Passive Ion Fluxes Research Articles

Development of potentiometric immunosensor for determination of live attenuated Varicella Vaccine: Potency and stability studies

Determination of Varicella vaccine's potency; containing live attenuated strain (Oka) of Varicella Zoster virus, has been limited to in vitro cell culture methods. In this study, a label free potentiometric biosensor has been developed for the first time and optimized to determine the content of varicella zoster virus. A passive ion-flux sensing platform has been developed using an anti-varicella monoclonal antibody and tetrabutyl ammonium bromide as a marker ion. The immunosensor has been optimized with respect to membrane diameter and concentration of the immobilized antibody. Linearity was achieved over a concentration range of 2.5–3.2 log PFU/dose with a LOD of 1.9 log PFU/dose. Potentiometric results were compared to the plaque-forming assay using the cell culture technique. The developed immunosensor was superior with respect to analysis time and cost without affecting critical analytical performance characteristics. Furthermore, in order to evaluate the stability indicating ability of the immunosensor, the effect of pH and temperature was investigated. Vaccine samples were subjected to forced degradation conditions; pH and elevated temperatures. Stability results showed the ability of immunosensor to differentiate between intact and degraded viral content. This would demonstrate the reliability of the immunosensor for evaluating the efficacy and stability of the vaccine.

Membrane-Active Molecular Machines.

Both biological and artificial membrane transporters mediate passive transmembrane ion flux predominantly via either channel or carrier mechanisms, tightly regulating the transport of materials entering and exiting the cell. One early elegant example unclassifiable as carriers or channels was reported by Smith who derivatized a phospholipid molecule into an anion transporter, facilitating membrane transport via a two-station relay mechanism (Smith et al. J. Am. Chem. Soc. 2008, 130, 17274-17275). Our journey toward blurring or even breaking the boundaries defined by the carrier and channel mechanisms starts in January of 2018 when seeing a child swinging on the swing at the playground park. Since then, I have been wondering whether we could build a nanoscale-sized molecular swing able to perform the swing function at the molecular level to induce transmembrane ion flux. Such research journey culminates in several membrane-active artificial molecular machines, including molecular swings, ion fishers, ion swimmers, rotors, tetrapuses and dodecapuses that permeabilize the membrane via swinging, ion-fishing, swimming, rotating, or swing-relaying actions, respectively. Except for molecular ion swimmers, these unconventional membrane transporters in their most stable states readily span across the entire membrane in a way akin to channels. With built-in flexible arms that can swing or bend in the dynamic membrane environment, they transport ions via constantly changing ion permeation pathways that are more defined than carriers but less defined than channels. Applying the same benzo-crown ether groups as the sole ion-binding and -transporting units, these transporters however differ immensely in ion transport property. While the maximal K+ transport activity is achieved by the molecular swing also termed "motional channel" that displays an EC50 value of 0.021 mol % relative to lipid and transports K+ ions at rate 27% faster than gramicidin A, the highest K+/Na+ selectivity of 18.3 is attained by the molecular ion fisher, with the highest Na+/K+ selectivity of 13.7 by the molecular dodecapus. Having EC50 values of 0.49-1.60 mol % and K+/Na+ values of 1.1-6.3, molecular rotors and tetrapuses are found to be generally active but weakly to moderately K+-selective. For molecular ion swimmers that contain 10 to 14 carbon atom alkyl linkers, they all turn out to be highly active (EC50 = 0.18-0.41 mol %) and highly selective (RK+/RNa+ = 7.0-9.5) transporters. Of special note are crown ether-appended molecular dodecapuses that establish the C60-fullerene core as an excellent platform to allow for a direct translation of solution binding affinity to transmembrane ion transport selectivity, providing a de novo basis for rationally designing artificial ion transporters with high transport selectivity. Considering remarkable cytotoxic activities displayed by molecular swings and ion swimmers, the varied types of existing and emerging unconventional membrane transporters with enhanced activities and selectivities eventually might lead to medical benefits in the future.

