RESUMO
Skin wound healing is a complex and organized biological process, and the dermal fibroblasts play a crucial role. α-Catenin is known to be involved in regulating various cellular signals, and its role in wound healing remains unclear. Here, we have identified the pivotal role of the α-catenin/FAK/YAP signaling axis in the proliferation and migration of dermal fibroblasts, which contributes to the process of skin wound healing. Briefly, when α-catenin was knocked down specifically in dermal fibroblasts, the wound healing rate is significantly delayed. Moreover, interfering with α-catenin can impede the proliferation and migration of dermal fibroblasts both in vitro and in vivo. Mechanistically, the overexpression of α-catenin upregulates the nuclear accumulation of YAP and transcription of downstream target genes, resulting in enhanced the proliferation and migration of dermal fibroblasts. Furthermore, the FAK Tyr397 phosphorylation inhibitor blocked the promoting effects of α-catenin on YAP activation. Importantly, the continuous phosphorylation mutation of FAK Tyr397 reversed the retardatory effects of α-catenin knockdown on wound healing, by increasing the vitality of fibroblasts. Likewise, α-catenin/FAK was validated as a therapeutic target for wound healing in the db/db chronic trauma model. In summary, our findings have revealed a novel mechanism by which α-catenin facilitates the function of fibroblasts through the activity of the FAK/YAP signaling axis. These findings define a promising therapeutic strategy for accelerating the wound healing process.
Assuntos
Fibroblastos , Cicatrização , alfa Catenina/genética , Mutação , Proliferação de CélulasRESUMO
BACKGROUND AND AIMS: Chronic liver diseases are associated with the development of liver fibrosis. Without treatment, liver fibrosis commonly leads to cirrhosis and HCC. FGF12 is an intracrine factor belonging to the FGF superfamily, but its role in liver homeostasis is largely unknown. This study aimed to investigate the role of FGF12 in the regulation of liver fibrosis. APPROACH AND RESULTS: FGF12 was up-regulated in bile duct ligation (BDL)-induced and CCL 4 -induced liver fibrosis mouse models. Expression of FGF12 was specifically up-regulated in nonparenchymal liver cells, especially in hepatic macrophages. By constructing myeloid-specific FGF12 knockout mice, we found that deletion of FGF12 in macrophages protected against BDL-induced and CCL 4 -induced liver fibrosis. Further results revealed that FGF12 deletion dramatically decreased the population of lymphocyte antigen 6 complex locus C high macrophages in mouse fibrotic liver tissue and reduced the expression of proinflammatory cytokines and chemokines. Meanwhile, loss-of-function and gain-of-function approaches revealed that FGF12 promoted the proinflammatory activation of macrophages, thus inducing HSC activation mainly through the monocyte chemoattractant protein-1/chemokine (C-C motif) receptor 2 axis. Further experiments indicated that the regulation of macrophage activation by FGF12 was mainly mediated through the Janus kinase-signal transducer of activators of transcription pathway. Finally, the results revealed that FGF12 expression correlates with the severity of fibrosis across the spectrum of fibrogenesis in human liver samples. CONCLUSIONS: FGF12 promotes liver fibrosis progression. Therapeutic approaches to inhibit macrophage FGF12 may be used to combat liver fibrosis in the future.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Camundongos , Humanos , Animais , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Cirrose Hepática/patologia , Fígado/patologia , Macrófagos/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BL , Células Estreladas do Fígado/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismoRESUMO
Diabetes is a chronic disease characterized by perturbed glucose and lipid metabolism, resulting in high blood glucose levels. Many complications induced by endothelial dysfunction can cause disability and even death of diabetic patients. Here, we found that the protein level of casein kinase 2α (CK2α) was increased in the endothelium of mice with type I diabetes (T1D) induced by streptozotocin (STZ) injection. Although a potential correlation between the protein level of CK2α and endothelial dysfunction in diabetes was established, the contribution of CK2α to the progression of endothelial dysfunction in diabetes remained largely unknown. By using CX4945 (a selective CK2α antagonist) and Si-csnk2a1 (small interfering RNA targeting CK2α), we found that inhibition of CK2α accelerated skin wound healing in T1D mice by promoting proliferation of endothelial cells. Administration of CX4945 or Si-csnk2a1 rescued the impaired Hedgehog signaling pathway in high glucose-treated human umbilical vein endothelial cells (HUVECs). Exploration of the underlying molecular mechanism revealed that the protective effect of CK2α inhibition on angiogenesis, which contributes to skin wound healing in diabetic mice, was blocked by administration of GANT61 (an inhibitor targeting the Hedgehog signaling pathway). Our findings establish CK2α as a regulator of endothelial dysfunction in diabetes and demonstrate that inhibition of CK2α accelerates skin wound healing in T1D mice by promoting endothelial cell proliferation via the Hedgehog signaling pathway.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Humanos , Animais , Camundongos , Proteínas Hedgehog , Caseína Quinase II , Proliferação de Células , Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana , CicatrizaçãoRESUMO
Liver fibrosis, which is characterized by excessive accumulation of extracellular matrix (ECM) primarily produced by hepatic stellate cells (HSCs), can eventually lead to cirrhosis. Fibroblast growth factor 18 (FGF18) mediates various biological activities. However, the precise role of FGF18 in the pathological process of liver fibrosis and the underlying mechanisms have not been elucidated. In this study, we found that FGF18 was markedly upregulated in carbon tetrachloride (CCl4)-induced fibrotic mouse liver tissues and transforming growth factor ß (TGF-ß) stimulated LX-2 cells. Furthermore, our studies demonstrated that overexpression of FGF18 in the liver significantly alleviated CCl4-induced fibrosis and inhibited the activation of HSCs, while exacerbated by HSC-specific deletion of FGF18. Mechanistically, FGF18 treatment dramatically activated Hippo signaling pathway by suppressing smoothened (SMO) both in vivo and in vitro. Moreover, the interaction between SMO and LATS1 was crucial for the FGF18 induced protective effects. In conclusion, these results indicated that FGF18 attenuates liver fibrosis at least partially via the SMO-LATS1-YAP signaling pathway and therefore may be a potential therapeutic target for liver fibrosis.
Assuntos
Células Estreladas do Fígado , Cirrose Hepática , Animais , Tetracloreto de Carbono/efeitos adversos , Tetracloreto de Carbono/metabolismo , Fatores de Crescimento de Fibroblastos , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Cutibacterium acnes (C. acnes) is a common commensal bacterium that is closely associated with the pathogenesis of acne. Fibroblast growth factor 21 (FGF21), as a favorable regulator of glucose and lipid metabolism and insulin sensitivity, was recently shown to exert anti-inflammatory effects. The role and mechanism of FGF21 in the inflammatory reactions induced by C. acnes, however, have not been determined. The present study shows that FGF21 in the dermis inhibits epidermal C. acnes-induced inflammation in a paracrine manner while it functions on the epidermal layer through a receptor complex consisting of FGF receptor 1 (FGFR1) and ß-Klotho (KLB). The effects of FGF21 in heat-killed C. acnes-induced HaCaT cells and living C. acnes-injected mouse ears were examined. In the presence of C. acnes, FGF21 largely counteracted the activation of Toll-like receptor 2 (TLR2), the downstream nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK) signaling pathways induced by C. acnes. FGF21 also significantly reduced the expression of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α. Taken together, these findings indicate that FGF21 suppresses C. acnes-induced inflammation and might be used clinically in the management and treatment of acne.
Assuntos
Acne Vulgar , Propionibacterium acnes , Acne Vulgar/microbiologia , Animais , Fatores de Crescimento de Fibroblastos/metabolismo , Inflamação/microbiologia , CamundongosRESUMO
BACKGROUND: The mechanism underlying endothelial dysfunction leading to cardiovascular disease in type 2 diabetes mellitus (T2DM) remains unclear. Here, we show that inhibition of histone deacetylase 3 (HDAC3) reduced inflammation and oxidative stress by regulating nuclear factor-E2-related factor 2 (Nrf2), which mediates the expression of anti-inflammatory- and pro-survival-related genes in the vascular endothelium, thereby improving endothelial function. METHODS: Nrf2 knockout (Nrf2 KO) C57BL/6 background mice, diabetic db/db mice, and control db/m mice were used to investigate the relationship between HDAC3 and Nrf2 in the endothelium in vivo. Human umbilical vein endothelial cells (HUVECs) cultured under high glucose-palmitic acid (HG-PA) conditions were used to explore the role of Kelch-like ECH-associated protein 1 (Keap1) -Nrf2-NAPDH oxidase 4 (Nox4) redox signaling in the vascular endothelium in vitro. Activity assays, immunofluorescence, western blotting, qRT-PCR, and immunoprecipitation assays were used to examine the effect of HDAC3 inhibition on inflammation, reactive oxygen species (ROS) production, and endothelial impairment, as well as the activity of Nrf2-related molecules. RESULTS: HDAC3 activity, but not its expression, was increased in db/db mice. This resulted in de-endothelialization and increased oxidative stress and pro-inflammatory marker expression in cells treated with the HDAC3 inhibitor RGFP966, which activated Nrf2 signaling. HDAC3 silencing decreased ROS production, inflammation, and damage-associated tube formation in HG-PA-treated HUVECs. The underlying mechanism involved the Keap1-Nrf2-Nox4 signaling pathway. CONCLUSION: The results of this study suggest the potential of HDAC3 as a therapeutic target for the treatment of endothelial dysfunction in T2DM. Video Abstract.
