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Thompson, Christopher Hal. "Identification and characterization of a peptide toxin inhibitor of ClC-2 chloride channels." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/26604.
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Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009.Committee Chair: McCarty, Nael; Committee Co-Chair: Harvey, Stephen; Committee Member: Hartzell, Criss; Committee Member: Kubanek, Julia; Committee Member: Lee, Robert. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Ekberg, Jenny. "Novel peptide toxin and protein modulators of voltage-gated ion channels /." [St. Lucia, Qld.], 2005. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe20102.pdf.
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Droctove, Laura. "Premières toxines Kunitz antagonistes du récepteur de type 2 à la vasopressine : étude pharmacodynamique et relations structure-activité." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS009/document.
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La mambaquarétine-1 (MQ-1), une toxine du mamba vert, est le tout premier peptide Kunitz à bloquer sélectivement l’activité du récepteur de type 2 à la vasopressine (V2R). Celui-ci contrôle la concentration finale des urines dans le rein. Impliqué dans plusieurs pathologies, son inhibition est actuellement considérée comme la meilleure stratégie thérapeutique dans le traitement de la polykystose rénale, une maladie génétique héréditaire. L’étude pharmacodynamique de MQ-1 sur des rats sains a confirmé son activité in vivo qui se traduit par un effet aquarétique dépendant de la dose. L’effet maximum est atteint 2 heures après injection intrapéritonéale et disparait avec un temps de demi-vie biologique variant de 1 à 4 heures selon la dose. L’administration quotidienne d’une faible dose a montré une accumulation de l’effet les trois premiers jours, avant un plateau, suggérant une activité résiduelle au-delà de 24 heures. Le criblage des trois autres venins de mambas ainsi qu’une analyse comparée des séquences peptidiques les plus proches dans les bases de données ont révélé l’existence d’un groupe phylogénétique de onze toxines Kunitz antagonistes de V2R. Une approche innovante, combinant tests de liaison de variants de MQ-1 et modélisation du complexe MQ-1-V2R, a permis de décrypter une partie du pharmacophore de la toxine. Les deux partenaires partagent une importante complémentarité ionique impliquant plusieurs boucles extracellulaires du récepteur, et une région hydrophobe de MQ-1 interagit au cœur de V2R à proximité de son site orthostérique supposé. Enfin, une première collaboration avec une industrie pharmaceutique a mis en évidence les points critiques à approfondir pour aboutir au développement thérapeutique de MQ-1Mambaquaretin-1 (MQ-1), a green mamba toxin, is the very first Kunitz peptide to selectively hinder the vasopressin type 2 receptor (V2R) activation. This receptor controls the final concentration of urine in kidneys. Involved in a number of pathologies, its inhibition is currently considered as the best therapeutic strategy in the treatment of polycystic kidney disease, a hereditary genetic disease. Pharmacodynamic study of MQ-1 carried out on healthy rats confirmed its in vivo activity which consists in inducing a dose-dependent aquaretic effect. Maximum effect is reached 2 hours after an intraperitoneal injection and disappears in a biological half-life ranging from 1 to 4 hours according to the dose. The daily injection of small quantities pointed to a cumulative effect over the first three days, leading to a plateau, which suggests a residual activity exceeding 24 hours. The screening of the three other mamba venoms along with a comparative analysis of the closest peptide sequences reported in databases revealed the existence of a phylogenetic group of eleven V2R antagonist Kunitz toxins. An innovative approach combining binding assays on MQ-1 variants and the modelling of the MQ-1-V2R complex has led to a partial deciphering of the pharmacophore of the toxin. The two partners share a significant ionic complementarity involving a number of extracellular loops of the receptor, and a hydrophobic region of MQ-1 interacts within V2R in the vicinity of its supposed orthosteric site. Lastly, a collaboration initiated with a pharmaceutical company brought out the need for the closer scrutiny of some crucial points to succeed in a therapeutic development of MQ-1
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Blagojevic, Mariana. "Epithelial cell death induced by Candidalysin, a cytolytic peptide toxin of Candida albicans." Thesis, King's College London (University of London), 2018. https://kclpure.kcl.ac.uk/portal/en/theses/epithelial-cell-death-induced-by-candidalysin-a-cytolytic-peptide-toxin-of-candida-albicans(7a2a83b3-dd43-472f-87b3-1d157687b440).html.
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Microbial infections contribute significantly to morbidity and mortality in humans. Fungi are often an under-represented component of the microbial communities that colonise mucosal surfaces. The most common human fungal pathogens are the Candida species, of which Candida albicans is the most prevalent. C. albicans has an asymptomatic carriage rate of approximately 60% in the human population, where it resides as a member of the microflora that colonises the mucosal surfaces of the body. C. albicans is a polymorphic fungus capable of growing in a number of distinct morphological forms. At mucosal surfaces, growth of C. albicans in the unicellular yeast form is typically associated with commensalism, whereas the production of filamentous hyphae is associated with fungal overgrowth and pathogenesis. In healthy individuals, the immune system functions to restrict the growth of C. albicans hyphae, preventing infection. However, in the absence of effective immune surveillance, C. albicans hyphae can invade the mucosal surfaces of the body, causing infection and tissue damage. Translocation of C. albicans across mucosal barriers and invasion of underlying tissues is a major risk-factor for the development of life-threatening systemic infection in immune-compromised individuals. The hyphae of C. albicans secrete Candidalysin, a toxin essential for epithelial damage and activation of mucosal immune responses. Cellular damage sustained during infection can often result in cell death by apoptosis, necrosis, necroptosis or pyroptosis. While cell death is often regarded as being beneficial for microbial pathogenesis, it is becoming increasingly clear that cell death can also influence host defence by initiating specific immune responses that contribute to microbial clearance. Collectively, these data demonstrate that oral epithelial cells respond to the secreted fungal toxin Candidalysin by triggering numerous cellular stress responses that are intimately linked with the induction of cellular death. Candidalysin was observed to induce necrosis, but not apoptosis, necroptosis or pyroptosis, and promoted inflammatory responses through a mechanism involving necrosis-dependent release of pro-IL-1β and pro-IL-18.
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Johnson, Stephen Roy. "A Biochemical And Pharmacological Characterization Of A Novel Neuroactive Peptide From The Neotropical Hunting Ant Dinoponera Australis." Available to subscribers only, 2009. http://proquest.umi.com/pqdweb?did=1879010761&sid=3&Fmt=2&clientId=1509&RQT=309&VName=PQD.
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Thesis (Ph. D.)--Southern Illinois University Carbondale, 2009."Department of Pharmacology." Keywords: Peptides, Toxins, Venom, Neuroactive peptide, Neurotoxins. Includes bibliographical references (p. 200-210). Also available online.
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Perez, Morales Tiara G. "Production of and Response to the Cannibalism Peptide SDP in Bacillus subtilis." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/4895.
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The Gram positive soil dwelling bacteria Bacillus subtilis produces spores when encountered with a low nutrient environment. However, B. subtilis can delay spore production by a mechanism known as cannibalism. Cannibalism is a process by which B. subtilis delays commitment to sporulation by killing a subpopulation of its cells. This process involves production of two toxins, SDP and SKF. SDP is a 42 amino acid peptide with a disulfide bond derived from the internal cleavage of its precursor protein pro-SdpC. pro-SdpC is part of the sdpABC operon. Production of extracellular SDP induces expression of the sdpRI operon. Encoded in this operon is the negative regulator SdpR and SdpI. SdpI is a dual function protein which acts both as a signal transduction protein and the immunity factor against SDP. The current model states that production of SDP is sensed via SdpI. SdpI will sequester SdpR to the membrane in response and allow for sdpRI expression. The aims of this dissertation are to establish the requirements for SDP production and its response via SdpI/SdpR during cannibalism.
Studies in Chapter II were carried out to determine the factors required for production of the antimicrobial peptide SDP. Site directed mutagenesis of the leader signal peptide sequence in pro-SdpC demonstrated that proper signal peptide cleavage was required for SDP production. Additional site directed mutants of the cysteine residues in pro-SdpC revealed that these are not required for SDP toxic activity. These studies also included deletions within the sdpABC operon and revealed that the two proteins of unknown function, SdpA and SdpB are required for SDP production. Using mass spectrometry analysis, we found that SdpA and SdpB together are required to produce the active 42 amino acid peptide SDP. Taken together we concluded that SDP production was a multi step process which required proteins encoded within the operon and additional processing supplemented in the cell.
In Chapter III we investigated the role of SdpI, specifically what residues were required for the signaling and immunity functions observed. Our initial screen, included site directed mutagenesis of highly conserved residues between the 4th and 5th transmembrane domains of SdpI. These resulted in over 20 SdpI mutants generated. From these, only two SdpI mutants had defects in either signal transduction or SDP immunity. Additional localized mutagenesis was used to isolate two other mutants in SdpI which only affected signal transduction or SDP immunity. SdpI signaling-immunity+ mutants presented a defect in SdpR membrane sequestration and sdpRIinduction. Our findings suggest these types of SdpI mutants may be important for the downstream effect of SdpR membrane sequestration. SdpI signaling+ immunity- mutants revealed defects in SDP protection. Some of the residues mutated were conserved in other SdpI homologs. Site directed mutagenesis of these conserved residues in the SdpI ortholog YfhL showed these are also required for SDP resistance. For the first time, we were able to identify mutations which affected only SDP immunity and gained further insight into how SdpI signaling-immunity+ mutants play a role during signal transduction.
In Chapter IV we initiated studies to define what regions of the negative regulator SdpR are important for its function during cannibalism. We employed localized mutagenesis to identify SdpR mutants which decreased sdpRIexpression even in the presence of inducing signal. We isolated three such SdpR mutants, referred to as super repressors. We expect these SdpR super repressors are unable to be sequestered to the membrane in the presence of SDP.
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Voegele, Alexis. "Study of the translocation mechanism of the cyaa toxin from bordetella pertussis." Thesis, Université de Paris (2019-....), 2019. https://theses.md.univ-paris-diderot.fr/VOEGELE_Alexis_va.pdf.
