Dissertations / Theses: 'Lectin'

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Lucca, Rosemeire Aparecida da Silva de. "Propriedades físico-químicas da lectina KM+ monitoradas por dicroismo circular (CD) e fluorescência. Estimativa do conteúdo de estrutura secundaria por CD." Universidade de São Paulo, 1994. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-02042014-100315/.

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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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Correia, Jorge Luis Almeida. "PurificaÃÃo, caracterizaÃÃo parcial e potencialidade biotecnolÃgica de trÃs lectinas de sementes de espÃcies de Leguminosae da subtribo Diocleinae." Universidade Federal do CearÃ, 2015. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=17238.

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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico
Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
Leguminosae is recognized by the large amount of isolated and characterized lectins, especially seeds. In this group stand out proteins extracted from species belonging to subtribe Diocleinae, the number of studies in different areas of knowledge. Lectins can be defined as proteins or glycoproteins which are not originated from a body's immune response and have the ability to recognize and bind reversibly to mono or oligosaccharides particular without, however, altering their chemical structures. Different works in our group are structurally haracterizing these proteins as well as elucidating some possible biotechnological applications for these molecules. In this sense, the objective of this study was to isolate and characterize lectins of different species of Leguminosae of Diocleinae subtribe and test them for toxicity to Artemia sp. naupilos, The effect on the smooth muscle of blood vessels and the detention lectin matrix agarose previously activated with cyanogenic bromide (CNBr). Were isolated and characterized the lectin Dioclea sclerocarpa, Dioclea lasiocarpa and Dioclea lasiophylla. Were also made circular dichroism studies on lectin Dioclea sclerocarpa and Dioclea lasiocarpa. The lectin Dioclea lasiophylla was tested against Artemia sp. order to assess their toxicity and was also immobilized on agarose matrix. We evaluated the effect of lectin Dioclea lasiocarpa in the smooth muscle of blood vessels. The knowledge gained from the three scientific articles published in this thesis is a major breakthrough in this promising field of study that has been continuously growing for biotechnological applications.
A famÃlia Leguminosae Ã reconhecida pela grande quantidade de lectinas isoladas e caracterizadas, especialmente de sementes. Neste grupo se destacam as proteÃnas extraÃdas de espÃcies pertencentes a subtribo Diocleinae, pela quantidade de estudos em diferentes Ãreas do conhecimento. Lectinas podem ser definidas como proteÃnas ou glicoproteÃnas que nÃo sÃo originadas a partir de uma resposta imunolÃgica do organismo e possuem a capacidade de reconhecer e se ligar reversivelmente a mono ou oligossacarÃdeos especÃficos sem, no entanto, alterar suas estruturas quÃmicas. Diferentes trabalhos no nosso grupo vÃm caracterizando estruturalmente essas proteÃnas, bem como elucidando algumas possÃveis aplicaÃÃes biotecnolÃgicas para essas molÃculas. Neste sentido, o objetivo deste trabalho foi isolar e caracterizar lectinas de diferentes espÃcies de Leguminosae da subtribo Diocleinae e testa-las com relaÃÃo Ã toxicidade para naupilos de Artemia sp., o efeito na musculatura lisa de vasos sanguÃneos e a imobilizaÃÃo de lectina em matriz de agarose previamente ativada com brometo cianogÃnico (CNBr). Foram isoladas e caracterizadas as lectinas de Dioclea sclerocarpa, Dioclea lasiocarpa e Dioclea lasiophylla. TambÃm foram feitos estudos de dicroÃsmo circular na lectina de Dioclea sclerocarpa e Dioclea lasiocarpa. A lectina de Dioclea lasiophylla foi testada contra Artemia sp.de forma a avaliar sua toxicidade e tambÃm foi imobilizada em matriz de agarose. Foi avaliado o efeito da lectina de Dioclea lasiocarpa na musculatura lisa de vasos sanguineos. O conhecimento acumulado a partir dos trÃs artigos cientÃficos publicados nesta tese constitui um grande avanÃo no neste campo promissor de estudo que vem em contÃnuo crescimento para aplicaÃÃo biotecnolÃgica.

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Dalvina, Correia da Silva Michele. "Aplicações biotecnológicas das lectinas ClaveLL (Cladonia verticillaris Lichen Lectin) E BmoLL (Bauhinia monandra Leaf Lectin)." Universidade Federal de Pernambuco, 2008. https://repositorio.ufpe.br/handle/123456789/1424.

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Lectinas são proteínas presentes em diferentes organismos, dos quais são isoladas; possuem origem não imune e habilidade para se ligarem a carboidratos ou glicoconjugados, através de sítios específicos; forças de interação eletrostática e a presença de íons metálicos podem influenciar o processo de ligação. Neste trabalho, foram avaliadas a lectina de folhas de Bauhinia monandra, BmoLL, e a lectina do líquen Cladonia verticillaris, ClaveLL. A investigação e conseqüente emprego biotecnológico de lectinas como proteínas com ação antimicrobiana e inseticida, bem como sua utilidade em histoquímica no estudo e diagnóstico de patologias, estimularam a realização desta Tese. As lectinas foram avaliadas quanto a potencial ação contra bactérias e espécies fúngicas do gênero Fusarium, como proteínas inseticidas para a espécie de cupins Nasutitermes corniger, e também como ferramentas histoquímicas para a investigação histopatológica dos hipocampos de pacientes com doença de Alzheimer. BmoLL e ClaveLL são ativas contra diferentes espécies de Fusarium (F. solani, F. lateritium, F. fusarioides, F. moniliforme e F. verticiloides com BmoLL; e Fusarium verticiloides, F. descemcellulare, F. fusarioides, F. oxysporum e F. moniliforme com ClaveLL) e são hábeis em aglutinar, como também inibir a proliferação de bactérias Gram-positivas e Gram-negativas. BmoLL e ClaveLL possuem ação não repelente e inseticida contra N. corniger. Em histoquímica de hipocampo, BmoLL (galactose-específica) reconhece o citoplasma neuronal e marca intensamente corpos amiláceos que ocorrem em abundância; ClaveLL (com elevada afinidade por N-acetil-D-glicosamina e glicoproteínas) reconhece intensamente células neuronais e corpos amiláceos e, mais importante, marca neurônios lesionados com emaranhados neurofibrilares ou com degeneração grânulovacuolar, degenerações que são típicas da doença de Alzheimer

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Correia, Jorge Luis Almeida. "Purificação, caracterização parcial e potencialidade biotecnológica de três lectinas de sementes de espécies de Leguminosae da subtribo Diocleinae." reponame:Repositório Institucional da UFC, 2015. http://www.repositorio.ufc.br/handle/riufc/18837.

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CORREIA, Jorge Luis Almeida. Purificação, caracterização parcial e potencialidade biotecnológica de três lectinas de sementes de espécies de Leguminosae da subtribo Diocleinae. 2015. 101 f. Tese (Doutorado em biotecnologia)- Universidade Federal do Ceará, Fortaleza-CE, 2015.
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Leguminosae is recognized by the large amount of isolated and characterized lectins, especially seeds. In this group stand out proteins extracted from species belonging to subtribe Diocleinae, the number of studies in different areas of knowledge. Lectins can be defined as proteins or glycoproteins which are not originated from a body's immune response and have the ability to recognize and bind reversibly to mono or oligosaccharides particular without, however, altering their chemical structures. Different works in our group are structurally haracterizing these proteins as well as elucidating some possible biotechnological applications for these molecules. In this sense, the objective of this study was to isolate and characterize lectins of different species of Leguminosae of Diocleinae subtribe and test them for toxicity to Artemia sp. naupilos, The effect on the smooth muscle of blood vessels and the detention lectin matrix agarose previously activated with cyanogenic bromide (CNBr). Were isolated and characterized the lectin Dioclea sclerocarpa, Dioclea lasiocarpa and Dioclea lasiophylla. Were also made circular dichroism studies on lectin Dioclea sclerocarpa and Dioclea lasiocarpa. The lectin Dioclea lasiophylla was tested against Artemia sp. order to assess their toxicity and was also immobilized on agarose matrix. We evaluated the effect of lectin Dioclea lasiocarpa in the smooth muscle of blood vessels. The knowledge gained from the three scientific articles published in this thesis is a major breakthrough in this promising field of study that has been continuously growing for biotechnological applications.
A família Leguminosae é reconhecida pela grande quantidade de lectinas isoladas e caracterizadas, especialmente de sementes. Neste grupo se destacam as proteínas extraídas de espécies pertencentes a subtribo Diocleinae, pela quantidade de estudos em diferentes áreas do conhecimento. Lectinas podem ser definidas como proteínas ou glicoproteínas que não são originadas a partir de uma resposta imunológica do organismo e possuem a capacidade de reconhecer e se ligar reversivelmente a mono ou oligossacarídeos específicos sem, no entanto, alterar suas estruturas químicas. Diferentes trabalhos no nosso grupo vêm caracterizando estruturalmente essas proteínas, bem como elucidando algumas possíveis aplicações biotecnológicas para essas moléculas. Neste sentido, o objetivo deste trabalho foi isolar e caracterizar lectinas de diferentes espécies de Leguminosae da subtribo Diocleinae e testa-las com relação à toxicidade para naupilos de Artemia sp., o efeito na musculatura lisa de vasos sanguíneos e a imobilização de lectina em matriz de agarose previamente ativada com brometo cianogênico (CNBr). Foram isoladas e caracterizadas as lectinas de Dioclea sclerocarpa, Dioclea lasiocarpa e Dioclea lasiophylla. Também foram feitos estudos de dicroísmo circular na lectina de Dioclea sclerocarpa e Dioclea lasiocarpa. A lectina de Dioclea lasiophylla foi testada contra Artemia sp.de forma a avaliar sua toxicidade e também foi imobilizada em matriz de agarose. Foi avaliado o efeito da lectina de Dioclea lasiocarpa na musculatura lisa de vasos sanguineos. O conhecimento acumulado a partir dos três artigos científicos publicados nesta tese constitui um grande avanço no neste campo promissor de estudo que vem em contínuo crescimento para aplicação biotecnológica.

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Colin, Sylvie. "Étude biochimique et spécifique d'une lectine extracellulaire impliquée dans la floculation de la levure kluyveromyces bulgaricus." Nancy 1, 1993. http://www.theses.fr/1993NAN10044.

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K. Bulgaricus flocule en milieu enrichi en calcium. Ce phénomène est lie à l'excrétion d'une lectine spécifique du galactose (kb-ml). Le fait que le trifluoroperazine inhibe la floculation alors que l'ophioboline A n'a pas d'effet suggère qu'une protéine kinase indépendante de la calmoduline pourrait être impliquée dans le mécanisme de la floculation. Kb-ml présente deux formes moléculaires de 250 et 65 kda respectivement kb-mli est la forme tetramérique de kb-MLII. Les deux formes kb-MLI et kb-MLII agglutinent les hématies mais la première a une activité spécifique 6 fois supérieure à la seconde. L'association de kb-MLII pour former kb-MLI dépend de la concentration en protéine et seuls, les agents dénaturants (sds, urée) peuvent l'inhiber. La proline, la glycine et l'alanine représentent 60% de la partie peptidique des deux formes. La partie osidique (13%) est constituée de chaines n-oligomannosidiques. Les deux formes montrent la même microhétérogénéité avec un produit majeur de phi 4,5 et deux mineures de phi 3,9 et 4,9. Kb-MLII a deux sites fixateurs des sucres avec un ka de 1,12. 10#6 m##1 pour le 4-methylumbelliferyl-d-gal. Kb-MLI et kb-MLII reconnaissent les glycannes de type n-acetyllactosamine et la structure glcnac b1-2 man est la mieux reconnue. La déphosphorylation des mannoprotéines de la paroi de la levure kb stimule l'interaction de ces molécules avec kb-MLI et kb-MLII. Ces résultats suggèrent que l'antigène somatique (glcnaca1-2 man) de la levure est un site de reconnaissances pour cette lectine et que la phosphorylation de la surface cellulaire agit comme un modulateur de la floculation de k. Bulgaricus

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Andersson, Pontus. "Comparison of Lectins and their suitability in Lectin Affinity Chromatography for isolation of Glycoproteins." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-417024.

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Virtually all extracellular proteins in humans are glycoproteins and likewise are many biopharmaceuticals. The glycosylation is directly correlated to biological function and stability of these proteins. The ability to isolate glycoproteins is thus of great importance in many applications. The most common isolation method for glycoproteins is affinity chromatography using lectins, a ubiquitous and versatile group of carbohydrate-binding proteins. The lectin Concanavalin A (ConA) has long been used for this purpose but suffers from undesired leakage into the eluate, causing an inquiry of alternative chromatography ligands or optimization of the ConA resin.In this study, a total of 20 different lectins, including ConA, were evaluated and compared in terms of suitability as ligands in affinity chromatography for glycoprotein isolation. The lectins’ binding to glycoproteins were studied, mainly through microtiter plate binding assays using a monoclonal IgG1 antibody and Conalbumin (Ovotransferrin). Further, sugar-specificities and potential eluting sugars for the lectins were examined through inhibition with eight different carbohydrates. Additionally, the glycoprotein binding and leakage of ConA columns were examined, and a potential leakagereducing treatment of ConA resin evaluated.ConA was found to be superior in binding to the investigated glycoproteins but exhibited a limited binding when immobilized to an agarose resin. This discrepancy is likely a consequence of structurally hidden glycans on the used glycoproteins and requirements of long residence time when used in a chromatographic setting. Binding competition with several sugars were investigated with a similar microtiter plate binding assay. This method displayed potential to predict the behaviour of sugars and their suitability as eluting agents in a chromatography column. The best eluting sugar for ConA was showed to be methylmannoside, ideally in combination with methylglucoside. Lastly, evaluation of ConA columns with a crosslinking glutaraldehyde-treatment showed that the ConA ligand leakage may be significantly reduced, although further studies and optimizations are needed.This study thus presents a repertoire of lectins and their differences in terms of glycoprotein-binding and sugar-specificity, as well as evaluations of ConA columns’ efficiency and potential leakage-prevention.

