RESUMEN
Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [35 S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [35 S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment.
Asunto(s)
Calgranulina B/metabolismo , Epitelio/metabolismo , Oviductos/metabolismo , Capacitación Espermática , Interacciones Espermatozoide-Óvulo , Reacción Acrosómica/efectos de los fármacos , Adulto , Animales , Sitios de Unión , Epitelio/efectos de los fármacos , Femenino , Humanos , Masculino , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oviductos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes/farmacología , Semen/efectos de los fármacos , Semen/metabolismo , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/metabolismoRESUMEN
Lichen planus (LP) is a chronic inflammatory mucocutaneous disease. The inflammatory status of LP may be related to S100A8 (myeloid-related protein 8; MRP8) activation of cytotoxic cells. The aims of this study were to evaluate S100A8 expression in skin lesions and the in vitro effects of S100A8 on CD8+ T cells and natural killer (NK) cells in LP. Increased levels of S100A8/S100A9 were detected in the skin lesions as well as in the sera of subjects with LP. S100A8 expression induced an increased cytotoxic response by peripheral blood CD8+CD107a+ T cells as well as by NK CD56bright cells in patients with LP. Increased expression of interleukin (IL)-1ß, tumour necrosis factor (TNF) and IL-6 in the CD8+ T cells of patients with LP was induced by S100A8, in contrast to the control group that produced IL- 10 and interferon (IFN) type I genes. These data suggest that, in individuals with LP, S100A8 may exert distinct immunomodulatory and cytotoxicity functions.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Calgranulina A/metabolismo , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Liquen Plano/metabolismo , Piel/metabolismo , Antígeno CD56/inmunología , Antígeno CD56/metabolismo , Linfocitos T CD8-positivos/inmunología , Calgranulina A/inmunología , Calgranulina B/inmunología , Calgranulina B/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Humanos , Mediadores de Inflamación/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Liquen Plano/genética , Liquen Plano/inmunología , Liquen Plano/patología , Proteína 1 de la Membrana Asociada a los Lisosomas/inmunología , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Transducción de Señal , Piel/inmunología , Piel/patología , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVES: We evaluated whether pancreatic main duct fluid can provide protein biomarkers with prognostic value. METHODS: Mass spectrometry proteomics was applied to as little as 20µL of fluid collected at the time of tumor surgical resection. Biomarker proteins identified for 27 patients were correlated with clinical outcomes. RESULTS: Thirteen patients had pancreatic ductal adenocarcinomas, 4 had intraductal papillary mucinous neoplasm with in situ adenocarcinoma, 5 had ampullary adenocarcinomas, 2 had intraductal papillary mucinous neoplasms, and 3 had benign diseases. In pathologic stage II or higher pancreatic ductal adenocarcinoma, moderate or high expression of S100A8 or S100A9 proteins was associated with a median disease recurrence-free survival of 5.8 months compared with 17.3 months in patients with low expression (P = 0.002). Median overall survival was 12.6 versus 27 months for patients with moderate to high versus low S100A8 and A9 expression (P = 0.02). CONCLUSIONS: This analysis suggests distinct proteomic signatures for pancreatic cancer. Patients in our study with elevated levels of S100A8 or A9 in the ductal fluid, a near absence of pancreatic enzymes, and high levels of mucins were found to have significantly worse prognosis. Although further validation is needed to corroborate these findings, analysis of pancreatic ductal fluid is a promising tool for identifying biomarkers of interest.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Mucinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patología , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patología , Humanos , Estimación de Kaplan-Meier , Espectrometría de Masas/métodos , Recurrencia Local de Neoplasia , Jugo Pancreático/metabolismo , Neoplasias Pancreáticas/patología , Pronóstico , Proteoma/metabolismo , Proteómica/métodosRESUMEN
The aim of this study was to identify feature genes that are associated with hereditary hemochromatosis (HHC; iron overload) in cardiac and skeletal muscle of mice. First, the expression profile GSE9726 was downloaded from Gene Expression Omnibus database which included 12 samples. Then the differentially expressed genes (DEGs) were identified by R language. Furthermore, the KUPS software was used to identify relationships in interactions among common DEGs in the cardiac and skeletal muscles. We then used the EASE software to obtain enriched pathways in a gene interaction network. Finally, we used the plugins of the Cytoscape software, i.e., Mcode and Bingo, to conduct module analysis. By comparing diseased and normal tissue samples, 5 and 6 genes in the cardiac and skeletal muscles, respectively, were identified as DEGs. We observed that the S100a8 and S100a9 genes were common DEGs in both tissues examined. In addition, we constructed an interaction network with common DEGs and their interacting components, and identified S100a8 and S100a9 as being associated with immune responses. In view of the relationship between the early stage of myelodysplastic syndrome and the immune system, we hypothesize that the expression of the S100a8 and S100a9 genes is a feature that can be used for diagnosis during the early stage of the myelodysplastic syndrome and that the 2 genes could be used as targets in treating this disease.
