Graph-based analysis of kinetics on multidimensional potential-energy surfaces.

The aim of this paper is twofold: one is to give a detailed description of an alternative graph-based analysis method, which we call saddle connectivity graph, for analyzing the global topography and the dynamical properties of many-dimensional potential-energy landscapes and the other is to give examples of applications of this method in the analysis of the kinetics of realistic systems. A Dijkstra-type shortest path algorithm is proposed to extract dynamically dominant transition pathways by kinetically defining transition costs. The applicability of this approach is first confirmed by an illustrative example of a low-dimensional random potential. We then show that a coarse-graining procedure tailored for saddle connectivity graphs can be used to obtain the kinetic properties of 13- and 38-atom Lennard-Jones clusters. The coarse-graining method not only reduces the complexity of the graphs, but also, with iterative use, reveals a self-similar hierarchical structure in these clusters. We also propose that the self-similarity is common to many-atom Lennard-Jones clusters.

Connectivity graph: multiple connectivity on potential energy surface does affect the dynamics.

A mapping called a connectivity graph is proposed to visualize the complicated networks between local minima via saddles on many-dimensional potential energy surfaces. With this mapping, the effect of the topography of a complex potential energy surface on the dynamics is studied in model funnel potentials and a Lennard-Jones cluster. We present strong evidence that energetically expensive but dynamically relevant saddles are indispensable for kinetic dynamics. The results show that the connectivity graphs provide a sound basis for understanding nonequilibrium dynamics.

Frameshift mutation in the collagen VI gene causes Ullrich's disease.

Patients with Ullrich's disease have generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. Recently, we found a deficiency of collagen VI protein in two patients with Ullrich's disease. In this study, we detected a homozygous 26 bp deletion in exon 14 of the collagen VI alpha 2 gene (COL6A2) in one patient. This mutation causes a frameshift and a premature termination codon, and results in a truncated collagen VI alpha 2 chain. Our data suggest that at least some cases of Ullrich's disease result from recessive mutations in COL6A2.

Microvascular endothelial abnormality in skeletal muscle from a patient with gastric cancer without dermatomyositis.

We found a microvascular endothelial abnormality in a biopsy specimen from the gastrocnemius muscle of a patient with gastric cancer, who had severe myalgia and angialgia in the calf region with the symptoms of thrombophlebitis. There were no definite findings of inflammatory myopathy in histochemical and immunohistochemical studies. Electron microscopic examination revealed the accumulation of abnormal mitochondria in the subsarcolemmal area, and a fair number of degenerating capillaries. Immunohistochemical analysis of procoagulant or anticoagulant factors revealed marked reduction of thrombomodulin (TM) expression on small vessels and capillaries. Although a reduction of TM on small vessels has been observed around perifascicular atrophic fibers in patients with dermatomyositis, histochemical findings of the present patient showed no perifascicular atrophy or severely degenerating fibers. These pathological findings in the patient may be related to a malignant neoplasm and may be one of the causes of disseminated intravascular coagulation (DIC), which is the main complication of malignant neoplasms. Further studies are necessary to determine whether the reduction of TM on the small vessels and capillaries in skeletal muscle is a predictor of some severe condition such as DIC or a rare pathological finding in some special condition such as scirrhous carcinoma with thrombophlebitis.

Experimental observation of further frequency upshift from dc to ac radiation converter with perpendicular dc magnetic field

A frequency upshift of a short microwave pulse is generated by the interaction between a relativistic underdense ionization front and a periodic electrostatic field with a perpendicular dc magnetic field. When the dc magnetic field is applied, further frequency upshift of 3 GHz is observed with respect to an unmagnetized case which has typically a GHz range. The radiation frequency depends on both the plasma density and the strength of the dc magnetic field, i.e., the plasma frequency and the cyclotron frequency. The frequency of the emitted radiation is in reasonable agreement with the theoretical values.

Characteristic expression of thrombomodulin in the muscle sarcoplasm in patients with the acute phase of rhabdomyolysis.

