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1
Payne, Tiffany Anne. "Analysis of dirhenium carboxylate : purine dinucleotide adducts." Scholarly Commons, 2006. https://scholarlycommons.pacific.edu/uop_etds/629.
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The discovery of cisplatin, cis-[pt(NH3)2Cl2], as an anticancer agent in 1969 by Rosenbert and his colleagues sparked interest in the area of metal complexes as chemotherapeutic agents. Anticancer dimetal complexes such as Re2(O2CCH2CH3)2Br4·2H2O are proposed to prevent replication of cancel cells by coordinating to the purine nucleobases in DNA. To investigate the interaction between dimetal compounds and DNA, dirhenium complexes coordinated to purine dinucleotides were isolated and analyzed. LC/MS, HPLC, 1H NMR, and UV-Visible spectroscopy were used to characterized complexes of Re2(O2CR)2X4·2H2O (R = CH3, CH2CH3; X= Cl, Br) with the purine dinucleotides dApG and dGpG. HPLC, UV-Vis, and 1H NMR are used to investigate the aquation of Re2(O2C2H3)2Cl4μ2H2O which may contribute to its biological activity.
Upon reaction of Re22C2H3)2Cl4μ2H2O with dApG or dGpG, the intact dirhenium:dinucleotide complex is observed by LC/MS after two days. In both of these reactions, dirhenium:GMP complexes are also observes.
1H NMR studies show the appearance of new resonances in the aromatic region that cannot be attributed to starting material or hydrolyzed DNA fragments. These resonances are proposed to result from the formation of dirhenium:dinucleotide complexes. Additionally, MS spectra support the conclusion that a complex between the dinuclear rhenium complex and the purine dinucleotides of dApG and dGpG is formed after two days. A dirhenium:nucleotide product is also formed as a result of the dinucleotide hydrolysis.
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Worthington, Rebecca A. (Ann). "Structure-function studies of P2X receptors." Thesis, The University of Sydney, 2001. https://hdl.handle.net/2123/27719.
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P2X receptors are fast, ATP-gated cation ion channels. To date seven subtypes of P2X receptors
have been cloned and identified, PZXH. The membrane topology of the P2X subunit consists of
intracellular amino- and carboxy-termini, two transmembrane spanning domains and a large
extracellular loop. Despite similar membrane topology, within this family of receptors the P2X
subtypes possess different functional characteristics. They exhibit different sensitivities to
agonists and antagonists, are modulated differently by extracellular ions, and have different pore
forming abilities. The regions that are responsible for differences in function between P2X
subtypes have not been elucidated.
This thesis aims to further knowledge regarding the relationship between the structure of the P2X
family and differences in the function of the various receptor subtypes.
Examination of the primary structure of the P2X receptor family led to the identification of
epitope regions suitable for antibody production. This suite of antibodies was tested for
specificity and the distribution of P2X receptors was examined in a range of rat tissues and two
cell lines.
The pathophysiological involvement of the P2X7 receptor was examined in B-CLL patients. Two
polymorphisms as well as a loss of function mutation were identified in both normal and
leukaemic populations.
The site of agonist binding is believed to be within the extracellular loop. Examination of the
primary structure of the human cytolytic receptor P2X7 led to the identification of two noncontiguous regions that could potentially be involved in binding ATP. Three amino acid residues
that lie within the extracellular loop were targeted and their involvement in ATP binding was
determined. Two lysine residues at positions 193 and 31 1 and a proline residue at 210 were each
exchanged with alanine. An abolition of function of human receptors with mutations at positions
193 or 311 was observed, consistent with a disruption of the ATP binding domain, although
alterations in transduction or gating cannot be dismissed. The P2X receptor appears to be
comprised of a trimeric subunit arrangement, and Hill coefficients of between 1 and 3 reported
for ATP binding suggest that there is more than one ATP binding site per functional receptor.
Modelling of the putative binding cleft of the hP2X7 subunit was performed and the residues
important for ATP binding were highlighted. The fimctional trimeric receptor appears to possess
three intersubunit ATP binding sites.
In an attempt to isolate regions of the extracellular domain that contribute to or control various
channel properties, chimaeras between subtypes P2X], P2X4 and P2X7 were constructed and their
properties examined. Each of the six chimaeras has been shown to be correctly inserted into the
cell membrane and functional. These constructs will continue to be investigated and form the
basis for extensive future work.
3
Chen, Xue Bin. "Excretion of purine derivatives by sheep and cattle and its use for the estimation of absorbed microbial protein." Thesis, University of Aberdeen, 1989. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=128449.
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The nucleic acids digested by ruminants are essentially of microbial origin. Absorbed purines are extensively degraded and excreted in urine as allantoin, uric acid, xanthine and hypoxanthine. The measurement of the urinary purine derivatives could be used to estimate the uptake of purines and hence that of microbial protein. 1) Automated methods for measurements of purine derivatives were improved. A HPLC method for determining total adenine and guanine was used to measure the purine contents of mixed rumen bacteria and the digestibility of microbial purines in sheep. 2) Endogenous excretions of purine derivatives by sheep and cattle of different physiological states were measured using animals nourished by intragastric infusions of VFA and casein. The species difference in and the effects of changes in protein supply on the endogenous excretion were studied. 3) A microbial nucleic acid extract was infused into the abomasum of lambs at 6 levels. The subsequent urinary excretion of purine derivatives was examined. The results suggested that exogenous purines were utilised by the sheep. A model is proposed to describe the relationship between purine derivative excretion and purine uptake. 4) Allantoin was infused intravenously to sheep and the changes in plasma concentration, nephric tubular re-absorption and urinary excretion of allantoin were studied. Results showed that plasma allantoin was rapidly removed and a constant proportion of the allantoin entering the blood was excreted in urine of sheep. 5) Secretion of allantoin and uric acid into the gut via saliva was quantified in sheep. The possible decomposition of the allantoin in the rumen by microbes in the rumen fluid and in the rumen wall of sheep was examined in vitro and in vivo. Allantoin infused into the rumen or abomasum of sheep and cattle was not recovered in urine. 6) A model for calculation of microbial protein available to sheep is proposed. It is suggested that a different model should be used for cattle. These models were applied in two feeding experiments with sheep and steers.
4
Anderson, Crystal Annette. "Reactivity of Re₂(CH₃COO)₂Cl₄·2H₂O with purine DNA dinucleotides." Scholarly Commons, 2005. https://scholarlycommons.pacific.edu/uop_etds/603.
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Covalent binding of dinuclear metal carboxylate compounds to purine DNA nucleobases has been shown to be a source of anticancer activity, and has resulted in intense research to understand the coordination of metal complexes to DNA. This investigation focuses on the formation of dirhenium metal:dinucleotide complexes of purine nucleobases. To our knowledge, complexes formed by the reaction of dinuclear rhenium metal compounds with dinucleotides have not been reported in the literature.
5
Rodrigues, Elisandra Márcia. "Estudos moleculares das fosforribosil pirofosfato sintetases." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-18062008-085454/.
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Fosforribosil pirofosfato (PRPP) sintetases são enzimas de central importância em diversas vias metabólicas em todas as células. A enzima PRPP sintetase humana é constituída por um complexo composto de três subunidades catalíticas (PRSI, PRSII e PRSIII) e por proteínas homólogas de 39 e 41 kDa denominadas PRS Associated Proteins (PAPs) cuja função é desconhecida. A importância da PRPP sintetase em humanos, tem sido documentada pela identificação de uma desordem associada ao cromossomo X, resultando em uma atividade aumentada da PRPP sintetase.Em conseqüência se percebem concentrações elevadas na dosagem de ácido úrico, purino nucleotídeos levando ao desenvolvimento de patogenias como a gota e problemas neurológicos. Nesse sentido, realizaram-se estudos moleculares com o complexo enzimático que compõem as PRPP sintetases. Foram obtidos clones do gene hprsI em vetor de expressão pET29a(+) e a enzima foi expressa em bactérias Escherichia coli cepa BL21 (DE3). A proteína recombinante hPRSI foi purificada depois de fracionamento com sulfato de estreptomicina, sulfato de amônio e em coluna cromatográfica de troca iônica. O raio hidrodinâmico e o pI da enzima foram determinados usando respectivamente a técnica de espalhamento dinâmico de luz e o sistema Fast de eletroforese. A enzima foi caracterizada quanto ao seu enovelamento através da técnica de Dicroísmo Circular, apresentando um espectro característico de estrutura enovelada, composta predominantemente por a-hélices. Os genes hprsII e hpap4l-1 que codificam respectivamente para as proteínas hPRSI1 e HPAP41-1 foram clonados no vetor de clonagem pCR4-TOPO. A proteína recombinante hPAP39 foi clonada em vetor pMAL-c2X em fusão com a proteína Maltose Binding Protein (MBP) e expressa em E. coli. A proteína hPAP39 está em fase de purificação e foi submetida ao experimento de Imunoblotting. Investigações estruturais destas enzimas poderão trazer informações fundamentais para o conhecimento da via biossintética, assim como para o desenvolvimento de inibidores específicos visando o tratamento das desordens a elas associadas.Phosphoribosyl pyrophosphate (PRPP) synthetases are enzymes of central importance in several metabolic pathways in all cells. The enzyme PRPP synthetase complex is composed of three catalytic subunitis (PRSI, PRSII and PRSIII) and homologous 39 and 41 kDa proteins termed PRPP synthetase-Associated Proteins (PAPs) which function is unknown. The importance of PRPP synthetase function in humans has been documented by the identification of an X chromosome-linked disorder associated with super activity of PRPP synthetase. As a consequence uric acid overproduction, purine nucleotide are observed resulting in the development of diseases such as gout and neurodevelopment impairment. In this line, molecular studies were done with the enzyme complex that constitutes the PRPP synthetases. Clones were obtained from the hprsI gene in the pET29a(+) expression vector and the enzyme was expressed in Escherichia coli BL21(DE3) bacterial. The recombinant human PRSI enzyme was purified, after streptomicine and ammonium sulfate fractionation and by anion exchange chromatography. The hPRSI hydrodynamic radius and pI were determined using, respectively, measures of Dynamic Light Scattering (DLS) and isoeletrophocusing electrophoresis. In addition, according to circular dichroism spectroscopy, hPRSI prevalent secondary structure is a-helix. The hprsí and hpap41-1 genes that codify, respectively, to hPRSII and hPAP41-1 proteins were cloned in pCR4-TOPO cloning vector. The recombinant protein hPAP39 was cloned in the pMAl-c2X expression vector in fusion with the Maltose Binding Protein (MBP) and expressed in E.coli. A purification protocol is been establish for the hPAP39 protein and is submitted by imunoblotting technique. Structural investigation of these enzymes will provide information about the biosynthetic pathway de novo of purine nucleotides, as well as to development of specific inhibitors aiming at the treatment of the associated disorders.
