Oscar Goñi

Common Features at the Start of the Neurodegeneration Cascade

A single-molecule study reveals that neurotoxic proteins share common structural features that may trigger neurodegeneration, thus identifying new targets for therapy and diagnosis.

Water status and quality improvement in high-CO2 treated table grapes

Unfreezable water (UFW) content in berry tissues (pulp, skin, seed) and rachis of table grape clusters stored at 0°C has been studied using differential scanning calorimetry. The effect of short exposure to high CO2 (20% CO2 for 3days) and the transfer to air were also studied. Water status of pulp tissues was related to the thawing behaviour and the structural characteristics, using low-temperature scanning electron microscopy (LT-SEM). The UFW content in all tissues increased rapidly in response to high CO2 while it remained stable or decreased in untreated clusters. The strong potential of this beneficial gaseous treatment for increasing the UFW content was also evident after transfer to air. The metabolic adjustment caused by exposure to high CO2, which reduced the amount of water available to be frozen, improved stored fruit quality, thus minimising structural damage and reducing water leakage associated with the freezing-thawing process.

Regulation of defense and cryoprotective proteins by high levels of CO2 in Annona fruit stored at chilling temperature

This study focuses on how the length of exposure to chilling temperature and atmosphere storage conditions regulate the hydrolytic activity and expression of chitinase (PR-Q) and 1,3-beta-glucanase (PR-2) isoenzymes in cherimoyas (Annona cherimola Mill.). Storage at 6 degrees C modified the expression of constitutive isoenzymes and induced the appearance of novel acidic chitinases, AChi26 and AChi24, at the onset of the storage period, and of a basic chitinase, BChi33, after prolonged storage. The induction of this basic isoenzyme was concomitant with the accumulation of basic constitutive 1,3-beta-glucanases. These low-temperature-induced chitinases modified the growth inhibition in vitro of Botrytis cinerea. Short-term high CO(2) treatment activated a coordinated response of acidic chitinases and 1,3-beta-glucanases after prolonged storage at chilling temperature. Moreover, the high in vitro cryoprotective activity of CO(2)-treated protein extracts was associated with the induction of two low molecular mass isoenzymes, AGlu19 and BChi14. Thus, exposure to high concentrations of CO(2) modified the response of fruit to low temperature, inducing the synthesis of cryoprotectant proteins such as specific pathogenesis-related isoenzymes that could be functionally associated with an increase in chilling tolerance in vivo.

Potent cryoprotective activity of cold and CO2-regulated cherimoya (Annona cherimola) endochitinase.

A cryoprotective chitinase (BChi14) was isolated and purified from the mesocarp of CO(2)-treated cherimoya fruit (Annona cherimola Mill.) stored at chilling temperature by anion exchange and chromatofocusing chromatography. This hydrolase was characterized as an endochitinase with a M(r) of 14.31 kDa and a pI of 8.26, belonging to the family 19 of glycosyl hydrolases (GH19). While it was stable over a wide pH range and active in a broad acidic pH range, it had an optimum pH of 7.0. Its optimum temperature was low, 35 degrees C, and it retained about 30% of its maximum activity at 5 degrees C. Moreover, BChi14 was relatively heat unstable and its activity was progressively lost at temperatures above 50 degrees C. Kinetic studies revealed many similarities with other plant endochitinases. However, BChi14 had high k(cat) (6.93 s(-1)) value for the fluorogenic substrate 4-MU-(GlcNAc)(3), reflecting its great catalytic efficiency. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k(cat) at 37 and 5 degrees C, and a low E(a) (11.32 kJ mol(-1)). In vitro functional studies indicated that BChi14 had no effect on the inhibition of Botrytis cinerea hyphal growth and no antifreeze activity, as shown by the thermal hysteresis analysis using differential scanning calorimetry. However, the purified endochitinase showed very strong cryoprotective activity against freeze-thaw inactivation of lactate dehydrogenase. The PD(50) was 12.5 times higher than that of the cryoprotective protein BSA, and 2 or 3 orders of magnitude greater than sucrose, comparable with that of most cryoactive plant dehydrins. These results, together with the consolidated microstructure and the integrity of CO(2)-treated mesocarp tissue, indicate that BChi14 is functionally implicated in the mechanisms underlying chilling tolerance activated by high CO(2) concentrations.

