RESUMEN

OBJECTIVES: Autoantibodies directed against cytosolic 5'-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5'-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis. MATERIALS AND METHODS: Data from various European inclusion body myositis registries were pooled. Anticytosolic 5'-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. RESULTS: Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5'-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients. INTERPRETATION: Differences were observed in clinical and histopathological features between anticytosolic 5'-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5'-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype.

Asunto(s)

5'-Nucleotidasa/inmunología , Autoanticuerpos/sangre , Fibras Musculares Esqueléticas/patología , Miositis por Cuerpos de Inclusión/sangre , Miositis por Cuerpos de Inclusión/diagnóstico , Edad de Inicio , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Citosol , Complejo IV de Transporte de Electrones/análisis , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Fibras Musculares Esqueléticas/química , Debilidad Muscular/etiología , Miositis por Cuerpos de Inclusión/patología , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Dispositivos de Autoayuda/estadística & datos numéricos , Tasa de Supervivencia , Factores de Tiempo

RESUMEN

This prospective study conducted in the Holy Family Municipal Hospital in Techiman, Ghana aimed to determine the incidence of wound infection following internal fixation of closed fractures in a municipal hospital in a developing country. Between May 2000 and February 2005, 194 patients were treated for closed fractures, implanting a total of 215 internal fixations. Patients were reviewed 10, 30 and 120 days after operation. In 141 (73%) patients, a follow-up of four months was achieved. Of all patients, six developed an infection, two deep and four superficial. The cumulative incidence of wound infection after internal fixation was 3.3%. This study demonstrates that the incidence of wound infection following internal fixation is comparable with hospitals in a temperate climate in industrialized countries. We therefore conclude that specific tropical risk factors play a minimal role in the development of wound infection.

Asunto(s)

Fijación Interna de Fracturas/efectos adversos , Fracturas Cerradas/cirugía , Hospitales Municipales , Infección de la Herida Quirúrgica/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Femenino , Fracturas Óseas/cirugía , Ghana/epidemiología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Infección de la Herida Quirúrgica/etiología

RESUMEN

BACKGROUND: Acute exacerbations are a characteristic clinical expression of chronic obstructive pulmonary disease (COPD). The objective of this study was to investigate the occurrence rate, management, and healthcare costs of exacerbations in patients with COPD in Dutch general practice. METHODS: Baseline data set from the COPD on Primary Care Treatment (COOPT) trial was used. Details on the occurrence and management of exacerbations were collected by systematic medical record review for the 2-year period preceding trial inclusion. RESULTS: The mean age of the 286 study subjects involved was 59.2 (SD 9.6) years, postbronchodilator FEV1 67.1% (SD 16.2) of predicted. Following ERS criteria, subjects suffered from: no (26%); mild (19%); moderate (40%); or severe (15%) airflow obstruction. The overall mean and median annual exacerbation rates were 0.88 (SD 0.79) and 0.5 (IQR 1.0), respectively. Exacerbation rate was not related to severity of airflow obstruction (p=0.628). Mean annual exacerbation costs per subject were 40 Euro, 53 Euro, 61 Euro and 92 Euro for the respective severity subgroups (p=0.012). The increase of costs in the more severe subgroups was mainly attributable to more physician consultations, diagnostic procedures, and prescription of reliever medication (e.g., bronchodilators, cough preparations). CONCLUSIONS: Occurrence of exacerbations did not depend on the severity of airflow obstruction, whereas the healthcare cost associated with exacerbations increased along with the severity of the disease.