6
Fong, Yuen Ting. "Quantitative structure retention relationships on using high-performance liquid chromatography." HKBU Institutional Repository, 2003. http://repository.hkbu.edu.hk/etd_ra/426.
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Liu, Jiangqiong. "Kinetic Studies of 6-Halopurine Nucleoside in SNAr Reactions; 6-(Azolyl, Alkylthio and Fluoro)-purine Nucleosides as Substrates for Suzuki Reactions." Diss., CLICK HERE for online access, 2007. http://contentdm.lib.byu.edu/ETD/image/etd1821.pdf.
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Wu, Joe. "HIF-1α in the Heart: Provision of Ischemic Cardioprotection and Remodeling of Nucleotide Metabolism." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etd/2450.
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In our studies we found that stabilized expression of HIF-1α in heart led to better recovery of function and less tissue death after 30 minutes of global ischemia, via mechanisms that preserve the mitochondrial polarization. Our group previously showed that HIF-1α conferred ischemic tolerance by allowing cardiomyocytes to use fumarate as an alternative terminal electron acceptor to sustain anaerobic mitochondrial polarization. The source of fumarate was identified as the purine nucleotide cycle (PNC). Here we discovered that HIF-1α upregulates AMP deaminase 2 (AMPD2), the entry point to the PNC. The combination of glycolysis and the PNC may protect the heart's nucleotide resources. We subsequently examined the effects that HIF-1α exerts on nucleotide metabolism in the ischemic heart. We found that HIF-1α expression reduces adenosine accumulation in the ischemic heart. As ATP is depleted during ischemia, AMP accumulates. Our results suggest that AMP metabolism is shunted towards AMPD2 rather than the adenosine producing 5'-nucleotidase pathway. Subsequently, we treated hearts with the PNC inhibitor hadacidin followed by 30 minutes of global ischemia. Inclusion of hadacidin reduced ATP and adenylate energy charge in the hearts. These findings allow us to propose that activity of the PNC prevents the F0F1 ATP synthase from consuming glycolytic ATP in order to maintain mitochondrial polarization during ischemia. Thus, the PNC provides ATP sparing effects and preserves the energy charge in the ischemic heart. The fact that ATP and adenylate energy charge is better preserved during the initial 20 minutes of ischemia in HIF-1α expressing hearts is supportive of our observation that HIF-1α upregulates the PNC. HIF-1α also upregulates adenosine deaminase, which degrades adenosine. The limitation of adenosine accumulation may help HIF-1α expressing hearts avoid toxicity due to chronic adenosine exposure. Finally, we found that HIF-1α induces the expression of the nucleotide salvage enzyme hypoxanthine phosphoribosyl transferase (HPRT). Upon reperfusion HPRT serves to reincorporate the nucleotide degradation product, hypoxanthine, into the adenylate pool and may prevent the production of reactive oxygen species. Collectively, HIF-1α robustly protects the heart from ischemic stress and it upregulates several pathways whose cardioprotective role may extend beyond the remodeling of nucleotide metabolism.
9
Barbosa, Sara Isabel Cadinha. "Compostos que interferem no metabolismo dos purina- e pirimidina-nucleótidos: utilização como agentes terapêuticos." Master's thesis, [s.n.], 2015. http://hdl.handle.net/10284/5160.
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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências FarmacêuticasO conteúdo deste trabalho será desenvolvido em dois temas principais, um referente à utilização de compostos que interferem no metabolismo dos purina- e pirimidinanucleótidos como agentes antineoplásicos e outro referente à sua utilização como agentes antivirais. A síntese dos nucleótidos envolve a construção de ácidos nucleicos e a inserção dos derivados de nucleótidos noutras vias bioquímicas, sendo responsável por inúmeras funções do metabolismo celular. Existem patologias que envolvem enzimas essenciais do metabolismo dos nucleótidos, o que levou à síntese de novos fármacos. As doenças oncológicas continuam a matar milhares de pessoas e um tratamento eficaz e com sucesso tem sido um desafio. O mesmo se passa com algumas infeções virais, nomeadamente infeções provocadas pelo HIV. Para contornar os obstáculos enfrentados na terapia destas doenças têm sido usados análogos de nucleótidos e/ou nucleósidos como agentes terapêuticos. Estes têm o propósito de inibir a síntese de novo dos nucleótidos em determinadas etapas, estando envolvidos na replicação e síntese do RNA e DNA nas células em divisão. Atuam por inibição específica de enzimas no metabolismo dos nucleótidos/nucleósidos ou ainda por incorporação no DNA ou no RNA. This study will be developed into two main subjects; one related to the use of compounds which interfere with the metabolism of purine- and pyrimidine- nucleotides as antineoplastic agents; another related to their use as antiviral agents. The nucleotides’ synthesis involves the construction of nucleic acids and the introduction of the nucleotides’ derivatives into other biochemical pathways and it is responsible for numerous functions of cellular metabolism. There are pathologies involving key enzymes from the nucleotides’ metabolism, which led to the synthesis of new drugs. Cancer is a disease that continues killing thousands of people, an effective and successful treatment has been a challenge. The same happens with some viral infections, mainly infections caused by HIV. To overcome the obstacles faced in the therapy of these diseases it has been used nucleotide and/or nucleoside analogues as therapeutic agents. These agents have the purpose of inhibiting the de novo nucleotide synthesis in certain steps, by being involved in RNA and DNA replication and synthesis in dividing cells. They act by specific enzymes inhibition in nucleotide/nucleoside metabolism and by incorporation into DNA or RNA.
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Wei, Shuang. "Modifications du metabolisme des nucleotides en relation avec la differenciation et en reponse a une irradiation dans des cellules tumorales humaines (doctorat : structure et fontionnement des systemes biologiques integres)." Paris 11, 1998. http://www.theses.fr/1998PA114846.
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Zhong, Minghong. "N9 Alkylation and Glycosylation of Purines; A Practical Synthesis of 2-Chloro-2'-deoxyadenosine." Diss., CLICK HERE for online access, 2004. http://contentdm.lib.byu.edu/ETD/image/etd433.pdf.
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Mantovani, Monique. "Adenylosuccinato lyase (ADSL) de leishmania major friedlin: sua relevância na via de recuperação de purino-nucleotídeos." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-09042008-133909/.
