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Clonagem, expressão e estudo de alguns cDNAs codificando proteínas estruturalmente relacionadas às alfa neurotoxinas da glândula de veneno da cobra coral Micrurus corallinus (Serpentes, Elapidae). / Cloning, expression and study of some cDNAs codifying proteins structurally related to the alpha neurotoxins of the venom gland from coral snake Micrurus corallinus (Serpentes, Elapidae).Alvaro Rossan de Brandão Prieto da Silva 28 January 2002 (has links)
De uma biblioteca de cDNA da glândula de veneno da cobra coral brasileira Micrurus corallinus foi isolada uma seqüência denominada NXH8. Esta seqüência de cDNA apresenta similaridade estrutural com a família de toxinas de serpentes em 'três dígitos' ricas em pontes dissulfeto. A subclasse melhor conhecida nesta família, são as alfa neurotoxinas. Uma outra seqüência distinta, denominada NXH1 e suas isoformas NXH3 e NXH7, foram isoladas anteriormente. Pertencem à mesma família de toxinas e estão presentes na mesma biblioteca. Alguns resultados da caracterização de NXH1, são utilizados neste estudo, em comparação com NXH8. Algumas características estruturais tornam a seqüência NXH8 diferente da classe usual das alfa neurotoxinas, vindo a constituir possivelmente uma nova subclasse da família. A proteína NXH8 foi expressa em diversos vetores de expressão em Escherichia coli. A proteína recombinante, expressa pelo vetor pRSET C - NXH8 foi utilizada para imunizar camundongos. O soro contra NXH8, assim como o soro anti - elapídico do Instituto Butantan, reconhece a toxina recombinante em ELISA e Western blot. O soro anti - NXH8 detecta apenas uma banda do veneno de M. corallinus em Western blot, mas apresenta reatividade cruzada com componentes do veneno de alguns elapídeos neotropicais e do velho mundo. Em contraste, dados anteriores demonstraram que o soro anti - NXH1 é específico para um componente único do veneno de M. corallinus. O veneno de M. corallinus tem alfa neurotoxinas que bloqueiam o receptor pós - sináptico nicotínico de acetilcolina nas membranas do músculo esquelético de ratos. O soro anti - NXH8 é capaz de impedir a ligação de componentes do veneno bruto a esses receptores. Já o soro contra NXH1 não apresenta a mesma capacidade inibitória. Isto indica que NXH8 tem afinidade pelo receptor nicotínico muscular de acetilcolina, ou que NXH8 compartilha de um epítopo neutralizante presente também nas alfa neurotoxinas do veneno da cobra coral M. corallinus. / A cDNA sequence encoding a putative new toxin, NXH8, was isolated from the cDNA library constructed from the venom gland of the Brazilian coral snake, Micrurus corallinus. This sequence shows a structural similarity with the snake toxin family known as 'three-fingered' toxins, a family of toxins with approximately 60 to 70 amino acids and usually 4 to 5 disulfide bonds. Irrespective of whether these proteins are functionally different, their amino acid sequences can be readily aligned, using 8 half-cystines as conserved elements, suggesting the presence of common structural features. The best known subclass of three-finger-type toxins are the curaremimetic toxins, also called alpha-neurotoxins, found in most venoms from Elapid and Sea snakes. Another toxin with a distinct sequence, known as NXH1 and its isoforms NXH3 and NXH7 had been previously isolated. They belong to the same family of toxins and were characterized from the same cDNA library. In the present study, a comparative biochemical, pharmacological and structural analyses of NXH1 and NXH8 were described. Few structural characteristics of NXH8 seem to indicate that it differs from the usual class of alpha neurotoxins, belonging, possibly, to a new subclass of 'three-finger' toxins. The NXH8 protein was expressed in various E. coli expression vectors and the resulted recombinant toxin from pRSETC-NXH8 plasmid was used as a "toxoid" for mice immunization. The anti - NXH8 sera, as well as the anti elapid sera from the Butantan Institute, recognized the recombinant toxin by both ELISA and Western blot assays. In contrast to the claim that anti - NXH1 sera is specific to one component of M. corallinuss venom, the anti NXH8 sera show cross reactivity to venom of some Neotropical and Old World elapids. The M. corallinus's venom contains alpha toxins, which inhibit post-synaptic nicotinic acetylcholine receptor of neonatal rat skeletal muscle membrane. The anti - NXH8 serum was capable of blocking the binding of the components of the crude venom to these receptors. In contrast, the anti NXH1 serum did not show this inhibitory effect. This indicates that either NXH8 presents affinity for muscular nicotinic acetylcholine receptor or it shares a neutralizing epitope also present in M. corallinuss alpha neurotoxins.
