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Membrane Pore

(151 References)

Abrami, L., M. Fivaz, et al. (2003). "Sensitivity of polarized epithelial cells to the pore-forming toxin aerolysin." Infect Immun 71(2): 739-46.

            Aerolysin is one of the major virulence factors produced by Aeromonas hydrophila, a human pathogen that produces deep wound infection and gastroenteritis. The toxin interacts with target mammalian cells by binding to the glycan core of glycosylphosphatidyl inositol (GPI)-anchored proteins and subsequently forms a pore in the plasma membrane. Since epithelial cells of the intestine are the primary targets of aerolysin, we investigated its effect on three types of polarized epithelial cells: Caco-2 cells, derived from human intestine; MDCK cells, a well-characterized cell line in terms of protein targeting; and FRT cells, an unusual cell line in that it targets its GPI-anchored proteins to the basolateral plasma membrane in contrast to other epithelial cells, which target them almost exclusively to the apical surface. Surprisingly, we found that all three cell types were sensitive to the toxin from both the apical and the basolateral sides. Apical sensitivity was always higher, even for FRT cells. In contrast, FRT cells were more sensitive from the basolateral than from the apical side to the related toxin Clostridium septicum alpha-toxin, which also binds to GPI-anchored proteins but lacks the lectin binding domain found in aerolysin. These observations are consistent with the notion that a shuttling mechanism involving low-affinity interactions with surface sugars allows aerolysin to gradually move toward the membrane surface, where it can finally encounter the glycan cores of GPI-anchored proteins.

Abramov, A. Y. and M. R. Duchen (2003). "Actions of ionomycin, 4-BrA23187 and a novel electrogenic Ca(2+) ionophore on mitochondria in intact cells." Cell Calcium 33(2): 101-12.

            We have used fluorescence digital imaging techniques to explore the actions of two groups of Ca(2+) ionophores: (i) ferutinin, an electrogenic naturally occurring ionophore, and (ii) the neutral ionophores 4-BrA23187 and ionomycin, on cytosolic [Ca(2+)] ([Ca(2+)](c)), mitochondrial [Ca(2+)] ([Ca(2+)](m)) and mitochondrial membrane potential (Deltapsi(m)) in HepG2 cells and primary hippocampal neurones in culture. 4-BrA23187 and ionomycin promoted the equilibration of [Ca(2+)] gradients between cellular compartments, including ER, mitochondria and cytosol. Thus, [Ca(2+)](c) and [Ca(2+)](m) increased together and then recovered in parallel on removal of the ionophore. In contrast, following a rise in [Ca(2+)](c) in response to ferutinin, [Ca(2+)](m) remained elevated for prolonged periods after the recovery of [Ca(2+)](c) levels despite washout of the compound. Both groups of Ca(2+) ionophores caused some mitochondrial depolarisation, although this was highly variable in degree. Mitochondrial depolarisation induced by ionomycin and 4-BrA23187 was often modest, independent of cyclosporin A (CsA), was suppressed in the absence of extracellular Ca(2+) and was enhanced by pre-incubation of cells with the inhibitor of the mitochondrial Ca(2+)/2Na(+)-exchanger, CGP37157, suggesting that the change in potential reflects the prior state of mitochondrial calcium loading. The mitochondrial depolarisation induced by ferutinin was not influenced by CGP37157 but was completely blocked by CsA, suggesting that it reflects opening of the mitochondrial permeability transition pore (mPTP). We suggest that ferutinin may provide a very valuable tool to promote mitochondrial calcium overload experimentally and to promote calcium-dependent opening of the mPTP.

Akao, M., B. O'Rourke, et al. (2003). "Differential actions of cardioprotective agents on the mitochondrial death pathway." Circ Res 92(2): 195-202.

            We examined the effect of cardioprotective agents on three distinct phases of the H2O2-induced response that leads to loss of mitochondrial membrane potential (DeltaPsi(m)) and cell death in cultured cardiac myocytes: (1) priming, consisting of calcium-dependent morphological changes in mitochondria (swelling and loss of cristae), with preserved DeltaPsi(m), (2) depolarization, the rapid DeltaPsi(m) depolarization caused by mitochondrial permeability transition pore (PTP) opening, and (3) cell fragmentation. The mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel opener diazoxide markedly decreased the likelihood that cells would undergo priming: many mitochondria remained fully polarized and morphologically intact. Diazoxide not only decreased the number of cells undergoing DeltaPsi(m) depolarization but also delayed the onset of DeltaPsi(m) loss, whereas it did not change the duration of depolarization in unprotected cells. The adenine nucleotide translocase inhibitor bongkrekic acid mimicked the effect of diazoxide to suppress priming, except that its effects were not blocked by the mitoK(ATP) channel blocker 5-hydroxydecanoate. In contrast, the PTP inhibitor cyclosporin A (CsA) did not prevent priming: neither latency for DeltaPsi(m) depolarization nor mitochondrial morphological changes were affected. However, CsA slowed the process of depolarization and blunted its severity. Importantly, coapplication of diazoxide and CsA exhibited additive effects, improving the efficacy of protection. Activation of mitoK(ATP) channels suppresses the cell death process at its earliest stage, by preserving mitochondrial integrity during oxidative stress. By virtue of its pharmacology and its phenotypic consequences, this mode of action is distinguishable from that of other cardioprotective interventions.

Akao, M., B. O'Rourke, et al. (2003). "Mechanistically distinct steps in the mitochondrial death pathway triggered by oxidative stress in cardiac myocytes." Circ Res 92(2): 186-94.

            Oxidative stress plays an important role in the pathogenesis of cardiovascular diseases. In the present study, we characterize three distinct phases of the H2O2-induced response, which leads to loss of mitochondrial membrane potential (DeltaPsi(m)) and subsequent cell death in cultured cardiac myocytes. (1) Priming: After H2O2 exposure (100 micromol/L), cells maintain a constant DeltaPsi(m) for the cell-to-cell specific latency but at the same time undergo progressive changes in inner mitochondrial membrane structure (swelling and loss of cristae by electron microscopy). An increase of matrix calcium is required, but not sufficient, for this process. (2) Depolarization: Priming is followed by sudden depolarization of DeltaPsi(m), which is mediated by mitochondrial permeability transition pore opening, as evidenced by the concomitant release of calcein from mitochondria. This process is rapid (<4 minutes), complete, and irreversible. The duration of depolarization is constant and does not depend on the length of the priming process in any given cell. (3) Fragmentation: Along with massive mitochondrial swelling and release of cytochrome c into the cytoplasm, cells undergo surface membrane alterations, such as exposure of phosphatidylserine and eventual loss of membrane integrity and cellular fragmentation. Thus, oxidant stress elicits reproducible and stereotyped responses in cardiac cells. The priming phase, during which mitochondria undergo major ultrastructural alterations but remain functional, represents a particularly attractive target for intervention in the prevention of cell death.

Alvarez, C., F. Casallanovo, et al. (2003). "Binding of sea anemone pore-forming toxins sticholysins I and II to interfaces-Modulation of conformation and activity, and lipid-protein interaction." Chem Phys Lipids 122(1-2): 97-105.

            Sticholysins I and II (St I and St II) are water-soluble toxins produced by the sea anemone Stichodactyla helianthus. St I and St II bind to biological and model membranes containing sphingomyelin (SM), forming oligomeric pores that lead to leakage of internal contents. Here we describe functional and structural studies of the toxins aiming at the understanding at a molecular level of their mechanism of binding, as well as their effects on membrane permeabilization. St I and St II caused potassium leakage from red blood cells and temperature-dependent hemolysis, the activation energy of the process being lower for the latter toxin. Protein intrinsic fluorescence measurements provided evidence for toxin binding to model membranes composed of 1:1 (mol:mol) egg phosphatidyl choline (ePC):SM. The fluorescence intensity increased and the maximum emission wavelength decreased as a result of binding. The changes were quantitatively different for both toxins. Circular dichroism spectra showed that both St I and St II exhibit a high content of beta-sheet structure and that binding to model membranes did not alter the toxin's conformation to a large extent. Changing the lipid composition by adding 5 mol% of negatively charged phosphatidic acid (PA) or phosphatidyl glycerol (PG) had small, but detectable, effects on protein conformation. The influence of lipid composition on toxin-induced membrane permeabilization was assessed by means of fluorescence measurements of calcein leakage. The effect was larger for ePC:SM bilayers containing 5 mol% of negative curvature-inducing lipids. Electron paramagnetic resonance (EPR) spectra of intercalated fatty acid spin probes carrying the nitroxide moiety at different carbons (5, 7, 12, and 16) evidenced the occurrence of lipid-protein interaction. Upon addition of the toxins, two-component spectra were observed for the probe labeled at C-12. The broader component, corresponding to a population of strongly immobilized spin probes, was ascribed to boundary lipid. The contribution of this component to the total spectrum was larger for St II than for St I. Moreover, it was clearly detectable for the C-12-labeled probe, but it was absent when the label was at C-16, indicating a lack of lipid-protein interaction close to the lipid terminal methyl group. This effect could be either due to the fact that the toxins do not span the whole bilayer thickness or to the formation of a toroidal pore leading to the preferential interaction with acyl chain carbons closer to the phospholipids head groups.

