Clinical and mutational spectrum of Japanese patients with Charcot-Marie-Tooth disease caused by GDAP1 variants.

BACKGROUND: Mutations in GDAP1 are responsible for heterogeneous clinical and electrophysiological phenotypes of Charcot-Marie-Tooth disease (CMT), with autosomal dominant or recessive inheritance pattern. The aim of this study is to identify the clinical and mutational spectrum of CMT patients with GDAP1 variants in Japan. MATERIALS AND METHODS: From April 2007 to October 2014, using three state-of-art technologies, we conducted gene panel sequencing in a cohort of 1,030 patients with inherited peripheral neuropathies (IPNs), and 398 mutation-negative cases were further analyzed with whole-exome sequencing. RESULTS: We identified GDAP1 variants from 10 patients clinically diagnosed with CMT. The most frequent recessive variant in our cohort (5/10), c.740C>T (p.A247V), was verified to be associated with a founder event. We also detected three novel likely pathogenic variants: c.928C>T (p.R310W) and c.546delA (p.E183Kfs*23) in Case 2 and c.376G>A (p.E126K) in Case 8. Nerve conduction study or sural nerve biopsy of all 10 patients indicated axonal type peripheral neuropathy. CONCLUSION: We identified GDAP1 variants in approximately 1% of our cohort with IPNs, and established a founder mutation in half of these patients. Our study originally described the mutational spectrum and clinical features of GDAP1-related CMT patients in Japan.

Laser-stimulated luminescence used to measure x-ray diffraction of a contracting striated muscle.

An integrating x-ray area detector that operates on the basis of laser-stimulated luminescence was used in a diffraction study of muscle contraction. The area detector has a dynamic range of 1 to 10(5), a sensitivity about 60 times greater with approximately 1/300 as much fog background as x-ray film. It is erasable and reusable but, like film, can integrate at a practically unlimited counting rate. The high sensitivity and wide dynamic range of the detector resulted in a sufficient reduction in the exposure time to make possible the recording of a clear x-ray diffraction pattern, with up to 2.0-nanometer axial spacing, from a contracting frog skeletal muscle in as little as 10 seconds with synchrotron radiation. During the isometric contraction of the muscle, most of the actin diffraction lines increased in intensity without noticeable changes in their peak positions. Changes also occurred in diffraction intensities from the myosin heads. The results indicate that during contraction the structure of the actin filaments differs from that in the rigor state, suggesting a possible structural change in the actin subunits themselves; the myosin heads during contraction retain the axial periodicity of the myosin filament and become aligned in a more perpendicular manner to the actin filaments.

Effect of 6-hydroxydopamine on [3H]-adenine nucleotide and [3H]-noradrenaline release from the guinea-pig taenia caecum evoked by electrical field stimulation, nicotine and perivascular nerve stimulation.

1 The effects of pretreatment of guinea-pigs with 6-hydroxydopamine (6-OHDA) on the release of [(3)H]-noradrenaline and [(3)H]-adenine nucleotide following electrical field, nicotine and perivascular nerve stimulation of guinea-pig taenia caecum were studied.2 High frequency electrical field stimulation (15,30 Hz) released both [(3)H]-noradrenaline and [(3)H]-adenine nucleotide but low frequency (0.5, 5 Hz) stimulation, producing comparable muscle relaxation led only to [(3)H]-noradrenaline release.3 6-OHDA (50-200 mg/kg) pretreatment inhibited the muscle relaxation and [(3)H]-noradrenaline release with electrical stimulation or with nicotine in isolated taenia but did not affect the release of [(3)H]-nucleotide.4 A low dose of 6-OHDA (50 mg/kg), completely inhibited the muscle relaxation and [(3)H]-noradrenaline release elicited by perivascular nerve stimulation.5 Both tissue noradrenaline content and [(3)H]-noradrenaline uptake were decreased to the same extent by low as well as high doses of 6-OHDA: noradrenaline content was reduced to 20% and uptake to 30% of the control value.6 Catecholamine fluorescence disappeared from tissue layers of the taenia after treatment with a high dose of 6-OHDA.7 In these experiments the inhibitory action of electrical stimulation and nicotine on the taenia can be correlated better with noradrenaline than with nucleotide release.

(3H)-adenosine nucleotide and (3H)-noradrenaline uptake by cold stored guinea-pig taenia caecum; mechanical effects and release of (3H)-adenosine nucleotide by noradrenaline, papaverine, and nitroglycerine.

