Selective susceptibility to nanosecond pulsed electric field (nsPEF) across different human cell types

Tumor ablation by nanosecond pulsed electric fields (nsPEF) is an emerging therapeutic modality. We compared nsPEF cytotoxicity for human cell lines of cancerous (IMR-32, Hep G2, HT-1080, and HPAF-II) and non-cancerous origin (BJ and MRC-5) under strictly controlled and identical conditions. Adherent cells were uniformly treated by 300-ns PEF (0–2000 pulses, 1.8 kV/cm, 50 Hz) on indium tin oxide-covered glass coverslips, using the same media and serum. Cell survival plotted against the number of pulses displayed three distinct regions (initial resistivity, logarithmic survival decline, and residual resistivity) for all tested cell types, but with differences in LD₅₀ spanning as much as nearly 80-fold. The non-cancerous cells were less sensitive than IMR-32 neuroblastoma cells but more vulnerable than the other cancers tested. The cytotoxic efficiency showed no apparent correlation with cell or nuclear size, cell morphology, metabolism level, or the extent of membrane disruption by nsPEF. Increasing pulse duration to 9 µs (0.75 kV/cm, 5 Hz) produced a different selectivity pattern, suggesting that manipulation of PEF parameters can, at least for certain cancers, overcome their resistance to nsPEF ablation. Identifying mechanisms and cell markers of differential nsPEF susceptibility will critically contribute to the proper choice and outcome of nsPEF ablation therapies.     

Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome

Platelet extracellular vesicles (PEVs) have emerged as potential mediators in intercellular communication. PEVs exhibit several activities with pathophysiological importance and may serve as diagnostic biomarkers. Here, imaging and analytical techniques were employed to unveil morphological pathways of the release, structure, composition, and surface properties of PEVs derived from human platelets (PLTs) activated with the thrombin receptor activating peptide (TRAP). Based on extensive electron microscopy analysis, we propose four morphological pathways for PEVs release from TRAP-activated PLTs: (1) plasma membrane budding, (2) extrusion of multivesicular α-granules and cytoplasmic vacuoles, (3) plasma membrane blistering and (4) “pearling” of PLT pseudopodia. The PLT extracellular vesiculome encompasses ectosomes, exosomes, free mitochondria, mitochondria-containing vesicles, “podiasomes” and PLT “ghosts”. Interestingly, a flow cytometry showed a population of TOM20⁺LC3⁺ PEVs, likely products of platelet mitophagy. We found that lipidomic and proteomic profiles were different between the small PEV (S-PEVs; mean diameter 103 nm) and the large vesicle (L-PEVs; mean diameter 350 nm) fractions separated by differential centrifugation. In addition, the majority of PEVs released by activated PLTs was composed of S-PEVs which have markedly higher thrombin generation activity per unit of PEV surface area compared to L-PEVs, and contribute approximately 60% of the PLT vesiculome procoagulant potency.     

Structural variations in wheat HKT1;5 underpin differences in Na<Superscript>+</Superscript> transport capacity

An important trait associated with the salt tolerance of wheat is the exclusion of sodium ions (Na⁺) from the shoot. We have previously shown that the sodium transporters TmHKT1;5-A and TaHKT1;5-D, from Triticum monoccocum (Tm) and Triticum aestivum (Ta), are encoded by genes underlying the major shoot Na⁺-exclusion loci Nax1 and Kna1, respectively. Here, using heterologous expression, we show that the affinity (K _m) for the Na⁺ transport of TmHKT1;5-A, at 2.66 mM, is higher than that of TaHKT1;5-D at 7.50 mM. Through 3D structural modelling, we identify residues D⁴⁷¹/a gap and D⁴⁷⁴/G⁴⁷³ that contribute to this property. We identify four additional mutations in amino acid residues that inhibit the transport activity of TmHKT1;5-A, which are predicted to be the result of an occlusion of the pore. We propose that the underlying transport properties of TmHKT1;5-A and TaHKT1;5-D contribute to their unique ability to improve Na⁺ exclusion in wheat that leads to an improved salinity tolerance in the field.     

