RESUMO
We have coined the term "alacrity" to describe the extraordinary diversity of B cell activation potentials, even among cells in a single B cell clone responding to a single antigen. The discovery of methodologies for B cell culture in limiting dilution allowed scientists to identify the source of cellular heterogeneity among cells of the immune system. Analyses of individual B cells set the stage for more detailed descriptions of the factors that diversify B cell functions, some of which will be expanded upon by partner articles in this B cell issue.
Assuntos
Anticorpos/história , Linfócitos B/imunologia , Seleção Clonal Mediada por Antígeno , Ativação Linfocitária , Animais , Anticorpos/classificação , Formação de Anticorpos , Técnicas de Cultura de Células/história , História do Século XX , Humanos , Camundongos , Linfócitos T/imunologiaRESUMO
BACKGROUND: The contribution of aberrant DNA methylation in silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations are reversible, it became of interest to determine the effects of the 5-aza-2'-deoxycytidine (DAC) demethylation therapy in breast cancer at different molecular levels. METHODS AND FINDINGS: Here we investigate a synoptic model to predict complete DAC treatment effects at the level of genes, microRNAs and proteins for several human breast cancer lines. The present study assessed an effective treatment dosage based on the cell viability, cytotoxicity, apoptosis and methylation assays for the investigated cell lines. A highly aggressive and a non-aggressive cell line were investigated using omics approaches such as MALDI-TOF MS, mRNA- and microRNA expression arrays, 2-D gel electrophoresis and LC-MS-MS. Complete molecular profiles including the biological interaction and possible early and late systematic stable or transient effects of the methylation inhibition were determined. Beside the activation of several epigenetically suppressed TSGs, we also showed significant dysregulation of some important oncogenes, oncomiRs and oncosuppressors miRNAs as well as drug tolerance genes/miRNAs/proteins. CONCLUSIONS: In the present study, the results denote some new molecular DAC targets and pathways based on the chemical modification of DNA methylation in breast cancer. The outlined approach might prove to be useful as an epigenetic treatment model also for other human solid tumors in the management of cancer patients.
Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Metilação de DNA , Epigênese Genética , MicroRNAs/fisiologia , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Decitabina , Eletroforese em Gel Bidimensional , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In this work we apply the Internal Standard-based analytical approach that we described in an earlier communication and here we demonstrate experimental results on functional associations among the hypervariably-expressed genes (HVE-genes). Our working assumption was that those genetic components, which initiate the disease, involve HVE-genes for which the level of expression is undistinguishable among healthy individuals and individuals with pathology. We show that analysis of the functional associations of the HVE-genes is indeed suitable to revealing disease-specific differences. We show also that another possible exploit of HVE-genes for characterization of pathological alterations is by using multivariate classification methods. This in turn offers important clues on naturally occurring dynamic processes in the organism and is further used for dynamic discrimination of groups of compared samples. We conclude that our approach can uncover principally new collective differences that cannot be discerned by individual gene analysis.
Assuntos
Perfilação da Expressão Gênica/métodos , Variação Genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise por Conglomerados , Interpretação Estatística de Dados , Doença/genética , Expressão Gênica , Perfilação da Expressão Gênica/normas , Redes Reguladoras de Genes , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Padrões de Referência , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Dahl salt-sensitive (DS) and salt-resistant (DR) inbred rat strains represent a well established animal model for cardiovascular research. Upon prolonged administration of high-salt-containing diet, DS rats develop systemic hypertension, and as a consequence they develop left ventricular hypertrophy, followed by heart failure. The aim of this work was to explore whether this animal model is suitable to identify biomarkers that characterize defined stages of cardiac pathophysiological conditions. The work had to be performed in two stages: in the first part proteomic differences that are attributable to the two separate rat lines (DS and DR) had to be established, and in the second part the process of development of heart failure due to feeding the rats with high-salt-containing diet has to be monitored. This work describes the results of the first stage, with the outcome of protein expression profiles of left ventricular tissues of DS and DR rats kept under low salt diet. Substantial extent of quantitative and qualitative expression differences between both strains of Dahl rats in heart tissue was detected. Using Principal Component Analysis, Linear Discriminant Analysis and other statistical means we have established sets of differentially expressed proteins, candidates for further molecular analysis of the heart failure mechanisms.
