Rozprawy doktorskie: „Phosphoribosyltransferase”

1

Catton, Gemma R. "Mechanistic studies on quinolinate phosphoribosyltransferase /". St Andrews, 2007. http://hdl.handle.net/10023/485.

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Catton, Gemma Rachel. "Mechanistic studies on quinolinate phosphoribosyltransferase". Thesis, University of St Andrews, 2008. http://hdl.handle.net/10023/485.

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Quinolinate phosphoribosyltransferase (QPRTase, EC 2.4.2.19) is an intriguing enzyme which appears to catalyse two distinct chemical reactions; transfer of a phosphoribosyl moiety from 5-phosphoribosyl-1-pyrophosphate to the nitrogen of quinolinic acid and decarboxylation at the 2-position to give nicotinic acid mononucleotide. The chemical mechanism of QPRTase is not fully understood. In particular, enzymatic involvement in the decarboxylation step is yet to be conclusively proven. QPRTase is neurologically important as it degrades the potent neurotoxin, quinolinic acid, implicated in diseases such as Huntington’s disease and AIDS related dementia. Due to its neurological importance and unusual chemistry the mechanism of QPRTase is important. Described here is a mechanistic study on human brain QPRTase. Human brain QPRTase was successfully expressed in E. coli BL21 (DE3) from the pEHISTEV-QPRTase construct and the protein was efficiently purified by nickel affinity chromatography. The crystal structure was solved using multiwavelength methods to a resolution of 1.9 Å. Human brain QPRTase was found to adopt an energetically stable hexameric arrangement. The enzyme was also found to exist as a hexamer during gel filtration under physiological conditions. Kinetic studies allowed the measurement of the kinetic parameters for quinolinic acid. The data gave a Km of 13.4 ± 1.0 μM and a Vmax of 0.92 ± 0.01 μM min-1. There was no evidence for cooperative binding of quinolinic acid to the six subunits of the QPRTase hexamer. The enzyme showed maximum activity at approximately pH 6. The active site of human brain QPRTase is a deep pocket with a highly positive electrostatic surface composed of three arginine residues, two lysine residues and one histidine residue. Mutation of these residues resulted in either complete loss or significant reduction in enzymatic activity showing they are important for binding and/or catalysis. A possible mechanism involving QPRTase in the decarboxylation of quinolinic acid mononucleotide was proposed. A series of quinolinic acid analogues were synthesised and tested as inhibitors of QPRTase. The inhibition studies highlighted some key interactions in the active site.

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Marinaki, Anthony Marin. "The biochemical and molecular basis of Hypoxanthine-guanine phosphoribosyltransferase deficiency". Doctoral thesis, University of Cape Town, 1996. http://hdl.handle.net/11427/26969.

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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.

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4

Murungi, Edwin Kimathi. "Purification and characterisation of plasmodium falciparum Hypoxanthine phosphoribosyltransferase". Thesis, University of the Western Cape, 2007. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_9199_1257245819.

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Malaria remains the most important parasitic disease worldwide. It is estimated that over 500 million infections and more that 2.7 million deaths arising from malaria occur each year. Most (90%) of the infections occur in Africa with the most affected groups being children of less than five years of age and women. this dire situation is exacerbated by the emrggence of drug resistant strains of Plasmodium falciparum. The work reported in this thesis focuses on improving the purification of PfHPRT by investigating the characteristics of anion exchange DE-52 chromatography (the first stage of purification), developing an HPLC gel filtration method for examining the quaternary structure of the protein and possible end stage purification, and initialcrystalization trials. a homology model of the open, unligaded PfHPRT is constructed using the atoomic structures of human, T.ccruz and STryphimurium HPRT as templates.

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5

Ford, Barry Noel. "Structure-function relationships in Chinese hamster adenine phosphoribosyltransferase". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ34263.pdf.

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6

Shahabuddin, Mohammed. "Molecular studies on the hypoxanthine phosphoribosyltransferase of Plasmodium falciparum". Thesis, University of Edinburgh, 1990. http://hdl.handle.net/1842/14382.

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Hypoxanthine phosphoribosyltransferase (EC 2.7.2.8) of P.falciparum has been studied for its biological properties and cellular location. The enzyme plays an important role in the parasite's life, and therefore is a putative target for chemotherapy against malaria. Due to the difficulty in obtaining large amounts of the enzyme from the parasite, it was over-expressed in E.coli, first as a fusion protein with E.co/i-j8-galactosidase. This facilitated the one step purification of the protein, using /3-galactosidase substrate affinity chromatography, for making antibodies against the enzyme. The antibodies thus made were used to investigate the cellular location, a prerequisite for a successful drug design against the parasite enzyme. Im-munofluorescent microscopy (IFA) and immunogold electron microscopy revealed that; 1. the enzyme is expressed at all the stages of the parasite's life, 2. the enzyme is concentrated in some vesicular bodies of unknown origin, 3. in the sporozoite, it may be released into the intrapellicular spaces. Subsequently, the enzyme was over-expressed directly in E.coli, as a non-fusion protein and retained its enzymatic activity. This opened the way to study the enzyme for its biochemical properties and structure-activity relationship. The active E.co/i-expressed P.falciparum HPRT compensated a S.typhimurium hpt mutation. The strain, named S.lyphimurium SH4 can now be used to screen large numbers of putative antimalarial drugs which might act against the parasite HPRT. This will be both simple and inexpensive. The cell free extract of induced SH4 was used to study some biochemical properties of the enzyme. Such a study confirmed the finding that unlike any other Plasmodia studied so far, P.falciparum HPRT can use xanthine as its substrate in addition to hypoxanlhine and guanine. However, competitive inhibition studies revealed that hypoxanthine is the most favourable substrate. The possible biological significance of such properties is discussed.

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7

Mittelstädt, Gerd Horst. "Allosteric regulation of the adenosine triphosphate phosphoribosyltransferase from campylobacter jejuni". Thesis, University of Canterbury. Chemistry, 2015. http://hdl.handle.net/10092/10799.

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The enzyme adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyses the first reaction of the histidine biosynthetic pathway. ATP-PRT also represents a metabolic control point, directing the flux of metabolites through this energetically expensive pathway. Two distinctly different forms of ATP-PRT exist, the long form and the short form, which differ in the presence of a C-terminal regulatory domain. In the short form, where this domain is absent, it is functionally replaced by a regulatory protein, called HisZ. ATP-PRT activity is modulated by two layers of regulation: active site inhibition by adenosine monophosphate, which reflects cellular energy levels, and pathway end product feedback inhibition by histidine. In the long form ATP-PRT histidine binds to the allosteric site at the regulatory domain, but the exact nature of the inhibitory mechanism is still debated. This thesis characterises a new member of the ATP-PRT long form from Campylobacter jejuni (CjeATP-PRT) and investigates the molecular mechanisms involved in the feed back inhibition by histidine. Chapter 2 describes the characterisation of the CjeATP-PRT including a detailed description of its crystal structure. The C. jejuni enzyme is similar to the previously described enzymes of the ATP-PRT long form, but exists only as hexameric species under experimental conditions, which contradicts previous assumptions that the hexamer is exclusively inactive. Chapter 3 investigates the catalytic apparatus of CjeATP-PRT by separating the catalytic and regulatory domains of the enzyme for individual study. The isolated catalytic portion of the enzyme, the CjeATP-PRT Core mutant, forms a dimeric species with very limited catalytic capabilities but high substrate and product affnities. The CjeATP-PRT Core characteristics suggest that it exists in a permanently inhibited conformation, highlighting the requirement of the regulatory domain not only for feedback regulation but also for enzyme function. Additionally this supports the evolutionary need for the recruitment of a regulatory apparatus. In chapter 4 a potential intramolecular communication pathway from the allosteric to the active site is probed by the generation of several single site mutations. One of these, CjeATP-PRT R216A, is completely insensitive to histidine inhibition, although this ligand is still able to bind at the allosteric site, which is consistent with the involvement of R216 in the allosteric signal communication. The catalytic abilities of CjeATP-PRT R216A are largely impaired, leading to the assumption that this mutation causes a permanent inhibitory response. In summary this thesis supports the existence of a simple physical regulatorymechanism for the feedback inhibition of the ATP-PRT long form, the change between two different hexamer conformations depending on the presence of the allosteric effector.

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Evans, Laura. "Investigating the role of nicotinamide phosphoribosyltransferase (NAMPT) in cartilage catabolism". Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/58335/.

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NAMPT (nicotinamide phosphoribosyltransferase) is a universally expressed protein elevated in the serum and synovial fluid of rheumatoid arthritis (RA) sufferers. NAMPT is a rate-limiting enzyme in the biosynthesis of (nicotinamide adenine dinucleotide) NAD+, an essential cellular coenzyme, and has also been shown to exert cytokine-like activities as a mediator of innate immunity. However little is currently known of the role of NAMPT in cartilage metabolism. In this thesis, the role and regulation of NAMPT was studied in a variety of model systems. Addition of exogenous (e)NAMPT to fibroblasts in vitro increased MMP-3 release, an effect attenuated by APO866. In vivo studies in a murine arthritis model showed APO866 treatment reduced arthritis index, with a slight reduction in synovial hyperplasia, inflammatory infiltrate and cartilage degradation. In vivo imaging with MMP activatable fluorescent probes showed a reduction in MMP activity in APO866-treated animals compared with placebo. Analyses of archived tissue indicated that APO866 reduced MMP gene expression in affected limbs in a time and dose dependent manner. In a cartilage explant culture system, APO866 significantly attenuated cytokine-mediated proteoglycan (PG) depletion and MMP-2 and -9 release. This was associated with a dose-dependent loss of cell viability. These effects were reversed by co-incubation with NAD+ metabolites NMN and NA, suggesting that cell survival was highly NAD+-dependent, and that cytokine-induced MMP expression could be NAMPT independent. Finally, in vitro studies in RA synovial fibroblasts revealed that intracellular (i)NAMPT is upregulated by cytokine stimulation, with concurrent upregulation of MMPs -1 and -3. The data emphasise the pleiotropic function of NAMPT in health and disease, and suggest a role for extracellular (but not intracellular) NAMPT in cartilage catabolism. Finally, NAMPT inhibition is highlighted as a promising therapeutic strategy for RA.

