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Funktionelle Analyse einer Familie von Oligopeptidtransportern des humanpathogenen Hefepilzes Candida albicans / Functional analysis of a family of oligopeptide transporters in the human pathogenic yeast Candida albicansReuß, Oliver Rainer January 2006 (has links) (PDF)
Der Hefepilz Candida albicans ist Teil der natürlichen Mikroflora auf den Schleimhäuten des Verdauungs- und Urogenitaltrakts der meisten gesunden Menschen. Allerdings kann C. albicans vor allem in immunsupprimierten Patienten auch schwerwiegende Infektionen verursachen. Diese reichen von oberflächlichen Mykosen bis hin zu lebensbedrohlichen systemischen Infektionen. C. albicans besitzt eine Reihe von Eigenschaften, die es diesem opportunistisch humanpathogenen Pilz ermöglichen unterschiedliche Wirtsgewebe zu kolonisieren und zu infizieren. Ein wichtiger Virulenzfaktor sind sekretorische Aspartylproteasen (SAPs), die von einer großen Genfamilie von zehn SAP-Genen codiert werden. Die SAPs werden während der Infektion differentiell exprimiert und übernehmen unterschiedliche Rollen im Infektionsverlauf. So tragen sie zur Adhärenz bei, können Wirtsbarrieren und Moleküle der Wirtsimmunabwehr zerstören oder liefern Nährstoffe, indem sie Proteine abbauen. Unter den zehn SAP-Genen ist SAP2 für ein Wachstum von C. albicans auf Proteinen als alleiniger Stickstoffquelle verantwortlich. Allerdings ist wenig über die Regulation der SAP2-Expression und über die Aufnahme der proteolytischen Abbauprodukte in die Zelle bekannt. In dieser Arbeit wurde eine Familie von Oligopeptidtransportern von C. albicans funktionell analysiert. Da aus früheren Arbeiten bekannt war, dass SAP2 durch Peptide mit mindestens acht Aminosäuren induziert werden kann, könnten einzelne Mitglieder dieser Familie neben der Transportfunktion auch eine Sensorfunktion für Peptide übernehmen und somit über einen Signalweg SAP2 induzieren. In der Genomsequenz von C. albicans wurden neben dem bereits beschriebenen OPT1-Gen sieben weitere Gene identifiziert, deren Genprodukte signifikante Homologie zu Opt1p aufwiesen und die deshalb als OPT2-OPT8 bezeichnet wurden. Um die Rolle dieser putativen Oligopeptidtransporter bei der SAP2-Induktion und beim Transport der durch Sap2p-Aktivität bereitgestellten proteolytischen Abbauprodukte zu untersuchen, wurden Mutanten hergestellt, in denen die OPT-Gene spezifisch deletiert waren. Zu diesem Zweck wurde eine Methode zur gezielten Geninaktivierung etabliert, die auf einem neuen, recycelbaren dominanten Selektionsmarker (caSAT1) beruht, der Resistenz gegen Nourseothricin verleiht. Die “SAT1-Flipping“-Strategie kann direkt in C. albicans Wildstämmen angewendet werden und umgeht somit alle Probleme, die mit der Verwendung von auxotrophen Ausgangsstämmen verbunden sind. Alle Mutanten, in denen jeweils eines der OPT-Gene inaktiviert war, verhielten sich wie der Wildtyp und zeigten keinen Wachstumsdefekt auf bovinem Serumalbumin (BSA) als alleiniger Stickstoffquelle, während eine sap2-Nullmutante unter diesen Bedingungen nicht wachsen kann. Somit ist kein einzelnes OPT-Gen für C. albicans notwendig, um auf BSA als alleiniger Stickstoffquelle zu wachsen. Dagegen zeigten opt123-Triplemutanten ähnlich wie die sap2-Mutante einen starken Wachstumsdefekt auf BSA als alleiniger Stickstoffquelle, der durch Reintegration einer intakten Kopie von OPT1, OPT2 oder OPT3 wieder aufgehoben werden konnte. Der Wachstumsdefekt der opt123-Triplemutanten war nicht auf eine fehlende Induktion von SAP2 zurückzuführen, sondern auf das Unvermögen dieser Mutanten, die durch den proteolytischen Abbau von BSA entstandenen Peptide zu transportieren. Mit Hilfe von Reportergenen konnte gezeigt werden, dass die einzelnen OPT-Gene differentiell exprimiert werden. Während keines der Gene in einem Vollmedium (YPD) exprimiert wurde, wurde eine starke Induktion von OPT1 und OPT3 in Gegenwart von BSA als alleiniger Stickstoffquelle beobachtet. Nach Expression von OPT4 und OPT5 unter Kontrolle des konstitutiven ADH1-Promotors in den opt123-Triplemutanten konnte deren Wachstumsdefekt auf BSA als alleiniger Stickstoffquelle ebenfalls kompensiert werden, während die zusätzliche Deletion dieser Gene in den dabei entstandenen opt1234-Quadruple- und opt12345-Quintuplemutanten den Wachstumsdefekt noch verstärkte. Diese Ergebnisse bestätigten, dass die Gene OPT1-OPT5 für funktionelle Oligopeptidtransporter codieren. Weitere Experimente zeigten, dass die Oligopeptidtransporter unterschiedliche Substratpräferenzen haben. Während das Tetrapeptid LWMR für Stämme, die spezifisch OPT3, OPT4, oder OPT5 exprimierten, ein besseres Substrat war als das Tetrapeptid LSKL, konnten Stämme, die spezifisch OPT2 exprimierten, das LSKL-Peptid verwerten, nicht aber das LWMR-Peptid. Experimente mit Peptiden definierter Länge und Zusammensetzung wiesen außerdem darauf hin, dass die Oligopeptidtransporter in der Lage sind, auch längere Peptide mit bis