Consensus channelome of dinoflagellates revealed by transcriptomic analysis sheds light on their physiology

Ion channels are membrane protein complexes mediating passive ion flux across the cell membranes. Every organism has a certain set of ion channels that define its physiology. Dinoflagellates are ecologically important microorganisms characterized by effective physiological adaptability, which backs up their massive proliferations that often result in harmful blooms (red tides). In this study, we used a bioinformatics approach to identify homologs of known ion channels that belong to 36 ion channel families. We demonstrated that the versatility of the dinoflagellate physiology is underpinned by a high diversity of ion channels including homologs of animal and plant proteins, as well as channels unique to protists. The analysis of 27 transcriptomes allowed reconstructing a consensus ion channel repertoire (channelome) of dinoflagellates including the members of 31 ion channel families: inwardly-rectifying potassium channels, two-pore domain potassium channels, voltage-gated potassium channels (Kv), tandem Kv, cyclic nucleotide-binding domain-containing channels (CNBD), tandem CNBD, eukaryotic ionotropic glutamate receptors, large-conductance calcium-activated potassium channels, intermediate/small-conductance calcium-activated potassium channels, eukaryotic single-domain voltage-gated cation channels, transient receptor potential channels, two-pore domain calcium channels, four-domain voltage-gated cation channels, cation and anion Cys-loop receptors, small-conductivity mechanosensitive channels, large-conductivity mechanosensitive channels, voltage-gated proton channels, inositole-1,4,5- trisphosphate receptors, slow anion channels, aluminum-activated malate transporters and quick anion channels, mitochondrial calcium uniporters, voltage-dependent anion channels, vesicular chloride channels, ionotropic purinergic receptors, animal volage-insensitive cation channels, channelrhodopsins, bestrophins, voltage-gated chloride channels H+/Cl- exchangers, plant calcium-permeable mechanosensitive channels, and trimeric intracellular cation channels. Overall, dinoflagellates represent cells able to respond to physical and chemical stimuli utilizing a wide range of Gprotein coupled receptors- and Ca2+-dependent signaling pathways. The applied approach not only shed light on the ion channel set in dinoflagellates, but also provided the information on possible molecular mechanisms underlying vital cellular processes dependent on the ion transport.

Optimization of polydopamine imprinted polymer for label free sensitive potentiometric determination of proteins: Application to recombinant human erythropoietin sensing in different matrices

The integration of molecular imprinted polymers (MIPs) with ion selective electrodes (ISE) has been an ideal platform for sensing of biological products. In this work, a facile MIP was constructed over the surface of PVC polymeric membrane using self-assembled dopamine (DA) as a monomer around recombinant human Erythropoietin (rhEPO) as a template. A label free passive ion flux MIP potentiometric sensor was fabricated using tetra butyl ammonium bromide as a marker ion. MIP preparation was optimized for critical factors (monomer concentration and polymerization time) that affect the thickness of the imprinted layer by central composite response surface design. The optimum condition for DA-MIP preparation was found to be 3.79 mg mL−1 DA for 18.14 h with desirability of 0.9919. A highly selective and sensitive potentiometric sensor was obtained by combining membrane surface minimization with superficial imprinting process strategy. The sensor has elicited selectivity for template molecule as confirmed by imprinting factor exceeding 11-fold the values obtained from closely related rhEPO analogues. The imprinted layer surface was characterized using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The quantitative determination of rhEPO was established over the range of 1.00–100.00 ng mL−1 (10-9 to 10-11 M) with a limit of detection of 0.33 ng mL−1. The sensor could classify rhEPO-α products in a similar attitude of natural antibodies. The potentiometric sensor was able to quantitate rhEPO in human serum with a limit of detection of 0.50 ng mL−1 and the bioanalytical assay was successfully validated. The proposed MIP biosensor elicited specificity for template recognition and selectivity in biological fluids without the need of immunoaffinity purification. Such approach shall plan the direction towards a portable sensor for doping disclosure in sports cheating.

Focus on Triple-Negative Breast Cancer: Potassium Channel Expression and Clinical Correlates.

