RESUMEN

BACKGROUND: Guanidine exchange factor (GEF)-catalysed activation of Rho proteins such as Cdc42 has been shown to have a crucial role in cellular transformation, malignant progression and invasion. We have previously shown that the HPV16 E6 oncoprotein binds to the PDZ domain protein Tax-interacting-protein 1 (Tip-1) and we now report identification and functional analysis of a novel Tip-1 binding GEF. METHODS: Yeast two-hybrid, in vitro pull-down, site-directed mutagenesis, semiquantitative PCR, co-immunoprecipitation and western blotting were used to identify/confirm novel Tip-1 binding partners and analyse cellular expression levels. In vitro kinetic analyses of recombinant proteins, siRNA gene silencing and in cell assays were used to measure Rho protein activation. RESULTS: Tax-interacting-protein 1 was shown to interact with ARHGEF16 by its carboxyl PDZ binding motif. Levels of ARHGEF16 were increased in transformed and immortalised cells expressing ectopic HPV16 E6 and Cdc42 was co-immunoprecipitated by ARHGEF16 in the presence of high-risk HPV E6. In vitro kinetic analysis confirmed that recombinant ARHGEF16 activates Cdc42 and this was increased by the addition of recombinant Tip-1 and E6. Cells expressing HPV16 E6 had higher levels of Cdc42 activation, which was decreased by siRNA silencing of either Tip-1 or ARHGEF16. CONCLUSION: These data suggest that HPV16 E6, Tip-1 and ARHGEF16 may cooperate to activate Cdc42 and support a potential link between the expression of HPV16 E6 and Cdc42 activation.

Asunto(s)

Factores de Intercambio de Guanina Nucleótido/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas Represoras/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Secuencia de Bases , Western Blotting , Cartilla de ADN , Silenciador del Gen , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Unión Proteica , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos Híbridos

RESUMEN

OBJECTIVE: Human Papillomavirus (HPV) 6 and 11 are the aetiological agents responsible for Recurrent Respiratory Papillomatosis (RRP). There is general consensus that HPV11 results in more aggressive disease compared to HPV6. METHOD: Pubmed was searched using the terms respiratory papillomatosis, HPV 6 and HPV11. Comparisons were made in the outcomes of HPV6 versus HPV11 positive RRP disease. RESULTS: There are numerous sub-types or variants of both HPV6 and HPV11. These sub-types have different activities at least in-vitro. The numbers of different HPV types within RRP tissue may be more extensive than initially appeared. This depends specifically upon the HPV types tested for. CONCLUSION: The clinical differences between HPV6 and HPV11 disease may not be accurately predictable as these viruses exist in numerous sub-types. Also, RRP tissue may contain more than one subtype or even be co-infected with other viruses that may influence outcome. In-vitro studies upon cell lines are a reasonable starting point for evaluation of these differences.

Asunto(s)

Papillomavirus Humano 11/aislamiento & purificación , Papillomavirus Humano 6/aislamiento & purificación , Papiloma/virología , Infecciones del Sistema Respiratorio/virología , Condiloma Acuminado/epidemiología , Condiloma Acuminado/virología , Genoma , Papillomavirus Humano 11/genética , Papillomavirus Humano 6/genética , Humanos , Laringoestenosis/cirugía , Virus Oncogénicos/genética , Procedimientos Quirúrgicos Otorrinolaringológicos/estadística & datos numéricos , Papiloma/epidemiología , Papiloma/cirugía , Recurrencia , Índice de Severidad de la Enfermedad , Traqueostomía/estadística & datos numéricos

RESUMEN

BACKGROUND: Constitutive activation of RhoA-dependent RhoA kinase (ROCK) signalling is known to promote cellular transformation and the ROCK inhibitor Y-27632 has the ability to suppress focus formation of RhoA transformed NIH3T3 cells. METHODS: Sixty-four novel structural analogues of Y27632 were synthesised and tested for their ability to persistently inhibit the transformation of NIH3T3 cells by Rho guanidine exchange factor 16 (ARHGEF16) or Ras. In vitro kinase inhibitor profiling, co-culture of transformed cells with non-transformed cells and a novel Lucifer yellow/PKH67 dye transfer method were used to investigate their mode of action. RESULTS: Four Y27632 analogues inhibited transformed focus formation that persisted when the compound was withdrawn. No toxicity was observed against either transformed or non-transformed cells and the effect was dependent on co-culture of these two cell types. In vitro kinase inhibitor profiling indicated that these compounds had reduced activity against ROCK compared with Y27632, targeting instead Aurora A (AURKA), p38 (MAPK14) and Hgk (MAP4K4). Dye transfer analysis showed they increased gap junction intercellular communication (GJIC) between transformed and non-transformed cells. CONCLUSIONS: These data are the first to suggest that transient blockade of specific kinases can induce a persistent inhibition of non-contact inhibited transformed colony formation and can also remove pre-formed colonies. These effects could potentially be mediated by the observed increase in GJIC between transformed and non-transformed cells. Selection of kinase inhibitors with this property may thus provide a novel strategy for cancer chemoprevention.

