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1.
Chembiochem ; 23(16): e202200265, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35748603

RESUMO

Fucoidan is a natural sulfated polysaccharide with a large range of biological activities including anticancer and anti-oxidation activities. Hepatocellular carcinoma is the fourth most common aggressive cancer type. The aim of this study was to investigate the bioactivity of free fucoidan versus its vectorization using nanoparticles (NPs) in human hepatoma cells, Huh-7. Iron oxide NPs were functionalized with fucoidan by a one-step surface complexation. NP cellular uptake was quantified by magnetic measurement at various extracellular iron concentrations. Cell invasion and migration were reduced with NPs while free fucoidan increases these events at low fucoidan concentration (≤0.5 µM). Concomitantly, a high decrease of reactive oxygen species production related with a decrease of the matrix metalloproteinase-9 activity and an increase of its expression was observed with NPs compared to free fucoidan. A proteomic analysis evidenced that some fucoidan regulated proteins appeared, which were related to protein synthesis, N-glycan processing, and cellular stress. To our knowledge, this is the first study which reveals such activity induced by fucoidan. These results pave the way for USPIO-fucoidan-NPs as potential theranostic nanotools for hepatocellular carcinoma treatment.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Polissacarídeos , Medicina de Precisão , Proteômica
2.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298982

RESUMO

Engineering of biomimetic motives have emerged as promising approaches to improving cells' binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface's receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5ß1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.


Assuntos
Marcadores de Afinidade , Materiais Biomiméticos , Fibronectinas , Oligopeptídeos , Fragmentos de Peptídeos , Proteínas Recombinantes de Fusão/química , Adsorção , Adesão Celular , Materiais Revestidos Biocompatíveis , Escherichia coli , Matriz Extracelular/química , Vetores Genéticos , Integrina alfa5beta1/metabolismo , Ligantes , Espectrometria de Massas , Poliestirenos , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
3.
Environ Res ; 182: 109116, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32069763

RESUMO

Glyphosate-based herbicides, such as Roundup®, are the most widely used non-selective, broad-spectrum herbicides. The release of these compounds in large amounts into the environment is susceptible to affect soil quality and health, especially because of the non-target effects on a large range of organisms including soil microorganisms. The soil filamentous fungus Aspergillus nidulans, a well-characterized experimental model organism that can be used as a bio-indicator for agricultural soil health, has been previously shown to be highly affected by Roundup GT Plus (R450: 450 g/L of glyphosate) at concentrations far below recommended agricultural application rate, including at a dose that does not cause any macroscopic effect. In this study, we determined alterations in the transcriptome of A. nidulans when exposed to R450 at a dose corresponding to the no-observed-adverse-effect level (NOAEL) for macroscopic parameters. A total of 1816 distinct genes had their expression altered. The most affected biological functions were protein synthesis, amino acids and secondary metabolisms, stress response, as well as detoxification pathways through cytochromes P450, glutathione-S-transferases, and ABC transporters. These results partly explain the molecular mechanisms underlying alterations in growth parameters detected at higher concentrations for this ascomycete fungus. In conclusion, our results highlight molecular disturbances in a soil fungus under conditions of apparent tolerance to the herbicide, and thus confirm the need to question the principle of "substantial equivalence" when applied to plants made tolerant to herbicides.


Assuntos
Aspergillus nidulans , Perfilação da Expressão Gênica , Glicina/análogos & derivados , Herbicidas , Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/metabolismo , Glicina/toxicidade , Herbicidas/toxicidade , Transcriptoma , Glifosato
5.
Exp Hematol Oncol ; 13(1): 41, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38622749

RESUMO

Despite major therapeutic advances for two decades, including the most recently approved anti-HER2 drugs, brain metastatic localizations remain the major cause of death for women with metastatic HER2 breast cancer. The main reason is the limited drug passage of the blood-brain barrier after intravenous injection and the significant efflux of drugs, including monoclocal antibodies, after administration into the cerebrospinal fluid. We hypothesized that this efflux was linked to the presence of a FcRn receptor in the blood-brain barrier. To overcome this efflux, we engineered two Fab fragments of trastuzumab, an anti-HER2 monoclonal antibody, and did a thorough preclinical development for therapeutic translational purpose. We demonstrated the safety and equal efficacy of the Fabs with trastuzumab in vitro, and in vivo using a patient-derived xenograft model of HER2 overexpressing breast cancer. For the pharmacokinetic studies of intra-cerebrospinal fluid administration, we implemented original rat models with catheter implanted into the cisterna magna. After intraventricular administration in rats, we demonstrated that the brain-to-blood efflux of Fab was up to 10 times lower than for trastuzumab, associated with a two-fold higher brain penetration compared to trastuzumab. This Fab, capable of significantly reducing brain-to-blood efflux and enhancing brain penetration after intra-cerebrospinal fluid injection, could thus be a new and original effective drug in the treatment of HER2 breast cancer brain metastases, which will be demonstrated by a phase I clinical trial dedicated to women in resort situations.

6.
Angiogenesis ; 16(3): 575-93, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23386104

RESUMO

Osteoprotegerin (OPG), a soluble tumour necrosis factor receptor superfamily member, inhibits RANKL-mediated osteoclastogenesis. We have previously reported that OPG enhances the proangiogenic properties of endothelial colony-forming cells (ECFCs) in vitro, and promotes vasculogenesis in vivo. Here we investigated how OPG promotes neovascularisation. Proteomic experiments showed that OPG pretreatment affected ECFCs protein expression in two ways, 23 spots being down-regulated and 6 upregulated. These spots corresponded to proteins involved in cell motility, adhesion, signal transduction and apoptosis. In keeping with these proteomic results, we found that OPG induced ECFCs adhesion to activated endothelium in shear stress conditions, promoting intermediate but not focal adhesion to fibronectin and collagen. Treatment with OPG induced a reorganization of the ECFCs cytoskeleton, with the emergence of cell protrusions characteristic of a migratory phenotype. These effects correlated with decreased FAK phosphorylation and enhanced integrin αVß3 expression. OPG drastically reduced caspase-3/7 activities and maintained ECFCs viability after 48 h of treatment. All these effects were significantly attenuated by ECFCs incubation with the CXCR4 antagonist AMD-3100, and by prior heparan sulphate proteoglycan disruption. The proangiogenic properties of OPG appeared to be mediated by the proteoglycan syndecan-1, although OPG 1-194 lacking its heparin-binding domain still had pro-vasculogenic effects in vitro and in vivo. These results suggest that OPG may interact with ECFCs by binding to HSPGs/syndecan-1, thereby induce an anti-adhesive effect and promoting ECFCs migration through a SDF-1/CXCR4 dependent pathway.


Assuntos
Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Osteoprotegerina/farmacologia , Benzilaminas , Caspase 3/metabolismo , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclamos , Citoesqueleto/efeitos dos fármacos , Células Endoteliais/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica/fisiologia , Compostos Heterocíclicos , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina alfaVbeta3/metabolismo , Neovascularização Fisiológica/fisiologia , Osteoprotegerina/metabolismo , Fosforilação/efeitos dos fármacos , Proteômica , Sindecana-1/metabolismo
7.
Proc Natl Acad Sci U S A ; 106(33): 14144-9, 2009 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-19666481

