RESUMEN
Identifying the steps involved in striatal development is important both for understanding the striatum in health and disease, and for generating protocols to differentiate striatal neurons for regenerative medicine. The most prominent neuronal subtype in the adult striatum is the medium spiny projection neuron (MSN), which constitutes more than 85% of all striatal neurons and classically expresses DARPP-32. Through a microarray study of genes expressed in the whole ganglionic eminence (WGE: the developing striatum) in the mouse, we identified the gene encoding the transcription factor Forkhead box protein P1 (FoxP1) as the most highly up-regulated gene, thus providing unbiased evidence for the association of FoxP1 with MSN development. We also describe the expression of FoxP1 in the human fetal brain over equivalent gestational stages. FoxP1 expression persisted through into adulthood in the mouse brain, where it co-localised with all striatal DARPP-32 positive projection neurons and a small population of DARPP-32 negative cells. There was no co-localisation of FoxP1 with any interneuron markers. FoxP1 was detectable in primary fetal striatal cells following dissection, culture, and transplantation into the adult lesioned striatum, demonstrating its utility as an MSN marker for transplantation studies. Furthermore, DARPP-32 expression was absent from FoxP1 knock-out mouse WGE differentiated in vitro, suggesting that FoxP1 is important for the development of DARPP-32-positive MSNs. In summary, we show that FoxP1 labels MSN precursors prior to the expression of DARPP-32 during normal development, and in addition suggest that FoxP1 labels a sub-population of MSNs that are not co-labelled by DARPP-32. We demonstrate the utility of FoxP1 to label MSNs in vitro and following neural transplantation, and show that FoxP1 is required for DARPP-32 positive MSN differentiation in vitro.
Asunto(s)
Diferenciación Celular/fisiología , Cuerpo Estriado , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Células-Madre Neurales/fisiología , Neuronas/citología , Neuronas/metabolismo , Proteínas Represoras/metabolismo , Animales , Animales Recién Nacidos , Proteínas Portadoras/metabolismo , Células Cultivadas , Cuerpo Estriado/citología , Cuerpo Estriado/embriología , Cuerpo Estriado/crecimiento & desarrollo , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Embrión de Mamíferos , Endodesoxirribonucleasas , Feto/citología , Factores de Transcripción Forkhead/genética , Técnicas In Vitro , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/trasplante , Proteínas Nucleares/metabolismo , Proteínas Represoras/genética , Estadísticas no ParamétricasRESUMEN
The purpose of this study was to evaluate the psychosocial impact of heart transplantation on spouses and compare the adjustment of spouses and their partners. Data were collected from 51 couples prior to transplant and 12 months posttransplant. The Psychosocial Adjustment to Illness Scale (PAIS) was used to measure adjustment in seven domains. Prior to the transplant, spouses experienced profound psychosocial distress related to their partner's illness. From pretransplant to posttransplant, PAIS total score decreased (lower scores indicate better adjustment) for spouses (M = 42, SD = 16, vs. M = 26, SD = 13). Spouses showed improvement in all domains. Posttransplant, patients' and spouses' PAIS total scores (overall adjustment) were not significantly different. Spouses reported more psychological distress than patients; patients reported more problems than spouses in vocational and domestic function. Study findings highlight the importance of sensitivity in the clinical setting to the distinct psychosocial needs of spouses.
Asunto(s)
Trasplante de Corazón/psicología , Esposos/psicología , Adaptación Psicológica , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The fusion of myoblasts to make multinucleate muscle fibres is central to muscle development. Recent work on Drosophila has identified two members of the immunoglobulin superfamily that have key roles in controlling the specificity of myoblast fusion.
Asunto(s)
Fusión Celular , Drosophila/embriología , Músculos/embriología , Animales , Músculos/citologíaRESUMEN
Aggregation and fusion of myoblasts to form myotubes is essential for myogenesis in many organisms. In Drosophila the formation of syncytial myotubes is seeded by founder myoblasts. Founders fuse with clusters of fusion-competent myoblasts. Here we identify the gene dumbfounded (duf) and show that it is required for myoblast aggregation and fusion. duf encodes a member of the immunoglobulin superfamily of proteins that is an attractant for fusion-competent myoblasts. It is expressed by founder cells and serves to attract clusters of myoblasts from which myotubes form by fusion.
