Wong Sui Ming. / Thesis (M.Ph.)--Chinese University of Hongkong, 1979. / Bibliography: leaves 131-138. / Chapter CHAPTER I --- Introduction: / Chapter I --- Aim of study --- p.1 / Chapter II --- Plan of study --- p.1 / Chapter CHAPTER II --- Phytochemical Screening / Chapter I --- Literature review --- p.5 / Chapter II --- Experimental --- p.14 / Chapter III --- Results / Chapter (A) --- Screening for saponin and sterol --- p.15 / Chapter (B) --- Screening for tannin and polyphenol / Chapter (C) --- Screening for anthraquinone --- p.16 / Chapter (D) --- Screening for flavonoid and leucoanthocyanin / Chapter (E) --- Screening for cardenolide and bufadienolide / Chapter (F) --- Screening for cyanogenic glycoside / Chapter (G) --- Screening for alkaloid --- p.18 / Chapter IV --- Conclusion --- p.19 / Chapter CHAPTER III --- Introduction to Anthraquinone & Triterpenoid / Chapter I --- Biogenesis --- p.20 / Chapter II --- Anthraquinone / Chapter (A) --- Introduction --- p.23 / Chapter (B) --- Biogenesis --- p.25 / Chapter (C) --- Properties --- p.28 / Chapter (D) --- Spectrometry --- p.33 / Chapter (E) --- Chromatography --- p.39 / Chapter III --- Triterpenoid --- p.42 / Chapter (A) --- Pentacyclic triterpenoid --- p.44 / Chapter (B) --- General properties --- p.47 / Chapter (C) --- IR Spectra --- p.48 / Chapter (D) --- PMR Spectra --- p.50 / Chapter (E) --- Mass Spectra --- p.56 / Chapter CHAPTER IV --- Structural Elucidation / Chapter I --- Separation Scheme --- p.63 / Chapter II --- Results and Discussions --- p.66 / Chapter (A) --- "Hydroxyanthrac,_uinone" / Chapter (i) --- Chrysophanol (Hl) --- p.69 / Chapter (ii) --- Physicon (H2) --- p.82 / Chapter (B) --- Triterpenoid / Chapter (i) --- Sophoradiol (T1) --- p.90 / Chapter (ii) --- Soyasapogenol-(B) (T0) --- p.106 / Chapter (C) --- Analysis of sugars --- p.119 / Chapter III --- Experimental --- p.120 / REFERENCES --- p.131

Pulchellina é uma proteína inativadora de ribossomo (RIP) isolada de sementes de Abrus pulchellus fragmento que codifica a cadeia A da pulchellina (PAC) foi clonado e inserido no vetor pGEX-5X para expressar a cadeia A recombinante (rPAC) como uma proteína de fusão em Escherichia coli. A análise da seqüência de aminoácidos mostrou que a rPAC apresenta uma alta identidade seqüencial (&#62 86%) com a cadeia A da abrina-c. A habilidade que a rPAC possui para depurinar rRNA em ribossomos de levedura também foi demonstrada em testes in vitro. Objetivando verificar a atividade tóxica do produto heterólogo, nós promovemos a associação in vitro da rPAC com a cadeia B recombinante da pulchellina (rPBC). Ambas as cadeias foram incubadas na presença de um sistema de redução/oxidação, originando um heterodímero ativo (rPAB). O rPAB apresentou uma massa molecular aparente de aproximadamente 60 kDa, similar a pulchellina nativa. As atividades tóxicas do rPAB e da pulchellina nativa foram comparadas através da injeção intraperitonial em camundongos, usando diferentes diluições de cada proteína. O rPAB foi capaz de matar 50% dos animais testados com doses de 45&#956g.kg-1. Nossos resultados mostraram que o heterodímero recombinante apresenta tanto toxicidade quanto um padrão conformacional similar a pulchellina nativa. Estudos usando cultura de tecidos também foram realizados com o objetivo de investigar a presença da pulchellina em calos obtidos a partir de sementes de A. pulchellus. Segmentos de cotilédones de sementes imaturas foram inoculados em meio MS suplementado com diferentes concentrações de auxina, citocinina e sacarose para promover a indução dos calos. A expressão da pulchellina nos calos foi monitorada através de RT-PCR e testes de atividade biológica. Os calos obtidos após 35 dias foram congelados, macerados e submetidos a extração de RNA total e proteínas. Um fragmento específico de DNA que codifica a cadeia A da pulchellina foi amplificado a partir do RNA total sugerindo a síntese da proteína nos calos. Isto foi confirmado no extrato bruto de calos, que mostrou atividade hemaglutinante contra sangue de coelho e uma alta toxicidade quando injetado via intraperitoneal em camundongos.O extrato bruto também foi submetido à cromatografia de afinidade em coluna de Sepharose-4B. A fração retida na coluna apresentou duas bandas protéicas quando analisadas em gel de poliacrinamida, sob condições desnaturantes, apresentando um padrão similar ao obtido com a pulchellina de semente. / Pulchellin is a type 2 ribosome-inactivating protein (RIP) isolated from seeds of the Abrus pulchellus tenuiflorus plant. The DNA fiagment encoding Pulchellin A-chain (PAC) was cloned and inserted in pGEX-5X to express the recombinant pulchellin Achain (rPAC) as a fusion protein in Escherichin coli. The deduced amino acid sequence analyses of the rPAC presented a high sequential identity (&#62 86%) with the A-chain of abrin-c. The ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. Intending to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reducedloxidized system, yielding an active heterodimer (rPAB). The rPAB showed an apparent molecular mass of about 60 D a similar to the native pulchellin. The toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection in mice using different dilutions. The rPAB was able to kill 50% of the tested mice with doses of 45&#956g.kg-1. Our results indicated that the recombinant heterodimer presented toxic activity and a conformational pattern similar to pulchellin. Studies using tissue cultures were also performed to investigate the presence of the pulchellin in callus established from seed explants of A. pulchellus. Cotyledon segments of immature seeds were inoculated in basal medium MS supplemented with different concentrations of auxin, citokinin and sucrose in order to determine the best callus induction. The pulchellin expression was monitored in callus cultures by RT-PCR and biological activity. The calli obtained aRer 35 days were freeze dried, macerated and submitted to extraction of total RNA and proteins. A specific DNA fragment codifying the A-chain pulchellin was amplified from callus RNA suggesting the synthesis of the protein. This was confirmed in the calli crude extract that showed haemagglutinating activity against rabbit blood cells and a high intraperitoneal toxicity to mice. The crude extract was also submitted to affinity chromatography on a Sepharose-4B column. The retained protein, showed to be composed by two main bands in polyacrylamide gel electrophoresis, in denaturating conditions, with a similar pattern to the results obtained with seeds pulchellin.

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.

Pulchellina é uma proteína inativadora de ribossomo (RIP) isolada de sementes de Abrus pulchellus fragmento que codifica a cadeia A da pulchellina (PAC) foi clonado e inserido no vetor pGEX-5X para expressar a cadeia A recombinante (rPAC) como uma proteína de fusão em Escherichia coli. A análise da seqüência de aminoácidos mostrou que a rPAC apresenta uma alta identidade seqüencial (&#62 86%) com a cadeia A da abrina-c. A habilidade que a rPAC possui para depurinar rRNA em ribossomos de levedura também foi demonstrada em testes in vitro. Objetivando verificar a atividade tóxica do produto heterólogo, nós promovemos a associação in vitro da rPAC com a cadeia B recombinante da pulchellina (rPBC). Ambas as cadeias foram incubadas na presença de um sistema de redução/oxidação, originando um heterodímero ativo (rPAB). O rPAB apresentou uma massa molecular aparente de aproximadamente 60 kDa, similar a pulchellina nativa. As atividades tóxicas do rPAB e da pulchellina nativa foram comparadas através da injeção intraperitonial em camundongos, usando diferentes diluições de cada proteína. O rPAB foi capaz de matar 50% dos animais testados com doses de 45&#956g.kg-1. Nossos resultados mostraram que o heterodímero recombinante apresenta tanto toxicidade quanto um padrão conformacional similar a pulchellina nativa. Estudos usando cultura de tecidos também foram realizados com o objetivo de investigar a presença da pulchellina em calos obtidos a partir de sementes de A. pulchellus. Segmentos de cotilédones de sementes imaturas foram inoculados em meio MS suplementado com diferentes concentrações de auxina, citocinina e sacarose para promover a indução dos calos. A expressão da pulchellina nos calos foi monitorada através de RT-PCR e testes de atividade biológica. Os calos obtidos após 35 dias foram congelados, macerados e submetidos a extração de RNA total e proteínas. Um fragmento específico de DNA que codifica a cadeia A da pulchellina foi amplificado a partir do RNA total sugerindo a síntese da proteína nos calos. Isto foi confirmado no extrato bruto de calos, que mostrou atividade hemaglutinante contra sangue de coelho e uma alta toxicidade quando injetado via intraperitoneal em camundongos.O extrato bruto também foi submetido à cromatografia de afinidade em coluna de Sepharose-4B. A fração retida na coluna apresentou duas bandas protéicas quando analisadas em gel de poliacrinamida, sob condições desnaturantes, apresentando um padrão similar ao obtido com a pulchellina de semente. / Pulchellin is a type 2 ribosome-inactivating protein (RIP) isolated from seeds of the Abrus pulchellus tenuiflorus plant. The DNA fiagment encoding Pulchellin A-chain (PAC) was cloned and inserted in pGEX-5X to express the recombinant pulchellin Achain (rPAC) as a fusion protein in Escherichin coli. The deduced amino acid sequence analyses of the rPAC presented a high sequential identity (&#62 86%) with the A-chain of abrin-c. The ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. Intending to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reducedloxidized system, yielding an active heterodimer (rPAB). The rPAB showed an apparent molecular mass of about 60 D a similar to the native pulchellin. The toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection in mice using different dilutions. The rPAB was able to kill 50% of the tested mice with doses of 45&#956g.kg-1. Our results indicated that the recombinant heterodimer presented toxic activity and a conformational pattern similar to pulchellin. Studies using tissue cultures were also performed to investigate the presence of the pulchellin in callus established from seed explants of A. pulchellus. Cotyledon segments of immature seeds were inoculated in basal medium MS supplemented with different concentrations of auxin, citokinin and sucrose in order to determine the best callus induction. The pulchellin expression was monitored in callus cultures by RT-PCR and biological activity. The calli obtained aRer 35 days were freeze dried, macerated and submitted to extraction of total RNA and proteins. A specific DNA fragment codifying the A-chain pulchellin was amplified from callus RNA suggesting the synthesis of the protein. This was confirmed in the calli crude extract that showed haemagglutinating activity against rabbit blood cells and a high intraperitoneal toxicity to mice. The crude extract was also submitted to affinity chromatography on a Sepharose-4B column. The retained protein, showed to be composed by two main bands in polyacrylamide gel electrophoresis, in denaturating conditions, with a similar pattern to the results obtained with seeds pulchellin.

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.

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.