RESUMEN

The dorsal striatum is involved in motor-response learning, but the extent to which distinct populations of striatal efferent neurons are differentially involved in such learning is unknown. Activity-regulated, cytoskeleton-associated (Arc) protein is an effector immediate-early gene implicated in synaptic plasticity. We examined arc mRNA expression in striatopallidal vs. striatonigral efferent neurons in dorsomedial and dorsolateral striatum of rats engaged in reversal learning on a T-maze motor-response task. Male Sprague-Dawley rats learned to turn right or left for 3 days. Half of the rats then underwent reversal training. The remaining rats were yoked to rats undergoing reversal training, such that they ran the same number of trials but ran them as continued-acquisition trials. Brains were removed and processed using double-label fluorescent in situ hybridization for arc and preproenkephalin (PPE) mRNA. In the reversal, but not the continued-acquisition, group there was a significant relation between the overall arc mRNA signal in dorsomedial striatum and the number of trials run, with rats reaching criterion in fewer trials having higher levels of arc mRNA expression. A similar relation was seen between the numbers of PPE(+) and PPE(-) neurons in dorsomedial striatum with cytoplasmic arc mRNA expression. Interestingly, in behaviourally activated animals significantly more PPE(-) neurons had cytoplasmic arc mRNA expression. These data suggest that Arc in both striatonigral and striatopallidal efferent neurons is involved in striatal synaptic plasticity mediating motor-response learning in the T-maze and that there is differential processing of arc mRNA in distinct subpopulations of striatal efferent neurons.

Asunto(s)

Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/genética , Aprendizaje/fisiología , Neostriado/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuronas Eferentes/metabolismo , Neuronas Eferentes/fisiología , ARN Mensajero/biosíntesis , Animales , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Aprendizaje por Laberinto/fisiología , Neostriado/citología , Ratas , Ratas Sprague-Dawley , Aprendizaje Inverso/fisiología , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/fisiología

RESUMEN

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

Asunto(s)

Factores de Ribosilacion-ADP/metabolismo , Proteínas del Citoesqueleto/química , Proteínas Activadoras de GTPasa/metabolismo , Fosfoproteínas/química , Secuencias de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Células CHO , Movimiento Celular , Células Cultivadas , Cricetinae , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Activación Enzimática , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Eliminación de Gen , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Microscopía por Video , Modelos Genéticos , Mutagénesis Sitio-Dirigida , Mutación , Paxillin , Fenotipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Seudópodos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Transfección

RESUMEN

Tryptases are serine proteases involved in mast cell-mediated inflammatory responses which represent potential targets of drugs against diseases such as asthma, arthritis and inflammatory bowel disease. In order to interpret pharmacodynamic data on the tryptase inhibitors undergoing clinical trials, we defined the genetic variability of the tryptase 1 (TPS1) and tryptase 2 (TPS2) loci by screening a reference population of 32 individuals representing three major ethnic groups (Caucasian, African American, Asian). Using overlapping PCR products, we resequenced the entire tryptase genes with the only exclusion of TPS2 intron 1 and 20 bp of TPS2 5' untranslated region included in exon 1 and we identified 21 novel single nucleotide polymorphisms in TPS1 and 17 single nucleotide polymorphisms plus a large polymorphic deletion in the TPS2 gene. We also compared the type, frequency and distribution of single nucleotide polymorphisms in TPS1 and TPS2 and we observed that the polymorphism frequency within these two loci is unexpectedly high (approximately 1 SNP every 90 bp) and that some of the allele frequencies differ significantly among the three ethnic groups. Based on differences observed in preclinical studies using a cynomolgus monkey (Macaca fascicularis) asthma model system, we investigated the difference between monkey and human tryptase genes in order to better understand the mechanism of action of our tryptase inhibitors.

