Rectal mucosal suction biopsy specimens from a series of 16 infants and children with cnstipation and other gastrointestinal problems, were stained with a sensitive acetylcholinesterase (AchE) method. In 8 cases there was an increase in numbers and sizes of cholinergic nerves in the lamine propria and musclaris mucosae and submucosae. These date demonstrate that an abnormal pattern of AchE reaction is diagnostic of aganglionic megacolon. Therefore these methods are useful for diagnosis of Hirschsprung's disease.

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Four proteases (designated Proteases I, II, III, and IV) were obtained from Clostridium Botulinum type A. Purification steps were precipitation with ammonium sulfate, DEAE-Sephadex A-25,Sephadex G-75 and affinity column chromatography. The affinity column was prepared from CNBr-activated Sepharose 4B gel by coupling with L-arginine methyl ester (AME). The molecular weights of these enzymes were estimated to be 40,000 (protease I), 25,000 (protease II), 31,000 (protease III) and 20,000 (protease IV) by gel filtration and by SDS-polyacrylamide gel electrophoresis. The activities for these proteases were inhibited by Cu^<++>, Sn^<++>, EDTA and PCMB. The activities of four enzymes were activated by Ca^<++>. Proteases III and IV showed similar chracteristic for metal ions and inhibitors and they were activated by Mg^<++>, cysteine, DFP and PMSF. Optimum pH for casein hydrolysis was 8.0 for protease I and 8.5 for proteases II, III and IV. Proteases II and III were stable at 40 C for 60 min but proteases I and IV did not. A number of previous reports (2,3,6,9,12,13) have documented the isolation and characterization of Clostridium botulinum proteases (Inukai. 1963,DasGupta. 1971,DasGupta & Sugiyama. 1973,Ohishi. 1977,Nakane. 1978). Inukai et al (6) isolated three proteases from Clostridium botulinum type A. Tjaberg (12,13) isolated two protease produced by C. botulinum type A. Nakane (9) isolated four enzymes from a proteolytic mutant of C. botulinum type E. We attempted the isolation of four proteolytic enzymes (designated Proteases I, II, III and IV) and an esterolytic enzyme (designated Protease V) from C. botulinum type A. In this study, we report the isolation and characterization of four proteolytic enzymes, which differes from the previous findings.

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A protease (Protease V ) with esterase activity for α-N-benzoyl-L-arginine ethyl ester (BAEE) was purified from Clostridium botulinum type A strain 190 by ammonium sulfate fraction, DEAE-Sephadex A-25,affinity chromatography and Sephadex G-50. The affinity column was prepared from Sepharose 4B gel by coupling with L-arginine methyl ester (AME). Protease V showed the greatest affinity for column at pH 7.0 and was eluted by the concentration of salt. The enzyme had a molecular weight of 67,000 as found by Sephadex G-75 gel filtration and by SDS-polyacrylamide gel electrophoresis. The enzyme had both activities for esterase and for amidase. The optlmum pH of esterase activity for BAEE and of amidase activity for α-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) was 7.0. The enzyme required dithiothreitol (DTT) for esterase activity and was activated by addition of I × l0^<-3>M Ca^&lt : ++>, Sn^<++> , Mg^<++> and K^+ . Cysteine, Cu^<++> , Fe^<++> and PCMB reduced the esterase activity by 40〜50%, but DFP did not. Several proteases have been isolated from cultures of C. botulinum (DasGupta, 1971,DasGupta & Sugiyama, 1972,Tjaberg, 1973,Ohishi, 1977,Nakane, 1978). DasGupta & Sugiyama (2,3) isolated a trypsin-like enzyme produced by C. botulinum type B. Ohishi et al (10) purified a sulfydryl-dependent protease (SHP) from a proteolytic strain of C. botulinum type F. Nakane (8) isolated four proteases (proteases I , II , III , and IV ) from a proteolytic mutant of C. botulinum type E. One of them, protease II hydrolyzed α-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and BAEE but not azocasein. The present report describes an isolation and characterization of a protease with esterase activity (designated protease V ) from C. botulinum type A.

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Der dritte Grundsatz enthalt die schwierigste Aufgabe in der Wissenschaftslehre ( 1794/5). Die ersten beiden Grundsatze sind nach Fichte unbedingt. Das bedeutet Absolutheit. Die Aufgabe des dritten liegt also darin, die beiden "Absoluten" zu synthesieren. Ist das aber moglich? Dies scheint logisch betrachtet unmoglich. So konzentrieren sich fast alle Fichte-Interpetation und Kritik ins Problem der Synthesis. Schon hat Hegel In 1801 kritisiert, daβ die Synthesis nicht moglich ist. Und wiederholt sich diese Kritik mannigfaltigerweise. Hier wird behandelt, ob diese Kriik oder Interpretation richtig ist. Dazu mussen Wir zuerst die Ableitungsweise des dritten Grundsatz nachgehen. Dann untersuchen wir dessen Sinn. Schlieβ lich wird die Kritik Hegels behandelt, weil sie den groβ en Einfluss auf fast alle Fichte-Interpretation ausgeubt hat. Durch diese Betrachtung wird gefolgert, daβ es eigentlich nur einen Grundsatz (d. I. Synthesis) gibt, daβ also die Frage, ob die Synthesis moglich ist, scheinbare Frage ist.

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Composition of dehydrocholate-neural red (DCNR) agar medium of Morse and Alire (J. Bacteriol. 76 : 270-271,1958) was modified. The modified DCNR agar proved to be more suitable for testing carbohydrate fermentation in several staphylococcal species including Staphylococcus aureus.

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Cultured tissues of mycobionts are obtained from the following 12 species of Umbilicaria lichens : Umbilicaria aprina, U. caroliniana, U. cylindrica, U decussata, U. esculenta, U. hyperborea, U. kisovana, U. muehlenbergii, U. pensylvanica, U. polyrrhiza, U. proboscidea, and U. vellea. These lichens (intact) are expected for medical usages (I.e.control against cancer and AIDS). Phycobionts ( Trebouxia) are also isolated from the following five Umbilicaria species : U. caroliniana, U. esculenta, U. hyperborea, U. muehlenbergii, and U. pensylvanica. Mycobionts grow separatedly from phycobionts in all 12 Umbilicaria species under at 15℃, and dark condition in MY-medium. My-cobionts of Umbilicaria caroliniana, U. muehlenbergii, and U. pensylvanica grow well under at 15℃ and dark conditions in MY-medium. On the contraly, mycobionts of the following three species scarcely grow : U. aprina, U. esculenta, , and U. proboscidea. Mycobionts of U. muehlenbergii and U. pensylvanica grow about 100 times their intial weight at 15℃ after 16 weeks culture.

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Among three 2ndary metablic substances which are detected in the intact thallus of Usnea flexilis Stirt. Protocetraric acid and salacinic acid are detected in both culture tissues and mycobiont colonies of U. flexilis by using TLC; howere, usnic acid is found only in its natural thallus. Production and amounts of 2ndary metablic substances in culture tissues or mycobiont colonies seem to be valiable during their culture time. Phycobints of U. flexilis are identifed as Trebouxia usneae (Hidreth et Ahmadjian) Gartner.

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