Effectiveness of Centruroides scorpion antivenom compared to historical controls.

BACKGROUND: Envenomation by North American scorpions of genus Centruroides is associated with a syndrome of neurotoxicity and respiratory compromise that disproportionately affects rural children. Severe scorpion envenomation is rare, which makes treatment difficult to study using conventional controlled clinical trials; and small-scale placebo-controlled trials conducted in tertiary centers are of limited generalizability to the community setting. Open label studies, although safer and easier to conduct, are of limited value unless a suitable comparator group is used. Historical controls may be appropriate when concurrent controls are not feasible or ethical. METHODS: A successful placebo-controlled, double-blind clinical trial design was adapted for community use in Arizona and Mexico. A comparator population was established by replacement of the placebo group with a retrospective cohort and preservation of criteria for inclusion, exclusion, dosing and endpoint assessment. Study endpoints were selected to demonstrate the clearest possible difference between treatment groups, while minimizing confounders. Results were summarized and endpoints were directly compared between groups and with the prior double-blind study. RESULTS: The clinical syndrome remained evident in 95.9% of the historical cohort (93/97) 4 h after admission, and their cumulative dose of midazolam given between baseline and discharge was 5.29 ± 8.68 mg/kg (range 0-62.8). Among 78 prospectively treated cases, none received midazolam and only 2 (2.8%) remained symptomatic at 4 h. Venom was detectable in the plasma of all antivenom recipients tested, and it dropped by 90% of baseline in 95% of cases studied. CONCLUSIONS: The results of this pragmatic study strongly support the findings of the double-blind, placebo controlled clinical trial of the same antivenom. Recipients of antivenom at rural sites improved at a rate similar to that in the intensive care (ICU) setting, and historical cases resolved at a rate similar to that for untreated ICU controls. Use of antivenom in the primary care setting appeared to be safe and effective and to eliminate the need for intensive care or for transport to a tertiary care center, in all subjects prospectively studied.

Contractile properties of muscle fibers from the deep and superficial digital flexors of horses.

Equine digital flexor muscles have independent tendons but a nearly identical mechanical relationship to the main joint they act upon. Yet these muscles have remarkable diversity in architecture, ranging from long, unipennate fibers ("short" compartment of DDF) to very short, multipennate fibers (SDF). To investigate the functional relevance of the form of the digital flexor muscles, fiber contractile properties were analyzed in the context of architecture differences and in vivo function during locomotion. Myosin heavy chain (MHC) isoform fiber type was studied, and in vitro motility assays were used to measure actin filament sliding velocity (V(f)). Skinned fiber contractile properties [isometric tension (P(0)/CSA), velocity of unloaded shortening (V(US)), and force-Ca(2+) relationships] at both 10 and 30°C were characterized. Contractile properties were correlated with MHC isoform and their respective V(f). The DDF contained a higher percentage of MHC-2A fibers with myosin (heavy meromyosin) and V(f) that was twofold faster than SDF. At 30°C, P(0)/CSA was higher for DDF (103.5 ± 8.75 mN/mm(2)) than SDF fibers (81.8 ± 7.71 mN/mm(2)). Similarly, V(US) (pCa 5, 30°C) was faster for DDF (2.43 ± 0.53 FL/s) than SDF fibers (1.20 ± 0.22 FL/s). Active isometric tension increased with increasing Ca(2+) concentration, with maximal Ca(2+) activation at pCa 5 at each temperature in fibers from each muscle. In general, the collective properties of DDF and SDF were consistent with fiber MHC isoform composition, muscle architecture, and the respective functional roles of the two muscles in locomotion.

Dynamic micro-Hall detection of superparamagnetic beads in a microfluidic channel.

We report integration of an InAs quantum well micro-Hall magnetic sensor with microfluidics and real-time detection of moving superparamagnetic beads. Beads moving within and around the Hall cross area result in positive and negative Hall voltage signals respectively. Relative magnitudes and polarities of the signals measured for a random distribution of immobilized beads over the sensor are in good agreement with calculated values and explain consistently the shape of the dynamic signal.

