Mechanism of the chemical composition changes of yuba prepared by a laboratory processing method.

Yuba is a filmlike soybean food made from heated soymilk that contains oil bodies (average diameter, 270 nm), particulate protein (>40 nm; average diameter, 70 nm), soluble protein (<40 nm), and carbohydrate (molecular size). Three varieties of soybean were used to make yuba. The carbohydrate in the remaining soymilk increased sharply while lipid increased a little. The particle size distributions of oil body showed the trend that smaller oil bodies were concentrated in the remaining soymilk, and the percentage of soluble protein in whole protein increased in the remaining soymilk. These results could be explained well with diffusion theory. The temperature gradient and concentration gradient originating from the heat treatment were considered to cause the net particle diffusion from the surface to the bottom soymilk. Lipids, which mainly exist as oil bodies, are easily incorporated into yuba films because a few of these less dense droplets diffuse downward, causing the lipid concentration in the soymilk to change a little. Carbohydrate at the surface quickly diffuses downward, causing the carbohydrate concentration increase in the soymilk beneath the developing yuba. Protein (particulate and soluble) in the soymilk was intermediate between lipid and carbohydrate.

Oxidized alginate-cross-linked alginate/gelatin hydrogel fibers for fabricating tubular constructs with layered smooth muscle cells and endothelial cells in collagen gels.

Hydrogel fibers that possessed a cell-adhesive surface and were degradable via enzymatic reactions were developed for fabricating tubular constructs with smooth muscle cell (SMC) and endothelial cell (EC) layers, similar to native blood vessels, in collagen gels. The fibers were prepared by soaking hydrogel fibers prepared from a solution of sodium alginate and gelatin containing bovine ECs (BECs) in medium containing oxidized alginate (AO). BECs soaked in 8.0% (w/v) AO showed no reduction in viability within 3 h of soaking. Furthermore, mouse SMCs (MSMCs) adhered and proliferated on the AO-cross-linked hydrogels. Based on these results, we prepared AO-cross-linked hydrogel fibers containing BECs, covered their surface with MSMCs, and embedded them in collagen gels. We then degraded the fibers using alginate lyase to obtain channels in the collagen gels. Histological analysis of the released ECs using a specific fluorescent dye revealed the formation of tubular structures with layered BECs and MSMCs.

Novel technique for fabricating double-layered tubular constructs consisting of two vascular cell types in collagen gels used as templates for three-dimensional tissues.

Double-layered tubular constructs consisting of two vascular cell types in collagen gels were fabricated using a previously developed technique [Takei et al., Biotechnol. Bioeng., 95, 1-7 (2006)]. Histological examination suggested that shear stress (4.0 dyn/cm2) on the luminal surface of the constructs induced morphological changes in their walls.

Isolation, cloning, and characterization of a novel phosphomannan-binding lectin from porcine serum.

Mannan-binding protein (MBP) is a C-type serum lectin that is an important constituent of the innate immune defense because it activates the complement system via the lectin pathway. While the pig has been proposed to be an attractive source of xenotransplantable tissues and organs, little is known about porcine MBP. In our previous studies, phosphomannan, but not mannan, was found to be an effective inhibitor of the C1q-independent bactericidal activity of newborn piglet serum against some rough strains of Gram-negative bacteria. In contrast, the inhibitory activities of phosphomannan and mannan were very similar in the case of MBP-dependent bactericidal activity against rough strains of Escherichia coli K-12 and S-16. Based on these findings, we inferred that an MBP-like lectin with slightly or completely different carbohydrate binding specificity might exist in newborn piglet serum and be responsible for the C1q-independent bactericidal activity. Herein we report that a novel phosphomannan-binding lectin (PMBL) of 33 kDa under reducing conditions was isolated from both newborn and adult porcine serum and characterized. Porcine PMBL functionally activated the complement system via the lectin pathway triggered by binding with both phosphomannan (P-mannan) and mannan, which, unlike MBP, was effectively inhibited by mannose 6-phosphate- or galatose-containing oligosaccharides. Our observations suggest that porcine PMBL plays a critical role in the innate immune defense from the newborn stage to adult-hood, and the establishment of a newborn piglet experimental model for the innate immune system studies is a valuable step toward elucidation of the physiological function and molecular mechanism of lectin pathway.

Exogenous expression of interferon-beta in cultured brain microvessel endothelial cells.

Brain microvessel endothelial cells (BMECs) make up the blood-brain barrier (BBB) and regulate the passage of therapeutic proteins as well as drugs from the cerebrovasucular circulation to the brain. In the present study, we transferred mouse or human interferon-beta (IFN-beta) gene via cationic liposomes into primary cultures of bovine BMECs developed as an in vitro model of the BBB. The gene-transferred BMECs secreted transiently a substantial amount of IFN activity more efficiently during the growth phase than at confluence. This was suggested to be due to a difference in the potential for plasmid incorporation between growing and confluent BMECs in a series of cell association experiments with (32)P-labelled plasmid DNA. Furthermore, when BMEC monolayers in Transwell plates were transfected with the IFN-beta-expression vectors from the upper side, IFN-beta was predominantly detected in the upper compartments, suggesting polarized secretion of the transgene products in BMEC monolayers. These findings provide important basic information about therapeutic secretory protein gene delivery to BMECs.