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Transient co-transfection of receptor cDNA and suitable reporter genes was used to study human glucocorticoid receptor (hGR) function in a neutral mammalian cell background. A variety of natural and synthetic steroids were analyzed for their ability to activate gene expression through the hGR and to bind to extracts of cells expressing the hGR cDNA. There was very good correlation between these two in vitro parameters for these compounds. Furthermore, correlation of these data with reported in vivo anti-inflammatory potencies was surprisingly close, with two exceptions. The in vitro data suggest an explanation for the discrepant compounds, consistent with published data on their metabolic fate in vivo. The co-transfection assay has utility as a quantitative predictor of in vivo glucocorticoid pharmacology.

All-trans retinoic acid (RA) has previously been shown to modulate the transcriptional properties of the retinoic acid receptor (RAR) and retinoid X receptor (RXR). The inability of all-trans RA to bind to RXR suggests that it may be metabolized to a more active high affinity ligand. We report here an experimental approach that has identified 9-cis RA as an RXR ligand. It is up to 40-fold more potent than all-trans RA in transfection assays and binds with high affinity. The production of 9-cis RA in cultured cells and the identification of this molecule in liver and kidney demonstrates the existence of this molecule in living organisms. The discovery of this novel hormone points to the key role retinoid metabolism may have in generating new signaling pathways.

Locomotion was compared in an intact and in-vitro preparation of the adult mudpuppy (Necturus maculatus). The intact animals walked on an aquatic treadmill while in-vitro preparations were made to walk with a bath application of the excitatory amino acid NMA (N-methyl DL-aspartate). EMG recordings of shoulder muscles (pectoralis, latissimus dorsi, dorsalis scapulae, and procoracohumeralis) and elbow muscles (brachialis and extensor ulnae) were obtained from intact animals while recordings were made from only the elbow muscles in-vitro. The in-vitro preparation required magnesium in the bath to initiate and maintain locomotion, consistent with an NMDA mediated response. Also consistent with an NMDA response was the finding that glycine potentiated the NMA induced locomotion in-vitro. The range of cycle durations seen in-vitro was well within the range seen in the intact animal, while gait analysis demonstrated the similarity of intact and in-vitro locomotor cycles. In spite of these very similar locomotor patterns there are interesting differences in the patterned output seen in-vitro, such as the absence of a second burst in the brachialis muscle in-vitro.

The relationship between motor unit force and the recorded voltage produced by activated muscle unit fibres (electromyogram, EMG) was examined in normal and reinnervated rat tibialis anterior muscles. The number, cross-sectional area, and radial distance from the recording electrode of muscle fibres in a given unit, obtained directly from a sample of glycogen-depleted motor units, were analysed in relation to the magnitude of the EMG signal produced by that unit. EMG peak to peak amplitude and area varied as approximately the square root of twitch force in both normal and reinnervated units. Furthermore, the EMG amplitude increased approximately as the total cross-sectional area of the motor unit (number of muscle fibres x the average cross-sectional area of the fibres) and inversely with approximately the square root of the distance of fibres from the recording electrodes on the surface of the muscle.

Hoffman (H) reflexes were elicited from the soleus muscle during treadmill walking in 21 spastic paretic patients. The soleus and tibialis anterior muscles were reciprocally activated during walking in most patients, much like that observed in healthy individuals. The pattern of H-reflex modulation varied considerably between patients, from being relatively normal in some patients to a complete absence of modulation in others. The most common pattern observed was a lack of H-reflex modulation through the stance phase and slight depression of the reflex in the swing phase, considerably less modulation than that of normal subjects under comparable walking conditions. The high reflex amplitudes during periods of the step cycle such as early stance seems to be related to the stretch-induced large electromyogram bursts in the soleus in some subjects. The abnormally active reflexes appear to contribute to the clonus encountered during walking in these patients. In three patients who were able to walk for extended periods, the effect of stimulus intensity was examined. Two of these patients showed a greater degree of reflex modulation at lower stimulus intensities, suggesting that the lack of modulation observed at higher stimulus intensities is a result of saturation of the reflex loop. In six other patients, however, no reflex modulation could be demonstrated even at very low stimulus intensities.

In an attempt to define the molecular basis of the functional diversity of K+ channels, we have isolated overlapping rat brain cDNAs that encoded a neuronal delayed rectifier K+ channel, K,4, that is structurally related to the Drosophila Shaw protein. Unlike previously characterized mammalian K+ channel genes, which each contain a single protein-coding exon, K,4 arises from alternative exon usage at a locus that also encodes another mammalian Shaw homolog, NGK2. Thus, the enormous diversity of K+ channels in mammals can be generated not just through gene duplication and divergence but also through alternative splicing of RNA.

In crosslinking experiments, [125I]endothelin-1 was treated with N-hydroxysuccinimidyl-4-azidobenzoate, purified by HPLC, allowed to bind to bovine aortic membranes and then photoactivated. Autoradiography of sodium dodecyl sulfate polyacrylamide gel electrophoretograms of the products of this reaction showed that a component of apparent Mr = 42,000 was specifically labelled by endothelin-1 under reducing conditions. Under nonreducing conditions, a small amount of 125I-labelled endothelin-1 specifically labelled a component of apparent Mr = 45,900 in the absence of crosslinking agent. Non-radiolabelled endothelin analogues with a wide range of binding affinities inhibited specific labelling of the Mr = 42,000 and 45,900 components in parallel over the concentration ranges which inhibited binding of radiolabelled endothelin. Specific labelling of these components was also observed in parallel in membranes from bovine heart and kidney. The components labelled in the presence and absence of crosslinker appear to be the same, and the small difference in apparent Mr in the labelled components is likely due to a difference in conformational constraints arising from the two labelling processes, with a true, corrected Mr of 43,400. Since the specific labelling of this component is related to physiologically relevant binding in several bovine tissues, we conclude that it is a component of the bovine endothelin receptor.

The effects of E4031, a new class III antiarrhythmic agent similar to sotalol, were tested in isometrically contracting rabbit papillary muscles and in anesthetized, open-chest dogs. In papillary muscles, E4031 caused a modest dose-dependent increase of 26 +/- 8% in developed tension and 38 +/- 8% in its maximal rate of rise. Since there was no significant change in the maximal rate of relaxation, the ratio between both maximal velocities increased from 0.92 +/- 0.03 to 1.19 +/- 0.10. Time to peak tension did not change significantly, whereas time to half relaxation increased from 72 +/- 3 to 85 +/- 4 ms. The effective refractory period in the rabbit papillary muscles increased from 179 +/- 10 to 414 +/- 45 ms. In the open-chest dog, the i.v. administration of E4031 did not induce significant changes in heart rate, mean arterial pressure, or left ventricular end diastolic pressure. +dP/dt increased from 1,839 +/- 162 to 2,470 +/- 247 mm Hg/s with no significant change in -dP/dt after 100 micrograms/kg of E4031. Consequently, (+dP/dt)/(-dP/dt) increased from 0.97 +/- 0.07 to 1.18 +/- 0.08. To further evaluate the effects of E4031 on myocardial relaxation, the time constant of isovolumic left ventricular pressure decay was measured by two different methods (tau 1 and tau 2) before and after administering 10 micrograms/kg E4031. Tau 1 increased from 27 +/- 1.8 to 33 +/- 1.6 ms and tau 2 increased from 30 +/- 2.3 to 41 +/- 3.3 ms.(ABSTRACT TRUNCATED AT 250 WORDS)

Most models of muscle have assumed that the series elastic element has a constant stiffness, although experimentally this is not true, either at the level of individual cross-bridges or that of the whole muscle. In this paper elastic and viscous elements are introduced whose properties vary systematically with activity levels as has been found experimentally. The effects of these nonlinear variations on the methods for optimally controlling a pair of antagonistic muscles are calculated. Although the nonlinearities add some complexity to the derivation and are important to the mechanism of force generation in the muscle, they do not qualitatively change the overall control of movement.

Control of an active above-knee prosthesis has been simulated for a selected gait activity using a hierarchical closed-loop method. An extension of finite-state control, referred to as artificial reflex control, was adopted at the strategic level of control. At the actuator level of control an optimal tracking method, based on dynamic programming, is applied. This deals mainly with the actuator level of control, but considers the interaction of the leg dynamics and the switching effects of artificial reflex control. Optimal tracking at the actuator level of the above-knee prosthesis reduces the on-off effects of finite-state methods, such as artificial reflex control. The proposed method can also be used for the design of prosthetic elements. Specific attention is paid to the limited torque and power in the prosthetic joint actuator, which are imposed by the principle of self-containment in the artificial leg. The hierarchical structure, integrating artificial reflex control and optimal tracking, can be used in real time, as estimated from the number of computer operations required for the suggested method.

Small, rapid stretches were applied to the soleus muscle during the stance phase of walking by lifting the forefoot with a pneumatic device. Stretch responses were induced in the soleus muscle by the disturbance. The amplitude and time course of the responses from the soleus muscle were a function of both the kinematics of the disturbance and the time in the step cycle when the disturbance was applied. The step cycle was divided into 16 equal time parts, and data obtained within each of these parts were averaged together. The electromyographic (EMG) response of the soleus muscle showed a time course that was similar to the time course of the angular velocity induced by the disturbance at the ankle. Three linear equations were used to predict the EMG response from the soleus muscle as a function of the angular kinematics of the disturbance: 1) velocity, 2) velocity and displacement, 3) velocity, displacement and acceleration. Introduction of a pure delay between the EMG and the kinematics substantially improved the predictions. Most of the variance (70%) in the EMG response could be accounted for by the velocity of the disturbance alone with an optimal delay (average 38 ms). Inclusion of a displacement term significantly increased the variance accounted for (85%), but further addition of an acceleration term did not. Since the velocity of the disturbance accounted for most of the variance, the reflex gain was estimated from the velocity coefficient. This coefficient increased in a ramp-like fashion through the early part of the stance phase, qualitatively similar to the increase in the H-reflex.(ABSTRACT TRUNCATED AT 250 WORDS)

The number of motor units in the thenar muscle group was calculated by dividing the surface electromyogram and twitch force, in maximal stimulation of the median nerve, by estimates of the average electromyogram and twitch force from single units. The following three techniques were used to estimate the average electromyogram from single units: spike-triggered averaging from units recorded with a needle electrode, intramuscular microstimulation of motor nerve branches, and graded whole nerve stimulation at the wrist. The first two techniques also provided independent estimates of motor unit numbers based on the average force generated by single units. The five estimates (three based on the electromyogram and two on force) ranged from 116 to 170 motor units in the thenar group. Correcting for cancellation when unit responses sum to form the compound action potential or twitch increased the estimated number of units, which ranged from 130 to 179. The estimates were not statistically different from one another but were substantially lower than some previous electrophysiological estimates based on graded whole nerve stimulation. The recruitment pattern of single units during whole nerve stimulation was recorded and simulated mathematically. The most likely reason for the higher estimates in previous studies using graded whole nerve stimulation is shown to be alternation of motor units. Potential errors in all the techniques are discussed and compared.

