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1.
Exp Mol Med ; 52(1): 66-78, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31956273

RESUMO

Patients with antiphospholipid syndrome (APS) are at high risk of developing venous and arterial thromboembolism (TE). The role of platelets in the pathogenesis of these prothrombotic conditions is not yet fully understood. The aim of this study was to gain mechanistic insights into the role of platelets in APS by comparing the platelet proteome between lupus anticoagulant (LA)-positive patients with (LA+ TE+) and without a history of TE (LA+ TE-) and healthy controls. The platelet proteome of 47 patients with LA, 31 with a history of TE and 16 without thrombotic history, and 47 healthy controls was analyzed by two-dimensional differential in-gel electrophoresis and mass spectrometry to identify disease-related proteins. Afterward, selected LA-related platelet proteins were validated by western blot and ELISA. Alterations of 25 proteins were observed between the study groups. STRING pathway analysis showed that LA-related protein profiles were involved in platelet activation, aggregation, and degranulation. For example, protein disulfide isomerase family members, enzymes that promote thrombosis, were upregulated in platelets and plasma of LA+ TE+ patients. Leukocyte elastase inhibitor (SERPINB1), an antagonist of neutrophil extracellular trap (NET) formation, was decreased in platelets of LA+ TE+ patients compared to healthy controls. Additionally, citrullinated histone H3, a NET-specific marker, was increased in plasma of LA+ TE+ patients. These findings suggest that decreased platelet SERPINB1 levels favor prothrombotic NETosis, especially in LA+ TE+ patients. Our findings reveal protein abundance changes connected to altered platelet function in LA-positive patients, thus suggesting a pathogenic role of platelets in thrombotic complications in APS.

2.
Thromb Haemost ; 19(10): 1642-1654, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31370072

RESUMO

INTRODUCTION: Blood platelets are increasingly recognized as modulators of leukocyte effector functions in various pathologies including acute lung injury (ALI). ALI is a life-threatening disease, caused by damage to the alveolar epi- and endothelium. Excessive accumulation of leukocytes leads to severe lung inflammation, resulting in impaired lung function and hypoxemia. OBJECTIVE: Since leukocyte migration is modulated by activated platelets and phosphatidylinositol 3-kinase (PI3K) signaling is involved in platelet function, we aimed to elucidate the effect of PI3K on platelet-mediated immune responses. MATERIALS AND METHODS: We generated a mouse model with a platelet-specific deletion of p85α, the most important regulatory subunit of the class IA PI3K, and evaluated platelet function and platelet-leukocyte interactions. Moreover, we analyzed the impact of platelet-specific p85α gene deficiency during sterile peritonitis and acid-induced ALI. RESULTS: In vitro analyses of platelets revealed that lack of p85α led to decreased downstream signaling and diminished expression of surface activation markers, for example, CD62P and CD63, as well as reduced platelet aggregation. Moreover, platelet PI3K essentially mediated direct interactions of platelets with monocytes and neutrophils. In mice, platelet-specific p85α deficiency prevented leukocyte infiltration into the peritoneum and the bronchoalveolar compartment during sterile peritonitis and ALI, respectively. Additionally, the release of the inflammatory cytokine interleukin-12/23 was diminished in platelet p85α-deficient mice during ALI. In contrast to PI3K, neither overexpression nor depletion of platelet phosphatase and tensin homolog, the endogenous antagonist of PI3K, significantly modulated platelet function. CONCLUSION: Our data indicate a crucial role of platelet PI3K signaling for leukocyte extravasation upon inflammatory stimuli in various diseases models.

3.
J Proteomics ; 178: 73-81, 2018 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-29278785

RESUMO

The incidence of Alzheimer's disease (AD) is higher in elderly women than in men. The molecular background of this gender-related risk, however, is largely unknown. In a previous proteomics study, we identified significantly elevated levels of monoamine oxidase-B and tropomyosin-1 in AD patients, together with significant changes of the genetic AD risk factors apolipoprotein E4 (APOE4) and glutathione S-transferase omega 1 (GSTO1), in platelets - a promising source for AD blood biomarkers. The present study aimed to investigate the gender-specificity as well as the disease-stage dependency of these biomarkers in AD patients and those with mild cognitive impairment (MCI). Tropomyosin-1 and monoamine oxidase-B protein levels were quantified by 2-D DIGE and 1-D Western blotting. Here, for the first time, we revealed a significant increase of 38&39kDa tropomyosin-1 protein levels in female but not male AD (+56%; p=0.008) and MCI patients (+46%; p=0.041) measured by 1-D WB. In contrast, levels of monoamine oxidase-B were, independently of gender, elevated in AD patients (+52%; p=0.009) but unaltered in MCI compared to control subjects. Moreover, we confirmed that APOE4-positive females are at a higher risk (OR=18.7; p=9.7E-09) of developing AD compared to APOE4-positive males (OR=6.5; p=5.9E-04). No gender-related effects were observed for GSTO1. SIGNIFICANCE: Platelet tropomyosin-1 constitutes a gender-related and stage-dependent protein in cognitive impairment. In contrast, platelet monoamine oxidase-B, frequently described to be increased in platelets and brains of AD patients, shows a gender-independent but stage-related increase since it is unaltered in MCI subjects. A blood biomarker test for this preceding stage of AD that considers gender-specificity is not yet available. The newly described AD-related platelet protein profiles might refine and facilitate routine diagnosis and enable early as well as tailored interventions.