zu mindestens acht Aminosäuren zu transportieren. Die Evolution einer Genfamilie, die für Oligopeptidtransporter mit unterschiedlicher Substratpräferenz codieren, hat deshalb vermutlich dazu beigetragen, dass C. albicans Proteine sehr effizient als Stickstoffquelle verwerten und sich an die Nahrungsbedingungen in verschiedenen Wirtsnischen optimal anpassen kann. / The yeast Candida albicans is a member of the microflora on mucosal surfaces of the gastrointestinal and urogenitary tract in most healthy people. However, in immunocompromised patients C. albicans can cause superficial as well as life-threatening systemic infections. C. albicans exhibits a variety of characteristics that enable this opportunistic human fungal pathogen to colonize and infect different host tissues. Among these virulence factors are secreted aspartic proteinases (SAPs), which are encoded by a family of ten SAP genes. The SAPs are differentially expressed during infection and play different roles in disease progression by contributing to adherence, by degrading tissue barriers and host defence molecules or by providing nutrients through the digestion of proteins. Of the ten SAP genes, SAP2 enables C. albicans to grow on proteins as a sole source of nitrogen. However, little is known about how SAP2 expression is regulated and how proteolytic products are taken up into the cell. In this work a family of oligopeptide transporters of C. albicans was functionally characterized. Since earlier studies had demonstrated that SAP2 expression can be induced by peptides of at least eight amino acids in length, oligopeptide transporters, in addition to transporting peptides, might also serve as sensors which in the presence of peptides activate a signalling pathway that induces SAP2 expression. Beside the already described OPT1 gene, seven additional genes were identified in the C. albicans genome sequence whose encoded products exhibit significant similarity to Opt1p and hence were designated as OPT2-OPT8. To elucidate the role of these putative oligopeptide transporters in SAP2 induction and in the uptake of proteolytic products provided by Sap2p activity, a series of mutants lacking specific OPT genes was constructed. For this purpose, a method for targeted gene inactivation was established that relies on the use of a new recyclable, dominant selection marker, caSAT1, which confers resistance to nourseothricin upon C. albicans transformants. The SAT1 flipping strategy can be used directly in C. albicans wild-type strains and, therefore, circumvents all problems related to the use of auxotrophic host strains. All knockout mutants lacking single OPT genes behaved like the wild-type parental strain and did not show a growth defect in a medium containing bovine serum albumin (BSA) as the sole nitrogen source, conditions in which a sap2 null mutant can not grow. Therefore, no single OPT gene is required for growth of C. albicans on BSA as the sole source of nitrogen. In contrast, opt123 triple mutants, similar to a sap2 mutant, had a severe growth defect on BSA as the sole nitrogen source, which could be rescued by reintroduction of an intact copy of either OPT1, OPT2 or OPT3. The poor growth of the opt123 triple mutants was not caused by failure to induce SAP2 expression but by the inability of these mutants to efficiently transport the peptides produced by proteolytic degradation of BSA into the cell. By using reporter genes it could be demonstrated that individual members of the OPT gene family are differentially expressed. While none of the OPT genes was detectably expressed in rich YPD medium, a strong induction of OPT1 and OPT3 was observed in the presence of BSA as the sole nitrogen source. Forced expression of OPT4 and OPT5 under control of the constitutive ADH1 promoter in the opt123 triple mutants also complemented their growth defect on BSA as a sole nitrogen source, whereas the additional deletion of these genes in the resulting opt1234 quadruple and opt12345 quintuple mutants exacerbated the growth defect. These results confirmed that at least OPT1-OPT5 encode functional oligopeptide transporters. Additional experiments showed that the individual oligopeptide transporters differ in their substrate preferences. While the tetrapeptide LWMR was a better substrate than the tetrapeptide LSKL for strains that specifically expressed OPT3, OPT4 or OPT5, strains specifically expressing OPT2 grew on the LSKL peptide, but not on the LWMR peptide. Furthermore, experiments with peptides of defined length and sequence suggested that the oligopeptide transporters are also able to transport longer peptides up to at least eight amino acids in length. Therefore, the evolution of a gene family encoding oligopeptide transporters with different substrate preferences probably contributed to the ability of C. albicans to efficiently utilize proteins as a nitrogen source and adapt to the nutritional conditions in different host niches.
