A novel gene delivery system in plants with calcium alginate micro-beads

We have produced micrometer-sized calcium alginate beads referred to as "bio-beads" that encapsulate plasmid DNA molecules carrying a reporter gene. In order to evaluate the efficiency of the bio-beads in mediating genetic transfection, protoplasts isolated from cultured tobacco cells (BY-2) were transfected with bio-beads containing a plasmid that carries the modified green fluorescent protein gene CaMV35S-sGFP. With the bio-beads treatment, approximately ten-fold higher GFP expression was observed after 24 h incubation compared to that with the conventional method using a naked plasmid solution. Transfection was up to 0.22% efficient. These results indicate that bio-beads have a possibility for efficient transformation in plants.     

Deletion and insertion of a 192-residue peptide in the active-site domain of glycosyl hydrolase family-2 beta-galactosidases

The monomeric multimetal-binding beta-galactosidase of Saccharopolyspora rectivirgula (srbg), a glycosyl hydrolase family-2 enzyme, has a unique sequence consisting of 192 amino acid residues with no similarity to known proteins. This 192-residue sequence (termed the "iota [iota] sequence") appears to be inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme (lacZbg). To assess the effects of the t sequence at specific sites of beta-galactosidase on the catalytic functioning and molecular properties of beta-galactosidase, deletion or insertion mutants of beta-galactosidases were constructed, expressed in LacZ- E. coli strains, and characterized: srbgdelta in which the iota sequence was deleted from srbg, and lacZbgI, in which the 192-residue iota sequence was inserted into the corresponding position (between Asp591 and Phe592) in the active-site domain of lacZbg. srbgdelta was a catalytically inactive, dimeric protein which retained multimetal-binding characteristics, suggesting that the iota sequence is very important for maintaining the structure necessary for the catalytic functioning and the monomeric structure of srbg but is not responsible for the unique metal ion requirements of srbg. On the other hand, lacZbgI existed as a mixture of a monomer, a tetramer, and higher multimers. The monomeric species was inactive, whereas the tetramer and other multimers were catalytically active (V(max )K(m) value, 25% of that of lacZbg) and highly specific for beta-D-galactoside. The tetrameric lacZbgI was activated by Mg2+ and Mn2+ with lowered metal affinities, and the stoichiometry of metal binding was unchanged from that of lacZbg. These results, along with the published stereo structure of lacZbg, suggest that, in lacZbgI, the inserted 192-residue iota peptide could fold independently of the lacZbg domains into a "sub-domain," lying distant from the active site and subunit interfaces.     

Development of effective detection method for Coxiella burnetii in mayonnaise by real-time PCR and investigation of C. burnetii contamination in commercial mayonnaise in Tokyo

A PCR method for the effective detection of Coxiella burnetii in commercially available mayonnaise was developed. Sample preparations were isolated from 50 g portions of each mayonnaise product by four successive extraction steps in phosphate buffer with 2.0 M NaCl. These extracts were then centrifuged at 20,000 x g for 60 min. DNA was isolated from the solution containing the precipitate with a commercial kit, and amplified quantitatively using real-time PCR that targeted the com1 region of C. burnetii. The recoveries of C. burnetii from 2 kinds of commercial mayonnaise specimens, with a baseline control of 1 x 10(7) particles of the Nine Mile phase II strain, were 85.0 +/- 6.0% and 72.0 +/- 0.4%, respectively. The determination limit of this method was 500 C. burnetii particles per 50 g of mayonnaise. The DNA specimens isolated from 50 different commercial mayonnaise samples sold in Tokyo using this method were amplified using both nested PCR and real-time PCR. No contamination by C. burnetii was detected in any of the mayonnaise samples.     

Shear properties of metal-free wooden load-bearing walls using plywood jointed with a combination of adhesive tape and wood dowels

In this study, the potential of wood-only (metal-free) load-bearing walls was proposed and tested based on the idea of using the same type of material throughout wherever possible while improving basic properties such as strength, sound insulation, and heat insulation. Specifically, a technique was tested of combining pressure sensitive adhesive tape and wood dowels in place of nails for load-bearing structures with panel reinforcement represented by wood-frame construction. For this paper, its strength was evaluated by conducting in-plane shear tests, which revealed that the initial rigidity and maximum load of the proposed test specimens were greater than that of specimens using nails for jointing. However, the shear load factor, which is calculated based on some strength parameters and which forms the foundation of housing strength design, was slightly lower than that of the nail-jointed specimen. This was because there was a large decline in post-collapse resistance and a lower calculated absorbed energy caused by brittleness. The early detachment of panels was also revealed as a problem in the tests.     

Large-scale genome reorganization in Saccharomyces cerevisiae through combinatorial loss of mini-chromosomes

A highly efficient technique, termed PCR-mediated chromosome splitting (PCS), was used to create cells containing a variety of genomic constitutions in a haploid strain of Saccharomyces cerevisiae. Using PCS, we constructed two haploid strains, ZN92 and SH6484, that carry multiple mini-chromosomes. In strain ZN92, chromosomes IV and XI were split into 16 derivative chromosomes, seven of which had no known essential genes. Strain SH6484 was constructed to have 14 mini-chromosomes carrying only non-essential genes by splitting chromosomes I, II, III, VIII, XI, XIII, XIV, XV, and XVI. Both strains were cultured under defined nutrient conditions and analyzed for combinatorial loss of mini-chromosomes. During culture, cells with various combinations of mini-chromosomes arose, indicating that genomic reorganization could be achieved by splitting chromosomes to generate mini-chromosomes followed by their combinatorial loss. We found that although non-essential mini-chromosomes were lost in various combinations in ZN92, one mini-chromosome (18kb) that harbored 12 genes was not lost. This finding suggests that the loss of some combination of these 12 non-essential genes might result in synthetic lethality. We also found examples of genome-wide amplifications induced by mini-chromosome loss. In SH6484, the mitochondrial genome, as well as the copy number of genomic regions not contained in the mini-chromosomes, was specifically amplified. We conclude that PCS allows for genomic reorganization, in terms of both combinations of mini-chromosomes and gene dosage, and we suggest that PCS could be useful for the efficient production of desired compounds by generating yeast strains with optimized genomic constitutions.     

