Counter Transport Research Articles

Selective removal of arsenic(V) from natural water using N-methyl-d-glucamine functionalized poly(propylene) membranes

Functionalized membranes for As(V) removal were synthesized by grafting glycidyl methacrylate in the fibrous and microporous host substrates, and subsequently reacting these precursor membranes with N-methyl-d-glucamine (NMDG). These membranes have been characterized and studied for As(V) removal from aqueous solution by the permeation method using dense membranes and flow-through method using fibrous membranes. In the permeation experiment, it was observed that As(V) could be transferred 60% from feed (ground water) to receiver compartment (NaOH) using the dense NMDG-membranes. The lower transfer of As(V) from feed to receiver compartment was due to counter transport of OH− ions from a receiver to a feed compartment. To address this problem, the removal of As(V) by sequential flow-through cycles of the feed and receiver across the fibrous NMDG-membrane was studied. In this mode, 92% of As(V) was sorbed in the fibrous NMDG-membrane in one cycle of 7.5min for 15mL feed. As(V) was deloaded to 66% by a single cycle of 0.3molL−1 NaOH. The HPLC method was also developed for quantifying As(V) content preconcentrated in a receiver solution. This method could be used for coupling desorption flow through cycle directly to the HPLC system. The reusability experiments showed that the membranes developed in the present work is highly stable and can be regenerated without affecting their As(V) removal efficiency.

Structure and mechanism of Zn2+-transporting P-type ATPases.

Zinc is an essential micronutrient for all living organisms. It is required for signalling and proper functioning of a range of proteins involved in, for example, DNA binding and enzymatic catalysis. In prokaryotes and photosynthetic eukaryotes, Zn(2+)-transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn(2+) and related elements. Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2·Pi) of ZntA from Shigella sonnei, determined at 3.2Å and 2.7Å resolution, respectively. The structures reveal a similar fold to Cu(+)-ATPases, with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn(2+) ions by the transporter. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including C392, C394 and D714. The pathway closes in the E2·Pi state, in which D714 interacts with the conserved residue K693, which possibly stimulates Zn(2+) release as a built-in counter ion, as has been proposed for H(+)-ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter transport. These findings suggest a mechanistic link between PIB-type Zn(2+)-ATPases and PIII-type H(+)-ATPases and at the same time show structural features of the extracellular release pathway that resemble PII-type ATPases such as the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and Na(+),K(+)-ATPase. These findings considerably increase our understanding of zinc transport in cells and represent new possibilities for biotechnology and biomedicine.

Influence of triphenyltetradecylphosphonium bromide on the Na+-Li+- countertransport rate in the erythrocyte membrane in patients with genetically different permeability of cell membranes to sodium

Aim. To study the influence of triphenyltetradecylphosphonium bromide [(PPh)3P+C14H29] Br- on the cell membranes permeability to Na+ by determining the rate of Na+-Li+-counter transport in erythrocyte membrane depending on it’s variable initial condition. Methods. Blood samples of 10 healthy volunteers with different Na+-Li+-counter transport rate distribution in erythrocyte membrane were analyzed: I quartile (5 subjects) - low permeability, III quartile (5 subjects) - moderately high permeability. Results. Na+-Li+-counter transport rate change in erythrocyte membrane under the influence of triphenyltetradecylphosphonium bromide depends on genetically determined initial membrane permeability. In subjects belonging to III quartile of Na+-Li+-counter transport the initial membrane permeability is high, and after administration of 0.001 and 0.005 μM concentrations of [(PPh)3P+C14H29] Br- membrane permeability to Na+ increased. In subjects belonging to I quartile of Na+-Li+-counter transport (low initial permeability) [(PPh)3P+C14H29] Br- did not alter membrane permeability to Na+. Conclusion. Triphenyltetradecylphosphonium bromide increases erythrocyte membrane permeability to Na+ in subjects belonging to III quartile of Na+-Li+-counter transport and dose not influence subjects belonging to I quartile of Na+-Li+-counter transport.

