Effect of electrical stimulation-induced muscle force and streptomycin treatment on muscle and trabecular bone mass in early-stage disuse musculoskeletal atrophy.

OBJECTIVES: The aim was to determine whether daily muscle electrical stimulation (ES) and streptomycin treatment would have positive or negative effects on trabecular bone mass in disuse rats. METHODS: Seven-week-old male F344 rats were randomly divided into five groups of eight animals each: an age-matched control group (CON); a sciatic denervation group (DN); a DN + direct electrical stimulation group (DN+ES); a DN + streptomycin treatment group (DN+SM); and a DN+ES+SM group. The tibialis anterior (TA) muscles in all ES groups were stimulated with 16mA at 10Hz for 30 min/day, six days/week, for one week. Bone volume and structure were evaluated using micro-CT, and histological examinations of the tibiae were performed. RESULTS: Direct ES significantly reduced the disuse-induced trabecular bone loss. Osteoid thickness were also significantly greater in the ES groups than in the DN group. Micro CT and histomorphological parameters were significantly lower in the DN+ES+SM group than in the DN+ES group, while there were no significant differences between the DN and DN+SM groups. CONCLUSIONS: These results suggest that ES-induced muscle force reduced trabecular bone loss, and streptomycin treatment did not induce bone loss, but attenuated the effects of ES-induced muscle force on reducing the loss of disused bone.

Electrical stimulation of denervated rat skeletal muscle retards trabecular bone loss in early stages of disuse musculoskeletal atrophy.

OBJECTIVES: We aimed to determine the intensity of muscle stimulation required to prevent structural failure as well as bone and skeletal muscle loss after denervation-induced disuse. METHODS: Seven-week-old rats (weight, 198-225 g) were randomly assigned to age-matched groups comprising control (CON), sciatic nerve denervation (DN) or direct electrical stimulation (ES) one day later [after denervation] with 4, 8 and 16 mA at 10 Hz for 30 min/day, six days/week, for one or three weeks. Bone architecture and mean osteoid thickness in histologically stained tibial sections and tension in tibialis anterior muscles were assessed at one and three weeks after denervation. RESULTS: Direct ES with 16 mA generated 23-30% maximal contraction force. Denervation significantly decreased trabecular bone volume fraction, thickness and number, connectivity density and increased trabecular separation in the DN group at weeks one and three. Osteoid thickness was significantly greater in the ES16 group at week one than in the DN and other ES groups. Trabecular bone volume significantly correlated with muscle weight. CONCLUSIONS: Relatively low-level muscle contraction induced by low-frequency, high-intensity electrical muscle stimulation delayed trabecular bone loss during the early stages (one week after DN) of musculoskeletal atrophy due to disuse.

Esophageal fibrovascular polyp removed by cervical esophagotomy.

We report a case of esophageal fibrovascular polyp (FVP) removed by cervical esophagotomy. The patient was a 74-year-old man in whom an intraesophageal mass was detected by a chest CT examination during a complete medical check-up. An upper gastrointestinal series showed a large, pedunculated, cervical esophageal mass for which our preoperative diagnosis was a FVP. We studied its features, as well as removal procedures in 45 patients in the literature. Most patients had marked symptoms, but ours had no complaints, and so this case may be a rare one. Various removal procedures were reported, but thoracotomy and esophagectomy are considered to be the inappropriate procedures since FVP is a benign disorder.

The expression patterns of Pax7 in satellite cells during overload-induced rat adult skeletal muscle hypertrophy.