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Peptides vipérins à activité anti-intégrines : intérêt dans le traitement des pathologies ischémiques de la rétine et les DMLA / Integrins inhibitor isolated from snake venom for the treatment of ischemic retinopathies and AMDMontassar, Fadoua 29 September 2017 (has links)
Les rétinopathies ischémiques et la forme humide de la dégénérescence maculaire liée à l’âge (DMLA) sont la principale cause de malvoyance respectivement chez les personnes en âge de travailler et les personnes agées. Les néovascularisations choroïdiennes (NVC) et rétiniennes et l’œdème maculaire associés à ces pathologies sont traités à l’aide de biomolécules qui ciblent uniquement la voie de signalisation des VEGF. Le développement de thérapies indépendantes de cette voie permettrait d’offrir aux patients résistants aux anti-VEGF une alternative thérapeutique pour préserver leur vision. Les intégrines αvβ3 et αvβ5, impliquées dans la néovascularisation oculaire, apparaissent ainsi comme une cible alternative intéressante. La Lébécetine (LCT), une lectine de type-C, de 30 kDa et de structure hétérodimèrique issue de venin de serpent Macrovipera lebetina interagit spécifiquement avec les intégrines α5β1 et αvβ3, αvβ5. La LCT a une activité anti-angiogénique in vitro sur des cellules endothéliales microvasculaire du cerveau humain (HBMEC) et in vivo sur le modèle de la membrane chorioallantoïde du poulet (CAM). Afin d’étudier son effet sur la néovascularisation oculaire, nous avons eu recours aux modèles d’angiogenèse ex vivo utilisant des explants aortique ou choroïdien cultivés en présence de LCT, puis son effet a été évalué in vivo dans un modèle de NVC chez la souris et également sur la néovascularisation rétinienne dans le modèle de rétinopathie induite par l’oxygène (RIO). Nos données démontrent qu’une injection unique de LCT est capable de réduire la NVC et rétinienne dans ces modèles sans affecter les vaisseaux quiescents matures indiquant un bon profil d’innocuité. / Ischemic retinopathies and the wet form of age-related macular degeneration (AMD) are characterized by devastating angiogenesis responsible for the majority of irreversible blindness. Current therapies include use of anti-VEGF agents to reduce choroidal neovascularization and edema. These treatments are effective in most cases, but spontaneous or acquired resistance to anti-VEGF highlight a need for additional alternative therapies. In recent years, pharmacological inhibition of αvβ3 and αvβ5, which regulate endothelial cell proliferation and stabilization, have emerged as new therapeutic tools for the treatment of these diseases. Lebecetin (LCT), a 30-kDa heterodimeric C-type lectin that is isolated from Macrovipera lebetina venom, interacts with α5β1 and αv-containing integrins (αvβ3, αvβ5). We previously showed that LCT has an anti-angiogenic effect in vitro on human brain microvascular endothelial cells (HBMEC) and in vivo in a chick chorioallontoic membrane assay (CAM). To evaluate the inhibitory effect of LCT on ocular angiogenesis, we cultured aortic and choroidal explants in the presence of LCT and analyzed the effect of LCT on choroidal neovascularization in the mouse CNV model and on retinal neovascularization in the oxygen induced retinopathy (OIR) model. Our data demonstrated that a single injection of LCT efficiently reduced choroidal and retinal neovascularization in these models with no significant effect on mature blood vessels predicting a good safety profile.
