京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19188号 / 工博第4065号 / 新制||工||1627(附属図書館) / 32180 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Chloroplasts of many species of hornworts (Anthocerotae) have a structure that resembles the pyrenoid of green algae but whether these two structures are homologous has not been determined. We utilized immunogold labelling on thin sections to determine the distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the major protein of algal pyrenoids, in sixteen hornwort species with and without pyrenoids. Several species (Phaeoceros laevis, Anthoceros punctatus, A. formosae, A. laminiferus, Folioceros fuciformis, Folioceros sp., Dendroceros tubercularis, D. japonicus, D. validus, Notothylas orbicularis, N. temperata, and Spaerosporoceros adscendens) have uniplastidic (or primarily uniplastidic) cells with large prominent multiple pyrenoids. In all of these species, the labelling is found exclusively in the pyrenoid and, with the exception of the Folioceros, Dendroceros, and Notothylas species, the labelling is randomly distributed throughout the pyrenoid. In the exceptional species, the pyrenoids have prominent pyrenoglobuli or other inclusions that are unlabelled. In Megaceros flagellaris and M. longispirus, the cells are multiplastidic (with the exception of the apical cell and some epidermal cells) and the chloroplasts lack pyrenoids. Anthoceros fusiformis and Phaeoceros coriaceus have primarily uniplastidic cells but the chloroplasts lack pyrenoids; only an area of stroma in the center of the plastid devoid of starch, reminiscent of a pyrenoid, is found. In all of the species lacking pyrenoids, RuBisCo is found throughout the stroma, including the stromal spaces made by the so-called channel thylakoids. No preferential accumulation of RuBisCo is found in the pyrenoid-like region in A. fusiformis and P. coriaceus. These data indicate that 1) the hornwort pyrenoid is homologous to algal pyrenoids in the presence of RuBisCo; 2) that at least some of the RuBisCo in the pyrenoid must represent an active form of the enzyme; and 3) that, in the absence of pyrenoids, the RuBisCo is distributed throughout the stroma, as in higher plants.

No description available.

Disabled-2 (Dab2) is an adapter protein that interacts with cell membranes and it is involved in several biological processes including endocytosis and platelet aggregation. During endocytosis, the Dab2 phosphotyrosine-binding (PTB) domain mediates protein binding to phosphatidylinositol 4,5-bisphosphate (PIP2) at the inner leaflet of the plasma membrane and helps co-localization with clathrin coats. Dab2, released from platelet alpha granules, inhibits platelet aggregation by binding to the °IIb? integrin receptor on the platelet surface through an Arg-Gly-Asp (RGD) motif located within the PTB domain. Alternatively, Dab2 binds sulfatides on the platelets surface, and this binding partition Dab2 in two pools (sulfatide and integrin receptor-bound states), but the biological consequences of lipid binding remain unclear. Dab2 binds sulfatides through two basic motifs located on its N-terminal region including the PTB domain (N-PTB). We have characterized the binding of Dab2 to micelles, which are widely used to mimic biological membranes. These micellar interactions were studied in the absence and presence of Dab2 lipid ligands, sulfatides and PIP2. By applying multiple biochemical, biophysical, and structural techniques, we found that whereas Dab2 N-PTB binding to PIP2 stabilized the protein but did not contribute to the penetration of the protein into micelles, sulfatides induced conformational changes and facilitated penetration of Dab2 N-PTB into micelles. This is in agreement with previous observation that sulfatides, but not PIP2, protect Dab2 N-PTB from thrombin cleavage. By studying the mechanism by which Dab2 targets membranes, we will have the opportunity to manipulate its function in different lipid-dependent biological processes. / Master of Science

