KCl Gradient Research Articles

Enhancing Ionic Selectivity and Osmotic Energy by Using an Ultrathin Zr‐MOF‐Based Heterogeneous Membrane with Trilayered Continuous Porous Structure

AbstractDesigning a nanofluidic membrane with high selectivity and fast ion transport property is the key towards high‐performance osmotic energy conversion. However, most of reported membranes can produce power density less than commercial benchmark (5 W/m2), due to the imbalance between ion selectivity and permeability. Here, we report a novel nanoarchitectured design of a heterogeneous membrane with an ultrathin and dense zirconium‐based UiO‐66‐NH2 metal–organic framework (MOF) layer and a highly aligned and interconnected branched alumina nanochannel membrane. The design leads to a continuous trilayered pore structure of large geometry gradient in the sequence from angstrom‐scale to nano‐scale to sub‐microscale, which enables the enhanced directional ion transport, and the angstrom‐sized (~6.6–7 Å) UiO‐66‐NH2 windows render the membrane with high ion selectivity. Consequently, the novel heterogeneous membrane can achieve a high‐performance power of ~8 W/m2 by mixing synthetic seawater and river water. The power density can be largely upgraded to an ultrahigh ~17.1 W/m2 along with ~48.5 % conversion efficiency at a 50‐fold KCl gradient. This work not only presents a new membrane design approach but also showcases the great potential of employing the zirconium‐based MOF channels as ion‐channel‐mimetic membranes for highly efficient blue energy harvesting.

Enhancing Ionic Selectivity and Osmotic Energy by Using an Ultrathin Zr-MOF-Based Heterogeneous Membrane with Trilayered Continuous Porous Structure.

Designing a nanofluidic membrane with high selectivity and fast ion transport property is the key towards high-performance osmotic energy conversion. However, most of reported membranes can produce power density less than commercial benchmark (5 W/m2), due to the imbalance between ion selectivity and permeability. Here, we report a novel nanoarchitectured design of a heterogeneous membrane with an ultrathin and dense zirconium-based UiO-66-NH2 metal-organic framework (MOF) layer and a highly aligned and interconnected branched alumina nanochannel membrane. The design leads to a continuous trilayered pore structure of large geometry gradient in the sequence from angstrom-scale to nano-scale to sub-microscale, which enables the enhanced directional ion transport, and the angstrom-sized (~6.6-7 Å) UiO-66-NH2 windows render the membrane with high ion selectivity. Consequently, the novel heterogeneous membrane can achieve a high-performance power of ~8 W/m2 by mixing synthetic seawater and river water. The power density can be largely upgraded to an ultrahigh ~17.1 W/m2 along with ~48.5 % conversion efficiency at a 50-fold KCl gradient. This work not only presents a new membrane design approach but also showcases the great potential of employing the zirconium-based MOF channels as ion-channel-mimetic membranes for highly efficient blue energy harvesting.

Advanced ion-selective wood membranes: Leveraging aligned cellulose nanofibers for enhanced osmotic energy conversion

Nanofluidic reverse electrodialysis (RED) is widely recognized as an exceptionally effective method for harvesting osmotic energy. Nevertheless, its progress has been impeded by the high costs and intricate fabrication processes associated with the required ion exchange membranes. The ion-selective wood membranes (ISWM) are derived directly from natural wood through a meticulously designed two-step chemical modification and densification process. The obtained ISWM with a highly charged surface effectively preserves the alignment of nanochannels from the cellulose fibers sourced in natural wood. Moreover, the laminated structure consisting of oriented nanofibers enhances ion conductivity by a factor of 4 compared to natural wood birch under low KCl concentrations. The charged and aligned nanochannels serve as nanofluidic conduits, facilitating selective ion transport. This, in turn, engenders efficient charge separation and the generation of an electrochemical potential difference. In energy harvesting systems such as ISWM-RED, the synergistic interplay between the electrochemical potential difference and ionic flux manifests in a remarkable output power density, reaching up to 658 mW m−2 to a salinity gradient of KCl at a magnitude of 50-fold. ISWM is an outstanding nanofluidic material that provides a cost-effective and easy-to-prepare solution for producing natural nanofluidic materials. Our investigation delineates a promising trajectory for developing high-performance nanofluidic RED devices tailored for efficient osmotic energy harvesting from renewable and abundant natural resources.

Molecular self-assembled cellulose enabling durable, scalable, high-power osmotic energy harvesting

In recent years, renewable cellulose-based ion exchange membranes have emerged as promising candidates for capturing green, abundant osmotic energy. However, the low power density and structural/performance instability are challenging for such cellulose membranes. Herein, cellulose-molecule self-assembly engineering (CMA) is developed to construct environmentally friendly, durable, scalable cellulose membranes (CMA membranes). Such a strategy enables CMA membranes with ideal nanochannels (∼7 nm) and tailored channel lengths, which support excellent ion selectivity and ion fluxes toward high-performance osmotic energy harvesting. Finite element simulations also verified the function of tailored nanochannel length on osmotic energy conversion. Correspondingly, our CMA membrane shows a high-power density of 2.27 W/m2 at a 50-fold KCl gradient and super high voltage of 1.32 V with 30-pair CMA membranes (testing area of 22.2 cm2). In addition, the CMA membrane demonstrates long-term structural and dimensional integrity in saline solution, due to their high wet strength (4.2 MPa for N-CMA membrane and 0.5 MPa for P-CMA membrane), and correspondingly generates ultrastable yet high power density more than 100 days. The self-assembly engineering of cellulose molecules constructs high-performance ion-selective membranes with environmentally friendly, scalable, high wet strength and stability advantages, which guide sustainable nanofluidic applications beyond the blue energy.

Isolation and purification of isocitrate lyase by chromatographic meth-ods from the liver of rats under conditions of alloxan-induced diabetes

The homogeneous preparation with isocitrate lyase activity was obtained from the peroxisomal fraction of rat hepatocytes using chromatographic methods. Male white inbred laboratory Wistar rats (Rattus norvegicus) were used as the object of the study. Experimental diabetes was induced by a single injection of a 5% alloxan solution. The development of diabetes was monitored by measuring blood glucose levels using a glucometer (SatellitePlus PKG-02.4). Blood sampling was carried out in the morning from the tail vein. On the 10th day of the experiment, laboratory animals were subjected to decapitation, previously euthanized with ether anaesthesia, to obtain liver tissue samples. For the isolation of a homogeneous preparation of isocitrate lyase (ICL, EC 4.1.3.1.), chromatographic methods were used. Purification included several stages: fractionation of the homogenate with ammonium sulphate, gel filtration on columns filled with Sephadex G-25 and ion exchange chromatography, which was the determining stage. The anion exchanger DEAE-Sephacel was used as a sorbent. This allowed to achieve a degree of purification of 12.12 for the first ICL isoform and 24.24 for the second ICL isoform. The specific activity for the first sample was 0.04 U/mg protein, and the yield was 26.3%. The specific activity value for the second form of ICL was 0.08 U/mg protein, and the yield was 47.4%. For elution from a column filled with DEAE-Sephacel was performed using step KCl gradient (60 mM – 200 mM). Identification of the resulting preparations was carried out spectrophotometrically, following the increase in the optical density, according to the Kornberg method (λ-324 nm). The presence of isoforms was determined by specific staining of the gel after PAGE electrophoresis. As a result of four-step purification, two isoforms with different from ICL (EC 4.1.3.1) from other sources electrophoretic mobility were obtained.

Porous Ti3C2Tx MXene Membranes for Highly Efficient Salinity Gradient Energy Harvesting.

Extracting osmotic energy through nanoporous membranes is an efficient way to harvest renewable and sustainable energy using the salinity gradient between seawater and river water. Despite recent advances of nanopore-based membranes, which have revitalized the prospect of blue energy, their energy conversion is hampered by nanomembrane issues such as high internal resistance or low selectivity. Herein, we report a lamellar-structured membrane made of nanoporous Ti3C2Tx MXene sheets, exhibiting simultaneous enhancement in permeability and ion selectivity beyond their inherent trade-off. The perforated nanopores formed by facile H2SO4 oxidation of the sheets act as a network of cation channels that interconnects interplanar nanocapillaries throughout the lamellar membrane. The constructed internal nanopores lower the energy barrier for cation passage, thereby boosting the preferential ion diffusion across the membrane. A maximum output power density of the nanoporous Ti3C2Tx MXene membranes reaches up to 17.5 W·m–2 under a 100-fold KCl gradient at neutral pH and room temperature, which is as high as by 38% compared to that of the pristine membrane. The membrane design strategy employing the nanoporous two-dimensional sheets provides a promising approach for ion exchange, osmotic energy extraction, and other nanofluidic applications.

