RESUMEN
Dolichol and natural rubber are representative cis-polyisoprenoids in primary and secondary metabolism, respectively. Their biosynthesis is catalyzed by cis-prenyltransferase (CPT) by sequential condensations of isopentenyl diphosphates (IPPs) to a priming molecule. Although prokaryotic CPTs have been well characterized, the mechanism of eukaryotic CPTs in cis-polyisoprene biosynthesis was only recently revealed. It was shown that eukaryotes have evolved a unique protein complex, comprised of CPT and CPT-binding protein (CBP), to synthesize cis-polyisoprenoids. In the context of this new discovery, we found discrepancies in literature for CPT or CBP biochemical assays and in vivo CPT complementation using rer2 (yeast CPT) yeast mutant. Our study here shows that rer2 revertants occur at a frequency that cannot be disregarded and are likely accountable for the results that cannot be explained by the CPT/CBP heteroprotein complex model. To make a stable mutant, SRT1 gene (secondary CPT expressed at a basal level in yeast) was additionally deleted in the rer2Δ mutant background. This stable rer2Δ srt1Δ strain was then used to individually or simultaneously express Arabidopsis CPT1 (AtCPT1, At2g17570) and CBP (AtLEW1, At1G11755). We found that the simultaneous expression of Arabidopsis CPT1 and AtLEW1 effectively complements the rer2Δ srt1Δ strain, whereas the individual expression of AtCPT1 alone or AtLEW1 alone failed to rescue the yeast mutant. Microsomes from the dual expresser showed an efficient incorporation of IPPs into cis-polyisoprenoid (30% in 2h). These results showed that the CPT/CBP heteroprotein complex model is valid in Arabidopsis thaliana. Experimental details of these results are described in this methodology paper.
Asunto(s)
Transferasas Alquil y Aril/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Dimetilaliltranstransferasa/genética , Dolicoles/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transferasas/genética , Transferasas Alquil y Aril/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Vías Biosintéticas , Dimetilaliltranstransferasa/metabolismo , Dolicoles/genética , Técnicas de Silenciamiento del Gen , Hemiterpenos/genética , Hemiterpenos/metabolismo , Mutación , Compuestos Organofosforados/metabolismo , Goma/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Metabolismo Secundario , Transferasas/metabolismoRESUMEN
Neuron-targeted nucleic acid delivery systems are important technologies for realizing the potential of gene therapy for nervous system disorders. However, neurons are difficult cells to transfect using non-viral vectors due in part to the specific and unique delivery challenges present in these cells. We have investigated several bioactive peptides for their ability to assist in overcoming delivery barriers in mammalian cells. We summarize here our recent progress in developing and applying peptide-modified polycations for nucleic acid delivery. In addition, we present data demonstrating the potential of using multicomponent, peptide-modified polycations for nucleic acid delivery to neurons.
