Specific interaction of arabinose residue in ginsenoside with egg phosphatidylcholine vesicles

The interaction of the specific sugar residue in ginsenosides with egg phosphatidylcholine vesicles was investigated by ESR spectrometry using phosphatidic acid spin-labeled at the polar head groups. Ginsenoside-Rc, which has an α-l-arabinofuranose residue and agglutinability toward egg yolk phosphatidylcholine vesicles (Fukuda, K. et al. (1985) Biochim. Biophys. Acta 820, 199–206), caused the restriction of the segmental motion of spin-labeled phosphatidic acid in egg phosphatidylcholine vesicles, indicating that the saponin interacted with the polar head groups of vesicles. Other ginsenosides-Rb₂, Rb₁, Rd and p-nitrophenyl glycoside derivatives which have less or no agglutinability were also investigated in the same manner. Only ginsenoside-Rb₂ and p-nitrophenyl α-l-arabinofuranoside which have the specific sugar residue (arabinose) showed a strong interaction with the polar head groups of vesicles. To gain an insight into the mechanism of agglutination by ginsenoside-Rc, the interaction with the fatty acyl groups was also studied by using phosphatidylcholine spin-labeled at the fatty acyl groups. Ginsenoside-Rc increased the order parameter of the spin-labeled phosphatidylcholine, indicating that the saponin was inserted into lipid bilayers. In other saponins investigated, only ginsenoside-Rb₂ interacted with the fatty acyl part of vesicles. The process of expression of agglutination by ginsenoside-Rc was discussed on the basis of the ESR studies.     

Lateral mobility of phospholipid molecules in thin liquid films

The Fluorescence Recovery After Photobleaching (FRAP) method was applied to measure the lateral mobility of the fluorescent lipid analog, dioctadecylindocarbocyanine perchlorate (Dil-C18), in microscopic thin liquid films (Foam Films (FFs)). The foam film structures were comprised of two phosphatidylcholine monolayers adsorbed at air/water interfaces which sandwiched a thin liquid core. Lateral diffusion of the DiI molecules in the plane of the monolayers was determined as a function of the thickness of the thin liquid core of the film between the FF monolayers. The results obtained indicated that the diffusion coefficient was strongly dependent both on the distance between the FF monolayers in the range 4 nm to 85 nm (corresponding to the FF thickness) and on the film type. The applicability of the FRAP method for studying the molecular mobility in phospholipid FFs was demonstrated. Considerable differences in the surface diffusion coefficient of Dil were observed, ranging between 2 × 10^–8 cm²/s and 22 × 10^–8 cm²/s in so called yellow, gray, common black and Newton black FFs. The effect of the presence of polyethylene glycol (PEG-400) in the liquid core of lecithin FFs on surface diffusion was also studied. The surface diffusion results from the FF studies were compared with data from black lipid membranes (BLMs). These structures are related in thickness terms but the molecular orientation in FFs is the reverse of that in BLMs. Correspondence to: Z. Lalchev     

Biosensor based on enzyme-catalysed degradation of thin polymer films

A biosensor based on the enzyme-catalysed dissolution of biodegradable polymer films has been developed. Three polymer-enzyme systems were investigated for use in the sensor: a poly(ester amide), which is degraded by the proteolytic enzyme alpha-chymotrypsin; a dextran hydrogel, which is degraded by dextranase; and poly(trimethylene) succinate, which is degraded by a lipase. Dissolution of the polymer films was monitored by Surface Plasmon Resonance (SPR). The rate of degradation was directly related to enzyme concentration for each polymer/enzyme couple. The poly(ester amide)/alpha-chymotrypsin couple proved to be the most sensitive over a concentration range from 4 x 10(-11) to 4 x 10(-7) mol l(-1) of enzyme. The rate of degradation was shown to be independent of the thickness of the poly(ester amide) films. The dextran hydrogel/dextranase couple was less sensitive than the poly(ester amide)/alpha-chymotrypsin couple but showed greater degradation rates at low enzyme concentrations. Enzyme concentrations as low as 2 x 10(-11) mol l(-1) were detected in less than 20 min. Potential fields of application of such a sensor system are the detection of enzyme concentrations and the construction of disposable enzyme based immunosensors, which employ the polymer-degrading enzyme as an enzyme label.     

Elongated films of polymer-dispersed liquid crystals as scattering polarizers

Elongated films of polymer dispersed liquid crystals have been experimentally investigated. The dependence of transmittance light polarization on elongation coefficient and temperature has been studied. The possibilities of practical applications of elongated films of polymer dispersed liquid crystals are discussed.     

