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The aim of this study was to investigate the roles of CD4+ T cells and transforming growth factor beta (TGFß1) in the pathological process of valvular hyperblastosis and fibrosis of patients with rheumatic heart disease (RHD). A total of 151 patients were enrolled, among whom, 78 patients were with RHD, and 73 were age and gender matched RHD negative patients. Blood samples and valve specimens were collected for analysis. Pathological changes and collagen fibers contents of valves were analyzed using HE and Masson staining. Percentage of peripheral blood CD4+ T cells was tested through flow cytometry. TGFß1 level in serum were identified by ELISA. CD4+ T cells infiltration and expression of TGFß1, p-p38, p-JNK, p-ERK in valves were detected by immunohistochemistry. The mRNA and protein levels of p38, JNK, ERK, TGFß1, I-collagen and α-SMA were detected by qRT-PCR and western blotting, respectively. The heart valve tissues of RHD patients showed higher degrees of fibrosis, calcification and lymphocytes infiltration, which were mainly CD4+ T cells. In addition, compared with control group, RHD patients had more total CD4+ T cells in peripheral blood and valve tissues. Expression of TGFß1, phosphorylation of JNK and p38, and synthesis of I-collagen in valve tissues of RHD patients were also significantly increased. Furthermore, we found a strong positive correlation between TGFß1 expression and phosphorylation of JNK and p38. CD4+ T cells, and fibrogenic cytokine TGFß1, which activate the intracellular MAPK signaling pathway may participate in the fibrosis of heart valve in RHD patients.
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Enfermedades de las Válvulas Cardíacas/genética , Estenosis de la Válvula Mitral/genética , Cardiopatía Reumática/genética , Factor de Crecimiento Transformador beta1/genética , Adulto , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/fisiología , Quinasas MAP Reguladas por Señal Extracelular/sangre , Quinasas MAP Reguladas por Señal Extracelular/genética , Femenino , Fibrosis/sangre , Fibrosis/genética , Fibrosis/patología , Regulación de la Expresión Génica/genética , Enfermedades de las Válvulas Cardíacas/sangre , Enfermedades de las Válvulas Cardíacas/patología , Humanos , MAP Quinasa Quinasa 4/sangre , MAP Quinasa Quinasa 4/genética , Sistema de Señalización de MAP Quinasas/genética , Masculino , Persona de Mediana Edad , Estenosis de la Válvula Mitral/sangre , Estenosis de la Válvula Mitral/patología , Cardiopatía Reumática/sangre , Cardiopatía Reumática/patología , Factor de Crecimiento Transformador beta1/sangre , Proteínas Quinasas p38 Activadas por Mitógenos/sangre , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Hypothyroidism is an endocrine disorder due to decreased thyroid hormone production. This endocrine disorder significantly affects the menstrual cycle and fertility. The aim of this present study was to assess the efficacy of Panax ginseng, one of traditional Chinese medicine, in ameliorating the gonadal hormonal dysfunction and lowering oxidative stress accompanied with hypothyroidism in adult female albino rats. After confirming regularity of the oestrus cycle in the female rats in this study, hypothyroidism was induced by using daily 5.0 mg kg-1 oral dose of Neo-mercazole. The hypothyroid rats were randomly grouped into two groups; hypothyroid group (H): did not received any treatment, group II (H+G) was treated with Panax ginseng extract for one months after hypothyroidism induction. Another two groups were included in the study, a negative control group (Euthyroid group) and a positive control group; received Panax ginseng extract only. Hypothyroidism resulted in irregularity of oestrus cycle accompanied with decrease in luteinizing hormone (LH), follicular stimulating hormone (FSH) and estradiol (E2), while prolactin (PRL), progesterone (P) and testosterone (T) hormone were significantly elevated. Hypothyroidism elevated capsae-3 and 8OH-deoxy guanosine expression and increased secretion of corticosterone and ERK1/2. This study showed that Panax ginseng improved hypothyroid-induced deterioration in trophic and gonadal hormones through free radicals' scavenger.
