Molecular évolution of RuBisCO subunits

The environmental conditions of our planet have been changing since its origin. For species’ survival, adaptation to the environment is crucial, for example through the adaptive evolution of photosynthesis. The appearance of the mechanism to concentrate CO2 has given some species a selective advantage under CO2-depleted conditions. C4 plants comprise one of the main groups of such species that have diverged from classical C3 plants and adapted to depletion of CO2 by modifying the cellular structures and biochemical cascades. Ribulose- 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), an enzyme which catalyzes the first step of CO2 fixation, has changed cellular location during C4 evolution. RuBisCO of C4 is surrounded by highly concentrated CO2, which prevents the loss of energy and CO2 caused by the affinity of the enzyme for both O2 and CO2. The intercellular gas composition surrounding RuBisCO directly influences the rate of photosynthesis because RuBisCO’s slow turnover rate is often the limiting factor for the rate of photosynthesis in higher plants. Therefore, RuBisCO has been considered as the determining factor of the photosynthetic rate and it has been thought to play an important role in plant adaptation to the environmental conditions. In previous studies, the evidence of adaptive evolution of RuBisCO has been detected by positive selection acting on the chloroplast rbcL gene encoding large subunits of RuBisCO (RBCL) in independent C4 lineages. The other subunit of RuBisCO, the small subunit (RBCS), has been reported to influence the catalytic efficiency, CO2 specificity, assembly, activity, and stability of RuBisCO. However, the evolution of its encoding nuclear gene rbcS is yet poorly studied. Therefore, I aimed to study the molecular evolution of rbcS in angiosperms. The rbcS gene is a multigene family and the number of gene copies is different between species. The phylogenetic tree of the rbcS gene reveals two lineages that may have originated from a duplication event before the divergence of land plants. Copies originating from ancient duplication events seem to have been removed, whereas the copies from recent events appear to be retained. This explains the observation in the rbcS tree that gene copies of the same species are more closely related to each other than ones from different species. I hypothesized that each rbcS gene copy of the same species may have different characteristics. I compared the interaction of rbcS and rbcL genes as well as the influence of different encoding RBCS subunits to the stability of RuBisCO by respectively testing coevolution between rbcS and each rbcL and by homology modelling of RuBisCO composed with a RBCS encoded by different rbcS copies. The results suggested that the interaction between RBCS and RBCL, and the influence on the overall stability of the enzyme, are the same among different rbcS copies. Therefore, I assumed that all the rbcS gene copies cannot be divergent because they need to be structurally compatible with RBCL. In general, when all the gene copies of a multigene family have the same characteristics, multiple gene copies of a species exist to maintain the number of transcripts at the same level as that of a single copy carrying species (dosage effect hypothesis). To test this hypothesis, I estimated the gene expression levels of each gene copy by using published transcriptome data. The results suggest that the gene expression level is similar between species carrying single and multiple copies. The results suggest that species carrying a higher gene copy number have a larger amount of RuBisCO. It has been reported that RuBisCO is degraded or down regulated under specific environmental stress. Thus, I conclude that plants living in such an environmental stress condition may need to synthesize more RuBisCO to prevent a shortage of the enzyme. To understand better the role of RBCS to cope with environmental changes, I tested the positive selection of the rbcS gene in species of Poaceae that have different photosynthetic types. Positive selection was detected all over the tree and the signal was not C4-specific. This suggests that the positive selection acting on the rbcS gene has not led to the shift of photosynthetic types. I assume that RBCS might be involved in the optimization of RuBisCO after the establishment of C4 photos nthesis type or after migration to new habitats that require different catalytic properties. -- Les conditions environnementales de notre planète ne cessent de changer depuis son origine. Pour survivre, il est crucial pour les espèces de s’adapter à leur environnement. Un exemple est l’évolution adaptative de la photosynthèse. L’apparition de mécanismes permettant de concentrer le CO2 a donné à certaines espèces un avantage sélectif lorsqu’elles font face à des conditions appauvries en CO2. Les plantes C4 constituent l’un des principaux groupes d’espèces qui ont divergé des plantes C3 classiques en s’adaptant en modifiant leurs