Interaction of phloretin with membranes: on the mode of action of phloretin at the water-lipid interface.

The interaction of phloretin with single lipid bilayers on a spherical support and with multilamellar vesicles was studied by differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR). The results indicated that phloretin interacts with the lipid layer and changes its structural parameters. In DSC experiments, phloretin in its neutral form strongly decreased the lipid phase transition temperature and slightly reduced the cooperativity of the phase transition within the lipid layer. In NMR measurements, phloretin led to an increase of the transverse relaxation time constant but had no effect on the spin-lattice relaxation time constant. The overall dipole moment of phloretin was experimentally determined and was found to be roughly 40% lower than has been published previously. This result suggested that the size of the dipole moment of phloretin does not provide such a high contribution to the effect of phloretin on the dipole potential of monolayers and bilayers as has been published previously. To understand the discrepancy between phloretin adsorption and dipole potential change, we performed computational conformational analysis of phloretin in the gas phase. The results showed that a wide distribution of the dipole moments of phloretin conformers exists, which mainly depends on the orientation of the OH moieties. The adsorption of phloretin as determined from its binding to solid supported bilayers differed from the one determined from dipole potential measurements on black lipid membranes. The difference between the phloretin dissociation constants of both types of experiments suggested a change of its dipole moment normal to the membrane surface in a concentration-dependent manner, which was in agreement with the results of the computational conformational analysis.

Interaction of phloretin with lipid monolayers: relationship between structural changes and dipole potential change.

Phloretin is known to adsorb to lipid surfaces and alters the dipole potential of lipid monolayers and bilayers. Its adsorption to biological and artificial membranes results in a change of the membrane permeability for a variety of charged and neutral compounds. In this respect phloretin represents a model substance to study the effect of dipole potentials on membrane permeability. In this investigation we studied the interaction of phloretin with monolayers formed of different lipids in the liquid-expanded and the condensed state. Phloretin integrated into the monolayers as a function of the aqueous concentration of its neutral form, indicated by an increase of the surface pressure in the presence of phloretin. Simultaneous recording of the surface potential of the monolayers allowed us to correlate the degree of phloretin integration and the phloretin-induced dipole potential change. Increasing the surface pressure decreased the phloretin-induced shift of the isotherms, but did not influence the phloretin-induced surface potential change. This means that phloretin adsorption to the lipid surface can occur without affecting the lipid packing. The surface potential effect of phloretin is accompanied by a change of the lipid dipole moment vector dependent on the lipid packing. This means that the relation between the surface potential change and the lipid packing cannot be described by a static model alone. Taking into account the deviations of the surface potential change versus molecular area isotherms of the experimental data to the theoretically predicted course, we propose a model that relates the area change to the dipole moment in a dynamic manner. By using this model the experimental data can be described much better than with a static model.

Study of sugar binding to the sucrose-specific ScrY channel of enteric bacteria using current noise analysis.

ScrY, an outer membrane channel of enteric Gram-negative bacteria, which confers to the bacteria the rapid uptake of sucrose through the outer membrane was reconstituted into lipid bilayer membranes and the current noise was investigated in the open and in the carbohydrate-induced closed state of the channel. The open state of the channel exhibited up to about 200 Hz 1/f-noise with a rather small spectral density. Upon addition of carbohydrates to the aqueous phase the current through the ScrY channels decreased in a dose-dependent manner. Simultaneously, the spectral density of the current noise increased drastically, which indicated interaction of the carbohydrates with the binding site inside the channel and its reversible block. The frequency dependence of the spectral density was of the Lorentzian type but very often two Lorentzians were observed, from which the slow one may not be related to carbohydrate binding. Analysis of the power density spectra of the second Lorentzian using a previously proposed simple model of carbohydrate binding allowed the evaluation of the on- and the off-rate constants for the carbohydrate association with the binding site inside the ScrY channel and of a mutant (ScrYDelta3-72), in which 70 amino acids at the N-terminus are deleted. The binding of carbohydrates to ScrY was compared to those of the closely related maltoporin channels of Escherichia coli and Salmonella typhimurium by assuming that only the time constant and spectral density of the high frequency Lorentzian is related to carbohydrate transport.

