RESUMEN
Although the 11.5 kDa Zn(2+)-binding protein (ZnBP, parathymosin-alpha) possesses a functional bipartite nuclear localization signal it was found in most tissues in the cytoplasm. The cultivation of freshly isolated rat hepatocytes for 24 hours under standard conditions was associated with an almost complete translocation of ZnBP from the cytoplasm to the nuclei. Here we demonstrate, that this translocation is negatively correlated with cell density. The translocation of ZnBP to the nucleus can be inhibited or abolished by inhibitors of protein synthesis (cycloheximide) or transcription (actinomycin D). Moreover, cycloheximide can induce a relocation of ZnBP to the cytoplasm when applied after the appearance of ZnBP in the nuclei. DMSO, an inhibitor of dedifferentiation of cultured hepatocytes, abolishes also the translocation of ZnBP into the nucleus. Thinly seeded cells keep their ZnBP in the cytoplasm if they are co-cultured with plasma membranes from Morris MH7777 hepatoma cells or antibodies against E-cadherin indicating the involvement of cell adhesion proteins. We have enriched a protein from the cytosol of fresh hepatocytes which inhibits the translocation of ZnBP, but not that of albumin-NLS into the nucleus in a permeabilized cell system. Such an activity could not be found in the cytoplasm of permanent cell lines which harbor ZnBP only in the nucleus. A model for the regulation of the nuclear import of ZnBP is proposed.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Hígado/citología , Timosina/análogos & derivados , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Secuencia de Bases , Transporte Biológico/efectos de los fármacos , Cadherinas/inmunología , Proteínas Portadoras/genética , Recuento de Células , Línea Celular , Membrana Celular/fisiología , Núcleo Celular/química , Núcleo Celular/efectos de los fármacos , Células Cultivadas , Citoplasma/química , Citosol/química , Dimetilsulfóxido/farmacología , Colorantes Fluorescentes , Hígado/química , Masculino , Microscopía Confocal , Datos de Secuencia Molecular , Proteínas/farmacología , Ratas , Ratas Wistar , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares/química , Timosina/metabolismo , Células Tumorales CultivadasRESUMEN
The 11.5-kDa zinc-binding protein (ZnBP, parathymosin-alpha), a potent inactivator of 1-phosphofructokinase, is found only in the cytoplasm of most tissues despite the presence of the putative nuclear localization signal PKRQKT. Recent reports on nuclear uptake of ZnBP could not exclude the participation of unspecific diffusion. We show here that wild-type ZnBP overexpressed in COS cells accumulates exclusively in the nucleus but that ZnBP with a mutated or deleted PKRQKT motif appears both in the nucleus and in the cytoplasm. In contrast, fusion proteins between ZnBP and parts of the endoplasmic reticulum protein calreticulin required the intact PKRQKT motif for nuclear import. The motif RKR, located nine amino acids upstream of the PKRQKT motif, is also involved in the active nuclear import of ZnBP. In contrast to rat hepatocytes and kidney cells in situ, which have ZnBP almost exclusively in the cytosol, we find ZnBP in Reuber H35 hepatoma cells and normal rat kidney cells only in the nuclei. Freshly isolated rat hepatocytes translocate their ZnBP to the nucleus in < 24 h during standard cell culture conditions.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Timosina/análogos & derivados , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Compartimento Celular , Línea Celular , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Ratas , Timosina/metabolismoRESUMEN
Purified phenobarbital-induced rat liver cytochrome P-450 was incorporated in a reconstituted system containing NADPH-cytochrome P-450 reductase, dilauroyl phosphatidyl choline and sodium cholate. This system was added to organ cultures of limb buds from mouse embryos on day 11 of gestation. Cyclophosphamide (100 micrograms per ml) was used as a "pre-teratogen" and activation was initiated by adding an NADPH-regenerating system. Due to extensive purification, toxicity of the enzyme preparations and residual solubilisation detergents could be greatly reduced. A reconstituted system containing 10-100 pmol cytochrome P-450 per ml without cyclophosphamide caused no noticeable interference with limb development. The same assay containing cyclophosphamide, however, resulted in a pronounced impairment of cartilage differentiation and in the formation of clearly abnormal structures, especially at the paw skeleton. The activity of the reconstituted system declined under the experimental conditions used, but some activating capacity towards cyclophosphamide was still demonstrable after about 2 h of incubation.
Asunto(s)
Ciclofosfamida/farmacocinética , Sistema Enzimático del Citocromo P-450/metabolismo , Animales , Biotransformación , Extremidades/embriología , Masculino , Ratones , Microsomas Hepáticos/enzimología , Técnicas de Cultivo de Órganos , Ratas , Ratas EndogámicasRESUMEN
Examples of a combined approach using in vivo as well as in vitro methods for the assessment of prenatal toxicity are presented. The topics discussed include the analysis of the possible embryotoxic potential of valproic acid (VPA), female sex hormones, bis(tri-n-butyltin) oxide (TBTO), and acyclovir and the problem of supplementing in vitro systems with drug-metabolizing activity.
