Browsing by Author "San Martin, R"

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    Control of plant-parasitic nematodes with extracts of Quillaja saponaria
    (BRILL ACADEMIC PUBLISHERS, 2005) San Martin, R; Magunacelaya, JC
    Aqueous extracts of the tree Quillaja saponaria Molina, containing triterpenoid saponins, polyphenols, salts and sugars, were tested against the most important plant-parasitic nematodes present in Chile: Xiphinema index, X. americanum s.l., Meloidogyne hapla, M. ethiopica, Pratylenchus thornei, Tylenchorhynchus sp., Criconemoides xenoplax and Helicotylenchus sp. Three different products were tested in the laboratory: i) QL 1000((R)) (whole extract containing saponin and non-saponin fractions); ii) QL ULTRA((R)) (saponin fraction); and iii) QL NS(R) (non-saponin fraction). The results showed that QL 1000((R)) had important nematicidal effects at economically attractive doses (e.g., 100 ppm). When used separately, QL ULTRA((R)) and QL NS(R) had minimum nematicidal effects. However, the combined use of both products gave similar results to QL 1000((R)), suggesting a synergy between the saponin and non-saponin fractions. Based on this, QL 1000((R)) was tested in commercial vineyards and table grapes in the Central region of Chile. The use of 301 ha(-1) showed a similar control and fruit yields to synthetic chemical nematicides.
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    Equilibrative nucleoside transporter 2 is expressed in human umbilical vein endothelium, but is not involved in the inhibition of adenosine transport induced by hyperglycaemia
    (W B SAUNDERS CO LTD, 2005) Aguayo, C; Casado, J; Gonzalez, M; Pearson, JD; San Martin, R; Casanello, P; Pastor Anglada, M; Sobrevia, L
    Human equilibrative, Na+-independent nucleoside transport is mediated by membrane proteins sensitive (system es, hENT1) or insensitive (system ei, hENT2) to nitrobenzylthioinosine (NBMPR). Gestational diabetes and elevated extracellular concentrations of D-glucose reduce adenosine transport in human umbilical vein endothelium (HUVEC). We studied hENT2 and hENT1 expression in HUVEC, and the effect of D-glucose on their activity and expression in HUVEC preincubated with 25 MM D-glucose (24 h). hENT2 and hENT1 mRNA were quantified by real-time reverse transcription polymerase chain reaction, and their proteins were detected by Western blotting. hENT2 and hENT1 proteins are co-expressed in HUVEC and are located at the plasma membrane, however, hENT2 was mainly cytoplasmatic and perinuclear in location. D-Glucose reduced hENT1 and hENT2 mRNA expression, but only hENT1 protein abundance at the plasma membrane. Adenosine transport was inhibited by D-glucose and NMBPR (1 mu M) in intact cells and membrane vesicles. Hypoxanthine inhibited adenosine transport in the absence or in the presence of 1 mu M NBMPR. D-Glucose reduced NBMPR maximal binding in intact cells, membrane vesicles, and plasma membrane fractions. In conclusion, the present study demonstrates that hENT2 and hENT1 are co-expressed in HUVEC, and even when adenosine transport is also mediated by hENT2, the hENT2-mediated transport activity is not involved in the D-glucose-induced down-regulation of total adenosine transport.
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    High D-glucose reduces promoter activity of human equilibrative nucleoside transporter 1 in human umbilical vein endothelium
    (2006) Farías Jofré, Marcelo Enrique; San Martin, R; Sobrevía Luarte, Luis Alberto
    Reduction of adenosine uptake by human equilibrative membrane transporters 1 (hENT1) in human umbilical vein endothelial cells (HUVEC) from gestational diabetes, or in HUVEC from normal pregnancies exposed to high extracellular D-glucose, is associated with reduced hENT1 mRNA expression. We studied the effect of high D-glucose on the transcriptional activity of the promoter region of SLC29A1 gene (for hENT1) in HUVEC. Methods: Cells were isolated and cultured in medium 199 (Ethics committee approval and informed patient consent were obtained). Fragments of SLC29A1 promoter (-3100, -2056, -1016 and -697 bp from ATG) were subcloned in pGL3 vector, upstream firefly luciferase reporter gene. Cells were co-transfected with hENT1-promoter constructs and pRL-TK vector by electroporation (320 V, 20 ms) and exposed to 5 or 25 mM D-glucose (24 hrs). Results: firefly/renilla luciferase activity was similar in all constructs transfected in 5 mM D-glucose. However, 25 mM D-glucose was associated with reduced transcriptional activity of sequences -697 to -1016 bp and -2056 to -3100 bp. Conclusions: These results suggest that the reduced hENT1 mRNA level detected in HUVEC exposed to high D-glucose could result from altered transcriptional activity of SLC29A1 promoter, most likely related to activation of repressor sequences of this gene.