Activation of glutamate transport evokes rapid glutamine release from perisynaptic astrocytes

Stimulation of astrocytes by neuronal activity and the subsequent release of neuromodulators is thought to be an important regulator of synaptic communication. In this study we show that astrocytes juxtaposed to the glutamatergic calyx of Held synapse in the rat medial nucleus of the trapezoid body (MNTB) are stimulated by the activation of glutamate transporters and consequently release glutamine on a very rapid timescale. MNTB principal neurones express electrogenic system A glutamine transporters, and were exploited as glutamine sensors in this study. By simultaneous whole-cell voltage clamping astrocytes and neighbouring MNTB neurones in brainstem slices, we show that application of the excitatory amino acid transporter (EAAT) substrate d-aspartate stimulates astrocytes to rapidly release glutamine, which is detected by nearby MNTB neurones. This release is significantly reduced by the toxins L-methionine sulfoximine and fluoroacetate, which reduce glutamine concentrations specifically in glial cells. Similarly, glutamine release was also inhibited by localised inactivation of EAATs in individual astrocytes, using internal DL-threo-β-benzyloxyaspartic acid (TBOA) or dissipating the driving force by modifying the patch-pipette solution. These results demonstrate that astrocytes adjacent to glutamatergic synapses can release glutamine in a temporally precise, controlled manner in response to glial glutamate transporter activation. Since glutamine can be used by neurones as a precursor for glutamate and GABA synthesis, this represents a potential feedback mechanism by which astrocytes can respond to synaptic activation and react in a way that sustains or enhances further communication. This would therefore represent an additional manifestation of the tripartite relationship between synapses and astrocytes.

Role of Endosomal Sodium/Hydrogen Exchangers NHE6 and NHE9 in a Neurobiological Model of Disease

Ionic and pH gradients must be tightly regulated for many aspects of neuronal signaling and synaptic cleft homeostasis, including neurotransmitter uptake and astrocytic clearance through transporters, receptor recycling, and communication through gap junctions. Recently, several independent studies have correlated a range of neurological disorders, including autism, X‐linked mental retardation, epilepsy, attention deficit hyperactivity disorder and addiction, to mutations in a subgroup of sodium‐hydrogen exchangers, NHE6 and NHE9. These transporters are widely expressed in the brain and localize to early (NHE6) and recycling endosomes (NHE9), where they are thought to mediate the electrically silent counter transport of cations with protons to regulate luminal pH and control cargo trafficking. We have tested the hypothesis that NHE6 and NHE9 share overlapping functions in regulating intravesicular pH to control the delivery and turnover of glial specific glutamate transporters at the synapse. We have shown that isoform‐specific overexpression of NHE9 alters trafficking with increased surface expression of general (transferrin) receptors and synapse specific (glutamate) transporters. These findings provide the first insight into the neurobiological basis of NHE dysfunction. We will further elucidate the molecular mechanisms by measuring the pH of the endosomes, as well as create single and double knock down lines of NHE6 and NHE9 to demonstrate their potential overlapping functions..

Ca2+ Binding Alters the Interdomain Flexibility between the Two Cytoplasmic Calcium-binding Domains in the Na+/Ca2+ Exchanger

The Na(+)/Ca(2+) exchanger (NCX) is a membrane protein, which catalyzes the counter transport of Na(+) and Ca(2+) ions across the plasma membrane, playing a key role in the maintenance of the intracellular Ca(2+) homeostasis in various cell types. NCX consists of a transmembrane part and a large intracellular loop. The activation of the NCX transport function requires the binding of Ca(2+) to two tandem C2 domains, CBD1 and CBD2, which are an integral part of the exchanger's intracellular loop. Although high-resolution structures of individual CBD1 and CBD2 are available, their interdomain structure and dynamics and the atomic level mechanism of allosteric Ca(2+)-regulation remains unknown. Here, we use solution NMR spectroscopy to study the interdomain dynamics of CBD12, a 32 kDa construct that contains both the CBD1 and CBD2 domains connected by a short linker. Analysis of NMR residual dipolar couplings shows that CBD12 assumes on average an elongated shape both in the absence and in the presence of Ca(2+). NMR (15)N relaxation data of the Apo state indicate that the two domains sample a wide range of relative arrangements on the nanosecond time scale. These arrangements comprise significantly non-linear interdomain orientations. Binding of Ca(2+) to CBD1 significantly restricts the interdomain flexibility, stabilizing a more rigid elongated conformation. These findings suggest a molecular mechanism for the role of CBD12 in the function of NCX.

