Search

Abstract: A novel system for liquid volume detection is reported in this paper. The system uses two lasers with different wavelengths to scan the entire height of tubes. By detecting the shape and position of the meniscus that is formed at the liquid-air interface, the volume of liquid in a tube of known geometry can be determined. To increase the accuracy of this level-based volume detection, the shape of the meniscus must be considered. The optical effect of the meniscus in such a system was modeled and its effect on the volume detection simulated. A laboratory prototype was built and the biggest difference between the measured power of the transmitted light through the meniscus and the simulated data was less than 8%. [Copyright &y& Elsevier]

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR's extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes. [ABSTRACT FROM AUTHOR]

Turgor pressure provides a sensitive indicator for irrigation scheduling. Leaf turgor pressure of Musa acuminate was measured by using the so-called leaf patch clamp pressure probe, i.e. by application of an external, magnetically generated and constantly retained clamp pressure to a leaf patch and determination of the attenuated output pressure Pp that is highly correlated with the turgor pressure. Real-time recording of Pp values was made using wireless telemetric transmitters, which send the data to a receiver base station where data are logged and transferred to a GPRS modem linked to an Internet server. Probes functioned over several months under field and laboratory conditions without damage to the leaf patch. Measurements showed that the magnetic-based probe could monitor very sensitively changes in turgor pressure induced by changes in microclimate (temperature, relative humidity, irradiation and wind) and irrigation. Irrigation effects could clearly be distinguished from environmental effects. Interestingly, oscillations in stomatal aperture, which occurred frequently below turgor pressures of 100 kPa towards noon at high transpiration or at high wind speed, were reflected in the Pp values. The period of pressure oscillations was comparable with the period of oscillations in transpiration and photosynthesis. Multiple probe readings on individual leaves and/or on several leaves over the entire height of the plants further emphasised the great impact of this non-invasive turgor pressure sensor system for elucidating the dynamics of short- and long-distance water transport in higher plants. [ABSTRACT FROM AUTHOR]

An advanced non-invasive, field-suitable and inexpensive leaf patch clamp pressure probe for online-monitoring of the water relations of intact leaves is described. The probe measures the attenuated output patch clamp pressure, Pp, of a clamped leaf in response to an externally applied input pressure, Pclamp. Pclamp is generated magnetically. Pp is sensed by a pressure sensor integrated into the magnetic clamp. The magnitude of Pp depends on the transfer function, Tf, of the leaf cells. Tf consists of a turgor pressure-independent (related to the compression of the cuticle, cell walls and other structural elements) and a turgor pressure-dependent term. Tf is dimensionless and assumes values between 0 and 1. Theory shows that Tf is a power function of cell turgor pressure Pc. Concomitant Pp and Pc measurements on grapevines confirmed the relationship between Tf and Pc. Pp peaked if Pc approached zero and assumed low values if Pc reached maximum values. The novel probe was successfully tested on leaves of irrigated and non-irrigated grapevines under field conditions. Data show that slight changes in the microclimate and/or water supply (by irrigation or rain) are reflected very sensitively in Pp. [ABSTRACT FROM AUTHOR]

The continuity of the xylem water columns was studied on 17- to 23-m tall birch trees (trunk diameter about 23 cm; first branching above 10 m) all year round. Fifty-one trees were felled, and 5-cm thick slices or 2-m long boles were taken at regular, relatively short intervals over the entire height of the trees. The filling status of the vessels was determined by (i) xylem sap extraction from trunk and branch pieces (using the gas bubble-based jet-discharge method and centrifugation) and from trunk boles (using gravity discharge); (ii) 1H nuclear magnetic resonance imaging of slice pieces; (iii) infusion experiments (dye, 86Rb+, D2O) on intact trees and cut branches; and (iv) xylem pressure measurements. This broad array of techniques disclosed no evidence for continuous water-filled columns, as postulated by the Cohesion–Tension theory, for root to apex directed mass transport. Except in early spring (during the xylem refilling phase) and after extremely heavy rainfall during the vegetation period, cohesive/mobile water was found predominantly at intermediate heights of the trunks but not at the base or towards the top of the tree. Similar results were obtained for branches. Furthermore, upper branches generally contained more cohesive/mobile water than lower branches. The results suggest that water lifting occurs by short-distance (capillary, osmotic and/or transpiration-bound) tension gradients as well as by mobilisation of water in the parenchymatic tissues and the heartwood, and by moisture uptake through lenticels. [ABSTRACT FROM AUTHOR]

