Alteration of the N-glycome of bovine milk glycoproteins during early lactation

Milk provides nutritional, immunological and developmental components for newborns. Whereas identification of such components has been performed by targeting proteins and free oligosaccharides, structural and functional analyses of the N-glycome of milk glycoproteins are scarce. In this study, we investigated, for the first time, the alterations of the bovine milk N-glycome during early lactation (1 day, 1, 2, 3 and 4 weeks postpartum), characterizing more than 80 N-glycans. The glycomic profile of colostrum on day 1 after calving differed substantially from that in other periods during early lactation. The proteins in colostrum obtained 1 day postpartum were more highly sialylated than milk samples obtained at other time points, and the N-glycolylneuraminic acid (Neu5Gc)/N-acetylneuraminic acid (Neu5Ac) ratio was significantly higher on day 1, showing a gradual decline with time. In order to dissect the N-glycome of colostrum, alterations of the N-glycosylation profile of major bovine milk proteins during the early lactation stage were elucidated, revealing that the alteration is largely attributable to qualitative and quantitative N-glycosylation changes of IgG, the major glycoprotein in colostrum. Furthermore, by preparing and analyzing IgGs in which the N-glycan structure and subtypes were well characterized, we found that the interaction between IgG and FcRn was not affected by the structure of the N-glycans attached to IgG. We also found that bovine FcRn binds IgG(2) better than IgG(1) , strongly suggesting that the role of FcRn in the bovine mammary gland is to recycle IgG(2) from the udder to blood, rather than to secrete IgG(1) into colostrum.     

Different binding sites for the neuropeptide Y Y1 antagonists 1229U91 and J-104870 on human Y1 receptors

The peptidic Y1 antagonist 1229U91 and the non-peptidic antagonist J-104870 have high binding affinities for the human Y1 receptor. These Y1 antagonists show anorexigenic effects on NPY-induced feeding in rats, although they have completely different structures and molecular sizes. To identify the binding sites of these ligands, we substituted amino acid residues of the human Y1 receptor with alanine and examined the abilities of the mutant receptors to bind the radio-labeled ligands. Alanine substitutions, F98A, D104A, T125A, D200A, D205A, L215A, Q219A, L279A, F282A, F286A, W288A and H298A, in the human Y1 receptor lost their affinity for the peptide agonist PYY, but not for 1229U91 and J-104870, while L303A and F173A lost affinity for 1229U91 and J-104870, respectively. N283A retained its affinity for 1229U91, but not for PYY and J-104870. Y47A and N299A retained their affinity for J-104870, but not for PYY and 1229U91. W163A and D287A showed no affinity for any of the three ligands. Taken together, these data indicate that the binding sites of 1229U91 are widely located in the shallow region of the transmembrane (TM) domain of the receptor, especially TM1, TM6 and TM7. In contrast, J-104870 recognized the pocket formed by TM4, TM5 and TM6, based on the molecular modeling of the Y1 receptor and J-104870 complex. In conclusion, 1229U91 and J-104870 have high affinities for Y1 receptors using basically different binding sites. D287 of the common binding site in the TM6 domain could be crucial for the binding of Y1 antagonists.     

Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells

The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues in vitro. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated in vitro by 3D MSC constructs incorporated with HUVECs.     

Protective effect of nasal immunization of influenza virus hemagglutinin with recombinant cholera toxin B subunit as a mucosal adjuvant in mice

To develop an efficient nasal influenza vaccine, influenza A and B virus HA with rCTB as a mucosal adjuvant were administered to mice intranasally. Serum anti-HA IgG and IgA antibody responses for both HA vaccines were significantly increased in the presence of rCTB. Higher HI and neutralizing antibody titers and higher mucosal IgA antibody responses in the respiratory tract were detected when rCTB was added than without rCTB. When mice were immunized with HA vaccine with or without rCTB and challenged by intranasal administration of mouse-adapted pathogenic influenza A virus, all mice immunized with HA plus rCTB survived for seven days without any inflammatory changes in the lungs, while not all the mice immunized with HA without rCTB survived, and all of them had lung consolidations. These results demonstrate that intranasal co-administration of rCTB as a mucosal adjuvant with influenza virus HA is necessary not only for the induction of systemic and mucosal HA antibodies, but also for the protection of mice from morbidity and mortality resulting from virus infection.     

Bacterial oxidation of polyethylene glycol.

