Relationship between Sarcomere Length and Active Force in Rabbit Papillary Muscle1

Isometric peak twitch force (stimulation frequency 0.5/s; 29.5–30.5°C) was correlated with sarcomere length in isolated papillary muscles of the rabbit. Sarcomere length was measured from photographic recordings (1.5 ms exposure time) performed at rest between contractions and at the time of isometric peak twitch force. The sarcomere length at rest was found to be relatively uniform throughout the preparation and to be linearly related to the overall muscle length within the range L_max- 0.85 L_max. The distribution of sarcomere lengths increased considerably as the muscle went from rest to activity. Studies of surface markers showed different degrees of shortening (or elongation) of individual segments along the length of the preparation. The mean resting sarcomere length at L_max (the optimum muscle length for force production) was 2.44±0.01 μm (grand mean ± S.E., 7 muscles). The mean active sarcomere length at L_max was 2.29 ± 0.04 /μm. Active force declined steeply as the muscle length was reduced below L_max. At a resting sarcomere length of 2.0 μm, active force was approximately 1/3 of the maximum. The observed differences between the length-tension relationships in myocardium (twitch responses) and skeletal muscle (tetanic contractions) are discussed on the basis of a length dependency of the activation process in cardiac muscle.     

The organization of titin (connectin) and nebulin in the sarcomeres: an immunocytolocalization study

Summary Monospecific polyclonal antibodies against two exceptionally large proteins, titin (a-T) and nebulin (a-N) isolated from rabbit skeletal muscles, were raised in guinea pig. Using an immuno-pre-embedding method, we have localized at the ultrastructural level of resolution the reactivity sites in skinned muscle fibres. At resting length a-T and a-N antibodies recognize epitopes which only partially overlap. a-T antibodies decorate mostly the A band with at least four clearly distinguished lines of reaction and one line in the I band, all near the A/I limit; a-N antibodies bind to the same region, but with wider areas of reaction in both A and I bands. To study whether the localization of these reaction sites varies according to the sarcomere length, skinned rabbit psoas fibres were incubated at sarcomere lengths ranging from maximum shortening to overstretching. The results indicate that lines decorated by a-T move away from the Z disc when the sarcomere is lengthened. With respect to the M line, the behaviour was biphasic. When the sarcomere was stretched up to about 2.8 m, the decorated lines maintain almost the same distance from the M line. When the sarcomere is stretched beyond 2.8 m, all a-T epitopes move away from the M line and the molecule behaves elastically. At resting length the a-N decoration appears to be localized on three large adjacent bands at the I, A/I and A level. The a-N line of reaction at the edge of the A band moves away from the Z discs as the sarcomere lengthens, while a second line which seems to be localized at the tip of the thin filament moves away from M line when the sarcomere lengthens. In non-overlapping sarcomeres a-N antibodies decorate only the tip of the thin filaments. Our results indicate that titin forms a polar filament connecting the M line to the Z line. In short sarcomeres, the filament seems to have some connections with structures of the A band, since titin epitopes do not move during stretching. These connections are lost at longer sarcomere lengths. On the other hand, our results suggest that nebulin is probably not a constituent of the titin filament.     

Mechanism of force enhancement during and after lengthening of active muscle: a temperature dependence study

The aim of the present study was to examine the temperature dependence of active force in lengthening and shortening muscle. Experiments were done, in vitro, on bundles of intact fibres (fibre length L₀?~2?mm; sarcomere length?~2.5???m) isolated from a rat fast muscle (flexor hallucis brevis) and a ramp length change of 5?C7?% L₀ was applied on the plateau of an isometric tetanic contraction. Ramp lengthening increased and ramp shortening decreased the muscle tension to new approximately steady levels in a velocity-dependent way. The isometric tension and the lower steady tension reached at a given shortening velocity, increased with warming from 10 to 35?°C and the relation between tension and reciprocal absolute temperature was sigmoidal. However, the tension?Ctemperature curve of shortening muscle was sharper and shifted to higher temperature with increased velocity. In contrast, the enhanced steady tension during lengthening at a given velocity was largely temperature-insensitive within the same temperature range; we hypothesize that the tension?Ctemperature curve may be shifted to lower temperatures in lengthening muscle. Consequently, when normalised to the isometric tension at each temperature, the tension during lengthening at a given velocity decreased exponentially with increase of temperature. The residual force enhancement that remains after ramp lengthening showed a similar behaviour and was markedly reduced in warming from 10 to 35?°C. The findings are consistent with the thesis that active force generation in muscle is endothermic and strain-sensitive; during shortening with a faster crossbridge cycle it becomes more pronounced, but during lengthening it becomes depressed as the cycle slows in a velocity-dependent way. The residual force enhancement may be caused by the same process in addition to non-crossbridge mechanism(s).     

The elementary force generation process probed by temperature and length perturbations in muscle fibres from the rabbit

Single chemically permeabilized fibres from rabbit psoas muscle were activated maximally at 5–6 °C and then exposed to a rapid temperature increase ('T-jump') up to 37 °C by passing a high-voltage pulse (40 kHz AC, 0.15 ms duration) through the fibre length. Fibre cooling after the T-jump was compensated by applying a warming (40 kHz AC, 200 ms) pulse. Tension and changes in sarcomere length induced by the T-jumps and by fast length step perturbations of the fibres were monitored. In some experiments sarcomere length feedback control was used. After T-jumps tension increased from ∼55 kN m⁻² at 5–6 °C to ∼270 kN m⁻² at 36–37 °C, while stiffness rose by ∼15 %, suggesting that at a higher temperature the myosin head generates more force. The temperature-tension relation became less steep at temperatures above 25°C, but was not saturated even at near-physiological temperature. Comparison of tension transients induced by the T-jump and length steps showed that they are different. The T-jump transients were several times slower than fast partial tension recovery following length steps at low and high temperature (phase 2). The kinetics of the tension rise after the T-jumps was independent of the preceding length changes. When the length steps were applied during the tension rise induced by the T-jump, the observed complex tension transient was simply the sum of two separate responses to the mechanical and temperature perturbations. This demonstrates the absence of interaction between these processes. The data suggest that tension transients induced by the T-jumps and length steps are caused by different processes in myosin cross-bridges.     

Unloaded shortening of skinned mammalian skeletal muscle fibres: effects of the experimental approach and passive force

Summary In rabbit, rat and human skinned skeletal muscle fibres the length-time relationship of isotonic releases was determined after maximal Ca²⁺ activation. Slack test experiments provided information about unloaded conditions. Force clamp experiments of different load were extrapolated for zero load and compared with the slack test data. The course length-time relationship for unloaded conditions was similar using both approaches. However, slack test data showed a triphasic shape which could be fitted by three straight lines (phase I, II, III), whereas the data of force clamp experiments exhibited a steady curved shape. Consequently, the instantaneous slopes differed in the two relationships, but the distance which was shortened during the time interval of phase II was similar in both approaches. The ratio between these unloaded shortening velocities resulting from force clamp and slack test experiments was 1.01 ± 0.05 (sd) (n=25). The effects of passive force on the velocity of fibre shortening was investigated in skinned rabbit muscle fibres using slack test experiments. A significant increase in the unloaded shortening velocity was observed when the sarcomere length of the fibres was increased to values which exhibited considerable amounts of passive force. The high reproducibility of the isotonic releases required in this study was achieved by improving some methodological details. Using these improved techniques an identity between the relative fibre and sarcomere shortening was observed during the isotonic releases.     