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Multifunctional oligopeptides as an artificial toolkit for molecular recognition eventsWich, Peter Richard Unknown Date (has links) (PDF)
Würzburg, Univ., Diss., 2009
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Novel Reactions of Nickel (II) - Oligopeptide Complexes with Dioxygen SpeciesTom, Rickey T. 04 1900 (has links)
The ability of simple oligopeptide complexes of nickel (II) to
react with various dioxygen intermediates was investigated. Under
physiological conditions, nickel (II)-histidine-containing
oligopeptides were found to dismutate superoxide anions and
disproportionate hydrogen peroxide. In the latter process,
chemiluminescence was generated and a strongly oxidizing intermediate
was detected capable of oxidizing uric acid, hydroxylating p-nitrophenol,
and damaging 2-deoxy-D-ribose. The generation of this
reactive intermediate likely occurs without the involvement of free
hydroxyl radicals derived from Haber-Weiss or Fenton-type reactions.
In addition, the Ni(II) complex of glycylglycyl-L-histidine (GGH) was
found to react with mollecular oxygen resulting in the oxidation of the
ligand. An attempt was made to relate these reactions to the
involvement: of the nickel(III)/(II) redox couple which was shown to
exist under physiological conditions. Similar reactivity was observed
for non-histidine-containing oligopeptides but higher pH values were
required.
The oligopeptides used not only represent biologically relevant
ligands but: the histidine containing oligopeptides mimics the specific
copper(II)/nickel(II) binding and transport site of human serum
albumin. The observations made in this study suggest some novel
mechanism for the deleterious effects associated with excessive lifelong
exposure to nickel compounds, especially in relation to cancer of
the respiratory tract. / Thesis / Master of Science (MS)
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NMR-spektroskopische Untersuchungen binärer Palladium-Peptid-Komplexe sowie ternärer Komplexe mit Desoxyribonucleotiden unter besonderer Berücksichtigung der Strukturänderung der Liganden bei der MetallkoordinationPark, Hye-Seo. Unknown Date (has links) (PDF)
Universiẗat, Diss., 2003--Dortmund.
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Les régulateurs transcriptionnels Rgg. Confirmation de leur implication dans des phénomènes de quorum-sensing et identification de leurs cibles. / RGG transcriptional regulators. Confirmation of their involvement in quorum-sensing phenomenon and identification of their targets.Fleuchot, Betty 06 December 2011 (has links)
La découverte d'un contexte génétique chez les streptocoques – codant un petit peptide hydrophobe (SHP) et un régulateur transcriptionnel appartenant à la famille Rgg –, suivi de l'étude d'un de ces loci chez S. thermophilus LMD-9, a conduit à l'hypothèse que les protéines régulatrices Rgg en association avec une phéromone putative SHP pourraient intervenir dans un mécanisme de type quorum-sensing (QS) chez les bactéries à Gram positif. La première partie de ma thèse a consisté à confirmer cette hypothèse sur le locus shp/rgg1358 de S. thermophilus LMD-9, espèce contenant le plus grand nombre de systèmes SHP/Rgg dans son génome. Pour ceci, les étapes impliquées dans un mécanisme de QS ont été étudiées : la sécrétion, la maturation et la détection à une concentration seuil de la phéromone, sa réimportation à l'intérieur de la cellule, son interaction avec un régulateur transcriptionnel et enfin l'interaction de la protéine régulatrice à l'ADN. Par l'utilisation d'approches génétiques et biochimiques, nous avons démontré l'existence d'un nouveau mécanisme de QS impliquant pour la première fois un régulateur transcriptionnel Rgg et une phéromone SHP, importée à l'intérieur de la cellule par le transporteur d'oligopeptides AmiCDEF. Le rôle de la protéase membranaire, Eep, a également été démontré dans la maturation de la phéromone, dont la forme mature a été déterminée par spectrométrie de masse et validée in vivo. Dans un second temps, nous avons exploré la fonctionnalité de ce nouveau mécanisme sur d'autres loci shp/rgg, dans le but d’étudier l'existence d’éventuels phénomènes de cross-talk entre les bactéries. L'étude de nouveaux loci, en système hétérologue chez S. thermophilus LMD-9, a permis d'étendre la fonctionnalité du mécanisme à deux systèmes SHP/Rgg de streptocoques pathogènes, à savoir S. agalactiae et S. mutans. En parallèle à ce travail de caractérisation, l'identification des régulons des systèmes SHP/Rgg a été entreprise. La construction d'un arbre phylogénétique des protéines Rgg-like a permis d'identifier 68 systèmes SHP/Rgg, que nous avons classés en trois groupes. L'analyse des régions promotrices des gènes shp a conduit à l'identification d'un site putatif de liaison des protéines Rgg à l'ADN spécifiques de chaque groupe SHP/Rgg. Une approche in silico a ensuite été menée afin de rechercher, dans les génomes séquencés de streptocoques, les gènes cibles putatifs. Alors que des cibles proximales ont été détectées pour les groupes II et III, des cibles distales ont été identifiées dans les groupes I et II. Actuellement, la validation de certaines cibles est en cours au laboratoire. A l'avenir, ce travail pourrait permettre le développement de petits peptides permettant d'optimiser l'utilisation de S. thermophilus en industries laitières et de réduire la virulence des streptocoques pathogènes. / The discovery of a genetic context – encoding a small hydrophobic peptide (SHP) and a transcriptional regulator belonging to the Rgg family (in nearly all streptococcal genomes) –, following by the study of one of this loci in S. thermophilus LMD-9, led to the hypothesis that the regulatory proteins Rgg in association with a putative pheromone SHP could define a novel quorum-sensing (QS) regulatory mechanism in Gram-positive bacteria. The first part of my PhD consisted to validate this hypothesis. For this purpose, we analyzed the SHP/Rgg system in all the steps that are commonly involved in QS mechanisms: (i) secretion of the putative pheromone, (ii) maturation of the pheromone, (iii) capture of the pheromone from the external environment at a threshold concentration, (iv) importation of the pheromone inside the cell and (v) interaction of the transcriptional regulator to the promoter regions of targeted genes. Experimentally, we focused on the so-called shp/rgg1358 locus of S. thermophilus LMD-9, which is the streptococcal species containing the largest number of shp/rgg pairs in its genome. By using genetic and biochemistry approaches, we uncovered a new QS mechanism that involves the pheromone SHP, the oligopeptide transporter AmiCDEF for the uptake of the pheromone and the transcriptional regulator Rgg for the control of target gene expression. Furthermore, we showed that the membrane protease Eep participates in the production of the mature pheromone, which has been identified by mass spectrometry. Once characterized, the second part of my PhD was to explore the functionality of this new QS system in other streptococcal strain or species, in order to determine if cross-reactivity phenomenon between streptococci can occur. By using heterologous expression in S. thermophilus LMD-9, we extended the functionality of the SHP/Rgg system to two pathogenic streptococcal species, i.e. S. agalactiae and S. mutans. The last part of my PhD consisted in identifying the regulon of all SHP/Rgg systems. Following the construction of a phylogenetic tree of the Rgg-like proteins in low GC Gram-positive bacteria, we identified 68 SHP/Rgg systems that we classified in three groups. Analyzing the promoter regions of all shp genes led to the identification of a putative Rgg DNA binding site specific to each SHP/Rgg group. An in silico approach was used to scan all sequenced streptococcal genomes for the three identified patterns. Whereas proximal target genes were detected for groups II and III, distal target genes were found in groups I and II. In addition, we uncovered that putative Rgg DNA binding sites can be localized in coding or non-coding region. Currently, validations are in progress. To sum-up, my PhD studies provided evidences that the Rgg proteins in association with small peptide pheromones define a new QS mechanism that seems to regulate the expression of distal and proximal genes in a species-dependent manner. Important insights should be obtained concerning a putative crosstalk among streptococci that involves the SHP/Rgg QS system. My studies may constitute a basis for the development of small peptides to optimize the use of S. thermophilus in dairy factories and reduce the virulence of pathogenic streptococci.