Assuntos
Diabetes Mellitus/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos Endogâmicos C57BL , NADPH Oxidase 4/antagonistas & inibidores , NADPH Oxidase 4/metabolismo , Substâncias Protetoras/farmacologia , Ligação Proteica/efeitos dos fármacosRESUMO
Metallothioneins (MTs) are intracellular cysteine-rich proteins, and their expressions are enhanced under stress conditions. MTs are recognized as having the ability to regulate redox balance in living organisms; however, their role in regulating osteoblast differentiation is still unclear. In this research, we found that the expression of MT3, one member of the MT protein family, was specifically upregulated in the differentiation process of C2C12 myoblasts treated with bone morphogenetic protein 4 (BMP4). Transfection with MT3-overexpressing plasmids in C2C12 cells enhanced their differentiation to osteoblasts, together with upregulating the protein expression of bone specific transcription factors runt-related gene 2 (Runx2), Osterix, and distal-less homeobox 5 (Dlx5). Additionally, MT3 knockdown performed the opposite. Further studies revealed that overexpression of MT3 decreased reactive oxygen species (ROS) production in C2C12 cells treated with BMP4, and MT3 silencing enhanced ROS production. Treating C2C12 cells with antioxidant N-acetylcysteine also promoted osteoblast differentiation, and upregulated Runx2/Osterix/Dlx5, while ROS generator antimycin A treatment performed the opposite. Finally, antimycin A treatment inhibited osteoblast differentiation and Runx2/Osterix/Dlx5 expression in MT3-overexpressing C2C12 cells. These findings identify the role of MT3 in osteoblast differentiation and indicate that MT3 may have interesting potential in the field of osteogenesis research.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Mioblastos/citologia , Proteínas do Tecido Nervoso/metabolismo , Osteoblastos/citologia , Osteogênese , Estresse Oxidativo , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Metalotioneína 3 , Camundongos , Mioblastos/metabolismo , Proteínas do Tecido Nervoso/genética , Osteoblastos/metabolismoRESUMO
Myocardial infarction (MI) remains a major health-related problem with high incidence and mortality rates. Oxidative stress plays an important role in myocardial ischemia injury and further leads to myocardial remodeling. Basic fibroblast growth factor (bFGF) is a member of the fibroblast growth factors that regulate a variety of biological functions. However the function of bFGF in myocardial infarction is still unknown. Here we aimed to investigate the role of bFGF and its underlying mechanism in ischemia heart and cardiomyocytes apoptosis. We found that bFGF treatment could significantly enhance the cardioprotective effects by reducing oxidative stress both in vivo and vitro. In addition, we found that bFGF activated Nrf2-mediated antioxidant defenses via Akt/GSK3ß/Fyn pathway. Furthermore, Nrf2 knockdown largely counteracted the protective effect of bFGF. In summary, our study suggested that bFGF could alleviate myocardial infarction injury and cardiomyocytes apoptosis via Nrf2.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Animais , Hipóxia Celular , Linhagem Celular , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Binge drinking is associated with increased cardiac autophagy, and often triggers heart injury. Given the essential role of autophagy in various cardiac diseases, this study was designed to investigate the role of autophagy in ethanol-induced cardiac injury and the underlying mechanism. Our study showed that ethanol exposure enhanced the levels of LC3-II and LC3-II positive puncta and promoted cardiomyocyte apoptosis in vivo and in vitro. In addition, we found that ethanol induced autophagy and cardiac injury largely via the sequential triggering of reactive oxygen species (ROS) accumulation, activation of c-Jun NH2-terminal kinase (JNK), phosphorylation of Bcl-2, and dissociation of the Beclin 1/Bcl-2 complex. By contrast, inhibition of ethanol-induced autophagic flux with pharmacologic agents in the hearts of mice and cultured cells significantly alleviated ethanol-induced cardiomyocyte apoptosis and heart injury. Elimination of ROS with the antioxidant N-acetyl cysteine (NAC) or inhibition of JNK with the JNK inhibitor SP600125 reduced ethanol-induced autophagy and subsequent autophagy-mediated apoptosis. Moreover, metallothionein (MT), which can scavenge reactive oxygen and nitrogen species, also attenuated ethanol-induced autophagy and cell apoptosis in MT-TG mice. In conclusion, our findings suggest that acute ethanol exposure induced autophagy-mediated heart toxicity and injury mainly through the ROS-JNK-Bcl-2 signaling pathway.
