Aspectos estruturais da mucilagem de Pereskia aculeata, Mill (ora pro nóbis)

Orientador: Dr. João Batista Chaves CorrêaTese (Doutorado) - Universidade Federal do Paraná, Curso de Pós-Graduação em BioquímicaInclui referências: p. 115-134Resumo: A extração aquosa, a 50°, do pó acetônico de folhas de Pereskia aculeata , deu origem a um material mucilaginoso que , após precipitação com cetavlon e tratamento de SEVAG, originou o polissacarídeo P. Esse, contém 3,5% de proteína e é constituído por unidades de arabinose, galactose, ramnose e ácido galacturônico numa relação molar de respectivamente 5,1: 8,2: 1,8: 1,0. A análise cromatográfica do polissacarídeo P, colorido com Azul de Procion, em coluna de Sephadex, bem como a de fracionamento em coluna de DEAE-celulose, indicaram que o mesmo é polidisperso. Os espectros de 13C-n.m.r em D2O das três principais frações obtidas em DEAE - celulose , F-2, F-3 e F-7 mostraram sinais correspondentes às unidades de arabinofuranose e ramnopiranose e, para F-2 e F-3, também de éster metílico. Além disso, esses espectros foram bem mais definidos do que aqueles do polissacarídeo P em D2O e em DMS0- 2H6, que apresentou cinco sinais de arabinofuranose, correspondentes aqueles da fração F-2, e que aparecem sobrepostos a alguns extremamente amplos. Esses últimos correspondem as unidades de galactose, cujo movimento molecular é limitado, provavelmente, devido a própria complexação dos polissacarídeos. Essa estrutura baseada em pontes de hidrogénio, foi, então, dissociada após a passagem através da coluna de DEAE-celulose, As técnicas convencionais de analise química indicaram que o polissacarídeo P, possui na cadeia principal unidades de -D galactopiraranose interligadas (1 - 4) e parcialmente substituídas em 0-3 por unidades de B - L arabinopiraranoses, que são por seu turno di- 0 - substituídas em 0-2 e 0-4 por outros terminais não redutores de a - L arabinopiraranoses. As unidades de ácido galacturônico estão presentes internamente ou como grupos terminais não redutores ligados (1 - 2) as unidades de ramnopiranose. Na análise por B - eliminação em meio alcalino, ficou demonstrado, que a ligação polissacarídeo - proteína, ocorre entre arabinose e hidroxiprolina, enquanto que o tratamento do polissacarídeo P com lítio metálico em etilenodiamina, originou um produto B - degradado, através das unidades de ácido galacturônico esterificadas. Além de outros constituintes o complexo arabinogalactana- proteína apresenta 25 moles% de grupos 0-acetil. A localização dos grupos 0-acetil, como foi determinada pelo método de BOUVENG, ocorre principalmente como 3 - 0 - acetil -arabinopiranose (12%), 3-0-aceti 1-ramnopiranose (11%) e 2 - 0 - aceti 1-gal actopiranose (3%) e foi determinada por g.l.c-m.s através da análise dos 0-metil alditóis acetatos em coluna de DB-210 que, mostrou ser a mais eficiente, para a separação dos derivados de arabinitol. A distribuição mais exata foi efetuada substituindo os grupos 0 - acetil por 0 - tri deuterometil, formando um polissacarídeo que foi sequencialmente 0 - metilado e convertido em acetatos de alditóis parcialmente deutero - 0 - metilados. A análise por g.l.c-m.s , interpretada juntamente com o mecanismo de fragmentação por i . e ., mostrou que no heteropolímero os grupos 0 - acetil estão distribuídos em mais do que uma posição de um mesmo açúcar. A solução aquosa do heteropolissacarídeo P que apresenta viscosidade máxima entre pH 4,4 -5,6 a temperatura ambiente, sofre redução dessa propriedade reolõgica, tanto pela presença de sais como pela variação do pH . Todavia, apresenta a mesma viscosidade em diferentes temperaturas.Abstract: 1. Aqueous extraction at 50° of the leaves of Pereskia aculeata gave rise to a mucilaginous material, which after Cetavlon precipitation and Sevag treatment, provided purified polysaccharide P containing 3.5% protein and units of arabinose, galactose, rhamnose and galacturonic acid in a 5.1: 8.2 :1.8: 1.0 molar ratio. Dyeing with Procion Blue, followed by fractionation on columns of Sephadex and of DEAE-cellulose showed that P was polydisperse. 2. DEAE- c e l1ulose column chromatography of P gave 3 o 13 main fractions F-2, F-3, and F-7. Their C-n.m.r spectra, in D2 O solution, were well defined with recognizable signals cor responding to arabinofuranosyl and rhamnopyranosyl units, and in the case of F-2 and F-3, methyl ester. These spectra were much better defined than those of P in D2 O and in DMS0- Hg, which consisted mainly of 5 sharp signals of arabinofuranose, corresponding to those of fractions F-2, superimposed on e x tremely broad ones. The latter arise from material consisting main ly of galactosyl units, whose molecular motion is limited probably by high complexation of the compoments p o lysaccharides .Such a structure based on hydrogen bonding was thus dissociated on passage through the DEAE-celulose column. 3. Overall chemical analysis of P , using conventional chemical techniques ,showed a main chain consisting of (1 4 )- inked-g-Q-galactopyranosyl units partly substituted at 0 - 3 with residues of B - L - a r abinopyranose , which are in turn, di-0- substituted at 0-2 and 0-4 with nonreducing end-groups of a-Larabinouranose . Galacturonic acid units are present as nonre ducing end-groups, or as internal ones linked (1 + 2 ) to rhamnopyranosyl residues. 4. The polysaccharide-protein linkage in P was found to consist of units of arabinose combined to hydrox y p r o 1 i n e , and the presence of galacturonic acid residues esterified with m e thyl groups was consistent with degradation via 6-elimination of polysaccharide P in ethylenediamine containing lithium. 5. Heteropolysaccharide P was also found to contain 25 moles % of 0-acetyl groups, which were located by the method of BOUVENG. £-Acetyl groups were replaced with those of 0-methyl and the partly 0-methylated polysaccharide converted to amixture of -methylalditol acetates, which was examined by g.l.cm. s , on DB-210, which is efficient in separating the arabinitol derivatives. Characterized were acetates of 3-0^-methyl arabinitol (12%), 3-0-methylrhamnitol (11%), and 2 -0-methylgalactitol (3%). A more exact distribution of 0-acetyl groups was achieved via a similar replacement, but incorporating trideute romethylation, followed by conversion to a mixture of corres ponding trideuteromethylated alditol acetates. G.l.c-m.s analy sis showed the location of the ester group in each type of struc: tural unit and that they occurred not in one position but are distributed throughout the available hydroxyl groups of each unit. 6 . Maximum viscosity pf polysaccharide P was observed at 25 and at pH 4.4 to 5.6 . Reduction of viscosity occurred on addition of salts, but is invariable with the temperature

