Up-regulation of 94-kDa glucose-regulated protein by hypoxia-inducible factor-1 in human endothelial cells in response to hypoxia

Hypoxic environment in solid tumor is known to favor cell survival and to initiate the formation of new capillaries. In this work, we identified by 2D gel analysis 94-kDa glucose-regulated protein (GRP94) as being upregulated in human endothelial cells in response to hypoxia. Three putative hypoxia responsive elements (HRE) were found in the GRP94 promoter. Competition experiments of HIF-1 DNA binding using specific probes containing each HRE sequence of the GRP94 promoter clearly evidenced that HIF-1 binds these sequences with high affinity. The human GRP94 promoter was then cloned upstream of the luciferase gene and showed enhanced activity in hypoxic conditions. Mutation of two of the three HREs present in this promoter completely inhibited the hypoxia-induced increase in luciferase activity.     

Pig muscle carnitine palmitoyltransferase I (CPTI beta), with low Km for carnitine and low sensitivity to malonyl-CoA inhibition, has kinetic characteristics similar to those of the rat liver (CPTI alpha) enzyme

The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of long-chain fatty acids. There are two well-characterized isotypes of CPTI: CPTIalpha (also known as L-CPTI) and CPTIbeta (also known as M-CPTI) that in human and rat encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition. Kinetic hallmarks of the CPTIalpha are high affinity for carnitine and low sensitivity to malonyl-CoA inhibition, while the opposite characteristics, low affinity for carnitine and high sensitivity to malonyl-CoA, are intrinsic to the CPTIbeta isotype. We have isolated the pig CPTIbeta cDNA which encodes for a protein of 772 amino acids that shares extensive sequence identity with human (88%), rat (85%), and mouse (86%) CPTIbeta, while the degree of homology with the CPTIalpha from human (61%), rat (62%), and mouse (60%) is much lower. However, when expressed in the yeast Pichia pastoris, pig CPTIbeta shows kinetic characteristics similar to those of the CPTIalpha isotype. Thus, the pig CPTIbeta, unlike the corresponding human or rat enzyme, has a high affinity for carnitine (K(m) = 197 microM) and low sensitive to malonyl-CoA inhibition (IC(50) = 906 nM). Therefore, the recombinant pig CPTIbeta has unique kinetic characteristics, which makes it a useful model to study the structure-function relationship of the CPTI enzymes.     

Hyperaccumulation of metals by Thlaspi caerulescens as affected by root development and Cd-Zn/Ca-Mg interactions

The aim of this work was to study, in a rhizobox experiment, the phytoextraction of metals by the hyperaccumulator plant Thlaspi caerulescens in relation to the heterogeneity of metal pollution. Six treatments were designed with soils containing various levels of metals. Homogeneous soils and inclusions of soils in other soil matrices were prepared in order to vary metal concentration and localization. Growth parameters of the plant (rosette diameter and shoot biomass) and localization of roots and shoot uptake of Zn, Cd, Ca, and Mg were determined after 10 weeks of growth. The plants grown on the polluted industrial soils provided a larger biomass and had lower mortality rates than those grown on the agricultural soil. Moreover, these plants accumulated more Zn and Cd (up to 17,516 and 375 mg kg(-1) DM, respectively) than plants grown on the agricultural soil (up to 7300 mg Zn kg(-1) and 83 mg Cd kg(-1) DM). The roots preferentially explored metal-contaminated areas. The exploration of polluted soil inclusions by the roots was associated with a higher extraction of metals. Zinc and Cd in the shoots of Thlaspi caerulescens were negatively correlated with Ca and Mg concentrations; however, the soil supply for these two elements was identical. This suggests that there is competition for the uptake of these elements and that Zn is preferentially accumulated.