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Muitas espécies de Leishmania são responsáveis por sérias doenças cutâneas e viscerais, que exibem alta incidência em regiões tropicais e subtropicais. Os medicamentos disponíveis são potencialmente carcinogênicos, requerem múltiplas administrações e a hospitalização do paciente. Um programa efetivo de desenvolvimento de novos fármacos requer a validação genética e bioquímica dos alvos. Neste contexto, uma das diferenças metabólicas mais marcantes entre os protozoários parasitas e o hospedeiro mamífero encontra-se na cadeia de síntese de purino-nucleotídeos. A enzima adenilosuccinato liase (ADSL), no hospedeiro mamífero, atua em duas etapas no metabolismo de purino-nucleotídeos: uma na via de síntese de novo e outra na via de recuperação. Esta característica particular da ADSL nos motivou a investigar como esta enzima poderia nos fornecer evidências sobre a evolução desta via metabólica em Kinetoplastida. Assim, os objetivos do presente trabalho foram validar a ADSL como potencial alvo terapêutico, através da técnica de RNA de interferência (RNAi), usando Trypanosoma brucei como modelo, bem como, caracterizar molecularmente o gene adsl-Lm e caracterizar bioquímica e estruturalmente a enzima recombinante de Leishmania major Friedlin (ADSL-Lm). Os resultados de RNAi demonstraram que a ADSL pode ser considerada um potencial alvo, uma vez que ela se apresentou essencial a viabilidade do parasita. Em relação à caracterização molecular do gene adsl-Lm suas regiões não traduzidas 5\'UTR e 3\'UTR foram definidas por RT-PCR, indicando que o RNA mensageiro maduro possui 2060 nucleotídeos. Análises com enzimas de restrição e eletroforese em campo pulsado (PFGE), seguidas de \"Southern blot\" revelaram que adsl-Lm trata-se de um gene de cópia simples e está localizado no cromossomo 4 deste parasita. O gene adsl-Lm foi clonado em um vetor de expressão e um protocolo de purificação da enzima recombinante foi estabelecido. A forma tetramérica da ADSL-Lm foi confirmada por eletroforese em gel nativo e por espalhamento dinâmico de luz (DLS). ADSL-Lm apresentou um pI experimental de 6,07 e exibiu atividade máxima no pH 8,5. Os parâmetros cinéticos de Km, Vmax , Kcat e eficiência catalítica (Kcat/Vm) foram determinados para o substrato adenilosuccinato. Experimentos de complementação funcional evidenciaram que ADSL-Lm foi capaz de complementar de maneira eficiente o genoma deficiente em ADSL da linhagem de E. coli JK268 (mutação purB58). Entretanto, esta complementação deve ocorrer na via de recuperação, uma vez que ensaios enzimáticos com o SAICAR (substrato da via de novo) mostraram que a enzima não retém atividade sobre este composto. Estes resultados indicam que provavelmente os tripanosomatídeos não representam um caso de perda da via de síntese de novo. Adicionalmente, ADSL-Lm foi cristalizada no grupo espacial tetragonal I4122, com parâmetros de cela unitária a= b= 130,023 \'angstron\', c= 316,826 \'angstron\', = = =90°. A estrutura cristalográfica da ADSL-Lm foi resolvida por SIRAS (substituição isomórfica simples com dispersão anômala) usando um cristal nativo (2.2 \'angstron\') e um derivado de Gadolínio. O monômero é composto por três domínios em um enovelamento típico das enzimas da superfamília de -eliminação e é constituído quase exclusivamente por hélices- . Para o sitio ativo, três subunidades são requeridas e os resíduos envolvidos são His 153 (ácido geral), His 231 (base geral), Gln 308, Asn 364 and Glu 369. Entretanto, sem um ligante (substrato ou inibidor) no sítio ativo torna-se difícil estudar detalhadamente as interações entre a enzima e seus dois substratos. Desta forma, experimentos de co-cristalização e modelagem molecular podem auxiliar nesta questão.Many species of Leishmania are responsible for serious visceral or skin diseases that exhibit high incidence in tropical and subtropical regions. The drugs currently employed in the treatment of parasitic diseases are potentially carcinogenic, often require prolonged treatment and patient hospitalization. An effective program of drug design requires the validation of the potential target. In this context, one of the most striking metabolic discrepancies between Trypanosomatidae and their human hosts is the purine nucleotide biosynthesis pathway. Adenylosuccinate lyase (ADSL) is a bifunctional enzyme that catalyses two non-sequential steps in this cycle (one in the de novo purine pathway and another in the salvage pathway). This particular ADSL feature motivated us to investigate if ADSL could give us information about purine biosynthesis evolution in Kinetoplastida. Hence, the present work is aimed to validate ADSL as a potential target using the RNAi (RNAi) technique, as well as to characterize the adsl gene and the recombinant enzyme from Leishmania major Friedlin (ADSL-Lm). The RNAi results proved that ADSL can be considered a potential target, because it is shown to be essential for parasite viability. Regarding the molecular characterization of the adsl-Lm gene, the mature mRNA transcript containing 2060 nucleotides was defined by 5\' and 3\'RT-PCR. Restriction analysis and Pulse Field Gel Electrophoresis (PFGE) followed by Southern Blot hybridizations showed that adsl-Lm is a single copy gene and is located in chromosome 4 of this parasite. The adsl-Lm gene was cloned into an expression vector and a purification protocol of the recombinant enzyme was established. The tetrameric form of the recombinant ADSL-Lm enzyme was confirmed by native gel electrophoresis and Dynamic Light Scattering. ADSL-Lm has an experimental pI of 6.07 and exhibited maximum enzymatic activity at pH 8.5. The kinetic parameters of Km, Vmax , Kcat and kinetic efficiency (Kcat/Vm) were obtained for adenylosuccinate substrate. Functional complementation experiments showed that the adsl-Lm gene can effectively complement the E. coli JK268 purB58 mutation. However, this complementation must be in the salvage pathway, because enzymatic assays were performed using SAICAR (de novo pathway substrate) and ADSL-Lm did not convert this compound into product. This result indicates that probably Trypanosomatidae is not an example of de novo purine nucleotide cycle lost. In addition, ADSL-Lm was crystallized in the tetragonal I4122 space group, with unit cell parameters a= b= 130,023 \'angstron\', c= 316,826 \'angstron\', = = =90° and diffracted beyond 2.2\'angstron\'. ADSL-Lm crystal structure has been determined by SIRAS (Single Isomorphous Replacement with Anomalous Dispersion), using both native and Gadolinium derivative crystals. The ADSL-Lm monomer is composed of three domains arranged in the elongated manner typical of enzymes in the p-elimination superfamily, and is constituted almost exclusively by helices. Three subunits are necessary to form the active site cleft and the residues His 153, His 231 (general bases), Gln 308, Asn 364 and Glu 369 are involved. Without a bound inhibitor or substrate, the specific contacts made by the enzyme to its two substrates cannot be analyzed in detail. Hence, co-crystallization and docking can help in this question.
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Boussiengui-Boussiengui, Gino. "Analysis of the role of relative nucleotide concentrations on the regulation of carbohydrate in higher plants." Thesis, Stellenbosch : Stellenbosch University, 2010. http://hdl.handle.net/10019.1/5473.
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Thesis (PhD (Genetics))--Stellenbosch University, 2010.ENGLISH ABSTRACT: The current understanding of the regulation of carbohydrate accumulation is still under investigation despite the tremendous work done in this subject. Purine and pyrimidine nucleotides have been implicated in many biochemical processes in plants. Amongst others, they are building blocks for nucleic acid synthesis, an energy source, precursors for the synthesis of primary products such as sucrose, polysaccharides, phospholipids, as well as secondary products. With the aim of placing adenine and uridine nucleotides in the context of sucrose and starch metabolism and carbon partitioning in higher plant, we have investigated the transcripts, enzymes and metabolites in carbohydrate metabolism and both de novo and salvage of purine and pyrimidine nucleotides in both sugarcane and tobacco tissues. For that purpose, adenylate kinase (ADK) and UMP synthase were chosen for silencing and over expression as they are rate limiting steps of de novo adenine and uridine nucleotides biosynthesis, respectively. Sugarcane with repressed ADK activity showed significant increase in both the starch and adenylate pools. Increase in starch content was highly correlated with reduced ADK activity. As a result of decreased ADK activity, the salvage pathway was up regulated via the increased activity of both adenosine kinase (AK) and adenine phosphoribosyl transferase (APRTase) which positively correlated with increase in adenine nucleotide contents. In addition hexose phosphates and ADP glucose, the committed substrate for starch biosynthesis positively correlated with changes in starch content. A high ratio of ATP/ADP was observed in all transgenic lines compared with the untransformed wild type and suggested to favour starch synthesis. Over expression of cytosolic ADK in tobacco demonstrated an expression of the enzyme where 2/3 of the total activity was in the direction of ADP production. As a result of over expression of ADK, starch content increased in all transgenic plants and positively correlated with changes in the activity of ADK. Despite changes in adenine nucleotide content, the salvage pathway was not activated and no significant changes in both AK and APRTase acivities were found between the transgenic and the untransformed plants. Sucrose synthase (SuSy) activity in breakdown direction positively correlated with changes in starch content suggesting a contribution in the starch accumulation in tobacco plants. In addition the ratio of ATP/ADP was low in all transgenic lines compared with the untransformed wild type. This was in line with the higher content in ADP compare to ATP in all transgenic lines and was supported by the over expression of ADK, and predominantly in the direction of ADP production. Repressed UMP synthase in transgenic sugarcane resulted in increases in sucrose, starch and uridinylate. UDP-glucose, hexose phosphates and uridinylate content positively correlated with changes in sucrose content. Transgenic lines had increased sucrose phosphate synthase (SPS) activity and low activity in SuSy, which suggests alteration of carbon flux toward sucrose. As a result of decreased UMP synthase activity, an up regulation of the salvage pathway was observed and predominantly via increased activity of uridine kinase (UK) which positively correlated with changes in the uridinylate pool. In addition to repressed UMP synthase activity, starch content and adenine nucleotides increased in transgenic lines. Tobacco plants transformed with a cytosolic UMP synthase demonstrated an over expression of the enzyme in all transgenic lines. As a result of over expression of UMP synthase, key metabolites were up regulated, amongst them sucrose. Increase in sucrose content positively correlated with both hexoses and hexose phosphates but not the uridinylate pool. SPS activity positively correlated with increase in sucrose content, and accounted for most of the sucrose synthesized in transgenic lines. Despite the increase in the adenylate pool, no significant changes were observed in starch content. The depletion level of UDP-glucose in all transgenic lines was a mere reflection of the higher activity of UDP glucose pyrophosphorylase (UGPase) in the formation of glucose-1-phosphate. In addition, no salvage pathway was up regulated in transgenic lines.
