Rocío Rodríguez

Steam-explosion of olive stones: hemicellulose solubilization and enhancement of enzymatic hydrolysis of cellulose

Olive stones (whole stones and seed husks in fragments) were processed by steam-explosion under different experimental conditions of temperature and time, 200-236 degrees C for 2-4 min, with or without previous acid impregnation with 0.1%, H2SO4 (w/w). This paper examines the solubilization of hemicelluloses and their molecular weight distribution. The subsequent enzymatic hydrolysis of the solid residue, using a preparation of cellulase, was also studied. The maximum yield of the pentosan recovered in the water solution was 63% pentose in the starting material for seed husk treated at 200 degrees C for 2 min (log R0 3.24) prior to acid-impregnation, or at 215 degrees C for 2 min (log R0 3.69) without acid, compared to 39% of the potential yield for whole stones pre-impregnated with acid under more severe conditions (at log R0 = 4.07). This indicates that the autohydrolysis of hemicellulose in seed husks when compared to whole stones is enhanced. The molecular weight distribution of profile sugars showed that the depolymerization of hemicelluloses is a function of the severity of the treatment. Steam-explosion improved the accessibility of the cellulose and increased the enzymatic hydrolysis yield after steam-explosion with respect to material without steam explosion (ball-milled material), although little increase in the extent of saccharification occurred when the alkali-soluble lignin was removed. Only when the substrate was post-treated with Na-chlorite was the enzymatic hydrolysis improved, the water-insoluble residue being almost completely hydrolyzed in 8 h of incubation.

Relationship between a 47-kDa cytoplasmic membrane polypeptide and nitrate transport in Anacystis nidulans.

The polypeptide composition of cytoplasmic membranes of the cyanobacterium Anacystis nidulans changes in response to variations in the nitrogen source available to the cells, differing specifically in the amount of a polypeptide of 47-kDa molecular mass. Synthesis of the polypeptide and expression of nitrate transport activity are repressed by ammonium. Transfer of ammonium-grown cells to a medium containing nitrate as the sole nitrogen source results in parallel development of the 47-kDa polypeptide and nitrate transport activity of the cells. These results suggest the involvement of the 47-kDa cytoplasmic membrane polypeptide in nitrate transport by A. nidulans.

Dietary fibre content of table olives processed under different European styles: study of physico-chemical characteristics

Some European varieties of olive fruit (Douro, Hojiblanca, Cassanese, Conservolia, Taggiasca, and Thasos), processed under different conditions (black oxidised, fermented in brine or dried by different methods) were analysed, their contents of moisture, fat and dietary fibre being quantified. The percentages of moisture and fat were very different between varieties due to the different processing conditions, although differences between samples of the same variety were much less. The content of dietary fibre was around 12% of the fresh weight, although in dried samples this percentage increased to around 20%. Some physico-chemical characteristics (water holding capacity, cation exchange capacity, and ion retention capacity) were measured. The water holding capacities were related to moisture content, dry samples having the lowest values. Processed olives had very low cation exchange capacity in comparison to other vegetables, exhibiting the same relationship between this characteristic and moisture content. Olive fibre retained more iron than calcium under the assay conditions. © 2000 Society of Chemical Industry

SODIUM-DEPENDENT NITRATE TRANSPORT AND ENERGETICS OF CYANOBACTERIA

Nitrate is a major source of nitrogen for organisms in aquatic environments. The first and least understood step in nitrate assimilation is the transport of this anion into the cell. Among the microalgae, cyanobacteria represent a convenient model system to study the transport of nitrate. Early studies on nitrate transport in the unicellular cyanobacterium Anacjstis (Sjnechococcus) showed endergonic accumulation of nitrate into the cells and, hence, the operation of an active nitrate transport system (Lara et al. 1987). Nitrate transport in Anacjstis has proved to have some unusual and interesting features. The nitrate transport activity was shown to be sensitive to the regulation exerted by products of both ammonium and CO, assimilation, thus providing evidence that photosynthetic nitrate assimilation in cyanobacteria is regulated at the level of substrate supply to the cell (Lara et al. 1987). The expression of the nitrate transport system was also shown to be under nitrogen control, being repressed when ammonium is used as the nitrogen source and derepressed upon transfer of the cells to media containing nitrate or no nitrogen source (Sivak et al. 1989). A 47-kDa polypeptide, which is a major plasma membrane component in nitrate-grown cells but is virtually absent from ammonium-grown cells, was identified as an essential component of the nitrate transporter (Omata et al. 1989, Sivak et al. 1989). More recent studies have provided evidence of the Na+ dependence of active nitrate transport, A,GNa+ appearing to be the driving force for a sodiumnitrate symport system (Rodriguez et al. 1992). In this article, the operation of such a sodiumdependent nitrate transport system is discussed in relation to the bioenergetics of cyanobacteria. A personal view, rather than a comprehensive review, on these matters is presented.

Ammonium-sensitive protein kinase activity in plasma membranes of the cyanobacterium Anacystis nidulans

Cytoplasmic membranes prepared from nitrate‐grown Anacystis nidulans cells exhibit a Mg2+‐dependent protein kinase activity able to phosphorylate in vitro plasma membrane polypeptides with molecular masses of 98, 93, 83, 47, 44 and 31 kDa. The protein kinase activity was inhibited in cytoplasmic membrane preparations from nitrate‐grown cells which had been exposed to ammonium for 5 min. Parallely, ammonium exposure also resulted in a more than two‐fold activation of an alkaline phosphatase activity present in the soluble fraction. These results are discussed in relation to the well‐known inhibition by ammonium of nitrate transport activity, and a hypothesis for the regulatory mechanism involved is presented.

Mechanism of sodium/nitrate symport in Anacystis nidulans R2

Abstract A kinetic study of sodium-dependent nitrate transport in the cyanobacterium Anacystis nidulans R2 has been performed by following intracellular accumulation of nitrate in intact cells of the mutant strain FM6, lacking nitrate reductase activity and unable, therefore, to reduce the transported nitrate. Initial transport rates were determined at different external fixed sodium and varying nitrate concentrations and, conversely, at different fixed nitrate and varying sodium concentrations. The resulting kinetic pattern has the best fit to a reaction mechanism model in which sodium nitrate is the substrate for the transporter and sodium behaves additionally as a non-essential activator of the system. Half-saturation constants for the substrate sodium nitrate (1.6 ± 0.2 μM) and the activator sodium (0.36 ± 0.04 mM) have been calculated. The operation of such a sodium/nitrate symport system, driven by the energy of the Δ \ gm Na + across the plasma membrane, provides the basis for energy coupling between uphill nitrate and downhill sodium transport into Anacystis cells.

Age-regulated function of autophagy in the mouse inner ear.

Autophagy is a highly conserved catabolic process essential for embryonic development and adult homeostasis. The autophagic machinery supplies energy by recycling intracellular components and facilitates the removal of apoptotic cells. In the inner ear, autophagy has been reported to play roles during early development in the chicken embryo and in the response to otic injury in the adult mouse. However, there are no studies on the expression of the autophagy machinery in the postnatal and adult inner ear. Insulin-like growth factor 1 (IGF-1) is one of the factors that regulate both otic development and cochlear postnatal maturation and function. Here, we hypothesised that autophagy could be one of the processes involved in the cochlear development and functional maturation. We report that autophagy-related genes (ATG) Becn1, Atg4g and Atg5 are expressed in the mouse cochlea, vestibular system and brainstem cochlear nuclei from late developmental stages to adulthood. Atg9 was studied in the mouse cochlea and showed a similar pattern. The presence of autophagic flux was confirmed by decreased sequestosome 1 (SQSTM1/p62) and increased relative levels of microtubule-associated protein light chain 3-II (LC3-II). Inner ear autophagy flux is developmentally regulated and is lower at perinatal stages than in the adult mouse, where an expression plateau is reached at the age of two-months, coinciding with the age at which full functional activity is reached. Expression is maintained in adult mice and declines after the age of twelve months. LC3B labelling showed that autophagy was primarily associated with spiral ganglion neurons. Over time, Igf1 wild type mice showed lower expression of genes coding for IGF-1 high affinity receptor and the family factor IGF-2 than null mice. Parallel analysis of autophagy machinery gene expression showed no significant differences between the genotypes over the lifespan of the null mice. Taken together, these results show that the autophagy machinery expression in the inner ear is regulated with age but is not compromised by the chronic absence of IGF-1. Our data also strongly support that the up-regulation of autophagy machinery genes is concomitant with the functional maturation of the inner ear.

C-Raf deficiency leads to hearing loss and increased noise susceptibility

The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K+ channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf+/− mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection.

Autophagy in the Vertebrate Inner Ear

Autophagy is a conserved catabolic process that results in the lysosomal degradation of cell components. During development, autophagy is associated with tissue and organ remodeling, and under physiological conditions it is tightly regulated as it plays a housekeeping role in removing misfolded proteins and damaged organelles. The vertebrate inner ear is a complex sensory organ responsible for the perception of sound and for balance. Cell survival, death and proliferation, as well as cell fate specification and differentiation, are processes that are strictly coordinated during the development of the inner ear in order to generate the more than a dozen specialized cell types that constitute this structure. Here, we review the existing evidence that implicates autophagy in the generation of the vertebrate inner ear. At early stages of chicken otic development, inhibiting autophagy impairs neurogenesis and causes aberrant otocyst morphogenesis. Autophagy provides energy for the clearing of dying cells and it favors neuronal differentiation. Moreover, autophagy is required for proper vestibular development in the mouse inner ear. The autophagy-related genes Becn1, Atg4g, Atg5, and Atg9, are expressed in the inner ear from late developmental stages to adulthood, and Atg4b mutants show impaired vestibular behavior associated to defects in otoconial biogenesis that are also common to Atg5 mutants. Autophagic flux appears to be age-regulated, augmenting from perinatal stages to young adulthood in mice. This up-regulation is concomitant with the functional maturation of the hearing receptor. Hence, autophagy can be considered an intracellular pathway fundamental for in vertebrate inner ear development and maturation.