Examination of the active center of a (1----3)-beta-D-glucanase preparation, zymolyase.
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Biomedical subjects
Publications and source records attributed to N Ohno.
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The antitumor beta-D-glucan [SSG, (1----3)-linked with a D-glucosyl group attached to position 6 of alternate units] produced by Sclerotinia sclerotiorum IFO 9395 afforded a gel in neutral aqueous solution which gave no 13C-n.m.r. signals. Signals appeared on the addition to the gel of sodium hydroxide to 0.15M and the line-width was narrowest near 0.25M base (gel-to-sol transition). SSG bound to Congo Red and shifted the lambda max in a manner similar to that of curdlan. Neutralisation of the sol regenerated the gel. SSG and regenerated SSG showed different c.p.-m.a.s. 13C-n.m.r. spectra, especially the signals for C-3 which appeared at 86 and 89 p.p.m., respectively. The c.p.-m.a.s. spectrum of regenerated SSG was similar to that of curdlan, suggesting a helical structure. Thus, SSG and regenerated SSG appear to have different conformations.
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The antitumor activity of a highly branched (1----3)-beta-D-glucan, SSG, purified from the liquid culture filtrate of Sclerotinia sclerotiorum IFO 9395 and its several derivatives were treated in ICR mice bearing Sarcoma 180 cells. SSG was effective by both systemic (intraperitoneal and intravenous) and local (intratumoral) administrations on the solid form of Sarcoma 180 in ICR mice and the mice acquired resistance to subsequent inoculation of Sarcoma 180. However, SSG was not effective on the ascites form Sarcoma 180. The pretreatment of ICR mice with carrageenan suppressed the antitumor activity, suggesting the involvement of macrophages on the antitumor activity. Derivatives prepared from SSG by periodate oxidation/borohydride reduction showed antitumor activity, but those obtained after acetylation, carboxymethylation and hydroxyethylation were less active. From these results, it is suggested that SSG is a useful antitumor glucan which modifies biological responses and can be used as a source for some antitumor derivatives.
The antitumor mechanism of grifolan NMF-5N, a beta-1,3-glucan obtained from mycelia of Grifola frondosa, was examined. Grifolan NMF-5N did not show direct cytocidal effect on cultured tumor cells. However, intraperitoneal injection of grifolan NMF-5N increased the number of peritoneal exudate cells and peritoneal adherent cells which showed cytostatic activity towards syngeneic tumor cells. In an in vivo assay, the administration of carrageenan, an inhibitor of macrophage function, reduced the antitumor activity of grifolan NMF-5N. The delayed-type hypersensitivity reaction was augmented in the grifolan NMF-5N-administered mice. The administration of NMF-5N augmented the induction of cytotoxic T cells but the antitumor activity of grifolan NMF-5N was reduced in athymic nu/nu mice. In addition, the treatment with anti-Thy 1,2 antibody and complement C' of spleen cells taken from mice which showed regression of tumor due to grifolan NMF-5N, reduced the neutralizing effect in Winn assay. These results suggested that grifolan NMF-5N shows antitumor activity via host-mediated mechanisms and both macrophages and T cells play important roles in the mechanisms.
Antitumor activity of grifolan NMF-5N, a beta-1,3-glucan obtained from mycelia of Grifola frondosa, was examined. Grifolan NMF-5N showed antitumor activities in allogeneic and syngeneic murine tumor systems. In the allogeneic tumor system, a potent antitumor activity over 95% was observed against the solid form of sarcoma 180 when grifolan NMF-5N was injected intraperitoneally (i.p.) at 25-200 micrograms/mouse daily for 10 successive days. In the syngeneic tumor systems, significant antitumor activities were observed against Meth A fibrosarcoma and MM 46 carcinoma by injection at 100 micrograms/mouse daily for 5 successive days, especially i.p. injection at day 7-11, when the tumor cells were inoculated subcutaneously (s.c.) on day 0. Moreover, when grifolan NMF-5N was injected i.p. every other week, significant antitumor activity was also observed. In addition, a single treatment with grifolan NMF-5N at 500 micrograms/mouse showed antitumor activities. Grifolan NMF-5N exhibited antitumor activities against these two syngeneic tumors by intraveneous (i.v.) injection. However, a marked inhibitory activity was observed by intratumorous (i.t.) injection against Meth A fibrosarcoma but not against MM46 carcinoma. These results suggest that antitumor activities of grifolan NMF-5N in murine syngeneic tumor systems depend on not only dosage but also injection routes and timing.
