ゼニゴケ葉緑体遺伝子と相同性を持つラン藻 Synechocystis PCC6803株のORF326、frxC およびORF469を標的にした変異の導入

ORF326, frxC and ORF469 of a transfomable cynobacterium, Synechcystis PCC6803, have sequence similarity with ORF465 on the choroplast genome of a livewort, Marchantia polymorpha, respectively. To elucidate their functions,targeted mutagenesis was performed by transformation with clened DNA in which the ORF was disrupted by insertion of a kanamycin resistancen gene cassette.Streak-purifications of a single colony of each transformant were repeatde to segregate homozygous mutants for disrupted copies, because Synechocystis PCC6803 was reported to have approximately 10 chromosomal DNA copies. Southern blot analysis revealed that mutants for ORF326 had not only disrupted ORF326 copies but also wild type ORF326 copies. This suggests that ORF326 is indispensable for growth under the mixotrophic growth condition used. However, mutants for frxC and mutants for ORF469 had only mutated copies, indicating that they dispensable for growth. Growth and chlorophyll a content of an ORF469-disrupted mutant were compared and chlorophyll a content of an ORF469-disrupted mutant were compared to those of wild type under mixotrophic growth condition, but no significant difference was detected. This indicates that ORF469 is required for neither normal growth nor chlorophyll biosynthesis under thie condition.ゼニゴケ葉緑体ORF316、frxCおよびORFと相同性を持つ、形質転換型ラン藻Synechocystis PCC6803株のORF326、frxCおよびORF469の機能についての情報を得るため、これらの欠損株の作製を行った。コード領域にカナマイシン耐性遺伝子カセットを挿入することにより不活性化された変異型のORF（オーブンリーディングフレーム）を持つプラスミドを用いてSynechocystis PCC6803株の形質転換を行い、カナマイシンを含む培地で選抜した。Synechocystis PCC6803株は約10コピーのクロモソームを持つが、サザンブロット解析の結果、ORF326については、変異型と野生型のORF326の双方を持つ株しか得られず、増殖に必要と推測された。一方frxCおよびORF469については、ともに全て変異型に置き換わった株が得られ、増殖には必要ないことが示された。さらにORF469欠損株を光照射下で培養し、増殖速度とクロロフィルa濃度を測定したが、いずれも野生株とほぼ同じであり、ORF469は、この条件下では、増殖やクロロフィル生合成に必要ないと推察された

Improved Adsorption of an Enterococcus faecalis Bacteriophage ΦEF24C with a Spontaneous Point Mutation

Some bacterial strains of the multidrug-resistant Gram-positive bacteria Enterococcus faecalis can significantly reduce the efficacy of conventional antimicrobial chemotherapy. Thus, the introduction of bacteriophage (phage) therapy is expected, where a phage is used as a bioagent to destroy bacteria. E. faecalis phage ΦEF24C is known to be a good candidate for a therapeutic phage against E. faecalis. However, this therapeutic phage still produces nonuniform antimicrobial effects with different bacterial strains of the same species and this might prove detrimental to its therapeutic effects. One solution to this problem is the preparation of mutant phages with higher activity, based on a scientific rationale. This study isolated and analyzed a spontaneous mutant phage, ΦEF24C-P2, which exhibited higher infectivity against various bacterial strains when compared with phage ΦEF24C. First, the improved bactericidal effects of phage ΦEF24C-P2 were attributable to its increased adsorption rate. Moreover, genomic sequence scanning revealed that phage ΦEF24C-P2 had a point mutation in orf31. Proteomic analysis showed that ORF31 (mw, 203 kDa) was present in structural components, and immunological analysis using rabbit-derived antibodies showed that it was a component of a long, flexible fine tail fiber extending from the tail end. Finally, phage ΦEF24C-P2 also showed higher bactericidal activity in human blood compared with phage ΦEF24C using the in vitro assay system. In conclusion, the therapeutic effects of phage ΦEF24C-P2 were improved by a point mutation in gene orf31, which encoded a tail fiber component

Molecular Breeding of Sweetpotato Carotenoids

Sweetpotato [sweet potato; Ipomoea batatas (L.) Lam.] is the seventh most valued food crop of the world. It has an inherent ability to grow under diverse agro-ecological and microclimatic zones ranging from tropical and subtropical zones to temperate areas with its tuberous roots enriched with the secondary metabolites of immense nutritional value. Among these, carotenoids are the most conspicuous one for having their use in nutritional, pharmaceutical, food, feed, aquaculture, and cosmetic industries. In food industries, carotenoids are used as food additives being antioxidants with attractive colors. Despite the immense economic importance, sweetpotato has received lesser attention in terms of its breeding with improved varieties. The conventional method of breeding by crossing has not been much successful due to the complexity of genome sterility and cross-incompatibility. Hence, the modern molecular breeding approaches, e.g. genetic, genomic, and metabolic (pathway) engineering, have been applied to this crop by some of researchers in Japan, Korea, and China to generate various cultivars with improved quantities and qualities of carotenoids. This has also opened a new gate for molecular breeders to engineer new sweetpotato cultivars enriched with carotenoids under current global scenario of dramatically rising climatic changes where novel food resources are bitterly needed, especially under alarmingly growing world population, the majority of which suffers from malnutrition

Cleavage of Hsp70.1 causes lysosomal cell death under stress conditions

Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Heat shock protein 70.1 (Hsp70.1) exhibits dual functions as a chaperone protein and a lysosomal membrane stabilizer. Since chaperone-mediated autophagy participates in the recycling of ∼30% cytosolic proteins, its disorder causes cell susceptibility to stress conditions. Cargo proteins destined for degradation such as amyloid precursor protein and tau protein are trafficked by Hsp70.1 from the cytosol into lysosomes. Hsp70.1 is composed of an N-terminal nucleotide-binding domain (NBD) and a C-terminal domain that binds to cargo proteins, termed the substrate-binding domain (SBD). The NBD and SBD are connected by the interdomain linker LL1, which modulates the allosteric structure of Hsp70.1 in response to ADP/ATP binding. After the passage of the Hsp70.1–cargo complex through the lysosomal limiting membrane, high-affinity binding of the positive-charged SBD with negative-charged bis(monoacylglycero)phosphate (BMP) at the internal vesicular membranes activates acid sphingomyelinase to generate ceramide for stabilizing lysosomal membranes. As the integrity of the lysosomal limiting membrane is critical to ensure cargo protein degradation within the acidic lumen, the disintegration of the lysosomal limiting membrane is lethal to cells. After the intake of high-fat diets, however, β-oxidation of fatty acids in the mitochondria generates reactive oxygen species, which enhance the oxidation of membrane linoleic acids to produce 4-hydroxy-2-nonenal (4-HNE). In addition, 4-HNE is produced during the heating of linoleic acid-rich vegetable oils and incorporated into the body via deep-fried foods. This endogenous and exogenous 4-HNE synergically causes an increase in its serum and organ levels to induce carbonylation of Hsp70.1 at Arg469, which facilitates its conformational change and access of activated μ-calpain to LL1. Therefore, the cleavage of Hsp70.1 occurs prior to its influx into the lysosomal lumen, which leads to lysosomal membrane permeabilization/rupture. The resultant leakage of cathepsins is responsible for lysosomal cell death, which would be one of the causative factors of lifestyle-related diseases

〔資 料〕 閉経後女性の体脂肪蓄積，筋肉量および骨密度低下の抑制を 目的とした食事条件を提案するための基礎研究 ―自発運動可能な卵巣摘除メスラットの下肢骨格筋重量，大腿骨骨密度および 走行運動レベルに対する食餌アミノ酸添加の影響―

The present study aims to review whether dietary modifications will prevent body fat accumulation, muscle mass wasting and bone mineral density loss in postmenopausal women who incorporate physical exercise into daily life.9-week-old ovariectomized Wistar strain female rats were divided into four groups: CA-Ex, Gln-Ex, Leu-Ex and CitD-Ex. Depending on which group they were in, the rats were given a 20％ casein protein based experimental diet supplemented with nothing （control diet, CA-Ex）; 5％ of L-glutamine （Gln-Ex）; 5％ of L-leucine （Leu-Ex）; or 2.5％ of L-citrulline＋2.5％ of D-serine （CitD-Ex）, each 11 g per day for 10 weeks. All of these rats were housed in individual cage with a running wheel for 10 weeks. A sham operation was carried out on another group of rats （Sham） and were given the same diet as CA-Ex, the control diet.Results were as follows:1） Uterus weights of the ovariectomized rats, that is group CA-Ex, Gln-Ex, Leu-Ex and CitD-Ex, appeared to show lower values than that of the group Sham. 2） No significant differences were observed in a）changes in body weight, b）blood analyses, c）liver, kidney, uterus and muscle weights, and d）femoral-bone mineral density in groups Gln-Ex, Leu-Ex, and CitD-Ex as compared to the group CA-Ex.3） Relatively higher running activity was observed in group CitD-Ex than that of group CA-Ex. This observation suggests that increasing dietary L-citrulline plus D-serine in postmenopausal rats may lead to an increase in physical activity. Further research is needed to understand the physiological and nutritional significance of the unexpected results that dietary amino acid may accelerate the physical activity

ゼニゴケミトコンドリアのリボソームタンパク質およびNADH脱水素酵素サブユニット遺伝子の構造と発現

本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである京都大学0048新制・課程博士博士(農学)甲第6068号農博第838号新制||農||697(附属図書館)学位論文||H7||N2791(農学部図書室)UT51-95-D387京都大学大学院農学研究科農芸化学専攻(主査)教授 大山 莞爾, 教授 駒野 徹, 教授 山田 康之学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDFA