Triple helix conformation of botryosphaeran, a (1 -> 3;1 -> 6)-beta-D-glucan produced by Botryosphaeria rhodina MAMB-05

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Lasiodiplodan, an exocellular (1 -> 6)-beta-d-glucan from Lasiodiplodia theobromae MMPI: production on glucose, fermentation kinetics, rheology and anti-proliferative activity

Lasiodiplodan, an exopolysaccharide of the (1 -> 6)-beta-d-glucan type, is produced by Lasiodiplodia theobromae MMPI when grown under submerged culture on glucose. The objective of this study was to evaluate lasiodiplodan production by examining the effects of carbon (glucose, fructose, maltose, sucrose) and nitrogen sources (KNO3, (NH4)(2)SO4, urea, yeast extract, peptone), its production in shake flasks compared to a stirred-tank bioreactor, and to study the rheology of lasiodiplodan, and lasiodiplodan's anti-proliferative effect on breast cancer MCF-7 cells. Although glucose (2.05 +/- A 0.05 g L-1), maltose (2.08 +/- A 0.04 g L-1) and yeast extract (2.46 +/- A 0.06 g L-1) produced the highest amounts of lasiodiplodan, urea as N source resulted in more lasiodiplodan per unit biomass than yeast extract (0.74 +/- A 0.006 vs. 0.22 +/- A 0.008 g g(-1)). A comparison of the fermentative parameters of L. theobromae MMPI in shake flasks and a stirred-tank bioreactor at 120 h on glucose as carbon source showed maximum lasiodiplodan production in agitated flasks (7.01 +/- A 0.07 g L-1) with a specific yield of 0.25 +/- A 0.57 g g(-1) and a volumetric productivity of 0.06 +/- A 0.001 g L-1 h(-1). A factorial 2(2) statistical design developed to evaluate the effect of glucose concentration (20-60 g L-1) and impeller speed (100-200 rpm) on lasiodiplodan production in the bioreactor showed the highest production (6.32 g L-1) at 72 h. Lasiodiplodan presented pseudoplastic behaviour, and the apparent viscosity increased at 60A degrees C in the presence of CaCl2. Anti-proliferative activity of lasiodiplodan was demonstrated in MCF-7 cells, which was time- and dose-dependent with an IC50 of 100 mu g lasiodiplodan mL(-1).

Efeito do 1,3/1,6 beta-glucano no sistema imune de cadelas submetidas a ovário-histerectomia

Pós-graduação em Medicina Veterinária - FCAV

Effects of β-(1→3,1→6)-d-glucan and density of diets on the blood profiles of immunologically challenged weaned piglets

An experiment was conducted to evaluate the effects of two levels of the β-(1→3,1→6)-d-glucan (0 and 500ppm) from yeast Saccharomyces cerevisiae and two levels of energy (3300 and 3450kcalMEkg(-1)) on the hematological, immunological and, biochemical profiles of thirty-six 21-days-old weaned piglets, challenged with 150μgkg(-1) of BW lipopolysaccharide (LPS) from Escherichia coli serotype 055:B5. The experimental design was a randomized complete block design in a 2×2 factorial arrangement with nine replicates per treatment and, one animal per experimental unit. The data were analyzed in accordance with the multivariate analysis procedure of SAS and, the treatment means of parametric and non-parametric data were compared by Bonferroni's test (P<0.05) and, by Dunn's test (P<0.05), respectively. The data of the blood profiles of alanine aminotransferase, alkaline phosphatase and, creatinine showed that LPS did not cause kidney or liver damage in the animals. The addition of beta-glucan in the diets did not prove the robustness of its effect and biological relevance when provided with low nutrient-density. However, its addition combined with the high-nutrient-density diets showed less marked hypoglobulinemia in piglets, which may have contributed to the decreasing of the synthesis of inflammatory mediators.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-beta-1,3-glucanase from Thermotoga petrophila

1,3-beta-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal beta-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90 degrees C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Efeito do exercício físico sobre a liberação de interleucina-1[beta], interleucina-6 e fator de necrose tumoral-[alfa] em homens portadores de doença pulmonar obstrutiva crônica