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Fertilidad , Hipotiroidismo/complicaciones , Infertilidad Femenina/prevención & control , Panax/química , Animales , Biomarcadores/sangre , Corticosterona/sangre , Ciclo Estral/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/sangre , Femenino , Hidrocortisona/sangre , Hipotiroidismo/sangre , Infertilidad Femenina/sangre , Infertilidad Femenina/tratamiento farmacológico , Infertilidad Femenina/patología , Estrés Oxidativo/efectos de los fármacos , Fitoterapia , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Ratas Wistar , Hormonas Tiroideas/sangreRESUMEN
Purpose TAK-733, an investigational, selective, allosteric MEK1/2 inhibitor, has demonstrated antitumor effects against multiple cancer cell lines and xenograft models. This first-in-human study investigated TAK-733 in patients with solid tumors. Methods Patients received oral TAK-733 once daily on days 1-21 in 28-day treatment cycles. Adverse events (AEs) were graded using the Common Terminology Criteria for AEs version 3.0. Response was assessed using RECIST v1.1. Blood samples for TAK-733 pharmacokinetics and pharmacodynamics (inhibition of ERK phosphorylation) were collected during cycle 1. Results Fifty-one patients received TAK-733 0.2-22 mg. Primary diagnoses included uveal melanoma (24 %), colon cancer (22 %), and cutaneous melanoma (10 %). Four patients had dose-limiting toxicities of dermatitis acneiform, plus fatigue and pustular rash in one patient, and stomatitis in one patient. The maximum tolerated dose was 16 mg. Common drug-related AEs included dermatitis acneiform (51 %), diarrhea (29 %), and increased blood creatine phosphokinase (20 %); grade ≥ 3 AEs were reported in 27 (53 %) patients. Median Tmax was 3 h; systemic exposure increased less than dose-proportionally over the dose range 0.2-22 mg. On day 21 maximum inhibition of ERK phosphorylation in peripheral blood mononuclear cells of 46-97 % was seen in patients receiving TAK-733 ≥ 8.4 mg. Among 41 response-evaluable patients, 2 (5 %) patients with cutaneous melanoma (one with BRAF L597R mutant melanoma) had partial responses. Conclusions TAK-733 had a generally manageable toxicity profile up to the maximum tolerated dose, and showed the anticipated pharmacodynamic effect of sustained inhibition of ERK phosphorylation. Limited antitumor activity was demonstrated. Further investigation is not currently planned.
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Antineoplásicos , Neoplasias del Colon/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas , Piridonas , Pirimidinonas , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias de la Úvea/tratamiento farmacológico , Adulto , Anciano , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias del Colon/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/sangre , Femenino , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Masculino , Dosis Máxima Tolerada , Melanoma/metabolismo , Persona de Mediana Edad , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridonas/efectos adversos , Piridonas/farmacocinética , Piridonas/farmacología , Piridonas/uso terapéutico , Pirimidinonas/efectos adversos , Pirimidinonas/farmacocinética , Pirimidinonas/farmacología , Pirimidinonas/uso terapéutico , Neoplasias Cutáneas/metabolismo , Resultado del Tratamiento , Neoplasias de la Úvea/metabolismo , Adulto JovenRESUMEN
BACKGROUND Astragaloside IV (AS-IV) has been shown to prevent ischemia-induced acute kidney injury (AKI) in rat models of ischemia and reperfusion. However, the effects of AS-IV on AKI during sepsis and endotoxinemia is unclear. The current study aimed to investigate the effects and molecular mechanisms of AS-IV on lipopolysaccharide (LPS)-induced AKI. MATERIAL AND METHODS Adult male CD-1 mice were randomly assigned into 6 groups (n=8/group): control group: mice were intraperitoneally (i.p.) injected with normal saline; LPS group (10 mg/kg, i.p.); low-dose AS-IV (25 mg/kg; gavage for 7 days) + LPS (i.p., 1 hour after last gavage) group; medial-dose AS-IV (50 mg/kg) + LPS group; high-dose AS-IV (100 mg/kg) + LPS group; high-dose AS-IV alone (100 mg/kg; gavage for 7 days) group. Blood samples were collected at 24 hours after LPS injection, and plasma uric acid and BUN were measured with colorimetric detection kits. The concentration of plasma tumor necrosis factor (TNF)-α and interleukin 1ß, renal p-extracellular signal-regulated kinases, and urinary albumin were evaluated by ELISA. The expression of CCR5 in renal tissue was evaluated by PCR and Western blotting. Concentrations of glutathione (GSH) and reactive oxygen species (ROS) in renal tissue were also measured. RESULTS AS-IV decreased LPS-stimulated production of blood TNF-α and IL-6, LPS-induced the expression of CCR5, and activation of ERK in the kidneys in a rodent model of endotoxinemia. AS-IV attenuated LPS-caused decreased GSH and increased ROS. It also attenuated LPS-induced increases in plasma uric acid, BUN, and urinary albumin. CONCLUSIONS AS-IV protects against AKI during bacterial endotoxinemia by attenuating expression of cytokines, CCR5, and p-ERK, and elevating anti-oxidative ability.
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Riñón/efectos de los fármacos , Riñón/patología , Saponinas/metabolismo , Saponinas/uso terapéutico , Triterpenos/metabolismo , Triterpenos/uso terapéutico , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/tratamiento farmacológico , Animales , Citocinas/metabolismo , Regulación hacia Abajo , Quinasas MAP Reguladas por Señal Extracelular/sangre , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Interleucina-6/sangre , Interleucina-6/metabolismo , Lipopolisacáridos/efectos adversos , Masculino , Ratones , Modelos Animales , Especies Reactivas de Oxígeno/metabolismo , Receptores CCR5/sangre , Receptores CCR5/metabolismo , Saponinas/farmacología , Triterpenos/farmacología , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: We previously determined that protein kinase C δ (PKCδ) regulates platelet function. However, the function of PKCδ in megakaryopoiesis is unknown. APPROACH AND RESULTS: Using PKCδ(-/-) and wild-type littermate mice, we found that deficiency of PKCδ caused an increase in white blood cells and platelet counts, as well as in bone marrow and splenic megakaryocytes (P<0.05). Additionally, the megakaryocyte number and DNA content were enhanced in PKCδ(-/-) mouse bone marrow after culturing with exogenous thrombopoietin compared with wild-type (P<0.05). Importantly, thrombopoietin-induced signaling was also altered with PKCδ deletion because both extracellular signal-regulated kinase and Akt308 phosphorylation were heightened in PKCδ(-/-) megakaryocytes compared with wild-type. Finally, PKCδ(-/-) mice recovered faster and had a heightened rebound thrombocytosis after thrombocytopenic challenge. CONCLUSIONS: These data suggest that PKCδ is an important megakaryopoietic protein, which regulates signaling induced by thrombopoietin and represents a potential therapeutic target.