The expression of thrombomodulin and neural cell adhesion molecule (NCAM) was studied immunocytochemically in biopsied muscle specimens from 10 patients with rhabdomyolysis with different etiologic factors, including 5 with malignant hyperthermia. We have already reported that thrombomodulin was expressed on regenerating muscle cell membranes as well as on vessel walls in patients with various neuromuscular diseases, including Duchenne muscular dystrophy, Becker muscular dystrophy and inflammatory myopathy. We found increased expression of thrombomodulin not only on the sarcolemma, but also in the sarcoplasm of a fair number of muscle fibers in the acute phase of rhabdomyolysis. The granular pattern of thrombomodulin expression in the sarcoplasm seems to be a characteristic finding in the acute phase of rhabdomyolysis. Most muscle fibers which expressed NCAM on the sarcolemma also expressed thrombomodulin. However, the muscle fibers which expressed thrombomodulin in the sarcoplasm did not express NCAM, and showed a degenerative appearance on electron microscopic examination. These results suggest that thrombomodulin is expressed in the sarcoplasm during the acute degeneration phase of rhabdomyolysis in addition to the expression on the sarcolemma during the muscle fiber regeneration as shown in our previous study, and the former process, which is characterized by the granular expression of thrombomodulin in the sarcoplasm, may be a characteristic finding in rhabdomyolysis.

An overexpression of fibroblast growth factor (FGF) and FGF receptor 4 in a severe clinical phenotype of facioscapulohumeral muscular dystrophy.

We evaluated the expression of a select panel of growth factors and their receptors, including fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2), platelet-derived growth factor (PDGF), FGF receptor 1 (FGF-R1), FGF receptor 3 (FGF-R3), FGF receptor 4 (FGF-R4), PDGF receptor alpha (PDGF-Ralpha), PDGF receptor beta (PDGF-Rbeta), and heparan sulfate proteoglycan (HSPG), in muscle biopsy specimens from nine facioscapulohumeral muscular dystrophy (FSHD) patients using immunohistochemistry. Two cases of Duchenne-type muscular dystrophy (DMD), two of Becker-type muscular dystrophy (BMD), and one of limb-girdle-type muscular dystrophy (LGMD) were also investigated. Widespread immunostaining for FGF-1 and FGF-2 on the sarcolemma and overexpression of FGF-R4 in endomysial and perimysial connective tissue were seen in one patient with a severe clinical phenotype of FSHD who had respiratory failure. Standard histochemistry in this patient revealed marked interstitial fibrosis and lobulated fibers. The overexpression of FGF and FGF-R4 in this severe FSHD case may be associated with the muscle fibrosis and disease severity.

Expression of thrombomodulin, caveolin-3, and desmin on muscle fibers in neuromuscular diseases.

The expression of thrombomodulin (TM) and caveolin-3 on biopsied muscle fibers from 17 patients with various neuromuscular diseases was studied immunocytochemically. We also compared the expression of TM and caveolin-3 with that of desmin, which is a marker for muscle fiber regeneration. Most TM-positive muscle fibers strongly expressed caveolin-3 and desmin. Our results suggest that TM is expressed and caveolin-3 is upregulated during the process of muscle fiber regeneration.

Multiple episodes of thrombosis in a patient with Becker muscular dystrophy with marked expression of utrophin on the muscle cell membrane.

We previously reported a patient with Becker muscular dystrophy (BMD) who exhibited a benign clinical phenotype and marked expression of utrophin on the muscle cell membrane. The patient developed multiple episodes of thrombosis (middle cerebral and femoral arteries) in the course of the disease. We re-examined the biopsy muscle specimen from the patient immunohistochemically as to the expression of procoagulant or anticoagulant factors. We found a lower expression of thrombomodulin on the muscle cell membrane in the BMD patient compared with other BMD or Duchenne muscular dystrophy (DMD) patients. Although utrophin up-regulation in muscle is thought to prevent the muscle wasting in dystrophin-deficient DMD or BMD, the data obtained in the present study indicate that up-regulated utrophin may have an unexpected influence on the function of the vascular or coagulation system.

Statistically significant differences in the number of CD24 positive muscle fibers and satellite cells between sarcoglycanopathy and age-matched Becker muscular dystrophy patients.

OBJECT: The aim of this study was to reveal variations in the patterns of expression of the cell surface proteins in regenerating fibers and those in the number of satellite cells to gain an understanding of the pathological processes involved in sarcoglycanopathy. METHODS: We have reported that there is a reduction of the beta-1 subunit of laminin, heparan sulfate proteoglycan (HSPG), and HCAM (CD44) in Japanese patients with sarcoglycanopathy. Here, we investigated immunohistochemically the expression of the neural cell adhesion molecule (NCAM), which is a marker for human regenerating muscle and satellite cell, and CD24, which appears to be expressed in the early stages of the regeneration process. PATIENTS: We investigated six Japanese patients with sarcoglycanopathy, and compared to age-matched Becker muscular dystrophy. RESULTS: We found that the incidences of muscle fibers with increased NCAM were not statistically different between the two groups. However, the incidences of muscle fibers with increased CD24 and those of NCAM positive satellite cells were very low in sarcoglycanopathy and were statistically different between sarcoglycanopathy and age-matched Becker muscular dystrophies. CONCLUSION: The poor expression of CD24 and the fewer satellite cells in sarcoglycanopathy without significant difference in the number of total regenerating fibers suggest that a different regeneration process is involved in sarcoglycanopathy compared to that in other types of muscular dystrophy.

Apoptosis following spinal cord injury in rats and preventative effect of N-methyl-D-aspartate receptor antagonist.

OBJECT: The aims of this study were to clarify the histological and histochemical changes associated with cell death in the spinal cord after acute traumatic injury and to examine the role of excitatory amino acid release mediated by N-methyl-D-aspartate (NMDA) receptors. METHODS: Following laminectomy, the spinal cord in 70 rats was injured at the T-9 level by applying extradural static weight-compression, in which a cylindrical compressor was used to induce complete and irreversible transverse spinal cord injury (SCI) with paralysis of the lower extremities. The injured rats were killed between 30 minutes and 14 days after injury, and the injured cord was removed en bloc. Rats that received NMDA receptor antagonist (MK-801) were killed at the same time points as those that received saline. The specimens were stained with hematoxylin and eosin, Nissl, and Klüver-Barrera Luxol fast blue and subjected to in situ nick-end labeling, a specific in situ method used to allow visualization of apoptosis. Thirty minutes post-SCI, a large hematoma was observed at the compressed segment. Six hours after injury, large numbers of dead cells that were not stained by in situ nick-end labeling were observed. Between 12 hours and 14 days postinjury, nuclei stained by using the in situ nick-end labeling technique were observed not only at the injury site but also in adjoining segments that had not undergone mechanical compression, suggesting that the delayed cell death was due to apoptosis. The number of cells stained by in situ nick-end labeling was maximum at 3 days postinjury. The results of electron microscopic examination were also consistent with apoptosis. In the MK-801-treated rats, the number of cells stained by in situ nick-end labeling was smaller than in nontreated rats at both 24 hours and 7 days after injury. CONCLUSIONS: These findings suggest that NMDA-receptor activation promotes delayed neuronal and glial cell death due to apoptosis.

Induction of apoptosis signal regulating kinase 1 (ASK1) after spinal cord injury in rats: possible involvement of ASK1-JNK and -p38 pathways in neuronal apoptosis.

The aims of this study were to clarify the mechanism of cell death by apoptosis in the spinal cord after traumatic injury, and to examine the role of the mitogen-activated protein kinase (MAPK) pathways in the transmission of apoptosis signals. The rat spinal cord, experimentally injured by extradural static weight-compression, was studied by hematoxylin and eosin staining, Nissl-staining, terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) staining, and immunostaining using polyclonal antibodies against Apoptosis Signal-regulating Kinase 1 (ASK1), c-Jun N-terminal kinase (JNK), and p38 MAPK. TUNEL-positive cells were present at all stages studied until 7 days after injury, and percentage positivity for these cells was maximal at 3 days after injury. Electron microscopic analysis revealed the occurrence of apoptosis in both neuronal cells and glial cells. TUNEL-positive glial cells were stained by oligodendrocyte-specific maker. Expression of ASK1 was maximal at 24 h after injury in the gray matter and at 3 days after injury in the white matter. Following the expression of ASK1, activated forms of JNK and p38 were observed in apoptotic cells detected by the TUNEL method. Colocalization of ASK1 and activated JNK or activated p38 was observed in the same cell. These findings suggest the involvement of the stress-activated MAPK pathways including ASK1 in the transmission of apoptosis signals after spinal cord injury.

Adult onset limb-girdle type mitochondrial myopathy with a mitochondrial DNA np8291 A-to-G substitution.

We analyzed mitochondrial DNA (mtDNA) from 7 patients in four families with adult onset limb-girdle type mitochondrial myopathy to clarify their genetic background. The patients, 2 men and 5 women, showed common clinical features, characterized by isolated skeletal myopathy, high serum creatine kinase level, ragged-red fibers and cytochrome c oxidase-defective fibers. Analysis of muscle biopsy specimens indicated that cytochrome c oxidase activity was decreased relative to that of citrate synthase in 5 of the 7 patients. Southern blotting and direct sequence analyses showed an A-to-G homoplasmic transition at np8291 and intergenic COII/tRNA (Lys) 9bp deletion in all patients. This substitution was detected in only 2 of 600 control individuals including healthy subjects and patients with other neuromuscular disorders; these 2 individuals had diabetes mellitus and myotonic dystrophy, respectively. Consequently, the mtDNA transition at np8291 was a rare polymorphism. However, the 7 patients we studied had identical clinical, pathological, biochemical, and genetic features. Therefore, limb-girdle type mitochondrial myopathy with this rare polymorphism may form a subgroup of adult onset mitochondrial myopathy.

An autopsy case of peripheral neuroepithelioma in posterior mediastinum with p53 point mutation.

An 18-year old male was admitted to our hospital complaining of back pain. His chest computed tomography showed a tumor in the posterior mediastinum. Open biopsy was performed, and a diagnosis of peripheral neuroepithelioma was made. No genetic abnormalities were detected in the DNA obtained from the biopsy specimen. He received chemotherapy and radiation several times. These treatment regimens were effective, but he relapsed 14 months later and died of respiratory failure due to tumor growth. Autopsy examination revealed a large tumor which occupied almost the entire right thoracic cavity, but there was no evidence of metastasis to other organs. Chromosomal translocation t(14;17) (q24;p12.2) and point mutation of exon 5 of the p53 gene were detected.

Accumulation of mitochondrial ATP synthase subunit c in muscle in a patient with neuronal ceroid lipofuscinosis (late infantile form).

We report a case late infantile neuronal ceroid lipofuscinosis (NCL). Abnormal granules were found in the skeletal muscle fibers, Schwann cells, perineurial cells, endothelial cells, fibroblasts, and perivascular smooth muscle cells in the sural nerve. Electron microscopy revealed that these granules showed fingerprint profiles, curvilinear profiles or membrane-bound membranous structures. Acid phosphatase reaction was increased in these cells. Immunohistochemical studies for mitochondrial ATP synthase subunit c showed a strong reaction in these cells, suggesting abnormal accumulation of subunit c. Immunohistochemistry for subunit c in muscle may be useful in the diagnosis of late infantile NCL.

Gnome--an Internet-based sequence analysis tool.

Gnome (GenomeNet Open Mail-service Environment) is a sequence analysis tool that enables an end-user to make use of several Internet- (mainly e-mail) based services with an easy-to-use graphical user interface. Users can conduct homology and motif searches, and database-entry retrieval against the latest databases by emitting search requests to and receiving their results form a search-server by e-mail. The search results are viewed and managed efficiently with this system. The Macintosh and X (Motif) versions of the Gnome client and the UNIX version of the Gnome server are available to academic users free of charge.

Chromosomal assignment of HepG2 3'-directed partial cDNA sequences by Southern blot hybridization using monochromosomal hybrid cell panels.

Large-scale sequencing of a 3'-directed cDNA library from the human liver cell line HepG2 has generated several hundred species of cDNA gene signatures, about 85% of which identify novel genes. They are useful molecular landmarks for human genome mapping. We used 160 of these novel signatures as probes for Southern hybridization to human DNA. We then identified the copy number of the corresponding genes and assigned them to chromosomes, with reference to monochromosomal hybrid cell mapping panels. The distribution profile of the expressing genes among chromosomes suggested that the expressing gene density is not uniform.