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1.
Int J Mol Sci ; 16(1): 1657-76, 2015 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-25588218

RESUMO

Silk fibroin (SF) is a protein with bulky hydrophobic domains and can be easily purified as sericin-free silk-based biomaterial. Silk fibroin modified chitosan nanoparticle (SF-CSNP), a biocompatible material, has been widely used as a potential drug delivery system. Our current investigation studied the bio-effects of the SF-CSNP uptake by liver cells. In this experiment, the characterizations of SF-CSNPs were measured by particle size analysis and protein assay. The average size of the SF-CSNP was 311.9 ± 10.7 nm, and the average zeta potential was +13.33 ± 0.3 mV. The SF coating on the SF-CSNP was 6.27 ± 0.17 µg/mL. Moreover, using proteomic approaches, several proteins involved in the ubiquitin proteasome pathway were identified by analysis of differential protein expressions of HepG2 cell uptake the SF-CSNP. Our experimental results have demonstrated that the SF-CSNP may be involved in liver cancer cell survival and proliferation.


Assuntos
Quitosana/metabolismo , Portadores de Fármacos/metabolismo , Fibroínas/metabolismo , Nanopartículas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Materiais Biocompatíveis/metabolismo , Linhagem Celular , Células Hep G2 , Humanos , Proteômica , Transdução de Sinais
2.
Analyst ; 137(21): 4921-33, 2012 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-22910856

RESUMO

The cell responses to biopolymer surface at the early adhesion stages can be critical for cell survival. The purpose of this research was to assess formation of hyaluronic acid (HA) biopolymer surface, the fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymer materials using a mass spectrometry-based profiling system. Surfaces were covered by multi-walled carbon nanotubes (CNT), chitosan (CS), and HA to increase the surface area, enhance the adhesion of biopolymer and promote the rate of cell proliferation. The amount of adhered fibroblasts on CNT/CS/HA electrodes of quartz crystal microbalance (QCM) were greatly exceeded those on other surfaces that were consistent with cell-count technique. Moreover, analyzing differential protein expressions of adhered fibroblasts on those biopolymer surfaces by proteomic approaches identified CD36, CD44, PP2A, and CDK9 as key proteins. To validate the influences of those four proteins on adhesions of fibroblasts on biopolymers, the cells were blocked by antibodies of the proteins and the adhesions of cells on the tested biopolymer surfaces were examined using a QCM technique, flow cytometric analysis and morphological observations. The results of significantly decreasing the weights and densities of the blocked fibroblasts adhering to CNT/CS/HA surfaces were obtained, and validate those proteins found by proteomic approaches. Utilizing mass spectrometry-based proteomics to evaluate cell adhesions on biopolymers is proposed.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Proteômica/métodos , Sequência de Aminoácidos , Bromodesoxiuridina/metabolismo , Antígenos CD36/química , Antígenos CD36/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Quitosana/química , Quinase 9 Dependente de Ciclina/química , Quinase 9 Dependente de Ciclina/metabolismo , Eletrodos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Receptores de Hialuronatos/química , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Espectrometria de Massas , Dados de Sequência Molecular , Nanotubos de Carbono/química , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Propriedades de Superfície
3.
J Biomed Biotechnol ; 2011: 901329, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21860587

RESUMO

The purpose of this paper was to characterize proteins secreted from the human nonpigmented ciliary epithelial (HNPE) cells, which have differentiated a rat retinal ganglion cell line, RGC-5. Undifferentiated RGC-5 cells have been shown to express several marker proteins characteristic of retinal ganglion cells. However, RGC-5 cells do not respond to N-methyl-D aspartate (NMDA), or glutamate. HNPE cells have been shown to secrete numbers of neuropeptides or neuroproteins also found in the aqueous humor, many of which have the ability to influence the activity of neuronal cells. This paper details the profile of HNPE cell-secreted proteins by proteomic approaches. The experimental results revealed the identification of 132 unique proteins from the HNPE cell-conditioned SF-medium. The biological functions of a portion of these identified proteins are involved in cell differentiation. We hypothesized that a differentiation system of HNPE cell-conditioned SF-medium with RGC-5 cells can induce a differentiated phenotype in RGC-5 cells, with functional characteristics that more closely resemble primary cultures of rat retinal ganglion cells. These proteins may replace harsh chemicals, which are currently used to induce cell differentiation.