A atividade física intensa pode induzir resposta inflamatória subclínica e aumento nos níveis plasmáticos de citocinas pró-inflamatórias. O objetivo deste estudo foi avaliar a relação entre a liberação de citocinas (IL-1β, IL-6, e TNF-α), o exercício físico agudo e o exercício regular em pacientes com doença pulmonar obstrutiva crônica (DPOC). Foram estudados 18 pacientes do sexo masculino com DPOC moderada a muito grave, divididos em dois grupos: 11 pacientes foram incluídos em programa de reabilitação pulmonar (RP) durante 8 semanas e 7 pacientes sem atividade física regular foram incluídos como grupo controle (C). Todos os pacientes realizaram espirometria, teste de exercício cardiopulmonar incremental máximo e teste de endurance em cicloergômetro com carga constante (60% da carga máxima do teste incremental) no início do projeto e após oito semanas. Foi coletado sangue venoso periférico para dosagem de citocinas, antes e 15 minutos após os testes de endurance (TE1 e TE2). IL-1β, IL-6, e TNF-α foram dosadas com kits ELISA específicos (Quantikine®, R&D Systems). Os pacientes submetidos à RP liberaram menos IL-1β que os controles após o treinamento (RP: TE1 0,96±0,66; TE2 -0,24±0,27 pg/ml; grupo C: TE1 -1,48±1,14; TE2 0,66±0,61 pg/ml; p=0,03). Não houve diferença significativa na liberação de IL-6 quando comparados os dois testes de endurance (RP: TE1 0,44±1,21; TE2 0,80±1,24 pg/ml; grupo C: TE1 0,88±0,85; TE2 0,78±0,95 pg/ml; p=0,68). Não foi observada diferença na liberação de IL-6 entre os dois grupos. Apenas cinco pacientes (quatro no grupo da RP) liberaram TNF-α e o exercício não modificou o seu padrão de liberação (RP: TE1 2,86±1,18; TE2 2,57±1,37pg/ml; grupo C: TE1 4,98; TE2 6,84 pg/ml; p=0,14). Não houve associação significativa entre intensidade de exercício e liberação de citocinas (IL-1β r=0,10; IL-6 r=-0,23). Houve maior liberação de IL-6 após o TE2 nos pacientes que apresentaram exacerbação da DPOC (exacerbados 9,59±1,32; estáveis 6,31±0,92 pg/ml; p=0,03) e não houve diferença nos níveis de IL-1β. Apenas pacientes com exacerbação da DPOC liberaram TNF-α (2,82±1,48 pg/ml). Concluiu-se que o exercício físico regular reduz a liberação de IL-1β e as exacerbações estimulam a liberação de IL-6 e TNF-α em pacientes com DPOC.

Enzymatic hydrolysis of botryosphaeran and laminarin by beta-1,3-glucanases produced by Botryosphaeria rhodina and Trichoderma harzianum Rifai

Botryosphaeran, a (1 -> 3; 1 -> 6)-beta-D-glucan produced by Botryosphaeria rhodina, and laminarin were hydrolysed by two fungal beta-glucanases predominantly of the 1,3-type produced by B. rhodina and Trichoderma harzianum Rifai grown on botryosphaeran as sole carbon source. Both beta-glucanase preparations presented different modes of attack on botryosphaeran and laminarin. Laminarin was hydrolysed to the extent of similar to 50% in 1 hand 100% within 24 h, and its hydrolysis products were mainly glucose and gentiobiose, and lesser amounts of laminaribiose and oligosaccharides of DP 3-4 during the early stages of hydrolysis, while botryosphaeran 'yielded mainly glucose and gentiobiose with some trisaccharide, but no laminaribiose or tetrasaccharide when hydrolysed by the T. harzianum enzyme. By contrast, B. rhodina beta-1,3-glucanases produced predominantly glucose during all stages of botryosphaeran hydrolysis. Some physicochemical properties of the 1,3- and 1,6-beta-glucanases, and beta-glucosidases contained in the two fungal P-glucanase preparations are also described for the first time. (c) 2006 Elsevier Ltd. All rights reserved.

Botryosphaeran, a new substrate for the production of beta-1,3-glucanases by Botryosphaeria rhodina and Trichoderma harzianum Rifai

Botryosphaeria rhodina and Trichoderma harzianum Rifai were grown on botryosphaeran (an exopolysaccharide (EPS) of the beta-1,3; 1,6-D-Glucan type produced by B. rhodina) as sole carbon source with the objective of producing beta-glucanases of the beta-type. Conditions for beta-1,3-glucanase production by T harzianum were examined by a statistical response surface method, and showed maximal enzyme production at 5 days growth in media containing 1.5 g/1 of EPS. Good agreement was obtained between the experimental values of beta-1, 3-glucanase activity and the corresponding values predicted by the mathernatical model. The crude beta-1,3-glucanase preparations were active towards a number of different beta-1,3-glucans and beta-glucosides. The mycelium of B. rhodina also proved to be a good substrate for beta-1,3-glucanase production by both fungal species. (c) 2005 Published by Elsevier Ltd.

Evaluation of the beta-glucanolytic enzyme complex of Trichoderma harzianum Rifai for the production of gluco-oligosaccharide fragments by enzymatic hydrolysis of 1,3;1,6-beta-D-glucans

Botryosphaeran, a new exopolysaccharide from the endophytic fungus Botryosphaeria rhodina MAMB-05, and algal laminarin were hydrolyzed by partially-fractionated enzymes of the beta-glucanolytic complex from Trichoderma harzianum Rifai. beta-Glucanase fractions (F-I and F-II) separated by gel permeation chromatography presented different modes of attack on botryosphaeran and laminarin. Botryosphaeran was hydrolyzed to the extent of 66% (F-I) and 98% (F-II) within 30 min, and its main hydrolysis products were gluco-oligosaccharides of DP >= 4, with lesser amounts of glucose, di- and tri-saccharides. The action of enzyme fractions I and II on laminarin resulted in 15% conversion to glucose, while the percentage of saccharification was radically different (70% for F-I and 25% for F-II). The different product arrays within the polysaccharide hydrolysates can be explained by the difference in the enzymes' specificities within each enzyme fraction, and the molecular structures of the polysaccharides and their complexity.

