RESUMEN
ADAMTS-2 is a metalloproteinase that plays a key role in the processing of fibrillar procollagen precursors into mature collagen molecules by excising the amino-propeptide. We demonstrate that recombinant ADAMTS-2 is also able to reduce proliferation of endothelial cells, and to induce their retraction and detachment from the substrate resulting in apoptosis. Dephosphorylation of Erk1/2 and MLC largely precedes the ADAMTS-2 induced morphological alterations. In 3-D culture models, ADAMTS-2 strongly reduced branching of capillary-like structures formed by endothelial cells and their long-term maintenance and inhibited vessels formation in embryoid bodies (EB). Growth and vascularization of tumors formed in nude mice by HEK 293-EBNA cells expressing ADAMTS-2 were drastically reduced. A similar anti-tumoral activity was observed when using cells expressing recombinant deleted forms of ADAMTS-2, including catalytically inactive enzyme. Nucleolin, a nuclear protein also found to be associated with the cell membrane, was identified as a potential receptor mediating the antiangiogenic properties of ADAMTS-2.
Asunto(s)
Proteínas ADAM/metabolismo , Inhibidores de la Angiogénesis/metabolismo , Neoplasias/metabolismo , Neovascularización Patológica , Procolágeno N-Endopeptidasa/metabolismo , Proteínas ADAM/genética , Proteínas ADAMTS , Proteína ADAMTS4 , Animales , Apoptosis/fisiología , Bovinos , Línea Celular , Proliferación Celular , Cuerpos Embrioides/metabolismo , Células Endoteliales/citología , Células Endoteliales/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Ratones , Ratones Noqueados , Ratones Desnudos , Neoplasias/patología , Neoplasias Experimentales , Procolágeno N-Endopeptidasa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiologíaRESUMEN
In mice, the Nkx6 genes are crucial to alpha- and beta-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in zebrafish, nkx6.1 and nkx6.2 are co-expressed at early stages in the first pancreatic endocrine progenitors, but that their expression domains gradually segregate into different layers, nkx6.1 being expressed ventrally with respect to the forming islet while nkx6.2 is expressed mainly in beta-cells. Knockdown of nkx6.2 or nkx6.1 expression leads to nearly complete loss of alpha-cells but has no effect on beta-, delta-, or epsilon-cells. In contrast, nkx6.1/nkx6.2 double knockdown leads additionally to a drastic reduction of beta-cells. Synergy between the effects of nkx6.1 and nkx6.2 knockdown on both beta- and alpha-cell differentiation suggests that nkx6.1 and nkx6.2 have the same biological activity, the required total nkx6 threshold being higher for alpha-cell than for beta-cell differentiation. Finally, we demonstrate that the nkx6 act on the establishment of the pancreatic endocrine progenitor pool whose size is correlated with the total nkx6 expression level. On the basis of our data, we propose a model in which nkx6.1 and nkx6.2, by allowing the establishment of the endocrine progenitor pool, control alpha- and beta-cell differentiation.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Islotes Pancreáticos/fisiología , Células Madre/citología , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Proteínas de Homeodominio/genética , Hibridación in Situ , Islotes Pancreáticos/citología , Microinyecciones , Oligonucleótidos Antisentido/farmacología , Factores de Transcripción/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Calsenilin/DREAM/Kchip3 is a neuronal calcium-binding protein. It is a multifunctional protein, mainly expressed in neural tissues and implicated in regulation of presenilin processing, repression of transcription, and modulation of A-type potassium channels. Here, we performed a search for new genes expressed during pancreatic development and have studied the spatiotemporal expression pattern and possible role of calsenilin in pancreatic development in zebrafish. We detected calsenilin transcripts in the pancreas from 21 somites to 39 hours postfertilization stages. Using double in situ hybridization, we found that the calsenilin gene was expressed in pancreatic endocrine cells. Loss-of-function experiments with anti-calsenilin morpholinos demonstrated that injected morphants have a significant decrease in the number of pancreatic endocrine cells. Furthermore, the knockdown of calsenilin leads to perturbation in islet morphogenesis, suggesting that calsenilin is required for early islet cell migration. Taken together, our results show that zebrafish calsenilin is involved in endocrine cell differentiation and morphogenesis within the pancreas.
