Maximum efficiency phothochemistry and potential photosynthetic index in melon plants (Cucumis melo) treated with low temperatures

La fluorescencia de la clorofila se utiliza para determinar la eficiencia fotoquímica de las plantas ante diferentes condiciones ambientales. Existen índices como Fv/Fm y PI abs que son indicadores indirectos del rendimiento cuántico del fotosistema II (PSII). El objetivo de este trabajo fue determinar la eficiencia fotoquímica máxima del PSII y el potencial fotosintético en plantas sometidas a bajas temperaturas. La experiencia se llevó a cabo en la EEA, Santiago del Estero y en la Facultad de Agronomía y Agroindustria, UNSE. Los tratamientos consistieron en plantas de melón (cv. Sweet Ball) sin estímulo de frío (testigo) y plantas con estímulo de frío durante la noche con rangos térmicos de 0ºC a 10ºC y de -3ºC a 0ºC. Se evaluó la eficiencia fotoquímica máxima (Fv/Fm), índice de potencial fotosintético (PI abs) y concentración de malondialdehido (MDA) en hoja. En las plantas estimuladas con frío se obtuvo menor Fv/Fm, PI abs e incrementos en la concentración de MDA.Measurements of the chlorophyll fluorescence is used to examine the photochemical efficiency of plants a wide range of environmental conditions. The quantum yield of non-cyclic electron transport is directly proportional to the efficiency of excitation of the reaction centers of Photosystem II (PS II) and can be determined by indexes such as Fv / Fm and PI abs. The aim of this study was to determine the maximum photochemical efficiency of PSII and photosynthetic potential in plants treated with low temperatures. The experiment was conducted at the Estación Experimental Agropecuaria (EEA) INTA y la Facultad de Agronomía y Agroindustria de la Universidad Nacional de Santiago del Estero (UNSE). Treatments consisted of melon plants (cv. Sweet Ball) without (control) and with low night temperatures between 10°C and 0ºC to -3ºC to 0°C. Photochemical efficiency (Fv / Fm), photosynthetic potential index (PI abs) and in turn MDA concentration increased.Fil: Rodriguez Torresi, A. O.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Tucumán-Santiago del Estero. Estación Experimental Agropecuaria Santiago del Estero; ArgentinaFil: Yonny, Melisa Evangelina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; ArgentinaFil: Nazareno, M.. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; ArgentinaFil: Galmarini, Claudio Romulo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Mendoza-San Juan. Estación Experimental Agropecuaria La Consulta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; ArgentinaFil: Bouzo, Carlos Alberto. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentin

XPF-Dependent DNA Breaks and RNA Polymerase II Arrest Induced by Antitumor DNA Interstrand Crosslinking-Mimetic Alkaloids

SummaryTrabectedin and Zalypsis are two potent anticancer tetrahydroisoquinoline alkaloids that can form a covalent bond with the amino group of a guanine in selected triplets of DNA duplexes and eventually give rise to double-strand breaks. Using well-defined in vitro and in vivo assays, we show that the resulting DNA adducts stimulate, in a concentration-dependent manner, cleavage by the XPF/ERCC1 nuclease on the strand opposite to that bonded by the drug. They also inhibit RNA synthesis by: (1) preventing binding of transcription factors like Sp1 to DNA, and (2) arresting elongating RNA polymerase II at the same nucleotide position regardless of the strand they are located on. Structural models provide a rationale for these findings and highlight the similarity between this type of DNA modification and an interstrand crosslink

Effect of foliar application of boron, zinc and calcium on onion seed production = Efecto de la aplicación foliar de boro, zinc y calcio en la producción de semilla de cebolla

La semilla de cebolla representa un alto porcentaje del valor económico total de semillas de hortalizas comercializado en el mundo. En Argentina, la producción de semilla de cebolla se realiza en San Juan y Mendoza y se utilizan cultivares de polinización abierta (OP) e híbridos de primera generación. Una variedad OP tiene buenos rendimientos de semilla, pero el rendimiento de semillas híbridas es errático y considerablemente más bajo. El rendimiento y la calidad de semilla dependen, entre otros factores, de la fertilización. Nutrientes tales como boro, zinc y calcio son esenciales para el crecimiento y el desarrollo de las plantas. La fertilización foliar permite suministrar nutrientes sobre órganos específicos cuando la etapa de crecimiento, la demanda interna y las condiciones ambientales limitan su entrega. El objetivo de este estudio fue evaluar el efecto de la aplicación foliar de boro, zinc y calcio, sobre el rendimiento y la calidad de semilla de cebolla. El estudio se realizó sobre la cv. Angaco INTA en una finca comercial ubicada en el departamento de Pocito, San Juan. Se utilizó un diseño completamente aleatorizado con cuatro repeticiones. Se evaluaron tres dosis de boro, de calcio y de zinc, más una combinación de los mismos y el testigo. Se concluye que la aplicación foliar de boro, tanto simple como combinado con zinc y calcio, produce un mayor cuajado de frutos, mientras que el calcio mejora el poder germinativo. No se observaron efectos significativos en el rendimiento de semillas.Onion seed represents a high percentage of the total economic value of vegetable seeds marketed worldwide. In Argentina, onion seed production is carried out in San Juan and Mendoza, where open-pollination (OP) cultivars and first generation hybrids are used. An OP variety has good seed yield, but the yield of hybrid seeds is erratic and considerably lower. Seed yield and quality depends, among other factors, on fertilization. Nutrients such as boron, zinc and calcium are essential for the growth and development of plants. Foliar fertilization allows the supply of nutrients to specific organs when the growth stage, internal demand and environmental conditions limit their delivery. The objective of this study was to evaluate the effect of the foliar application of boron, zinc and calcium, on the yield and quality of onion seed. The study was conducted on cv. Angaco INTA in a commercial farm located in the department of Pocito, San Juan. A completely randomized design with four replications was used. Three doses of boron, calcium and zinc, plus a combination of the three nutrients and the control were evaluated. The foliar application of boron, both simple and combined with zinc and calcium, produces a greater fruit set, while calcium improves germination. No significant effects on seed yield were observed.EEA San JuanFil: Gabri Martín, Carlos Guillermo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria San Juan; Argentina.Fil: Gabri Martín, Carlos Guillermo. Universidad Nacional de San Juan. Departamento de Agronomía. Facultad de Ingeniería; Argentina.Fil: Gabri Martín, Carlos Guillermo. Universidad Nacional de Cuyo. Facultad de Ciencia Agrarias; ArgentinaFil: Ibañez, Antonio Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria San Juan; Argentina.Fil: Güell, J. M. Universidad Nacional de San Juan. Departamento de Agronomía. Facultad de Ingeniería; Argentina.Fil: Galmarini, Claudio Rómulo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria La Consulta; Argentina.Fil: Galmarini, Claudio Rómulo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

c-Jun N-terminal kinase phosphorylation is a biomarker of plitidepsin activity

Plitidepsin is an antitumor drug of marine origin currently in Phase III clinical trials in multiple myeloma. In cultured cells, plitidepsin induces cell cycle arrest or an acute apoptotic process in which sustained activation of c-Jun N-terminal kinase (JNK) plays a crucial role. With a view to optimizing clinical use of plitidepsin, we have therefore evaluated the possibility of using JNK activation as an in vivo biomarker of response. In this study, we show that administration of a single plitidepsin dose to mice xenografted with human cancer cells does indeed lead to increased phosphorylation of JNK in tumors at 4 to 12 h. By contrast, no changes were found in other in vitro plitidepsin targets such as the levels of phosphorylated-ERK, -p38MAPK or the protein p27KIP1. Interestingly, plitidepsin also increased JNK phosphorylation in spleens from xenografted mice showing similar kinetics to those seen in tumors, thereby suggesting that normal tissues might be useful for predicting drug activity. Furthermore, plitidepsin administration to rats at plasma concentrations comparable to those achievable in patients also increased JNK phosphorylation in peripheral mononuclear blood cells. These findings suggest that changes in JNK activity provide a reliable biomarker for plitidepsin activity and this could be useful for designing clinical trials and maximizing the efficacy of plitidepsin.This work has been partially supported by grants (Programa Cenit, CEN-20091016, SAF2010-18302 and Fondo Europeo de Desarrollo Regional-Instituto de Salud Carlos III, RD12/0036/0021) from Ministerio de Economíay Competitividad of Spain.S

Irvalec Inserts into the Plasma Membrane Causing Rapid Loss of Integrity and Necrotic Cell Death in Tumor Cells

Irvalec is a marine-derived antitumor agent currently undergoing phase II clinical trials. In vitro, Irvalec induces a rapid loss of membrane integrity in tumor cells, accompanied of a significant Ca2+ influx, perturbations of membrane conductivity, severe swelling and the formation of giant membranous vesicles. All these effects are not observed in Irvalec-resistant cells, or are significantly delayed by pretreating the cells with Zn2+. Using fluorescent derivatives of Irvalec it was demonstrated that the compound rapidly interacts with the plasma membrane of tumor cells promoting lipid bilayer restructuration. Also, FRET experiments demonstrated that Irvalec molecules localize in the cell membrane close enough to each other as to suggest that the compound could self-organize, forming supramolecular structures that likely trigger cell death by necrosis through the disruption of membrane integrity

PM060184, a new tubulin binding agent with potent antitumor activity including P-glycoprotein over-expressing tumors

PM060184 belongs to a new family of tubulin-binding agents originally isolated from the marine sponge Lithoplocamia lithistoides. This compound is currently produced by total synthesis and is under evaluation in clinical studies in patients with advanced cancer diseases. It was recently published that PM060184 presents the highest known affinities among tubulin-binding agents, and that it targets tubulin dimers at a new binding site. Here, we show that PM060184 has a potent antitumor activity in a panel of different tumor xenograft models. Moreover, PM060184 is able to overcome P-gp mediated resistance in vivo, an effect that could be related to its high binding affinity for tubulin. To gain insight into the mechanism responsible of the observed antitumor activity, we have characterized its molecular and cellular effects. We have observed that PM060184 is an inhibitor of tubulin polymerization that reduces microtubule dynamicity in cells by 59%. Interestingly, PM060184 suppresses microtubule shortening and growing at a similar extent. This action affects cells in interphase and mitosis. In the first case, the compound induces a disorganization and fragmentation of the microtubule network and the inhibition of cell migration. In the second case, it induces the appearance of multipolar mitosis and lagging chromosomes at the metaphase plate. These effects correlate with prometaphase arrest and induction of caspase-dependent apoptosis or appearance of cells in a multinucleated interphase-like state unrelated to classical apoptosis pathways. Taken together, these results indicate that PM060184 represents a new tubulin binding agent with promising potential as an anticancer agent.This work was supported by grants BIO2010-16351 (JFD), CAM S2010/BMD-2457 (JFD), CAM S2010/BMD-2353 (JMA), BFU2011-23416 (JMA) and PharmaMar-CSIC contracts. BP had a contract from Comunidad de Madrid

New interfacial microtubule inhibitors of marine origin, PM050489/PM060184, with potent antitumor activity and a distinct mechanism

We have investigated the target and mechanism of action of a new family of cytotoxic small molecules of marine origin. PM050489 and its dechlorinated analogue PM060184 inhibit the growth of relevant cancer cell lines at subnanomolar concentrations. We found that they are highly potent microtubule inhibitors that impair mitosis with a distinct molecular mechanism. They bind with nanomolar affinity to unassembled αβ-tubulin dimers, and PM050489 binding is inhibited by known Vinca domain ligands. NMR TR-NOESY data indicated that a hydroxyl-containing analogue, PM060327, binds in an extended conformation, and STD results define its binding epitopes. Distinctly from vinblastine, these ligands only weakly induce tubulin self-association, in a manner more reminiscent of isohomohalichondrin B than of eribulin. PM050489, possibly acting like a hinge at the association interface between tubulin heterodimers, reshapes Mg2+-induced 42 S tubulin double rings into smaller 19 S single rings made of 7 ± 1 αβ-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulans map to β-tubulin Asn100, suggesting a new binding site different from that of vinblastine at the associating β-tubulin end. Inhibition of assembly dynamics by a few ligand molecules at the microtubule plus end would explain the antitumor activity of these compounds, of which PM060184 is undergoing clinical trials.We wish to thank J. M. Fernandez Sousa (PharmaMar) for useful discussions and support, E. Hamel (NCI) for providing eribulin, C. Scazzocchio and G. Diallinas for useful advice on mutant screening, H. N. Arst for advice on mutant screening and mapping and for kindly providing strains MAD3688 and MAD4655, T. J. Fitzgerald (A&M University) for MTC and C. Alfonso (CIB) for AUC analysis. We also thank Rhône Poulenc Rorer Aventis for supplying docetaxel and Matadero Municipal Vicente de Lucas de Segovia for providing the calf brains for tubulin purification. B.P. had a contract from Comunidad de Madrid, and A.C. had a Ramon y Cajal contract, J.R.-S. had a fellowship from “Programa de Cooperación Científica entre el Ministerio de Ciencia, Tecnologías y Medio Ambiente de la República de Cuba (CITMA) y el CSIC”. This work was supported by grants BIO2010-16351 (J.F.D.), BQU2009-08536 (J.J.-B.), CAM S2010/BMD-2457 (J.F.D.), CAM S2010/BMD-2353 (J.J.-B., J.M.A.), IPT-2011-0752-900000 and BIO2012-30965 (M.A.P.), BFU2011-23416 (J.M.A.) and PharmaMar-CSIC contracts

The PARP inhibitor olaparib enhances the sensitivity of Ewing sarcoma to trabectedin

Producción CientíficaRecent preclinical evidence has suggested that Ewing Sarcoma (ES) bearing EWSR1-ETS fusions could be particularly sensitive to PARP inhibitors (PARPinh) in combination with DNA damage repair (DDR) agents. Trabectedin is an antitumoral agent that modulates EWSR1-FLI1 transcriptional functions, causing DNA damage. Interestingly, PARP1 is also a transcriptional regulator of EWSR1-FLI1, and PARPinh disrupts the DDR machinery. Thus, given the impact and apparent specificity of both agents with regard to the DNA damage/DDR system and EWSR1-FLI1 activity in ES, we decided to explore the activity of combining PARPinh and Trabectedin in in vitro and in vivo experiments. The combination of Olaparib and Trabectedin was found to be highly synergistic, inhibiting cell proliferation, inducing apoptosis, and the accumulation of G2/M. The drug combination also enhanced γH2AX intranuclear accumulation as a result of DNA damage induction, DNA fragmentation and global DDR deregulation, while EWSR1-FLI1 target expression remained unaffected. The effect of the drug combination was corroborated in a mouse xenograft model of ES and, more importantly, in two ES patient-derived xenograft (PDX) models in which the tumors showed complete regression. In conclusion, the combination of the two agents leads to a biologically significant deregulation of the DDR machinery that elicits relevant antitumor activity in preclinical models and might represent a promising therapeutic tool that should be further explored for translation to the clinical setting.Ministerio de Economía y Competitividad (PI081828)Ministerio de Economía y Competitividad (RD06/0020/0059 )Ministerio de Economía y Competitividad (RD12/0036/0017)Ministerio de Economía y Competitividad (PT13/0010/0056

Zalypsis has in vitro activity in acute myeloid blasts and leukemic progenitor cells through the induction of a DNA damage response

[EN]Although the majority of patients with acute myeloid leukemia initially respond to conventional chemotherapy, relapse is still the leading cause of death, probably because of the presence of leukemic stem cells that are insensitive to current therapies. We investigated the antileukemic activity and mechanism of action of zalypsis, a novel alkaloid of marine origin. The activity of zalypsis was studied in four acute myeloid leukemia cell lines and in freshly isolated blasts taken from patients with acute myeloid leukemia before they started therapy. Zalypsis-induced apoptosis of both malignant and normal cells was measured using flow cytometry techniques. Gene expression profiling and western blot studies were performed to assess the mechanism of action of the alkaloid. Zalypsis showed a very potent antileukemic activity in all the cell lines tested and potentiated the effect of conventional antileukemic drugs such as cytarabine, fludarabine and daunorubicin. Interestingly, zalypsis showed remarkable ex vivo potency, including activity against the most immature blast cells (CD34(+) CD38(-) Lin(-)) which include leukemic stem cells. Zalypsis-induced apoptosis was the result of an important deregulation of genes involved in the recognition of double-strand DNA breaks, such as Fanconi anemia genes and BRCA1, but also genes implicated in the repair of double-strand DNA breaks, such as RAD51 and RAD54. These gene findings were confirmed by an increase in several proteins involved in the pathway (pCHK1, pCHK2 and pH2AX). The potent and selective antileukemic effect of zalypsis on DNA damage response mechanisms observed in acute myeloid leukemia cell lines and in patients' samples provides the rationale for the investigation of this compound in clinical trials