RESUMEN
PURPOSE: The purpose of this project was to configure the parameters for optimal comparison of image quality differences between the prototype imaging technique, read-out-segmented EPI multi-shot (RESOLVE) and the clinically used single-shot EPI (SS-EPI) MRI sequence for Diffusion Weighted Imaging (DWI) in a saline phantom, followed by a normal rat brain. METHODS: Saline phantom (4.2 × 4.2 cm2 ) and a rat brain were imaged on the 1.5T ESPREE (Siemens, Germany) MRI magnet using a wrist coil. Both the SS-EPI and RESOLVE DWI were optimized with equal imaging parameters such as slice thickness/slice gap = 3.0/0 mm, field of view = 210 mm, phase resolution = 100%, and diffusion b values = 0, 600 s/mm2 while other parameters were given similar settings such as the base resolution, which had to be interpolated for the SS-EPI to 96i to match the RESOLVE as it was unable to be set at 192. Image quality was compared in terms of signal intensity, distortion, % ghosting and Apparent Diffusion Co-efficient (ADC) measurements from each sequence. RESULTS: A marked enhancement of overall image quality with superior detail was observed in each of the scans from the RESOLVE sequence when compared to the SS-EPI. Comparing the efficiency of both sequences in terms of image quality, RESOLVE demonstrated a better stable image with minimal distortion and ghosting. The phantom dimensions obtained from RESOLVE images matched the actual dimensions perfectly. RESOLVE images had signal intensities significantly higher than (2.4×) the SS-EPI images (p=0.0008) and â¼0.6 % times less ghosting compared to SS-EPI (p=0.82) which also exhibited distortion with reduced phantom dimensions (4.05 × 3.28 cm) from original. The ADC values computed were almost identical to be (p=0.32) from SS-EPI (2.21mm2 /sec) and RESOLVE (2.19mm2 /sec). CONCLUSIONS: The RESOLVE sequence has significantly superior image quality compared to SS-EPI at 1.5 T with reasonable scan times. DWI with RESLOVE acquisition scheme can be highly beneficial in obtaining distortion free images in routine clinical studies to characterize tumors and other pathological mechanisms with much greater detail than the current clinically used SS-EPI.
RESUMEN
S100B, a glial-secreted protein, is believed to play a major role in neurodegeneration in Alzheimer's disease, Down syndrome, traumatic brain injury, and spinocerebellar ataxia type 1 (SCA1). SCA1 is a trinucleotide repeat disorder in which the expanded polyglutamine mutation in the protein ataxin-1 primarily targets Purkinje cells of the cerebellum. Currently, the exact mechanism of S100B-mediated Purkinje cell damage in SCA1 is not clear. However, here we show that S100B may act via the activation of the receptor for advanced glycation end product (RAGE) signaling pathway, resulting in oxidative stress-mediated injury to mutant ataxin-1-expressing neurons. To combat S100B-mediated neurodegeneration, we have designed a selective thermally responsive S100B inhibitory peptide, Synb1-ELP-TRTK. Our therapeutic polypeptide was developed using three key elements: (1) the elastin-like polypeptide (ELP), a thermally responsive polypeptide, (2) the TRTK12 peptide, a known S100B inhibitory peptide, and (3) a cell-penetrating peptide, Synb1, to enhance intracellular delivery. Binding studies revealed that our peptide, Synb1-ELP-TRTK, interacts with its molecular target S100B and maintains a high S100B binding affinity as comparable with the TRTK12 peptide alone. In addition, in vitro studies revealed that Synb1-ELP-TRTK treatment reduces S100B uptake in SHSY5Y cells. Furthermore, the Synb1-ELP-TRTK peptide decreased S100B-induced oxidative damage to mutant ataxin-1-expressing neurons. To test the delivery capabilities of ELP-based therapeutic peptides to the cerebellum, we treated mice with fluorescently labeled Synb1-ELP and observed that thermal targeting enhanced peptide delivery to the cerebellum. Here, we have laid the framework for thermal-based therapeutic targeting to regions of the brain, particularly the cerebellum. Overall, our data suggest that thermal targeting of ELP-based therapeutic peptides to the cerebellum is a novel treatment strategy for cerebellar neurodegenerative disorders.