Asunto(s)
Sistema Endocrino/embriología , Sistema Endocrino/metabolismo , Proteínas de Interacción con los Canales Kv/metabolismo , Páncreas/embriología , Páncreas/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Interacción con los Canales Kv/genética , Mutación/genética , Hormonas Pancreáticas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Tretinoina/metabolismo , Pez Cebra/genéticaRESUMEN
Dopamine (DA) facilitates male sexual behavior and modulates aromatase activity in the quail preoptic area (POA). Aromatase neurons in the POA receive dopaminergic inputs, but the anatomical substrate that mediates the behavioral and endocrine effects of DA is poorly understood. Intracellular recordings showed that 100 microm DA hyperpolarizes most neurons in the medial preoptic nucleus (80%) by a direct effect, but depolarizes a few others (10%). DA-induced hyperpolarizations were not blocked by D1 or D2 antagonists (SCH-23390 and sulpiride). Extracellular recordings confirmed that DA inhibits the firing of most cells (52%) but excites a few others (24%). These effects also were not affected by DA antagonists (SCH-23390 and sulpiride) but were blocked by alpha2-(yohimbine) and alpha1-(prazosin) noradrenergic receptor antagonists, respectively. Two dopamine-beta-hydroxylase (DBH) inhibitors (cysteine and fusaric acid) did not block the DA-induced effects, indicating that DA is not converted into norepinephrine (NE) to produce its effects. The pK(B) of yohimbine for the receptor involved in the DA- and NE-induced inhibitions was similar, indicating that the two monoamines interact with the same receptor. Together, these results demonstrate that the effects of DA in the POA are mediated mostly by the activation of alpha2 (inhibition) and alpha1 (excitation) adrenoreceptors. This may explain why DA affects the expression of male sexual behavior through its action in the POA, which contains high densities of alpha2-noradrenergic but limited amounts of DA receptors. This study thus clearly demonstrates the existence of a cross talk within CNS catecholaminergic systems between a neurotransmitter and heterologous receptors.
Asunto(s)
Dopamina/farmacología , Área Preóptica/fisiología , Receptores Adrenérgicos/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Antagonistas Adrenérgicos/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Coturnix , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Dopamina beta-Hidroxilasa/metabolismo , Antagonistas del GABA/farmacología , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microelectrodos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Norepinefrina/farmacología , Área Preóptica/efectos de los fármacos , Área Preóptica/metabolismo , Receptores Adrenérgicos/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Tetrodotoxina/farmacología , Yohimbina/farmacocinéticaRESUMEN
Retinoids play important roles in diverse cellular processes including growth, cell differentiation and vision. Many natural and synthetic retinoids are used as drugs in dermatology and oncology. A large amount of data has been accumulated on the cellular activity of different synthetic retinoids. They are stabilized and transported inside the cell cytoplasm by binding and transport proteins, such as cellular retinol-binding proteins and cellular retinoic acid binding proteins (CRABPs). The structures of human CRABP II in complex with two different synthetic retinoids, Ro13-6307 and Ro12--7310 (at 2.1 and 2.0 A resolution, respectively) and of bovine CRABP I in complex with a retinobenzoic acid, Am80 (at 2.8 A resolution) are described. The binding affinities of human CRABP I and II for the retinoids studied here have been determined. All these compounds have comparable binding affinities (nanomolar range) for both CRABPs. Apart from the particular interactions of the carboxylate group of the retinoids with specific protein groups, each structure reveals characteristic interactions. Studying the atomic details of the interaction of retinoids with retinoid-binding proteins facilitates the understanding of the kinetics of retinoid trafficking inside the cytoplasm.