Direct measurement of the interaction of carnosine and its analogs with free radicals]

An ESR study of interactions of carnosine and its derivatives with free radicals has been carried out. In model systems the spin trap OH. radical adduct generation has been shown to decrease significantly in the presence of carnosine in a pronounced concentration-dependent manner. A comparative study of effects of some other histidine-containing dipeptides on this process has revealed a similarity in anserine, homocarnosine, and acetylcarnosine actions.     

An EPR study of the interfacial properties of phosphatidylcholine vesicles with different lipid chain lengths

The hydrophobic spin probe 2,2,6,6-tetramethyl-piperidin-1-oxyl-4-yl octadecanoate (TEMPO-stearate) is used to study the interfacial properties of a variety of phosphatidylcholine vesicles. Since the spin probe exhibits a fast motional electron paramagnetic resonance (EPR) spectrum above the phase transition, the EPR spectrum of the spin probe is analyzed by nonlinear least-squares spectral fitting. EPR spectral line fitting provides high precision spectral parameters, which can be used to construct a detailed picture of the dynamics of the probe and its environment. The hyperfine coupling spacing is used to estimate the effective water concentration in the polar shell of vesicles, while the rotational correlation times give the information on the motion of the spin probe. The effective water concentration of the polar shell of dimyristoyl-phosphatidylglycerol (DMPG) vesicles is greater on average by about 4.0M than the effective water concentration of the polar shell of dimyristoyl-phosphatidylcholine (DMPC) vesicles. The effective water concentration decreases by about 0.5M for an increase of two carbons in the chain, and increases noticeably with hydrocarbon chain unsaturation, which is in good agreement with literature values. The nitroxide moiety rotates preferentially along the N-O bond, that is, parallel to its hydrocarbon chain.     

Interaction of gentamicin with phosphatidylserine/phosphatidylcholine mixtures in adsorption monolayers and thin liquid films: morphology and thermodynamic properties

Gentamicin possesses strong adverse actions like oto and nephrotoxicity. The latter is a result of strong gentamicin–acid phospholipid interactions, resulting in cell fusion, fission, etc., ions as calcium interact with gentamicin and effectively deter its toxicity. In this work, the interactions of gentamicin and Ca²⁺ with phosphatidylserine/phosphatidylcholine (PS/PC) mixtures of different ratio are experimentally characterized. Special attention is paid to bridge thermodynamic and morphological properties of adsorption monolayers and thin liquid films (TLFs) composed of these lipid mixtures. Our results show that gentamicin decreases the stability of common black TLFs formed of pure PS coupled with suppression of lipid surface adsorption to the monolayers at the air–water interface; also, gentamicin reveals effects of lowering of lipid spreading on the interface and significant loss of material during monolayer cycling, increase of condensed phase, and organization of dense net-like domain monolayer texture. Gentamicin addition results in opposite effects for films formed of DPPC/PS (95:5) mixture. It increases the stability of Newton black TLFs formed by DPPC/PS correlated with faster and stronger surface adsorption and better surface spreading; also, gentamicin lowers the amount of condensed phase and organization of domains of smaller size. We also showed that Ca²⁺ itself decreases the stability of common black TLFs formed of PS accompanied with weaker surface adsorption, formation of higher amounts of condensed phase and organization of domains. In our experiments, Ca²⁺ softens, even deters, the effects of gentamicin on both PS and DPPC/PS films.     

Direct observation of the distribution of fluorescent probes in phosphatidylcholine/cholesterol vesicles using flow microfluorometry

