Juan Carlos Díaz Ricci

Enhanced resistance to Botrytis cinerea mediated by the transgenic expression of the chitinase gene ch5B in strawberry

Plants of strawberry (cultivar Pájaro) were transformed with three defense related genes: ch5B, gln2 and ap24 using Agrobacterium tumefaciens. The ch5B gene encodes for a chitinase from Phaseolus vulgaris, while gln2 and ap24 encode for a glucanase and a thaumatin-like protein, respectively, both from Nicotiana tabacum. Sixteen transgenic lines expressing one or a combination of two defense genes were obtained. Phytopathological tests showed that two transgenic lines expressing only the ch5B gene displayed high levels of resistance to gray mold disease (Botrytiscinerea). The resistance was correlated with the presence of the foreign protein CH5B and the increase of chitinolytic activity in leaves. However, resistance toward Colletotrichum acutatum, the etiological agent of the anthracnose disease, was not enhanced in the transgenic plants. These results suggest that the ch5B gene can be used to introduce transgene-mediated resistance to gray mold in strawberry, due to the lack of natural resistance to this disease in the crop.

On-line monitoring of galactoside conjugates and glycerol by flow injection analysis

A biosensor attached to a flow injection analysis (FIA) system was developed for the automatic determination of galactoside conjugates and glycerol. The biosensor was based on the enzymatic reaction of galactose oxidase (GalOD) using galactose, raffinose, lactose and glycerol as substrates. GalOD converts galactoside conjugates to galactohexodialdose conjugates and glycerol to glyceraldehyde with formation of hydrogen peroxide and consumption of oxygen. Variation of dissolved oxygen in the carrier was estimated utilizing an amperometric oxygen probe. The FIA system consisted in a multichannel peristaltic pump, an injection valve and an electronic transducer which were controlled by the CAFCA software. Stability of the enzyme and optimal working condition were investigated. Optimum pH for the immobilized enzymes under these experimental conditions was 7.4 and the enzyme retained 80% of the original activity after two months of use. Studies on the dynamical response of the biosensor showed that the elapsed time between two successive injections could be as short as 120 s without signal deterioration when the flow rate was 2 ml/min and 50 μl of injection volume. Sensitivity of the biosensor was higher for galactose followed by raffinose, lactose and glycerol. The sensor showed linear response between 0.2 and 2 mM for galactose, 0.5 and 6 mM for raffinose, 25 and 250 mM for lactose, and 2 and 200 mM for glycerol.

Global Sensitivity Analysis in dynamic metabolic networks

Abstract In this work, we have performed global sensitivity analysis through variance based techniques to determine parameters to be estimated in a kinetic metabolic network. Sensitivity indices have been calculated for each parameter [1] to provide a proper measure for their influence on model outputs, regardless of model nonlinearity and non-additivity. The global sensitivity analysis has been carried out on a large-scale nonlinear differential algebraic system representing a dynamic model for the Embden-Meyerhof-Parnas pathway, the phosphotransferase system and the pentose phosphate pathway of Escherichia coli K12 W3110 [2]. As a result, sixteen parameters from the complex metabolic network under study have been selected to be estimated.

Pathogen-Induced Accumulation of an Ellagitannin Elicits Plant Defense Response

In an incompatible interaction between Colletotrichum fragariae and strawberry plants, the accumulation of phenolic compounds in plant leaves was observed. A particularly abundant penta-esterified ellagitannin that accumulated in response to pathogen attack was identified as 1-0-galloyl-2,3;4,6-bis-hexahydroxydiphenoyl-β-d-glucopyranose (HeT) by mass spectroscopy and nuclear magnetic resonance. Foliar application of purified HeT prior to inoculation with a virulent pathogen was shown to increase resistance toward C. acutatum in strawberry plants and to Xanthomonas citri subsp. citri in lemon plants. The induced resistance in strawberry was associated with a rapid oxidative burst, callose deposition, a transient increase of salicylic acid in phloem, and induction of gene expression responsive to salicylic acid. Results obtained suggested that HeT could be a common plant defense response molecule capable of inducing pathogen resistance in different plant species.

Effect of arsenic in endochondral ossification of experimental animals.

Arsenic (As) toxicity is a global health problem affecting millions of people, the most toxic forms being Arsenites [As(III)] and Arsenates [As(V)]. Arsenic intoxication can occur through different exposure routes. The aim of the present work was to determine the effect of As on endochondral ossification and bone remodeling in experimental animals, by means of biochemical, histologic, and histomorphometric determinations. Sixteen male Wistar rats, 100g body weight (b.w.), were divided into two groups: experimental group (n=8), treated with 10mg/l of NaAsO(2) in their drinking water, receiving 0.21mg/kgb.w./day during 45 days; and control group (n=8) remained untreated. On day 45, blood samples were obtained by cardiac puncture to perform hematologic blood counts and biochemical determination. The animals were killed, the tibiae, femurs, kidneys and livers were resected, fixed in formalin and processed histologically. Tibia and femur sections were obtained and stained with H&E. The following histomorphometric parameters were determined on tibia and femur sections: bone volume (BV/TV), thickness of growth plate cartilage (GPC.Th) and thickness of hypertrophic zone (HpZ.Th). Biochemical determinations showed that experimental animals exhibited neutrophilia and a decrease in lymphocytes and monocytes. As levels were below 1 microg/dl in both groups. The femur sections of the experimental group showed (1) a statistically significant increase in total growth cartilage plate thickness (p<0.05) at the expense of the hypertrophic zone (p<0.05); (2) subchondral trabecular bone sealed to the growth plate with a non-significant increase in primary spongiosa bone volume. These results suggest that As alters endochondral ossification.