Comparison of the tension responses to ramp shortening and lengthening in intact mammalian muscle fibres: crossbridge and non-crossbridge contributions

We examined the tension responses to ramp shortening and lengthening over a range of velocities (0.1–5 L ₀/s) and at 20°C and 30°C in tetanized intact fibre bundles from a rat fast (flexor hallucis brevis) muscle; fibre length (L ₀) was 2.2 mm and sarcomere length ∼2.5 μm. The tension change during ramp releases as well as ramp stretches showed an early transition (often appearing as an inflection) at 1–4 ms; the tension change at this transition and the length change at which it occurred increased with velocity. A second transition, indicated by a more gradual reduction in slope, occurred when the length had changed by 14–28 nm per half-sarcomere; the tension at this transition increased with lengthening velocity towards a plateau and it decreased with shortening velocity towards zero tension. The velocity dependence of the time to the transitions and the length change at the transitions showed some asymmetries between shortening and lengthening. Based on analyses of the velocity dependence of the tension and modelling, we propose that the first transition reflects the tension change associated with the crossbridge power stroke in shortening, or with the reversal of the power stroke in lengthening. Modelling shows that the reduction in slope at the second transition occurs when most of the crossbridges (myosin heads) that were attached at the start of the ramp become detached. After the second transition, the tension reaches a steady level in the model whereas the tension continues to increase during lengthening and continues to decrease during shortening in the experiments; this continuous tension change is seen at a wide range of initial sarcomere lengths and when active force is reduced by the myosin inhibitor, BTS. The continuous tension decline during shortening is not abolished by caffeine, but the rate of decline is reduced when the active force is depressed by BTS. We propose that stiffening of non-crossbridge visco-elastic elements upon activation contributes to the continuous tension rise during lengthening and the release of such tension and Ca-insensitive deactivation contribute to the tension decline during shortening in muscle fibres.     

Speed of 10 MHz sound in canine aortic wall: Effects of temperature,storage and formalin soaking

The speed of 10 MHz sound in excised canine thoracic aortic wall was measured over a temperature range from 23°C to 37°C. The effect of storage (for 24h) in Ringer's lactate solution upon the speed of sound was investigated. The effect of soaking in 10% formalin solution was also investigated. At 37°C, the speed of 10 MHz sound was 1·59 mm μs⁻¹ in fresh samples. There was negligible difference between the speed of sound in fresh and 24h samples at 37°C. The temperature dependence of the speed of sound in aortic wall was slight. The effect of formalin soaking was to decrease the speed of sound very slightly at temperatures approaching 37°C, raising questions as to the validity of a model formalin stiffening for evaluation of the ultrasonic tissue signature in aortic wall.     

Artifactual changes in the haematocrit buffy coat of stored anti-coagulated blood samples of farm animals

The buffy coat percentage (BCP) of centrifuged anti-coagulated blood is routinely clinically used to estimate the total leukocyte count (TLC). There had been observations in our clinical laboratory of BCPs that do not correlate with TLC, especially in blood samples in which centrifugation were delayed. This study therefore investigated the artifactual changes that occur in the BCP of stored anti-coagulated blood samples of farm animals. The BCPs of blood samples from a total of 16 cattle, 18 goats, 15 pigs, and 16 chickens were determined immediately upon blood collection to obtain the baseline value (BV). The blood samples were then divided into three parts and stored at 5°C (3–7°C), 30°C (27.5–32.5°C), and 37°C (35.5–38.5°C). Further BCP determinations on the samples were carried out at 24-h intervals for 72 h (3 days). Results showed that there were statistically significant increases (p < 0.05) in BCP of cattle blood samples at all temperatures of storage as from the 48th hour of storage onwards, in goat blood samples stored at 5°C and 37°C at the 72nd hour of storage and as from the 48th hour of storage in the goat blood samples stored at 30°C, in pig blood samples as from the 24th hour of storage onwards for all the storage temperatures, and in chicken blood samples stored at 30°C and 37°C as from the 48th hour of storage onwards. However, clinically significant increases (BCP > 1.5%) occurred only in cattle and chicken blood samples stored at 30°C and 37°C at hour 72 of storage and in pig blood samples stored at 30°C and 37°C as from the 48th hour of storage onwards. It was concluded that statistically significant increases in BCP occur in the blood of cattle, goats, pigs, and chicken during storage, but clinically significant increases that could lead to a false notion of leukocytosis do occur in the BCP of stored anti-coagulated blood of cattle, pigs, and chicken when the blood is stored/kept at 30°C and 37°C.     

