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Several lipopeptidyl nucleoside antibiotics that inhibit bacterial translocase I (MraY) involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. A-90289 and muraminomicin are the two representative antibiotics that belong to this family. Bioinformatic analysis of the biosynthetic A-90289 gene clusters revealed that five enzymes are likely involved in the assembly and attachment of the aminoribosyl unit. These enzymes of A-90289 are functionally assigned by in vitro characterization. The results reveal a unique ribosylation pathway that highlighted by uridine-5′-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor. Muraminomicin, which has a structure similar to A-90289, holds the distinction in that both ribose units are 2-deoxy sugars. The biosynthetic gene cluster of muraminomicin has been identified, cloned and sequenced, and bioinformatic analysis revealed a minimum of 24 open reading frames putatively involved in the biosynthesis, resistance, and regulation of muraminomicin. Similar to the A-90289 pathway, fives enzymes are still likely involved in the assembly of the 2,5-dideoxy-5-aminoribose saccharide unit, and two are now functionally assigned and characterized: Mra20, a 5′-amino-2′,5′-dideoxyuridine phosphorylase and Mra23, a UTP:5-amino-2,5-dideoxy-α-D-ribose-1-phosphate uridylyltransferase. The cumulative results are consistent with the incorporation of the ribosyl appendage of muraminomicin via the archetypical sugar biosynthetic pathway that parallels A-90289 biosynthesis

Chronic, noncommunicable diseases such as chronic fatigue syndrome (also known as myalgic encephalomyelitis) are rapidly becoming a worldwide epidemic that profoundly affects public health and productivity. Chronic fatigue syndrome (CFS) is characterised by severe and debilitating fatigue and although its etiology is still unknown, recent studies have found considerable evidence that mitochondrial dysfunction and oxidative stress might be responsible for the underlying energy deficit in these patients. Adenine and pyridine nucleotides could be used as potential biomarkers for energy related disorders such as chronic fatigue syndrome because of their various functions in the energy and redox pathways. The first part of this study focussed on developing a liquid chromatography electrosprayionisation tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of these nucleotides in blood samples. Due to the instability of nucleotides in biological matrices it was also necessary to find a suitable extraction method that would be able to stop enzymatic activity via protein precipitation. Out of the four extraction methods investigated during this study, deproteinisation of whole blood samples with perchloric acid produced the highest nucleotide abundances. Although nucleotide standards were found to be stable in perchloric acid, nucleotide levels in blood samples were not stabilised by addition of perchloric acid. The second part of this study consisted of measuring the nucleotide levels in blood samples of controls and possible CFS patients in order to test the proof of concept of the new LCESI- MS/MS method. Despite changes in the nucleotide levels due to perchloric acid and problems with nucleotide instability, it was still possible to distinguish between the two groups based on the results obtained with the new LC-ESI-MS/MS method. The newly developed LC-ESI-MS/MS method proved to be reliable and adequate for nucleotide quantification in whole blood samples, thus the aim of this study was achieved. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Die Umsetzung des Park-Nucleotids zu Lipid I in der bakteriellen Zellwandbiosynthese wird durch das Enzym MraY katalysiert. Dieses Enzym ist ein interessantes, bisher ungenutztes Target für neuartige Antibiotika. In der vorliegenden Dissertation wurde das fluoreszenzmarkierte Park-Nucleotid, welches für MraY-Assays geeignet ist, totalsynthetisch dargestellt. Die Synthese konnte ausgehend von kommerziell erhältlichem N-Acetylglucosamin in 14 Stufen mit einer Gesamtausbeute von 4 % erfolgreich durchgeführt werden. Dabei wurden einige Schritte auf Basis einer literaturbekannten Synthese gründlich optimiert und neue Wege zur Darstellung des Peptids und anschließender Kupplung mit einem Kohlenhydrat Baustein gefunden. Im Zuge dieser neuen Syntheseroute wurde die Uridindiphosphat-N-Acetylmuraminsäure, als Substrat für MurC-Assays, synthetisiert. Dies gelang in einer achtstufigen Synthese ausgehend von