Single center method: a computational tool for ionization and electronic excitation studies of molecules.

We discuss the recent progress in the development of the single center (SC) method for computation of highly-delocalized discrete and partial photoelectron wave continuous functions of molecules. Basic equations of the SC method are presented, and an efficient scheme for the numerical solution of a system of coupled Hartree-Fock equations for a photoelectron is described. Several illustrative applications of the method to photoionization and electronic excitation processes in diatomic molecules are considered. Thereby, we demonstrate its potential for theoretically studying angularly resolved molecular photoionization processes.

Symmetry-forbidden electronic state interference observed in angularly resolved NO+ (A 1Π) deexcitation spectra of the N*O(2σ(-1)2π(2)) resonance.

Quantum mechanical interference between different pathways in inner-shell resonance excitation-deexcitation spectra is a realization of a double-slit experiment on the atomic scale. If the intermediate inner-shell resonances are of different symmetries, this interference is symmetry forbidden in the solid-angle-averaged or magic-angle-recorded deexcitation spectra. It has, however, been suggested that interference may by observable in off-magic-angle-recorded spectra. Here, we prove this interference in angularly resolved deexcitation spectra of the 2σ(-1)2π(2)(2Δ,2Σ±) resonances of N*O by a quantitative comparison between ab initio calculations and experiment.

Neutral dissociation of the I, I', and I" vibronic progressions of O2.

It is suggested that the main mechanism for neutral dissociation of the I, I('), and I(") vibronic progressions in O(2) is due to their interaction with the vibrational continuum of the 1pi(u) (-1)(A (2)Pi(u))3ssigma(g) (3)Pi(u)(v(epsilon)) Rydberg state (J state) leading to the formation of the O(2p(4) (3)P)+O( *)(2p(3)((4)S)3s (3)S) fragments. In order to justify this, the O I 2p(3)((4)S)3s (3)S-->2p(4) (3)P fluorescence emission cross section following the neutral dissociation of the O(2) 1pi(u) (-1)(a (4)Pi(u))4ssigma(g)/3ddelta(g)/3dsigma(g) (3)Pi(u)(v) Rydberg states is simulated in the exciting-photon energy range of 14.636-16.105 eV. The results of high-resolution measurements (H. Liebel et al., J. Phys. B 34, 2581 (2001)) can be reproduced if a small adjustment of the computed potential curve of the J state is applied. Non-Franck-Condon resonant intensity distributions of the I, I('), and I(") progressions observed in the experiment are qualitatively explained by the presence of the O(2) 1pi(g) (-1)(X (2)Pi(g))npsigma(u)/nfsigma(u)/nfdelta(u) (3)Pi(u) perturber states. Present calculations allow to decide between two different assignments of the I, I('), and I(") states available in literature.

Effects on capacitance by overexpression of membrane proteins.

Functional Channelrhodopsin-2 (ChR2) overexpression of about 10(4)channels/mum(2) in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C(m)). The C(m) increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C(m) of 1.15+/-0.08 microF/cm(2) in ChR2-expressing cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (0.79+/-0.02 microF/cm(2)). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C(m) (0.85+/-0.07 microF/cm(2)) as compared to non-expressing control cells (0.70+/-0.03 microF/cm(2)). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins.

Dielectric spectroscopy of Schizosaccharomyces pombe using electrorotation and electroorientation.

Two complementary AC electrokinetic techniques electrorotation (ER) and electroorientation (EO) enabled the dielectric characterization of the rod-shaped fission yeast Schizosaccharomyces pombe. The use of microstructured electrodes allowed both ER and EO measurements to be performed over wide ranges of field frequency and medium conductivity. Due to their layered structure, living S. pombe cells exhibited up to three well resolved peaks in their ER spectra and also two distinct orientations, i.e., parallel or perpendicular to the imposed linear field. Heat treatment and enzymatic protoplast isolation led to dramatic changes in the electrokinetic behavior of fission yeast. Application of the theoretical models linking the ER and EO spectra yielded the dielectric parameters of the major structural units of S. pombe cells (cell wall, plasma membrane and cytosol). The dielectric characterization of yeasts has an enormous impact in biotechnology and biomedicine, because electric field pulse techniques (electrofusion and electropermeabilization) are widely used for production of transgenic yeast strains of economic importance. The present study also showed that combined ER and EO measurements can be employed as a powerful diagnostic tool for analyzing changes in yeast structure and physiology upon exposure to various stress conditions.

Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells.

The effects of ionic composition and conductivity of the medium on electropermeabilization of the plasma membrane of mammalian cells were studied. Temporal and spatial uptake of propidium iodide (PI) into field-treated cells was measured by means of flow cytometry, spectrofluorimetry and confocal laser scanning microscopy. Murine myeloma cells were electropulsed in iso-osmolar solutions. These contained 10-100 micrograms ml-1 PI at different conductivities (0.8-14 mS cm-1) and ionic strengths, adjusted by varying concentrations of K+, Na+, Cl- and SO4(2-). Field-induced incorporation of PI into reversibly permeabilized cells was (almost) independent of ionic composition and strength (at a fixed medium conductivity), but increased dramatically with decreasing medium conductivity at a fixed field strength. The time-course of PI uptake (which apparently reflected the resealing process of the membrane) could be fitted by single-exponential curve (relaxation time about 2 min in the absence of Ca2+) and was independent of medium conductivity and composition. These and other data suggested that the expansion of the 'electroleaks' during the breakdown process is field-controlled and depends, therefore, on the (conductivity-dependent) discharging process of the permeabilized membrane. The threshold field strength for dye uptake increased with increasing K+ concentration (about 0.6 kV cm-1 in K(+)-free, NaCl-containing medium and about 0.9 kV cm-1 in 30 mM KCl-containing medium). Also, the spatial uptake pattern of PI shifted from an asymmetric permeation through the cell hemisphere facing the anode to a more symmetric uptake through both hemispheres. These results suggested that the generated potential is superimposed on the (K(+)-dependent) resting membrane potential. Taking this into account, the breakdown voltage of the membrane was estimated to be about 1 V.

Photohemolysis sensitized by psoralen: reciprocity law is not fulfilled.

The effect of the intensity of ultraviolet-A (UV-A) radiation (366 nm) on delayed photohemolysis sensitized by psoralen (PUV-A hemolysis) was studied. It was shown that PUV-A hemolysis induced by UV-A radiation at low fluence rate (20 W m-2) develops according to the well-known colloid-osmotic mechanism: there was no threshold dose of PUV-A treatment. After irradiation all the cells were hemolysed. The rate of PUV-A hemolysis was proportional to the square of the fluence. Hemolysis was delayed in the presence of sucrose. When the fluence rate of UV-A radiation was increased to 150 W m-2, the character of PUV-A hemolysis changed drastically. A threshold fluence appeared, below which PUV-A hemolysis was not induced. At fluences slightly exceeding the threshold, only part of the cells in the suspension were lysed. The dependence of the portion of hemolysing cells on fluence was S-shaped. Increasing the fluence resulted in complete (100%) hemolysis. The rate of complete hemolysis decreased at higher fluences, but was many-fold higher than the rate of low-intensity PUV-A hemolysis at equal fluences. The main features of high intensity PUV-A hemolysis (dependences on fluence and temperature, effect of sucrose) were the same for the hemolysis induced by the addition of previously photooxidized psoralen. We suggest that high intensity PUV-A hemolysis is induced with participation of photooxidized psoralen as an intermediate.

Fe2+ ions and reduced glutathione--chemical activators of psoralen-sensitized photohaemolysis.

The influence of Fe2+ ions and reduced glutathione (GSH) on the haemolysis of erythrocytes photosensitized (366 nm) by psoralen (PUVA haemolysis) was investigated. PUVA haemolysis was induced by the low fluence rate (24 W m-2) and high fluence (greater than 150 W m-2) of UV-A irradiation. It has been shown that Fe2+ ions and GSH activated PUVA haemolysis at both fluence rates of irradiation. PUVA haemolysis activation by Fe2+ ions was more pronounced than that by GSH. It is supposed that activation caused by Fe2+ ions and GSH is connected with their ability to reduce lipid peroxides or psoralen peroxides with the subsequent formation of free radicals. The regeneration of endogenous Fe2+ by reduced glutathione is also possible.

[Relationship between absorption and fluorescence excitation spectra of psoralens and coumarins and their concentration]. / Zavisimost' spektrov pogloshcheniia i vozbuzhdeniia fluorestsentsii psoralenov i kumarinov ot kontsentratsii.

The shape of absorption spectra is changed and their maxima are red shifted with an increase of furocoumarin (psoralen, 8-methoxypsoralen and angelicin) concentration. Fluorescence excitation spectra of psoralen, 7-methoxycoumarin and 7-hydroxy-4-methylocumarin do not depend on the concentration in solutions. They are similar or coincident with absorption spectra of the most concentrated solutions. One may conclude the existence of different forms of furocoumarin and coumarin aggregates in solution. From the coincidence or similarity of fluorescence excitation spectra and absorption spectra in the most concentrated solutions it may be proposed that only the aggregated forms of psoralens and coumarins (the dimers or associates of higher order) are able to emit fluorescence.

[Effect of antioxidants on the psoralen photo-oxidation process]. / Vliianie antioksidantov na protsess fotookisleniia psoralena.

Psoralens are capable of photosensitizing oxidation of unsaturated fatty acids due to the two-stage mechanism. During the first (light) stage psoralen solution in ethanol undergoes photooxidation under UV-irradiation (366 nm). At the second (dark) stage the addition of photooxidized psoralen (POP) to the aqueous solution of liposomes is followed by lipid oxidation. Antioxidants inhibited the UV-stage, but did not influence the dark one. Neither spectrophotometry, nor spectrofluorometry could detect photoproducts of psoralen involved in the two-stage oxidation of lipids. However, mixing of ethanol solution of POP with water resulted in the flash of chemiluminescence. The inhibition constants by antioxidants of photoproducts formation which are active in the two-stage oxidation of lipids were estimated by chemiluminescence. Stern--Volmer's constants for antioxidants: 2,6-dimethyl-3,5-diacetyl-1,4-dihydropyridine (DHP), 6-hydroxy-2,2,5,7,8-pentamethylchroman (chromanol--C1), water soluble sodium phenozan and butilated hydroxytoluen (ionol) appeared to be (7.4 +/- 2.2) X 10(3) M-1, (4.4 +/- 1.0) X 10(3) M-1, (3.3 +/- 0.7) X 10(3) M-1, (4.5 +/- 2.5) X 10(2) M-1, respectively. The biological importance of these two-stage oxidation photosensitized by furocoumarins is discussed.

[Antioxidant inhibition of hemolysis induced by photooxidized psoralen]. / Ingibirovanie antioksidantami gemoliza, indutsirovannogo fotookislennym psoralenom.

Antioxidants butylated hydroxytoluene++ (ionol) and 2,2,5,7,8-pentamethyl-chromanol-6-(alpha-T-0) were shown to inhibit hemolysis induced by the ethanolic solution of photooxidized psoralen (POP). This inhibition appeared to be the same whether antioxidants were present during irradiation or were added immediately after photooxidation. Therefore antioxidants do not influence POP formation, but inhibit the interaction of POP degradation products in water with biomolecules. Possibility of POP participation in phototoxic effects of furocoumarins is discussed.

[Dark oxidation of unsaturated lipids and dihydroxy phenylalanine by photooxidized furocoumarins]. / Temnovoe okislenie nenasyshchennykh lipidov i dioksifenilalanina fotookislennymi furokumarinami.

Well-known skin photosensitizers furocoumarins--psoralen, angelio in and 8-methoxypsoralen were shown to photosensitize oxidation of unsaturated lipids and dihydroxyphenylalanine via a two-step mechanism. At the first step (UV-irradiation at 366 nm) photooxidation of furocoumarins occurred, at the second step--the photooxidized furocoumarins oxidized unsaturated lipids or dihydroxyphenylalanine in darkness. The photochemical activity of furocoumarins in the two-step reaction decreased as follows: psoralen greater than angelicin greater than 8-methoxypsoralen. Similar photoreactions appear to participate in induction of phototoxic effects of furocoumarins.

Leaf patch clamp pressure probe measurements on olive leaves in a nearly turgorless state.

