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1.
It has been proposed, based on theoretical considerations, that the strain rate-dependent viscoelastic response of cartilage reduces local tissue and cell deformations during cyclic compressions. However, experimental studies have not addressed the in situ viscoelastic response of chondrocytes under static and dynamic loading conditions. In particular, results obtained from experimental studies using isolated chondrocytes embedded in gel constructs cannot be used to predict the intrinsic viscoelastic responses of chondrocytes in situ or in vivo. Therefore, the purpose of this study was to investigate the viscoelastic response of chondrocytes in their native environment under static and cyclic mechanical compression using a novel in situ experimental approach. Cartilage matrix and chondrocyte recovery in situ following mechanical compressions was highly viscoelastic. The observed in situ behavior was consistent with a previous study on in vivo chondrocyte mechanics which showed that it took 5-7min for chondrocytes to recover shape and volume following virtually instantaneous cell deformations during muscular loading of the knee in live mice. We conclude from these results that the viscoelastic properties of cartilage minimize chondrocyte deformations during cyclic dynamic loading as occurs, for example, in the lower limb joints during locomotion, thereby allowing the cells to reach mechanical and metabolic homeostasis even under highly dynamic loading conditions. 相似文献
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The mechanical environment is an important factor affecting the maintenance and adaptation of articular cartilage, and thus the function of the joint and the progression of joint degeneration. Recent evidence suggests that cartilage deformation caused by mechanical loading is directly associated with deformation and volume changes of chondrocytes. Furthermore, in vitro experiments have shown that these changes in the mechanical states of chondrocytes correlate with a change in the biosynthetic activity of cartilage cells. The purpose of this study was to apply our knowledge of contact forces within the feline patellofemoral joint to quantify chondrocyte deformation in situ under loads of physiological magnitude. A uniform, static load of physiological magnitude was applied to healthy articular cartilage still fully intact and attached to its native bone. The compressed cartilage was then chemically fixed to enable the evaluation of cartilage strain, chondrocyte deformation and chondrocyte volumetric fraction. Patella and femoral groove articular cartilages differ in thickness, chondrocyte aspect ratio, and chondrocyte volumetric fraction in both magnitude and depth distribution. Furthermore, when subjected to the same compressive loads, changes to all of these parameters differ in magnitude and depth distribution between patellar and femoral groove articular cartilage. This evidence suggests that significant chondrocyte deformation likely occurs during in vivo joint loading, and may influence chondrocyte biosynthetic activity. Furthermore, we hypothesise that the contrasts between patella and femoral groove cartilages may explain, in part, the site-specific progression of osteoarthritis in the patellofemoral joint of the feline anterior cruciate ligament transected knee. 相似文献
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Transport between the cytoplasm and the nucleus 总被引:1,自引:0,他引:1
Summary Active transport of proteins and RNAs across the nuclear-pore complex (NPC) is mediated by a family of related transport receptors which shuttle between the cytoplasm and the nucleoplasm. A number of import and export pathways have been described. Some transport substrates require adapters which mediate association with certain transporters. The transport receptors specifically bind to a recognition signal within the transport substrate or adapter, pass the NPC in one direction, and deliver their cargo to the other side of the nuclear envelope. The Ran GTPase is the crucial regulator of bidirectional transport. Ran-modulating proteins establish an asymmetric intracellular distribution of Ran. As a result, Ran is mainly bound to GTP in the nucleus and to GDP in the cytoplasm. Evidently, RanGTP regulates binding and release of the transport substrates by binding to the transport receptors in the nucleus as well as the transport direction across the NPC. However, little is known about the molecular mechanism of translocation through the NPC. 相似文献
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BACKGROUND: The motion and redistribution of intracellular organelles is a fundamental process in cells. Organelle motion is a complex phenomenon that depends on a large number of variables including the shape of the organelle, the type of motors with which the organelles are associated, and the mechanical properties of the cytoplasm. This paper presents a study that characterizes the diffusive motion of mitochondria in chondrocytes seeded in agarose constructs and what this implies about the mechanical properties of the cytoplasm. METHOD OF APPROACH: Images showing mitochondrial motion in individual cells at 30 s intervals for 15 min were captured with a confocal microscope. Digital image correlation was used to quantify the motion of the mitochondria, and the mean square displacement (MSD) was calculated. Statistical tools for testing whether the characteristic motion of mitochondria varied throughout the cell were developed. Calculations based on statistical mechanics were used to establish connections between the measured MSDs and the mechanical nature of the cytoplasm. RESULTS: The average MSD of the mitochondria varied with time according to a power law with the power term greater than 1, indicating that mitochondrial motion can be viewed as a combination of diffusion and directional motion. Statistical analysis revealed that the motion of the mitochondria was not uniform throughout the cell, and that the diffusion coefficient may vary by over 50%, indicating intracellular heterogeneity. High correlations were found between movements of mitochondria when they were less than 2 microm apart. The correlation is probably due to viscoelastic properties of the cytoplasm. Theoretical analysis based on statistical mechanics suggests that directed diffusion can only occur in a material that behaves like a fluid on large time scales. CONCLUSIONS: The study shows that mitochondria in different regions of the cell experience different characteristic motions. This suggests that the cytoplasm is a heterogeneous viscoelastic material. The study provides new insight into the motion of mitochondria in chondrocytes and its connection with the mechanical properties of the cytoplasm. 相似文献
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Alterations in the mechanical properties of the human chondrocyte pericellular matrix with osteoarthritis 总被引:4,自引:0,他引:4
In articular cartilage, chondrocytes are surrounded by a pericellular matrix (PCM), which together with the chondrocyte have been termed the "chondron." While the precise function of the PCM is not know there has been considerable speculation that it plays a role in regulating the biomechanical environment of the chondrocyte. In this study, we measured the Young's modulus of the PCM from normal and osteoarthritic cartilage using the micropipette aspiration technique, coupled with a newly developed axisymmetric elastic layered half-space model of the experimental configuration. Viable, intact chondrons were extracted from human articular cartilage using a new microaspiration-based isolation technique. In normal cartilage, the Young's modulus of the PCM was similar in chondrons isolated from the surface zone (68.9 +/- 18.9 kPa) as compared to the middle and deep layers (62.0 +/- 30.5 kPa). However, the mean Young's modulus of the PCM (pooled for the two zones) was significantly decreased in osteoarthritic cartilage (66.5 +/- 23.3 kPa versus 41.3 +/- 21.1 kPa, p < 0.001). In combination with previous theoretical models of cell-matrix interactions in cartilage, these findings suggest that the PCM has an important influence on the stress-strain environment of the chondrocyte that potentially varies with depth from the cartilage surface. Furthermore, the significant loss of PCM stiffness that was observed in osteoarthritic cartilage may affect the magnitude and distribution of biomechanical signals perceived by the chondrocytes. 相似文献
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Shuttling of galectin-3 between the nucleus and cytoplasm 总被引:6,自引:0,他引:6
In previous studies, we documented that galectin-3 (M(r) approximately 30,000) is a pre-mRNA splicing factor. Recently, galectin-3 was identified as a component of a nuclear and cytoplasmic complex, the survival of motor neuron complex, through its interaction with Gemin4. To test the possibility that galectin-3 may shuttle between the nucleus and the cytoplasm, human fibroblasts (LG-1) were fused with mouse fibroblasts (3T3). The monoclonal antibody NCL-GAL3, which recognizes human galectin-3 but not the mouse homolog, was used to monitor the localization of human galectin-3 in heterodikaryons. Human galectin-3 localized to both nuclei of a large percentage of heterodikaryons. Addition of the antibiotic leptomycin B, which inhibits nuclear export of galectin-3, decreased the percentage of heterodikaryons showing human galectin-3 in both nuclei. In a parallel experiment, mouse 3T3 fibroblasts, which express galectin-3, were fused with fibroblasts derived from a mouse in which the galectin-3 gene was inactivated. Mouse galectin-3 localized to both nuclei of a large percentage of heterodikaryons. Again, addition of leptomycin B restricted the presence of galectin-3 to one nucleus of a heterodikaryon. The results from both heterodikaryon assays suggest that galectin-3 can exit one nucleus, travel through the cytoplasm, and enter the second nucleus, matching the definition of shuttling. 相似文献
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J. Brachet 《Protoplasma》1967,63(1-3):86-87
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Kevin C. Haudek Kimberly J. Spronk Patricia G. Voss Ronald J. Patterson John L. Wang Eric J. Arnoys 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
