Marek Langner

Anthocyanin Extracts with Antioxidant and Radical Scavenging Effect

Abstract The antioxidative activity of three anthocyanin pigments, extracted from the fruits of chokeberry, honeysuckle and sloe, were studied. Lipid oxidation in the liposome membrane, induced by UV radiation, was evaluated with a thiobarbituric acid-reactive substances assay. The antioxidant efficiency of the studied compounds follows this sequence: chokeberry > sloe > honeysuckle. The extract concentrations at which a 50% reduction of phosphatidylcho line oxidation was observed, were respectively: 48, 54 and 60 mg/1. The end products of lipid membrane oxidation were evaluated using HPLC. It was found that the antioxidative potency of anthocyanin extracts is concentration-dependent. As shown by E P R technique the efficiency of the extracts to eliminate free radicals from the solution follows the order of the antioxidant activity.

The application of fluorescence correlation spectroscopy in detecting DNA condensation

We report the application of fluorescence correlation spectroscopy (FCS) in characterizing conformational changes (condensation) of chemically well-defined DNA plasmids. The plasmids: pHbetaAPr-1-neo (10 kbp, contour length 3.4 microm) and pBluescript SKt (2.96 kbp, contour length 1.02 microm) were imaged by a confocal fluorescence microscope using two fluorescent probes: ethidium bromide (EtBr) and propidium iodide (PrIo). It became clear that the DNA molecule exhibits discrete conformational change between the coil and globule states with the addition of a small amount (the order of magnitude being 10(-5) M) of cationic surfactant, spermine and hexadecyltrimethyl ammonium bromide (HTAB). When the concentrations of both condensing agents are smaller than 6.0x10(-6) M and 2.0 x 10(-6) M for the 10 and 2.96 kbp, both plasmids are in the extended coil state with diffusion constants D(10 kbp)=9.6 x 0(-13) m(2) s(-1) and D(2.96 kbp)=2.5x10(-12) m(2) s(-1), respectively. When the condensing agent in a concentration higher than 1.10 x 10(-5) M is added to pHbetaAPr-1-neo (10 kbp), plasmids are in the condensed globular state and their diffusion constants are D(10 kbp)=8.0 x 10(-12) m(2) s(-1) (spermine) and D(10 kbp)=5.5x10(-12) m(2) s(-1) (HTAB). The globular state of the pBluescript SKt (2.96 kbp) plasmids is characterized by diffusion constants equal to D(2.96 kbp)=9.2x10(-12) m(2) s(-1) (spermine) and D(2.96 kbp)=8.2x10(-12) m(2) s(-1) (HTAB).

Effect of phenyltin compounds on lipid bilayer organization

Phenyltin compounds are known to be biologically active. Their chemical structure suggests that they are likely to interact with the lipid fraction of cell membranes. Using fluorescence and NMR techniques, the effect of phenyltin compounds on selected regions of model lipid bilayers formed from phosphatidylcholine was studied. The polarization of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) dipalmitoyl-L-phosphatidylethanolamine and desorption of praseodymium ions was used to probe the polar region, whereas the polarization of 1 - (4 - trimethylammoniumphenyl) - 6 - phenyl -1,3,5-hexatriene p-toluenesulfonate measured the hydrophobic core of the membrane. In addition, changes in the N-(5-fluoresceinthiocarbanoly)dipalmitoyl - L - α - phosphatidyl -ethanolamine fluorescence intensity indicated the amount of charge introduced by organotin compounds to the membrane surface. There were no relevant changes of measured parameters when tetraphenyltin was introduced to the vesicle suspension. Diphenyltin chloride causes changes of the hydrophobic region, whereas the triphenyltin chloride seems to adsorb in the headgroup region of the lipid bilayer. When the hemolytic activity of phenyltin compounds was measured, triphenyltin chloride was the most effective whereas diphenyltin chloride was much less effective. Tetraphenyltin causes little damage. Based on the presented data, a correlation between activity of those compounds to hemolysis (and toxicity) and the location of the compound within the lipid bilayer could be proposed. In order to inflict damage on the plasma membrane, the compound has to penetrate the lipid bilayer. Tetraphenyltin does not partition into the lipid fraction; therefore its destructive effect is negligible. The partition of the compound into the lipid phase is not sufficient enough, by itself, to change the structure of the lipid bilayer to a biologically relevant degree. The hemolytic potency seems to be dependent on the location of the compound within the lipid bilayer. Triphenyltin chloride which adsorbs on the surface of the membrane, causes a high level of hemolysis, whereas diphenyltin chloride, which penetrates much deeper, seems to have only limited potency.

Propidium Iodide and PicoGreen as Dyes for the DNA Fluorescence Correlation Spectroscopy Measurements

Many experimental designs, in which nucleic acid conformational changes are of interest, require reliable fluorescence labeling. The appropriate fluorescence probe should have suitable optical properties and, more importantly, should not interfere with the investigated processes. In order to avoid chemical modifications the fluorescence label needs to be associated with nucleic acid via weak non-covalent interactions. There are a number of fluorescent probes that change their fluorescent properties (i.e. their quantum yield and/or spectral characteristics) upon association with nucleic acid. Such probes are frequently used to detect, visualize and follow processes involving nucleic acid and its conformational changes. In order to obtain reliable data regarding macromolecule or aggregate topology a detailed knowledge of probe–nucleic acid interactions on the molecular level is needed. In this paper we show that the association of propidium iodide with DNA alters its conformation and that it selectively labels plasmid fragments and/or its subpopulations in a concentration-dependent meaner. Another dye, PicoGreen, exhibits better properties. It labels nucleic acid uniformly and without any concentration-dependent artifacts.

Fluorescence Techniques for Determination of the Membrane Potentials in High Throughput Screening

The characterization of small molecules requires identification and evaluation of several predictive parameters, when selecting compounds for pharmacological applications and/or determining their toxicity. A number of them are correlated with the compound interaction with biological membranes and/or capacity to cross them. The knowledge of the extent of adsorption, partition coefficient and permeability along with the compound ability to alter membrane properties are critical for such studies. Lipid bilayers are frequently used as the adequate experimental models of a biological membrane despite their simple structure and a limited number of components. A significant number of the biologically relevant lipid bilayer properties are related to its electrostatics. Three electrostatic potentials were defined for the lipid bilayer; the intrinsic or induced surface electrostatic potential, the dipole potential and the membrane potential. Each of them was measured with dedicated methodologies. The complex measurement protocols and technically demanding instrumentation made the development of efficient HTS approaches for complete characterization of membrane electrostatics practically impossible. However, the rapid development of fluorescence techniques accompanied by rapid growth in diversity and number of dedicated fluorescent probes enabled characterization of lipid bilayer electrostatics in a moderately simple manner. Technically advanced, compact and automated workstations, capable of measuring practically all fluorescence parameters, are now available. Therefore, the proper selection of fluorescent probes with measuring procedures can be designed to evaluate drug candidates in context of their ability to alter membrane electrostatics. In the paper we present a critical review of available fluorescence methods, useful for the membrane electrostatics evaluation and discuss the feasibility of their adaptation to HTS procedures. The significance of the presented methodology is even greater considering the rapid growth of advanced drug formulations, where electrostatics is an important parameter for production processes and pharmacokinetics of the product. Finally, the potential of the membrane electrostatics to emerge as a viable pharmacological target is indicated and fluorescence techniques capable to evaluate this potential are presented.

The effect of spermine on plasmid condensation and dye release observed by fluorescence correlation spectroscopy.

Abstract We demonstrate that fluorescence correlation spectroscopy (FCS) can be employed to follow the conformational changes of DNA molecules induced by the addition of a cationic condensing compound (spermine). In our experiments the plasmid pHβAPr-1-neo (10 kbp; contour length 3.4 m) was labeled with propidium iodide (PrIo) and then titrated with spermine to induce its condensation. When spermine was applied at concentrations above 5 M (spermine/DNAphosphate =0.375), the diffusion time of the labeled plasmid dropped from 15 ms down to 3 ms (its diffusion coefficient, D, increased from 1.010 exp 12 square m/s to 6.010 exp 12 square m/s). The application of spermine was also accompanied by decreasing count rate and particle number, reflecting the dyes dissociation. The data presented show that FCS may become a valuable tool in studying supramolecular aggregate formation, especially when association is followed by a change in the hydrodynamic size of the resulting complex.