Despite improvements in early diagnosis and treatment, breast cancer is still a major health problem worldwide. Among breast cancer subtypes, the most challenging and harder to treat is represented by triple-negative molecular subtype. Due to its intrinsic features this subtype cannot be treated neither with hormonal therapy (since it does not express estrogen or progesterone receptors) nor with epidermal growth factor receptor 2 (HER2) inhibitors (as it does not express high levels of this protein). For these reasons, the standard of care for these patients is represented by a combination of surgery, radiation therapy and chemotherapy. In this scenario, searching for novel biomarkers that might help both in diagnosis and therapy is mandatory. In the last years, it was shown that different families of potassium channels are overexpressed in primary breast cancers. The altered ion channel expression may be useful for diagnostic and therapeutic purposes due to some peculiar characteristics of this class of molecules. Ion channels are defined as pore-forming transmembrane proteins regulating passive ion fluxes in the cells. Ion channels represent good potential markers since, being localized at the plasma membrane level, their detection and block with specific drugs and antibodies might be fast and tunable. This review focuses on triple-negative breast cancers and recapitulates the current knowledge about potassium channels' clinical relevance and their potential use in the clinical setting, for triple-negative breast cancer diagnosis and therapy.

Ionoregulatory aspects of the hypoxia-induced osmorespiratory compromise in the euryhaline Atlantic killifish (Fundulus heteroclitus): the effects of salinity.

The osmorespiratory compromise is a physiological trade-off between the characteristics of the gill that promote respiratory gas exchange and those that limit passive flux of ions and water with the environment. In hypoxia, changes in gill blood flow patterns and functional surface area that increase gas transfer can promote an exacerbation in ion and water flux. Our goal was to determine whether the osmorespiratory compromise is flexible, depending on environmental salinity (fresh, isosmotic and sea water) and oxygen levels (hypoxia) in euryhaline killifish, Fundulus heteroclitus Plasma ion concentrations were minimally affected by hypoxia, indicating a maintenance of osmoregulatory homeostasis. In freshwater killifish, hypoxia exposure reduced branchial Na+/K+-ATPase and NEM-sensitive ATPase activities, as well as diffusive water flux rates. Unidirectional Na+ influx and Na+ efflux decreased during hypoxia in freshwater, but net Na+ flux remained unchanged. Net loss rates of Cl-, K+ and ammonia were also attenuated in hypoxia, suggesting both transcellular and paracellular reductions in permeability. These reductions appeared to be regulated phenomena as fluxes were restored immediately in normoxia. Na+ flux rates increased during hypoxia in 11 ppt, but decreased in 35 ppt, the latter suggesting a similar response to hypoxia to that in freshwater. In summary, freshwater and seawater killifish experience a reduction in gill permeability, as seen in other hypoxia-tolerant species. Fish acclimated to isosmotic salinity increased Na+ influx and efflux rates, as well as paracellular permeability in hypoxia, responses in accord with the predictions of the classic osmorespiratory compromise.

Label-Free Potentiometric Ion Flux Immunosensor for Determination of Recombinant Human Myelin Basic Protein: Application to Downstream Purification from Transgenic Milk.

Recombinant human myelin basic protein (rhMBP) produced in the milk of transgenic cows was found exclusively associated with milk caseins. This hindered its direct determination without extensive sample pretreatment. Here, a label-free potentiometric immunosensor was developed and validated for the determination of rhMBP. An ion flux was generated under zero-current based on surface blocking of the polymeric membrane ion-selective electrode by anti-hMBP antibody and tetrabutylammonium bromide as a marker ion. The immunosensor was successfully employed in the quantitative determination of hMBP in the range of 0.10-20.00 μg/mL with a limit of detection of 50.00 ng/mL. The applicability of the passive ion flux immunosensor for determination of target analyte in complex matrices was investigated. Downstream purification of rhMBP from the milk of transgenic cows was achieved using cation exchange chromatography, immobilized metal affinity chromatography, and immunoaffinity chromatography. The specificity of the immunosensor along with matrix effect of milk proteins were demonstrated. Results obtained using the rhMBP immunosensor were further cross-validated using an orthogonal testing protocol assembled of RP-HPLC and SE-HPLC. It should be noted that the proposed ion flux immunosensor provided a feasible and specific tool for monitoring rhMBP concentration/purity, immunogenic activity, and stability. Such approach provides an attractive economic alternative to sophisticated biosensors required for in-process quality control of biopharmaceutical products.