Asunto(s)
Degeneración Nerviosa/prevención & control , Factores de Crecimiento Nervioso/antagonistas & inhibidores , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/farmacología , Oligopéptidos/farmacología , Proteínas/farmacología , Proteínas S100/antagonistas & inhibidores , Animales , Proteína CapZ , Línea Celular , Diseño de Fármacos , Humanos , Immunoblotting , Ratones , Fármacos Neuroprotectores/metabolismo , Oligopéptidos/metabolismo , Fragmentos de Péptidos , Péptidos , Unión Proteica/fisiología , Proteínas/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100 , TemperaturaRESUMEN
Many different amphiphilic compounds cause an increase in the fluid-phase endocytosis rates of cells in parallel with a decrease in membrane-cytoskeleton adhesion. These compounds, however, do not share a common chemical structure, which leaves the mechanism and even site of action unknown. One possible mechanism of action is through an alteration of inositol lipid metabolism by modifying the cytoplasmic surface of the plasma membrane bilayer. By comparing permeable amphiphilic amines used as local anesthetics with their impermeable analogs, we find that access to the cytoplasmic surface is necessary to increase endocytosis rate and decrease membrane-cytoskeleton adhesion. In parallel, we find that the level of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in the plasma membrane is decreased and cytoplasmic Ca(2+) is increased only by permeable amines. The time course of both the decrease in plasma membrane PIP(2) and the rise in Ca(2+) parallels the decrease in cytoskeleton-membrane adhesion. Inositol labeling shows that phosphatidylinositol-4-phosphate levels are increased by the permeable anesthetics, indicating that lipid turnover is increased. Consistent with previous observations, phospholipase C (PLC) inhibitors block anesthetic effects on the PIP(2) and cytoplasmic Ca(2+) levels, as well as the drop in adhesion. Therefore, we suggest that PLC activity is increased by amine anesthetics at the cytoplasmic surface of the plasma membrane, which results in a decrease in membrane-cytoskeleton adhesion.
Asunto(s)
Anestésicos Locales/farmacología , Membrana Celular/efectos de los fármacos , Citoesqueleto/metabolismo , Fosfolipasas de Tipo C/metabolismo , Células 3T3 , Anestésicos Locales/química , Animales , Antipsicóticos/química , Antipsicóticos/farmacología , Calcio/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Clorpromazina/química , Clorpromazina/farmacología , Activación Enzimática , Estrenos/farmacología , Proteínas Fluorescentes Verdes , Rayos Láser , Lidocaína/análogos & derivados , Lidocaína/farmacología , Proteínas Luminiscentes/metabolismo , Ratones , Micromanipulación , Microscopía Confocal/métodos , Estructura Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolípidos/metabolismo , Pirrolidinonas/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
Elastin-like polypeptides (ELPs) composed of a VPGXG repeat undergo a reversible phase transition in aqueous solution. They are hydrophilic and soluble in aqueous solution below their transition temperature (T(t)), but they become hydrophobic and aggregate when the temperature is raised above their T(t). In this study, we examine the quantitative uptake of a fluorescence-labeled, thermally responsive ELP as a function of ELP concentration between 5 and 15 microM in solution in response to hyperthermia by three cultured cancer cell lines. Flow cytometry of fluorescein-ELP conjugates showed that hyperthermia enhanced the cellular uptake of the thermally responsive ELP in human ovarian carcinoma cells (SKOV-3) and in HeLa cells at a concentration of 10 microM or higher, but not at a concentration of 5 microM, as compared with the uptake of a thermally inactive ELP control. In FaDu cells, hyperthermia stimulated uptake of the thermally responsive ELP at all solution concentrations of ELP between 5 and 15 microM. In particular, a >2-fold greater uptake of thermally responsive ELP compared with the thermally inactive control ELP was observed for FaDu cells at a solution concentration of 15 microM in heated cells. Confocal fluorescence microscopy of tumor cells incubated with a rhodamine conjugate of the thermally responsive ELP showed that the cytoplasm was uniformly stained below the T(t). Above the T(t), fluorescent particles were observed in the cytoplasm, suggesting that these particles are aggregates of the thermally responsive polypeptide resulting from the ELP phase transition. These studies demonstrate that the endocytotic uptake of a thermally responsive ELP is significantly enhanced by the thermally triggered phase transition of the polypeptide.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Elastina/farmacocinética , Hipertermia Inducida , Neoplasias Ováricas/metabolismo , Péptidos/farmacocinética , Carcinoma de Células Escamosas/terapia , Portadores de Fármacos , Elastina/química , Femenino , Citometría de Flujo , Colorantes Fluorescentes , Calor , Humanos , Cinética , Microscopía Confocal , Neoplasias Ováricas/terapia , Péptidos/química , Fracciones Subcelulares/metabolismo , Células Tumorales CultivadasRESUMEN
Binding interactions between the plasma membrane and the cytoskeleton define cell functions such as cell shape, formation of cell processes, cell movement, and endocytosis. Here we use optical tweezers tether force measurements and show that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) acts as a second messenger that regulates the adhesion energy between the cytoskeleton and the plasma membrane. Receptor stimuli that hydrolyze PIP2 lowered adhesion energy, a process that could be mimicked by expressing PH domains that sequester PIP2 or by targeting a 5'-PIP2-phosphatase to the plasma membrane to selectively lower plasma membrane PIP2 concentration. Our study suggests that plasma membrane PIP2 controls dynamic membrane functions and cell shape by locally increasing and decreasing the adhesion between the actin-based cortical cytoskeleton and the plasma membrane.
Asunto(s)
Membrana Celular/enzimología , Citoesqueleto/metabolismo , Fosfatidilinositol 4,5-Difosfato/fisiología , Sistemas de Mensajero Secundario/fisiología , Células 3T3 , Actinas/metabolismo , Animales , Metabolismo Energético/fisiología , Proteínas de la Membrana/metabolismo , Ratones , Complejos Multienzimáticos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Polímeros/metabolismo , Unión Proteica/fisiología , Transducción de Señal/fisiología , Estrés MecánicoRESUMEN
Cell spreading and motility require the extension of the plasma membrane in association with the assembly of actin. In vitro, extension must overcome resistance from tension within the plasma membrane. We report here that the addition of either amphiphilic compounds or fluorescent lipids that expanded the plasma membrane increased the rate of cell spreading and lamellipodial extension, stimulated new lamellipodial extensions, and caused a decrease in the apparent membrane tension. Further, in PDGF-stimulated motility, the increase in the lamellipodial extension rate was associated with a decrease in the apparent membrane tension and decreased membrane-cytoskeleton adhesion through phosphatidylinositol diphosphate hydrolysis. Conversely, when membrane tension was increased by osmotically swelling cells, the extension rate decreased. Therefore, we suggest that the lamellipodial extension process can be activated by a physical signal (perhaps secondarily), and the rate of extension is directly dependent upon the tension in the plasma membrane. Quantitative analysis shows that the lamellipodial extension rate is inversely correlated with the apparent membrane tension. These studies describe a physical chemical mechanism involving changes in membrane-cytoskeleton adhesion through phosphatidylinositol 4,5-biphosphate-protein interactions for modulating and stimulating the biochemical processes that power lamellipodial extension.
