Evidence on antimicrobial properties and mode of action of a chitosan obtained from crustacean exoskeletons on Pseudomonas syringae pv. tomato DC3000.

Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) causes bacterial speck of tomato, a widely spread disease that causes significant economical losses worldwide. It is representative of many bacterial plant diseases for which effective controls are still needed. Despite the antimicrobial properties of chitosan has been previously described in phytopathogenic fungi, its action on bacteria is still poorly explored. In this work, we report that the chitosan isolated from shrimp exoskeletons (70 kDa and 78 % deacetylation degree) exerts cell damage on Pto DC3000. Chitosan inhibited Pto DC3000 bacterial growth depending on its concentration, medium-pH, and presence of metal ion (Mg(+2)). Biochemical and cellular changes resulting in cell aggregation and impaired bacterial growth were also viewed. In vivo studies using fluorescent probes showed cell aggregation, increase in membrane permeability, and cell death, suggesting the chitosan antibacterial activity is due to its interaction as a polycation with Pto DC3000 membranes. Transmission electron microscopic analysis revealed that chitosan also caused morphological changes and damage in bacterial surfaces. Also, the disease incidence in tomato inoculated with Pto DC3000 was significantly reduced in chitosan pretreated seedlings, revealing a promising action of chitosan as nontoxic biopesticide in tomato plants. Indeed, a wider comprehensive knowledge of the mechanism of action of chitosan in phytopathogenic bacterial cells will increase the chances of its successful application to the control of spread disease in plants.

Chitooligosaccharides as novel ingredients of fermented foods.

Chitooligosaccharides (COSs) have been clinically evaluated for their immunostimulating effects after oral intake. Similar to dietary supplements, prebiotics and biopreservatives, these water-soluble bioactives are easily incorporated into dairy products and beverages. Notwithstanding, the use of COS in fermented foods would be limited by its antimicrobial properties. In order to study the interaction with yoghurts as a model of fermented food, the effects of COS on chemical composition, viability, morphology and metabolism of lactic acid bacteria, fatty acid profiles and conjugated linoleic acid (CLA) were assessed over 28 days and after chemical digestion. There were no significant differences between the nutritional composition of controls and yoghurts supplemented with concentrations up to 0.1% w/w of COS. However, the acidification of milk decreased at 0.5% (p < 0.05) and the formation of yoghurt failed at 3.0%, without affecting viable counts. Lipid hydrolysis of yoghurts supplemented with 0.1% COS was not affected by chemical digestion. No significant differences were found between CLA percentages of controls and supplemented yoghurts after digestion. Although the nutritional composition, fatty acids and viable counts were not significantly modified after COS supplementation, the present study shows that COS diminishes bacterial acidification at concentrations higher than 0.1%, thus limiting the amounts that could be added to yoghurt.

Evolution towards hormone independence of the MXT mouse mammary tumor is associated with a gradual change in its estrogen receptor molecular polymorphism.

Using a method based on [3H]tamoxifenaziridine ([3H]TAZ) labeling, sequential immunoadsorption with anti-ER monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and fluorography, we observed a striking change inthe estrogen receptor (ER) electrophoresis pattern of the transplantable MXT mouse mammary tumor. Early, ER "rich" tumors (approximately 100 fmol/mg prot) displayed classical cytosolic 67 and 50 KDa bands. These bands disappeared in favor of a "cytosolic" 35 KDa band during progression towards undifferentiated ER "poor" tumors (approximately 25 fmol/mg prot). Although we can not rule out that this 35 KDa peptide results from in vivo ER proteolysis, it seems unique in view of the following: 1. It is immunoadsorbed not only by an anti-ER monoclonal antibody (H-222) directed to the hormone-binding domain, but also by an anti-ER monoclonal antibody (H-226) which interacts with an epitope in the A/B region close to the DNA-binding domain and is mainly exposed under activation conditions. 2. It does not bind [3H]estradiol([3H]E2) and a tentative to restore its [3H]E2 binding capacity with calmodulin and ATP was unsuccessful. The observation of similar approximately 35 KDa ERs in the nuclear fraction of early tumor transplants and in control uterus suggests that this peptide is already in an activated form. Structural alterations of ER and/or associated "anchorage" nuclear proteins may beat the origin of its cytosolic localization. Moreover, the fact that the addition of calmodulin and ATP to late MXT transplants cytosols fails to increase their [3H]E2 binding capacity indicates that the low ER content of these tumors does not result from a deficiency in the phosphorylation status of the receptor.