Biophysical characteristics of TRIC-A and TRIC-B channels and their regulation of RYR2

Trimeric intracellular cation channels (TRIC-A and TRIC-B), found in the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes, are thought to provide countercurrents to balance Ca2+-movements across the SR, but there is also evidence that they physically interact with ryanodine receptors (RYR). We therefore investigated if TRIC channels could modulate the single-channel function of RYR2 after incorporation of vesicles isolated from HEK293 cells expressing TRIC-A or TRIC-B with RYR2 into artificial membranes under voltage clamp. We also examined the gating and conductance properties of TRIC channels. Co-expression of RYR2 with either TRIC-A or TRIC-B significantly altered the gating behavior of RYR2; however, co-expression with TRIC-A was particularly effective at potentiating the activating effects of cytosolic Ca2+. Fusing membrane vesicles containing TRIC-A or TRIC-B together with RYR2 into bilayers produced large currents of rapidly gating current fluctuations of multiple amplitudes. In 740 cytosolic/210 luminal mM KCl gradient, current-voltage relationships of macroscopic currents revealed average reversal potentials (Erev) of -13.67 ± 9.02 (n = 7), -2.11 ± 3.84 (n = 11), and 13.19 ± 3.23 (n = 13, **, P = 0.0025) from vesicles from RYR2 only, RyR2 + TRIC-A, or RyR2 + TRIC-B cells, respectively. Thus, with the incorporation of TRIC channels, the Erevs depart further from the calculated Erev for ideally selective cation channels than occurs when vesicles from RYR2-only cells are incorporated, suggesting that TRIC channels are permeable to both K+ and Cl-. In conclusion, our results indicate that both TRIC-A and TRIC-B regulate the gating of RYR2, but that TRIC-A has greater capacity to stimulate the RYR2 opening. The results also suggest that TRIC channels may be relatively nonselective ion channels being permeable to both cations and anions. This property would enable TRIC channels to be versatile providers of counter-ion current throughout the SR of many cell types.

Effect of Auxin (IAA) on the Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles.

In contrast to the well-studied effect of auxin on the plasma membrane K+ channel activity, little is known about the role of this hormone in regulating the vacuolar K+ channels. Here, the patch-clamp technique was used to investigate the effect of auxin (IAA) on the fast-activating vacuolar (FV) channels. It was found that the macroscopic currents displayed instantaneous currents, which at the positive potentials were about three-fold greater compared to the one at the negative potentials. When auxin was added to the bath solution at a final concentration of 1 µM, it increased the outward currents by about 60%, but did not change the inward currents. The imposition of a ten-fold vacuole-to-cytosol KCl gradient stimulated the efflux of K+ from the vacuole into the cytosol and reduced the K+ current in the opposite direction. The addition of IAA to the bath solution with the 10/100 KCl gradient decreased the outward current and increased the inward current. Luminal auxin reduced both the outward and inward current by approximately 25% compared to the control. The single channel recordings demonstrated that cytosolic auxin changed the open probability of the FV channels at the positive voltages to a moderate extent, while it significantly increased the amplitudes of the single channel outward currents and the number of open channels. At the positive voltages, auxin did not change the unitary conductance of the single channels. We suggest that auxin regulates the activity of the fast-activating vacuolar (FV) channels, thereby causing changes of the K+ fluxes across the vacuolar membrane. This mechanism might serve to tightly adjust the volume of the vacuole during plant cell expansion.

Nanopore Resistive Pulse Sensing with Multiple Alpha-Hemolysin Pores Improves the Detection Limit of Microrna

