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Modelin-5 isoforms were used to gain an insight into the effects of amidation on antimicrobial selectivity. When tested against Escherichia coli, amidation increased toxicity 10-fold (MIC = 31.25 μM) while showing limited increased... more
Modelin-5 isoforms were used to gain an insight into the effects of amidation on antimicrobial selectivity. When tested against Escherichia coli, amidation increased toxicity 10-fold (MIC = 31.25 μM) while showing limited increased hemolytic activity (2% lysis). Our results show that both the amidated and non-amidated peptides had a disordered structure in aqueous solution (<18% helical) and folded to form helices at the membrane interface (for example, >43% in the presence of DMPC). The stabilization of the helical structure by amidation has previously been shown to play a key role in increasing antibacterial efficacy. The presence of cholesterol in the membrane increases the packing density (C(s)(-1) values 25-33 mN m(-1)) and so prevents the peptide from forming stable association with the membrane, which is evidenced by the higher binding coefficient (K(d)) in the presence of cholesterol: 57.70 μM for Modelin-5-COOH and 35.64 μM for Modelin-5-CONH(2) compared to the presence of E. coli lipid extract (10 μM), which would prevent local concentration of the peptide at the bilayer interface as seen by reduction in monolayer interaction. This in turn would be predicted to inhibit activity.
Research Interests: Biochemistry, Thermodynamics, Protein Folding, Membranes, Kinetics, and 17 moreProtein Stability, Humans, Circular Dichroism, Escherichia coli, Cholesterol, Animals, Liposomes, Anti-Bacterial Agents, Antimicrobial Cationic Peptides, Amides, Protein Secondary Structure Prediction, Microbial Sensitivity Tests, Erythrocytes, Sus Scrofa, Drug Stability, Biochemistry and cell biology, and Hemolysis
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ABSTRACT It is becoming increasingly clear that plants, ranging from across the plant kingdom produce anionic host defence peptides (AHDPs) with potent activity against a wide variety of human cancers cells. In general, this activity... more
ABSTRACT It is becoming increasingly clear that plants, ranging from across the plant kingdom produce anionic host defence peptides (AHDPs) with potent activity against a wide variety of human cancers cells. In general, this activity involves membrane partitioning by AHDPs, which leads to membranolysis and / or internalization to attack intracellular targets such as DNA. Several models have been proposed to describe these events including: the toroidal pore and Shai-Matsuzaki-Huang (SHM) mechanisms but, in general, the mechanisms underpinning the membrane interactions and anticancer activity of these peptides are poorly understood. Plant AHDPs with anticancer activity can be conveniently discussed with reference to two groups: cyclotides, which possess cyclic molecules stabilized by cysteine knot motifs, and other ADHPs that adopt extended and α-helical conformations. Here, we review research into the anticancer action of these two groups of peptides along with current understanding of the mechanisms underpinning this action.
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To gain insight into the effects of amidation on the mechanism of membrane interaction, we studied two peptides modelin-5-COOH and modelin-5-CONH(2) and found they exhibit high surface activities (23.2 and 27.1 mN/m, respectively). When... more
To gain insight into the effects of amidation on the mechanism of membrane interaction, we studied two peptides modelin-5-COOH and modelin-5-CONH(2) and found they exhibit high surface activities (23.2 and 27.1 mN/m, respectively). When they were tested against Escherichia coli, amidation was seen to increase efficacy approximately 10-fold. Our results demonstrated that both peptides adopted low levels of α-helix in solution (<20%); however, in the presence of E. coli lipid extract, modelin-5-CONH(2) had a greater propensity (69%) than modelin-5-COOH (32%) to generate α-helical structure. The binding coefficient for both peptides was ∼10 μM, and the Hill coefficient approximated 1, suggesting that for both peptides the interactions with E. coli membranes were monomeric and comparable in strength. The peptides showed a clear preference for anionic lipid, with monolayer data showing that enhanced levels of helicity were associated with a greater pressure change (∼6 mN/m). Use of fluorescein-phosphatidylethanolamine showed the amidated version was able to generate greater levels of membrane disruption, which was confirmed by thermodynamic analysis. The data would imply that both peptides are able to initially bind to bilayer structures, but upon binding, the amidation stabilizes helix formation. This would be expected to help overcome a key rate-limiting step and generate higher local concentrations of peptide at the bilayer interface, which in turn would be predicted to increase efficacy.
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Page 1. Editorial Annual review David A Phoenix ... We will also need to look more closely at Europe, possibly mak ing representation through the European Communities Biological Association (ECBA) Council to strengthen sales in these... more
Page 1. Editorial Annual review David A Phoenix ... We will also need to look more closely at Europe, possibly mak ing representation through the European Communities Biological Association (ECBA) Council to strengthen sales in these countries. ...
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Using inverted Escherichia coli inner membrane vesicles we have analyzed the phosphatidylglycerol dependence of translocation of an OmpF-Lpp fusion protein carrying a signal sequence with varying positive charge at the N terminus and a... more
Using inverted Escherichia coli inner membrane vesicles we have analyzed the phosphatidylglycerol dependence of translocation of an OmpF-Lpp fusion protein carrying a signal sequence with varying positive charge at the N terminus and a hydrophobic core of varying length. It is shown that there is a direct relationship between the phosphatidylglycerol requirement of translocation and the requirement within the translocation process for positive charges on the signal sequence. This provides further evidence that the negative head group of the lipid is required for functional interaction with the positively charged N terminus of the signal sequence.
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ABSTRACT Antisense and antigene oligonucleotides bind mRNA to prevent translation or bind DNA to prevent gene transcription, respectively. In this chapter, we review recent progress in developing these agents for antimicrobial use. A... more
ABSTRACT Antisense and antigene oligonucleotides bind mRNA to prevent translation or bind DNA to prevent gene transcription, respectively. In this chapter, we review recent progress in developing these agents for antimicrobial use. A particular focus of this research over the last decade has been the small RNA pathways in eukaryotes, which have been identified as a major mechanism of gene regulation and are finding diverse applications in antimicrobial therapy either by regulating immune reactions or by acting as immune effectors. For example, in insects, the antiviral response has been shown to involve small interfering RNA (siRNA), which is able to induce gene silencing through nucleotide-sequence-specific RNA degradation mechanisms that result in the suppression of gene expression. It has been proposed that siRNA may be used as a tool to control insect immunity and hence act as pest control agents. In another study, using mouse models chronically infected with Mycobacterium tuberculosis, siRNA was used to silence the gene tgfβ1 which encodes an immunoregulatory cytokine. This treatment led to an increased expression of antimicrobial mediators, which effectively reduced the bacterial load of the mice. Furthermore, it has been suggested that these agents could be used to inhibit microbial resistance mechanisms at the nucleic acid level, thereby helping to address a problem of growing significance in multiple drug resistant (MDR) drugs. This interruption in the expression of resistance genes could restore susceptibility to key antibiotics, which would be co-administered with the antisense compound.
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Modelin-5-CONH2, a synthetic antimicrobial peptide, was used to gain an insight into species-selective haemolytic activity. The peptide displayed limited haemolytic activity against sheep (12%), human (2%), and pig (2%) erythrocytes. Our... more
Modelin-5-CONH2, a synthetic antimicrobial peptide, was used to gain an insight into species-selective haemolytic activity. The peptide displayed limited haemolytic activity against sheep (12%), human (2%), and pig (2%) erythrocytes. Our results show that Modelin-5-CONH2 had a disordered structure in the presence of vesicles formed from sheep, human, and pig erythrocyte lipid extract (<26% helical) yet folded to form helices in the presence of a phosphatidylcholine (PC) membrane interface (e.g. >42% in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine). Monolayer studies showed a strong correlation between anionic lipid content and monolayer insertion and lysis inducing surface pressure changes of 9.17 mN m(-1) for 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine compared with PC monolayers, which induced pressure changes of ca. 3 mN m(-1). The presence of cholesterol in the membrane is shown to increase the packing density as the PC:sphingomyelin (SM) ratio increases s...
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Maximin H5 (MH5) is an amphibian antimicrobial peptide specifically targeting Staphylococcus aureus. At pH 6, the peptide showed an improved ability to penetrate (ΔΠ = 6.2 mN m(-1)) and lyse (lysis = 48%) Staphylococcus aureus membrane... more
Maximin H5 (MH5) is an amphibian antimicrobial peptide specifically targeting Staphylococcus aureus. At pH 6, the peptide showed an improved ability to penetrate (ΔΠ = 6.2 mN m(-1)) and lyse (lysis = 48%) Staphylococcus aureus membrane mimics, which incorporated physiological levels of lysylated phosphatidylglycerol (Lys-PG, 60%), compared to that at pH 7 (ΔΠ = 5.6 mN m(-1) and lysis = 40% at pH 7) where levels of Lys-PG are lower (40%). The peptide therefore appears to have optimal function at pH levels known to be optimal for the organism's growth. MH5 killed S. aureus (minimum inhibitory concentration of 90 μM) via membranolytic mechanisms that involved the stabilization of α-helical structure (approximately 45-50%) and showed similarities to the "Carpet" mechanism based on its ability to increase the rigidity (Cs(-1) = 109.94 mN m(-1)) and thermodynamic stability (ΔGmix = -3.0) of physiologically relevant S. aureus membrane mimics at pH 6. On the basis of theoretic...