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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.
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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.
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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 (≤0.5â 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.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Polisacáridos , Medicina de Precisión , ProteómicaRESUMEN
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.
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Linfocitos B/metabolismo , Cambio de Clase de Inmunoglobulina/genética , Inmunoglobulina E/metabolismo , Nociceptores/metabolismo , HumanosRESUMEN
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.
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Marcadores de Afinidad , Materiales Biomiméticos , Fibronectinas , Oligopéptidos , Fragmentos de Péptidos , Proteínas Recombinantes de Fusión/química , Adsorción , Adhesión Celular , Materiales Biocompatibles Revestidos , Escherichia coli , Matriz Extracelular/química , Vectores Genéticos , Integrina alfa5beta1/metabolismo , Ligandos , Espectrometría de Masas , Poliestirenos , Unión Proteica , Dominios Proteicos , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
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.
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Aspergillus nidulans , Perfilación de la Expresión Génica , Glicina/análogos & derivados , Herbicidas , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/metabolismo , Glicina/toxicidad , Herbicidas/toxicidad , Transcriptoma , GlifosatoRESUMEN
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.
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Aspergillus nidulans/efectos de los fármacos , Proteínas Fúngicas/biosíntesis , Glicina/análogos & derivados , Herbicidas/toxicidad , Proteómica/métodos , Suelo/química , Animales , Aspergillus nidulans/metabolismo , Ecosistema , Glicina/toxicidad , Nivel sin Efectos Adversos Observados , GlifosatoRESUMEN
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.
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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.
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Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Osteoprotegerina/farmacología , Bencilaminas , Caspasa 3/metabolismo , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclamas , Citoesqueleto/efectos de los fármacos , Células Endoteliales/fisiología , Quinasa 1 de Adhesión Focal/metabolismo , Regulación de la Expresión Génica/fisiología , Compuestos Heterocíclicos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Integrina alfaVbeta3/metabolismo , Neovascularización Fisiológica/fisiología , Osteoprotegerina/metabolismo , Fosforilación/efectos de los fármacos , Proteómica , Sindecano-1/metabolismoRESUMEN
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.
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Chaperonina 60/sangre , Neoplasias Colorrectales/diagnóstico , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Western Blotting , Antígeno CA-19-9/sangre , Antígeno Carcinoembrionario/sangre , Neoplasias Colorrectales/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Electroforesis Bidimensional Diferencial en GelRESUMEN
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.
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Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos , Microtúbulos/metabolismo , Paclitaxel/farmacología , Proteoma/metabolismo , Septinas/metabolismo , Moduladores de Tubulina/farmacología , Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Proteómica/métodos , Tubulina (Proteína)/metabolismo , Regulación hacia ArribaRESUMEN
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.
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Péptidos beta-Amiloides/líquido cefalorraquídeo , Electroforesis Capilar/métodos , Péptidos/líquido cefalorraquídeo , Péptidos beta-Amiloides/química , Calibración , Estudios de Casos y Controles , Electroforesis en Gel de Poliacrilamida , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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.
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Citoesqueleto de Actina/metabolismo , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Miocitos Cardíacos/metabolismo , Animales , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Masculino , Contracción Muscular , Miocardio/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Sarcómeros/metabolismoRESUMEN
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.
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Colon/química , Bromuro de Cianógeno/química , Proteínas/análisis , Proteómica/métodos , Animales , Bovinos , Fraccionamiento Químico , Electroforesis en Gel Bidimensional/métodos , Humanos , Peso Molecular , Membrana Mucosa/química , Proteínas/química , Reproducibilidad de los Resultados , Albúmina Sérica Bovina/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Electroforesis en Gel Bidimensional/métodos , Proteómica/métodos , Precursor de Proteína beta-Amiloide/genética , Animales , Apolipoproteínas E/metabolismo , Western Blotting/métodos , Encéfalo/patología , Diagnóstico por Imagen/métodos , Modelos Animales de Enfermedad , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
The troponin (Tn) complex is composed of troponin T, troponin C and troponin I. The cardiac isoform of TnI (cTnI) is modified and released in blood of patients with cardiovascular diseases as a heterogeneous mixture of free, complexed and posttranslationally modified forms. With the aim to determine later, whether specific forms of cTnI could be associated with the different pathologies leading to cTnI release, the cTnI forms present in the plasma from 64 patients with acute myocardial infarction (AMI) have been analysed by SELDI-TOF MS using anti-TnI mAbs coupled to PS20 ProteinChips arrays. Upfront immunoaffinity enrichment using anti-cTnI 19C7 mAb allowed us to detect cTnI and bis-phosphorylated cTnI in 11/12 and 9/12 analyses respectively, as well as truncated cTnI in plasma with concentration of cTnI as low as 8 ng/mL. Cardiac troponin C (cTnC) and covalent TnIC complex were also found in pools of plasma with higher concentrations of cTnI. MAb 19C7-affinity SELDI-TOF MS analysis performed after immunopurification of one pool of AMI plasma with anti-free cTnI, anti-cTnC, and anti-phosphorylated cTnI mAbs indicated that intact and bis-phosphorylated cTnI were mostly under the free form. Besides, a 18 718 m/z peak could correspond to a truncated phosphorylated form initially complexed with cTnC.
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Infarto del Miocardio/sangre , Isoformas de Proteínas/análisis , Troponina I/análisis , Western Blotting , Humanos , Fosforilación , Análisis por Matrices de Proteínas , Isoformas de Proteínas/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Troponina I/metabolismoRESUMEN
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.
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Hipertensión Renovascular/genética , Hipertensión Renovascular/metabolismo , Proteínas/genética , Proteómica , Animales , Arteriolas/metabolismo , Arteriolas/patología , Electroforesis en Gel de Poliacrilamida , Hipertensión Renovascular/patología , Espectrometría de Masas , Fenotipo , Ratas , Ratas Endogámicas Lew , Circulación RenalRESUMEN
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.
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Riñón/irrigación sanguínea , Músculo Liso Vascular/metabolismo , Proteómica , Troponina T/biosíntesis , Animales , Arteriolas/metabolismo , Diferenciación Celular , Electroforesis en Gel Bidimensional , Hipertensión Renovascular/genética , Hipertensión Renovascular/metabolismo , Masculino , Ratas , Ratas Endogámicas Lew , Renina/biosíntesis , Renina/genética , Sistema Renina-Angiotensina/fisiología , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tinción con Nitrato de Plata , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Troponina T/análisis , Troponina T/genéticaRESUMEN
5'-azacytidine (AZC) is a potent DNA demethylating agent used clinically for treatment of patients with malignant hemopathies. We have previously shown that AZC induces a halt in cell growth and a decrease of cell activity, without affecting cell viability. We have also shown using proteomics, that 35 polypeptides were differentially expressed in a cytoplasmic fraction. The aim of this study was to provide a more complete picture of modifications in AZC-treated cells using cell membrane preparations. Therefore the protein pattern changes following AZC treatment of the cell line DG75 were studied on a detergent-solubilized fraction obtained from these membranes. Results showed that 49 proteins were differentially expressed in the membrane fraction. Seven polypeptides were down-regulated, while 42 were up-regulated. The identity of most of these differentially expressed proteins was determined by mass spectrometry (liquid chromatography-tandem mass spectrometry or matrix-assisted laser desorption/ionization-time of flight), and the identified proteins were grouped based on cellular function and participation in biochemical and signaling pathways.