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Megacariocitos/citología , Megacariocitos/enzimología , Proteína Quinasa C-delta/deficiencia , Trombocitopenia/sangre , Trombocitopenia/enzimología , Trombopoyesis/fisiología , Animales , Células de la Médula Ósea/citología , Quinasas MAP Reguladas por Señal Extracelular/sangre , Recuento de Leucocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Recuento de Plaquetas , Proteína Quinasa C-delta/sangre , Proteína Quinasa C-delta/genética , Proteínas Proto-Oncogénicas c-akt/sangre , ARN Mensajero/sangre , ARN Mensajero/genética , Transducción de Señal , Bazo/citología , Trombocitopenia/inmunología , Trombopoyesis/genética , Trombopoyetina/sangre , Regulación hacia ArribaRESUMEN
OBJECTIVE: The activities of two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), correlate with disease severity in SLE patients. Whether they are also associated with long-term organ damage is unknown. The aim of the present work was to determine whether impaired expression and activity of ERK and JNK correlate with long-term damage in SLE. METHODS: The expression of ERK and JNK and their phosphorylated active forms was determined by western blot analysis four times during the first year of follow-up in peripheral blood mononuclear cells from 36 SLE patients. A correlation analysis was performed between ERK and JNK expression and longterm organ damage estimated by the SLICC/ARC Damage Index (SDI) 4 years later. RESULTS: Mean levels of ERK and JNK activities during the first year correlated with long-term organ damage severity (r = 0.38 and r = 0.35, respectively; P = 0.05). Overall JNK expression increased with the severity of chronic damage (P = 0.01; P = 0.05 for SDI score 2 and 3, respectively). In contrast, overall ERK expression significantly decreased in patients with maximal organ damage (SDI score 3) compared with patients with an SDI score of 2 (P = 0.03). The ERK/JNK ratio decreased by approximately 40% and 30% in patients with an SDI score of 3 as compared with patients without organ damage and healthy controls, respectively. CONCLUSION: These results demonstrate that early activation of ERK and JNK along with decreased overall ERK expression and reduced ERK/JNK ratio may predict the severity of long-term organ damage in SLE patients.
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Quinasas MAP Reguladas por Señal Extracelular/sangre , Proteínas Quinasas JNK Activadas por Mitógenos/sangre , Lupus Eritematoso Sistémico/enzimología , Adulto , Anciano , Biomarcadores/sangre , Células Cultivadas , Activación Enzimática , Femenino , Estudios de Seguimiento , Glucocorticoides/uso terapéutico , Humanos , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Fosforilación , Prednisona/uso terapéutico , Pronóstico , Índice de Severidad de la EnfermedadRESUMEN
This study investigated the inhibitory effects of oligoporin A on platelet aggregation and the mechanism of its action on downstream signaling molecules. Oligoporin A was isolated from the fruiting bodies of Oligoporus tephroleucus (Polyporaceae). The anti-platelet activities of oligoporin A were studied using rat platelets. The effects of oligoporin A on intracellular Ca(2+) mobilization, ATP release, production of the cyclic nucleotides cAMP and cGMP, extracellular signal-regulated kinase (ERK) 2 phosphorylation, and fibrinogen binding to active integrin α(II)(b)ß(3) were assessed. Oligoporin A, but not oligoporins B and C, inhibited collagen-induced platelet aggregation in a concentration-dependent manner. Interestingly, oligoporin A did not affect ADP- and thrombin-induced platelet aggregations, which act on different types of membrane receptors. Granule secretion analysis demonstrated that oligoporin A significantly and dose-dependently reduced collagen-induced ATP release and intracellular Ca(2+) mobilization. Additionally, oligoporin A induced the dynamic increase in cAMP and cGMP. Increased cGMP production was further confirmed by the simultaneous production of nitric oxide. Pretreatment with oligoporin A significantly blocked collagen-induced ERK2 phosphorylation. Finally, oligoporin A vaguely diminished the binding of fibrinogen to its cognate receptor, integrin α(II)(b)ß(3). The results indicate that oligoporin A inhibits only collagen-induced platelet aggregation mediated through the modulation of downstream signaling molecules. Oligoporin A may be beneficial against cardiovascular disease provoked by aberrant platelet activation.