Asunto(s)

Amidas/farmacología , Comunicación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Piridinas/farmacología , Amidas/síntesis química , Amidas/química , Animales , Aurora Quinasa A , Aurora Quinasas , Línea Celular Transformada , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Clonación Molecular/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Ratones , Células 3T3 NIH , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/síntesis química , Piridinas/química , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo , Quinasa de Factor Nuclear kappa B

Asunto(s)

Carcinoma/radioterapia , Tolerancia a Radiación , Proteínas Represoras , Neoplasias del Cuello Uterino/radioterapia , Animales , Relación Dosis-Respuesta en la Radiación , Femenino , Humanos , Masculino , Ratones , Ratones SCID , Neoplasias Experimentales/radioterapia , Proteínas Oncogénicas Virales , Proteínas E7 de Papillomavirus , Factores Sexuales

RESUMEN

Topical 5-aminolevulinic acid-based photodynamic therapy (PDT) has produced complete response rates of >90% for nonmelanoma skin carcinomas, which are mostly human papillomavirus (HPV) negative. Using a similar treatment protocol, we observed a short-term response in only one third (10 of 32) of high-grade vulval intraepithelial neoplasia (VIN 2-3) lesions. Unifocal lesions were found more responsive than multifocal and pigmented lesions. Animal model studies have suggested that long-term PDT response involves an immune reaction in which CTLs play a crucial role. In this study, we have assessed: (a) HPV infection; (b) HLA expression; and (c) immune infiltrating cells in VIN biopsies from responders and nonresponders to determine whether these factors may limit response to topical 5-aminolevulinic acid-based PDT. Tissues from normal vulva (n = 9), vulval carcinoma (n = 11), and VIN (32 patients from which 19 pre- and 43 post-PDT biopsies were taken) were investigated for immune cell infiltration and HLA class I expression by immunohistochemistry and HPV infection by PCR. There was a greater likelihood of HPV positivity associated with a lack of response of VIN to PDT (P = 0.002), and VIN nonresponders were more likely to show HLA class I loss compared with responders (P = 0.030). HLA class I down-regulation was significantly greater in the carcinomas (82%, total loss) than the VIN (28%, 19%, total loss; and 9%, allele loss; P = 0.004). None of the cases with class I down-regulation responded to PDT, whereas 3 of 6 (50%) of cases that showed total class I loss subsequently developed superficial invasion. Compared with normal vulval skin, VIN lesions showed increased infiltration by CD4 (T-helper) and CD68 (macrophages) but not CD1a (Langerhans cells) or CD8 (CTLs). There was, however, a significant increase of CD8 infiltration in posttreatment VIN responders compared with nonresponders (P = 0.0001). These data clearly support the contention that high-risk HPV infection and lack of cell-mediated immunity may play a role in the observed poor response of lower genital lesions to topical PDT.

Asunto(s)

Antígenos HLA/inmunología , Papillomaviridae , Fotoquimioterapia , Neoplasias de la Vulva/inmunología , Neoplasias de la Vulva/virología , Adolescente , Adulto , Anciano , Ácido Aminolevulínico/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , ADN Viral/análisis , Femenino , Antígenos HLA/biosíntesis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Células de Langerhans/inmunología , Persona de Mediana Edad , Papillomaviridae/genética , Infecciones por Papillomavirus/complicaciones , Fármacos Fotosensibilizantes/uso terapéutico , Infecciones Tumorales por Virus/complicaciones , Neoplasias de la Vulva/tratamiento farmacológico