Assembling of xyloglucans and lectin onto si wafers and onto amino-terminated surfaces

Immobilization of xyloglucans extracted from Hymenaea coubaril (HXG) and Tamarindus indica seeds (TXG) on Si/SiO2 wafers or amino-terminated wafers from aqueous solution at concentration of 0.5 g L-1 and pH 3.5 has been investigated by means of ellipsometry and atomic force microscopy (AFM) measurements. Experiments were carried out under equilibrium conditions (adsorption) and non-equilibrium conditions (casting). Under equilibrium conditions neither TXG nor HXG chains adsorbed from solution onto Si/SiO2 surfaces, indicating that negatively charged SiO- groups on the surface do not attract XG chains. Casting TXG and HXG solutions onto Si/SiO2 surfaces led to layers (2.4 ± 0.4) nm and (3.8 ± 0.9) nm thick, respectively. TXG and HXG adsorbed onto amino-terminated surfaces forming layers (1.0 ± 0.1) nm and (1.3 ± 0.1) nm thick, respectively. Upon casting solutions of TXG and HXG onto amino-terminated surfaces, aggregates and fibrils appeared more frequently on the surface, increasing the mean thickness and roughness values. Regardless the substrate, HXG chains tended to form thicker layers than TXG chains did. This trend can be explained with basis on the molecular characteristics of HXG, namely, higher molecular weight and persistence length. The adsorption isotherms of concanavalin A (Con A) onto HXG- and TXG-covered amino-terminated wafers presented maximum adsorbed amount of (3.3 ± 0.3) mg m-2. AFM images shown Con A molecules as small entities densely packed on the surface. The presence of fibrils and aggregates was observed only when the TXG and HXG surfaces were prepared by casting. There ConA molecules adsorbed predominantly on regions free of fibrils and aggregates