der geschützten N-Acetylmuraminsäure in einer Gesamtausbeute von 15 %. Die Muraymycine, eine Klasse von natürlichen Nucleosid-Antibiotika, inhibieren das Enzym MraY effizient. In dieser Dissertation wurde der Grundstein für die Synthese möglichst vielfältiger vereinfachter Muraymycin Analoga gelegt, welche potentielle Inhibitoren für MraY darstellen. Die Harnstoffpeptidseitenkette wurde variiert, um den Einfluss dieses Strukturbausteins auf die Inhibitor-Aktivität intensiver untersuchen zu können. Der Ersatz der anspruchsvoll zu synthetisierenden Aminosäure Epicapreomycidin durch die kommerziell erhältlichen Aminosäuren L-Lysin, L-Ornithin, L-Arginin, L-Alanin und L-Methionin konnte die Synthese der Harnstoffpeptidkette stark vereinfachen. Des Weiteren wurde durch eine stereospezifische Synthese die geschützte 2S,3S-Diaminohexansäure dargestellt, um das in den natürlichen Muraymycinen enthaltene L-Hydroxyleucin, welches häufig mit einer Fettsäure verestert ist, zu ersetzten. Dieses sollte durch den Austausch einer Esterbindung durch eine Amidbindung zu einer Stabilisierung der Muraymycin Derivate führen. Unter Verwendung des in dieser Dissertation beschriebenen Lysin-haltigen Harnstoffpeptids wurden vor kurzen vereinfachte Muraymycin-Analoga synthetisiert und IC50 Werte mittels eines fluoreszenzbasierten MraY-Assays bestimmt. Diese stark vereinfachten Verbindungen weisen bereits gute Inhibitor Aktivitäten auf. Durch die Kombination der verschiedenen, in dieser Arbeit synthetisierten Harnstoffpeptide und Aminosäuren mit unterschiedlichen Uridinderivaten kann zukünftig eine große Anzahl diverser potentieller Inhibitoren dargestellt werden.

Cette thèse a premièrement permis le développement d’une méthode de détection non-enzymatique de l’ADN. Les techniques enzymatiques couramment utilisées, comme la Polymerase Chain Reaction (PCR), échouent souvent dans l’analyse d’échantillons anciens ou transformés. L’ADN subit en effet de nombreuses dégradations post mortem, parmi lesquelles certaines bloquent les enzymes ADN-polymérases. Notre méthode combine hybridation et détection par SERRS (Surface Enhanced Resonant Raman Scattering), permettant la détection et la quantification de séquences d’ADN dégradées réfractaires à l’analyse par PCR. De nouvelles perspectives s’ouvrent donc en paléogénétique. Cette thèse aborde également le rôle des surfaces minérales dans l’origine des acides nucléiques. Les surfaces minérales pourraient avoir piégé et concentré les briques élémentaires de ces biopolymères, contribuant ainsi à leur construction. Les travaux existants se sont concentrés sur des minéraux comme la montmorillonite, qui n’était cependant pas abondante à l’Hadéen/Archéeen. La minéralogie de la Terre primitive aurait plutôt été dominée par les phyllosilicates ferromagnésiens. Nous avons étudié l’adsorption de nucléotides sur des minéraux plus cohérents avec le contexte géologique, en variant les paramètres environnementaux. Ce travail permet de préciser le mécanisme d’adsorption des nucléotides sur les surfaces minérales, ainsi que les conditions de l’origine du matériel génétique. / The first goal of this thesis was the development of a non-enzymatic DNA detection method. Current enzymatic techniques such as Polymerase Chain Reaction (PCR) often fail in analyzing ancient or processed samples. Indeed DNA undergoes numerous post-mortem degradations, among which some are known to block the bypass of DNA-polymerases. Our method combines hybridization and SERRS (Surface Enhanced Resonant Raman Scattering) spectroscopy, and allows the detection and quantification of degraded DNA sequences that are refractory to PCR analysis. This novel detection method therefore opens new perspectives, especially in paleogenetics. This thesis also aims at studying the role of mineral surfaces in the origin of nucleic acids. Mineral surfaces might have trapped and concentrated the elementary bricks of those biopolymers, thus contributing in their formation. Previous work has focused on minerals such as montmorillonite, although it might not have been abundant during the Hadean/Archean. The primitive Earth’s mineralogy would have been preferentially dominated by Fe-Mg rich phyllosilicates. We have therefore studied the adsorption of nucleotides on minerals we think are relevant to the geological context, and have varied the environmental conditions. This work allows characterizing the adsorption mechanism of nucleotides on mineral surfaces, as well as environmental conditions of the origin of genetic material.