RESUMEN

INTRODUCTION: The serine protease inhibitor Serpin 2A is highly expressed in ex vivo bipotent granulocyte/macrophage progenitor cells and in cultured myeloid stem cells. The gene undergoes rapid down-regulation as these cells are induced to differentiate, and constitutive expression in cultured myeloid stem cells retards maturation. Serpin 2A is also expressed in T cells as a consequence of activation. We now report analysis of the upstream regulatory elements that control Serpin 2A transcription. MATERIALS AND METHODS: Using primer extension and rapid amplification of cDNA ends the transcription start site of the Serpin 2A gene was mapped, and a 1.2 Kb genomic upstream fragment cloned and sequenced. Promoter activity and protein binding of deletion and site-directed mutant constructs were analysed by transient transfection and by electrophoretic mobility shift assays. RESULTS: A minimal promoter fragment was identified with high activity dependent on NF-kappa and Moloney murine leukaemia enhancer factor LVa binding sites in both myeloid stem cells and activated T cells. NF-kappa was shown to be the main DNA binding protein in T cells, whereas that in haematopoietic stem cells appears to be novel. CONCLUSION: Serpin 2A promoter activity in T cells is due predominantly to NF-kappa binding to its consensus site. Activity in haematopoietic stem cells appears to be mediated by a novel protein, which recognises the NF-kappa consensus only in the context of flanking sequences. This concise regulatory element may be of potential value in gene therapeutic applications.

Asunto(s)

Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Activación de Linfocitos/genética , Regiones Promotoras Genéticas , Serpinas/genética , Linfocitos T/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Mapeo Cromosómico , Secuencia de Consenso , Cósmidos , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Exones/genética , Genes Reporteros , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , FN-kappa B/metabolismo , Especificidad de Órganos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Serpinas/biosíntesis , Linfocitos T/inmunología , Transcripción Genética , Transfección

RESUMEN

CD105 (endoglin), a receptor for transforming growth factor beta (TGFbeta), is highly expressed in tissue-cultured, activated endothelial cells in vitro and in tissues undergoing angiogenesis in vivo. The absence of CD105 in knockout mice leads to their death from defective vascular development, but the role of CD105 in the modulation of angiogenesis has not been elucidated. TGFbeta1 is a well-recognized regulator of angiogenesis. Using an antisense approach, we have shown that inhibition of CD105 protein translation in cultured human endothelial cells enhances the ability of TGFbeta1 to suppress growth and migration in these cells. The ability of endothelial cells to form capillary tubes was evaluated by the use of a 3-dimensional collagen matrix system where TGFbeta1 not only reduced the length of capillary-like structures, but also caused massive mortality in CD105-deficient cells compared to control cultures. These results provide direct evidence that CD105 antagonizes the inhibitory effects of TGFbeta1 on human vascular endothelial cells and that normal cellular levels of CD105 are required for the formation of new blood vessels.

Asunto(s)

Endotelio Vascular/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Molécula 1 de Adhesión Celular Vascular/fisiología , Animales , Antígenos CD , Secuencia de Bases , Muerte Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Cartilla de ADN , Endoglina , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Ratones , Neovascularización Fisiológica/fisiología , Oligonucleótidos Antisentido/farmacología , ARN Mensajero/metabolismo , Receptores de Superficie Celular , Factor de Crecimiento Transformador beta/farmacología , Molécula 1 de Adhesión Celular Vascular/genética

RESUMEN

The p16(INK4a) tumor suppressor inhibits cyclin-dependent kinases (CDK4 and CDK6). Here we report the isolation of a novel gene, SEI-1, whose product (p34(SEI-1)) appears to antagonize the function of p16(INK4a). Addition of p34(SEI-1) to cyclin D1-CDK4 renders the complex resistant to inhibition by p16(INK4a). Expression of SEI-1 is rapidly induced on addition of serum to quiescent fibroblasts, and ectopic expression of p34(SEI-1) enables fibroblasts to proliferate even in low serum concentrations. p34(SEI-1) seems to act as a growth factor sensor and may facilitate the formation and activation of cyclin D-CDK complexes in the face of inhibitory levels of INK4 proteins.

