Evaluation of Rhamnetin as an Inhibitor of the Pharmacological Effect of Secretory Phospholipase A2

Rhamnetin (Rhm), 3-O-methylquercetin (3MQ), and Rhamnazin (Rhz) are methylated derivatives of quercetin commonly found in fruits and vegetables that possess antioxidant and anti-inflammatory properties. Phospholipase A2 (PLA2) displays several important roles during acute inflammationtherefore, this study aimed at investigating new compounds able to inhibit this enzyme, besides evaluating creatine kinase (CK) levels and citotoxicity. Methylated quercetins were compared with quercetin (Q) and were incubated with secretory PLA2 (sPLA2) from Bothrops jararacussu to determine their inhibitory activity. Cytotoxic studies were performed by using the J774 cell lineage incubated with quercertins. In vivo tests were performed with Swiss female mice to evaluate decreasing paw edema potential and compounds' CK levels. Structural modifications on sPLA2 were made with circular dichroism (CD). Despite Q and Rhz showing greater enzymatic inhibitory potential, high CK was observed. Rhm exhibited sPLA2 inhibitory potential, no toxicity and, remarkably, it decreased CK levels. The presence of 3OH on the C-ring of Rhm may contribute to both its anti-inflammatory and enzymatic inhibition of sPLA2, and the methylation of ring A may provide the increase in cell viability and low CK level induced by sPLA2. These results showed that Rhm can be a candidate as a natural compound for the development of new anti-inflammatory drugs.Universidade Estadual Paulista (UNESP)Universidade Federal de São Paulo (UNIFESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed São Paulo, Postgrad Program Food Nutr & Hlth, BR-11015020 São Paulo, BrazilUniv Estadual Paulista, Biosci Inst, BR-11330900 São Paulo, BrazilBrazil Univ, Prorector Res, BR-08230030 São Paulo, BrazilUniv São Paulo, Pathol Lab Infect Dis LIM50, Dept Pathol, Sch Med, BR-01246903 São Paulo, BrazilUniv Fed São Paulo, Postgrad Program Food Nutr & Hlth, BR-11015020 São Paulo, BrazilWeb of Scienc

TsTX-V, uma 'alfa'-toxina do nosso mundo : estrutura e estudo do efeito sobre o mecanismo de secreção de insulina

