RESUMEN
OBJECTIVES: To present clinical and cytopathological features of nine cases of chordomas, diagnosed over 9 years and confirmed by brachyury (T) immunostaining. METHODS: Conventional cytological smears, stained with Papanicolaou and May-Grünwald Giemsa, along with corresponding histopathological (n = 8) and immunostained sections (n = 8) were reviewed. Immunohistochemical staining was performed on tissue sections by polymer detection technique. RESULTS: Nine tumours occurred in seven males and two females, with age ranging from 36 to 72 years (average = 58.7), in the sacrum (seven) and spine (two). On fine needle aspiration cytology, five cases were either diagnosed with or diagnosed with a suggestion of a chordoma, while three cases were diagnosed with chordoma as a differential diagnosis. On review, smears were moderately cellular, comprising myxoid stroma (9/9), epithelioid cells (9/9), physaliphorous cells (8/9), including binucleation (7/9), prominent nucleolisation (2/9), pleomorphic cells (2/9) and intranuclear inclusions (3/9). Immunohistochemically, tumour cells expressed cytokeratin (4/4), pan cytokeratin (4/4), epithelial membrane antigen (8/8), S100 protein (6/8) and brachyury (8/8). Five patients underwent surgical excision, including two who underwent adjuvant radiotherapy (RT) and four patients who underwent RT. During follow-up (n = 8), a single patient developed recurrence and another presented with metastatic lesions. Finally, five patients were alive with disease (7-53 months); a single patient was free of disease (4 months), and two patients died of disease; the latter cases displayed pleomorphic cells and intranuclear inclusions. CONCLUSIONS: Chordomas can be primarily diagnosed by fine needle aspiration cytology in a typical clinicoradiological setting with a combination of key cytomorphological features. Pleomorphic cells and intranuclear inclusions are associated with a relatively aggressive subtype. An exact diagnosis has treatment implications and requires confirmation by brachyury immunostaining.
Asunto(s)
Cordoma/diagnóstico , Citodiagnóstico , Proteínas Fetales/aislamiento & purificación , Proteínas de Dominio T Box/aislamiento & purificación , Adulto , Anciano , Biopsia con Aguja Fina , Cordoma/genética , Cordoma/patología , Femenino , Proteínas Fetales/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Persona de Mediana Edad , Mucina-1/genética , Proteínas de Dominio T Box/genéticaRESUMEN
Proteomics has brought with it the hope of identifying novel biomarkers for the fetal aneuploidies. This hope is built on the ability of proteomic technologies, such as two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients combined with protein identification by mass spectrometry (MS). The large dynamic range of plasma necessitates the effective removal of abundant plasma proteins to allow analysis of the lower concentration analytes. There are many factors that make this research very challenging, beginning with standardization of sample collection, consistent sample preparation, and continuing through the entire analytical process. Therefore, reproducible sample complexity reduction methods such as depletions or fractionations are an essential first step in biomarker discovery experiments. For qualitative and quantitative evaluation of the proteome, the fluorescent dye stains offer several advantages over traditional staining methods. The sensitivity of the fluorescent dye stains such as SYPRO Ruby is comparable with that of silver staining and also has a broad dynamic range, which allows accurate protein quantification being compatible with MS methods for protein identification. Despite its limitations for proteome analysis 2-DE is currently the workhorse for proteomics. Taking into account the factors such as cost, availability and ease of use, 2-DE electrophoresis is one of the most apposite approaches toward the methodical characterization of proteomes.