Asunto(s)

Mastocitos/enzimología , Polimorfismo Genético , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Animales , Quimasas , Marcadores Genéticos , Humanos , Macaca fascicularis , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Triptasas

RESUMEN

The UDP-glucuronosyltransferases (UGTs) comprise a large family of proteins capable of detoxifying a wide variety of both endogenous and exogenous substrates. The primary function of this gene superfamily is to catalyze the glycosylation of substrates such as biogenic amines, steroids, bile acids, phenolic compounds and various other pharmacologically relevant compounds, including numerous carcinogens, toxic environmental pollutants and prescription drugs. This conjugation increases the solubility of these compounds, allowing them to be excreted more readily through hepatic or renal mechanisms. This paper describes the genomic characterization and chromosomal localization of three UGT2B genes which together comprise part of a large cluster of related sequences, including pseudogenes found on human chromosome 4q13. A genomic map spanning approximately 500-1000 kb of this region reveals the presence of three previously described UGT2B genes, at least two previously uncharacterized pseudogenes and a significant number of remnant gene fragments and places UGT2B4 between UGT2B7 and UGT2B15. Additionally, access to a large reference DNA bank allowed us to calculate allele frequencies for two UGT2B SNPs: D85R in UGT2B15 and Q458D in UGT2B4 amongst 803 unrelated individuals representing five ethnic populations. The data presented here suggest a recent evolutionary history of gene duplication, mutation and rearrangement. Furthermore, they suggest that a re-evaluation of the current description of the UGT2B gene family with respect to the number of specific genes, degree of allelic diversity and molecular evolution may be necessary.

Asunto(s)

Cromosomas Humanos Par 4/genética , Glucuronosiltransferasa/genética , Familia de Multigenes , Evolución Biológica , Mapeo Cromosómico , Etnicidad/genética , Frecuencia de los Genes , Biblioteca Genómica , Humanos , Hibridación Fluorescente in Situ , Isoenzimas/genética , Polimorfismo Genético , Regiones Promotoras Genéticas , Seudogenes

RESUMEN

Activin A and Transforming Growth Factor-beta (TGF-beta) are members of a common family of cytokines that bind to and stimulate serine/threonine kinase receptors. Activin A and TGF-beta are important during embryonic development exerting both positive and negative effects on cell growth. In the adult organism, they function in processes such as tissue repair, cellular proliferation, and differentiation. Although activin A and TGF-beta often induce opposite functional outcomes in specific cells; proliferation or differentiation, both were found to stimulate the formation of actin stress fibers and focal adhesions in serum-starved rat aortic smooth muscle (RASM) cells. These structural changes were accompanied by phosphorylation of the focal adhesion proteins, paxillin, and p130(cas). Similar cytoskeletal and biochemical changes were observed with the vasoactive agonist angiotensin II. Activation of the ERK/MAP kinase pathway has been implicated in the migration in certain cell types. However, while activin A, TGF-beta, and angiotensin II all stimulated ERK activity in RASM cells, only activin A and angiotensin II stimulated migration. TGF-beta failed to illicit a chemotactic response. Furthermore, pharmacologic inhibition of MEK activity failed to block migration in response to activin A and angiotensin II, indicating RASM migration can occur independent of ERK activity. These results suggest that TGF-beta and activin A share several signaling pathways with angiotensin II leading to cytoskeletal remodeling and ERK activation, but there are distinct differences regarding the effect of these agonists on cellular migration.

Asunto(s)

Aorta/citología , Moléculas de Adhesión Celular/metabolismo , Citoesqueleto/efectos de los fármacos , Inhibinas/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Músculo Liso Vascular/efectos de los fármacos , Proteínas , Factor de Crecimiento Transformador beta/farmacología , Actinas/metabolismo , Activinas , Angiotensina II/farmacología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Proteína Sustrato Asociada a CrK , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Activación Enzimática/efectos de los fármacos , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , MAP Quinasa Quinasa 1 , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/metabolismo , Paxillin , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Ratas , Proteína p130 Similar a la del Retinoblastoma , Transducción de Señal/efectos de los fármacos

RESUMEN

Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.

Asunto(s)

Ancirinas/metabolismo , Proteínas Portadoras/fisiología , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/fisiología , Proteínas de Unión al GTP/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Factores de Ribosilacion-ADP , Secuencia de Aminoácidos , Animales , Ancirinas/genética , Sitios de Unión , Células CHO , Células COS , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular , Cricetinae , Proteínas de Unión al ADN/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal , GTP Fosfohidrolasas , Proteínas Activadoras de GTPasa , Péptidos y Proteínas de Señalización Intracelular , Proteínas con Dominio LIM , Datos de Secuencia Molecular , Paxillin , Proteínas Tirosina Quinasas/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Fracciones Subcelulares , Vinculina/metabolismo , Proteína de Unión al GTP cdc42 , Quinasas p21 Activadas

RESUMEN

The proliferative capacity of T cells infiltrating human tumors is known to be impaired, possibly through their interaction with tumor. Here we demonstrate that soluble products derived from renal cell carcinoma (RCC-S) explants but not normal kidney can inhibit an IL-2-dependent signaling pathway that is critical to T cell proliferation. A major target of the immunosuppression was the IL-2R-associated protein tyrosine kinase, Janus kinase 3 (Jak3). RCC-S suppressed basal expression of Jak3 and its increase following stimulation with anti-CD3/IL-2. Jak3 was most sensitive to suppression by RCC-S; however, reduction in expression of p56(lck), p59(fyn), and ZAP-70 was observed in some experiments. Expression of other signaling elements linked to the IL-2R (Jak1) and the TCR (TCR-zeta, CD3-epsilon, and phospholipase C-gamma) were minimally affected. In naive T cells, RCC-S also partially blocked induction of IL-2R alpha-, beta- and gamma-chain expression when stimulating via the TCR/CD3 complex with anti-CD3 Ab. To determine whether RCC-S suppressed IL-2-dependent signaling, primed T cells were employed since RCC-S had no effect on IL-2R expression but did down-regulate Jak3 expression and, to a lesser degree, p56(lck) and p59(fyn). Reduction in Jak3 correlated with impaired IL-2-dependent proliferation and signal transduction. This included loss of Jak1 kinase tyrosine phosphorylation and no induction of the proto-oncogene, c-Myc. These findings suggest that soluble products from tumors may suppress T cell proliferation through a mechanism that involves down-regulation of Jak3 expression and inhibition of IL-2-dependent signaling pathways.

Asunto(s)

Carcinoma de Células Renales/inmunología , Neoplasias Renales/inmunología , Proteínas Tirosina Quinasas/inmunología , Receptores de Interleucina-2/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Escape del Tumor , División Celular/inmunología , Citotoxicidad Inmunológica , Humanos , Janus Quinasa 3 , Activación de Linfocitos/inmunología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proto-Oncogenes Mas , Receptores de Interleucina-2/antagonistas & inhibidores , Células Tumorales Cultivadas

RESUMEN

Members of the Janus (JAK) protein tyrosine kinase family including JAK3 have recently emerged as important components in cytokine signal transduction. Mutations of JAK3 have been found in a number of patients who present with severe combined immunodeficiency. To facilitate the further identification of JAK3-SCID patients and to understand the structure of JAK3 better, we undertook the determination of the genomic sequence, organization, and chromosomal localization of the JAK3 gene. The JAK3 gene was found to consist of 19 exons and 18 introns. Interestingly, the organization of the kinase-(JH1) and pseudokinase-(JH2) domains were found to be dissimilar. In addition, the JAK3 gene was localized to human chromosome 19p13.1. These data should facilitate the identification of patients with this new form of immunodeficiency and will provide insight into the structure of this kinase.

Asunto(s)

Cromosomas Humanos Par 19 , Proteínas Tirosina Quinasas/genética , Mapeo Cromosómico , Clonación Molecular , Exones , Humanos , Intrones , Janus Quinasa 3 , Datos de Secuencia Molecular

