RESUMEN
This study determined the occurrence of legionellae in private houses for which there were no available data on aquatic environments other than the water supply system. From June 2013 to November 2014, we collected 138 water and 90 swab samples from aquatic environments in 19 houses. Legionella DNA was detected via a loop-mediated isothermal amplification assay in 66 (47·8%) water and 17 (18·9%) swab samples. High Legionella DNA detection rates were observed in water samples from washing machines and aquariums. Legionella spp. was isolated from 9 (6·5%) water and 3 (3·3%) swab samples. Legionella pneumophila SG 1 was detected from the outlet water of a bathtub spout and a bath sponge. Use of amoebic co-culture effectively increased legionellae and Legionella DNA detection rates from all sample types. A logistic regression analysis revealed that the heterotrophic plate count was significantly related to Legionella contamination. Our findings indicate that there is a risk of legionellosis from exposure to Legionella spp. in a variety of aquatic environments in residential houses. Control measures for legionellae in houses should include frequent cleaning and disinfecting to reduce heterotrophic bacteria in water and, where possible, preventing aerosolization from aquatic environments.
Asunto(s)
Agua Potable/microbiología , Legionella/aislamiento & purificación , Legionelosis/epidemiología , ADN Bacteriano/aislamiento & purificación , Japón/epidemiología , Legionelosis/microbiología , Modelos Logísticos , Riesgo , Análisis de Secuencia de ADN , Abastecimiento de AguaRESUMEN
INTRODUCTION: A high incidence of thyroid dysfunction is reported in patients with HIV or HCV mono-infection. We have conducted a periodic medical examination including the thyroid function for haemophilic patients with HIV/HCV co-infection due to contaminated blood products. METHODS: We examined the thyroid function (as assessed by the FT3, FT4 and TSH levels) in 45 haemophilic patients, including thyroglobulin and auto-antibody, antithyroglobulin antibody, antithyroid peroxidase antibody and anti-TSH receptor antibody in 28 patients. RESULTS: All the patients were males (median age: 42 years; range: 29-66). The median values of thyroid function were FT3 3.36 pg mL(-1) , FT4 1.125 ng mL(-1) and TSH 1.65 µIU mL(-1) . Five patients (11.1%) had high TSH levels. In 28 patients in whom the presence of auto-antibodies was examined, the median age was 47 years of age. The median value of thyroglobulin was 16 ng mL(-1) and two patients showed high levels of thyroglobulin. The presence of anti-TSH receptor antibody of all the patients was negative, but one patient (3.5%) was positive of antithyroid peroxidase antibody and antithyroglobulin antibody. CONCLUSIONS: Since 0.68-3.6% of the general healthy population is reported to show hypothyroidism, our data showed that the proportion of hypothyroidism in haemophilic patients with HIV/HCV co-infection was more frequent than that of the normal population.
Asunto(s)
Autoanticuerpos/sangre , Coinfección/diagnóstico , Infecciones por VIH/diagnóstico , VIH/fisiología , Hemofilia A/diagnóstico , Hepacivirus/fisiología , Hepatitis C/diagnóstico , Hipotiroidismo/diagnóstico , Glándula Tiroides/fisiología , Adulto , Anciano , Coinfección/epidemiología , Infecciones por VIH/epidemiología , Hemofilia A/epidemiología , Hepatitis C/epidemiología , Humanos , Hipotiroidismo/epidemiología , Japón/epidemiología , Masculino , Persona de Mediana Edad , Prevalencia , Tiroglobulina/sangreRESUMEN
AIM: In Japan, surgery for Graves' disease (GD), which is considered to be a radical therapy, has been restricted by various guidelines. Nevertheless, some patients benefit from surgery. We sought to identify a reasonable operative method for GD by comparing the efficacy and safety among patients undergoing different extents of thyroidectomy. METHODS: A total of 162 patients underwent thyroidectomy for GD between 2003 and 2012 in our department. We compared the clinical factors among those who underwent subtotal thyroidectomy (ST), near-total thyroidectomy (NTT), and total thyroidectomy (TT). RESULTS: The ST, NTT, and TT groups included 111, 21, and 30 patients, respectively. The patient sex, period between disease onset and surgery, and preoperative thyroidal function were not substantially different among the three groups. With regard to surgical variables, the duration of surgery, amount of blood loss, and postoperative length of hospitalization were not substantially different among the three groups. Postoperative recurrent laryngeal nerve (RLN) palsy was transient in all cases, but the rate was significantly higher in the TT group compared to the other two groups (P<0.001). The incidences of transient hypocalcemia and permanent hypoparathyroidism were not substantially different among the groups. The proportion of patients who required the postoperative administration of levothyroxine was significantly lower in the ST group compared to the TT and NTT groups. Hyperthyroidism recurrence was noted in eight patients in the ST group (7.2%). CONCLUSION: NTT for GD is thus considered to be a reasonable operative method regarding both efficacy and safety.
