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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.
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Músculos Abdominais/irrigação sanguínea , Adesão Celular , Células Endoteliais/efeitos dos fármacos , Leucócitos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Linhagem Celular , Técnicas de Cocultura , Células Endoteliais/enzimologia , Ativação Enzimática , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Fatores de TempoAssuntos
COVID-19 , SARS-CoV-2 , Humanos , Peru , Reinfecção , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Leukocyte recruitment is one of the most important cellular responses to tissue damage. Leukocyte extravasation is exquisitely regulated by mechanisms of selective leukocyte-endothelium recognition through adhesion proteins in the endothelial cell surface that recognize specific integrins in the activated leukocytes. A similar mechanism is used by tumor cells during metastasis to extravasate and form a secondary tumor. Nitric oxide (NO) has been classically described as an anti-inflammatory molecule that inhibits leukocyte adhesion. However, the evidence available shows also a positive role of NO in leukocyte adhesion. These apparent discrepancies might be explained by the different NO concentrations reached during the inflammatory response, which are highly modulated by the expression of different nitric oxide synthases, along the inflammatory response and by changes in their subcellular locations.
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Este estudio muestra los significados expresados por tres parejas del mismo género (cuatro mujeres y dos hombres) sobre la crianza de sus hijas e hijos, quienes tenían entre uno y tres años de edad. Desde un enfoque hermenéutico-interpretativo, analizamos entrevistas narrativas, resultando en las siguientes categorías: Importancia de verbalizar y explicar; Reglas, límites y consecuencias; Enseñanza y aprendizaje de valores y habilidades sociales; Mantener una relación cercana; Rasgos/atributos personales valorados (en las y los niños). Encontramos diversas funciones asumidas en su labor como educadores, que se vincularon de forma dinámica con lo que esperaban y valoraban en cuanto a aprendizaje y desarrollo. Fue importante para ellos/as complacer, dar gusto y satisfacer las necesidades de sus niñas y niños, sin que esto significara descuidar una alimentación saludable, establecer y mantener reglas y rutinas, así como enseñar habilidades acordes a su edad. Un significado compartido giró en torno al lenguaje y comunicación, pues para los adultos era importante explicar: desde rutinas, hasta verbalizar sentimientos, o hacer explícito su tipo de familia (tener dos padres o dos madres); siempre considerando lo que puede comprenderse a esa edad. También fue importante para los adultos que sus hijos e hijas fueran alegres y sintieran su amor, manteniendo una relación cercana: cálida, lúdica y afectuosa. Concluimos que estos padres gais y madres lesbianas significaron a la crianza como una tarea amorosa, ardua, permanente y dirigida a formar sujetos con derechos, con quienes se podía negociar y llegar a acuerdos, aun siendo niños y niñas
This study shows the meanings expressed by three Mexican same-gender couples (four women and two men) about the upbringing of their daughters and sons, who were aged between one and three years. From a hermeneutic-interpretative approach, we analysed narrative interviews, resulting in the following categories: Importance of verbalizing and expressing; Rules, limits and consequences; Teaching and learning values and social skills; Maintain a warm relationship; Personal traits/attributes valued (in children). We found that fathers and mothers played different roles in their work as educators, which were linked dynamically with what they expect and value in terms of learning and development. It was important for them to please, to indulge and to satisfy their children's needs, without this neglecting a healthy diet, establishing and maintaining rules and routines, as well as teaching values and skills according to their age. A shared meaning revolved around language and communication: for adults it was important explaining routines, verbalizing feelings, or making explicit their type of family (having two fathers or two mothers); always considering what can be understood at that age. It was also important for our participants that their sons and daughters felt happy and loved through a close, warm, playful and affectionate relationship. We conclude that these gay parents and lesbian mothers meant the upbringing as a loving, arduous and permanent task, committed to forming subjects with rights, with whom they could negotiate and reach agreements
Assuntos
Humanos , Características da Família , Poder Familiar , Idioma , Sexo , Ensino , Família , Núcleo Familiar , Comunicação , Emoções , Aprendizagem , Amor , MéxicoRESUMO
Rhizobia are nitrogen-fixing symbionts of plants. Their genomes frequently contain large plasmids, some of which are able to perform conjugative transfer. Plasmid pSfr64a from Sinorhizobium fredii GR64 is a conjugative plasmid, whose transfer is regulated by quorum sensing genes encoded by itself (traR64a, traI64a), in the symbiotic plasmid pSfr64b (traR64b, traI64b), and in the chromosome (ngrI). Also, transfer of pSfr64b requires quorum sensing elements encoded in this plasmid (traR64b, traI64b), in pSfr64a (traR64a), and in the chromosome (ngrI). These results demonstrate that pSfr64a and the symbiotic plasmid depend on each other for conjugative transfer. Plasmid pSfr64a from S. fredii GR64 is unable to transfer from the genomic background of Rhizobium etli CFN42. Our results show that the relaxase of pRet42a is able to process the oriT of pSfr64a, and viceversa, underlining their functional similarity and suggesting that in addition to the external signals, the "cytoplasmic environment" may pose a barrier to plasmid dissemination, even if the plasmids are functional in other aspects.