Asunto(s)

Costos de la Atención en Salud , Enfermedad Pulmonar Obstructiva Crónica/economía , Anciano , Análisis de Varianza , Antitusígenos/uso terapéutico , Broncodilatadores/uso terapéutico , Distribución de Chi-Cuadrado , Tos/tratamiento farmacológico , Recolección de Datos , Medicina Familiar y Comunitaria , Femenino , Humanos , Masculino , Persona de Mediana Edad , Países Bajos , Enfermedad Pulmonar Obstructiva Crónica/clasificación , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Ensayos Clínicos Controlados Aleatorios como Asunto , Pruebas de Función Respiratoria , Estaciones del Año

RESUMEN

WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors. The physiological role of WSX-1, which is expressed mainly in T cells, was investigated in gene-targeted WSX-1-deficient mice. IFN-gamma production was reduced in isolated WSX-1(-/-) T cells subjected to primary stimulation in vitro to induce Th1 differentiation but was normal in fully differentiated and activated WSX-1(-/-) Th1 cells that had received secondary stimulation. WSX-1(-/-) mice were remarkably susceptible to Leishmania major infection, showing impaired IFN-gamma production early in the infection. However, IFN-gamma production during the later phases of the infection was not impaired in the knockout. WSX-1(-/-) mice also showed poorly differentiated granulomas with dispersed accumulations of mononuclear cells when infected with bacillus Calmette-Guerin (BCG). Thus, WSX-1 is essential for the initial mounting of Th1 responses but dispensable for their maintenance.

Asunto(s)

Leishmania major , Leishmaniasis Cutánea/inmunología , Receptores de Citocinas/fisiología , Células TH1/inmunología , Animales , Diferenciación Celular , División Celular , Células Cultivadas , Granuloma/patología , Sistema Hematopoyético/fisiología , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-4/biosíntesis , Interleucina-4/genética , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/patología , Sistema Linfático/inmunología , Ratones , Ratones Noqueados , Mycobacterium bovis , ARN Mensajero/biosíntesis , Receptores de Citocinas/genética , Receptores de Interleucina , Tuberculosis/patología

RESUMEN

In this report, we tested whether ectopic overexpression of a cell surface receptor cDNA could be used to explore the physiological roles of that receptor. We generated c-mpl overexpressing animals by reconstituting mice with retroviral vector-transduced bone marrow (BM) cells. We observed that platelet counts in the c-mpl overexpressing mice failed to recover to normal levels and remained at <200 x 10(6)/mL post-transplantation, while platelet numbers in the control mice returned to > 800 x 10(6)/mL by 4 weeks post-transplantation. However, platelet counts in the c-mpl overexpressing mice could be stimulated to normal levels after administration of rhMGDF. No significant changes in peripheral leukocyte counts were observed, although the number of CFU-E, GM-CFC, and CFC-multi were reduced two- to threefold in the BM of the c-mpl overexpressing mice. In addition, enhanced erythropoiesis was observed in the c-mpl overexpressing mice. The mpl receptors on erythroid cells were functional as demonstrated by tyrosine-phosphorylation of mpl receptor on RBC and by in vitro erythroid colony-formation in response to MGDF stimulation, respectively. These results suggested that ectopically expressed mpl receptors competed for ligand in vivo leading to an insufficient amount of circulating thrombopoietin (Tpo) for the development of megakaryocytic lineage. These results further suggest that, in addition to sequestering circulating Tpo, overexpression of the mpl receptor on erythroid progenitors may directly contribute to enhanced erythropoiesis in vivo. Our studies demonstrate that ectopic overexpression of a receptor by retroviral-mediated gene transfer provides an approach to explore the biological roles of novel receptors.

Asunto(s)

Eritropoyesis , Hematopoyesis , Megacariocitos/citología , Proteínas de Neoplasias , Proteínas Proto-Oncogénicas/biosíntesis , Receptores de Citocinas , Animales , Ensayo de Unidades Formadoras de Colonias , ADN Complementario/genética , Vectores Genéticos/genética , Factores de Crecimiento de Célula Hematopoyética/farmacología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/virología , Recuento de Leucocitos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Proteínas Proto-Oncogénicas/genética , Provirus/genética , Receptores de Trombopoyetina , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/fisiología , Proteínas Recombinantes/farmacología , Retroviridae/genética , Trombopoyetina/farmacología , Trombopoyetina/fisiología , Transfección