1. Cold storage (2 degrees C) treatment progressively reduced noradrenaline uptake by the taenia caecum of the guinea-pig. After 15 days of cold treatment, [(3)H]-noradrenaline uptake by tissue was reduced to about 10% of control. On the other hand, prolonged cold storage failed to decrease [(3)H]-adenosine uptake by the taenia caecum.2. Cocaine (10 muM) inhibited noradrenaline uptake by about 82% but nucleoside uptake was inhibited by 20%. Cocaine treatment failed to decrease the residual noradrenaline uptake in the cold stored strips (more than 10 days). Phenoxybenzamine (1 muM) or oligomycin (1 mug/ml) treatment decreased the tissue adenosine uptake to about 34% and 28% of the respective controls.3. Based on thin layer chromatography, it was estimated that approximately 68% of [(3)H]-adenosine was converted into and retained as [(3)H]-ATP in the fresh tissues, a small fraction was accountable as [(3)H]-adenosine (18%) but virtually no [(3)H]-AMP, [(3)H]-cyclic AMP or [(3)H]-ADP was detected. Similar distribution of radioactivity of nucleotides was observed in tissues cold stored for 8 days.4. The inhibition of the mechanical activity of taenia by noradrenaline (10 muM), papaverine (100 muM) and nitroglycerine (100 muM) was accompanied by [(3)H]-adenine nucleotide but not [(3)H]-noradrenaline release. Treatment with phentolamine and propranolol (both 1 muM) had no effect on the adenine nucleotide release elicited by nicotine and electrical field stimulation, whereas such treatment reduced the inhibitory action of both stimuli.5. These results suggest that the nucleotide release after application of electrical and chemical stimulation may be from an extraneurogenic source. Thus, we conclude that ATP or a related nucleotide is not the chemical transmitter of the non-adrenergic inhibition in the taenia caecum of the guinea-pig.

(3H)-adenine nucleotide and (3H)-noradrenaline release evoked by electrical field stimulation, perivascular nerve stimulation and nicotine from the taenia of the guinea-pig caecum.

1. The release of [(3)H]-noradrenaline and adenine nucleotide evoked by electrical field stimulation (20-60 V, 30 Hz), perivascular nerve stimulation (20-80 V, 60 Hz) and nicotine (10, 100 muM) was studied in the taenia of the guinea-pig caecum under various conditions.2. Electrical stimulation at high intensity (60 V) caused the release of [(3)H]-adenine nucleotide; however, the inhibitory action of electrical stimulation was proportional to [(3)H]-noradrenaline release.3. The intensity of the inhibitory effect of stimulation of the perivascular nerves was directly related to [(3)H]-noradrenaline release and not associated with the release of [(3)H]-nucleotide.4. Cold storage for more than 8 days, cooling (19 degrees C) or tetrodotoxin treatment (1 mug/ml) abolished the inhibitory responses to electrical stimulation and to nicotine. After these treatments, nicotine and electrical stimulation elicited only contractions; the release of [(3)H]-noradrenaline, but not that of [(3)H]-adenine nucleotide, was inhibited.5. The dissociation of the inhibitory effects of electrical stimulation and nicotine from [(3)H]-nucleotide release does not support the hypothesis that ATP or a related nucleotide is the humoral transmitter of the non-adrenergic inhibition in the taenia of the guinea-pig caecum.

Effect of isoprenaline and phenylephrine on the adenosine 3',5'-monophosphate content and mechanical activity of cold-stored and fresh taenia caecum from the guinea-pig.

1. Cold storage treatment of the guinea-pig taenia caecum had a greater inhibitory effect on the isoprenaline-induced relaxation than that induced by phenylephrine. Prolonged cold storage (12-14 days) almost abolished the effect of isoprenaline but only reduced the phenylephrine effect. The ED50 of cyclic adenosine 3',5'-monophosphate (cyclic AMP) that elicited muscle relaxation was not altered by the prolonged cold storage. 2. After cold storage treatment, tissue cyclic AMP content was decreased; however, isoprenaline still caused a dose-dependent increase in the cyclic AMP level. The threshold dose of isoprenaline for cyclic AMP accumulation was the same in fresh and cold-stored preparations. 3. In the fresh preparation, the onset of the isoprenaline (10(-6)M)-induced relaxation preceded the increase in tissue cyclic AMP. 4. Isoprenaline, phenylephrine, adrenaline and noradrenaline at doses (ED50) sufficient to induce muscle relaxation did not always increase the cyclic AMP level. 5. Similarly, the responses to papaverine and nitroglycerine were not accompanied by an increase in cyclic AMP. 6. The adenylate cyclase and phosphodiesterase (low and high Km) activities of taenia caecum were not attenuated by the prolonged cold storage. 7. Propranolol inhibited both the isoprenaline-induced relazation and cyclic AMP accumulation; however, the pA2 values were significantly different for the two events. 8. Based on these results, both the relaxation and cyclic AMP accumulation caused by isoprenaline are mediated by activation of beta-adrenoceptors but are independent phenomena.

