Search Results (41)

Gramicidin S (GS), one of the first discovered antimicrobial peptides, still shows strong antibiotic activity after decades of clinical use, with no evidence of resistance. The relatively high hemolytic activity and narrow therapeutic window of GS limit its use in topical applications. Encapsulation and targeted delivery may be the way to develop the internal administration of this drug. The lipid composition of membranes and non-covalent interactions affect GS’s affinity for and partitioning into lipid bilayers as monomers or oligomers, which are crucial for GS activity. Using both differential scanning calorimetry (DSC) and FTIR methods, the impact of GS on dipalmitoylphosphatidylcholine (DPPC) membranes was tested. Additionally, the combined effect of GS and cholesterol on membrane characteristics was observed; while dipalmitoylphosphatydylglycerol (DPPG) and cerebrosides did not affect GS binding to DPPC membranes, cholesterol significantly altered the membrane, with 30% mol concentration being most effective in enhancing GS binding. The effect of star-like dextran-polyacrylamide D-g-PAA(PE) on GS binding to the membrane was tested, revealing that it interacted with GS in the membrane and significantly increased the proportion of GS oligomers. Instead, calcium ions affected GS binding to the membrane differently, with independent binding of calcium and GS and no interaction between them. This study shows how GS interactions with lipid membranes can be effectively modulated, potentially leading to new formulations for internal GS administration. Modified liposomes or polymer nanocarriers for targeted GS delivery could be used to treat protein misfolding disorders and inflammatory conditions associated with free-radical processes in cell membranes. Full article

We have investigated the morphology of two-dimensional monolayers of gramicidin-D (GD) and alamethicin (Al) formed on the water surface by the dropping method (DM) using surface tension measurement (STm), Brewster angle microscopy (BAM), and atomic force microscopy (AFM). Dynamic light scattering (DLS) revealed that GD in alcoholic solutions formed a dimeric helical structure. According to the CD and NMR spectroscopies, GD molecules existed in dimer form in methanol and lipid membrane environments. The STm results and BAM images revealed that the GD dimer monolayer was in a liquid expanded (LE) state, whereas the Al monolayer was in a liquid condensed (LC) state. The limiting molecular area (A₀) was 6.2 ± 0.5 nm² for the GD-dimer and 3.6 ± 0.5 nm² for the Al molecule. The AFM images also showed that the molecular long axes of both the GD-dimer and Al were horizontal to the water surface. The stability of each monolayer was confirmed by the time dependence of the surface pressure (π) observed using the STm method. The DM monolayer preparation method for GD-dimer and Al peptide molecules is a useful technique for revealing how the model biological membrane’s components assemble in two dimensions on the water surface. Full article

Perturbations in bilayer material properties (thickness, lipid intrinsic curvature and elastic moduli) modulate the free energy difference between different membrane protein conformations, thereby leading to changes in the conformational preferences of bilayer-spanning proteins. To further explore the relative importance of curvature and elasticity in determining the changes in bilayer properties that underlie the modulation of channel function, we investigated how the micelle-forming amphiphiles Triton X-100, reduced Triton X-100 and the H_II lipid phase promoter capsaicin modulate the function of alamethicin and gramicidin channels. Whether the amphiphile-induced changes in intrinsic curvature were negative or positive, amphiphile addition increased gramicidin channel appearance rates and lifetimes and stabilized the higher conductance states in alamethicin channels. When the intrinsic curvature was modulated by altering phospholipid head group interactions, however, maneuvers that promote a negative-going curvature stabilized the higher conductance states in alamethicin channels but destabilized gramicidin channels. Using gramicidin channels of different lengths to probe for changes in bilayer elasticity, we found that amphiphile adsorption increases bilayer elasticity, whereas altering head group interactions does not. We draw the following conclusions: first, confirming previous studies, both alamethicin and gramicidin channels are modulated by changes in lipid bilayer material properties, the changes occurring in parallel yet differing dependent on the property that is being changed; second, isolated, negative-going changes in curvature stabilize the higher current levels in alamethicin channels and destabilize gramicidin channels; third, increases in bilayer elasticity stabilize the higher current levels in alamethicin channels and stabilize gramicidin channels; and fourth, the energetic consequences of changes in elasticity tend to dominate over changes in curvature. Full article