RESUMEN

Immature myeloid cells have been shown to transduce signals through a carboxyl-terminally truncated isoform of Stat5. This functionally distinct signal transducer and activator of transcription isoform is generated through a unique protein-processing event. Evaluation of numerous cell lines has determined that there is a direct correlation between the expression of truncated Stat5 and protease activity. Moreover, protease activity is found only in the myeloid and not in lymphoid progenitors. To further characterize the protease small quantities have been purified to near homogeneity. Studies on this purified material indicate that the protease has an apparent molecular mass of approximately 25 kDa and is active over a wide range of pH values. The protease will also cleave both activated (i.e. tyrosine-phosphorylated) and inactivate Stat5. Although this activity is sensitive to phenylmethylsulfonyl fluoride, it is notably not sensitive to several other serine protease inhibitors. Additional studies have led to the identification of the unique site where the protease cleaves Stat5. Mutagenesis of this site renders Stat5 resistant to cleavage. Consistent with the model that Stat5 cleavage is important for early myeloid development, introduction of a "non-cleavable" isoform of Stat5 into FDC-P1 cells (a myeloid progenitor line) leads to significant phenotypic changes.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Endopeptidasas/metabolismo , Proteínas de la Leche , Transactivadores/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/enzimología , Diferenciación Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/genética , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Ratones , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Factor de Transcripción STAT5 , Transducción de Señal , Transactivadores/genética

RESUMEN

Two interesting representatives of a new generation of platinum-based cytostatic drugs that are currently being tested in clinical trials are lobaplatin [1,2-diaminomethylcyclobutane platinum(II) lactate] and oxaliplatin [1,2-diaminocyclohexane platinum (II) oxalate]. Since little is known about the DNA adduct formation of these compounds, we studied their formation in DNA in vitro in calf thymus DNA and in cells. The major adducts formed in vitro were the Pt-GG and Pt-AG intrastrand crosslinks. The latter adducts could be detected using a recently developed 32P-postlabelling method. Using both this assay and atomic absorption spectroscopy, it was shown that there is a substantially higher rate of the in vitro adduct formation by cisplatin, compared with lobaplatin and oxaliplatin. Platinum concentrations required to obtain 90% cell kill during a 2 h incubation of A2780 cells were 15 microM for cisplatin and oxaliplatin and 22 microM for lobaplatin. Using an antiserum originally raised against cisplatin-treated DNA, we were also able to detect platinum-DNA adducts induced by lobaplatin and oxaliplatin. Maximal nuclear staining for all three compounds was observed after a 4 h post-incubation period. The nuclear staining level induced by cisplatin was about 10-fold higher than after lobaplatin and oxaliplatin treatment. GG and AG adducts, measured by 32P-postlabelling, also showed maximum levels at about 4 h after treatment. Relative GG peak levels were 4:1:3 for cisplatin, lobaplatin and oxaliplatin, respectively. The ratios of GG over AG intrastrand crosslinks in the A2780 cells were not significantly different for the various compounds. In conclusion, the 32P-postlabelling technique has been shown to be appropriate for adduct analysis, not only for the classical Pt compounds cisplatin and carboplatin but also for novel platinum compounds like lobaplatin and oxaliplatin. Results indicated large differences in reactivity of the latter compounds to DNA in vitro, compared with cisplatin. This difference was smaller in cells, suggesting enhancement of adduct formation by certain cellular mechanisms and/or compounds. From these studies, no conclusions can be drawn with respect to the cytotoxicity of the different Pt-GG and Pt-AG intrastrand crosslinks formed by these compounds.