RESUMO

In the heart, cAMP is a key regulator of excitation-contraction coupling and its biological effects are mainly associated with the activity of protein kinase A (PKA). The aim of this study was to investigate the contribution of the cAMP-binding protein Epac (Exchange protein directly activated by cAMP) in the regulation of the contractile properties of rat ventricular cardiac myocytes. We report that both PKA and Epac increased cardiac sarcomere contraction but through opposite mechanisms. Differently from PKA, selective Epac activation by the cAMP analog 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT) reduced Ca(2+) transient amplitude and increased cell shortening in intact cardiomyocytes and myofilament Ca(2+) sensitivity in permeabilized cardiomyocytes. Moreover, ventricular myocytes, which were infected in vivo with a constitutively active form of Epac, showed enhanced myofilament Ca(2+) sensitivity compared to control cells infected with green fluorescent protein (GFP) alone. At the molecular level, Epac increased phosphorylation of 2 key sarcomeric proteins, cardiac Troponin I (cTnI) and cardiac Myosin Binding Protein-C (cMyBP-C). The effects of Epac activation on myofilament Ca(2+) sensitivity and on cTnI and cMyBP-C phosphorylation were independent of PKA and were blocked by protein kinase C (PKC) and Ca(2+) calmodulin kinase II (CaMKII) inhibitors. Altogether these findings identify Epac as a new regulator of myofilament function.


Assuntos
Citoesqueleto de Actina/metabolismo , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Miócitos Cardíacos/metabolismo , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Masculino , Contração Muscular , Miocárdio/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Sarcômeros/metabolismo
8.
Biomedicines ; 10(12)2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36551773

RESUMO

Macrophages play a key role in the inflammatory phase of wound repair and foreign body reactions-two important processes in the Masquelet-induced membrane technique for extremity reconstruction. The macrophage response depends largely on the nature of the biomaterials implanted. However, little is known about the influence of the macrophage microenvironment on the osteogenic properties of the induced membrane or subsequent bone regeneration. We used metakaolin, an immunogenic material, as an alternative spacer to standard polymethylmethacrylate (PMMA) in a Masquelet model in rats. Four weeks after implantation, the PMMA- and metakaolin-induced membranes were harvested, and their osteogenic properties and macrophage microenvironments were investigated by histology, immunohistochemistry, mass spectroscopy and gene expression analysis. The metakaolin spacer induced membranes with higher levels of two potent pro-osteogenic factors, transforming growth factor-ß (TGF-ß) and bone morphogenic protein-2 (BMP-2). These alternative membranes thus had greater osteogenic activity, which was accompanied by a significant expansion of the total macrophage population, including both the M1-like and M2-like subtypes. Microcomputed tomographic analysis showed that metakaolin-induced membranes supported bone regeneration more effectively than PMMA-induced membranes through better callus properties (+58%), although this difference was not significant. This study provides the first evidence of the influence of the immune microenvironment on the osteogenic properties of the induced membranes.

9.
Proteomics ; 11(19): 3877-86, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21761557

RESUMO

Cell resistance to low doses of paclitaxel (Taxol) involves a modulation of microtubule (MT) dynamics. We applied a proteomic approach based on 2-DE coupled with MS to identify changes in the MT environment of Taxol-resistant breast cancer cells. Having established a proteomic pattern of the microtubular proteins extracted from MDA-MB-231 cells, we verified by Western blotting that in resistant cells, α- and ß-tubulins (more specifically the ßIII and ßIV isotypes) increased. Interestingly, four septins (SEPT2, 8, 9 and 11), which are GTPases involved in cytokinesis and in MT/actin cytoskeleton organization, were overexpressed and enriched in the MT environment of Taxol-resistant cells compared to their sensitive counterpart. Changes in the MT proteome of resistant cells also comprised increased kinesin-1 heavy chain expression and recruitment on MTs while dynein light chain-1 was downregulated. Modulation of motor protein recruitment around MTs might reflect their important role in controlling MT dynamics via the organization of signaling pathways. The identification of proteins previously unknown to be linked to taxane-resistance could also be valuable to identify new biological markers of resistance.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Microtúbulos/metabolismo , Paclitaxel/farmacologia , Proteoma/metabolismo , Septinas/metabolismo , Moduladores de Tubulina/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Proteômica/métodos , Tubulina (Proteína)/metabolismo , Regulação para Cima
10.
Anal Chem ; 83(5): 1696-703, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21314132

