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1.
Proc Natl Acad Sci U S A ; 108(12): 4846-51, 2011 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-21383202

RESUMEN

The final stage of cytokinesis is abscission, the cutting of the narrow membrane bridge connecting two daughter cells. The endosomal sorting complex required for transport (ESCRT) machinery is required for cytokinesis, and ESCRT-III has membrane scission activity in vitro, but the role of ESCRTs in abscission has been undefined. Here, we use structured illumination microscopy and time-lapse imaging to dissect the behavior of ESCRTs during abscission. Our data reveal that the ESCRT-I subunit tumor-susceptibility gene 101 (TSG101) and the ESCRT-III subunit charged multivesicular body protein 4b (CHMP4B) are sequentially recruited to the center of the intercellular bridge, forming a series of cortical rings. Late in cytokinesis, however, CHMP4B is acutely recruited to the narrow constriction site where abscission occurs. The ESCRT disassembly factor vacuolar protein sorting 4 (VPS4) follows CHMP4B to this site, and cell separation occurs immediately. That arrival of ESCRT-III and VPS4 correlates both spatially and temporally with the abscission event suggests a direct role for these proteins in cytokinetic membrane abscission.


Asunto(s)
Citocinesis/fisiología , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Perros , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Endosomas/genética , Humanos , Factores de Transcripción/genética
2.
Biochim Biophys Acta ; 1793(5): 893-902, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19027042

RESUMEN

Many model systems and measurement tools have been engineered for observing and quantifying the effect of mechanics on cellular response. These have contributed greatly to our current knowledge of the molecular events by which mechanical cues affect cell biology. Cell responses to the mechanical properties of type 1 collagen gels are discussed, followed by a description of a model system of very thin, mechanically tunable collagen films that evoke similar responses from cells as do gel systems, but have additional advantages. Cell responses to thin films of collagen suggest that at least some of the mechanical cues that cells can respond to in their environment occur at the sub-micron scale. Mechanical properties of thin films of collagen can be tuned without altering integrin engagement, and in some cases without altering topology, making them useful in addressing questions regarding the roles of specific integrins in transducing or mitigating responses to mechanical cues. The temporal response of cells to differences in ECM may provide insight into mechanisms of signal transduction.


Asunto(s)
Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Modelos Biológicos , Animales , Línea Celular , Matriz Extracelular/química , Microscopía de Fuerza Atómica , Transducción de Señal/fisiología , Estrés Mecánico
3.
Langmuir ; 26(5): 3629-36, 2010 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-20104910

RESUMEN

The mechanical cues that adherent cells derive from the extracellular matrix (ECM) can effect dramatic changes in cell migration, proliferation, differentiation, and apoptosis. Model ECMs composed of collagen fibrils formed from purified collagen are an important experimental system to study cell responses to mechanical properties of the ECM. Using a self-assembled model system of a film composed of 100-200 nm diameter collagen fibrils overlaying a bed of smaller fibrils, we have previously demonstrated changes in cellular response to systematically controlled changes in mechanical properties of the collagen. In this study, we describe an experimental and modeling approach to calculate the elastic modulus of individual collagen fibrils, and thereby the effective stiffness of the entire collagen thin film matrix, from atomic force microscopy force spectroscopy data. These results demonstrate an approach to the analysis of fundamental properties of thin, heterogeneous, and organic films and add further insights into the mechanical and topographical properties of collagen fibrils that are relevant to cell responses to the ECM.


Asunto(s)
Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Nanoestructuras , Fenómenos Biomecánicos , Módulo de Elasticidad , Matriz Extracelular/metabolismo , Modelos Biológicos , Reproducibilidad de los Resultados
4.
Langmuir ; 26(17): 14111-7, 2010 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-20666411

RESUMEN

Type I collagen fibrillar thin films have been prepared on hydrophobic recovered poly(dimethylsiloxane) (PDMS) surfaces and inside of irreversibly sealed PDMS microfluidic devices. Fibrillar films prepared on PDMS surfaces have been characterized with optical microscopy and atomic force microscopy and compared with films prepared using more traditional bulk methods on thiol-coated gold substrates. Collagen fibril films formed after 18 h of incubation on PDMS surfaces were observed to have similar underlying film thicknesses (15 nm), fibril size (67 nm), fibril coverage (45%), and physiologically supermolecular structure when compared to films on gold substrates. Collagen fibrils formed within devices were also determined to be usable across physiologically relevant cell perfusion rates. To validate the utility of these collagen fibril thin films for cell culture applications, vascular smooth muscle cells are shown to attach to collagen fibrils and exhibit cell spread areas equivalent to those seen on collagen fibrils created via bulk cell culture methods on thiol-coated gold substrates. These results extend the use and benefits of collagen fibril thin films into microfluidic-based cellular studies.


Asunto(s)
Colágeno Tipo I/química , Dimetilpolisiloxanos/química , Membranas Artificiales , Técnicas Analíticas Microfluídicas/métodos , Tamaño de la Partícula , Propiedades de Superficie
5.
J Phys Chem B ; 110(1): 33-5, 2006 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-16471493

RESUMEN

We show that mixing zwitterionic lipids with up to 20% mole % cationic lipids produces gel-phase supported lipid bilayers that are morphologically free of defects detectable using noncontact mode atomic force microscopy (AFM). This contrasts with the observation of massive defects when anionic lipid was added, and also when no charged lipid was added. Infrared measurements of headgroup orientation in the presence of cationic lipid show that the mean headgroup orientation changes only minimally when temperature is lowered from the fluid phase to the gel phase. This is consistent with a tentative explanation, based on simple electrostatic arguments, in which cationic lipids "stitch" the bilayers together. On the functional side, this study demonstrates a simple method by which to minimize defects in gel-supported phospholipid bilayers.


Asunto(s)
Geles/química , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Microscopía de Fuerza Atómica/métodos , Transición de Fase , Sensibilidad y Especificidad , Electricidad Estática , Temperatura
6.
Nucleic Acids Res ; 29(7): 1534-8, 2001 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-11266555

RESUMEN

The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20-50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to gamma-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of approximately 2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the alpha parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.


Asunto(s)
Proteínas de Unión al ADN/genética , Neoplasias de la Próstata/genética , ARN Catalítico/genética , Secuencia de Bases , División Celular/genética , División Celular/efectos de la radiación , Citomegalovirus/genética , Reparación del ADN , Relación Dosis-Respuesta en la Radiación , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Proteínas Fluorescentes Verdes , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/patología , ARN Catalítico/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinasa Rad51 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/efectos de la radiación
7.
Nucleic Acids Res ; 30(2): E1, 2002 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-11788727

RESUMEN

Cellular survival following ionising radiation-mediated damage is primarily a function of the ability to successfully detect and repair DNA double-strand breaks (DSBs). Previous studies have demonstrated that radiosensitivity, determined as a reduction in colony forming ability in vitro, may be related to the incorrect repair (misrepair) of DSBs. The novel rapid dual fluorescence (RDF) assay is a plasmid-based reporter system that rapidly assesses the correct rejoining of a restriction-enzyme produced DSBs within transfected cells. We have utilised this novel assay to determine the fidelity of DSB repair in the prostate tumour cell line LNCaP, the bladder tumour cell line MGH-U1 and a radiosensitive subclone S40b. The two bladder cell lines have been shown in previous studies to differ in their ability to correctly repair plasmids containing a single DSB. Using the RDF assay we found that a substantial portion of LNCaP cells [80.4 +/- 5.3(standard error)%] failed to reconstitute reporter gene expression; however, there was little difference in this measure of DSB repair fidelity between the two bladder cell lines (48.3 +/- 3.5% for MGH-U1; 39.9 +/- 8.2% for S40b). The RDF assay has potential to be developed to study the relationship between DSB repair fidelity and radiosensitivity as well as the mechanisms associated with this type of repair defect.


