POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex

Human telomere length is controlled by a negative feedback loop based on the binding of TRF1 to double-stranded telomeric DNA. The TRF1 complex recruits POT1, a single-stranded telomeric DNA-binding protein necessary for cis-inhibition of telomerase. By mass spectrometry, we have identified a new telomeric protein, which we have named POT1-interacting protein 1 (PIP1). PIP1 bound both POT1 and the TRF1-interacting factor TIN2 and could tether POT1 to the TRF1 complex. Reduction of PIP1 or POT1 levels with shRNAs led to telomere elongation, indicating that PIP1 contributes to telomere length control through recruitment of POT1.     

The effect of changed visual feedback on intention tremor in multiple sclerosis

In patients with multiple sclerosis (MS), intention tremor amplitude is enhanced during the visually guided compared to the memory guided motor tasks. In the present study, the effect of visual feedback on intention tremor was investigated during visually guided wrist step-tracking tasks. Specifically, visual feedback of the hand was provided either instantly or averaged over different time windows. Thirteen MS patients with intention tremor and 14 healthy controls performed the wrist step-tracking task, while the visual representation of the actual hand position was displayed instantly or averaged over time windows of 150, 250 and 350 ms. It has been found in the patient group that, in association with a decreased initial error and decreased tremor amplitude on the screen, the amplitude of the actual performed tremor also decreased when visual feedback was changed. The tremor reduction was not different between conditions with manipulated feedback, although delays in presenting visual feedback of the hand position increased when the time window was larger. The reduction in overall tremor amplitude was unlikely related to other factors, such as eye fixation deficits or the speed of the primary hand movement. These results suggest that hand tremor severity is dependent on the visual feedback of position and movement errors.     

The intrinsically disordered N-terminal region of mouse DNA polymerase alpha mediates its interaction with POT1a/b at telomeres

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.     

Human POT1 is required for efficient telomere C-rich strand replication in the absence of WRN

Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.     

Stimulus features underlying reduced tremor suppression with temporally patterned deep brain stimulation

Deep brain stimulation (DBS) provides dramatic tremor relief when delivered at high-stimulation frequencies (more than ～100 Hz), but its mechanisms of action are not well-understood. Previous studies indicate that high-frequency stimulation is less effective when the stimulation train is temporally irregular. The purpose of this study was to determine the specific characteristics of temporally irregular stimulus trains that reduce their effectiveness: long pauses, bursts, or irregularity per se. We isolated these characteristics in stimulus trains and conducted intraoperative measurements of postural tremor in eight volunteers. Tremor varied significantly across stimulus conditions (P < 0.015), and stimulus trains with pauses were significantly less effective than stimulus trains without (P < 0.002). There were no significant differences in tremor between trains with or without bursts or between trains that were irregular or periodic. Thus the decreased effectiveness of temporally irregular DBS trains is due to long pauses in the stimulus trains, not the degree of temporal irregularity alone. We also conducted computer simulations of neuronal responses to the experimental stimulus trains using a biophysical model of the thalamic network. Trains that suppressed tremor in volunteers also suppressed fluctuations in thalamic transmembrane potential at the frequency associated with cerebellar burst-driver inputs. Clinical and computational findings indicate that DBS suppresses tremor by masking burst-driver inputs to the thalamus and that pauses in stimulation prevent such masking. Although stimulation of other anatomic targets may provide tremor suppression, we propose that the most relevant neuronal targets for effective tremor suppression are the afferent cerebellar fibers that terminate in the thalamus.     

