In vitro influence of photodynamic antimicrobial chemotherapy on staphylococcus aureus by using phenothiazines derivatives associated with laser/LED Light

ABSTRACT The aim of this study was to evaluate the effect of photodynamic antimicrobial chemotherapy (PACT) using phenothiazinium dyes -PTZ irradiated with red laser (ʎ660nm) or red-orange LED (ʎ632±2nm) on Staphylococcus aureus in vitro. triplicate tests were performed in 10 groups: control, Laser (L1 + P -and L2 + P -) bacterial suspensions were irradiated only with laser energy 2.4 and 4.8 J/cm 2 respectively, (Led1 + P -and Led2 + P -) irradiated only with LED energy 2.4 and 4.8 J/cm 2 respectively, (L1 + P + and L2 + P + ) irradiated with laser in the presence of 1μg/ml of photosensitizer, (Led1 + P + and Led2 + P + ) irradiated with LED in the presence of 1μg/ml of photosensitizer and finally (L -P + ) only in the presence of PTZ dye. Bactericidal effect of the PACT was assessed by counting colony-forming units. The results showed no significant difference on regards different energy densities on group PACT for both lights. PACT groups (L2 + P + and Led2 + P + ) compared to the Control showed significant reduction of CFUs. LED/Laser groups (L2 + P -and Led2 + P -) compared to control and PTZ groups showed also significant differences as groups LED/Laser (4.8J/cm 2 ) increased the average of CFUs. Although the results of this study have shown a reduction in average number of colonyforming units by the appropriate Laser or LED-dye treatment combination, it this topic requires further investigation

Evaluation of the Allergenicity Potential of TcPR-10 Protein from Theobroma cacao

Background: The pathogenesis related protein PR10 (TcPR-10), obtained from the Theobroma cacao-Moniliophthora perniciosa interaction library, presents antifungal activity against M. perniciosa and acts in vitro as a ribonuclease. However, despite its biotechnological potential, the TcPR-10 has the P-loop motif similar to those of some allergenic proteins such as Bet v 1 (Betula verrucosa) and Pru av 1 (Prunus avium). The insertion of mutations in this motif can produce proteins with reduced allergenic power. The objective of the present work was to evaluate the allergenic potential of the wild type and mutant recombinant TcPR-10 using bioinformatics tools and immunological assays. Methodology/Principal Findings: Mutant substitutions (T10P, I30V, H45S) were inserted in the TcPR-10 gene by sitedirected mutagenesis, cloned into pET28a and expressed in Escherichia coli BL21(DE3) cells. Changes in molecular surface caused by the mutant substitutions was evaluated by comparative protein modeling using the three-dimensional structure of the major cherry allergen, Pru av 1 as a template. The immunological assays were carried out in 8-12 week old female BALB/c mice. The mice were sensitized with the proteins (wild type and mutants) via subcutaneous and challenged intranasal for induction of allergic airway inflammation. Conclusions/Significance: We showed that the wild TcPR-10 protein has allergenic potential, whereas the insertion of mutations produced proteins with reduced capacity of IgE production and cellular infiltration in the lungs. On the other hand, in vitro assays show that the TcPR-10 mutants still present antifungal and ribonuclease activity against M. perniciosa RNA. In conclusion, the mutant proteins present less allergenic potential than the wild TcPR-10, without the loss of interesting biotechnological properties. (Résumé d'auteur

The identification of proteoglycans and glycosaminoglycans in archaeological human bones and teeth

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeleto ns. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k2 = 21±1 s−1) was much higher than the HNE deacylation step (k3 = 0.57±0.05 s−1). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k1 2.4-fold and reducing k−1 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k2 value, whereas the k3 value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs

Colorectal cancer desmoplastic reaction up-regulates collagen synthesis and restricts cancer cell invasion

During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. the accumulation of an extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread; therefore the study of desmoplasia is of vital importance. Stromal fibroblasts surrounding tumors are activated to myofibroblasts and become the primary producers of ECM during desmoplasia. the composition, density and organization of this ECM accumulation play a major role on the influence desmoplasia has upon tumor cells. in this study, we analyzed desmoplasia in vivo in human colorectal carcinoma tissue, detecting an up-regulation of collagen I, collagen IV and collagen V in human colorectal cancer desmoplastic reaction. These components were then analyzed in vitro co-cultivating colorectal cancer cells (Caco-2 and HCT116) and fibroblasts utilizing various co-culture techniques. Our findings demonstrate that direct cell-cell contact between fibroblasts and colorectal cancer cells evokes an increase in ECM density, composed of unorganized collagens (I, III, IV and V) and proteoglycans (biglycan, fibromodulin, perlecan and versican). the desmoplastic collagen fibers were thick, with an altered orientation, as well as deposited as bundles. This increased ECM density inhibited the migration and invasion of the colorectal tumor cells in both 2D and 3D co-culture systems. Therefore this study sheds light on a possible restricting role desmoplasia could play in colorectal cancer invasion.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Bioquim, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04044020 São Paulo, BrazilFac Med ABC, Dept Pathol, Santo Andre, SP, BrazilFac Med ABC, Dept Gastrosurg, Santo Andre, SP, BrazilUniv Wisconsin, Dept Pathol & Lab Med, Madison, WI 53792 USAUniversidade Federal de São Paulo, Dept Bioquim, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04044020 São Paulo, BrazilFAPESP: 2007/59801-1Web of Scienc

Proteoglycans extracted from ancient bones and teeth.

<p>PGs were extracted from various types of archaeological human bone samples (femur, humerus, radius, tibia and ulna) identified as juvenile, adolescent or adult, and also archaeological adult teeth and a modern wisdom tooth. Extracted PGs were analyzed by agarose gel electrophoresis in PDA buffer, and stained by amide black followed by toluidine blue. Proteins are stained in blue and GAG chains in purple. CS: chondroitin sulfate; DS: dermatan sulfate; HS: heparan sulfate; St: standard (CS, DS and HS extracted from shark cartilage).</p

Disaccharide content of the CS population extracted from archaeological human adolescent femur was characterized by strong anion exchange liquid chromatography.

<p>(A) C6S standard profile; (B) C4S/C6S Standard profile; and (C) profile of CS extracted from archaeological human adolescent femur. (a) Peak representing the C6S disaccharide; and (b) peak representing the C4S disaccharide.</p