Asunto(s)
ADN/metabolismo , Neuronas/metabolismo , Péptidos/química , Polietileneimina/química , Transfección/métodos , Animales , Transporte Axonal , ADN/química , Endosomas/metabolismo , Células PC12 , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , RatasRESUMEN
BACKGROUND: Desmoglein (Dsg) enzyme-linked immunosorbent assay (ELISA) is a highly sensitive and specific method to detect anti-Dsg3 and anti-Dsg1 IgG autoantibodies in pemphigus vulgaris (PV) and pemphigus foliaceus (PF), respectively. Whereas ELISA index values fluctuate in parallel with disease activity, ELISA positivity during clinical remission has been observed. OBJECTIVE: To determine the prevalence of positive Dsg ELISA index values during clinical remission. To ascertain how positive Dsg ELISA scores during remission compare with those during active disease. METHODS: Dsg ELISA was performed on serum samples of PV and PF patients taken during remission (lesion-free >or= 3 months on Asunto(s)
Autoanticuerpos/sangre
, Desmogleínas/inmunología
, Inmunoglobulina G/inmunología
, Pénfigo/inmunología
, Autoanticuerpos/inmunología
, Ensayo de Inmunoadsorción Enzimática
, Femenino
, Humanos
, Masculino
, Pénfigo/tratamiento farmacológico
, Prednisolona/uso terapéutico
, Sensibilidad y Especificidad
RESUMEN
D-raf, a Drosophila homolog of the raf proto-oncogene, has diverse functions throughout development and is transcribed in a wide range of tissues, with high levels of expression in the ovary and in association with rapid proliferation. The expression pattern resembles those of S phase genes, which are regulated by E2F transcription factors. In the 5'-flanking region of D-raf, four sequences (E2F sites 1-4) similar to the E2F recognition sequence were found, one of them (E2F site 3) being recognized efficiently by Drosophila E2F (dE2F) in vitro. Transient luciferase expression assays confirmed activation of the D-raf gene promoter by dE2F/dDP. Expression of Draf-lacZ was greatly reduced in embryos homozygous for the dE2F mutation. These results suggest that dE2F is likely to be an important regulator of D-raf transcription.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Drosophila , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Proto-Oncogénicas c-raf/genética , Transactivadores , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Animales , Secuencia de Bases , Unión Competitiva , Sondas de ADN/genética , Sondas de ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción E2F , Embrión no Mamífero/metabolismo , Genes Reporteros/genética , Hibridación in Situ , Mutación/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta/genética , Proteína 1 de Unión a Retinoblastoma , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
This study demonstrates for the first time the effects of IL-1beta on the regulation of protein production as well as MUC2 gene transcription in cultured human airway epithelial cells. The effect of IL-1beta on the regulation of MUC2 protein was determined by flow cytometric analysis. The expression level of MUC2 induced by IL-1beta increased in a dose-dependent manner. MUC2 transcripts were detected after 2 h of exposure to IL-1beta and reached maximal level after 8 h. Actinomycin D experiments indicated that the IL-1beta-mediated MUC2 expression was controlled by transcriptional regulation. Both RT-PCR and FACS analysis showed that budesonide concomitantly attenuated IL-1beta mediated MUC2 gene as well as protein production levels. Use of the glucocorticoid receptor antagonist, RU-486, restored the inhibitory effect of budesonide on the IL-1beta-mediated MUC2 protein as well as gene. The data suggest that IL-1beta up-regulates MUC2 gene by transcriptional regulation and that budesonide suppresses the IL-1beta-medicated MUC2 expression via decreased transcriptional activation.
Asunto(s)
Regulación de la Expresión Génica , Interleucina-1/metabolismo , Pulmón/citología , Mucinas/metabolismo , Broncodilatadores/farmacología , Budesonida/farmacología , Separación Celular , Dactinomicina/farmacología , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Antagonistas de Hormonas/farmacología , Humanos , Mifepristona/farmacología , Mucina 2 , Mucinas/genética , Inhibidores de la Síntesis del Ácido Nucleico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Activación Transcripcional , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The Drosophila raf (D-raf) gene promoter contains a recognition consensus sequence for Drosophila STAT (D-STAT). By band mobility shift assay, we detected a factor binding to the D-STAT-recognition sequence in extracts of cultured Drosophila cells treated with vanadate peroxide. UV-cross-linking analyses suggested the size of the binding factor to be almost same as that of D-STAT. Furthermore, the binding activity was increased in cells cotransfected with HOP and D-STAT expression plasmids. These results strongly suggest that D-STAT binds to the D-STAT recognition sequence in the D-raf gene promoter. Transient luciferase expression assay using Schneider 2 cells indicated that the D-raf gene promoter is activated by D-STAT through the D-STAT-binding site. Furthermore, analyses with transgenic flies carrying Draf-lacZ fusion genes with and without mutations in the D-STAT-binding site pointed to an important role in D-raf gene promoter activity throughout development. We also found that the D-STAT-binding site is required for injury-induced activation of the D-raf gene promoter. Here we propose that D-STAT can participate in regulation of the mitogen-activated protein kinase cascade through D-raf gene activation.