Atabay, H. I., F. Aydin, et al. (2003). "The prevalence of Arcobacter spp. on chicken carcasses sold in retail markets in Turkey, and identification of the isolates using SDS-PAGE." Int J Food Microbiol 81(1): 21-8.

            In this study, the prevalence of Arcobacter spp. on chicken carcasses sold in various retail markets in Turkey was investigated. The isolates were characterized and identified using various phenotypic and molecular tests. The membrane filtration technique employing 0.45-microm pore size membrane filters laid onto a nonselective blood agar was used after enrichment in Oxoid Arcobacter Enrichment Broth (AEB) to examine a total of 75 chicken carcasses (44 fresh and 31 frozen). Species level identification was performed using SDS-PAGE of whole-cell proteins and a recently developed multiplex-PCR assay. All isolates were identified as Arcobacter butzleri. Of the 44 fresh chicken carcasses examined, 42 (95%) were positive for A. butzleri. A. butzleri was also recovered from seven (23%) of the 31 frozen carcasses examined.

Banke, T. G. and S. F. Traynelis (2003). "Activation of NR1/NR2B NMDA receptors." Nat Neurosci 6(2): 144-52.

            N-methyl-D-aspartate (NMDA) receptors are highly expressed in the central nervous system and are involved in excitatory synaptic transmission as well as synaptic plasticity. Despite considerable structural and biophysical research, the mechanism behind activation of the NMDA receptor is still poorly understood. By analyzing patch clamp recordings of one channel activated by glutamate, we determined the burst structure and open probability for recombinant rat NR1/NR2B receptors. We used partial agonists at the glutamate and glycine binding sites to show that at least two kinetically distinct subunit-associated conformational changes link co-agonist binding to the opening of the NMDA receptor pore. These data suggest that NR1 and NR2B subunits, respectively, undergo a fast and slow agonist-dependent conformational change that precedes opening of the pore. We propose a new working model of receptor activation that can account for macroscopic as well as microscopic NMDA receptor properties.

Bates, M., M. Burns, et al. (2003). "Dynamics of DNA molecules in a membrane channel probed by active control techniques." Biophys J 84(4): 2366-72.

            The dynamics of single-stranded DNA in an alpha-Hemolysin protein pore was studied at the single-molecule level. The escape time for DNA molecules initially drawn into the pore was measured in the absence of an externally applied electric field. These measurements revealed two well-separated timescales, one of which is surprisingly long (on the order of milliseconds). We characterized the long timescale as being associated with the binding and unbinding of DNA from the pore. We have also found that a transmembrane potential as small as 20 mV strongly biased the escape of DNA from the pore. These experiments have been made possible due to the development of a feedback control system, allowing the rapid modulation of the applied force on individual DNA molecules while inside the pore.

Bhatnagar, A., R. Kumar, et al. (2003). "Differential pyridine nucleotide coenzyme binding to the beta-subunit of the voltage-sensitive K(+) channel: a mechanism for redox regulation of excitability?" Chem Biol Interact 143-144: 613-20.

            The pore-forming subunits of the voltage-sensitive K(+) channel (K(v)) associate with ancillary beta-subunits that regulate inactivation and voltage-dependence of the channel. The beta-subunits are members of the aldo-keto reductase (AKR) superfamily. We have previously demonstrated that recombinant K(v)beta2.1 displays tight binding to NADP(H). The protein also binds NAD(H), but with less affinity. To assess the physiological significance of this binding, we examined how pyridine nucleotides regulate the K(v)beta-mediated inactivation of K(+) channels. Transient transfection of COS-7 cells with an pIRES-hrGFP vector containing the Kvalpha1.5 cDNA led to the appearance of the K(v)1.5 protein in the membrane fraction and large non-inactivating potassium currents were recorded from the transfected cells. No such currents were observed in cells transfected with the empty vector alone or with K(v)beta1.3 (AKR6A3), which was localized to the cytoplasm. In contrast, K(v)beta1.3 co-transfected with Kvalpha1.5 was localized to the membrane, suggesting high affinity binding of the two proteins. Moreover, the K currents recorded from cells transfected with both K(v)alpha1.5 and K(v)beta1.3 displayed pronounced inactivation. Inclusion of 1 mM NAD(+) in the internal solution of the patch pipette abolished K(v)beta-induced inactivation of K(v)1.5 currents, but did not affect the non-inactivating currents recorded from cells transfected with K(v)1.5 alone, indicating that in the absence of K(v)beta, NAD(+) does not affect the activity of K(v)alpha. The inactivating currents recorded from cells expressing both K(v)alpha1.5 and K(v)beta1.3 were unaffected by the inclusion of 0.1 mM NADPH in the pipette solution. Together, these data suggest that NADPH and NAD(+) impart different conformational states to the K(v)beta protein and that only the NADPH bound K(v)beta imparts inactivation to non-inactivating K(+) currents. Thus, differential binding of pyridine nucleotide coenzymes to K(v)beta could regulate membrane potential and excitability as a function of the cellular redox state. Because NAD(+)/NADPH ratio is sensitive to oxygen concentration, the differential changes in K(v)beta-mediated inactivation of K currents by NAD(+) and NADPH could represent an oxygen-sensing mechanism.

Boldt, W., M. Klapperstuck, et al. (2003). "Glu496Ala polymorphism of human P2X7 receptor does not affect its electrophysiological phenotype." Am J Physiol Cell Physiol 284(3): C749-56.

            A glutamate to alanine exchange at amino acid position 496 of the human P2X(7) receptor was recently shown to be associated with a loss of function in human B lymphocytes in terms of ATP-induced ethidium(+) uptake, Ba(2+) influx, and induction of apoptosis (Gu BJ, Zhang WY, Worthington RA, Sluyter R, Dao-Ung P, Petrou S, Barden JA, and Wiley JS. J Biol Chem 276: 11135-11142, 2001). Here we analyzed the effect of the Glu(496) to Ala exchange on the channel properties of the human P2X(7) receptor expressed in Xenopus oocytes with the two-microelectrode voltage-clamp technique. The amplitudes of ATP-induced whole cell currents characteristic of functional expression, kinetic properties including ATP concentration dependence, and permeation behavior were not altered by this amino acid exchange. Also in HEK293 cells, the Ala(496) mutant mediated typical P2X(7) receptor-dependent currents like the parent Glu(496) hP2X(7) receptor. Because the function of the P2X(7) receptor as an ATP-gated channel for small cations including Ba(2+) remained unaffected by this mutation, we conclude that Glu(496) plays a critical role in pore formation but does not determine the ion channel properties of the human P2X(7) receptor.

Bonev, B. B., Y. H. Lam, et al. (2003). "Effects of the Eukaryotic Pore-Forming Cytolysin Equinatoxin II on Lipid Membranes and the Role of Sphingomyelin." Biophys J 84(4): 2382-92.

            Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by (31)P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20-40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state (31)P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The (2)H and (31)P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.

Breukink, E., H. E. Van Heusden, et al. (2003). "Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes." J Biol Chem.

            The peptidoglycan layers surrounding bacterial membranes are essential for bacterial cell survival and provide an important target for antibiotics. Many antibiotics have mechanisms of action that involve binding to Lipid II, the prenyl chain linked donor of the peptidoglycan building blocks. One of these antibiotics, the pore-forming peptide nisin, uses Lipid II as a receptor molecule to increase its antimicrobial efficacy dramatically. Nisin is the first example of a targeted membrane permeabilizing peptide antibiotic. However, it was not known whether Lipid II functions only as a receptor to recruit nisin to bacterial membranes, thus increasing its specificity for bacterial cells, or whether it also plays a role in pore formation. We have developed a new method to produce large amounts of Lipid II and variants thereof so that we can address the role of the lipid-linked disaccharide in nisin's activity. We show here that Lipid II is not only the receptor for nisin, but an intrinsic component of the pore formed by nisin, and we present a new model for the pore-complex that includes Lipid II.

Burnouf, T., M. Radosevich, et al. (2003). "Nanofiltration of single plasma donations: feasibility study." Vox Sang 84(2): 111-9.