Determination of Ion Channel and Pump Fluxes for Chinese Hamster Ovary Cells using Rubidium Uptake Measurement

Cells expend a significant portion of their energy and have considerable apparatus to control the flow ions across the cell membrane. Ions concentrations have also been demonstrated to be important to processes such as apoptosis and implicated in monitoring cells metabolism for biopharmaceutical production. Accurate quantitative modelling of ion transport is challenging since the densities of ion channels and pumps are known for relatively few cells. In this work the ion channels and pumps fluxes for CHO cells were estimated using Rubidium uptake measurements combined with Ouabain inhibition of pumps.Attached CHO cells were incubated in a buffer containing 5.4 mM Rb and in some cases Ouabain was also added to the buffer in different concentrations over a period of two hours to inhibit the Na+-K+ ATPase pump to study passive and active ion fluxes across the membrane.Using Inductively Coupled Plasma Optical Emission Spectrometry, the Rb content of the lysed cells after two hours of incubation was 10.6 mg/L, which is a result of Rb transfer through the channels and pumps. From this, the Rb flux through the active and passive pathways is estimated to be 8.5×10−12 mol.cm−2.s−1.The Rb entry through the diffusion pathway was estimated using Ouabain to inhibit the Na+-K+ ATPase activity. With 5 mM concentration of Ouabain the inhibition was maximized and the Rb uptake is reduced by 80% compared to the control. From this result the Rb flux through the channels is estimated to be 2.8×10−12 mol.cm−2.s−1.The obtained results were used in temporal ion concentration simulations using methodology of Hernandez 2000. These simulations are in agreement with the work where ion pumps were inhibited via ATP production inhibition in Mitochondria using Oligomycin (B. Saboktakin Rizi, Biomicrofluidics, 2014).

Electrical signals in higher plants: Mechanisms of generation and propagation

Local stimulation induces generation and propagation of electric signals in higher plants. Noninvasive stimulus induces an action potential and damaging influences lead to the variation potential. The mechanism of the generation of an action potential is rather complex in nature and is associated with both activation of ion channels (Ca2+, Cl–, and K+) and transient change in the activity of the plasma membrane H+-ATPase. Generation of the variation potential, the duration of which is considerably longer than that of the action potential, is based on transient inactivation of the electrogenic pump; however, passive ion fluxes also contribute to such process, which causes qualitative similarity of the mechanisms of action potential and variation potential generation. Propagation of electrical signals mainly occurs in conducting bundles; thus, transfer of an action potential is associated with vascular parenchyma and sieve elements, while the variation potential is connected to the xylem vessels. The mechanism of the distribution the action potential is similar to nerve impulse transmission, while generation of the variation potential is induced by transfer of a chemical substance, whose propagation is accelerated by a hydraulic wave.

A Thermodynamic Model of Monovalent Cation Homeostasis in the Yeast Saccharomyces cerevisiae.

Cationic and heavy metal toxicity is involved in a substantial number of diseases in mammals and crop plants. Therefore, the understanding of tightly regulated transporter activities, as well as conceiving the interplay of regulatory mechanisms, is of substantial interest. A generalized thermodynamic description is developed for the complex interplay of the plasma membrane ion transporters, membrane potential and the consumption of energy for maintaining and restoring specific intracellular cation concentrations. This concept is applied to the homeostasis of cation concentrations in the yeast cells of S. cerevisiae. The thermodynamic approach allows to model passive ion fluxes driven by the electrochemical potential differences, but also primary or secondary active transport processes driven by the inter- play of different ions (symport, antiport) or by ATP consumption (ATPases). The model—confronted with experimental data—reproduces the experimentally observed potassium and proton fluxes induced by the external stimuli KCl and glucose. The estimated phenomenological constants combine kinetic parameters and transport coefficients. These are in good agreement with the biological understanding of the transporters thus providing a better understanding of the control exerted by the coupled fluxes. The model predicts the flux of additional ion species, like e.g. chloride, as a potential candidate for counterbalancing positive charges. Furthermore, the effect of a second KCl stimulus is simulated, predicting a reduced cellular response for cells that were first exposed to a high KCl stimulus compared to cells pretreated with a mild KCl stimulus. By describing the generalized forces that are responsible for a given flow, the model provides information and suggestions for new experiments. Furthermore, it can be extended to other systems such as e.g. Candida albicans, or selected plant cells.

Electomechanical Model for Non-Excitable Cells

Mechanical and electrical properties of cells are crucial in regulating a wide range of cellular processes, such as motility, cancer metastasis, and embryogenesis. Cell size and membrane potential are both highly regulated properties in eukaryotic, non-excitable cells. The proposed model explains the interplay between mechanical, electrical, and biochemical properties of a cell, and can have important implications for pathology and medicine. The combination of osmosis-driven water flow, passive ion flux, ion pumping, signaling, and cytoskeletal forces provide a mechanism for how cells control their volume and membrane potential. For example, the model predicts that cell volume increases during depolarization and decreases slightly during hyperpolarization. Whole cell voltage clamp experiments provide a technique for measuring cell volume while controlling membrane potential. Experiments done on different eukaryotic, non-excitable cancer cell lines validate the model.

The role of Ca2+, H+, and Cl− ions in generation of variation potential in pumpkin plants

The contributions of Ca2+, H+, and Cl− in generation of variation potentials (VP) in 3- to 4-week-old pumpkin (Cucurbita pepo L., cv. Mozoleevskaya) plants were assessed. During VP generation, transient alkalinization of the medium around the stem was recorded with a potentiometric method. The pH changes were kinetically similar to the electric potential changes and were apparently due to temporal suppression of the plasma-membrane electrogenic H+ pump. These data and the observed inhibition of VP in the stem zone treated locally with a metabolic inhibitor (NaN3) indicate that the VP generation is related to the reversible suppression of the H+-pump. The anion channel blocker (ethacrynic acid) decelerated significantly the front slope of VP and reduced the VP amplitude. A short-term increase in external Cl− concentration around the stem was observed during potential transients representing the VP front slope and the pulses integrated into VP. The removal of Ca2+ from extracellular medium inhibited the VP generation. It is proposed that Ca2+ plays a role in activation of anion channels and in the H+-pump inactivation. The VP generation is probably determined by a complex mechanism, with contributions from passive ion fluxes (Ca2+, Cl−) moving along the electrochemical gradients and from changes in the electrogenic pump activity.

Mathematical model of interactions between cell volume regulation and transport in cortical TAL cells

We developed a mathematical model of a cortical thick ascending limb (cTAL) cell to study interactions between ion transport and cell volume (CV) regulation. Because the NKCC2 transporter mediates apical Na+ uptake, and its activity is regulated by CV‐dependent kinases, we hypothesize that transport‐related CV changes should result in compensatory regulation of NKCC2 activity and Na+ reabsorption. The cTAL model is based on mass conservation and includes models of Na+‐K+‐ATPase, the NKCC2 (A‐isoform) and KCC1 cotransporters, and passive ion fluxes. CV‐sensitive regulation of the NKCC2 and KCC1 activities is represented with empirical functions. Model parameters were chosen to yield potentials, cytosolic [ion], and ionic fluxes consistent with published data. To test our hypothesis, we simulated published experiments which measured the relationship between short‐circuit current (Isc) and luminal [NaCl]. Simulations were done with and without NKCC2 and KCC1 regulation. The agreement between the predicted and measured responses was significantly improved, particularly at lumen [NaCl] > 75 mM, by inclusion of NKCC2 and KCC1 regulation. Moreover, the gain that provided the best fit to the Isc curve yielded regulatory volume decreases consistent with published data. These findings support the concept that CV regulatory responses modulate Na+ transport in cTAL cells.Supported by NIH grant DK‐42091.