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La toxine adénylcyclase (CyaA) est un des principaux facteurs de virulence produite par Bordetella pertussis, l’agent de la coqueluche. CyaA a l’unique capacité de transloquer son domaine catalytique directement à travers la membrane plasmique. Puis le domaine catalytique lie la calmoduline (CaM) pour produire de grandes quantités d’AMPc, conduisant à l’intoxication de la cellule. Bien que plusieurs modèles aient été proposés, le mécanisme moléculaire et les forces impliquées dans la translocation de CyaA restent peu connus. Un gradient de calcium, un potentiel de membrane et des acylations post-traductionnelles sont requis pour la translocation de CyaA. Pendant mon doctorat, je me suis principalement intéressé au processus de translocation. Il a été montré précédemment que la suppression de la région de translocation abolit le passage du domaine catalytique. Dans cette région, le peptide P454 (résidus 454 à 484 de CyaA) a été identifié et montre des propriétés membranaires, i.e interaction avec la membrane, repliement en hélice α au contact de la membrane et perméabilisation membranaire. Nous avons étudié le rôle de P454 dans le processus de translocation. Nous avons observé que des lipides fluides et chargés négativement favorisent l’insertion de P454 dans les membranes. Le peptide possède deux arginines qui sont impliquées dans ses activités membranaires. P454 possède aussi la capacité de transloquer à travers la membrane et de former un complexe avec la CaM. Nous avons identifié plusieurs résidus de P454 impliqués dans la liaison à la membrane et la CaM. Dans le contexte de la toxine entière, ces résidus sont essentiels pour la translocation du domaine catalytique et la production d’AMPc. On propose un modèle de translocation dans lequel le segment P454 de la région de translocation déstabilise la membrane, favorisant sa translocation. Dans le cytosol, le segment P454 est piégé par la CaM et le complexe pourrait agir comme une force tirant le domaine catalytique à travers la membrane. Nous avons aussi montré que la liaison à la CaM du peptide liant la CaM dans le domaine catalytique induit des effets allostériques qui stabilisent le site catalytique, permettant la catalyse rapide d’ATP en AMPc. La pertinence de ces résultats pour la translocation et l’activation de CyaA sont discutéesThe adenylate cyclase toxin (CyaA) is one of the major virulence factor produced by Bordetella pertussis, the causative agent of whopping cough. CyaA has the unique capacity to translocate its catalytic domain directly across the plasma membrane. Then, the catalytic domain binds to calmodulin (CaM) to produce high levels of cAMP, leading to cell intoxication. Although several models have been proposed, the molecular mechanism and the forces involved in the translocation of CyaA remain elusive. The calcium gradient, the membrane potential across the plasma membrane and post-translational acylation are required for an efficient CyaA translocation. During my PhD, I mainly investigated the translocation process. It has been previously shown that deletion of the translocation region abolishes the delivery of the catalytic domain into the cytosol of target cells. In this region, the peptide P454 (residues 454 to 484 of CyaA) was identified and exhibits membrane-active properties related to antimicrobial peptides, i.e membrane interaction, α-helical folding upon membrane insertion and membrane permeabilization. We have investigated the role of P454 on the translocation process. We observed that negatively charged and fluidic membrane favor P454 membrane insertion. The peptide contains two arginine residues that are critically involved in its membrane-active properties. We further identified that P454 exhibits the intrinsic propensity to translocate across lipid bilayers and forms a stable complex with CaM. We identified several residues from P454 involved in both membrane interaction and CaM binding. We showed in the context of the full-length CyaA toxin that these residues are essential for the efficient translocation of the catalytic domain into the cell and production of cAMP. We propose a translocation model in which the membrane-active P454 segment from the translocation region destabilizes the membrane, favoring its translocation. In the cytosol, the P454 segment is trapped by CaM and the formation of the complex may act as a driving force pulling the catalytic domain across the plasma membrane. We further showed that CaM binding to the main CaM-binding site in the catalytic domain induces local and long-range allosteric effects that stabilize the enzymatic site, allowing fast ATP catalysis to cAMP, leading to host subversion. The relevance of these results for the translocation and activation of CyaA are discussed
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Wernecke, Julia [Verfasser]. "Biophysical characterisation of the fungal peptide toxin Ece1-III and its interaction with lipid membranes / Julia Wernecke." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2016. http://d-nb.info/1121535852/34.
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Tisseyre, Céline. "La maurocalcine : substance naturelle d'intérêt thérapeutique." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV047/document.
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La maurocalcine (MCa) est une toxine de 33 acides aminés initialement issue du venin du scorpionScorpio maurus palmatus, et est considérée comme faisant partie de la famille des CPP(Cell Penetrating Peptides) depuis de nombreuses années déjà. La MCa présente donc un intérêtthérapeutique certain dans le domaine de la délivrance intracellulaire de cargos, et lestravaux exposés ici cherchent à caractériser au mieux les propriétés de pénétration de la moléculenative ainsi que celle de certains de ses variants.Après avoir quantifié l’internalisation de plusieurs variants tronqués (linéaires), j’ai pu mettreen évidence le fait que tous ces analogues testés ont une capacité à être internalisés bien plusélevée que celle des CPP de référence (notamment Tat et la pénétratine). Parmi ces variants,l’analogue MCaUF1−9 présente l’avantage d’un temps de rétention relativement élevé au seindes cellules, ainsi que d’une accumulation légèrement accrue en environnement acide (ce quiadvient lors de la formation tumeurs solides). Ce nouveau CPP possède donc un certain potentielthérapeutique mais l’étude de la MCa native, remarquablement stable in vivo, reste plusque jamais d’actualitéMaurocalcine (MCa) is a 33-mer toxin originally isolated from the venom of the scorpioScorpio maurus palmatus, and has been considered as a cell-penetrating peptide (CPP) for severalyears. MCa presents a therapeutic interest for the intracellular delivery of cargoes, andthis thesis aims to characterise the cell penetration properties of the native molecule as well assome of its variants’.After quantifying several truncated (linear) variants’ internalisation, I have been able tohighlight the fact that all of those analogs possess a higher internalization ability than those ofstandard CPP (especially Tat and penetratin). Among those variants, the analog MCaUF1−9 hasa relatively high rentention time within cells, as well as a slightly increased accumulation whenin an acidic environment (which occurs during solid tumours formation). This new CPP showsa certain therapeutic potential but the study of nativeMCa, remarkably stable in vivo, remainsa priority
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Höfs, Sarah [Verfasser], Bernhard Gutachter] Hube, Joachim [Gutachter] Ernst, and Kerstin [Gutachter] [Voigt. "Identification of Candidalysin : a Candida albicans peptide toxin involved in epithelial damage / Sarah Höfs ; Gutachter: Bernhard Hube, Joachim Ernst, Kerstin Voigt." Jena : Friedrich-Schiller-Universität Jena, 2016. http://d-nb.info/1177608758/34.
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Bernedo-Navarro, Robert Alvin 1975. "Obtenção de peptídeos com capacidade inibitória da ação citotoxigênica das toxinas Stx de Escherichia colia partir de bibliotecas de phage display." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314705.
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Orientador: Tomomasa YanoTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Escherichia coli produtora de toxina de Shiga (STEC) é um grupo de importantes patógenos para humanos. Essas bactérias são relacionadas a várias doenças, como por exemplo, Síndrome Urêmica Hemolítica e produzem potentes toxinas denominadas toxinas de Shiga. Essas toxinas, tanto Stx1 quanto Stx2, compartilham um receptor celular comum, a globotriaosilceramida (Gb3) e exibem a mesma atividade biológica intracelular. O desenvolvimento de novos agentes neutralizantes dos danos induzidos por Stx pode representar uma estratégia promissora para o tratamento das doenças causadas por STEC em humanos. No presente estudo, nós desenvolvemos peptídeos sintéticos que exibem atividade neutralizante contra a citotoxicidade induzida por Stx tanto in vitro quanto in vivo e, além disso, que se ligam eficientemente ao receptor Gb3. O peptídeo P12-26 compete eficientemente com Stx2 para a ligação ao Gb3 in vitro. Além disso, os peptídeos PC7-12, P12-26 e PC7-30 inibiram a citotoxicidade de Stx1 e Stx2 em células Vero. Nós observamos que o peptídeo PC7-30 em forma de loop e o peptídeo P12-26 que é linear produziram as maiores porcentagens de inibição de Stx1 e Stx2 em células Vero, respectivamente. No entanto, o peptídeo P12-26 não inibiu a letalidade em camundongos, enquanto que o peptídeo PC7-30 inibiu a letalidade causada pela toxina Stx1. Nossos resultados indicam que os peptídeos P12-26 e PC7-30 são candidatos promissores para o desenvolvimento de agentes terapêuticos contra as doenças em seres humanos causadas por STEC
Abstract: Shiga toxin-producing Escherichia coli strains are important pathogens for humans. These bacteria are linked with severe diseases such as hemolytic uremic syndrome and produce potent known as Shiga toxins. These toxins, Stx1 and Stx2, share a common cellular receptor called globotriaosylceramide (Gb3) and exhibit the same intracellular biological activity. The development of new neutralizing agents for Stx-induced damage may represent a promising strategy for the treatment of diseases caused by STEC infections. In this study, we developed synthetic peptides that exhibit neutralizing activity against Stxinduced cytotoxicity both in vitro and in vivo and that bind efficiently to the Gb3 receptor. The peptide P12-26 competed efficiently with Stx2 for binding to Gb3 in vitro. Moreover, the peptides PC7-12, P12-26 and PC7-30 inhibited the cytotoxicity of Stx1 and Stx2 in Vero cells. We observed that the loop-constrained peptide PC7-30 and linear peptide P12-26 produced higher percentages of inhibition of Stx1 and Stx2 in Vero cells, respectively. However, the peptide P12-26 did not inhibit lethality in mice, whereas the loopconstrained peptide PC7-30 inhibited the lethality caused by Stx1. Our results indicate that the peptides P12-26 and PC7-30 are promising candidates for the development of therapeutic agents against diseases caused by STEC in humans
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular
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Arnett, Eusondia A. "Neutrophil products inhibit LLO secretion and activity, and Listeria monocytogenes intracellular growth." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1374064718.
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Hickey, Ashley N. "Expression of CTB-proinsulin in transgenic chloroplasts." Honors in the Major Thesis, University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1088.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
Burnett College of Biomedical Sciences
Molecular and Microbiology
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Marvin, Laure. "Etude de toxines peptidiques du venin de la mygale Scodra griseipes." Rouen, 1997. http://www.theses.fr/1997ROUES016.
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Scodra griseipes est une mygalomorphe agressive de la famille des Theraphosidae. L'étude de la neurotoxicité de son venin, réalisée sur des souris blanches, situe celui-ci parmi les venins de mygale les plus toxiques testés chez les mammifères. La séparation des différents constituants du venin a été menée par chromatographie d'exclusion stérique (SE-HPLC) afin d'isoler une fraction présentant des symptômes identiques à ceux provoqués par le venin brut. Cette fraction a ensuite été purifiée par RP-HPLC, qui conduit à 7 nouvelles fractions dont la pureté a été contrôlée par électrophorèse capillaire (CZE). La réincorporation de ces fractions dans des bicouches lipidiques planes a permis de mettre en évidence une activité ionophore pour 6 d'entre elles. Leur masses moléculaires ont été déterminées par spectrométrie de masse en mode ionisation électrospray (ESI-MS). Les masses mises en évidence sont comprises entre 3775 et 4547 Da. Des études plus approfondies d'une fraction issue de la RP-HPLC, la toxine SGTx1, pure à 95%, ont montré une activité ionophore avec dépendance au voltage mais aucune activité antibactérienne. L'analyse par ESI-MS a permis de déterminer : 1) sa masse : 3775 Da ; 2) présence de 5 sites basiques ; 3) 62 protons échangeables ; 4) 2 fragments : Thr et Phe. La détermination de la séquence primaire s'est effectuée par dégradation d'Edman et a conduit à un peptide de 34 acides aminés dont 6 cystéines : TCRYLFGGCKTTADCCKHLACRSDGKYCAWDGTF. L'analogie des séquences primaires de SGTx1 avec les toxines du venin de deux mygales Grammostola spatulata (HaTx2, HaTx1, omégaGsTxSIA) et de Brachypelma smithii (TxP5) est frappante et montre jusqu'à 80% d'analogie. Les données obtenues ouvrent des perspectives en ce qui concerne la structure secondaire de SGTx1. En effet, une ébauche de celle-ci a été débutée grâce au dichroïsme circulaire (DC), à la RMN et aux calculs prédictionnels de structure (programme PHD) ; ces résultats montrent la présence d'hélice α avec une dominance de feuillet β.
