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1.
J Sci Med Sport ; 22(7): 763-768, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30922550

RESUMO

OBJECTIVES: Participants of ultramarathon events experience a complex interaction of psychophysiological stressors. Therefore, the purpose of this study was to investigate the role of trait emotional intelligence (trait EI) on mood states and serum cortisol responses to a 80.5km treadmill ultramarathon. DESIGN: Twelve participants completed an 80.5km time-trial on a motorised treadmill in the fastest possible time. METHODS: Participants' trait EI was measured prior to the trial. A mood state questionnaire was completed prior (baseline: within two weeks of treadmill ultramarathon), immediately prior (pre: within 30min of commencing treadmill ultramarathon), at 40.25km (halfway: during standardised 10min rest period to allow for venous blood sampling) and on completion of 80.5km (post: immediately on completion of treadmill ultramarathon), along with serum cortisol concentrations measured at the same time points. RESULTS: Completion time was 09:00:18±01:14:07 (hhmmss). Significant increase in serum cortisol and total mood disturbance (TMD) was observed throughout the treadmill ultramarathon (p<0.05). Participants with higher trait EI displayed a higher post cortisol concentration (p=0.01) with no change in TMD, compared to those with low trait EI who displayed a significant increase in TMD between pre and halfway (p=0.02). CONCLUSIONS: The treadmill ultramarathon elicited a significant increase in serum cortisol concentration, which was significantly greater in those with a higher trait EI. Those individuals with higher trait EI were more effective at managing their mood, with little change total mood disturbance and perceived effort compared to those with lower trait EI.


Assuntos
Afeto/fisiologia , Atletas/psicologia , Inteligência Emocional/fisiologia , Corrida/fisiologia , Corrida/psicologia , Estresse Psicológico/psicologia , Adulto , Biomarcadores/sangue , Teste de Esforço/métodos , Feminino , Humanos , Hidrocortisona/sangue , Masculino , Consumo de Oxigênio/fisiologia , Inquéritos e Questionários
2.
Sci Rep ; 7(1): 17542, 2017 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-29235533

RESUMO

We describe a novel generic method to derive the unknown endogenous concentrations of analyte within complex biological matrices (e.g. serum or plasma) based upon the relationship between the immunoassay signal response of a biological test sample spiked with known analyte concentrations and the log transformed estimated total concentration. If the estimated total analyte concentration is correct, a portion of the sigmoid on a log-log plot is very close to linear, allowing the unknown endogenous concentration to be estimated using a numerical method. This approach obviates conventional relative quantification using an internal standard curve and need for calibrant diluent, and takes into account the individual matrix interference on the immunoassay by spiking the test sample itself. This technique is based on standard additions for chemical analytes. Unknown endogenous analyte concentrations within even 2-fold diluted human plasma may be determined reliably using as few as four reaction wells.


Assuntos
Imunoensaio/métodos , Peptídeos beta-Amiloides/sangue , Análise Química do Sangue/métodos , Calibragem , Feminino , Humanos , Hidrocortisona/sangue , Modelos Lineares , Masculino , Fragmentos de Peptídeos/sangue , Reprodutibilidade dos Testes
3.
BMC Res Notes ; 4: 281, 2011 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-21834984