Asunto(s)
Enfermedad de Graves/cirugía , Tiroidectomía/métodos , Adolescente , Adulto , Anciano , Femenino , Humanos , Japón , Tiempo de Internación , Masculino , Persona de Mediana Edad , Hemorragia Posoperatoria/etiología , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Tiroidectomía/efectos adversos , Resultado del TratamientoRESUMEN
PURPOSE: The effects of astaxanthin (Ax) on the in vitro development of bovine embryos cultured under heat stress were investigated in combination with the assessment of its cellular accumulation and action on mitochondrial membrane potential (ΔΨm). METHODS: Bovine ≥8-cell embryos were collected on day 3 after in vitro fertilization and exposed to single (day 4) or repeated (day 4 and 5) heat stress (10 h/day at 40.5 °C). Ax was added into culture medium under the repeated heat stress and blastocyst development was evaluated. The cellular uptake of Ax in embryos was examined using bright-field and confocal laser-scanning microscopy, and high-performance liquid chromatography. The relationship between Ax and mitochondria localization was assessed using MitoTracker dye. The effects of Ax on ΔΨm were investigated using JC-1 dye. RESULTS: Blastocyst development in the repeated heat stress treatment decreased significantly (P < 0.05) compared with those in single heat stress or normal thermal treatment. The addition of Ax into culture medium did lead to a significant recovery in blastocyst development in the repeated heat-treated group. Ax was detected in cytoplasm of embryos and observed to colocalize with mitochondria. Ax recovered ΔΨm in embryos that was decreased by the heat treatment. CONCLUSIONS: Ax ameliorated the heat stress-induced impairment of blastocyst development. Our results suggest that the direct action of Ax on mitochondrial activity via cellular uptake is a mechanism of the ameliorating effects.
Asunto(s)
Blastocisto/efectos de los fármacos , Respuesta al Choque Térmico/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Animales , Disponibilidad Biológica , Blastocisto/citología , Blastocisto/fisiología , Bovinos/embriología , Células Cultivadas , Evaluación Preclínica de Medicamentos , Técnicas de Cultivo de Embriones/métodos , Desarrollo Embrionario/efectos de los fármacos , Femenino , Fertilización In Vitro , Fibrinolíticos/farmacocinética , Fibrinolíticos/farmacología , Respuesta al Choque Térmico/fisiología , Técnicas de Maduración In Vitro de los Oocitos , Mitocondrias/fisiología , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/fisiología , Xantófilas/farmacocinética , Xantófilas/farmacologíaRESUMEN
BACKGROUND: Outcomes of liver resection for hepatocellular carcinoma (HCC) have improved owing to better surgical techniques and patient selection. Portal hypertension may influence outcome but the preoperative definition and role of portal hypertension are far from clear. The aim of this study was to elucidate the influence of portal venous pressure (PVP) measured directly during surgery on outcomes of liver resection in patients with HCC. METHODS: Patients who had resection of HCC between 1997 and 2009, and who underwent direct measurement of PVP immediately after laparotomy were enrolled. These patients were divided into groups with high (at least 20 cmH(2)O) and low (less than 20 cmH(2)O) PVP. The influence of PVP on overall and recurrence-free survival was analysed and prognostic factors were identified. RESULTS: A total of 177 patients were enrolled, 129 in the low-PVP group and 48 in the high-PVP group. The 5-year overall survival rate (63·7 versus 31 per cent; P < 0·001) and recurrence-free survival rate (52·5 versus 12 per cent; P < 0·001) were significantly higher in patients with low PVP. In multivariable analysis, two or more tumours, tumour diameter at least 5 cm, high PVP, grade B liver damage and Hepatic Activity Index (HAI) grade 7 or more were significant predictors of poorer survival after liver resection. Two or more tumours, tumour diameter at least 5 cm and HAI grade 7 or more were significant predictors of poorer recurrence-free survival. CONCLUSION: High PVP was associated with poor long-term outcome after liver resection for HCC.