AIM: Activated satellite cells (SCs) have the ability to reacquire a quiescent, undifferentiated state. Pax7 plays a crucial role in allowing activated SCs to undergo self-renewal. Because the increase in the SC population is induced during overload-induced skeletal muscle hypertrophy, it is possible that Pax7-regulated SC self-renewal is involved in the modulation of the SC population during the functional overload of skeletal muscles. However, the characteristics of the expression patterns of Pax7 in SCs during the functional overload of adult skeletal muscles are poorly understood. METHODS: Using immunohistochemical approaches, we examined the temporal and spatial expression patterns of Pax7 expressed in SCs during the functional overloading of rat skeletal muscles. RESULTS: The time course of Pax7 expression in SCs was similar to that of the expression of the differentiation regulatory factor myogenin during the early stage of functional overload. However, the percentage of SCs that expressed Pax7 was markedly higher than that of the SCs that expressed myogenin. Coexpression of Pax7 and myogenin was not detected in SCs. In addition, the expression of cyclin-dependent kinase inhibitor p21, which regulates cell cycle arrest and differentiation, was not detected in Pax7-positive SCs. CONCLUSION: These results suggest that Pax7-regulated self-renewal of SCs may be induced during the early stage of functional overload and may contribute to modulating the SC population in hypertrophied muscles. Furthermore, it was suggested that the numbers of SCs which underwent self-renewal may be higher than that of SCs which were provided as the additional myonuclei for hypertrophying myofibres.

Monitoring of cytomegalovirus reactivation after allogeneic stem cell transplantation: comparison of an antigenemia assay and quantitative real-time polymerase chain reaction.

Cytomegalovirus (CMV) antigenemia and quantitative real-time polymerase chain reaction (PCR) were compared for monitoring of CMV reactivation after allogeneic stem cell transplantation. The number of CMV antigen-positive cells by the antigenemia assay and the level of CMV DNA by real-time PCR correlated well. The sensitivity and specificity of the antigenemia assay was 55.4% and 95.5%, respectively, using real-time PCR as the reference standard. The probability of positive antigenemia at day 100 was 76.5%, with a median of first detection at day 37 in 51 patients, compared with a positive PCR of 84.3% and day 33, respectively. When HLA-identical sibling donor transplant recipients and other donor transplant recipients were analyzed separately, there was no difference between the two tests. However, temporal patterns of first detection of CMV antigen-positive cells and CMV DNA differed between HLA-identical and alternative recipients; patients without CMV (29%) or with sporadic positive PCR results (14%) were more common in HLA-identical sibling transplants, whereas patients with simultaneous antigenemia and positive PCR occurred more in alternative transplants (48%). Two of 51 patients (4%) developed CMV colitis despite antigenemia-guided prophylaxis, but both were successfully treated with ganciclovir. Although PCR is more sensitive than antigenemia, both tests are useful in the early detection of CMV after allogeneic stem cell transplantation.

Eccentric exercise-induced morphological changes in the membrane systems involved in excitation-contraction coupling in rat skeletal muscle.

1. Physiological evidence suggests that excitation-contraction (E-C) coupling failure results from eccentric contraction-induced muscle injury because of structural and morphological damage to membrane systems directly associated with the E-C coupling processes within skeletal muscle fibres. In this study using rats, we observed the ultrastructural features of the membrane systems of fast-twitch (FT) and slow-twitch (ST) muscle fibres involved in E-C coupling following level and downhill running exercise. Our aim was to find out whether mechanically mediated events following eccentric exercise caused disorder in the membrane systems involved in E-C coupling, and how soon after exercise such disorder occurred. We also compared the morphological changes of the membrane systems between ST and FT muscle fibres within the same muscles. 2. Single muscle fibres were dissected from triceps brachii muscles of male Fischer 344 rats after level or downhill (16 deg decline) motor-driven treadmill running (18 m min(-1), 5 min running with 2 min rest interval, 18 bouts). All single muscle fibres were histochemically classified into ST or FT fibres. The membrane systems were visualized using Ca(2+)-K(3)Fe(CN)(6)-OsO(4) techniques, and observed by high voltage electron microscopy (120-200 kV). 3. There were four obvious ultrastructural changes in the arrangement of the transverse (t)-tubules and the disposition of triads after the downhill running exercise: (1) an increase in the number of longitudinal segments of the t-tubule network, (2) changes in the direction and disposition of triads, (3) the appearance of caveolar clusters, and (4) the appearance of pentads and heptads (close apposition of two or three t-tubule elements with three or four elements of terminal cisternae of the sarcoplasmic reticulum). The caveolar clusters appeared almost exclusively in the ST fibres immediately after downhill running exercise and again 16 h later. The pentads and heptads appeared almost exclusively in the FT fibres, and their numbers increased dramatically 2-3 days after the downhill running exercise. 4. The eccentric exercise led to the formation of abnormal membrane systems involved in E-C coupling processes. These systems have unique morphological features, which differ between ST and FT fibres, even within the same skeletal muscle, and the damage appears to be concentrated in the FT fibres. These observations also support the idea that eccentric exercise- induced E-C coupling failure is due to physical and chemical disruption of the membrane systems involved in the E-C coupling process in skeletal muscle.

Quantitative monitoring of circulating Epstein-Barr virus DNA for predicting the development of posttransplantation lymphoproliferative disease.

Epstein-Barr virus (EBV)-DNA was quantitatively measured to assess posttransplantation virus reactivation by real-time polymerase chain reaction (PCR). In the first retrospective analysis of a 7-year-old boy with lymphoproliferative disease (LPD) after an unrelated cord blood transplantation, serum EBV-DNA progressively increased to 4 x 10(5) copies/mL. EBV load was then prospectively monitored in peripheral blood from posttransplantation patients. The second case was an 8 year-old boy with aplastic anemia who received a CD34+ cell transplantation. This patient died of LPD with the progression of pulmonary nodules. EBV-DNA increased to 4 x 10(4) copies/mL after the control of cytomegalovirus reactivation. On the other hand, EBV-DNA was undetectable (<200 copies/mL) in the series of all 58 samples from 10 patients who did not develop LPD after hematopoietic stem cell transplantation. Sequential monitoring of circulating EBV-DNA by quantitative PCR may be a useful indicator for predicting the development of posttransplantation LPD.

Synthesis, structural characterization and antimicrobial activities of 4- and 6-coordinate nickel(II) complexes with three thiosemicarbazones and semicarbazone ligands.

To investigate the relationship between antimicrobial activities and the molecular structures of nickel(II) complexes with thiosemicarbazone and semicarbazone ligands, nickel(II) complexes with ligands Hmtsc, Hatsc, Hasc and H2dmtsc, were prepared and characterized by elemental analysis, FT-IR, 1H and 13C NMR spectroscopies, magnetic susceptibility measurements, UV-Vis absorption spectra, TG/DTA and single-crystal X-ray analysis. Their antimicrobial activities were evaluated by the MIC against four bacteria (B. subtilis, S. aureus, E. coli and P. aeruginosa), two yeasts (C. albicans and S. cerevisiae) and two molds (A. niger and P. citrinum). The 4-coordinate, diamagnetic nickel(II) complexes showed antimicrobial activities which were different from those of free ligands or the starting nickel(II) compounds; [Ni(mtsc)(OAc)] 1 showed selective and effective antimicrobial activities against two Gram-positive bacteria (B. subtilis and S. aureus) and modest activities against a yeast (S. cerevisiae), [Ni(mtsc)Cl] 3 exhibited moderate activities against a Gram-positive bacterium (S. aureus), and [Ni(atsc)(OAc)] 5 showed modest activities against two Gram-positive bacteria (B. subtilis and S. aureus). On the other hand, the 6-coordinate, paramagnetic nickel(II) complexes with two protonated or deprotonated ligands ([Ni(mtsc)2] 2, [Ni(atsc)(mtsc)] 4, [Ni(atsc)2] 6, [Ni(Hatsc)2](NO3)(2)7, [Ni(Hatsc)2]Cl(2)8 and [Ni(Hasc)2](OAc)(2)9) and the sterically crowded 4-coordinate, diamagnetic nickel(II) complex ([Ni(dmtsc)] 10) did not inhibit the growth of the test organisms. The structure-activity correlation in this series of nickel(II) complexes was discussed based on their ligand-replacement abilities.