Asunto(s)
Receptores de Ácido Retinoico/química , Retinoides/química , Animales , Benzoatos/química , Bovinos , Cristalografía por Rayos X , Etretinato/análogos & derivados , Etretinato/química , Ácidos Grasos Insaturados/química , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Unión Proteica , Proteínas Recombinantes/química , Tetrahidronaftalenos/químicaRESUMEN
We report the discovery of an Antirrhinum MADS-box gene, FARINELLI (FAR), and the isolation of far mutants by a reverse genetic screen. Despite striking similarities between FAR and the class C MADS-box gene PLENA (PLE), the phenotypes of their respective mutants are dramatically different. Unlike ple mutants, which show homeotic conversion of reproductive organs to perianth organs and a loss of floral determinacy, far mutants have normal flowers which are partially male-sterile. Expression studies of PLE and FAR, in wild-type and mutant backgrounds, show complex interactions between the two genes. Double mutant analysis reveals an unexpected, redundant negative control over the B-function MADS-box genes. This feature of the two Antirrhinum C-function-like genes is markedly different from the control of the inner boundary of the B-function expression domain in Arabidopsis, and we propose and discuss a model to account for these differences. The difference in phenotypes of mutants in two highly related genes illustrates the importance of the position within the regulatory network in determining gene function.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Estructuras de las Plantas/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Clonación Molecular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Fertilidad , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox/genética , Genes de Plantas/genética , Genes de Plantas/fisiología , Proteínas de Dominio MADS , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación/genética , Fenotipo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estructuras de las Plantas/genética , Estructuras de las Plantas/fisiología , Plantas Modificadas Genéticamente , Plantas Tóxicas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Nicotiana/genética , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
The identity and developmental pattern of the four organ types constituting the flower is governed by three developmental functions, A, B and C, which are defined by homeotic genes and established in two adjacent whorls. In this report we morphologically and genetically characterise mutants of two genes, STYLOSA (STY) and FISTULATA (FIS) which control floral homeotic meristem- and organ-identity genes and developmental events in all floral whorls. The morphology of the reproductive organs in the first and second whorls of sty fis double mutant flowers indicate that the two genes are part of the mechanism to prevent ectopic expression of the C-function in the perianth of wild-type flowers. This is verified by the detection of the expansion of the expression domain of the class C gene PLENA (PLE) towards the perianth. Interestingly, in the second whorl of sty and fis mutants, spatial differences in stamenoid features and in the pattern of ectopic expression of the PLE gene were observed. This suggests that, with respect to the negative control of PLE, petals are composed of two regions, a lateral and a central one. Mutation in ple is epistatic to most of the sty/fis-related homeotic defects. PLE, however, is not the primary target of STY/FIS control, because dramatic reduction of expression of FIMBRIATA, meristem identity genes (FLORICAULA and SQUAMOSA) and of class B organ identity genes (GLOBOSA) occur before changes in the PLE expression pattern. We propose that STY/FIS are hierarchically high-ranking genes that control cadastral component(s) of the A-function. SQUAMOSA as a potential target of this control is discussed. Retarded growth of second whorl organs, subdivision of third whorl primordia and the failure to initiate them in sty/fis mutants may be mediated by the FIMBRIATA gene.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Genes Homeobox/genética , Genes de Plantas/genética , Proteínas de Dominio MADS , Desarrollo de la Planta , Regulación del Desarrollo de la Expresión Génica/genética , Genotipo , Proteínas de Homeodominio/genética , Hibridación in Situ , Meristema/genética , Microscopía Electrónica de Rastreo , Morfogénesis , Mutación , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Fenotipo , Proteínas de Plantas/genética , Plantas/genética , ARN Mensajero/análisisRESUMEN
Regulatory mechanisms controlling basic aspects of floral morphogenesis seem to be highly conserved among plant species. The class B organ identity genes, which are required to establish the identity of organs in the second (petals) and third (stamens) floral whorls, are a good example of such conservation. This work compares the function of two similar class B genes in the same genetic background. The DEFICIENS (DEF) gene from Antirrhinum, including its promoter, was transformed into Arabidopsis and compared in function and expression with the Arabidopsis class B genes APETALA3 (AP3) and PISTILLATA (PI). The DEF gene was expressed in the second, third, and fourth whorls, as was PI. Functionally, DEF could replace AP3 in making petals and stamens. The DEF gene's AP3-like function and PI-like expression caused transformation of fourth-whorl carpels to stamens. Like AP3, all aspects of DEF function in Arabidopsis required a functional PI protein. Surprisingly, DEF could not replace the AP3 protein in properly maintaining AP3 transcripts (autoregulation). Our data allow us to revise the current model for class B autoregulation and propose a hypothesis for the evolution of class B gene expression in dicotyledonous plants.