Formation, drug-release kinetics and solution-stability of N-acetyl-N-carboxymethyl chitosan nanoparticles as potential drug carriers

Nano-aggregates of N-acetyl-N-carboxymethyl chitosan (NCac) were studied at 0.5 mg.mL-1 using pyrene fluorimetry analysis in 0.1 mol.L-1 phosphate buffer (pH 7.4). The size and morphology of the aggregates were determined by dynamic light scattering and atomic force microscopy. The stability of the particles for periods up to 20 h in buffer was determined. Camptothecin was entrapped in the particles using various methods and the rate constant for drug release (k) was determined. Lower k values indicate strong interactions between the drug and the hydrophobic core of the polymeric micelles

Time-dependent viscometry study of endoglucanase action on xyloglucan: A real-time approach

AbstractHydrolysis of xyloglucan from Tamarindus indica and Hymenaea courbaril seeds with endoglucanase (EGII), which randomly breaks the (1→4)-linked β-glycosidic bonds of the polymer chain, was monitored in real time using time-dependent viscometry analysis (TDV). For both samples there was a decrease in the intrinsic viscosity ([η]), viscosity average molar mass (Mv), radius de gyration (Rg) and persistence length (Lp) immediately after the addition of the enzyme. It was observed the formation of oligosaccharides and oligomers composed of ∼2 units, up to 140min. Galactose-containing side chains two positions away from the non-substituted glucose, modulated the action of EGII, and the complete hydrolysis of the XG oligomers occurred after 24h. The results demonstrate for the first time the real-time degradation of xyloglucan as well the macromolecular and oligosaccharide composition during the EGII hydrolysis process

DESENVOLVIMENTO DE IMUNÓGENO CONJUGADO DE LIPOPOLISSACARÍDEO DE Pseudomonas Aeruginosa E TOXÓIDE TETÂNICO

O lipopolissacarídeo (LPS) de Pseudomonas aeruginosa foi conjugado ao toxóide tetânico (TT, toxina inativada do Clostridium tetani) utilizando a técnica de aminação redutiva direta, para o desenvolvimento de uma vacina conjugada (LPS-TT). Avaliou-se a resposta imune in vivo após aplicações intraperitoneais do imunógeno LPS-TT em camundongos Swiss, administrando-se 4 aplicações de LPS-TT com intervalos entre as doses de 14 dias. Avaliou-se o título de anticorpos anti-Pseudomonas produzidos pela técnica de soroaglutinação microscópica. Os grupos que foram imunizados com LPS-TT e com LPS+TT (não conjugado quimicamente) apresentaram resposta de anticorpos aglutinantes superior ao controle imunizado apenas com LPS, e a resposta após o reforço vacinal dos animais imunizados com o conjugado LPS-TT foi 567% superior ao dos animais imunizados com LPS

Efeito das xiloglucanas de sementes e derivados no crescimento de Arabidopsis thaliana.

