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Atividade biolÃgica das lectinas de sementes de erythrina fusca e velutina, de algas marinhas hypnea musciformes, bryothamnion seaforthii e triquetrum e do produto natural diterpeno casbano, em culturas de pseudomonas aeruginosa / Biological activity of lectins from Erythrina velutina and fusca, seaweed Hypnea musciformes, bryothamnion seaforthii and triquetrum and casbano diterpene natural product, in cultures of Pseudomonas aeruginosaRicardo Hideo Togashi 24 February 2010 (has links)
FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Neste trabalho avaliamos a atividade biolÃgica de lectinas de sementes de Erythrina
fusca e velutina, de algas marinhas Hypnea musciformes, Bryothamnion seaforthii e
triquetrum e do diterpeno casbano, um produto natural isolado de Croton nepetaefolius,
sobre Pseudomonas aeruginosa ATCC 10145. Foi comparada a aÃÃo in vitro das 5
lectinas e do diterpeno casbano, sobre colÃnias de P. aeruginosa, em placas de
poliestireno. Investigada a aÃÃo das lectinas de alga marinha H.musciforme, de sementes
de Erythrina velutina, e do diterpeno casbano, no processo de formaÃÃo do biofilme
bacteriano de P.aeruginosa, em placas de poliestireno; e identificado entre as lectinas de
E.velutina, H.musciforme e diterpeno casbano, aquele com maior potencial de aplicaÃÃo
no controle do crescimento de colÃnias de P. aeruginosa. As lectinas testadas nÃo foram
capazes de inibir o crescimento e a formaÃÃo de biofilme de Pseudomonas aeruginosa
nas condiÃÃes experimentadas. Por outro lado, diterpeno casbano, na concentraÃÃo de
500 μg/mL em 18 horas, foi capaz de inibir o crescimento de P. aeruginosa em 40%,
comparado ao controle positivo. Esta inibiÃÃo foi observada atà uma concentraÃÃo de
125 μg/mL. Entretanto, nÃo foi observada inibiÃÃo da formaÃÃo do biofilme da P.
aeruginosa nas concentraÃÃes utilizadas neste estudo. / In this study the biological activity of seeds lectins from Erythrina velutina and fusca,
marine algae Hypnea musciformis, Bryothamnion seaforthii and triquetrum and the
diterpene casbane, a natural product isolated from Croton nepetaefolius was evaluated
upon Pseudomonas aeruginosa ATCC 10145. We compared the in vitro effect of lectins
and diterpene casbane on colonies of P. aeruginosa in microtiter plates. Investigated the
action of lectins from marine algae H. musciforme of seeds of Erythrina velutina, and
diterpeno casbano in the process of formation of P. aeruginosa biofilm on polystyrene
plates, and identified among lectins: E. velutina, H. musciforme and diterpene casbane,
the one with greater potential for application in controlling the growth of colonies of
P. aeruginosa. The lectins tested were able to inhibit growth and biofilm formation of
P. aeruginosa in the studied conditions. Moreover, diterpene casbane at a concentration
of 500 mg/mL in 18 hours, was able to inhibit the growth of P. aeruginosa in 40%,
compared to positive control. This inhibition was observed until a concentration of 125
mg/mL. However, the inhibition of biofilm formation of P. aeruginosa there was no
observed at the concentrations used in this study.