Asunto(s)
Factores Quimiotácticos/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Proteínas del Ojo , Proteínas de Insectos/metabolismo , Proteínas de la Membrana , Proteínas Musculares , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Moléculas de Adhesión Celular Neuronal/clasificación , Agregación Celular , Fusión Celular , Factores Quimiotácticos/clasificación , Factores Quimiotácticos/genética , Factores Quimiotácticos/fisiología , ADN Complementario , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Expresión Génica , Proteínas de Insectos/clasificación , Proteínas de Insectos/genética , Proteínas de Insectos/fisiología , Mesodermo/metabolismo , Datos de Secuencia Molecular , Músculos/citología , Fenotipo , Ratas , Homología de Secuencia de Aminoácido , Células Madre/citologíaRESUMEN
It is unknown how the general patterning mechanisms that subdivide the mesoderm initiate different pathways of cell differentiation. One route to understanding these events is to isolate and analyse genes specifically expressed early in this differentiation process. I have therefore undertaken a novel molecular screen in Drosophila in a systematic search for such genes. The approach utilised subtractive hybridisation coupled to directional cDNA library construction. Libraries were made from as little as 20 microg total RNA isolated from hand-picked embryos of defined stage of development and genotype. In a one-step procedure, the subtraction was 6.5- to 7.25-h wild-type embryos minus 6.5- to 7.25-h twist (twi) zygotic mutant embryos. A two-step procedure in which maternally expressed sequences were subtracted from each of these cDNA libraries, before subtracting twi from wild-type, increased the subtraction efficiency. It resulted in a cDNA population enriched more than 100-fold for mesodermal cDNAs. This was screened by determination of the embryonic expression pattern of each clone in a high throughput procedure and then DNA sequencing. The method, which is comprehensive and does not discriminate against rarer cDNAs, is generally applicable and calculations show that it would work for just 10 embryos. Analysis of one clone, Dmeso18E, that encodes a putative nuclear protein and fulfils the screen's aims is described. It is novel and its expression is mesoderm-specific, twi-dependent, and early during somatic, visceral, and heart muscle differentiation. Two pivotal regulators of mesoderm development and gene expression are Dmef2 and tinman (tin). Analysis of Dmeso18E expression revealed new aspects to their roles: there are effects of Dmef2 on developing muscle much earlier than hitherto believed, and there is tin-independent gene expression in, and invagination of, prospective midgut visceral muscle cells. Dmeso18E expression is regulated by Dmef2, although some expression is Dmef2-independent. The tin-independent and Dmef2-independent expression of Dmeso18E indicates that it either occupies a link between twi and genes like tin and Dmef2 or it lies in a parallel pathway of gene activation.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila/embriología , Drosophila/genética , Genes de Insecto , Proteínas de Insectos/genética , Proteínas Musculares/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Femenino , Regulación del Desarrollo de la Expresión Génica , Biblioteca de Genes , Hibridación in Situ , Factores de Transcripción MEF2 , Masculino , Mesodermo/metabolismo , Datos de Secuencia Molecular , Mutación , Factores Reguladores Miogénicos , ARN/genética , Activación Transcripcional , Cigoto/metabolismoRESUMEN
mef2 genes encode alternatively spliced transcription factor isoforms that function in muscle differentiation in both Drosophila and vertebrates. Drosophila mef2 (Dmef2) has been shown to be required for the differentiation of a variety of distinct muscle types. However, many possible aspects of its function in muscle remain unexplored. There has also been no analysis in vivo of the activity of different MEF2 isoforms in any species. Our investigation centred on the role of different levels of DMEF2 in the Drosophila embryo in regulating diverse events of muscle differentiation and on the functional significance of Dmef2 alternative splicing. We used the GAL4/UAS system to