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Estudo comparativo do veneno botrópico de referência em relação ao veneno das serpentes Bothrops jararaca nascidas em cativeiro no Laboratório de Herpetologia do Instituto Butantan. / Comparative study between Bothropic reference venom and venom from Brazilian Bothrops jararaca snake born in captivity in the Herpetology Laboratory of Butantan Institute.Iasmim Baptista de Farias 29 September 2016 (has links)
Em 1987 o Instituto Nacional de Controle de Qualidade em Saúde iniciou o uso do Veneno Botrópico de Referência Nacional (VBRN), que é a primeira extração das serpentes B. jararaca recém-chegadas da natureza. Em 10 anos notou-se uma queda de 67,65% na recepção de serpentes ao Instituto Butantan (IB), e na distribuição geográfica, resultando em uma maior heterogeneidade na composição do VBRN. Assim, comparamos os venenos das serpentes nascidas em cativeiro (VP) do Laboratório de Herpetologia do IB com o VBRN, para verificar a possibilidade de incorporar o VP na preparação dos lotes do VBRN. O VP mostrou-se estatisticamente semelhante ao VBRN nos testes de dosagem de proteínas, SDS-PAGE, 2DE, cromatografia, atividade fosfolipásica, ELISA, Western blotting, dose mínima coagulante (plasma) e hemorrágica, enquanto para as atividades de L-aminoácido oxidase, caseinolítica, zimografia e dose mínima coagulante (fibrinogênio) o VP mostrou-se diferente do VBRN. Para as doses letais e efetiva dos venenos o pool do VP com VBRN foi mais eficaz do que os VP e o VBRN sozinhos. / In 1987, The National Institute of Quality Control in Health in Brazil (INCQS) established the Brazilian Reference Bothrops Venoms (BRBV), which should be composed of the first extractions of newcomers wild snakes. In 10 years there has been a decrease of 67.65% in the reception snakes to Butantan Institute (IB), and geographic distribution, resulting in a higher heterogeneity in BRBV composition. So, we compared the venom samples of snakes born in captivity (SVP) of IB Herpetology Laboratory with BRBV to check the possibility of incorporating the SVP in the preparation of batches of BRBV. SVP was statistically similar to BRBV the proteins dosage, SDS-PAGE, 2DE, chromatography, phospholipase, ELISA, Western blotting, minimum coagulant dose (plasma) and hemorrhage dose, while for L-amino acid oxidase activity, caseinolytic, zymography and minimum coagulant dose (fibrinogen) SVP was shown to be different from BRBV. For effective and lethal doses of pool with SVP and BRBV it was more effective than SVP and BRBV alone.
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"Characterization of Red Diamondback Rattlesnake Venom Proteins on Cell Death and Function"Ebrahimian, Venus January 2013 (has links)
No description available.
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Planejamento baseado na estrutura da metaloprotease BPMP-I e avalia??o de tiossemicarbazonas ativas contra a pe?onha da serpente Bothrops pauloensis / Structure-based planning Of BPMP-I metalloprotease and evaluation Of thiosemicarbazones active against The snake venom Bothrops PauloensisFerreira, Francis Barbosa 04 August 2016 (has links)
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Previous issue date: 2016-08-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / In this work, semi and thiosemicarbazones selected from the LaDMol-QM library, were used
to study their interactions with a metalloproteinase from the snake Bothrops pauloensis
(BpMP-I) by molecular modelling and enzymatic inhibition assays with the toxin. The
crystalographic structure of BaPI (PDB code: 2W12) was used as a mold to build the 3D
model of BpMP-I by homology modeling. The theorical model of BpMP-I showed good
quality parameters and was used in a subsequent molecular modeling study. The
thiossemicarbazones showed better molecular docking results and in vitro enzymatic
inhibitions assays than semicarbazones. Studies by semi-empirical methods indicate a positive
enthalpy of interaction, suggesting that the enzyme inhibition by these compounds must be a
entropy-driven process. The results were used together to select the LDQM-IN-23 compound
and propose rationally designed modifications to improve the interactions with the toxin. The
study of the catalytic site of BpMP-I showed that there is an adjacent pocket with amino
groups of the peptide bonds available for interaction. All results were used together to design
structural changes, aiming the enhancing of the interaction with toxin. Therefore, was
proposed the insertion of the carboxyl group with different spacers, containing 2 (LDQM-IN-
23b) and 3 methylene groups (LDQM-IN-23c). The docking results and semi-empiric
optimization showed that there was a considerable improvement in the interaction for the
modified compounds. The modified compounds were synthesized and tested for biological
and enzymatic inhibition activity. It was observed that the IC50 values have improved: the
original molecule, LDQM-IN-23 has an IC50 of 3,011 ?M and the modified molecules have
IC50 of 79.12 (LDQM-IN-23b) and 1.77 ?M (LDQM-IN-23c). These molecules were tested
for inhibition of hemorrhagic activity induced by Bothropoidin, a P-III class
metalloproteinase, and by the B. pauloensis whole snake venom. The three molecules can
inhibit the hemorrhagic activity induced by isolated toxin and whole venom, and LDQM-IN-
23c showed higher efficiency compared with the other two, and in a rate of 1:10 (w/w
venom/inhibitor) the inhibition of the hemorrhagic activity was 100%. A molecular docking
study of this lead compound with Snake Venom Metalloproteases (SVMPs) from different