Asunto(s)
Carcinoma Hepatocelular/cirugía , Neoplasias Hepáticas/cirugía , Presión Portal/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/fisiopatología , Supervivencia sin Enfermedad , Femenino , Hepatectomía/mortalidad , Humanos , Cuidados Intraoperatorios/métodos , Cuidados Intraoperatorios/mortalidad , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/fisiopatología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/mortalidad , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Helicobacter bilis is considered to be a causative factor in the pathogenesis of biliary cancer. This study investigated the prevalence of H. bilis colonization of the biliary system of patients with pancreaticobiliary maljunction (PBM). METHODS: Bile juice and biliary tissue samples were collected from 17 patients with PBM and 27 controls who had benign biliary disease without PBM. DNA extracted from each biliary sample was subjected to polymerase chain reaction (PCR) analysis for H. bilis and Helicobacter pylori. RESULTS: PCR assays revealed that 12 of the 17 patients with PBM were positive for H. bilis DNA, compared with eight of 27 patients without PBM (P = 0.009). Among patients with PBM, H. bilis DNA was identified in six of eight children, including a 2-month-old infant, and in six of nine adults. The high prevalence of H. bilis DNA in the biliary system of patients with PBM was independent of age, sex, common bile duct dilatation, configuration of the pancreatic and bile ducts, and amylase activity in bile. CONCLUSION: H. bilis colonization of the biliary system is extremely common in patients with PBM. This may point to a role in the pathogenesis of biliary cancer.
Asunto(s)
Conductos Biliares/anomalías , Neoplasias del Sistema Biliar/microbiología , Infecciones por Helicobacter , Helicobacter/aislamiento & purificación , Conductos Pancreáticos/anomalías , ARN Bacteriano/análisis , Adolescente , Adulto , Anciano , Bilis/microbiología , Sistema Biliar/microbiología , Estudios de Casos y Controles , Niño , Preescolar , Electroforesis , Femenino , Helicobacter/genética , Humanos , Lactante , Masculino , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
Using functional magnetic resonance imaging (fMRI), we compared the cerebral activity during bilateral light fist-clenching and light-teeth clenching to provide more information on the central processing mechanisms underlying awake bruxism. Fourteen subjects participated in our study. Statistical comparisons were used to identify brain regions with significant activation in the subtraction of light fist clenching and light teeth clenching activity minus baseline. Participants also evaluated the perceived effort of clenching for each task, using a visual analogue scale of 0-100, after fMRI was performed. Bilateral light fist-clenching significantly activated the bilateral sensorimotor cortex, while light teeth-clenching was significantly associated with activation of the bilateral sensorimotor cortex, supplementary motor area, dorsolateral prefrontal cortex, and posterior parietal cortex. The VAS scores for fist clenching and teeth clenching were not significantly different. As light teeth-clenching activates a more extensive cortical network compared with light fist-clenching, we suggest that the teeth clenching may induce a more complex cerebral activity compared with the performance of a hand motor task. The clinical significance of these findings remains unknown but could perhaps be related to the propensity to trigger awake bruxism.