Assuntos
Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas/metabolismo , Proteoma/química , Proteômica/métodos , Células Ganglionares da Retina/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Meios de Cultivo Condicionados , Bases de Dados de Proteínas , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/classificação , Fenótipo , Proteínas/química , Proteínas/classificação , Proteoma/metabolismo , Ratos , Células Ganglionares da Retina/citologia , Espectrometria de Massas em Tandem
4.
Rapid Commun Mass Spectrom ; 25(19): 2851-62, 2011 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21913264

RESUMO

Bladder cancer is the most common urological cancer with higher incidence rate in the endemic areas of Blackfoot disease (BFD) in southern Taiwan. The aim of this study was to utilize the proteomic approach to establish urinary protein patterns of bladder cancer. The experimental results showed that most patients with bladder cancer had proteinuria or albuminuria. The urine arsenic concentrations of bladder cancer patients in BFD areas were significantly higher than those patients from non-BFD areas. In the proteomic analysis, the urinary proteome was identified by nano-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (nano-HPLC/ESI-MS/MS) followed by peptide fragmentation pattern analysis. We categorized 2782 unique proteins of which 89 proteins were identified with at least three unique matching peptide sequences. Among these 89 proteins, thirteen of them were not found in the control group and may represent proteins specific for bladder cancer. In this study, three proteins, SPINK5, ADAM28 and PTP1, were also confirmed by Western blotting and showed significant differential expression compared with the control group. ADAM28 may be used as a possible biomarker of bladder cancer.


Assuntos
Proteínas ADAM/urina , Biomarcadores Tumorais/urina , Carcinoma de Células de Transição/urina , Espectrometria de Massas em Tandem/métodos , Neoplasias da Bexiga Urinária/urina , Proteínas ADAM/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Arsênio/urina , Biomarcadores Tumorais/biossíntese , Carcinoma de Células de Transição/metabolismo , Estudos de Casos e Controles , Cromatografia Líquida/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/urina , Doenças Vasculares Periféricas , Proteínas Secretadas Inibidoras de Proteinases/urina , Proteínas/análise , Proteínas/classificação , Proteinúria/urina , Proteoma/análise , Proteômica/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Inibidor de Serinopeptidase do Tipo Kazal 5 , Taiwan , Neoplasias da Bexiga Urinária/metabolismo
5.
J Mater Sci Mater Med ; 22(6): 1383-91, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21479636

RESUMO

Self-assembled monolayers (SAMs) on coinage metallic material can provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition and other interfacial phenomena. Recently, a bio-sensing system has been produced by analysis of the attachment of antibody using alkanethiols, to form SAMs on the face of Au-quartz crystal microbalance (QCM) surfaces. In this study, the attachment of anti-α-fetoprotein monoclonal antibody to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water-soluble N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as coupling agents. Surface analyses were utilized by X-ray photoelectron spectroscopy and atomic force microscopy. The quantization of immobilized antibody was characterized by the frequency shift of QCM and the radioactivity change of ¹²5I labeled antibody. The limit of detection and linear range of the calibration curve of the QCM method were 15 ng/ml and 15-850 ng/ml. The correlation coefficients of α-fetoprotein concentration between QCM and radioimmunoassay were 0.9903 and 0.9750 for the standards and serum samples, respectively. This report illustrates an investigation of SAMs for the preparation of covalently immobilized antibody biosensors.


