Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第16879号 / 工博第3600号 / 新制||工||1544(附属図書館) / 29554 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 金谷 利治, 教授 長谷川 博一, 教授 辻井 敬亘 / 学位規則第4条第1項該当

Abstract Collagen prolyl 4-hydroxylase is the key enzyme in the biosynthesis of collagens, a family of extracellular matrix proteins. Vertebrate collagen prolyl 4-hydroxylases are α2β2 tetramers, the β subunit being identical to the multifunctional protein disulphide isomerase (PDI). Several isoforms of the catalytic α subunit have been identified in various organisms. Prolyl 4-hydroxylases have also been isolated from plants, where they hydroxylate proline-rich structural glycoproteins of the cell walls. The structural and functional properties of the peptide-substrate-binding domain of human collagen prolyl 4-hydroxylase are characterized here. Data obtained from NMR studies indicate that the domain consists of five α helices and one short β strand, this structure being quite different from those of other proline-rich peptide-binding modules. Several residues involved in the binding of a short synthetic peptide were also identified by NMR. Kd values for the binding of several synthetic peptides to the α(I) and α(II) domains were determined by surface plasmon resonance and isothermal calorimetry, and the results indicated that the binding properties of the type I and type II collagen prolyl 4-hydroxylase tetramers can mainly be explained by the binding of peptides to this domain rather than to the catalytic domain. The peptide-substrate-binding domain of human type I collagen prolyl 4-hydroxylase was also crystallized. The crystals were well ordered and diffracted to at least 3 Å, the asymmetric unit most probably containing a domain dimer. The genome of Arabidopsis thaliana was found to encode at least six putative prolyl 4-hydroxylase polypeptides, one of which was cloned and characterized here as a recombinant protein. All the catalytically critical residues identified in animal prolyl 4-hydroxylases were also conserved in this plant prolyl 4-hydroxylase, and their mutagenesis led to inactivation of the enzyme. The recombinant plant enzyme was effective in hydroxylating poly(L-proline) and several synthetic proline-rich peptides. Surprisingly, contrary to previous reports on plant prolyl 4-hydroxylases, the collagen-like peptides were found to be good substrates, the enzyme preferentially hydroxylating prolines in the Y positions of the -X-Y-Gly- triplets, thus resembling the vertebrate collagen prolyl 4-hydroxylases even in this respect. The recombinant plant prolyl 4-hydroxylase also hydroxylated peptides representing the N and C-terminal hydroxylation sites present in the hypoxia-inducible transcription factor α. The fact that these peptides contain only one proline residue indicated that a poly(L-proline) type II conformation was not required for hydroxylation.

Hyperuricemia and local temperature changes in the joints of the extremities are known to be responsible, in part, for the development of gouty arthritis. No satisfactory explanation is yet available for (1) the selective deposition of monosodium urate monohydrate (MSUM) crystals in connective tissues (2) the increased incidence of gout in the later years of life and (3) the increased incidence of MSUM crystal deposition in connective tissues after trauma and in joints with preexisting disease. It is possible that the alterations in composition of the non-fibrillar matrix of cartilage and synovial fluid which are thought to occur with ageing, trauma or preexisting disease, may predispose these tissues to crystal deposition. The objectives of this study were to determine the effect of the cartilage and synovial fluid components, chondroitin sulfate, hyaluronic acid, proteoglycan monomer, proteoglycan aggregate, phospholipids and albumin on the growth of MSUM. The degradation of MSUM solutions was studied under sterile and non-sterile conditions to determine the possible causes of degradation and to define the time span of crystal growth experiments. The rate of degradation of MSUM solutions increased with an increase in temperature. The concentration of MSUM in solution fell sharply after autoclaving and solutions stored in containers with rubber closures showed greater degradation of MSUM than autoclaved solutions stored in all-glass containers. Rubber stoppers apparently absorbed MSUM from solution. The degradation of MSUM solutions was thought to be due to both bacterial consumption and chemical decomposition in non-sterile solutions but was due only to chemical decomposition in sterile solutions. The aqueous solubility of MSUM was determined at different temperatures and in the presence of varying concentrations of sodium chloride. Sodium chloride suppressed MSUM solubility. The aqueous solubility of MSUM was also determined in the presence of several connective tissue components at 37°. Chondroitin sulfate (CS) decreased the saturation solubility of MSUM probably due to the sodium present in the CS samples. Proteoglycan aggregate, proteoglycan monomer, hyaluronic acid an albumin resulted in very slight increases in the solubility of MSUM. The growth kinetics of MSUM was studied using the seeded growth technique. An equation of the general form: [formula omitted] was used to determine the overall growth rate constant, [formula omitted]. Linear plots of the integrated form of the second order growth equation gave the best fit between the points and gave reasonably constant values for [formula omitted] determined at a given initial supersaturation concentration and varying seed amounts. An induction period or a period of slow growth was observed at both the initial supersaturation concentrations studied. The length of the induction period was inversely proportional to the added seed amount. Differing concentrations of additives were included in the growth medium and K' determined. Chondroitin sulfate (CS) significantly increased the growth rate constant for MSUM growth. However, the proportion of CS decreases in aged and osteoarthritic cartilage and thus a decreasing proportion of a growth accelerator is unlikely to be a factor in the deposition of MSUM in cartilage. CS has been found in the synovial fluid of arthritic joints and may act as an MSUM growth accelerator in this medium. Hyaluronic acid (HA) and albumin caused significant inhibition of the growth of MSUM crystals. This effect may be due to the adsorption of these molecules onto the MSUM seed crystals resulting in the poisoning of the active growth sites on the crystal surface. Cartilage HA and synovial fluid albumin levels are increased in aged and/or diseased joints. Increased proportions of growth inhibitors do not offer likely explanations of crystal deposition in joint tissues. At concentrations of 0.1-1.0 mg mL⁻¹ proteoglycan monomer (PGM) and proteoglycan aggregate (PGA) slightly increased the MSUM growth rate constant but this increase was statistically insignificant. The two phospholipids, phosphatidylcholine and phospha-tidylserine increased the growth rate constant of MSUM. Phosphatidylserine, however, did not significantly increase the growth rate constant at the concentrations studied. It is possible that the raised levels of phospholipids in aged or diseased cartilage and synovial fluid could accelerate the growth of MSUM crystals resulting in MSUM deposition in these tissues. / Pharmaceutical Sciences, Faculty of / Graduate

