Thesis (doctoral)--Universität Hamburg, 1980.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyses the first step in purine salvage. A complete deficiency of the enzyme results in the devastating neurological symptoms of the Lesch-Nyhan syndrome. The Lesch-Nyhan syndrome is characterised by purine overproduction leading to, hyperuricemia and gout and a central nervous system disorder characterised by severe, spasticity, choreoathetosis, mental retardation and compulsive self-mutilatory behaviour, A partial deficiency of the enzyme results in purine overproduction, gout and occasionally, mild neurological symptoms. Patients are spared the compulsive self-mutilation of the Lesch-Nyhan syndrome. The major part of the thesis consists of the characterisation of the molecular defects in nine patients with the Lesch-Nyhan syndrome. The polymerase chain reaction was used to amplify reverse transcribed HPRT mRNA. The coding region of the amplified HPRT cDNA was either directly sequenced, or cloned and sequenced. All the mutations characterised were insertion or deletion events which resulted in premature termination of the predicted protein. Three patients were found to have a deletion of exon 7, two patients had single base insertions, while two patients appeared to have a complete deletion of the HPRT gene. An insertion in one patient was the result of a mutation within. intron 6 which created a new splice donor site. The new splice donor site in concert with a cryptic splice acceptor resulted in the creation of a new exon. A deletion of exons 2, 3 and 4 in another patient was found to lead to the alternative splicing of exon 5. These unusual splice junction mutations provided in viva support for the exon definition model of pre-mRNA splicing.

The patient (T.K.), was first diagnosed as having a partial hypoxanthine-guanine phosphoribosyltransferase deficiency in 1978 when he was 20 years old. At presentation, he complained of a colicky loin-pain which radiated into his groin, and that he had had dark urine for a month. He was shown to have haematuria and urate crystalluria, and had a serum urate of 0.8mmol/l (reference range 0.12-0.5mmol/l). The diagnosis was confirmed by demonstrating a haemolysate hypoxanthine-guanine phosphoribosyltransferase activity of 550μU/mg Hb (reference range 1680-2480μU/mg Hb). Studies to determine whether the low hypoxanthine-guanine phosphoribosyltransferase activity was caused by an altered Kₘ of the enzyme for one of its substrates, showed that there was substrate inhibition of the enzyme activity by hypoxanthine. This thesis examines the patient and the variant HPRT at three levels. Firstly, a detailed and comprehensive study of the kinetic properties of the variant enzyme was made. The novel feature of the kinetics is the presence of substrate inhibition by the purine bases, with a true Kᵢ value for hypoxanthine of 80± 20μM, and a normal value for the true Kₘ. The pattern of substrate inhibition is characteristic of that associated with the formation of a dead-end complex and double inhibition experiments indicate that the form of this complex is enzyme-hypoxanthine-PPi. These unusual kinetic properties provided an opportunity to study the order of substrate binding in a way not possible for the normal enzyme and showed an ordered sequential reaction mechanism. Some limited protein-structural studies were performed and showed an altered electrophoretic mobility for the variant enzyme in non-denaturing gels. Secondly, the purine metabolic pathways in cultured cells, derived from T.K., from a patient with the Lesch-Nyhan syndrome, and from normal individuals, were studied. The cells were labelled with precursors of the de novo or of the salvage pathways, usually in the presence of a reference label, and sometimes in the presence of inhibitors of the various steps in the purine metabolic pathways. Hypoxanthine salvage was about 10% of that of control cultures. The growth of cells in a variety of selective media was also studied. Thirdly, as physician in charge of T.K., I could monitor the progress of his hyperuricaemia and observe the effects of therapy throughout the duration of this project.

Thesis (Ph. D.)--Virginia Commonwealth University, 2008. / Prepared for: School of Pharmacy. Bibliography: leaves 170-178. Available online via the internet.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves 120-139).

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 135 pages. Includes Vita. Includes bibliographical references.