Asunto(s)
Encéfalo/fisiología , Mano/fisiología , Imagen por Resonancia Magnética , Músculos Masticadores/fisiología , Contracción Muscular/fisiología , Diente/fisiología , Adulto , Fenómenos Biomecánicos , Bruxismo/fisiopatología , Femenino , Fuerza de la Mano/fisiología , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Corteza Motora/fisiología , Destreza Motora/fisiología , Lóbulo Parietal/fisiología , Esfuerzo Físico/fisiología , Corteza Prefrontal/fisiología , Corteza Somatosensorial/fisiología , Factores de TiempoRESUMEN
TGF-beta 1 controls the expression of numerous genes, including early response and cellular matrix genes. However, the signal-transducing mechanism underlying this regulation of gene expression is not fully understood. In this study, we investigated whether redox regulation plays a role in the TGF-beta 1 signal transduction in the mouse osteoblastic cell line (MC3T3-E1). The overall intracellular oxidized state of the cells, when measured using 2',7'-dichlorofluorescin diacetate by laser-scanning confocal microscopy, was increased transiently after the addition of TGF-beta 1. This increase was abolished by the addition of oxygen radical scavengers such as catalase and N-acetylcysteine. In a variant cell line lacking the TGF-beta 1 receptor, the intracellular oxidized state was not modulated by treatment with TGF-beta 1. We then examined the expression of early growth response-1 (egr-1) gene, which is inducible by TGF-beta 1 and H2O2. Radical scavengers inhibited the induction of egr-1 by TGF-beta 1, but not that by 12-O-tetradecanoylphorbol-13 acetate. A nuclear run-on assay indicated that this inhibition was at the transcriptional level. From transient expression experiments using chloramphenicol acetyltransferase gene linked to serially deleted egr-1 gene 5'-upstream region, the CArG element in the 5' flanking region of egr-1 was identified as an essential sequence in the transcriptional activation for both TGF-beta 1 and H2O2 stimulation. These findings suggest that H2O2 acts as a mediator for the TGF-beta 1-induced transcription of egr-1 gene.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Peróxido de Hidrógeno/farmacología , Proteínas Inmediatas-Precoces , Osteoblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Células 3T3 , Acetilcisteína/farmacología , Animales , Secuencia de Bases , Catalasa/farmacología , Línea Celular Transformada , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa/biosíntesis , Proteína 1 de la Respuesta de Crecimiento Precoz , Depuradores de Radicales Libres , Expresión Génica , Genes ras , Humanos , Ratones , Datos de Secuencia Molecular , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Proteínas Recombinantes/farmacología , Secuencias Reguladoras de Ácidos Nucleicos , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Transfección , Dedos de ZincRESUMEN
Cell cycle-dependent phosphorylation and nuclear import of the tumorigenic transcription factor viral Jun (v-Jun) were investigated in chicken embryo fibroblasts. Nuclear accumulation of v-Jun but not of cellular Jun (c-Jun) is cell cycle dependent, decreasing in G1 and increasing in G2. The cell cycle-dependent regulation of v-Jun was mapped to a single serine residue at position 248 (Ser248), adjacent to the nuclear localization signal (NLS). Ser248 of v-Jun represents an amino acid substitution, replacing cysteine of c-Jun. It was shown by peptidase digestion and immunoprecipitation with antibody to the NLS that v-Jun is phosphorylated at Ser248 in the cytoplasm but not in the nucleus. This phosphorylation is high in G1 and low in G2. Nuclear accumulation of v-Jun is correlated with underphosphorylation at Ser248. The regulation of nuclear import by phosphorylation was also examined using NLS peptides with Ser248 of v-Jun. Phosphorylation of the serine inhibited nuclear import mediated by the NLS peptide in vivo and in vitro. The protein kinase inhibitors staurosporine and H7 stimulated but the phosphatase inhibitor okadaic acid inhibited nuclear import mediated by the NLS peptide. The cytosolic activity of protein kinases phosphorylating Ser248 increased in G0 and decreased during cell cycle progression, reaching a minimum in G2, whereas phosphatase activity dephosphorylating Ser248 was not changed. These results show that nuclear import of v-Jun is negatively regulated by phosphorylation at Ser248 in the cytoplasm in a cell cycle-dependent manner.
