Selective attenuation of metabolic branch of insulin receptor down-signaling by high glucose in a hepatoma cell line, HepG2 cells

The effects of a high concentration of glucose on the insulin receptor-down signaling were investigated in human hepatoma (HepG2) cells in vitro to delineate the molecular mechanism of insulin resistance under glucose toxicity. Treatment of the cells with high concentrations of glucose (15-33 mm) caused phosphorylation of serine residues of the insulin receptor substrate 1 (IRS-1), leading to reduced electrophoretic mobility of it. The phosphorylation of IRS-1 with high glucose treatment was blocked only by protein kinase C (PKC) inhibitors. The high glucose treatment attenuated insulin-induced association of IRS-1 and phosphatidylinositol 3-kinase and insulin-stimulated phosphorylation of Akt. A metabolic effect of insulin, stimulation of glycogen synthesis, was also inhibited by the treatment. In contrast, insulin-induced association of Shc and Grb2 was not inhibited. Treatment of the cells with high glucose promoted the translocation of PKCepsilon and PKCdelta from the cytosol to the plasma membrane but not that of other PKC isoforms. Finally, PKCepsilon and PKCdelta directly phosphorylated IRS-1 under cell-free conditions. We conclude that a high concentration of glucose causes phosphorylation of IRS-1, leading to selective attenuation of metabolic signaling of insulin. PKCepsilon and PKCdelta are involved in the down-regulation of insulin signaling, and they may lie in a pathway regulating the phosphorylation of IRS-1.     

Pigment cell-specific expression of the tyrosinase gene in ascidians has a different regulatory mechanism from vertebrates

Tyrosinase is the key enzyme required for the synthesis of melanin pigments. Sequence comparison and functional analysis of the 5' upstream regions of vertebrate tyrosinase genes have revealed the importance of conserved E-box motifs in regulating their specific expression in pigment cells, optic cup-derived retinal pigment epithelium (RPE) and neural crest-derived melanocytes. In ascidians (more basal protochordates), two pigment cells that resemble vertebrate RPE cells are formed and specifically express the orthologous tyrosinase gene (HrTyr) in the cerebral vesicle located at the anterior end of the neural tube. To define regulatory sequences required for pigment cell-lineage-specific expression of HrTyr during embryogenesis, a series of mutations of the 5' upstream region of HrTyr were fused to the lacZ reporter gene and were microinjected into fertilized eggs. We found that the -152bp upstream of the translational start site is essential for expression in pigment cell precursors of tailbud-stage embryos. Further, additional positive and unique restriction elements were identified in the region up to -1.8kb. Surprisingly, in the -152bp minimal promoter or in other regions with regulatory activities, there are no E-box motifs or sequences correlating with other conserved elements regulating vertebrate tyrosinase promoters. The possibility that Pax proteins regulate HrTyr expression is also discussed.     

The mouse ortholog of EFHC1 implicated in juvenile myoclonic epilepsy is an axonemal protein widely conserved among organisms with motile cilia and flagella

The gene product of EFHC1 recently implicated in juvenile myoclonic epilepsy (JME) was found to be a homolog of Chlamydomonas axonemal protein Rib72, whose homologs are present in a wide variety of organisms that have motile cilia and flagella. Western blot analyses and immunofluorescence localization of the mouse ortholog mRib72-1/Efhc1 indicated that it is indeed abundantly present in sperm flagella and tracheal cilia but only in a small amount in the brain. It is not present in immotile primary cilia. These observations raise the possibility that malfunction of motile cilia is involved in the development of JME.     

CIBEX: center for information biology gene expression database

We describe the current status of the gene expression database CIBEX (Center for Information Biology gene EXpression database, http://cibex.nig.ac.jp), with a data retrieval system in compliance with MIAME, a standard that the MGED Society has developed for comparing and data produced in microarray experiments at different laboratories worldwide. CIBEX serves as a public repository for a wide range of high-throughput experimental data in gene expression research, including microarray-based experiments measuring mRNA, serial analysis of gene expression (SAGE tags), and mass spectrometry proteomic data.     

Involvement of p42/44 MAPK and RhoA protein in augmentation of ACh-induced bronchial smooth muscle contraction by TNF-alpha in rats.

Bronchial asthma is characterized by chronic inflammation of airway tissues and nonspecific airway hyperresponsiveness (AHR), but the underlying mechanisms of AHR have yet to be elucidated. Recently, tumor necrosis factor-alpha (TNF-alpha) has been identified as a proinflammatory cytokine that might be important in the hyperresponsiveness of airway tissue. We have investigated the effects of SB-203580 (a p38 MAPK inhibitor), U-0126 (an inhibitor of p42/44 MAPK activation), and cycloheximide (an inhibitor of protein synthesis) on TNF-alpha-augmented ACh-induced bronchial smooth muscle contraction. We have also investigated the phosphorylation of p42/44 MAPK and upregulation of RhoA protein by TNF-alpha. Treatment of rat bronchial smooth muscles with TNF-alpha (300 and 1,000 ng/ml for 24 h) resulted in a significant upward shift in the concentration-response curve to ACh, but not to high K(+), compared with control tissues. The effect of TNF-alpha was completely blocked by pretreatment with U-0126 or cycloheximide, but not with SB-203580. Immunoblotting demonstrated that p42/44 MAPK was phosphorylated and RhoA protein was increased in bronchial tissue by TNF-alpha. Furthermore, the TNF-alpha-induced upregulation of RhoA protein was abolished by U-0126 pretreatment. In conclusion, we suggest that TNF-alpha might be one of the important mediators involved in the pathogenesis of augmented bronchial smooth muscle contractility in AHR. For the first time, we have demonstrated that augmentation of ACh-induced contractile response evoked by TNF-alpha was mediated by synthesis of protein, such as RhoA, through activation of p42/44, but not p38 MAPK, in rat bronchial smooth muscle.