RESUMO

BACKGROUND: Protein biomarker studies are currently hampered by a lack of measurement standards to demonstrate quality, reliability and comparability across multiple assay platforms. This is especially pertinent for immunoassays where multiple formats for detecting target analytes are commonly used. FINDINGS: In this pilot study a generic panel of six non-human protein standards (50 - 10^7 pg/mL) of varying abundance was prepared as a quality control (QC) material. Simulated "normal" and "diseased" panels of proteins were prepared in pooled human plasma and incorporated into immunoassays using the Meso Scale Discovery® (MSD®) platform to illustrate reliable detection of the component proteins. The protein panel was also evaluated as a spike-in material for a model immunoassay involving detection of ovarian cancer biomarkers within individual human plasma samples. Our selected platform could discriminate between two panels of the proteins exhibiting small differences in abundance. Across distinct experiments, all component proteins exhibited reproducible signal outputs in pooled human plasma. When individual donor samples were used, half the proteins produced signals independent of matrix effects. These proteins may serve as a generic indicator of platform reliability.Each of the remaining proteins exhibit differential signals across the distinct samples, indicative of sample matrix effects, with the three proteins following the same trend. This subset of proteins may be useful for characterising the degree of matrix effects associated with the sample which may impact on the reliability of quantifying target diagnostic biomarkers. CONCLUSIONS: We have demonstrated the potential utility of this panel of standards to act as a generic QC tool for evaluating the reproducibility of the platform for protein biomarker detection independent of serum matrix effects.

4.
J Immunol Methods ; 362(1-2): 176-9, 2010 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-20688072

RESUMO

Cell surface protein profiling is a generic tool to determine the quality of cultured mammalian cell lines. Flow cytometry is commonly employed as the screening technique of choice, but it consumes a large quantity of cells. We have evaluated an electrochemiluminescent (ECL)-based platform, the Meso Scale Discovery (MSD) Sector 6000 Imager, for cell surface protein detection with fewer than 100 cells per well. The detection of CD13 on human foreskin fibroblast (HFF) cells was evaluated using indirect detection of the reverse-phase immunoassays in 96-well and 384-well plate formats. The study has shown robust cell surface detection of CD13 on HFF cells using the 384-well plate format, with a LOD of 15.3 ± 6.5 cells (n=3). The 96-well plate format exhibited far greater variability between experiments, such that the best LOD obtained was 42.5 cells per well. We have demonstrated, with our model system, the feasibility to perform cell-binding assays with as few as 15 cells per well of a 384-well microplate using the MSD platform.


Assuntos
Antígenos CD13/metabolismo , Técnicas Eletroquímicas/métodos , Fibroblastos/metabolismo , Medições Luminescentes/métodos , Células Cultivadas , Técnicas Eletroquímicas/instrumentação , Fibroblastos/citologia , Humanos , Medições Luminescentes/instrumentação , Masculino
5.
J Immunol Methods ; 302(1-2): 1-12, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15993890

RESUMO

Recent developments in microarray technology have made it possible to perform immunoassays in a multiplexed format. This ability is highly desirable given the potential for greater throughput analysis. In spite of the obvious advantages, a number of issues arise as a result of multiplexing the reactions. In this study, we have assessed the performance characteristics that are associated with the transfer of technology from a uniplexed to a multiplexed format. Two solution phase array platforms were chosen for this study: the commercially available Luminex(100) xMap system (Austin, Texas, USA) and the UltraPlex technology devised by SmartBead Technologies Ltd. (Cambridge, UK). For this comparative study, a test for the presence of six autoantibodies in a selection of human patient serum samples was chosen as a model system. The multiplexed Luminex xMap and SmartBead UltraPlex assays were generally comparable. However, both systems generated some results that were at variance with those obtained by ELISA. The different methods used for the assignment of the cut-off levels for each of the assays within any given platform was identified as the major source of these non-concordant results. The present study demonstrates that array platforms have the potential to be used in immunodiagnostics providing that suitable cut-off levels are established.


Assuntos
Análise Serial de Proteínas/métodos , Anticorpos/análise , Anticorpos/sangue , Antígenos Nucleares/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/tendências , Humanos , Soros Imunes/análise , Microesferas , Análise Serial de Proteínas/tendências
7.
Biochem Soc Symp ; (72): 39-45, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15649128

RESUMO

MDCK (Madin-Darby canine kidney) cells represent a good model of polarized epithelium to investigate the signals involved in the apical targeting of proteins. As reported previously, GPI (glycosylphosphatidylinositol) anchors mediate the apical sorting of proteins in polarized epithelial cells through their interaction with lipid rafts. However, using a naturally N-glycosylated and GPI-anchored protein, we found that the GPI anchor does not influence the targeting of the protein. It is, in fact, the N-glycans that signal the protein to the apical surface. In the present review, the role of N-glycans and GPI anchors as apical signals is discussed along with the putative mechanisms involved.


