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The effects of neuroinflammation on the subventricular zone neurogenic compartment following Theiler's Murine Encephalomyelitis virus infection and its regulation by Galectin-3James, Rachel Elizabeth January 2012 (has links)
The subventricular zone (SVZ) is an adult neurogenic niche that contains multipotent stem/progenitor cells that may be a viable target for remyelination in Multiple Sclerosis. In response to demyelination, SVZ progenitors are recruited into myelin lesions. Currently, the effect of inflammation on the endogenous brain stem cell compartment remains poorly characterised. Theiler's murine encephalomyelitis virus (TMEV) induces a demyelinating disease in susceptible SJL/J mice, due to persistence of the virus, which models the chronic progressive form of multiple sclerosis. In contrast, virus is rapidly cleared in TMEV resistant C57BL/6 mice. This viral clearance is critically dependent on infiltration of CD4<sup>+</sup> and CD8<sup>+</sup> T cells during the first 3-14 days after infection, through the blood vessels and meninges. This project aimed to characterise the impact of TMEV induced inflammation on SVZ homeostasis. In both strains of mice the most pronounced and consistent inflammation in the CNS was observed in periventricular regions and in particular, the SVZ, which showed targeted infection by the TMEV virus. However, the time course and kinetics for infiltration at 3,7 and 14 days post infection have very different profiles between resistant C57BL/6 and susceptible SJL mice. Using RT-PCR arrays and ELISA I have shown that these differences in T cell infiltration to the SVZ may be due to much higher chemokine and cytokine expression levels in B6 mice. TMEV infection decreases SVZ cell proliferation and results in a loss of neuroblast numbers. Galectin-3 (Gal-3) is a β-galactoside binding protein that is constitutively expressed specifically in the SVZ. Following TMEV infection Gal-3 levels are significantly upregulated in the SVZ, with higher expression in B6 mice compared to SJL mice. Primary SVZ astrocytes secrete extracellular Galectin-3 at much higher levels than cortical astrocytes. Galectin-3 is a pro-inflammatory mediator which upon secretion is able to activate immune and inflammatory signaling events and amplify pro-inflammatory cytokine production. Both SJL and B6 Gal-3<sup>-/-</sup> KO mice have decreased expression of CCL2, CCL5 CXCL10 and CCL8 chemokines in the SVZ after TMEV infection. Deletion of Gal-3 prevents the loss of SVZ proliferation and in B6 mice decreases hematopoietic cell infiltration and enhances ectopic neuroblast emigration. These data implicate Galectin- 3 as a novel regulator of the SVZ inflammatory response and may provide a new target for regulating T cell CNS immigration in autoimmune disease.
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Evidência da dualidade funcional de galectina-3 no crescimento de melanoma murino / Evidence for a dual role of galectin-3 in murine melanoma growthAndrade, Luciana Nogueira de Sousa 17 April 2007 (has links)
Tumores são definidos como microambientes compostos não só pelas células malignas, mas também por células endoteliais, fibroblastos e leucócitos, que promovem o crescimento tumoral e a angiogênese. Galectina-3, uma proteína que se liga a b- galactosídeos, é abundantemente expressa por monócitos/macrófagos, dentre outros leucócitos. Inúmeras evidências sugerem que galectina-3 atua como uma molécula reguladora da resposta inflamatória. Tendo em vista que o infiltrado inflamatório pode promover a progressão de tumores, o objetivo do presente trabalho foi avaliar se galectina-3, expressa tanto pela célula tumoral como pelas células estromais, modula o crescimento de melanoma. Para tal, células de melanoma murino Tm1 foram transfectadas com o gene de galectina-3. Ambos clones celulares (galectina-3 positivos e negativos) foram injetados na intrafáscia ou no subcutâneo de camundongos (fêmeas) C57BL/6 selvagens e/ou nocautes para o gene de galectina-3 para análise da implantabilidade e crescimento tumoral. Com relação à implantabilidade, não foi observado diferenças no estabelecimento de uma massa tumoral proliferativa em animais selvagens inoculados com células Tm1 transfectadas ou não com o gene de galectina-3 em animais selvagens. Em relação a taxa de crescimento dos tumores, nenhum animal nocaute inoculado com células Tm1 galectina-3 positivas apresentou tumores de dimensões mensuráveis até o 11º dia pós-inóculo. Independente do nível de expressão de galectina- 3 pela célula tumoral, os tumores originados nos animais nocautes apresentavam menor massa em gramas comparados ao grupo selvagem, sugerindo que galectina-3 expressa pelas células estromais promove o crescimento tumoral. Ainda, os tumores originados nos animais nocautes e no grupo selvagem inoculado com células Tm1 galectina-3 positivas apresentavam menor extensão de área necrótica do que os animais selvagens inoculados com células Tm1 galectina-3 negativas. Interessantemente, os animais selvagens e nocautes inoculados com células Tm1 galectina-3 positivas apresentaram tumores com menor área vascular e menor número de estruturas vasculares funcionais quando comparados aos animais selvagens inoculados com células Tm1 galectina-3 negativas. A análise de expressão gênica nos tumores mostrou que os níveis relativos de RNAm de VEFG (fator de crescimento de endotélio vascular) foram menores nos animais inoculados com células Tm1 galectina-3 positivas em relação aos inoculados com células Tm1 galectina-3 negativas, indicando que galectina-3 expressa pelas células tumorais atua como uma molécula anti-angiogênica. Finalizando, o presente trabalho sugere que galectina-3 pode atuar como uma molécula pró- ou anti-tumoral, dependendo do tipo celular que a expressa no microambiente tumoral. / Tumors have been described as microenvironments composed not only by malignant cells, but also by endothelial cells, fibroblasts and leukocytes, which can promote tumor growth and angiogenesis. Galectin-3, a b-galactoside binding protein, is expressed by monocytes/macrophages and others leukocytes. In fact, several lines of evidence suggest that galectin-3 act as master regulators of the inflammatory response. Based on the fact that the inflammatory infiltrate can promote tumor progression, the proposal of this study was to evaluate if galectin-3, either from tumor or stromal cells could modulate melanoma growth. Tm1 murine melanoma cell line was transfected with the galectin-3 gene. Both clones (galectin-3 negative and positive) were injected in the foot pad or subcutaneous in female C57BL/6 wild-type (WT) and galectin-3 knock-out (KO) mice to tumor engraftment and growth analysis. There was no difference in the tumor engraftment between animas injected with Tm1 galectin-3 positive or negative cells. In addition, any knock-out mice injected with galectin-3 positive cells had measurable tumors up to day 11 post inoculation. Regardless the galectin-3 expression level in the melanoma cell, tumors from galectin-3 KO mice were smaller than those from WT animals, suggesting that galectin-3 expressed by stromal cells promotes tumor growth. Moreover, tumor necrotic area was smaller in KO mice and in wild-type animals injected with Tm1 galectin-3 positive cells compared to wild type animals injected with Tm1 galectin-3 negative cells. Interestingly, both vascular area and the number of functional vessels in animals injected with galectin-3 positive Tm1 cells were smaller in WT as well as in KO mice compared to the same animals injected with galectin-3 negative Tm1 cells. Gene expression analysis showed that VEGF (vascular endothelial growth factor) mRNA levels were smaller in wild type animals injected with Tm1 galectin-3 positive cells compared to those injected with Tm1 galectin-3 negative cells, indicating that galectin-3 expressed by tumor cells can act as an anti-angiogenic molecule. The present study suggests that galectin-3 can act either as a pro or antitumoral molecule, depending on which type of cell (tumoral or stromal) this lectin is expressed within tumor microenvironment.
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Avaliação da interação entre galectina-1 e zinco e suas potenciais implicações estruturais e funcionais / Evaluation of the interaction between Galectin-1 and Zinc and their potential structural and functional implicationsSilveira, Willian Abraham da 01 July 2011 (has links)
Introdução: A Galectina-1 (Gal-1) é uma proteína multifuncional capaz de reconhecer, de modo específico, glicanas compostas por resíduos de -galactosídeos, por meio de domínios de reconhecimento de carboidrato (CRD). A Gal-1 é um homodímero de 14.900 daltons, pI = 5.6, apresenta uma topologia molecular do tipo jelly-roll composto por duas folhas- anti-paralelas. Além disso, esta proteína não apresenta peptídeo sinal e possui 6 cisteínas, 7 ácidos glutâmicos, 9 ácidos aspárticos e 4 histinas por monômero. A Gal-1 liga-se a diferentes moléculas biológicas contidas nas superfícies celulares, núcleo e componentes da matriz extracelular. O zinco é um importante metal em sistemas biológicos. Aproximadamente 10% do proteoma humano é potencialmente capaz de complexar zinco. Este íon exibe propriedades adequadas tanto para funções catalíticas, quanto estruturais em proteínas. Os sítios de ligação a zinco, nas proteínas, podem ser divididos em catalíticos, estruturais, co-catalíticos e sítios na interface protéica. Geralmente, os resíduos de cisteína, histidina, ácido glutâmico e ácido aspártico são alvos preferênciais de interação com Zn. Há na literatura dados que mostram a interação da Gal-1 humana com íons orgânicos, porém não há relatos sobre a interação Gal-1/Zn . Objetivos: O presente trabalho teve como objetivo avaliar a existência e as implicações da interação entre o íon Zn2+ e a proteína Gal-1. Materiais e Métodos: Foi efetuada a produção, purificação e padronização do uso das formas dimérica e monomérica da Gal-1 recombinante humana. A interação Gal-1/Zn foi avaliada através de ensaios biofísicos e biológicos. A análise in vitro e in silico dos paramêtros biofísicos, foi feita através de espectrofluorimetria, de dicroísmo circular, de ensaio de precipitação, do método GRID e por dinâmica molecular. A análise in vitro dos parâmetros biológicos, foi realizada por meio de ensaio de hemaglutinação e interação com laminina por ELISA. Resultados e Discussão: A adição de ZnCl2 numa solução de Gal-1 causa aumento da emissão por fluorescência do triptofano e uma alteração para o vermelho, altera o espectro de dicroísmo circular e causa precipitação protéica da Gal-1. Estes eventos ocorreram de forma seletiva e dependente da concentração desse íon. As análises in silico indicam que o provável sítio de complexação Zn/Gal-1 é distinto do CRD e é formado pelos aminoácidos Glu-15, Asp-92 e Asp-134, assumindo a conformação trigonal bipiramidal e tendo número de coordenação igual a 5. Conclusão: As análises biofísicas in vitro e in silico, nos indicam que a Galectina-1 tem a capacidade de se complexar com o íon Zn2+. / Introduction: Galectin-1 (Gal-1) is a multifunctional protein that specifically recognizes glycans with -galactosides through carbohydrate recognition domains (CRD). Gal-1 is a homodimeric protein of 14.900daltons, pI=5.6, shows a jelly-roll molecular topology composed of two anti-parallels - sheet, has no signal peptide and contains 6 cysteines, 7 glutamic acids, 9 aspartic acids and 4 histidines per monomer. This lectin binds to different biological molecules contained in the cell surface, nucleus and extracellular matrix components. Zinc is an important metal in biological systems because can participate in the maintenance of protein structure and biological activity. Usually, cysteine , histidine, glutamic acid and aspartic acid residues are preferential targets for interaction with Zn. Approximately 10% of the human proteome is potentially capable to forming complexes with Zn. The Zn2+ ion exhibits properties suitable for both catalytic and structural protein functions. Proteins zinc binding sites can be divided into catalytic, structural, co-catalytic and protein interface sites.There are