AFRIKAANSE OPSOMMING: Die huidige beskikbare inligting in verband met die reguleering van koolhidraat akkumulasie word steeds ondersoek ten spyte van die groot hoeveelheid navorsing wat reeds in hierdie verband gedoen is. Purien en pirimidien nukleotide speel ‘n rol in baie biochemiese prosesse in plante. Onder andere is hulle boublokke vir nukleïensuur sintese, ‘n energie bron, voorlopers vir die sintese van primêre produkte soos byvoorbeeld sukrose, polisakkariede, fosfolipiede, asook sekondêre produkte. Met die vooruitsig om adenine- en uridiennukleotide in verband te plaas met sukrose en stysel metabolisme en koolstof afskorting in plante, ondersoek ons hier die transkripte, ensieme en metaboliete in koolhidraat metabolisme in beide de novo en berging van purien en pirimidien nukleotide in suikerriet asook tabak weefsel. Vir hierdie doel is adenilaatkinase (ADK) en UMP-sintase gekies vir uitskakeling en ooruitdrukking, juis omdat hulle tempo vermindering stappe van de novo adenine- en uridiennukleotide biosintese is. Suikerriet met onderdrukte ADK aktiwiteit wys betekenisvolle vermeerdering in beide die stysel en adenilaat poele. Verhoging in styselinhoud was hoogs gekorreleerd met verminderde ADK aktiwiteit. As gevolg van ‘n vermindering in ADK aktiwiteit, is die bergingspad opwaards gereguleer via die vermeerdering van beide adenosienkinase (AK) en adenien-fosforibosieltransferase (APRTase) aktiwiteit wat positief korreleer met die vermeerdering in adeniennukleotied-inhoud. Addisioneel word hexosefosfate en ADP-glukose, die toegewysde substraat vir stysel biosintese, positief gekorreleer met veranderinge in styselinhoud. ‘n Hoë verhouding van ATP/ADP was geobserveer in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe en blyk stysel sintese te begunstig. Ooruitdrukking van sitologiese ADK in tabak demonstreer die uitdrukking van die ensiem waar 2/3 van die totale aktiwiteit in die rigting van ADP produksie was. As ‘n resultaat van ooruitdrukking van ADK, word stysel inhoud vermeerder in alle transgeniese plante en positief gekorreleer met die verandering in die aktiwiteit van ADK. Ten spyte van veranderinge in adeniennukleotide inhoud was die bergingspad nie geaktiveer nie en geen betekenisvolle veranderinge in beide AK en APRTase aktiwiteit was gevind tussen die transgeniese en nie-transgeniese plante nie. Sukrose sintese (SuSy) aktiwiteit tydens afbreking korreleer positief met die veranderinge in stysel inhoud en dui moontlik op ‘n bydrae in die stysel akkumulasie in tabak plante. Verder was die verhouding van ATP/ADP laag in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe. Hierdie bevinding word ondersteun deur die hoër inhoud in ADP in vergelyking met ATP in alle transgeniese lyne en word verder ondersteun deur die ooruitdrukking van ADK, hoofsaaklik in die rigting van ADP produksie. Onderdrukte UMP-sintase in transgeniese suikerriet lei tot verhogings in sukrose, stysel en uridienilaat. UDP-glukose, hexose-fosfate en uridienilaat inhoud korreleer positief met die verandering in sukrose inhoud. Transgeniese lyne het verhoogde sukrose-fosfaatsintase (SPS) aktiwiteit en lae SuSy aktiwiteit wat dui op ‘n verandering in koolstof vloei in die rigting van sukrose. As gevolg van die afname in UMP-sintese aktiwiteit, word ‘n verhoogde reguleering van die bergingspad gesien, en dít hoofsaaklik via verhoogde aktiwiteit in uridienkinase (UK) wat positief korreleer met veranderinge in die uridienilaat poel. Addisioneel tot die onderdrukking van UMP-sintase was stysel inhoud en adenine- nucleotides in transgeniese lyne verhoog. Tabak plante wat getransformeer is met sitologiese UMP-sintase demonstreer verhoogde uitdrukking van die ensiem in al die transgeniese lyne. As ‘n resultaat van ooruitdrukking van UMP-sintase is sleutel metaboliete, onderandere sucrose, oorgereguleer. ‘n Verhoging in sukrose inhoud korreleer positief met beide hexose en hexose-fosfate maar nie met die uridienilaat poel nie. SPS aktiwiteit korreleer positief met die verhoging in sukrose inhoud en verklaar die meeste van die sukrose vervaardig in transgeniese lyne. Ten spyte van die verhoging in die adenilaat poel word geen noemenswaardige veranderinge gesien in die stysel inhoud nie. Die uitputtingsvlak van die UDP-glukose in alle transgeniese lyne was slegs ‘n aanduiding van die hoër aktiwiteit van UDP-glukose pirofosforilase (UGPase) in die formasie van glukose-1-fosfaat. Verder was geen bergingspad opgereguleer in die transgeniese lyne nie.
The South African Sugarcane Research Institute and the Gabonese Government who provided the financial support for this work
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Barbeiro, Hermes Vieira. ""Avaliação temporal da regulação do tônus vascular e da produção de superóxido induzido por purinas em aorta isolada de ratos Wistar endotoxêmicos"." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/5/5160/tde-05092005-120436/.
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Pacientes sépticos podem evoluir para choque séptico, destes 40% sobrevivem. Caracterizamos o modelo experimental, avaliamos fatores envolvidos na inflamação e avaliamos a modulação causada por purinas (ATP/ADP) na quantificação de superóxido (O2-) e na reatividade vascular da aorta isolada. Os resultados sugerem que na aorta isolada de animais endotoxêmicos, ATP e ADP aumentam a síntese de óxido nítrico (NO), porém somente o ATP reduz a biodisponibilidade de O2-, provavelmente pelo reacoplamento da NO sintase endotelialSeptic patients can evolve for septic shock and 40% of these survive. We characterize the experimental model we evaluate involved factors in the inflammation and evaluate the modulation caused by purines (ATP/ADP) in the superoxide quantification (O2-) and in the vascular reactivity of isolated aorta. The results suggest that in isolated aorta of endotoxemics rats, ATP and ADP increase the endothelial nitric oxide synthase (NOS) however just ATP reduces the bio availability of O2-, probably for the re-couples of the endothelial NOS synthase
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Smith, James. "Molecular discrimination analysis for purine nucleotide binding sites." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620247.
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Wolfe, Kara. "The Role of Purine Nucleotide Metabolism in Renal Cell Carcinoma Migration." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1563872926565308.
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Carrique, Loic. "Études des relations structure-fonctionactivité d’enzymes de Plasmodium falciparum pour la conception et la synthèse de nouvelles molécules antipaludiques." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1109.
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Plasmodium falciparum est responsable de la forme la plus grave de paludisme avec plus de 600 000 décès par an. L'absence de vaccin efficace, combinée à l'émergence de résistances aux traitements récurrents, exige le développement de nouvelles molécules. Afin de limiter ces résistances, il est nécessaire de cibler de nouvelles voies métaboliques indispensables à la survie du parasite. Ce travail de thèse repose sur l'étude de deux voies métaboliques essentielles au parasite que sont la voie de recyclage des bases puriques et la voie de biosynthèse des ancres glycosylphosphatidylinositol (GPI).En ce qui concerne la voie de recyclage des bases puriques, la détermination des structures cristallines de l' « IMP specific 5‘-nucleotidase » (PfISN1) associée aux études biochimiques et biophysiques (SAXS, EM, MALS…), a permis de préciser le mécanisme d'action fournissant ainsi une base solide pour la mise au point d'inhibiteurs. Une banque de plus 3000 composés a été criblée par Fluorimétrie à Balayage Différentiel et les effets des molécules sélectionnées seront évalués sur l'enzyme et sur la croissance du parasite en culture.Quatre cibles thérapeutiques potentielles appartenant à la voie de biosynthèse des ancres GPI ont été sélectionnées. L'utilisation de plusieurs systèmes d'expression disponibles au laboratoire (bactérie, levure, acellulaire en germe de blé) ou via des plateformes européennes pour l‘expression en cellules de mammifères HEK293T (Oxford), de cellules BHK21 transfectées avec le virus de la vaccine modifié, T7-MVA, (Strasbourg) ou la plateforme ESPRIT (Grenoble) ont permis de passer outre les difficultés rencontrées pour exprimer les protéines d'intérêt. L'une des quatre cibles, la mannose-1-phosphate guanylyltransférase (PfMPG), a pu être exprimée de manière suffisante quantitativement et qualitativement pour une caractérisation biochimique et structurale. Une analyse par SAXS et cristallographie aux rayons X a été réaliséePlasmodium falciparum is responsible for the most severe form of malaria with more than 600,000 deaths per year. The lack of an effective vaccine, combined with the emergence of drug resistant parasites, necessitates the development of new drugs. In order to limit these resistances, it is necessary to target new metabolic pathways essential for parasite survival. This thesis work is based on the study of two metabolic pathways essential to the parasite, the purine salvage pathways and the glycosylphosphatidylinositol (GPI) anchor biosynthesis pathway.Concerning the purine salvage pathway, the determination of the crystal structures of the IMP -specific 5'-nucleotidase (PfISN1) associated with biochemical and biophysical studies (SAXS, EM, MALS, etc.) have allowed to propose a reaction mechanism, thereby providing a solid basis for the conception and development of inhibitors. A library of more than 3000 compounds was screened by Differential Scanning Fluorimetry and the selected molecules will be evaluated for their inhibitory effect on the enzyme and on the growth of parasites in culture.Four potential therapeutic targets belonging to the GPI anchor biosynthesis pathway were selected. The use of several in-house available expression systems (bacteria, yeast, and acellular wheat germ) as well as European platforms for the expression in HEK293T mammalian cells (Oxford), in BHK21 cells transfected with the modified vaccinia virus, T7-MVA, (Strasbourg) or the ESPRIT platform (Grenoble) has allowed us to overcome the difficulties encountered on obtaining the selected protein targets. One of the four targets has been expressed in sufficient amount and quality for biochemical- and structural characterization, namely the mannose-1-phosphate guanylyltransferase (PfMPG). SAXS and X-ray crystallography analyses have been carried out
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Green, Stephen Mark. "Purine nucleotide biosynthesis in Escherichia coli K12 : molecular biology of the purB gene." Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316409.
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Gray, Alexander. "Uptake and metabolism of purine nucleotide and pyridoxal phosphate precursors in Trypanosoma brucei brucei." Thesis, University of Edinburgh, 1988. http://hdl.handle.net/1842/18920.
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Frame, Andrew Scott. "The synthesis of some imidazole nucleosides as potential inhibitors of de novo purine nucleotide biosynthesis." Thesis, University of Lincoln, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386384.
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Amara, Francis Morigua. "The significance of purine salvage pathway enzymes in mutagenesis and nucleotide metabolism in friend erythroleukaemia cells." Thesis, University of Ulster, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.281331.
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Wilkinson, Yvonne Annette. "Nucleotide pool changes resulting from purine and pyromidine salvage pathway enzyme deficiency in friend erythroleukaemia cells." Thesis, University of Ulster, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.258030.
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Baccolini, Chiara [Verfasser]. "Analysis of in vivo purine nucleotide catabolism in Arabidopsis thaliana with focus on nucleoside hydrolase 2 / Chiara Baccolini." Hannover : Gottfried Wilhelm Leibniz Universität Hannover, 2019. http://d-nb.info/1193177146/34.