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CrisÃstomo, ClÃbia Vieira. "PolissacarÃdeo endospÃrmico de Bauhinia pentandra: caracterizaÃÃo e estudo de interaÃÃo com lectinas." Universidade Federal do CearÃ, 2008. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=4498.

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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
Devido Ãs suas diferentes propriedades quÃmicas tais como capacidade de formar soluÃÃes viscosas ou gÃis em meio aquoso o isolamento e caracterizaÃÃo de galactomananas tÃm sido de grande importÃncia Nesse trabalho a galactomanana do endosperma da semente de Bauhinia pentandra foi isolada e caracterizada, apresentando-se homogÃnea por GPC Este polissacarideo foi demonstrado ser uma galactomanana clÃssica formada por uma cadeia linear de manose unidas por ligaÃÃes β(1-4) com substituiÃÃes de galactose em ligaÃÃo α(1-6) com uma proporÃÃo Man:Gal de 2 5.1 e viscosidade intrÃnseca em Ãgua de 10 1 dL/g A galactomanana foi avaliada quanto Ã capacidade de interagir com lectinas galactose ligante O polissacarÃdeo foi tratado com epicloridrina e o material obtido foi utilizado para a montagem de coluna cromatogrÃfica de afinidade Extratos ricos em lectinas de Artocarpus incisa Artocarpus integrifÃlia e Bauhinia pentandra foram aplicados e fraÃÃes lectinicas purificadas foram obtidas A capacidade da galactomanana de B. pentandra em reter a lectina (LBp) da mesma semente foi comparada com a matriz de galactomanana de Adenantera pavonina Caesalpinea pulcherrima Sophora japonica e com a matriz comercial Sepharose 4B Apesar da galactomanana de B. pentandra ter apresentado a menor capacidade de retenÃÃo frente Ãs demais, ela mostrou-se semelhante Ã matriz comercial sendo viÃvel a sua utilizaÃÃo
Due the chemicals properties diferences, such as the ability to make viscous solution or aqueous gels, the study of the galactomanans has been too important. In this study, the endospermic galactomannans from seeds of Bauhinia pentandra was isolated and partially characterized. This polysaccharide is a classical galalactomannan constituted by a linear chain of mannose linked by β(1-4) linkages with galactose substituintions linked by α(1-6) linkages, resulting in a Man:Gal ratio of 2.5:1, and water intrinsic viscosity equal to 10.1 dL/g. Galactomannan was evaluated in ability to interact with galactose-binding lectins. The polysaccharide was treated with epichlorohidrine and the material obtained was utilized to make the affinity chromatography matrix. Lectin-rich extracts from Artocarpus incisa, Artocarpus integrifolia and Bauhinia pentandra were applied and lectin fractions were obtained, thus, the affinity matrix showed to be efficient to isolate them. The retention capacity of the galactomannan from B. pentandra was compared with galactomannan matrix from Adenantera pavonina, Caesalpinea pulcherrima, Sophora japonica and commercial matrix of Sepharose 4B in regards to the isolation of the lectin from B. pentandra (LBp). Although the galactomannan matrix had been showed the smallest retention capacity in comparision with the others, it is equivalent to the commercial matrix, enabling your utilization

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Santori, Fabio. "Lectin affinity chromatography of monosaccharides." Thesis, University of Bath, 2002. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.760807.

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Nascimento, Antônia Sâmia Fernandes do. "Lectines recombinantes d'algues rouges marines Hypnea musciformis (Wulfen) J.V. Lamouroux et Bryothamnion triquetrum (S.G. Gmelin) M. Howe : production hétérologue et caractérisation biochimique." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV052/document.

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Les gènes synthétiques des lectines des algues marines rouges Hypnea musciformis (HML) et Bryothamnion triquetrum (BTL) ont été clonés dans différents vecteurs et transformés dans plusieurs souches bactériennes d’expression. Les lectines recombinantes ont été obtenues dans la fraction soluble de cultures bactérienne dans les souches d’Escherichia coli Rosettagami 2 (DE3) pour rHML et BL21 (DE3) pour rBTL. Les tests d'hémagglutination ont montré que rHML et rBTL sont capables d’agglutiner les érythrocytes de lapin avec un bon pouvoir hémagglutinant mais seulement après traitement à la trysine et la papaine indiquant que leur ligands ne sont pas directement accessibles à la surface des hématies. Les propriétés hémagglutinantes de rHML et de rBTL confirment le repliement correct et l'état fonctionnel des protéines. La caractérisation de leur spécificité sur puces à sucres a montré une spécificité stricte de HML, BTL et rBTL pour les oligosaccharides complexes comportant la fucosylation (α1-6) du noyau, avec une préférence particulière pour les N-glycanes non bissectés, bi- et tri-antennés de chaîne courte. La présence d’acide sialique à l'extrémité non réductrice du glycane favorise la reconnaissance de ces glycanes. C'est la première caractérisation des lectines d’algues rouges par puce à sucres. Des expériences de STD-RMN menées sur BTL, ont montré son interaction avec un octasaccharide au niveau de noyau fucosylé (α1-6). L'activité toxique des lectines sauvages et recombinantes a été évaluée contre Artemia sp. et contre des cellules d'adénocarcinome du poumon (A549). Dans les essais de cytoxicité, HML, rHML, BTL et rBTL n’ont montré aucune toxicité contre Artemia sp. et seulement HML et rHML ont montré une cytotoxicité faibles contre les cellules d’adénocarcinome du poumon (A549). Le premier cristal de rBTL a été obtenu à l'aide d'un robot de cristallisation et diffractait à 15 Å
Synthetic genes from the red marine algae Hypnea musciformis (HML) and Bryothamnion triquetrum (rBTL) were cloned into different vectors and transformed into several bacterial expression strains. The recombinant lectins were obtained from the soluble fraction of bacterial cultures using Escherichia coli Rosettagami 2 (DE3) strain for rHML and E. coli BL21 (DE3) strain for rBTL. Haemagglutination tests showed that rHML and rBTL are able to agglutinate rabbit erythrocytes with strong haemagglutination activity only after treatment with papain and trysine indicating that their ligands are not directly accessible at the cell surface. The haemagglutinating properties of rHML and rBTL confirm the correct folding and functional state of the proteins. A study of the specificity of these lectins by glycan array was conducted. HML, BTL and rBTL showed a restricted specificity for complex N-glycans with core (α1-6) fucose. A more detailed analysis of the specificity of these lectins showed a preference for non bisecting N-glycans, bi- and tri-antennary branching sugars with short chains. Addition of Sialic acid at the non-reducing end of N-glycans favors their recognition by the lectins. This is the first characterization of lectins from red algae by glycan array. An interaction between BTL and a core (α1-6) fucosylated octasaccharides was also observed by STD-NMR. The toxic activity of wild and recombinant lectins were evaluated against Artemia sp. and the human lung adenocarcinoma cell line (A549). In cytotoxicity assays, HML, rHML, BTL and rBTL showed no toxicity against Artemia sp. Only HML and rHML showed a low cytotoxic activity against cell line (A549). The first crystal of rBTL was obtained in micro-scale level using a robot and diffracted at 15 Å

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10

Sicard, Delphine. "Caractérisation par microscopie à force atomique des arrangements protéine/sucre impliquant la lectine PA-IL de la bactérie pseudomonas aeruginosa." Phd thesis, Ecole Centrale de Lyon, 2012. http://tel.archives-ouvertes.fr/tel-00904559.

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La bactérie Pseudomonas aeruginosa est un pathogène opportuniste responsable de graves infections chez les personnes affaiblies immunitairement. Présentant des souches résistantes aux antibiotiques, une nouvelle approche thérapeutique est en cours de développement avec pour objectif l'inhibition des facteurs de virulence de la bactérie. Lors de son processus d'infection, le pathogène utilise les lectines pour reconnaître et se lier de manière spécifique aux glycoconjugués des cellules-hôtes en formant une interaction lectine/glycoconjugué. Plus particulièrement, la lectine PA-IL, spécifique du galactose, a été étudiée. A l'aide de glycomimétique, il semble possible de bloquer l'action de la lectine en créant une interaction lectine/glycomimétique. Pour développer cette approche, de nombreux glycocluster sont donc été élaborés et leur affinité avec la lectine PA-IL a été évaluée par plusieurs méthodes de caractérisation (SPR, HIA, ELLA, puce à sucre,...).Dans ce projet de thèse, nous avons cherché à visualiser par microscopie à force atomique (AFM) l'arrangement des complexes lectine PA-IL/glycocluster formés pour trois glycoclusters différents. Nous avons ainsi pu montrer l'influence du cœur du glycocluster et des bras-espaceurs sur l'arrangement des complexes. Suivant le glycocluster, l'arrangement prend la forme de filaments 1D,de structures dentelées avec des bras sinueux ou encore de larges structures compactes. Dans le cas des filaments, la résolution de nos images AFM nous a permis d'identifier les lectines à l'intérieur même de la structure filaire. Nous avons aussi démontré, en observant les lectines seules, l'existence d'une interaction lectine/lectine. De plus, des expériences ont été menées pour déterminer les conditions expérimentales appropriées à leur observation à l'air et en milieu liquide.

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11

Duarte, Christiane Eliza Motta. "Brassica oleracea lectin: Isolation, characterization, and functional assessment of the first lectin with MATH domains." Universidade Federal de Viçosa, 2016. http://www.locus.ufv.br/handle/123456789/9366.

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Lectinas estão envolvidas em uma ampla variedade de processos biológicos, inclusive atuando como agentes imunomoduladores capazes de ativar a imunidade inata. Nós purificamos e caracterizamos uma nova lectina de couve-flor (Brassica oleracea ssp. botrytis – BOL) através de três etapas sequenciais de cromatografia, cuja pureza foi confirmada por SDS-PAGE. Além disso, avaliamos o papel dessa lectina na imunidade inata por meio de um ensaio de fagocitose, produção de H 2 O 2 e NO. BOL foi caracterizada como uma proteína não glicosilada com uma massa molecular de ~ 34 kDa em SDS-PAGE. Para otimizar o processo de obtenção da lectina e possibilitar um estudo mais aprofundado de sua estrutura e função, realizou-se a clonagem molecular e expressão heteróloga de BOL. Para isso utilizamos RNA total extraído de plântulas de couve-flor e isolamos a sequência de 1053 pb do cDNA codificante. Ferramentas de bioinformática foram utilizadas para determinar a sequência promotora de 1000 pb de Bol, a qual revelou vários elementos cis-regulatórios conhecidos por estarem envolvidos em várias condições de estresse na planta. A análise comparativa tecido-específica da expressão de Bol demonstrou níveis mais elevados do transcrito nas folhas, em comparação aos tecidos do caule e da raiz. A análise da sequência de aminoácidos e o alinhamento com proteínas homólogas deduzidas nos permitiu determinar que a proteína madura é constituída por 301 aminoácidos. A estrutura tridimensional predita confirmou que esta lectina apresenta uma estrutura em forma de cúpula com dois domínios MATH. Este é o primeiro relato do isolamento, clonagem e expressão bacteriana de uma lectina com domínios MATH e pode ser de interesse significativo para compreender o papel regulador desta proteína como agente imunoestimulante bem como para à fisiologia da própria planta.
Lectins are involved in a wide range of biological mechanisms, including act as immunomodulatory agent able to activate the innate immunity. We purified and characterized a new lectin from cauliflower (Brassica oleracea ssp. botrytis - BOL) by three sequential chromatographic steps and confirmed the purity by SDS-PAGE. Additionally, we evaluated the role of the lectin in innate immunity by a phagocytosis assay, production of H 2 O 2 and NO. BOL was characterized as a non-glycosylated protein with a molecular mass of ~34 kDa in SDS-PAGE. To optimize the process of the lectin obtaining and allow further study of their structure and function, the molecular cloning and heterologous expression of BOL were carried out. Using total RNA extracted from cauliflower seedlings a Bol coding cDNA sequence of 1053 bp was isolated. Bioinformatics tools were used to determine a promoter sequence of 1000 bp of Bol which revealed several key cis-regulatory elements known to be involved in various plant stresses. Comparative expression analysis of tissue specific Bol demonstrated the highest transcript levels in leaves, as compared to stem and root tissues. Analysis of amino acid sequence and alignment with deduced homologous proteins allowed us to determine that the mature protein comprises 301 amino acids. Predicted three-dimensional structure confirmed that this lectin had an overall dome-like structure with two MATH-domains. This is the first report of isolation, cloning and bacterial expression of a lectin with MATH-domains and may be of significant interest to understand the regulatory role of this protein as immunostimulatory agent as well as to the physiology of the plant itself.

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12

Odom-Crespo, Eric William. "F-type lectins biochemical, genetic, and topological characterization of a novel lectin family in lower vertebrates /." College Park, Md. : University of Maryland, 2004. http://hdl.handle.net/1903/1437.

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Thesis (Ph. D.) -- University of Maryland, College Park, 2004.
Thesis research directed by: Marine-Estuarine-Environmental Sciences. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

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13

Presanis, Julia. "The lectin pathway of complement activation." Thesis, University of Oxford, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413519.

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14

Krarup, Anders. "The lectin pathway of complement activation." Thesis, University of Oxford, 2007. http://ora.ox.ac.uk/objects/uuid:46255854-bfba-4d57-9185-3e6ed970a2db.

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The complement system is an important immune system mechanism involved in both the recognition and elimination of invading pathogens. It is activated by three different pathways: The classical pathway, which relies on binding of C1, and results in the cleavage of C4 and C2 through activation of C1r and C1s; the alternative pathway that relies on the spontaneous hydrolysis of C3 and the lectin pathway. The lectin pathway is activated by binding of Mannan-binding lectin (MBL) or the ficolins (L-ficolin, H-ficolin and M-ficolin) to microbial binding motifs, and the subsequent activation of the MBL-associated serine proteases (MASP) 1/ 2/ 3. Of these MASP2 has been identified as the enzyme responsible for the activation of complement by C4 and C2 cleavage. The work presented here will focus on four different aspects of the lectin pathway: specificity and stoichiometry of the L-ficolin protein complex, expression of H-ficolin, substrate characterization for MASP1 and investigation of the prothrombin activation potential of MASP2. L-ficolin binding specificity was investigated using glycan array technology, and it was found that L-ficolin, instead of recognizing single monosaccharides like MBL, instead binds to extended oligosaccharide structures. The binding to these was dependent not only on the presence of acetyl groups, but also on their orientation in space. It was also found that L-ficolin in serum is found as a multimeric protein complex composed of 18 polypeptide chains and associated with one MASP dimer. The expression of H-ficolin resulted in the generation of a stable mammalian cell line producing oligomerized and biologically functional H-ficolin. MASP1 substrate specificity was investigated by two different procedures. Firstly fractionated plasma was subjected to MASP1 treatment in an attempt to identify a plasma protein substrate. This did not yield any substrate candidates, since only cleavage of the protease inhibitor α-2-macroglobulin could be detected. Additionally the thrombin-like activity of MASP1 was investigated through cleavage experiments done with factor XIII and fibrinogen. These experiments showed that the factor XIII cleavage site for MASP1 and thrombin is identical. This was also found for the fibrinogen β-chain but not for the α-chain showing that MASP1 interaction with fibrinogen is distinct from that of thrombin. An earlier observation that MASP2 was capable of activating prothrombin and generating thrombin was further characterized. Here it was shown that the activation of prothrombin by MASP2 is identical to that by factor Xa, which is the enzyme undertaking this role in the coagulation system, and that the activation can result in deposition of fibrin on the surface upon which MASP2 is bound. The prothrombin activation potential of MASP2 was also utilized to develop a new MASP2 activity assay, which was shown to be capable of measuring MASP2 activity, when MASP2 is bound, via MBL (or L-ficolin) to appropriate surfaces.

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15

HRIBERNIK, NIVES. "DECOYS FOR DISRUPTING CARBOHYDRATE-LECTIN INTERACTIONS." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/871720.

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Carbohydrates are biomolecules used for presentation of biochemical signals on the cell surface. The interactions between carbohydrates and lectins are involved in numerous physiological and pathophysiological processes, therefore understanding how the information is encoded in the structures of sugars, how is it read out by carbohydrate binding proteins (lectins) and furthermore how to control or alter the flow of this information is of high importance. To approach this challenge various principles are used, taking advantage of carbohydrate chemistry, supramolecular chemistry and rational protein engineering to deliver test compounds that can be glycans, glycomimetics or lectin-based. The goal of this thesis was to develop both glycomimetics and lectin constructs able to interfere with carbohydrate-lectin interactions. In the first part (at the University of Milano), the focus was set on the synthesis of glycomimetic compounds. Considering, that the major disadvantages of utilizing carbohydrates in studies are their metabolic instability and difficult synthesis, we created a new class of enzymatically and hydrolytically stable glycomimetics with the general structure of N-linked-pseudo-thio-glycosides. To simplify their synthesis, we developed a new synthetic approach in which the formation of the thiol from glycosyl thioacetate and subsequent opening of the aziridine are combined in a one-pot reaction. We then investigated the scope of this reaction on various mono- and disaccharides. Surprisingly, thio-derivatives of β-glucose, β-galactose, β-fucose, β-lactose and β-N-acetylglucosamine underwent anomeric isomerization under the reaction conditions employed. On the other hand, tiho-derivatives of α-mannose, α-rhamnose and α-Neu5Ac were configurationally stable under the reaction conditions. Therefore, to unravel the mystery of the anomeric isomerization of sugar derivatives containing anomeric sulphur we conducted an in depth literature search on this topic and performed mechanistic studies to better understand the cause of anomerization observed during the one-pot aziridine opening reactions. β-Glucosyl thioacetate was selected as a model substrate for our studies. Through a series of experiments, we confirmed that the anomerization under the experimental conditions (Et2NH, DMF) occurs through the intermediate thiolate. Furthermore, we carefully investigated the tuning of the reaction conditions to improve the anomeric ratio to synthetically useful levels. The second part of the project (at Utrecht University) was a shift from carbohydrates towards lectins. We aimed towards making new flexible multisite lectin constructs. As a model, we chose cyanovirin-N, a lectin with broad antiviral activity, due to its ability to bind to high-mannose glycans on enveloped viruses. In the first step towards making new peptide constructs we aimed to identify the minimum sequence of this lectin still able to bind carbohydrates, to be the used for multisite presentation. We did so by shortening the sequence of cyanovirin-N to domain B that bears the high affinity carbohydrate binding site. We then compared preparation processes of this peptide, synthesis versus recombinant production from bacteria. Finally, secondary structure and glycan binding ability of the new peptide constructs were investigated.

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16

Neto, Cornevile Correia. "ProduÃÃo e purificaÃÃo parcial de IgY anti-lectina de sementes de Canavalia brasiliensis (ConBr) (Leguminosae) em galinhas poedeiras." Universidade Federal do CearÃ, 2015. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=16386.

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nÃo hÃ
Lectins are a group of proteins present in various living organisms, particularly in plants and especially in Leguminosae seeds. Among this family, a species that stands out for having large amount of lectin in the seed is the Canavalia brasiliensis. One approach for the investigation of lectins on biological systems is the detection there of by techniques involving antibody. Such techniques may include from classical immunochemical assays (immunodiffusion, immunoelectrophoresis and rocketimunoeletroforese) to ELISA experiments and advanced microscopy techniques. In this sense, antibodies become biotechnological tools invaluable since they are capable of specifically recognizing epitopes of the target molecules. Immunoglobulins (Igs) are proteins present in high concentration in the blood plasma of mammals. They are vectors of humoral immunity, with the primary function to join the foreign antigens to the individual so as to neutralize them. In the case of poultry the main immunoglobulin class (IgYs) can be found not only in blood, but also in egg yolk. This fact enables fast and efficient purification of these molecules facilitating its use as biotechnological inputs. Thus, this study aimed to produce antibodies against the lectin extracted from Canavalia brasiliensis seed (ConBr) (Fabaceae) in laying hens, implementing production methodology of new biotechnological inputs for the study of lectins. For this laying hens were immunized with 200 or 400μg of lectin Canavalia brasiliensis (ConBr) for 15 weeks at 10-day intervals. Collected eggs from vaccinated birds had their isolated and precipitated yolk in 0-30% fraction with ammonium sulfate for partial purificaÃÃoo of IgYs. Confirmation of the presence of antibodies was made by immunodiffusion assays agarose gel. The fraction showed 0-30%, from the first week after the immunization, capable of recognizing ConBr IgYs. This profile appeared in all groups over the 15-week experiment. The production of specific IgYs against the lectin from Canavalia brasiliensis (ConBr) proved feasible and remained throughout the experiment, possibly in reasonable quantities. In this sense, IgYs produced in this work provide a powerful biotechnological tool to be made available for future studies.
As lectinas constituem um grupo de proteÃnas presentes em diversos organismos vivos, sobretudo em vegetais e especialmente em sementes de Leguminosae. Dentre essa famÃlia, uma espÃcie que se destaca por possuir grande quantidade de lectina na semente Ã a Canavalia brasiliensis. Uma das possÃveis abordagens para a investigaÃÃo de lectinas em sistemas biolÃgicos Ã a detecÃÃo das mesmas atravÃs de tÃcnicas que envolvam anticorpos. Tais tÃcnicas podem incluir desde ensaios clÃssicos de imunoquÃmica (imunodifusÃo, imunoeletroforese e rocketimunoeletroforese), atÃ experimentos de ELISA e tÃcnicas avanÃadas de microscopia. Neste sentido, os anticopos tornam-se ferramentas biotecnolÃgicas de grande valia uma vez que sÃo capazes de reconhecer especificamente epÃtopos das molÃculas alvo. As imunoglobulinas (Igs) são proteínas presentes em grande concentração no plasma sanguÃneo de mamÃferos. São os vetores da imunidade humoral, tendo como função principal unir-se aos antígenos estranhos ao indivÃduo, de modo a neutralizá-los. No caso das aves a principal classe de imunoglobulinas (IgYs) pode ser encontrada nÃo somente no sangue mas tambÃm nas gemas dos ovos. Esse fato permite purificaÃÃo rÃpida e eficiente dessas molÃculas facilitando seu uso como insumos biotecnolÃgicos. Dessa forma, este trabalho teve por objetivo produzir anticorpos contra a lectina extraÃda da semente de Canavalia brasiliensis (ConBr) (Leguminosae) em galinhas poedeiras, implementando metodologia de produÃÃo de novos insumos biotecnolÃgicos para o estudo de lectinas. Para tanto galinhas poedeiras foram imunizadas com 200 ou 400μg da lectina de Canavalia brasiliensis (ConBr) por 15 semanas em intervalos de 10 dias. Ovos coletados das aves imunizadas tiveram suas gemas isoladas e precipitadas na fraÃÃo 0-30% com sulfato de amÃnio para a purificaÃÃo parcial das IgYs. A confirmaÃÃo da presenÃa dos anticorpos foi feita por ensaios de imunodifusÃo em gel de agarose. A fraÃÃo 0-30% apresentou, a partir da primeira semana apÃs a imunizaÃÃo, as IgYs capazes de reconhecer ConBr. Esse perfil se apresentou em todos os grupos ao longo das 15 semanas de experimento. A produÃÃo das IgYs especÃficas contra a lectina de Canavalia brasiliensis (ConBr) se mostrou viÃvel e permaneceu durante todo experimento, possivelmente em quantidades razoÃveis. Neste sentido, as IgYs produzidas nesse trabalho constituem uma potente ferramenta biotecnolÃgica a ser disponibilizada para estudos futuros.

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17

Peron, Gabriela. "Tabhys: um peptídeo com atividade lectínica extraído de Tabernaemontana hystrix." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17147/tde-11112015-155331/.

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Lectinas são proteínas que possuem pelo menos um domínio não catalítico que se liga reversível e especificamente a um monossacarídeo ou oligossacarídeo. A capacidade de ligação a diferentes tipos de açúcares torna essas moléculas ferramentas úteis no estudo de diversos processos celulares específicos. Embora as lectinas de plantas sejam amplamente estudadas, aquelas referentes à família Apocynaceae ainda são pouco exploradas. Resultados prévios obtidos pelo nosso grupo de pesquisa mostraram que extratos brutos de súber do caule da apocinácea Tabernaemontana hystrix Steud apresenta atividade hemaglutinante. Além de aglutinar eritrócitos do sistema ABO, a putativa aglutinina foi capaz de estimular a síntese de RNAm de IL-6 e TGF- beta em células esplênicas de camundongos. À vista disso, no presente projeto tivemos como objetivo identificar, caracterizar bioquimicamente e avaliar o possível potencial imunoestimulador da aglutinina de T. hystrix. Os extratos de T. hystrix obtidos por meio da farinha de raspas do súber apresentaram atividade hemaglutinante, o que não foi observado no extrato do caule destituído de súber e no extraído das folhas. Para comprovar que se tratava da atividade observada anteriormente, obtivemos a inibição da hemaglutinação com a glicoproteína fetuína, mas não houve inibição por monossacarídeos. Foi determinado um protocolo de isolamento da hemaglutinina com precipitação do extrato do súber com sulfato de amônio, cuja atividade foi recuperada no material precipitado na faixa de 30 a 60% de saturação, seguido de cromatografias sequenciais por (1) interação hidrofóbica (HiTrap Octyl), (2) troca catiônica (HiTrap SP), (3) fase reversa (EC Nucleosil C18) e (4) afinidade (Blue Sepharose). Nessas colunas a atividade foi recuperada do (1) material não retido e dos eluatos (2 e 4) com 1M e 0,5M de NaCl, respectivamente, e (3) 83% de acetonitrila. Esse protocolo produziu uma preparação homogênea contendo um peptídeo cuja análise eletrofóretica revelou massa molecular (MM) aproximada de 3kDa e concentração hemaglutinante mínima de 50g/mL. A fim de determinar se esse peptídeo formava estrutura quaternária (dímeros, tetrâmetros, etc.), característica da maioria das lectinas de plantas, submeteu-se a preparação a uma eletroforese em gel nativo (PAGE), não sendo observadas mudanças na MM do peptídeo e nem a presença de outras moléculas com MM maiores que pudessem estar associadas a ele, o que sugere que a aglutinina de T. hystrix (denominado aqui de Tabhys) é um peptídeo de MM aproximada de 3kDa. O fato da heveína, um dos peptídeos lectínicos com atividade antifúngica mais estudado, ter especificidade por quitina nos motivou a tentar o isolamento do peptídeo em coluna desse polissacarídeo. Observou-se atividade hemaglutinante e presença de peptídeo com MM de 3kDa no material eluído com Ácido acético a 0,1M da coluna de quitina. Curiosamente, nenhuma de nossas preparações foram capazes de inibir o crescimento do fungo Trichophyton rubrum. O peptídeo purificado foi testado quanto a sua capacidade em induzir a proliferação celular e a produção de citocinas em células esplênicas murinas. Os resultados dos ensaios de RT-PCR em tempo real e citometria de fluxo demonstraram que o a aglutinina de T. hystrix não foi capaz de estimular a proliferação de linfócitos, entretanto, induziu o aumento de mensagem para a citocina TGF-beta, cujo pico de produção ocorreu em célula estimuladas com 37ng/mL. Neste estudo, relatamos a presença de um peptídeo no extrato de T. hystrix com atividade hemaglutinante, o que é relativamente raro e novo. Devido a isso, este estudo pode proporcionar novas perspectivas e paradigmas nos estudos das lectinas a nível molecular e estrutural.
Lectins are proteins that have at least one non-catalytic domain that binds specifically and reversibly to a monosaccharide or oligosaccharide. This ability to bind to different types of sugars makes these molecules useful tools in the study of various specific cellular processes. Although the plant lectins are widely studied, those belong to Apocynaceae family are still little explored. Previous results obtained by our research group showed that bark crude extracts from Tabernaemontana hystrix Steud (Apocynaceae) had hemagglutination activity. Besides to agglutinate erythrocytes from ABO blood group system, the putative agglutinin induced the synthesis of IL-6 and TGF-beta mRNA in mouse spleen cells. Here we aim to identify, characterize biochemically and evaluate the possible immunostimulatory potential of T. hystrix agglutinin. The haemagglutination activity was obtained from crude extracts of bark flour, but not of flours of stems without bark and leaves. The activity of the bark extract was similar to that from the previous study, since the haemagglutination was inhibited by the glycoprotein fetuin, but not by monosaccharides. An isolation protocol was determined by using ammonium sulfate precipitation, with haemagglutination activity recovered in the range of 30-60% of saturation, and sequential chromatography procedures: (1) hydrophobic interaction (HiTrap Octyl), (2) cation-exchange (HiTrap SP), (3) reverse phase (EC Nucleosil) and (4) affinity (BlueSepharose) chromatography. From these columns the activity was recovered in the (1) unbound material, and eluates (2 and 4) with 1M and 0,5M of NaCl, respectively, and (3) 83% acetonitrile. On the basis of electrophoresis analysis, the protocol produced a preparation comprised of only band corresponding a peptide with molecular weight (MW) of about 3-kDa, with minimum haemagglutination concentration of 50g/ml. To determine if this molecule arrangement had a quaternary structure arrangement, a feature of most known lectins, we submitted the preparation to a native electrophoresis. Because there was neither change in migration pattern nor presence of molecules of higher molecular mass, we suggested that T. hystrix peptide (Tabhys) is a peptide with MW of about 3-kDa. Since hevein, which is a most studied lectin-like peptide with antifungal activity, binds specifically to chitin, we performed an affinity chromatography in the chitin column with bark extract. We observed haemagglutination activity and the presence of peptide with MW of 3-kDa in the material bound to column and eluted with 0,1M acetic acid. Curiously, this peptide was not able to inhibit the growth of the fungus Trichophyton rubrum. Thereafter, when the purified peptide was used to stimulate murine spleen cells, we detected the expression of TGF-beta message, with a peak production obtained in cell stimulated with 37 ng/mL of Tabhys. In the current study, we isolated a peptide from crude extract of T. hystrix bark with haemagglutination activity, providing new perspectives in molecular and structural researches of peptide lectins.

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Angeli, Anthony. "Conception, synthèse, et évaluation de l'affinité de glyco-oligonucléotides et glycoclusters contre les lectines I et II de Pseudomonas aeruginosa." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT194/document.

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Pseudomonas aeruginosa (PA) est une bactérie présentant des résistances aux antibiotiques toute particulière. Elle est aujourd’hui largement impliquée dans de nombreuses maladies nosocomiales et dans l’infection de patients immunodéprimés. Les lectines sont des glycoprotéines formant des interactions faibles et réversibles avec les saccharides, et elles sont impliquées dans les mécanismes de protection et de virulence de la bactérie. Afin d’augmenter l’affinité des sucres pour celle-ci nous utilisons la multivalence conduisant à des leurres moléculaires ciblant les lectines I et II de PA. Notre stratégie consiste à synthétiser des glycoclusters conjugués à une étiquette ADN permettant leur criblage par des puces à ADN. Nous décrivons le design, la synthèse et l’assemblage des différents blocs de construction composant les glyco-oligonucléotides en s’appuyant sur la chimie "Click" (CuAAc) et la chimie oligonucléotidique. Le criblage des glyco-oligonucléotides est complété par des études de modélisation moléculaire et par RMN STD. Les meilleurs composés issus du criblage sont synthétisés sans leur étiquette ADN afin d’évaluer leur capacité inhibitrice de la formation du biofilm par PA
Pseudomonas aeruginosa (PA) is a bacteria with a strong resistance to antibiotics.It’s an opportunistic germ involved in nosocomial infections and the infection of immunocompromised patients. Lectins are glycoproteins that bind saccharides reversibly with a low affinity. They are involved in bacteria protection mechanisms and virulence. In order to increase the affinity of saccharides to PA lectins I and II, we used multivalence and synthesized molecular decoys. Our strategy involves the synthesis of glycoclusters conjugated with a DNA tag in order to easily screen them on DNA arrays. We described here the design, the synthesis and the assembly of different building blocks allowing the synthesis of glyco-oligonucleotides,based on "Click" chemistry (CuAAc) and oligonucleotide chemistry. The screening of theglyco-oligonucleotides was completed by studies of molecular modelisation and by STD NMR.The best compounds from the screening were synthesized without the DNA tag in order to evaluate their abilities of inhibiting the biofilm formation of PA

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19

Gadelha, Carlos Alberto de Almeida. "CristalizaÃÃo, estrutura tridimensional e atividade biolÃgica de uma lectina de sementes de Canavalia maritima." Universidade Federal do CearÃ, 2005. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=8320.

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nÃo hÃ
Cristais da lectina de sementes de Canavalia maritima (ConM) nativa foram obtidos pelo mÃtodo da difusÃo de vapor em gota suspensa com soluÃÃo do reservatÃrio contendo tampÃo Hepes 0,1M pH 8.43 com 4% v/v de polietilenoglicol 400 e 2M de sulfato de amÃnio. AtravÃs de âsoakingâ dos cristais com ligantes glicÃdicos foram produzidos cristais da lectina complexada aos aÃucares maltose e trealose. Os cristais obtidos de ConM nativa difrataram a uma resoluÃÃo mÃxima de 1,56 Ã, pertencendo ao grupo primitivo ortorrÃmbico, P21212 com parÃmetros de cÃlula de a=67.9, b=71.0, c=97.6 Ã e apresentando um dÃmero na unidade assimÃtrica (VM = 2,4 A3 Da-1). Com base nas densidades eletrÃnicas dos resÃduos da estrutura tridimensional resolvida, constatou-se que a sequencia de Perez et al (1991) apresentava erros, que quando corrigidos e comparados com a seqÃÃncia de ConA, aumentaram para 98% o grau de similaridade entre as duas lectinas. A estrutura de ConM nativa exibe um alto grau de enovelamento terciÃrio, tÃpico das lectinas de leguminosas, com visÃvel conservaÃÃo do sÃtio de ligaÃÃo a metais e loops envolvidos na oligomerizaÃÃo molecular. Embora a especificidade e o nÃmero de pontes de hidrogÃnio formadas na interaÃÃo com os dissacarÃdeos maltose e trealose seja a mesma, estruturalmente ocorrem diferenÃas no padrÃo de formaÃÃo de pontes de hidrogÃnio, principalmente na distribuiÃÃo do nÃmero de pontes de hidrogÃnio entre os dois resÃduos de glicose dos dissacarÃdeos. Os ensaios de atividade biolÃgica mostraram que o efeito relaxante concentraÃÃo-dependente da lectina de sementes de Canavalia maritima em anÃis de aorta isolados ocorre provavelmente atravÃs da interaÃÃo com um sÃtio de ligaÃÃo lectina-especÃfico presente no endotÃlio, resultando em liberaÃÃo de Ãxido nÃtrico.
Crystals of Canavalia maritima seeds lectin (ConM) were obtained using the vapor diffusion method in hanging drop by sparse matrix with reservatory solution containing 0,1 M HEPES, pH 8,43, 4% Polyethylene Glycol 400 and 2 M ammonium sulfate. The soaking technic was used to produce lectin crystals complexed with the carbohydrates maltose and trehalose. ConM native crystals diffracted to 1.56 Ã resolution, belonging to the primitive orthorhombic space group P21212, with unit-cell parameters a=67.9, b=71.0, c=97.6 Ã, consisting in a dimer in the asymmetric unit (VM = 2,4 A3 Da-1), with two metal binding sites per monomer, and loops involved in the molecular oligomerization. The structure was solved by standard molecular replacement techniques. The wrong amino acids residues previously suggested by manual sequencing by Perez et al. (1991) are now determinated as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196 and S202. These modifications confer 98% of similarity between ConM, ConA and ConBr. ConM structure presents a high level of tertiary protein folding, typically of legume lectins. Despite the specificity and the numbers of hydrogen bonds formed in the interaction with the disaccharides maltose and trehalose be the same, structurally changes occurs in the pattern of formation of the hydrogen bonds, especially in the distribution in the number of hydrogen bonds between the two glucose residues of the disaccharides. Our biological activity data show that the relaxant concentration-dependent effect of the seed lectin of Canavalia maritima on isolated aortic rings probably occurs via interaction with a specific lectin-binding site on the endothelium, resulting in a release of nitric oxide.

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20

Teixeira, Claudener Souza. "DeterminaÃÃo da estrutura tridimensional de uma lectina de sementes de Canavalia maritima (Aublet) complexada com o polifenol antioxidante resveratrol." Universidade Federal do CearÃ, 2015. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=15090.

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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
O resveratrol Ã um polifenol antioxidante natural encontrado especialmente na epicarpo da uva, em nozes e na romÃ o qual pode inibir a ativaÃÃo de mediadores prÃ-inflamatÃrio e citocinas na fase precoce de expressÃo do gene. JÃ Ã bem conhecido que as lectinas sÃo proteÃnas de ligaÃÃo de aÃÃcares que atuam tanto como molÃculas prÃ- e anti-inflamatÃrias. Assim, o objetivo do presente trabalho foi o de verificar a ligaÃÃo de um composto de polifenol com uma lectina de Canavalia maritima (ConM) com base na sua capacidade para inibir processos prÃ-inflamatÃrios. Para alcanÃar este objetivo, a ConM foi purificada e cristalizada, uma soluÃÃo de 5 mM de resveratrol 5 mM foi utilizada para soaking durante 2 horas de incubaÃÃo. Os cristais obtidos pertencem ao grupo espacial monoclÃnico C2, o refinamento final resultou em um Rfactor de 16,0% e uma Rfree de 25,5%. Resveratrol se liga na rÃgida atravÃs de pontes de hidrogÃnio e interaÃÃo hidrofÃbica com aminoÃcidos que compÃem a quinta e sexta fita-β da folha-β rÃgida da ConM. A ConM complexada com o resveratrol inibiu a oxidaÃÃo do DPPH, mostrando atividade sinÃrgica entre a proteÃna e o ligante com a relaÃÃo mais eficaz de 2:3. Foi verificado ainda que o sÃtio de ligaÃÃo a carboidratos nÃo estÃ diretamente relacionado Ã atividade antioxidante. Ã a interaÃÃo entre ConM e resveratrol, que indica o sinergismo destas duas molÃculas em agir como agentes sequestrantes de radicais livres que podem estarem relacionados a capacidade de reduÃÃo do processo inflamatÃrio atravÃs da inibiÃÃo de muitos mediadores prÃ-inflamatÃrios por lecitinas.
Resveratrol is a natural antioxidant polyphenol found especially in grape epicarp, walnuts, and pomegranates, which can inhibit the activation of proinflammatory mediators and cytokines at the early gene expression stage. It is well known that lectins are sugar-binding proteins that act as both pro- and anti-inflammatory molecules. Thus, the objective of this work was to verify the binding of a polyphenol compound with a lectin of Canavalia maritima (ConM) based on their ability to inhibit pro-inflammatory processes. To accomplish this, ConM was purified and crystallized, and resveratrol was soaked at 5 mM for 2 hours of incubation. The crystal belongs to the monoclinic space group C2, the final refinement resulted in an Rfactor of 16.0% and an Rfree of 25.5%. Resveratrol binds in the rigid β-sheet through H-bonds and hydrophobic interaction with amino acids that compose the fifth and sixth β-strands of the rigid β-sheet of ConM. The ConM and resveratrol inhibited DPPH oxidation, showing synergic activity with the most effective ratio of 2:3 and carbohydrate binding site is not directly related to antioxidant activity. It is the interaction between ConM and resveratrol that indicates the synergism of these two molecules in acting as free radicals scavengers and in reducing the inflammatory process through the inhibition of many pro-inflammatory events.

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Felix, Wagner Pereira. "Produção heteróloga de frutalina em Pichia pastoris." reponame:Repositório Institucional da UFC, 2008. http://www.repositorio.ufc.br/handle/riufc/18810.

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FELIX, Wagner Pereira. Produção heteróloga de frutalina em Pichia pastoris. 2008. 113 f. Tese (Doutorado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2008.
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Frutalin, the Artocarpus incise alfa-D-galactose binding seed lectin was expressed and processed in a heterologous system for protein expression, using the metilotrophic yeast Pichia pastoris. In order to obtain the proposed objectives, three strategies were followed: identify the frutalin gene from the genomic DNA, obtained from the leaves; obtain, from the GenScript Corporation, the synthetic frutalin polypeptide chains gene, optimized for expression in Pichia system; and isolate frutalin gene, from mRNA present in fresh seeds in order to obtain the cDNA, using the RT-PCR technique. The obtained genes were inserted in the Pichia genome, in order to evaluate the lectin expression. While in the first strategy only the beta chain gene was obtained, in the other two the complete frutalin gene was obtained. The best results were obtained from the third strategy. The expression process was temperature dependent, with the optimum at 18 οC and the recombinant frutalin showed an apparent molecular mass of 17 kDa, which suggest the presence of both the linker peptide and the histidine tag.
A frutalina, lectina α-D-galactose ligante, foi expressa e processada num sistema heterólogo para expressão de proteínas utilizando a levedura metilotrófica Pichia pastoris. Para atingir os objetivos esperados foram desenvolvidas três estratégias: identificar o gene que codifica para a frutalina a partir do DNA genômico obtido de folhas de A. incisa; sintetizar, por uma empresa especializada, o gene que codifica para as cadeias polipeptídicas da frutalina, otimizando-o para a expressão em P. pastoris e isolar o gene que codifica para a frutalina, a partir do mRNA presente em sementes maduras e frescas, para obtenção do cDNA utilizando a técnica da RT-PCR. Enquanto na primeira estratégia, foi obtido apenas o gen da cadeia beta, nas duas outras estratégias o gen completo foi obtido. No entretanto, a estratégia que mostrou melhor expressão da frutalina recombinante em P. pastoris foi a da obtenção do gene que codifica para a frutalina, a partir do mRNA. A expresão foi tempertura dependente e a frutalina recombinante apresentou uma massa molecular aparente de 17 kDa, sugerindo a presença do peptídeo de ligação e da cauda de histidina.

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22

Filho, JoÃo Garcia Alves. "Cloning, sequencing and partial characterization of Vatairea Macrocarpa lectin related genes." Universidade Federal do CearÃ, 2008. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=8149.

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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
No presente trabalho Ã feita a descriÃÃo de trÃs genes distintos que codificam lectinas ou proteÃnas relacionadas Ã lectina de Vatairea macrocarpa. As sequÃncias foram obtidas pela amplificaÃÃo de DNA genÃmico e cDNA de folhas utilizando primers semi-degenerados construÃdos a partir da informaÃÃo da sequÃncia de aminoÃcidos da lectina VML depositada no GenBank. O resultado do sequenciamento revelou a presenÃa de trÃs contigs. O contig1 corresponde Ã lectina VML desde que se assuma que a lectina depositada VML contenha heterogeneidades ou ambiguidades decorrentes na degradaÃÃo de Edman. A traduÃÃo dos contigs 2 e 3 mostram identidade de sequÃncia de 77% quando comparadas com VML. As sequÃncias, apesar de apresentar regiÃes conservadas, mostram diferenÃas de aminoÃcidos nos sÃtios de N-glicosilaÃÃo, sÃtios de ligaÃÃo a carboidrato e metais alÃm da presenÃa de resÃduos de cisteÃna sugerindo que tais proteÃnas podem ter outras atividades biolÃgicas. A anÃlise da sequÃncia obtida pelo 3â RACE se mostrou complementar ao contig3. Sendo assim, a sequÃncia hÃbrida contig3/contigA possui 2 resÃduos de cisteÃna alÃm de revelar diferenÃas de aminoÃcidos na regiÃo C-terminal quando alinhada com outras lectinas de leguminosas. AnÃlises filogenÃticas revelaram que os contigs observados formam um grupo monofiletico e tem alta similaridade com as lectinas de Sohora japonica e Robinia pseudoacacia, alÃm da proteÃna relacionada Ã lectina de Cladrastis lutea.
In this paper is made a description of three distinct genes that encode Vatairea macrocarpa lectin and related proteins. The sequences were obtained by amplification of genomic DNA and cDNA of leaves using semi - degenerate primers constructed from the information of the amino acid sequence of VML lectin deposited in GenBank. The result of sequencing rev eals the presence of three different genes, called contig 1, 2 and 3 . The VML lectin corresponds to contig1 long as one assumes that the lectin contains heterogeneities deposited VML or ambiguities arising in the Edman degradation . The translation of cont igs 2 and 3 show sequence identity of 77% compared to VML. Sequences, despite having conserved regions show differences in amino acid N - glycosylation sites, carbohydrate binding sites and metals and the presence of cysteine residues suggests that these pro teins may have other biological activities . The analysis of the sequence obtained by 3 'RACE proved complementary to contig3. Thus, the sequence contig3/contigA hybrid has two cysteine residues in addition to revealing differences in amino acid C - terminal region when aligned with other legume lectins. Phylogenetic analysis revealed that the observed contigs form a monophyletic group and has high similarity with lectins from Robinia pseudoacacia Sohora japonica and, in addition to the lectin - related protein Cladrastis lutea .

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23

Pourceau, Gwladys. "Mise au point de nouvelles méthodes de conjugaison oligonucléotide/sucre et développement d'un microsystème d'analyse des interactions lectine/sucre." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20224.

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Les interactions entre les sucres et les lectines sont généralement l'étape clé dans de nombreux phénomènes biologiques et pathologiques. Malgré leu r importance cruciale, ces interactions sont paradoxalement caractérisées par des constantes d'affinité faibles et nécessite une multiprésentation des motifs saccharidiques pour être significatives. Cette augmentation est appelée "effet cluster". En outre, les techniques d'analyse actuellement utilisées en laboratoire nécessitent des quantités importantes de produits, ce qui est difficilement compatible avec les méthodes de synthèse actuelle. Pour pallier ces difficultés, une approche originale basée sur l'utilisation conjointe de glycooligonucléotide et de puces à ADN a été proposée. Les glycoconjugués basés sur des squelettes phosphodiesters et couplés à des séquences d'ADN ont été synthétisés en utilisant la chimie des oligonucléotides, couplée à la "click chemistry". La séquence d'ADN quant à elle a permis l'ancrage sur une puce à ADN et donc la mesure de leur affinité vis-à-vis de différentes lectines. Ce manuscrit rapporte le développement des nouvelles méthodologies de synthèse des glycooligonucléotides ainsi que la préparation de nombreux glycoconjugués originaux, dont l'affinité pour différentes lectines a été mesurée via l'utilisation de la puce à ADN. L'influence de plusieurs paramètres a été étudiée: le nombre de résidus, l'arrangement spatial, la lipophilie etc. Il s'avère que l'arrangement spatial semble être l'un des points les plus importants dans la mise au point d'un glycoconjugué
The interactions between carbohydrates and lectins are generally the "key step" in many biological and pathological phenomena. Despite their importance, these interactions are paradoxically characterized by low affinity constants and requires multipresence of saccharide to be significant. This increase is called "cluster effect". In addition, the analysis techniques currently used in the laboratory requires large quantities of products, which is hardly compatible with the current methods of synthesis. To circumvent these difficulties, a original approach based on the combined use of glycooligonucleotides and DNA microarrays has been proposed. Glycoconjugates based on phosphodiester skeletons linked to DNA sequences have been synthesized using the chemistry of oligonucleotides, coupled with the "click chemistry". The DNA sequence has allowed the anchoring on a DNA chip and therefore the measurement of their affinity versus different lectins.This manuscript reports the development of new synthetic methodologies for the glycooligonucleotides synthesis and the preparation of many original glycoconjugates, whose affinity for various lectins was measured through the use of DNA microarray. The influence of several parameters was studied: the number of residues, the spatial arrangement, etc. lipophilicity. The spatial arrangement appears to be one of the most important parameters in the development of a glycoconjugate

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24

Paniccia, Rocco. "Detection of nucleoplasmic glycoconjugates using lectin cytochemistry." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=59968.

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The lectins Ulex europaeus 1 (UEA 1), Ricinus communis 1 (RCA 1), Wheat Germ Agglutinin (WGA), and Lens culinaris agglutinin (LCA) which are specific for L-fucose, D-galactose, N-acetylglucosamine, and D-mannose respectively, have been used to localize cytochemically the presence of these sugar residues in certain human, mammalian, and amphibian cell types. Thin sections of Lowicryl K4M-embedded human distal colon, rat duodenum, and frog dorsal root ganglion were incubated with UEA 1-gold, RCA 1-gold, WGA/ovomucoid-gold, or LCA-gold. UEA-1, RCA-1, and LCA binding sites were present in the nuclei of human colonic cells, rat duodenal goblet and columnar cells and frog dorsal root ganglion neurons, satellite cells, and Schwann cells. Most of the nuclear binding sites were localized in the euchromatin compartment, while extranuclear labelling was largely restricted to sites known to contain glycoproteins. WGA binding sites were present in the nuclei of rat duodenum columnar cells and frog dorsal root ganglion neurons, satellite cells, and Schwann cells, but no labelling of human colonic cell nuclei was observed. Thus, we report the presence of these sugars in the nuceloplasm of human colonic cells (with the exception of WGA), rat duodenal villous columnar cells, and frog dorsal root ganglion cells examined.

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25

Legge, Glen Barry. "Structural studies of a C-type lectin." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627613.

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26

Robinson, Mark Alexander. "LEAPT : lectin-directed enzyme activated prodrug therapy." Thesis, University of Oxford, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433562.

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Tregear, James W. "The lectin gene family of Ricinus communis." Thesis, University of Warwick, 1989. http://wrap.warwick.ac.uk/101217/.

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The aim of this project has been to isolate, characterise and investigate the expression patterns of the various members of the castor bean lectin gene family. Genomic Southern blotting experiments showed that the gene family contains approximately eight members, of which two appear to be ricin-like. Castor bean genomic DNA was cloned into a bacteriophage lambda vector and the resulting recombinant clones screened with a ricin cDNA probe. Seventeen positive clones were isolated, amongst which at least five different groups could be recognised, on the basis of Southern blotting data. Four different lambda clones were selected for further analysis. One of the clones was found to contain a functional ricin gene with an identical restriction pattern to that of the ricin cDNA, but none of the clones appears to contain an authentic Riclnus communis agglutinin (RCA I) gene. DNA sequencing and RNAse protection data showed that three of the clones analysed contain lectin pseudogenes. The expression pattern of the functional ricin gene in pCBG3Hl (lambda clone 10) was investigated at the transcriptional level using RNAse protection. The results obtained show that the mRNA transcribed from this gene accumulates during the late (post-testa) stages of seed development. The pCBG3Hl ricin gene appears to use multiple cap and poly(A) sites. The developmental profile of lectin gene transcript levels observed in this study is similar to the patterns previously observed at the protein and translatable mRNA levels. This indicates a close correlation between the accumulation of the lectin proteins and the amounts of steady state transcripts. DNA sequencing enabled the identification of putative transcriptional regulatory elements in the promoter of the pCBG3Hl ricin gene, including an element resembling the RY repeat previously implicated in seed-specific gene expression. Ultimately, it is hoped that studies of this type will make it possible for the network of regulatory processes governing the expression pattern of the castor bean lectin genes to be unravelled.

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Thakur, A. K. "Study of a lectin from ganoderma lucidum." Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 2007. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/2591.

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Dommett, Rachel Mary. "Structural and functional studies of mannose binding lectin (MBL) and the lectin pathway of complement in children with cancer." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444151/.

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Infection remains a major cause of morbidity and hospitalisation in children receiving chemotherapy treatment for cancer. Among patients with the same diagnosis and treatment regimen, not all suffer equally from infectious complications. This suggests that as yet unidentified host factors may contribute to increased susceptibility to infection. Deficiency of mannose binding lectin (MBL), a pattern recognition receptor of the innate immune system, has been proposed as one such factor but clinical studies have been inconclusive. MBL works in concert with MBL associated serine proteases (MASPs) to activate the lectin pathway of complement. Functional activity of the pathway has not been investigated in children with cancer to date. Children undergoing chemotherapy were recruited to cross sectional observational and longitudinal studies with details of febrile neutropenia (FN) episodes recorded prospectively. MBL gene polymorphisms were characterized by heteroduplexing and reverse hybridization. MBL protein and MASP levels were measured and functional activity of the lectin pathway was quantified. High oligomer structure of MBL was analysed by Western blotting. Transcriptional regulation of MBL in response to infection and inflammation was studied. The MBL promoter was cloned and its activity investigated using luciferase assays. A major finding is that individuals in possession of MBL variant alleles suffered from more frequent and longer episodes of FN over the study period compared to wildtype individuals. Functional analysis enabled identification of specific deficiencies within the pathway in patients deemed MBL sufficient by their genotype and protein level. Changes in MASP levels were noted in response to chemotherapy and complement function was observed to increase during FN episodes. Structural analysis revealed variability in MBL higher order oligomer structure during the acute phase of FN and promoter studies further highlighted the complexity of MBL regulation. The results presented in this thesis provide further evidence that MBL deficiency increases the frequency and the duration of FN in the largest paediatric cancer cohort studied to date. The complex interplay between other pathway components appears crucial to MBL function but their influence on clinical outcome is not yet fully understood.

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30

Togashi, Ricardo Hideo. "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." Universidade Federal do CearÃ, 2010. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=5455.

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FundaÃÃo de Amparo Ã Pesquisa do Estado do CearÃ
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 atÃ 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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Fazanaro, Fabiano. "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." Universidade Federal do CearÃ, 2010. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=5702.

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FundaÃÃo de Amparo Ã Pesquisa do Estado do CearÃ
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 poderÃ 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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MIRANDA, Ileana Costa. "Avalia??o lectino-histoqu?mica de f?gado e linfonodo mesent?rico de b?falos mantidos em pastagens de Brachiaria spp." Universidade Federal Rural do Rio de Janeiro, 2015. https://tede.ufrrj.br/jspui/handle/jspui/1349.

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CAPES
Animals grazing Brachiaria spp commonly present foamy macrophages isolated or grouped in the liver, and crystals within biliary ducts. The pathogenesis of formation and the nature of the material stored on these cells, however, are not completely known. Through lectin histochemistry evaluation, steroidal saponins (secondary glycosylated metabolites) have been identified in the crystals and within the cytoplasm of the foam cells, which are probably liable for damage the liver leading to accumulation of phylloerythrin. This study aims to standardize the use of lectin histochemistry to detect glycosylated metabolites in tissues of buffaloes kept in different Brachiaria spp pastures in Brazil. Fragments of liver and mesenteric lymph node from 40 animals were analyzed: 10 buffaloes that were kept in predominant pasture of B. decumbens for 12 months; 10 buffaloes that were kept in pasture with a predominance of B. brizantha for 18 months; 10 buffaloes that were kept in pasture of B. brizantha for approximately four years; and, as a negative control, 10 buffaloes that were maintained in native pasture without Brachiaria spp since birth. Fourteen lectins were tested (Con-A, SBA, WGA, DBA, UEA, RCA, PNA, GSL-I, PSA, LCA, PHA-E, PHA-L, SJA and SWGA), in a total of 1120 evaluated fragments. Previous studies demonstrated that PNA showed great binding reactivity for foamy macrophages in bovine and ovine. In the present study, SWGA presented high specificity and marked binding reactivity for foamy macrophages; WGA, GSL, PHA-E and PHA-L showed moderate to marked reactivity but low specificity for foamy macrophages; the other lectins didn't show significant reactivity or specificity. It remains unclear why there is this difference in lectins binding reactivity to foamy macrophages; it is suggested that divergences may occur depending on the species of Brachiaria ingested, the plant growth stage, the type and proportion of saponins stored in the plant due to seasonality, the differences in the metabolism of animal species, the presence of photosensitivity, the clinical course of the disease and the plant intake time. Moreover, there was no significant reactivity difference between the collected fragments of animals that grazed in B. decumbens for 12 months and B. brizantha for 18 months. However, the decreased presence of foamy macrophages and its lectin histochemical binding in animals that fed on B. brizantha for a longer time indicates that the animals can pass through an adaptation process according to the the plant intake time. Lectin histochemistry analysis can be used to characterize the material stored in foamy macrophages present in liver and mesenteric lymph node of buffaloes that graze in Brachiaria spp pastures and helps to clarify the pathogenesis of these cells.
Animais que se alimentam em pastos de Brachiaria spp comumente apresentam macr?fagos espumosos isolados ou agrupados no f?gado, al?m de cristais no interior de ductos biliares. A patog?nese da forma??o e a natureza do material armazenado nestas c?lulas, contudo, ainda n?o s?o completamente conhecidas. Atrav?s da avalia??o lectino-histoqu?mica, saponinas esteroidais (metab?litos glicosilados secund?rios) t?m sido identificadas nos cristais e no citoplasma das c?lulas espumosas, que provavelmente s?o respons?veis por danificar o f?gado e levar ao ac?mulo de filoeritrina. Por meio deste trabalho, objetivou-se padronizar e caracterizar a utiliza??o da lectino-histoqu?mica na detec??o de metab?litos glicosilados nos tecidos de b?falos mantidos em diferentes pastos de Brachiaria spp no Brasil. Fragmentos de f?gado e linfonodo mesent?rico de 40 animais foram analisados: 10 b?falos mantidos em pastagem predominante de B. decumbens por aproximadamente 12 meses; 10 b?falos mantidos em pastagem predominante de B. brizantha por aproximadamente 18 meses; 10 b?falos mantidos em pastagem de B. brizantha por aproximadamente quatro anos; e, como controle negativo, 10 b?falos mantidos em pastagem livre de Brachiaria spp desde o nascimento. Quatorze lectinas foram testadas (Con-A, SBA, WGA, DBA, UEA, RCA, PNA, GSL-I, PSA, LCA, PHA-E, PHA-L, SJA e SWGA), em um total de 1120 fragmentos avaliados. Estudos anteriores demonstraram que a lectina PNA possui marcada reatividade para macr?fagos espumosos de bovinos e ovinos. No presente estudo, a lectina SWGA apresentou acentuada reatividade e alta especificidade para macr?fagos espumosos; WGA, GSL, PHA-E e PHA-L mostraram moderada a acentuada reatividade, mas baixa especificidade aos macr?fagos espumosos; as outras lectinas n?o apresentaram reatividade ou especificidade significativas. Ainda n?o se sabe exatamente a que atribuir a diferen?a de reatividade aos macr?fagos espumosos. Sugere-se que diverg?ncias ocorram em fun??o da esp?cie de Brachiaria ingerida, da fase de crescimento da planta, do tipo e propor??o dos glicoconjugados armazenados na planta em decorr?ncia da ?poca do ano, das diferen?as no metabolismo da esp?cie do animal em quest?o, da presen?a de fotossensibiliza??o, da evolu??o cl?nica da doen?a e do tempo de ingest?o da planta. N?o houve diferen?a de marca??o significativa entre os fragmentos coletados de animais que se alimentaram de B. decumbens por 12 meses e B. brizantha por 18 meses. Por?m, a diminui??o da presen?a e marca??o lectino-histoqu?mica dos macr?fagos espumosos nos tecidos dos b?falos que ingeriram B. brizantha durante mais tempo indica que os animais podem passar por um processo de adapta??o de acordo com o tempo de ingest?o da planta. A avalia??o lectino-histoqu?mica pode ser utilizada para caracterizar o material armazenado em macr?fagos espumosos presentes no f?gado e linfonodo mesent?rico de b?falos que se alimentam em pastagens de Brachiaria spp e ajuda na compreens?o da patog?nese de forma??o destas c?lulas.

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SILVA, Flávio de Oliveira. "Atividade moduladora da lectina isolada das sementes de Canavalia brasiliensis." Universidade Federal Rural de Pernambuco, 2012. http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/4585.

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Lectins are proteins that bind specifically and reversibly to carbohydrates, showing several biological effects. In this work, we carried out a literature review about biological effects of lectin extracted from seeds of Canavalia brasiliensis (ConBr), a plant present in the northeastern and southern Brazil, which is popularly known as wild bean of Ceará. In addition, we carried out a study to analyze the modulating activity of ConBr on murine splenocytes, verifying its effect on cell viability and proliferation, cytokine and nitric oxide (NO) production. We have also performed a study to evaluate ConBr effect on B16F10 murine melanoma cells by analyzing the inhibition of cell proliferation and migration as well as apoptosis induction and synthesis of cytokines and NO. The results show that ConBr induced at concentrations of 2.5, 5.0 and 10 μg/ml promoted the proliferation of splenocytes, with high cell viability. Furthermore, the concentration of 10 μg/ml induced cytokine production and nitric oxide on B16F10 murine melanoma, it was observed that ConBr inhibited tumor cell proliferation inducing apoptosis. It was also observed nitric oxide and IL-12 production by B16F10 cells under stimulus. ConBr lectin possesses a biotechnological potential use as a mitogen and anti-tumor agent.
As lectinas são proteínas que apresentam a capacidade de se ligar de maneira específica e reversível a carboidratos, exibindo distintos efeitos biológicos. Neste trabalho, realizou-se uma revisão de literatura sobre os efeitos biológicos da lectina extraída das sementes da Canavalia brasiliensis (ConBr), uma planta presente no Nordeste e Sul do Brasil, que é conhecida popularmente como feijão bravo do Ceará. Além disso, realizou-se um estudo para analisar a atividade moduladora da ConBr sobre esplenócitos murinos, verificando-se sua ação sobre a proliferação e viabilidade celular, produção de citocinas e óxido nítrico (NO). Realizou-se também, um estudo para avaliar o efeito da ConBr sobre células B16F10 de melanoma murino, analisando-se a inibição da proliferação e migração celular, bem como a indução de apoptose e síntese de citocinas e NO. Os resultados demostraram que a ConBr induziu nas concentrações de 2.5, 5.0 e 10 μg/ml promoveu a proliferação de esplenócitos, com alto índice de viabilidade celular. Além disso, a concentração de 10 μg/ml induziu a produção de citocinas e óxido nítrico. Em células B16F10 de melanoma murino, observou-se que a ConBr inibiu a proliferação das células tumorais promovendo apoptose celular. Verificou-se ainda, a produção de óxido nítrico e da citocina IL-12 pelas células submetidas ao estímulo. A lectina ConBr possue um potencial uso biotecnológico como mitógeno e agente antitumoral.

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Alves, Filho João Garcia. "Clonagem, sequenciamento e caracterização parcial dos genes relacionados à lectina de Vatairea macrocarpa." reponame:Repositório Institucional da UFC, 2008. http://www.repositorio.ufc.br/handle/riufc/18177.

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ALVES FILHO, João Garcia. Clonagem, sequenciamento e caracterização parcial dos genes relacionados à lectina de Vatairea macrocarpa. 2008. 81 f. Dissertação (Mestrado)-Universidade Federal do Ceará, Fortaleza-CE, 2008.
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In this paper is made a description of three distinct genes that encode Vatairea macrocarpa lectin and related proteins. The sequences were obtained by amplification of genomic DNA and cDNA of leaves using semi - degenerate primers constructed from the information of the amino acid sequence of VML lectin deposited in GenBank. The result of sequencing rev eals the presence of three different genes, called contig 1, 2 and 3 . The VML lectin corresponds to contig1 long as one assumes that the lectin contains heterogeneities deposited VML or ambiguities arising in the Edman degradation . The translation of cont igs 2 and 3 show sequence identity of 77% compared to VML. Sequences, despite having conserved regions show differences in amino acid N - glycosylation sites, carbohydrate binding sites and metals and the presence of cysteine residues suggests that these pro teins may have other biological activities . The analysis of the sequence obtained by 3 'RACE proved complementary to contig3. Thus, the sequence contig3/contigA hybrid has two cysteine residues in addition to revealing differences in amino acid C - terminal region when aligned with other legume lectins. Phylogenetic analysis revealed that the observed contigs form a monophyletic group and has high similarity with lectins from Robinia pseudoacacia Sohora japonica and, in addition to the lectin - related protein Cladrastis lutea.
No presente trabalho é feita a descrição de três genes distintos que codificam lectinas ou proteínas relacionadas à lectina de Vatairea macrocarpa. As sequências foram obtidas pela amplificação de DNA genômico e cDNA de folhas utilizando primers semi-degenerados construídos a partir da informação da sequência de aminoácidos da lectina VML depositada no GenBank. O resultado do sequenciamento revelou a presença de três contigs. O contig1 corresponde à lectina VML desde que se assuma que a lectina depositada VML contenha heterogeneidades ou ambiguidades decorrentes na degradação de Edman. A tradução dos contigs 2 e 3 mostram identidade de sequência de 77% quando comparadas com VML. As sequências, apesar de apresentar regiões conservadas, mostram diferenças de aminoácidos nos sítios de N-glicosilação, sítios de ligação a carboidrato e metais além da presença de resíduos de cisteína sugerindo que tais proteínas podem ter outras atividades biológicas. A análise da sequência obtida pelo 3’ RACE se mostrou complementar ao contig3. Sendo assim, a sequência híbrida contig3/contigA possui 2 resíduos de cisteína além de revelar diferenças de aminoácidos na região C-terminal quando alinhada com outras lectinas de leguminosas. Análises filogenéticas revelaram que os contigs observados formam um grupo monofiletico e tem alta similaridade com as lectinas de Sohora japonica e Robinia pseudoacacia, além da proteína relacionada à lectina de Cladrastis lutea.