The concentration profile of microRNAs in body fluids has been related to a variety of diseases, including specific cancers. By conjugation with a complementary DNA probe, specific miRNA species can be enumerated by nanopore resistive pulsing. This is possible because a miRNA-DNA probe duplex temporarily blocks a nanopore of ∼1.5 nm diameter, causing a decrease in the electrical current by displacing electrolyte ions. Under the influence of the electrophoretic driving force, the duplex dissociates after ∼10-1000 ms, and both the miRNA and the DNA molecules transit through the pore. The pore is now open again and can capture another duplex, with the frequency of the duplex pore translocations corresponding to the miRNA concentration in the solution. Biological porins are attractive because of their reproducible self-assembly, but the lipid bilayer matrix prevents the application of voltages >120 mV. It has previously been shown that the electrophoretic driving force can also be enhanced by an electrolyte concentration gradient of 0.5 / 4 M KCl, but few pulses were observed below 1 nM duplex concentration. Here, we demonstrate that the frequency can be further increased by incorporating multiple pores in the same bilayer. With an electrolyte gradient of 0.2 / 1 M KCl, it was possible to perform resistive pulse sensing with up to 100 alpha-hemolysin pores in an aperture-suspended DPhPC bilayer. We characterize the effect of electrolyte salt gradients on the stability of bilayer-incorporated alpha-hemolysin and quantify the resistive pulses for duplex concentrations in the range 0.01-100 nM. The achieved ∼10-fold increase in frequency, with respect to single-pore sensing with a 0.5 / 4 M KCl gradient, demonstrates the potential of multi-pore sensing to achieve miRNA detection at clinically relevant concentrations.

Unraveling the Anomalous Surface-Charge-Dependent Osmotic Power Using a Single Funnel-Shaped Nanochannel.

Nanofluidic osmotic power, which converts a difference in salinity between brine and fresh water into electricity with nanoscale channels, has received more and more attention in recent years. It is long believed that to gain high-performance osmotic power, highly charged channel materials should be exploited so as to enhance the ion selectivity. In this paper, we report counterintuitive surface-charge-density-dependent osmotic power in a single funnel-shaped nanochannel (FSN), violating the previous viewpoint. For the highly charged nanochannel, the performance of osmotic power decreases with a further increase in its surface charge density. With increasing pH (surface charge density), the FSN enables a local maximum power density as high as ∼3.5 kW/m2 in a 500 mM/1 mM KCl gradient. This observation is strongly supported by our rigorous model where the equilibrium chemical reaction between functional carboxylate ion groups on the channel wall and protons is taken into account. The modeling reveals that for a highly charged nanochannel, a significant increase in the surface charge density amplifies the ion concentration polarization effect, thus weakening the effective salinity ratio across the channel and undermining the osmotic power generated.

α-Synuclein Interaction with and Translocation by the MspA Porin

We have recently reported (Hoogerheide et al., Biophysical Journal 2018, 114, 772-776) the use of “selectivity tags” to track the translocation of a non-uniformly charged polypeptide through the mitochondrial VDAC nanopore on the single molecule level. We now demonstrate the generality of the selectivity tag technique with another widely used, robust β-barrel nanopore, the mycobacterial porin MspA. First, we show that α-Synuclein (α-Syn), a naturally occurring, intrinsically disordered polypeptide associated with Parkinson's disease pathology and mitochondrial bioenergetics, can interact with the bilayer-reconstituted D90N/D91N/D93N/D118R/E139K/D134R MspA pore (“MspA”). α-Syn causes a discrete ∼80% blockage of the MspA current, with the blockage on-rate rising exponentially with the applied field. The residence time, on the other hand, displays a crossover behavior, indicating that at voltages >50 mV the entire α-Syn polymer passes through the pore. Second, threading α-Syn, which has a “diblock copolymer” structure with regions of opposite uniform charges at its ends, through MspA strongly modulates the electrostatics and ionic selectivity of MspA's constriction, with an increased sensitivity compared to the original findings reported for the VDAC nanopore (Hoogerheide et al., 2018). Under conditions of 0.2/1 M KCl gradient across the membrane, the α-Syn interactions with MspA produce readily detectable current sub-levels, which correspond to differently charged regions of α-Syn polypeptide as it interacts and threads through the pore and allow for direct single-molecule detection of α-Syn translocation success. Third, we employ the selectivity tag technique to observe the effect of various co-solvents (e.g. salt species) on the process of α-Syn-MspA translocation.

Increased dwell time and occurrence of dsDNA translocation events through solid state nanopores by LiCl concentration gradients.