snake species and genera showed that this molecule can effectivelly interact with these
SVMPs. / Neste trabalho, foram utilizadas semi e tiossemicarbazonas, selecionadas na quimioteca do
LaDMol-QM (Dequim-UFRRJ), para o estudo das intera??es destas com o s?tio ativo de uma
metaloprotease da pe?onha da serpente Bothrops pauloensis por modelagem molecular e
ensaios de inibi??o da atividade enzim?tica e biol?gica sobre a toxina. A estrutura
cristalogr?fica de uma metaloprotease (BaPI) complexada com um inibidor (um
peptideomim?tico) (c?digo PDB 2W12) foi utilizada como molde para a constru??o do
modelo 3D da metaloprotease da pe?onha de B. pauloensis (BpMP-I). O modelo 3D te?rico
da BpMP-I, in?dito para esta toxina, apresentou bons par?metros de qualidade, sendo
considerado adequado para estudos de planejamento de ligantes baseado na estrutura. As
tiossemicarbazonas obtiveram melhores resultados, quando comparados com os resultados
das semicarbazonas, tanto para os ensaios de docagem molecular quanto para estudos de
inibi??o da atividade enzim?tica in vitro. Estudos por m?todos semiemp?ricos indicam uma
entalpia de intera??o positiva, sugerindo que a inibi??o enzim?tica por estes compostos deve
ser um processo controlado entropicamente. Os resultados foram utilizados para selecionar o
derivado LDQM-IN-23 e propor modifica??es estruturais planejadas racionalmente, visando
melhorar a intera??o deste com a toxina. O estudo do s?tio catal?tico da metaloprotease
mostrou que esta possui uma cavidade adjacente com grupos amino das liga??es pept?dicas
dispon?veis para intera??o. Foi proposta, ent?o, a inser??o de um grupo carboxilato com
diferentes espa?adores, 2 (LDQM-IN-23b) e 3 grupos metileno (LDQM-IN-23c). Os
resultados de docagem e otimiza??o semi-emp?rica mostraram que houve uma melhora
consider?vel na intera??o dos ligantes modificados, os quais foram sintetizados e testados
para as atividades de inibi??o enzim?tica e biol?gica. Na inibi??o enzim?tica, houve melhora
da CI50 com o aumento do espa?ador. O composto LDQM-IN-23 tem CI50 de 3011,00 ?M e
os compostos modificados possuem a CI50 de 79,12 (LDQM-IN-23b) e 1,77 ?M (LDQM-IN-
23c). Estes compostos foram testados para a inibi??o da atividade hemorr?gica in vivo
induzida pela Botropoidina, uma metaloprotease da classe P-III, e pela pe?onha bruta de B.
pauloensis. Os tr?s compostos conseguiram inibir a atividade hemorr?gica induzida pela
toxina isolada e pela pe?onha, sendo que o composto LDQM-IN-23c mostrou maior
efici?ncia, quando comparado com os outros dois, e para a propor??o de 1:10 (m/m
pe?onha/inibidor) a inibi??o da atividade foi de 100%. Foi realizado um estudo de docagem
deste composto l?der com outras metaloproteases de pe?onha de serpentes (SVMPs ? Snake
Venom Metalloproteinases), de esp?cies e g?neros diferentes, mostrando que este ligante
consegue interagir com outras SVMPs e ? um candidato para inibir a atividade hemorr?gica
de SVMPs presentes na pe?onha, n?o s? de B. pauloensis, mas de outras serpentes
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Conformationally Constrained Nucleosides : Design, Synthesis, and Biochemical Evaluation of Modified Antisense OligonucleotidesVarghese, Oommen P. January 2007 (has links)
This thesis is concerned with synthesis, structure and biochemical analysis of chemically modified oligonucleotides with potential therapeutic applications. The three types of chemical modifications described here are: (a) A North-East locked 1',2'-azetidine nucleoside (b) A North locked 2',4'-cyanomethylene bridged nucleoside and (c) A 2',4'-aza-ENA-T nucleoside. The synthesis of the 1',2'-azetidine fused nucleosides was described using two different approaches. A highly strained 2',4'-cyanomethylene locked nucleoside was synthesized but could not be converted to the phosphoramidite derivative due to instability during derivatization. The key cyclization step in the aza-ENA-T nucleoside synthesis gave rise to two separable diastereomers due to chirality at the exocyclic nitrogen. Conversion of diastereomer 55 to 56 occurred with a large free energy of activation (ΔG‡ = 23.4 kcal mol-1 at 298 K in pyridine-d5). Of the two isomers the equatorial NH product was more stable than the axial one due to reduced 1,3 diaxial interactions. As a result, all NH axial product was converted to the equatorial isomer during subsequent steps in the synthesis. NMR and ab initio experiments confirmed the North-East structure of the 1',2'-azetidine locked nucleoside and North conformation of aza-ENA-T locked nucleosides with a chair conformation of the piperidine ring. The amino modified nucleosides were incorporated into different positions of a 15mer oligonucleotide. The azetidine modified AONs did not form stable duplexes with complementary RNA (ΔTm ~-1 to -4 °C), but they performed better than previously synthesized isosequential 1',2'-oxetane modified oligonucleotides. The 2',4'-aza-ENA-T modified oligonucleotide, on the other hand, showed excellent target affinity with complementary RNA (ΔTm ~+4 °C). The azetidine and aza-ENA-T modified oligonucleotides showed significant stability in the presence of human serum and snake venom phosphodiesterase (3'-exonuclease) as compared to the unmodified native sequence. The singly modified 15mer oligonucleotides were also subjected to RNase H promoted digestion in order to evaluate their potential as effective antisense agents. The effective enzyme activity (kcat/Km) was found to be lower in the modified AONs due to reduced enzyme-substrate binding. However, the catalytic activity of RNase H with these modified-AON:RNA duplexes were higher than observed with the native duplex.
