Adhesion and stress relaxation forces between melanoma and cerebral endothelial cells

Mechanical parameters play a crucial role in proper cellular functions. This article examines the process of the appearance and breaking of adhesion forces during contact between the confluent cerebral endothelial cell layer and a melanoma cell attached to a tipless cantilever. This adhesion is the initial phase of melanoma transmigration through the endothelial cell layer. Taking the force measurement, if the contact was prolonged for several seconds, a decrease in the load force was observed, which corresponds to stress relaxation of the cells. The dependence of adhesion force and stress relaxation on dwell time showed a saturation-like behavior. These stress relaxation curves could be fitted with the sum of two exponentials, suggesting that two independent processes take place simultaneously. The breakup of the adhesion during the retraction of the cantilever with the attached melanoma cell is not continuous but shows jumps. Between living endothelial and melanoma cells, a minimum jump size of about 20 pN could be determined. The minimum jump is independent of the dwell time and load force. It seems to be the elementary binding force between these two cell types. In case of fixed endothelial cells, the adhesion force was strongly decreased and the jumps disappeared, whereas the stress relaxation did not show considerable change upon fixation.     

Determination of thermodynamics and kinetics of RNA reactions by force

Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force.     

Helix nucleation kinetics from molecular simulations in explicit solvent

We study the reversible folding/unfolding of short Ala and Gly-based peptides by molecular dynamics simulations of all-atom models in explicit water solvent. A kinetic analysis shows that the formation of a first alpha-helical turn occurs within 0.1-1 ns, in agreement with the analyses of laser temperature jump experiments. The unfolding times exhibit Arrhenius temperature dependence. For a rapidly nucleating all-Ala peptide, the helix nucleation time depends only weakly on temperature. For a peptide with enthalpically competing turn-like structures, helix nucleation exhibits an Arrhenius temperature dependence, corresponding to the unfolding of enthalpic traps in the coil ensemble. An analysis of structures in a "transition-state ensemble" shows that helix-to-coil transitions occur predominantly through breaking of hydrogen bonds at the helix ends, particularly at the C-terminus. The temperature dependence of the transition-state ensemble and the corresponding folding/unfolding pathways illustrate that folding mechanisms can change with temperature, possibly complicating the interpretation of high-temperature unfolding simulations. The timescale of helix formation is an essential factor in molecular models of protein folding. The rapid helix nucleation observed here suggests that transient helices form early in the folding event.     

Effects of inorganic phosphate on endothermic force generation in muscle.

Using a rapid (ca. 0.2 ms) laser temperature jump technique, the rate of endothermic force generation was examined in single-skinned (rabbit psoas) muscle fibres when they were exposed to different levels of inorganic phosphate (a product released during ATP hydrolysis in active muscle). The steady force is reduced by increased phosphate but the apparent rate constant of force generation induced by a standard temperature jump (from ca. 9 degrees C to ca. 12 degrees C) increases two- to threefold when the phosphate added is increased from zero to ca. 25 mM. The increase in the apparent rate constant also exhibits saturation at higher phosphate levels and the relation is hyperbolic. Detailed examination of the data, particularly in relation to our pressure release experiments, leads to a scheme for the molecular steps involved in phosphate release and force generation in active muscle fibres, where phosphate release from attached cross-bridges involves three reversible and sequentially faster molecular steps. Step one is a moderately slow, pre-force generation step that probably represents a transition of cross-bridges from non-specific to stereospecific attached states. Step two is moderately fast and represents endothermic cross-bridge force generation (temperature sensitive) and step three is a very rapid phosphate release. Such a scheme accommodates findings from a variety of different studies, including pressure perturbation experiments and other studies where the effect of phosphate on muscle force was studied.     

Quantitative measurements of force and displacement using an optical trap.

We combined a single-beam gradient optical trap with a high-resolution photodiode position detector to show that an optical trap can be used to make quantitative measurements of nanometer displacements and piconewton forces with millisecond resolution. When an external force is applied to a micron-sized bead held by an optical trap, the bead is displaced from the center of the trap by an amount proportional to the applied force. When the applied force is changed rapidly, the rise time of the displacement is on the millisecond time scale, and thus a trapped bead can be used as a force transducer. The performance can be enhanced by a feedback circuit so that the position of the trap moves by means of acousto-optic modulators to exert a force equal and opposite to the external force applied to the bead. In this case the position of the trap can be used to measure the applied force. We consider parameters of the trapped bead such as stiffness and response time as a function of bead diameter and laser beam power and compare the results with recent ray-optic calculations.     

