Tsuyoshi Totsuka

Differential adaptation of growth and differentiation factor 8/myostatin, fibroblast growth factor 6 and leukemia inhibitory factor in overloaded, regenerating and denervated rat muscles.

Mice genetically deficient in growth and differentiation factor 8 (GDF8/myostatin) had markedly increased muscle fiber numbers and fiber hypertrophy. In the regenerating muscle of mice possessing FGF6 mutation, fiber remodeling was delayed. Although myostatin and FGF6 may be important for the maintenance, regeneration and/or hypertrophy of muscle, little work has been done on the possible role of these proteins in adult muscle in vivo. Using Western blot and immunohistochemical analysis, we investigated, in rats, the distribution of myostatin, FGF6 and LIF proteins between slow- and fast-type muscles, and the adaptive response of these proteins in mechanically overloaded muscles, in regenerating muscles following bupivacaine injection and in denervated muscles after section of the sciatic nerve. The amounts of myostatin and LIF protein were markedly greater in normal slow-type muscles. In the soleus muscle, myostatin and LIF proteins were detected at the site of the myonucleus in both slow-twitch and fast-twitch fibers. In contrast, FGF6 protein was selectively expressed in normal fast-type muscles. Mechanical overloading rapidly enhanced the myostatin and LIF but not FGF6 protein level. In the regenerating muscles, marked diminution of myostatin and FGF6 was observed besides enhancement of LIF. Denervation of fast-type muscles rapidly increased the LIF, but decreased the FGF6 expression. Therefore, the increased expressions of myostatin and LIF play an important role in muscle hypertrophy following mechanical overloading. The marked reduction of FGF6 in the hypertrophied and regenerating muscle would imply that FGF6 regulates muscle differentiation but not proliferation of satellite cells and/or myoblasts.

Polymerization of plasmodium actin

Abstract 1. (1)|On the addition of monovalent cations, plasmodium G-actin is polymerized to a fibrous polymer, termed plasmodium F-actin. The F-actin has a high viscosity, and shows a positive flow birefringence. During polymerization, 1 mole of inorganic phosphate is liberated from 1 mole of G-actin. Thus: G-actin-ATP ⇄ ATP 0.1 M KCl F-actin-ADP + P i Kinetic and structural analyses shows the F-actin to be a microfilament, 60 A in diameter, having a two-stranded helical structure with a half pitch of about 290 A containing about 13 spherical units. 2. (2)|Addition of divalent cations forms another kind of polymer, termed Mg-polymer, which has a relatively low viscosity. During the polymerization, 1 mole of inorganic phosphate is liberated from 1 mole of G-actin, suggesting the reaction: G-actin-ATP ⇄ ATP 2 mM MgCl 2 Mg-polymer-ADP + P i Moreover, this polymer itself seems to have ATP-splitting activity. Mg-polymer appears as a globular aggregate in electron micrographs, with a diameter of about 100–600 A. 3. (3)|F-Actin and Mg-polymer are depolymerized to G-actin by removing salts from their solutions in the presence of ATP and cysteine, for example, by dialysis. The resultant G-actin may be polymerized again to one of the two polymer forms. Moreover, it is suggested that F-actin and Mg-polymer can transform into each other directly, not passing through the G-actin state. 4. (4)|Plasmodium G-actin can be copolymerized with rabbit striated muscle G-actin on the addition of monovalent cations or divalent cations.

A possible role for BDNF, NT-4 and TrkB in the spinal cord and muscle of rat subjected to mechanical overload, bupivacaine injection and axotomy.