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Conjugação Genética , Plasmídeos/genética , Percepção de Quorum , Sinorhizobium fredii/fisiologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Mutação , Rhizobium/fisiologia , SimbioseRESUMO
We investigated whether short term high fructose intake may induce early hepatic dysfunction in rats and to test whether allopurinol treatment may have beneficial effects. Twenty male Sprague-Dawley rats received 20% fructose in drinking water (10 treated with allopurinol and 10 received vehicle) and 10 control rats received tap water. After 14 days, the hepatic response to an acute fructose load was evaluated, and in fasted animals, respirometry studies in freshly isolated mitochondria were performed. In fasting rats, we did not find differences in systemic or hepatic uric acid and triglyceride concentrations among the groups, but mitochondrial respiratory control rate was significantly decreased by high fructose feeding and correlated with a reduced expression of Complex I, as well as decreased aconitase-2 activity. On the other hand, in fructose fed rats, an acute fructose load increased systemic and hepatic uric acid, triglycerides and oxidative stress. Fructose feeding was also associated with fructokinase and xanthine oxidase overexpression and increased liver de novo lipogenesis program (fatty acid synthase (FAS) and cell death-inducing DFFA-like effector C (CIDEC) overexpression, ATP citrate lyase (ACL) and acetyl coA carboxylase (ACC) overactivity and decreased AMP-activated protein kinase (AMPk) and endothelial nitric oxide synthase (eNOS) activation). Allopurinol treatment prevented hepatic and systemic alterations. These data suggest that early treatment with xanthine oxidase inhibitors might provide a therapeutic advantage by delaying or even halting the progression of non-alcoholic fatty liver disease (NAFLD).
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Alopurinol/farmacologia , Frutose/antagonistas & inibidores , Lipogênese/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Administração Oral , Alopurinol/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Frutose/administração & dosagem , Frutose/farmacologia , Masculino , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Ratos , Ratos Sprague-DawleyRESUMO
La cuantificación de las hormonas esteroides en moluscos se lleva a cabo con diferentes técnicas utilizando la hemolinfa o gónadas. Lobatus gigas es un gasterópodo de interés comercial en el Caribe, indexado en CITES como una especie protegida. Los estudios hormonales de esta especie aún no están disponibles. Por lo tanto, el objetivo de este estudio fue determinar la presencia de las hormonas esteroides 17ß - estradiol y progesterona en L. gigas a través de un método no invasivo y comparar dos técnicas para su cuantificación. Cada dos meses en un período de un año, se recolectaron las heces de diez organismos en Xel-Há, Quintana Roo, México. Las muestras se analizaron con cromatografía líquida de alta resolución y EIA. Los valores más altos de ambas hormonas se presentaron de marzo a septiembre y disminuyeron en noviembre y enero. La comparación de las concentraciones obtenidas con HPLC y EIA, mostró que los resultados son similares para el 17β-estradiol (Passing - Bablock r = 0.673; -0.17 ng / ml). En contraste, los resultados de la progesterona con ambas técnicas no mostraron ajuste (Passing - Bablock r = 0.389; -1.43 ng / ml). Nuestros resultados sugieren que la técnica de EIA es adecuada para el estudio de hormonas en esta especie. El conocimiento generado permitirá monitorear y seleccionar organismos reproductores que se encuentren acondicionados en laboratorios y de esta manera no incidir en la recolecta de masas ovígeras silvestres.