Temperature jump study of charge translocation during the bacteriorhodopsin photocycle

Temperature jump experiments were carried out on purple membranes oriented and fixed in polyacrylamide gel. With green background illumination a relaxation of the photocurrent after an infrared laser pulse could be observed. To simulate the temperature jump signals different models of the bacteriorhodopsin photocycle were tested. The parameters of these models were obtained by measuring absorbance changes and photocurrent after excitation with a 575-nm laser flash.

A model with a temperature-dependent branching before the M state turned out to be satisfying. Other models, especially those with a late branching or without branching, could not reproduce the temperature jump measurements.

     

Reliability and validity of a linear position transducer for measuring jump performance

This study determined the reliability and validity of a linear position transducer to measure jump performance by comparing the mean force, peak force, and time-to-peak force measurements with data obtained simultaneously with a force platform. Twenty-five men performed squat, countermovement, and drop jumps with the linear transducer connected from a waist belt and base, which were placed upon a force platform. The Pearson correlation coefficients across the 3 jumps for the mean force (r = 0.952-0.962), peak force (r = 0.861-0.934), and time-to-peak force (r = 0.924-0.995) were high, providing evidence that the linear-transducer and force-platform measurements were similar. The trial-to-trial reliability of the jumps measured by the linear position transducer gave an intraclass correlation coefficient of 0.924-0.975 for mean force, 0.977-0.982 for peak force, and 0.721-0.964 for time-to-peak force. The coefficients of variation were 2.1-4.5% for mean force, 2.5-8.4% for peak force, and 4.1-11.8% for time-to-peak force. Our findings showed that the calculations derived from the linear transducer were very similar to those of the force platform and hence provided evidence of the validity of this method. The data from the linear transducer were also shown to be reliable. Therefore, this method of calculating force may provide a cost-effective alternative to the force platform for measuring this variable.     

A model system for studying the adsorption of a hydrophobic mutagen to dietary fibre

The adsorption of 1,8-dinitropyrene (DNP) to alpha-cellulose has been studied as a model system for examining the adsorption of a hydrophobic mutagen to dietary fiber. Most of the DNP rapidly disappeared from an aqueous solution and partitioned between the glass wall of the test tube and the alpha-cellulose. Factors affecting DNP distribution included (i) the time of incubation, (ii) the final concentration of the solvent, dimethyl sulphoxide, in which the DNP has been dissolved, and (iii) the relative concentrations of DNP and alpha-cellulose. We suggest that this model system could be applied to other mutagens, and that alpha-cellulose would provide a useful standard fiber to permit inter-laboratory comparisons.     

The jump of the click beetle (Coleoptera, Elateridae)—a preliminary study

A preliminary account is given of the jump of the click beetle, Athous haemorrhoidalis (F.). The jump is normally made from an inverted position. It involves a jack-knifing movement whereby a prosternal peg is slid very rapidly down a smooth track into a mesosternal pit. The muscles which produce this movement are allowed to build up tension by a friction hold on the dorsal side of the peg. The anatomy of this jumping mechanism is briefly described. Ciné recording showed that the jump was usually nearly vertical and could exceed 0.3m in height; the beetle normally rotated several times head over tail during a jump. The jump was produced by a very rapid upwards movement of the beetle's centre of gravity during the jack-knifing action. In a typical jump, a 4 × 10⁻⁵ kg beetle could be subjected to an upwards acceleration of 3800 m/s⁻² (380 g). The minimum work done and the power output of the muscles causing jumping have been calculated. A simple mechanical model has been constructed to simulate a jump, and several possible ways in which the jumping mechanism could operate have been discussed.     

Identification of Phytopathogenic Virus Strains by their Luminescent Properties

A quick test has been developed to identify phytopathogenic virus strains by variations in their photoluminescence parameters. When heated, the virus suspension shows a jumpwise drop or rise in fluorescence intensity caused by conformational changes in the macromolecules of the virion protein capsules. The relative value of the jump in fluorescence intensity and the temperature value at which the jump occurs are essentially strain-specific. The minimum time needed for one test is from 15 to 20 minutes.     

