CC1021 Sigma-AldrichHuman Integrin αVβ3, Octyl-β-D-glucopyranoside Formulation

The L-galactose (Smirnoff-Wheeler) pathway represents the major route to L-ascorbic acid (vitamin C) biosynthesis in higher plants. Arabidopsis thaliana VTC2 and its paralogue VTC5 function as GDP-L-galactose phosphorylases converting GDP-L-galactose to L-galactose-1-P, thus catalyzing the first committed step in the biosynthesis of L-ascorbate. Here we report that the L-galactose pathway of ascorbate biosynthesis described in higher plants is conserved in green algae. The Chlamydomonas reinhardtii genome encodes all the enzymes required for vitamin C biosynthesis via the L-galactose pathway. We have characterized recombinant C. reinhardtii VTC2 as an active GDP-L-galactose phosphorylase. C. reinhardtii cells exposed to oxidative stress show increased VTC2 mRNA and L-ascorbate levels. Genes encoding enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, manganese superoxide dismutase, and dehydroascorbate reductase) are also up-regulated in response to increased oxidative stress. These results indicate that C. reinhardtii VTC2, like its plant homologs, is a highly regulated enzyme in ascorbate biosynthesis in green algae and that, together with the ascorbate recycling system, the L-galactose pathway represents the major route for providing protective levels of ascorbate in oxidatively stressed algal cells.

Integrin alpha(v)beta(3) is overexpressed on neoendothelial cells and frequently on tumor cells. We have developed a peptide-like scaffold (regioselectively addressable functionalized template, RAFT), which holds four cyclo(-RGDfK-) (cRGD) motifs and proved that this molecule (called regioselectively addressable functionalized template-arginine-glycine-aspartic acid, RAFT-RGD) targets integrin alpha(v)beta(3) in vitro and in vivo. Using fluorescence correlation spectroscopy (FCS), we measured the constant of affinity (K(D)) of the RAFT-RGD for purified integrins. K(D) values rose from 3.87 nmol/l for RAFT-RGD to 41.70 nmol/l for cyclo(-RGDfK-). In addition, RAFT-RGD inhibited alpha(v)beta(3) lateral mobility in the cell membrane, probably due to the formation of integrin clusters as demonstrated by fluorescence recovery after photobleaching (FRAP). This was confirmed by electronic microscopy data, which established the formation of molecular complexes containing two integrins in the presence of RAFT-RGD but not cRGD or regioselectively addressable functionalized template-arginine-alanine- aspartic acid (RAFT-RAD). Using an enzyme-linked immunosorbent assay (ELISA), we proved that 1 micromol/l RAFT-RGD increased by 79% alpha(v)beta(3) internalization via clathrin-coated vesicles. Conversely, cRGD was internalized without modifying alpha(v)beta(3) internalization. Although RGD has been known for >20 years, this is the first study to formerly establish the relationships among multimeric presentation, increased affinity, and subsequent integrin-mediated cointernalization. These results strongly support the rationale for using multimeric RGD-peptides as targeting vectors for imaging, diagnosis, or therapy of cancers.