The Experts below are selected from a list of 120 Experts worldwide ranked by ideXlab platform
Robert E. W. Hancock - One of the best experts on this subject based on the ideXlab platform.
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Structure-based design of an indolicidin Peptide analogue with increased protease stability.
Biochemistry, 2003Co-Authors: Annett Rozek, Jonpaul S Powers, Carol L Friedrich, Robert E. W. HancockAbstract:Indolicidin is an Antimicrobial Cationic Peptide with broad-spectrum activity isolated from bovine neutrophils. An indolicidin analogue CP-11, ILKKWPWWPWRRK-NH(2), with improved activity against Gram-negative bacteria had increased positive charge and amphipathicity while maintaining the short length of the parent molecule. The structure of CP-11 in the presence of dodecylphosphocholine (DPC) micelles was determined using nuclear magnetic resonance spectroscopy. CP-11 was found to be an amphipathic molecule with a U-shaped backbone bringing the N- and C-termini in close proximity. On the basis of this close proximity, a cyclic disulfide-bonded Peptide cycloCP-11, ICLKKWPWWPWRRCK-NH(2), was designed to stabilize the lipid-bound structure and to increase protease resistance. The three-dimensional structure of cycloCP-11 was determined under the same conditions as for the linear Peptide and was found to be similar to CP-11. Both CP-11 and cycloCP-11 associated with phospholipid membranes in a similar manner as indicated by circular dichroism and fluorescence spectra. The minimal inhibitory concentrations of CP-11 and cycloCP-11 for a range of bacteria differed by no more than 2-fold, and they were nonhemolytic at concentrations up to 256 microg/mL. Cyclization was found to greatly increase protease stability. The half-life of cycloCP-11 in the presence of trypsin was increased by 4.5-fold from 4 to 18 min. More importantly, the antibacterial activity of cycloCP-11, but not that of CP-11, in the presence of trypsin was completely retained up to 90 min since the major degradation product was equally active. A structural comparison of CP-11 and cycloCP-11 revealed that the higher trypsin resistance of cycloCP-11 may be due to the more compact packing of lysine and tryptophan side chains. These findings suggest that cyclization may serve as an important strategy in the rational design of Antimicrobial Peptides.
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structure based design of an indolicidin Peptide analogue with increased protease stability
Biochemistry, 2003Co-Authors: Annett Rozek, Jonpaul S Powers, Carol L Friedrich, Robert E. W. HancockAbstract:Indolicidin is an Antimicrobial Cationic Peptide with broad-spectrum activity isolated from bovine neutrophils. An indolicidin analogue CP-11, ILKKWPWWPWRRK-NH2, with improved activity against Gram-negative bacteria had increased positive charge and amphipathicity while maintaining the short length of the parent molecule. The structure of CP-11 in the presence of dodecylphosphocholine (DPC) micelles was determined using nuclear magnetic resonance spectroscopy. CP-11 was found to be an amphipathic molecule with a U-shaped backbone bringing the N- and C-termini in close proximity. On the basis of this close proximity, a cyclic disulfide-bonded Peptide cycloCP-11, ICLKKWPWWPWRRCK-NH2, was designed to stabilize the lipid-bound structure and to increase protease resistance. The three-dimensional structure of cycloCP-11 was determined under the same conditions as for the linear Peptide and was found to be similar to CP-11. Both CP-11 and cycloCP-11 associated with phospholipid membranes in a similar manner as i...
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role of pseudomonas aeruginosa phop phoq in resistance to Antimicrobial Cationic Peptides and aminoglycosides
Microbiology, 2000Co-Authors: Emma L A Macfarlane, Agnieszka Kwasnicka, Robert E. W. HancockAbstract:Resistance to the polyCationic antibiotic polymyxin B and expression of the outer-membrane protein OprH in the opportunistic pathogen Pseudomonas aeruginosa both involve the PhoP-PhoQ two-component regulatory system. The genes for this system form an operon with oprH, oprH-phoP-phoQ, that responds to Mg2+ starvation and PhoP levels. In this study, the Mg2+-regulated promoter for this operon was mapped upstream of oprH by primer-extension experiments. An oprH::xylE-GmR mutant H855 was constructed and measurement of the catechol 2,3-dioxygenase activity expressed from this transcriptional fusion provided evidence for a second, weak promoter for phoP-phoQ. Wild-type P. aeruginosa PAO1 strain H103 was found to exhibit Mg2+-regulated resistance to the α-helical Antimicrobial Cationic Peptide CP28 in addition to its previously characterized resistance to polymyxin B. Resistance to this Peptide was unchanged in the OprH-null mutant H855 and a PhoP-null mutant H851. In contrast, PhoQ-null mutant H854 demonstrated constitutive CP28 resistance. Northern blot analysis revealed constitutive expression of phoP in this strain, implicating PhoP-PhoQ in the resistance of P. aeruginosa to Cationic Peptides. Furthermore, all three null-mutant strains demonstrated increased resistance to the aminoglycoside antibiotics streptomycin, kanamycin and amikacin. Two additional mutant strains, H895 and H896, were constructed that carried unmarked deletions in oprH and were found to exhibit aminoglycoside susceptibility equivalent to that of the wild-type. This result provided definitive evidence that OprH is not involved in P. aeruginosa aminoglycoside resistance and that the changes in resistance in strain H855 and a previously reported oprH mutant were due to polar effects on phoP-phoQ rather than loss of OprH expression. A role for PhoP-PhoQ in resistance to aminoglycosides is envisaged that is distinct from that in resistance to Cationic Peptides and polymyxin B.
