逆遺伝学および比較トランスクリプトーム解析を用いたヒラタケリグニン分解不全変異株の特性評価

京都大学0048新制・課程博士博士(農学)甲第22854号農博第2437号新制||農||1082(附属図書館)学位論文||R2||N5314(農学部図書室)京都大学大学院農学研究科地域環境科学専攻(主査)教授 本田 与一, 教授 田中 千尋, 准教授 坂本 正弘学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDGA

The Cytoplasmic N,n\u27-Diacetylchitobiase Gene From Vibrio Parahaemolyticus: Sequence Analysis, Protein Secretion, and Secretion System Development.

The nucleotide sequence of the gene encoding the cytoplasmic N,N\sp\prime-diacetylchitobiase from Vibrio parahaemolyticus has been determined. The deduced peptide sequence surprisingly has minimum evolutionary relationship to two other reported N,N\sp\prime-diacetylchitobiases from vibrios, except for highly conserved regions which are also homologous with lysosomal beta-hexosaminidases from eukaryotes including humans. In contrast, the other two sequenced chitobiases from vibrios are much more closely related to each other. This 85 kDa cytoplasmic protein, as revealed from the sequence, appears to be a unique protein, lacking a signal sequence and genetically distant from other known enzymes of similar function. This is consistent with its limited substrate specificity to small N-acetylglucosamine terminated oligosaccharides. The signal peptide of an extracellular endochitinase from V. parahaemolyticus causes the mature chitinase to be efficiently secreted through the double membranes of both Gram negative V. parahaemolyticus, and of Escherichia coli JM101 when the gene is cloned therein. By using recombinant PCR, this signal sequence was fused in frame to the chitobiase coding sequence, and active chitobiase was found in the E. coli culture medium. Two secretion vectors were developed during this study for the secretion of wild type proteins. PCR fragment of a structural gene can be inserted in frame with the signal sequence, which is under the strong trc promoter. The secretion system has been tested using the cytoplasmic chitobiase gene as a model system. Restriction sites and complete DNA sequence are not required for the structural gene to be cloned. The chitobiase and the original chitinase as cloned for secretion were stabilized by EDTA added to the medium. This observation may prove generally useful for protecting cloned and secreted proteins in E. coli

Issues and Countermeasures of Enterprise Compliance Management in China

Compliance management is an effective method to reduce compliance risks of organizations by requiring the organizations to meet the applicable laws and regulations, industry standards, organization standards, group standards, contract, effective governance principles and ethics. The enterprise compliance management can improve the chances of commercial success of the enterprise. This paper discovered the existing issues in the compliance management in China and proposed the countermeasures to deal with these problems, which is based on the survey of existing situation of Chinese enterprise compliance management

Ultrasound-targeted microbubble destruction enhances AAV mediated gene transfection: human RPE cells in vitro and the rat retina in vivo

The present study was performed to investigate the efficacy and safety of Ultrasound-targeted microbubble destruction (UTMD) mediated rAAV2-EGFP to cultured human retinal pigment epithelium (RPE) cells _in vitro_ and the rat retina _in vivo_. _In vitro_ study, cultured human RPE cells were exposed to US under different conditions with or without microbubbles. Furthermore, the effect of UTMD to rAAV2-EGFP itself and the cells were evaluated. _In vivo_ study, gene transfer was examined by injecting rAAV2-EGFP into the subretinal space of the rats with or without microbubbles and then exposed to US. We investigated EGFP expression _in vivo_ via stereomicroscopy and performed quantitative analysis by Axiovision 3.1 software. HE staining and frozen sections were used to observe tissue damage and location of EGFP gene expression. _In vitro_ study, the transfection efficiency of rAAV2-EGFP increased 74.85% under the optimal UTMD conditions. Furthermore, there was almost no cytotoxicity to the cells and rAAV2-EGFP itself. _In vivo_ study, UTMD could be used safely to enhance and accelerate transgene expression of the retina. Fluorescence expression was mainly located in the layer of retina. UTMD is a promising method for gene delivery to the retina

Extremal properties of the first eigenvalue and the fundamental gap of a sub-elliptic operator

We consider the problems of extreming the first eigenvalue and the fundamental gap of a sub-elliptic operator with Dirichlet boundary condition, when the potential $V$ is subjected to a $p$-norm constraint. The existence results for weak solutions, compact embedding theorem and spectral theory for sub-elliptic equation are given. Moreover, we provide the specific characteristics of the corresponding optimal potential function

Glutaredoxin 2 Prevents H2O2-Induced Cell Apoptosis by Protecting Complex I Activity in the Mitochondria

Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200 μM hydrogen peroxide (H2O2) for 24 h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) could protect cells against H2O2-induced damage while Grx2 knockdown (KD) showed the opposite effect. Under the same conditions, H2O2 treatment caused 50% inactivation of complex I activity in control cells (vector only), 75% in Grx2 KD cells but only 20% in Grx2 OE cells. This antiapoptotic function of Grx2 is specific as rotenone, a complex I specific inhibitor, could block this Grx2-mediated protection of complex I activity. Immunoprecipitation study also revealed that Grx2 co-precipitated with complex I in the mitochondrial lysate. Thus, the mechanism of Grx2 protection against H2O2- induced apoptosis is likely associated with its ability to preserve complex I

The Important Functions of GSH-Dependent Enzyme Glutaredoxin 2 (Grx2)

Reactive oxygen species (ROS) are generated at a very high rate throughout our lives as part of normal aerobic life. Glutathione (GSH), normally an antioxidant molecule that scavenges free radicals, oxidizes to form glutathione mixed disulfide (GSSG). As the GSSG/GSH ratio increases, GSSG naturally adds to other proteins, causing protein glutathionylation. Protein glutathionylation, defined as the reversible formation of a mixed disulfide (PSSG) between protein thiols (P-SH) and glutathione (GSH), appears to be the most important mode of thiol oxidation. In my chapter, we will discuss the important roles of GSH and GSH-dependent enzymes in health and disease, with the emphasis on glutaredoxin and thioredoxin systems. Their structures, catalytic reaction mechanisms, major physiological functions, and associations with diseases will be summarized in my chapter. We will also mention how GSH-dependent enzymes play a role in each major organ systems including the nervous, cardiovascular, immune, and visual system

Some controllability results of a class of N-dimensional parabolic equations with internal single-point degeneracy

This paper investigates the controllability of a class of $N$-dimensional degenerate parabolic equations with interior single-point degeneracy. We employ the Galerkin method to prove the existence of solutions for the equations. The analysis is then divided into two cases based on whether the degenerate point $x=0$ lies within the control region $\omega_0$ or not. For each case, we establish specific Carleman estimates. As a result, we achieve null controllability in the first case $0\in\omega_0$ and unique continuation and approximate controllability in the second case $0\notin\omega_0$