Epstein-Barr virus (EBV) load and cytokine gene expression in activated T cells of chronic active EBV infection.

To identify the role of T cells in chronic active Epstein-Barr virus (EBV) infection, EBV and cytokine gene expression was quantified by use of real-time polymerase chain reaction (PCR) among 6 patients who fulfilled the diagnostic criteria for chronic active EBV infection. Four of these patients showed clonal expansion of EBV-infected T cells. Quantitative PCR for EBV DNA in peripheral blood of patients with symptomatic chronic active EBV infection showed higher copy numbers of virus (mean, 1.45 x 10(5) copies/mL) than were seen in blood from patients with infectious mononucleosis (3.08 x 10(3) copies/mL) or with EBV-associated hemophagocytosis (2.95 x 10(4) copies/mL). Fractionated CD3(+) HLA-DR(+) cells from patients with chronic active EBV infection contained higher copy numbers than did CD3(+) HLA-DR(-) cells. Quantitative PCR for cytokines revealed that interferon-gamma, interleukin (IL)-2, IL-10, and transforming growth factor-beta genes were expressed at higher levels in HLA-DR(+) than in HLA-DR(-) T cells. These results suggest that activated T cells in chronic active EBV infection expressed high levels of EBV DNA and both Th1 and Th2 cytokines. EBV-infected T cells may contribute to the unbalanced cytokine profiles of chronic mononucleosis.

Differential response of the membrane systems involved in excitation-contraction coupling to early and later postnatal denervation in rat skeletal muscle.

We compared the morphological features of the membrane systems involved in excitation-contraction (E-C) coupling during early postnatal development stages in rat skeletal muscles (tibialis anterior) denervated either at birth or 7 days after birth. Four obvious structural changes are observed in the arrangement of the transverse (T) tubule network and the disposition of triads following early postnatal denervation: (1) an increase in the longitudinal segments of the T tubule network, (2) changes in the direction and disposition of triads, (3) the appearance of caveolae clusters, (4) the appearance of pentads and heptads (i.e. a close apposition of two or three T tubule elements with three or four elements of terminal cisternae of sarcoplasmic reticulum). The increased presence of longitudinal T tubules parallels the loss of cross striations, and this in turn is due to misalignment of the myofibrils. The clusters of caveolae appear almost exclusively in muscle fibres denervated at birth, and pentads and heptads are more frequently observed in muscles denervated at 7 days. The differential growth of muscle fibres in response to denervation leads to the formation of abnormal membrane systems involved in the E-C coupling with very unique morphological features, which differ from the case of denervation in adult muscle fibres.

Differential normalization of mucosal interleukin-8 and interleukin-6 activity after Helicobacter pylori eradication.

There is differential resolution of mucosal infiltration with neutrophils and mononuclear cells following successful Helicobacter pylori eradication. We investigated the effects of H. pylori eradication on mucosal interleukin-8 (IL-8) and IL-6 activity in relation to the resolution of H. pylori-associated gastritis. Eighty-one duodenal ulcer patients with H. pylori infection received dual- or triple-treatment eradication therapy, and mucosal biopsy specimens obtained at the initial and follow-up endoscopic examinations were cultured in vitro for 24 h. The levels of IL-8 and IL-6 were measured by enzyme-linked immunosorbent assays. In the 42 patients in whom H. pylori eradication failed, there was little change in the numbers of neutrophils and mononuclear cells infiltrating the mucosa and in IL-8 and IL-6 activity. In the 39 patients in whom H. pylori was eradicated, there was normalization both in the numbers of infiltrating neutrophils and in mucosal IL-8 activity, which was evident within 1 month following therapy. In contrast, there was a gradual resolution of mononuclear cell infiltration over a 6-month period, accompanied by a gradual normalization in IL-6 levels. Addition of H. pylori to cultures of mucosal tissues induced a significant increase in IL-8 activity in both uninfected control subjects and patients from whom H. pylori was eradicated. However, this introduction yielded a significant increase in IL-6 activity only in the latter group. This study indicates a dichotomy in the changes of mucosal IL-8 and IL-6 activity after H. pylori eradication. The rapid normalization of IL-8 after H. pylori eradication and the ability of H. pylori cells to stimulate IL-8 in control tissues indicate that IL-8 induction is a part of the innate (nonimmune) responses to this organism. In contrast, the results of experiments analyzing IL-6 activity in cultured mucosal tissues suggest that the gradual resolution of mucosal IL-6 activity and mononuclear infiltration after successful eradication observed in vivo may reflect gradually diminishing residual immune responses against H. pylori.

Mucosal chemokine activity in Helicobacter pylori infection.

We examined secretion, mRNA expression, and histologic localization of interleukin-8 (IL-*) and growth-related gene product-alpha (GRO alpha) in the Helicobacter pylori-infected gastric antral mucosa. Antral biopsies were obtained from an area of endoscopically intact mucosa. Significantly higher levels of IL-8 and GRO alpha were secreted in organ cultures from patients with H. pylori infection, and their elevation was prominent in patients with duodenal ulcer. There was a significant association between these alpha-chemokine levels and histologic grades of activity, inflammation, and H. pylori density. In fresh antral biopsies, IL-8 and GRO alpha mRNA expression was detected more frequently in H. pylori-infected patients compared with those without infection. Immunofluorescence microscopy showed localization of IL-8 and GRO alpha proteins in gastric epithelial cells and infiltrating CD68+ macrophages. In the chemotaxis assay, a significant positive correlation was found between neutrophil migration induced by the organ culture supernatants and their contents of IL-8 and GRO alpha. After H. pylori eradication, a significant decrease was observed in IL-8 and GRO alpha levels detected in organ cultures. In conclusion, mucosal alpha-chemokine activity correlates well with histologic severity of H. pylori-associated antral gastritis and can be used to predict the effects of H. pylori eradication therapy.

Morphological changes in the triads and sarcoplasmic reticulum of rat slow and fast muscle fibres following denervation and immobilization.

We observed the morphological features of the membrane systems (sarcoplasmic reticulum, transverse tubules and triads) involved with the excitation-contraction coupling in rat soleus and extensor digitorum longus muscle following two disuse protocols: denervation and immobilization. The immobilized positions were: maximum dorsal flexor (soleus were stretched and extensor digitorum longus were shortened), maximum plantar flexor (soleus were shortened and extensor digitorum longus were stretched), and midway between the dorsal flexor and plantar flexor. The arrangement of the membrane systems was disordered following both disuse conditions. Increases in transverse tubule network were apparent; there were clearly more triads than in normal fibres, and pentadic and heptadic structures (i.e., a close approximation of two or three transverse tubule elements with three or four elements of terminal cisternae of sarcoplasmic reticulum) were frequently appeared following both denervation and immobilization. The most notable difference between the influence of denervation and immobilization on the membrane systems is the time at which the pentads and heptads appeared. They appeared much earlier (1 week after denervation) in denervated than in immobilized (3 or 4 weeks after immobilization) muscle fibres. On the other hand, the frequency of pentads and heptads is clearly related to the fibre type (significantly higher in extensor digitorum longus) and to extent of atrophy. The different influences of immobilization in each leg position suggest that disuse, but with neurotrophic factor(s), influences on the membrane systems were affected by sarcomere length, and the neurotrophic factor(s) and muscle activity were not always necessary to form new membrane systems in disuse skeletal muscle fibres.

Influences of sarcomere length and selective elimination of myosin filaments on the localization and orientation of triads in rat muscle fibres.