structures cellulaires et cascades biochimiques. La ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) – une enzyme catalysant la première étape de fixation de CO2 – a changé de localisation cellulaire durant l’évolution du mode de fixation du carbone C4. La RuBisCO des plantes C4 est localisée dans un compartiment caractérisé par une haute concentration en CO2, évitant ainsi la perte d’énergie et de CO2 causée par l’affinité de l’enzyme pour deux substrats: le CO2 et le O2. L’environnement gazeux intracellulaire auquel est confrontée la RuBisCO influence directement le taux de photosynthèse, car son faible taux de renouvellement par rapport à d’autres enzymes photosynthétiques constitue souvent le facteur limitant le taux de photosynthèse chez les plantes supérieures. De ce fait, la RuBisCO est considérée comme le facteur déterminant le taux de photosynthèse et jouant un rôle important dans l’adaptation des plantes aux conditions environnementales. De précédentes études démontrèrent l’évolution adaptative de la RuBisCO par sélection positive agissant sur le gène chloroplastique rbcL – qui code pour la grande sous-unité de la RuBisCO (RBCL) – dans des lignées indépendantes de plantes C4. Il a été démontré que l’autre sous-unité de la RuBisCO – la petit sous-unité (RBCS) – influence l’efficacité catalytique, la spécificité de liaison au CO2, l’assemblement, l’activité et la stabilité de la RuBisCO. Néanmoins, l’évolution du gène codant pour cette sous-unité – le gène nucléaire rbcS – n’a été que très peu étudiée jusqu’à présent. Par conséquent, le but de mon projet est d’étudier l’évolution moléculaire du gène rbcS chez les Angiospermes. Le gène rbcS fait partie d’une famille de gènes multiples et son nombre de copies varie selon les espèces. Des arbres phylogénétiques se basant sur rbcS ont révélé deux lignées provenant potentiellement d’un événement de duplication ayant eu lieu avant la divergence des plantes terrestres. Les copies provenant d’anciens événements de duplication semblent avoir été éliminées, alors que les copies provenant d’événements récents de duplications paraissent avoir été conservées. Cela explique que les copies de rbcS provenant d’une même espèce soient plus proches phylogénétiquement les unes des autres que des copies provenant d’espèces différentes. Je mets en avant l’hypothèse que chaque copie du gène rbcS de la même espèce pourrait avoir différentes caractéristiques. J’ai comparé l’interaction entre les gènes rbcS et rbcL ainsi que l’influence des différentes sous-unités RBCS à la stabilité de la RuBisCO en testant respectivement la coévolution entre rbcS et chaque rbcL et en modélisant par homologie la RuBisCO composée par une sous-unité RBCS codée par différentes copies du gène rbcS. Les résultats suggèrent que l’interaction entre chaque rbcS et rbcL et l’influence sur la stabilité générale de l’enzyme est similaire entre les différentes copies de rbcS. En conséquence, je présume que les différentes copies du gène rbcS ne peuvent pas être divergentes car il est nécessaire qu’elles soient compatibles structurellement avec la sous-unité RBCL. En général, lorsque toutes les copies de gènes provenant d’une même famille de gènes multiples ont les mêmes caractéristiques, les différentes copies de gènes permettent de maintenir la même quantité d’éléments transcrits en comparaison avec une espèce ne possédant qu’une copie du gène (hypothèse « d’effet de dosage »). Afin de tester cette hypothèse, j’ai estimé le niveau d’expression pour chaque copie de gène de la même espèce en me basant sur des données transcriptomiques déjà publiées. Les résultats suggèrent que le niveau d’expression des gènes est similaire entre les espèces ayant une ou plusieurs copies du gène. De ce fait, l’hypothèse d’effet de dosage n’est pas applicable dans le cadre de l’évolution de rbcS. Les résultats suggèrent que les espèces ayant un plus grand nombre de copies du gène disposent également d’une plus grande quantité de RuBisCO. Il a été rapporté que la RuBisCO se dégrade ou est régulée négativement dans des conditions de stress spécifiques. Par conséquent, je présume que les plantes vivant dans de telles conditions environnementales stressantes doivent synthétiser plus de RuBisCO pour éviter une pénurie de l’enzyme. Pour mieux comprendre le rôle de RBCS face aux changements environnementaux, j’ai testé la sélection positive du gène rbcS chez des espèces de Poacées ayant différents mécanismes photosynthétiques. Une sélection positive a été détectée chez toutes les espèces et le signal n’était pas spécifique aux espèces à système C4. Cela suggère que la sélection positive agissant sur le gène rbcS n’est pas responsable du changement de type de photosynthèse. Je présume que RBCS ne serait donc pas impliquée dans la transition C3 à C4, mais que cette sous-unité pourrait être impliquée dans l’optimisation de la RuBisCO après l’établissement de la photosynthèse de type C4 ou après la migration vers de nouveaux habitats nécessitant différentes propriétés catalytiques

