RESUMEN
In recent years, inflammatory disorders have emerged as a significant concern for human health. Through ongoing research on anti-inflammatory agents, alpinetin has shown promising anti-inflammatory properties, including involvement in epigenetic modification pathways. As a crucial regulator of epigenetic modifications, Mecp2 may play a role in modulating the epigenetic effects of alpinetin, potentially impacting its anti-inflammatory properties. To test this hypothesis, two key components, p65 (a member of NF-KB family) and p300 (a type of co-activator), were screened by the expression profiling microarray, which exhibited a strong correlation with the intensity of LPS stimulation in mouse macrophages. Meanwhile, alpinetin demonstrates the anti-inflammatory properties through its ability to disrupt the synthesis of p65 and its interaction with promoters of inflammatory genes, yet it did not exhibit similar effects on p300. Additionally, Mecp2 can inhibit the binding of p300 by attaching to the methylated inflammatory gene promoter induced by alpinetin, leading to obstacles in promoter acetylation and subsequently impacting the binding of p65, ultimately enhancing the anti-inflammatory capabilities of alpinetin. Similarly, in a sepsis mouse model, it was observed that homozygotes overexpressing Mecp2 showed a greater reduction in organ damage and improved survival rates compared to heterozygotes when administered by alpinetin. However, blocking the expression of DNA methyltransferase 3A (DNMT3A) resulted in the loss of Mecp2's anti-inflammatory assistance. In conclusion, Mecp2 may augment the anti-inflammatory effects of alpinetin through epigenetic 'crosstalk', highlighting the potential efficacy of a combined therapeutic strategy involving Mecp2 and alpinetin for anti-inflammatory intervention.
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Antiinflamatorios , Epigénesis Genética , Flavanonas , Proteína 2 de Unión a Metil-CpG , Regiones Promotoras Genéticas , Proteína 2 de Unión a Metil-CpG/metabolismo , Proteína 2 de Unión a Metil-CpG/genética , Animales , Flavanonas/farmacología , Epigénesis Genética/efectos de los fármacos , Ratones , Antiinflamatorios/farmacología , Células RAW 264.7 , Metilación de ADN/efectos de los fármacos , Lipopolisacáridos/farmacología , Factor de Transcripción ReIA/metabolismo , Sepsis/tratamiento farmacológico , Sepsis/genética , Sepsis/metabolismo , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Inflamación/tratamiento farmacológico , Inflamación/patología , Inflamación/genética , Inflamación/metabolismo , ADN Metiltransferasa 3A/metabolismo , Masculino , Proteína p300 Asociada a E1A/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genéticaRESUMEN
This study aims to analyze the potential biomarkers using bioinformatics technology, explore the pathogenesis, and investigate potential Chinese herbal ingredients for the Clear cell renal cell carcinoma (ccRCC), which could provide theoretical basis for early diagnosis and effective treatment of ccRCC. The gene expression datasets GSE6344 and GSE53757 were obtained from the Gene Expression Omnibus database to screen differentially expressed genes (DEGs) involved in ccRCC carcinogenesis and disease progression. Enrichment analyses, protein-protein interaction networks construction, survival analysis and herbal medicines screening were performed with related software and online analysis platforms. Moreover, network pharmacology analysis has also been performed to screen potential target drugs of ccRCC and molecular docking analysis has been used to validate their effects. Total 274 common DEGs were extracted through above process, including 194 up-regulated genes and 80 down-regulated genes. The enrichment analysis revealed that DEGs were significantly focused on multiple amino acid metabolism and HIF signaling pathway. Ten hub genes, including FLT1, BDNF, LCP2, AGXT2, PLG, SLC13A3, SLC47A2, SLC22A8, SLC22A7, and SLC13A3, were screened. Survival analysis showed that FLT1, BDNF, AGXT2, PLG, SLC47A2, SLC22A8, and SLC12A3 were closely correlated with the overall survival of ccRCC, and AGXT2, SLC47A2, SLC22A8, and SLC22A7 were closely associated with DFS. The potential therapeutic herbs that have been screened were Danshen, Baiguo, Yinxing, Huangqin and Chuanshanlong. The active compounds which may be effective in ccRCC treatment were kaempferol, Scillaren A and (-)-epigallocatechin-3-gallate.