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    Hypoxia increases equilibrative nucleoside transporter 2 activity by a transcriptional independent mechanism in human umbilical vein endothelium
    (2006) Torres, A; San Martin, R; Farías Jofré, Marcelo Enrique; Sobrevía Luarte, Luis Alberto; Casanello Toledo, Paola Cecilia
    Low oxygen tension (hypoxia) reduces adenosine transport in several types of mammalian cells. Adenosine transport is mediated by human equilibrative nucleoside transporter 1 (hENT1) and hENT2 in human umbilical vein endothelium (HUVEC), a fetal cell type that grows under 5% O2 (ie. normoxia for this cell type). We studied whether hypoxia alters hENT2 expression and activity in HUVEC. Methods: Cells were cultured (0-24 h) in 5% or 2% O2 (hypoxia), and [3H]adenosine uptake (125 and 500 μM, 4 μCi/ml, 20 s, 37°C) was measured in absence or presence of 100 nM nitrobenzylthioinosine (NBMPR, hENT1 inhibitor). hENT2 mRNA was quantified by real time RT-PCR, and protein abundance was determined by Western blot. SLC29A2 (for hENT2) promoter activity was measured following transfection (electroporation, 320 V, 30 ms) with pGL3 basic plasmid (firefly/renilla luciferase reporter gene) carrying -1477 bp and -587 bp of the promoter sequence. Results: Hypoxia reduced hENT2 mRNA expression (~55%), and promoter activity (~50%), but did not alter hENT2 protein abundance. Adenosine uptake via hENT2 was increased (2-fold) in hypoxia. Conclusions: Adenosine uptake via hENT2 may be modulated by post-translational mechanisms in hypoxia in HUVEC. Supported by FONDECYT 1030781/1030607/7050030. A Torres holds a School of Medicine research fellowship, and M Farías holds a CONICYT-PhD fellowship.
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    Role of adenosine transport in gestational diabetes-induced L-arginine transport and nitric oxide synthesis in human umbilical vein endothelium
    (WILEY, 2004) Vasquez, G; Sanhueza, F; Vasquez, R; Gonzalez, M; San Martin, R; Casanello, P; Sobrevia, L
    Gestational diabetes is associated with increased L-arginine transport and nitric oxide (NO) synthesis, and reduced adenosine transport in human umbilical vein endothelial cells (HUVEC). Adenosine increases endothelial L-arginine/NO pathway via A(2) purinoceptors in HUVEC from normal pregnancies. It is unknown whether the effect of gestational diabetes is associated with activation of these purinoceptors or altered expression of human cationic amino acid transporter 1 (hCAT-1) or human equilibrative nucleoside transporter I (hENT1), or endothelial NO synthase (eNOS) in HUVEC. Cells were isolated from normal or gestational diabetic pregnancies and cultured up to passage 2. Gestational diabetes increased hCAT-1 mRNA expression (2.4-fold) and activity, eNOS mRNA (2.3-fold), protein level (2.1-fold), and phosphorylation (3.8-fold), but reduced hENT1 mRNA expression (32%) and activity. Gestational diabetes increased extracellular adenosine (2.7 mum), and intracellular L-arginine (1.9 mm) and L-citrulline (0.7 mm) levels compared with normal cells (0.05 mum, 0.89 mm, 0.35 mm, respectively). Incubation of HUVEC from normal pregnancies with 1 mum nitrobenzylthioinosine (NBMPR) mimicked the effect of gestational diabetes, but NBMPR was ineffective in diabetic cells. Gestational diabetes and NBMPR effects involved eNOS, PKC and P42/44(mapk) activation, and were blocked by the A(2a) purinoceptor antagonist ZM-241385. Thus, gestational diabetes increases the L-arginine/NO pathway involving activation of mitogen-activated protein (MAP) kinases, protein kinase C (PKC) and NO cell signalling cascades following activation of A(2a) purinoceptors by extracellular adenosine. A functional relationship is proposed between adenosine transport and modulation Of L-arginine transport and NO synthesis in HUVEC, which could be determinant in regulating vascular reactivity in diabetes mellitus.