The treatment of rubber tree (Hevea brasiliensis) bark with chloro-2-ethyl phosphonic acid (ethrel), an ethylene-releasing chemical, induced, after a lag period of 13 to 21 hours, a marked increase in the total adenine nucleotides (essentially ATP and ADP) of latex cells. This rise in the latex adenylate pool was concomitant with a marked decrease in the [ATP]/[ADP] ratio without significant changes in the adenylate energy charge. The apparent equilibrium constant for the adenylate kinase, which appeared to behave as a key enzyme in maintaining the adenylate energy charge in the latex, was considerably reduced, probably as a consequence of the alkalinization of the latex cytosol induced by the treatment with ethrel. To reduce the "sink effect" and activation of the metabolism induced in Hevea bark by regular tapping, the latex was collected by micropuncture (few drops) at increasing distance (5-50 centimeters) above and below an ethrel-treated area on the virgin bark of resting trees. The effect of ethrel was shown to spread progressively along the trunk. The increase in the adenylate pool (essentially ATP) was detectable as early as 24 hours after the bark treatment and was maximum after 6 or 8 days, 5 centimeters as well as 50 centimeters above and below the stimulated bark ring. The correlative vacuolar acidification and cytosolic alkalinization, i.e. the increase in the transtonoplast ApH, induced in the latex cells by ethrel were shown to be concomitant with the rise in ATP content of the latex. This suggests that the tonoplast H+-pumping ATPase, which catalyzes vacuolar acidification in the latex, is directly and essentially under the control of the availability of its substrate (i.e. ATP) in the latex. The results are discussed in relation to energydependent activation of metabolism, and increased rubber production, as induced by the stimulation of rubber trees with ethrel.

1. The in vitro metabolism of alpha-dihydroergocryptine (DHEC, Almirid), an ergot-derived dopamine agonist for the treatment of Parkinson's disease, has been studied in cultured cell lines following incubation with DHEC. Human hepatocytes as well as two sets of metabolically competent cell lines expressing one single human cytochrome P450 (1A1, 1A2, 1B1, 2A6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4) were used. 2. Mono- and dihydroxy metabolites of DHEC could only be detected in the culture media of the cell line expressing human cytochrome CYP3A4. The same metabolites were found in the media of cultured human hepatocytes derived from three different donors. After 24-h incubation with 1 microM DHEC, approximately 60% mono- and approximately 20% dihydroxy metabolites were detected, i.e. approximately 80% of DHEC was metabolized. Further, DHEC demonstrated an inhibitory effect on CYP3A4-mediated testosterone metabolism and additionally could induce CYP3A4 and CYP2E1 mRNA when added at 10 microM to cultured human hepatocytes. 3. The data suggest that DHEC metabolism in humans is primarily mediated by the CYP3A4 isoform. The results are in accordance with findings derived from other ergot alkaloids.

Lutoids represent a lysosomal microvacuolar compartment of rubber-tree (Hevea brasiliensis) latex. We observed acidification of isolated vesicles after imposing an outward Mg2" diffusion gradient and dissipation of a preformed pH gradient in the presence of exogenous Mg2". These the conclusion that this process is catalyzed by an electroneutral Mg2+/2H' antiporter. This is the first indication of the existence of such an antiporter in a plant tonoplast. MATERIALS AND METHODS Plant MaterialLatex was obtained from rubber tree (Hevea brasiliensis) of the Institut de Recherches sur le Caoutchouc plantations at Bimbresso, Abidjan, Cote d'Ivoire. Lutoids were isolated from latex by centrifugation (40,000g, 60 min), and washed five times in buffer containing 300 mm mannitol, 50 mm HepesTris at pH 7.5, and then lyophilized (16). Resuspension of lyophilized lutoids with a Potter homogenizer gives tight vesicles with functional (H+)ATPase (16). In this study, resuspension medium contained 5 mm Hepes-BTP2 (impermeant buffer) at the indicated pH, 300 mm mannitol, and MgSO4 or (NH4)2SO4 at the indicated concentrations. Control experiments were performed with liposomes from soybean lipids; 40 mg of soybean lipids were dispersed by vigorous mixing on a Vortex mixer in the presence of glass beads in 1 mL of the same buffer as the one used for lutoid vesicles, for 15 min under argon. Afterward, the liposome suspension was clarified by sonication for 15 min under argon in a Bransonic sonicator bath. Fluorescence MeasurementsFluorescence experiments were carried out with an SLMAminco SFP 500 spectrofluorometer. Acidification of lutoids was monitored at 300C using the permeant fluorescent ACMA probe at 415/485 nm excitation/emission wavelengths. Assay medium (2 mL) contained 5 mm Hepes-BTP (pH 8.5 unless otherwise indicated), 1 mg/mL of BSA (fraction V), 300 mt mannitol, and 1 ,UM ACMA. When the assay medium also contained Mg2" at equilibrium, an H+ gradient across lutoid vesicles (25 ,g/mL of protein) was generated by the addition of a saturating EDTA concentration. A proton gradient could also be generated by diluting 200-fold lutoid vesicles preloaded with Mg2" (as indicated) or 25 mm 2Abbreviations: BTP, bis-tris-propane; ACMA, 9-amino-6-chloro-2-methoxyacridine; VH+, initial rate of quenching; FO, initial fluorescence; AF, increase in the fluorescence. 255www.plantphysiol.org on May 10, 2018 -Published by Downloaded from

