Dissertations / Theses on the topic 'Abrus precatorius'

A schistosomose causada por Schistosoma mansoni é uma das parasitoses mais prevalentes em humanos, sendo o seu hospedeiro intermediário um molusco de água doce da espécie Biomphalaria glabrata. Para o controlo desta parasitose, além do tratamento dos doentes infetados, também é importante que as populações de moluscos sejam controladas. Visando encontrar estratégias de controlo dos moluscos, este estudo pretende clarificar o papel de alguns extratos vegetais enquanto inibidores do desenvolvimento do parasita na fase intra molusco. Neste contexto, o presente estudo teve como objetivo principal avaliar a atividade biológica de extratos de folhas de duas plantas da Guiné-Bissau (Abrus precatorius e Khaya senegalensis) sobre Biomphalaria glabrata infetada com Schistosoma mansoni. Os parâmetros avaliados foram a sobrevivência/mortalidade, oviposição, eliminação de cercárias e a sua viabilidade no hospedeiro definitivo (murganho), assim como a presença de danos no DNA dos moluscos através da técnica de Comet Assay. Embora os obtidos resultados sejam bastante preliminares, parecem indicar que os extratos de folhas de Abrus precatorius e Khaya senegalensis não apresentam atividade moluscicida uma vez que ocorreu uma baixa taxa mortalidade nos grupos de moluscos expostos aos extratos e esta ocorreu ao longo do bioensaio e não nas primeiras 24 horas de exposição. Através da técnica de Comet assay foi possível verificar que o DNA dos moluscos expostos aos extratos apresentava danos, mas não significativos. Em relação ao grupo de moluscos infetados com S.mansoni e expostos aos extratos, só os que foram expostos ao extrato de A. precatorius e a DMSO apresentaram danos significativos no DNA. No que diz respeito à libertação de cercárias demonstrou que a exposição aos extratos induziu uma maior libertação de cercárias por parte dos moluscos quando comparativamente ao grupo de moluscos só infetados com S.mansoni ,no entanto estas diferenças não se apresentam como significativas. Em relação à oviposição, os extratos em si parecem não ser responsáveis pelo aumento do número médio de posturas produzidas por B.glabrata. Embora tenham existido diferenças no número médio de posturas produzidas entre os vários grupos experimentais, estas só foram significativas no grupo de moluscos infetados e expostos ao extrato de K.senegalensis e no grupo de moluscos infetados mas não expostos a nenhum extrato (neste caso no controlo para os moluscos expostos ao extrato de K. senegalensis). Por outro lado, os extratos parecem afetar a viabilidade das formas infetantes, quer para o hospedeiro definitivo (cercárias), quer para o hospedeiro intermediário (miracídios).
Schistosomiasis, caused by Schistosoma mansoni, is one of the most prevalent parasitic diseases in humans, its intermediate host is a freshwater mollusc from the species Biomphalaria glabrata. For the control of this disease, besides treatment of infected patients, it is also important that the mollusc populations are controlled. In order to find strategies for control of molluscs, this study aimed to investigate the activity of plant extracts as inhibitors of parasite development in mollusc. The main objective was to evaluate the biological activity of extracts from the leaves of two plants from Guinea-Bissau (Abrus precatorius and Khaya senegalensis) in Biomphalaria glabrata exposed to Schistosoma mansoni. The parameters evaluated were survival / mortality, oviposition, elimination of cercariae and its viability on the definitive host (mouse) as well as DNA damage in the molluscs through the technique of Comet Assay. Although the results are very preliminary, they seem to indicate that the extracts from leaves of Abrus precatorius and Khaya senegalensis don’t have molluscicidal activity, as the mortality rate was low in the groups of molluscs exposed to the extracts and mortality occurred later in the bioassay and not during the first 24 hours post-exposure. Through the technique of Comet assay we found that the DNA from molluscs exposed to extracts showed damage but that it was not significant. Regarding the group of molluscs infected with S.mansoni and exposed to the extracts, only those exposed to the extract of A. precatorius and DMSO showed significant DNA damage. Exposure to extracts induced an increase in the release of cercariae by the molluscs when compared to the group of molluscs infected only with S.mansoni, however these differences were not statistically significant. It was not detected an increase in the average number of eggs produced by B.glabrata exposed to the extracts. Although there were differences in the mean number of eggs produced between the various experimental groups, they were only significant between the group of molluscs infected and exposed to the extract from Khaya senegalensis and in the group infected but not exposed to any of the extracts (specifically in the control for the molluscs exposed to the extract of Khaya senegalensis). On the other hand the extracts appear to affect the viability of the infective forms in the definitive host (cercariae), and in the intermediate host (miracidia).

