Biomedical subjects

Eberhard Neumann

Publications and source records attributed to Eberhard Neumann.

7 recordsLinked to original sources

PubMed2006

Nonlinear current-voltage relationship of the plasma membrane of single CHO cells.

The application of electric field pulses to Chinese Hamster Ovary (CHO) cells causes membrane electroporation (MEP). If a voltage or current ramp is applied to the cellular membrane of a single CHO cell, the membrane conductance increases nonlinearly with field strength reaching saturation. In particular, the kinetics of the induced conductance changes represents the data basis for the interpretation in terms of underlying structural changes. The current/voltage characteristic is found to be continuous, but displays occasionally a sharp increase in the conductance. The step-like increases are interpreted to reflect the formation of one (or more) larger pore(s). The analysis of current clamp data yields pores of radius (r(p)) in the range of 2.5< or =r(p)/nm< or =20; the pores of the voltage clamp data are in the range of 2.5< or =r(p)/nm< or =55. The larger pores occur predominantly during hyperpolarising and less frequently during depolarising conditions, respectively. The different kinetics of pore formation in the hyperpolarising condition, where the inward field increases, and the depolarising condition, where the inward field first decreases and then increases in the opposite direction, suggests structural asymmetry with respect to the direction of the electric membrane field. At the required higher voltage, the effect of the resting potential is negligibly small.

Animals↗

PubMed2005

Adsorption of DNA and electric fields decrease the rigidity of lipid vesicle membranes.

The adsorption of calf-thymus DNA-fragments of 300 +/- 50 base pairs (bp) to the outer membrane monolayer of unilamellar lipid vesicles in the presence of Ca2+ ions has been quantified by the standard method of chemical relaxation spectrometry using polarized light. The vesicles of radius a = 150 +/- 45 nm are prepared from bovine brain extract type III containing 80-85% phosphatidylserine (PS) and palmitoyl-oleoyl-phosphatidylcholine (POPC) in the molar ratio PS : 2POPC; total lipid concentration [L(t)] = 1 mM in 1 mM HEPES buffer, pH 7.4 at T = 293 K (20 degrees C). The turbidity relaxations of vesicle suspensions, at the wavelength lambda = 365 nm at two characteristic electric field strengths are identified as electroelongation of the whole vesicle coupled to smoothing of thermal membrane undulations and membrane stretching, and at higher fields, to membrane electroporation (MEP). The elongation kinetics indicates that the DNA adsorption renders the membrane more flexible and prone to membrane electroporation (MEP). Remarkably, it is found that the Ca-mediated adsorption of DNA (D) decreases both, bending rigidity kappa and stretching modulus K, along an unique Langmuir adsorption isotherm for the fraction of bound DNA at the given Ca concentration [Ca(t)] = 0.25 mM. The characteristic chemo-mechanical parameter of the isotherm is the apparent dissociation equilibrium constant K(D,Ca) = 100 +/- 10 microM (bp) of the ternary complex DCaB of DNA base pairs (bp) and Ca binding to sites B on the outer vesicle surface. Whereas both kappa and K decrease in the presence of high electric fields (E), the key parameter K(D,Ca) is independent of E in the range 0 < or = E/(kV cm(-1)) < or = 40.

Adsorption↗

PubMed2005

Cholesterol reduces membrane electroporation and electric deformation of small bilayer vesicles.

Electric fields, similar in the order of magnitude of the natural membrane fields of cellular lipid/protein membranes, and chemical relaxation spectrometry can be used as tools to quantify the rigidifying effect of cholesterol in membranes. Small unilamellar vesicles of radius a=50+/-3 nm, prepared form phosphatidylcholine, phosphatidylserine and phosphatidyl-glycerol in the molar ratio 1:1:1 and containing the optical lipid probe molecule 2-(3-diphenyl-hexatrienyl) propanoyl)-1-palmitoyl-sn-glycerol-3-phosphocholine (beta-DPH pPC), serve as examples for curved lipid membranes. The data of electrooptical turbidity and absorbance relaxations at the wavelength lambda=365 nm are analysed in terms of membrane bending rigidity kappa and membrane stretching modulus K. Both kappa and K increase with increasing mole fraction x of cholesterol up to x=0.5. The cholesterol induced denser packing of the lipids reduces the extent of both membrane electroporation (ME) and electroelongation of the vesicles. Further on, cholesterol in the lipid phase and sucrose in the aqueous suspension reduce the extent of membrane undulation and electro-stretching.

Animals↗

PubMed2005

Interfacial ternary complex DNA/Ca/lipids at anionic vesicle surfaces.

The electroporative transfer of gene DNA and other bioactive substances into tissue cells by electric pulses gains increasing importance in the new disciplines of electrochemotherapy and electrogenetherapy. The efficiency of the electrotransfer depends crucially on the adsorption of the gene DNA and oligonucleotides to the plasma cell membranes. Here it is shown that the adsorption of larger oligonucleotides such as fragments (ca. 300 bp) of sonicated calf-thymus DNA, to anionic lipids of unilamellar vesicles (diameter Phi=300+/-90 nm) is greatly enhanced by divalent cations such as Ca(2+)-ions. Applying centrifugation, bound and free DNA are monitored optically at the wavelength lambda=260 nm. Using arsenazo III as a Ca(2+)-indicator and atomic absorption spectroscopy (AAS), Ca(2+)-titrations of DNA and vesicles yield the individual equilibrium constants of Ca(2+)- and DNA-binding not only for the binary complexes: Ca/lipids, Ca/DNA and DNA/lipids, respectively, but also for the various processes to form the ternary complex DNA/Ca/lipids. The data provide the basis for goal-directed optimization protocols for the adsorption and thus efficient electrotransfer of oligonucleotides and polynucleotides into cells.

Anions↗

PubMed2002

Temperature jump kinetic study of the stability of apo-calmodulin.

Temperature-jump relaxation spectrometry has been used to study the unfolding properties of Ca(2+)-free Drosophila calmodulin from 278 to 336 K, monitored by absorption of Tyr-138. The T-jump amplitude data are well fitted throughout with a melting temperature T(m) = 315.7 K, deltaH(o)(m) = 140.5 kJ mol(-1) and deltaC(p)(o) = 3.28 kJ K(-1) mol(-1), giving deltaG(o)(293) = 7.36 kJ mol(-1) for the C-domain, in good agreement with other data. The relaxation rate observed (time range 1 micros-1 ms) obeys a simple two-state kinetic mechanism throughout. The activation energy for unfolding is nearly temperature-independent, in contrast to that for refolding, and hence the transition state is relatively compact, resembling the folded state, and the relaxation time, tau, shows complex temperature dependence. The domain unfolding is a two-state process occurring with tau of approximately 100 micros at the T(m). At 296 K, when the C-domain is approximately 6% unfolded, k(unfolding) approximately 305 s(-1), k(refolding) approximately 4660 s(-1) and tau approximately 200 micros. This closely resembles the rate and extent of a reported C-domain exchange process, inferred from NMR line-broadening at 296 K. The inherent instability of the apo-C-domain of calmodulin indicates that the unfolded form significantly contributes to the physical properties of apo-calmodulin at normal temperatures, and this instability is enhanced by low ionic strength conditions.

Animals↗

PubMed2002

Ionic conductivity of electroporated lipid bilayer membranes.

The ionic conductivity of lipid membrane pores has been theoretically analysed in terms of electrostatic interactions of the transported ions with the low-dielectric pore wall for a commonly encountered case of unequal concentrations of electrolyte on the two sides of curved lipid membranes. Theoretical analysis of the data on the conductivity of the electroporated membrane of lipid vesicles (Lecithin 20%) of radius a=90 nm yields the molar energy of interaction of a small monovalent ion with a pore wall w(0)=9+/-1 RT (or w(0)=22+/-kJ mol(-1)), corresponding to a mean pore radius of (-)r(p)=0.56+/-0.05 nm. The proposed theoretical approach provides a tool for the analysis and description of the nonlinear current-voltage dependencies in membrane pores and channels.

Electric Conductivity↗

PubMed2002

Digression on membrane electroporation for drug and gene delivery.

Membrane electroporation (ME) defines an electrical technique to render lipid membranes porous and permeable, transiently and reversibly, by external voltage pulses. Although there are numerous applications of ME to manipulate cells, organelles and tissues in cell biology, biotechnology and medicine, yet the molecular mechanism of ME is only slowly being understood. A general chemical- thermodynamical approach for the quantitative description of cell membrane electroporation has been developed to provide the framework to quantitatively rationalize electroporative cell transformation and electroporative uptake of drug-like dyes into cells, as well as electrolyte efflux from salt-filled electroporated vesicles. Mechanistically, the electroporative transfer of gene and drug-like dyes involves the coupling between an interactive contact formation of the permeates with the cell surface membrane and the structural electroporation-resealing cycle C <--> (P) where C is the closed and (P) represents a number of different porated membrane states, respectively. The experimentally accessible concentration fraction f(p) = [(P)] / ([C] + [(P)]) of porous states is related to thermodynamic and electro-mechanic parameters such as temperature and the electric field strength, membrane rigidity or curvature. The results of the theoretical approach, mainly based on electrooptical data of lipid vesicles, have been successfully used to analyze single cells and to specify conditions for the practical purpose of direct electroporative gene transfer and drug delivery, in particular in the new medical disciplines of electroporative chemotherapy and electroporative gene vaccination.

Animals↗