RESUMO

Nitric oxide (NO) is a key factor in inflammation. Endothelial nitric oxide synthase (eNOS), whose activity increases after stimulation with proinflammatory cytokines, produces NO in endothelium. NO activates two pathways: 1) soluble guanylate cyclase-protein kinase G and 2) S-nitrosylation (NO-induced modification of free-thiol cysteines in proteins). S-nitrosylation affects phosphorylation, localization, and protein interactions. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce a fast leukocyte adhesion, which suggests that NO might positively regulate leukocyte adhesion. We tested the hypothesis that eNOS-induced NO promotes leukocyte adhesion through the S-nitrosylation pathway. We stimulated leukocyte adhesion to endothelium in vitro and in vivo using tumor necrosis factor-α (TNF-α) as proinflammatory agonist. ICAM-1 changes were evaluated by immunofluorescence, subcellular fractionation, immunoprecipitation, and fluorescence recovery after photobleaching (FRAP). Protein kinase Cζ (PKCζ) activity and S-nitrosylation were evaluated by Western blot analysis and biotin switch method, respectively. TNF-α, at short times of stimulation, activated the eNOS S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-α-induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-α-induced NO also produced S-nitrosylation and phosphorylation of PKCζ, association of PKCζ with ICAM-1, and ICAM-1 phosphorylation. The inhibition of PKCζ blocked leukocyte adhesion induced by TNF-α. Mass spectrometry analysis of purified PKCζ identified cysteine 503 as the only S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS S-nitrosylation-dependent mechanism that induces leukocyte adhesion and suggests that S-nitrosylation of PKCζ may be an important regulatory step in early leukocyte adhesion in inflammation.NEW & NOTEWORTHY Contrary to the well-established inhibitory role of NO in leukocyte adhesion, we demonstrate a positive role of nitric oxide in this process. We demonstrate that NO induced by eNOS after TNF-α treatment induces early leukocyte adhesion activating the S-nitrosylation pathway. Our data suggest that PKCζ S-nitrosylation may be a key step in this process.

Assuntos

RESUMO

Glioblastoma is a highly aggressive brain tumor, characterized by the formation of dysfunctional blood vessels and a permeable endothelial barrier. S-nitrosylation, a post-translational modification, has been identified as a regulator of endothelial function. In this work we explored whether S-nitrosylation induced by glioblastoma tumors regulates the endothelial function. As proof of concept, we observed that S-nitrosylation is present in the tumoral microenvironment of glioblastoma in two different animal models. Subsequently, we measured S nitrosylation and microvascular permeability in EAhy296 endothelial cells and in cremaster muscle. In vitro, conditioned medium from the human glioblastoma cell line U87 activates endothelial nitric oxide synthase, causes VE-cadherin- S-nitrosylation and induces hyperpermeability. Blocking Interleukin-8 (IL-8) in the conditioned medium inhibited S-nitrosylation of VE-cadherin and hyperpermeability. Recombinant IL-8 increased endothelial permeability by activating eNOS, S-nitrosylation of VE-cadherin and p120, internalization of VE-cadherin and disassembly of adherens junctions. In vivo, IL-8 induced S-nitrosylation of VE-cadherin and p120 and conditioned medium from U87 cells caused hyperpermeability in the mouse cremaster muscle. We conclude that eNOS signaling induced by glioma cells-secreted IL-8 regulates endothelial barrier function in the context of glioblastoma involving S-nitrosylation of VE-cadherin and p120. Our results suggest that inhibiting S-nitrosylation may be an effective way to control and/or block damage to the endothelial barrier and prevent cancer progression.

RESUMO

S-nitrosylation, the modification by nitric oxide of free sulfhydryl groups in cysteines, has become an important regulatory mechanism in carcinogenesis and metastasis. S-nitrosylation of targets in tumor cells contributes to metastasis regulating epithelial to mesenchymal transition, migration and invasion. In the tumor environment, the role of S-nitrosylation in endothelium has not been addressed; however, the evidence points out that S-nitrosylation of endothelial proteins may regulate angiogenesis, adhesion of tumor cells to the endothelium, intra and extravasation of tumor cells and contribute to metastasis.

Assuntos

RESUMO

We tested the hypothesis that platelet-activating factor (PAF) induces S-nitrosylation of vasodilator-stimulated phosphoprotein (VASP) as a mechanism to reduce microvascular endothelial barrier integrity and stimulate hyperpermeability. PAF elevated S-nitrosylation of VASP above baseline levels in different endothelial cells and caused hyperpermeability. To ascertain the importance of endothelial nitric oxide synthase (eNOS) subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced S-nitrosylation of VASP in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Reconstitution of VASP knockout myocardial endothelial cells with cysteine mutants of VASP demonstrated that S-nitrosylation of cysteine 64 is associated with PAF-induced hyperpermeability. We propose that regulation of VASP contributes to endothelial cell barrier integrity and to the onset of hyperpermeability. S-nitrosylation of VASP inhibits its function in barrier integrity and leads to endothelial monolayer hyperpermeability in response to PAF, a representative proinflammatory agonist.NEW & NOTEWORTHY Here, we demonstrate that S-nitrosylation of vasodilator-stimulated phosphoprotein (VASP) on C64 is a mechanism for the onset of platelet-activating factor-induced hyperpermeability. Our results reveal a dual role of VASP in endothelial permeability. In addition to its well-documented function in barrier integrity, we show that S-nitrosylation of VASP contributes to the onset of endothelial permeability.