Batch culture of Candida utilis in a medium deprived of a phosphorous source

SummaryCandida utilis var. major NRRL-Y-1084 was grown in a defined medium without a phosphorous (P) source. During the exponential phase, cells divided according to a specific growth rate of 0.32 h-1, which is lower than the usual rate for a balanced medium (0.4–0.6 h-1). The relative P content of the biomass decreased from 2.70% to 0.75% over a period of 6 h, including 2 h of cell division arrest. At the end of this period there was another interruption of cell division. After that, multiplication restarted at a considerably lower rate and it deviated slightly from the exponential pattern. The stationary phase began when biomass P content reached 0.4%–0.5%, slowly decreasing afterwards to 0.25–0.20%. Biomass synthesis was less affected than cell division by the relative decrease of endogenous P, the two processes differing partially in their kinetics. Cell lysis started shortly before the stationary phase and affected about 20% of the population by the end of the assay. RNA and P content of the resulting biomass were 2.4% and 0.25% respecitvely, P being mainly incorporated to RNA.The relationship of biomass production to glucose uptake was very low, probably because the marked P deficiency called for an increase in energy consumption for growth and specially for maintenance. Compared with yeasts grown in a balanced medium, 40% increase in glycogen was observed, whereas no mean changes in the content of cell wall carbohydrates (glucan and mannan) and that of “true protein” were found.

Identification and characterisation of a novel class I endo-β-1,3-glucanase regulated by salicylic acid, ethylene and fungal pathogens in strawberry

The identification of a full length cDNA encoding an endo-β-1,3-glucanase (FaOGBG-5) from strawberry (Fragaria×ananassa Duch) is reported. The analysis of the deduced amino acid sequence of FaOGBG-5 showed that it shares typical structural features and a high degree of identity with other plant β-1,3-glucanases of the class I. The expression of FaOGBG-5 in plants infected with a virulent isolate of Colletotrichum acutatum and an avirulent isolate of Colletotrichum fragariae was examined. Induction of expression was observed with both pathogens but exhibited a delayed high expression with the virulent one. Additionally, the accumulation of FaOGBG-5 transcripts was also observed after treatments with the stress related hormones salicylic acid and ethylene. Results obtained suggest that the β-1,3-glucanase encoded by FaOGBG-5 may be implicated in plant defence against biotic and abiotic stress.

Parameter Estimation in Kinetic Models for Large Scale Metabolic Networks with Advanced Mathematical Programming Techniques

Abstract In this work, we formulate a parameter estimation problem for a large-scale dynamic metabolic network. The DAE system represents the dynamic model for the Embden-Meyerhof-Parnas pathway, the phosphotransferase system and the pentose-phosphate pathway of Escherichia coli K-12 W3110 (Chassagnole et al., 2002), with modifications on several enzyme kinetics and the addition of fermentation reactions. Model parameters have been estimated based on recently published experimental data for this strain. Most sensitive parameters have been ranked by performing global sensitivity analysis on the dynamic metabolic network (Di Maggio et al., 2009a,b). Eleven kinetic parameters, including maximum reaction rates, inhibition and half-saturation constants, have been estimated with good agreement with available experimental data.

Determination of the optimal conditions for the continuous culture of Candida utilis in sugarcane stillage

SummaryCandida utilis was continuously cultured in sugarcane stillage without any supplementation, but the yeast utilized the stillage nitrogen only partially. As a result, the cell biomass production was low and the residual chemical oxygen demand (COD), as well as the total nitrogen content of the effluent were high. The addition of 2–4 g/l ammonium phosphate and small amounts of Fe2+, Cu2+, and Zn2+, markedly enhanced both the everall nitrogen uptake and cell biomass production, decreased COD, and a higher critical dilution rate was attained. No substantial differences were found when pH was kept at 5.0 or allowed to evolve freely. The optimal temperature range was between 30 and 35°C.

The ellagitannin HeT induces electrolyte leakage, calcium influx and the accumulation of nitric oxide and hydrogen peroxide in strawberry

HeT (1-0-galloyl-2,3; 4,6-bis-hexahydroxydiphenoyl-β-D-glucopyranose) is a penta-esterified ellagitannin obtained from strawberry leaves. Previous studies have shown that foliar application of HeT prior to inoculation with a virulent pathogen increases the resistance toward Colletotrichum acutatum in strawberry plants and to Xanthomonas citri subsp. citri in lemon plants. In this work we report that HeT induces an immediate leak of electrolytes, the hyperpolarization of the cellular membrane, a rapid Ca2+ influx to the cytoplasm during the first few seconds, which in turn modulates the accumulation of nitric oxide 5 min after treatment. At longer times, a biphasic accumulation of H2O2 with peaks at 2 and 5 h post treatment could be observed. In addition, HeT elicited the increase of alternative oxidase capacity during the first 12 h post treatment.