Asunto(s)
Calgranulina A/metabolismo , Calgranulina B/metabolismo , Hemocromatosis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Calgranulina A/genética , Calgranulina B/genética , Estudios de Casos y Controles , Redes Reguladoras de Genes , Estudios de Asociación Genética , Hemocromatosis/metabolismo , Humanos , Ratones , Músculo Esquelético/metabolismo , Miocardio/metabolismo , TranscriptomaRESUMEN
Proteinase-activated receptors (PAR) are widely recognized for their modulatory properties in inflammatory and immune responses; however, their direct role on phagocyte effector functions remains unknown. S100A9, a protein secreted during inflammatory responses, deactivates activated peritoneal macrophages, and its C-terminal portion inhibits spreading and phagocytosis of adherent peritoneal cells. Herein, the effect of PAR1 and PAR2 agonists was investigated on spreading and phagocytosis by adherent peritoneal cells, as well as the ability of murine C-terminal of S100A9 peptide (mS100A9p) to modulate this effect. Adherent peritoneal cells obtained from mouse abdominal cavity were incubated with PAR1 and PAR2 agonists and spreading and phagocytosis of Candida albicans particles were evaluated. PAR1 agonists increased both the spreading and the phagocytic activity, but PAR2 agonists only increased the spreading index. mS100A9p reverted both the increased spreading and phagocytosis induced by PAR1 agonists, but no interference in the increased spreading induced by PAR2 agonists was noticed. The shorter homologue peptide to the C-terminal of mS100A9p, corresponding to the H(92)-E(97) region, also reverted the increased spreading and phagocytosis induced by PAR1 agonists. These findings show that proteinase-activated receptors have an important role for spreading and phagocytosis of adherent peritoneal cells, and that the peptide corresponding to the C-terminal of S100A9 protein is a remarkable candidate for use as a novel compound to modulate PAR1 function.
Asunto(s)
Calgranulina B/química , Forma de la Célula/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Peritoneo/citología , Fagocitosis/efectos de los fármacos , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calgranulina B/metabolismo , Bovinos , Adhesión Celular , Tamaño de la Célula/efectos de los fármacos , Masculino , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Peritoneo/metabolismo , Receptor PAR-1/agonistas , Receptor PAR-1/química , Receptor PAR-2/agonistas , Receptor PAR-2/química , Homología de Secuencia de Aminoácido , Zinc/metabolismoRESUMEN
Calcium-binding protein S100A9 (MRP-14) induces antinociceptive effect in an experimental model of painful sensibility and participates of antinociception observed during neutrophilic peritonitis induced by glycogen or carrageenan in mice. In this study, the direct antinociceptive role of the protein S100A9 in neutrophilic cell-free exudates obtained of mice injected with glycogen was investigated. Mice were intraperitoneally injected with a glycogen solution, and after 4, 8, 24, and 48 hours, either the pattern of cell migration of the peritoneal exudate or the nociceptive response of animals was evaluated. The glycogen-induced neutrophilic peritonitis evoked antinociception 4 and 8 hours after inoculation of the irritant. Peritoneal cell-free exudates, collected in different times after the irritant injection, were transferred to naive animals which were submitted to the nociceptive test. The transference of exudates also induced antinociceptive effect, and neutralization of S100A9 activity by anti-S100A9 monoclonal antibody totally reverted this response. This effect was not observed when experiments were made 24 or 48 hours after glycogen injection. These results clearly indicate that S100A9 is secreted during glycogen-induced neutrophilic peritonitis, and that this protein is responsible by antinociception observed in the initial phase of inflammatory reaction. Thus, these data reinforce the hypothesis that the calcium-binding protein S100A9 participates of the endogenous control of inflammatory pain.
