Dissection of the effects induced by VEGF165, VEGF121 and Semaphorin 3A in endothelial cells

2007/2008The development of new blood vessels is a complex and highly regulated process that requires the coordinated action of different growth factors. Vascular Endothelial Growth Factor (VEGF) is a powerful inducer of angiogenesis, acting through the metabolic activation, proliferation and migration of endothelial cells. The major angiogenic member of the VEGF family is VEGF-A, a gene which is transcribed to give rise to at least 4 major splicing variants, of 206, 189, 165 and 121 amino acids in humans. The angiogenic effect of the most abundant isoform (VEGF165) is basically mediated by its interaction with VEGFR2 (KDR) and VEGFR1 (Flt-1) as well as with the co-receptor Neuropilin-1 (NP-1). The shortest isoform (VEGF121) binds VEGFR1 and VEGFR2 only, but not NP-1. NP-1, together with PlexinA1, also acts as a receptor of Semaphorin 3A (Sema3A), a factor also involved in vascular patterning, besides its very well known role in axonal guidance. The aim of this project has been the detailed analysis of the peculiarity/specificity of the effect of VEGF165, VEGF121 and Sema3A in endothelial cells. We first exploited an Adeno Associated Virus (AAV)-based gene delivery to unravel the in vivo effect of these cytokines. In particular, we observed that the prolonged expression of the main isoform of VEGF, VEGF165, acted as a powerful inducer of angiogenesis, stimulating the development of both a larger capillary network and the formation of an impressive new set of arterioles. Most surprisingly, an unexpected effect of VEGF165 was also the recruitment to the sites of its expression, of a vast set of mononuclear cells. These cells derived from the bone marrow and expressed a broad set of monocytic markers (CD45+, CD11b+); their presence correlated with the formation of arterioles and the maturation of the VEGF-induced vasculature. Strikingly, VEGF121 (the shorter form unable to bind NP-1) was, on the contrary, unable to induce maturation of the newly formed capillaries to mature arteries and to recruit cells, an observation that suggested that these monocytes might be essential in blood vessel maturation. We found that cell recruitment both in vitro and in vivo strictly depends on NP-1 and that Sema3A, a NP-1 activator, acts as a powerful recruiter of these cells. The expression of VEGF and Sema3A receptors by CD11b+ cells together with the VEGFR2 interaction with NP-1, indicate that these cells could be target of a peculiar VEGF and/or Sema3A induced signalling pathway. Taken together, these results prompted us to develop an in vitro model to unravel the signalling features elicited by the VEGF165, VEGF121 and Sema3A on endothelial cells. In particular, we wanted to dissect these pathways through a proteomic approach involving the detection of the phosphoproteome of endothelial cells expressing specific receptor after treatment with the various ligands of interest. Due to the large amount of growth factors required for this kind of experimentation, we first established a Baculovirus-based system to generate the recombinant factors. For this purpose, we engineered suitable plasmids encoding VEGF165, VEGF121 and Sema3A endowed of a cleavable histidine tag at the N-terminus. Once the recombinant protein expression conditions were set up, we moved to optimize protein purification by affinity chromatography, together with the removal of the tag. The functionality of baculovirus-expressed VEGF165 and VEGF121 was ascertained by the analysis of VEGF receptor phosphorylation, as well as proliferation assays on endothelial cells; a cell contraction assay confirmed that the recombinant Sema3A produced was as active as protein expressed in mammalian cells. To dissect the differential signalling induced by each of the recombinant factors, we explored their transduction pathways in endothelial cell lines expressing the main receptors (KDR and NP1). We set up the lysis conditions and bi-dimensional SDS-PAGE, together with the optimal phospho-proteome staining. The findings obtained, together with phospho-enrichment approaches, strongly supported that peculiar signalling pathways exist in endothelial cells, selectively triggered by VEGF165, VEGF121 and Sema3A, as demonstrated by the differential pattern of phosphorylated proteins induced by the recombinant proteins. Furthermore, we first demonstrated a close similarity in the phospho-tyrosine proteome induced by VEGF165 and Sema3A, at least in the cellular system examined.198

Use of Essential Oils to Inhibit Alicyclobacillus Acidoterrestris: A Short Overview of the Literature

Essential oils (EOs) are promising and friendly antimicrobials for the prolongation of the shelf life of many foods. They have been extensively used to inhibit spoiling and pathogenic microorganisms of many kinds of products like fruit juices and acidic drinks. Therefore, they could be used successfully to control the germination of spores of Alicyclobacillus acidoterrestris, that finds in these products an optimal environment for growth. This paper reports a brief overview of the literature available, focusing on the effects of EOs toward alicyclobacilli

Selection of Yeasts as Starter Cultures for Table Olives: A Step-by-Step Procedure

The selection of yeasts intended as starters for table olives is a complex process, including a characterization step at laboratory level and a validation at lab level and factory-scale. The characterization at lab level deals with the assessment of some technological traits (growth under different temperatures and at alkaline pHs, effect of salt, and for probiotic strains the resistance to preservatives), enzymatic activities, and some new functional properties (probiotic traits, production of vitamin B-complex, biological debittering). The paper reports on these traits, focusing both on their theoretical implications and lab protocols; moreover, there are some details on predictive microbiology for yeasts of table olives and on the use of multivariate approaches to select suitable starters

Use of Chitosan to Prolong Mozzarella Cheese Shelf Life

This study was undertaken to evaluate the feasibility of using chitosan, a natural antimicrobial substance, to improve the preservation of a very perishable cheese. The effectiveness of chitosan to inhibit the growth of spoilage microorganisms in Mozzarella cheese was studied during refrigerated storage. A lactic acid/chitosan solution was added directly to the starter used for Mozzarella cheese manufacturing. Mozzarella cheese samples were stored at 4 degrees C for about 10 d and microbial populations as well as the pH were monitored. Results demonstrated that chitosan inhibited the growth of some spoilage microorganisms such as coliforms, whereas it did not influence the growth of other microorganisms, such as Micrococcaceae, and lightly stimulated lactic acid bacteria