Using the gramicidin A channel as a molecular probe, we show that tubulin binding to planar lipid membranes changes the channel kinetics—seen as an increase in the lifetime of the channel dimer—and thus points towards modification of the membrane’s mechanical properties. The effect is more pronounced in the presence of non-lamellar lipids in the lipid mixture used for membrane formation. To interpret these findings, we propose that tubulin binding redistributes the lateral pressure of lipid packing along the membrane depth, making it closer to the profile expected for lamellar lipids. This redistribution happens because tubulin perturbs the lipid headgroup spacing to reach the membrane’s hydrophobic core via its amphiphilic α-helical domain. Specifically, it increases the forces of repulsion between the lipid headgroups and reduces such forces in the hydrophobic region. We suggest that the effect is reciprocal, meaning that alterations in lipid bilayer mechanics caused by membrane remodeling during cell proliferation in disease and development may also modulate tubulin membrane binding, thus exerting regulatory functions. One of those functions includes the regulation of protein–protein interactions at the membrane surface, as exemplified by VDAC complexation with tubulin. Full article

Background and Objectives: Gramicidin, a bactericidal antibiotic used in dermatology and ophthalmology, has recently garnered attention for its inhibitory actions against cancer cell growth. However, the effects of gramicidin on ovarian cancer cells and the underlying mechanisms are still poorly understood. We aimed to elucidate the anticancer efficacy of gramicidin against ovarian cancer cells. Materials and Methods: The anticancer effect of gramicidin was investigated through an in vitro experiment. We analyzed cell proliferation, DNA fragmentation, cell cycle arrest and apoptosis in ovarian cancer cells using WST-1 assay, terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL), DNA agarose gel electrophoresis, flow cytometry and western blot. Results: Gramicidin treatment induces dose- and time-dependent decreases in OVCAR8, SKOV3, and A2780 ovarian cancer cell proliferation. TUNEL assay and DNA agarose gel electrophoresis showed that gramicidin caused DNA fragmentation in ovarian cancer cells. Flow cytometry demonstrated that gramicidin induced cell cycle arrest. Furthermore, we confirmed via Western blot that gramicidin triggered apoptosis in ovarian cancer cells. Conclusions: Our results strongly suggest that gramicidin exerts its inhibitory effect on cancer cell growth by triggering apoptosis. Conclusively, this study provides new insights into the previously unexplored anticancer properties of gramicidin against ovarian cancer cells. Full article

Fungi represents a rich repository of taxonomically restricted, yet chemically diverse, secondary metabolites that are synthesised via specific metabolic pathways. An enzyme’s specificity and biosynthetic gene clustering are the bottleneck of secondary metabolite evolution. Trichoderma harzianum M10 v1.0 produces many pharmaceutically important molecules; however, their specific biosynthetic pathways remain uncharacterised. Our genomic-based analysis of this species reveals the biosynthetic diversity of its specialised secondary metabolites, where over 50 BGCs were predicted, most of which were listed as polyketide-like compounds associated clusters. Gene annotation of the biosynthetic candidate genes predicted the production of many medically/industrially important compounds including enterobactin, gramicidin, lovastatin, HC-toxin, tyrocidine, equisetin, erythronolide, strobilurin, asperfuranone, cirtinine, protoilludene, germacrene, and epi-isozizaene. Revealing the biogenetic background of these natural molecules is a step forward towards the expansion of their chemical diversification via engineering their biosynthetic genes heterologously, and the identification of their role in the interaction between this fungus and its biotic/abiotic conditions as well as its role as bio-fungicide. Full article

