Iron derivatives from casein hydrolysates as a potential source in the treatment of iron deficiency

The properties of an Fe(3+)-peptide complex containing 5.6% Fe, obtained by the reaction of ferric chloride with an enzymatic hydrolysate of casein, are described. The major site of iron binding corresponds primarily to the carboxylate groups and to a lesser extent to the peptide bonds. The Fe(3+)-peptide complex is insoluble at acid pH and completely soluble at neutral to alkaline pH. When soluble, the Fe(3+) is tightly bound to the complex peptide mixture but can be displaced and complexed by a low molecular weight ligand such as cysteine. Its efficacy in relation to iron sulfate was compared in rats. Both iron sources were administrated in Milli-Q water by gastric gavage to male Wistar rats (180-200 g) after an 18 h fast with water ad libitum. Fe(3+) from the Fe(3+)-peptide complex was transferred to the blood in a dose-dependent manner (1-8 mg of Fe/kg), and the serum iron levels were significantly higher (p < 0.001) than in a similar group of rats treated with iron sulfate. In the comparative kinetics experiments, the rats received 4 mg of Fe/kg. Both iron sources presented maximum absorption, as indicated by the elevation of serum iron levels, 30 min after administration, and the AUC(0)(-->2h) of the Fe(3+)-peptide complex was significantly higher (p < 0.05) than that observed with iron sulfate. The simultaneous administration of free peptides (0-192 mg) with the Fe(3+)-peptide complex or iron sulfate did not modify the extent of absorption of iron from both sources, suggesting that the absorption is due to the complex formed and probably not to exchange reactions in the gastrointestinal tract. In the hemoglobin repletion experiments carried out on newly weaned rats with anemia induced by a low-iron diet, supplementation of the diet with the the Fe(3+)-peptide complex was as efficient as supplementation with iron sulfate in the conversion from diet to hemoglobin iron. These results, taken together, suggest that the Fe(3+)-peptide complex is a potential compound for use as an iron source in biological situations.     

Two-week Toxicity of Multi-walled Carbon Nanotubes by Whole-body Inhalation Exposure in Rats

To evaluate pulmonary toxicity of multi-walled carbon nanotubes (MWCNTs), F344 rats of both sexes were exposed by inhalation to 0.2, 1 or 5 mg/m³ MWCNT aerosol for 6 h/day, 5 days/week for 2 weeks using a whole-body exposure system. At the end of the 2-week exposure period, one-half of the rats were necropsied, and at the end of an additional 4-week postexposure period, the remaining rats were necropsied. MWCNTs were deposited in the lungs of all MWCNT-exposed groups and mostly remained in the lungs throughout the 4-week postexposure period. Granulomatous changes in the lung were found in the rats exposed to 5 mg/m³ MWCNTs, and these changes were slightly aggravated at the end of the 4-week postexposure period. In the bronchoalveolar lavage fluid (BALF), the numbers of neutrophils, percentages of bi- and multinucleated alveolar macrophages, levels of ALP activity and concentrations of total protein and albumin were elevated in the rats exposed to 1 and 5 mg/m³ MWCNTs. At the end of the 4-week postexposure period, the values of the BALF parameters tended to remain elevated. In addition, goblet cell hyperplasias in the nasal cavity and nasopharynx were observed in the rats exposed to 1 and 5 mg/m³ MWCNTs, but these lesions had largely regressed by the end of the postexposure period. Based on the histopathological and inflammatory changes, the no-observed-adverse-effect level (NOAEL) for inhalation of MWCNTs for 2 weeks was 0.2 mg/m³.     

Effects of a combination of hinokitiol (β‐thujaplicin) and an organic acid mixture on ruminal fermentation in heifers fed a high‐grain diet

This study evaluated the effects of hinokitiol (a natural antibacterial compound extracted from Thujopsis dolabrata var. hondai) and an organic acid mixture (citrate content 50%) on ruminal fermentation. Antibacterial properties were examined by measuring minimal inhibitory concentration. Hinokitiol at 1.56 µg/mL or an organic acid mixture at 1600 µg/mL inhibited Streptococcus bovis growth. The combination of 0.78 µg/mL hinokitiol and 200 µg/mL of an organic acid mixture also inhibited S. bovis growth. Both hinokitiol and the hinokitiol and an organic acid mixture combination showed strong antibacterial properties on Gram‐positive bacteria such as S. bovis, but relatively weak antibacterial activities on Gram‐negative bacteria such as Megasphaera elsdenii. Three ruminally cannulated heifers were fed a bloat‐producing diet containing barley, pelleted alfalfa meal, soybean meal and salt without long‐cut roughage to investigate the ruminal characteristics in vivo. Feeding to heifers a bloat‐producing diet containing 7.8 mg/kg hinokitiol and 0.2% of an organic acid mixture significantly decreased the increase in stable ingesta volume. Hinokitiol or an organic acid mixture did not affect ruminal volatile fatty acids, protozoa and bacteria. These results suggest that a combination of hinokitiol and an organic acid mixture might reduce frothy bloat in cattle fed high‐grain diets.     