Occurrence of widespread motor-unit firing correlations in muscle contractions: their role in the generation of tremor and time-varying voluntary force

The firing behavior of motor units (MUs) of the first dorsal interrosseus muscle of the hand was examined during both constant-force and varying-force (sinusoidal or broadband random variations) isometric contractions in healthy adults. The emphasis was on the analysis of MU synchrony with an efficient and sensitive method. In static contractions, widespread and strong MU firing correlations, with the MUs in phase, were present at the frequency of muscle tremor, when the tremor was regular (narrowband) and large. MU correlations could also exist in contractions where the tremor of a subject was irregular (broadband) overall, but they were generally weak. These correlations were at the frequency of the subject's regular tremor, and the corresponding distinct tremor component was sometimes discernible within the broad tremor-band. In contrast, the MUs did not show any such correlations in the case of purely irregular and small tremor. On the basis of these observations, it is concluded that the rhythms in the force contributions of the last- recruited, large MUs, which fire near their threshold rate, compose the broadband frequency content of physiological muscle tremor in every contraction. Within this band, there is an additional distinct tremor component when MU correlations are present. For widespread and strong MU correlations, this component dominates and constitutes the observed regular tremor. In dynamic contractions, the firing of all MUs was modulated in the frequency band of both the sinusoidal and the complex variations of the force. The MU modulations showed a time-lead over the force variations and were strongly correlated both to these variations and among themselves. Thus widespread and strong correlations of MU firing modulations seem to provide a mechanism for generation of time-varying voluntary force, under general dynamic conditions. Finally, when regular tremor was present in dynamic contractions, widespread and fairly strong MU correlations also existed at the tremor frequency. It is concluded that at least two mechanisms can cause widespread MU synchrony, and they can act in parallel. They involve two types of correlated inputs to the alpha-motoneurons (presumably from the muscle spindles and the cortex), whose effects combine at the level of the membrane potential of the cells.     

Determining posture from physiological tremor

The measurement of body and limb posture is important to many clinical and research studies. Current approaches either directly measure posture (e.g., using optical or magnetic methods) or more indirectly measure it by integrating changes over time (e.g., using gyroscopes and/or accelerometers). Here, we introduce a way of estimating posture from movements without requiring integration over time and the resulting complications. We show how the almost imperceptible tremor of the hand is affected by posture in an intuitive way and therefore can be used to estimate the posture of the arm. We recorded postures and tremor of the arms of volunteers. By using only the minor axis in the covariance of hand tremor, we could estimate the angle of the forearm with a standard deviation of about 4° when the subject's elbow is resting on a table and about 10° when it is off the table. This technique can also be applied as a post hoc analysis on other hand-position data sets to extract posture. This new method allows the estimation of body posture from tremor, is complementary to other techniques, and so can become a useful tool for future research and clinical applications.     

NEK6‐mediated phosphorylation of human TPP1 regulates telomere length through telomerase recruitment

Shelterin component TPP1 plays critical roles in chromosome end protection and telomere length regulation. Specifically, TPP1 contains an OB‐fold domain that provides an interface to recruit telomerase. However, it remains largely unknown how telomerase recruitment is regulated by cell cycle regulators. We show that TPP1 interacts with the cell cycle regulator kinase NEK6 in human cells. We found that NEK6‐mediated phosphorylation of TPP1 Ser255 in G2/M phase regulates the association between telomerase activity and TPP1. Furthermore, we found evidence that POT1 negatively regulates TPP1 phosphorylation because the level of Ser255 phosphorylation was elevated when telomeres were elongated by a POT1 mutant lacking its OB‐fold domains. Ser255 is located in the intervening region between the telomerase‐recruiting OB‐fold and the POT1 recruitment domains. Ser255 and the surrounding amino acids are conserved among vertebrates. These observations suggest that a region adjacent to the OB‐fold domain of TPP1 is involved in telomere length regulation via telomerase recruitment.     