Adsorption of Phenyltin Compounds onto Phosphatidylcholine/Cholesterol Bilayers

Phenyltin compounds are known to be biologically active and, whan widely spread, are potentially hazardous. As their chemical structure suggests, they interact with the lipid fraction of the cell membrane. Their effect on the model phosphatidylcholine/cholesterol bilayer has been studied using fluorescence and 1H NMR techniques. The change in the fluorescein-PE fluorescence intensity indicates the amount of charge added by phenyltin compounds to the membrane surface. Although the presence of cholesterol alone does not alter membrane interface properties measured with fluorescein-PE, 1H NMR measurements show that lipid mobility is altered throughout the hydrophobic core of the membrane. Cholesterol in the phosphatidylcholine bilayer does not alter tetraphenyltin interaction with the membrane, though the effect of diphenyltin dichloride, penetrating deeply into the hydrophobic core of the membrane, is reduced when the amount of cholesterol in the membrane is increased, suggesting decreased compound adsorption. Triphenyltin chloride has a qualitatively different effect on the lipid bilayer, when observed using this fluorescence technique. The adsorption of triphenyltin onto the phosphatidylcholine/cholesterol membrane induces a lateral phase separation of membrane components. Since triphenyltin chloride is known to be adsorbed onto the interface of the lipid bilayer, this separation mechanism must originate in this region and does not seem to be electrostatic in origin. 1H NMR measurements have confirmed the observation that these two active phenyltin compounds interact with the phosphatidylcholine/cholesterol membrane differently, disrupting different regions of the bilayer to a different degree. Copyright

Combined Effect of Surface Electrostatic Charge and Poly(ethyl glycol) on the Association of Liposomes with Colon Carcinoma Cells

Particulate drug formulations are considered to be a means that may improve the pharmacokinetics and biodistribution of active compounds. By using them, drug distribution is determined solely by the properties of the carrier. The surface properties of such supramolecular aggregates determine how they will interact with various biological structures. Among others, surface electrostatic charge and surface grafted polymers are considered to be among the major factors affecting its interaction with proteins and cells. In this article, we present experimental evidence that properly selected surface electrostatic charge and grafted polymers can alter the association of liposomes with colon cancer cells. The dependence of the adsorption of liposomes onto the cell surface on the quantity and length of surface grafted polymers for a certain surface charge density exhibits a distinct maximum. For example, when liposomes were formed with 20 mol% of DOTAP, PE-PEG350 increased liposome adsorption by up to 6 mol%. This adsorption maximum depends on both polymer length and charge type. Results presented in this article show that the interaction of liposomes with colon cancer cells can be tuned by a proper combination of liposome surface electrostatics and surface grafted polymers.

Lipid-binding role of βII-spectrin ankyrin-binding domain

It is known that erythroid and non‐erythroid spectrins binding of vesicles and monolayers containing PE proved sensitive to inhibition by red blood cell ankyrin. We now show that the bacterially‐expressed recombinant peptides representing βII(brain)‐spectrins ankyrin‐binding domain and its truncated mutants showed lipid‐binding activity, although only those containing a full‐length amino terminal fragment showed high to moderate affinity towards phospholipid mono‐ and bilayers and a substantial sensitivity of this binding to inhibition by ankyrin. These results are in accordance with our published data on βI‐spectrins ankyrin‐binding domain [Hryniewicz‐Jankowska A, et al. Mapping of ankyrin‐sensitive, PE/PC mono‐ and bilayer binding site in erythroid beta‐spectrin. Biochem J 2004;382:677–85]. Moreover, we tested also the effect of transient transfection of living cells of several cell‐lines with vectors coding for GFP‐conjugates including βII and also βI full‐length ankyrin‐binding domain and their truncated fragments on the membrane skeleton organization. The transfection with constructs encoding full‐length ankyrin‐binding domain of βII and βI spectrin resulted in increased aggregation of membrane skeleton and its punctate appearance in contrast to near normal appearance of membrane skeleton of cells transiently transfected with GFP control or construct encoding ankyrin‐binding domain truncated at their N‐terminal region. Our results therefore indicate the importance of N‐terminal region for lipid‐binding activity of the β‐spectrin ankyrin‐binding domain and its substantial role in maintaining the spectrin‐based skeleton distribution.

The effect of the lipid-binding site of the ankyrin-binding domain of erythroid β-spectrin on the properties of natural membranes and skeletal structures

It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of β-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482–94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na+K+ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the β-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.