Assuntos
Técnicas Biossensoriais/métodos , Lipossomas Unilamelares/química , alfa-Fetoproteínas/análise , Algoritmos , Técnicas Biossensoriais/instrumentação , Eletroquímica/métodos , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Microscopia de Força Atômica , Modelos Biológicos , Concentração Osmolar , Espectroscopia Fotoeletrônica , Técnicas de Microbalança de Cristal de Quartzo/métodos , Propriedades de Superfície , Lipossomas Unilamelares/metabolismo , alfa-Fetoproteínas/imunologia
6.
Anal Bioanal Chem ; 395(3): 729-35, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19669733

RESUMO

In the summer of 2008, serious illnesses and deaths of babies in China were linked to melamine-tainted powdered infant formula. Melamine contains several metabolites, such as ammeline, ammelide, and cyanuric acid, and has been used for the adulteration of foods or milk to increase their apparent protein content. It is assumed that melamine and its metabolites are absorbed in the gastrointestinal tract, and precipitate in the kidney to form crystals. A new tolerable daily intake of 0.2 mg kg(-1) body weight was adapted by the World Health Organization in 2008. This paper reviews the variety of analytical methods that have been used for the analysis of melamine in food. The limit of detection of these various methods is 0.05-100 ppm. The maximum acceptable concentration in food has been set at 50 ppb by the US FDA. A fast and ultrasensitive procedure for screening, detection, and characterization of melamine and its derivative compounds needs to be established. Currently, mass-spectrometry technologies provide an alternative to derivatization for regulatory analysis of food.


Assuntos
Contaminação de Alimentos/análise , Triazinas/análise , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Lactente , Espectrometria de Massas em Tandem/métodos , Triazinas/efeitos adversos
7.
Anal Bioanal Chem ; 388(3): 637-43, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17447055

RESUMO

Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. Analysis of human serum from HCC patients using two-dimensional gel electrophoresis (2DE) combined with nano-high-performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC-ESI-MS/MS) identified fourteen different proteins differentially expressed between HCC patients and the control group. Twelve proteins were up-regulated and two down-regulated. By using nano-HPLC-MS/MS system to analyze proteome in human serum, 317 proteins were identified, twenty-nine of which to high confidence levels (protein matched at last two unique peptide sequences). Of these twenty-nine proteins, six were present only in HCC patients and may serve as biomarkers for HCC.


Assuntos
Biomarcadores Tumorais/sangue , Proteínas Sanguíneas/análise , Carcinoma Hepatocelular/sangue , Neoplasias Hepáticas/sangue , Proteômica/métodos , Biomarcadores Tumorais/biossíntese , Proteínas Sanguíneas/biossíntese , Carcinoma Hepatocelular/diagnóstico , Cromatografia Líquida de Alta Pressão , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Humanos , Neoplasias Hepáticas/diagnóstico , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
8.
Biomed Res Int ; 2017: 4051763, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28127555

RESUMO

Micelles, with the structure of amphiphilic molecules including a hydrophilic head and a hydrophobic tail, are recently developed as nanocarriers for the delivery of drugs with poor solubility. In addition, micelles have shown many advantages, such as enhanced permeation and retention (EPR) effects, prolonged circulation times, and increased endocytosis through surface modification. In this study, we measured the critical micelle concentrations, diameters, stability, and cytotoxicity and the cell uptake of micelles against hepatic cells with two kinds of hydrophilic materials: PEG-PCL and HA-g-PCL. We used 131I as a radioactive tracer to evaluate the stability, drug delivery, and cell uptake activity of the micelles. The results showed that HA-g-PCL micelles exhibited higher drug encapsulation efficiency and stability in aqueous solutions. In addition, the 131I-lipiodol loaded HA-g-PCL micelles had better affinity and higher cytotoxicity compared to HepG2 cells.


Assuntos
Sistemas de Liberação de Medicamentos , Óleo Etiodado/administração & dosagem , Radioisótopos do Iodo/administração & dosagem , Compostos Radiofarmacêuticos/administração & dosagem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Meios de Contraste/administração & dosagem , Meios de Contraste/farmacocinética , Meios de Contraste/toxicidade , Portadores de Fármacos/química , Estabilidade de Medicamentos , Óleo Etiodado/farmacocinética , Óleo Etiodado/toxicidade , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/efeitos da radiação , Humanos , Ácido Hialurônico/análogos & derivados , Interações Hidrofóbicas e Hidrofílicas , Radioisótopos do Iodo/farmacocinética , Radioisótopos do Iodo/toxicidade , Micelas , Tamanho da Partícula , Poliésteres , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/toxicidade , Solubilidade
9.
Eur J Obstet Gynecol Reprod Biol ; 129(1): 31-5, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16580118

