RESUMEN
Glucocorticoids (GCs) have been widely used in clinical treatment as anti-inflammatory, anti-shock and immunosuppressive medicines. However, the effect of excessive GCs on immune response and metabolism of kidney remains unclear. Here, we profiled the gene expression of kidney from mice with high-dose dexamethasone (DEX) treatment. A total of 1193 differentially expressed genes (DEGs) were screened in DEX treatment group compared with the saline group, including 715 down- regulated and 478 up-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of these DEGs showed extracellular matrix (ECM)-receptor interaction, cell adhesion molecules signaling pathway were significantly enriched, and that the vast majority of DEGs were involved in monocarboxylic acid metabolism, leukocyte cell-cell adhesion and fatty acid metabolism. Gene set enrichment analysis (GSEA) revealed that DEGs were strongly associated with immune-response and cell adhesion gene sets, such as Fc γ R-mediated phagocytosis, leukocyte transendothelial migration, T-cell receptor signaling pathway, cell adhesion, ECM-receptor interaction and focal adhesion-associated pathways. KEGG pathway analysis of differentially expressed kinases (DEKs) showed T-cell receptor and forkhead box class O signaling pathway were enriched. Furthermore, we found multiple protein kinases expression were dysregulated greatly after dexamethasone treatment, including classical effector of GCs stimulation-serum and GC-regulated kinase. These protein kinases are involved in multiple signaling pathways in mice kidney, such as mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. We profiled the gene expression of the kidney from high-dose dexamethasone-treated mice and provided important information for further study the mechanism of side effects of GCs in clinical therapy.
Asunto(s)
Antiinflamatorios/efectos adversos , Dexametasona/efectos adversos , Riñón/metabolismo , Metabolismo/efectos de los fármacos , Proteínas Quinasas/biosíntesis , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/inmunología , Movimiento Celular/efectos de los fármacos , Biología Computacional , Dexametasona/administración & dosificación , Dexametasona/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Inmunidad/efectos de los fármacos , Inflamación/metabolismo , Inyecciones Intraperitoneales , Riñón/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Morphine is a potent opioid analgesic with high propensity for the development of antinociceptive tolerance. Morphine antinociception and tolerance are partially regulated by the midbrain ventrolateral periaqueductal gray (vlPAG). However, the majority of research evaluating mu-opioid receptor signaling has focused on males. Here, we investigate kinase activation and localization patterns in the vlPAG following acute and chronic morphine treatment in both sexes. Male and female mice developed rapid antinociceptive tolerance to morphine (10 mg/kg i.p.) on the hot plate assay, but tolerance did not develop in males on the tail flick assay. Quantitative fluorescence immunohistochemistry was used to map and evaluate the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2), protein kinase-C (PKC), and protein kinase-A (PKA). We observed significantly greater phosphorylated ERK 1/2 in the vlPAG of chronic morphine-treated animals which co-localized with the endosomal marker, Eea1. We note that pPKC is significantly elevated in the vlPAG of both sexes following chronic morphine treatment. We also observed that although PKA activity is elevated following chronic morphine treatment in both sexes, there is a significant reduction in the nuclear translocation of its phosphorylated substrate. Taken together, this study demonstrates increased activation of ERK 1/2, PKC, and PKA in response to repeated morphine treatment. The study opens avenues to explore the impact of chronic morphine treatment on G-protein signaling and kinase nuclear transport.
