KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.     

Efficient and rapid purification of drug- and gene-carrying bio-nanocapsules, hepatitis B virus surface antigen L particles, from Saccharomyces cerevisiae

Bio-nanocapsules (BNCs) are hollow particles (approx. 50 nm diameter) consisting of hepatitis B virus surface antigen (HBsAg) large (L, pre-S1+pre-S2+S) proteins embedded in a unilamellar liposome, sharing the same transmembrane S region with an immunogen of hepatitis B vaccine (i.e., HBsAg small (S) protein particle). BNCs can incorporate drugs and genes into the hollow space and systemic administration of the BNCs can deliver the products to human liver via the human hepatocyte-specific receptor within the pre-S (pre-S1+pre-S2) region displayed on BNC's surface. Thus, BNCs are expected to offer efficient and safe non-viral nanocarriers to deliver human liver-specific genes and drugs. To date, BNCs have been purified from the crude extract of BNC-overexpressing yeast cells by fractionation with polyethylene glycol followed by one CsCl equilibrium and two sucrose density gradient ultracentrifugation steps. However, the process was inefficient in terms of yield and time, and was not suitable for mass production because of the ultracentrifugation step. Furthermore, trace contamination with yeast-derived proteinases degraded the pre-S region, which is indispensable for liver-targeting, during long-term storage. In this study, we developed a new purification method involving heat treatment and sulfated cellulofine column chromatography to facilitate rapid purification, completely remove proteinases, and enable mass production. In addition, the BNCs were functional for at least 14 months after lyophilization with 5% (w/v) sucrose as an excipient. This new process will significantly contribute to the development of forthcoming BNC-based nanomedicines as well as hepatitis B vaccines.     

Tumor-selective,adenoviral-mediated GFP genetic labeling of human cancer in the live mouse reports future recurrence after resection

We have previously developed a telomerase-specific replicating adenovirus expressing GFP (OBP-401), which can selectively label tumors in vivo with GFP. Intraperitoneal (i.p.) injection of OBP-401 specifically labeled peritoneal tumors with GFP, enabling fluorescence visualization of the disseminated disease and real-time fluorescence surgical navigation. However, the technical problems with removing all cancer cells still remain, even with fluorescence-guided surgery. In this study, we report imaging of tumor recurrence after fluorescence-guided surgery of tumors labeled in vivo with the telomerase-dependent, GFP-containing adenovirus OBP-401.. Recurrent tumor nodules brightly expressed GFP, indicating that initial OBP-401-GFP labeling of peritoneal disease was genetically stable, such that proliferating residual cancer cells still express GFP. In situ tumor labeling with a genetic reporter has important advantages over antibody and other non-genetic labeling of tumors, since residual disease remains labeled during recurrence and can be further resected under fluorescence guidance.Key words: green fluorescent protein, adenovirus, cancer labeling, in situ, fluorescence-guided surgery, recurrence, detection     

Glycomics of a novel type-2 N-acetyllactosamine-specific lectin purified from the feather star, Oxycomanthus japonicus (Pelmatozoa: Crinoidea)

A lectin - designated OXYL for the purposes of this study that strongly recognizes complex-type oligosaccharides of serum glycoproteins - was purified from a crinoid, the feather star Oxycomanthus japonicus, the most basal group among extant echinoderms. OXYL was purified through a combination of anion-exchange and affinity chromatography using Q-sepharose and fetuin-sepharose gel, respectively. Lectin was determined to be a 14-kDa polypeptide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions. However, 14-kDa and 28-kDa bands appeared in the same proportion under non-reducing conditions. Gel permeation chromatography showed a 54-kDa peak, suggesting that lectin consists of four 14-kDa subunits. Divalent cations were not indicated, and stable haemagglutination activity was demonstrated at pH 4-12 and temperatures below 60°C. Surface plasmon resonance analysis of OXYL against fetuin showed k(ass) and k(diss) values of 1.4×10(-6)M(-1)s(-1) and 3.1×10(-3)s(-1), respectively, indicating that it has a strong binding affinity to the glycoprotein as lectin. Frontal affinity chromatography using 25 types of prydylamine-conjugated glycans indicated that OXYL specifically recognizes multi-antennary complex-type oligosaccharides containing type-2 N-acetyllactosamines (Galβ1-4GlcNAc) if α2-3-linked sialic acid is linked at the non-reducing terminal. However, type-1 N-acetyllactosamine (Galβ1-3GlcNAc) chains and α2-6-linked sialic acids were never recognized by OXYL. This profiling study showed that OXYL essentially recognizes β1-4-linkage at C-1 position and free OH group at C-6 position of Gal in addition to the conservation of N-acetyl groups at C-2 position and free OH groups at C-3 position of GlcNAc in N-acetyllactosamine. This is the first report on glycomics on a lectin purified from an echinoderm belonging to the subphylum Pelmatozoa.     