Quantification of steroid hormones in molluscs is performed with different techniques, using the hemolymph or gonads. Lobatus gigas is a Caribbean gastropod of commercial interest indexed in CITES as a protected species. Hormonal studies of this species are as yet unavailable. The objective of this study is to determine the presence of the steroid hormones 17ß - estradiol and progesterone in L. gigas using a non-invasive method, and to compare two techniques for their quantification. Every two months over the course of one year, the feces of ten organisms were collected in Xel-Ha park Quintana Roo, México. The samples were analyzed with High resolution liquid chromatography and Enzyme-linked immunosorbent assays. The values of both hormones were highest during the months of March to September then decreased during November and January. Comparison of the concentrations obtained with HPLC and EIA, presented similar results for 17β-estradiol (Passing - Bablock r = 0.673; mean differences -0.17 ng / ml). In contrast, the progesterone results with both techniques showed no adjustment (Passing - Bablock r = 0.389; mean differences -1.43 ng / ml). Our results suggest that the enzyme-linked immunosorbent assay is suitable for the study of hormones in L. gigas. The knowledge generated will allow the monitoring and selection of breeding organisms that are conditioned in laboratories and thus will not affect the collection of wild egg masses.
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Hypercoagulable state is linked to cancer progression; however, the precise role of the coagulation cascade is poorly described. Herein, we examined the contribution of a hypercoagulative state through the administration of intravenous Coagulation Factor Xa (FXa), on the growth of solid human tumors and the experimental metastasis of the B16F10 melanoma in mouse models. FXa increased solid tumor volume and lung, liver, kidney and lymph node metastasis of tail-vein injected B16F10 cells. Concentrating on the metastasis model, upon coadministration of the anticoagulant Dalteparin, lung metastasis was significantly reduced, and no metastasis was observed in other organs. FXa did not directly alter proliferation, migration or invasion of cancer cells in vitro. Alternatively, FXa upon endothelial cells promoted cytoskeleton contraction, disrupted membrane VE-Cadherin pattern, heightened endothelial-hyperpermeability, increased inflammatory adhesion molecules and enhanced B16F10 adhesion under flow conditions. Microarray analysis of endothelial cells treated with FXa demonstrated elevated expression of inflammatory transcripts. Accordingly, FXa treatment increased immune cell infiltration in mouse lungs, an effect reduced by dalteparin. Taken together, our results suggest that FXa increases B16F10 metastasis via endothelial cell activation and enhanced cancer cell-endothelium adhesion advocating that the coagulation system is not merely a bystander in the process of cancer metastasis.
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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.
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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.
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Neoplasias da Mama/metabolismo , Metástase Neoplásica/fisiopatologia , Neovascularização Patológica/fisiopatologia , Proteínas/metabolismo , Animais , Endotélio Vascular/metabolismo , Humanos , Nitratos/metabolismo , Nitrosação , Proteínas/químicaRESUMO
Glioblastoma (GBM) is the brain tumor with the worst prognosis composed of a cell subpopulation called Glioblastoma Stem-like Cells (GSCs) responsible for tumor recurrence mediated by cell invasion. GSCs persist in a hypoxic microenvironment which promotes extracellular adenosine production and activation of the A3 Adenosine Receptor (A3AR), therefore, the aim of this study was to determine the role of extracellular adenosine and A3AR on GSCs invasion under hypoxia. GSCs were obtained from a U87MG cell line and primary cultures of GBM patients, and then incubated under normoxia or hypoxia. Gene expression was evaluated by RNAseq, RT-qPCR, and western blot. Cell migration was measured by spreading and transwell boyden chamber assays; cell invasion was evaluated by Matrigel-coated transwell, ex vivo brain slice, and in vivo xenograft assays. The contribution of A3AR on cell migration/invasion was evaluated using the A3AR antagonist, MRS1220. Extracellular adenosine production was higher under hypoxia than normoxia, mainly by the catalytic action of the prostatic acid phosphatase (PAP), promoting cell migration/invasion in a HIF-2-dependent process. A3AR blockade decreased cell migration/invasion and the expression of Epithelial-Mesenchymal Transition markers. In conclusion, high levels of extracellular adenosine production enhance cell migration/invasion of GSCs, through HIF-2/PAP-dependent activation of A3AR under hypoxia.