Asunto(s)
Daño del ADN/genética , Reparación del ADN/genética , Plásmidos/genética , Plásmidos/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Vejiga Urinaria/genética , Supervivencia Celular , Análisis Mutacional de ADN/métodos , Citometría de Flujo , Fluorescencia , Genes Reporteros , Humanos , Masculino , Plásmidos/química , Tolerancia a Radiación , Reproducibilidad de los Resultados , Factores de Tiempo , Transfección , Células Tumorales Cultivadas
8.
Cancer Res ; 54(5): 1194-7, 1994 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-8118805

RESUMEN

Dietary intervention to prevent colon cancer is a major health issue. At present it is not clear which dietary factors modify colon cancer risk. Caloric restriction reduces the incidence of many spontaneous and carcinogen-induced tumors in rodents, but its role in human carcinogenesis is unknown. The relationships of body mass index (BMI), body composition, and resting metabolic rate (RMR) to colon cancer risk are also undefined. In this study involving obese persons, we measured the effect of reducing caloric intake on rectal cell proliferation, a biomarker in colon carcinogenesis, and studied the relation of BMI, body composition, and RMR to rectal cell proliferation. Colonic cell proliferation was measured in rectal biopsies from persons weighing more than 130% of ideal body weight. Follow-up biopsies were performed in patients who enrolled in and completed a 16-week behavior modification weight-reduction program in which caloric intake was reduced. Baseline measurements included body composition by total body electrical conductance, RMR, and BMI. Rectal biopsies were processed for autoradiography following incubation with [3H]thymidine. Epithelial proliferation measurements were evaluable in 35 persons at baseline and in 8 persons before and after caloric restriction. Before caloric restriction, mean (+/- SD) BMI was 38 +/- 4 kg/m2 and percentage of body fat 41 +/- 2%. Subjects reduced their caloric intake by a mean of 34 +/- 4% and their weight by 8.6 +/- 1%. Caloric restriction resulted in a 39% reduction in whole-crypt labeling index (P < 0.001) and a 57% reduction in upper crypt labeling index (P < 0.05) without reduction in crypt depth. Labeling index was unrelated to BMI, RMR, or body composition. We conclude that caloric restriction reduced rectal cell proliferation measurements--intermediate biomarkers related to colon carcinogenesis. BMI, RMR, and body composition were unrelated to colonic proliferation. Caloric restriction may have a role in colon cancer prevention.


Asunto(s)
Colon/citología , Neoplasias del Colon/prevención & control , Dieta Reductora , Ingestión de Energía , Obesidad/dietoterapia , Obesidad/patología , Adulto , Anciano , Biopsia , División Celular/fisiología , Colon/fisiología , Neoplasias del Colon/dietoterapia , Neoplasias del Colon/etiología , Células Epiteliales , Epitelio/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Obesidad/complicaciones , Recto/citología , Recto/fisiología
9.
Biotechnol Prog ; 28(4): 1069-78, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22619183

RESUMEN

Descriptive terms are often used to characterize cells in culture, but the use of nonquantitative and poorly defined terms can lead to ambiguities when comparing data from different laboratories. Although recently there has been a good deal of interest in unambiguous identification of cell lines via their genetic markers, it is also critical to have definitive, quantitative metrics to describe cell phenotypic characteristics. Quantitative metrics of cell phenotype will aid the comparison of data from experiments performed at different times and in different laboratories where influences such as the age of the population and differences in culture conditions or protocols can potentially affect cellular metabolic state and gene expression in the absence of changes in the genetic profile. Here, we present examples of robust methodologies for quantitatively assessing characteristics of cell morphology and cell-cell interactions, and of growth rates of cells within the population. We performed these analyses with endothelial cell lines derived from dolphin, bovine and human, and with a mouse fibroblast cell line. These metrics quantify some characteristics of these cells lines that clearly distinguish them from one another, and provide quantitative information on phenotypic changes in one of the cell lines over large number of passages.


Asunto(s)
Técnicas Citológicas/métodos , Células Endoteliales/citología , Fibroblastos/citología , Microscopía Fluorescente/métodos , Microscopía de Contraste de Fase/métodos , Animales , Bovinos , Línea Celular , Proliferación Celular , Tamaño de la Célula , Delfines , Células Endoteliales/química , Fibroblastos/química , Humanos , Cinética , Ratones , Fenotipo
11.
Biomaterials ; 30(29): 5486-96, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19640581

RESUMEN

The enzyme tissue transglutaminase 2 (TG2) appears to play an important role in several physiological processes such as wound healing, the progression of cancer and of vascular disease. Additionally, TG2 has been proposed as a means of stabilizing collagen extracellular matrix (ECM) scaffolds for tissue engineering applications. In this report, we examined the effect of TG2 treatment on the mechanical properties of the ECM, and associated cell responses. Using a model ECM of fibrillar collagen, we quantitatively examined vascular smooth muscle cell (vSMC) response to untreated, or TG2 treated collagen. We show that cells respond to TG2 treated collagen with increased spreading, an increase in contractile response as indicated by elevated F-actin polymerization and myosin light chain phosphorylation, and increased proliferation, without apparent changes in integrin specificity or matrix topography. Comparative atomic force microscopy loading studies indicate that TG2 treated fibrils are 3 times more resistant to shearing force from an AFM tip than untreated fibrils. The data suggest that TG2 treatment of collagen increases matrix mechanical stiffness, which apparently alters the contractile and proliferative response of vSMC.


Asunto(s)
Células Endoteliales/fisiología , Colágenos Fibrilares/química , Contracción Muscular/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Transducción de Señal/fisiología , Transglutaminasas/administración & dosificación , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Proteínas de Unión al GTP , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Proteína Glutamina Gamma Glutamiltransferasa 2 , Ratas , Transducción de Señal/efectos de los fármacos , Ingeniería de Tejidos/métodos , Transglutaminasas/química
12.
Biomaterials ; 30(35): 6687-94, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19762078

RESUMEN

Cells within tissues derive mechanical anchorage and specific molecular signals from the insoluble extracellular matrix (ECM) that surrounds them. Understanding the role of different cues that extracellular matrices provide cells is critical for controlling and predicting cell response to scaffolding materials. Using an engineered extracellular matrix of Type I collagen we examined how the stiffness, supramolecular structure, and glycosylation of collagen matrices influence the protein levels of cellular FAK and the activation of myosin II. Our results show that (1) cellular FAK is downregulated on collagen fibrils, but not on a non-fibrillar monolayer of collagen, (2) the downregulation of FAK is independent of the stiffness of the collagen fibrils, and (3) FAK levels are correlated with levels of tyrosine phosphorylation of the collagen adhesion receptor DDR2. Further, siRNA depletion of DDR2 blocks FAK downregulation. Our results suggest that the collagen receptor DDR2 is involved in the regulation of FAK levels in vSMC adhered to Type I collagen matrices, and that regulation of FAK levels in these cells appears to be independent of matrix stiffness.


Asunto(s)
Regulación hacia Abajo , Matriz Extracelular/fisiología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Colágeno/metabolismo , Receptores Mitogénicos/metabolismo , Animales , Células Cultivadas , Colágeno Tipo I/química , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Receptores con Dominio Discoidina , Matriz Extracelular/química , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/análisis , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Glicosilación , Ratas , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Colágeno/genética , Receptores Mitogénicos/genética
13.
Nano Lett ; 7(2): 531-5, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17298021

RESUMEN

We show that water-soluble fullerenes accumulate on the surface of zwitterionic and cationic supported bilayers to different extents. We propose on the basis of bilayer thicknesses, phase-transition temperatures, and fullerene movement that the water-soluble fullerenes do not penetrate into the hydrocarbon tails of supported bilayers. These findings are important to toxicity issues concerning fullerene materials and the development of decorated lipid bilayers for future drug delivery or sensor application.


