Biomedical subjects
Publications and source records attributed to Y Uesaka.
A simple method for purification of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and for purification of cholera toxin using immobilized D-galactose column is described. A single run of column chromatography yielded homogeneous toxin as demonstrated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis.
We used transcranial magnetic stimulation on nine normal volunteers to establish an effective way to stimulate the leg area of the motor cortex. Three types of coils: a large figure-eight coil, small figure-eight coil, and a round coil were used. Surface electromyographic activities were recorded from the left tibialis anterior muscle, and the latencies and amplitudes compared with those obtained by anodal electrical stimulation. The most stable responses were obtained when the large figure-eight coil was centered over the vertex and backward current was run through it or when the round coil was centered two to three centimeters anterior to the vertex with left-flowing current in it at the posterior widening. The latencies obtained under these stimulation conditions were the same as those obtained by electrical stimulation. We conclude that direct activation of the pyramidal cells occurs in the leg area of the motor cortex in all forms of magnetic and electrical stimulation.
Biochemical and physiological traits of a collection of strains of Vibrio cholerae O139 Bengal isolated from India, Bangladesh, and Thailand showed that these strains formed a phenotypically homogeneous group with identical characteristics that were essentially similar to those of the O1 serogroup. Resistance to 150 micrograms of the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine) and Mukherjee's El Tor phage 5 and classical phage IV and the nonagglutinability of the strains with O1 antiserum were the only discernible differences between the O139 and O1 serogroups. Extensive serological characterization further revealed the O139 serogroup to be distinct from the existing 138 serogroups of V. cholerae. Antiserum raised against the O139 serogroup required absorption with the R reference strain CA385 and with the reference strain representing serogroup O22 to remove cross-reacting agglutinins. All of the 223 representative strains of V. cholerae O139 examined hybridized with DNA probes specific for the cholera toxin (CT) gene, zonula occludens toxin gene, and El Tor hemolysin gene but not with the probe specific for the heat-stable enterotoxin gene. The amount of CT present in stool samples of patients infected with the O139 serogroup was higher than that found in stools of patients infected with O1 El Tor, and this echoed findings that the amount of CT produced by O139 strains in vitro was higher than that produced by the O1 El Tor strains. The nucleotide sequences of the genes encoding the A and B subunits of CT of the O139 serogroup were identical to the sequences reported for the CT gene of O1 El Tor. The CT gene of O139 strains could be amplified by using primers developed for detection of the CT gene of the O1 serogroup by a PCR assay, which could also be used to detect the CT gene in stool samples of patients infected with strains of the O139 serogroup.
Hyaline-like inclusions (HLIs) and skein-like inclusions (SLIs) were recently observed in the lower motor neurons in amyotrophic lateral sclerosis (ALS). HLIs mainly consist of randomly arranged neurofilaments and abnormally large (about 15 nm) fibrils, while SLIs comprise bundles of the abnormally large fibrils alone. Although the abnormal fibrils have been reported to be ubiquitinated in both types of inclusions, their final catabolic destination in the neurons is unknown. Electron microscopic observation of the lower motor neurons in an ALS case revealed several neurons containing SLIs in addition to ones containing HLIs. Bundles of SLIs, which were located close to HLIs or were observed by themselves without an HLI, were frequently associated with flattened cisterns; some were wrapped, some were completely sequestrated, and some were demarcated together with a portion of moderately electron dense cytoplasm by cisterns or double-membraned units. These features resemble reported early stages in the formation of autolysosomes, and thus indicate that at least some of the bundles of SLIs or parts of them in the lower motor neurons in ALS undergo degradation through the autolysosomal pathway.
We investigated somatosensory evoked magnetic fields (SEFs) and somatosensory evoked potentials (SEPs) in patients with myoclonus epilepsy. The median nerve was stimulated at the wrist, and responses were recorded over the contralateral hemisphere. The source of the enlarged cortical component of the giant SEF was localized on the post-central sensory cortex. The P1 component of the giant cortical response was composed mainly of a tangentially oriented dipole at area 3b. This is the first magnetoencephalographic analysis of an abnormally enlarged somatosensory cortical response.
