The inhibition of the binding of ¹²⁵I-labeled Clostridium botulinum type C neurotoxin to synaptosomes by unlabeled toxin indicated that there were two kinds of receptors on the synaptosomal membrane. The dissociation constants (K_d) were calculated as 79 pM and 35 nM from the concentration of unlabeled toxin that induced half-displacement of bound ¹²⁵I-toxin. These values agree satisfactorily with the values obtained from direct binding experiments (Agui, T, , B., Oguma, K., Iida, H., & Kubo, S. (1983) J. Biochem. 94, 521-527).
The inhibition of the binding of ¹²⁵I-toxin to synaptosomes and N-acetylneuraminyl(α2-3)galactosyl(β1-3)N-acetylgalactosaminyl(β1-4) [N-acetylneuraminyl(α2-8)N-acetylneuraminyl(α2-3)]galactosyl(β1-4)glucosyl(β1-1)ceramide (GT_1b) by unlabeled heavy chain indicated that heavy chain facilitates the binding of toxin to synaptosomes and GT_1b. The synaptosomal and heavy chain complex K_d values were estimated as 12 nM and 24 μM.
Monoclonal antibodies C-9 and CA-12 recognized the binding sites to GT_1b and synaptosomes, respectively. Antigenic determinants against the two antibodies are presumably partially overlapping, and the overlapping area seems to be essential to the reaction between toxin and C-9 antibody.

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The binding of Clostridium botulinurn type C neurotoxin to rat brain synaptosomes was determined by the use of ¹²⁵I-neurotoxin. The binding was independent of the incubation temperature (0°C and 37°C) and was equilibrated in 10min. The dose dependence of ¹²⁵I-toxin binding to synaptosomes at 0°C showed that there were two kinds of toxin receptors on the synaptosomal membrane; the association constants and maximum binding values were 1.05×10¹⁰ M^-1, 5.25×10^-13 mol/mg of synaptosomal protein and 5.00×108 M^-1, 5.00×10^-12 mol/mg of synaptosomal protein, respectively.
When the incubation of toxin with synaptosomes was continued at 37°C after ¹²⁵I-toxin had been pre-incubated with synaptosomes at 0°C for 10 min, the displacement of labeled toxin by the addition of excess amounts of unlabeled toxin decreased slightly with increasing incubation time, and finally 0.4% of the bound ¹²⁵I-toxin was not displaced from synaptosomes. The binding of ¹²⁵I-toxin to synaptosomes was inhibited by anti-heavy chain IgG and a monoclonal antibody which neutralized toxin and recognized heavy chain. These results suggest that the binding sites of toxin to synaptosomes are localized on heavy chain and a small amount of the bound toxin is incorporated into the synaptosomal membrane or synaptosomes.

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The binding characteristics of Clostridium botulinum neurotoxins of types B, C₁, and F to gangliosides was studied by thin layer chromatography plate and microtiter plate methods at low (10mM NaCI in 10mM Tris-HCI buffer, pH 7.2) or high (150mM NaCI in 10mM Tris-HCI buffer, pH 7.2) ionic strengths and at 0 or 37°C. The three types of toxins bound exclusively to three kinds of gangliosides, GD_1a, GD_1b, and GT_1b, in both the thin layer chromatography plate and the microtiter plate methods. Type C₁, toxin bound to the three gangliosides under all the conditions, while type B and F toxins bound only at low ionic strength and 37°C. At low ionic strength, the binding kinetics for the three toxins was monophasic in Scatchard plots, and the association constants obtained in the microtiter plate system were 2-4×10⁸M^-1. In contrast, the binding kinetics of type C₁ toxin in high ionic strength was biphasic in the Scatchard plot, and two association constants were obtained in the microtiter plate system. The heavy chain facilitated the binding of the toxin to the gangliosides.
These results indicate that different types of botulinum toxins bind to the gangliosides under different optimal conditions and that gangliosides may not be the common receptor for all types of botulinum toxins. The gangliosides may bind to type C₁ toxin together with other potential receptor (s) on synaptosomal membranes.