RESUMO

OBJECTIVE: To determine whether there is a correlation between adiponectin levels and glucose levels in women screened for gestational diabetes mellitus by means of a 50-g oral glucose challenge test, and to examine the difference in adiponectin levels between women who tested negative, and those who tested false positive. We further calculated the correlations between adiponectin levels and glucose levels, body mass index, gestational age and maternal age. METHODS: A case control study included 171 mothers with negative or false positive results in the 1-h 50-g glucose challenge test at 24-28 gestational weeks. Serum adiponectin levels were determined by radioimmunoassay at the time of the glucose challenge test. RESULTS: There was a significant difference between women who tested negative at screening, and those who tested false positive with respect to age, prepregnancy body weight and body mass index, and adiponectin levels. Correlation analysis showed adiponectin levels to be negatively correlated to glucose levels (r=-0.193, P=0.011). To examine the association between glucose levels, adiponectin levels and demographic variables, multiple linear regression analysis was carried out. Prepregnancy body mass index and age accounted for 14.6% of the variance in the glucose challenge test. Adiponectin levels did not contribute independently to the variation in glucose levels. A further multiple linear regression analysis was undertaken to investigate the association between adiponectin levels and age, prepregnancy body mass index and glucose levels in the glucose challenge test. In this regression model, prepregnancy body mass index and age explained 12.1% of the variance in adiponectin levels. CONCLUSIONS: In conclusion, our study indicated a negative correlation between adiponectin and glucose levels in women screened for gestational diabetes mellitus by a glucose challenge test. We further found that maternal age and body mass index were independent risk factors for a false positive glucose challenge test. Reduced adiponectin levels in women who tested false positive on the glucose challenge test were dependent on advanced age and higher body mass index.


Assuntos
Glicemia/metabolismo , Índice de Massa Corporal , Diabetes Gestacional/diagnóstico , Teste de Tolerância a Glucose , Adiponectina/sangue , Adulto , Fatores Etários , Reações Falso-Positivas , Feminino , Humanos , Gravidez
10.
Biomed Res Int ; 2016: 1917394, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28053978

RESUMO

The microenvironment of neuron cells plays a crucial role in regulating neural development and regeneration. Hyaluronic acid (HA) biomaterial has been applied in a wide range of medical and biological fields and plays important roles in neural regeneration. PC12 cells have been reported to be capable of endogenous NGF synthesis and secretion. The purpose of this research was to assess the effect of HA biomaterial combining with PC12 cells conditioned media (PC12 CM) in neural regeneration. Using SH-SY5Y cells as an experimental model, we found that supporting with PC12 CM enhanced HA function in SH-SY5Y cell proliferation and adhesion. Through RP-nano-UPLC-ESI-MS/MS analyses, we identified increased expression of HSP60 and RanBP2 in SH-SY5Y cells grown on HA-modified surface with cotreatment of PC12 CM. Moreover, we also identified factors that were secreted from PC12 cells and may promote SH-SY5Y cell proliferation and adhesion. Here, we proposed a biomaterial surface enriched with neurotrophic factors for nerve regeneration application.


Assuntos
Adesão Celular/efeitos dos fármacos , Ácido Hialurônico/administração & dosagem , Neuroblastoma/metabolismo , Engenharia Tecidual , Animais , Proliferação de Células/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Chaperonina 60/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Proteínas Mitocondriais/biossíntese , Chaperonas Moleculares/biossíntese , Regeneração Nervosa/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Neurônios/metabolismo , Neurônios/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/biossíntese , Células PC12 , Ratos
11.
Environ Health Perspect ; 113(11): 1645-50, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16263525