both misexpress and overexpress individual DMEF2 isoforms and to rescue the different aspects of the Dmef2 mutant phenotype. Ectopic ectodermal expression of DMEF2 activated muscle gene expression and inhibited epidermal differentiation. Overexpression of DMEF2 in the mesoderm disrupted differentiation of the somatic and visceral muscle and the heart. The use of different DMEF2 levels in the rescue experiments revealed an activity range compatible with differentiation of the different muscle types: the consequence of too little or too much DMEF2 activity was disrupted differentiation. These rescue experiments also revealed that distinct DMEF2 thresholds are required for different properties within a cell and also for different cells within a muscle type and for different muscle types. Finally, each isoform functioned equivalently in these experiments, including in the stringent test of rescue of the Dmef2 mutant phenotype.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/embriología , Músculos/embriología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Empalme Alternativo , Animales , Tipificación del Cuerpo , Diferenciación Celular , Proteínas de Drosophila , Drosophila melanogaster/genética , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Genes de Insecto , Biblioteca Genómica , Factores de Transcripción MEF2 , Mesodermo/fisiología , Músculos/citología , Factores Reguladores Miogénicos , Fenotipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Mapeo RestrictivoRESUMEN
Recent studies have substantially advanced our understanding of the transcriptional program regulating development of the different muscle types in Drosophila. For body wall muscle, a pathway can now be drawn that links the transcription factor Dorsal, inherited from the egg, with the differentiated-muscle protein tropomyosin.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Músculos/embriología , Transcripción Genética , Animales , Proteínas de Unión al ADN/genética , Factores de Transcripción MEF2 , Factores Reguladores Miogénicos , Proteínas Nucleares/genética , Factores de Transcripción/genética , Proteína 1 Relacionada con TwistRESUMEN
Experiments manipulating the level of the basic helix-loop-helix transcription factor Twist in the Drosophila embryo have revealed a novel role for this protein in a 'myogenic switch' during early development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Músculos/embriología , Proteínas Nucleares/genética , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Drosophila/embriología , Drosophila/genética , Proteínas de Drosophila , Secuencias Hélice-Asa-Hélice , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Proteína 1 Relacionada con TwistRESUMEN
Analysis of flies with mutations in the gene encoding the D-mef2 transcription factor identifies it as a controller of differentiation in multiple muscle cell types; it is the first such gene to be described.
Asunto(s)
Proteínas de Unión al ADN/genética , Drosophila/genética , Músculos/embriología , Factores de Transcripción/genética , Animales , Diferenciación Celular/genética , Proteínas de Unión al ADN/fisiología , Drosophila/embriología , Proteínas de Drosophila , Genes de Insecto , Factores de Transcripción MEF2 , Músculos/citología , Factores Reguladores Miogénicos , Factores de Transcripción/fisiologíaRESUMEN
We describe a group of Drosophila cDNAs that encode MADs box proteins and which are members of the MEF2 (myocyte enhancer-binding factor 2) family of transcription factors. Drosophila has a single MEF2 gene, DMEF2, that is alternatively spliced to produce different transcripts and which is expressed in the mesodermal primordium before gastrulation. The mechanisms responsible for the subsequent subdivision of the mesoderm are unknown. However, DMEF2 may play a role in this important event because our experiments show that it is a downstream target for twist and that its early expression pattern modulates as the mesoderm is organising into cell groupings with distinct fates. DMEF2 is also expressed in both the segregating primordia and the differentiated cells of the somatic, visceral and heart musculature. It is the only known gene expressed in these three main types of muscle throughout differentiation.