The electrical and contractile properties of hand muscles in a selected population of quadriplegic subjects were studied intensively before and after EMG biofeedback. Spontaneously active motor units and units that could only be slowly and weakly activated were observed in these subjects, in addition to units that were voluntarily activated normally. This suggests a considerable overlap of surviving motor neurons to a single muscle that are below, near or above the level of a lesion. Despite the common occurrence of polyphasic potentials and other signs of neuromuscular reinnervation, the average twitch tension of single motor units in hand muscles of quadriplegic subjects was not significantly different from that in control subjects. Nor did it increase after biofeedback training that typically increased the peak surface EMG by a factor of 2-5 times. The percentage of spontaneously active units was also constant. The surface EMG may be increased during biofeedback by using higher firing rates in motor units that can already be activated, rather than by recruiting previously unavailable motor units.

The number of motor units in the thenar muscle group was estimated in 11 patients with cervical spinal cord injuries. The surface electromyogram and twitch force, in response to maximal stimulation of the median nerve was divided by the average surface electromyogram and twitch of single units. The average single unit size was obtained by intramuscular microstimulation of motor nerve branches and by graded whole nerve stimulation, which provided three independent estimates, two based on the electromyogram and one based on force. The motor unit estimates from the patients covered a wide range. Some had essentially normal motor units both in numbers and contractile properties, while others had varying reductions in numbers of units. Those patients who showed a large reduction in motor unit numbers also had greatly enlarged units, which produced an average of up to sixfold the normal force. These enlarged units summed to produce maximal compound action potentials and twitches that were sometimes indistinguishable from normal. Magnetic resonance imaging scans of the cervical spine obtained from some patients provided independent evidence that patients with low motor unit counts had sustained direct injury to the anterior aspect of the spinal cord at the relevant segmental levels. Some patients showed a normal number of motor units long after the injury. No evidence of transneuronal degeneration could be demonstrated in the thenar group in these patients with the current techniques.

In open-chest anesthetized dogs, the time constant of isovolumic left ventricular pressure decay increased following the intravenous administration of either E4031, a class III antiarrhythmic agent which acts by K+ channel blockade, or DPI 201-106 (DPI), a cardiotonic agent which acts by delaying Na+ channel inactivation. In addition to prolonging cardiac refractoriness, both E4031 and DPI increased left ventricular +dP/dt but without significantly altering -dP/dt. Consequently, the value of the ratio (+dP/dt)/(-dP/dt) increased. There were no significant changes in heart rate, mean arterial pressure, or left ventricular end diastolic pressure. Since both E4031 and DPI prolonged the action potential duration (APD) and the refractory period, and slowed relaxation in vivo, the possibility of a causal link between these effects was further investigated under in vitro conditions. In isometrically contracting rabbit papillary muscles, E4031 and DPI increased peak developed tension (DT) and its maximal rate of rise (+T). Since the maximal rate of fall of DT (-T) did not increase by the same factor that +T increased, the value of the ratio +T/-T increased. Time to half relaxation increased, whereas time to peak tension was not significantly changed by either E4031 or DPI. These negative lusitropic effects produced by E4031 or DPI were not observed when equivalent increases in contractility were produced by increasing the extracellular Ca2+ concentration. The effective refractory period measured in the papillary muscles increased following superfusion with either of the two drugs, consistent with their known ability to increase APD. A causal link between the prolongation of APD and the negative lusitropic effects of E4031 and DPI is postulated as the possible mechanism.

1. The extent to which an active, human motoneuron pool can be inhibited via short-latency inhibitory pathways was studied by stimulating the common peroneal nerve and recording the inhibition of on-going soleus electromyographic (EMG) activity. The responses were compared at the same EMG level during walking and tonic voluntary activity to determine whether the inhibition was task dependent. 2. In both tasks the amount of inhibition (measured as the depression in rectified, filtered, and averaged EMG activity) increased approximately linearly with the amount of motor activity, as determined from the mean EMG level before stimulation (correlation coefficient greater than or equal to 0.9). No difference in the amount of inhibition was found between the two tasks at the same stimulus and EMG levels. 3. Previously published studies based on the H-reflex method have reported that the amount of inhibition decreases with the amount of motor activity. On the contrary, single-unit studies and the present results suggest that segmental inhibitory reflexes retain their capacity to mediate a rapid reduction of motoneuronal discharge during voluntary activity. This inhibition may be important in regulating the amount of activity early in the stance phase of walking and during the transition from stance to the swing phase. 4. Analytic results are derived in an APPENDIX that should be of general interest in interpreting the inhibition of motor units from a peristimulus time histogram (PSTH). The linear correlation between inhibition and level of voluntary activity can be explained if newly recruited units are strongly inhibited by the stimulus, whereas previously active motor units are inhibited relatively less, as their firing rate increases with increasing background activity.

The antiarrhythmic actions of high-dose intravenous (i.v.) lidocaine infusions were assessed in conscious dogs with spontaneous ventricular ectopy subacutely (48 h) after anterior myocardial infarction and in anesthetized dogs with ventricular tachyarrhythmias inducible by programmed ventricular stimulation at 4-11 days after anterior myocardial infarction. In conscious dogs administered cumulative doses of lidocaine at 48 h after myocardial infarction, a significant reduction in the frequency of spontaneous ventricular ectopic complexes (from 61 +/- 12 to 11 +/- 9% of total complexes) occurred only after administration of 10 mg/kg i.v. lidocaine. In anesthetized postinfarction dogs responding to baseline programmed stimulation with ventricular tachyarrhythmias, lidocaine administration (6 mg/kg i.v. loading dose + 100 micrograms/kg/min i.v. maintenance infusion) resulted in a selective increase in infarct zone conduction time (53.0 +/- 5.6 to 60.5 +/- 6.2 msec; p less than 0.05), increases in infarct zone relative refractory periods (RRPs 182 +/- 5 to 193 +/- 5 ms; p less than 0.05), and effective refractory periods (ERPs 156 +/- 4 to 165 +/- 3 ms; p less than 0.05), and an increase in noninfarct zone ERP (154 +/- 5 to 166 +/- 8 ms; p less than 0.05). The induction of ventricular arrhythmias by programmed stimulation was suppressed by lidocaine (6 mg/kg + 100 micrograms/kg/min i.v.) in 5 of 10 postinfarction animals tested, with an additional 3 animals displaying favorable stabilizations of induced arrhythmias.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Reflex responses during walking were elicited in humans by stimulation of the tibial nerve at the ankle. The stimulus intensity was controlled by monitoring the M-wave from an intrinsic foot muscle. Responses were observed in the ipsilateral tibialis anterior (TA), soleus (SO), and rectus femoris (RF) muscles. The most reproducible responses were observed at a middle latency between 50 and 90 ms. The responses were most likely of cutaneous origin, because they closely resembled the responses to stimulation of a purely cutaneous nerve, the sural nerve. 2. A reversal in the direction of the middle latency response from excitation to inhibition was observed for the first time within single muscles during walking. Evidence for a reversal was seen in all three muscles examined and in all seven subjects. 3. The reflex reversal could not be elicited in standing. An inhibition whose amplitude varied in a linear fashion with stimulus intensity and background activation level was always observed at middle latency. The responses elicited during standing resembled those during the stance phase of walking. The two tasks shared some common movement goals and appeared to make use of similar reflex pathways.

Electro-mechanical devices can help a variety of patients with motor disabilities. Surface EMG from remaining muscles in an amputated arm can be used to control powered electronic hands, wrists and elbows. Sensory signals such as knee angle and ankle torque can be used to control the visco-elastic properties of a knee joint for above-knee amputees. Finally, percutaneous electrodes can be used to stimulate paralyzed muscles to replace hand function in quadriplegics and leg function in paraplegics. This article summarizes recent progress in each of these areas.

The antiarrhythmic efficacy of a new and potent class III agent E4031 [1-[2-(6-methyl-2-pyridyl)-ethyl]-4-(4- methylsulfonylaminobenzoyl)piperidine] was evaluated in several canine models of recent myocardial infarction. In anesthetized dogs with baseline inducible ventricular arrhythmias studied 4-10 days after anterior myocardial infarction, 30-300 micrograms/kg i.v. E4031 suppressed induction of ventricular tachyarrhythmias by programmed ventricular stimulation in 7 of 10 animals tested, while significantly prolonging refractoriness in both noninfarcted and infarcted ventricular myocardium. The incidence of lethal ischemic ventricular arrhythmias developing in response to acute posterolateral myocardial ischemia in the presence of previous anterior infarction was reduced from 10 of 10 (100%) in a vehicle pretreatment group to 3 of 10 (30%, p less than 0.01) in an E4031 (300 micrograms/kg intravenously, i.v.) pretreatment group. Neither the sizes of the underlying anterior myocardial infarctions (26.9 +/- 3.7 vs. 33.2 +/- 2.1% of left ventricle) nor the times to development of acute posterolateral myocardial ischemia (43 +/- 11 vs. 40 +/- 8 min) differed significantly between the vehicle and E4031 pretreatment groups, respectively, suggesting that the reduction in the incidence of lethal ischemic arrhythmias in the E4031 pretreatment group was not due to smaller underlying, electrically unstable myocardial substrates nor to a delay in onset of the acute ischemic insult. In conscious dogs with spontaneous ventricular ectopy at 48 h after myocardial infarction and in anesthetized dogs with no baseline inducible arrhythmias at 4-10 days after myocardial infarction, E4031 (30-3,000 micrograms/kg i.v.) produced no facilitation or aggravation of spontaneous or inducible ventricular arrhythmias. These findings suggest that pharmacologic agents such as E4031 that increase ventricular refractoriness (class III electrophysiologic activity) may provide significant protection against development of malignant ischemic ventricular arrhythmias in the setting of previous myocardial infarction.

cDNAs encoding a delayed-rectifier-type K+ channel were cloned from both neonatal rat heart and ovariectomized, diethylstilbestrol-primed rat uterus by using the polymerase chain reaction. Both clones have nucleotide sequences identical to that encoding the rat kidney IsK channel [Takumi, T., Ohkubo, H. & Nakanishi, S. (1988) Science 242, 1042-1045] and encode a putative protein of 130 amino acids. Injection of RNA transcripts of the cDNAs into Xenopus oocytes resulted in the expression of a slowly activating, voltage-dependent K+ current. An antisense oligonucleotide, derived from the sequence of the clone, specifically inhibited the expression of the slow, outward current observed in cells injected with mRNAs isolated from the parent tissues (i.e., kidney, heart, and uterus), indicating that the cloned gene underlies the major K+ current expressed from RNA isolated from these tissues.

