Biomedical subjects

E Pate

Publications and source records attributed to E Pate.

At least 37 records · Page 2Linked to original sources

PubMed1995

The relative distribution of T cell subsets is altered in Jamaican children infected with human T cell lymphotropic virus type I.

Early childhood infection with human T cell lymphotropic virus type I (HTLV-I) has been suggested to be involved in the pathogenesis of infective dermatitis and adult T cell leukemia/lymphoma. Since only a very small percentage of HTLV-I-infected children develop disease later in life, identification of early interim markers for persons at risk for developing disease would enable monitoring and might provide insight into the pathophysiology of the various diseases associated with HTLV-I infection. A cross-sectional study analyzed T cell subsets in 35 HTLV-I-seronegative and 16 HTLV-I-seropositive Jamaican children 11-31 months old. HTLV-I seropositivity was associated with an increase in the mean percentage of CD4 cells expressing HLA-DR, a marker for T cell activation (P = .02). This increase was positively correlated with duration of infection (r = .74, P = .009). These data demonstrate perturbation of regulatory cells of the immune system in HTLV-I-infected children.

Breast Feeding↗

PubMed1994

A model of the release of myosin heads from actin in rapidly contracting muscle fibers.

We describe a model that relates the maximum shortening velocity of a muscle fiber, Vm, to the kinetics of the dissociation of a myosin head from actin. At Vm, the positive work exerted by cross-bridges attached in the powerstroke must be balanced by cross-bridges that have been carried by movement of the filaments into a region where they exert a negative force. This balance allows one to relate Vm and the rate of cross-bridge detachment. Studies of actomyosin kinetics suggest that at high substrate, detachment should be limited by a slow protein isomerization (approximately 50 s-1) that precedes ADP release. This rate is too slow to be easily accommodated in existing models. However, a slow rate for cross-bridge dissociation, similar to that of the isomerization, is predicted if previous models are modified to include rapid detachment of cross-bridges that have been carried so far into the negative force region that their free energy exceeds that of the detached state. The model also explains another aspect of muscle contraction: at high shortening velocities, the observed rate of ATP hydrolysis is low, because a cross-bridge can interact with multiple actin binding sites before releasing the hydrolysis products and binding another ATP.

Actins↗

PubMed1994

Temperature dependence of the inhibitory effects of orthovanadate on shortening velocity in fast skeletal muscle.

We have investigated the effects of the orthophosphate (P(i)) analog orthovanadate (Vi) on maximum shortening velocity (Vmax) in activated, chemically skinned, vertebrate skeletal muscle fibers. Using new "temperature-jump" protocols, reproducible data can be obtained from activated fibers at high temperatures, and we have examined the effect of increased [Vi] on Vmax for temperatures in the range 5-30 degrees C. We find that for temperatures < or = 20 degrees C, increasing [Vi] inhibits Vmax; for temperatures > or = 25 degrees C, increasing [Vi] does not inhibit Vmax. Attached cross-bridges bound to Vi are thought to be an analog of the weakly bound actin-myosin.ADP-P(i) state. The data suggest that the weakly bound Vi state can inhibit velocity at low temperature, but not at high temperature, with the transition occurring over a narrow temperature range of < 5 degrees C. This suggests a highly cooperative interaction. The data also define a Q10 for Vmax of 2.1 for chemically skinned rabbit psoas fibers over the temperature range of 5-30 degrees C.

Actins↗

PubMed1994

Demonstration of human T lymphotropic virus type I (HTLV-I)-specific T cell responses from seronegative and polymerase chain reaction-negative persons exposed to HTLV-I.

Human T lymphotropic virus type I (HTLV-I) is a human retrovirus etiologically linked to adult T cell leukemia and the progressive chronic neurologic disease HTLV-I-associated myelopathy/tropical spastic paraparesis. Described is a method that measures the production of interleukin-2 from HTLV-I synthetic peptide-stimulated peripheral blood lymphocytes (PBL) of HTLV-I-infected persons. The peptides correspond to immunogenic regions of the HTLV-I Env and Tax proteins. Significantly, this assay demonstrated T cell responses to these HTLV-I peptides from coded PBL samples in 7 of 19 HTLV-I-seronegative polymerase chain reaction-negative persons known to have been exposed to HTLV-I but in none of 16 matched controls without risk factors for exposure (P = .007). The implications of this finding are discussed.