The aim of the diploma thesis was to find optimal conditions of high pressure liquid chromatography for the detection and quantification of two common nucleotides, namely adenosine diphosphate and adenosine triphosphate, as well as to perform an analysis of these in real life samples of citrus fruits and plant extracts. Further aim of the project was to determine the limits of detection and quantification of adenosine diphosphate and adenosine triphosphate under the optimized conditions and using these to compare the sensitivity of given detectors. To achieve this HPLC-UV, capillary HPLC-DAD and HPLC-MS apparatus were used. With the help of HPLC with UV detection and capillary HPLC with diode array detector, the calibration curves of the mixture of analytes were measured and the limits of detection as well as quantification of adenosine diphosphate and adenosine triphosphate were determined. Separation of the analytes up to the base line using HPLC-UV and capillary HPLC-DAD was achieved under the conditions of ion pairing chromatography. Column C18 was chosen as an appropriate column. The mobile phase included phosphate buffer, acetonitrile and tetrabutylammonium bisulphate as an ion pairing reagent. The separation was performed with gradient elution. Conditions for analysis using LC-MS were...

L’infarctus du myocarde (IC) représente plus de 15 % de la mortalité mondiale liés aux maladies cardiovasculaires (MC). En absence d’une rapide reperfusion des coronaires occluses, aucune intervention thérapeutique n’est capable de limiter les effets délétères de l’IC. Un des moyens les plus efficaces de la cardioprotection, étudié en recherche, est le préconditionnement cardiaque ischémique (PCI). L’adénosine libérée au cours du PCI active via ces récepteurs P1 les voies de cardioprotection. Des études mettent en évidence également l’implication des purinorécepteurs P2Y dans la cardioprotection. Les récepteurs P2Y2, 4 et 6 sont les plus étudiés dans la cardioprotection. Les récepteurs P2Y sont sensibles aux nucléotides comme l’ATP et l’UTP libérés au cours de l’ischémie. Parmi les récepteurs P2Y seul le récepteur P2Y11 est doublement couplé à l’adénylate cyclase et à la phospholipase C (PLC). Il est également le seul récepteur dont le polymorphisme génétique, induisant une perte du signal métabotropique, prédispose à plus de risques d’IC et d’inflammation dans toutes les catégories d’âge indépendamment des facteurs de risques variables de l’IC (Amisten et al., 2007). Ceci suggère un important rôle du récepteur P2Y11 dans la prévention primaire et comme cible thérapeutique de l’infarctus du myocarde. Le β-NAD, nucléotide à base de pyridine, est libéré au cours de l’ischémie comme les médiateurs de cardioprotection notamment l’adénosine, l’ATP et l’UTP. Cependant, aucune étude ne s’est intéressée spécifiquement aux rôles du P2Y11 et des nucléotides pyridiniques, comme médiateurs, dans la cardioportection alors que le NAADP, un métabolite du β-NAD, est rapporté comme agoniste (Moreschi et al., 2008) du récepteur P2Y11. Dans ce travail, nous avons mis en évidence pour la première fois l’augmentation des concentrations interstitielles des métabolites du β-NAD comme le NAADP au cours de l’ischémie. L’augmentation des concentrations du NAADP décrit une cinétique comparable à celle de l’adénosine. Le NAADP extracellulaire ([NAADP]e), appliqué avant un cycle d’ischémie/reperfusion (I/R), déclenche une cardioprotection envers les effets délétères de l’I/R. En effet, le [NAADP]e améliore les fonctions contractiles, réduit les contractures, les arythmies de reperfusion et la taille de l’infarctus, dans un modèle d’I/R cardiaque chez le rat. Ce préconditionnement pharmacologique induit par le [NAADP]e implique les récepteurs P2Y11-like. Dans un modèle de cardiomyocytes, nous mettons en évidence l’activité métabotropique spécifique du récepteur P2Y11-like déclenché par le [NAADP]e. Le [NAADP]e déclenche via le récepteur P2Y11-like l’activation de la PKCε, ERK1/2, AKT et GSK-3β, des protéines kinases de prosurvie cellulaire, ce qui explique ses effets cardioprotecteurs. La phosphorylation des protéines de prosurvie cellulaire nécessite la médiation de