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
PURPOSE: The link between dietary fiber intake and Non-alcoholic fatty liver disease (NAFLD) is under exploration, yielding inconsistent findings. Considering the limitations of previous research and the significance of dietary fiber in hepatic steatosis, this study investigates the association between dietary fiber intake and Controlled Attenuation Parameter (CAP) among 5935 participants from the National Health and Nutrition Examination Survey (NHANES). MATERIALS AND METHODS: Multivariable regression was used to evaluate the association between dietary fiber intake and CAP. Smoothed curve fitting and threshold effect analysis techniques were applied to illustrate non-linear relationships. RESULTS: After adjusting for other variables, a negative correlation emerged between dietary fiber intake and CAP. Subgroup analysis by gender and race/ethnicity revealed a sustained negative association between dietary fiber intake and CAP among females and Whites. Additionally, an inverted U-shaped relationship was observed between dietary fiber intake and CAP among women and other race, with inflection points at 13.80 g/day and 33.45 g/day, respectively. CONCLUSION: Our research indicates that in the majority of Americans, there is an inverse relationship between dietary fiber intake and hepatic steatosis. This relationship exhibits an inverted U-shaped curve in women and other race, with a threshold effect. The findings of this study hold potential significance for clinical nutrition interventions, personalized dietary guidance, and advancing research into the diet-disease mechanism relationship.
Assuntos
Fibras na Dieta , Hepatopatia Gordurosa não Alcoólica , Inquéritos Nutricionais , Humanos , Fibras na Dieta/administração & dosagem , Feminino , Masculino , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Pessoa de Meia-Idade , Adulto , Estados Unidos/epidemiologia , Fatores SexuaisRESUMO
BACKGROUND: Type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease frequently coexist and share pathophysiological manifestations. This study aimed to explore the association between T2DM status and prevalence of liver steatosis and fibrosis, identified using the controlled attenuation parameter and liver stiffness measurement attained via liver ultrasound transient elastography. METHODS: This was a cross-sectional analysis of data collected in the National Health and Nutrition Examination Survey for 2017-2018. Multivariable logistic regression model was used to evaluate the association between T2DM and prevalence of liver steatosis and fibrosis. Subgroup analyses, stratified by sex age, race, and body mass index (BMI), were further performed. RESULTS: Of the 2,780 participants aged ≥ 40 years enrolled, 749 had T2DM, and 2,031 did not. After adjustment for potential confounders, T2DM was associated with a higher prevalence of liver steatosis (OR = 1.7, 95% CI, 1.3-2.1). This T2DM-related prevalence was higher among women (OR = 1.8, 95% CI, 1.3-2.5) and in the non-Hispanic Black (OR = 1.8, 95% CI, 1.1-3.0), other race (OR = 1.9, 95% CI, 1.2-3.0), and BMI < 25 kg/m2 (OR = 2.0, 95% CI, 1.1-3.8) groups. T2DM was also associated with a significantly higher prevalence of fibrosis (OR = 2.0, 95% CI, 1.5-2.7), with this association being more prominent for the other race (OR = 2.9, 95% CI, 1.5-5.5) and BMI < 25 kg/m2 (OR = 3.3, 95% CI: 1.3-8.8) groups. CONCLUSIONS: Our findings indicated a positive association between T2DM status and prevalence of hepatic steatosis and fibrosis. This association was more prominent for individuals with a BMI < 25 kg/m2 and was influenced by race-specific effects.
