As Proteínas Inativadoras de Ribossomos (RIPs) compreendem uma família de enzimas que inibem a síntese protéica através da depurinação de uma adenina específica do RNA ribossomal. Os membros desta família são classificados como RIPs do tipo I, quando possuem somente o RNA-N-Glicosidase e RIPs do tipo II quando além do domínio glicosidase, também apresentam um domínio de lectina. As RIPs foram mais estudadas em plantas, onde a ricina e a aglutinina, ambas RIP do tipo II de mamona (Ricinus communis), estão entre as primeiras descritas. O presente trabalho teve o objetivo de identificar parálogos da ricina e aglutinina, bem como RIPs do tipo I de mamona e analisar as suas relações filogenéticas. Além disso, validar o uso de 14 potenciais genes de referência para qRT-PCR em cinco estádios do desenvolvimento da semente de mamona. O padrão de expressão gênica por RT-qPCR de todas RIPs de mamona identificadas, também foram analisados nestes mesmos estádios. Um total de 18 genes de RIPs foi identificado em mamona (Rcom RIPs), dos quais 10 foram classificados como do tipo II e 8 do tipo I. As topologias das árvores filogenéticas sugerem que as Rcom RIPs foram originadas a partir de múltiplos eventos de duplicação gênica. Dois modelos evolutivos foram propostos para a radiação das Rcom RIPs, baseados em processos de fusão gênica associado ou não a eventos de duplicação parcial. Os genes Act 2/7, EF β, Ubi, TIP e UBC foram os que apresentaram perfil de expressão mais estável e foram selecionados para subsequente normalização dos dados de expressão das Rcom RIPs. Os genes que codificam as Rcom RIPI 3, 4, 5, 7 e 8 e as Rcom RIPII 1, 2, 4, 5, 6 e 8 são transcritos em sementes, sendo que a Rcom RIPII 1 (ricina) e a Rcom RIPII 2 (aglutinina) foram as mais expressas. O presente trabalho apresenta um modelo evolutivo das Rcom RIPs, o qual pode ser extrapolado para outras espécies de plantas. Este trabalho também demonstra o primeiro esforço para a padronização de genes de referência para RT-qPCR em mamona e o primeiro que apresenta a expressão outras Rcom RIPs, além da ricina e aglutinina. / Ribosome inactivating proteins (RIPs) comprise a family of enzymes that inhibit protein synthesis, after depurination of an adenine-specific ribosomal RNA. The members of this family are classified as type I RIPs, which have a RNA-Nglycosidase domain and type II RIPs encompassing a RNA-N-glycosidase and a lectin domain.The RIPs were more studied in plants, where ricin and agglutinin, both type II RIP of castor bean (Ricinus communis), were the first to be described. This work aimed to: 1) identifine paralogous of ricin and agglutinin, as well as the type I RIPs of castor bean; 2) analyze their phylogenetic relationships; 3) validate the use of 14 potential housekeeping genes for qRT-PCR for five developmental stages of R. communis seeds; 4) analyze the pattern of gene expression by RTqPCR of all RIPs castor identified in these same stages. A total of 18 genes that encode RIPs were identified in castor bean (Rcom RIPs), 10 of which were classified as type II and 8 as type I. The phylogenetic trees topologies suggest that Rcom RIPs were originated from multiple events of gene duplications. Two evolutionary models have been proposed for the radiation of Rcom RIPs based on gene fusion processes associated or not to events of partial duplication. The genes Act 2/7, EF β, Ubi, TIP and UBC presented the more stable expression profile and were selected for further RT- qPCR normalization experiments. The Rcom RIPI 3, 4, 5, 7 and 8 and Rcom RIPI 1, 2, 4, 5, 6 and 8 genes are actively transcribed in seeds, whereas the Rcom RIPI 1 (ricin) and Rcom RIPI 2 (agglutinin) were the most expressed. This paper presents an evolutionary model of Rcom RIPs, which can be extrapolated to other plant species. Also, corresponds to the first effort to standardize housekeeping genes for RT-qPCR in castor bean and the first that shows the expression Rcom RIPs, other than ricin and agglutinin.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / As lectinas de algas marinhas possuem vÃrias aplicaÃÃes farmacolÃgicas. O objetivo deste trabalho foi caracterizar parcialmente a estrutura e avaliar os efeitos em modelos clÃssicos de inflamaÃÃo da lectina da alga marinha verde Caulerpa cupressoides. A lectina de Caulerpa cupressoides (LCc) foi extraÃda com tampÃo Tris-HCl 25 mM, pH 7,5 e isolada por cromatografia de troca iÃnica em coluna de DEAE-celulose. A caracterizaÃÃo estrutural parcial apresentou uma sequÃncia aminoterminal com 31 resÃduos de aminoÃcidos, obtida de acordo com o mÃtodo de degradaÃÃo de Edman, enquanto que na espectroscopia de ressonÃncia magnÃtica nuclear unidimensional 1H para a LCc purificada por Sephadex G-100 e obtida por DEAE-celulose, foi demostrado uma similaridade entre os sinais obtidos em ambos os espectros. A atividade anti-inflamatÃria foi avaliada em ratos Wistar machos (n=6), utilizando o modelo de edema de pata induzidos por carragenana (700 μg/pata), dextrana (500 μg/pata), histamina (100 μg/pata), serotonina (20 μg/pata) ou bradicinina (30 μg/pata). Grupos de animais foram submetidos ao tratamento com LCc (0,1; 1,0 ou 10,0 mg/kg; i.v.), 30 min antes do estÃmulo inflamatÃrio. Foi avaliado o envolvimento da LCc (1,0 mg/kg) na via da Heme oxigenase-1. Foram utilizados grupos que receberam tratamento com LCc ligada ao inibidor mucina (8,0 mg/kg; i.v.) ou somente mucina (8,0 mg/kg; i.v.) e dexametasona (1,0 mg/kg; s.c.) foram utilizados como controles. O efeito edematogÃnico de LCc foi avaliado aplicando as doses de 0,1; 1,0 ou 10 mg/kg (i.pl.). No ensaio de edema de pata induzido por carragenana, LCc reduziu a formaÃÃo de edema sendo confirmado pela determinaÃÃo dos nÃveis teciduais de mieloperoxidase. A LCc (1,0 mg/kg) ligada a mucina nÃo apresentou efeito anti-inflamatÃrio no edema de pata induzido por carragenana, exceto na primeira hora apÃs o estÃmulo. No edema induzido por dextrana, LCc tambÃm inibiu o edema osmÃtico. Apenas a dose de 1,0 mg/kg foi utilizada no edema induzido por histamina reduzindo em 40% o edema no intervalo de 30 min LCc (1,0 mg/kg), entretanto, nÃo reduziu a formaÃÃo de edema induzido por serotonina ou bradicinina. AlÃm disso, na anÃlise histolÃgica do tecido subplantar, LCc (1,0 e 10,0 mg/kg) foi capaz de reduzir a migraÃÃo celular. Na presenÃa de ZnPP IX (3,0 mg/kg; s.c.), LCc perdeu sua capacidade inibir o edema induzido por carragenana, exercendo seu mecanismo de aÃÃo anti-inflamatÃrio atravÃs do envolvimento da via da HO-1. Enquanto que na imunohistoquÃmica, LCc (10 mg/kg) reduziu a expressÃo de IL-1β, porÃm ocorreu intensa marcaÃÃo das citocinas TNF-α e IL-6, alÃm da expressÃo de HO-1, nos grupos tratados com a mesma dose de LCc. Com relaÃÃo ao efeito edematogÃnico, LCc foi capaz de induzir intenso processo inflamatÃrio com efeito dose-dependente. No entanto, o edema induzido com a dose de 10 mg/kg de LCc foi foi inibido por indometacina, meclizina, pentoxifilina