Seasonal variations in osmolality and components of xylem sap in tall birch trees were determined using several techniques. Xylem sap was extracted from branch and trunk sections of 58 trees using the very rapid gas bubble-based jet-discharge method. The 5-cm long wood pieces were taken at short intervals over the entire tree height. The data show that large biphasic osmolality gradients temporarily exist within the conducting xylem conduits during leaf emergence (up to 272 mosmol·kg−1 at the apex). These gradients (arising mainly from glucose and fructose) were clearly held within the xylem conduit as demonstrated by 1H NMR imaging of intact twigs. Refilling experiments with benzene, sucrose infusion, electron and light microscopy, as well as 1H NMR chemical shift microimaging provided evidence that the xylem of birch represents a compartment confined by solute-reflecting barriers (radial: lipid linings/lipid bodies; axial: presumably air-filled spaces). These features allow transformation of osmolality gradients into osmotic pressure gradients. Refilling of the xylem occurs by a dual mechanism: from the base (by root pressure) and from the top (by hydrostatic pressure generated by xylem-bound osmotic pressure). The generation of osmotic pressure gradients was accompanied by bleeding. Bleeding could be observed at a height of up to 21 m. Bleeding rates measured at a given height decreased exponentially with time. Evidence is presented that the driving force for bleeding is the weight of the static water columns above the bleeding point. The pressure exerted by the water columns and the bleeding volume depend on the water-filling status of (communicating) vessels. [ABSTRACT FROM AUTHOR]

A high-precision pressure probe is described which allows non-invasive online-monitoring of the water relations of intact leaves. Real-time recording of the leaf water status occurred by data transfer to an Internet server. The leaf patch clamp pressure probe measures the attenuated pressure, Pp, of a leaf patch in response to a constant clamp pressure, Pclamp. Pp is sensed by a miniaturized silicone pressure sensor integrated into the device. The magnitude of Pp is dictated by the transfer function of the leaf, Tf, which is a function of leaf patch volume and ultimately of cell turgor pressure, Pc, as shown theoretically. The power function Tf=f(Pc) theoretically derived was experimentally confirmed by concomitant Pp and Pc measurements on intact leaflets of the liana Tetrastigma voinierianum under greenhouse conditions. Simultaneous Pp recordings on leaflets up to 10 m height above ground demonstrated that changes in Tf induced by Pc changes due to changes of microclimate and/or of the irrigation regime were sensitively reflected in corresponding changes of Pp. Analysis of the data show that transpirational water loss during the morning hours was associated with a transient rise in turgor pressure gradients within the leaflets. Subsequent recovery of turgescence during the afternoon was much faster than the preceding transpiration-induced water loss if the plants were well irrigated. Our data show the enormous potential of the leaf patch clamp pressure probe for leaf water studies including unravelling of the hydraulic communication between neighbouring leaves and over long distances within tall plants (trees). [ABSTRACT FROM PUBLISHER]

Abstract: Functional Channelrhodopsin-2 (ChR2) overexpression of about 104 channels/μm2 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±0.08μF/cm2 in ChR2-expressing cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (0.79±0.02μF/cm2). However, even after complete loss of microvilli under strong hypoosmolar conditions (100mOsm), the ChR2-expressing cells still exhibited a significantly larger C m (0.85±0.07μF/cm2) as compared to non-expressing control cells (0.70±0.03μF/cm2). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins. [Copyright &y& Elsevier]