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Carcinoma de Células Renales , Carcinoma , Neoplasias Renales , Humanos , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Factor Neurotrófico Derivado del Encéfalo , Simulación del Acoplamiento Molecular , Farmacología en Red , Biomarcadores , Biología Computacional , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Miembro 3 de la Familia de Transportadores de Soluto 12RESUMEN
BACKGROUND: Advances in next-generation sequencing technologies are changing the ways cancer diagnosis and treatment, which leads to a new branch of precision medicine: "Precision Oncology". This study aims to deliver a structured overview to carry out a bibliometric analysis of precision oncology research over the past 10 years retrospectively. METHODS: Bibliometric methods including clustering analysis and co-occurrence visualized study were conducted based on publications of academic databases Web of Science Main Collection from 1st January 2012, to 31st December 2021. This study analyzed the information about related research outputs, countries, institutions, authors, cited papers, and hot topics. RESULTS: 7163 papers related to precision oncology were identified. Since 2014, the number of articles has proliferated, and oncology precision has attracted significant attention from scholars worldwide in recent years. The USA leads the research in this field, and the League of European Research Universities is the primary research institution. Research institutions from Asia paid more attention to this field through high-level international cooperation. Besides, there are still many issues expected to be explored and evaluated correctly. Such as the considerable uncertainty that pharmacogenomic methods have no significant influence on patient outcomes. CONCLUSIONS: Precision oncology serves as an essential method in clinical treatment, and is closely related to biological study, including biochemistry, molecular and genetics, advanced technology, and pharmacology discovery. The future research prospect would be the broad involvement of social participation and global cooperation in oncology precision research to acquire better results via the balance of technology and public health policy.
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Bibliometría , Neoplasias , Humanos , Estudios Retrospectivos , Oncología Médica , Neoplasias/terapia , Medicina de PrecisiónRESUMEN
Furin is a proprotein convertase that regulates the activation and the inactivation of multiple proteins including matrix metalloproteinases, integrins, and cytokines. It is a serine endoprotease that localizes to the plasma membrane and can be secreted into the extracellular space. The role of furin in regulating inflammation in isolated canine airway smooth muscle tissues was investigated. The treatment of airway tissues with recombinant furin (rFurin) inhibited the activation of Akt and eotaxin secretion induced by IL-13, and it prevented the IL-13-induced suppression of smooth muscle myosin heavy chain expression. rFurin promoted a differentiated phenotype by activating ß1-integrin proteins and stimulating the activation of the adhesome proteins vinculin and paxillin by talin. Activated paxillin induced the binding of Akt to ß-parvin IPP [integrin-linked kinase (ILK), PINCH, parvin] complexes, which inhibits Akt activation. Treatment of tissues with a furin inhibitor or the depletion of endogenous furin using shRNA resulted in Akt activation and inflammatory responses similar to those induced by IL-13. Furin inactivation or IL-13 caused talin cleavage and integrin inactivation, resulting in the inactivation of vinculin and paxillin. Paxillin inactivation resulted in the coupling of Akt to α-parvin IPP complexes, which catalyze Akt activation and an inflammatory response. The results demonstrate that furin inhibits inflammation in airway smooth muscle induced by IL-13 and that the anti-inflammatory effects of furin are mediated by activating integrin proteins and integrin-associated signaling complexes that regulate Akt-mediated pathways to the nucleus. Furin may have therapeutic potential for the treatment of inflammatory conditions of the lungs and airways.
