RESUMEN
The availability of bromodomain and extra-terminal inhibitors (BETi) has enabled translational epigenetic studies in cancer. BET proteins regulate transcription by selectively recognizing acetylated lysine residues on chromatin. BETi compete with this process leading to both downregulation and upregulation of gene expression. Hypoxia enables progression of triple negative breast cancer (TNBC), the most aggressive form of breast cancer, partly by driving metabolic adaptation, angiogenesis and metastasis through upregulation of hypoxia-regulated genes (for example, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A). Responses to hypoxia can be mediated epigenetically, thus we investigated whether BETi JQ1 could impair the TNBC response induced by hypoxia and exert anti-tumour effects. JQ1 significantly modulated 44% of hypoxia-induced genes, of which two-thirds were downregulated including CA9 and VEGF-A. JQ1 prevented HIF binding to the hypoxia response element in CA9 promoter, but did not alter HIF expression or activity, suggesting some HIF targets are BET-dependent. JQ1 reduced TNBC growth in vitro and in vivo and inhibited xenograft vascularization. These findings identify that BETi dually targets angiogenesis and the hypoxic response, an effective combination at reducing tumour growth in preclinical studies.
Asunto(s)
Azepinas/farmacología , Anhidrasa Carbónica IX/metabolismo , Hipoxia/metabolismo , Neovascularización Patológica , Triazoles/farmacología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Animales , Anhidrasa Carbónica IX/genética , Línea Celular Tumoral , Análisis por Conglomerados , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Regiones Promotoras Genéticas , Unión Proteica , Esferoides Celulares , Transcriptoma , Neoplasias de la Mama Triple Negativas/genética , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bovine leukaemia virus (BLV) is an oncogenic retrovirus that causes B-cell lymphocytosis and in the terminal stage of the disease lymphosarcoma. The comparison of the previously published BLV provirus sequence from Belgium, Australia and Japan showed that the protease gene (prt) of the Australian and the Japanese isolate contain a nucleotide deletion when compared to the Belgian isolate. Because all these proviruses were isolated from tumour tissue, the prt gene of functionally active and infectious proviruses from peripheral blood leucocytes (PBLs) of BLV-infected cattle and from BLV-infected fetal lamb kidney cells were sequenced. The only variations between these sequences and the Belgian isolate consist of nucleotide substitutions. The delection of one nucleotide of the prt gene of the Japanese and the Australian BLV tumour isolate caused a changed reading frame and a premature translational stop. It was shown that the Japanese provirus is non-infectious in transfected cell culture and in injected sheep. To analyse the impact of the prt mutation on viral protein expression and infectivity, the prt region of the Japanese provirus was exchanged with the prt region from the Belgian provirus. The resulting pBLVprtbelg was infectious in transfected cells and enabled the expression of gag and gag-precursor proteins. One sheep was injected with this mutated provirus and became positive in BLV-PCR, but no seroconversion was developed. The prt mutation of the Japanese tumour isolates was shown to be responsible for the loss of infectivity and changed viral expression. These results and the occurrence of this mutation in only two isolates from lymphosarcoma indicate a possible relation between the prt mutation and the induction of cell transformation.
Asunto(s)
Leucosis Bovina Enzoótica/virología , Eliminación de Gen , Regulación Viral de la Expresión Génica , Virus de la Leucemia Bovina/genética , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Cartilla de ADN , ADN Viral/aislamiento & purificación , Endopeptidasas/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/veterinaria , Biosíntesis de Proteínas , Sistemas de Lectura/genética , Alineación de Secuencia/veterinaria , Ovinos , Proteínas Virales/químicaRESUMEN
According to the contextual change theory of memory loss, spontaneous forgetting reflects a retrieval impairment due to subtle and unprogrammed shifts in environmental cues over a retention interval. However, Riccio, Richardson, and Ebner (1984) noted an apparent paradox in this model; specifically, laboratory studies inducing explicit shifts in contextual cues found less disruption of performance as retention intervals increased. Bouton, Nelson, and Rosas (1999) critiqued several of the claims made by Riccio et al. and concluded that the contextual cue theory is still a valid account of spontaneous forgetting. In this comment, the authors address the 3 major criticisms offered by Bouton et al., point out an inconsistency in their argument, and conclude that the original paradox still poses problems for the contextual change theory of forgetting.
