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Investigation of the Effect of Dimerization on Human α-Galactosidase ActivityDooley, Scott R 01 January 2014 (has links) (PDF)
Fabry disease is an X-linked lysosomal storage disease that results from a deficiency in the enzyme α-galactosidase (α-GAL). α-GAL hydrolyzes α-galactosides, and patients with Fabry disease suffer from an accumulation of these undegraded substrates. Human α-GAL naturally occurs as a homodimer, as determined through SEC and crystallographic analysis. This means its quaternary structure consists of two identical α-GAL subunits that are associated together into a single unit. Other species, such as rice, produce a monomeric form of α-GAL, consisting of only a single subunit.
If α-GAL is functional as both a homodimer and monomer, then how does homodimerization affect the activity of human α-GAL? This can be answered through two model systems. First, a monomeric form of human α-GAL can be produced, testing the activity of human α-GAL in a monomeric state. A variant of α-GAL was engineered (called α-GALF273G/W277G) that appeared promising. Secondly, another system can be produced capable of stabilizing one active site of the dimer and testing the other active site for activity. Another lysosomal enzyme, α-N-acetylgalactosaminidase (α-NAGAL), shares 46% amino acid sequence identity and share 11 of 13 active site residues. Previously, an α-GAL variant (called α-GALE203S/L206A) was produced, that maintained the antigenicity of α-GAL, but had acquired the enzymatic specificity of α-N-acetylgalactosaminidase (α-NAGAL). A heterodimeric form of α-GAL can be produced combining one subunit of α-GAL with the engineered variant. The engineered site can be stabilized, while the wild-type site can be tested for activity. SEC analysis suggests α-GALF273G/W277G is a monomer, and its kinetic properties are reported. Evidence shows monomeric α-GAL could be useful as an improved enzyme replacement therapy. Western blotting and activity assays suggest the presence of the α-GAL/ α-GALE203S/L206A heterodimer.
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Liquid Scintillation Analysis of the Main Environmental Radioisotopes in Air Using a Liquid Collector with Absorption Properties / 吸収溶剤を用いた空気中主要環境放射能の液体シンチレーション分析Kato, Takahisa 23 March 2023 (has links)
京都大学 / 新制・論文博士 / 博士(工学) / 乙第13545号 / 論工博第4208号 / 新制||工||1984(附属図書館) / (主査)教授 米田 稔, 教授 松井 康人, 准教授 福谷 哲 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM
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Transport of Deuterium-Labeled Tocopherols During PregnancyAcuff, Robert V., Dunworth, Robert G., Webb, Lisa W., Lane, Jonathan R. 01 January 1998 (has links)
With use of deuterium-labeled isotopes of RRR-and all-rac-α-tocopheryl acetate, the transport of vitamin E in pregnancy was evaluated to determine whether the placenta discriminates between these compounds. Fifteen pregnant subjects were recruited 5 d before delivery to receive 15, 30, 75, 150, or 300 mg vitamin E/d in capsules containing d3-RRR-α-tocopheryl acetate and d6-all-rac-α-tocopheryl acetate (1:1, by wt). Maternal blood was obtained before dosing, at hospital admission, and at parturition. Cord blood samples were obtained at parturition. Deuterium-labeled and unlabeled tocopherol contents were determined by gas chromatography-mass spectrometry in plasma and lipoproteins (chylomicrons, VLDL, LDL, and HDL). Maternal plasma and lipoproteins obtained at delivery had higher concentrations of d3-RRR-α- tocopherol than d6-all-rac-α-tocopherol regardless of the vitamin E dose administered (P < 0.05). Cord plasma at delivery also had higher concentrations of d3-RRR-α-tocopherol than d6-all-rac-α-tocopherol in plasma irrespective of the dose administered (P < 0.05). In lipoproteins isolated from cord blood, tocopherol concentrations were greatest in the HDL fraction (P < 0.05), whereas in maternal blood they were greatest in the LDL fraction (P < 0.05). We conclude that the placental-fetal unit, the fetal liver, or both further discriminate between RRR- and all-rac-α-tocopherol.
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Protein Kinase C-Mediated Contractile Response of the Rat Vas DeferensAbraham, S. T., Rice, Peter J. 06 August 1992 (has links)
The role of protein kinase C (PKC) in mediating contractile responses in the rat vas deferens was studied. Phorbol-12,13-di-acetate (PDA) in the presence of 20 mM K+ elicited a concentration-dependent response with an EC50 of 190 nM. The non-PKC activator 4α-phorbol (2 μM) was unable to elicit contraction in 20 mM K+ buffer. Incubation of rat vas deferens with the PKC inhibitor iso-H7 (30 μM) attenuated the response to norepinephrine (NE) ane neurokinin A, with maximal effects depressed to 42 and 39% of control, respectively. Responses to 60 mM K+ and 2 μM PDA (20 mM K+) was also significantly inhibited by iso-H7. In the presence of 2 μM PDA and 20 mM K+, the NE concentration-effect curve was shifted 3,6-fold to the right of the control curve in a parallel manner. 4α-Phorbol (20 mM K+) at the same concentration did not produce this effect. These results suggest a significant role for PKC in the contractile response of the rat vas deferens.