RESUMO

We report a CE-LIF method for the separation and detection of five synthetic amyloid-ß peptides corresponding to an important family of CSF-biomarkers in the context of Alzheimer disease (AD). The presumed most relevant peptides (Aß1-42, Aß1-40, and Aß1-38) that may support the differentiation between AD and healthy patients or other dementias were successfully detected in CSF by incorporating an immunoconcentration step prior to CE analysis of derivatized peptides. We labeled the Aß peptides with a fluoroprobe dye before CE-LIF analysis. This reagent reacts with the amino groups of lysine residues and produced mostly ditagged Aß peptides under the proposed experimental conditions. The labeling reaction displayed similar efficiency with each one of the five different synthetic Aß peptides that were tested. The limit of detection of the CE-LIF method approached 280 attomoles of injected synthetic labeled Aß peptides. We obtained excellent correlation between peak areas and peptide concentrations from 35 nM to 750 nM. For the detection of Aß peptides in human CSF samples, we enriched the peptides by immunoprecipitation prior to the CE-LIF analysis. The comparison of the CE-LIF profiles obtained from CSF samples from 3 AD patients and 4 non-demented control subjects indicated noticeable differences, suggesting that this method, which relies on a multibiomarker approach, may have potential as a clinical diagnostic test for AD.


Assuntos
Peptídeos beta-Amiloides/líquido cefalorraquidiano , Eletroforese Capilar/métodos , Peptídeos/líquido cefalorraquidiano , Peptídeos beta-Amiloides/química , Calibragem , Estudos de Casos e Controles , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
JCI Insight ; 6(24)2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34727095

RESUMO

Nociceptors, the high-threshold primary sensory neurons that trigger pain, interact with immune cells in the periphery to modulate innate immune responses. Whether they also participate in adaptive and humoral immunity is, however, not known. In this study, we probed if nociceptors have a role in distinct airway and skin models of allergic inflammation. In both models, the genetic ablation and pharmacological silencing of nociceptors substantially reduced inflammatory cell infiltration to the affected tissue. Moreover, we also found a profound and specific deficit in IgE production in these models of allergic inflammation. Mechanistically, we discovered that the nociceptor-released neuropeptide substance P helped trigger the formation of antibody-secreting cells and their release of IgE. Our findings suggest that nociceptors, in addition to their contributions to innate immunity, play a key role in modulating the adaptive immune response, particularly B cell antibody class switching to IgE.


Assuntos
Linfócitos B/metabolismo , Switching de Imunoglobulina/genética , Imunoglobulina E/metabolismo , Nociceptores/metabolismo , Humanos
12.
Environ Sci Pollut Res Int ; 24(33): 25933-25946, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28940012

RESUMO

Roundup® is a glyphosate-based herbicide (GBH) used worldwide both in agriculture and private gardens. Thus, it constitutes a substantial source of environmental contaminations, especially for water and soil, and may impact a number of non-target organisms essential for ecosystem balance. The soil filamentous fungus Aspergillus nidulans has been shown to be highly affected by a commercial formulation of Roundup® (R450), containing 450 g/L of glyphosate (GLY), at doses far below recommended agricultural application rate. In the present study, we used two-dimensional gel electrophoresis combined to mass spectrometry to analyze proteomic pattern changes in A. nidulans exposed to R450 at a dose corresponding to the no-observed-adverse-effect level (NOAEL) for macroscopic parameters (31.5 mg/L GLY among adjuvants). Comparative analysis revealed a total of 82 differentially expressed proteins between control and R450-treated samples, and 85% of them (70) were unambiguously identified. Their molecular functions were mainly assigned to cell detoxification and stress response (16%), protein synthesis (14%), amino acid metabolism (13%), glycolysis/gluconeogenesis/glycerol metabolism/pentose phosphate pathway (13%) and Krebs TCA cycle/acetyl-CoA synthesis/ATP metabolism (10%). These results bring new insights into the understanding of the toxicity induced by higher doses of this herbicide in the soil model organism A. nidulans. To our knowledge, this study represents the first evidence of protein expression modulation and, thus, possible metabolic disturbance, in response to an herbicide treatment at a dose that does not cause any visible effect. These data are likely to challenge the concept of "substantial equivalence" when applied to herbicide-tolerant plants.