No description available.

No description available.

No description available.

Semicrystalline ionomers are an important class of polymers that are utilized in a wide range of applications. The particular end-use applications of these materials are determined by their chemical, physical, and thermomechanical properties which are directly related to their crystallization behavior. It is therefore critical to identify structure-property relationships for these materials. Sulfonated syndiotactic polystyrene (SsPS) is used as a model semicrystalline ionomer and two approaches are utilized to control the rate of crystallization of the SsPS ionomer in the presence of ionic aggregates. The first approach investigates the effect of the incorporation of nanoscale particles, montmorillonite clay, on the crystallization behavior of SsPS. The morphology of the ionomer clay hybrids were studies via TEM and WAXD while the crystallization behavior of SsPS in the presence of the clay was evaluated using DSC. It was found that the SsPS matrix containing 5 wt.% organically-modified clay crystallized more rapidly than the sPS homopolymer containing the same clay content. This behavior is attributed to the presence of homogenously dispersed nanoscale clay platelets that act as nucleation sites distributed throughout the ionomer matrix. The second approach that employed involved the manipulation of SsPS ionomer architecture via a controlled placement of the ionic sulfonate groups along the polymer backbone. A post-polymerization sulfonation technique was developed to place the sulfonate groups along the homopolymer backbone in a non-random fashion leading to a pseudo-block ionomer architecture. The crystallization behavior of the non-randomly sulfonated SsPS ionomer is compared to randomly sulfonated SsPS using differential scanning calorimetry. The morphologies of the two ionomers were studied using SALLS and SAXS. We have found that the non-randomly sulfonated SsPS ionomer crystallizes much more rapidly than the randomly sulfonated ionomer. The more rapid crystallization behavior of the non-random ionomer to the presence of longer sequences of unsulfonated homopolymer that are able to readily organize into crystalline structures than the random SsPS ionomer containing the same ionic content. / Ph. D.

The applicability of the conventional Hoffman-Weeks (HW) linear extrapolation for the determination of the equilibrium melting temperatures of semicrystalline polymers is critically reviewed. It is shown that the linear extrapolation of observed melting temperatures cannot, in general, provide a reliable estimate of the equilibrium melting temperature. A more rigorous nonlinear HW analysis is proposed in this dissertation, which yields more accurate estimates of the equilibrium melting temperatures for semicrystalline polymers. The proposed nonlinear HW analysis is successfully applied to the cases of isotactic polypropylene and poly(ethylene oxide). The predicted initial lamellar thickness as a function of the crystallization temperature matches well with experimental results and/or literature values. Results based on the nonlinear HW analysis are consistent with those obtained from the analysis of the temperature dependence of the crystal growth rates. The general applicability of the Lauritzen Hoffman (LH) secondary nucleation theory is also addressed for isotactic polypropylene and poly(ethylene oxide). While the LH theory provides an excellent account of the temperature dependence of spherulitic growth rates and ratio of nucleation constants for different regimes, it appears not to yield a meaningful value for the substrate length, L, for poly(ethylene oxide). In a second project, the effects of structural and topological constraints on the morphology, melting and crystallization behavior of ethylene/styrene copolymers are investigated. During cooling from the melt, the longest ethylene sequences crystallize into lamellae in the primary crystallization process, while the shorter ethylene sequences are suggested to form fringed micelles in the secondary crystallization process. Kinetic studies indicate that secondary crystallization is characterized by an Avrami exponent of ½ which is consistent with a one dimensional, diffusion controlled growth. The increase in the melting temperature of secondary crystals with crystallization time is tentatively explained by a decrease in the molar conformational entropy of the remaining amorphous fraction as a result of secondary crystallization, although the possible role of an increase of crystal lateral dimensions with time cannot be rigorously ruled out. / Ph. D.

M.S.

No / The thermodynamic status of -carbamazepine has been clarified using equilibrium solubility measurements, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), heated X-ray powder diffraction (XRPD), and temperature-controlled X-ray scattering techniques. -Carbamazepine is the least stable of the three well-characterized anhydrous polymorphs of carbamazepine at 25°C. In addition, it was confirmed that -carbamazepine undergoes an exothermic transition to -carbamazepine at 130°C. The novel technique of time-resolved simultaneous small- and wide-angle X-ray scattering has been successfully applied to monitor this transition in situ. It was concluded that -carbamazepine has a monotropic relationship with -carbamazepine.