<p> The ɣ-radiosensitivity of haploid and diploid Oedogonium cardiacum cells was measured and compared to other cell lines. With the doubling of the chromosome complement, the Do value doubled, but the extrapolation number decreased four-fold. A general conclusion was drawn from the results that at all doses of ɣ-radiation, the diploids were more resistant than the haploids. A new radiation technique was used and compared to that used routinely in the laboratory. The further use of the technique was not recommended since the data obtained with the diploid line only was not as reliable as one would like.</p> <p> The degree of synchrony of Oe. cardiacum zoospore cultures was measured using cell division as the biological end-point and a mathematical expression, the percent phasing, as the index of synchrony. It was intended that this research problem be secondary to the radiation studies. The percent phasing values were determined for cells growing in two inorganic media and in the presence of an inhibitor, hydroxyurea. However the degree of synchrony was not improved beyond that of the routine laboratory procedure. Attempts to improve the size of the synchronous populations collected also proved unsuccessful.</p> / Thesis / Master of Science (MSc)

Background: Hemophilia A is X-linked recessive congenital bleeding disorder. Exogenous infusion of FVIII is the treatment of choice in hemophilia A patients. However, inhibitor development remains the major problem in management of Hemophilia A. It has been showed that IVIG has immunomodulatory effects and it has been being used in the treatment of several autoimmune and inflammatory disorders. Here, we investigated the effect of co-administration of FVIII with IVIG on the development of inhibitor in naive and previously immunized hemophilia A mice. Methods: Initially, hemophiliac mice were immunized by weekly intraperitoneal injection of human recombinant FVIII (rFVIII). The mice then were treated, either by rFVIII/IVIG co-injection or rFVIII alone. In the other experimental group, naive hemophiliac mice were treated with rFVIII/IVIG co-injection for four weeks followed by injection of either rFVIII or rFVIII/IVIG. Plasma's anti-FVIII Ab titer was measured using ELISA. Results: Weekly injection of rFVIII led to the development of anti-FVIII Ab in all previously untreated mice. Treatment of those immunized mice with rFVIII/IVIG co-injection did not reduce the level of pre-existing Ab. On the other hand, naive mice treated with rFVIII/IVIG co-injection showed significantly less Ab titer compared to the mice received rFVIII alone after 4 weeks (mean Ab titre of 1 compared to 39, in rFVIII/IVIG co-injection and rFVIII groups respectively). Although the rFVIII/IVIG-treated mice developed immune response following the injection of rFVIII alone, Ab titer in those that kept receiving rFVIII/IVIG co-injection remained lower compared to other groups during the whole twelve weeks of the experiment. Conclusions: Co-injection of rFVIII with IVIG decreased the anti-FVIII immune response in previously untreated hemophilia A mice. These findings suggest that IVIG co-administration can be effective in management of hemophilia A patients at risk of inhibitor development. / Thesis / Master of Applied Science (MASc)

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No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23765号 / 医博第4811号 / 新制||医||1056(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 辻川 明孝, 教授 寺田 智祐, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A novel partial agonist of the exchange protein activated by cAMP isoform 1 (EPAC1), I942, was recently discovered and shown to reduce the guanine exchange factor activity of cAMP-bound EPAC1 to approximately 10% relative to cAMP activation. However, the inhibition mechanism of I942 remains unknown. Here, we utilize NMR spectroscopy to probe the inhibitory I942 - EPAC1 interactions at atomic resolution. The EPAC1 - I942 interface was mapped through intermolecular NOEs measured by 15N and 13C filtered NOESY-HSQC experiment. Intermolecular NOE mapping combined with other protein NMR methods, such as saturation transfer difference, transfer Nuclear Overhauser Effect spectroscopy and chemical shift mapping, we revealed that I942 interacts with the phosphate binding cassette (PBC) and base binding region (BBR) of the EPAC1 cyclic nucleotide binding (CNB) domain, similar to cAMP. The PBC controls the conformation of the hinge region, and subsequently, allosterically shifts the hinge region between its active/inactive states. Molecular dynamics simulation based on the NMR spectroscopy data revealed that EPAC1-CNB adopts an intermediate conformation between its inactive and active states, which explains the partial agonist nature of I942. / Thesis / Master of Science (MSc) / The exchange protein activated by cAMP (EPAC) is a receptor for the classical secondary messenger cAMP. EPAC is present in multiple human systems and plays a pivotal role in the development of a wide range of diseases. In this study, we aim to establish the inhibition mechanism of a novel small molecule EPAC inhibitor/partial agonist I942 using NMR spectroscopy with the goal of achieving a better understanding of EPAC inhibition and paving the way for new small molecule EPAC inhibitors that can potentially treat EPAC-related diseases such as heart failure and diabetes.