Adrenergic mechanism in cold stored taenia caecum after rewarming.

After cold storage of guinea-pig taenia strips for 4 days at 2 degrees C, structures with specific catecholamine fluorescence were not observed. Upon incubation in Krebs Ringer medium (37 degrees C) the fluorescence in the cold stored tissue reappeared and the density of fluorescence was comparable to that of fresh preparations. The specific fluorescence was affected following reserpine or 6-hydroxydopamine treatment; however, the rewarming process restored fluorescence only in the reserpine-treated tissue. The cold storage treatment (4 and 7 days) also decreased the tissue noradrenaline and dopamine contents and dopamine-beta-hydroxylase activity and inhibited the [3H-noradrenaline release from the tissue, all of which were not restored by the incubation procedure. Moreover, in these cold stored taenia the inhibitory effect to nicotine and perivascular nerve stimulation were not manifested. Therefore, the reappearance of specific fluorescence in the cold stored taenia strips after rewarming does not assure functional recovery of adrenergic nerve activity.

Comparison of three different assays for the assessment of ciguatoxin in fish tissues: radioimmunoassay, mouse bioassay and in vitro guinea pig atrium assay.

Moderate correlation was shown between the following tests: RIA: mouse bioassay (r = 0.58); RIA: guinea pig atrium assay (r = 0.62); guinea pig atrium assay: mouse bioassay (r = 0.62). All three assay procedures showed good correlation when ciguatoxin was present in tissues in high concentrations. The results also suggest that more than one structurally related toxin may be present in the fish tissues.

Application of the "imaging plate" to TEM image recording.

The "imaging plate" is a highly sensitive image recording plate for X-ray radiography, which is coated with photo-stimulable phosphor. The imaging plate is exposed to electrons in a transmission electron microscope. Its fundamental properties (sensitivity, dynamic range and sharpness) have been estimated in detail. Also, the image quality of the imaging plate for some specimens in a transmission electron microscope has been estimated. As a result, it has been ascertained that the imaging plate has superior properties and high practicability as an image recording material in a transmission electron microscope.

Determination of the antigen/epitope that is recognized by human monoclonal antibody CLN-IgG.

Ever since the development of human monoclonal antibody CLN-IgG in 1982, we anticipated the identification of the antigen that is recognized by this antibody. Despite its scarce expression on the cell surface, susceptibility to proteolytic enzymes and adherence to experimental equipment, we finally succeeded in determining the antigen moiety that is recognized by this antibody by means of CLN-IgG conjugated column affinity chromatography, two-dimensional electrophoresis, MALDI-TOF/MS and use of glioblastoma cell line U-251MG. The antigen was found to be vimentin, a cytoskeletal protein, and we succeeded in determining a 79 amino acids sequence of the epitope which turned out to comprise a part of the c2 (coil 2 of the central rod) domain of vimentin.

Imaging plate illuminates many fields.

The erasable phosphor imaging plate developed for medical radiography has found new uses in the laboratory. X-ray diffraction, protein crystallography and autoradiography have all benefited from this technology transfer from the clinic.

Non-linear summation of unit synaptic potentials in spinal motoneurones of the cat.

1. Monosynaptic excitatory post-synaptic potentials (EPSPs) produced in spinal motoneurones of the cat by stimulation of a single afferent fibre were recorded with intracellular electrodes.2. In total, seventy-three triceps surae motoneurones were studied with stimulation of thirty-six different afferent fibres.3. The mean amplitude of the EPSPs evoked by single afferent impulses ranged from 0.06 to 2.0 mV with an average of 0.27 mV.4. The mean number of unit EPSPs responding to a single afferent impulse (m) was calculated from the number of failures. The values ranged from 0.7 to more than 5. About 10% of the sample showed no failure of synaptic response in about 200 consecutive trials. The m values for these synaptic responses were estimated to range from 5 to 15.5. In the majority of tests, the observed amplitude fluctuations of monosynaptic EPSPs evoked by stimulation of a single fibre were less than those expected from Poisson's law. This discrepancy may be accounted for by non-linear summation of the unit EPSPs at dendritic synaptic sites.6. It is suggested that the synaptic responses initiated at different sites of a motoneurone may summate linearly at the soma, although summation of unit EPSPs is non-linear at individual synaptic sites.

