Voltage-activated ionic channels and conductances in embryonic chick osteoblast cultures

Summary Patch-clamp measurements were made on osteoblast-like cells isolated from embryonic chick calvaria. Cell-attachedpatch measurements revealed two types of high conductance (100–250 pS) channels, which rapidly activated upon 50–100 mV depolarization. One type showed sustained and the other transient activation over a 10-sec period of depolarization. The single-channel conductances of these channel types were about 100 or 250 pS, depending on whether the pipettes were filled with a low K⁺ (3mm) or high K⁺ (143mm) saline, respectively. The different reversal potentials under these conditions were consistent with at least K⁺ conduction. Whole-cell measurements revealed the existence of two types of outward rectifying conductances. The first type conducts K⁺ ions and activates within 20–200 msec (depending on the stimulus) upon depolarizing voltage steps from <–60 mV to >–30 mV. It inactivates almost completely with a time constant of 2–3 sec. Recovery from inactivation is biphasic with an initial rapid phase (1–2 sec) followed by a slow phase (>20 sec). The second whole-cell conductance activates at positive membrane potentials of >+50 mV. It also rapidly turns on upon depolarizing voltage steps. Activation may partly disappear at the higher voltages. Its single channels of 140 pS conductance were identified in the whole cell and did conduct K⁺ ions but were not highly Cl^– or Na⁺ selective. The results show that osteoblasts may express various types of voltage controlled ionic channels. We predict a role for such channels in mineral metabolism of bone tissue and its control by osteoblasts.     

Characterization of glucoamylase adsorption to raw starch

The adsorption of Aspergillus niger glucoamylase forms (GA-I and GA-II) to raw corn starch was studied as a function of pH, ionic strength, and temperature. A three-parameter model was developed to account for the specific and nonspecific adsorption of GA-I to starch. The adsorption of the GA-II form to raw starch was weak and independent of the pH and ionic strength of the mixture. GA-I was bound strongly to the starch surface, with association constant values ranging from 2 to 5 × 10⁶ M⁻¹. Maximum adsorption capacities (saturation concentrations) Q_max for GA-I were affected by pH, inonic strength, and temperature and varied between 1.6 and 4.3 mg protein g⁻¹ starch. The tightly bound GA-I could be specifically eluted from the starch surface with maltose, maltodextrin, or soluble starch. The adsorption of GA-II to starch in the presence of acarbose (glucoamylase activity inhibitor) indicated that the active site participates minimally in the adsorption process. The comparison of the distribution coefficients of GA-I and GA-II showed that the starch-binding domain, present only in GA-I, increases the affinity of GA-I for starch by two orders of magnitude.     

Use of cyclodextrins as chiral selectors for direct resolution of the enantiomers of fluoxetine and its metabolite norfluoxetine by HPLC

The goal of this work is to investigate the direct chromatographic separation of the enantiomers of fluoxetine and its active metabolite norfluoxetine. The liquid chromatographic retention behavior of these enantiomers on a β-cyclodextrin bonded-phase column was investigated with respect to mobile phase composition, pH, ionic strength, and solvent selectivity. Relationships were established between these factors and the three most important chromatographic parameters: retention time, resolution, and selectivity. Most of the evidence suggests that the unique selectivity of this column isdue to inclusion complex formation, which provides the physical basis for enantiomeric resolution. After these studies a set of optimum chromatographic conditions was chosen for the simultaneous separation/determination of a mixture of the four enantiomers using fluorescence detector. © 1993 Wiley-Liss, Inc.     

Hydration, protons and onset of physiological activities in maize seeds

Dry maize ( Zea mays L.) seed components, namely, embryo and endosperm, provide model materials for studies on water-dependent mechanisms in cellular function. We explored the thermodynamics of hydration for both tissues, along with their dielectric behavior, as a function of water content. In addition, we evaluated the direct current (DC) conductivity due to water protons. Our data on embryo tissue show large enthalpic and entropic peaks at water content [h, in g H₂O (g dry sampie)⁻¹] around 0.08 g g⁻¹, indicating very tight binding and ordering of water molecules. With increasing water content both enthalpy and entropy decrease, and the completion of primary hydration requires h ∼ 0.26 g g⁻¹. Data for endosperm tissue show the absence of such an enthalpic peak and a reduced degree of ordering for h < 0.10 g g⁻¹. The DC protonic conductivity shows explosive growth above a threshold hydration level h_c= 0.082 g g⁻¹ and h_c= 0.12 g g⁻¹, for embryo and endosperm, respectively. Protonic conduction can be considered within the framework of a percolation modell characterized by a hydration threshold and by a power law increase in conductivity with further hydration. The critical exponent of the power law is in agreement with theory for a two-dimensional percolative process. This percolative water-assisted behavior reflects the presence of an extended network of water molecules adsorbed on the surface of proteins and/or membranes inside cells. We consider this percolative protonic conduction as being a prerequisite to respiration processes.     

A whole-cell patch clamp technique which minimizes cell dialysis

Summary A variant of the whole-cell patch clamp technique is described which allows measurement of whole-cell ionic currents in small cells while minimizing cell dialysis with the pipette solution. The technique involves the application of negative pressure to the inside of small (< 1 µm) tip diameter pipettes placed on the cell surface to achieve high resistance seals and membrane rupture. The technique has been used successfully in a variety of different types of cells to study membrane currents carried by Ca and K, currents generated by exchange carriers as well as electrical coupling between cells. Overall, the technique seems well suited for the study of ionic currents in small cells, and provides an alternative to conventional patch clamping techniques which necessitate intracellular dialysis.     

Preliminary investigation of a bioelectrochemical sensor for the detection of phenol vapours

This work investigates the feasibility of constructing a bioelectrochemical sensor that can operate directly in gases. A series of experiments are described, resulting in a sensor that is responsive to phenol vapours. The sensor was based on ionically conducting films that incorporate a biological redox catalyst at the surface of an array of interdigitated microband electrodes. Exposure to phenol vapour drives the bioelectrochemical reaction, providing a basis for a current signal under constant potential conditions. Ionic materials included Nafion and films based on tetrabutylammonium toluene-4-sulphonate (TBATS). The quasi-reversible electrode reaction of potassium hexacyanoferrate (II) within TBATS was investigated as a function of drying time. E^o′ and K⁰ were determined at a TBATS modified microdisc electrode under steady-state conditions. Drying time (water loss) from the TBATS film had the effect of increasing the film ionic strength. It was shown that as the film ionic strength increased, E⁰′ for potassium hexacyanoferrate (II) shifts toward positive potentials (because of ion pairing) and there was a corresponding increase in the heterogeneous rate constant K⁰. The latter effect was attributed to increasing ion-ion (cation-ferrocyanide ion) interactions as the film dried and the enhancing effect this had on the prevention of surface poisoning reactions at the electrode. These factors are discussed in relation to sensor design.     

Erythrocyte stability under imposed fields

Lysis of erythrocytes offers an unique opportunity to probe the fine structure of the bilayer as a function of its state of energization. Critical monitoring of the volumes, ion fluxes and related measures in erythrocytes exposed to a variety of milieu and treatments showed that one can critically distinguish the nature of the prelytic perturbations and the proximate forces actually responsible for the disruption of the membranes among surface charge density, elastic energy etc.     

Is it possible to predict the salinity of Iberian salt lakes from their conductivity?

Conductivity measures have been widely used to estimate salinity, an obvious feature of salt lakes. Regression analysis using log-log linear model provides an adequate approach to the study of the relationship between both variables. However, actual use of that kind of statistical model suffers too frequently from lack of correctness. We have studied conductivity-salinity relationships for 69 salt lakes (128 samples) on the Iberian peninsula. Despite the satisfactory fit of the data to the model (r² = 0.88), predictive efficiency is shown to be low. This feature, which is likely to be common to this relationship anywhere, is mainly related to heterogeneity in ionic composition.     

Osmoregulation in a nectar-feeding insect, the carpenter bee Xylocopa capitata: water excess and ion conservation

Abstract The liquid diet and high metabolic water production during flight in the carpenter bee Xylocopa capitata Smith 1854 (Hymenoptera, Anthophoridae) causes a water excess, and this is exacerbated by a low dietary intake of ions. The nectar and pollen of the preferred food-plants, Virgilia divaricata Adamson and Podalyria calyptrata Willd., and other Fabaceae had low levels of sodium. Analyses of the bees and their body fluids showed that the bees have an exceptionally low Na content, and Na homeostasis seems to depend on recycling almost all Na which enters the rectum. The copious dilute urine (137 mOsm) had Na and K concentrations of only 3.4 and 7.0 ITIM, respectively. Isolated preparations of Xylocopa Malpighian tubules secreted a fluid with a K concentration 10 times that of the haemolymph. This means that recycling of K is as important as that of Na, and the bulk of K resorption probably occurs passively in the ileum. This study is the first to examine hymenopteran Malpighian tubules. Their stimulation by cAMP is indicative of the presence of a diuretic hormone in Xylocopa.