Makoto Kunishige

Oxidative damage to skeletal muscle DNA from patients with mitochondrial encephalomyopathies

To estimate the oxidative damage to skeletal muscle DNA in mitochondrial encephalomyopathies, we studied the amount of 8-hydroxy-deoxyguanosine (8-OH-dG) and the localization of superoxide dismutase (SOD) in the skeletal muscles of patients with progressive external ophthalmoplegia (PEO) or Kearns-Sayre syndrome (KSS). The molar ratio of 8-OH-dG/deoxyguanosine in skeletal muscle from PEO or KSS patients was significantly higher than the control value. The ratio from patients with polymyositis or Duchennes muscular dystrophy was not significantly elevated. Immunohistochemical staining for both Mn-SOD and Cu,Zn-SOD showed pronounced staining in the subsarcolemmal and intermyofibrillar regions of cytochrome-oxidase-negative ragged red fibers of KSS or PEO muscles. Our findings suggest that overproduction of 8-OH-dG and mitochondrial dysfunction with gene deletions are associated with each other in muscle cells of patients with PEO or KSS, and that free radicals may play an important role in the pathophysiology of mitochondrial encephalomyopathies.

Oxidative stress–associated mitochondrial dysfunction in corticosteroid‐treated muscle cells

We analyzed the effects of corticosteroid on mitochondrial membrane potentials (ΔΨm), generation of reactive oxygen species (ROS), and apoptosis in a human rhabdomyosarcoma cell line, RD, and a dopaminergic neuroblastoma cell line, SH‐SY5Y. The cell lines were cultured in the presence or absence of dexamethasone and superoxide dismutase (SOD) for up to 1 week. Dexamethasone treatment increased ΔΨm, ROS generation, and apoptosis in proliferating RD cells. Treatment with SOD attenuated ROS generation and apoptosis, but not ΔΨm. The increase in ΔΨm seemed to be the primary effect of dexamethasone on proliferating RD cells, which is probably mediated by mitochondrial transcription. In differentiated RD cells, but not differentiated SH‐SY5Y cells, dexamethasone treatment showed a delayed effect of interfering with the ΔΨm and increasing ROS generation and apoptosis. Since these changes disappeared in the presence of SOD, dexamethasone primarily induced ROS generation, resulting in apoptosis. We speculate that this mechanism provides the basis of a pathophysiological model of corticosteroid myopathy. Muscle Nerve 30:49–54,2004

Clinical, pathological, and genetic features of limb‐girdle muscular dystrophy type 2A with new calpain 3 gene mutations in seven patients from three Japanese families

We report on the clinical, pathological, and genetic features of 7 patients with limb‐girdle muscular dystrophy type 2A (LGMD2A) from three Japanese families. The mean age of onset was 9.7 ± 3.1 years (mean ± SD), and loss of ambulance occurred at 38.5 ± 2.1 years. Muscle atrophy was predominant in the pelvic and shoulder girdles, and proximal limb muscles. Muscle pathology revealed dystrophic changes. In two families, an identical G to C mutation at position 1080 the in calpain 3 gene was identified, and a frameshift mutation (1796insA) was found in the third family. The former mutation results in a W360R substitution in the proteolytic site of calpain 3, and the latter in a deletion of the Ca2+‐binding domain.

Beneficial Effect of Steroid Pulse Therapy on Acute Viral Encephalitis

Corticosteroids are often used in the treatment of acute viral encephalitis, although the efficacy of corticosteroid therapy has not been proven. We examined the effects of high-dose corticosteroid therapy on acute viral encephalitis in 5 patients with progressive disturbances of consciousness. In 3 patients who were treated within 5 days after the onset of illness, pulse therapy dramatically reduced the degree of consciousness disturbance. They became alert within 24 h, and then neurological symptoms gradually improved. Corticosteroid therapy in the other 2 patients, in whom treatment was started more than 3 weeks after the onset of illness, was not as effective, but repeated therapy at 2-week intervals resulted in complete recovery. These findings suggest that high-dose corticosteroid therapy is effective, particularly for disturbances of consciousness, an important prognostic factor in acute viral encephalitis.