The technique of flow microfluorometry has been extended to the study of small lipid complexes to assess either the lipid (hydrophobic) or aqueous (hydrophilic) compartments of selected natural or model membrane systems. sn-1-Palmitoyl-sn-2-oleoyl-phosphatidylcholine/cholesterol unilamellar vesicles, averaging 268 nm in diameter and containing varying concentrations of the synthetic lipophile probe, sn-1-palmitoyl-sn-2-12-[N-4-nitrobenzo-2-oxa-1,3-diazole]-aminocaproyl-phosphatidylcholine (NBD-PC), were analyzed using an Ortho Series 50-H Cytofluorograf and an Ortho 2150 computer system. NBD-labeled vesicles were analyzed for green fluorescence and the intensity of scattered light, the later being analyzed both at low angle (2–5°) and at 90° to the incident beam. At the high amplification required for vesicle detection, background signals from the sheath buffer, nonspecific laser light, and electronic noise were observed. However, this background noise signal was removed by appropriately setting a discriminator window. Profiles of signals falling within this region were then constructed. For the settings selected, more than 98% of data recorded could be attributed to observations on vesicles. Size information from the intensity of scattered light was obtained by comparison of the sample with fluorescent microspheres after correcting for the particle-scattering function difference between hollow and solid spheres and for refractive index differences. Additionally, cytograms and profiles were constructed for vesicles containing 5 m 6-carboxyfluorescein, 3′,6′-dihydroxy-3-oxospiro(isobenzofuran-1 (3H),9′-(9H)xanthen)-6-carboxylic acid, trapped in the aqueous core. Thus, the utility of flow microfluorometry has been extended to much smaller particle populations than studied previously by this technique. It has significant potential for studying several important properties of selected populations of vesicles and lipoproteins including (i) the size and fluorescence distribution of particles, (ii) the equilibrium distribution of probes among different size populations and among different domains within populations, (iii) the time dependence of probe transfer from a specific labeled population to a specific unlabeled population, (iv) the time dependence of vesicle fusion (combining aqueous compartments), and (v) sorting particles which are labeled differently.     

High-resolution micromechanical measurement in real time of forces exerted by living cells

The aim of this study was to compare uniaxial traction forces exerted by different cell types using a novel sensor design and to test the dependence of measured forces on cytoskeletal integrity. The sensor design detects forces generated between 2 contact points by cells spanning a gap. The magnitude of these forces varied according to cell type and were dependent on cytoskeletal integrity. The response time for drug-induced cytoskeletal disruption also varied between cell types: dermal fibroblasts exerted the greatest forces and had the slowest drug response times; EBV-transformed epithelial cells also had slow cytoskeletal depolymerisation times but exerted the lowest forces overall. Conversely, lung epithelial tumor cells exerted low forces but had the fastest depolymerisation drug response. These results provide proof of principle for a new design of force-measurement sensor based on optical interferometry, an approach that can be used to study cytoskeletal dynamics in real time.     

Inhibition of phosphatidylcholine synthesis is associated with excitotoxic cell death in cerebellar granule cell cultures

Summary.  Glucose deprivation (GD) enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. Neither 70 min of GD, a treatment that depletes cell energy resources, nor exposure to 20 μM glutamate (GLU) for 30 min, induce significant cell death in cultures of cerebellar granule cells. However, the combined treatment with GLU and GD induces choline (Cho) release before excitotoxic cell death. We investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (PC) synthesis. We found that exposure to GLU for 30 min, to GD for 70 min, and to the combination of both, inhibited PC synthesis at the end of treament by 71%, 92% and 91%, respectively. The inhibition of PC synthesis was accompanied by a decrease in the incorporation of [³H]Cho into phosphocholine and by an increase of the intracellular content of free [³H]Cho, indicating that these treatments inhibit the synthesis of PC by inhibiting choline kinase activity. However, only the combined treatment with GLU and GD induced a prolonged inhibition of PC synthesis that extented after the end of treatment. These results show that excitotoxic death is associated with sustained inhibition of PC synthesis and suggest that this effect of the combined treatment with GLU and GD on PC synthesis is produced by an action on an enzymatic step downstream of choline kinase activity. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

Transition of a liquid crystalline phosphatidylcholine bilayer to the gel phase in a vesicle reduces the internal aqueous volume

The liquid crystalline to gel phase transition in phospholipid bialyers is associated with a marked reduction in the area per phospholipid molecule. Geometric considerations based on published data suggest that this decrease in molecular area is accompanied by a reduction in the internal aqueous volume trapped within a unilamellar bilayer vesicle. This volume reduction, which depends upon the shape of the vesicle, is shown to be between 23 and 60 percent. We have observed a 25 to 30 percent reduction in the internal aqueous volume of unilamellar vesicles about 700 Å in diameter formed from dipalmitoylphosphatidylcholine using the self-quenching of 6-carboxyfluorescein trapped within this compartment.     

The transbilayer movement of phosphatidylcholine in vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane

Vesicles have been prepared from 18 : 1_c/18 : 1_c-phosphatidylcholine with or without purified glycophorin or partially purified band 3 (obtained by organomercurial gel chromatography). The vesicles have been characterized by freeze-fracture electron microscopy, binding studies to DEAE-cellulose, ³¹P-NMR and K⁺ trap measurements. Pools of phosphatidylcholine available for exchange have been investigated using phosphatidylcholine exchange protein from bovine liver. The protein-containing vesicles both exhibit exchangeable pools larger than the fraction of phosphatidylcholine in the outer monolayer, whereas in the protein-free vesicles the exchangeable pool is consistent with the outer monolayer. The results indicate that both glycophorin and the partially purified band 3 preparation enhance the transbilayer movement of phosphatidylcholine.     

Applications of thin layer chromatography,high performance liquid chromatography and mass spectrometry in the fermentation and isolation of the antibiotic nybomycin

Summary Thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and mass spectrometry (MS) methods have been developed for the analysis of the antibiotic nybomycin, its derivatives deoxynybomycin and nybomycin acetate, during the fermentation and isolation of nybomycin. Using a quantitative HPLC based assay, the time course of nybomycin production (nybomycin titers) in 1000 liter fermentations was determined. Desorption chemical ionization mass spectrometry (DCI/MS) of standard nybomycin samples, fermentation broth samples and purified fractions suggested the co-production of deoxynybomycin which was not reported previously from this organism. TLC and HPLC were used to confirm the presence of deoxynybomycin in the crude extracts of fermentation broths.     

Thin liquid films and monolayers of DMPC mixed with PEG and phospholipid linked PEG

In this work thin liquid films (TLFs) and monolayers at the air/water interface formed by dimyristoylphosphatidylcholine (DMPC) and by DMPC mixed with poly ethylene glycols (PEGs) and dimyristoylphosphatidylethanolamine (DMPE) linked PEGs were studied. Film forming dispersions were composed of two types of particles: liposomes and micelles. TLFs stability, threshold concentration C _t (i.e., the minimum one for stable film formation), and hydrodynamic behavior were measured. At equivalent conditions, DMPC films were Newton black films (real bilayers), while DMPE-PEGs films were much thicker with free water between the monolayers. DMPE-PEG addition to DMPC films caused both C _t decrease (depending on PEG moiety length and Mw) and change of TLF formation mechanism. TLFs’ hydrodynamic behavior also strongly depended on DMPE-PEG content and Mw. It was observed that thinning of the DMPC and DMPE-PEGs films continued to different film types and thickness, being much thicker for the latter films. Addition of free PEGs (PEG-200/6000) did not alter TLF type or stability, but changed TLF thinning time, confirming that free PEGs with Mw<8000 could not penetrate in the membrane and alter “near-membrane” water layer viscosity. Monolayer studies showed improved formation kinetics of both adsorbed and spread films, decrease of surface tension (equilibrium and dynamic), and of film compression/decompression histeresis area in DMPE-PEGs monolayers compared with DMPC pure films. Our study shows that combining the models of phospholipid TLFs and monolayers provide the opportunity to investigate the properties of membrane surface and to clarify some mechanisms of its interactions with membrane-active agents.     

Structural damage of Bacillus subtilis biofilms using pulsed laser interaction with gold thin films

There is a huge interest in developing strategies to effectively eliminate biofilms due to their negative impact in both industrial and clinical settings. In this study, structural damage was induced on two day‐old B. subtilis biofilms using the interaction of 532 nm pulsed laser with gold thin films. Radiant exposure of 225 mJ/cm² induced distinct changes on the surface structure and overall morphology of the matured biofilms after laser irradiation. Moreover, at the radiant exposure used, changes in the colour and viscosity of the biofilm were observed which may indicate a compromised extracellular matrix. Irradiated biofilms in the presence of gold film also showed strong propidium iodide signal which implies an increase in the number of dead bacterial cells after laser treatment. Thus, this laser‐based technique is a promising approach in targeting and eradicating matured biofilms attached on surfaces such as medical implants.

     

Interaction of heme proteins with poly(propyleneimine) dendrimers in layer-by-layer assembly films under different pH conditions

With the isoelectric point at pH 7.4, hemoglobin (Hb) has net positive surface charges at pH 5.0 and overall negative charges at pH 9.0, and is essentially neutral at pH 7.0. The fifth-generation poly(propyleneimine) (PPI) dendrimer is usually positively charged in aqueous solution. The {PPI/Hb}n films under different pH conditions have been successfully fabricated on various solid surfaces by the layer-by-layer assembly technique, and the growth of films was monitored by ultraviolet-visible (UV-vis) spectroscopy, quartz crystal microbalance (QCM), and cyclic voltammetry (CV). Not only was the negatively charged Hb at pH 9.0 alternately adsorbed with positively charged PPI onto solid substrates by electrostatic attraction between them, but the positively charged Hb at pH 5.0 was also successfully assembled with like charged PPI into layer-by-layer {PPI/Hb(pH 5.0)}n films. For the latter, the localized electrostatic interaction or the charge reversal of proteins on PPI surface may be the main driving force. For {PPI/Hb(pH 7.0)}n films, however, the hydrophobic/hydrophilic interaction may play a more important role in the assembly, making the amount of adsorbed Hb even less than that of {PPI/Hb(pH 5.0)}n films. For comparison, negatively charged catalase (Cat) at pH 8.0 was used to assemble layer-by-layer films with positive PPI, but {PPI/Cat}n films showed quite different properties from {PPI/Hb}n films. UV-vis and infrared (IR) spectroscopy, QCM, ellipsometry, and voltammetry were utilized to characterize the {PPI/protein}n films. The results suggest that the proteins in the multilayer films retain their near-native structure and display good voltammetric response for heme Fe(III)/Fe(II) redox couples at underlying pyrolytic graphite (PG) electrodes. Electrocatalysis of oxygen and hydrogen peroxide based on direct electrochemistry of heme proteins at {PPI/protein}n film electrodes was also demonstrated.     

Direct measurement of Gag-Gag interaction during retrovirus assembly with FRET and fluorescence correlation spectroscopy

During retrovirus assembly, the polyprotein Gag directs protein multimerization, membrane binding, and RNA packaging. It is unknown whether assembly initiates through Gag-Gag interactions in the cytosol or at the plasma membrane. We used two fluorescence techniques-two-photon fluorescence resonance energy transfer and fluorescence correlation spectroscopy-to examine Rous sarcoma virus Gag-Gag and -membrane interactions in living cells. Both techniques provide strong evidence for interactions between Gag proteins in the cytoplasm. Fluorescence correlation spectroscopy measurements of mobility suggest that Gag is present in large cytosolic complexes, but these complexes are not entirely composed of Gag. Deletion of the nucleocapsid domain abolishes Gag interactions and membrane targeting. Deletion of the membrane-binding domain leads to enhanced cytosolic interactions. These results indicate that Gag-Gag interactions occur in the cytosol, are mediated by nucleocapsid domain, and are necessary for membrane targeting and budding. These methods also have general applicability to in vivo studies of protein-protein and -membrane interactions involved in the formation of complex macromolecular structures.     

The potential for interaction of hydrochlorothiazide with garlic in rats

The present study was undertaken to determine the pharmacokinetic and pharmacodynamic interaction of hydrochlorothiazide (HCTZ) with garlic homogenate (GH), in rats. The influence of garlic on pharmacokinetics of HCTZ was studied by HPLC method, while pharmacodynamic interaction was studied using diuretic activity, ECG and BP changes and isoproterenol (ISO) induced myocardial injury. HCTZ was given orally at 10 mg/kg and GH was administered at three different doses of 125, 250 and 500 mg/kg, p.o. The CK-MB, LDH, SOD, catalase and histopathological studies were carried out. The administration of HCTZ in GH pretreated rats found to decrease the QRS duration, RR interval, QT segment, systolic blood pressure, heart rate, serum potassium level, serum LDH and serum CK-MB activities significantly. The diuretic effect of HCTZ was significantly increased in presence of GH; however, kaliuresis was significantly reduced in presence of GH 250 mg/kg. Histopathological studies of heart tissue reveal the protective effect of GH 250 mg/kg in presence or absence of HCTZ during ISO stress to myocardium. The pharmacokinetic studies show that GH increases the bioavailability and half-life, along with decrease in clearance and elimination rate of HCTZ when administered orally. It was concluded that careful addition of garlic in moderate doses might result in beneficial effect during treatment of hypertension in patients with myocardial stress as garlic causes substantial fall in excretion of potassium when compared to HCTZ alone treatment in rats. This could be important in reducing the dose of HCTZ to achieve enhanced therapeutic effect with minimal adverse effect.     

The effect of thermal treatment on antibacterial properties of nanostructured TiO<Subscript>2</Subscript>(N) films illuminated with visible light

This work focuses on the photocatalytic performances and antibacterial activity of nitrogen doped TiO₂ nanosystems with three and five layers obtained by a sol-gel route, followed by thermal treatment in oxygen or ammonia atmosphere at temperatures between 400 and 1000°C. Subsequently, the antibacterial activity of the obtained nanosystems on the Escherichia coli cells are determined and discussed. The obtained results show a significant dependence of the functional performances on the system’s composition. In particular, the antimicrobial activity of nitrogen-doped TiO₂ films is correlated with the temperature of thermal treatment and illumination time with visible artificial light.