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Improved Derivatives of Bactenecin, a Cyclic Dodecameric Antimicrobial Cationic Peptide
Antimicrobial Agents and Chemotherapy, 1999Co-Authors: Manhong Wu, Robert E. W. HancockAbstract:Both linear and cyclic derivatives of the cyclic 12-amino-acid Antimicrobial Peptide bactenecin were designed based on optimization of amphipathicity and charge location. In general, increasing the number of positive charges at the N and C termini and adding an extra tryptophan residue in the loop not only increased the activities against both gram-positive and gram-negative bacteria but also broadened the Antimicrobial spectrum.
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Interaction of the Cyclic Antimicrobial Cationic Peptide Bactenecin with the Outer and Cytoplasmic Membrane
Journal of Biological Chemistry, 1999Co-Authors: Manhong Wu, Robert E. W. HancockAbstract:Abstract Bactenecin, a 12-amino acid Cationic Antimicrobial Peptide from bovine neutrophils, has two cysteine residues, which form one disulfide bond, making it a cyclic molecule. To study the importance of the disulfide bond, a linear derivative Bac2S was made and the reduced form (linear bactenecin) was also included in this study. Circular dichroism spectroscopy showed that bactenecin existed as a type I β-turn structure regardless of its environment, while the reduced form and linear bactenecin adopted different conformations according to the lipophilicity of the environment. Bactenecin was more active against the Gram-negative wild type bacteria Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium than its linear derivative and reduced form, while all three Peptides were equally active against the outer membrane barrier-defective mutants of the first two bacteria. Only the two linear Peptides showed activity against the Gram-positive bacteria Staphylococcus epidermidis and Enterococcus facaelis. Bactenecin interacted well with the outer membrane and its higher affinity forE. coli UB1005 lipopolysaccharide and improved ability to permeabilize the outer membrane seemed to account for its better Antimicrobial activity against Gram-negative bacteria. The interaction of bactenecin with the cytoplasmic membrane was determined by its ability to dissipate the membrane potential by using the fluorescence probe 3,3-dipropylthiacarbocyanine and an outer membrane barrier-defective mutant E. coli DC2. It was shown that the linear derivative and reduced form were able to dissipate the membrane potential at much lower concentrations than bactenecin despite the similar minimal inhibitory concentrations of all three against this barrier-defective mutant.
Debabrata Ghosh - One of the best experts on this subject based on the ideXlab platform.
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In-vitro effects of the Antimicrobial Peptide Ala8,13,18-magainin II amide on isolated human first trimester villous trophoblast cells
Reproductive Biology and Endocrinology, 2011Co-Authors: Jayasree Sengupta, Meraj A. Khan, Berthold Huppertz, Debabrata GhoshAbstract:Background Research on Antimicrobial Cationic Peptides (AMPs) has gained pace toward using their potential to replace conventional antibiotics. These Peptides preferentially interact with negatively charged membrane lipids typically seen in bacteria and thereby lead to membrane perturbations and membrane dysfunction. However, one possible disadvantage of AMP drugs is their potential for toxicity, especially to those cells which display externalization of negatively charged moieties to the outer leaflet of the plasma membrane during the process of syncytialization. Human placental villous trophoblast is one such cell type. Indeed, intra-vaginal administration of an Antimicrobial Cationic Peptide Ala8,13,18-magainin II amide (AMA) which is a synthetic analogue of magainin 2 derived from Xenopus frog has been observed to result in inhibition of pregnancy establishment in monkeys. However, only little is known about the cellular behavior of early placental cytotrophoblasts (CTB) in the presence of Cationic Antimicrobial Peptides. It is believed that suitable cell culture approaches using AMA as a representative alpha-helical AMP may yield tangible knowledge in this regard.