Asunto(s)

Antineoplásicos/metabolismo , Cisplatino/metabolismo , Ciclobutanos/metabolismo , Aductos de ADN/análisis , Compuestos Organoplatinos/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Oxaliplatino , Soluciones , Células Tumorales Cultivadas

RESUMEN

p36 (also termed annexin II) is a 39 kDa Ca2+/phospholipid-binding, membrane-associated protein that is a protein-tyrosine kinase substrate. We report here studies of the noncoding exons of p36, which combined with our earlier studies of the coding exons, allow us to conclude that the murine p36 gene is 34 kb in length with 14 exons. Comparison of the genes coding for mouse and human p36 (annexin II) and mouse, rat and human p35 (annexin I) and pigeon cp35 (an annexin I-related protein) shows strong genomic structural conservation supporting the hypothesis that these genes had a common ancestor. Both human and murine p36 mRNAs were found to be alternatively spliced in their 5' noncoding region. In both cases exon 2 is a cassette exon, which is present in a small fraction of p36 mRNAs. In type 1 mouse p36 mRNA the first noncoding 44 base exon 1 is joined to exon 3, the first of the 12 coding exons. In type 2 mRNA a 70 base noncoding exon (exon 2) is inserted between exon 1 and exon 3. Type 1 mRNA was present in all cell types studied as revealed by Northern analysis and primer extension, whereas type 2 mRNA could only be detected by RACE or PCR, indicating that it is of very low abundance. The major transcription start site of the mouse p36 gene was mapped by primer extension to be 61 bp upstream of the AUG initiation codon, which corresponds to type 1 mRNA, The murine p36 gene enhancer/promoter region contains a putative TATA box and several other potential regulatory sequences. The two alternatively-spliced human p36 mRNAs differ by the presence or absence of a noncoding 81 base exon (exon 2) inserted after exon 1, with exon 2-containing mRNAs representing approximately 10% of total p36 mRNA. The 300 bp spanning the promoter and exons 1-3 of the human and murine p36 genes show strong sequence homology immediately before and after the major transcription start site except in the region corresponding to exon 2, where homology is more limited.

Asunto(s)

Empalme Alternativo , Anexina A2/genética , Exones , ARN Mensajero/genética , Células 3T3 , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , ADN Complementario , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Secuencias Reguladoras de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Transcripción Genética

RESUMEN

Megakaryocyte growth and development factor (MGDF) has recently been identified as a ligand for the c-mpl receptor. Using retroviral-mediated gene transfer, MGDF has been overexpressed in mice to evaluate the systematic effects due to chronic exposure to this growth factor. MGDF overexpressing mice had more rapid platelet recovery than control mice after transplantation. Following this recovery, the platelet levels continued increasing to fourfold to eightfold above normal baseline levels and remained elevated (five-fold above control mice) in these animals, which are alive and well at more than 4 months posttransplantation. Increased megakaryocyte numbers were detected in a number of organs in these mice including bone marrow, spleen, liver, and lymph nodes. Prolonged overexpression of MGDF led to decreased marrow hematopoiesis, especially erythropoiesis, with a shift to extramedullary hematopoiesis in the spleen and liver. All the MGDF overexpressing mice analyzed to date developed myelofibrosis and osteosclerosis, possibly induced by megakaryocyte and platelet produced cytokines. No significant effect on other hematopoietic lineages was seen in the MGDF overexpressing mice, showing that the stimulatory effect of MGDF in vivo is restricted to the megakaryocyte lineage.

Asunto(s)

Megacariocitos/citología , Receptores de Citocinas/fisiología , Receptores Inmunológicos/fisiología , Trombopoyetina/fisiología , Animales , Trasplante de Médula Ósea , Diferenciación Celular , División Celular , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Masculino , Megacariocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Osteosclerosis/etiología , Osteosclerosis/patología , Mielofibrosis Primaria/etiología , Mielofibrosis Primaria/patología , Receptores de Citocinas/genética , Receptores Inmunológicos/genética , Retroviridae/genética , Bazo/citología , Bazo/trasplante , Trombocitosis/etiología , Trombopoyetina/genética

RESUMEN

Recently, the ligand for c-mpl, a member of the family of cytokine receptors, was cloned and found to be a physiologic regulator of platelet homeostasis. We report that megakaryocyte growth and development factor (MGDF, thrombopoietin [TPO], c-mpl ligand ) induces differentiation in a majority of mpl-transfected 32D cells, while interleukin (IL)-3 is exclusively mitogenic in this system. MGDF differentiation, as measured by decreased proliferation, changes in cellular morphology, increased adherence, and downregulation of very late antigen (VLA)-4, is dominant over IL-3 proliferation. MGDF induces tyrosine-phosphorylation of mpl, JAK2, SHC, SHPTP-1 (HCP, motheaten) and SHPTP-2 (Syp, PTP-1D) within 30 seconds of stimulation, as well as of vav and MAPK with slightly delayed kinetics. A fraction of mpl and JAK2 is preassociated, and the stoichiometry of this complex is unaltered by cytokine stimulation. After MGDF stimulation, we detect interactions among SHC, grb2, SHPTP-1, SHPTP-2, and the mpl/JAK2 complex. IL-3 induces phosphorylation of the above proteins with the exception of mpl and also causes weak JAK1 phosphorylation. Although similar in composition, the MGDF- and IL-3-induced complexes of signal transducers appear to be assembled in different configurations, especially with respect to SHPTP-2. Both MGDF and IL-3 induce tyrosine phosphorylation of STAT3 (APRF) and STAT5 (MGF), with MGDF favoring STAT3 while IL-3 predominantly causes STAT5 phosphorylation. In addition, some proteins become tyrosine-phosphorylated in response to MGDF only, suggesting that we may have detected differentiation-specific signal transducers. These include a number of high-molecular-weight proteins (140 to 200 kD) and one 28-kD protein that becomes tyrosine-phosphorylated only briefly.

Asunto(s)

Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-3/farmacología , Megacariocitos/citología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transducción de Señal/fisiología , Trombopoyetina/farmacología , Trombopoyetina/fisiología , Secuencia de Aminoácidos , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Perros , Activación Enzimática/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Ratones , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Proteínas Quinasas/metabolismo , Receptores de Interleucina-3/efectos de los fármacos , Receptores de Interleucina-3/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Trombopoyetina/efectos de los fármacos , Trombopoyetina/genética , Factores de Transcripción/metabolismo , Transfección

RESUMEN

Within the group of DNA alkylation products, phosphotriesters (PTE) are among the most stable lesions. Hence, alkyl PTE are attractive biomarkers for DNA alkylation monitoring purposes. We have developed a 32P-postlabelling method for the analysis of both methyl and ethyl PTE in DNA. Since PTE bonds are not cleaved by any known DNA degrading enzyme, they are easily obtainable as PTE dinucleoside monophospates. A purification step, separating the PTE dinucleoside monophosphates from interfering compounds, such as mono- or oligonucleotides resulting from incomplete digestion of DNA, was developed using Waters C18 Sep-Pak cartridges. Phosphotriester dinucleoside monophosphates themselves are not a substrate for phosphorylation by polynucleotide kinase. Polynucleotide kinase probably requires a negative charge on the phosphate closest to the 5'-end. Therefore, prior to the post-labelling step they have to be converted into either phosphodiester dinucleoside monophosphates or 3'-phosphate alkylated mononucleotides by treatment with alkali. For analysis of the labelled compounds we developed a two-step procedure, combining TLC and HPLC, that gave very straightforward information on the composition of the rather complex mixture. The detection limit is approximately fmol PTE.

Asunto(s)

Daño del ADN , ADN/química , Marcaje Isotópico/métodos , Organofosfatos/análisis , Alquilación , Secuencia de Bases , ADN/metabolismo , Reparación del ADN , Datos de Secuencia Molecular , Radioisótopos de Fósforo

RESUMEN

Despite the importance of blood platelets in health and disease, the mechanisms regulating their formation within megakaryocytes are unknown. We generated mice lacking the hematopoietic subunit (p45) of the heterodimeric erythroid transcription factor NF-E2. Unexpectedly, NF-E2-/- mice lack circulating platelets and die of hemorrhage; their megakaryocytes show no cytoplasmic platelet formation. Though platelets are absent, serum levels of the growth factor thrombopoietin/MGDF are not elevated above controls. Nonetheless, NF-E2-/- megakaryocytes proliferate in vivo in response to thrombopoietin administration. Thus, as an essential factor for megakaryocyte maturation and platelet production, NF-E2 must regulate critical target genes independent of the action of thrombopoietin. These findings provide insight into the genetic analysis of megakaryocyte maturation and thrombopoiesis.