Phosphatidylinositol 3′-Kinase Is a Critical Mediator of Interferon-γ-Induced Increases in Enteric Epithelial Permeability

The epithelial lining of mucosal surfaces acts as a barrier to regulate the entry of antigen and pathogens. Nowhere is this function of the contiguous epithelium more important than in the gut, which is continually exposed to a huge antigenic load and, in the colon, an immense commensal microbiota. We assessed the intracellular signaling events that underlie interferon (IFN) gamma-induced increases in epithelial permeability using monolayers of the human colonic T84 epithelial cell line. Confluent epithelial monolayers on semipermeable supports were treated with IFNgamma (20 ng/ml), and barrier function was assessed 48 h later by measuring transepithelial electrical resistance (TER: reflects passive ion flux), fluxes of (51)Cr-EDTA and horseradish peroxidase (HRP), and transcytosis of noninvasive, nonpathogenic Escherichia coli (strain HB101). Exposure to IFNgamma decreased barrier function as assessed by all four markers. The phosphatidylinositol 3'-kinase (PI-3K) inhibitors, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride] and wortmannin, did not affect baseline permeability characteristics but completely blocked the drop in TER, increased fluxes of (51)Cr-EDTA and HRP, and significantly reduced E. coli transcytosis evoked by IFNgamma. In addition, use of the pan-protein kinase C (PKC) inhibitor, bisindolylmaleimide I (5 muM), but not rottlerin (blocks PKCdelta), partially ameliorated the drop in TER and inhibited increased E. coli transcytosis. Addition of the PI-3K and PKC inhibitors to epithelia 6 h after IFNgamma exposure still prevented the increase in paracellular permeability but not E. coli transcytosis. Thus, IFNgamma-induced increases in epithelial paracellular and transcellular permeability are critically dependent on PI-3K activity, which may represent an epithelial-specific target to treat immune-mediated loss of barrier function.

Claudins 6, 9, and 13 are developmentally expressed renal tight junction proteins.

The adult proximal tubule is a low-resistance epithelium where there are high rates of both active transcellular and passive paracellular NaCl transport. We have previously demonstrated that the neonatal rabbit and rat proximal tubule have substantively different passive paracellular transport properties than the adult proximal tubule, which results in a maturational change in the paracellular passive flux of ions. Neonatal proximal tubules have a higher P(Na)/P(Cl) ratio and lower chloride and bicarbonate permeabilities than adult proximal tubules. Claudins are a large family of proteins which are the gate keepers of the paracellular pathway, and claudin isoform expression determines the permeability characteristics of the paracellular pathway. Previous studies have shown that claudins 1, 2, 3, 4, 5, 7, 8, 10, 11, 12, 15, and 16 are expressed in the adult mouse kidney. To determine whether there are developmental claudin isoforms, we compared the claudin isoforms present in the neonatal and adult kidney using RT-PCR to detect mRNA of claudin isoforms. Claudin 6, claudin 9, and claudin 13 were either not expressed or barely detectable in the adult mouse kidney using traditional PCR, but were expressed in the neonatal mouse kidney. Using real-time RT-PCR, we were able to detect a low level of claudin 6 mRNA expression in the adult kidney compared with the neonate, but claudin 9 and claudin 13 were only detected in the neonatal kidney. There was the same maturational decrease in these claudin proteins with Western blot analysis. Immunohistochemistry showed high levels of expression of claudin 6 in neonatal proximal tubules, thick ascending limb, distal convoluted tubules, and collecting ducts in a paracellular distribution but there was no expression of claudin 6 in the adult kidney. Using real-time RT-PCR claudin 6 and 9 mRNA were present in 1-day-old proximal convoluted tubules and were virtually undetectable in proximal convoluted tubules from adults. Claudin 13 was not detectable in neonatal or adult proximal convoluted tubules. In summary, we have identified developmentally expressed claudin isoforms, claudin 6, claudin 9, and claudin 13. These paracellular proteins may play a role in the maturational changes in paracellular permeability.

Analysis of a Model Describing the Dynamics of Intracellular Ion Composition in Biological Cells

An electrophysiological model describing the dynamics of the intracellular ion concentration and the membrane potential (Vm) in biological cells is presented. The model links passive ion fluxes through channels of sodium, potassium and chloride to active ion fluxes generated by the sodium potassium pump. To model the interaction of Vm to the ionic fluxes Kirchhoff current law is used. Only one Vm-dependent permeability as represented by an inwardly rectifying potassium channel (IKR) is incorporated. It is shown that the resulting system of ordinary differential equations is degenerate. Decomposition of the system into noninteracting subsystems allows a dynamically independent description of the currents of sodium and potassium in relation to Vm. Physical and mathematical arguments for the decomposition into subsystems are presented. Analysis of the model show hysteresis properties that can account for the experimentally-observed bistability in skeletal and heart muscles fibers.