la PKA, de la PLC et de la src. Le NF546, un nouvel agoniste sélectif du récepteur P2Y11, déclenche une activité métabotropique semblable à celle du [NAADP]e, confirmant ainsi l’existence fonctionnelle du récepteur P2Y11-like au niveau des cardiomyocytes de rat. L’activation du récepteur P2Y11-like, que ce soit par le [NAADP]e ou le NF546, induit une augmentation des concentrations intracellulaires du β-NAD et de ses métabolites, le NADP, le NAADP, le NAAD et l’ADP ribose cyclique. Le β-NAD, le NAADP comme le NAAD, ont été impliqués dans le déclenchement au niveau intracellulaire des voies de cardioprotection comme la voie des sirtuines et de l’autophagie. Ces mécanismes peuvent être complementaires de l’effet cardioprotecteur du [NAADP]e. L’ensemble de nos données conforte le rôle cardioprotecteur du récépteur P2Y11 et suggère que le NAADP interstitiel accumulé au cours de l’ischémie pourrait avoir un rôle de facteur paracrine de survie cellulaire améliorant la survie des cardiomyocytes au cours des évènements ischémiques. / Myocardial infarction (MI) accounts for more than 15 % of global deaths related to cardiovascular diseases (CD). In the absence of a prompt reperfusion of occluded coronary arteries, none of therapeutic interventions is able to limit the deleterious effects of MI. One of the most effective means of cardioprotection, studied in research, is the ischemic cardiac preconditioning (ICP). Adenosine released during ICP triggers, via P1 receptors, cardioprotective effects. Involvement of the purinoceptors (P2Y) in cardioprotective effects has been also reported. P2Y2,4,6 receptors, the most studied P2Y receptors in cardioprotection, are activated by the nucleotides released during ischemia such as ATP and UTP. Among the P2Y receptors, P2Y11 is dually coupled to Gs and Gq proteins and is the single P2Y receptor which has been linked, via a genetic polymorphism, to an increased risk of acute myocardial infarction and elevated levels of C-reactive protein in humans in all age groups (Amisten et al., 2007). This suggests for the P2Y11 receptor an important role in primary prevention and as a therapeutic target of the P2Y11 receptor for myocardial infarction. β-NAD, a pyridine nucleotide, is released during ischemia like adenosine, ATP and UTP. However, no study has focused on the roles of P2Y11 receptor and pyridine nucleotide in mediating cardioprotective effects while NAADP, a β-NAD metabolite, has been reported as an agonist (Moreschi et al., 2008) of the P2Y11 receptor. In this work, we show, for the first time, increased interstitial concentrations of NAADP during ischemia. Interstitial kinetics of NAADP is similar to adenosine. Using a pharmacological preconditioning protocol, triggered by extracellular NAADP ([NAADP]e) before a prolonged ischemia/reperfusion (I/R), rat hearts rapidly recovered post-ischemic contractile function and displayed attenuated contracture, infarct size and arrhythmogenesis. This pharmacological preconditioning involves the P2Y11-like receptor. In cardiomyocytes culture, [NAADP]e induces specific metabotropic response of the P2Y11-like receptor. [NAADP]e triggers via the P2Y11-like receptor prosurvival protein kinases activation such as PKCε, ERK1/2, AKT and GSK-3β which explains its protective effects. Phosphorylation of prosurvival protein kinases requires the mediation of PKA, PLC and src. As with [NAADP]e, the NF546, a new selective agonist of P2Y11 receptor, triggers a metabotropic activity, thus confirming the functional existence of P2Y11-like receptor in rat cardiomyocytes. Activation of the P2Y11-like receptor, either by [NAADP]e or NF546 induced an increase in intracellular concentrations of β-NAD and its metabolites, NADP, NAADP, NAAD and cyclic ADP ribose. More, β-NAD, NAADP as well as NAAD have been involved in activation of cardioprotective pathways such as sirtuine pathways and autophagy. Taken together, our data demonstrate for the first time that the P2Y11 receptor mediates cardioprotective effects induced by [NAADP]e. NAADP is released during ischemia suggesting that [NAADP]e may act as a paracrine survival factor, prolonging cardiomyocytes lifespan during ischemic events.

