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J. Michael Conlon - One of the best experts on this subject based on the ideXlab platform.
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Evidence from peptidomic analysis of skin secretions that allopatric populations of Xenopus gilli (Anura:Pipidae) constitute distinct lineages
Peptides, 2015Co-Authors: Milena Mechkarska, Laurent Coquet, Hubert Vaudry, J. Michael Conlon, Thierry Jouenne, Jérôme Leprince, G John MeaseyAbstract:The International Union for Conservation of Nature (IUCN) Endangered Cape Platanna Xenopus gilli inhabits disjunct ranges at the tip of Cape Peninsula and near the town of Kleinmond on opposite sides of False Bay in the extreme southwest of Africa. Peptidomic analysis of host-defense peptides in norepinephrine-stimulated skin secretions from frogs from the Cape Peninsula range resulted in the identification of two Magainins, two peptide glycine-leucine-amide (PGLa) peptides, two xenopsin-precursor fragment (XPF) peptides, nine caerulein-precursor fragment (CPF) peptides, and a peptide related to peptide glycine-glutamine (PGQ) previously found in an extract of Xenopus laevis stomach. The primary structures of the peptides indicate a close phylogenetic relationship between X. gilli and X. laevis but only Magainin-1, PGLa and one CPF peptide are identical in both species. Consistent with previous data, the CPF peptides show the greatest antimicrobial potency but are hemolytic. There are appreciable differences in the expression of host-defense peptide genes in frogs from the population of animals sampled near Kleinmond as peptides corresponding to Magainin-G2, XPF-G1, XPF-G2, and four CPF peptides, present in secretions from the Cape Peninsula frogs, were not identified in the skin secretions from Kleinmond frogs. Conversely, PGLa-G3, XPF-G3, and three CPF peptides were identified in the Kleinmond frogs but not in the Cape Peninsula animals. The data support the conclusion from morphometric analyses and comparisons of the nucleotide sequences of mitochondrial genes that the disjunct populations of X. gilli have undergone appreciable genetic, morphological, and phenotypic divergence.
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Host-Defense Peptides with Therapeutic Potential from Skin Secretions of Frogs from the Family Pipidae
Pharmaceuticals, 2014Co-Authors: J. Michael Conlon, Milena MechkarskaAbstract:Skin secretions from frogs belonging to the genera Xenopus, Silurana, Hymenochirus, and Pseudhymenochirus in the family Pipidae are a rich source of host-defense peptides with varying degrees of antimicrobial activities and cytotoxicities to mammalian cells. Magainin, peptide glycine-leucine-amide (PGLa), caerulein-precursor fragment (CPF), and xenopsin-precursor fragment (XPF) peptides have been isolated from norepinephrine-stimulated skin secretions from several species of Xenopus and Silurana. Hymenochirins and pseudhymenochirins have been isolated from Hymenochirus boettgeri and Pseudhymenochirus merlini. A major obstacle to the development of these peptides as anti-infective agents is their hemolytic activities against human erythrocytes. Analogs of the Magainins, CPF peptides and hymenochirin-1B with increased antimicrobial potencies and low cytotoxicities have been developed that are active (MIC < 5 μM) against multidrug-resistant clinical isolates of Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Stenotrophomonas maltophilia and Klebsiella pneumoniae. Despite this, the therapeutic potential of frog skin peptides as anti-infective agents has not been realized so that alternative clinical applications as anti-cancer, anti-viral, anti-diabetic, or immunomodulatory drugs are being explored.
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Peptidomic analysis of skin secretions demonstrates that the allopatric populations of Xenopus muelleri (Pipidae) are not conspecific.
Peptides, 2011Co-Authors: Milena Mechkarska, Jay D. King, Eman Ahmed, Laurent Coquet, Hubert Vaudry, Thierry Jouenne, Jérôme Leprince, J. Michael ConlonAbstract:Abstract Mueller's clawed frog Xenopus muelleri (Peters 1844) occupies two non-contiguous ranges in east and west Africa. The phylogenetic relationship between the two populations is unclear and it has been proposed that the western population represents a separate species. Peptidomic analysis of norepinephrine-stimulated skin secretions from X. muelleri from the eastern range resulted in the identification of five antimicrobial peptides structurally related to the Magainins (Magainin-M1 and -M2), xenopsin-precursor fragments (XPF-M1) and caerulein-precursor fragments (CPF-M1 and -M2) previously found in skin secretions of other Xenopus species. A cyclic peptide (WCPPMIPLCSRF.NH 2 ) containing the RFamide motif was also isolated that shows limited structural similarity to the tigerinins, previously identified only in frogs of the Dicroglossidae family. The components identified in skin secretions from X. muelleri from the western range comprised one Magainin (Magainin-MW1), one XPF peptide (XPF-MW1), two peptides glycine-leucine amide (PGLa-MW1 and -MW2), and three CPF peptides (CPF-MW1, -MW2 and -MW3). Comparison of the primary structures of these peptides suggest that western population of X. muelleri is more closely related to X. borealis than to X. muelleri consistent with its proposed designation as a separate species. The CPF peptides showed potent, broad-spectrum activity against reference strains of bacteria (MIC 3–25 μM), but were hemolytic against human erythrocytes.
