Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS): novel compound heterozygous mutations in the SACS gene.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disorder first described among French Canadians in Quebec. To date, 24 mutations have been reported in the SACS gene of ARSACS patients. The authors report a clinical and genetic analysis of a Japanese family with ARSACS with novel compound heterozygous mutations in the SACS gene (N161fsX175, L802P). The phenotype is similar to that of previously reported ARSACS patients.

Elemental diet modulates the growth of Clostridium difficile in the gut flora.

BACKGROUND AND AIMS: Tube feeding is regarded as a risk factor for Clostridium difficile-associated diarrhoea. Recently, we reported that C. difficile toxin was frequently found in patients receiving an elemental diet. The present study was conducted to clarify whether elemental diets are associated with the growth of C. difficile in the gut flora. METHODS: C. difficile was cultured for 72 h in various concentrations of elemental diet containing 3% thioglycollate, and the growth rate or activity of C. difficile was evaluated by Gram stain or by measuring optical density at 560 nm. Faecal samples from 10 healthy adults were cultured in elemental diet + 3% thioglycollate. RNA was extracted from faeces with glass powder, which can eliminate PCR inhibitors, and mRNA of C. difficile toxin B was measured by reverse transcription PCR. RESULTS: Maximum OD560 value during culture in thioglycollate-containing elemental diet was 2.4 times higher than that in thioglycollate alone (P = 0.0163). Viability of C. difficile was decreased in thioglycollate but not in thioglycollate-containing elemental diet. Toxin B mRNA was detected in five faecal samples (50%) before culture and in all samples after culture. CONCLUSIONS: Our results suggest that an elemental diet can modulate the growth of C. difficile in the gut flora.

CSF hypocretin levels in Guillain-Barré syndrome and other inflammatory neuropathies.

CSF hypocretin-1 was measured in 28 Guillain-Barré syndrome (GBS), 12 Miller-Fisher syndrome, 12 chronic inflammatory demyelinating polyneuropathy (CIDP), and 48 control subjects. Seven GBS subjects had undetectably low hypocretin-1 levels (<100 pg/mL). Hypocretin-1 levels were moderately reduced in an additional 11 GBS, 5 Miller-Fisher syndrome, and 1 CIDP subject. Low levels in GBS occurred early in the disease and were associated with upper CNS level abnormalities.

Time course of polyglutamine aggregate body formation and cell death: enhanced growth in nucleus and an interval for cell death.

Polyglutamine (polyQ) aggregate bodies are a hallmark of dentatorubral-pallidoluysian atrophy and related neurodegenerative disorders, although the relationship between aggregate body formation and cell death is not clear. We analyzed the kinetics of polyQ aggregate formation and the time intervals for cell death, tracking individual cells using fluorescence video microscopy, for the first time. Expanded polyQ tracts of atrophin-1 with or without nuclear localization signal (NLS) labeled with green fluorescent protein (GFP) were constructed, Q57NLS/GFP and Q56/GFP, respectively. All of the Q57NLS/GFP aggregate bodies were in nuclei, and all of the Q56/GFP aggregate bodies were in cytoplasm. Aggregates of Q56/GFP were larger than those of Q57NLS/GFP. Surprisingly, a kinetic analysis showed that the latter grew 5.37 times faster than the former. The time interval between transfection and cell death was shorter in Q57NLS/GFP, but the time between the end of the rapid growing phase of aggregation and the start of the cell death process did not show a significant difference. Aggregate growth was confirmed to correspond to the accumulated free polyQ by the time of starting aggregation. These findings suggest that aggregate body formation induced by expanded polyQ stretches is a self-limiting process and is enhanced by factor(s) in nuclei, whereas it is not tightly bound to the cell death process.

Human muscle protein degradation in vitro by eosinophil cationic protein (ECP).

To clarify the role of tissue eosinophils in and around inflammatory foci, we purified eosinophil cationic protein (ECP) and examined its effect on muscle protein degradation in vitro. Eosinophil cationic protein was purified from the buffy coat of blood from healthy volunteers. Myofibrillar, soluble sarcoplasmic, and membrane-associated cytoskeletal proteins were fractionated from latissimus dorsi muscle obtained by orthopedic procedures done on a patient with no neurologic abnormalities. After incubation of these fractions with purified ECP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed. Eosinophil cationic protein degraded the myofibrillar proteins, especially the myosin heavy chain (MHC) and alpha-actinin. It also degraded membrane-associated cytoskeletal proteins dystrophin and spectrin, whereas soluble sarcoplasmic proteins did not undergo proteolysis. Quantitative analysis of the MHC degradation showed that the ECP reaction was dose-dependent and that the optimal pH was 7.0. Protein degradation was not inhibited by heparin or the protease inhibitors leupeptin, E-64, and pepstatin A. Our results suggest that ECP functions in the degradation of myofibrillar and membrane-associated cytoskeletal proteins, indicating that tissue eosinophils have a specific role in muscle fiber degradation in some myopathies associated with numerous tissue eosinophils, such as eosinophilic myositis, eosinophilic myalgia syndrome, and eosinophilic endocardial disease.