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Each year in the U.S., approximately 7-8 million patients with non-traumatic chest pain visit hospital emergency departments (ED) for medical evaluation. It is estimated that approximately 2-5% of these patients are experiencing acute cardiac ischemia, but due to the shortcomings of current test methods, they are incorrectly diagnosed and discharged without appropriate treatment provided, thus leading to poor patient outcome and potential medical malpractice litigation.The goals of this research were to evaluate plasma samples for potential biomarker(s) of acute cardiac ischemia prior to heart tissue necrosis, and to ultimately develop a rapid method for detection of the potential biomarker(s) in human plasma. Initial experiments were performed using the mouse model, with subsequent evaluations on human plasma samples using high performance liquid chromatographic ultraviolet detection (HPLC-UV). The final phase of this research involved the development of a rapid luminometer test method (An HPLC-UV detection method was developed and utilized for inosine, hypoxanthine and other adenosine triphosphate (ATP) catabolic by-products in Krebs-Henseleit (Krebs) buffer solution, with analysis on perfusate samples from isolated mouse hearts undergoing 20 min acute global ischemia. The HPLC-UV method was modified for subsequent use on human plasma samples, obtained from hospital emergency department (ED) patients presenting with non-traumatic chest pain (potential acute cardiac ischemia) and from healthy normal individuals. The HPLC-UV (component quantification) and HPLC-MS (component identification) test methods utilized C18 column technology, mobile phases consisting of aqueous trifluoroacetic acid (0.05% TFA in deionized water pH 2.2, v/v) and methanol gradient to achieve component separation, with both utilizing simple sample preparations (e.g. direct injection of Krebs perfusate samples and centrifugal membrane filtration on plasma samples).Results of the animal experiments using isolated mouse hearts undergoing 20 min acute global ischemia demonstrated significant levels of endogenous inosine effluxed from the heart tissue, indicating its use as a potential candidate biomarker of acute cardiac ischemia. The HPLC results from human plasma representing ED non-traumatic chest pain patients demonstrated elevated levels of inosine (hypoxanthine precursor) and significant levels of hypoxanthine, which provided additional support for the use of these candidate biomarker(s) as a potential diagnostic tool for the initial acute cardiac ischemic event, prior to heart tissue necrosis.The final phase of this research focused on the development of a rapid, simple and sensitive chemiluminescence test method. Using a microplate luminometer with direct injectors and continuous mixing, the measurement of inosine and hypoxanthine in human plasma was achieved for healthy normal individuals and on patients with confirmed acute MI, with an analysis time of less than 5 minutes. The utility of this rapid luminescence technique would be the potential use at point-of-care (POC) services (e.g. hospital clinical laboratory or emergency medical services) as part of the initial ED treatment protocol on patients presenting with non-traumatic chest pain and signs/symptoms of acute myocardial ischemia or acute MI.

An estimated 1,735,350 new cancer diagnosis and 609,640 cancer related deaths are predicted to occur in the United States in 2018. To improve patient prognosis, biomarkers are needed to identify cancer in early stages. When diagnosed at an early stage, cancer is more likely to respond to treatments and patients have a higher survival rate. Consequently, there is an ever-present need to identify biomarkers that can aid in the detection of cancer. Additionally, there is a paradigm shift in the field of cancer treatment towards immunotherapy. Traditional cancer treatments include chemotherapy, radiation, and hormone therapy and are not cancer-specific, which leads to bystander effects on the patient<&trade>s normal organs that often harm the patient and create unnecessary hardship. To alleviate this, immunotherapy utilizes a patient<&trade>s own immune cells to attack and destroy cancer cells via cancer-specific biomarkers. These biomarkers are ideally on the surface of cancer cells and absent from the patient<&trade>s normal cells to avoid healthy tissue destruction. With this new therapy, there is a recent push to find surface antigens for immunotherapy techniques.This dissertation describes the characterization of HPRT as a diagnostic and therapeutic biomarker for the detection and possible treatment of hematological and solid malignancies. We describe the general upregulation of HPRT upon malignancy and show that this elevation in protein expression is independent of stage, which indicates that it would be useful as an early stage diagnostic companion tool. We have preliminarily linked the elevation in HPRT to a mutation in one of its prime transcription factors, p53. Specific mutation in p53 called Gain of Function mutations have shown to influence salvage pathway enzyme expression, and we have shown that mutations in p53 are relevant to the elevated levels of HPRT within several cancer types. In addition, we also found that HPRT associates significantly with the membrane of several cancer cell lines as well as patient samples. We found that HPRT has insignificant expression on normal cells, which suggests it may be useful as a targetable biomarker for immunotherapy. Throughout our analysis, we also determined that HPRT might have a role in immune regulation as an elevation of the protein correlates to the decrease of several pro-inflammatory genes involved in immune activation. The knowledge gained from the data presented in this dissertation have opened up new functions for HPRT outside of simple nucleotide production and have confirmed that HPRT has a unique role in cancer that has not been previously reported.

Thesis (Ph.D). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2009/06/08). Includes bibliographical references.