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Duckworth, Megan Jane Medical Sciences Faculty of Medicine UNSW. "Characterisation of the xanthineguanine phosphoribosyltransferase of helicobacter pylori as a potential therapeutic target". Publisher:University of New South Wales. Medical Sciences, 2008. http://handle.unsw.edu.au/1959.4/43418.

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Helicobacter pylori infects more than half of the global population and causes gastric disorders. The increasing development of antibiotic resistance by the bacterium continues to limit treatment options. The identification and characterisation of novel therapeutic targets are necessary for successful future treatment of the infection. One potential target for therapeutic intervention is the gpt gene encoded by hp0735 (jhp0672) in H. pylori strain 26695 (J99). This gene produces a putative xanthine-guanine phosphoribosyltransferase (XGPRTase), an enzyme of the purine salvage synthesis pathway. This project employed theoretical, molecular and biochemical approaches to investigate features of H. pylori gpt and XGPRTase that will serve to ascertain their therapeutic potential. The production of a functional XGPRTase by H. pylori was investigated in cell-free extracts, and the kinetic parameters of this activity were compared to those of purified rXGPRTase enzyme. The three 6-oxopurine substrates were recognised by rXGPRTase and allosteric kinetics were observed for some substrates of the enzyme in cell-free extracts and for purified enzyme. These observations indicate complex regulation and an influence of cellular interactions on activity. Bioinformatics were employed to analyse XGPRTase phylogeny, and threading techniques used to build a structural model of XGPRTase. The enzyme is significantly divergent from the equivalent mammalian enzyme, and modelling identified specific features of the enzyme. Molecular approaches were utilised to analyse the essential role of gpt in H. pylori survival. These included insertional inactivation of the gpt in wild-type H. pylori strains and in mutants possessing a complementing copy of the gene present at the rdxA locus. No mutants were recovered with inactivated gpt possibly as a result of pleiotropic effects. Plasmid-mediated complementation was attempted employing IPTG-inducible shuttle vectors and did not yield any mutants. Further characterisation of H. pylori XGPRTase was performed by determining the effects of nucleotide monophosphates and purine analogues on enzyme activity. Inhibition by GMP was observed in all cases, however differences in the inhibition by other nucleotide monophosphates were found between cell-free extracts and the recombinant enzyme. Inhibition of rXGPRTase activity was observed by the purine analogue 6-mercaptopurine ribose, a compound that previously has been shown to inhibit H. pylori growth in culture.

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10

Phehane, Vuyisile Ntosi. "The expression and drug targeting of parasitic hypoxanthine-guanine phosphoribosyltransferase (HGPRT)". Doctoral thesis, University of Cape Town, 2002. http://hdl.handle.net/11427/2701.

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Bibliography: leaves 231-243.
We have expressed and purified human, two forms of P. falciparum, and Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HPRT) in E. coli using the pET expression system. The cDNA encoding the ORF of HPRT was amplified by PCR and transformed into E. coli cells using standard methods. Expression was induced by IPTG and reached about 13% of the total cell protein for all four proteins. The HPRTs were purified by nickel affinity chromatography most of the expressed protein could be isolated from the crude supernatant fraction in a soluble form. Human HPRT was active, with activity levels in the region of 38 umoles GMP min⁻¹ mg⁻¹ at 37 ⁰C, which is comparable to published literature values.

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11

Sintra, Pisco João. "Studies on the mechanism of allosteric regulation of M. tuberculosis ATP-phosphoribosyltransferase". Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10046004/.

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Allosteric regulation is an efficient way of controlling enzymatic activity. In Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, ATPphosphoribosyltransferase (ATP-PRT) catalyses the first and committed step of the biosynthesis of L-histidine (L-His). L-His biosynthetic pathway is essential for Mtb and is absent in humans, making ATP-PRT an attractive target for the development of novel antibiotics. ATP-PRT is a hexamer in solution and, like other enzymes regulated via ferrodoxin-like (FL) domains, interconverts between an open active and a closed inactive conformation. Binding of L-His, its feedback allosteric inhibitor, shifts the equilibrium towards the closed state. The exact mechanisms of allosteric control of ATP-PRT activity are currently poorly understood. Likewise, it is unknown if perturbations in the allosteric regulation of ATP-PRT would be detrimental to Mtb. In this work, I characterised the allosteric regulation of ATP-PRT by L-His employing enzymology, biophysics, native mass spectrometry and X-ray crystallography. Using analogues of L-His, I showed that the allosteric inhibition is specific for L-His. A new targeted compound screen for possible allosteric regulators was developed and led to the discovery of 3-(2-Thienyl)-L-alanine (TIH) as an allosteric activator. Kinetic and structural analyses revealed uncoupling between ATP-PRT activity and conformational changes. In order to understand the molecular basis of allosteric regulation of ATP-PRT by L-His, I generated and analysed ATP-PRT variants with point mutations in the allosteric site. These mutations had a direct effect on ATP-PRT stability, activity and allosteric regulation by L-His and TIH. Overall, the results of this work show a complex allosteric regulation of ATPPRT by L-His while revealing the possibility of allosteric activation. Results with the ATP-PRT variants will guide us towards a detailed characterization of the allosteric control of ATP-PRT and its importance for Mtb, which might have implications to other FL domain-containing enzymes and prove useful for drug discovery.

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Colgin, Lorel Melanie. "Spontaneous mutations in aging human renal epithelia in vivo /". Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/6318.

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Junior, Alécio Antonio Pimenta. "Estudos estruturais e correlação com a síndrome urolitíase de mutantes da adenina fosforribosiltransferase humana". Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-29082011-095840/.

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A 2,8-DHA Urolitíase é uma doença resultante de uma desordem hereditária que leva a deficiência de atividade da enzima APRT do grupo das PRTases. Até o momento, foram encontradas 18 mutações em pacientes, das quais 7 são missense. O presente trabalho dedica-se ao estudo funcional e estrutural dessas 7 mutações e da deleção ΔF173. Construções dos mutantes D65V, L110P, M136T, R67Q, R89Q, I112F e F173G foram obtidas no vetor pET-29a(+) e clonados em E. coli. Os protocolos de expressão e purificação foram estabelecidos, onde as enzimas foram obtidas após uma etapa cromatográfica na coluna CHTTM Hidroxiapatita Cerâmica no grau de pureza necessário. A única exceção foi o mutante I112F que se mostrou insolúvel durante a expressão. Um ensaio cinético acoplado através de kit para detecção de PPi foi padronizado e permitiu a caracterização cinética da hAPRT nativa e de seu mutantes. Os mutantes apresentaram perdas de eficiência de uma ordem de magnitude, com exceção do F173G, cujos valores foram comparáveis aos da hAPRT nativa. Ensaios de cristalização foram realizados para todos os mutantes e resultaram em cristais para os mutantes F173G, R67Q e R89Q. Esses cristais foram coletados na linha de Cristalografia de Macromoléculas 2 no LNLS. Os conjuntos de dados estão sendo analisados e as estruturas estão em fase final de refinamento.
2,8-DHA Urolithiasis is a disease resulting from an inherited disorder that leads to deficiency of APRT, an enzyme of the PRTases group. So far, 18 mutations were found in patients, 7 of which are missense. This work is dedicated to the functional and structural study of these 7 mutations and the deletion ΔF173. Mutants constructions D65V, L110P, M136T, R67Q, R89Q, I112F and F173G were obtained in the vector pET-29a (+) and cloned in E. coli. The expression and purification protocols were established and the enzymes were obtained after 1 chromatographic step using the CHTTM Hydroxyapatite Ceramics column in the required purity. The only exception was the mutant I112F that resulted insoluble during expression. A coupled kinetic assay using PPi detection kit was standardized and allowed the kinetic characterization of native hAPRT and its mutants. The mutants had an efficiency loss of an order of magnitude, with the exception of F173G, whose values were comparable to those of native hAPRT. Crystallization trials were performed for all mutants and resulted in crystals for mutants F173G, R67Q and R89Q. These crystals were collected on Macromolecules Crystallography 2 line at LNLS. The datasets are being analyzed and the structures are in the final stages of refinement.

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Nelson, Aileen A. "The significance of adenine phosphoribosyltransferase for DNA excision repair processes in friend erythroleukaemia cells". Thesis, University of Ulster, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241676.

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Mbewe, Boniface. "Cloning, expression, purification and drug targeting of Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT)". Doctoral thesis, University of Cape Town, 2005. http://hdl.handle.net/11427/2696.

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Includes bibliographical references.
The research concerns sub-cloning the gene for HGXPRT from Plasmodium falciparum from a vector with a His-tag facility to one without, expression of the protein in E. coli, and purification. On an analytical scale (40 ml culture), a purification procedure was developed that involves extraction of contaminating proteins by anion exchange chromatography (HGXPRT does not bind under the conditions used), followed by Reactive Red 120 agarose affinity chromatography.

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GIMBERT, LUC. "A propos d'un nouveau cas de lithiase de 2,8 dihydroxyadenine par deficit complet en adenine phosphoribosyltransferase et de son traitement par une dissolution in situ". Aix-Marseille 2, 1989. http://www.theses.fr/1989AIX20401.

