Effect of tissue viscoelasticity and adjacent phase-changed microbubbles on vaporization process and direct growth threshold of nanodroplet in an ultrasonic field.

Understanding the behavior of nanodroplets converted into microbubbles with applied ultrasound is an important problem in tumor therapeutical and diagnostic applications. In this study, a comprehensive model is proposed to investigate the vaporization process and the direct growth threshold of the nanodroplet by following the vapor bubble growth, especially attention devoted to the effect of tissue viscoelasticity and adjacent phase-changed microbubbles (PCMBs). It is shown that the ultrasonic energy must be sufficiently strong to counterbalance the natural condensation of the vapor bubble and the tissue stiffness-inhibitory effect. The softer tissue with a lower shear modulus favors the vaporization process, and the nanodroplet has a lower direct growth threshold in the softer tissue. Moreover, the adjacent PCMBs show a suppression effect on the vaporization process due to the negative value of the secondary Bjerknes force, implying an attractive force, preventing the nanodroplet from escaping from the constraint of the adjacent PCMBs. However, according to the linear scattering theory, the attractive force signifies that the constraint is weak, causing the direct growth threshold to increase in the range of 0.09-0.24 MPa. The weak increase in threshold demonstrates that the direct growth threshold is relatively unaffected by the adjacent PCMBs. The prediction results of our model are in good agreement with the experiment results obtained by the echo enhancement method, in which the threshold is relatively independent of the intermediate concentration. The findings presented here provide physical insight that will be further helpful in understanding the complex behavior of the nanodroplet responses to ultrasound in practical medical applications.

Cysteine 159 delineates a hinge region of the alternating access monocarboxylate transporter 1 and is targeted by cysteine-modifying inhibitors.

Monocarboxylate transporter isoforms 1-4, MCT, of the solute carrier SLC16A family facilitate proton-coupled transport of l-lactate. Growth of tumors that exhibit the Warburg effect, that is, high rates of anaerobic glycolysis despite availability of oxygen, relies on swift l-lactate export, whereas oxygenic cancer cells import circulating l-lactate as a fuel. Currently, MCTs are viewed as promising anticancer targets. Small-molecule inhibitors have been found, and, recently, high-resolution protein structures have been obtained. Key questions, however, regarding the exact binding sites of cysteine-modifying inhibitors and the substrate translocation cycle lack a conclusive experimental basis. Here, we report Cys159 of the ubiquitous human MCT1 to reside in a critical hinge region of the alternating access-type transporter. We identified Cys159 as the binding site of the organomercurial pCMBS. The inhibitory effect of pCMBS was proposed to be indirect via modification of the chaperone basigin. We provide evidence that pCMBS locks MCT1 in its outward open conformation in a wedge-like fashion. We corroborated this finding using smaller cysteine-modifying reagents that size-dependently inhibited l-lactate transport. The smallest modifiers targeted additional cysteines as shown by a C159S mutant. We found a Cys399/Cys400 pair to constitute the second hinge of the transporter that tolerated only individual replacement by serine. The hinge cysteines, in particular the selectively addressable Cys159, provide natural anchors for placing probes into MCTs to report, for instance, on the electrostatics or hydration upon binding of the transported l-lactate substrate and the proton cosubstrate.

Integrin-mediated cell adhesion requires extracellular disulfide exchange regulated by protein disulfide isomerase.

Cell adhesion to extracellular matrix, mediated by integrin receptors, is crucial for cell survival. Receptor-ligand interaction involves conformational changes in the integrin by a mechanism not fully elucidated. In addition to several direct evidence that there is disulfide re-arrangement of integrins, we previously demonstrated a role for extracellular thiols and protein disulfide isomerase (PDI) in integrin-mediated functions using platelets as model system. Exploring the possible generality of this mechanism, we now show, using three different nucleated cells which depend on adhesion for survival, that non-penetrating blockers of free thiols inhibit α2ß1 and α5ß1 integrin-mediated adhesion and that disulfide exchange takes place in that process. Inhibiting extracellular PDI mimics thiol blocking. Transfection with WT or enzymatically inactive PDI increased their membrane expression and enhanced cell adhesion, suggesting that PDI level is a limiting factor and that the chaperone activity of the enzyme contributes to adhesion. Exogenously added PDI also enhanced adhesion, further supporting the limiting factor of the enzyme. These data indicate that: a) Dependence on ecto-sulfhydryls for integrin-mediated adhesion is not exclusive to the platelet; b) PDI is involved in integrin-mediated adhesion, catalyzing disulfide bond exchange; c) PDI enhances cell adhesion by both its oxidoreductase activity and as a chaperone.