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AvaliaÃÃo in vitro da interferÃncia de lectinas vegetais e do diterpeno casbano isolado de Croton nepataefolius sobre o crescimento de formas planctÃnicas e biofilmes de Pseudomonas aeruginosa / In vitro evaluation of the interference of plant lectins and the diterpene isolated from Croton casbano nepataefolius on the growth of planktonic forms and biofilms of Pseudomonas aeruginosaFabiano Fazanaro 26 February 2010 (has links)
FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Este trabalho mostra as atividades biolÃgicas de lectinas isoladas de sementes de Vatairea macrocarpa e de Vatairea guianensis e do composto vegetal diterpeno casbano, isolado do Croton nepetaefolius, sobre o crescimento de Pseudomonas aeruginosa (ATCC 9027), causadora de otite externa. Comparou-se a aÃÃo in vitro das duas lectinas e do composto vegetal diterpeno casbano sobre culturas de P. aeruginosa em placas de poliestireno. As cÃlulas bacterianas foram testadas tanto em sua forma planctÃnica como na de biofilme. As lectinas testadas nÃo foram capazes de inibir o crescimento da forma planctÃnica e a formaÃÃo de biofilme da P. aeruginosa nas condiÃÃes experimentais. Por outro lado, o diterpeno casbano foi capaz de inibir o crescimento de P. aeruginosa na forma planctÃnica, nas concentraÃÃes de 500, 250 e 125 Âg/mL. Entretanto, nÃo foi observada inibiÃÃo da formaÃÃo do biofilme da P. aeruginosa nas concentraÃÃes utilizadas neste estudo. O diterpeno casbano isolado de Croton nepetaefolius poderà ser utilizado, apÃs a realizaÃÃo de outros estudos, como ferramenta biotecnolÃgica antimicrobiana sobre as formas planctÃnicas de P. aeruginosa / This work shows the biological activities of lectins isolated from Vatairea macrocarpa and Vatairea guianensis seeds and the vegetable compound diterpen casban, isolated from Croton nepetaefolius on the growth of Pseudomonas aeruginosa (ATCC 9027) that causes otites externa. The in vitro activity of the two lectins and vegetable compound casbane diterpene were compared on cultures of P. aeruginosa in polystyrene microplates. The bacterial cells were tested such in planktonic as in biofilm forms. The lectins tested were not capable to inhibit the growth and biofilm production of P. aeruginosa in the experimental conditions. On the other hand, the casbane diterpene was capable to inhibit the growth of planctonic forms of P. aeruginosa at the concentrations of 500, 250 and 125 Âg/mL. However, the inhibition of biofilm production was not observed at the same concentrations. The casbane diterpene isolated from Croton nepetaefolius can be used, after the realization of other studies, as an antibiotic biotechnological tool on planktonic forms of P. aeruginosa
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ResoluÃÃo da estrutura tridimensional da lectina de Dioclea violacea Mart para estudo da relaÃÃo estrutura-funÃÃo / Three-dimensional structure of lectin from Dioclea violacea for study structure/function relationMaria JÃlia Barbosa Bezerra 17 February 2011 (has links)
Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As lectinas da subtribo Diocleinae pertencem à famÃlia de Leguminosas e sÃo caracterizadas pela alta homologia entre suas sequÃncias de aminoÃcidos. Apesar da alta similaridade estrutural o mesmo nÃo acontece nas atividades biolÃgicas das lectinas dessa famÃlia. Estudos mostram que a modificaÃÃo de poucos aminoÃcidos em suas sequÃncias à capaz de provocar grandes alteraÃÃes nas atividades biolÃgicas dessas lectinas, dessa forma o entendimento mais detalhado das estruturas tridimensionais dessas proteÃnas à essencial para a anÃlise da relaÃÃo entre estrutura e funÃÃo. A lectina de Dioclea violacea foi purificada por cromatografia de afinidade em coluna de Sephadex G-50. A proteÃna foi cristalizada na presenÃa do ligante X-Man e os cristais foram obtidos pelo mÃtodo de difusÃo de vapor em matriz esparsa por gota suspensa. Foram utilizando os kit âcristal screenâ I e II da Hampton Research e a condiÃÃo que obteve melhor cristal foi a condiÃÃo 33 do kit I composta por 4M Formato de sÃdio. O cristal foi difratado a 2,61à e apresenta grupo espacial I222 com cela unitÃria com dimensÃes de a = 61,34, b = 66,11, c = 106,69à e Ãngulos de α = β = γ = 90Â. Foi observada