Solid-state nanopore based biosensors are cost effective, high-throughput engines for single molecule detection of biomolecules with the added benefit of size modification. Progress in the translation of the science into a viable diagnostic tool is impeded by inadequate sensitivity of data acquisition systems in detection of fast DNA translocations through the pore. To combat this, slowing the transport of DNA through the nanopore by use of various media or by altering experimental parameters is common. Applying a concentration gradient of KCl in the experimental ionic solution has been shown to effectively prolong dwell times as well as increase the capture rate of DNA by the nanopore. Our previous work has corroborated the ability of LiCl ionic solution to slow down the transport of dsDNA through the nanopore by up to 10-fold through cation-DNA interactions. However, this drastically reduced the event occurrence frequency, thus hindering the efficacy of this system as a reliable biosensor downstream. Here, we present the use of a concentration gradient of lithium chloride ionic solution to increase the event frequency of single molecule dsDNA translocation through a solid state nanopore. By using 0.5M/3M LiCl on the cis/trans chambers respectively, average dwell times experienced up to a 3-fold increase when compared to experiments run in symmetric 1 M LiCl. Additionally, experiments using the 0.5 M/3 M displayed a greater than 10-fold increase in event frequency, confirming the capture propensity of the asymmetric conditions.

Slowed Down Double-Stranded DNA Transport through Solid-State Nanopores by using a Lithium Chloride Concentration Gradient

Nanopore biosensors represent the future of diagnostic devices as they are low-cost, high-throughput engines for single molecule detection. Solid-state nanopores are particularly attractive since their size can be controlled to detect a wide range of analytes in solution. However, progress in the field is impeded by inadequate sensitivity of data acquisition systems in detection of fast DNA translocations through the pore. One way to overcome this is by slowing translocation of DNA through the nanopore by use of various media or by altering experimental parameters. Applying a concentration gradient of KCl in the experimental buffer has been shown to effectively prolong dwell times by creating a free-energy barrier that DNA molecules have to overcome. In addition to this, the concentration gradient enhances the magnitude of the local potential, Vr, increasing the capture rate of DNA and thereby increasing event frequency. Our previous work has demonstrated the ability of LiCl buffer to slow down the transport of dsDNA through the nanopore by up to 10-fold through cation/DNA interactions. However, this drastically reduced the event frequency, which can affect the efficacy of this system as a reliable biosensor downstream. Here, we present the use of a concentration gradient of Lithium Chloride buffer to increase the event frequency of dsDNA translocation and further increase dwell time to enhance detection of single molecules through a solid state nanopore. By using 0.5M/3M LiCl on the cis/trans chambers respectively, average dwell times experienced up to a 3-fold increase when compared to experiments run in symmetric 1M LiCl. Additionally, experiments using the 0.5M/3M displayed a greater than 10-fold increase in event frequency, confirming the capture propensity of the asymmetric conditions.

Salt Gradient Modulation of MicroRNA Translocation through a Biological Nanopore.

In resistive pulse sensing of microRNA biomarkers, selectivity is achieved with polynucleotide-extended DNA probes, with the unzipping of a miRNA-DNA duplex in the nanopore recorded as a resistive current pulse. As the assay sensitivity is determined by the pulse frequency, we investigated the effect of cis/trans electrolyte concentration gradients applied over α-hemolysin nanopores. KCl gradients were found to exponentially increase the pulse frequency, while reducing the preference for 3'-first pore entry of the duplex and accelerating duplex unzipping, all manifestations of an enhanced electrophoretic force. Unlike silicon nitride pores, a counteracting contribution from electro-osmotic flow along the pore wall was not apparent. Significantly, a gradient of 0.5/4 M KCl increased the pulse frequency ∼60-fold with respect to symmetrical 1 M KCl, while the duplex dwell time in the nanopore remained acceptable for pulse detection and could be extended by LiCl addition. Steeper gradients caused lipid bilayer destabilization and pore instability, limiting the total number of recorded pulses. The 8-fold KCl gradient enabled a linear relationship between pulse frequency and miRNA concentration for the range of 0.1-100 nM. This work highlights differences between biological and solid-state nanopore sensing and provides strategies for subnanomolar miRNA quantification with bilayer-embedded porins.

Vacuolar ion channels in the liverwort Marchantia polymorpha: influence of ion channel inhibitors

Main conclusionPotassium-permeable slow activating vacuolar channels (SV) and chloride-permeable channels in the vacuole of the liverwortMarchantia polymorphawere characterized in respect to calcium dependence, selectivity, and pharmacology.The patch-clamp method was used in the study of ion channel activity in the vacuoles from the liverwort Marchantia polymorpha. The whole-vacuole recordings allowed simultaneous observation of two types of currents—predominant slow activated currents recorded at positive voltages and fast activated currents recorded at negative voltages. Single-channel recordings carried out in the gradient of KCl indicated that slow activated currents were carried by potassium-permeable slowly activating vacuolar channels (SV) and fast activated currents—by chloride-permeable channels. Both types of the channels were dependent in an opposite way on calcium, since elimination of this ion from the cytoplasmic side caused inhibition of SV channels, but the open probability of chloride-permeable channels even increased. The dependence of the activity of both channels on different types of ion channel inhibitors was studied. SV channels exhibited different sensitivity to potassium channel inhibitors. These channels were insensitive to 3 mM Ba2+, but were blocked by 3 mM tetraethyl ammonium (TEA). Moreover, the activity of the channels was modified in a different way by calcium channel inhibitors. 200 µM Gd3+ was an effective blocker, but 50 µM ruthenium red evoked bursts of the channel activity resulting in an increase in the open probability. Different effectiveness of anion channel inhibitors was observed in chloride-permeable channels. After the application of 100 µM Zn2+, a decrease in the open probability was recorded but the channels were still active. 50 µM DIDS was more effective, as it completely blocked the channels.

A Glutathione S-transferase Elutes with Glyoxalase-I (Gly-I) During Purification of Gly-I from Maize ( Zea mays L.) Seedlings

In this study an attempt was taken to purify Glyoxalase-I (Gly-I: E.C., 4.4.1.5), from maize seedlings. Both green and non-green parts of 7 day old maize seedlings were used as plant materials. Crude proteins were precipitated by 65% (NH4)2SO4, and dialyzed overnight. The dialyzate was applied on DEAE-cellulose chromatography and eluted with linear gradient of KCl from 0 to 0.2 M. In both cases, Gly-I eluted at approximately 85 mM of KCL. The active Gly-I fractions were pooled and applied on a hydroxylapatite chromatography and eluted with 0-40 mM potassium-phosphate buffer, but the eluted fractions showed very poor activity. Therefore, the active pooled fraction of DEAE-chromatography was then applied directly on affinity chromatography (S-hexyl glutathione-agarose) for final purification and eluted with 1.2 mM of S-hexyl glutathione. The purified protein from green and non-green part had specific activity of 33.23 and 39.25 μmol min-1 mg-1 protein, respectively, along with recovery of 1.47 and 162, respectively, and yield of 83.11 and 68.15, respectively. In SDS-PAGE, the active purified affinity fraction was found to move with another protein. The spectrophotometric analysis of high active Gly-I fractions from DEAE-cellulose and affinity chromatography showed GST [another detoxifying enzyme (E.C., 2.5.1.18)] activity. This result suggested that one of the adjacent protein bands in SDS-PAGE was due to presence of a GST in Gly-I fraction.

Mitochondrial large-conductance potassium channel from Dictyostelium discoideum

In the present study, we describe the existence of a large-conductance calcium-activated potassium (BKCa) channel in the mitochondria of Dictyostelium discoideum. A single-channel current was recorded in a reconstituted system, using planar lipid bilayers. The large-conductance potassium channel activity of 258±12pS was recorded in a 50/150mM KCl gradient solution. The probability of channel opening (the channel activity) was increased by calcium ions and NS1619 (potassium channel opener) and reduced by iberiotoxin (BKCa channel inhibitor). The substances known to modulate BKCa channel activity influenced the bioenergetics of D. discoideum mitochondria. In isolated mitochondria, NS1619 and NS11021 stimulated non-phosphorylating respiration and depolarized membrane potential, indicating the channel activation. These effects were blocked by iberiotoxin and paxilline. Moreover, the activation of the channel resulted in attenuation of superoxide formation, but its inhibition had the opposite effect. Immunological analysis with antibodies raised against mammalian BKCa channel subunits detected a pore-forming α subunit and auxiliary β subunits of the channel in D. discoideum mitochondria. In conclusion, we show for the first time that mitochondria of D. discoideum, a unicellular ameboid protozoon that facultatively forms multicellular structures, contain a large-conductance calcium-activated potassium channel with electrophysiological, biochemical and molecular properties similar to those of the channels previously described in mammalian and plant mitochondria.

Comparative Investigation of Glutathione S-transferase (GST) in Different Crops and Purification of High Active GSTs from Onion (Allium cepa L.)