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Sera from patients with poststreptococcal glomerulonephritis (PSGN) known to have antibodies to proteoglycans were studied for the presence of antibodies against other basement membrane (BM) components. BM collagen (type IV) was isolated in the native state by extracting bovine anterior lens capsule (ALC) with 0.5 M acetic acid. The 7-S (collagenous) domain and the NC-1 (noncollagenous) domain of type IV collagen were obtained after bacterial collagenase digestion of ALC followed by gel filtration. Laminin was isolated from the mouse EHS tumor and fibronectin from human plasma. Immunologic studies, using an ELISA and electroimmunoblot, revealed the presence of antibodies that reacted with intact, native type IV collagen and the 7-S collagenous domain of this molecule. Reaction with the NC-1 (noncollagenous) domain was minimal, and not higher than that obtained with control sera. Laminin reaction strongly with the patients' sera, but fibronectin did not. Unlike sera from patients with Goodpasture syndrome, which contain antibodies primarily against the NC-1 (noncollagenous) domain of type IV collagen, sera from patients with acute PSGN contain antibodies against all the major macromolecular components of BM. This difference in immunologic reactivity may account for the observed differences in the pathologic picture at the glomerular level.
The correlation between murine lymphocytes blastoid transformation and alkaline phosphatase (AL-P) activity was examined. When murine spleen cells were stimulated with lipopolysaccharide, a B cell mitogen, for 48 h, the AL-P activity was induced in a dose dependent manner. Similarly, stimulation of the spleen cells with Concanavalin A (Con A), a T cell mitogen, induced the AL-P activity. However, when the thymocytes were treated with Con A, only the [3H]dThd uptake was increased. Other B cell mitogens, such as lipid A and fungal B cell mitogens, and T cell mitogen, Pusum sativum agglutinin (PSA) also induced the AL-P activity of spleen cells. Kinetics of [3H]dThd uptake and induction of AL-P was similar. Correlation coefficients between [3H]dThd uptake and AL-P activity were r = 0.98789, p less than 0.01 for LPS, r = 0.99530, p less than 0.01 for lipid A and r = 0.62595, p less than 0.001 for the fungal B cell mitogens. The AL-P activity of the spleen cell was also induced by treatment with Con A sup, which was a supernatant fluid prepared by spleen cell cultured with Con A for 48 h, containing various lymphokines. These findings suggest that the AL-P activity was induced in the case of B lymphocyte blast transformation when stimulated directly with B cell mitogen or indirectly via lymphokines with T cell mitogen. Measurement of the AL-P activity would be a useful method to assay murine blastoid lymphocytes.
The antitumor activity of a branched beta-1,3-glucan "grifolan LE" purified from liquid cultures of Grifola frondosa (Ohno et al. Chem. Pharm. Bull., 34, 1709-1715 (1986] was examined on an allogeneic murine tumor system. By intraperitoneal (i.p.) administration (100-200 micrograms/mouse/d X 5) at days 1 to 9 from the tumor transplantation, grifolan LE showed marked inhibitory activity on the growth of solid form sarcoma 180 in ICR mice. Significant activity was also observed in intravenous (i.v.) or intratumoral (i.t.) administrations. However, the oral (p.o.) administration of grifolan LE was not effective. I.p. administration of grifolan LE at a dose of 100 micrograms/mouse/d X 5 before the tumor transplantation showed significant inhibition of tumor growth. I.p. administration of grifolan LE at day +11 to +19 was also effective. Grifolan LE was not effective on the ascites form of sarcoma 180. The pretreatment of sarcoma 180 cell with grifolan LE in vitro did not affect tumor growth. The mice cured from the solid form of sarcoma 180 by administration of grifolan LE had the ability to reject the same tumor cell. From these results, it is suggested that the antitumor activity of grifolan LE occurred by modification of biological responses.