Studies on xyloglucan (XG) extracted from Hymenaea courbaril L. (jatoba) seeds showed that this biopolymer has biological activity that enhanced wheat coleoptiles growth. In apple tree micropropagation, the culture medium containing XG combined with agar induced a higher multiplication rate, rooting rate and root length than medium solidified with agar only. The purpose of this study was to determine the effect of XG from jatobá seeds extracted from jatoba seeds collected in Sinope/MT (XGS) and Cuiabá/MT (XGC), and from XGC hydrolysed with a cellulase (XGCH), as well from Tamarindus indica seeds (XGT) collected in Bahia/BA, on the growth of in vitro cultured Arabidopsis thaliana plantlets. In the first experiment, XGCH (0.25, 25 and 250 nM) or XGC (0.5, 50 and 500 nM) were added to a liquid half-strength MS medium. In the second experiment, XGs from several origins were compared: XGC (500 nM), XGS (1200 nM) and XGT (800 nM), using culture medium solidified with 6 g.L-1agar. Arabidopsis thaliana L. seeds germinated in Petri plates for 4 to 5 days were transferred to culture media containing the different concentrations of XGs and cultured in a growing room. When the plantlets were cultured in a liquid medium, their growth was very slow in the presence of XGC and XGCH at the highest concentration tested, and it was faster at the lowest concentration. In the semi-solid culture medium, XGs also reduced growth. It was concluded that XGs can play a biological role in Arabidopsis thaliana (L.) Heynh. plantlets, stimulating or inhibiting the root system growth and the lateral root formation. These opposite effects varied according to the plant specie that furnished the seeds containing XG, as well as the place where the seeds were collected, to the XG form used (hydrolyzed or not) and to its concentration in the culture media. Estudos realizados com xiloglucanas (XG) extraídas de sementes de Hymenaea courbaril L. (jatobá) mostraram que esse biopolímero apresenta atividade biológica, promovendo o crescimento de coleóptilos de trigo. Na micropropagação da macieira, foi mostrado que o meio de cultura contendo uma mistura de XG e ágar leva à formação de maior número de brotações com raízes e maior comprimento dessas que o meio solidificado com ágar. O objetivo do presente trabalho foi verificar o efeito da XG de sementes de jatobá coletadas em Sinop/MT (XGS) e em Cuiabá/MT (XGC) e da XGC hidrolisada com uma celulase (XGCH), além da XG de sementes de Tamarindus indica L. (XGT) coletadas na Bahia/BA, no crescimento de plântulas de Arabidopsis thaliana (L.) Heynh. cultivadas in vitro. No primeiro experimento, XGCH (0,25; 25 e 250 nM) ou XGC (0,5; 50 e 500 nM) foram acrescentadas a um meio MS ½ líquido. O segundo experimento comparou as XGs das diferentes fontes: XGC (500 nM), XGS (1200 nM) e XGT (800 nM), usando meio de cultura solidificado com 6 g.L-1 de ágar. Sementes de Arabidopsis thaliana recém-germinadas em placas de petri, durante 4-5 dias, foram colocadas nos meios de cultura com as diferentes concentrações de XGs, em sala de crescimento, para obtenção de plântulas. Quando plântulas foram cultivadas em meio de cultura líquido, houve diminuição do crescimento das raízes na presença da XGC e de XGCH na concentração mais alta e o efeito foi inverso na concentração mais baixa. Em meio de cultura semi-sólido, as XGs também inibiram o crescimento. Concluiu-se que as XGs apresentam papel biológico nas plântulas de Arabidopsis thaliana, estimulando ou inibindo o crescimento do sistema radicular e a formação de raízes laterais. Esses efeitos opostos variaram em função da espécie vegetal doadora das sementes contendo XG, do local de coleta das sementes, da forma da XG usada (hidrolisada ou não) e da sua concentração nos meios de cultura