a presenÃa de um monÃmero na unidade assimÃtrica contendo 42,04% de solvente no cristal. A substituiÃÃo molecular foi feita utilizando a estrutura da lectina de Dioclea rostrata (PDB: 2ZBJ). O refinamento final obteve Rfactor de 0,23 e Rfree de 0,27 com ausÃncia aminoÃcidos em regiÃes nÃo permitidas do grÃfico de Ramachandran. O ligante X-Man foi co-cristalizado e sua estrutura foi observada perfeitamente encaixada no domÃnio de reconhecimento a carboidratos. Em relaÃÃo ao equilÃbrio dÃmero tetrÃmero, a lectina de Dioclea violacea apresenta o aminoÃcido HIS 131 e a posiÃÃo da HIS 51 à semelhante ao mesmo aminoÃcido da Dioclea grandiflora, caracterizando esta lectina como tetrÃmero mesmo em pH baixo. Foram feitas comparaÃÃes entre as atividades biolÃgicas de outras lectinas do gÃnero Dioclea e as distÃncias entre os resÃduos do sÃtio de ligaÃÃo a carboidrato. Essa analise mostrou que a variaÃÃo nessas distÃncias influi no efeito e eficÃcia da atividade vasorelaxante em aorta de ratos. / Lectins from subtribe Diocleinae belong to the family from Leguminosae and are characterized by high homology among their amino acid sequences. Despite this high structural similarity the same is not true in the biological activities from this lectin family. Studies show that the modification of a few amino acids can cause large changes in biological activities of these lectins. Thus more detailed understanding of three dimensional structures of these proteins is essential for structure/function analyses. The Dioclea violacea lectin was purified by affinity chromatography on a column of Sephadex G-50. The protein was crystallized in the presence of the ligand X-Man and the crystals were obtained by the vapor diffusion method in hanging drop by sparse matrix. Crystallization kits âCrystal Screen I and IIâ from Hampton Research were used to obtain protein crystals and the better condition was 33 from kit I4 M sodium formate. The crystal was diffracted to 2.61à with space group I222 with unit cell dimensions a = 61.34; b = 66.11; c = 106.69à and α = β = γ = 90Â. It was observed the presence of a monomer in asymmetric unit containing 42.04% of solvent in the crystal. The molecular replacement was made using Dioclea rostrata structure (PDB: 2ZBJ). The final refinement obtained Rfactorof 0.23 and Rfree of 0.27 in absence of any amino acid in regions not allowable in Ramachandran plot. The structure of X-Man was co-crystallized and observed perfectly placed in the carbohydrate recognition domain. Regarding the balance of the dimmer-tetramer associations of plant lectins, the lectin from Dioclea violacea has the amino acid HIS 131 and the position of HIS 51 is similar to the same amino acid in Dioclea grandiflora lectin, characterizing these lectins as a tetramer even at low pH. It was made comparisons between the differences in biological activities of other lectins from Diocleainae and the distances of the residues from carbohydrate site. It was observed that differences in biological activities in vasorelaxant effects on vascular smooth muscle are probably related to the distances between the residues that compose the carbohydrate domain.
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Characterization Of Carbohydrate Specificity And Primary Structure Of the B-cell Mitogen, Artocarpin, From Artocarpus Integrifolia SeedsGeetha Rani, P 05 1900 (has links) (PDF)
No description available.
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Syk Kinase Is Required for Collaborative Cytokine Production Induced Through Dectin-1 and Toll-Like ReceptorsDennehy, Kevin, Ferwerda, Gerben, Faro-Trindade, Inês, Pyz, Elwira, Willment, Janet A., Taylor, Philip R., Kerrigan, Ann, Tsoni, S. Vicky, Gordon, Siamon, Meyer-Wentrup, Friederike, Adema, Gosse J., Kullberg, Bart Jan, Schweighoffer, Edina, Tybulewicz, Victor, Mora-Montes, Hector M., Gow, Neil A.R., Williams, David L., Netea, Mihia G., Brown, Gordon D. 01 February 2008 (has links)
Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal β-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF,MIP-1α andMIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kB (IkB), enhancing NFkB nuclear translocation. These findings establish the first example of Syk-and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.