reports in the literature that shows the interaction between galectin-1 and organic ions. However, were not found reports about Zn-Gal-1 complexes. Objective: The aim of this study was to evaluate the existence and implications of the interaction between galectin-1 and Zn2+ ion. Materials and Methods: Human recombinant Gal-1 (monomer and dimmer) was obtained and purified. Also, the conditions for the use of Gal-1 were standardized. The interaction Zn/Gal-1 was assessed by biophysical an biological procedures. The analysis in vitro and in silico was made by spectrofluorimetry, circular dichroism, precipitation test, method of GRID, and molecular dynamics. The in vitro analysis of biological parameters were performed by hemmaglutination and laminin binding (ELISA) tests. Results and Discussion: The addition of ZnCl2 in Gal-1 solution causes increased fluorescence emission of tryptophan-70 and a red shift, alters the circular dichroism spectrum and causes precipitation of Gal-1 protein. These events occurred in a selective manner dependent of Zinc concentration. The in silico analysis indicates that the probable site of Zn/Gal-1 complexation is distinct from the CRD and is formed by the amino acids Glu-15, Asp-92 and Asp-134, assuming trigonal bipyramidal conformation and with coordination number equal to 5 . Conclusion: The biophysical in vitro and in silico findings suggests that Galectin-1 has the ability to complex with the Zn2+ ion.
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Influência da galectina-3 na resposta de neutrófilos a patógenos periodontais / Influence of galectin-3 on neutrophil response to periodontal pathogensGarcia, Rudan Paraíso 04 March 2016 (has links)
Galectina-3, uma proteína que se liga a -galactosídeos, é expressa por neutrófilos e inúmeras evidências indicam que esta molécula atua como uma possível reguladora da resposta imune. Sabe-se que galectina-3 ao ligar com LPS pode levar a formação de oligômeros, que podem alterar o limiar de ativação de células da resposta imune inata. Apesar de existirem diversos estudos que mostram a influência de galectina-3 na resposta de neutrófilos frente a componentes bacterianos, os resultados são em sua maioria contraditórios e inconclusivos. Para elucidar a influência da galectina-3 na reposta imune inata a patógenos periodontais, o presente trabalho avaliou a atividade antimicrobiana in vitro de neutrófilos, isolados de camundongos selvagens (WT) ou geneticamente deficientes de galectina-3 (Gal-3KO), previamente estimulados com LPS de Aa e Pg. Os resultados não evidenciaram diferenças significativas no número de unidades formadoras de colônia (UFC) recuperadas das culturas de neutrófilos provenientes de animais deficientes de galectina-3 e do grupo controle (WT). Contudo, a estimulação de neutrófilos com LPS por 18 horas levou a redução no número de UFC recuperadas das culturas, quando comparado com as culturas estimuladas com LPS por apenas 3 horas. / Galectin-3, a protein that binds -galactosides, is expressed by neutrophils and numerous evidences indicate that this molecule acts as a possible regulator of the immune response. It is known that galectin-3 binding to LPS can lead to the formation of oligomers and thus changing the activation threshold of cells of the innate immune response. Although there are several studies that show the influence of galectin-3 in neutrophil response against bacterial components, the results are conflicting and inconclusive in their majority. To elucidate the influence of galectin-3 in the innate immune response to periodontal pathogens, the present study evaluated the in vitro antimicrobial activity of neutrophils, isolated from wild-type or galectin-3 deficient mice, previously stimulated with LPS of Aa and Pg. The results showed no significant differences in the number of colony forming units (CFU) recovered from cultured galectin-3 deficient neutrophils or control group. However, in a 18 hours time course of LPS stimulation, we observed reduction in the number of CFU, when compared to 3 hours of LPS stimulation.