Asunto(s)
Ciclo Celular , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteína Oncogénica p65(gag-jun)/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Alcaloides/farmacología , Secuencia de Aminoácidos , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Citoplasma/metabolismo , Éteres Cíclicos/farmacología , Fibroblastos , Isoquinolinas/farmacología , Datos de Secuencia Molecular , Señales de Localización Nuclear , Proteínas Nucleares/química , Ácido Ocadaico , Proteína Oncogénica p65(gag-jun)/química , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Fosfoserina/metabolismo , Piperazinas/farmacología , Proteínas Quinasas/metabolismo , EstaurosporinaRESUMEN
After the Fukushima Daiichi Nuclear Power Plant accident, the radiation dose for first responders was not evaluated accurately due to lack of the monitoring data. It has been important to evaluate a radiation dose for workers in emergency response at a nuclear accident. In this study, a new device which can evaluate both of external and internal exposure doses was developed and the performance of various environmental radiation monitors including commercially available monitors were tested and compared from the viewpoint of an environmental monitoring at emergency situation. Background counts of the monitors and the ambient dose equivalent rate were measured in Fukushima Prefecture. The detection limit for beta particles was evaluated by the method of ISO11929. The sensitivity for gamma-rays of the dust monitor using a ZnS(Ag) and a plastic scintillator was high, but that of the external exposure monitor using a silicon photodiode with CsI(Tl) crystal was relatively low. The detection limit ranged 190-280 Bq m-3 at 100 µSv h-1, exceeding the detection limit of 100 Bq m-3 in the minimum requirement by the National Regulation Authority in Japan. Use of the shielding with lead is necessary to achieve the minimum requirement. These results indicate that the dust monitor using a ZnS(Ag) scintillator and a plastic scintillator is suitable for the external exposure monitor and the developed internal exposure monitor is for the internal exposure monitor at emergency situation among the evaluated monitors. In the future study, the counting efficiency, the relative uncertainty and the performance of the detection for alpha particles will be evaluated, and it will be considered which type of a monitor is suitable after taking the portability into account.
Asunto(s)
Contaminación Ambiental/análisis , Rayos gamma , Monitoreo de Radiación/instrumentación , Conteo por Cintilación/instrumentación , Humanos , Dosis de Radiación , Sulfuros/química , Compuestos de Zinc/químicaRESUMEN
A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.
Asunto(s)
Proteínas de Caenorhabditis elegans , Isoenzimas , Familia de Multigenes , Músculos/enzimología , Proteína Quinasa C/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Proteínas Portadoras , Línea Celular , ADN , Expresión Génica , Ratones , Datos de Secuencia Molecular , Forbol 12,13-Dibutirato/metabolismo , Proteína Quinasa C/biosíntesis , Proteína Quinasa C/genética , Proteína Quinasa C-theta , Receptores de Droga/metabolismo , Mapeo Restrictivo , Alineación de SecuenciaRESUMEN
Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKC zeta in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKC zeta to associate specifically with the GLUT4 compartments and that PKC zeta together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKC zeta and GLUT4 recycled independently of one another. To further establish the importance of PKC zeta in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKC zeta (DNPKC zeta) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKC zeta was associated with a marked increase in the activity of this isoform. The overexpressed, active PKC zeta coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKC zeta caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKC zeta induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKC zeta disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKC zeta regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.