Assuntos
Doença de Alzheimer/sangue , Plaquetas/metabolismo , Disfunção Cognitiva/sangue , Tropomiosina/metabolismo , Apolipoproteína E4/sangue , Plaquetas/química , Feminino , Glutationa Transferase/sangue , Humanos , Masculino , Monoaminoxidase/metabolismo , Fatores Sexuais
4.
Clin Chem Lab Med ; 56(5): 796-802, 2018 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-29220880

RESUMO

BACKGROUND: Apolipoprotein E (APOE) is a key player in lipid transport and metabolism and exists in three common isoforms: APOE2, APOE3 and APOE4. The presence of the E4 allelic variant is recognized as a major genetic risk factor for dementia and other chronic (neuro)degenerative diseases. The availability of a validated assay for rapid and reliable APOE4 classification is therefore advantageous. METHODS: Biochip array technology (BAT) was successfully applied to identify directly the APOE4 status from plasma within 3 h, through simultaneous immunoassay-based detection of both specific APOE4 and total APOE levels. RESULTS: Samples (n=432) were first genotyped by polymerase chain reaction (PCR), and thereafter, using BAT, the corresponding plasma was identified as null, heterozygous or homozygous for the E4 allele by calculating the ratio of APOE4 to total APOE protein. Two centers based in Austria and Ireland correctly classified 170 and 262 samples, respectively, and achieved 100% sensitivity and specificity. CONCLUSIONS: This chemiluminescent biochip-based sandwich immunoarray provides a novel platform to detect rapidly and accurately an individual's APOE4 status directly from plasma. The E4 genotype of individuals has been shown previously to affect presymptomatic risk, prognosis and treatment response for a variety of diseases, including Alzheimer's disease. The biochip's potential for being incorporated in quantitative protein biomarker arrays capable of analyzing disease stages makes it a superior alternative to PCR-based APOE genotyping and may deliver additional protein-specific information on a variety of diseases in the future.


Assuntos
Apolipoproteína E4/sangue , Imunoensaio , Medições Luminescentes , Idoso , Idoso de 80 Anos ou mais , Alelos , Apolipoproteína E4/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase
5.
Oncotarget ; 7(26): 39108-39117, 2016 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-27259244

RESUMO

Approximately 30 million people currently suffer from late-onset Alzheimer's disease (LOAD) worldwide. Twin studies demonstrated that 60 to 80% of LOAD is genetically determined, 20% of which remaining unassigned. This case-control study included 118 cognitively healthy controls, 52 patients with mild cognitive impairment (MCI; the pre-stage of LOAD) and 71 LOAD patients. The participants were genotyped for the genetic LOAD marker apolipoprotein E4 (APOE4) and the single-nucleotide polymorphism rs4925 in glutathione S-transferase omega-1 (GSTO1). Additive logistic regression showed a novel, statistically significant association of the major allele GSTO1*C with MCI (OR1.9; p = 0.032). However, identification of significant SNP-disease relations required well-defined study groups. When classifying participants solely by the short Mini Mental State examination (MMSE), the associations of GSTO1*C and the reference marker APOE4 with MCI were cancelled. Moreover, even identifying only the control group by MMSE nullified a statistically significant association (OR1.8; p = 0.045) between GSTO1*C and LOAD. In contrast, these statistical relations were retained when the detailed Consortium to Establish a Registry for Alzheimer's Disease (CERAD-Plus) test battery was used. Hence, besides proposing rs4925 as a genetic marker for cognitive impairment, this work also emphasized the importance of carefully characterized controls in addition to well-diagnosed patients in case-control studies.


Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Glutationa Transferase/genética , Testes Neuropsicológicos , Idoso , Idoso de 80 Anos ou mais , Alelos , Apolipoproteína E4/genética , Estudos de Casos e Controles , Disfunção Cognitiva , Feminino , Marcadores Genéticos , Predisposição Genética para Doença , Variação Genética , Genótipo , Humanos , Masculino , Testes de Estado Mental e Demência , Mutação de Sentido Incorreto , Análise de Regressão
6.
Sci Rep ; 5: 16662, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26567861

RESUMO

We previously showed that, when peripheral blood mononuclear cells (PBMCs) were stressed with ionizing radiation, they released paracrine factors that showed regenerative capacity in vitro and in vivo. This study aimed to characterize the secretome of PBMCs and to investigate its biologically active components in vitro and vivo. Bioinformatics analysis revealed that irradiated PBMCs differentially expressed genes that encoded secreted proteins. These genes were primarily involved in (a) pro-angiogenic and regenerative pathways and (b) the generation of oxidized phospholipids with known pro-angiogenic and inflammation-modulating properties. Subsequently, in vitro assays showed that the exosome and protein fractions of irradiated and non-irradiated PBMC secretome were the major biological components that enhanced cell mobility; conversely, secreted lipids and microparticles had no effects. We tested a viral-cleared PBMC secretome, prepared according to good manufacturing practice (GMP), in a porcine model of closed chest, acute myocardial infarction. We found that the potency for preventing ventricular remodeling was similar with the GMP-compliant and experimentally-prepared PBMC secretomes. Our results indicate that irradiation modulates the release of proteins, lipid-mediators and extracellular vesicles from human PBMCs. In addition our findings implicate the use of secretome fractions as valuable material for the development of cell-free therapies in regenerative medicine.


Assuntos
Exossomos/metabolismo , Leucócitos Mononucleares/metabolismo , Proteoma/análise , Doença Aguda , Animais , Apoptose/efeitos da radiação , Linhagem Celular , Movimento Celular , Micropartículas Derivadas de Células/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos da radiação , Peroxidação de Lipídeos/efeitos da radiação , Lipídeos/análise , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/veterinária , Análise de Sequência com Séries de Oligonucleotídeos , Proteoma/efeitos da radiação , Radiação Ionizante , Regeneração/fisiologia , Suínos , Transcriptoma/efeitos da radiação , Remodelação Ventricular
7.
Electrophoresis ; 36(21-22): 2837-2840, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26332751

RESUMO

Serine protease inhibitors (serpin) have therapeutic potential in a variety of pathogenic processes, ranging from thrombosis and altered immune response to liver cirrhosis. To investigate the physiological effects of protein C inhibitor (PCI, serpinA5), its gene was inactivated in a mouse model, resulting in male infertility. In the present report, 2D differential gel electrophoresis was utilized to investigate the molecular mechanisms for PCI involvement in male reproduction. Comparing the testes proteomes of three PCI-knockout mice with three wild types demonstrated similar patterns with the exception of a massive upregulation of prostaglandin reductase 1 (tenfold; p < 0.002) and the complete shifts in the molecular weights of serpinA1C and serpinA3K. All these PCI-dependent proteome changes were immunologically verified. Unbiased proteome analysis indicated that inactivation of serpinA5 strongly influenced both the protein species pattern of other A-clade serpins as well as prostaglandin metabolism in the testes.

8.
Acta Neuropathol ; 128(5): 665-77, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25248508

RESUMO

Alzheimer's disease (AD), a multifactorial neurodegenerative condition caused by genetic and environmental factors, is diagnosed using neuropsychological tests and brain imaging; molecular diagnostics are not routinely applied. Studies have identified AD-specific cerebrospinal fluid (CSF) biomarkers but sample collection requires invasive lumbar puncture. To identify AD-modulated proteins in easily accessible blood platelets, which share biochemical signatures with neurons, we compared platelet lysates from 62 AD, 24 amnestic mild cognitive impairment (aMCI), 13 vascular dementia (VaD), and 12 Parkinson's disease (PD) patients with those of 112 matched controls by fluorescence two-dimensional differential gel electrophoresis in independent discovery and verification sets. The optimal sum score of four mass spectrometry (MS)-identified proteins yielded a sensitivity of 94 % and a specificity of 89 % (AUC = 0.969, 95 % CI = 0.944-0.994) to differentiate AD patients from healthy controls. To bridge the gap between bench and bedside, we developed a high-throughput multiplex protein biochip with great potential for routine AD screening. For convenience and speed of application, this array combines loading control-assisted protein quantification of monoamine oxidase B and tropomyosin 1 with protein-based genotyping for single nucleotide polymorphisms (SNPs) in the apolipoprotein E and glutathione S-transferase omega 1 genes. Based on minimally invasive blood drawing, this innovative protein biochip enables identification of AD patients with an accuracy of 92 % in a single analytical step in less than 4 h.


Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/diagnóstico , Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Análise Serial de Proteínas/métodos , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Apolipoproteínas E , Transtornos Cognitivos/etiologia , Disfunção Cognitiva , Feminino , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Masculino , Espectrometria de Massas , Monoaminoxidase/sangue , Monoaminoxidase/genética , Testes Neuropsicológicos , Fenótipo , Estatísticas não Paramétricas , Tropomiosina/sangue , Tropomiosina/genética
9.
Br J Pharmacol ; 171(24): 5708-27, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25091578

RESUMO

BACKGROUND AND PURPOSE: Despite new therapeutic approaches, metastatic melanomas still have a poor prognosis. Statins reduce low-density lipoprotein cholesterol and exert anti-inflammatory and anti-proliferative actions. We have recently shown that simvastatin triggers an apoptotic burst in human metastatic melanoma cells by the synthesis of an autocrine factor. EXPERIMENTAL APPROACH: The current in vitro study was performed in human metastatic melanoma cell lines (A375, 518a2) and primary human melanocytes and melanoma cells. The secretome of simvastatin-stressed cells was analysed with two-dimensional difference gel electrophoresis and MS. The signalling pathways involved were analysed at the protein and mRNA level using pharmacological approaches and siRNA technology. KEY RESULTS: Simvastatin was shown to activate a stress cascade, leading to the synthesis of 15-deoxy-12,14-PGJ2 (15d-PGJ2 ), in a p38- and COX-2-dependent manner. Significant concentrations of 15d-PGJ2 were reached in the medium of melanoma cells, which were sufficient to activate caspase 8 and the mitochondrial pathway of apoptosis. Inhibition of lipocalin-type PGD synthase, a key enzyme for 15d-PGJ2 synthesis, abolished the apoptotic effect of simvastatin. Moreover, 15d-PGJ2 was shown to bind to the fatty acid-binding protein 5 (FABP5), which was up-regulated and predominantly detected in the secretome of simvastatin-stressed cells. Knockdown of FABP5 abolished simvastatin-induced activation of PPAR-γ and amplified the apoptotic response. CONCLUSIONS AND IMPLICATIONS: We characterized simvastatin-induced activation of the 15d-PGJ2 /FABP5 signalling cascades, which triggered an apoptotic burst in melanoma cells but did not affect primary human melanocytes. These data support the rationale for the pharmacological targeting of 15d-PGJ2 in metastatic melanoma.


Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Melanócitos/efeitos dos fármacos , Melanoma/metabolismo , Prostaglandina D2/análogos & derivados , Sinvastatina/farmacologia , Comunicação Autócrina , Caspase 8/efeitos dos fármacos , Caspase 8/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a Ácido Graxo/efeitos dos fármacos , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Oxirredutases Intramoleculares/antagonistas & inibidores , Lipocalinas/antagonistas & inibidores , Melanócitos/metabolismo , Metástase Neoplásica , Prostaglandina D2/metabolismo
10.
Acta Neuropathol Commun ; 2: 65, 2014 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-24934666

RESUMO

Peripheral biomarkers play an indispensable role in quick and reliable diagnoses of any kind of disease. With the population ageing, the number of people suffering from age-related diseases is expected to rise dramatically over the coming decades. In particular, all types of cognitive deficits, such as Alzheimer's disease, will increase. Alzheimer's disease is characterised mainly by coexistence of amyloid plaques and neurofibrillary tangles in brain. Reliable identification of such molecular characteristics antemortem, however, is problematic due to restricted availability of appropriate sample material and definitive diagnosis is only possible postmortem. Currently, the best molecular biomarkers available for antemortem diagnosis originate from cerebrospinal fluid. Though, this is not convenient for routine diagnosis because of the required invasive lumbar puncture. As a consequence, there is a growing demand for additional peripheral biomarkers in a more readily accessible sample material. Blood platelets, due to shared biochemical properties with neurons, can constitute an attractive alternative as discussed here. This review summarises potential platelet Alzheimer's disease biomarkers, their role, implication, and alteration in the disease. For easy comparison of their performance, the Hedge effect size was calculated whenever data were available.


Assuntos
Doença de Alzheimer/sangue , Biomarcadores/sangue , Plaquetas/metabolismo , Animais , Humanos
11.
J Proteomics ; 94: 540-51, 2013 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-24284060

RESUMO

UNLABELLED: Accurate biomarker quantification requires carefully chosen normalisation procedures. When single proteins are used as loading controls (LCs), it is crucial that their expressional stability must be known. Platelets are an important biomarker source, especially for neurological diseases. We performed a systematical analysis of the platelet proteome to identify proteins suitable as LCs, using the 2-D DIGE system. We first screened a healthy population (n=137), aged between 18 and 104years, to find proteins with small coefficients of total variation (CVtot), herein termed low biological variation proteins (LBVP). Thereafter, expressional stability was verified in 101 patients suffering from Alzheimer's- (AD), Parkinson's- disease, vascular dementia or schizophrenia. Interestingly, traditional LCs such as tubulin beta-1 and GAPDH, were not found amongst LBVP. The least variable protein, calculated over all 238 individuals, was 14-3-3 gamma, with a CVtot of 9.3%, showing no gender, age or disease dependency. The normalisation capability of 14-3-3 gamma was superior to traditional LC in quantifying Western blot signals of the platelet AD-biomarker Monoamine Oxidase B of patient versus controls. Similar results were obtained with HepG2 cells, treated in vitro with DNA-methyltransferase inhibitor 5-aza-2'deoxicytidine. Finally, we provide a list of alternative normalisation candidates for accurate biomarker quantification. BIOLOGICAL SIGNIFICANCE: This paper suggests a considerable list of platelet proteins with a lower biological variation than well known "housekeeping" proteins like GAPDH and tubulin. Spot abundances of found proteins are middle ranged and unaffected by gender, age and certain diseases. Hence, listed proteins might be valuable normalisation candidates used additionally or alternatively. Platelet's least variable protein 14-3-3 gamma is validated as normalisation protein in platelet biomarker quantification. Furthermore 14-3-3 gamma is demonstrated to be also stable expressed by in HepG2, cells others than platelets, when treated by DNA methylation inhibitor.


Assuntos
Proteínas 14-3-3/metabolismo , Plaquetas/metabolismo , Western Blotting/normas , Proteômica/normas , Proteínas 14-3-3/química , Actinas/química , Actinas/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Biomarcadores/química , Biomarcadores/metabolismo , Plaquetas/química , Demência Vascular/metabolismo , Feminino , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Células Hep G2 , Humanos , Masculino , Pessoa de Meia-Idade , Monoaminoxidase/química , Monoaminoxidase/metabolismo , Doença de Parkinson/metabolismo , Esquizofrenia/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
12.
J Proteomics ; 75(18): 5848-60, 2012 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-22885077

RESUMO

High biological variation in protein expression represents a major challenge in clinical proteomics. In a study based on 2D-DIGE, we found that the standardised abundance of only a few proteins varied by more than 50%. While some of the highest variable proteins in platelets of 52 healthy elderly were of plasmatic origin, such as albumin or haptoglobin, absence of several other high-abundant plasma proteins strongly suggests that plasma-derived proteins represent an integral part of the platelet proteome. Amongst the highly variable platelet-derived proteins, two spots were both identified as GSTO1 and assigned to either the wild-type or mutant isoform of SNP A140D. Remarkably, when the spots were considered within the respective genotype groups, their CV decreased to about the median variation. Albeit 2D-DIGE allowed correct genotyping, two individuals seemed to be GSTO1*A140 deficient. Probing 2D-Western blots with novel mAb, however, detected A140 protein as additional spot at pH 8.1, caused by the SNPs E155del and E208K. In contrast to previous studies, we show that GSTO1 protein is expressed in vivo, despite the deletion E155. Our data indicate that incorporation of exogenous proteins and genetic polymorphisms of endogenous proteins represent the main source of extreme biological variation in the platelet proteome.


Assuntos
Plaquetas/química , Proteínas Sanguíneas/genética , Glutationa Transferase/genética , Proteoma/análise , Idoso , Idoso de 80 Anos ou mais , Feminino , Frequência do Gene , Glutationa Transferase/sangue , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Proteômica , Eletroforese em Gel Diferencial Bidimensional
13.
J Proteomics ; 75(6): 1792-802, 2012 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-22227400

RESUMO

Quantitative proteomic comparisons require a sufficient number of samples to reach an acceptable level of significance. But 2D gel electrophoresis commonly results in incomplete data sets due to spots with missing values reducing thereby the number of parallel measurements for individual proteins. Here we investigated how many missing values per spot can be tolerated. The number of spots in common between all gels was found to decrease with the number of parallel gels in a non-linear fashion. Increasing numbers of missing values were associated with a moderate increase in the quantitative variation of spot volumes. Based on the missing value pattern in 20 gels we performed an analysis of the multiple testing power for the hypothetical scenario of a comparative 2DE study with six or twelve parallel gels. The calculation considered the statistical power of the individual spot as well as the number of spots included in the analysis. The power increased with inclusion of spots with higher number of missing values and showed an optimum at a specific minimum number of spot replicates. The results suggest that proteins with missing values can be included in a univariate analysis as long as a sufficient number of parallel gels are made.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas/análise , Proteômica/métodos , Proteínas Sanguíneas/isolamento & purificação , Humanos , Processamento de Imagem Assistida por Computador/métodos , Reprodutibilidade dos Testes , Projetos de Pesquisa , Estatística como Assunto
14.
J Proteomics ; 75(7): 2080-92, 2012 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-22270014

RESUMO

Monoamine oxidase-B (Mao-B) catalysing the breakdown of the neurotransmitter dopamine, is known to be involved in the pathophysiology of Parkinson's (PD) and Alzheimer's disease (AD). Increased brain Mao-B activity is associated with AD. This alteration can also be seen in platelets, albeit the cause has hitherto remained elusive. To gain a deeper understanding of the etiology of AD, the platelet proteome was characterised, (2D DIGE pH6-9, including Mao-B) from 150 individuals: 34 AD, 13 vascular dementia, 15 non-demented PD patients, 49 matched controls, 18 oldest old and 21 young individuals. One significant change was noted after applying false discovery rate with the upregulation of the Mao-B expression (30% adjusted P value<0.001; effect size 1.31) in AD compared to age- and sex-matched controls. In contrast, Mao-B levels were unchanged in PD to matched controls. Western blot and mRNA analyses verified these findings. Moreover, Mao-B concentration correlated with age in the cognitive healthy individuals (r=0.53; P<0.001) and PD patients but not in those suffering from AD (r=-0.03; P=0.874). Mao-B activity correlated with the increased Mao-B protein expression in AD (r=0.81; P=0.016). We suggest that Mao-B platelet protein level may serve as a biomarker for age-related dementia, especially AD.


Assuntos
Doença de Alzheimer/metabolismo , Plaquetas/metabolismo , Regulação Enzimológica da Expressão Gênica , Monoaminoxidase/biossíntese , Doença de Parkinson/metabolismo , Proteoma/biossíntese , Regulação para Cima , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Dopamina/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
15.
J Neural Transm (Vienna) ; 118(5): 653-62, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21424576

RESUMO

Studies investigating the impact of high meat intake on cognition have yielded contradictory results as some show improved cognitive performance, whereas others report an increase of risk factors for dementia. However, few studies were designed to directly assess the effect of a high protein (HP) diet on both cognitive performance and corresponding biochemical parameters. A randomised intervention study was conducted with 23 healthy males (aged 19-31 years) to investigate the effects of a usual (UP) versus a HP diet on cognitive function and on the platelet proteome a well-established model for neurons. The study individuals were assigned to either a UP diet (15% energy) or a HP diet (30% energy) for 3 weeks with controlled intake of food and beverages. Blood samples were taken along with measurements of cognitive functions at the beginning and at the end of the intervention period. Among 908 reproducibly studied platelet proteins only the level of monoamine oxidase B (MaoB), a neurotransmitter degrading enzyme, decreased by 26% significantly (adjusted P value < 0.05) due to the HP diet. In addition, we found a correlation (r = 0.477; P < 0.02) between the decrease of MaoB expression and the shortened reaction time (cognitive function) which is in accordance with reports that dementia patients show increased MaoB activity. Plasma vitamin B(12) concentration was increased by the HP diet and correlates inversely with platelet MaoB expression (r = -0.35; P < 0.02). Healthy young males on a HP diet showed improved cognitive function and counteract well-known dementia biomarkers such as platelet MaoB and components of the methylation cycle such as vitamin B(12) and homocysteine.