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Galectin-3 regulation of non small cell lung cancer growthKouverianou, Eleni January 2014 (has links)
Galectin-3 is a β-galactoside binding lectin expressed in tumour cells and macrophages and has been associated with increased malignancy in a variety of cancers. Previous work has shown that galectin-3 is an important regulator of macrophage function, promoting an alternative (M2) phenotype which potentiates chronic inflammation and fibrosis. Tumour associated macrophages (TAMs) adopt an M2 phenotype and are thought to promote tumour growth by down regulating T cell effector function and promoting angiogenesis. This project examines the hypothesis that host galectin-3 promotes lung cancer growth and spread. In order to test this hypothesis, Lewis Lung Carcinoma tumour growth and metastasis was investigated in strain matched mice either expressing or deficient in galectin-3. The Lewis Lung Carcinoma cell line (LLC1) is a spontaneous lung carcinoma line, derived from C57BL/6 mice, which readily forms tumours when transplanted. Furthermore, LLC1 cells were stably transfected with a Luciferase expressing vector in order to assist detection of tumour growth and metastasis in vivo. An orthotopic model of LLC1 growth suggested that galectin-3-/- animals do not support lung carcinoma growth and spread. This finding was confirmed by a subcutaneous model of cancer growth, where it was found that wild type animals display a higher proportion of macrophages expressing a prototypic M2 marker around tumour sites compared to galectin-3-/- animals. M2-promoting cytokine transcripts were also reduced in galectin-3-/- mice. Additionally, tumours of wild type mice were more invasive and presented more mature blood vessels compared to galectin-3-/- mice. To specifically address the role of recruited cells on tumour growth, metastasis and the inflammation profile around tumour sites, in relation to galectin-3 expression, bone marrow cells (BMCs) were transplanted from wild type to galectin-3-/- mice and vice versa. It was shown that galectin-3 positive BMCs restore the wild type phenotype of tumour growth in galectin-3-/- mice, while galectin-3 deficient BMCs impair tumour growth in wild type animals. Furthermore, macrophage ablation experiments demonstrated incapacity for tumour establishment in the absence of macrophages. A series of experiments investigating reported inhibition of galectin-3 by modified citrus pectin (MCP) via competitive inhibition did not provide conclusive results. MCP had no effect in vivo, but was able to inhibit LLC1 cell growth in vitro. Most importantly though, results were inconclusive as to whether galectin-3 binds MCP. Some ligand displacement was seen, but direct binding of the molecules could not be shown. In general, the results obtained demonstrate a strong pro-tumoural effect of galectin-3 on growth, tissue invasion and metastasis of LLC1 tumours via an increased proportion of Ym1-expressing macrophages around tumour sites. It was shown that macrophages are key cells for tumour initiation and that BMC phenotype in relation to galectin-3 expression determines the phenotype of tumour development in subcutaneous and orthotopic LLC1 models. Therefore, galectin-3 has a strong regulatory effect on tumour phenotype and could present a key target in the management of lung carcinomas.