Assuntos
Autofagia , Cardiomiopatia Alcoólica/enzimologia , Etanol , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/enzimologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cardiomiopatia Alcoólica/patologia , Cardiotoxicidade , Células Cultivadas , Modelos Animais de Doenças , Predisposição Genética para Doença , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Ratos Sprague-Dawley , Transdução de Sinais , Fatores de TempoRESUMO
Catalase is an antioxidative enzyme that converts hydrogen peroxide (H2 O2 ) produced by superoxide dismutase from highly reactive superoxide (O2- ) to water and oxygen molecules. Although recent findings demonstrate that catalase, autophagy and the nuclear factor κB (NF-κB) signalling pathway are centrally involved in diabetic cardiomyopathy (DCM), the interplay between the three has not been fully characterized. Thus, the mechanism responsible for catalase-mediated protection against heart injury in diabetic mice was investigated in this study, as well as the role of NF-κB-p65 in the regulation of autophagic flux was investigated in this study. Western blot analysis revealed that catalase inhibited NF-κB activity and decreased LC3-II (microtubule-associated protein 1 light chain 3) and beclin-1 (Atg6) expression. Furthermore, up-regulation of autophagy was detrimental for cardiac function in diabetic mice. Catalase overexpression reduced the level of NF-κB subunit in the nucleus, where it initiates autophagy through activation of the key autophagy gene BECN1. To evaluate the role of the NF-κB pathway in diabetes-induced autophagy, Bay11-7082, an NF-κB inhibitor, was injected into diabetic mice, which suppressed NF-κB and attenuated diabetes-induced autophagy and myocardial apoptosis. In agreement with the in vivo results, Bay11-7082 also inhibited high-glucose-induced activation of NF-κB and the up-regulation of LC3-II and beclin-1 expression in H9c2 cells. In addition, high-glucose-induced activation of autophagic flux and apoptosis were largely attenuated by p65 siRNA, suggesting that catalase ameliorates diabetes-induced autophagy, at least in part by increasing the activity of the NF-κB pathway and p65-mediated transcription of BECN1.
Assuntos
Proteína Beclina-1/genética , Catalase/genética , Diabetes Mellitus Experimental/genética , Cardiomiopatias Diabéticas/genética , Proteínas Associadas aos Microtúbulos/genética , Fator de Transcrição RelA/genética , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Catalase/metabolismo , Linhagem Celular , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Cardiomiopatias Diabéticas/induzido quimicamente , Cardiomiopatias Diabéticas/enzimologia , Cardiomiopatias Diabéticas/patologia , Regulação da Expressão Gênica , Glucose/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Nitrilas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Estreptozocina , Sulfonas/farmacologia , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Transcrição GênicaRESUMO
A Schiff base compound derived from naphthalene has been synthesized and characterized as an Al3+ -selective fluorescent probe. The chemosensor (L) exhibits high selectively for Al3+ in aqueous solution, even in the presence of biologically relevant cations such as Na+ , K+ , Ca2+ , Mg2+ , Pb2+ and several transition metal ions. There was no observed interference from anions like Br- , Cl- , HSO3- , SO32- , S2 O32- , NO2- , CO32- and AC- . The lowest detection limit for the chemosensor L was found to be 1.89 × 10-8 M with a linear response towards Al3+ over a concentration range of 5 × 10-6 to 4 × 10-5 M. Furthermore, the proposed chemosensor has been used for imaging of Al3+ in two different types of cells with satisfying results, which further demonstrates its value for practical application in biological systems.