Reactivity of anti-Nipah virus monoclonal antibodies to formalin-fixed, paraffin-embedded lung tissues from experimental Nipah and Hendra virus infections

The immunohistochemical reactivity of seven clones of mouse monoclonal antibodies raised to Nipah virus antigens were investigated using formalin-fixed, paraffin embedded porcine and equine lung tissues from experimental Nipah and Hendra virus infection, respectively. Either microwave irradiation or enzymatic digestion effectively unmasked the viral antigens in formalin-fixed, paraffin-embedded tissue sections. Four clones showed positive reaction to both Nipah virus-infected porcine lung tissue and Hendra virus-infected equine lung tissue. Two clones (11F6 and 13A5) reacted with Nipah virus-infected porcine lung tissue, but not with Hendra virus-infected equine lung tissue. These Nipah virus-specific monoclonal antibodies may therefore be useful for immunohistological diagnosis of Nipah virus infection and for further research on Nipah virus pathogenesis.     

Experimental assessment of the pathogenicity of H5N1 influenza A viruses isolated in Japan

We examined the pathogenicity for chickens of two H5N1 avian influenza viruses isolated in Japan, A/chicken/ Yamaguchi/7/2004 (Ck/Yamaguchi/7/04) isolated from outbreaks in commercial layer chickens, and A/duck/Yokohama/aq10/ 2003 (Dk/Yokohama/aq10/03) isolated from duck meat imported from China. All chickens inoculated intranasally with either strain died, and the viruses were reisolated from all organs examined. However, both the mean time of onset of clinical signs and the mean death time of Ck/Yamaguchi/7/04 were shorter than those of Dk/Yokohama/aq10/03.     

Advanced molecular immunoassay system for immunobiotic lactic acid bacteria using a transfectant of Toll-like receptor 2

Toll‐like receptor 2 (TLR2) is a receptor for a variety of microbial components, and it also mediates activation signals in the cell relating to the innate immune system. In order to evaluate the precise molecular immunoregulation by various strains of lactic acid bacteria (LAB) via TLR2, the swine TLR2 (sTLR2)‐expressing transfectant was constructed using human embryonic kidney (HEK) 293 cells. It is demonstrated that intact immunobiotic LAB can induce immune responses through TLR2, and that different nuclear factor‐κB (NF‐κB) activities of various strains can be accurately detected by sTLR2‐expressing HEK293 cells. Furthermore, cellular activation of NF‐κB via TLR2 is reflected in enhanced binding and uptake of LAB. The sTLR2‐expressing HEK293 cells were also useful for characterizing the expression pattern of type I helper T (Th1) and type II helper T (Th2) cytokines by the stimulation of immunobiotic LAB. These results suggest that sTLR2‐expressing HEK293 cells may be useful in certain molecular immunoassay systems for producing new physiologically functional foods with intestinal immunomodulatory abilities, such as the maintenance of Th1/Th2 polarization.     

Feed intake, lactation performance, blood metabolites and fertility in early lactation dairy cows grazing a timothy pasture

The present study was conducted to investigate feed intake, milk yield, milk composition, blood metabolites and fertility in early lactation dairy cows grazing a timothy pasture. Fourteen multiparous Holstein cows that calved between 20 May and 19 July were used over a 3‐year period. The stocking rate was 3.6–4.3 cow/ha. Concentrates were fed separately at 9.5–11.5 kg/day per cow (dry matter basis) from 1 to 13 weeks postparturition. Herbage intake was estimated using chromium oxide as an indigestible marker. The mean contents of crude protein, total digestible nutrients and neutral detergent fiber of pasture during the 3‐year study period were 22.3%, 71.8% and 51.7%, and those of total diet were 18.9%, 77.3% and 40.3%, respectively. The mean herbage dry matter intake was 13.0 kg/day from 2 to 13 weeks postparturition during the study, total dry matter intake was 23.7 kg/day, the total digestible nutrients sufficiency rate was 105%, milk yield was 39.7 kg/day, and milk fat percentage was 3.30%. The decrease in bodyweight postparturition was slight. Urea nitrogen concentrations in serum were below 18.3 mg/dL. The mean days to first estrus and days open were 36 and 104 days, respectively. These results indicate that energy deficiency, decrease in bodyweight and fertility in early lactation barely occur when high producing dairy cows are fed enough grazing grass and suitable concentrates.     

Identification of sialyl oligosaccharides including an oligosaccharide nucleotide in colostrum of an addax (Addax nasomaculatus) (Subfamily Antelopinae)

Mammalian milk/colostrum usually contains milk oligosaccharides along with the predominant lactose. Although milk oligosaccharides of a variety of Bovidae species including cow, sheep and goat have been characterized, those of the addax, an Antelopinae species of the Bovidae, have not as yet been clarified. In this study, several sialyl oligosaccharides were purified from a sample of addax colostrum and characterized as follows: Neu5Ac(α2‐8)Neu5Ac(α2‐3)Gal(β1‐4)Glc, Neu5Gc(α2‐8)Neu5Gc(α2‐3)Gal(β1‐4)Glc, Neu5Ac(α2‐3)Gal(β1‐4)Glc, Neu5Ac(α2‐6)Gal(β1‐4)GlcNAc, Neu5Gc(α2‐3)Gal(β1‐4)Glc, Neu5Gc(α2‐6)Gal(β1‐4)Glc, Neu5Gc(α2‐6)Gal(β1‐4)GlcNAc. In addition, an oligosaccharide nucleotide Neu5Gc(α2‐6)Gal(β1‐4)GlcNAcα1‐UDP was characterized. Molecular species of a variety of sialyl oligosaccharides found in milk and colostrum of these Bovidae were compared.     

Chemical characterization of milk oligosaccharides of the common wombat (Vombatus ursinus)

Previous structural characterizations of marsupial milk oligosaccharides have been performed in the tammar wallaby, red kangaroo, koala, common brushtail possum and the eastern quoll. To clarify the homology and heterogeneity of milk oligosaccharides among marsupial species, which could provide information on their evolution, the oligosaccharides of wombat milk carbohydrate were characterized in this study. Neutral and acidic oligosaccharides were isolated from the carbohydrate fractions of two samples of milk of the common wombat and characterized by ¹H‐nuclear magnetic resonance spectroscopy. The structures of six neutral saccharides were found to be Gal(β1‐4)Glc (lactose), Gal(β1‐3)Gal(β1‐4)Glc (3'‐galactosyllactose), Gal(β1‐3)Gal(β1‐3)Gal(β1‐4)Glc (3',3”‐digalactosyllactose), Gal(β1‐3)Gal(β1‐3)Gal(β1‐3)Gal(β1‐4)Glc, Gal(β1‐3)Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc (galactosyl lacto‐N‐novopentaose I) and Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc (lacto‐N‐novooctaose), while those of six acidic saccharides were Neu5Ac(α2‐3)Gal(β1‐3)Gal(β1‐4)Glc. (sialyl 3'‐galactosyllactose), Neu5Ac(α2‐3)Gal(β1‐3)Gal(β1‐3)Gal(β1‐4)Glc (sialyl 3',3”‐digalactosyllactose), Neu5Ac(α2‐3)Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc (sialyl lacto‐N‐novopentaose a), Gal(β1‐3)[Neu5Ac(α2‐3)Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc (sialyl lacto‐N‐novopentaose c), Neu5Ac(α2‐3)Gal(β1‐3)Gal(β1‐3)Gal(β1‐3)Gal(β1‐4)Glc,, Neu5Ac(α2‐3)Gal(β1‐3)Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc and Gal(β1‐3)Gal(β1‐3)[Neu5Ac(α2‐3)Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc. In addition, small amounts of sulfated oligosaccharides but no oligosaccharides containing Neu5Gc or α(2–6) linked Neu5Ac were detected.     

Development of molecular immunoassay system for probiotics via toll-like receptors based on food immunology

Recent interest has focused on the importance of intestinal immunity for the host defense, but to date, not much is known about the underlying mechanisms. The toll‐like receptor (TLR) family plays an important role in host defense through recognizing bacterial pathogen‐associated molecular patterns. Our recent research on the physiological function of food products has investigated the immunoregulatory effects of probiotic lactic acid bacteria (LAB) via TLR. Studies of swine, which often substitute for a human model, have demonstrated intestinal immunoregulation by the probiotic LAB mediated by TLR in the gut. On the basis of our study, efforts have also been made to develop a molecular immunoassay system for probiotic LAB and find novel immunostimulatory DNA sequences from probiotics and high potential immunobiotic LAB strains via TLR signaling. These findings may provide important clues at the molecular level on TLR signal transduction pathways and recognition mechanisms for the ligands. They also provide impetus to further delineate the activation mechanism of the innate immune response. In addition to identifying immunoregulatory factor immunogenics from LAB, a better understanding of intestinal immune regulation through cytokine networks holds out promise for basic food immunology research and the development of immunobiotic foods to prevent specific diseases.