Use of D-glucose-fenpiclonil conjugate as a potent and specific inhibitor of sucrose carriers.

Until now, specific inhibitors of sucrose carriers were not available. This led us to study the properties of the recently synthesized D-glucose-fenpiclonil conjugate (D-GFC). This large amphiphilic glucoside exhibited an extremely low phloem systemicity in contrast to L-amino acid-fenpiclonil conjugates. Using Ricinus seedlings, the effect of D-GFC on 0.5 mM [14C]sucrose (Suc), 3-O-[3H]methylglucose, and [3H]glutamine uptake by cotyledon tissues was compared with that of p-chloromercuribenzenesulfonic acid (PCMBS). D-GFC dramatically inhibited H+-Suc symport at the same concentrations as PCMBS (0.5 and 1 mM), but in contrast to the thiol reagent, it did not affect 3-O-methylglucose and glutamine transport, nor the acidification of the incubation medium by cotyledon tissues. Similarly, 0.5 mM D-GFC inhibited active Suc uptake by Vicia faba leaf tissues and by Saccharomyces cerevisiae cells transformed with AtSUC2, a gene involved in Suc phloem loading in Arabidopsis, by approximately 80%. The data indicated that D-GFC was a potent inhibitor of Suc uptake from the endosperm and of Suc phloem loading. It is the first chemical known to exhibit such specificity, at least in Ricinus, and this property permitted the quantification of the two routes involved in phloem loading of endogenous sugars after endosperm removal.

Sugar uptake in the Aril of litchi fruit depends on the apoplasmic post-phloem transport and the activity of proton pumps and the putative transporter LcSUT4.

The post-phloem unloading pathway and the mechanism of sugar accumulation remain unclear in litchi fruit. A combination of electron microscopy, transport of phloem-mobile symplasmic tracer (carboxyfluorescein, CF) and biochemical and molecular assays was used to explore the post-phloem transport pathway and the mechanism of aril sugar accumulation in litchi. In the funicle, where the aril originates, abundant plasmodesmata were observed, and CF introduced from the peduncle diffused to the parenchyma cells. In addition, abundant starch and pentasaccharide were detected and the sugar concentration was positively correlated with activities of sucrose hydrolysis enzymes. These results clearly showed that the phloem unloading and post-phloem transport in the funicle were symplastic. On the other hand, imaging of CF showed that it remained confined to the parenchyma cells in funicle tissues connecting the aril. Infiltration of both an ATPase inhibitor [eosin B (EB)] and a sucrose transporter inhibitor [p-chloromercuribenzene sulfonate (PCMBS)] inhibited sugar accumulation in the aril. These results indicated an apoplasmic post-phloem sugar transport from the funicle to the aril. Although facilitated diffusion might help sucrose uptake from the cytosol to the vacuole in cultivars with high soluble invertase, membrane ATPases in the aril, especially tonoplast ATPase, are crucial for aril sugar accumulation. The expression of a putative aril vacuolar membrane sucrose transporter gene (LcSUT4) was highly correlated with the sugar accumulation in the aril of litchi. These data suggest that apoplasmic transport is critical for sugar accumulation in litchi aril and that LcSUT4 is involved in this step.

Uptake and metabolic effects of salicylic acid on the pulvinar motor cells of Mimosa pudica L.

In this paper, the salicylic acid (o-hydroxy benzoic acid) (SA) uptake by the pulvinar tissues of Mimosa pudica L. pulvini was shown to be strongly pH-dependent, increasing with acidity of the assay medium. This uptake was performed according to a unique affinity system (K(m) = 5.9 mM, V(m) = 526 pmol mgDW(-1)) in the concentration range of 0.1-5 mM. The uptake rate increased with increasing temperature (5-35 °C) and was inhibited following treatment with sodium azide (NaN3) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), suggesting the involvement of an active component. Treatment with p-chloromercuribenzenesulfonic acid (PCMBS) did not modify the uptake, indicating that external thiol groups were not necessary. KCl, which induced membrane depolarization had no significant effect, and fusicoccin (FC), which hyperpolarized cell membrane, stimulated the uptake, suggesting that the pH component of the proton motive force was likely a driving force. These data suggest that the SA uptake by the pulvinar tissues may be driven by two components: an ion-trap mechanism playing a pivotal role and a putative carrier-mediated mechanism. Unlike other benzoic acid derivatives acting as classical respiration inhibitors (NaN3 and KCN), SA modified the pulvinar cell metabolism by increasing the respiration rate similar to CCCP and 2,4-dinitrophenol (DNP). Furthermore, SA inhibited the osmoregulated seismonastic reaction in a pH dependent manner and induced characteristic damage to the ultrastructural features of the pulvinar motor cells, particularly at the mitochondrial level.