RESUMO

The aim of this study is to examine the association between transplacental exposure to dioxins/polychlorinated biphenyls (PCBs) and thyroid and growth hormones in newborns. We recruited 118 pregnant women, between 25 and 34 years of age, at the obstetric clinic. Personal data collected included reproductive and medical histories and physical factors. Clinicians gathered placental and umbilical cord serum upon delivery and carefully scored the 118 newborns, making both structural and functional assessments. We analyzed placentas for 17 polychlorinated dibenzo-p-dioxins and dibenzofurans and 12 dioxin-like PCB congeners with the World Health Organization-defined toxic equivalent factors, and six indicator PCBs by high-resolution gas chromatography and high-resolution mass spectrometry. We analyzed thyroid and growth hormones from cord serum using radioimmunoassay. Insulin-like growth factor (IGF)-1, IGF-binding globulin-3, and thyroxine x thyroid-stimulating hormone (T4 x TSH) were significantly associated with increased placental weight and Quetelet index (in kilograms per square meter; correlation coefficient r = 0.2-0.3; p < 0.05). Multivariate analyses showed independently and significantly decreased free T4 (FT4) x TSH with increasing non-ortho PCBs (r = -0.2; p < 0.05). We suggest that significant FT4 feedback alterations to the hypothalamus result from in utero exposure to non-ortho PCBs. Considering the vast existence of bioaccumulated dioxins and PCBs and the resultant body burden in modern society, we suggest routine screening of both thyroid hormone levels and thyroid function in newborns.


Assuntos
Poluentes Ambientais/toxicidade , Exposição Materna , Bifenilos Policlorados/toxicidade , Tiroxina/metabolismo , Adulto , Benzofuranos/análise , Benzofuranos/toxicidade , Dibenzofuranos Policlorados , Monitoramento Ambiental , Poluentes Ambientais/análise , Feminino , Sangue Fetal/química , Humanos , Recém-Nascido , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Placenta/química , Bifenilos Policlorados/análise , Dibenzodioxinas Policloradas/análogos & derivados , Dibenzodioxinas Policloradas/análise , Dibenzodioxinas Policloradas/toxicidade , Gravidez , Taiwan , Tireotropina/metabolismo
12.
J Steroid Biochem Mol Biol ; 91(1-2): 11-20, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15261303

RESUMO

The significance of steroidogenic factor 1 (SF-1) in adrenal steroidogenesis was studied using adrenocortical cell lines transformed with a dominant negative mutant of SF-1. Constitutive expression of the mutant did not only impair the activity of endogenous SF-1 but also diminish its own expression, suggesting that SF-1 was under autoregulation. Inhibition of the endogenous SF-1 activity significantly reduced basal and inducible transcription of CYP17, CYP21B and CYP11B1, but exhibited little effects on StAR and CYP11A1 expression. Stimulating the transformed cells with potassium and cAMP freed CYP11B2 from the mutant-caused transcriptional inhibition, whereas the transformation abolished induction of CYP17 by both stimulants. Consistent with the transcriptional changes of steroidogenic genes, basal and inducible synthesis of cortisol and androgens drastically declined in the transformed cell lines. The relief of CYP11B2 repression following the potassium and cAMP stimulation removed the restraint the mutant exerted on aldosterone synthesis, and resulted in aldosterone overproduction in the stimulated transformed cells. SF-1 also plays a role in regulating the adrenocorticotrophic hormone (ACTH) responsiveness of the adrenocortical cells. Inhibition of SF-1 activity significantly decreased basal expression of ACTH receptor and its induction by potassium and cAMP.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Fatores de Transcrição/biossíntese , Hormônio Adrenocorticotrópico/metabolismo , Western Blotting , Linhagem Celular , Linhagem Celular Transformada , AMP Cíclico/metabolismo , Ativação Enzimática , Fatores de Transcrição Fushi Tarazu , Regulação da Expressão Gênica , Genes Dominantes , Proteínas de Homeodomínio , Humanos , Modelos Biológicos , Mutação , Plasmídeos/metabolismo , Potássio/metabolismo , Cloreto de Potássio/farmacologia , RNA/metabolismo , Receptores da Corticotropina/biossíntese , Receptores Citoplasmáticos e Nucleares , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroide 11-beta-Hidroxilase/biossíntese , Esteroide 17-alfa-Hidroxilase/biossíntese , Fator Esteroidogênico 1 , Esteroides/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção
13.
Clin Chim Acta ; 348(1-2): 87-93, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15369740