The development of novel imidazopyridines, which includes both tetrahydroimidazo[1,5-a]pyridine (rIMP) and imidazo[1,5-a]pyridine (IMP) was investigated using conventional and microwave induced procedures that afforded compounds at high yield of 88-96%. rIMP was synthesized using a two-step procedure that involved the microwave synthesis of IMP, then the reduction of the pyridine moiety of the fused imidazopyridine rings using 10% Pd/C and hydrazine monohydrate. The microwave synthesis of imidazopyridines involved the one pot reaction of 2-benzoylpyridine, substituted benzaldehyde and ammonium formate in acetic acid under open vessel microwave conditions, which resulted in products within 40 minutes. Novel PEG-IMP development, involved the synthesis of ethylene glycol tethered benzaldehydes and IMPs using traditional Williamson etherification synthesis, which afforded products at a high yield of 92-95%. We have then shown IMP and rIMP roles in its antiproliferative property towards PCa cells, specificity in inhibiting PI3K isoforms, and structural motif’s interaction with different residues in the kinase binding domain of the class I PI3K isoforms. The antiproliferative property towards PC3 cells shows increased activity with compounds containing pyridyl group on carbon 3 of the imidazo[1,5-a]pyridine parent moiety with signs of toxicity to PC3 within 24 hours of incubation and at 1 μM of the parent compound. Furthermore, the IMPs were tested against five prostate cellular lines: PC3, RWPE1, D145, LNCaP and LNCaP C81. IMPs showed little activity towards RWPE1 and increased activity towards PC3 cells. We determined that functionalizing the phenyl group at position 1 increased the efficacy of rIMP compared to the IMP. After showing increased toxicity to PC3 cells, it was important to investigate the mechanism in which IMP pose toxicity towards PC3 cells. The biochemical assay showed that rIMP was more effective in inhibiting PI3Kα isoform compared to both pan inhibitor wortmannin and IMP. Both IMP and rIMP inhibited more than 60% of PI3Kγ isoform activity at nanomolar concentrations. After showing IMPs affinity to PI3K isoforms, we investigated the binding interactions rIMP and IMP towards the PI3K isoforms using MOE molecular modeling software.

Inwardly rectifying potassium (Kir) channels establish and regulate the resting membrane potential of excitable cells in the heart, brain and other peripheral tissues. Phosphatidylinositol- 4,5-bisphosphate (PIP2) is a key direct activator of ion channels, including Kir channels. Gasotransmitters, such as carbon monoxide (CO), have been reported to regulate the activity of Kir channels by altering channel-PIP2 interactions. We tested, in a model system, the effects and mechanism of action of another important gasotransmitter, hydrogen sulfide (H2S) thought to play a key role in cellular responses under ischemic conditions. Direct administration of sodium hydrogen sulfide (NaHS), as an exogenous H2S source, and expression of cystathionine γ-lyase (CSE), a key enzyme that produces endogenous H2S in specific brain tissues, resulted in comparable current inhibition of several Kir2 and Kir3 channels. A “tag switch” assay provided biochemical evidence for sulfhydration of Kir3.2 channels. The extent of H2S regulation depended on the strength of channel-PIP2 interactions: H2S regulation was attenuated when strengthening channel-PIP2 interactions and was increased when channel-PIP2 interactions were weakened by depleting PIP2 levels via different manipulations. These H2S effects took place through specific cytoplasmic cysteine residues in Kir3.2 channels, where atomic resolution structures with PIP2 gives us insight as to how they may alter channel-PIP2 interactions. Mutation of these residues abolished H2S inhibition, and reintroduction of specific cysteine residues into the background of the mutant lacking cytoplasmic cysteine residues, rescued H2S inhibition. Molecular dynamics simulation experiments provided mechanistic insights as to how sulfhydration of specific cysteine residues could lead to changes in channel-PIP2 interactions and channel gating.

In this diploma work changes of initial and total activities and content of Rubisco in beech and Norway spruce were studied. The plants were cultivated in conditions with ambient CO2 concentration (350 mol·mol–1) and elevated CO2 concentration (700 mol·mol–1). Three series of samples (at the beginning, in the middle and at the end of growing season) were taken. Initial and total Rubisco activities were measured spectrophotometrically and activation state was calculated. Rubisco content was determined by SDS–PAGE method. Rubisco activity in beech cultivated in elevated CO2 concentration decreased during the whole growing season while in beech growing in ambient CO2 concentration Rubisco activity decreased up to middle of growing season and then increased. Rubisco content in beech in ambient CO2 concentration slightly increased and in beech in elevated CO2 concentration decreased up to middle of growing season and then increased. Rubisco activities in Norway spruce both in ambient and elevated CO2 concentration decreased. Rubisco content in Norway spruce in ambient CO2 concentration decreased but in Norway spruce in elevated CO2 concentration first decreased and then increased.

No description available.