Spatial and temporal dynamics of jasmonate synthesis and accumulation in Arabidopsis in response to wounding.

A new metabolite profiling approach combined with an ultrarapid sample preparation procedure was used to study the temporal and spatial dynamics of the wound-induced accumulation of jasmonic acid (JA) and its oxygenated derivatives in Arabidopsis thaliana. In addition to well known jasmonates, including hydroxyjasmonates (HOJAs), jasmonoyl-isoleucine (JA-Ile), and its 12-hydroxy derivative (12-HOJA-Ile), a new wound-induced dicarboxyjasmonate, 12-carboxyjasmonoyl-l-isoleucine (12-HOOCJA-Ile) was discovered. HOJAs and 12-HOOCJA-Ile were enriched in the midveins of wounded leaves, strongly differentiating them from the other jasmonate metabolites studied. The polarity of these oxylipins at physiological pH correlated with their appearance in midveins. When the time points of accumulation of different jasmonates were determined, JA levels were found to increase within 2-5 min of wounding. Remarkably, these changes occurred throughout the plant and were not restricted to wounded leaves. The speed of the stimulus leading to JA accumulation in leaves distal to a wound is at least 3 cm/min. The data give new insights into the spatial and temporal accumulation of jasmonates and have implications in the understanding of long-distance wound signaling in plants.

Synthesis of chemically functionalized superparamagnetic nanoparticles as delivery vectors for chemotherapeutic drugs.

Superparamagnetic iron oxide nanoparticles (SPIONs) are in clinical use for disease detection by MRI. A major advancement would be to link therapeutic drugs to SPIONs in order to achieve targeted drug delivery combined with detection. In the present work, we studied the possibility of developing a versatile synthesis protocol to hierarchically construct drug-functionalized-SPIONs as potential anti-cancer agents. Our model biocompatible SPIONs consisted of an iron oxide core (9-10 nm diameter) coated with polyvinylalcohols (PVA/aminoPVA), which can be internalized by cancer cells, depending on the positive charges at their surface. To develop drug-functionalized-aminoPVA-SPIONs as vectors for drug delivery, we first designed and synthesized bifunctional linkers of varied length and chemical composition to which the anti-cancer drugs 5-fluorouridine or doxorubicin were attached as biologically labile esters or peptides, respectively. These functionalized linkers were in turn coupled to aminoPVA by amide linkages before preparing the drug-functionalized-SPIONs that were characterized and evaluated as anti-cancer agents using human melanoma cells in culture. The 5-fluorouridine-SPIONs with an optimized ester linker were taken up by cells and proved to be efficient anti-tumor agents. While the doxorubicin-SPIONs linked with a Gly-Phe-Leu-Gly tetrapeptide were cleaved by lysosomal enzymes, they exhibited poor uptake by human melanoma cells in culture.

Loss of insulin synthesis and beta cell survival induced by oxidized LDL require activation of reticulum endoplasmic stress: a mechanism countered by HDL

Elevated circulating concentrations in modified LDL-cholesterol particles (e.g. oxidised LDL) and low levels in HDL increase not only the risk for diabetic patients to develop cardiovascular diseases but also may contribute to development and progression of diabetes by directly having adverse effects on β-cells. Chronic exposure of β-cells to 2 mM human oxidised LDL-cholesterol (oxLDL) increases the rate of apoptosis, reduce insulin biosynthesis and the secretory capacity of the cells in response to nutrients. In line with the protective role, HDL efficiently antagonised the harmful effects of ox- LDL, suggesting that low levels of HDL would be inefficient to protect β-cells against oxLDL attack in patients. Activation of endoplasmic reticulum (ER) stress is pointed out to contribute to β-cell dysfunction elicited by environmental stressors. In this study we investigated whether activation of ER stress is required for oxLDL to mediate detrimental effects on β-cells and we tested the potential antagonist properties of HDL: The mouse MIN6 insulin-secreting cells were cultured with 2 mM of LDL-cholesterol preparation (native or in vitro oxidized) in the presence or absence of 1 mM of HDL-cholesterol or the ER stress inhibitor 4-phenylbutyrate (4-PBA): Prolonged exposure of MIN6 cells to 2 mM oxLDL-cholesterol for 48 hours led to an increase in expression of ER stress markers such as ATF4, CHOP and p58 and stimulated the splicing of XBP-1 whereas, induction of these markers was not observable in the cells cultured with native LDL. Treatment of the cells with the 4-PBA chemical chaperone molecule efficiently blocked activation of the ER stress markers induced by oxLDL. The latter mediates β-cell dysfunction and apoptosis by diminishing the expression of islet brain 1 (IB1) and Bcl2. The levels of these two proteins were preserved in the cells that were co-treated with oxLDL and the 4-PBA. Consistent with this result we found that blockade of ER stress activation alleviated the loss of insulin synthesis and abolished apoptosis evoked by oxLDL. However incubation of the cells with 4-PBA did not prevent impairment of insulin secretion elicited by oxLDL, indicating that ER stress is not responsible for the oxLDL-mediated defect of insulin secretion. Co-incubation of the cells with HDL mimicked the effects of 4-PBA on the expression of IB1 and Blc2 and thereby counteracted oxLDL attacks on insulin synthesis and cell survivals. We found that HDL efficiently inhibited activation of the ER stress mediated by oxLDL: These data highlight the contribution of the ER stress in the defects of insulin synthesis and cell survivals induced by oxLDL and emphasize the potent role of HDL to counter activation of the oxLDL-mediated ER-stress activation:

Involvement of a transforming-growth-factor-beta-like molecule in tumor-cell-derived inhibition of nitric-oxide synthesis in cerebral endothelial cells.

Nitric oxide (NO) has been shown to exert cytotoxic effects on tumor cells. We have reported that EC219 cells, a rat-brain-microvessel-derived endothelial cell line, produced NO through cytokine-inducible NO synthase (iNOS), the induction of which was significantly decreased by (a) soluble factor(s) secreted by DHD/PROb, an invasive sub-clone of a rat colon-carcinoma cell line. In this study, the DHD/PROb cell-derived NO-inhibitory factor was characterized. Northern-blot analysis demonstrated that the induction of iNOS mRNA in cytokine-activated EC219 cells was decreased by PROb-cell-conditioned medium. When DHD/PROb cell supernatant was fractionated by affinity chromatography using Con A-Sepharose or heparin-Sepharose, the NO-inhibitory activity was found only in Con A-unbound or heparin-unbound fractions, respectively, indicating that the PROb-derived inhibitory factor was likely to be a non-glycosylated and non-heparin-binding molecule. Pre-incubation of DHD/PROb-cell supernatant with anti-TGF-beta neutralizing antibody completely blocked the DHD/PROb-derived inhibition of NO production by EC219 cells. Addition of exogenous TGF-beta 1 dose-dependently inhibited NO release by EC219 cells. The presence of active TGF-beta in the DHD/PROb cell supernatant was demonstrated using a growth-inhibition assay. Moreover, heat treatment of medium conditioned by the less invasive DHD/REGb cells, which constitutively secreted very low levels of active TGF-beta, increased both TGF-beta activity and the ability to inhibit NO production in EC219 cells. Thus, DHD/PROb colon-carcinoma cells inhibited NO production in EC219 cells by secreting a factor identical or very similar to TGF-beta.