Neurotrophins play a crucial role in the regulation of survival and the maintenance of specific functions for various populations of neurons. Neurotrophin-4 (NT-4) is most abundant in skeletal muscle, and is thought to promote sciatic nerve sprouting, inhibit agrin-induced acetylcholine receptor (AChR) clustering, evoke postsynaptic potentiation and induce mitochondrial proliferation. Using Western blot analysis, immunoprecipitation and immunohistochemistry, we investigated the distribution of NT-4 in slow- and fast-type muscles. We also tested the adaptive response of this protein in the mechanically overloaded muscle, in the regenerating muscle following bupivacaine injection and in the denervated muscle. Additionally, we investigated whether TrkB phosphorylation in the spinal cord and in the sciatic nerve occurs through the interaction with BDNF or NT-4 when the innervating muscle is damaged. Markedly more NT-4 was expressed in fast-type muscles compared with the slow types. TrkB protein was more frequently observed around the edge of myofibers (neuromuscular junction) of the soleus muscle compared with the gastrocnemius muscle. TrkB tyrosine phosphorylation occurred in the spinal cord but not in the sciatic nerve 24 h after bupivacaine injection of the innervating muscle. At the same time, the amount of TrkB co-precipitating with BDNF was markedly increased in the spinal cord. A rapid activation of TrkB (1-8 h) was also observed in the spinal cord after axotomy,while the amount of TrkB co-precipitating with NT-4 was markedly lower after axotomy. These results indicate that NT-4 is preferentially distributed in fast-type muscles. Furthermore, by interacting with BDNF and NT-4, the TrkB in the spinal cord may be important for the survival of motoneurons and outgrowth of injured peripheral axons following muscle damage.

The adaptive response of MyoD family proteins in overloaded, regenerating and denervated rat muscles

Using Western blot analysis, we investigated whether the amount of myogenic regulatory factors differs in slow-type and fast-type muscles. In addition, we examined the adaptive response of myogenic regulatory factor protein in the overloaded rat muscles by the ablation of synergists, in the regenerating muscles following bupivacaine injection and in the denervated muscle. The amount of myogenin protein in the slow-type muscle was markedly greater. In contrast, the proteins MyoD and Myf-5 were selectively accumulated in the fast-type muscles. A gradual down-regulation of MyoD and Myf-5 proteins was detected in the denervated fast-type muscles, but not in the myogenin protein content. A rapid down-regulation of myogenic regulatory factor protein was observed both of the mechanically overloaded and in the regenerating muscles. These results indicate that the fast-type-specific gene expression in muscle is modulated by MyoD and Myf-5 proteins and suggest that myogenin protein plays an important role in the reconstruction of damaged neuromuscular connections.

Differential adaptations of insulin-like growth factor-I, basic fibroblast growth factor, and leukemia inhibitory factor in the plantaris muscle of rats by mechanical overloading: an immunohistochemical study

Abstract We investigated changes in several growth factors in the rat plantaris muscle produced by mechanical overloading by ablation of synergists using immunohistochemistry. At 1 and 3 days post surgery, the insulin-like growth factor-I (IGF-I) level was slightly increased in the cytosol and markedly increased in the invading cells of the extracellular space. Thereafter, the IGF-I immunoreactivity evoked by overloading rapidly decreased to the normal level. The level of leukemia inhibitory factor (LIF), which was not shown to change at 1 day post surgery, was increased in the cytosol at 3, 5, 7 and 10 days and at 2 weeks. Basic fibroblast growth factor (bFGF) immunoreactivity did not change during the entire period of overloading (1 day–3 weeks post surgery). These results indicate that the elevations of the levels of IGF-I and LIF show differential time course in the plantaris muscle subjected to functional overload. Furthermore, bFGF appears not to be related to the compensatory hypertrophy produced by overloading.

The adaptive response of transforming growth factor-β2 and -βRII in the overloaded, regenerating and denervated muscles of rats

Abstract Using a muscle cell line and satellite cell cultures, it has been shown that transforming growth factor-β (TGF-β) has a powerful inhibitory effect on myoblast replication and differentiation. However, little work has been done on the possible role of TGF-β in adult muscle in vivo. Using Western blot and immunohistochemical analyses, we investigated normal distribution of TGF-β2 and TGF-βRII proteins between slow and fast-type muscles, and the adaptive response of these proteins in the mechanically overloaded muscles, in the regenerating muscles following bupivacaine injection and in the denervated muscle after section of sciatic nerve. Slight TGF-β2 immunoreactivity was detected both in slow- and fast-type muscles of mature rat. The amount of TGF-βRII protein was markedly greater in fast-type muscles. In the overloaded muscle, immunohistochemical analysis showed a marked increase in TGF-β2 immunoreactivity in the mononuclear cells (probably endothelial and perithelial or smooth muscle cells of endomysial capillaries) of the extracellular space at 3 and 6 days post surgery. Rapid increase of TGF-β2 protein and concomitant decrease of the receptor (TGF-βRII) were observed in the mechanically overloaded and regenerating muscles. On the other hand, denervation of slow- and fast-type muscles showed a rapid increase in TGF-β2 protein, but did not elicit a concomitant decrease of TGF-βRII. These results indicate that TGF-βRII is preferentially distributed in fast-type muscles. Furthermore, TGF-β2 may play an important role in muscle hypertrophy and regeneration by the usage of TGF-βRII.