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Lorenz, Veronika. "Komplexe Veränderungen in der Genexpression der Ecto-Nukleosid-5-Triphosphat-Diphosphohydrolase bei Hypoxanthin-Phosphoribosyltransferase-Defizienz". kostenfrei, 2008. http://www.opus-bayern.de/uni-regensburg/volltexte/2009/1207/.

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Cookson, Tammie Violet Marie. "Probing the active site of anthranilate phosphoribosyltransferase from Mycobacterium tuberculosis to facilitate novel drug development". Thesis, University of Canterbury. Chemistry, 2013. http://hdl.handle.net/10092/9049.

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Caused by the organism Mycobacterium tuberculosis (Mtu), the globally distributed disease tuberculosis was responsible for the deaths of 1.4 million people in 2011. Anthranilate phosphoribosyltransferase (AnPRT) is an enzyme that catalyses the second committed step of the tryptophan biosynthetic pathway within Mtu, and is a promising target for antibiotics. This research aimed to further understand the mechanics of the AnPRT active site, in order to provide useful information towards AnPRT drug design. AnPRT inhibition and alternate substrates were investigated as well as variant AnPRT proteins, the results of which aided in unravelling a complex active site mechanism and illuminating several decisive inhibition strategies.

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Elangovan, Venkateswaran Ramamoorthi, Sara M. Camp, Gabriel T. Kelly, Ankit A. Desai, Djanybek Adyshev, Xiaoguang Sun, Stephen M. Black, Ting Wang i Joe G. N. Garcia. "Endotoxin- and Mechanical Stress–Induced Epigenetic Changes in the Regulation of the Nicotinamide Phosphoribosyltransferase Promoter". UNIV CHICAGO PRESS, 2016. http://hdl.handle.net/10150/622492.

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Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.

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Gaillard, Catherine. "Contribution a l'etude du role des genes d'adenine phosphoribosyltransferase dans le metabolisme des cytokinines chez les plantes". Paris 11, 1997. http://www.theses.fr/1997PA112034.

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Les cytokinines sont impliquees dans de nombreux aspects de la croissance et du developpement des plantes. Nous avons entrepris de cloner les genes impliques dans l'interconversion entre les formes base, riboside et ribotide des cytokinines dans le but d'elucider leur role dans le transport des cytokinines et la regulation de leurs teneurs dans les plantes. Cette interconversion peut etre assuree par des enzymes de la voie de recuperation des purines. L'apt (adenine phosphoribosyltransferase), qui convertit l'adenine en amp, agit egalement sur les cytokinines. La conversion de la benzyladenine en benzyladenosine-5'-monophosphate est fortement reduite chez le mutant bm3 d'arabidopsis thaliana deficient en activite apt. Ce mutant est male sterile, presente un retard de croissance et est difficile a regenerer en culture in vitro. Trois adnc differents codant pour des apts ont ete identifies chez a. Thaliana. Leur cartographie, comparee a celle du locus bm3, nous a permis de determiner que la mutation affecte le gene apt1. Le sequencage de l'allele apt1-3 du mutant bm3 a revele une mutation ponctuelle au site d'epissage en 5' du troisieme intron. Le gene apt2 a ete caracterise. Il est peu exprime. L'etude de l'expression d'un gene rapporteur gus place sous le controle du promoteur du gene apt2 dans des plantes d'arabidopsis a ete amorcee afin de preciser son profil d'expression. Contrairement a la proteine apt1, la proteine apt2 presente une affinite plus importante pour les cytokinines que pour l'adenine. Des plantes d'a. Thaliana contenant des constructions exprimant des arnm anti-sens du gene apt2 ne presentent pas de phenotype particulier. Des etudes de complementation du mutant apt1-3 ont ete entreprises avec des constructions visant a surexprimer le gene apt2 ou a l'exprimer sous la dependance du promoteur du gene apt1. Ces constructions ont ete introduites dans des plantes d'arabidopsis de type sauvage. Le croisement des transformants primaires contenant les constructions sens et anti-sens avec le mutant apt1-3 permettra d'etudier l'influence du gene apt2 dans le contexte ou le gene apt1 est mute. Afin de faciliter l'etude biochimique de la gametogenese male, la recherche de phenocopies males steriles du mutant apt1-3 a ete entreprise chez nicotiana plumbaginifolia via les strategies de cosuppression et d'expression d'arn anti-sens. Pour cela, deux adnc de n. Plumbaginifolia codant pour des apts ont ete clones par complementation fonctionnelle d'un mutant d'e. Coli. Ces adnc ont ete sequences et sont respectivement homologues aux genes apt1 et apt2 d'a. Thaliana

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LoCoco, Peter M. "Pharmacological Stimulation of Nicotinamide Phosphoribosyltransferase with P7c3-A20 as a Protective Strategy for Paclitaxel-Induced Peripheral Neuropathy". Thesis, The University of Texas Health Science Center at San Antonio, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10273903.

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Improvements in anticancer pharmacotherapy over the past 40 years have led to a steady increase in the number of cancer survivors worldwide. The clinical effectiveness of anticancer agents like the microtubule-stabilizing agent, paclitaxel, ultimately led to their adoption into standard of care regimens for most cancers. What makes these drugs so effective is how they bind to their respective targets to disrupt fundamental cellular processes. For example, by binding to β-tubulin, paclitaxel induces polymerization and stabilization of cellular microtubules, leading to impairments in cellular functions like mitosis and intracellular transport. While an ingenious approach to kill cancer cells, microtubules are ubiquitous in all cell types. Consequently, paclitaxel has a burdensome side effect profile due to its effects on noncancerous cells. The most prevalent nonhematologic side effect is chemotherapy-induced peripheral neuropathy, which arises due to paclitaxel-induced damage to peripheral afferent sensory neurons. Cancer patients with peripheral neuropathy often develop debilitating neuropathic pain and numbness that diminish everyday quality of life. Symptoms intensify as treatment progresses and can persist for months and years beyond treatment. There is no effective treatment or prevention for chemotherapy-induced peripheral neuropathy. Instead, patients must dose de-escalate or discontinue life-saving chemotherapy, which subsequently worsens cancer prognosis. Thus, there is a compelling need to identify novel therapeutic options to prevent or treat chemotherapy-induced peripheral neuropathy so to improve both anticancer treatment and patient quality of life. The goal of this dissertation is to evaluate the neuroprotective efficacy of a first-in-class stimulator of nicotinamide phosphoribosyltransferase, P7C3-A20, against paclitaxel-induced peripheral neurotoxicity. Nicotinamide phosphoribosyltransferase is the rate-limiting enzyme in the salvage pathway for nicotinamide adenine dinucleotide, an indispensable redox biomolecule that drives energy production. We first developed an aggressive model of paclitaxel-induced peripheral neuropathy in adult male rats. Treatment with a near maximally-tolerated dose of paclitaxel produced significant, but recoverable weight loss and leukopenia. Paclitaxel-treated rats exhibited differentially altered nociceptive thresholds to noxious stimuli, including the development of persistent allodynia to mechanical and cold stimulation as well as transient hyposensitivity to heat stimulation. Toxicity associated with paclitaxel treatment required that 25% of the rats be removed from the study. Histological analysis determined that paclitaxel triggered degeneration of intraepidermal nerve fibers and up-regulated expression of activating transcription factor 3 in nuclei of neuron cell bodies residing in the lumbar dorsal root ganglia. Remarkably, daily treatment with P7C3-A20 prevented mechanical allodynia and heat hypoalgesia, and reduced the cold allodynia associated with paclitaxel treatment. P7C3-A20 also prevented intraepidermal nerve fiber degeneration and partially decreased activating transcription factor 3 expression in lumbar dorsal root ganglia neurons. A randomized, double-blind trial determined that P7C3-A20, and another analogue, P7C3-S321, dose-dependently decreased paclitaxel-induced neuropathic pain and intraepidermal nerve fiber loss. Furthermore, P7C3-A20 improved indices of general health and prevented premature death that normally arose because of overt toxicity caused by paclitaxel. P7C3-A20 displayed superior neuroprotective efficacy, while an inhibitor of poly(adenosine diphosphate-ribose) polymerase was completely ineffective. P7C3-A20 stimulated nicotinamide adenine dinucleotide production in vitro following induction of damage with either hydrogen peroxide or paclitaxel, but not under normal conditions. Additionally, P7C3-A20 in vivo stimulated nicotinamide adenine dinucleotide recovery in the hindpaw and sciatic nerve of rats treated with paclitaxel. FK866 blocked P7C3-A20-mediated nicotinamide adenine dinucleotide production in vitro and the neuroprotective effects on peripheral nociceptive neurons in vivo. Although treatment with either nicotinamide or a subthreshold dose of P7C3-A20 alone was ineffective, the combination produced neuroprotection against paclitaxel that was equivalent to that of a maximal dose of P7C3-A20. We also investigated the effects of P7C3-A20 on cancer cell proliferation and on the anticancer efficacy of paclitaxel. Only MDA-MB-231 breast cancer cells demonstrated a slight increase in proliferation by P7C3-A20, but enhanced growth of implanted MDA-MB-231 tumor xenografts was not observed. Furthermore, P7C3-A20 did not diminish the antiproliferative effects of paclitaxel, despite preventing the development of mechanical allodynia in the tumored mice. In conclusion, these studies discovered robust neuroprotective efficacy of P7C3-A20 against paclitaxel -induced peripheral neurotoxicity, likely through the enhancement of nicotinamide phosphoribosyltransferase-mediated recovery of nicotinamide adenine dinucleotide. Based on these results, clinical investigation of P7C3-A20 as a potential treatment option for chemotherapy-induced peripheral neuropathy may be warranted.