Asunto(s)
Analgésicos/farmacología , Calgranulina B/farmacología , Neutrófilos/metabolismo , Peritonitis/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Calgranulina B/inmunología , Calgranulina B/metabolismo , Movimiento Celular/efectos de los fármacos , Sistema Libre de Células/química , Glucógeno/administración & dosificación , Glucógeno/toxicidad , Leucocitos/citología , Leucocitos/efectos de los fármacos , Masculino , Ratones , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Dimensión del Dolor/métodos , Peritonitis/inducido químicamente , Factores de TiempoRESUMEN
BACKGROUND AND PURPOSE: S100A9 protein induces anti-nociception in rodents, in different experimental models of inflammatory pain. Herein, we investigated the effects of a fragment of the C-terminus of S100A9 (mS100A9p), on the hyperalgesia induced by serine proteases, through the activation of protease-activated receptor-2 (PAR2). EXPERIMENTAL APPROACH: Mechanical and thermal hyperalgesia induced by PAR2 agonists (SLIGRL-NH2 and trypsin) was measured in rats submitted to the paw pressure or plantar tests, and Egr-1 expression was determined by immunohistochemistry in rat spinal cord dorsal horn. Calcium flux in human embryonic kidney cells (HEK), which naturally express PAR2, in Kirsten virus-transformed kidney cells, transfected (KNRK-PAR2) or not (KNRK) with PAR2, and in mouse dorsal root ganglia neurons (DRG) was measured by fluorimetric methods. KEY RESULTS: mS100A9p inhibited mechanical hyperalgesia induced by trypsin, without modifying its enzymatic activity. Mechanical and thermal hyperalgesia induced by SLIGRL-NH2 were inhibited by mS100A9p. SLIGRL-NH2 enhanced Egr-1 expression, a marker of nociceptor activation, and this effect was inhibited by concomitant treatment with mS100A9p. mS100A9p inhibited calcium mobilization in DRG neurons in response to the PAR2 agonists trypsin and SLIGRL-NH2, but also in response to capsaicin and bradykinin, suggesting a direct effect of mS100A9 on sensory neurons. No effect on the calcium flux induced by trypsin or SLIGRL in HEK cells or KNRK-PAR2 cells was observed. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that mS100A9p interferes with mechanisms involved in nociception and hyperalgesia and modulates, possibly directly on sensory neurons, the PAR2-induced nociceptive signal.
Asunto(s)
Analgésicos/metabolismo , Calgranulina B/metabolismo , Hiperalgesia/prevención & control , Analgésicos/farmacología , Animales , Calcio/metabolismo , Calgranulina B/farmacología , Línea Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Humanos , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Nociceptores/efectos de los fármacos , Nociceptores/metabolismo , Oligopéptidos , Dimensión del Dolor , Umbral del Dolor/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/metabolismo , Ratas , Ratas Wistar , Receptor PAR-2/agonistas , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Sustancia P/metabolismo , Transfección , TripsinaRESUMEN
OBJECTIVE AND DESIGN: In the present study, the effect of a synthetic peptide (H(92)-G(102)) identical to the C-terminus of murine S100A9 (mS100A9p) was investigated on adherent peritoneal cell function. MATERIALS AND METHODS: For in vitro assays, peritoneal cells were obtained from the abdominal cavity of mice and incubated, with the different concentrations of mS100A9p, for 1 h, and then their spreading and phagocytosis activities were evaluated. For ex-vivo assays, cells obtained from animals treated for 1 h with the peptide were submitted to the mannose-receptor phagocytosis assay. Shorter homologue peptides to the C-terminus of mS100A9p were also evaluated on in vitro phagocytosis assays of Candida albicans particles. RESULTS: mS100A9p reduced both the spreading index and phagocytic activity, in vitro and ex-vivo, independent of the receptor evaluated. The homologue peptide corresponding to the H(92)-E(97) region of mS100A9p, the zinc-binding motif, was responsible for such an effect. CONCLUSION: These results suggest a modulator effect of the C-terminus of S100A9 protein on the function of adherent peritoneal cells.