Foram utilizados 160 leitões recém-desmamados, com peso médio inicial de 6,43 ± 0,71 kg, com o objetivo de avaliar os efeitos dos nucleotídeos sobre o desempenho, a frequência de diarreia, a morfometria de órgãos, a histologia do epitélio intestinal e a microbiota intestinal. Foram testados cinco tratamentos em um experimento em blocos casualizados, com oito repetições (blocos) por tratamento e quatro animais por unidade experimental (dois machos castrados e duas fêmeas). Os tratamentos foram: dieta basal à base de milho, farelo de soja, derivados lácteos e plasma com inclusão de 120 ppm de clorohidroxiquinolina (antimicrobiano) e dieta basal contendo 0, 100, 150 e 200 ppm de nucleotídeos. Para a determinação das variáveis de desempenho, os animais foram pesados ao 1º, 14º e 34º dia de experimentação e foram quantificadas as rações fornecidas e desperdiçadas. Para avaliar a frequência de diarreia, foram observadas presença e ausência de diarreia na baia, diariamente, no período da manhã. Ao final do experimento, um animal de cada baia (unidade experimental) foi abatido para avaliação da morfometria de órgãos (estômago vazio, intestino delgado vazio, pâncreas, fígado e baço) e da histologia do epitélio intestinal (altura e largura de vilosidade, profundidade de cripta, relação altura de vilosidade: profundidade de cripta), além da coleta de amostras do conteúdo do duodeno e do jejuno para avaliação da microbiota intestinal dos leitões (mesófilos, Gram positivos totais, Gram negativos totais, Lactobacillus spp., Escherichia coli, Salmonella spp., Staphylococcus aureus e Clostridium perfringens). No período de 1 a 14 dias de experimentação, as variáveis de desempenho não foram influenciadas (P>0,05) pelos tratamentos. Para o período total (1 a 34 dias), houve efeito linear benéfico da inclusão dos nucleotídeos no peso final (P=0,005; P34 = 0,0033X + 23,657, R2 = 0,87) e no ganho diário de peso (P=0,008; GDP = 0,0002X + 0,4955, R2 = 0,83) dos animais. Os leitões alimentados com o antibiótico tiveram menor (P=0,049) frequência de diarreia comparados aos animais alimentados com nucleotídeos no período de 1 a 14 dias de experimentação. Por outro lado, no período de 1 a 34 dias de experimentação, os tratamentos não afetaram (P>0,05) a frequência de diarreia, a morfometria de órgãos, a histologia do epitélio intestinal e a microbiota intestinal. Assim, de maneira geral, a inclusão de até 200 ppm de nucleotídeos em dietas complexas para leitões recém-desmamados melhora o desempenho dos animais, sem afetar a frequência de diarreia, a morfometria de órgãos, a histologia do epitélio intestinal e a microbiota intestinal de leitões na fase de creche. / The purpose of this study was to evaluate the effects of dietary nucleotide levels on performance, occurrence of diarrhea, organ morphometry, intestinal histology and intestinal microbiota of weanling pigs fed complex diets containing corn, soybean meal, milk products, and spray-dried plasma. One hundred and sixty 21d-weaned pigs, averaging 6.43 ± 0.71 kg BW, were used in a randomized complete block design experiment with 5 treatments, 8 replications (blocks) per treatment and 4 animals per experimental unit (pen). The treatments were: basal diet with 120 ppm of chloro-hydroxyquinoline (antimicrobial treatment), and basal diet with 0, 100, 150, and 200 ppm of nucleotides. The ADG, ADFI, G:F and occurrence of diarrhea were calculated during 1 to 14 and 1 to 34 d of experimental period. A day after the end of the experimental period, an animal from each pen was slaughtered to evaluate of organ morphometry (empty stomach, empty small intestine, pancreas, liver and spleen), intestinal histology (villus height, villus width, crypt depth and villus height-to-crypt depth ratio), and intestinal microbiota (mesophiles, Gram-positive bacteria, Gram-negative bacteria, Lactobacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp., and Clostridium perfringens). During 1-14 d of experimental period, performance was not affected (P>0.05) by treatments. For the total experimental period (1-34 d), beneficial linear effects of dietary levels of nucleotides on final BW (P=0.005; BW = 0.0033X + 23.657, R2 = 0.87) and ADG (P = 0.008; ADG = 0.0002X + 0.4955, R2 = 0.83) were observed, but not (P>0.05) on ADFI and G:F. Pigs fed antimicrobial treatment had lower (P=0.049) occurrence of diarrhea from d 1 to 14 than those fed nucleotide treatments. However, for the total experimental period (1-34 d), treatments did not affect (P>0.05) occurrence of diarrhea, organ morphometry, intestinal histology and intestinal microbiota. Therefore, adding up to 200 ppm of dietary nucleotides to complex diets for weanling pigs showed beneficial effect on growth performance, without affecting organ morphometry, intestinal histology, intestinal microbiota and occurrence of diarrhea of nursery pigs.