Asunto(s)
Aneuploidia , Proteínas Fetales/metabolismo , Diagnóstico Prenatal/métodos , Proteómica , Biomarcadores/sangre , Electroforesis en Gel Bidimensional , Femenino , Proteínas Fetales/genética , Proteínas Fetales/aislamiento & purificación , Colorantes Fluorescentes , Humanos , Intercambio Materno-Fetal , Embarazo , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Formins are key regulators of actin cytoskeletal dynamics that constitute a diverse protein family that is present in all eukaryotes examined. They typically consist of more than 1000 amino acids and are defined by the presence of two conserved regions, namely the formin homology 1 and 2 domains. Additional conserved domains comprise a GTPase-binding domain for activation, a C-terminal autoregulation motif and an N-terminal recognition domain. In this study, the N-terminal region (residues 1-339) of the human formin homology domain-containing protein 1 (FHOD1) was purified and crystallized from 20%(w/v) PEG 4000, 10%(v/v) glycerol, 0.3 M magnesium chloride and 0.1 M Tris-HCl pH 8.0. Native crystals belong to space group P1, with unit-cell parameters a = 35.4, b = 73.9, c = 78.7 A, alpha = 78.2, beta = 86.2, gamma = 89.7 degrees. They contain two monomers of FHOD1 in the asymmetric unit and diffract to a resolution of 2.3 A using a synchrotron-radiation source.
Asunto(s)
Proteínas Fetales/química , Proteínas Nucleares/química , Fragmentos de Péptidos/química , Cristalización , Proteínas Fetales/aislamiento & purificación , Forminas , Humanos , Proteínas Nucleares/aislamiento & purificación , Fragmentos de Péptidos/aislamiento & purificación , Estructura Terciaria de ProteínaRESUMEN
Proteomic patterns from an ordered cDNA library of mouse fetal thymus origin of an overall complexity of 1536 clones are described. Patterns have been analyzed at 96, 12, or 8 clones in a mixture, or as individual clones. Clones yield in some instances a single spot, in other cases a complex cluster or family of spots is formed. The determination of the clonal address (a six-digit number, indicating the interception of three pooling dimensions) by inspection of pools of 8, 12 or 16 clones is a reliable approach; nevertheless a complete proof consists in retrieving the clone and then submitting to transcription, translation and proteomic analysis. The spot clusters are meaningful clone identifiers; cluster components (families of polypeptides) are characteristic of individual clones and are independent of clones coexisting (and being co-expressed) in a given pool. A 'cluster' originates from a single cloned message and might be due to post-translational modification (offered by the reticuleocyte machinery) or as a result of programmed degradation. Thirteen clones or families of clonal products are shown, and the heterogeneity of the 'appearance' of clones is documented. In about half, the assignment of a clonal polypeptide product to a storage well position has failed; this might be due to a variety of considerations elaborated herein. The arguments are presented, that analysis of abundant proteins of a cell (activated lymphocyte) comprises 'classical proteomics', but for the analysis of the rare molecular species of proteins, Poissonian approaches of replicable material have to be used.
Asunto(s)
ADN Complementario/genética , Proteínas Fetales/genética , Proteoma/genética , Timo/metabolismo , Animales , Clonación Molecular , Electroforesis en Gel Bidimensional , Proteínas Fetales/aislamiento & purificación , Biblioteca de Genes , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Biosíntesis de Proteínas , Proteoma/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
We have characterized NORPEG, a novel gene from human retinal pigment epithelial cells (ARPE-19), in which its expression is induced by all-trans-retinoic acid. Two transcripts ( approximately 3 and approximately 5 kilobases in size) have been detected for this gene, which is localized to chromosome band 5p13.2-13.3. Placenta and testis showed the highest level of expression among various human tissues tested. Six ankyrin repeats and a long coiled-coil domain are present in the predicted sequence of the NORPEG protein, which contains 980 amino acid residues. This approximately 110-kDa protein was transiently expressed in COS-7 cells as a FLAG fusion protein and immunolocalized to the cytoplasm. Confocal microscopic analysis of the NORPEG protein in ARPE-19 cells showed threadlike projections in the cytoplasm reminiscent of the cytoskeleton. Consistent with this localization, the expressed NORPEG protein showed resistance to solubilization by Triton X-100 and KCl. An ortholog of NORPEG characterized from mouse encoded a protein that showed 91% sequence similarity to the human NORPEG protein. The expression of Norpeg mRNA was detected in mouse embryo at embryonic day 9.5 by in situ hybridization, and the expression appears to be developmentally regulated. In adult mouse, the highest level of expression was detected in the seminiferous tubules of testis.
