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All living organisms are charged with repair after injury particularly at epithelial barrier sites, but in some cases this response leads instead to structural remodeling and long-term disease. Identifying the molecular and cellular control of this divergence is key to disease modification. In that regard, stress kinase control of epithelial stem cells is a rational entry point for study. Here we examine the potential for mitogen-activated protein kinase 13 (MAPK13) regulation of epithelial stem cells using models of respiratory viral injury and post-viral lung disease. We show that Mapk13 gene-knockout mice handle acute infectious illness as expected but are protected against structural remodeling manifest as basal-epithelial stem cell (basal-ESC) hyperplasia-metaplasia, immune activation, and mucinous differentiation. In corresponding cell models, Mapk13-deficiency directly attenuates basal-ESC growth and organoid formation. Extension to human studies shows marked induction/activation of basal-cell MAPK13 in clinical samples of comparable remodeling found in asthma and COPD. Here again, MAPK13 gene-knockdown inhibits human basal-ESC growth in culture. Together, the data identify MAPK13 as a control for structural remodeling and disease after epithelial injury and as a suitable target for down-regulation as a disease-modifying strategy.
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Subacute spinal cord injury (SCI) displays a complex pathophysiology associated with pro-inflammation and ensuing tissue damage. Microglia, the resident innate immune cells of the CNS, in concert with infiltrating macrophages, are the primary contributors to SCI-induced inflammation. However, subpopulations of activated microglia can also possess immunomodulatory activities that are essential for tissue remodeling and repair, including the production of anti-inflammatory cytokines and growth factors that are vital for SCI recovery. Recently, reports have provided convincing evidence that sex-dependent differences exist in how microglia function during CNS pathologies and the extent to which these cells contribute to neurorepair and endogenous recovery. Herein we employed flow cytometry and immunohistochemical methods to characterize the phenotype and population dynamics of activated innate immune cells within the injured spinal cord of age-matched male and female rats within the first week (7 days) following thoracic SCI contusion. This assessment included the analysis of pro- and anti-inflammatory markers, as well as the expression of critical immunomodulatory kinases, including P38 MAPK, and transcription factors, such as NFκB, which play pivotal roles in injury-induced inflammation. We demonstrate that activated microglia from the injured spinal cord of female rats exhibited a significantly diminutive pro-inflammatory response, but enhanced anti-inflammatory activity compared to males. These changes included lower levels of iNOS and TLR4 expression but increased levels of ARG-1 and CD68 in females after SCI. The altered expression of these markers is indicative of a disparate secretome between the microglia of males and females after SCI and that the female microglia possesses higher phagocytic capabilities (increased CD68). The examination of immunoregulatory kinases and transcription factors revealed that female microglia had higher levels of phosphorylated P38Thr180/Tyr182 MAPK and nuclear NFκB pp50Ser337 but lower amounts of nuclear NFκB pp65Ser536, suggestive of an attenuated pro-inflammatory phenotype in females compared to males after SCI. Collectively, this work provides novel insight into some of the sex disparities that exist in the innate immune response after SCI and indicates that sex is an important variable when designing and testing new therapeutic interventions or interpretating positive or negative responses to an intervention.