The first part of this work describes the synthesis of a new histone deacetylase (HDAC) inhibitor (HDI). HDAC enzymes modify core histones, influence nucleosome structure and change gene transcription by removing the acetyl groups from lysine residues on proteins. HDIs are showing exciting potential as a new class of drugs for cancer and a variety of other diseases. A new HDAC inhibitor based on the hydroxamic acid motif has been synthesized. Two characteristic structural features were incorporated into the design of the novel inhibitor. A cyclic peptide mimetic of known structure was fused to a hydroxamic acid moiety through an aliphatic chain. The HDAC inhibitor provided significant inhibitory activity against HDACs with an IC50 value of 46 ± 15 nM, and against HDAC8 with an IC50 value of 208 ± 20 nM. The potent HDAC inhibitory activity of the HDAC inhibitor demonstrates the importance of the rim recognition region in the design of HDIs. The hydrophobic cyclic turn mimic allows the formation of a tight complex between HDI and HDAC enzymes. The second part of this work is to synthesize secondary structure mimics and incorporate them into the helix-turn-helix (HTH) motif. One of the important methods to study the conformation of the biologically active peptides is to incorporate the rigid peptidomimetics into the relevant peptides. Important information can be obtained from the study of conformationally constrained peptides. HTH proteins are well characterized and found in many organisms from prokaryotes to eukaryotes. The relatively small size, simple structure, and significance in stabilizing tertiary structures make the HTH peptide an attractive target to mimic. Both a Gly HTH turn mimic and a Ser HTH turn mimic were synthesized using stereoselective hydrogenation and macrocyclization starting from unnatural amino acids in a yield of 33% and 14%, respectively. The synthesis of Fmoc protected HTH turn mimics allowed incorporation into HTH peptides using Fmoc chemistry on solid phase. The incorporation of the HTH turn mimics into the peptides proved to be challenging, either by sequential elongation or by segment condensation. Alternative peptide synthesis strategies were employed in attempts to solve the problems. / Ph. D.

No / Background: In previous experiments, we showed that heparin oligosaccharides inhibit the angiogenic cytokine fibroblast growth factor-2. Here, we present the first in vivo study of size-fractionated heparin oligosaccharides in four models of angiogenesis that are progressively less dependent on fibroblast growth factor-2. Experimental Design: Heparin oligosaccharides were prepared using size-exclusion gel filtration chromatography and characterized through depolymerization and strong anion exchange high-performance liquid chromatography. Size-defined oligosaccharides (20 mg/kg/d) were given to mice bearing s.c. sponges that were injected with fibroblast growth factor-2 (100 ng/d). After 14 days, octasaccharides and decasaccharides reduced the microvessel density to levels below control. In a second experiment, HEC-FGF2 human endometrial cancer cells that overexpress fibroblast growth factor-2 were implanted in a hollow fiber placed s.c. in vivo. Oligosaccharides were given at 20 mg/kg/d for 2 weeks and the data again showed that octasaccharides significantly reduced microvessel density around the fiber (P = 0.03). In a more complex model, where angiogenesis was induced by a broad spectrum of growth factors, including vascular endothelial growth factor, we implanted H460 lung carcinoma cells in hollow fibers and treated the animals with oligosaccharides at 20 mg/kg/d over 3 weeks. Octasaccharides reduced the microvessel density to that of control. Preliminary investigation of 6-O-desulfated heparins showed that these also had antiangiogenic activity. Results: Finally, we examined the inhibitory potential of hexasaccharides and octasaccharides given at 20 mg/kg/d and these inhibited the growth of H460 lung carcinoma in vivo. At clinically attainable concentrations, significant anticoagulation (activated partial thromboplastin time, anti-factor Xa, and anti-factor IIa) was not observed in vitro unless species containing 16 saccharide residues were investigated. Conclusions: Thus, our preclinical data show that heparin octasaccharides represent novel antiangiogenic compounds that can be given without the anticoagulant effects of low molecular weight heparin.