Orientador: Sergio MarangoniDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As neurotoxinas escorpiônicas têm sido classificadas em duas grandes categorias: as a -toxinas (encontradas em espécies do Velho Mundo) que retardam a inativação dos canais de sódio e as ß-toxinas (provenientes de espécies do Novo Mundo), que induzem um estado persistente de despolarização das membranas de células excitáveis (neurônios). A TsTX-V foi obtida por cromatografia de troca iônica em CM-Cellulose 52 e teve seu grau de pureza confirmado por cromatografia de fase reversa em HPLC e por eletroforese em PAGE-SDS Tricina. A estrutura primária da TsTX-V foi determinada por degradação automática de Edman, a partir da proteína pura carboximetilada e reduzida e de seus digestos peptídicos obtidos por Tripsina e Protease V8. Sua seqüência, 64 resíduos de aminoácidos e uma massa molecular, calculada através da somatória dos resíduos, de 7,2 kDa, contendo oito resíduos de cisteina. Testes eletrofisiológicos realizados em nervo vago de coelho mostraram que a toxina TsTX-V (0,03µg/mL) induz um prolongamento do potencial de ação das fibras B do nervo vago devido ao retardo na inativação do canal de Na+. A 0,3µg/mL ela induz também uma despolarização do nervo. Esses efeitos são irreversíveis e podem ser abolido por tetrodoxina (200 - 500 mM), mas não por um aumento da concentração de potássio no meio externo. Estes resultados demosntraram que TsTX-V é uma a-toxina (Arantes et aI., 1994). Em ilhotas de Langerhans, isoladas de ratos, a TsTX-V (a-toxina) potencializou a secreção de insulina, na ausência ou na presença de 8,3 mM de glicose. A Ts-g potencializou a secreção de insulina por 8,3 mM de glicose. A toxina TsTX-V (5,6 mg/ ml) induziu um aumento do efluxo de 86Rb+ cerca de 2,0 a 2,4 vezes em relação ao induzido por 8,3 mM de glicose em ilhotas de Langerhans. Este efeito foi persistente e lentamente reversível. Efeito similar foi observado em presença de (110 mM) de veratridina, substância que retarda a inativação de canais de sódio sensíveis a voltagem. Estes dados sugerem que a toxina TsTX-V prolonga o período de despolarização das células B, ativando indiretamente os canais de K+ dependente de voltagem, mantendo, desta forma, a permeabilidade da membrana ao K+, medida indiretamente pelo efluxo de 86Rb+, mateve-se elevada.. A estrutura tridimensional da toxina TsTX-V foi determinada através de modelagem molecular, utilizando-se para isso os dados cristalográficos da toxina CsE-V3, cujas coordenadas estão depositadas em banco de dados, e da toxina AaH II. De um modo geral, ela tem as feições típicas das neurotoxinas de escorpiões, como a formação em a.-hélice e as três folhas b antiparalelas, mas diferindo quanto à disposição das alças J e B, e da região N-terminal e Carboxi terminal, quando comparados com a toxina do Androctonus autralis Hector (AaH II). O dendograma obtido através das similaridades e coincidências de resíduos e do alinhamento das estruturas secundárias mostra que as toxinas do Novo Mundo podem ser subdivididas em três subgrupos: duas (beta) b e uma (alfa) aAbstract: Antimamals scorpion neurotoxins are classified in two groups: a-toxins (from the Old World species) that induce a slowing down of the inactivation of the sodium channel, and b-toxins (from the New World species), that induce a persistant depolarization of excitable cell membranes. TsTX-V was obtained by ion-exchange chromatography on CM-Cellulose 52 and its of purity was confirmed by reverse phase chromatography and Tricine SDS-PAGE. Primary structure of TsTX-V has been determined by automatic Edman degradation from the reduced and carboxymethyled protein and digestic peptide obtained by Protease V8 and Tripsin. Its sequence shows, a total of 64 aminoacid residues, a calculated molecular weight of 7200.and eight cysteine residues. Electrophysiological assay performed on the vagus nerve of rabit, showed that TsTX-V (0,03 ).mg/mL) induce a persistent depolarized state during long time on B fibers, this effect was abolished by addition of tetrodoxin (200 - 500 mM) but not abolished by high externaI potassium depolarization. Those effects characterized TsTX-V as a-toxins.(Arantes et al, 1994) On isolated rat islets of Langerhans TsTX-V induced insulin secretion in both absecence or presence os glucose (8,3 mM)presence of glucose 8,3 mM, but in absence any effect was observed. However, Tsg (b toxin) potentiated insulin secretion in the presence of glucose 8,3 mM. TsTX-V (5,6 mg/mL) induced a 86Rb+ outflow increase, 2,0~2,4 fold the rate of the marker outflow in the presence of 8,3 mM glucose. This effect was persistent and slowly reversible, showing similarity to that induced by 100 mM veratridine, an agent that prolong the open period of Na+ channel, delaying their inactivation. This suggested that, by extending the depolarized period, TsTX-V indirectly affect b cells voltage dependent K+ channels, thus increasing K+ permeability. Homology studies performed with TsTX-V and other scorpion neurotoxins revealed common folding among them, for example the presence of highly conserved regions and residues and the presence of eight cystein residues at same locations. The three-dimensional structure of TsTX-V was determined by modeling from crystalographic determined structure of CsE-V3 and AaH II. TsTX-V has conserved a and b structure present in other scorpion neurotoxins. Basically its three-dimensional structure is similar to CsE-V3 and AaH lI, but differs in the folding patterns in the J and B loops. Computer simulation performed on the three-dimensional structure obtained by molecular modeling, shows that the C-terminal and N-terminal loops have a great degree of freedom. On the other hand J and B loops did not show great diferences in its leght spatial disposition whem compared with atoxic fraction TsTX-VI or Ts-g (b toxins)MestradoBioquimicaMestre em Ciências Biológica

Miotoxinas PLA2 "like" de Bothrops pirajai : caracterização molecular e funcional