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Traumatismos de la Médula Espinal , Ratas , Animales , Masculino , Femenino , Traumatismos de la Médula Espinal/patología , Inmunidad Innata , Inflamación/patología , Antiinflamatorios , Factores de TranscripciónRESUMEN
Novel series of pyrazolo[3,4-b]pyridines 9a-j and 14a-f were prepared via a one-pot three-component reaction. Compounds 9a-j were synthesized by the reaction of 3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-5-amine (4) with benzoyl acetonitriles 3a,b and aldehydes 5a-e, whereas the spiro derivatives 14a-f were synthesized by the reaction of pyrazole derivative 4 with 3a-c and indoline-2,3-diones 10a,b. Screening of the antiproliferative activity of 9a-j and 14a-f revealed that 14a and 14d were the most potent analogues against HepG2 and HeLa cells, with IC50 = 4.2 and 5.9 µM, respectively. Moreover, compounds 9c and 14a could promote cell cycle disturbance and apoptosis in HepG2 cells, as evidenced by DNA flow cytometry and Annexin V-FITC/PI assays. Cell cycle analysis of 9c and 14a indicated a reduction in HepG2 cells in the G1 phase, with arrest in the S phase and the G2/M phase, respectively. Also, 9c and 14a are good apoptotic inducers in the HepG2 cell line. Furthermore, compounds 9h and 14d stood out as the most efficient antiproliferative agents in the NCI 60-cell line panel screening, with mean GI % equal to 60.3% and 55.4%, respectively. Additionally, 9c, 9h, 14a, and 14d showed good inhibitory action against the cellular pathway regulator p38α kinase, with IC50 = 0.42, 0.41, 0.13, and 0.64 µM, respectively. A docking study was carried out on the p38α kinase active site, showing a binding mode comparable to that of reported p38 mitogen-activated protein kinase inhibitors. These newly discovered pyrazolo[3,4-b]pyridines could be considered as potential candidates for the development of newly targeted anticancer agents.
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Antineoplásicos/farmacología , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Pirazoles/farmacología , Piridinas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HeLa , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Pirazoles/síntesis química , Pirazoles/química , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
In response to foreign or endogenous stimuli, both microglia and astrocytes adopt an activated phenotype that promotes the release of pro-inflammatory mediators. This inflammatory mechanism, known as neuroinflammation, is essential in the defense against foreign invasion and in normal tissue repair; nevertheless, when constantly activated, this process can become detrimental through the release of neurotoxic factors that amplify underlying disease. In consequence, this study presents the anti-inflammatory and immunomodulatory properties of o-orsellinaldehyde, a natural compound found by an in silico approach in the Grifola frondosa mushroom, in astrocytes and microglia cells. For this purpose, primary microglia and astrocytes were isolated from mice brain and cultured in vitro. Subsequently, cells were exposed to LPS in the absence or presence of increasing concentrations of this natural compound. Specifically, the results shown that o-orsellinaldehyde strongly inhibits the LPS-induced inflammatory response in astrocytes and microglia by decreasing nitrite formation and downregulating iNOS and HO-1 expression. Furthermore, in microglia cells o-orsellinaldehyde inhibits NF-κB activation; and potently counteracts LPS-mediated p38 kinase and JNK phosphorylation (MAPK). In this regard, o-orsellinaldehyde treatment also induces a significant cell immunomodulation by repolarizing microglia toward the M2 anti-inflammatory phenotype. Altogether, these results could partially explain the reported beneficial effects of G. frondosa extracts on inflammatory conditions.
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Rutin is a flavonoid with antioxidant property. It has been shown to exert cardioprotection against cardiomyocyte hypertrophy. However, studies regarding its antihypertrophic property are still lacking, whether it demonstrates similar antihypertrophic effect to its metabolite, quercetin. Hence, this study aimed to investigate the effects of both flavonoids on oxidative stress and mitogen-activated protein kinase (MAPK) pathway in H9c2 cardiomyocytes that were exposed to angiotensin II (Ang II) to induce hypertrophy. Cardiomyocytes were exposed to Ang II (600 nM) with or without quercetin (331 µM) or rutin (50 µM) for 24 h. A group given vehicle served as the control. The concentration of the flavonoids was chosen based on the reported effective concentration to reduce cell hypertrophy or cardiac injury in H9c2 cells. Exposure to Ang II increased cell surface area, intracellular superoxide anion level, NADPH oxidase and inducible nitric oxide synthase activities, and reduced cellular superoxide dismutase activity and nitrite level, which were similarly reversed by both rutin and quercetin. Rutin had no significant effects on phosphorylated proteins of extracellular signal-related kinases (ERK1/2) and p38 but downregulated phosphorylated c-Jun N-terminal kinases (JNK1/2), which were induced by Ang II. Quercetin, on the other hand, had significantly downregulated the phosphorylated proteins of ERK1/2, p38, and JNK1/2. The quercetin inhibitory effect on JNK1/2 was stronger than the rutin. In conclusion, both flavonoids afford similar protective effects against Ang II-induced cardiomyocyte hypertrophy, but they differently modulate MAPK pathway.
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Angiotensina II/toxicidad , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hipertrofia/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mioblastos Cardíacos/metabolismo , Quercetina/farmacología , Rutina/farmacología , Animales , Antioxidantes/farmacología , Células Cultivadas , Hipertrofia/inducido químicamente , Hipertrofia/tratamiento farmacológico , Hipertrofia/patología , Proteínas Quinasas Activadas por Mitógenos/genética , Mioblastos Cardíacos/citología , Mioblastos Cardíacos/efectos de los fármacos , NADPH Oxidasas/metabolismo , Óxido Nítrico/metabolismo , Fosforilación , Ratas , Especies Reactivas de Oxígeno/metabolismo , Vasoconstrictores/toxicidadRESUMEN
Background: Calcineurin inhibitors (CNIs) such as cyclosporine A and tacrolimus are commonly used after renal transplantation to suppress the immune system. In lymphoid cells, cyclosporine A acts via the calcineurin/nuclear factor of activated T-cell (NFAT) axis. In non-lymphoid cells, such as kidney epithelial cells, cyclosporine A induces calcineurin inhibitor toxicity. It is unknown via which off-targets cyclosporine A induces calcineurin inhibitor toxicity in kidney epithelial cells. Methods: To measure a compound's potential to induce nephrotoxicity, the expression of the surrogate marker Fn14 was measured by flow cytometry. Compounds were tested for their potential to induce Fn14 either chemically or plasmid-mediated. Mice were injected with various compounds, and changes in nephrotoxic gene expression levels of the kidney epithelial cells were then analyzed. Results: Fn14 is specifically upregulated due to calcineurin inhibitor toxicity inducing agents. Inhibition of the NFAT axis showed no increase of the Fn14 expression on the surface of kidney cells. However, inhibition of p38 MAPK, phosphoinositide-3-kinase (PI3K)/Akt, protein kinase C (PKC), and inhibitor of nuclear factor-κB (IκB) kinase (IKK) showed clear induction of Fn14 and increased expressions of nephrotoxic, inflammatory, and fibrotic genes in vitro and in vivo. Conclusions: These findings show that cyclosporine A acts independently of NFAT on kidney epithelial cells. Moreover, inhibition of serine/threonine protein kinases mimics cyclosporine A's activity on kidney epithelial cells. This mimicking effect indicates that these protein kinases are off-targets of cyclosporine A and damage structural renal cells when inhibited and therefore contributes likely to the development and progression of calcineurin inhibitor toxicity.
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Vascular pathologies, such as thrombosis or atherosclerosis, are leading causes of death worldwide and are strongly associated with the dysfunction of vascular endothelial cells. In this context, the extracellular endonuclease Ribonuclease 1 (RNase1) acts as an essential protective factor in regulation and maintenance of vascular homeostasis. However, long-term inflammation causes strong repression of RNase1 expression, thereby promoting endothelial cell dysfunction. This inflammation-mediated downregulation of RNase1 in human endothelial cells is facilitated via histone deacetylase (HDAC) 2, although the underlying molecular mechanisms are still unknown. Here, we report that inhibition of c-Jun N-terminal kinase by small chemical compounds in primary human endothelial cells decreased physiological RNase1 mRNA abundance, while p38 kinase inhibition restored repressed RNase1 expression upon proinflammatory stimulation with tumor necrosis factor alpha (TNF-α) and poly I:C. Moreover, blocking of the p38 kinase- and HDAC2-associated kinase casein kinase 2 (CK2) by inhibitor as well as small interfering RNA (siRNA)-knockdown restored RNase1 expression upon inflammation of human endothelial cells. Further downstream, siRNA-knockdown of chromodomain helicase DNA binding protein (CHD) 3 and 4 of the nucleosome remodeling and deacetylase (NuRD) complex restored RNase1 repression in TNF-α treated endothelial cells implicating its role in the HDAC2-containing repressor complex involved in RNase1 repression. Finally, chromatin immunoprecipitation in primary human endothelial cells confirmed recruitment of the CHD4-containing NuRD complex and subsequent promoter remodeling via histone deacetylation at the RNASE1 promoter in a p38-dependent manner upon human endothelial cell inflammation. Altogether, our results suggest that endothelial RNase1 repression in chronic vascular inflammation is regulated by a p38 kinase-, CK2-, and NuRD complex-dependent pathway resulting in complex recruitment to the RNASE1 promoter and subsequent promoter remodeling.
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INTRODUCTION: CacyBP/SIP is a multifunctional protein present in various mammalian tissues, among them in brain. Recently, it has been shown that CacyBP/SIP exhibits phosphatase activity towards ERK1/2 and p38 kinases. OBJECTIVES: The aim of our study was to analyze the localization and level of CacyBP/SIP and its substrates, phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated p38 (p-p38) kinases, in an intact and transected rat spinal cord. METHODS: To achieve our goals we have performed Western blot/densitometric analysis and double immunofluorescence staining using rat spinal cord tissue, intact and after total transection at different time points. RESULTS: We have observed a decrease in the level of CacyBP/SIP and an increase in the level of p-ERK1/2 and of p-p38 in fragments of the spinal cord excised 1 and 3 months after transection. Moreover, immunofluorescence staining has shown that CacyBP/SIP, p-ERK1/2 or p-p38 co-localized with a neuronal marker, NeuN, and with an oligodendrocyte marker, Olig2. CONCLUSION: The inverse correlation between CacyBP/SIP and p-ERK1/2 or p-p38 levels suggests that CacyBP/SIP may dephosphorylate p-ERK1/2 and p-p38 kinases and be involved in neural plasticity following spinal cord injury.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Animales , Femenino , Fosforilación/fisiología , Ratas , Ratas Wistar , Traumatismos de la Médula Espinal/patologíaRESUMEN
Patients with metastatic breast cancer (MBC) have limited therapeutic options and novel treatments are critically needed. Prior research implicates tumor-induced mobilization of myeloid cell populations in metastatic progression, as well as being an unfavorable outcome in MBC; however, the underlying mechanisms for these relationships remain unknown. Here, we provide evidence for a novel mechanism by which p38 promotes metastasis. Using triple-negative breast cancer models, we showed that a selective inhibitor of p38 (p38i) significantly reduced tumor growth, angiogenesis, and lung metastasis. Importantly, p38i decreased the accumulation of myeloid populations, namely, myeloid-derived suppressor cells (MDSCs) and CD163+ tumor-associated macrophages (TAMs). p38 controlled the expression of tumor-derived chemokines/cytokines that facilitated the recruitment of protumor myeloid populations. Depletion of MDSCs was accompanied by reduced TAM infiltration and phenocopied the antimetastatic effects of p38i. Reciprocally, p38i increased tumor infiltration by cytotoxic CD8+ T cells. Furthermore, the CD163+ /CD8+ expression ratio inversely correlated with metastasis-free survival in breast cancer, suggesting that targeting p38 may improve clinical outcomes. Overall, our study highlights a previously unknown p38-driven pathway as a therapeutic target in MBC.
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Antineoplásicos/farmacología , Carcinogénesis/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células Mieloides/efectos de los fármacos , Células Mieloides/patología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Carcinogénesis/efectos de los fármacos , Carcinogénesis/metabolismo , Línea Celular Tumoral , Quimiocinas/metabolismo , Citocinas/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCID , Ratones Transgénicos , Células Mieloides/metabolismo , Células Supresoras de Origen Mieloide/efectos de los fármacos , Células Supresoras de Origen Mieloide/metabolismo , Células Supresoras de Origen Mieloide/patología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Receptores de Superficie Celular/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
We have previously reported that stable expression of a dominant negative Death Receptor 5 (dnDR5) in skeletal myoblasts results in decreased basal caspase activity and decreased mRNA and protein expression of the muscle regulatory transcription factor MyoD in growth medium (GM), resulting in inhibited differentation when myoblasts are then cultured in differentiation media (DM). Further, this decreased level of MyoD mRNA was not a consequence of altered message stability, but rather correlated with decreased acetylation of histones in the distal regulatory region (DRR) of the MyoD extended promoter known to control MyoD transcription. As serum response factor (SRF) is the transcription factor known to be responsible for basal MyoD expression in GM, we compared the level of SRF binding to the non-canonical serum response element (SRE) within the DRR in parental and dnDR5 expressing myoblasts. Herein, we report that stable expression of dnDR5 resulted in decreased levels of serum response factor (SRF) binding to the CArG box in the SRE of the DRR. Total SRF expression levels were not affected, but phosphorylation indicative of SRF activation was impaired. This decreased SRF phosphorylation correlated with decreased phosphorylation-induced activation of p38 kinase. Moreover, the aforementioned signaling events affected by expression of dnDR5 could be appropriately recapitulated using either a pharmacological inhibitor of caspase 3 or p38 kinase. Thus, our results have established a signaling pathway from DR5 through caspases to p38 kinase activation, to SRF activation and the basal expression of MyoD.
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Prostaglandin A2-AcMe (1) and Prostaglandin A2 (2) were isolated from the octocoral Plexaura homomalla and three semisynthetic derivatives (3-5) were then obtained using a reduction protocol. All compounds were identified through one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) experiments. Additionally, evaluation of in vitro cytotoxic activity against the breast (MDA-MB-213) and lung (A549) cancer cell lines, in combination with enzymatic activity and molecular docking studies with the enzymes p38α-kinase, Src-kinase, and topoisomerase IIα, were carried out for compounds 1-5 in order to explore their potential as inhibitors of cancer-related molecular targets. Results showed that prostaglandin A2 (2) was the most potent compound with an IC50 of 16.46 and 25.20 µg/mL against MDA-MB-213 and A549 cell lines, respectively. In addition, this compound also inhibited p38α-kinase in 49% and Src-kinase in 59% at 2.5 µM, whereas topoisomerase IIα was inhibited in 64% at 10 µM. Enzymatic activity was found to be consistent with molecular docking simulations, since compound 2 also showed the lowest docking scores against the topoisomerase IIα and Src-kinase (-8.7 and -8.9 kcal/mol, respectively). Thus, molecular docking led to establish some insights into the predicted binding modes. Results suggest that prostaglandin 2 can be considered as a potential lead for development inhibitors against some enzymes present in cancer processes.
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Antozoos , Antineoplásicos/farmacología , Prostaglandinas/farmacología , Células A549/efectos de los fármacos , Animales , Línea Celular Tumoral/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Océanos y MaresRESUMEN
Primary tumor-derived factors (TDFs) act upon normal cells to generate a pre-metastatic niche, which promotes colonization of target organs by disseminated malignant cells. Here we report that TDFs-induced activation of the p38α kinase in lung fibroblasts plays a critical role in the formation of a pre-metastatic niche in the lungs and subsequent pulmonary metastases. Activation of p38α led to inactivation of type I interferon signaling and stimulation of expression of fibroblast activation protein (FAP). FAP played a key role in remodeling of the extracellular matrix as well as inducing the expression of chemokines that enable lung infiltration by neutrophils. Increased activity of p38 in normal cells was associated with metastatic disease and poor prognosis in human melanoma patients whereas inactivation of p38 suppressed lung metastases. We discuss the p38α-driven mechanisms stimulating the metastatic processes and potential use of p38 inhibitors in adjuvant therapy of metastatic cancers.
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Neoplasias Pulmonares , Transducción de Señal , Fibroblastos/patología , Humanos , Pulmón/patología , Neoplasias Pulmonares/patología , Proteínas QuinasasRESUMEN
P38alpha kinase plays an important role in the regulation of both cell stress response and cell fate. In this study, we report that p38alpha kinase-deficient embryonic stem cells exhibit a higher production of reactive oxygen species (ROS) in contrast to their wild-type counterpart. Analysis of the expressions of NADPH oxidases (NOXs) and dual oxidases, crucial enzymes involved in intracellular ROS formation, shows NOX2/gp91phox is over-expressed in p38alpha deficient cells. The particular increase in superoxide formation was confirmed by the specific detection of hydroethidine derivate 2-hydroxyethidium. ROS formation decreased when the level of NOX2 was silenced by siRNA in p38alpha deficient cells. These data suggest the importance of p38alpha kinase in the regulation of ROS metabolism in embryonic stem cells and the significance of the observed phenomena of cancer cell-like phenotypes, which is discussed.
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Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , NADPH Oxidasa 2/metabolismo , Superóxidos/metabolismo , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Mitocondrias/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/genética , NADPH Oxidasa 2/genéticaRESUMEN
BACKGROUND: Twenty one amide compounds possessing phenoxy/benzyloxy/pyridinyl groups have been synthesized by benzoylation of respective amines in presence of base with moderate to encouraging yields. Upon confirmation of structure, compounds were subjected for p38 kinase inhibitory, anti-inflammatory, antimicrobial and antitubercular activities. METHOD: Anti-inflammatory activity was determined using carrageenan induced rat paw edema model while p38 kinase inhibitory activity was studied using ELISA method and serial dilution method was employed to determine MICs. Two compounds 4g and 4n showed over 30% p38 kinase inhibitory activity at 10 µM and best anti-inflammatory activity was found for compounds 4g, 4i, 4n and 4o which exhibited to reduce paw edema over 70%. Compound 4b was observed to be the most potent against gram +ve organisms with MIC value of 1.6 µG/mL and compound 4u displayed potent antibacterial activity against gram negative organisms. CONCLUSION: Most encouraging antitubercular activity was noticed for compounds 4u, 4r and 4k with 6.25, 12.5 and 12.5 µG/mL Further, in order to know the binding site interactions, a docking simulations of compounds was performed. These preliminary results will certainly show fruitful directions to improve the activities of compounds.
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Antibacterianos/farmacología , Antiinflamatorios no Esteroideos/farmacología , Antifúngicos/farmacología , Benzamidas/farmacología , Edema/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Antifúngicos/síntesis química , Antifúngicos/química , Benzamidas/síntesis química , Benzamidas/química , Carragenina , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Edema/inducido químicamente , Hongos/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Ratas , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Matrix metalloproteinases (MMPs) are known to play an important role in the degradation of the extracellular matrix and the pathological progression of osteoarthritis (OA). The natural polyphenolic compound rosmarinic acid (Ros. A) has been shown to suppress the inhibitory activity of matrix metalloproteinases (MMPs). However, the effects of Ros. A on OA have not been investigated. METHODS: In the current study, primary articular chondrocytes were cultured from rabbit articular cartilage and treated with Ros. A. Phenotypic characterization was performed by western blotting to assess specific markers, prostaglandin E2 (PGE2) assays, and alcian blue staining to measure sulfated-proteoglycan production. RESULTS: We report that in rabbit articular chondrocytes, Ros. A increased type II collagen, sulfated-proteoglycan, cyclooxygenase-2 (COX-2), and PGE2 production in a dose- and time-dependent manner. Furthermore, Ros. A suppressed the expression of MMP-13. In addition, treatment with Ros A activated extracellular signal-regulated kinase (ERK)-1/2 and p38 kinase signaling pathways. Inhibition of MMP-13 enhanced Ros. A-induced type II collagen expression and sulfated-proteoglycan synthesis but COX-2 and PGE2 production were unchanged. Ros. A-mediated up-regulation of ERK phosphorylation was abolished by the MEK inhibitor, PD98059, which prevented induction of the associated inflammatory response. Inhibition of p38 kinase with SB203580 enhanced the increase in type II collagen expression via Ros. A-mediated down-regulation of MMP-13. CONCLUSIONS: Results suggest that ERK-1/2 regulates Ros. A-induced inflammation and that p38 regulates differentiation by inhibiting MMP-13 in rabbit articular chondrocytes.
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Condrocitos/inmunología , Cinamatos/metabolismo , Ciclooxigenasa 2/genética , Depsidos/metabolismo , Inflamación , Metaloproteinasa 13 de la Matriz/genética , Regulación hacia Arriba , Animales , Cartílago Articular/inmunología , Ciclooxigenasa 2/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Conejos , Ácido RosmarínicoRESUMEN
BACKGROUND: TGFß1, a decisive regulator of megakaryocyte maturation and platelet formation, has previously been shown to up-regulate both, store operated Ca2+ entry (SOCE) and Ca2+ extrusion by Na+/Ca2+ exchange. The growth factor thus augments the increase of cytosolic Ca2+ activity ([Ca2+]i) following release of Ca2+ from intracellular stores and accelerates the subsequent decline of [Ca2+]i. The effect on SOCE is dependent on a signaling cascade including p38 kinase, serum & glucocorticoid inducible kinase SGK1, and nuclear factor NFκB. The specific Na+/Ca2+ exchanger isoforms involved and the signalling regulating the Na+/Ca2+ exchangers remained, however elusive. The present study explored, whether TGFß1 influences the expression and function of K+ insensitive (NCX) and K+ sensitive (NCKX) Na+/Ca2+ exchangers, and aimed to shed light on the signalling involved. METHODS: In human megakaryocytic cells (MEG01) RT-PCR was performed to quantify NCX/NCKX isoform transcript levels, [Ca2+]i was determined by Fura-2 fluorescence, and Na+/Ca2+ exchanger activity was estimated from the increase of [Ca2+]i following switch from an extracellular solution with 130 or 90 mM Na+ and 0 mM Ca2+ to an extracellular solution with 0 Na+ and 2 mM Ca2+. K+ concentration was 0 mM for analysis of NCX and 40 mM for analysis of NCKX. RESULTS: TGFß1 (60 ng/ml, 24 h) significantly increased the transcript levels of NCX1, NCKX1, NCKX2 and NCKX5. Moreover, TGFß1 (60 ng/ml, 24 h) significantly increased the activity of both, NCX and NCKX. The effect of TGFß1 on NCX and NCKX transcript levels and activity was significantly blunted by p38 kinase inhibitor Skepinone-L (1 µM), the effect on NCX and NCKX activity further by SGK1 inhibitor GSK-650394 (10 µM) and NFκB inhibitor Wogonin (100 µM). CONCLUSIONS: TGFß1 markedly up-regulates transcription of NCX1, NCKX1, NCKX2, and NCKX5 and thus Na+/Ca2+ exchanger activity, an effect requiring p38 kinase, SGK1 and NFκB.
Asunto(s)
Proteínas Inmediatas-Precoces/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Benzoatos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Calcio/metabolismo , Línea Celular , Dibenzocicloheptenos/farmacología , Flavanonas/farmacología , Humanos , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Inmediatas-Precoces/genética , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Microscopía Fluorescente , FN-kappa B/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Intercambiador de Sodio-Calcio/genética , Transcripción Genética/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
N-3 polyunsaturated fatty acids (PUFAs) improve endothelial function. The arachidonic acid-derived metabolites (epoxyeicosatrienoic acids (EETs)) are part of the endothelial hyperpolarization factor and are vasodilators independent of nitric oxide. However, little is known regarding the regulation of EET concentration by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in blood vessels. Sprague-Dawley rats were fed either a control or fish oil diet for 3 weeks. Compared with the control, the fish oil diet improved acetylcholine-induced vasodilation and reduced the protein expression of soluble epoxide hydrolase (sEH), a key EET metabolic enzyme, in aortic strips. Both DHA and EPA suppressed sEH protein expression in rat aorta endothelial cells (RAECs). Furthermore, the concentration of 4-hydroxy hexenal (4-HHE), a lipid peroxidation product of n-3 PUFAs, increased in n-3 PUFA-treated RAECs. In addition, 4-HHE treatment suppressed sEH expression in RAECs, suggesting that 4-HHE (derived from n-3 PUFAs) is involved in this phenomenon. The suppression of sEH was attenuated by the p38 kinase inhibitor (SB203580) and by treatment with the antioxidant N-acetyl-L-cysteine. In conclusion, sEH expression decreased after n-3 PUFAs treatment, potentially through oxidative stress and p38 kinase. Mild oxidative stress induced by n-3 PUFAs may contribute to their cardio-protective effect.
Asunto(s)
Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Células Endoteliales/efectos de los fármacos , Epóxido Hidrolasas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Acetilcolina/farmacología , Alimentación Animal/análisis , Animales , Antígenos CD , Aorta/efectos de los fármacos , Cadherinas , Suplementos Dietéticos , Ácidos Docosahexaenoicos/química , Ácido Eicosapentaenoico/química , Células Endoteliales/metabolismo , Epóxido Hidrolasas/genética , Aceites de Pescado/química , Análisis de los Alimentos , Genes Supresores de Tumor , Proteínas Nucleares , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Arteria Renal/citología , Vasodilatación/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
p38α is a Ser/Thr protein kinase involved in a variety of cellular processes and pathological conditions, which makes it a promising pharmacological target. Although the activity of the enzyme is highly regulated, its molecular mechanism of activation remains largely unexplained, even after decades of research. By using state-of-the-art molecular dynamics simulations, we decipher the key elements of the complex molecular mechanism refined by evolution to allow for a fine tuning of p38α kinase activity. Our study describes for the first time the molecular effects of different regulators of the enzymatic activity, and provides an integrative picture of the activation mechanism that explains the seemingly contradictory X-ray and NMR data.
Asunto(s)
Activación Enzimática , Simulación de Dinámica Molecular , Proteínas Quinasas p38 Activadas por Mitógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
Mitogen-activated protein (MAP) kinases are important players in cellular signaling pathways. Recently, it has been shown that CacyBP/SIP serves as a phosphatase for one of the MAP kinases, ERK1/2. Through dephosphorylation of this kinase CacyBP/SIP modulates the transcriptional activity of Elk-1 and the activity of the CREB-BDNF pathway. In this work, using NB2a cell lysate and recombinant proteins, we show that CacyBP/SIP binds and dephosphorylates another member of the MAP kinase family, p38. Analysis of recombinant full-length CacyBP/SIP and its three major domains, N-terminal, middle CS and C-terminal SGS, indicates that the middle CS domain is responsible for p38 dephosphorylation. Moreover, we show that CacyBP/SIP might be implicated in response to oxidative stress. Dephosphorylation of phospho-p38 by CacyBP/SIP in NB2a cells treated with hydrogen peroxide is much more effective than in control ones. In conclusion, involvement of CacyBP/SIP in the regulation of p38 kinase activity, in addition to that of ERK1/2, might point to the function of CacyBP/SIP in pro-survival and pro-apoptotic pathways.