Hepatic microsomal epoxide hydrase. Chemical evidence for a single polypeptide chain.

Highly purified hepatic microsomal epoxide hydrase, which had been purified in the presence of proteolytic enzyme inhibitors, was subjected to carboxypeptidase Y digestion, automated Edman degradation, and carbohydrate analysis. Carboxypeptidase Y digestion resulted in the near stoichiometric release of leucine, the COOH-terminal amino acid. Automated Edman degradation permitted the identification of the first 20 amino acid residues of epoxide hydrase. Methionine was identified as the NH2-terminal residue. The NH2-terminal region of epoxide hydrase is similar in hydrophobicity to the NH2-terminal precursor segments of several secretory proteins and the NH2-terminal regions of several microsomal cytochromes P-450. Carbohydrate analyses of the enzyme revealed the presence of 0.5 to 1.0 mol of mannose/50,000 g of protein. These results provide evidence for the presence of a single polypeptide chain in our purified enzyme preparations and suggest that there may be only one enzymic form of epoxide hydrase in microsomes from phenobarbital-treated rats.     

Meta-Modeling of Methylprednisolone Effects on Glucose Regulation in Rats

A retrospective meta-modeling analysis was performed to integrate previously reported data of glucocorticoid (GC) effects on glucose regulation following a single intramuscular dose (50 mg/kg), single intravenous doses (10, 50 mg/kg), and intravenous infusions (0.1, 0.2, 0.3 and 0.4 mg/kg/h) of methylprednisolone (MPL) in normal and adrenalectomized (ADX) male Wistar rats. A mechanistic pharmacodynamic (PD) model was developed based on the receptor/gene/protein-mediated GC effects on glucose regulation. Three major target organs (liver, white adipose tissue and skeletal muscle) together with some selected intermediate controlling factors were designated as important regulators involved in the pathogenesis of GC-induced glucose dysregulation. Assessed were dynamic changes of food intake and systemic factors (plasma glucose, insulin, free fatty acids (FFA) and leptin) and tissue-specific biomarkers (cAMP, phosphoenolpyruvate carboxykinase (PEPCK) mRNA and enzyme activity, leptin mRNA, interleukin 6 receptor type 1 (IL6R1) mRNA and Insulin receptor substrate-1 (IRS-1) mRNA) after acute and chronic dosing with MPL along with the GC receptor (GR) dynamics in each target organ. Upon binding to GR in liver, MPL dosing caused increased glucose production by stimulating hepatic cAMP and PEPCK activity. In adipose tissue, the rise in leptin mRNA and plasma leptin caused reduction of food intake, the exogenous source of glucose input. Down-regulation of IRS-1 mRNA expression in skeletal muscle inhibited the stimulatory effect of insulin on glucose utilization further contributing to hyperglycemia. The nuclear drug-receptor complex served as the driving force for stimulation or inhibition of downstream target gene expression within different tissues. Incorporating information such as receptor dynamics, as well as the gene and protein induction, allowed us to describe the receptor-mediated effects of MPL on glucose regulation in each important tissue. This advanced mechanistic model provides unique insights into the contributions of major tissues and quantitative hypotheses for the multi-factor control of a complex metabolic system.     

Global gene expression analysis to identify molecular markers of uterine receptivity and embryo implantation

Infertility and spontaneous pregnancy losses are an enduring problem to women's health. The establishment of pregnancy depends on successful implantation, where a complex series of interactions occurs between the heterogeneous cell types of the uterus and blastocyst. Although a number of genes are implicated in embryo-uterine interactions during implantation, genetic evidence suggests that only a small number of them are critical to this process. To obtain a global view and identify novel pathways of implantation, we used a dual screening strategy to analyze the expression of nearly 10,000 mouse genes by microarray analysis. Comparison of implantation and interimplantation sites by a conservative statistical approach revealed 36 up-regulated genes and 27 down-regulated genes at the implantation site. We also compared the uterine gene expression profile of progesterone-treated, delayed implanting mice to that of mice in which delayed implantation was terminated by estrogen. The results show up-regulation of 128 genes and down-regulation of 101 genes after termination of the delayed implantation. A combined analysis of these experiments showed specific up-regulation of 27 genes both at the implantation site and during uterine activation, representing a broad diversity of molecular functions. In contrast, the majority of genes that were decreased in the combined analysis were related to host immunity or the immune response, suggesting the importance of these genes in regulating the uterine environment for the implanting blastocyst. Collectively, we identified genes with recognized roles in implantation, genes with potential roles in this process, and genes whose functions have yet to be defined in this event. The identification of unique genetic markers for the onset of implantation signifies that genome-wide analysis coupled with functional assays is a promising approach to resolve the molecular pathways required for successful implantation.     

Target genes of peroxisome proliferator-activated receptor gamma in colorectal cancer cells

Activation of the nuclear hormone peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits cell growth and promotes differentiation in a broad spectrum of epithelial derived tumor cell lines. Here we utilized microarray technology to identify PPARgamma gene targets in intestinal epithelial cells. For each gene, the induction or repression was seen with two structurally distinct PPARgamma agonists, and the change in expression could be blocked by co-treatment with a specific PPARgamma antagonist. A majority of the genes could be regulated independently by a retinoid X receptor specific agonist. Genes implicated in lipid transport or storage (adipophilin and liver fatty acid-binding protein) were also activated by agonists of PPAR subtypes alpha and/or delta. In contrast, PPARgamma-selective targets included genes linked to growth regulatory pathways (regenerating gene IA), colon epithelial cell maturation (GOB-4 and keratin 20), and immune modulation (neutrophil-gelatinase-associated lipocalin). Additionally, three different genes of the carcinoembryonic antigen family were induced by PPARgamma. Cultured cells treated with PPARgamma ligands demonstrated an increase in Ca(2+)-independent, carcinoembryonic antigen-dependent homotypic aggregation, suggesting a potential role for PPARgamma in regulating intercellular adhesion. Collectively, these results will help define the mechanisms by which PPARgamma regulates intestinal epithelial cell biology.