Stool specimens obtained from 123 hospitalized patients with acute secretory diarrhea admitted to the Infectious Diseases Hospital, Calcutta, India, were examined for isolation of Vibrio cholerae O1 by direct or enrichment plating on selective media for cholera toxin (CT) by bead enzyme-linked immunosorbent assay (bead-ELISA) and for the CT gene by polymerase chain reaction (PCR). V. cholerae O1 was isolated either by direct culture or by enrichment culture from 70 stool specimens, all of which gave positive results by PCR. Eleven specimens which were culture negative and bead-ELISA positive also gave positive results by PCR. In addition, 13 more specimens which were negative by both the culture method and bead-ELISA, were positive by PCR. With the combined results of both the culture method and the CT bead-ELISA, a confirmed laboratory diagnosis of cholera could be made from 81 stool specimens, while the combined results of the three methods, including PCR, yielded a positive result for 94 specimens examined. From these data, we conclude that PCR provides a more sensitive and specific assay for rapid diagnosis of cholera than currently available methods.
A 65-year-old woman with facial erythema and hypergammaglobulinemia developed excessive fatigability. A diagnosis of Lambert-Eaton myasthenic syndrome (LEMS) was made from electrophysiological studies. She had symptoms and laboratory data compatible with probable Sjögren's syndrome. Skin biopsy revealed the histological findings of discoid lupus erythematosus. Treatment with 3,4-diaminopyridine resulted in the improvement of fatigability. LEMS should be recognized as a treatable complication of systemic autoimmune diseases.
A bead-enzyme linked immunosorbent assay (bead-ELISA) for detection and quantification of cholera toxin (CT) in broth cultures of Vibrio cholerae O1 has been developed. Under optimal buffer and pH conditions the bead-ELISA could consistently detect 40 pg/ml of CT. None of the ingredients of commonly used media for in vitro culture of V. cholerae O1 hindered the performance of the bead-ELISA. Evaluation of the sensitivity and specificity of the bead-ELISA against the commonly used reversed passive latex agglutination (RPLA) test for detection of CT was performed using a collection of 239 strains of V. cholerae O1 (including both biotypes and serotypes) which were examined by a gene probe encoding for the A1 subunit of CT. Although both the assays were highly specific, the bead-ELISA was more sensitive than the RPLA. Quantification of CT by the bead-ELISA revealed that the concentration of CT produced by the strains of V. cholerae O1 which were negative by the RPLA was lower than 1 ng/ml and therefore below the minimum detection ability of the RPLA. The bead-ELISA is a simple, specific and highly sensitive assay for routine detection of CT and is recommended for routine use in clinical microbiology laboratories.
A highly sensitive bead enzyme-linked immunosorbent assay (bead ELISA) for detection of cholera toxin (CT) was evaluated for direct detection of CT from stool specimens of patients with acute secretory diarrhea. Of the 75 stool samples examined, 59 yielded biochemically, and serologically confirmed strains of Vibrio cholerae O1. The bead ELISA was positive for CT in stool supernatants in 50 (84.7%) of the 59 samples from which V. cholerae O1 was isolated. In addition, the bead ELISA was positive for three stool specimens which were negative by culture. The free CT present in 48 of the 50 stool samples positive by culture for V. cholerae O1 and for CT by bead ELISA was completely absorbed by anti-CT immunoglobulin G. All of the 59 strains of V. cholerae O1 biotype eltor isolated in this study produced in vitro CT. The concentration of CT present in the bead ELISA-positive stool samples ranged between 26 pg/ml and greater than 100 ng/ml. This evaluation study demonstrates that the bead ELISA is a sensitive and simple method for direct detection of CT in nonsterile stool samples, and we recommend routine use of this assay for detection of CT in stool samples and culture supernatants in clinical and reference laboratories.
We report the results of a clinical trial of Magnetoencephalography (MEG) on spike foci in patients with epilepsy, which was performed from December 1990 to June 1991 at The University of Tokyo Hospital. Fifty patients with focal epilepsy; 26 primary epilepsy, 24 secondary epilepsy (7 brain tumor, 4 arteriovenous malformation, 4 encephalitis, 3 porencephaly, 2 arachnoid cyst, 1 brain abscess, 1 hemimegaloencephaly, 1 Lance-Adams syndrome, 1 hygroma), and ten normal subjects were enrolled in this study. MEG data were recorded using a 37-channel biomagnetometer system SMI-1001 (BTi Magnes, Biomagnetic Technologies, Inc., San Diego). A simultaneous 19-channel EEG recording with linked-ear reference was also obtained. The overall study was completed safely and none of the normal subjects showed abnormal paroxysmal MEG activity. Two patients showed interictal EEG spikes which would not have been noticed without first noting the presence of corresponding prominent MEG spikes. On the whole, the MEG signal seemed to have a wider frequency bandwidth than EEG. In most cases, the source localization predicted by MEG corresponded well with the EEG findings. The relative accuracy of MEG spike source localization was estimated to be within a cubic centimeter from the cases which showed tightly clustered localization of individual spikes. High-pass filtering reduced interference by superimposed slow wave activity, thereby improving the localization of spike sources. These results demonstrate that 37-channel biomagnetometer system could be a useful tool for analyzing epileptic spike sources.