Temperature dependence for anammox bacteria enriched from freshwater sediments

The anoxic ammonium oxidation (anammox) process has been regarded as an attractive alternative process to treat wastewater containing high ammonium concentrations. By the implementation of anammox process at moderately low temperatures (<25°C), the anammox process will be applied to more various industrial wastewater treatments. In this study, we established enrichment cultures of anammox bacteria from freshwater sediments by using an up-flow column reactor equipped with porous polyester nonwoven fabric at moderately low temperatures. Their nitrogen conversion rates reached 0.07-0.26kg-N/m(3)/d. Phylogenetic analysis based on 16S rRNA gene from enrichment cultures revealed the presence of various anammox bacteria affiliated with unknown anammox bacteria as well as known anammox candidates, i.e., Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia fulgida, Candidatus Scalindua wagneri. Anammox bacterial populations were influenced by enrichment conditions, i.e., seed sediments and temperature.     

Tiliroside, a glycosidic flavonoid, inhibits carbohydrate digestion and glucose absorption in the gastrointestinal tract

Scope Recent studies have reported that tiliroside, a glycosidic flavonoid, possesses anti‐diabetic activities. In the present study, we investigated the effects of tiliroside on carbohydrate digestion and absorption in the gastrointestinal tract. Methods and results This study showed that tiliroside inhibits pancreatic α‐amylase (IC₅₀ = 0.28 mM) in vitro. Tiliroside was found as a noncompetitive inhibitor of α‐amylase with K_i values of 84.2 μM. In male ICR mice, the increase in postprandial plasma glucose levels was significantly suppressed in the tiliroside‐administered group. Tiliroside treatment also suppressed hyperinsulinemia after starch administration. Tiliroside administration inhibited the increase of plasma glucose levels in an oral glucose tolerance test, but not in an intraperitoneal glucose tolerance test. In human intestinal Caco‐2 cells, the addition of tiliroside caused a significant dose‐dependent inhibition of glucose uptake. The inhibitory effects of both sodium‐dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) inhibitors (phlorizin and phloretin, respectively) on glucose uptake were significantly inhibited in the presence of tiliroside, suggesting that tiliroside inhibited glucose uptake mediated by both SGLT1 and GLUT2. Conclusion These findings indicate that the anti‐diabetic effects of tiliroside are at least partially mediated through inhibitory effects on carbohydrate digestion and glucose uptake in the gastrointestinal tract.     

A method for the determination of acrylamide in a broad variety of processed foods by GC-MS using xanthydrol derivatization

A novel GC-MS method was developed for the determination of acrylamide, which is applicable to a variety of processed foods, including potato snacks, corn snacks, biscuits, instant noodles, coffee, soy sauces and miso (fermented soy bean paste). The method involves the derivatization of acrylamide with xanthydrol instead of a bromine compound. Isotopically labelled acrylamide (d?-acrylamide) was used as the internal standard. The aqueous extract from samples was purified using Sep-Pak? C?? and Sep-Pak? AC-2 columns. For amino acid-rich samples, such as miso or soy sauce, an Extrelut? column was used for purification or extraction. After reaction with xanthydrol, the resultant N-xanthyl acrylamide was determined by GC-MS. The method was validated for various food matrices and showed good linearity, precision and trueness. The limit of detection and limit of quantification ranged 0.5-5 and 5-20?μg?kg?1), respectively. The developed method was applied as an exploratory survey of acrylamide in Japanese foods and the method was shown to be applicable for all samples tested.     

The ability of gallate and pyrogallol moieties of catechins to inhibit P-glycoprotein function

The role of structure–activity relationships in the ability of catechins to inhibit P-glycoprotein (P-gp) function was investigated with respect to gallate and pyrogallol moieties. Experiments using octyl derivatives of gallic acid indicated that the gallate moiety required the catechol group and a neighboring carbonyl group to inhibit P-gp. On the other hand, the pyrogallol moiety appeared to require three hydroxyl groups to inhibit P-gp, according to comparisons between (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG). The difference in the number of hydroxyl groups that gallate or pyrogallol moieties required for P-gp inhibition, was due to the presence of a carbonyl group. The P-gp inhibition by catechins was restricted by their hydrophobicity. The pyrogallol moiety of EGC did not appear to inhibit P-gp because of its low hydrophobicity. The P-gp inhibitory activity of EGCG was speculated to be increased by the addition of long carbon chains to the C3″of gallate moieties.     

Elasto-plastic analysis of orthogonally stiffened plates with initial imperfections under uniaxial compression

In general, the stiffened plates consisting of steel plate elements are unavoidably accompanied by initial imperfections such as residual stresses and initial deflections, which have considerable effects on their ultimate strength. Therefore, it is needed for designing them to develop more rational method taking the ultimate strength influenced by initial imperfections into account rather than the conventional design method being on the basis of the linear elastic buckling theory.From this point of view, this study aims to evaluate rigorously the ultimate strength of orthogonally stiffened plate with initial imperfections under uniaxial in-plane compression. The elasto-plastic finite element method is applied to attain this purpose. By a happy combination of modal analytical technique and conventional finite element method, much reduction of the degree of freedom can be expected to be realized herewith. Some numerical calculations are performed by means of this rigorous method to examine the exactness of the analysis. Moreover, the numerical results are compared with the experimental ones.