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Bradykinin and Tumor Necrosis Factor-α Alter Albumin Transport in Vivo: A Comparative StudySaulpaw, Charles E., Joyner, William L. 01 November 1997 (has links)
These studies indicate that tumor necrosis factor-α (TNFα) alters albumin permeability and unlike bradykinin (BK) the increased albumin permeability lasts for the duration of the application. Neither agonist requires the presence of white blood cells or other blood-borne substances to produce this inflammatory response. These experiments were completed in the in situ, microcannulated, perfused venules of the mesentery in the anesthetized hamster. Albumin transport was measured using intravital fluorescence microscopy, TRITC-labeled albumin, and densitometric tracking. Further, by varying the intravascular pressure, the hydraulic (L(p)(1 - σ)) and diffusive permeability (P0) coefficients of these microvessels were determined. Both BK and TNFα produced an increase in albumin flux, which was dependent upon the dose and time domains. This response was present when the agonists were given by either intra- or extravascular presentation. Both hydraulic coupling and microvascular permeability were increased by BK and TNFα. TNFα increased albumin permeability rapidly and its effect lasted as long as TNFα was present, whereas the increased albumin transport by BK was biphasic. The results implicate a dynamic modification in the microvascular wall to these inflammatory agonists and the mechanism(s) for transduction in the endothelium are quite different.
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Conjugate Additions and Transposition of the Allylic Alcohols of Enol Ethers of 1, 2-Cyclohexanedione.Otoo, Barnabas 18 December 2010 (has links) (PDF)
A variety of protected enolic forms of 1, 2-cyclohexanedione was prepared as substrates for conjugate addition studies using organocopper reagents. The sequence involved the enol ether preparation via the enolate, alkylation with an organometalic reagent, and oxidative rearrangement with pyridinium chlorochromate followed by the conjugate addition reactions. Protection of 1, 2-cyclohexanedione was achieved by reacting with chloro tert-butyldimethyl silane and subjected to alkylation. Steric problems were encountered and so an alternative protective group the methoxymethyl acetal was prepared and studied. Alkylation of these derivatives was successful; however, the oxidation was problematic and although evidence for rearrangement was observed in one case, it did not provide the desired ketone.
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微生物代謝産物サッカロスリオリドBに着想を得たプロドラッグ化に関する研究竹中, 慧 23 March 2022 (has links)
京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23848号 / 薬博第855号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 掛谷 秀昭, 教授 高須 清誠, 教授 大野 浩章 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM
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FBXL16 promotes breast cancer cell growth and diminishes fulvestrant responsiveness by stabilizing ERα proteinShah, Krushangi Nirav 17 May 2022 (has links)
No description available.
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Asymmetric Dihydroxylation and Aziridination of Allenes and Related ChemistryLiu, Renmao 11 May 2007 (has links) (PDF)
A novel method for asymmetric synthesis of α-hydroxy ketone with excellent regio- and stereoselectivity has been established by the systematic investigation of asymmetric dihydroxylation of allenes. The efficiency of kinetic resolution of racemic allenes was also investigated by using the AD reaction on both 1,3-disubstituted and trisubstituted allenes. Steric effects, electronic effects and allene substitution are also discussed. Aziridines were formed by copper-catalyzed intramolecular nitrene addition to alkenes. The carbamate group was used as the tether between the alkene and the nitrene. Subsequent nucleophilic attack of the aziridine was accomplished using RSH, R2NH, N3-,or ROH as the nucleophile. This addition was found to be regio- and stereoselective. This methodology has provided a new strategy for the stereoselective construction of three adjacent functional groups, in particular the 1,2 diamino-3-hydroxy unit. The rhodium-catalyzed intramolecular aziridination of allenic N-sulfonyloxy carbamates has been established. Efficient ring opening of these bicyclic compounds may provide synthetic utility in organic chemistry. The intramolecular aziridination of allenic sulfamate esters was tested on a single example to afford in situ a ring opened product.
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Chronic AMP-Activated Protein Kinase Activation and a High-Fat Diet Have an Additive Effect on Mitochondria in Rat Skeletal MuscleFillmore, Natasha 02 July 2010 (has links) (PDF)
Factors that stimulate mitochondrial biogenesis in skeletal muscle include AMPK, calcium, and circulating FFAs. Chronic treatment with either AICAR, a chemical activator of AMPK, or increasing circulating FFAs with a high fat diet increases mitochondria in rat skeletal muscle. The purpose of this study was to determine whether the combination of chronic chemical activation of AMPK and high fat feeding would have an additive effect on skeletal muscle mitochondria levels. We treated Wistar male rats with a high fat diet (HF), AICAR injections (AICAR), or a high fat diet and AICAR injections (HF+AICAR) for six weeks. At the end of the treatment period, markers of mitochondrial content were examined in white quadriceps, red quadriceps, and soleus muscles, predominantly composed of unique muscle-fiber types. In white quadriceps, there was a cumulative effect of treatments on LCAD, cytochrome c, and PGC-α protein, as well as on citrate synthase and β-HAD activity. In contrast, no additive effect was noted in the soleus and in the red quadriceps only β-HAD activity increased additively. The additive increase of mitochondrial markers observed in the white quadriceps may be explained by a combined effect of two separate mechanisms: high fat diet-induced post transcriptional increase in PGC-α protein and AMPK mediated increase in PGC-α protein via a transcriptional mechanism. These data show that chronic chemical activation of AMPK and a high fat diet have a muscle type specific additive effect on markers of fatty acid oxidation, the citric acid cycle, the electron transport chain, and transcriptional regulation.
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