The metabolism of polyethylene glycol (PEG) was investigated with a synergistic, mixed culture of Flavobacterium and Pseudomonas species, which are individually unable to utilize PEGs. The PEG dehydrogenase linked with 2,6-dichlorophenolindophenol was found in the particulate fraction of sonic extracts and catalyzed the formation of a 2,4-dinitrophenylhydrazine-positive compound, possibly an an aldehyde. The enzyme has a wide substrate specificity towards PEGs: from diethylene glycol to PEG 20,000 Km values for tetraethylene glycol (TEG), PEG 400, and PEG 6,000 were 11, 1.7, and 15 mM, respectively. The metabolic products formed from TEG by intact cells were isolated and identified by combined gas chromatography-mass spectrometry as triethylene glycol and TEG-monocarboxylic acid plus small amounts of TEG-dicarboxylic acid, diethylene glycol, and ethylene glycol. From these enzymatic and analytical data, the following metabolic pathway was proposed for PEG: HO(CH2CH2O)nCH2CH2OH leads to HO(CH2CH2O)nCH2CHO leads to HO(CH2CH2O)nCH2COOH leads to HO(CH2CH2O)n-1CH2CH2OH.     

Total Synthesis of Nodakenetin and Marmesin

Nodakenetin and marmesin were synthesized from β-resorcylaldehyde through 2-(α-hy-droxyisopropyl)-6-hydroxycoumaran.     

Effect of Water Content on the Glass Transition Temperature of Calcium Maltobionate and its Application to the Characterization of Non-Arrhenius Viscosity Behavior

To understand the fundamental physical properties of calcium maltobionate (MBCa), its water sorption isotherm, glass transition temperature (T _g), and viscosity (η) were investigated and compared with those of maltobionic acid (MBH) and maltose. Although amorphous maltose crystalized at water activity (a _w) higher than 0.43, MBCa and MBH maintained an amorphous state over the whole a _w range. In addition, MBCa had a higher T _g and greater resistance to water plasticizing than MBH and maltose. These properties of MBCa likely originate from the strong interaction between MBCa and water induced by electrostatic interactions. Moreover, the effects of temperature and water content on η of an aqueous MBCa solution were evaluated, and its behavior was described using a semi-empirical approach based on a combination of T _g extrapolated by the Gordon-Taylor equation and a non-Arrhenius formula known as the Vogel–Fulcher–Tammann equation. This result will be useful for understating the effect of MBCa addition on the solution’s properties.     

Analysis of terminal sugar moieties and species-specificities of acrosome reaction-inducing substance in Xenopus (ARISX)

The acrosome reaction of Xenopus sperm is triggered by the acrosome reaction-inducing substance in Xenopus (ARISX), an oviductal pars recta-derived, sugar-rich substance decorated on the entire surface of the vitelline envelope (VE) during ovulation. Here we addressed the functional importance of the sugar moiety in ARISX. Among various lectins examined, soybean agglutinin and Dolichos biflorus agglutinin were shown to abolish the acrosome reaction-inducing activity of ARISX present in pars recta extract or on the VE, indicating the importance of the terminal alpha-N-acetylgalactosamine residue for the function of ARISX. Consistently, the acrosome reaction-inducing activity was not affected by proteinase K digestion, in spite of the simultaneous shift of ARISX to a smaller molecular weight. Indirect immunofluorescence microscopic examinations showed that ARISX was distributed as two types of structures on VE; thick fiber-like materials and thin filamentous materials, and that a new structure appeared on the fertilization envelope instead of the thin filamentous materials. Sperm from several amphibian species were subjected to an in vitro assay during induction of the acrosome reaction with ARISX. The resulting limited population of sperm from a non-Xenopus species underwent acrosome reaction, implying a weak species-specificity of ARISX.     

Hydrogen production by termite gut protists: characterization of iron hydrogenases of Parabasalian symbionts of the termite Coptotermes formosanus

Cellulolytic flagellated protists in the guts of termites produce molecular hydrogen (H(2)) that is emitted by the termites; however, little is known about the physiology and biochemistry of H(2) production from cellulose in the gut symbiotic protists due to their formidable unculturability. In order to understand the molecular basis for H(2) production, we here identified two genes encoding proteins homologous to iron-only hydrogenases (Fe hydrogenases) in Pseudotrichonympha grassii, a large cellulolytic symbiont in the phylum Parabasalia, in the gut of the termite Coptotermes formosanus. The two Fe hydrogenases were phylogenetically distinct and had different N-terminal accessory domains. The long-form protein represented a phylogenetic lineage unique among eukaryotic Fe hydrogenases, whereas the short form was monophyletic with those of other parabasalids. Active recombinant enzyme forms of these two Fe hydrogenases were successfully obtained without the specific auxiliary maturases. Although they differed in their extent of specific activity and optimal pH, both enzymes preferentially catalyzed H(2) evolution rather than H(2) uptake. H(2) evolution, at least that associated with the short-form enzyme, was still active even under high hydrogen partial pressure. H(2) evolution activity was detected in the hydrogenosomal fraction of P. grassii cells; however, the vigorous H(2) uptake activity of the endosymbiotic bacteria compensated for the strong H(2) evolution activity of the host protists. The results suggest that termite gut symbionts are a rich reservoir of novel Fe hydrogenases whose properties are adapted to the gut environment and that the potential of H(2) production in termite guts has been largely underestimated.