Kunihiro Yoshida

Domain-specific mutations in TGFB1 result in Camurati-Engelmann disease

Camurati-Engelmann disease (CED, MIM 131300) is an autosomal dominant, progressive diaphyseal dysplasia characterized by hyperosteosis and sclerosis of the diaphyses of long bones. We recently assigned the CED locus to an interval between D19S422 and D19S606 at chromosome 19q13.1–q13.3 (ref. 2), which two other groups confirmed. As the human transforming growth factor-β1 gene (TGFB1) is located within this interval, we considered it a candidate gene for CED.

An X-ray structural study of human ceruloplasmin in relation to ferroxidase activity

Abstract The role of ceruloplasmin as a ferroxidase in the blood, mediating the release of iron from cells and its subsequent incorporation into serum transferrin, has long been the subject of speculation and debate. However, a recent X-ray crystal structure determination of human ceruloplasmin at a resolution of around 3.0 Å, in conjunction with studies associating mutations in the ceruloplasmin gene with systemic haemosiderosis in humans, has added considerable weight to the argument in favour of a ferroxidase role for this enzyme. Further X-ray studies have now been undertaken involving the binding of the cations Co(II), Fe(II), Fe(III), and Cu(II) to ceruloplasmin. These results give insights into a mechanism for ferroxidase activity in ceruloplasmin. The residues and sites involved in ferroxidation are similar to those proposed for the heavy chains of human ferritin. The nature of the ferroxidase activity of human ceruloplasmin is described in terms of its three-dimensional molecular structure.

Progression of dystrophic features and activation of mitogen‐activated protein kinases and calcineurin by physical exercise, in hearts of mdx mice

We have previously demonstrated that calcineurin and p38 mitogen‐activated protein kinase (MAPK) are up‐regulated in the hearts of mdx mice. However, the degree of up‐regulation observed was variable, which may reflect variable levels of daily physical activities among the mice. To investigate whether or not exercise affects dystrophic features and activates intracellular signaling molecules in mdx hearts, we subjected mdx and C57BL/10 mice to treadmill exercise and examined intracellular signaling molecules in cardiac muscles, at the protein level. The heart to body weight ratio was significantly increased in exercised mdx mice. Histopathology in exercised mdx hearts showed extensive infiltration of inflammatory cells, together with increases in interstitial fibrosis and adipose tissues, all of which were not observed either in exercised C57BL/10 or non‐exercised mdx hearts. Phosphorylated p38 MAPK, phosphorylated extracellular signal‐regulated kinase 1/2 and calcineurin, but not phosphorylated c‐Jun N‐terminal kinase 1, were up‐regulated in exercised mdx hearts compared to exercised C57BL/10 or non‐exercised mdx hearts. These data suggest that physical exercise accelerates the dystrophic process through activation of intracellular signaling molecules in dystrophin‐deficient hearts.

Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights

Aceruloplasminemia is an autosomal recessive disease of iron overload associated with mutation(s) in the ceruloplasmin gene. We report here a new case of aceruloplasminemia in a woman who is a compound heterozygote for two new mutations. Besides this novel genotypic profile, this observation provides new insights on: (i) iron metabolism with normal erythroid repartition, in the absence of serum non-transferrin-bound iron and with an increase of 59Fe plasma clearance; (ii) hepatic abnormalities associated with the presence of iron-free foci; (iii) the therapeutic management of the disease, chronic subcutaneous infusion of deferrioxamine being remarkably effective at reducing hepatic iron overload.

Activation and localization of matrix metalloproteinase-2 and -9 in the skeletal muscle of the muscular dystrophy dog (CXMDJ).