Asunto(s)
Inducción Enzimática/efectos de los fármacos , Morfina/farmacología , Sustancia Gris Periacueductal/efectos de los fármacos , Sustancia Gris Periacueductal/enzimología , Proteínas Quinasas/biosíntesis , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Tolerancia a Medicamentos , Femenino , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Dimensión del Dolor/efectos de los fármacos , Proteína Quinasa C/metabolismo , Transporte de Proteínas , Caracteres Sexuales , Proteínas de Transporte Vesicular/biosíntesis , Proteínas de Transporte Vesicular/genéticaRESUMEN
SR1 is a dual-function sRNA from Bacillus subtilis. It inhibits translation initiation of ahrC mRNA encoding the transcription activator of the arginine catabolic operons. Base-pairing is promoted by the RNA chaperone CsrA, which induces a slight structural change in the ahrC mRNA to facilitate SR1 binding. Additionally, SR1 encodes the small protein SR1P that interacts with glyceraldehyde-3P dehydrogenase A to promote binding to RNase J1 and enhancing J1 activity. Here, we describe a new target of SR1, kinA mRNA encoding the major histidine kinase of the sporulation phosphorelay. SR1 and kinA mRNA share 7 complementary regions. Base-pairing between SR1 and kinA mRNA decreases kinA translation without affecting kinA mRNA stability and represses transcription of the KinA/Spo0A downstream targets spoIIE, spoIIGA and cotA. The initial interaction between SR1 and kinA mRNA occurs 10 nt downstream of the kinA start codon and is decisive for inhibition. The sr1 encoded peptide SR1P is dispensable for kinA regulation. Deletion of sr1 accelerates sporulation resulting in low quality spores with reduced stress resistance and altered coat protein composition which can be compensated by sr1 overexpression. Neither CsrA nor Hfq influence sporulation or spore properties.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Biosíntesis de Proteínas , Proteínas Quinasas/genética , ARN Pequeño no Traducido/fisiología , Bacillales/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Emparejamiento Base , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Proteínas Quinasas/biosíntesis , Estabilidad del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/metabolismo , Esporas Bacterianas/química , Esporas Bacterianas/genética , Esporas Bacterianas/fisiología , Factores de Transcripción/metabolismoRESUMEN
Background: Mixed lineage kinase domain-like pseudokinase (MLKL), one of the main downstream components of the necroptosis or programmed necrosis has recently been demonstrated in advanced atherosclerotic lesions. However, its precise role in the atherosclerosis pathogenesis still requires more elucidation. Our study was set to delineate both the changes in peripheral MLKL gene expression and its influence on disease severity in atherosclerotic patients with and without type 2 diabetes mellitus. Methods: The study involved 50 patients (20 non-diabetics and 30 diabetics) undergoing coronary artery bypass graft and 20 apparently healthy controls. Taqman RT-PCR was used to quantify MLKL mRNA expression levels, while ELISA was employed to estimate serum insulin and high sensitivity C-reactive protein (hsCRP) levels. Results: Compared with the control group, MLKL gene was up regulated significantly in cardiovascular diseases (CVD; p ≤ 0.001). Higher MLKL expression was demonstrated in diabetic CVD group than non-diabetic group (p < 0.05). Correlation studies reported positive associations between MLKL and markers of dyslipidemia, inflammation, and insulin resistance. Multiple regression analysis revealed that FBG levels, hsCRP levels, and total white blood cells count were significant predictors for MLKL levels. Receiver operating characteristic curve showed a significant diagnostic value of MLKL for CVD. Moreover, regression analysis demonstrated that MLKL and hsCRP were independent predicting factors for the severity of CVD. Conclusion: MLKL is linked to hallmarks of atherosclerosis and could explain increased cardiovascular risk in diabetic patients. Thus, it can be a potential drug target for treatment of atherosclerotic patients.
Asunto(s)
Aterosclerosis/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteínas Quinasas/biosíntesis , Aterosclerosis/epidemiología , Aterosclerosis/genética , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/genética , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Proteínas Quinasas/genéticaRESUMEN
Sharp eyespot, caused by necrotrophic fungus Rhizoctonia cerealis, is a serious fungal disease in wheat (Triticum aestivum). Certain wall-associated receptor kinases (WAK) mediate resistance to diseases caused by biotrophic/hemibiotrophic pathogens in several plant species. Yet, none of wheat WAK genes with positive effect on the innate immune responses to R. cerealis has been reported. In this study, we identified a WAK gene TaWAK7D, located on chromosome 7D, and showed its positive regulatory role in the defense response to R. cerealis infection in wheat. RNA-seq and qRT-PCR analyses showed that TaWAK7D transcript abundance was elevated in wheat after R. cerealis inoculation and the induction in the stem was the highest among the tested organs. Additionally, TaWAK7D transcript levels were significantly elevated by pectin and chitin treatments. The knock-down of TaWAK7D transcript impaired resistance to R. cerealis and repressed the expression of five pathogenesis-related genes in wheat. The green fluorescent protein signal distribution assays indicated that TaWAK7D localized on the plasma membrane in wheat protoplasts. Thus, TaWAK7D, which is induced by R. cerealis, pectin and chitin stimuli, positively participates in defense responses to R. cerealis through modulating the expression of several pathogenesis-related genes in wheat.
