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.

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.

Large-format imaging plate and weissenberg camera for accurate protein crystallographic data collection using synchrotron radiation.

Off-line and on-line protein data-collection systems using an imaging plate as a detector are described and their components reported. The off-line scanner IPR4080 was developed for a large-format imaging plate ;BASIII' of dimensions 400 x 400 mm and 400 x 800 mm. The characteristics of this scanner are a dynamic range of 10(5) photons pixel(-1), low background noise and high sensitivity. A means of reducing electronic noise and a method for finding the origin of the noise are discussed in detail. A dedicated screenless Weissenberg camera matching IPR4080 with synchrotron radiation was developed and installed on beamline BL6B at the Photon Factory. This camera can attach one or two sheets of 400 x 800 mm large-format imaging plate inside the film cassette by evacuation. The positional reproducibility of the imaging plate on the cassette is so good that the data can be processed by batch job. Data of 93% completeness up to 1.6 A resolution were collected on a single axis rotation and the value of R(merge) becomes 4% from a tetragonal lysozyme crystal using a set of two imaging-plate sheets. Comparing two types of imaging plates, the signal-to-noise ratio of the ST-VIP-type imaging plate is 25% better than that of the BASIII-type imaging plate for protein data collection using 1.0 and 0.7 A X-rays. A new on-line protein data-collection system with imaging plates is specially designed to use synchrotron radiation X-rays at maximum efficiency.

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)

A survey of ciguatera: assessment of Puako, Hawaii, associated with ciguatera toxin epidemics in humans.

A survey for the assessment of the ciguatera problem has been determined in Puako, South Kohala, on the Island of Hawaii. This is in the area of persistent ciguateric outbreaks during the months of January through March, caused by a specific species of fish (Cheilinus rhodochrous, red rose wrasse, or po'ou). Analyses of algae, Gambierdiscus toxicus, and various species of fish, including herbivores and carnivores, gave positive indications of Puako as a potential ciguateric area. Algae associated with Gambierdiscus toxicus blooms and the dinoflagellate itself were found in transects A and D. Transects A and D showed 291 G. toxicus per gram of Tolycarpidia glomurata and 9 G. toxicus per gram of Turbinaria sp. with epiphytic Jania sp., respectively. No G. toxicus was found in transects B and C. This may be attributed to the low salinity from intrusion of freshwater in this vicinity. Examinations of the fish, kole, manini, Hawaiian kole, roi, and po'ou by the solid-phase immunoassay showed 89% of fish in the borderline and positive categories from all transects. Extracts of viscera and flesh showed high levels of toxicity in mouse (13 of 23 deaths), particularly in the viscera (gut) of both herbivores and carnivores. The guinea pig atrial analysis generally showed a few ciguatoxin-like, but most were nonciguateric type responses. The data presented in this Puako survey showed evidence of toxic fish associated with ciguatoxin-like and most probably other toxins, either polyethers or non-polyethers as yet unidentified.

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.

Molecular cloning and nucleotide sequencing of the Arthrobacter dextranase gene and its expression in Escherichia coli and Streptococcus sanguis.

A bacterial strain, which assimilated dextran and water-insoluble glucan produced by Streptococcus mutans, was isolated from soil. The bacterium produced and secreted potent dextranase activity, which was identified as Arthrobacter sp. and named CB-8. The dextranase was purified and some enzymatic properties were characterized. The enzyme efficiently decomposed the water-insoluble glucan as well as dextran. A gene library from the bacteria was constructed with Escherichia coli, using plasmid pUC19, and clones producing dextranase activity were selected. Based on the result of nucleotide sequencing analysis, it was deduced that the dextranase was synthesized in CB-8 cells as a polypeptide precursor consisting of 640 amino acid residues, including 49 N-terminal amino acid residues which could be regarded as a signal peptide. In the E. coli transformant, the dextranase activity was detected mostly in the periplasmic space. The gene for the dextranase was introduced into Streptococcus sanguis, using an E. coli-S. sanguis shuttle vector that contained the promoter sequence of a gene for glucosyltransferase derived from a strain of S. mutans. The active dextranase was also expressed and accumulated in S. sanguis cells.

Evaluation of the stick enzyme immunoassay in Caranx sp. and Seriola dumerili associated with ciguatera.

The stick enzyme immunoassay (S-EIA) using monoclonal antibody to ciguatoxin (MAb-CTX) was used to examine clinically implicated fish and to pre-screen two species of fish, Caranx sp. (ulua or jack) and Seriola dumerili (kahala or amberjack), supplied by sports fishermen. All of the clinically implicated fish from the Department of Health gave S-EIA values greater than or equal to 1.3. The Caranx sp. and Seriola dumerili considered safe (less than or equal to 1.2 value) and consumed after testing gave no false-negative results. The S-EIA procedure using MAb-CTX proved to be specific, sensitive, and simple to use in the laboratory. It also proved to be useful in screening two large carnivorous fish for ciguatoxin and related polyethers prior to consumption.

Causitive toxin(s) in the death of two Atlantic dolphins.

Following the death of two Atlantic dolphins in a lagoon in March of 1989, the Hawaiian fishes in the lagoon were examined as a potential source of toxin(s). This study reports the findings of the causitive toxin(s) involved, utilizing the stick enzyme immunoassay (S-EIA) and the mouse and guinea pig atrium assays. The S-EIA proved effective in screening the toxic fishes (mullet, wrasse, manini, and aholehole). Following extraction, the major toxin was found in the viscera of these fishes, as confirmed in the mouse assay. The most toxic level was shown in the viscera of the mullet (13.2 mouse units/mg of extract). The viscera of the wrasse, aholehole, and manini also showed high levels of the toxic substance. The guinea pig atrium assay showed the presence of a potent Na+ channel inhibitor, characteristic of tetrodotoxin and saxitoxin. The toxin was also demonstrated in low levels in the dolphin liver and gut content and in the sand and algae extracts from the lagoon. This is the first report of this type of toxin in Hawaii.

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.

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.

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.

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).

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.

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.

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.

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.