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1.
Plast Reconstr Surg Glob Open ; 7(8): e2339, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31592376

RESUMO

Chronic inflammation during morbid obesity significantly alters cutaneous tissue. Large weight loss achieved after bariatric surgery minimizes or halts damage caused by metabolic syndrome, but further deteriorates the clinical condition of skin. Postbariatric skin flaccidity produces major difficulties to plastic surgery. In this study, we analyzed differences in protein composition of the skin between patients with morbid obesity and those after large weight loss and established correlations between differentially expressed proteins and clinical characteristics of postbariatric skin tissue, to improve body contouring surgery techniques. METHODS: Skin fragments were removed from the abdomen of 32 patients, who were allocated into 3 groups: morbidly obese, large weight loss without surgery, and postbariatric surgery. Samples were subjected to proteomic analysis, and the protein profiles of the groups were compared. Six differentially expressed proteins of clinical interest were validated by immunohistochemistry and statistical analysis. RESULTS: Comparative analyses confirmed differences in protein profile of the skin between morbidly obese and large weight loss groups. A persistent increase in inflammatory markers such as haptoglobin was observed in all groups and decrease in the expression of collagen XIV, which regulates the physical properties of cutaneous tissue, was observed in the postbariatric group. CONCLUSIONS: High expression of haptoglobin associated with the decrease of Collagen XIV, vinculin, and periplakin in the groups after major weight losses, mainly postbariatric, confirm that the inflammatory lesion remains active in the skin and causes changes in its structural organization, with serious repercussions on its clinical characteristics and physical properties.

2.
BMC Med Genomics ; 11(1): 73, 2018 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-30157864

RESUMO

BACKGROUND: Lymph node metastasis is one of the most important prognostic factors in head and neck squamous cell carcinomas (HNSCCs) and critical for delineating their treatment. However, clinical and histological criteria for the diagnosis of nodal status remain limited. In the present study, we aimed to characterize the proteomic profile of lymph node metastasis from HNSCC patients. METHODS: In the present study, we used one- and two-dimensional electrophoresis and mass spectrometry analysis to characterize the proteomic profile of lymph node metastasis from HNSCC. RESULTS: Comparison of metastatic and non-metastatic lymph nodes showed 52 differentially expressed proteins associated with neoplastic development and progression. The results reinforced the idea that tumors from different anatomical subsites have dissimilar behaviors, which may be influenced by micro-environmental factor including the lymphatic network. The expression pattern of heat shock proteins and glycolytic enzymes also suggested an effect of the lymph node environment in controlling tumor growth or in metabolic reprogramming of the metastatic cell. Our study, for the first time, provided direct evidence of annexin A1 overexpression in lymph node metastasis of head and neck cancer, adding information that may be useful for diagnosing aggressive disease. CONCLUSIONS: In brief, this study contributed to our understanding of the metastatic phenotype of HNSCC and provided potential targets for diagnostic in this group of carcinomas.


Assuntos
Perfilação da Expressão Gênica , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Proteômica , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Idoso , Feminino , Neoplasias de Cabeça e Pescoço/genética , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética
3.
J Proteome Res ; 16(4): 1542-1555, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28317380

RESUMO

Yellow fever virus (YFV) replication is highly dependent on host cell factors. YFV NS4B is reported to be involved in viral replication and immune evasion. Here interactions between NS4B and human proteins were determined using a GST pull-down assay and analyzed using 1-DE and LC-MS/MS. We present a total of 207 proteins confirmed using Scaffold 3 Software. Cyclophilin A (CypA), a protein that has been shown to be necessary for the positive regulation of flavivirus replication, was identified as a possible NS4B partner. 59 proteins were found to be significantly increased when compared with a negative control, and CypA exhibited the greatest difference, with a 22-fold change. Fisher's exact test was significant for 58 proteins, and the p value of CypA was the most significant (0.000000019). The Ingenuity Systems software identified 16 pathways, and this analysis indicated sirolimus, an mTOR pathway inhibitor, as a potential inhibitor of CypA. Immunofluorescence and viral plaque assays showed a significant reduction in YFV replication using sirolimus and cyclosporine A (CsA) as inhibitors. Furthermore, YFV replication was strongly inhibited in cells treated with both inhibitors using reporter BHK-21-rep-YFV17D-LucNeoIres cells. Taken together, these data suggest that CypA-NS4B interaction regulates YFV replication. Finally, we present the first evidence that YFV inhibition may depend on NS4B-CypA interaction.