Asunto(s)
Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Arabidopsis/ultraestructura , Microscopía Electrónica de Rastreo , Fenotipo , Plantas Modificadas Genéticamente , ARN Mensajero/genéticaRESUMEN
Flowers of the temperature-sensitive DEFICIENS (DEF) mutant, def-101, display sepaloid petals and carpelloid stamens when grown at 26 degrees C, the non-permissive temperature. In contrast, when cultivated under permissive conditions at 15 degrees C, the morphology of def-101 flowers resembles that of the wild type. Temperature shift experiments during early and late phases of flower development revealed that second and third whorl organ development is differentially sensitive to changes in DEF expression. In addition, early DEF expression seems to control the spatially correct initiation of fourth whorl organ development. Reduction of the def-101 gene dosage differentially affects organogenesis in adjacent whorls: at the lower temperature development of petals in the second whorl and initiation of carpels in the centre of the flower is not affected while third whorl organogenesis follows the mutant (carpelloid) pattern. The possible contribution of accessory factors to organ-specific DEF functions is discussed. In situ analyses of mRNA and protein expression patterns during def-101 flower development at 15 degrees C and at 26 degrees C support previously proposed combinatorial regulatory interactions between the MADS-box proteins DEF and GLOBOSA (GLO), and provide evidence that the autoregulatory control of DEF and GLO expression by the DEF/GLO heterodimer starts after initiation of all organ primordia. Immunolocalisation revealed that both proteins are located in the nucleus. Interestingly, higher growth temperature affects the stability of both the DEF-101 and GLO proteins in vivo. In vitro DNA binding studies suggest that the temperature sensitivity of the def-101 mutant is due to an altered heterodimerisation/DNA-binding capability of the DEF-101 protein, conditioned by the deletion of one amino acid within the K-box, a protein region thought to be involved in protein-protein interaction. In addition, we introduce a mutant allele of GLO, glo-confusa, where insertion of one amino acid impairs the hydrophobic carboxy-terminal region of the MADS-box, but which confers no strong phenotypic changes to the flower. The strong mutant phenotype of flowers of def-101/glo-conf double mutants when grown in the cold represents genetic evidence for heterodimerisation between DEF and GLO in vivo. The potential to dissect structural and functional domains of MADS-box transcription factors is discussed.
Asunto(s)
Genes Homeobox , Genes de Plantas , Plantas/genética , Secuencia de Bases , Expresión Génica , Técnicas In Vitro , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Mutación , Desarrollo de la Planta , Plantas/anatomía & histologíaRESUMEN
BACKGROUND: Retinoic acid (RA) plays a fundamental role in diverse cellular activities. Cellular RA binding proteins (CRABPs) are thought to act by modulating the amount of RA available to nuclear RA receptors. CRABPs and cellular retinol-binding proteins (CRBPs) share a unique fold of two orthogonal beta-sheets that encapsulate their ligands. It has been suggested that a trio of residues are the prime determinants defining the high specificity of CRBPs and CRABPs for their physiological ligands. RESULTS: Bovine/murine CRABP I and human CRABP II have been crystallized in complex with their natural ligand, all-trans-RA. Human CRABP II has also been crystallized in complex with a synthetic retinoid, 'compound 19'. Their structures have been determined and refined at resolutions of 2.9 A, 1.8 A and 2.2 A, respectively. CONCLUSIONS: The retinoid-binding site in CRABPs differs significantly from that observed in CRBP. Structural changes in three juxtaposed areas of the protein create a new, displaced binding site for RA. The carboxylate of the ligand interacts with the expected trio of residues (Arg132, Tyr134 and Arg111; CRABP II numbering). The RA ligand is almost flat with the beta-ionone ring showing a significant deviation (-33 degrees) from a cis conformation relative to the isoprene tail. The edge atoms of the beta-ionone ring are accessible to solvent in a suitable orientation for presentation to metabolizing enzymes. The bulkier synthetic retinoid causes small conformational changes in the protein structure.
Asunto(s)
Receptores de Ácido Retinoico/química , Retinoides/química , Tretinoina/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
The expression of the Antirrhinum gene FIL2 is affected in mutants of the homeotic transcription factor DEFICIENS. Northern and Western blot analyses showed that FIL2 in wild-type Antirrhinum flowers is expressed weakly in the petals and more abundantly in the reproductive organs; the gene is active in the filaments and anthers of stamens, and in the stigma and transmitting tissue of the carpels. The FIL2 protein is glycosylated with high mannose type glycan chains and is located in the middle lamella of the extracellular matrix. The amino acid sequence contains 10 tandem repeats, the composition of which is similar to the Leucine-Rich Repeat (LRR) motif found in mammals, Drosophila and yeast. The possibility that FIL2 might be a component of a cellular signalling mechanism, involving LRR-mediated protein-protein interactions is discussed.