This review summarizes selected studies on galectin-3 (Gal3) as an example of the dynamic behavior of a carbohydrate-binding protein in the cytoplasm and nucleus of cells. Within the 15-member galectin family of proteins, Gal3 (Mr ∼ 30,000) is the sole representative of the chimera subclass in which a proline- and glycine-rich NH2-terminal domain is fused onto a COOH-terminal carbohydrate recognition domain responsible for binding galactose-containing glycoconjugates. The protein shuttles between the cytoplasm and nucleus on the basis of targeting signals that are recognized by importin(s) for nuclear localization and exportin-1 (CRM1) for nuclear export. Depending on the cell type, specific experimental conditions in vitro, or tissue location, Gal3 has been reported to be exclusively cytoplasmic, predominantly nuclear, or distributed between the two compartments. The nuclear versus cytoplasmic distribution of the protein must reflect, then, some balance between nuclear import and export, as well as mechanisms of cytoplasmic anchorage or binding to a nuclear component. Indeed, a number of ligands have been reported for Gal3 in the cytoplasm and in the nucleus. Most of the ligands appear to bind Gal3, however, through protein–protein interactions rather than through protein–carbohydrate recognition. In the cytoplasm, for example, Gal3 interacts with the apoptosis repressor Bcl-2 and this interaction may be involved in Gal3's anti-apoptotic activity. In the nucleus, Gal3 is a required pre-mRNA splicing factor; the protein is incorporated into spliceosomes via its association with the U1 small nuclear ribonucleoprotein (snRNP) complex. Although the majority of these interactions occur via the carbohydrate recognition domain of Gal3 and saccharide ligands such as lactose can perturb some of these interactions, the significance of the protein's carbohydrate-binding activity, per se, remains a challenge for future investigations. 相似文献
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The nuclear lamina is a protein meshwork that lines the nuclear envelope in metazoan cells. It is composed largely of a polymeric assembly of lamins, which comprise a distinct sequence homology class of the intermediate filament protein family. On the basis of its structural properties, the lamina originally was proposed to provide scaffolding for the nuclear envelope and to promote anchoring of chromatin and nuclear pore complexes at the nuclear surface. This viewpoint has expanded greatly during the past 25 years, with a host of surprising new insights on lamina structure, molecular composition and functional attributes. It has been established that the self-assembly properties of lamins are very similar to those of cytoplasmic intermediate filament proteins, and that the lamin polymer is physically associated with components of the cytoplasmic cytoskeleton and with a multitude of chromatin and inner nuclear membrane proteins. Cumulative evidence points to an important role for the lamina in regulating signaling and gene activity, and in mechanically coupling the cytoplasmic cytoskeleton to the nucleus. The significance of the lamina has been vaulted to the forefront by the discovery that mutations in lamins and lamina-associated polypeptides lead to an array of human diseases. A key future challenge is to understand how the lamina integrates pathways for mechanics and signaling at the molecular level. Understanding the structure of the lamina from the atomic to supramolecular levels will be essential for achieving this goal. 相似文献
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Nuclear transport is an energy-dependent process mediated by saturable receptors. Import and export receptors are thought to recognize and bind to nuclear localization signals or nuclear export signals, respectively, in the transported molecules. The receptor-substrate interaction can be direct or mediated by an additional adapter protein. The transport receptors dock their cargoes to the nuclear pore complexes (NPC) and facilitate their translocation through the NPC. After delivering their cargoes, the receptors are recycled to initiate additional rounds of transport. Because a transport event for a cargo molecule is unidirectional, the transport receptors engage in asymmetric cycles of translocation across the NPC. The GTPase Ran acts as a molecular switch for receptor-cargo interaction and imparts directionality to the transport process. Recently, the combined use of different in vitro and in vivo approaches has led to the characterization of novel import and export signals and to the identification of the first nuclear import and export receptors. 相似文献