Asunto(s)

Plaquetas/fisiología , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Megacariocitos/fisiología , Trombopoyetina/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Células Sanguíneas/citología , Células de la Médula Ósea , Diferenciación Celular , ADN/análisis , Cartilla de ADN , Proteínas de Unión al ADN/genética , Células Precursoras Eritroides/patología , Factores de Unión al ADN Específico de las Células Eritroides , Megacariocitos/patología , Megacariocitos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Factor de Transcripción NF-E2 , Subunidad p45 del Factor de Transcripción NF-E2 , Reacción en Cadena de la Polimerasa , Trombocitopenia , Factores de Transcripción/genética

RESUMEN

We developed a sensitive 32P-postlabeling method for the detection of bifunctional intrastrand crosslinks d(Pt-GpG) and d(Pt-ApG) in DNA in vitro and in vivo. After enzymatic digestion of DNA the positively charged platinum adducts were purified from unplatinated products, using strong cation exchange chromatography. Subsequently the samples were deplatinated with cyanide, because platinated dinucleotides are very poor substrates for polynucleotide kinase. The excess of cyanide was removed using Sep-pak C18 cartridges, and the resulting dinucleoside monophosphates d(GpG) and d(ApG) were subsequently postlabelled. Analysis of the postlabelling mixture was performed by a combined TLC and HPLC-procedure. Good correlations with existing methods (AAS, immunocytochemistry and ELISA) were found in DNA samples treated in vitro and in vivo with cis- or carboplatin. The detection limit of the assay was 1 adduct/10(7) nucleotides in a 10 micrograms DNA sample.

Asunto(s)

Aductos de ADN/análisis , Marcaje Isotópico/métodos , Platino (Metal)/análisis , Animales , Carboplatino/farmacología , Cromatografía por Intercambio Iónico , Cisplatino/farmacología , ADN/efectos de los fármacos , Aductos de ADN/aislamiento & purificación , Femenino , Radioisótopos de Fósforo , Platino (Metal)/química , Ratas , Ratas Sprague-Dawley , Sensibilidad y Especificidad

RESUMEN

The interleukin (IL)-3 family of cytokines mediates its numerous effects on myeloid growth and maturation by binding a family of related receptors. It has been shown recently that IL-3 induces the activation of two distinct cytoplasmic signal transducing factors (STFs) that are likely to mediate the induction of immediate early genes. In immature myeloid cells, IL-3 activates STF-IL-3a, which comprises two tyrosine-phosphorylated DNA binding proteins of 77 and 80 kDa. In mature myeloid cells, IL-3 and granulocyte-macrophage colony-stimulating factor activate STF-IL-3b, which consists of a 94 and 96 kDa tyrosine-phosphorylated DNA binding protein. Peptide sequence data obtained from the purified 77 and 80 kDa proteins (p77 and p80) indicate that they are closely related but are encoded by distinct genes. Both peptide and nucleotide sequence data demonstrate that these two proteins are the murine homologs of ovine mammary gland factor (MGF)/Stat5. The peptide data also indicate that p77 and p80 are phosphorylated on tyrosine 699, a position analogous to the tyrosine that is phosphorylated in Stat1 and Stat2 in response to interferon. Additionally, antiserum raised against bacterially expressed p77/p80 recognizes the 94 and 96 kDa protein components of STF-IL-3b, suggesting that these may be additional isoforms of Stat5. These studies indicate that the IL-3 family of ligands is able to activate multiple isoforms of the signal transducing protein Stat5.