Adult↗

PubMed1994

Effect of series elasticity on delay in development of tension relative to stiffness during muscle activation.

Experimental data have indicated that during activation, the attachment of myosin to actin, measured by mechanical stiffness, precedes tension generation by 10-30 ms. Using computer simulation, we have investigated the effect of a series elastic element on the lag between stiffness and tension development during muscle activation. Two versions of the two-state cross-bridge model originally proposed by Huxley and a three-state model were considered. After simulated activation, stiffness and tension increased with rates that were strongly dependent on the series elastic strain. In the absence of a series elastic element, the rise in stiffness preceded, lagged, or was coincident with the increase in tension, depending on the model. For large elastic strains, tension lagged stiffness for all models. Lags of 10-30 ms could be obtained with elastic strains of 0.3-1% of the muscle length. This is a realistic value in experiments without sarcomere length servocontrol, suggesting that series elasticity may be an important contributor to the experimentally observed lag between tension and stiffness.

Animals↗

PubMed1993

The use of differing nucleotides to investigate cross-bridge kinetics.

We have investigated the ability of the nucleotides GTP, CTP, and 1-N6-etheno-2-aza-ATP (aza-ATP) to support contraction of chemically skinned rabbit psoas fibers. Working at 10 degrees C, millimolar concentrations of all nucleotides relaxed fibers in the absence of calcium. In active fibers, GTP served as a very poor substrate with isometric tension, isometric GTPase rate, and maximum shortening velocity (Vmax) all less than 10% of those obtained with ATP. Aza-ATP was only a slightly better substrate. CTP, on the other hand, was an effective substrate with mechanical parameters which were 65-100% those obtained with ATP, and with a hydrolysis rate that exceeded that of ATP. For all three ligands, Vmax followed Michaelis-Menten saturation behavior with values for Km which were from 2.5 to 12 times greater than that for ATP, showing that the analogs bound slowly to myosin in the fibers. Increasing concentrations of orthophosphate inhibited tension with CTP, to a lesser extent with aza-ATP, but not all with GTP. A combination of the mechanical data obtained in fibers with the kinetic data obtained in solution (White, H.D., Belknap, B., and Jiang, W. (1993) J. Biol. Chem. 268, 10039-10045) is used to better define the actomyosin interaction in fibers.

Adenosine Triphosphate↗

PubMed1993

Determination of the myosin step size from mechanical and kinetic data.

During muscle contraction, work is generated when a myosin cross-bridge attaches to an actin filament and exerts a force on it through some power-stroke distance, h. At the end of this power stroke, attached myosin heads are carried into regions where they exert a negative force on the actin filament (the drag stroke) and where they are released rapidly from actin by ATP binding. Although the length of the power stroke remains controversial, average distance traversed in the drag-stroke region can be determined when one knows both rate of cross-bridge dissociation and filament-sliding velocity. At maximum contraction velocity, the average force exerted in the drag stroke must balance that exerted in the power stroke. We discuss here a simple model of cross-bridge interaction that allows one to calculate the force exerted in the drag stroke and to relate this to the power-stroke distance h traversed by cross-bridges in the positive-force region. Both the rate at which myosin can be dissociated from actin and the velocity at which an actin filament can be translated have been measured for a series of myosin isozymes and for different substrates, producing a wide range of values for each. Nonetheless, we show here that the rate of myosin dissociation from actin correlates well with the velocity of filament sliding, providing support for the simple model presented and suggesting that the power stroke is approximately 10 nm in length.

Actins↗

PubMed1993

Synthesis of non-nucleotide ATP analogues and characterization of their chemomechanical interaction with muscle fibres.

