Thermal Stability of the Human Immunodeficiency Virus Type 1 (HIV-1) Receptors, CD4 and CXCR4, Reconstituted in Proteoliposomes

BACKGROUND: The entry of human immunodeficiency virus (HIV-1) into host cells involves the interaction of the viral exterior envelope glycoprotein, gp120, and receptors on the target cell. The HIV-1 receptors are CD4 and one of two chemokine receptors, CCR5 or CXCR4. METHODOLOGY/PRINCIPAL FINDINGS: We created proteoliposomes that contain CD4, the primary HIV-1 receptor, and one of the coreceptors, CXCR4. Antibodies against CD4 and CXCR4 specifically bound the proteoliposomes. CXCL12, the natural ligand for CXCR4, and the small-molecule CXCR4 antagonist, AMD3100, bound the proteoliposomes with affinities close to those associated with the binding of these molecules to cells expressing CXCR4 and CD4. The HIV-1 gp120 exterior envelope glycoprotein bound tightly to proteoliposomes expressing only CD4 and, in the presence of soluble CD4, bound weakly to proteoliposomes expressing only CXCR4. The thermal stability of CD4 and CXCR4 inserted into liposomes was examined. Thermal denaturation of CXCR4 followed second-order kinetics, with an activation energy (E(a)) of 269 kJ/mol (64.3 kcal/mol) and an inactivation temperature (T(i)) of 56°C. Thermal inactivation of CD4 exhibited a reaction order of 1.3, an E(a) of 278 kJ/mol (66.5 kcal/mol), and a T(i) of 52.2°C. The second-order denaturation kinetics of CXCR4 is unusual among G protein-coupled receptors, and may result from dimeric interactions between CXCR4 molecules. CONCLUSIONS/SIGNIFICANCE: Our studies with proteoliposomes containing the native HIV-1 receptors allowed an examination of the binding of biologically important ligands and revealed the higher-order denaturation kinetics of these receptors. CD4/CXCR4-proteoliposomes may be useful for the study of virus-target cell interactions and for the identification of inhibitors

Differentially expressed genes during malting and correlation with malting quality phenotypes in barley (Hordeum vulgare

Breeding for malting quality is an important goal of malting barley breeding programs. Malting quality is a complex phenotype that combines a large number of interrelated components, each of which shows complex inheritance. Currently, only a few genes involved in determining malting quality have been characterized. We combined transcript profiling with phenotypic correlations to identify candidate genes for malting quality. The Barley1 GeneChip® array containing 22,792 probe sets was used to conduct transcript profiling of genes expressed in several different stages of malting of four malting cultivars. Genes that were differentially expressed in comparisons between different malting stages relative to ungerminated seed, as well as in comparisons between malting cultivars in the same malting stage were identified. Correlation analysis of 723 differentially expressed genes with malting quality phenotypes showed that 11–102 of these genes correlated with six malting quality phenotypes. Genes involved in carbohydrate metabolism were among the positively correlated genes. Genes for protein and lipid metabolism, cell wall organization and biogenesis, and genes involved in stress and defense response also correlated with malting quality phenotypes. Expressed sequence tags (ESTs) were generated from a ‘malting-gene enriched’ cDNA library made by suppression subtractive hybridization between malted and ungerminated seeds of ‘Morex’. Eleven percent of the ESTs had no significant homology with sequences in the databases, suggesting that there may be other malting-related genes not represented in the barley gene chip array. The results provide candidate genes for malting quality phenotypes that need to be functionally validated