Asunto(s)
Memoria/fisiología , Animales , HumanosRESUMEN
In a retrospective study, the long term outcome of the modified Watson-Jones tenodesis according to Lemberger and Kramer was determined using a questionnaire, clinical examination, radiographic data, including stress views, measurement of plantar pressure distribution, and peroneal reaction times on a tilt board. Twenty-five male patients (mean age, 34 years) with a mean followup of 12 years from surgery were available for examination. Eighteen patients (72%) were classified clinically as having excellent or good results. The higher presence of osteophytes in the surgically treated ankle in comparison with the opposite side indicated the progression of arthrosis with time, but this finding could not be related to the reconstruction method. Anterior drawer and talar tilt were reduced significantly in comparison with the preoperative stress radiographs. No differences in plantar pressure distribution were seen between the patients' surgically treated and nonsurgically treated feet. The peroneal reaction times of the peroneus brevis and peroneus longus muscles were significantly shorter in the surgically treated foot compared with the opposite side. It was concluded that the modified Watson-Jones tenodesis effectively corrected lateral ankle instability with no clinical deterioration with time and no influence on gait.
Asunto(s)
Articulación del Tobillo , Inestabilidad de la Articulación/cirugía , Transferencia Tendinosa , Adolescente , Adulto , Fenómenos Biomecánicos , Estudios de Seguimiento , Marcha , Humanos , Inestabilidad de la Articulación/fisiopatología , Masculino , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
237 cattle of a dairy herd in Syria were tested for anti-BLV antibody by the ELISA. 194 animals were additionally examined by the agar gel immunodiffusions test (AGID) on BLV antibodies and 100 by polymerase chain reaction (PCR) for BLV provirus. BLV specific antibodies were determined by means of AGID and ELISA at 62.9% and 69.2% of the examined animals, respectively. Using the PCR method the BLV provirus was detected in 89% of the investigated cattle. Only one ELISA seropositive animal was negative for BLV provirus. The results show the high BLV contamination of this herd and lead to the presumption of wide spread enzootic bovine leukosis in Syria. In the case of the diagnosis of BLV-infection, the PCR-technique compared to the serological tests proved to be much more sensitive. By the detection of BLV antibody, the ELISA showed a higher sensitivity than the AGID and in this way, is advisable as a method of choice for screening investigations. Restriction enzyme and sequence analysis of PCR-amplificates demonstrate that different BLV provirus variants (A, B and C) in the examined herd occur, where the variant C which a high similarity to an Australian BLV provirus isolates showed, occurred most frequently at 92.5%.
Asunto(s)
Leucosis Bovina Enzoótica/diagnóstico , Virus de la Leucemia Bovina/aislamiento & purificación , Animales , Bovinos , Leucosis Bovina Enzoótica/epidemiología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Inmunodifusión/métodos , Reacción en Cadena de la Polimerasa/métodos , Pruebas Serológicas/métodos , Siria/epidemiologíaRESUMEN
A typical infection with bovine leukemia virus (BLV) induces a permanent antibody (Ab) response with high titers against BLV-antigens. In the last few years atypical courses of infection with low or transient BLV-Ab-titers or even lack of any detectable BLV-Ab-titers in animals with BLV-provirus integration have been described. This makes it difficult to eliminate BLV infection from herds using serological assays only. Whether or not polymerase chain reaction (PCR) is a useful tool to complement serological Ab-assays in BLV-eradication in herds was clarified in three ways: (i) different DNA-quick-preparations of blood were examined in nested PCR, (ii) cows of a BLV infected herd that was involved in a national eradication program were investigated for 6 months und (iii) BLV-provirus-variants occurring in this herd were differentiated. The results show, that even by using PCR it was not possible to detect all infected animals all the time and that eradication of BLV from this herd was not completed in this short time. The PCR is useful for the investigation of herds and more sensitive than ELISA. PCR using LTR-primers (34 positive cattle) was more sensitive than PCR with env-primers (30 positive cattle). Using PCR 34 BLV infected cattle were detected of which only 21 reacted in ELISA. Restriction enzyme analysis or sequence analysis of PCR-amplificates allowed the detection of virus variants and conclusions about the way of infection. PCR should be used for BLV-eradication in cattle herds with low BLV-incidence, for the investigation of new outbreaks or tumor cases in long term BLV free herds and for investigation of breeding cattle.