Assuntos
Aspergillus nidulans/efeitos dos fármacos , Proteínas Fúngicas/biossíntese , Glicina/análogos & derivados , Herbicidas/toxicidade , Proteômica/métodos , Solo/química , Animais , Aspergillus nidulans/metabolismo , Ecossistema , Glicina/toxicidade , Nível de Efeito Adverso não Observado , Glifosato
13.
FASEB J ; 18(3): 585-6, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14715693

RESUMO

Renovascular hypertension is characterized by stenosis of the renal artery and high plasma renin levels. The renal phenotype is characterized by high levels of renin in the hypoperfused kidney due to the recruitment of renin-producing cells along the afferent arterioles. This increase in myoepithelioïd cells is due mainly to the differentiation of existing smooth muscle cells with acquisition of a secretory phenotype. To understand the molecular mechanisms involved in this recruitment, we used the established rat model of renovascular hypertension known as the two-kidney, one-clip model in the Lewis rat. Renal arterioles were isolated using magnetized iron suspension. Differential proteomic analysis was performed using 2-D polyacrylamide gel electrophoresis followed by mass spectrometry. Comparative analysis of soluble proteins extracted from afferent arterioles of clipped and contralateral kidneys showed 14 proteins significantly differentially expressed by at least a factor of 2. These proteins were identified by mass spectrometry. The most striking protein revealed by proteomics is troponin T, which is down-regulated in the afferent arterioles of the clipped kidney. Confocal microscopy showed that troponin T is specific of the smooth muscle phenotype and absent in the myoepithelioïd phenotype. Our data suggest that troponin T is only present in renal smooth muscle cells.


Assuntos
Rim/irrigação sanguínea , Músculo Liso Vascular/metabolismo , Proteômica , Troponina T/biossíntese , Animais , Arteríolas/metabolismo , Diferenciação Celular , Eletroforese em Gel Bidimensional , Hipertensão Renovascular/genética , Hipertensão Renovascular/metabolismo , Masculino , Ratos , Ratos Endogâmicos Lew , Renina/biossíntese , Renina/genética , Sistema Renina-Angiotensina/fisiologia , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Coloração pela Prata , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Troponina T/análise , Troponina T/genética
14.
Stem Cells Int ; 2015: 283796, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26161094

RESUMO

By using a high internal phase emulsion process, elastomeric poly(ε-caprolactone urethane) (PCLU) scaffolds were designed with pores size ranging from below 150 µm to 1800 µm and a porosity of 86% making them suitable for bone tissue engineering applications. Moreover, the pores appeared to be excellently interconnected, promoting cellularization and future bone ingrowth. This study evaluated the in vitro cytotoxicity of the PCLU scaffolds towards human mesenchymal stem cells (hMSCs) through the evaluation of cell viability and metabolic activity during extract test and indirect contact test at the beginning of the scaffold lifetime. Both tests demonstrated that PCLU scaffolds did not induce any cytotoxic response. Finally, direct interaction of hMSCs and PCLU scaffolds showed that PCLU scaffolds were suitable for supporting the hMSCs adhesion and that the cells were well spread over the pore walls. We conclude that PCLU scaffolds may be a good candidate for bone tissue regeneration applications using hMSCs.

15.
Cancer Cell Int ; 1(1): 2, 2001 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-11983026

RESUMO

BACKGROUND: 5-AzaCytidine (AzaC) is a DNA demethylating drugs that has been shown to inhibit cell growth and to induce apoptosis in certain cancer cells. Induced expression of the galectin1 (Gal1) protein, a galactoside-binding protein distributed widely in immune cells, has been described in cultured hepatoma-derived cells treated with AzaC and this event may have a role in the effect of the drug. According to this hypothesis, we investigated the effect of AzaC and Gal1 on human lymphoid B cells phenotype. METHODS: The effect of AzaC and Gal1 on cell growth and phenotype was determined on the Burkitt lymphoma cell line BL36. An immunocytochemical analysis for detection of Gal1 protein expression was performed in AzaC-treated cells. To investigate the direct effects of Gal1, recombinant Gal1 was added to cells. RESULTS: Treatment of lymphoid B cells with AzaC results in: i) a decrease in cell growth with an arrest of the cell cycle at G0/G1 phase, ii) phenotypic changes consistent with a differentiated phenotype, and iii) the expression of p16, a tumor-suppressor gene whose expression was dependent of its promoter demethylation, and of Gal1. A targeting of Gal 1 to the plasma membrane follows its cytosolic expression. To determine which of the effects of AzaC might be secondary to the induction of Gal1, recombinant Gal1 was added to BL36 cells. Treated cells displayed growth inhibition and phenotypic changes consistent with a commitment toward differentiation. CONCLUSIONS: Altered cell growth and expression of the cell surface plasma cell antigen, CD138 are detectable in BL36 cells treated by AzaC as well as by Gal1. It seems that AzaC-induced Gal1 expression and consequent binding of Gal1 on its cell membrane receptor may be, in part, involved in AzaC-induced plasmacytic differentiation.

16.
J Chromatogr B Analyt Technol Biomed Life Sci ; 771(1-2): 197-209, 2002 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-12015999

RESUMO

Advances in genomics have led to the accumulation of an unprecedented amount of data, giving rise to a new field in biochemistry, proteomics. We used a combination of two dimensional gel electrophoresis, analysis and annotation using third-generation software, and mass spectrometry to establish the proteome maps of lymphoblastoid B-cells, a prerequisite for analysis of drug effects and lymphocyte cell diseases. About 1200 protein spots were detected and characterised in terms of their isoelectric point, molecular mass and expression. The present status of proteomic technologies, as well as a description of the usefulness of human hematopoietic cells proteomic database are discussed.


Assuntos
Bases de Dados de Proteínas , Linfócitos/química , Proteoma , Eletroforese em Gel de Poliacrilamida
17.
Therapie ; 59(1): 13-20, 2004.
Artigo em Francês | MEDLINE | ID: mdl-15199662

RESUMO

Renovascular hypertension is characterised by stenosis of the renal artery and high plasma renin levels due to the recruitment of renin-producing cells along the afferent arterioles. This increase in myoepithelioid cells is mainly a result of the differentiation of existing smooth muscle cells with acquisition of a secretory phenotype. To understand the molecular mechanisms involved in this recruitment, we used the model of renovascular hypertension known as the two-kidney, one-clip model in the Lewis rat. Renal arterioles were isolated using magnetised iron suspension. Differential proteomic analysis was performed using two-dimensional electrophoresis gel followed by mass spectrometry for identification. The most striking protein revealed by proteomics is troponin T, which is down-regulated in the afferent arterioles of the clipped kidney. Confocal microscopy showed that troponin T is specific to the smooth muscle phenotype and absent in the myoepithelioid phenotype.


Assuntos
Hipertensão Renovascular/genética , Hipertensão Renovascular/metabolismo , Proteínas/genética , Proteômica , Animais , Arteríolas/metabolismo , Arteríolas/patologia , Eletroforese em Gel de Poliacrilamida , Hipertensão Renovascular/patologia , Espectrometria de Massas , Fenótipo , Ratos , Ratos Endogâmicos Lew , Circulação Renal
18.
FEBS J ; 278(24): 4845-59, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21973086

