First results from KamLAND: evidence for reactor antineutrino disappearance.

KamLAND has measured the flux of nu;(e)'s from distant nuclear reactors. We find fewer nu;(e) events than expected from standard assumptions about nu;(e) propagation at the 99.95% C.L. In a 162 ton.yr exposure the ratio of the observed inverse beta-decay events to the expected number without nu;(e) disappearance is 0.611+/-0.085(stat)+/-0.041(syst) for nu;(e) energies >3.4 MeV. In the context of two-flavor neutrino oscillations with CPT invariance, all solutions to the solar neutrino problem except for the "large mixing angle" region are excluded.

Augmentation of protective immune responses against viral infection by oral administration of schizophyllan.

An oral administration of fungal polysaccharide schizophyllan has augmented protective immune responses to Sendai virus infection in mice and the rodshaped DNA virus of Penaeus japonicus (RV-PJ) infection in Kuruma shrimps. When schizophyllan was administered orally at a dose of 50 or 100 mg/kg body weight per day, the survival rates after virus challenge were significantly higher than those of the control groups. High phagocytic activities were observed in the haemocytes of the schizophyllan-fed shrimps.These results suggest that schizophyllan confers effective protection against viral infection by increasing antiviral immune responses, and that it could be used to boost immunity to virus infection in animals or in invertebrates.

Epitope analysis using anti-oligosaccharide (G4) monoclonal antibody.

A murine monoclonal antibody recognizing (1-->6)-beta-D-glucopyranosyl laminaritriose (G4) was prepared by immunizing BALB/c mice with G4-bovine serum albumin conjugate and fusing the splenocytes with mouse myeloma cells. The monoclonal antibody (IgM) provoked by the cloned cells showed low reactivity with schizophyllan, an antitumor polysaccharide, but notable reactivity with some low-molecular-weight schizophyllans. This antibody was useful for determination of the epitope of several polysaccharides. The extent of reactivity of this monoclonal antibody was related only to the molecular weight of schizophyllan.

An improved sandwich ELISA method for the determination of immunoreactive schizophyllan (SPG).

As it is important to determine the optimal serum concentration of schizophyllan (SPG) when it is used as an anti-cancer drug, we devised a solid-phase ELISA. We also developed a sandwich ELISA using murine anti-SPG monoclonal antibody as the first antibody and rabbit anti-SPG serum as the second antibody in order to improve the detection sensitivity. This assay was able to determine SPG concentrations over 1.0 ng/ml and the absorbance at 490 nm was directly proportional to the SPG concentration. SPG in rabbit serum, obtained after intravenous and intramuscular injection (SPG; 10 mg/kg), was determined by this sandwich ELISA. Furthermore, the sensitivity of this ELISA method was compared with that of the Limulus test. The Limulus test is able to detect SPG in physiological saline (pH 6.3) at concentrations greater than 1.0 microgram/ml, but the sensitivity increased when SPG was dissolved in alkaline solution (pH 12.0), enabling SPG to be measured almost down to 1.0 ng/ml. These data suggest that our sandwich ELISA may be used for the measurement of SPG in blood or tissue.

An antitumor, branched (1-->3)-beta-D-glucan from a water extract of fruiting bodies of Cryptoporus volvatus.

A water-soluble, (1-->6)-branched (1-->3)-beta-D-glucan (H-3-B) was isolated from a hot-water extract of the fruiting bodies of the fungus, Cryptoporus volvatus (Basidiomycetes). Enzymatic analysis using exo-(1-->3)-beta-D-glucanase and methylation analysis indicated that this polysaccharide has a main chain composed of beta-(1-->3)-linked D-glucopyranosyl residues, and single, beta-(1-->6)-linked D-glucopyranosyl residues attached as side chains to, on average, every fourth sugar residue of the main chain. This structure was confirmed by 13C NMR spectra of the glucan in Me2SO-d6. The weight-average molecular weight (Mw) of H-3-B was determined to be 44.0 x 10(4) by gel permeation chromatography equipped with a low-angle laser-light-scattering photometer. The electron microscopic observations showed that H-3-B and its sonicated sample (S-H-3-B, Mw = 13.7 x 10(4)) can be described as linear worm-like chains. The mass per unit length for native and sonicated H-3-B was determined to be 1750 and 1780 g mol-1 nm-1, respectively, from the contour lengths obtained by electron microscopy and the molecular weights. These values are in good agreement with that expected for the triple stranded structure. A sample denatured in 0.1 M NaOH and subsequently renatured by neutralization showed a mixture of linear and cyclic structures, and larger aggregates with less well-defined morphology. The H-3-B and S-H-3-B had antitumor activity against the Sarcoma 180 tumor.

The inhibitory effect of sulfated schizophyllans and related oligosaccharides on coagulation and fibrinolysis.

Sulfated schizophyllans and sulfated oligosaccharides were prepared and their inhibitory effects on coagulation and fibrinolysis were examined. Some sulfated schizophyllans, which had a sulfur content of more than 10%, prolonged prothrombin time (PT) and thrombin time (TT). Interestingly, some sulfated schizophyllans and related oligosaccharides were able to inhibit plasmin toward fibrinolysis and amidolysis.