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Furina/farmacología , Inflamación/prevención & control , Integrinas/metabolismo , Interleucina-13/toxicidad , Músculo Liso/efectos de los fármacos , Tráquea/efectos de los fármacos , Animales , Perros , Humanos , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Integrinas/genética , Músculo Liso/inmunología , Músculo Liso/metabolismo , Músculo Liso/patología , Transducción de Señal , Tráquea/inmunología , Tráquea/metabolismo , Tráquea/patologíaRESUMEN
Mechanical tension and humoral stimuli can induce transitions in airway smooth muscle phenotype between a synthetic inflammatory state that promotes cytokine secretion and a differentiated state that promotes the expression of smooth muscle phenotype-specific proteins. When tissues are maintained under high tension, Akt activation and eotaxin secretion are suppressed, but expression of the differentiation marker protein, smooth muscle myosin heavy chain (SmMHC), is promoted. When tissues are maintained under low tension, Akt activation and eotaxin secretion are stimulated, and the differentiated phenotype is suppressed. We hypothesized that mechanical stimuli are differentially transduced to Akt-mediated signaling pathways that regulate phenotype expression by α-parvin and ß-parvin integrin-linked kinase/PINCH/parvin (IPP) signaling complexes within integrin adhesomes. High tension or ACh triggered paxillin phosphorylation and the binding of phospho-paxillin to ß-parvin IPP complexes. This inhibited Akt activation and promoted SmMHC expression. Low tension or IL-4 did not elicit paxillin phosphorylation and triggered the binding of unphosphorylated paxillin to α-parvin IPP complexes, which promoted Akt activation and eotaxin secretion and suppressed SmMHC expression. Expression of a nonphosphorylatable paxillin mutant or ß-parvin depletion by siRNA promoted the inflammatory phenotype, whereas the depletion of α-parvin promoted the differentiated phenotype. Results demonstrate that phenotype expression is regulated by the differential interaction of phosphorylated and unphosphorylated paxillin with α-parvin and ß-parvin IPP complexes and that these complexes have opposite effects on the activation of Akt. Our results describe a novel molecular mechanism for transduction of mechanical and humoral stimuli within integrin signaling complexes to regulate phenotype expression in airway smooth muscle.
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Actinina/genética , Mecanotransducción Celular , Músculo Liso/metabolismo , Paxillin/genética , Proteínas Proto-Oncogénicas c-akt/genética , Tráquea/metabolismo , Acetilcolina/farmacología , Actinina/metabolismo , Animales , Quimiocina CCL11/genética , Quimiocina CCL11/metabolismo , Perros , Femenino , Regulación de la Expresión Génica , Interleucina-4/genética , Interleucina-4/metabolismo , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/genética , Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Paxillin/metabolismo , Fenotipo , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/metabolismo , Tráquea/efectos de los fármacosRESUMEN
The effects of mechanical forces and focal adhesion kinase (FAK) in regulating the inflammatory responses of airway smooth muscle (ASM) tissues to stimulation with interleukin (IL)-13 were investigated. Canine tracheal tissues were subjected to different mechanical loads in vitro, and the effects of mechanical load on eotaxin secretion and inflammatory signaling pathways in response to IL-13 were determined. Eotaxin secretion by tissues in response to IL-13 was significantly inhibited in muscles maintained at a higher (+) load compared with those at a lower (-) load as assessed by ELISA, and Akt activation was also reduced in the higher (+) loaded tissues. Conversely the (+) mechanical load increased activation of the focal adhesion proteins FAK and paxillin in the tissues. The role of FAK in regulating the mechanosensitive responses was assessed by overexpressing FAK-related nonkinase in the tissues, by expressing the FAK kinase-dead mutant FAK Y397F, or by treating tissues with the FAK inhibitor PF-573228. FAK inactivation potentiated Akt activity and increased eotaxin secretion in response to IL-13. FAK inhibition also suppressed the mechanosensitivity of Akt activation and eotaxin secretion. In addition, FAK inactivation suppressed smooth muscle myosin heavy chain expression induced by the higher (+) mechanical load. The results demonstrate that the imposition of a higher mechanical load on airway smooth muscle stimulates FAK activation, which promotes the expression of the differentiated contractile phenotype and suppresses the synthetic phenotype and the inflammatory responses of the muscle tissue.