RESUMO

Colorectal cancer (CRC) is a major public health issue worldwide, and novel tumor markers may contribute to its efficient management by helping in early detection, prognosis or surveillance of disease. The aim of our study was to identify new serum biomarkers for CRC, and we followed a phased biomarker discovery and validation process to obtain an accurate preliminary assessment of potential clinical utility. We compared colonic tumors and matched normal tissue from 15 CRC patients, using two-dimensional difference gel electrophoresis (2D-DIGE), and identified 17 proteins that had significant differential expression. These results were further confirmed by western blotting for heat shock protein (HSP) 60, glutathione-S-transferase Pi, α-enolase, T-complex protein 1 subunit ß, and leukocyte elastase inhibitor, and by immunohistochemistry for HSP60. Using mAbs raised against HSP60, we developed a reliable (precision of 5-15%) and sensitive (0.3 ng·mL(-1)) immunoassay for the detection of HSP60 in serum. Elevated levels of HSP60 were found in serum from CRC patients in two independent cohorts; the receiver-operating characteristic curve obtained in 112 patients with CRC and 90 healthy controls had an area under the curve (AUC) of 0.70, which was identical to the AUC of carcinoembryonic antigen. Combination of serum markers improved clinical performance: the AUC of a three-marker logistic regression model combining HSP60, carcinoembryonic antigen and carbohydrate antigen 19-9 reached 0.77. Serum HSP60 appeared to be more specific for late-stage CRC; therefore, future studies should evaluate its utility for determining prognosis or monitoring therapy rather than early detection.


Assuntos
Chaperonina 60/sangue , Neoplasias Colorretais/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Western Blotting , Antígeno CA-19-9/sangue , Antígeno Carcinoembrionário/sangue , Neoplasias Colorretais/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Eletroforese em Gel Diferencial Bidimensional
19.
Electrophoresis ; 29(20): 4158-68, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18924100

RESUMO

Proteomic approaches including high-resolution 2-DE are providing the tools needed to discover disease-associated biomarkers in complex biological samples. Although 2-DE is an extremely powerful approach to analyze the proteome, the separation of proteins with extreme molecular masses still remains an issue requiring improvement. Because high molecular mass (HMM) proteins larger than 150 kDa have already been observed to be differentially expressed in several pathologies such as cancer, we developed an original strategy to analyze this part of the proteome that is not easily separated by 2-DE in polyacrylamide gels. This strategy is based on the 2-DE separation of cyanogen bromide (CNBr) fragments of purified HMM protein fractions, and combines techniques including SEC fractionation, TCA precipitation, CNBr cleavage, 2-DE and MS analysis. The method was first tested on a model protein, the BSA. Preliminary results obtained using colonic tissues led to the identification of six HMM proteins with M(r) comprised between 163 and 533 kDa in their reduced state. These results demonstrated that our CNBr/2-DE approach should provide a powerful tool for identification of new biomarkers larger than 150 kDa.


Assuntos
Colo/química , Brometo de Cianogênio/química , Proteínas/análise , Proteômica/métodos , Animais , Bovinos , Fracionamento Químico , Eletroforese em Gel Bidimensional/métodos , Humanos , Peso Molecular , Mucosa/química , Proteínas/química , Reprodutibilidade dos Testes , Soroalbumina Bovina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
Neurobiol Aging ; 28(3): 357-70, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16519965

RESUMO

We used a beta-amyloid precursor protein (APP) transgenic (Tg) mouse model that displays some of the typical Alzheimer-associated pathological features to study the brain proteoma associated with amyloid plaque deposition. Two groups (male and female) of 14-month-old Tg mice were compared with their wild type littermates. We used differential 2D electrophoresis coupled with mass spectrometry to generate one of the first complete image of changes in brain protein expression occurring in this well-recognized model of Alzheimer's disease (AD). We identified 15 different proteins, which are significantly regulated in this pathology (p<0.05, > or =1.5-fold variation in expression comparing with the wild type samples). These comprise a number of proteins that were already known to be implicated in AD and neurodegeneration, as well as several proteins which relationship with AD had not been shown before. Identified proteins were grouped according to their biological key pathways. Results obtained are discussed in view of existing bibliographic data on human AD transcriptoma and proteoma.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Eletroforese em Gel Bidimensional/métodos , Proteômica/métodos , Precursor de Proteína beta-Amiloide/genética , Animais , Apolipoproteínas E/metabolismo , Western Blotting/métodos , Encéfalo/patologia , Diagnóstico por Imagem/métodos , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
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