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Therapeutic Cancer Vaccines Targeting Molecules Associated with Tumor AngiogenesisFemel, Julia January 2014 (has links)
Induction of an endogenous antibody response by therapeutic vaccination could provide an alternative to cost-intensive monoclonal antibody-based treatments for cancer. Since the target of a cancer vaccine will most likely be a self-antigen, self-tolerance of the immune system must be circumvented. Using fusion proteins consisting of the self-antigen to be targeted and a part derived from a foreign antigen, it is possible to break tolerance against the self-antigen. Furthermore, a potent adjuvant is required to support an immune response against a self-molecule. Currently no adjuvant suitable for this purpose is approved for use in humans. This thesis describes the development of a therapeutic vaccine targeting the vasculature of tumors. As tumor cells have developed strategies to escape immune surveillance, targeting of molecules associated with the tumor stroma is an interesting alternative. The alternatively spliced extra domain-A and B (ED-A and ED-B) of fibronectin and the glycan-binding protein galectin-1 are selectively expressed during events of tumor angiogenesis. We have designed recombinant proteins to target ED-B, ED-A and galectin-1, containing bacterial thioredoxin (TRX) as a non-self part, resulting in TRX-EDB, TRX-EDA and TRX-Gal-1. Vaccination against ED-B induced anti-ED-B antibodies and inhibited growth of subcutaneous fibrosarcoma. Immunization against ED-A decreased tumor burden and reduced the number of lung metastases in the MMTV-PyMT model for metastatic mammary carcinoma in a therapeutic setting. Analysis of the tumor tissue from ED-B and ED-A-immunized mice indicated an attack of the tumor vasculature by the immune system. Finally, we show that galectin-1 immunization reduced tumor burden and increased leukocyte numbers in the tumor tissue. Galectin-1 is pro-angiogenic and immunosuppressive, and therefore allows simultaneous targeting of fundamental characteristics of tumorigenesis. We furthermore show that the biodegradable squalene-based Montanide ISA 720 combined with CpG oligo 1826 (M720/CpG) is at least as potent as Freund’s adjuvant with respect to breaking self-tolerance, when comparing several immunological parameters. Freund’s is a potent but toxic adjuvant used in the majority of preclinical studies. The work presented in this thesis shows that therapeutic cancer vaccines targeting the tumor vasculature are a feasible and promising approach for cancer therapy.
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Glykobiologie nádorů hlavy a krku / Glycobiology of the head and neck cancerSzabo, Pavol January 2012 (has links)
Povrch buněk je bohatě pokryt oligosacharidy, které jsou v plazmalemě ukotvené pomocí proteinů a lipidů. Oligosacharidy zprostředkují vzájemnou vazbu mezi buňkami nebo vazbu buněk k složkám extracelulární matrix. Galektiny jsou živočišné lektinů které mají afinitu k oligosacharidům obsahujícím β-galaktózu. Jsou to multifaktoriální proteiny, které se účastňují řady reakcí v organizmu, jako jsou mezibuněčné interakce, interakce buněk s mezibuněčnou hmotou, proliferace i apoptóza a sestřih pre-mRNA. Proteiny po translaci procházejí různými strukturálnimy úpravami, které mají vliv na jejich funkci. Galektin-3 je možný prognostický ukazatel u nádorů vycházejících z vrstevnatých dlaždicových epitelů je fosforylován na N-konci. Prokázali jsme, že tato posttranslační modifikace nemá vliv na jeho vazebnou reaktivitu. Jiný endogenní lektin, galektin-1 je charakteristickou molekulou nádorového stromatu a granulační tkáně hojícího se poranění. Zjistili jsme, že galektin-1 indukuje na TGF-β nezávislý in vitro přechod normálních fibroblastů na myofibroblasty včetně produkce sítě extracelulární matrix bohaté fibronektinem a galektinem-1. Tento poznatek je využitelný v terapii hojení ran a v tkáňovém inženýrství. Dnes je jasné, že nádorové stroma ovlivňuje i biologické vlastnosti nádoru (lokální agresivita,...