Yes / The polysialyltransferases (polySTs) catalyse the polymerisation of polysialic acid, which plays an important role in tumour metastasis. While assays are available to assess polyST enzyme activity, there is no methodology available specifically optimised for identification and quantitative evaluation of potential polyST inhibitors. The development of an HPLC-fluorescence-based enzyme assay described within includes a comprehensive investigation of assay conditions, including evaluation of metal ion composition, enzyme, substrate and acceptor concentrations, temperature, pH, and tolerance to DMSO, followed by validation using known polyST inhibitors. Thorough analysis of each of the assay components provided a set of optimised conditions. Under these optimised conditions, the experimentally observed Ki value for CMP, a competitive polyST inhibitor, was strongly correlated with the predicted Ki value, based on the classical Cheng-Prusoff equation [average fold error (AFE) = 1.043]. These results indicate that this assay can provide medium-throughput analysis for enzyme inhibitors with high accuracy, through determining the corresponding IC50 values with substrate concentration at the KM, without the need to perform extensive kinetic studies for each compound. In conclusion, an in vitro cell-free assay for accurate assessment of polyST inhibition is described. The utility of the assay for routine identification of potential polyST inhibitors is demonstrated, allowing quantitative measurement of inhibition to be achieved, and exemplified through assessment of full competitive inhibition. Given the considerable and growing interest in the polySTs as important anti-metastatic targets in cancer drug discovery, this is a vital tool to enable preclinical identification and evaluation of novel polyST inhibitors. / Yorkshire Cancer Research, Wellcome Trust

The WHO-designated neglected-disease pathogen Chlamydia trachomatis (CT) is a gram-negative bacterium responsible for the most frequently diagnosed sexually transmitted infection worldwide. CT infections can lead to infertility, blindness and reactive arthritis, among others. CT acts as an infectious agent by its ability to evade the immune response of its host, which includes the impairment of the NF-κB mediated inflammatory response and the Mcl1 pro-apoptotic pathway through its deubiquitylating, deneddylating and transacetylating enzyme ChlaDUB1 (Cdu1). Expression of Cdu1 is also connected to host cell Golgi apparatus fragmentation, a key process in CT infections. Cdu1 may this be an attractive drug target for the treatment of CT infections. However, a lead molecule for the development of novel potent inhibitors has been unknown so far. Sequence alignments and phylogenetic searches allocate Cdu1 in the CE clan of cysteine proteases. The adenovirus protease (adenain) also belongs to this clan and shares a high degree of structural similarity with Cdu1. Taking advantage of topological similarities between the active sites of Cdu1 and adenain, a target-hopping approach on a focused set of adenain inhibitors, developed at Novartis, has been pursued. The thereby identified cyano-pyrimidines represent the first active-site directed covalent reversible inhibitors for Cdu1. High-resolution crystal structures of Cdu1 in complex with the covalently bound cyano-pyrimidines as well as with its substrate ubiquitin have been elucidated. The structural data of this thesis, combined with enzymatic