Asunto(s)
Movimiento Celular/fisiología , Orgánulos/fisiología , Células 3T3 , Animales , Membrana Celular/metabolismo , Membrana Celular/fisiología , Ácido Desoxicólico/farmacología , Detergentes/farmacología , Colorantes Fluorescentes/metabolismo , Ratones , Fosfolípidos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Seudópodos/fisiologíaRESUMEN
When membrane-attached beads are pulled vertically by a laser tweezers, a membrane tube of constant diameter (tether) is formed. We found that the force on the bead (tether force) did not depend on tether length over a wide range of tether lengths, which indicates that a previously unidentified reservoir of membrane and not stretch of the plasma membrane provides the tether membrane. Plots of tether force vs. tether length have an initial phase, an elongation phase, and an exponential phase. During the major elongation phase, tether force is constant, buffered by the "membrane reservoir." Finally, there is an abrupt exponential rise in force that brings the tether out of the trap, indicating depletion of the membrane reservoir. In chick embryo fibroblasts and 3T3 fibroblasts, the maximum tether lengths that can be pulled at a velocity of 4 microm/s are 5.1 +/- 0. 3 and 5.0 +/- 0.2 microm, respectively. To examine the importance of the actin cytoskeleton, we treated cells with cytochalasin B or D and found that the tether lengths increased dramatically to 13.8 +/- 0.8 and 12.0 +/- 0.7 microm, respectively. Similarly, treatment of the cells with colchicine and nocodazole results in more than a twofold increase in tether length. We found that elevation of membrane tension (through osmotic pressure, a long-term elevation of tether force, or a number of transitory increases) increased reservoir size over the whole cell. Using a tracking system to hold tether force on the bead constant near its maximal length in the exponential phase, the rate of elongation of the tethers was measured as a function of tether force (membrane tension). The rate of elongation of tethers was linearly dependent on the tether force and reflected an increase in size of the reservoir. Increases in the reservoir caused by tension increases on one side of the cell caused increases in reservoir size on the other side of the cell. Thus, we suggest that cells maintain a plasma membrane reservoir to buffer against changes in membrane tension and that the reservoir is increased with membrane tension or disruption of the cytoskeleton.
Asunto(s)
Membrana Celular/fisiología , Fibroblastos/citología , Células 3T3 , Actinas/fisiología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Tamaño de la Célula/efectos de los fármacos , Embrión de Pollo , Colchicina/farmacología , Citocalasinas/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Fibroblastos/efectos de los fármacos , Soluciones Hipotónicas , Cinética , Rayos Láser , Ratones , Microesferas , Nocodazol/farmacología , Concentración Osmolar , Presión Osmótica , Estimulación FísicaRESUMEN
Mitosis in mammalian cells is accompanied by a dramatic inhibition of endocytosis. We have found that the addition of amphyphilic compounds to metaphase cells increases the endocytosis rate even to interphase levels. Detergents and solvents all increased endocytosis rate, and the extent of increase was in direct proportion to the concentration added. Although the compounds could produce a variety of different effects, we have found a strong correlation with a physical alteration in the membrane tension as measured by the laser tweezers. Plasma membrane tethers formed by latex beads pull back on the beads with a force that was related to the in-plane bilayer tension and membrane- cytoskeletal adhesion. We found that as cells enter mitosis, the membrane tension rises as the endocytosis rate decreases; and as cells exited mitosis, the endocytosis rate increased as the membrane tension decreased. The addition of amphyphilic compounds decreased membrane tension and increased the endocytosis rate. With the detergent, deoxycholate, the endocytosis rate was restored to interphase levels when the membrane tension was restored to interphase levels. Although biochemical factors are clearly involved in the alterations in mitosis, we suggest that endocytosis is blocked primarily by the increase in apparent plasma membrane tension. Higher tensions inhibit both the binding of the endocytic complex to the membrane and mechanical deformation of the membrane during invagination. We suggest that membrane tension is an important regulator of the endocytosis rate and alteration of tension is sufficient to modify endocytosis rates during mitosis. Further, we postulate that the rise in membrane tension causes cell rounding and the inhibition of motility, characteristic of mitosis.
Asunto(s)
Membrana Celular/fisiología , Endocitosis/fisiología , Mitosis/fisiología , Células 3T3 , Animales , Fenómenos Biomecánicos , Membrana Celular/efectos de los fármacos , Movimiento Celular/fisiología , Tamaño de la Célula/fisiología , Ácido Desoxicólico/farmacología , Detergentes/farmacología , Dextranos , Endocitosis/efectos de los fármacos , Fluoresceínas , Colorantes Fluorescentes , Células HeLa , Humanos , Indoles , Ratones , Mitosis/efectos de los fármacos , Modelos Biológicos , Compuestos de Piridinio , Compuestos de Amonio CuaternarioRESUMEN
We have used a novel alpha-iodoketone spin-label (IKSL) to study myosin head orientation and cross-bridge dynamics in the putative pre-powerstroke state. Possible perturbation of the cross-bridge cycle by the label was assayed by the sinusoidal analysis method (Kawai and Brandt, 1980; Kawai and Zhao, 1993), which determines the rate constants of the elementary steps in the cycle. A comparison of the rates obtained from unlabeled and IKSL fibers revealed small (10-20%) changes in the ATP hydrolysis rate and in the rate constants of the elementary steps. The labeling induced small changes (< 13%) in the distribution of the cross-bridges among the various intermediate states. Pre-powerstroke cross-bridges were induced by aluminum fluoride in the presence of Ca2+ and ATP. In this state, force development is inhibited, but a large proportion (40%) of the cross-bridges are still attached to the thin filament. We have used conventional electron paramagnetic resonance to measure orientation, and have found that the pre-powerstroke heads are as disordered as in relaxation. Their mobility, as determined by saturation transfer electron paramagnetic resonance, was significantly restricted. Assuming that stiffness is proportional to the fraction of strongly attached heads, the rotational correlation time of the bound heads is estimated to be tau r = approximately 150-400 microseconds.