Asunto(s)
Leucina/análisis , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario , Genes de Plantas , Inmunohistoquímica , Datos de Secuencia Molecular , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/química , Plantas , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo , Transcripción GenéticaRESUMEN
GLOBOSA (GLO) is a homeotic gene whose mutants show sepaloid petals and carpelloid stamens. The similarity of Glo mutants to those of the DEFICIENS (DEFA) gene suggests that the two genes have comparable functions in floral morphogenesis. The GLO cDNA has been cloned by virtue of its homology to the MADS-box, a conserved DNA-binding domain also contained in the DEFA gene. We have determined the structure of the wild type GLO gene as well as of several glo mutant alleles which contain transposable element insertions responsible for somatic and germinal instability of Glo mutants. Analyses of the temporal and spatial expression patterns of the DEFA and GLO genes during development of wild type flowers and in flowers of various stable and unstable defA and glo alleles indicate independent induction of DEFA and GLO transcription. In contrast, organ-specific up-regulation of the two genes in petals and stamens depends on expression of both DEFA and GLO. In vitro DNA-binding studies were used to demonstrate that the DEFA and GLO proteins specifically bind, as a heterodimer, to motifs in the promoters of both genes. A model is presented which proposes both combinatorial and cross-regulatory interactions between the DEFA and GLO genes during petal and stamen organogenesis in the second and third whorls of the flower. The function of the two genes controlling determinate growth of the floral meristem is also discussed.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes Homeobox , Proteínas de Homeodominio/genética , Proteínas de Plantas/genética , Plantas/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN , Expresión Génica , Genes de Plantas , Hibridación in Situ , Datos de Secuencia Molecular , Mutación , Desarrollo de la Planta , Especificidad de la Especie , Transcripción GenéticaRESUMEN
UNLABELLED: Two serum calcitonin assays with sensitivities < or = 10 pg/mL were compared to our standard radioimmunoassay (sensitivity 100 pg/mL) in multiple endocrine neoplasia type 2A (MEN 2A) screening. Values from the Nichols displacement radioimmunoassay averaged 38% higher than values from the CIS immunoradiometric assay; values from both were highly correlated, r = 0.845. In three individuals, both of the newer assays revealed abnormalities in pentagastrin tests three to four years before abnormalities were detected by the standard assay. Pentagastrin tests after total thyroidectomy were assayed by the newer methods in patients with medullary thyroid carcinoma (MTC) diagnosed at initial testing (group I); in patients with early MTC diagnosed by prospective screening (group II); and in patients with pure C-cell hyperplasia detected by prospective screening (group III). At least 64% of group I, at least 25% of group II, but none of group III had detectable postoperative C-cell function. CONCLUSIONS: 1) The previous estimate of 12 years as median age of onset of C-cell disease in MEN 2A is probably three to four years too old. 2) Patients diagnosed with early MTC by screening had not necessarily skipped a preneoplastic phase of C-cell hyperplasias. At least some early disease was not detected by the standard assay. Higher sensitivity assay should improve screening for C-cell disease by earlier disease detection. 3) Biochemical cure by thyroidectomy after the development of MTC is not as frequent as previously thought, but the apparent cure rate of pure C-cell hyperplasia remains 100%.
Asunto(s)
Calcitonina/sangre , Neoplasia Endocrina Múltiple/complicaciones , Enfermedades de la Tiroides/diagnóstico , Adolescente , Niño , Femenino , Humanos , Hiperplasia , Masculino , Radioinmunoensayo , Sensibilidad y Especificidad , Enfermedades de la Tiroides/complicaciones , Enfermedades de la Tiroides/patología , Glándula Tiroides/patologíaRESUMEN
We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.
Asunto(s)
Cromatina/ultraestructura , ADN/metabolismo , Plantas/metabolismo , Ribosomas/metabolismo , Inmunohistoquímica , Cariotipificación , Microscopía Electrónica , Conformación de Ácido Nucleico , Ribosomas/ultraestructuraRESUMEN
A basic phosphoprotein defined by a monoclonal antibody named AF5 was found to be highly abundant in human hepatocellular carcinoma by Western immunoblotting. Under the same conditions, the levels of this phosphoprotein were low or undetectable in normal liver extracts. The AF5 antibody was used to screen a cDNA expression library of a human hepatoma cell line named FOCUS. A 960-base-pair cDNA was isolated and found to be a partial cDNA encoding the human protein-tyrosine kinase substrate p36, also known as lipocortin II. p36 expression was highly abundant in hepatocellular carcinomas at both the transcript and protein levels. Its expression was not induced significantly during rat liver regeneration following a partial hepatectomy. These results suggest that the induction of p36 expression is associated with malignant transformation of hepatocytes. p36 was previously shown to be phosphorylated upon transformation of normal fibroblasts by retroviral oncogenes without significant modulation of expression. We report here the initial description of the association of increased p36 expression with malignant transformation.
