Channel-like Pathway Research Articles

Carboxylated carbon nanotubes can serve as pathways for molecules in sandwich-like two-phase organic-water systems

Molecular dynamics simulations of pristine and carboxylated (6,6) carbon nanotubes (CNTs) immersed in water-chloroform, water-formaldehyde and water-nitromethane mixtures were performed at ambient conditions. The selected co-solvents differed in their degree of polarity (chloroform < formaldehyde < nitromethane) and proticity (formaldehyde is a polar aprotic solvent, while nitromethane is a polar protic solvent). The hydrophobic character of the CNTs was altered via functionalizing one of the nanotube endings with three −COO− groups, or by decorating both endings with three −COO− groups on each side. The immiscibility of water with chloroform and nitromethane leads to a sandwich-like liquid-liquid structure of the solvent mixture. In both cases the pristine CNT preferred to be fully burred inside the organic phase, while partial or complete carboxylation of the CNT openings oriented the nanotube in such a way as to form a channel-like pathway through the organic layer (i.e. −COO− groups touching aqueous phase and the hydrophobic part immersed in the organic phase). Since the nonpolar chloroform molecules were too large to enter the interior of the (6,6) CNT, the nanotube presented a pathway for water transport between the layers. In the case of water-formaldehyde mixture (no phase separation) the hydrophobic character of the CNT was the only decisive factor for the preferential solvation of the tube. Here, −COO− groups were predominantly hydrated, while formaldehyde molecules preferentially solvated the hydrophobic parts of the CNT's exterior and interior.

Modeling selective intergranular oxidation of binary alloys.

Intergranular attack of alloys under hydrothermal conditions is a complex problem that depends on metal and oxygen transport kinetics via solid-state and channel-like pathways to an advancing oxidation front. Experiments reveal very different rates of intergranular attack and minor element depletion distances ahead of the oxidation front for nickel-based binary alloys depending on the minor element. For example, a significant Cr depletion up to 9 μm ahead of grain boundary crack tips was documented for Ni-5Cr binary alloy, in contrast to relatively moderate Al depletion for Ni-5Al (∼100 s of nm). We present a mathematical kinetics model that adapts Wagner's model for thick film growth to intergranular attack of binary alloys. The transport coefficients of elements O, Ni, Cr, and Al in bulk alloys and along grain boundaries were estimated from the literature. For planar surface oxidation, a critical concentration of the minor element can be determined from the model where the oxide of minor element becomes dominant over the major element. This generic model for simple grain boundary oxidation can predict oxidation penetration velocities and minor element depletion distances ahead of the advancing front that are comparable to experimental data. The significant distance of depletion of Cr in Ni-5Cr in contrast to the localized Al depletion in Ni-5Al can be explained by the model due to the combination of the relatively faster diffusion of Cr along the grain boundary and slower diffusion in bulk grains, relative to Al.

Organic Pollutant Clustered in the Plant Cuticular Membranes: Visualizing the Distribution of Phenanthrene in Leaf Cuticle Using Two-Photon Confocal Scanning Laser Microscopy

Plants play a key role in the transport and fate of organic pollutants. Cuticles on plant surfaces represent the first resistance for the uptake of airborne toxicants. In this study, a confocal scanning microscope enhanced with a two-photon laser was applied as a direct and noninvasive probe to explore the in situ uptake of a model pollutant, phenanthrene (PHE), into the cuticular membrane of a hypostomatic plant, Photinia serrulata. On the leaf cuticle surfaces, PHE forms clusters instead of being evenly distributed. The PHE distribution was quantified by the PHE fluorescence intensity. When PHE concentrations in water varying over 5 orders of magnitude were applied to the isolated cuticle, the accumulated PHE level by the cuticle was not vastly different, whether PHE was applied to the outer or inner side of the cuticle. Notably, PHE was found to diffuse via a channel-like pathway into the middle layer of the cuticle matrix, where it was identified to be composed of polymeric lipids. The strong affinity of PHE for polymeric lipids is a major contributor of the fugacity gradient driving the diffusive uptake of PHE in the cuticular membrane. Membrane lipids constitute important domains for hydrophobic interaction with pollutants, determining significant differentials of fugacities within the membrane microsystem. These, under unsteady conditions, contribute to enhance net transport and clustering along the z dimension. Moreover, the liquid-like state of polymeric lipids may promote mobility by enhancing the diffusion rate. The proposed "diffusive uptake and storage" function of polymeric lipids within the membrane characterizes the modality of accumulation of the hydrophobic contaminant at the interface between the plant and the environment. Assessing the capacity of fugacity of these constituents in detail will bring about knowledge of contaminant fate in superior plants with a higher level of accuracy.

