Transcriptional regulation of the human soluble epoxide hydrolase gene EPHX2.

Soluble epoxide hydrolase (sEH) is a multifunctional protein encoded by the EPHX2 gene. The biological functions and enzyme kinetics of sEH have been extensively investigated, however, little is known about its transcriptional regulation and mechanisms of tissue specific expression. Here, a luciferase gene based reporter assay was used to identify the minimal promoter and cis regulatory elements of EPHX2. The minimal promoter was identified as a GC-rich region between nts -374 and +28 with respect to the putative transcriptional start site. A reporter plasmid carrying this minimal promoter showed higher or similar activities in comparison to a reporter plasmid carrying nts -5,974 to +28 of EPHX2 in 9 human cell lines that were tested. Sp1 binding sites that are involved in augmenting the minimal promoter activity of EPHX2 were identified by nested deletion analysis, site-specific mutation, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay.

Juvenile hormone (JH) esterase: why are you so JH specific?

Juvenile hormone esterases (JHEs) from six insects belonging to three orders (Lepidoptera, Coleoptera, and Diptera) were compared in terms of their deduced amino acid sequence and biochemical properties. The four lepidopteran JHEs showed from 52% to 59% identity to each other and about 30% identity to the coleopteran and dipteran JHEs. The JHE of Manduca sexta was remarkably resistant to the addition of organic co-solvents and detergent; in some cases, it demonstrated significant activation of activity. Trifluoromethylketone (TFK) inhibitors with chain lengths of 8, 10 or 12 carbons were highly effective against both lepidopteran and coleopteran JHEs. The coleopteran JHE remained sensitive to TFK inhibitors with a chain length of 6 carbons, whereas the lepidopteran JHEs were significantly less sensitive. When the chain was altered to a phenethyl moiety, the coleopteran JHE remained moderately sensitive, while the lepidopteran JHEs were much less sensitive. The lepidopteran and coleopteran JHEs did not show dramatic differences in specificity to alpha-naphthyl and rho-nitrophenyl substrates. However, as the chain length of the alpha-naphthyl substrates increased from propionate to caprylate, there was a trend towards reduced activity. The JHE of M. sexta was crystallized and the properties of the crystal suggest a high-resolution structure will follow.

A rapid protocol for construction and production of high-capacity adenoviral vectors.

High-capacity adenoviral vectors (HC-AdVs) lacking all viral coding sequences were shown to result in long-term transgene expression and phenotypic correction in small and large animal models. It has been established that HC-AdVs show significantly reduced toxicity profiles compared with early-generation adenoviral vectors. Furthermore, with capsid-modified HC-AdV becoming available, we are just starting to understand the full potential of this vector system. However, for many researchers, the wide-scale use of HC-AdV is hampered by labor-intensive and complex production procedures. Herein, we provide a feasible and detailed protocol for efficient generation of HC-AdV. We introduce an efficient cloning strategy for the generation of recombinant HC-AdV vector genomes. Infection and amplification of the HC-AdV are performed in a spinner culture system. For purification, we routinely apply cesium chloride gradients. Finally, we describe various methods for establishing vector titers. Generation of high-titer HC-AdV can be achieved in 3 weeks.

Development of a high-throughput screen for soluble epoxide hydrolase inhibition.

Mammalian soluble epoxide hydrolase (sEH) represents a highly promising new target for drug development. Chemical inhibition of this enzyme in animal models was shown to treat hypertension and vascular inflammation as well as related syndromes. Existing sEH inhibitors are relatively potent and specific. However, the low solubility and relatively fast metabolism of described sEH inhibitors make them less than therapeutically efficient, stating the need for novel inhibitor structures. Therefore, a series of alpha-cyanoester and alpha-cyanocarbonate epoxides were evaluated as potential human sEH (HsEH) substrates for the high-throughput screen (HTS) of compound libraries. (3-Phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl)-methyl ester (PHOME), which displayed the highest aqueous stability and solubility, was selected for the development of an HTS assay with long incubation times at room temperature. Concentrations of HsEH and PHOME were optimized to ensure assay sensitivity, reliability, and reproducibility. Assay validation, which employed these optimized concentrations, resulted in good accuracy (60-100%) and high precision (<7% relative standard deviation). In addition, an overall Z' value of 0.7 proved the system's robustness and potential for HTS. The developed assay system will be a valuable tool to discover new structures for the therapeutic inhibition of sEH to treat various cardiovascular diseases.

Fluorescent substrates for soluble epoxide hydrolase and application to inhibition studies.

Inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of disorders resulting from hypertension and vascular inflammation. A spectrophotometric assay (4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate, NEPC) is currently used to screen libraries of chemicals; however this assay lacks the required sensitivity to differentiate the most potent inhibitors. A series of fluorescent alpha-cyanoester and alpha-cyanocarbonate epoxides that produce a strong fluorescent signal on epoxide hydrolysis by both human and murine sEH were designed as potential substrates for an in vitro inhibition assay. The murine enzyme showed a broad range of specificities, whereas the human enzyme showed the highest specificity for cyano(6-methoxy-naphthalen-2-yl)methyl trans-[(3-phenyloxiran-2-yl)methyl] carbonate. An in vitro inhibition assay was developed using this substrate and recombinant enzyme. The utility of the fluorescent assay was confirmed by determining the IC(50) values for a series of known inhibitors. The new IC(50) values were compared with those determined by spectrophotometric NEPC and radioactive tDPPO assays. The fluorescent assay ranked these inhibitors on the basis of IC(50) values, whereas the NEPC assay did not. The ranking of inhibitor potency generally agreed with that determined using the tDPPO assay. These results show that the fluorescence-based assay is a valuable tool in the development of sEH inhibitors by revealing structure-activity relationships that previously were seen only by using the costly and labor-intensive radioactive tDPPO assay.