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

dc.contributor.advisorHarley, Eric Hen_ZA
dc.contributor.authorShires, Karen Lesleyen_ZA
dc.date.accessioned2018-01-29T06:45:52Z
dc.date.available2018-01-29T06:45:52Z
dc.date.issued1994en_ZA
dc.description.abstractA 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.identifier.apacitationShires, 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/11427/27031en_ZA
dc.identifier.chicagocitationShires, 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/11427/27031en_ZA
dc.identifier.citationShires, K. 1994. Citrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide production. University of Cape Town.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
dc.identifier.urihttp://hdl.handle.net/11427/27031
dc.identifier.vancouvercitationShires 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/11427/27031en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Chemical Pathologyen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherChemical Pathologyen_ZA
dc.subject.otherCitrulline - Metabolismen_ZA
dc.subject.otherFibroblasts - chemistryen_ZA
dc.subject.otherNitric Oxide - metabolismen_ZA
dc.titleCitrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide productionen_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMSc (Med)en_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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