RESUMEN
The arcuate nucleus of the hypothalamus is a primary site for sensing blood borne nutrients and hormonal messengers that reflect caloric status. To identify novel energy homeostatic genes, we examined RNA extracts from the microdissected arcuate nucleus of fed and 48-h fasted rats using oligonucleotide microarrays. The relative abundance of 118 mRNA transcripts was increased and 203 mRNA transcripts was decreased during fasting. One of the down-regulated mRNAs was ankyrin-repeat and suppressor of cytokine signalling box-containing protein 4 (Asb-4). The predicted structure of Asb-4 protein suggested that it might encode an intracellular regulatory protein, and therefore its mRNA expression was investigated further. Reverse transcription quantitative polymerase chain reaction was used to validate down-regulation of Asb-4 mRNA in the arcuate nucleus of the fasted Sprague-Dawley rat (relative expression of Asb-4 mRNA: fed = 4.66 +/- 0.26; fasted = 3.96 +/- 0.23; n = 4, P < 0.01). Down-regulation was also demonstrated in the obese fa/fa Zucker rat, another model of energy disequilibrium (relative expression of Asb-4 mRNA: lean Zucker = 3.91 +/- 0.32; fa/fa = 2.93 +/- 0.26; n = 5, P < 0.001). In situ hybridisation shows that Asb-4 mRNA is expressed in brain areas linked to energy homeostasis, including the arcuate nucleus, paraventricular nucleus, dorsomedial nucleus, lateral hypothalamus and posterodorsal medial amygdaloid area. Double in situ hybridisation revealed that Asb-4 mRNA colocalises with key energy homeostatic neurones. In the fed state, Asb-4 mRNA is expressed by 95.6% of pro-opiomelanocortin (POMC) neurones and 46.4% of neuropeptide Y (NPY) neurones. By contrast, in the fasted state, the percentage of POMC neurones expressing Asb-4 mRNA drops to 73.2% (P < 0.001). Moreover, the density of Asb-4 mRNA per fasted POMC neurone is markedly decreased. Conversely, expression of Asb-4 mRNA by NPY neurones in the fasted state is modestly increased to 52.7% (P < 0.05). Based on its differential expression, neuroanatomical distribution and colocalisation, we hypothesise that Asb-4 is a gene involved in energy homeostasis.
Asunto(s)
Repetición de Anquirina/genética , Núcleo Arqueado del Hipotálamo/fisiología , Ayuno/fisiología , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Clonación Molecular , ADN Complementario , Conducta Alimentaria/fisiología , Homeostasis/genética , Hibridación in Situ , Masculino , Obesidad/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero , Ratas , Ratas Sprague-Dawley , Ratas Zucker , Transcripción Genética/fisiologíaRESUMEN
Although most drugs target proteins, the proteome has remained largely untapped for the discovery of drug targets. The sequencing of the human genome has had a tremendous impact on proteomics and has provided a framework for protein identification. There is currently substantial interest in implementing proteomics platforms for drug target discovery. Although the field is still in the early stages, current proteomic tools include a variety of technologies that could be implemented for large-scale protein expression analysis of cells and tissues, leading to discovery of novel drug targets. Proteomics uniquely allows delineation of global changes in protein expression patterns resulting from transcriptional and post-transcriptional control, post-translational modifications and shifts in proteins between different cellular compartments. Some of the current technologies for proteome profiling and the application of proteomics to the analysis of leukemias by our group are reviewed.
Asunto(s)
Antineoplásicos/uso terapéutico , Perfilación de la Expresión Génica/métodos , Leucemia/tratamiento farmacológico , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteoma/genética , Biotecnología/métodos , Diseño de Fármacos , Humanos , Biología Molecular , Técnicas de Diagnóstico MolecularRESUMEN
ICF (immunodeficiency, centromeric region instability and facial anomalies) is a recessive disease caused by mutations in the DNA methyltransferase 3B gene (DNMT3B). Patients have immunodeficiency, chromosome 1 (Chr1) and Chr16 pericentromeric anomalies in mitogen-stimulated lymphocytes, a small decrease in overall genomic 5-methylcytosine levels and much hypomethylation of Chr1 and Chr16 juxtacentromeric heterochromatin. Microarray expression analysis was done on B-cell lymphoblastoid cell lines (LCLs) from ICF patients with diverse DNMT3B mutations and on control LCLs using oligonucleotide arrays for approximately 5600 different genes, 510 of which showed a lymphoid lineage-restricted expression pattern among several different lineages tested. A set of 32 genes had consistent and significant ICF-specific changes in RNA levels. Half of these genes play a role in immune function. ICF-specific increases in immunoglobulin (Ig) heavy constant mu and delta RNA and cell surface IgM and IgD and decreases in Ig(gamma) and Ig(alpha) RNA and surface IgG and IgA indicate inhibition of the later steps of lymphocyte maturation. ICF-specific increases were seen in RNA for RGS1, a B-cell specific inhibitor of G-protein signaling implicated in negative regulation of B-cell migration, and in RNA for the pro-apoptotic protein kinase C eta gene. ICF-associated decreases were observed in RNAs encoding proteins involved in activation, migration or survival of lymphoid cells, namely, transcription factor negative regulator ID3, the enhancer-binding MEF2C, the iron regulatory transferrin receptor, integrin beta7, the stress protein heme oxygenase and the lymphocyte-specific tumor necrosis factor receptor family members 7 and 17. No differences in promoter methylation were seen between ICF and normal LCLs for three ICF upregulated genes and one downregulated gene by a quantitative methylation assay [combined bisulfite restriction analysis (COBRA)]. Our data suggest that DNMT3B mutations in the ICF syndrome cause lymphogenesis-associated gene dysregulation by indirect effects on gene expression that interfere with normal lymphocyte signaling, maturation and migration.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Síndromes de Inmunodeficiencia/genética , Mutación , ARN/metabolismo , Línea Celular , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 16/genética , Metilación de ADN , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Linfocitos/patología , Proteínas de la Membrana/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Regiones Promotoras Genéticas , Síndrome , ADN Metiltransferasa 3BRESUMEN
A novel proteomic approach for probing cell and tissue proteome, which combines liquid phase protein separations with microarray technology has been developed. Proteins in cell and tissue lysates or in cellular subfractions are separated using any one of a number of separation modes which may consist of ion exchange liquid chromatography (LC), reverse phase LC, carrier ampholyte based separations, e.g. the use of Rotofor, affinity based separations, or gel based separations. Each first-dimension fraction obtained using one separation mode can be further resolved using one or more of the other separation modes to yield either purified protein in solution or liquid fractions with substantially reduced protein complexity. The advantage of a liquid based separation system is that proteins in hundreds of individual fractions can be arrayed directly and used as targets for a variety of probes. Constituent proteins in reactive fractions are identified by mass spectrometry and may be further resolved to determine the nature of the reactive protein(s). We present in this report initial data based on microarray analysis of individual Rotofor fractions obtained from lung adenocarcinoma cell line A549 lysates which have been probed with antibodies against specific proteins.
Asunto(s)
Biomarcadores de Tumor/análisis , Perfilación de la Expresión Génica/métodos , Proteínas de Neoplasias/análisis , Neoplasias/metabolismo , Proteoma/análisis , Anticuerpos Antineoplásicos/inmunología , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/inmunología , Biomarcadores de Tumor/inmunología , Western Blotting , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Fluorescencia , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas de Neoplasias/inmunología , Neoplasias/inmunología , Neoplasias/patología , Proteoma/inmunología , Sensibilidad y Especificidad , Células Tumorales CultivadasRESUMEN
PURPOSE: We used a proteomics-based approach to identify tumor proteins that elicit a humoral response in breast carcinoma and that may occur as circulating antigens. EXPERIMENTAL DESIGN: The breast cell line SUM-44 was used as a source of tumor cell proteins for two-dimensional PAGE (2-D PAGE) and for Western blot analysis in which individual sera were analyzed for primary antibodies. RESULTS: Sera from 30 newly diagnosed patients with breast cancer were screened for IgG antibodies to tumor cell proteins. Sera from 116 patients with other cancers and from 25 healthy subjects served as controls. Restricted reactivity against a set of three proteins, identified by mass spectrometry as isoforms of a novel oncogenic protein that regulates RNA-protein interaction (designated RS/DJ-1), was observed in four patients with breast cancer, but not in healthy subjects. The identity was further confirmed by Western blotting with specific antibodies. RS/DJ-1 was found to be secreted in the breast cell line SUM-44, which led us to determine whether RS/DJ-1 was found in circulation in breast cancer. Interestingly, unlike in controls, RS/DJ-1 was readily detectable in sera from 37% of newly diagnosed patients with breast cancer. CONCLUSION: The presence of autoantibodies and/or circulating RS/DJ-1 protein in sera from patients with breast cancer may have clinical utility.
