RESUMEN
We herein report a 61-year-old man who sustained injury after a 2-m fall and developed mediastinitis. He presented to another hospital two days after the fall and was transferred to our hospital four days after the fall with a fever and dysphagia. Computed tomography revealed osteophytes on the second and third thoracic vertebrae and free air in the mediastinum, indicating esophageal perforation. Emergent surgery was performed. Intraoperatively, a longitudinal esophageal tear was identified. We stress the importance of being aware of the possibility of osteophyte-related esophageal perforation in patients with a history of a fall. A delayed diagnosis affects the prognosis.
Asunto(s)
Perforación del Esófago , Mediastinitis , Osteofito , Heridas no Penetrantes , Perforación del Esófago/complicaciones , Perforación del Esófago/diagnóstico por imagen , Humanos , Masculino , Mediastinitis/complicaciones , Mediastinitis/diagnóstico por imagen , Persona de Mediana Edad , Osteofito/complicaciones , Osteofito/diagnóstico por imagen , Osteofito/cirugía , Vértebras Torácicas/diagnóstico por imagen , Vértebras Torácicas/cirugía , Heridas no Penetrantes/complicaciones , Heridas no Penetrantes/diagnóstico por imagenRESUMEN
It has been reported that the amount of intermediate water, defined as water molecules loosely bound to a material, is a useful index of the material's bio-inert properties. Polyethylene glycol (PEG) is a well-known biocompatible polymer with a large amount of intermediate water. Many researchers have showed that PEGylated nanoparticles are passively accumulated in tumor tissues owing to their enhanced permeability and retention (EPR) effects. Dendrimers are regularly branched polymers with highly controllable size and structure, which can be exploited as potent drug carriers. In this study, we investigated the tripartite relationship among the PEG density, the hydration state, and the passive tumor targeting property, using PEGylated dendrimers. The fully PEGylated dendrimer, PEG64-den, showed similar hydration behavior to PEG and a passive tumor targeting property. In contrast, the hydration state of the partly PEGylated dendrimer, PEG5-den, was different from that of PEG64-den, and the passive tumor targeting property was not observed. This is the first report to show the hydration state of a drug carrier as well as discuss a relationship between the hydration state and biodistribution.
Asunto(s)
Dendrímeros , Neoplasias , Portadores de Fármacos , Humanos , Neoplasias/tratamiento farmacológico , Polietilenglicoles , Distribución TisularRESUMEN
ALG-2, a member of the penta-EF-hand protein family, interacts Ca²+-dependently with a COPII component, Sec31A. In this study, we first established HeLa cells stably expressing green fluorescent protein-fused ALG-2 (GFP-ALG-2) and red fluorescent protein-fused Sec31A (Sec31A-RFP). After inducing Ca²+-mobilization, the cytoplasmic distribution of GFP-ALG-2 changed from a diffuse to a punctate pattern, which extensively overlapped with the Sec31A-RFP-positive structures, indicating that ALG-2 is recruited to the endoplasmic reticulum exit sites (ERES) in living cells. Next, overlay experiments with biotin-labeled ALG-2 were done to dissect the ALG-2 binding site (ABS). They revealed that a sequence comprising amino acid residues 839-851 in the Pro-rich region was necessary and sufficient for direct binding to ALG-2. Finally, fluorescence recovery after photobleaching analysis indicated that the ABS deletion reduced the high-affinity population of Sec31A to the ERES, suggesting that the ABS is one of the key determinants of the retention kinetics of Sec31A at ERES.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Imagen de Lapso de Tiempo/métodos , Proteínas de Transporte Vesicular/metabolismo , Sitios de Unión , Vesículas Cubiertas por Proteínas de Revestimiento , Células HeLa , Humanos , Cinética , Proteínas Luminiscentes , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de FusiónRESUMEN
Alix and TSG101, known to physically interact with each other, have Pro-rich regions that are bound by ALG-2 Ca2+-dependently. We investigated the role of ALG-2 in the Alix-TSG101 association by pulldown assays using Strep-tagged Alix and its various mutants. The ALG-2-binding site was required for the Ca2+-dependent pulldown of TSG101 using HEK293T cells, whereas the PSAP sequence, a binding motif for the UEV domain of TSG101, was dispensable. Alix-TSG101 association was not observed using ALG-2-knockdown cells but became detectable by addition of the purified recombinant ALG-2 protein in the assay mixtures. Exogenous expression of mGFP-fused ALG-2 also restored the pulldown capability of Strep-Alix, but an alternatively spliced shorter ALG-2 isoform and a dimerization-defective mutant were incompetent. Based on the X-ray crystal structure model showing the presence of one ligand-binding site in each molecule of an ALG-2 dimer, we propose that Ca2+-loaded ALG-2 bridges Alix and TSG101 as an adaptor protein.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Factores de Transcripción/genéticaRESUMEN
During the manufacture of cell- and tissue-based products, such as engineered cartilage for autologous chondrocyte implantation, maximizing the number of cells isolated from donor tissue substantially improves the productivity of these products. The method used for agitating tissues with digestive fluid and enzymes can considerably affect both the quality and quantity of isolated cells. This study aimed to investigate the effectiveness of a rotation/revolution-type agitator for chondrocyte isolation following the enzymatic digestion of rat costal cartilage. Cartilage tissue cut into 1 mm3-thick sections was equally divided between two groups and placed in 50-mL conical tubes; sections in both groups were digested using 0.1 mg/mL liberase TH (collagenase/thermolysin) at 37 °C for 4 h with either rotation/revolution or conventional orbital agitation method. Compared with using conventional orbital agitator, using the rotation/revolution-type agitator resulted in a significant (>two-fold) increase in the number of isolated cells. In subsequent primary cultures, chondrocytes obtained by rotation/revolution agitation showed superior initial attachment to tissue culture dish on day 1 and 2 compared with those obtained by conventional agitation; however, no differences in cell proliferation or cartilage-related molecule expression patterns were observed between cells derived from either method after 3 days of subculture. These findings suggested that there are no disadvantages to the proposed rotation/revolution agitation method. Rotation/revolution-type agitators are a promising apparatus for preparing chondrocytes for primary cultures and cartilage tissue engineering.
