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1.
Anal Biochem ; 439(1): 50-61, 2013 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-23583820

RESUMEN

Quartz crystal microbalances (QCMs) measure mass on the nanogram (ng) scale. We built novel QCMs as toxicity biosensors incorporating living cells. Human endothelial cells or canine macrophages were equilibrated on QCM crystal surfaces until stable oscillation frequencies occurred. Vehicle or sodium azide (NaN3) (25-100 mM) was added to these QCMs while continuously collecting crystal oscillation frequency data. At these doses, NaN3 alters mitochondrial membrane permeability and causes mitochondrial swelling and intrinsic apoptosis. Our studies demonstrated no frequency change in QCMs with untreated cells or without cells but NaN3. If NaN3 was added to either cell type within QCMs, 5 to 8 min later increases in oscillation frequency (Δf) occurred (400-1600 Hz) that correlated with dose. All frequency changes reverted to baseline by 15 min. In parallel, during the first 30 min, no change in cell or nuclear areas, or in actin or microtubule distributions, was detected. Yet, mitochondrial size and membrane permeability increased significantly during, but not after, 5 to 8 min. Viability studies confirmed dose-dependent toxicity that was predicted and proportionate to the 5- to 8-min Δf. These studies confirm that cell-based QCMs can detect early events in intrinsic apoptosis and reveal unique kinetic information about events occurring within subcellular structures in response to toxins.


Asunto(s)
Vasos Sanguíneos/citología , Vasos Sanguíneos/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Tecnicas de Microbalanza del Cristal de Cuarzo , Azida Sódica/toxicidad , Animales , Apoptosis/efectos de los fármacos , Citotoxinas/toxicidad , Perros , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Humanos , Pulmón/citología , Tamaño Mitocondrial/efectos de los fármacos , Permeabilidad/efectos de los fármacos
2.
Proc Natl Acad Sci U S A ; 107(8): 3351-5, 2010 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-19966310

RESUMEN

Urodeles and fetal mammals are capable of impressive epimorphic regeneration in a variety of tissues, whereas the typical default response to injury in adult mammals consists of inflammation and scar tissue formation. One component of epimorphic regeneration is the recruitment of resident progenitor and stem cells to a site of injury. Bioactive molecules resulting from degradation of extracellular matrix (ECM) have been shown to recruit a variety of progenitor and stem cells in vitro in adult mammals. The ability to recruit multipotential cells to the site of injury by in vivo administration of chemotactic ECM degradation products in a mammalian model of digit amputation was investigated in the present study. Adult, 6- to 8-week-old C57/BL6 mice were subjected to midsecond phalanx amputation of the third digit of the right hind foot and either treated with chemotactic ECM degradation products or left untreated. At 14 days after amputation, mice treated with ECM degradation products showed an accumulation of heterogeneous cells that expressed markers of multipotency, including Sox2, Sca1, and Rex1 (Zfp42). Cells isolated from the site of amputation were capable of differentiation along neuroectodermal and mesodermal lineages, whereas cells isolated from control mice were capable of differentiation along only mesodermal lineages. The present findings demonstrate the recruitment of endogenous stem cells to a site of injury, and/or their generation/proliferation therein, in response to ECM degradation products.


Asunto(s)
Factores Biológicos/farmacología , Quimiotaxis , Matriz Extracelular/metabolismo , Células Madre Multipotentes/efectos de los fármacos , Regeneración/efectos de los fármacos , Amputación Quirúrgica , Animales , Factores Biológicos/aislamiento & purificación , Factores Biológicos/metabolismo , Proliferación Celular , Matriz Extracelular/química , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Células Madre Multipotentes/fisiología , Cicatrización de Heridas
3.
Anal Biochem ; 421(1): 164-71, 2012 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-22119070

RESUMEN

During transformation of a normal cell to a cell capable of forming a cancerous growth, cellular morphology, the cytoskeleton, and focal contacts undergo significant changes. These changes should be capable of being characterized via real-time monitoring of the dynamic cell adhesion process and viscoelastic properties of cells. Here, we describe use of the quartz crystal microbalance (QCM) to distinguish the dynamic cell adhesion signatures of human normal (HMEC) versus malignant (MCF-7) mammary epithelial cells. The significantly reduced QCM responses (changes in frequency [Δf] and motional resistance ΔR) of MCF-7 cells compared with those of HMECs mirror the cancer cells' morphological features as observed via optical microscope. We analyzed the initial 2-h cell adhesion kinetics, suggesting cell-cell cooperativity for HMECs and no or weak cell-cell interactions for MCF-7 cells. We propose that changes of the ΔR/Δf ratio, which we term the cell viscoelastic index (CVI), reflect the establishment of cytoskeleton structure and dynamic viscoelastic properties of living cells. The CVI decreases significantly on initiation of cell to surface interactions as cells establish their cytoskeletal structures. During the cell adhesion process, MCF-7 cells were consistently softer, exhibiting up to a 2.5-fold smaller CVI when compared with HMECs.