Assuntos
Alumínio/análise , Corantes Fluorescentes/química , Naftalenos/química , Espectrometria de Fluorescência/métodos , Animais , Corantes Fluorescentes/síntese química , Concentração de Íons de Hidrogênio , Limite de Detecção , Espectroscopia de Ressonância Magnética , Imagem Molecular/métodos , Estrutura Molecular , Células PC12 , Ratos , Bases de Schiff , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
In order to achieve an easy, rapid and sensitive protocol to detect proteins in polyacrylamide gel, an advanced negative detection method comparable to silver stain is described. When a gel was incubated with Phloxine B and followed by the development in acidic solution, the zones where forming protein-dye complex were selectively transparent, unlike opaque gel background. Within 50 min after electrophoresis, down to 0.1-0.4 ng of gel-separated proteins (similar with silver stain) could be observed, without labor-intensive and time-consuming procedure. Comparing with the most common negative stain method, Imidazole-zinc stain, Phloxine B stain has been shown higher sensitivity and distinct contrast between the transparent protein bands/spots and opaque background than those; furthermore, it is no longer necessary to concern about retention time of observation. This technique may provide a sensitive and practical choice for proteomics researches.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Azul de Eosina I/química , Animais , Bovinos , Humanos , Coloração pela Prata/métodosRESUMO
Catalase is an antioxidant enzyme that specifically catabolizes hydrogen peroxide (H2O2). Overexpression of catalase via a heart-specific promoter (CAT-TG) was reported to reduce diabetes-induced accumulation of reactive oxygen species (ROS) and further prevent diabetes-induced pathological abnormalities, including cardiac structural derangement and left ventricular abnormity in mice. However, the mechanism by which catalase overexpression protects heart function remains unclear. This study found that activation of a ROS-dependent NF-κB signaling pathway was downregulated in hearts of diabetic mice overexpressing catalase. In addition, catalase overexpression inhibited the significant increase in nitration levels of key enzymes involved in energy metabolism, including α-oxoglutarate dehydrogenase E1 component (α-KGD) and ATP synthase α and ß subunits (ATP-α and ATP-ß). To assess the effects of the NF-κB pathway activation on heart function, Bay11-7082, an inhibitor of the NF-κB signaling pathway, was injected into diabetic mice, protecting mice against the development of cardiac damage and increased nitrative modifications of key enzymes involved in energy metabolism. In conclusion, these findings demonstrated that catalase protects mouse hearts against diabetic cardiomyopathy, partially by suppressing NF-κB-dependent inflammatory responses and associated protein nitration.
Assuntos
Catalase/metabolismo , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Miocárdio/enzimologia , Miocárdio/patologia , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Cardiotônicos/metabolismo , Linhagem Celular , Inflamação/patologia , Camundongos Transgênicos , Nitrilas/farmacologia , Nitrosação , Especificidade de Órgãos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Tirosina/análogos & derivados , Tirosina/biossínteseRESUMO
A new hydrazide derivative was synthesized and used for the first time as a specific, brief, and economical probe to selectively visualize glycoproteins in 1-D and 2-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with high sensitivity. The detection limit of the newly developed staining method is 2- and 4-fold higher than that of the widely used Pro-Q Emerald 300 and 488 stains, respectively.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Glicoproteínas/análise , Hidrazinas/química , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência MolecularRESUMO
A fluorescent quenching detection method for phosphoproteins in SDS-PAGE by using calconcarboxylic acid (CCA) was described. In this method, the fluorescence intensity of CCA was greatly increased with the presence of Al(3+) in the gel background, while in zones where phosphoproteins are located this intensity was absent because of fluorescence quenching phenomenon through the formation of CCA-Al(3+) -phosphoprotein appended complex. Approximately 4-8 ng of phosphoproteins can be selectively detected within 1 h (1D SDS-PAGE), which is similar to that of the most commonly used Pro-Q Diamond stain. The specificity of this novel technique for phosphoproteins was confirmed by dephosphorylation, Western blot, and LC-MS/MS analysis, respectively. Furthermore, to better understand the newly developed method, the detection mechanism of CCA stain was explored by fluorescent spectrometry. According to the results, it is believed that CCA stain may provide a new choice for selective, economical, MS compatible, and convenient visualization of gel-separated phosphoproteins.