Asunto(s)
Fosfoproteínas/biosíntesis , Proteínas Quinasas/metabolismo , Células Tumorales Cultivadas/metabolismo , Anticuerpos Monoclonales , Northern Blotting , Western Blotting , Carcinoma Hepatocelular , Línea Celular , Clonación Molecular , Biblioteca de Genes , Humanos , Técnicas para Inmunoenzimas , Hígado/metabolismo , Neoplasias Hepáticas , Mapeo Peptídico , Fosfoproteínas/genética , Especificidad por SustratoRESUMEN
Depending on the author and the animal or plant origin of the material under study, the term "nucleolonema" is used in different contexts and thus indicates nucleolar ultrastructures that are different. In this paper, we attempt to clarify this state of affairs and to propose a definition for the plant cell nucleolonema.
Asunto(s)
Región Organizadora del Nucléolo/ultraestructura , Plantas/ultraestructura , Animales , ADN Ribosómico/ultraestructura , Especificidad de la EspecieRESUMEN
Previous studies have suggested that molecular species larger than the mature calcitonin (CT) are produced by tumors of different origin. In order to study these species, we developed a monoclonal immunoradiometric assay for calcitonin precursors (CT-pr). This assay was based on both monoclonal antibody KC01 directed to the 1-11 region of katacalcin and monoclonal antibody CT08 directed to the 11-17 portion of CT. The sensitivity of this monoclonal immunoradiometric assay for CT-pr was less than 100 pg/ml. Only one of 131 healthy subjects had CT-pr serum levels greater than 100 pg/ml; this value was therefore selected as the standard serum value in healthy individuals. CT-pr was present in the serum of seven of ten patients with advanced renal failure and in that of 21 of 52 patients (40%) with benign liver disease but was undetectable in sera of patients with other benign diseases. The serum CT-pr level was correlated with that of mature CT in patients with medullary carcinoma of the thyroid. In contrast, the serum CT-pr level was frequently elevated in the absence of a detectable CT level in patients with various malignant tumors and, particularly, in those with either tumors of the neuroendocrine system (60%) or hepatocellular carcinomas (62%). CT-pr was detected in tumor extract from a patient with a hepatocellular carcinoma. Moreover, hybridization experiments with total RNA extracted from this tumor demonstrated the presence of RNAs hybridizing with complementary DNA encoding for common region, calcitonin, and katacalcin sequences. These results show that CT precursors are excreted by numerous cancers and might well be useful biological markers for the follow-up of productive tumors.
Asunto(s)
Calcitonina/sangre , Neoplasias/sangre , Anticuerpos Monoclonales , Calcitonina/genética , Carcinoma Hepatocelular/genética , Carcinoma de Células Pequeñas/genética , Femenino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/genética , Neoplasias/genética , Embarazo/sangre , Precursores de Proteínas/sangre , ARN Neoplásico/genéticaRESUMEN
A Mr 50,000 cell surface protein antigen (p50) was identified on a human hepatocellular carcinoma derived cell line (FOCUS) by two monoclonal antibodies (SF 31 and SF 90). This antigen was subsequently shown to be expressed in vivo in human hepatocellular carcinoma. All 18 tumors tested by Western immunoblotting demonstrated high levels of p50 with undetectable amounts observed in the adjacent normal liver counterparts. Further characterization revealed that p50 is a monomeric polypeptide with a neutral pI (6.5-7.2) and appears not to be glycosylated. The cellular localization was determined by direct antibody binding to intact cells, immunoprecipitation of 125I-labeled cell surface proteins, and Western immunoblotting of subcellular fractions. p50 was found on the cell surface as well as in the cytoplasm. In vitro monoclonal antibody binding studies indicate that the protein is expressed in all human malignant cells (n = 34) tested thus far regardless of the embryonic tissue of origin and the degree of differentiation. p50 was present at very low levels in normal tissues with the notable exception of high expression in adrenal glands. The protein is conserved in mammalian evolution since a similar protein was also found in bovine adrenals. The molecular characteristics and the pattern of expression of p50 indicate that this normal adrenal protein is associated with the transformed phenotype.