Tomio Yabe

Portable sulphotransferase domain determines sequence specificity of heparan sulphate 3-O-sulphotransferases

3-O-Sulphates are the rarest substituent of heparan sulphate and are therefore ideally suited to the selective regulation of biological activities. Individual isoforms of heparan sulphate D-glucosaminyl 3-O-sulphotransferase (3-OST) exhibit sequence-specific action, which creates heparan sulphate structures with distinct biological functions. For example, 3-OST-1 preferentially generates binding sites for anti-thrombin, whereas 3-OST-3 isoforms create binding sites for the gD envelope protein of herpes simplex virus 1 (HSV-1), which enables viral entry. 3-OST enzymes comprise a presumptive sulphotransferase domain and a divergent N-terminal region. To localize determinants of sequence specificity, we conducted domain swaps between cDNA species. The N-terminal region of 3-OST-1 was fused with the sulphotransferase domain of 3-OST-3(A) to generate N1-ST3(A). Similarly, the N-terminal region of 3-OST-3(A) was fused to the sulphotransferase domain of 3-OST-1 to generate N3(A)-ST1. Wild-type and chimaeric enzymes were transiently expressed in COS-7 cells and extracts were analysed for selective generation of binding sites for anti-thrombin. 3-OST-1 was 270-fold more efficient at forming anti-thrombin-binding sites than 3-OST-3(A), indicating its significantly greater selectivity for substrates that can be 3-O-sulphated to yield such sites. N3(A)-ST1 was as active as 3-OST-1, whereas the activity of N1-ST3(A) was as low as that of 3-OST-3(A). Analysis of Chinese hamster ovary cell transfectants revealed that only 3-OST-3(A) and N1-ST3(A) generated gD-binding sites and conveyed susceptibility to infection by HSV-1. Thus sequence-specific properties of 3-OSTs are defined by a self-contained sulphotransferase domain and are not directly influenced by the divergent N-terminal region.

The Bovine Lactophorin C-Terminal Fragment and PAS6/7 Were Both Potent in the Inhibition of Human Rotavirus Replication in Cultured Epithelial Cells and the Prevention of Experimental Gastroenteritis

Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. We have found that high-Mr glycoprotein fraction (F1) from cow’s milk whey has potent inhibitory activity against human rotavirus (HRV) in cell culture. The present study was undertaken to identify and characterize the components responsible for this inhibitory activity. F1 was initially heated at 95 °C for 30 min, rendering milk antibodies inert, subjected to ammonium sulfate fractionation, and then resolved by two-dimensional polyacrylamide gel electrophoresis. After electroelution, we found that a heat-stable milk protein lactophorin C-terminal fragment (LP16) and bovine milk fat globule membrane protein PAS6/7 strongly inhibited the replication of HRV MO strains in MA104 cells. Furthermore, we found that prophylactic oral administration of F1 once before inoculation of the HRV MO strain obviously prevented the development of diarrhea in vivo. These non-immunoglobulin components are a promising candidate for a prophylactic food additive against HRV infection.

In vitro and in vivo evaluation of the efficacy of bovine colostrum against human rotavirus infection.

We found that skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal cows at 6–7 d after parturition exhibited high potency in inhibiting replication of human rotavirus (HRV) in vitro. Furthermore, prophylactic oral administration of SCBLC once before inoculation of HRV prevented the development of diarrhea in suckling mice in vivo. SCBLC from normal cows might be useful in the prevention of HRV-induced severe gastroenteritis in immunocompromised hosts.

Bovine κ-casein inhibits human rotavirus (HRV) infection via direct binding of glycans to HRV.