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Summary The kinetics of the nuclear and cytoplasmic fluorescence response to glycolytic substrate were studied in ascites cells in culture (EL2 cells) and radiation giants (EL2G) maintained under a variety of conditions, using a beam-splitter supplemented microfluorimeter which allows fluorescence recording simultaneously with microelectrophoretic addition of substrate. A sequence of fluorescence pulses which resemble closely the curve of formation and disappearance of the enzyme-substrate complex were obtained upon repetitive additions of substrate. The pulses were analyzed in terms of peak fluorescence response (PFR), duration of steady state, halftime of fluorescence rise and decay (tin1/2off), number of consecutive cycles elicited, etc. There is a considerable parallelism in the kinetics of the nuclear and cytoplasmic fluorescence after addition of glycolytic substrate, over the whole time course of consecutive pulses. However in untreated, Amytal- or Rotenone-perfused cells the peak magnitudes of the cytoplasmic fluorescence are significantly lower and the cytoplasmic pulses are damped earlier than the nuclear upon repeated additions of substrate. In Amytal-grown EL2 cells there is a drop of PFR and a prolongation of t
1/2off in the nucleus and cytoplasm, which persists when the cells are transferred to an Amytal-free medium. However, if the cells are maintained for longer periods in Amytal, the nuclear fluorescence tends to return to control level, while the cytoplasmic pulses remain small and are easily damped. In T3- or Amytal + T3 -grown EL2 cells the cytoplasmic fluorescence instead of dropping like in the controls, follows the nuclear level over the whole time course of repeated pulses, and can even exceed the nuclear. Comparable phenomena are observed in T3 -and Insulin-grown radiation giants When the amount of substrate is varied, starting from levels which can barely elicit a response the magnitude of the fluorescence response (integrated fluorescence pulse) in the cytoplasm and nucleus follows a sigmoid curve which can be interpreted as a function of allosteric enzymes.List of Abbreviations EL2
mouse Ehrlich ascites cells in tissue culture
- EL2G
giant tissue culture mouse Ehrlich ascites cells obtained by X-irradiation
- EL2T
giant tissue culture EL2 cells obtained by treatment with Trenimon (2.3.5-Tris aethyleniminobenzochinon-1.4)
- G6P
glucose-6-phosphate
- FDP
fructose-1.6-diphosphate
- 6 PG
6-phospho-gluconate
- UDPG
uridine-5-diphosphoglucose
- AMP
adenosine-5-monophosphate
- ADP
adenosine-5-diphosphate
- G1P
D-glucose-1-phosphate
- PFR
peak fluorescence response
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t
1/2off
halftime of fluorescence rise and decay
- T3
triidothyronine 相似文献
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Size-dependent DNA mobility in cytoplasm and nucleus 总被引:20,自引:0,他引:20
Lukacs GL Haggie P Seksek O Lechardeur D Freedman N Verkman AS 《The Journal of biological chemistry》2000,275(3):1625-1629
The diffusion of DNA in cytoplasm is thought to be an important determinant of the efficacy of gene delivery and antisense therapy. We have measured the translational diffusion of fluorescein-labeled double-stranded DNA fragments (in base pairs (bp): 21, 100, 250, 500, 1000, 2000, 3000, 6000) after microinjection into cytoplasm and nucleus of HeLa cells. Diffusion was measured by spot photobleaching using a focused argon laser spot (488 nm). In aqueous solutions, diffusion coefficients of the DNA fragments in water (D(w)) decreased from 53 x 10(-8) to 0.81 x 10(-8) cm(2)/s for sizes of 21-6000 bp; D(w) was related empirically to DNA size: D(w) = 4.9 x 10(-6) cm(2)/s.[bp size](-0.72). DNA diffusion coefficients in cytoplasm (D(cyto)) were lower than D(w) and depended strongly on DNA size. D(cyto)/D(w) decreased from 0.19 for a 100-bp DNA fragment to 0.06 for a 250-bp DNA fragment and was <0.01 for >2000 bp. Diffusion of microinjected fluorescein isothiocyanate (FITC) dextrans was faster than that of comparably sized DNA fragments of 250 bp and greater. In nucleus, all DNA fragments were nearly immobile, whereas FITC dextrans of molecular size up to 580 kDa were fully mobile. These results suggest that the highly restricted diffusion of DNA fragments in nucleoplasm results from extensive binding to immobile obstacles and that the decreased lateral mobility of DNAs >250 bp in cytoplasm is because of molecular crowding. The diffusion of DNA in cytoplasm may thus be an important rate-limiting barrier in gene delivery utilizing non-viral vectors. 相似文献
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A new in situ model of partially digested growth plate cartilage suitable for patch clamp study of membrane currents of chondrocytes from various differentiation stages was developed. Thin sections of growth plate were enzyme digested to expose intact membranes of chondrocytes previously covered by extracellular matrix. This treatment dramatically increased the success rate of tight-seal formation from virtually 0% up to 40%. Whole-cell patch clamp recording revealed a delayed outward rectifying current as the major macroscopic current in chondrocytes of all differentiation stages. This current was sensitive to tetraethylammonium chloride and reversed polarity at a membrane potential close to the equilibrium potential of K+. Chondrocytes at resting stage expressed a much smaller K+ current than the proliferative and hypertrophic chondrocytes. When the current amplitudes were normalized for the cell membrane area, proliferative cells expressed a significantly higher outward current density. 相似文献
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