Assuntos
Polaridade Celular/fisiologia , Polissacarídeos/metabolismo , Transdução de Sinais/fisiologia , Animais , Transporte Biológico Ativo , Linhagem Celular , Dipeptidases/metabolismo , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Microdomínios da Membrana/metabolismo
8.
J Cell Sci ; 117(Pt 21): 5079-86, 2004 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-15456847

RESUMO

The glycosyl-phosphatidylinositol (GPI) anchor mediates the apical sorting of proteins in polarised epithelial cells through its interaction with lipid rafts. Here we investigated the signals required for the apical targeting of the naturally N-glycosylated and GPI-anchored membrane dipeptidase by selective point mutation to remove the GPI anchor addition signal or the sites for N-linked glycosylation, or both. Activity assays, immunoblotting and immunofluorescence microscopy revealed that the constructs lacking the GPI anchor were secreted from Madin-Darby canine kidney (MDCK) cells, whereas those retaining the GPI anchor were attached at the cell surface, irrespective of the glycosylation status. Wild-type membrane dipeptidase was expressed preferentially on the apical surface of both MDCK and CaCo-2 cells. By contrast, the GPI-anchored construct lacking the N-glycans was targeted preferentially to the basolateral surface of both cell types. In constructs lacking the GPI anchor, the N-glycans also targeted the protein to the apical surface. Both the apically targeted, glycosylated and the basolaterally targeted, unglycosylated GPI-anchored forms of the protein were located in detergent-insoluble lipid rafts. These data indicate that it is the N-glycans, not the association of the GPI anchor with lipid rafts, which determine apical targeting of an endogenously N-glycosylated, GPI-anchored protein in polarised epithelial cells.


Assuntos
Células Epiteliais/metabolismo , Polissacarídeos/química , Animais , Sítios de Ligação , Western Blotting , Células CACO-2 , Linhagem Celular , Membrana Celular , DNA Complementar/metabolismo , Cães , Glicosilação , Glicosilfosfatidilinositóis/química , Humanos , Microdomínios da Membrana , Microscopia Confocal , Microscopia de Fluorescência , Mutação Puntual , Estrutura Terciária de Proteína , Transporte Proteico , Transdução de Sinais
9.
Int J Biochem Cell Biol ; 35(6): 944-54, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12676178

RESUMO

Emerging data have provided evidence for the presence of a local renin-angiotensin system (RAS) in the pancreas, which play a role in the regulation of pancreatic microcirculation, thus affecting islet hormonal secretion. The present study aimed, therefore, at elucidating the presence and changes of angiotensin-converting enzyme (ACE) using reverse transcription-polymerase chain reaction (RT-PCR) and a specific assay for ACE activity using the internally quenched fluorogenic substrate Meoc-DL-Amp-Gly-Lys(epsilon -DNP)-Gln-OH. RT-PCR clearly demonstrated the expression of ACE mRNA in the pancreas. ACE activity was markedly and significantly increased by chronic hypoxia and by acute pancreatitis when compared with that of their respective control pancreas. Addition of captopril, a specific inhibitor for ACE, completely blocked the ACE activity both in the control and experimental groups. All these data suggest that increased activity of pancreatic ACE in chronic hypoxia and acute pancreatitis could have implications for pancreatic physiology and pathophysiology.


Assuntos
Hipóxia/metabolismo , Pâncreas/enzimologia , Pancreatite/metabolismo , Peptidil Dipeptidase A/metabolismo , Doença Aguda , Animais , Doença Crônica , Regulação Enzimológica da Expressão Gênica , Hipóxia/genética , Pancreatite/genética , Peptidil Dipeptidase A/genética , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
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