The non-invasive leaf patch clamp pressure (LPCP) probe measures the attenuated pressure of a leaf patch, P(p) , in response to an externally applied magnetic force. P(p) is inversely coupled with leaf turgor pressure, P(c) , i.e. at high P(c) values the P(p) values are small and at low P(c) values the P(p) values are high. This relationship between P(c) and P(p) could also be verified for 2-m tall olive trees under laboratory conditions using the cell turgor pressure probe. When the laboratory plants were subjected to severe water stress (P(c) dropped below ca. 50 kPa), P(p) curves show reverse diurnal changes, i.e. during the light regime (high transpiration) a minimum P(p) value, and during darkness a peak P(p) value is recorded. This reversal of the P(p) curves was completely reversible. Upon watering, the original diurnal P(p) changes were re-established within 2-3 days. Olive trees in the field showed a similar turnover of the shape of the P(p) curves upon drought, despite pronounced fluctuations in microclimate. The reversal of the P(p) curves is most likely due to accumulation of air in the leaves. This assumption was supported with cross-sections through leaves subjected to prolonged drought. In contrast to well-watered leaves, microscopic inspection of leaves exhibiting inverse diurnal P(p) curves revealed large air-filled areas in parenchyma tissue. Significantly larger amounts of air could also be extracted from water-stressed leaves than from well-watered leaves using the cell turgor pressure probe. Furthermore, theoretical analysis of the experimental P(p) curves shows that the propagation of pressure through the nearly turgorless leaf must be exclusively dictated by air. Equations are derived that provide valuable information about the water status of olive leaves close to zero P(c) .

A combined patch-clamp and electrorotation study of the voltage- and frequency-dependent membrane capacitance caused by structurally dissimilar lipophilic anions.

Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA(-)), tetraphenylborate (TPB(-)) and [W(2)(CO)(10)(S(2)CH)](-), the putative lipophilic anion B(CF(3))(4)(-) and three new heterocyclic W(CO)(5) derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA(-), TPB(-) and B(CF(3))(4)(-) increased and that of W(CO)(5) derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA(-) and W(CO)(5) derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB(-) and B(CF(3))(4)(-) no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.

Mechanisms of electrically mediated cytosolic Ca2+ transients in aequorin-transformed tobacco cells.

Cytosolic Ca(2+) changes induced by electric field pulses of 50-micros duration and 200-800 V/cm strength were monitored by measuring chemiluminescence in aequorin-transformed BY-2 tobacco cells. In Ca(2+)-substituted media, electropulsing led to a very fast initial increase of the cytosolic Ca(2+) concentration reaching a peak value within <100-200 ms. Peaking of [Ca(2+)](cyt) was followed by a biphasic decay due to removal of Ca(2+) (e.g., by binding and/or sequestration in the cytosol). The decay had fast and slow components, characterized by time constants of approximately 0.5 and 3-5 s, respectively. Experiments with various external Ca(2+) concentrations and conductivities showed that the fast decay arises from Ca(2+) fluxes through the plasmalemma, whereas the slow decay must be assigned to Ca(2+) fluxes through the tonoplast. The amplitude of the [Ca(2+)](cyt) transients increased with increasing field strength, whereas the time constants of the decay kinetics remained invariant. Breakdown of the plasmalemma was achieved at a critical field strength of approximately 450 V/cm, whereas breakdown of the tonoplast required approximately 580 V/cm. The above findings could be explained by the transient potential profiles generated across the two membranes in response to an exponentially decaying field pulse. The dielectric data required for calculation of the tonoplast and plasmalemma potentials were derived from electrorotation experiments on isolated vacuolated and evacuolated BY-2 protoplasts. The electrorotation response of vacuolated protoplasts could be described in terms of a three-shell model (i.e., by assuming that the capacitances of tonoplast and plasmalemma are arranged in series). Among other things, the theoretical analysis together with the experimental data show that genetic manipulations of plant cells by electrotransfection or electrofusion must be performed in low-conductivity media to minimize release of vacuolar Ca(2+) and presumably other vacuolar ingredients.

Swelling-activated pathways in human T-lymphocytes studied by cell volumetry and electrorotation.