The Mg2+/2H+ antiporter recently described on lutoid membrane (Z. Amalou, R. Gibrat, C. Brugidou, P. Trouslot, J. d'Auzac [1992] Plant Physiol 100 255-260) was solubilized by odylglucoside and reconstituted into soybean liposomes using the detergent dilution method. Magnesium efflux or influx experiments were used to generate a H+ influx or efflux, respectively, monitored with the fluorescent probe 9-amino-6-chloro-2-methoxyacridine. Both experiments gave saturable H+ fluxes as a function of internal or external Mg2+ concentrations with similar kinetic parameters K, and Vmax. The K, value for Mgz+ (about 2 mM) was identical to that previously found in lyophilized-resuspended lutoid (reference therein), whereas the V,, value was 14-fold higher. Since only 10% of the initial proteins were recovered in proteoliposomes, and electrophoretic patterns of the two kinds of vesicles differed significantly, it was inferred that the increase in Vmax was due essentially to an enrichment of the protein antiporter in the reconstituted fraction, owing to a selective effect of octylglucoside at both solubilization and reconstitution steps. None of the various divalent cations used could dissipate the pH gradient of control liposomes of soybean lipids, unless the divalent/H+ exchanger A23187 was added, whereas a rapid dissipation of the pH gradient was observed with reconstituted proteoliposomes from lutoid proteins, with the cation selectivity sequence ZnZ+ > CdZ+ > Mg2+ in the millimolar concentration range. The divalent ions CaZ+, BaZ+, and MnZ+ were incapable of generating a H+ efflux in reconstituted proteoliposomes, whereas both MgZ+/H+ and CaZ+/H+ exchanges were observed in lyophilized-resuspended lutoids. Therefore, the lutoid membrane seems to contain separate h4g2+/H+ and CaZ+/H+ transport systems, the latter being eliminated during the solubilization/ reconstitution of lutoid membrane proteins.

There is an acute need for biomarkers at every phases of drug development from selecting preclinical models in pharmacogenomic studies to enrollment of patients in clinical trials. However, their identification remains extremely challenging due to the limited availability of clinical samples. In contrast, standard tumor models such as cell lines are available but are genetically relatively far from patient tumors. Use patient-derived xenografts (PDX) for anticancer agent-testing is of increasing interest due to their closer similarity to patient tumors compared to cell lines. Over the last 30 years, we have established a collection of 400 PDX covering more than 30 different cancer types. PDX models have been extensively characterized using the microarray or next-generation sequencing technologies for gene expression, copy number variations and whole-exome mutations. Biomarker research is now possible using these data in combination with drug response data from in vivo or in vitro 2D or 3D assays routinely performed on-site with large panels of 100-200 PDX. We present here a fully integrated bioinformatics pipeline dedicated to biomarker discovery in which the complete molecular profiles of our PDX have been systematically tested for association with drug sensitivity. To identify the biomarkers associated with drug response, several statistical tests have been performed. Drug response data were treated either as continuous variables using the Spearman or Wilcoxon tests, or as categorical variables (with two groups of responders and non-responders) using the LIMMA, t-test or Fisher exact test. Given that high throughput data frequently leads to large biomarker lists, we used specific filters to narrow down the list of candidates by defining thresholds based on corrected p-values, by intersecting results from different tests, or by integrating the tumor type into the statistical tests. Since sensitivity to anticancer agents is often multi-factorial, we also used integrative approaches that combined gene mutations, copy number loss and lack of gene expression for association with drug response. Finally, significant biomarkers were visualized using clustering heatmaps and enrichment GO/pathway approaches to get more insight in their biological function. Using a selection of several datasets of PDX drug responses to chemotherapeutics and targeted therapies (targeting RTK/RAS/RAF and PI3K/MTOR pathways and using specific compounds such as Vemurafenib, Erlotinib or Cetuximab), we demonstrate the efficacy of our approach to retrieve biomarkers of known clinical utility. Using these datasets we also could address the questions of model panel sizes, molecular data type and tumor subtype representation, and show how more accurate biomarkers can be validated using an independent dataset of samples. The development of strategies for testing anticancer agents using PDX in mouse clinical trials, or high throughput in vitro 2D, 3D screening approaches coupled to a more systematic biomarker research should significantly contribute to early biomarker identification and facilitate drug development. Citation Format: Bruno Zeitouni, Anne-Lise Peille, Zakia Amalou, Thomas Metz, Heinz-Herbert Fiebig, Vincent Vuaroqueaux. A systematic patient-derived xenograft based solution for pre-clinical biomarker discovery. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A20.