RESUMEN
Human mesenchymal stem cells (hMSCs) are adult multipotent cells that have high therapeutic potential due to their immunological properties. They can be isolated from several different tissues with bone marrow (BM) being the most common source. Because the isolation procedure is invasive, other tissues such as human umbilical cord vein (UCV) have been considered. However, their interchangeability remains unclear. In the present study, total protein extracts of BM-hMSCs and UCV-hMSCs were quantitatively compared using gel-LC-MS/MS. Previous SAGE analysis of the same cells was re-annotated to enable comparison and combination of these two data sets. We observed a more than 63% correlation between proteomic and transcriptomic data. In silico analysis of highly expressed genes in cells of both origins suggests that they can be modulated by microRNA, which can change protein abundance. Our results showed that MSCs from both tissues shared high similarity in metabolic and functional processes relevant to their therapeutic potential, especially in the immune system process, response to stimuli, and processes related to the delivery of the hMSCs to a given tissue, such as migration and adhesion. Hence, our results support the idea that the more accessible UCV could be a potentially less invasive source of MSCs.
Asunto(s)
Células de la Médula Ósea/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteoma/análisis , Transcriptoma , Venas Umbilicales/citología , Adulto , Células Cultivadas , Cromatografía Liquida/métodos , Humanos , Proteoma/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
HnRNP K protein is a heterogeneous nuclear ribonucleoprotein which has been proposed to be involved in the leukemogenesis of acute promyelocytic leukemia (APL), as well as in differentiation induced by all-trans retinoic acid (ATRA). We previously demonstrated a connection between SET and hnRNP K function in head and neck squamous cell carcinoma (HNSCC) cells related to splicing processing. The objective of this study was to characterize the participation of hnRNP K and SET proteins in ATRA-induced differentiation in APL. We observed higher (5- to 40-fold) levels of hnRNP K and SET mRNA in APL patients at the diagnosis phase compared with induction and maintenance phases. hnRNP K knockdown using short-hairpin RNA led to cell death in ATRA-sensitive NB4 and resistant NB4-R2 cells by apoptosis with SET cleavage. In addition, hnRNP K knockdown increased granulocytic differentiation in APL cells, mainly in NB4-R2 with ATRA. hnRNP K knockdown had an effect similar to that of treatment with U0126 (an meiosis-specific serine/threonine protein kinase/ERK inhibitor), mainly in NB4-R2 cells. SET knockdown in APL cells revealed that apoptosis induction in cells with hnRNP K knockdown occurred by SET cleavage rather than by reduction in SET protein. Transplantation of NB4-R2 cells into nude mice confirmed that arsenic trioxide (ATO) combined with U0126 has higher potential against tumor progression when compared to ATO. Therefore, hnRNP K/SET and ERK are potential therapeutic targets for both antineoplastic leukemia therapy and relapsed APL patients with ATRA resistance.
Asunto(s)
Leucemia Promielocítica Aguda , Animales , Trióxido de Arsénico/metabolismo , Trióxido de Arsénico/uso terapéutico , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Ratones , Ratones Desnudos , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
Aim: Histone acetylation and methylation control gene expression. We investigated the impact of SET knockdown on histone methylation status and the consequences for the miRNAs levels in oral squamous cell carcinoma (OSCC). Methods: OSCC cells with and without SET knockdown were analyzed by quantitative real-time PCR to determine miRNA levels, and by immunoreactions to histone modifications. Results: The knockdown of SET increased the levels of histone H4K20me2 and miR-137. Still, SET protein binds to the miR-137 promoter region. The transfection of miR-137 mimic reduced the KI67 and Rb proteins and proliferation of OSCC cells. Conclusion: Our results show for the first time a relationship between SET and histone methylation associated with the control of miRNA expression and KI67 and Rb as targets of miR-137 in OSCC.
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Proteínas de Unión al ADN/fisiología , Chaperonas de Histonas/fisiología , Histonas/metabolismo , MicroARNs/genética , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Chaperonas de Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Antígeno Ki-67/metabolismo , Metilación , MicroARNs/metabolismo , Neoplasias de la Boca/patología , Proteína de Retinoblastoma/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patologíaRESUMEN
Melanoma is responsible for most deaths among skin cancers and conventional and palliative care chemotherapy are limited due to the development of chemoresistance. We used proteomic analysis to identify cellular responses that lead to chemoresistance of human melanoma cell lines to cisplatin. A systems approach to the proteomic data indicated the participation of specific cellular processes such as oxidative phosphorylation, mitochondrial organization and homeostasis, as well as the unfolded protein response (UPR) to be required for the survival of cells treated with cisplatin. Prohibitin (PHB) was among the proteins consistently accumulated, interacting with the functional clusters associated with resistance to cisplatin. We showed PHB accumulated at different levels in melanoma cell lines under stressing stimuli, such as (i) treatment with temozolomide (TMZ), dacarbazine (DTIC) and cisplatin; (ii) serum deprivation; (iii) tunicamycin, an UPR inducer. Prohibitin accumulated in the mitochondria of melanoma cells after cisplatin and tunicamycin treatment and its de novo accumulation led to chemoresistance melanoma cell lines. In contrast, PHB knock-down sensitized melanoma cells to cisplatin and tunicamycin treatment. We conclude that PHB participates in the survival of cells exposed to different stress stimuli, and can therefore serve as a target for the sensitization of melanoma cells to chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Proteínas Represoras/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Melanoma/genética , Melanoma/patología , Prohibitinas , Proteómica , Proteínas Represoras/genética , Tunicamicina/farmacologíaRESUMEN
Mesenchymal stem or stromal cells (MSCs) are of great interest in biomedical sciences and disease treatment because of their multipotency and wide range of applications for tissue repair and suppression of the immune system. Proteomic analysis of these unique cells has contributed to the identification of important pathways utilized by MSCs to differentiate into distinct tissues as well as important proteins responsible for their special function in vivo and in vitro. However, comparison of proteomic studies in MSCs still suffers from the heterogeneity of MSC preparations. In addition, as proteomics technology advances, several studies can be revisited in order to increase the depth of analysis and, therefore, elucidate more refined mechanisms involved in MSC functionalities. Here, we present detailed protocols to obtain MSCs, as well as protocols to perform in-depth profiling and quantification of alterations in MSC proteomes.
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Células Madre Mesenquimatosas/metabolismo , Proteómica/métodos , Venas Umbilicales/citología , Diferenciación Celular , Proliferación Celular , Femenino , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/citología , EmbarazoRESUMEN
UNLABELLED: Tumor fluid samples have emerged as a rich source for the identification of ovarian cancer in the context of proteomics studies. To uncover differences among benign and malignant ovarian samples, we performed a quantitative proteomic study consisting of albumin immunodepletion, isotope labeling with acrylamide and in-depth proteomic profiling by LC-MS/MS in a pool of 10 samples of each histological type. 1135 proteins were identified, corresponding to 505 gene products. 223 proteins presented associated quantification and the comparative analysis of histological types revealed 75 differentially abundant proteins. Based on this, we developed a panel for targeted proteomic analysis using the multiple reaction monitoring (MRM) method for validation of 51 proteins in individual samples of high-grade serous ovarian tumor fluids (malignant) and benign serous cystadenoma tumor fluids. This analysis showed concordant results in terms of average amounts of proteins, and APOE, SERPINF2, SERPING1, ADAM17, CD44 and OVGP1 were statistically significant between benign and malignant group. The results observed in the MRM for APOE were confirmed by western blotting, where APOE was more abundant in malignant samples. This molecular signature can contribute to improve tumor stratification and shall be investigated in combination with current biomarkers in larger cohorts to improve ovarian cancer diagnosis. BIOLOGICAL SIGNIFICANCE: Despite advances in cancer research, ovarian cancer has a high mortality and remains a major challenge due to a number of particularities of the disease, especially late diagnosis caused by vague clinical symptoms, the cellular and molecular heterogeneity of tumors, and the lack of effective treatment. Thus, efforts are directed to better understand this neoplasia, its origin, development and, particularly the identification and validation of biomarkers for early detection of the disease in asymptomatic stage. In the present work, we confirmed by MRM method in individual ovarian tumor fluid samples the regulation of 27 proteins out of 33 identified in a highthroughput study. We speculate that the presence and/or differential abundance observed in tumor fluid is a cooperation primarily of high rates of secretion of such tumor proteins to extra tumor environment that will at the end accumulate in plasma, and also the accumulation of acute-phase proteins throughout the entire body. On top of that, consideration of physiological influences in the interpretation of expression observed, including age, menopause status, route-of-elimination kinetics and metabolism of the tumor marker, coexisting disease, hormonal imbalances, life-style influences (smoking, alcoholism, obesity), among others, are mandatory to enable the selection of good protein tumor marker candidates for extensive validation.