Asunto(s)
ADN Viral/química , Leucosis Bovina Enzoótica/diagnóstico , Virus de la Leucemia Bovina/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Secuencia de Bases , Bovinos , ADN Viral/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Datos de Secuencia Molecular , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Infection of cattle with the bovine leukemia virus (BLV) results in a strong permanent antibody response to the BLV antigens some weeks after infection. However, cattle may carry provirus and not have detectable antibody titers. To prove the occurrence of different BLV provirus variants in German cattle and to study the influence of special BLV variants on the immunoreaction, a 444-bp fragment of the env gene of 35 naturally BLV infected animals was analyzed. Seven different groups of BLV provirus variants were found on the basis of restriction fragment length polymorphism. Three BLV provirus variant groups and five additionally sequenced BLV isolates showed a high similarity to BLV provirus isolates from other geographical areas. The variation in nucleotide sequence of the five BLV isolates compared with nine previously sequenced BLV isolates ranged up to 5. 3%. While BLV provirus variant groups A, C, D, E, F, and G were clearly related to agar-gel immunodiffusion test (AGID)- and enzyme-linked immunosorbent assay (ELISA)-positive animals, BLV provirus variant group B was solely found in permanent AGID- and ELISA-negative or in transient ELISA-positive animals. Altogether, these results indicate that special BLV provirus variants may be responsible for atypical forms of BLV infection in cattle.
Asunto(s)
Enfermedades de los Bovinos/virología , Leucosis Bovina Enzoótica/virología , Virus de la Leucemia Bovina/genética , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Variación Genética , Datos de Secuencia MolecularRESUMEN
The extent of the heterogeneity of drug utilization among NSAID users has not been extensively studied. We studied the longitudinal prescribing and switching patterns of NSAID users in a 1-year follow-up study in four German pharmacies. The study population consisted of 526 persons with an average age of 57 years. We observed that the legend duration of prescription increased with age; 14.3 days for patients aged 44 or younger to 25.1 days for persons 75 years or older, and was dependent on disease chronicity; 16.0 days for acutely ill persons compared to 23.9 days for chronic patients. The average legend duration also varied between different types of NSAIDs, from 18.0 days for ibuprofen to 29.1 days for tiaprofen. Switching from one type of NSAID to another proved to be related to the legend duration of prescription and patient characteristics such as compliance with NSAID therapy, duration of the disease and the effectiveness (poor tolerability or insufficient effect) of the NSAID therapy reported by the patients 4 weeks after recruitment. We conclude that NSAID users cannot be viewed as an homogeneous group of patients with respect to exposure time to the drug. This heterogeneity should be considered in the exposure definition, the 'time-window' design of observational studies dealing with risk comparisons in particular.
RESUMEN
Topical non-steroidal anti-inflammatory drugs NSAID account for two-thirds of the most frequently prescribed NSAIDs in Germany. Nevertheless, the extent of use and the benefit of local NSAIDs are viewed critically. We described the use of topical NSAIDs in people with systemic NSAID therapy. The study population consisted of 526 people with an average age of 57 years; two-thirds of the patients were women. We observed that the elderly (60 years and older) and patients with osteo- and/or rheumatoid arthritis had an increased likelihood of being treated with both topical and systemic NSAIDs. We suggest that the frequent use of topical NSAIDs in these patients is based on their supportive therapeutic effect rather than on their efficacy. Further research is needed in order to investigate the potential beneficial effects of topical NSAIDs in NSAID therapy.
Asunto(s)
Antiinflamatorios no Esteroideos/administración & dosificación , Administración Tópica , Artritis Reumatoide/tratamiento farmacológico , Utilización de Medicamentos , Femenino , Alemania , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Osteoartritis/tratamiento farmacológico , FarmacoepidemiologíaRESUMEN
The practical application of polymerase chain reaction (PCR) for the diagnosis of bovine leukaemia virus (BLV) infections in naturally infected cattle was evaluated. Compared to serological tests the PCR was definitely found to be a more sensitive method, yielding the highest number of positive results (10% more compared to enzyme-linked immunosorbent assay, (ELISA), and 17.7% more compared to agar-gel immunodiffusion, (AGID)). In testing cattle from herds with BLV incidence under 5%, out of 52 provirus positive cattle only 43 were correctly identified by ELISA. When compared to AGID only 37 of the 52 PCR positive animals were correctly identified. Of 18 cattle imported from the Slovak Republic and kept in a quarantine stable, four were found to be BLV provirus positive by PCR, while serological tests indicated one animal positive and three negative. Therefore, it is impossible to prevent the spread of the infection from one country to another by serological testing only. Moreover, it is feasible to identify animals with changing antibody titres correctly by PCR. Using PCR we were also able to distinguish BLV infected from uninfected calves that were serologically positive due to colostral antibodies. Higher sensitivity of BLV provirus detection by PCR was achieved using env gene rather than tax gene specific primers. Negative results by PCR in cases of positive serological reactions are still possible, as shown in case of one adult animal. These findings indicate that PCR is a highly sensitive method and might be successfully used and economically advantageous for different practical applications in detection of BLV infection in naturally infected cattle.
Asunto(s)
ADN Viral/análisis , Leucosis Bovina Enzoótica/diagnóstico , Virus de la Leucemia Bovina/genética , Reacción en Cadena de la Polimerasa/veterinaria , Animales , Secuencia de Bases , Bovinos , Cartilla de ADN/análisis , Cartilla de ADN/química , Cartilla de ADN/genética , ADN Viral/sangre , ADN Viral/genética , Leucosis Bovina Enzoótica/epidemiología , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Alemania/epidemiología , Inmunodifusión/métodos , Inmunodifusión/normas , Inmunodifusión/veterinaria , Incidencia , Virus de la Leucemia Bovina/aislamiento & purificación , Estudios Longitudinales , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/normas , Sensibilidad y Especificidad , Eslovaquia/epidemiologíaRESUMEN
Polymerase chain reaction (PCR) has been used for direct detection of bovine leukemia virus (BLV) proviral DNA in cattle, but it is still mainly used for experimental research. One bottleneck for routine diagnosis of BLV by PCR has always been the isolation and purification of DNA. We compare the use of not purificated with highly-purified DNA in the PCR-based diagnosis of BLV infection. DNA extracted from whole blood by chloroform extraction (CP-DNA) and DNA prepared only by osmotic shock, washing, heating and freezing procedures (RPoS-DNA), were utilized. Fifteen cattle well characterized serologically were investigated for BLV-provirus with PCR using this different DNA preparations. With both methods all but one investigated animal were correctly identified. It was estimated that in case of CP-DNA PCR 10 BLV-provirus copies were sufficient to obtain a positive result. The sensitivity of RPoS-DNA PCR was similar. Because of the greater practicability of the latter technique we used it in a small field study with ten cattle. All serologically positive animals were correctly identified by the PCR. In addition one seronegative animal was found to carry BLV-provirus. Therefore RPoS-DNA PCR might be a good tool for the routine diagnosis of BLV-infected cattle.
Asunto(s)
Leucosis Bovina Enzoótica/diagnóstico , Virus de la Leucemia Bovina/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Animales , Anticuerpos Antivirales/sangre , Bovinos , Cartilla de ADN , ADN Viral/análisis , Leucosis Bovina Enzoótica/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Reacción en Cadena de la Polimerasa/veterinaria , Reproducibilidad de los ResultadosRESUMEN
Chicken egg yolk antibodies (lgY) play an increasing role as alternative to mammalian polyclonal antibodies. They are widely used in biomedical research, for diagnostics, prophylaxis, and therapy of diseases. The extraction steps of IgY from egg yolk must be simple, with high output of purified antibodies. The aim of the present study was a comparison of different purification methods of egg yolk antibodies. The results of eight extraction methods of IgY and method combinations were investigated by PAGE and densitometric analysis. It has been demonstrated, that the IgY preparation with dextran sulfate is very effective, quick and simple to perform. It is well-suited in combination with other methods, e.g. ammonium sulfate precipitation.
Asunto(s)
Yema de Huevo/inmunología , Inmunoglobulinas/aislamiento & purificación , Animales , Densitometría , Electroforesis en Gel de Poliacrilamida , Femenino , Indicadores y Reactivos , Colorantes de Rosanilina , Tinción con Nitrato de PlataRESUMEN
The mechanism of BLV-induced tumorigenesis has not been clear up to now. Changes of viral protein expression in infected cells may be involved in the molecular events leading to BLV-induced leukaemogenesis. In this study Western blot investigations of cells transfected with plasmid DNA containing the complete Japanese BLV tumour clone provirus demonstrate that this provirus is unable to express gag and env proteins. Following this an attempt was made to express the genes from this provirus in eukaryotic and prokaryotic cells using the phagemid pBK-RSV (Stratagene), but not as fusion proteins. The protein patterns expressed from the 5' and the 3' region of the BLV genome were compared with those of FLK/BLV cells. The results indicate that there is a defect in this provirus located in the genome region between the gag and env gene.
Asunto(s)
Regulación Viral de la Expresión Génica , Virus de la Leucemia Bovina/genética , Provirus/genética , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Estructurales Virales/genética , Animales , Bovinos , Línea Celular , Virus de la Leucemia Bovina/metabolismo , Provirus/metabolismo , Transfección , Proteínas Reguladoras y Accesorias Virales/biosíntesis , Proteínas Estructurales Virales/biosíntesisRESUMEN
The selection of animals infected with the bovine leukaemia virus (BLV) is performed by the immunological detection of antibodies against the virus, commonly using the antigen gp51. Furthermore, research is being carried out to develop protective vaccines against BLV that have gp51 as their main component. Taking both of these factors into account, it is clear that there will be an increasing requirement for the virus antigen gp51 for some time to come. The permanently BLV-infected foetal lamb kidney cell line FLK/BLV (and its sublines) has been proved to be the most useful culture for the mass production of the virus antigen. Stable cell lines producing higher quantities of BLV antigen have not been established, either by subcloning of the FLK/BLV or by infection of other permanent cells with BLV. Here, a report is made on efforts to increase the expression of gp51 in BLV-infected cells via the additional expression of homologous transactivating virus protein tax. Selectable tax expression vectors that integrate into the host cell genome were constructed using BL provirus DNA fragments. Highly productive FLK/BLV cells were transfected with these vectors. Following selection with G 418, gp51-producing cell lines were established and tested for their productivity for several months. Some tax-vector-containing cell lines have produced 1.5-2 times more gp51 than the highly productive parental control cell line FLK/BLV 44-1.
Asunto(s)
Antígenos Virales/biosíntesis , Productos del Gen tax/biosíntesis , Virus de la Leucemia Bovina/inmunología , Animales , Antígenos Virales/genética , Línea Celular , Chlorocebus aethiops , Criopreservación , Regulación Viral de la Expresión Génica , Productos del Gen tax/genética , Genes pX , Virus de la Leucemia Bovina/genética , Virus de la Leucemia Bovina/metabolismo , Ovinos , Células VeroRESUMEN
Serum samples from wild birds of 21 species were investigated for antibodies against a broad range of pathogenic microorganisms. Using routine diagnostic methods, antibodies against a choice of relevant viral and bacterial agents were detected. The epidemiology and the significance of infections in both wild birds and farm poultry are discussed.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Anticuerpos Antivirales/sangre , Infecciones Bacterianas/veterinaria , Enfermedades de las Aves/inmunología , Virosis/veterinaria , Animales , Animales Domésticos , Animales Salvajes , Animales de Zoológico , Infecciones Bacterianas/epidemiología , Infecciones Bacterianas/inmunología , Enfermedades de las Aves/epidemiología , Aves , Alemania/epidemiología , Virosis/epidemiología , Virosis/inmunologíaRESUMEN
The validity of the Rorschach as an instrument to assess impulsivity was examined in a sample of 55 adolescent psychiatric inpatients. The Rorschach variables considered to be related to impulsivity (D, Adjusted D, M, Afr, X + %, FC:CF+C, and L) were used to predict performance on the Gordon Diagnostic System (GDS). Only the discriminant function for the GDS Delay Task (which assesses the ability to formulate response strategies and to benefit from feedback) was statistically significant, with a 76.36% correct classification of subjects. The Rorschach variables with the highest correlations within the discriminant function were D, FC:CF+C, and M. The results of this study appeared to provide some support for the utility of the Rorschach in the assessment of impulsivity.