Preparation of N-2-Nitrophenylsulfenyl Imino Peptides and Their Catalyst-Controlled Diastereoselective Indolylation

N-2-Nitrophenylsulfenyl (Nps) imino dipeptides bearing various functional groups were successfully prepared via MnO2-mediated oxidation and then subjected to diastereoselective indolylation. Each diastereomer of the adduct was selectively obtained from the same substrates using the appropriate chiral phosphoric acid catalysts. These transformations would be useful for synthesizing non-canonical amino acid-containing peptides as novel drug candidates

Artificial O2 Carrier with Antioxidant Activities

A covalent core–shell structured protein cluster composed of hemoglobin (Hb) at the center and human serum albumins (HSA) at the periphery, Hb-HSAm, is an artificial O2 carrier that can function as a red blood cell substitute. Here we described the preparation of a novel Hb-HSA3 cluster with antioxidant activities and its O2 complex stable in aqueous H2O2 solution. We used an approach of incorporating a Pt nanoparticle (PtNP) into the exterior HSA unit of the cluster. A citrate reduced PtNP (1.8 nm diameter) was bound tightly within the cleft of free HSA with a binding constant (K) of 1.1×107 M−1, generating a stable HSA-PtNP complex. This platinated protein showed high catalytic activities for dismutations of superoxide radical anions (O2•–) and hydrogen peroxide (H2O2), i.e., superoxide dismutase and catalase activities. Also, Hb-HSA3 captured PtNP into the external albumin unit (K = 1.1×107 M−1), yielding an Hb-HSA3(PtNP) cluster. The association of PtNP caused no alteration of the protein surface net charge and O2 binding affinity. The peripheral HSA-PtNP shell prevents oxidation of the core Hb, which enables the formation of an extremely stable O2 complex, even in H2O2 solution

Conformational Study of Polypeptide Chains Grafted on the Surface of Polylactide Latex Particle

Polylactide (PLA) latex particle covered with polypeptide chains were prepared by means of solvent exchange method from PLA and PLA-block-polypeptide block copolymer solutions. PLA segment of the block copolymer and PLA homopolymer formed a core of the particle, and the polypeptide segment of the block copolymer, which is designed as tightly fixed biodegradable emulsifier, formed corona around the particle surface. This picture was supported by the fact that zeta-potential of PLA latex particle covered with polypeptide segment was different from that of bare PLA particle because of the presence of the ionizable group in the polypeptide chains. To clarify the effect of the ionizable group on conformation of the polypeptide chain, the relation between the polypeptide chain length and the area occupied by the single block chain was evaluated. The result that the occupied area per a polypeptide chain was linearly increased with the increase in the polypeptide chain length indicates that the polypeptide chains trail on the particle surface and did not take helical structures

卵巣明細胞癌と類内膜癌の鑑別に関するMRIについての知見

BACKGROUND: Common cancerous histological types associated with endometriosis are clear cell carcinoma (CCC) and endometrioid carcinoma (EC). CCC is regarded as an aggressive, chemoresistant histological subtype. Magnetic resonance imaging (MRI) offers some potential advantages to diagnose ovarian tumors compared with ultrasonography or computed tomography. This study aimed to identify MRI features that can be used to differentiate between CCC and EC. METHODS: We searched medical records of patients with ovarian cancers who underwent surgical treatment at Nara Medical University Hospital between January 2008 and September 2018; we identified 98 patients with CCC or EC who had undergone preoperative MRI. Contrasted MRI scans were performed less than 2 months before surgery. Patients were excluded from the study if they had no pathology, other pathological subtype of epithelial ovarian cancer, and/or salvage treatment for recurrence and metastatic ovarian cancer at the time of study initiation. Clinically relevant variables that were statistically significant by univariate analysis were selected for subsequent multivariate regression analysis to identify independent factors to distinguish CCC from EC. RESULTS: MRI of CCC and EC showed a large cystic heterogeneous mixed mass with mural nodules protruding into the cystic space. Univariate logistic regression analysis revealed that the growth pattern (broad-based nodular structures [multifocal/concentric sign] or polypoid structures [focal/eccentric sign]), surface irregularity (a smooth/regular surface or a rough/irregular/lobulated surface), "Width" of mural nodule, "Height-to-Width" ratio (HWR), and presence of preoperative ascites were factors that significantly differed between CCC and EC. In the multivariate logistic regression analysis, the growth pattern of the mural nodule (odds ratio [OR] = 0.69, 95% confidence interval [CI]: 0.013-0.273, p = 0.0004) and the HWR (OR = 3.71, 95% CI: 1.128-13.438, p = 0.036) were independent predictors to distinguish CCC from EC. CONCLUSIONS: In conclusion, MRI data showed that the growth pattern of mural nodules and the HWR were independent factors that could allow differentiation between CCC and EC. This finding may be helpful to predict patient prognosis before operation.博士（医学）・乙第1433号・令和元年9月27日© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated

超短高次高調波による希ガス原子の多光子二重イオン化過程の光電子コインシデンス分光

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 山内 薫, 東京大学教授 大越 慎一, 東京大学教授 佃 達哉, 東京大学教授 合田 圭介, 東京大学特任准教授 原野 幸治University of Tokyo(東京大学

Asymmetric Synthesis of α‐Amino Phosphonic Acids using Stable Imino Phosphonate as a Universal Precursor

A practical method for synthesizing chiral α-amino phosphonic acid derivatives was developed. Readily available and stable N-o-nitrophenylsulfenyl (Nps) imino phosphonate was utilized as a substrate for a highly enantioselective Friedel–Crafts-type addition of indole or pyrrole nucleophiles catalyzed by chiral phosphoric acid. The resulting adduct was easily converted to N-9-fluorenylmethyloxycarbonyl (Fmoc) amino phosphonic acid, which is useful for synthesizing peptides containing an amino phosphonic acid

子宮内膜側に発症する (Subtypel) 子宮腺筋症は黄体ホルモン療法による多量性器出血の危険因子である

We aimed to retrospectively analyze the risk factors of a continuous dienogest (DNG) therapy for serious unpredictable bleeding in patients with symptomatic adenomyosis. This is a retrospective study based on data extracted from medical records of 84 women treated with 2 mg of DNG orally each day between 2008 and 2017. 47 subjects were excluded from the original analyses due to an inadequate subcategorization into subtype I and subtype II and a lack of hemoglobin levels. The influence of various independent variables on serious unpredictable bleeding was assessed. Of the 37 eligible patients who received the continuous DNG therapy, 14 patients experienced serious unpredictable bleeding. Univariate analysis revealed that the serious bleeding group had subtype I adenomyosis (P = 0.027). There was no correlation between age, parity, minimum hemoglobin level before treatment, previous endometrial curettage, and duration of DNG administration, or uterine or adenomyosis size and the serious bleeding. A DNG-related serious unpredictable bleeding is associated with the structural type of adenomyosis (subtype I) in patients with symptomatic adenomyosis.博士（医学）・甲第800号・令和3年9月29日© The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Investigation of biomarkers in a rare case of fulminant necrotizing enterocolitis in a preterm infant

We encountered a very rare case of fulminant necrotizing enterocolitis (F-NEC) in a preterm male baby. The course of NEC and sepsis in this case was clearly different from the usual course. After onset at 14 days of life, catheter-related bloodstream infection was first assumed, and antibiotics and γ-globulin administration were started. However, 12 hours after onset, the baby's abdominal distension increased remarkably, and his entire abdominal wall turned red to purple. Escherichia coli were isolated from the blood culture, but the catheter tip culture was negative. Exchange transfusion was performed 32 hours after onset, but no significant changes were observed in the baby's general condition, and he died 46 hours after onset. The acute phase reactants of CRP and α1-acid glycoprotein increased, but haptoglobin did not. Although IL-1β and TNFα increased as expected with sepsis, IL-6, IL-8, IL-10, and G-CSF however increased to a greater extent than expected. From the above, we diagnosed the development of intestinal necrosis as a result of widespread intestinal ischemia, and that sepsis was associated with this poor condition