Asunto(s)
Anomalías Inducidas por Medicamentos/patología , Aciclovir/toxicidad , Embrión de Mamíferos/efectos de los fármacos , Estrógenos/toxicidad , Progesterona/toxicidad , Teratógenos , Compuestos de Trialquiltina/toxicidad , Ácido Valproico/toxicidad , Animales , Desinfectantes/toxicidad , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos/métodos , RatasRESUMEN
Acyclovir [9-(2-hydroxyethoxymethyl)guanine] interfered with embryonic development in vitro when assessed with the "whole-embryo" culture technique. The "no-observed-effect level" was at 10 microM acyclovir; Minor impairment of embryonic development (retarded development of ear anlagen) was observed in vitro at 25 microM acyclovir in the culture medium. At high concentrations (100 or 200 microM) development of the ear anlagen was largely inhibited. At concentrations of 50 microM acyclovir or higher, additional disturbances of embryonic differentiation in vitro became obvious, resulting in gross structural abnormalities, especially of the brain (telencephalon); Histological examinations confirmed and extended these observations: at 100 microM acyclovir alterations of the neuroepithelium of the ventricles were pronounced, the telencephalon had developed poorly or was almost completely absent, and necroses were seen in the ear anlagen, the maxillary branch and within the somites; In a limb bud culture (mouse embryos, starting with day 11 of gestation) acyclovir interfered with the differentiation of cartilaginous bone anlagen at concentrations of 200 microM and more in the culture medium. A concentration of 100 microM induced no significant effect. Thus, this organ culture system is less sensitive to the action of acyclovir when compared with whole-embryo culture; Contrary to the results achieved with acyclovir, physiological nucleosides (2'-deoxyguanosine and 2'-deoxyadenosine) did not interfere with embryonic development in vitro even at the highest concentration tested (500 microM).
Asunto(s)
Aciclovir/toxicidad , Embrión de Mamíferos/efectos de los fármacos , Anomalías Inducidas por Medicamentos/etiología , Animales , Sistema Nervioso Central/anomalías , Sistema Nervioso Central/efectos de los fármacos , Técnicas de Cultivo , Desoxiadenosinas/toxicidad , Desoxiguanosina/toxicidad , Relación Dosis-Respuesta a Droga , Oído/anomalías , Oído/efectos de los fármacos , Extremidades/efectos de los fármacos , Femenino , Técnicas de Cultivo de Órganos , Ratas , Ratas EndogámicasAsunto(s)
Anomalías Inducidas por Medicamentos/etiología , Proyectos de Investigación , Teratógenos/análisis , Toxicología/métodos , Animales , Biotransformación , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Técnicas de Cultivo/métodos , Evaluación Preclínica de Medicamentos , Embrión de Mamíferos/efectos de los fármacos , Desarrollo Embrionario y Fetal/efectos de los fármacos , Reacciones Falso Positivas , Ratones , Morfogénesis/efectos de los fármacos , Técnicas de Cultivo de Órganos/métodos , Solubilidad , Teratógenos/metabolismoRESUMEN
The alkylating agent acetoxymethyl-methylnitrosamine (DMN-OAc) triggers preferential left-sided paw defects in mice following IP administration on either day 11 or 12 of pregnancy. Predominantly, ectrodactyly and hypoplasia of the left paws were found. In an organ culture system, using limb buds of 11-day-old mouse embryos, differentiation is severely impaired following addition of 2 microM DMN-OAc to the culture medium. Left and right limbs are equally affected. In contrast, when DMN-OAc is administered in vivo to the dams with subsequent culturing of the limb buds, growth and differentiation of the left limb buds is more affected when compared to the right. Furthermore, DNA alkylation experiments were performed: in vitro, following addition of (14C)-DMN-OAc (2.3 microM) to the culture medium, the DNA alkylation rate of the limb bud DNA is determined. In vivo, following IP administration of 10 mg/kg DMN-OAc to the dams on day 11 of pregnancy, the extent of DNA alkylation of whole-embryo DNA is similar. However, the DNA alkylation rate of separately pooled left and right limb buds exhibits a two-fold difference according to the different teratogenic susceptibility. The results obtained with both in vivo and in vitro systems are consistent with the thesis that a certain amount of DNA alkylation in the tissue of the embryos is the initial step of alkylating agent-induced teratogenicity.
Asunto(s)
Anomalías Inducidas por Medicamentos/fisiopatología , Alquilantes/toxicidad , Dimetilnitrosamina/análogos & derivados , Deformidades Congénitas del Pie , Anomalías Inducidas por Medicamentos/etiología , Animales , Carcinógenos/toxicidad , Dimetilnitrosamina/toxicidad , Femenino , Dedos/anomalías , Miembro Anterior , Edad Gestacional/efectos de los fármacos , Miembro Posterior , Metilnitrosourea/toxicidad , Ratones , EmbarazoRESUMEN
A sudden matting of hair after shampooing of a hairpiece is reported, which led the patient to a lawsuit. Chemical analyses showed that hairs were weathered and led to a possible explanation of the curious phenomenon.