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Adenosina/metabolismo , Neoplasias Encefálicas/metabolismo , Movimento Celular , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptor A3 de Adenosina/metabolismo , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Células-Tronco Neoplásicas/patologia , Receptor A3 de Adenosina/genética , Transdução de Sinais , Células Tumorais Cultivadas , Hipóxia Tumoral , Microambiente TumoralRESUMO
The permeability of endothelial cells is regulated by the stability of the adherens junctions, which is highly sensitive to kinase-mediated phosphorylation and endothelial nitric oxide synthase (eNOS)-mediated S-nitrosylation of its protein components. Solid tumors can produce a variety of factors that stimulate these signaling pathways leading to endothelial cell hyperpermeability. This generates stromal conditions that facilitate tumoral growth and dissemination. Galectin-8 (Gal-8) is overexpressed in several carcinomas and has a variety of cellular effects that can contribute to tumor pathogenicity, including angiogenesis. Here we explored whether Gal-8 has also a role in endothelial permeability. We show that recombinant Gal-8 activates eNOS, induces S-nitrosylation of p120-catenin (p120) and dissociation of adherens junction, leading to hyperpermeability of the human endothelial cell line EAhy926. This pathway involves focal-adhesion kinase (FAK) activation downstream of eNOS as a requirement for eNOS-mediated p120 S-nitrosylation. This suggests a reciprocal, yet little understood, regulation of phosphorylation and S-nitrosylation events acting upon adherens junction permeability. In addition, glutathione S-transferase (GST)-Gal-8 pull-down experiments and function-blocking ß1-integrin antibodies point to ß1-integrins as cell surface components involved in Gal-8-induced hyperpermeability. Endogenous Gal-8 secreted from the breast cancer cell line MCF-7 has similar hyperpermeability and signaling effects. Furthermore, the mouse cremaster model system showed that Gal-8 also activates eNOS, induces S-nitrosylation of adherens junction components and is an effective hyperpermeability agent in vivo. These results add endothelial permeability regulation by S-nitrosylation as a new function of Gal-8 that can potentially contribute to the pathogenicity of tumors overexpressing this lectin.
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Junções Aderentes/metabolismo , Galectinas/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Glutationa Transferase , Humanos , Células MCF-7 , Masculino , Camundongos , Fosforilação/fisiologiaRESUMO
Leishmaniasis is a neglected tropical disease caused by different species of protozoan parasites of the genus Leishmania. Dogs have been proven as primary hosts of the parasite. Cases of cutaneous leishmaniasis in humans caused by Leishmania mexicana have been reported in Sinaloa; however, the vectors and hosts involved in the epidemiology of the parasite in northwestern Mexico are still unknown. Given the public health implications of this parasite's domestic hosts regarding the permanence and transmission of the disease to humans, the objective of the present study was to detect and determine the species of Leishmania that caused the first three cases of autochthonous canine leishmaniasis in the state of Sinaloa, Mexico. Three domestic dogs showing symptoms similar to canine leishmaniasis were identified, including chronic eye inflammation, corneal opacity, ocular exudate, emaciation and hyporexia. DNA was extracted from venous blood of the infected animals using a commercial kit. The internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was amplified by specific primers for Leishmania from the extracted DNA, and the PCR products were digested with the restriction enzyme HaeIII. In addition, PCR products were subjected to automated sequencing. Molecular analysis showed that the infecting species was L. mexicana. This is the first report of autochthonous canine leishmaniasis caused by L. mexicana in Sinaloa, Mexico. Further studies are required to identify the species that serve as vectors and other wild and domestic hosts of the parasite, as well as to determine if there are more species of Leishmania circulating in Sinaloa.
