Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 67-84).

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: antimicrobial peptides; protegrins; synthetic genes. Includes bibliographical references (p. 56-57).

published_or_final_version / Zoology / Master / Master of Philosophy

<p>Antimicrobial peptides (AMPs) are part of the innate immune system that is widely distributed in nature, acting as a defense mechanism against invading microorganisms. AMPs have potent antimicrobial activity against a range of microorganisms including fungi, bacteria and viruses. In view of growing multidrug resistance, AMPs are increasingly being viewed as potential therapeutic agents with a novel mechanism of action. Mastoparan is a natural, highly positively charged AMP derived from the venom of wasps. It was originally of interest based on its inherent mast cell degranulation activity. Previously, mastoparan has been shown to exhibit antimicrobial activity in vitro however these studies have been limited in scope. Here we hypothesize that mastoparan possess the capacity to be a potent broad spectrum antibacterial agent including activity against multidrug resistant bacteria. </p><p>We examined the scope of antibacterial activity exhibited by mastoparan using a variety of antimicrobial susceptibility tests and have utilized a bacterial skin infection (S. aureus) model to determine the potential of mastoparan to serve as a therapeutic agent. We tested mastoparan against 4 Gram-positive clinical isolates (e.g., S. aureus, and E. faecium), 9 Gram-negative clinical isolates (e.g., E. coli, P. aeruginosa, and B. cepacia), and 4 multidrug resistant clinical isolates (e.g., MRSA, ESBL E.coli, and ESBL K. pneumonia). These studies reveal that mastoparan exhibits broad spectrum activity against both Gram-negative (MIC: 1.9 - 125 &mug/ml) and Gram-positive (MIC: 15.6 - 125 &mug/ml) bacteria and against multidrug resistant bacteria (MIC: 7.8 - 125 &mug/ml). We also demonstrated that mastoparan disrupts the bacterial membrane, exhibits fast acting antibacterial activity and is highly effective against both multiplying and non-multiplying bacteria. Furthermore, we have shown that mastoparan demonstrates efficacy as a topical antimicrobial agent reducing lesion size by up to 79% and the amount of bacteria recovered from skin lesions by up to a 98% reduction. Based on these results we conclude that mastoparan is a highly effective antibacterial agent and is therefore a potential alternative to currently antibiotics. Mastoparan offers a promising new therapeutic option for treating bacterial infections.</p> / Dissertation

No description available.

Much of what is known about the role of the ERBB family in cellular biology and in cancer has to do with canonical downstream signaling cascades and modifications associated with their trafficking and degradation. The focus on canonical activity, while important, ignores a rapidly expanding number of separate, arguably equally important functions for which there is emerging knowledge. These include the translocation of ERBB family members to non-canonical sub-cellular locations including the nucleus and mitochondria. Of current interest is the elucidation of fate determination mechanisms for these proteins. How is one ERBB receptor designated to traffic to the nucleus or mitochondria, versus degradative lysosomes? A portion of the work presented here addresses the potential role of ubiquitin in EGFR nuclear translocation, a role which ubiquitin has been shown to play in the context of other proteins. This work demonstrates that while ubiquitinated EGFR can be translocated from the plasma membrane to the nucleus in response to ligand, efficient ubiquitination is not essential for this process. This work also broadens the potential roles for ubiquitin to include those involving EGFR nuclear biology. Additional work described aimed to exploit what was already known regarding the diverse roles played by the EGFR juxtamembrane domain in the non-canonical activities of EGFR and the ERBBs. This work involves the creation and evaluation of an EGFR juxtamembrane domain derived peptide designed to competitively interact with endogenous ERBB domains and inhibit their function in cancer cells. Termed EJ1, treatment induces cell death and promotes the formation of inactive ERBB dimers and reduces ERBB activation. While inactivating CaMKII signaling, myosin light chain dependent cell blebbing occurs, coincident with the induction of cell death. EJ1 also directly translocates to mitochondria, potentially contributing to a loss of mitochondrial membrane potential and production of reactive oxygen species. Finally, treatment of mouse models of breast cancer with EJ1 results in the inhibition of tumor growth and metastasis, without observable side-effects/toxicities. These data demonstrate that a portion of the ERBB juxtamembrane domain, used as an intracellular decoy, can affect tumor growth and metastasis through ERBB-dependent and ERBB-independent mechanisms, representing a novel anti-cancer therapeutic.

Few weeks after birth, a switch in peptide elongation factor 1As from EF-1alpha/EF1A-1 to S1/EF1A-2 occurs in brain neurons, heart and skeletal muscles of mammalians. In order to elucidate the reason behind this switch, I studied the expression of both homologous proteins during muscle differentiation and apoptosis and, documented the relation between peptide elongation factors and caspase-3 activation. I found that during in vitro muscle differentiation of L6 myoblasts, a switch in peptide elongation factors 1A occurs as physiologically observed in skeletal muscles. While EF-1alpha/EF1A-1 is expressed in replicating myoblasts, S1/EF1A-2 is solely found in differentiated myotubes where it replaces EF-1alpha/EFIA-1 as the major elongation factor. Similarly, upon serum deprivation-induced apoptosis, a reversion in peptide elongation factors 1A is observed: EF-1alpha/EF1A-1 replaces S1/EF1A-2 and becomes the major form of elongation factor 1A present in dying myotubes. This switch correlates in myotubes with the activation of caspase-3 protein, a cysteine protease involved in apoptosis. When L6 myotubes constitutively express S1/EF1A-2 as caused by adenoviral gene transfer, they become resistant to serum deprivation-induced apoptosis. In contrast, when L6 myotubes are transfected with EF-1alpha/EF1A-1 gene, they die more rapidly from serum deprivation-induced apoptosis than control cells. Transfection using anti-sense EF-1alpha/EF1A-1 gene protects myotubes from apoptotic cell death. Thus, both elongation factor 1As exert opposing effect on muscle survival: while EF-1alpha/EF1A-1 accelerates apoptotic cell death, S1/EF1A-2 protects muscles against apoptosis. / I found that skeletal muscles are the only tissues where, despite the constitutive expression of caspase-3 mRNA, the protein can be absent. Furthermore, I found that while immediately after birth, caspase-3 protein is present in skeletal muscles, a few weeks afterwards, the protein cannot be detected by Western blotting. In skeletal muscle, this change correlates with the observed switch in peptide elongation factors from EF-1alpha/EF1A-1 to S1/EF1A-2 and suggests that caspase-3 is translationally regulated in skeletal muscles. The laboratory previously reported that while EF-1alpha/EF1A-1 protein reappears; S1/EF1A-2 protein becomes absent from regenerating muscles. However, once tissue regeneration is completed, the situation returns to normal as EF-1alpha/EF1A-1 disappears and S1/EF1A-2 reappears to become the only type 1A elongation factor expressed in muscle. / In conclusion, I found that the developmental switch observed in peptide elongation factors from EF-1alpha/EF1A-1 to S1/EF1A-2 partly serves to protect muscle cells from apoptosis. Thus, I am the first to identify a noncanonical function for S1/EF1A-2. (Abstract shortened by UMI.)

Stroke is a pathological condition that causes extensive brain damage. During ischemic stroke, an excess of the excitatory neurotransmitter glutamate exerts many deleterious effects, which leads to cellular damage and cell death, a phenomenon appropriately termed excitotoxicity. Among the events triggered is the activation of the enzyme calpain, a protease whose action is dependent on the intracellular concentration of calcium, which is known to be elevated during excitotoxicity. In this thesis, I hypothesize that neuroprotective drugs can be better accumulated into stroke-affected regions by utilizing the actions of calpain. The extent of calpain activation was first investigated, and it was found to increase over time in both in vitro and in vivo models of stroke. Different amino acid sequences recognized and cleaved by calpain were then incorporated into the neuroprotective Tat-GluR2/3Y peptide. Although in vivo detection of modified Tat-GluR2/3Y peptides was unsuccessful due to technical difficulties, the accumulation of the therapeutic 3Y peptide fragments in neurons under excitotoxic conditions in vitro was found to increase with the CP-3 peptide, a peptide that is a modified version of the Tat-GluR2/3Y, with a sequence cleavable by calpain from the protein Collapsin Response Mediator Protein-3 (CRMP-3). These results suggest that it is possible to concentrate therapeutic agents into stroke-affected neurons, and this may translate into enhanced neuroprotective properties in both in vitro and in vivo animal stroke models.

Amino aldehydes are important building blocks in organic synthesis. However, due to the innate propensity for condensation to occur upon combination of aldehydes and amines, uprotected amino aldehydes are unstable. One exception to this is the existence of dimeric aziridine aldehydes. We have shown that the enhanced stability observed with these unprotected aziridine aldehydes is due to their dimeric nature. In addition, we have shown that reversible dimer dissociation plays a key role in the kinetics and stereoselectivity of subsequent reactions. Reductive amination with the unprotected amino aldehyde dimers occurs without double addition or epimerization. The resulting aziridine conjugates were used towards a convergent synthesis of aminomethylene peptidomimetics, by aziridine ring opening with C-terminal peptide thioacids. We have shown that we can also utilize the aziridine aldehydes towards the assembly of branched peptides.

Cationic antimicrobial peptides (CAPs) are small peptides that constitute an important defence against microbial pathogens. Certain CAPs also possess anticancer properties. NRC-03 and NRC-07 are pleurocidins derived from winter and yellowtail flounder, respectively. The purpose of this investigation was to study the anticancer properties of NRC-03 and NRC-07. NRC-03 and NRC-07 killed breast cancer cells, including P-glycoprotein-overexpressing cells, in a time-dependent manner that peaked at 4 h. NRC-03 and NRC-07 lysed breast cancer cells by a mechanism that involved cell binding, mitochondrial destabilization, nuclear localization, and significant membrane damage. Interestingly, NRC-07, but not NRC-03, caused DNA fragmentation. NRC-03 and NRC-07 killed normal human epithelial cells, but did not kill endothelial cells or fibroblasts, or lyse human erythrocytes. NRC-03, and to a lesser extent NRC-07, had chemo-sensitizing properties, suggesting promise for their inclusion in combinational treatment regimens. Importantly, intratumoural injections of NRC-03 or NRC-07 inhibited tumour growth in a mouse model of breast cancer. Fetal bovine serum dose-dependently reduced cell killing by NRC-03. NRC-03 was degraded in human and mouse serum, which limited its potency. NRC-03- and NRC-07-induced cytotoxicity correlated with expression of several different negatively-charged molecules, rationalizing the generation of [D]-NRC-03, which carries the same positive charge as NRC-03, and was more potent but less selective for cancer cells than NRC-03. [D]-NRC-03 was also cytolytic and exhibited in vivo anticancer properties. To further test the clinical potential of NRC-03- and NRC-07-resistant cells were generated. NRC-03 and NRC-07 bound to resistant cells to a lesser extent than parental cells and were phenotypically distinct. Importantly, NRC-03- and NRC-07-resistant cells were killed by chemotherapeutic drugs, as well as [D]-NRC-03. These studies demonstrate that NRC-03, NRC-07, and [D]-NRC-03 are cytolytic peptides that kill breast cancer cells in vitro and in vivo. While more potent than NRC-03, [D]-NRC-03 requires further modification to minimize its toxicity toward normal cells. Although cancer cells may become resistant to NRC-03 and NRC-07 over time, resistant cells are still killed by other cytotoxic drugs, thereby reinforcing the value of adding these peptides to combinational regimens for the treatment of breast cancer.