Studies on the Effect of Salts on the Channel Activity of Kissper, a Kiwi Fruit Peptide

Fruits and vegetables are known to possess health benefits: indeed, they contain a large number of bioactive molecules with a positive impact on human health. Dietary proteins possess specific biological properties which make these components potential ingredients of functional or health-promoting foods. Many of these properties are attributed to physiologically-active peptides encrypted in protein molecules. Kissper, a 39-residue peptide isolated from kiwi fruit (Actinidia deliciosa), derives from the processing of kiwellin, a well-represented novel allergenic protein present in the edible part of the fruit. Kissper has six cysteine residues forming three disulfide bridges. This cysteine pattern is similar, but not identical, to those observed in some plant and animal proteins, including toxins involved in defence mechanisms. We show that kissper forms ion channels in DOPS:DOPE:POPC PLMs in different media, such as KCl, potassium gluconate, potassium citrate and potassium phosphate monobasic. Kissper's channels show a strong voltage dependence in all media used, and an estimated pore diameter in the range from 1.9 to 7.6 A in KCl medium. Finally, kissper's pores shift selectivity from anions to neutral to cations, depending on the nature of the salt solutions. Therefore we propose the kissper as a novel nutraceutical component of kiwi fruit that may have a positive impact on human health due to its channel-like pathways which modulate membrane permeability. The kissper action might promote the adsorption of substances, in particular, within the gastrointestinal tract, helping digestion, preventing constipation and harmful accumulation of metabolites.

Forms and sand transport in shallow hydraulic fractures in residual soil

Four sand-filled hydraulic fractures were created at a depth of 1.5 m, and the vicinities of the fractures were excavated and mapped in detail. All the fractures were shaped like slightly asymmetric saucers between 4.5 and 7.0 m across that were roughly flat-lying in their centers and curved upward to dip between 10° and 15° along their peripheries. Three different colors of sand were injected in sequence to trace the relative ages of the sand in the fracture. The first sand to be injected remained in the vicinity of the injection casing, whereas the last sand moved rapidly to the leading edge. Sand transport occurred through localized, channel-like pathways that extended from the injection casing and then branched into multiple paths as they approached the leading edge. At least four branching pathways of different ages were identified in one fracture, suggesting that this represents a fundamental mechanism of sand transport in these shallow fractures. A theoretical model was developed by adapting Franc2d, a code well-known in structural mechanics, to predict the propagation paths of curved hydraulic fractures at shallow depths. The model predicts fracture forms that are remarkably similar to those in field exposures when properties typical of field conditions are used.Key words: hydraulic fracture, field test, mechanics, modeling.

EFFECTS OF ECONAZOLE ON Ca2+ LEVELS IN AND THE GROWTH OF HUMAN PROSTATE CANCER PC3 CELLS

1. Econazole is used clinically as an antifungal drug with many different in vitro effects. However, the effects of econazole on prostate cancer cells are unknown. The effects of econazole on intracellular Ca2+ concentrations ([Ca2+]i) in and the proliferation of human PC3 prostate cancer cells was explored in the present study using fura-2 and tetrazolium as fluorescent dyes. 2. At a concentration of 0.1 micromol/L, econazole started to increase [Ca2+]i in a concentration-dependent manner. The econazole-induced increase in [Ca2+]i was reduced by 48% by removal of extracellular Ca2+, suggesting that the econazole-induced increase in [Ca2+]i was composed of extracellular Ca2+ influx and intracellular Ca2+. 3. This econazole-induced Ca2+ influx was via an L-type Ca2+ channel-like pathway. In Ca2+-free medium, 1 micromol/L thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic increase in [Ca2+]i, after which the effect of econazole to increase [Ca2+]i was substantially inhibited. Conversely, pretreatment with 5 micromol/L econazole to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+. 4. The phospholipase C (PLC) inhibitor U73122 (2 micromol/L) abolished the increase in [Ca2+]i induced by 10 micromol/L ATP (a Ca2+ mobilizer that needs inositol 1,4,5-trisphosphate). 5. Overnight incubation with 1-30 micromol/L econazole inhibited proliferation of PC3 cells in a concentration-dependent manner. 6. These findings suggest that, in PC3 cells, econazole increases [Ca2+]i by stimulating Ca2+ influx into cells and Ca2+ release from the endoplasmic reticulum via a PLC-independent mechanism. Econazole is cytotoxic at submicromolar concentrations.

Novel Properties of a Mouse γ-Aminobutyric Acid Transporter (GAT4)

We expressed the mouse gamma-aminobutyric acid (GABA) transporter GAT4 (homologous to rat/ human GAT-3) in Xenopus laevis oocytes and examined its functional and pharmacological properties by using electrophysiological and tracer uptake methods. In the coupled mode of transport (Na+/ Cl-/GABA cotransport), there was tight coupling between charge flux and GABA flux across the plasma membrane (2 charges/GABA). Transport was highly temperature-dependent with a temperature coefficient (Q10) of 4.3. The GAT4 turnover rate (1.5 s(-l); -50 mV, 21 degrees C) and temperature dependence suggest physiological turnover rates of 15-20 s(-1). No uncoupled current was observed in the presence of Na+. In the absence of external Na+, GAT4 exhibited two distinct uncoupled currents. (i) A Cl- leak current (ICl(leak)) was observed when Na+ was replaced with choline or tetraethylammonium. The reversal potential of (ICl(leak)) followed the Cl- Nernst potential. (ii) A Li+ leak current (ILi(leak)) was observed when Na+ was replaced with Li+. Both leak currents were inhibited by Na+, and both were temperature-independent (Q10 approximately 1). The two leak modes appeared not to coexist, as Li+ inhibited (ICl(leak)). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na+/Cl-/GABA cotransport, ILi(leak), and ICl(leak), (Ki approximately 30 microM), and the voltage-induced presteady-state charge movements (Ki approximately 440 microM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jicroumps revealed that slow Na+ binding to the transporter is followed by rapid GABA-induced translocation of the ligands across the plasma membrane. Thus, Na+ binding and associated conformational changes constitute the rate-limiting steps in the transport cycle.

Fluorescence Resonance Energy Transfer Analysis of Protein Translocase: SecYE FROM THERMUS THERMOPHILUS HB8 FORMS A CONSTITUTIVE OLIGOMER IN MEMBRANES

SecY and SecE are the two principal translocase subunits that create a channel-like pathway for the transit of preprotein across the bacterial cytoplasmic membrane. Here we report the cloning, expression, and purification of the SecYE complex (TSecYE) from a thermophilic bacterium, Thermus thermophilus HB8. Purified TSecYE can be reconstituted into proteoliposomes that function in T. thermophilus SecA (TSecA) dependent preprotein translocation. After the mixing of TSecYE derivatives labeled with either a donor or an acceptor fluorophore during reconstitution, fluorescence resonance energy transfer experiments demonstrated that 2 or more units of TSecYE in the lipid bilayer associate to form a largely non-exchangeable oligomeric structure.

Internal aluminum block of plant inward K(+) channels.

Aluminum (Al) inhibits inward K(+) channels (K(in)) in both root hair and guard cells, which accounts for at least part of the Al toxicity in plants. To understand the mechanism of Al-induced K(in) inhibition, we performed patch clamp analyses on K(in) in guard cells and on KAT1 channels expressed in Xenopus oocytes. Our results show that Al inhibits plant K(in) by blocking the channels at the cytoplasmic side of the plasma membrane. In guard cells, single-channel recording revealed that Al inhibition of K(in) occurred only upon internal exposure. Using both "giant patch" recording and single-channel analyses, we found that Al reduced KAT1 open probability and changed its activation kinetics through an internal membrane-delimited mechanism. We also provide evidence that a Ca(2)+ channel-like pathway that is sensitive to antagonists verapamil and La(3)+ mediates Al entry across the plasma membrane. We conclude that Al enters plant cells through a Ca(2)+ channel-like pathway and inhibits K(+) uptake by internally blocking K(in).