Functional interaction between telomere protein TPP1 and telomerase

Human chromosome end-capping and telomerase regulation require POT1 (Protection of Telomeres 1) and TPP1 proteins, which bind to the 3′ ssDNA extension of human telomeres. POT1–TPP1 binding to telomeric DNA activates telomerase repeat addition processivity. We now provide evidence that this POT1–TPP1 activation requires specific interactions with telomerase, rather than it being a DNA substrate-specific effect. First, telomerase from the fish medaka, which extends the same telomeric DNA primer as human telomerase, was not activated by human POT1–TPP1. Second, mutation of a conserved glycine, Gly100 in the TEN (telomerase essential N-terminal) domain of TERT, abolished the enhancement of telomerase processivity by POT1–TPP1, in contrast to other single amino acid mutations. Chimeric human–fish telomerases that contained the human TEN domain were active but not stimulated by POT1–TPP1, showing that additional determinants of processivity lie outside the TEN domain. Finally, primers bound to mouse POT1A and human TPP1 were activated for extension by human telomerase, whereas mPOT1A–mTPP1 was most active with mouse telomerase, indicating that these mammalian telomerases have specificity for their respective TPP1 proteins. We suggest that a sequence-specific interaction between TPP1 in the TPP1–POT1–telomeric DNA complex and the G100 region of the TEN domain of TERT is necessary for high-processivity telomerase action.     

Exploring the functional significance of physiological tremor: A biospectroscopic approach

The functional significance of physiological tremor - the high frequency (8-12 Hz), low amplitude oscillation that occurs during the maintenance of steady limb postures - is not known. Often tremor, perhaps because of its pathological manifestations, is considered a source of unwanted noise in the system, something to be damped out or controlled. An examination of the phase relationship between tremor and rapid voluntary finger movement in normal subjects suggests a very different view. In four experiments in which tremor displacement and accompanying electromyographic activity were simultaneously monitored, we show a clear and systematic relationship between tremor and movement initiation. Empirically obtained frequency distributions of tremor peak-to-movement initiation time were most closely aligned to a probability density function (derived via numerical integration techniques) that assumed movements were initiated when the muscle-joint system possessed peak momentum. This relationship - evaluated by Chi-square goodness-of-fit tests - was evident regardless of whether the movements were self-paced (Experiments 1 and 2) or in response to an auditory reaction time signal (Experiments 3 and 4). The addition of a load to the finger in Experiments 2 and 4, though tending to reduce tremor frequency, did not prove disruptive, nor did a fractionated reaction time analysis reveal any significant inertial contribution to the maintenance of the phase relationship. These data are consistent with an emerging view that the motor control system is sensitive to its own dynamics, and suggest that under certain conditions normal physiological tremor is a potentially exploitable oscillation intrinsic to the motor system.     

Neurophysiology of orthostatic tremor. Influence of transcranial magnetic stimulation

A 74-year-old patient suffers from painful muscle cramps when he stands since 30 years. He has no visible tremor but 16 Hz burst activity on EMG, indicating orthostatic tremor. Previous diagnosis was hysteria, stiff person syndrome or dystonia. This shows that EMG during standing should be part of the examination of patients with stiff muscles or muscle cramps. Tremor was not strictly orthostatic. It appeared in back muscles while sitting, when the patient supported a weight with outstretched arms. Phase between muscles differed between normal standing and standing on heels. Subthreshold transcranial magnetic stimulation modulated timing of the tremor bursts and inhibited them at higher intensity stimulation.     

Differential modulation of tremor and pulsatile control of human jaw and finger by experimental muscle pain

Resting tremor is seen in both the limbs and in the trigeminal motor system. These rhythmical perturbations are the result of alternating activation of antagonistic muscles, and these increase in amplitude during slow, voluntary movements. In the present study, we examined the effect of experimental muscle pain on finger and jaw tremor. The tremor in the mandible and in the middle finger was measured on separate occasions, at rest and during two constant-velocity movements. Pain was then induced by the infusion of hypertonic saline into a jaw-closing muscle (masseter) or into a finger extensor muscle (extensor digitorum longus, EDL). During masseter pain, the power at the peak tremor frequency of the mandible decreased significantly both when the jaw was at rest and during voluntary jaw movements at two velocities. In contrast, pain in EDL resulted in a significant increase in the power of finger tremor only during the two speeds of voluntary movement. No change in the peak tremor frequency was seen in either the finger or the jaw during pain. The most likely explanation for these data is that muscle pain tonically modulates the amplitude of the outputs from the central "pulsatile control" generators that drive the alternating activation of antagonistic muscles which produce tremor at rest and during movements. This modulation is in the opposite direction for systems controlling jaw and hand, suggesting a specific interaction of the nociceptive afferents with separate central oscillators. Electronic Publication     

Primordial odontogenic tumor occurred in the maxilla with unique calcifications and its crucial points for differential diagnosis

Primordial odontogenic tumor (POT) is a newly classified, mixed epithelial and mesenchymal odontogenic tumor, with only 17 reported cases to date. Herein, we report a case of POT that occurred in the right maxilla of a 10-year-old boy and reveal unique features in comparison with those previously reported. Radiologically, the lesion presented as a well-defined, unilocular radiolucency with notable radiopaque foci on the periphery. Microscopically, the tumor was mainly composed of dental papilla-like myxoid fibrous connective tissue, largely surrounded by non-keratinized squamous epithelium with numerous calcified particles, and partly enclosed by inner enamel epithelium-like columnar cells and enamel organ-like structures accompanied with cuboidal and/or stellate reticulum-like cells. Immunohistochemically, the epithelium tested positive for cytokeratin 14 and 19. Moreover, amelogenin and ameloblastin, matrix proteins relating to enamel formation, were positive in the covering epithelium. The tumor was enucleated as a whole, and no recurrence was recorded thereafter. Although the presence of numerous calcified particles was unique, we diagnosed this lesion as POT based on the above-described features. Furthermore, we emphasize the importance of the differential diagnosis of POT and other odontogenic tumors that resemble corresponding tooth germ components.     

Tremorgenesis: a new conceptual scheme using reciprocally innervated circuit of neurons

Neural circuits controlling fast movements are inherently unsteady as a result of their reciprocal innervation. This instability is enhanced by increased membrane excitability. Recent studies indicate that the loss of external inhibition is an important factor in the pathogenesis of several tremor disorders such as essential tremor, cerebellar kinetic tremor or parkinsonian tremor. Shaikh and colleagues propose a new conceptual scheme to analyze tremor disorders. Oscillations are simulated by changing the intrinsic membrane properties of burst neurons. The authors use a model neuron of Hodgkin-Huxley type with added hyperpolarization activated cation current (I_h), low threshold calcium current (I_t), and GABA/glycine mediated chloride currents. Post-inhibitory rebound is taken into account. The model includes a reciprocally innervated circuit of neurons projecting to pairs of agonist and antagonist muscles. A set of four burst neurons has been simulated: inhibitory agonist, inhibitory antagonist, excitatory agonist, and excitatory antagonist. The model fits well with the known anatomical organization of neural circuits for limb movements in premotor/motor areas, and, interestingly, this model does not require any structural modification in the anatomical organization or connectivity of the constituent neurons. The authors simulate essential tremor when I_h is increased. Membrane excitability is augmented by up-regulating I_h and I_t. A high level of congruence with the recordings made in patients exhibiting essential tremor is reached. These simulations support the hypothesis that increased membrane excitability in potentially unsteady circuits generate oscillations mimicking tremor disorders encountered in daily practice. This new approach opens new perspectives for both the understanding and the treatment of neurological tremor. It provides the rationale for decreasing membrane excitability by acting on a normal ion channel in a context of impaired external inhibition.     

