Ikuko Yokota

Influence of the pattern size of micropatterned scaffolds on cell morphology, proliferation, migration and F-actin expression

To determine how the three-dimensional (3D) shape of scaffolds influences cell functions, 3D micropatterned scaffolds of various sizes were fabricated on a silicon substrate. The micropatterns were equilateral triangular pores with 3-20 μm long sides, and all had the same pore ratio (total pore area per unit area) and depth. The patterns only differed in terms of their 2D size. Such scaffolds have not been previously generated, and thus the effects of pattern size on cell functions have not been addressed. NIH-3T3 cells were cultured on these micropatterned scaffolds, and their morphology, proliferation rate, migration rate, and level of F-actin expression were assessed. Cells became more rounded and F-actin expression decreased as the pattern size of the scaffold decreased. Relationships were also demonstrated between pattern size and cell proliferation and migration. These results suggest that the pattern size of 3D micropatterned scaffolds affects the level of mechanical stress that cells experience, and thereby influences F-actin expression, cell morphology, cell proliferation and cell migration.

Quantitative O-Glycomics by Microwave-Assisted β-Elimination in the Presence of Pyrazolone Analogues

O-Linked glycosylation of serine/threonine residues is a posttranslational modification of proteins and is essential for protein recognition and lipid functions on cell surfaces and within cells. The characterization of differently structured O-linked glycans (O-glycans) is particularly challenging because there is no known endoglycosidase for such groups. Therefore, chemical digestion approaches have been widely used; however, it is sometimes difficult to suppress unwanted side reactions. Recently, we reported a novel O-glycomics procedure using β-elimination in the presence of pyrazolone analogues (BEP). In the present study, we describe a microwave (MW)-assisted BEP procedure for rapid and quantitative O-glycomic analysis. Following optimization of the reaction conditions, the MW-assisted BEP reaction substantially improved the recovery of total O-glycans from model glycoproteins (PSM) and the reaction time was reduced from 16 to 2 h. Combined with sequential solid-phase extractions, this MW-assisted BEP procedure enabled O-glycomic analyses of various biological samples.

Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method

Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma membrane lipid raft, which is clustered with sphingolipids, sterols, and specific proteins, and thereby contributes to membrane physical properties and specific recognition sites for various biological events. These bioactive GSL molecules consequently affect the pathophysiology and pathogenesis of various diseases. Thus, altered expression of GSLs in various diseases may be of importance for disease-related biomarker discovery. However, analysis of GSLs in blood is particularly challenging because GSLs are present at extremely low concentrations in serum/plasma. In this study, we established absolute GSL-glycan analysis of human serum based on endoglycoceramidase digestion and glycoblotting purification. We established two sample preparation protocols, one with and the other without GSL extraction using chloroform/methanol. Similar amounts of GSL-glycans were recovered with the two protocols. Both protocols permitted absolute quantitation of GSL-glycans using as little as 20 μl of serum. Using 10 healthy human serum samples, up to 42 signals corresponding to GSL-glycan compositions could be quantitatively detected, and the total serum GSL-glycan concentration was calculated to be 12.1–21.4 μM. We further applied this method to TLC-prefractionated serum samples. These findings will assist the discovery of disease-related biomarkers by serum GSL-glycomics.

Modulation of surface stiffness and cell patterning on polymer films using micropatterns

Here, a new technology was developed to selectively produce areas of high and low surface Youngs modulus on biomedical polymer films using micropatterns. First, an elastic polymer film was adhered to a striped micropattern to fabricate a micropattern-supported film. Next, the topography and Youngs modulus of the film surface were mapped using atomic force microscopy. Contrasts between the concave and convex locations of the stripe pattern were obvious in the Youngs modulus map, although the topographical map of the film surface appeared almost flat. The concave and convex locations of a polymer film supported by a different micropattern also contrasted clearly. The resulting Youngs modulus map showed that the Youngs modulus was higher at convex locations than at concave locations. Hence, regions of high and low stiffness can be locally generated based on the shape of the micropattern supporting the film. When cells were cultured on the micropattern-supported films, NIH3T3 fibroblasts preferentially accumulated in convex regions with high Youngs moduli. These findings demonstrate that this new technology can regulate regions of high and low surface Youngs modulus on a cellular scaffold with high planar resolution, as well as providing a method for directing cellular patterning.

Impact of the Niemann–Pick c1 Gene Mutation on the Total Cellular Glycomics of CHO Cells

Niemann-Pick disease type C (NPC) is an autosomal recessive lipid storage disorder, and the majority of cases are caused by mutations in the NPC1 gene. In this study, we clarified how a single gene mutation in the NPC1 gene impacts the cellular glycome by analyzing the total glycomic expression profile of Chinese hamster ovary cell mutants defective in the Npc1 gene (Npc1 KO CHO cells). A number of glycomic alterations were identified, including increased expression of lactosylceramide, GM1, GM2, GD1, various neolacto-series glycosphingolipids, and sialyl-T (O-glycan), which was found to be the major sialylated protein-bound glycan, as well as various N-glycans, which were commonly both fucosylated and sialylated. We also observed significant increases in the total amounts of free oligosaccharides (fOSs), especially in the unique complex- and hybrid-type fOSs. Treatment of Npc1 KO CHO cells with 2-hydroxypropyl-β-cyclodextrin (HPBCD), which can reduce cholesterol and glycosphingolipid (GSL) storage, did not affect the glycomic alterations observed in the GSL-, N-, and O-glycans of Npc1 KO CHO cells. However, HPBCD treatment corrected the glycomic alterations observed in fOSs to levels observed in wild-type cells.