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Plaquetas/efectos de los fármacos , AMP Cíclico/sangre , GMP Cíclico/sangre , Quinasas MAP Reguladas por Señal Extracelular/sangre , Glucósidos/farmacología , Triterpenos/farmacología , Adenosina Difosfato/sangre , Adenosina Difosfato/farmacología , Animales , Plaquetas/enzimología , Plaquetas/metabolismo , Calcio/sangre , Fibrinógeno/farmacología , Glucósidos/sangre , Humanos , Masculino , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Triterpenos/sangreRESUMEN
BACKGROUND: Aberrant signalling along the p21ras/MAP kinase pathway has been demonstrated in systemic lupus erythematosus (SLE). OBJECTIVE: To determine whether expression and activity of the MAP kinases ERK and JNK reflect disease activity in patients with SLE. METHODS: Blood samples of 42 outpatients with SLE were prospectively collected during four consecutive visits. The control group included 20 healthy subjects. Disease activity was assessed using the SLE Disease Activity Index (SLEDAI). Expression of total ERK and JNK kinases and their active forms (pERK and pJNK) was determined in whole protein lysates of peripheral blood mononuclear cells. RESULTS: The mean levels of the active kinases pERK and pJNK were significantly increased in patients with active disease (SLEDAI 4-20) as compared with patients with inactive disease (SLEDAI 0-3), p = 0.04, as well as with healthy controls, p = 0.03 and p = 0.003 for pERK and pJNK, respectively. The percentage of activated forms of ERK and JNK of the total expression of these MAP kinases was also gradually increased, reaching 50% for pERK and >40% for pJNK in patients with SLE with moderate-to-severe disease (SLEDAI 7-20), p = 0.005, p = 0.005 and p = 0.02, p = 0.05 as compared with controls and inactive patients, respectively. A decrease of more than three SLEDAI points was associated with a significant reduction in the expression of both total and activated forms of ERK and JNK, p = 0.03, p = 0.01, respectively. CONCLUSIONS: The results show that ERK and JNK activity reflects disease activity in patients with SLE. These MAP kinases may serve as additional tools for the evaluation of disease activity and management of these patients.
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Quinasas MAP Reguladas por Señal Extracelular/sangre , Lupus Eritematoso Sistémico/enzimología , MAP Quinasa Quinasa 4/sangre , Adulto , Anciano , Biomarcadores/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Índice de Severidad de la EnfermedadRESUMEN
Acute myeloid leukemia (AML) is a malignant disorder of hematopoietic stem and progenitor cells, characterized by accumulation of immature blasts in the bone marrow and peripheral blood of affected patients. Standard induction therapy leads to complete remission in approximately 50% to 75% of patients. In spite of favorable primary response rates, only 20% to 30% of patients enjoy long-term disease free survival. Identifying proteins involved in prognosis is important for proposing biomarkers that can aid in the clinical management of the disease. The aim of this study was to construct a protein-protein interaction (PPI) network based on serum proteins associated with unfavorable prognosis of AML, and analyze the biological pathways underlying molecular complexes in the network. We identified 16 candidate serum proteins associated with unfavorable prognosis (in terms of poor response to treatment, poor overall survival, short complete remission, and relapse) in AML via a search in the literature: IL2RA, FTL, HSP90AA1, D2HGDH, PLAU, COL18A1, FGF19, SPP1, FGA, PF4, NME1, TNF, ANGPT2, B2M, CD274, LGALS3. The PPI network was constructed with Cytoscape using association networks from String and BioGRID, and Gene Ontology enrichment analysis using the ClueGo pluggin was performed. The central protein in the network was found to be PTPN11 which is involved in modulating the RAS-ERK, PI3K-AKT and JAK-STAT pathways, as well as in hematopoiesis, and in the regulation of apoptotic genes. Therefore, a dysregulation of this protein and/or of the proteins connected to it in the network leads to the defective activation of these signaling pathways and to a reduction in apoptosis. Together, this could cause an increase in the frequency of leukemic cells and a resistance to apoptosis in response to treatment.