Asunto(s)
Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Mama/citología , Mama/fisiología , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Adhesión Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/patología , Células Cultivadas , Elasticidad , Células Epiteliales/citología , Células Epiteliales/fisiología , Femenino , Humanos , Viscosidad
4.
Int J Hyperthermia ; 27(7): 682-97, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21992561

RESUMEN

PURPOSE: HER-2 is in the EGF tyrosine kinase receptor family, overexpressed by many human cancers and minimally expressed by normal adult tissues. HER-2 expression in human cancers is correlated with reduced survival, increased metastasis, reduced apoptosis and increased proliferation. Herceptin is a humanised mouse antibody that targets and inactivates HER-2. In the present study, Herceptin was used to deliver ferric oxide-enriched nanoparticles to HER-2(+) cancer cells. If exposed to alternating magnetic field (AMF), the nanoparticles heat. We tested the ability of AMF-activated Herceptin-directed nanoparticles to selectively kill HER-2(+) human cancer cells. METHODS: Herceptin-conjugated nanoparticles were incubated with normal human mammary epithelial cells (HMEC)(HER-2(-)) or malignant human mammary epithelial cells (SK-BR-3)(HER-2(+)). Cells were stained to detect Herceptin or iron and the kinetics of binding quantified. Once conditions were optimised for binding, cells were exposed to either antibody-directed or non-antibody-conjugated nanoparticles, washed and sham-treated or exposed to AMF and cell death quantified. RESULTS: SK-BR-3 cells bound Herceptin-directed nanoparticles in increasing amounts over 3 h but did not retain non-antibody conjugated nanoparticles. HMECs did not retain either nanoparticles. SK-BR-3 cells with bound Herceptin-directed-nanoparticles, exposed to AMF, died by apoptosis, quantifiable by Live/Dead and nuclear morphology assays and released LDH. Sham-treated SK-BR-3 cells with Herceptin-directed nanoparticles, HMECs with either nanoparticles, with or without AMF treatment, exhibited no increase in toxicity above baseline cell death using these three assays. CONCLUSIONS: These studies demonstrate Herceptin-directed nanoparticles can selectively kill HER-2(+) cancer cells via hyperthermia after AMF activation.


Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Neoplasias de la Mama/terapia , Hipertermia Inducida/métodos , Campos Magnéticos , Nanopartículas/uso terapéutico , Apoptosis , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Compuestos Férricos/administración & dosificación , Humanos , Receptor ErbB-2/metabolismo , Trastuzumab
5.
Part Fibre Toxicol ; 8: 4, 2011 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-21266033

RESUMEN

BACKGROUND: Numerous engineered nanomaterials (ENMs) exist and new ENMs are being developed. A challenge to nanotoxicology and environmental health and safety is evaluating toxicity of ENMs before they become widely utilized. Cellular assays remain the predominant test platform yet these methods are limited by using discrete time endpoints and reliance on organic dyes, vulnerable to interference from ENMs. Label-free, continuous, rapid response systems with biologically meaningful endpoints are needed. We have developed a device to detect and monitor in real time responses of living cells to ENMs. The device, a living cell quartz crystal microbalance biosensor (QCMB), uses macrophages adherent to a quartz crystal. The communal response of macrophages to treatments is monitored continuously as changes in crystal oscillation frequency (Δf). We report the ability of this QCMB to distinguish benign from toxic exposures and reveal unique kinetic information about cellular responses to varying doses of single-walled carbon nanotubes (SWCNTs). RESULTS: We analyzed macrophage responses to additions of Zymosan A, polystyrene beads (PBs) (benign substances) or SWCNT (3-150 µg/ml) in the QCMB over 18 hrs. In parallel, toxicity was monitored over 24/48 hrs using conventional viability assays and histological stains to detect apoptosis. In the QCMB, a stable unchanging oscillation frequency occurred when cells alone, Zymosan A alone, PBs alone or SWCNTs without cells at the highest dose alone were used. With living cells in the QCMB, when Zymosan A, PBs or SWCNTs were added, a significant decrease in frequency occurred from 1-6 hrs. For SWCNTs, this Δf was dose-dependent. From 6-18 hrs, benign substances or low dose SWCNT (3-30 µg/ml) treatments showed a reversal of the decrease of oscillation frequency, returning to or exceeding pre-treatment levels. Cell recovery was confirmed in conventional assays. The lag time to see the Δf reversal in QCMB plots was linearly SWCNT-dose dependent. Lastly, the frequency never reversed at high dose SWCNT (100-150 µg/ml), and apoptosis/necrosis was documented in conventional 24 and 48 hr-assays. CONCLUSION: These data suggest that the new QCMB detects and provides unique information about peak, sub-lethal and toxic exposures of living cells to ENMs before they are detected using conventional cell assays.