Os pacientes que usam lítio (Li) para tratamento do transtorno bipolar freqüentemente apresentam poliúria e deficiência de concentração urinária, sintomas do Diabetes Insipidus nefrogênico (DIN). Animais tratados com Li apresentam baixos níveis de produção de adenosina monofosfato cíclico (AMPc) em resposta ao hormônio antidiurético (HAD). O Sildenafil (Sil), um inibidor da fosfodiesterase 5 (PDE5), eleva os níveis intracelulares de guanosina monofosfato cíclico (GMPc), levando a inserção de aquaporina 2 (AQP2) na membrana plasmática das células do ducto coletor. Portanto, inibidores de PDE podem promover a inserção de AQP2 na membrana plasmática mesmo sem a ativação do receptor de HAD, indicando a participação de uma via alternativa mediada pelo GMPc. Nós investigamos o efeito do Sil na expressão renal das proteínas de membrana AQP2, UT-A1, NKCC2, NHE3, P-ENaC em ratos com DIN induzido pelo Li. Ratos Wistar foram divididos nos seguintes grupos: grupo controle, recebendo dieta alimentar normal durante quatro semanas; grupo Li, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas; grupo Li + Sil, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas e 200 mg por quilo de dieta de Sil a partir da segunda semana; grupo Sil, recebendo dieta alimentar normal durante a primeira semana e a partir da segunda semana recebendo dieta normal com 200 mg de Sil por quilo de dieta. Os animais do grupo Li desenvolveram poliúria, diminuição da osmolalidade urinária e diminuição da expressão da AQP2. No grupo Li+Sil, o Sil foi capaz de reverter parcialmente a poliúria, diminuir o clearance de água livre, aumentar a osmolalidade urinária e aumentar a expressão da AQP2. A expressão de UTA1 foi completamente normalizada com o tratamento com Sil. A expressão das proteínas NKCC2 e NHE3 apresentaram-se aumentadas no grupo tratado com Li, e o Sil não foi capaz de reverter tal alteração. Além disso, o tratamento com Sil reverteu completamente o aumento da resistência vascular renal. Assim, concluímos que o tratamento com Sil em ratos com DIN melhora a poliúria, aumenta a smolalidade urinária e diminui o clearance de água livre pelo aumento da expressão de AQP2 e UT-A1. O tratamento com Sil pode ser benéfico para pacientes que sofrem com DIN induzida pelo Li. / Patients taking lithium to treat bipolar disorder often present polyuria and urinary concentrating defect. In addition, lithium-treated animals present lower cyclic adenosine monophosphate production in response to vasopressin. Sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, elevates intracellular cyclic guanosine monophosphate (cGMP) levels, leading to plasma membrane accumulation of aquaporin 2 (AQP2). Therefore, PDE inhibitors might induce AQP2 membrane insertion even without vasopressin receptor activation by activating a parallel cGMP-mediated signal transduction pathway. We investigated the effect of sildenafil on renal expression of AQP2, UT-A1, sodium/hydrogen exchanger (NHE3), type 1 bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), and the epithelial sodium channel alpha subunit (P-ENaC). Wistar rats received lithium (40 mmol/kg food) or not for 4 weeks (Li or control), some rats also receiving sildenafil (200 mg/kg food) in weeks 2-4, with or without lithium (Li+Sil or Sil). In Li+Sil rats, urine output was markedly lower, as was water free clearance, whereas urine osmolality was higher. Semiquantitative immunoblotting revealed the following: AQP2 expression was partially normalized; UT-A1 expression was completely normalized; expression of NKCC2 and NHE3 was significantly higher in Li rats (although not significantly different between Li+Sil rats and Li rats); and P-ENaC protein expression was unaltered in all groups. Sildenafil treatment completely reversed the lithium-induced increase in renal vascular resistance. In conclusion, sildenafil treatment of lithium-induced nephrogenic diabetes insipidus (NDI) improves polyuria, increases urinary osmolality, and decreases free water clearance via upregulation of renal AQP2 and UT-A1. Sildenafil treatment could be beneficial in patients with lithium-induced NDI.

Este trabalho objetivou investigar a existência de polimorfismos de seqüência nucleotídica (SNPs "single nucleotide polymorphisms") em fragmentos genômicos de cana-de-açúcar homólogos a genes de resistência. Para tal, iniciadores sintéticos homólogos às extremidades de uma seqüência expressa identificada (ESTs "expressed sequence tag") como homóloga ao gene Xa1 de arroz (EST SCCCCL3080E03.g) e outra ao gene Rp1-D de milho (EST SCCCCL3080B03.g) foram utilizados para amplificar, em duplicata, fragmentos genômicos de variedades de cana-de-açúcar. Em seguida, os fragmentos foram clonados e seqüenciados. No caso do primeiro EST, foram obtidas seqüências de 119 insertos de uma variedade resistente (SP804966) e 156 de uma variedade suscetível (SP80180) a Xanthomonas albilineans. No segundo caso, foram obtidas seqüências de 167 insertos de uma terceira variedade resistente (R570) e 135 de outra suscetível (SP811763) a Puccinia melanocephala. Para cada EST considerado, as seqüências foram comparadas por meio do programa DNA Sequencher 3.0 (Gene Codes Corporation, Ann Arbor, MI). Nesta análise, somente foram consideradas seqüências reproduzíveis, isto é, que ocorreram nas duas repetições. No caso de ambos ESTs, a comparação de seqüências entre variedades possibilitou a identificação de quatro a seis fragmentos distintos. A comparação entre variedades, por sua vez, revelou a existência de até duas seqüências comuns. Presume-se que estas seqüências reproduzíveis e confirmadas por meio de digestões de clones representativos de cada uma com enzimas de restrição, correspondam a seqüências alélicas. Iniciadores foram sintetizados com base em polimorfismos encontrados entre os diferentes alelos. No entanto, tentativas de amplificar alelos específicos por meio destes iniciadores se revelaram infrutíferas. Tal insucesso deve-se possivelmente à condição polialélica da cana-de-açúcar, que faz com que polimorfismos entre dois alelos sejam compensados por monomorfismos entre estes e os demais alelos. Assim, a utilização de SNPs como marcadores moleculares baseados em PCR em cana-de- açúcar mostra-se mais complexa quando comparada a plantas diplóides. / The objective of this work was to investigate the existence of single nucleotide polymorphisms (SNPs) in genomic fragments homologous to resistance genes in sugarcane. For this purpose, primers were designed based on the sequence of the extremities of an identified expressed sequence tag (ESTs) similar to the Xa1 gene of rice (EST SCCCCL3080E03.g) and another to the maize Rp1-D gene (EST SCCCCL3080B03.g). These primers were used to amplify genomic fragments of sugarcane varieties in duplicate. The fragments were then cloned and sequenced. In the case of first EST, sequences of 119 inserts from a resistant variety (SP804966) and 156 from a susceptible variety (SP80180) to Xanthomonas albilineans were analyzed. In the case of the second EST, sequences of 167 inserts from a third variety (R570) and 135 of a fourth one (SP811763), resistant and susceptible respectively to Puccinia melanocephala, were analyzed. Sequences were compared using the program DNA Sequencher 3.0 (Gene Codes Corporation, Ann Arbor, MI). In this analysis, only reproducible sequences were considered, that is, sequences that occurred in both replicates. Four to six different sequences were identified within varieties whereas comparisons among varieties revealed the existence of up to two sequences in common. These reproducible sequences, which were further confirmed through digestion of representative clones with appropriate restriction enzymes, could correspond to allelic sequences. Primers were designed based on the SNPs detected among these so-called alleles. However, attempts to amplify specific alleles using these primers were unsuccessful. This could be due to the polyallelic condition of sugarcane in which polymorphisms between two alleles could be compensated by monomorphisms between these and other alleles. Thus the use of SNPs as PCR-based molecular markers is not as straightforward in sugarcane as in diploid species.