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Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Músculo Liso/enzimología , Estrés Mecánico , Animales , Quimiocina CCL11/metabolismo , Perros , Activación Enzimática/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Proteínas Fluorescentes Verdes/metabolismo , Inflamación/metabolismo , Inflamación/patología , Interleucina-13/metabolismo , Ratones , Modelos Biológicos , Músculo Liso/efectos de los fármacos , Músculo Liso/patología , Cadenas Pesadas de Miosina/metabolismo , Paxillin/metabolismo , Fenotipo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinolonas/farmacología , Sulfonas/farmacología , Tráquea/patologíaRESUMEN
KEY POINTS: In airway smooth muscle, tension development caused by a contractile stimulus requires phosphorylation of the 20 kDa myosin light chain (MLC), which activates crossbridge cycling and the polymerization of a pool of submembraneous actin. The p21-activated kinases (Paks) can regulate the contractility of smooth muscle and non-muscle cells, and there is evidence that this occurs through the regulation of MLC phosphorylation. We show that Pak has no effect on MLC phosphorylation during the contraction of airway smooth muscle, and that it regulates contraction by mediating actin polymerization. We find that Pak phosphorylates the adhesion junction protein, paxillin, on Ser273, which promotes the formation of a signalling complex that activates the small GTPase, cdc42, and the actin polymerization catalyst, neuronal Wiskott-Aldrich syndrome protein (N-WASP). These studies demonstrate a novel role for Pak in regulating the contractility of smooth muscle by regulating actin polymerization. ABSTRACT: The p21-activated kinases (Pak) can regulate contractility in smooth muscle and other cell and tissue types, but the mechanisms by which Paks regulate cell contractility are unclear. In airway smooth muscle, stimulus-induced contraction requires phosphorylation of the 20 kDa light chain of myosin, which activates crossbridge cycling, as well as the polymerization of a small pool of actin. The role of Pak in airway smooth muscle contraction was evaluated by inhibiting acetylcholine (ACh)-induced Pak activation through the expression of a kinase inactive mutant, Pak1 K299R, or by treating tissues with the Pak inhibitor, IPA3. Pak inhibition suppressed actin polymerization and contraction in response to ACh, but it did not affect myosin light chain phosphorylation. Pak activation induced paxillin phosphorylation on Ser273; the paxillin mutant, paxillin S273A, inhibited paxillin Ser273 phosphorylation and inhibited actin polymerization and contraction. Immunoprecipitation analysis of tissue extracts and proximity ligation assays in dissociated cells showed that Pak activation and paxillin Ser273 phosphorylation triggered the formation of an adhesion junction signalling complex with paxillin that included G-protein-coupled receptor kinase-interacting protein (GIT1) and the cdc42 guanine exchange factor, ßPIX (Pak interactive exchange factor). Assembly of the Pak-GIT1-ßPIX-paxillin complex was necessary for cdc42 and neuronal Wiskott-Aldrich syndrome protein (N-WASP) activation, actin polymerization and contraction in response to ACh. RhoA activation was also required for the recruitment of Pak to adhesion junctions, Pak activation, paxillin Ser273 phosphorylation and paxillin complex assembly. These studies demonstrate a novel role for Pak in the regulation of N-WASP activation, actin dynamics and cell contractility.
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Actinas/fisiología , Contracción Muscular/fisiología , Músculo Liso/fisiología , Paxillin/fisiología , Tráquea/fisiología , Quinasas p21 Activadas/fisiología , Animales , Perros , Femenino , Masculino , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Polimerizacion , Proteína Neuronal del Síndrome de Wiskott-Aldrich/fisiología , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
Craniofacial reconstruction recreates a facial outlook from the cranium based on the relationship between the face and the skull to assist identification. But craniofacial structures are very complex, and this relationship is not the same in different craniofacial regions. Several regional methods have recently been proposed, these methods segmented the face and skull into regions, and the relationship of each region is then learned independently, after that, facial regions for a given skull are estimated and finally glued together to generate a face. Most of these regional methods use vertex coordinates to represent the regions, and they define a uniform coordinate system for all of the regions. Consequently, the inconsistence in the positions of regions between different individuals is not eliminated before learning the relationships between the face and skull regions, and this reduces the accuracy of the craniofacial reconstruction. In order to solve this problem, an improved regional method is proposed in this paper involving two types of coordinate adjustments. One is the global coordinate adjustment performed on the skulls and faces with the purpose to eliminate the inconsistence of position and pose of the heads; the other is the local coordinate adjustment performed on the skull and face regions with the purpose to eliminate the inconsistence of position of these regions. After these two coordinate adjustments, partial least squares regression (PLSR) is used to estimate the relationship between the face region and the skull region. In order to obtain a more accurate reconstruction, a new fusion strategy is also proposed in the paper to maintain the reconstructed feature regions when gluing the facial regions together. This is based on the observation that the feature regions usually have less reconstruction errors compared to rest of the face. The results demonstrate that the coordinate adjustments and the new fusion strategy can significantly improve the craniofacial reconstructions.
