Influence of icing on muscle regeneration after crush injury to skeletal muscles in rats

The influence of icing on muscle regeneration after crush injury was examined in the rat extensor digitorum longus. After the injury, animals were randomly divided into nonicing and icing groups. In the latter, ice packs were applied for 20 min. Due to the icing, degeneration of the necrotic muscle fibers and differentiation of satellite cells at early stages of regeneration were retarded by ～1 day. In the icing group, the ratio of regenerating fibers showing central nucleus at 14 days after the injury was higher, and cross-sectional area of the muscle fibers at 28 days was evidently smaller than in the nonicing group. Besides, the ratio of collagen fibers area at 14 and 28 days after the injury in the icing group was higher than in the nonicing group. These findings suggest that icing applied soon after the injury not only considerably retarded muscle regeneration but also induced impairment of muscle regeneration along with excessive collagen deposition. Macrophages were immunohistochemically demonstrated at the injury site during degeneration and early stages of regeneration. Due to icing, chronological changes in the number of macrophages and immunohistochemical expression of transforming growth factor (TGF)-β1 and IGF-I were also retarded by 1 to 2 days. Since it has been said that macrophages play important roles not only for degeneration, but also for muscle regeneration, the influence of icing on macrophage activities might be closely related to a delay in muscle regeneration, impairment of muscle regeneration, and redundant collagen synthesis.     

Simulation of ATP metabolism in cardiac excitation–contraction coupling

To obtain insights into the mechanisms underlying the membrane excitation and contraction of cardiac myocytes, we developed a computer model of excitation–contraction coupling (Kyoto model: Jpn. J. Physiol. 53 (2003) 105). This model was further expanded by incorporating pivotal reactions of ATP metabolism; the model of mitochondrial oxidative phosphorylation by Korzeniewski and Zoladz (Biophys. Chem. 92 (2001) 17). The ATP-dependence of contraction, and creatine kinase and adenylate kinase were also incorporated. After minor modifications, the steady-state condition was well established for all the variables, including the membrane potential, contraction, and the ion and metabolite concentrations in sarcoplasmic reticulum, mitochondria and cytoplasm. Concentrations of major metabolites were close to the experimental data. Responses of the new model to anoxia were similar to experimental results of the P-31 NMR study in whole heart. This model serves as a prototype for developing a more comprehensive model of excitation–contraction–metabolism coupling.     

MRI-Based Analysis of Intracerebral Hemorrhage in Mice Reveals Relationship between Hematoma Expansion and the Severity of Symptoms

Intracerebral hemorrhage (ICH) is featured by poor prognosis such as high mortality rate and severe neurological dysfunction. In humans, several valuables including hematoma volume and ventricular expansion of hemorrhage are known to correlate with the extent of mortality and neurological dysfunction. However, relationship between hematoma conditions and the severity of symptoms in animal ICH models has not been clarified. Here we addressed this issue by using 7-tesla magnetic resonance imaging (MRI) on collagenase-induced ICH model in mice. We found that the mortality rate and the performance in behavioral tests did not correlate well with the volume of hematoma. In contrast, when hemorrhage invaded the internal capsule, mice exhibited high mortality and showed poor sensorimotor performance. High mortality rate and poor performance in behavioral tests were also observed when hemorrhage invaded the lateral ventricle, although worsened symptoms associated with ventricular hemorrhage were apparent only during early phase of the disease. These results clearly indicate that invasion of the internal capsule or the lateral ventricle by hematoma is a critical determinant of poor prognosis in experimental ICH model in mice as well as in human ICH patients. MRI assessment may be a powerful tool to refine investigations of pathogenic mechanisms and evaluations of drug effects in animal models of ICH.     

Influence of Structural Symmetry on Protein Dynamics

Structural symmetry in homooligomeric proteins has intrigued many researchers over the past several decades. However, the implication of protein symmetry is still not well understood. In this study, we performed molecular dynamics (MD) simulations of two forms of trp RNA binding attenuation protein (TRAP), the wild-type 11-mer and an engineered 12-mer, having two different levels of circular symmetry. The results of the simulations showed that the inter-subunit fluctuations in the 11-mer TRAP were significantly smaller than the fluctuations in the 12-mer TRAP while the internal fluctuations were larger in the 11-mer than in the 12-mer. These differences in thermal fluctuations were interpreted by normal mode analysis and group theory. For the 12-mer TRAP, the wave nodes of the normal modes existed at the flexible interface between the subunits, while the 11-mer TRAP had its nodes within the subunits. The principal components derived from the MD simulations showed similar mode structures. These results demonstrated that the structural symmetry was an important determinant of protein dynamics in circularly symmetric homooligomeric proteins.     