Asunto(s)
Proteínas del Citoesqueleto/biosíntesis , Proteínas del Ojo/biosíntesis , Epitelio Pigmentado Ocular/fisiología , Factores de Transcripción , Tretinoina/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Mapeo Cromosómico , Cromosomas Humanos Par 5 , Proteínas del Citoesqueleto/genética , Proteínas del Ojo/genética , Proteínas Fetales/aislamiento & purificación , Regulación de la Expresión Génica , Biblioteca de Genes , Humanos , Hibridación Fluorescente in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Túbulos Seminíferos , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Distribución TisularRESUMEN
Basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) proteins form dimeric transcription factors to mediate diverse biological functions including xenobiotic metabolism, hypoxic response, circadian rhythm and central nervous system midline development. The Ah receptor nuclear translocator protein (ARNT) plays a central role as a common heterodimerization partner. Herein, we describe a novel, embryonically expressed, ARNT interacting protein (AINT) that may be a member of a larger coiled-coil PAS interacting protein family. The AINT C-terminus mediates interaction with the PAS domain of ARNT in yeast and interacts in vitro with ARNT and ARNT2 specifically. AINT localizes to the cytoplasm and overexpression leads to non-nuclear localization of ARNT. A dynamic pattern of AINT mRNA expression during embryogenesis and cerebellum ontogeny supports a role for AINT in development.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Fetales/metabolismo , Receptores de Hidrocarburo de Aril , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Cerebelo/embriología , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario y Fetal , Proteínas Fetales/genética , Proteínas Fetales/aislamiento & purificación , Expresión Génica , Secuencias Hélice-Asa-Hélice , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ratones , Proteínas Asociadas a Microtúbulos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Mensajero , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Transducción de Señal , Fracciones Subcelulares , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
To investigate molecular mechanisms of lung organogenesis, we used representational difference analysis to search for glucocorticoid-inducible genes in developing lung in a fetal rat model. Messenger RNA prepared from fetal and adult rat lung was used to prepare "representative amplicons." Adult-lung complementary DNA (cDNA) amplicons were used as "driver" in successive rounds of subtractive hybridization/amplification to isolate target fetal lung-specific cDNAs. A single clone, which was conserved and had near-perfect homology to eight human/rodent expressed sequence tags, was used as template for 5' and 3' rapid amplification of cDNA ends and SPICE (system for polymerase chain reaction amplification of cDNA ends) reactions to obtain the 3.6-kb cDNA, LGL2 (Genbank, AF 110195) encoding a deduced polypeptide (lgl2) of 963 amino acids. Northern analysis confirmed that LGL2 is differentially expressed in fetal lung (maximal during the pseudoglandular stage, gestational Days 14 to 16), induced by glucocorticoid, and enriched in epithelium relative to the mesenchyme. LGL2 was also detected in human fetal lung at gestational Week 16 as well as in human and rat fetal brain, heart, intestine, and kidney. We mapped LGL2 to chromosome 1p33-34.2. Comparison with sequences in the genome database identified lgl2 as a member of the karyopherin-beta family of nuclear import proteins, with greatest homology to transportin SR. Maximal expression of LGL2 in the pseudoglandular stage of development is coordinate with that of key transcription factors that regulate prominent signal transduction pathways in fetal lung organogenesis. We propose a role for lgl2 in nuclear import of transcription factors that regulate signal transduction during fetal lung development.