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Etude du système de communication cellulaire NprR-NprX au sein du groupe Bacillus cereus / Study of the cell-cell communication system NprR-NprX in the Bacillus cereus groupDubois, Thomas 05 March 2012 (has links)
Chez les bactéries sporulantes du genre Bacillus, des mécanismes importants tels que la sporulation et la virulence sont régulés par des systèmes de communication cellulaire qui impliquent des peptides de signalisation et des régulateurs de la famille RNPP (Rap, NprR, PlcR, PrgX). L'objectif de mon travail de thèse a été de déterminer le rôle du régulateur NprR chez les bactéries du groupe B. cereus. Ce travail se divise en trois parties complémentaires. La première partie a consisté à montrer que NprR est impliqué dans un système de communication cellulaire. Nous avons montré que NprR est un régulateur transcriptionnel de début de phase stationnaire qui est dépendant du peptide de signalisation NprX. Associé à NprX, NprR active la transcription du gène nprA qui code pour une protéase extracellulaire. Nous avons démontré que le peptide NprX est sécrété, maturé puis réimporté dans la cellule bactérienne par deux systèmes d'oligopeptide perméase (Opp et Npp). Une fois dans la cellule, la forme mature de NprX (vraisemblablement l'heptapeptide SKPDIVG) se lie à NprR et permet la transcription du gène nprA. Nous avons ensuite cherché à déterminer la fonction de ce régulateur au cours du cycle infectieux de B. thuringiensis (Bt) chez l'insecte. Nous avons montré que NprR est actif après la mort de l'insecte et permet aux bactéries de survivre, sous forme de cellules végétatives, dans les cadavres. Une analyse transcriptomique indique que NprR régule l'expression d'au moins 41 gènes qui codent notamment pour des enzymes dégradatives et un locus de gènes impliqués dans la production d'un peptide synthétisé de façon non ribosomique (la kurstakine). Nous avons démontré que les gènes codant pour les enzymes dégradatives s'expriment spécifiquement après la mort de l'hôte et que les produits de ces gènes sont essentiels pour hydrolyser différents substrats (protéines, lipides, chitine), ce qui suggère que Bt a un mode de vie nécrotrophe dans le cadavre. La kurstakine est essentielle pour la survie de Bt pendant son développement nécrotrophe et nous avons montré que cette molécule est nécessaire pour le swarming et la formation de biofilm. Par ailleurs, un mutant du gène nprR ne se développe pas et ne sporule pas efficacement dans le cadavre. L'ensemble de nos résultats indiquent que le necrotrophisme est un mode de vie hautement régulé, qui est essentiel dans le cycle infectieux de Bt car il contribue à la transmission horizontale de ce micro-organisme. Enfin, nous avons étudié la régulation de l'expression des gènes nprR et nprX. Nous avons montré que les gènes nprR-nprX sont co-transcrits à partir d'un promoteur dépendant de sigma-A (PA) situé en amont du gène nprR. La transcription à partir de ce promoteur débute lors de l'entrée en phase stationnaire et est contrôlée par deux régulateurs transcriptionnels: CodY et PlcR. Le répresseur CodY pourrait se lier à l'ADN en amont du promoteur PA et réprimer la transcription des gènes nprR-nprX pendant la phase exponentielle de croissance. Au début de la phase stationnaire, le contrôle négatif de CodY est levé et PlcR active la transcription de nprR-nprX en se liant à une boîte PlcR située en amont de PA. Nos résultats indiquent que nprX est également transcrit indépendamment de nprR à partir de deux promoteurs, PH et PE, respectivement dépendant de sigma-H et sigma-E. Les deux promoteurs permettent d'assurer la transcription de nprX en phase stationnaire tardive alors que la transcription à partir du promoteur PA est achevée. Cette étude met en évidence le role clé des régulateurs CodY, PlcR and Spo0A dans la régulation de l'expression des gènes nprR-nprX. / In sporulating Bacillus, major processes like virulence gene expression and sporulation are regulated by communication systems involving signaling peptides and regulators of the RNPP family. In this work, we investigated the role of one such regulator, NprR, in bacteria of the Bacillus cereus group. This work can be divided into three complementary parts.The first part consisted to demonstrate that NprR is involved in a quorum-sensing system. We showed that NprR is a transcriptional regulator whose activity depends on the NprX signalling peptide. In association with NprX, NprR activates the transcription of an extracellular protease gene (nprA) during the first stage of the sporulation process. We demonstrated that the NprX peptide is secreted, processed and then reimported within the bacterial cell by two oligopeptide permease systems (Opp and Npp). Once inside the cell, the mature form of NprX, presumably the SKPDIVG heptapeptide, directly binds to NprR allowing nprA transcription. The second part was to explore the function of NprR during the infectious cycle of B. thuringiensis (Bt). We showed that NprR is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes encoding degradative enzymes or involved in the synthesis of a non-ribosomal peptide named kurstakin. The degradative enzymes include chitinases, proteases and lipases. The corresponding genes are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We showed that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. Altogether, our results show that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to horizontal transmission. Finally, the last part of my PhD consisted to study the regulation of nprR and nprX expression. We showed that the nprR-nprX genes are cotranscribed from a sigma A-dependent promoter (PA) located upstream from nprR. The transcription from PA starts at the onset of the stationary phase and is controlled by two transcriptional regulators: CodY and PlcR. The nutritional repressor CodY binds a DNA target site upstream from PA and represses nprR-nprX transcription during the exponential growth phase. At the onset of the stationary phase, the negative control of CodY is relieved and PlcR activates nprR-nprX transcription by binding a PlcR box located upstream from PA. We showed that nprX is also transcribed independently of the nprR transcription from two promoters, PH and PE, dependent on sigma-H and sigma-E, respectively. Both promoters ensure nprX transcription during late stationary phase and sporulation while transcription from PA is complete. This study highlights the key role played by CodY, PlcR and Spo0A in nprR-nprX transcription.
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Identificação de um gene de Leishmania major relacionado à resistência a ciclosporina A / -Marco, Rogério Milton De 20 August 2004 (has links)
A Ciclosporina A (CsA) faz parte de um grupo de oligopeptidios cíclicos e hidrofóbicos, produzida naturalmente por fungos. Este composto além de ser um potente imunossupressor pode também apresentar uma série de outros efeitos fisiológicos como atividade antiinflamatória e antialérgica, ou mesmo efeitos anti fúngicos e antiparasitários. Partimos de uma genoteca genômica de L. (L.) major cepa Friedlin A1 (LmFA1) construída no cosmídio cLHYG, que teve o seu DNA transfectado em promastigotas para em seguida as células serem submetidas aos experimentos de superexpressão gênica em presença de concentrações crescentes de CsA. Foram selecionados dois loci capazes de conferir resistência a CsA. Um desses loci (contendo cerca de 30 - 40 kb) foi mapeado e caracterizado após análises do perfil de digestão com diferentes enzimas de restrição, e da transfecção de algumas deleções em células LmFA1, que após amplificação gênica, foram submetidas a testes funcionais em presença de CsA. Apesar de valores baixos dos índices de resistência obtidos com os testes funcionais, foi possível identificar nesse locus um gene que após seqüenciamento parcial de nucleotídeos e análises comparativas no banco genômico de leishmânia, revelou a presença de uma fase aberta de leitura contendo 1 844 aminoácidos que codifica para uma proteína de membrana pertencente a família das transportadoras ABC e que parece estar relacionada com a resistência a CsA. Essa proteína, denominada LmABC-CR1, possui alta identidade com uma transportadora ABC de L. (L.) tropica (95,5%), e baixa identidade (27,2%) com uma outra transportadora ABC de LmFA1 isolada por nosso grupo e relacionada com a resistência a Pentamidiana (PRP1). A análise molecular dos DNAs das espécies, L. (L.) amazonensis, L. (V.) braziliensis, L. (L.), chagasi e L. (L.) major demonstrou um relativo polimorfismo do gene LmABC-CR1 nestas espécies. O papel dessa nova proteína na resistência a CsA frente a diferentes espécies de leishmânia deverá ser melhor estudado / Cyclosporin A (CsA) is a cyclic hydrophobic oligopeptide naturally produced by fungus. Besides this drug is usually known as a potent immunosupressor, it also presents a couple of other physiologic activities as anti-inflammatory, anti-allergic, or even acting as fungicidal and anti-parasitic. Starting from an L. (L.) major Friedlin A1 (LmFA1) genomic library constructed into the cLHYG shuttle vector, DNA was transfected on promastigotes, and cells submitted to over expression in the presence of increasing concentrations of CsA. Two loci were selected capable to render cells CsA resistance after transfection.One of those loci (containing about 30 - 40 kb) was mapped after analyses of the restriction enzymes pattern, and characterized by transfection of its deletions in LmFA1 cells, which after gene amplification, were submitted to functional tests in the presence of CsA. In spite of low of resistance indexes values after functional tests, it was possible to identify a gene that after partial nucleotides sequencing and comparison with leishmania genomic bank data indicated the presence of an open reading containing 1 844 amino acids codifying a membrane protein from the ABC transporter family that seems to be related with CsA resistance (LmABC-CR1). LmABC-CR1 presents high identity (95,5%) with a L. (L.) tropica ABC transporter protein, and low identity (27,2%) with another LmFA1 ABC transporter (PRP1) isolated by us, and related with Pentamidine resistance. Southern blot analysis with L. (L.) amazonensis, L. (V.) braziliensis, L. (L.), chagasi and L. (L.) major genomic DNA shown a relative polymorphism of LmABC-CR1 gene in this species. The role of this new protein on the CsA resistance in leishmania species should be further studied