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Avaliação “in vitro” do efeito antitumoral e antiangiogênico de uma metaloprotease isolada da peçonha de Bothrops pauloensisGuimarães, Denise de Oliveira 16 September 2016 (has links)
O câncer de mama é uma neoplasia altamente maligna e continua a ser a segunda principal causa de mortalidade entre as mulheres. Os efeitos antitumorais de metaloproteinases e desintegrinas de veneno de serpentes têm sido investigados em vários tipos de células tumorais. Neste estudo, foram avaliados os efeitos antitumorais e anti-angiogênicos induzidos pela Bothropoidina, uma metaloproteinase desintegrina-like isolada da peçonha de Bothrops pauloensis em células de câncer de mama humano MDA-MB-231 e células endoteliais. Após 24 horas de tratamento com 100pg/mL de Bothropoidina foi constatado um efeito citotóxico moderado de 30% em MDA-MB-231 contra 10% de citotoxicidade em MCF10A (uma linha de células da mama não tumorigênica), uma diferença significativa que sugere uma possível preferência desta proteína por alvos em células tumorais. Observou-se apoptose e apoptose tardia após tratamento com Bothropoidina (10pg/mL e 40pg/mL) em células MDA-MB-231. Além disso, esta toxina não só inibiu a adesão de células MDA-MB-231 de uma forma dose dependente, como a migração celular em aproximadamente 45%. Bothropoidina reduziu a viabilidade e adesão de células endoteliais em Matrigel e inibiu a angiogênese in vitro estimulada por bFGF em Matrigel, mostrando um número de vasos formados significativamente menor em relação ao controle. Os resultados demonstraram que Bothropoidina tem um potente efeito antitumoral e antiangiogênico in vitro, representando uma ferramenta biotecnológica para elucidar o efeito antitumoral de metaloproteinases desintegrinas-like em células cancerígenas. / Breast cancer is a highly malignant carcinoma and remains the second leading cause of mortality among women. The antitumor effects of metalloproteinases and disintegrins from snake venom on various types of cancer cells have been investigated. In this study, we evaluated the antitumor and antiangiogenic effects on MDA-MB-231 human breast cancer cells and endothelial cells induced by Bothropoidin, a disintegrin-like metalloproteinase isolated from Bothrops pauloensis snake venom. At 24h after treatment at 100pg/mL, Bothropoidin exerted a moderate cytotoxic effect of 30% on MDA-MB-231 versus 10% cytotoxicity against MCF10A (a non-tumorigenic breast cell line), a significant difference that suggests a possible preference by this protein for targets in cancer cells. Early and late apoptosis of MDA-MB-231 was observed after Bothropoidin treatment (10gg/mL and 40gg/mL). Furthermore, this toxin inhibited not only the adhesion of MDA-MB-231 cells in a dose-dependent manner but also cell migration by approximately 45%. In addition, Bothropoidin decreased endothelial cells viability and adhesion in Matrigel and inhibited in vitro angiogenesis in Matrigel stimulated by bFGF, showing significantly fewer formed vessels. The results demonstrated that Bothropoidin has potent in vitro antitumor and antiangiogenic effect and represents a biotechnological tool for elucidating the antitumor effect of disintegrins-like metalloproteinases in cancer cells. / Dissertação (Mestrado)
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