Recently we reported that N-glycans on the ␤-propeller domain of the integrin ␣5 subunit (S-3,4,5) are essential for ␣5␤1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type ␣5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the ␣5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type ␣5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the ␣5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.Glycosylation is a crucial post-translational modification of most secreted and cell surface proteins (1). Glycosylation is involved in a variety of physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion. It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2, 3).Integrins comprise a family of receptors that are important for cell adhesion. The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin ␣5␤1 is well known as a fibronectin (FN) 3 receptor. The interaction between integrin ␣5 and FN is essential for cell migration, cell survival, and development (5-8). In addition, integrins are N-glycan carrier proteins. For example, ␣5␤1 integrin contains 14 and 12 putative N-glycosylation sites on the ␣5 and ␤1 subunits, respectively. Several studies suggest that N-glycosylation is essential for functional integrin ␣5␤1. When human fibroblasts were cultured in the presence of 1-deoxymannojirimycin, which prevents N-linked oligosaccharide processing, immature ␣5␤1 integrin appeared on the cell surface, an...

Owing to the increasing importance of genomic information, obtaining genomic DNA easily from biological specimens has become more and more important. This article proposes an efficient method for obtaining genomic DNA from nail clippings. Nail clippings can be easily obtained, are thermostable and easy to transport, and have low infectivity. The drawback of their use, however, has been the difficulty of extracting genomic material from them. We have overcome this obstacle using the protease solution obtained from Cucumis melo. The keratinolytic activity of the protease solution was 1.78-fold higher than that of proteinase K, which is commonly used to degrade keratin. With the protease solution, three times more DNA was extracted than when proteinase K was used. In order to verify the integrity of the extracted DNA, genotype analysis on 170 subjects was performed by both PCR-RFLP and Real Time PCR. The results of the genotyping showed that the extracted DNA was suitable for genotyping analysis. In conclusion, we have developed an efficient extraction method for using nail clippings as a genome source and a research tool in molecular epidemiology, medical diagnostics, and forensic science.

Trans fatty acid may impair fetal growth and infant neurodevelopment, but the quantity in a placenta and human tissues remains unknown. To address the issue, a simple and reliable method of quantification is needed. We established a method of quantifying trans-octadecenoic acids (trans-6,8,9,11 18:1 fatty acids, TOAs), a major component of trans fatty acid, in human tissue samples, and then determined the TOAs level in the placenta. Oleic acid (OA) (C18:1(9c)) was measured by isotope dilution gas chromatography-mass spectrometry, and the TOAs level was subsequently calculated based on the ratio of the peak areas for TOAs and OA (TOAs/OA) in the mass chromatogram. Lipids were extracted from 28 human placentas at different gestational ages from 28 to 41 weeks, and the TOAs and OA levels were measured. In method validation, the limit of detection for elaidic acid (trans-9,18:1 fatty acid), a major component of TOAs, was 0.57 ng, and linearity of calibration ranging from 7.7 to 68.0 μg/g placenta for TOAs. In human placenta analysis, the TOAs level was significantly higher in term (n = 15, 40.2 ± 9.7 μg/g placenta) than in preterm placentas (n = 13, 18.9 ± 7.4 μg/g placenta) (p < 0.001), while OA levels were similar in term (n = 15, 863 ± 132 μg/g placenta) and preterm (n = 13, 743 ± 283 μg/g placenta) placentas (p = 0.15). TOAs accumulate in the placenta as pregnancy progresses and have a fate different from that of OA in vivo. To our knowledge, this is the first report of TOA quantification in human tissue samples. Copyright © 2017 John Wiley & Sons, Ltd.

Aging increases free radical generation and lipid oxidation and, thereby, mediates neurodegenerative diseases. As the brain is rich in lipids (polyunsaturated fatty acids), the antioxidative system plays an important role in protecting brain tissues from oxidative injury. The changes in microtubule formation and antioxidative enzyme activities have been investigated in rat pheochromocytoma PC12 cells exposed to various concentrations of phosphatidylcholine hydroperoxides (PCOOH). We measured three typical antioxidative enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). The microtubule assembly system was dependent on the antioxidative enzyme system in cells exposed to oxidative stress. The activities of the three enzymes increased in a PCOOH exposure-dependent manner. In particular, the changes in the activity as a result of PCOOH exposure were similar in the three antioxidative enzymes. This is the first report indicating the compatibility between the tubulin-microtubule and antioxidative enzyme systems in cells that deteriorate as a result of phospholipid hydroperoxide administration from an exterior source. The descending order of sensitivity of the three enzymes to PCOOH is also discussed.

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