Asunto(s)
Óxidos N-Cíclicos , Contracción Muscular/fisiología , Miosinas/química , Miosinas/fisiología , Marcadores de Spin , Compuestos de Aluminio/farmacología , Animales , Fenómenos Biofísicos , Biofisica , Calcio/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Fluoruros/farmacología , Técnicas In Vitro , Cinética , Modelos Biológicos , Contracción Muscular/efectos de los fármacos , Músculos Psoas/química , Músculos Psoas/efectos de los fármacos , Músculos Psoas/fisiología , ConejosRESUMEN
Conventional EPR studies of muscle fibers labeled with a novel alpha-iodoketo spin label at Cys-707 of the myosin head revealed substantial internal domain reorganization on the addition of ADP to rigor fibers. The spin probes that are well-ordered in the rigor state become disordered and form two distinct populations. These orientational changes do not correspond to rotation of the myosin catalytic domain as a whole because other probes (maleimide and iodoacetamide nitroxides attached to the same Cys-707 of myosin head) report only a small (5-10 degrees) torsional rotation and little or no change in the tilt angle [Ajtai et al. (1992) Biochemistry 31, 207-17; Fajer (1994) Biophys. J. 66, 2039-50]. In the presence of ADP, the labeled domain becomes more flexible and executes large-amplitude microsecond motions, as measured by saturation-transfer EPR with rates (tau r = 150 microseconds) intermediate between the rotations of detached (tau r = 7 microseconds) and rigor heads (tau r = 2500 microseconds). This finding contrasts with an absence of global motion of the myosin head in ADP (tau r = 2200 microseconds) as reported by the maleimide spin label. Our results imply that the myosin head in a single chemical state (AM.ADP) is capable of attaining many internal configurations, some of which are dynamic. The presence of these slow structural fluctuations might be related to the slow release of the hydrolysis products of actomyosin ATPase.
Asunto(s)
Adenosina Difosfato/farmacología , Miosinas/química , Adenosina Trifosfato/farmacología , Animales , Sitios de Unión , Óxidos N-Cíclicos , Espectroscopía de Resonancia por Spin del Electrón , Conformación Proteica , Conejos , Marcadores de SpinRESUMEN
We have determined the orientation and dynamics of the putative pre-power stroke crossbridges in skinned muscle fibers labeled with maleimide spin-label at Cys-707 of myosin. Orientation was measured using electron paramagnetic resonance (EPR) and mobility by saturation transfer EPR. The crossbridges are trapped in the pre-power stroke conformation in the presence of aluminum fluoride, Ca, and ATP. In agreement with data published for unlabeled fibers (Chase et al., 1994), spin-labeled muscle fibers display 42.5% of rigor stiffness, without the generation of force. The trapped crossbridges are as disordered as the relaxed heads, but their microsecond dynamics are significantly restricted. Modeling of the immobile fraction (35%), in terms of attached heads as estimated from stiffness, suggests that the bound heads rotate with a correlation time tau r = 150-400 microseconds, as compared to tau r = 3 microseconds for the heads in relaxed fibers. These "strongly" attached myosin heads, at orientations other than in rigor, are a candidate for the state from which head rotation generates force, as postulated by H. E. Huxley (1969). Ordering of the heads may well be the structural event driving the generation of force.