Epitope analysis of peanut allergen Ara h1 with human monoclonal IgM antibody 92-2

A human-mouse hybridoma clone 92-2 secreting IgM-class human monoclonal antibody to peanut allergen protein Ara h1 was established. To detect antibody-binding sequences on Ara h1, we synthesized a series of peptides of the Ara h1 protein on a multi-pin apparatus for the pin-peptide ELISA. The 92-2 human monoclonal antibody was found to recognize a sequence of GREGEQEWGTPGSHVREETS. Further analysis with shorter pin-peptides with eight amino acid-long showed that the sequence of QEWGTPGS was an essential linear sequence of this epitope. When the QEW part of the sequence was replaced by alanine, the 92-2 monoclonal antibody did not bind to the substituted peptide, showing that those amino acids play an important role in the binding of the 92-2 monoclonal antibody.     

Local risks and global impacts considering plant-specific functions and constraints: a case study of metal parts cleaning

Background, aim, and scope  

To achieve sustainable development in industrial processes, attributed chemical risks as well as environmental impacts should be managed. Such non-monetary issues have been analyzed by scientific assessment methodologies such as various risk assessment (RA) and life cycle assessment (LCA) procedures. Local risks to be addressed in RA are microenvironments, including the workplace and neighborhood. Although a comprehensive interpretation of such risks is necessitated in industrial decision making, no practical method has been developed to interpret various types of risk with sufficient understandings of plant-specific functions and constraints. Because elaborate model-based approaches are inevitable for practical process development, actual case studies on chemical risks and detailed plant-specific functions and constraints should be performed. Manufacturing processes require that metal parts must be cleaned in preparation for surface treatments or the completion of metal processing. The significant amount of cleansing agents utilized in cleaning processes has become an issue in Japan. Almost all cleaning processes in Japan are carried out by small- and medium-sized enterprises (SMEs). Machinery processes have not been systematically analyzed in terms of chemical risks and, in addition, the environmental management skills of SMEs are generally far behind those of large enterprises. The objective of this study is to reveal the relationships between chemical risks and plant-specific conditions for a practical risk reduction carried out by industrial decision makers. For this purpose, we aimed at the analysis of such relationships in metal-cleaning processes. Through this analysis, the correlation between local risks and global impacts were discussed in terms of plant-specific conditions.     

Genetic networks regulated by ASYMMETRIC LEAVES1 (AS1) and AS2 in leaf development in Arabidopsis thaliana: KNOX genes control five morphological events

The asymmetric leaves 1 ( as1 ) and as2 mutants of Arabidopsis thaliana exhibit pleiotropic phenotypes. Expression of a number of genes, including three class-1 KNOTTED -like homeobox ( KNOX ) genes ( BP , KNAT2 and KNAT6 ) and ETTIN / ARF3 , is enhanced in these mutants. In the present study, we attempted to identify the phenotypic features of as1 and as2 mutants that were generated by ectopic expression of KNOX genes, using multiple loss-of-function mutations of KNOX genes as well as as1 and as2 . Our results revealed that the ectopic expression of class-1 KNOX genes resulted in reductions in the sizes of leaves, reductions in the size of sepals and petals, the formation of a less prominent midvein, the repression of adventitious root formation and late flowering. Our results also revealed that the reduction in leaf size and late flowering were caused by the repression, by KNOX genes, of a gibberellin (GA) pathway in as1 and as2 plants. The formation of a less prominent midvein and the repression of adventitious root formation were not, however, related to the GA pathway. The asymmetric formation of leaf lobes, the lower complexity of higher-ordered veins, and the elevated frequency of adventitious shoot formation on leaves of as1 and as2 plants were not rescued by multiple mutations in KNOX genes. These features must, therefore, be controlled by other genes in which expression is enhanced in the as1 and as2 mutants.     

Differential expression of ACC oxidase genes during low-pH-induced root hair formation in lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.) seedlings

Root hair formation is induced in lettuce seedlings when the seedlings are transferred from a liquid medium at pH 6.0 to one at pH 4.0. Auxin, ethylene, and light are also required for the induction of root hair formation. To investigate the mechanism by which ethylene production is regulated during root hair formation, we isolated three 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase genes (Ls-ACO1, 2, and 3) from lettuce, each of which exists as a single copy in the genome. Analysis of the deduced amino acid sequences of the three ACO proteins as well as a phylogenetic analysis revealed that Ls-ACO3 was the most divergent among the ACO family. Northern hybridization analyses revealed that the mRNA levels of Ls-ACO2, but not Ls-ACO1 and Ls-ACO3, increased in the primary root after the transfer to a pH 4.0 medium. Addition of ACC or indole-3-acetic acid (IAA) to the pH 6.0 medium induced root hair formation, and a concomitant accumulation of Ls-ACO2 mRNA was observed. In contrast, the mRNA levels of Ls-ACO1 and Ls-ACO3 were unaffected by either ACC or IAA treatment. Furthermore, white light irradiation of dark-grown seedlings following the transfer to pH 4.0 medium induced the accumulation of all three ACO mRNAs. However, accumulation of Ls-ACO2 mRNA was also observed in non-irradiated seedlings, suggesting that the expression of Ls-ACO2 was induced not by light but by low pH. These results suggest that among the differentially regulated ACO genes, Ls-ACO2 plays a key role in ethylene production during low-pH-induced root hair formation in lettuce.     