Assuntos
Plaquetas/enzimologia , Cognição/fisiologia , Alimentos Fortificados , Monoaminoxidase/sangue , Proteínas/administração & dosagem , Adulto , Eletroforese em Gel Bidimensional/métodos , Jejum/sangue , Homocisteína/sangue , Humanos , Masculino , Metilação , Testes Neuropsicológicos , Proteômica/métodos , Desempenho Psicomotor/fisiologia , Tempo de Reação , Método Simples-Cego , Estatística como Assunto , Estatísticas não Paramétricas , Regulação para Cima/fisiologia , Vitamina B 12/sangue , Adulto Jovem
16.
Clin Nutr ; 30(3): 303-11, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21239090

RESUMO

BACKGROUND: Recommendations to use other criteria than N-balance for defining protein requirements have been proposed. However, little evidence to support other measures such as physiological functions is available. OBJECTIVE: To investigate the effects of a usual (UP) versus a high protein (HP) diet on muscle function, cognitive function, quality of life and biochemical regulators of protein metabolism. DESIGN: A randomised intervention study was conducted with 23 healthy males (aged 19-31 yrs). All subjects consumed a Usual Protein (UP) diet (1.5 g protein/kg BW) for a 1-wk run-in period before the intervention period where they were assigned to either a UP or a High Protein (HP) diet (3.0 g protein/kg BW) for 3-wks with controlled intake of food and beverages. Blood and urine samples were taken along with measurements of physiological functions at baseline and at the end of the intervention period. RESULTS: The HP group improved their reaction time significantly compared with the UP group. Branched chain amino acids and phenylalanine in plasma were significantly increased following the HP diet, which may explain the improved reaction time. CONCLUSION: Healthy young males fed a HP diet improved reaction time. No adverse effects of the HP diet were observed. This trial was registered at www.clinicaltrials.gov as NCT00621231.


Assuntos
Cognição , Proteínas na Dieta/uso terapêutico , Carne , Músculo Esquelético/fisiologia , Adolescente , Adulto , Aminoácidos de Cadeia Ramificada/sangue , Dinamarca , Dieta , Proteínas na Dieta/efeitos adversos , Humanos , Masculino , Carne/efeitos adversos , Necessidades Nutricionais , Fenilalanina/sangue , Tempo de Reação , Método Simples-Cego , Adulto Jovem
17.
Atherosclerosis ; 212(1): 153-60, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20684828

RESUMO

OBJECTIVE: Oxidative stress and systemic inflammation negatively affect several protective functions of high density lipoproteins (HDL) and oxidative modification of HDL by the inflammation-derived oxidant hypochlorite converts HDL into a potent platelet agonist. Therefore it was the aim of this work to clarify if these platelet-activating effects result from specific binding of hypochlorite-oxidized HDL (hyp-OxHDL) to the platelet surface and to identify responsible receptors. METHODS: Binding and functional studies were performed with hyp-OxHDL in absence and presence of (potential) competitors in normal and CD36-deficient human platelets. Platelet aggregation was quantified by light transmission aggregometry. Surface expression of CD62P, phosphatidylserine and CD40L was quantified by flow cytometry. RESULTS: Binding studies reveal that hyp-OxHDL show specific and saturable high-affinity binding to the platelet surface. Hyp-OxHDL trigger platelet aggregation and in a dose dependent way provoke the release of significant amounts of CD40L as well as phosphatidylserine on the platelet surface. Blocking specific binding of hyp-OxHDL to the platelet surface interferes with the ability of hyp-OxHDL to stimulate human platelets. CD36-deficient human platelets show markedly reduced binding of hyp-OxHDL. Upon addition of hypochlorite-oxidized HDL, CD36-deficient platelets do not aggregate and completely fail to release CD40L or phosphatidylserine. CONCLUSIONS: From these results we conclude that specific binding of hyp-OxHDL to platelet CD36 is essential for the proinflammatory and procoagulant effects of hyp-OxHDL shown within this work. The contribution of other receptors besides CD36 to specific binding of hyp-OxHDL to the platelet membrane appears to be minimal, at best.


Assuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Antígenos CD36/sangue , Ácido Hipocloroso/química , Mediadores da Inflamação/sangue , Inflamação/sangue , Lipoproteínas HDL/sangue , Oxidantes/química , Ligação Competitiva , Plaquetas/imunologia , Antígenos CD36/deficiência , Ligante de CD40/sangue , Sinalização do Cálcio , Citometria de Fluxo , Humanos , Inflamação/imunologia , Masculino , Oxirredução , Selectina-P/sangue , Fosfatidilserinas/sangue , Agregação Plaquetária , Testes de Função Plaquetária , Ligação Proteica , Espécies Reativas de Oxigênio/sangue , Fatores de Tempo
18.
Atherosclerosis ; 213(1): 129-34, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20701912