Asunto(s)
Proteínas Fetales/biosíntesis , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hidrocortisona/farmacología , Pulmón/embriología , Proteínas Nucleares/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Mapeo Cromosómico , Cromosomas Humanos Par 1/genética , ADN Complementario/genética , Escherichia coli , Etiquetas de Secuencia Expresada , Proteínas Fetales/química , Proteínas Fetales/genética , Proteínas Fetales/aislamiento & purificación , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Edad Gestacional , Humanos , Hibridación Fluorescente in Situ , Pulmón/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Conformación Proteica , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/biosíntesis , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transducción de Señal , Especificidad de la Especie , Técnica de Sustracción , beta CarioferinasRESUMEN
A novel member of the Formin/Diaphanous family of proteins was cloned and characterized. A 4kB mRNA is ubiquitously expressed but is found in abundance in the spleen. FHOS (Formin Homologue Overexpressed in Spleen) contains a 3414bp open reading frame and encodes for an approximately 128kDa protein. FHOS has sequence homology to Diaphanous and Formin proteins within the Formin Homology (FH)1 and FH2 domains. FHOS also contains a coiled-coil, a collagen-like domain, two nuclear localization signals, and several potential PKC and PKA phosphorylation sites. FHOS-specific antiserum was generated and used to determine that FHOS is a predominantly cytoplasmic protein and is expressed in a variety of human cell lines. FHOS was mapped to chromosome 16q22 between framework markers WI-5594 and WI-9392.
Asunto(s)
Proteínas Fetales/química , Proteínas Fetales/aislamiento & purificación , Proteínas Nucleares/química , Proteínas Nucleares/aislamiento & purificación , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Northern Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 16/genética , Clonación Molecular , Femenino , Proteínas Fetales/biosíntesis , Forminas , Células HL-60 , Humanos , Proteínas de Microfilamentos , Datos de Secuencia Molecular , Proteínas Nucleares/biosíntesis , ARN/análisis , Alineación de Secuencia , Bazo/metabolismo , Células Tumorales CultivadasRESUMEN
Trace levels of 5-methylcytosine persist in the DNA of mouse embryonic stem cells that are homozygous for null mutations in Dnmt1 , the gene for the one previously recognized metazoan DNA methyltransferase. This residual 5-methylcytosine may be the product of a candidate second DNA methyltransferase, Dnmt2, that has now been identified in human and mouse. Dnmt2 contains all the sequence motifs diagnostic of DNA (cytosine-5)-methyltransferases but appears to lack the large N-terminal regulatory domain common to other eukaryotic methyltransferases. Dnmt2 is more similar to a putative DNA methyltransferase of the fission yeast Schizosaccharomyces pombe than to Dnmt1. Dnmt2 produces multiple mRNA species that are present at low levels in all tissues of human and mouse and is not restricted to those cell types known to be active in de novo methylation. The human DNMT2 gene was mapped to chromosome 10p12-10p14 in a panel of radiation hybrids. Dnmt2 is a candidate for the activity that methylates newly integrated retroviral DNA and maintains trace levels of 5-methylcytosine in the DNA of embryonic stem cells homozygous for null mutations in Dnmt1.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/aislamiento & purificación , Proteínas Fúngicas/genética , Isoenzimas/aislamiento & purificación , Ratones/genética , Schizosaccharomyces/enzimología , 5-Metilcitosina , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Mapeo Cromosómico , Cromosomas Humanos Par 10/genética , Citosina/análogos & derivados , Citosina/análisis , ADN (Citosina-5-)-Metiltransferasas/química , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , ADN Complementario/genética , Evolución Molecular , Proteínas Fetales/química , Proteínas Fetales/genética , Proteínas Fetales/aislamiento & purificación , Proteínas Fúngicas/química , Expresión Génica , Marcación de Gen , Humanos , Isoenzimas/química , Isoenzimas/genética , Ratones/embriología , Datos de Secuencia Molecular , Schizosaccharomyces/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Células Madre/enzimologíaRESUMEN
A membrane hyaluronate-binding protein from cerebral cortex of human embryonic brain (22-24 weeks) was purified by affinity, ion exchange chromatographies and gel-filtration. While gel-filtration analysis the protein had Mm 250 kDa. Electrophoresis under reduction conditions in the presence of DS-Na revealed a major band with Mm 85 kDa and two minor binds with Mm 68 and 36 kDa. The isolated protein did not react with antibodies against known hyaluronate-binding and other proteins with similar mass. The results show that a new membrane hyaluronate-binding protein was isolated and purified from human embryonic brain.
Asunto(s)
Corteza Cerebral/metabolismo , Proteínas Fetales/metabolismo , Ácido Hialurónico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Corteza Cerebral/embriología , Proteínas Fetales/aislamiento & purificación , Humanos , Proteínas de la Membrana/aislamiento & purificación , Proteínas del Tejido Nervioso/aislamiento & purificación , Unión ProteicaRESUMEN
The adhesion molecule BEN/SC1/DM-GRASP (BEN) is a marker in the developing chicken nervous system that is also expressed on the surface of embryonic and adult hematopoietic cells such as immature thymocytes, myeloid progenitors, and erythroid progenitors. F84.1 and KG-CAM, two monoclonal antibodies to rat neuronal glycoproteins with similarity to BEN, cross-react with an antigen on rat hematopoietic progenitors, but F84.1 only also recognizes human blood cell progenitors. We have defined the antigen recognized by F84.1 as the hematopoietic cell antigen (HCA). HCA expression was detected on 40% to 70% of CD34+ fetal and adult bone marrow cells and mobilized peripheral blood cells. Precursor cell activity for long-term in vitro bone marrow cell culture was confined to the subset of CD34+ cells that coexpress HCA. HCA is expressed by the most primitive subsets of CD34+ cells, including all rhodamine 123(lo), Thy-1+, and CD38(-/lo) CD34+ adult bone marrow cells. HCA was also detected on myeloid progenitors but not on early B-cell progenitors. We also describe here the cloning and characterization of cDNAs encoding two variants of the human HCA antigen (huHCA-1 and huHCA-2) and of a cDNA clone encoding rat HCA (raHCA). The deduced amino acid sequences of huHCA and raHCA are homologous to that of chicken BEN. Recombinant proteins produced from either human or rat HCA cDNAs were recognized by F84.1, whereas rat HCA but not human HCA was recognized by antirat KG-CAM. Expression of either form of huHCA in CHO cells conferred homophilic adhesion that could be competed with soluble recombinant huHCA-Fc. The molecular cloning of HCA and the availability of recombinant HCA should permit further evaluation of its role in human and rodent hematopoiesis.
Asunto(s)
Antígenos de Superficie/aislamiento & purificación , Células de la Médula Ósea , Moléculas de Adhesión Celular Neuronal , Células Madre Hematopoyéticas/inmunología , Adulto , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD , Antígenos CD34/análisis , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/genética , Médula Ósea/embriología , Pollos , Clonación Molecular , Secuencia de Consenso , ADN Complementario/genética , Proteínas Fetales/biosíntesis , Proteínas Fetales/genética , Proteínas Fetales/aislamiento & purificación , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Neuronas/inmunología , Neuronas/metabolismo , Especificidad de Órganos , Ratas , Proteínas Recombinantes de Fusión/inmunología , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Several peptide growth factors, including members of the fibroblast growth factor (FGF) superfamily, are potential inducers of mesoderm in vertebrates. Receptor binding of basic FGF (FGF-2) is promoted by cell surface or extracellular matrix proteoglycans. The substantial biosynthesis of proteoglycans by embryonic cells (called embryoglycans) and their potential role as ligands for growth factor receptors led us to examine the role of embryoglycans that carry the developmentally regulated oligosaccharide epitope TEC 1, in the binding of FGF-2 to cultured rabbit inner cell masses (ICMs). Culture of isolated ICMs in the presence of FGF-2 gave rise to well delimited colonies with migrating cells at the periphery. In these cells, TEC 1 staining shifts from a punctate pattern over the entire membrane, to an apical, finely granular distribution with some internalization. This shift occurs after 96 hours in culture. Here we show that: (1) migrating cells are mesoderm-like in phenotype; (2) antibodies against TEC 1 blocked FGF-2 mediated differentiation in vitro; (3) antibodies against TEC 1 selectively blocked binding of FGF-2 to ectodermal receptors and, vice versa, the binding of TEC 1-specific antibodies to ectodermal cells can be competed by excess FGF-2; (4) the same switch in TEC 1 staining patterns was observed in vivo, between the day 7 and the day 9 rabbit embryo. These data suggest the involvement of defined species of embryonic cell surface epitopes in the regulation of FGF-2 receptor binding. Moreover, this proposed binding activity is temporally restricted to ectodermal cells and disappears early during differentiation. Thus, the apical TEC 1 redistribution can be considered as the earliest indicator of mesoderm formation.
Asunto(s)
Inducción Embrionaria , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Mesodermo/fisiología , Oligosacáridos/metabolismo , Proteoglicanos/metabolismo , Proteínas de Dominio T Box , Animales , Variación Antigénica , Antígenos de Diferenciación , Blastocisto , Proteínas de Unión al ADN/aislamiento & purificación , Ectodermo/efectos de los fármacos , Epítopos , Proteínas Fetales/aislamiento & purificación , Técnica del Anticuerpo Fluorescente , Oligosacáridos/inmunología , Unión Proteica/efectos de los fármacos , Proteoglicanos/inmunología , Conejos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Vimentina/aislamiento & purificaciónRESUMEN
By screening with monoclonal antibodies (mAbs) raised against growth cone membrane fraction from fetal porcine brains, we have identified a 230 kDa antigen, termed p230. Western blot analysis of extracts from various tissues demonstrated that p230 is specifically expressed in brains, in which its expression is temporally restricted; it was especially prominent in the embryonic and the early postnatal stage, and decreased to subdetectable levels in the adult brain. Further characterization of p230 revealed that it is a peripherally-membrane associated, cell surface protein produced by astrocytes. Neurite outgrowth of E18 rat cerebral cortex neurons cultured on a monolayer of astrocytes was significantly reduced in the presence of anti-p230 polyclonal antibody. Partial amino acid sequences of p230 purified from fetal porcine brains were highly homologous to an extracellular matrix protein, tenascin-C. These lines of evidence suggest that p230, a tenascin-C-like molecule present in fetal porcine brains, plays important roles during early brain development, particularly in growth cone guidance.
Asunto(s)
Astrocitos/citología , Encéfalo/citología , Encéfalo/metabolismo , Corteza Cerebral/citología , Proteínas Fetales/aislamiento & purificación , Proteínas Fetales/fisiología , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/fisiología , Neuritas/fisiología , Neuronas/citología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Astrocitos/metabolismo , Química Encefálica , Células Cultivadas , Corteza Cerebral/metabolismo , Embrión de Mamíferos , Proteínas Fetales/biosíntesis , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Neuritas/metabolismo , Neuronas/metabolismo , Ratas , Ratas Wistar , Sensibilidad y Especificidad , Homología de Secuencia de Aminoácido , Porcinos , Tenascina/genética , Tenascina/metabolismoRESUMEN
We have used a functional assay to identify a putative T-box transcription factor (Xombi) that has the ability to induce sites of invagination in the ectoderm of Xenopus embryos that resemble the blastopore lip. Maternal Xombi transcript is first localized to the oocyte's vegetal cortex and cytoplasm, early sources of mesoderm and endoderm-inducing signals. Soon after zygotic transcription begins, there is a wave of Xombi expression, beginning in dorsal mesoderm and then extending to lateral and ventral mesoderm, that precedes and parallels blastopore lip formation at the border between the mesoderm and endoderm. Transcripts encoding brachyury, Xwnt8 and goosecoid colocalize with Xombi transcripts within the marginal zone; ectopic expression of Xombi induces expression of all three mesodermal genes. In ectodermal explants, Xombi expression is induced by the secreted mesoderm inducers activinA, activinB, Xnrl and eFGF, and by brachyury, another Xenopus T-box containing gene. The time course and location of Xombi expression, its biological activities and the partial dependence of Xombi expression and blastopore lip formation on fibroblast growth factor (FGF) signaling suggest that Xombi contributes to a traveling wave of morphogenesis and differentiation during Xenopus gastrulation.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas de Unión al ADN/genética , Inducción Embrionaria/genética , Gástrula , Proteínas de Homeodominio , Mesodermo , Proteínas Represoras , Proteínas de Dominio T Box , Factores de Transcripción/genética , Proteínas de Xenopus , Proteínas de Pez Cebra , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Ectodermo/metabolismo , Proteínas Fetales/aislamiento & purificación , Factores de Crecimiento de Fibroblastos/metabolismo , Proteína Goosecoide , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Unión Proteica , Proteínas Proto-Oncogénicas/aislamiento & purificación , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/metabolismo , Proteínas Wnt , Xenopus/embriologíaRESUMEN
We have cloned the gene for a novel Ets-related transcription factor, new Ets-related factor (NERF), from human spleen, fetal liver, and brain. Comparison of the deduced amino acid sequence of NERF with those of other members of the Ets family reveals that the level of homology to ELF-1, which is involved in the regulation of several T- and B-cell-specific genes, is highest. Homologies are clustered in the putative DNA binding domain in the middle of the protein, a basic domain just upstream of this domain, and several shorter stretches of homology towards the amino terminus. The presence of two predominant NERF transcripts in various fetal and adult human tissues is due to at least three alternative splice products, NERF-1a, NERF-1b, and NERF-2, which differ in their amino termini and their expression in different tissues. Only NERF-2 and ELF-1, and not NERF-1a and NERF-1b, function as transcriptional activators of the lyn and blk gene promoters, although all isoforms of NERF bind with affinities similar to those of ELF-1 to a variety of Ets binding sites in, among others, the blk, lck, lyn, mb-1, and immunoglobulin H genes and are expressed at similar levels. Since NERF and ELF-1 are coexpressed in B and T cells, both might be involved in the regulation of the same genes.
Asunto(s)
Proteínas de Unión al ADN/química , Genes , Factores de Transcripción/química , Factores de Transcripción/aislamiento & purificación , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Línea Celular Transformada , Chlorocebus aethiops , Clonación Molecular , Proteínas Fetales/aislamiento & purificación , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Células HeLa/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Nucleares , Especificidad de Órganos , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Secuencias Reguladoras de Ácidos Nucleicos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Activación Transcripcional , Familia-src Quinasas/genéticaRESUMEN
The mouse Brachyury gene (T) is required in notochord differentiation and posterior mesoderm formation during axial development. We have isolated the chick homologue of T(Ch-T) and determined its putative protein sequence and expression pattern during embryogenesis. Ch-T is expressed in the epiblast close to and within the primitive streak, in early migrating mesoderm and in the notochord. In later stages Ch-T expression is found in the tail bud and in the entire notochord. The notochord expression ceases in an anterior-posterior wave when the formation of the body anlage is completed. This pattern is consistent with those reported for the expression of the mouse T gene and the T homologues of Xenopus laevis and zebrafish, suggesting that the mechanisms of embryonic pattern formation are highly conserved in all vertebrates. The N-terminal half of Ch-T shows a very high degree of sequence identity with the corresponding region of mouse T which has DNA-binding activity, and with the N-terminal half of Xenopus (Xbra) and zebrafish (Ntl) T protein. Finally, we have analyzed the effects of activin A on Ch-T induction and axis formation. Localized activin A treatment of prestreak blastoderms results in ectopic Ch-T expression that correlates with formation of second primitive streaks or with repositioning of the site of single streak origin (Cooke et al., 1994). These results strengthen the previous evidence that Brachyury activation is an early response to axis-inducing signals in vivo.