To probe the substrate requirements for the actomyosin chemomechanical interaction, the effects of a series of eight new non-nucleotide ATP analogues on actomyosin-catalysed hydrolysis rates and on fibre mechanics have been investigated. These analogues have substitutions of new functional groups at the 2- and 4-positions of the ATP analogues, 2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate (NANTP), and 3-[(4-nitrophenyl)amino]propyl triphosphate (PrNANTP). Previous work has shown NANTP but not PrNANTP will support active tension and shortening in skinned muscle fibres in a manner almost identical to ATP. Here all 2- and 4-phenyl substituted analogues had myosin subfragment 1 (S1) NTPase hydrolysis rates higher than ATP and the rates were stimulated by addition of actin. In general, the replacement of the 4-azido group of NANTP with -H, -NO2 or -NH2 had small effects on fibre mechanics while replacement of 2-NO2 group with -H or -NH2 dramatically lowered the ability of the new analogues to support active tension and shortening. All PrNANTP-based analogues were ineffective in supporting active tension or shortening. We found no correlation between S1 or actoS1 NTPase rates and any mechanical parameters. However, for all analogues there was a strong correlation between the maximal velocity of shortening (Vmax) and isometric tension (P0). A three-state, chemomechanical model is proposed in which the analogues effect the transition rate into a strongly-bound, force-producing crossbridge state to account for this correlation. These studies identify 2-[(2-nitrophenyl)amino]ethyl triphosphate as the chemically simplest ATP analogue which closely mimics the effect of ATP in skinned muscle fibres.

Actins↗

PubMed1993

A model of stress relaxation in cross-bridge systems: effect of a series elastic element.

Many experimental protocols employed in the study of muscle mechanics use tension transients as a probe of the magnitudes of the kinetic rates in the underlying cross-bridge dynamics. These transients could potentially be modified by the elastic elements that exist both within the fiber and at the points of attachment to the experimental apparatus. To better understand the magnitude of such modifications, we have used computer simulation to investigate the transients that would be expected for cross bridges acting on an actin filament attached to an elastic element. The original model of cross-bridge mechanics by A.F. Huxley was used (Prog. Biophys. 7: 255-318, 1957). After an isometric equilibrium is achieved, a tension transient is produced by changing the dissociation rate constant, g1, while holding the attachment rate constant, f1, fixed. This decreases the number of attached, force-producing cross bridges. We find that the tension transients are markedly slowed by the presence of even a few (> or = 2) nanometers of series elastic strain per half-sarcomere. Thus some rate constants inferred from mechanical transients (e.g., those induced by caged ligands) may underestimate the actual kinetic rates of the cross-bridge processes.

Animals↗

PubMed1992

Contraction of glycerinated rabbit slow-twitch muscle fibers as a function of MgATP concentration.

We have measured the isometric tension and force-velocity relationships of glycerinated rabbit slow-twitch semimembranosus muscle as a function of MgATP concentration ([MgATP]) and have compared the results with those obtained previously from fast-twitch psoas muscle. We find that isometric tension decreases as [MgATP] increases. The magnitude of the decrease is not as great as observed in psoas. Maximum shortening velocity (Vmax) exhibits classical Michaelian saturation behavior with respect to [MgATP] with a Michaelis constant (Km) for half-maximal velocity of 18 microM and a value at saturating [MgATP] of 0.6 muscle lengths/s. Similar values were observed in fibers from soleus, another slow-twitch muscle. The corresponding values in rabbit psoas muscle are 150 microM and 1.6 lengths/s. Compared with psoas, in semimembranosus muscle Km decreases by a factor of approximately 10, whereas Vmax decreases by about a factor of 3. Thus, although in a nonphysiological regime, at low [MgATP], a "fast" muscle actually has a lower shortening velocity than a "slow" muscle.

Adenosine Triphosphate↗

PubMed1991

Simulation of stochastic processes in motile crossbridge systems.

