シロイヌナズナのヒスチジン生合成経路に関する研究

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・論文博士博士(農学)乙第10046号論農博第2216号新制||農||774(附属図書館)学位論文||H11||N3232(農学部図書室)UT51-99-D250(主査)教授 關谷 次郎, 教授 天知 輝夫, 教授 佐藤 文彦学位規則第4条第2項該当Doctor of Agricultural ScienceKyoto UniversityDFA

Isolation and characterization of mutations affecting expression of the Δ9- fatty acid desaturase gene, OLE1, in Saccharomyces cerevisiae

AbstractExpression of the Δ9- fatty acid desaturase gene, OLE1, of Saccharomyces cerevisiae is negatively regulated transcriptionally and post-transcriptionally by unsaturated fatty acids. In order to isolate mutants exhibiting irregulation of OLE1 expression, we constructed an OLE1p–PHO5 fusion gene as a reporter consisting of the PHO5 gene encoding repressible acid phosphatase (rAPase) under the control of the OLE1 promoter (OLE1p). By EMS mutagenesis, we isolated three classes of mutants, pfo1, pfo2 and pfo3 (positive regulatory factor for OLE1) mutants, which show decreased rAPase activity under derepression conditions (absence of oleic acid). Analysis of the transcription of OLE1 in these pfo mutants revealed that pfo1 and pfo3 mutants have a defect in the regulation of OLE1 expression at the transcriptional level while pfo2 mutants were suggested to have a mutation affecting OLE1 expression at a post-transcriptional step. In addition, four other classes of mutants, nfo1, nfo2, nfo3 and nfo4 (negative factor for OLE1) mutants that have mutations causing strong expression of the OLE1p–PHO5 fusion gene under repression conditions (presence of oleic acid), were isolated. Results of Northern analysis of OLE1 as well as OLE1p-PHO5 transcripts in nfo mutants suggested that these mutations occurred in genes encoding global repressors. We also demonstrated that TUP1 and SSN6 gene products are required for full repression of OLE1 gene expression, by showing that either tup1 or ssn6 mutations greatly increase the level of the OLE1 transcript

L-PGDS-produced PGD2 in premature, but not in mature, adipocytes increases obesity and insulin resistance

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the production of PGD2 in adipocytes and is selectively induced by a high-fat diet (HFD) in adipose tissue. In this study, we investigated the effects of HFD on obesity and insulin resistance in two distinct types of adipose-specific L-PGDS gene knockout (KO) mice: fatty acid binding protein 4 (fabp4, aP2)-Cre/L-PGDS flox/flox and adiponectin (AdipoQ)-Cre/L-PGDS flox/flox mice. The L-PGDS gene was deleted in adipocytes in the premature stage of the former strain and after maturation of the latter strain. The L-PGDS expression and PGD2 production levels decreased in white adipose tissue (WAT) under HFD conditions only in the aP2-Cre/L-PGDS flox/flox mice, but were unchanged in the AdipoQ-Cre/L-PGDS flox/flox mice. When fed an HFD, aP2-Cre/L-PGDS flox/flox mice significantly reduced body weight gain, adipocyte size, and serum cholesterol and triglyceride levels. In WAT of the HFD-fed aP2-Cre/L-PGDS flox/flox mice, the expression levels of the adipogenic, lipogenic, and M1 macrophage marker genes were decreased, whereas those of the lipolytic and M2 macrophage marker genes were enhanced or unchanged. Insulin sensitivity was improved in the HFD-fed aP2-Cre/L-PGDS flox/flox mice. These results indicate that PGD2 produced by L-PGDS in premature adipocytes is involved in the regulation of body weight gain and insulin resistance under nutrient-dense conditions

Standard fluorescent imaging of live cells is highly genotoxic

Fluorescence microscopy is commonly used for imaging live mammalian cells. Here, we describe studies aimed at revealing the potential genotoxic effects of standard fluorescence microscopy. To assess DNA damage, a high throughput platform for single cell gel electrophoresis is used (e.g., the CometChip). Light emitted by three standard filters was studied: (a) violet light [340–380 nm], used to excite DAPI and other blue fluorophores, (b) blue light [460–500 nm] commonly used to image green fluorescent protein (GFP) and Calcein AM, and (c) green light [528–553 nm], useful for imaging red fluorophores. Results show that exposure of samples to light during imaging is indeed genotoxic even when the selected wavelengths are outside the range known to induce significant damage levels. Shorter excitation wavelengths and longer irradiation times lead to higher levels of DNA damage. We have also measured DNA damage in cells expressing enhanced GFP or stained with Calcein AM, a widely used green fluorophore. Data show that Calcein AM leads to a synergistic increase in the levels of DNA damage and that even cells that are not being directly imaged sustain significant DNA damage from exposure to indirect light. The nature of light-induced DNA damage during imaging was assessed using the Fpg glycosylase, an enzyme that enables quantification of oxidative DNA damage. Oxidative damage was evident in cells exposed to violet light. Furthermore, the Fpg glycosylase revealed the presence of oxidative DNA damage in blue-light exposed cells for which DNA damage was not detected using standard analysis conditions. Taken together, the results of these studies call attention to the potential confounding effects of DNA damage induced by standard imaging conditions, and identify wavelength, exposure time, and fluorophore as parameters that can be modulated to reduce light-induced DNA damage.National Institutes of Health (U.S.) (Grant 5-UO1-ES016045)National Institutes of Health (U.S.) (grant P30-ES002109)National Institutes of Health (U.S.) (Grant 1-R21-ES019498)National Institutes of Health (U.S.) (Grant R43-ES021116-01)National Institute of Environmental Health Sciences (NIEHS Training Grant in Environmental Toxicology number T32-ES007020

Synthesis of Novel Farnesoid X Receptor Agonists and Validation of Their Efficacy in Activating Differentiation of Mouse Bone Marrow-Derived Mesenchymal Stem Cells into Osteoblasts

The modulators of farnesoid X receptor (FXR), a bile acid receptor, regulate various biological processes including bile acid metabolism, and are associated with the control of fatty liver and osteoporosis. Thus, the control of FXR activity and development of FXR modulators are critical not only for research, but also for clinical application. In this study, we synthesized novel FXR agonists 1–4 possessing isoxazole and N-substituted benzimidazole moieties, and compared their effects on osteoblast differentiation with the known FXR agonists, chenodeoxycholic acid and a synthetic compound, GW4064. Two (3 and 4) of the four novel FXR agonists 1–4 showed high specificities for FXR. Computer-assisted modeling suggested that the binding of the FXR agonist 3 with ligand binding domain of FXR was similar to GW4064. FXR was expressed in mouse bone marrow-derived mesenchymal stem cell (MSC)-like ST2 cells (ST-2 MSCs). The FXR agonists activated the BMP-2-induced differentiation of ST-2 MSCs into osteoblasts and enhanced the expression of RUNX2. Moreover, the potency of the FXR agonist 3 was comparable to GW4064 in promoting osteoblast differentiation of ST-2 MSCs. These results indicate that FXR activation enhanced the BMP-2-induced differentiation of MSCs into osteoblasts through activating RUNX2 expression. FXR could be a potential therapeutic target for the treatment of bone diseases such as osteoporosis

Prostaglandins as PPARγ Modulators in Adipogenesis

Adipocytes and fat cells play critical roles in the regulation of energy homeostasis. Adipogenesis (adipocyte differentiation) is regulated via a complex process including coordinated changes in hormone sensitivity and gene expression. PPARγ is a ligand-dependent transcription factor and important in adipogenesis, as it enhances the expression of numerous adipogenic and lipogenic genes in adipocytes. Prostaglandins (PGs), which are lipid mediators, are associated with the regulation of PPARγ function in adipocytes. Prostacyclin promotes the differentiation of adipocyte-precursor cells to adipose cells via activation of the expression of C/EBPβ and δ. These proteins are important transcription factors in the activation of the early phase of adipogenesis, and they activate the expression of PPARγ, which event precedes the maturation of adipocytes. PGE2 and PGF2α strongly suppress the early phase of adipocyte differentiation by enhancing their own production via receptor-mediated elevation of the expression of cycloxygenase-2, and they also suppress the function of PPARγ. In contrast, PGD2 and its non-enzymatic metabolite, Δ12-PGJ2, activate the middle-late phase of adipocyte differentiation through both DP2 receptors and PPARγ. This paper focuses on potential roles of PGs as PPARγ modulators in adipogenesis and regulators of obesity

Inhibition of Prostaglandin F2α Receptors Exaggerates HCl-Induced Lung Inflammation in Mice

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe respiratory disorders that are caused by aspiration, sepsis, trauma, and pneumonia. A clinical feature of ALI/ARDS is the acute onset of severe hypoxemia, and the mortality rate, which is estimated at 38–50%, remains high. Although prostaglandins (PGs) are detected in the bronchoalveolar lavage fluid of patients with ALI/ARDS, the role of PGF2α in ALI remains unclear. We aimed to clarify the role of PGF2α/PGF2α receptor (FP) signaling in acid-induced ALI using an FP receptor antagonist, AL8810. Intratracheal injection of hydrochloric acid (HCl) increased neutrophil migration into the lungs, leading to respiratory dysfunction. Pre-administration of AL8810 further increased these features. Moreover, pre-treatment with AL8810 enhanced the HCl-induced expression of pro-inflammatory cytokines and neutrophil migratory factors in the lungs. Administration of HCl decreased the gene expression of lung surfactant proteins, which was further reduced by co-administration of AL8810. Administration of AL8810 also increased lung edema and reduced mRNA expression of epithelial sodium channel in the lungs, indicating that AL8810 reduced fluid clearance. Furthermore, AL8810 also increased lipopolysaccharide-induced expression of adhesion molecules such as intracellular adhesion molecule-1 and E-selectin in human umbilical vein endothelial cells. These results indicate that inhibition of FP receptors by AL8810 exacerbated HCl-induced ALI