Evaluation of different conditions to enhance the performances of Lactobacillus pentosus OM13 during industrial production of Spanish-style table olives

The main objective was to set up a methodology to improve the high volume production of green table olives, cv. Nocellara del Belice. Lactobaccillus pentosus OM13 was applied during three different industrial processes of table olives as follows: trial one (IOP1) was subjected to an addition of lactic acid until a brine level of pH 7.0 was reached; trial two (IOP2) subjected to same addition of lactic acid as in trial one plus nutrient adjuvant; and trial three (IOP3) subjected to same addition of lactic acid as in trial one, but with the strain L. pentosus OM13 acclimatized in brine for 12 h before inoculation. These trials were compared against two untreated controls (spontaneously fermented and addition of L. pentosus OM13 only). Within the third day of fermentation, the pH of the brines decreased significantly, reaching pH 4.85 for trial three, pH 5.15 for trial two, and pH 5.92 for trial one. The pH of both controls decreased more slowly, and had values below pH 5.0 only after the fifteenth day of fermentation (control one) and the sixty-fifth day of fermtation (control two). Trial three reached the highest lactic acid bacteria (LAB) concentration on the third day of fermentation. After six days of fermentation, all trials showed similar values of LAB counts that were significantly higher compared to control number one. The result from genotypic identification showed that L. pentosus OM13 was the most frequently isolated in the inoculated trials. Lactobacillus plantarum, Lactobacillus coryniformis and Pediococcus pentosaceous were also detected at very low concentrations. Homoguaiacol, 2-butanol, 4-ethylphenol, phenylethyl alcohol and 4-ethylphenol were the volatile organic compounds detected at the highest levels in all experimental trials. Trial three showed a higher concentration of squalene that was not detected in other trials. The highest sensory scores of green olive aroma and overall satisfaction were found for all experimental olives, especially for those of trial one and trial two, that differed significantly from the untreated controls. This study provides evidence that the addition of lactic acid, nutrient adjuvants and, most importantly, the acclimatization of LAB cells significantly shortens the acidification process of olive brine, and improves safety and sensory quality. Shorter acidification processes result in a more rapid transformation of table olives, with reduced commodity loss and lower costs of production compared to conventional manufacturing protocols

Neural influence on muscle dystrophin. Protein localization and gene expression.

Abstract a Congress

Normal and pathological neuro-muscular development in man and animal models

The differentiation of muscle cells during development and pathologically-induced alterations results in the variation of some important indexes; among these are the isoforms of myosin, the acetylcholine receptor subunits and the levels and molecular forms of acetylcholinesterase (AChE). We have studied the variations in AChE activity and molecular forms in different experimental conditions: 1) Denervation and reinnervation process in rat muscles. 2) Maturation of myogenic mouse cells (early myoblast, late myoblast, and satellite cells). 3) Muscular distrophy in genetically dystrophic mice. 4) Non-dystrophic child myopathies. The results of our experiments indicate that: 1) The most complex asymmetric molecular form (A12) appears to represent a significant index of the establishment of synaptic contacts. 2) The study of the levels of A12 form in the cells and their culture medium in different experimental conditions supports the hypothesis that the three different myogenic cells are endowed with different maturation programs. 3) The AChE molecular forms pattern is altered in dystrophic mouse muscles, showing a selective and marked reduction of one particular form (the tetrameric one inserted in the area of the membrane probably near the acetylcholine receptor). 4) The complete disappearance of high and medium molecular forms of AChE is a characteristic of 60% of "myopathic non-dystrophic" patients. This last observation enables the differentiation of two distinct groups within a population of subjects affected by pathologies of a hitherto indistinctly defined aetiology

Acetylcholinesterase modulates neurite outgrowth on fibronectin

Acetylcholinesterase (AChE) has been reported to be involved in the modulation of neurite outgrowth. To understand the role played by different domains, we transfected neuroblastoma cells with three constructs containing the invariant region of AChE, differing in the exon encoding the C-terminus and therefore in AChE cellular fate and localization. All isoforms increased neurite extension, suggesting the involvement of the invariant domain [A. De Jaco, G. Augusti-Tocco, S. Biagioni, Alternative AChE molecular forms exhibit similar ability to induce neurite outgrowth, J. Neurosci. Res. 70 (2002) 756-765]. The peripheral anionic site (PAS) is encoded by invariant exons and represents the domain involved in non-cholinergic functions of AChE. Masking of PAS with fasciculin results in a significant decrease of neurite outgrowth in all clones overexpressing AChE. A strong reduction was also observed when clones were cultured on fibronectin. Treatment of clones with fasciculin, therefore masking PAS, abolished the fibronectin-induced reduction. The inhibition of the catalytic site cannot revert the fibronectin effect. Finally, when clones were cultured on fibronectin in the presence of heparin, a ligand of fibronectin, the inhibitory effect was completely reversed. Our results indicate that PAS could directly or indirectly mediate AChE/fibronectin interactions. © 2007 Elsevier Inc. All rights reserved

Hepatocyte growth factor and cell migration in neurogenesis

Hepatocyte growth factor (HGF) is a pleiotropic factor which activates cell proliferation, differentiation and migration of various cell types. Its action is mediated by a receptor, Met, endowed with tyrosine kinase activity, which activates a complex signalling cascade and mediates the diverse cell responses. Although HGF action has been first demonstrated on epithelial cells, HGF and Met receptor expression have also been described in the developing and adult mammalian brain. In the developing brain HGF and Met expression areas are coincident with the migratory paths of precursor cells. These data will be reviewed together with cell culture data, which directly support a role for HGF in precursor cell migration and neuron differentiation

β-Catenin and actin reorganization in HGF/SF response of ST14A cells

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic factor that activates proliferation, differentiation, and migration of various cell types. Its action is mediated by c-Met, a receptor endowed with tyrosine kinase activity that activates complex signaling cascades and mediates diverse cell responses. Although HGF action was first demonstrated in epithelial cells, expression of HGF and c-Met receptor has also been described in developing and adult mammalian brain. In the developing central nervous system, areas of HGF and c-Met expression are coincident with the migratory pathway of precursor cells. In the present article we report that the interaction between c-Met and HGF/SF in striatal progenitor ST14A cells triggers a signaling cascade that induces modification of cell morphology, with decreased cell-cell interactions and increased cell motility; in particular, we analyzed the reorganization of the actin cytoskeleton and the delocalization of β-catenin and N-cadherin. The testing of other neurotrophic factors (NGF, BDNF, NT3, and CNTF) showed that the observed modifications were peculiar to HGF. We show that phosphoinositide 3-kinase inhibitor treatment, which blocks cell scattering induced by HGF/SF, does not abolish actin and β-catenin redistribution. The effects of HGF/SF on primary spinal cord cell cultures were also investigated, and HGF/SF was found to have a possible motogenic effect on these cells. The data reported suggest that HGF could play a role in the early steps of neurogenesis as a motogenic factor. © 2007 Wiley-Liss, Inc

CNTF VARIANTS WITH INCREASED BIOLOGICAL POTENCY AND RECEPTOR SELECTIVITY DEFINE A FUNCTIONAL SITE OF RECEPTOR INTERACTION

Human CNTF is a neurocytokine that elicits potent neurotrophic effects by activating a receptor complex composed of the ligand-specific alpha-receptor subunit (CNTFR alpha) and two signal transducing proteins, which together constitute a receptor for leukemia inhibitory factor (LIFR). At high concentrations, CNTF can also activate the LIFR and possibly other cross-reactive cytokine receptors in the absence of CNTFR alpha. To gain a better understanding of its structure-function relationships and to develop analogs with increased receptor specificity, the cytokine was submitted to affinity maturation using phage display technology. Variants with greatly increased CNTFR alpha affinity were selected from a phage-displayed library of CNTF variants carrying random amino acid substitutions in the putative D helix, Selected variants contained substitutions of the wild-type Gln167 residue, either alone or in combination with neighboring mutations. These results provide evidence for an important functional role of the mutagenized region in CNTFR alpha binding. Affinity enhancing mutations conferred to CNTF increased potency to trigger biological effects mediated by CNTFR alpha and enhanced neurotrophic activity on chicken ciliary neurons. In contrast, the same mutations did not potentiate the CNTFR alpha-independent receptor actions of CNTF, These CNTF analogs thus represent receptor-specific superagonists, which should help to elucidate the mechanisms underlying the pleiotropic actions of the neurocytokine

Muscle acetylcholinesterase in childhood myopathies

The activities and the molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were examined in 28 biopsies of quadriceps femoris muscle from children with a myopathic non-dystrophic disease. These cases were compared with biopsies from 7 children with a neurogenic damage, 14 children with muscular dystrophy and 12 controls. All the biopsies, histochemically stained for AChE, showed no endplates; electron microscopy of muscle fibers from diseased biopsies revealed a diffuse AChE reaction on the fiber surfaces which was not associated with any endplate structure. The AChE activities in NaCl/Triton X-100 extracts from the three groups of patients were all more or less the same, and average levels were similar to those evidenced in controls. The complete disappearance of heavy and medium forms of AChE was noted in 60% of myopathic non-dystrophic patients. We never observed the pattern characteristic to these patients in the biopsies from neurogenic and dystrophic patients or from controls, which displayed a high variability in the profiles of AChE molecular forms

Membrane acetylcholinesterase in murine muscular dystrophy in vivo and in cultured myotubes

Murine muscular dystrophy is characterized by a reduction of the 10S molecular form of acetylcholinesterase (AChE); this reduction occurs in both strains of dystrophic mice and at the time of the phenotypic appearance of the disease. In the present study we have analyzed the biochemical features, the cellular distribution and the developmental appearance of the AChE alteration. Sequential extractions with low salt, detergent and high salt revealed that this alteration affects only membrane-bound forms (those requiring Triton X-100 for solubilization), while both the low salt soluble and the high salt soluble forms appeared almost identical in normal and dystrophic muscles. Specific activity, sensitivity to different ions, pH dependence and Km were found to be identical in the enzymes from normal and dystrophic muscles, suggesting that the catalytic site of the 10S form is probably not altered. Further analysis, by non-denaturing gel electrophoresis, of the detergent soluble forms separated by sedimentation, revealed a single band for the 4S, a doublet for the 6S and three bands for the 10S peaks, indicating the existence of charge heterogeneity in AChE molecular forms. The corresponding molecular forms from dystrophic muscles behaved identically upon electrophoresis: the residual activity in the detergent soluble 10S form could still be separated into three bands, comigrating with their normal counterparts. Neuraminidase treatment resulted in a reduction of migration of both the 6S and 10S derived bands, but not of the 4S species, showing that sialic acid is added only to polymeric forms. Interestingly, the reduction of the 10S form appears to be linked to a developmental stage not reached in cell cultures, as cultured myotubes from muscles of dystrophic mice contained normal amounts of membrane-bound AChE forms. The molecular mechanism underlying the reduction of the tetrameric membrane bound AChE form in dystrophic muscle and the possible functional consequences are discussed