Protein kinase Czeta and glucose uptake.

Protein kinase Czeta (PKCzeta) is a member of the PKC family, serving downstream of insulin receptor and phosphatidylinositol (PI) 3-kinase. Many evidences suggest that PKCzeta plays a very important role in activating glucose transport response. Not only insulin but also glucose and exercise can activate PKCzeta through diverse pathways. PKCzeta activation and activity are impaired with insulin resistance in muscle and adipose tissues of type II diabetes individuals, but heightened in liver tissue, wherein it also increases lipid synthesis mediated by SREBP-1c (sterol-regulatory element-binding protein). Many studies have focused on linkage between PKCzeta and GLUT4 translocation and activation. Exploring the molecular mechanisms and pathways by which PKCzeta mediates glucose transport will highlight the insulin-signaling pathway.

Atypical protein kinase C in glucose metabolism.

Type 2 diabetes mellitus is a multigenic disease with evident genetic predisposition, and complex pathogenesis in which environmental and genetic factors interact. The disorder of body utilization glucose is a crucial reason for causing diabetes. Atypical PKCs, belonging to Ser/Thr protein kinase, have many important biological functions in vivo, and may be involved in the pathogenesis of diabetes mellitus. APKCs participate in glucose metabolism by regulating glucose transport and absorption, glycogen synthesis, and insulin secretion. The exact mechanism by which aPKCs participate in glucose metabolism remains unclear. So far, the clarification of which will be helpful for the prevention and cure of type 2 diabetes.

[Expression and identification of recombinant arresten in Pichia pastoris].

Arresten as a endogenous inhibitor of angiogenesis originated from the carboxyl-terminal 223 amino acids fragment of the non-collagen domain in alpha1 chain of human collagen IV. In order to get the soluble arresten with biological activity, the cDNA of arresten was cloned and expressed in Pichia pastoris. The produced arresten cDNA was amplified by PCR using primer P1:5'-AGGCCCCGATGGGTTGC-3', primer P2:5'-CTATAAG GCACTTTACGGTTTC-3'. The PCR products was cloned into pGEM-T vector, and sequenced. The arresten cDNA from pGEM-T vector was recombined with vector pPIC9 as pPIC9-arresten, used to transform E. coli DH5alpha, and the inserted arresten cDNA confirmed by agarose electrophoresis and sequencing. pPIC9-arresten was linearized by Sac I. Pichia pastoris GS115 was treated with PEG1000 (followed Invitrogen' s specification); transformed with linear recombined pPIC9-arresten. Pichia pastoris GS115 was culured on MD mediun, single clone was selected and the DNA from the single clone was extracted, used as template, characterized by PCR using the second pair primers P3:5'-CGCTCGAGAAAAGATCTGTTGATC-3', P4:5'-GCCCCGG ATCCTTATGTTCTFCTCATACAG-3'. The polynucleotides CTCGAGAAAAGA used as marker sequence was inserted into primer P3 for signal peptidase to cleave off the signal sequence correctively. The recombined Pichia pastoris GS115 was selected according to the results of PCR, cultured on MM and MD media and then in the BMGY media using methanol as inducer. Expressed arresten was analysed by SDS-PAGE. The soluble arresten expressed by Pichia pastoris gave apparent molecular weight in SDS-PAGE consistent with that calculated, and in matrigel gel it showed inhibitary activity on the tubulation of endothelial cell ECV-304 induced by tumor cell MDA-MB-435S. These results showesd arresten with biological activity is expressed successfully in Pichia pastoris GS115.