Internal Aluminum Block of Plant Inward K+ Channels

Aluminum (Al) inhibits inward K(+) channels (K(in)) in both root hair and guard cells, which accounts for at least part of the Al toxicity in plants. To understand the mechanism of Al-induced K(in) inhibition, we performed clamp analyses on K(in) in guard cells and on KAT1 channels expressed in Xenopus oocytes. Our results show that Al inhibits plant K(in) by blocking the channels at the cytoplasmic side of the plasma membrane. In guard cells, single-channel recording revealed that Al inhibition of K(in) occurred only upon internal exposure. Using both giant patch recording and single-channel analyses, we found that Al reduced KAT1 open probability and changed its activation kinetics through an internal membrane-delimited mechanism. We also provide evidence that a Ca(2)+ channel-like pathway that is sensitive to antagonists verapamil and La(3)+ mediates Al entry across the plasma membrane. We conclude that Al enters plant cells through a Ca(2)+ channel-like pathway and inhibits K(+) uptake by internally blocking K(in).

Salivary Histatin 5 Induces Non-lytic Release of ATP fromCandida albicans Leading to Cell Death

Salivary histatins are potent in vitro antifungal proteins and have promise as therapeutic agents against oral candidiasis. We performed pharmacological studies directed at understanding the biochemical basis of Hst 5 candidacidal activity. Three inhibitors of mitochondrial metabolism: carbonyl cyanide p-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5 killing of Candida albicans, while not inhibiting cellular ATP production. In contrast, Hst 5 caused a drastic reduction of C. albicans intracellular ATP content, which was a result of an efflux of ATP. Carbonyl cyanide p-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5-induced ATP efflux, thus establishing a correlation between ATP release and cell killing. Furthermore, C. albicans cells were respiring and had polarized membranes at least 80 min after ATP release, thus implying a non-lytic exit of cellular ATP in response to Hst 5. Based on evidence that transmembrane ATP efflux can occur in the absence of cytolysis through a channel-like pathway and that released ATP can act as a cytotoxic mediator by binding to membrane purinergic receptors, we evaluated whether extracellular ATP released by Hst 5 may have further functional role in cell killing. Consistent with this hypothesis, purinergic agonists BzATP and adenosine 5'O-(thiotriphosphate) induced loss of C. albicans cell viability and purinergic antagonists prevented Hst 5 killing.

A conformation of Na(+)-K+ pump is permeable to proton

The Na(+)-K+ pump is thought to operate through a two-conformation (E1-E2) transport cycle in which the cation binding sites are accessible only from one side at a time. Using Na(+)-loaded Xenopus oocytes in which Na(+)-K+ pumps were overexpressed by injection of cRNA of the Xenopus Na(+)-K+ pump alpha-and beta-sub units, we observed a Na(+)-K+ pump-mediated (ouabain-sensitive) inward current in the absence of other transportable cations, except H+, in the external solution. This inward current was strongly inwardly rectifying, pH dependent, and larger at acid pH. Under conditions favoring a large ouabain-sensitive inward current, we observed a ouabain-sensitive intracellular acidification, and the amplitude of the acidification was significantly related to the ouabain-sensitive current, indicating that this current was carried by protons. The reversal potential of the ouabain-sensitive current was dependent on external pH as expected for a proton-conductive pathway. We conclude that in the absence of external K+ the Na(+)-K+ pump can mediate a large inward electrogenic transport of proton. This is most easily explained by the hypothesis that the E2 conformation of the Na(+)-K+ pump with cation binding sites exposed to the outside is accessible to protons from both sides and thus provides a channellike pathway for protons.

Expression of a channel-like pathway for adenosine transport in leishmania donovani promastigotes

L. donovani promastigotes (MHOM/ET/67/HA3) transport adenosine by a route that is sensitive to inhibition by a nonspecific channel blocker, propranolol. At the logarithmic and stationary phases of growth, the transport of 1μ m-3H-adenosine was significantly inhibited (40–50%) by 100 μM-propranolol. In contrast, a strain of Leishmania donovani promastigotes clonally selected to grow in defined medium was only slightly (approximately 10%) inhibited at the logarithmic but not at the stationary phase. These results suggest that the differences in expression of adenosine in the parasites previously reported, may be related to uptake by a channel-like pathway in the promastigotes.

Langmuir-Blodgett films composed of cationic amphiphile complexed with triblock polyanion with a biomembrane-mimicking, channellike pathway for transporting ions

Polyion complexes composed of newly synthesized triblock styrene-p-sulfonate/methacrylic acid copolymers (S n M m S n ) and a double-chain, cationic amphiphile formed stable surface monolayers, which were affected by pH in the subphase. The permeability of the hydrophilic permeants (KCl and tetramethylammonium bromide) across the LB films of those monolayers (10 layers) was reversibly switched on and off by pH change, while the permeability of the hydrophobic permeant (hexyltrimethylammonium bromide) with long alkyl chain was controlled by the phase transition of the component monolayers rather than by pH change

PH-Induced permeation control of a polyion complexed Langmuir-Blodgett film with a channel-like pathway of poly(methacrylic acid) segment

pH-Induced permeation control of a polyion complexed Langmuir-Blodgett film with a channel-like pathway of poly(methacrylic acid) segment

Coupling of proton flow and rotation in the bacterial flagellar motor: stochastic simulation of a microscopic model

The rotatory motor of bacterial flagella is driven by a transmembrane electrochemical gradient of protons. A model of the flagellar motor is analysed, which is based on the notion that protons passing through the motor use a channel-like pathway formed by ligand groups located partly on the rotor, partly on the stator. Proton translocation is linked to the displacement of stator elements which are elastically bound to the cell wall. The model is described by a cyclic sequence of translocation steps and proton binding and release reactions. Stochastic simulations of the model are carried out in which transitions between the states of the reaction cycle are treated as random events. In this way the rotation frequency can be predicted as a function of experimental variables such as driving force and viscous load. Furthermore, the effects of microscopic parameters such as the transition frequencies of stator elements and the force constant of elastic coupling on the dynamic properties of the motor can be studied. The model allows for intrinsic uncoupling (“slippage”) resulting from translocation steps without associated rotational movement. It is shown that mechanistic information can be obtained by studying random fluctuations of rotational speed.

Inhibition of transepithelial osmotic water flow by blockers of the glucose transporter

On the basis of evidence derived mostly from human erythrocytes, it has been suggested that water traverses cell membranes through membrane-spanning proteins such as the anion channel or the glucose transporter acting as water pores. However, specific inhibitors of such permeation processes have not been found to block water transport, and hence a precise identification of the water route has not been possible so far. We have investigated this issue by characterizing the osmotic flows across a fluid-transporting epithelium, the rabbit corneal endothelium. The rate of such flows was monitored continuously as a function of time. We confirmed prior findings of (a) an inhibition by PCMBS on osmotic water flow, and (b) lack of inhibition by DTNB and DIDS. On the other hand, we have found for the first time that several blockers of glucose facilitated diffusion, namely, phloretin (2 mM), phloridzin (2 mM), diallyldiethylstilbestrol (0.1 mM), cytochalasin B (20 μg/ml), and ethylidene- d-glucose (200 mM), all clearly inhibit osmotic flow. Our evidence is consistent with the hypothesis that both water and glucose may traverse these cell membranes through the same channel-like pathway contained in the glucose transporter membrane-spanning protein.

Computational studies of the interaction of myoglobin and xenon

Computational studies are used to investigate the energies of xenon binding to myoglobin and to describe pathways through the protein interior for a metmyoglobin-xenon complex. Empirical energy calculations indicate a favorable enthalpic contribution of 0.6 to 4.2 kcal/mol to xenon binding for four experimentally determined xenon sites. These calculated enthalpies help to explain the different xenon occupancies observed experimentally. A fifth site, modeled in place of the iron co-ordinated water molecule in the distal cavity, is also predicted to bind xenon. The largest contribution to the binding energy is from van der Waals' interactions with smaller contributions from polarization and protein strain terms. Ligand trajectory calculations as well as a new geometric algorithm define a connecting network of channel-like pathways through the static protein structure. One or two pathways appear to lead most easily from each major internal cavity to the protein surface. The importance of these channels in protein dynamics and their implications as routes for ligand motion are discussed.

Demonstration of percellular channels in glomerular podocytes in aminonucleoside nephrosis

Nephrotic syndrome was experimentally induced by a single i.v. injection of puromycin. Permeability of the visceral epithelial wall of glomeruli was examined with electron microscope. 30–60 seconds prior to fixation, ferritin solution was injected into the urinary space of superficially located glomeruli either by direct micropuncture of the urinary space or by retrograde filling of the urinary space via the corresponding tubule. Experimental evidence is presented for formation of channel-like pathways of ferritin penetration through the epithelial layer. Since these structures in nephrotic glomeruli are surrounded by a single cell only, we would like to propose the termpercellular channel for this phenomenon.