Asunto(s)
Resistencia a la Enfermedad , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/microbiología , Proteínas de Plantas , Proteínas Quinasas , Rhizoctonia/crecimiento & desarrollo , Triticum , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/genética , Triticum/enzimología , Triticum/genética , Triticum/microbiologíaRESUMEN
The factors influencing Leydig cell maturity and the acquisition of functional capacity are incompletely defined. Here we analyzed the constant light (LL) influence on Leydig cells' endocrine function during reproductive maturation. Rats were exposed to LL from P21 to P90. Data were collected at juvenile (P35), peri/pubertal (P42, P49), and adult (P90) stages of life. The results proved the effect of LL on rats' physiology by changing of bimodal voluntary activity pattern into free-running. Additionally, the peripheral clock in Leydig cells changed in LL condition, indicating disturbed rhythm: the positive element (Bmal1) increased in pre-/pubertal but decreased in the adult period, while negative elements (Per2 and Reverba) were increased. The effects of LL were most prominent in puberty: pituitary genes encoding gonadotropic hormones (Cga, Lhb, Fshb) decreased; serum corticosterone increased, while serum androgens and mass of testicular and sex accessory organs reduced; markers of Leydig cells maturity/differentiation (Insl3, Lhcgr) and steroidogenesis-related genes (Scarb1, Star, Cyp11a1, Cyp17a1) decreased; the steroidogenic and energetic capacity of the Leydig cell mitochondria decreased; the mtDNA copy number reduced, and mitochondrial dynamics markers changed: fusion decreased (Opa1 and Mfn2), and mitophagy increased (Pink1). In adults, the negative effect of LL on mitochondrial function and steroidogenic capacity persists in adult Leydig cells while other parameters reached control values. Altogether, the results indicate that LL slows down Leydig cells' maturation by reducing the endocrine and energy capacity of cells leading to the delay of reproductive development.
Asunto(s)
Corticosterona/sangre , Sistema Endocrino/fisiología , Células Intersticiales del Testículo/metabolismo , Luz , Adenosina Trifosfato/metabolismo , Andrógenos/farmacología , Animales , Peso Corporal , Diferenciación Celular , ADN Mitocondrial/metabolismo , GTP Fosfohidrolasas/biosíntesis , Hormona Luteinizante/sangre , Masculino , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Proteínas Mitocondriales/biosíntesis , Tamaño de los Órganos , Hipófisis/efectos de los fármacos , Proteínas Quinasas/biosíntesis , Ratas , Ratas Wistar , Maduración Sexual , Esteroides/metabolismo , Testosterona/sangreRESUMEN
Circular RNAs (circRNAs) have emerged as vital regulators in the chemoresistance of diverse human tumors, including ovarian cancer. In the present study, we attempted to explore the function of circ_CELSR1 in paclitaxel resistance of ovarian cancer. Quantitative real-time PCR (qRT-PCR) was conducted for the expression of circ_CELSR1, miR-149-5p and salt inducible kinase 2 (SIK2). Cell Counting Kit-8 (CCK-8) assay was performed to evaluate the half-maximal inhibitory concentration (IC50) of paclitaxel and cell viability. Colony formation assay was adopted for cell colony formation. Flow cytometry analysis was conducted to analyze cell cycle process and apoptosis. Western blot assay was utilized to determine the protein levels. RNA immunoprecipitation (RIP) and dual-luciferase reporter assays were conducted to verify the association between miR-149-5p and circ_CELSR1 or SIK2. Murine xenograft model assay was carried out to determine the effect of circ_CELSR1 in paclitaxel resistance in vivo. Circ_CELSR1 was upregulated in paclitaxel-resistant ovarian cancer tissues and cells. Circ_CELSR1 knockdown enhanced paclitaxel sensitivity and cell apoptosis and repressed cell viability, colony formation and cell cycle process in paclitaxel-resistant ovarian cancer cells. For mechanism analysis, circ_CELSR1 could positively modulate SIK2 expression via sponging miR-149-5p. MiR-149-5p inhibition effectively restored the impacts of circ_CELSR1 knockdown on paclitaxel resistance and cell progression in paclitaxel-resistant ovarian cancer cells. MiR-149-5p overexpression suppressed paclitaxel resistance and cell progression in paclitaxel-resistant ovarian cancer cells by interacting with SIK2. In addition, circ_CELSR1 silencing impeded paclitaxel resistance of ovarian cancer in vivo. Circ_CELSR1 improved the resistance of ovarian cancer to paclitaxel by regulating miR-149-5p/SIK2 axis.
Asunto(s)
Resistencia a Antineoplásicos/fisiología , MicroARNs/biosíntesis , Neoplasias Ováricas/patología , Paclitaxel/farmacología , Proteínas Quinasas/biosíntesis , ARN Circular/biosíntesis , Animales , Apoptosis/fisiología , Ciclo Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Regulación hacia Arriba/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
An abundance of protein structures has been solved in the last six decades that are paramount in defining the function of such proteins. For unsolved protein structures, however, predictions based on sequence and phylogenetic similarity can be useful for identifying key domains of interaction. Here, we describe expression and purification of a recombinant plant LRR-RLK ectodomain MIK1 using a modified baculovirus-mediated expression system with subsequent N-linked glycosylation analysis using LC-MS/MS and computational sequence-based analyses. Though highly ubiquitous, glycosylation site specificity and the degree of glycosylation influenced by genetic and exogenous factors are still largely unknown. Our experimental analysis of N-glycans on MIK1 identified clusters of glycosylation that may explicate the regions involved in MIK1 ectodomain binding. Whether these glycans are necessary for function is yet to be determined. Phylogenetic comparison using multiple sequence alignment between MIK1 and other LRR-RLKs, namely TDR in Arabidopsis thaliana, revealed conserved structural motifs that are known to play functional roles in ligand and receptor binding.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Expresión Génica , Filogenia , Proteínas Quinasas , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Quinasas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Invasion of hepatocytes by Plasmodium sporozoites initiates the pre-erythrocytic step of a malaria infection. Subsequent development of the parasite within hepatocytes and exit from them is essential for starting the disease-causing erythrocytic cycle. Identification of signaling pathways that operate in pre-erythrocytic stages provides insight into a critical step of infection and potential targets for chemoprotection from malaria. We demonstrate that P. berghei homologs of Calcium Dependent Protein Kinase 1 (CDPK1), CDPK4 and CDPK5 play overlapping but distinct roles in sporozoite invasion and parasite egress from hepatocytes. All three kinases are expressed in sporozoites. All three are required for optimal motility of sporozoites and consequently their invasion of hepatocytes. Increased cGMP can compensate for the functional loss of CDPK1 and CDPK5 during sporozoite invasion but cannot overcome loss of CDPK4. CDPK1 and CDPK5 expression is downregulated after sporozoite invasion. CDPK5 reappears in a subset of late stage liver stages and is present in all merosomes. Chemical inhibition of CDPK4 and depletion of CDPK5 in liver stages implicate these kinases in the formation and/or release of merosomes from mature liver stages. Furthermore, depletion of CDPK5 in merosomes significantly delays initiation of the erythrocytic cycle without affecting infectivity of hepatic merozoites. These data suggest that CDPK5 may be required for the rupture of merosomes. Our work provides evidence that sporozoite invasion requires CDPK1 and CDPK5, and suggests that CDPK5 participates in the release of hepatic merozoites.
Asunto(s)
Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Malaria/epidemiología , Merozoítos/enzimología , Plasmodium berghei/enzimología , Proteínas Quinasas/biosíntesis , Proteínas Protozoarias/biosíntesis , Esporozoítos/enzimología , Animales , Eritrocitos/enzimología , Eritrocitos/parasitología , Femenino , Células Hep G2 , Humanos , Hígado/enzimología , Hígado/parasitología , Malaria/patología , RatonesRESUMEN
BACKGROUND: Rheumatoid arthritis (RA) is an inflammatory disease, which seriously affects human joints. This study aimed to detect the changes in the expression of long non-coding RNA growth arrest-specific transcript 5 (GAS5) in peripheral blood mononuclear cells (PBMCs) derived from patients with RA and healthy controls (HC), as well as analyze the correlation between GAS5 and clinical indicators of RA. Also, the role and mechanism of GAS5 in regulating the AMP-activated protein kinase (AMPK) pathway in RA was further assessed. METHODS: The PBMCs were isolated from the RA patients. Next, GAS5 expression was detected in RA PBMCs by quantitative real-time polymerase chain reaction, and its diagnostic value on RA was determined by receiver operating characteristic curves (ROC). The levels of interleukin (IL)-6 and IL-17 were detected via enzyme-linked immunosorbent assay. The expressions of total and phosphorylated AMPK as well as p38MAPK were determined with Western blot. RESULTS: GAS5 was down-regulated in RA PBMCs, and consequently serves as a potential diagnostic marker for RA (sensitivity, 90%; specificity, 80%; area under the curve, 0.89). Further, GAS5 negatively regulated erythrocyte sedimentation rate, C-reactive protein, Disease Activity Score of 28 joints and antibodies against cyclic citrullinated peptide, as well as the IL-6 and IL-17 levels of RA PBMCs. Similarly, GAS5 was observed to activate the AMPK pathway. CONCLUSION: GAS5 activated the AMPK pathway, while it negatively regulated the expression of cytokines IL-6 and IL-17.