The underlying stochastic nature of many models of the actomyosin interaction should result in fluctuations in both force and shortening velocity. In classical experimental approaches involving intact or glycerinated muscle preparations these fluctuations are too small to resolve owing to the large numbers of crossbridges involved. However, new experimental techniques allow mechanical measurements to be made in systems in which small numbers of myosin heads act on a single actin filament, or small numbers of kinesin molecules act on a single tubulin filament. In these systems, stochastic effects should be evident. To understand better the nature of the expected stochastic effects, we have used computer simulation to investigate the fluctuations predicted by the original model for muscle crossbridge mechanics proposed by A.F. Huxley. We consider three situations: (1) the translation of actin or tubulin filaments by myosin or kinesin motors immobilized on a fixed substrate, (2) the production of tension by ensembles of immobilized myosin which involve the displacement of an elastic load, and (3) the fluctuations in axial displacement of a single, bipolar myosin thick filament interacting with actin filaments as in a sarcomere. In all three cases, fluctuations are clearly evident in simulations involving small numbers of motors. For case (1), we show that translation velocities can vary with crossbridge density. Whether one motor translates a filament faster, slower or at the same speed as many motors depends on the relative magnitudes of the attachment and detachment rate functions. Analytical expressions are provided to quantitate this relationship. For case (2), we show that fluctuations predicted assuming perfectly isometric conditions differ form those observed when the 'isometric state' is achieved against an elastic load. 'Elastic damping' of the fluctuations in the system results from the presence of many attached motors. In case (3) we show that in spite of the presence of stochastic fluctuations which can destabilize the uniformity of filament overlap in a sarcomere, the magnitude of thick filament displacement is less than might be anticipated over time periods of in vivo contraction. Taken together, these simulations allow one to better interpret experimental data in terms of current models of motor function.

Actin Cytoskeleton↗

PubMed1991

Mechanics of glycerinated muscle fibers using nonnucleoside triphosphate substrates.

We have investigated the ability of the photoaffinity, nonnucleotide ATP analogues, 2-[(4-azido-2-nitrophenyl) amino] ethyl triphosphate (NANTP) and 2-[(4-azido-2-nitrophenyl) amino] propyl triphosphate (PrNANTP), to support active contraction in glycerinated rabbit psoas fibers. At millimolar concentrations, in the absence of calcium, both analogues relaxed fibers. In the presence of calcium, MgNANTP produced isometric tension and stiffness that were one-half to two-thirds the values obtained in MgATP. Maximum shortening velocity and the calcium-activated, myofibrillar catalyzed rate of hydrolysis were approximately the same for MgNANTP as for MgATP. With MgNANTP as the substrate, increasing concentrations of the diphosphate analogue, MgNANDP, inhibited shortening velocity but did not change isometric tension. The addition of increased concentrations of orthophosphate (P) decreased tension while shortening velocity increased. Thus, the effects of the hydrolysis products of NANTP were quite similar to those observed previously for ADP and P in the presence of MgATP. Taken together, these observations show that MgNANTP binds to, and functions in the active site of myosin in a manner quite analogous to MgATP. Thus, the aryl azido group should serve as a valid photoaffinity label for the purine portion of the active site. In contrast, MgPrNANTP, which differs from MgNANTP only in an extra CH2 spacer between the nitrophenyl ring and the triphosphate moiety did not support isometric tension or active shortening in the presence of calcium. Fiber stiffness increased in the presence of calcium and MgPrNANTP, with a calcium-activated, myofibrillar MgPrNANTPase which was about half that obtained with MgATP. Thus, in the presence of MgPrNANTP, cross-bridges appeared to be cycling through states that were attached to actin, but not producing force.

Adenosine Triphosphate↗

PubMed1989

Addition of phosphate to active muscle fibers probes actomyosin states within the powerstroke.

We have measured the effect of phosphate (Pi) on the tension and maximum shortening velocity of permeable rabbit psoas fibers. Work in a number of laboratories has established that addition of phosphate (0-25 mM) to active muscle fibers at physiological MgATP concentrations decreases isometric tension with little effect on the maximum shortening velocity. Here we extend these results to a wider range of Pi concentrations and to low MgATP concentrations. Low levels of Pi (approx. 150 microM-200 microM) were obtained by using sucrose phosphorylase and sucrose to reduce contaminating Pi in the solutions used to activate the fiber, and high levels (52-73 mM) were obtained by replacing acetate with Pi as the principal anion. In an activating solution containing either 50 microM or 4 mM MgATP, pH 6.2 or 7.0, isometric tension declines linearly with the logarithm of Pi concentration. Although the isometric tension decreases with increasing concentrations of H+ or MgATP, the slope of relative isometric tension as a function of log[Pi] is the same at the two values of pH and [MgATP]. At pH 7 and 4 mM MgATP, the velocity of contraction increased slightly as Pi increased from 0.2 to 52 mM. At 50 microM MgATP the velocity decreased slightly as Pi increased from 0.2 to 10 mM with a substantial decrease as Pi increased from 10 to 52 mM. These results are discussed in terms of models of cross-bridge energetics. The observation that force declines linearly with the logarithm of [Pi] is compatible with models in which a major force producing state occurs subsequent to Pi release.(ABSTRACT TRUNCATED AT 250 WORDS)

Actomyosin↗

PubMed1989

A model of crossbridge action: the effects of ATP, ADP and Pi.