e dexametasona. Portanto, a LCc quando parcialmente caracterizada apresentou na sua sequÃncia aminoterminal uma identidade de 43% com a proteÃna da alga verde unicelular Chlamydomonas reinhardtii e apresentou propriedades anti- e prÃ-inflamatÃrias, sendo considerada um agente terapÃutico em potencial para estudos futuros nos processos inflamatÃrios. / Lectins seaweed have various pharmacological applications. This work was partially characterize the structure and evaluate the effects on classical models of inflammation lectin of the green seaweed Caulerpa cupressoides. The Caulerpa cupressoides lectin (CcL) was extracted with Tris-HCl 25 mM, pH 7.5 and isolated by ion exchange chromatography on DEAE-cellulose. Structural characterization showed a partial aminoterminal sequence with 31 amino acid residues, obtained according to the method of Edman degradation, while in nuclear magnetic resonance spectroscopy to the CcL 1H-NMR purified by Sephadex G-100 and DEAE-cellulose obtained by was demonstrated similarity between signals obtained in both spectra. The anti-inflammatory activity was evaluated in male Wistar rats (n = 6), using the model of paw edema induced by carrageenan (700 μg/paw), dextran (500 μg/paw), histamine (100 μg/paw), serotonin (20 μg/paw) or bradykinin (30 μg/paw). Groups of animals were treated with CcL (0.1, 1.0 or 10.0 mg/kg, i.v.) 30 min before the inflammatory stimulus. Was evaluated the involvement of CcL (1.0 mg/kg) towards heme oxygenase-1. Groups were used that were treated with inhibitor CcL linked mucin (8.0 mg/kg, i.v.) or only mucin (8.0 mg/kg, i.v.) and dexamethasone (1.0 mg/kg, s.c.) were used as controls. The effect edematogenic CcL was evaluated by applying the doses of 0.1, 1.0 or 10 mg/kg (i.pl.). In the trial of paw edema induced by carrageenan, CcL reduced edema formation was confirmed by determining tissue levels of myeloperoxidase. CcL (1.0 mg/kg) showed no mucin linked to anti-inflammatory effect on the paw edema induced by carrageenan, except in the first hour after stimulation. In dextran-induced edema, CcL also inhibited the osmotic swelling. Only the dose of 1.0 mg/kg was used in reducing histamine-induced edema by 40% the edema within 30 min CcL (1.0 mg/kg), however, did not reduce the edema induced by serotonin or bradykinin. Besides, in the histological analysis of tissue subplantar, CcL (1.0 and 10.0 mg/kg) was able to reduce cell migration. In the presence of ZnPP IX (3.0 mg/kg, s.c.), CcL has lost its ability to inhibit the carrageenan-induced edema, exerting its mechanism of action anti-inflammatory pathway through the involvement of HO-1. While in immunohistochemistry, CcL (10 mg/kg) reduced the expression of IL-1β, but intense staining occurred cytokines TNF-α and IL-6, and expression of HO-1 in the groups treated with the same dose of CcL. In relation the effect edematogenic, CcL was able to induce intense inflammatory process with dose-dependent effect. However, the induced edema at a dose of 10 mg/kg was CcL was inhibited by indomethacin, meclizine, pentoxifylline and dexamethasone. Therefore, when the CcL partially characterized presented in its aminoterminal sequence of 43% identity with the protein from unicellular green alga Chlamydomonas reinhardtii and showed anti-and pro-inflammatory therapeutic agent is considered a potential for future studies in inflammatory processes.

Type-II Ribosome Inactivating Proteins from Abrus precatorius: Cytotoxicity and Mechanism of Cell Death A/B toxins produced by bacteria and plants are among the deadliest molecules known. The plant type-II ribosome inactivating proteins (RIPs) are prototype of A/B toxins. They are two subunit proteins with a toxic A subunit that harbors an RNA N-glycosidase activity and a lectin like B subunit which allows toxin entry into cells. The toxicity of A chain is due to its RNA-N-glycosidase activity which cleaves the bond between the ribose sugar and the adenine at position 4324 as demonstrated in rat liver ribosomes. The B- chain, a lectin, binds to the cell surface receptors terminating in galactose sugars and allows toxin entry into cells. The seeds of the subtropical climber Abrus precatorius contain two RIPs: the potent toxic lectin abrin and the relatively less toxic Abrus agglutinin. The toxic property of RIPs has widespread applications in the field of agriculture and medicine. The cells of our body commit suicide in response to genetic or environmental cues by the process, apoptosis or programmed cell death which results in the safe clearance of the dead cells without affecting the extra-cellular milieu. Apoptosis is essential for development, tissue homeostasis, and defense against pathogens. It involves the interplay of multiple pathways that are initiated and executed by a family of proteases termed caspases. Several plant type-I and type-II RIPs as well as bacterial toxins have been shown to induce apoptosis in cultured cell lines. Though many agents that inhibit macromolecular synthesis in cells induce DNA fragmentation and morphological changes associated with apoptosis, the link between protein synthesis inhibition by these toxins and apoptosis remains elusive. Though extensive studies have been carried out on several RIPs for e.g. ricin and shiga toxin, only few reports are available in literature on the mechanisms of toxicity exhibited by abrin, a type-II RIP, of South-East Asian origin. Earlier studies from the laboratory have focused on the sensitivity and mechanism of abrin induced cell death in Jurkat, a cell line of haematopoietic lineage and its variants. In the same direction, the objectives of my study were: (1) To delineate the structure-function relationship of Abrus agglutinin-I in comparison with abrin, (2) To establish monoclonal antibodies to the A subunit of abrin, analyzing their neutralizing effect on abrin toxicity in vitro and in vivo and (3) To delineate the pathway and determine the kinetics of apoptosis induced by abrin on cell lines of epithelial lineage. The thesis will be presented in three four chapters. The first chapter, ‘Introduction’, begins with a brief history of RIPs, followed by the description of their distribution and classification. The transport of toxins which is a unique property of this class of proteins is discussed in detail and supported with appropriate figures. Also, information pertaining to the structure of abrin and apoptosis induced by RIPs is written in brief. In the second chapter of the thesis the structural and functional studies of Abrus agglutinin-I (APA-I) as compared to abrin are discussed. Abrin and APA-I share a high degree of homology, however, previous reports by Liu et al., indicate that APA-I is many fold less toxic in cell free systems as compared to abrin. In our studies, APA-I was found to be less toxic on cultured cell lines. The IC50 value of protein synthesis inhibition by abrin was found to be 0.4 ng/ml for both Jurkat and MCF-7 cell lines. A 