Asunto(s)
Biomarcadores de Tumor/análisis , Detección Precoz del Cáncer/métodos , Proteínas de Neoplasias/análisis , Neoplasias Ováricas/química , Neoplasias Ováricas/diagnóstico , Proteómica/métodos , Adulto , Anciano , Líquidos Corporales/química , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/patología , Factores de Riesgo , Índice de Severidad de la EnfermedadRESUMEN
Lectin from a leaf of Erythrina indica was isolated by affinity chromatography on Lactamyl-Seralose 4B. Lectin gave a single band in polyacrylamide gel electrophoresis (PAGE). In SDS-gel electrophoresis under reducing and non-reducing conditions Erythrina indica leaf lectin (EiLL) split into two bands with subunit molecular weights of 30 and 33 kDa, whereas 58 kDa was obtained for the intact lectin by gel filtration on Sephadex G-100. EiLL agglutinated all human RBC types, with a slight preference for the O blood group. Lectin was found to be a glycoprotein with a neutral sugar content of 9.5%. The carbohydrate specificity of lectin was directed towards D-galactose and its derivatives with pronounced preference for lactose. EiLL had pH optima at pH 7.0; above and below this pH lectin lost sugar-binding capability rapidly. Lectin showed broad temperature optima from 25 to 50 degrees C; however, at 55 degrees C EiLL lost more than 90% of its activity and at 60 degrees C it was totally inactivated. The pI of EiLL was found to be 7.6. The amino acid analysis of EiLL indicated that the lectin was rich in acidic as well as hydrophobic amino acids and totally lacked cysteine and methionine. The N-terminal amino acids were Val-Glu-Thr-IIe-Ser-Phe-Ser-Phe-Ser-Glu-Phe-Glu-Ala-Gly-Asn-Asp-X-Leu-Thr-Gln-Glu-Gly-Ala-Ala-Leu-. Chemical modification studies of both EiLL and Erythrina indica seed lectin (EiSL) with phenylglyoxal, DEP and DTNB revealed an absence of arginine, histidine and cysteine, respectively, in or near the ligand-binding site of both lectins. Modification of tyrosine with NAI led to partial inactivation of EiLL and EiSL; however, total inactivation was observed upon NBS-modification of two tryptophan residues in EiSL. Despite the apparent importance of these tryptophan residues for lectin activity they did not seem to have a direct role in binding haptenic sugar as D-galactose did not protect lectin from inactivation by NBS.
Asunto(s)
Erythrina/química , Galactosa/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/aislamiento & purificación , Semillas/química , Secuencia de Aminoácidos , Reacciones Cruzadas/inmunología , Fluorescencia , Hemaglutinación , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Hojas de la Planta/química , Lectinas de Plantas/antagonistas & inhibidores , Lectinas de Plantas/inmunología , Subunidades de Proteína/química , Análisis de Secuencia de Proteína , Espectrometría de Fluorescencia , Termodinámica , VolumetríaRESUMEN
The primary structure of the 142 residue Glossoscolex paulistus d-chain hemoglobin has been determined from Edman degradation data of 11 endo-Glu-C peptides and 11 endo-Lys-C peptides, plus the results of Edman degradation of the intact globin. Tryptophan occupies positions 15, 33 and 129. Homology modeling allowed us to assign the positions of these Trp residues relative to the heme and its environment. The reference coordinates of the indole rings (average coordinates of the C(varepsilon2) and C(delta2) atoms) for W15 and W129 were 16.8 and 18.5 A, respectively, from the geometric center of the heme, and W33 was located in close proximity to the heme group at a distance which was approximately half of that for W15 and W129. It was possible to identify three rotamers of W33 on the basis of electrostatic and Van der Waals energy criteria. The calculated distances from the center of the heme were 8.3, 8.4 and 9.1 A for Rot1, Rot2 and Rot3, respectively. Radiationless energy transfer from the excited indole to the heme was calculated on the basis of Förster theory. For W33, the distance was more important than the orientation factor, kappa(2), due to its proximity to the heme. However, based on kappa(2), Rot2 (kappa(2)=0.945) was more favorable for the energy transfer than Rot1 (kappa(2)=0.433) or Rot3 (kappa(2)=0.125). In contrast, despite its greater distance from the heme, the kappa(2) of W129 (2.903) established it as a candidate to be more efficiently quenched by the heme than W15 (kappa(2)=0.191). Although the Förster approach is powerful for the evaluation of the relative efficiency of quenching, it can only explain pico- and sub-nanosecond lifetimes. With the average lifetime,
Asunto(s)
Hemoglobinas Anormales/química , Oligoquetos/química , Triptófano/química , Secuencia de Aminoácidos , Animales , Cisteína/metabolismo , Fluorescencia , Modelos Moleculares , Datos de Secuencia Molecular , Oligoquetos/metabolismo , Conformación Proteica , Serina Endopeptidasas/metabolismo , Espectrometría de FluorescenciaRESUMEN
Dendritic cells (DCs) are highly specialized antigen-presenting cells that play an essential role in the immune response. We used the proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry to identify the protein changes that occur during differentiation of DCs from monocytes (Mo) stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 (GM-CSF/IL-4) and during the maturation of immature DCs stimulated with lipopolysaccharide. Sixty-three differentially expressed proteins (+/- two-fold) were unambiguously identified with sequence coverage greater than 20%. They corresponded to only 36 different proteins, because 11 were present as 38 electrophoretic forms. Some proteins such as tropomyosin 4 and heat shock protein 71 presented differentially expressed electrophoretic forms, suggesting that many of the changes in protein expression that accompany differentiation and maturation of DCs occur in post-translationally modified proteins. The largest differences in expression were observed for actin (21-fold in Mo), Rho GDP-dissociation inhibitor 2 (20-fold in Mo), vimentin (eight-fold in immature DCs), lymphocyte-specific protein 1 (12-fold in mature DCs) and thioredoxin (14-fold in mature DCs). Several proteins are directly related to functional and morphological characteristics of DCs, such as cytoskeletal proteins (cytoskeleton rearrangement) and chaperones (antigen processing and presentation), but other proteins have not been assigned specific functions in DCs. Only a few proteins identified here were the same as those reported in proteomic studies of DCs, which used different stimuli to produce the cells (GM-CSF/IL-4 and tumor necrosis factor-alpha). These data suggest that the DC protein profile depends on the stimuli used for differentiation and especially for maturation.
Asunto(s)
Diferenciación Celular/fisiología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-4/farmacología , Lipopolisacáridos/farmacología , Proteoma/análisis , Animales , Células Dendríticas/citología , Perfilación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Monocitos/fisiología , Procesamiento Proteico-Postraduccional , Proteínas/análisisRESUMEN
A lectin was isolated from the saline extract of Erythrina speciosa seeds by affinity chromatography on lactose-Sepharose. The lectin content was about 265 mg/100g dry flour. E. speciosa seed lectin (EspecL) agglutinated all human RBC types, showing no human blood group specificity; however a slight preference toward the O blood group was evident. The lectin also agglutinated rabbit, sheep, and mouse blood cells and showed no effect on horse erythrocytes. Lactose was the most potent inhibitor of EspecL hemagglutinating activity (minimal inhibitory concentration (MIC)=0.25 mM) followed by N-acetyllactosamine, MIC=0.5mM, and then p-nitrophenyl alpha-galactopyranoside, MIC=2 mM. The lectin was a glycoprotein with a neutral carbohydrate content of 5.5% and had two pI values of 5.8 and 6.1 and E(1%)(1 cm) of 14.5. The native molecular mass of the lectin detected by hydrodynamic light scattering was 58 kDa and when examined by mass spectroscopy and SDS-PAGE it was found to be composed of two identical subunits of molecular mass of 27.6 kDa. The amino acid composition of the lectin revealed that it was rich in acidic and hydroxyl amino acids, contained a lesser amount of methionine, and totally lacked cysteine. The N-terminal of the lectin shared major similarities with other reported Erythrina lectins. The lectin was a metaloprotein that needed both Ca(2+) and Mn(2+) ions for its activity. Removal of these metals by EDTA rendered the lectin inactive whereas their addition restored the activity. EspecL was acidic pH sensitive and totally lost its activity when incubated with all pH values between pH 3 and pH 6. Above pH 6 and to pH 9.6 there was no effect on the lectin activity. At 65 degrees C for more than 90 min the lectin was fairly stable; however, when heated at 70 degrees C for 10 min it lost more than 80% of its original activity and was totally inactivated at 80 degrees C for less than 10 min. Fluorescence studies of EspecL indicated that tryptophan residues were present in a highly hydrophobic environment, and binding of lactose to EspecL neither quenched tryptophan fluorescence nor altered lambda(max) position. Treating purified EspecL with NBS an affinity-modifying reagent specific for tryptophan totally inactivated the lectin with total modification of three tryptophan residues. Of these residues only the third modified residue seemed to play a crucial role in the lectin activity. Addition of lactose to the assay medium did not provide protection against NBS modification which indicated that tryptophan might not be directly involved in the binding of haptenic sugar D-galactose. Modification of tyrosine with N-acetylimidazole led to a 50% drop in EspecL activity with concomitant acetylation of six tyrosine residues. The secondary structure of EspecL as studied by circular dichroism was found to be a typical beta-pleated-sheet structure which is comparable to the CD structure of Erythrina corallodendron lectin. Binding of lactose did not alter the EspecL secondary structure as revealed by CD examination.
Asunto(s)
Fabaceae/metabolismo , Galactosa/metabolismo , Hemaglutinación , Lectinas/química , Lectinas/aislamiento & purificación , Semillas/metabolismo , Animales , Calcio/farmacología , Carbohidratos/química , Cromatografía , Cromatografía en Agarosa , Dicroismo Circular , Concanavalina A/farmacología , Cisteína/química , Ácido Edético/farmacología , Electroforesis en Gel de Poliacrilamida , Eritrocitos/efectos de los fármacos , Caballos , Humanos , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Lectinas/farmacología , Manganeso/farmacología , Espectrometría de Masas , Ratones , Estructura Terciaria de Proteína , Conejos , Ovinos , Espectrometría de Fluorescencia , Temperatura , Triptófano/química , Tirosina/química , Rayos UltravioletaRESUMEN
We describe the preparation of pancreatic enzymes hydrolysate of milk whey proteins containing low levels of aromatic amino acids. Pancreatin and trypsin/chymotrypsin (6.3 percent w/w protein) when used to hydrolyze whey proteins for 27 h at 37±2 °C, released 74 percent of the Phe, 100 percent of the Tyr and 100 percent of the Trp as free amino acids. Most of the free aromatic amino acids present in 2 kg hydrolysate were separated from the remaining peptides and other amino acids by gel filtration on a 15 liter Sephadex G-25 column eluted with 5 percent acetic acid at 60 liters 'h POT. -1' 25°C. The product, recovered in 37 percent yield, contained 0.70 mmol Phe, 0.41 mmol Tyr, and <0.01mmol Trp/100mmol recovered amino acids. The hydrolysate had a general amino acid composition similar to the whey proteins from which it was prepared and could be use as a nitrogen source for patients with phenylketonuria or tyrosinemia after the addition of appropriate aromatic amino acids...
Asunto(s)
Errores Innatos del Metabolismo , Fenilalanina Hidroxilasa , Fenilcetonurias , Tirosinemias , Clarificación Química , Cromatografía en Gel , Filtración/métodosRESUMEN
Este conversatorio trata sobre el proceso de evaluación de los artículos científicos para su publicación en el área biomédica, destacando aspectos como integridad de la literatura científica, calidad en los datos, significación de la investigación, etc. Describe la experiencia del Brazilian Journal of Medical and Biological Research en la evaluación de la literatura científica.
This lecture discusses the evaluation process of scientific articles submitted for publication in the biomedical area, pointing to issues such as integrity of the scientific literature, quality of data, relevance of theresearch etc. It also describes the Brazilian Journal of Medical and Biological Research experience in the scientific literature evaluation.
RESUMEN
Com o objetivo de estimar a prevalência de cistinuria numa área localizada no sul do Brasil, foram coletadas amostras ocasionais de urina de 827 pacientes com litíase urinária da regiäo de Porto alegre, Rio Grande do Sul. As amostras de urina foram submetidas ao teste do cianeto-nitroprussiato, à cromatografia em camada delgada de aminoácidos e à determinaçäo quantitativa dos aminoácidos por cromatografia de troca iônica. Foram detectados 7 pacientes com cistinúria na amostra estudada (0,84%). Ainda que essa freqüência näo seja superior a observada em diversos outros estudos, a identificaçäo de vários casos de cistinúria mostra que a doença existe no sul do Brasil, näo sendo muito rara entre pacientes com litíase urinária. Como é uma doença tratável e facilmente detectável por um método de triagem simples e econômico, os autores sugerem que o teste de triagem para cistinúria seja realizado, também na nossa regiäo, em todos os pacientes com litíase urinária. Os autores säo de opiniäo que o protocolo empregado no presente trabalho oferece uma abordagem racional para a triagem de cistinúria num país com as características do Brasil