RESUMO

BACKGROUND: Quantitative immunochromatographic strip (ICS) assay can facilitate clinical diagnosis by providing more information than traditional qualitative or semiquantitative strip assay. METHODS: We constructed a human serum alpha-fetoprotein (AFP) measurement system by combining semiquantitative ICS tests and artificial neural network (ANN) image analysis system [immunochromatographic strip analyzed by artificial neural network image analysis system (IAIS)]. After ICS tests completed, the AFP concentration can be obtained from analysis of IAIS software. The equipment required are commercial semiquantitative AFP dipstick, optical scanner, centrifuge (option), personal computer, and software of IAIS. RESULTS: The serum AFP concentrations measured by IAIS were strongly correlated (r = 0.9971) with that by RIA. Using Bland-Altman analysis, the IAIS achieved clinical acceptable limits of agreement in comparison with RIA. The within-run precision of IAIS, expressed as coefficients of variation (CV), at 81.7 ng/ml was 1.50% and, at 244.4 ng/ml, was 1.09%. The measurement of serum AFP by IAIS can be completed in 20 min. CONCLUSIONS: The newly constructed quantitative immunochromatographic strip assay (IAIS) is a simple, rapid, and reliable method for serum AFP measurement. With the simple equipment required, the IAIS can be performed outside the laboratory and is ideal for outpatient or point-of-care AFP testing.


Assuntos
Biomarcadores Tumorais/análise , Redes Neurais de Computação , Fitas Reagentes , alfa-Fetoproteínas/análise , Humanos , Radioimunoensaio/métodos , Sensibilidade e Especificidade
14.
J Biomed Mater Res A ; 71(1): 90-7, 2004 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-15368258

RESUMO

Self-assembled monolayers (SAMs) on coinage metal provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition, and other interfacial phenomena. Recently, the bonding of enzyme to SAMs of alkanethiols onto Au electrode surfaces was exploited to produce a bio-sensing system. In this work, the attachment of trypsin to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling agent. Experimental results have revealed that the X-ray Photoelectron Spectroscopy (XPS) C1s core levels at 286.3 and 286.5 eV (C with N), 288.1 eV (amide bond), and 289.3 eV (carboxyl) illustrate the immobilization of trypsin. These data were also in good agreement with Fourier-Transformed Infrared Reflection-Attenuated Total Reflection (FTIR-ATR) spectra for the peaks valued at 1659.4 cm(-1) (amide I) and 1546.6 cm(-1) (amide II). Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) observations, analytical results have demonstrated the platelet proteins digestion of the immobilized trypsin on the functionalized SAMs surface. For such surfaces, platelet proteins were digested on the trypsin-immobilized SAMs surface, which shows the enzyme digestion ability of the immobilized trypsin. The terminal groups of the SAMs structure can be further functionalized with biomolecules or antibodies to develop surface-base diagnostics, biosensors, or biomaterials.


Assuntos
Plaquetas/química , Perfilação da Expressão Gênica , Análise Serial de Proteínas/instrumentação , Proteoma , Tripsina/metabolismo , Plaquetas/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
15.
Eur J Obstet Gynecol Reprod Biol ; 108(1): 50-3, 2003 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-12694970

RESUMO

OBJECTIVES: To study the correlation between amniotic fluid leptin levels and maternal serum leptin levels during the early second trimester, and to determine whether the ratios of amniotic fluid leptin levels to maternal serum leptin levels are elevated in pregnant women who subsequently develop preeclampsia. STUDY DESIGN: Samples from 120 pregnant women were included in this prospective study, of which 20 were from pregnant women who subsequently developed preeclampsia and 100 were from normal pregnant women. Both the amniotic fluid and the maternal serum leptin levels were ascertained by radioimmunoassay (RIA). RESULTS: A strong correlation between amniotic fluid leptin levels and maternal serum leptin levels was observed in both preeclamptic and normal pregnant women. In addition, the ratios of amniotic fluid leptin levels to maternal serum leptin levels were positively correlated to amniotic fluid leptin levels, but negatively correlated to maternal serum leptin levels. Furthermore, the ratios of amniotic fluid leptin levels to maternal serum leptin levels in preeclamptic women were significantly higher than those in normal pregnant women. CONCLUSIONS: Amniotic fluid leptin levels correlated with maternal serum leptin levels during the early second trimester. The ratios of amniotic fluid leptin levels to maternal serum leptin levels were elevated in preeclamptic women. However, the maternal serum leptin levels themselves showed no such elevation. Therefore, this elevated ratio may be a marker at the early stage of pregnancy in preeclamptic women.


