Yoshiyasu Matsuo

Bacteriocin and Hemolysin from Streptococcus faecium

The bacteriocin and hemolysin produced by a Streptococcus faecium strain were partially separated after gel filtration on Ultrogel AcA-22. The molecular weight of bacteriocin was approximately 300,000, and that of hemolysin was 220,000. Non-bacteriocinogenic mutants retained the hemolytic activity. Production of hemolysin required glucose, whereas that of bacteriocin did not.

Enzymological heterogeneity of influenza B virus neuraminidase demonstrated by the fluorometric assay method

The neuraminidase activity of 26 strains of influenza B virus isolated from all over the world was investigated colorimetrically, using fetuin as a substrate, and fluorometrically, using 4-methylumbelliferyl(4-MU)-N-Ac-alpha-D-neuraminide as a substrate, with special reference to enzymological heterogeneity. The activity of influenza A viral neuraminidases and of a commercially available pure viral one was strongly inactivated by either ethylenediaminetetraacetic acid or glycoletherdiaminetetraacetic acid, when measured by the fluorometric assay method, whereas that of influenza B ones was not at all. However, the viral neuraminidases of both influenza A and B viruses were found to be calcium ion-dependent by the colorimetric assay method. A difference in the catalytic rate between the two assay methods was observed with influenza B viral neuraminidase to a much greater extent as compared with that of influenza A. A difference in substrate specificity of these enzymes was demonstrated to be due to a difference in the degree of competitive inhibition by N-acetylneuraminic acid. These findings strongly suggest that enzymological heterogeneity in influenza B viral neuraminidase may be attributed to delicate structural differences, between the enzymes of influenza A and B viruses demonstratable only by the fluorometric neuraminidase assay method using 4-MU-N-Ac-alpha-D-neuraminide as a substrate.

Isolation of Mycoplasma and Ureaplasma Species from Raccoon Dogs ( Nyctereutes procyonoides viverrinus)

Mycoplasma spp. were isolated from five wild raccoon dogs (Nyctereutes procyonoides viverrinus). On the basis of biochemical properties and serological tests, nine isolates were identified as Mycoplasma edwardii and four were similar to a possibly new Mycoplasma sp. represented by strain LM2 which is negative for both glucose fermentation and arginine hydrolysis. In addition, ureaplasmas were detected from these animals. Ureaplasmas were compared serologically with ureaplasma strains isolated from human, monkey, cattle, goat, sheep, cat, chicken and dog and cross-reacted with one of four serological groups of canine ureaplasmas.

Purification and Characterization of β‐Glucuronidase Inhibitor from Mycobacterium tuberculosis

Factors inhibitory to β‐glucuronidase were found in the culture filtrate and in a bacillary extract of Mycobacterium tuberculosis H37Rv grown for 6 weeks on Sauton medium. The inhibitors were purified by ammonium sulfate fractionation, treatment with n‐butanol and streptomycin, and chromatography on DEAE‐Sepharose CL‐6B. Two inhibitors were obtained from the culture filtrate. The molecular weights were estimated to be 25,500 and 15,500 by gel filtration on a Sephadex G‐75 column. Three inhibitors were purified from the bacillary extract, two of which were similar to those from the culture filtrate. The molecular weight of the third inhibitor was 21,000. However, the molecular weight of all the denatured inhibitors was 8,600 in the presence of sodium dodecyl sulfate.

Further Studies of the Use of Cycloheximide for Cultivating Mycobacterium lepraemurium in Cell Culture

The advantage of using cycloheximide for cultivating Mycobacterium lepraemurium in cell culture was further demonstrated. Continuous multiplication of the bacillus in successive subcultures was obtained in MFP, HEp‐2 and Vero cells maintained in culture medium containing 0.1 μg of cycloheximide per ml. Growth characteristics were comparable to those observed in the cultures of A31 cells previously reported. The procedure was simple and convenient. Comparable results, however, have not been obtained in cultures of other established cell lines, HeLa 229, L, MDCK, and Neuro‐2a.

In Vitro Growth of Mycobacterium lepraemurium under the Influence of Macrophage Cultures

Elongation and limited multiplication of Mycobacterium lepraemurium was observed extracellularly when the bacilli spotted on a coverslip were placed face to face with cultures of mouse peritoneal macrophages adhering to the inside of a test tube held at an angle of 15°. There was no doubt that certain growth‐promoting but unstable factors were released from the macrophages.