Depression of mechanical performance by active shortening during twitch and tetanus of vertebrate muscle fibres

Shortening during activity of frog single muscle fibres caused a graded depression of the contractile force that persisted for 800–900 ms during a partially fused or completely fused tetanus. The depression of force was not associated with a change of the shortening velocity at zero load. Passive shortening performed just before stimulation had no effect on the subsequent course of contraction. The decrease in isometric force produced by shortening was not significantly affected by a stretch applied immediately before or after the shortening phase. For a given amount of shortening the depressant effect during a fused tetanus was 8–28% of that produced during a twitch. The effect was substantially reduced, both during twitch and tetanus, in the presence of 0.5 mM caffeine. The length dependence of the movement effect was studied between 1.7 and 2.9 μm sarcomere spacing. Maximum depression of force (in per cent of the control at each length) was obtained at 2.1–2.2 μm sarcomere length, the effect being steadily reduced at shorter and more extended lengths. The Q₁₀ of the depressant effect was 0.95±0.16 (S.D.). The features of the movement effect are consistent with a true deactivation of the contractile system as would occur if shortening reduced the binding of activator calcium to the regulatory proteins of the myofilaments.     

Contribution of damped passive recoil to the measured shortening velocity of skinned rabbit and sheep muscle fibres

Summary Maximum shortening velocities of skinned fibres from rabbit psoas and sheep extensor digitorum longus muscles were measured by the slack test and by extrapolating force-velocity curves to zero load. Both overall muscle velocity and sarcomere velocity were measured with each method. Maximum sarcomere velocity measured by the slack test was not significantly different from that assessed from the force-velocity curves (p> 0.1). Maximum overall muscle velocity measured from the slack test was significantly (p > 0.001) and substantially (62% rabbit, 83% sheep) greater than maximum sarcomere velocity. The difference is attributed to damped recoil of the series elastic elements contributing to the overall muscle velocity. The extent and time course of this damped recoil in isotonic steps was assessed from comparisons of overall muscle length and sarcomere length records during isotonic steps. When the records were shifted and scaled so that they superposed during the late stages of isotonic shortening, there was a substantial difference between the early parts of the records. This difference was reduced by about half in association with the step and the remaining half declined at a diminishing rate following the step, lasting longer with lower loads. This result is explained by about half of the series elastic element behaving as a viscoelastic element and half being undamped. With steps to the lowest isotonic loads, which averaged 6.7% of isometric force in sheep and 9.5% in rabbit, the total series elastic element recoil (both damped and undamped) averaged 3.4% and 2.7% of fibre segment length, respectively, in sheep and rabbit. The rapid series elastic element recoil at zero load, assessed from the slack test, was approximately 50% higher, indicating a substantial series compliance at low forces. The contribution of an additional, longer lasting, damped series elastic element recoil to the overall muscle velocity can explain the greater maximum velocity that is frequently found with the slack test.     

Sarcomere length changes during end-held (isometric) contractions in intact mammalian (rat) fast and slow muscle fibres

The sarcomere length change, within a 2 mm region, during end-held isometric contractions in intact rat fast and slow muscle fibre bundles was investigated at 20°C and an initial sarcomere length of 2.68 m using He–Ne laser diffraction. In some experiments, the fibre segment displacement was monitored with markers (pieces of human hair) placed at regular intervals on the surface of the muscle fibre bundles. The sarcomere length changes, monitored near the proximal end of the bundle (transducer end), during tetanic contractions were similar to those previously reported in frog muscle fibres. Thus, throughout the tension plateau, sarcomere length remained constant (and shortened) but showed evidence of non-uniform sarcomere behaviour (further shortening) during the rapid tension relaxation phase. Such non-uniform behaviour was not seen during twitch contractions. During a twitch contraction, sarcomeres at the proximal end shortened rapidly at first and continued to shorten – or remained shortened – until the tension had relaxed to between 20–23% of its peak value before lengthening back to the original length. The maximum twitch sarcomere shortening (mean ± SEM) was 5.9 ± 0.2% (n = 16) in fast and 5.4 ± 0.3% (n = 14) in slow fibre bundles at 20°C; sarcomere shortening near body temperature (35°C) was greater, 8.8 ± 0.2% (n = 7) in fast and 8.1 ± 0.2% (n = 5) in slow fibre bundles. Increasing the initial sarcomere length of a preparation decreased the extent of sarcomere shortening and reducing the amount of sarcomere shortening, by sarcomere length clamping, markedly increased the peak twitch tension without significantly altering the twitch time course. When examined at different positions along muscle fibres, a sarcomere shortening was observed along much of the fibre length in most preparations. However, in about a third of the preparations some sarcomere lengthening was recorded in the distal end, but its amplitude was too small to accommodate the fibre shortening elsewhere. Complementary data were obtained using the surface marker technique. The displacement was largest and in opposite – but fibre shortening – direction in the markers placed 0.5–1.0 mm away from the two tendon attachments; the markers placed at or near the centre of the fibre bundle showed the least amount of displacement. The findings suggest that the compliant region, where lengthening occurs, is at fibre ends, i.e. near myotendinous junction.     