A simple model with myofilament compliance predicts activation-dependent crossbridge kinetics in skinned skeletal fibers.

The contribution of thick and thin filaments to skeletal muscle fiber compliance has been shown to be significant. If similar to the compliance of cycling cross-bridges, myofilament compliance could explain the difference in time course of stiffness and force during the rise of tension in a tetanus as well as the difference in Ca(2+) sensitivity of force and stiffness and more rapid phase 2 tension recovery (r) at low Ca(2+) activation. To characterize the contribution of myofilament compliance to sarcomere compliance and isometric force kinetics, the Ca(2+)-activation dependence of sarcomere compliance in single glycerinated rabbit psoas fibers, in the presence of ATP (5.0 mM), was measured using rapid length steps. At steady sarcomere length, the dependence of sarcomere compliance on the level of Ca(2+)-activated force was similar in form to that observed for fibers in rigor where force was varied by changing length. Additionally, the ratio of stiffness/force was elevated at lower force (low [Ca(2+)]) and r was faster, compared with maximum activation. A simple series mechanical model of myofilament and cross-bridge compliance in which only strong cross-bridge binding was activation dependent was used to describe the data. The model fit the data and predicted that the observed activation dependence of r can be explained if myofilament compliance contributes 60-70% of the total fiber compliance, with no requirement that actomyosin kinetics be [Ca(2+)] dependent or that cooperative interactions contribute to strong cross-bridge binding.

Dimethyl sulphoxide enhances the effects of P(i) in myofibrils and inhibits the activity of rabbit skeletal muscle contractile proteins.

In the catalytic cycle of skeletal muscle, myosin alternates between strongly and weakly bound cross-bridges, with the latter contributing little to sustained tension. Here we describe the action of DMSO, an organic solvent that appears to increase the population of weakly bound cross-bridges that accumulate after the binding of ATP, but before P(i) release. DMSO (5-30%, v/v) reversibly inhibits tension and ATP hydrolysis in vertebrate skeletal muscle myofibrils, and decreases the speed of unregulated F-actin in an in vitro motility assay with heavy meromyosin. In solution, controls for enzyme activity and intrinsic tryptophan fluorescence of myosin subfragment 1 (S1) in the presence of different cations indicate that structural changes attributable to DMSO are small and reversible, and do not involve unfolding. Since DMSO depresses S1 and acto-S1 MgATPase activities in the same proportions, without altering acto-S1 affinity, the principal DMSO target apparently lies within the catalytic cycle rather than with actin-myosin binding. Inhibition by DMSO in myofibrils is the same in the presence or the absence of Ca(2+) and regulatory proteins, in contrast with the effects of ethylene glycol, and the Ca(2+) sensitivity of isometric tension is slightly decreased by DMSO. The apparent affinity for P(i) is enhanced markedly by DMSO (and to a lesser extent by ethylene glycol) in skinned fibres, suggesting that DMSO stabilizes cross-bridges that have ADP.P(i) or ATP bound to them.

2-deoxy-ATP enhances contractility of rat cardiac muscle.