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Orthologs of Magainin, PGLa, procaerulein-derived, and proxenopsin-derived peptides from skin secretions of the octoploid frog Xenopus amieti (Pipidae)
Peptides, 2010Co-Authors: J. Michael Conlon, Mohammed A Meetani, Nadia Al-ghaferi, Eman Ahmed, Jérôme Leprince, Per F. NielsenAbstract:Abstract The Volcano clawed frog Xenopus amieti Kobel, du Pasquier, Fischberg, and Gloor, 1980, with a chromosome number of 2 n = 72, is believed to have undergone two rounds of genome duplication since evolving from a diploid ancestor. Nine peptides with differential antimicrobial activity against Escherichia coli and Staphylococcus aureus were isolated from norepinephrine-stimulated skin secretions of X. amieti that showed structural similarity to peptides previously isolated from the tetraploid frog X . laevis (2 n = 36) and the diploid frog Silurana (formerly Xenopus ) tropicalis (2 n = 20). Two peptides (Magainin-AM1 and -AM2) are othologous to the Magainins, two peptides (PGLa-AM1 and -AM2) orthologous to peptide glycine–leucine-amide, four peptides (CPF-AM1, -AM2, -AM3, -AM4) orthologous to caerulein-precursor fragments, and one peptide (XPF-AM1) structurally similar to xenopsin-precursor fragments were characterized. CFP-AM1 (GLGSVLGKALKIGANLL.NH 2 ) was the most potent peptide present in the secretions and Magainin-AM2 (GVSKILHSAGKFGKAFLGEIMKS) was the most abundant. The data indicate that nonfunctionalization has been the most common fate of duplicated antimicrobial peptide genes following the polyploidization events in the X. amieti lineage. However, the very low antimicrobial activity of the Magainin-AM1 and PGLa-AM2 paralogs suggests the possibility that certain peptides may have evolved toward a new, as yet undetermined, function (neofunctionalization).
Michael Zasloff - One of the best experts on this subject based on the ideXlab platform.
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Ciba Foundation Symposium 186 - Antimicrobial Peptides - Potential therapeutic applications of Magainins and other antimicrobial agents of animal origin.
Ciba Foundation symposium, 2007Co-Authors: Leonard S. Jacob, Michael ZasloffAbstract:Magainins are a family of linear, amphipathic, cationic antimicrobial peptides, 21 to 27 residues in length, found in the skin of Xenopus laevis. They kill microbial targets through disruption of membrane permeability. They exhibit selectivity, on the basis of their affinity for membranes which contain accessible acidic phospholipids, a property characterizing the cytoplasmic membranes of many species of bacteria. Magainins are broad-spectrum antimicrobial agents exhibiting cidal activity against Gram-negative and Gram-positive bacteria, fungi and protozoa. In addition these peptides lyse many types of murine and human cancer cells at concentrations 5-10-fold lower than normal human cells. Because of their selectivity, broad spectrum, low degree of bacterial resistance and ease of chemical synthesis, Magainins are being developed as human therapeutic agents. The most advanced candidate is MSI-78, a 22-residue Magainin analogue. This peptide is currently in human Phase IIb/III clinical trials in studies intended to evaluate its efficacy as a topical agent for the treatment of impetigo. Preclinical studies have demonstrated that analogues of Magainin exhibit activity in vivo against malignant melanoma and ovarian cancer cells in mouse models. Intravenous administration of several Magainin analogues has been shown to treat effectively systemic Escherichia coli infections in the mouse.
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Mechanism of Synergism between Antimicrobial Peptides Magainin 2 and PGLa
Biochemistry, 1998Co-Authors: Katsumi Matsuzaki, Michael Zasloff, Osamu Murase, Yasuyuki Mitani, Ken’ya Akada, Shuji Yoneyama, Koichiro MiyajimaAbstract:The antimicrobial peptides Magainin 2 and PGLa, discovered in the skin of the African clawed frog, Xenopus laevis, exhibit marked synergism [Westerhoff, H. V., Zasloff, M., Rosner, J. L., Hendler, R. W., de Waal, A., Vaz Gomes, A., Jongsma, A. P. M., Riethorst, A., and Juretic, D., Eur. J. Biochem. 228, 257-264 (1995)], although the mechanism is not yet clear. They are believed to kill bacteria by permeabilizing membranes. In this study, we examined the interactions of these peptides in lipid bilayers. PGLa, like Magainin 2, preferentially interacts with acidic lipids, forming an amphipathic helix. The peptide induces the release of a water-soluble dye, calcein, entrapped within liposomes. The coexistence of Magainin 2 enhances membrane permeabilization, which is maximal at a 1:1 molar ratio. Fluorescence experiments using L18W-PGLa revealed that both peptides form a stoichiometric 1:1 complex in the membrane phase with an association free energy of -15 kJ/mol. Single amino acid mutations in Magainin 2 significantly altered the synergistic activity, suggesting that precise molecular recognition is involved in complex formation. The complex as well as each component peptide form peptide-lipid supramolecular complex pores, which mediate the mutually coupled transbilayer transport of dye, lipid, and the peptide per se. The rate of pore formation rate is in the order complex >/= PGLa > Magainin 2, whereas the pore lifetime is in the order Magainin 2 > complex > PGLa. Therefore, the synergism is a consequence of the formation of a potent heterosupramolecular complex, which is characterized by fast pore formation and moderate pore stability.
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Structure and dynamics of the antibiotic peptide PGLa in membranes by solution and solid-state nuclear magnetic resonance spectroscopy.
Biophysical Journal, 1998Co-Authors: Burkhard Bechinger, Michael Zasloff, Stanley J. OpellaAbstract:PGLa, a 21-residue member of the Magainin family of antibiotic peptides, is shown to be helical between residues 6 and 21 when associated with detergent micelles by multidimensional solution nuclear magnetic resonance (NMR) spectroscopy. Solid-state NMR experiments on specifically 15N-labeled peptides in oriented phospholipid bilayer samples show that the helix axis is parallel to the plane of the bilayers. 15N solid-state NMR powder pattern line shapes obtained on unoriented samples demonstrate that the amino-terminal residues are highly mobile and that the fluctuations of backbone sites decrease from Ala6 toward the carboxy terminus. The powder pattern observed for 15N-labeled Ala20 is essentially that expected for a rigid site. These findings are similar to those for the 23-residue Magainin2 peptide in membrane environments.
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Functional synergism of the Magainins PGLa and Magainin-2 in Escherichia coli, tumor cells and liposomes
European journal of biochemistry, 1995Co-Authors: Hans V. Westerhoff, Michael Zasloff, Richard W. Hendler, J. Lee Rosner, Anthony De Waal, Ana Vaz Gomes, Ans P. M. Jongsma, Albert Riethorst, Davor JuretićAbstract:Xenopus laevis skin secretion contains a mixture of Magainins, which are small positively charged oligopeptides with antimicrobial activity. In this study, we show that two of these peptides, i.e. Magainin-2 and PGLa, are much more active in biological functions when added together than when added alone. This synergy applies for the antimicrobial activity of these peptides, and for the toxic effects on tumor cells. We show that this peptide combination is also synergistic when permeabilizing protein-free liposomes for glucose, when dissipating the membrane potential in cytochrome oxidase liposomes and Escherichia coli, and, reversibly, when stimulating respiration in the liposomes. The occurrence of synergy in these diverse systems (complex and simple) suggests that the biological synergy results from synergy in the primary activity of the Magainin peptides, namely the permeabilization of free-energy transducing membranes, possibly by forming a multimeric transmembrane pore of mixed peptide composition. The antimicrobial activity of X. laevis skin secretions may be greatly enhanced by the application of this binary weapon.
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Magainin oligomers reversibly dissipate DeltamuH in cytochrome oxidase liposomes.
Biochemistry, 1994Co-Authors: Davor Juretić, Michael Zasloff, Richard W. Hendler, Frits Kamp, Winslow S. Caughey, Hans V. WesterhoffAbstract:Magainin peptides present in the skin of Xenopus laevis and identified as antimicrobial agents are shown to decrease the membrane potential in cytochrome oxidase liposomes. They also released respiratory control with a third or higher order concentration dependence. Respiratory control was restored by proteolytic digestion of the added Magainin. The amount of Magainin required for half-maximal stimulation of respiration was proportional to lipid concentration. At appreciably higher concentrations Magainins inhibited uncoupled respiration. The results are discussed in terms of a model in which most of the added Magainin adsorbs as a monomer to the membranes but equilibrates with a multimeric pore that causes rather general permeability of membranes. The ensuing ion permeation dissipates membrane potential and stimulates respiration.
Masahito Yamazaki - One of the best experts on this subject based on the ideXlab platform.
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Mechanism of Initial Stage of Pore Formation Induced by Antimicrobial Peptide Magainin 2.
Langmuir : the ACS journal of surfaces and colloids, 2018Co-Authors: Moynul Hasan, Mohammad Abu Sayem Karal, Victor Levadnyy, Masahito YamazakiAbstract:Antimicrobial peptide Magainin 2 forms pores in lipid bilayers, a property that is considered the main cause of its bactericidal activity. Recent data suggest that tension or stretching of the inner monolayer plays an important role in Magainin 2-induced pore formation in lipid bilayers. Here, to elucidate the mechanism of Magainin 2-induced pore formation, we investigated the effect on pore formation of asymmetric lipid distribution in two monolayers. First, we developed a method to prepare giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC), and lyso-PC (LPC) in the inner monolayer and of DOPG/DOPC in the outer monolayer. We consider that in these GUVs, the lipid packing in the inner monolayer was larger than that in the outer monolayer. Next, we investigated the interaction of Magainin 2 with these GUVs with an asymmetric distribution of LPC using the single GUV method, and found that the rate constant of Magainin 2-induced pore formatio...