We have explored a model of crossbridge kinetics that explains many of the effects on steady-state muscle contraction of ligands that bind to the nucleotide site on myosin. The mathematical model follows the basic framework for crossbridge function first established by A. F. Huxley. In the model, detached crossbridges initially bind in a weakly attached, A.M.D.Pi state (A, actin; M, myosin; D, ADP; Pi, orthophosphate) at the beginning of the region of positive force production. Pi release then results in transition to a strongly-bound A.M.D state, as has been suggested by other investigators from both biochemical and mechanical data. Mg2+ ADP release and subsequent crossbridge detachment due to Mg2+ ATP binding to the A.M state occur at the end of the region of positive force production. Work in a number of laboratories has now defined the effects on steady-state contraction of variations in the concentrations of Mg2+ ATP, Mg2+ ADP and Pi. These data provide valuable constraints that can be used to further refine current models. The maximum velocity of shortening (V max) and ATPase activity of muscle fibres exhibit classical saturation behaviour with respect to Mg2+ ATP concentration, with Mg2+ ADP acting as a competitive inhibitor. The model can reproduce this behaviour. The model also explains the observations that increasing [Mg2+ ATP] decreases isometric tension and increasing [Mg2+ ADP] increases tension. As the concentration of Pi increases, model predictions suggest that tension should decrease approximately as log[Pi], that ATPase activity should decrease less than tension and that V max should be almost unchanged, as has been found experimentally. The model also demonstrates that the connection between the parameters of contraction and the free energy of hydrolysis of Mg2+ ATP can be complex.

Actomyosin↗

PubMed1989

A cascading development model for amphibian embryos.

The mesodermal tissue of some amphibian gastrula develops into a dorsal-to-ventral sequence of notochord, somite, pronephros, and lateral plate cell types. The cellular proportions regulate with respect to embryo size. The dorsal blastoporal lip appears to function as an organizer for the embryo. The transplantation of a donor lip to the ventral side of a host causes a second, opposed embryo to form and the system commits similar total proportions of cells as do normally developing embryos. Transplantation of donor somite to the ventral side of a host causes a reduction in the proportion of host somite developed. A modified reaction-diffusion system governing embryo development is proposed. Developmental simulations consistent with experimental observations are presented and analyzed. The results suggest that the degree of somite inhibition is positively correlated with the size of the somite transplant. Further predictions are that sufficiently large somite transplants would induce ectopic, ventral pronephros to form and ventral pronephros transplants would inhibit host pronephros development.

Amphibians↗

PubMed1988

Energetics of the actomyosin bond in the filament array of muscle fibers.

The interaction between actin and myosin in the filament array of glycerinated muscle fibers has been monitored using paramagnetic probes and mechanical measurements. Both fiber stiffness and the spectra of probes bound to a reactive sulfydral on the myosin head were measured as the actomyosin bond was weakened by addition of magnesium pyrophosphate (MgPPi) and glycerol. In the absence of MgPPi, all myosin heads are attached to actin with oriented probes. When fibers were incubated in buffers containing MgPPi, a fraction of the probes became disordered, and this effect was greater in the presence of glycerol. To determine whether the heads with disordered probes were detached from actin, spin-labeled myosin subfragment-1 (MSL-S1) was diffused into unlabeled fibers, and the fractions bound to actin and free in the medium were correlated with the oriented and disordered spectral components. These experiments showed that the label was oriented when MSL-S1 was attached to actin in a ternary complex with the ligand and that all heads with disordered probes were detached from actin. Thus the fraction of oriented labels could be used to determine the fraction of heads attached to actin in a fiber in the presence of ligand. The fraction of myosin heads attached to actin decreased with increasing [MgPPi], and in the absence of glycerol approximately 50% of the myosin heads were dissociated at 3.3 mM ligand with little change in fiber stiffness. In the presence of 37% glycerol plus ligand, up to 80% of the heads could be detached with a 50% decrease in fiber stiffness. The data indicate that there are two populations of myosin heads in the fiber. All the data could be fit with a model in which one population of myosin heads (comprising approximately 50% of the total) sees an apparent actin concentration of 0.1 mM and can be released from actin with little change in fiber stiffness. A second population of myosin heads (approximately 50%) sees a higher actin concentration (5 mM) and is only released in the presence of both glycerol and ligand.