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Hojící se rána jako model pro studium buněčných interakcí. / Healing wound as a model for the study of cell interactionsGál, Peter January 2015 (has links)
Healing wound as a model for the study of cell interactions Abstract Galectins play an important role in the processes of cell proliferation, differentiation, migration and extracellular matrix formation. Furthermore, galectins are able to transfer cellular signals and to participate in cell interaction. It has been proven that galectins play an important role in the microenvironment formation of a tumor and/or healing wound. This study demonstrated significant role of galectins, in particular Galectin-1, in wound healing and cell interactions (endothelial cells, fibroblasts and keratinocytes) forming a part of the granulation tissue and tumor stroma. We have demonstrated that the extracellular matrix rich on Galectin-1 creates a suitable environment for the cultivation of keratinocytes. Galectin-1 also induces differentiation of fibroblasts into myofibroblasts. The knowledge of above mentioned processes is important to better understand the complexity of cancer biology and its parallel to wound healing. Key words: tissue repair, regeneration, galectin, tumor
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Influência da galectina-3 na resposta de neutrófilos a patógenos periodontais / Influence of galectin-3 on neutrophil response to periodontal pathogensRudan Paraíso Garcia 04 March 2016 (has links)
Galectina-3, uma proteína que se liga a -galactosídeos, é expressa por neutrófilos e inúmeras evidências indicam que esta molécula atua como uma possível reguladora da resposta imune. Sabe-se que galectina-3 ao ligar com LPS pode levar a formação de oligômeros, que podem alterar o limiar de ativação de células da resposta imune inata. Apesar de existirem diversos estudos que mostram a influência de galectina-3 na resposta de neutrófilos frente a componentes bacterianos, os resultados são em sua maioria contraditórios e inconclusivos. Para elucidar a influência da galectina-3 na reposta imune inata a patógenos periodontais, o presente trabalho avaliou a atividade antimicrobiana in vitro de neutrófilos, isolados de camundongos selvagens (WT) ou geneticamente deficientes de galectina-3 (Gal-3KO), previamente estimulados com LPS de Aa e Pg. Os resultados não evidenciaram diferenças significativas no número de unidades formadoras de colônia (UFC) recuperadas das culturas de neutrófilos provenientes de animais deficientes de galectina-3 e do grupo controle (WT). Contudo, a estimulação de neutrófilos com LPS por 18 horas levou a redução no número de UFC recuperadas das culturas, quando comparado com as culturas estimuladas com LPS por apenas 3 horas. / Galectin-3, a protein that binds -galactosides, is expressed by neutrophils and numerous evidences indicate that this molecule acts as a possible regulator of the immune response. It is known that galectin-3 binding to LPS can lead to the formation of oligomers and thus changing the activation threshold of cells of the innate immune response. Although there are several studies that show the influence of galectin-3 in neutrophil response against bacterial components, the results are conflicting and inconclusive in their majority. To elucidate the influence of galectin-3 in the innate immune response to periodontal pathogens, the present study evaluated the in vitro antimicrobial activity of neutrophils, isolated from wild-type or galectin-3 deficient mice, previously stimulated with LPS of Aa and Pg. The results showed no significant differences in the number of colony forming units (CFU) recovered from cultured galectin-3 deficient neutrophils or control group. However, in a 18 hours time course of LPS stimulation, we observed reduction in the number of CFU, when compared to 3 hours of LPS stimulation.
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Avaliação da interação entre galectina-1 e zinco e suas potenciais implicações estruturais e funcionais / Evaluation of the interaction between Galectin-1 and Zinc and their potential structural and functional implicationsWillian Abraham da Silveira 01 July 2011 (has links)
Introdução: A Galectina-1 (Gal-1) é uma proteína multifuncional capaz de reconhecer, de modo específico, glicanas compostas por resíduos de -galactosídeos, por meio de domínios de reconhecimento de carboidrato (CRD). A Gal-1 é um homodímero de 14.900 daltons, pI = 5.6, apresenta uma topologia molecular do tipo jelly-roll composto por duas folhas- anti-paralelas. Além disso, esta proteína não apresenta peptídeo sinal e possui 6 cisteínas, 7 ácidos glutâmicos, 9 ácidos aspárticos e 4 histinas por monômero. A Gal-1 liga-se a diferentes moléculas biológicas contidas nas superfícies celulares, núcleo e componentes da matriz extracelular. O zinco é um importante metal em sistemas biológicos. Aproximadamente 10% do proteoma humano é potencialmente capaz de complexar zinco. Este íon exibe propriedades adequadas tanto para funções catalíticas, quanto estruturais em proteínas. Os sítios de ligação a zinco, nas proteínas, podem ser divididos em catalíticos, estruturais, co-catalíticos e sítios na interface protéica. Geralmente, os resíduos de cisteína, histidina, ácido glutâmico e ácido aspártico são alvos preferênciais de interação com Zn. Há na literatura dados que mostram a interação da Gal-1 humana com íons orgânicos, porém não há relatos sobre a interação Gal-1/Zn . Objetivos: O presente trabalho teve como objetivo avaliar a existência e as implicações da interação entre o íon Zn2+ e a proteína Gal-1. Materiais e Métodos: Foi efetuada a produção, purificação e padronização do uso das formas dimérica e monomérica da Gal-1 recombinante humana. A interação Gal-1/Zn foi avaliada através de ensaios biofísicos e biológicos. A análise in vitro e in silico dos paramêtros biofísicos, foi feita através de espectrofluorimetria, de dicroísmo circular, de ensaio de precipitação, do método GRID e por dinâmica molecular. A análise in vitro dos parâmetros biológicos, foi realizada por meio de ensaio de hemaglutinação e interação com laminina por ELISA. Resultados e Discussão: A adição de ZnCl2 numa solução de Gal-1 causa aumento da emissão por fluorescência do triptofano e uma alteração para o vermelho, altera o espectro de dicroísmo circular e causa precipitação protéica da Gal-1. Estes eventos ocorreram de forma seletiva e dependente da concentração desse íon. As análises in silico indicam que o provável sítio de complexação Zn/Gal-1 é distinto do CRD e é formado pelos aminoácidos Glu-15, Asp-92 e Asp-134, assumindo a conformação trigonal bipiramidal e tendo número de coordenação igual a 5. Conclusão: As análises biofísicas in vitro e in silico, nos indicam que a Galectina-1 tem a capacidade de se complexar com o íon Zn2+. / Introduction: Galectin-1 (Gal-1) is a multifunctional protein that specifically recognizes glycans with -galactosides through carbohydrate recognition domains (CRD). Gal-1 is a homodimeric protein of 14.900daltons, pI=5.6, shows a jelly-roll molecular topology composed of two anti-parallels - sheet, has no signal peptide and contains 6 cysteines, 7 glutamic acids, 9 aspartic acids and 4 histidines per monomer. This lectin binds to different biological molecules contained in the cell surface, nucleus and extracellular matrix components. Zinc is an important metal in biological systems because can participate in the maintenance of protein structure and biological activity. Usually, cysteine , histidine, glutamic acid and aspartic acid residues are preferential targets for interaction with Zn. Approximately 10% of the human proteome is potentially capable to forming complexes with Zn. The Zn2+ ion exhibits properties suitable for both catalytic and structural protein functions. Proteins zinc binding sites can be divided into catalytic, structural, co-catalytic and protein interface sites.There are reports in the literature that shows the interaction between galectin-1 and organic ions. However, were not found reports about Zn-Gal-1 complexes. Objective: The aim of this study was to evaluate the existence and implications of the interaction between galectin-1 and Zn2+ ion. Materials and Methods: Human recombinant Gal-1 (monomer and dimmer) was obtained and purified. Also, the conditions for the use of Gal-1 were standardized. The interaction Zn/Gal-1 was assessed by biophysical an biological procedures. The analysis in vitro and in silico was made by spectrofluorimetry, circular dichroism, precipitation test, method of GRID, and molecular dynamics. The in vitro analysis of biological parameters were performed by hemmaglutination and laminin binding (ELISA) tests. Results and Discussion: The addition of ZnCl2 in Gal-1 solution causes increased fluorescence emission of tryptophan-70 and a red shift, alters the circular dichroism spectrum and causes precipitation of Gal-1 protein. These events occurred in a selective manner dependent of Zinc concentration. The in silico analysis indicates that the probable site of Zn/Gal-1 complexation is distinct from the CRD and is formed by the amino acids Glu-15, Asp-92 and Asp-134, assuming trigonal bipyramidal conformation and with coordination number equal to 5 . Conclusion: The biophysical in vitro and in silico findings suggests that Galectin-1 has the ability to complex with the Zn2+ ion.
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