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Bases de Datos Factuales , Huesos Faciales/diagnóstico por imagen , Antropología Forense/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Modelos Biológicos , Cráneo/diagnóstico por imagen , Tomografía Computarizada por Rayos X/métodos , Adolescente , Adulto , Anciano , Cara , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
The aim of this study was to manage diabetes with medicinal plants (Gymnema sylvestre, Artemisia absinthium and Citillus colocynthis) in human patients with type II diabetes. Thirty two patients of type II diabetes from both sexes of 30-60 years age were registered for this study and distributed them into four groups, each having 8 patients. Capsules of each, Gymnema sylvestre, Artemisia absinthium and Citrullus colocynthis were given to patients twice a day for 30 days in 1 g per day dosage and investigated for glucose, triglyceride (TGL) and cholesterol level. Gymnema sylvestre reduced 37% glucose, 5% TGL, 13% cholesterol and 19% low desity lipoproteins (LDL) level in diabetic individuals. Citrullus colocynth reduced glucose, cholesterol and TGL and HDL-cholesterol level by 35, 6, 6, and 5%, respectively. Artemisia absinthium reduced 3% high desity lipoproteins (HDL) and 6% LDL level. From results, it can be concluded that the powdered Gymnema sylvestre, Citrulus colocynthis, and Artemisia absinthium possess good anti-diabetic features, however these herbal products had no significant effect on lipid profiles of the diabetic human.
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Artemisia absinthium , Glucemia/análisis , Citrullus colocynthis , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Gymnema sylvestre , Fitoterapia , Adulto , Diabetes Mellitus Tipo 2/sangre , Femenino , Humanos , Lipoproteínas HDL/sangre , Lipoproteínas LDL/sangre , Masculino , Persona de Mediana EdadRESUMEN
Vascular remodeling is closely related to hypertension, atherosclerosis, and restenosis after PCI. Considerable evidence indicates that the activation and proliferation of adventitial fibroblasts play key roles in vessel injury. The inflammatory response and high expression of connexins contribute to adventitial remodeling. Therefore, reducing inflammation reaction and connexins expression in adventitia may become a new target to prevent vascular remodeling. Yiqihuoxuejiedu formula, composed of TCM therapeutic principle of supplementing qi, activating blood and detoxification, can inhibit restenosis after intimal injury. To further investigate the effect of Yiqihuoxuejiedu formula on inflammation and connexins, we established a carotid artery injury model. In model rats, hyperplasia in the intima was mild but obvious in the adventitia; CRP heightened; expressions of MCP-1, CD68, and Cx43 increased. Yiqihuoxuejiedu formula relieved intimal hyperplasia and adventitial area, obviously diminished the expressions of CD68 and Cx43 in the adventitia, and reduced CRP but did not lower MCP-1. These results indicated that Yiqihuoxuejiedu formula inhibited vascular remodeling especially adventitial hyperplasia by reducing the inflammation reaction including lowering macrophages infiltration and systemic nonspecific inflammatory response and also restraining gap junction connexins leading to less communication among cells. This study provides new ideas and methods for the prevention and treatment of vascular remodeling.
RESUMEN
Recent studies have demonstrated a novel molecular mechanism for the regulation of airway smooth muscle (ASM) contraction by RhoA GTPase. In ASM tissues, both myosin light chain (MLC) phosphorylation and actin polymerization are required for active tension generation. RhoA inactivation dramatically suppresses agonist-induced tension development and completely inhibits agonist-induced actin polymerization, but only slightly reduces MLC phosphorylation. The inhibition of MLC phosphatase does not reverse the effects of RhoA inactivation on contraction or actin polymerization. Thus, RhoA regulates ASM contraction through its effects on actin polymerization rather than MLC phosphorylation. Contractile stimulation of ASM induces the recruitment and assembly of paxillin, vinculin, and focal adhesion kinase (FAK) into membrane adhesion complexes (adhesomes) that regulate actin polymerization by catalyzing the activation of cdc42 GTPase by the G-protein-coupled receptor kinase-interacting target (GIT) - p21-activated kinase (PAK) - PAK-interacting exchange factor (PIX) complex. Cdc42 is a necessary and specific activator of the actin filament nucleation activator, N-WASp. The recruitment and activation of paxillin, vinculin, and FAK is prevented by RhoA inactivation, thus preventing cdc42 and N-WASp activation. We conclude that RhoA regulates ASM contraction by catalyzing the assembly and activation of membrane adhesome signaling modules that regulate actin polymerization, and that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to a contractile agonist.