            BACKGROUND AND OBJECTIVES: Major technical developments have been made in recent years to improve the quality and safety of human plasma for transfusion and fractionation. The present study was performed to assess, for the first time, the feasibility of applying a nanofiltration process, using 75-nm and 35-nm mean pore size membranes (Planova(R) 75N and Planova(R) 35N), to human plasma. MATERIALS AND METHODS: Ten apheresis plasma units were obtained from 10 plasma donors. Within 4 h of collection, plasma was subjected to leucoreduction and filtration (using 75-nm and 35-nm mean pore size membranes) at 35 degrees C, at less than 1 bar pressure. Aliquots of plasma were taken at all steps of the filtration procedure and numerous plasma quality parameters were measured. In addition, six hepatitis C virus (HCV)-positive plasma donations were experimentally subjected to the same filtration sequence and subsequently assessed by RNA polymerase chain reaction (PCR) and branched-chain DNA-quantification assays. RESULTS: Leucoreduced plasma can be reproducibly nanofiltered onto a sequence of 75-nm and 35-nm membranes, at a flow rate of 450 ml/h and a temperature of 35 +/- 0.5 degrees C. Some protein dilution, or loss, was found during filtration, but the plasma filtered through membranes with a mean pore size of 75 nm and 35 nm met in vitro specifications for use in transfusion or fractionation. There were no signs of activation of the coagulation system. HCV-positive plasma donations became negative, as judged by PCR and branched-chain DNA assay results, after filtration through the 35-nm membrane. CONCLUSIONS: It is possible to apply a 75 + 35-nm filtration process to leucoreduced human plasma. This technology may have important future benefits in improving the quality and safety of plasma, by removing blood cell debris and infectious agents.

Campanucci, V. A., I. M. Fearon, et al. (2003). "A novel O2-sensing mechanism in rat glossopharyngeal neurones mediated by a halothane-inhibitable background K+ conductance." J Physiol.

            Modulation of K(+) channels by hypoxia is a common O2-sensing mechanism in specialised cells. More recently, acid-sensitive TASK-like background K(+) channels, which play a key role in setting the resting membrane potential, have been implicated in O2-sensing in certain cell types. Here, we report a novel O2 sensitivity mediated by a weakly pH-sensitive background K(+) conductance in nitric oxide synthase (NOS)-positive neurones of the glossopharyngeal nerve (GPN). This conductance was insensitive to 30 mM TEA, 5 mM 4-aminopyridine (4-AP) and 200 micro M Cd(2+), but was reversibly inhibited by hypoxia (O2 tension (PO2) = 15 mmHg), 2-5 mM halothane, 10 mM barium and 1 mM quinidine. Notably, the presence of halothane occluded the inhibitory effect of hypoxia. Under current clamp, these agents depolarised GPN neurones. In contrast, arachidonic acid (5-10 micro M) caused membrane hyperpolarisation and potentiation of the background K(+) current. This pharmacological profile suggests the O2-sensitive conductance in GPN neurones is mediated by a class of background K(+) channels different from the TASK family; it appears more closely related to the THIK (tandem pore domain halothane-inhibited K(+)) subfamily, or may represent a new member of the background K(+) family. Since GPN neurones are thought to provide NO-mediated efferent inhibition of the carotid body (CB), these channels may contribute to the regulation of breathing during hypoxia via negative feedback control of CB function, as well as to the inhibitory effect of volatile anaesthetics (e.g. halothane) on respiration.

Cans, A. S., N. Wittenberg, et al. (2003). "Artificial cells: unique insights into exocytosis using liposomes and lipid nanotubes." Proc Natl Acad Sci U S A 100(2): 400-4.

            Exocytosis is the fundamental process underlying neuronal communication. This process involves fusion of a small neurotransmitter-containing vesicle with the plasma membrane of a cell to release minute amounts of transmitter molecules. Exocytosis is thought to go through an intermediate step involving formation of a small lipid nanotube or fusion pore, followed by expansion of the pore to the final stage of exocytosis. The process of exocytosis has been studied by various methods; however, when living cells are used it is difficult to discriminate between the molecular effects of membrane proteins relative to the mechanics of lipid-membrane-driven processes and to manipulate system parameters (e.g., membrane composition, pH, ion concentration, temperature, etc.). We describe the use of liposome-lipid nanotube networks to create an artificial cell model that undergoes the later stages of exocytosis. This model shows that membrane mechanics, without protein intervention, can drive expansion of the fusion pore to the final stage of exocytosis and can affect the rate of transmitter release through the fusion pore.

Carrillo, C., J. A. Teruel, et al. (2003). "Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin." Biochim Biophys Acta 1611(1-2): 91-7.

            Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactant and possesses several other interesting biological activities. This work deals with the molecular mechanism of membrane permeabilization by incorporation of surfactin. The surfactin-induced vesicle contents leakage was monitored by following release of carboxyfluorescein entrapped into unilamellar vesicles made of palmitoyloleoylphosphatidylcholine (POPC). The effect of the addition of cholesterol, dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylethanolamine (POPE) was also checked. It was observed that surfactin was able to induce content leakage at concentrations far below the onset surfactin/lipid ratio for membrane solubilization to occur, which in our system was around 0.92. Electron microscopy showed that vesicles were present after addition of surfactin at a ratio below this value, whereas no vesicles could be observed at ratios above it. Cholesterol and POPE attenuated the membrane-perturbing effect of surfactin, whereas the effect of DPPC was to promote surfactin-induced leakage, indicating that bilayer sensitivity to surfactin increases with the lipid tendency to form lamellar phases, which is in agreement with our previous observation that surfactin destabilizes the inverted-hexagonal structure. Fourier-transform infrared spectroscopy (FTIR) was used to specifically follow the effect of surfactin on different parts of the phospholipid bilayer. The effect on the Cz.dbnd6;O stretching mode of vibration of POPC indicated a strong dehydration induced by surfactin. On the other hand, the Cz.sbnd;H stretching bands showed that the lipopeptide interacts with the phospholipid acyl chains, resulting in considerable membrane fluidization. The reported effects could be useful to explain surfactin-induced 'pore' formation underlying the antibiotic and other important biological actions of this bacterial lipopeptide.

Cavalier-Smith, T. (2003). "Genomic reduction and evolution of novel genetic membranes and protein-targeting machinery in eukaryote-eukaryote chimaeras (meta-algae)." Philos Trans R Soc Lond B Biol Sci 358(1429): 109-34.

            Chloroplasts originated just once, from cyanobacteria enslaved by a biciliate protozoan to form the plant kingdom (green plants, red and glaucophyte algae), but subsequently, were laterally transferred to other lineages to form eukaryote-eukaryote chimaeras or meta-algae. This process of secondary symbiogenesis (permanent merger of two phylogenetically distinct eukaryote cells) has left remarkable traces of its evolutionary role in the more complex topology of the membranes surrounding all non-plant (meta-algal) chloroplasts. It took place twice, soon after green and red algae diverged over 550 Myr ago to form two independent major branches of the eukaryotic tree (chromalveolates and cabozoa), comprising both meta-algae and numerous secondarily non-photosynthetic lineages. In both cases, enslavement probably began by evolving a novel targeting of endomembrane vesicles to the perialgal vacuole to implant host porter proteins for extracting photosynthate. Chromalveolates arose by such enslavement of a unicellular red alga and evolution of chlorophyll c to form the kingdom Chromista and protozoan infrakingdom Alveolata, which diverged from the ancestral chromalveolate chimaera. Cabozoa arose when the common ancestor of euglenoids and cercozoan chlorarachnean algae enslaved a tetraphyte green alga with chlorophyll a and b. I suggest that in cabozoa the endomembrane vesicles originally budded from the Golgi, whereas in chromalveolates they budded from the endoplasmic reticulum (ER) independently of Golgi-targeted vesicles, presenting a potentially novel target for drugs against alveolate Sporozoa such as malaria parasites and Toxoplasma. These hypothetical ER-derived vesicles mediated fusion of the perialgal vacuole and rough ER (RER) in the ancestral chromist, placing the former red alga within the RER lumen. Subsequently, this chimaera diverged to form cryptomonads, which retained the red algal nucleus as a nucleomorph (NM) with approximately 464 protein-coding genes (30 encoding plastid proteins) and a red or blue phycobiliprotein antenna pigment, and the chromobiotes (heterokonts and haptophytes), which lost phycobilins and evolved the brown carotenoid fucoxanthin that colours brown seaweeds, diatoms and haptophytes. Chromobiotes transferred the 30 genes to the nucleus and lost the NM genome and nuclear-pore complexes, but retained its membrane as the periplastid reticulum (PPR), putatively the phospholipid factory of the periplastid space (former algal cytoplasm), as did the ancestral alveolate independently. The chlorarachnean NM has three minute chromosomes bearing approximately 300 genes riddled with pygmy introns. I propose that the periplastid membrane (PPM, the former algal plasma membrane) of chromalveolates, and possibly chlorarachneans, grows by fusion of vesicles emanating from the NM envelope or PPR. Dinoflagellates and euglenoids independently lost the PPM and PPR (after diverging from Sporozoa and chlorarachneans, respectively) and evolved triple chloroplast envelopes comprising the original plant double envelope and an extra outermost membrane, the EM, derived from the perialgal vacuole. In all metaalgae most chloroplast proteins are coded by nuclear genes and enter the chloroplast by using bipartite targeting sequences - an upstream signal sequence for entering the ER and a downstream chloroplast transit sequence. I present a new theory for the four-fold diversification of the chloroplast OM protein translocon following its insertion into the PPM to facilitate protein translocation across it (of both periplastid and plastid proteins). I discuss evidence from genome sequencing and other sources on the contrasting modes of protein targeting, cellular integration, and evolution of these two major lineages of eukaryote 'cells within cells'. They also provide powerful evidence for natural selection's effectiveness in eliminating most functionless DNA and therefore of a universally useful non-genic function for nuclear non-coding DNA, i.e. most DNA in the biosphere, and dramatic examples of genomic reduction. I briefly argue that chloroplast replacement in dinoflagellates, which happened at least twice, may have been evolutionarily easier than secondary symbiogenesis because parts of the chromalveolate protein-targeting machinery could have helped enslave the foreign plastids.

Chanturiya, A., J. Yang, et al. (2003). "New cationic lipids form channel-like pores in phospholipid bilayers." Biophys J 84(3): 1750-5.

            Two representatives of a new class of cationic lipids were found to have high pore-forming activity in planar bilayer membranes. These molecules, called BHHD-TADC and BHTD-TADC, have qualitatively similar effects on phospholipid membranes. Addition of 2.5-5 micro M of either of them to the membrane bathing solutions resulted in formation of long-lived anion-selective pores with conductance in the range 0.1-2 nS in 0.1 M KCl. Pore formation was found to be dependent on the potential applied to the membrane. When negative potential was applied to membrane at the side of addition, the rate of pore formation was much lower compared to when the positive potential was applied. Dependence of pore formation on compound concentration was highly nonlinear, indicating that this process requires assembly of molecules in the membrane. Addition of any of these compounds on both sides of the membrane increased the efficiency of pore formation by one to two orders of magnitude. Pore formation was strongly pH dependent. Although pores were formed with high efficiency at pH 6.5, only occasional fluctuations of membrane conductance were observed at pH 7.5. Possible mechanisms of new compounds biological activity are discussed.

Choat, B., M. Ball, et al. (2003). "Pit membrane porosity and water stress-induced cavitation in four co-existing dry rainforest tree species." Plant Physiol 131(1): 41-8.

            Aspects of xylem anatomy and vulnerability to water stress-induced embolism were examined in stems of two drought-deciduous species, Brachychiton australis (Schott and Endl.) A. Terracc. and Cochlospermum gillivraei Benth., and two evergreen species, Alphitonia excelsa (Fenzal) Benth. and Austromyrtus bidwillii (Benth.) Burret., growing in a seasonally dry rainforest. The deciduous species were more vulnerable to water stress-induced xylem embolism. B. australis and C. gillivraei reached a 50% loss of hydraulic conductivity at -3.17 MPa and -1.44 MPa, respectively; a 50% loss of hydraulic conductivity occurred at -5.56 MPa in A. excelsa and -5.12 MPa in A. bidwillii. To determine whether pit membrane porosity was responsible for greater vulnerability to embolism (air seeding hypothesis), pit membrane structure was examined. Expected pore sizes were calculated from vulnerability curves; however, the predicted inter-specific variation in pore sizes was not detected using scanning electron microscopy (pores were not visible to a resolution of 20 nm). Suspensions of colloidal gold particles were then perfused through branch sections. These experiments indicated that pit membrane pores were between 5 and 20 nm in diameter in all four species. The results may be explained by three possibilities: (a) the pores of the expected size range were not present, (b) larger pores, within the size range to cause air seeding, were present but were rare enough to avoid detection, or (c) pore sizes in the expected range only develop while the membrane is under mechanical stress (during air seeding) due to stretching/flexing.

Cohen-Addad, J. P., A. Prunelet, et al. (2003). "Acrylonitrile-sodium methallylsulfonate copolymer. DSC approach to membrane porosity of foam and hollow fibers." Biomaterials 24(1): 173-9.

            The porosity of membranes formed from acrylonitrile-sodium methallylsulfonate copolymer was characterized from the analysis of the depression of the melting point of absorbed water. Membranes were obtained either as a foam or as a hollow fiber; the foam consisted of interconnected macrocavities (mean diameter about equal to 1mm) while the hollow fiber was a symmetric membrane used for blood ultrafiltration. Differential scanning calorimetry (DSC) of water revealed both the Gaussian distribution of pore sizes and correspondingly, their mean size: 5.2nm for the pores through the walls separating macrocavities in the foam and 5.6 and 10.6nm associated with two distributions representing nearly equal amounts of absorbed water, for the hollow fiber. In addition to DSC, the water magnetic relaxation showed that the isothermal dehydration of the foam was due to the deswelling of macrocavities while the increasing amount of absorbed water in pores reflects its strong interaction with the polymer.

Cole, D. S. and B. P. Morgan (2003). "Beyond lysis: how complement influences cell fate." Clin Sci (Lond).

            Complement is a central component of the innate immune system involved in protection against pathogens. For many years, complement has been known to cause death of targets either indirectly by attracting and activating phagocytes or directly by formation of a membrane pore, the membrane attack complex. More recently, it has been recognised that complement may cause other 'non-classical' effects that may not directly be aimed at killing of pathogens. Products of complement activation collaborate with the adaptive immune system to enhance responses to antigens. The membrane attack complex of complement, apart from lysing cells, can also trigger diverse events in target cells that include cell activation, proliferation, resistance to subsequent complement attack and either resistance to, or induction of, apoptosis. Various complement products play important roles in signalling for clearance by phagocytes of apoptotic self cells. Here we review some of these 'non-classical' activities of complement and stress the roles they may play in maintaining the integrity of the organism.

Collins, R. F., R. C. Ford, et al. (2003). "Three-Dimensional Structure of the Neisseria meningitidis Secretin PilQ Determined from Negative-Stain Transmission Electron Microscopy." J Bacteriol 185(8): 2611-7.

            The PilQ secretin from the pathogenic bacterium Neisseria meningitidis is an integral outer membrane protein complex which plays a crucial role in the biogenesis of type IV pili. We present here the first three-dimensional structure of this type of secretin at 2.5-nm resolution, obtained by single-particle averaging methods applied to the purified protein complex visualized in a negative stain. In projection, the PilQ complex is circular, with a donut-like appearance. When viewed from the side it has a rounded, conical profile. The complex was demonstrated to have 12-fold rotational symmetry, and this property was used to improve the quality of the density map by symmetry averaging. The dominant feature of the structure is a cavity, 10 nm deep, within the center of the molecule. The cavity is funnel-shaped in cross section, measures 6.5 nm in diameter at the top of the complex, and tapers to a closed point, effectively blocking formation of a continuous pore through the PilQ complex. These results suggest that the complex would have to undergo a conformational change in order to accommodate an assembled pilus fiber of diameter 6.5 nm running through the outer membrane.

Colombo, G., S. J. Marrink, et al. (2003). "Simulation of MscL Gating in a Bilayer under Stress." Biophys J 84(4): 2331-7.

            The initial stages of the gating of the mechanoselective channel of large conductance from Mycobacterium tuberculosis have been studied in atomic detail using molecular dynamics simulation techniques. A truncated form of the protein complex embedded in a palmitoyloleoylphosphatidylcholine lipid bilayer and surrounded by explicit water was simulated under different pressure conditions to mimic the effects of tension and compression within the membrane on the protein. As a direct result of lateral tension being applied to the membrane, an increase in the tilt of a subset of the transmembrane helices was observed. This in turn led to the enlargement of the pore and the disruption of the hydrophobic gate consisting of residues Ile-14 and Val-21. The simulations suggest that opening occurs in a sequential staged process. Such a mechanism could explain the partial opening or staged conductance observed in patch-clamp experiments using related large conductance mechanosensitive channel complexes.

Connon, C. J., R. D. Young, et al. (2003). "P2X(7) Receptors Are Redistributed on Human Monocytes after Pore Formation in Response to Prolonged Agonist Exposure." Pharmacology 67(3): 163-8.

            The P2X(7) cell surface receptor is responsible for adenosine triphosphate-dependent lysis of immune cells following the formation of non-selective membrane pores. This study examined changes in P2X(7) receptor distribution following agonist treatment. Human THP-1 monocytes were exposed to the agonist 2'-3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate for 1-40 min after which P2X(7) receptors were immunogold labelled. The number and distribution of P2X(7) receptors on the cell membrane were quantified using transmission electron microscopy. Increasing the time of agonist exposure resulted in clustering of P2X(7) receptors, a decrease in individual P2X(7) receptors and no change in the total number of P2X(7) receptors. These results suggest that pore formation does not involve further insertion or initial movement of receptors in the membrane but that P2X(7) receptors do cluster together after prolonged activation.

Cronin, N. B., A. O'Reilly, et al. (2003). "Binding of the anticonvulsant drug lamotrigine and the neurotoxin batrachotoxin to voltage-gated sodium channels induces conformational changes associated with block and steady-state activation." J Biol Chem 278(12): 10675-82.