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Mechanism of Initial Stage of Pore Formation Induced by Antimicrobial Peptide Magainin 2
2018Co-Authors: Moynul Hasan, Mohammad Abu Sayem Karal, Victor Levadnyy, Masahito YamazakiAbstract:Antimicrobial peptide Magainin 2 forms pores in lipid bilayers, a property that is considered the main cause of its bactericidal activity. Recent data suggest that tension or stretching of the inner monolayer plays an important role in Magainin 2-induced pore formation in lipid bilayers. Here, to elucidate the mechanism of Magainin 2-induced pore formation, we investigated the effect on pore formation of asymmetric lipid distribution in two monolayers. First, we developed a method to prepare giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC), and lyso-PC (LPC) in the inner monolayer and of DOPG/DOPC in the outer monolayer. We consider that in these GUVs, the lipid packing in the inner monolayer was larger than that in the outer monolayer. Next, we investigated the interaction of Magainin 2 with these GUVs with an asymmetric distribution of LPC using the single GUV method, and found that the rate constant of Magainin 2-induced pore formation, kp, decreased with increasing LPC concentration in the inner monolayer. We constructed a quantitative model of Magainin 2-induced pore formation, whereby the binding of Magainin 2 to the outer monolayer of a GUV induces stretching of the inner monolayer, causing pore formation. A theoretical equation defining kp as a function of Magainin 2 surface concentration, X, reasonably explains the experimental relationship between kp and X. This model quantitatively explains the effect on kp of the LPC concentration in the inner monolayer. On the basis of these results, we discuss the mechanism of the initial stage of Magainin 2-induced pore formation
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Stretch-activated pore of the antimicrobial peptide, Magainin 2.
Langmuir : the ACS journal of surfaces and colloids, 2015Co-Authors: Mohammad Abu Sayem Karal, Jahangir Md. Alam, Tomoki Takahashi, Victor Levadny, Masahito YamazakiAbstract:Antimicrobial peptide Magainin 2 forms pores in lipid membranes and induces membrane permeation of the cellular contents. Although this permeation is likely the main cause of its bactericidal activity, the mechanism of pore formation remains poorly understood. We therefore investigated in detail the interaction of Magainin 2 with lipid membranes using single giant unilamellar vesicles (GUVs). The binding of Magainin 2 to the lipid membrane of GUVs increased the fractional change in the area of the membrane, δ, which was proportional to the surface concentration of Magainin 2, X. This indicates that the rate constant of the Magainin 2- induced two-state transition from the intact state to the pore state greatly increased with an increase in δ. The tension of a lipid membrane following aspiration of a GUV also activated Magainin 2-induced pore formation. To reveal the location of Magainin 2, the interaction of carboxyfluorescein (CF)-labeled Magainin 2 (CF-Magainin 2) with single GUVs containing a water-soluble fluorescent probe, AF647, was investigated using confocal microscopy. In the absence of tension due to aspiration, after the interaction of Magainin 2 the fluorescence intensity of the GUV rim due to CF-Magainin 2 increased rapidly to a steady value, which remained constant for a long time, and at 4−32 s before the start of leakage of AF647 the rim intensity began to increase rapidly to another steady value. In contrast, in the presence of the tension, no increase in rim intensity just before the start of leakage was observed. These results indicate that Magainin 2 cannot translocate from the outer to the inner monolayer until just before pore formation. Based on these results, we conclude that a Magainin 2-induced pore is a stretch-activated pore and the stretch of the inner monolayer is a main driving force of the pore formation.
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Kinetic pathway of antimicrobial peptide Magainin 2-induced pore formation in lipid membranes.
The journal of physical chemistry. B, 2010Co-Authors: Yukihiro Tamba, Victor Levadny, Hirotaka Ariyama, Masahito YamazakiAbstract:The pore formation in lipid membranes induced by the antimicrobial peptide Magainin 2 is considered to be the main cause for its bactericidal activity. To reveal the mechanism of the pore formation, it is important to elucidate the kinetic pathway of Magainin 2-induced pore formation in lipid membranes. In this report, to examine the change in pore size over time during pore formation which can monitor its kinetic pathway, we investigated the rate of the leakage of various sized fluorescent probes through the Magainin 2-induced pores in single giant unilamellar vesicles (GUVs) of 50% dioleoylphosphatidylglycerol (DOPG)/50% dioleoylphosphatidylcholine (DOPC) membrane. Magainin 2- induced leakage of Texas-Red dextran 10 000, Texas-Red dextran 3000, and Alexa-Fluor trypsin inhibitor occurred in two stages; a transient rapid leakage in the initial stage followed by a stage of slow leakage. In contrast, Magainin 2 induced a transient, but very small (10−20%), leakage of fluorescent probes of a larger size such...