20-1000 fold difference was observed in the sensitivity of these cell lines to APA-I. The extent of apoptosis induced by APA-I in A3I9.2 a caspases-8 mutant Jurkat variant cell line was comparable to abrin indicating that the apoptosis induction by APA-I might not be through the extrinsic pathway. instead, our studies showed that APA-I induced apoptosis followed the mitochondrial pathway of cell death, in a caspase dependent manner similar to that of abrin. Unlike other agglutinins like wheat germ agglutinin, the agglutinating ability of the agglutinin-I had no role in the apoptosis induced. Protein synthesis inhibition appeared to be mandatory for the apoptosis induced by APA-I. The reason for the decreased toxicity of agglutinin-I became apparent on the analysis of the crystal structure of agglutinin-I obtained by us in comparison to that of the reported structure of abrin. The substitution of Asn200 in abrin with Pro199 in agglutinin-I seems to be a major cause for the decreased toxicity. This perhaps is not a consequence of any kink formation by Pro residue in the helical segment, as reported by others earlier but due to fewer interactions that proline can possibly have with the bound substrate. Passive immuno-neutralization by administration of neutralizing antibodies is widely used as therapy against poisoning by various toxins. In case of type-II RIPs like ricin, antibodies to the toxic subunit were proven to have better protective efficacy than those to the lectin subunit. Neutralizing antibodies to abrin are not reported in literature. Therefore, a panel of monoclonal antibodies (mAbs) to the recombinant A chain of abrin was developed in our laboratory and characterized, which is presented in the third chapter of the thesis. Of these, D6F10 a high affinity antibody, exhibited neutralizing effect on abrin induced cytotoxicity on different cell lines tested. Antibodies may neutralize biological toxins in multiple ways; our studies suggested that mAb D6F10 interferes in the earliest event i.e. attachment of the toxin to the cell surface. Significantly, with the administration of mice with mAb D6F10 the prophylactic effect of the mAb could be demonstrated. In chapter 4, the sensitivity, kinetics of proteins synthesis inhibition and the mechanism of abrin induced cell death in cell lines of epithelial lineage is presented. Both sensitivity and kinetics of MCF-7/pv, Ovcar3, and T47D cells appeared comparable while, a variant culture of MCF-7 over-expressing caspases-3 was 50 times more sensitive to abrin. There was no significant difference in the binding of abrin between MCF-7/pv and MCF-7/C3+ cells. Previous studies in our laboratory indicated that abrin induced apoptosis is a caspases-3 dependent process. Also, in several systems it has been shown that caspases-3 is an indispensable molecule for apoptotic cell death. To test the absolute requirement of caspase-3, we examined abrin-induced apoptosis in a human breast cancer cell line MCF-7/pv reportedly deficient in caspases-3. Unlike other molecules like cisplatin, apoptosis induced by abrin in the MCF- 7/pv cells was found to be caspase -3 independent. However faster kinetics of apoptosis is observed, indicating that there is amplification of the apoptotic signals in the presence of caspases-3 resulting in an early onset of DNA fragmentation. The kinetics of protein synthesis inhibition and apoptosis follows similar kinetics in Jurkat cells while there is a time lapse between the two events in epithelial cells. Even with very high concentrations of abrin no detectable apoptosis was observed within 24 h in epithelial cells. The onset of fragmentation occurs after 24 h in the cell lines tested as opposed to Jurkat where it is observed as early as 6 h. Inhibition of caspases rescued the toxins from DNA fragmentation suggesting that the toxin does not cause direct nuclear damage in the cell line which does not involve the activation of caspases.

No description available.

ANDRADE, Igo Renan Albuquerque de. Destoxificação da torta de mamona por produtos químicos alternativos. 2015. 91 f. Tese (Doutorado em Zootecnia)-Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Vitor Campos (vitband@gmail.com) on 2016-10-06T23:04:56Z No. of bitstreams: 1 2015_tese_iraandrade.pdf: 1066084 bytes, checksum: d0e22653e500e6aad6267d895bef9366 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-10-07T17:45:49Z (GMT) No. of bitstreams: 1 2015_tese_iraandrade.pdf: 1066084 bytes, checksum: d0e22653e500e6aad6267d895bef9366 (MD5) / Made available in DSpace on 2016-10-07T17:45:49Z (GMT). No. of bitstreams: 1 2015_tese_iraandrade.pdf: 1066084 bytes, checksum: d0e22653e500e6aad6267d895bef9366 (MD5) Previous issue date: 2015 / The objectives of the present study were to evaluate the use of alternative chemical sources in the degradation and inactivation of ricin and Ricinus communis agglutinin from castor cake; the use of an easily applicable methodology able to precisely identify the biological activity of the lectins present in the castor cake; and the influence of the detoxification process on the nutritional values of the castor cake and estimate the maximum sale price of detoxified cakes so that their use in the concentrate feed replacing soybean meal can be viable. The use efficiency of ten chemical compounds (calcitic limestone, magnesian limestone, sodium chloride, potassium chloride, dicalcium phosphate, monodicalcium phosphate, calcium hydroxide, sodium hydroxide, calcium oxide, and urea) in the inactivation of lectins from castor cake was evaluated, using four different volumes of water. Detoxification analyses were performed by visual observation and densitometry of electrophoresis gels, in addition to hemagglutination assays. After satisfactory results were obtained with the treatments using calcium oxide and sodium hydroxide, analyses of chemical composition; crude protein fractionation; rumen degradation of dry matter, crude protein, and neutral detergent fiber; and intestinal digestibility of crude protein were performed. Next, aiming to analyze the viability of using detoxified castor cake in concentrate diets completely replacing soybean meal, different diets were formulated to estimate its maximum sale costs. Alkaline treatments calcium oxide and sodium hydroxide provided complete inactivation of the lectins from castor cakes. Reagent concentration and water volume were decisive to the inactivation efficiency, with detoxification being effective when 60 or 90 g sodium hydroxide were used with water volumes equal to or greater than 1500 mL/kg castor cake or calcium oxide at the concentration of 90 g with 2500 or 3000 mL water/kg of cake. Chemical detoxification treatments directly influenced the profile of the different nitrogen fractions that compose the dietary crude protein; the rumen degradation parameters of dry matter, crude protein, and neutral detergent fiber; as well as the intestinal digestibility of