Increasing compliance to instructions in the squat jump

The purposes of this investigation were to evaluate the occurrence of a small amplitude counter-movement (SACM) in SJ (squat jump) trials of elite athletes to determine the efficacy of gross observation and the use of a portable position transducer to determine whether or not a SACM occurred. The subjects (N = 30, 20.1 +/- 3.0 years, 199.0 +/- 8.4 cm, and 87.2 +/- 9.5 kg) were a combination of high-performance (National Team) and elite athletes (Olympian) from the sports of athletics, swimming, and volleyball. All subjects performed SJ trials on a force platform, with a linear position transducer attached to a bar placed across the shoulders. Subjects performed the SJ from a depth that allowed for a 90 degrees knee angle, with the subject's instructed to maintain a 3-second isometric hold preceding the concentric action of the jump. One hundred twenty-five SJ trials were observed for a SACM and analyzed (using the force plate data and position transducer data) for a SACM. Of the 125 SJ trials, 69 trials (55.2%) were observed to have a SACM by the researchers. In the remaining 56 trials, 43 of these trials contained a force unload (>/=10% body mass) before initiation of the concentric action, indicating a SACM. Of the 119 SJ trials where a force unload was observed and detected by the force-time graph, 118 (99.2%) of these trials also showed a change in displacement using the displacement-time graph from the linear position transducer. The results of this study indicate that achieving compliance to protocol in the SJ is difficult, and that gross observation is inadequate in detecting a SACM in the SJ. From a practical perspective, these results suggest that using a force plate or a linear position transducer would allow the strength and conditioning coach to ensure compliance to instructions in the SJ.     

Endothermic force generation in fast and slow mammalian (rabbit) muscle fibers.

Isometric tension responses to rapid temperature jumps (T-jumps) of 3-7 degrees C were examined in single skinned fibers isolated from rabbit psoas (fast) and soleus (slow) muscles. T-jumps were induced by an infrared laser pulse (wavelength 1.32 microns, pulse duration 0.2 ms) obtained from a Nd-YAG laser, which heated the fiber and bathing buffer solution in a 50-microliter trough. After a T-jump, the temperature near the fiber remained constant for approximately 0.5 s, and the temperature could be clamped for longer periods by means of Peltier units assembled on the back trough wall. A T-jump produced a step decrease in tension in both fast and slow muscle fibers in rigor, indicating thermal expansion. In maximally Ca-activated (pCa approximately 4) fibers, the increase of steady tension with heating (3-35 degrees C) was approximately sigmoidal, and a T-jump at any temperature induced a more complex tension transient than in rigor fibers. An initial (small amplitude) step decrease in tension followed by a rapid recovery (tau(1); see Davis and Harrington, 1993) was seen in some records from both fiber types, which presumably was an indirect consequence of thermal expansion. The net rise in tension after a T-jump was biexponential, and its time course was characteristically different in the two fibers. At approximately 12 degrees C the reciprocal time constants for the two exponential components (tau(2) and tau(3), respectively, were approximately 70.s(-1) and approximately 15.s(-1) in fast fibers and approximately 20.s(-1) and approximately 3.s(-1) in slow fibers. In both fibers, tau(2) ("endothermic force regeneration") became faster with an increase in temperature. Furthermore, tau(3) was temperature sensitive in slow fibers but not in fast fibers. The results are compared and contrasted with previous findings from T-jump experiments on fast fibers. It is observed that the fast/slow fiber difference in the rate of endothermic force generation (three- to fourfold) is considerably smaller than the reported differences in the "phosphate release steps" (> 30-fold).     

Variation of muscle stiffness with force at increasing speeds of shortening

Single frog skeletal muscle fibers were attached to a servo motor and force transducer by knotting the tendons to pieces of wire at the fiber insertions. Small amplitude, high frequency sinusoidal length changes were then applied during tetani while fibers contracted both isometrically and isotonically at various constant velocities. The amplitude of the resulting force oscillation provides a relative measure of muscle stiffness. It is shown from an analysis of the transient force responses observed after sudden changes in muscle length applied both at full and reduced overlap and during the rising phase of short tetani that these responses can be explained on the basis of varying numbers of cross bridges attached at the time of the length step. Therefore, the stiffness measured by the high frequency length oscillation method is taken to be directly proportional to the number of cross bridges attached to thin filament sites. It is found that muscle stiffness measured in this way falls with increasing shortening velocity, but not as rapidly as the force. The results suggest that at the maximum velocity of shortening, when the external force is zero, muscle stiffness is still substantial. The findings are interpreted in terms of a specific model for muscle contraction in which the maximum velocity of shortening under zero external load arises when a force balance is attained between attached cross bridges some of which are aiding and others opposing shortening. Other interpretations of these results are also discussed.     