Asunto(s)

Proteínas de Unión al ADN/biosíntesis , Interleucina-3/farmacología , Proteínas de la Leche , Transducción de Señal/efectos de los fármacos , Linfocitos T/fisiología , Transactivadores/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/fisiología , Biblioteca de Genes , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Ratones , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fosfotirosina , Factor de Transcripción STAT5 , Homología de Secuencia de Aminoácido , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Transactivadores/aislamiento & purificación , Transactivadores/fisiología , Tirosina/análogos & derivados , Tirosina/metabolismo

RESUMEN

The reactivity of the N-acetoxy metabolite of 2-amino-5-phenylpyridine (Phe-P-1), a pyrolysis product of phenylalanine, towards 2'-deoxyguanosine (dG), 2'-deoxyguanosine-3'-monophosphate (dGMP) and DNA was studied and compared with that of the ortho-methyl derivative. Reaction of 2-acetoxyamino-5-phenylpyridine (N-OAc-APP) with dG resulted in substitution at the 8-position of this nucleoside by the ortho carbon of the amine. The major reaction, however, was acetylation of dG. In contrast, 2-acetoxyamino-3-methyl-5-phenyl-pyridine (N-OAc-MeAPP) mainly attacked the 8-position of dG by the exocyclic nitrogen and hardly any acetylation of the nucleoside occurred. The adducts were chromatographically isolated and characterized by their mass and NMR spectra. Upon reaction of N-acetoxy compounds with DNA and dGMP, formation of the same adducts was observed, besides the formation of minor amounts of unidentified compounds, as was established by 32P-postlabelling analysis. The amount of DNA-bound amine, formed by the interaction of N-OAc-APP with DNA, was approximately 15 times smaller than that observed after the reaction with the corresponding ortho-methyl derivative under the same conditions.

Asunto(s)

Aminopiridinas/metabolismo , Aminopiridinas/toxicidad , Carcinógenos/metabolismo , Carcinógenos/toxicidad , Animales , Bovinos , Cromatografía Liquida , Aductos de ADN/biosíntesis , Daño del ADN , Nucleótidos de Desoxiguanina/metabolismo , Desoxiguanosina/metabolismo , Espectrometría de Masas , Relación Estructura-Actividad

RESUMEN

Thrombocytopenia is a condition of multiple etiologies affecting the megakaryocyte lineage. To perturb this lineage in transgenic mice, the tsA58 mutation of the simian virus 40 large tumor antigen was targeted to megakaryocytes using the platelet factor 4 promoter. Ten of 17 transgenic lines generated exhibited low platelet levels, each line displaying a distinct, heritable level of thrombocytopenia. Within a line, the degree of the platelet reduction correlated directly with transgene zygosity. The platelet level could be further reduced by the inactivation of one copy of the endogenous retinoblastoma gene. Western blot analysis detected large tumor antigen protein in the most severely affected lines; less affected lines were below the level of detection. Platelets and megakaryocytes from thrombocytopenic mice exhibited morphological abnormalities. Mice with either normal or reduced platelet levels developed megakaryocytic malignancies with a mean age of onset of about 8 months. There was no correlation between severity of thrombocytopenia and onset of malignancy. These mice provide a defined genetic model for thrombocytopenia, and for megakaryocytic neoplasia, and implicate the retinoblastoma protein in the process of megakaryocyte differentiation.