Asunto(s)
Fulerenos/química , Membrana Dobles de Lípidos/química , Dimiristoilfosfatidilcolina/química , Microscopía de Fuerza Atómica , Miristatos/química , Nanotecnología/métodos , Compuestos de Amonio Cuaternario/química , Solubilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Termodinámica , Agua
14.
Biophys J ; 91(8): 2919-27, 2006 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-16877517

RESUMEN

Poly-L-lysine-induced morphological changes in liquid phase supported bilayers consisting of mixed anionic/zwitterionic and neat zwitterionic headgroup phospholipids were studied with atomic force microscopy and epifluorescence microscopy. Results obtained from these studies indicate that poly-L-lysine can induce domains, defects, and aggregate structures on both mixed bilayers and strictly zwitterionic bilayers. The structures formed on liquid phase supported bilayers were observed to be immobile from a timescale of 50 ms to several minutes. We propose that poly-L-lysine of sufficient length interacts with the mica substrate and phospholipids to create the stationary structures noted.


Asunto(s)
Membrana Dobles de Lípidos/química , Fosfolípidos/química , Polilisina/química , Geles , Iones , Microscopía de Fuerza Atómica
15.
Biophys J ; 88(3): 2154-64, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15596519

RESUMEN

We utilize in situ, temperature-dependent atomic force microscopy to examine the gel-fluid phase transition behavior in supported phospholipid bilayers constructed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. The primary gel-fluid phase transition at T(m) occurs through development of anisotropic cracks in the gel phase, which develop into the fluid phase. At approximately 5 degrees C above T(m), atomic force microscopy studies reveal the presence of a secondary phase transition in all three bilayers studied. The secondary phase transition occurs as a consequence of decoupling between the two leaflets of the bilayer due to enhanced stabilization of the lower leaflet with either the support or the water entrained between the support and the bilayer. Addition of the transmembrane protein gramicidin A or construction of a highly defected gel phase results in elimination of this decoupling and removal of the secondary phase transition.


Asunto(s)
Membrana Dobles de Lípidos/química , Fluidez de la Membrana , Microscopía de Fuerza Atómica/métodos , Fosfolípidos/análisis , Fosfolípidos/química , Geles/análisis , Geles/química , Membrana Dobles de Lípidos/análisis , Sustancias Macromoleculares/análisis , Sustancias Macromoleculares/química , Conformación Molecular , Transición de Fase , Temperatura
16.
J Cell Physiol ; 172(3): 306-13, 1997 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9284950

RESUMEN

Retinoic acid inhibits proliferation of hormone-dependent, but not hormone-independent breast cancer cells. Retinoic acid-induced changes in cellular proliferation and differentiation are associated with disturbances in growth factor signaling and frequently with changes in protein kinase C expression. PKC delta, epsilon, and zeta are expressed in both hormone-dependent (T-47D) and hormone-independent (MDA-MB-231) cell lines. Retinoic acid arrested T-47D proliferation, induced PKC alpha expression and concomitantly repressed PKC zeta expression. The changes in PKC alpha and PKC zeta reflect retinoic acid-induced changes in mRNA. In contrast, retinoic acid had no effect on growth, or PKC expression in MDA-MB-231 cells. Growth arrest and the induction of PKC alpha, but not the reduction in PKC zeta, resulted from selective activation of RAR alpha. In total, these results support an important role for PKC alpha in mediating the anti-proliferative action of retinoids on human breast carcinoma cells.


