Effects of Piptoporus betulinus ethanolic extract on the proliferation and viability of melanoma cells and models of their cell membranes

Piptoporus betulinus is a fungus known for its medicinal properties. It possesses antimicrobial, anti-inflammatory, and anti-cancer activity. In this study, several tests were performed to evaluate the cytotoxic effect of the ethanolic extract of Piptoporus betulinus on two melanoma human cell lines, WM115 primary and A375 metastatic cell lines, as well as Hs27 human skin fibroblasts. The extract proved to affect cancer cells in a dose-dependent manner, and at the same time showed a low cytotoxicity towards the normal cells. The total phenolic content (TPC) was determined spectrophotometrically by the Folin-Ciocalteu method (F-C), and the potential antioxidant activity was measured by ferric-reducing antioxidant power (FRAP) assay. One of the active compounds in the extract is betulin. It was isolated and then its cytotoxic activity was compared to the results obtained from the Piptoporus betulinus extract. To further understand the mechanism of action of the extract’s anticancer activity, tests on model cell membranes were conducted. A model membrane of a melanoma cell was designed and consisted of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, disialoganglioside-GD(1a) and cholesterol: DMPC:GD(1a):chol (5:2:3 mole ratio). Changes in a Langmuir monolayer were observed and described based on Π-A(mol) isotherm and compressibility modulus changes. LB lipid bilayers were deposited on a hydrophilic gold substrate and analyzed by IR and X-ray photoelectron spectroscopy. Our study provides new data on the effect of Piptoporus betulinus extract on melanoma cells and its impact on the model of melanoma plasma membranes

Study of the potential driven changes in a collagen film self-assembled on a polycrystalline gold electrode surface

International audienceThe influence of the potential applied to the polycrystalline gold electrode on the adsorption state and structure of collagen molecules were studied by means of electrochemistry, in situ ellipsometry and in situ polarization modulation infrared reflection-absorption spectroscopy. At the macroscopic level, potential and the corresponding charge accumulated on the gold electrode determine the adsorption process of the collagen film on the Au electrode surface. The protein film is stable on the electrode surface at potentials close to the potential of zero charge. The protein film sustains a large negative potential drop (Delta phi(M vertical bar S) >= -0.5 V) whereas it is unstable at a small positive potential drop (Delta phi(M vertical bar S) approximate to 0.1 V) across the film. Positive net charge accumulated on the Au surface causes electrostatic repulsions of collagen molecules bearing a positive net charge. Loosening of water and destabilization of the protein film reflect repulsions between the Au electrode and the collagen molecules. In contrast, at charge densities between -8 < sigma(M) < -20 mu C cm(-2) electrostatic attractions between the electrode surface and the protein molecule appear. A stable, well hydrated collagen film is formed on the Au surface. Higher negative charges accumulated on the electrode surface lead to swelling on the protein film by water and lead to the desorption of the protein film from the Au surface. In situ spectroelectrochemical studies show that at the molecular level neither the secondary structure nor the orientation of collagen molecules are affected by electrical potentials. Independently of the applied potential and electric fields acting on the film the collagen molecule maintains its native structure. Collagen molecules adsorbed on the Au surface form a heterogeneous film with well-defined structure and unusual stability at the molecular level

A Langmuir monolayer study of the action of phospholipase A2 on model phospholipid and mixed phospholipid-GM1 ganglioside membranes

Polarization-modulation infrared reflection-absorption spectroscopy, surface pressure measurements and thermodynamic analysis were used to study enzymatic hydrolysis of lipid monolayers at the air/water interface. The Ca2+-requiring pork pancreatic phospholipase A2 was used as a catalyst. The substrates were pure 1,2-dilauroyl-sn-glycero-3-phosphocholine or mixed 1,2-dilauroyl-sn-glycero-3-phosphocholine – monosialotetrahexosylganglioside Langmuir films. The physicochemical properties of the monolayers were established with the aim of a correlation with enzyme activity. The infrared spectra were acquired upon the advancement of the catalysis; the latter was studied at a controlled surface pressure and area of the film. Changes of the intensity and frequency of different infrared signals characteristic for the two lipids were correlated with modification of the properties of the monolayer due to hydrolysis. The amide I signal characteristic for peptides permitted detecting the enzyme adsorbed at the interface. The thermodynamic and infrared results indicate that monosialotetrahexosylganglioside increases H-bonding of the lipid polar heads in the films. This effect, which may be responsible for the low activity of phospholipase A2 in the mixed films, could be used for developing enzyme-resistant lipid systems

Submolecular Structure and Orientation of Oligonucleotide Duplexes Tethered to Gold Electrodes Probed by Infrared Reflection Absorption Spectroscopy: Effect of the Electrode Potentials

Unique electronic and ligand recognition properties of the DNA double helix provide basis for DNA applications in biomolecular electronic and biosensor devices. However, the relation between the structure of DNA at electrified interfaces and its electronic properties is still not well understood. Here, potential-driven changes in the submolecular structure of DNA double helices composed of either adenine-thymine (dAdT)₂₅ or cytosine-guanine (dGdC)₂₀ base pairs tethered to the gold electrodes are for the first time analyzed by <i>in situ</i> polarization modulation infrared reflection absorption spectroscopy (PM IRRAS) performed under the electrochemical control. It is shown that the conformation of the DNA duplexes tethered to gold electrodes via the C₆ alkanethiol linker strongly depends on the nucleic acid sequence composition. The tilt of purine and pyrimidine rings of the complementary base pairs (dAdT and dGdC) depends on the potential applied to the electrode. By contrast, neither the conformation nor orientation of the ionic in character phosphate–sugar backbone is affected by the electrode potentials. At potentials more positive than the potential of zero charge (pzc), a gradual tilting of the double helix is observed. In this tilted orientation, the planes of the complementary purine and pyrimidine rings lie ideally parallel to each other. These potentials do not affect the integral stability of the DNA double helix at the charged interface. At potentials more negative than the pzc, DNA helices adopt a vertical to the gold surface orientation. Tilt of the purine and pyrimidine rings depends on the composition of the double helix. In monolayers composed of (dAdT)₂₅ molecules the rings of the complementary base pairs lie parallel to each other. By contrast, the tilt of purine and pyrimidine rings in (dGdC)₂₀ helices depends on the potential applied to the electrode. Such potential-induced mobility of the complementary base pairs can destabilize the helix structure at a submolecular level. These pioneer results on the potential-driven changes in the submolecular structure of double stranded DNA adsorbed on conductive supports contribute to further understanding of the potential-driven sequence-specific electronic properties of surface-tethered oligonucleotides

Local control of protein binding and cell adhesion by patterned organic thin films

Control of the cell adhesion and growth on chemically patterned surfaces is important in an increasing number of applications in biotechnology and medicine, for example implants, in-vitro cellular assays, and biochips. This review covers patterning techniques for organic thin films suitable for site-directed guidance of cell adhesion to surfaces. Available surface patterning techniques are critically evaluated, with special emphasis on surface chemistry that can be switched in time and space during cultivation of cells. Examples from the authors' laboratory include the use of cell-repellent self-assembled monolayers (SAM) terminated by oligoethylene glycol (OEG) units and the lifting of the cell repellent properties by use of electrogenerated Br2/HOBr which can be performed with positionable microelectrodes. Structural changes of the SAM were analyzed by polarization-modulated infrared reflection absorption spectroscopy (PM IRRAS). Use of a soft array system of individually addressable microelectrodes enables formation of flexible and complex patterns in a short time and has the potential for further acceleration of probe-induced local manipulation of cell adhesion

How do Antimicrobial Peptides Interact with the Outer Membrane of Gram-Negative Bacteria? Role of Lipopolysaccharides in Peptide Binding, Anchoring, and Penetration

Gram-negative bacteria possess a complex structural cell envelope that constitutes a barrier for antimicrobial peptides that neutralize the microbes by disrupting their cell membranes. Computational and experimental approaches were used to study a model outer membrane interaction with an antimicrobial peptide, melittin. The investigated membrane included di[3-deoxy-d-manno-octulosonyl]-lipid A (KLA) in the outer leaflet and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in the inner leaflet. Molecular dynamics simulations revealed that the positively charged helical C-terminus of melittin anchors rapidly into the hydrophilic headgroup region of KLA, while the flexible N-terminus makes contacts with the phosphate groups of KLA, supporting melittin penetration into the boundary between the hydrophilic and hydrophobic regions of the lipids. Electrochemical techniques confirmed the binding of melittin to the model membrane. To probe the peptide conformation and orientation during interaction with the membrane, polarization modulation infrared reflection absorption spectroscopy was used. The measurements revealed conformational changes in the peptide, accompanied by reorientation and translocation of the peptide at the membrane surface. The study suggests that melittin insertion into the outer membrane affects its permeability and capacitance but does not disturb the membrane’s bilayer structure, indicating a distinct mechanism of the peptide action on the outer membrane of Gram-negative bacteria