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Hyland, Paula Lisa. "Sequence analysis of the adenine phosphoribosyltransferase gene locus in wild-type and thymidine kinase-deficient friend erythroleukaemia cells". Thesis, University of Ulster, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390158.

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Caldas, Victor Emanoel Armini. "Adenina fosforibosiltransferase de Schistosoma mansoni: proposta de detalhamento do mecanismo catalítico por dinâmica molecular". Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-19102011-141742/.

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A Adenina Fosforibosiltransferase (APRT E.C. 2.4.2.7) pertence à família de enzimas Fosforibosil Transferases (PRTase) do Tipo I , que catalisa a conversão reversível de Adenina e 5-fosfo-α-D-ribose-1-difosfato (PRPP) em difosfato e adenosina monofosfato, um importante precursor energético da célula. A APRT integra a via de salvação de purinas, única forma de suprir o balanço de purinas em Schistosoma mansoni. Este trabalho apresenta o isolamento, clonagem, expressão heteróloga e purificação da APRT de S. mansoni a fim de caracterizá-la quanto seus parâmetros físico-químicos. Não se obtendo cristais de proteína, foram elaborados modelos tridimensionais por homologia para estudos de dinâmica molecular e avaliação conformacional via tCONCOORD. A estrutura de APRT humana foi usada como controle nas simulações. Os dados computacionais e de biologia molecular foram comparados entre si para validação mútua e, verificou-se que a análise cuidadosa de dados computacionais é capaz de fornecer informações críticas sobre a APRT, auxiliando e guiando os estudos experimentais. Ainda, as simulações de dinâmica molecular foram capazes de evidenciar a abertura de loops do sítio ativo, explicitar a importância da análise de rotâmeros em modelos, permitindo, então, rearranjar da forma correta resíduos erroneamente modelados. Por fim, um estudo envolvendo mecanismo catalítico sugere a participação de uma molécula de água abstraindo o próton ligado ao N9 da adenina e, para efeito de comparação, um mecanismo alternativo independente desta participação também foi descrito. Ambas as observações expandem a corrente visão sobre o mecanismo catalítico de APRTs.
The Adenine Phoshoribosyltransferase (APRT E.C.2.4.2.7) belongs to the Phosphoribosyltransferase enzyme family (PRTase) of type I which catalyzes the reversible conversion of adenine and 5-phospho-α-D-ribose-1-diphosphate (PRPP) in diphosphate and adenosine monophosphate, an important source of this compound for the cell. APRT is involved in the purine salvage pathway, the only way that Schistosoma mansoni has to supply its purine demands. This work shows the isolation, cloning, heterologous expression and the purification of APRT from S. mansoni in order to establish its physical chemical and kinetics parameters. Since crystallographic structure was not obtained, we built homology models to perform molecular dynamics experiments and conformational evaluation via tCONCOORD. The human APRT structure was used as control in the simulation experiments. The computational and molecular biology data were compared for consistency and the detailed analysis of the former allowed us to improve experimental manipulation of APRT. The molecular dynamics experiments showed the opening of the loops of the catalytic binding site and the key function of rotamers. Finally, we were able to suggest that a water molecule may catch the proton of adenine N9. Both observations improve the current view of APRT catalytic mechanism.

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Livingstone, Emma Kathrine. "Allosteric Regulation of the First Enzyme in Histidine Biosynthesis". Thesis, University of Canterbury. Chemistry, 2015. http://hdl.handle.net/10092/10470.

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The ATP-PRTase enzyme catalyses the first committed step of histidine biosynthesis in archaea, bacteria, fungi and plants.1 As the catalyst of an energetically expensive pathway, ATP-PRTase is subject to a sophisticated, multilevel regulatory system.2 There are two families of this enzyme, the long form (HisGL) and the short form (HisGS) that differ in their molecular architecture. A single HisGL chain comprises three domains. Domains I and II house the active site of HisGL while domain III, a regulatory domain, forms the binding site for histidine as an allosteric inhibitor. The long form ATP-PRTase adopts a homo-hexameric quaternary structure.3,4 HisGS comprises a similar catalytic core to HisGL but is devoid of the regulatory domain and associates with a second protein, HisZ, to form a hetero-octameric assembly.5 This thesis explores the allosteric regulation of the short form ATP-PRTase, as well as the functional and evolutionary relationship between the two families. New insight into the mode allosteric inhibition of the short form ATP-PRTase from Lactococcus lactis is reported in chapter two. A conformational change upon histidine binding was revealed by small angle X-ray scattering, illuminating a potential mechanism for the allosteric inhibition of the enzyme. Additionally, characterisation of histidine binding to HisZ by isothermal titration calorimetry, in the presence and absence of HisGS, provided evidence toward the location of the functional allosteric binding site within the HisZ subunit. Chapter three details the extensive effort towards the purification of the short form ATP-PRTase from Neisseria menigitidis, the causative agent of bacterial meningitis. This enzyme is of particular interest as a potential target for novel, potent inhibitors to combat this disease. The attempts to purify the long form ATP-PRTase from E. coli, in order to clarify earlier research on the functional multimeric state of the enzyme, are also discussed. Chapter four reports the investigation of a third ATP-PRTase sequence architecture, in which hisZ and hisGS comprise a single open reading frame, forming a putative fusion enzyme. The engineering of two covalent linkers between HisZ and HisGS from L. lactis and the transfer of the regulatory domain from HisGL to HisGS, is also discussed, in an attempt to delineate the evolutionary pathway of the ATP-PRTase enzymes. Finally, the in vivo activity of each functional and putative ATP-PRTase was assessed by E. coli BW25113∆hisG complementation assays.

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Dantas, Deyse de Souza. "Caracterização estrutural e bioquimica da hipoxantina-guanina-xantina fosforribosiltransferase". [s.n.], 2008. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314756.

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Orientadores: Gonçalo Amarante Guimarães Pereira, Francisco Javier Medrano Martin
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-11T15:38:56Z (GMT). No. of bitstreams: 1 Dantas_DeysedeSouza_D.pdf: 4937353 bytes, checksum: 5c9b26f6ef8ed34e974ca6c8bb0708f2 (MD5) Previous issue date: 2008
Resumo: Os genes que codificam para a 6-oxopurina fosforribosiltransferase (HPRT, EC2.4.2.8) dos organismos Pyrococcus horikoshii e Schistosoma mansoni foram clonados em vetores de expressão. As proteínas foram expressas e purificadas em larga escala no sistema de expressão de Escherichia coli. Estudos cinéticos mostraram que a enzima de P. horikoshii é capaz de usar hipoxantina, guanina e xantina. Os dois primeiros substratos apresentam uma eficiência catalítica semelhante. A xantina apresenta um valor menor (ao redor de 20 vezes mais baixo), mas a constante catalítica é comparável com a da hipoxantina. A proteína não foi capaz de se ligar a GMP-agarose, mas sim pode ligar o outro substrato da reação reversa, pirofosfato inorgânico, com baixa afinidade (Kd = 4,7 ± 0,1 mM). Os dados de espalhamento dinâmico de luz, gel filtração e espalhamento de raios X a baixo ângulo mostram que esta proteína é um homohexâmero em solução. Este hexâmero é compacto e resistente à ação limitada de enzimas proteolíticas. Estudos de estabilidade frente a agentes químicos mostraram que a proteína é bastante estável resistindo os efeitos da uréia sem desenovelar completamente com a maior concentração do agente (8,0 M). Os dados obtidos com cloreto de guanidina mostraram que a proteína possui, no mínimo, um estado intermediário de desnaturação, como mostram os diferentes perfis obtidos com as diferentes técnicas usadas nos estudos de desnaturação. Os dados preliminares obtidos com a HPRT de S. mansoni mostraram que, na presença da cauda de histidinas, a proteína está presente como um octâmero alongado. Mas, muito provavelmente ela deve ser um tetrâmero. A presença de caudas fusionadas nas proteínas recombinantes pode afetar a estrutura e função das proteínas
Abstract: The genes that code for the 6-oxopurine phosphoribosyltransferase (HPRT, EC2.4.2.8) from the organisms Pyrococcus horikoshii and Schistosoma mansoni were cloned in expression vectors. The proteins were expressed and purified in large scale in the de Escherichia coli expression system. Kinetic studies showed that the enzyme from P. horikoshii is able to use hypoxanthine, guanine and xanthine. The first two substrates show a similar catalytic efficiency. Xanthine show a lower value (around 20 times), but the catalytic constant is comparable to that of hypoxanthine. The protein was unable to bind to GMP-agarose, but was able to bind the other substrate of the reverse reaction, inorganic pyrophosphate, with low affinity (Kd = 4.7 ± 0.1 mM). Dynamic light scattering, gel filtration and small angle X-ray scattering data show that the protein is a homohexamer in solution. This hexamer is compact and resistant to the limited action of proteolytic enzymes. Stability studies with chemical agents showed that the protein is very stable being able to stand the effects of urea without unfolding completely at the highest agent concentration (8.0 M). Data obtained with guanidine hydrochloride showed that the protein presents, at least, one unfolding intermediate state, as can be seen with the different profiles obtained with different techniques used in the unfolding studies. Preliminary data obtained from HPRT from S. mansoni showed that in the presence of the histidine tag, is present as a long octamer. But, most likely it should be a tetramer. The presence of histidine tag fused to the recombinant proteins could affect the structure and function of proteins
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular

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26

Lecomte, Isabelle. "Métabolisme purique chez le pécher. Recherche d'enzymes marqueurs de potentialités de croissance des bourgeons : étude de l'adénine phosphoribosyltransferase et de l'adénosine nucleosidase". Clermont-Ferrand 2, 1999. http://www.theses.fr/1999CLF22165.