Preferential gray matter involvement in dengue myelitis

Dengue virus (DV) belongs to the family Flaviviridae , and its infection is characterized by dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Some flaviviruses, such as Japanese encephalitis, St. Louis encephalitis, Murray Valley encephalitis, and West Nile viruses, have the common feature of neurovirulence in the human CNS, including poliomyelitis-like syndrome.1 We report a patient with dengue fever who developed gray matter myelitis. A 42-year-old Japanese man working in Indonesia was admitted to a local hospital because of fever (38° C). Three days after admission, he had weakness in both legs and retained urine, and he was transferred to Singapore General Hospital. On admission, he was febrile and had a generalized macular rash. There was flaccid paralysis of the lower limbs with absent knee and ankle jerks. Sensation to pinprick was decreased below T6 segment. Laboratory examination revealed thrombocytopenia. Results of CSF examination were as follows: cells, 201/mm3 (mononuclear cell dominant); glucose, 1.6 mmol/L; and protein, 100 …

Functional association between nicotinic acetylcholine receptor and sarcomeric proteins via actin and desmin filaments

By affinity chromatography utilizing α‐cobrotoxin from digitonin‐solubilized fractions of rabbit skeletal muscle, we found that many proteins are associated with the nicotinic acetylcholine receptor (AChR). In addition to the proteins we previously reported to bind to AChR (including dystrophin‐dystrophin‐associated protein (DAP) complex, utrophin, rapsyn, and actin; Mitsui et al. [1996] Biochem. Biophys. Res. Commun.224:802–807), α‐actinin, desmin, myosin, tropomyosin, troponin T, and titin are also identified to be associated with AChR. Alkaline treatment or Triton X‐100 solubilization released dystrophin‐DAP complex, utrophin, and rapsyn from the AChR fraction, while actin and desmin remained associated. These findings demonstrate that AChR is supported primarily by a submembranous organization of actin and desmin filaments, and is linked to sarcomeric proteins via these filaments. To further investigate whether the association has any functional role, we studied the effect of acetylcoline on ATPase activity of the AChR fraction. Acetylcholine (0.5–4 μM) significantly activated Mg2+‐ATPase activity of digitonin‐solubilized AChR fraction (P < 0.05). Furthermore, we found that desmin as well as actin activated myosin Mg2+‐ATPase activity. From these findings, it is suggested that desmin and actin form a submembranous organization in the postsynaptic region, and function as mediators of excitation of AChR to the sarcomeric contraction system. J. Cell. Biochem. 77:584–595, 2000.

Thyrotoxicosis Masked by Diabetic Ketoacidosis: A fatal complication

OBJECTIVE The link between hyperglycemia and the complications of diabetes is unknown. It is still discussed whether oxidative stress precedes or merely reflects diabetic complications. To search for a familial predisposition to oxidative stress, we investigated indexes of glucose and lipid metabolism, markers of plasma and cell lipid oxidation, a marker of oxidant-induced protein damage, and the effects of oxygen radicals on erythrocytes (or red blood cells [RBCs]) of patients with type 1 diabetes and their relatives. RESEARCH DESIGN AND METHODS We recruited 30 type 1 diabetic subjects (10 without diabetic complications, 10 with retinopathy, and 10 with nephropathy), 36 nondiabetic siblings, 37 nondiabetic parents of type 1 diabetic subjects, and 3 control groups of healthy subjects without a family history of diabetes. Levels of blood creatinine, glucose, HbA(1c), cholesterol, triglycerides, lipoprotein(a) (Lp[a]), fibrinogen, malondialdehyde (MDA), and advanced oxidation protein products were determined. The RBC response to oxidative stress (3-h incubation at 37 degrees C with or without a radical generating system) was evaluated by measuring RBC glutathione (GSH), RBC-MDA, and hemolysis. RESULTS Diabetic patients had higher levels of blood glucose (P < 0.001), HbA(1c) (P < 0.001), Lp(a) (P < 0.01), and fibrinogen (P < 0.05) than control subjects. Siblings of diabetic patients had higher Lp(a) levels (P < 0.001). Parents had higher levels of plasma glucose (P < 0.05) and Lp(a) (P < 0.01). Plasma and RBC-MDA were significantly elevated in diabetic subjects and relatives compared with control subjects. Basal RBC-GSH was lower in diabetic subjects (P < 0.01). In diabetic subjects, incubations of cells caused a decrease in RBC-GSH of a lesser degree than that in control subjects, but they caused a significant increase in hemolysis. Among relatives, hemolysis was increased both at baseline and after incubation. Plasma MDA levels were associated with blood glucose, creatinine, and fibrinogen levels (multiple r = 0.5, P < 0.001), and basal RBC-MDA levels were associated with plasma Lp(a), fibrinogen, and plasma MDA levels (r = 0.6, P < 0.001). Basal RBC-GSH content correlated with serum glucose and RBC-MDA production (r = 0.3, P < 0.01). CONCLUSIONS Our study is the first to present evidence that markers of lipoprotein metabolism (Lp[a]), oxidative stress (plasma and RBC-MDA), and cellular fragility (hemolysis) are abnormal in nondiabetic relatives of type 1 diabetic subjects, thereby supporting the view that familial elements of diabetes even precede the onset of diabetes. It seems reasonable that the same biological markers considered major predictors of cardiovascular disease can also trace familial susceptibility to type 1 diabetes, just as they have been associated with the development of type 2 diabetes.