Clinical and electromyographic characteristics of tremor in patients with generalized anxiety disorder

The tremor in patients with anxiety was supposed to be an enhanced physiological tremor. However no detailed clinical and electromyographic examination of tremor in patients with anxiety has been published. It has been also supposed that propranolol or benzodiazepines may influence the tremor The aim of this investigation was to establish the clinical and electromyographic pattern of tremor in patients with anxiety and to compare the effects of propranolol and alprazolam treatment. One hundred and twenty patients with tremor and generalized anxiety disorder were investigated. Tremor was scored clinically by the Webster Tremor Scale. Electromyographic examination of tremor activity from antagonistic hand muscles was performed. Sixty patients were treated with alprazolam and propranolol, in an open, crossover design, while the other sixty patients received no particular treatment for the tremor The patients were randomly assigned to treatment with propranolol, alprazolam, or received no tremor treatment. Our results revealed a postural and kinetic tremor with characteristics of an enhanced physiological tremor. Tremor involved only upper limbs and no other body parts. Treatment with propranolol or alprazolam had similar favorable effect on tremor. In conclusion tremor in generalized anxiety disorder is an enhanced physiological tremor The kinetic tremor disturbs patients and particular tremor treatment with propranolol or alprazolam should be applied.     

Oral administration of proteinase inhibitor II from potatoes reduces energy intake in man

The effect of a proteinase inhibitor extracted from potatoes (POT II) which increases CCK release, on food intake was examined in 11 lean subjects. They received 1.5 g POT II in a high-protein soup vehicle (70 kcal), the soup vehicle alone, or a no-soup control five minutes before being presented with a lunchtime test meal, according to a double-blind, within-subjects design. Consuming the soup alone led to a nonsignificant 3% reduction in energy intake. The addition of 1.5 g POT II to the soup significantly reduced energy intake by a further 17.5%. Premeal ratings of motivation to eat and food preferences did not predict the reduction in energy intake by the proteinase inhibitor. These findings suggest that endogenous CCK may be important in the control of food intake and that proteinase inhibition may have therapeutic potential for reducing food intake.     

Characterization of POT1 tumor predisposition syndrome: Tumor prevalence in a clinically diverse hereditary cancer cohort

PurposeGermline variants in POT1 have been implicated in predisposition to melanoma, sarcoma, and glioma in limited studies. Here, we determine the prevalence of cancer types in individuals with POT1 pathogenic variants (PVs) undergoing multigene panel testing (MGPT) for a broad variety of cancer indications.MethodsWe performed a retrospective review of data provided on clinical documents from individuals with POT1 PVs identified via MGPT over a 5-year period. Tumor prevalence in POT1 PV heterozygotes was compared with MGPT-negative wild-type (WT) controls using χ² test.ResultsPOT1 PVs were identified in 227 individuals. POT1 PV and WT (n = 13,315) cohorts had a similar proportion of reported tumors (69.6% and 69.2%, respectively); however, POT1 PV heterozygotes were more likely to be diagnosed with multiple tumors (18.9% vs 8.7%; P < .001). Compared with POT1 WT, we identified a significant increase in melanoma (odds ratio 7.03; 95% CI 4.7-10.5; P < .001) and sarcoma (odds ratio 6.6; 95% CI 3.1-13.9; P < .001).ConclusionThis analysis of the largest POT1 PV cohort to date validates the inclusion of POT1 in hereditary cancer MGPT and has the potential to impact clinical management recommendations, particularly for patients and families at risk for melanoma and sarcoma.     

A POT1 mutation implicates defective telomere end fill-in and telomere truncations in Coats plus

Coats plus (CP) can be caused by mutations in the CTC1 component of CST, which promotes polymerase α (polα)/primase-dependent fill-in throughout the genome and at telomeres. The cellular pathology relating to CP has not been established. We identified a homozygous POT1 S322L substitution (POT1^CP) in two siblings with CP. POT1^CP induced a proliferative arrest that could be bypassed by telomerase. POT1^CP was expressed at normal levels, bound TPP1 and telomeres, and blocked ATR signaling. POT1^CP was defective in regulating telomerase, leading to telomere elongation rather than the telomere shortening observed in other telomeropathies. POT1^CP was also defective in the maintenance of the telomeric C strand, causing extended 3′ overhangs and stochastic telomere truncations that could be healed by telomerase. Consistent with shortening of the telomeric C strand, metaphase chromosomes showed loss of telomeres synthesized by leading strand DNA synthesis. We propose that CP is caused by a defect in POT1/CST-dependent telomere fill-in. We further propose that deficiency in the fill-in step generates truncated telomeres that halt proliferation in cells lacking telomerase, whereas, in tissues expressing telomerase (e.g., bone marrow), the truncations are healed. The proposed etiology can explain why CP presents with features distinct from those associated with telomerase defects (e.g., dyskeratosis congenita).     

Effect of deep brain stimulation on amplitude and frequency characteristics of rest tremor in Parkinson’s disease

The effect of chronic high frequency deep brain stimulation (DBS) on rest tremor was investigated in subjects with Parkinson’s disease (PD). Eight PD subjects with high amplitude tremor (Group 1) and eight PD subjects with low amplitude tremor (Group 2, used as a reference group) were examined by a clinical neurologist and tested with a velocity laser to quantify time and frequency domain characteristics of tremor. Possible rebound effects in rest tremor when DBS was stopped for 60 min were also explored. Participants received DBS of the internal globus pallidus (GPi) (n=7), the subthalamic nucleus (STN) (n=6) or the ventrointermediate nucleus of the thalamus (Vim) (n=3). Tremor was recorded with a velocity laser under two conditions of DBS (on–off) and two conditions of medication (l-Dopa on–off). Correlations between clinical and experimental results for tremor amplitude was 0.70 with no medication and no stimulation. In Group 1, DBS decreased tremor amplitude but also increased spectral concentration and median frequency significantly. Under medication, the changes in tremor with and without stimulation were not statistically significant (Group 1). When stimulation was stopped for 60 min, a rebound in tremor amplitude was observed and median frequency remained stable in Group 1. None of the comparisons examined produced significant effects in Group 2. Taken together, these results suggest that beyond its effect on tremor amplitude DBS acted also on tremor frequency and did not modify tremor characteristics in subjects with low amplitude tremor.     

The effects of load and force on tremor at the normal human elbow joint

1. Spontaneous flexion-extension tremor of the elbow was recorded in normal human subjects while they exerted a flexing force against different loads.2. Increases in the flexing force up to (1/2) or (3/4) of the subject's maximum were accompanied by increases in the amplitude of the tremor. A further increase to very large forces was associated with a decrease in tremor.3. When the subject flexed against a spring, the frequency of the tremor was related to the properties of the spring; with increasingly stiff springs the tremor frequency increased up to about 12 Hz. With the stiffest springs, however, the tremor became irregular and very small in amplitude.4. Weights added to the limb at the wrist reduced the frequency of tremor.5. By using appropriate combinations of spring and mass, the principal tremor frequency could be adjusted between 2 and 12 Hz, higher in some subjects. Within this range the frequency(2) was approximately proportional to the spring stiffness/mass, just as it is in a spring-mass system, though the forearm provided some of this mass, and some of the spring-like resistance was in the flexor muscles.6. When by suitable loading the tremor frequency was brought between 8 and 12 Hz, and the subject forcibly flexed his elbow, the tremor became regular and large in amplitude. This was a form of limited instability in the stretch reflex which arose because a powerful reflex response to extension of the elbow acted back on the flexor muscles after a delay.7. When the limb was so loaded that its natural frequency was outside this 8-12 Hz range, it behaved as a filter, and in response to any disturbing noise it oscillated mainly at frequencies close to the natural frequencies of either the mechanical system or the stretch reflex.8. This filtering function of the limb with its stretch reflex probably plays an important part in the control of normal movement and tremor.9. The large flexing forces that were here associated with vigorous tremor normally occur when a limb supports a large mass. Such a mass would give the mechanical system within the limb a low natural frequency; inconveniently large oscillations in the 8-12 Hz range would therefore seldom occur.