Effects of N-acetylcysteine on isolated mouse skeletal muscle: contractile properties,temperature dependence,and metabolism

The effects of the general antioxidant N-acetylcysteine (NAC) on muscle function and metabolism were examined. Isolated paired mouse extensor digitorum longus muscles were studied in the absence or presence of 20 mM NAC. Muscles were electrically stimulated to perform 100 isometric tetanic contractions (300 ms duration) at frequencies resulting in ～85 % of maximal force (70–150 Hz at 25–40 °C). NAC did not significantly affect peak force in the unfatigued state at any temperature but significantly slowed tetanic force development in a temperature-dependent fashion (e.g., time to 50 % of peak tension averaged 35?±?2 ms [control] and 37?±?1 ms [NAC] at 25 °C vs. 21?±?1 ms [control] and 52?±?6 ms [NAC, P?<?0.01] at 40 °C). During repeated contractions, NAC maximally enhanced peak force by the fifth tetanus at all temperatures (by ～30 %). Thereafter, the effect of NAC disappeared rapidly at high temperatures (35–40 °C) and more slowly at the lower temperatures (25–30 °C). At all temperatures, the enhancing effect of NAC on peak force was associated with a slowing of relaxation. NAC did not significantly affect myosin light chain phosphorylation at rest or after five contractions (～50 % increase vs. rest). After five tetani, lactate and inorganic phosphate increased about 20-fold and 2-fold, respectively, both in control and NAC-treated muscles. Interestingly, after five tetani, the increase in glucose 6-P was ～2-fold greater, whereas the increase in malate was inhibited by ～75 % with NAC vs. control, illustrating the metabolic effects of NAC. NAC slightly decreased the maximum shortening velocity in early fatigue (five to seven repeated tetani). These data demonstrate that the antioxidant NAC transiently enhances muscle force generation by a mechanism that is independent of changes in myosin light chain phosphorylation and inorganic phosphate. The slowing of relaxation suggests that NAC enhances isometric force by facilitating fusion (i.e., delaying force decline between pulses). The initial slowing of tension development and subsequent slowing of relaxation suggest that NAC would result in impaired performance during a high-intensity dynamic exercise.     

Shortening induced deactivation of skinned fibres of frog and mouse striated muscle

The depressant effect of active shortening, previously established in intact muscle fibres, was studied during calcium induced contractures of chemically skinned fibres from the semitendinosus muscle of Rana temporaria and the psoas muscle of the mouse. The decrease in contractile activity was determined by comparing the rate of force redevelopment (at a given tension level) after a large (test) and a small (control) release step. Under standard experimental conditions (ionic strength: frog 135 mM, mouse 190 mM; Ca²⁺ 3.0 μM; Mg²⁺: frog 25 μM, mouse 100 μM; MgATP^2-: frog 1.0 mM, mouse 2.0 mM) active shortening of 0.15 μm per sarcomere (in excess of control release) reduced the contractile activity by approximately 50% of the control in both frog and mouse muscle fibres. Full contractile activity was regained within 4 s during isometric activity after the shortening phase. The depressant effect of shortening was steadily reduced, to almost complete disappearance of the effect, by increasing the free calcium concentration within the range 1.5–12.0 μM. Similarly, an increase in ionic strength from 105 to 235 mM reduced the depressant effect by approximately 40%. In contrast, there was a progressive enhancement of the shortening effect as the magnesium ion concentration was increased from 25 to 590 μM. It is proposed that interaction between the myosin cross-bridges and the thin filament during sarcomere shortening leads to a decrease in troponin-calcium binding resulting in a temporary deactivation of the contractile system.     