Assuntos
Diabetes Mellitus Tipo 2 , Hepatopatia Gordurosa não Alcoólica , Adulto , Estudos Transversais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/patologia , Feminino , Fibrose , Humanos , Fígado/patologia , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/epidemiologia , Cirrose Hepática/etiologia , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Inquéritos Nutricionais , PrevalênciaRESUMO
BACKGROUND: Osteoarthritis (OA) is a leading cause of disability and a source of societal costs among older adults, especially with an increasingly obese population. However, very few published studies have investigated the burden of knee and hip OA due to a high body mass index (BMI). Therefore, this study aimed to systematically summarize the trends of knee and hip OA due to a high BMI in China and the USA between 1990 and 2019. METHODS: Data from the Global Burden of Disease Study 2019 were used to estimate the age-standardized prevalence, disability-adjusted life years (DALYs) rates of knee and hip OA, and the burden of knee and hip OA due to a high BMI by sex. Joinpoint regression analysis was used to determine the temporal trend changes in the related DALYs rates of knee and hip OA. RESULTS: The trends in the DALYs rates of knee OA due to a high BMI have shown a steady and large increase in China, while the trends first showed an increase followed by a large decrease near 2001-2005 and finally a steady increase in the USA. The trends in the DALYs rates of hip OA due to a high BMI have shown a steady and large increase in both men and women in China and the USA from 1990 to 2019. For the comparison by age categories from 30 to 34 years to 90-94 years in 2019, the age-standardized DALYs rates due to a high BMI first increased and then decreased after 60-64 years and 70-74 years in both men and women from China and the USA for knee OA, respectively. For hip OA, the age-standardized DALYs rates first increased and then decreased after 70-74 years in both men and women from China and the USA. CONCLUSIONS: The burden of knee and hip OA due to a high BMI is substantially increasing in China and the USA in recent years. Researchers and health policy makers should assess the changing patterns of high BMI on the burden of OA and devise corresponding weight-control strategies.
Assuntos
Osteoartrite do Quadril , Osteoartrite do Joelho , Adulto , Idoso , Índice de Massa Corporal , China/epidemiologia , Efeitos Psicossociais da Doença , Anos de Vida Ajustados por Deficiência , Feminino , Carga Global da Doença , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Quadril/diagnóstico , Osteoartrite do Quadril/epidemiologia , Osteoartrite do Joelho/diagnóstico , Osteoartrite do Joelho/epidemiologiaRESUMO
BACKGROUND: Increasingly, studies have addressed the possible association between type 2 diabetes mellitus (T2DM) and osteoarthritis (OA), although this remains an issue of controversy. Our aim in this study was to investigate the association between T2DM and OA among 7781 adults ≥50 years of age, through a cross-sectional analysis of the National Health and Nutrition Examination Survey from 2011 through 2018. METHODS: Amultivariable logistic regression model was used to evaluate the association between T2DM or prediabetes status and OA, with subgroup analyses performed, stratified by age, sex, body mass index (BMI), and race. RESULTS: Of the 7781 participants enrolled, 1567 (20.1%) had T2DM, 3131 (40.3%) had prediabetes, and 3083 (39.6%) were normal. A significant positive association was identified between T2DM and OA, after adjusting for age, sex, and race. This association, however, was no longer significant after adjusting for BMI. In the fully-adjusted model, there were no significant association between T2DM and OA. CONCLUSIONS: Our findings showed no association between T2DM and OA. The association between T2DM and OA can be strongly confounded by BMI.
Assuntos
Diabetes Mellitus Tipo 2 , Osteoartrite , Adulto , Índice de Massa Corporal , Estudos Transversais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiologia , Humanos , Pessoa de Meia-Idade , Inquéritos Nutricionais , Osteoartrite/epidemiologiaRESUMO
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: Hemoglobin A1c (HbA1c) is the product of a non-enzymatic chemical reaction between hemoglobin (Hb) and glucose. However, the association between Hb and HbA1c remains to be fully elucidated in view of the controversial findings reported to date. Therefore, our aim in this study was to evaluate the association between Hb levels within the normal range and HbA1c levels among Chinese non-diabetes adults using cross-sectional data from the China Health and Nutrition Survey 2009. METHODS: Our analysis was based on the data of 1659 non-diabete adults 20-49 years of age. Multivariable linear models were applied to examine the association between Hb and HbA1c levels. Subgroup analyses stratified by age and sex were also performed. RESULTS: The association between Hb and HbA1c levels was positive in the unadjusted model (ß =0.020, 95% CI: 0.008, 0.032). However, this association did not remain significant when the regression model was minimally adjusted for age and sex (ß =0.006, 95% CI: - 0.014, 0.024); this association became negative when the model was further adjusted for covariates whose effect estimates of HbA1c levels more than 10% (ß = - 0.042, 95% CI: - 0.064, - 0.020). The association remained negative on subgroup analyses stratified by age (20-34 years: ß = - 0.052, 95% CI: - 0.091, - 0.013; 35-49 years: ß = - 0.041, 95% CI: - 0.068, - 0.014) and sex (men: ß = - 0.042, 95% CI: - 0.074, - 0.010; women: ß = - 0.042, 95% CI: - 0.073, - 0.012) when controlling for covariates. CONCLUSIONS: Our findings revealed that Hb levels within the normal range were negatively associated with HbA1c levels among Chinese non-diabetes adults. Confounding factors, such as red blood cell counts can affect the association between Hb and HbA1c levels.