            Voltage-gated sodium channels are dynamic membrane proteins characterized by rapid conformational changes that switch the molecule between closed resting, activated, and inactivated states. Sodium channels are specifically blocked by the anticonvulsant drug lamotrigine, which preferentially binds to the channel pore in the inactivated open state. Batrachotoxin is a lipid-soluble alkaloid that causes steady-state activation and binds in the inner pore of the sodium channel with overlapping but distinct molecular determinants from those of lamotrigine. Using circular dichroism spectroscopy on purified voltage-gated sodium channels from Electrophorus electricus, the secondary structures associated with the mixture of states present at equilibrium in the absence of these ligands were compared with specific stabilized states in their presence. As the channel shifts to open states, there appears to be a significant change in secondary structure to a more alpha-helical conformation. The observed changes are consistent with increased order involving the S6 segments that form the pore, the domain III-IV linker, and the P-loops that form the outer pore and selectivity filter. A molecular model has been constructed for the sodium channel based on its homology with the pore-forming regions of bacterial potassium channels, and automated docking of the crystal structure of lamotrigine with this model produces a structure in which the close contacts of the drug are with the residues previously identified by mutational studies as forming the binding site for this drug.

Dempsey, C. E., S. Ueno, et al. (2003). "Enhanced membrane permeabilization and antibacterial activity of a disulfide-dimerized magainin analogue." Biochemistry 42(2): 402-9.

            A cysteine substitution analogue of magainin-2 amide (magainin-F12W, N22C; denoted here as mag-N22C), and a disulfide-linked dimer prepared by air oxidation [(mag-N22C)(2)], were compared in their ability to release carboxyfluorescein (CF) from 100-nm large unilamellar vesicles (LUV) and to kill the Gram negative bacteria Stenotrophomonas maltophilia and Escherichia coli. The disulfide-dimerized peptide showed enhanced permeabilization and antimicrobial activity, when compared with the monomeric peptide, that was particularly marked at very low peptide concentrations. The enhanced CF-releasing activity of the dimer at low concentrations in vesicles results from (i) enhanced binding to negatively charged membrane surfaces and (ii) a low concentration dependence for permeabilization in the dimer compared to the monomer. The unique properties of the dimeric peptide suggest a role for structural diversity of antimicrobial peptides in frog skin, including the recent identification of a heterodimer composed of disulfide-linked amphipathic helical peptides [Batista et al. (2001) FEBS Lett. 494, 85-89]. Disulfide-dimerization of pore-forming, positively charged, amphipathic helical peptides may be a useful general approach to the generation of peptide antimicrobials having activity at very low concentrations.

Dernick, G., G. Alvarez De Toledo, et al. (2003). "Exocytosis of single chromaffin granules in cell-free inside-out membrane patches." Nat Cell Biol 5(4): 358-62.

            In chromaffin cells, exocytosis of single granules and properties of the fusion pore - the first connection between vesicular lumen and extracellular space - can be studied by cell-attached patch amperometry, which couples patch-clamp capacitance measurements with simultaneous amperometric recordings of transmitter release. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.

Dopico, A. M. (2003). "ETHANOL SENSITIVITY OF BKCa CHANNELS CLONED FROM ARTERIAL SMOOTH MUSCLE DOES NOT REQUIRE THE PRESENCE OF THE {beta}1 SUBUNIT." Am J Physiol Cell Physiol.

            Ethanol (EtOH) inhibition of large conductance, Ca(++)-activated K(+) (BKCa) channel activity in aortic myocytes may contribute to the direct contraction of aortic smooth muscle produced by acute exposure to alcohol. In this tissue, BKCa channels consist of pore forming (alpha, encoded by the slo gene) and modulatory (beta1) subunits. Here, modulation of aortic smooth muscle BKCa channels by acute alcohol was explored by expressing bslo subunits in Xenopus oocytes, in the absence and presence of beta1 subunits, and studying channel responses to clinically relevant concentrations of EtOH in excised membrane patches. Overall, average values of bslo channel activity NPo)in response to acute EtOH (3-200 mM) mildly decrease when compared to pre-EtOH, isosmotic control values. However, data show qualitative heterogeneity in bslo channel responses to EtOH at all concentrations tested. In the majority of patches (42 out of 71 patches, i.e., 59%) a reversible reduction in NPo is observed. In this subset, the maximal effect is obtained with 100 mM EtOH, at which NPo reaches 46.2+/-9% of control. The presence of beta1 subunits, which determines channel sensitivity to dihydrosoyaponin-I and 17-betaestradiol, fails to modify EtOH action on bslo channels. EtOH reduction of bslo channel activity results from a marked increase in the mean closed time. While the voltage-dependence of gating remains largely unaffected, the apparent effectiveness of Ca(++) to gate the channel is decreased by EtOH. These changes occur in the absence of modifications of channel conduction properties. In conclusion, a new molecular mechanism that may contribute to EtOH-induced aortic smooth muscle contraction has been identified and characterized: a functional interaction between EtOH and the bslo subunit and/or its lipid microenvironment, which leads to a decrease in BKCa channel activity.

Erxleben, C., C. Gomez-Alegria, et al. (2003). "Modulation of cardiac Ca(V)1.2 channels by dihydropyridine and phosphatase inhibitor requires Ser-1142 in the domain III pore loop." Proc Natl Acad Sci U S A 100(5): 2929-34.

            Dihydropyridine-sensitive, voltage-activated calcium channels respond to membrane depolarization with two distinct modes of activity: short bursts of very short openings (mode 1) or repetitive openings of much longer duration (mode 2). Here we show that both the dihydropyridine, BayK8644 (BayK), and the inhibitor of SerThr protein phosphatases, okadaic acid, have identical effects on the gating of the recombinant cardiac calcium channel, Ca(V)1.2 (alpha(1)C). Each produced identical mode 2 gating in cell-attached patches, and each prevented rundown of channel activity when the membrane patch was excised into ATP-free solutions. These effects required Ser or Thr at position 1142 in the domain III pore loop between transmembrane segments S5 and S6, where dihydropyridines bind to the channel. Mutation of Ser-1142 to Ala or Cys produced channels with very low activity that could not be modulated by either BayK or okadaic acid. A molecular model of Ca(V)1.2 indicates that Ser-1142 is unlikely to be phosphorylated, and thus we conclude that BayK binding stabilizes mode 2 gating allosterically by either protecting a phospho Ser/Thr on the alpha(1)C subunit or mimicking phosphorylation at that site.

Estacion, M., J. S. Weinberg, et al. (2003). "Blockade of maitotoxin-induced endothelial cell lysis by glycine and L-alanine." Am J Physiol Cell Physiol 284(4): C1006-20.

            The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs) is a model for oncotic/necrotic cell death. The cascade is initiated by an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)), which is followed by the biphasic uptake of vital dyes. The initial phase of dye entry reflects activation of large pores and correlates with surface membrane bleb formation; the second phase reflects cell lysis. In the present study, the effect of the cytoprotective amino acid glycine was examined. Glycine had no effect on MTX-induced change in [Ca(2+)](i) or on the first phase of vital dye uptake but produced a concentration-dependent (EC(50) approximately 1 mM) inhibition of the second phase of dye uptake. No cytoprotective effect was observed with l-valine, l-proline, or d-alanine, whereas l-alanine was equieffective to glycine. Furthermore, glycine had no effect on MTX-induced bleb formation. To test the hypothesis that glycine specifically blocks formation of a lytic "pore," the loss of fluorescence from BAECs transiently expressing GFP and concatemers of GFP ranging in size from 27 to 162 kDa was examined using time-lapse videomicroscopy. MTX-induced loss of GFP was rapid, correlated with the second phase of dye uptake, and was relatively independent of molecular size. The MTX-induced loss of GFP from BAECs was completely blocked by glycine. The data suggest that the second "lytic" phase of MTX-induced endothelial cell death reflects formation of a novel permeability pathway that allows macromolecules such as GFP or LDH to escape, yet can be prevented by the cytoprotective agents glycine and l-alanine.

Ferber, M., A. Sporning, et al. (2003). "A novel conus peptide ligand for K+ channels." J Biol Chem 278(4): 2177-83.

            Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na(+) and Ca(2+) channels have been characterized, relatively few have been identified that interact with K(+) channels. We describe a novel Conus peptide that interacts with the Shaker K(+) channel, kappaM-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although kappaM-conotoxin RIIIK is structurally similar to the mu-conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K(+) channels. Studies using Shaker K(+) channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe(425) and Thr(449), drastically reduced toxin affinity. Based on the Shaker results, a teleost homolog of the Shaker K(+) channel, TSha1 was identified as a kappaM-conotoxin RIIIK target. Binding of kappaM-conotoxin RIIIK is state-dependent, with an IC(50) of 20 nm for the closed state and 60 nm at 0 mV for the open state of TSha1 channels.

Ferroni, S., P. Valente, et al. (2003). "Arachidonic acid activates an open rectifier potassium channel in cultured rat cortical astrocytes." J Neurosci Res 72(3): 363-372.

            A pathophysiological increase in free arachidonic acid (AA) is thought to regulate the channel-mediated astrocytic swelling occurring in several brain injuries. We report that in cultured rat type-1 cortical astrocytes, exposure to 10 &mgr;M AA activates an open rectifier K(+) channel, which exhibits many similarities with TREK/TRAAK members of the two-pore-domain K(+) channel family KCNK. Patch-clamp experiments showed that the current developed with a long latency and was preceded by a depression of the previously described outward rectifier K(+) conductance. Pharmacologic studies indicate that the K(+) open rectifier was differentially sensitive to classic K(+)-channel blockers (quinine, quinidine, tetraethylammonium, and barium) and was inhibited potently by gadolinium ions. The activation of this K(+) current occurred independently of the AA metabolism as pharmacologic inhibition of the lipoxygenase, cyclooxygenase, and cytochrome P450 epoxygenase signaling cascades did not alter the AA effect. Moreover, neither the neutralization of the NADPH-oxidase pathway nor scavenging intracellular free radicals modified the AA response. Finally, the AA-induced K(+) current was unaffected by protein kinase C inhibitors. The activation mechanism of the K(+) open rectifier was through an extracellular interaction of AA with the plasma membrane. RT-PCR analysis revealed that the AA-induced K(+) conductance was mediated likely by TREK-2 channels. Collectively, the results demonstrate that in cultured cortical astrocytes, pathological levels of AA directly activate an open rectifier K(+) channel, which may play a role in the control of K(+) homeostasis under pathophysiological conditions. Copyright 2003 Wiley-Liss, Inc.

Forouhar, F., W. N. Huang, et al. (2003). "Structural basis of membrane-induced cardiotoxin A3 oligomerization." J Biol Chem.

            Cobra cardiotoxins (CTXs) have previously been shown to induce membrane fusion of vesicles formed by phospholipids such as cardiolipin or sphingomyelin. It can also form a pore in membrane bilayers containing anionic lipid such as phsphatidylserine or phosphatidylglycerol. Herein, we show that the interaction of CTX with negatively-charged lipids causes CTX dimerization, an important intermediate for the eventual oligomerization of CTX during the CTX-induced fusion and pore formation process. The structural basis of the lipid-induced oligomerization of CTX A3, a major CTX from Naja atra, is then illustrated by the crystal structure of CTX A3 in complex with sodium dodecyl sulfate (SDS), SDS likely mimics anionic lipids of the membrane, under micelle condition at 1.9 A resolution. The crystal packing reveals distinct SDS free and SDS rich regions, in the latter of which two types of interconnecting CTX A3 dimers, D1 and D2, and several SDS molecules can be identified to stabilize D1 and D2 by simultaneously interacting with residues at each dimer interface. When the three CTX-SDS complexes in the asymmetric unit are overlaid, the orientation of CTX A3 monomers relative to the SDS molecules in the crystal is strikingly similar to that of the toxin with respect to model membranes as determined by NMR and FTIR methods. These results not only illustrate how lipid-induced CTX dimer formation may be transformed into oligomers either as inverted micelles of fusion intermediates or as membrane pore of anionic lipid bilayers, but also underscore a potential role for SDS in X-ray diffraction study of protein-membrane interactions in the future.

Gallucci, E., D. Meleleo, et al. (2003). "Magainin 2 channel formation in planar lipid membranes: the role of lipid polar groups and ergosterol." Eur Biophys J 32(1): 22-32.

            Magainin 2, a polycationic peptide, displays bactericidal and tumoricidal activity, presumably interacting with negatively charged phospholipids in the membrane hosts. In this work, we investigate the role played by the lipid head-group in the interactions and self-association of magainin 2 during pore formation in lipid bilayers. Two methods are used: single-channel and macroscopic incorporation into planar lipid membranes. Single-channel incorporation showed that magainin 2 did not interact with zwitterionic membranes, while the addition of negatively charged dioleoylphosphatidylglycerol to the membrane leads to channel formation. On the other hand, magainin 2 did not form channels in membranes made up of dioleoylphosphatidylserine (DOPS), although the addition of ergosterol to DOPS membranes leads to channel formation. This finding could indicate that ergosterol may be a possible target of magainin 2 in fungal membranes. Further support for this hypothesis comes from experiments in which the addition of ergosterol to palmitoyloleoylphosphatidylcholine membranes induced channel formation. Besides the role of negatively charged membranes, this study has shown that magainin 2 also forms channels in membranes lacking heads, such as monoolein and oxidized cholesterol, indicating an interaction of magainin 2 with acyl chains and cholesterol, respectively. This finding provides further evidence that peptide binding and assembly in lipid membranes is a complex process driven by electrostatic and/or hydrophobic interactions, depending on the structure of the peptide and the membrane composition.

Gessner, G. and S. H. Heinemann (2003). "Inhibition of hEAG1 and hERG1 potassium channels by clofilium and its tertiary analogue LY97241." Br J Pharmacol 138(1): 161-71.

            1 We investigated the inhibition of hEAG1 potassium channels, expressed in mammalian cells and Xenopus oocytes, by several blockers that have previously been reported to be blockers of hERG1 channels. 2 In the whole-cell mode of mammalian cells, LY97241 was shown to be a potent inhibitor of both hEAG1 and hERG1 channels (IC(50) of 4.9 and 2.2 nM, respectively). Clofilium, E4031, and haloperidol apparently inhibited hEAG1 channels with lower potency than hERG1 channels, but they cannot be considered hERG1-specific. 3 The block of hEAG1 channels by LY97241 and clofilium was time-, use-, and voltage-dependent, best explained by an open-channel block mechanism. 4 Both drugs apparently bind from the intracellular side of the membrane at (a) specific site(s) within the central cavity of the channel pore. They can be trapped by closure of the activation gate. 5 In inside-out patches from Xenopus oocytes, hEAG1 block by clofilium was stronger than by LY97241 (IC(50) of 0.8 and 1.9 nM, respectively). In addition, hEAG1 block by clofilium was much faster than by LY97241 although there was no difference in the voltage dependence of the on-rate of block. 6 Physico-chemical differences of clofilium and the weak base LY97241 determine the access of the drugs to the binding site and thereby the influence of the recording mode on the apparent block potencies. This phenomenon must be considered when assessing the inhibitory action of drugs on ion channels. British Journal of Pharmacology (2003) 138, 161-171. doi:10.1038/sj.bjp.0705025

Gomis-Ruth, F. X., A. Dessen, et al. (2003). "The Matrix Protein VP40 from Ebola Virus Octamerizes into Pore-like Structures with Specific RNA Binding Properties." Structure (Camb) 11(4): 423-33.

            The Ebola virus membrane-associated matrix protein VP40 is thought to be crucial for assembly and budding of virus particles. Here we present the crystal structure of a disk-shaped octameric form of VP40 formed by four antiparallel homodimers of the N-terminal domain. The octamer binds an RNA triribonucleotide containing the sequence 5'-U-G-A-3' through its inner pore surface, and its oligomerization and RNA binding properties are facilitated by two conformational changes when compared to monomeric VP40. The selective RNA interaction stabilizes the ring structure and confers in vitro SDS resistance to octameric VP40. SDS-resistant octameric VP40 is also found in Ebola virus-infected cells, which suggests that VP40 has an additional function in the life cycle of the virus besides promoting virus assembly and budding off the plasma membrane.

Gong, X. and P. Linsdell (2003). "Coupled movement of permeant and blocking ions in the CFTR chloride channel pore." J Physiol.

            The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore is blocked in a voltage-dependent manner by a broad range of anionic substances added to the cytoplasmic side of the membrane. Here we investigate the origin of the voltage dependence of block by intracellular Au(CN)2(-), a highly permeant lyotropic anion which also acts as a high-affinity blocker of Cl(-) permeation. Not only the affinity, but also the voltage dependence of block by intracellular Au(CN)2(-) ions is strongly dependent on extracellular Cl(-) concentration; following replacement of most extracellular Cl(-) by glucose or by impermeant anions, block by Au(CN)2(-) shows greatly weakened voltage dependence. This suggests that coupled movement of Au(CN)2(-) and Cl(-) ions within the pore contributes to the voltage dependence of block. This explanation requires that interactions between different anions take place within the pore, implying simultaneous binding of multiple anions to intrapore sites. Other anions are able to substitute for extracellular Cl(-) and interact with intracellular Au(CN)2(-) ions. Analysis of the effects of different extracellular anions on the apparent affinity and voltage dependence of block by intracellular Au(CN)2(-) ions suggests that extracellular anions do not need to permeate through the channel in order to destabilize Au(CN)2(-) binding within the pore, implying that this destabilizing effect results from binding to an externally accessible site in the permeation pathway. We propose that multiple anions can bind simultaneously within the CFTR channel pore, and that repulsive interactions between bound anions speeds anion exit from the pore.

Gordon, J., C. H. Wu, et al. (2003). "Beta2-microglobulin induces caspase-dependent apoptosis in the CCRF-HSB-2 human leukemia cell line independently of the caspase-3, -8 and -9 pathways but through increased reactive oxygen species." Int J Cancer 103(3): 316-27.

            Exogenous beta(2)-microglobulin (beta(2)m) induces significant apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line as detected by DNA fragmentation, DAPI staining and annexin V binding assay. beta(2)m treatment induced the release of cytochrome c and apoptosis-inducing factor (AIF) from the mitochondria, but no change in mitochondrial membrane potential (DeltaPsim) was observed during apoptosis, suggesting that cytochrome c may be released through a mechanism independent of mitochondrial permeability transition (MPT) pore formation. Moreover, the beta(2)m-induced release of cytochrome c and AIF from the mitochondria in CCRF-HSB-2 cells was caspase-independent, since Z-VAD-fmk, a general inhibitor of caspases, did not block the release of these factors. However, Z-VAD-fmk treatment significantly blocked beta(2)m-induced apoptosis, while Western blot analysis revealed that caspases-1, -2, -3, -6, -7, -8 and -9 are not activated during beta(2)m-induced apoptosis in these cells. These results collectively indicate that a post-mitochondrial caspase-dependent mechanism is involved in beta(2)m-induced apoptosis. Moreover, beta(2)m significantly enhanced the production of reactive oxygen species (ROS) during 12-48 hr treatment, and beta(2)m-induced apoptosis was almost totally inhibited in cells pre-treated with the antioxidant N-acetylcysteine (NAC), providing evidence that beta(2)m-induced apoptosis in CCRF-HSB-2 cells is ROS-dependent. Therefore, these results reveal that beta(2)m-induced apoptosis in CCRF-HSB-2 cells may occur through an unknown caspase-dependent and ROS-dependent mechanism(s) that is associated with cytochrome c and AIF release from mitochondria, but is independent of the caspase -3, -8 and -9 pathways.

Green, D. M., C. P. Johnson, et al. (2003). "The C-terminal domain of myosin-like protein 1 (Mlp1p) is a docking site for heterogeneous nuclear ribonucleoproteins that are required for mRNA export." Proc Natl Acad Sci U S A 100(3): 1010-5.

            For mRNA to be transported from the nucleus to the cytoplasm, it must travel from the site of transcription through the nuclear interior to the nuclear pore. Studies in Saccharomyces cerevisiae have suggested a relationship between poly(A) RNA trafficking and myosin-like protein 1 (Mlp1p), a nuclear-pore associated protein that is homologous to the mammalian Tpr (translocated promoter region) protein [Kosova, B., Pante, N., Rollenhagen, C., Podtelejnikov, A., Mann, M., Aebi, U., and Hurt, E. (2000) J. Biol. Chem. 275, 343-350]. We identified a yeast two-hybrid interaction between the C-terminal globular domain of Mlp1p and Nab2p, a shuttling heterogeneous nuclear ribonucleoprotein that is required for mRNA export. Coimmunoprecipitation confirms that Nab2p also interacts with full-length Mlp1p and in vitro binding experiments show that Nab2p binds directly to the C-terminal domain of Mlp1p. In addition, our experiments reveal that the C-terminal domain of Mlp1p is both necessary and sufficient to cause accumulation of poly(A) RNA and Nab2p in the nucleus. We propose a model where Mlp1p acts as a checkpoint at the nuclear pore by interacting with export-competent ribonucleoprotein complexes through its C-terminal globular domain. This study identifies Nab2p as a heterogeneous nuclear ribonucleoprotein found in complex with Mlp1p and begins to delineate the path that mRNA travels from the chromatin to the nuclear pore.

Griffis, E. R., S. Xu, et al. (2003). "Nup98 localizes to both nuclear and cytoplasmic sides of the nuclear pore and binds to two distinct nucleoporin subcomplexes." Mol Biol Cell 14(2): 600-10.

            The vertebrate nuclear pore is an enormous structure that spans the double membrane of the nuclear envelope. In yeast, most nucleoporins are found symmetrically on both the nuclear and cytoplasmic sides of the structure. However, in vertebrates most nucleoporins have been localized exclusively to one side of the nuclear pore. Herein, we show, by immunofluorescence and immunoelectron microscopy, that Nup98 is found on both sides of the pore complex. Additionally, we find that the pore-targeting domain of Nup98 interacts directly with the cytoplasmic nucleoporin Nup88, a component of the Nup214, Nup88, Nup62 subcomplex. Nup98 was previously described to interact with the nuclear-oriented Nup160, 133, 107, 96 complex through direct binding to Nup96. Interestingly, the same site within Nup98 is involved in binding to both Nup88 and Nup96. Autoproteolytic cleavage of the Nup98 C terminus is required for both of these binding interactions. When cleavage is blocked by a point mutation, a minimal eight amino acids downstream of the cleavage site is sufficient to prevent most binding to either Nup96 or Nup88. Thus, Nup98 interacts with both faces of the nuclear pore, a localization in keeping with its previously described nucleocytoplasmic shuttling activity.

Gruszecki, W. I., M. Gagos, et al. (2003). "Organization of antibiotic amphotericin B in model lipid membranes. A mini review." Cell Mol Biol Lett 8(1): 161-70.

            Amphotericin B (AmB) is a polyene antibiotic frequently applied in the treatment of fungal infections. According to the general understanding, the mode of action of AmB is directly related to the molecular organization of the drug in the lipid environment, in particular to the formation of pore-like molecular aggregates. Electronic absorption and fluorescence techniques were applied to investigate formation of molecular aggregates of AmB in the lipid environment of liposomes and monomolecular layers formed at the argon-water interface. It appears that AmB dimers, stabilized by van der Waals interactions, are present in the membrane environment along with the aggregates formed by a greater number of molecules. Linear dichroism measurements reveal that AmB is distributed between two fractions of molecules, differently oriented with respect to the bilayer. Molecules in one fraction remain parallel to the plane of the membrane and molecules in the other one are perpendicular. Scanning Force Microscopy imaging of the surface topography of the monolayers formed with AmB in the presence of lipids reveals formation of pore-like structures characterized by the external diameter close to 17 A and the internal diameter close to 6 A. All the findings are discussed in terms of importance of the molecular organization of AmB in the pharmacological action, as well as of the toxic side effects of the drug.

Guo, D., Y. Ramu, et al. (2003). "Mechanism of Rectification in Inward-rectifier K+ Channels." J Gen Physiol 121(4): 261-76.

            Rectification in inward-rectifier K+ channels is caused by the binding of intracellular cations to their inner pore. The extreme sharpness of this rectification reflects strong voltage dependence (apparent valence is approximately 5) of channel block by long polyamines. To understand the mechanism by which polyamines cause rectification, we examined IRK1 (Kir2.1) block by a series of bis-alkyl-amines (bis-amines) and mono-alkyl-amines (mono-amines) of varying length. The apparent affinity of channel block by both types of alkylamines increases with chain length. Mutation D172N in the second transmembrane segment reduces the channel's affinity significantly for long bis-amines, but only slightly for short ones (or for mono-amines of any length), whereas a double COOH-terminal mutation (E224G and E299S) moderately reduces the affinity for all bis-amines. The apparent valence of channel block increases from approximately 2 for short amines to saturate at approximately 5 for long bis-amines or at approximately 4 for long mono-amines. On the basis of these and other observations, we propose that to block the channel pore one amine group in all alkylamines tested binds near the same internal locus formed by the COOH terminus, while the other amine group of bis-amines, or the alkyl tail of mono-amines, "crawls" toward residue D172 and "pushes" up to 4 or 5 K+ ions outwardly across the narrow K+ selectivity filter. The strong voltage dependence of channel block therefore reflects the movement of charges carried across the transmembrane electrical field primarily by K+ ions, not by the amine molecule itself, as K+ ions and the amine blocker displace each other during block and unblock of the pore. This simple displacement model readily accounts for the classical observation that, at a given concentration of intracellular K+, rectification is apparently related to the difference between the membrane potential and the equilibrium potential for K+ ions rather than to the membrane potential itself.

Gurkan, C. and D. J. Ellar (2003). "Expression of the Bacillus thuringiensis Cyt2Aa1 toxin in Pichia pastoris using a synthetic gene construct." Biotechnol Appl Biochem.

            B. thuringiensis delta-endotoxins are membrane-active, pore-forming proteins with highly specific insecticidal activities. In addition to a well-established role in the biological control of a wide variety of crop pests and disease vectors, these toxins also have great potential for the development of anti-tumour agents called immunotoxins (ITs), chimeric molecules consisting of a cell-binding ligand coupled to a toxin or its subunits. The ultimate goal of our study was the recombinant production of such ITs based on the Cyt2Aa1 toxin from B. thuringiensis subspecies kyushuensis. We explored the use of P. pastoris for recombinant IT production because earlier attempts in our laboratory using the E. coli expression system or various chemical conjugation strategies yielded only low levels of functional product. However, our initial attempts were not successful because the A+T-rich bacterial cyt2Aa1 gene contained fortuitous polyadenylation sites, causing premature transcription termination in this yeast. Accordingly, we designed and constructed a synthetic cyt2Aa1 gene (syncyt2Aa1) optimised for heterologous expression in P. pastoris. This was achieved by increasing the overall G+C-content of the bacterial cyt2Aa1 while changing its codon usage to that preferred by the methylotrophic yeast. Here we describe in detail the design, synthesis, and requisite PCR repair of syncyt2Aa1, then present analyses of recombinant Cyt2Aa1 expression in P. pastoris using this synthetic gene. Following the results presented in this paper, the syncyt2Aa1 gene was also successfully used for the recombinant production of a Cyt2Aa1-based IT in the same expression host (Gurkan, C. and Ellar, D. J. Protein Expr. Purif., in the press).

