Yoko Kobayashi

A point mutation in the mitochondrial tRNALeu(UUR) gene in melas (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes)

Mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike episode (MELAS) is a major group of heterogeneous mitochondrial disorders. To identify the defective gene, mitochondrial DNA from a patient with MELAS was sequenced by using amplified DNA fragments as sequencing templates. In 14.1 kbp determined out of 16.6 kbp of the whole mitochondrial gene, at least 21 nucleotides were different from those of a control human mitochondrial DNA. One of the substitutions was a transition of A to G in the tRNA(Leu) (UUR) gene at Cambridge nucleotide number 3,243. This nucleotide is conserved not only in many mitochondrial tRNAs but in most cytosolic tRNA molecules. An Apa I restriction site was gained by the substitution of this nucleotide. The Apa I digestion of the amplified DNA fragment revealed that all independent 6 patients had G at nucleotide number 3,243 in their mitochondrial DNAs, but none of 11 control individuals had G at this position. This result strongly suggests that the mutation in the mitochondrial tRNALeu gene causes MELAS.

Some human B and T cell epitopes of bovine serum albumin, the major beef allergen

Bovine serum albumin (BSA) is the major beef allergen. Since IgE and T cell recognitions are central to the specific immune response to allergens, the identification and immunologic characterization of B and T cell epitopes of BSA represent important steps in the development of treatments for beef allergy. Prior to our experiments, we hypothesized that BSA-specific antibodies and T cells react primarily with sequential epitopes in which the amino acid sequences differ greatly between bovine and human albumin. To clarify this hypothesis, 16 peptides corresponding to such regions were synthesized as candidate epitopes. Among them, at least two regions, aa336-345 and aa451-459, were found to be B cell (IgE-binding) epitopes. In inhibition ELISA experiments, EYAV (aa338-341) and LILNR (aa453-457) bound to patient IgE antibodies and were found to be the cores of the IgE-binding epitopes. Three regions, DDSPDLPKLKPDPNTLC (aa107-123), PHACYTSVFDKLKHLVDEP (aa364-382), and LSLILNRLC (aa451-459), were found to induce T cell proliferation in more than half of the patients tested. Of interest was that these three regions were also recognized by B cells. Information concerning human B and T cells epitopes can contribute greatly to the elucidation of the etiology of beef allergy.

Long-term effect of repeated lidocaine injections into the external oblique for upper camptocormia in Parkinson's disease

BACKGROUND Parkinsons disease (PD) is occasionally complicated by camptocormia. In a previous study, we classified camptocormia into upper and lower types based on the inflection point, and reported that lidocaine injection into the external oblique muscle, but not into the internal oblique or rectus abdomen, improved upper camptocormia in PD. The effect of a single lidocaine injection disappeared over a period of few days. In this study, we used repeated lidocaine injections into the external oblique for 4-5 days and evaluated the effects of such treatment for up to 90 days. METHODS The study subjects were 12 patients with PD and upper camptocormia who were treated with repeated lidocaine injections into the bilateral external oblique followed by rehabilitation. The effect of treatment was evaluated by measuring the angle of truncal flexion before and after the injection. Patients who showed improvement with repeated injections were evaluated during a 90-day period. RESULTS Eight out of 12 patients showed significant improvement in posture after a single lidocaine injection. However, the effect subsided several days after treatment. Repeated injections produced long-term improvement in 9 out of 12 patients, which was maintained during the 90-day observation period in eight of these patients. CONCLUSIONS Our results showed that repeated lidocaine injections into the external oblique improved upper camptocormia, and that the effect was maintained in the majority of patients during the 90-day observation period, indicating that repeated lidocaine injections into the external oblique have therapeutic effect on upper camptocormia in patients with Parkinsons disease.

The mutant mitochondrial genes in mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) were selectively amplified through generations

Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is one of the clinical phenotypes of mitochondrial myopathies characterized by stroke-like episodes (Pavlakis et al 1984). For the cytogenetic analyses, we isolated respiratory-deficient and normal myogenic cell lines from muscle of a patient with MELAS by transfecting replication origin-defective SV40 DNA (Nakamigawa et al 1988; Shimoizumi et al t989). By sequencing mitochondriat DNA (mtDNA) of these two clones, we identified a transition of A to G in the mitochondrial tRNA-Leu(UUR) gene at Cambridge nucleotide number 3243 only in the mtDNA of the respiratorydeficient clone, and concluded that this base transition was associated with MELAS (Kobayashi et al 1990, 1991). Another research group reported the same point mutation by analysing mtDNA of muscle tissues of patients (Goto et al 1990). This indicates that MELAS is associated with mutation in mtDNA like other mitochondrial myopathies, such as Kearns-Sayre syndrome or progressive external ophthalmoplegia in which deletions of mtDNA were detected (Holt et al 1988, 1989; Moraes et al 1989), and myoclonus epilepsy associated with ragged-red fibres in which a base transition in mitochondrial tRNA-Lys gene was reported (Shoffner et al 1990; Yoneda et al 1990). Being different from the nuclearly coded gene mutations, there should exist certain unknown cytogenetic processes for a mutation in one mtDNA to become dominant and produce the patient. To analyse the processes of amplification and distribution of the mutant mtDNA in MELAS, the relative proportion of the mutant mtDNA was quantitatively studied in patients and family members as well as in various tissues. We used PCR-amplified mtDNA fragments after assessing the validity of the method in the quantification of the mutant mtDNA by using normal and the mutant mtDNA cloned from respiratory-deficient and normal myogenic cells.