Dynamic locomotor function in normals and patients with vertigo.

Gait analysis was performed in patients with various vestibular systems using a tactile sensor. There were 4 patients with vestibular neuronitis, 6 patients with large acoustic neuroma and 6 patients with spino-cerebellar degeneration (SCD). Gait phase related parameters such as stance, swing and double support were studied to assess gait stability. Also the area ratio of trajectories of center of force during stance and progression of foot pressure were checked. The calculated value of each variable became high in pathological cases compared with normal controls, and the highest value was obtained in the SCD group. As regards the effect of visual deprivation on stability of gait, the most striking change was found in the large acoustic neuroma group. In a case with a unilateral lesion such as vestibular neuronitis and large acoustic neuroma, foot pressure was greater on the lesion side, especially during gait with eyes closed. As for the foot pressure progression curve, the SCD group showed the most irregular pattern in general, although there were some individual variations. Those results could reflect a functional disorder of the gait control system caused by each disease. Significance of gait analysis is also discussed.

A novel de novo mutation in the desmin gene causes desmin myopathy with toxic aggregates.

OBJECTIVE: To determine the cause and pathogenic mechanisms of a 21-year-old patient's cardioskeletal myopathy. The patient's muscle atrophy and weakness began in distal parts of limbs; cardiac and facial muscles were later involved. BACKGROUND: Desmin myopathy is a skeletal myopathy often associated with cardiomyopathy, caused by mutations in the desmin gene and characterized by desmin accumulation in affected muscle fibers, a leading marker of myofibrillar myopathies. Two kinds of deletions and seven missense mutations in the desmin gene have been identified. METHODS: Clinical examination, electron microscopy of muscle tissue, two-dimensional gel electrophoresis, DNA sequencing, restriction enzyme analysis, and gene transfection were performed. RESULTS: Electron microscopy showed disruption of sarcomeres at Z discs and electron-dense aggregates in biopsied skeletal and heart muscle. Two-dimensional gel electrophoresis of the patient's skeletal muscle proteins showed massive accumulation of desmin. The authors identified a novel desmin mutation, L385P in one allele in the carboxyl end of the rod domain 2B in the patient's leukocytes and skeletal muscle; neither parent had the mutation. Serologic study and DNA markers confirmed the de novo mutation. A peptide harboring desmin rod domains 2A and 2B with L385P tagged with green fluorescent protein induced cytoplasmic aggregates, nuclear DNA condensation, and cell death. CONCLUSIONS: A novel de novo mutation, L385P, causes desmin myopathy. An expression study indicated the toxic effect of the L385P mutation.

Increased lysosome-related proteins in the skeletal muscles of distal myopathy with rimmed vacuoles.

Investigators have speculated that the degenerative process in distal myopathy with rimmed vacuoles (DMRV) mainly involves the lysosomal system. To investigate possible protein abnormalities related to intracellular lysosomal proteolytic pathways in DMRV-affected muscles, we performed immunohistochemical analyses of certain proteins in muscle biopsy specimens obtained from patients with various neuromuscular diseases, including DMRV, muscular dystrophy, polymyositis, and amyotrophic lateral sclerosis, and in normal human muscles specimens. Immunohistochemically, most muscle fibers in normal control specimens showed little or no reaction for clathrin and alpha- and gamma-subunits of adaptin-constituted adaptin proteins (AP)-1 and AP-2, respectively. Abnormal increases in these proteins were demonstrated mainly in the cytoplasm of atrophic fibers or in necrotic fibers in all diseased specimens. Particularly in DMRV-affected muscles, alpha- and gamma-adaptins were often observed inside or on the rims of vacuoles and in the cytoplasm of vacuolated fibers. Abnormal increases in Golgi-zone protein were also demonstrated in DMRV muscles. The rims of rimmed vacuoles were negative for kinectin, an endoplasmic reticulum-binding protein. Positive staining for both proteins, however, was sometimes seen inside the vacuoles in DMRV-affected fibers. These results suggest increased endocytosis at the plasma membrane as well as secretion involving transport from the trans-Golgi network of the Golgi apparatus in DMRV. Accumulation of various lysosome-related proteins within the rimmed vacuoles indicates at least some of these vacuoles may be autolysosomes.

