Preparation of tartary buckwheat protein product and its improving effect on cholesterol metabolism in rats and mice fed cholesterol-enriched diet.

Tartary buckwheat protein product (TBP) was prepared from buckwheat flour by alkali extraction and isoelectric precipitation. The protein content of TBP was 45.8%, and its amino acid composition of TBP was similar to that of common buckwheat protein product (BWP). SDS-PAGE analysis showed that the protein profile of TBP was partially different from that of BWP. TBP contained more quercetin (1710 mg/100 g) than BWP (5.4 mg/100 g), while there was a small difference in the contents of rutin between them. In experiment 1, the consumption of BWP and TBP at 20% net protein level for 13 d caused 32% and 25% reductions in serum cholesterol of rats fed cholesterol, respectively, when compared to the consumption of casein (P < 0.05). The reduction of serum cholesterol by BWP and TBP was associated with enhanced excretion of fecal neutral sterols. In experiment 2, the consumption of BWP and TBP for 27 d caused 62% and 43% reductions in the lithogenic index in mice fed cholesterol, respectively (P < 0.05). The reduction in lithogenic index was associated with enhanced excretion of fecal bile acids. Taken together, these results suggest a potential source of TBP as a functional food ingredient as well as BWP.

Serological diversity demonstrable by a set of monoclonal antibodies to eight serotypes of the mutans streptococci.

A set of monoclonal antibodies were prepared by the conventional cell fusion of myeloma cells (SP2/0-Ag14) with spleen cells from BALB/c mice immunised with whole cells of a strain of mutans streptococci. Their specificities were examined against 35 reference strains of mutans streptococci, 34 reference strains of other oral streptococci and 8 reference strains of other microorganisms often inhabiting the oral cavity. Specificity was examined by enzyme immunoassay using whole cells. A total of 52 strains, consisting of 19 strains isolated in Japan, 19 strains isolated in Italy and 14 strains isolated in England, were characterised by conventional physiological and biochemical tests and then serotyped by the use of 8 monoclonal antibodies with different specificities. They were also confirmed by guanine-plus-cytosine contents of their nucleic acid and DNA-DNA hybridisation test. The results indicated that all monoclonal antibodies are useful for identification of 8 serotypes of the mutans streptococci responsible for dental caries. They also suggest the existence of more serological varieties among mutans species.

Stronger suppression of plasma cholesterol and enhancement of the fecal excretion of steroids by a buckwheat protein product than by a soy protein isolate in rats fed on a cholesterol-free diet.

We investigated the effects of a buckwheat protein product (BWP), soy protein isolate (SPI) and casein on the plasma cholesterol level and fecal steroid excretion in rats fed on a cholesterol-free diet. The consumption of BWP suppressed plasma cholesterol by enhancing the fecal excretion of both neutral and acidic steroids. These effects of BWP were stronger than those of SPI.

A buckwheat protein product suppresses 1,2-dimethylhydrazine-induced colon carcinogenesis in rats by reducing cell proliferation.

This study was conducted to examine the effect of consumption of buckwheat protein product (BWP) on 1,2-dimethylhydrazine (DMH)-induced colon tumor in rats. Male growing Sprague-Dawley rats were fed diets containing either casein or BWP (net protein level, 200 g/kg; n = 20/group) for 124 d. The rats were gavaged weekly with DMH (20 mg/kg body) for the first 8 wk. Food intake and growth were unaffected by dietary manipulation. Dietary BWP caused a 47% reduction in the incidence of colonic adenocarcinoma (P < 0.05), but did not affect the incidence of colonic adenomas. BWP intake tended to reduce the number of colon adenocarcinomas (P = 0.16). Consumption of BWP significantly reduced cell proliferation and expression of c-myc and c-fos proteins in colonic epithelium. The results suggest that dietary BWP has a protective effect against DMH-induced colon carcinogenesis in rats by reducing cell proliferation.

Combined effect of dietary calcium and iron on colonic aberrant crypt foci, cell proliferation and apoptosis, and fecal bile acids in 1,2-dimethylhydrazine-treated rats.

To investigate the combined effect of Ca and Fe on colon carcinogenesis, cell proliferation, apoptosis and fecal bile acids, male Wistar rats were fed the diet containing 5 g Ca/kg (normal Ca) or 15 g Ca/kg (excessive Ca) with 45 mg Fe/kg (normal Fe) or 500 mg Fe/kg (excessive Fe) for 32 days, and given an injection of 1,2-dimethylhydrazine on day 4. Supplemental Ca reduced colonic aberrant crypt foci (ACF), especially in excess Fe group. Excessive Fe elevated the ACF, especially in the normal Ca diet. When the Ca intake was high, excessive Fe caused no influence on the ACF. Alteration of colonic ACF was associated with those of liver and serum Fe concentration. Also, colonic cell proliferation and concentration of deoxycholic acid (DCA) in fecal water-soluble fraction were reduced by supplementation of dietary Ca, but unaffected by that of dietary Fe. Supplementation of Ca and/or Fe elevated colonic cell apoptosis. The results suggest that dietary Ca markedly suppresses colon ACF in the Fe-overloaded rats through altering Fe status, and that supplemental Ca lowers colonic cell proliferation and fecal DCA in the water-soluble fraction and elevates colonic cell apoptosis irrespective of Fe status.

