Characterization of the enzymatic and nonenzymatic reaction of 13-oxooctadecadienoic acid with glutathione

The enzymatic oxygenation of linoleic acid leads to the production of 13-hydroxyoctadecadienoic acid (13-HODE). Subsequent dehydrogenation of 13-HODE by the NAD(+)-dependent 13-HODE dehydrogenase results in the formation of the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). These oxidized derivatives of linoleic acid have been shown to be involved in several cellular regulatory processes. In the present study, we have examined the enzymatic and nonenzymatic reaction of 13-OXO with glutathione (GSH) and N-acetylcysteine (N-AcCySH). Nonenzymatic reaction rates were determined spectrophotometrically and exhibited a pH optimum of 9.0 which is consistent with attack of a thiolate anion. Product formation was evaluated by reverse-phase HPLC which showed formation of one major product upon reaction with either GSH or N-AcCySH. The HPLC-purified products were examined by FAB MS as well as one- and two-dimensional NMR. The products, with either GSH or N-AcCySH, were found to consist of an equal mixture of two diastereomers arising from addition of a thiolate to the 9 position of 13-OXO. Using GSH as the thiol, the reaction was also shown to be catalyzed by rat glutathione transferase 8-8. In the case of the enzymatic reaction there is stereoselective product formation. Furthermore, submicromolar concentrations of the 13-OXO-GSH conjugate were shown to significantly inhibit glutathione transferase activity in HT-29 homogenates. These investigations provide insight into the potential metabolic disposition of linoleate oxygenation products.     

CD94/NKG2 inhibitory receptor complex modulates both anti-viral and anti-tumoral responses of polyclonal phosphoantigen-reactive V gamma 9V delta 2 T lymphocytes

Viral, bacterial, protozoal, and cancer-associated Ags elicit strong responses in human gammadelta T lymphocytes. The majority of these cells in the peripheral blood express the Vgamma9Vdelta2-encoded TCR and recognize nonpeptidic phosphoantigens without an apparent MHC restriction. We have shown that Vgamma9Vdelta2 T cells express the inhibitory CD94/NKG2 receptor for HLA class I molecules. The anti-CD94 mAb inhibits 1) the Vgamma9Vdelta2 T cell proliferation in response mycobacterial phosphoantigens and 2) the HIV-induced Vgamma9Vdelta2 T cell expansion. Vgamma9Vdelta2 T cells stimulated with nonpeptidic mycobacterial antigens produce IFN-gamma and TNF-alpha. Signaling through the CD94/NKG2 receptor interferes with the synthesis of these cytokines. The CD94/HLA class I interaction is also involved in the cytotoxic activity of Vgamma9Vdelta2 T cells. The Vgamma9Vdelta2 T cell regulation through the CD94 receptor may be important for the potentially dual function in innate immunity, i.e., 1) NK-like and 2) TCR ligand-induced cytolytic activities.     

Differences in polymeric proteins among grains in spring wheat spikes

A uniform amount and size distribution of polymeric proteins within grains in a spike might determine the stability of wheat quality. Two cultivars were grown to maturity in solution culture in a climate chamber. Nitrogen (N) in the form of nitrate was added daily and replaced with ¹⁵N before harvest. Plants were harvested during grain development. Protein composition and relationships of labelled N in grains from different spikelets within the spike were determined. Higher percentages of large unextractable polymeric proteins (%‐LUPP) and total unextractable polymeric proteins (%‐TUPP) were found in the lower‐ and uppermost spikelets in the spike compared with the middle ones for cv. WL, but not for cv. Sport. Both cultivars showed variations in the percentage of large unextractable monomeric proteins (%‐LUMP) and total SDS‐extractable protein (Tote) in the spikelets within the spike. The amount of total SDS‐unextractable protein (Totu) did not vary for either of the cultivars. The spikelets within the spike showing high and low %‐LUMP and Tote at maturity showed a similar behaviour shortly after flowering in cv. WL, but not in cv. Sport. The N concentration of SDS and sonicated extracts varied along the spikelets of the spike for both cultivars. The atom‐% excess ¹⁵N decreased in cv. Sport SDS‐extractable and ‐unextractable proteins and cv. WL albumins + globulins, gliadins and glutenins from grains at different spikelet positions along the spike. Copyright © 2005 Society of Chemical Industry     

Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment

A fundamental obstacle in gene therapy for cancer treatment is the specific delivery of an anticancer gene product to a solid tumor. Although several strategies exist to control gene expression once a vector is directly introduced into a tumor, as yet no systemic delivery system exists that specifically targets solid tumors. Nonpathogenic, obligate anaerobic bacteria of the genus Clostridium have been used experimentally as anticancer agents because of their selective growth in the hypoxic regions of solid tumors after systemic application. In this report we further describe a novel approach to cancer gene therapy in which genetically engineered clostridia are used as tumor-specific vectors for the delivery of antitumor genes. We have introduced into a strain of C. beijerinckii the gene for an E. coli nitroreductase known to activate the nontoxic prodrug CB 1954 to a toxic anticancer drug. Nitroreductase produced by these clostridia enhanced the killing of tumor cells in vitro by CB 1954, by a factor of 22. To demonstrate the specificity of this approach for tumor targeting, we intravenously injected the inactive spore form of C. beijerinckii, which upon transition to a reproductive state will express the E. coli nitroreductase gene. Nitroreductase activity was detectable in 10 of 10 tumors during the first 5 days after intravenous injection of inactive clostridial spores, indicating a rapid transition from spore to reproductive state. Tumors harboring clostridial spores which did not possess the E. coli nitroreductase gene were devoid of nitroreductase activity. Most importantly, E. coli nitroreductase protein was not found in a large survey of normal mouse tissues following intravenous injection of nitroreductase containing clostridia, strongly suggesting that obligate anaerobic bacteria such as clostridia can be utilized as highly specific gene delivery vectors for cancer therapy.