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Effects of Surface Charge Density of Lipid Membranes on the Pore Formation Induced by Antimicrobial Peptide Magainin 2: the Single GUV Method Study
2010Co-Authors: Yukihiro Tamba, Hirotaka Ariyama, Masahito YamazakiAbstract:Interactions of antimicrobial peptides with lipid membranes have been investigated using a suspension of many small liposomes, and their details remain unclear. Recently we have proposed a novel method, the single GUV method; we observe and measure physical properties of single GUVs, and analyze these results over many single GUVs statistically, which will provide much new information that cannot be obtained by the conventional LUV suspension method [e.g.,1,2]. Using the single GUV method, we have succeeded in revealing elementary processes of the pore formation in lipid membranes induced by Magainin 2 [3]. In this report, to elucidate the mechanism of the Magainin 2-induced pore formation, we investigated the effect of surface charge density of membranes on the pore formation. To change the surface charge density, we used GUVs of mixture membranes of negatively charged DOPG and electrically neutral DOPC, and controlled the DOPG concentration (mol%) in the membrane. The experiments were done in 10 mM PIPES (pH 7.0), 150 mM NaCl (buffer A) at 26 °C. We investigated the interaction of Magainin 2 with single 60%DOPG/ 40% DOPC-GUVs containing the fluorescent dye, calcein, by fluorescence microscopy using the single GUV method. Low concentrations (0.5−5 μM) of Magainin 2 caused the rapid leakage of calcein from single GUVs without change of the GUV structure, showing directly that Magainin 2 forms pores in the membrane (Fig.1A,B) [3]. The rapid leakage of calcein from a GUV started stochastically, and once it began the complete leakage occurred rapidly (Fig.1C). The fraction of leaked GUV among the observed single GUVs, PLS, increased with time. On the other hand, Magainin 2 did not induce the leakage of calcein from single 30%DOPG/70%DOPC-GUVs at less than 10 μM. However, higher concentrations (≥10 μM) of Magainin 2 induced a similar rapid leakage of calcein, indicating that the pores were formed in the membrane. The rapid leakage of calcein from a GUV started stochastically. Figure 2A shows the dependence of PLS after the interaction of Magainin 2 with a single GUV for 6min on the Magainin 2 concentration in the buffer for various GUVs with a different surface charge density. PLS of DOPG/DOPC-GUVs with a same surface charge density increased with an increase in Magainin 2 concentration. The Magainin 2 concentration at PLS = 0.5 increased with a decrease in the surface charge density. We can consider that the amount of Magainin 2 bound with the membrane interface of GUVs decreased with a decrease in the surface charge density in the presence of the same Magainin 2 concentration in the buffer, due to the decrease in the electrostatic attraction of Magainin 2 with the membranes. Using the Gouy-Chapman theory, (A) (1) (2) (3)
Milena Mechkarska - One of the best experts on this subject based on the ideXlab platform.
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Evidence from peptidomic analysis of skin secretions that allopatric populations of Xenopus gilli (Anura:Pipidae) constitute distinct lineages
Peptides, 2015Co-Authors: Milena Mechkarska, Laurent Coquet, Hubert Vaudry, J. Michael Conlon, Thierry Jouenne, Jérôme Leprince, G John MeaseyAbstract:The International Union for Conservation of Nature (IUCN) Endangered Cape Platanna Xenopus gilli inhabits disjunct ranges at the tip of Cape Peninsula and near the town of Kleinmond on opposite sides of False Bay in the extreme southwest of Africa. Peptidomic analysis of host-defense peptides in norepinephrine-stimulated skin secretions from frogs from the Cape Peninsula range resulted in the identification of two Magainins, two peptide glycine-leucine-amide (PGLa) peptides, two xenopsin-precursor fragment (XPF) peptides, nine caerulein-precursor fragment (CPF) peptides, and a peptide related to peptide glycine-glutamine (PGQ) previously found in an extract of Xenopus laevis stomach. The primary structures of the peptides indicate a close phylogenetic relationship between X. gilli and X. laevis but only Magainin-1, PGLa and one CPF peptide are identical in both species. Consistent with previous data, the CPF peptides show the greatest antimicrobial potency but are hemolytic. There are appreciable differences in the expression of host-defense peptide genes in frogs from the population of animals sampled near Kleinmond as peptides corresponding to Magainin-G2, XPF-G1, XPF-G2, and four CPF peptides, present in secretions from the Cape Peninsula frogs, were not identified in the skin secretions from Kleinmond frogs. Conversely, PGLa-G3, XPF-G3, and three CPF peptides were identified in the Kleinmond frogs but not in the Cape Peninsula animals. The data support the conclusion from morphometric analyses and comparisons of the nucleotide sequences of mitochondrial genes that the disjunct populations of X. gilli have undergone appreciable genetic, morphological, and phenotypic divergence.
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Host-Defense Peptides with Therapeutic Potential from Skin Secretions of Frogs from the Family Pipidae
Pharmaceuticals, 2014Co-Authors: J. Michael Conlon, Milena MechkarskaAbstract:Skin secretions from frogs belonging to the genera Xenopus, Silurana, Hymenochirus, and Pseudhymenochirus in the family Pipidae are a rich source of host-defense peptides with varying degrees of antimicrobial activities and cytotoxicities to mammalian cells. Magainin, peptide glycine-leucine-amide (PGLa), caerulein-precursor fragment (CPF), and xenopsin-precursor fragment (XPF) peptides have been isolated from norepinephrine-stimulated skin secretions from several species of Xenopus and Silurana. Hymenochirins and pseudhymenochirins have been isolated from Hymenochirus boettgeri and Pseudhymenochirus merlini. A major obstacle to the development of these peptides as anti-infective agents is their hemolytic activities against human erythrocytes. Analogs of the Magainins, CPF peptides and hymenochirin-1B with increased antimicrobial potencies and low cytotoxicities have been developed that are active (MIC < 5 μM) against multidrug-resistant clinical isolates of Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Stenotrophomonas maltophilia and Klebsiella pneumoniae. Despite this, the therapeutic potential of frog skin peptides as anti-infective agents has not been realized so that alternative clinical applications as anti-cancer, anti-viral, anti-diabetic, or immunomodulatory drugs are being explored.
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hybridization between the african clawed frogs xenopus laevis and xenopus muelleri pipidae increases the multiplicity of antimicrobial peptides in skin secretions of female offspring
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 2012Co-Authors: Milena Mechkarska, Mohammed A Meetani, Pawel Michalak, Zalman Vaksman, Koji Takada, Michael J ConlonAbstract:Abstract Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of the common clawed frog Xenopus laevis (Daudin, 1802) and Mueller's clawed frog Xenopus muelleri (Peters, 1844) with the corresponding distribution in skin secretions from the parent species. A total of 18 peptides were identified in secretions from the hybrid frogs. Eleven peptides (Magainin-1, Magainin-2, CPF-1, CPF-3, CPF-4, CPF-5, CPF-6, CPF-7, XPF-1, XPF-2, and PGLa) were identified in secretions of both the hybrids and X. laevis . Four peptides (Magainin-M1, XPF-M1, CPF-M1, and tigerinin-M1) were previously found in skin secretions of X. muelleri but Magainin-M2 and CPF-M2 from X. muelleri were not detected. Three previously undescribed peptides (Magainin-LM1, PGLa-LM1, and CPF-LM1) were purified from the secretions of the hybrid frogs that were not detected in secretions from either X. laevis or X. muelleri . Magainin-LM1 differs from Magainin-2 from X. laevis by a single amino acid substitution (Gly 13 → Ala) but PGLa-LM1 and CPF-LM1 differ appreciably in structure from orthologs in the parent species. CPF-LM1 shows potent, broad-spectrum antimicrobial activity and is hemolytic. The data indicate that hybridization increases the multiplicity of skin host-defense peptides in skin secretions. As the female F1 hybrids are fertile, hybridization may represent an adaptive strategy among Xenopus species to increase protection against pathogenic microorganisms in the environment.
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Peptidomic analysis of skin secretions demonstrates that the allopatric populations of Xenopus muelleri (Pipidae) are not conspecific.
Peptides, 2011Co-Authors: Milena Mechkarska, Jay D. King, Eman Ahmed, Laurent Coquet, Hubert Vaudry, Thierry Jouenne, Jérôme Leprince, J. Michael ConlonAbstract:Abstract Mueller's clawed frog Xenopus muelleri (Peters 1844) occupies two non-contiguous ranges in east and west Africa. The phylogenetic relationship between the two populations is unclear and it has been proposed that the western population represents a separate species. Peptidomic analysis of norepinephrine-stimulated skin secretions from X. muelleri from the eastern range resulted in the identification of five antimicrobial peptides structurally related to the Magainins (Magainin-M1 and -M2), xenopsin-precursor fragments (XPF-M1) and caerulein-precursor fragments (CPF-M1 and -M2) previously found in skin secretions of other Xenopus species. A cyclic peptide (WCPPMIPLCSRF.NH 2 ) containing the RFamide motif was also isolated that shows limited structural similarity to the tigerinins, previously identified only in frogs of the Dicroglossidae family. The components identified in skin secretions from X. muelleri from the western range comprised one Magainin (Magainin-MW1), one XPF peptide (XPF-MW1), two peptides glycine-leucine amide (PGLa-MW1 and -MW2), and three CPF peptides (CPF-MW1, -MW2 and -MW3). Comparison of the primary structures of these peptides suggest that western population of X. muelleri is more closely related to X. borealis than to X. muelleri consistent with its proposed designation as a separate species. The CPF peptides showed potent, broad-spectrum activity against reference strains of bacteria (MIC 3–25 μM), but were hemolytic against human erythrocytes.
Yukihiro Tamba - One of the best experts on this subject based on the ideXlab platform.
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Kinetic pathway of antimicrobial peptide Magainin 2-induced pore formation in lipid membranes.
The journal of physical chemistry. B, 2010Co-Authors: Yukihiro Tamba, Victor Levadny, Hirotaka Ariyama, Masahito YamazakiAbstract:The pore formation in lipid membranes induced by the antimicrobial peptide Magainin 2 is considered to be the main cause for its bactericidal activity. To reveal the mechanism of the pore formation, it is important to elucidate the kinetic pathway of Magainin 2-induced pore formation in lipid membranes. In this report, to examine the change in pore size over time during pore formation which can monitor its kinetic pathway, we investigated the rate of the leakage of various sized fluorescent probes through the Magainin 2-induced pores in single giant unilamellar vesicles (GUVs) of 50% dioleoylphosphatidylglycerol (DOPG)/50% dioleoylphosphatidylcholine (DOPC) membrane. Magainin 2- induced leakage of Texas-Red dextran 10 000, Texas-Red dextran 3000, and Alexa-Fluor trypsin inhibitor occurred in two stages; a transient rapid leakage in the initial stage followed by a stage of slow leakage. In contrast, Magainin 2 induced a transient, but very small (10−20%), leakage of fluorescent probes of a larger size such...
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Effects of Surface Charge Density of Lipid Membranes on the Pore Formation Induced by Antimicrobial Peptide Magainin 2: the Single GUV Method Study
2010Co-Authors: Yukihiro Tamba, Hirotaka Ariyama, Masahito YamazakiAbstract:Interactions of antimicrobial peptides with lipid membranes have been investigated using a suspension of many small liposomes, and their details remain unclear. Recently we have proposed a novel method, the single GUV method; we observe and measure physical properties of single GUVs, and analyze these results over many single GUVs statistically, which will provide much new information that cannot be obtained by the conventional LUV suspension method [e.g.,1,2]. Using the single GUV method, we have succeeded in revealing elementary processes of the pore formation in lipid membranes induced by Magainin 2 [3]. In this report, to elucidate the mechanism of the Magainin 2-induced pore formation, we investigated the effect of surface charge density of membranes on the pore formation. To change the surface charge density, we used GUVs of mixture membranes of negatively charged DOPG and electrically neutral DOPC, and controlled the DOPG concentration (mol%) in the membrane. The experiments were done in 10 mM PIPES (pH 7.0), 150 mM NaCl (buffer A) at 26 °C. We investigated the interaction of Magainin 2 with single 60%DOPG/ 40% DOPC-GUVs containing the fluorescent dye, calcein, by fluorescence microscopy using the single GUV method. Low concentrations (0.5−5 μM) of Magainin 2 caused the rapid leakage of calcein from single GUVs without change of the GUV structure, showing directly that Magainin 2 forms pores in the membrane (Fig.1A,B) [3]. The rapid leakage of calcein from a GUV started stochastically, and once it began the complete leakage occurred rapidly (Fig.1C). The fraction of leaked GUV among the observed single GUVs, PLS, increased with time. On the other hand, Magainin 2 did not induce the leakage of calcein from single 30%DOPG/70%DOPC-GUVs at less than 10 μM. However, higher concentrations (≥10 μM) of Magainin 2 induced a similar rapid leakage of calcein, indicating that the pores were formed in the membrane. The rapid leakage of calcein from a GUV started stochastically. Figure 2A shows the dependence of PLS after the interaction of Magainin 2 with a single GUV for 6min on the Magainin 2 concentration in the buffer for various GUVs with a different surface charge density. PLS of DOPG/DOPC-GUVs with a same surface charge density increased with an increase in Magainin 2 concentration. The Magainin 2 concentration at PLS = 0.5 increased with a decrease in the surface charge density. We can consider that the amount of Magainin 2 bound with the membrane interface of GUVs decreased with a decrease in the surface charge density in the presence of the same Magainin 2 concentration in the buffer, due to the decrease in the electrostatic attraction of Magainin 2 with the membranes. Using the Gouy-Chapman theory, (A) (1) (2) (3)
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The size of the pore in lipid membranes induced by antimicrobial peptide Magainin 2
2009 International Symposium on Micro-NanoMechatronics and Human Science, 2009Co-Authors: Hirotaka Ariyama, Victor Levadny, Yukihiro Tamba, Masahito YamazakiAbstract:Antimicrobial peptides found and isolated from a wide variety of organisms have an activity to kill bacteria. The target of these peptides is thought to be the lipid membrane region of the bacterial and fungal biomembranes. Using the single GUV (giant unilamellar vesicle) method, we have succeeded in revealing the elementary processes of the pore formation in lipid membranes induced by antimicrobial peptide, Magainin 2. The statistical analysis of the pore formation in a GUV over many “single GUVs” enabled us to estimate the rate constant of the Magainin 2-induced pore formation in lipid membranes. In this report, to reveal the size of the Magainin 2-induced pores in lipid membranes, we investigated the interactions of Magainin 2 with single GUVs containing various sizes of fluorescent probes. Under the conditions with no photobleaching of fluorescent probes, we investigated the interaction of Magainin 2 with single GUVs of 50% dioleoylphosphatidylglycerol (DOPG)/ 50% dioleoylphosphatidylcholine(DOPC) membrane containing various sizes of fluorescent probes in 10 mM PIPES (pH 7.0), 150 mM NaCl (buffer A). Magainin 2 induced a transient (less than 10 s), but very small (10–20 %) leakage of Texas-Red dextran 40,000 (TRD-40k), Texas-Red dextran 70,000 (TRD-70k), and FITC-BSA, although the same concentrations of Magainin 2 induced a complete leakage of calcein. In contrast, the Magainin 2-induced leakage of Texas-Red dextran 10,000 (TRD-10k) and Texas-Red dextran 3,000 (TRD-3k) had two phases; the transient rapid leakage in the initial stage and the following slow leakage. These results indicate that in the initial stage of the Magainin 2-induced pore formation, the size of the pore was large and then transformed into a small, steady size. The radius of the transient, large pore in the initial stage is larger than 6.4 nm (i.e., the Stokes-Einstein radius, R SE , of TRD-70k) and also that the radius of the small steady pore in the final stage is smaller than 3.5 nm (i.e., R SE of FITC-BSA). The amount of the leakage of TRD-10k in the initial stage increased with an increase in Magainin 2 concentration. This result indicates that the radius of the large pore in the initial stage increased with an increase in Magainin 2 concentration. We discuss these results from a point of view of the pore formation.
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Effects of Surface Charge Density of Lipid Membranes on the Pore Formation Induced by Magainin 2
2007 International Symposium on Micro-NanoMechatronics and Human Science, 2007Co-Authors: Yukihiro Tamba, Shah Md. Masum, Masahito YamazakiAbstract:Interactions of antimicrobial peptides with lipid membranes have been investigated using a suspension of small liposomes, and their details remain unclear. Using the single GUV method, we have succeeded in revealing elementary processes of the pore formation in lipid membranes induced by Magainin 2 in our previous paper (Biochemistry, 44, 15823, 2005). In this report, to elucidate the mechanism of the Magainin 2-induced pore formation, we investigated the effect of surface charge density of membranes on the pore formation. To change the surface charge density, we controlled negatively charged DOPG concentration in DOPG/DOPC membrane from 30 to 60 mol%. We found that, in all kinds of GUVs, Magainin 2 induced a rapid leakage of calcein from single GUVs, showing that Magainin 2 formed pores in the membrane. For GUVs with the same charge density, the fraction of leaked GUV, PLS, increased with time, and PLS at a fixed time increased with Magainin 2 concentration. The Magainin 2 concentration at PLS = 0.5 at a fixed time increased with a decrease in the surface charge density, indicating that higher concentrations of Magainin 2 in a buffer were required to induce the pore formation in GUVs with lower surface charge density. Using the Gouy-Chapman theory, we obtained Magainin 2 concentration in the membrane interface, assuming the intrinsic binding constant of Magainin 2 with DOPG/DOPC membranes. On the basis of these results, we discuss the role of Magainin 2 concentration in the membrane interface in the Magainin 2-induced pore formation.
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Single giant unilamellar vesicle method reveals effect of antimicrobial peptide Magainin 2 on membrane permeability.
Biochemistry, 2005Co-Authors: Yukihiro Tamba, Masahito YamazakiAbstract:It is thought that Magainin 2, an antimicrobial peptide, acts by binding to lipid membranes. Recent studies using a suspension of large unilamellar vesicles (LUVs) indicate that Magainin 2 causes gradual leakage from LUVs containing negatively charged lipids. However, the details of the characteristics of the membrane permeability and the mechanism of pore formation remain unclear. In this report, we investigated the interaction of Magainin 2 with single giant unilamellar vesicles (GUVs) composed of a dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol mixture (50% DOPG/50% DOPC GUVs) containing the fluorescent dye, calcein, by phase contrast, fluorescence microscopy using the single GUV method. Low concentrations (3−10 μM) of Magainin 2 caused the rapid leakage of calcein from single GUVs but did not disrupt the liposomes or change the membrane structure, showing directly that Magainin 2 forms membrane pores through which calcein leaked. The rapid leakage of calcein from a GUV started stochastic...