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Chez les plantes pérennes, il existe une relation entre la régulation du contenu en nucléosides triphosphates après un traitement exogène à l'adénosine et l'aptitude à la croissance des bourgeons (test nucléotides). Chez le pécher, l'adénosine exogène est préférentiellement recyclée en nucléotides adenyliques par une voie indirecte faisant intervenir une adénosine nucleosidase et une adénine phosphoribosyltransferase. Pendant plusieurs périodes automno-hivernales successives, l'examen de l'activité de ces enzymes a été réalisé au niveau des bourgeons végétatifs et floraux de cette espèce végétale. Un accroissement de l'activité adénosine nucleosidase des le mois d'octobre suivi d'une décroissance rapide en décembre, au moment de la levée de dormance, a été constaté. Cette baisse d'activité spécifique n'ayant pas lieu en conditions de privation de froid, l'activité adénosine nucleosidase peut être considérée comme un marqueur de la levée de dormance des bourgeons de pécher. L'activité aprtase augmente régulièrement dans les deux types de bourgeons du mois d'octobre au mois de février ou mars, ou elle atteint un maximum. Cette évolution fait d'elle un marqueur potentiel de la période de post-dormance des bourgeons. Afin de préciser les rôles respectifs de ces enzymes, leurs purifications ont été entreprises, mais seule l'aprtase a pu être purifiée à homogénéité et faire l'objet d'une étude plus approfondie. Sa masse moléculaire est estimée à 27,0 0,1 kda. Son ph optimal d'activité est de 8,5 et sa vitesse de réaction dépend strictement de la présence de cations divalents (mn 2 + > mg 2 +). Elle présente de fortes spécificité et affinité vis à vis de l'adénine (km = 0,74 m), mais des cytokinines comme la zeatine (km = 0,3 mm) et la benzyladenine sont également des substrats potentiels. Le km mesure vis à vis du prpp est de 20 m. L'adp et l'amp sont seuls inhibiteurs de la réaction, l'inhibition par l'amp étant de type compétitif vis à vis du prpp. L'obtention d'anticorps polyclonaux anti-aprtase de pécher a permis d'effectuer en parallèle des mesures de la quantité d'enzyme présente dans les bourgeons pendant la période automno-hivernale. Cette étude démontre que l'élévation de l'activité spécifique aprtase observée au cours de cette période est corrélée à une synthèse accrue de l'enzyme dans les bourgeons végétatifs et floraux. Grace à l'utilisation des anticorps, une observation des évolutions de la localisation intra tissulaire de l'enzyme a également été réalisée. Une forte présence d'aprtase dans le tissu sous-jacent au bourgeon, confirmée par la mesure d'une activité spécifique élevée dans ce territoire, caractérise un bourgeon végétatif dormant. Un marquage intense au niveau des anthères en décembre correspond au moment où apparait la méiose du bourgeon floral. Ces résultats ont permis une meilleure appréhension des rôles respectifs de l'adénosine nucleosidase et de l'adénine phosphoribosyltransferase dans le déterminisme physiologique de la dormance

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Grant, Donna. "Mutations in the hypoxanthine phosphoribosyltransferase (hprt) gene in T lymphocytes from arthritis patients and in human B lymphoid cell lines exposed to nitric oxide-donating drugs". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape8/PQDD_0001/MQ46576.pdf.

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Kallies, Mathias Sebastian [Verfasser], i George [Akademischer Betreuer] Iliakis. "The effect of high linear energy transfer ionizing radiation on mutation and reversion events at the hypoxanthin-guanin-phosphoribosyltransferase locus / Mathias Sebastian Kallies ; Betreuer: George Iliakis". Duisburg, 2017. http://d-nb.info/1133478794/34.

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Petitgas, Céline. "Etude des mécanismes pathogéniques de la maladie de Lesch-Nyhan en relation avec le système dopaminergique chez un organisme modèle, Drosophila melanogaster". Thesis, Paris Sciences et Lettres (ComUE), 2019. http://www.theses.fr/2019PSLET049.

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L’adénine phosphoribosyltransférase (APRT) et l’hypoxanthine-guanine phosphoribosyltransférase (HGPRT) sont deux enzymes majeures impliquées dans le recyclage des purines chez les Mammifères, une voie métabolique essentielle permettant la récupération des bases puriques dérivées de l’alimentation ou de la dégradation des nucléotides. La voie de sauvetage des purines est en effet moins coûteuse en énergie que la voie de synthèse de novo et son dysfonctionnement induit diverses pathologies. En particulier, des mutations héréditaires supprimant l’activité de l’HGPRT sont associées à la maladie de Lesch-Nyhan (MLN), une pathologie infantile rare liée à l’X caractérisée par une hyperuricémie et de graves troubles neurocomportementaux tels que dystonie, spasticité et automutilations. Des études ont montré que les patients présentent une diminution significative des taux de dopamine dans les ganglions de la base, sans que l’on comprenne clairement le lien entre la neurotransmission dopaminergique et l’absence d’activité de l’HGPRT. L’objectif de cette thèse a donc été d’étudier la relation entre le sauvetage des purines et le système dopaminergique chez un organisme modèle, la drosophile, afin de mieux comprendre les mécanismes impliqués dans la MLN. La drosophile n’a pas d’homologue de l’HGPRT, ce qui suggère que l’homologue de l’APRT, désignée Aprt, est l’unique enzyme de recyclage des purines chez cet organisme. Nos travaux montrent que des drosophiles mutantes déficientes en Aprt présentent des défauts physiologiques en partie comparables à ceux associés à l’absence d’HGPRT chez l’Homme, notamment un taux élevé d’acide urique ainsi que des altérations des marqueurs dopaminergiques et des troubles neurocomportementaux. A l’inverse, des perturbations génétiques du système dopaminergique induisent une diminution de l’expression et de l’activité de l’Aprt. Nos résultats confirment ainsi la conservation d’un lien physiologique entre le recyclage des purines et le système dopaminergique chez la drosophile, et indiquent de plus que cette régulation fait intervenir la signalisation adénosinergique. Ce nouveau modèle pourrait donc s’avérer utile à terme pour identifier de nouvelles cibles thérapeutiques permettant d’améliorer le traitement de cette maladie dramatique
Adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are two major enzymes involved in purine recycling in mammals, an essential metabolic pathway that allows the recovery of free purine bases derived from diet or the degradation of nucleotides. The purine salvage pathway is indeed less energy costly than de novo purine synthesis and its dysfunction induces various pathologies. In particular, inherited mutations suppressing HGPRT enzyme activity are associated with Lesch-Nyhan disease (LND), a rare X-linked metabolic and neurophysiological disorder in children, characterized by hyperuricemia and severe neurobehavioural disturbances such as dystonia, spasticity and compulsive self-injury. Studies have shown that LND patients have markedly reduced dopamine levels specifically in the basal ganglia, but the mechanisms linking the lack of HPRT activity and dopaminergic neurotransmission have not been elucidated to date. In this thesis work, we have been studying the relation between purine metabolism and the dopaminergic system in a model organism, Drosophila, with the aim to find new clues about the mechanisms involved in LND. No HGPRT homologue is present in the Drosophila genome, which suggests that the APRT homologue, named Aprt, is the only purine-recycling enzyme in this organism. Our work shows that Aprt-deficient flies have defects partly comparable to those associated with HGPRT deficiency in humans, notably an increase in uric acid levels, as well as alterations in dopaminergic markers and neurobehavioural defects. Conversely, genetic disruptions of the dopaminergic system decrease the expression and activity level of Aprt. Our results therefore confirm the conservation of a physiological link between purine recycling and the dopaminergic system in Drosophila, and further indicate that this regulation requires adenosinergic signaling. This new model could therefore prove valuable to find new therapeutic targets and possibly improve the cure of this dramatic disease

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Monzani, Paulo Sérgio. "Estrutura cristalográfica da enzima hipoxantina-guanina fosforibosiltransferase (HGPRT) de Leishmania tarentolae complexada com GMP". Universidade de São Paulo, 2003. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-24062008-163341/.

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O presente trabalho teve como objetivos a clonagem, expressão e purificação da proteína HGPRT de Leishimania tarentolae, para a caracterização e cristalização dessa enzima, a fim do seu estudo estrutural e funcional. O gene da HGPRT foi amplificado a partir de uma biblioteca genômica de Leishmania tarentolae da cepa UC Lambda ZAP Express BamHI-Sal3A I. Esse gene foi clonado no vetor de expressão pET29a(+) e usado na transformação da bactéria Escherichia coli BL21 (DE3). Esse sistema de expressão apresentou uma superexpressão da proteína recombinante, que foi purificada em cromatografia de forma iônica, com o uso da coluna de troca anônica POROS 20HQ. A proteína purificada foi utilizada para sua caracterização, como a determinação das constantes cinéticas (Km, Vmax e Kcat) para os diferentes substratos. A proteína foi encontrada como dímero em solução e o pI de aproximadamente 8,2. Além disso, essa proteína foi utilizada nos ensaios de cristalização pelo método de difusão de vapor em gotas suspensas. Os cristais obtidos foram utilizados nos testes da difração de raios-X sendo coletado um conjunto de dados no Laboratório Nacional de Luz Síncroton. Os dados de difração de raios-X foram processados com o uso do pacote de programa HKL. A resolução da estrutura cristalográfica foi obtida pelo método de substituição molecular através do programa AmoRe. Para o refinamento da estrutura foram utilizados os programas de refinamento automático CNS e REFMAC, e a manipulação na estação gráfica foi realizada através do programa O. A estrutura da HGPRT foi resolvida a 2,1 Å de resolução com os fatores de concordância Rwork e Rfree finais de 17,34 e 21,53%, respectivamente.
The aims of this work were the cloning, expression and purification of the protein HGPRT from Leishimania tarentolae, for the characterization and crystallization of the enzyme for it structural and functional study. The full-length hgprt gene was isolated from a leishmania tarentolae UC strain Lambda ZAP Express BamHI-Sal3A I genomic library. This gene was cloned in the expression vector pET29a+, and used in the transformation of the bacteria Escherichia coli BL21(DE3). This expression system presented a super-expression of the recombinant protein, which was purified by ionic change chromatography, utilizing the anionic change column POROS 20HQ. The purified protein was utilized for its characterization, including its kinetics constants (Km, Vmax e Kcat) for different substrates. The protein was found to be a dimmer in solution with pI close to 8.2. Besides, this protein was used in the crystallization assays by the steam diffusion in suspended drop technique. The obtained crystals were utilized in the x-ray diffraction studies. The data collection was performed in the Laboratório Nacional de Luz Síncroton. The X-ray diffraction data were processed utilizing the package program HKL. The crystallographic structure resolution was obtained by the molecular replacement method from the AmoRe program. For the structure refinement, the automatic refinement programs CNS and REFMAC were used, and the graphic manipulation was made through the O program. The structure of the HGPRT was resolved with 2,1 Å of resolution and final agreement factors Rwork and Rfree of 17,34% and 21,53%, respectively.