Asunto(s)
Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transcripción Genética , Animales , Animales Modificados Genéticamente , Células Cultivadas , Relación Dosis-Respuesta a Droga , Drosophila/inmunología , Proteínas de Drosophila , Femenino , Galactósidos/metabolismo , Regulación de la Expresión Génica , Peróxido de Hidrógeno/farmacología , Indoles/metabolismo , Quinasas Janus , Luciferasas/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Modelos Genéticos , Plásmidos , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Tiempo , Factores de Transcripción , Activación Transcripcional , Transfección , Rayos Ultravioleta , Vanadatos/farmacología , beta-Galactosidasa/metabolismoRESUMEN
The DRE/DREF system plays an important role in transcription of DNA replication genes such as those encoding the 180 and 73 kDa subunits of DNA polymerase alpha as well as that for encoding PCNA. In this study, we found two sequences homologous to DRE (5'-TATCGATA-3') in the 5'-flanking region (-370 to -357 with respect to the transcription initiation site) of the D-raf gene and confirmed transcriptional activity through gel mobility shift assays, transient CAT assays, and spatial patterns of lacZ expression in transgenic larval tissues carrying D-raf and lacZ fusion genes. Further, we demonstrated that the D-raf gene is another target of the Zerknüllt (Zen) protein with observation of D-raf repression by Zen protein in cultured cells and its ectopic expression in the dorsal region of the homozygous zen mutant embryo. The evidence of DRE/DREF involvement in regulation of the D-raf gene obtained in this study strongly supports the idea that the DRE/DREF system is responsible for the coordinated regulation of cell proliferation-related genes in Drosophila.
Asunto(s)
Replicación del ADN , Proteínas de Drosophila , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Factores de Transcripción/genética , Transcripción Genética , Animales , Cloranfenicol O-Acetiltransferasa/genética , Drosophila melanogaster , Proteínas de Homeodominio/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-raf , Proteínas Represoras/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
The purpose of this study was to develop a new nursing unit which can meet changing health care needs, enhance patients' satisfaction and nurses' job satisfaction, and finally guarantee quality nursing care with present manpower. For this, one medical unit was selected as a unit for quality care. And one medical unit which is similar in staffing and patients' characteristics was selected as a control unit. To assess present problems and identify the remedies to the problems a hospital-wide survey and a workshop were performed. According to the survey results, educational programs and improvement of the facilities and equipment supply system, managerial support for interdepartmental cooperation and intensification of bed-side nursing care were adopted as main principles for operating model unit. This model unit was operated for 3 months from Sep. 1, 1992 to Nov. 30, 1992. To evaluate the effectiveness of the model unit, direct/indirect nursing care hours, patients' satisfaction to nursing care, nurses' job satisfaction, and quality care index were measured. Direct/indirect nursing care hours were compared with that of the control unit, and patients' and nurses' satisfaction and quality care index were measured before and after operating model unit and compared with each other. The results of the study were as follows; 1. In the model unit mean direct nursing care hours per each shift was 146.88 minutes and indirect nursing care hours was 354.72 minutes. The ratio of the direct nursing care hour to indirect nursing hour was 29.6:70.4 and that of the control unit was 26.9:73.1. Direct nursing care hour in model unit was longer than that of the control unit. But, the difference was not significant. In subcategories of direct nursing care, the time spent in mobility and exercise, conservation of body temperature, hygiene, and communication and health education were longer than that of the control unit. 2. Indirect nursing care hour in model unit was shorter than that of the control unit. But, the difference was not significant. In subcategories of indirect nursing care, the time spent in drug management and ward arrangement was shorter than that of the control unit. 3. Patients' satisfaction to nursing care was increased significantly after operating the model unit (T = -3.48, P = 0.002) and satisfaction to subcategories of physical comfort measure, psychological care, and unit management components were significantly higher than before.(ABSTRACT TRUNCATED AT 400 WORDS)