Axial dispositions and conformations of myosin crossbridges along thick filaments in relaxed and contracting states of vertebrate striated muscles by X-ray fiber diffraction

X-ray diffraction patterns from live vertebrate striated muscles were analyzed to elucidate the detailed structural models of the myosin crown arrangement and the axial disposition of two-headed myosin crossbridges along the thick filaments in the relaxed and contracting states. The modeling studies were based upon the previous notion that individual myosin filaments had a mixed structure with two regions, a "regular" and a "perturbed". In the relaxed state the distributions and sizes of the regular and perturbed regions on myosin filaments, each having its own axial periodicity for the arrangement of crossbridge crowns within the basic period, were similar to those reported previously. A new finding was that in the contracting state, this mixed structure was maintained but the length of each region, the periodicities of the crowns and the axial disposition of two heads of a crossbridge were altered. The perturbed regions of the crossbridge repeat shifted towards the Z-bands in the sarcomere without changing the lengths found in the relaxed state, but in which the intervals between three successive crowns within the basic period became closer to the regular 14.5-nm repeat in the contracting state. In high resolution modeling for a myosin head, the two heads of a crossbridge were axially tilted in opposite directions along the three-fold helical tracks of myosin filaments and their axial orientations were different from each other in perturbed and regular regions in both states. Under relaxing conditions, one head of a double-headed crossbridge pair appeared to be in close proximity to another head in a pair at the adjacent crown level in the axial direction in the regular region. In the perturbed region this contact between heads occurred only on the narrower inter-crown levels. During contraction, one head of a crossbridge oriented more perpendicular to the fiber axis and the partner head flared axially. Several factors that significantly influence the intensities of the myosin based-meridional reflections and their relative contributions are discussed.     

Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta

Human β-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of α- and β-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using α-1,6-mannosyltransferase-deficient (och1Δ) yeast as the host. Genes encoding the α- and β-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (αα) and HexB (ββ). A total of 57 mg of β-hexosaminidase isozymes, of which 13 mg was HexA (αβ), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the β-subunit. The purified HexA was treated with α-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ± 0.1 and 1.7 ± 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the β-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.     

Enrichment of longevity phenotype in mtDNA haplogroups D4b2b, D4a, and D5 in the Japanese population

We report new results from the re-analysis of 672 complete mitochondrial (mtDNA) genomes of unrelated Japanese individuals stratified into seven equal sized groups by the phenotypes: diabetic patients, diabetic patients with severe angiopathy, healthy non-obese young males, obese young males, patients with Alzheimer’s disease, patients with Parkinson’s disease and centenarians. Each phenotype had 96 samples over 27 known haplogroups: A, B4a, B4b, B4c, B*, B5, D*, F1, F2, M*, M7a, M7b, M8, M9, D4a, D4b1, D4b2, D4d, D4e, D4g, D4h, D5, G, Z, M*, N9a, and N9b. A t-test comparing the fraction of samples in a haplogroup to healthy young males showed a significant enrichment of haplogroups D4a, D5, and D4b2 in centenarians. The D4b2 enrichment was limited to a subgroup of 40 of 61 samples which had the synonymous mutation 9296C > T. We identified this cluster as a distinct haplogroup and labeled it as D4b2b. Using an exhaustive procedure, we constructed the complete list of “mutation patterns” for centenarians and showed that the most significant patterns were in D4a, D5, and D4b2b. We argue that if a selection for longevity appeared only once, it was probably an autosomal event which could be dated to after the appearance of the D mega-group but before the coalescent time of D4a, D5, and D4b2b. Using a simple procedure, we estimated that this event occurred 24.4 ± 0.9 kYBP. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Gabriela Alexe and Noriyuki Fuku are joint first authors.     

Rapid and onsite BOD sensing system using luminous bacterial cells-immobilized chip

A BOD monitoring system based on a bio-chip which immobilized luminous bacterium in micrometer-order holes were arrayed and fabricated by micro-machine techniques, was developed. The acrylic chip (3 cmx3 cm) comprises nine micro-holes (diameter: 700 microm or 1 mm, depth: 100 microm) arranged in a three by three array. Cells of the marine luminous bacterium, Photobacterium phosphoreum IFO 13896, which was grown at 15 degrees C for 15 h, were immobilized with 3% or 15% sodium alginate gel. BOD standard solutions or actual sample solution (approximately 10 microl) was fallen onto the cell-arrayed chip, and then the chip was incubated at 25 degrees C for 25 min. After incubation, bioluminescence from the each hole was gray-scaled and measured by a chemi-imager or newly developed onsite-type-monitoring system using a digital camera and a mobile-type personal computer. BOD values less than 16 ppm could detect by the chip, in particular, linear relationship at the concentrations between 0 and 16 ppm could be observed when luminous cells were immobilized with 3% sodium alginate gel. Steady bioluminescence was observed on the chip in the presence of BOD standard solution (GGA solution) which contained mineral elements. Furthermore, simultaneous detection of BOD values in various samples could be employed in the single chip. These results showed that the monitoring system with bio-chip could achieve high-through-put and onsite BOD detection. Our newly developed onsite-type BOD detection system which was used a digital camera and a (mobile) laptop computer was applied to measure and detect organic pollution due to biodegradable substances in wastewater treatment system. The same performance as the chemi-imager system was obtained for data of bioluminescence. The obtained BOD values showed a similar correlation with that of the conventional method for BOD determination (BOD5). These results suggested for successful achievement of high-though-put and onsite detection of BOD in practical.