Selection of Promising Bacterial Strains as Potential Tools for the Bioremediation of Olive Mill Wastewater

The main objective of this paper was the selection of promising bacterial strains to be used as potential tools to remove phenols in olive mill wastewater (OMW) or in other food wastes. Therefore, 12 OMW samples were analyzed and 119 isolates were collected. After a preliminary screening on a medium containing vanillic and cinnamic acids, three isolates were selected to evaluate their viability in presence of different compounds (cinnamic, vanillic and caffeic acids, rutin, tyrosol and oleuropein) and a possible bioremediation effect. The isolates generally survived with phenols added and exerted a significant bioremediation activity in some samples (reduction of phenols by 20%). The last step was focused on the evaluation of the combined effects of pH, cinnamic and vanillic acids on the viability of a selected isolate (13M); the combination of the acids exerted a strong effect on the target, but alkaline pH played a protective role

Metabiotic effects of Fusarium spp. on Escherichia coli O157:H7 and Listeria monocytogenes on raw portioned tomatoes.

The metabiotic effects of Fusarium proliferatum, F. avenaceum, and F. oxysporum on Escherichia coli O157:H7 and Listeria monocytogenes in fresh tomatoes were investigated. Tomatoes were preinoculated with the molds and incubated at 15 degrees C for 7 days; then they were inoculated separately with the pathogens, packaged in air and modified atmosphere (5% O2, 30% CO2, and 65% N2), and stored at 4, 8, and 12 degrees C for 9 days. The cell loads of pathogens and lactic acid bacteria and the pH were evaluated periodically. The data were modeled through some different mathematical models to assess the shoulder length, i.e., the time before the beginning of the exponential death phase, the 1-log reduction time (s), and the pathogen death time (deltastand). The preinoculation of tomatoes with the molds enhanced the survival of E. coli O157:H7 by prolonging shoulder length and 8 parameters; this effect, however, was not observed for L. monocytogenes. pH values did not undergo significant changes within the storage time, and the lactic acid bacteria increased from 5 to 7 log CFU/g, without significant differences among the storage temperatures or the packaging atmospheres. The results of this research showed that the use of fresh tomatoes colonized by fusaria (even if the contamination is not visible) could increase significantly the risk of outbreaks due to some pathogens that could be on the surface of fruits and vegetables as a result of cross-contamination at home or incorrect postharvest operations

Modelling the Survival of Escherichia coli O157:H7 on Raw Portioned Tomatoes, Inoculated with Aspergillus fumigatus and Emericella nidulans

The metabiotic interactions occurring among two fungi (Aspergillus fumigatus and Emericella nidulans) and Escherichia coli O157:H7 on raw portioned tomatoes were studied. Tomatoes, preinoculated with the moulds and inoculated with the pathogen, were packaged in air and stored at 4, 8 and 12∘C for 9 days; pathogen cell number and pH were monitored throughout the storage and the data were modeled using three different equations (Geeraerd, Weibull, and modified Weibull), to assess the shoulder length, the 1-log reduction time, and the death time. Both A. fumigatus and E. nidulans increased the survival of E. coli O157:H7 through the prolongation of the shoulder length; in contrast, the death time was significantly increased. The results of this paper suggested that the metabiotic interactions aspergilli/E. coli O 157:H7 could be of public concern, as the consumption of tomatoes (or other fruits and vegetables) contaminated both by the moulds and the pathogen is a possible scenario

Immobilization of lysozyme on polyvinylalcohol films for active packaging applications.

A new technique for the immobilization of lysozyme onto the surface of polyvinylalcohol films is presented. The active compound was sprayed along with a suitable bonding agent onto the surface of the cross-linked polymeric matrix. Active compound release tests determined the amount of lysozyme immobilized on the film surface. With the use of Micrococcus lysodeikticus, the antimicrobial activity of the films was determined and the results correlated with the amount of immobilized lysozyme. This new technique was effective for immobilizing the enzyme, and the developed films were active against the test microorganism. Results were compared with those obtained with a different immobilizing technique, in which the active compound was bound into the bulk of the polymeric film. As expected, the surface-immobilized lysozyme films have a higher antimicrobial activity than bulk-bound films

Two Nonthermal Technologies for Food Safety and Quality-Ultrasound and High Pressure Homogenization: Effects on Microorganisms, Advances, and Possibilities: A Review.

Some nonthermal technologies have gained special interest as alternative approaches to thermal treatments. High pressure homogenization (HPH) and ultrasound (US) are two of the most promising approaches. They rely upon two different modes of action, although they share some mechanisms or ways of actions (mechanic burden against cells, cavitation and micronization, primary targets being the cell wall and the membrane, temperature and pressure playing important roles for their antimicrobial potential, and their effect on cells can be either positive or negative). HPH is generally used in milk and dairy products to break lipid micelles, whereas US is used for mixing and/or to obtain active compounds of food. HPH and US have been tested on pathogens and spoilers with different effects; thus, the main goal of this article is to describe how US and HPH act on biological systems, with a focus on antimicrobial activity, mode of action, positive effects, and equipment. The article is composed of three main parts: (i) an overview of US and HPH, with a focus on some results covered by other reviews (mode of action toward microorganisms and effect on enzymes) and some new data (positive effect and modulation of metabolism); (ii) a tentative approach for a comparative resistance of microorganisms; and (iii) future perspectives