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Lemin, David. "Synthèse d'analogues des ligands naturels de récepteurs nicotiniques et purinergiques." Doctoral thesis, Universite Libre de Bruxelles, 2004. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211158.
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Cette thèse s’inscrit dans le cadre de l’étude de la relation structure-activité d’analogues de ligands naturels de récepteurs nicotiniques et purinergiques. Ce travail se divise en deux parties.Dans la première partie de cette thèse, nous avons réalisé la synthèse d’analogues de la 11-homosédinone, alcaloïde isolé de la plante Sedum acre, qui présente une activité agoniste sur différents récepteurs nicotiniques du système nerveux central. Les différents analogues ont été synthétisé par application de la méthoxylation anodique pour introduire succesivement deux substituants en postion 2 et 6 d’un noyau pipéridinique. Les analogues synthétisés se différencient par la nature du noyau aromatique, la présence d’un groupement méthyle sur l’atome d’azote de la pipéridine et l’oxydation du sustituant en position 2. Ce travail a notamment permis de montré l’importance du groupement N-méthyle vis-à-vis de l’activité des analogues. Nous avons également pu mettre en évidence que l’introduction d’un halogène sur le noyau aromatique diminuait l’activité de l’analogue sur le récepteur a7 tout en augmentant l’acitivité sur le récepteur a4b2 et que l’introduction d’un noyau furanique permettait d’augmenter la sélectivité vis-à-vis du récepteur a4b2 tandis que l’introduction sur le noyau aromatique d’un groupement nitro ou méthoxy conduit à une perte totale de l’activité.
Dans la seconde partie de cette thèse, nous avons réalisé la synthèse d’analogues de la dATP, afin d’évaluer leur effet agoniste sur le récepteur P2Y11, impliqué dans différents mécanismes de différentiation cellulaire, dont notamment celui de la maturation des cellules leucémiques HL60 en cellules de type neutrophile. Les analogues synthétisés se différencient de la dATP par la présence d’un groupement méthylène ou dichlorométhylène entre les phosphores b et g de la chaîne polyphosphate, ainsi que par l’estérification de l’alcool en position 3’ du sucre. Ce travail a pu confirmer que les analogues en série 2’-désoxy conduisent à de meilleures activités que ceux de la série 2’-OH. Nous avons également pu montrer que l’estérification de la position 3’ conduit à une diminution de l’activité agoniste, à l’exception du groupement a-naphtoyle qui conduit à une augmentation significative de l’activité sur P2Y11.
Doctorat en sciences, Spécialisation chimie
info:eu-repo/semantics/nonPublished
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Thompson, Nadine. "Single Nucleotide Polymorphisms in the Folypoly-gamma-glutamate synthetase Gene." VCU Scholars Compass, 2006. http://scholarscompass.vcu.edu/etd/672.
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Folic acid is an essential vitamin utilized in the one-carbon metabolism pathway for the synthesis of purine and thymidine nucleotides, which are necessary for cell growth and proliferation. As a result, the enzymes that participate in the metabolism of folic acid have been good targets for cancer chemotherapy. Folylpoly-γ-glutamate synthetase (FPGS) is an enzyme in the folate metabolism pathway that catalyzes the addition of glutamic acid to the naturally occurring folates, thereby allowing the retention of folate cofactors in cells. Similarly, in the case of cancer chemotherapy, antifolates, such as Lometrexol and Tomudex are retained in cells through the activity of FPGS. Consequently, any single nucleotide polymorphisms (SNPs) that exist in the fpgs gene may decrease or increase the cytotoxicity of antifolates and, ultimately, the clinical response rate to antifolate therapy. The goal of this project is to define the position and frequency of single nucleotide polymorphisms (SNPs) in the mRNA made from the fpgs gene from peripheral blood of one hundred normal individuals. Six Polymerase Chain Reaction (PCR) primers were designed to amplify the gene as three overlapping pieces and four primers were designed for sequencing of the three PCR products. In this study, we found polymorphic sites at nucleotides 64, 123, 253, 423, 1334 and 1781. The majority of the samples (49/88) expressed rnRNA with point mutations on at least one allele at base 64, while 8 samples had a SNP at base 123. At nucleotide 123, 6 samples expressed the heterozygote GIA genotype, and one sample expressed the homozygote A/A allele at this site. At nucleotide 423, two samples expressed a G allele and also the common C allele. While the SNPs at nucleotide 64, 123, and 423 caused a silent or conservative mutation in the gene, in sample 82, a mutation C253T produced an amino acid change from an arginine to tryptophan, which may cause a functional change in the fpgs protein, due to the significant change in size and charge of the wild type amino acid. Similarly, sample 26 expessed a homozygote T/T allele at nucleotide 1334 instead of the common C/C allele expressed in the remaining samples. This point mutation caused a valine to alanine amino acid change. We also detected a SNP that is expressed after the stop codon in sample 40.
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Faheem, Muhammad. "Structure-functional analysis of a novel cell wall modifying autoproteolytic enzyme and crystallographic fragment screening for Schistosoma mansoni purine nucleotide phophorylases." Universidade Cat??lica de Bras??lia, 2016. https://bdtd.ucb.br:8443/jspui/handle/tede/2260.
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Metagenomics techniques are now widely used for the search of new valuable enzymes of interest and other biotechnological products. Sophistication in the second-generation sequencing has significantly facilitated metagenomics technique for collection of huge amount of microbial genomic data. One of the current focuses in science is to seek the interpretation and transformation of the collected genomic data into functional proteomics data. Combination of structural biology and genomic data is one way to achieve such goal. In this study we have assessed a novel bacterial protein selected on a screen for activity on carbohydrates in a microbial metagenomic library from the gut of Capra hircus. Initial sequence analysis of the open reading frame (ORF) for this selected novel bacterial protein indicated that it could be annotated as an uncharacterized novel bacterial cell wall modifying enzyme. Sequence analysis of the protein has shown that it carries three domains: an N-terminus cysteine protease, a peptidoglycan binding (PGBD) and a C-terminus Src-Homology 3 (SH3) bacterial domain. Later with homology modeling we have observed that it carries an additional N-terminus domain with LCI fold. We have successfully cloned, expressed and purified this Capra hircus putative cysteine protease (PCP). Autoproteolytic activity has been observed for PCP, which was inhibited with protease inhibitors cocktail. We have observed that the autoproteolytic activity is carried either by the second or third domain of PCP. This protein has shown cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most of bacterial cell wall modifying enzymes. Ampicillin binding to PCP was further evaluated with fluorimetric analysis. PCP structure was modeled by homology modeling with good validation statistics and in agreement with circular dichroism data. The domains of PCP have conserved LCI, Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), PGBD and SH3 folds. It has a conserved active site dyad, Cys100 and His161, which is a signature of cysteine proteases. Furthermore, the overall architecture of the model was assembled in SAXS generated density map. Initial protein crystals are also obtained for the last two domains, which diffracted to very low resolution.
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Jain, Nishant Rajkumar. "Characterization and functional analysis of the P2Y₂R gene promoter." Diss., Columbia, Mo. : University of Missouri-Columbia, 2006. http://hdl.handle.net/10355/4629.
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Thesis (M.S.)--University of Missouri-Columbia, 2006.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 21, 2009) Includes bibliographical references.
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Ahn, Jae Suk. "Regulation of P2Y₂ nucleotide receptor expression in salivary glands." free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3012944.
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Park, Minjung Kang. "P2 nucleotide receptors during postnatal development of rat salivary glands." free to MU campus, to others for purchase, 1999. http://wwwlib.umi.com/cr/mo/fullcit?p9946285.
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Landon, Linda A. Neighbors. "Salivary gland P2 nucleotide receptors : structure and function studies /." free to MU campus, to others for purchase, 1998. http://wwwlib.umi.com/cr/mo/fullcit?p9904855.
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Romieu, Anthony. "Synthèse d'oligonucléotides modifiés comportant des lésions radio-induites des bases puriques et pyrimidiques." Université Joseph Fourier (Grenoble), 1999. http://www.theses.fr/1999GRE10140.
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Divers facteurs comme des agents oxydants ou cancerigenes, les rayonnement ultraviolets et ionisants, peuvent engendrer des modifications des bases de l'adn. Afin d'evaluer les consequences biologiques et physico-chimiques de ces dommages, l'obtention de courts fragments d'adn (oligonucleotides), de sequence definie (20 a 50 bases de long) et comportant une ou plusieurs modifications en des sites bien precis est primordiale. La synthese oligonucleotidique est aujourd'hui la methode de choix pour preparer de tels composes modeles. Ce travail est consacre a la preparation de fragments d'adn synthetiques contenant des nucleosides modifies formes lors de la radiolyse ou de la photosensibilisation des acides nucleiques. La premiere partie de cette these concerne la preparation d'un synthon phosphoramidite de la 5-hydroxy-2-desoxycytidine et l'incorporation de ce dernier dans des oligonucleotides de synthese de 14 a 33 bases de long. La deuxieme partie (chapitres iii et iv) concerne la synthese et l'incorporation de lesions radio-induites originales : les cyclonucleosides. Les deux diastereoisomeres (5r)- et (5s)- des 5,8-cyclopurine-2-desoxyribonucleosides ont ete inseres separement dans differents oligonucleotides (3 a 22 bases de long) en utilisant la chimie phosphoramidite classique. L'incorporation de la (5s, 6s)-5,6-cyclo-5,6-dihydrothymidine a egalement ete effectuee. La structure particuliere de ce cyclonucleosides (perte du caractere aromatique de l'heterocycle azote) ainsi que sa faible reactivite (determinee au cours de nos experiences) nous ont contraint au developpement d'une strategie de synthese radicalement differente de celle utilisee pour les 5,8-cyclopurine-2-desoxyribonucleosides. La troisieme partie de ce travail est consacre a la preparation d'un synthon phosphoramidite pour la 4-hydroxy-8-oxo-7,8-dihydro-2-desoxyguanosine. Au cours de l'une des etapes de synthese de ce precurseur, nous avons pu facilement separer les deux diastereoisomeres (4r)- et (4s)- de ce nucleoside modifie. Ils ont ete incorpores separemment dans les fragments d'adn synthetiques; l'epimerisation de la position c-4 n'etant pas observee au cours de la synthese sur support solide et lors de l'etape de deprotection ammoniacale. La derniere partie de ce manuscrit concerne la preparation d'oligonucleotides (2 a 9 bases de long) contenant un nucleoside modifie precurseur du radical 5-(2-desoxyuridilyl)methyle : la 5-(phenylthiomethyl)-2-desoxyuridine. Ces substrats ont ete utilises dans des etudes mecanistiques ayant pour but de preciser la reactivite de cet intermediaire radicalaire. Pour chacun des dommages incorpores une attention toute particuliere a ete portee a l'integrite des oligonucleotides synthetises. L'utilisation de differentes methodes analytiques (spectrometrie de masse et analyses clhp et maldi-tof des digestions enzymatiques) a permis de demontrer la purete des produits obtenus.
