A simple method for efficiently establishing 8-azaguanine-resistant mutant human leukemia and myeloma cell lines.

A simple and convenient method for efficiently establishing 8-azaguanine-resistant mutant leukemia and myeloma cell lines (for example, the T cell lines Jurkat and CCRF-CEM, human myeloid/macrophage-like cell lines HL60 and U937, Burkitt lymphoma line Raji and the human myeloma line RPMI 8226), is described. The method relies on culturing the cell lines in RPMI 1640 medium containing 8-azaguanine and supplemented with 15% heat-inactivated fetal calf serum and large amounts of amino acids and vitamins, and removes the necessity for pretreatment with mutagenic reagents such as ethyl methylsulfonate or X-irradiation. The possibility of obtaining mutant cell lines using the method described here is about 15 times greater than using media without high levels of amino acids and vitamins. Hybridomas produced between mitogen-activated human peripheral blood lymphocytes and an 8-azaguanine-resistant Jurkat mutant cell line (established by this method) were shown to produce soluble T cell-derived macrophage activating factor (MAF)-like material.

Single radial complement fixation test using complement film. Assay of the antibody response to strain and type specific antigens of influenza virus.

A stabilized modification of the single radial complement fixation test in gel (SRCF) was developed for detecting influenza antibodies. The principle of the test is the use of a single-step procedure with the following reagents: (1) Agarose plate containing influenza antigen and antibody coated erythrocytes (EA). (2) Thin plastic film coated with dried complement. By filling the wells cut in the agar with the heat inactivated serum samples and covering the agar surface with the complement film, a zone of unlysed cells surrounded by a haemolytic area appears after overnight incubation for 16-18 h at 4 degrees C and 1-2 h at 37 degrees C. The squares of the zone diameter were measured for estimating the antibody quantity by using CF(S) and virion antigen of influenza virus, and the type-specific antibody was demonstrated by using CF(S) antigen, while the strain-specific antibody was demonstrated by using virion antigen. An excellent correlation was demonstrated for antibody titres between conventional CF and SRCF with CF(S) antigen, on the one hand, and, between conventional HI and SRCF with virion antigen, on the other.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of polyphenol oxidase from Trametes sp. MS39401.

Two polyphenol oxidases (EC 1.14.18.1), P-1 and P-2, were purified as electrophoretically homogeneous proteins from the culture filtrate of Trametes sp. MS39401 by acetone precipitation and column chromatographies on DEAE-Sephadex A-50, Sephadex G-150 and hydroxylapatite. P-1 was purified 34-fold with a yield of 4.2%, while P-2 was purified 37-fold with a yield of 20.7%. The molecular masses of P-1 and P-2 were estimated to be 61 kDa and 90 kDa, respectively, by gel filtration. The isoelectric points of P-1 and P-2 were 3.4 and 2.7, respectively. The optimum pH range of both enzymes was 4.5-5.0 at 45 degrees C. The optimum temperature of both enzymes was 55 degrees C at pH 5.0. P-1 was stable at pH 5.0-7.5 and temperatures up to 60 degrees C. P-2 was stable at pH 3.0-7.5 and temperatures up to 50 degrees C. The thermostability of P-1 was comparable to that of the PM1 laccase of basidiomycetes, which was reported to be the most stable among basidiomycete laccases. Both enzymes were active toward various phenolic compounds and aminophenols. However, they lacked activity toward l-tyrosine. The K(m) values for (+)-catechin were 0.19 mM for P-1 and 0.67 mM for P-2. Both enzymes were appreciably inactivated by Hg(2+) and Sn(2+). Significant activation of neither enzyme was observed in the presence of metal ions and reagents. Both enzymes were significantly inhibited by copper-chelating agents, reducing agents and N-bromosuccinimide. Carbon monoxide caused appreciable inactivation of neither enzyme, so it is suggested that P-1 and P-2 belong to the group of laccases.