Assuntos
Povo Asiático , Glicemia/metabolismo , Hemoglobinas Glicadas/metabolismo , Hemoglobinas/metabolismo , Vigilância da População , Inquéritos e Questionários , Adulto , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Vigilância da População/métodos , Valores de Referência , Adulto JovemRESUMO
OBJECTIVE: To evaluate the correlation between continuation of glucocorticoid (GC) treatment and risk of femoral head collapse in patients with glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) after hip-preserving interventions. METHODS: The cohort included patients with GIONFH who had received a hip-preserving intervention between 1 January 2014 and 1 April 2016. All hips were at the non-collapse stage. The independent variable and the dependent variable were postoperative GC use and femoral head collapse (≥3â¯mm), respectively. Multivariate Cox proportional hazards regression were performed to estimate the association of the variables after adjusting for other covariates. RESULTS: A total of 27 hips (24 patients) were included for the final data analysis of which 6 hips out of 18 developed femoral head collapse (≥3â¯mm) in patients who discontinued taking GC postoperatively and 6 hips out of 9 developed collapse (≥3â¯mm) in those requiring GC treatment postoperatively. In the fully adjusted Cox proportional hazards model, the risk of femoral head collapse (≥3â¯mm) was higher in participants requiring postoperative GC use than those not requiring GC (hazard ratio, HR 3.7, 95% confidence interval, CI 1.1-13.0). CONCLUSION: The results of this study demonstrated that patients with GIONFH who continued GC treatment postoperatively had a significantly increased risk of femoral head collapse (≥3â¯mm) compared to those who discontinued use of GC.
Assuntos
Necrose da Cabeça do Fêmur , Glucocorticoides , Cabeça do Fêmur/diagnóstico por imagem , Necrose da Cabeça do Fêmur/induzido quimicamente , Necrose da Cabeça do Fêmur/diagnóstico por imagem , Necrose da Cabeça do Fêmur/epidemiologia , Glucocorticoides/efeitos adversos , Quadril , Humanos , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Calcium is a requisite nutrient in maintaining bone health. High calcium intake has been recommended for osteoporosis prevention. However, the relationship of dietary calcium intake with bone mineral density (BMD) in the elderly remained controversial. We aimd to evaluate the association between them in older adults. Using data from the Nutrition and Health Examination Survey (NHANES, 2001-2006), we conducted multivariate logistic regression analyses to explore the association of dietary calcium intake with lumbar BMD in older adults. Subgroup analyses were also performed. We included 2904 older adults aged ≥ 60 (43.63% men) for analysis. Higher dietary calcium intake was associated with greater lumbar BMD after adjusting for other covariates. In the subgroup analysis stratified by sex, this positive association existed in women, but not in men. In conclusion, for older adults aged ≥ 60, higher dietary calcium intake is associated with a higher lumbar BMD in women, but not in men.
Assuntos
Densidade Óssea , Cálcio da Dieta/administração & dosagem , Idoso , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inquéritos Nutricionais , Caracteres SexuaisRESUMO
OBJECTIVE: To study the value of body fat mass measured by bioelectrical impedance analysis (BIA) in predicting abnormal blood pressure and abnormal glucose metabolism in children. METHODS: Stratified cluster sampling was used to select the students aged 6-16 years, and a questionnaire survey and physical examination were performed. The BIA apparatus was used to measure body fat mass. Body mass index (BMI), body fat mass index (FMI), and fat mass percentage (FMP) were calculated. Fasting blood glucose level were measured. RESULTS: A total of 14â293 children were enrolled, among whom boys accounted for 49.89%. In boys and girls, the percentile values (P60, P65, P70, P75, P80, P85, P90, P95) of FMI and FMP fitted by the LMS method were taken as the cut-off values. Based on the receiver operating characteristic curve analysis, the P70 values with a better value in predicting abnormal blood pressure and blood glucose metabolism were selected as the cut-off values for excessive body fat. When FMI or FMP was controlled below P70, the incidence of abnormal blood pressure or abnormal glucose metabolism may be decreased in 8.25%-43.24% of the children. CONCLUSIONS: The evaluation of obesity based on FMI and FMP has a certain value in screening for hypertension and hyperglycemia in children, which can be further verified in the future prevention and treatment of obesity and related chronic diseases in children.
