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1.
J Pain Res ; 12: 2875-2889, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31686904

RESUMO

Objective: Neuropathic pain and fibromyalgia are two common and poorly understood chronic pain conditions that lack satisfactory treatments, cause substantial suffering and societal costs. Today, there are no biological markers on which to base chronic pain diagnoses, treatment choices or to understand the pathophysiology of pain for the individual patient. This study aimed to investigate cerebrospinal fluid (CSF) protein profiles potentially associated with fibromyalgia and neuropathic pain. Methods: CSF samples were collected from 25 patients with neuropathic pain (two independent sets, n=14 patients for discovery, and n=11 for verification), 40 patients with fibromyalgia and 134 controls without neurological disease from two different populations. CSF protein profiling of 55 proteins was performed using antibody suspension bead array technology. Results: We found increased levels of apolipoprotein C1 (APOC1) in CSF of neuropathic pain patients compared to controls and there was a trend for increased levels also in fibromyalgia patients. In addition, levels of ectonucleotide pyrophosphatase family member 2 (ENPP2, also referred to as autotaxin) were increased in the CSF of fibromyalgia patients compared to all other groups including patients with neuropathic pain. Conclusion: The increased levels of APOC1 and ENPP2 found in neuropathic pain and fibromyalgia patients may shed light on the underlying mechanisms of these conditions. Further investigation is required to elucidate their role in maintaining pain and other main symptoms of these disorders.

2.
Sci Rep ; 9(1): 8324, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31171813

RESUMO

There is a strong need for procedures that enable context and application dependent validation of antibodies. Here, we applied a magnetic bead assisted workflow and immunoprecipitation mass spectrometry (IP-MS/MS) to assess antibody selectivity for the detection of proteins in human plasma. A resource was built on 414 IP experiments using 157 antibodies (targeting 120 unique proteins) in assays with heat-treated or untreated EDTA plasma. For each protein we determined their antibody related degrees of enrichment using z-scores and their frequencies of identification across all IP assays. Out of 1,313 unique endogenous proteins, 426 proteins (33%) were detected in >20% of IPs, and these background components were mainly comprised of proteins from the complement system. For 45% (70/157) of the tested antibodies, the expected target proteins were enriched (z-score ≥ 3). Among these 70 antibodies, 59 (84%) co-enriched other proteins beside the intended target and mainly due to sequence homology or protein abundance. We also detected protein interactions in plasma, and for IGFBP2 confirmed these using several antibodies and sandwich immunoassays. The protein enrichment data with plasma provide a very useful and yet lacking resource for the assessment of antibody selectivity. Our insights will contribute to a more informed use of affinity reagents for plasma proteomics assays.

3.
Exp Hematol ; 75: 11-20, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31154069

RESUMO

Translational regulation plays a critical role in erythropoiesis, as it reflects the translational needs of enucleated mature erythroid cells in the absence of transcription and the large translational demands of balanced globin chain synthesis during erythroid maturation. In addition, red blood cells need to respond quickly to changes in their environment and the demands of the organism. Translational regulation occurs at several levels in erythroid cells, including the differential utilization of upstream open reading frames during differentiation and in response to signaling and the employment of RNA-binding proteins in an erythroid cell-specific fashion. Translation initiation is a critical juncture for translational regulation in response to environmental signals such as heme and iron availability, whereas regulatory mechanisms for ribosome recycling are consistent with recent observations highlighting the importance of maintaining adequate ribosome levels in differentiating erythroid cells. Translational deregulation in erythroid cells leads to disease associated with ineffective erythropoiesis, further highlighting the pivotal role translational regulation in erythropoiesis plays in human physiology and homeostasis. Overall, erythropoiesis has served as a unique model that has provided invaluable insight into translational regulation.


Assuntos
Diferenciação Celular/fisiologia , Células Eritroides/metabolismo , Eritropoese/fisiologia , Biossíntese de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Células Eritroides/citologia , Heme/metabolismo , Humanos , Ferro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo
4.
BMC Res Notes ; 11(1): 390, 2018 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-29898783

RESUMO

ΟBJECTIVE: To construct mammalian expression vectors for the N- or C-terminal tagging of proteins with a tandem affinity tag comprised of the biotinylatable Avi tag and of a triple FLAG tag. RESULTS: We constructed and tested by transient transfections mammalian expression vectors for the co-expression from a single plasmid of N- or C-terminally tagged proteins bearing a tandem affinity tag comprised of the biotinylatable Avi tag and of a triple FLAG tag separated by a tobacco etch virus (TEV) protease cleavage site, together with a mammalian codon-optimized BirA biotin ligase fused to green fluorescent protein. We also describe platform vectors for the N- or C-terminal AVI-TEV-FLAG tagging of any complementary DNA of choice. These vectors offer versatility and efficiency in the application of metabolic biotinylation tandem affinity tagging of nuclear proteins in mammalian cells.


Assuntos
Marcadores de Afinidade , Biotinilação/métodos , Vetores Genéticos , Animais , Células HEK293 , Humanos , Camundongos , Plasmídeos , Coelhos , Ratos
5.
Mol Biol Cell ; 23(19): 3873-81, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22875994

RESUMO

Epithelial-to-mesenchymal transition (EMT) is a key process in cancer progression and metastasis, requiring cooperation of the epidermal growth factor/Ras with the transforming growth factor-ß (TGF-ß) signaling pathway in a multistep process. The molecular mechanisms by which Ras signaling contributes to EMT, however, remain elusive to a large extent. We therefore examined the transcriptional repressor Ets2-repressor factor (ERF)-a bona fide Ras-extracellular signal-regulated kinase/mitogen-activated protein kinase effector-for its ability to interfere with TGF-ß-induced EMT in mammary epithelial cells (EpH4) expressing oncogenic Ras (EpRas). ERF-overexpressing EpRas cells failed to undergo TGF-ß-induced EMT, formed three-dimensional tubular structures in collagen gels, and retained expression of epithelial markers. Transcriptome analysis indicated that TGF-ß signaling through Smads was mostly unaffected, and ERF suppressed the TGF-ß-induced EMT via Semaphorin-7a repression. Forced expression of Semaphorin-7a in ERF-overexpressing EpRas cells reestablished their ability to undergo EMT. In contrast, inhibition of Semaphorin-7a in the parental EpRas cells inhibited their ability to undergo TGF-ß-induced EMT. Our data suggest that oncogenic Ras may play an additional role in EMT via the ERF, regulating Semaphorin-7a and providing a new interconnection between the Ras- and the TGF-ß-signaling pathways.


Assuntos
Antígenos CD/fisiologia , Células Epiteliais/fisiologia , Glândulas Mamárias Animais/citologia , Proteínas Repressoras/fisiologia , Semaforinas/fisiologia , Proteínas ras/metabolismo , Substituição de Aminoácidos , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Sistema de Sinalização das MAP Quinases , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Transcriptoma , Fator de Crescimento Transformador beta/metabolismo
6.
Genomics ; 100(2): 93-101, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22659238

RESUMO

National/ethnic mutation databases aim to document the genetic heterogeneity in various populations and ethnic groups worldwide. We have previously reported the development and upgrade of FINDbase (www.findbase.org), a database recording causative mutations and pharmacogenomic marker allele frequencies in various populations around the globe. Although this database has recently been upgraded, we continuously try to enhance its functionality by providing more advanced visualization tools that would further assist effective data querying and comparisons. We are currently experimenting in various visualization techniques on the existing FINDbase causative mutation data collection aiming to provide a dynamic research tool for the worldwide scientific community. We have developed an interactive web-based application for population-based mutation data retrieval. It supports sophisticated data exploration allowing users to apply advanced filtering criteria upon a set of multiple views of the underlying data collection and enables browsing the relationships between individual datasets in a novel and meaningful way.


Assuntos
Bases de Dados Genéticas , Grupos Étnicos/genética , Frequência do Gene , Genoma Humano , Mutação , Alelos , Mapeamento Cromossômico , Biologia Computacional/métodos , Marcadores Genéticos , Genética Populacional/métodos , Humanos , Armazenamento e Recuperação da Informação , Internet , Farmacogenética , Software , Interface Usuário-Computador
7.
J Immunol ; 188(11): 5521-7, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22547704

RESUMO

IFN-ß and the CD40L (CD154) share important roles in the antiviral and antitumor immune responses. In this study, we show that CD40 receptor occupancy results in IFN-ß upregulation through an unconventional "feed-forward" mechanism, which is orchestrated by canonical NF-κB and involves the sequential de novo synthesis of IFN regulatory factor (IRF)1 and Viperin (RSAD2), an IRF1 target. RelA (p65) NF-κB, IRF1, and Viperin-dependent IRF7 binding to the IFN-ß promoter largely controls its activity. However, full activation of IFN-ß also requires the parallel engagement of noncanonical NF-κB2 signaling leading to p52 recruitment to the IFN-ß promoter. These data define a novel link between CD40 signaling and IFN-ß expression and provide a telling example of how signal propagation can be exploited to ensure efficient regulation of gene expression.


