Comparative risk of common peroneal nerve injury in far anteromedial portal drilling and transtibial drilling in anatomical double-bundle ACL reconstruction.

PURPOSE: The purpose of this study was to investigate the risk of common peroneal nerve injury in FM drilling as compared to transtibial drilling in anatomical double-bundle ACL reconstruction. METHODS: Ten cadaveric knees without ligament injury or significant arthritis were used for this study. Knees were secured at 90° and 120° of flexion. In transtibial drilling groups, a guide pin was drilled through either the anteromedial bundle (AMB) or posterolateral bundle (PLB) tibial insertion site to either the AMB or PLB femoral insertion site (tibial insertion site-femoral insertion site: AM-AM, PL-PL, PL-AM and AM-PL). In FM drilling groups (FM-AM and FM-PL),the pin was drilled at the AMB or PLB femoral insertion site through the FM. We measured the shortest distance between the point at which the pin ran through the lateral cortex of the femur and the ipsilateral common peroneal nerve at a knee flexion of 90° and 120°. RESULTS: At a knee flexion of 90°, the shortest mean distance to the common peroneal nerve was 15.3 mm in the FM-PL group, 13.4 mm in the FM-AM group, 27.9 mm in the PL-PL group, 30.8 mm in the AM-AM group, 37.8 mm in the PL-AM group and 29.5 mm in the AM-PL group. At a knee of flexion 120°, the mean distance was 17.3 mm in the FM-PL group, 18.1 mm in the FM-AM group, 32.2 mm in the PL-PL group, 36.6 mm in the AM-AM group, 38.0 mm in the PL-AM group and 35.2 mm in the AM-PL group. Significant differences were observed between 90° and 120° of knee flexion in the FM-AM, PL-PL, AM-AM and AM-PL groups (P < 0.05). Significant differences were observed at flex 90° between the FM-AM group and AM-AM group, and between the FM-AM group and PL-AM group. Significant differences were observed at flex 120° between the FM-AM group and AM-AM group, between the FM-AM group and PL-AM group and between the FM-PL group and AM-PL group. CONCLUSION: The distance to the peroneal nerve in FM drilling was significantly longer at 120° than at 90° of knee flexion. Therefore, the risk of peroneal injury using FM drilling should decrease at a higher angle of knee flexion.

Molecular motors: thermodynamics and the random walk.

The biochemical cycle of a molecular motor provides the essential link between its thermodynamics and kinetics. The thermodynamics of the cycle determine the motor's ability to perform mechanical work, whilst the kinetics of the cycle govern its stochastic behaviour. We concentrate here on tightly coupled, processive molecular motors, such as kinesin and myosin V, which hydrolyse one molecule of ATP per forward step. Thermodynamics require that, when such a motor pulls against a constant load f, the ratio of the forward and backward products of the rate constants for its cycle is exp [-(DeltaG + u(0)f)/kT], where -DeltaG is the free energy available from ATP hydrolysis and u(0) is the motor's step size. A hypothetical one-state motor can therefore act as a chemically driven ratchet executing a biased random walk. Treating this random walk as a diffusion problem, we calculate the forward velocity v and the diffusion coefficient D and we find that its randomness parameter r is determined solely by thermodynamics. However, real molecular motors pass through several states at each attachment site. They satisfy a modified diffusion equation that follows directly from the rate equations for the biochemical cycle and their effective diffusion coefficient is reduced to D-v(2)tau, where tau is the time-constant for the motor to reach the steady state. Hence, the randomness of multistate motors is reduced compared with the one-state case and can be used for determining tau. Our analysis therefore demonstrates the intimate relationship between the biochemical cycle, the force-velocity relation and the random motion of molecular motors.

Fluctuations in sliding motion generated by independent and random actions of protein motors.