Orientador: Sergio MarangoniTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As serpentes do gênero Bothrops incluem várias espécies, que são amplamente distribuídas na América do Sul e do Norte. Entre as proteínas bioativas do veneno de Bothrops, as Fosfolipases Az (PLAz,E.c. 3.1.14) e as miotoxinas PLAz"like" destacam-se como seus componentes majoritários. Fosfolipases Az são enzimas cálcio dependentes que hidrolisam a ligação 2-éster do 1,2 diacil-3sn fosfoglicerídeo (Chang et aI., 1994, Shimohigachi et aI., 1995, Ogawa, et aI., 1996). Estas enzimas são encontradas em muitos tecidos, principalmente no suco pancrácio de mamíferos,no veneno de serpentes e insetos. Enzimas PLAz são classificados dentro de quatro grupos (I, lI, III e IV) de acordo com sua origem extracelular ou intracelular, de acordo com sua estrutura primária e pontes de sulfeto (Denis et aI., 1994). Nesta tese, trabalhos com o veneno total de Bothrops pirajai e suas frações fosfolipases Az miotóxicas. Num primeiro instante, nos desenvolvemos uma nova estratégia de purificaçãodestas miotoxinasem HPLC(HPLCde fase reversa, troca iônicae exclusão molecular) e em cromatografia convencional de baixa pressão (CM-Sepharose). Em nossas condições experimentais, observamos que o HPLCde fase reversa (RP HPLC)tem vantagens sobre o método convencional, em relação ao tempo da corrida cromatográfica, a resolução dos picos e a manutenção da integridade molecular da PLAz. O primeiro trabalho feito por Toyama et aI., (1995) apresenta os resultados da purificação da principal miotoxina PLAz"Iike" (MPI e MPII) do veneno total de Bothrops pirajai, usando uma única etapa cromatográfica em HPLCde fase reversa. Este novo protocolo de purificação restringe a quantidade de proteína a ser colocada na coluna algumas mg. O rendimento final é 35% melhor do que na cromatografia de troca iônica de baixa pressão. Em Soares et aI., (1998), propusemos um uma outra alternativa de purificação das miotoxinas PLAz "like" majoritárias usando coluna convencional, uma vez que o RP HPLC não aceita grandes quantidades de amostras. No procedimento convencional,a eluição das miotoxinasPLAz"like" era eluídausando acetato de amônia em altas concentraçõe se altos valor de pH. Atroca do acetato de amônio por bicarbonato de amônia permitiu o uso de tampões com baixo pH e baixas concentrações salinas. Outros venenos de Bothrops jararacussu, Bothrops asper, Bothrops atrox, Bothrops pirajai, Bothrops moojeni, Bothrops alternatus e Bothrops (Bothriopsis) bilineata foram fracionados usando procedimentos simplificados baseados na cromatografia em CMSepharose a pH 8.0 ou em RP HPLC. O uso de NH4HC03ou de acetonitrila como tampões nestes procedimentos cromatográficosdescritos acima, tem outras vantagens como a omissãode desalificaçãoe facilidadena liofilização. Isto permite uma melhorrecuperação das proteínas eluídas e redução do tempo de corrida. O método em CM-Sepharosee RP-HPLCdescritos aqui são recomendados para escalas preparativas e analíticas, respectivamente. Nos métodos previamente descritos usam dois ou mais passos cromatográficos para o isolamento de miotoxinas. Em adição as colunas preparativas wBondapack C18 mostraram também ser conveniente para a purificação de PLA2S. Ambos os métodos descritos aqui separam os componentes principaisdo veneno usando unicamente passo cromatográfico. Os perfis cromatográficos mostraram importantes diferenças no conteúdo de miotoxinas destes venenos. O veneno de B. alternatus, B. atrox e Bothriopsis bilineata não contem a miotoxina principal encontrada em outros venenos. O sequenciamento de aminoácidos dos primeiros 50 resíduos da região N-terminal destas miotoxinas PLA2"like" mostram uma homologia de 90 a 96% com outras miotoxinas botropicas. Todas as miotoxinas isoladas induzem edema de pata, aumentando o nível de creatinina quinase pancreática e indução da mionecrose junto com a infiltraçãode células polimorfonucleares. Nesta tese, nos apresentamos o isolamento, purificação e a determinação da estrutura primária de três miotoxinas fosfolipase A2"like" do veneno de Bothrops pirajai, denominadas como PrTX-I,PrTX-IIe MP-III4R. PrTX-I ou Piratoxina I foi isolado por Mancuso et ai., (1995), e completamente seqüênciada por Toyama et ai., (1998). PrTX-Ié a principal PLA2 miotóxica encontrado no veneno total de Bothrops pirajai, composto por 121 resíduos de aminoácidos, uma DLso de cerca de 8mg/kg em camundongos e uma dose edematogênica mínima de 39.5 :t 1.8 Ilg. A modificação química da His 48 da PrTX-I pelo p-BPB praticamente aboliu sua atividade biológica. PrTX-II ou Piratoxina II foi a segunda miotoxina importante isolada do veneno de Bothrops pirajai que foi sequenciada por Toyama et aI., (1999, submetido). Tem 121 resíduos de aminoácido de baixa atividade PLA2 devido substituição do Asp49 por Lys49e alteração do "Ioop" de ligação do cálcio pela substituição de Gly32 por Leu32 e outras modificações foram importantes para a perda da flexibilidadedo sítio de ligação do cálcio. PrTX-II tem somente um único amino ácido modificado (D132 para A132) em relação a PrTX-I esta mudança confere a PrTX-II um ligeiro caráter básico. A MP-III 4R foi a terceira miotoxina isolada do veneno de Bothrops pirajai. Esta miotoxina PLA2 "Iike" tem uma atividade PLA2moderada se comparada com outras enzimas encontradas em venenos Crotálicos. MP-III4R é um raro exemplode PLA2 com atividade fosfolipase A2, anticoagulante e miotóxica. Sua atividade PLA2 moderada é devido a substituição do resíduo E53 pelo K53 e seu efeito anticoagulante é devido a atividade PLA2.Amiotoxicidade não é devido a sua atividade catalítica. O alinhamento de amino ácidos da PrTX-I,PrTX-II mostra um alto nível (95%) de homologia sequencial entre esta miotoxina e outras PLA2botropicas. Contudo, estes valores diminuem para 80% para as não botropicas e para 70-75% para as PLA2Asp49. Ambas toxinas foram caracterizadas como miotoxinas potentes e tem um atividade PLA2 residual. MP-III 4R mostra uma homologia seqüencial de mais de 50% com outras PLA2 D-49. Maso alinhamento de aminoácidos da MP-III4R com PLA2K-49diminui para cerca de 60% de homologia. PrTX-II e PrTX-III foram cristalizados e difratados usando uma resulução de 2.04 e 2.7 A. Recentemente, a