Assuntos
Antígenos CD40/fisiologia , Retroalimentação Fisiológica , Interferon beta/biossíntese , NF-kappa B/fisiologia , Transdução de Sinais/imunologia , Neoplasias da Bexiga Urinária/imunologia , Antígenos CD40/genética , Carcinoma/genética , Carcinoma/imunologia , Carcinoma/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Regulação da Expressão Gênica/imunologia , Células HEK293 , Humanos , Interferon beta/genética , Transdução de Sinais/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
8.
IEEE Trans Inf Technol Biomed ; 15(6): 806-12, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22113338

RESUMO

An increasing number of studies have profiled gene expressions in tumor specimens using distinct microarray platforms and analysis techniques. One challenging task is to develop robust statistical models in order to integrate multi-platform findings. We compare some methodologies on the field with respect to estrogen receptor (ER) status, and focus on a unified-among-platforms scale implemented by Shen et al. in 2004, which is based on a Bayesian mixture model. Under this scale, we study the ER intensity similarities between four breast cancer datasets derived from various platforms. We evaluate our results with an independent dataset in terms of ER sample classification, given the derived gene ER signatures of the integrated data. We found that integrated multi-platform gene signatures and fold-change variability similarities between different platform measurements can assist the statistical analysis of independent microarray datasets in terms of ER classification.


Assuntos
Mineração de Dados/métodos , Bases de Dados Genéticas , Análise em Microsséries/métodos , Modelos Moleculares , Modelos Estatísticos , Receptores Estrogênicos/análise , Integração de Sistemas , Inteligência Artificial , Teorema de Bayes , Neoplasias da Mama/genética , Simulação por Computador , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Reprodutibilidade dos Testes
9.
J Cell Biol ; 192(3): 391-9, 2011 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-21282461

RESUMO

CD40, a tumor necrosis factor (TNF) receptor family member, is widely recognized for its prominent role in the antitumor immune response. The immunostimulatory effects of CD40 ligation on malignant cells can be switched to apoptosis upon disruption of survival signals transduced by the binding of the adaptor protein TRAF6 to CD40. Apoptosis induction requires a TRAF2-interacting CD40 motif but is initiated within a cytosolic death-inducing signaling complex after mobilization of receptor-bound TRAF2 to the cytoplasm. We demonstrate that receptor-interacting protein 1 (RIP1) is an integral component of this complex and is required for CD40 ligand-induced caspase-8 activation and tumor cell killing. Degradation of the RIP1 K63 ubiquitin ligases cIAP1/2 amplifies the CD40-mediated cytotoxic effect, whereas inhibition of CYLD, a RIP1 K63 deubiquitinating enzyme, reduces it. This two-step mechanism of apoptosis induction expands our appreciation of commonalities in apoptosis regulatory pathways across the TNF receptor superfamily and provides a telling example of how TNF family receptors usurp alternative programs to fulfill distinct cellular functions.


Assuntos
Apoptose/imunologia , Ligante de CD40/metabolismo , Carcinoma/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais/imunologia , Carcinoma/genética , Carcinoma/imunologia , Caspase 8/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Células HeLa , Humanos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo
10.
Biosens Bioelectron ; 26(4): 1588-92, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20728330

RESUMO

The detection of DNA hybridization using capacitive readout and a biosensor array of ultrathin Si membranes is presented. The biosensor exploits the ability of the ultrathin membranes to deflect upon surface stress variations caused by biological interactions. Probe DNA molecules are immobilized on the membrane surface and the surface stress variations during hybridization with their complementary strands force the membrane to deflect and effectively change the capacitance between the flexible membrane and the fixed substrate. The sensor array comprises 256 such sensing sites thus allowing the concurrent sensing of multiple DNA mutations. The biosensor and its performance for the detection of complementary DNA strands are demonstrated using beta-thalassemia oligonucleotides. The experimental results show that the presented sensors are able to detect DNA hybridization and to discriminate single nucleotide mismatches.


Assuntos
Técnicas Biossensoriais/instrumentação , Análise Mutacional de DNA/instrumentação , DNA/química , DNA/genética , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Desenho de Equipamento , Humanos , Microtecnologia , Mutação , Hibridização de Ácido Nucleico , Oligonucleotídeos/genética , Silício , Propriedades de Superfície , Talassemia beta/genética
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