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Kerscher, Berhard Gerhard Richard. "Characterisation of the C-type lectin receptor Clecsf8." Thesis, University of Aberdeen, 2016. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=230779.

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C-type lectin-like receptors (CTLRs) play critical roles in immunity and homeostasis by recognising a variety of microbial or endogenous ligands. Clecsf8 is a member of the Dectin-2 family of CTLRs. Clecsf8 shares important similarities with its relatives Mincle and Dectin-2, such as the lack of an integral signalling motif and a single, calcium dependent ligand binding domain. They were shown to associate with the FcRγ adaptor, which is essential for receptor surface expression and downstream signalling. Recent publications revealed an important role for Clecsf8 in anti-mycobacterial immunity. It was reported to recognise the mycobacterial cord factor (TDM), similar to the related CTLR Mincle, as well as a possible role in candidiasis. In this study, we characterised the underlying mechanism of Clecsf8 expression in a context of mycobacterial disease. The generation of novel anti-Clecsf8 monoclonal antibodies allowed us to characterise the expression of Clecsf8 in detail in homeostasis and inflammation in murine models in vivo and culture systems in vitro. We found Clecsf8 to be predominantly expressed on monocytes/macrophages and neutrophils within e. g. the peritoneal cavity, blood and bone marrow. Notably, Clecsf8 was expressed only weakly in the lung, but strongly upregulated in a pulmonary mycobacterial infection model. In vitro, Clecsf8 expression on elicited macrophages was strongly induced upon treatment with microbial stimuli in a Myd88- and Mincle dependent manner. Interestingly, surface expression of Clecsf8 in a murine fibroblast cell line was greatly enhanced by co-transfection of Mincle, but not another related CTLR, Dectin-2. Notably, we confirmed mycobacteria as a ligand of CLECSF8, but found no role for the receptor in Candida immunity. In conclusion, Clecsf8 is a myeloid expressed, mycobacterial receptor, showing significant interdependence with Mincle and is regulated through the Myd88 pathway.

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36

Li, Mi. "Preliminary crystallization and characterization of lectin-sugar complex." Thesis, Georgia Institute of Technology, 1992. http://hdl.handle.net/1853/27569.

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37

Ma, Long. "Synthesis and biological properties of cytotoxic lectin conjugates." Thesis, University of Liverpool, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485905.

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Proliferative vitreoretinopathy (PVR) is an anomalous cell proliferation condition that occurs after surgery on the retina. 5-Fluorouracil (5-FU) has been shown to be effective in reducing the incidence ofPVR in animal models and recently been used as a clinic adjunctive treatment of PVR. However, the results of such treatment have been disappointing, largely because these drugs are quickly metabolized within the eye minimizing their long-term effectiveness; high doses ofthese drugs may also be toxic to the retina. Agaricus bisporus lectin (ABL) is obtained from t~e edible common mushroom and has been shown to have reversible, dose-dependent and specific anti- proliferation activity in vitro. The aim ofthis research project is to conjugate 5-FU and ABL together by using chemical conjugation methods to give potentially potent and specific anti-proliferative activity for the treatment ofPVR. The first part of this project is to study the protein structure of the ABL, which is to find the suitable residues as conjugation targets. ABL is found as a homotetramer protein and each ofthe four subunits has a molecular mass of 16,053.8 Da. There are one cysteine and five lysines for each of the lectin subunits. The free -SH of cysteine, free -NH2 of lysine and the N-terminal amino groups are conjugation targets. The second part of the project is to modify the 5-FU with either amino or azido functional groups preparing derivatives of 5-fluorouridine which can then be conjugated to ABL. The third part of the work describes the conjugation of the modified 5-FU with ABL by using SMCC (succinirnidyI4-(N-maleimidomethyl) cyclohexane-l-carboxylate) cross linker, aconitic acid anhydrate linker, 'click' chemistry as well as the photochemistry method separately, followed by characterization by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and the biological test. MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) is also attempted for further characterization.

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38

Christou, Charita. "C-type lectin-like receptors and their interactions." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.509908.

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39

杜鈺輝 and Yuk-fai To. "Mannose binding lectin in hepatitis B virus infection." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31225214.

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40

Deb, Sipra. "Structural studies on the Dolichos biflorus seed lectin." Thesis, University of Portsmouth, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310414.

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Koshi, Yoichiro. "Development of New Chemical Methods toward Lectin Engineering." 京都大学 (Kyoto University), 2008. http://hdl.handle.net/2433/57283.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(工学)
甲第13851号
工博第2955号
新制||工||1436(附属図書館)
26067
UT51-2008-C767
京都大学大学院工学研究科合成・生物化学専攻
(主査)教授濵地格, 教授森泰生, 教授白川昌宏
学位規則第4条第1項該当

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42

Simpson, Jonathan Robert Henry. "SERS-based nanoparticle biodetection using carbohydrate-lectin interactions." Thesis, University of Strathclyde, 2016. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=27026.

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Endogenous biological processes including cellular recognition, motility and differentiation together with infection often result from carbohydrate-based interactions. Investigation into glycobiological interactions using sugar-coated nanoparticles are the basis for the research described herein. Metallic nanoparticles were coated with a variety of thiol-based linker molecules. Heterobifunctional PEG (carboxyl/thiol) molecules were found to be most successful in preventing non-specific aggregation. The carboxylic acid functionality of the PEG molecules used allowed for subsequent coupling of a variety of carbohydrates to the nanoparticle surface. This resulted in the production of glyconanoparticles with unique surface functionality, for example, glucose or galactose. Additionally, functionalising the particles with Raman reporter molecules (RRMs) resulted in the measurement of surface enhanced Raman scattering (SERS) signals. Aggregation of the glyconanoparticles in the presence of a variety of carbohydrate-binding proteins (lectins) was measured via changes in the extinction profile, size and the SERS response of those particles. Nanoparticle aggregation was used for the sensitive detection of plant lectins, including the Concanavalin A and Jacalin lectin and also bacterial lectins including cholera toxin B subunit (CTB). CTB was detected sensitively, selectively and rapidly by using glyconanoparticles coated in a mixture of different carbohydrates (mixed-monolayers of galactose and N-acetylneuraminic acid). Detection was possible in both buffer and synthetic freshwater conditions, demonstrating the use of these glyconanoparticles in detecting a target in complex samples. By exploiting the reversible nature of carbohydrate-lectin interactions, it was possible to use the glyconanoparticles together with the lectin ConA to develop a glucose sensor. This performed effectively across the physiological range and into the hypo/hyperglycaemic regions in buffer conditions. Finally, the glyconanoparticles were used for the detection of plant and bacterial lectins on glass substrates by initially developing a sandwich SERS assay with a view to eventually creating SERS-based carbohydrate microarrays.

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To, Yuk-fai. "Mannose binding lectin in hepatitis B virus infection /." Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk:8888/cgi-bin/hkuto%5Ftoc%5Fpdf?B23435987.

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MEDVE, LAURA ANETT. "SYNTHESIS OF SELECTIVE C-TYPE LECTIN ANTAGONIST GLYCOMIMETICS." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/584203.

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In this thesis we have tackled the problem of identifying high-affinity, selective CLR ligands with two different approaches: in the first approach, we synthesized a diversity library and screened it in microarray format against a group of human CLRs; in the second approach we proceeded to the computer-aided design of a high affinity ligand for the dendritic cell CLR DC-SIGN. In the first approach, we prepared a mannosylated (based on the structure of Manalpha1-2Man) and fucosylated glycomimetic library that was printed on microarray chips. We optimized and validated the proper presentation of the glycomimetics with plant, fungal and bacterial lectins of known specificity. The glycomimetic chip was then screened against available human CLRs and the results were further assessed in complementary assays, such as SPR. In the second approach we targeted the dendritic cell-specific receptor DC-SIGN, which is responsible for the uptake and dissemination of numerous infectious pathogens, such as HIV-1, Dengue, Ebola, M. tuberculosis, etc. Selective inhibitors of the lectin have high potential as anti-microbial therapeutics, but so far, only a handful of monovalent ligands have reached low micromolar affinities. A virtual fragment screening campaign indicated a novel, exploitable binding pocket near the primary sugar binding site of DC-SIGN. Glycomimetic ligands previously developed in our group were functionalized in the appropriate position to reach this pocket, and modified with various substituents in order to form additional interactions. One of the highest affinity DC-SIGN ligands up-to-date was synthesized and X-ray structure analysis proved the in silico predicted interactions with the amino acid residues around the new binding site.

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45

Griffin, Raymond Leonard. "Distribution of N-acetyl-Î±-D-galactosamine (GalNAc) in normal and malignant oral epithelium." Thesis, University of the West of England, Bristol, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271046.

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In this project, the N-acetyl-ex-D-galactosamine (GalNAc) binding lectin from the green marine alga Codium fragile ssp. tomentosoides (C. fragile) was purified and techniques developed to label the lectin for visualisation by light and electron microscopy and electrophoresis. This represents the first time a histochemical reagent has been developed from a marine alga. The new reagent was initially assessed for transmission electron microscopy using human blood group A1 erythrocytes. A novel method gave a topographical view, and showed the distribution of gold particles on the surface of erythrocytes. The pattern of C. fragile lectin binding to pig normal oral epithelium was studied in the environmental scanning electron microscope to avoid charging artefact, using paraffin wax sections of pig normal epithelium stained with C. fragile lectin-gold conjugate enhanced with silver. X-ray micro analysis demonstrated lectin binding on the plasma membrane surface of epithelial cells at cells to cell contacts suggesting binding to cellular adhesion molecules. Biotinylated lectins binding GalNAc were used to investigate, identify and compare the binding of lectins in pig normal oral epithelium and altered glycoconjugates in cultured malignant cells from human oral tumours, using lectin histochemistry in the light microscope. Lectin from C. fragile was compared with Dolichos biflorus, a lectin from plants, and Helix pomatia (HP A) from snails. Although each lectin binds GalNAc it was shown that their binding pattern to pig normal oral epithelium was different, demonstrating that these lectins could be used to identify altered cellular glycosylation in the normal cellular maturation process. Cultured human oral tumour cells from different grades of malignancy were investigated using this panel of lectins. Binding of GalNAc specific lectins to cultured tumour cells changed in relation to their level of differentiation. This discriminating capability of GalNAc specific lectins offers exciting potential as indicators of tumour prognosis in human oral epithelial tumours. The lectins from C. fragile and HP A gave very similar staining results using histochemistry. Binding of these lectins to cell membrane glycoproteins was investigated using electrophoresis to show that C. fragile lectin binds to more and different cell membrane glycoproteins than lectin from HP A, but did not bind to purified CD44, excluding this adhesion molecule as a candidate for binding these lectins.

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Grundy, Gabrielle Jane. "The structure and function of human soluble CD23." Thesis, King's College London (University of London), 2001. https://kclpure.kcl.ac.uk/portal/en/theses/the-structure-and-function-of-human-soluble-cd23(1c2f66b1-b335-4192-892f-bbbf3afde432).html.

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47

Bezerra, Maria JÃlia Barbosa. "ResoluÃÃo da estrutura tridimensional da lectina de Dioclea violacea Mart para estudo da relaÃÃo estrutura-funÃÃo." Universidade Federal do CearÃ, 2011. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=5883.

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As lectinas da subtribo Diocleinae pertencem Ã 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 Ã capaz de provocar grandes alteraÃÃes nas atividades biolÃgicas dessas lectinas, dessa forma o entendimento mais detalhado das estruturas tridimensionais dessas proteÃnas Ã 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 2,61Ã e apresenta grupo espacial I222 com cela unitÃria com dimensÃes de a = 61,34, b = 66,11, c = 106,69Ã 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 Ã 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 2.61Ã with space group I222 with unit cell dimensions a = 61.34; b = 66.11; c = 106.69Ã 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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48

Fontenelle, Thais Pontes Carvalho. "Caracterização estrutural e atividade anti-inflamatória da lectina da alga marinha vermelha Bryothamnion triquetrum." reponame:Repositório Institucional da UFC, 2012. http://www.repositorio.ufc.br/handle/riufc/14977.

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FONTENELLE, T. P. C. Caracterização estrutural e atividade anti-inflamatória da lectina da alga marinha vermelha Bryothamnion triquetrum. 2012. 78 f. Dissertação (Mestrado em Bioquímica) - Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2012.
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The great biodiversity of seaweeds and their occurrence in almost all environments on the planet demonstrate the importance of such beings to ecosystems. One example is the extraction of potentially active biomolecules such as lectins, which are proteins that have been subject of research worldwide. This is due to its impressive ability to bind specifically to carbohydrates or substances containing them, providing an immense field of biological application. This work purpose was to produce lectin from the red seaweed Bryothamnion triquetrum (Lec), evaluate its secondary structure using spectroscopic techniques, and investigate their anti-inflammatory properties in mice. Initially, the lectin was purified by precipitation with ammonium sulfate (0-60) and ion exchange chromatography, and obtained the first peak (PI). This peak was selected by hemagglutination activity thus obtaining pure lectin from Bryothamnion triquetrum, verified by SDS-PAGE. The secondary structure of lectin was analyzed by circular dichroism spectroscopic techniques, against to pH extremes (4.0 to 11.0), and compared to the spectrum of its native structure, and fluorescence against different temperatures, ranging from 25° to 75°, each 10° interval. In biological activities assessment, groups of animals were subjected to pretreatment with the lectin (1.5 and 10mg/kg, ip) and indomethacin (10mg/kg) 30 min before the inflammatory stimulus. The anti-inflammatory activity in mice was evaluated through the assays of peritonitis induced by carrageenan - Cg (500 μg / cav) and paw edema induced by Cg (500 μg / paw), and dextran (500 μg / paw). Extremes of pH (4.0 and 11.0) did not alter the protein secondary structure. Fluorescence data (280nm and 295nm) also showed stability, and aromatic amino acids presence in their structure. Lectin inhibited cell migration in the induced peritonitis by Cg and induced paw edema by Cg and dextran. Thus, it is concluded that the lectin obtained from the Bryothamnion triquetrum DEAE PI shows stable structure related to pH and temperature variation. Regarding biological properties tested, the lectin showed anti-inflammatory effects in models used, and it can be considered a molecule with potential for further studies of inflammation.
A grande diversidade biológica das algas marinhas e sua ocorrência em praticamente todos os ambientes do planeta demonstram a importância desses seres para os ecossistemas. Um deles é a extração de biomoléculas potencialmente ativa como as lectinas, que são proteínas que têm sido alvo de pesquisas no mundo inteiro. Isso se deve à sua impressionante capacidade de ligar-se especificamente a carboidratos ou a substâncias que os contêm, propiciando um imenso campo de aplicação biológica. O objetivo deste trabalho foi produzir a lectina da alga marinha vermelha Bryothamnion triquetrum (Lec), avaliar sua estrutura secundária a partir de técnicas espectroscópicas e investigar as suas propriedades anti-inflamatórias em camundongos. Primeiramente a lectina foi purificada por precipitação com sulfato de amônio (0-60) e cromatografia de troca – iônica, sendo recolhido o primeiro pico (PI). Este pico foi selecionado através da atividade de hemaglutinação obtendo assim a lectina pura a partir de Bryothamnion triquetrum, verificada por SDS-PAGE. A lectina foi analisada quanto a sua estrutura secundária por técnicas espectroscópicas de dicroísmo circular, frente a extremos de pH (4,0 – 11,0) e comparada ao espectro da sua estrutura nativa, e fluorescência frente a diferentes temperaturas que variaram de 25° a 75° em intervalo de 10°. Na avaliação das atividades biológicas, grupos de animais foram submetidos ao pré-tratamento com a Lectina (1,5 e10mg/kg; i.p.) e indometacina (10mg/kg), 30 min antes do estímulo inflamatório. A atividade anti-inflamatória em camundongos foi avaliada através dos ensaios de peritonite induzida por carragenina - Cg (500 μg/cav) e de edema de pata induzidos por Cg (500 μg/pata); dextrano (500 μg/pata). Extremos do pH (4,0 e 11,0) não alterou a estrutura secundária da proteína. Os dados de fluorescência (280nm e 295nm), mostraram também uma estabilidade, além da presença de aminoácidos aromáticos na sua estrutura. A Lectina inibiu a migração celular na peritonite induzida por Cg e os edemas de pata induzidos por Cg e dextrano. Deste modo, conclui-se que a Lectina obtida do PI da DEAE de Bryothamnion triquetrum apresentou estrutura estável em relação a variação de pH e temperatura. Em relação às propriedades biológicas testadas, a Lectina apresentou efeitos anti-inflamatórios nos modelos empregados, podendo ser considerada uma molécula com potencial para estudos mais aprofundados de inflamação.

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49

Felix, Wagner Pereira. "ProduÃÃo heterÃloga de frutalina em Pichia pastoris." Universidade Federal do CearÃ, 2008. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=2314.

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A frutalina, lectina α-D-galactose ligante, foi expressa e processada num sistema heterÃlogo para expressÃo de proteÃnas utilizando a levedura metilotrÃfica Pichia pastoris. Para atingir os objetivos esperados foram desenvolvidas trÃs estratÃgias: identificar o gene que codifica para a frutalina a partir do DNA genÃmico obtido de folhas de A. incisa; sintetizar, por uma empresa especializada, o gene que codifica para as cadeias polipeptÃdicas da frutalina, otimizando-o para a expressÃo em P. pastoris e isolar o gene que codifica para a frutalina, a partir do mRNA presente em sementes maduras e frescas, para obtenÃÃo do cDNA utilizando a tÃcnica da RT-PCR. Enquanto na primeira estratÃgia, foi obtido apenas o gen da cadeia beta, nas duas outras estratÃgias o gen completo foi obtido. No entretanto, a estratÃgia que mostrou melhor expressÃo da frutalina recombinante em P. pastoris foi a da obtenÃÃo do gene que codifica para a frutalina, a partir do mRNA. A expresÃo foi tempertura dependente e a frutalina recombinante apresentou uma massa molecular aparente de 17 kDa, sugerindo a presenÃa do peptÃdeo de ligaÃÃo e da cauda de histidina.
Frutalin, the Artocarpus incise alfa-D-galactose binding seed lectin was expressed and processed in a heterologous system for protein expression, using the metilotrophic yeast Pichia pastoris. In order to obtain the proposed objectives, three strategies were followed: identify the frutalin gene from the genomic DNA, obtained from the leaves; obtain, from the GenScript Corporation, the synthetic frutalin polypeptide chains gene, optimized for expression in Pichia system; and isolate frutalin gene, from mRNA present in fresh seeds in order to obtain the cDNA, using the RT-PCR technique. The obtained genes were inserted in the Pichia genome, in order to evaluate the lectin expression. While in the first strategy only the beta chain gene was obtained, in the other two the complete frutalin gene was obtained. The best results were obtained from the third strategy. The expression process was temperature dependent, with the optimum at 18 οC and the recombinant frutalin showed an apparent molecular mass of 17 kDa, which suggest the presence of both the linker peptide and the histidine tag.

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50

Silva, Luana Maria Castelo Melo. "Atividades antinociceptiva e antiinflamatÃria da lectina da alga marinha vermelha Pterocladiella capilacea (S.G. Gmelin) Santelices & Hommersand." Universidade Federal do CearÃ, 2008. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=2313.

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Lectinas de algas marinhas tÃm-se mostrado importantes ferramentas biotecnolÃgicas. Objetivou-se estudar as atividades antinociceptivas e antiinflamatÃrias da lectina da alga marinha vermelha Pterocladiella capillacea (Pc). A Pc, apresentando atividade hemaglutinante contra eritrÃcitos tripsinizados de coelho, foi obtida a partir da aplicaÃÃo do extrato protÃico total em cromatografia de troca iÃnica em coluna de DEAE-celulose seguida da cromatografia de afinidade em coluna de goma de guar. A seguir, foi utilizada nos ensaios de nocicepÃÃo e inflamaÃÃo, utilizando camundongos machos Swiss e ratos machos Wistar, respectivamente. Pc (0,9; 8,1 ou 72,9 mg/kg; i.v) foi administrada 30 min antes de cada estÃmulo nocigÃnico, ou seja, antes da injeÃÃo i.p de Ãcido acÃtico a 0,8% (10 μL/mL), da injeÃÃo intraplantar de formalina a 1% (20 μL/pata) ou do teste da Placa quente (51Â1 ÂC), e comparada a animais nÃo tratados ou prÃ-tratados com Indometacina ou Morfina, ambas a 5 mg/kg; s.c. Observou-se que a Pc (0,9; 8,1 ou 72,9 mg/kg) reduziu significantemente o nÃmero de contorÃÃes abdominais induzidas pelo Ãcido acÃtico em 29,2%; 39,3%, e 51,9%, respectivamente. Pc (72,9 mg/kg) tambÃm reduziu (p<0,05) a fase 1 (neurogÃnica) e a fase 2 (inflamatÃria) observadas apÃs administraÃÃo da formalina, em 58% e 87%, respectivamente. Entretanto, a Pc (72,9 mg/kg) nÃo foi capaz de reduzir a nocicepÃÃo observada no teste da Placa Quente, quando comparada Ã morfina. Os efeitos antinociceptivos da Pc foram abolidos quando a Pc foi prÃ-incubada com a glicoproteÃna mucina (1,25 mg/mL), inibidora de sua atividade hemaglutinante. Sugere-se, portanto, que a atividade antinociceptiva da Pc possa ser predominante via inibiÃÃo de mecanismos perifÃricos. Assim, seguiram-se os ensaios de induÃÃo da migraÃÃo neutrofÃlica para cavidade peritoneal ou do edema de pata de ratos por Carragenana (Cg-tipo l; 500 g/cavidade ou pata), onde observou-se que a administraÃÃo da Pc (8,1 mg/kg; i.v) 30 min antes da Cg reduziu significativamente a contagem do nÃmero de neutrÃfilos em 84%. No entanto, a Pc nÃo foi capaz de prevenir o edema de pata induzido pela Cg. Desta forma, sugere-se que esta proteÃna foi capaz de reduzir o mecanismo de migraÃÃo de neutrÃfilos, possivelmente ligando-se Ã molÃculas especÃficas celulares, como por exemplo, selectinas. Para confirmar sua seguranÃa, a PC (8,1 mg/kg) foi administrada em camundongos diariamente e no 7Â dia foram coletadas amostras sanguÃneas para dosagens de urÃia e transaminases (TGO e TGP), e pesados rins e fÃgado. Observou-se que a Pc nÃo causou alteraÃÃes significativas, sugerindo portanto, ser segura no perÃodo de administraÃÃo avaliado. Dessa forma, considerando os dados em conjunto, conclui-se que a Pc possui propriedades antinociceptiva e antiinflamatÃria com aÃÃo perifÃrica.
Marine algae lectins had been showing important biotechnical tools. Our objectives were to study the antinociceptive and anti-inflammatory activities of the lectin from the marine red alga Pterocladiella capillacea (Pc). The Pc, presenting haemagglutinating activity against trypsin-treated erytrocytes from rabbit, was purified by application of crude extract (0.025 M Tris-HCl buffer, pH 7.5) on ion exchange chromatography on DEAE-cellulose followed by affinity chromatography on guar-gum column. To proceed, it was used in the nocicepÃÃo and inflammation assays, using male Swiss mice and male Wistar rats, respectively. Pc (0.9; 8.1 or 72.9 mg/kg; i.v) it was administered 30 min before each challenge, that is, before the injection i.p of acetic acid 0.8% (10 μl/mL), of the intraplantar injection of 1% formalin (20 μL/paw) or of the Hot Plate test (52Â1 ÂC), and compared to non treated animals or to pre-treated by Indomethacin or Morphine, both at 5 mg/kg; s.c. It was observed that the Pc (0.9; 8.1 or 72.9 mg/kg) reduced significantly the number of writhes induced by acetic acid (29.2%; 39.3%, and 51.9%, respectively). Pc (72.9 mg/kg) also reduced (p<0.05) the 1st phase (neurogenic) and the 2nd phase (inflammatory) observed after administration of the formalin (58% and 87%, respectively). However, the Pc (72.9 mg/kg) was not capable to reduce the nociception evaluated by Hot Plate test, compared to morphine. These antinociceptive effects were abolished when the Pc was pre-incubated with mucin (1.25 mg/mL), inhibitory glycoprotein of its haemagglutinating activity. Therefore, it is suggested that the antinociceptive activity of the Pc can be predominant by inhibition of peripheric mechanisms. After this, was realized the assays of neutrophil migration for peritoneal cavity or of the paw edema of mice by Carragenan (Cg-type l; 500 g/cavity or paw), where was observed that the administration of the Pc (8,1 mg/kg) 30 min before Cg reduced the neutrophil counts significantly by 84%. However, Pc was not capable to prevent the paw edema induced by Cg. This way, it is suggested that this protein was capable to reduce neutrophil migration by previous mechanism to migration, possibly linking to cellular specific molecules as, for example, selectins. Then, to confirm its safety, the Pc (8.1 mg/kg) was administered daily in mice and observed their behaviors, and at the 7th day, sanguine samples were collected for urea and transaminases (TGO and TGP) dosages, and heavy kidneys and liver. It was observed that Pc did not cause significant alterations, suggesting be safety for the administration period. Considering the data together, it is ended that the Pc possesses antinociceptive and anti-inflammatory properties with peripheral action.

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