Effect of Temperature on the Metabolism of Proteoglycans in Explants of Bovine Articular Cartilage

The turnover of proteoglycans in bovine articular cartilage was determined in explant cultures, maintained at 32°C or 37°C. Both the rate of proteoglycan synthesis and the release of newly synthesized proteoglycans were decreased in cultures incubated at 32°C compared to 37°C.

At both temperatures the newly synthesized proteoglycans were similar in hydrodynamic size and chain length of the glycosaminoglycans. However, the ratio of 6-sulfated disaccharides over 4-sulfated disaccharides of the newly synthesized glycosaminoglycans, differed less from the endogenous ratio at 32°C than at 37°C.

At both temperatures, the incorporated ³⁵S-sulfate is released from explants in two pools. Twenty-three percent of the ³⁵S-radiolabel was released into the culture medium during an initial short phase (t_1/2 = 1.1 day at 32°C, 1.3 day at 37°C), 77% had a much longer half-life. The lowered temperature markedly decreased the release of ³⁵S-sulfate with a slow turnover (t_1/2 = 60 days at 32°C, 38 days at 37°C).     

Mechano-Elastic Properties of Human Muscles at Different Temperatures

The effect of changes in the muscle temperature on their ability to store elastic energy was studied by having 5 trained subjects perform maximal vertical jumps on a force platform, with and without counter movement, at muscle temperatures between about 32°C and 37°C. The results showed that the heights of vertical jumps were considerably reduced at lowered temperature, but the gain in height after a counter movement in the form of a jump down from a height of 0.4 m over the force platform, was significantly higher in the cold condition. To test whether this was due to an increased stiffness of the muscles, experiments with imposed sinusoidal length variations at 14 Hz were performed. Δforce ×Δlength^-1 (i.e. stiffness) increased with isometric tension independent of muscle temperature. Experiments in which the rate of tension development and relaxation in voluntary maximal isometric contractions were measured at different muscle temperatures showed that maximal isometric tension changed by less than 1 % per degree but the rate of tension development and relaxation by 3–5 % and 5 % per degree, respectively, in the temperature range studied (30° to 40°). These data may be explained by the hypothesis that the series elastic components of the active muscle are located in the cross-bridges between myosin and actin filaments. The storage of elastic energy would be enhanced if the rate of breaking of these bridges were decreased at lower temperatures.     

Prediction of the In Vivo Force–Velocity Relationship of Slow Human Skeletal Muscle from Measurements in Myofibers

Direct measurement of the in vivo contractile properties of an individual muscle cannot be made in humans. The objective of this study was to predict the force–velocity (F–V) properties of slow human skeletal muscle for the in vivo temperature of 37 °C from F–V measurements in type I myofibers. Specifically, to quantitatively link myofiber measurements, which must be conducted at relatively low temperatures, to in vivo properties, the temperature-dependence of contractile properties must be modeled. We estimated the kinetic parameters of a crossbridge model within 15–30 °C from F–V measurements recorded in the myofibers of one subject, extrapolated their values at 37 °C, and then predicted the in vivo shortening and lengthening F–V curves. The prediction for maximal shortening velocity was 2.2 ± 0.2 fiber lengths per second and that for saturation force during lengthening was 2.3 ± 0.2 times isometric force. These estimates agree with previously reported in vivo measurements but are substantially different than those used in muscle models for many musculoskeletal simulations. The results from this study indicate that during low levels of muscle activation when slow motor units are primarily recruited, musculoskeletal models should consider having F–V properties that reflect the contractile properties of type I myofibers.     

Muscle performance and electromyogram activity of the lower leg muscles with different levels of cold exposure

The purpose of this study was to evaluate the relationship between different levels of body cooling and muscle performance decrement and to study the motor co-ordination of the working agonist–antagonist muscle pair of the lower leg. Eight volunteer male subjects dropped from a 40-cm bench on to a force plate and performed a maximal rebound jump (stretch–shortening cycle). The jumps were performed after 60-min exposures to 27°C, 20°C, 15°C and 10°C. In comparison to those at 27°C, all the exposures to lower temperatures decreased the flight time of the jump, average force production and take-off velocity in a dose-dependent manner. The changes in electromyogram (EMG) activity also behaved in a dose-dependent manner. During preactivity and stretch phases the integrated EMG (iEMG) activity of the agonist muscle (triceps surae) increased due to cooling (at 10°C, P?P?P?P?相似文献   

Successful maintenance of suckling rat ileum in organ culture

Ileum from rats 4, 9, 11, 12, and 15 days old can best be maintained for 24 hours in a system using Hanks' Balanced Salt Solution without fetal calf serum, at 25°C and 21% O₂. Suckling rat duodenum and jejunum were difficult to maintain well for 24 hours in this system or a variety of other systems that were tried. A temperature of 37°C hastened deterioration of duodenum, jejunum or ileum. With ileum, ³H-thymidine and ¹⁴C-leucine were increasingly incorporated into DNA and protein over the 24-hour period. Light microscopy, as well as scanning and transmission electron microscopy, showed very good preservation of the ileum after 24 hours. The addition to the medium of hydrocortisone, 1 μm, and thyroxine, 0.01 μm, alone or in combination, did not change DNA or protein synthesis, or morphology, possibly because of the relatively short (24 hour) time period. Our organ culture system emphasizes the differences between suckling rat ileum and the rest of the intestine, and provides a new tool for evaluating, over a 24-hour period, the developing rat small intestine.     

Constitutive fragile sites 1p31, 3p14, 6q26, and 16q23 and their use as controls for false-negative results with the fragile(X)

Fragile(X) estimations in fragile(X)-mental retardation hemizygotes or heterozygotes can become falsely negative in stored blood (lymphocytes). This was shown in blood stored (before culture) at 4°C, room temperature (25°C), 37°C, and 39°C for 1–4 days. After storage, blood was cultured in Ham's F10-5% FC serum with 0.1 μM FUdR and scored for fra(X) and the constitutive fragile sites at 3p14 and 6q26. It was found that the proportion of cells expressing the fragile(X) and the 3p14 site varied inversely with the temperature and time of storage. In addition, 50 patients and controls were scored for the three latter sites after routine 72–96-hr culture in F 10–0.05 or 0.1 μM FUdR. The 3p14 site was detected in every individual tested in a mean ± S.D. of 11.3 ± 3.2% of cells (0.1 μM FUdR). It was found that this site was FUdR dose dependent whereas the 6q26 site was not. The 3p14 (but not the 6q26) site is therefore suitable as a control site for the FUdR effect. It is proposed that repeat studies are necessary when less then 4% 3p14 sites are present in specimens from males referred for fra(X) estimation. Other constitutive fragile sites (eg, 1p31 and 16q23) can also be used.     

Connecting filament mechanics in the relaxed sarcomere

By examining the mechanical properties of single unactivated myofibrils it has been shown that shortening and stretching of sarcomeres occurs in stepwise fashion, and that steps are seen also in the relaxed state (Yang et al. (1998) Biophys J 74: 1473–1483; Blyakhman et al. (2001) Biophys J 81: 1093–1100; Nagornyak et al. (2004) J. Muscle. Res. Cell Motil. 25: 37–43). The latter are inevitably associated with connecting filaments. Here, we carried out measurements on single myofibrils from rabbit psoas muscle to investigate steps in unactivated specimens in more detail. Myofibrils were stretched and released in ramp-like fashion. For the single sarcomere the time course of length change was consistently stepwise. We found that in the unactivated myofibrils, step size depended on initial sarcomere length, diminishing progressively with increase of initial sarcomere length, whereas in the case of activated sarcomeres, step size was consistently 2.7 nm.