Actins↗

PubMed1988

The inhibition of rabbit skeletal muscle contraction by hydrogen ions and phosphate.

1. The effects of phosphate and protons on the mechanics and energetics of muscle contraction have been investigated using glycerinated rabbit psoas muscle. 2. Fibres were fully activated by addition of Ca2+ (pCa 4-5) at 10 degrees C. The velocities of contraction were measured in isotonic load clamps, and the velocities of unloaded fibres were measured by applying a series of step changes in fibre length. Fibre ATPase activity was monitored using an enzyme system to couple ADP production to reduced nicotinamide-adenine dinucleotide (NADH) and measuring the depletion of NADH by optical density. 3. At pH 7.0 and 3 mM-phosphate, isometric tension (P0) was 13.2 +/- 0.9 N/cm (mean +/- S.E.M., n = 10 observations), the maximum contraction velocity (Vmax) was 1.63 +/- 0.05 lengths/s (n = 5) and the ATPase activity was 1.27 +/- 0.12 s-1 myosin head-1 (n = 35). Increasing phosphate from 3 to 20 mM at pH 7.0 does not affect Vmax, causes a small decrease in the ATPase activity (15-20%) and decreases P0 by approximately 20%. Changing pH from 7 to 6 at 3 mM-phosphate decreases P0 by 45% and both Vmax and ATPase activity by 25-30%. The effects of changing both pH and phosphate were approximately additive for all parameters measured. The inhibition of these parameters by low pH and high concentration of phosphate was reversible. 4. The force-velocity relation was fitted by the Hill equation using a non-linear least-squares method. The value of the parameter which describes the curvature, a/P0, was 0.20. The curvature of the force-velocity relation was not changed by addition of phosphate or by changes in pH. 5. These data provide information on both the kinetics of the actomyosin interaction and on the process of muscle fatigue. The data are consistent with models of cross-bridge kinetics in which phosphate is released within the powerstroke in a step involving a rapid equilibrium between states. The inhibition by protons is more complex, and may involve less specific effects on protein structure. 6. During moderate fatigue of living skeletal muscle, MgATP concentration is known to remain approximately constant at 4 mM, phosphate to increase from 3 to 20 mM, and protons from 0.1 to 1 microM. The data suggest that much of the inhibition of P0 observed during moderate fatigue can be explained by the increased levels of phosphate and protons, and that much of the inhibition of fibre Vmax and ATPase activity can be explained by the increase in protons.

Adenosine Triphosphatases↗

PubMed1988

The effects of pyrophosphate on myosin heads and on fiber stiffness.

We have investigated the effect of magnesium pyrophosphate (MgPPi) on the orientation and stiffness of myosin heads in glycerinated muscle fibers. MgPPi is known to bind to the nucleotide site of myosin and to weaken the bond between myosin and actin. The spectra of paramagnetic probes attached to the reactive sulfhydryl on the myosin head were used to monitor the orientation of the head. Addition of 3 mM MgPPi to rigor fibers resulted in a shift of approximately 50% of the myosin heads from a state in which the probes are highly oriented to a state in which the probes are disordered. Experiments in which MgPPi dissociated labeled subfragment-1 from unlabeled fibers support the conclusion that the disordered probes are on myosin heads that are not attached to actin. Addition of MgPPi causes only a small decrease in fiber stiffness, measured by step changes in fiber length. We conclude that up to 50% of the myosin heads can be dissociated from actin with little change in muscle stiffness. Thus stiffness is not a linear measure of the fraction of myosin heads attached to actin. We present a model in which one head of the pair of a myosin molecule binds more weakly to actin and can be dissociated preferentially by the binding of MgPPi.

Animals↗