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GTP Fosfohidrolasas/metabolismo , Pulmón/metabolismo , Contracción Muscular , Músculo Liso/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Actinas/metabolismo , Humanos , Transducción de SeñalRESUMEN
Vinculin localizes to membrane adhesion junctions in smooth muscle tissues, where its head domain binds to talin and its tail domain binds to filamentous actin, thus linking actin filaments to the extracellular matrix. Vinculin can assume a closed conformation, in which the head and tail domains bind to each other and mask the binding sites for actin and talin, and an open activated conformation that exposes the binding sites for talin and actin. Acetylcholine stimulation of tracheal smooth muscle tissues induces the recruitment of vinculin to the cell membrane and its interaction with talin and actin, which is required for active tension development. Vinculin phosphorylation at Tyr(1065) on its C terminus increases concurrently with tension development in tracheal smooth muscle tissues. In the present study, the role of vinculin phosphorylation at Tyr(1065) in regulating the conformation and function of vinculin during airway smooth muscle contraction was evaluated. Vinculin constructs with point mutations at Tyr(1065) (vinculin Y1065F and vinculin Y1065E) and vinculin conformation-sensitive FRET probes were expressed in smooth muscle tissues to determine how Tyr(1065) phosphorylation affects smooth muscle contraction and the conformation and cellular functions of vinculin. The results show that vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation and that Tyr(1065) phosphorylation regulates the conformation and scaffolding activity of the vinculin molecule. We conclude that the phosphorylation of vinculin at tyrosine 1065 provides a mechanism for regulating the function of vinculin in airway smooth muscle in response to contractile stimulation.
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Membrana Celular/metabolismo , Contracción Muscular/fisiología , Músculo Liso/metabolismo , Tráquea/metabolismo , Vinculina/metabolismo , Acetilcolina/farmacología , Actinas/genética , Actinas/metabolismo , Animales , Membrana Celular/genética , Agonistas Colinérgicos/farmacología , Perros , Contracción Muscular/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Estructura Terciaria de Proteína , Talina/genética , Talina/metabolismo , Vinculina/genéticaRESUMEN
The activation of the small GTPase RhoA is necessary for ACh-induced actin polymerization and airway smooth muscle (ASM) contraction, but the mechanism by which it regulates these events is unknown. Actin polymerization in ASM is catalyzed by the actin filament nucleation activator, N-WASp and the polymerization catalyst, Arp2/3 complex. Activation of the small GTPase cdc42, a specific N-WASp activator, is also required for actin polymerization and tension generation. We assessed the mechanism by which RhoA regulates actin dynamics and smooth muscle contraction by expressing the dominant negative mutants RhoA T19N and cdc42 T17N, and non-phosphorylatable paxillin Y118/31F and paxillin ΔLD4 deletion mutants in SM tissues. Their effects were evaluated in muscle tissue extracts and freshly dissociated SM cells. Protein interactions and cellular localization were analyzed using proximity ligation assays (PLA), immunofluorescence, and GTPase and kinase assays. RhoA inhibition prevented ACh-induced cdc42 activation, N-WASp activation and the interaction of N-WASp with the Arp2/3 complex at the cell membrane. ACh induced paxillin phosphorylation and its association with the cdc42 GEFS, DOCK180 and α/ßPIX. Paxillin tyrosine phosphorylation and its association with ßPIX were RhoA-dependent, and were required for cdc42 activation. The ACh-induced recruitment of paxillin and FAK to the cell membrane was dependent on RhoA. We conclude that RhoA regulates the contraction of ASM by catalyzing the assembly and activation of cytoskeletal signaling modules at membrane adhesomes that initiate signaling cascades that regulate actin polymerization and tension development in response to contractile agonist stimulation. Our results suggest that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to agonist -induced smooth muscle contraction.
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Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Contracción Muscular/fisiología , Músculo Liso/metabolismo , Transducción de Señal/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Acetilcolina/metabolismo , Acetilcolina/farmacología , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Sustitución de Aminoácidos , Animales , Membrana Celular/genética , Pollos , Citoesqueleto/genética , Perros , Humanos , Contracción Muscular/efectos de los fármacos , Músculo Liso/citología , Mutación Missense , Paxillin/genética , Paxillin/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.