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Biomarcadores de Tumor/genética , Proteínas Sanguíneas/genética , Regulación Leucémica de la Expresión Génica , Redes Reguladoras de Genes , Leucemia Mieloide Aguda/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Proteínas Sanguíneas/metabolismo , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Supervivencia sin Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/sangre , Quinasas MAP Reguladas por Señal Extracelular/genética , Ontología de Genes , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Quinasas Janus/sangre , Quinasas Janus/genética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/mortalidad , Anotación de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/sangre , Fosfatidilinositol 3-Quinasas/genética , Pronóstico , Mapeo de Interacción de Proteínas , Proteína Tirosina Fosfatasa no Receptora Tipo 11/sangre , Proteínas Proto-Oncogénicas c-akt/sangre , Proteínas Proto-Oncogénicas c-akt/genética , Inducción de Remisión , Factores de Transcripción STAT/sangre , Factores de Transcripción STAT/genética , Transducción de Señal , Proteínas ras/sangre , Proteínas ras/genéticaRESUMEN
BACKGROUND: Thrombin belongs to the most potent platelet agonists and activates human platelets through GPIbα and two protease activated receptors (PARs), PAR1 and PAR4. However, the details of thrombin receptor system, especially the role of PAR4 on human platelet activation is still not clear. OBJECTIVES: We found a significant difference in PAR4-activating peptide (PAR4-AP)-induced, but not PAR1-AP, platelet aggregation between healthy Japanese subjects. Sequencing analysis revealed a single nucleotide change in PAR4 gene F2RL3 (SNP rs773902) leading to Ala120Thr variant. To elucidate the role of PAR4 in human platelet activation, we examined if platelet activation induced by PAR4-AP may be associated with PAR4 genotype. METHODS: Platelets from 202 healthy Japanese volunteers were genetically analyzed and determined the genotype frequency of rs773902. Agonist induced platelet aggregation, integrin αIIbß3 activation, granule release, Ca2+ mobilization, and activation of ERK and MLC were evaluated. The specificity of effects observed in platelets was confirmed in 293T cells transfected PAR4-Thr120 or Ala120. RESULTS: The frequencies of PAR4 variant Thr/Thr120, Ala/Thr120, and Ala/Ala 120 were 5.9, 37.1, and 57.0%, respectively. Platelets with Thr/Thr120 showed significantly higher reactivity in PAR4-AP-induced platelet aggregation, αIIbß3 activation and granule release compared to platelets with Ala/Ala120. PAR4-AP induced higher Ca2+ mobilization and ERK activation in platelets with Thr/Thr120 than Ala/Ala120. Ca2+ mobilization and ERK activation were also increased in 293T cells transfected with PAR4-Thr120 compared to Ala120. CONCLUSION: Our data suggested that PAR4-AP-induced platelet reactivity between PAR4 rs773902 was associated with altered intensity of Ca2+ mobilization and ERK activation.
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Plaquetas/fisiología , Calcio/sangre , Péptidos/farmacología , Activación Plaquetaria/fisiología , Agregación Plaquetaria/fisiología , Receptores de Trombina/agonistas , Receptores de Trombina/sangre , Plaquetas/efectos de los fármacos , Plaquetas/enzimología , Plaquetas/metabolismo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/sangre , Humanos , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Polimorfismo de Nucleótido Simple , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , TransfecciónRESUMEN
Reactive oxygen species (ROS)-induced injury has been shown to occur during the reperfusion phase of ischemia-reperfusion and ROS are known to induce signaling events. We hypothesized that oxygen sensing in endothelial cells is also dependent on internal redox changes during hypoxia and that endothelial cells respond to changing oxygen environments via signaling, switching to an inflammatory phenotype. Endothelial cells exposed to relative hypoxia or the mitochondrial inhibitors rotenone, antimycin A, or FCCP show loss of mitochondrial membrane potential. During hypoxia, an increase in cytoplasmic ROS and glutathione S-transferase activity occurred, suggesting changes in intracellular redox state, mimicked with rotenone or FCCP but inhibited by antimycin A. Phosphorylation of stress-responsive mitogen-activated protein kinases occurred in hypoxia and was rapid and prolonged. Phosphorylation was inhibited by vitamin C, N-acetyl cysteine, or antimycin A. Chelation of intracellular calcium inhibits phosphorylation but the mitochondrial transition pore inhibitor cyclosporin A had no effect. Reoxygenation caused a further round of signaling, which was rapid but transient. Functionally, adhesion of neutrophils after hypoxia-reoxygenation under flow is ROS, P-selectin, and MAPK dependent. Therefore, changes in cellular signaling and phenotype are abrogated by ROS scavengers and suggest their use as therapeutic agents in ischemia-reperfusion.
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Hipoxia de la Célula/fisiología , Endotelio Vascular/fisiología , Quinasas MAP Reguladas por Señal Extracelular/sangre , Sistema de Señalización de MAP Quinasas/fisiología , Neutrófilos/fisiología , Consumo de Oxígeno/fisiología , Especies Reactivas de Oxígeno/metabolismo , Antimicina A/farmacología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Hipoxia de la Célula/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Glutatión Transferasa/metabolismo , Humanos , Potenciales de la Membrana/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/fisiología , Neutrófilos/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Rotenona/farmacología , Venas UmbilicalesRESUMEN
Propionate is a short-chain fatty acid produced under normal physiological conditions in the rumen of cattle. It is also involved in the inflammatory process and neutrophil function via calcium release, reactive oxygen species and intracellular pH (pH(i)) changes. This study examined the effect of propionate on the pH(i) of bovine neutrophils; specifically if pH(i) changes are controlled by calcium flux, and the mitogen-activated protein kinase (MAPK) pathway. Propionate caused rapid intracellular acidification and sustained alkalinization in bovine neutrophils loaded with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), a fluorescent indicator of pH(i). The acidification phase seems to be controlled by intracellular calcium release and p38 MAPK pathway. The pH recovery phenomenon was mediated by an amiloride-sensitive Na+/H+ exchanger and H+ channel, and was inhibited by UO126 (an ERK1/2 MAPK phosphorylation inhibitor), Gö6850 (a PKC inhibitor) and calcium chelating. Ionomycin, a calcium ionophore, induced intracellular acidification and sustained alkalinization. The intracellular acidification was strongly inhibited by BAPTA-AM (an intracellular calcium chelator) and SB203580 (a p38 MAPK inhibitor). In addition, the intracellular alkalinization was reduced by EGTA (a calcium chelator), UO126, LY294002 (a PI3K inhibitor) and Gö6850. Propionate did not increase superoxide production, however it reduced the superoxide production induced by platelet-activating factor (PAF), and increased the release of superoxide induced by ionomycin. Our results suggest that propionate-induced intracellular acidification is mediated by intracellular calcium release and p38 MAPK activation, and that pH recovery is controlled via ERK1/2 MAPK, PKC and calcium entry in bovine neutrophils.
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Calcio/sangre , Bovinos/sangre , Quinasas MAP Reguladas por Señal Extracelular/sangre , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Propionatos/farmacología , Proteína Quinasa C/sangre , Animales , Butadienos/farmacología , Quelantes/farmacología , Cromonas/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Femenino , Concentración de Iones de Hidrógeno , Imidazoles/farmacología , Indoles/farmacología , Ionomicina/farmacología , Maleimidas/farmacología , Morfolinas/farmacología , Neutrófilos/enzimología , Nitrilos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Espectrometría de Fluorescencia/veterinaria , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/sangreRESUMEN
P-cresol is a well-known uremic toxin and environmental toxicant that may affect platelet functions. In this study, p-cresol (1-5 µM) inhibited the arachidonic acid (AA)-induced platelet aggregation, with 47% and 82% of inhibition at concentrations of 2 and 5 µM, respectively. Under similar experimental condition, p-cresol showed little effect on the U46619-induced platelet aggregation. p-cresol (<500 µM) revealed no discernable cytotoxicity to platelets as analyzed by quantification of lactate dehydrogenase release. Antiplatelet effect of p-cresol was related to inhibition of thromboxane A(2) (TXA(2)) and prostaglandin D(2) (PGD(2)) formation. P-cresol (2-100 µM) partly inhibited the AA-induced reactive oxygen species (ROS) production as well as the extracellular signal-regulated kinase (ERK1/2) and p38 phosphorylation in platelets. P-cresol further inhibited the AA-induced aggregation of rabbit platelet-rich plasma (PRP) with an IC50 of 2 µM and aggregation of human PRP (IC50 = 13.6 µM). Intravenous administration of p-cresol (250-1000 nmole) into mice effectively suppressed the ex vivo platelet aggregation, whereas showed little effect on the value of RBC, hemoglobin (HGB), hematocrit, MCV, MCH, MCHC, platelets and lymphocyte counts. These results indicate that in acute p-cresol-poisoning and long-term exposure to cresol as in severe uremic patients, p-cresol may potentially inhibit blood clot formation and lead to hemorrhagic disorders via inhibition of platelet aggregation, ROS production, ERK/p38 activation and TXA(2) production.
Asunto(s)
Plaquetas/efectos de los fármacos , Cresoles/toxicidad , Contaminantes Ambientales/toxicidad , Quinasas MAP Reguladas por Señal Extracelular/sangre , Inhibidores de Agregación Plaquetaria/toxicidad , Agregación Plaquetaria/efectos de los fármacos , Especies Reactivas de Oxígeno/sangre , Transducción de Señal/efectos de los fármacos , Tromboxano A2/sangre , Uremia/inducido químicamente , Proteínas Quinasas p38 Activadas por Mitógenos/sangre , Animales , Recuento de Células Sanguíneas , Plaquetas/enzimología , Relación Dosis-Respuesta a Droga , Activación Enzimática , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Fosforilación , Pruebas de Función Plaquetaria , Prostaglandina D2/sangre , Conejos , Factores de Tiempo , Uremia/sangreRESUMEN
Cytokine-induced inflammation is involved in the pathogenesis of type 2 diabetes mellitus (DM). We investigated plasma concentrations and ex vivo production of cytokines and chemokines, and intracellular signalling molecules, mitogen-activated protein kinases (MAPK) in T helper (Th) cells and monocytes in 94 type 2 diabetic patients with or without nephropathy and 20 healthy controls. Plasma concentrations of inflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-18 and chemokine CCL2 in patients with diabetic nephropathy (DN) were significantly higher than control subjects, while IL-10, CXCL8, CXCL9, CXCL10 and adiponectin concentrations of DN were significantly higher than patients without diabetic nephropathy (NDN) and control subjects (all P < 0.05). Plasma concentrations of TNF-alpha, IL-6, IL-10, IL-18, CCL2, CXCL8, CXCL9, CXCL10 and adiponectin exhibited significant positive correlation with urine albumin : creatinine ratio in DN patients. The percentage increases of ex vivo production of IL-6, CXCL8, CXCL10, CCL2 and CCL5 upon TNF-alpha activation were significantly higher in both NDN and DN patients than controls (all P < 0.05). The percentage increases in IL-18-induced phosphorylation of extracellular signal-regulated kinase (ERK) in Th cells of NDN and DN were significantly higher than controls (P < 0.05), while the percentage increase in TNF-alpha-induced phosphorylation of p38 MAPK in monocytes and IL-18-induced phosphorylation of p38 MAPK in Th cells and monocytes were significantly higher in NDN patients than controls. These results confirmed that the aberrant production of inflammatory cytokines and chemokines and differential activation of MAPK in different leucocytes are the underlying immunopathological mechanisms of type 2 DM patients with DN.
Asunto(s)
Citocinas/sangre , Diabetes Mellitus Tipo 2/inmunología , Nefropatías Diabéticas/inmunología , Proteínas Quinasas Activadas por Mitógenos/sangre , Adiponectina/sangre , Adulto , Células Cultivadas , Quimiocinas/biosíntesis , Quimiocinas/sangre , Citocinas/biosíntesis , Quinasas MAP Reguladas por Señal Extracelular/sangre , Femenino , Humanos , Interleucina-18/inmunología , Masculino , Persona de Mediana Edad , Monocitos/enzimología , Fosforilación , Linfocitos T Colaboradores-Inductores/enzimología , Factor de Necrosis Tumoral alfa/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/sangreRESUMEN
In insects, phagocytosis is an important innate immune response against pathogens and parasites, and several signal transduction pathways regulate this process. The focal adhesion kinase (FAK)/Src and mitogen activated protein kinase (MAPK) pathways are of central importance because their activation upon pathogen challenge regulates phagocytosis via haemocyte secretion and activation of the prophenoloxidase (proPO) cascade. The goal of this study was to explore further the mechanisms underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme-linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate that (1) phagocytosis of bacteria (both Gram-negative and Gram-positive) is dependent on RGD-binding receptors, FAK/Src and MAPKs, (2) latex bead phagocytosis is RGD-binding-receptor-independent and dependent on FAK/Src and MAPKs, (3) lipopolysaccharide internalization is RGD-binding-receptor-independent and FAK/Src-independent but MAPK-dependent and (4) in unchallenged haemocytes in suspension, FAK, Src and extracellular signal-regulated kinase (ERK) signalling molecules participating in phagocytosis show both a functional and a physical association. Overall, this study has furthered knowledge of FAK/Src and MAPK signalling pathways in insect haemocyte immunity and has demonstrated that distinct signalling pathways regulate the phagocytic activity of biotic and abiotic components in insect haemocytes. Evidently, the basic phagocytic signalling pathways among insects and mammals appear to have remained unchanged during evolution.
Asunto(s)
Ceratitis capitata/inmunología , Hemocitos/inmunología , Fagocitosis/inmunología , Transducción de Señal/inmunología , Animales , Escherichia coli/inmunología , Quinasas MAP Reguladas por Señal Extracelular/sangre , Proteína-Tirosina Quinasas de Adhesión Focal/sangre , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/inmunología , Inmunidad Celular , Lipopolisacáridos/inmunología , Microesferas , ARN Bicatenario/genética , Staphylococcus aureus/inmunología , Familia-src Quinasas/sangreRESUMEN
OBJECTIVE: Maintenance of postinjury T-lymphocyte immune paralysis or anergy could result from failure to activate costimulatory receptors during T-cell receptor activation and/or from chronic stimulation of a competing set of elevated corepressor receptors. Our objective was to assess whether elevated posttrauma T-lymphocyte surface expression of corepressor receptors was associated with immunodepressed lymphocyte responses and corresponded to increased inhibitory and decreased activating signal transduction molecules. DESIGN: Prospective observational study. SETTING: University trauma intensive care unit and research laboratory. PATIENTS: Sixty-one severe thermal and mechanical trauma patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Isolated trauma patients' and controls' peripheral blood T cells were assayed for negative and positive costimulation receptor expression. These receptor expression levels were compared (flow cytometry) between the two groups and correlated to T-cell levels of inhibitory and activating signal transduction molecules and proliferation capacity. Patients' proliferation hyporesponsive (anergic) T cells had increased expression of novel inhibitory receptors (corepressors) PD-1 (p < .05) and CD47 (p < .05) vs. patients' T-cell proliferation competent or controls' T cells. Patients' T-cell CD152 (CTLA-4) expression was also elevated vs. controls. Only patients' anergic T cells had simultaneously increased levels of the inhibitory signal transduction proteins, c-Cbl, a ubiquitin-ligase (p < .01) and SHP-1, a phosphatase (p < .01), concomitant to depressed phosphorylation of the activating signal kinases Erk, Zap70, and CD3Euro. T-cell receptor complex phosphorylation and activation of the interleukin-2 pivotal transcriptional complex protein CREB were also simultaneously depressed as c-Cbl and SHP-1 were elevated. CONCLUSIONS: Up-regulated corepressor receptor expression is novelly shown to characterize trauma patients' anergic T cells and correlate with predominance of inhibitory overactivating signal transduction molecules during T-cell stimulation. This could contribute to postinjury immunosuppression.