A buckwheat protein product suppresses gallstone formation and plasma cholesterol more strongly than soy protein isolate in hamsters.

This study was conducted to investigate the effects of a buckwheat protein product (BWP) on plasma cholesterol, gallbladder bile composition and fecal steroid excretion in hamsters fed diets with 5 g/kg cholesterol. Diets also contained 200 g/kg of casein, soy protein isolate (SPI) or BWP as protein sources. After 2 wk, plasma and liver concentrations of cholesterol in the hamsters fed BWP were significantly lower than those in the hamsters fed casein and SPI. The molar proportion of cholesterol in gallbladder bile was significantly lower in the BWP group than in the other groups, whereas that of bile acids was slightly higher in the BWP group (P

Characterization of serotype II polysaccharide antigen of group E streptococci using a monoclonal antibody.

A Monoclonal antibody (MAb II-T) specific for serotypes II and V Group E streptococci (GES) was prepared by fusing myeloma cells with spleen cells of mice immunized with whole cells of a serotype II strain. MAb II-T reacted in an enzyme immunoassay (EIA) with whole cells of both serotypes and reacted in gel diffusion test with autoclaved-saline extraction of serotypes II and V. The extract was purified by DEAE-Sephadex A-25, followed by treatment with proteinase K, and further by chromatography with a Sephadex G-200 column. The purified polysaccharide (PS) antigen contained 98.6% carbohydrate and 1.4% protein, but no detectable phosphorus. In hapten inhibition tests using various sugars, D-mannosamine markedly inhibited the precipitin reaction. These results indicated that the antigenic determinant might have a structure similar to D-mannosamine.

Chromatographic study of spirosin by use of monoclonal antibodies to spirosin of Yersinia enterocolitica.

Two monoclonal antibodies (MAbs) reacting with spirosins from Enterobacteriaceae were obtained in a course of screening MAbs to spirosin from Yersinia enterocolitica SYT-11-72 (YE72). The antibodies were designated MAbs-S44 and S50. They were IgG2b and IgG2a, respectively, both with kappa light chains. On Western blotting after limited proteolysis of YE72 spirosin with Staphylococcus aureus V8 protease, they reacted markedly with peptide fragments of 27 and 35 kDa, suggesting the presence of an antigenic determinant on the fragments. When supernatant cell lysate from Escherichia coli K12 was chromatographed on DEAE-cellulose and Sepharose CL-6B columns successively, a 96-kDa protein with alcohol dehydrogenase (ADH) activity was always associated with reactivity to MAb-S50. These findings combined with N-terminal amino acid sequences clearly indicate the identity of spirosin to ADH in E. coli.

Immunochemical study of polysaccharide antigen in Streptococcus sobrinus and Streptococcus downei with a cross-reactive monoclonal antibody.

A monoclonal antibody (mAb h-448) was prepared after cell fusion of mouse myeloma cells (SP2/0-Ag-14) to the spleen cells of mice immunised with serotype h strain (MF25) of Streptococcus downei. The antibody (IgM class) reacted in enzyme immunoassay only with whole cells as well as purified polysaccharide (PS) antigen of Streptococcus sobrinus (types d and g) and Streptococcus downei (serotype h), but not with cells or purified PS antigen from any other serotypes of the mutans group of streptococci. mAb h-448 also quantitatively precipitated in solution with the purified antigens. Competitive hapten inhibition tests demonstrated that beta-methylgalactopyranoside inhibited the reaction most strongly. Although rhamnose also showed a substantial inhibitory effect, the results of this study indicate that the antigenic determinant of the PS antigen has a structure similar to the beta-methylgalactopyranoside molecule.

Monoclonal antibodies to spirosin of Yersinia enterocolitica and analysis of the localization of spirosome by use of them.

Two hybridomas producing monoclonal antibodies (MAbs) were prepared by fusing myeloma cells (Sp2/0-Ag14) with mouse spleen cells immunized with purified spirosin from Yersinia enterocolitica SYT-11-72 (YE72). The antibodies produced by them were designated MAbs-S5 and S27. They were IgG2a and IgG1, respectively, both with kappa light chains. MAbs-S5 and S27 reacted specifically with spirosin from YE72. On Western blotting after limited proteolysis with Staphylococcus aureus V8 protease, YE72 spirosin revealed peptide fragments of 35 and 37 kDa reacting markedly with MAb-S5, which suggested the presence of an antigenic determinant on these fragments. By cellular fractionation of YE72 and subsequent EIA and Western blot analysis, spirosome was shown to be present in the cytoplasm of YE72.