Asunto(s)
Cartílago/fisiología , Separación Celular/instrumentación , Condrocitos/citología , Rotación , Técnicas de Cultivo de Tejidos/instrumentación , Ingeniería de Tejidos , Animales , Cartílago/citología , Cartílago/crecimiento & desarrollo , Proliferación Celular , Separación Celular/métodos , Células Cultivadas , Colagenasas/metabolismo , Diseño de Equipo , Ratas , Ratas Sprague-Dawley , Costillas/citología , Termolisina/metabolismo , Técnicas de Cultivo de Tejidos/métodos , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodosRESUMEN
ALG-2, a prototypic member of the penta-EF-hand protein family, interacts with Alix at its C-terminal Pro-rich region containing four tandem PXY repeats. Human phospholipid scramblase 3 (PLSCR3) has a similar sequence (ABS-1) in its N-terminal region. In the present study, we found that ALG-2 interacts with PLSCR3 expressed in HEK293 cells in a Ca(2+)-dependent manner by co-immunoprecipitation, pulldown with glutathione S-transferase (GST) fused ALG-2 and an overlay assay using biotin-labeled ALG-2. The GST fusion protein of an alternatively spliced isoform of ALG-2, GST-ALG-2(DeltaGF122), pulled down green fluorescent protein (GFP)-fused PLSCR3 but not GFP Alix. Deletion of a region containing ABS-1 was not sufficient to abrogate the binding. A second ALG-2-binding site (ABS-2) was essential for interaction with ALG-2(DeltaGF122). Real-time interaction analyses with a surface plasmon resonance biosensor using synthetic oligopeptides and recombinant proteins corroborated direct Ca(2+)-dependent binding of ABS-1 to ALG-2 and that of ABS-2 to ALG-2 as well as to ALG-2(DeltaGF122). The sequence of ABS-2 contains multiple prolines and two phenylalanines, among which Phe(49) was found to be critical, because its substitution with Ala or Tyr caused a loss of binding ability by pulldown assays using oligopeptide-immobilized beads. ALG-2-interacting proteins were classified into two groups based on binding ability to ALG-2(DeltaGF122): (i) isoform-non-interactive (ABS-1) types, including Alix, annexin A7, annexin A11, and TSG101 and (ii) isoform-interactive (ABS-2) types including PLSCR3, PLSCR4 and Sec31A. GST-pulldown assays using single amino acid-substituted ALG-2 mutants revealed differences in binding specificities between the two groups, suggesting structural flexibility in ALG-2-ligand complex formation.
Asunto(s)
Empalme Alternativo/fisiología , Sustitución de Aminoácidos , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Anexina A7/genética , Anexina A7/metabolismo , Anexinas/genética , Anexinas/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Sitios de Unión/genética , Proteínas de Unión al Calcio/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas de Transferencia de Fosfolípidos/genética , Unión Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Intracellular localization of the penta-EF-hand Ca2+-binding protein ALG-2 in HeLa cells was investigated by immunofluorescent confocal microscopy using a polyclonal antibody. In addition to its presence in the nucleus, ALG-2 was found to be distributed in a punctate pattern in the cytoplasm, where it was partly co-stained with an endoplasmic reticulum (ER) exit site marker p125. In vitro GST pull down analysis demonstrated that ALG-2 and its alternatively spliced isoform interact with the COPII component Sec31A in a Ca2+-dependent manner, and a biotin-labeled ALG-2 overlay assay revealed direct binding of ALG-2 to Sec31A. Biochemical and immunofluorescent microscopic analyses showed that ALG-2 was enriched at the Sec31A-localizing membrane compartments upon stimulation with the Ca2+ ionophore A23187. In contrast, treatment of cells with the membrane-permeant Ca2+ chelator BAPTA-AM led to a dispersion of ALG-2 throughout the cells and to a significant loss of Sec31A in the perinuclear region. These findings establish Sec31A as a novel target for ALG-2 and provide a framework for studies on the roles of ALG-2 in ER-Golgi transport.