Assuntos
Líquido Amniótico/química , Leptina/análise , Leptina/sangue , Pré-Eclâmpsia/metabolismo , Adulto , Amniocentese , Índice de Massa Corporal , Feminino , Idade Gestacional , Humanos , Pré-Eclâmpsia/sangue , Gravidez , Segundo Trimestre da Gravidez , Análise de Regressão
16.
Biomed Res Int ; 2014: 209469, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24818131

RESUMO

The purpose of this study was to develop the pathway of silk fibroin (SF) biopolymer surface induced cell membrane protein activation. Fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymer material using a mass spectrometry-based profiling system. The surface was covered by multiwalled carbon nanotubes (CNTs) and SF to increase the surface area, enhance the adhesion of biopolymer, and promote the rate of cell proliferation. The amount of adhered fibroblasts on CNTs/SF electrodes of quartz crystal microbalance (QCM) greatly exceeded those on other surfaces. Moreover, analyzing differential protein expressions of adhered fibroblasts on the biopolymer surface by proteomic approaches indicated that CD44 may be a key protein. Through this study, utilization of mass spectrometry-based proteomics in evaluation of cell adhesion on biopolymer was proposed.


Assuntos
Materiais Biocompatíveis/farmacologia , Fibroínas/farmacologia , Proteínas/metabolismo , Proteômica , Animais , Bromodesoxiuridina/metabolismo , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Eletrodos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Ontologia Genética , Receptores de Hialuronatos/metabolismo , Imuno-Histoquímica , Espectrometria de Massas , Microscopia de Força Atômica , Nanotubos de Carbono/química , Técnicas de Microbalança de Cristal de Quartzo , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
17.
Clin Chim Acta ; 420: 54-61, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23078852

RESUMO

BACKGROUND: Samples originating from body fluids often contain a complex mixture of inorganic salts, buffers, chaotropic agents, surfactant/detergents, preservatives, and other solubilizing agents. The presence of those contaminants often precludes direct analysis by mass spectrometry. Urine, a blood filtrate produced by the urinary system, is an ideal bio-sample and a rich source of biomarkers for diagnostic information. METHODS: To enhance our understanding of urinary proteome, the urine proteins were prepared by magnetic nanoparticles (MNPs) combined with MACS separation column system and then identified by reverse phase nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (RP-nano-HPLC-ESI-MS/MS) followed by peptide fragmentation pattern. RESULTS: Experimental results have revealed that the better protein identification for the demonstration of bovine serum albumin (BSA) in artificial urine. Using this cleanup approach, a total of 542 peptides, corresponding to 282 unique proteins, were identified from human urine samples, in which 54 proteins have higher confidence levels. Indeed, this study has revealed that some biological factors might be increased along with aging, such as up-regulation of immunoproteins. CONCLUSIONS: The present study was designed to establish optimal techniques to develop a proteomic map of urinary proteins, and a cleanup method that greatly simplifies this sample preparation process was proposed.


Assuntos
Técnicas de Química Analítica/métodos , Magnetismo , Espectrometria de Massas , Nanopartículas , Proteoma , Adulto , Idoso , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estrutura Molecular , Análise Serial de Proteínas , Proteinúria/patologia , Soroalbumina Bovina
18.
Clin Chim Acta ; 420: 99-103, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23078848

