Purification,characterization and developmental expression of pig liver PSP

We have isolated a perchloric acid-soluble protein designated as PL-PSP from the post-mitochondria supernatant fraction of pig liver. It is soluble in 5% perchloric acid and purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The PL-PSP showed approximately 80–90% homology with PSP isolated from rat liver (RL-PSP) with its partial amino acid sequences. The protein has a molecular mass of approximately 14 kDa which was slightly higher than that of RL-PSP. It inhibited protein synthesis in a rabbit reticulocyte lysate system. The expression of PL-PSP was predominant in liver, kidney and duodenum, and was also expressed in stomach, lung and brain. PL-PSP expression in liver increased from the 1st day to the 1st month. Thus, our findings are the first report on the presence of a PSP in porcine tissues which may be involved in the regulation of cellular growth and differentiation.     

Reaction mechanism for the diastereocontrolled synthesis of phosphorothioate DNA by the oxazaphospholidine approach

Reaction mechanisms for the diastereoselective synthesis of deoxyribonucleoside 3'-cyclic phosphoramidites as well as dinucleoside phosphite intermediates by the oxazaphospholidine approach were analyzed by means of ab initio molecular orbital calculations at the HF/6-31G* level. These reactions are essential for the diastereoselective synthesis of phsophorothioate DNA.     

Diastereocontrolled synthesis of phosphorothioate DNA via an oxazaphospholidine approach using a novel class of activators, dialkyl(cyanomethyl)ammonium salts

Dialkyl(cyanomethyl)ammonium salts 1 were synthesized and used as a novel class of activators for the stereospecific condensations of diastereopure nucleoside 3'-O-oxazaphospholidines with a nucleoside. This new oxazaphospholidine method could efficiently produce both (Rp)- and (Sp)-dinucleoside phosphorothioates.     

Inhibitory effect of hyperglycemia on insulin-induced Akt/protein kinase B activation in skeletal muscle

To determine the molecular mechanism underlying hyperglycemia-induced insulin resistance in skeletal muscles, postreceptor insulin-signaling events were assessed in skeletal muscles of neonatally streptozotocin-treated diabetic rats. In isolated soleus muscle of the diabetic rats, insulin-stimulated 2-deoxyglucose uptake, glucose oxidation, and lactate release were all significantly decreased compared with normal rats. Similarly, insulin-induced phosphorylation and activation of Akt/protein kinase B (PKB) and GLUT-4 translocation were severely impaired. However, the upstream signal, including phosphorylation of the insulin receptor (IR) and insulin receptor substrate (IRS)-1 and -2 and activity of phosphatidylinositol (PI) 3-kinase associated with IRS-1/2, was enhanced. The amelioration of hyperglycemia by T-1095, a Na(+)-glucose transporter inhibitor, normalized the reduced insulin sensitivity in the soleus muscle and the impaired insulin-stimulated Akt/PKB phosphorylation and activity. In addition, the enhanced PI 3-kinase activation and phosphorylation of IR and IRS-1 and -2 were reduced to normal levels. These results suggest that sustained hyperglycemia impairs the insulin-signaling steps between PI 3-kinase and Akt/PKB, and that impaired Akt/PKB activity underlies hyperglycemia-induced insulin resistance in skeletal muscle.     

p110beta is up-regulated during differentiation of 3T3-L1 cells and contributes to the highly insulin-responsive glucose transport activity

Activation of p85/p110 type phosphatidylinositol kinase is essential for aspects of insulin-induced glucose metabolism, including translocation of GLUT4 to the cell surface and glycogen synthesis. The enzyme exists as a heterodimer containing a regulatory subunit (e.g. p85alpha) and one of two widely distributed isoforms of the p110 catalytic subunit: p110alpha or p110beta. In the present study, we compared the two isoforms in the regulation of insulin action. During differentiation of 3T3-L1 cells into adipocytes, p110beta was up-regulated approximately 10-fold, whereas expression of p110alpha was unaltered. The effects of the increased p110 expression were further assessed by expressing epitope tagged p110beta and p110alpha in 3T3-L1 cells using adenovirus transduction systems, respectively. In vitro, the basal lipid kinase activity of p110beta was lower than that of p110alpha. When p110alpha and p110beta were overexpressed in 3T3-L1 adipocytes, exposing cells to insulin induced each of the subunits to form complexes with p85alpha and tyrosine-phosphorylated IRS-1 with similar efficiency. However, whereas the kinase activity of p110beta, either endogenous or exogeneous, was markedly enhanced by insulin stimulation, only very small increases of the activity of p110alpha were observed. Interestingly, overexpression of p110beta increased insulin-induced glucose uptake by 3T3-L1 cells without significantly affecting basal glucose transport, whereas overexpression of p110alpha increased both basal and insulin-stimulated glucose uptake. Finally, microinjection of anti-p110beta neutralizing antibody into 3T3-L1 adipocytes abolished insulin-induced translocation of GLUT4 to the cell surface almost completely, whereas anti-p110alpha neutralizing antibody did only slightly. Together, these findings suggest that p110beta plays a crucial role in cellular activities evoked acutely by insulin.