RESUMEN
The epithelial to mesenchymal transition (EMT) is known to involve several physiological and pathological phenomena. In this study, we utilized a microplate measurement system (MMS) approach based on the deflection of a flexible micro-cantilever to measure cell stiffness (in Pa) and adhesion force (in nN) of a single cell during EMT with nN resolution. Our results demonstrated that after transforming growth factor-ß1 (TGF-ß1) induced EMT (tEMT), NMuMG cells became stiffer due to thicker and more abundant F-actin and displayed stronger vinculin accumulation after long-term cell-substrate adhesion. The MMS could distinguish differences in compressive stiffness (219 ± 10 and 287 ± 14 Pa), tensile stiffness (114 ± 14 and 132 ± 12 Pa), and adhesion force (150 ± 42 and 192 ± 31 nN) between cells before and after tEMT. However, without proper development of the F-actin structure and adequate adherent time, the mechanical differences were diminished. After tEMT, the cells with increased stiffness and a cell-substrate adhesion force benefited by migrating more rapidly and had more invasiveness. Thus, this technology has the potential to benefit research focused on cancer diagnosis, drug development, and cell-substrate interactions.
Asunto(s)
Actinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Fenómenos Mecánicos , Microtecnología/instrumentación , Factor de Crecimiento Transformador beta1/farmacología , Animales , Fenómenos Biomecánicos , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Citocalasina D/farmacología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Ratones , Vinculina/metabolismoRESUMEN
Dopa-responsive dystonia (DRD) is induced by a deficiency of GTP cyclohydrolase I (GCH) and has a postulated autosomal dominant inheritance with a low penetrance. G201E is a dominant DRD mutation. Recombinant G201E mutant protein possessed very low enzyme activity. When G201E was expressed in eukaryotic cells, only a small amount of GCH protein could be detected. In baby hamster kidney cells, G201E protein was synthesized normally but was degraded rapidly in pulse-chase experiments. More interestingly, G201E dramatically decreased the level of wild-type protein and GCH activity in cotransfection studies. Therefore, G201E exerts a dominant-negative effect on the wild-type protein, probably going through an interaction between them. We also showed that L79P but not R249S (a recessive DRD mutation) had a dominant-negative effect. Through the dominant-negative mechanism, a single mutation could decrease GCH activity to less than 50% of normal. This study not only explains the inheritance of DRD but also increases the understanding of genetic diseases associated with multiple subunit proteins.
Asunto(s)
Dihidroxifenilalanina/uso terapéutico , Distonía/genética , GTP Ciclohidrolasa/deficiencia , GTP Ciclohidrolasa/genética , Animales , Cricetinae , Distonía/tratamiento farmacológico , Riñón/patología , Mutación/genéticaRESUMEN
A unique structure and in situ localization of E proteins were demonstrated in cultured neurons infected with neurovirulent and aneurovirulent strains of local Japanese encephalitis virus (JEV). Dilated rough endoplasmic reticulum (rER) containing smooth membrane structures (SMS) was continuous with the outer membrane of the nuclear envelope. These membranes were found to be connected to unique dense bodies, membrane vesicle structures (MVS). The de novo formation of SMS, annulate lamellae, and the appearance of MVS indicated proliferation of the membranous system in response to JEV infection. E proteins were possibly assembled in the virions in the nuclear envelope or rER or on the plasma membrane. The interconnections between MVS, rER, and the nuclear envelope and immunogold labeling of E proteins on the MVS provided strong evidence that MVS serve as a reservoir of JEV components during virus assembly.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie)/fisiología , Virus de la Encefalitis Japonesa (Especie)/ultraestructura , Neuronas/virología , Proteínas del Envoltorio Viral/análisis , Proteínas del Envoltorio Viral/ultraestructura , Animales , Carcinoma Embrionario , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Ratones , Microscopía Inmunoelectrónica , Neuroblastoma , Neuronas/citología , Tretinoina/farmacología , Células Tumorales Cultivadas , Replicación ViralRESUMEN
GTP cyclohydrolase I (GTPCH) catalyzes the rate-limiting step of tetrahydrobiopterin (BH4) biosynthesis. GTPCH has been associated with two clinically distinct human diseases: the recessive hyperphenylalaninemia (HPA) and the dominant dopa-responsive dystonia (DRD). We found a recessive GTPCH mutation (R249S, 747C-->G in a dystonia patient. Her PHA-stimulated mononuclear blood cells had a normal amount of GTPCH mRNA, but low GTPCH activity. Arginine 249 is located at the C-terminus of GTPCH, outside the catalytic site. E. coli expressed recombinant R249S mutant protein possessed normal enzyme activity and kinetics. However, in transfected eukaryotic cells, R249S mutant protein expression level was lower than the wild-type protein. Therefore, this is suspected to be a destabilizing mutation. Our data suggest that DRD could be either dominantly or recessively inherited, and the inheritance might be determined by the mechanism of mutation.
Asunto(s)
Distonía/genética , GTP Ciclohidrolasa/genética , Sustitución de Aminoácidos , Línea Celular , Niño , ADN/química , ADN/genética , Análisis Mutacional de ADN , ADN Recombinante/genética , Dihidroxifenilalanina/uso terapéutico , Distonía/tratamiento farmacológico , Distonía/enzimología , Escherichia coli/genética , Femenino , GTP Ciclohidrolasa/deficiencia , GTP Ciclohidrolasa/metabolismo , Regulación Enzimológica de la Expresión Génica , Genes Recesivos , Células HeLa , Homocigoto , Humanos , Mutación , Plásmidos , Mutación Puntual , ARN Mensajero/sangre , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
By Reid's functional test of erythrocyte deformability, the filtration time of 1 ml 5% erythrocyte suspension through a 5 micron nucleopore membrane was measured from 18 normal people and 19 patients with thalassemia. In the control group, the filtration time at 37.0 degrees C and pH 7.4 was 3.26 +/- 0.38 sec. Erythrocyte deformability was constant while blood temperature stayed between 35.0 degrees C and 42.0 degrees C. However the erythrocyte filtration time prolonged as the blood became alkaline or incubated for more than 2 hours. The filtration time of blood was 4.56 +/- 1.02 sec for thalassemia patients, significantly different (p less than 0.01) as comparing to the normal control group. Poor deformability in diseased blood could be explained by membrane defect. Erythrocyte deformability is a good rheological indicator if the physiological conditions of blood is controlled well.