Biomedical subjects

Tadao Saito

Publications and source records attributed to Tadao Saito.

13 recordsLinked to original sources

PubMed2004

Characterization of oligosaccharides in milk of bearded seal (Erignathus barbatus).

Carbohydrates were extracted from milk of a bearded seal, Erignathus barbatus (Family Phocidae). Free neutral oligosaccharides were separated by gel filtration, anion-exchange chromatography and preparative thin layer chromatography, while free acidic oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by 1H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows; lactose, 2'-fucosyllactose, lacto-N-fucopentaose IV, difucosyl lacto-N-neohexaose and difucosyl decasaccharide which contained a lacto-N-neohexaose unit as well as an additional Gal(beta1-4)GlcNAc(beta1-3) unit and two residues of non-reducing Fuc(alpha1-2). The acidic oligosaccharides were thought to contain an Neu5Ac(alpha2-6) residue linked to GlcNAc or a sulfate linked to Gal at OH-3. The sialyl oligosaccharides and sulfated oligosaccharides had a lacto-N-neohexaose unit and two non-reducing Fuc(alpha1-2) residues and some of them had in addition one or two Gal(beta1-4)GlcNAc(beta1-3) units. The milk oligosaccharides of the bearded seal were compared to those of the harbour seal, which had been studied previously.

Animals↗

PubMed2004

Cloning and characterization of Swine interleukin-17, preferentially expressed in the intestines.

Interleukin-17 (IL-17), initially reported as CTLA-8, is a proinflammatory cytokine produced mainly by activated T cells. In the present study, the cDNA of a swine IL-17 (PoIL-17) gene was cloned from activated neonatal thymocytes, and the recombinant PoIL-17 (rPoIL-17) was biologically characterized. The complete open reading frame (ORF) of PoIL-17 contains 462-bp coding deduced 153 amino acid residues, with a calculated molecular weight of 17.3 kDa. The amino acid sequence showed 72.9%, 64.9%, 64.7%, 60.1%, and 47.4% similarities with that of human, rat, mouse, Herpesvirus saimiri ORF 13, and chicken, respectively. The six cysteine residues conserved over species including the virus were observed in PoIL-17. We successfully prepared the recombinant mature form of PoIL-17 and analyzed its biologic activities for swine splenocytes. RT-PCR analysis revealed a marked upregulation of expression of IL-1beta, IL-8, tumor necrosis factor-alpha (TNF-alpha), granulocyte colony-stimulating factor (G-CSF), and monocyte chemotactic protein-1 (MCP-1) mRNA expression in splenocytes treated with 100 ng/ml rPoIL-17 for 3 h. Furthermore, a swine chemokine, alveolar macrophage-derived neutrophil chemotactic factor II (AMCF-II), which was classified into the CXC subfamily was also augmented in mRNA level. This evidence indicates that recombinat PoIL-17 expressed in Escherichia coli was biologically active and exerted similar effects to those of a human (HuIL-17) and murine IL-17 (MuIL-17). The PoIL-17 mRNA is strongly expressed in the adult heart, skin, and, interestingly, intestinal tissues, including mesenteric lymph nodes but is restricted in neonatal tissues by using real-time quantitative RT-PCR. The gene sequence and biologically active recombinat protein for PoIL-17 will be useful for elucidation of the role of IL-17 in the regulation of intestinal immune responses.

Amino Acid Sequence↗

PubMed2004

Structural and functional differences in two cyclic bacteriocins with the same sequences produced by lactobacilli.

Lactobacillus gasseri LA39 and L. reuteri LA6 isolated from feces of the same human infant were found to produce similar cyclic bacteriocins (named gassericin A and reutericin 6, respectively) that cannot be distinguished by molecular weights or primary amino acid sequences. However, reutericin 6 has a narrower spectrum than gassericin A. In this study, gassericin A inhibited the growth of L. reuteri LA6, but reutericin 6 did not inhibit the growth of L. gasseri LA39. Both bacteriocins caused potassium ion efflux from indicator cells and liposomes, but the amounts of efflux and patterns of action were different. Although circular dichroism spectra of purified bacteriocins revealed that both antibacterial peptides are composed mainly of alpha-helices, the spectra of the bacteriocins did not coincide. The results of D- and L-amino acid composition analysis showed that two residues and one residue of D-Ala were detected among 18 Ala residues of gassericin A and reutericin 6, respectively. These findings suggest that the different D-alanine contents of the bacteriocins may cause the differences in modes of action, amounts of potassium ion efflux, and secondary structures. This is the first report that characteristics of native bacteriocins produced by wild lactobacillus strains having the same structural genes are influenced by a difference in D-amino acid contents in the molecules.