1. The two types of fusimotor neurones, dynamic and static, can be differentiated by their effects on muscle spindle afferents. We have recorded the activity of muscle spindle primary afferents from the intercostal nerves of anaesthetized or decerebrate cats. A 4 Hz sinusoidal stretch was applied to the muscle containing the spindles of interest before and after crushing the nerve proximal to the recording site to eliminate fusimotor effects. The relative activity of the dynamic and static fusimotor neurones was inferred from the change in the spindle afferents' response. 2. Some areas of intercostal muscle normally showed phasic activity linked to respiration, where as other areas of intercostal muscle showed no EMG activity under our experimental conditions. In areas of intercostal muscle lacking EMG activity, the afferents' mean rate was higher and the modulation around the mean was lower at all phases of the breathing cycle when the efferent supply was intact. This result suggests the muscle spindles were receiving a steady level of static fusimotor activity. 3. Spindle primary afferents from regions of intercostal muscle that were typically recruited during respiration had an additional increase in mean rate and modulation around the mean rate in phase with the EMG activity. This is suggestive of phasic activation of dynamic fusimotor neurones in addition to static fusimotor discharge. 4. Thus, the two types of fusimotor neurones can be activated separately by the CNS to control the sensitivity of muscle spindles. The regional differences in the recruitment patterns of fusimotor neurones parallels the functional specializations of different areas of the intercostal muscles. The temporal modifications of fusimotor activity during each respiratory cycle means that the segmental reflex gain will vary in those intercostal muscles that are active during respiration. 5. These findings regarding the CNS recruitment of the two types of fusimotor neurones during respiration are similar to those reported for the hindlimb extensors during locomotion, but differ from those reported for jaw muscles during chewing. This may reflect differing control strategies being used by the CNS to meet the unique demands of the various rhythmical movements.

Movements against a variety of loads are relatively invariant in form. These movements are controlled in general by antagonistic groups of muscles. In this paper optimal control strategies are computed for coupling antagonistic muscles so as to minimize deviations from a desired trajectory. Simulations are presented for linear and nonlinear "decision functions" linking control of the two muscles for a variety of movements in a way that may be compared with experimental observations.

Muscle stiffness increases faster than muscle force during the rising phase of a tetanic contraction, and decreases more slowly during the falling phase. Different models of the stiffness arising from series, parallels, and crossbridge elasticity were compared to determine whether they could account quantitatively for the observed time course of force and stiffness. Data for slow and fast twitch mouse muscles at temperatures from 6 to 37 degrees C (Stein and Gordon, Can. J. Physiol. Pharmacol. 64, 1236-1244, 1986) and for single frog muscle fibers (Cecchi et al., Contractile Mechanisms in Muscle, pp. 641-655. Plenum, New York, 1984) were compared. The results showed that a good fit to the data for mouse muscles could be obtained with a model in which: (1) a nonlinear series elasticity contributed significantly to stiffness; (2) the attached crossbridges went from a stiff, force-generating state to a stiff, non-force-generating state; and (3) the rate of transition between these two states increased abruptly at the onset of relaxation. The increased transition rate probably arises from the internal rearrangement in which some sarcomeres shorten at the expense of other sarcomeres, once the muscle begins to relax. A significant series elasticity was not required for the frog data, but a pre-tension state was then needed to obtain a good fit.

We have analyzed several lots of epidermal growth factor (EGF) purified from murine submaxillary glands including "receptor grade" EGF from Collaborative Research and EGF from Boehringer Mannheim Biochemicals. New England Nuclear uses "receptor grade" EGF to produce 125I-labeled EGF. Though these reagents are reported to be homogeneous, we found them to be a mixture of six species. A method was developed to separate this mixture into its component parts. The individual components were chemically characterized and tested for biological potency. N-terminal sequence analysis of the unfractionated EGF-mixture reveals three different sequences starting with residues 1, 2, or 3 of the mature peptide. Each component exhibited different degrees of mitogenic and EGF receptor binding activity indicating that the N-terminal region contributes to the biological response. The species representing the complete EGF peptide is the most active species in all biological assays. A rapid method for purification of homogeneous complete EGF from commercial EGF preparations is described.

Rodent fibroblasts infected with the ts371 Kirsten murine sarcoma virus (KiMuSV) are temperature sensitive for the maintenance of transformation because of the production of an abnormal p21 protein. We cloned the ts371 KiMuSV provirus from the genome of a conditionally transformed nonproducer cell line, ts371 KiMuSV NRK clone 5 (T. Y. Shih, M. O. Weeks, H. A. Young, and E. M. Scolnick, J. Virol. 31:546-556, 1979). The molecularly cloned virus had 1,000-fold lower transformed focus-forming activity at 39 degrees C than at 34 degrees C. The ts371-v-Ki-ras gene differed from the wild type (wt) by a single point mutation, resulting in the substitution of arginine for glutamine at amino acid residue 43 of the encoded p21. A second difference from the published sequence for wt v-Ki-ras (N. Tsuchida, T. Ryder, and E. Ohtsubo, Science 217:937-939, 1982) at amino acid residue 37 was found. However, on sequencing the wt v-Ki-ras in this region, we found that it also contained a glutamate at residue 37. Preliminary characterization of bacterially expressed wt and ts371-v-Ki-ras p21 proteins is discussed.

The contractile and electrical properties of motor units in the first dorsal interosseous muscle of the hand have been studied in 26 patients with ulnar neuropathies and motor neurone disease (amyotrophic lateral sclerosis). Among patients with unilateral pressure or entrapment ulnar neuropathies, there was a tendency for the twitch tensions for single motor units to be smaller, while the surface EMG amplitudes were generally larger in the affected hands. Very large EMG amplitudes but normal size twitch tensions were observed among the motor neurone disease patients, indicating that, in general, motor units enlarged by sprouting are less efficient contractile units than units of normal physiological size.