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 $$ {\text{B}}{\left( {{\text{CF}}_{3} } \right)}^{ - }_{4} $$ 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 $$ {\text{B}}{\left( {{\text{CF}}_{3} } \right)}^{ - }_{4} $$ 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 $$ {\text{B}}{\left( {{\text{CF}}_{3} } \right)}^{ - }_{4} $$ 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. [ABSTRACT FROM AUTHOR]

Abstract: Giant HEK293 cells of 30–65μm in diameter were produced by three-dimensional multi-cell electrofusion in 75mOsm 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 75mOsm. 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±0.1μF/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 > 50μm) was found to be between 12.8 and 16.1μS/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. [Copyright &y& Elsevier]

Abstract: Alginate-based microencapsulation is a promising method for long-term maintenance of cellular and membrane function of the cells and tissue fragments required for in vitro and in vivo biosensors, for tissue engineering and particularly for immunoisolation of non-autologous transplants. Microcapsules of high mechanical strength and optimum permeability can be produced by injection of BaCl2 crystals into alginate droplets before they come into contact with external Ba2+. A key requirement is that the system parameters (number of crystals, speed of the crystal stream etc.) are properly adjusted according to the mannuronic and guluronic acid ratio and the average molecular mass of the alginate as well as to the diameter of the microcapsules. Robust, reliable, rapid and low-cost validation tools are, therefore, needed for assurance of the microcapsule quality. Here, we describe a novel three-dimensional (3-D) dark-field microscopy that allows the real-time measurement of the number and spatial distribution of the injected Ba2+ ions throughout the microcapsules after treatment with sulphate. This novel method requires only a conventional microscope equipped with three polarising filters and a double aperture stop. In contrast to confocal laser scanning microscopy images, peripherally attached BaSO4 precipitates can clearly be distinguished from internal ones. The data also demonstrate that several steps of the alginate gelling process must be improved before such immunoisolation can be used in patients. [Copyright &y& Elsevier]

Abstract: The Na+/H+ antiporter NhaA is the main Na+ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA''s electrogenicity, its specificity for Li+ and Na+ and its pronounced pH dependence in the range pH 6.5–8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na+ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent K m values were 11 mM for Na+ and 7.3 mM for Li+ at basic pH and 180 mM for Na+ and 50 mM for Li+ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent K m as well as turnover (V max). [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary Skin biopsies were taken from four body sites (head, thorax, flank and perineum) of three male entire Beagles and the primary hair follicles were isolated. Culture conditions were established to keep the hair follicles growing for up to 7 days. Additionally, hair follicles were incubated in supplemented medium (containing insulin, transferrin, glutamine and sodium selenite) with or without the addition of testosterone (T) (1, 10 or 100 ng/ml) or oestradiol-17β (E2β ) (0.01, 0.1 or 1 ng/ml), respectively and the daily growth of hair follicles was measured. In vitro daily growth of hair follicles from the thorax was stimulated by the low concentration of both hormones, but the growth of those from the flank was inhibited by the high concentration of both hormones. Hair follicles from the head were positively influenced by the lowest concentration of T and the medium concentration of E2β . The daily growth of hair follicles from the perineum was not significantly influenced by either hormone. [ABSTRACT FROM AUTHOR]

Reports on the electrophysiological characterization of specific interactions between bacterial sensory rhodopsins and their transducers. Use of the oocyte expression system from Xenopus laevis; Expression of pSRII and pHtrII; Effect of the transducers on the photocurrent; Current/voltage behavior of the sensory rhodopsins.

Comments on the suggestion that the major conformer responsible for ion movement across membranes is a double-stranded dimer. Background on the establishment of the identity of the channel structure; Reason that the membrane-spanning channels are not double-stranded dimers.