Fatal reducing body myopathy. ultrastructural and immnunohistochemical observations

Two female infants who developed normally during infancy began to have progressive muscle hypotonia and weakness from 2 years 10 months and 2 years 3 months of ages, respectively. Both patients had rapidly progressive muscle weakness with death from respiratory failure at 4 years 11 months and 3 years 9 months, respectively. In addition to mild inflammation in their muscle biopsies, the most striking finding was the presence of numerous reducing bodies (RB) in almost all degenerating fibers. By electron microscopy, these bodies consisted of fine granular material, usually located around the degenerating nucleus. These bodies showed no immunohistochemical reaction to antibodies against structural, cytoskeletal and membrane proteins and a histone-specific antibody against nuclei and chromosomes. They were occasionally positively stained with a ubiquitin antibody. Although the origin of these bodies remains unknown, they appeared to be related to active myofibrillar degeneration, probably resulting from primary nuclear degeneration.

The α1β1 heterodimer of ATP synthase

Abstract The α 3 β 3 hexamer was reconstituted from the α and β subunits of TF 1 portion of ATP synthase of thermophilic bacterium (Kagawa et al. (1989) FEBS Lett. 249 , 67). The α 1 β 1 heterodimer of ATP synthase was isolated by high performance liquid chromatography (HPLC) of the α 3 β 3 hexamer in the presence of AT(D)P-Mg. On polyacrylamide gel electrophoresis, both bands corresponding to the dimer and hexamer showed ATPase activity. The α 1 β 1 dimer was dissociated into the equal amounts of the α and β monomers by sodium dodecyl sulfate. The α and β monomers were practically inactive. The α 2 and β 2 homodimers were not detected by electrophoresis and HPLC.

Hyperphenylalaninaemia due to impaired dihydrobiopterin biosynthesis: Leukocyte function and effect of tetrahydrobiopterin therapy

We have described the clinical and biochemical status of two patients with tetrahydrobiopterin (BH4) deficiency due to impaired dihydrobiopterin biosynthesis. BH4 administration appeared to improve the mental and psychological status more than did neurotransmitter replacement therapy alone. This enhancement of activities of daily life was seen with a dose of BH4 as low as 1.25 mg kg−1 day−1. Granulocyte adherence capacity was below normal and recovered after BH4 therapy in both patients. B-cell differentiation capacity was altered either before or after therapy.

Validation of the Japanese translation of the Dysphagia Handicap Index

Background We developed, and examined the reliability and validity of, a Japanese version of the Dysphagia Handicap Index (DHI; DHI-J), which is a self-reported measure to assess the quality of life (QOL) of individuals with dysphagia. Participants and methods The DHI-J was developed via the back-translation method: the DHI was translated into Japanese and then translated back into English by a native English speaker. The back translation was discussed with and approved by the DHI’s lead author. A total of 229 patients (119 males, 110 females; median age: 66 years) who underwent videofluorography at our hospital between January and December 2013 and 65 controls (23 males, 42 females; median age: 44 years) were included in the study. All the subjects completed the DHI-J and self-reported their dysphagia severity. Twenty-three patients repeated the procedure 1 week later. Patients’ swallowing function was classified as “normal”, “moderately impaired”, or “severely impaired”, and the DHI-J total scores were compared between the severity groups. Results The internal consistency of the DHI-J was high (Cronbach’s α=0.95), as was the test–retest reliability of the 23 patients who answered the questionnaire twice (intraclass correlation coefficient =0.98, P<0.01). The DHI-J total score and its three subscale scores were significantly higher among the patients than among controls. A significant correlation (ρ=0.85) was observed between the DHI-J total score and self-reported dysphagia severity score. Regarding the comparison of DHI-J scores by severity groups, the DHI-J total scores significantly differed between the normal and moderately impaired groups, and the normal and severely impaired groups. However, the moderately and severely impaired groups showed no significant difference in scores. Conclusion The DHI-J is a reliable and valid questionnaire for assessing the QOL of patients with dysphagia. However, we did not survey patients with cerebrovascular diseases; thus, the questionnaire must be validated for that patient group.