A case of gliomatosis cerebri was studied with positron emission tomography (PET). Carbon-11-L-methionine (11C-Met) accumulated in the diffusely infiltrative tumorous area more widely and accurately than the lesion detected by conventional x-ray computerized tomography (CT) or magnetic resonance (MR) imaging. Autopsy findings three months after the time of the PET study showed good anatomical correspondence between the extent of densely aggregated tumor cells and the region with high uptake of 11C-Met. PET may offer an innovative approach in the delineation of gliomatosis cerebri, which has not been clearly recognized by CT or MR.
A 75-year-old woman was admitted to our hospital complaining of right hypochondrial pain. Echo sonography and computed tomography demonstrated a large tumor with irregular internal density in the right lobe of the liver. Angiography revealed a moderately hypervascular tumor. She was treated with transcatheter arterial embolization. Three weeks later, the tumor ruptured. She died of accompanying acute myocardial infarction seven months after the onset of the illness. Autopsy revealed primary fibrosarcoma of the liver. The tumor appearance varied from firm whitish to soft myxomatous. A part of the tumor showed hemorrhagic necrosis. There was no intrahepatic metastasis. The tumor tissue was composed of spindle shaped cells and immunohistochemically stained with vimentin.
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A gastric carcinoma associated with marked leukocytosis is reported. The case involved a 70-year-old male who was admitted to Akita Kumiai General Hospital because of general fatigue and vertigo in October, 1989. A palpable, hard, uneven mass was found in the epigastrium. Further, an upper gastrointestinal series revealed a large filling defect in the gastric body. Also, an endoscopic examination disclosed an uneven mass, and specimens that were biopsied showed an adenocarcinoma. The white blood cell (WBC) count was 45,800/microliter and had a 96% presence of neutrophils, and an examination of the bone marrow showed excessive myeloid hyperplasia. The WBC gradually increased, reaching up to 67,800/microliter. Finally, an EIA assay of the serum colony stimulating factor (CSF) disclosed a marked increase. After a gastrectomy, the WBC promptly decreased to the normal range and the CSF activity in a supernatant of a tumor-cell-conditioned medium showed a higher value. Thus, this tumor was diagnosed as being a CSF-producing gastric carcinoma.
To establish a rapid method for detection of Haemophilus influenzae, an antiserum against H. influenzae (Anti-HibOMP) was prepared by immunization of rabbits with crude outer membrane proteins (OMP) of H. influenzae type b. Various isolates of H. influenzae including typable and nontypable strains and other species were tested by ELISA and Western blot assay with Anti-HibOMP. The results are as follows: 1. Anti-HibOMP reacted to all of the OMPs from 18 H. influenzae isolates which contained typable and nontypable strains by Western blot assay. Molecular weights of these OMPs were about 24, 27, 31, 34, 39 and 45 kilo-dalton. This result suggests that all H. influenzae isolates have identical antigenic proteins on their outer membranes. 2. It was found that 172 out of 179 (96%) culture suspensions of H. influenzae isolates including 66 typable and 113 nontypable showed positive result by ELISA with Anti-HibOMP. 3. To define the cross-reactivity of Anti-HibOMP, 20 species (111 isolates) other than H. influenzae were tested by the ELISA. All isolates were negative with exception of a portion of H. parainfluenzae and Staphylococcus aureus producing Protein A. The cross-reactions to H. parainfluenzae and S. aureus were removed by absorption of Anti-HibOMP with formalinized cells of H. parainfluenzae and reduction of the antibody to F(ab)2 with pepsin digestion respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
A highly sensitive sandwich enzyme-linked immunosorbent assay to detect bacterial toxins was developed. Fab' of anti-toxin IgG was conjugated with horseradish peroxidase by the maleimide method and tetramethylbenzidine was used as substrate. As the solid phase, a 6.5 mm diameter polystyrene bead was used and this was coated with the anti-toxin IgG. The entire assay could be completed within 3.5 hr. The sensitivity of this bead-ELISA was found to be quite high with various bacterial toxins: less than 20 pg/ml for thermostable direct hemolysin of Vibrio parahaemolyticus, less than 60 pg/ml for Shiga toxin, less than 20 pg/ml for VT2 (Shiga-like toxin II) of Escherichia coli, less than 200 pg/ml for heat-labile enterotoxin of E. coli, and less than 6 pg/ml for cholera enterotoxin.
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