Methodological concerns for determining power output in the jump squat

The purpose of this study was to investigate the validity of power measurement techniques during the jump squat (JS) utilizing various combinations of a force plate and linear position transducer (LPT) devices. Nine men with at least 6 months of prior resistance training experience participated in this acute investigation. One repetition maximums (1RM) in the squat were determined, followed by JS testing under 2 loading conditions (30% of 1RM [JS30] and 90% of 1RM [JS90]). Three different techniques were used simultaneously in data collection: (a) 1 linear position transducer (1-LPT); (b) 1 linear position transducer and a force plate (1-LPT + FP); and (c) 2 linear position transducers and a force place (2-LPT + FP). Vertical velocity-, force-, and power-time curves were calculated for each lift using these methodologies and were compared. Peak force and peak power were overestimated by 1-LPT in both JS30 and JS90 compared with 2-LPT + FP and 1-LPT + FP (p 相似文献   

Correlation between ground reaction force and tibial acceleration in vertical jumping

Modern electronics allow for the unobtrusive measurement of accelerations outside the laboratory using wireless sensor nodes. The ability to accurately measure joint accelerations under unrestricted conditions, and to correlate them with jump height and landing force, could provide important data to better understand joint mechanics subject to real-life conditions. This study investigates the correlation between peak vertical ground reaction forces, as measured by a force plate, and tibial axial accelerations during free vertical jumping. The jump heights calculated from force-plate data and accelerometer measurements are also compared. For six male subjects participating in this study, the average coefficient of determination between peak ground reaction force and peak tibial axial acceleration is found to be 0.81. The coefficient of determination between jump height calculated using force plate and accelerometer data is 0.88. Data show that the landing forces could be as high as 8 body weights of the jumper. The measured peak tibial accelerations ranged up to 42 g. Jump heights calculated from force plate and accelerometer sensors data differed by less than 2.5 cm. It is found that both impact accelerations and landing forces are only weakly correlated with jump height (the average coefficient of determination is 0.12). This study shows that unobtrusive accelerometers can be used to determine the ground reaction forces experienced in a jump landing. Whereas the device also permitted an accurate determination of jump height, there was no correlation between peak ground reaction force and jump height.     

Micropatterning and characterization of electrospun poly(ε‐caprolactone)/gelatin nanofiber tissue scaffolds by femtosecond laser ablation for tissue engineering applications

Experimental investigations aimed at assessing the effectiveness of femtosecond (FS) laser ablation for creating microscale features on electrospun poly(ε‐caprolactone) (PCL)/gelatin nanofiber tissue scaffold capable of controlling cell distribution are described. Statistical comparisons of the fiber diameter and surface porosity on laser‐machined and as‐spun surface were made and results showed that laser ablation did not change the fiber surface morphology. The minimum feature size that could be created on electrospun nanofiber surfaces by direct‐write ablation was measured over a range of laser pulse energies. The minimum feature size that could be created was limited only by the pore size of the scaffold surface. The chemical states of PCL/gelatin nanofiber surfaces were measured before and after FS laser machining by attenuated total reflectance Fourier transform infrared (ATR‐FTIR) spectroscopy and X‐ray photoelectron spectroscopy (XPS) and showed that laser machining produced no changes in the chemistry of the surface. In vitro, mouse embryonic stem cells (mES cells) were cultured on as‐spun surfaces and in laser‐machined microwells. Cell densities were found to be statistically indistinguishable after 1 and 2 days of growth. Additionally, confocal microscope imaging confirmed that spreading of mES cells cultured within laser‐machined microwells was constrained by the cavity walls, the expected and desired function of these cavities. The geometric constraint caused statistically significant smaller density of cells in microwells after 3 days of growth. It was concluded that FS laser ablation is an effective process for microscale structuring of these electrospun nanofiber tissue scaffold surfaces. Biotechnol. Bioeng. 2011; 108:116–126. © 2010 Wiley Periodicals, Inc.     

Validity of the Myotest® in measuring force and power production in the squat and bench press

The purpose of this study was to verify the concurrent validity of a bar-mounted Myotest? instrument in measuring the force and power production in the squat and bench press exercises when compared to the gold standard of a computerized linear transducer and force platform system. Fifty-four men (bench press: 39-171 kg; squat: 75-221 kg) and 43 women (bench press: 18-80 kg; squat: 30-115 kg) (age range 18-30 years) performed a 1 repetition maximum (1RM) strength test in bench press and squat exercises. Power testing consisted of the jump squat and the bench throw at 30% of each subject's 1RM. During each measurement, both the Myotest? instrument and the Celesco linear transducer of the directly interfaced BMS system (Ballistic Measurement System [BMS] Innervations Inc, Fitness Technology force plate, Skye, South Australia, Australia) were mounted to the weight bar. A strong, positive correlation (r) between the Myotest and BMS systems and a high correlation of determination (R2) was demonstrated for bench throw force (r = 0.95, p < 0.05) (R2 = 0.92); bench throw power (r = 0.96, p < 0.05) (R2 = 0.93); squat jump force (r = 0.98, p < 0.05) (R2 = 0.97); and squat jump power (r = 0.91, p < 0.05) (R2 = 0.82). In conclusion, when fixed on the bar in the vertical axis, the Myotest is a valid field instrument for measuring force and power in commonly used exercise movements.     

Infantile acid maltase deficiency

The loss of normal ultrastructure of skeletal muscle during the relentless course of infantile acid maltase deficiency (AMD) is re-examined in the light of the lysosomal rupture hypothesis. This hypothesis suggests that movement and increased myofibril rigidity during contraction cause lysosomes in muscle to rupture and release glycogen and other lysosomal contents to a much greater extent than do lysosomes in other cell types in cases of infantile AMD. Muscle fibers are destroyed, while macrophages and other cells mostly accumulate glycogen in storage lysosomes without being destroyed. When morphological stages of fiber destruction are placed in a sequential series, from fibers most like normal infant muscle to those with only remnants of muscle ultrastructure, the earliest stages seen contain intact storage lysosomes. Intermediate stages exhibit ruptured lysosomal membranes and free glycogen as well as glycogen in lysosomes. Loss of myofibrillar material and loss of glycogen occur in later stages of fiber destruction. Membrane-enclosed glycogen and mitochondria are relatively protected from the process of destruction. The electron-microscopic observations support the lysosomal rupture hypothesis and are compatible with the original proposal of Hers, that the disease results from a deficiency of a single lysosomal enzyme. Secondary changes other than muscle fiber destruction probably relate to disrupted control mechanisms and the nature of muscle as a specialized cell. At least two different mechanisms could contribute to the loss of contractile activity and myofibrillar structure.     

Laser Temperature-Jump Technique for Relaxation Studies of the Ionic Conductances in Myelinated Nerve Fibers

A temperature-jump technique for single nodes of Ranvier has been developed using a pulsed laser system. The temperature perturbation was accomplished by firing the laser beam obtained from a neodymium rod through the solution surrounding a single node. The temperature step was achieved within 1 msec using the laser in the normal mode of operation. During the voltage-clamped steady-state current a temperature jump from 4°C increased the current to a new steady-state value within the time course of the T-jump. This finding suggests that the maximum potassium permeability P_K has a rapid relaxation time and that the steady-state value of n (the value of potassium permeability divided by its maximum value) is relatively independent of temperature. T-jumps applied during the voltage-clamped sodium currents showed that the sodium permeability changed with a relaxation time that was also shorter than the duration of the normal mode laser output. T-jumps observed during a hyperpolarization or at the resting potential showed no detectable conductance change. When a T-jump immediately preceded a voltage clamp pulse the technique was then used to investigate the effect of changes in the steady-state temperature on the ionic conductances. It was found that the magnitude of the change in membrane current due to a T-clamp was directly related to the level of cathodal polarization.     

Influence of sensor size on the accuracy of in-vivo ligament and tendon force measurements.

In-vivo tendon forces are commonly measured using transducers, which detect tension in the tendon fibers. A poorly understood source of measurement errors is the difference in stress distribution within the tendon between experimental and transducer calibration conditions. The objective of this study was to investigate this source of error, and to determine whether these errors could be minimized by proper selection of transducer size. The study was conducted using the infrapatellar ligament (patellar tendon) of New Zealand White rabbits. Tendon force was measured with two different size implantable force transducers (IFTs), one Wide and one Narrow, and by a strain gaged load cell in series with the tendon. Tests were conducted at five different loading conditions selected to produce five different stress distributions within the tendon. One loading condition corresponded to a typical post-experiment calibration, and the data from that condition were used to develop a calibration equation for the transducer. The errors that resulted from using this calibration were determined by comparing the tendon force measured by the in-series load cell with the force predicted from the IFT output using the calibration equation. Changes in stress distribution produced measurement errors up to 64 N with the Narrow IFT but only 24 N with the Wide IFT. We found the measurement error was dependent on sensor width. Our results support the hypothesis that measurement errors can be caused by differences in tendon stress distribution between calibration and experimental conditions. We further showed that these errors can be minimized by using an IFT, which samples the tension in a large percentage of the tendon fibers. Information from this study can be used for selection of an appropriately sized implantable force transducer for measuring tendon and ligament force.