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Acetilcolina/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/fisiología , Tráquea/fisiología , Vinculina/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Pollos , Colinérgicos/farmacología , Perros , Humanos , Integrinas/metabolismo , Talina/metabolismoRESUMEN
BACKGROUND: Recent guidelines on iron deficiency anaemia (IDA) have confirmed the aetiological role of Helicobacter pylori (H pylori), but the relationship still remains controversial. METHODS: Starting in May 2009, searches of the following databases were undertaken: Medline (1966 to April 2009), Embase (1980 to April 2009), the Cochrane library (1800 to June 2008), Cochrane Central Register of Controlled Trials, Premedline, Healthstar, CBMdisc and the Chinese National Knowledge Infrastructure Database (January 1970 to April 2009). Changes in haemoglobin (Hb) concentrations and serum ferritin (SF) concentrations were recorded for intervention and control groups. The meta-analysis used random effect models and subgroup analyses were performed to explain heterogeneity. RESULTS: Eight studies met the inclusion criteria. All studies were performed in Asia, an area with a high incidence of IDA and H pylori. The pooled analysis of eight studies showed that H pylori eradication therapy can improve IDA, since changes in Hb and SF concentrations in the intervention groups were higher than in controls. The weighted mean difference (WMD) of Hb was 12.88 g/l (95% CI 6.03 to 19.74 g/l, p<0.00001); the WMD of SF was 10.05 mug/l (95% CI 5.48 to 14.63 mug/l, p<0.00001). CONCLUSIONS: H pylori eradication therapy combined with iron administration is more effective than iron administration alone for the treatment of IDA. Eradication therapy has different effects on adults and children. Bismuth based triple therapy has a better response in terms of increased Hb and SF concentrations than proton pump inhibitor (PPI) based triple therapy.
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Anemia Ferropénica/tratamiento farmacológico , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori , Adolescente , Adulto , Anciano , Antibacterianos/uso terapéutico , Bismuto/uso terapéutico , Niño , Quimioterapia Combinada , Humanos , Persona de Mediana Edad , Inhibidores de la Bomba de Protones/uso terapéutico , Ensayos Clínicos Controlados Aleatorios como Asunto , Resultado del Tratamiento , Adulto JovenRESUMEN
AIM: To perform a meta-analysis of observational studies and randomized controlled trials (RCTs) on the association between Helicobacter pylori (H. pylori) and iron deficiency anemia (IDA). METHODS: A defined search strategy was used to search Medline, Embase, the Cochrane Library, Clinical Trials, Cochrane Central Register of Controlled Trials, Premedline and Healthstar. Odds ratio (OR) was used to evaluate observational epidemiology studies, and weighted mean difference (WMD) was used to demonstrate the difference between control and intervention groups. RESULTS: Fifteen observational studies and 5 RCTs were identified and used for calculation. The pooled OR for observational studies was 2.22 (95% CI: 1.52-3.24, P < 0.0001). The WMD for hemoglobin (HB) was 4.06 g/L (95% CI: -2.57-10.69, P = 0.01), and the WMD for serum ferritin (SF) was 9.47 mug/L (95% CI: -0.50-19.43, P < 0.0001). Results were heterogeneous for all comparisons. CONCLUSION: This meta-analysis on observational studies suggests an association between H. pylori and IDA. In RCTs, eradication of H. pylori can improve HB and SF levels but not significantly.
Asunto(s)
Anemia Ferropénica/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , Adolescente , Adulto , Anciano , Anemia Ferropénica/sangre , Anemia Ferropénica/tratamiento farmacológico , Antibacterianos/uso terapéutico , Biomarcadores/sangre , Distribución de Chi-Cuadrado , Niño , Preescolar , Medicina Basada en la Evidencia , Femenino , Ferritinas/sangre , Infecciones por Helicobacter/sangre , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/tratamiento farmacológico , Hemoglobinas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Ensayos Clínicos Controlados Aleatorios como Asunto , Medición de Riesgo , Factores de Riesgo , Resultado del Tratamiento , Adulto JovenRESUMEN
The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.