BackgroundMatrix metalloproteinases (MMPs) are key regulatory molecules in the formation, remodeling and degradation of all extracellular matrix (ECM) components in both physiological and pathological processes in various tissues. The aim of this study was to examine the involvement of gelatinase MMP family members, MMP-2 and MMP-9, in dystrophin-deficient skeletal muscle. Towards this aim, we made use of the canine X-linked muscular dystrophy in Japan (CXMDJ) model, a suitable animal model for Duchenne muscular dystrophy.MethodsWe used surgically biopsied tibialis cranialis muscles of normal male dogs (n = 3) and CXMDJ dogs (n = 3) at 4, 5 and 6 months of age. Muscle sections were analyzed by conventional morphological methods and in situ zymography to identify the localization of MMP-2 and MMP-9. MMP-2 and MMP-9 activity was examined by gelatin zymography and the levels of the respective mRNAs in addition to those of regulatory molecules, including MT1-MMP, TIMP-1, TIMP-2, and RECK, were analyzed by semi-quantitative RT-PCR.ResultsIn CXMDJ skeletal muscle, multiple foci of both degenerating and regenerating muscle fibers were associated with gelatinolytic MMP activity derived from MMP-2 and/or MMP-9. In CXMDJ muscle, MMP-9 immunoreactivity localized to degenerated fibers with inflammatory cells. Weak and disconnected immunoreactivity of basal lamina components was seen in MMP-9-immunoreactive necrotic fibers of CXMDJ muscle. Gelatinolytic MMP activity observed in the endomysium of groups of regenerating fibers in CXMDJ did not co-localize with MMP-9 immunoreactivity, suggesting that it was due to the presence of MMP-2. We observed increased activities of pro MMP-2, MMP-2 and pro MMP-9, and levels of the mRNAs encoding MMP-2, MMP-9 and the regulatory molecules, MT1-MMP, TIMP-1, TIMP-2, and RECK in the skeletal muscle of CXMDJ dogs compared to the levels observed in normal controls.ConclusionMMP-2 and MMP-9 are likely involved in the pathology of dystrophin-deficient skeletal muscle. MMP-9 may be involved predominantly in the inflammatory process during muscle degeneration. In contrast, MMP-2, which was activated in the endomysium of groups of regenerating fibers, may be associated with ECM remodeling during muscle regeneration and fiber growth.

Dystonia and parkinsonism in GM1 type 3 gangliosidosis.

GM1 gangliosidosis is due to β‐galactosidase deficiency. Only patients with type 3 disease survive into adulthood and develop movement disorders. Clinical descriptions of this form are rare, particularly in non‐Japanese patients. We describe four new patients and systematically analyze all previous reports found by a literature search and contacts with the authors for additional information. Generalized dystonia remained the predominant feature throughout the disease course and was often associated with akinetic–rigid parkinsonism. GM1 gangliosidosis must be considered as a cause of early‐onset generalized dystonia, particularly in patients with short stature and skeletal dysplasia.

Iron overload and antioxidative role of perivascular astrocytes in aceruloplasminemia

Aceruloplasminemia (ACP) is an inherited disorder of iron metabolism caused by the lack of ceruloplasmin activity; the neuropathological hallmarks are excessive iron deposition, neuronal loss, bizarrely deformed astrocytes, and numerous ‘grumose or foamy spheroid bodies (GFSBs)’. We histopathologically examined two autopsied ACP brains, and observed for the first time that GFSBs form in clusters at the ends of perivascular astrocytic foot processes. Both the deformed astrocytes and the GFSBs contained ferric iron and were intensely immunolabelled with antibodies against the antioxidant proteins ferritin and manganese superoxide dismutase (Mn SOD). Ceruloplasmin is largely produced by perivascular astrocytes in the central nervous system and exhibits a ferroxidase activity that inhibits iron‐associated lipid peroxidation and hydroxyl radical formation; therefore, the lack of ceruloplasmin causes direct oxidative stress on astrocytes. The intense immunolabelling of ferritin and Mn SOD most likely reflects a defensive response to iron‐mediated oxidative stress. This study suggests that astrocytes play key roles in iron trafficking and the detoxification of iron‐mediated free radicals at the blood–brain barrier and in the parenchyma in ACP brain. The antioxidative ability of astrocytes is one of their essential neuroprotective effects, and the decompensation of this ability may lead to secondary neuronal cell death in ACP.