Monoclonal antibody to proteoglycan derived from Grifola frondosa (Maitake).

A murine monoclonal antibody (MAb) was prepared by immunizing BALB/c mice with a proteoglycan fraction derived from Grifola frondosa (Maitake mushroom), followed by the hybridization of spleen cells with mouse myeloma cells. The MAb (subclass; Ig G2b), designated MPG2, reacted with schizophyllan (SPG), curdlan, scleroglucan, laminarin and lentinan, but not with dextran, pullulan, mannan and xylan. Immunohistochemistry (ABC-GO method) showed that MAb MPG2 reacted with lysosomal proteoglycan and (1-->6)-beta-branched laminaritriose taken up by rabbit peritoneal macrophages. These results suggest that this MAb may recognize mainly (1-->3)-beta-D-glucan, and may be useful for determining the immunological properties of Grifola frondosa-derived proteoglycan.

Detection of immunoreactive schizophyllan by solid-phase enzyme-linked immunosorbent assay.

In order to detect immunoreactive schizophyllan (SPG), one of the beta-1,3-glucans in solution, a murine anti-schizophyllan monoclonal antibody, SPG1-HS, was used as a primary antibody for solid-phase enzyme-linked immunosorbent assay (ELISA). Polysaccharide-bovine serum albumin (BSA), and polysaccharide-bovine hemoglobin (Hb) conjugate were prepared as an antigen. The absorbance at 490 nm in ELISA was directly proportional to the SPG concentration in SPG-BSA or SPG-Hb conjugates, allowing the accurate measurement of SPG at concentrations of more than 1.0 microgram/ml. Also, this ELISA detected SPG in SPG-BSA and SPG-Hb conjugates in human serum in a similar manner. These results suggest that this ELISA is applicable for the determination of SPG concentration in SPG-BSA and SPG-Hb conjugates in human tissue and blood.

Preparation of mouse-human chimeric antibody to an embryonic carbohydrate antigen, Lewis Y.

Stage-specific embryonic antigen-1 (SSEA-1) is a well-known carbohydrate antigen that is specifically expressed on the surface of cancer cells as well as embryonic cells. In this study, starting with a previously established hybridoma producing a monoclonal antibody (named H18A) to Lewis Y antigen, which is closely related to SSEA-1, we cloned genomic DNA encoding active variable regions both of heavy and light chains of the antibody. Sequence analysis showed that VH and V kappa genes of H18A were in the VH 7183 family and V kappa C1 family, respectively. A transfected cell line named HC-H18A-7 expressing a recombinant chimeric H18A composed of mouse-derived antigen-binding variable regions and human-derived constant regions was established. The chimeric H18A was purified to homogeneity and shown to bind purified Lewis Y antigen with the same dose-response curve as the original H18A. The chimeric H18A looks more promising for clinical application than the original mouse-derived H18A because its antigenicity is expected to be reduced.

Preparation of polyclonal antibodies to an anti-tumor (1----3)-beta-D-glucan, schizophyllan.

Antibodies against Schizophyllan (SPG) or SPG conjugated with bovine serum albumin (BSA) were raised in rabbits by multiple subcutaneous immunization. The titer of SPG-reactive antibodies in the antiserum to SPG-BSA conjugate was significantly higher than in the antiserum to SPG as assessed by enzyme-linked immunosorbent assay (ELISA). The SPG-reactive antibodies purified by affinity chromatography using a Protein A-Sepharose CL-4B column interacted with the SPG-BSA conjugate spotted on a nitrocellulose membrane filter in a dose-dependent manner, but the anti-SPG antibodies did not interact with BSA. Immunocytochemical staining has also shown that the anti-SPG antibodies reacted with SPG taken up by mouse peritoneal macrophages.

Immunopharmacological study of sulfated schizophyllan (SPG). I.--Its action as a mitogen and anti-HIV agent.

Sulfated schizophyllans of differing sulfur content were prepared and their mitogenic activities and effects on human immunodeficiency virus (HIV) were investigated in vitro. The sulfated schizophyllans were found to possess mitogenic activity. Anti-viral activity was evaluated in terms of cytopathic effect, and by the fluorescence antibody technique, reverse transcriptase assay and cell proliferation assay. Schizophyllans with a higher sulfur content showed potent anti-HIV effects on Molt-4 clone No. 8 or MT-4 cells infected with HIV as well as on lymphocytes from AIDS patients. It is suggested that the sulfur content of schizophyllans is the most important factor for inhibiting growth of HIV, rather than molecular weight or kinds of sugar component. Thus, sulfated schizophyllans appear applicable as anti-HIV drugs.

Further characterization of sulfated homopolysaccharides as anti-HIV agents.

Fucoidan and dextran sulfate showed anti-HIV activities against mononuclear cells from AIDS patients, and they abrogated HIV reverse transcriptase (RT) activity by interacting with the HIV envelope in the membranes of target cells. Furthermore, they showed a synergistic effect with azidothymidine (AZT).