Small organic solutes, including sugar derivatives, amino acids, etc., contribute significantly to the osmoregulation of mammalian cells. The present study explores the mechanisms of swelling-activated membrane permeability for electrolytes and neutral carbohydrates in Jurkat cells. Electrorotation was used to analyze the relationship between the hypotonically induced changes in the electrically accessible surface area of the plasma membrane (probed by the capacitance) and its permeability to the monomeric sugar alcohol sorbitol, the disaccharide trehalose, and electrolyte. Time-resolved capacitance and volumetric measurements were performed in parallel using media of different osmolalities containing either sorbitol or trehalose as the major solute. Under mild hypotonic stress in 200 mOsm sorbitol or trehalose solutions, the cells accomplished regulatory volume decrease by releasing cytosolic electrolytes presumably through pathways activated by the swelling-mediated retraction of microvilli. This is suggested by a rapid decrease of the area-specific membrane capacitance C(m) (microF/cm2). The cell membrane was impermeable to both carbohydrates in 200 mOsm media. Whereas trehalose permeability remained also very poor in 100 mOsm medium, extreme swelling of cells in a strongly hypotonic solution (100 mOsm) led to a dramatic increase in sorbitol permeability as evidenced by regulatory volume decrease inhibition. The different osmotic thresholds for activation of electrolyte release and sorbitol influx suggest the involvement of separate swelling-activated pathways. Whereas the electrolyte efflux seemed to utilize pathways preexisting in the plasma membrane, putative sorbitol channels might be inserted into the membrane from cytosolic vesicles via swelling-mediated exocytosis, as indicated by a substantial increase in the whole-cell capacitance C(C) (pF) in strongly hypotonic solutions.

Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system.

Giant HEK293 cells of 30-65 microm in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1+/-0.1 microF/cm2 for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 microm) was found to be between 12.8 and 16.1 microS/cm2, which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system.

Surviving high-intensity field pulses: strategies for improving robustness and performance of electrotransfection and electrofusion.

Electrotransfection and electrofusion, both widely used in research and medical applications, still have to face a range of problems, including the existence of electroporation-resistant cell types, cell mortality and also great batch-to-batch variations of the transfection and fusion yields. In the present study, a systematic analysis of the parameters critical for the efficiency and robustness of electromanipulation protocols was performed on five mammalian cell types. Factors examined included the sugar composition of hypotonic pulse media (trehalose, sorbitol or inositol), the kinetics of cell volume changes prior to electropulsing, as well as the growth medium additives used for post-pulse cell cultivation. Whereas the disaccharide trehalose generally allowed regulatory volume decrease (RVD), the monomeric sugar alcohols sorbitol and inositol inhibited RVD or even induced secondary swelling. The different volume responses could be explained by the sugar selectivity of volume-sensitive channels (VSC) in the plasma membrane of all tested cell types. Based on the volumetric data, highest transfection and fusion yields were mostly achieved when the target cells were exposed to hypotonicity for about 2 min prior to electropulsing. Longer hypotonic treatment (10-20 min) decreased the yields of viable transfected and hybrid cells due to (1) the cell size reduction upon RVD (trehalose) or (2) the excessive losses of cytosolic electrolytes through VSC (inositol/sorbitol). Doping the plasma membrane with lipophilic anions prevented both cell shrinkage and ion losses (probably due to VSC inhibition), which in turn resulted in increased transfection and fusion efficiencies.

Towards a medically approved technology for alginate-based microcapsules allowing long-term immunoisolated transplantation.

The concept of encapsulated-cell therapy is very appealing, but in practice a great deal of technology and know-how is needed for the production of long-term functional transplants. Alginate is one of the most promising biomaterials for immunoisolation of allogeneic and xenogeneic cells and tissues (such as Langerhans islets). Although great advances in alginate-based cell encapsulation have been reported, several improvements need to be made before routine clinical applications can be considered. Among these is the production of purified alginates with consistently high transplantation-grade quality. This depends to a great extent on the purity of the input algal source as well as on the development of alginate extraction and purification processes that can be validated. A key engineering challenge in designing immunoisolating alginate-based microcapsules is that of maintaining unimpeded exchange of nutrients, oxygen and therapeutic factors (released by the encapsulated cells), while simultaneously avoiding swelling and subsequent rupture of the microcapsules. This requires the development of efficient, validated and well-documented technology for cross-linking alginates with divalent cations. Clinical applications also require validated technology for long-term cryopreservation of encapsulated cells to maintaining a product inventory in order to meet end-user demands. As shown here these demands could be met by the development of novel, validated technologies for production of transplantation-grade alginate and microcapsule engineering and storage. The advances in alginate-based therapy are demonstrated by transplantation of encapsulated rat and human islet grafts that functioned properly for about 1 year in diabetic mice.