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Mellor, Ian R. "The biophysics of peptide ion channels." Thesis, University of Nottingham, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335759.
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St, Pierre Liam Daniel. "Identification and comparative analysis of novel factors from the venom gland of the coastal taipan (Oxyuranus scutellatus) and related species." Thesis, Queensland University of Technology, 2005. https://eprints.qut.edu.au/16677/1/Liam_St_Pierre_Thesis.pdf.
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Snake venoms are a complex mixture of polypeptide and other molecules that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Amongst the most potently toxic venoms in the world are those of the Australian venomous snakes, which belong almost exclusively to the elapid family. Their venoms posses a number of unique properties by which they target the mammalian cardiovascular and neuromuscular systems and are the focus for the identification of novel pharmacologically interesting compounds which may be of diagnostic or therapeutic benefit. Although much is known about the biochemical properties of Australia snake venoms as a whole, little research attention has focused upon individual components at the molecular level. This thesis describes the cloning, characterisation and comparative analysis of a number of unique toxins from the venom gland of the coastal taipan (Oxyuranus scutellatus) and a total of seven other related Australian snakes. These include the factor X- and factor V-like components of a prothrombin activator that causes a highly coagulable state in mammals. Comparative analysis of the sequences identified in this study, along with recombinant expression of an active form of the factor X-like component, provides important information on the structural, functional and evolutionary relationships of these molecules. Numerous other toxins were similarly identified and characterised including a pseudechetoxin-like protein, multiple phospholipase A2 enzymes and neurotoxin isoforms as well as vasoactive venom natriuretic peptides. Identified transcripts included not only toxin sequences but also other cellular peptides implicated in toxin processing, including a calglandulin-like protein. This thesis is the first description of the majority of these molecules at either the cDNA or protein level, and provides a means to study the activity of individual components from snake venoms and probe their function within the systems they specifically target. This study represents the most detailed and comprehensive description to date of the cloning and characterisation of different genes associated with envenomation from Australian snakes.
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St, Pierre Liam Daniel. "Identification and comparative analysis of novel factors from the venom gland of the coastal taipan (Oxyuranus scutellatus) and related species." Queensland University of Technology, 2005. http://eprints.qut.edu.au/16677/.
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Snake venoms are a complex mixture of polypeptide and other molecules that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Amongst the most potently toxic venoms in the world are those of the Australian venomous snakes, which belong almost exclusively to the elapid family. Their venoms posses a number of unique properties by which they target the mammalian cardiovascular and neuromuscular systems and are the focus for the identification of novel pharmacologically interesting compounds which may be of diagnostic or therapeutic benefit. Although much is known about the biochemical properties of Australia snake venoms as a whole, little research attention has focused upon individual components at the molecular level. This thesis describes the cloning, characterisation and comparative analysis of a number of unique toxins from the venom gland of the coastal taipan (Oxyuranus scutellatus) and a total of seven other related Australian snakes. These include the factor X- and factor V-like components of a prothrombin activator that causes a highly coagulable state in mammals. Comparative analysis of the sequences identified in this study, along with recombinant expression of an active form of the factor X-like component, provides important information on the structural, functional and evolutionary relationships of these molecules. Numerous other toxins were similarly identified and characterised including a pseudechetoxin-like protein, multiple phospholipase A2 enzymes and neurotoxin isoforms as well as vasoactive venom natriuretic peptides. Identified transcripts included not only toxin sequences but also other cellular peptides implicated in toxin processing, including a calglandulin-like protein. This thesis is the first description of the majority of these molecules at either the cDNA or protein level, and provides a means to study the activity of individual components from snake venoms and probe their function within the systems they specifically target. This study represents the most detailed and comprehensive description to date of the cloning and characterisation of different genes associated with envenomation from Australian snakes.
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Chaivimol, Jittra. "The study of peptide toxins from freshwater cyanobacteria." Thesis, University of Warwick, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389448.
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Benites, Thais Azevedo [UNESP]. "Peptídeos sintéticos no estudo do sistema toxina-antitoxina ParE/ParD." Universidade Estadual Paulista (UNESP), 2017. http://hdl.handle.net/11449/151105.
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O sistema ParE-ParD é um sistema Toxina-Antitoxina (TA) do tipo II (composto por duas proteínas) encontrado no plasmídeo RK2 de uma gama de bactérias. A antitoxina ParD (9kDa) é capaz de neutralizar a citotoxicidade da toxina ParE, pela formação de um complexo estável, e também é eficaz na auto-repressão do operon parDE. A toxina (12kDa) apresenta atividade citotóxica no processo de replicação do DNA por interferir diretamente na ação da DNA girase. Estudos prévios sugeriram que a região C-terminal da antitoxina é responsável pelo processo de interação com ParE. Embora esta toxina possa ser encontrada em um grande número de microrganismos, ainda apresenta mecanismos de citotoxicidade e funções celulares a serem elucidadas. Neste contexto, este trabalho teve como objetivo a tentativa de expressão das duas proteínas ParE e ParD, bem como o design e a síntese de peptídeos análogos da antitoxina, para a realização de estudos de interação molecular, a fim de encontrar uma estrutura mínima de ParD capaz de inativar a função toxica de ParE. Com base nas informações estruturais, obtidas por modelagem e dinâmica molecular, quatro sequências peptídicas análogas de ParD foram projetadas e sintetizadas pela metodologia da fase sólida. As sequências foram analisadas e purificadas por cromatografia líquida de alta eficiência e caracterizadas por espectrometria de massas. Os estudos de interação foram realizados através de ensaios de cromatografia de afinidade e supressão de fluorescência. A fluorescência intrínseca de ParEAC2 foi suprimida pelos análogos de ParD (ParDTB1, ParDTB3, ParDTB5 e ParDTB6), evidenciando a formação de complexos estáveis entre as espécies, resultados confirmados pelos ensaios de cromatografia de afinidade. Resultados semelhantes foram obtidos empregando a proteína ParD obtida por expressão heteróloga. Com base nos resultados obtidos, foi possível concluir que o análogo ParDTB1 representa uma estrutura peptídica mínima com potencial para neutralizar o efeito da toxina ParE.
The ParE-ParD system is a toxin-antitoxin (TA) type II module (composed of two proteins) of the plasmid RK2 of a range of bacteria. The ParD antitoxin (9 kDa) is able to neutralize the cytotoxicity of the ParE toxin by forming a stable complex and is effective in the auto repression of the parDE operon. The toxin (12 kDa) exhibits cytotoxic activity by blocking DNA replication, acting directly in the DNA gyrase action. Previous studies have been suggest that the C-terminal region of the antitoxin is responsible for the interaction process with ParE. Although this toxin can be find in a large number of microorganisms, still have cytotoxicity mechanisms and cellular functions to be elucidate. In this context, this work aimed at the expression of ParE and ParD proteins, as well as the design and synthesis of antitoxin analog peptides, to perform molecular interaction studies in order to find a minimum ParD structure able to inactivate the toxic function of ParE. Based on the structural information obtained by modeling and molecular dynamics, four analogous peptide sequences of ParD were designed and synthesized by the solid phase methodology. The peptide sequences were analyzed and purified by high performance liquid chromatography and characterized by mass spectrometry. Interaction studies were performed by affinity chromatography and fluorescence suppression assays. The intrinsic fluorescence of ParEAC2 was suppressed by ParD analogs (ParDTB1, ParDTB3, ParDTB5 and ParDTB6) addition, evidencing the formation of stable complexes between the species, results confirmed by the affinity chromatography assays. Similar results were obtained using ParD protein obtained by heterologous expression. Based on the results obtained, it was possible to conclude that the ParDTB1 analog represents a minimal peptide structure with potential to neutralize the effect of the ParE toxin.
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Fletcher, David Ian. "Synthesis and structure-activity studies of novel potassium ion channel blockers." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265183.
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Marcello, Alessandro. "Escherichia coli heat-labile enterotoxin : a multipurpose delivery system for peptides and epitopes." Thesis, University of Kent, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387151.
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Li, Feng. "Studies on inhibition against anthrax lethal toxin." Oklahoma City : [s.n.], 2010.
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Coyle, Sadie Marie. "Investigations of microcystins (cyanobacterial peptide toxins) : detection, purification and analysis." Thesis, Robert Gordon University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360091.
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Souissi, Wided. "Cytocidal activity of Cry41Aa, an anticancer toxin from Bacillus thuringiensis." Thesis, University of Sussex, 2018. http://sro.sussex.ac.uk/id/eprint/80446/.
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Bacillus thuringiensis (Bt) is a gram positive spore forming bacterium which produces intracellular protein crystals toxic to a wide variety of insect larvae and is the most commonly used biological pesticide worldwide. More recently, Bt crystal proteins known as parasporins have been discovered, that have no known insecticidal activity but target some human cancer cells exhibiting strong cytocidal activities with different toxicity spectra and varied activity levels. Amongst these parasporins, parasporin-3 most closely resembles the commercially used insecticidal toxins and presents the narrowest activity spectrum, showing moderate cytotoxicity against only two cancer cell lines, HL-60 (Human promyelocytic leukemia cells) and HepG2 (Human liver cancer cells). Parasporin-3, also called Cry41Aa, has only been shown to exhibit cytocidal activity towards these two cell lines after being proteolytically cleaved. In order to understand this activation mechanism various mutations were made at the N- or C-terminal region of the protein and the toxicity against both HepG2 and HL-60 cell lines was evaluated. Our results indicate that only N-terminal cleavage is required for activation and that N-terminally deleted mutants show some toxicity without the need for proteolytic activation. Furthermore we have shown that the level of toxicity towards the two cell lines depends on the protease used to activate the toxin. Proteinase K-activated toxin was significantly more toxic towards HepG2 and HL-60 than trypsin-activated toxin. N-terminal sequencing of activated toxins showed that this difference in toxicity is associated with a difference of just two amino acids (serine and alanine at positions 59 and 60 respectively) which we hypothesize occlude a binding motif. Preliminary work carried out on binding showed a lack of correlation between binding and toxicity since toxin binds to both susceptible and non-susceptible cancer cell lines. In an attempt to better understand the mechanism of action of Cry41Aa against these cells, we evolved resistance in HepG2 through repeated exposure to increasing doses of the toxin. Morphological, physiological and genetic characteristics of the resistant cell line were compared with susceptible cells. Toxin was shown to bind to resistant HepG2 similarly to the susceptible line. RNA sequencing identified AQP9 as a potential mediator of resistance but extensive investigations failed to show a direct link. The involvement of certain intracellular signalling pathways were also investigated in order to understand cell responses to the toxin and showed that in response to the toxin p38, but not ERK1/2, is activated and in a dose dependent manner.
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Patchett, John Colin. "Cellular diversity in Microcystis aeruginosa : implications with respect to peptide toxins." Thesis, University of Warwick, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398715.
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Marquet, Fabrice. "Action de la sorbine sur les sécrétions induites par le VIP et la toxine cholérique à différents niveaux de l'intestin chez le rat, in vivo." Lyon 1, 1998. http://www.theses.fr/1998LYO1T222.
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Baker, Keren Julie. "Effects of peptide toxins on the Ca'2'+-ATPase of sarcoplasmic reticulum." Thesis, University of Southampton, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296260.