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Saïag, Bernard. "Modulation de l'etat contractile du muscle lisse vasculaire par les purines, les pyrimidines et leurs recepteurs." Rennes 1, 1989. http://www.theses.fr/1989REN1A052.
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Etude de la modulation de l'etat contractile des muscles lisses arteriels (arteres caudale et femorale du rat) et veineux (veines saphene et maxillaire interne du chien) par des molecules de nature purique (adenosine. . . Atp) ou pyrimidique (utp)
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Tikk, Meelis. "Peptides and ribonucleotides in fresh meat as a function of aging in relation to sensory attributes of pork /." Uppsala : Dept. of Food Science, Swedish University of Agricultural Sciences, 2008. http://epsilon.slu.se/200886.pdf.
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Liu, Wanbo. "Molecule recognition of nucleic acids, nucleosides, nucleotides, and their derivatives." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/150.
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It has long been known that the efficiency of anticancer drugs is limited by the emergence of resistance due to the evolving repair of such DNA lesions in malignant cells. Therefore, development of pharmaceutical agents, which can interfere with the DNA repair pathways, may represent a novel approach to enhance the cytotoxic effects of chemotherapy by reducing drug resistance. Abasic sites (AP sites) are the key intermediates in the BER pathway and promising targets for BER inhibition. In chapter 2, we report the synthesis of two small molecules specifically targeting at AP sites and the evaluation of their activity in terms of interstrand crosslinking formation. Our results show no covalent adduct is induced, which is due to the weak DNA binding affinity. In chapter 3, we try to use TFOs to deliver the interstrand crosslinking moiety to the AP site in a sequence specific manner. Two modified phosphoramidites were synthesized and incorporated into the 5' end of TFOs. The activity was evaluated by using various biophysical and biochemical experiments. The work reported in chapter 4 is focused on the G-quadruplex structure formed in the guanine rich telomeric sequence. Many studies have shown G4 ligands can induce and stabilize G-quadruplex within telomere region and inhibit the activity of telomerase that is overexpressed in 80-90% of cancer cells. Our results indicate that phenanthroline based metal complexes, Ni(Phen) 2 , have strong binding affinity and selectivity towards G-quadruplex over duplex DNA. The effect of Ni(Phen) 2 on telomerase activity and cytotoxicity towards cancer cells was also investigated. Calixarenes containing DNA building units such as nucleotides, nucleosides, and nucleobases have recently aroused much interest because of their versatile applications. In chapter 5, we report the synthesis of calix[4]arenes ( 5.11-5.14 ) functionalized with a single nucleobase (thymine, adenine, guanine, or cytosine) at the upper rim via click chemistry. Their complexation with alkali metal ions was examined using MALDI-TOF mass spectrometry and their molecular interactions were determined using 1 H NMR. All calix[4]arene derivatives show good complexation with alkali metal ions with apparent selectivity. The results also reveal that nucleobase-calix[4]arenes are capable of self-association in CDC1 3 and calix[4]arenes bearing complementary nucleobases can bind to each other via base pairing.
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Cheviet, Thomas. "Ciblage d'enzymes du métabolisme purique chez Plasmodium falciparum : Conception et étude de molécules bioactives." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTS121.
Full textAbstract:
Le paludisme, problème de santé publique mondial, est dû à plusieurs parasites possédant la caractéristique de n'avoir qu'une voie de biosynthèse nucléotidique : la voie de récupération. Dans le cadre d'une conception rationnelle d'inhibiteurs d'enzymes impliqués dans le cycle purique chez Plasmodium Falciparum (un des parasites responsable du paludisme), plusieurs composés ont donné des résultats prometteurs. Le projet de thèse sera consacré d'une part à l'optimisation structurale de ces composé-hits en interaction avec une équipe de biologistes (SAR) et une équipe de structuralistes (relation structure fonction activité), et d'autre part à la mise au point de méthodes de dosages LC/MS/MS pour identifier et étudier les cibles biologiques, et révéler l'impact des nouveaux composés sur le métabolisme puriqueMalaria, a global public health problem, is due to several parasites which are characterized by presenting only one nucleotide biosynthesis pathway : the recovery path. As part of a rational design of inhibitors of enzymes involved in purine cycle of Plasmodium Flaciparum (one of parasites responsible for malaria), several compounds have shown promising results. This PhD project will focus firstly on the structural optimization of these hits, in interaction with a team of biologists (SAR) and of structuralists (structure-fonction-activity relationship), and secundly on the development of LC/MS/MS dosage methods to identify and study the biological targets, and reveal the impact of the omptimized compounds on the purine metabolism
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Gu, Baijun. "THE P2X7 RECEPTOR OF HUMAN LEUKOCYTES." Thesis, The University of Sydney, 2003. http://hdl.handle.net/2123/501.
Full textAbstract:
Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.
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Gu, Baijun. "THE P2X7 RECEPTOR OF HUMAN LEUKOCYTES." University of Sydney. Medicine, 2003. http://hdl.handle.net/2123/501.
Full textAbstract:
Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.
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Rayala, Ramanjaneyulu. "Design and Synthesis of Novel Nucleoside Analogues: Oxidative and Reductive Approaches toward Synthesis of 2'-Fluoro Pyrimidine Nucleosides." FIU Digital Commons, 2015. http://digitalcommons.fiu.edu/etd/2172.
Full textAbstract:
Fluorinated nucleosides, especially the analogues with fluorine atom(s) in the ribose ring, have been known to exert potent biological activities. The first part of this dissertation was aimed at developing oxidative desulfurization-fluorination and reductive desulfonylation-fluorination methodologies toward the synthesis of 2'-mono and/or 2',2'-difluoro pyrimidine nucleosides from the corresponding 2'-arylthiopyrimidine precursors. Novel oxidative desulfurization-difluorination methodology was developed for the synthesis of α,α-difluorinted esters from the corresponding α-arylthio esters, wherein the arylthio group is present on a secondary internal carbon. For the reductive desulfonylation studies, cyclic voltammetry was utilized to measure the reduction potentials at which the sulfone moiety of substrates can be cleaved.
The 5-bromo pyrimidine nucleosides and 8-bromo purine nucleosides act as crucial intermediates in various synthetic transformations. The second part of the present dissertation was designed to develop a novel bromination methodology using 1,3-dibromo-5,5-dimethylhydantoin (DBH). Various protected and deprotected pyrimidine and purine nucleosides were converted to their respective C5 and C8 brominated counterparts using DBH. The effect of Lewis acids, solvents, and temperature on the efficiency of bromination was studied. Also, N-bromosuccinimide (NBS) or DBH offered a convenient access to 8-bromotoyocamycin and 8-bromosangivamycin.
Third part of this research work focuses on the design and synthesis of 6-N-benzylated derivatives of 7-deazapurine nucleoside antibiotics, such as tubercidin, sangivamycin and toyocamycin. Target molecules were synthesized by two methods. First method involves treatment of 7-deazapurine substrates with benzylbromide followed by dimethylamine-promoted Dimroth rearrangement. The second method employs fluoro-diazotization followed by SNAr displacement of the 6-fluoro group by a benzylamine. The 6-N-benzylated 7-deazapurine nucleosides showed type-specific inhibition of cancer cell proliferation at micromolar concentrations and weak inhibition of human equilibrative nucleoside transport protein (hENT1).
In the fourth part of this dissertation, syntheses of C7 or C8 modified 7-deazapurine nucleosides, which might exhibit fluorescent properties, were undertaken. 8-Azidotoyocamycin was synthesized by treatment of 8-bromotoyocamycin with sodium azide. Strain promoted click chemistry of 8-azidotoyocamycin with cyclooctynes gave the corresponding 8-triazolyl derivatives. Alternatively, 7-benzotriazolyl tubercidin was synthesized by iodine catalyzed CH arylation of tubercidin with benzotriazole.
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Mbongwa, Hlengiwe Prosperity. "Characterisation of the SULT1A1 polymorphism in a South African Tswana population group / y Hlengiwe P. Mbongwa." Thesis, North-West University, 2010. http://hdl.handle.net/10394/4225.
Full textAbstract:
This dissertation brings to the fore the “Characterization of the SULT1A1 polymorphism in a
South Africa Tswana population group.” The primary experimental group studied came from
South African homogeneous Tswana individuals who participated voluntarily in an ongoing
large-scale epidemiological Prospective Urban and Rural Epidemiological (PURE) study the
North-West University (Potchefstroom Campus) participates in, as one of the 16 low- middleand
high-income countries across the world.