the rumen-undegradable protein. Chemical detoxification treatments changed the nutritional value and the use of the dietary protein by the ruminant, reducing the nutritional value of the cakes and providing feedstuffs with different nutritional characteristics. Castor cakes treated with calcium oxide allowed a higher level of replacement of soybean meal as compared with the cakes treated with sodium hydroxide. In economic terms, the cakes treated with calcium oxide allowed a higher sale price of the product in relation to those treated with sodium hydroxide. / O presente estudo foi conduzido com o objetivo de avaliar o uso de fontes químicas alternativas na degradação e inativação da ricina e Ricinus communis aglutinina da torta de mamona; o uso de uma metodologia de fácil aplicação, capaz de identificar com precisão a atividade biológica das lectinas presentes na torta de mamona; bem como verificar a influência do processo de destoxificação sobre os valores nutritivos da torta de mamona e estimar o custo máximo de venda das tortas destoxificadas, para que seja viável a sua utilização na ração concentrada, em substituição ao farelo de soja. Foi avaliada a eficácia do uso de dez produtos químicos: calcário calcítico, calcário magnesiano, cloreto de sódio, cloreto de potássio, fosfato bicálcico, fosfato monobicálcico, hidróxido de cálcio, hidróxido de sódio, óxido de cálcio e ureia, na destoxificação das lectinas da torta de mamona, utilizando quatro diferentes volumes de água. As análises de destoxificação foram realizadas através da observação visual e por densitometria de géis de eletroforese, além de ensaios de hemaglutinação. Após verificados resultados satisfatórios dos tratamentos que utilizaram óxido de cálcio e hidróxido de sódio, foram realizadas análises de composição química, fracionamento da proteína bruta, degradação ruminal da matéria seca, proteína bruta e fibra em detergente neutro, além da digestibilidade intestinal da proteína bruta. Em seguida, com o intuito de analisar a viabilidade de uso da torta de mamona destoxificada em rações concentradas, substituindo totalmente o farelo de soja, foram formuladas diferentes rações, buscando estimar os custos máximos de comercialização das mesmas. Verificou-se que os tratamentos químicos alcalinos, óxido de cálcio e hidróxido de sódio, promoveram completa inativação das lectinas das tortas de mamona. A concentração do reagente e o volume de água foram determinantes para a eficácia de tal inativação, sendo a destoxificação eficaz ao se utilizar 60 ou 90 g de hidróxido de sódio em volumes de água iguais ou superiores a 1500 mL/kg de torta de mamona ou óxido de cálcio na concentração de 90 g de reagente com 2500 ou 3000 mL de água/kg de torta de mamona. Os tratamentos químicos de destoxificação influenciaram diretamente o perfil das diferentes frações nitrogenadas que compõe a proteína bruta dietética, assim como os parâmetros de degradação ruminal da matéria seca, proteína bruta e fibra em detergente neutro, bem como a digestibilidade intestinal da proteína bruta não degradável no rúmen. Os tratamentos químicos de destoxificação alteraram o valor nutritivo e o aproveitamento da proteína dietética pelo animal ruminante, observando diminuição do valor nutritivo das tortas e proporcionando alimentos com diferentes características nutricionais. As tortas de mamona tratadas com óxido de cálcio permitiram um nível de substituição do farelo de soja mais elevado, quando comparados às tortas de mamona tratadas com hidróxido de sódio. Em termos econômicos, verificou-se que as tortas tratadas com óxido de cálcio permitiram um maior preço de venda da mesma em relação às tortas tratadas com hidróxido de sódio.

The journey of structural studies on lectins, starting with ConA in the 70s, has crossed many milestones. Lectins, multivalent carbohydrate-binding proteins of non-immune origin, specifically bind diverse sugar structures. They have received considerable attention in recent times on account of the realization of the importance of protein-sugar interactions, especially at the cell surface, in biological recognition. They occur in plants, animals, fungi, bacteria and viruses. Plant lectins constitute about 40% of the lectins of known structure. They can be classified into five structural groups, each characterized by a specific fold. Among them, legume lectins constitute the most extensively investigated group. Basic Winged bean lectin (WBAI) is a glycosylated, homodimeric, legume lectin with Mr 58000. The structure of WBAI complexed with methyl-a-galactose, determined earlier in this laboratory, provided information about the oligomeric state and the carbohydrate specificity of the lectin in terms of lectin-monosaccharide interactions. The present work was initiated to understand the carbohydrate specificity of the lectin, especially at the oligosaccharide level, with special reference to its blood group specificity. The hanging drop method was used for crystallizing WBAI and its complexes. Intensity data were collected on Mar Research imaging plates mounted on Rigaku RU-200 or ULTRAX-18 X-ray generators. The data were processed using DENZO and SCALEPACK of HKL suite of programs. The structure factors from the processed data were calculated using TRUNCATE of CCP4 suite of programs. The molecular replacement program AMoRe was used for structure solution. Structure refinement was carried out using the CNS software package. Model building was done using the molecular graphics program O. INSIGHT II, ALIGN, CONTACT and PROCHECK of CCP4 were used for the analysis and validation of the refined structures. WBAI exhibits differential affinity for different monosaccharide derivatives of galactose. In order to elucidate the structural basis for this differential affinity, the crystal structures of the complexes of basic winged bean lectin with galactose, 2-methoxygalactose, N-acetylgalactosamine and methyl-a-N-acetylgalactosamine have been determined. Lectin-sugar interactions involve four hydrogen bonds and a stacking interaction in all of them. In addition, a N-H O hydrogen bond involving the hydroxyl group substituted at C2 exists in the galactose and 2-methoxygalactose complexes. The additional hydrophobic interaction, involving the methyl group, in the latter leads to the higher affinity of the methyl derivative. In the lectin - N- acetylgalactosamine complex the N-H O hydrogen bond is lost, but a compensatory hydrogen bond involving the oxygen atom of the acetamido group is formed. In addition, the CH3 moiety of the acetamido group is involved in hydrophobic interactions. Consequently, the 2-methyl and the acetamido derivatives of galactose have nearly the same affinity for the lectin. The methyl group, a-linked to the galactose, takes part in additional hydrophobic interactions. Therefore, methyl-a- N-acetylgalactosamine has higher affinity than N-acetylgalactosamine to the lectin. The structures of basic winged