Effects of balance training using wobble boards in the elderly

Few studies have examined balance training of elderly people using wobble boards. This study assessed the effects of wobble board balance training on physical function in institutionalized elderly people. This study examined 23 subjects (age 84.2 ± 5.9 years) who lived in a nursing home. The exercise program for the training group comprised balance training standing on a wobble board for 9 weeks, twice a week. In all, 11 training group subjects and 11 control group subjects completed this study. After 9 weeks, standing time on a wobble board, standing time on a balance mat, and maximum displacement distance of anterior-posterior center of pressure in the training group were significantly greater than those of the control group. Frequency analysis revealed that the power spectrum in 0.1-0.2 Hz significantly increased in the training group. These results suggest that wobble board training is effective for elderly people to improve their standing balance, by which they frequently control their center of gravity and maintain a standing posture on unstable surface conditions.     

Walking strategies during the transition between level and hill surfaces

Healthy young adults transition between level and hill surfaces of various angles while walking at fluctuating speeds. These surface transitions have the potential to decrease dynamic balance in both the anterior-posterior and medial-lateral directions. Hence, the purpose of the current study was to analyze modifications in temporal-spatial parameters during hill walking transitions. We hypothesized that in comparison with level walking, the transition strides would indicate the adoption of a distinct gait strategy with a greater base of support. Thirty-four participants completed level and hill trials on a walkway with a 15-degree portable ramp apparatus. We collected data during 4 transition strides between level and ramp surfaces. In support of our hypothesis, compared with level walking, the base of support was 20% greater during 3 out of the 4 transition strides. In short, our results illustrate that healthy young adults did adopt a distinct gait strategy different from both level and hill walking during transitions strides.     

High levels of human recombinant cyclooxygenase-1 expression in mammalian cells using a novel gene amplification method

We report the expression of a high level of human cyclooxygenase-1 (hCOX-1) in mammalian cells using a novel gene amplification method known as the IR/MAR gene amplification system. IR/MAR-plasmids contain a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) and amplify autonomously without a specific induction process. In this study, the IR/MAR-plasmid pΔBN.AR1 was cotransfected with pCAG-COX1, which expresses hCOX-1, into human HEK293T cells, and G418 and blasticidin S double-resistant cells were obtained in about 1month. Real-time PCR and Western blotting revealed that the expressions of hCOX-1 mRNA and protein in both polyclonal and monoclonal cells were remarkably higher than those in only pCAG-COX1-transfected control cells. Southern blotting demonstrated the amplification of the hCOX-1 gene, and the copy number of clone #43 obtained by the cotransfection of pΔBN.AR1 and pCAG-COX1 was more than 20 copies per cell, though that of clone #14 obtained without using the IR/MAR plasmid pΔBN.AR1 was only two copies. These results indicate that a high level of hCOX-1 expression was achieved as a result of hCOX-1 gene amplification. Furthermore, the crude extract from clone #43 showed a strong COX-1 activity, and the activity was inhibited by the representative COX-1 inhibitor indomethacin, with an IC(50) value of 36nM. These results demonstrate that the IR/MAR gene amplification system is a simple but useful method for generating highly productive mammalian cells.     

Generation of micronuclei during interphase by coupling between cytoplasmic membrane blebbing and nuclear budding

Micronucleation, mediated by interphase nuclear budding, has been repeatedly suggested, but the process is still enigmatic. In the present study, we confirmed the previous observation that there are lamin B1-negative micronuclei in addition to the positive ones. A large cytoplasmic bleb was found to frequently entrap lamin B1-negative micronuclei, which were connected to the nucleus by a thin chromatin stalk. At the bottom of the stalk, the nuclear lamin B1 structure appeared broken. Chromatin extrusion through lamina breaks has been referred to as herniation or a blister of the nucleus, and has been observed after the expression of viral proteins. A cell line in which extrachromosomal double minutes and lamin B1 protein were simultaneously visualized in different colors in live cells was established. By using these cells, time-lapse microscopy revealed that cytoplasmic membrane blebbing occurred simultaneously with the extrusion of nuclear content, which generated lamin B1-negative micronuclei during interphase. Furthermore, activation of cytoplasmic membrane blebbing by the addition of fresh serum or camptothecin induced nuclear budding within 1 to 10 minutes, which suggested that blebbing might be the cause of the budding. After the induction of blebbing, the frequency of lamin-negative micronuclei increased. The budding was most frequent during S phase and more efficiently entrapped small extrachromosomal chromatin than the large chromosome arm. Based on these results, we suggest a novel mechanism in which cytoplasmic membrane dynamics pulls the chromatin out of the nucleus through the lamina break. Evidence for such a mechanism was obtained in certain cancer cell lines including human COLO 320 and HeLa. The mechanism could significantly perturb the genome and influence cancer cell phenotypes.