Phospholipid subclass specific alterations in the passive ion permeability of membrane bilayers: separation of enthalpic and entropic contributions to transbilayer ion flux.

Alterations in phospholipid class, subclass, and individual molecular species contribute to the diversity of biologic membranes, but their effects on membrane passive ion permeability have not been systematically studied. Herein, we developed a simple and efficient fluorescence technique based upon the loss of valinomycin-inducible membrane potential to characterize the passive flux of ions across phospholipid bilayers. Detailed kinetic characterization of ion flux across membrane bilayers composed of discrete chemical entities demonstrated that the class, subclass, and individual molecular species of each phospholipid have substantive effects on membrane passive ion permeability properties. Increasing the degree of unsaturation in either the sn-1 or sn-2 aliphatic chains in phosphatidylcholine markedly enhanced transmembrane ion flux, with over 10-fold differences in the first-order rate constant manifested in molecular species containing four double bonds in comparison to those possessing three double bonds (e.g., kapp = 0.0014 min-1 for 1-octadec-9'-enoyl-2-octadec-9', 12'-dienoyl-sn-glycero-3-phosphocholine (18:1-18:2 phosphatidylcholine) while kapp = 0.021 min-1 for 1,2-dioctadec-9', 12'-dienoyl-sn-glycero-3-phosphocholine (18:2-18:2 phosphatidylcholine)). Moreover, although the apparent first-order rate constants for transmembrane ion flux in vesicles composed of phosphatidylcholine or plasmanylcholine containing palmitate at the sn-1 position and arachidonate at the sn-2 position were similar (kapp = 0.04 min-1 at 22 degreesC for both), the kapp for corresponding vesicles composed of plasmenylcholine was 20-fold less (kapp = 0.002 min-1 at 22 degreesC). Examination of the temperature dependence of passive membrane ion permeability demonstrated that altered ion flux across membranes composed of choline glycerophospholipids was primarily due to entropic effects without substantial changes in the activation energy for ion translocation. For example, Ea = 19.7 +/- 0.5 and 20.7 +/- 0.6 kcal.mol-1 for 1-hexadecanoyl-2-eicosa-5',8',11', 14'-tetraenoyl-sn-glycero-3-phosphocholine (16:0-20:4 phosphatidylcholine) and 1-O-(Z)-hexadec-1'-enyl-2-eicosa-5',8',11', 14'-tetraenoyl-sn-glycero-3-phosphocholine (16:0-20:4 plasmenylcholine), respectively, while their difference in the entropies of activation (DeltaS) was 4.3 +/- 0.5 cal.mol-1.K-1. Collectively, these results identify substantial differences in the membrane passive ion permeability properties of phospholipid classes, subclasses, and molecular species present in biologic membranes of eukaryotic cells and identify entropic alterations as an important contributor to these differences.

Towards the quantitative traits regulation: fountain theory implications in comparative and developmental biology.

A fountain mechanism of quantitative regulation of gene expression level during development is proposed. The mechanism is based on postulated ability of a special class of RNA molecules, so called fountain RNAs (fRNAs), to induce passive and selective ionic channels in the internal nuclear membrane. Ions diffuse via channel from the nuclear lumen into the chromatin compartment. An RNA-dependent battery of ion channels is assumed to produce <<a fountain>> of ions in close vicinity to the corresponding genes. An ion atmosphere, in its turn, locally changes the chromatin configuration and effectiveness of transcription and processing of transcripts. Hence this mechanism can be used to change genes productivity. It is a basic mechanism of quantitative traits regulation. A passive selective ion flux periodically stops after a threshold ion concentration induces local chromatin compactization and arrests the activity of ion channels in a given chromatin compartment. This process serves as a basis for many cellular biorhythms that are relatively temperature-independent because of the passive nature of ion channel. It is postulated that eukaryotes became eukaryotes just to obtain this fountain mechanism that allows them to perform gradual quantitative modulation of corresponding genes expression levels. The fountain mechanism is partly responsible for dominance and heterosis, X-chromosome inactivation, gene position effects, and some other epigenetic events. It plays an important role in embryonic and post-embryonic development. A significant portion of the former <<junk>> DNA can be referred to as fDNA involved in the proposed mechanism functioning. Genomic rearrangements of fDNA could lead to micro- and macroevolutionary changes in the animal and plant kingdoms. The pivotal evolutionary function of transposons could reside in their ability to contain and relocate fDNA along the chromosomes.