The cells of living organisms are surrounded by the biological membranes that form a barrier between the internal and external environment of the cells. Cell membranes serve as barriers and gatekeepers. They protect cells against the entry of undesirable substances and are the first line of interaction with foreign particles. Therefore, it is very important to understand how substances such as particulate matter (PM) interact with cell membranes. To investigate the effect of PM on the electrical properties of biological membranes, a series of experiments using a black lipid membrane (BLM) technique were performed. L-α-Phosphatidylcholine from soybean (azolectin) was used to create lipid bilayers. PM samples of different diameters (<4 (SRM-PM4.0) and <10 μm (SRM-PM10) were purchased from The National Institute of Standards and Technology (USA) to ensure the repeatability of the measurements. Lipid membranes with incorporated gramicidin A (5 pg/mL) ion channels were used to investigate the effect of PM on ion transport. The ionic current passing through the azolectin membranes was measured in ionic gradients (50/150 mM KCl on cis/trans side). In parallel, the electric membrane capacitance measurements, analysis of the conductance and reversal potential were performed. Our results have shown that PM at concentration range from 10 to 150 μg/mL reduced the basal ionic current at negative potentials while increased it at positive ones, indicating the interaction between lipids forming the membrane and PM. Additionally, PM decreased the gramicidin A channel activity. At the same time, the amplitude of channel openings as well as single channel conductance and reversal potential remained unchanged. Lastly, particulate matter at a concentration of 150 μg/mL did not affect the electric membrane capacity to any significant extent. Understanding the interaction between PM and biological membranes could aid in the search for effective cytoprotective strategies. Perhaps, by the use of an artificial system, we will learn to support the consequences of PM-induced damage. Full article

This study used an experimental design approach to optimize an HPLC method for the simultaneous determination of three pharmaceutical residues (triamcinolone, nystatin, and gramicidin) in industrial wastewater samples. The goal of using an experimental design approach was to maximize the method performance through separation enhancement and shortening the time of analysis and/or minimizing the environmental effects through the reduction in wastes and sample treatment. To achieve this goal, two steps were performed: a full factorial screening design for the three chromatographic variables, and optimization design using central composite design to select the optimum conditions that accomplished the highest resolution between adjacent peaks within a minimum run time of less than 5 min. The optimal chromatographic conditions derived from Minitab software using the desirability function were applied. Separation was carried out on a Zorbax C18 column (250 mm × 4.6, 5 μm) with gradient elution of a mobile phase composed of methanol and 0.25 M potassium dihydrogen phosphate buffer (pH 3.6) at different UV detections. For the validation of the developed HPLC method, ICH guidelines were followed, and the obtained results were found to be in compliance with the acceptance criteria. Linearity was over the concentration range of 1.00–25.00 μg/mL for triamcinilone and nystatin and 10.00–50.00 µg/mL for gramicidin. The proposed method was successfully applied to quantify the three studied pharmaceutical compounds in rinsing wastewater samples. Full article

Cationic and hydrophilic coatings based on casting and drying water dispersions of two different nanoparticles (NPs) onto glass are here described and evaluated for antimicrobial activity. Discoid cationic bilayer fragments (BF) surrounded by carboxy-methylcellulose (CMC) and poly (diallyl dimethyl ammonium) chloride (PDDA) NPs and spherical gramicidin D (Gr) NPs dispersed in water solution were cast onto glass coverslips and dried, forming a coating quantitatively evaluated against Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. From plating and colony forming units (CFU) counting, all strains interacting for 1 h with the coatings lost viability from 10⁵ to 10⁶, to zero CFU, at two sets of Gr and PDDA doses: 4.6 and 25 μg, respectively, or, 0.94 and 5 μg, respectively. Combinations produced broad spectrum, antimicrobial coatings; PDDA electrostatically attached to the microbes damaging cell walls, allowing Gr NPs interaction with the cell membrane. This concerted action promoted optimal activity at low Gr and PDDA doses. Further washing and drying of the deposited dried coatings showed that they were washed out so that antimicrobial activity was no longer present on the glass surface. Significant applications in biomedical materials can be foreseen for these transient coatings. Full article

Finding an effective drug to prevent or treat COVID-19 is of utmost importance in tcurrent pandemic. Since developing a new treatment takes a significant amount of time, drug repurposing can be an effective option for achieving a rapid response. This study used a combined in silico virtual screening protocol for candidate SARS-CoV-2 PL^pro inhibitors. The Drugbank database was searched first, using the Informational Spectrum Method for Small Molecules, followed by molecular docking. Gramicidin D was selected as a peptide drug, showing the best in silico interaction profile with PL^pro. After the expression and purification of PL^pro, gramicidin D was screened for protease inhibition in vitro and was found to be active against PL^pro. The current study’s findings are significant because it is critical to identify COVID-19 therapies that are efficient, affordable, and have a favorable safety profile. Full article