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In-vitro effects of the Antimicrobial Peptide Ala8,13,18-magainin II amide on isolated human first trimester villous trophoblast cells
Reproductive Biology and Endocrinology, 2011Co-Authors: Jayasree Sengupta, Meraj A. Khan, Berthold Huppertz, Debabrata GhoshAbstract:Background Research on Antimicrobial Cationic Peptides (AMPs) has gained pace toward using their potential to replace conventional antibiotics. These Peptides preferentially interact with negatively charged membrane lipids typically seen in bacteria and thereby lead to membrane perturbations and membrane dysfunction. However, one possible disadvantage of AMP drugs is their potential for toxicity, especially to those cells which display externalization of negatively charged moieties to the outer leaflet of the plasma membrane during the process of syncytialization. Human placental villous trophoblast is one such cell type. Indeed, intra-vaginal administration of an Antimicrobial Cationic Peptide Ala8,13,18-magainin II amide (AMA) which is a synthetic analogue of magainin 2 derived from Xenopus frog has been observed to result in inhibition of pregnancy establishment in monkeys. However, only little is known about the cellular behavior of early placental cytotrophoblasts (CTB) in the presence of Cationic Antimicrobial Peptides. It is believed that suitable cell culture approaches using AMA as a representative alpha-helical AMP may yield tangible knowledge in this regard. Methods Immunocytochemical (ICC) analyses using confocal microscopy (n = 6 for each treatment sub-group) and Western blot (WB) method (n = 5 for each treatment sub-group) of CTB differentiation based on synthesis of beta-hCG and hPL, and apoptosis based on apoptosis-associated cytokeratin 18 neo-epitope (CK18f) were performed for CTB isolated from human first trimester placental villi and grown in serum-free primary culture for 24 h, 48 h and 96 h on rat-tail collagen with and without AMA (1000 ng/ml). Moreover, secretion of beta-hCG and hPL into conditioned media from isolated CTB grown in vitro for 24 h, 48 h and 96 h (n = 6/each sub-group) with and without AMA was examined using enzyme immunoassays. Furthermore, TUNEL assay, and cell viability based on LDH leakage into medium (n = 6/each sub-group) were assessed to examine the phenomenon of cell death with time and administration of AMA. Results CTB in serum-free primary culture showed increased (P < 0.05) level of synthesis and secretion of beta-hCG and hPL with time, and higher (P < 0.05) level of cellular cytokeratin 18 neo-epitope and number of TUNEL-positive cells, and LDH activity in conditioned medium at 96 h of culture. Exposure of CTB to AMA resulted in lower (P < 0.05) level of synthesis and secretion of beta-hCG and hPL, as well as, an increase (P < 0.05) of cellular cytokeratin 18 neo-epitope and number of TUNEL-positive cells, and LDH activity in conditioned medium at 96 h as compared to the control treatment. Conclusions Administration of AMA resulted in attenuation of differentiation, enhancement in apoptosis and loss of viability in early placental villi trophoblast cells in primary culture. Thus, it appears that administration of alpha-helical AMP may adversely affect the process of placentation and pregnancy outcome.
Santosh Misra - One of the best experts on this subject based on the ideXlab platform.
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Transgenic Potatoes Expressing a Novel Cationic Peptide are Resistant to Late Blight and Pink Rot
Transgenic Research, 2004Co-Authors: Milan Osusky, Lubica Osuska, Robert E. Hancock, Santosh MisraAbstract:Potato is the world’s largest non-cereal crop. Potato late blight is a pandemic, foliar wasting potato disease caused by Phytophthora infestans , which has become highly virulent, fungicide resistant, and widely disseminated. Similarly, fungicide resistant isolates of Phytophthora erythroseptica , which causes pink rot, have also become an economic scourge of potato tubers. Thus, an alternate, cost effective strategy for disease control has become an international imperative. Here we describe a strategy for engineering potato plants exhibiting strong protection against these exceptionally virulent pathogens without deleterious effects on plant yield or vigor. The small, naturally occurring Antimicrobial Cationic Peptide, temporin A, was N-terminally modified (MsrA3) and expressed in potato plants. MsrA3 conveyed strong resistance to late blight and pink rot phytopathogens in addition to the bacterial pathogen Erwinia carotovora . Transgenic tubers remained disease-free during storage for more than 2 years. These results provide a timely, sustainable, effective, and environmentally friendly means of control of potato diseases while simultaneously preventing storage losses.