Assuntos
Regulação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Ventrículos do Coração/metabolismo , Proteínas Musculares/biossíntese , Proteoma/biossíntese , Animais , Modelos Animais de Doenças , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Proteômica/métodos , Ratos , Ratos Endogâmicos Dahl , Cloreto de Sódio na Dieta/efeitos adversos , Cloreto de Sódio na Dieta/farmacologiaRESUMO
Analysis of DNA, RNA, and proteins for downstream genetic, epigenetic, transcriptomic, and proteomic analysis holds an important place in the field of medical care and life science. This is often hampered by the limited availability of sample material. For this reason, there exists an increasing interest for simultaneous isolation of DNA, RNA and proteins from a single sample aliquot. Several kit-systems allowing such a procedure have been introduced to the market. We present an approach using the AllPrep method for simultaneous isolation of DNA, RNA and proteins from several human specimens, such as whole blood, buffy coat, serum, plasma and tissue samples. The quantification and qualification of the isolated molecular species were assessed by different downstream methods: NanoDrop for measuring concentration and purity of all molecular species; DNA and RNA LabChip for fractionation analysis of nucleic acids; quantitative PCR for quantification analysis of DNA and RNA; thymidine-specific cleavage mass array on MALDI-TOF silico-chip for epigenetic analysis; Protein LabChip and two-dimensional (2D) gel electrophoresis for proteomic analysis. With our modified method, we can simultaneously isolate DNA, RNA and/or proteins from one single sample aliquot. We could overcome to some method limitations like low quality or DNA fragmentation using reamplification strategy for performing high-throughput downstream assays. Fast and easy performance of the procedure makes this method interesting for all fields of downstream analysis, especially when using limited sample resources. The cost-effectiveness of the procedure when material is abundantly available has not been addressed. This methodological improvement enables to execute such experiments that were not performable with standard procedure, and ensures reproducible outcome.
Assuntos
DNA , Epigênese Genética , Perfilação da Expressão Gênica , Proteínas , Proteoma/análise , RNA , Linhagem Celular , DNA/análise , DNA/isolamento & purificação , Eletroforese em Gel Bidimensional/métodos , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , Proteínas/análise , Proteínas/isolamento & purificação , Proteômica/métodos , RNA/análise , RNA/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Genome-scale microarray experiments for comparative analysis of gene expressions produce massive amounts of information. Traditional statistical approaches fail to achieve the required accuracy in sensitivity and specificity of the analysis. Since the problem can be resolved neither by increasing the number of replicates nor by manipulating thresholds, one needs a novel approach to the analysis. This article describes methods to improve the power of microarray analyses by defining internal standards to characterize features of the biological system being studied and the technological processes underlying the microarray experiments. Applying these methods, internal standards are identified and then the obtained parameters are used to define (i) genes that are distinct in their expression from background; (ii) genes that are differentially expressed; and finally (iii) genes that have similar dynamical behavior.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Animais , Camundongos , Padrões de ReferênciaRESUMO
Methodological evaluation of the proteomic analysis of cardiovascular-tissue material has been performed with a special emphasis on establishing examinations that allow reliable quantitative analysis of silver-stained readouts. Reliability, reproducibility, robustness and linearity were addressed and clarified. In addition, several types of normalization procedures were evaluated and new approaches are proposed. It has been found that the silver-stained readout offers a convenient approach for quantitation if a linear range for gel loading is defined. In addition, a broad range of a 10-fold input (loading 20-200 microg per gel) fulfills the linearity criteria, although at the lowest input (20 microg) a portion of protein species will remain undetected. The method is reliable and reproducible within a range of 65-200 microg input. The normalization procedure using the sum of all spot intensities from a silver-stained 2D pattern has been shown to be less reliable than other approaches, namely, normalization through median or through involvement of interquartile range. A special refinement of the normalization through virtual segmentation of pattern, and calculation of normalization factor for each stratum provides highly satisfactory results. The presented results not only provide evidence for the usefulness of silver-stained gels for quantitative evaluation, but they are directly applicable to the research endeavor of monitoring alterations in cardiovascular pathophysiology.
Assuntos
Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Regulação da Expressão Gênica , Coração/fisiologia , Miocárdio/metabolismo , Proteoma , Proteômica/métodos , Animais , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Ratos , Ratos Endogâmicos Dahl , Ratos Wistar , Especificidade da EspécieRESUMO
Most of the biologically relevant data on cardiomyocytes are derived from isolated cells under conditions that are, to some extent, altered compared to the natural milieu of the functional heart. The handling procedure of the dissection, isolation, and short-term culturing induces changes in the cells such that the subsequently measured parameters (among others, the protein synthesis) reflect the actual experimental conduct rather than the intrinsic properties of these terminally differentiated cells. Although it is known that the protein synthetic machinery of isolated cardiomyocytes is operational and functional, the biosynthetic yield of human cardiomyocytes in the natural milieu of the trabeculae remains to be established, with a special emphasis to clarify whether the protein synthesis includes just a limited set of polypeptides or it encompasses all cellular constituents. Knowledge on this issue is a prerequisite for achieving further advances in our understanding of heart remodeling related to hypertrophy in particular, and for attempting interventions leading to repair of damaged heart in general. The experimental system of "organ bath" enables simultaneous registration of contractile forces of portions of cardiac muscle tissue (and other myocyte-containing tissues) and biosynthetic labeling of newly synthesized cellular constituents. The organ bath methodology was adapted for this project such as enabling to measure molecular changes in response to in vitro applied stimuli. Instead of Krebs-Henseleit-solution, as used in classical protocols of organ bath studies, we utilized cell culture media suitable to experimental conditions related to metabolic labeling. Proteome patterns established by performing two-dimensional gel electrophoresis of the extracts from biosynthetically labeled trabeculae revealed that cardiomyocytes synthesize the full spectrum of cellular proteins. Proteomic silver-stain readout was used to obtain samples for spot excisions, as material suitable for mass spectrometric analysis. Protein spots were identified both from the excised spots and also by matching with the in-house- and www-databases (Swiss-Prot/High-Performance Heart). From our findings that protein synthesis in terminally differentiated cardiomyocytes is not confined just to the synthesis of those structures needed for the post-mitotic house-keeping functions, we conclude that this model might serve as a valid experimental system to study and elucidate the effects of various pharmacological compounds under conditions where physiology (contractile forces) and biochemistry (protein synthesis) of intact human heart tissue are monitored simultaneously.
Assuntos
Átrios do Coração/patologia , Proteômica/métodos , Actinas/química , Técnicas de Cultura de Células/métodos , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Coração/fisiologia , Átrios do Coração/metabolismo , Humanos , Cinética , Espectrometria de Massas/métodos , Contração Muscular , Miocárdio/metabolismo , Peptídeos/química , Proteoma , Coloração pela PrataRESUMO
Whether or not there are molecular differences, at the intra- and extracellular level, between aortic dilatation in patients with bicuspid (BAV) and those with a tricuspid aortic valve (TAV) has remained controversial for years. We have performed 2-dimensional gel electrophoresis and mass spectrometry coupled with dephosphorylation and phosphostaining experiments to reveal and define protein alterations and the high abundant structural phosphoproteins in BAV compared to TAV aortic aneurysm samples. 2-D gel patterns showed a high correlation in protein expression between BAV and TAV specimens (n=10). Few proteins showed significant differences, among those a phosphorylated form of heat shock protein (HSP) 27 with significantly lower expression in BAV compared to TAV aortic samples (p=0.02). The phosphoprotein tracing revealed four different phosphoproteins including Rho GDP dissociation inhibitor 1, calponin 3, myosin regulatory light chain 2 and four differentially phosphorylated forms of HSP27. Levels of total HSP27 and dually phosphorylated HSP27 (S78/S82) were investigated in an extended patient cohort (n=15) using ELISA. Total HSP27 was significantly lower in BAV compared to TAV patients (p=0.03), with no correlation in levels of phospho-HSP27 (S78/S82) (p=0.4). Western blots analysis showed a trend towards lower levels of phospho-HSP27 (S78) in BAV patients (p=0.07). Immunohistochemical analysis revealed that differences in HSP27 occur in the cytoplasma of VSMC's and not extracellularly. Alterations in HSP27 may give early evidence for intracellular differences in aortic aneurysm of patients with BAV and TAV. Whether HSP27 and the defined phosphoproteins have a specific role in BAV associated aortic dilatation remains to be elucidated.
Assuntos
Aneurisma Aórtico/metabolismo , Valva Aórtica/metabolismo , Proteínas de Choque Térmico/metabolismo , Valva Mitral/metabolismo , Fosfoproteínas/metabolismo , Proteômica , Valva Tricúspide/metabolismo , Estudos de Coortes , Eletroforese em Gel Bidimensional , Feminino , Proteínas de Choque Térmico/química , Humanos , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/química , Fosforilação , Proteoma/química , Serina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Bicuspid aortic valve is often associated with lesions of the ascending aorta, which differ histologically from those in tricuspid valve patients. We undertook proteomic analyses to assess differences at the proteome level. Aortic samples were collected from 20 patients undergoing aortic valve and/or ascending aortic replacement; 9 had a bicuspid valve: 5 with aortic aneurysm (diameter > 50 mm) and 4 without dilation; 11 had a tricuspid valve: 6 with aortic aneurysm and 5 without dilation. Patients with histologically proven connective tissue disorders were excluded. Samples were dissected, solubilized, and subjected to 2-dimensional gel electrophoresis. Gel patterns showed an average of 580 protein spots in samples from bicuspid valve patients, and 564 spots in those with tricuspid valves. Comparative analysis revealed a correlation coefficient of 0.93 for protein expression in the bicuspid valve group compared to the tricuspid group. Three protein spots were significantly over-expressed and 4 were significantly down-regulated in the bicuspid group compared to the tricuspid group. The lowest correlation in protein expression was between non-dilated aortic tissues. These differences between aortic tissues of bicuspid and tricuspid valve patients suggest that mechanisms of aortic dilation might differ, at least in part, between such patients.
Assuntos
Aorta/química , Aneurisma Aórtico/metabolismo , Insuficiência da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/metabolismo , Valva Aórtica/anormalidades , Cardiopatias Congênitas/complicações , Proteínas/análise , Proteômica , Adulto , Idoso , Aorta/cirurgia , Aneurisma Aórtico/etiologia , Aneurisma Aórtico/cirurgia , Valva Aórtica/cirurgia , Insuficiência da Valva Aórtica/etiologia , Insuficiência da Valva Aórtica/cirurgia , Estenose da Valva Aórtica/etiologia , Estenose da Valva Aórtica/cirurgia , Implante de Prótese Vascular , Dilatação Patológica , Eletroforese em Gel Bidimensional , Feminino , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/cirurgia , Implante de Prótese de Valva Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Proteômica/métodosRESUMO
Proteomics describes, analogous to the term genomics, the study of the complete set of proteins present in a cell, organ, or organism at a given time. The genome tells us what could theoretically happen, whereas the proteome tells us what does happen. Therefore, a genomic-centered view of biologic processes is incomplete and does not describe what happens at the protein level. Proteomics is a relatively new methodology and is rapidly changing because of extensive advances in the underlying techniques. The core technologies of proteomics are 2-dimensional gel electrophoresis, liquid chromatography, and mass spectrometry. Proteomic approaches might help to close the gap between traditional pathophysiologic and more recent genomic studies, assisting our basic understanding of cardiovascular disease. The application of proteomics in cardiovascular medicine holds great promise. The analysis of tissue and plasma/serum specimens has the potential to provide unique information on the patient. Proteomics might therefore influence daily clinical practice, providing tools for diagnosis, defining the disease state, assessing of individual risk profiles, examining and/or screening of healthy relatives of patients, monitoring the course of the disease, determining the outcome, and setting up individual therapeutic strategies. Currently available clinical applications of proteomics are limited and focus mainly on cardiovascular biomarkers of chronic heart failure and myocardial ischemia. Larger clinical studies are required to test whether proteomics may have promising applications for clinical medicine. Cardiovascular surgeons should be aware of this increasingly pertinent and challenging field of science.
Assuntos
Doenças Cardiovasculares/metabolismo , Procedimentos Cirúrgicos Cardiovasculares , Proteômica , Animais , Biomarcadores/análise , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/cirurgia , Sistema Cardiovascular/metabolismo , Cardiopatias/diagnóstico , Cardiopatias/metabolismo , Cardiopatias/cirurgia , Humanos , Miocárdio/químicaRESUMO
Deficiency in a protein called signaling lymphocytic activation molecule-associated protein (SAP) causes X-linked lymphoproliferative disease (XLP) and helper T cell-dependent antibody defects. To identify proteins regulated by SAP, we performed proteomic analyses of SAP deficient vs wild type T cells. Our results reveal protein species whose abundances are profoundly altered by SAP. Our work therefore identifies candidate cellular mediators of SAP-dependent T cell help.
Assuntos
Antígenos de Diferenciação de Linfócitos T/química , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Proteoma/análise , Proteoma/genética , Linfócitos T/metabolismo , Animais , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Mapeamento de Peptídeos , Proteômica/métodos , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Cardiovascular research of the past decades dealt with classical pathophysiological descriptions, then shifted toward the identification of relevant receptors, and then proceeded to the analysis of signal transduction pathways. Most recently, hand in hand with the achievements of the human genome project, the research has gone down the road toward molecular biological "disease gene(s) mapping". The application of proteome research will attempt to close the gap between genomic (and genetic) analysis and the physiological research. The rich source of heart surgery specimens represents an excellent starting point in data acquisition of proteomic context. Furthermore, animal models of cardiovascular diseases and deficiencies are considered, and will be explored. Examples of results from feasibility studies are given, with the emphasis on quantitative evaluation of proteomic components, hoping to discover co-regulated sets of proteins that are involved in any particular disease state. Identification of new, not yet discovered proteins will be pursued, though the emphasis of this work will be on the definition of characteristic sets of expressed proteins, which in turn might be able to delimit the state of disease and prognosis of therapy outcome. Besides the systematic issues, this paper refers to a number of methodological questions, like the comparison of the proteins detected by staining procedures and proteins detected in models in which biosynthetic labeling is applicable.
Assuntos
Sistema Cardiovascular , Proteoma , Animais , Aorta/patologia , Autorradiografia , Proteínas Sanguíneas/metabolismo , Linhagem Celular , Mapeamento Cromossômico , Análise por Conglomerados , Estudos de Coortes , Eletroforese em Gel Bidimensional , Cardiopatias/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Focalização Isoelétrica , Espectrometria de Massas , Camundongos , Miocárdio/metabolismo , Ratos , Coloração pela PrataRESUMO
The notion of functional proteomics (description of changes in protein expression during differentiation of cells) and structural proteomics (elucidation of the primary structure of the components in the proteomic pattern) is reviewed. Quantitative and qualitative aspects of the proteome analysis are discussed and evaluated. A list of projects is given that are of high priority for the elucidation of the proteome. This includes, e.g., the study of the repertoire of the proteome and the description of molecular pathways during the clonal cell replication. The paper re-evaluates a number of methodological issues, such as the comparison of the use of Ampholines and Immobilines, various staining procedures and labeling procedures.
Assuntos
Proteômica , Autorradiografia , Linhagem Celular , Eletroforese em Gel Bidimensional , Sensibilidade e EspecificidadeRESUMO
In this paper we focus on the detection of specific state of protein phosphorylation within a complex protein mixture separated by two-dimensional gel electrophoresis followed by immunoblotting. The availability of antibodies that specifically recognize the phosphorylated residue(s) of proteins make this approach feasible as exemplified by the study of the regulatory mechanisms of the cell cycle. The major advantage of the presented approach is its relative simplicity and sensitivity that allows specific detection of protein phosphorylation and distinguishes different phosphorylation sites of target protein. Current findings demonstrate that this method represents a reasonable alternative to the use of other tools to study protein phosphorylation.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas/metabolismo , FosforilaçãoRESUMO
Lymphocytes are the best-studied higher eukaryote cells. In this report, quantitative relationships of the protein components in resting cell, blast cell and plasma cell types are evaluated. The comparison of these cell types leads to the conclusion that resting cells synthesize about one-twentieth of the protein species as compared to blast cells. Blast cells seem to be metabolically the most robust lymphocyte type. Plasma cells are geared towards synthesis of one main product (antibody in B plasma cells), while most of the synthesis of other protein species (including those for housekeeping and repair) decreases as the messages decay. Although the data presented in this communication allow a meaningful comparison of three cell populations, they are far from providing a full picture. Both silver staining and radiofluorography depict only proteins of high or intermediate abundance. Silver staining misses most proteins present at <10 000 copies/cell, while radiofluorography misses all those proteins with slow turnover (and those with no methionine residue in their sequence). The detection of 1100 spots in the blast cell-related radiofluorograph includes visualization of some 97-99% of protein mass, but some 3900 polypeptide species in the remaining 1-3% of protein mass will pass undetected. This protein mass (0.7-2 pg) reflects some 2500-7500 copies of each of those 3900 polypeptide species that are present in the cell below the detection limit. The work emphasizes that full understanding of cellular function can be achieved only if quantitative aspects of cell inventory are considered.
RESUMO
Denervated but not innervated skeletal muscles secrete polypeptides that are involved in neuromuscular synapse formation. With the aim of identifying such components, metabolically labeled polypeptides in extracts from denervated and innervated muscles were submitted to two-dimensional gel electrophoresis, and the abundance of individual molecular species was compared. Consistent differences between the proteomic maps from the two sources of muscles were seen. Likewise, proteomic maps of polypeptides from organ culture media conditioned by chronically denervated muscles and by control muscles revealed consistent differences, but the abundance of material within individual spots from conditioned media was not sufficient for analysis by mass spectrometry. Since it was not possible to match the patterns from muscle extracts and from conditioned media, it has been established that extract of Sol8 muscle cells was a satisfactory source of material for analysis. From 1,200 spots identified on the proteomic map from Sol8 cells by image analysis, some 140 have been defined by mass spectrometric analysis. In order to identify the components that are shared by secreted molecules from denervated muscles and Sol8 cells, a mixture of extracts from the two sources was co-electrophoresed and a shared proteomic pattern was established by visualization of metabolically labeled spots from the conditioned medium and of silver stained spots from the Sol8 cells. More than 100 spots sharing x/y coordinate localization could be seen on the pattern. Of these, fourteen were among those identified by mass spectrometry. It is concluded that co-electrophoresis of radioactively labeled polypeptides from conditioned media with extracts from Sol8 cells can be used to mark in the proteome of Sol8 cells those polypeptides that are secreted at low abundance by adult muscles. Their higher abundance in Sol8 cells opens the possibility for further scrutiny of spots by mass spectrometry or by microsequencing.
Assuntos
Proteínas Musculares/análise , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Junção Neuromuscular/fisiologia , Proteômica/métodos , Animais , Extratos Celulares/química , Linhagem Celular , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Camundongos , Denervação Muscular , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Esquelético/citologia , Junção Neuromuscular/citologia , Junção Neuromuscular/metabolismo , Técnicas de Cultura de Órgãos , Proteoma/análise , Proteoma/química , Proteoma/metabolismoRESUMO
We assessed mast cell influence on eosinophils, the prominent cells in late and chronic allergic reactions, by comparing the proteomic pattern of eosinophils incubated with mast cells, tumor necrosis factor alpha (TNF-alpha) or granulocyte macrophage colony stimulating factor (GM-CSF). Eosinophils were incubated with the human mast cell line HMC-1 cellular sonicate and their survival and GM-CSF production were evaluated. For proteomic studies, eosinophils were cultured with HMC-1 sonicate, TNF-alpha or GM-CSF in the presence of [(35)S]methionine, solubilized and submitted to isolelectric focusing separation and sodium dodecyl sulfate polyacrylamide gel electrophoresis in the ISODALT system, followed by radiofluorography and computer image analysis. HMC-1-incubated eosinophils displayed increased survival partly mediated by mast cell-associated TNF-alpha, and produced GM-CSF. Metabolically labeled eosinophils incubated with either HMC-1, TNF-alpha or GM-CSF released eosinophil peroxidase. Comparison of two-dimensional gel spots from the eosinophils revealed that each of the three activating signals yielded a distinctly different proteomic pattern of labeled polypeptides. GM-CSF provided the strongest signal and the highest rate of protein synthesis (1,018 spots) followed by TNF-alpha (747 spots) and HMC-1 sonicate (611 spots). A portion of spots differed both in terms of quality and quantity. Although each stimulus induced similar functional effects, the resulting biosynthetic programs of the eosinophils greatly differed. The presented proteomic analysis is the first step in the exploration of molecular mechanisms involved in eosinophil activation.
Assuntos
Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Perfilação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Mastócitos/fisiologia , Proteômica/métodos , Fator de Necrose Tumoral alfa/farmacologia , Comunicação Autócrina , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Eosinófilos/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Humanos , Mastócitos/química , Proteoma/metabolismoRESUMO
The work presented here attempts to consolidate our knowledge on cellular transcriptome and proteome. It takes into account that a typical activated cell (lymphocyte) contains 40 000 mRNA molecules at any time, and it represents about 5000 different molecular species of transcripts. Such a cell has about 1 000 000 000 protein molecules, some of them being present at 10 000 000 copies while others at a very low copy number (say 1 to 10 copies per cell). By studying cell free expression of individual cDNA clones (or pools of known complexity) we address to those rare molecular components that will remain undetected by the current analytical means. For our analysis we use cell free translation systems (wheat germ or rabbit reticulocyte origin) and we study polypeptide products originating from intact, or restriction endonuclease-treated cDNA clones. We conclude that in most instances expressed genes yield transcript(s) that translate into several, and often very numerous families of polypeptide species. In our ISODALT two-dimensional gel system we characterize the proteomic profile of the clonal polypeptide families in terms of their molecular mass, charge, multiple products, and appearance.