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Göransson, Anna-Lena. "The Alzheimer Aβ Peptide : Identification of Properties Distinctive for Toxic Prefibrillar Species." Licentiate thesis, Linköpings universitet, Molekylär Bioteknik, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-76741.
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Proteins must have specific conformations to function correctly inside cells. However, sometimes they adopt the wrong conformation, causing dysfunction and disease. A number of amyloid diseases are caused by misfolded proteins that form amyloid fibrils. One such disease is Alzheimer’s disease (AD). The protein involved in this deadly disease is the amyloid β (Aβ) peptide. The formation of soluble prefibrillar oligomeric Aβ species has been recognized as an important factor in the development of AD. The aim of work described in this thesis was to investigate which properties of these oligomeric species can be linked to toxicity. We approached this task by comparing the aggregation behavior and biophysical properties of aggregates formed by variants of the Aβ peptide that have been shown to differ in neurotoxicity when expressed in the central nervous system (CNS) of Drosophila melanogaster. A combined set involving different fluorescent probes was used in parallell with transmission electron microscopy. The toxicity of species formed during the aggregation process was examined by exposing human SH-SY5Y neuroblastoma cells to Aβ aggregates. We deduced that there is a correlation between cell toxicity and the propensity of the Aβ peptide to form small prefibrillar assemblies at an early stage of aggregation in vitro. Moreover, these prefibrillar species were characterized by their ability to be recognized by pentamer formyl thiophene acetic acid (p-FTAA) and the presence of exposed hydrophobic patches. We also found that larger aggregates did not induce cell death.
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Rippa, Sonia. "Interactions peptaïbols/plantes : étude de l'effet de l'alaméthicine, toxine ou éliciteur de réactions de défense, chez la plante modèle Arabidopsis thaliana." Compiègne, 2007. http://www.theses.fr/2007COMP1688.
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Les peptaïbols sont des peptides hélicoïdaux amphipathiques d'origine fongique connus pour former des pores permettant le passage des ions dans les membranes plasmiques. Des travaux récents ont mis en évidence leurs propriétés élicitrices sur certains végétaux. Nous avons étudié l'effet du peptaïbol alaméthicine sur Arabidopsis thaliana. Ce peptide induit à concentration modérée des réponses physiologiques ayant des traits communs avec la réponse hypersensible végétale. A forte concentration, la molécule est toxique et induit rapidement un clivage caractéristique des ARN ribosomiques. Les propriétés de l'alaméthicine in planta sont vraisemblablement liées à son interaction avec la membrane plasmique, la présence d'acide a-aminoisobutyrique dans sa séquence et sa résistance aux protéases végétales. L'utilisation d'un analogue fluorescent de l'alaméthicine permet d'envisager l'existence d'une cible intracellulaire secondairePeptaibols are helicoidal amphipathic peptides produced by soil fungi. They form pores allowing the passage of ions through plasmic membranes. Recent work highlighted their elicitor properties in plants. We studied the effect of the peptaibol alamethicin on Arabidopsis thaliana. Moderate concentrations of alamethicin induce physiological effects belonging to the plant hypersensitive response. Higher concentrations are toxic and quickly induce a characteristic ribosomal RNA cleavage. The properties of alamethicin in plants are probably related to its interaction with the plasmic membrane, to the presence of a-aminoisobutyric acid in its sequence and to its resistance to plant proteases. The use of a fluorescent analogue of alamethicin might suggest the existence of a secondary intracellular target
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Péré-Védrenne, Christelle. "Etude de la Cytolethal Distending Toxin B des Hélicobacters dans l’inflammation et la carcinogenèse digestive." Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0404/document.
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La démonstration du rôle de la CDT (« Cytolethal Distending Toxin ») de Helicobacterhepaticus dans le développement de l’hépatocarcinome murin fait de cette toxine un candidatpertinent dans l'activation de processus pro-cancéreux. Comme la toxine CagA de Helicobacterpylori, la sous-unité active CdtB de la CDT pourrait être une oncoprotéine. Nous avons étudié lerôle de la CdtB des Hélicobacters dans l’inflammation et la carcinogenèse digestive via unestratégie lentivirale d’expression constitutive ou conditionnelle de la CdtB ou de son mutant pourl’activité DNase. Nous avons réalisé une étude du transcriptome et montré que la CdtB deH. hepaticus induisait une réponse inflammatoire en surexprimant des cytokines, chimiokines,peptides antimicrobiens et en activant la voie du NF-κB des cellules épithéliales. La CdtB réguleégalement l’expression et la localisation nucléaire du facteur de transcription et oncogène MafB.Ces résultats ont été confirmés pour la CdtB de Helicobacter pullorum. Des expériencesd'infection des cellules avec des souches sauvages et mutées pour la CDT (deH. hepaticus & H. pullorum) ont permis de valider les résultats obtenus et de les attribuer à laCdtB et notamment à son activité DNase. Nous avons aussi développé un nouveau modèle dexénogreffes de cellules épithéliales inductibles pour l’expression de la CdtB de H. hepaticus.Dans ce modèle, la CdtB, en plus de ses effets déjà connus, retarde la croissance tumorale,induit l’apoptose, la sénescence et la surexpression du marqueur nucléaire de prolifération,Ki-67, suggérant la survie cellulaire. L’ensemble de ces résultats fournit de nouveaux argumentsen faveur du potentiel oncogénique de la CDTThe demonstration of the role of the Cytolethal Distending Toxin (CDT) of Helicobacter hepaticusin the development of hepatocarcinoma in mice, makes this toxin a relevant candidate in theactivation of precancerous processes. As in the case of the CagA toxin of Helicobacter pylori, theCdtB active subunit of CDT could be an oncoprotein. We studied the role of Helicobacter CdtB ininflammation and digestive carcinogenesis using a lentiviral strategy for constitutive or conditionalexpression of the CdtB subunit or its corresponding DNase mutant. We conducted a study of thetranscriptome and showed that CdtB induced an inflammatory response by overexpressingcytokines, chemokines, antimicrobial peptides and activating the NF-kB pathway in epithelialcells. The CdtB also regulated the expression and nuclear localization of the transcription factorand oncogene MafB. These results were confirmed for the CdtB of Helicobacter pullorum.Infection of cells with wild type strains and the corresponding CDT-mutant strains (of H. hepaticus& H. pullorum) were used to validate the results and to attribute the effects to the CdtB and, inparticular, to its DNase activity. We also developed a novel epithelial cell xenograft model toevaluate the inducible expression of H. hepaticus CdtB. In this model, the CdtB, in addition to itspreviously well-known effects, delayed tumor growth, induced apoptosis, senescence and theoverexpression of nuclear proliferation marker, Ki-67, suggesting cell survival. All of these resultsprovide new arguments in favor of the oncogenic potential of the CDT
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Tonk, L. "Impact of environmental factors on toxic and bioactive peptide production by harmful cyanobacteria." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2007. http://dare.uva.nl/document/51378.
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Pedoussaut, Sylvie. "Immunisation sérique et mucosale contre la toxine cholérique à l'aide de peptides synthétiques : mise en évidence d'un héptapeptide immunogénique en absence de porteur." Paris 5, 1988. http://www.theses.fr/1988PA05P615.
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Radzey, Hanna Agnes. "Synthesis of fluorescent toxin and nucleotide derivatives to specifically address membrane proteins." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://hdl.handle.net/11858/00-1735-0000-0022-6055-5.
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Bodles, Angela M. "Studies on physiochemical and toxic properties of peptides implicated in Alzheimer's disease." Thesis, Queen's University Belfast, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343089.
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Serra, Vincent. "Influence de l'association C3b-toxine tétanique sur la production de peptides immunogéniques." Université Joseph Fourier (Grenoble), 1998. http://www.theses.fr/1998GRE10046.
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La proteine c3 est impliquee dans de nombreux mecanismes de defense de l'organisme contre des elements etrangers pathogenes. C3 participe a la reponse immune naturelle : element clef du syteme du complement, elle intervient dans l'elimination des complexes immuns. Cette proteine participe egalement a la reponse immune specifique : son influence dans l'activation b a clairement ete etablie. Un role dans l'appretement et la presentation de l'ag a egalement ete suggere. Mon projet de recherche a vise a determiner l'influence de c3b sur la generation de peptides antigeniques de la tt au cours de l'appretement dans la cpa. Je me suis d'abord interesse a mieux definir les effets de l'association de c3b a la toxine tetanique (tt) dans la presentation par les cpa aux cellules t. Les complexes tt-c3b activent tous les clones t specifiques des epitopes p2 et p30 a des doses 100 fois moins importantes, par rapport a la tt libre. Cette meilleure efficacite de presentation des cpa ne resulte pas, dans mon systeme experimentale, d'une modification de neosynthese de molecules hla-dr ou de b7. Mon etude s'est portee dans un deuxieme temps sur l'analyse de l'influence des complexes tt-c3b sur la stabilite en sds des molecules hla-dr. L'appretement de complexes tt-c3b permet la synthese de 2 fois plus de formes hla-dr1 compactes qu'avec la tt libre. Ces resultats demontrent que la proteine c3b modifie une ou plusieurs etapes de l'appretement de l'ag qui lui est associe. La production de formes hla-dr compactes en presence de complexes tt-c3b est preferentiellement observee dans les compartiments tardifs de la voie endocytaire, de type lysosomal. Je me suis enfin efforce de determiner les sequences des peptides de la tt naturellement generes par une cpa et associes aux molecules hla-dr, ainsi que d'analyser l'influence de l'association de c3b. L'appretement des complexes tt-c3b permet de generer des epitopes t differents de ceux de la tt, dont la quantite (ou l'immunogenicite) est responsable d'une meilleure activation des clones t utilises. Ces resultats suggerent donc un role direct de c3b dans la generation des epitopes t au cours de l'appretement de la tt, en favorisant la production de peptides immunogeniques differents, capables de se lier aux molecules hla-dr et d'activer de facon plus importante les clones t specifiques.
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Gauckler, Philipp Alexander [Verfasser]. "Identifizierung inhibitorischer Peptide gegen die Toxine von Clostridium difficile aus humanem Hämofiltrat / Philipp Alexander Gauckler." Ulm : Universität Ulm, 2018. http://d-nb.info/1162193549/34.
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Rocha, Camila Aguiar [UNESP]. "Peptídeos derivados da toxina ParE: síntese, estrutura e estudos de inibição de DNA topoisomerases." Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/110373.