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Transgenic Potatoes Expressing a Novel Cationic Peptide are Resistant to Late Blight and Pink Rot
Transgenic Research, 2004Co-Authors: Milan Osusky, Lubica Osuska, Robert E. Hancock, William W. Kay, Santosh MisraAbstract:Potato is the world’s largest non-cereal crop. Potato late blight is a pandemic, foliar wasting potato disease caused by Phytophthora infestans , which has become highly virulent, fungicide resistant, and widely disseminated. Similarly, fungicide resistant isolates of Phytophthora erythroseptica , which causes pink rot, have also become an economic scourge of potato tubers. Thus, an alternate, cost effective strategy for disease control has become an international imperative. Here we describe a strategy for engineering potato plants exhibiting strong protection against these exceptionally virulent pathogens without deleterious effects on plant yield or vigor. The small, naturally occurring Antimicrobial Cationic Peptide, temporin A, was N-terminally modified (MsrA3) and expressed in potato plants. MsrA3 conveyed strong resistance to late blight and pink rot phytopathogens in addition to the bacterial pathogen Erwinia carotovora . Transgenic tubers remained disease-free during storage for more than 2 years. These results provide a timely, sustainable, effective, and environmentally friendly means of control of potato diseases while simultaneously preventing storage losses.
Naresh Kumar - One of the best experts on this subject based on the ideXlab platform.
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Synthesis, characterization and in vitro activity of a surface-attached Antimicrobial Cationic Peptide.
Biofouling, 2009Co-Authors: Renxun Chen, Nerida Cole, Mark D. P. Willcox, Josephine Park, Riaz Rasul, Elizabeth A. Carter, Naresh KumarAbstract:Infection associated with implanted biomaterials is common and costly and such infections are extremely resistant to antibiotics and host defenses. Consequently, there is a need to develop surfaces which resist bacterial adhesion and colonization. The broad spectrum synthetic Cationic Peptide melimine has been covalently linked to a surface via two azide linkers, 4-azidobenzoic acid (ABA) or 4-fluoro-3-nitrophenyl azide (FNA), and the resulting surfaces characterized by X-ray photoelectron spectroscopy and contact angle measurements. The quantity of bound Peptide was estimated by a modified Bradford assay. The Antimicrobial efficacy of the two melimine-modified surfaces against Pseudomonas aeruginosa and Staphylococcus aureus was compared by scanning electron microscopy (SEM) and fluorescence microscopy. Attachment of melimine via ABA gave an approximately 4-fold greater quantity of melimine bound to the surface than attachment via FNA. Surfaces melimine-modified by either attachment strategy showed signi...
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synthesis characterization and in vitro activity of a surface attached Antimicrobial Cationic Peptide
Biofouling, 2009Co-Authors: Renxun Chen, Nerida Cole, Mark D. P. Willcox, Josephine Park, Riaz Rasul, Elizabeth A. Carter, Naresh KumarAbstract:Infection associated with implanted biomaterials is common and costly and such infections are extremely resistant to antibiotics and host defenses. Consequently, there is a need to develop surfaces which resist bacterial adhesion and colonization. The broad spectrum synthetic Cationic Peptide melimine has been covalently linked to a surface via two azide linkers, 4-azidobenzoic acid (ABA) or 4-fluoro-3-nitrophenyl azide (FNA), and the resulting surfaces characterized by X-ray photoelectron spectroscopy and contact angle measurements. The quantity of bound Peptide was estimated by a modified Bradford assay. The Antimicrobial efficacy of the two melimine-modified surfaces against Pseudomonas aeruginosa and Staphylococcus aureus was compared by scanning electron microscopy (SEM) and fluorescence microscopy. Attachment of melimine via ABA gave an approximately 4-fold greater quantity of melimine bound to the surface than attachment via FNA. Surfaces melimine-modified by either attachment strategy showed significantly reduced bacterial adhesion for both strains of bacteria. P. aeruginosa exposed to ABA-melimine and FNA-melimine surfaces showed marked changes in cell morphology when observed by SEM and a reduction of approximately 15-fold (p < 0.001) in the numbers of adherent bacteria compared to controls. For the ABA-melimine surface there was a 33% increase in cells showing damaged membranes (p = 0.0016) while for FNA-melimine there was no significant difference. For S. aureus there were reductions in bacterial adhesion of approximately 40-fold (p < 0.0001) and 5-fold (p = 0.008) for surfaces modified with melimine via ABA or FNA, respectively. There was an increase in cells showing damaged membranes on ABA-melimine surfaces of approximately 87% (p = 0.001) compared to controls, while for FNA-melimine there was no significant difference observed. The data presented in this study show that melimine has excellent potential for development as a broad spectrum Antimicrobial coating for biomaterial surfaces. Further, it was observed that the efficacy of Antimicrobial activity is related to the method of attachment.
