Interaction effect of PTEN and CDKN1B chromosomal regions on prostate cancer linkage

The tumor suppressor functions of PTEN and CDKN1B have been extensively characterized. Recent data from mouse models suggest that, for some organs, the combined action of both PTEN and CDKN1B has a stronger tumor suppressor function than each alone; for the prostate, heterozygous knockout of both genes leads to 100% penetrance for prostate cancer. To assess whether such an interaction contributes to an increased risk of prostate cancer in humans, we performed a series of epistatic PTEN and CDKN1B interaction analyses in a collection of 188 high-risk hereditary prostate cancer families. Two different analytical approaches were performed; a nonparametric linkage (NPL) regression analysis that simultaneously models allele sharing at these two regions in all families, and an ordered subset analysis (OSA) that assesses linkage evidence at a target region in a subset of families based on the magnitude of allele sharing at the reference region. The strongest evidence of interaction effect was observed at 10q23-24 and 12p11-13 from both the NPL regression analysis (P=0.0002) in all families and the OSA analyses in subsets of families. A LOD-delta of 3.15 (P=0.01) was observed at 10q23-24 among 54 families with the highest NPL scores at 12p11-13, and a LOD-delta of 2.63 (P=0.02) was observed at 12p11-13 among 34 families with the highest NPL scores at 10q23-24. The evidence for the interaction was stronger when using additional fine-mapping markers in the PTEN (10q23) and CDKN1B (12p13) regions. Our data are consistent with epistatic interactions between the PTEN and CDKN1B genes affecting risk for prostate cancer and demonstrate the utility of modeling epistatic effects in linkage analysis to detect susceptibility genes of complex diseases.Jianfeng Xu and Carl D. Langefeld contributed equally to this work     

Dynamic coupling between the SH2 domain and active site of the COOH terminal Src kinase, Csk

The SH2 domain is required for high catalytic activity in the COOH-terminal Src kinase (Csk). Previous solution studies suggest that a short peptide sequence, the SH2-kinase linker, provides a functional connection between the active site and the distal SH2 domain that could underlie this catalytic phenomenon. Substitutions in Phe183 (tyrosine, alanine, and glycine), a critical hydrophobic residue in the linker, result in large decreases in substrate turnover and large increases in the K(m) for ATP. Indeed, F183G possesses kinetic parameters that are similar to that for a truncated form of Csk lacking the SH2 domain, suggesting that a single mutation disrupts communication between this domain and the active site. Based on equilibrium and stopped-flow fluorescence experiments, the elevated K(m) values for the mutants are due to changes in the rates of phosphoryl transfer and not to reduced ATP-binding affinities. Based on hydrogen-deuterium exchange experiments, glycine substitution reduces flexibility in several polypeptide regions in Csk, tyrosine substitution increases flexibility, and alanine substitution leads to mixed effects compared to wild-type. Normal mode analysis indicates that Phe183 and its environment are under strain, a theoretical finding that supports the results of mutations. Overall, the data indicate that domain-domain interactions, controlled through the SH2-kinase linker, provide a dynamic balance within the Csk framework that is ideal for efficient phosphoryl transfer in the active site.     

Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates

The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid concentration decreased by more than half. These changes in the wine and distillate composition had a pronounced effect on the solvent or chemical aroma (associated with ethyl acetate and iso-amyl acetate) and the herbaceous and heads-associated aromas of the final distillate and the solvent or chemical and fruity or flowery characters of the Chenin blanc wines. This study establishes the concept that the overexpression of acetyltransferase genes such as ATF1 could profoundly affect the flavor profiles of wines and distillates deficient in aroma, thereby paving the way for the production of products maintaining a fruitier character for longer periods after bottling.     

Design of a control system for biotransformation of toxic substrates: toluene hydroxylation by Pseudomonas putida UV4

Using the hydroxylation of toluene to toluene cis-glycol by Pseudomonas putida UV4 as an example, the design of a feed-back control system for the addition of a toxic, poorly water-soluble substrate to a fed-batch biotransformation is described. In kinetic studies the reaction followed Michaelis-Menten behavior until toxic toluene concentrations were reached (2.4 mM), above which irreversible denaturation of the biocatalyst was observed. An algorithm, based on a system mass balance, was used to maintain the aqueous phase toluene concentration in the desired range for zero order kinetics. The mass balance required accurate and rapid analysis of the product and reactant in both the liquid and the vapor phase. Various analytical methods were considered and the effect of the sampling and analysis time on the response of the control system was examined.     

CD44v10 interaction with Rho-kinase (ROK) activates inositol 1,4,5-triphosphate (IP3) receptor-mediated Ca2+ signaling during hyaluronan (HA)-induced endothelial cell migration

Aortic endothelial cells (GM7372A) express a major cell adhesion molecule, CD44v10, which binds the extracellular matrix component, hyaluronan (HA), at its external domain and interacts with various signaling molecules at its cytoplasmic domain. In this study, we have determined that CD44v10 and Rho-Kinase (ROK) are physically associated as a complex in vivo. Using a recombinant fragment of ROK (in particular, the pleckstrin homology [PH] domain) and in vitro binding assays, we have detected a specific binding interaction between the PH domain of ROK and the cytoplasmic domain of CD44. Scatchard plot analysis indicates that there is a single high-affinity CD44 binding site in the PH domain of ROK with an apparent dissociation constant (Kd) of 1.76 nM, which is comparable to CD44 binding (Kd approximately 1.56 nM) to intact ROK. These findings suggest that the PH domain is the primary ROK binding region for CD44. Furthermore, HA binding to GM7372A cells promotes RhoA-mediated ROK activity, which, in turn, increases phosphorylation of three different inositol 1, 4, 5-trisphosphate receptors (IP(3)Rs) [in particular, subtype 1 (IP(3)R1), and to a lesser extent subtype 2 (IP(3)R2) and subtype 3 (IP(3)R3)] all known as IP(3)-gated Ca(2+) channels. The phosphorylated IP(3)R1 (but not IP(3)R2 or IP(3)R3) is enhanced in its binding to IP(3) which subsequently stimulates IP(3)-mediated Ca(2+) flux. Transfection of the endothelial cells with ROK's PH cDNA significantly reduces ROK association with CD44v10, and effectively inhibits ROK-mediated phosphorylation of IP(3)Rs and IP(3)R-mediated Ca(2+) flux in vitro. The PH domain of ROK also functions as a dominant-negative mutant in vivo to block HA-dependent, CD44v10-specific intracellular Ca(2+) mobilization and endothelial cell migration. Taken together, we believe that CD44v10 interaction with ROK plays a pivotal role in IP(3)R-mediated Ca(2+) signaling during HA-mediated endothelial cell migration.