Dans le but de cerner in vitro l'impact de la photosynthese sur les voies metaboliques essentielles, un souchier de lignees cellulaires photomixotrophes & heterotrophes a ete etabli, sous forme de cals et de suspensions cellulaires, pour une plante medicinale tropicale: abrus precatorius l. (leguminosae). La consommation, en cours de culture, des macroelements majeurs du milieu (no#3, nh#4, pi, so#4) a ete mesuree. Cette etude a permis de mettre en evidence des differentes importantes entre les besoins nutritionnels pour chaque element. De plus, une caracterisation partielle de deux enzymes impliquees dans l'assimilation de l'azote mineral: nitrate reductase & glutamine synthetase, a ete realisee. Afin de mieux connaitre les phenomenes biochimiques lies a la photomixotrophie, l'activite de deux carboxylases (ribulose 1,5 bisphosphate carboxylase -rubisco- & phosphoenolpyruvate carboxylase -pepc-) a ete mesuree sur les suspensions cellulaires. Nous avons suivi l'activite de ces 4 systemes enzymatiques (nr, gs, rubisco & pepc) au cours de cultures conduites sous differentes conditions (hetero/mixotrophie) a l'aide de deux systemes de culture (erlenmeyers ou bioreacteur experimental). Cette etude est completee par une caracterisation immunologique de ces enzymes, permettant de degager quelques donnees concernant leur biosynthese et leur regulation

Pine rocklands are endangered ecosystems unique to south Florida, the Bahamas and Cuba. As a result of their karstic calcium carbonate­rich soil, these systems are limited in phosphorus and nitrogen, making symbiotic associations critical to plant growth. Four leguminous species (Cajanus cajan, Chamaecrista fasciculata, Tephrosia angustissima, and Abrus precatorious) were used to determine the relationship between rhizobial partners and plant performance, and the symbiosis related gene nifH was amplified to characterize the diversity of rhizobial symbionts. Plants were grown in soils from four different south Florida pine rocklands, and a salinity treatment was added to determine how storm surge and sea level rise could affect this symbiotic relationship. While plant performance and nodulation were highly impacted by soil type, salinity did not represent a significant effect. Phylogenetic analysis determined that all four plant species were found to associate with Bradyrhizobium spp. and no rhizobial shift between salinity treatment and soil type was found.

碩士
國立臺灣大學
生化學研究所
84
A protease inhibitor was isolated from the seeds of Abrus precatorius using ion exchang chromatography on DEAE-cellulose, gel filtration on Sephadex G-75 and affinity chromatography on immobilized trypsin.The purified inhibitor is a Bowman-Birk type and designated as APTI. APTI is a double-headed protease inhibitor because it inhi- bits both the trypsin and chymotrypsin activities.The complete amino acid sequence was deduced from the analysis of peptides derived from the reduced and S-carboxymethylated protein by cleaving lysyl endopeptidase and thermolysin.This inhibitor com- prises 77 amino acid residues including seven disulfide bridges. The putative reactive inhibitor sites of APTI are at Lys(27)-Ser (28) and Leu(52)-Ser(53) for trypsin and chymotrypsin,respectively .The amino acid sequence,the invariant amino acid residues and the reactive inhibitor sites of APTI resemble to those of the Bowman- Birk double-headed protease inhibitor.APTI consists of four anti- parallel structures and probably formed by two tandem repeat domains.

The present study indicates that Abrus precatorius was found to be the potential source for lectins. The lectin called Abrus agglutinin was isolated and characterized from white Abrus seed by the lactamyl sepharose 4B chromatography followed by SDS-PAGE. Lectin bind with the sepharose bead and it was separated out by the process of dialysis. The concentration of protein was measured. Agglutinins are sensitive to RBC of blood. Haemagglutination assay is another method for tittering protein based on their ability to attach to molecules present on the surface of red blood cells. The protein was identified by the Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis. The relative molecular weight of agglutinin was found 33kDa, 29kDa which corresponds to A chain and B chain of Agglutinin.

The study was done to isolate lectin from red seeds of Abrus precatorius. The protein was purified by using affinity chromatography. The activity of the lectin was determined by haemagglutinin assay and the purity of the protein was tested by Sodium dodecyl sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The activity of lectins is quantified by their ability to agglutinate erythrocyte. The eluted protein exhibited agglutinating activity when reacted against different types of fresh erythrocytes. In SDS-PAGE in affinity 2 bands were observed of 34kD and 31kD of heterotetrameric agglutinin.

博士
國立臺灣大學
生化學研究所
88
Abrus agglutinin (AAG), a low-toxicity protein from the plant Abrus precatorius, is less lethal than abrina (ABRa) in mice (LD50, 5 mg/kg vs. 20 μg/kg body weight). The nucleotide sequence analysis of a cDNA clone encoding the full-length AAG showed an open reading frame with 1,641 base pairs, corresponding to 547 amino acid residue preproprotein containing a signal peptide and a linker region (2 amino acid residues) between AAG-A and AAG-B subunits. AAG had high homology to ABRa (77.8 %). The 13 amino acid residues involved in catalytic function, which are highly conserved among ABRs and ricins, were also conserved within AAG-A. The protein synthesis inhibitiory activity of AAG-A (IC50, 3.5 nM) was weaker than that of ABRa-A (0.05 nM). Molecular modeling followed by site-directed mutagenesis showed that IC50 of ABRa-A (N200P) and AGG-A (P199N) was dretemined to be 2.3 nM and 0.53 nM, respectively. It suggested that the Pro199 residue of AAG-A, located in the amphiphilic helix H and corresponding to the Asn200 residue of ABRa-A, can induce bending of helix H. This bending would presumably affect the binding of AAG-A to its target sequence, GpApGpAp, in the tetraloop structure of the 28S rRNA subunit and could be one of the major factors contributing to the relatively weak protein synthesis inhibitory activity and toxicity of AAG.

Tese de mestrado em Química Farmacêutica e Terapêutica apresentada à Universidade de Lisboa através da Faculdade de Farmácia, 2007
O presente estudo foi efectuado sobre Abrus precatorius L., Fabaceae, originária da Ásia, que se encontra difundida em várias regiões tropicais e subtropicais do globo onde a planta tem vários usos etnobotânicos incluindo etnomédicos e teve como objectivo contribuir para o aprofundamento do seu estudo químico e biológico, tendo, neste âmbito, sido efectuado um estudo fitoquímico bioguiado por ensaios de actividade antimicrobiana sobre as raízes e um estudo fitoquímico sobre as folhas desta espécie, colhidas na Guiné-Bissau. Foi demonstrada a actividade antimicrobiana in vitro contra Enterococcus faecalis, Staphylococcus aureus, Candida albicans e Cladosporium cucumerinum de um extracto clorofórmico das raízes. O estudo bioguiado deste extracto permitiu localizar três dos constituintes total ou parcialmente responsáveis pela actividade biológica do extracto, tendo em sequência sido identificados dois deles, a abruquinona A e B. Estes compostos mostraram-se activos contra os quatro microrganismos supramencionados e também contra Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella dysenteriae e Vibrio cholerae. Staphylococcus aureus foi a bactéria contra a qual o extracto e as abruquinonas A e B mostraram maior actividade. Para além destes dois compostos foi também identificado o alcalóide abrina nesta parte da planta. Nas folhas de A. precatorius foram identificados dois dos constituintes maioritários, o abrusósido C e abrusósido D e também o alcalóide trigonelina. Foram também isolados e são propostas estruturas para dois constituintes ainda não descritos nesta espécie, pertencentes à classe dos flavonóides, nomeadamente ao grupo das flavonas metiladas, o qual é caracterizado pelas suas interessantes actividades biológicas.
The here presented study is a contribution to a deeper chemical and biological knowledge of Abrus precatorius L. - a Fabaceae originated from Asia and widespread throughout several tropical and sub-tropical regions. Different ethnobotanical uses have been reported for this species, ethnomedical uses included. Results of our phytochemical study on the roots of Abrus precatorius, collected in Guiné-Bissau, showed the in vitro antibacterial activity of its chloroform extract against Enterococcus faecalis and Staphylococcus aureus and its in vitro antifungical activity against Candida albicans and Cladosporium cucumerinum. A bioguided study of this extract, by means of antimicrobial activity assays, allowed the isolation of three components total or partially responsible for its biological activity, two of them later identified as abruquinone A and abruquinone B. These compounds were active not only against the above mentioned four microorganisms, but also against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella dysenteriae and Vibrio cholerae. The greatest activity revealed both by the extract and the abruquinones A e B was against Staphylococcus aureus. We have also identified the alkaloid abrine in this plant part. Results of our phytochemical study on the leaves of Abrus precatorius, also collected in Guiné-Bissau, allowed the identification of two major components - abrusoside C and abrusoside D as well as the alkaloid trigonelline. We have also isolated two other methylated flavones not yet described in this species, of which we hereby propose the structures.
Fundação Calouste Gulbenkian (FCG) e Centro de Estudos de Ciências Farmacêuticas da Faculdade de Farmácia da Universidade de Lisboa (financiamento do projecto de Investigação no âmbito do programa de Financiamento Plurianual das Unidades de ID da Fundação para a Ciência e a Tecnologia).