estrutura tridimensional da PrTX-IIfoi resolvida e mostrou uma estrutura diméricaAbstract: The genus Bothrops comprises severaI species, which are widely distributed in South and North America. Among the bioactive proteins from Bothrops venoms, the phospholipase A2(PLA2,E.C.3.1.14) and PLA2-likemyotoxin are outstanding as their major components. Phospholipase A2Sare calcium-dependent enzymes which hydrolyze the 2 ester bonds of 1,2 diacyl-3sn phosphoglycerides (Chang et aI., 1994, Shimohigachi et aI., 1995, Ogawa, et aI., 1996). They are found in most tissues, mainly in the pancreatic juice of mammals, venom of snakes and insects. PLA2enzymes are classified onto four groups (I, II, III and IV), according to their extracellular or intracellular origin, their primary structure and disulfide bonding (Denis et aI., 1994). In this thesis, we work with Bothrops pirajai whole venom and its myotoxic phospholipase A2fractions. At first time, we develop a new strategy of purification of this myotoxinon the HPLC(reverse phase, ion exchange, and molecular exclusion) and in the convention low-pressure chromatography (CM-Sepharose). In our experimental condition, we observed that Reverse Phase HPLC(RP HPLC) has advantageous on the conventional method about the time of chromatographic run, the resolution of some proteins and preservation of integrity of PLA2 molecule. The first work made by Toyama et aI., (1995) presents the results of the purification of the main myotoxin PLA2"Iike" (MPI and MPII) from the whole venom of Bothrops piraja.,using only chromatographicstep on the RPHPLC. This novel purification protocol restricts the amount of protein to be loaded on each column in few mg. The final yield is 35% better than to in low-pressure ion exchange chromatography. In the Soares et aI., (1998), we proposed to increase the purification grade of main PLA2 "like" myotoxin using conventional column, because the RP HPLC does not accept great amount of samples. In the conventional procedure, the elution of the PLA2 "like" myotoxin was eluted using ammonium acetate at high salt and pH values. The exchange of the ammonium acetate by ammonium bicarbonate allowed using low pH and ionic salt concentration. PrTX-II or Piratoxin II was a second important myotoxin from Bothrops pirajai that has been sequecianated by Toyama (Submitted). It has 121 amino acid residues and has low PLA2activity arose of the substitution of Asp49 by Lys49and alteration of the calcium binding loop sequence by replacement of Gly32 by Leu32 and other modification were important for 1055of the flexibility the calcium ion binding site. PrTX-II I have only one amino acid change (0132 to A132) to PrTX-I, this change confer to PrTX-II a slight basic character. The MP-III 4R was thirty myotoxin isolated from the Bothrops pirajai venom. This PLA2 like myotoxinhasa moderate PLA2activity if comparedto other enzymesfound in the Crotalic venom. MP-III 4R is a rare example of PLA2 with phospholipase A2, anticoagulant and myotoxic activities. Its moderate PLA2activity is due to the replacement of E53 by K53 and its anticoagulant effects is due to that PLA2activity. The myotoxicity is not due to the catalytic activity. The aminoacid alignment of PrTX-I, PrTX-II shows a high levei(95%) of sequential homology between this myotoxin and other bothropic Lys-49 PLA2. However, these values fali to 80% for nonbothropic and to 70-75% for the Asp 49 PLA2S. 80th toxins were characterized as very potent myotoxin and have a residual PLA2 activity. MP-III 4R 049 exhibit a sequence homology with other 0-49 PLA2up 75%. But the amino acid alignment of MP-III 4R with K-49 PLA2falls to around 60% of homology. PrTX-II and PrTX-III were crystallizedand were diffractedat resolution of 2.04 and 2.7 of resolution, respectively. Recently, the three-dimensional structure of PrTX-II was solved and showed a dimeric structure. Other venoms from Bothropsjararacussu, Bothrops asper, Bothrops atrox, Bothrops pirajai, Bothrops moojeni, Bothrops alternatus and Bothrops (Bothriopsis) bilineata were fractionated using a simplified procedure based on ion-exchange chromatography on CMSepharose at pH 8.0 or reverse phase HPLC. The use of NH4HCO3or acetonitrile as the buffer in these chromatographic procedure described above, has other advantages as omissionof desalting and easy for freeze-drying. This allows a best recoveryof the proteins eluted and reduction of run time. The CM-Sepharose and the RP-HPLC methods described here are recommended for preparative and analytical purpose, respectively. Previous reported methods use two or more chromatographic steps for the isolation of myotoxins. In addition, the preparative WBondapackC18 column was also useful for the purificationof PLA2S. Both methods described here allow separating the major components of the venom using an only one chromatographic step. The resulting elution profiles showed important differences in the myotoxin content of these venoms. The venoms from B. alternatus, B. atrox and Bothriopsis bilineata did not contain the major myotoxin found in the other venoms. The amino acid sequence of the first 50 residues of the N-terminal region of the PLA2-like myotoxins showed a homology of 90-96% with other botropic myotoxins. All of the myotoxins isolated induced rat paw edema, increased the levei of plasma creatine kinase and produced myonecrosis together with polymorphonuclear cell infiltration. In this thesis, we present the isolation; purification and determination of primary structure of three phospholipase A2"like" myotoxin from the Bothrops pirajai snake venom denominated as PrTX-I, PrTX-II and MP-III 4R. PrTX-I or Piratoxin I was firstly isolated by Mancuso et aI., (1995), and full sequenced by Toyama et aI., (1998). PrTX-Iis the main miotoxic PLA2found in the whole Bothrops pirajai venom, composed by 121 amino acid residues, a DLso around 8mg/kg in mice and a minimal edematogenic dose of 39.5 :t 1.8 /.lg. The chemical modification of His-48 of PrTX-I by p-BPB practically destroyed its biological activityDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Edema Induced by a Crotalus durissus terrificus Venom Serine Protease (Cdtsp 2) Involves the PAR Pathway and PKC and PLC Activation