RESUMEN

Cytokines that bind to the interleukin-2 (IL-2) receptor common gamma chain (gamma c), including IL-2, IL-4, IL-7, IL-9, and IL-15, are important for the growth and differentiation of T and B lymphocytes, natural killer cells, macrophages, and monocytes. These cytokines have overlapping biological effects that in part result from the use of the shared receptor subunit gamma c. Recently it has become clear that these cytokines activate a number of important intracellular signaling molecules, including the Janus kinases JAK1 and JAK3 and members of the transcription factor family of signal transducers and activators of transcription (STATs). The discovery of these signaling pathways has led to important new insights into their role in lymphocyte maturation, as it has emerged that mutations in the genes encoding both gamma c and JAK3 result in similar forms of severe combined immunodeficiency (SCID). In this review we examine the structure and function of cytokine receptors and the signaling pathways involved in their regulation of gene expression. Furthermore, we discuss recent advances that have led to a better understanding of how cytokines elicit intracellular responses, as well as their role in normal lymphoid development.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Interleucina-2/fisiología , Proteínas Tirosina Quinasas/fisiología , Receptores de Citocinas/fisiología , Transducción de Señal , Transactivadores/fisiología , Transformación Celular Neoplásica/genética , Mapeo Cromosómico , Precursores Enzimáticos/fisiología , Genes , Humanos , Interleucina-12/fisiología , Péptidos y Proteínas de Señalización Intracelular , Janus Quinasa 3 , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Linfocitos/fisiología , Fosfotirosina/fisiología , Factor de Transcripción STAT1 , Inmunodeficiencia Combinada Grave/fisiopatología , Quinasa Syk , Dominios Homologos src , Familia-src Quinasas/fisiología

RESUMEN

Males with X-linked severe combined immunodeficiency (XSCID) have defects in the common cytokine receptor gamma chain (gamma c) gene that encodes a shared, essential component of the receptors of interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. The Janus family tyrosine kinase Jak3 is the only signaling molecule known to be associated with gamma c, so it was hypothesized that defects in Jak3 might cause an XSCID-like phenotype. A girl with immunological features indistinguishable from those of XSCID was therefore selected for analysis. An Epstein-Barr virus (EBV)-transformed cell line derived from her lymphocytes had normal gamma c expression but lacked Jak3 protein and had greatly diminished Jak3 messenger RNA. Sequencing revealed a different mutation on each allele: a single nucleotide insertion resulting in a frame shift and premature termination in the Jak3 JH4 domain and a nonsense mutation in the Jak3 JH2 domain. The lack of Jak3 expression correlated with impaired B cell signaling, as demonstrated by the inability of IL-4 to activate Stat6 in the EBV-transformed cell line from the patient. These observations indicate that the functions of gamma c are dependent on Jak3 and that Jak3 is essential for lymphoid development and signaling.

Asunto(s)

Linfocitos B/inmunología , Proteínas Tirosina Quinasas/fisiología , Inmunodeficiencia Combinada Grave/enzimología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Transformada , Femenino , Mutación del Sistema de Lectura , Ligamiento Genético , Humanos , Lactante , Interleucina-4/farmacología , Janus Quinasa 3 , Datos de Secuencia Molecular , Fenotipo , Mutación Puntual , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina/fisiología , Factor de Transcripción STAT6 , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunología , Transducción de Señal , Transactivadores/metabolismo , Cromosoma X

RESUMEN

We describe here a new member of the kinesin superfamily in Drosophila, KLP3A (Kinesin-Like-Protein-at-3A). The KLP3A protein localizes to the equator of the central spindle during late anaphase and telophase of male meiosis. Mutations in the KLP3A gene disrupt the interdigitation of microtubules in spermatocyte central spindles. Despite this defect, anaphase B spindle elongation is not obviously aberrant. However, cytokinesis frequently fails after both meiotic divisions in mutant testes. Together, these findings strongly suggest that the KLP3A presumptive motor protein is a critical component in the establishment or stabilization of the central spindle. Furthermore, these results imply that the central spindle is the source of signals that initiate the cleavage furrow in higher cells.

Asunto(s)

División Celular/fisiología , Drosophila/fisiología , Cinesinas/fisiología , Meiosis/fisiología , Espermatocitos/fisiología , Huso Acromático/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Compartimento Celular , Clonación Molecular , Drosophila/citología , Drosophila/ultraestructura , Proteínas de Drosophila , Femenino , Fertilidad , Genes de Insecto/genética , Immunoblotting , Inmunohistoquímica , Cinesinas/genética , Cinesinas/inmunología , Masculino , Microscopía por Video , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Datos de Secuencia Molecular , Mutación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Caracteres Sexuales , Espermatocitos/ultraestructura , Huso Acromático/ultraestructura

RESUMEN

Classical Northern blot analysis for measuring mRNA requires too many cells to be practical for cell sorting. Yet, measurement of gene expression in small subsets within a heterogeneous population of cells is often desired. The PCR in combination with prior reverse transcription (RT-PCR) of the mRNA of interest provides a means for measuring gene expression using as few as one cell. When RT-PCR is performed, the reliability of the data can be highly subjective due to the efficiency of both RT and PCR steps. This subjectivity can be eliminated by a technique for quantitating specific RNA molecules using an internal RNA competitive reference standard (RNA-CRS), which is identical to the sequence of interest except for a deletion of 80 bases. Here we illustrate a strategy for quantitative PCR using a RNA-CRS, synthesized solely using nonplasmid-based PCR techniques. The competitive reaction consists of a constant quantity of wild-type mRNA (from 100-1000 cells) added individually to tubes containing a serially decreasing amount of RNA-CRS. The RT-PCR is performed on these samples, then the resulting product is analyzed by gel electrophoresis and densitometry. The procedure for preparing the RNA-CRS and subsequent RT-PCR steps are described in detail.

Asunto(s)

Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/análisis , Animales , Secuencia de Bases , Células Cultivadas , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Estándares de Referencia , Familia-src Quinasas

Asunto(s)

Macrófagos/citología , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/genética , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Expresión Génica/efectos de los fármacos , Cinética , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Proto-Oncogenes

Asunto(s)

Citometría de Flujo/métodos , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Neoplasias de la Mama/genética , Cartilla de ADN/genética , ADN de Neoplasias/genética , Femenino , Citometría de Flujo/normas , Gastrinas/genética , Amplificación de Genes , Expresión Génica , Humanos , Hibridación Fluorescente in Situ/métodos , Datos de Secuencia Molecular , Oncogenes , Mutación Puntual , Reacción en Cadena de la Polimerasa/normas , Estándares de Referencia

RESUMEN

We have shown that there are two forms of progenitor cells for macrophages. The first is characterized by a short lag period (about 1 day) before initiating the cell cycle, forms large colonies, is found in the bone marrow, and is in the nonadherent fraction. The second progenitor cell, found primarily in the adherent cell fraction of bone marrow and in peripheral tissues, forms small colonies after 14 days. We investigated the effect of combining interleukin-6 (IL-6) with colony-stimulating factor 1 (CSF-I) on macrophage proliferation. We found that IL-6 inhibited the proliferation of both types of progenitor cells, as well as more differentiated macrophages. This inhibitory effect was reversible because macrophages could initiate a proliferative response after removal of IL-6 from the culture medium. The introduction of anti-IL-6 into macrophage cultures containing IL-6 allowed proliferation, indicating that the effect was IL-6 specific. These results suggest that IL-6 may play a regulatory role in vivo by suppressing the production of bone marrow and tissue macrophages.

Asunto(s)

Interleucina-6/fisiología , Macrófagos/citología , Células de la Médula Ósea , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Proteínas Recombinantes/fisiología

Asunto(s)

Papio/genética , Paternidad , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético/genética , Animales , Estudios de Evaluación como Asunto , Femenino , Humanos , Masculino , Oligodesoxirribonucleótidos/genética , Linaje

RESUMEN

Foreign genes were expressed in liver and skin cells of live mice by using a new apparatus to accelerate DNA-coated microprojectiles into tissues. After introduction of a plasmid in which the firefly luciferase gene was controlled by the human beta-actin promoter, luciferase activity was detectable for up to 14 days in mouse tissues (skin and liver). In situ hybridization histochemistry revealed that microprojectiles penetrated through multiple cell layers without evidence of tissue injury and that 10-20% of the cells in the bombarded area expressed the foreign gene. An advantage of the new design is that internal organs, such as liver, can be transfected without subjecting the tissue to a vacuum. This procedure potentially is applicable to a wide variety of tissues and cell types for studies of transcriptional control elements and for expression of foreign proteins in intact animals.

