Identification of Key Uric Acid Synthesis Pathway in a Unique Mutant Silkworm Bombyx mori Model of Parkinson’s Disease

Plasma uric acid (UA) levels decrease following clinical progression and stage development of Parkinson’s disease (PD). However, the molecular mechanisms underlying decreases in plasma UA levels remain unclear, and the potential to apply mutagenesis to a PD model has not previously been discovered. We identified a unique mutant of the silkworm Bombyx mori (B.mori) op. Initially, we investigated the causality of the phenotypic “op” by microarray analysis using our constructed KAIKO functional annotation pipeline. Consequently, we found a novel UA synthesis-modulating pathway, from DJ-1 to xanthine oxidase, and established methods for large-scale analysis of gene expression in B. mori. We found that the mRNA levels of genes in this pathway were significantly lower in B. mori op mutants, indicating that downstream events in the signal transduction cascade might be prevented. Additionally, levels of B.mori tyrosine hydroxylase (TH) and DJ-1 mRNA were significantly lower in the brain of B. mori op mutants. UA content was significantly lower in the B. mori op mutant tissues and hemolymph. The possibility that the B. mori op mutant might be due to loss of DJ-1 function was supported by the observed vulnerability to oxidative stress. These results suggest that UA synthesis, transport, elimination and accumulation are decreased by environmental oxidative stress in the B. mori op mutant. In the case of B. mori op mutants, the relatively low availability of UA appears to be due both to the oxidation of DJ-1 and to its expenditure to mitigate the effects of environmental oxidative stress. Our findings are expected to provide information needed to elucidate the molecular mechanism of decreased plasma UA levels in the clinical stage progression of PD.     

CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor

CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy.     

Synthesis and biological evaluation of novel tryptoline derivatives as indoleamine 2,3-dioxygenase (IDO) inhibitors

Indoleamine 2,3-dioxygenase (IDO) plays a significant role in several disorders such as Alzheimer’s disease, age-related cataracts and tumors. A series of novel tryptoline derivatives were synthesized and evaluated for their inhibitory activity against IDO. Substituted tryptoline derivatives (11a, 11c, 11e, 12b and 12c) were demonstrated to be more potent than known inhibitor MTH-Trp. Suzuki–Miyaura cross-coupling reaction of 11a–d with phenylboronic acid proceeded in high yields. In most cases, C5 and C6 substitutions on the corresponding indole ring were well tolerated. The tryptoline derivative 11c is a promising chemical lead for the discovery of novel IDO inhibitors.     

Volatile Flavor Components of Dried Bonito (Katsuobushi) II. From Neutral Fraction

The aqueous extract of dried bonito (Katsuobushi) was distilled under reduced pressure. The resulting distillate with diethyl ether and the extract was separated into acidic, phenolic, basic and neutral fractions. The neutral fraction was further fractionated into ten sub-fractions by silica gel column chromatography. All these sub-fractions were analyzed by gas chromatography and gas chromatography-mass spectrometry.

One hundred and sixty-five compounds were identified and 12 compounds were tentatively identified from the neutral fraction. Among them, 111 compounds were newly identified as flavor components of Katsuobushi.     

Exercise ameliorates high-fat diet-induced impairment of differentiation of adipose-derived stem cells into neuron-like cells in rats

Adipose-derived stem cells (ADSCs) can differentiate into neurons under particular conditions. It remains largely unknown whether this differentiation potential is affected by physical conditions such as obesity, which modulates the functions of adipose tissue. In this study, we determined the impact of either a 9-week high-fat diet (60% fat; HFD) or 9-week exercise training on the differentiation potential of ADSCs into neuron-like cells in male Wistar rats. Rats were randomly assigned to a normal diet-fed (ND-SED) group, HFD-fed (HFD-SED) group, or exercise-trained HFD-fed group (HFD-EX). After a 9-week intervention, ADSCs from all groups differentiated into neuron-like cells. Expression of neuronal marker proteins (nestin, βIII-tubulin, and microtubule-associated protein 2 [MAP2]) and the average length of cell neurites were lower in cells from HFD-SED rats than in other groups. Instead, protein expression of COX IV and Cyt-c, the Bax/Bcl-2 and LC3-II/I ratio, and the malondialdehyde level in culture medium were higher in cells from HFD-SED rats. No significant difference between ND-SED and HFD-EX rats was observed, except for the average length of cell neurites in MAP2. Thus, HFD impaired the differentiation potential of ADSCs into neuron-like cells, which was accompanied by increases in apoptotic activity and oxidative stress. Importantly, exercise training ameliorated the HFD-induced impairment of neurogenesis in ADSCs. The adipose tissue microenvironment could influence the differentiation potential of ADSCs, a source of autologous stem cell therapy.     