We consider theoretical fluctuations in the in vitro sliding movement of individual cytoskeletal filaments generated by an ensemble of protein motors whose actions are assumed to be statistically independent and random. We show that the mean square deviation of the sliding distances of a filament for a given period of time around their average is proportional to the inverse of the filament length. This result provides a basis for an experimental test of the general assumption on the independent and random actions of protein motors.

Small molecular mass inhibitor of growth of MDCK cells derived from pig spinal cord.

We have discovered cell growth inhibitory activity in a salt extract of pig spinal cords. The growth inhibitory factor was purified by gel-filtration, ion-exchange and high performance liquid chromatography. Incubation of MDCK cells with the inhibitor arrested their locomotion within half an hour, suppressed their proliferation, and caused them to become round. The round cells that were still attached to the culture plate were alive. Upon removal of the inhibitor these cells flattened out and resumed locomotion and proliferation. The inhibitor was 100 times less effective on CHO-K1 cells. The reversible effects of the inhibitor on MDCK cells and its little effects on CHO-K1 cells indicate that the inhibitory activity is not due to a non-specific toxic mechanism. The inhibitor was both heat-stable and resistant to several chemical treatments, including proteases. Its behavior upon ion exchange chromatography suggested that it was positively charged at neutral pH, whilst its molecular mass was estimated to be 350 or larger by gel-filtration FPLC analysis. The inhibitory fraction reacted extensively with fluorescamine, suggesting that the inhibitory factor has amine groups, which are a possible candidate for its positive charges. Since spermine and spermidine, unlike the inhibitor in the present study, irreversibly inhibited the growth of the MDCK cells, the inhibitory activity in the present study is thus not due to contamination by these polyamines. Our experiments also support that the inhibitor is not a peptide.

A protein friction model of the actin sliding movement generated by myosin in mixtures of MgATP and MgGTP in vitro.

The sliding movement of an actin filament generated by myosin heads with MgGTP bound is much slower than that by those with MgATP bound. Nonetheless, there is a report that the actin sliding velocity at low (11-21 microM) MgATP concentrations is increased by the addition of MgGTP in a range of 1-3 mM, although the actin sliding velocity at these MgATP concentrations is larger than the maximum sliding velocity attained in the presence of MgGTP alone. The convex rise in the velocity was called "mutual sensitization of MgATP and MgGTP" in the report. Here we propose a theoretical model to account for the mutual sensitization of MgATP and MgGTP. The model is an extension of a protein friction model, accommodating the presence of two different substrates and assuming the presence of motile and non-motile myosins. This new model is in accord with the characteristics of the actin/myosin sliding movement experimentally observed in mixtures of MgATP and MgGTP. Comparison of the model with the experimental results implies that the non-motile and motile myosins are those with the "converse and correct" orientations of their heads with respect to the direction of the actin sliding movement in vitro.

Synchronous firing patterns of a set of insect neurosecretory cells.

The number of active cells in each synchronous firing event in a set of 10 neurosecretory cells in the silkmoth Bombyx mori was estimated from the amplitude and waveform of compound action potentials. One to 10 cells discharged an action potential within a period of 30 ms and one to two or nine to 10 units became active more frequently in a synchronous firing event. Numbers of active cells fluctuated like a sequence of pseudo random numbers, though the same number of cells tended to fire in two successive firing events of a short interval. These patterns suggest that electrical coupling may mediate synchronous firings in the insect neurosecretory cell system.

Fluctuation correlation in the sliding movement generated by protein motors in vitro.

The fluctuation in the sliding distance of cytoskeletal filaments driven to move by protein motors in vitro does not depend on the filament length. This is in sharp contrast to the case of Brownian movement of filamentous particles in their longitudinal directions, in which the positional fluctuation is proportional to the inverse of the length (L) of filaments. This latter 1/L dependence is a direct consequence of the central limit theorem: the statistical independence and randomness of the solvent molecule collisions with filaments, the collisions of which cause the random Brownian movement. The above length-independence in the sliding distance fluctuation found in the in vitro motility indicates the presence of correlation in the fluctuation. A possible explanation for the correlation is to assume that there is an extended time-correlation in the sliding movement, a correlation which could be produced by the actions on a sliding filament of protein motors with their heads randomly oriented in the in vitro motility assay system. We have checked this possibility by using long myosin thick filaments of molluscan smooth muscles, on which myosin heads are uniformly oriented, and have found that even with such myosin filaments with oriented myosin heads, the positional fluctuation of actin sliding distance does not depend on the actin filament length. This result thus indicates that the actions of protein motors on a sliding filament are not statistically independent or random, so that the positional fluctuation of filaments in the motor-generated sliding movement does not depend on the filament length.

Length dependence of displacement fluctuations and velocity in microtubule sliding movement driven by sea urchin sperm outer arm beta dynein in vitro.

We have studied the dependence on microtubule length of sliding velocity and positional fluctuation from recorded trajectories of microtubules sliding over sea urchin sperm outer arm beta dynein in a motility assay in vitro. The positional fluctuation was quantified by calculating the mean-square displacement deviation from the average, the calculation of which yields an effective diffusion coefficient. We have found that (1) the sliding velocity depends hyperbolically on the microtubule length, and (2) the effective diffusion coefficients do not depend on the length for sufficiently long microtubules. The length dependence of the sliding velocity indicates that the duty ratio, defined as the force producing period over the total cycle time of beta dynein interaction with microtubule, is very small. The length independence of the effective diffusion coefficient indicates that there is a correlation in the sliding movement fluctuation of microtubules.

Force production by chemically crosslinked myosin-actin crossbridges in rabbit skinned fibers in response to MgATP depletion.

In order to study the contractile property of myosin crossbridges attached to thin filaments, myosin heads were crosslinked to the filaments at their interface in single skinned rabbit psoas fibers with a zero-length chemical crosslinker, 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide (EDC). The results obtained show that a partially crosslinked single fiber produces a large rigor-like force when MgATP is depleted from the myofibrillar space. Such crosslinked fibers contain two types of crosslinked myosin heads: one with one of the two heads of the myosin molecule crosslinked to actin with the other head uncrosslinked; the other has both heads crosslinked to actin. The results of this work suggest that a crosslinked myosin head of the former type produces a much larger force than the latter type.

Monte Carlo study for fluctuation analysis of the in vitro motility driven by protein motors.

The fluctuation properties of the sliding movement of an individual cytoskeletal filament driven by protein motors in vitro can be analyzed by calculating the mean-square deviation of the displacement from the average within its single trajectory. For this purpose, a Monte Carlo simulation was used to define the conditions and limitations of a method for smoothing (curved) noisy trajectories without affecting either the steady or fluctuation characteristics inherent to the individual filament sliding movement. By applying the method to real experimental trajectory data, we show that an effective diffusion coefficient from displacement fluctuations of a sliding filament can be obtained from its single noisy trajectory even when it is curved.

Fluctuation in the microtubule sliding movement driven by kinesin in vitro.

We studied the fluctuation in the translational sliding movement of microtubules driven by kinesin in a motility assay in vitro. By calculating the mean-square displacement deviation from the average as a function of time, we obtained motional diffusion coefficients for microtubules and analyzed the dependence of the coefficients on microtubule length. Our analyses suggest that 1) the motional diffusion coefficient consists of the sum of two terms, one that is proportional to the inverse of the microtubule length (as the longitudinal diffusion coefficient of a filament in Brownian movement is) and another that is independent of the length, and 2) the length-dependent term decreases with increasing kinesin concentration. This latter term almost vanishes within the length range we studied at high kinesin concentrations. From the length-dependence relationship, we evaluated the friction coefficient for sliding microtubules. This value is much larger than the solvent friction and thus consistent with protein friction. The length independence of the motional diffusion coefficient observed at sufficiently high kinesin concentrations indicates the presence of correlation in the sliding movement fluctuation. This places significant constraint on the possible mechanisms of the sliding movement generation by kinesin motors in vitro.