Assuntos
Compostos Azo/química , Eletroforese em Gel de Poliacrilamida/métodos , Corantes Fluorescentes/química , Naftóis/química , Fosfoproteínas/análise , Sequência de Aminoácidos , Eletroforese em Gel Bidimensional , Dados de Sequência Molecular , Fosfoproteínas/química , Sensibilidade e EspecificidadeRESUMO
In order to obtain an easy and rapid protocol to visualize phosphoproteins in SDS-PAGE, a fluorescent detection method named 8-Quinolinol (8-Q) stain is described. 8-Q can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion (Al(3+) ) to the phosphate groups on the proteins and the metal chelating property of 8-Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4â¼8 ng of α-casein and ß-casein, 16â¼32 ng of ovalbumin and κ-casein which is more sensitive than Stains-All but less sensitive than Pro-Q Diamond. The protocol of 8-Q requires only 70 min in 0.75 mm mini-size or 1.0 mm large-size gels with five changes of solutions without destaining step; Pro-Q takes at least 250 min with 11 changes of solutions. In addition, the new method was confirmed by the study of dephosphorylation and LC-MS/MS, respectively. The approach to visualize phosphoprotein utilizing 8-Q could be an alternative to simplify the analytical operations for phosphoproteomics research.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Corantes Fluorescentes/química , Oxiquinolina/química , Fosfoproteínas/análise , Espectrometria de Massas , Fosfoproteínas/química , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Chemical doping has been demonstrated to be an effective way to realize new functions of graphene as metal-free catalyst in energy-related electrochemical reactions. Although efficient catalysis for the oxygen reduction reaction (ORR) has been achieved with doped graphene, its performance in the hydrogen evolution reaction (HER) is rather poor. In this study we report that nitrogen and sulfur co-doping leads to high catalytic activity of nanoporous graphene in HER at low operating potential, comparable to the best Pt-free HER catalyst, 2D MoS2 . The interplay between the chemical dopants and geometric lattice defects of the nanoporous graphene plays the fundamental role in the superior HER catalysis.
RESUMO
Single-atom nickel dopants anchored to three-dimensional nanoporous graphene can be used as catalysts of the hydrogen evolution reaction (HER) in acidic solutions. In contrast to conventional nickel-based catalysts and graphene, this material shows superior HER catalysis with a low overpotential of approximately 50 mV and a Tafel slope of 45 mV dec(-1) in 0.5 M H2SO4 solution, together with excellent cycling stability. Experimental and theoretical investigations suggest that the unusual catalytic performance of this catalyst is due to sp-d orbital charge transfer between the Ni dopants and the surrounding carbon atoms. The resultant local structure with empty C-Ni hybrid orbitals is catalytically active and electrochemically stable.
RESUMO
For analysis of nitration modification of α oxoglutarate dehydrogenase (α-OGDH) induced by diabetes, a targeted proteomics strategy was developed through the use of Skyline. All peptides containing Y and W of the target proteins were nitrated in silico and output to produce parallel reaction monitoring (PRM) or SRM method for nitration analysis. A nitrated casein mixture was used as standard protein to assess the feasibility of this method. The results demonstrated the availability of this strategy for nitration identification, and subsequently this method was used to analyze the nitration of α-OGDH from myocardial tissue extracts of diabetic mouse. The PRM method was primarily generated by Skyline for identification of the actual nitrated peptides from all possible nitrated peptides of α-OGDH due to the complexity of α-OGDH. The PRM-based data were analyzed by SEQUEST, and transitions of the identified nitrated peptides were used to develop an SRM method for relative quantitation of nitration degree. The nitration degree of α-OGDH for diabetic mouse is higher than that for control mouse, indicating that α-OGDH of the diabetic mouse suffered from more intense oxidative damage. We believe that this approach for obtaining information regarding nitration will facilitate the study of other PTMs in complex mixtures.
Assuntos
Diabetes Mellitus/enzimologia , Complexo Cetoglutarato Desidrogenase/química , Nitratos/análise , Proteômica/métodos , Sequência de Aminoácidos , Animais , Caseínas/química , Diabetes Mellitus/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Miocárdio/química , Miocárdio/enzimologia , Miocárdio/metabolismo , Nitratos/metabolismo , Estresse Oxidativo , Peptídeos/análise , Peptídeos/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
A fluorescent staining technique, using selective chelation with fluorophore and metal ion to the phosphate groups of phosphoproteins in SDS-PAGE is described. As a fluorescent dye and a metal ion, Fura 2 pentapotassium salt and Al(3+) were employed, respectively. The staining method, Fura 2 stain, has sensitivities of 16-32 ng of α-casein and ß-casein, 62 ng of ovalbumin, phosvitin, and κ-casein using an ultraviolet transilluminator. Furthermore, Fura 2 stain is able to carry out continuative double detection of total proteins and phosphoproteins on the same gel within 3.5 h. Consequently, selective phosphoprotein and total protein detections could be obtained without other poststaining. Considering the low cost, simplicity, and speed, Fura 2 staining may provide great practicalities in routine phosphoproteomics research.