Complicated paraneoplastic neurological syndromes: a report of two patients with small cell or non-small cell lung cancer

Paraneoplastic neurological syndromes are frequently associated in patients with small cell lung cancer (SCLC) and antineuronal antibodies are involved in the autoimmune mechanism. Multiple syndromes are sometimes complicated in a single patient with SCLC. However, little is known about non-SCLC-associated neurological manifestations. We report two patients with complicated paraneoplastic neurological syndromes. Patient 1 showed paraneoplastic limbic encephalitis (PLE), paraneoplastic sensory neuropathy (PSN) and Lambert-Eaton myasthenic syndrome (LEMS) associated with SCLC. Patient 2 developed opsoclonus-ataxia and probable PLE associated with non-SCLC. Analysis of various antineuronal antibodies revealed that anti-Hu and P/Q-type voltage-gated calcium channel (VGCC) antibodies were positive in Patient 1 but any antibodies were not in Patient 2. Brain MRI demonstrated high intensity signals in temporal lobes particularly on fluid-attenuated inversion recovery (FLAIR) or diffusion-weighted images. These findings suggest that complicated paraneoplastic neurological syndromes occur in non-SCLC as well as SCLC and that unidentified antineuronal autoantibodies may underlie the pathophysiology.

Overexpressions of myoglobin and antioxidant enzymes in ragged-red fibers of skeletal muscle from patients with mitochondrial encephalomyopathy.

To determine the relationship between myoglobin (Mb) and the defense system against reactive oxygen species in various myopathies, we performed immunohistochemical analyses of Mb and various antioxidant enzymes, including manganese superoxide dismutase (Mn‐SOD), copper zinc SOD (CuZn‐SOD), catalase (CAT), and glutathione peroxidase (GSH‐Px). Biopsied muscle specimens were obtained from patients with chronic progressive external ophthalmoplegia (CPEO), Kearns–Sayre syndrome (KSS), Duchenne muscular dystrophy (DMD), and polymyositis (PM). In patients with CPEO/KSS, stainings of Mb, SOD, CAT, and GSH‐Px in nonatrophic ragged‐red fibers (RRFs) were more intense than those in non‐RRFs. These pronounced stainings corresponded to ragged‐red lesions. The staining intensities of these antioxidant enzymes were significantly correlated with that of Mb (P < 0.001). Atrophic RRFs in specimens from patients with CPEO/KSS showed intense stainings of these antioxidant enzymes but not intense staining of Mb. In specimens from patients with DMD/PM, the antioxidant enzymes but not Mb were overexpressed in degenerative fibers. These results suggest that oxidative stress is associated with Mb expression specifically in mitochondrial diseases. The antioxidant enzymes seem to be upregulated to protect against muscle damage in nonatrophic RRFs. However, the Mb‐mediated oxidative damage may become more extensive and result in further mitochondrial dysfunction and progressive atrophy of RRF with impaired upregulation of Mb. Muscle Nerve 28: 484–492, 2003

Interferon Alpha-2a Therapy for Disseminated Intravascular Coagulation in a Patient with Blue Rubber Bleb Nevus Syndrome A Case Report

The authors present a sixteen-year-old girl with blue rubber bleb nevus syndrome (BRBNS) associated with disseminated hemangiomas involving the skin, oral cavity, skeletal muscle, and cerebrum. Although she denied neurologic symptoms, magnetic resonance imaging of the brain demonstrated dilatated cerebral veins and the Chiari I malformation. Examination of hemostasis revealed disseminated intravascular coagula tion (DIC) manifesting as Kasabach-Merritt syndrome, with the potential for life-threat ening bleeding or thrombosis in the central nervous system. Since successful management of life-threatening hemangiomas with interferon alpha-2a (IFN α-2a) has been reported, the authors administered IFN α-2a with an improvement in hemostasis. These findings suggest that IFN α-2a therapy is beneficial for relieving the life-threatening consumptive coagulopathy associated with BRBNS.