Asunto(s)

Antígenos Transformadores de Poliomavirus/genética , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis , Leucemia Experimental/genética , Megacariocitos/citología , Trombocitopenia/genética , Animales , Secuencia de Bases , Diferenciación Celular , Cartilla de ADN/química , Femenino , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , ARN Mensajero/genética , Proteína de Retinoblastoma/genética

RESUMEN

The mouse Pim-1 gene encodes two cytoplasmic serine-threonine-specific protein kinases. The gene has been found to be activated (overexpressed) by retroviral insertion in hematopoietic tumors in mice. Transgenic mice that overexpress Pim-1 (E mu-Pim-1) have a low incidence of spontaneous T-cell lymphomas and an increased susceptibility to Moloney murine leukemia virus and N-ethyl-N-nitrosourea-induced lymphomas. Apart from a slight enlargement of the spleen, no abnormalities were found in prelymphomatous transgenic mice. Inactivation of the Pim-1 gene in the germline of mice resulted in mice with a surprisingly subtle phenotype. Therefore, we investigated whether subtle effects of the absence of Pim-1 could be made visible during in vitro culturing of hematopoietic cells. We found that bone marrow-derived mast cells (BMMC) lacking Pim-1 had a distinct growth disadvantage when grown on interleukin (IL)-3, but not when stimulated by the factors IL-4, IL-9, or Steel factor (SF). This indicates a role for Pim-1 as a modulator of the IL-3 signal transduction pathway.

Asunto(s)

Células de la Médula Ósea , Interleucina-3/fisiología , Mastocitos/química , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/deficiencia , Animales , Diferenciación Celular , División Celular/efectos de los fármacos , Células Cultivadas , Factores de Crecimiento de Célula Hematopoyética/farmacología , Tejido Linfoide/citología , Ratones , Ratones Mutantes , Proteínas Proto-Oncogénicas c-pim-1 , Factor de Células Madre

RESUMEN

The Pim-1 gene has frequently been found activated by proviral insertion in haematopoietic tumors in mice. The fact that overexpression of Pim-1 can contribute to lymphomagenesis was formally proven by overexpressing a Pim-1 transgene in lymphoid cells. The transgene induces a low incidence of T cell lymphomas and an increased susceptibility to chemically (ENU) and virally (MoMuLV) induced lymphomas. The mouse Pim-1 gene encodes two cytoplasmic protein-serine/threonine kinases. Northern analysis shows the highest expression to be in haematopoietic tissues, especially early in development. High expression has also been noted in testis and ES cells. Expression can be induced by growth factors and mitogens. The gene is evolutionarily highly conserved. Inactivation of both Pim-1 alleles in ES cells or mice did not reveal any obvious abnormalities. In order to look more closely for possible haematopoietic abnormalities specific growth factor response were studied in vitro. The IL-3 response of bone marrow-derived mast-cell cultures (BMMC) was found to be severely impaired in mast cells derived from Pim-1 deficient mice.

Asunto(s)

Células de la Médula Ósea , Mastocitos/citología , Ratones Transgénicos/genética , Proteínas Serina-Treonina Quinasas , Proto-Oncogenes/fisiología , Alelos , Animales , Secuencia de Bases , Médula Ósea/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Genotipo , Interleucina-3/farmacología , Mastocitos/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-pim-1 , Proto-Oncogenes/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

We have developed a highly sensitive 32P-postlabelling assay for the detection of alkylphosphotriesters in DNA using selective enzymatic DNA hydrolysis, sample preparation by C18 Sep-Pak solid phase extraction and product analysis by HPLC. The assay provides information on the quantity of the alkylphosphotriesters and gives some clues with respect to their identity.

Asunto(s)

ADN/química , Radioisótopos de Fósforo , Alquilación , Animales , Bovinos , ADN/análisis , Daño del ADN , Estudios de Evaluación como Asunto , Humanos , Hidrólisis , Técnicas In Vitro , Métodos , Fosforilación , Ratas

RESUMEN

The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.

Asunto(s)

Reparación del ADN/genética , Replicación del ADN/genética , Nucleótidos de Desoxiguanina/genética , Desoxiguanosina/análogos & derivados , 8-Hidroxi-2'-Desoxicoguanosina , Secuencia de Bases , Nucleótidos de Desoxiguanina/metabolismo , Desoxiguanosina/genética , Desoxiguanosina/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Pruebas de Mutagenicidad , Oligodesoxirribonucleótidos/genética , Plásmidos/genética