P-type ATPases at a glance

P-type ATPases are a large family of integral membrane transporters that are of vital importance in all kingdoms of life. In eponymous distinction from the other main classes of transport ATPase – the FF1 (F-), the vacuolar (V-) and the ATP-binding cassette (ABC-) type – the P-type ATPases form

Maintenance of antioxidant defenses of brain cells: plasma membrane glutamate transporters and beyond

Oxidative stress is invariably involved in neurodegenerative insults as a contributing mechanism in various forms of cell death (Bains and Shaw 1997). If strategies have to be developed for protection against oxidative injury, a better understanding of how antioxidant defenses are maintained in the brain is required. In this issue, leading scientists in the field present the state of the art concerning how precursors of the main brain antioxidant glutathione are delivered to brain cells, and explore regulatory mechanisms to identify new neuroprotective strategies against neurodegenerative insults. The essence of these articles comes from lectures presented at the 11th International Congress on Amino Acids, Peptides and Proteins held in Vienna, August 3–7, 2009. The meeting was organized by Pr. G. Lubec and the session on glutamate, its transporters and antioxidant defenses by Dr. L. Had-Aissouni. Glutathione (GSH) is the main brain antioxidant molecule. It is a tripeptide composed of glutamate, cysteine and glycine. Substrates for GSH synthesis may be provided by direct transport of constituent amino acids from the extracellular milieu. In this context, cysteine is thought to be the limiting precursor for GSH synthesis. Indeed in the extracellular milieu it is oxidized into cystine and intracellularly (in the reduced form, cysteine) it is present at a lower concentration, compared to the other constituent of GSH in most cells. However, none of these amino acids is indispensable, so they can also be synthesized in cells from other amino acids, providing that the necessary metabolic pathways are present (McBean 2011). Moreover, released GSH or conjugates are substrates for the ectoenzyme gamma-glutamyltransferase (EC 2.3.2.2) forming dipeptides such as cysteinylglycine or gamma-glutamylcysteine that, after extracellular hydrolysis, may provide substrates for GSH synthesis. Dipeptides may also be transported intracellularly in some cells and in the case of gammaglutamylcysteine used to bypass the first step of GSH synthesis (Dringen 2000). Due to the different pathways that cells may use to obtain substrates for GSH synthesis, it is very unlikely that the activity of a single amino acid transport system is required to sustain GSH synthesis. However, data obtained in culture as well as in vivo suggest that plasma membrane glutamate transporters may be a determinant for maintenance of antioxidant defenses in the brain. Before cloning of the cDNA coding for amino acid transporter proteins or subunits, transport systems were identified and characterized according to functional criteria such as transport mechanism (exchange, uniport, cotransport, counter transport, primary or secondary active transport) substrate specificity (that is generally rather large) and ionic dependence (mainly sodium dependent or independent). These characteristics were first used to name the corresponding transport systems generally using the first letter of the preferred substrate or x for anionic amino acids or y for cationic amino acids, sodium-dependent transport systems being designated in upper case while sodium-independent transport system are generally designated in lower case. Moreover, the charge of the transported substrates may also be indicated in superscript (0, ?, or -) while the first letter of the preferred substrate may be indicated in subscript when not directly used to name the transport system. According to this nomenclature, glutamate was found to be transported inside cells in sodium-independent as well as sodium-dependent L. Had-Aissouni (&) Developmental Biology Institute of Marseille-Luminy, CNRS/Aix-Marseille University, Luminy Campus-Case 907, 13288 Marseille cedex 9, France e-mail: laurence.had-aissouni@univmed.fr

Functional Evaluation of Autism-Related Mutations in the Na+ (k+)/ H+ Exchanger NHE9

A number of neurological disorders including autism, have been linked to mutations in a subgroup of intracellular Na+(K+)/H+ exchangers, which include NHE6 and NHE9 that localize to recycling and late endosomes respectively. These are thought to mediate the electrically silent counter transport of cations with protons. Yeast ortholog, Nhx1, has been shown to regulate endosomal pH and control vesicular trafficking. Here, we examine the functional consequence of missense mutations in NHE9 associated with autism, using yeast as a first approach. Further, we extend our observations from yeast to a glial cell model, providing the first insight into the neurobiological basis of NHE dysfunction. Previously, a 3-dimensional model of the human NHE1 was constructed using the crystal structure of NhaA, Na+/H+ antiporter from Escherichia coli, as template. As the endosomal NHE display significant similarity to NHE1, we used the NHE1 model as a template and used a unique modeling approach to model these other human transporters. Three NHE9 mutations (S438P, L236S, V176I) could be readily extrapolated to yeast Nhx1 from sequence conservation. We used site-directed mutagenesis to replace the yeast residues with the human equivalent, as well as the disease-associated mutation. Mutants were expressed in nhx1 deletion background and evaluated for pH, salt and trafficking phenotypes. While S43P and L236S mutations led to loss of function phenotypes in yeast, unexpectedly, V167I had no discernable effect. The latter may reveal isoform specific functions that remain to be identified or may call to question the functional contribution to the disease phenotype. In a parallel approach, these NHE9 mutations were expressed in glial cells for functional evaluation in a mammalian cell model. We observed altered trafficking and surface expression of glutamate transporters, consistent with elevated glutamate levels reported in patient brains.

The counter transport of heavy metals ion through cellulose acetate (CA) plastisizer membrane made from mulberry xylem

The counter transport of heavy metals ion through cellulose acetate (CA) plastisizer membrane made from mulberry xylem

Lack of association of sodium-lithium counter transport with microalbuminuria in young diabetics of Bangladesh

Background: Raised sodium-lithium counter transport activity (SLCT) in red blood cells (RBC) is claimed to be an early marker of nephropathy in type 1 and type 2 diabetic patients. This study aimed to investigate whether SLCT changes parallel with early nephropathy in young diabetics of Bangladesh. Materials and Methods: Thirty-five newly diagnosed, untreated, young and normotensive Bangladeshi diabetics without ketoacidosis, family history of hypertension and renal disease, and 19 age and body mass index (BMI) matched healthy controls without family history of hypertension and diabetes were studied to investigate whether their blood glucose, C-peptide, lipids and microalbuminuria were related with abnormality of SLCT activity. Results: Diabetic subjects had extremely variable degree of serum glucose (mmol/L) (mean ± SD) (fasting 16.6 5.8; postprandial 29.7 ± 7.5) and C-peptide (pmol/L) [median (range) (fasting 0.289 (0.028–0.994); postprandial 0.379 (0.053–2.424)], significantly higher (P 2.6 (mean + 2SD of control) was found in seven (20%) diabetics with no difference of SLCT between normoalbuminuric and microalbuminuric diabetics. Conclusions: SLCT activity in RBC is not a predictor of nephropathy in young diabetics of Bangladesh and the development of nephropathy is mainly related to glycemic and insulin status. SLCT may be elevated by marked hypoinsulinemia even without hypertension or family history of hypertension.

The effect of the counter anion on the transport of thiourea in a PVC-based polymer inclusion membrane using Capriquat as carrier

This paper describes the effect of four counter anions (CH3COO−, Cl−, NO3−, ClO4−) of the trioctylmethylammonium (TOMA) cation on the rate of solvent extraction of thiourea and its transport across poly(vinyl chloride) (PVC)-based polymer inclusion membranes (PIMs). The membranes also contained 2-nitrophenyl octyl ether (NPOE) as the plasticizer while chloroform was used as diluent in the solvent extraction studies. It is demonstrated that the counter anion affects substantially the rate of membrane transport and the degree of extraction follows the order: CH3COO− > Cl− > NO3− ≫ ClO4−. The transport rate is negligible for the perchlorate anion. This order is consistent with thiourea interacting with the counter anion through hydrogen bonding to form a heteroconjugate anion. A mathematical model of the membrane transport of thiourea assuming ideal mixing in the feed and receiver solutions and Fickian diffusion of the heteroconjugate anion has been developed and fitted to the experimental transport data. This has allowed the determination of the diffusion coefficient of the heteroconjugate anion (5.28 × 10−11 m2 s−1) and the thiourea membrane extraction constants (CH3COO−: 3.41; Cl−: 2.61 × 10−1; NO3−: 2.26 × 10−2 m3 mol−1) which follow the same order as the solvent extraction constants (CH3COO−: 2.42; Cl−: 6.63 × 10−1; NO3−: 4.26 × 10−1; ClO4−: 8.31 × 10−2 m3 mol−1). The exceedingly low ability of PIMs containing TOMA-ClO4 to extract and transport thiourea suggests that these membranes can potentially overcome the difficulties in the PIM separation of gold(III) which are associated with the counter transport of thiourea.

Extraction and recovery of lignosulfonate from its aqueous solution using bulk liquid membrane

The paper presents an experimental study on the coupled transport of lignosulfonate (LS) through bulk liquid membrane (BLM) and thereby to identify the best set of solvent, operating conditions and mode of transport that would yield optimum performance of the BLM. Trioctylamine (TOA) is used as carrier. Among various solvents, tested for the above purpose, dichloroethane is found to be the best. The effects of operating condition, viz. pH, temperature, and carrier concentration, on the equilibrium distribution of LS are investigated. The effects of temperature, stirring of aqueous and organic phases, stirring speed, carrier concentration, initial feed and strip phase concentration on the separation of LS using BLM are also studied. It is observed that transport of LS can be enhanced by increasing the temperature and stirring speed of feed phase. Stirring of strip phase has no appreciable effects on the transport of LS. With increase in initial feed concentration the initial rate of the transport of LS is higher but continues for a longer time. Recovery of LS is much higher in co-transport mode in comparison to counter transport mode. Application of 1.25 M NaOH as stripping solution gives high recovery (70%) and high strip flux (70% of feed flux).

Experimental study on dealcoholization of wine by osmotic distillation process

The types of stripping solutions and influences of operating parameters including feed and stripping velocities, system temperatures on the ethanol removal of ethanol solution and wine by using osmotic distillation (OD) process were investigated along with the mass transfer study. The experiments were performed by the membrane contactor unit using 0.2 μm pore size diameter microporous PVDF hollow fibers. Three different types of stripping solutions (pure water, 50% (w/w) glycerol, 40% (w/w) CaCl 2) were used to compare the dealcoholization performance of the process. Aroma loss study was also carried out by using ethyl acetate and iso-amyl alcohol solutions to represent the major aroma compounds presenting in the real wine. Long-term tests for ethanol solution and wine were also performed in order to evaluate the dealcoholization performance of OD process. Using water as a stripper presented more promising result compared with the others because it provided higher ethanol flux and lower counter transport of water due to the water activity differences. The results of the study showed that the ethanol flux and ethanol removal performance were enhanced by the increase of feed and stripping solution velocities and system temperatures. Aroma components were significantly lost during the process operation. The ethanol concentration in the wine can be reduced to roughly 34% of the initial concentration for 360 min operation.

Dynamic compartmentalization of protein tyrosine phosphatase receptor Q at the proximal end of stereocilia: Implication of myosin VI‐based transport

Hair cell stereocilia are apical membrane protrusions filled with uniformly polarized actin filament bundles. Protein tyrosine phosphatase receptor Q (PTPRQ), a membrane protein with extracellular fibronectin repeats has been shown to localize at the stereocilia base and the apical hair cell surface, and to be essential for stereocilia integrity. We analyzed the distribution of PTPRQ and a possible mechanism for its compartmentalization. Using immunofluorescence we demonstrate that PTPRQ is compartmentalized at the stereocilia base with a decaying gradient from base to apex. This distribution can be explained by a model of transport directed toward the stereocilia base, which counteracts diffusion of the molecules. By mathematical analysis, we show that this counter transport is consistent with the minus end-directed movement of myosin VI along the stereocilia actin filaments. Myosin VI is localized at the stereocilia base, and exogenously expressed myosin VI and PTPRQ colocalize in the perinuclear endosomes in COS-7 cells. In myosin VI-deficient mice, PTPRQ is distributed along the entire stereocilia. PTPRQ-deficient mice show a pattern of stereocilia disruption that is similar to that reported in myosin VI-deficient mice, where the predominant features are loss of tapered base, and fusion of adjacent stereocilia. Thin section and freeze-etching electron microscopy showed that localization of PTPRQ coincides with the presence of a dense cell surface coat. Our results suggest that PTPRQ and myosin VI form a complex that dynamically maintains the organization of the cell surface coat at the stereocilia base and helps maintain the structure of the overall stereocilia bundle.

Electron Crystallography Reveals Plasticity within the Drug Binding Site of the Small Multidrug Transporter EmrE

EmrE is a Small Multidrug Resistance transporter (SMR) family member that mediates counter transport of protons and hydrophobic cationic drugs such as tetraphenylphosphonium (TPP+), ethidium, propidium and dequalinium. It is thought that the selectivity of the drug binding site in EmrE is defined by two negatively charged glutamate residues within a hydrophobic pocket formed from six of the α-helices, three from each monomer of the asymmetric EmrE homodimer. It is not apparent how such a binding pocket accommodates drugs of various sizes and shapes or whether the conformational changes that occur upon drug binding are identical for drugs of diverse chemical nature. Here, using electron cryomicroscopy of EmrE two-dimensional crystals we have determined projection structures of EmrE bound to three structurally different planar drugs, ethidium, propidium and dequalinium. Using image analysis and rigorous comparisons between these density maps and the density maps of the ligand-free and TPP+-bound forms of EmrE, we identify regions within the transporter that adapt differentially depending on the type of ligand bound. We show that all three planar drugs bind at the same pocket within the protein as TPP+. Furthermore, our analysis indicates that, while retaining the overall fold of the protein, binding of the planar drugs is accompanied by small rearrangements of the transmembrane domains that are different to those that occur when TPP+ binds. The regions in the EmrE dimer that are remodelled surround the drug binding site and include transmembrane domains from both monomers.

Facilitated transport of copper through bulk liquid membranes containing different carriers: compared kinetic study

The discharge of heavy metals into the environment is a serious problem facing numerous industries. Heavy metals tend to accumulate in living organisms causing various diseases and disorders. So the search for extraction techniques to remove those heavy metals are of increasing interest. Liquid membranes have shown great potential in this way, especially in cases where metal concentrations are relatively low and other techniques cannot be applied efficiently. A comparative kinetic study of the facilitated counter transport of copper ion through bulk liquid membranes containing D2EHPA, CYANEX 272 and LIX 984N as carriers and protons as counter ions is carried out is this paper. The transport kinetic was analysed by means of a kinetic model involving two consecutive irreversible first-order reactions. The rate constants of the extraction and stripping reactions and the maximum transport fluxes of copper(II) through the bulk liquid membrane were determined for the three carriers.

Calcium transporters and signalling in smooth muscles

Two P-type Ca transporters, the plasma membrane Ca-ATPase (PMCA) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA), play a crucial role in maintaining Ca homeostasis, controlling contractility and contributing to excitably and cell signalling in smooth muscle cells. There is considerable structural homology between the two Ca-ATPases; they both have transmembrane spanning regions, have similar ATP-phosphorylated intermediaries, counter transport protons and are regulated by several second messengers. They both also exist in several isoforms and have many splice variants, which presumably impart some of their tissue specific functions. We describe the relative contribution of PMCA and the Na–Ca exchanger to Ca efflux in relaxation to smooth muscle, including recent data from transgenic mice, which has begun to elucidate the specific contributions of individual isoforms to Ca signalling. We then consider Ca release and uptake into the SR in smooth muscle. Experiments investigating the distribution of SERCA in smooth muscle cells have provided new insights into control of SR luminal Ca, and the effects of SR Ca load on signalling, is discussed. This is followed by a detailed consideration of the interactions between the surface membrane and SR membrane pumps, exchangers and ion channels in smooth muscle, along with their distribution to caveolae and cholesterol-rich membrane domains. Where relevant the importance of these functions to health and disease are noted. We conclude that the dynamic changes in splice variants expressed, constituents of membrane microdomains and environment of the sub-sarcolemmal space, close to the SR, need to be the focus of future research, so that the full importance of Ca transporters to smooth muscle signalling cascades can be better understood.

A suite of sucrose transporters expressed in coats of developing legume seeds includes novel pH‐independent facilitators

A suite of newly discovered sucrose transporter genes, PsSUF1, PsSUF4, PvSUT1 and PvSUF1, were isolated from the coats of developing pea (Pisum sativum L.) and bean (Phaseolus vulgaris L.) seeds. Sequence analysis indicated that deduced proteins encoded by PsSUF1, PvSUT1 and PvSUF1 clustered in a separate sub-group under sucrose transporter Clade I, whereas the deduced protein encoded by PsSUF4 clustered in Clade II. When expressed in yeast, these genes were shown to encode sucrose transporters with apparent Michaelis Menten constant (Km) values ranging from 8.9 to 99.8 mm. PvSUT1 exhibited functional characteristics of a sucrose/H+ symporter. In contrast, PsSUF1, PvSUF1 and PsSUF4 supported the pH- and energy independent transport of sucrose that was shown to be bi-directional. These transport properties, together with that of counter transport, indicated that PsSUF1, PvSUF1 and PsSUF4 function as carriers that support the facilitated diffusion of sucrose. Carrier function was unaffected by diethylpyrocarbonate and by maltose competition, suggesting that the sucrose binding sites of these transporters differed from those of known sucrose/H+ symporters. All sucrose transporters were expressed throughout the plant and, of greatest interest, were co-expressed in cells considered responsible for sucrose efflux from seed coats. The possible roles played by the novel facilitators in sucrose efflux from seed coats are discussed.

Diclofenac Transport through Stagnant Sandwich and Supported Liquid Membrane Systems

The removal of a drug, diclofenac (DCF), from aqueous media by using a supported liquid membrane (SLM) and a stagnant sandwich LM (SSwLM) has been investigated. The two systems were compared in terms of flux and stability. Optimal chemical conditions were determined by means of liquid-liquid extraction tests. In the transport tests, the obtained results indicated a higher initial flux J 0 (2.4 vs 1.3 mmol/h·m 2 ) and a faster transport (counter transport reached at 1.75 vs 26.3 h) for the SLM system than for the SSwLM system. The diclofenac transport in the SLM from the feed to the strip was quite complete after 3 h, whereas in the SSwLM, only 39% of the initial drug was recovered in the strip phase after 30 h. Higher system stability was obtained using the SSwLM system (> 120 vs 47.5 h). The overall results obtained in this work showed that liquid membrane systems can effectively be used to remove pharmaceuticals present at low concentration in wastewaters.