Asunto(s)

Actinas/genética , Genes , Ingeniería Genética/instrumentación , Hígado/enzimología , Luciferasas/genética , Plásmidos , Regiones Promotoras Genéticas , Piel/enzimología , Animales , Escarabajos/enzimología , Escarabajos/genética , Femenino , Ingeniería Genética/métodos , Ratones , Ratones Endogámicos , Hibridación de Ácido Nucleico , Especificidad de Órganos

RESUMEN

Flow cytometric determination of viable versus nonviable cells in fixed samples can be accomplished by utilizing the irreversible binding of photoactivated ethidium monoazide (EMA). EMA is a positively charged molecule which is excluded by cells with intact membranes (viable cells), included by cells with damaged membranes, and can be photochemically crosslinked to nucleic acids using visible light. EMA fluorescence can be excited using a standard argon laser operating at 488 nm and is able to be distinguished from fluorescein and phycoerythrin. Fixation is important when analyzing cells from a potentially infectious origin. EMA is photochemically crosslinked and therefore unable to leak out of cells when removed from the extracellular media, unlike propidium iodide (PI) or other viability stains, which were heretofore commonly used. We demonstrate the usefulness of EMA in combination with fluoresceinated and phycoerythrin labeled monoclonal antibodies in immunophenotyping. The photoaffinity labeling technique allows for a quick and efficient means of identifying nonviable cells which cannot be distinguished on the basis of light-scattering properties.

Asunto(s)

Marcadores de Afinidad , Azidas , Supervivencia Celular , Citometría de Flujo , Colorantes Fluorescentes , Anticuerpos Monoclonales , Fijadores/farmacología , Humanos , Inmunofenotipificación , Rayos Láser , Manejo de Especímenes

RESUMEN

The hemodynamic response to maximal exercise was determined in sedentary and trained rats with a chronic myocardial infarction (MI) produced by coronary artery ligation and in rats that underwent sham operations (SHAM). Infarct size in the MI groups of rats comprised 28-29% of the total left ventricle and resulted in both metabolic and hemodynamic changes that suggested that these animals had moderate compensated heart failure. The training regimen used in the present study produced significant increases in maximal O2 uptake (VO2max) when expressed in absolute terms (ml/min) or when normalized for body weight (ml.min-1.kg-1) and consisted of treadmill running at work loads that were equivalent to 70-80% of the animal's VO2max for a period of 60 min/day, 5 days/wk over an 8- to 10-wk interval. This training paradigm produced two major cardiocirculatory adaptations in the MI rat that had not been elicited previously when using a training paradigm of a lower intensity. First, the decrement in the maximal heart rate response to exercise (known as "chronotropic incompetence") found in the sedentary MI rat was completely reversed by endurance training. Second, the downregulation of cardiac myosin isozyme composition from the fast ATPase V1 isoform toward the slower ATPase (V2 and V3) isoforms in the MI rat was partially reversed by endurance training. These cardiac adaptations occurred without a significant increase in left ventricular pump function as an increase in maximal cardiac output (Qmax) and maximal stroke volume (SVmax) did not occur in the trained MI rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Adaptación Fisiológica , Hemodinámica , Infarto del Miocardio/fisiopatología , Resistencia Física , Animales , Gasto Cardíaco , Masculino , Miocardio/enzimología , Miosinas/metabolismo , Consumo de Oxígeno , Ratas , Ratas Endogámicas , Volumen Sistólico

RESUMEN

Basal plasma hydroxyproline was measured in 104 male Navy Seal candidates 1 week into their intense physical training program, which lasted 7 weeks, and correlated to the incidence of connective tissue injuries incurred later in the training program. Eleven subjects (10.6%) were diagnosed as having connective tissue injuries. Those subjects with connective tissue injuries had a significantly higher (P less than 0.05) mean plasma hydroxyproline value (4.02 micrograms/ml) than subjects without injury (3.10 micrograms/ml). The majority of graduates (75%) had plasma hydroxyproline values less than 3.3 micrograms/ml. These graduates represented the strongest and most enduring injury-free subjects. Of the subject pool who incurred connective tissue injuries, only 27% had plasma hydroxyproline values less than 3.3 micrograms/ml. The majority of the injured subjects (73%) had plasma hydroxyproline values greater than or equal to 3.3 micrograms/ml. In conclusion, there is a relationship between initial training basal plasma hydroxyproline levels and connective tissue injuries later incurred in an intense physical training program. These data suggest that elevated plasma hydroxyproline levels may represent a risk factor associated with connective tissue injuries.

Asunto(s)

Traumatismos en Atletas/etiología , Hidroxiprolina/sangre , Educación y Entrenamiento Físico , Adolescente , Adulto , Tejido Conectivo , Humanos , Masculino , Medicina Naval , Factores de Riesgo