Min S. Chang

TARGETING EXPRESSION WITH LIGHT USING CAGED DNA

In this report, we describe the inactivation and site-specific light induction of plasmid expression using a photosensitive caging compound. Plasmids coding for luciferase were caged with 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) and transfected into ∼1-cm diameter sites of the skin of rats with particle bombardment. Skin sites transfected with caged plasmids did not express luciferase. However, subsequent exposure of transfected skin sites to 355-nm laser light induced luciferase expression in proportion to the amount of light. Liposome transfection of HeLa cells with DMNPE-caged green fluorescent protein (GFP) plasmids showed similar results. Caging DNA with DMNPE blocks expression at the level of transcription, since in vitro production of mRNA from linearized GFP plasmid was also blocked by caging and subsequently restored by exposure to light. Under the reaction conditions of these experiments, our absorbance data indicate that each DMNPE-caged GFP plasmid contains ∼270 caging groups. In addition to inhibition and subsequent restoration of plasmid bioactivity, the presence and photocleavage of this relatively small number of cage groups also alters electrophoretic mobility of plasmids and optical absorption characteristics. This light-induced expression strategy provides a new means to target the expression of genetic material with spatial and temporal specificity.

Purification and Molecular Cloning of an 8R-Lipoxygenase from the Coral Plexaura homomalla Reveal the Related Primary Structures of R- and S-Lipoxygenases

Lipoxygenases that form S configuration fatty acid hydroperoxides have been purified or cloned from plant and mammalian sources. Our objectives were to characterize one of the lipoxygenases with R stereospecificity, many of which are described in marine and freshwater invertebrates. Characterization of the primary structure of an R-specific enzyme should help provide a new perspective to consider the enzyme-substrate interactions that are the basis of the specificity of all lipoxygenases. We purified an 8R-lipoxygenase of the prostaglandin-containing coral Plexaura homomalla by cation and anion exchange chromatography. This yielded a colorless enzyme preparation, a band of ∼100 kDa on SDS-polyacrylamide gel electrophoresis, and turnover numbers of 4000 min−1 of 8R-lipoxygenase activity in peak chromatographic fractions. The full-length cDNA was cloned by PCR using peptide sequence from the purified protein and by 5′- and 3′-rapid amplification of cDNA ends. The cDNA encodes a polypeptide of 715 amino acids, including over 70 amino acids identified by peptide microsequencing. A peptide presequence of 52 amino acids is cleaved to give the mature protein of 76 kDa; the difference from the estimated size by SDS-PAGE implies a post-translational modification of the P. homomalla enzyme. All of the iron-binding histidines of S-lipoxygenases are conserved in the 8R-lipoxygenase. However, the C-terminal amino acid is a threonine, as opposed to the isoleucine that provides the carboxylate ligand to the iron in all known S-lipoxygenases. These results establish that the 8R-lipoxygenase is related in primary structure to the S-lipoxygenases. A model of the basis of R and S stereospecificity is described.

Investigation of a Second 15S-Lipoxygenase in Humans and its Expression in Epithelial Tissues

The first reports of a lipoxygenase in animal cells, the 12S-lipoxygenase of platelets, were published in 1974–75 (Hamberg and Samuelsson, 1974; Nugteren, 1975). At nearly the same time, Rapoport and colleagues recognized the existence of the 15S-lipoxygenase in reticulocytes (Schewe et al., 1975), and discovery of the leukocyte 5S-lipoxygenase was reported in the following year (Borgeat et al., 1976). Subsequent metabolism studies and molecular analyses indicated the occurrence of the same enzymes in selected other tissues; for example, both the 12S-and 15S-lipoxygenases were expressed also in skin (Nugteren and Kivits, 1987; Hussain et al., 1994; Takahashi et al., 1993). For the past two decades it had appeared that the three enzymes could account for all known lipoxygenase activities in man.

Inhibition of RhoA Signaling with Increased Bves in Trabecular Meshwork Cells

PURPOSE Blood vessel epicardial substance (Bves) is a novel adhesion molecule that regulates tight junction (TJ) formation. TJs also modulate RhoA signaling, which has been implicated in outflow regulation. Given that Bves has been reported in multiple ocular tissues, the authors hypothesize that Bves plays a role in the regulation of RhoA signaling in trabecular meshwork (TM) cells. METHODS Human TM cell lines NTM-5 and NTM-5 transfected to overexpress Bves (NTM-w) were evaluated for TJ formation, and levels of occludin, cingulin, and ZO-1 protein were compared. Assays of TJ function were carried out using diffusion of sodium fluorescein and transcellular electrical resistance (TER). Levels of activated RhoA were measured using FRET probes, and phosphorylation of myosin light chain (MLC-p), a downstream target of RhoA, was assessed by Western blot analysis. RESULTS Overexpression of Bves led to increased TJ formation in NTM-5 cells. Increased TJ formation was confirmed by increased occludin, cingulin, and ZO-1 protein. Functionally, NTM-w cells showed decreased permeability and increased TER compared with NTM-5 cells, consistent with increased TJ formation. NTM-w cells also exhibited decreased levels of active RhoA and lower levels of MLC-p than did NTM-5 cells. These findings support a TJ role in RhoA signaling. CONCLUSIONS Increased Bves in TM cells leads to increased TJ formation with decreased RhoA activation and decreased MLC-p. This is the first report of a regulatory pathway upstream of RhoA in TM cells. In TM tissue, RhoA has been implicated in outflow regulation; thus, Bves may be a key regulatory molecule in aqueous outflow.

Molecular Cloning of a Second Human 15S-Lipoxygenase and its Murine Homologue, an 8S-Lipoxygenase

The three well recognized lipoxygenases in humans are best known for their occurrence in different types of blood cells (1). And these three enzymes appear to have distinct biological roles. The leukocyte 5S-lipoxygenase clearly functions in the initiation of a metabolic pathway; the leukotriene products have distinct cell receptors and mediate proin-flammatory activities. The platelet 12S lipoxygenase synthesizes 12S-HpETE, and following reduction by cellular peroxidase, the hydroperoxide is converted efficiently and almost exclusively to the hydroxy derivative, 12S-HETE. The 12S-HETE may be a signaling molecule involved in cell-cell communication. Overall, however, there is no strong consensus on the biological role of this highly specific 12S-lipoxygenase product.