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Development and implementation of a FT-ICR mass spectrometer for the investigation of ion conformations of peptide sequence isomers containing basic amino acid residues by gas-phase hydrogen/deuterium exchangeMarini, Joseph Thomas 30 September 2004 (has links)
The gas-phase hydrogen/deuterium (H/D) exchange of protonated di- and tripeptides containing a basic amino acid residue has been studied with a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Bimolecular reactions are monitored as a function of time providing exchange efficiencies and temporal distributions for the peptide ions. Results from these experiments indicated that position of the basic residue within the peptide (i.e. N-terminal, internal, or C-terminal) influences gas-phase H/D exchange, suggesting unique peptide ion conformations. The FT-ICR mass spectrometer employed for these gas-phase H/D exchange studies was modified from its original design. Instrument modifications include development of an internal matrix assisted laser desorption ionization (MALDI) source for peptide protonation. In addition, a two-section cell was utilized, allowing control of ion motion and factors affecting gas-phase ion molecule reactions. Systems investigated in these gas-phase H/D exchange studies are peptides containing the same amino acid residues but different sequences. These sequence isomers display dissimilar reaction efficiencies and temporal distributions for deuterium incorporation depending on the primary structure of the peptide ion. Specifically, [M+H]+ peptide ions containing a N-terminal basic residue demonstrate unique H/D exchange behavior when compared to their internal and C-terminal counterparts. These differences are attributed to dissimilar intramolecular bridging interactions involved with inductive stabilization of the charge site. Gas-phase H/D exchange of peptide sequence isomers was also probed with various deuterium reagents. Findings suggest that different reagents also influence H/D exchange reaction rate efficiencies and temporal distributions. These dissimilarities are ascribed to relative gas-phase basicity and proposed mechanistic exchange differences for the deuterium reagents.
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Development and implementation of a FT-ICR mass spectrometer for the investigation of ion conformations of peptide sequence isomers containing basic amino acid residues by gas-phase hydrogen/deuterium exchangeMarini, Joseph Thomas 30 September 2004 (has links)
The gas-phase hydrogen/deuterium (H/D) exchange of protonated di- and tripeptides containing a basic amino acid residue has been studied with a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Bimolecular reactions are monitored as a function of time providing exchange efficiencies and temporal distributions for the peptide ions. Results from these experiments indicated that position of the basic residue within the peptide (i.e. N-terminal, internal, or C-terminal) influences gas-phase H/D exchange, suggesting unique peptide ion conformations. The FT-ICR mass spectrometer employed for these gas-phase H/D exchange studies was modified from its original design. Instrument modifications include development of an internal matrix assisted laser desorption ionization (MALDI) source for peptide protonation. In addition, a two-section cell was utilized, allowing control of ion motion and factors affecting gas-phase ion molecule reactions. Systems investigated in these gas-phase H/D exchange studies are peptides containing the same amino acid residues but different sequences. These sequence isomers display dissimilar reaction efficiencies and temporal distributions for deuterium incorporation depending on the primary structure of the peptide ion. Specifically, [M+H]+ peptide ions containing a N-terminal basic residue demonstrate unique H/D exchange behavior when compared to their internal and C-terminal counterparts. These differences are attributed to dissimilar intramolecular bridging interactions involved with inductive stabilization of the charge site. Gas-phase H/D exchange of peptide sequence isomers was also probed with various deuterium reagents. Findings suggest that different reagents also influence H/D exchange reaction rate efficiencies and temporal distributions. These dissimilarities are ascribed to relative gas-phase basicity and proposed mechanistic exchange differences for the deuterium reagents.
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Identificação de um gene de Leishmania major relacionado à resistência a ciclosporina A / -Rogério Milton De Marco 20 August 2004 (has links)
A Ciclosporina A (CsA) faz parte de um grupo de oligopeptidios cíclicos e hidrofóbicos, produzida naturalmente por fungos. Este composto além de ser um potente imunossupressor pode também apresentar uma série de outros efeitos fisiológicos como atividade antiinflamatória e antialérgica, ou mesmo efeitos anti fúngicos e antiparasitários. Partimos de uma genoteca genômica de L. (L.) major cepa Friedlin A1 (LmFA1) construída no cosmídio cLHYG, que teve o seu DNA transfectado em promastigotas para em seguida as células serem submetidas aos experimentos de superexpressão gênica em presença de concentrações crescentes de CsA. Foram selecionados dois loci capazes de conferir resistência a CsA. Um desses loci (contendo cerca de 30 - 40 kb) foi mapeado e caracterizado após análises do perfil de digestão com diferentes enzimas de restrição, e da transfecção de algumas deleções em células LmFA1, que após amplificação gênica, foram submetidas a testes funcionais em presença de CsA. Apesar de valores baixos dos índices de resistência obtidos com os testes funcionais, foi possível identificar nesse locus um gene que após seqüenciamento parcial de nucleotídeos e análises comparativas no banco genômico de leishmânia, revelou a presença de uma fase aberta de leitura contendo 1 844 aminoácidos que codifica para uma proteína de membrana pertencente a família das transportadoras ABC e que parece estar relacionada com a resistência a CsA. Essa proteína, denominada LmABC-CR1, possui alta identidade com uma transportadora ABC de L. (L.) tropica (95,5%), e baixa identidade (27,2%) com uma outra transportadora ABC de LmFA1 isolada por nosso grupo e relacionada com a resistência a Pentamidiana (PRP1). A análise molecular dos DNAs das espécies, L. (L.) amazonensis, L. (V.) braziliensis, L. (L.), chagasi e L. (L.) major demonstrou um relativo polimorfismo do gene LmABC-CR1 nestas espécies. O papel dessa nova proteína na resistência a CsA frente a diferentes espécies de leishmânia deverá ser melhor estudado / Cyclosporin A (CsA) is a cyclic hydrophobic oligopeptide naturally produced by fungus. Besides this drug is usually known as a potent immunosupressor, it also presents a couple of other physiologic activities as anti-inflammatory, anti-allergic, or even acting as fungicidal and anti-parasitic. Starting from an L. (L.) major Friedlin A1 (LmFA1) genomic library constructed into the cLHYG shuttle vector, DNA was transfected on promastigotes, and cells submitted to over expression in the presence of increasing concentrations of CsA. Two loci were selected capable to render cells CsA resistance after transfection.One of those loci (containing about 30 - 40 kb) was mapped after analyses of the restriction enzymes pattern, and characterized by transfection of its deletions in LmFA1 cells, which after gene amplification, were submitted to functional tests in the presence of CsA. In spite of low of resistance indexes values after functional tests, it was possible to identify a gene that after partial nucleotides sequencing and comparison with leishmania genomic bank data indicated the presence of an open reading containing 1 844 amino acids codifying a membrane protein from the ABC transporter family that seems to be related with CsA resistance (LmABC-CR1). LmABC-CR1 presents high identity (95,5%) with a L. (L.) tropica ABC transporter protein, and low identity (27,2%) with another LmFA1 ABC transporter (PRP1) isolated by us, and related with Pentamidine resistance. Southern blot analysis with L. (L.) amazonensis, L. (V.) braziliensis, L. (L.), chagasi and L. (L.) major genomic DNA shown a relative polymorphism of LmABC-CR1 gene in this species. The role of this new protein on the CsA resistance in leishmania species should be further studied
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