Ultrastructural features of internal membrane systems directly concerned with the excitation-contraction coupling were observed in chemically skinned muscle bundles prepared from Wistar rat extensor digitorum longus muscle to clarify two questions: (1) whether triads localization and orientation are influenced by the sarcomere length and (2) whether triads localisation and orientation are influenced by the selective elimination of myosin filaments. The distance between triads and Z-lines depends on the sarcomere length: it increase with sarcomere length. There is a highly significant (p < 0.01) positive correlation between sarcomere length and the distance between triads and Z-line. The distance between Z-line and triads is dependent on sarcomere length, but the width of junctional gap remains constant when the sarcomere length was changed. Incubation in a concentration of KCI, which dissolves the myosin filaments. The localization and orientation of triads was not altered by the elimination of myosin filaments, however, the distance between the Z-line and triads becomes shorter when the myosin filaments was completely eliminated. There were significant differences (p < 0.01) between control and myosin filament eliminated fibres in the distances between Z-lines and triads (over 2 microns). These results indicate that the distance between triads and Z-lines depend on the sarcomere length and that there may be some connection(s) between triads and the myofibrils. There is that the elastic component responsible for tethering the triads in their normal position is interrupted either because it is normally attached to the myosin filaments, or because it is extracted by the conditions that dissociate myosin filaments.

Postnatal changes in cell body size and oxidative enzyme activity of spinal motoneurons innervating the rat tibialis anterior muscle.

The cell body size and succinate dehydrogenase (SDH) activity of spinal motoneurons innervating the superficial and deep regions of the tibialis anterior muscle were studied in rats ranging in postnatal age from 3 to 11 weeks, by retrograde neuronal labeling using fluorescent neuronal tracers. The motoneurons innervating the tibialis anterior muscle were located primarily at the L4 spinal cord segment and those innervating the superficial and deep regions of the muscle were distributed throughout the entire extent of the motoneuron pool. The distribution of the motoneurons during postnatal development was similar to that observed in the adult animal. The mean cell body size of the motoneurons innervating the superficial region of the muscle in rats from 5 to 11 weeks of age was greater than that innervating the deep region at corresponding ages. The mean SDH activity of the motoneurons innervating the deep region of the muscle increased during postnatal development, while there were no changes in the mean SDH activity of those innervating the superficial region during this period. At 11 weeks of age, the motoneurons innervating the deep region of the muscle had a higher mean SDH activity than those innervating the superficial region. An inverse relationship between cell body size and SDH activity of motoneurons innervating both the superficial and deep regions of the muscle was observed, independent of age. These results indicate that motoneurons innervating the superficial and deep regions of the rat tibialis anterior muscle have different developmental patterns with regard to cell body size and SDH activity.

Differences in ultrastructural and metabolic profiles within the same type of fibres in various muscles of young and adult rats.

Single fibres from tibialis anterior, extensor digitorum longus, gastrocnemius and soleus muscles in young (4-week-old) and adult (35-week-old) Wistar male rats were classified into three types on the basis of their enzyme-histochemical features: slow-twitch oxidative (SO), fast-twitch oxidative and glycolytic (FOG) and fast-twitch glycolytic (FG) fibres. Ultrastructural (volume density of mitochondria: Vmt and Z line width) and metabolic (phosphofructokinase: PFK and succinate dehydrogenase: SDH activities) profiles were measured. PFK activity in all types of fibres was higher in adult rats, and the difference between the two age-groups (adult/young) was largest between FG, FOG and SO fibres respectively. SDH activity and Vmt were lower in adult rats in a similar way in all fibres. A significant positive correlation was observed between the Vmt and SDH activity in both age-groups. This positive correlation was very specific in fast-twitch and slow-twitch fibres. Changes in the Vmt did not relate directly to the changes in fibre cross-sectional area. The overall pattern indicates that glycolytic capacity of fast-twitch fibres in flexor muscles (TA and EDL) is higher than in extensor muscles (GC and SOL), and that oxidative capacity of all types of fibre in extensor muscles is higher than in flexor muscles. These profiles were changed by growth, and may be related to the specific differences in pattern of activity of each skeletal muscle, and may reflect differences in the recruitment order of different muscles.

[Fiber types and some contractile properties of extensor digitorum longus and soleus muscles in different animal species].

We studied the fiber types and contractile properties of the extensor digitorum longus (EDL) and soleus (SOL) muscles from young adult mice, rats and guinea pigs, and the correlation between these two parameters. Individual fibers in both muscles were classified as fast-twitch glycolytic (FG), fast-twitch oxidative glycolytic (FOG) or slow-twitch oxidative (SO) fibers according to Peter et al., and type II B, II A, or I fibers according to Brooke & Kaiser. Contractile properties were measured in situ at 37 degrees C. The isometric twitch contraction time (CT) and one-half relaxation time (1/2 RT) tended to be shortened in proportion to the area occupied by type II fibers, and type II B fibers. However, the differences between CT and fiber types were not always uniform among the three species. The CT of the rat EDL, in spite of its higher proportion of type II B fibers about 10% was the same as that of the guinea-pig EDL. The SOL of the mouse, composed of about 50% type I (SO) fibers, had a CT about as short as that of the EDL. In the case of the classification by Peter et al., the relationship between the percentage of subgroups of fast-twitch fibers and the CT or 1/2 RT, but not the resistance to fatigue, was not obvious. The resistance to fatigue tended to be enhanced in proportion to the area occupied by FOG in the EDL and by SO (type I) in the SOL. These results suggest that the contractile properties of individual fibers identified histochemically are distinct among animal species, producing interspecies differences in fiber types along with different contractile properties. However, it may be possible to compare the difference between fiber types and CT or 1/2 RT in the classification based on the pH lability of myosin ATPase, and also the difference between fiber types and resistance to fatigue in the classification based on the oxidative enzyme.

Deterioration induced by physiological concentration of calcium ions in skinned muscle fibres.

The deteriorating effect of microM order of Ca2+ on skinned frog skeletal muscle fibres was studied from the view point of the digestion of proteins by calcium-activated neutral protease (CANP). Tension developed in solutions containing no MgATP (rigor solution) decreased irreversibly with the addition of Ca2+ in quantities of more than 0.1 microM. Low temperature was seen to suppress (Q10 greater than 4), and neutral pH to maximize, this decrease in tension. In rigor solution containing Ca2+, SDS electrophoresis indicated that a 95 k dalton component (alpha-actinin) was released from the fibre; electron micrography showed the disappearance of Z-lines. These results suggest that one of the causes for decrease in rigor tension is the proteolytic activity of CANP, and its inhibitors were shown to be quite useful in experiments on skinned fibre.

Radial stiffness of frog skinned muscle fibers in relaxed and rigor conditions.

Radial stiffness in various conditions of mechanically skinned fibers of semitendinosus muscle of Rana catesbeiana was determined by compressing the fiber with polyvinylpyrrolidone (PVP K-30, Mr = 40,000) in incubating solution. The change in width (D) of fibers with increasing and decreasing PVP concentrations was highly reproducible at a range 0-6% PVP. Radial stiffness of relaxed fibers was almost independent of the sarcomere length. On the other hand, radial stiffness of rigor fibers showed a linear relation against the sarcomere length. These results indicate that cross-bridge attachment would be a major factor in the increase of the radial stiffness. Radial stiffness of relaxed and rigor fibers was (2.14 +/- 0.52) X 10(4) N/m2 (mean +/- SD) and (8.76 +/- 2.04) X 10(4) N/m2, respectively, at the relative fiber width (D/D0) of 0.92, where D0 denotes the fiber width in the rigor solution at 0% PVP. Radial stiffness of a fiber in a rigor solution containing pyrophosphate (PPi) was between those of relaxed and rigor fibers, i.e., (4.76 +/- 0.86) X 10(4) N/m2 at D/Do of 0.92. In PPi and rigor solutions, radial stiffness reversibly increased to around 150 and 130%, respectively, in the presence of 10(-6) M Ca2+. To explain these results, especially the Ca2+-induced change in the radial stiffness, some factor in addition to the number of attached cross-bridges has to be taken into account. The variation of radial stiffness under various conditions will be discussed in relation to the possible manner of cross-bridge attachment.