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Townsend, Michelle Hannah. "The Clinical Significance of HPRT as a Diagnostic and Therapeutic Biomarker for Hematological and Solid Malignancies". Thesis, Brigham Young University, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10846744.

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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’s normal organs that often harm the patient and create unnecessary hardship. To alleviate this, immunotherapy utilizes a patient’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’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.

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Montigny, Jacky de. "Ura5 et ura10, deux genes codant pour deux isoenzymes a activite omp pyrophosphorylase chez la levure saccharomyces cerevisiae : structure, expression et regulation". Strasbourg 1, 1988. http://www.theses.fr/1988STR13198.

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Mitchell, Shaneice Renee. "Preclinical evaluation of NAMPT inhibitor KPT-9274 in Acute Myeloid Leukemia". The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1546527486477125.

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Fattori, Ana Carolina Maragno. "Efeitos da imunização com Adenosina Quinase (AK) e Hipoxantina-Guanina Fosforibosiltransferase (HGPRT) recombinantes de Schistosoma mansoni : controle da infecção murina". Universidade Federal de São Carlos, 2016. https://repositorio.ufscar.br/handle/ufscar/7924.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
The mansoni schistosomiasis is the most important of human helminthiasis. Despite advances in its control this disease continues to spread to new geographical areas. It currently affects more than 250 million people. However, limited options are available for and Praziquantel is the drug of choice. Various authors have been searching new drugs and vaccines to control schistosomiasis. This study aimed to evaluate the effects of a prior immunization with recombinant enzymes of Schistosoma mansoni: Adenosine Kinase (AK) and Hypoxanthine-guanine Phosphoribosyltransferase (HGPRT), which are important for parasite purine metabolism, as well as a MIX of these enzymes, and subsequent challenge with cercariae of the parasite in the control of murine infection. Female Balb/c mice were divided into 5 groups. The groups were enzyme-immunized in three doses and 15 days after the last immunization, animals were infected with S. mansoni. After infection in the 47º day egg count were carried in mice faeces and in the 48º day mice were sacrificed for evaluation of leukocyte numbers (blood and peritoneal cavity), worm burden, antibodies production, cytokines quantification and histopathological analysis of the liver of these animals. Our results strongly suggest that, immunization with a MIX originated in these animals reduction in the number of eggs in faeces by 46% when compared with the animals of the infected group. Animals of the groups immunized with AK, HGPRT and/or MIX seem to induce a reduction in the number of eosinophils in the peritoneal cavity when compared to the animals of the infected group. Concerning worm burden, the animals of the MIX group presented greater reduction (31.27%) when compared to the animals of the infected group. The animals of the immunized groups, AK, HGPRT and/or MIX were capable of producing IgG1 antibodies and IgE anti the enzymes and anti the parasite proteins. The animals of the immunized group MIX showed a slight increase in IL-4 production and observed reduction of IL-10, and in the HGPRT group induced a slight increase on IFN-γ production when in compared with the infected group. In addition, the animals of the AK group showed a decrease in the number of hepatic granulomas in tissue (44,55%) and the eggs present in liver (42,31%). Therefore, it suggests that immunization with these enzymes can contributes to schistosomiasis control, as well as it might helps to modulate experimental infection inducing reduction of physiopathology of this parasitosis.
A esquistossomose mansônica é a mais importante das helmintíases humanas. Apesar dos avanços no seu controle continua se espalhando para novas áreas geográficas. Atualmente afeta mais de 250 milhões de pessoas. Entretanto, opções limitadas estão disponíveis para o tratamento da doença e o único fármaco de escolha é o Praziquantel. Assim, vários estudos têm sido propostos para encontrar novos fármacos e vacinas para combater a esquistossomose. Dessa forma, o presente estudo teve como proposta avaliar os efeitos da imunização prévia com as enzimas recombinantes de Schistosoma mansoni Adenosina Quinase (AK) e Hipoxantina-Guanina Fosforibosiltransferase (HGPRT), que participam do metabolismo de purinas do parasito, bem como com o MIX das duas enzimas, e posterior desafio com cercárias do parasito, para o controle da infecção murina. Camundongos fêmea Balb/c foram divididos em 5 grupos. Os grupos imunizados receberam três doses das enzimas e após 15 dias da última imunização, os animais foram infectados com S. mansoni. Após a infecção, no 47° dia foi realizada a contagem de ovos nas fezes e no 48° dia foi realizada a eutanásia dos animais para avaliação de resposta leucocitária (sangue e lavado da cavidade peritoneal), carga parasitária, produção de anticorpos, quantificação de citocinas e análise histopatológica do fígado desses animais. Os resultados demonstraram que, a imunização com o MIX promoveu nesses animais redução do número de ovos nas fezes de 46% quando comparado com os animais do grupo somente infectado. Os animais dos grupos imunizados com AK, HGPRT e/ou MIX apresentaram diminuição na quantidade de eosinófilos na cavidade peritoneal quando comparados com os animais do grupo somente infectado. Em relação à carga parasitária, os animais do grupo imunizado com o MIX apresentaram maior redução (31,27%) quando comparados aos animais do grupo somente infectado. Os animais dos grupos imunizados com AK, HGPRT e/ou MIX foram capazes de produzir anticorpos IgG1 e IgE anti as enzimas e anti as proteínas do parasito. Os animais do grupo imunizado com o MIX apresentaram aumento discreto de IL-4 e foi observada redução de IL-10, e no grupo imunizado com HGPRT houve aumento discreto de IFN-γ, quando comparados com os animais do grupo somente infectado. Além disso, os animais do grupo imunizado com AK apresentaram redução do número de granulomas hepáticos (44,55%) e de ovos no fígado (42,31%), quando comparados com o grupo somente infectado. Assim, sugere-se que a imunização com essas enzimas pode contribuir para o controle da esquistossomose, bem como auxiliar na modulação da infecção experimental, induzindo redução da fisiopatologia desta parasitose.

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Romanello, Larissa. "Estudos das enzimas adenosina kinase isoforma 1, hipoxantina-guanina fosforibosiltransferase isoformas 1, 2 e 3, adenilsuccinato liase, adenilsuccinato sintetase de Schistosoma mansoni". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-27102016-102455/.