Asunto(s)

Proteínas Portadoras/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogénicas , Transactivadores , Animales , Fenómenos Fisiológicos Sanguíneos , Células COS , Ciclo Celular/fisiología , Células Cultivadas , Chlorocebus aethiops , Cromosomas Humanos Par 19/genética , Medio de Cultivo Libre de Suero , Quinasa 4 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina/antagonistas & inhibidores , ADN Complementario/genética , Activación Enzimática , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Genes , Células HeLa , Humanos , Saccharomyces cerevisiae , Factores de Transcripción , Transfección , Técnicas del Sistema de Dos Híbridos

RESUMEN

Cytotoxic T cells and early haemopoietic progenitors share the expression of a number of specific genes. Of these, granzyme B has attracted particular interest because of its role in inducing apoptosis during cytotoxic T cell-mediated target cell killing, and its potential role in the mobilisation and homeostasis of haemopoietic stem cells. Studies of granzyme B regulation should therefore yield valuable information concerning the molecular control of these processes, and also identify elements capable of directing gene expression to two cell types of relevance to gene therapy. Here we show that proximal regulatory elements already known to direct promoter activity in T cells are similarly active in haemopoietic progenitors. However, this activity is not strictly specific, since the promoter regions also direct low levels of reporter gene expression in fibroblasts. More importantly, we also report the presence of two previously unidentified clusters of DNaseI hypersensitive sites upstream from the murine granzyme B gene, and show that these regions impart both increased transcriptional activity and the appropriate cell type specificity on the granzyme B promoter. These upstream regulatory regions are therefore likely to play a key role in the coordination of granzyme B expression in vivo.

Asunto(s)

Células Madre Hematopoyéticas/inmunología , Regiones Promotoras Genéticas , Serina Endopeptidasas/genética , Linfocitos T/inmunología , Animales , Línea Celular , Clonación Molecular , Cósmidos , Desoxirribonucleasa I/metabolismo , Granzimas , Masculino , Ratones

RESUMEN

We have identified a gene that has a high level of mRNA expression in undifferentiated, multipotential hematopoietic cells (FDCP-Mix) and that downregulates both transcript and protein, as these cells are induced to differentiate into mature myeloid cells. Sequence analysis of this gene has identified it as a serine protease inhibitor EB22/3 (serpin 2A). Constitutive expression of serpin 2A in FDCP-Mix cells was associated with an increase in the clonogenic potential of the cells and with a delay in the appearance of fully mature cells in cultures undergoing granulocyte macrophage differentiation when compared with control cells. Serpin 2A was also found to be expressed in bone marrow-derived bipotent granulocyte macrophage progenitor cells (GM-colony forming cell [CFC]), but not in erythrocyte progenitor cells from day 15 fetal liver. Expression of serpin 2A also showed a marked up regulation during the activation of cytotoxic suppressor CD8+ T cells, with a clear lag between the appearance of transcript and detection of protein.

Asunto(s)

Linfocitos T CD8-positivos/metabolismo , Genes , Células Madre Hematopoyéticas/metabolismo , Activación de Linfocitos , Serpinas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Línea Celular , Regulación de la Expresión Génica , Vectores Genéticos , Granulocitos , Células Madre Hematopoyéticas/clasificación , Hígado/embriología , Hígado/metabolismo , Macrófagos , Ratones , Datos de Secuencia Molecular , Serpinas/biosíntesis , Serpinas/genética , Transfección

Asunto(s)

Aziridinas , Benzoquinonas , Clonación Molecular/métodos , Reactivos de Enlaces Cruzados , Hibridación de Ácido Nucleico , Animales , Aziridinas/metabolismo , Benzoquinonas/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , ADN Complementario/genética , ADN Complementario/metabolismo , Biblioteca de Genes , Humanos , ARN/aislamiento & purificación , ARN/metabolismo , ARN Mensajero/aislamiento & purificación

RESUMEN

We describe a method of global PCR amplification of cDNA such that the strand sense is maintained. The products of this process are random primed fragments ranging in size from 100 to 500 bp which facilitates uniform PCR amplification of total cDNA. Directional incorporation of a T7 RNA polymerase initiator/promoter sequence allows efficient synthesis of total sense RNA from this material and the use of a biotinylated primer permits the separation of single-stranded cDNA. Isolation of these products from different cell types provides a renewable source of target single-stranded cDNA and driver RNA from limited cell numbers and we demonstrate their use for subtractive hybridisation cloning of differentially expressed cDNAs.

Asunto(s)

Clonación Molecular , Cartilla de ADN , ADN Complementario/genética , Reacción en Cadena de la Polimerasa/métodos , Northern Blotting , Línea Celular , ADN Complementario/aislamiento & purificación , ADN de Cadena Simple/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/genética , Regiones Promotoras Genéticas , Proteínas Virales

RESUMEN

The product encoded by the latent membrane protein (LMP) gene of Epstein-Barr virus (EBV) has been implicated as a transforming protein by a number of studies. We have examined the effects of LMP expression in FDCP-mix cells, a growth factor-dependent multipotential murine 'stem cell' line. Our studies show that LMP reduces the generation of clonogenic cells and leads to the production of cells expressing a marker (lysozyme M) characteristic of mature monocytes and macrophages. Furthermore, cells expressing LMP are compromised in their ability to produce mature neutrophils. These data suggest that expression of LMP in primitive cells can modulate their self-renewal and differentiation potential and provide evidence in support of the suggestion that EBV may be involved in some of the maturation defects of haemopoiesis.