The primary aspect investigated was the comprehensive profile of the single nucleotide
polymorphism (SNP) and copy number variation (CNP) of the SULT1A1 gene. Using the PCRbased
RFLP method, SULT1A1 genotypes, and allele frequency distributions in an
experimental group of 1 867 individuals were determined. According to the literature this is by
far the largest and most homogeneous group from which such information has been acquired to
date. The SULT1A1*1, SULT1A1*1/*2 and SULT1A1*2 genotypes were found to be present at
a percentage of 43.76, 47.12 and 9.11 respectively. In comparison to similar studies in other
population groups, results from this study indicate that there are ethnic differences in the
SULT1A1 genotypes incidence. Asian group differs from Caucasian and Tswana groups
because of its exceptionally high prevalence of individuals with the SULT1A1*1 genotype and a
very low incidence of the SULT1A1*2 genotype. The SULT1A1*1 genotype profiles of
Caucasian and Tswana groups were comparable, but notable differences were observed for the
SULT1A1*2 genotype.
Using a quantitative multiplex PCR method for the CNV study, the numbers of copies of the
SULT1A1 gene in the Tswana population were determined, and the results showed 1 to ~5
copies: only 0.65% of the subjects had a single copy, whereas 59.69% of the subjects had 3 or
more copies. This result shows a significant discrepancy between the Caucasian-American
samples, which showed that only 26% from that group had more than three copies. However,
there is a significant relationship with the African-American population, which presented 63%
with 3 or more copies. This finding confirms results from a much smaller African-American
study, and suggests a possible genetic link between the African Tswana and the heritage of the
African-Americans. These findings were submitted for publication to the South African Journal
of Science, as that journal specializes in publication of new knowledge that has a regional focus
on Africa. Simultaneous phenotypic consequences of the SNP and CNP of the SULT1A1 gene, as well as
the thermo-stable and thermo-labile forms of the sulfotransferases were determined. For this,
the formation of [35S]-4-nitrophenyl sulphate from 4-nitrophenol and [35S]-3’-phosphoadenosine-
5’-phosphosulfate ([35S]-PAPS) in platelet homogenates were measured, with the data
normalized to a common platelet count. This investigation required fresh blood for enzyme
activity. These samples came from 98 Caucasian subjects who voluntarily participated in this
part of the study. The experimental data presented a unique challenge to develop a statistical
model to accommodate the complexity of the distribution of the data in the phenotype and
genotype components, which could be achieved by the development of a mixed model. The
model indicated that product formation increased through increasing copy number, but did not
differ for SULT1A1*1 and SULT1A1*1/*2. However, the rate of increase in product for the
thermo-stable forms of the SULTs was greater than that of thermo-labile forms. In contrast, copy
number effect for SULT1A1*2 differed considerably from that of the other two genotypes. Since
genotype is also a significant factor, it was concluded from Tukey post-hoc tests that the
population group means for product formation differ significantly (for all levels). These results
are presently being prepared for publication in an accredited international journal.
Finally, perturbations in 23 biochemical parameters measured in the PURE study were
analyzed as a function of the SULT1A1 SNP and CNP were evaluated. No group separation in
this regard could be found. It could be shown however, that sulfonation of the iodothyronines,
which are endogenous substrates for the SULTs, was influenced by the SULT1A1 genotype.
The relative concentrations in plasma of the sulphonated iodothyronines may be expressed as
T2S > T3S >> T4S, which coincides with the substrate preference of the SULT1A1 enzymes.
This observation may, however, only be qualitatively interpreted as (1) the targeted
metabolomics mass spectrometric method used for the quantitative analysis of these
substances needs further development, and (2) the influence of deiodonation was not taken into
account in these studies. In conclusion, three perspectives are given at the end of the thesis
which might be considered for further investigations.Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.
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WEBER, M. ELISABETH. "Transporteurs de pyrimidines chez saccharomyces cerevisiae : sequence de deux genes et prediction de structure des proteines correspondantes." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13197.
Full text
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Chou, Chih-Hung, and 周志宏. "Simultaneous analysis of purine nucleotides by capillary zone electrophoresis." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/73264684403892474127.
Full textAbstract:
碩士國立暨南國際大學
應用化學系
93
Purine nucleosides, purine nucleotides and their derivatives not only provide with the ability of energy conversion but are released from the neurons and react with the receptor on another cell to transmit signal. In order to monitor the concentrations of extracellular purine nucleosides and purine nucleotides in the process of neurotransmitting, one method of capillary zone electrophoresis was developed to analyze purine deoxynucleosides dA and dG, purine nucleosides A and G, purine nucleotides AMP, ADP, ATP, GMP, GDP and GTP, deoxynucleotides dAMP, dADP, dATP, dGMP, dGTP and cyclic nucleotides cAMP and cGMP in the microdialysate. EDTA and water were added into the microdialysate before sample injection. Borax with b-CD was served as background electrolyte. This method could resolve the nucleosides and nucleotides in 24 minutes. The correlation coefficient of each nucleotide was higher than 0.9967. Validation test has been carried out. The relative standard deviation (RSD) of the area ratio of each nucleotide to internal standard cGMP was less than 8.9%, and that of migration time was less than 1.2%. Therefore, the method could be applied to the study of extracellular purine nucleotides.
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Zhao, Xiaoqin S. "Purine nucleotide-induced seizures in rat prepiriform cortex." Thesis, 1994. http://hdl.handle.net/1957/35537.
Full text
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Richards, SM. "The release of purine and pyrimidine nucleotide catabolites from working blood vessels." Thesis, 1993. https://eprints.utas.edu.au/21380/1/whole_RichardsStephenMichael1994_thesis.pdf.
Full textAbstract:
This study examined the release of purine and pyrimidine nucleotide catabolites from perfused rat hindlimb and mesenteric arcade, and incubated aortas in response to vasoconstriction, stretch-induced tone and skeletal muscle contraction.
The release of uric acid and uracil from non-recirculating, perfused rat hindlimb was stimulated from 0.95 and 0.4nmol/min per g, respectively, by a factor of 2-5fold by noradrenaline, vasopressin or angiotensin II, in a dose-dependent manner, coinciding with increases in V02 and perfusion pressure. Angiotensin-mediated increases were blocked by the nitrovasodilator nitroprusside. Skeletal muscle contraction increased the release of inosine and hypoxanthine but did not alter uracil or uric acid release. Perfused mesenteric arcade and incubated aortas released nucleotide catabolites in response to serotonin and tension respectively. The main products released from aortas were uridine, cytidine, urate and hypoxanthine. Following vasoconstriction pyrimidine compounds represented 23-38 % of the nucleotide catabolites found in venous effluent or incubation medium from hindlimb, mesentery or aorta. Pyrimidine nucleotides represented 25 % of the intracellular nucleotides in aorta. In contrast, only 2.9% of the hindlimb nucleotide pool consisted of pyrimidine nucleotides. It was concluded that nucleotide catabolite release during vasoconstriction derived from vascular tissue.
The possibility that uracil derived from the breakdown of UTP released with a vasomodulatory function was examined. UTP was a potent vasodilator in the perfused rat hindlimb, opposing noradrenaline- and angiotensin-mediated vasoconstriction. Exogenous UTP was rapidly catabolized by perfused hindlimb to non-vasoactive products, UMP, uridine and uracil. α, β-MethyleneADP, an ecto-5'nucleotidase inhibitor, blocked extracellular UMP degradation during infusion of UTP, but failed to reduce uracil or uridine release and did not lead to the accumulation of UMP during angiotensin infusion. Hence, extracellular UTP degradation is unlikely to be the source of hindlimb uracil release.
Lactate release from hindlimb correlated well with uracil release during vasoconstriction, vasodilation or cyanide poisoning (r=0.831; P<0.001) but not during B-adrenergic stimulation. High [Ca2+], (≅10mM) but not K+-depolarization, stimulated nucleotide catabolite release from aortas incubated under tension. This data provides evidence of a link between glycolysis, raised cytoplasmic [Na+] and pyrimidine release.
Pyrimidine nucleotide (but not RNA) levels were reduced in hindlimb and aorta after intensive pyrimidine release. However, the relationship between catabolite release and tissue nucleotide depletion was not stoichiometric, suggesting that pyrimidine de novo synthesis rates may be high in vascular tissue. Radiolabel from [3H]cytidine entered aortic uracil nucleotides, suggesting that uridine may derive from cytosine nucleotides. Aorta homogenate UTP hydrolysis was stimulated by 10-150mM Na+ in the absence of other cations was separate from Na+/K+-ATPase and not ouabain inhibited. Highest activity was observed with UTP and the activity was probably intracellular but not soluble.
Overall the study shows: (i) that high workloads result in the release of nucleotide catabolites from VSM, of which a high proportion are pyrimidine compounds; (ii) that nucleotide catabolites released in response to vascular work are likely to be generated intracellularly; (iii) and that pyrimidine nucleotide catabolite generation and release from VSM is associated with raised glycolysis and high intracellular [Na+].
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Stathis, Christos George. "Factors Influencing Muscle Purine Nucleotide Metabolism." 2006. http://eprints.vu.edu.au/581/1/581contents.pdf.
Full textAbstract:
The experiments in this thesis were designed to investigate the factors influencing the metabolism of purine nucleotides in human skeletal muscle, plasma and urine. Study one investigated the influence of the number of intermittent sprint bouts and the subsequent accumulation of plasma purines and urinary purine excretion. Study two investigated the influence of sprint training on urinary purine loss following intense exercise. Study three examined the influence of allopurinol on urinary purine loss after repeated sprint exercise in humans. The final study examined the combined effects of allopurinol and sprint training on purine nucleotide metabolism in humans at rest.
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Stathis, Christos. "The effect of sprint training on human skeletal muscle purine nucleotide metabolism." Thesis, 1994. https://vuir.vu.edu.au/15387/.
Full textAbstract:
The major focus of this thesis was to examine the effect of sprint training on skeletal muscle purine nucleotide (PnN) metabolism. Specifically, this thesis studied the effects of sprint training on skeletal muscle PnN and metabolites of
purine catabolism at rest, during a 30 s "all out" cycle bout and after 3 min of recovery. Furthermore, the accumulation of purine degradation prcducts in the plasma (ammonia, hypoxanthine) during exercise, and in recovery from a 30 s "all out" sprint was also examined. Finally, this thesis studied the influence of sprint training on performance variables pertinent to sprint exercise.
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Bagshaw, Andrew. "An investigation of links between simple sequences and meiotic recombination hotspots : a thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular and Cellular Biology at the University of Canterbury /." 2008. http://hdl.handle.net/10092/1597.
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Nkosi, Thokozani Clement. "Regulation of kinases by synthetic imidazoles, nucleotides and their deuterated analogues." Diss., 2015. http://hdl.handle.net/10500/20129.
Full textAbstract:
Deuteration is the replacement of a hydrogen atom by deuterium atom in a molecule. The replacement begins at the most acidic hydrogen in the molecule. In ATP, the deshielded hydrogen is C8-H which is the first replaced during deuteration. During ATP deuteration some of the ATP is hydrolysed to ADP concurrently. Using kinetic analysis, it was confirmed that the ATP hydrolysis that occurs is 1st order in ATP concentration, while the hydrogen replacement is 2nd order. The ATP and its C8 deuterated analogue were tested against three enzymes shikimate kinase (SK), acetate kinase (AK) and glutamine synthetase (GS) to determine if a kinetic isotope effect (KIE) exists in these systems. With AK and GS, the KIED increased as the KIEH decreased, while with SK the KIED decreased as the KIEH increased as the concentration of the ATP or deuterated analogue increased. Deuteration of imidazole and purine compounds reduced the specific activity of AK or SK at low concentrations in an enzyme-catalysed reaction. From a library of imidazole-containing compounds that inhibited SK, three compounds were selected and their IC50 values were determined on the SK-catalysed reaction. These compounds show a differential potency and efficiency between their protonated and deuterated analogues when compared in a 1:1 mixture. Synthesized purines incorporating three different substituents at N-9 were tested against AK or SK for their ability to lower the specific activity of the enzymes usedPhysics
M. Sc. (Physics)
48
Stathis, Christos. "Factors Influencing Muscle Purine Nucleotide Metabolism." Thesis, 2006. https://vuir.vu.edu.au/581/.
Full textAbstract:
The experiments in this thesis were designed to investigate the factors influencing the metabolism of purine nucleotides in human skeletal muscle, plasma and urine. Study one investigated the influence of the number of intermittent sprint bouts and the subsequent accumulation of plasma purines and urinary purine excretion. Study two investigated the influence of sprint training on urinary purine loss following intense exercise. Study three examined the influence of allopurinol on urinary purine loss after repeated sprint exercise in humans. The final study examined the combined effects of allopurinol and sprint training on purine nucleotide metabolism in humans at rest.
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Hung, Jaw-Shian, and 洪照先. "Characterization of purine 5'-nucleotidase from Hep G2 and Sf9." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/71588476312823025931.
Full textAbstract:
碩士國立暨南國際大學
應用化學系
94
The 5’-nucleotidaes (5’-NT) are involved in the regulation different physiological activation of nucleosides, nucleotides, and their analogues in organs. Therefore 5’-NT maintains balanced nucleotide pools, so it is one of the important phosphohydrolases. The traditional enzyme assays of 5’-NT are quite complicated, and need more sample. In order to develop non-radioactive assays to characterize the 5’-NTs from Hep G2 and Sf9, adenosine, hypoxanthine, inosine and AMP were separated by capillary zone electrophoresis. For the assay of 5’-NT, the standards were effectively separated in only 6 minutes. Addition of acetonitrile and sodium chloride to sample buffer could enhance sample stacking. The correlation coefficient of Ado、hXan and I were 0.9998, 0.9997, and 0.9993, respectively. The concentration limits of detection of Ado、hXan and I were 2.49 M、2.79M and 4.36 M, respectively. In the precision test, all relative standard deviations of migration time and area ratio were smaller than 1.60 % and 7.47 %, respectively. This method was applied to characterize the 5’-NT related to AMP metabolism. The kinetics of the 5'-NTs from Hep G2 and Sf9 followed Hill equation. It suggested that these 5’-NTs were regulated by allosteric effectors. The 5’-NT from Hep G2 showed higher affinity to AMP compared to that from Sf9; however, the latter showed a larger maximum velocity than the former.
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"Expressions of cyclic nucleotide-gated ionic conductances in rat cerebellar purkinje neurons =: 大鼠小腦浦肯野細胞環核苷酸門控離子通道的表達." 2005. http://library.cuhk.edu.hk/record=b5892479.
Full textAbstract:
Tsoi Sze Man.Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (leaves 82-104).
Text in English; abstracts in English and Chinese.
Tsoi Sze Man.
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Overview of study --- p.1
Chapter 1.2 --- Cerebellum --- p.2
Chapter 1.2.1 --- General Structure of cerebellum --- p.3
Chapter 1.2.2 --- Cell types of cerebellar cortex --- p.4
Chapter 1.2.2.1 --- Basket cells --- p.5
Chapter 1.2.2.2 --- Stellate cells --- p.6
Chapter 1.2.2.3 --- Purkinje cells --- p.6
Chapter 1.2.2.4 --- Granule cells --- p.7
Chapter 1.2.2.5 --- Golgi cells --- p.8
Chapter 1.2.2.6 --- Unipolar brush cells --- p.9
Chapter 1.2.2.7 --- Deep cerebellar nuclear neurons --- p.11
Chapter 1.2.3 --- The neuronal circuitry of the cerebellum --- p.12
Chapter 1.2.4 --- Cerebellar function --- p.14
Chapter 1.3 --- Cyclic nucleotide-gated (CNG) channels --- p.16
Chapter 1.3.1 --- Molecular characterization of CNG channels --- p.16
Chapter 1.3.2 --- Functional properties of CNG channels --- p.19
Chapter 1.3.3 --- Expression of CNG channels in brain --- p.21
Chapter 1.3.4 --- CNG channel and neuronal plasticity --- p.23
Chapter 1.4 --- Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels --- p.26
Chapter 1.4.1 --- Molecular characterization of HCN channels --- p.27
Chapter 1.4.2 --- Functional properties of HCN channels and Ih current --- p.29
Chapter 1.4.3 --- Modulation by cyclic nucleotides --- p.31
Chapter 1.4.4 --- Expression of HCN channels in brain --- p.33
Chapter 1.4.5 --- Physiological roles of Ih current in central nervous system --- p.35
Chapter 1.5 --- Aims of study --- p.38
Chapter Chapter 2 --- Material and Methods --- p.39
Chapter 2.1 --- Immunohistochemistry Experiments --- p.39
Chapter 2.1.1 --- Animal preparation --- p.39
Chapter 2.1.2 --- Tissue preparation --- p.39
Chapter 2.1.3 --- Primary and secondary antibodies --- p.40
Chapter 2.1.4 --- Immunofluroescence staining --- p.41
Chapter 2.1.5 --- Confocal laser scanning microscopy and data processing --- p.41
Chapter 2.2 --- Whole cell patch clamp recordings --- p.42
Chapter 2.2.1 --- Brain slice preparation and identification of the cerebellar Purkinje neurons --- p.42
Chapter 2.2.2 --- Whole cell voltage- and current-clamp recordings --- p.43
Chapter 2.2.3 --- Drug solutions and delivery --- p.44
Chapter 2.2.4 --- Statistical analysis --- p.45
Chapter Chapter 3 --- Expression of Various Cyclic Nucleotide-Gated (CNG) Channel Subunits in Rat Cerebellum --- p.46
Chapter 3.1 --- Introduction --- p.46
Chapter 3.2 --- Results --- p.46
Chapter 3.2.1 --- Immunoreactivity of CNGA1 in cerebellum --- p.46
Chapter 3.2.2 --- Immunoreactivity of CNGA2 in cerebellum --- p.47
Chapter 3.2.3 --- Immunoreactivity of CNGA3 in cerebellum --- p.47
Chapter 3.3 --- Discussion --- p.48
Chapter Chapter 4 --- Expression of Various Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channel Subunits in Rat Cerebellum --- p.53
Chapter 4.1 --- Introduction --- p.53
Chapter 4.2 --- Results --- p.53
Chapter 4.2.1 --- Immunoreactivity of HCN 1 in cerebellum --- p.53
Chapter 4.2.2 --- Immunoreactivity of HCN2 in cerebellum --- p.55
Chapter 4.2.3 --- Immunoreactivity of HCN3 in cerebellum --- p.55
Chapter 4.2.4 --- Immunoreactivity of HCN4 in cerebellum --- p.55
Chapter 4.3 --- Discussion --- p.55
Chapter Chapter 5 --- Electrophysiological Recordings of Cyclic Nucleotide-Gated Ionic Conductance in Rat Cerebellar Purkinje Neurons --- p.59
Chapter 5.1 --- Introduction --- p.59
Chapter 5.2 --- Results --- p.59
Chapter 5.2.1 --- Effect of cyclic nucleotides on the membrane potential of cerebellar Purkinje neurons --- p.59
Chapter 5.2.2 --- Ionic conductance of the cyclic nucleotide-induced inward current --- p.61
Chapter 5.2.3 --- The mechanism of the cyclic nucleotide-induced inward current --- p.61
Chapter 5.2.3.1 --- Site of action --- p.62
Chapter 5.2.3.2 --- Involvement of CNG channels and HCN channels --- p.63
Chapter 5.2.3.3 --- Involvement of protein kinase A (PKA) and protein kinase G (PKG) --- p.65
Chapter 5.2.3.4 --- Involvement of inwardly rectifying potassium (Kir) channels and transient receptor potential (TRP) channels --- p.65
Chapter 5.2.4 --- Effect of cyclic nucleotides on Ih current in Purkinje neurons --- p.67
Chapter 5.3 --- Discussion --- p.68
Chapter Chapter 6 --- Concluding remarks References --- p.78
References --- p.82