Glutathione S-transferases (GSTs; EC 2.5.1.18) are abundant proteins encoded by a highly divergent ancient gene family with protective functions through detoxification and non-catalytic roles as carriers of cytotoxins. In this work, we investigated the GST activities in seedlings of 38 crops to obtain a plant with high active GST for further study. In screening of 38 crops, onion seedling showed the highest GST activity (483.54 nmol min-1mg-1 protein) followed by wheat(372.89 nmol min-1mg-1 protein), barley (253.44 nmol min-1mg-1 protein), rice (244.12 nmol min-1mg-1 protein) and proso millet (173.34 nmol min-1mg-1 protein). Carrot seedling showed the lowest activity (3.63 nmol min-1mg-1 protein).In onion plants, both root and leaf showed high GST activity. Onion bulb GSTs were separated DEAE cellulose column chromatography, and purified by affinity chromatography (S-hexyl glutathione-agarose). Three GST peaks were found to elute at 67, 107 and 140mM of KCl gradient solution, and were named as GSTa, GSTb and GSTc. Among the three GSTs, GSTa, GSTb and GSTc contained 9.58, 61.45 and 28.97% of total activity, respectively. In purification, GSTa, GSTb and GSTc had specific activities of 9075, 17259 and 19868 nmol min-1 mg-1 protein, respectively, along with yield of 2.48, 3.17 and 1.28,and purification fold of 15.8, 29.9 and 34.5, respectively. The purity and molecular mass of the fraction was examined by SDS-PAGE. The silver staining of the purified GSTa, GSTb and GSTc indicated that final product of GSTb and GSTc were highly purified and migrated as a single band on SDS-PAGE with an apparent molecular mass of 27 kDa. However, GSTa eluted with Glyoxalase-I (Gly-I) and to purify GSTa, more methodological application are suggested.

Selective evaluation of high density lipoprotein from mouse small intestine by an in situ perfusion technique

The small intestine (SI) is the second-greatest source of HDL in mice. However, the selective evaluation of SI-derived HDL (SI-HDL) has been difficult because even the origin of HDL obtained in vivo from the intestinal lymph duct of anesthetized rodents is doubtful. To shed light on this question, we have developed a novel in situ perfusion technique using surgically isolated mouse SI, with which the possible filtration of plasma HDL into the SI lymph duct can be prevented. With the developed method, we studied the characteristics of and mechanism for the production and regulation of SI-HDL. Nascent HDL particles were detected in SI lymph perfusates in WT mice, but not in ABCA1 KO mice. SI-HDL had a high protein content and was smaller than plasma HDL. SI-HDL was rich in TG and apo AIV compared with HDL in liver perfusates. SI-HDL was increased by high-fat diets and reduced in apo E KO mice. In conclusion, with our in situ perfusion model that enables the selective evaluation of SI-HDL, we demonstrated that ABCA1 plays an important role in intestinal HDL production, and SI-HDL is small, dense, rich in apo AIV, and regulated by nutritional and genetic factors.

Ion permeability of artificial membranes evaluated by diffusion potential and electrical resistance measurements (719.1)

A novel model of artificial membranes that provides efficient assistance in teaching the origins of diffusion potentials is proposed. These membranes are made of polycarbonate filters fixed to 12 mm plastic rings and then saturated with a mixture of creosol and n‐decane. Electrical resistance and potential difference across these membranes can be easily measured using low‐cost volt‐ohmmeter and home‐made Ag/AgCl electrodes. The advantage of the model is the lack of ionic selectivity of the membrane, which can be modified by introduction of different ionophores to the organic liquid mixture. Membrane treated with the mixture containing valinomycin generates voltages from ‐53 mV to ‐25 mV in the presence of a 10‐fold KCl gradient (in‐to‐out) and from ‐79 mV to ‐53 mV in the presence of a bi‐ionic KCl/NaCl gradient (in‐to‐out). This latter bi‐ionic gradient potential reverses to a value from +9 mV to +20 mV when monensin is present in the organic liquid mixture. Thus, the model can be build stepwise, i.e. all factors leading to the development of diffusion potentials can be introduced sequentially, helping to understand the quantitative relationships of ionic gradients and differential membrane permeability in the generation of cell electrical signals.Grant Funding Source: Supported by FER (ULB)