Asunto(s)
Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Isoenzimas/genética , Neoplasias Hormono-Dependientes/patología , Proteína Quinasa C/genética , Tretinoina/farmacología , Benzoatos/farmacología , Northern Blotting , Western Blotting , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica , Humanos , Isoenzimas/metabolismo , Neoplasias Hormono-Dependientes/enzimología , Neoplasias Hormono-Dependientes/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C-alfa , Proteína Quinasa C-delta , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Retinoides/farmacología , Tetrahidronaftalenos/farmacología , Células Tumorales Cultivadas
17.
EMBO Rep ; 2(7): 609-14, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11454737

RESUMEN

Colon cancer cells frequently display minisatellite instability (MIN) or chromosome instability (CIN). While MIN is caused by mismatch repair defects, the lesions responsible for CIN are unknown. The observation that CIN cells fail to undergo mitotic arrest following spindle damage suggested that mutations in spindle checkpoint genes may account for CIN. However, here we show that CIN cells do undergo mitotic arrest in response to spindle damage. Although the maximum mitotic index achieved by CIN lines is diminished relative to MIN lines, CIN cells clearly have a robust spindle checkpoint. Consistently, mutations in spindle checkpoint genes are rare in human tumours. In contrast, the adenomatous polyposis coli (APC) gene is frequently mutated in CIN cells. Significantly, we show here that expression of an APC mutant in MIN cells reduces the mitotic index following spindle damage to a level observed in CIN cells, suggesting that APC dysfunction may contribute to CIN.


Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Aneuploidia , Ciclo Celular/fisiología , Neoplasias del Colon/genética , Genes cdc/fisiología , Huso Acromático/fisiología , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Antineoplásicos/farmacología , Cromosomas/metabolismo , Neoplasias del Colon/patología , Neoplasias del Colon/fisiopatología , Citometría de Flujo , Genes APC , Humanos , Microscopía Fluorescente , Índice Mitótico , Nocodazol/farmacología , Huso Acromático/efectos de los fármacos , Huso Acromático/genética , Células Tumorales Cultivadas
18.
Anal Chem ; 74(5): 1157-64, 2002 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-11924978

RESUMEN

Vapor adsorption into porous ultrathin films on a gold surface is investigated with in situ surface plasmon resonance (SPR) and polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS). The thin films are prepared by the electrostatic self-assembly of oppositely charged poly(L-lysine) (PL) and silica nanoparticles on a chemically modified gold surface. Characterization with ex situ SPR and PM-IRRAS demonstrates the buildup of multiple PL/SiO2 bilayers as well as an excellent correlation between the quantitative results from these two techniques. In situ vapor adsorption experiments with these thin films show evidence of porosity, reproducibility, and rapid reversibility. Exposure to acetone vapor (P/P0 = 0.032) causes the film to adsorb 9% acetone by volume, which corresponds to coverage of approximately one-half of the silica nanoparticle surface area. In situ PM-IRRAS provides much information about the molecular interactions occurring in the film upon adsorption or desorption of vapors. Dosing with a mixture of vapors leads to a competition for adsorption into the film, and PM-IRRAS results show that acetone slightly outcompetes nitromethane. These experiments with nanoparticle thin films demonstrate the advantages of using in situ PM-IRRAS for studying reversible adsorption in the presence of vapor mixtures.

19.
J Am Chem Soc ; 126(37): 11420-1, 2004 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-15366871

RESUMEN

Dendrimers with molecular weights ranging from ca. 2700 to 11 000 and from 16 to 64 homoallyl ether end groups were cross-linked using the Grubbs ring-closing metathesis reaction. A combination of SEC, MALDI-TOF-MS, and AFM were used to characterize the cross-linked nanoparticles. The data suggest a significant decrease in volume with cross-linking and a concomitant increase in rigidity, both of which can be controlled independently with a fair degree of precision.


Asunto(s)
Éteres/química , Polímeros/química , Reactivos de Enlaces Cruzados/química , Éteres/síntesis química , Peso Molecular , Nanotecnología , Tamaño de la Partícula , Polímeros/síntesis química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
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