Effect of seed xyloglucans and derivates on the growth of Arabidopsis thaliana

Estudos realizados com xiloglucanas (XG) extra\ueddas de sementes de Hymenaea courbaril L. (jatob\ue1) mostraram que esse biopol\uedmero apresenta atividade biol\uf3gica, promovendo o crescimento de cole\uf3ptilos de trigo. Na micropropaga\ue7\ue3o da macieira, foi mostrado que o meio de cultura contendo uma mistura de XG e \ue1gar leva \ue0 forma\ue7\ue3o de maior n\ufamero de brota\ue7\uf5es com ra\uedzes e maior comprimento dessas que o meio solidificado com \ue1gar. O objetivo do presente trabalho foi verificar o efeito da XG de sementes de jatob\ue1 coletadas em Sinop/MT (XGS) e em Cuiab\ue1/MT (XGC) e da XGC hidrolisada com uma celulase (XGCH), al\ue9m da XG de sementes de Tamarindus indica L. (XGT) coletadas na Bahia/BA, no crescimento de pl\ue2ntulas de Arabidopsis thaliana (L.) Heynh. cultivadas in vitro. No primeiro experimento, XGCH (0,25; 25 e 250 nM) ou XGC (0,5; 50 e 500 nM) foram acrescentadas a um meio MS \ubd l\uedquido. O segundo experimento comparou as XGs das diferentes fontes: XGC (500 nM), XGS (1200 nM) e XGT (800 nM), usando meio de cultura solidificado com 6 g.L-1 de \ue1gar. Sementes de Arabidopsis thaliana rec\ue9mgerminadas em placas de petri, durante 4-5 dias, foram colocadas nos meios de cultura com as diferentes concentra\ue7\uf5es de XGs, em sala de crescimento, para obten\ue7\ue3o de pl\ue2ntulas. Quando pl\ue2ntulas foram cultivadas em meio de cultura l\uedquido, houve diminui\ue7\ue3o do crescimento das ra\uedzes na presen\ue7a da XGC e de XGCH na concentra\ue7\ue3o mais alta e o efeito foi inverso na concentra\ue7\ue3o mais baixa. Em meio de cultura semi-s\uf3lido, as XGs tamb\ue9m inibiram o crescimento. Concluiu-se que as XGs apresentam papel biol\uf3gico nas pl\ue2ntulas de Arabidopsis thaliana, estimulando ou inibindo o crescimento do sistema radicular e a forma\ue7\ue3o de ra\uedzes laterais. Esses efeitos opostos variaram em fun\ue7\ue3o da esp\ue9cie vegetal doadora das sementes contendo XG, do local de coleta das sementes, da forma da XG usada (hidrolisada ou n\ue3o) e da sua concentra\ue7\ue3o nos meios de cultura.Studies on xyloglucan (XG) extracted from Hymenaea courbaril L. (jatoba) seeds showed that this biopolymer has biological activity that enhanced wheat coleoptiles growth. In apple tree micropropagation, the culture medium containing XG combined with agar induced a higher multiplication rate, rooting rate and root length than medium solidified with agar only. The purpose of this study was to determine the effect of XG from jatob\ue1 seeds extracted from jatoba seeds collected in Sinope/MT (XGS) and Cuiab\ue1/MT (XGC), and from XGC hydrolysed with a cellulase (XGCH), as well from Tamarindus indica seeds (XGT) collected in Bahia/BA, on the growth of in vitro cultured Arabidopsis thaliana plantlets. In the first experiment, XGCH (0.25, 25 and 250 nM) or XGC (0.5, 50 and 500 nM) were added to a liquid half-strength MS medium. In the second experiment, XGs from several origins were compared: XGC (500 nM), XGS (1200 nM) and XGT (800 nM), using culture medium solidified with 6 g.L-1agar. Arabidopsis thaliana L. seeds germinated in Petri plates for 4 to 5 days were transferred to culture media containing the different concentrations of XGs and cultured in a growing room. When the plantlets were cultured in a liquid medium, their growth was very slow in the presence of XGC and XGCH at the highest concentration tested, and it was faster at the lowest concentration. In the semi-solid culture medium, XGs also reduced growth. It was concluded that XGs can play a biological role in Arabidopsis thaliana (L.) Heynh. plantlets, stimulating or inhibiting the root system growth and the lateral root formation. These opposite effects varied according to the plant specie that furnished the seeds containing XG, as well as the place where the seeds were collected, to the XG form used (hydrolyzed or not) and to its concentration in the culture media