Salt Tolerance, Ion Relations and the Effect of Root Medium on the Response of Citrus to Salinity

This paper describes the ion relations of two citrus genotypes, Etrog citron (Citrus medica L.), a salt-sensitive genotype, and Rangpur lime (Citrus reticulata var. austera hybrid?), a salt-tolerant genotype, under conditions of high NaCl concentrations. Root morphology was modified by establishing seedlings for 6 months in solution or sand culture. When established in sand culture, both genotypes displayed a high efficiency, relative to plants established in solution culture, to exclude Na from leaves, but differed in their capacities to exclude Cl; this capacity was much lower in Etrog citron than Rangpur lime. The net Cl root to shoot transport rates over 7 weeks for Etrog citron and Rangpur lime, treated with 50 mol m-3 NaCl, were 0.25 and 0.07 μmol g-1 root FW h-1, respectively. The net transport rates of Cl in Etrog citron were 10 times those in Rangpur lime between weeks 4 and 7. In Rangpur lime Cl reached pseudo steady-state levels in root and leaf tissue by week 4, thereafter, the net Cl root to shoot transport rate of Rangpur lime decreased while the rates in Etrog citron increased. The accumulation of Na and Cl in leaves of Rangpur lime, in contrast to Etrog citron, was not affected by transpiration. The high salt tolerance of Rangpur lime appears to be associated with low rates of net uptake and transport of Cl, feedback control of net root to shoot transport by Cl levels in the shoot, a low interaction between water flow and Cl and Na fluxes, and a reduction in the leafiroot ratio in response to salinity. When plants were established in solution culture the pioneer roots became elongated and the normal development of the laterals into a fibrous root system was suppressed. Plants of both genotypes, previously established in solution culture and then treated with 50 mol m-3 NaCl, accumulated levels of Na and Cl in leaves up to 200-400 mol m-3. Under these conditions the rates of net Cl root to shoot transport over the 7 weeks were 0.53 and 0.56 μmol g-1 root FW h-1 for Etrog citron and Rangpur lime, respectively. These high rates of Cl uptake and transport were attributed to changes in root permeability and increases in passive ion fluxes. Keywords: citrus, root morphology, root medium, transpiration, Cl, Na, salt accumulation, salinity.

Chloride is preferentially accumulated in a subpopulation of dendrites and periglomerular cells of the main olfactory bulb in adult rats.

GABA is predominantly an inhibitory transmitter. Mediated by GABAA receptors, GABA opens chloride channels, induces a passive flux of chloride ions, which is usually directed from extracellular to intracellular space, and hyperpolarizes postsynaptic neurons. Recent electrophysiological data suggested that GABA may also depolarize neurons and exert excitatory actions. However, it remained unclear whether excitatory GABA effects are based on reversed transmembrane chloride gradient due to modifications in extracellular or intracellular chloride concentrations. Here, the first histochemical evidence is provided for local redistribution of chloride in the CNS of healthy adult rats. Olfactory bulbs were examined using freeze substitution, silver trapping of chloride and intensification techniques at light and electron microscopic level. The chloride content of precipitates was evidenced by electron spectroscopic imaging using a CEM 902 (Zeiss) electron microscope. Chloride concentration was high in a subpopulation of some periglomerular cell bodies and isolated dendritic profiles, while it seemed to be very low in certain parts of the glomerular neuropil including intercellular clefts. Data suggest that reversed chloride gradients can be demonstrated by cytochemical methods, and may be responsible for excitatory GABA effects on selected periglomerular neurons and dendrites in the olfactory glomeruli. Conditions leading to chloride redistribution in the CNS of normal adult rats remain to be determined.