The cell wall porin of Nocardia farcinica: biochemical identification of the channel-forming protein and biophysical characterization of the channel properties.

A channel-forming protein was identified in cell wall extracts of the Gram-positive, strictly aerobic bacterium Nocardia farcinica. The cell wall porin was purified to homogeneity and had an apparent molecular mass of about 87 kDa on tricine-containing SDS-PAGE. When the 87 kDa protein was boiled for a longer time in sodium dodecylsulphate (SDS) it dissociated into two subunits with molecular masses of about 19 and 23 kDa. The 87 kDa form of the protein was able to increase the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine (PC) phosphatidylserine (PS) mixtures by the formation of ion-permeable channels. The channels had on average a single-channel conductance of 3.0 nS in 1M KCl, 10mM Tris-HCl, pH8, and were found to be cation selective. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted in an asymmetric voltage dependence. The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated point charge effects on the channel properties. The analysis of the single-channel conductance data in different salt solutions using the Renkin correction factor, and the effect of negative charges on channel conductance suggested that the diameter of the cell wall porin is about 1.4-1.6nm. Channel-forming properties of the cell wall porin of N. farcinica were compared with those of mycobacteria and corynebacteria. The cell wall porins of these members of the order Actinomycetales share common features because they form large and water-filled channels that contain negative point charges.

The adsorption of phloretin to lipid monolayers and bilayers cannot be explained by langmuir adsorption isotherms alone.

Phloretin and its analogs adsorb to the surfaces of lipid monolayers and bilayers and decrease the dipole potential. This reduces the conductance for anions and increases that for cations on artificial and biological membranes. The relationship between the change in the dipole potential and the aqueous concentration of phloretin has been explained previously by a Langmuir adsorption isotherm and a weak and therefore negligible contribution of the dipole-dipole interactions in the lipid surface. We demonstrate here that the Langmuir adsorption isotherm alone is not able to properly describe the effects of dipole molecule binding to lipid surfaces--we found significant deviations between experimental data and the fit with the Langmuir adsorption isotherm. We present here an alternative theoretical treatment that takes into account the strong interaction between membrane (monolayer) dipole field and the dipole moment of the adsorbed molecule. This treatment provides a much better fit of the experimental results derived from the measurements of surface potentials of lipid monolayers in the presence of phloretin. Similarly, the theory provides a much better fit of the phloretin-induced changes in the dipole potential of lipid bilayers, as assessed by the transport kinetics of the lipophilic ion dipicrylamine.

Effect of toluene inhalation on the liver of rats--dependence on sex dose and exposure time.

Groups of CFY rats were exposed to toluene inhalation as follows: both males and females to 1000 mg/m3 6 h daily five times a week for 6 months; only males to 3500 mg/h3 8 h daily every day for 6 months; and only males to 1500, 3000 and 6000 mg/m3 8 h daily for 4 weeks. Control groups were exposed to air inhalation under identical conditions. Toluene was found to inhibit growth but to cause no abnormal light-microscopical changes. Hepatic changes were: (i) Signs of compensation such as increased relative liver weight, SER proliferation; increased succinate dehydrogenase activity; a decrease in glycogen content; increased cytochrome P-450 and b5 concentration; increased hepatic aniline hydroxylase and aminopyrine N-demethylase activity. (ii) Non-specific subcellular effect was observed in a small number of hepatocytes, namely RER dilatation, separation of ribosomes, mitochondria of variable shapes, an increased number of autophagous bodies. As regards indicators of the hepatic function, BSP retention decreased, GOT and GPT activity did not change. The changes were observed in both sexes, were dose-dependent and reversible, and showed no--or only a slight--dependence on exposure time. Chronic toluene exposure has no specific hepatotoxic effect leading to chronic liver disease.