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Doenças do Cão/parasitologia , Leishmania mexicana/isolamento & purificação , Leishmaniose Cutânea/veterinária , Animais , Cães , Leishmania mexicana/genética , Leishmaniose Cutânea/parasitologia , Reação em Cadeia da PolimeraseRESUMO
The aim of this research was to explore the elements that configure the quality of care among three Mexican same-sex planned families: two female-parented families (through donor insemination) and a male-parented one (through adoption). The first family consisted of two mothers and a 3-year-old daughter; the second one had two mothers and a 1.5-year-old set of boy twins and the third family consisted of two fathers and a 2-year-old girl. It was assumed that Ainsworth's notions of quality of care organization are useful in order to understand caregiver-child attachment relationships, regardless of the parents' sexual orientation. A collective case study was selected due to the fact that these families shared their "unconventionality" (i.e., parents were not heterosexual) and the fact that they were planned, but each one constituted a particular case with a unique configuration. Four trained independent observers used the q-sort methodology (Maternal Behavior Q-Sort and Attachment Q-Sort) to describe parents' and children's behavior, respectively. The findings showed that parents were highly sensitive and all children used them as a secure base. To provide an in-depth examination of which elements configure the quality of care, a semi-structured interview with each parent was carried out. Through a thematic analysis, an over-arching theme named Affections and Emotions was identified, together with six subthemes: (1) Creating an affective environment; (2) Being available; (3) Acknowledging and expressing emotions; (4) Perceiving, interpreting and responding adequately to the child's real self; (5) Taking the child's perspective into account; and (6) Agreeing on roles and dividing the tasks. In order to showcase the particular configuration of gay parenting, the male-headed family narrative is reported in detail, because gay parents have been perceived as violating traditional gender roles as well as the hegemonic model of masculinity. The findings were consistent with the notion of quality of care as proposed by Ainsworth and her collaborators. The implications of the methodological device and research regarding same-sex planned families are discussed so as to understand the organization of the caregiving environment.
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We present here the high-quality complete genome sequences of eight strains of Rhizobium-nodulating Phaseolus vulgaris Comparative analyses showed that some of them belonged to different genomic and evolutionary lineages with common symbiotic properties. Two novel symbiotic plasmids (pSyms) with P. vulgaris specificity are reported here.
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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.
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Permeabilidade Capilar/fisiologia , Moléculas de Adesão Celular/metabolismo , Cisteína/metabolismo , Células Endoteliais/fisiologia , Proteínas dos Microfilamentos/metabolismo , Óxido Nítrico/metabolismo , Fosfoproteínas/metabolismo , Vasculite/metabolismo , Animais , Capilares , Bovinos , Células Cultivadas , Humanos , Mediadores da Inflamação/metabolismoRESUMO
Current knowledge about rhizobial diversity patterns in non-nodule habitats is scarce, limiting our understanding of basic aspects of rhizobial ecology like competitiveness for nodule occupancy and host effects on community structure. We used a combination of cultivation-dependent and independent approaches to analyse alpha and beta diversity patterns of Rhizobiaceae communities from a conserved seasonally dry tropical forest site in central Mexico and two nearby agricultural fields. Lineage-specific recA amplicon libraries were generated from soil DNA and their sequences compared with those from root surface and nodule isolates recovered in trapping experiments from two native Acacia species and two Phaseolus vulgaris cultivars. Rarefaction analyses revealed that Rhizobiaceae diversity in soils is larger than on root surfaces, and smallest in nodules. A 'rare biosphere'-like distribution of species was found in the three habitats. Multivariate statistical analyses demonstrated that the plant genus exerted a stronger influence than the land-usage regime on the diversity of rhizobia associated with hosts. Rhizobium etli was the dominant Rhizobiaceae found in the soil libraries. It dominated nodulation of Acacia spp. and predominately harboured symbiovar mimosae-like nodC genes. A novel Rhizobium lineage (Rsp1) dominated bean nodulation. Specialist and generalist genotypes for host nodulation were detected in both species.