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Investigating the role of the Dendritic Cell Immunoactivating Receptor in the Immune Response during Pneumocystis murinaMthembu, Nontobeko F 25 September 2020 (has links)
Pneumocystis jirovecii causes fungal pneumonia in immunocompromised patients and can be fatal if left untreated. The global mortality rate is estimated to be over 200 000 in AIDS patients. In non-AIDS patients there is an estimated mortality rate of 50 000 cases. This rate is increasing in developed countries, attributed to an increase in disorders which require immunotherapy. These include hematologic malignancies, organ transplant, inflammatory disorders and pre-existing lung disease. Immediate immunity is initiated by receptors that recognize pathogen associated molecular patterns on the surface of pathogenic fungi. Specifically, C-type lectin receptors (CLRs) have been shown to be the principal initiators of innate immune response during fungal infection. Limited studies have focused on the role of CLRs in Pneumocystis infection. Dectin1and Mincle have been shown to recognise Pneumocystis surface antigens with Dectin-1 recognizing β-glucans on the Pneumocystis cell wall leading to an effective immune response. However, the role of a newly described CLR, the Dendritic Cell Immunoactivating Receptor (DCAR) remains undefined. For this reason, we investigated the potential role of this receptor in a mouse model of Pneumocystis murina infection. Wild type and DCAR-deficient C57BL/6 mice were infected with P. murina organisms via intratracheal instillation. Fungal burden was measured in the lung using quantitative Polymerase Chain Reaction. DCAR-deficient mice had a significantly reduced burden compared to wild type mice at Day 7 and 14 post-infection. To identify the immune components involved in pathogen clearance in these mice we measured cellular recruitment and cytokine production at both early (48 hours) and late (7, 14 and 21 days) time points. Flow cytometry analysis showed an increase in alveolar macrophage, dendritic cells, inflammatory monocytes, eosinophils and T cell recruitment to the lung. While ELISA showed increased levels of IL-1β and IFN-γ at 48 hours, and later on in infection IL-1β and IL-12p40 levels were also elevated. Histology analysis determined the localization of the recruited cells, and v interestingly showed an increase in mucus production at day 21 in DCARdeficient mice. In conclusion, we have identified DCAR deficiency as a potential driver of protective immunity in mice during P. murina infection. This may be associated with increased levels of IL-1β in DCAR-deficient mice. Furthermore, DCAR may also be important in adaptive inflammatory response regulation, as DCAR-deficient mice have increased cellular recruitment and mucus production later in infection. The mechanism by which the deletion of this receptor affords these mice the ability to efficiently clear P. murina remains to be determined.
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Expression Of Gal/galnac Lectin Of Entamoeba Histolytica In Transgenic Chloroplasts To Develop A Vaccine For AmebiasisChebolu, Seethamahalakshmi 01 January 2005 (has links)
Amebiasis, also defined as invasive intestinal and extra intestinal amebiasis, is caused by Entameoba histolytica, an invasive protozoan parasite. World Health Organization (WHO) has reported that approximately 50 million people are infected each year causing an estimated 40 to 100 thousand deaths annually. Entameoba histolytica ranks only second to malaria as a protozoan cause of death. Amebiasis occurs world wide but people living in Central and South America, Africa and Asia are the majority to suffer from morbidity and mortality. The enteric parasite has no zoonotic reservoirs and insect vectors for its transmission and infects humans and non-human primates. Therefore, anti-amebic vaccine could completely eradicate the disease. Entamoeba histolytica invades tissue and causes the disease in series of events. The disease is caused when the cyst form of the parasite is ingested with contaminated food or water. After excysting in the small intestine to form the trophozoite, the parasite adheres to the colonic mucus and epithelial cells through interaction of Gal/GalNAc lectin, an amebic surface adhesin with the host glycoconjugates. The parasite then secrets the proteolytic enzymes that disrupt the intestinal mucus and epithelial barrier facilitating tissue penetration. The trophozoite then kills the host epithelial and immune cells. Also, it resists the host's immune response causing the prolonged infection called the invasive amebiasis and causes colon or liver abscess. The symptoms include gradual onset of abdominal pain, diarrhea and bloody stools. Also, it can form cysts that are excreted with stools to start new cycle. The parasite recognition of the host glycoconjugates plays an important role in the pathogenesis. Therefore, the Gal/GalNAc lectin could be a possible vaccine candidate. The Gal/GalNAc lectin is composed of a 260-kDa heterodimer of disulfide-linked heavy (170 kDa) and light (35 kDa) subunits, which is non-covalently associated with an intermediate sub-unit of 150 kDa. The only recognized Carbohydrate recognition domain (CRD) was found in the heavy sub-unit. The CRD of the lectin is the potential target for colonization blocking vaccines and drugs. Preliminary studies have shown that the recombinant fragments of cysteine-rich region of LecA (lectin) containing the CRD (carbohydrate recognition domain) of the GalNAc lectin conferred protection against amebiasis. Therefore, production of LecA in plants using chloroplast genetic engineering would result in low cost vaccine because of high expression levels of vaccine antigens, and elimination of the cold-chain (low temperature, storage & transportation), hospitals and health professionals for their delivery. The LecA protein was expressed in transgenic chloroplasts of Nicotiana tabacum var. Petit havana by transforming the chloroplast genome using the LecA gene (1755 bp) by homologous recombination. The pLD-CtV has trnI and trnA genes that are used as flanking sequences for homologous recombination and the constitutive 16s rRNA promoter to regulate transcription. The aadA gene conferring spectinomycin resistance has been used for selection and gene10 regulatory sequence from T7 bacteriophage to enhance translation. The chloroplast integration of LecA was confirmed by PCR and Southern blot analysis. The expression of LecA protein in transgenic chloroplasts was analyzed by immunoblot analysis using anti-LecA antibodies. Maximum expression levels of LecA up to 6.3 % of the total soluble protein were observed in the old leaves. The evaluation of the immune response in animal model is underway. This is the first report of expression of LecA in a plant system.
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Analysis of Porcine Kupffer Cell Recognition of Human ErythrocytesBurlak, Christopher, II January 2003 (has links)
No description available.
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Genetic and Immunological Analyses of a Brucella abortus Protein Exhibiting Lectin-like PropertiesVemulapalli, Tracy H. 16 February 2000 (has links)
Brucella abortus is a facultative, intracellular zoonotic pathogen, which can cause undulant fever in humans and abortion in cattle. Despite all of the progress in brucellosis research, there are still many unanswered questions regarding the molecular mechanisms involved in the pathogenesis of Brucella infections. To better understand the Brucella antigens involved in virulence and/or immunity, genetic and immunologic characterization of a 16 kDa protein of B. abortus was performed. Using PCR methods, the gene encoding the 16 kDa protein was cloned and sequenced. PCR and Southern blot analysis revealed that the gene is conserved among the 6 nomen species of Brucella. Overexpression of this protein in B. abortus vaccine strain RB51 was achieved using Brucella groE and sodC promoters as well as its own promoter. Protection and clearance studies were performed in mice to determine the role of this protein in Brucella immunity and pathogenesis. Inoculation with either strain RB51 overexpressing the 16 kDa protein or a DNA vaccine encoding the 16 kDa protein gene failed to provide significant protection. No difference was noted between the splenic clearance of B. abortus strain 2308 and its recombinant overexpressing the 16 kDa protein. A mutant of strain 2308 (2308D16) was created by disrupting the 16 kDa protein's gene with a chloramphenicol resistance cassette. Western blot analysis indicated that the O antigen profile of strain 2308D16 differed