Human rotavirus (HRV) is a major etiologic agent of severe infantile gastroenteritis. κ-Casein (κ-CN) from both human and bovine mature milk has been reported to have anti-HRV activity; however, the mechanism of this activity is poorly understood. The present study examined the molecular basis for the protective effect of bovine κ-CN derived from late colostrum (6-7 d after parturition) and from mature milk. Among the components of casein, κ-CN is the only glycosylated protein that has been identified. Therefore, we investigated whether the glycan residues in κ-CN were involved in the anti-HRV activity. Desialylated CN obtained by neuraminidase treatment exhibited anti-HRV activity, whereas deglycosylated CN obtained by o-glycosidase treatment lacked antiviral activity, indicating that glycans were responsible for the antiviral activity of CN. Furthermore, an evanescent-field fluorescence-assisted assay showed that HRV particles directly bound to heated casein (at 95°C for 30 min) in a viral titer-dependent manner. Although the heated κ-CN retained inhibitory activity in a neutralization assay, the activity was weaker than that observed before heat treatment. Our findings indicate that the inhibitory mechanism of bovine κ-CN against HRV involves direct binding to viral particles via glycan residues. In addition, heat-labile structures in κ-CN may play an important role in maintenance of κ-CN binding to HRV.

Ce3+-induced exopolysaccharide production by Bradyrhizobium sp. MAFF211645

Ce³+, a rare earth element (REE), has been widely used in high-technology industries. Despite the importance of Ce³+ in the fields of chemistry and physics, the role of Ce³+ in biology has been ignored. To investigate physiological effects of Ce³+ on microorganisms, we screened microorganisms that showed peculiar growth in the presence of Ce³+. We isolated a free-living soil bacterium that produced exopolysaccharide (EPS) around its colonies on 1/100 nutrient agar with 30 μM CeCl₃ or 1.0% D-mannitol. The bacterium was identified as Bradyrhizobium sp. by morphological, biochemical, and physiological tests as well as 16S rDNA sequence analysis. La³+, Pr³+, and Nd³+ also induced EPS production in large quantities, while Sm³+ did in small amounts. However, other heavier REEs from Eu³+ to Lu³+, and metals such as Na+, Al³+, K+, Ca²+, V³+, Cr³+, Co²+, Ni²+, Sr²+, Ba²+, and Pb²+ did not induce EPS production. The mean molecular weight of EPS was estimated to be approximately 1 x 10⁶ by Sepharose CL-4B column chromatography. TLC revealed that EPS was composed of L-rhamnose. Quantitative analysis of alditol acetate derivatives of acid hydrolyzate of EPS by GLC revealed that EPS was composed of more than 95% L-rhamnose, indicating that this EPS was a rhamnan. The spectrum of FT-IR of the rhamnan demonstrated that L-rhamnose residues in the rhamnan were α-linked. GC/MS analysis of methylated alditol acetate derivatives of the rhamnan demonstrated that it was composed of main chain α-(1→4)-linked L-rhamnopyranosyl residues. From spectral analyses of ¹H-NMR and FT-IR, EPS produced in the presence of 1.0% D-mannitol was found to be structurally similar to rhamnans.

The Multiplicity of N-Glycan Structures of Bovine Milk 18 kDa Lactophorin (Milk GlyCAM-1)

Lactophorin is a heat-stable phosphoglycoprotein, also known as milk glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1). Bovine 18 kDa lactophorin was purified by heparin affinity chromatography from cow’s milk whey. Its N-glycans were obtained by proteomic techniques, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by in-gel digestion with peptide-N4-(N-acetyl-β-glucosaminyl)-asparagine amidase (PNGase F). The released N-glycans were derivatized with 2-aminopryridine (PA) and analyzed by matrix-assisted laser desorption ionization quadruple ion trap time of flight mass spectrometry (MALDI-QIT-TOF MS). Among the MS analyzed peaks, 15 peaks were found to be N-glycan molecules as detected by MS2 analysis. These glycans consisted of mono-sialylated bi-, tri-, and tetra-antennary complex-type N-glycans carrying Gal-GlcNAc (LacNAc) or GalNAc-GlcNAc (LacdiNAc) with and without core-fucose.