Asunto(s)
Artritis Reumatoide/genética , Leucocitos Mononucleares/metabolismo , Proteínas Quinasas/genética , ARN Largo no Codificante/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Adulto , Anciano , Animales , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Leucocitos Mononucleares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteínas Quinasas/biosíntesis , ARN/genética , ARN/metabolismo , ARN Largo no Codificante/biosíntesis , Sinoviocitos/metabolismo , Sinoviocitos/patología , Adulto JovenRESUMEN
Parkinson's disease (PD) is a devastating neurodegenerative disease that leads to motor deficits and selective destruction of nigrostriatal dopaminergic neurons. PD is typically treated by dopamine replacement agents; however, dopamine replacement loses effectiveness in the later stages of the disease. Here, we describe the neuroprotective effects of the omega-3 fatty acid docosahexaenoic acid (DHA) in the medial forebrain bundle 6-hydroxydopamine (6-OHDA) model of advanced-stage PD in rats. We show that daily administration of DHA protects against core symptoms of PD, including deficits in postural stability, gait integrity, and dopamine neurochemistry in motor areas of the striatum. Our results also demonstrate that DHA increases striatal dopamine synthesis via phosphorylation of the rate-limiting catecholamine synthesizing enzyme tyrosine hydroxylase, in a manner dependent on the second messenger-linked protein kinases PKA and PKC. We also show that DHA specifically reverses dopamine loss in the nigrostriatal pathway, with no effect in the mesolimbic or mesocortical pathways. This suggests that DHA is unlikely to produce pharmacotherapeutic or adverse effects that depend on dopamine pathways other than the nigrostriatal pathway. To our knowledge, previous reports have not examined the effects of DHA in such an advanced-stage model, documented that the dopamine synthesizing effects of DHA in vivo are mediated through the activation of protein kinases and regulation of TH activity, or demonstrated specificity to the nigrostriatal pathway. These novel findings corroborate the beneficial effects of omega-3 fatty acids seen in PD patients and suggest that DHA provides a novel means of protecting patients for dopamine neurodegeneration.
Asunto(s)
Cuerpo Estriado/enzimología , Ácidos Docosahexaenoicos/uso terapéutico , Dopamina/biosíntesis , Trastornos Parkinsonianos/enzimología , Trastornos Parkinsonianos/prevención & control , Proteínas Quinasas/biosíntesis , Animales , Cuerpo Estriado/efectos de los fármacos , Ácidos Docosahexaenoicos/farmacología , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Oxidopamina/toxicidad , Trastornos Parkinsonianos/inducido químicamente , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
AIMS: 1,8-Cineole is a plant-derived monoterpene and a major constituent of Eucalyptus essential oil. Previously, we demonstrated that 1,8-cineole inhibited hepatocellular carcinoma (HCC) HepG2 cell growth. However, the underlying mechanisms remain unknown. Here, we evaluated the mechanisms of action of 1,8-cineole and the potential benefits of its combination with anticancer compounds harboring "anti-senescence" properties in HepG2 cells. MAIN METHODS: Cell viability was determined by the MTT assay. Cell cycle was assessed through flow cytometry (FC) and western blot (WB). Senescence was determined by the SA-ß-galactosidase assay, and apoptosis by caspase-3 activity, WB, and TUNEL. MAPKs (ERK, JNK, and p38), AMPK, and Akt/mTOR were analyzed by WB. Reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were evaluated by FC and fluorescence microscopy. KEY FINDINGS: 1,8-Cineole inhibited cell proliferation by promoting G0/G1 arrest. While 1,8-cineole was unable to trigger apoptosis, it induced cellular senescence. 1,8-Cineole promoted ROS production, ΔΨm depolarization, AMPK, ERK, and p38 activation and mTOR inhibition. Antioxidants, like N-acetyl-L-cysteine and vitamins, prevented HepG2 cell growth inhibition and senescence induced by 1,8-cineole. Pre-incubation with 1,8-cineole sensitized HepG2 cells to the anti-senescence compounds, quercetin, simvastatin, U0126, and SB202190. Combinations of 1,8-cineole and each compound synergistically inhibited cell viability, and combined treatment with 1,8-cineole and simvastatin induced apoptosis. SIGNIFICANCE: 1,8-Cineole induces G0/G1 arrest and senescence in HepG2 cells through oxidative stress and MAPK, AMPK, and Akt/mTOR pathways, and sensitizes cells to anti-senescence drugs, suggesting that 1,8-cineole has potential as an antineoplastic and adjuvant compound in combination with anti-senescence drugs in HCC therapy.
Asunto(s)
Puntos de Control del Ciclo Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Eucaliptol/farmacología , Fase G1/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Antioxidantes/farmacología , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Eucaliptol/administración & dosificación , Células Hep G2 , Humanos , Proteínas Quinasas/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: Scutellarin, an herbal compound, can effectively suppress the inflammatory response in activated microglia/brain macrophage(AM/BM) in experimentally induced cerebral ischemia; however, the underlying mechanism for this has not been fully clarified. We sought to elucidate if scutellarin would exert its anti-inflammatory effects on AM/BM through the MAPKs pathway. MATERIALS AND METHODS: Western blot and immunofluorescence labeling were used to determine the expression of the MAPKs pathway in AM/BM in rats subjected to middle cerebral artery occlusion (MCAO) also in lipopolysaccharide (LPS)-activated BV-2 microglia in vitro. Furthermore, expression of p-p38 along with that of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta(IL-1ß), and inducible nitric oxide synthase (iNOS) in LPS-activated microglia subjected to pretreatment with p38 inhibitor SB203580, p38 activator sc-201214, scutellarin, or a combination of them was evaluated. FINDINGS: Scutellarin markedly attenuated the expression of p-p38, p-JNK in AM/BM in MCAO rats and in vitro. Conversely, p-ERK1/2 expression level was significantly increased by scutellarin. Meanwhile, scutellarin suppressed the expression of proinflammatory mediators including iNOS, TNF-α, and IL-1ß in AM/BM. More importantly, SB203580 suppressed p-p38 protein expression level in LPS-activated BV-2 microglia that was coupled with decreased expression of proinflammatory mediators (TNF-α, iNOS) in LPS-activated BV-2 microglia. However, p38 activator sc-201214 increased expression of proinflammatory mediators TNF-α, iNOS, and IL-1ß. Interestingly, the decreased expression of both proinflammatory markers by p38 MAPK inhibitor and increased expression of proinflammatory markers by p38 MAPK activator were compatible with that in BV-2-activated microglia pretreated with scutellarin. CONCLUSIONS: The results suggest that scutellarin down-regulates the expression of proinflammatory mediators in AM/BM through suppressing the p-JNK and p-p38 MAPKs. Of note, the anti-inflammatory effect of p38 MAPK inhibitor and scutellarin is comparable. Besides, p38 MAPKs activator reverses the effect of scutellarin. Additionally, scutellarin increases p-ERK1/2 expression that may be neuroprotective.
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Antiinflamatorios no Esteroideos/farmacología , Apigenina/farmacología , Glucuronatos/farmacología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Microglía/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Apigenina/uso terapéutico , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucuronatos/uso terapéutico , Imidazoles/farmacología , Infarto de la Arteria Cerebral Media/patología , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/genética , Piridinas/farmacología , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Recent discoveries revealed several types of programmed necrosis, such as necroptosis, ferroptosis, pyroptosis, etc. Necroptosis is mediated by signaling complexes with receptor-interacting protein kinases (RIPs) and mixed lineage kinase domain-like protein (MLKL). Here, we described an MLKL mediated non-canonical necroptosis through reactive oxygen species (ROS) in lung cancer cells triggered by a natural compound, tanshinol A (TSA). Morphologically, TSA-induced necrotic cell death is characterized by increased cell volume, transparent of cytoplasm, and rupture of the cell membrane. Biochemically, it induces intracellular ATP depletion and PI penetration. Molecularly, TSA-induced cell death is mediated by MLKL but independent of RIP1 and RIP3. Furthermore, TSA induces MLKL phosphorylation and membrane translocation, and cytosolic calcium accumulation. However, calcium shows no effect on TSA-induced cell death. Especially, TSA induces intracellular ROS generation, which was found to be the upstream of MLKL. Collectively, our data indicated that TSA triggers a novel type of programmed necrosis mediated by MLKL in lung cancer cells, which might have therapeutic potentials for lung cancer treatment.
Asunto(s)
Ácidos Cafeicos/farmacología , Neoplasias Pulmonares/metabolismo , Necroptosis/efectos de los fármacos , Proteínas Quinasas/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Células A549 , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Inducción Enzimática/efectos de los fármacos , Humanos , Neoplasias Pulmonares/patología , Fosforilación/efectos de los fármacosRESUMEN
The liver fluke Fasciola hepatica causes fasciolosis, a foodborne zoonosis affecting humans and livestock worldwide. A reliable quantification of gene expression in all parasite life stages relevant for targeting by anthelmintics in the mammalian host is fundamental. The aim of this study was to define a set of stably expressed reference genes for qRT-PCR in Fasciola studies. We determined the expression stabilities of eight candidate reference genes by the algorithms NormFinder, geNorm, BestKeeper, and comparative ΔCT method. The most stably expressed reference genes for the comparison of intra-mammalian life stages were glutamyl-prolyl-tRNA synthetase (Fheprs) and tubulin-specific chaperone D (Fhtbcd). The two best reference genes for analysis of in vitro-cultured juveniles were Fhtbcd and proteasome subunit beta type-7 (Fhpsmb7). These genes should replace the housekeeping gene gapdh which is used in most Fasciola studies to date, but in fact was differentially expressed in our analysis. Based on the new reference genes, we quantified expression of five kinases (Abl1, Abl2, PKC, Akt1, Plk1) discussed as targets in other parasitic flatworms. Distinct expression patterns throughout development were revealed and point to interesting biological functions. We like to motivate using this set of validated reference genes for future F. hepatica research, such as studies on drug targets or parasite development.
Asunto(s)
Fasciola hepatica , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Proteínas del Helminto , Proteínas Quinasas , Animales , Fasciola hepatica/enzimología , Fasciola hepatica/genética , Proteínas del Helminto/biosíntesis , Proteínas del Helminto/genética , Humanos , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/genéticaRESUMEN
Brassinosteroids (BRs) regulate plant growth and stress responses. BRASSINOSTEROID-INSENSITIVE 1 (BRI1) is a BR receptor that perceives BRs and subsequently activates BR signaling. However, how BR contents and BRI1 expression levels affect the drought resistance of tomato requires further investigation. Here, we exogenously applied 24-epibrassinolide (EBR) and brassinazole (Brz) to tomato plants and generated different transgenic tomato SlBRI1 overexpression lines to study the drought stress response. Our results showed that EBR application 3 days before drought stress increased the contents of BRs and decreased abscisic acid (ABA) and reactive oxygen species (ROS), after which stomatal aperture and drought resistance eventually increased. Brz application reduced the drought resistance. Astonishingly, overexpression of 35S:SlBRI1, which increased BR signaling intensity, led to slightly improved contents of ABA and ROS and ultimately reduced both stomatal aperture and drought resistance. Moreover, plants expressing SlBRI1 driven by a stress-inducible promoter (Atrd29A) also exhibited reduced plant drought resistance. In all cases, enhancing the BR signaling intensity reduced antioxidant enzyme activity and reduced the expression of drought stress-related genes, ultimately compromising the drought resistance. Additionally, SlBRI1 mutants with altered brassinolide sensitivity (abs), which was weak BR signaling, exhibited significantly increased drought resistance. Therefore, our results reveal that BR contents positively regulated tomato drought resistance and that BR signaling intensity via BRI1 was negatively related to the drought resistance. These imply that the increased drought resistance in response to BRs is a newly discovered BR signaling branch that is located downstream of BRs and that differs from that of BRI1.