Jayasree Sengupta - One of the best experts on this subject based on the ideXlab platform.
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In-vitro effects of the Antimicrobial Peptide Ala8,13,18-magainin II amide on isolated human first trimester villous trophoblast cells
Reproductive Biology and Endocrinology, 2011Co-Authors: Jayasree Sengupta, Meraj A. Khan, Berthold Huppertz, Debabrata GhoshAbstract:Background Research on Antimicrobial Cationic Peptides (AMPs) has gained pace toward using their potential to replace conventional antibiotics. These Peptides preferentially interact with negatively charged membrane lipids typically seen in bacteria and thereby lead to membrane perturbations and membrane dysfunction. However, one possible disadvantage of AMP drugs is their potential for toxicity, especially to those cells which display externalization of negatively charged moieties to the outer leaflet of the plasma membrane during the process of syncytialization. Human placental villous trophoblast is one such cell type. Indeed, intra-vaginal administration of an Antimicrobial Cationic Peptide Ala8,13,18-magainin II amide (AMA) which is a synthetic analogue of magainin 2 derived from Xenopus frog has been observed to result in inhibition of pregnancy establishment in monkeys. However, only little is known about the cellular behavior of early placental cytotrophoblasts (CTB) in the presence of Cationic Antimicrobial Peptides. It is believed that suitable cell culture approaches using AMA as a representative alpha-helical AMP may yield tangible knowledge in this regard.
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In-vitro effects of the Antimicrobial Peptide Ala8,13,18-magainin II amide on isolated human first trimester villous trophoblast cells
Reproductive Biology and Endocrinology, 2011Co-Authors: Jayasree Sengupta, Meraj A. Khan, Berthold Huppertz, Debabrata GhoshAbstract:Background Research on Antimicrobial Cationic Peptides (AMPs) has gained pace toward using their potential to replace conventional antibiotics. These Peptides preferentially interact with negatively charged membrane lipids typically seen in bacteria and thereby lead to membrane perturbations and membrane dysfunction. However, one possible disadvantage of AMP drugs is their potential for toxicity, especially to those cells which display externalization of negatively charged moieties to the outer leaflet of the plasma membrane during the process of syncytialization. Human placental villous trophoblast is one such cell type. Indeed, intra-vaginal administration of an Antimicrobial Cationic Peptide Ala8,13,18-magainin II amide (AMA) which is a synthetic analogue of magainin 2 derived from Xenopus frog has been observed to result in inhibition of pregnancy establishment in monkeys. However, only little is known about the cellular behavior of early placental cytotrophoblasts (CTB) in the presence of Cationic Antimicrobial Peptides. It is believed that suitable cell culture approaches using AMA as a representative alpha-helical AMP may yield tangible knowledge in this regard. Methods Immunocytochemical (ICC) analyses using confocal microscopy (n = 6 for each treatment sub-group) and Western blot (WB) method (n = 5 for each treatment sub-group) of CTB differentiation based on synthesis of beta-hCG and hPL, and apoptosis based on apoptosis-associated cytokeratin 18 neo-epitope (CK18f) were performed for CTB isolated from human first trimester placental villi and grown in serum-free primary culture for 24 h, 48 h and 96 h on rat-tail collagen with and without AMA (1000 ng/ml). Moreover, secretion of beta-hCG and hPL into conditioned media from isolated CTB grown in vitro for 24 h, 48 h and 96 h (n = 6/each sub-group) with and without AMA was examined using enzyme immunoassays. Furthermore, TUNEL assay, and cell viability based on LDH leakage into medium (n = 6/each sub-group) were assessed to examine the phenomenon of cell death with time and administration of AMA. Results CTB in serum-free primary culture showed increased (P < 0.05) level of synthesis and secretion of beta-hCG and hPL with time, and higher (P < 0.05) level of cellular cytokeratin 18 neo-epitope and number of TUNEL-positive cells, and LDH activity in conditioned medium at 96 h of culture. Exposure of CTB to AMA resulted in lower (P < 0.05) level of synthesis and secretion of beta-hCG and hPL, as well as, an increase (P < 0.05) of cellular cytokeratin 18 neo-epitope and number of TUNEL-positive cells, and LDH activity in conditioned medium at 96 h as compared to the control treatment. Conclusions Administration of AMA resulted in attenuation of differentiation, enhancement in apoptosis and loss of viability in early placental villi trophoblast cells in primary culture. Thus, it appears that administration of alpha-helical AMP may adversely affect the process of placentation and pregnancy outcome.