bean lectin-sugar complexes provide a framework for examining the relative affinity of galactose and galactosamine for the lectins that bind to them. The complexes also lead to a structural explanation for the blood group specificity of basic winged bean lectin, in terms of its monosaccharide specificity. The Tn-determinant (GalNAc-a-O-Ser/Thr) is a human specific tumor associated carbohydrate antigen. Having epithelial origin, it is expressed in many carcinogenic tumors including breast, prostate, lung and pancreatic cancers. The crystal structure of WBAI in complex with GalNAc-a-O-Ser (Tn-antigen) has been elucidated, in view of its relevance to diagnosis and prognosis of various human cancers. The Gal moiety occupies the primary binding site and makes interactions similar to those found in other Gal/GalNAc specific legume lectins. The nitrogen and oxygen atoms of the acetamido group of the sugar make two hydrogen bonds with the protein atoms whereas its methyl group is stabilized by hydrophobic interactions. A water bridge formed between the terminal oxygen atoms of the serine residue of the Tn-antigen and the side chain oxygen atom of Asn128 of the lectin increase the affinity of the lectin for Tn-antigen compared to that for GalNAc. A comparison with the available structures reveals that while the interactions of the glyconic part of the antigen are conserved, the mode of stabilization of the serine residue differs and depends on the nature of the protein residues in its vicinity. The structure provides a qualitative explanation for the thermodynamic parameters of the formation of the complex of the lectin with Tn-antigen. Modelling studies indicate the possibility of an additional hydrogen bond with the lectin when the antigen is part of a glycoprotein. WBAI binds A-blood group substance with higher affinity and B-blood group substance with lesser affinity. It does not bind the O substance. The crystal structures of the lectin, complexed with A -reactive and B - reactive di and tri saccharides, have been determined. In addition, the complexes of the lectin with fucosylated A- and B-trisaccharides and with a variant of the A-trisaccharide have been modelled. These structures and models provide valuable insights into the structural basis of blood group specificities. All the four carbohydrate binding loops of the lectin contribute to the primary combining site while the loop of variable length contributes to the secondary binding site. In a significant advance to the current understanding, the interactions at the secondary binding site also contribute substantially, albeit in a subtle manner, to determine the blood group specificity. Compared to the interactions of the B- trisaccharide with the lectin, the third sugar residue of the A -reactive trisaccharide forms an additional hydrogen bond with a lysine residue in the variable loop. In the former, the formation of such a hydrogen bond is prevented by a shift in the orientation of the third sugar resulting from an internal hydrogen bond in it. The formation of this bond is also facilitated by an interaction dependent change in the rotamer conformation of the lysyl residue of the variable loop. Thus, the difference in the interactions at the secondary site is generated by coordinated movements in the ligand as well as the protein. A comparison of the crystal structure and the model of the complex involving the variant of the A-trisaccharide results in the delineation of the relative contributions of the interactions at the primary and the secondary sites in determining blood group specificity. At the disaccharide level, WBAI exhibits higher affinity for á1-3 linked Gal/GalNAc containing oligosaccharides, compared to that of other á linked oligosaccharides. With an objective of understanding the preferential binding of WBAI for á 1-3 linked Gal/GalNAc containing oligosaccharides, crystal structure of the complexes of the lectin with Galá1-4Gal, Galá1-4GalâEt and Galá1-6Gal have been determined. The reducing sugar of the disaccharides with linkages other than á1-3 binds to the lectin through a water bridge whereas the same sugar moiety with á 1-3 linkage makes direct interactions with the loop L4 of the protein. The modelling study on the complex of the lectin with Galá1-2Gal further upholds this observation. Different structures involving WBAI, reported earlier and presented here, were used to investigate the plasticity of the lectin. The front curved â-sheet, which nestles the metal binding region and on which the carbohydrate binding loops are perched, is relatively rigid. On the contrary, the flat back â-sheet, involved in the quaternary association in legume lectins, is flexible. This flexibility is probably necessary to account for the variation in quaternary structure. With the results presented in this thesis, 14 crystal structures of WBAI, in the free form and in complex with different sugars, have been reported, all from this laboratory. It is now, perhaps, appropriate to examine the new information and insights gained from these investigations, on the structure and function of the lectin. Earlier X-ray studies of WBAI contributed substantially in establishing that legume lectins are a family of proteins in which small alterations in essentially the same tertiary structure lead to large alterations in quaternary association. Structural studies on WBAI, particularly those reported here, also contributed to the elucidation of the nuances of carbohydrate recognition by lectins. A comparative study of the available structures also revealed the flexible and rigid regions of the protein. The study of the influence of covalently linked sugars on the structure of Erythrina corallodendron lectin (ECorL), a homolog of WBAI, is the content of appendix of the thesis. The three-dimensional structure of the recombinant form of Erythrina Corallodendron lectin(rECorL) complexed with lactose, has been elucidated by X-ray crystallography. Comparison of this non-glycosylated structure with that of the native glycosylated lectin reveals that the tertiary and quaternary structures are identical in the two forms, with local changes observed at one of the glycosylation sites(Asn17). These changes take place in such a way that hydrogen bonds with the neighbouring protein molecules in rECorL compensate those made by the glycan with the protein in ECorl. contrary to an earlier report, this study demonstrates that the glycan attached to the lectin does not influence the oligomeric state of the lectin. Identical interactions between the lectin and the non-covalently bound lactose in the two forms indicate, in line with earlier reports, that glycosylation does not affect the carbohydrate specificity of the lectin. The present study, the first of its kind involving a glycosylated protein with a well defined glycan and the corresponding deglycosylated form, provides insights into the structural aspects of protein glycosylation.

Ribosome Inactivating Proteins (RIPs) are protein or glycoprotein toxins that bring about the arrest of protein synthesis by directly interacting with and inactivating the ribosomes. Such toxins are in general, of plant origin and differ from bacterial toxins that inhibit protein synthesis by mechanisms other than ribosome inactivation. After the toxins had been in the centre of interest in biomedical research for a couple of decades in the end of 19th century, the scientific community largely lost interest in the plant toxins. Interest in these toxins was revived when it was found that they are more toxic to tumor cells when compared to normal cells. Based on their structure RIPs can be classified into three types: Type I RIPs – They consist exclusively of a single RNA-N-glycosidase chain of ~30kDa. Type II RIPs – They consists of chain-A comparable to type I RIPs linked by a disulfide bridge to an unrelated chain-B, which has carbohydrate binding activity. The molecular weight of the type II RIPs is ~60kDa. Type III RIPs – Besides the classical type II RIPs a 60kDa RIP (called JIP60) has been identified in barley (Hordeum vulgare) that consists of chain-A resembling type I RIPs linked to an unrelated chain-B with unknown function. In addition to these classes of RIPs there is another group of toxins called four subunit toxins, whose structure is almost similar to type II RIPs, but are made up of two such subunits linked by non-covalent interactions forming tetramers having two A- and two B-chains. The definition and classification of these toxins is not so clear as they are frequently referred to as agglutinins or lectins (e.g Abrus precatorius agglutinins I and II, Ricinus communis agglutinin etc.), having red blood cell (RBC) agglutinating activity. However they have been found to be less toxic and better agglutinins when compared with type II RIPs. The present thesis reports the crystal structure of a type II RIP, Abrus precatorius agglutinin-I (APA-I) from the seeds of Abrus precatorius plant. The protein was purified from the plant seed and crystallized. The crystal structure was solved by molecular replacement method. Preliminary crystals of abrus agglutinin were obtained almost thirty years ago and unsuccessful attempts to solve the crystal Structure of APA-I were made almost five years ago by other groups. The structure solution of API-I was obtained at 3.5 Å using synchrotron data set collected at room temperature from a single crystal. Crystal structure is already known for Abrin, another type II RIP isolated from the same seeds. Abrin and APA-I have similar therapeutic indices for the treatment of experimental mice with tumors, but APA-I has much lower toxicity, with lethal dose (LD50) being 5mg/kg of body weight when compared with Abrin-a (LD50 = 20 μg/kg of body weight). The striking difference in the toxicity shown by Abrin and its agglutinin (APA-I) encouraged us to look at the structure function relationship of these proteins, which might prove to be useful in the design and construction of immunotoxins. As apparent from the comparative study, the reduced toxicity of APA-I can be attributed to fewer interactions it can possibly have with the substrate due to the presence of Pro199 at the binding site and not due to any kink formed in the helix due to the presence of praline as reported by other groups. In recent years, these plant RIPs which inhibit protein synthesis have become a subject of intense investigation not only because of the possible role played by them in synthesizing immunotoxins that are used in cancer therapy but also because they serve as model system for studying the molecular mechanism of transmembrane translocation of proteins. In silico docking studies were carried out in search of inhibitors that could modulate the toxicity of RIPs. Many adenine like ringed compounds were studied in order to identify them as novel inhibitors of Abrin-a molecule and facilitate detailed analyis of protein ligand complex in various ways to ascertain their potential as ligands. In addition, the structural analysis of conformationally constrained, α β-dehydrophenylalanine containing dipeptides is carried out. While there are several studies of molecular self assembly of peptides containing coded amino acids, not much work has been done on molecular assembly formation utilizing non-coded amino acids. The non-coded amino acid used in the analysis is a member of α β-dehydroamino acids. These are the derivatives of protein amino acids with a double bond between Cα And Cβ atoms and are represented by a prefix symbol ‘Δ’. They are frequently found in natural peptides of microbial and fungal origins. The presence of α , β-dehydroamino acid residues in bioactive peptides confers altered bioactivity as well as an increased resistance to enzymatic degradation. Thus, α, β-dehydroamino acid residues, in particular α, β-dehydrophenylalaine(ΔPhe) has become one of the most promising residues in the study of structure-activity relationships of biologically important peptides. The utilization of in the molecular self assembly ΔPhe in the molecular self assembly offers in added benfit in terms of variey and stability. Taking advantage of the conformation constraining property of the ΔPhe residue, its incorporation in three dipeptide molecules has been probed. In this thesis the crystal structures of the following designed dipeptide are reported.(I). +H3N-Phe-ΔPhe-COO˙ (FΔF); (II). +H3N-Val-ΔPhe- COO˙ (VΔF); +H3N-Ala-ΔPhe-COO˙ (AΔF). The peptides were found to be in the zwitterionic conformation and two (I, II) of the three dipeptides have resulted in tubular structures of dimensions in the nanoscale range. Chapter 1 starts with a brief introduction of RIPs, their classification and overall fold, with Abrin-a as example. A brief mention is made about how the protein is translocated in the cell and the depurination mechanism. Chapter 2 presents the purification of APA-I from the seeds of Abrus precatorius plant, the crystallization of APA-I, X-ray intensity data collection on these crystals and processing of data sets for APA-I. Chapter 3 details the structure determination of tetramer Abrus precatorius agglutinin-I,(APA-I), using the molecular replacement method, iterative model building and refinement and the quality of final protein structure model. Chapter 4 details the crystal structure of Abrus precatorius agglutinin-I (APA-I), the comparison of primary and secondary structure of APA-I with Abrin-a and the structural insights into the reduced toxicity in relation to Abrin-a and future prospects. Chapter 5 deals with the in-silico modeling of Abrin-a inhibitors using the docking method. Abrin-a is being tested extensively for the design of therapeutic immunotoxins. Chapter 6 deals with the self-assembly of dipeptides containing conformationally constrained amino acid, α. β -dehydrophenylalanine (ΔF).