Snake venom serine proteases (SVSPs) represent an essential group of enzymatic toxins involved in several pathophysiological effects on blood homeostasis. Some findings suggest the involvement of this class of enzymatic toxins in inflammation. In this paper, we purified and isolated a new gyroxin isoform from the Crotalus durissus terrificus (Cdt) venom, designated as Cdtsp 2, which showed significant proinflammatory effects in a murine model. In addition, we performed several studies to elucidate the main pathway underlying the edematogenic effect induced by Cdtsp 2. Enzymatic assays and structural analysis (primary structure analysis and three-dimensional modeling) were closely performed with pharmacological assays. The determination of edematogenic activity was performed using Cdtsp 2 isolated from snake venom, and was applied to mice treated with protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, dexamethasone (Dexa), antagonists for protease-activated receptors (PARs), or saline (negative control). Additionally, we measured the levels of cyclooxygenase 2 (COX-2), malondialdehyde (MDA), and prostaglandin E2 (PGE2). Cdtsp 2 is characterized by an approximate molecular mass of 27 kDa, an isoelectric point (pI) of 4.5, and significant fibrinolytic activity, as well as the ability to hydrolyze Nα-benzoyl-l-arginine 4-nitroanilide (BAPNA). Its primary and three-dimensional structures revealed Cdtsp 2 as a typical snake venom serine protease that induces significant edema via the metabolism of arachidonic acid (AA), involving PARs, PKC, PLC, and COX-2 receptors, as well as inducing a significant increase in MDA levels. Our results showed that Cdtsp 2 is a serine protease with significant enzymatic activity, and it may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. In this article, we provide a new perspective for the role of SVSPs beyond their effects on blood homeostasis