Asunto(s)
Proteínas de Unión al ADN/genética , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes de Insecto , Músculos/embriología , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Diferenciación Celular/fisiología , Clonación Molecular , Proteínas de Drosophila , Corazón/embriología , Factores de Transcripción MEF2 , Mesodermo/metabolismo , Datos de Secuencia Molecular , Músculos/citología , Músculos/metabolismo , Miocardio/citología , Miocardio/metabolismo , Factores Reguladores Miogénicos , Vísceras/embriología , Vísceras/metabolismoRESUMEN
Two alternatively spliced products of the human E2A gene, E12 and E47, encode helix-loop-helix DNA-binding proteins. Here we describe the isolation of two Xenopus cDNAs; one, XE12, is structurally similar to human E12 and the other contains a sequence similar to E47. Transcripts of both cDNAs are present at all the stages of Xenopus development tested and in all regions of the embryo. The DNA binding properties of in vitro translated XE12 are indistinguishable from those of human E12. We have shown previously that an embryonic muscle DNA-binding activity, EMF1, that binds to a promoter sequence required for the expression of the cardiac actin gene, contains the Xenopus myogenic factor XMyoD. Here we show that it also contains protein that interacts with an anti-E12 antiserum, suggesting that XE12 and XMyoD proteins, or very similar ones, are present in EMF1. We have addressed the functional role of XE12 in muscle gene transcription in Xenopus embryos by injecting in vitro synthesized RNA into the two cell embryo. Overexpression of XE12 and XMyoD augments by greater than 10-fold the ectopic activation of the endogenous cardiac actin gene that can be produced by XMyoD alone. Our DNA binding results strongly suggest that this effect is mediated through a direct interaction of the XE12-XMyoD complex with specific sites in the cardiac actin promoter. We suggest that XE12 is functionally important in muscle gene activation in embryonic development.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Musculares/genética , Músculos/fisiología , Xenopus/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero/fisiología , Expresión Génica , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Proteínas Musculares/metabolismo , Proteína MioD , Oligodesoxirribonucleótidos , Biosíntesis de Proteínas , Conformación Proteica , Empalme del ARN , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Xenopus/embriologíaRESUMEN
We identify the "M region" of the muscle-specific Xenopus cardiac actin gene promoter from -282 to -348 as necessary for the embryonic expression of a cardiac actin-beta-globin reporter gene injected into fertilized eggs. Four DNA-binding activities in embryo extracts, embryonic M-region factors 1-4 (EMF1-4), are described that interact specifically with this region. One of these, EMF1, is detected in extracts from microdissected somites, which differentiate into muscle, but not in extracts from the adjacent neurectoderm, which differentiates into a variety of other cell types. Moreover, EMF1 is detected in embryo animal caps induced to form mesoderm, which includes muscle, and in which the cardiac actin gene is activated, but not in uninduced animal caps. EMF1 is also first detectable when cardiac actin transcripts begin to accumulate; therefore, both its temporal and spatial distributions during Xenopus development are consistent with a role in activating cardiac actin expression. Two lines of evidence suggest that EMF1 contains the myogenic factor Xenopus MyoD (XMyoD): (1) XMyoD synthesized in vitro can bind specifically to the same site as EMF1; and (2) antibodies raised against XMyoD bind to EMF1. DNA-binding studies indicate that EMF1 may be a complex between XMyoD and proteins found in muscle and other tissues. Our results suggest that the myogenic factor XMyoD, as a component of somite EMF1, regulates the activation of the cardiac actin gene in developing embryonic muscle by binding directly to a necessary region of the promoter.
Asunto(s)
Actinas/genética , Proteínas de Unión al ADN/genética , Mesodermo/metabolismo , Proteínas Musculares/genética , Músculos/embriología , Regiones Promotoras Genéticas , Xenopus laevis/embriología , Actinas/biosíntesis , Animales , Secuencia de Bases , ADN/química , Ectodermo/química , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Músculos/metabolismo , Proteína MioD , Xenopus laevis/genéticaRESUMEN
The CArG box is an essential promoter sequence for cardiac muscle actin gene expression in Xenopus embryos. To assess the role of the CArG motif in promoter function during Xenopus development, the DNA-binding activities present in the embryo that interact with this sequence have been investigated. A family of four Embryo CArG box1 Factors (ECFs) was separated by a 2-step fractionation procedure. These factors were distinct from the previously described C-ArG box binding activity Serum Response Factor (SRF). ECF1 was the most prominent binding activity in cardiac actin-expressing tissues, and bound the CArG box in preference to a Serum Response Element (SRE). SRF was also detectable in muscle, but it bound preferentially to an SRE. The properties of ECF3 were similar to those of ECF1, but it was much less prominent in cardiac actin-expressing tissues. The properties of the two other factors were distinctive: ECF2 was of relatively low affinity and high abundance, whilst ECF4 bound non-specifically to ends of DNA. The binding activity (or activities) that interacted with the CArG box was found to be influenced by both the concentrations of the other CArG box binding activities and the sequence of the site. Although there was no evidence for a muscle-specific CArG box binding activity, the properties of ECF1 suggest that it could play a role in the expression of the cardiac actin gene during Xenopus development.