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000773281.pdf: 6334511 bytes, checksum: ac228b59d3dede16f09efc26274c2ef6 (MD5)O sistema toxina-antitoxina ParE-ParD é um sistema bacteriano de morte póssegregacional codificado numa ampla gama de hospedeiros. ParE é uma proteína pequena, de aproximadamente 12 kDa codificada pelo gene parE. ParD é uma antitoxina de cerca de 9 kDa, codificada pelo gene parD, capaz de complexar com ParE e neutralizá-la. Estudos têm mostrado o envolvimento da enzima DNA girase no processo de morte celular pela ParE, entretanto, não se tem disponível na literatura nenhuma evidência de inibição desta proteína sobre a atividade da topoisomerase IV. Embora encontrada numa ampla diversidade de microorganismos, ParE não possui função celular totalmente elucidada e seu mecanismo de citotoxicidade permanece ainda desconhecido. Com base na estrutura primária da proteína ParE de E. coli e nos escassos dados disponíveis para esta toxina, peptídeos miméticos foram racionalmente projetados visando compreender o mecanismo de inibição de ParE sobre a atividade da DNA girase, bem como, avaliar a ação destes miméticos na atividade da Topoisomerase IV e da Topo II humana. Estas sequências peptídicas foram sintetizadas pela metodologia de fase sólida, purificadas e analisadas por cromatografia líquida de alta eficiência e caracterizadas por espectrometria de massas. A capacidade de inibição destes peptídeos miméticos sobre a atividade das topoisomerases foi testada por ensaios de eletroforese em gel. Os peptídeos que apresentaram melhores resultados de inibição sobre a atividade das topoisomerases foram o ParERM3 e o ParEC3, projetados para conter os resíduos de aminoácidos L61-R100 e L69-R100, respectivamente, presentes na porção C-terminal da estrutura da proteína ParE de Escherichia coli. A sequência “LNIES” (L101 a S105), quando presente nos peptídeos miméticos atenuou a toxicidade dos mesmos frente às topoisomerases, provavelmente por interferir na interação peptídeo-topoisomerase. Embora não existam...
The toxin-antitoxin system ParE-ParD is a bacterial system of post-segregational death encoded in a wide range of hosts. ParE is a small protein of approximately 12 kDa encoded by parE gene. ParD is an antitoxin about 9 kDa, encoded by the parD gene, able of complexing with ParE and neutralize it. Studies have shown the involvement of the enzyme DNA gyrase in the cell death process by ParE, however, is not available in the literature any evidence of inhibition of this protein on the activity of topoisomerase IV. Although found in a wide variety of micro-organisms, the ParE function has not been fully elucidated and its cytotoxic mechanism remains unknown. Based on the primary structure of the E. coli ParE and the limited data available for this toxin, mimetic peptides were rationally designed aiming at understanding the mechanism of inhibition of ParE on the activity of DNA gyrase, as well as on the topoisomerase IV and human topoisomerase II activities. These peptides sequences were synthesized by solid phase methodology, purified and analyzed by high performance liquid chromatography and characterized by mass spectrometry. The ability of these mimetics to inhibit the activity of topoisomerases was tested by electrophoresis on agarose gel. The peptides that showed better results on the inhibition activity of topoisomerases were ParERM3 and ParEC3 , designed to contain the L61-R100 and L69-R100 amino acids sequence, respectively, found of the C-terminal portion of the ParE structure of Escherichia coli. The LNIES sequence (L101 to S105), when present in the mimetic peptides, attenuated the toxicity of the peptides on topoisomerases activity, probably by interfering in the topoisomerase - peptide interactions. Despite the absence of data in the literature regarding the toxicity of ParE protein on the topo IV and human topo II activities, the peptides ParERM3 and ParEC3 showed better inhibitory activity on these enzymes when compared...
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Rocha, Camila Aguiar. "Peptídeos derivados da toxina ParE : síntese, estrutura e estudos de inibição de DNA topoisomerases /." Araraquara, 2014. http://hdl.handle.net/11449/110373.
Full textAbstract:
Orientador: Reinaldo MarchettoBanca: Saulo Santesso Garrido
Banca: Clóvis Ryuichi Nakaie
Resumo: O sistema toxina-antitoxina ParE-ParD é um sistema bacteriano de morte póssegregacional codificado numa ampla gama de hospedeiros. ParE é uma proteína pequena, de aproximadamente 12 kDa codificada pelo gene parE. ParD é uma antitoxina de cerca de 9 kDa, codificada pelo gene parD, capaz de complexar com ParE e neutralizá-la. Estudos têm mostrado o envolvimento da enzima DNA girase no processo de morte celular pela ParE, entretanto, não se tem disponível na literatura nenhuma evidência de inibição desta proteína sobre a atividade da topoisomerase IV. Embora encontrada numa ampla diversidade de microorganismos, ParE não possui função celular totalmente elucidada e seu mecanismo de citotoxicidade permanece ainda desconhecido. Com base na estrutura primária da proteína ParE de E. coli e nos escassos dados disponíveis para esta toxina, peptídeos miméticos foram racionalmente projetados visando compreender o mecanismo de inibição de ParE sobre a atividade da DNA girase, bem como, avaliar a ação destes miméticos na atividade da Topoisomerase IV e da Topo II humana. Estas sequências peptídicas foram sintetizadas pela metodologia de fase sólida, purificadas e analisadas por cromatografia líquida de alta eficiência e caracterizadas por espectrometria de massas. A capacidade de inibição destes peptídeos miméticos sobre a atividade das topoisomerases foi testada por ensaios de eletroforese em gel. Os peptídeos que apresentaram melhores resultados de inibição sobre a atividade das topoisomerases foram o ParERM3 e o ParEC3, projetados para conter os resíduos de aminoácidos L61-R100 e L69-R100, respectivamente, presentes na porção C-terminal da estrutura da proteína ParE de Escherichia coli. A sequência "LNIES" (L101 a S105), quando presente nos peptídeos miméticos atenuou a toxicidade dos mesmos frente às topoisomerases, provavelmente por interferir na interação peptídeo-topoisomerase. Embora não existam...
Abstract: The toxin-antitoxin system ParE-ParD is a bacterial system of post-segregational death encoded in a wide range of hosts. ParE is a small protein of approximately 12 kDa encoded by parE gene. ParD is an antitoxin about 9 kDa, encoded by the parD gene, able of complexing with ParE and neutralize it. Studies have shown the involvement of the enzyme DNA gyrase in the cell death process by ParE, however, is not available in the literature any evidence of inhibition of this protein on the activity of topoisomerase IV. Although found in a wide variety of micro-organisms, the ParE function has not been fully elucidated and its cytotoxic mechanism remains unknown. Based on the primary structure of the E. coli ParE and the limited data available for this toxin, mimetic peptides were rationally designed aiming at understanding the mechanism of inhibition of ParE on the activity of DNA gyrase, as well as on the topoisomerase IV and human topoisomerase II activities. These peptides sequences were synthesized by solid phase methodology, purified and analyzed by high performance liquid chromatography and characterized by mass spectrometry. The ability of these mimetics to inhibit the activity of topoisomerases was tested by electrophoresis on agarose gel. The peptides that showed better results on the inhibition activity of topoisomerases were ParERM3 and ParEC3 , designed to contain the L61-R100 and L69-R100 amino acids sequence, respectively, found of the C-terminal portion of the ParE structure of Escherichia coli. The "LNIES" sequence (L101 to S105), when present in the mimetic peptides, attenuated the toxicity of the peptides on topoisomerases activity, probably by interfering in the topoisomerase - peptide interactions. Despite the absence of data in the literature regarding the toxicity of ParE protein on the topo IV and human topo II activities, the peptides ParERM3 and ParEC3 showed better inhibitory activity on these enzymes when compared...
Mestre
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Benites, Thais Azevedo. "Peptídeos sintéticos no estudo do sistema toxina-antitoxina ParE/ParD /." Araraquara, 2017. http://hdl.handle.net/11449/151105.
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Orientador: Reinaldo MarchettoBanca: Henrique da Silva
Banca: Luiz Carlos Bertucci Barbosa
Resumo: O sistema ParE-ParD é um sistema Toxina-Antitoxina (TA) do tipo II (composto por duas proteínas) encontrado no plasmídeo RK2 de uma gama de bactérias. A antitoxina ParD (9kDa) é capaz de neutralizar a citotoxicidade da toxina ParE, pela formação de um complexo estável, e também é eficaz na auto-repressão do operon parDE. A toxina (12kDa) apresenta atividade citotóxica no processo de replicação do DNA por interferir diretamente na ação da DNA girase. Estudos prévios sugeriram que a região C-terminal da antitoxina é responsável pelo processo de interação com ParE. Embora esta toxina possa ser encontrada em um grande número de microrganismos, ainda apresenta mecanismos de citotoxicidade e funções celulares a serem elucidadas. Neste contexto, este trabalho teve como objetivo a tentativa de expressão das duas proteínas ParE e ParD, bem como o design e a síntese de peptídeos análogos da antitoxina, para a realização de estudos de interação molecular, a fim de encontrar uma estrutura mínima de ParD capaz de inativar a função toxica de ParE. Com base nas informações estruturais, obtidas por modelagem e dinâmica molecular, quatro sequências peptídicas análogas de ParD foram projetadas e sintetizadas pela metodologia da fase sólida. As sequências foram analisadas e purificadas por cromatografia líquida de alta eficiência e caracterizadas por espectrometria de massas. Os estudos de interação foram realizados através de ensaios de cromatografia de afinidade e supressão de fluorescência. ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The ParE-ParD system is a toxin-antitoxin (TA) type II module (composed of two proteins) of the plasmid RK2 of a range of bacteria. The ParD antitoxin (9 kDa) is able to neutralize the cytotoxicity of the ParE toxin by forming a stable complex and is effective in the auto repression of the parDE operon. The toxin (12 kDa) exhibits cytotoxic activity by blocking DNA replication, acting directly in the DNA gyrase action. Previous studies have been suggest that the C-terminal region of the antitoxin is responsible for the interaction process with ParE. Although this toxin can be find in a large number of microorganisms, still have cytotoxicity mechanisms and cellular functions to be elucidate. In this context, this work aimed at the expression of ParE and ParD proteins, as well as the design and synthesis of antitoxin analog peptides, to perform molecular interaction studies in order to find a minimum ParD structure able to inactivate the toxic function of ParE. Based on the structural information obtained by modeling and molecular dynamics, four analogous peptide sequences of ParD were designed and synthesized by the solid phase methodology. The peptide sequences were analyzed and purified by high performance liquid chromatography and characterized by mass spectrometry. Interaction studies were performed by affinity chromatography and fluorescence suppression assays. The intrinsic fluorescence of ParEAC2 was suppressed by ParD analogs (ParDTB1, ParDTB3, ParDTB5 and ParDTB6) add... (Complete abstract click electronic access below)
Mestre
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Monteiro, Sandra Maria Nunes. "Efficacy of glutamine, peptides and vitamins A and E supplementation on diarrheal disease induced by methotrexate and cholera toxin: improvement of intestinal barrier function." Universidade Federal do CearÃ, 2004. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=596.