Asunto(s)
Proteínas de Unión al ADN/genética , Embrión no Mamífero/metabolismo , Proteínas Fetales/genética , Inhibinas/farmacología , Proteínas de Dominio T Box , Activinas , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas Fetales/biosíntesis , Proteínas Fetales/aislamiento & purificación , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Ratones , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
It was observed in the course of other studies that rat fetal lung extracts inhibited proliferation of fetal lung cells in culture. The purpose of the present study was to isolate and characterize this cytostatic factor. It was found that fetal lungs contained a 16 kDa cytostatic factor and its concentration was twofold greater in fetal lungs of diabetic rats compared with control rats. This fetal lung cytostatic protein (FLCP) was purified by reversed-phase, heparin-affinity and gel filtration high-performance liquid chromatography and SDS-PAGE. The purified protein was electroblotted onto polyvinylidene difluoride membrane and subjected to sequence analysis. The amino-terminal sequence of this fetal lung cytostatic protein was P E P A K S A P A P X K G I G K Q X X K A X X K A ... and showed significant homology with histone H2B; however, the amino acid composition of FLCP suggested that it may be structurally distinct from histone H2B. Ion-spray mass spectrometry suggested that FLCP was made up of at least two species of the protein with molecular weights of 13,776.1 and 14,007.3 and was different from the molecular weight of rat histone H2B predicted by its cDNA sequence. The concentration of FLCP, based on amino acid compositions, was 0.32 nmol/g and 0.83 nmol/g wet fetal lung from non-diabetic and diabetic rats respectively. These findings suggest that the fetal rat lung produces a regulatory factor bearing considerable homology with but possibly different from histone H2B and that fetal lung immaturity during diabetic pregnancy might be contributed to by an increase in this factor.
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Diabetes Mellitus Experimental/metabolismo , Proteínas Fetales/aislamiento & purificación , Pulmón/embriología , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Diabetes Mellitus Experimental/embriología , Electroforesis en Gel de Poliacrilamida , Proteínas Fetales/genética , Histonas/genética , Pulmón/química , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de AminoácidoRESUMEN
Mutations at the mouse limb deformity (ld) locus result in defects of growth and patterning of the limb and kidney during embryonic development. The gene responsible for this phenotype is large and complex, with the capacity to generate a number of alternatively spliced messenger RNA transcripts encoding nuclear protein isoforms called "formins." We have made polyclonal antibodies to specific formin peptides and have confirmed the authenticity of the antibodies' reactivity, using cell lines derived from mice with molecularly defined mutations at the ld locus. In addition, we have used these antibodies to detect and characterize polypeptides encoded by both wild-type and mutant ld alleles. In so doing, we show that a formin isoform (i) is modified by posttranslational phosphorylation at serine and threonine residues and (ii) when present in a crude nuclear extract, is retained by DNA-cellulose.
Asunto(s)
Proteínas Fetales/biosíntesis , Deformidades Congénitas de las Extremidades , Proteínas Nucleares/biosíntesis , Fosfoproteínas/biosíntesis , Células 3T3 , Alelos , Animales , Línea Celular , Cromatografía de Afinidad , ADN , Electroforesis en Gel de Poliacrilamida , Extremidades/embriología , Proteínas Fetales/genética , Proteínas Fetales/aislamiento & purificación , Forminas , Heterocigoto , Homocigoto , Riñón/anomalías , Riñón/embriología , Ratones , Ratones Mutantes , Proteínas de Microfilamentos , Peso Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Biosíntesis de Proteínas , Transcripción Genética , TransfecciónRESUMEN
Chromosomal mosaicism in chorionic villus samples (CVS) may arise from different sources, such as clonal diversity within the chorionic tissue or contamination with maternal cells. To determine the origin of karyotyped cells, we compared the immunocytochemical features of mitotic cells in CVS long-term cultures with histological sections of their tissue of origin, i.e. chorionic villi. Immunolabelling of intermediate filaments specific for epithelial cells (cytokeratin) and mesenchymal cells (vimentin) established that mitoses yielded from CVS long-term cultures indeed stem from independently growing clones derived from both the epithelial and mesenchymal parts of the chorionic villi. Thus, mosaicism in CVS cultures may reflect true genetic heterogeneity within the biopsy. However, epithelial chorionic cells undergo in vitro metaplasia leading to co-expression of cytokeratins and vimentin. Fetal-specific immune markers (beta-HCG and SP1-glycoprotein) are not reliably expressed in CVS cell culture.