Water-soluble extracts from defatted sesame seed flour show antioxidant activity in vitro

Defatted white and gold sesame seed flour, recovered as a byproduct after sesame oil extraction, was extracted with 70% ethanol to obtain polar-soluble crude extracts. The in vitro antioxidant activity of the extract was evaluated by DPPH free radical scavenging activity and oxygen radical absorbing capacity (ORAC). The polar-soluble crude extracts of both sesame seed types exhibited good antioxidant capacity, especially by the ORAC method with 34,720 and 21,700 μmol Trolox equivalent/100g of white and gold sesame seed extract, respectively. HPLC, butanol extraction, and UPLC-MS analyses showed that different compounds contributed to the antioxidant activity of the polar-soluble crude extracts. Sesaminol glycosides were identified in the butanol-soluble fractions; whereas, purified water-soluble fraction contained ferulic and vanillic acids. This study shows that hydrophilic antioxidants in the purified water-soluble fraction contributed to the antioxidant activity of white and gold sesame seed polar-soluble crude extracts.

Skimmed, sterilized, and concentrated bovine late colostrum promotes both prevention and recovery from intestinal tissue damage in mice

Bovine colostrum is a rich source of tissue repair and growth factors, and inhibits gastrointestinal injury induced by the side effects of nonsteroidal antiinflammatory drugs (NSAID), such as indomethacin. Nonsteroidal antiinflammatory drugs are drugs with analgesic and antipyretic effects, but in higher doses they have inflammatory effects. The pathogenesis of small intestinal damage caused by NSAID is unclear. The present study was performed to investigate the antiinflammatory effects of skimmed, sterilized, and concentrated bovine late colostrum on intestinal injury induced by side effects of NSAID, and then to identify the active ingredient in the colostrum for intestinal tissue. In Japan, the sale of bovine colostrum within 5 d after parturition is prohibited by law. Therefore, we focused on bovine late colostrum obtained from healthy lactating cows 6 to 7 d after parturition. Proliferation of small intestine epithelial cells was stimulated in mice fed the colostrum for 1 wk. With regard to indomethacin-induced enteropathy, both prefeeding and postfeeding with colostrum facilitated growth of the intestinal villi, indicating preventive and healing effects. Furthermore, to identify the active ingredient in the colostrum responsible for this effect, the casein and whey fractions were prepared from the colostrum and fed to normal mice. Only the colostrum casein fraction stimulated intestinal villus elongation, whereas the whey fraction and mature milk casein showed no such effect. Taken together, these observations indicate that the skimmed, sterilized, and concentrated bovine late colostrum, especially the casein fraction, could be used to treat the injurious effects of NSAID in the intestine and could be effective for treatment of other ulcerative conditions in the bowel, suggesting that the colostrum has therapeutic potential for intestinal inflammation.

A highly efficient construction of GM1 epitope tetrasaccharide and its conjugation with KLH

GM1 epitope tetrasaccharide was synthesized by a condensation of sialyl-α(2–3)-gal acceptor and gal-β(1–3)-GalN donor in a highly efficient manner. After introduction of mercaptohexanol to the tetrasaccharide, it was coupled to maleimide-activated KLH carrier protein to give the desired GM1 epitope-KLH conjugate.

An Antioxidant of Dried Chili Pepper Maintained Its Activity through Postharvest Ripening for 18 Months

The antioxidant properties of hot-water extracts from a dried chili pepper were maintained through the postharvest ripening process at 10 °C for 18 months. In order to isolate the antioxidant from the ripe pepper, we fractionated hot-water extracts by size-exclusion gel chromatography. A certain fraction showed antioxidative activity via the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity assay. Structural analysis by reversed-phase high performance liquid chromatography (HPLC), LC-MS, and nuclear magnetic resonance (NMR) revealed that the antioxidant was a known compound, p-coumaryl alcohol. This study indicates that an effective antioxidant in chili pepper sustains its antioxidative effects during the postharvest ripening process.