Assuntos

RESUMO

The present work shows the characterization of Phaseolus acutifolius variety latifolius, on which little research has been published, and provides detailed information on the corresponding lectin. This protein was purified from a semi-domesticated line of white tepary beans from Sonora, Mexico, by precipitation of the aqueous extract with ammonium sulfate, followed by affinity chromatography on an immobilized fetuin matrix. MALDI TOF analysis of Phaseolus acutifolius agglutinin (PAA) showed that this lectin is composed of monomers with molecular weights ranging between 28 and 31 kDa. At high salt concentrations, PAA forms a dimer of 63 kDa, but at low salt concentrations, the subunits form a tetramer. Analysis of PAA on 2D-PAGE showed that there are mainly three types of subunits with isoelectric points of 4.2, 4.4, and 4.5. The partial sequence obtained by LC/MS/MS of tryptic fragments from the PAA subunits showed 90-100% identity with subunits from genus Phaseolus lectins in previous reports. The tepary bean lectin showed lower hemagglutination activity than Phaseolus vulgaris hemagglutinin (PHA-E) toward trypsinized human A and O type erythrocytes. The hemagglutination activity was inhibited by N-glycans from glycoproteins. Affinity chromatography with the immobilized PAA showed a high affinity to glycopeptides from thyroglobulin, which also has N-glycans with a high content of N-acetylglucosamine. PAA showed less mitogenic activity toward human lymphocytes than PHA-L and Con A. The cytotoxicity of PAA was determined by employing three clones of the 3T3 cell line, demonstrating variability among the clones as follows: T4 (DI50 51.5 µg/mL); J20 (DI50 275 µg/mL), and N5 (DI50 72.5 µg/mL).

Assuntos

RESUMO

El presente trabajo es una revisión de la literatura, mas reciente sobre Sistemas Adhesivos a Esmalte y Dentina. Su objetivo principal es exponer a los sistemas adhesivos no como un proceso mecánico; sino como el resultado del conocimiento e investigación científica. De esta forma el empleo de estos materiales se orienta a los sustratos adhesivos y a las propiedades (químico-mecánicas) de los elementos que componen el sistema, cualquiera que sea su presentación comercial. En cuanto a Esmalte y Dentina se puede decir, que están sujetos a variables Extrínsecas e Intrínsecas, las cuales de no ser controladas adecuadamente, comprometen la longevidad de las restauraciones, ya que se encuentran inmersos en un medio húmedo que favorece el fenómeno de hidrólisis; que visto en una línea de tiempo se puede traducir en nano filtración y consecuentemente en sensibilidad post-operatoria. El éxito de los Sistemas Adhesivos, radica en: Crear una capa hibrida de calidad, que promueva una Interfase Sellada, que no dependa de la longitud de los tapones de resina, sino del intimo contacto de ambos sustratos, favorecido directamente por un sustrato con alta energía superficial y un adhesivo con baja tensión superficial, capaz de humectar la red de fibras colágenas.

This work is a review of the most recent literature on Enamel and Dentin Adhesive Systems. Its main objective is to expose adhesive systems not as a mechanical process; but as the result of scientific knowledge and research. In this way, the use of these materials is oriented towards the adhesive substrates and the (chemical-mechanical) properties of the elements that make up the system, whatever their commercial presentation. Regarding Enamel and Dentin, it can be said that they are subject to Extrinsic and Intrinsic variables, which, if not adequately controlled, compromise the longevity of the restorations, since they are immersed in a humid environment that favors the hydrolysis phenomenon; that seen in a timeline can be translated into nanofiltration and consequently in post-operative sensitivity. The success of Adhesive Systems lies in: Creating a quality hybrid layer that promotes a Sealed Interface, which does not depend on the length of the resin plugs, but on the intimate contact of both substrates, directly favored by a substrate with high surface energy and an adhesive with low surface tension, capable of wetting the network of collagen fibers.

Assuntos

RESUMO

Smith and naylor's techniques has been successfully used by many authors around the world to recover inorganic particles from the lungs. In this short report we compare the results of counting inorganic particles recovered from human lung tissue by two different methods: Smith and Naylor's technique and a modification to it. We used ferruginous bodies (FB) as markers for inorganic particles and we compared the results of the FB counts in both methods. In the traditional technique the interface formed in the 1:1 ethanol-chloroform mixture is discharged and FB are counted only in the formed pellet. Our modification also quantified FB in the interface fraction. Post.mortem samples from each of the lung lobes were taken from 22 individuals, totalling 198 sample. Each samples. Each samples was digested in liquid commercial bleach and processed by both techniques. our results showed that the modified technique was more sensitive in the detection of FB than the traditional method, with 16 out of 22 individuals showing positive identification against 14 out of 22, respectively. Furthermore, the modified technique proved not only to be nore sensitive, but also almost twice as accurate: 49 FB/gvs. 26.7 FB/g of dry tissue

Assuntos