QUEIROZ, Ismael Nilo Lino de. Caracterização estrutural preliminar e efeitos na inflamação da lectina da alga marinha verde Caulerpa cupressoides. 115 f. : Dissertação (Mestrado em Bioquímica) - Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará Fortaleza-CE, 2013. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-05-23T13:42:40Z No. of bitstreams: 1 2013_dis_inlqueiroz.pdf: 2778929 bytes, checksum: 1ecf927693bc8e91aabb5a746c3dfbce (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-07-05T19:19:39Z (GMT) No. of bitstreams: 1 2013_dis_inlqueiroz.pdf: 2778929 bytes, checksum: 1ecf927693bc8e91aabb5a746c3dfbce (MD5) / Made available in DSpace on 2016-07-05T19:19:39Z (GMT). No. of bitstreams: 1 2013_dis_inlqueiroz.pdf: 2778929 bytes, checksum: 1ecf927693bc8e91aabb5a746c3dfbce (MD5) Previous issue date: 2013 / Lectins seaweed have various pharmacological applications. This work was partially characterize the structure and evaluate the effects on classical models of inflammation lectin of the green seaweed Caulerpa cupressoides. The Caulerpa cupressoides lectin (CcL) was extracted with Tris-HCl 25 mM, pH 7.5 and isolated by ion exchange chromatography on DEAE-cellulose. Structural characterization showed a partial aminoterminal sequence with 31 amino acid residues, obtained according to the method of Edman degradation, while in nuclear magnetic resonance spectroscopy to the CcL 1H-NMR purified by Sephadex G-100 and DEAE-cellulose obtained by was demonstrated similarity between signals obtained in both spectra. The anti-inflammatory activity was evaluated in male Wistar rats (n = 6), using the model of paw edema induced by carrageenan (700 μg/paw), dextran (500 μg/paw), histamine (100 μg/paw), serotonin (20 μg/paw) or bradykinin (30 μg/paw). Groups of animals were treated with CcL (0.1, 1.0 or 10.0 mg/kg, i.v.) 30 min before the inflammatory stimulus. Was evaluated the involvement of CcL (1.0 mg/kg) towards heme oxygenase-1. Groups were used that were treated with inhibitor CcL linked mucin (8.0 mg/kg, i.v.) or only mucin (8.0 mg/kg, i.v.) and dexamethasone (1.0 mg/kg, s.c.) were used as controls. The effect edematogenic CcL was evaluated by applying the doses of 0.1, 1.0 or 10 mg/kg (i.pl.). In the trial of paw edema induced by carrageenan, CcL reduced edema formation was confirmed by determining tissue levels of myeloperoxidase. CcL (1.0 mg/kg) showed no mucin linked to anti-inflammatory effect on the paw edema induced by carrageenan, except in the first hour after stimulation. In dextran-induced edema, CcL also inhibited the osmotic swelling. Only the dose of 1.0 mg/kg was used in reducing histamine-induced edema by 40% the edema within 30 min CcL (1.0 mg/kg), however, did not reduce the edema induced by serotonin or bradykinin. Besides, in the histological analysis of tissue subplantar, CcL (1.0 and 10.0 mg/kg) was able to reduce cell migration. In the presence of ZnPP IX (3.0 mg/kg, s.c.), CcL has lost its ability to inhibit the carrageenan-induced edema, exerting its mechanism of action anti-inflammatory pathway through the involvement of HO-1. While in immunohistochemistry, CcL (10 mg/kg) reduced the expression of IL-1β, but intense staining occurred cytokines TNF-α and IL-6, and expression of HO-1 in the groups treated with the same dose of CcL. In relation the effect edematogenic, CcL was able to induce intense inflammatory process with dose-dependent effect. However, the induced edema at a dose of 10 mg/kg was CcL was inhibited by indomethacin, meclizine, pentoxifylline and dexamethasone. Therefore, when the CcL partially characterized presented in its aminoterminal sequence of 43% identity with the protein from unicellular green alga Chlamydomonas reinhardtii and showed anti-and pro-inflammatory therapeutic agent is considered a potential for future studies in inflammatory processes. / As lectinas de algas marinhas possuem várias aplicações farmacológicas. O objetivo deste trabalho foi caracterizar parcialmente a estrutura e avaliar os efeitos em modelos clássicos de inflamação da lectina da alga marinha verde Caulerpa cupressoides. A lectina de Caulerpa cupressoides (LCc) foi extraída com tampão Tris-HCl 25 mM, pH 7,5 e isolada por cromatografia de troca iônica em coluna de DEAE-celulose. A caracterização estrutural parcial apresentou uma sequência aminoterminal com 31 resíduos de aminoácidos, obtida de acordo com o método de degradação de Edman, enquanto que na espectroscopia de ressonância magnética nuclear unidimensional 1H para a LCc purificada por Sephadex G-100 e obtida por DEAE-celulose, foi demostrado uma similaridade entre os sinais obtidos em ambos os espectros. A atividade anti-inflamatória foi avaliada em ratos Wistar machos (n=6), utilizando o modelo de edema de pata induzidos por carragenana (700 μg/pata), dextrana (500 μg/pata), histamina (100 μg/pata), serotonina (20 μg/pata) ou bradicinina (30 μg/pata). Grupos de animais foram submetidos ao tratamento com LCc (0,1; 1,0 ou 10,0 mg/kg; i.v.), 30 min antes do estímulo inflamatório. Foi avaliado o envolvimento da LCc (1,0 mg/kg) na via da Heme oxigenase-1. Foram utilizados grupos que receberam tratamento com LCc ligada ao inibidor mucina (8,0 mg/kg; i.v.) ou somente mucina (8,0 mg/kg; i.v.) e dexametasona (1,0 mg/kg; s.c.) foram utilizados como controles. O efeito edematogênico de LCc foi avaliado aplicando as doses de 0,1; 1,0 ou 10 mg/kg (i.pl.). No ensaio de edema de pata induzido por carragenana, LCc reduziu a formação de edema sendo confirmado pela determinação dos níveis teciduais de mieloperoxidase. A LCc (1,0 mg/kg) ligada a mucina não apresentou efeito anti-inflamatório no edema de pata induzido por carragenana, exceto na primeira hora após o estímulo. No edema induzido por dextrana, LCc também inibiu o edema osmótico. Apenas a dose de 1,0 mg/kg foi utilizada no edema induzido por histamina reduzindo em 40% o edema no intervalo de 30 min LCc (1,0 mg/kg), entretanto, não reduziu a formação de edema induzido por serotonina ou bradicinina. Além disso, na análise histológica do tecido subplantar, LCc (1,0 e 10,0 mg/kg) foi capaz de reduzir a migração celular. Na presença de ZnPP IX (3,0 mg/kg; s.c.), LCc perdeu sua capacidade inibir o edema induzido por carragenana, exercendo seu mecanismo de ação anti-inflamatório através do envolvimento da via da HO-1. Enquanto que na imunohistoquímica, LCc (10 mg/kg) reduziu a expressão de IL-1β, porém ocorreu intensa marcação das citocinas TNF-α e IL-6, além da expressão de HO-1, nos grupos tratados com a mesma dose de LCc. Com relação ao efeito edematogênico, LCc foi capaz de induzir intenso processo inflamatório com efeito dose-dependente. No entanto, o edema induzido com a dose de 10 mg/kg de LCc foi foi inibido por indometacina, meclizina, pentoxifilina e dexametasona. Portanto, a LCc quando parcialmente caracterizada apresentou na sua sequência aminoterminal uma identidade de 43% com a proteína da alga verde unicelular Chlamydomonas reinhardtii e apresentou propriedades anti- e pró-inflamatórias, sendo considerada um agente terapêutico em potencial para estudos futuros nos processos inflamatórios.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O presente estudo foi conduzido com o objetivo de avaliar o uso de fontes quÃmicas alternativas na degradaÃÃo e inativaÃÃo da ricina e Ricinus communis aglutinina da torta de mamona; o uso de uma metodologia de fÃcil aplicaÃÃo, capaz de identificar com precisÃo a atividade biolÃgica das lectinas presentes na torta de mamona; bem como verificar a influÃncia do processo de destoxificaÃÃo sobre os valores nutritivos da torta de mamona e estimar o custo mÃximo de venda das tortas destoxificadas, para que seja viÃvel a sua utilizaÃÃo na raÃÃo concentrada, em substituiÃÃo ao farelo de soja. Foi avaliada a eficÃcia do uso de dez produtos quÃmicos: calcÃrio calcÃtico, calcÃrio magnesiano, cloreto de sÃdio, cloreto de potÃssio, fosfato bicÃlcico, fosfato monobicÃlcico, hidrÃxido de cÃlcio, hidrÃxido de sÃdio, Ãxido de cÃlcio e ureia, na destoxificaÃÃo das lectinas da torta de mamona, utilizando quatro diferentes volumes de Ãgua. As anÃlises de destoxificaÃÃo foram realizadas atravÃs da observaÃÃo visual e por densitometria de gÃis de eletroforese, alÃm de ensaios de hemaglutinaÃÃo. ApÃs verificados resultados satisfatÃrios dos tratamentos que utilizaram Ãxido de cÃlcio e hidrÃxido de sÃdio, foram realizadas anÃlises de composiÃÃo quÃmica, fracionamento da proteÃna bruta, degradaÃÃo ruminal da matÃria seca, proteÃna bruta e fibra em detergente neutro, alÃm da digestibilidade intestinal da proteÃna bruta. Em seguida, com o intuito de analisar a viabilidade de uso da torta de mamona destoxificada em raÃÃes concentradas, substituindo totalmente o farelo de soja, foram formuladas diferentes raÃÃes, buscando estimar os custos mÃximos de comercializaÃÃo das mesmas. Verificou-se que os tratamentos quÃmicos alcalinos, Ãxido de cÃlcio e hidrÃxido de sÃdio, promoveram completa inativaÃÃo das lectinas das tortas de mamona. A concentraÃÃo do reagente e o volume de Ãgua foram determinantes para a eficÃcia de tal inativaÃÃo, sendo a destoxificaÃÃo eficaz ao se utilizar 60 ou 90 g de hidrÃxido de sÃdio em volumes de Ãgua iguais ou superiores a 1500 mL/kg de torta de mamona ou Ãxido de cÃlcio na concentraÃÃo de 90 g de reagente com 2500 ou 3000 mL de Ãgua/kg de torta de mamona. Os tratamentos quÃmicos de destoxificaÃÃo influenciaram diretamente o perfil das diferentes fraÃÃes nitrogenadas que compÃe a proteÃna bruta dietÃtica, assim como os parÃmetros de degradaÃÃo ruminal da matÃria seca, proteÃna bruta e fibra em detergente neutro, bem como a digestibilidade intestinal da proteÃna bruta nÃo degradÃvel no rÃmen. Os tratamentos quÃmicos de destoxificaÃÃo alteraram o valor nutritivo e o aproveitamento da proteÃna dietÃtica pelo animal ruminante, observando diminuiÃÃo do valor nutritivo das tortas e proporcionando alimentos com diferentes caracterÃsticas nutricionais. As tortas de mamona tratadas com Ãxido de cÃlcio permitiram um nÃvel de substituiÃÃo do farelo de soja mais elevado, quando comparados Ãs tortas de mamona tratadas com hidrÃxido de sÃdio. Em termos econÃmicos, verificou-se que as tortas tratadas com Ãxido de cÃlcio permitiram um maior preÃo de venda da mesma em relaÃÃo Ãs tortas tratadas com hidrÃxido de sÃdio. / The objectives of the present study were to evaluate the use of alternative chemical sources in the degradation and inactivation of ricin and Ricinus communis agglutinin from castor cake; the use of an easily applicable methodology able to precisely identify the biological activity of the lectins present in the castor cake; and the influence of the detoxification process on the nutritional values of the castor cake and estimate the maximum sale price of detoxified cakes so that their use in the concentrate feed replacing soybean meal can be viable. The use efficiency of ten chemical compounds (calcitic limestone, magnesian limestone, sodium chloride, potassium chloride, dicalcium phosphate, monodicalcium phosphate, calcium hydroxide, sodium hydroxide, calcium oxide, and urea) in the inactivation of lectins from castor cake was evaluated, using four different volumes of water. Detoxification analyses were performed by visual observation and densitometry of electrophoresis gels, in addition to hemagglutination assays. After satisfactory results were obtained with the treatments using calcium oxide and sodium hydroxide, analyses of chemical composition; crude protein fractionation; rumen degradation of dry matter, crude protein, and neutral detergent fiber; and intestinal digestibility of crude protein were performed. Next, aiming to analyze the viability of using detoxified castor cake in concentrate diets completely replacing soybean meal, different diets were formulated to estimate its maximum sale costs. Alkaline treatments calcium oxide and sodium hydroxide provided complete inactivation of the lectins from castor cakes. Reagent concentration and water volume were decisive to the inactivation efficiency, with detoxification being effective when 60 or 90 g sodium hydroxide were used with water volumes equal to or greater than 1500 mL/kg castor cake or calcium oxide at the concentration of 90 g with 2500 or 3000 mL water/kg of cake. Chemical detoxification treatments directly influenced the profile of the different nitrogen fractions that compose the dietary crude protein; the rumen degradation parameters of dry matter, crude protein, and neutral detergent fiber; as well as the intestinal digestibility of the rumen-undegradable protein. Chemical detoxification treatments changed the nutritional value and the use of the dietary protein by the ruminant, reducing the nutritional value of the cakes and providing feedstuffs with different nutritional characteristics. Castor cakes treated with calcium oxide allowed a higher level of replacement of soybean meal as compared with the cakes treated with sodium hydroxide. In economic terms, the cakes treated with calcium oxide allowed a higher sale price of the product in relation to those treated with sodium hydroxide.

1) Aims To explore the ultrastructure of interstitial cells in the upper lamina propria of the human bladder, to describe the spatial relationships and to investigate cell-cell contacts. 2) Methods Focused ion beam scanning electron microscopy (FIB-SEM), 3-View SEM and confocal laser scanning microscopy were used to analyze the 3D ultrastructure of the upper lamina propria in male and female human bladders. 3) Results 3View-SEM image stacks as large as 59µm x 59µm x 17µm (xyz) at a resolution of 16nm x 16nm x 50 nm and high resolution (5nm x 5nm x 10nm) FIB-SEM stacks could be analyzed. Interstitial cells with myoid differentiation (mIC) and fibroblast like interstitial cells (fIC) were the major cell types in the upper lamina propria. The flat, sheet-like ICs were oriented strictly parallel to the urothelium sheet-like morphology. No spindle shaped cells were present. We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria. 4) Conclusions Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions.