L-Fucose-containing arabinogalactan-protein in radish leaves.

The carbohydrate moieties of arabinogalactan-proteins (AGPs) have β-(1 → 3)-galactan backbones to which side chains of (1 → 6)-linked β-Gal residues are attached through O-6. Some of these side chains are further substituted with other sugars. We investigated the structure of L-Fuc-containing oligosaccharides released from the carbohydrate moieties of a radish leaf AGP by digestion with α-L-arabinofuranosidase, followed by exo-β-(1 → 3)-galactanase. We detected a series of neutral β-(1 → 6)-galactooligosaccharides branching variously at O-3 of the Gal residues, together with corresponding acidic derivatives terminating in 4-O-methyl-GlcA (4-Me-GlcA) or GlcA at the non-reducing terminals. In neutral oligosaccharides with degree of polymerization (dp) mainly higher than 10, L-Fuc groups were attached through L-Ara residues as the sequence, α-L-Fucp-(1 → 2)-α-L-Araf-(1 →. This sequence was verified by isolation of the pentasaccharide α-L-Fuc-(1 → 2)-α-L-Araf-(1 → 3)-β-Gal-(1 → 6)-β-Gal-(1 → 6)-Gal upon digestion of the higher oligosaccharides with endo-β-(1 → 6)-galactanase. By contrast, in lower polymerized (predominantly dp 4) acidic oligosaccharides, L-Fuc groups were attached directly at the non-reducing terminals through α-(1 → 2)-linkages, resulting in the release of the tetrasaccharides, α-L-Fucp-(1 → 2)-β-GlcA-(1 → 6)-β-Gal-(1 → 6)-Gal and α-L-Fucp-(1 → 2)-β-4-Me-GlcA-(1 → 6)-β-Gal-(1 → 6)-Gal. In long acidic oligosaccharides with dp mainly higher than 13, L-Fuc groups localized on branches were attached to the uronic acids directly and/or L-Ara residues as in the neutral oligosaccharides.The authors would like to thank Prof. M. Hisamatsu, Mie University, Tsu, Japan, for a gift of cyclic β-(1→2)-glucan. This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant-in-Aid for Scientific Research no. 23570048 to Y.T. and no. 24114006 to Y.T. and T.K.). Support was also provided by BBSRC Sustainable Bioenergy Centre: Cell wall sugars program (Grant No. BB/G016240/1) to P.D.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.carres.2015.07.00

Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis.

Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility

Thinking Globally But Acting Locally

The Center for Asian Business and Alpha Kappa Psi presented a special lecture on Wednesday, December 5th featuring Yoshimi Inaba, chairman of Toyota Motor Sales, U.S.A., Inc. and executive advisor for Toyota Motor Corporation. The title of his lecture was based on one of Toyota\u27s key principles, “Thinking Globally but Acting Locally.” Toyota’s Japanese-oriented way of thinking has adapted to fit the American culture, and that is one of the reasons why the company is so successful. During the first half hour of the lecture, Mr. Inaba provided an overview of Toyota’s global presence and impact on the automobile industry. He shared the key principles that make Toyota one of the biggest and most successful automobile companies in the world - reliability and durability. Mr. Inaba said he encourages his employees in each region to focus on being the best company in their local area rather than the entire world. The last half hour of the lecture was spent answering questions from the audience. Questions ranged from “How did you handle the Toyota recall?” to “What’s your favorite Toyota model?” Students were treated to free pizza and soda following the event. Mr. Inaba is chairman of Toyota Motor Sales (TMS), U.S.A., Inc., Toyota’s U.S. sales, marketing, distribution and customer service arm in Torrance, Calif. He also serves as an executive advisor of Toyota Motor Corporation (TMC), Toyota’s parent company in Japan. Mr. Inaba is responsible for Toyota’s sales, marketing and external affairs operations in the U.S. He began his career with TMC in 1968 and has reached many achievements, such as being named to TMC’s Board of Directors, becoming president of TMS, becoming senior managing director at TMC, and being named executive VP. In 2007, he was appointed president and CEO of Central Japan International Airport Co., Ltd., and a senior advisor to the board of TMC. In 2009, Mr. Inaba returned to TMC in his current capacity. He has a degree in economics from Kyoto University and a MBA from Northwestern University\u27s Kellogg School of Management.https://digitalcommons.lmu.edu/ybminlectureseries/1026/thumbnail.jp