RESUMO

OBJECTIVE: OxLDL represents a central player in atherogenesis and has been shown to activate human blood platelets. In light of the pivotal role of CD40L in inflammation, it was the aim of this work to clarify if platelet-activating effects of oxidized LDL result in surface exposure and liberation of CD40L and to explore the role of platelet scavenger receptor CD36 in this process. METHODS: Binding and functional studies were performed with hypochlorite-oxidized LDL in absence and presence of (potential) competitors in normal and CD36-deficient human platelets. To determine functional effects of hypochlorite-oxidized LDL on human platelets, formation of reactive oxygen species, intraplatelet calcium, CD40L and CD62P as well as platelet aggregation were quantified. RESULTS: Addition of OxLDL to resting human platelets results in intracellular calcium flux, platelet aggregation and surface expression of CD62P. OxLDL triggers the formation of intracellular reactive oxygen species and surface exposure of CD40L, with both being sensitive to the NADPH oxidase inhibitor apocynin. In CD36-deficient human platelets, functional effects as well as high affinity binding of hypochlorite-oxidized LDL appears to be significantly reduced compared with platelets positive for CD36. CONCLUSIONS: Our results prove a prominent--however, not exclusive--role of CD36 in platelet binding of hypochlorite-oxidized LDL. CD36 appears to be the major receptor responsible for hypochlorite-oxidized LDL-induced platelet activation that accumulates in the release of CD40L. As platelets represent the major source of CD40L, our findings emphasize an important pro-inflammatory role of platelets, especially in conditions of oxidative stress.


Assuntos
Antígenos CD36/metabolismo , Ligante de CD40/metabolismo , Ácido Hipocloroso/farmacologia , Lipoproteínas LDL/química , Oxigênio/química , Espécies Reativas de Oxigênio , Aterosclerose/metabolismo , Plaquetas/metabolismo , Membrana Celular/metabolismo , Citometria de Fluxo , Humanos , Cinética , Estresse Oxidativo , Agregação Plaquetária , Receptores Depuradores
19.
J Proteome Res ; 9(2): 1041-9, 2010 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-20073474

RESUMO

The only treatment of end-stage renal disease patients undergoing chronic dialysis is kidney transplantation. However, about half of graft recipients encounter organ loss within ten years after renal transplantation. There is emerging evidence that the presence of alloreactive antibodies against non-HLA antigens in the serum of the recipient prior transplantation is associated with higher incidence of chronic rejection. However, the molecular identity of these antigens is largely unknown. To determine the most common non-HLA antigens, we tested lymphocytic extracts from 20 healthy volunteers with sera of 28 patients on the transplantation waiting list by Western blotting. There was a group of five proteins that was recognized by most sera. Using patient's own lymphocytes revealed that autoimmunity plays a minor role in this recognition. Two-dimensional Western blotting experiments followed by mass spectrometry identified the antigens as tubulin beta chain, vimentin, lamin-B1, and Rho GDP-dissociation inhibitor 2. A detailed analysis of vimentin expression revealed that the antigenic 60 kDa isoform is underrepresented in patient's lymphocytes in comparison to those of healthy volunteers. The study revealed that preformed alloreactive antibodies are directed against a small number of specific protein isoforms. Our findings could provide a basis for future improvement of donor-recipient matching.


Assuntos
Isoanticorpos/sangue , Falência Renal Crônica/imunologia , Diálise Renal , Adulto , Idoso , Western Blotting , Feminino , Antígenos HLA/imunologia , Humanos , Isoanticorpos/imunologia , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Peso Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
Acta Neuropathol ; 118(1): 181-95, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19259691

RESUMO

Proteomic analysis enables us to identify dementia-related protein profiles of both genetic and environmental origins. In this review, current proteomics technologies are described including many examples of clinical proteomics studies. Many of these studies present only results of the discovery phase. Progression to the validation phase was achieved by developing more advanced proteomics technologies such as fluorescence two-dimensional differential gel electrophoresis or isobaric tagging for relative and absolute protein quantification. These technologies will lead to the design of several new Alzheimer's disease-related protein panels for the analysis of CSF. On these new panels, established markers such as tau and Abeta42 will be used in combination with novel markers, for example beta-2-microglobulin, brain-derived neurotrophic factor 1 and fragments of cystatin C. However, there are still limitations to using proteomic assays. The preparation of homogeneous sample material is difficult due the complexity of brain tissue. Laser capture microdissection and recently developed more sensitive proteomics methods, for example fluorescence saturation labelling, will overcome these limitations. Combining proteomics with approaches at the level of the genome and transcriptome followed by interpretation by systems biology will soon shed further light on dementia-related pathogenesis.


Assuntos
Doença de Alzheimer/metabolismo , Demência/metabolismo , Proteômica/métodos , Biomarcadores , Cromatografia Líquida/métodos , Eletroforese em Gel Bidimensional/métodos , Humanos , Espectrometria de Massas/métodos , Estresse Oxidativo
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