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O Schistosoma mansoni, parasita responsável pela esquistossomose (barriga dágua), doença que afeta cerca de 300 milhões de pessoas em todo mundo, não possui a via de síntese de purinas, dependendo integralmente da via de salvação de purinas para seu suprimento dessas bases. Uma vez que a terapia se resume a administração de um único fármaco, o praziquantel, diversos casos de resistência do parasita a esse medicamento foram reportadas, sendo assim esta via tem sido citada como alvo potencial para o desenvolvimento de novos fármacos contra a doença. As enzimas adenosina kinase (AK), hipoxantina-guanina fosforibosiltransferase (HGPRT), adenilsuccinato liase (ADSL) e adenilsuccinato sintetase (ADSS) são enzimas chave desta via. Este trabalho faz parte de um projeto maior que visa a obtenção de todas as estruturas das enzimas envolvidas na via de salvação de purinas de Schistosoma mansoni. O cDNA correspondente às enzimas foi amplificado e clonado no vetor de expressão pOPIN; as enzimas AK isoforma 1, HGPRT isoforma 1 e ADSL foram expressas em E. coli Lemo21(DE3) e HGPRT isoforma 3 em E. coli B834(DE3); purificadas em coluna de cobalto agarose por afinidade, concentradas e cristalizadas no kit de cristalização Morpheus (Molecular Dimensions) no Oxford Protein Production Facility (OPPF) em Harwell UK. As coletas de dados por difração de raio-X foram realizadas no Síncrotron Diamond Light Source (DLS) - UK. Foram coletadas duas estruturas de ADSL, a 2.36Å de resolução em complexo com AMP e 2.14Å na forma Apo. A análise das estruturas revelou uma estrutura tetramérica bastante conservada entre as ADSLs, sendo este estado de oligomerização requerido, uma vez que resíduos de três das quatro subunidades compõem o sítio ativo. Apesar do sítio ativo ser altamente conservado entre SmADSL e ADSL humana, a interface dimérica dessas enzimas tem se apresentado suficientemente distintas, o que pode representar um potencial alvo para o desenvolvimento de um inibidor. O ensaio de atividade enzimática de ADSL revelou uma reação endotérmica, indicando que a contribuição da entropia relacionada a grande quantidade de moléculas de água presentes no sítio ativo é importante para a reação cinética. Após diversos experimentos de otimização dos cristais de HGPRT1 e aproximadamente 200 cristais testados, foi obtida uma estrutura em complexo com IMP a 2.8Å de resolução. A análise da estrutura revelou uma estrutura tetramérica. Apesar das subunidades não compartilharem o sítio ativo, este estado de oligomerização é requerido, uma vez que resíduos que compõem o sítio ativo também estão envolvidos em interações na interface dimérica, orientando o resíduo invariável Arg206 na direção do sítio ativo. Foram identificadas quatro mutações na região do sítio ativo entre SmHGPRT e HGPRT humana: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. Desta forma, a obtenção das estruturas contribui para o entendimento bioquímico desta via essencial para o parasita e de como este pode ser seletivamente privado de recursos.
Schistosoma mansoni is the parasite responsible for schistosomiasis, disease that affects about 300 million people worldwide, and does not have the purine de novo pathway, depending entirely on the purine salvage pathway to supply its demands on purines. Currently, both direct treatment and most disease control initiatives, rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, has stimulated efforts to develop new drugs for the treatment of schistosomiasis. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenosine kinase (AK), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenylosuccinate lyase (ADSL), adenylosuccinate synthetase (ADSS) are key enzymes in this pathway. This work is part of a larger project aimed at obtaining all the structures of enzymes involved in purine salvage pathway of Schistosoma mansoni. The cDNA corresponding to the enzymes was amplified and cloned in vector pOPIN, AK isoform 1, HGPRT isoform 1 and ADSL were expressed in E. coli Lemo 21 (DE3) and HGPRT isoform 3 in E. coli B834(DE3); purified in cobalt agarose column, concentrated and crystallized in several conditions of the Morpheus (Molecular Dimensions) crystallization kit at the Oxford Protein Production Facility (OPPF) in Harwell UK. The data collection by xray diffraction were performed at Diamond Light Source UK. Two ADSL structures were obtained, ADSL in complex with AMP at 2.36Å resolution and ADSL Apo form at 2.14Å The analysis revealed a tetrameric structure highly conserved between ADSLs, and this oligomerization state is required since residues three of the four subunits comprise the active site. Despite the active site being highly conserved between human ADSL and SmADSL, the dimeric interface of these enzymes it has been shown sufficiently distinct, which may represent a potential target for the development of an inhibitor. The ADSL enzymatic activity assay showed an endothermic reaction, indicating the contribution of the entropy related to the large quantity of water molecules present in the active site is important for the reaction kinetics. After several optimization experiments of HGPRT1 crystals and about 200 crystals tested was obtained a structure in complex with IMP at 2.8Å resolution. The structure analysis revealed a tetrameric structure. Despite the subunits do not share the active site, this oligomerization state is required, since residues that make up the active site are also involved in interactions in dimeric interface, guiding the invariable residue Arg206 toward the active site. Four mutations were identified in the region of the active site between SmHGPRT and human HGPRT: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. These structures increase the important structural information available about the Schistosoma mansoni purine salvage pathway and how it can be selectively private resources.

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Pétriacq, Pierre. "Etude de la biosynthese du nad chez les plantes : conséquences physiologiques de sa manipulation chez Arabidopsis thaliana". Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112188/document.

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Porteur redox intervenant dans nombre de processus métaboliques, le NAD (nicotinamide adénine dinucléotide) est central pour les cellules vivantes. Outre son importance dans le métabolisme oxydoréductif, des données récentes suggèrent fortement d’autres rôles importants pour le NAD dans la signalisation cellulaire. Un système inductible d’enrichissement en NAD en surproduisant la quinolinate phosphoribosyltransférase (QPT) d’Escherichia coli chez Arabidopsis thaliana a permis de mettre en évidence l’implication du NAD dans les mécanismes spécifiques de défenses qui régissent les interactions plante-pathogène. Par ailleurs, une dérégulation de la synthèse de NAD sur l’étape enzymatique catalysée par la QPT endogène d’Arabidopsis thaliana souligne le rôle critique du NAD dans la balance C/N des plantes, en particulier en bouleversant l’assimilation de l’azote en conditions photorespiratoires. Ces travaux nous ouvrent à une nouvelle compréhension des mécanismes de signalisation impliquant le NAD dans les grandes fonctions métaboliques des plantes
Plant development and functions are underpinned by redox reactions which depend on cofactors such as pyridine nucleotides as nicotinamide adenine dinucleotide (NAD). Beside its redox properties, NAD has recently been implicated in cellular signalling. An inducible system based on Escherichia coli quinolinate phosphoribosyltransferase (QPT) overproduction in transgenic Arabidopsis thaliana was set up as a convenient experimental technique to raise NAD content. This build-up highlights the involvement of NAD in plant-pathogen specific defense mechanisms. Furthermore, manipulating endogenous Arabidopsis thaliana QPT levels was used to deregulate NAD production. Such an approach points out the critical role of NAD in C/N interactions by shaking up nitrogen assimilation upon photorespiratory conditions. These results pave the way for a new understanding of signalling mechanisms involving NAD in plants major metabolic functions

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Ramakrishnan, Sathiya. "Structural, functional and evolutionary studies on 6-oxopurine phosphoribosyltransferases (PRTases) /". St. Lucia, Qld, 2002. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16106.pdf.

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Neris, Débora Meira. "Efeito da imunização com enzimas recombinantes do metabolismo de nucleotídeos de Schistosoma mansoni sobre o desenvolvimento da esquistossomose mansônica experimental". Universidade Federal de São Carlos, 2012. https://repositorio.ufscar.br/handle/ufscar/7018.

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Universidade Federal de Sao Carlos
Schistosimiasis mansoni is a neglected chronic parasitic disease that affects thousands of people worldwide, caused by the trematode Schistosoma mansoni. In the infected host the disease is characterized by the presence of granuloma, imunnopathological response of the cellular infiltration against egg antigens. Thus, the host-parasite relation favors hepatosplenomegaly, acite and hepatic fibrosis. Current chemotherapy is based on the use of Praziquantel (PZQ), used against all species of Schistosoma spp for over 30 years. The main issue is that the PZQ is practically inactive against immature schistosomula and favors the resistance growth of the existent lineages, which makes the study for new drugs and vaccines that can contribute to the control of this disease even more urgent. One of the paths on the search for new therapeutic targets is the study of essential enzymes to the S. mansoni. In particular, it is known that the enzymes Adenylate Kinase 1 and 2 (ADK), Uridine cytidine Kinase 1 and 2 (UCK), Hypoxanthine guanine phosphoribosyltransferase (HGPRT) e Purine nucleoside phosphorilase 1 (PNP) are found on the metabolic pathways of the parasite s nucleotide, participating in the metabolism of purines and pyrimidines. Our goals in this study were to assess the immunization with these enzymes, using the S. mansoni cercariae infected murine model, and subsequently analyze the acting in oviposition and growth of adult worms. Our results showed that the immunization in Balb/c mice with the UCK enzyme was capable of inducing a specific immune response, which favored a significant reduction of the parasitic load (adult worms). However, it was not possible to observe significant reduction in the number of eliminated eggs. Regarding the immunization with PNP and HGPRT enzymes, the mice had a reduction in the number of eggs per gram of feces. The data obtained are considered interesting and can be new targets for immunotherapy against schistosomiasis mansoni. Thereby, new assays must be made with different dosages of the enzymes for a better assessment on how these enzymes modulate the parasitic load through the eggs reduction, reduction in the adult worms retrieving, as well as the antibody levels during the murine infection by the S.mansoni.
A esquistossomose mansônica é uma doença parasitária, crônica e negligenciada que afeta milhares de pessoas ao redor do mundo, causada pelo trematódeo Schistosoma mansoni. No hospedeiro infectado a doença é caracterizada pela presença do granuloma, resultado imunopatológico do infiltrado celular contra antígenos dos ovos A quimioterapia atual é baseada no uso do Praziquantel (PZQ), usado contra todas as espécies de Schistosoma spp há mais de 30 anos. O principal problema é que o PZQ é praticamente inativo contra esquistossomulos imaturos e favorece o desenvolvimento de resistência das linhagens existentes. Um dos caminhos na busca por novos alvos terapêuticos é o estudo de enzimas que são essenciais para o S. mansoni. Em especial, sabe-se que as enzimas Adenilato Quinase 1 e 2 (ADK), Uridina Citidina Quinase 1 e 2 (UCK), Hipoxantina-guanina fosforibosiltransferase (HGPRT) e Purina Nucleosídeo Fosforilase 1 (PNP) são encontradas nas vias metabólicas de nucleotídeos do parasito, participando do metabolismo de purinas e pirimidinas. A estratégia de utilizar enzimas do parasito na esquistossomose mansônica murina foi de avaliar uma resposta induzida por estas enzimas quando aplicadas em camundongos BALB/c e desafiados com cercarias de S. mansoni. Desta forma, avaliamos a fase crônica de camundongos imunizados e infectados com S. mansoni, onde foram analisadas amostras parasitológicas, hematológicas, sorológicas e fluidos da cavidade peritoneal. Nosso objetivo neste estudo foi avaliar a imunização com essas enzimas, usando o modelo murino infectado com cercarias de S. mansoni e posteriormente avaliar a ação na oviposição e desenvolvimento de vermes adultos. Nossos resultados demonstraram que a imunização em camundongos Balb∕c com a enzima UCK foi capaz de induzir uma resposta imune específica, a qual favoreceu a diminuição significativa da carga parasitária (vermes adultos). No entanto, não foi possível observar redução significativa no número de ovos eliminados. Em relação à imunização com as enzimas PNP e HGPRT os camundongos que receberam as imunizações com PNP e HGPRT tiveram redução no número de ovos por grama de fezes. Os dados obtidos são considerados interessantes e podem ser considerados novos alvos para a imunoterapia contra a esquistosomose mansônica. Dessa forma, novos ensaios deverão ser realizados com diferentes doses das enzimas para melhor avaliar como essas enzimas modulam a carga parasitária através da redução de ovos, diminuição na recuperação de vermes adultos, assim como os níveis de anticorpos durante a infecção murina pelo S. mansoni.