In modern science Nanotechnology is a ablaze field for the researchers.Nanoparticles having a size of 1-100 nm in one dimension, is used significantly concerning medical chemistry, atomic physics, and all other known fields. Nanoparticles are used immensely due to its small size, orientation, physical properties,which are reportedly shown to change the performance of any other material which is in contact with these tiny particles. These particles can be prepared easily by different chemical, physical, and biological approaches. But the biological approach is the most emerging approach of preparation, because, this method is easier than the other methods, ecofriendly and less time consuming. The semiconductor ZnO has gained substantial interest in the research community in part because of its large exciton binding energy 60 meV which could lead to lasing action based on exciton recombination even above room temperature. The Green synthesis was done by using the aqueous solution of Abrus precatorius seeds extract and zinc acetate. A fixed ratio of plant extract to metal ion was prepared and the color change was observed which proved the formation of nanoparticles. The nanoparticles were characterized by UV-vis Spectrophotometer, FTIR, DLS, Zeta Analysis, XRD, and SEM. The particles synthesized were of the size ranging from 90-500 nm.

碩士
台北醫學院
藥學研究所
74
BCG ，OK-432等免疫增強劑，能刺激活化巨噬細胞之抗癌作用，已受今日醫藥界之矚 目。1975年美國Sloan Kettering 研究所之Dr. L. J. Old 及E. A. Carswell等，首 先報告腫瘤壞死因子（Tumor Necrosis Factor; TNF）會引起腫瘤細胞壞死，進而使 腫瘤消失。 雞母珠血球凝集素（Abrus Agglutinin；AAG ）及雞母珠毒蛋白（Abrin ；Ab），於 1970年代即被證明具有抗癌作用，其抗癌機制，除直接細胞毒外，尚可加強免疫機能 ，可知雞母珠為一特殊抗癌物質，但毒性太大，至今尚未用於臨床。TNF 沒有種之特 異性，且對正常細胞沒有毒性，倘能經由雞母珠誘導TNF 應用於臨床，將有助於抗癌 新藥之開發及植物資源之利用。 本研究分三部份：1.經由體內生物檢定，探討雞母珠毒蛋白誘發腫瘤壞死因子之活性 ，及其最低腫瘤壞死條件。2.經由驗外細胞毒檢定方式，探討TNS 之誘導機制，及其 TNS 活性之力價，同時設定一經濟省時之實驗方法。3.TNS 對肝功能和末稍血液白血 球分類之影響。 由本研究得知AAG 可當第一刺激物質priming agent ，Ab可當第二刺激物（elicitin g agent ）誘導產生TNF ，其導致最佳腫瘤壞死條件為AAG 200ng/mouse ，Ab 20ng/ mouse ，Ab給予2 小時後採血，以0.4ml 之血清，靜脈注射至擔癌老鼠，可得最明顯 腫瘤壞死現象，且TNF 無種之特異性，對肝功能及末稍血球中沐巴球百分比無明顯差 異，而其體外百分之五十細胞毒之力價為26LU/ml 。 本研究以AAG 、Ab誘導TNF 類物質，但此物質是否與Old 所報導之TNF 為同一物質， 需更進一步純化及生化特性之確認。

碩士
中國醫藥學院
藥物化學研究所
81
Hsiang-ssu-tzu is first described in Pen-Tsao-Kan-Mu, and used as a folk-medicine in Chinese for the treatments of laryngitis, hepatitis and bronchitis. In this study, the isolation and purification of the active constituents of the roots of Hsiang-ssu-tzu have been performed mainly with the aid of the physiological tests of antiplatelet aggregation. From the chloroform fraction of the methanolic crude extract,two known compounds, named abruquinone A and B, and three new compounds, designed as abruquinone D,E and F,have been isolated and characterized. All of them are isoflavan- quinone in structure. The results of the evaluations of the activities of the five compounds indicate that abruquinone A, B and D showed remarkable inhibitory effect on the platelet aggregation. On the other hand , due to the applications in folk-medicine, the antiinflammatory activity of the five compounds has also been evaluated. the result shows that abruquinone A, B, D and F exhibited strong antiinflammatory effect.

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.

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.

博士
國立清華大學
物理學系
97
In the first part, nine crystal structures of imidazole compounds have been determined by X-ray crystallography. Their coordination geometry and crystal packing are described. This part of work helped the author to be accustomed to crystallography. In the second part, the structure of agglutinin from Abrus precatorius has been determined by X-ray crystallography. The structure and the activity as a ribosome-inactivating protein (RIP) have been compared with abrin-a from Abrus precatorius. With docking study, ASN200 of abrin-a was found to be hydrogen bonded with G4323 of 28SRNA, while corresponding PRO199 of agglutinin is a non-polar residue. This may explain the lower toxicity of agglutinin than abrin-a, despite of similarity in secondary structure and the activity cleft of two RIPs.

碩士
國立臺灣大學
生化學研究所
88
Ribosome-inactivating proteins (RIPs) inhibit protein synthesis and kill the cells by inactivating the ribosome. The mechanism by which RIPs kill cells involves their highly specific N-glycosidase, which is able to hydrolyze the glycosidic bond of the adenine 4324 in 28S rRNA of rat 60S ribosomal subunit and reduces the binding affinity of elongation factor 2 (EF-2) to ribosome and arrests the elongation step of translation. Several ribosomal proteins (RPs), L9, P0, P1, and P2, in the ribosomal target site of RIPs and three RIPs, ricin (RIC), abrin (ABR), and Abrus precatorius agglutinin (AAG) were expressed by an E. coli expression system. The relationship between RIPs and RPs was studied by pull-down assay. The results apparently indicated that RPs were able to interact with RIPs. RIC, ABR, and AAG were preferred to interacting with P1, P0, and L9, respectively. In conclusion, relative affinities of ABR interacting with L9, P0, P1, and P2 are 1.00, 4.46, 0.42, and 0.48, respectively; however, those of RIC interacting with L9, P0, P1, and P2 are 1.00, 4.44, 2.13, and 0.10, respectively. In addition, those of AAG interacting with L9, P0, P1, and P2 are 1.00, 0.03, 0.09, and 0.02.This difference results from the variety of structures of three RIPs and different binding orientation between RIPs and RPs. The binding regions on the ABR were mapped by deletion methods. Only L9 interacted with ABR-I-II which was the deletion of domain 3 of ABR. ABR-II-III, domain 1 of ABR deleted, still interacted with L9 strongly while P0, P1, and P2 interacted moderately. It suggests that ABR domain 1 contributed to the interaction between ABR and L9.

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).

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).

Abrin is a potent toxin obtained from the seeds of Abrus precatorius. It is a heterodimeric glycoprotein consisting of an A and a B subunit linked together by a disulfide bond. The toxicity of the protein comes from the A subunit harboring RNA-N-glycosidase activity which cleaves the glycosidic bond between the ribose sugar and the adenine at position 4324 in 28S rRNA. The depurination of a specific adenine residue at position 4324 results in loss of conformation of the 28S rRNA at the α sarcin/ricin loop to which elongation factor-2 (EF-2) binds, during the transloction step of translation, leading to inhibition of protein synthesis. The B subunit of abrin is a galactose specific lectin. The lectin activity enables the toxin to gain entry inside cells on binding to receptors with terminal galactose. After entering cells, a few molecules of abrin reach the endoplasmic reticulum (ER) via the retrograde transport, where the disulfide bond between the A and the B subunits gets cleaved. Then the A chain escapes into the cytosol where it binds to its target, the α-sarcin loop of the 28S ribosomal RNA and inhibits protein synthesis. Apart from inhibition of protein synthesis, exposure of cells to abrin leads to the loss of mitochondrial membrane potential (MMP) resulting in the activation of caspases and finally apoptosis. However, whether apoptosis is dependent on the inhibition of protein synthesis has not been elucidated. The major objectives of this study are therefore to delineate the signaling pathways involved in abrin-induced apoptosis. The thesis is divided into 4 Chapters: Chapter 1. provides a overview of the general properties of RIPs, with a brief history, classification, trafficking and biological activities of the toxins. This chapter also discusses their potential use in bio-warfare and the treatments available for management of toxicity. Chapter 2 and 3 discuss the results obtained on studies aimed at gaining insights into the signaling pathways involved in abrin-induced apoptosis. Chapter 4 focuses on the research carried out to understand the mechanisms of neutralization of abrin by the mAb D6F10. Towards the first objective, chapter 2 elucidates the role of endoplasmic reticulum (ER) stress signaling in abrin-induced apoptosis using the human T-cell line, Jurkat as a model system. It could be concluded that the inhibition of protein synthesis by the catalytic A subunit of abrin could result in accumulation of unfolded proteins in the ER leading to ER stress which triggers the unfolded protein response (UPR) pathway. The ER resident trans-membrane sensors IRE1 (Inositol-requiring enzyme 1), PERK (PKR-like ER kinase) and ATF6 (Activating transcription factor 6) are the important players of UPR in mammalian cells. These sensors inhibit translation and increase the levels of chaperones to restore protein homeostasis. However, if the ER stress is prolonged, apoptotic pathways get activated to remove severely damaged cells in which protein folding defects cannot be resolved. Recent studies have shown that endoplasmic reticulum (ER) stress induces apoptosis by activating initiater caspases such as caspase-2 and -8 which eventually trigger mitochondrial membrane potential loss and activation of downstream effector capases-9 and -3. Phosphorylation of eukaryotic initiation factor 2α and upregulation of CHOP [CAAT/enhancer binding protein (C/EBP) homologous protein], important players involved in ER stress signaling by abrin, suggested activation of ER stress in the cells. ER stress is also known to induce apoptosis via stress kinases such as p38 MAPK and JNK. Activation of both the pathways was observed upon abrin treatment and found to be upstream of the activation of caspases. However, abrin-induced apoptosis was found be dependent on p38 MAPK but not JNK. We also observed that abrin induced activation of caspase-2 and caspase-8 and triggered Bid cleavage leading to mitochondrial membrane potential loss and thus connecting the signaling events from ER stress to mitochondrial death machinery. Few toxins belonging to the family of ribosome inactivating proteins such as Shiga toxin have been observed to induce DNA damage in human endothelial cells and activate p53/ATM-dependent signaling pathway in mammalian cells. To further investigate the role of abrin on activation of DNA damage signaling pathway, we analysed the phosphorylation of H2AX and ATM, which are markers for double strand DNA breaks. We observed phosphorylation of H2AX and ATM upon abrin treatment but not when cells were pretreated with the broad spectrum pan caspase inhibitor. This study suggested that the DNA damage observed was an indirect effect of caspase-activated DNase. We concluded from the studies in chapter 2 that inhibition of protein synthesis by abrin can trigger endoplasmic reticulum stress leading to mitochondria-mediated apoptosis. Further studies were conducted to understand the dependence of ER stress on inhibition of protein synthesis and are presented in chapter 3. For this study, we have used an active site mutant of abrin A chain (R167L) which exhibits lower protein synthesis inhibitory activity than the wild type abrin A chain. Recombinant wild type and mutant abrin A chains were expressed in E.coli and purified. Since, abrin A chain requires the B chain for internalization into cells, both wild type and mutant abrin A chains were conjugated to native ricin B chain to generate a hybrid toxin. Next, we have compared the toxic effects of the two conjugates in cells. The rate of inhibition of protein synthesis mediated by the mutant ricin B-rABRA (R167L) conjugate was slower than that of the wild type ricin B-rABRA conjugate but it could trigger ER stress leading to mitochondrial mediated apoptosis in cells though delayed, suggesting that inhibition of protein synthesis is the major factor contributing to abrin-mediated apoptosis. Abrin is extremely lethal and considered as a potential agent for use in biological warfare. Currently, there are no antidotes or effective therapies available for abrin poisoning. Antibody based antitoxins function by either preventing toxin binding to cell surface receptors or by translocation. Antibodies against the B chain of RIPs function by inhibiting the binding of B chain of the toxin to cells, whereas the exact mechanism by which antibodies against A chain function is still not clear. The only known neutralizing monoclonal antibody against abrin A chain, namely, D6F10, was generated in our laboratory and was shown to rescue cells and mice from abrin intoxication. Earlier experiments with confocal microscopy suggested that mAb D6F10 could internalize in HeLa cells along with abrin, suggesting that the antibody can function intracellularly. Chapter 4 discusses the work carried out to delineate the mechanism of intracellular neutralization of abrin by the mAb D6F10. We observed significant reduction in binding and delay in abrin internalization in the presence of the neutralizing monoclonal antibody (mAb) D6F10. Considering that the majority of the abrin after internalization is removed by lysosomal degradation, we studied the fate of abrin in the presence of mAb D6F10. Confocal images did not show any difference in the distribution of abrin in the lysosomes in the absence or presence of antibody. However, the antibody remained persistently colocalized with abrin in the cells, suggesting that the antibody might inhibit enzymatic activity of abrin at its cellular site of action.

Abrin is a potent toxin obtained from the seeds of Abrus precatorius. It is a heterodimeric glycoprotein consisting of an A and a B subunit linked together by a disulfide bond. The toxicity of the protein comes from the A subunit harboring RNA-N-glycosidase activity which cleaves the glycosidic bond between the ribose sugar and the adenine at position 4324 in 28S rRNA. The depurination of a specific adenine residue at position 4324 results in loss of conformation of the 28S rRNA at the α sarcin/ricin loop to which elongation factor-2 (EF-2) binds, during the transloction step of translation, leading to inhibition of protein synthesis. The B subunit of abrin is a galactose specific lectin. The lectin activity enables the toxin to gain entry inside cells on binding to receptors with terminal galactose. After entering cells, a few molecules of abrin reach the endoplasmic reticulum (ER) via the retrograde transport, where the disulfide bond between the A and the B subunits gets cleaved. Then the A chain escapes into the cytosol where it binds to its target, the α-sarcin loop of the 28S ribosomal RNA and inhibits protein synthesis. Apart from inhibition of protein synthesis, exposure of cells to abrin leads to the loss of mitochondrial membrane potential (MMP) resulting in the activation of caspases and finally apoptosis. However, whether apoptosis is dependent on the inhibition of protein synthesis has not been elucidated. The major objectives of this study are therefore to delineate the signaling pathways involved in abrin-induced apoptosis. The thesis is divided into 4 Chapters: Chapter 1. provides a overview of the general properties of RIPs, with a brief history, classification, trafficking and biological activities of the toxins. This chapter also discusses their potential use in bio-warfare and the treatments available for management of toxicity. Chapter 2 and 3 discuss the results obtained on studies aimed at gaining insights into the signaling pathways involved in abrin-induced apoptosis. Chapter 4 focuses on the research carried out to understand the mechanisms of neutralization of abrin by the mAb D6F10. Towards the first objective, chapter 2 elucidates the role of endoplasmic reticulum (ER) stress signaling in abrin-induced apoptosis using the human T-cell line, Jurkat as a model system. It could be concluded that the inhibition of protein synthesis by the catalytic A subunit of abrin could result in accumulation of unfolded proteins in the ER leading to ER stress which triggers the unfolded protein response (UPR) pathway. The ER resident trans-membrane sensors IRE1 (Inositol-requiring enzyme 1), PERK (PKR-like ER kinase) and ATF6 (Activating transcription factor 6) are the important players of UPR in mammalian cells. These sensors inhibit translation and increase the levels of chaperones to restore protein homeostasis. However, if the ER stress is prolonged, apoptotic pathways get activated to remove severely damaged cells in which protein folding defects cannot be resolved. Recent studies have shown that endoplasmic reticulum (ER) stress induces apoptosis by activating initiater caspases such as caspase-2 and -8 which eventually trigger mitochondrial membrane potential loss and activation of downstream effector capases-9 and -3. Phosphorylation of eukaryotic initiation factor 2α and upregulation of CHOP [CAAT/enhancer binding protein (C/EBP) homologous protein], important players involved in ER stress signaling by abrin, suggested activation of ER stress in the cells. ER stress is also known to induce apoptosis via stress kinases such as p38 MAPK and JNK. Activation of both the pathways was observed upon abrin treatment and found to be upstream of the activation of caspases. However, abrin-induced apoptosis was found be dependent on p38 MAPK but not JNK. We also observed that abrin induced activation of caspase-2 and caspase-8 and triggered Bid cleavage leading to mitochondrial membrane potential loss and thus connecting the signaling events from ER stress to mitochondrial death machinery. Few toxins belonging to the family of ribosome inactivating proteins such as Shiga toxin have been observed to induce DNA damage in human endothelial cells and activate p53/ATM-dependent signaling pathway in mammalian cells. To further investigate the role of abrin on activation of DNA damage signaling pathway, we analysed the phosphorylation of H2AX and ATM, which are markers for double strand DNA breaks. We observed phosphorylation of H2AX and ATM upon abrin