Asunto(s)

Antígenos Virales/fisiología , Transformación Celular Viral/genética , Células Madre Hematopoyéticas/fisiología , Herpesvirus Humano 4/fisiología , Proteínas de la Membrana/fisiología , Proteínas de la Matriz Viral/fisiología , Animales , Antígenos Virales/genética , Diferenciación Celular/fisiología , Línea Celular Transformada , Herpesvirus Humano 4/genética , Proteínas de la Membrana/genética , Fenotipo , Proteínas de la Matriz Viral/genética

RESUMEN

Using the technique of differential cDNA library screening, we have molecularly cloned a gene that is highly expressed in an undifferentiated myeloid multipotent and growth factor-dependent stem cell line (FDCP-Mix) and that downregulates as these cells are induced to differentiate along monocytic, granulocytic, and erythroid cell lineages. Sequence analysis of this gene has shown homology with a previously cloned gene, cytotoxic cell protease 1 (CCP1 or Granzyme 'B'), that has been shown to be expressed only in thymocytes, activated T cells, a mast cell line, and peritoneal exudate leukocytes. In situ hybridization, Northern blot analysis, and nuclear run-off assay has confirmed that expression of CCP1 is restricted to the phenotypically primitive multipotent undifferentiated. FDCP-Mix cells that are undergoing self-renewal in the presence of growth factors such as interleukin-3.

Asunto(s)

Diferenciación Celular , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Sustancias de Crecimiento/farmacología , Interleucina-3/farmacología , Serina Endopeptidasas/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Línea Celular , Clonación Molecular , Biblioteca de Genes , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Granzimas , Interleucina-6/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , ARN/genética , ARN/aislamiento & purificación , Proteínas Recombinantes/farmacología , Células Madre , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología

Asunto(s)

Sondas de ADN/química , Hibridación de Ácido Nucleico , Reactivos de Enlaces Cruzados

RESUMEN

We have used an antiserum raised against a purified heparan sulfate proteoglycan (HSPG) preparation isolated from rat liver to screen a lambda gt11 expression library and have obtained overlapping cDNA clones that contain the full-length coding sequence of an HSPG core protein capable of spanning the plasma membrane. The open reading frame of the rat cDNA encodes a protein of 211 amino acids. The predicted protein sequence (23 kDa) has a high degree of homology with the published partial sequence of the human lung fibroblast HSPG, fibroglycan. The deduced protein sequence contains a 24-amino acid transmembrane domain and a 33-amino acid cytoplasmic domain, both of which are identical with the corresponding regions of human fibroglycan and are highly homologous to the human, hamster, and mouse epithelial HSPG, syndecan. The putative ectodomain, which has 85% homology to fibroglycan, contains three possible glycosaminoglycan attachment sites that may be occupied by heparan sulfate chains. The major 49-kDa core protein in the liver HSPG preparation was found to be reactive to an antibody that specifically recognizes the cytoplasmic domain of fibroglycan. We have used the full-length cDNA clone to analyze the expression of this transmembrane core protein gene in whole tissues and several epithelial and fibroblastoid cell lines. It hybridizes to three mRNA species in all cell and tissue types examined, but in liver, isolated hepatocytes, and kidney, an additional 0.8-kilobase mRNA was detected. The three common messages arise from differential use of alternative polyadenylation sites, whereas the fourth tissue-restricted RNA species represents a related gene transcript. The rat equivalent of human fibroglycan therefore appears to be the major transmembrane proteoglycan in liver, and its widespread expression in many diverse tissues and cells suggests that it plays an important role in cellular interactions.

