Biochemical differences in the skin of two blueberries (Vaccinium corymbosum) varieties with contrasting firmness: Implication of ions, metabolites and cell wall related proteins in two developmental stages.

The pursuit of firmer and better-quality blueberries is a continuous task that aims at a more profitable production. To this end it is essential to understand the biological processes linked to fruit firmness, which may diverge among tissues. By contrasting varieties with opposing firmness, we were able to elucidate events that, taking place at immature stage, lay the foundation to produce a firmer ripe fruit. A deep analysis of blueberry skin was carried out, involving diverse comparative approaches including proteomics and metabolomics coupled to immunolocalization assays. In'O'Neal' (low firmness) enhanced levels of aquaporins, expansins and pectin esterases at the green stage were found to be critical in distinguishing it from 'Emerald' (high firmness). The latter featured higher levels of ABA, low methyl esterified pectins in tricellular junctions and high levels of catechin at this stage. Meanwhile, in 'Emerald' 's ripe fruit epicarp, several mechanisms of cell wall reinforcement such as calcium and probably boron bridges, appear to be more prominent than in 'O'Neal'. This study highlights the importance of cell wall reorganization and structure, abundance of specific metabolites, water status, and hormonal signalling in connection to fruit firmness. These findings result particularly valuable in order to improve the fertilization procedures or in the search of molecular markers related with firmness.

Proteomic and metabolomic approaches unveil relevant biochemical changes in carbohydrate and cell wall metabolisms of two blueberry (Vaccinium corymbosum) varieties with different quality attributes.

Quality maintenance in rapidly decaying fruit such as blueberries (Vaccinium corymbosum) is of essential importance to guarantee the economic success of the crop. Fruit quality is a multifaceted subject that encompasses flavor, aroma, visual and physical issues as main factors. In this paper we report an ample characterization of different biochemical and physical aspects in two varieties (O'Neal and Emerald) of blueberries that differ in firmness, aspect, flavor and harvesting times, at two different phenological stages (fruit set vs. ripe), with the intention of unveiling how the metabolic signature of each contributes to their contrasting quality. To this effect a metabolomic, ionomic and proteomic approach was selected. The results presented here show marked differences in several variables at the two stages and between varieties. Emerald is an early variety with a large, good taste and firm fruit, while O'Neal is soft, medium sized and very sweet. Proteomic data comparison between both cultivars showed that, at fruit set, processes related with the response to inorganic compounds and small molecule metabolisms are relevant in both varieties. However, solute accumulation (mainly amino acids and organic acids), enzymes related with C: N balance, water transport and cell wall recycling are enhanced in Emerald. In ripe fruit, Emerald showed an enrichment of proteins associated with TCA, nitrogen, small molecules and cell wall in muro recycling processes, while mannitol and fatty acid metabolism were enhanced in the soft variety. The measured variation in metabolite levels gave strong support to the precedent results. This study suggests that at fruit set, a composite scenario of active metabolic recycling of the cell wall, improved C: N balance and solute accumulation give place to a more efficient carbon and water resource management. During the ripe stage, an increased and efficient in muro and metabolic recycling of the cell wall, added to enhanced inositol and secondary metabolism may be responsible for a best turgor conservation in Emerald. These findings may yield clues for improvements in fertilization practices, as well as to assist the guided development of new varieties based on biochemical quality.

Purification and Structural and Kinetic Characterization of the Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase from the Crassulacean Acid Metabolism Plant, Pineapple.

Pyrphosphate-dependent phosphofructokinase (PFP) was purified to electrophoretic homogeneity from illuminated pineapple (Ananas comosus) leaves. The purified enzyme consists of a single subunit of 61.5 kD that is immunologically related to the potato tuber PFP [beta] subunit. The native form of PFP likely consists of a homodimer of 97.2 kD, as determined by gel filtration. PFP's glycolytic activity was strongly dependent on pH, displaying a maximum at pH 7.7 to 7.9. Gluconeogenic activity was relatively constant between pH 6.7 and 8.7. Activation by Fru-2,6-bisphosphate (Fru-2,6-P2) was dependent on assay pH. In the glycolytic direction, it activated about 10-fold at pH 6.7, but only 2-fold at pH 7.7. The gluconeogenic reaction was only weakly affected by Fru-2,6-P2. The true substrates for the PFP forward and reverse reactions were Fru-6-phosphate and Mg-pyrophosphate, and Fru-1,6-P2, orthophosphate, and Mg2+, respectively. The results suggest that pineapple PFP displays regulatory properties consistent with a pH-based regulation of its glycolytic activity, in which a decrease in cytosolic pH caused by nocturnal acidification during Crassulacean acid metabolism, which could curtail its activity, is compensated by a parallel increase in its sensitivity to Fru-2,6-P2. It is also evident that the [beta] subunit alone is sufficient to confer PFP with a high catalytic rate and the regulatory properties associated with activation by Fru-2,6-P2.

Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase. A resonance energy transfer study.

Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R-NH2 group, presumably the epsilon-NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. 11, 1535-1558 (1962)] provides clear evidence that a pH 8 eight R-NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non-essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s). One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isothiocyanate. In the doubly labeled protein non-radiative single-singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (+/- 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8-1.1, 1.9-2.1, 2.9-3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8-5.1 nm from the donor site and third site being 6.4 nm apart from the donor. Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein-bound dansyl chloride and eosin isothiocyanate was observed. Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate-binding site with one lysyl residue crucial for activity.

Craneosinostosis (1980-1985): evolución conceptual y terapéutica / Craniosynostosis (1980-1985): conceptual and therapeutic evolution

Se analiza una serie de 122 craneosinostosis intervenidas en el Servicio de Neurocirugía Pediátrica del Instituto de Neurocirugía de Santiago durante el período 1980-1985 (incluyendo 82 casos personales). Su comparación con una serie similar presentada y publicada en 1970 (3), permite apreciar una notoria disminución en la edad de diagnóstico y tratamiento junto al desarrollo de nuevas técnicas quirúrgicas. Se enfatiza y se intentan esquematizar las distintas alteraciones óseas, encefálicas y las alteraciones dinámicas de la circulación de LCR junto a las alteraciones estéticas como una manera de lograr una comprensión más global del problema y planear un tratamiento quirúrgico más racional y de orientación etiológica