MYZOBDELLA PLATENSIS (HIRUNDINIDA: PISCICOLIDAE) IS TRUE PARASITE of BLUE CRABS (CRUSTACEA: PORTUNIDAE)

Leeches exhibit a marked scope of diversity, including different kinds of symbiosis. The aim of the present study was to demonstrate through biochemical and histological analysis that a species of piscicolid leech, Myzobdella platensis, is a true parasite of blue crabs, feeding on their hemolymph and using them as a site for cocoon deposition. In a total of 48 blue crabs collected on October 2007 at 3 sites of the Sao Vicente Estuary, 12 specimens were infested with leeches. Callinectes bocourti (n = 7) was the most infested species with leeches and cocoons; it was chosen for biochemical and histological assays. The immunoblotting assays showed a positive reaction of the proteins in the intestinal samples of leeches collected from crabs using antihemocyanin polyclonal antibody of Ampullaria canaliculata. In addition. leech intestinal samples were recognized by antihemolymph polyclonal antibody of nonparasitized blue crabs. Histological sections of leech gut showed hemocytes and a granular matrix similar to those found in crab blood vessels. Collectively, this evidence strongly suggests a parasitic interaction between the leech M. platensis and the blue crab C. bocourti, in which the former utilizes the latter as a site for cocoon deposition and possibly for dispersal similar to that proposed for Myzobdella lugubris in Callinectes sapidus in North America.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Evaluation of Macroalgae Sulfated Polysaccharides on the Leishmania (L.) amazonensis Promastigote