Asunto(s)
Glucosa/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Proteína Quinasa C/metabolismo , Serina/metabolismo , Animales , Transporte Biológico , Fraccionamiento Celular , Células Cultivadas , Activación Enzimática , Expresión Génica , Transportador de Glucosa de Tipo 4 , Membranas Intracelulares/metabolismo , Músculo Esquelético/citología , Fosforilación , Proteína Quinasa C/genética , Proteínas R-SNARE , RatasRESUMEN
In the DNA binding domain of microphthalmia-associated transcription factor (MITF), four mutations are reported: mi, Mi wh, mi ew, and mi or. MITFs encoded by the mi, Mi wh, mi ew, and Mi or mutant alleles (mi-MITF, Mi wh-MITF, Mi ew-MITF, and Mi or-MITF, respectively) interfered with the DNA binding of wild-type MITF, TFE3, and another basic helix-loop-helix leucine zipper protein in vitro. Polyclonal antibody against MITF was produced and used for investigating the subcellular localization of mutant MITFs. Immunocytochemistry and immunoblotting revealed that more than 99% of wild-type MITF and Mi wh-MITF located in nuclei of transfected NIH 3T3 and 293T cells. In contrast, mi-MITF predominantly located in the cytoplasm of cells transfected with the corresponding plasmid. When the immunoglobulin G (IgG)-conjugated peptides representing a part of the DNA binding domain containing mi and Mi wh mutations were microinjected into the cytoplasm of NRK49F cells, wild-type peptide and Mi wh-type peptide-IgG conjugate localized in nuclei but mi-type peptide-IgG conjugate was detectable only in the cytoplasm. It was also demonstrated that the nuclear translocation potential of Mi or-MITF was normal but that Mi ew-MITF was impaired as well as mi-MITF. In cotransfection assay, a strong dominant negative effect of Mi wh-MITF against wild-type MITF-dependent transactivation system on tyrosinase promoter was observed, but mi-MITF had a small effect. However, by the conjugation of simian virus 40 large-T-antigen-derived nuclear localization signal to mi-MITF, the dominant negative effect was enhanced. Furthermore, we demonstrated that the interaction between wild-type MITF and mi-MITF occurred in the cytoplasm and that mi-MITF had an inhibitory effect on nuclear localization potential of wild-type MITF.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/metabolismo , Leucina Zippers , Ratones , Ratones Mutantes , Factor de Transcripción Asociado a Microftalmía , Datos de Secuencia Molecular , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the alpha, delta, eta, and zeta isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the alpha, delta, and eta isoforms. Overexpression of the eta isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G1 arrest. The eta-overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the eta-overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the eta isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative eta isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1alpha,25-dihydroxyvitamin D3, and a high concentration of Ca2+. Among the isoforms examined, the delta isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the alpha and zeta isoforms did not. These findings indicate that the eta and delta isoforms of PKC are involved crucially in squamous cell differentiation.
Asunto(s)
Adenovirus Humanos/genética , Transformación Celular Viral , Isoenzimas/biosíntesis , Queratinocitos/citología , Proteína Quinasa C/biosíntesis , Animales , Células COS , Ciclo Celular , Diferenciación Celular , División Celular , Células Cultivadas , Cósmidos , Inducción Enzimática , Vectores Genéticos , Genoma Viral , Humanos , Queratinocitos/enzimología , Cinética , Ratones , Fosforilación , Proteína Quinasa C-delta , Conejos , Proteínas Recombinantes/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).
Asunto(s)
Retículo Endoplásmico/enzimología , Isoenzimas/biosíntesis , Proteína Quinasa C/biosíntesis , Secuencia de Aminoácidos , Animales , Anticuerpos , Carcinoma de Células Escamosas , Línea Celular , Chlorocebus aethiops , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/ultraestructura , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Isoenzimas/análisis , Riñón , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/inmunología , Proteína Quinasa C/análisis , Transfección , Células Tumorales CultivadasRESUMEN
Tissues of normal human gastric mucosae and 15 advanced gastric carcinomas were studied immunohistologically for the presence of receptors for epidermal growth factor (EGF) by use of a murine monoclonal antibody (528IgG), which reacts with the binding domain of human EGF receptor. On normal gastric mucosae, only parietal cells showed positive staining. On cancer tissues, definite staining was observed in 9 of 15 cases. Their staining intensities were variable and weaker in general compared to those of either gastric parietal cells or normal tonsilar squamous epithelium. No apparent correlation of EGF receptor staining with the grade of histologic differentiation or lymph node metastases of these gastric carcinomas was noted.
Asunto(s)
Carcinoma/metabolismo , Receptores ErbB/metabolismo , Mucosa Gástrica/metabolismo , Neoplasias Gástricas/metabolismo , Anciano , Anticuerpos Monoclonales , Carcinoma/patología , Epitelio/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Gástricas/patologíaRESUMEN
Three parameters were evaluated as diagnostic of the malignant potential of cultured rat liver epithelial cells: cytology, growth in soft agar, and production of extracellular plasminogen activator. A total of 22 tumorigenic and nontumorigenic cultures from 15 cell lines were sent coded from their originators to two different laboratories for the evaluation of these three parameters. Cytologic diagnosis and growth in soft agar were reliable means of determining the malignant potential of the cultured cells. However, the production of extracellular plasminogen activator showed little correlation with tumorigenicity. Of cytologic properties evaluated, the two that correlated best with malignant potential were increased cytoplasmic basophilia and and increased nuclear:cytoplasmic ratio.
Asunto(s)
Activadores Plasminogénicos/biosíntesis , Agar , Animales , División Celular , Línea Celular , Transformación Celular Neoplásica , Epitelio/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Experimentales/patología , RatasRESUMEN
The present study was undertaken to investigate the possibility that 12-O-tetradecanoylphorbol-13-acetate (TPA) is active as a tumor promoter in human skin. Human epidermal and dermal cells were isolated from the skin of normal subjects by trypsinization and separation of the epidermis from the dermis. Cells in primary culture were exposed to a wide range of TPA concentrations (0.001 to 1000 ng/ml) for various time intervals, and its effect on DNA synthesis, sugar uptake, and polyamine synthesis was measured. Results obtained using human cells were compared with those for the corresponding cells isolated from Sencar mice. In human epidermal cells, TPA did not stimulate but instead inhibited DNA synthesis and uptake of 2-deoxy-D-glucose (DG), a glucose analogue. Inhibition of DNA synthesis could be detected at a dose of TPA as low as 0.1 ng/ml, while at 10 ng/ml DNA synthesis was 50 to 70% of the control. Inhibition of DG uptake depended on concentration of and length of exposure to TPA. Exposure to TPA (10 ng/ml) for 3 hr resulted in a 35% inhibition of DG uptake. Furthermore, exposure of human epidermal cells to TPA under various conditions, including the use of uncultured, freshly isolated cells and of a low-calcium medium, did not result in induction of ornithine decarboxylase, a key enzyme in the synthesis of polyamine. Teleocidin B, a tumor promoter, structurally unrelated to TPA but with an ornithine decarboxylase inducibility in mouse skin similar to that of TPA, also failed to induce ornithine decarboxylase activity in human epidermal cells. Mouse epidermal cells reacted differently from human epidermal cells on addition of TPA. DNA synthesis, sugar uptake, and polyamine synthesis were all stimulated. DG uptake alone was stimulated in human and mouse dermal cells treated with TPA.
Asunto(s)
Replicación del ADN/efectos de los fármacos , Desoxiazúcares/metabolismo , Desoxiglucosa/metabolismo , Ornitina Descarboxilasa/genética , Forboles/toxicidad , Piel/metabolismo , Acetato de Tetradecanoilforbol/toxicidad , Animales , Transporte Biológico Activo/efectos de los fármacos , Células Cultivadas , Inducción Enzimática , Humanos , Cinética , Ratones , Piel/efectos de los fármacosRESUMEN
The presence of specific binding sites for phorbol esters was demonstrated in human epidermal and dermal cells in culture by assay of binding of [3H]phorbol-12,13-dibutyrate (PDBU) to intact cells. The specificity of the binding was shown by displacement of the binding with biologically active tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate, teleocidin B, and mezerein, but not with inactive derivatives. The equilibrium binding data were analyzed by the Scatchard method and fitted by a straight line to the model of a single class of binding sites. Human epidermal cells bound PDBU with a Kd of 28 nm at 3.7 X 10(6) molecules per cell, while human dermal cells bound PDBU with a Kd of 27 nm at 2.1 X 10(6) molecules per cell. These values were compared with those of epidermal and dermal cells of mice. Although mouse cells showed the same affinity as did human cells, mouse epidermal cells bound one-third as much as human epidermal cells, and mouse dermal cells bound one-fifth as much as human dermal cells. When precultured with unlabeled PDBU for 24 hr, [3H]PDBU binding decreased time dependently in all cells except human epidermal cells. Thus, the binding of phorbol esters to human epidermal cells is unique in that there are a large number of binding sites compared with mouse epidermal cells, and there is no down regulation.