Reactivity of Limulus amoebocyte lysate towards (1----3)-beta-D-glucans

The structure activity relationship for beta-D-glucans for the gelation of the amoebocyte lysates of the horseshoe crab (Limulus) has been investigated. beta-D-Glucans that had no (1----3) linkages induced little or no gelation. The (1----3)-beta-D-glucans curdlan (unbranched), grifolan (approximately 33% branched), schizophyllan (approximately 33% branched), lentinan (approximately 40% branched). SSG (approximately 50% branched), and OL-2 (approximately 66% branched) induced significant gelation. The optimum concentration for gelation was correlated with the content of branching. Single chain (rather than a triple helix) conformation and higher molecular weight were associated with higher reactivity.     

Establishment of Autoantibody-Producing Cell Lines from Peripheral Blood Lymphocytes of Patients with Systemic Lupus Erythematosus

Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.     

Induction of supermelanin synthesis and morphological changes in interspecific reconstituted cells and its reversal by tumor promoter

Chloramphenicol-resistant (CAPr) reconstituted cells and cybrids were isolated by fusion of karyoplasts (or intact cells) of mouse amelanotic melanoma B16 cells with cytoplasts of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) -deficient, CAPr rat myoblastic cells, L6TG.CAPr, and double selection in HAT medium containing CAP. Reconstituted cells or cybrids exhibited unique cellular arrangement, and about one third of the isolated clones expressed high tyrosinase activity and marked melanin synthesis, although the parental mouse cells expressed low tyrosinase activity and the parental rat cells did not express tyrosinase activity. These phenotypic changes have been stable for more than a year. The phenotypic reversions of these clonal cells were induced by treatment with a tumor promoter. There were changes in the morphology of the treated cells to that of the mouse B16 cells and extinction of tyrosinase activity and melanin synthesis in pigmented clonal cells. These phenotypic changes and reversions induced by a promoter were repeatedly reversible.     

Structural examination of antitumour, water-soluble glucans from Grifora umbellata by use of four types of glucanase.

Antitumour glucans (GU) from the fungus Grifora umbellata have been subjected to periodate oxidation, Smith degradation, methylation analysis, and treatment with endo-(1 leads to 6)-beta-D-, endo-(1 leads to 3)-beta-D-, and exo-(1 leads to 3)-beta-D-glucanases, and alpha-amylase; the following structural features were revealed. GU-2 contains a backbone involving (1 leads to 6)-beta- and () leads to 3)-beta linkages, and two kinds of branches involving (1 leads to 6)-beta and (1 leads to 4)-alpha linkages. GU-3 has a (1 leads to 3)-beta-linked backbone and branches involving (1 leads to 6)-beta linkages or (1 leads to 4)-alpha and (1 leads to 6)-beta linkages. GU-4 also contains a (1 leads to 3) beta-D-glucan backbone and a small number of (1 leads to 6)-beta-linked branches. Probable structural units of these glucans are proposed.     

Cytotoxicity of cysteine in culture media.

When added to Eagle's Minimum Essential Medium supplemented with 10% bovine serum (MEM-10BS), 1mM cysteine was highly toxic to cultured cells. This toxicity was eliminated by (a) preincubation of the medium at 37 degrees C for 24 hr before use, or (b) presence of 5mM pyruvate. Similar results were obtained with freshly prepared CMRL 1066 supplemented with 10% bovine serum (CMRL-10BS), which contains 1.5 mM cysteine as an original ingredient. Medium L 15 supplemented with 10% bovine serum (L-10BS), which contains both 1 mM cysteine and 5 mM pyruvate, supported cell growth. On incubation of MEM-10BS supplemented with 1 mM cysteine (MEM-10BS-1CySH) or CMRL-10BS without cells for one day, the cysteine concentrations decreased to about one-tenth or less of the original concentrations. The cysteine concentration in L-10BS did not decrease so much on similar incubation. Pyruvate reduced the rate of disappearance of the cysteine in MEM-10BS-1CySH or CMRL-10BS as assayed with p-chloromercuribenzoate, although less than that in L-10BS. This effect of pyruvate was concentration dependent. These paradoxical effects of pyruvate on cysteine, i.e. the reduction of its cytotoxicity and the stabilization as an SH compound, are probably due to the formation of a dissociable complex between these two compounds, which is not cytotoxic and resistant to oxidation.     

An endo-(1→6-β-D-glucanase from Mucor hiemalis

An endo-(1→6)-β-D-glucanase (EC 3.2.1), isolated from the culture filtrate of Mucor hiemalis, was purified by ammonium sulphate fractionation and gel filtration. The homogeneity of the enzyme was confirmed by disc electrophoresis. The enzyme had a wide range of temperature and pH stability, high substrate specificity, and an action pattern of the endo-type.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.