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Yu-ChingKo i 柯兪靖. "Nicotinamide Phosphoribosyltransferase (NAMPT) expression in Periodontitis and Peri-implantitis". Thesis, 2016. http://ndltd.ncl.edu.tw/handle/kb99jn.

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Campbell, Elizabeth. "Nicotinamide phosphoribosyltransferase interaction with polyphenolic modulator in the presence of ATP". Thesis, 2019. https://hdl.handle.net/2144/36172.

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Nicotinamide adenine dinucleotide (NAD) is critical in human physiology, the molecule is part of redox reactions vital to metabolism and it is a substrate for multiple signaling proteins in the body. During times of stress in the body, which if chronic can lead to lifelong illness, NAD consumption by signaling proteins is increased and NAD concentration can be depleted. Age related illnesses are often the result of compounding stresses placed on the body over time. A novel way to approach understanding age-related illness, such as diabetes type II and neurodegenerative diseases, is through investigation of NAD modulation. The importance of NAD is exemplified by the redundancy of routes in which NAD is produced in vivo. The predominant pathway in humans is the salvage pathway. The rate-limiting enzyme of the salvage pathway and key component of this research is Nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is a 55 kDa homodimer. It contains autophosphorylation and transferase activity which leads to the recycling of NAD in the body. The enzyme’s reaction happens sequentially with autophosphorylation increasing the enzyme’s affinity for the next reactant, phosphoribosyl pyrophosphate (PRPP). When PRPP binds the phosphorylated enzyme the affinity for NAM increases and promotes the conversion of NAM to NNM. Due to the physiological importance of NAD, NAMPT has become an enzyme of interest in multiple fields including metabolism, oncology, and neurology. Identification of NAMPT modulators would aid the understanding of NAMPT’s physiological role as well as NAD modulation in disease. Through years of research, the Almeida Lab at Rhode Island College has identified a small polyphenolic compound, Compound 20, which consistently enhances NAMPT’s activity. Compound 20 is part of the anthocyanin family. Its modulation of NAMPT activity has been studied through enzyme activity coupled fluorescence assays. To move forward with investigations of Compound 20 as an allosteric enhancer of NAMPT, the physical interaction between the enzyme and compound needed to be confirmed. This research aimed to confirm binding between Compound 20 and NAMPT via fluorescence polarization assays. Further, it examined the hypothesis that the autophosphorylation of NAMPT enhanced Compound 20’s binding with the enzyme. Analysis of fluorescence polarization values revealed Compound 20 and NAMPT bind with a high affinity, while there is a low binding affinity between phosphorylated enzyme, NAMPTPO4, and Compound 20. In conclusion, Compound 20’s binding to NAMPT was confirmed. Further the hypothesis that the phosphorylation of NAMPT increased Compound 20’s binding affinity for the enzyme was disproved. Alternative hypotheses were investigated, and it is more likely Compound 20 binds prior to the enzyme’s phosphorylation and reaction onset. Overall this research corroborates Compound 20 as an allosteric modulator of NAMPT. The identification of this enhancer may lead to novel insights on NAMPT’s physiological role, as well insights on the effects of enhanced NAD concentration in human diseases.

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Chen, Shiu-Min, i 陳秀敏. "The study of relationship between hypoxanthine-guanine phosphoribosyltransferase activity and gout or uric acid". Thesis, 2001. http://ndltd.ncl.edu.tw/handle/02530974026597526444.

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碩士
高雄醫學大學
公共衛生學研究所
89
Abstract There are two aboriginal tribes (Bunun, Paiwan) and one non-aboriginal tribe (Fukien-Taiwan) included as the study population. The subjects were used to explore the relationship between the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and gout disease or uric acid level. Comparison the difference of HPRT activity between aborigines and non-aborigines and the related factors are the study major aims. A total of 172 samples included in this study. A questionnaire was completed by the subjects to obtain the social demographic data, and a whole blood was drawn to perform the biochemical and HPRT activity analysis. A screening method for detection of HPRT activity in peripheral blood mononuclear cells (PBMCs) was used by high-performance liquid chromatography (HPLC). The results showed that： (1)The mean HPRT activities of subjects with gout and non-gout were 6.76±3.69 nmol/106 cell/h and 7.36±3.50 nmol/106 cell/h, respectively, and they did not make difference in statistics. (2)The mean HPRT activities of subjects aborigines and non-aborigines were 6.90±3.45 nmol/106 cell/h and 7.32±3.85 nmol/106 cell/h, respectively, and they did not different. (3)The mean HPRT activity of women with gout was 5.48±2.89 nmol/106 cell/h that had significant difference to femeal contol was 7.94±3.78 nmol/106 cell/h. (4)The related factors of enzyme activity were amount smoking, history of gout,and triglyceride. The study’s conclusion was the relationship of HPRT activity and gout or uric acid level had not to arrive at significant, and the reason was maybe we could not avoide counfounding of treatment with gouty drug. Key word: HPRT, HPLC, GOUT, URIC ACID

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Schwab, Thomas [Verfasser]. "Zusammenhang zwischen Quartärstruktur, Stabilität und katalytischer Aktivität am Beispiel der Anthranilat-Phosphoribosyltransferase / vorgelegt von Thomas Schwab". 2010. http://d-nb.info/1008881627/34.

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Deuß, Miriam [Verfasser]. "Untersuchungen zur Struktur, Funktion und Dynamik der Anthranilat-Phosphoribosyltransferase aus Sulfolobus solfataricus / vorgelegt von Miriam Deuß". 2007. http://d-nb.info/984762493/34.

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Wang, Chiung-Pei, i 王瓊珮. "Mutation Spectra Induced by Safrole in Hypoxanthine-guanine Phosphoribosyltransferase Locus of Chinese Hamster Ovary K1 Cells". Thesis, 2000. http://ndltd.ncl.edu.tw/handle/93030672516238781842.

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碩士
國立陽明大學
藥理學研究所
88
Safrole, an essential oil that has been used in cosmetics and as a food flavoring agents, is classified as a rodent hepatocarcinogen. The carcinogenicity of safrole is mediated through 1'-hydroxysafrole formation, sulfonated to an unstable sulfuric acid ester, and consequently forming stable safrole-DNA adducts. DNA adduct is known to be an initial step of mutagenesis and carcinogenesis. In Taiwan, Piper betle inflorescence contains high concentration of safrole (15 mg/g fresh weight), and may contribute to human exposure (420 microM of safrole in saliva) while chewing betel quid with Piper betle inflorescence. We have documented that safrole has potential to form stable safrole-dG adducts in humans oral cancer tissue following betel quid chewing, which may contribute to oral carcinogenesis. However, the role of safrole-DNA adduct in cancer is not known. To gain insight into the mechanisms of safrole-induced mutations, this study has determined the types and location (spectrum) of mutations induced in the coding region of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene by 1'-hydroxysafrole, the reactive metabolite of safrole. CHO-K1 and HFW cells were exposed to 1'-hydroxysafrole(0~1000 microM) for 24 h. Using 32P-postlabeling technique, this study demonstrated that safrole forms the major premutagenic lesion, N2-(trans-isosafrol-3’-yl)deoxyguanosine. The IC50 of 1'-hydroxysafrole in CHO-K1 and HFW cells were 16.1 and 43.2 microM, respectively. The 1'-hydroxysafrole induced safrole-DNA adduct level is significantly correlated to hprt mutation frequency induced by 1'-hydroxysafrole. hprt mRNA from mutant clones were reverse transcribed to cDNA, amplified by PCR and examined for mutations by DNA sequencing. The result indicated 19/23 safrole-induced mutants contained a single base pair substitution. The majority of the mutations at G: C base pair occurred at sites with G in the nontranscribed strand (16/19). G:C to A:T transitions were the most common type of base pair substitution (9/19). The result reflected the specific types of DNA damage produced by safrole. Results from this study further demonstrated the relationship between safrole-DNA adduct and safrole-induced mutagenesis.

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Lorenz, Veronika [Verfasser]. "Komplexe Veränderungen in der Genexpression der Ecto-Nukleosid-5'-Triphosphat-Diphosphohydrolase bei Hypoxanthin-Phosphoribosyltransferase-Defizienz / vorgelegt von Veronika Lorenz". 2009. http://d-nb.info/993681751/34.

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Do, Hong-Hai, i Erhard Rahm. "Flexible Integration of Molecular-Biological Annotation Data: The GenMapper Approach". 2004. https://ul.qucosa.de/id/qucosa%3A32457.

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Molecular-biological annotation data is continuously being collected, curated and made accessible in numerous public data sources. Integration of this data is a major challenge in bioinformatics. We present the GenMapper system that physically integrates heterogeneous annotation data in a flexible way and supports large-scale analysis on the integrated data. It uses a generic data model to uniformly represent different kinds of annotations originating from different data sources. Existing associations between objects, which represent valuable biological knowledge, are explicitly utilized to drive data integration and combine annotation knowledge from different sources. To serve specific analysis needs, powerful operators are provided to derive tailored annotation views from the generic data representation. GenMapper is operational and has been successfully used for large-scale functional profiling of genes.

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Xie, Fu-Bin, i 謝富彬. "Effect of O6-alkylguaninetransferase on the mutational spectrum induced by N-methyl-N?nitro-N-nitrosoguanidine in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase in Chinese hamster ovary cells". Thesis, 1993. http://ndltd.ncl.edu.tw/handle/79164470862931025267.

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