Gramicidin A (gA) is a linear antimicrobial peptide that can form a channel and specifically conduct monovalent cations such as H⁺ across the lipid membrane. The antimicrobial activity of gA is associated with the formation of hydroxyl free radicals and the imbalance of NADH metabolism, possibly a consequence caused by the conductance of cations. The ion conductivity of gramicidin A can be blocked by Ca²⁺ ions. However, the effect of Ca²⁺ ions on the antimicrobial activity of gA is unclear. To unveil the role of Ca²⁺ ions, we examined the effect of Ca²⁺ ions on the antimicrobial activity of gramicidin A against Staphylococcus aureus (S. aureus). Results showed that the antimicrobial mechanism of gA and antimicrobial activity by Ca²⁺ ions are concentration-dependent. At the low gA concentration (≤1 μM), the antimicrobial mechanism of gA is mainly associated with the hydroxyl free radical formation and NADH metabolic imbalance. Under this mode, Ca²⁺ ions can significantly inhibit the hydroxyl free radical formation and NADH metabolic imbalance. On the other hand, at high gA concentration (≥5 μM), gramicidin A acts more likely as a detergent. Gramicidin A not only causes an increase in hydroxyl free radical levels and NAD⁺/NADH ratios but also induces the destruction of the lipid membrane composition. At this condition, Ca²⁺ ions can no longer reduce the gA antimicrobial activity but rather enhance the bacterial killing ability of gramicidin A. Full article

Herein, we report that chromone-containing allylmorpholines can affect ion channels formed by pore-forming antibiotics in model lipid membranes, which correlates with their ability to influence membrane boundary potential and lipid-packing stress. At 100 µg/mL, allylmorpholines 1, 6, 7, and 8 decrease the boundary potential of the bilayers composed of palmitoyloleoylphosphocholine (POPC) by about 100 mV. At the same time, the compounds do not affect the zeta-potential of POPC liposomes, but reduce the membrane dipole potential by 80–120 mV. The allylmorpholine-induced drop in the dipole potential produce 10–30% enhancement in the conductance of gramicidin A channels. Chromone-containing allylmorpholines also affect the thermotropic behavior of dipalmytoylphosphocholine (DPPC), abolishing the pretransition, lowering melting cooperativity, and turning the main phase transition peak into a multicomponent profile. Compounds 4, 6, 7, and 8 are able to decrease DPPC’s melting temperature by about 0.5–1.9 °C. Moreover, derivative 7 is shown to increase the temperature of transition of palmitoyloleoylphosphoethanolamine from lamellar to inverted hexagonal phase. The effects on lipid-phase transitions are attributed to the changes in the spontaneous curvature stress. Alterations in lipid packing induced by allylmorpholines are believed to potentiate the pore-forming ability of amphotericin B and gramicidin A by several times. Full article

Gramicidin (Gr) nanoparticles (NPs) and poly (diallyl dimethyl ammonium) chloride (PDDA) water dispersions were characterized and evaluated against Gram-positive and Gram-negative bacteria and fungus. Dynamic light scattering for sizing, zeta potential analysis, polydispersity, and colloidal stability over time characterized Gr NPs/PDDA dispersions, and plating and colony-forming units counting determined their microbicidal activity. Cell viabilities of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in the presence of the combinations were reduced by 6, 7, and 7 logs, respectively, at 10 μM Gr/10 μg·mL⁻¹ PDDA, 0.5 μM Gr/0. 5μg·mL⁻¹ PDDA, and 0.5 μM Gr/0.5 μg·mL⁻¹ PDDA, respectively. In comparison to individual Gr doses, the combinations reduced doses by half (S. aureus) and a quarter (C. albicans); in comparison to individual PDDA doses, the combinations reduced doses by 6 times (P. aeruginosa) and 10 times (C. albicans). Gr in supported or free cationic lipid bilayers reduced Gr activity against S. aureus due to reduced Gr access to the pathogen. Facile Gr NPs/PDDA disassembly favored access of each agent to the pathogen: PDDA suctioned the pathogen cell wall facilitating Gr insertion in the pathogen cell membrane. Gr NPs/PDDA differential cytotoxicity suggested the possibility of novel systemic uses for the combination. Full article

Previous research found that the six major cyclodecapeptides from the tyrothricin complex, produced by Brevibacillus parabrevis, showed potent activity against chloroquine sensitive (CQS) Plasmodium falciparum. The identity of the aromatic residues in the aromatic dipeptide unit in cyclo-(D-Phe¹-Pro²-(Phe³/Trp³)-D-Phe⁴/D-Trp⁴)-Asn⁵-Gln⁶-(Tyr⁷/Phe⁷/Trp⁷)-Val⁸-(Orn⁹/Lys⁹)-Leu¹⁰ was proposed to have an important role in activity. CQS and resistant (CQR) P. falciparum strains were challenged with three representative cyclodecapeptides. Our results confirmed that cyclodecapeptides from tyrothricin had significantly higher antiplasmodial activity than the analogous gramicidin S, rivaling that of CQ. However, the previously hypothesized size and hydrophobicity dependent activity for these peptides did not hold true for P. falciparum strains, other than for the CQS 3D7 strain. The Tyr⁷ in tyrocidine A (TrcA) with Phe³-D-Phe⁴ seem to be related with loss in activity correlating with CQ antagonism and resistance, indicating a shared target and/or resistance mechanism in which the phenolic groups play a role. Phe⁷ in phenycidine A, the second peptide containing Phe³-D-Phe⁴, also showed CQ antagonism. Conversely, Trp⁷ in tryptocidine C (TpcC) with Trp³-D-Trp⁴ showed improved peptide selectivity and activity towards the more resistant strains, without overt antagonism towards CQ. However, TpcC lead to similar parasite stage inhibition and parasite morphology changes than previously observed for TrcA. The disorganization of chromatin packing and neutral lipid structures, combined with amorphous hemozoin crystals, could account for halted growth in late trophozoite/early schizont stage and the nanomolar non-lytic activity of these peptides. These targets related to CQ antagonism, changes in neural lipid distribution, leading to hemozoin malformation, indicate that the tyrothricin cyclodecapeptides and CQ share a target in the malaria parasite. The differing activities of these cyclic peptides towards CQS and CQR P. falciparum strains could be due to variable target interaction in multiple modes of activity. This indicated that the cyclodecapeptide activity and parasite resistance response depended on the aromatic residues in positions 3, 4 and 7. This new insight on these natural cyclic decapeptides could also benefit the design of unique small peptidomimetics in which activity and resistance can be modulated. Full article

The soil-borne species Aneurinibacillus migulanus (A. migulanus) strains Nagano and NCTC 7096 were shown to be potent biocontrol agents active against several plant diseases in agricultural and forest ecosystems. Both strains produce the cyclic peptide gramicidin S (GS) that was described as the main weapon inhibiting some gram-negative and gram-positive bacteria and fungus-like organisms along with the production of biosurfactant and hemolysis activities. However, the contribution of the cyclic peptide gramicidin S (GS) to the biocontrol ability of A. migulanus has never been studied experimentally. In this paper, using a mutant of the A. migulanus Nagano strain (E1 mutant) impaired in GS biosynthesis we evaluated the contribution of GS in the biocontrol potential of A. migulanus against Phytophthora spp. The two strains of A. migulanus, Nagano and NCTC 7096, were tested in a pilot study for the inhibition of the growth of 13 Phytophthora species in dual culture assays. A. migulanus Nagano was significantly more inhibitory than NCTC 7096 to all species. Additionally, using apple infection assays, P. rosacearum MKDF-148 and P. cryptogea E2 were shown to be the most aggressive on apple fruits displaying clear infection halos. Therefore, the three A. migulanus strains, Nagano, NCTC 7096, and E1, were used in apple infection experiments to check their effect on infection ability of these two Phytophthora species. Treatment with A. migulanus Nagano significantly reduced the severity of symptoms in apple fruits compared with NCTC 7096. A. migulanus E1 mutant showed total loss of biocontrol ability suggesting that GS is a major actor in the biocontrol ability of A. migulanus Nagano strain. Full article