Chimeric gene library construction by a simple and highly versatile method using recombination-dependent exponential amplification

A simple and efficient method for the construction of chimeric gene libraries termed RDA-PCR (recombination-dependent exponential amplification polymerase chain reaction) was developed by modifying polymerase chain reaction. A chimeric gene library is generated from homologous parental genes with additional primer-annealing sequences at their "heads" and "tails". Two primers ("skew primers") are designed to exclusively anneal to either the heads of maternal genes or the tails of paternal genes. During the RDA-PCR, short annealing/extension periods facilitate homologous recombination. The chimeric sequences can be exponentially amplified to form the chimeric gene library, whereas parental sequences without crossovers are not amplified. As a model, we constructed a chimeric gene library of yellow and green fluorescent protein (yfp and gfp, respectively). The crossover point profile of RDA-PCR clones was compared with those obtained by (modified) family shuffling. PCR restriction fragment polymorphism (PCR-RFLP) analysis of the RDA-PCR clones showed a high content of chimeric genes in the library, whereas family shuffling required the modification using skew primers for selective enrichment of chimeric sequences. PCR-RFLP analysis also indicated that the crossover points of RDA-PCR chimeras were distributed over the entire protein-coding region. Moreover, as few as 2 bp of the continual identity of nucleotides were found at the crossover points at high frequency (30% of the tested clones), suggesting that RDA-PCR resulted in a higher diversity in crossover points than family shuffling.     

Evaluation of the influence of muscle deactivation on other muscles and joints during gait motion

When any muscle in the human musculoskeletal system is damaged, other muscles and ligaments tend to compensate for the role of the damaged muscle by exerting extra effort. It is beneficial to clarify how the roles of the damaged muscles are compensated by other parts of the musculoskeletal system from the following points of view: From a clinical point of view, it will be possible to know how the abnormal muscle and joint forces caused by the acute compensations lead to further physical damage to the musculoskeletal system. From the viewpoint of rehabilitation, it will be possible to know how the role of the damaged muscle can be compensated by extra training of the other muscles. A method to evaluate the influence of muscle deactivation on other muscles and joints is proposed in this report. Methodology based on inverse dynamics and static optimization, which is applicable to arbitrary motion was used in this study. The evaluation method was applied to gait motion to obtain matrices representing (1) the dependence of muscle force compensation and (2) the change to bone-on-bone contact forces. These matrices make it possible to evaluate the effects of deactivation of one of the muscles of the musculoskeletal system on the forces exerted by other muscles as well as the change to the bone-on-bone forces when the musculoskeletal system is performing the same motion. Through observation of this matrix, it was found that deactivation of a muscle often results in increment/decrement of force developed by muscles with completely different primary functions and bone-on-bone contact force in different parts of the body. For example, deactivation of the iliopsoas leads to a large reduction in force by the soleus. The results suggest that acute deactivation of a muscle can result in damage to another part of the body. The results also suggest that the whole musculoskeletal system must go through extra retraining in the case of damage to certain muscles.     

Simultaneous determination of prednisolone, prednisone, cortisol, and cortisone in plasma by GC-MS: estimating unbound prednisolone concentration in patients with nephrotic syndrome during oral prednisolone therapy

Individual variability of the pharmacokinetics of prednisolone based on the unbound concentration in plasma is of significant clinical consideration. The unbound concentrations of prednisolone were measured in 10 patients with nephrotic syndrome, two patients with systemic lupus erythematosus, and one patient with dermatomyositis by examining protein bindings of prednisolone on one or more occasions during prednisolone treatment. In this study, plasma concentrations of prednisolone, prednisone, cortisol, and cortisone were simultaneously analyzed by GC-MS by using stable isotope-labeled internal standards. Equilibrium dialysis was employed to accurately estimate the unbound fractions of prednisolone in plasma. The unbound fraction of prednisolone changed depending on plasma total prednisolone concentration and plasma albumin concentration. The unbound fraction of prednisolone (Y) is calculated: Y=(-0.0101x' + 0.0736) x + 10.23, where x' is the plasma albumin concentration and x is the total prednisolone concentration. The estimated concentrations of unbound prednisolone by using the above equation were in good agreement with the measured concentrations of unbound prednisolone. Since the protein binding of prednisolone did not change in the presence of prednisone (114.0 ng/ml), it appeared that prednisone produced from the therapeutic dose of prednisolone did not affect the unbound fraction of prednisolone.