Two cationic antimicrobial peptides, ESF I-GR7, and pleurocidin, were assessed for their stability in plant intercellular fluid, the targeted locale for their expression in transgenic plants. Incubation of ESFI-GR7 and pleurocidin with intercellular fluid (ICF) extracted from sugarcane, tomato and tobacco leaves reduced their biotoxicity towards various pathogens, namely Camobacterium mobile DMSO and Xanthomollas campestris. It was concluded that it may be necessary to modify the aminoacid structures of the peptides in order to ensure that endogenous proteases would not degrade the peptides once expressed in a transgenic environment. The presence of pleurocidin was detected in transgenic sugarcane transformed (in a previous study) with pleurocidin gene cloned into the pUBI 510 plasmid. ICF was extracted from four month old transgenic Saccharum hybrid species (sugarcane). Western blotting verified the presence of the transgenic protein in crude protein extracts. Immunogold labelling and transmission electron microscopy were performed to investigate the localisation of transgenic pleurocidin. The peptide was localized predominantly in the intercellular spaces and cell wall sugarcane leaves. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.

No description available.

Vaccination with immunogenic peptides offers a safe and specific way of inducing protection against pathogens, however as of yet there are no peptide-based vaccines available. The limitations on the therapeutic use of peptides are due to their poor immunogenicity and short life span in vivo. Peptide delivery systems act to circumvent these issues. The aims of this research were to investigate the ability of virus-like particles (VLP) from Rabbit haemmorhagic disease virus (RHDV) to deliver immunogenic peptides, to characterize the immune response to these particles, and to investigate whether baculovirus could also act as a delivery system. The vaccine peptides HAT (representing a T helper cell epitope) and HAB (representing the major B cell epitope) derived from the haemagglutinin antigen of influenza virus A/PR/8/34 were used as a model to investigate the ability of these virus particles to act as delivery vehicles to the immune system. A scheme for the production and purification of RHDV VLP was established. Expression of the capsid protein from RHDV in a serum-free recombinant baculovirus system using suspension cultures of up to 200 ml, and separation by isopycnic centrifugation on cesium chloride gradients led to high yields of purified RHDV VLP. Up to 20 mg of pure VLP could be obtained from an 800 ml culture of insect cells infected with recombinant baculovirus. In vitro testing revealed that RHDV VLP carrying the peptide HAT as a genetic fusion were processed by dendritic cells (DC), and that this peptide could be presented to induce activation of T cells. However, the purified RHDV VLP alone were not able to induce significant upregulation of cell activation markers CD40, CD86, and CD80. A preliminary in vivo study revealed that when RHDV VLP carrying the HAT peptide were delivered by an intraperitoneal injection in the absence of adjuvant, the immune response to the peptide was weak, therefore the route of delivery and the use of immune adjuvants with the VLP were optimised. Five different routes of delivery and two different immune adjuvants were compared. VLP were delivered through subcutaneous, intraperitoneal, transcutaneous, intramuscular and intranasal routes. Delivery of the VLP through each of these routes resulted in potent serum antibody responses. However, the strongest antibody responses were elicited when the VLP were delivered through the intraperitoneal or intranasal routes. Of these two routes, intranasal delivery gave the best mucosal responses at the lung surface, and was therefore chosen as the route of delivery for subsequent trials. CpG DNA and the wild-type baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) were tested as adjuvants for the RHDV VLP. These two adjuvants gave similar results, both acting to enhance a T[H]1 type response against the VLP, characterized by significantly increased levels of serum IgG2a and enhanced IFN-γ production. Two approaches were then tested: using the RHDV VLP as a peptide carrier with a CpG adjuvant, and using baculovirus particles directly as self-adjuvanting carriers for vaccine peptides. HAT and HAB peptides were chemically coupled to RHDV VLP. Mice that were vaccinated with these VLP mixed with a CpG adjuvant were able to raise low levels of specific antibody in the serum against influenza, and specific IgA against influenza was detected in the lung. These results indicated that, though the immune responses raised were modest, the RHDV VLP was able to deliver the vaccine peptides to the immune system. HAT and HAB peptides were chemically coupled to baculovirus particles. When mice were immunized with the baculovirus carrying the vaccine peptides, they raised significant levels of IgG1 (p<0.001) and IgG2a (p<0.05) against influenza in the serum, when compared to peptide delivered alone. A significant level of influenza-specific IgA was also detected in the lung at 10 ng/ml in the mice that received the baculovirus coupled with peptide. Analysis of splenocyte cytokines showed that these mice also responded to restimulation with IFN-γ production at around 100 pg/ml. This research revealed that RHDV VLP are able to act as carriers for vaccine peptides, however there are some limitations to their use with the HAT and HAB model peptides. It also showed that baculovirus can be rapidly modified to carry vaccine peptides by chemical conjugation, and that these peptides can be delivered to induce specific systemic and mucosal immunity, raising both B cell and cell mediated responses. Both virus particles have potential as components for new strategies for vaccination.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

No description available.