Usefulness of MALDI/TOF mass spectrometry of immunoprecipitated serum variant transthyretin in the diagnosis of familial amyloid polyneuropathy.

A matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometry (MS) system was used to detect variant transthyretin (TTR) in immunoprecipitated serum TTR molecules obtained from 6 patients with familial amyloid polyneuropathy (FAP) who were already proven not to have ATTR Val30Met. This simple and quick method showed six different patterns of mass spectra of TTR-related immunoprecipitates from these patients, and in each patient the clearly identified characteristic doublet-shaped ion peaks consisted of normal and variant TTR apart from each other peak with a mass difference between them. DNA sequencing confirmed that the patterns of variant TTR corresponded respectively to ATTR Val30Leu, ATTR Phe33Val, ATTR Asp38Ala, ATTR Ser50Arg, ATTR Ala97Gly and ATTR Ala97Ser. ATTR Asp38Ala and ATTR Ala97Ser are previously unknown variants of TTR leading to the development of FAP. ATTR Phe33Val was found in a Chinese FAP patient and ATTR Ala97Ser in a Taiwanese. Serum analysis using immunoprecipitation and MALDI/TOF MS system can provide useful information when investigating FAP patients with diverse types of variant TTR.

Matrix metalloproteinase-2 ablation in dystrophin-deficient mdx muscles reduces angiogenesis resulting in impaired growth of regenerated muscle fibers

Matrix metalloproteases (MMPs) are a family of endopeptidases classified into subgroups based on substrate preference in normal physiological processes such as embryonic development and tissue remodeling, as well as in various disease processes via degradation of extracellular matrix components. Among the MMPs, MMP-9 and MMP-2 have been reported to be up-regulated in skeletal muscles in the lethal X-linked muscle disorder Duchenne muscular dystrophy (DMD), which is caused by loss of dystrophin. A recent study showed that deletion of the MMP9 gene in mdx, a mouse model for DMD, improved skeletal muscle pathology and function; however, the role of MMP-2 in the dystrophin-deficient muscle is not well known. In this study, we aimed at verifying the role of MMP-2 in the dystrophin-deficient muscle by using mdx mice with genetic ablation of MMP-2 (mdx/MMP-2(-/-)). We found impairment of regenerated muscle fiber growth with reduction of angiogenesis in mdx/MMP-2(-/-) mice at 3 months of age. Expression of vascular endothelial growth factor-A (VEGF-A), an important angiogenesis-related factor, decreased in mdx/MMP-2(-/-) mice at 3 months of age. MMP-2 had not a critical role in the degradation of dystrophin-glycoprotein complex (DGC) components such as β-dystroglycan and β-sarcoglycan in the regeneration process of the dystrophic muscle. Accordingly, MMP-2 may be essential for growth of regenerated muscle fibers through VEGF-associated angiogenesis in the dystrophin-deficient skeletal muscle.

Follow-up of three patients with a large in-frame deletion of exons 45–55 in the Duchenne muscular dystrophy (DMD) gene

We review the clinical status of skeletal involvement and cardiac function in three unrelated patients harboring an in-frame deletion of exons 45 to 55 in the DMD gene followed up for 2 to 7 years. Two younger patients diagnosed as having X-linked dilated cardiomyopathy (XLDCM) developed congestive heart failure without overt skeletal myopathy. Heart failure recurred after viral infection but responded well to diuretics and angiotensin-converting enzyme inhibitors. One older patient diagnosed with Becker muscular dystrophy showed limb-girdle muscular atrophy and weakness at the age of 50, but did not have any cardiac symptoms. Skeletal muscle involvement in each patient remained unchanged, and cardiac function did not worsen in any of the patients during the study. In a younger XLDCM patient, the amount and molecular weight of mutant dystrophin were equally slightly decreased in both skeletal and cardiac muscles. Immunostaining for dystrophin and dystrophin-associated proteins was slightly reduced in both skeletal and cardiac muscle, with no discernible difference between the two. The phenotype of this dystrophinopathy can manifest as XLDCM in younger patients; however, careful attention to cardiac management may result in a favorable prognosis.