Asunto(s)
Actinas/genética , Proteínas de Unión al ADN/metabolismo , Desarrollo de Músculos , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Unión Competitiva , Fraccionamiento Celular , ADN/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Nucleares/metabolismo , Factor de Respuesta Sérica , XenopusRESUMEN
cDNA clones encoding the sequence-specific DNA binding protein, serum response factor (SRF), have been isolated from a Xenopus laevis neurula library and their nucleotide sequence determined. The Xenopus SRF (SRFX) gene produces multiple-sized transcripts, present at 10(5) copies per unfertilized egg. A similar level is detected in the embryo during early cleavage, but SRFX transcripts accumulate rapidly following gastrulation. The protein they encode is similar in sequence to human SRF in its central and carboxy-terminal regions, but possesses a divergent amino-terminal portion. We have previously described a Xenopus embryo sequence-specific binding activity that recognized the CArG motif of the cardiac actin gene promoter. Here we show that the DNA-binding characteristics of synthetic SRFX are indistinguishable from those of the embryo factor. Moreover, antiserum raised against the synthetic SRFX recognizes this factor. Together, these results establish that the same factor binds to elements required for constitutive transcription in Xenopus oocytes, muscle-specific gene expression in Xenopus embryos and serum-responsive transcription in cultured amphibian cells.
Asunto(s)
Proteínas de Unión al ADN/genética , Embrión no Mamífero/fisiología , Proteínas Nucleares/genética , Envejecimiento , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Óvulo/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Ácido Nucleico , Factor de Respuesta Sérica , Programas Informáticos , Xenopus laevisRESUMEN
We describe the isolation and complete sequence of the Xenopus c-fos proto-oncogene. c-fos expression throughout Xenopus development was analysed using a homologous probe derived from the cloned gene. c-fos RNA is accumulated during oogenesis to reach a plateau of 2 x 10(5) transcripts per stage VI oocyte, suggesting an unusual stability of the c-fos message. The amount of RNA per embryo decreases substantially after fertilisation to reach a level corresponding to less than 0.1 molecule per cell at the tailbud stage. Subsequently, at the swimming tadpole stage, the amount of c-fos mRNA increases; an increase that is correlated with the start of skeleton formation. In the newly metamorphosed froglet, c-fos mRNA shows a marked tissue-specific distribution, with the highest level in intestine and lowest in gall bladder, lung and spleen. We also demonstrate that the Xenopus c-fos gene is serum-inducible in Xenopus cultured cells, a property attributable to a promoter sequence known as the Serum Response Element (SRE). A protein activity (indistinguishable from Serum Response Factor) in both whole cell and nuclear Xenopus embryo extracts binds specifically to the SRE and is present at an approximately constant level throughout early development. Our results suggest roles for c-fos in aspects of both the rapid cell proliferation and cell differentiation characteristic of early Xenopus development.
Asunto(s)
Expresión Génica/fisiología , Oogénesis/genética , Proto-Oncogenes/fisiología , Xenopus laevis/genética , Animales , Secuencia de Bases , Northern Blotting , Células Cultivadas , Embrión no Mamífero/química , Intestinos/análisis , Datos de Secuencia Molecular , ARN Mensajero/análisisRESUMEN
Promoter sequences required for activation of the Xenopus cardiac actin gene in embryonic muscle were analysed by micro-injecting chimeric actin/beta-globin genes into the two-cell Xenopus embryo. Transcription was monitored during subsequent differentiation of embryonic muscle and non-muscle tissues. The effect of a variety of mutations including internal deletions and linker scan mutations between -64 and -396 within the cardiac actin promoter were tested. This region contains four copies of a conserved motif, the CArG box, common to vertebrate striated muscle acting gene promoters. In the Xenopus cardiac actin gene, the most proximal of these motifs (CArG box 1) located at -80, was essential for muscle-specific transcription. Other CArG motifs could functionally substitute for CArG box 1 when placed in this position. CArG boxes 3 and 4 bound the same activity in a neurula embryo nuclear extract as CArG box 1 and the amount of this binding activity was constant through early development.
