Diversity and adaptation of soil fungi in an ecosystem with contamination originating from a phosphate fertilizer plant

In the vicinity of a former phosphate fertilizer production plant, high phosphate contents with up to 463 mg phosphate per kilogram of soil dry matter were found 13 years after closing the plant while pH is only slightly elevated at pH 7 to 8. The deposited phosphate is seen to be moved to deeper soil horizons. The effect on soil microbiota was analyzed with respect to soil respiration, fungal biomass and cultivation of benomyl resistant and ligninolytic fungi. Increasing numbers and diversity of soil fungi were found with distance from the former emittent. This was confirmed by investigation of mycorrhization rates of mycotrophic, ectomycorrhizal birch trees. We tested for adaptation by growth and phosphate acquisition on soil extract media. The best growth was seen on the media containing highest phosphate concentrations showing no in vitro growth inhibition. In contrast to previous findings, however, more polyphosphate granula were seen on soil extract media from distant sites, although phosphate concentration was lowest in these media

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 [W2(CO)10(S2CH)]−, the putative lipophilic anion \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{B}}{\left( {{\text{CF}}_{3} } \right)}^{ - }_{4}$$\end{document} 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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{B}}{\left( {{\text{CF}}_{3} } \right)}^{ - }_{4}$$\end{document} 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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{B}}{\left( {{\text{CF}}_{3} } \right)}^{ - }_{4}$$\end{document} 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

Electroporation-Induced Electrosensitization

BACKGROUND: Electroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs). Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs. METHODOLOGY/PRINCIPAL FINDINGS: We focused first on the role of EP rate for membrane permeabilization and lethal effects in mammalian cells. The rate was varied from 0.001 to 2,000 Hz while keeping other parameters constant (2 to 3,750 pulses of 60-ns to 9-µs duration, 1.8 to 13.3 kV/cm). The efficiency of all EP treatments was minimal at high rates and started to increase gradually when the rate decreased below a certain value. Although this value ranged widely (0.1-500 Hz), it always corresponded to the overall treatment duration near 10 s. We further found that longer exposures were more efficient irrespective of the EP rate, and that splitting a high-rate EP train in two fractions with 1-5 min delay enhanced the effects severalfold. CONCLUSIONS/SIGNIFICANCE: For varied experimental conditions, EPs triggered a delayed and gradual sensitization to EPs. When a portion of a multi-pulse exposure was delivered to already sensitized cells, the overall effect markedly increased. Because of the sensitization, the lethality in EP-treated cells could be increased from 0 to 90% simply by increasing the exposure duration, or the exposure dose could be reduced twofold without reducing the effect. Many applications of electroporation can benefit from accounting for sensitization, by organizing the exposure either to maximize sensitization (e.g., for sterilization) or, for other applications, to completely or partially avoid it. In particular, harmful side effects of electroporation-based therapies (electrochemotherapy, gene therapies, tumor ablation) include convulsions, pain, heart fibrillation, and thermal damage. Sensitization can potentially be employed to reduce these side effects while preserving or increasing therapeutic efficiency

Isolation of guard cells from fresh epidermis using a piezo-power micro-dissection system with vibration-attenuated needles

The Eppendorf Piezo-Power Microdissection (PPMD) system uses a tungsten needle (MicroChisel) oscillating in a forward-backward (vertical) mode to cut cells from surrounding tissue. This technology competes with laser-based dissection systems, which offer high accuracy and precision, but are more expensive and require fixed tissue. In contrast, PPMD systems can dissect freshly prepared tissue, but their accuracy and precision is lower due to unwanted lateral vibrations of the MicroChisel. Especially in tissues where elasticity is high, these vibrations can limit the cutting resolution or hamper the dissection. Here we describe a cost-efficient and simple glass capillary–encapsulation modification of MicroChisels for effective attenuation of lateral vibrations. The use of modified MicroChisels enables accurate and precise tissue dissection from highly elastic material

Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies

The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems

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

A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins

The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin

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