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The action of botulinum neurotoxin type C₁, on the release of acetylcholine from rat brain synaptosomes was studied by using anti-toxin heavy chain Fab and anti-toxin light chain Fab. The toxin was bound to synaptosomes at 0°C for 10 min, in which [¹⁴C] acetylcholine had been accumulated previously. The toxin-binding synapto-somes were pre-incubated at 37°C, and the release of acetylcholine was determined after the synaptosomes had been incubated in 25 mM KCl-incubation medium for 20 min at 37°C. Inhibition of [¹⁴C] acetylcholine release from the synaptosomes was observed with increasing pre-incubation time and toxin concentration, and the maximum inhibition was seen after preincubation for at least 15 min, which was called the “lag time.” The toxin-binding synaptosomes were reacted with anti-toxin heavy chain and anti-toxin light chain Fabs at 0°C for 1.5 min before preincubation of the synaptosomes at 37°C. Both Fabs reversed the acetylcholine release inhibi-tion by the toxin. However, when the Fabs were added during the pre-incubation time at 37°C, they showed less restoration with increasing pre-incubation time. The restoration was completely abolished if the Fabs were added to the synapto-somes after the first half of the “lag time.” On the other hand, when ¹²⁵I-labeled toxin-binding synaptosomes were reacted with the Fabs at 0°C for 1.5 min before pre-incubation of the synaptosomes at 37°C, anti-heavy chain Fab removed ¹²⁵I-toxin from the synaptosomes, but anti-light chain Fab did not. However, if the Fabs were added to toxin-binding synaptosomes during the pre-incubation time at 37°C, the Fabs could not remove ¹²⁵I-toxin from the synaptosomes, and thesynaptosomes retained more labeled toxin with increasing pre-incubation time. These results suggest that there are three distinct steps in the inhibition of acetyl-choline release from synaptosomes by botulinum neurotoxin. The first is binding, which is reversible, temperature-independent, and mediated by the heavy chain of the toxin. The second is temperature-dependent internalization, that takes place in the first half of the “lag time, ” in which both the chains are internalized into synaptosomes. The third is the development of toxicity, which requires the latter half of the “lag time.”

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Since 1962, Shuto Expressway has been playing an important role in enhancing mobility in Tokyo area, offering novel landscape in the city at the same time. This paper attempts to clarify the transition of technical method to treat landscape, and social image toward its view, and their correlations.
In earlier times, view of Shuto Expressway was taken with positive image of modernism with its elegant shape formed by structural design. In early 1970's its image turned negative by pollution problem and several methods aimed to weaken its visual tension. Recently, the image turned positive again. Verbal technique made parts of Expressway of “the Sights”, and visitors discovered its essential beauty from novel angle. That caused us to fmd morphological interest in it.

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We report a female patient with dermatomyositis whose erythematous lesions were successfully treated with topical tacrolimus. In spite of the usually good response of muscle symptoms to systemic corticosteroid, the skin lesions of dermatomyositis often remain resistant to conventional therapy. The skin lesions of our patient were markedly improved after four weeks of topical application of tacrolimus. The clinical efficacy of tacrolimus has been described for other skin disorders, but tacrolimus ointment is also useful for treating the skin lesions of dermatomyositis.

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Two fragments with molecular weights of 105, 000 (fragment I) and 58, 000 (fragment II) were separated chromatographically from each other after Clostridium botulinum type A derivative toxin adsorbed onto a QAE-Sephadex column was treated with dithiothreitol and urea. They were antigenic and formed crossing precipitin lines against anti-derivative toxin in agar gel diffusion tests. Upon removal of dithiothreitol and urea by dialysis, the two fragments reassembled to reconstruct the derivative toxin molecule.

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  The aim of this study is to examine the cytocompatibility of new cobalt-29 chromium-6 molybdenum alloys prepared by addition of a small amount of zirconium or carbon. Four kinds of Co-based alloy, containing 0.05 mass% of zirconium (0.05Zr-CCM), 0.08 mass% of zirconium (0.08Zr-CCM), 0.09 mass% of carbon (0.09C-CCM) and 0.18 mass% of carbon (0.18C-CCM), were prepared. The tests for cell adhesion, colony formation and cell growth were carried out according to a procedure of JIS T0301 by use of L929 cells.
   By addition of ziruconium to cobalt-based alloy, some cytocompatibilities were improved. in cell adhesion and colony formation in the direct contact tests.
   In 0.05Zr-CCM, the rates of cell adhesion and colony formation in the direct contact tests were improved in 0.05Zr-CCM but not in the other alloys. The colony formation rate in the static extract solution was improved in 0.08Zr-CCM. In the dynamic extract solution, the rate was decreased in 0.09C-CCM and increased in 0.18C-CCM.
   These results indicate that the cytocompatibility of Co-based alloy is changeable by addition of a small amount of zirconium or carbon.

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微生物の産生する毒素タンパク質はヒトおよび動物に対する殺傷力が強く,古くからその構造と機能および防圧が研究の対象となってきた.すなわち,その作用が微量で発現することから,(1)酵素の阻害,特にカスケード反応への関与,(2)細胞膜の攪乱,(3)イオン輸送の攪乱および(4)高次生体制御系の攪乱など細胞間および細胞内情報伝達系への関与が予想されてきた.一方,毒素の単離・精製が進み,その構造と機能に関する知見が蓄積されるにつれて,毒素の特異的な作用を積極的に利用し生体機能の解明に役立てる気運が高まりつつある. ボツリヌス神経毒素は嫌気菌Clostridium botulinumの産生する毒素タンパク質で高次生体制御系を攪乱する.ここでは,本毒素の構造と機能に関する知見をまとめるとともに,本毒素による生体機能の解析への応用について考察する.

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細胞融合によるボツリヌス毒素に対する単クローン抗体作製の過程で,大分子量の抗体存在が予想されたので,これを製精して性状を調べた. 単クローン抗体を含む腹水をSephadex G-200でゲルろ過し,IgG・IgM間に溶出される画分を,抗原(毒素)を結合させたカラムに負荷して,目的物質を分離製精した. 製精標品は,disc電気泳動で移動率の異なる3バンドを示し,すべての成分が抗体活性を有していた. SDS電気泳動では,245,000, 22,000, 188,000, 152,000, 135,000, 105,000, 75,000, 67,000, 52,000,および25,000のバンドが検出された. 2-メルカプトエノタール処理により,135,000以上のバンドは消失し,通常IgGの重鎖と軽鎖の他に,120,000, 82,000および67,000のバンドが検出された. 標品はゲル内沈降反応で抗IgG抗体と反応したが,抗IgM抗体および抗IgA抗体とは反応しなかった. 以上の結果から,細胞融合では通常IgGの他に,大分子IgGも産生されていることが示唆された.

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  We evaluated the biocompatibility of cobalt-29 chromium-6 molybdenum alloy by analyzing disorders of the membranes of cells of the murine fibroblast L929 line. L929 cells were incubated with 10% fetal bovine serum at 37°C for 7 days in Eagle's minimum essential medium in the presence of either an extract of this alloy, or titanium extract, or vanadium ions. No morphological changes in the cells were observed in medium containing cobalt-29 chromium-6 molybdenum alloy extract or titanium extract, but a remarkable change was observed in the medium containing vanadium ion as a positive control. From the enzyme activity of the culture supernatant we determined the amount of lactate dehydrogenase (LDH) that leaked through the cell membranes. The amount that leaked from cells incubated in the presence of either of the metal extracts or vanadium ion decreased on day 1 and then increased with incubation time from day 2 onward. There was only a small increase in leakage in the media containing the metal extracts but a marked increase in the medium containing vanadium ion. These results indicate that the cell membrane remained nearly unaffected by Co-29Cr-6Mo alloy.

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The secretory response of the exocrine pancreas via endogenous secretin (IRS) by intraduodenal instillation of hydrochloric acid (HCl) and various monocarboxylic acid solutions was studied in anesthetized piglets. The secretion induced by HCl solutions of various concentrations containing 250mM NaCl occurred when pH of the solutions was lower than 1.5. After instillation of the HCl solution of pH 1.0, juice flow and protein output increased 26 times and 9 times, respectively, as compared with basal levels. Such pancreatic responses paralleled an increase in plasma IRS concentration in the portal vein. The pancreatic response induced by a lactic acid solution occurred when pH of the solutions was lower than 3.8. The juice flow and protein output stimulated by a lactic acid solution of 250mM and pH 2.0 were 16 and 8 times higher than the basal levels. The responses to the lactic acid solution of pH 2.0 increased concentration dependently, and were followed by an increase in IRS concentration in the portal vein. The pancreatic exocrine responses induced by other monocarboxylic acid solutions (250mM) of pH 2.0 were in the following order: formic acid>lactic acid>pyruvic acid_??_acetic acid>butyric acid>propionic acid. Lactamide, an analogous substance of lactic acid, did not evoke any pancreatic secretion. The results indicate the possibility that pancreatic exocrine response induced by HCl is dependent upon hydrogen ion, while the response induced by monocarboxylic acid is not always dependent on dissociation constant of acid.

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Three cases of laryngeal tuberculosis that were experienced in the Department of Otolaryngology, General Hospital, are reported.
Although it has been said that laryngeal tuberculosis decreased recently in number, they were still reported sometimes, and a case per 2-3 years had visited our out-patient clinic. We did not think that laryngeal tuberculosis was a very rare case.
All three cases had suffered from lung tuberculosis, but they all had been examined regulary, and they had thought that it had already been cured. But laryngeal tuberculosis had recurred due to intraductal infection.
As it is necessary to make differential diagnosis of laryngeal tuberculosis from carcinoma, histopathological examination is important.

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Congenital factor XIII (FXIII) deficiency is a rare congenital hemorrhagic disorder. Since FXIII is an essential factor in pregnancy, pregnant patients with congenital FXIII deficiency should receive adequate replacement of FXIII concentrate. We report here on a 19-year-old pregnant woman with congenital FXIII deficiency. Despite regular replacement of FXIII concentrate, FXIII activity decreased rapidly after 32 weeks of gestation and she had to receive a high-dose of FXIII concentrate until delivery. Careful monitoring of FXIII activity is needed in patients with congenital FXIII deficiency, because FXIII activity may decrease rapidly in late pregnancy.

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