Thesis (Ph. D.)--School of Chemistry and Biochemistry, Georgia Institute of Technology, 2004. Directed by Suzanne B. Shuker. / Includes bibliographical references.

Antibiotics have been used widely for the treatment of bacterial infections for over half a century. However, the emergence of resistance to antibiotics has aroused public health concern, leading to the development of antimicrobial peptides (AMPs) as potential alternative therapeutic agents against bacterial infections. AMPs are naturally found in many species and have important roles in our innate immune defense systems. AMPs are usually cationic amphipathic peptides with membrane destabilizing property. They have a relatively broad spectrum of antimicrobial activity and pathogens are less likely to develop resistance against AMPs. The major challenge of using AMPs as therapeutic agents is their toxicity towards mammalian cells. The biological stability of AMPs to protease in human body is another concern. To address the latter problem, instead of the naturally occur L-enantiomers, Denantiomeric AMPs were introduced to enhance their stability. This study aimed to test the hypothesis that the D-enantiomeric AMPs are more resistant than the Lenantiomeric AMPs against proteolytic degradation. Three pairs of synthetic D-/LAMPs (D-LAO160-P13/LAO160-P12; D-LAO160-H/LAO160-H; and D-LAK-120-HP13/LAK-120-HP13) were employed to test for their stability when treated with trypsin, serum and gastric fluid, and the samples were analyzed by high performance liquid chromatography (HPLC). Generally, all the D-enantiomeric AMPs were found to be resistant towards proteolysis. Besides, to compare the cytotoxicity of D-/LAMPs, MTT and LDH assays of the D/L-LAK120-HP13 pair were carried out on two different cell lines, A549 cells (human lung adenocarcinoma epithelial cells) and RAW264.7 cells (mouse macrophage cells). Significant difference in cytotoxicity of D-LAK120-HP13 and LAK120-HP13 on RAW264.7 cells were obtained from MTT assay, but not in LDH assays or on A549 cells. Further analysis has to be done to validate the findings obtained from this research. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

A common strategy employed to destroy harmful bacteria is to disrupt the bacterial membrane through the action of pore-forming anti-microbial peptides. The manner in which the peptides arrange themselves spatially to form a pore in the membrane, which is important for understanding both the mechanism of pore formation and pore function, is a topic of current debate. We contrast the response of a model membrane bilayer to the presence of solid, cylindrical nanoparticle insertions, when the bilayer is composed of persistent worm-like chains and when it is composed of flexible Gaussian chains. We use self-consistent field theory, with the appropriate single-chain propagator, to describe the amphiphilic star-like triblock copolymers composing the membrane and the solvent. The nanoparticle surfaces are designed to have patches that prefer either the solvent or the tail groups of the copolymers, and the nanoparticles are fixed in space. Using this model with polymers in the lamellar phase, we investigate the question of pore-formation, nanoparticle insertion and hydrophobic mismatch in lipid bilayers and the effect that chain rigidity has on these particular interactions. We find that the main effect of increased chain rigidity is that it increases the free energy scaling and the significance of the energy barriers associated with these pore-forming processes. These results demonstrate the importance of using a more realistic persistent chain when modelling pore formation. / NSERC, CFI, SHARCNET

Copy of author's previously published works inserted. / Bibliography: leaves 183-220. / xiii, 226 leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Three antimicrobial peptides, maculatin 1.1, uperin 3.6 and caerin 4.1 have been isolated from the respective skin glands of the Australian amphibians Litoria genimaculata, Uperoleia mjobergii and Litoria caerulea. To gain a deeper insight into their mechanisms of action, three dimensional structural studies have been conducted using circular dichroism, two-dimensional nuclear resonance and computer modelling techniques. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is also discussed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 2000

Thesis (Ph.D.) -- University of Adelaide, Dept. of Chemistry, 2000. / Copies of author's previously published articles inserted. Includes bibliographical references.