Asunto(s)
Antígenos de Neoplasias/sangre , Neoplasias de la Mama/sangre , Proteoma/análisis , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Autoanticuerpos/sangre , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/metabolismo , Western Blotting , Neoplasias de la Mama/patología , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Neoplasias/sangre , Neoplasias/patología , Proteoma/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales CultivadasRESUMEN
Molecular classification of tumors based on their gene expression profiles promises to significantly refine diagnosis and management of cancer patients. The establishment of organ-specific gene expression patterns represents a crucial first step in the clinical application of the molecular approach. Here, we report on the gene expression profiles of 154 primary adenocarcinomas of the lung, colon, and ovary. Using high-density oligonucleotide arrays with 7129 gene probe sets, comprehensive gene expression profiles of 57 lung, 51 colon, and 46 ovary adenocarcinomas were generated and subjected to principle component analysis and to a cross-validated prediction analysis using nearest neighbor classification. These statistical analyses resulted in the classification of 152 of 154 of the adenocarcinomas in an organ-specific manner and identified genes expressed in a putative tissue-specific manner for each tumor type. Furthermore, two tumors were identified, one in the colon group and another in the ovarian group, that did not conform to their respective organ-specific cohorts. Investigation of these outlier tumors by immunohistochemical profiling revealed the ovarian tumor was consistent with a metastatic adenocarcinoma of colonic origin and the colonic tumor was a pleomorphic mesenchymal tumor, probably a leiomyosarcoma, rather than an epithelial tumor. Our results demonstrate the ability of gene expression profiles to classify tumors and suggest that determination of organ-specific gene expression profiles will play a significant role in a wide variety of clinical settings, including molecular diagnosis and classification.
Asunto(s)
Adenocarcinoma/genética , Neoplasias del Colon/genética , Perfilación de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Ováricas/genética , Adenocarcinoma/clasificación , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Biomarcadores de Tumor/metabolismo , Neoplasias del Colon/clasificación , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Diagnóstico Diferencial , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/clasificación , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Ováricas/clasificación , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patologíaRESUMEN
Astrocytomas are heterogeneous intracranial glial neoplasms ranging from the highly aggressive malignant glioblastoma multiforme (GBM) to the indolent, low-grade pilocytic astrocytoma. We have investigated whether DNA microarrays can identify gene expression differences between high-grade and low-grade glial tumors. We compared the transcriptional profile of 45 astrocytic tumors including 21 GBMs and 19 pilocytic astrocytomas using oligonucleotide-based microarrays. Of the approximately 6800 genes that were analyzed, a set of 360 genes provided a molecular signature that distinguished between GBMs and pilocytic astrocytomas. Many transcripts that were increased in GBM were not previously associated with gliomas and were found to encode proteins with properties that suggest their involvement in cell proliferation or cell migration. Microarray-based data for a subset of genes was validated using real-time quantitative reverse transcription-PCR. Immunohistochemical analysis also localized the protein products of specific genes of interest to the neoplastic cells of high-grade astrocytomas. Our study has identified a large number of novel genes with distinct expression patterns in high-grade and low-grade gliomas.
Asunto(s)
Astrocitoma/genética , Astrocitoma/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Glioblastoma/genética , Glioblastoma/patología , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Tumorales CultivadasRESUMEN
The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis as well as leads for therapy. The purpose of this study was to identify proteins that commonly induce a humoral response in lung cancer by using a proteomic approach and to investigate biological processes that may be associated with the development of autoantibodies. Aliquots of solubilized proteins from a lung adenocarcinoma cell line (A549) and from lung tumors were subjected to two-dimensional PAGE, followed by Western blot analysis in which individual sera were tested for primary antibodies. Sera from 54 newly diagnosed patients with lung cancer and 60 patients with other cancers and from 61 noncancer controls were analyzed. Sera from 60% of patients with lung adenocarcinoma and 33% of patients with squamous cell lung carcinoma but none of the noncancer controls exhibited IgG-based reactivity against proteins identified as glycosylated annexins I and/or II. Immunohistochemical analysis showed that annexin I was expressed diffusely in neoplastic cells in lung tumor tissues, whereas annexin II was predominant at the cell surface. Interestingly, IL-6 levels were significantly higher in sera of antibody-positive lung cancer patients compared with antibody-negative patients and controls. We conclude that an immune response manifested by annexins I and II autoantibodies occurs commonly in lung cancer and is associated with high circulating levels of an inflammatory cytokine. The proteomic approach we have implemented has utility for the development of serum-based assays for cancer diagnosis as we report in this paper on the discovery of antiannexins I and/or II in sera from patients with lung cancer.
Asunto(s)
Anexina A1/inmunología , Anexina A2/inmunología , Anticuerpos Antineoplásicos/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Interleucina-6/sangre , Neoplasias Pulmonares/inmunología , Proteínas de Neoplasias/inmunología , Secuencia de Aminoácidos , Anexina A1/química , Anexina A1/genética , Anexina A2/química , Anexina A2/genética , Anticuerpos Antineoplásicos/sangre , Autoanticuerpos/sangre , Autoantígenos/química , Autoantígenos/genética , Western Blotting , Proteína C-Reactiva/análisis , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Células Pequeñas/sangre , Carcinoma de Células Pequeñas/inmunología , Electroforesis en Gel Bidimensional , Perfilación de la Expresión Génica , Glicosilación , Humanos , Sueros Inmunes , Interleucina-1/sangre , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/genética , Datos de Secuencia Molecular , Neoplasias/sangre , Neoplasias/inmunología , Procesamiento Proteico-Postraduccional , Factor de Necrosis Tumoral alfa/análisisRESUMEN
The human TAX-1 gene encodes a Mr 135,000 glycoprotein that is transiently expressed on the surface of a subset of neurons during development and is involved in neurite outgrowth. The TAX-1 gene has been mapped to a region on chromosome 1 that has been implicated in microcephaly and the Van der Woude syndrome. Using restriction landmark genome scanning to search for amplified genes in gliomas, we found TAX-1 to be amplified in 2 high-grade gliomas among a group of 26 gliomas investigated. Real-time reverse transcription-quantitative PCR analysis detected high levels of TAX-1 mRNA in glial tumors, even in the absence of TAX-1 gene amplification. Immunohistochemical analysis revealed abundant levels of TAX-1 in neoplastic glial cells of glioblastoma multiforme tumors. Because glial tumors are highly invasive and in view of the role of TAX-1 in neurite outgrowth, we investigated the potential role of TAX-1 in glioma cell migration. Using an in vitro assay, we found that the migration of glioma tumor cells is profoundly reduced in the presence of either an anti-TAX-1 antibody or a TAX-1 antisense oligonucleotide. Our findings suggest that TAX-1 plays a role in glial tumorigenesis and may provide a potential target for therapeutic intervention.
Asunto(s)
Neoplasias Encefálicas/genética , Moléculas de Adhesión Celular Neuronal/genética , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glicoproteínas de Membrana/genética , Northern Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Moléculas de Adhesión Celular Neuronal/biosíntesis , Movimiento Celular/fisiología , Contactina 2 , Regulación hacia Abajo , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Inmunohistoquímica , Glicoproteínas de Membrana/biosíntesis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. We have utilized two independent approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14(+) blood monocytes and their derived DCs. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays complementary to 6300 human genes showed that approximately 40% of the genes were expressed in DCs. A total of 255 genes (4%) were found to be regulated during DC differentiation or maturation. Most of these genes were not previously associated with DCs and included genes encoding secreted proteins as well as genes involved in cell adhesion, signaling, and lipid metabolism. Protein analysis of the same cell populations was done using two-dimensional gel electrophoresis. A total of 900 distinct protein spots were included, and 4% of them exhibited quantitative changes during DC differentiation and maturation. Differentially expressed proteins were identified by mass spectrometry and found to represent proteins with Ca(2+) binding, fatty acid binding, or chaperone activities as well as proteins involved in cell motility. In addition, proteomic analysis provided an assessment of post-translational modifications. The chaperone protein, calreticulin, was found to undergo cleavage, yielding a novel form. The combined oligonucleotide microarray and proteomic approaches have uncovered novel genes associated with DC differentiation and maturation and has allowed analysis of post-translational modifications of specific proteins as part of these processes.
Asunto(s)
Células Dendríticas/fisiología , Perfilación de la Expresión Génica , Secuencia de Aminoácidos , Diferenciación Celular/genética , Células Dendríticas/citología , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , ProteomaRESUMEN
There is currently substantial interest in the identification of human tumor antigens for diagnosis and immunotherapy of cancer. We have implemented a proteomic approach for the identification of tumor proteins that elicit a humoral response in cancer patients, which we have applied to neuroblastoma. Proteins from neuroblastoma tumors and cell lines were separated by two-dimensional PAGE and transferred to poly(vinylidene difluoride) membranes. Sera from 23 newly diagnosed patients with neuroblastoma, from 12 newly diagnosed children with other solid tumors, and from 13 normal individuals were screened for IgG and IgM autoantibodies against neuroblastoma proteins by means of Western blot analysis. Sera from 11 patients with neuroblastoma and from 1 patient with a primitive neuroectodermal tumor, but none of the other controls exhibited IgG-based reactivity against a protein constellation with an estimated Mr 50,000. NH2-terminal sequence and mass spectrometric analysis identified the major constituents of this constellation as beta-tubulin isoforms I and III. The IgG antibodies were additionally characterized to be of the subclass IgG1. Neuroblastoma patient sera that contained anti-beta-tubulin IgG antibodies also contained IgM antibodies specific against the full-length beta-tubulin molecule and against COOH-terminal beta-tubulin cleavage products. Neuroblastoma patient sera that reacted with beta-tubulin I and III isoforms in neuroblastoma tissues did not react with beta-tubulin I and III isoforms found in normal brain tissue. Our findings indicate the occurrence of beta-tubulin peptides in neuroblastoma, which are immunogenic. The occurrence of immunogenic peptides in neuroblastoma may have utility in diagnosis and in immunotherapy of this aggressive childhood tumor.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Neoplasias Encefálicas/metabolismo , Neuroblastoma/metabolismo , Tubulina (Proteína)/sangre , Tubulina (Proteína)/química , Adolescente , Western Blotting , Neoplasias Encefálicas/sangre , Niño , Preescolar , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Lactante , Recién Nacido , Masculino , Neuroblastoma/sangre , Tinción con Nitrato de Plata , Tubulina (Proteína)/metabolismo , Células Tumorales CultivadasRESUMEN
A novel two-dimensional liquid-phase separation method was developed that is capable of resolving large numbers of cellular proteins. The proteins are separated by pI using isoelectric focusing in the first dimension and by hydrophobicity using nonporous reversed-phase HPLC in the second dimension (IEF-NP RP HPLC). Proteins were mapped using original software in order to create a protein pattern analogous to that of the 2-D PAGE image. RP HPLC peaks are represented by bands of different intensity in the 2-D image, according to the intensity of the peaks eluting from the HPLC. Each peak was collected as the eluent of the HPLC separation in the liquid phase. The proteins collected were identified using proteolytic enzymes, MALDI-TOF MS and MSFit database searching. Using IEF-NP RP HPLC, approximately 700 bands were resolved in a pI range from 3.2 to 9.5 and 38 different proteins with molecular weights ranging from 12,000 to 75,000 were identified. In comparison to a 2-D gel separation of the same human erythroleukemia cell line lysate, the IEF-NP RP HPLC produced improved resolution of low mass and basic proteins. In addition, the proteins remained in the liquid phase throughout the separation, thus making the entire procedure highly amenable to automation and high throughput. It is demonstrated that IEF-NP RP HPLC provides a viable alternative to the 2-D gel separation method for the screening of protein profiles.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Focalización Isoeléctrica/métodos , Proteínas de Neoplasias/aislamiento & purificación , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales CultivadasRESUMEN
Some of the acute actions of insulin may be mediated by an enzyme-modulating inositol phosphate glycan, produced by the insulin-sensitive hydrolysis of a glycosyl phosphatidylinositol that is structurally similar to a membrane protein anchor. An inositol glycan fragment from the structurally characterized Trypanosoma brucei variant surface glycoprotein glycosyl phosphatidylinositol anchor inhibits isoproterenol-stimulated lipolysis in intact rat epididymal adipocytes in a manner mechanistically similar to that of insulin. To explore these effects in more detail, we evaluated the effects of this glycan on protein phosphorylation. Isoproterenol stimulates the phosphorylation of a 70-kilodalton (kDa) protein in these cells. Like insulin, the glycan fragment specifically attenuates the phosphorylation state of the phosphoprotein. In purified adipocyte cytosol, the glycan stimulates the dephosphorylation on serine residues of a 70-kDa protein in a time- and dose-dependent fashion. The glycan-dependent dephosphorylation of the 70-kDa phosphoprotein is unaffected by addition of trifluoroperazine, an inhibitor of serine/threonine phosphatase-2B, but is blocked by the addition of okadaic acid, an inhibitor of serine/threonine phosphatase-1 and -2A. The differential sensitivities of this dephosphorylation reaction to polycations, which activate phosphatase-2A, and phosphorylated inhibitor 1, which blocks phosphatase-1, suggest that dephosphorylation of the 70-kDa protein results from the specific activation of a type 1 serine/threonine phosphatase in adipocytes, providing a mechanistic basis for the insulin-mimetic effects of the inositol glycan.
Asunto(s)
Adipocitos/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Fosfatos de Inositol/farmacología , Fosfoproteínas/metabolismo , Polisacáridos/farmacología , Trypanosoma brucei brucei/metabolismo , Adipocitos/citología , Animales , Relación Dosis-Respuesta a Droga , Éteres Cíclicos/farmacología , Fosfatos de Inositol/metabolismo , Isoproterenol/farmacología , Masculino , Ácido Ocadaico , Fosforilación , Polisacáridos/metabolismo , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares , Trypanosoma brucei brucei/químicaRESUMEN
The L1 larvae of the parasitic nematode Trichinella spiralis invade skeletal muscle and initiate a process that has been interpreted to represent skeletal muscle dedifferentiation. In this process, the infected region of the muscle cell is converted into a unique structure, called the Nurse cell. The nematode T. spiralis can survive for tens of years within the cytoplasm of the Nurse cell and secretes proteins into the cytoplasm that are believed to play a role in mediating the Nurse cell formation or maintenance. We have cloned a cDNA encoding the T. spiralis-derived, 43-kDa secreted protein. Structural analysis of the predicted 344-amino acid sequence revealed an N terminally located signal peptide and a potential helix-loop-helix motif in the main body of the protein. Antibodies raised against the 43-kDa recombinant protein were used in immunocytolocalizations of T. spiralis-infected skeletal muscle sections. These antibodies strongly stained the Nurse cell nuclei and the nematode itself. Specific, though slightly weaker staining also occurred in the Nurse cell cytoplasm. In Western blots, the antibodies react with the 43-kDa protein but also detected at least two other T. spiralis-secreted proteins. DNA hybridizations reveal at least one additional 43-kDa-related sequence encoded in the T. spiralis genome. We conclude that either the 43-kDa protein and/or a closely related 43-kDa family member is secreted into the muscle and translocates to the muscle-derived nuclei. This model may provide insights into the mechanisms involved in Nurse cell formation.
Asunto(s)
Glicoproteínas/metabolismo , Trichinella/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , Núcleo Celular/metabolismo , ADN , Glicoproteínas/química , Glicoproteínas/genética , Inmunohistoquímica , Datos de Secuencia Molecular , Conformación Proteica , ARN/análisisRESUMEN
Some of the acute actions of insulin may be mediated by an enzyme-modulating inositol phosphate glycan, produced by the insulin-sensitive hydrolysis of glycosyl-phosphatidylinositol (GPI) that is structurally similar to a membrane protein anchor. An inositol glycan fragment from the structurally characterized Trypanosoma brucei variant surface glycoprotein GPI anchor is evaluated for insulin-mimetic antilipolytic activity. The fragment specifically and dose-dependently inhibits isoproterenol-stimulated lipolysis. Like the effect of insulin, glycan-induced antilipolysis is blocked by the low Km cAMP phosphodiesterase inhibitor imazodan (CI-914) and the serine/threonine phosphatase inhibitor, okadaic acid, suggesting that the activation of both cAMP phosphodiesterase and serine/threonine protein phosphatases are necessary. Moreover, this fragment causes a specific and dose-dependent inhibition of both microsomal glucose-6-phosphatase (EC 3.1.3.9) and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activity. Additionally, direct addition of the glycan to hepatocytes caused marked inhibition of glucose production from pyruvate. These results suggest that the direct modification of the activities of these two gluconeogenic enzymes by an inositol glycan may play a role in the inhibition of glucose output by insulin and provide the first evidence for the insulin-mimetic properties of a chemically characterized inositol glycan.
Asunto(s)
Tejido Adiposo/metabolismo , Fructosa-Bifosfatasa/antagonistas & inhibidores , Glucosa-6-Fosfatasa/antagonistas & inhibidores , Glucolípidos/química , Fosfatos de Inositol/aislamiento & purificación , Fosfatos de Inositol/farmacología , Lipólisis/efectos de los fármacos , Hígado/metabolismo , Fosfatidilinositoles/química , Polisacáridos/aislamiento & purificación , Polisacáridos/farmacología , Trypanosoma brucei brucei/química , Glicoproteínas Variantes de Superficie de Trypanosoma/química , Tejido Adiposo/efectos de los fármacos , Animales , Carbohidratos/farmacología , Células Cultivadas , Citosol/enzimología , Gluconeogénesis/efectos de los fármacos , Glucolípidos/farmacología , Glicosilfosfatidilinositoles , Cinética , Hígado/enzimología , Masculino , Microsomas Hepáticos/enzimología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fosfatidilinositoles/farmacología , Pronasa , Ratas , Ratas Endogámicas , Glicoproteínas Variantes de Superficie de Trypanosoma/aislamiento & purificaciónRESUMEN
We have studied the role of microtubules and actin filaments in the biogenesis of epithelial cell surface polarity, using influenza hemagglutinin and vesicular stomatitis G protein as model apical and basolateral proteins in infected Madin-Darby canine kidney cells. Addition of colchicine or nocodazole to confluent monolayers at concentrations sufficient to completely disassemble microtubules did not affect the asymmetric budding of influenza or vesicular stomatitis virus and only slightly reduced the typical asymmetric surface distribution of their envelope proteins, despite extensive cytoplasmic redistribution of the Golgi apparatus. Alteration of microtubular function by taxol or dissociation of actin filaments by cytochalasin D also failed to have a significant effect. Furthermore, neither colchicine nor cytochalasin D pretreatment blocked the ability of subconfluent Madin-Darby canine kidney cells to sustain polarized budding of influenza virus a few hours after attachment to the substrate. Our results indicate that domain-specific microtubule or actin filament "tracks" are not responsible for the vectorial delivery of apically or basolaterally directed transport vesicles. In conjunction with currently available evidence, they are compatible with a model in which receptors in the cytoplasmic aspect of apical or basolateral regions provide vectoriality to the transport of vesicles carrying plasma membrane proteins to their final surface localization.
Asunto(s)
Citoesqueleto de Actina/fisiología , Citoesqueleto/fisiología , Epitelio/ultraestructura , Glicoproteínas de Membrana , Microtúbulos/fisiología , Proteínas del Envoltorio Viral , Actinas/fisiología , Alcaloides/farmacología , Animales , Bencimidazoles/farmacología , Compartimento Celular/efectos de los fármacos , Línea Celular , Membrana Celular/metabolismo , Colchicina/farmacología , Citocalasina D , Citocalasinas/farmacología , Perros , Exocitosis , Hemaglutininas Virales/metabolismo , Riñón/citología , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Nocodazol , Orthomyxoviridae , Paclitaxel , Tubulina (Proteína)/fisiología , Virus de la Estomatitis Vesicular Indiana , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
To study the biogenetic pathway of influenza hemagglutinin (HA), a model apical glycoprotein, in polarized epithelial MDCK cells, anti-HA antibodies were added to the basolateral surface during influenza infection. In monolayers grown on collagen gels influenza and VSV plaque development was blocked only when the antibodies were added to the respective budding surface. Addition of anti-HA antibodies to the basal medium of monolayers grown on nitrocellulose filter chambers neither resulted in HA-coupled transport of antibody nor inhibited HA migration to the apical surface. These results indicate that the bulk of HA is vectorially inserted into the apical surface of MDCK cells by polarized exocytosis. Other apical proteins in epithelia may use a similar mechanism during biogenesis.