Asunto(s)
Brasinoesteroides/metabolismo , Resistencia a la Enfermedad , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/biosíntesis , Proteínas Quinasas/biosíntesis , Transducción de Señal , Solanum lycopersicum , Deshidratación , Solanum lycopersicum/enzimología , Solanum lycopersicum/genética , Proteínas de Plantas/genética , Proteínas Quinasas/genéticaRESUMEN
Necroptosis is a programmed form of necrotic cell death, which is tightly regulated by the necroptotic signaling pathway containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL) protein. In addition to the RIP1-RIP3-MLKL axis, other factors regulating necroptosis are still largely unknown. Here a cell-based small-molecule screening led to the finding that BET inhibitors protected cells from necroptosis in the TNFα/Smac-mimetic/Z-VAD-FMK (TSZ)-induced cell necroptosis model. Mechanistic studies revealed that BET inhibitors acted by downregulating MLKL expression. Further research demonstrated that BRD4, IRF1, P-TEFb, and RNA polymerase II formed a transcription complex to regulate the expression of MLKL, and BET inhibitors interfered with the transcription complex formation. In necroptosis-related disease model, the BET inhibitor JQ-1 showed promising therapeutic effects. Collectively, our studies establish, for the first time, BRD4 as a new epigenetic factor regulating necroptosis, and highlight the potential of BET inhibitors in the treatment of necroptosis-related diseases.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Quinasas/biosíntesis , Factores de Transcripción/metabolismo , Animales , Azepinas/farmacología , Proteínas de Ciclo Celular/genética , Línea Celular , Células HEK293 , Células HT29 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necroptosis/fisiología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Factores de Transcripción/genética , Transfección , Triazoles/farmacología , Células U937RESUMEN
Oral squamous cell carcinoma (OSCC) is the most common malignant cancer, with high mortality rates in advanced stages. Recent studies have shown that the expression of ALPK1 mRNA and its inhibitory differentiation function are associated with cancer progression. However, the expression and clinicopathologic features of ALPK1 in OSCC remain unexplored. Herein, the authors investigated the expression patterns of ALPK1 in 39 matched OSCC patients and examined the relationship between ALPK1 protein expression and clinicopathologic factors using immunohistochemical scores. Using Western blot analysis, ALPK1 expression was found to be significantly higher in tumor tissues than that in nontumor tissues. Through an immunoreactive scoring system, a significantly higher number of advanced-stage tumor size T4 and lymph node metastasis N2 exhibited higher ALPK1 expression levels than that exhibited by T1/T2/T3 tumors and N0/N1. In addition, ALPK1 protein expression was aberrant in malignant oral cancer cell lines compared with that in pre-malignant oral epithelial cells, whereas minimal expression was observed in normal oral epithelial cells. Knockdown of ALPK1 resulted in a significant reduction in cell growth, migration, and invasion capacity in vitro. Consequently, expression of N-cadherin and vimentin decreased in ALPK1-deficient cells. Thus, these results suggest that ALPK1 serves as a potential biomarker and target for OSCC development in late stages.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Carcinoma de Células Escamosas/enzimología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Proteínas Quinasas/biosíntesis , Neoplasias de la Lengua/enzimología , Adulto , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Cadherinas/genética , Cadherinas/inmunología , Cadherinas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Queratinocitos/inmunología , Queratinocitos/metabolismo , Queratinocitos/patología , Metástasis Linfática/genética , Metástasis Linfática/inmunología , Metástasis Linfática/patología , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Estadificación de Neoplasias , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Neoplasias de la Lengua/genética , Neoplasias de la Lengua/inmunología , Neoplasias de la Lengua/patología , Vimentina/genética , Vimentina/inmunología , Vimentina/metabolismoRESUMEN
Carboxyl-terminal repeat domain (CTD) of the largest subunit Rpb1 of RNA polymerace II is essential for transcription regulation. Heptapeptide repeat of CTD of Rpb1 is phosphorylated by carboxyl-terminal repeat domain kinase (CTDK-I), composed of CTK1, CTK2 and CTK3, in order to regulate transcription and transcription associated processes. The yeast specific protein CTK3 binds to cyclin CTK2 to form a heterodimer serving as a regulational factor to control CTK1 activity by binding to CTK1. Structural information of CTK2-CTK3 complex is yet to be elucidated. Here, we report the co-expression of CTK2-CTK3 complex from Saccharomyces cerevisiae with N-terminal His6-tag in CTK3 in Escherichia coli (E. coli), purification of the complex by four chromatographic steps and crystallization of the complex as well as the diffraction data collection and processing. This study provides some essential information and a guide for structural and functional study of CTK2-CTK3 complex and CTDK-I in the future.