Assuntos
Ciclofilina A/metabolismo , Proteínas/genética , Replicação Viral/genética , Vírus da Febre Amarela/genética , Ciclofilina A/genética , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Biologia de Sistemas , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Vírus da Febre Amarela/patogenicidade
4.
Protein Pept Lett ; 21(1): 56-62, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-23919378

RESUMO

The eukaryotic translation initiation factor 3, subunit L (eIF3L) is one of the subunits of the eIF3 complex, an accessory protein of the Polymerase I enzyme and may have an important role in the Flavivirus replication by interaction with a viral non-structural 5 protein. Considering the importance of eIF3L in a diversity of cellular functions, we have produced the recombinant full-length eIF3L protein in Escherichia coli and performed spectroscopic and in silico analyses to gain insights into its hydrodynamic behavior and structure. Dynamic light scattering showed that eIF3L behaves as monomer when it is not interacting with other molecular partners. Circular dichroism experiments showed a typical spectrum of α-helical protein for eIF3L, which is supported by sequence-based predictions of secondary structure and the 3D in silico model. The molecular docking with the K subunit of the eIF3 complex revealed a strong interaction. It was also predicted several potential interaction sites in eIF3L, indicating that the protein is likely capable of interacting with other molecules as experimentally shown in other functional studies. Moreover, bioinformatics analyses showed approximately 8 putative phosphorylation sites and one possible N-glycosylation site, suggesting its regulation by post-translational modifications. The production of the eIF3L protein in E. coli and structural information gained in this study can be instrumental for target-based drug design and inhibitors against Flavivirus replication and to shed light on the molecular mechanisms involved in the eukaryotic translation initiation.


Assuntos
Fator de Iniciação 3 em Eucariotos/análise , Fator de Iniciação 3 em Eucariotos/genética , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Dicroísmo Circular , Clonagem Molecular , Escherichia coli/genética , Fator de Iniciação 3 em Eucariotos/química , Glicosilação , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Fosforilação , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína
5.
PLoS One ; 7(12): e50517, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23227181

RESUMO

The prediction of tumor behavior for patients with oral carcinomas remains a challenge for clinicians. The presence of lymph node metastasis is the most important prognostic factor but it is limited in predicting local relapse or survival. This highlights the need for identifying biomarkers that may effectively contribute to prediction of recurrence and tumor spread. In this study, we used one- and two-dimensional gel electrophoresis, mass spectrometry and immunodetection methods to analyze protein expression in oral squamous cell carcinomas. Using a refinement for classifying oral carcinomas in regard to prognosis, we analyzed small but lymph node metastasis-positive versus large, lymph node metastasis-negative tumors in order to contribute to the molecular characterization of subgroups with risk of dissemination. Specific protein patterns favoring metastasis were observed in the "more-aggressive" group defined by the present study. This group displayed upregulation of proteins involved in migration, adhesion, angiogenesis, cell cycle regulation, anti-apoptosis and epithelial to mesenchymal transition, whereas the "less-aggressive" group was engaged in keratinocyte differentiation, epidermis development, inflammation and immune response. Besides the identification of several proteins not yet described as deregulated in oral carcinomas, the present study demonstrated for the first time the role of cofilin-1 in modulating cell invasion in oral carcinomas.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Cofilina 1/metabolismo , Neoplasias Bucais/metabolismo , Proteômica , Idoso , Carcinoma de Células Escamosas/patologia , Cofilina 1/genética , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Feminino , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Metástase Linfática , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Invasividade Neoplásica
6.
Rev Assoc Med Bras (1992) ; 58(3): 366-75, 2012.
Artigo em Inglês, Português | MEDLINE | ID: mdl-22735231

RESUMO

Proteomic approach has allowed large-scale studies of protein expression in different tissues and body fluids in discrete conditions and/or time points. Recent advances of methodologies in this field have opened new opportunities to obtain relevant information on normal and abnormal processes occurring in the human body. In the current report, the main proteomics techniques and their application to human disease study are reviewed.


Assuntos
Líquidos Corporais/química , Doença/genética , Proteômica/métodos , Cromatografia em Gel , Eletroforese em Gel Bidimensional , Humanos , Espectrometria de Massas
7.
Rev. Assoc. Med. Bras. (1992) ; 58(3): 366-375, May-June 2012. ilus
Artigo em Português | LILACS | ID: lil-639563

RESUMO

A abordagem proteômica tem permitido estudos em larga escala da expressão proteica em diferentes tecidos e fluidos corporais, em condições e/ou momentos distintos. O recente progresso de metodologias nessa área tem aberto novas oportunidades para obtenção de informações relevantes sobre processos normais e anormais que ocorrem no organismo humano. No presente artigo, é feita uma revisão das principais técnicas proteômicas e de suas aplicações no estudo de doenças humanas.