A cryoprotective and cold-adapted 1,3-β-endoglucanase from cherimoya (Annona cherimola) fruit

A 1,3-β-glucanase with potent cryoprotective activity was purified to homogeneity from the mesocarp of CO2-treated cherimoya fruit (Annona cherimola Mill.) stored at low temperature using anion exchange and chromatofocusing chromatography. This protein was characterized as a glycosylated endo-1,3-β-glucanase with a Mr of 22.07kDa and a pI of 5.25. The hydrolase was active and stable in a broad acidic pH range and it exhibited maximum activity at pH 5.0. It had a low optimum temperature of 35°C and it retained 40% maximum activity at 5°C. The purified 1,3-β-glucanase was relatively heat unstable and its activity declined progressively at temperatures above 50°C. Kinetic studies revealed low k(cat) (3.10±0.04 s(-1)) and Km (0.32±0.03 mg ml(-1)) values, reflecting the intermediate efficiency of the protein in hydrolyzing laminarin. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k(cat) at both 37 and 5°C, and a low Ea (6.99 kJ mol(-1)) within this range of temperatures. In vitro functional studies indicated that the purified 1,3-β-glucanase had no inhibitory effects on Botrytis cinerea hyphal growth and no antifreeze activity, as determined by thermal hysteresis analysis using differential scanning calorimetry. However, a strong cryoprotective activity was observed against freeze-thaw inactivation of lactate dehydrogenase. Indeed, the PD50 was 8.7 μg ml(-1) (394 nM), 9.2-fold higher (3.1 on a molar basis) than that of the cryoprotective protein BSA. Together with the observed accumulation of glycine-betaine in CO2-treated cherimoya tissues, these results suggest that 1,3-β-glucanase could be functionally implicated in low temperature-defense mechanism activated by CO2.

Two cold-induced family 19 glycosyl hydrolases from cherimoya (Annona cherimola) fruit: An antifungal chitinase and a cold-adapted chitinase

Two cold-induced chitinases were isolated and purified from the mesocarp cherimoyas (Annona cherimola Mill.) and they were characterised as acidic endochitinases with a Mr of 24.79 and 47.77kDa (AChi24 and AChi48, respectively), both family 19 glycosyl hydrolases. These purified chitinases differed significantly in their biochemical and biophysical properties. While both enzymes had similar optimal acidic pH values, AChi24 was enzymatically active and stable at alkaline pH values, as well as displaying an optimal temperature of 45°C and moderate thermostability. Kinetic studies revealed a great catalytic efficiency of AChi24 for oligomeric and polymeric substrates. Conversely, AChi48 hydrolysis showed positive co-operativity that was associated to a mixture of different functional oligomeric states through weak transient protein interactions. The rise in the AChi48 kcat at increasing enzyme concentrations provided evidence of its oligomerisation. AChi48 chitinase was active and stable in a broad acidic pH range, and while it was relatively labile as temperatures increased, with an optimal temperature of 35°C, it retained about 50% of its maximal activity from 5 to 50°C. Thermodynamic characterisation reflected the high kcat of AChi48 and the remarkably lower ΔH(‡), ΔS(‡) and ΔG(‡) values at 5°C compared to AChi24, indicating that the hydrolytic activity of AChi48 was less thermodependent. In vitro functional studies revealed that AChi24 had a strong antifungal defence potential against Botrytis cinerea, whereas they displayed no cryoprotective or antifreeze activity. Hence, based on biochemical, thermodynamic and functional data, this study demonstrates that two acidic endochitinases are induced at low temperatures in a subtropical fruit, and that one of them acts in an oligomeric cold-adapted manner.