Asunto(s)
Técnicas Biosensibles/métodos , Macrófagos/efectos de los fármacos , Monitoreo Fisiológico/métodos , Nanotubos de Carbono/toxicidad , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Macrófagos/patología , Macrófagos/fisiología , Fagocitosis/efectos de los fármacos , Poliestirenos/toxicidad , Cuarzo , Zimosan/toxicidad
6.
Anal Biochem ; 384(1): 86-95, 2009 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-18926794

RESUMEN

We have created thin films by cyclic voltammetry (CV) electropolymerizations of the following phenolic functional group-based monomers: phenol; tyrosineamide; the tetrapeptide RGDY-containing the integrin membrane adhesion protein recognition tripeptide RGD; RDGY, a nonsense control tetrapeptide; and 1:3 mixtures of tyrosineamide with the two tetrapeptide monomers. The film formation process and description of the film properties were obtained by repetitive CV cycling using the oscillating quartz frequency shift, Deltaf, and motional resistance shift, DeltaR, parameters obtained with the electrochemical quartz crystal microbalance technique. Only the poly(phenol) film exhibited close chain packing-based self-limiting behavior, where all film synthesis ceased after approximately 7 CV cycles. All other films continued to form by electropolymerization with successive CV cycles out to the maximum cycle number (30 cycles) we measured. All of the films exhibited little energy dissipation behavior. Using the quartz crystal microbalance, we next compared the time course of cell attachment with the washed films and demonstrated that cells bound best to films in the following order: RGDY sense peptide:tyrosineamide films>RDGY nonsense peptide:tyrosineamide films=tyrosineamide films>phenol films. Cell enumeration after washing and trypsinization revealed firm protein-based cell attachment to the underlying extracellular matrix for the RGDY-containing films. These sense peptide films bound and retained two- to fivefold as many cells as the other films, with cells exhibiting a normal morphology. These results suggest the operation of specific cell attachment to the electropolymerized films via the RGD binding site for cellular integrin membrane proteins. The electropolymerization method we studied here forms a cassette system for creating electropolymerized films tailored to specific attachment of different cell types by varying the electropolymerized Y(tyrosine)-containing recognition peptide.


Asunto(s)
Electroquímica/métodos , Oligopéptidos/química , Polímeros/química , Tirosina/química , Sitios de Unión , Biomimética , Técnicas Biosensibles , Oligopéptidos/metabolismo
7.
Molecules ; 13(11): 2704-16, 2008 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-18978700

RESUMEN

Catechins, naturally occurring flavonoids derived from wine and green tea, are known to exhibit multiple health benefits. Epigallocatechin gallate (EGCG) is one of the most widely investigated catechins, but its efficacy in cancer therapy is still inconsistent and limited. The poor stability of EGCG has contributed to the disparity in the reported anti-cancer activity and other beneficial properties. Here we report an innovative enzymatic strategy for the oligomerization of catechins (specifically epicatechin) that yields stable, water-soluble oligomerized epicatechins with enhanced and highly specific anti-proliferative activity for human breast cancer cells. This one-pot oxidative oligomerization is carried out in ambient conditions using Horseradish Peroxidase (HRP) as a catalyst yielding water-soluble oligo(epicatechins). The oligomerized epicatechins obtained exhibit excellent growth inhibitory effects against human breast cancer cells with greater specificity towards growth-inhibiting cancer cells as opposed to normal cells, achieving a high therapeutic differential. Our studies indicate that water-soluble oligomeric epicatechins surpass EGCG in stability, selectivity and efficacy at lower doses.


Asunto(s)
Catequina/química , Catequina/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Catequina/metabolismo , Línea Celular Tumoral , Dicroismo Circular , Dimerización , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Estructura Molecular
8.
Int J Radiat Biol ; 82(8): 549-59, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16966182