Proteomic approach has allowed large-scale studies of protein expression in different tissues and body fluids in discrete conditions and/or time points. Recent advances of methodologies in this field have opened new opportunities to obtain relevant information on normal and abnormal processes occurring in the human body. In the current report, the main proteomics techniques and their application to human disease study are reviewed.


Assuntos
Humanos , Líquidos Corporais/química , Doença/genética , Proteômica/métodos , Cromatografia em Gel , Eletroforese em Gel Bidimensional , Espectrometria de Massas
8.
BMC Med Genomics ; 3: 14, 2010 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-20441585

RESUMO

BACKGROUND: The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression. METHODS: The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells. RESULTS: We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR. CONCLUSIONS: A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Bucais/metabolismo , Proteoma/metabolismo , Anexina A5/metabolismo , Apoptose , Proliferação de Células , Regulação para Baixo , Eletroforese em Gel Bidimensional , Fibroblastos/metabolismo , Genômica , Células Hep G2 , Humanos , Queratinas/metabolismo , Neoplasias Bucais/genética , Hibridização de Ácido Nucleico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Estromais/metabolismo , Vimentina/metabolismo
9.
Cancer Biomark ; 8(2): 95-107, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21896997

RESUMO

Several body fluids have been evaluated as new sources for cancer biomarker discovery. In this context, salivary and serum proteomics seem promising diagnostic and predictive tools for head and neck diseases. In the present study, we performed a proteomic analysis of saliva and serum from patients presenting head and neck squamous cell carcinoma (HNSCC) and compared the results before and after therapy. In saliva of cancer patients, we observed an altered protein profile, including over-expression of PLUNC and zinc-alpha-2-glycoprotein. Both proteins may contribute to control tumor growth and, therefore, represent targets for new analysis. We also detected serotransferrin and a modified transthyretin form with altered levels in serum from patients. Comparing preoperative and postreatment samples, the results showed that the protein profile after treatment reverted to a pattern closer to those observed for controls. These results add information on the role of secreted proteins in the cancer process and emphasize the potential of saliva and serum analysis for diagnosis and monitoring of HNSCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma/terapia , Neoplasias de Cabeça e Pescoço/terapia , Saliva/metabolismo , Soro/metabolismo , Idoso , Idoso de 80 Anos ou mais , Carcinoma/radioterapia , Carcinoma/cirurgia , Feminino , Glicoproteínas/metabolismo , Neoplasias de Cabeça e Pescoço/radioterapia , Neoplasias de Cabeça e Pescoço/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/metabolismo , Proteômica , Proteínas de Plasma Seminal/metabolismo , Resultado do Tratamento , Glicoproteína Zn-alfa-2
10.
J Biochem Mol Biol ; 39(2): 216-22, 2006 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-16584638

RESUMO

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.


Assuntos
Carcinoma de Células Escamosas/química , Eletroforese em Gel Bidimensional/métodos , Neoplasias de Cabeça e Pescoço/química , Linfonodos/química , Proteínas/análise , Soluções Tampão , Células , Detergentes/química , Eletroforese em Gel Bidimensional/normas , Humanos , Focalização Isoelétrica , Proteínas de Neoplasias/análise , Solubilidade , Tempo
11.
Rev. bras. hematol. hemoter ; 24(1): 41-44, mar. 2002.
Artigo em Português | LILACS | ID: lil-338285

RESUMO

Alteraçöes genéticas em que a mutaçäo de aminoácidos nas globinas afeta a estrutura da molécula tornando-a instável säo classificadas como hemoglobinas instáveis. Devido à grande diversidade dos pontos de mutaçöes por substituiçöes e deleçöes de aminoácidos, as formas de instabilizaçäo se apresentam muito variadas. A hemoglobina Köln é a variante instável descrita com maior freqüência na literatura e a terceira descoberta no Brasil, as outras säo Hb Niterói e Hb Hasharon. Anemia moderada, icterícia e presença de urina escura caracterizam as manifestaçöes clínicas da Hb Köln. Em programa de triagem neonatal identificamos uma criança com suspeita de heterozigose para hemoglobina Köln, confirmada por procedimentos eletroforéticos e HPLC. Avaliaçöes por diferentes metodologias laboratoriais e estudo familiar auxiliam no diagnóstico precoce, possibilitando minimizar os sintomas decorrentes da hemoglobina anormal e a realizaçäo do aconselhamento genético e educacional destas alteraçöes hereditárias


Assuntos
Hemoglobinas Anormais , Urina , Hemoglobinas , Triagem Neonatal , Diagnóstico Precoce , Aminoácidos , Mutação
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