from that of strain 2308. Mice cleared strain 2308D16 faster than strain 2308 indicating the potential attenuation of the disruption mutant. Purified 16 kDa protein was obtained by overexpressing it in E. coli via the pRSET expression system. Western blotting results initially identified this protein as an immunoglobulin-binding protein. Hemagglutination assay revealed that the 16 kDa protein exhibits lectin-like properties. Preliminary studies using hemagglutination inhibition identified mannose as a possible sugar to which the 16 kDa protein can interact. The lectin-like properties exhibited by the 16 kDa protein appears to influence smooth lipopolysaccharide production, and thereby may be involved in virulence. / Master of Science
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Propriedades físico-químicas da lectina KM+ monitoradas por dicroismo circular (CD) e fluorescência. Estimativa do conteúdo de estrutura secundaria por CD / Physico-chemical properties of lectin KM+ monitored by circular dichroism (CD) and fluorescence. Estimative of secondary structure content by CDLucca, Rosemeire Aparecida da Silva de 01 July 1994 (has links)
Uma nova lectina extraída da semente de Artocarpus integrifólia, denominada KM+ foi recentemente descrita. KM+ e haptotática para neutrófilos, promove a aglutinação de hemácias dos grupos A, B, 0, estimula a proliferação de linfócitos do baço de camundongos e liga-se em α D-manose, α metil manosidio e α D-glicose. Esta lectina é composta por quatro monômeros, com peso molecular de 13.150 daltons cada, unidos por interações não covalentes. KM+ contem 1,8% de carboidratos e apresentou quatro isoformas com pontos isoelétricos entre 4,2 e 5,2. Este trabalho teve como objetivos estudar modificações estruturais de KM+ em função de parâmetros como temperatura, força iônica, pH, agentes desnaturantes, ligação com D-manose, monitoradas por dicroísmo circular (CD) e fluorescência. CD também foi utilizado para estimar o conteúdo de estrutura secundaria de KM+, utilizando-se dois programas descritos na literatura: SSE (Secondary Structure Estimation), que utiliza o método dos mínimos quadrados para a estimativa da estrutura secundaria e obtenção dos espectros básicos, baseados nos dados cristalográficos de proteínas de .estrutura resolvida; CCA (Convex Constraint Analisys) que utiliza o algoritmo simplex e a partir dos espectros de CD das proteínas de referencia calcula os espectros das componentes básicas. Para a estimativa das frações de estrutura secundária o segundo método utiliza o programa Lincomb. Os espectros de CD foram registrados no intervalo de 185 a 260 nm. O conteúdo em estrutura secundária, estimado pelo programa SSE foi: 0% de α-hélice, 41% de folha β, 27% de volta β e 32,3 de estrutura desordenada; pelo programa CCA foi: 1% de α-hélice, 35% de folha β anti-paralela, 21% de volta β e/ou folha β paralela, 15% de contribuições de aromáticos e/ou ligações dissulfeto, 28% de estrutura desordenada. Os desvios médios quadráticos para os programas SSE e CCA foram 12% e 1%, respectivamente. Portanto a lectina KM+ é principalmente constituída por estruturas tipo folha β e tipo desordenada. A curva calculada pelo programa CCA foi mais bem estimada, pois tem o desvio médio quadrático 12 vezes menor que o do programa SSE. Este resultado, provavelmente ocorre devido aos seguintes fatores: (i) no programa CCA, o espectro da proteína a ser analisada e alinhado com os espectros das proteínas de referência, influenciando no calculo dos espectros básicos; (ii) maior número de proteínas com estrutura β no grupo de referência do programa CCA. A estabilidade de KM+ em função da temperatura tem comportamento diferente em tampão sódio fosfato (PBS) daquele observado em água. Em PBS, quando a amostra esta a 70°C, a forma do espectro de CD mostrou-se consistente com um espectro de proteína desnaturada. Comumente, um espectro de proteína desnaturada caracteriza-se pela perda da estrutura secundaria predominante e aumento da estrutura desordenada. Em água, também a 70°C, na região da estrutura β (216 nm) surge uma nova banda e na região da estrutura desordenada (195 nm) aparece uma banda com valores positivos mimetizando um espectro da estrutura α-hélice. Esta diferença de comportamento pode ser devida à força iônica. A desorganização promovida na molécula de KM+ por cloreto de guanidina foi