11.2 (LOD=2.87), 14q11.2-q13.2 (LOD=2.33), and 20p12.1-p11.2 (LOD=2.33). Although linkage to these regions has received little attention, these regions are included in or partially overlap the 10 regions reported by Lewis et al. that passed the two aggregate criteria of a meta-analysis. Results of the present study—which, to our knowledge, is the first genomewide analysis of schizophrenia in ASPs of a single Asian ethnicity that is comparable to the analyses done of ASPs of European descent—indicate the existence of schizophrenia susceptibility loci that are common to different ethnic groups but that likely have different ethnicity-specific effects.     

Adrenomedullin induces matrix metalloproteinase-2 activity in rat aortic adventitial fibroblasts

Background

The delicate balance of the extracellular matrix (ECM) determines the stiffness of the vascular wall, and adventitial fibroblasts are involved in ECM formation by synthesizing and degrading matrix proteins. In the present study, we examined the effect of the bioactive peptide adrenomedullin (AM) on activity and expression of matrix metalloproteinases (MMPs) in cultured aortic adventitial fibroblasts.

Methods and results

In cultured adventitial fibroblasts isolated from aorta of adult Wistar rats, 10⁻⁶ mol/L angiotensin II (Ang II) significantly (p < 0.05) down-regulated MMP-2 activity as determined by in vitro gelatin zymography. In contrast, 10⁻⁷ mol/L synthetic rat AM significantly (p < 0.05) stimulated zymographic MMP-2 activity by 23%, increasing intracellular cAMP, and AM abolished the action of Ang II, augmenting the MMP-2 activity. Similarly, Ang II down-regulated MMP-2 protein expression assessed by Western blotting, whereas AM increased it. Furthermore, 8-bromo-cAMP, an analogue of cAMP, mimicked the effect of AM, and H-89, an inhibitor for protein kinase A (PKA), significantly decreased the basal and AM-induced MMP-2 activity.

Conclusion

This study provides a new insight into the biological action of AM and its intracellular signaling system of cAMP/PKA stimulating the matrix degrading enzyme MMP-2, suggesting an important role for this molecule in modulating ECM deposition in the adventitial layer.     

Rapid degradation of Cdt1 upon UV-induced DNA damage is mediated by SCFSkp2 complex

Cdt1 is a licensing factor for DNA replication, the function of which is tightly controlled to maintain genome integrity. Previous studies have indicated that the cell cycle-dependent degradation of Cdt1 is triggered at S phase to prevent re-replication. In this study, we found that Cdt1 is degraded upon DNA damage induced by either UV treatment or gamma-irradiation (IR). Although the IR-triggered degradation of Cdt1 was caffeine-insensitive, the UV-triggered degradation of Cdt1 was caffeine-sensitive. This indicates that the cells treated with UV utilize the checkpoint pathway, which differs from that triggered by IR. A recent study has suggested that Cdt1 is phosphorylated, ubiquitylated, and degraded at the G(1)/S boundary in the normal cell cycle. Treatment with MG132, a proteasome inhibitor, inhibited the degradation of Cdt1 and resulted in the accumulation of the phosphorylated form of Cdt1 after UV treatment. In the case of UV treatment, phosphorylation of Cdt1 induced the recruitment of Cdt1 to a SCF(Skp2) complex. Moreover, ectopic overexpression of Cdt1 after UV treatment interfered the inhibition of DNA synthesis. These results indicate that Cdt1 is a target molecule of the cell cycle checkpoint in UV-induced DNA damage.     

Production of Man5GlcNAc2-type sugar chain by the methylotrophic yeast Ogataea minuta

The methylotrophic yeast Ogataea minuta IFO 10746 was selected as a suitable strain for producing human-compatible glycoproteins by means of analyses of its cell-wall mannoproteins. First, the OmURA3 gene encoding an orotidine-5'-phosphate decarboxylase was cloned and disrupted to generate a host strain with a uracil auxotrophic marker. Second, both the promoters and the terminators from the OmAOX1 gene encoding an alcohol oxidase for an inducible promoter, or those from the OmTDH1 gene encoding a glyceraldehyde-3-phosphate dehydrogenase for a constitutive promoter, were isolated to construct an expression vector system for heterologous genes. Next, the OmOCH1 gene encoding a starting enzyme with alpha-1,6-mannosyltransferase activity to form a backbone of the N-linked outer sugar chain peculiar to yeast was disrupted, and an alpha-1,2-mannosidase gene from Aspergillus saitoi with an endoplasmic reticulum retention signal (HDEL) under the control of the OmAOX1 promoter was introduced to convert the sugar chain to Man5GlcNAc2 in O. minuta. As a result, we succeeded in breeding a new methylotrophic yeast, O. minuta, producing a Man5GlcNAc2-high-mannose-type sugar chain as a prototype of a human-compatible sugar chain. We also elucidate here the usefulness of the strategy for producing human-compatible sugar chains in yeast.