RESUMEN

PURPOSE: The purpose of the study was to examine the optimal time of exposure and dose of heat and ionizing radiation that results in the killing of human cancer cells in vitro via apoptosis vs. necrosis. MATERIALS AND METHODS: Human mammary carcinoma, colorectal carcinoma and normal bovine capillary endothelial (BCE) cell lines were subjected to 20 Gy ionizing radiation and 6, 12, 24, and 72 h later assessed for apoptosis using detection of apoptotic bodies and caspase assays. Necrosis was detected by loss of cells from the surface and lactate dehydrogenase (LDH) release. The colorectal carcinoma cells were subjected to hyperthermia using temperatures ranging from 39 - 44 degrees C for 5, 15 or 45 min. exposures and at varying times post-treatment, apoptosis and necrosis were measured. RESULTS: In response to ionizing radiation, none of the cells underwent necrosis and some cell types apoptosed 24 and 72 h posttreatment. The colorectal cancer cells exhibited a steady increase of apoptosis at 6, 12, and 24 h. When these cells were exposed to 40 degrees C for 5 min, caspases increased within 6 h and a significant fraction (50%) of cells apoptosed. If the time of exposure to 40 degrees C was increased to 15 or 45 min, 80% and 100% of the dying cells apoptosed, respectively. A temperature of 39 degrees C did not cause cell death even after 45 min exposures. If heat was elevated to 42 or 44 degrees C, increased necrosis was observed with a corresponding decrease in apoptosis. CONCLUSIONS: These studies reveal time and temperature dependent in vitro cell responses to ionizing radiation and water-bath hyperthermia.


Asunto(s)
Apoptosis/efectos de la radiación , Carcinoma/patología , Carcinoma/fisiopatología , Células Endoteliales/fisiología , Células Endoteliales/efectos de la radiación , Calor , Radiación Ionizante , Animales , Bovinos , Células Cultivadas , Relación Dosis-Respuesta en la Radiación , Células Endoteliales/citología , Células HT29 , Humanos , Dosis de Radiación , Factores de Tiempo
9.
Assay Drug Dev Technol ; 3(1): 77-88, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15798398

RESUMEN

Taxanes are used for the treatment of many human cancers, as first- and second-line chemotherapeutics. In the course of treatment many patients develop resistance or hypersensitivity to one form of taxane and require a different taxane to rescue the therapeutic benefit of the drug. There is currently no method to reliably predict tumor responses to taxanes prior to therapy or when resistance or hypersensitivity develops. We adapted the quartz crystal microbalance (QCM) biosensor technique to study responses of human mammary epithelial tumor cells to taxanes. Studies indicate that stable frequency and resistance levels are reached at 24 h. Cells in the QCM can then be treated with taxanes and responses monitored in real time via frequency and resistance changes reflecting alterations of cell mass distribution and viscoelastic properties. Distinct shifts in frequency and resistance accurately predicted apoptosis or resistance to treatment, as determined in parallel convention assays. QCM analysis accurately predicted docetaxel was more effective than paclitaxel and MCF-7 cells were more resistant to taxanes compared to MDA-MB-231 cells. These studies suggest "signature" patterns for taxane responsivity could be compared to those of patient biopsy samples to predict therapy outcome prior to treatment for initial therapy or to rescue therapy efficacy.


Asunto(s)
Apoptosis/efectos de los fármacos , Bioensayo/métodos , Técnicas Biosensibles/métodos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/fisiopatología , Hidrocarburos Aromáticos con Puentes/administración & dosificación , Recuento de Células/métodos , Resistencia a Antineoplásicos , Taxoides/administración & dosificación , Antineoplásicos/administración & dosificación , Bioensayo/instrumentación , Técnicas Biosensibles/instrumentación , Recuento de Células/instrumentación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Diseño de Equipo , Análisis de Falla de Equipo , Estudios de Factibilidad , Humanos , Miniaturización , Transductores
10.
Radiat Res ; 161(2): 174-84, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14731072

RESUMEN

Radiation-induced changes in capillaries constitute a basic injury in the pathogenesis of chronic radiation damage to the heart, lung, liver, kidney and brain. It is important to identify new radioprotectors for capillary endothelial cells for use during radiotherapy to minimize normal tissue damage and possibly to increase the deliverable dose. Previously we demonstrated that exposure to ionizing radiation (10 Gy) results in death of bovine adrenal capillary endothelial cells in confluent monolayers by apoptosis. We also showed that retinoids inhibit the growth of endothelial cells, induce their differentiation, down-regulate matrix metalloproteinase (MMP) production, and up-regulate tissue inhibitors of matrix metalloproteinases (TIMPs). In the present studies, we demonstrated that radiation (10 Gy) induced an immediate increase in the amounts and activation of MMP1 and MMP2 in the cell fraction and medium of bovine capillary endothelial cells followed by an incidence of apoptosis. We also obtained data indicating that radiation-induced apoptosis can be inhibited by exposing bovine capillary endothelial cells to all-trans-retinol or all-trans-retinoic acid for 6 days before irradiation, even when the vitamins were removed 24 h before irradiation. Finally, we determined that inhibition of MMPs by TIMP was sufficient to block radiation-induced apoptosis, suggesting that the mechanism of protection by retinoids is through the alteration of levels of MMPs and TIMPs produced by the cells.


Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Células Endoteliales/efectos de los fármacos , Células Endoteliales/efectos de la radiación , Metaloproteinasas de la Matriz/metabolismo , Retinoides/farmacología , Inhibidor Tisular de Metaloproteinasa-1/farmacología , Animales , Bovinos , Células Cultivadas , Relación Dosis-Respuesta en la Radiación , Células Endoteliales/citología , Células Endoteliales/fisiología , Activación Enzimática/efectos de la radiación , Inhibidores de la Metaloproteinasa de la Matriz , Tolerancia a Radiación/efectos de los fármacos , Protectores contra Radiación/farmacología , Tretinoina/farmacología , Vitamina A/farmacología
11.
Biotechnol Prog ; 19(3): 987-99, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-12790666

RESUMEN

The quartz crystal microbalance (QCM) technique has been applied to the real time monitoring of endothelial cell (EC) adhesion and spreading on the QCM gold surface. We previously showed that the measured QCM Deltaf and DeltaR shifts were due to cells adhering to the gold crystal surface, requiring proteolytic enzyme treatment to be removed from the surface, in order for the Deltaf and DeltaR shifts to return to zero. In the present report, we demonstrate the quantitative dependence and saturation of the measured Deltaf and DeltaR shifts on the number of firmly attached ECs as measured by electronic counting of the cells. We demonstrate through a light microscope simulation experiment that the different Deltaf and DeltaR regions of the QCM temporal response curve correspond to the incident ECs contacting the surface, followed by their adhesion and spreading, which reflect cellular mass distribution and cytoskeletal viscoelasticity changes. Also, we demonstrate that the dose response curve of Deltaf and DeltaR values versus attached EC number is more sensitive and possesses less scatter for the hydrophilically treated surface compared to the native gold surface of the QCM. For both surfaces, a Deltaf and DeltaR versus trypsinized, attached EC number plot 1 h post-seeding exhibits a sigmoid curve shape whereas a similar plot 24 h post-seeding exhibits a hyperbolic curve shape. This number dependence suggests cell-cell cooperativity in the initial cell adhesion and spreading processes. These QCM data and our interpretation are corroborated by differences in cell appearance and spreading behavior we observed for ECs in a light microscope fluorescence simulation experiment of the cell density effect. For a stably attached EC monolayer at 24 h post-addition, steady-state Deltaf and DeltaR values are higher and exhibit saturation behavior for both the hydrophilically treated gold surface as compared to the untreated surface. The steady-state 24 h Deltaf and DeltaR values of stably attached ECs are shifted from the 1 h attached ECs. The 24 h values are characteristic of a more energy-dissipative structure. This is consistent with the time-dependent elaboration of surface contacts in anchorage-dependent ECs via the attachment of intregrins to underlying extracellular matrix. It is also in agreement with the known energy dissipation function of the ECs that cover the interior of blood vessels and are exposed to continuous pulsatile blood flow.


Asunto(s)
Adhesión Celular/fisiología , Comunicación Celular/fisiología , Movimiento Celular/fisiología , Electrodos , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Transductores , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/fisiología , Bovinos , Adhesión Celular/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Recuento de Células , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Materiales Biocompatibles Revestidos/farmacología , Electroquímica/instrumentación , Electroquímica/métodos , Endotelio Vascular/efectos de los fármacos , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Tripsina/farmacología
12.
Toxicology ; 299(2-3): 99-111, 2012 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-22634321

RESUMEN

The lactate dehydrogenase (LDH) assay accurately quantifies cytotoxicity of chemicals via the measurement of LDH released from damaged cells. In the assay, LDH catalyzes formation of a reporter chromophore that can be quantified spectrophotometrically at its 490 nm peak, a standard assay, and related to the released LDH concentration. However, certain engineered nanomaterials have been reported to produce aberrant values, resulting in inaccurate assessment of toxicity as measured by LDH levels in media. We studied this effect spectroscopically by measuring unexpected changes in the complete visible spectrum of the product chromophore resulting from using either purified LDH or LDH from lysed cells in the presence of varying concentrations of single walled carbon nanotubes (SWCNTs) or carbon nanohorns (SWCNH-oxs). Basically, at constant LDH concentrations, the 490 nm product peak decreased with increasing carbon nanotube concentration, while the 580 nm peak increased to a lesser extent and the maximum absorbing wavelength increased. The product chromophore spectrum was altered in different ways by potential interactions with a number of components in the reaction mixture including: BSA, LDH, SWCNTs, SWCNT-oxs, or various combinations of these species. We propose to improve the accuracy of the LDH assay when evaluated in the presence of varying concentrations of these carbon nanostructures by use of both the 490 and 580 nm peak absorbances combined via regression analysis. Our results indicate that molecular probes of cytotoxicity must be assessed individually for accuracy in the presence of engineered nanomaterials.