Disparate Na+-requirement of taurocholate and indocyanine green uptake by isolated hepatocytes

Summary Uptake of both taurocholate and indocyanine green (ICG) by isolated rat hepatocytes was saturable and obeyed Michaelis-Menten kinetics. Only uptake of taurocholate exhibited Na⁺-dependence, a phenomenon compatible with active membrane transport. These findings suggest that the mechanisms responsible for hepatocellular uptake of bile acids and anionic dyes differ fundamentally.This work was supported by the Swiss National Science Foundation.The authors thank Mr Th. Weber for technical assistance.     

Platelet-derived growth factor receptor beta identifies mesenchymal stem cells with enhanced engraftment to tissue injury and pro-angiogenic property

Mesenchymal stem cells (MSCs) are heterogeneous likely consisting of subpopulations with various therapeutic potentials. Here we attempted to acquire a subset of MSCs with enhanced effect in wound healing. We found that human placental MSCs expressing platelet-derived growth factor (PDGF) receptor (PDGFR)-β exhibited greater proliferation rates and generated more colony-forming unit-fibroblast (CFU-F), compared to PDGFR-β^? MSCs. Notably, PDGFR-β⁺ MSCs expressed higher levels of pro-angiogenic factors such as Ang1, Ang2, VEGF, bFGF and PDGF. When 10⁶ GFP-expressing MSCs were topically applied into excisional wounds in mice, PDGFR-β⁺ MSCs actively incorporated into the wound tissue, resulting in enhanced engraftment (3.92 ± 0.31 × 10⁵ remained in wound by 7 days) and accelerated wound closure; meanwhile, PDGFR-β^? MSCs tended to remain on the top of the wound bed with significantly fewer cells (2.46 ± 0.26 × 10⁵) engrafted into the wound, suggesting enhanced chemotactic migration and engraftment of PDGFR-β⁺ MSCs into the wound. Real-Time PCR and immunostain analyses revealed that the expression of PDGF-B was upregulated after wounding; transwell migration assay showed that PDGFR-β⁺ MSCs migrated eightfold more than PDGFR-β^? MSCs toward PDGF-BB. Intriguingly, PDGFR-β⁺ MSC-treated wounds showed significantly enhanced angiogenesis compared to PDGFR-β^? MSC- or vehicle-treated wounds. Thus, our results indicate that PDGFR-β identifies a subset of MSCs with enhanced chemotactic migration to wound injury and effect in promoting angiogenesis and wound healing, implying a greater therapeutic potential for certain diseases.     

Increased Na+-H+ exchanger activity in the ileal brush-border membrane of spontaneously hypertensive rats

In the present study, we have examined the intestinal Na⁺ transport, through the Na⁺-H⁺ exchanger, in ileal brush-border membrane vesicles (BBMV) isolated from spontaneously hypertensive rats (SHR), and normotensive Wistar Kyoto (WKY) rats as a control group. Na⁺ uptake into ileal BBMV was stimulated in the presence of a proton gradient (pH 5.5 inside/pH 7.5 outside) in SHR and WKY rats, resulting in a transient accumulation (overshoot) in both groups of rats. No overshoot was observed in the absence of a pH gradient. The magnitude of the accumulation was significantly higher in SHR than in WKY rats. Uptake of Na⁺ at equilibrium was identical in the presence and the absence of a proton gradient and was not changed in SHR. The use of amiloride inhibited pH gradient-driven Na⁺ uptake in a dose-dependent manner with a Ki of 90 μM and 100 μM for SHR and WKY rats, respectively. The relationship between proton gradient-driven Na⁺ uptake and external Na⁺ concentration was saturable and conformed to Michaelis-Menten kinetics in both SHR and WKY rats. Lineweaver-Burk analysis of the pH gradient-driven Na⁺ uptake indicated values of V_max that were significantly increased in SHR compared to WKY rats (11.4±0.55 nmol/mg/8 s vs. 4.96±0.78 nmol/mg/8 s for SHR and WKY rats, respectively). In contrast, similar K_m values for Na⁺ were found between SHR and WKY rats (4.0±0.2 mM vs. 4.9±0.6 mM for SHR and WKY rats, respectively). These studies show derangement in ileal BBMV Na⁺ transport of SHR, which is characterized by increased Na⁺-H⁺ exchanger activity. Received 18 December 1996; received after revision 3 February 1997; accepted 7 February 1997     

Inhibition of glucose transport in human erythrocytes by 2,3-dioxoindole (Isatin)

10 mM isatin (2,3-dioxoindole) inhibited glucose influx into human erythrocytes by over 30%. The inhibition is of the competitive type, where the affinity constant (K_t) was increased from 5.71 (control) to 11.11 mM in the presence of isatin with no change in V_max (130 nmol/min/ml packed cells). The observed inhibition of sugar transport by isatin was not mediated through membrane–SH groups accessible to iodoacetate, iodoacetamide, DTNB, DNP or sodium arsenite. Isatin inhibited sugar transport in the presence of 2 mM harmaline, an alkaloid inhibitor of Na⁺, K⁺–ATP_ase activity. The inhibition was non additive which suggests that these two compounds interact with the same or a similar site on the erythrocyte membrane.     

Depolarizing action of K-strophantine and K-strophantoside on isolated frog skin

Résumé Après une rapide revue de la littérature, les auteurs rendent compte des résultats de leur étude sur l'action dépolarisante de la strophantine K et du strophantoside K sur la peau isolée de la grenouille. Les résultats démontrent, selon les auteurs, que la strophantine K et le strophantoside K ont la même action sur les potentiels de la peau de la grenouille que sur le myocarde. L'action dépolarisante peut être mise en corrélation avec la sortie des K⁺ions et le transport actif des Na⁺ ions dans la membrane des cellules épithéliales de la peau de la grenouille.      

The effects of triaminopyrimidine on the short circuit current and transmembrane potentials of the isolated rat visceral yolk sac in vitro

Summary Intracellular potentials in the cells from 17.5-day old rat visceral yolk sacs were measured by a glass microelectrode. When penetrated from the maternal side, the cells have potentials of about 50.2±1.9 mV (inside negative) which were reduced by increasing the external K⁺ concentration and increased by removing Na⁺ ions from the bathing fluid. Triaminopyrimidine (TAP) which inhibited Na⁺ transport caused a dose-dependent depolarization of the cell membrane. The depolarization was dependent on the presence of extracellular Ca²⁺ ions. It is proposed that TAP may inhibit Na⁺ transport by increasing the intracellular concentration of calcium ions.This work was supported by the University of Hong Kong (grant number 335. 034.5105).Acknowledgment. Triaminopyrimidine was synthesized by Dr. Barbara Roth of the Wellcome Research Laboratories.     

Enhanced excitability in locust muscle fibres induced by calcium free saline

Summary The membrane of locust muscle fibres normally exhibits a graded electrical response to outward current pulses of increasing strength. On removal of Ca⁺⁺ ions from the external medium, these fibres are shown to exhibit depolarizing membrane responses of variable time course and duration. These responses are abolished in Na⁺-free solutions, and by the addition of Mn⁺⁺ ions.D.A.M. was the recipient of a Royal Society and Science Research Council European Exchange Fellowship. We thank the Centre for Overseas Pest Research, Kensington for supplying the locusts.     

Phosphatidylinositol-3,5-bisphosphate lipid-binding-induced activation of the human two-pore channel 2

Mammalian two-pore channels (TPCs) are activated by the low-abundance membrane lipid phosphatidyl-(3,5)-bisphosphate (PI(3,5)P₂) present in the endo-lysosomal system. Malfunction of human TPC1 or TPC2 (hTPC) results in severe organellar storage diseases and membrane trafficking defects. Here, we compared the lipid-binding characteristics of hTPC2 and of the PI(3,5)P₂-insensitive TPC1 from the model plant Arabidopsis thaliana. Combination of simulations with functional analysis of channel mutants revealed the presence of an hTPC2-specific lipid-binding pocket mutually formed by two channel regions exposed to the cytosolic side of the membrane. We showed that PI(3,5)P₂ is simultaneously stabilized by positively charged amino acids (K203, K204, and K207) in the linker between transmembrane helices S4 and S5 and by S322 in the cytosolic extension of S6. We suggest that PI(3,5)P₂ cross links two parts of the channel, enabling their coordinated movement during channel gating.     

Functional and structural determinants of reverse operation in the pH-dependent oligopeptide transporter PepT1

The functional and structural basis of reverse operation of PepT1 has been studied in Xenopus oocytes expressing the wild-type and mutated forms of this protein. Using brief pulses from a negative holding potential, wild-type and Arg282 mutants exhibit outward currents in the presence of Gly-Gln. The reversal potential of these currents is affected by both pH and substrate concentration, confirming coupled transport in the wild type and in the mutants as well. Long-lasting voltage and current-clamp experiments show that the outward currents are only temporary, and reflect accumulation and/or depletion effects near the membrane. The ability to operate in reverse mode was confirmed in all isoforms by intracellular injection of substrate. The role of Arg282 and Asp341 in the reverse transport was also investigated using charged substrates. Positive Lys-Gly (but not Gly-Lys) showed enhanced transport currents in the Arg282 mutants. In contrast, negative Gly-Asp and Asp-Gly elicited modest currents in all isoforms.     

Ion and metabolite transport in the chloroplast of algae: lessons from land plants

Chloroplasts are endosymbiotic organelles and play crucial roles in energy supply and metabolism of eukaryotic photosynthetic organisms (algae and land plants). They harbor channels and transporters in the envelope and thylakoid membranes, mediating the exchange of ions and metabolites with the cytosol and the chloroplast stroma and between the different chloroplast subcompartments. In secondarily evolved algae, three or four envelope membranes surround the chloroplast, making more complex the exchange of ions and metabolites. Despite the importance of transport proteins for the optimal functioning of the chloroplast in algae, and that many land plant homologues have been predicted, experimental evidence and molecular characterization are missing in most cases. Here, we provide an overview of the current knowledge about ion and metabolite transport in the chloroplast from algae. The main aspects reviewed are localization and activity of the transport proteins from algae and/or of homologues from other organisms including land plants. Most chloroplast transporters were identified in the green alga Chlamydomonas reinhardtii, reside in the envelope and participate in carbon acquisition and metabolism. Only a few identified algal transporters are located in the thylakoid membrane and play role in ion transport. The presence of genes for putative transporters in green algae, red algae, diatoms, glaucophytes and cryptophytes is discussed, and roles in the chloroplast are suggested. A deep knowledge in this field is required because algae represent a potential source of biomass and valuable metabolites for industry, medicine and agriculture.     

The ancient claudin Dni2 facilitates yeast cell fusion by compartmentalizing Dni1 into a membrane subdomain

Dni1 and Dni2 facilitate cell fusion during mating. Here, we show that these proteins are interdependent for their localization in a plasma membrane subdomain, which we have termed the mating fusion domain. Dni1 compartmentation in the domain is required for cell fusion. The contribution of actin, sterol-dependent membrane organization, and Dni2 to this compartmentation was analysed, and the results showed that Dni2 plays the most relevant role in the process. In turn, the Dni2 exit from the endoplasmic reticulum depends on Dni1. These proteins share the presence of a cysteine motif in their first extracellular loop related to the claudin GLWxxC(8–10 aa)C signature motif. Structure–function analyses show that mutating each Dni1 conserved cysteine has mild effects, and that only simultaneous elimination of several cysteines leads to a mating defect. On the contrary, eliminating each single cysteine and the C-terminal tail in Dni2 abrogates Dni1 compartmentation and cell fusion. Sequence alignments show that claudin trans-membrane helixes bear small-XXX-small motifs at conserved positions. The fourth Dni2 trans-membrane helix tends to form homo-oligomers in Escherichia plasma membrane, and two concatenated small-XXX-small motifs are required for efficient oligomerization and for Dni2 export from the yeast endoplasmic reticulum. Together, our results strongly suggest that Dni2 is an ancient claudin that blocks Dni1 diffusion from the intercellular region where two plasma membranes are in close proximity, and that this function is required for Dni1 to facilitate cell fusion.     

Pressure-induced variations of K+-permeability as related to a possible reversible electrical breakdown in human erythrocytes

Summary Above a hydrostatic pressure of about 600 b a pronounced reversible increase in the net K⁺-efflux from human erythrocytes is observed. The effect is explained in terms of an electro-mechanical compression of the membrane, resulting in a reversible breakdown of the membrane.Acknowledgments. This work was supported by a grant from the Sonderforschungsbereich 160 to U.Z. and by grant No.2.4511.76 from the FRFC to R.G.     

Na+,K+-ATPase as a docking station: protein–protein complexes of the Na+,K+-ATPase

The Na⁺,K⁺-ATPase, or sodium pump, is well known for its role in ion transport across the plasma membrane of animal cells. It carries out the transport of Na⁺ ions out of the cell and of K⁺ ions into the cell and thus maintains electrolyte and fluid balance. In addition to the fundamental ion-pumping function of the Na⁺,K⁺-ATPase, recent work has suggested additional roles for Na⁺,K⁺-ATPase in signal transduction and biomembrane structure. Several signaling pathways have been found to involve Na⁺,K⁺-ATPase, which serves as a docking station for a fast-growing number of protein interaction partners. In this review, we focus on Na⁺,K⁺-ATPase as a signal transducer, but also briefly discuss other Na⁺,K⁺-ATPase protein–protein interactions, providing a comprehensive overview of the diverse signaling functions ascribed to this well-known enzyme.     

Membrane-shed vesicles from the parasite <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>: characterization and their association with cell interaction

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor–ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.     

Sulindac sulfide suppresses 5-lipoxygenase at clinically relevant concentrations

Sulindac is a non-selective inhibitor of cyclooxygenases (COX) used to treat inflammation and pain. Additionally, non-COX targets may account for the drug’s chemo-preventive efficacy against colorectal cancer and reduced gastrointestinal toxicity. Here, we demonstrate that the pharmacologically active metabolite of sulindac, sulindac sulfide (SSi), targets 5-lipoxygenase (5-LO), the key enzyme in the biosynthesis of proinflammatory leukotrienes (LTs). SSi inhibited 5-LO in ionophore A23187- and LPS/fMLP-stimulated human polymorphonuclear leukocytes (IC₅₀ ≈ 8–10 μM). Importantly, SSi efficiently suppressed 5-LO in human whole blood at clinically relevant plasma levels (IC₅₀ = 18.7 μM). SSi was 5-LO-selective as no inhibition of related lipoxygenases (12-LO, 15-LO) was observed. The sulindac prodrug and the other metabolite, sulindac sulfone (SSo), failed to inhibit 5-LO. Mechanistic analysis demonstrated that SSi directly suppresses 5-LO with an IC₅₀ of 20 μM. Together, these findings may provide a novel molecular basis to explain the COX-independent pharmacological effects of sulindac under therapy.     

The effects of caffeine on physiological functions and mental performance

Summary Polygraphic monitoring of several physiological variables was done throughout an experiment investigating the effects of caffeine on mental performance. The experiment started with a mental maze learning task. Then the subjects were given the test beverages according to the group design (CC group (N=16)300 mg caffeine in decaffeinated coffee, DC group (N=16): decaffeinated coffee, WW group (N=8): warm water, and NB group (N=8): no beverage). The experiment continued with a letter cancellation task which was followed by a second mental maze learning task. The caffeine treated subjects differed from the other groups by increased regularity of letter cancellation performance, as indicated by decreases in intraindividual variance. They also differed from the other groups by a slight but significant acrodermal vasoconstriction. No intergroup differences were obtained for mental maze learning, heart rate, respiration, muscle tension, and skin conductance. The results suggest therefore that the drug at this dose level improves behavioral routine and speed rather than cognitive functions and that the vegetative side effects are minimal.