The sulfated polysaccharides from Solieria filiformis (Sf), Botryocladia occidentalis (Bo), Caulerpa racemosa (Cr) and Gracilaria caudata (Gc) were extracted and extensively purified. These compounds were then subjected to in vitro assays to evaluate the inhibition of these polysaccharides on the growth of Leishmania (L.) amazonensis promastigotes. Under the same assay conditions, only three of the four sulfated polysaccharides were active against L. amazonensis, and the polysaccharide purified from Cr was the most potent (EC50 value: 34.5 μg/mL). The polysaccharides derived from Bo and Sf demonstrated moderate anti-leishmanial activity (EC50 values of 63.7 μg/mL and 137.4 μg/mL). In addition, we also performed in vitro cytotoxic assays toward peritoneal macrophages and J774 macrophages. For the in vitro cytotoxicity assay employing J774 cells, all of the sulfated polysaccharides decreased cell survival, with CC50 values of 27.3 μg/mL, 49.3 μg/mL, 73.2 μg/mL, and 99.8 μg/mL for Bo, Cr, Gc, and Sf, respectively. However, none of the sulfated polysaccharides reduced the cell growth rate of the peritoneal macrophages. These results suggest that macroalgae contain compounds with various chemical properties that can control specific pathogens. According to our results, the assayed sulfated polysaccharides were able to modulate the growth rate and cell survival of Leishmania (L.) amazonensis promastigotes in in vitro assays, and these effects involved the interaction of the sulfated polysaccharides on the cell membrane of the parasites

Extracellular matrix of porcine pericardium: Biochemistry and collagen architecture

Pericardial tissue has been used to construct bioprostheses employed in the repair of different kinds of injuries, mostly cardiac. However, calcification and mechanical failure have been the main causes of the limited durability of cardiac bioprostheses constructed with bovine pericardium. In the course of this work, a study was conducted on porcine fibrous pericardium, its microscopic structure and biochemical nature. The general morphology and architecture of collagen were studied under conventional light and polarized light microscopy. The biochemical study of the pericardial matrix was conducted according to the following procedures: swelling test, hydroxyproline and collagen dosage, quantification of amino acids in soluble collagen, component extraction of the extracellular matrix of the right and left ventral regions of pericardium with different molarities of guanidine chloride, protein and glycosaminoglycan (GAG) dosage, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and total GAG analysis. Microscopic analysis showed collagen fibers arranged in multidirectionally oriented layers forming a closely knit web, with a larger number of fibers obliquely oriented, initiating at the lower central region toward the upper left lateral relative to the heart. No qualitative differences were found between proteins extracted from the right and left regions. Likewise, no differences were found between fresh and frozen material. Protein dosages from left frontal and right frontal pericardium regions showed no significant differences. The quantities of extracted GAGs were too small for detection by the method used. Enzymatic digestion and electrophoretic analysis showed that the GAG found is possibly dermatan sulfate. The proteoglycan showed a running standard very similar to the small proteoglycan decorin

“Biotecnological war” uma ferramenta de avaliação conceitual e de percepção para o ensino de biotecnologia e química de proteínas para os alunos de graduação em ciências biológicas

Biochemistry in general is practically unanimous as a discipline with a high degree of difficulty, complex and "boring". So, practical and creative play games as teaching methodology has been disseminated in several disciplines of biological sciences. “Biotecnological war” board game is a proposal that was initially conceived as an alternative complementary tool for biochemistry teaching of proteins and peptides, challenging students, aiming to review concepts transmitted in classroom, stimulating student’s abilities, such as their creativity, competitiveness, resource management and making possible to correlate biochemistry importance of proteins and peptides as new products. This game proved to be an excellent tool for complementary evaluation of students, which besides stimulating teamwork, also stimulated "a strong competitive spirit" within the classroom, which allowed to analyze students' perception in relation to the theme and mainly articulated group work.A bioquímica no geral é taxada como uma disciplina com alto grau de dificuldade, complexa e “chata”. Por outro lado, a aplicação de jogos lúdicos práticos e criativos como metodologia de ensino vem se disseminando em várias disciplinas em ciências biológicas. O jogo de tabuleiro “Biotecnological war” é uma proposta pensada como uma ferramenta complementar para o ensino de bioquímica de proteínas e peptídeos, desafiando os discentes, visando rever conceitos transmitidos em sala de aula, e estimulando aptidões dos estudantes, como sua criatividade, competitividade e gestão de recursos. Possibilitando ainda correlacionar a importância da bioquímica de proteínas e peptídeos com o desenvolvimento de produtos. Este jogo se mostrou uma excelente ferramenta de avaliação complementar dos alunos, e além de estimular o trabalho em equipe, também estimulou “um forte espírito competitivo”, o que permitiu analisar a percepção dos alunos em relação ao tema e principalmente o trabalho em grupo