Amino Acid Sequence↗

PubMed2004

A new assay using surface plasmon resonance (SPR) to determine binding of the Lactobacillus acidophilus group to human colonic mucin.

A new binding assay to investigate the mechanism of adhesion of lactic acid bacteria to the human intestine was established by the surface plasmon resonance technique using a biosensor BIACORE1000. Cells of 26 strains of the Lactobacillus acidophilus group as analytes were eluted onto a sensor chip on which were immobilized biotinylated A-trisaccharide polymer probes having human A-type antigen [(GalNAcalpha1-3(Fucalpha1-2)Gal)-] or human colonic mucin of blood type A (HCM-A) as ligands. In the first screening, high adhesive affinity to the A-trisaccharide BP-probe was observed in L. acidophilus OLL2769, L. crispatus JCM8778, LA205 and LA206. In the second screening, which used HCM-A, only L. acidophilus OLL2769 and L. crispatus JCM8778 were selected as adhesive strains with specific binding ability to human A-antigen. The results indicated that some strains of the L. acidophilus group could recognize and bind the sugar chain of A-antigen structure on HCM.

Bacterial Adhesion↗

PubMed2004

Dextran from Leuconostoc mesenteroides augments immunostimulatory effects by the introduction of phosphate groups.

The immunological effects of phosphorylated dextran (in which phosphate groups were chemically introduced) on murine splenocytes were examined. When dextran produced by Leuconostoc mesenteroides was phosphorylated by a reaction with polyphosphoric acid in formamide solution for 48 h, the degree of phosphorylation of dextran was maximal. The highest phosphorus content (1.7%, wt/wt) was observed in 40 kDa of dextran. The mitogenic response of murine splenocytes was enhanced by the phosphorylated dextran, but its activity was not related to its molecular weight. A strong response was detected at a concentration of 10 to 500 microg/ml, and the highest activity was obtained 48 h after stimulation. Phosphorylated dextran was characterized as a B-cell-specific mitogen. The expressions of CD86 on CD8alpha- CD11c- and CD8alpha- CD11c+ cells were augmented by phosphorylated dextran. The levels of mRNA expression of gamma interferon and interleukin-10 on murine splenocytes were also increased by the stimulation. These results demonstrate that dextran exerts immunostimulation by the introduction of phosphate groups.

Animals↗

PubMed2003

Immunostimulatory oligonucleotide, CpG-like motif exists in Lactobacillus delbrueckii ssp. bulgaricus NIAI B6.

The present study was conducted to find an immunostimulatory oligonucleotide derived from yogurt starter cultures. The chromosomal DNA was purified from nine strains of Lactobacillus delbrueckii ssp. bulgaricus and six strains of Streptococcus thermophilus. An immunostimulatory ability of the DNA was examined in a proliferation of peyer's patch and splenic B cells. Only the DNA from L. bulgaricus NIAI B6 induced a significant proliferation of both cells. When the DNA was cloned and amplified using PCR, the mitogenic activities to B cells were significantly increased by 13 of 135 DNA clones. Ten homologous nucleotide sequences were found as possible oligonucleotide sequences of mitogens, and were then chemically synthesized (sOL-LB1 to sOL-LB10). One CpG-like motif (sOL-LB7; 5'-CGGCACGCTCACGATTCTTG-3') was identified as an immunostimulatory oligonucleotide, but it did not contain palindromic CpG structure known as a B cell-specific mitogen. The sOL-LB7 substantially bound to B cells and increased the CD69 positive cells in peyer's patch cells. This study demonstrated that L. bulgaricus NIAI B6 was a good candidate of a starter culture for the production of new functional foods, "Bio-Defense Foods".

Animals↗

PubMed2003

Swine Toll-like receptor 9(1) recognizes CpG motifs of human cell stimulant.

Complementary DNA (cDNA) encoding swine Toll-like receptor 9 (sTLR9) was isolated from Peyer's patches (Pps) of gut-associated lymphoid tissue (GALT). The complete open reading frame (ORF) of sTLR9 contains 3093 bp coding deduced 1030 amino acid residues. The amino acid sequence of sTLR9 was characterized by a signal peptide followed by multiple leucine-rich repeats, a transmembrane sequence and a cytoplasmic domain homologous to that of the human interleukin-1 receptor (TIR). The sTLR9 showed a higher amino acid identity with humans (81.8%) and felis catus (86.7%) than mice (74.9%). The HEK293T cells transfected with pCXN2.1-FLAG DNA containing the sTLR9 cDNA were expressed sTLR9 as a membrane-bound molecules, which were reactive with anti-sTLR9 rabbit polyclonal antibody. Moreover, the transfectant was responsible for the CpG oligo DNA. sTLR9 was preferentially expressed in Pps and mesenteric lymph nodes (MLNs), and its degree was approximately three times higher than a spleen but weak in the other tissues by the real-time quantitative PCR analyses. The strong expression of sTLR9 in Pps and MLNs and its recognizing CpG DNA for human cell stimulant are shown first in this study, which may help in understanding the intestinal immune system mediated by a bacterial DNA through TLR9.

Amino Acid Sequence↗

PubMed2003

Differences in oligosaccharide pattern of a sample of polar bear colostrum and mid-lactation milk.

Although the concentrations of carbohydrate in the colostrum and in the mid-lactation milk of polar bear (Ursus maritimus) were similar, the oligosaccharide patterns differed. The colostrum sample contained Neu5Ac(alpha2-3)Gal(beta1-4)Glc (3'-N-acetylneuraminyllactose), GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc (A-tetrasaccharide), Fuc(alpha1-2)Gal(beta1-4)Glc (2'-fucosyllactose) and Gal(beta1-4)Glc (lactose). The mid-lactation milk contained Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)[Fuc(alpha1-3)]Glc (B-pentasaccharide), GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)[Fuc(alpha1-3)]Glc (A-pentasaccharide), Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc (B-tetrasaccharide), A-tetrasaccharide, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc (3-fucosylisoglobotriose), Gal(alpha1-3)Gal(beta1-4)Glc (isoglobotriose) and lactose. The dominant saccharides in the colostrum were 3'-N-Acetylneuraminyllactose and lactose, whereas isoglobotriose was the dominant saccharide in the mid-lactation milk in which lactose was only a minor component. Isoglobotriose, which had previously been found to be a dominant saccharide in mature milk from the Ezo brown bear, the Japanese black bear and the polar bear, was not found in the polar bear colostrum.

Animals↗

PubMed2003

Chemical characterization of the oligosaccharides in milk of high Arctic harbour seal (Phoca vitulina vitulina).

Carbohydrates were extracted from high Arctic harbour seal milk, Phoca vitulina vitulina (family Phocidae). Free neutral oligosaccharides were separated by gel filtration and preparative thin layer chromatography, while free sialyl oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by 1H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows: lactose, 2'-fucosyllactose, lacto-N-neotetraose, lacto-N-neohexaose, monofucosyl lacto-N-neohexaose and difucosyl lacto-N-neohexaose. Thus, all of the neutral saccharides contained lactose or lacto-N-neotetraose or lacto-N-neohexaose as core units and/or non-reducing alpha(1-2) linked fucose. These oligosaccharides have also been found in hooded seal milk. The structures of the silalyl oligosaccharides were: monosialyl lacto-N-neohexaose, monosialyl monofucosyl lacto-N-neohexaose, monosialyl difucosyl lacto-N-neohexaose and disialyl lacto-N-neohexaose. These oligosaccharides contained lacto-N-neohexaose as core units, and one or two alpha(2-6) linked Neu5Ac, and/or non-reducing alpha(1-2) linked Fuc. The Neu5Ac residues were found to be linked to GlcNAc or penultimate Gal residues. The acidic oligosaccharides are the first to have been characterized in the milk of any species of seal.

Animals↗

PubMed2003

Composition and oligosaccharides of a milk sample of the giant panda, Ailuropoda melanoleuca.

A milk sample from a captive giant panda (Ailuropoda melanoleuca), obtained at 13 days postpartum, contained 7.1% protein, 1.6% carbohydrate, 10.4% lipid and 0.9% ash. The ratio of casein to whey proteins was 5.0:2.1. Sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) of the whey protein fraction showed the presence of at least two major proteins other than alpha-lactalbumin and beta-lactoglobulin. SDS-PAGE and urea-gel electrophoresis showed that alphas-casein is not a major component. The proportions of triacylglycerol, cholesterol, cholesterol esters and phospholipid were 90.5, 5.3, 0.96 and 3.1%, of the total lipid, respectively. The dominant saccharide in the panda milk was Gal(alpha1-3)Gal(beta1-4)Glc (isoglobotriose). The milk contained, in addition, lesser amounts of lactose, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc (fucosyl isoglobotriose), Neu5Ac(alpha2-3)Gal(beta1-4)Glc (3'-N-acetylneuraminyl-lactose), Neu5Ac(alpha2-6)Gal(beta1-4)Glc (6'-N-acetylneuraminyl-lactose) and Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc.

Animals↗

PubMed2003

Determination of each neutral oligosaccharide in the milk of Japanese women during the course of lactation.

Using reverse-phase HPLC after pyridylamination, we quantified the concentrations of major neutral oligosaccharides in the milk of sixteen Japanese women collected at 4, 10, 30 and 100 d postpartum. In colostrum and mature milk (30 d lactation), lacto-N-fucopentaose (LNFP) I was the most abundant oligosaccharide, followed by 2'-fucosyllactose (2'-FL) + lacto-N-difucotetraose (LNDFT), LNFP II + lacto-N-difucohexaose II (LNDFH II), and 3-fucosyllactose (3-FL). Together these accounted for 73 % of the total weight of neutral oligosaccharides in colostrum and mature milk. Changes in concentration occurred during the course of lactation. LNFP I and 2'-FL + LNDFT increased from 4 to 10 d postpartum, and then declined by 100 d. LNFP II + LNDFH II steadily increased during the first 30 d and then declined. In contrast, 3-FL increased steadily throughout the entire 100 d of study. Large differences were observed between our data and previously published data in Italian women, in terms of both the concentration and temporal changes of each oligosaccharide. These differences may be caused by different assay methodology, although racial differences cannot be ruled out.

Adult↗

PubMed2002

A novel immunostimulating aspect of Lactobacillus gasseri: induction of "Gasserokine" as chemoattractants for macrophages.

The chemotactic activity of the culture supernatants from 14 strains of Lactobacillus acidophilus and L. gasseri was examined for murine macrophages. Significant macrophage chemotactic activity was observed in three strains of L. acidophilus and all strains of L. gasseri. The highest activity was observed in the supernatant (1131-sup) from 24-h cultures of L. gasseri JCM1131T. The chemotactic factor from 1131-sup, designated as "Gasserokine", was purified by the C18 reverse phase and ion-exchange chromatography. The purity of Gasserokine was checked by HPLC with the reverse-phase mode. The chemotactic activity of Gasserokine was also observed for human monocytes. The macrophage chemotaxis induced by L. gasseri JCM1131T culture supernatants was discovered to be a new biological function exerted by probiotic lactic acid bacteria. Therefore, the activity is expected to be used for one of the functional parameters in the immunomodulating properties of probiotic lactic acid bacteria.

Animals↗

PubMed2002

Chemical characterization of the oligosaccharides in beluga (Delphinapterus leucas) and Minke whale (Balaenoptera acutorostrata) milk.

Carbohydrates were extracted from the milk of a beluga, Delphinopterus leucas (family Odontoceti), and two Minke whales, Balaenoptera acutorostrata (Family Mysticeti), sampled late in their respective lactation periods. Free oligosaccharides were separated by gel filtration and then neutral oligosaccharides were purified by preparative thin layer chromatography and gel filtration, while acidic oligosaccharides were purified by ion-exchange chromatography, gel filtration and high performance liquid chromatography (HPLC). Their structures were determined by 1H-NMR. In one of the Minke whale milk samples, lactose was a dominant saccharide, with Fuc(alpha1-2)Gal(beta1-4)Glc(2'-fucosyllactose), Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc(lacto-N-neotetraose), GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc(A-tetrasaccharide), Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (para lacto-N-neohexaose), Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (sialyl lacto-N-neotetraose), Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LST c) and Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (sialyl para lacto-N-neohexaose) also being found in the milk. The second Minke whale sample contained similar amounts of lactose, 2'-fucosyllactose and A-tetrasaccharide, but no free sialyl oligosaccharides. Sialyl lacto-N-neotetraose and sialyl para lacto-N-neohexaose are novel oligosaccharides which have not been previously reported from any mammalian milk or colostrum. These and other oligosaccharides of Minke whale milk may have biological significance as anti-infection factors, protecting the suckling young against bacteria and viruses. The lactose of Minke whale milk could be a source of energy for them. The beluga whale milk contained trace amounts of Neu5Ac(alpha2-3)Gal(beta1-4)Glc(3'-N-acetylneuraminyllactose), but the question of whether it contained free lactose could not be clarified. Therefore, lactose may not be a source of energy for suckling beluga whales.

Animals↗