Massive accumulation of M and H subunits of neurofilament proteins in spinal motor neurons of neurofilament deficient Japanese quail, Quv.

Quiver (Quv) is a non-sense mutation of neurofilament protein L subunit (NF-L) that causes neurofilament deficiency with preserved microtubules in Japanese quail. Anti-NF-M and anti-NF-H mAbs stained cell bodies of motor neurons in Quv embryo spinal cords much more intense than those in control spinal cords. Volume of motor neurons in Quv spinal cords increased to 2.3 times of control motor neurons. Immunoblot of Quv spinal cords revealed a relative increase in non- and hypo-phosphorylated NF-M and NF-H, and a decrease in the total amount of NFs. Quv sciatic nerves showed faintly reacted phosphorylated NF-M and NF-H. These results suggest that deficiency of assembled neurofilament results in decreased axonal transport of NFs and accumulation of NFs in cell bodies of spinal motor neurons.

Pontine atrophy in spinocerebellar ataxia type 6.

To investigate the clinical range of spinocerebellar ataxia type 6 (SCA6), we screened CAG repeat expansion in the voltage-dependent alpha 1A calcium channel gene (CACNL1A4) in 71 ataxic patients in 60 families; 54 patients in 43 families with hereditary ataxia and 17 sporadic patients. Thirteen patients with SCA6 were detected to have elongated CAG in CACNL1A4. Of these, 7 patients had been diagnosed as having hereditary cerebellar cortical atrophy, and 6 patients had been found to have sporadic occurrence. One patient showed distinct pontine atrophy with prominent horizontal or oblique gaze nystagmus which is an unusual feature in sporadic olivopontocerebellar atrophy. For the efficient screening of SCA6, we would propose testing CAG repeat expansion in CACNL1A4, in patients with one of two markers: (1) horizontal or oblique gaze nystagmus without other eye movement disorders, (2) pure cerebellar atrophy, even if occurrence is sporadic. We should note that the pontine atrophy could also be caused by CAG repeat expansion in CACNL1A4.

[Hereditary ataxias in Akita prefecture].

To provide a genetic survey of hereditary ataxia, we performed PCR screening of SCA1, SCA2, MJD1 (SCA 3), SCA6, DRPLA, with 71 patients in 61 families living in Akita prefecture (1,205,571 population in 1997) in Japan. Of 71 patients in 61 families, 18 MJD1, 14 SCA6, 5 DRPLA, 1 SCA1 and 1 SCA2 patients were detected. Eighty percent of autosomal dominant inherited spinocerebellar degeneration (AD-SCD) including 7 spoladic patients genetically diagnosed as AD-SCD was MJD1 (45.7%) and SCA6 (34.3%). These suggest the prevalence rate of hereditary ataxias in Akita prefecture; 1.5 and 1.2/100,000 of MJD1 and SCA6, respectively. Only one patient of SCA1 was detected, which was frequently reported in Hokkaido and Tohoku area in Japan.

Tauroursodeoxycholic acid enhances phagocytosis of the cultured rat Kupffer cell.

BACKGROUND: Ursodeoxycholic acid is used in the treatment of acute and chronic intrahepatic cholestasis because it ameliorates cholestasis and protects hepatocytes. However, few studies have examined the effect of bile acids on the function of Kupffer cells. METHODS: The effect of various bile acids on cultured rat Kupffer cells was studied in terms of phagocytic activity in response to latex particles and morphological alterations. Video-enhanced differential interference contrast microscopy was used. RESULTS: Taurochenodeoxycholic acid and taurodeoxycholic acid reduced the number of latex particles incorporated into Kupffer cells, but taurocholic and tauroursodeoxycholic acids enhanced phagocytosis of latex particles. Inhibition of phagocytosis by taurochenodeoxycholic acid or taurodeoxycholic acid was essentially dose dependent. Tauroursodeoxycholic acid also enhanced phagocytosis by Kupffer cells in which phagocytosis had been reduced by pretreatment with taurochenodeoxycholic acid or taurodeoxycholic acid. Incorporated latex particles had a distinct translocation speed of 0.084+/-0.024 microm/s (mean maximum speed+/-SD); the speed was in the same range with tauroursodeoxycholic acid treatment. Tauroursodeoxycholic acid induced a 56% expansion of cytoplasm, associated with increased ruffling and movement of intracellular organelles. CONCLUSIONS: These observations suggest that tauroursodeoxycholic acid enhances membrane trafficking without changing translocation speed.