Asunto(s)
Conducta Impulsiva/diagnóstico , Prueba de Rorschach/estadística & datos numéricos , Trastornos de Adaptación/diagnóstico , Trastornos de Adaptación/psicología , Adolescente , Atención , Trastorno por Déficit de Atención con Hiperactividad/diagnóstico , Trastorno por Déficit de Atención con Hiperactividad/psicología , Niño , Trastornos de la Conducta Infantil/diagnóstico , Trastornos de la Conducta Infantil/psicología , Trastorno Depresivo/diagnóstico , Trastorno Depresivo/psicología , Femenino , Hospitalización , Humanos , Conducta Impulsiva/psicología , Control Interno-Externo , Masculino , Determinación de la Personalidad/estadística & datos numéricos , Desempeño PsicomotorAsunto(s)
Trastornos de la Personalidad/clasificación , Inventario de Personalidad/estadística & datos numéricos , Prueba de Rorschach/estadística & datos numéricos , Adolescente , Femenino , Identidad de Género , Hospitalización , Humanos , Masculino , Trastornos de la Personalidad/diagnóstico , Trastornos de la Personalidad/psicología , PsicometríaRESUMEN
We report a functional and molecular analysis of nine oncocytic tumors of the human thyroid. In all the abundance of mitochondria observed ultrastructurally was accompanied by an increase in enzymatic activities of respiratory complexes 1 (NADH dehydrogenase), 11 (succinate dehydrogenase) IV (cytochrome c oxidase), and V (ATPase). Western blot analysis failed to detect uncoupling protein in the tumors. The elevated respiratory enzyme activities were paralleled by an increase in the mitochondrial DNA content. Restriction analysis of mitochondrial DNA gave no indication of heteroplasmy or other gross alterations. We conclude that the mitochondrial proliferation in oncocytic tumors is probably not the result of a compensatory mechanism for the deficiency in enzyme complexes of the mitochondrial respiratory chain.
Asunto(s)
Adenoma/ultraestructura , Mitocondrias/metabolismo , Neoplasias de la Tiroides/ultraestructura , Adenosina Trifosfatasas/metabolismo , Western Blotting , Enzimas de Restricción del ADN , ADN Mitocondrial/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Histocitoquímica , Humanos , Microscopía Electrónica , Mitocondrias/ultraestructura , NADH Deshidrogenasa/metabolismo , Succinato Deshidrogenasa/metabolismoRESUMEN
[14C]Di-n-octyltin dichloride ([14C]DOTC) was administered by oral gavage to male and female rats. After 96 h hepatic and thymic DNA was isolated. All DNA fractions were radioactive, but analysis of DNA hydrolysates by HPLC revealed that the radioactivity was incorporated via biosynthesis and was not due to adduct formation. The limit of detection for adduct formation, expressed in units of the covalent binding index (CBI = mumol chemical bound per mol nucleotides/mmol chemical applied per kg body wt.) was approximately 0.2 for liver DNA and about 0.7 for thymus DNA. This maximum possible DNA-binding ability is about 30,000 times lower than the corresponding value for the strong carcinogen, aflatoxin B1. In addition, [14C]DOTC did not bind covalently to calf thymus DNA in the presence or absence of rat liver S9 or to DNA of V79 Chinese hamster cells. This study therefore gives no indication for genotoxic activity of DOTC mediated by DNA binding.
Asunto(s)
Daño del ADN , ADN/metabolismo , Compuestos Orgánicos de Estaño/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Bovinos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , ADN/efectos de los fármacos , Femenino , Técnicas In Vitro , Hígado/metabolismo , Masculino , Mutágenos/metabolismo , Compuestos Orgánicos de Estaño/toxicidad , Ratas , Ratas Endogámicas , Timo/metabolismoRESUMEN
A bacterial expression vector was constructed to encode a fusion protein which had, at its carboxy terminus, a polypeptide encoded within the 5' proximal open reading frame of the coronavirus MHV-JHM mRNA 4. This polypeptide was isolated and used to produce an antiserum. The antiserum reacted specifically with a 15,000 Mr polypeptide synthesized in MHV-JHM-infected cells, or in vitro translations of infected cell poly(A) RNA enriched for mRNA 4. These results demonstrate the translational activity of mRNA 4 during infection, identify conclusively the translation product and provide a means to investigate the synthesis and function of this protein.