Asunto(s)

Heparitina Sulfato/genética , Hígado/metabolismo , Proteoglicanos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , Membrana Celular/metabolismo , Clonación Molecular , ADN/genética , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Proteoglicanos de Heparán Sulfato , Hígado/citología , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Pruebas de Precipitina , ARN Mensajero/genética , Ratas

RESUMEN

The major effect of granulocyte colony-stimulating factor (G-CSF) is to induce neutrophilia in previously untreated animals or after chemotherapy or marrow transplantation in humans, primates and rodents. In addition, it has been reported that migration of committed progenitor cells to the blood occurs during G-CSF therapy. In this article, by using sex mismatched transplants and a molecular probe for Y-chromosome specific DNA sequences, we show that among the peripheral blood cells during G-CSF therapy are substantial numbers of primitive stem cells capable of (1) reconstituting the hematopoietic system in the long term, and (2) making a contribution to the lymphoid populations of the thymus, in radiation ablated recipients. These data suggest that blood from patients treated with G-CSF may provide a convenient source of the most primitive stem cells for autologous or allogeneic bone marrow transplantation.

Asunto(s)

Trasplante de Médula Ósea/patología , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/fisiología , Animales , Trasplante de Médula Ósea/fisiología , Femenino , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/fisiología , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Hematopoyesis/efectos de los fármacos , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Masculino , Ratones , Neutrófilos/efectos de los fármacos

RESUMEN

The complete coding sequence of the Epstein-Barr virus strain B95-8 latent membrane protein (LMP) was cloned using a Raji cell cDNA library and genomic B95-8 DNA. The clone was characterized by sequencing and then used to make a recombinant vaccinia virus. This virus (VLMP) was shown to express a relatively high level of LMP in an authentic fashion. Antisera raised in rabbits against VLMP were shown to react with B95-8 LMP as well as cross-reacting with a 50K cellular protein.

Asunto(s)

Antígenos Virales/genética , Regulación de la Expresión Génica , Herpesvirus Humano 4/genética , Virus Vaccinia/genética , Proteínas de la Matriz Viral , Animales , Anticuerpos Antivirales/análisis , Antígenos Virales/inmunología , Secuencia de Bases , Clonación Molecular , Reacciones Cruzadas , ADN/genética , ADN Viral/genética , Genes Virales , Herpesvirus Humano 4/inmunología , Humanos , Datos de Secuencia Molecular , Conejos , Recombinación Genética , Células Tumorales Cultivadas , Virus Vaccinia/inmunología

RESUMEN

The multicopy mouse Y chromosome DNA probe 80Y/B (1) has been used to probe genomic DNA isolated from male and female bone marrow cells, mixed together in known ratios. It was found that in a mixture with a ratio of 1 male:200 female marrow cells the male cells could readily be detected by this method. The potential use of this technique for assessing repopulation kinetics of marrow transplantation is discussed.

Asunto(s)

Trasplante de Médula Ósea , Sondas de ADN , Cromosoma Y , Animales , Médula Ósea/análisis , Quimera , Femenino , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/análisis , Masculino , Ratones , Hibridación de Ácido Nucleico

RESUMEN

The distribution of N-sulphate groups within fibroblast heparan sulphate chains was investigated. The detergent-extractable heparan sulphate proteoglycan from adult human skin fibroblasts, radiolabelled with [3H]glucosamine and [35S]sulphate, was coupled to CNBr-activated Sepharose 4B. After partial depolymerization of the heparan sulphate with nitrous acid, the remaining Sepharose-bound fragments were removed by treatment with alkali. These fragments, of various sizes, but all containing an intact reducing xylose residue, were fractionated on Sephacryl S-300 and the distribution of the 3H and 35S radiolabels was analysed. A decreased degree of sulphation was observed towards the reducing termini of the chains. After complete nitrous acid hydrolysis of the Sepharose-bound proteoglycan, analysis of the proximity of N-sulphation to the reducing end revealed the existence of an extended N-acetylated sequence directly adjacent to the protein-linkage sequence. The size of this N-acetylated domain was estimated by gel filtration to be approximately eight disaccharide units. This domain appears to be highly conserved, being present in virtually all the chains derived from this proteoglycan, implying the existence of a mechanism capable of generating such a non-random sequence during the post-polymeric modification of heparan sulphate. Comparison with the corresponding situation in heparin suggests that different mechanisms regulate polymer N-sulphation in the vicinity of the protein-linkage region of these chemically related glycosaminoglycans.

Asunto(s)

Proteoglicanos Tipo Condroitín Sulfato , Fibroblastos/análisis , Glicosaminoglicanos , Heparitina Sulfato , Proteoglicanos , Acetilación , Conformación de Carbohidratos , Secuencia de Carbohidratos , Carbohidratos/análisis , Células Cultivadas , Cromatografía en Gel , Proteoglicanos de Heparán Sulfato , Humanos , Unión Proteica , Sulfatos/análisis