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Antígenos CD/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Quemaduras/inmunología , Antígeno CD47/metabolismo , Anergia Clonal/inmunología , Transducción de Señal/fisiología , Linfocitos T/inmunología , Heridas y Lesiones/inmunología , Adulto , Antígenos CD/sangre , Antígenos de Diferenciación/sangre , Proteína de Unión a CREB/sangre , Antígeno CTLA-4 , Quinasas MAP Reguladas por Señal Extracelular/sangre , Femenino , Citometría de Flujo , Humanos , Tolerancia Inmunológica/inmunología , Unidades de Cuidados Intensivos , Péptidos y Proteínas de Señalización Intracelular/sangre , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Fosforilación , Receptor de Muerte Celular Programada 1 , Estudios Prospectivos , Proteína Tirosina Fosfatasa no Receptora Tipo 6/sangre , Proteínas Tirosina Quinasas/sangre , Proteínas Proto-Oncogénicas c-cbl/sangre , ARN Polimerasa I , Proteína Tirosina Quinasa ZAP-70/sangreRESUMEN
1. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) manifest pleiotropic effects that may contribute to their therapeutic efficacy. However, the mechanism of the beneficial action of statins on cardiac hypertrophy and fibrosis remains unclear. We have now investigated this action of pitavastatin in Dahl salt-sensitive (DS) rats. 2. The DS rats progressively develop marked hypertension when fed a diet containing 8% NaCl from 7 weeks of age. These animals exhibited pronounced cardiac hypertrophy and fibrosis, as well as upregulation of fetal-type cardiac gene expression at 12 weeks of age, compared with DS rats fed a diet containing 0.3% NaCl. The abundance of mRNAs for collagen types I and III, angiotensin-converting enzyme, transforming growth factor-beta1 and connective tissue growth factor was also increased in the heart of rats on the high-salt diet. 3. Treatment of rats on the high-salt diet with a non-antihypertensive dose of pitavastatin (0.3 or 1 mg/kg per day) from 7 to 12 weeks of age attenuated the development of cardiac hypertrophy and fibrosis, as well as inhibiting the upregulation of cardiac gene expression. Pitavastatin also blocked the translocation of RhoA to the membrane fraction of the left ventricle and RhoA activation, as well as the phosphorylation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)-1 and ERK-2 and an increase in the DNA binding activity of serum response factor (SRF) in the heart induced by the high-salt diet. 4. These findings suggest that the effects of pitavastatin on load-induced cardiac hypertrophy and fibrosis are independent of its cholesterol-lowering action and may be mediated, at least in part, through inhibition of RhoA-ERK-SRF signalling.
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Cardiomegalia/tratamiento farmacológico , Quinasas MAP Reguladas por Señal Extracelular/sangre , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Hipertensión/complicaciones , Miocardio/patología , Quinolinas/uso terapéutico , Transducción de Señal/fisiología , Proteína de Unión al GTP rhoA/sangre , Envejecimiento/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Colágeno/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Quinasas MAP Reguladas por Señal Extracelular/genética , Fibrosis/patología , Expresión Génica/efectos de los fármacos , Hipertensión/inducido químicamente , Hipertensión/fisiopatología , Hipertrofia Ventricular Izquierda/patología , Hipertrofia Ventricular Izquierda/prevención & control , Immunoblotting , Lípidos/sangre , Masculino , Ratas , Ratas Endogámicas Dahl , Transducción de Señal/efectos de los fármacos , Cloruro de Sodio , Proteína de Unión al GTP rhoA/genéticaRESUMEN
PURPOSE: This phase II study of sorafenib, an oral multikinase inhibitor that targets Raf kinase and receptor tyrosine kinases, assessed efficacy, toxicity, pharmacokinetics, and biomarkers in advanced hepatocellular carcinoma (HCC) patients. METHODS: Patients with inoperable HCC, no prior systemic treatment, and Child-Pugh (CP) A or B, received continuous, oral sorafenib 400 mg bid in 4-week cycles. Tumor response was assessed every two cycles using modified WHO criteria. Sorafenib pharmacokinetics were measured in plasma samples. Biomarker analysis included phosphorylated extracellular signal regulated kinase (pERK) in pretreatment biopsies (immunohistochemistry) and blood-cell RNA expression patterns in selected patients. RESULTS: Of 137 patients treated (male, 71%; median age, 69 years), 72% had CP A, and 28% had CP B. On the basis of independent assessment, three (2.2%) patients achieved a partial response, eight (5.8%) had a minor response, and 46 (33.6%) had stable disease for at least 16 weeks. Investigator-assessed median time to progression (TTP) was 4.2 months, and median overall survival was 9.2 months. Grade 3/4 drug-related toxicities included fatigue (9.5%), diarrhea (8.0%), and hand-foot skin reaction (5.1%). There were no significant pharmacokinetic differences between CP A and B patients. Pretreatment tumor pERK levels correlated with TTP. A panel of 18 expressed genes was identified that distinguished "nonprogressors" from "progressors" with an estimated 100% accuracy. CONCLUSION: Although single-agent sorafenib has modest efficacy in HCC, the manageable toxicity and mechanisms of action support a role for combination regimens with other anticancer agents.