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A rapid and simple assay method for determining neuraminidase activity was investigated fluorometrically with 4-methylumbelliferyl-N-Ac-α-D-neuraminide as a substrate. The time required for performance was less than 3 hr and the activity could be simply measured with a fluorescent spectrophotometer. Neuraminidase activity in culture supernatant of MDCK cell infected with a small quantity of influenza A/Aichi/2/68 virus was demonstrated by the fluorometric assay 24 hr after infection, when no viral cytopathogenic effect could be observed and no viral multiplication could be demonstrated by the hemagglutination test and the colorimetric neuraminidase assay method using fetuin as a substrate. The result was that the minimum number of virion detectable by the fluorometric neuraminidase assay method was approximately 10^3 order.

Alpha protein antigen isolated from culture filtrate of Mycobacterium tuberculosis by Yoneda and Fukui (1961) was a cross-reacting material among mycobacteria. Purified alpha protein of M. tuberculosis obtained by a series of procedures with gel filtration, ion-exchange chromatographies, and chromatofocusing. Alpha protein of Mycobacterium intracellulare was purified by an affinity chromatography on anti M. tuberculosis-alpha serum conjugated sepharose. Alpha antigen of M. tuberculosis possessed specific antigenic determinants for both M. tuberculosis and M. bovis, and so did alpha antigen of M. intracellulare for both M. intracellulare and M. avium. Extracts from M. kansasii, M. scrofulaceum, M. gordonae, M. xenopi and M. gastri showed alpha antigenicity somewhat different from those of M. tuberculosis and M. intracellulare. Alpha antigen was not detected in extracts from M. nonchromogenicum, M. terrae and M. triviale.

The interaction of mycobacterial lipase inhibitor (MLI), isolated from culture supernatant fluid of Mycobacterium tuberculosis strain H37Rv, and lipase from guinea pig peritoneal macrophages (GP-PMφs) was investigated fluorimetrically by the modified lipase assay system which had previously been proposed. Two peaks of lipase activity were observed in the enzyme preparation from GPPMφs. The activity of MLI against lipase from GP-PMφs was significantly high at acidic pH less than 5.0, and the pattern of inhibition was non-competitive. Two types of lipase were isolated from the enzyme solution prepared from GPPMφs by an ion-exchange chromatography on a DEAE-Sepharose column. One of these acted only at pH 4.5 and was considered to be a lysosomal acid lipase, but another showed the activity at both pH 4.5 and 7.0. The former was four times more sensitive to the activity of MLI than the latter as well as the crude enzyme preparation.

Extracellular release and subsequent decrease of intracellular lysosomal enzymes of guinea pig polymorphonuclear leukocytes (PMNLs) by influenza virus were observed. Total enzyme activity was also decreased in all the enzymes assayed. After incubation of the PMNLs with influenza virus at 37°C for 20 min, the degree in decrease of total enzyme activity varied from enzyme to enzyme: 50% in myeloperoxidase (MPO), 20% in acid phosphatase, 10% in N-Ac-β-glucosaminidase and β-glucuronidase and 5% in lysozyme, respectively. Since MPO assumed to play a critical role in chemiluminescent response of luminol-enhanced system, the dysfunction in oxidative metabolism of PMNLs induced by influenza virus seems to be attributed to intraphagosomal and/or extracellular inactivation of MPO of PMNLs during the process of direct stimulation of respiratory burst.

Some basic conditions for fluorometric measurement of lipase activity of guinea pig peritoneal macrophages were investigated using 4-methylumbelliferyl-oleate as a substrate. The most adequate condition for the assay is as follows: i ) Enzyme preparation recommended is the supernatant of ultrasonicate after freezing and thawing of guinea pig peritoneal macrophages suspended in distilled water. ii) Final concentration of buffered substrate should be adjusted at 0.1 mM in acetate buffer, pH 4.5, free from Triton X-100. iii) Reaction is terminated by addition of 50 mM Tris-HCl buffer, pH 8.6, instead of 50 mM glycine buffer, pH 10.4.

Effect of influenza virus on luminol-enhanced chemiluminescent response of guinea pig polymorphonuclear leukocytes (PMNL) was investigated in order to elucidate a possible mechanism by which influenza virus modified the oxidative metabolism of PMNL. Influenza viruses, strains A/USSR/92/77(H1N1) and B/Kanagawa/3/76(B), rapidly stimulated oxidative burst of resting PMNL and the peak chemiluminescence initiated by them was 5 to 15 times as high as that induced by PBS(—), while the peak chemiluminescent response induced by phorbol myristate acetate (PMA) delayed and the values were counted to be approximately 40% of that of PBS(—)-incubated cells. No significant changes were observed in both activities to enhance chemiluminescent response of resting PMNL and to depress PMA-induced chemiluminescent response when influenza virus with heatinactivated neuraminidase activity at 56°C for 20 min was used. Moreover, neither initiation of oxidative burst of PMNL nor depression of PMA-induced chemiluminescent response was observed with purified neuraminidase extracted from influenza virus. It seems likely that viral neuraminidase is at least not a critical component to modify the oxidative metabolism of guinea pig PMNL.

β-Glucuronidase inhibitor extracted and purified from culture filtrate of Mycobacterium tuberculosis H37Rv significantly reduced the microbicidal activity of guinea pig peritoneal macrophages against Candida parapsilosis. It did not have any effect on the antimicrobial action of polymorphonuclear cells against Staphylococcus aureus 209P.

The fluorometric neuraminidase assay method using 4-methylumbelliferyl-N-Ac-α-D-neuraminide as a substrate was investigated for the purpose of ascertaining its validity as a procedure for influenza viral neuraminidase antibody inhibition test. The enzyme antibody inhibition titer obtained by the fluorometric assay system was far weaker than that obtained by the standard colorimetric neuraminidase assay method using fetuin as a substrate. Although solubilization of viral neuraminidase spikes with Triton X-100 increased the enzyme inhibition of antiserum to some extent, there is little possibility of using the fluorometric assay system for the purpose.

A method for determining intracellular content of adenosine triphosphate (ATP) in the cultured cells was investigated with a firefly luciferase reagent, and a micro assay method was established. The method minimized the size of both samples and luciferase reagent employed. The per cell amount of intracellular ATP of HeLa 229 and McCoy cells was estimated to be 7.20×10-11 M arid 8.74×10-11 M, respectively. The method could be applied to a direct measurement of the ATP content in cultured cells grown in each well of a 96-well micro tissue culture plate.