Tau expression in denervated rat muscles.

We studied whether denervation affects the expression of tau, in particular phosphorylated tau, and how it is degraded in rat soleus muscles. Immunoblot analysis showed a high molecular weight, approximately 110 kDa (big tau), in normal muscle. Tau levels increased significantly in denervated muscles treated with chloroquine (a lysosomotrophic agent) and in untreated ones, as compared to levels of similarly treated contralateral, innervated muscles. Most of the tau in the innervated and denervated muscles was phosphorylated. Immunohistochemically, tau and beta-tubulin colocated in the sarcoplasm of innervated, saline-treated (intact) muscle, but the staining intensities were very weak. Both proteins, however, were expressed extensively in these areas in the denervated muscles from saline-treated rats. In the denervated muscle of chloroquine-treated rats there were numerous autophagic vacuoles in the sarcoplasm, and phosphorylated-tau accumulation was marked within these vacuoles, indicative that tau first was taken into autophagic, vacuoles by nonselective autophagy then degraded via the lysosomal as well as the nonlysosomal calpain system. Our findings suggest that phosphorylated big tau accumulates with beta-tubulin in denervated muscular atrophy, possibly in order to maintain or preserve the integrity of the muscle fiber during progressive atrophy or regeneration.

[Muscle atrophy in isolated ACTH deficiency].

We analyzed muscle area in CT and muscle pathology in a patient with isolated ACTH deficiency who started with the difficulty of elevation of both arms. Cortisol treatment resulted in full recovery from severe muscle atrophy and contracture of major joints. Change of volume of major muscles in arm, thigh and calf was followed. Major muscles were identified in CT and the area of each muscle was calculated with computer assistance. The increase of total muscle area in sequential 3 times in CT was up to 74% after prednisolone treatment. This indicates that the deficiency of cortisol resulted in 42% reduction of muscle volume. This also suggests that reduction of muscle volume induces the limitation of range of motion of shoulder joint. ATPase of muscle biopsy revealed the influence on fiber type proportion; type 1 : type 2A : type 2B = 29.6 : 6.0 : 64.4% and 35.7 : 17.6 : 46.7% in pre-treatment and post-treatment of cortisol, respectively. Mean diameters of muscle fibers in type 1, type 2A and type 2B was 41.8, 41.8, 39.1 microns and 46.2, 44.0, 37.2 microns in pre-treatment and post-treatment of cortisol, respectively. These suggest that deficiency of glucocorticoid introduces the reduction of the activity of the motor neurons innervating type 1 and type 2A muscle fibers.

Kinesin and cytoplasmic dynein in spinal spheroids with motor neuron disease.

Kinesin and cytoplasmic dynein are two major molecular motors responsible for fast axonal transport. As visualized by immunohistochemistry with monoclonal antibodies, both motors were found to be distributed throughout the cell bodies, dendrites and axons of motor neurons in normal human spinal cords. Large axonal swellings, spheroids, in the spinal cords of patients with motor neuron disease showed massive accumulation of kinesin co-localized with highly phosphorylated neurofilaments. Of 114 spheroids in five spinal cords, 87% were stained heavily with the three anti-kinesin antibodies used in this study. Cytoplasmic dynein was scarce or absent in most of the spheroids. These findings suggest that kinesin selectively accumulates in the spheroids of motor neuron axons, causing disturbance of the machinery for anterograde fast axonal transport in motor neuron disease.

Portal-systemic encephalopathy from a spontaneous gastrorenal shunt diagnosed by three-dimensional computed tomography and treated effectively by percutaneous vascular embolization.

A 67-year-old man with a portal-systemic shunt confirmed by three-dimensional computed tomography (3D-CT) was successfully treated by percutaneous vascular embolization. The patient had aggravated loss of memory, disorientation, and hyperammonemia. A gastrorenal shunt 16 mm in diameter was found by 3D-CT reconstructed by helical computed tomography (CT). Embolization was performed only in the shunt percutaneously through the inferior vena cava. One year after the embolization, no recurrence of portal-systemic encephalopathy and no portal hypertension have appeared, and the clinical course has been good.