treatment but not when cells were pretreated with the broad spectrum pan caspase inhibitor. This study suggested that the DNA damage observed was an indirect effect of caspase-activated DNase. We concluded from the studies in chapter 2 that inhibition of protein synthesis by abrin can trigger endoplasmic reticulum stress leading to mitochondria-mediated apoptosis. Further studies were conducted to understand the dependence of ER stress on inhibition of protein synthesis and are presented in chapter 3. For this study, we have used an active site mutant of abrin A chain (R167L) which exhibits lower protein synthesis inhibitory activity than the wild type abrin A chain. Recombinant wild type and mutant abrin A chains were expressed in E.coli and purified. Since, abrin A chain requires the B chain for internalization into cells, both wild type and mutant abrin A chains were conjugated to native ricin B chain to generate a hybrid toxin. Next, we have compared the toxic effects of the two conjugates in cells. The rate of inhibition of protein synthesis mediated by the mutant ricin B-rABRA (R167L) conjugate was slower than that of the wild type ricin B-rABRA conjugate but it could trigger ER stress leading to mitochondrial mediated apoptosis in cells though delayed, suggesting that inhibition of protein synthesis is the major factor contributing to abrin-mediated apoptosis. Abrin is extremely lethal and considered as a potential agent for use in biological warfare. Currently, there are no antidotes or effective therapies available for abrin poisoning. Antibody based antitoxins function by either preventing toxin binding to cell surface receptors or by translocation. Antibodies against the B chain of RIPs function by inhibiting the binding of B chain of the toxin to cells, whereas the exact mechanism by which antibodies against A chain function is still not clear. The only known neutralizing monoclonal antibody against abrin A chain, namely, D6F10, was generated in our laboratory and was shown to rescue cells and mice from abrin intoxication. Earlier experiments with confocal microscopy suggested that mAb D6F10 could internalize in HeLa cells along with abrin, suggesting that the antibody can function intracellularly. Chapter 4 discusses the work carried out to delineate the mechanism of intracellular neutralization of abrin by the mAb D6F10. We observed significant reduction in binding and delay in abrin internalization in the presence of the neutralizing monoclonal antibody (mAb) D6F10. Considering that the majority of the abrin after internalization is removed by lysosomal degradation, we studied the fate of abrin in the presence of mAb D6F10. Confocal images did not show any difference in the distribution of abrin in the lysosomes in the absence or presence of antibody. However, the antibody remained persistently colocalized with abrin in the cells, suggesting that the antibody might inhibit enzymatic activity of abrin at its cellular site of action.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2015
De acordo com fontes oficiais de vários países, as plantas tóxicas demonstraram ser responsáveis por numerosos casos de intoxicação em todo o Mundo. A beleza destas plantas oculta a sua toxicidade e o seu desconhecimento potencia situações de intoxicação especialmente em crianças. Em Portugal, o conhecimento destas plantas é ainda escasso, e o facto de não existirem muitos estudos do género no país, faz com que seja por importante fazer a caracterização de algumas espécies de plantas com interesse toxicológico, nomeadamente espécies autóctones em Portugal e que com alguma frequência provocam casos de intoxicação no ser Humano. Serão também referidas algumas espécies não autóctones em Portugal, mas que pela sua toxicidade e letalidade devem ser também ser conhecidas. Os animais, tanto domésticos como de exploração são também afetados por esta realidade, a fome e a curiosidade estão muitas vezes na origem das intoxicações. As espécies em destaque ao longo desta monografia de revisão são: Atropa beladona, Conium maculatum, Datura stramonium, Mandragora autumnalis, Nicotiana glauca, Taxus baccata, Ricinus communis, Nerium oleander, Digitalis purpurea, Hyoscyamun niger, Abrus precatoris, Thevetia peruviana e Strychnos nux- vomica. Estas espécies apesar de tóxicas estão atualmente a ser investigadas para o tratamento de certas doenças, nomeadamente o cancro. A oleandrina, por exemplo, tem demonstrado ser uma mais-valia no tratamento de vários tipos de cancro.