Role of marination, natural antimicrobial compounds, and packaging on microbiota during storage of chicken tawook.

The aim of this study was to investigate the antimicrobial effect of marination, natural antimicrobials, and packaging on the microbial population of chicken tawook during storage at 4°C. Chicken meat was cut into 10 g cubes and marinated. The chicken was then mixed individually with 0.5% or 1% (w/v) vanillin (VA), ß-resorcylic acid (BR), or eugenol (EU), and stored under aerobic (AP) or vacuum (VP) packing at 4°C for 7 d. The marinade decreased microbial growth as monitored by total plate count, yeast and mold, lactic acid bacteria, and Pseudomonas spp. by about 1 log cfu/g under AP. The combination of marinade and antimicrobials under AP and VP decreased growth of spoilage-causing microorganisms by 1.5 to 4.8 and 2.3 to 4.6 log cfu/g, respectively. Change in pH in VP meat was less than 0.5 in all treated samples including the control. Marination decreased the lightness of the meat (L*) and significantly (p < 0.05) increased the redness (A*) and yellowness (B*). Overall acceptability was highest for marinated samples with 0.5% BR.

A high-throughput microfluidic assay for SH2 domain-containing inositol 5-phosphatase 2.

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) is a potential drug target for the treatment of type 2 diabetes. This enzyme serves as a negative regulator of insulin-mediated signal transduction by catalyzing the dephosphorylation of the second messenger lipid molecule phosphatidylinositol 3,4,5-triphosphate. Traditionally, assays for phosphoinositide phosphatases such as SHIP2 have relied on radiolabeled phosphatidylinositol-containing lipid membranes and chromatographic separation of labeled phospholipid substrate from product by thin-layer chromatography. We have expressed and purified catalytically active phosphatase domain constructs of SHIP2 from Escherichia coli and developed a sensitive and antibody- or binding protein-independent assay for SHIP2 amenable to high-throughput screening of phosphoinositide phosphatases or phosphoinositide kinases. This microfluidic assay, with Z' values approximately 0.8, is based upon the difference in mobility within an electric field between a fluorophore-labeled phosphatidylinositol 3,4,5-triphosphate substrate and the corresponding 3,4-bisphosphate product. High-throughput screening of a 91,060-member compound library in 384-well format resulted in the identification of SHIP2 inhibitors.

Kinetic mechanism of AKT/PKB enzyme family.

AKT/PKB is a phosphoinositide-dependent serine/threonine protein kinase that plays a critical role in the signal transduction of receptors. It also serves as an oncogene in the tumorigenesis of cancer cells when aberrantly activated by genetic lesions of the PTEN tumor suppressor, phosphatidylinositol 3-kinase, and receptor tyrosine kinase overexpression. Here we have characterized and compared kinetic mechanisms of the three AKT isoforms. Initial velocity studies revealed that all AKT isozymes follow the sequential kinetic mechanism by which an enzyme-substrate ternary complex forms before the product release. The empirically derived kinetic parameters are apparently different among the isoforms. AKT2 showed the highest Km value for ATP, and AKT3 showed the highest kcat value. The patterns of product inhibition of AKT1, AKT2, and AKT3 by ADP were all consistent with an ordered substrate addition mechanism with ATP binding to the enzymes prior to the peptide substrate. Further analysis of steady state kinetics of AKT1 in the presence of dead-end inhibitors supported the finding and suggested that the AKT family of kinases catalyzes reactions via an Ordered Bi Bi sequential mechanism with ATP binding to the enzyme prior to peptide substrate and ADP being released after the phosphopeptide product. These results suggest that ATP is an initiating factor for the catalysis of AKT enzymes and may play a role in the regulation AKT enzyme activity in cells.