Full textAbstract:
CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorA desnutriÃÃo, desidrataÃÃo e agentes antineoplÃsicos, tais como o metotrexato (MTX), sÃo imediatos causadores das doenÃas diarrÃicas. Objetivando investigar a mucosite intestinal (MI) induzida por MTX, a eficÃcia das soluÃÃes de reidrataÃÃo oral (SRO) acrescidas com glutamina (Gln), alanilglutamina (Ala-Gln), Hyprol 4107 (HYP) e hyfoama (HYFO) utilizou-se a perfusÃo intestinal. E para a suplementaÃÃo com vitaminas A (VITA) e E (VITE), Gln e peptÃdeos, utilizou-se as avaliaÃÃes morfomÃtricas, metabÃlicas e a de permeabilidade intestinal [(teste lactulose/manitol (L/M)], em camundongos (n=344) e em coelhos (n=72). A mucosite intestinal por MTX (2,75 mg/kg/24h s.c., durante 3 dias) foi validada em camundongos, atravÃs da constataÃÃo do quadro diarrÃico e do prejuÃzo no estado nutricional (-1.89Â0,52 g) estabelecido apÃs o 3o dia de tratamento. A anÃlise morfomÃtrica demonstrou achatamento de vilos (364,8Â19.9 mm) e hiperplasia de criptas (251Â19.2 mm) intestinais com aumento do nÃmero de apoptoses (7.48Â1, 23/cripta), no duodeno e no jejuno. A taxa L/M no grupo MTX foi maior c que no controle (0,35). A perfusÃo intestinal com toxina da cÃlera aumentou a secreÃÃo de Na+ (-12,8Â1.4 mEq/g/min), Cl- (-12,9Â4,6 mEq/g/min) e de Ãgua (-0,02 Â0,04 ml/g/min). As SRO acrescidas de Gln e peptÃdeos, restauram este efeito secretÃrio. A suplementaÃÃo de Gln e peptÃdeos, VITA e VITE, melhoram o ganho ponderal, a ingestÃo de raÃÃo, as alteraÃÃes morfolÃgicas e a apoptose, no modelo de mucosite intestinal. Nenhuma alteraÃÃo foi observada no padrÃo de mitoses nas criptas intestinais, apÃs o 3o dia de tratamento com MTX. A suplementaÃÃo nutricional reduziu na mucosite, a elevaÃÃo das taxas de L/M (0,35 para 0,73) nas vias paracelular e transcelular. Nossos resultados sugerem que, o restabelecimento da barreira morfofuncional, induzido pela suplementaÃÃo, foi obtido atravÃs de um aumento do transporte de cÃtions (Na+ e K+) na mucosa, do fornecimento de substrato energÃtico para o enterÃcito, inibiÃÃo da apoptose (0,25Â0,05), de cÃlulas indiferenciadas incremento no potencial redox e reduÃÃo da peroxidaÃÃo lipÃdica das cÃlulas intestinais diferenciadas. EntÃo sugerimos que o uso de SRO acrescidas de Gln e peptÃdeos podem prevenir a desnutriÃÃo e reduzir as doenÃas diarrÃicas, e a suplementaÃÃo com VITE e VITA melhorar a morfo fisiologia da barreira intestinal
A desnutriÃÃo, desidrataÃÃo e agentes antineoplÃsicos, tais como o metotrexato (MTX), sÃo imediatos causadores das doenÃas diarrÃicas. Objetivando investigar a mucosite intestinal (MI) induzida por MTX, a eficÃcia das soluÃÃes de reidrataÃÃo oral (SRO) acrescidas com glutamina (Gln), alanilglutamina (Ala-Gln), Hyprol 4107 (HYP) e hyfoama (HYFO) utilizou-se a perfusÃo intestinal. E para a suplementaÃÃo com vitaminas A (VITA) e E (VITE), Gln e peptÃdeos, utilizou-se as avaliaÃÃes morfomÃtricas, metabÃlicas e a de permeabilidade intestinal [(teste lactulose/manitol (L/M)], em camundongos (n=344) e em coelhos (n=72). A mucosite intestinal por MTX (2,75 mg/kg/24h s.c., durante 3 dias) foi validada em camundongos, atravÃs da constataÃÃo do quadro diarrÃico e do prejuÃzo no estado nutricional (-1.89Â 0,52 g) estabelecido apÃs o 3o dia de tratamento. A anÃlise morfomÃtrica demonstrou achatamento de vilos (364,8Â 19.9 Âm) e hiperplasia de criptas (251Â 19.2 Âm) intestinais com aumento do nÃmero de apoptoses (7.48Â 1, 23/cripta), no duodeno e no jejuno. A taxa L/M no grupo MTX foi maior c que no controle (0,35). A perfusÃo intestinal com toxina da cÃlera aumentou a secreÃÃo de Na+ (-12,8Â 1.4 ÂEq/g/min), Cl- (-12,9Â 4,6 ÂEq/g/min) e de Ãgua (-0,02 Â 0,04 ml/g/min). As SRO acrescidas de Gln e peptÃdeos, restauram este efeito secretÃrio. A suplementaÃÃo de Gln e peptÃdeos, VITA e VITE, melhoram o ganho ponderal, a ingestÃo de raÃÃo, as alteraÃÃes morfolÃgicas e a apoptose, no modelo de mucosite intestinal. Nenhuma alteraÃÃo foi observada no padrÃo de mitoses nas criptas intestinais, apÃs o 3o dia de tratamento com MTX. A suplementaÃÃo nutricional reduziu na mucosite, a elevaÃÃo das taxas de L/M (0,35 para 0,73) nas vias paracelular e transcelular. Nossos resultados sugerem que, o restabelecimento da barreira morfofuncional, induzido pela suplementaÃÃo, foi obtido atravÃs de um aumento do transporte de cÃtions (Na+ e K+) na mucosa, do fornecimento de substrato energÃtico para o enterÃcito, inibiÃÃo da apoptose (0,25Â 0,05), de cÃlulas indiferenciadas incremento no potencial redox e reduÃÃo da peroxidaÃÃo lipÃdica das cÃlulas intestinais diferenciadas. EntÃo sugerimos que o uso de SRO acrescidas de Gln e peptÃdeos podem prevenir a desnutriÃÃo e reduzir as doenÃas diarrÃicas, e a suplementaÃÃo com VITE e VITA melhorar a morfo fisiologia da barreira intestinal
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Porcu, Bernardi Elisabeth. "Cyclotetrapeptides analogues de la chlamydocine et de l'HC-toxine à visée thérapeutique." Montpellier 2, 1991. http://www.theses.fr/1991MON20153.
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La chlamydocine et l'hc-toxine sont des cyclotetrapeptides naturels presentant in vitro une activite cytostatique tres importante. Celle-ci, directement liee a la presence d'un residu aoe portant sur sa chaine laterale un groupe alkylant cetoepoxydique, n'est pas retrouvee lors des tests effectues in vivo. Notre travail consiste en la synthese, la caracterisation et l'etude des activites biologiques, de cyclotetrapeptides analogues de la chlamydocine et de l'hc-toxine. Pour ces analogues, l'aoe est remplace par une lysine dont l'amine laterale est substituee par divers groupes alkylants, dans le but d'obtenir des produits antitumoraux actifs in vivo. La cyclisation, etape cle de ces syntheses, a fait l'objet d'etudes d'optimisation: l'utilisation du depc, dans le dmf, en milieu tres dilue, a permis d'atteindre un excellent rendement de cyclisation (70%). Selon la nature du groupe alkylant couple aux cyclotetrapeptides, nous avons obtenu des analogues de trois types: amidoepoxydique, chloroethylnitrosouree (cnu), moutarde a l'azote. Nous avons obtenu un analogue cnu de la chlamydocine presentant une excellente activite cytostatique in vitro, sur tests l1210. Les analyses conformationnelles des cyclotetrapeptides prepares montrent qu'ils adoptent dans le chloroforme la meme unique conformation caracterisant la chlamydocine, l'hc-toxine et, plus generalement, ce type de cyclotetrapeptides
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Barbosa, Luiz Carlos Bertucci [UNESP]. "Peptídeos derivados da toxina bacteriana ParE: síntese, estrutura e ação inibitória sobre a atividade de topoisomerases." Universidade Estadual Paulista (UNESP), 2012. http://hdl.handle.net/11449/100731.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O sistema ParE-ParD é um sistema toxina-antitoxina bacteriano, sendo ParE a toxina e ParD a antitoxina. ParD é capaz de neutralizar a ação de ParE formando um complexo com o mesmo, o qual é eficaz na autorregulação do operon parDE. Estudos têm mostrado que a atividade tóxica de ParE ocorre inibindo a atividade da DNA girase, mas nenhum efeito desta proteína sobre a atividade da topoisomerase IV foi observado até hoje. Baseando-se na estrutura primária da toxina ParE de Escherichia coli, bem como nos escassos dados em relação a esta toxina, a meta deste trabalho foi a obtenção de peptídeos sintéticos baseados nesta proteína a fim de avaliar as sequências de aminoácidos responsáveis pela interação com as diferentes topoisomerases bacterianas, além de tentar isolar uma sequência polipeptídica com potencial atividade inibitória sobre essas enzimas. Utilizando modelagem molecular por homologia, um modelo tridimensional para toxina ParE de E. coli foi obtido e validado. Com base nos dados estruturais inferidos a partir do modelo da estrutura tridimensional de ParE, 12 peptídeos foram racionalmente desenhados e sintetizados pela metodologia da fase sólida. Ensaios de inibição in vitro da atividade de superenovelamento de DNA pela girase e de relaxamento de DNA pela topoisomerase IV foram realizados e indicaram que os peptídeos ParE3 (ParE 80-100), ParE8 (ParE 61-105), ParE10 (ParE 61-87) e ParE12 (ParE 61-79) atuam como bons inibidores de ambas enzimas. Ensaios de fluorescência intrínseca e anisotropia de fluorescência, empregando peptídeos sintéticos derivados de ParE, evidenciaram que o processo de inibição da atividade da DNA girase pela toxina ParE deve ocorrer por interação com a proteína GyrA da enzima. Foi iniciado neste trabalho os primeiros testes usando lipossomas como veículos...
The operon parDE encode a toxin-antitoxin system formed by ParE toxin and its antitoxin ParD. ParD is able to neutralize ParE action and is effective in autoregulation of the operon. Studies have shown that the toxic activity of the ParE occurs by inhibiting the activity of DNA gyrase, but no effect of this protein on the activity of topoisomerase IV has been observed yet. Based on the primary structure of the Escherichia coli ParE toxin, as well as the scarce data of this toxin, the aim of this work was to obtain synthetic peptides based on this protein in order to assess the amino acid sequences responsible for interaction with the bacterial topoisomerases, besides trying to isolate a polypeptide sequence with potential inhibitory activity against these enzymes. Using molecular homology modeling, a three-dimensional model for E. coli ParE toxin was obtained and validated. Based on structural data inferred from ParE threedimensional model, 12 peptides were rationally designed and synthesized by solid-phase method. Tests of inhibition of the supercoiling reaction of the DNA gyrase and inhibition of DNA relaxation by topoisomerase IV were performed and indicated that the peptides ParE3 (ParE 80-100), ParE8 (ParE 61-105), ParE10 (ParE 61 -87) and ParE12 (ParE 61-79) act as good inhibitors of both enzymes. Intrinsic fluorescence and fluorescence anisotropy assays, using synthetic peptides derived from ParE showed that inhibition process of activity of the DNA gyrase by ParE toxin must occur by interaction with the GyrA protein. In this work was started the first tests using liposomes as carrier vehicles for bioactive peptides derived from ParE. The peptides were efficiently encapsulated in soybean phosphatidyl choline liposomes. It was observed, although reduced, inhibition of bacterial growth when peptides encapsulated in liposomes were... (Complete abstract click electronic access below)
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Barbosa, Luiz Carlos Bertucci. "Peptídeos derivados da toxina bacteriana ParE : síntese, estrutura e ação inibitória sobre a atividade de topoisomerases /." Araraquara, 2012. http://hdl.handle.net/11449/100731.