típica de desnaturação. o máximo da emissão de fluorescência, da KM+ em PBS pH 7,2, foi a 328 nm, característico de resíduos de triptofano protegidos do solvente. Este máximo mudou para 340 nm em pH 10,5. Este resultado indica mudanças no ambiente químico do triptofano neste pH. O deslocamento para a região do vermelho indica, que em pH. os resíduos de triptofano estio em maior contato com o solvente. O número de sitios ligantes de D-manose J)a molécula de KM+, foi estimado pela supressão da fluorescência promovida pelo D-manose. Esta estimativa foi baseada na suposição de que todos os sítios ligantes de D-manose estivessem próximos aos resíduos de triptofano. A relação encontrada foi de 2 moles de D-manose/mol de KM+ / Recently a new lectin, KM+, isolated from Artocarpus integrifolia seeds was described. KM+ induces neutrophil migration, agglutination of human red blood cells, proliferation of mouse spleen cells and binding with monosacharides D-mannose, D-glicose and α-metil mannoside. This glycoprotein is composed of four monomers, assembled by non covalent bonds, has 500 aminoacids residues/mol, with a Molecular Weight of 52,000 Daltons and 1.8% of carbohydrates [27]. In this work structural changes of KM+ was studied as a function of temperature, pH, chemical denaturing agents as well as the binding with D-mannose. These changes were monitored by circular dichroism (CD) and fluorimetry. Circular Dichroism (CD) spectroscopy was used for the analysis of the secondary structure of KM+ in solution due do its capacity to indicate the presence and to estimate the proportion of α-helix, β-sheet, β-turn and unordered conformations. This measurent can be regarded as a function of the relative orientation of the chromophores responsible for their chiroptical activity. CD spectroscopy is also one of the methods of choice for monitorization of conformational changes in proteins as a function of solvents, pH, temperature, ionic strength and specific or non specific binding. Two programs which are in use for estimation of secondary structure: SSE, using the linear least squares method and CCA, using the simplex method, were evaluated in the present work. SSE uses a set of proteins with known X-ray data as the basis for evaluation while CCA uses only pure proteins experimental CD spectra. Fluorescence spectroscopy is very useful to monitore of protein conformational changes in solution due to the presence of intrinsic fluorophores. Fluorescence Measurements were performed at 25°C. Samples were excited at 280 nm and the emission was monitored in the range 290-450 nm. The maximum emission as a function of pH was at pH 7.0. The wavelength for maximum emission changed from 328 nm at pH 7.0 to 340 nm at pH 10.5. CD spectra were recorded over the range of 185 up to 260 nm. The Secondary structure content estimated by SSE program was: 0% α-helix, 41% β-sheet, 26% β-turn and 32% random with RMS of 12% and CCA program was: 1% α-helix, 35% antiparallel β-sheet, 21% β-turn and/or parallel B-sheet, 28% random, 15% aromatics contributions and dissulfide linkages with RMS of 1%. The fractions of secondary structure obtained when using CCA program were more consistent than those of SSE program. The simulation by CCA program was better probably due to its desconvolution of the spectral contribution of the common secondary structures using experimental CD curves of proteins. The stability of KM+, in PBS, as a function of temperature changes above 55°C but only at 70°C the shape of the CD spectrum is consistent with the loss of the native ordered secondary structure that should accompany protein unfolding. CD spectra of KM+ in water showed conformational changes as a function of temperature was not consistent with denaturated proteins. The unfolding of KM+ by GdnCl and SDS resulted in CD spectroscopic changes: consistent with the increased random structure and disappearance of beta sheet. Using the two denaturing agents together GdnCl and temperature, the denaturation was observed at lower decreased both GdnCl concentration and at lower temperature. The estimation of the number of binding sites for D-mannose was obtained through the fluorescence intensity decrease due to a quenching effect of D-mannose and showed that the stoichiometry of binding was 2 moles of D-mannoseimol of lectin
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