Asunto(s)
L-Lactato Deshidrogenasa/análisis , Nanotubos de Carbono/química , Espectrofotometría Ultravioleta/métodos , Pruebas de Toxicidad/métodos , Animales , Línea Celular , Perros , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Microscopía Electrónica de Transmisión , Nanotubos de Carbono/ultraestructura
13.
Med Eng Phys ; 32(9): 1065-73, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20708956

RESUMEN

Previous research on vertebrate limb regeneration indicates there are several mediating factors involved during the re-growth process. These factors are both biochemical and biophysical. While the phenomenon of adult limb regeneration does not occur naturally in mammalian species, prior research has focused mainly on biochemical modes of stimulating tissue growth and regeneration. The BioDome was aimed at developing a new experimental tool to permit the more systematic study of the impact of biophysical and biochemical factors on mammalian tissue regeneration. The BioDome is a multi-component sleeve assembly that encompasses the wound site of an amputated murine digit and provides an environment conducive to tissue regeneration. The studies showed that the BioDome was effective in supporting early stages of murine digit tip regeneration when combined with a porcine urinary bladder matrix (UBM) pepsin digest and electrical stimulation. The hydrated inner environment of the BioDome influenced regeneration, with additional effects seen with the application of electrical stimulation and pharmacological treatments.


Asunto(s)
Fenómenos Bioquímicos , Fenómenos Biofísicos , Diseño de Equipo/métodos , Regeneración Tisular Dirigida/instrumentación , Modelos Biológicos , Animales , Estimulación Eléctrica , Extremidades/cirugía , Masculino , Ratones , Pepsina A/metabolismo , Vejiga Urinaria/metabolismo
14.
Radiat Res ; 173(6): 748-59, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20518654

RESUMEN

Numerous in vitro and in vivo studies have shown that the endothelial cells of the microvasculature of the lung and kidney are damaged by exposure to ionizing radiation, and this sustained endothelial cell injury is involved in the early and late radiation effects observed in these tissues. It is well accepted that ionizing radiation causes the generation of reactive oxygen species during exposure that results in damage to DNA and cellular organelles. It is more controversial, however, whether additional biochemical events or long-lived radicals occur and persist postirradiation that amplify and initiate new forms of cellular damage. Two families of Eukarion (EUK) compounds have been synthesized that possess superoxide dismutase (SOD), catalase and peroxidase activities. The Mn porphyrins are available orally whereas the salen Mn complexes are administered by injection. In the present study we have examined the ability of these SOD/catalase mimetics to prevent apoptosis of endothelial cells when administered 1 h postirradiation (mitigation). A range of salen Mn complex (EUK-189 and EUK-207) and Mn porphyrins (EUK-418, -423, -425, -450, -451, -452, -453) were used to treat endothelial cells 1 h after the cells received 2-20 Gy ionizing radiation in vitro. Two lead compounds, EUK-207 at a dose of 30 microM and EUK-451 at a dose of 10 microM, exhibited low toxicity and mitigated radiation-induced apoptosis. Future animal studies will test whether these compounds protect when administered after radiation exposure as would be done after a radiological accident or a terrorism event.


Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Materiales Biomiméticos/farmacología , Catalasa/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/efectos de la radiación , Superóxido Dismutasa/metabolismo , Animales , Materiales Biomiméticos/administración & dosificación , Materiales Biomiméticos/síntesis química , Materiales Biomiméticos/toxicidad , Caspasas/biosíntesis , Caspasas/metabolismo , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Células Endoteliales/citología , Células Endoteliales/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Necrosis/inducido químicamente , Compuestos Organometálicos/administración & dosificación , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/farmacología , Compuestos Organometálicos/toxicidad , Pruebas de Toxicidad
15.
Regen Med ; 5(2): 201-20, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20210581

RESUMEN

METHOD: We injected two drugs that modify the epigenome, the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) and the histone deacetylase inhibitor trichostatin A (TSA), alone or in combination, into C57Bl/6 mice subjected to amputation through the mid-second phalanx of the third digit. Wound-site tissue was collected. RESULTS: We observed increased staining of the stem cell markers Rex1 (Zfp42) and stem cell antigen-1 at digit amputation sites from drug-treated mice. Samples from 5-aza-dC plus TSA and TSA treated mice also showed increased proliferating cell nuclear antigen staining, a measure of cell proliferation. Drug treatments increased Msx1, but not Cyp26a1 or ALDH1a2 (RALDH2) mRNA. CONCLUSION: 5-aza-dC and TSA treatments stimulated cell proliferation at the amputation site, possibly via increased expression of genes involved in digit development and regeneration.


Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Metiltransferasas/antagonistas & inhibidores , Regeneración/efectos de los fármacos , Dedos del Pie/fisiología , Amputación Quirúrgica , Animales , Azacitidina/farmacología , Femenino , Miembro Anterior/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Ratones , Ratones Endogámicos C57BL , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Coloración y Etiquetado
16.
Matrix Biol ; 29(8): 690-700, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20797438

RESUMEN

Most adult mammals heal without restorative replacement of lost tissue and instead form scar tissue at an injury site. One exception is the adult MRL/MpJ mouse that can regenerate ear and cardiac tissue after wounding with little evidence of scar tissue formation. Following production of a MRL mouse ear hole, 2mm in diameter, a structure rapidly forms at the injury site that resembles the amphibian blastema at a limb amputation site during limb regeneration. We have isolated MRL blastemal cells (MRL-B) from this structure and adapted them to culture. We demonstrate by RT-PCR that even after continuous culturing of these cells they maintain expression of several progenitor cell markers, including DLK (Pref-1), and Msx-1. We have isolated the underlying extracellular matrix (ECM) produced by these MRL-B cells using a new non-proteolytic method and studied the biological activities of this cell-free ECM. Multiplex microELISA analysis of MRL-B cell-free ECM vs. cells revealed selective enrichment of growth factors such as bFGF, HGF and KGF in the matrix compartment. The cell-free ECM, degraded by mild enzyme treatment, was active in promoting migration and proliferation of progenitor cells in vitro and accelerating wound closure in a mouse full thickness cutaneous wound assay in vivo. In vivo, a single application of MRL-B cell matrix-derived products to full thickness cutaneous wounds in non-regenerative mice, B6, induced re-growth of pigmented hair, dermis and epidermis at the wound site whereas scar tissue replaced these tissues at wound sites in mice treated with vehicle alone. These studies suggest that matrix-derived products can stimulate regenerative healing and avert scar tissue formation in adult mammals.


Asunto(s)
Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/citología , Matriz Extracelular/metabolismo , Fragmentos de Péptidos/farmacología , Células Madre/citología , Cicatrización de Heridas/efectos de los fármacos , Estructuras Animales/anatomía & histología , Estructuras Animales/citología , Animales , Antígenos de Diferenciación/genética , Movimiento Celular/fisiología , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Oído/patología , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Expresión Génica/genética , Heparina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Pepsina A/antagonistas & inhibidores , Pepsina A/metabolismo , Fragmentos de Péptidos/uso terapéutico , Piel/patología , Células Madre/metabolismo , Tenascina/genética , Tenascina/metabolismo , Cicatrización de Heridas/fisiología , Heridas y Lesiones/patología
17.
Tissue Eng Part A ; 15(3): 605-14, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18652541

RESUMEN

Biologic scaffolds composed of extracellular matrix (ECM) are utilized in numerous regenerative medicine applications to facilitate the constructive remodeling of tissues and organs. The mechanisms by which the host remodeling response occurs are not fully understood, but recent studies suggest that both constituent growth factors and biologically active degradation products derived from ECM play important roles. The objective of the present study was to determine if degradation of ECM scaffold materials in vitro by methods that are biochemically and physiologically relevant can yield products that possess chemotactic and/or mitogenic activities for fully differentiated mammalian endothelial cells and undifferentiated multipotential progenitor cells. ECM harvested from porcine urinary bladder was degraded enzymatically with pepsin/hydrochloric acid or papain. The ECM degradation products were tested for chemoattractant properties utilizing either 48-well chemotaxis filter migration microchambers or fluorescence-based filter migration assays, and were tested for mitogenic properties in cell proliferation assays. Results showed that ECM degradation products possessed chemotactic and mitogenic activities for multipotential progenitor cells and that the same degradation products inhibited both chemotaxis and proliferation of differentiated endothelial cells. These findings support the concept that degradation products of ECM bioscaffolds are important modulators of the recruitment and proliferation of appropriate cell types during the process of ECM scaffold remodeling.


Asunto(s)
Movimiento Celular , Matriz Extracelular/metabolismo , Animales , Bioensayo , Linaje de la Célula , Proliferación Celular , Quimiotaxis , Células Endoteliales/citología , Humanos , Ratones , Papaína/metabolismo , Pepsina A/metabolismo , Células Madre/citología , Sus scrofa
18.
Anal Biochem ; 361(1): 77-92, 2007 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-17161375

