1.
Pasupuleti, Mukesh, et al.
(författare)
End-tagging of ultra-short antimicrobial peptides by W/F stretches to facilitate bacterial killing
2009
Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 4:4, s. e5285-
Tidskriftsartikel (refereegranskat) abstract
BACKGROUND: Due to increasing resistance development among bacteria, antimicrobial peptides (AMPs), are receiving increased attention. Ideally, AMP should display high bactericidal potency, but low toxicity against (human) eukaryotic cells. Additionally, short and proteolytically stable AMPs are desired to maximize bioavailability and therapeutic versatility. METHODOLOGY AND PRINCIPAL FINDINGS: A facile approach is demonstrated for reaching high potency of ultra-short antimicrobal peptides through end-tagging with W and F stretches. Focusing on a peptide derived from kininogen, KNKGKKNGKH (KNK10) and truncations thereof, end-tagging resulted in enhanced bactericidal effect against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Through end-tagging, potency and salt resistance could be maintained down to 4-7 amino acids in the hydrophilic template peptide. Although tagging resulted in increased eukaryotic cell permeabilization at low ionic strength, the latter was insignificant at physiological ionic strength and in the presence of serum. Quantitatively, the most potent peptides investigated displayed bactericidal effects comparable to, or in excess of, that of the benchmark antimicrobial peptide LL-37. The higher bactericidal potency of the tagged peptides correlated to a higher degree of binding to bacteria, and resulting bacterial wall rupture. Analogously, tagging enhanced peptide-induced rupture of liposomes, particularly anionic ones. Additionally, end-tagging facilitated binding to bacterial lipopolysaccharide, both effects probably contributing to the selectivity displayed by these peptides between bacteria and eukaryotic cells. Importantly, W-tagging resulted in peptides with maintained stability against proteolytic degradation by human leukocyte elastase, as well as staphylococcal aureolysin and V8 proteinase. The biological relevance of these findings was demonstrated ex vivo for pig skin infected by S. aureus and E. coli. CONCLUSIONS/SIGNIFICANCE: End-tagging by hydrophobic amino acid stretches may be employed to enhance bactericidal potency also of ultra-short AMPs at maintained limited toxicity. The approach is of general applicability, and facilitates straightforward synthesis of hydrophobically modified AMPs without the need for post-peptide synthesis modifications.
2.
Ringstad, Lovisa, et al.
(författare)
An electrochemical study into the interaction between complement-derived peptides and DOPC mono- and bilayers.
2008
Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 24:1, s. 208-216
Tidskriftsartikel (refereegranskat) abstract
Electrochemical methods employing the hanging mercury drop electrode were used to study the interaction between variants of the complement-derived antimicrobial peptide CNY21 (CNYITELRRQH ARASHLGLAR) and dioleoyl phosphatidylcholine (DOPC) monolayers. Capacitance potential and impedance measurements showed that the CNY21 analogues investigated interact with DOPC monolayers coating the mercury drop. Increasing the peptide hydrophobicity by substituting the two histidine residues with leucine resulted in a deeper peptide penetration into the hydrophobic region of the DOPC monolayer, indicated by an increase in the dielectric constant of the lipid monolayer (Deltaepsilon = 2.0 after 15 min interaction). Increasing the peptide net charge from +3 to +5 by replacing the histidines by lysines, on the other hand, arrests the peptide in the lipid head group region. Reduction of electroactive ions (Tl+, Pb2+, Cd2+, and Eu3+) at the monolayer-coated electrode was employed to further characterize the types of defects induced by the peptides. All peptides studied permeabilize the monolayer to Tl+ to an appreciable extent, but this effect is more pronounced for the more hydrophobic peptide (CNY21L), which also allows penetration of larger ions and ions of higher valency. The results for the various ions indicate that charge repulsion rather than ion size is the determining factor for cation penetration through peptide-induced defects in the DOPC monolayer. The effects obtained for monolayers were compared to results obtained with bilayers from liposome leakage and circular dichroism studies for unilamellar DOPC vesicles, and in situ ellipsometry for supported DOPC bilayers. Trends in peptide-induced liposome leakage were similar to peptide effects on electrochemical impedance and permeability of electroactive ions for the monolayer system, demonstrating that formation of transmembrane pores alone does not constitute the mechanism of action for the peptides investigated. Instead, our results point to the importance of local packing defects in the lipid membrane in close proximity to the adsorbed peptide molecules.
3.
Ringstad, Lovisa, et al.
(författare)
Composition Effect on Peptide Interaction with Lipids and Bacteria : Variants of C3a Peptide CNY21
2007
Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 92:1, s. 87-98
Tidskriftsartikel (refereegranskat) abstract
The effect of peptide hydrophobicity and charge on peptide interaction with model lipid bilayers was investigated for the C3a-derived peptide CNY21 by fluorescence spectroscopy, circular dichroism, ellipsometry, z-potential, and photon correlation spectroscopy measurements. For both zwitterionic and anionic liposomes, the membrane-disruptive potency for CNY21 variants increased with increasing net positive charge and mean hydrophobicity and was completely lost on elimination of all peptide positive charges. Analogous effects of elimination of the peptide positive net charge in particular were found regarding bacteria killing for both Pseudomonas aeruginosa and Bacillus subtilis. The peptides, characterized by moderate helix content both in buffer and when attached to the liposomes, displayed high adsorption for the net positively charged peptide variants, whereas adsorption was nonmeasurable for the uncharged peptide. That electrostatically driven adsorption represents the main driving force for membrane disruption in lipid systems was also demonstrated by a drastic reduction in both liposome leakage and peptide adsorption with increasing ionic strength, and this salt inactivation can be partly avoided by increasing the peptide hydrophobicity. This increased electrolyte resistance translates also to a higher antibacterial effect for the hydrophobically modified variant at high salt concentration. Overall, our findings demonstrate the importance of the peptide adsorption and resulting peptide interfacial density for membrane-disruptive effects of these peptides.
4.
Ringstad, Lovisa, et al.
(författare)
Effects of topology, length, and charge on the activity of a kininogen-derived peptide on lipid membranes and bacteria
2007
Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1768:3, s. 715-727
Tidskriftsartikel (refereegranskat) abstract
Effects of topology, length, and charge on peptide interactions with lipid bilayers was investigated for variants of the human kininogen-derived peptide HKH20 (HKHGHGHGKHKNKGKKNGKH) by ellipsometry, CD, fluorescence spectroscopy, and z-potential measurements. The peptides display primarily random coil conformation in buffer and at lipid bilayers, and their lipid interaction is dominated by electrostatics, the latter evidenced by higher peptide adsorption and resulting membrane rupture for an anionic than for a zwitterionic membrane, as well as by strongly reduced adsorption and membrane rupture at high ionic strength. At sufficiently high peptide charge density, however, electrostatic interactions contribute to reducing the peptide adsorption and membrane defect formation. Truncating HKH20 into overlapping 10 amino acid peptides resulted in essentially eliminated membrane rupture and in a reduced amount peptide charges pinned at the lipid bilayer. Finally, cyclic HKH20 was found to be less efficient than the linear peptide in causing liposome rupture, partly due to a lower adsorption. Analogous results were found regarding bactericidal effects.
5.
Ringstad, Lovisa
(författare)
Interaction Between Antimicrobial Peptides and Phospholipid Membranes : Effects of Peptide Length and Composition
2009
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Due to increasing problems with bacterial resistance development, there is a growing need for identifying new types of antibiotics. Antimicrobial peptides constitute an interesting group of substances for this purpose, since they are believed to act mainly by disrupting the bacterial membrane, which is a fast and non-specific mechanism. In order to understand the details on this action simplified phospholipid model membranes based on liposomes, monolayers and bilayers, were employed in this thesis. By in situ ellipsometry studies on supported lipid bilayers in combination with leakage from liposomes it was found that peptide-induced membrane rupture to a great extent is related to peptide adsorption. The peptide activity and mechanism of action is highly dependent on peptide properties such as length, topology, charge, and hydrophobicity. Electrostatic interactions are crucial for peptide adsorption, whereas α-helix formation is of less importance, demonstrated by the dominating peptide conformation being random coil both in absence and presence of membranes, as investigated by circular dichroism. Comparable effects were observed in both mono- and bilayer systems, showing that formation of transmembrane structures is no prerequisite for membrane rupture by complement-derived peptides. Electrochemical studies on these peptides further demonstrated that hydrophobic interactions facilitate peptide penetration into the membrane, causing defects in close proximity to the peptides, while strong electrostatic interactions arrest the peptide in the headgroup region. Increasing the peptide hydrophobicity, by e.g., tryptophan end-tagging, also increases salt resistance. Good correlations were found between model membrane investigations and antibacterial activity towards both Gram-negative and Gram-positive bacteria, showing that membrane rupture is a key mechanism of action for the peptides investigated. In addition, for all peptides investigated cell toxicity is low.
6.
Rodríguez, M, et al.
(författare)
Reduction of atherosclerotic nanoplaque formation and size by Ginkgo biloba (EGb 761) in cardiovascular high-risk patients
2007
Ingår i: Atherosclerosis. - : Elsevier BV. - 0021-9150 .- 1879-1484. ; 192:2, s. 438-444
Tidskriftsartikel (refereegranskat) abstract
Coating a silica surface with the isolated lipoprotein receptor proteoheparan sulfate (HS-PG) from arterial endothelium and vascular matrices and adding both the atherogenic VLDL/IDL/LDL lipid fraction in its native composition and Ca2+ ions, we could observe in vitro the earliest stages of atherosclerotic plaque development by ellipsometric techniques (patent EP 0 946 876). This so-called nanoplaque formation is represented by the ternary aggregational complex of the HS-PG receptor, lipoprotein particles and calcium ions. The model was validated in several clinical studies on statins in cardiovascular high-risk patients. In eight patients who had undergone an aortocoronary bypass operation, the reduction of atherosclerotic nanoplaque formation amounted to 11.9±2.5% (p<0.0078) and of nanoplaque size to 24.4±8.1% (p<0.0234), respectively, after a 2-month therapy with Ginkgo biloba extract (2× 120mg daily, EGb 761). Additionally, superoxide dismutase (SOD) activity was upregulated by 15.7±7.0% (p<0.0391), the quotient oxLDL/LDL lowered by 17.0±5.5% (p<0.0234) and lipoprotein(a) concentration decreased by 23.4±7.9% (p<0.0234) in the patients’ blood. The concentration of the vasodilating substances cAMP and cGMP was augmented by 37.5±9.1% (p<0.0078) and 27.7±8.3% (p<0.0156), respectively. A multiple regression analysis between the patients’ VLDL/IDL/LDL lipoprotein fraction applied in the ellipsometry measurements as well as the further risk factors oxLDL/LDL and Lp(a) on the one hand and changes in nanoplaque formation on the other hand reveals a basis for a mechanistic explanation of nanoplaque reduction under ginkgo treatment. The atherosclerosis inhibiting effect is possibly due to an upregulation in the body's own radical scavenging enzymes and an attenuation of the risk factors oxLDL/LDL and Lp(a).
7.
Siegel, Günter, et al.
(författare)
The importance of scavenging reactive oxygen species in angi-aging medicine
2009
Ingår i: Engineering in Life Sciences. - : Wiley. - 1618-0240 .- 1618-2863. ; 9:5, s. 363-375
Tidskriftsartikel (refereegranskat) abstract
In a pilot Study, we had reported on the beneficial effects of Ginkgo biloba (EGb 761) on arteriosclerotic nanoplaque formation and size in cardiovascular high-risk patients who had undergone an aortocoronary bypass operation. Briefly, nanoplaque formation and size, the ratio oxLDL/LDL, and the highly atherothrombotic lipoprotein(a) concentration were substantially reduced, while superoxide dismutase activity and the blood concentration of the vasodilating substances cAMP and cGMP were Upregulated. Since the arteriosclerosis prophylactic and well-aging promotive impact of Ginkgo extract has been proven in this pilot study, we wanted to confirm these beneficial effects through a second observational clinical trial. The measurable variables formerly used were additionally supplemented by a wide, novel biomarker spectrum, through which the latest parameters and markers of plaque stability and progression, oxidative stress, and inflammation were available. In eleven patients with metabolic syndrome in the initial stage, the reduction of arteriosclerotic nanoplaque formation amounted to 14.3 +/- 2.9% (p<0.0077) and of nanoplaque size to 23.4 +/- 3.7% (p<0.0004), respectively, after 2-(SOD) and glutathione peroxidase (GPx) activities were upregulated by 19.6 +/- 10.0% (p<0.0785) and 11.6 +/- 2.3% (p<0.001), respectively, the quotient oxLDL/LDL lowered by 21.0 +/- 4.3% (p<0.002), and lipoprotein(a) concentration decreased by 26.3 +/- 4.8% (p<0.001) in the patients' blood. The concentration of cAMP and cGMP was augmented by 43.5 +/- 12.0% (p<0.001) and 32.9 +/- 10.4% (p<0.001), respectively. Surprisingly, we found a lowering of the serum Ca2+ concentration by 5.4 +/- 1.6% (p<0.0076) from 2.37 +/- 0.03 to 2.24 +/- 0.04 mmol/L (p<0.0069). Apart from an additional vasodilatory effect, the lowered extracellular Ca2+ concentration affects Could show a favourable development of the biomarkers 8-iso-PGF(2 alpha), oxLDL/LDL, SOD, GPx (oxidative stress), hs-CRP, MPO, TNF alpha TGF beta(1) (inflammatory status) and MMP-9 (plaque stability). The markers selected here are suited to provide a comprehensive risk profile for the prevention of arteriosclerosis. Finally, a multiple regression analysis reveals a basis for a mechanistic explanation of nanoplaque reduction under Ginkgo treatment. The arteriosclerosis inhibiting effect is due to an attenuation of the risk factors oxLDL/LDL, Lp(a), and [Ca2+](o) as well as to a significant increase in the vasodilator cAMP and cGMP concentration. Thus, Ginkgo with its pleiotropic effects should be assigned a fixed rank among the anti-aging medical therapeutics as a prophylactic measure, especially in patients with early-stage metabolic syndrome.
8.
Silvander, Mats, et al.
(författare)
A new water-based topical carrier with polar skin-lipids
2006
Ingår i: Lipids in Health and Disease. - 1476-511X. ; 5, s. 12-
Tidskriftsartikel (refereegranskat) abstract
A new water-based topical formulation is presented that aims at providing good penetration properties for both lipophilic and hydrophilic drugs with as small a disturbance of the skin barrier function as possible. The formulation contains dispersed lipids in a ratio resembling that of human skin. The capacity to deliver is addressed in this first study while the mild effect on skin will be presented later. Three variations of the lipid formulation were investigated by use of pigskin in vitro diffusion cell. The hydrophilic 5(6)-carboxyfluorescein (CF) and the lipophilic acridine orange 10-nonyl bromide (AO) were used as model drug substances. The results showed that the delivery properties of the new formulation exceeded that of the references (vaseline and xanthan gum gel). The effect was largest for lipophilic AO where all lipid matrix formulations were superior in amount detected in the skin. The results for the hydrophilic CF were also promising. Especially efficient was the lipid formulation containing the non-ionic adjuvants tetra ethylene glycol monododecyl ether and polyoxyethylene 23 dodecyl ether. The additional in vivo study suggests that the used in vitro model has qualitative bearing on relevant in vivo situations.
9.
Sonesson, Andreas, et al.
(författare)
Antifungal activity of C3a and C3a-derived peptides against Candida
2007
Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1768:2, s. 346-353
Tidskriftsartikel (refereegranskat) abstract
Antimicrobial peptides are generated during activation of the complement system [Nordahl et al. Proc. Natl. Acad. Sci. U. S. A. 2004, 101:16879-16884]. Here we show that the anaphylatoxin C3a exerts antimicrobial effects against the yeast Candida. Fluorescence microscopy and electron microscopy analysis demonstrated that C3a-derived peptides bound to the cell surface of Candida, and induced membrane perturbations and release of extracellular material. Various Candida isolates were found to induce complement degradation, leading to generation of C3a. Arginine residues were found to be critical for the antifungal and membrane breaking activity of a C3a-derived antimicrobial peptide, CNY21 (C3a; Cys57–Arg77). A CNY21 variant with increased positive net charge displayed enhanced antifungal activity. Thus, C3a-derived peptides can be utilized as templates in the development of peptide-based antifungal therapies.
10.
Strömstedt, Adam A., et al.
(författare)
Effect of lipid headgroup composition on the interaction between melittin and lipid bilayers
2007
Ingår i: Journal of Colloid and Interface Science. - : Elsevier BV. - 0021-9797 .- 1095-7103. ; 311:1, s. 59-69
Tidskriftsartikel (refereegranskat) abstract
The effect of the lipid polar headgroup on melittin-phospholipid interaction was investigated by cryo-TEM, fluorescence spectroscopy, ellipsometry, CD, electrophoresis and photon correlation spectroscopy. In particular, focus was placed on the effect of the lipid polar headgroup on peptide adsorption to, and penetration into, the lipid bilayer, as well as on resulting colloidal stability effects for large unilamellar liposomes. The effect of phospholipid headgroup properties on melittin-bilayer interaction was addressed by comparing liposomes contg. phosphatidylcholine, -acid, and -inositol at varying ionic strength. Increasing the bilayer neg. charge leads to an increased liposome tolerance toward melittin which is due to an electrostatic arrest of melittin at the membrane interface. Balancing the electrostatic attraction between the melittin pos. charges and the phospholipid neg. charges through a hydration repulsion, caused by inositol, reduced this surface arrest and increased liposome susceptibility to the disruptive actions of melittin. Furthermore, melittin was demonstrated to induce liposome structural destabilization on a colloidal scale which coincided with leakage induction for both anionic and zwitterionic systems. The latter findings thus clearly show that coalescence, aggregation, and fragmentation contribute to melittin-induced liposome leakage, and that detailed mol. analyses of melittin pore formation are incomplete without considering also these colloidal aspects.