Asunto(s)
Actinas/genética , Regiones Promotoras Genéticas , Transcripción Genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN/genética , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Músculos/metabolismo , Miocardio/metabolismo , Xenopus laevis/embriologíaRESUMEN
Embryonic induction, a process in which the differentiation of a cell is determined by its proximity to other kinds of cells, is of major importance in animal development. We review here what is known of the steps by which a muscle-specific actin gene is first activated by embryonic induction in early amphibian embryos.
Asunto(s)
Diferenciación Celular , Inducción Embrionaria , Músculos/embriología , Actinas/biosíntesis , Animales , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Genes , Corazón/embriología , Activación Transcripcional , Xenopus/embriologíaRESUMEN
The expression of the proto-oncogene c-myc during oogenesis and embryonic development was followed by in situ hybridization using a cytological protocol adapted to amphibian embryos. The c-myc RNA was highly expressed in the cytoplasm of young oocytes and was further diluted during oocyte growth without specific localization. From the neurula stage on, new myc transcripts were detected and the whole embryo appeared positive with antisense myc RNA probes relative to control sense RNA probes. In addition, a spatial localization of high levels of the transcript was also observed in specific areas of the developing embryo, including the epidermis, gill buds, optic vesicles and lens placodes. These observations might indicate a specific role of the c-myc gene during the differentiation of these tissues. Alternatively, this high level of myc expression might prevent such tissues from entering into terminal differentiation during the growth of the embryo.
Asunto(s)
Embrión no Mamífero/fisiología , Oncogenes , Oocitos/fisiología , Oogénesis , Animales , Autorradiografía , Embrión no Mamífero/citología , Técnicas de Sonda Molecular , Oocitos/citología , Xenopus laevisRESUMEN
Comparisons were made between transformed S49 T-lymphoma cells and normal murine thymocytes in their polyphosphoinositides, inositol polyphosphates and cytosolic free calcium concentrations ([Ca2+]i), and the effects of the T-cell mitogen concanavalin A (Con A) on these properties. 1. The ratios of the polyphosphoinositides to phosphatidylinositol in both exponential-phase S49 cells and mitogen-stimulated thymocytes (G1 phase) were greater than in quiescent (G0-phase) thymocytes. 2. In response to Con A, the amount of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in S49 cells decreased slightly (17% in 30 min), and this was sufficient to account for the small amounts of inositol phosphates that accumulated. In contrast, it has been shown previously that Con A stimulates a rapid resynthesis of PtdInsP2 in thymocytes and the amounts of inositol phosphates released rapidly exceed the steady-state amount of the PtdInsP2 precursor [Taylor, Metcalfe, Hesketh, Smith & Moore (1984) Nature (London) 312, 462-465]. 3. The [Ca2+]i did not differ significantly in S49 cells and thymocytes before the addition of Con A, and the increases in [Ca2+]i in response to Con A were similar in both types of cell. 4. The [Ca2+]i increase in response to Con A was inhibited by similar concentrations of intracellular cyclic AMP (2-10 microM) in S49 cells and thymocytes, suggesting that similar regulatory mechanisms act on this response in both types of cell. The data demonstrate that the basal [Ca2+]i and phosphoinositide metabolism is similar in both the normal cells and their transformed counterparts. In addition, they suggest that the activated Con A receptors generate very similar signals in the two cell types, and that any perturbations of primary signal transduction to the secondary phosphoinositide and [Ca2+]i responses in the S49 phenotype are quantitative rather than qualitative.