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Biodiversidade , Phaseolus/microbiologia , Rhizobiaceae/isolamento & purificação , Nódulos Radiculares de Plantas/microbiologia , Microbiologia do Solo , Ecossistema , Genótipo , México , Phaseolus/fisiologia , Filogenia , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Rhizobiaceae/classificação , Rhizobiaceae/genética , Rhizobiaceae/fisiologia , Nódulos Radiculares de Plantas/fisiologia , Especificidade da Espécie , SimbioseRESUMO
Fructose in sweetened beverages (SB) increases the risk for metabolic and cardiorenal disorders, and these effects are in part mediated by a secondary increment in uric acid (UA). Rodents have an active uricase, thus requiring large doses of fructose to increase plasma UA and to induce metabolic syndrome and renal hemodynamic changes. We therefore hypothesized that the effects of fructose in rats might be enhanced in the setting of uricase inhibition. Four groups of male Sprague-Dawley rats (n = 7/group) were studied during 8 wk: water + vehicle (V), water + oxonic acid (OA; 750 mg/k BW), sweetened beverage (SB; 11% fructose-glucose combination) + V, and SB + OA. Systemic blood pressure, plasma UA, triglycerides (TG), glucose and insulin, glomerular hemodynamics, renal structural damage, renal cortex and liver UA, TG, markers of oxidative stress, mitDNA, fructokinase, and fatty liver synthase protein expressions were evaluated at the end of the experiment. Chronic hyperuricemia and SB induced features of the metabolic syndrome, including hypertension, hyperuricemia, hyperglycemia, and systemic and hepatic TG accumulation. OA alone also induced glomerular hypertension, and SB alone induced insulin resistance. SB + OA induced a combined phenotype including metabolic and renal alterations induced by SB or OA alone and in addition also acted synergistically on systemic and glomerular pressure, plasma glucose, hepatic TG, and oxidative stress. These findings explain why high concentrations of fructose are required to induce greater metabolic changes and renal disease in rats whereas humans, who lack uricase, appear to be much more sensitive to the effects of fructose.
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Bebidas/efeitos adversos , Frutose/efeitos adversos , Nefropatias/etiologia , Estresse Oxidativo/efeitos dos fármacos , Circulação Renal/efeitos dos fármacos , Urato Oxidase/metabolismo , Animais , Fígado Gorduroso/etiologia , Frutoquinases/metabolismo , Glucose/efeitos adversos , Hipertrofia/etiologia , Hiperuricemia/induzido quimicamente , Resistência à Insulina , Rim/efeitos dos fármacos , Rim/patologia , Nefropatias/enzimologia , Nefropatias/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Síndrome Metabólica/etiologia , Ácido Oxônico , Ratos , Ratos Sprague-Dawley , Urato Oxidase/antagonistas & inibidores , Ácido Úrico/metabolismo , Vasoconstrição/efeitos dos fármacosRESUMO
Nitric oxide (NO) is a key factor in inflammation as it regulates microvascular permeability, leukocyte adhesion and wound healing. This mini-review addresses mainly spatial and temporal requirements of NO regulatory mechanisms, with special emphasis on S-nitrosation. Endothelial nitric oxide synthase (eNOS)-derived NO induces S-nitrosation of p120 and ß-catenin, particularly in response to platelet-activating factor (PAF), and through traffic and interactions at the adherens junction promotes endothelial hyperpermeability. S-nitrosation is a determinant in vascular processes such as vasodilation and leukocyte-endothelium interactions. Interestingly, NO decreases leukocytes adhesion to endothelium, but the mechanisms are unknown. Advances in NO molecular biology and regulation may serve as a basis for the development of new therapeutic strategies in the treatment of diseases characterized by inflammation such as ischemia-reperfusion injury, stroke, cancer and atherosclerosis.
RESUMO
RATIONALE: Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of endothelial nitric oxide synthase (eNOS) to cytosol and stimulation of eNOS-derived nitric oxide signaling cascade. The mechanisms by which nitric oxide signaling regulates permeability at adherens junctions are still incompletely understood. OBJECTIVE: We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins. METHODS AND RESULTS: We measured PAF-stimulated SNO of ß-catenin and p120-catenin (p120) in 3 cell lines: ECV-eNOSGFP, EAhy926 (derived from human umbilical vein), and postcapillary venular endothelial cells (derived from bovine heart endothelium) and in the mouse cremaster muscle in vivo. SNO correlated with diminished abundance of ß-catenin and p120 at the adherens junction and with hyperpermeability. Tumor necrosis factor-α increased nitric oxide production and caused similar increase in SNO as PAF. To ascertain the importance of eNOS subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced SNO of ß-catenin and p120 and significantly diminished association between these proteins in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Inhibitors of nitric oxide production and of SNO blocked PAF-induced SNO and hyperpermeability, whereas inhibition of the cGMP pathway had no effect. Mass spectrometry analysis of purified p120 identified cysteine 579 as the main S-nitrosated residue in the region that putatively interacts with vascular endothelial-cadherin. CONCLUSIONS: Our results demonstrate that agonist-induced SNO contributes to junctional membrane protein changes that enhance endothelial permeability.