Assuntos
Tecido Adiposo , Adolescente , Pressão Sanguínea , Composição Corporal , Índice de Massa Corporal , Criança , Impedância Elétrica , Feminino , Glucose , Humanos , MasculinoRESUMO
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
We report here a rapid and sensitive technique for negative visualization of protein in 1D and 2D SDS-PAGE by using 2', 7'-dichlorofluorescein (DCF), which appeared as transparent and colorless bands in an opaque gel matrix background. For DCF stain, down to 0.1-0.2 ng protein could be easily visualized within 7 min by only two steps, and the staining is fourfold more sensitive than that of Eosin Y (EY) negative stain and glutaraldehyde (GA) silver stain, and eightfold more sensitive than that of the commonly used imidazole-zinc (IZ) negative stain. Furthermore, DCF stain provided good reproducibility, linearity, and MS compatibility compared with those of IZ stain. In addition, the potential staining mechanism was investigated by colorimetric experiment and molecular docking, and the results demonstrated that the interaction between DCF and protein occurs mainly via van der waals force, electrostatic interaction, and hydrogen bonding.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Fluoresceínas/análise , Coloração Negativa/métodos , Proteínas/análise , Colorimetria/métodos , Humanos , Simulação de Acoplamento Molecular/métodos , Proteômica/métodosRESUMO
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 this study, a new hydrazide derivative (UGF202) was synthesized and introduced as a highly sensitive and selective fluorescent probe to pre-stain glycoproteins in 1D and 2D SDS-PAGE. As low as 0.5-1 ng glycoproteins (transferrin, α1-acid glycoprotein, avidin) could be selectively detected, which is comparable to that of Pro-Q Emerald 300 stain, one of the most sensitive and commonly used glycoprotein staining kit. In addition, the specificity of the newly developed method was confirmed by the study of de-glycosylation, glycoproteins affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that UGF202 pre-stain can provide an alternative for the visualization of gel-separated glycoproteins.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Corantes Fluorescentes/química , Glicoproteínas/análise , Hidrazinas/química , Avidina/análise , Cromatografia Líquida , Eletroforese em Gel Bidimensional/métodos , Corantes Fluorescentes/síntese química , Humanos , Hidrazinas/síntese química , Orosomucoide/análise , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem , Transferrina/análiseRESUMO
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 sensitive and simple technique was developed for the visualization of gel-separated lipopolysaccharides by using a hydrazide derivative, UGF202. As low as 0.5-1 ng total LPS could be detected by UGF202 stain, which is 2- and 16-fold more sensitive than that of the commonly used Pro-Q Emerald 300 and Keenan et al. developed silver stain, respectively. The results indicated that UGF202 stain could be a good choice for LPS determination in polyacrylamide gels.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Corantes Fluorescentes/química , Lipopolissacarídeos/análise , Processamento de Imagem Assistida por Computador , Modelos Lineares , Lipopolissacarídeos/química , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
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
A new fluorescent prestaining method for gel-separated glycoproteins in 1D and 2D SDS-PAGE was developed by using 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH). The prestained gels were readily imaged after electrophoresis without any time-consuming steps needed for poststain. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be selectively detected, which is comparable to the most commonly used Pro-Q Emerald 488 glycoprotein stain. In addition, subsequent study of deglycosylation, glycoprotein affinity chromatography, and LC-MS/MS analysis were performed to confirm the specificity of the newly developed method. As a result, BH prestain provides a new choice for quick, sensitive, specific, economical, and MS compatible visualization of gel-separated glycoproteins.