Citrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide production

 

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dc.contributor.advisor Harley, Eric H en_ZA
dc.contributor.author Shires, Karen Lesley en_ZA
dc.date.accessioned 2018-01-29T06:45:52Z
dc.date.available 2018-01-29T06:45:52Z
dc.date.issued 1994 en_ZA
dc.identifier.citation Shires, K. 1994. Citrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide production. University of Cape Town. en_ZA
dc.identifier.uri http://hdl.handle.net/11427/27031
dc.description.abstract A citrullinemic fibroblast cell line was used in this study to investigate two biochemical pathways involving citrulline. In the first section, the genetic mutation responsible for the argininosuccinate synthetase (-ASS) deficiency (1-5% activity) in this cell line was investigated. PCR analysis of the ASS cDNA revealed that the mRNA coding region (1236bp) was intact, showing no signs of major rearrangements. The ASS cDNA (1307bp) was cloned and sequenced and showed the presence of a single base mutation at position 1045bp, which represented a G->A transition. This mutation resulted in a glycine -> serine amino acid substitution at position 324 in the ASS subunit protein sequence. Although this glycine residue was not found to occur in any potential substrate binding sites, it was shown to be highly conserved among species, indicating a possible role of this amino acid in ASS catalytic activity. In the second section, the presence of the nitric oxide pathway in fibroblasts was investigated. Inducible nitric oxide synthase activity was assayed by measuring the production of ¹⁴C-citrulline from ¹⁴C-arginine after cytokine stimulation. By using the citrullinemic cell line (ASS deficient) any citrulline that may be produced by this pathway would accumulate, allowing detection. Under the assay conditions that were tested, no detectable ¹⁴C-citrulline was formed. Evidence suggests that human fibroblasts have the potential to synthesise nitric oxide, although a more sensitive assay system may need to be employed (longer cytokine activation, nitrite/nitrate detection). en_ZA
dc.language.iso eng en_ZA
dc.subject.other Chemical Pathology en_ZA
dc.subject.other Citrulline - Metabolism en_ZA
dc.subject.other Fibroblasts - chemistry en_ZA
dc.subject.other Nitric Oxide - metabolism en_ZA
dc.title Citrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide production en_ZA
dc.type Master Thesis
uct.type.publication Research en_ZA
uct.type.resource Thesis en_ZA
dc.publisher.institution University of Cape Town
dc.publisher.faculty Faculty of Health Sciences en_ZA
dc.publisher.department Division of Chemical Pathology en_ZA
dc.type.qualificationlevel Masters
dc.type.qualificationname MSc (Med) en_ZA
uct.type.filetype Text
uct.type.filetype Image
dc.identifier.apacitation Shires, K. L. (1994). <i>Citrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide production</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Chemical Pathology. Retrieved from http://hdl.handle.net/11427/27031 en_ZA
dc.identifier.chicagocitation Shires, Karen Lesley. <i>"Citrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide production."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Chemical Pathology, 1994. http://hdl.handle.net/11427/27031 en_ZA
dc.identifier.vancouvercitation Shires KL. Citrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide production. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Chemical Pathology, 1994 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/27031 en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Shires, Karen Lesley AB - A citrullinemic fibroblast cell line was used in this study to investigate two biochemical pathways involving citrulline. In the first section, the genetic mutation responsible for the argininosuccinate synthetase (-ASS) deficiency (1-5% activity) in this cell line was investigated. PCR analysis of the ASS cDNA revealed that the mRNA coding region (1236bp) was intact, showing no signs of major rearrangements. The ASS cDNA (1307bp) was cloned and sequenced and showed the presence of a single base mutation at position 1045bp, which represented a G->A transition. This mutation resulted in a glycine -> serine amino acid substitution at position 324 in the ASS subunit protein sequence. Although this glycine residue was not found to occur in any potential substrate binding sites, it was shown to be highly conserved among species, indicating a possible role of this amino acid in ASS catalytic activity. In the second section, the presence of the nitric oxide pathway in fibroblasts was investigated. Inducible nitric oxide synthase activity was assayed by measuring the production of ¹⁴C-citrulline from ¹⁴C-arginine after cytokine stimulation. By using the citrullinemic cell line (ASS deficient) any citrulline that may be produced by this pathway would accumulate, allowing detection. Under the assay conditions that were tested, no detectable ¹⁴C-citrulline was formed. Evidence suggests that human fibroblasts have the potential to synthesise nitric oxide, although a more sensitive assay system may need to be employed (longer cytokine activation, nitrite/nitrate detection). DA - 1994 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 1994 T1 - Citrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide production TI - Citrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide production UR - http://hdl.handle.net/11427/27031 ER - en_ZA


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