To investigate the kinetic parameters of the crossbridge cycle that regulate force and shortening in cardiac muscle, we compared the mechanical properties of cardiac trabeculae with either ATP or 2-deoxy-ATP (dATP) as the substrate for contraction. Comparisons were made in trabeculae from untreated rats (predominantly V1 myosin) and those treated with propylthiouracil (PTU; V3 myosin). Steady-state hydrolytic activity of cardiac heavy meromyosin (HMM) showed that PTU treatment resulted in >40% reduction of ATPase activity. dATPase activity was >50% elevated above ATPase activity in HMM from both untreated and PTU-treated rats. V(max) of actin-activated hydrolytic activity was also >50% greater with dATP, whereas the K(m) for dATP was similar to that for ATP. This indicates that dATP increased the rate of crossbridge cycling in cardiac muscle. Increases in hydrolytic activity were paralleled by increases of 30% to 80% in isometric force (F(max)), rate of tension redevelopment (k(tr)), and unloaded shortening velocity (V(u)) in trabeculae from both untreated and PTU-treated rats (at maximal Ca(2+) activation), and F-actin sliding speed in an in vitro motility assay (V(f)). These results contrast with the effect of dATP in rabbit psoas and soleus fibers, where F(max) is unchanged even though k(tr), V(u), and V(f) are increased. The substantial enhancement of mechanical performance with dATP in cardiac muscle suggests that it may be a better substrate for contractility than ATP and warrants exploration of ribonucleotide reductase as a target for therapy in heart failure.

Viscosity and solute dependence of F-actin translocation by rabbit skeletal heavy meromyosin.

We tested the hypothesis that solvent viscosity affects translocation of rhodamine phalloidin-labeled F-actin by rabbit skeletal heavy meromyosin (HMM). When viscosity was increased using either glycerol, fructose, sucrose, or dextran (1.5, 6.0, or 15-20 kDa mol mass), there was little or no effect on the fraction of moving filaments, whereas sliding speed decreased in inverse proportion to viscosity. The results could be explained neither by an effect of osmotic pressure at high solute concentrations nor by altered solvent drag on the actin filament. Elevated viscosity inhibited HMM ATPase activity in solution, but only at much higher viscosities than were needed to reduce sliding speed. Polyethylene glycols (300, 1,000, or 3,000 mol wt) also inhibited speed via elevated viscosity but secondarily inhibited by enhancing electrostatic interactions. These results demonstrate that a diffusion-controlled process intrinsic to cross-bridge cycling can be limiting to actomyosin function.

Contractile properties of rabbit psoas muscle fibres inhibited by beryllium fluoride.

The structure of truncated, recombinant Dictyostelium myosin motor domain complexed with Mg.ADP and slowly dissociating analogues of Pi has previously been characterized as two main states (S1-MgADP plus BeFx vs. A1F4- or Vi). The BeFx bound state is thought to mimic the weak actin-binding M.ATP complex, while the states with A1F4- or Vi bound mimic the M.ADP.Pi state. While the effects of A1F4- and Vi on fibre mechanics have been previously described (Chase et al., 1994, 1993), the effects of BeFx have not been characterized in detail. At pCa 4.5 (12 degrees C), we measured (i) steady-state isometric tension, (ii) stiffness (KS; 1 kHz sinusoids), and (iii) unloaded shortening velocity (Vu; slack test) in single skinned muscle fibres from rabbit psoas. Results were compared when tension was inhibited with either BeFx or 2,3-butanedione-monoxime (BDM) or modulated by altering myoplasmic [Ca2+]. With 3 mM total fluoride, 1 mM BeFx inhibited both tension and KS by approximately 50% (compared to 7-10 mM BDM and 50-100 microM A1F4-). Increasing [BeFx] to 10 mM further reduced tension to approximately 15% P0, but had little further effect on KS; with BDM and altered [Ca2+], KS scaled more proportionately with tension. Inhibition of tension and KS by BeFx was more rapidly reversible, compared with slow recovery from tension inhibition with A1F4- or Vi. Vu exhibited a complex dependence on [BeFx], being relatively unaffected by concentrations < or = 1 mM, and becoming inhibited steeply for [BeFx] above this level. With BDM, Vu co-varied more directly with force. Our results suggest that BeFx may induce a different cross-bridge state in fibres than do A1F4- or Vi, but all three analogues of Pi form complexes that mimic crossbridge states that follow ATP hydrolysis.

Regulation of skeletal muscle tension redevelopment by troponin C constructs with different Ca2+ affinities.