Terpinen-4-ol and alpha-terpineol (tea tree oil components) inhibit the production of IL-1 beta, IL-6 and IL-10 on human macrophages

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Parâmetros ecocardiográficos e variabilidade da frequência cardíaca em fêmeas caninas com sepse de ocorrência natural devido à piometra e suas correlações com valores séricos de TNF-alfa, IL-1 beta, IL-6, IL-10 e proteína C reativa

Pós-graduação em Medicina Veterinária - FCAV

alpha,beta-Unsaturated Diazoketones as Platforms in the Asymmetric Synthesis of Hydroxylated Alkaloids. Total Synthesis of 1-Deoxy-8,8a-diepicastanospermine and 1,6-Dideoxyepicastanospermine and Formal Synthesis of Pumiliotoxin 251D

A versatile and concise approach for the stereoselective synthesis of mono-, di-, and trihydroxylated indolizidines is presented in four to six steps from Cbz-prolinal and a diazophosphonate. The key steps involved a Wolff rearrangement, followed by a stereoselective dihydroxylation/epoxidation reaction, from an alpha,beta-unsaturated diazoketone. The strategy also permits extension to the synthesis of many natural hydroxylated indolizidine alkaloids as demonstrated in the formal synthesis of pumiliotoxin 251D.

Biochemische Charakterisierung des Exopolysaccharids von Pediococcus parvulus B399, sowie dessen Hydrolyse durch eine neue beta-1,3-Glucanase aus Delftia sp. MV01

In der vorliegenden Arbeit wurde die erste β-1,3-Glucanase aus Delftia beschrieben. Es konnte gezeigt werden, dass das Enzym unter anderem gegen das nur schwer zu hydrolysierende Exopolysaccharid aus Pediococcus parvulus wirkte. rnrnIm Einzelnen wurde zunächst das Exopolysaccharid aus Pediococcus parvulus B399 aus einem eigens zusammengestellten β-Glucan-Synthesemedium (Medium M) isoliert und gereinigt. Anschließend erfolgte eine umfassende Charakterisierung des Biopolymers. Hierzu gehörten neben der sauren Hydrolyse zur Bestimmung der Monomerzusammensetzung des Polymers, auch spektroskopische Methoden, darunter 1H und 13C-NMR. Mithilfe der NMR-Spektroskopie konnte die Struktur des Exopolysaccharids aus Pediococcus parvulus B399 bestimmt werden. Es handelte sich hierbei ebenfalls um ein β-1,3(1,2)-Glucan, wie es bereits für Pediococcus parvulus 2.6 beschrieben wurde. Darüber hinaus wurde erstmals ein ATR-FTIR-Spektrum für ein Exopolysaccharid aus Pediokokken gezeigt. Über GPC-Messungen konnte auch die molekulare Größe des β-1,3(1,2)-Glucans aus Pediococcus parvulus B399 bestimmt werden. Es wurde nachgewiesen, dass sich das Exopolysaccharid bei Anzucht in Medium M aus einer hochmolekularen Fraktion (5*106 g/mol) und vier niedermolekularen Fraktionen (347; 818; 10048 und 20836 g/mol) zusammensetzte. Neben der strukturellen Charakterisierung, wurde das Exopolysaccharid auch rheologisch untersucht. Dabei konnte festgestellt werden, dass es sich durch seine schwach gelbildenen Eigenschaften auch zum Einsatz in der Lebensmittelindustrie als Stabilisator, Fettersatzmittel oder ähnliches eignen würde. Die erwähnte gelbildende Netzwerkstruktur konnte für das Exopolysaccharid aus Pediococcus parvulus B399 auch erstmals im AFM bestätigt werden. rnEin weiterer Teil der Arbeit umfasste ein breites Screeningverfahren nach einem geeigneten Organismus, der das Exopolysaccharid aus Pediococcus parvulus B399 effektiv hydrolysieren sollte. Aus einer Anreicherungskultur des Termitendarms (Wenzel et al., 2002), konnte Delftia sp. MV01 isoliert werden. Dieser Organismus produzierte bei Wachstum in β glucanhaltigem Medium (Exopolysaccharid aus Pediococcus parvulus B399, sowie weitere kommerziell erhältliche β-1,3-Glucane) eine Glucanase, die in folgenden Schritten konventionell gereinigt und charakterisiert wurde.