RESUMEN

The quartz crystal microbalance (QCM) was used to create piezoelectric whole-cell biosensors utilizing either living endothelial cells (ECs) or the metastatic human mammary cancer cell line MDA-MB-231 adhering to the gold QCM surface under in vitro growth conditions. We utilized the whole-cell QCM biosensors for the detection of the effects of varying concentrations of the microtubule binding drugs taxol and nocodazole by measuring changes in the QCM steady state frequency (Deltaf) and motional resistance (DeltaR), shift values. Using 0.11-50 microM nocodazole, we observed the Deltaf shift values of the biosensors, consisting of 20,000 ECs, to decrease significantly in magnitude (nearly 100%) to a limiting value, in a dose-dependent fashion, over a 5- to 6-h incubation period following drug addition. This effect is consistent with nocodazole's known disruption of intracellular microtubules. On the other hand, 10 microM taxol caused little alteration in Deltaf over the same time period, consistent with its microtubule hyperstabilization effect. When the EC QCM biosensor Deltaf shift values were normalized by the number of ECs found firmly attached to the QCM surface via trypsin removal and electronic counting, the dose curve was shifted to lower nocodazole concentrations, resulting in a more sensitive drug biosensor. The kinetics of the Deltaf decrease with increasing nocodazole concentrations measured by the EC QCM biosensor was found to be similar at all drug concentrations and was well fit by a single first-order exponential decay equation. For all nocodazole doses, t(0.5) was invariant, averaging t(0.5)=0.83+/-0.14 h. These data demonstrate that a single dynamic sensing system within the cell, the microtubules, is disrupted by the addition of nocodazole and this process is sensed by the cell QCM biosensor. This interpretation of the data was confirmed by a fluorescence light microscopy investigation of ECs undergoing treatment with increasing nocodazole doses using a fluorescent antibody to alpha-tubulin. These studies revealed a corresponding loss of the spread morphology of the cells, concomitant with a rearrangement of the extended native microtubules into increasingly large aggregates with the cells eventually lifting from the surface in significant numbers at 50 microM. At 6 microM nocodazole, partial reversibility of the EC QCM biosensor was demonstrated. These results indicate that the EC QCM biosensor can be used to detect and study EC cytoskeleton alterations and dynamics. We suggest the potential of this cellular biosensor for the real-time identification or screening of all classes of biologically active drugs or biological macromolecules that affect cellular attachment and cellular spreading, regardless of their molecular mechanism of action.


Asunto(s)
Técnicas Biosensibles/métodos , Citoesqueleto/metabolismo , Nocodazol/farmacología , Paclitaxel/farmacología , Animales , Aorta , Neoplasias de la Mama , Bovinos , Técnicas de Cultivo de Célula , Línea Celular , Línea Celular Tumoral , Citoesqueleto/efectos de los fármacos , Endotelio Vascular , Femenino , Humanos , Cinética , Microscopía Fluorescente/métodos , Cuarzo , Tubulina (Proteína)/análisis
19.
Anal Biochem ; 343(1): 23-34, 2005 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-15979557

RESUMEN

A quartz crystal microbalance (QCM) cell biosensor utilizing living endothelial cells (ECs) or human breast cancer cells (MCF-7) adhering to the gold QCM surface was used to study the relative contributions of the cells and their underlying extracellular matrix (ECM) to the measured QCM Deltaf and DeltaR shifts. The ECM represents a natural biomaterial that is synthesized by the cells to enable their attachment to surfaces. We followed the detachment of the ECs or MCF-7 cells from their ECM using a nonproteolytic method and were able to apportion the total frequency, Deltaf, decrease of the biosensor into contributions from cell attachment and from the intact underlying ECM. We also demonstrated that the Deltaf shift remaining after EC removal corresponds to ECM as determined by light microscopic visualization of the stained protein. During the process of cell detachment, we observed a novel transient increase in viscoelastic behavior expressed as a transient increase in the motional resistance, DeltaR, parameter. Then we showed via a simulation experiment using ECs stained with fluorescent rhodamine-labeled phalloidin, an actin stain, that the transient viscoelastic increase correlated with cellular stress exhibited by the cells during removal with ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'- tetraacetic acid. Prior to cells lifting from their ECM, the attached ECs rearrange their actin microfilaments first into peripheral stress fibers and second into internal aggregates, to maintain cell-cell connectivity, retain their spread morphology, and attempt to adhere more tightly to their underlying ECM. The decrease in DeltaR following its transient rise corresponds to cells finally losing their attachment focal points and lifting from the ECM. We also characterized the normalized f shifts, -Delta(Deltaf)(ECM)/attached cell and -Delta(Deltaf)(cells)/attached cell, as a function of varying the number of adherent cells. Finally, we demonstrate that the underlying native ECM biomaterial, from which all cells have been removed, does not exhibit any significant level of energy dissipation, in contrast to the cells when they are attached to the ECM.


Asunto(s)
Técnicas Biosensibles , Citoesqueleto/metabolismo , Células Endoteliales/fisiología , Matriz Extracelular/metabolismo , Cuarzo , Línea Celular , Elasticidad , Células Endoteliales/citología , Humanos , Cuarzo/química , Estrés Mecánico
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