Full textAbstract:
Orientador: Reinaldo MarchettoBanca: Eduardo Maffud Cilli
Banca: Wilton Rogério Lustri
Banca: Vani Xavier de Oliveira Junior
Banca: Herida Regina Nunes Salgado
Resumo: O sistema ParE-ParD é um sistema toxina-antitoxina bacteriano, sendo ParE a toxina e ParD a antitoxina. ParD é capaz de neutralizar a ação de ParE formando um complexo com o mesmo, o qual é eficaz na autorregulação do operon parDE. Estudos têm mostrado que a atividade tóxica de ParE ocorre inibindo a atividade da DNA girase, mas nenhum efeito desta proteína sobre a atividade da topoisomerase IV foi observado até hoje. Baseando-se na estrutura primária da toxina ParE de Escherichia coli, bem como nos escassos dados em relação a esta toxina, a meta deste trabalho foi a obtenção de peptídeos sintéticos baseados nesta proteína a fim de avaliar as sequências de aminoácidos responsáveis pela interação com as diferentes topoisomerases bacterianas, além de tentar isolar uma sequência polipeptídica com potencial atividade inibitória sobre essas enzimas. Utilizando modelagem molecular por homologia, um modelo tridimensional para toxina ParE de E. coli foi obtido e validado. Com base nos dados estruturais inferidos a partir do modelo da estrutura tridimensional de ParE, 12 peptídeos foram racionalmente desenhados e sintetizados pela metodologia da fase sólida. Ensaios de inibição in vitro da atividade de superenovelamento de DNA pela girase e de relaxamento de DNA pela topoisomerase IV foram realizados e indicaram que os peptídeos ParE3 (ParE 80-100), ParE8 (ParE 61-105), ParE10 (ParE 61-87) e ParE12 (ParE 61-79) atuam como bons inibidores de ambas enzimas. Ensaios de fluorescência intrínseca e anisotropia de fluorescência, empregando peptídeos sintéticos derivados de ParE, evidenciaram que o processo de inibição da atividade da DNA girase pela toxina ParE deve ocorrer por interação com a proteína GyrA da enzima. Foi iniciado neste trabalho os primeiros testes usando lipossomas como veículos... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The operon parDE encode a toxin-antitoxin system formed by ParE toxin and its antitoxin ParD. ParD is able to neutralize ParE action and is effective in autoregulation of the operon. Studies have shown that the toxic activity of the ParE occurs by inhibiting the activity of DNA gyrase, but no effect of this protein on the activity of topoisomerase IV has been observed yet. Based on the primary structure of the Escherichia coli ParE toxin, as well as the scarce data of this toxin, the aim of this work was to obtain synthetic peptides based on this protein in order to assess the amino acid sequences responsible for interaction with the bacterial topoisomerases, besides trying to isolate a polypeptide sequence with potential inhibitory activity against these enzymes. Using molecular homology modeling, a three-dimensional model for E. coli ParE toxin was obtained and validated. Based on structural data inferred from ParE threedimensional model, 12 peptides were rationally designed and synthesized by solid-phase method. Tests of inhibition of the supercoiling reaction of the DNA gyrase and inhibition of DNA relaxation by topoisomerase IV were performed and indicated that the peptides ParE3 (ParE 80-100), ParE8 (ParE 61-105), ParE10 (ParE 61 -87) and ParE12 (ParE 61-79) act as good inhibitors of both enzymes. Intrinsic fluorescence and fluorescence anisotropy assays, using synthetic peptides derived from ParE showed that inhibition process of activity of the DNA gyrase by ParE toxin must occur by interaction with the GyrA protein. In this work was started the first tests using liposomes as carrier vehicles for bioactive peptides derived from ParE. The peptides were efficiently encapsulated in soybean phosphatidyl choline liposomes. It was observed, although reduced, inhibition of bacterial growth when peptides encapsulated in liposomes were... (Complete abstract click electronic access below)
Doutor
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Nash, Ian Alun. "The development of solid phase strategies and methodologies : the synthesis of polyamine toxins and peptide nucleic acids." Thesis, University of Nottingham, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321373.
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Boitel, Brigitte. "La reconnaissance T d'un peptide de la toxine tétanique présenté par la molécule de classe II HLA-DR : illustration des bases moléculaires des intéractions TCRαβ / peptide / CMH." Paris 6, 1993. http://www.theses.fr/1993PA066726.
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Campos, Lucélia de Almeida. "Isolamento e caracterização da delta toxina do veneno de Crotalus durissus terrificus." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/85/85131/tde-14052012-135435/.
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O veneno de C. d. terrificus tem sido descrito como sendo de pouca complexidade, tendo 4 frações caracterizadas, convulxina, giroxina. crotoxina e crotamina. O presente trabalho visou o isolamento e caracterização da Delta toxina cuja existência havia sido aventada em trabalhos anteriores. Após a realização de uma varredura de tampões em uma coluna de exclusão molecular Superdex-75 acoplada a um sistema FPLC, na presença de três diferentes tampões, chegou-se a uma condição ideal de fracionamento do veneno crotálico. Em seqüência realizou-se a segunda etapa de purificação em sistema HPLC em uma coluna C4, onde foi possível identificar o pico de interesse. O pico puro passou por análises em MALDI-ToF sendo sua massa estimada em 14.074,92 Da, Quando analisado por eletroforese em gel de poiiacrilamida, a delta toxina apresentou massa molecular de cerca de 14 kDa e uma migração anômala, Por eletroforese 2D, a proteína apresentou caráter ácido, com pl entre 4 e 5 e massa molecular de aproximadamente 42 kDa, revelando \"spots\" muito semelhantes podendo ser isoformas com características de uma proteína glicosiiada. Após digestão dos spots com tripsina, os fragmentos foram confrontados com o banco de dados do \"swissprot\", mostrando alto grau de homologia \"até 43% de cobertura\" com a troca ri na, um ativador de protrombina do veneno de Tropidechis carinatus, esses dados foram confirmados com a análise de aminoácidos. De posse desses resultados, optou-se por testar a capacidade da fração purificada de ativar fator X e II, usando substratos sintéticos. Os resultados apontaram para uma ativação direta do fator X, uma vez que não houve ativação do fator II, atividade que também não foi detectada no veneno total. A mesma se mostrou um potente ativador da agregação de forma direta, uma vez que os ensaios de agregação plaquetária foram realizados com plaquetas lavadas, logo na ausência de fatores séricos. Quando os ensaios de agregação foram realizados na presença de alguns inibidores observou-se que nem a atividade metalo proteinase, nem a serino proteinase, tampouco um domínio lectina estavam envolvidos no processo, uma vez que EDTA, benzamidina e D-galactose não inibiram a atividade da proteína. No presente trabalho isolamos a Delta toxina do veneno de C. d. terrificus. A mesma se comportou como previsto por Vital Brazil em 1980, eluindo na posição por ele aventada, sendo uma proteína ativadora de Fator X que ativa agregação plaquetária mesmo em concentrações muito baixas e de massa molecular de 40 kDa levando nos a crer se tratar de um homotrímero cujos componentes são unidos por ligações fracas.The Crotalus durissus terrificus venom has been so far described as being of low complexity, with four major components described: convulxin, gyroxin, crotoxin and crotamine. In recent studies, other components of this venom were characterized as, for example, an analgesic factor. In 1980, Vital Brazil predicted the existence of a toxin which could be involved in platelet aggregation, and named it delta toxin. However, this toxin has never been isolated or characterized. The aim of the present work was to purify and characterize this toxin. After FPLC size exclusion chromatography followed by reverse phase HPLC, an homogeneous fraction was obtained, with a molecular weight of 14,074.92 Da. When analyzed by SOS-PAGE, this toxin presented an anomalous behavior, with a molecular weight of 14 kDa, while in 2D gels, spots around 40 kDa and with an isoelectrical point between 4 and 5 were observed suggesting isoforms with glicosilation microheterogeneity. After trypsin digestion, the fragments were submitted to the swissprot databank showing high homology (43% coverage, 15 matching peptides) with trocarin, a prothrombin activator from Tropidechis carinatus. These data were further confirmed by aminoacid analysis. The toxin was tested for its ability to activate factor II and X using synthetic substrates. Our data indicate a direct activation of factor X. The same toxin also behaved as a potent direct platelet aggregation activator on washed platelets. Assays with specific inhibitors indicate that neither metalloproteinase, nor serinoproteinase or t lectin domains are involved in the aggregating activity, since EDTA, benzamidin and D-galactose did not inhibit the toxin. In the present work, we were able to identify, purify and characterize a new toxin from the brazilian rattlesnake. It behaved as predicted by Vital-Brazil and displayed direct factor X activating properties, also inducing platelet aggregation, even at low concentrations. Our data also indicate that it is probably a homotrimer with the subunities linked by hydrophobic and/or electrostatic interactions.
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Elgar, Dale. "Ion selectivity and membrane potential effects of two scorpion pore-forming peptides / D. Elgar." Thesis, North-West University, 2005. http://hdl.handle.net/10394/991.
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Parabutoporin (PP) and opistoporin 1 (OP1) are cation, a-helical antimicrobial peptides isolated
from the southern African scorpion species, Parabuthus schlechteri and Opistophthalmus carinatus,
respectively. Along with their antimicrobial action against bacteria and fungi, these peptides show
pore-forming properties in the membranes of mammalian cells. Pore-formation and ion selectivity in
cardiac myocytes were investigated by measuring the whole cell leak current by means of the patch
clamp technique. Pore-formation was observed as the induction of leak currents. Ion selectivity of
the pores was indicated by the shift of the reversal potential (E,,,) upon substitution of intra (K' with
CS' and CI- with aspartate) and extracellular (Na' with NMDG') ions. Results were compared with
the effect of gramicidin A used as a positive control for monovalent cation selective pores. PP and
OP I induced a fluctuating leak current and indicate non-selectivity of PP and OP1-induced pores.
An osmotic protection assay to determine estimated pore size was performed on the cardiac
myocytes. PP and OP1-induced pores had an estimate pore size of 1.38-1.78 nm in diameter.
The effect of PP and OP1 on the membrane potential (MP) of a neuroblastoma cell line and cardiac
myocytes was investigated. TMRM was used to mark the MP fluorescently and a confocal
microscope used to record the data digitally. The resting membrane potential (RMP) of the
neuroblastoma cells was calculated at -38.3 f 1.9 mV. PP (0.5 uM) and OP1 (0.5-1 uM) depolarized
the entire cell uniformly to a MP of -1 1.9 k 3.9 mV and -9.4 k 1.9 mV, respectively. This occurred
after 20-30 min of peptide exposure. In the case of the cardiac myocytes depolarization was induced
to -39.7 f 8.4 mV and -32.6 f 5.2 mV by 0.5-1 uM PP and 1.5-2.5 uM OPl, respectively.Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2006.
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Cardoso, Kiara Carolina 1979. "Transcriptoma da glândula venenífera da serpente Bothrops alternatus (urutu) e caracterização molecular e bioquímica parcial da dipeptidilpeptidase IV." [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/312156.