The development of a neocentromere-based minichromosome gene delivery system

© 2011 Dr. Yoshimi InabaGene therapy has the potential to cure human genetic diseases permanently by introducing correct sets of therapeutic genes into patient cells. Multiple vector systems have been generated for gene therapy applications. They include human engineered chromosomes (HECs) that can overcome many of the hurdles presented by other gene delivery systems such as viral vector systems. HECs are fully functional chromosomes containing the minimal genomic content required for correct mitotic function, and have the capacity to carry large transgene or gene clusters. The neocentromere of marker chromosome mardel(10) provides a highly desirable source for HEC construction. The mardel(10) neocentromere-based minichromosomes (NC-MiCs) were generated as a gene delivery vector system, and developed for use in gene therapy applications. This study focused on (1) the purification of NC-MiC to achieve efficient NC-MiC delivery into target cells, and (2) the integration of a therapeutic gene into NC-MiC to demonstrate the potential of NC-MiCs as gene delivery vectors. A chromosome soup was prepared from a HT1080-derived cell line containing NC-MiC1 (3.5Mb) and subsequently subjected to flow-sorting. A high purity level of NC-MiC1 chromosome was obtained. The same approaches were applied for purifying NC-MiC6.1 (1.4Mb), which has a greater potential for use in gene therapy because of its smaller size and the presence of a site specific sequence for targeted gene integration. By applying multiple-gates and parameters on the sorting of NC-MiC6.1, the chromosome was enriched further by 27 times in the sorted samples, opening the way for the preparation and use of minichromosomes carrying less background genetic material. Success in the isolation of NC-MiC minichromosome using flow-sorter has not been previously described, and there has been no published report on the isolation or enrichment of minichromosomes as small as 1.4Mb. While comparing NC-MiC6.1 quantity in the sorted samples, we developed a novel DNA quantification method. It is relatively simple and quick and, importantly, more sensitive than currently available DNA quantification methods. This method, which is based on DOP-PCR, can be widely used to measure any DNA source. It can also be used as internal controls for genomic qPCR experiments, such as ChIP and Microarray experiments. Additional chromosome labelling using the interaction of lac operator (lacO) and lac repressor (LacI) was attempted to improve the isolation of specific chromosomes by flow-sorting. This lacO-LacI interaction was also utilised in coupling with magnetic bead for affinity purification of target chromosomes. Although isolation of specific chromosomes was not fully achieved in this study using these approaches, both approaches showed a potential use for chromosome purification. To demonstrate the utility of NC-MiCs as gene delivery vectors, the frataxin gene (FXN gene) was chosen as a model therapeutic gene for treatment of Friedreich ataxia by gene therapy. Targeted integration of a BAC containing FXN gene (FXN-BAC) into NC-MiC6.1 can be achieved using FRT/Flp site-specific recombination. The incorporation of the FRT sequence into FXN-BAC was successfully modified by homologous recombination. Due to time constraint, a small number of attempts to co-transfect FXN-BAC and the Flp expression vector into NC-MiC6.1 line were performed but to date no positive clone was obtained. Time constraint has also did not allow the transfer of NC-MiC into various cell lines in this study. In summary, this study has established the feasibility of using high-resolution flow-sorting methods to purify or significantly enrich the purity of minichromosomes as small as 1.4-3.5Mb. The study has also tested additional potential methods for further improving the purification of the very small chromosomes, established a new DNA quantification method, and begun testing the targeted integration of disease-relevant genes into the minichromosomes