In this study, Naegleria fowleri, a pathogenic amoeba and the causative agent of Primary Amebic Meningoencephalitis (PAM), was utilized to determine the presence or absence of classically conserved Golgi molecules featured in the expression of a Golgi apparatus. Previous studies concluded no Golgi expression via light microscopy and transmission electron microscopy, but a recent report on Naegleria gruberi indicated the presence of dispersed Golgi tubules. Non-pathogenic species of the Naegleria genus such as Naegleria gruberi 30540 and Naegleria lovaniensis 30569 were utilized in Western immunoblot analysis compared to reduced whole-cell lysate proteins of two strains of N. fowleri and Vero CCL-81, Chlorocebus sp. kidney epithelial cells, which were utilized as a positive control for Golgi expression. N. fowleri and N. lovaniensis whole-cell lysates had indications of a 110 kDa reduced protein, associated with the predicted molecular weights of the beta-COPI subunit of the COPI cis-Golgi vesicular transport complex with further Western immunoblot indication of a weak band around 25 kDa corresponding to rabbit polyclonal antibodies specific for ARF1. Serial Dilutions of Wheat Germ Agglutinin Alexa Fluor 488TM were performed on Vero cells, Naegleria fowleri 30894, and N. gruberi 30540 with 1:100 dilution of recommended stock dilution of WGA 488 determined for utilization in sequential immunofluorescence. Sequential immunofluorescence with Wheat Germ Agglutinin Alexa Fluor 488TM and then blocked with 3% BSA:PBS [wt/vol] dilution with subsequent incubation in rabbit anti-beta-COPI primary 1:250, and 1:1000 of Alexa Fluor 594 goat anti-rabbit secondary antibody exposure showed strong indications of organized cis- and trans-punctate Golgi body markers in close association in individual and dividing cells of Naegleria fowleri and conserved Golgi expression in the positive control Vero cells, but further experiments are necessary to verify this finding with N. fowleri.

Endotoxin or Lipopolysaccharide (LPS) is the invariant structural component of gram negative bacterial outer membranes and is the chief causative factor of Sepsis or endotoxic shock. Sepsis is a syndrome that has very high mortality rates even in this age of excellent therapeutics and critical patient care. The treatment for sepsis till date remains nonspecific and supportive due to lack of effective anti-endotoxic drugs. Sepsis is initiated when the circulating bacteria shed LPS from their cell envelopes. Shed LPS aggregates are recognized by LPS binding proteins and receptors, which activate the host's immune system. Uncontrolled and excessive stimulation of the host's immune system precipitates endotoxic shock which in advanced cases involving multiple system organ failure inevitably lead to patient's death. Many strategies have been tested out to combat this deadly affliction. One of the attractive clinical modalities in sepsis treatment is the use of peptides as LPS sequestering anti-endotoxic drugs. A classical peptide antibiotic of this class is Polymyxin B (PMB) a cyclic cationic acylated molecule, that recognizes LPS with a very high affinity. This thesis describes kinetics and thermodynamics of PMB-LPS interactions and applies these parameters over a framework of different models so as to gain insights into the structure-function relationships that govern the interactions of this peptide with endotoxin(s). Classical biophysical techniques like fluorescence, circular dichroism spectroscopy, stopped flow kinetics, titration calorirnetry (ITC) and the relatively new technique of Surface Plasmon Resonance (SPR) have been employed to dissect out the mechanism of the range of non-covalent forces that are involved in peptide-endotoxin recognition. Certain proteins that exhibit LPS binding activity have also been studied to gains insight about their mode of action. Implications of these studies for designing peptides that have better anti-endotoxic properties are also highlighted. The first chapter introduces and highlights the clinical features of sepsis. It also attempts to shed light on the LPS mediated signal transduction pathway that leads to endotoxic shock. This chapter also briefly explains the roles of many LPS receptors that are present in the human system and their specific roles in the signal transduction pathways. The second part of this chapter deals with the role of cationic peptides as anti-endotoxic drugs. Certain key functional aspects of these peptides, which impart in them, the desirable property of LPS recognition have also been discussed The second chapter describes the kinetic studies undertaken to unravel the exact mechanism of LPS-PMB interaction. The studies reveal that PMB recognizes LPS in a biphasic manner, with the second, unimolecular isomerization step of the reaction being the rate-limiting step. The initial reaction is shown to be influenced by the presence of salt in the reaction medium. The dissociation phase of this interaction also shows a biphasic pattern. These data allow us to speculate upon the exact mechanism by which PMB is able to recognize LPS. The studies also shed light on some structural aspects that govern and confer such high LPS binding activity to PMB. Based on these a model has been proposed to explain this recognition (C.J. Thomas et al, 1998). The second chapter discuses the mode of action of various PMB analogs. These analogs have been chosen in terms of their mode of action as well as their structural similarly to PMB. The affinities of these analogs to LPS and lipid A were quantified using the Surface plasmon resonance (SPR) method. SPR, a technique that relies on the quantification of change in mass during a binary binding process occurring between an immobilized entity and a flowing ligand, is a rapid and sensitive method to measure biologically relevant interactions. SPR studies provide us with the binding constants and thermodynamic parameters that allow evaluation of the affinities of these peptides towards LPS (C.J.Thomas and A.Surolia, 1999). The third chapter discusses a hitherto unknown mode by which PMB acts on a LPS lamellae. The results of this study wherein the binding affinities of PMB and its analogs were performed on monolayers and tethered liposomes, show that PMB is able to remove specifically LPS or lipid A from monolayers or bilayer assemblies such as tethered liposomes. The exact mode of action of PMB is deciphered in the light of these new studies, which allow us to posit on the observed efficacy of PMB in neutralizing the endotoxin as compared to peptides with nearly similar affinities for LPS (C.J Thomas et al 1999). In the fourth chapter a series of 23 residue peptides, based on the sequence corresponding to the anti-sense strand of magainin gene have been synthesized. Magainin an amphiphilic helical peptide obtained from frog skins plays a vital role in the innate immune defense mechanisms of these organisms. It also exhibits LPS binding activity that makes it an attractive target as an anti-endotoxic drug. Biochemical and biophysical characterization of these peptides reveal that they have the tendency to perturb both the inner and the outer membranes of E.coli. The peptides are amphiphilic and have helical structure in a membrane bound environment. Three of the peptides tested have high affinities for lipid A that approach the values shown by PMB. The kinetic parameters obtained by stopped flow and SPR studies in conjunction with the therrnodynamic parameters obtained using ITC studies allow us to highlight the key structural features that need to be exhibited by peptides that are designed to be LPS recognizers. The studies also project the fact that ionic forces play an important role in the initial recognition of LPS by these peptides. Fortification of the might of these ionic charges increases affinity for LPS where as the hydrophobic residues that interact at the next phase of binding are more amenable to disruptions in contiguity. These factors are discussed using the helical wheel diagram that shows the clear amphiphilicity displayed by these peptides. (C.J Thomas et al Manuscript under preparation, 2000) Chapter six discusses the mode of action of certain LPS binding proteins. Limulus anti endotoxic factor (LALF) plays a vital role in the innate immune based defense systems of the horseshoe crab. Galectin-3 is a metal ion independent, galactosc binding Icctin of human origin with unknown functions. Both these phylogcntically-unrclatcd proteins exhibit LPS/lipid A recognizing properties. ITC and SPR studies have been used to determine the binding constants displayed by these proteins for lipid A. LALF bind to lipid A with very high affinity than compared to Galectin-3 and is also able to take away selectively lipid A from both monolayers and tethered liposomes. Galectin-3 does not show this property of LALF, which might account for its lowered affinities. Also structurally LALF has amphiphilic nature that confers high lipid A binding activity, which is clearly lacking in Galectin-3. These studies in conjunction with the knowledge gained from the study of LPS-PMB interaction stress on the importance of amphiphilicity in LPS recognition. (C.J Thomas et al Manuscript under preparation, 2000). The final chapter is a general discussion that attempts to collate all these kinetic and thermodynamic observations in the pursuit of designing small easily manipulatable peptides that exhibit high LPS binding activity. These studies are aimed to act as rough guidelines to the design of LPS sequestering peptides that might have better therapeutic and pharmacokinetic properties. The appendix to the main body of work presented in thesis are two pieces of work pertaining to the elucidation the kinetics and mechanism of sugar lectin interactions, when sugars are presented as glycolipids in monolayers or bilaycrs liposomes. Mode of the presentation of sugars at cell-surfaces in the form of glycolipids as ligands influence their recognition by macromolecular receptors like lectins. Appendix 1 is a study of the mode of action of Ulex europeus I lectin binding to H-fucolipid containing tethered liposomes, by SPR. Fucosylated sugars are often used as key markers in histochemical analysis of malignant cancerous tissues. Ulex lectin plays a vital role as a marker for identification of these tissues. The kinetics and thermodynamic parameters that are obtained in this study throw some light on the mode of recognition of glycolipid receptor by Ulex europeus I lectin (C.J Thomas and A. Surolia 2000). Appendix 2 is a study, that attempts to quantify the initial kinetic parameters that correlate the recognition of glycolipid receptors with their inclination at the membrane surface and the influence of charge on them by soyabean agglutinin (SBA), Abrus agglutinin I and II. Studies on the soyabean agglutinin-globoside interaction highlights the divalent cation mediated reorientation of these receptors on their accessibility and recognition to the agglutinin. The divalent cations are speculated to orient the oligosaccharide head groups in a spatial geometry that allows a heightened kinetics of their interaction by SBA. These studies reveal that the reorganization of the binding pocket of a lectin can also have a profound influence on ihc rates of recognition of a glycospingolipid ligand by a lectin as exemplified by Abrus agglutinin II- GM1 interactions (C.J Thomas ct al, Manuscript under preparation).

Epidemiological, experimental, and clinical observations provide support for a colorectal cancer chemopreventive role for aspirin. We have evaluated the effects of aspirin on proliferation biomarkers in normal-risk and high-risk human subjects for colorectal cancer. Colorectal biopsies were obtained at baseline and at 24h after 28 daily doses of 81mg of aspirin from 13 high-risk and 15 normal-risk subjects for colorectal cancer. We evaluated aspirin's effects on proliferating cell nuclear antigen (PCNA) immunohistochemistry and epithelial mucin histochemistry using the lectin, Amaranthus caudatus agglutinin (ACA) in crypt sections from rectal biopsies. The baseline whole crypt PCNA LIs differed significantly between normal-risk and high-risk subjects. PCNA LIs are not affected by 28 days of aspirin at 81mg daily. ACA LIs are decreased by 28 days of aspirin at 81mg daily in both normal-risk and high-risk subjects. Aspirin's effects on ACA LIs may have mechanistic and biological implications that deserve further attention. PCNA and ACA LIs are not useful as proliferation biomarkers for aspirin's chemopreventive activity in morphologically normal human colorectal mucosa.

<p>Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction. </p><p>Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state <i>in vitro</i> and <i>in vivo</i>. </p><p>Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids.</p><p>An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented. </p>

Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction. Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state in vitro and in vivo. Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids. An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented.