Large-scale preparation and glycan characterization of sialylglycopeptide from bovine milk glycomacropeptide and its bifidogenic properties.

Bovine glycomacropeptide (GMP) is a 7,000-Da glycopolypeptide released from κ-casein during cheese making. The O-glycan chains linked to GMP have many biological activities, but their utilization for nutraceutical products is limited due to their low content. To concentrate the functional glycan chains of GMP, we prepared sialylglycopeptide concentrate (SGC) from GMP-containing whey protein concentrate via proteolytic digestion of peptide chains and concentration of sialylglycopeptide by ultrafiltration using membranes with a molecular weight cut-off of 1,000 Da. The abundant saccharides detected in the prepared SGC were N-acetylneuraminic acid (Neu5Ac: 32.3% wt/wt), N-acetylgalactosamine (11.3%), and galactose (10.2%), which constitute O-glycans attached to GMP. The Neu5Ac content in SGC was found concentrated at approximately 4.8-fold of its content in GMP-containing whey protein concentrate (6.8%). Structural analysis of O-glycopeptides by liquid chromatography tandem mass spectrometry identified 88 O-glycopeptides. Moreover, O-acetylated or O-diacetylated Neu5Ac was detected in addition to the previously characterized O-glycans of GMP. Quantitative analysis of O-glycan in SGC by fluorescence labeling of chemically released O-glycan revealed that a disialylated tetrasaccharide was the most abundant glycan (76.6% of the total O-glycan). We further examined bifidogenic properties of SGC in vitro, which revealed that SGC served as a more potent carbon source than GMP and contributes to the growth-promoting effects on certain species of bifidobacteria. Overall, our study findings indicate that SGC contains abundant O-glycans and has a bifidogenic activity. Moreover, the protocol for the preparation of SGC described herein is relatively simple, providing a high yield of glycan, and can be used for large-scale preparation.

TransCAIP: A Live 3D TV system using a camera array and an integral photography display with interactive control of viewing parameters.

The system described in this paper provides a real-time 3D visual experience by using an array of 64 video cameras and an integral photography display with 60 viewing directions. The live 3D scene in front of the camera array is reproduced by the full-color, full-parallax autostereoscopic display with interactive control of viewing parameters. The main technical challenge is fast and flexible conversion of the data from the 64 multicamera images to the integral photography format. Based on image-based rendering techniques, our conversion method first renders 60 novel images corresponding to the viewing directions of the display, and then arranges the rendered pixels to produce an integral photography image. For real-time processing on a single PC, all the conversion processes are implemented on a GPU with GPGPU techniques. The conversion method also allows a user to interactively control viewing parameters of the displayed image for reproducing the dynamic 3D scene with desirable parameters. This control is performed as a software process, without reconfiguring the hardware system, by changing the rendering parameters such as the convergence point of the rendering cameras and the interval between the viewpoints of the rendering cameras.

Comparison of a lateral-flow immunochromatography assay with real-time reverse transcription-PCR for detection of human metapneumovirus.

A lateral-flow immunochromatography (IC) assay for the detection of human metapneumovirus (hMPV) has been developed by using two mouse monoclonal antibodies to the nucleocapsid protein of hMPV. The purpose of this study was to compare the virus detection rate in nasopharyngeal secretions by the IC assay with that by real-time reverse transcription-PCR (RT-PCR). We collected nasopharyngeal swab samples from 247 children with respiratory symptoms in Sapporo, Japan, during the period from April to July 2007. Sixty-eight of the 247 children were positive for hMPV by real-time RT-PCR. When the real-time RT-PCR was used as the reference standard, the IC assay results were positive for 48 of the 68 real-time RT-PCR-positive children (70.6% sensitivity) and 8 of the 179 real-time RT-PCR-negative children (95.5% specificity). Although the sensitivity of the IC assay is lower than that of real-time RT-PCR, the IC assay is a rapid and useful test for the diagnosis of hMPV infections in children.

Crystal structure of a novel zinc-binding ATP sulfurylase from Thermus thermophilus HB8.

ATP sulfurylase (ATPS) is a ubiquitous enzyme that catalyzes the transfer of the adenylyl group from ATP to inorganic sulfate, producing adenosine 5'-phosphosulfate (APS) and pyrophosphate. The crystal structure of ATPS from Thermus thermophilus HB8 (TtATPS, 347 amino acid residues) in complex with APS was determined at 2.5 A resolution. TtATPS is composed of three domains [domain I (residues 1-134), domain II (residues 135-290), and domain III (residues 291-347)], like the Riftia pachyptila symbiont ATPS, but lacks a fourth domain present in ATPSs from the yeast Saccharomyces cerevisiae and from the fungus Penicillium chrysogenum. TtATPS forms a dimer in the crystal, and the manner of subunit association is different from that observed in dimeric R. pachyptila symbiont ATPS and in the hexameric S. cerevisiae and P. chrysogenum ATPSs. APS is located in the active site of TtATPS, which contains several motifs (QXRN, HXXH, and GRD) conserved in ATPSs. Unexpectedly, TtATPS binds one metal ion per subunit in domain III. XAFS measurement of the crystal and the Bijvoet difference Fourier map unambiguously characterized the metal ion as a zinc ion. The zinc ion is tetrahedrally coordinated by Cys294, Cys297, Cys306, and His310, and could not be removed from the protein by treatment with EDTA. The zinc ion binding site is far from the active site. Because all four residues coordinated to the zinc ion are conserved in the ATPSs from thermophilic bacteria such as Archaeoglobus fulgidus, Pyrococcus abyssi, and Sulfolobus solfataricus, zinc ion chelation may contribute to the thermal stability of these ATPSs.

Overproduction, crystallization and preliminary X-ray diffraction analysis of probable ATP sulfurylase from Thermus thermophilus HB8.

ATP sulfurylase catalyzes the reaction of inorganic sulfate with ATP, producing adenosine-5'-phosphosulfate (APS) and pyrophosphate. A probable ATP sulfurylase (MW = 38.8 kDa) from Thermus thermophilus HB8 was overproduced in Escherichia coli and purified. It was crystallized in the presence of 5 mM APS by the batch method. The crystal was monoclinic, space group P2(1), with unit-cell parameters a = 68.8, b = 61.2, c = 128.6 A, beta = 95.4 degrees. Diffraction to better than 2.5 A resolution was obtained using synchrotron radiation at SPring-8. The asymmetric unit most probably contains two subunits (V(M) = 3.48 A(3) Da(-1)).