RESUMO

BACKGROUND: The thyroid hormone, thyroxine (T4), is a tyrosine-based hormone produced by the thyroid gland, which is essential in regulating a number of biological processes, including growth, neurodevelopment, carbohydrate metabolism, oxygen consumption and protein synthesis. Data on human thyroid hormone metabolism were gathered since the middle of the 1970s mainly by the use of radioactive iodinated ((125)I or (131)I) hormones. METHODS: We describe an isotope dilution-matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method for the simultaneous determination of endogenous thyroid hormone and its (13)C6-labeled analogue in serum. The (13)C6-thyroxine ((13)C6-T4) was used as an internal standard; T4 and its isotopically labeled analogue were measured in the selected reaction monitoring mode for the transitions from m/z 777.8 to 732.1 and from m/z 784.2 to 738.1, respectively. RESULTS: Serum samples were prepared and concentrated by a solid-phase extraction chromatography method. The recovery rate was measured by (125)I-T4 and can be up to 82.8±2.8%. The detection limit and linear range for T4 were 5 ng/ml and 5-400 ng/ml, respectively. The correlation coefficient (R(2)) between radioimmunoassay (RIA) and isotope dilution-MALDI-TOF MS for detection of serum T4 was 0.982. CONCLUSION: This assay has a good relationship against a commercial RIA and the isotope dilution-mass spectrometry method and may serve as a reference method for quantitative analysis of T4.


Assuntos
Técnicas de Diluição do Indicador , Radioimunoensaio/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tiroxina/sangue , Humanos , Limite de Detecção , Estrutura Molecular
19.
J Proteomics ; 75(12): 3617-29, 2012 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-22554909

RESUMO

Alzheimer's disease (AD) is the most common cause of dementia of late life. To enhance our understanding of AD proteome, the serum proteins were analyzed using two-dimensional gel electrophoresis (2DE) combined with nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC-ESI-MS/MS) followed by peptide fragmentation patterning. In this study, six protein spots with differential expression were identified. Five up-regulated proteins were identified as actin, apolipoprotein A-IV (Apo A-IV), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-1-antitrypsin (AAT), and antithrombin-III (AT-III); one protein, activity-dependent neuroprotector homeobox protein (ADNP) was down-regulated in AD patients. These proteins with differential expression in the serum may serve as potential indicators of AD. Our results suggested that ADNP may play an important role in slowing the progression of clinical symptoms of AD.


Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/diagnóstico , Cromatografia Líquida de Alta Pressão/métodos , Proteínas de Homeodomínio/sangue , Proteínas do Tecido Nervoso/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
20.
Fertil Steril ; 88(2): 401-5, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17335820

RESUMO

OBJECTIVE: To test the hypothesis that plasma visfatin concentrations will be higher in women with polycystic ovary syndrome (PCOS) than in women without PCOS. DESIGN: Clinical study. SETTING: University hospital. PATIENT(S): A total of 52 women were evaluated. Twenty-six had PCOS, and the remaining 26 were healthy women with regular menstrual cycles who served as control subjects. INTERVENTION(S): Plasma visfatin concentrations were analyzed with the use of RIA. MAIN OUTCOME MEASURE(S): Serum concentrations of FSH, LH, TSH, PRL, T, insulin, E(2), and visfatin. RESULT(S): Plasma visfatin concentrations were significantly higher in the PCOS group (336.8 +/- 50.2 ng/mL) than in the healthy control group (282.4 +/- 43.3 ng/mL). Logistic regression analysis indicated a significant association between odds ratio (OR) values of PCOS and visfatin levels (OR = 2.81; 95% confidence interval [CI], 2.74-2.90), T (OR = 3.39; 95% CI, 2.85-4.16), and LH levels (OR = 3.49; 95% CI, 2.79-4.56). There was no correlation between plasma visfatin concentrations and T, insulin, and LH levels or age in either the PCOS group or the control group. We observed that plasma visfatin levels were positively correlated with body mass index in the PCOS group (r = 0.396, r(2) = 0.157) but not in the control group (r = -0.328, r(2) = 0.108). CONCLUSION(S): Our data show that women with PCOS exhibit higher plasma visfatin levels than control subjects of similar body mass index. Further studies are required to clarify the etiology and effects of hypervisfatinemia in women with PCOS.


Assuntos
Citocinas/sangue , Síndrome do Ovário Policístico/sangue , Adulto , Índice de Massa Corporal , Estudos de Casos e Controles , Feminino , Humanos , Nicotinamida Fosforribosiltransferase
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