Asunto(s)
Actinas/química , Cofilina 1/metabolismo , Regulación de la Expresión Génica , Músculo Liso/metabolismo , Tráquea/metabolismo , Acetilcolina/metabolismo , Actinas/metabolismo , Animales , Inhibidores de la Calcineurina , Calcio/metabolismo , Perros , Electroforesis en Gel Bidimensional , Modelos Biológicos , Contracción Muscular , Fosforilación , Plásmidos/metabolismoRESUMEN
Phenotypic changes in airway smooth muscle occur with airway inflammation and asthma. These changes may be induced by alterations in the extracellular matrix that initiate signaling pathways mediated by integrin receptors. We hypothesized that integrin-linked kinase (ILK), a multidomain protein kinase that binds to the cytoplasmic tail of beta-integrins, may be an important mediator of signaling pathways that regulate the growth and differentiation state of airway smooth muscle. We disrupted signaling pathways mediated by ILK in intact differentiated tracheal muscle tissues by depleting ILK protein using ILK antisense. The depletion of ILK protein increased the expression of the smooth muscle differentiation marker genes myosin heavy chain (SmMHC), SM22alpha, and calponin and increased the expression of SmMHC protein. Conversely, the overexpression of ILK protein reduced the mRNA levels of SmMHC, SM22alpha, and calponin and SmMHC protein. Analysis by chromatin immunoprecipitation showed that the binding of the transcriptional regulator serum response factor (SRF) to the promoters of SmMHC, SM22alpha, and calponin genes was increased in ILK-depleted tissues and decreased in tissues overexpressing ILK. ILK depletion also increased the amount of SRF that localized within the nucleus. ILK depletion and overexpression, respectively, decreased and increased the activation of its downstream substrate protein kinase B (PKB/Akt). The pharmacological inhibition of Akt activity also increased SRF binding to the promoters of smooth muscle-specific genes and increased expression of smooth muscle proteins, suggesting that ILK may exert its effects by regulating the activity of Akt. We conclude that ILK is a critical regulator of airway smooth muscle differentiation. ILK may mediate signals from integrin receptors that control airway smooth muscle differentiation in response to alterations in the extracellular matrix.
Asunto(s)
Antígenos de Diferenciación/biosíntesis , Regulación de la Expresión Génica , Cadenas beta de Integrinas/metabolismo , Proteínas Musculares/biosíntesis , Músculo Liso/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Asma/enzimología , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/enzimología , Perros , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Elementos de Respuesta , Transducción de Señal/efectos de los fármacos , Tráquea/enzimologíaRESUMEN
We have previously demonstrated that p68 RNA helicase, as an essential human splicing factor, acts at the U1 snRNA and 5' splice site (5'ss) duplex in the pre-mRNA splicing process. To further analyze the function of p68 in the spliceosome, we generated two p68 mutants (motif V, RGLD to LGLD, and motif VI, HRIGR to HLIGR). ATPase and RNA unwinding assays demonstrated that the mutations abolished the RNA-dependent ATPase activity and RNA unwinding activity. The function of p68 in the spliceosome was abolished by the mutations, and the mutations also inhibited the dissociation of U1 from the 5'ss, while the mutants still interacted with the U1-5'ss duplex. Interestingly, the nonactive p68 mutants did not prevent the transition from prespliceosome to the spliceosome. The data suggested that p68 RNA helicase might actively unwind the U1-5'ss duplex. The protein might also play a role in the U4.U6/U5 addition, which did not require the ATPase and RNA unwinding activities of p68. In addition, we present evidence here to demonstrate the functional role of p68 RNA helicase in the pre-mRNA splicing process in vivo. Our experiments also showed that p68 interacted with unspliced but not spliced mRNA in vivo.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Quinasas/metabolismo , ARN Helicasas/metabolismo , Empalme del ARN/genética , Empalmosomas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Línea Celular Tumoral , ARN Helicasas DEAD-box , Humanos , Mutación/genética , Proteínas Quinasas/química , Proteínas Quinasas/genética , ARN Helicasas/química , ARN Helicasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Empalmosomas/química , Transcripción Genética/genética , Trioxsaleno/farmacologíaRESUMEN
An extracellular lipase (EC 3.1.1.3) from Geotrichum marinum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDS-PAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V(max) and Km were 1000 micromol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40 degrees C. At 6 mM, Na+, K+, Ca2+, and Mg2+ stimulated activity, but Na+ and K+ at 500 mM and Fe2+ and Mn2+ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position.