An analysis of the reflex output of a motoneuron pool in response to a Ia-afferent input is presented. The analysis is based upon a model of the motoneuron pool which includes the subthreshold behavior of motoneurons (integration of synaptic inputs) and the statistical distribution of the motoneurons according to their resting conductance. The latter feature allows for the orderly recruitment of the motoneurons in the order of low resting conductance to high resting conductance. The number of active motoneurons (i.e. the excitation level) is determined by the balance of the excitatory and inhibitory conductances acting on the pool. The reflex output in response to a Ia-EPSP is computed at various excitation levels and with different amounts of presynaptic inhibition. The reflex output is the same for a given excitation level, regardless of the mixture of excitatory and inhibitory postsynaptic conductances used to produce that excitation level. In contrast, presynaptic inhibition markedly affects the relation between reflex output and excitation level.

The force produced by cat muscles over time with two stimuli separated by a short interval is approximately three times that produced by a twitch of cat muscles. This facilitation of force production by a second stimulus involves both increases in magnitude and duration of the contraction. Increased magnitude is relatively more important in the fast-twitch plantaris muscle, whereas increased duration is more important in the slow-twitch soleus muscle. The facilitation decays in an approximately exponential manner with the interval between stimuli, having a time constant between one and two times the twitch contraction time in different muscles. If a third stimulus is added, the greatest facilitation is seen at intervals longer than the twitch contraction time. The drug Dantrolene, which specifically reduces Ca++ release from the sarcoplasmic reticulum, eliminates the delayed peak in facilitation with three stimuli. Associated with the increases in force with one or more stimuli are increases in muscle stiffness, which can be measured with small, brief stretches and releases that do not alter the time-course of contraction. The stiffness of soleus muscle reaches a peak after the peak in force. The increasing stiffness of the muscle can considerably facilitate transmission of force generated internally, in addition to any facilitation arising from Ca++-release mechanisms.

The atrophy of cutaneous (sural) and muscle (medial gastrocnemius) nerves proximal to a ligation were studied in cats for periods up to 9 months, using light and electron microscopy, conduction velocity measurements and computer simulations. As atrophy proceeds, nerve fibres become increasingly non-circular. Cross-sectional areas of axons and fibres (axon + myelin) were measured. The diameters of equivalent circles (having the same axon and fibre cross-sectional area) were then calculated. A linear relation was found between axon diameter and fibre diameter, but the slope decreased as atrophy continued. This indicates that the axon cross-sectional area decreases relatively more than the total fibre area. Reduction in conduction velocity correlates more closely with reduction in axon diameter than fibre (axon + myelin) diameter. The ratio of the inner (axon) perimeter to the outer (myelin) perimeter remains constant at or near the optimal value of 0.6 for conduction in all groups of fibres at all periods of atrophy. Furthermore, the thickness of the myelin remains constant for a given perimeter over the entire period of atrophy studied. This suggests that the number of turns of myelin and the length of each turn remain unchanged during peripheral nerve atrophy. A simple geometric model explains how this can occur without gaps developing between the axon and myelin or between the turns of myelin. The Frankenhaeuser-Huxley equations for conduction in myelinated nerve fibres predict changes in conduction velocity similar to those observed, if the axons atrophy without changes in myelin. The advantages of this mode of atrophy are discussed.

Neural cuffs, implanted around various hindlimb nerves (sural, common peroneal, posterior tibial), were used to deliver brief stimulus trains to unrestrained cats walking on a treadmill. The resulting perturbations of the step cycle were evaluated by analyzing the EMG bursts recorded from the ankle extensors and by high speed cinematography. It was found that relatively weak stimulation (1.4 to 2 X T) of the posterior tibial nerve was very effective in eliciting a prolongation of the flexion phase provided the stimuli were applied just prior to the expected onset of the ankle extensor EMG burst. This ipsilateral hyperflexion was correlated with a prolongation of the contralateral extension. The same stimuli applied during stance usually evoked a yielding of the stimulated leg and a prolongation of the ongoing contralateral stance. In addition to these flexor and extensor reflex effects, it was found that low threshold stimulation of the sural and common peroneal nerves resulted in a powerful reflex activation of the ankle extensors. In contrast, stimulation of the gastrocnemius-soleus nerve (a muscle nerve) produced no discernible behavioral effects, even for stimuli at 3 X T, indicating that the observed reflexes are probably mediated by cutaneous afferents. The results were largely confirmed in experiments using the same cuffs implanted in spontaneously walking premammilliary cats.

Recently, we developed a model based on experimental data, which includes a pair of antagonistic muscles, a general load against which the muscles act, feedback pathways from muscle sense organs and spinal inhibitory circuits involving 1A interneurons and Renshaw cells. Descending inputs can activate the model through combinations of inputs to alpha-motoneurons, gamma-motoneurons (via intrafusal muscles and their feedback pathways) or the 1A interneurons. The role of each of these connections is analysed here in terms of its effect on the response of the muscles to impulse inputs, with particular interest in the effects on the overall stability of the systems. Increasing muscle stiffness or feedback from muscle receptors tends to produce high frequency oscillations. Coactivation of alpha- and gamma-motoneurons can lead to cancellation of oscillations, because of delays in the effects of gamma-motoneurons on contraction. Connections of 1A inhibitory interneurons onto antagonist motoneurons accentuate the oscillations. Inhibitory connections from Renshaw cells onto alpha-motoneurons tend to prevent oscillations, whereas the concentrations onto gamma-motoneurons may produce them.

A patient who had been treated with large doses of thyroid hormone for several years developed features of secondary hypothyroidism after thyroid hormone withdrawal. These findings were low serum T4 (3.8 micrograms/dl), T3 (23 ng/dl), and a failure of serum TSH to rise after TRH injection. Serum PRL values rose normally after TRH administration, and evaluation of other pituitary hormones was normal. When retested 3 months later, at which time the serum T4 was 5.5 micrograms/dl, the patient was somewhat less hypothyroid and there was an exaggerated TSH response to exogenous TRH, indicating recovery of pituitary TSH reserve. Indirect assessment of endogenous TRH reserve capacity was consistent with impairment of endogenous TRH activity. Repeat studies performed 7 months later indicated some improvements in this indirect assessment of endogenous TRH reserve capacity but a continued exaggerated TSH response to exogenous TRH administration. Further testing at 28 months revealed a serum T4 value of 7.8 micrograms/dl and a serum T3 value of 141 ng/dl. At this time, the TSH response to TRH was normal and the patient was considered fully recovered. A causal relationship between high doses of thyroid hormone and the presumptive impairment of endogenous TRH reserve is suggested.

Medial gastrocnemius and sural nerves in one hindlimb of the cat were transected and prevented from regenerating. After periods ranging from 29-273 days, compound action potentials were recorded from axotomised and contralateral control nerves. The amplitude and integrated area of action potentials decreased and conduction velocity slowed following axotomy. The area under compound action potentials generated by stimulating sensory fibres declined significantly faster than that generated by stimulating motor fibres. Analysis of changes in whole nerve conduction velocity distributions showed that the velocities of fast conducting sensory fibres decreased at the most rapid rate. The conduction velocities of motor fibres and slow sensory fibres declined at significantly slower rates. The loss of electrical activity in the largest sensory nerve fibres following axotomy, may play a role in determining the faster rate at which their action potentials deteriorate.

1. Physiological and histochemical properties of the cat ankle extensor muscles, the lateral and medial gastrocnemius, and the soleus were studied after cross-reinnervation by flexor motoneurons. 2. Tibial and common peroneal nerves were cut and cross-united in the popliteal fossa of 2- to 6-mo-old cats. Eighteen to 24 mo later, single motor units were isolated by dissection and stimulation of ventral root filaments and classified into four types: fast-twitch, fatigable (FF), fast-twitch with intermediate fatigue resistance (FI), fast-twitch, fatigue-resistant (FR), and slow, fatigue-resistant (S). Muscle fibers were classified as fast glycolytic (FG), fast, oxidative glycolytic (FOG), and slow oxidative (SO) on the basis of histochemical staining. 3. Although motor-unit force was normally well correlated with the size of the innervating motor axon in the cross-reinnervated muscles, the force of different unit types overlapped considerably. The reinnervated motor units also showed a higher than normal degree of fatigability. 4. The range of muscle unit forces in cross-reinnervated triceps surae muscles was the same as in the normally innervated triceps surae muscles. This range is 2-3 times greater than the flexor muscles, which the common peroneal nerve normally supplies. The range of contraction speed of units in the cross-reinnervated extensor muscles was comparable to that in the flexor muscles, consistent with a motoneuron-specific determination of muscle speed (28). 5. SO and FOG muscle fibers were found in all reinnervated triceps surae muscles, but FG fibers were only found in reinnervated medial gastrocnemius (MG) and lateral gastrocnemius (LG) muscles, consistent with previous findings of the resistance of soleus muscles to complete conversion (10, 16, 20, 21). Type grouping of muscle fibers was characteristic of the reinnervated muscles. 6. Reinnervated SO muscle fibers were larger than the corresponding fibers in normally innervated muscles as were the estimated number of muscle fibers innervated by slow motor axons. Nonetheless, the force generated by the S motor units remained relatively smaller than FR and FF units. The relative contributions of the number, cross-sectional area and specific tension to the force generation of reinnervated motor units are discussed.

The purpose of this study was to examine whether a microneurography electrode could record from and then selectively stimulate the same afferent fiber in cat sural and tibial nerves. Fiber activity was recorded distally with microneurography needle electrodes and proximally with hook electrodes. Records from the hook electrodes allowed the waveform shape and latency following natural stimulation to be compared with that produced by electrical stimuli delivered through the needle electrode. The action potentials from approximately 50% of the recorded fibers failed to propagate beyond the needle electrode. When propagation did occur and the comparison could be made, only 25% of the afferents recorded could be selectively stimulated. This relatively low success rate suggests that a typical microneurography needle is not particularly good for selectively stimulating identified afferents. Thus in human experiments, attempts to microstimulate identified afferents may often include stimulation of unidentified alternative, or additional, afferent fibers at thresholds of sensory perception.

With the use of a computer-controlled torque motor, experiments were carried out on 11 patients with essential tremor and 13 with parkinsonian tremor to determine the effect of mechanical displacements at the wrist joint on the established pattern of tremor. Analysis of the timing of tremor bursts in electromyographic recordings before and following the stimuli revealed that the phase of essential tremor could be readily reset by external perturbations. In the majority of the parkinsonian patients the same type of stimuli had very little effect on the pattern of tremor. Differences between predicted and actual times of occurrence of tremor bursts following the stimuli were used to calculate a normalized resetting index, with 0 representing no resetting and 1, complete resetting. For the patients with essential tremor the mean resetting index was 0.64 +/- 0.14 (SD); for the parkinsonian patients the generation of essential tremor. Reflex mechanisms are less important in parkinsonian tremor, which is more likely dependent on oscillations originating in the central nervous system.

1. Pairs of brief stretches or a series of stretches at random intervals (Poisson process) were applied to a slow (soleus) and a fast (plantaris) muscle in decerebrate cats to analyze the nonlinear effects of one stretch on the reflex responses to subsequent stretches. Neural activity, electromyogram (EMG), and force were recorded. The reflex responses due to stretch were compared with reflexes as a result of electrical stimulation of nerves. Nonlinearities of muscle were also examined in the absence of reflexes. Short-latency neural activity produced by the stimuli at all intervals studied was quite constant, so changes in sensory activity cannot account for the nonlinearities. Three phases of nonlinear interactions were observed, and mechanisms for these nonlinearities are suggested. 2. For short intervals (less than 100 ms) following a stretch the force and EMG produced by a second stretch is depressed. This early depression could be due to the after hyperpolarization of the motoneuron cel body or to synaptic mechanisms, since the depression of EMG is seen with electrical stimulation of Ia sensory, but not alpha-motor axons. In addition, a second stretch can disrupt the reflex contraction produced by the first stretch if it occurs at a time when new actomyosin bonds are not readily formed. Because of this force suppression, the total reflex force produced in response to two stretches may be less than the response to a single stretch. 3. For intervals between 100 and 300 ms the force and EMG produced by a second stretch is enhanced. This potentiation is also seen with electrical stimulation of large sensory but not motor axons and could result from a synchronization of motoneuronal excitability cycles. It is more prominent in the homogeneous (soleus) muscle than the mixed (plantaris) muscle, probably because the motoneuron cell bodies will reach a period of high excitability at more nearly the same time in the homogeneous muscle. 4. For longer intervals the force produced by a second stretch is reduced even when the EMG is close to control values. This late depression is also observed with electrical stimulation of cut motor axons and therefore arises from the contractile properties of muscles. 5. With a random series of stretches, the same time course of nonlinear interactions is observed. However, as the mean rate of the random stretches is increased, the average response of the reflex decreases. Thus, the stretch reflex will be most effective in correcting for occasional perturbations to a movement, rather than for continuously varying disturbances.

Chronic recording is possible from nerve fibers which have grown through holes in an insulating medium (regeneration electrodes) or which are enclosed by an insulating sheath (cuff electrodes). Use of three electrodes in a balanced configuration permits good rejection of electromyographic (EMG) signals and other sources of electrical interference (fluorescent lights, 60 Hz signals from the mains, etc.). Equations are derived and tested for predicting the amplitude and form of the signals expected for a given cuff length and diameter. These equations can be used to design electrode units optimally for a given application. Finally, the use of transformers permits the neural signals to be carefully matched to the recording apparatus and further optimizes the neural signal-to-noise and signal-to-EMG ratios. Use of these methods in several physiological and clinical applications, as well as potential abuses, are discussed.

Methods are described for analyzing the patterns of sensory activity in peripheral nerves during unrestrained behaviour. In locomotion, nerves supplying ankle extensors and flexors typically show two bursts of sensory activity per step cycle. Variations in sensory input may be important in modulating the ongoing oscillation. Altered sensory input may also change the phase of an ongoing oscillation (as in human essential tremor) or may only have a transient reflex effect (as in Parkinsonian tremor). Various types of interactions can be analyzed using a model of the peripheral neuromuscular system and its central connections. In general, the following categories are suggested for sensory actions on neural oscillations: (1) modulation of (a) the amplitude or (b) the frequency; (2) resetting (i.e., a maintained change in the timing of an oscillation without changes in frequency); (3) repatterning an oscillation (a) over several cycles (e.g., gait changes in locomotion) or (b) within a cycle or two (e.g., the stumble corrective reaction); and (4) gating an oscillation on or off.

With regular trains of stimuli at a high frequency, the contribution of each stimulus to the force generated over time declines from the second to about the tenth stimulus, but then begins to increase again. This late increase is referred to as tetanic potentiation in analogy with the post-tetanic potentiation of the twitch after such a period of stimulation. With regular trains of stimuli at a low frequency, a progressive decrease in the essentially unfused twitches (negative staircase) is observed in the slow soleus muscle of the cat, while a progressive increase (positive staircase) is observed for the fast plantaris muscle. The time constant for the approximately exponential changes observed is on the order of 10 s. Random trains of stimuli were applied at intermediate frequencies and analyzed in terms of general methods of analysis for nonlinear systems. Systematic decreases in the magnitude and increases in the time course of the average tension per stimulus were observed with increasing mean rates of stimulation. Similar changes were observed for short intervals between stimuli within a given random train at a constant mean rate. These changes can be described in terms of an early depression and a later facilitation described in the previous papers in this series.

The peristimulus time histogram (psth) provides a means of correlating the discharges of neurones with other events. The cumulative sum (cusum) derived from the psth facilitates the detection of small changes in the psth that may be obscured by random fluctuations in counts. The cusum integrates differences from the mean control level of counts in the psth. Any signal in the data that is related to the stimulus appears as a slope in the cusum. Psth's constructed from the rhythmic discharges of single neurones are shown to contain periodical fluctuations in counts that arise from refractoriness. This periodicity results in a cusum which deviates less from the horizontal line than predicted from a Poisson distribution of points. The more regular the spike train, i.e., the lower the coefficient of variation of the distribution of interspike intervals, the flatter is the cusum. The theory of stochastic point processes is used to derive an algorithm for calculating the best approximation of variance of the cusum. Significance limits set at 3 standard deviations of the cusum are shown to provide a good fit to cusums for unit discharges over a wide range of coefficients of variation (0.09-0.60).