In maximally activated skinned fibers, the rate of tension redevelopment (ktr) following a rapid release and restretch is determined by the maximal rate of cross-bridge cycling. During submaximal Ca2+ activations, however, ktr regulation varies with thin filament dynamics. Thus, decreasing the rate of Ca2+ dissociation from TnC produces a higher ktr value at a given tension level (P), especially in the [Ca2+] range that yields less than 50% of maximal tension (Po). In this study, native rabbit TnC was replaced with chicken recombinant TnC, either wild-type (rTnC) or mutant (NHdel), with decreased Ca2+ affinity and an increased Ca2+ dissociation rate (koff). Despite marked differences in Ca2+ sensitivity (>0.5 DeltapCa50), fibers reconstituted with either of the recombinant proteins exhibited similar ktr versus tension profiles, with ktr low (1-2 s-1) and constant up to approximately 50% Po, then rising sharply to a maximum (16 +/- 0.8 s-1) in fully activated fibers. This behavior is predicted by a four-state model based on coupling between cross-bridge cycling and thin filament regulation, where Ca2+ directly affects only individual thin filament regulatory units. These data and model simulations confirm that the range of ktr values obtained with varying Ca2+ can be regulated by a rate-limiting thin filament process.

Ca2+ and cross-bridge-induced changes in troponin C in skinned skeletal muscle fibers: effects of force inhibition.

Changes in skeletal troponin C (sTnC) structure during thin filament activation by Ca2+ and strongly bound cross-bridge states were monitored by measuring the linear dichroism of the 5' isomer of iodoacetamidotetramethylrhodamine (5'IATR), attached to Cys98 (sTnC-5'ATR), in sTnC-5'ATR reconstituted single skinned fibers from rabbit psoas muscle. To isolate the effects of Ca2+ and cross-bridge binding on sTnC structure, maximum Ca2+-activated force was inhibited with 0.5 mM AlF4- or with 30 mM 2,3 butanedione-monoxime (BDM) during measurements of the Ca2+ dependence of force and dichroism. Dichroism was 0.08 +/- 0.01 (+/- SEM, n = 9) in relaxing solution (pCa 9.2) and decreased to 0.004 +/- 0.002 (+/- SEM, n = 9) at pCa 4.0. Force and dichroism had similar Ca2+ sensitivities. Force inhibition with BDM caused no change in the amplitude and Ca2+ sensitivity of dichroism. Similarly, inhibition of force at pCa 4.0 with 0.5 mM AlF4- decreased force to 0.04 +/- 0.01 of maximum (+/- SEM, n = 3), and dichroism was 0.04 +/- 0.03 (+/- SEM, n = 3) of the value at pCa 9.2 and unchanged relative to the corresponding normalized value at pCa 4.0 (0.11 +/- 0.05, +/- SEM; n = 3). Inhibition of force with AlF4- also had no effect when sTnC structure was monitored by labeling with either 5-dimethylamino-1-napthalenylsulfonylaziridine (DANZ) or 4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). Increasing sarcomere length from 2.5 to 3.6 microm caused force (pCa 4.0) to decrease, but had no effect on dichroism. In contrast, rigor cross-bridge attachment caused dichroism at pCa 9.2 to decrease to 0.56 +/- 0.03 (+/- SEM, n = 5) of the value at pCa 9. 2, and force was 0.51 +/- 0.04 (+/- SEM, n = 6) of pCa 4.0 control. At pCa 4.0 in rigor, dichroism decreased further to 0.19 +/- 0.03 (+/- SEM, n = 6), slightly above the pCa 4.0 control level; force was 0.66 +/- 0.04 of pCa 4.0 control. These results indicate that cross-bridge binding in the rigor state alters sTnC structure, whereas cycling cross-bridges have little influence at either submaximum or maximum activating [Ca2+].

Compliant realignment of binding sites in muscle: transient behavior and mechanical tuning.

The presence of compliance in the lattice of filaments in muscle raises a number of concerns about how one accounts for force generation in the context of the cross-bridge cycle--binding site motions and coupling between cross-bridges confound more traditional analyses. To explore these issues, we developed a spatially explicit, mechanochemical model of skeletal muscle contraction. With a simple three-state model of the cross-bridge cycle, we used a Monte Carlo simulation to compute the instantaneous balance of forces throughout the filament lattice, accounting for both thin and thick filament distortions in response to cross-bridge forces. This approach is compared to more traditional mass action kinetic models (in the form of coupled partial differential equations) that assume filament inextensibility. We also monitored instantaneous force generation, ATP utilization, and the dynamics of the cross-bridge cycle in simulations of step changes in length and variations in shortening velocity. Three critical results emerge from our analyses: 1) there is a significant realignment of actin-binding sites in response to cross-bridge forces, 2) this realignment recruits additional cross-bridge binding, and 3) we predict mechanical behaviors that are consistent with experimental results for velocity and length transients. Binding site realignment depends on the relative compliance of the filament lattice and cross-bridges, and within the measured range of these parameters, gives rise to a sharply tuned peak for force generation. Such mechanical tuning at the molecular level is the result of mechanical coupling between individual cross-bridges, mediated by thick filament deformations, and the resultant realignment of binding sites on the thin filament.

Calcium regulation of tension redevelopment kinetics with 2-deoxy-ATP or low [ATP] in rabbit skeletal muscle.

The correlation of acto-myosin ATPase rate with tension redevelopment kinetics (k(tr)) was determined during Ca(+2)-activated contractions of demembranated rabbit psoas muscle fibers; the ATPase rate was either increased or decreased relative to control by substitution of ATP (5.0 mM) with 2-deoxy-ATP (dATP) (5.0 mM) or by lowering [ATP] to 0.5 mM, respectively. The activation dependence of k(tr) and unloaded shortening velocity (Vu) was measured with each substrate. With 5.0 mM ATP, Vu depended linearly on tension (P), whereas k(tr) exhibited a nonlinear dependence on P, being relatively independent of P at submaximum levels and rising steeply at P > 0.6-0.7 of maximum tension (Po). With dATP, Vu was 25% greater than control at Po and was elevated at all P > 0.15Po, whereas Po was unchanged. Furthermore, the Ca(+2) sensitivity of both k(tr) and P increased, such that the dependence of k(tr) on P was not significantly different from control, despite an elevation of Vu and maximal k(tr). In contrast, lowering [ATP] caused a slight (8%) elevation of Po, no change in the Ca(+2) sensitivity of P, and a decrease in Vu at all P. Moreover, k(tr) was decreased relative to control at P > 0.75Po, but was elevated at P < 0.75Po. These data demonstrate that the cross-bridge cycling rate dominates k(tr) at maximum but not submaximum levels of Ca(2+) activation.

Effect of viscosity on mechanics of single, skinned fibers from rabbit psoas muscle.

Muscle contraction is highly dynamic and thus may be influenced by viscosity of the medium surrounding the myofilaments. Single, skinned fibers from rabbit psoas muscle were used to test this hypothesis. Viscosity within the myofilament lattice was increased by adding to solutions low molecular weight sugars (disaccharides sucrose or maltose or monosaccharides glucose or fructose). At maximal Ca2+ activation, isometric force (Fi) was inhibited at the highest solute concentrations studied, but this inhibition was not directly related to viscosity. Solutes readily permeated the filament lattice, as fiber diameter was unaffected by added solutes (except for an increased diameter with Fi < 30% of control). In contrast, there was a linear dependence upon 1/viscosity for both unloaded shortening velocity and also the kinetics of isometric tension redevelopment; these effects were unrelated to either variation in solution osmolarity or inhibition of force. All effects of added solute were reversible. Inhibition of both isometric as well as isotonic kinetics demonstrates that viscous resistance to filament sliding was not the predominant factor affected by viscosity. This was corroborated by measurements in relaxed fibers, which showed no significant change in the strain-rate dependence of elastic modulus when viscosity was increased more than twofold. Our results implicate cross-bridge diffusion as a significant limiting factor in cross-bridge kinetics and, more generally, demonstrate that viscosity is a useful probe of actomyosin dynamics.

Skeletal muscle regulatory proteins enhance F-actin in vitro motility.

Using an in vitro motility assay, we have investigated the effects of rabbit skeletal muscle regulatory proteins, troponin and tropomyosin, on the gliding of F-actin filaments or F-actin filaments containing these regulatory proteins. We demonstrate that Ca2+ does not affect the motility of F-actin gliding on HMM, but does in the presence of skeletal muscle tropomyosin and troponin. We conclude that Ca2+ affects motility through troponin because, like F-actin, F-actin-Tm filaments show no Ca(2+)-dependence to their gliding speeds. Furthermore, there is a large enhancement of the gliding speed (about 75%) in the presence of skeletal muscle tropomyosin, troponin + saturating Ca2+ over that seen with F-actin filaments. This enhancement is not due to the action of tropomyosin alone as skeletal muscle tropomyosin without troponin enhances the speed little (about 5%) over that of F-actin. Thus troponin confers Ca2+ sensitivity to the motility and, additionally, potentiates motility greatly along with tropomyosin in the presence of saturating Ca2+. When [HMM] is varied, the decline in speed of F-actin seen at low HMM density is changed little by tropomyosin in the F-actin-Tm filaments. These data show that the skeletal regulatory proteins interact with F-actin to enhance the interaction with HMM particularly in the presence of troponin and saturating Ca2+ and enhance the gliding speed in the in vitro motility assay as they potentiate the ATPase activity in the isolated proteins. This enhancement of speed in the motility assay cannot be ascribed to tropomyosin alone.

Evidence for platelet-activating factor receptor subtypes on human polymorphonuclear leukocyte membranes.

Platelet-activating factor (PAF) is a potent phospholipid mediator that acts through specific cell surface receptors. The existence of PAF receptor subtypes has been suggested by functional and radioligand binding studies in a variety of cells and tissues. This report addresses this issue more directly and demonstrates differences between specific PAF receptors in human polymorphonuclear leukocytes (PMNs) and COS-7 cells transfected with the cloned human PAF receptor gene. The presence of more than one receptor in human PMNs is supported by three different studies. First, the Kd from the saturation isotherms for the binding of [3H]WEB 2086 on PMNs was 7-fold larger (Kd = 29.2 nM) than the kinetic Kd (4.2 nM). Second, the pseudo-Hill slope determined from the saturation experiments with PMNs was significantly lower than unity (0.69 +/- 0.05 SEM), and the saturation Kd values for transfected COS-7 (Kd = 9.6 nM) and PMN membranes were significantly different. These results contrasted with those for the transfected COS-7 cells, which showed a Kd from the saturation isotherms similar to that of the kinetic Kd (3.2 nM) and a pseudo-Hill slope that was not different from 1.0. Third, when the radiolabeled ligand [3H]WEB 2086 was increased in concentration from 10 to 50 nM in inhibition experiments with the human PMN membranes, the Ki increased, indicative of binding mainly to receptors with lower affinity. These results suggest that PAF receptor subtypes exist in human PMNs based on distinct radioligand binding characteristics from the human cloned PAF receptor.

Calcium regulation of skeletal muscle thin filament motility in vitro.

Using an in vitro motility assay, we have investigated Ca2+ regulation of individual, regulated thin filaments reconstituted from rabbit fast skeletal actin, troponin, and tropomyosin. Rhodamine-phalloidin labeling was used to visualize the filaments by epifluorescence, and assays were conducted at 30 degrees C and at ionic strengths near the physiological range. Regulated thin filaments exhibited well-regulated behavior when tropomyosin and troponin were added to the motility solutions because there was no directed motion in the absence of Ca2+. Unlike F-actin, the speed increased in a graded manner with increasing [Ca2+], whereas the number of regulated thin filaments moving was more steeply regulated. With increased ionic strength, Ca2+ sensitivity of both the number of filaments moving and their speed was shifted toward higher [Ca2+] and was steepest at the highest ionic strength studied (0.14 M gamma/2). Methylcellulose concentration (0.4% versus 0.7%) had no effect on the Ca2+ dependence of speed or number of filaments moving. These conclusions hold for five different methods used to analyze the data, indicating that the conclusions are robust. The force-pCa relationship (pCa = -log10[Ca2+]) for rabbit psoas skinned fibers taken under similar conditions of temperature and solution composition (0.14 M gamma/2) paralleled the speed-pCa relationship for the regulated filaments in the in vitro motility assay. Comparison of motility results with the force-pCa relationship in fibers suggests that relatively few cross-bridges are needed to make filaments move, but many have to be cycling to make the regulated filament move at maximum speed.

Calmidazolium alters Ca2+ regulation of tension redevelopment rate in skinned skeletal muscle.

To examine if the Ca2(+)-binding kinetics of troponin C (TnC) can influence the rate of cross-bridge force production, we studied the effects of calmidazolium (CDZ) on steady-state force and the rate of force redevelopment (ktr) in skinned rabbit psoas muscle fibers. CDZ increased the Ca2(+)-sensitivity of steady-state force and ktr at submaximal levels of activation, but increased ktr to a greater extent than can be explained by increased force alone. This occurred in the absence of any significant effects of CDZ on solution ATPase or in vitro motility of fluorescently labeled F-actin, suggesting that CDZ did not directly influence cross-bridge cycling. CDZ was strongly bound to TnC in aqueous solutions, and its effects on force production could be reversed by extraction of CDZ-exposed native TnC and replacement with purified (unexposed) rabbit skeletal TnC. These experiments suggest that the method of CDZ action in fibers is to bind to TnC and increase its Ca2(+)-binding affinity, which results in an increased rate of force production at submaximal [Ca2+]. The results also demonstrate that the Ca2(+)-binding kinetics of TnC influence the kinetics of ktr.

Effect of intracellular pH on force development depends on temperature in intact skeletal muscle from mouse.

The cellular mechanism of muscle fatigue is still in debate. Opposite conclusions regarding the role of intracellular pH (pHi) in fatigue have been drawn from skinned fiber vs. isolated perfused muscle studies. Because these experiments are typically performed at different temperatures, we tested the hypothesis that temperature alters the effects of pH on force. Tetanic force of isolated mouse extensor digitorum longus was measured at temperatures between 13 and 25 degrees C in either normocapnia (5% CO2) or hypercapnia (25% CO2). Hypercapnia decreased pHi (monitored by 31P nuclear magnetic resonance spectroscopy) by the same amount at both 15 and 25 degrees C. However, inhibition of force by hypercapnia was greater at the lower temperature. A similar pattern of temperature-dependent inhibition of force by pH was observed in glycerinated fibers from rabbit psoas at maximum Ca2+ activation. We conclude that temperature differences are responsible for disparate conclusions on the role of pHi in muscle fatigue. Based on our results, we suggest that changes in pHi may have little or no role in the loss in force production associated with muscular fatigue at physiological temperatures.

Regional mapping of the human platelet-activating factor receptor gene (PTAFR) to 1p35-->p34.3 by fluorescence in situ hybridization.

The human platelet-activating factor cell-surface receptor (PTAFR) is a G protein-coupled receptor thought to contribute to many atopic and inflammatory diseases and, perhaps, to the growth of some neoplasms. Exploring the possibility that the PTAFR might be involved in the genetic predisposition to any disease requires knowledge of its chromosomal localization. In this paper we have used a 20-kb human genomic fragment containing the coding sequence of the cloned PTAFR to determine the regional chromosomal localization of the gene. Using fluorescence in situ hybridization, the localization of the human PTAFR gene was mapped to 1p35-->p34.3.