Inward flux of lactate⁻ through monocarboxylate transporters contributes to regulatory volume increase in mouse muscle fibres.

Mouse and rat skeletal muscles are capable of a regulatory volume increase (RVI) after they shrink (volume loss resultant from exposure to solutions of increased osmolarity) and that this RVI occurs mainly by a Na-K-Cl-Cotransporter (NKCC)-dependent mechanism. With high-intensity exercise, increased extracellular osmolarity is accompanied by large increases in extracellular [lactate⁻]. We hypothesized that large increases in [lactate⁻] and osmolarity augment the NKCC-dependent RVI response observed with a NaCl (or sucrose)-induced increase in osmolarity alone; a response that is dependent on lactate⁻ influx through monocarboxylate transporters (MCTs). Single mouse muscle fibres were isolated and visualized under light microscopy under varying osmolar conditions. When solution osmolarity was increased by adding NaLac by 30 or 60 mM, fibres lost significantly less volume and regained volume sooner compared to when NaCl was used. Phloretin (MCT1 inhibitor) accentuated the volume loss compared to both NaLac controls, supporting a role for MCT1 in the RVI response in the presence of elevated [lactate⁻]. Inhibition of MCT4 (with pCMBS) resulted in a volume loss, intermediate to that seen with phloretin and NaLac controls. Bumetanide (NKCC inhibitor), in combination with pCMBS, reduced the magnitude of volume loss, but volume recovery was complete. While combined phloretin-bumetanide also reduced the magnitude of the volume loss, it also largely abolished the cell volume recovery. In conclusion, RVI in skeletal muscle exposed to raised tonicity and [lactate⁻] is facilitated by inward flux of solute by NKCC- and MCT1-dependent mechanisms. This work demonstrates evidence of a RVI response in skeletal muscle that is facilitated by inward flux of solute by MCT-dependent mechanisms. These findings further expand our understanding of the capacities for skeletal muscle to volume regulate, particularly in instances of raised tonicity and lactate⁻ concentrations, as occurs with high intensity exercise.

Cysteine substitutions define etomidate binding and gating linkages in the α-M1 domain of γ-aminobutyric acid type A (GABAA) receptors.

Etomidate is a potent general anesthetic that acts as an allosteric co-agonist at GABAA receptors. Photoreactive etomidate derivatives labeled αMet-236 in transmembrane domain M1, which structural models locate in the ß+/α- subunit interface. Other nearby residues may also contribute to etomidate binding and/or transduction through rearrangement of the site. In human α1ß2γ2L GABAA receptors, we applied the substituted cysteine accessibility method to α1-M1 domain residues extending from α1Gln-229 to α1Gln-242. We used electrophysiology to characterize each mutant's sensitivity to GABA and etomidate. We also measured rates of sulfhydryl modification by p-chloromercuribenzenesulfonate (pCMBS) with and without GABA and tested if etomidate blocks modification of pCMBS-accessible cysteines. Cys substitutions in the outer α1-M1 domain impaired GABA activation and variably affected etomidate sensitivity. In seven of eight residues where pCMBS modification was evident, rates of modification were accelerated by GABA co-application, indicating that channel activation increases water and/or pCMBS access. Etomidate reduced the rate of modification for cysteine substitutions at α1Met-236, α1Leu-232 and α1Thr-237. We infer that these residues, predicted to face ß2-M3 or M2 domains, contribute to etomidate binding. Thus, etomidate interacts with a short segment of the outer α1-M1 helix within a subdomain that undergoes significant structural rearrangement during channel gating. Our results are consistent with in silico docking calculations in a homology model that orient the long axis of etomidate approximately orthogonal to the transmembrane axis.

Movement of NH3 through the human urea transporter B: a new gas channel.

Aquaporins and Rh proteins can function as gas (CO2 and NH3) channels. The present study explores the urea, H2O, CO2, and NH3 permeability of the human urea transporter B (UT-B) (SLC14A1), expressed in Xenopus oocytes. We monitored urea uptake using [¹4C]urea and measured osmotic water permeability (Pf) using video microscopy. To obtain a semiquantitative measure of gas permeability, we used microelectrodes to record the maximum transient change in surface pH (ΔpHS) caused by exposing oocytes to 5% CO2/33 mM HCO3⁻ (pHS increase) or 0.5 mM NH3/NH4⁺ (pHS decrease). UT-B expression increased oocyte permeability to urea by >20-fold, and Pf by 8-fold vs. H2O-injected control oocytes. UT-B expression had no effect on the CO2-induced ΔpHS but doubled the NH3-induced ΔpHS. Phloretin reduced UT-B-dependent urea uptake (Jurea*) by 45%, Pf* by 50%, and (- ΔpHS*)NH3 by 70%. p-Chloromercuribenzene sulfonate reduced Jurea* by 25%, Pf* by 30%, and (ΔpHS*)NH3 by 100%. Molecular dynamics (MD) simulations of membrane-embedded models of UT-B identified the monomeric UT-B pores as the main conduction pathway for both H2O and NH3 and characterized the energetics associated with permeation of these species through the channel. Mutating each of two conserved threonines lining the monomeric urea pores reduced H2O and NH3 permeability. Our data confirm that UT-B has significant H2O permeability and for the first time demonstrate significant NH3 permeability. Thus the UTs become the third family of gas channels. Inhibitor and mutagenesis studies and results of MD simulations suggest that NH3 and H2O pass through the three monomeric urea channels in UT-B.

Importance of cysteine residues in the thyroid hormone transporter MCT8.

The thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) is crucial for brain development as demonstrated by the severe psychomotor retardation in patients with MCT8 mutations. MCT8 contains 10 residues of the reactive amino acid cysteine (Cys) whose functional roles were studied using the Cys-specific reagent p-chloromercurybenzenesulfonate (pCMBS) and by site-directed mutagenesis. Pretreatment of JEG3 cells with pCMBS resulted in a dose- and time-dependent decrease of subsequent T3 uptake. Pretreatment with dithiothreitol did not affect TH transport or its inhibition by pCMBS. However, pCMBS inhibition of MCT8 was reversed by dithiothreitol. Inhibition of MCT8 by pCMBS was prevented in the presence of T3. The single and double mutation of C481A and C497A did not affect T3 transport, but the single mutants were less sensitive and the double mutant was completely insensitive to pCMBS. Similar effects on MCT8 were obtained using HgCl2 instead of pCMBS. In conclusion, we have identified Cys481 and Cys497 in MCT8 as the residues modified by pCMBS or HgCl2. These residues are probably located at or near the substrate-recognition site in MCT8. It remains to be investigated whether MCT8 function is regulated by modification of these Cys residues under pathophysiological conditions.

Phloem unloading follows an extensive apoplasmic pathway in cucumber (Cucumis sativus L.) fruit from anthesis to marketable maturing stage.

The phloem unloading pathway remains unclear in fruits of Cucurbitaceae, a classical stachyose-transporting species with bicollateral phloem. Using a combination of electron microscopy, transport of phloem-mobile symplasmic tracer carboxyfluorescein, assays of acid invertase and sucrose transporter, and [(14)C]sugar uptake, the phloem unloading pathway was studied in cucumber (Cucumis sativus) fruit from anthesis to the marketable maturing stage. Structural investigations showed that the sieve element-companion cell (SE-CC) complex of the vascular bundles feeding fruit flesh is apparently symplasmically restricted. Imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the vascular bundles in the whole fruit throughout the stages examined. A 37 kDa acid invertase was located predominantly in the cell walls of SE-CC complexes and parenchyma cells. Studies of [(14)C]sugar uptake suggested that energy-driven transporters may be functional in sugar trans-membrane transport within symplasmically restricted SE-CC complex, which was further confirmed by the existence of a functional plasma membrane sucrose transporter (CsSUT4) in cucumber fruit. These data provide a clear evidence for an apoplasmic phloem unloading pathway in cucumber fruit. A presumption that putative raffinose or stachyose transporters may be involved in soluble sugars unloading was discussed.

Structure of the M2 transmembrane segment of GLIC, a prokaryotic Cys loop receptor homologue from Gloeobacter violaceus, probed by substituted cysteine accessibility.

GLIC is a homopentameric proton-gated, prokaryotic homologue of the Cys loop receptor family of neurotransmitter-gated ion channels. Recently, crystal structures of GLIC hypothesized to represent an open channel state were published. To explore the channel structure in functional GLIC channels, we tested the ability of p-chloromercuribenzenesulfonate to react with 30 individual cysteine substitution mutants in and flanking the M2 channel-lining segment in the closed state (pH 7.5) and in a submaximally activated state (pH 5.0). Nine mutants did not tolerate cysteine substitution and were not functional. From positions 10' to 27', p-chloromercuribenzenesulfonate significantly modified the currents at pH 7.5 and 5.0 in all mutants except H234C (11'), I235C (12'), V241C (18'), T243C (20'), L245C (22'), and Y250C (27'), which were not functional, except for 12'. Currents for P246C (23') and K247C (24') were only significantly altered at pH 5.0. The reaction rates were all >1000 m(-1) s(-1). The reactive residues were more accessible in the activated than in the resting state. We infer that M2 is tightly associated with the adjacent transmembrane helices at the intracellular end but is more loosely packed from 10' to the extracellular end than the x-ray structures suggest. We infer that the charge selectivity filter is in the cytoplasmic half of the channel. We also show that below pH 5.0, GLIC desensitizes on a time scale of minutes and infer that the crystal structures may represent a desensitized state.

Optimal metabolic regulation of the mammalian heart Na(+)/Ca(2+) exchanger requires a spacial arrangements with a PtdIns(4)-5kinase.

In inside-out bovine heart sarcolemmal vesicles, p-chloromercuribenzenesulfonate (PCMBS) and n-ethylmaleimide (NEM) fully inhibited MgATP up-regulation of the Na(+)/Ca(2+) exchanger (NCX1) and abolished the MgATP-dependent PtdIns-4,5P2 increase in the NCX1-PtdIns-4,5P2 complex; in addition, these compounds markedly reduced the activity of the PtdIns(4)-5kinase. After PCMBS or NEM treatment, addition of dithiothreitol (DTT) restored a large fraction of the MgATP stimulation of the exchange fluxes and almost fully restored PtdIns(4)-5kinase activity; however, in contrast to PCMBS, the effects of NEM did not seem related to the alkylation of protein SH groups. By itself DTT had no effect on the synthesis of PtdIns-4,5P2 but affected MgATP stimulation of NCX1: moderate inhibition at 1mM MgATP and 1µM Ca(2+) and full inhibition at 0.25mM MgATP and 0.2µM Ca(2+). In addition, DDT prevented coimmunoprecipitation of NCX1 and PtdIns(4)-5kinase. These results indicate that, for a proper MgATP up-regulation of NCX1, the enzyme responsible for PtdIns-4,5P2 synthesis must be (i) functionally competent and (ii) set in the NCX1 microenvironment closely associated to the exchanger. This kind of supramolecular structure is needed to optimize binding of the newly synthesized PtdIns-4,5P2 to its target region in the exchanger protein.

Characterization of the PCMBS-dependent modification of KCa3.1 channel gating.

Intermediate conductance, calcium-activated potassium channels are gated by the binding of intracellular Ca(2+) to calmodulin, a Ca(2+)-binding protein that is constitutively associated with the C terminus of the channel. Although previous studies indicated that the pore-lining residues along the C-terminal portion of S6 contribute to the activation mechanism, little is known about whether the nonluminal face of S6 contributes to this process. Here we demonstrate that the sulfhydral reagent, parachloromercuribenze sulfonate (PCMBS), modifies an endogenous cysteine residue predicted to have a nonluminal orientation (Cys(276)) along the sixth transmembrane segment (S6). Modification of Cys(276) manipulates the steady-state and kinetic behavior of the channel by shifting the gating equilibrium toward the open state, resulting in a left shift in apparent Ca(2+) affinity and a slowing in the deactivation process. Using a six-state gating scheme, our analysis shows that PCMBS slows the transition between the open state back to the third closed state. Interpreting this result in the context of the steady-state and kinetic data suggests that PCMBS functions to shift the gating equilibrium toward the open state by disrupting channel closing. In an attempt to understand whether the nonluminal face of S6 participates in the activation mechanism, we conducted a partial tryptophan scan of this region. Substituting a tryptophan for Leu(281) recapitulated the effect on the steady-state and kinetic behavior observed with PCMBS. Considering the predicted nonluminal orientation of Cys(276) and Leu(281), a simple physical interpretation of these results is that the nonluminal face of S6 forms a critical interaction surface mediating the transition into the closed conformation, suggesting the nonluminal C-terminal portion of S6 is allosterically coupled to the activation gate.

Association of the novel non-AT1, non-AT2 angiotensin binding site with neuronal cell death.

We have discovered a non-AT(1), non-AT(2) angiotensin binding site in rodent and human brain membranes, which, based on its pharmacological/biochemical properties and tissue distribution, is different from angiotensin receptors and key proteases processing angiotensins. In this study, the novel angiotensin binding site was localized to a specific brain cell type by using radioligand receptor binding assays. Our results indicate that the novel binding site is expressed in mouse primary cortical neuronal membranes but not in primary cortical astroglial and bEnd.3 brain capillary endothelial cell membranes. Whole-cell binding assays in neurons showed that the binding site faces the outer side of the plasma membrane. Consistent with our previous observations, the novel binding site was unmasked by the sulfhydryl reagent p-chloromercuribenzoate. This effect had a bell-shaped curve and was reversed by reduced glutathione, indicating that the function of the binding site might be regulated by the redox state of the environment. Density of the novel binding site measured by saturation binding assays was significantly increased in neuronal membranes of cells challenged in four in vitro models of cell death (oxygen-glucose deprivation, sodium azide-induced hypoxia, N-methyl-D-aspartate neurotoxicity, and hydrogen peroxide neurotoxicity). In addition, our in vivo data from developing mouse brains showed that the density of the novel angiotensin binding site changes similarly to the pattern of neuronal death in maturating brain. This is the first time that evidence is provided on the association of the novel angiotensin binding site with neuronal death, and future studies directed toward understanding of the functions of this protein are warranted.

Evidence for functional interaction of plasma membrane electron transport, voltage-dependent anion channel and volume-regulated anion channel in frog aorta.

Frog aortic tissue exhibits plasma membrane electron transport (PMET) owing to its ability to reduce ferricyanide even in the presence of mitochondrial poisons, such as cyanide and azide. Exposure to hypotonic solution (108 mOsmol/kg H2O) enhanced the reduction of ferricyanide in excised aortic tissue of frog. Increment in ferricyanide reductase activity was also brought about by the presence of homocysteine (100 microM dissolved in isotonic frog Ringer solution), a redox active compound and a potent modulator of PMET. Two plasma-membrane-bound channels, the volume-regulated anion channel (VRAC) and the voltage-dependent anion channel (VDAC), are involved in the response to hypotonic stress. The presence of VRAC and VDAC antagonists-tamoxifen, glibenclamide, fluoxetine and verapamil, and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), respectively-inhibited this enhanced activity brought about by either hypotonic stress or homocysteine. The blockers do not affect the ferricyanide reductase activity under isotonic conditions. Taken together, these findings indicate a functional interaction of the three plasma membrane proteins, namely, ferricyanide reductase (PMET), VDAC and VRAC.

Reactions of nitrite in erythrocyte suspensions measured by membrane inlet mass spectrometry.

The reactions of nitrite with deoxygenated human erythrocytes were examined using membrane inlet mass spectrometry to detect the accumulation of NO in an extracellular solution. In this method an inlet utilizing a silicon rubber membrane is submerged in cell suspensions and allows NO to pass from the extracellular solution into the mass spectrometer. This provides a direct, continuous, and quantitative determination of nitric oxide concentrations over long periods without the necessity of purging the suspension with inert gas. We have not observed accumulation of NO compared with controls on a physiologically relevant time scale and conclude that, within the limitations of the mass spectrometric method and our experimental conditions, erythrocytes do not generate a net efflux of NO after the addition of millimolar concentrations of nitrite. Moreover, there was no evidence at the mass spectrometer of the accumulation of a peak at mass 76 that would indicate N(2)O(3), an intermediate that decays into NO and NO(2). Inhibition of red cell membrane anion exchangers and aquaporins did not affect these processes.

Glimepiride reduces the expression of PrPc, prevents PrPSc formation and protects against prion mediated neurotoxicity in cell lines.

BACKGROUND: A hallmark of the prion diseases is the conversion of the host-encoded cellular prion protein (PrP(C)) into a disease related, alternatively folded isoform (PrP(Sc)). The accumulation of PrP(Sc) within the brain is associated with synapse loss and ultimately neuronal death. Novel therapeutics are desperately required to treat neurodegenerative diseases including the prion diseases. PRINCIPAL FINDINGS: Treatment with glimepiride, a sulphonylurea approved for the treatment of diabetes mellitus, induced the release of PrP(C) from the surface of prion-infected neuronal cells. The cell surface is a site where PrP(C) molecules may be converted to PrP(Sc) and glimepiride treatment reduced PrP(Sc) formation in three prion infected neuronal cell lines (ScN2a, SMB and ScGT1 cells). Glimepiride also protected cortical and hippocampal neurones against the toxic effects of the prion-derived peptide PrP82-146. Glimepiride treatment significantly reduce both the amount of PrP82-146 that bound to neurones and PrP82-146 induced activation of cytoplasmic phospholipase A(2) (cPLA(2)) and the production of prostaglandin E(2) that is associated with neuronal injury in prion diseases. Our results are consistent with reports that glimepiride activates an endogenous glycosylphosphatidylinositol (GPI)-phospholipase C which reduced PrP(C) expression at the surface of neuronal cells. The effects of glimepiride were reproduced by treatment of cells with phosphatidylinositol-phospholipase C (PI-PLC) and were reversed by co-incubation with p-chloromercuriphenylsulphonate, an inhibitor of endogenous GPI-PLC. CONCLUSIONS: Collectively, these results indicate that glimepiride may be a novel treatment to reduce PrP(Sc) formation and neuronal damage in prion diseases.

Membrane bound pyrophosphatase and P-type adenosine triphosphatase of Leishmania donovani as possible chemotherapeutic targets: similarities and differences in inhibitor sensitivities.

The activities of inorganic pyrophosphatase (PPase) and adenosine triphosphatase (ATPase) were studied in the plasma membrane of Leishmania donovani promastigotes and amastigotes. It was shown that the specific activity of PPase was greater than that of ATPase in the promastigote plasma membrane. We characterized H+-PPase present in the plasma membrane of L. donovani and investigated its possible role in the survival of promastigote and amastigote. PPase activity was stimulated by K+ and sodium orthovanadate and inhibited by pyrophosphate analogs (imidodiphosphate and alendronate), KF, N,N'-dicyclohexylcarbodiimide (DCCD), thiol reagents (p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), and phenylarsine oxide (PAO)), the ABC superfamily transport modulator verapamil, and also by the F(1)F(o)-ATPase inhibitor quercetin. ATPase activity was stimulated by K+ and verapamil, inhibited by DCCD, PCMBS, NEM, sodium azide, sodium orthovanadate, and quercetin, and was unaffected by PAO. We conclude that there are significant differences within promastigote, amastigote, and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase transporter as a putative target for rational drug design.

Salicylic acid transport in Ricinus communis involves a pH-dependent carrier system in addition to diffusion.

Despite its important functions in plant physiology and defense, the membrane transport mechanism of salicylic acid (SA) is poorly documented due to the general assumption that SA is taken up by plant cells via the ion trap mechanism. Using Ricinus communis seedlings and modeling tools (ACD LogD and Vega ZZ softwares), we show that phloem accumulation of SA and hydroxylated analogs is completely uncorrelated with the physicochemical parameters suitable for diffusion (number of hydrogen bond donors, polar surface area, and, especially, LogD values at apoplastic pHs and Delta LogD between apoplast and phloem sap pH values). These and other data (such as accumulation in phloem sap of the poorly permeant dissociated form of monohalogen derivatives from apoplast and inhibition of SA transport by the thiol reagent p-chloromercuribenzenesulfonic acid [pCMBS]) lead to the following conclusions. As in intestinal cells, SA transport in Ricinus involves a pH-dependent carrier system sensitive to pCMBS; this carrier can translocate monohalogen analogs in the anionic form; the efficiency of phloem transport of hydroxylated benzoic acid derivatives is tightly dependent on the position of the hydroxyl group on the aromatic ring (SA corresponds to the optimal position) but moderately affected by halogen addition in position 5, which is known to increase plant defense. Furthermore, combining time-course experiments and pCMBS used as a tool, we give information about the localization of the SA carrier. SA uptake by epidermal cells (i.e. the step preceding the symplastic transport to veins) insensitive to pCMBS occurs via the ion-trap mechanism, whereas apoplastic vein loading involves a carrier-mediated mechanism (which is targeted by pCMBS) in addition to diffusion.