Analysis of synaptic efficacy in spinal motoneurones from 'quantum' aspects.

1. Synaptic responses of triceps surae motoneurones of the cat following stimulation of single afferent fibres were examined by intracellular recording techniques.2. The mean quantum content (m) of monosynaptic excitatory postsynaptic potentials (EPSPs) was independent of the type of motoneurone recorded and of the afferent fibre stimulated. There was no significant difference in m value between homonymous and heteronymous synapses.3. A positive correlation was found between the amplitude of unit EPSPs and the input resistance of motoneurones. The difference in the amplitude of unit EPSPs appears to be responsible for the higher synaptic efficacy in slow-conducting motoneurones than in fast-conducting motoneurones.4. There was no significant difference in the time course of monosynaptic EPSPs evoked by impulses from homonymous and heteronymous afferent fibres.5. The ratio of monosynaptic connexions from a given afferent fibre was significantly greater on to homonymous than to heteronymous motoneurones. It is concluded that the difference in efficacy between homonymous and heteronymous synaptic transmission is due to the difference in the number of afferent fibres converging upon these motoneurones.

New computed radiography using scanning laser stimulated luminescence.

This short paper summarizes the basic concept and the technology of a new computed radiographic system which uses an energy-storage phosphorus panel called "Imaging Plate" as an image sensor. The "Imaging Plate" can be used to obtain radiographs in exactly the same way as the screen-film combination is used in conventional radiography. The system eliminates the drawbacks of conventional screen-film radiography by combining digital image processing and digitization of the x-ray energy pattern utilizing scanning laser stimulated luminescence.

Computed radiography utilizing scanning laser stimulated luminescence.

A new system of computed radiography that is based on new concepts and the latest computer technologies has been developed. This system eliminates the drawbacks of conventional screen-film radiography. The basic principle of the system is the conversion of the x-ray energy pattern into digital signals utilizing scanning laser stimulated luminescence (SLSL).

Effects of ciguatoxin and maitotoxin on the isolated guinea pig atria.

The effects of ciguatoxin (CT) and maitotoxin (MT) were studied on the isolated atria of the guinea pig. Application of CT evoked a prolonged positive inotropic effect on the electrically-driven atria and both inotropic and chronotropic effects on the spontaneously-beating right atria. The response to MT in contrast was biphasic. At low concentration (10(-8) Gm per ml) MT enhanced the atrial contractility, while at higher concentrations (5 x 10(-8) Gm per ml) it depressed the atria and frequently to a standstill. The results indicate that CT and MT have a direct action on the contractile mechanism of the heart and their effects are readily distinguishable.

Post-tetanic hyperpolarization produced by an electrogenic pump in dorsal spinocerebellar tract neurones of the cat.

1. Hyperpolarization following single and repetitive excitation of dorsal spinocerebellar tract (DSCT) neurones of the cat was studied by intracellular recording.2. Hyperpolarization following an antidromic action potential consisted of an initial, brief phase (undershoot) and a late, prolonged phase. The latter hyperpolarization was independent of the membrane potential, whereas the former was reversed in polarity by hyperpolarizing pulses applied across the DSCT cell membrane.3. DSCT neurones showed a prolonged hyperpolarization after a train of antidromic action potentials. The amplitude and duration of the hyperpolarization were dependent on the number and the frequency of action potentials. A similar hyperpolarization was observed following a train of impulses evoked by depolarizing pulses applied through the intracellular electrode.4. There was no detectable conductance change during the post-tetanic hyperpolarization. The latter showed no reversal potential when the membrane potential was altered.5. The half-decay time of the post-tetanic hyperpolarization was lengthened when the cord temperature was lowered. The temperature coefficient (Q(10)) was 2.4 within the range of 31-40 degrees C.6. The amplitude of the undershoot following each action potential was assumed to provide a criterion for the accumulation of the extracellular K(+). Alterations in the amplitude of undershoots during repetitive excitation suggested that the duration of post-tetanic hyperpolarization depends on the accumulation of extracellular K(+) as well as of intracellular Na(+) associated with a train of impulses.7. It is suggested that post-tetanic hyperpolarization is produced by an electrogenic sodium pump. A possible significance of such a hyperpolarization in impulse coding is discussed.

Characterization of marine toxin(s) in Myripristis sp. by immunological, mouse toxicity, and guinea pig assays.

Myripritis sp. (squirrelfish) has been assessed for toxicity by 1) the stick-enzyme immunoassay (S-EIA); 2) mouse toxicity bioassay; and 3) guinea pig atrial assay. Analysis of Myripristis flesh with MAb-CTX and MAb-OA showed that with every fish examined, the reaction with MAb-OA was considerably higher. The mean S-EIA value for MAB-OA was 2.9 +/- 0.8 while the mean for MAb-CTX was 1.7 +/- 0.5. The data strongly suggests that Myripristis sp. appear to contain okadaic acid-like toxins and/or mixed with CTX. Five fractions of the flesh extracts were obtained by silica gel chromatography. These included 100% CHC1(3), 10% MeOH/CHC1(3), 50% MeOH-CHC1(3), 100% MeOH, and 80% MeOH/H2O. The 100% CHC1(3) eluate proved to be the most toxic (mouse killed in 32 minutes) and the 10% MeOH-CHC1(3) fraction killed in approximately 48 hours. The remaining fractions showed a significantly lower toxicity level in mice. In the guinea pig atria examinations, extracts of Myripristis flesh, gut, and crustacea (from the gut) were studied. Extracts of the gut and crustacea showed strong sodium channel blockage, while the flesh extracts showed a weak inotropic effect, characteristic of okadaic acid. The latter response was only blocked by verapamil, but not with tetrodotoxin (10(-5) M) or the adrenergic blockers (10(-5)M). The data from this study suggest toxic compound(s) not previously reported, in the non-polar fraction of Myripristis flesh having a sodium channel blockage effect.

Studies on the quantitative autoradiography. II. Radioluminography for quantitative autoradiography of 3H.

The process of obtaining 3H-autoradiographic (ARG) images has been expected for a long time. A few X-ray films with no protection layer are commercially available, however, they do not give a reliable image which can be quantitated and can give good contrast. We tried to fabricate a 3H-type sensor which has no protection layer on a highly sensitive sensor, and called it Imaging plate (IP). The IP is composed of one of the specially designed photo-stimulable phosphors containing of BaFX: Eu2+(X = Cl, Br or I) crystals. Results indicated a good contrast and reliable whole-body ARG image of 3H-labelled glucose with trial IP, which has never been obtained with any X-ray films even if these were subjected to a long exposure time. The ARG image can be displayed by either black-and-white hard copy or a colored one with the digital display representing relative intensity of photostimulated luminescence (PSL) without back ground (BG) or relative intensity concentration ((PSL-BG)/S), where S was equivalent to 100 pixels (1 mm2). Similarly to the experimental results of 14C, the linear relationship relative intensity and radioactivity of the 3H standard sources was demonstrated with a very wide range of 10(2) to 10(5) dpm/mg upon the exposure for 7d. Also the relationship between relative intensity and relative exposure (radioactivity x exposure time) was linear within the latitude of relative intensity 10(1) to 10(5) (PSL-BG). The trial IP was particularly effective for the quantitative autoradiography of TLC plates.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative anticonvulsant activity and neurotoxicity of clobazam, diazepam, phenobarbital, and valproate in mice and rats.

The 1.5-benzodiazepine (clobazam), the 1,4-benzodiazepine (diazepam), and two nonbenzodiazepine antiepileptic drugs (phenobarbital and valproate) were evaluated in mice and rats with a battery of well-standardized anticonvulsant test procedures. The results obtained indicate that clobazam and valproate exhibit a wider range of experimental anticonvulsant activity than either diazepam or phenobarbital. Except for clobazam by the maximal electroshock seizure (MES) test in rats, clobazam and valproate are effective in nontoxic doses against MES and all four chemically induced seizures (Metrazol, bicuculline, picrotoxin, and strychnine). Clobazam is effective by the MES test in rats only in doses that exceed the median minimal toxic dose. Phenobarbital is effective against all of the above tests, but minimal toxic doses must be employed to prevent strychnine seizures. Diazepam, on the other hand, is effective in nontoxic doses against seizures induced by Metrazol, bicuculline, and picrotoxin, but protects animals from maximal electroshock and strychnine seizures only when given in toxic doses. When compared on the basis of protective indices (PI = TD50/ED50) calculated from intraperitoneal data, the PIs for clobazam were 1.6 to 13 times higher than those for diazepam. Overall, except for the MES test in rats, the PIs for clobazam were from 1.5 to 44 times higher than those for any of the other three substances. With respect to the MES test in rats, the PI for clobazam was 10.8 times higher than that for diazepam; however, the PIs for phenobarbital and valproate were 3.5 and 4.4 times higher, respectively, than that for clobazam. These data suggest that the spectrum of anticonvulsant activity for the 1,5-benzodiazepine (clobazam) is superior to that for the 1,4-benzodiazepine (diazepam). Also, the broad experimental profile of anticonvulsant activity of clobazam agrees well with its reported broad clinical efficacy.