Map-based cloning of a fertility restorer gene, Rf-1, in rice (Oryza sativa L.)

A rice nuclear gene, Rf-1, restores the pollen fertility disturbed by the BT-type male sterile cytoplasm, and is widely used for commercial seed production of japonica hybrid varieties. Genomic fragments carrying Rf-1 were identified by conducting chromosome walking and a series of complementation tests. Isolation and analysis of cDNA clones corresponding to the fragments demonstrated that Rf-1 encodes a mitochondrially targeted protein containing 16 repeats of the 35-aa pentatricopeptide repeat (PPR) motif. Sequence analysis revealed that the recessive allele, rf-1, lacks one nucleotide in the putative coding region, presumably resulting in encoding a truncated protein because of a frame shift. Rice Rf-1 is the first restorer gene isolated from cereal crops that has the property of reducing the expression of the cytoplasmic male sterility (CMS)-associated mitochondrial gene like many other restorer genes. The present findings may facilitate not only elucidating the mechanisms of male sterility by the BT cytoplasm and its restoration by Rf-1 but also isolating other restorer genes from cereal crops, especially rice.     

Transient exposure to ethylene stimulates cell division and alters the fate and polarity of hypocotyl epidermal cells

After transient exposure to the gaseous hormone ethylene, dark-grown cucumber (Cucumis sativus) hypocotyls developed unusual features. Upon ethylene's removal, the developing epidermis showed significant increases in cell division rates, producing an abundance of guard cells and trichomes. These responses to ethylene depended on the stage of development at the time of ethylene exposure. In the upper region of the hypocotyl, where cells were least differentiated at the onset of ethylene treatment, complex, multicellular protuberances formed. Further down the hypocotyl, where stomata and trichomes were beginning to develop at the onset of ethylene exposure, an increase in the number of stomata and trichomes was observed. Stomatal complexes developing after the ethylene treatment had a significant increase in the number of stomatal subsidiary cells and the number of cells per trichome increased. Analysis of division patterns in stomatal complexes indicated that exposure to ethylene either suspended or altered cell fate. Ethylene also altered cell division polarity, resulting in aberrant stomatal complexes and branched trichomes. To our knowledge, the results of this study demonstrate for the first time that transient treatment with physiological concentrations of ethylene can alter cell fate and increase the propensity of cells to divide.     

Structural consequences of target epitope-directed functional alteration of an antibody. The case of anti-hen lysozyme antibody,HyHEL-10

Decreased affinity of an antibody for a mutated epitope in an antigen can be enhanced and reversed by mutations in certain antibody residues. Here we describe the crystal structures of (a) the complex between a naturally mutated proteinaceous antigen and an antibody that was mutated and selected in vitro, and (b) the complex between the normal antigen and the mutated antibody. The mutated and selected antibody recognizes essentially the same epitope as in the wild-type antibody, indicating successful target site-directed functional alteration of the antibody. In comparing the structure of the mutated antigen-mutant antibody complex with the previously established structure of the wild-type antigen-wild-type antibody complex, we found that the enhanced affinity of the mutated antibody for the mutant antigen originated not from improvements in local complementarity around the mutated sites but from subtle and critical structural changes in nonmutated sites, including an increase in variable domain interactions. Our findings indicate that only a few mutations in the antigen-binding region of an antibody can lead to some structural changes in its paratopes, emphasizing the critical roles of the plasticity of loops in the complementarity-determining region and also the importance of the plasticity of the interaction between the variable regions of immunoglobulin heavy and light chains in determining the specificity of an antibody.