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Orientador: Stephen HyslopTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: O estudo do transcriptoma de bibliotecas de cDNA da glândula venenífera de serpentes, realizado a partir da análise de ESTs (expressed sequence tags), tem se mostrado útil na identificação de genes expressos neste tecido, inclusive no gênero Bothrops, responsável pela maioria dos acidentes ofídicos no Brasil. Neste trabalho utilizamos uma abordagem transcriptômica para analisar a composição gênica da glândula venenífera da serpente Bothrops alternatus, uma espécie encontrada no sudeste e sul do Brasil, Uruguai, norte da Argentina e leste do Paraguai. Também clonamos e caracterizamos parcialmente a enzima dipeptidilpeptidase IV (DPP IV), uma enzima que cliva peptídeos com prolina ou alanina na penúltima posição em sua porção N-terminal e que tem sido detectada em diversas peçonhas ofídicas. A construção de bibliotecas de cDNA usando métodos convencionais de clonagem, sequenciamento e análise bioinformática resultou em 5,350 ESTs que foram reunidas em 838 contigs and 4512 singletons. Pesquisas a partir de bancos de dados relevantes (BLAST) mostraram 30% de hits e 70% no-hits. Os transcritos relacionados a toxinas correspondem a 23% do total de transcritos e 78% dos hits, respectivamente. A análise por ontologia gênica (GO) detectou genes relacionados ao metabolismo geral, transcrição, tradução, processamento, degradação de polipeptídeos, funções estruturais, e regulação celular. Os principais grupos de toxinas identificados foram metaloproteinases (81%), peptídeos potenciadores da bradicinina/peptídeos natriuréticos do tipo C (8,8%), fosfolipases 'A IND. 2' ('PLA IND. 2'; 5,6%), serinoproteinases (1.9%) e lectinas do tipo C (1,5%). As metaloproteinases eram quase queexclusivamente da classe PIII, com poucas da classe PII e nenhuma da classe PI. As 'PLA IND. 2' eram todas ácidas; nenhuma 'PLA IND. 2' básica foi detectada. Outras toxinas encontradas incluíram a L-aminoácido oxidase, proteínas secretadas ricas em cisteína, DPP IV, hialuronidase, toxinas three-finger e ohanina. Foram identificadas duas proteínas não-tóxicas, a tioredoxina e a Dusp6 (fosfatase de dupla especificidade) que mostraram alto grau de similaridade a proteínas semelhantes de outras serpentes. Também foram observados polimorfismos de nucleotídeos únicos (SNPs - single-nucleotide polymorphisms), microssatélites, transposons e repetições invertidas, todos os quais podem contribuir de alguma forma para a multiplicidade de toxinas na glândula. Estes resultados mostram que a glândula venenífera de B. alternatus possui as principais classes de toxinas encontradas em estudos transcriptômicos e proteômicos de outras espécies botrópicas. ... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital
Abstract: Transcriptomic studies of snake venom gland cDNA based on the analysis of expressed sequence tags (ESTs) have been useful in identifying the genes expressed in this organ in a variety of species, including the genus Bothrops, which is responsible for most venomous snakebites in Brazil. In this work, we used a transcriptomic approach to analyze the gene composition of the venom gland of Bothrops alternatus (urutu), a species found in southeastern and southern Brazil, Uruguay, northern Argentina e eastern Paraguay. We also cloned and partially characterized dipeptidylpeptidase IV (DPP IV), an enzyme that cleaves peptides with proline or alanine as the penultimate residue in the N-terminal region and has been identified in several snake venoms. A cDNA library constructed using conventional methods of cloning, sequencing and bioinformatic analysis yielded 5,350 ESTs that formed 838 contigs and 4512 singletons. Databank BLAST searches yielded 30% hits and 70% no-hits. Toxin-related transcripts accounted for 23% of the total transcripts and 78% of the hits. Gene ontology analysis detected genes related to general metabolism, transcription, translation, processing, polypeptide degradation, structural functions and cellular regulation. The main toxin groups identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases 'A IND. 2' ('PLA IND. 2'; 5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively class PIII, with few class PII and no class PI enzymes. The 'PLA IND. 2' were all acidic; no basic 'PLA IND. 2' were detected. Other toxins identified included L-amino acid oxidase, cysteine-rich secretory proteins, DPP IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and a dual specificity phosphatase (Dusp6), shared high sequence homology with similar proteins from other snakes. Single-nucleotide polymorphisms (SNPs), microsatellites, transposons and inverted repeats were also observed and may contribute to the toxin diversity of the gland. These results show that the venom gland of B. alternatus contains the major toxin classes identified in transcriptomic and proteomic studies of other Bothrops species. The predominance of class PIII metalloproteinases agrees with the hemorrhagic activity of this venom, while the low content of serine proteinases and C-type lectins could account for the less intense coagulopathy observed after envenoming by this species. The lack of basic 'PLA IND. 2' agrees with the lower myotoxicity of this venom compared to other Bothrops species. ... Note: The complete abstract is available with the full electronic digital thesis or dissertations
Doutorado
Farmacologia
Doutor em Farmacologia
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Jin, Sha. "Membrane interaction of amyloid–beta (1–42) peptide induces membrane remodeling and benefits the conversion of non–toxic Aβ species into cytotoxic aggregate." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2016. http://dx.doi.org/10.18452/17632.
Full textAbstract:
Das Amyloid-beta Peptid (Ab) ist der Hauptbestandteil der extrazellulären Plaques bei der Alzheimerschen Krankheit. Das Ziel der vorliegenden Arbeit ist es, die Mechanismen der Wechselwirkungen des Ab mit der Plasmamembran und der nachfolgenden zellulären Aufnahme aufzuklären. Die Aggregation, die zelluläre Aufnahme und die Zytotoxizität von Ab42 wurden durch Verwendung von fluoreszenzmarkierten Ab42 in einem Neuroblastomzellkulturmodell untersucht. Sowohl bei Inkubation mit Monomeren als auch mit Aggregaten wurde in den Zellen Ab42 detektiert. Dabei binden Ab42 Monomere und kleine Aggregate zunächst an die Zellmembran. Allerdings erfolgt keine direkte Aufnahme von Monomeren in die Zelle. Erst nach Ausbildung von Aggregaten mit geordneter Sekundärstruktur wurde Ab42 in den endozytotischen Vesikel detektiert. Voraussetzung für den an der Membran ablaufenden Aggregationsprozess ist, dass die Monomere oberhalb einer kritischen Konzentration anwesend sind, um eine Bildung von beta-Faltblatt-Strukturen (bF) und entsprechenden Aggregaten zu ermöglichen. Ab42 Aggregate, die sich durch eine bF auszeichneten, benötigten keine kritische Schwellenkonzentration für die endozytotische Aufnahme. Eng mit der Aufnahme von Ab42 Aggregaten war die Veränderung des zellulären Metabolismus verbunden. Um die Wechselwirkung zwischen Ab und der Membrannäher zu charakterisieren, wurden Modellmembransystemen einschl. riesigen Membranvesikeln genutzt. Dabei wurde beobachtet, dass sowohl Ab42 als auch Ab40 Einstülpungen in der Membran induzieren können. Kleine Aggregate beider Isoformen, die noch keine bF aufweisen, interagierten bevorzugt mit der ungeordneten Lipidphase und induzierten dabei eine negative Membrankrümmung. Diese Beobachtungen legen den Schluss nahe, dass möglicherweise das Ab selbst den endozytotischen Prozess unterstützt oder diesen sogar einleiten könnte. Dies könnte auch auf eine mögliche physiologische Funktion von Ab Aggregaten, die nicht toxisch sind, hindeuten.The accumulation of Amyloid beta peptide 1-42 (Ab42) in extracellular plaques is one of the pathological hallmarks of Alzheimer’s disease. Several studies have suggested that a cellular reuptake of Ab42 may be a crucial step in its cytotoxicity, but mechanisms of Ab-membrane interaction and subsequent cellular uptake are not yet understood. The first aim of the present study is to answer the question whether aggregate formation is a prerequisite or a consequence of Ab-membrane interaction and of Ab endocytosis. We visualized aggregate formation of fluorescently labeled Ab42 by Förster resonance energy transfer and tracked its internalization by human neuroblastoma cells. Both monomeric and aggregated Ab42 entered the cells, however, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed beta-sheet-rich (bS) aggregates to form. By uncoupling membrane binding from internalization, we found that Ab42 monomers as well as small aggregate species bound rapidly to the plasma membrane and formed bS aggregates. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with inhibition of cellular metabolism activities. Our data therefore imply that the formation of bS aggregates at the cell membrane is a prerequisite for Ab42 uptake and cytotoxicity. The second aim of the study is to investigate the Ab-membrane interaction in vitro by using giant unilamellar vesicles and giant plasma membrane vesicles as model membrane systems. We found that both Ab isoforms, Ab42 and Ab40, interacted with the liquid disordered phase of model membranes. Early aggregation intermediates, which did not yet bind to the amyloiddophilic dye Thioflavin T, induced negative membrane curvature. The ability of Ab to induce membrane deformation suggests that Ab may facilitate its own endocytosis. It also hints at a possible physiological function of non-toxic Ab aggregate species.
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Quercini, Leila. "The antimicrobial peptide SET-M33. Characterization of back-up molecules for the setup of novel antibiotics and development of a medical device for removing bacterial toxins from blood." Doctoral thesis, Università di Siena, 2019. http://hdl.handle.net/11365/1070198.
Full textAbstract:
The increasing frequency of multidrug-resistant (MDR) and extensively drug- resistant pathogens creates an urgent need for new antimicrobial drugs and new strategies for the treatment of infectious diseases The peptide SET-M33 is currently under preclinical development for the set-up of a new antibacterial agent to treat infections caused by Gram-negative pathogens. It is a cationic non-natural peptide synthetized in a tetra-branched form that makes it more resistant to degradation in biological fluids. SET-M33 has broadly shown high antimicrobial activity, both in vitro and in vivo, anti-inflammatory activity through selective LPS neutralization, low haemolytic activity, lack of immunogenicity and ability to eradicate biofilms.
Here we report the construction of a new SET-M33-based device for the selective removal of bacterial toxins such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA) from a biological fluid of patient with sepsis. In such method the peptide is covalently attached to a solid support through its
C- terminus, optionally with the interposition of a linker, and is used to capture the toxins.
In an attempt to produce back-up molecules, SET-M33 was synthesized with amino acids in D configuration instead of traditional L residues (SET-M33D). This isomeric version shows a therapeutic activity in vitro and in vivo and immunomodulatory activity in vitro against pro-inflammatory cytokines induced by Gram-negative E. Coli and Gram-positive S. aureus bacteria. Furthermore, the results of peptide’s toxicity in vitro and in vivo suggest that SET-M33D exhibits a low selectivity for eukaryotic cells while it is able to target bacteria cell efficaciously.
Lastly, we report the synthesis of the peptide in the two-branched form (SET- M33DIM) and its characterization in terms of stability, antimicrobial toxicity and efficacy in vitro, ex vivo and in vivo, anti-inflammatory proprieties and mechanism of action. Compared to the tetra-branched molecule, SET- M33DIM shows a similar antibacterial activity in vitro for strains of K. pneumonia and E. coli, a moderate activity against P. aeruginosa, and no activity against strains of the Gram-positive S. aureus. In vivo the peptide is able to delay significantly signs of acute infection in animal lethally infected with P. aeruginosa. Its toxicity profile results improved respect to SET-M33L more than 20 fold when tested on eukaryotic cells and a haemolysis degree not more than 22%, even using a concentration which is 200-fold respect to the MIC. Moreover, SET-M33DIM exhibits a strong capability to neutralize LPS, inhibiting the expression of inflammatory cytokines and enzymes in macrophages.