Enzyme electrode formed by evaporative concentration and its performance characterization

A highly concentrated immobilized enzyme layer was formed on a small working electrode, and the behavior of the electrode as an amperometric sensor was examined. To this end, a super-hydrophobic layer was formed in an area other than the sensitive area by using polytetrafluoroethylene (PTFE) beads. A small droplet of an enzyme solution containing glucose oxidase (GOD) and bovine serum albumin (BSA) was placed on the sensitive area, concentrated by evaporation, and crosslinked with glutaraldehyde. With the same enzyme activity per unit area, the current density increased with smaller working electrodes. Also, the current density increased with higher enzyme loadings up to a limiting value. In addition, the linear range of the calibration plot was expanded to higher glucose concentrations. The enzyme electrode fabricated by the novel method was incorporated in a micro-flow channel. Compared with large enzyme electrodes with the same enzyme activity per unit area, smaller electrodes showed a significant increase in the current density and a decrease in the flow dependence. The conversion efficiency could be improved by narrowing the flow channel and increasing the number of electrodes, which was comparable with a large electrode placed in a shallow flow channel.     

Design, synthesis, and biological activity of folate receptor-targeted prodrugs of thiolate histone deacetylase inhibitors

Aiming to develop selective anticancer drugs, we designed and synthesized three disulfides bearing a folic acid moiety as candidate folate receptor (FR)-targeted prodrugs of thiolate histone deacetylase inhibitors. Among them, compound 1 displayed growth-inhibitory activity toward folate receptor-positive MCF-7 breast cancer cells. The activity of 1 was significantly reduced by free folic acid, suggesting that cellular uptake of 1 is mediated by FR.     

Molecular interactions of a soluble gibberellin receptor, GID1, with a rice DELLA protein, SLR1, and gibberellin

GIBBERELLIN INSENSITIVE DWARF1 (GID1) encodes a soluble gibberellin (GA) receptor that shares sequence similarity with a hormone-sensitive lipase (HSL). Previously, a yeast two-hybrid (Y2H) assay revealed that the GID1-GA complex directly interacts with SLENDER RICE1 (SLR1), a DELLA repressor protein in GA signaling. Here, we demonstrated, by pull-down and bimolecular fluorescence complementation (BiFC) experiments, that the GA-dependent GID1-SLR1 interaction also occurs in planta. GA(4) was found to have the highest affinity to GID1 in Y2H assays and is the most effective form of GA in planta. Domain analyses of SLR1 using Y2H, gel filtration, and BiFC methods revealed that the DELLA and TVHYNP domains of SLR1 are required for the GID1-SLR1 interaction. To identify the important regions of GID1 for GA and SLR1 interactions, we used many different mutant versions of GID1, such as the spontaneous mutant GID1s, N- and C-terminal truncated GID1s, and mutagenized GID1 proteins with conserved amino acids replaced with Ala. The amino acid residues important for SLR1 interaction completely overlapped the residues required for GA binding that were scattered throughout the GID1 molecule. When we plotted these residues on the GID1 structure predicted by analogy with HSL tertiary structure, many residues were located at regions corresponding to the substrate binding pocket and lid. Furthermore, the GA-GID1 interaction was stabilized by SLR1. Based on these observations, we proposed a molecular model for interaction between GA, GID1, and SLR1.     

The GID1-mediated gibberellin perception mechanism is conserved in the Lycophyte Selaginella moellendorffii but not in the Bryophyte Physcomitrella patens

In rice (Oryza sativa) and Arabidopsis thaliana, gibberellin (GA) signaling is mediated by GIBBERELLIN-INSENSITIVE DWARF1 (GID1) and DELLA proteins in collaboration with a GA-specific F-box protein. To explore when plants evolved the ability to perceive GA by the GID1/DELLA pathway, we examined these GA signaling components in the lycophyte Selaginella moellendorffii and the bryophyte Physcomitrella patens. An in silico search identified several homologs of GID1, DELLA, and GID2, a GA-specific F-box protein in rice, in both species. Sm GID1a and Sm GID1b, GID1 proteins from S. moellendorffii, showed GA binding activity in vitro and interacted with DELLA proteins from S. moellendorffii in a GA-dependent manner in yeast. Introduction of constitutively expressed Sm GID1a, Sm G1D1b, and Sm GID2a transgenes rescued the dwarf phenotype of rice gid1 and gid2 mutants. Furthermore, treatment with GA(4), a major GA in S. moellendorffii, caused downregulation of Sm GID1b, Sm GA20 oxidase, and Sm GA3 oxidase and degradation of the Sm DELLA1 protein. These results demonstrate that the homologs of GID1, DELLA, and GID2 work in a similar manner in S. moellendorffii and in flowering plants. Biochemical studies revealed that Sm GID1s have different GA binding properties from GID1s in flowering plants. No evidence was found for the functional conservation of these genes in P. patens, indicating that GID1/DELLA-mediated GA signaling, if present, differs from that in vascular plants. Our results suggest that GID1/DELLA-mediated GA signaling appeared after the divergence of vascular plants from the moss lineage.     

Phylogeographic structure and late Quaternary population history of the Japanese oak Quercus mongolica var. crispula and related species revealed by chloroplast DNA variation

Generally, oaks dominate the broadleaf deciduous forests in Japan. The genetic variation in 6 cpDNA regions (trnT-trnL, trnL-trnF, atpB-rbcL, and trnH-psbA speacers, trnL intron, and matK gene) with regard to the Japanese oak (Quercus mongolica var. crispula) and 3 related species in the section Prinus (Q. serrata, Q. dentata and Q. aliena) was investigated in 598 trees belonging to 44 populations distributed throughout the Japanese archipelago. Additional samples were collected from Korea, China, and Russia (Sakhalin). Thirteen haplotypes (I to XIII) were identified on the bases of 15 nucleotide substitutions and 3 indels. Haplotypes I and II were discovered in northeastern Japan, whereas haplotypes III to IX were distributed in southwestern Japan. The boundary distinguishing these 2 groups was located in central Japan coincident with the Itoigawa-Shzuoka tectonic line. Haplotype I was also found in Sakhalin, whereas haplotypes VI, VII, VIII, X, XI, XII, and XIII were found in Korea and China. Four oak species in the same location shared identical haplotypes, suggesting cpDNA introgression by occasional hybridization. Both the values of total haplotype diversity (HT) and haplotype diversity within populations (HS) in Q. mongolica var. crispula were higher in the southwestern populations than in the northeastern populations. A haplotype network indicated that haplotype VI is the ancestral haplotype. The presence of identical haplotypes in Korea, China, and Japan suggested that the haplotypes diversified on the Eurasian continent before the last glacial period. The difference in genetic structure between the northeastern and southwestern regions indicates a difference in the history of migration and recolonization in Japan during the last glacial period.     

Degradation of arylarsenic compounds by microorganisms

Microorganisms were not directly accumulated when soil contaminated to about 0.5 mM with diphenylarsinic acid (DPAA) was used as the sole source of carbon. However, using toluene as the carbon source yielded several isolates, which were then used in cultivation with DPAA as the sole source of carbon. By these methods, Kytococcus sedentarius strain NK0508, which can grow in up to 0.038 mM DPAA, was isolated. The toxicity of DPAA retarded the growth of K. sedentarius and the direct accumulation of DPAA-utilizing microorganisms from environmental samples. This strain can utilize about 80% of DPAA and phenylarsonic acid as the sole source of carbon for 3 days. Degradation products of DPAA were determined to be cis, cis, muconate and arsenic acid. When K. sedentarius was cultivated with methylphenylarsinic acid and diphenylmethylarsine, about 90% and 10% degradation of the two compounds, respectively, were observed. Diphenylmethylarsine oxide, possibly synthesized by methylation of DPAA, was detected as one of the transformation products. These results suggest that degradation is initiated by splitting of the phenyl groups from the arylarsenic compounds with subsequent hydroxylation of the phenyl groups and ring opening to yield cis, cis, muconate.     

Evidence that PI3K, Rac, Rho, and Rho kinase are involved in basic fibroblast growth factor-stimulated fibroblast-Collagen matrix contraction

Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated floating collagen matrix contraction is independent of Rho kinase while platelet-derived growth factor (PDGF)-stimulated contraction is Rho kinase-dependent. The current studies were carried out to determine the signaling mechanisms of basic fibroblast growth factor (bFGF)-stimulated fibroblast-collagen matrix contraction. Both bFGF and LPA promoted equally collagen matrix contraction well. Three different inhibitors, LY294002 for phosphatidylinositol-3-kinase (PI3K), C3 exotransferase for Rho and Y27632 for Rho kinase, suppressed the bFGF-stimulated fibroblast-collagen matrix contraction. With bFGF stimulation, fibroblasts spread with prominent stress fiber network formation and focal adhesions. In the presence of Rho kinase inhibitor, focal adhesions and stress fibers were mostly lost. We demonstrated that bFGF stimulation for fibroblast caused transient Rac and Rho activation but did not activate Cdc42. In addition, bFGF enhanced fibroblast migration in wound healing assay. The present study implicates PI3K, Rac, Rho, and Rho kinase as being involved in bFGF-stimulated collagen matrix contraction. The elucidation of bFGF-triggered signal transduction may be an important clue to understand the roles of bFGF in wound healing.     

Allelic diversity in the merozoite surface protein 1 gene of Plasmodium falciparum on Palawan Island, the Philippines

Allelic diversity of the Plasmodium falciparum merozoite surface protein 1 gene (msp1) is mainly generated by meiotic recombination at the mosquito stage. We investigated recombination-based allelic diversity of msp1 in a P. falciparum population from Palawan Island, the Philippines, where malaria transmission is moderate. We identified the 5' recombinant types, 3' sequence types and msp1 haplotypes (unique combinations of 5' recombinant type and 3' sequence type), and compared them with those of P. falciparum from the Solomon Islands, where malaria transmission is high. The mean number of 5' recombinant types per patient in Palawan was 1.44, which is comparable to the number for the Solomon Islands (1.41). The Palawan parasite population had 15 msp1 haplotypes, whereas the Solomon Islands population had only 8 haplotypes. The Palawan population showed strong linkage disequilibrium between polymorphic blocks/sites within msp1, which is comparable to the results for the Solomon Islands. These findings support our hypothesis that the extent of allelic diversity of msp1 is determined not only by the transmission intensity but also by the number of msp1 alleles prevalent in the local parasite population and the extent of mixed-allele infections. Contribution of a high prevalence of the chloroquine (CQ)-sensitive allele of P. falciparum CQ resistance transporter (pfcrt) to the relatively high msp1 diversity in the Palawan population is discussed.