Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 205-223). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 1998. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Abstract Lysyl hydroxylase (EC1.14.11.4, LH) catalyzes post-translationally the hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. Hydroxylysyl residues may also be glycosylated by hydroxylysyl galactosyltransferase (EC 2.4.1.50, GT) or galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66, GGT) to form galactosylhydroxylysyl or glucosylgalactosylhydroxylysyl residues, structures unique to collagen. Three LH isoenzymes (LH1, LH2a/2b, LH3) have been characterized so far. We analyzed mRNA levels of these isoforms, as well as the mRNAs of the main collagen types (I, III, IV, V) and the α subunit of PH-4 in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. The mRNA levels of LH1, LH2, and the α subunit of PH-4 showed significant correlation with each other whereas LH3 correlated with none. No correlation was observed between the LH isoforms and individual collagen types. Three human LH isoforms were expressed in different expression systems. The purified recombinant protein produced by LH3 cDNA was found to be the only one possessing LH, GT and GGT activities. The molecular weight of the partially purified LH3 expressed in Sf9 or Cos-7 cells corresponded to about 85 kDa whereas that in E.coli cells was about 81 kDa probably due to a deficiency of glycosylation in bacterial cells. The recombinant protein of C. elegans LH cDNA was expressed in a cell-free translation system and in E.coli cells. The data indicated that the glycosyltransferase activities, GT and GGT, were also associated with this gene product. The sequence alignment of LH isoforms from different species revealed that there are 29 amino acids conserved between human LH3, mouse LH3 and C. elegans LH sequences and scattered evenly in the molecule, but differing from those of LH1 and LH2. In vitro mutagenesis data showed that the amino acids important for the glycosyltransferase activities were located at the amino-terminal part of the molecule, being separate from the LH active site. Mutation of a conserved LH3 specific, non-disulfide linked cysteine to isoleucine caused a dramatic reduction in GT and GGT activity but had no effect on LH activity. Mutations of the amino-terminal DxD motif (D187-191) characteristic of many glycosyltransferases eliminated both GT and GGT activities, showing the importance of this motif for collagen glycosyltransferases and suggesting that it might serve as the Mn2+ binding site in the molecule.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22496号 / 農博第2400号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5276(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 菅原 達也, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The aim of the proposed diploma thesis is to optimize the procedure of collagen isolation from animal tissues and methods for molecular characterization of isolates. In experimental part, isolations of collagens from animal tissues (chicken, hen, carp, and pork skin) were performed. Total protein concentration was determined by Biuret method and by TNBSA method. Both methods were used also in order to study physiological and thermal stability of the isolates under model conditions. Isolated collagens were characterized at molecular level using PAGE–SDS and microfluidic electrophoresis. Further, amino acids composition and microelement content were analyzed. Finally, the stability of isolated collagens in several types of simulated physiological and pathological conditions was tested too. During isolation relatively sufficient collagen yields were obtained (about 15 % of purified collagen per original mass of biological material). In all analyzed tissues Type I collagen was found. Thermal stability of individual samples differed according to biological source type and tissue age. Viscosimetry measurements confirmed higher stability in collagen samples of older animals. Collagenase exhibited the lowest degradation effect to bovine collagen, while selected mixed microbial hydrolases differed according to enzyme preparative type. Incubation of collagen isolates with selected human pathogens confirmed higher resistance of bovine collagen to biofilm formation when compared with the chicken one.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Type I collagen, which consists of two alpha1 chains [α1(I), COL1A1 ] and one alpha2 chain [α2(I), COL1A2 ], is the most abundant member of the collagen family. Interferon-gamma (IFN-γ), which is both an important pro-inflammatory and a anti fibrotic cytokine, coordinately suppresses both α1(I) and α2(I) production primarily at the transcriptional level. Previous work identified a regulatory factor for X-box (RFX) binding site in the first exon of the collagen α2(I) gene. RFX5 complex, consisting of three proteins (RFX5, RFXB, and RFXAP), as well as class II transactivator (CIITA), activates major histocompatibility complex class II (MHC II) transcription during IFN-γ stimulation. This thesis demonstrates that these factors mediate repression of collagen gene transcription in response to IFN-γ. All three RFX5 complex proteins were required for maximum repression of COL1A2 promoter activity. Mutations in regions of RFX5 proteins important for complex formation either reversed repression of collagen transcription or activated collagen transcription, presumably due to dominant negative effects. CIITA, recruited to the collagen transcription start site by RFX5, repressed collagen gene transcription mainly through its acidic and proline-serine-threonine rich domains. Repression of the collagen promoter by CIITA was enhanced in the presence of the RFX5 complex. Inhibition of IFN-γ induced expression of CIITA by RNA interference (RNAi) led to partial alleviation of collagen repression and MHC II activation. IFN-γ decreased collagen transcription at the same time that it increased the expression of RFX5/CIITA complex. In addition, IFN-γ increased the expression of RFXB, but decreased the expression of an RFXB splice variant, RFXBSV. RFXBSV reversed collagen repression by IFN-γ. Both RFX5 and CIITA were present in a large repressor/co-repressor complex containing histone deacetylase (HDAC) and Sin3 proteins. IFN-γ promoted the recruitment of RFX5/CIITA complex as well as HDAC2/Sin3B to the collagen transcription start site, which resulted in the deacetylation of histories surrounding this site. IFN-γ also blocked the occupancy of RNA polymerase II (Pol II) on the collagen transcription start site in conjunction with the increase in CIITA binding. Taken together, these data identify the RFX5/CIITA complex as a repressor of collagen gene transcription. / 2031-01-01

Call number: LD2668 .T4 1957 F73 / Master of Science

published_or_final_version / abstract / Orthopaedics and Traumatology / Master / Master of Philosophy

No description available.

No description available.