A convenient bioassay for NGF using a new subline of PC12 pheochromocytoma cells (PC12D).

A useful bioassay for nerve growth factor (NGF) has been developed, based on the rapid outgrowth of neurites (within 24 h) from cells of a new subclone of PC12 cells (PC12D) in response to NGF. The sensitivity is similar to that of other bioassay systems that the the sensory ganglia of chick embryos of primed PC12 cells. The assay is readily adaptable for the purification of NGF and for the determination of levels of NGF in tissue, as shown by a comparison of results from this assay to the data obtained by an immunological assay.

A factor protecting mammalian [75Se]SeCys-tRNA is different from EF-1α

In Escherichia coli, an elongation factor (EF‐Tu‐like) specific to SeCys‐tRNA, SELB, has been identified; however, a mammalian counterpart of SELB has not been reported to date. We searched for and found this factor in bovine liver extracts using the assay of [75Se]SeCys‐tRNA protecting activity against alkaline hydrolysis (SePF activity). We found SePF activity in the protein extracts of the precipitate (microsomal fraction) collected at 150,000 × g from bovine liver. The proteins were separated by Sephacryl S‐300 chromatography, and the SePF and EF‐1α activities were found in the same fraction, indicating that SePF and EF‐1α have the same molecular mass (approximately 50 kDa). We then chromatographed this active fraction using CM‐Sephadex C‐25 columns. The SePF activity was eluted after the peak of EF‐1α activity. This result indicated that this SePF activity was not dependent on EF‐1α. In addition to performing these two chromatographies, we investigated pure EF‐1α from Bombyx mori but could not detect any SePF activity in B. mori EF‐1α. Thus we showed that the SePF activity in bovine liver differs from that of EF‐1α in eukaryotes. Therefore the factor protecting [75Se]SeCys‐tRNA in bovine liver is not EF‐1α and must be a SELB‐like factor.

ATPase activity of plasmodium actin polymer formed in the presence of Mg2

Abstract 1. In the presence of Mg2+, plasmodium actin forms a polymer different from F-actin. This polymer, termed Mg-polymer, has an ATPase activity. Its specific activity is about one-hundredth of the activity of myosin from muscle or plasmodium and of the same order as that of muscle F-actin under sonic vibration. 2. The formation of Mg-polymer and its ATPase activity are not due to modification of the protein in the preparation procedure. The ATPase activity is not due to contamination in the preparation but is closely related to the amount of Mg-polymer formed. 3. The ATPase activity of Mg-polymer is markedly inhibited by high concentration of ATP (about 1 mM) in the presence of KCl; but it is not inhibited in the absence of KCl. 4 Mg-polymer releases its bound [14C]ADP in a cold ATP solution. The half time of the release is about 10 min at 22°, which is of the same order as the rate of ATP splitting. This is understandable if both the ATP splitting and the ADP exchange are associated with the same cyclic process of intrapolymer reaction occurring everywhere along the Mg-polymer. 5. Copolymers of plasmodium and muscle G-actins are formed in the presence of Mg2+. The ATPase activity of copolymers decreases rapidly with increasing content of muscle actin; it is suggested that interaction of neighboring plasmodium actin molecules in the polymer is required for the ATPase activity.

Electric birefringence of myosin subfragments

Electric birefringence measurements have been made on aqueous solutions of myosin subfragments, heavy meromyosin, subfragments 1 and 2 (S-1 and S-2). All of these showed positive electric birefringence. Heavy meromyosin and S-2 showed a large intrinsic Kerr constant. From the analysis of the build up and decay process of the birefringence, the contribution of the slow induced dipole moment was concluded in heavy meromyosin and S-2, although the existence of the permanent dipole moment was not completely excluded. The decay process of the birefringence of heavy meromyosin was found to consist of two components; the fast one of which had a relaxation time of the same order as that of S-1. This is probably due to the presence of a flexible hinge in heavy meromyosin.