Tension relaxation induced by pulse photolysis of caged ATP in partially crosslinked fibers from rabbit psoas muscle.

Muscle contractile force is thought to be generated by ATP-induced conformational changes in myosin crossbridges. In the present study, we investigated the response to ATP binding of force-bearing, attached cross-bridges. For this investigation, skinned fibers, in which myosin heads were in part covalently crosslinked to thin filaments with a zero-length crosslinker, were prepared. Caged ATP [the P3-1-(2-nitro)phenylethyl ester of ATP] was then pulse-photolyzed in these crosslinked fibers, which retained ATP-induced "rigor" tension, and then the subsequent tension changes were followed at 14-16 degrees C and ionic strengths of 0.1-2 M. A rapid tension decrease was observed after the photolysis in the partially crosslinked fibers. The rate of the decrease was not any different from that in the uncrosslinked fibers compared at ionic strength of 0.2 M. This and other results thus indicate a kinetic similarity in the crosslinked and uncrosslinked crossbridges in response to ATP binding. These findings also suggest that ATP-induced structural changes take place in the attached crossbridges at a rate similar to that of the ATP-induced dissociation of crossbridges from thin filaments.

Trinitrophenylation of the reactive lysine residue in double-headed myosin in the presence of PP.

Lys-83 in the heavy chain of rabbit skeletal muscle myosin is rapidly and stoichiometrically modified by trinitrobenzene sulfonate. Other authors claimed that the half-stoichiometric trinitrophenylation of Lys-83 in myosin in the presence of PPi was correlated to a Pro/Ser microheterogeneity at the 78th residue position in the heavy chain [Miyanishi, T., Maita, T., Matsuda, G., & Tonomura, Y. (1982) J. Biochem. 91, 1845-1853]. However, our recent studies with chymotryptic subfragment 1 (S1) instead of myosin showed no such correlation between the half-stoichiometric trinitrophenylation and the Pro/Ser microheterogeneity [Komatsu, H. & Tawada, K. (1993) J. Biol. Chem. 268, 16974-16978]. Since the global structure of the head portion of myosin is different from that of chymotryptic S1 that lacks DTNB light chain, it could be argued that the difference is due to the structural difference between chymotryptic S1 and myosin. We hence reexamined the situation with myosin, and obtained the same results as found with S1: (i) Lys-83 in myosin was half-stoichiometrically trinitrophenylated in the presence of PPi, although it was stoichiometrically modified in the absence of PPi; (ii) there was a Pro/Ser microheterogeneity at the 78th position in the myosin heavy chain, which was not correlated to the half-stoichiometric trinitrophenylation of Lys-83 in the presence of PPi.

Microheterogeneity around the reactive lysine residue in the myosin heavy chain from rabbit skeletal muscle.

A molecule of myosin subfragment 1 (S1) from rabbit skeletal muscle has one highly reactive lysine residue, Lys-83 (RLR), in the heavy chain, which is rapidly and stoichiometrically modified by trinitrobenzenesulfonate. Our previous kinetic study (Komatsu, H., Emoto, Y., and Tawada K. (1993) J. Biol. Chem. 268, 7799-7808) showed that although MgPPi reduces the maximum number of trinitrophenylated RLR down to about 0.5 mol/mol of S1, this half-stoichiometric modification of RLR is not the result of any heterogeneity in the primary structure of S1. However, this result conflicts with a previous report in which the half-stoichiometric trinitrophenylation has been reported to be related to Pro/Ser microheterogeneity at the 78th residue position in the heavy chain of rabbit skeletal muscle myosin. To resolve the conflict, we isolated peptides containing both the 78th residue and RLR from the two different preparations of S1, one whose RLR was trinitrophenylated in the presence of MgPPi and the other whose RLR was not trinitrophenylated in the presence of MgPPi, and then we determined the amino acid sequences of the peptides. We found the same Pro/Ser microheterogeneity at the 78th position in the peptides from these S1s and thus concluded that this microheterogeneity has no correlation to the half-stoichiometric trinitrophenylation of RLR.

Half-stoichiometric trinitrophenylation of myosin subfragment 1 in the presence of pyrophosphate or adenosine diphosphate.

A molecule of myosin subfragment 1 (S1) from skeletal muscle has one reactive lysine residue (RLR) that is rapidly and stoichiometrically modified by trinitrobenzene sulfonate (TNBS). Previous studies on the RLR modification with TNBS in the presence of inorganic pyrophosphate (PPi) by others suggested the non-identical nature of the two myosin heads which is still controversial. To resolve this issue, we studied the effects of PPi and ADP on the trinitrophenylation reaction over a wide range of ligands more systematically, by using S1 containing the alkali 1 light chain. We herein show that MgPPi or MgADP reduces the maximum number of trinitrophenylated RLRs down to about 0.5 mol/mol of S1 and that this half-stoichiometric modification of RLRs is not the result of heterogeneity in the primary structure of S1. Instead, our results suggest the existence of two almost equimolar, different conformations of S1.PPi and S1.ADP. Furthermore, we show evidence that the two different conformations are in a slow equilibrium in the presence of TNBS. We also studied the effects of the stoichiometric and half-stoichiometric RLR modification on the EDTA- and Mg-ATPase activities of S1 and found no evidence for the functional heterogeneity of the myosin active site.

Dynamical role of "protein friction" in the sliding movement of protein motors in vitro.

When protein motors interact with a sliding cytoplasmic-filament through a weak-binding interaction (thus, without ATP splitting), this interaction cycle results in friction opposing the sliding movement. The friction is owing to the flexible nature of the heads of these motors as globular proteins. Under a certain condition, the friction becomes proportional to the sliding velocity. This viscous-like friction by protein motor is called protein friction. Since the protein friction is more than 10 times larger than the hydrodynamic viscous drag, we propose that the sliding velocity in the in vitro motility system is limited when the active sliding force generated by protein motors is balanced by the protein friction. The model of the protein friction hypothesis is consistent with many experimental data of the in vitro motility systems such as those of mixture experiments with different myosins and the ATP-concentration dependence of the sliding velocity. By relating the coefficient of the protein friction to the diffusion coefficient, we show that the model is consistent with the data on the one-dimensional Brownian movement of a microtubule on a dynein-coated glass surface in the presence of vanadate and ATP. The model also shows that the Brownian movement is driven directly by the thermally-generated structural fluctuations of the dynein heads rather than the atomic collision of solvent molecules. Thus, the model implies that the thermal structural fluctuations of the protein motor heads underlie the ATP-induced sliding movement by protein motors and hence protein motors are a Brownian actuator.

Effects of ionic strength on force transients induced by flash photolysis of caged ATP in covalently crosslinked rabbit psoas muscle fibers.

Single fibers from glycerinated rabbit psoas muscle were treated with 1-ethyl-3[3-(dimethylamino) propyl] carbodiimide (EDC), after rigor was induced, to crosslink myosin heads to actin. The optimally pre-stretched (approximately 1.8%), partially crosslinked fibers produce a large force when MgATP is depleted, and this force is abolished when MgATP is reintroduced, even in high ionic strength solution of 0.5 M (Tawada et al. 1989). We investigated the rate of force decay in the crosslinked, force-producing fibers using pulse photolysis of caged ATP (Goldman et al. 1984). The decay of force was fast, the rate of which depending both on the ionic strength and on the amount of ATP released (0.2-2.2 mM) with the second-order rate constant of 0.5-1 x 10(5) M-1s-1 at the ionic strength of 0.5 M. At high ionic strength (1-2M) force decayed at lower rate. At low ionic strength (0.1-0.2 M), however, force decayed more rapidly, but force redeveloped subsequently, which is probably caused by uncrosslinked myosin heads.