assays and covalent docking studies, provide valuable insights into Cdu1s activity, substrate recognition, active site pocket flexibility and potential hotspots for ligand interaction. Structure-informed drug design permitted the optimization of this cyano-pyrimidine based scaffold towards HJR108, the first molecule of its kind specifically designed to disrupt the function of Cdu1. The structures of potentially more potent and selective Cdu1 inhibitors are herein proposed. This thesis provides important insights towards our understanding of the structural basis of ubiquitin recognition by Cdu1, and the basis to design highly specific Cdu1 covalent inhibitors. / Der Krankheitserreger Chlamydia trachomatis (CT) - ein gramnegatives Bakterium - ist verantwortlich für die häufigste sexuell übertragene Infektionskrankheit weltweit, die CT basierte Chlamydiose. Sie wird von der Weltgesundheitsorganisation zu den vernachlässigten Krankheiten gezählt. CT Infektionen können unter anderem zu Unfruchtbarkeit, Erblindung und reaktiver Arthritis führen. CT agiert als Krankheitserreger mittels seiner Fähigkeit, die Immunantwort des Wirts zu umgehen. Dies umfasst unter anderem die Schwächung und Störung der NF-κB vermittelten Entzündungsantwort und des Mcl1 pro-Apoptoseweges über ihr deubiquitinierendes, deneddylierendes und trans-acetylierendes Enzym ChlaDub1 (Cdu1). Die Expression von Cdu1 ist aber auch mit der Fragmentierung des Golgi-Apparates des Wirtes verknüpft, ein Schlüsselprozess bei Infektionen mit CT. Cdu1 ist daher vermutlich ein attraktives Zielprotein für die Entwicklung von Wirkstoffen, um CT Infektionen zu behandeln. Eine Leitstrukturverbindung zur Entwicklung neuer wirksamer Inhibitoren war bislang jedoch noch nicht bekannt. Sequenzvergleiche und phylogenetische Untersuchungen verorten Cdu1 im CE Clan der Cysteinproteasen. Die Adenovirus-Protease (Adenain) gehört ebenfalls diesem Clan an und besitzt strukturelle Ähnlichkeit mit Cdu1. Unter Ausnutzung der topologischen Ähnlichkeiten der aktiven Zentren von Cdu1 und Adenain wurde ein Target-Hopping Ansatz mit einem klar definierten und fokussierten Satz von bei Novartis entwickelten Adenain-Inhibitoren verfolgt. Die hierbei identifizierten Cyano-Pyrimidine stellen die ersten kovalenten Inhibitoren von Cdu1 dar, die an das aktive Zentrum von Cdu1 binden und es direkt adressieren. Hochauflösend wurden Kristallstrukturen sowohl von Komplexen von Cdu1 mit kovalent gebundenen Cyano-Pyrimidinen als auch mit Cdu1’s natürlichem Substrat Ubiquitin bestimmt. Die Kristallstrukturdaten dieser Doktorarbeit in Kombination mit Enzymassays und kovalenten Docking-Studien liefern wertvolle Hinweise bezüglich der Aktivität des Enzyms, der molekularen Substraterkennung, der Flexibiliät der Proteintasche rund um das aktive Zentrum und potentielle Hotspots für die Wechselwirkung mit Liganden. Ein strukturbasiertes Wirkstoffdesign erlaubte die Optimierung des Cyano-Pyrimidin-basierten Molekülgerüstes, die zu der Entwicklung der HJR108 Verbindung führte. Es ist das erste Molekül seiner Art, das speziell dazu entworfen wurde Cdu1 zu inhibieren. Strukturen potentiell noch wirksamerer und selektiver Cdu1 Inhibitoren werden in dieser Arbeit vorgeschlagen. Diese Dissertationsschrift liefert somit wertvolle Beiträge zum Verständnis der strukturellen Grundlagen der molekularen Erkennung von Ubiquitin durch Cdu1 und Hinweise, die die Entwicklung hoch-spezifischer kovalenter Cdu1 Inhibitoren erlauben sollten.