Pathogenicity for mice and swine of Erysipelothrix isolates from the tonsils of healthy cattle

The pathogenicity of 79 Erysipelothrix isolates from bovine tonsils for mice and swine was determined. Five (6.3%) isolates were lethal for mice. These isolates belonged to serovars 1b (one isolate), 2 (2), 19 (1) and 21 (1). The 50% lethal dose values of the isolates ranged from 0.33 to 5x10(2) CFUs in mice. Twenty Erysipelothrix isolates (25.3%) were weakly virulent inducing only emaciation while 12 (15.2%) inducing emaciation and ruffled hair. In swine, clinical signs of varying severity were observed. Four isolates were virulent, capable of inducing localized or generalized urticarial lesions accompanied with a rise in body temperature after intradermal inoculation. One isolate each of serovars 1b, 2 and 19 was highly virulent, capable of inducing generalized urticarial lesions while another Erysipelothrix isolate of serovar 2 induced only a localized urticarial lesion at the site of inoculation. Another isolate of serovar 1b induced itching and irritation without obvious urticarial lesion at the site of inoculation. On the other hand, one isolate of serovar 21 and two other isolates of serovar 2 could not induce experimentally any clinical sign of erysipelas other than rise in body temperature. There was a rise in growth agglutination (GA) titer of serum in all the inoculated swine. These observations suggest that Erysipelothrix isolates from cattle are pathogenic for mouse and swine, and may also be pathogenic for other animals and humans.     

Identification of tumor-initiating cells in a canine hepatocellular carcinoma cell line

Tumor-initiating cells (TICs) or cancer stem cells (CSCs), a small subset of tumor cells, are involved in tumor initiation, progression, recurrence and metastasis. In human hepatocellular carcinoma (HCC), TICs are enriched with cell surface markers and have the ability to self-renew and differentiate tumors at a high frequency. We established a canine HCC cell line, HCC930599, and analyzed it for stem and progenitor cell marker expression using flow cytometry. HCC930599 showed high CD44 and CD29, moderate CD90, and low CD133, CD34, CD24, CD117, and CD13 expression. CD90⁺CD44⁺ and CD90⁻CD44⁺ cells were characterized using the in vitro sphere assay and an in vivo transplant model. CD90⁺CD44⁺ cells acquired enhanced self-renewal capacity, proliferative activity and tumourigenicity compared with CD90⁻CD44⁺ cells, suggesting that TICs exist in the HCC930599 cell line and that CD90 is a marker for enriched TICs. Understanding TIC characteristics may help elucidate hepatic carcinogenesis and HCC therapy development.     

Heat-induced formation of intermolecular disulfide linkages between thaumatin molecules that do not contain cysteine residues

Thaumatin, a sweet protein that contains no cysteine residues and eight intramolecular disulfide bonds, aggregates upon heating at pH 7.0 above 70 degrees C, and its sweetness thereby disappears. The aggregate can be solubilized by heating in the presence of both thiol reducing reagent and SDS. This molecular aggregation depended on the protein concentration during heating and was suppressed by the addition of N-ethylmaleimide or iodoacetamide, indicating a thiol-catalyzed disulfide interchange reaction between heat-denatured molecules. An amino acid analysis of the aggregates suggested that the cysteine and lysine residues were reduced, and the formation of a cysteine residue and a lysinoalanine residue was confirmed. The reduction and formation of these residues stoichiometrically satisfied the beta-elimination of a cystine residue. The disulfide interchange reaction was catalyzed by cysteine; that is, a free sulfhydryl residue was formed via beta-elimination of a disulfide bond. Intermolecular disulfide bonds were probably formed between thaumatin molecules upon heating at pH 7.0, which led to the aggregation of thaumatin molecules.     

Use of epichlorohydrin-treated chitosan resin as an adsorbent to isolate kappa-casein glycomacropeptide from sweet whey

This study was undertaken to develop a method to isolate glycomacropeptide (GMP), a bioactive compound, from sweet whey by using chitosan resins as anion exchangers. Shrimp shells were used to prepare two chitosan (polyglucosamine) resins, one with the primary amine (-NH(2)) (resin A) and the other with the secondary amine (-NH-) (resin B) as the major functional group. These resins were tested as adsorbents for the isolation of GMP from sweet whey, and the results obtained were compared with those obtained with commercial anion exchangers. The most important finding in this experiment was that the GMP binding capacity of resin A was much higher than that of resin B. Resin A may be the anion exchanger to be tested for industrial scale production of GMP. Amino acid analysis of the GMP-depleted whey fraction suggests that this product can replace sweet whey as an ingredient in various food products including infant formulas, bakery products, and beverages.