Han, J., C. Gnatenco, et al. (2003). "Background and tandem-pore potassium channels in magnocellular neurosecretory cells of the rat supraoptic nucleus." J Physiol 546(Pt 3): 625-39.

            Magnocellular neurosecretory cells (MNCs) were isolated from the supraoptic nucleus of rat hypothalamus, and properties of K(+) channels that may regulate the resting membrane potential and the excitability of MNCs were studied. MNCs showed large transient outward currents, typical of vasopressin- and oxytocin-releasing neurons. K(+) channels in MNCs were identified by recording K(+) channels that were open at rest in cell-attached and inside-out patches in symmetrical 150 mM KCl. Eight different K(+) channels were identified and could be distinguished unambiguously by their single-channel kinetics and voltage-dependent rectification. Two K(+) channels could be considered functional correlates of TASK-1 and TASK-3, as judged by their single-channel kinetics and high sensitivity to pH(o). Three K(+) channels showed properties similar to TREK-type tandem-pore K(+) channels (TREK-1, TREK-2 and a novel TREK), as judged by their activation by membrane stretch, intracellular acidosis and arachidonic acid. One K(+) channel was activated by application of pressure, arachidonic acid and alkaline pH(i), and showed single-channel kinetics indistinguishable from those of TRAAK. One K(+) channel showed strong inward rectification and single-channel conductance similar to those of a classical inward rectifier, IRK3. Finally, a K(+) channel whose cloned counterpart has not yet been identified was highly sensitive to extracellular pH near the physiological range similar to those of TASK channels, and was the most active among all K(+) channels. Our results show that in MNCs at rest, eight different types of K(+) channels can be found and six of them belong to the tandem-pore K(+) channel family. Various physiological and pathophysiological conditions may modulate these K(+) channels and regulate the excitability of MNCs.

He, L. and J. J. Lemasters (2003). "Heat shock suppresses the permeability transition in rat liver mitochondria." J Biol Chem.

            Heat shock proteins inhibit apoptotic and necrotic cell death in various cell types. However, the specific mechanism underlying protection by heat shock proteins remains unclear. To test the hypothesis that heat shock proteins inhibit cell death by blocking opening of mitochondrial permeability transition (MPT) pores, mitochondria from heat-preconditioned rat livers were isolated by differential centrifugation. Heat shock inhibited MPT pore opening induced by 50 mM CaCl2 plus 5 mM HgCl2 or 1 mM mastoparan and by 200 mM CaCl2 alone. Half maximal swelling was delayed 15 min or more after heat shock compared to control. Heat shock also increased the threshold of unregulated (Ca2+-independent and cyclosporin A-insensitive) MPT pore opening induced by higher doses of HgCl2 and mastoparan. Heat shock treatment decreased mitochondrial reactive oxygen species formation by 27% but did not change mitochondrial respiration, membrane potential, Ca2+-uptake, or total glutathione in mitochondrial and cytosolic extracts of liver. Western blot analysis showed that mitochondrial Hsp25 increased, whereas Hsp10, Hsp60, Hsp70, Hsp75, cyclophilin D and voltage dependent anion channel did not change after heat shock. These results indicate that heat shock causes resistance to opening of MPT pores, which may contribute to heat shock protection against cellular injury.

Heinz, C., H. Engelhardt, et al. (2003). "The Core of the Tetrameric Mycobacterial Porin MspA Is an Extremely Stable beta -Sheet Domain." J Biol Chem 278(10): 8678-85.

            MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the cell wall and is the prototype of a new family of tetrameric porins with a single central pore of 10 nm in length. Infrared and circular dichroism spectroscopy revealed that MspA consists mainly of antiparallel beta-strands organized in a coherent domain. Heating to 92 and 112 degrees C was required to dissociate the MspA tetramer and to unfold the beta-sheet domain in the monomer, respectively. The stability of the MspA tetramer exceeded the remarkable stability of the porins of Gram-negative bacteria for every condition tested and was not reduced in the presence of 2% SDS and at any pH from 2 to 14. These results indicated that the interactions between the MspA subunits are different from those in the porins of Gram-negative bacteria and are discussed in the light of a channel-forming beta-barrel as a core structure of MspA. Surprisingly, the channel activity of MspA in 2% SDS and 7.6 m urea at 50 degrees C was reduced 13- and 30-fold, respectively, although the MspA tetramer and the beta-sheet domain were stable under those conditions. Channel closure by conformational changes of extracellular loops under those conditions is discussed to explain these observations. This study presents the first experimental evidence that outer membrane proteins not only from Gram-negative bacteria but also from mycobacteria are beta-sheet proteins and demonstrates that MspA constitutes the most stable transmembrane channel protein known so far. Thus, MspA may be of special interest for biotechnological applications.

Hocquet, D., C. Vogne, et al. (2003). "MexXY-OprM Efflux Pump Is Necessary for Adaptive Resistance of Pseudomonas aeruginosa to Aminoglycosides." Antimicrob Agents Chemother 47(4): 1371-5.

            Exposure of Pseudomonas aeruginosa to aminoglycosides frequently selects for recalcitrant subpopulations exhibiting an unstable, "adaptive" resistance to these antibiotics. In this study, we investigated the implication in the phenomenon of MexXY-OprM, an active efflux system known to export aminoglycosides in P. aeruginosa. Immunoblotting experiments demonstrated that the transporter MexY, but not the outer membrane pore OprM, was overproduced during the post-drug exposure adaptation period in wild-type strain PAO1. Furthermore, MexY production was dependent upon the degree of bacterial exposure to gentamicin (drug concentration). In contrast to parental strain PAO1, mutants defective in MexXY or in OprM were unable to develop adaptive resistance. Altogether, these results indicate that the resistance process requires the rapid production of MexXY and the interaction of these proteins with the constitutively produced component OprM.

Hoenderop, J. G., T. Voets, et al. (2003). "Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6." Embo J 22(4): 776-85.

            The molecular assembly of the epithelial Ca(2+) channels (TRPV5 and TRPV6) was investigated to determine the subunit stoichiometry and composition. Immunoblot analysis of Xenopus laevis oocytes expressing TRPV5 and TRPV6 revealed two specific bands of 75 and 85-100 kDa, corresponding to the core and glycosylated proteins, respectively, for each channel. Subsequently, membranes of these oocytes were sedimented on sucrose gradients. Immuno blotting revealed that TRPV5 and TRPV6 complexes migrate with a mol. wt of 400 kDa, in line with a tetrameric structure. The tetrameric stoichiometry was confirmed in an electrophysiological analysis of HEK293 cells co-expressing concatemeric channels together with a TRPV5 pore mutant that reduced Cd(2+) sensitivity and voltage-dependent gating. Immuno precipitations using membrane fractions from oocytes co-expressing TRPV5 and TRPV6 demonstrated that both channels can form heteromeric complexes. Expression of all possible heterotetrameric TRPV5/6 complexes in HEK293 cells resulted in Ca(2+) channels that varied with respect to Ca(2+)-dependent inactivation, Ba(2+) selectivity and pharmacological block. Thus, Ca(2+)-transporting epithelia co-expressing TRPV5 and TRPV6 can generate a pleiotropic set of functional heterotetrameric channels with different Ca(2+) transport kinetics.

Hosy, E., A. Vavasseur, et al. (2003). "The Arabidopsis outward K+ channel GORK is involved in regulation of stomatal movements and plant transpiration." Proc Natl Acad Sci U S A.

            Microscopic pores present in the epidermis of plant aerial organs, called stomata, allow gas exchanges between the inner photosynthetic tissue and the atmosphere. Regulation of stomatal aperture, preventing excess transpirational vapor loss, relies on turgor changes of two highly differentiated epidermal cells surrounding the pore, the guard cells. Increased guard cell turgor due to increased solute accumulation results in stomatal opening, whereas decreased guard cell turgor due to decreased solute accumulation results in stomatal closing. Here we provide direct evidence, based on reverse genetics approaches, that the Arabidopsis GORK Shaker gene encodes the major voltage-gated outwardly rectifying K(+) channel of the guard cell membrane. Expression of GORK dominant negative mutant polypeptides in transgenic Arabidopsis was found to strongly reduce outwardly rectifying K(+) channel activity in the guard cell membrane, and disruption of the GORK gene (T-DNA insertion knockout mutant) fully suppressed this activity. Bioassays on epidermal peels revealed that disruption of GORK activity resulted in impaired stomatal closure in response to darkness or the stress hormone azobenzenearsonate. Transpiration measurements on excised rosettes and intact plants (grown in hydroponic conditions or submitted to water stress) revealed that absence of GORK activity resulted in increased water consumption. The whole set of data indicates that GORK is likely to play a crucial role in adaptation to drought in fluctuating environments.

Huynh, N. T., R. A. Ffrench, et al. (2003). "Transmembrane T-cell receptor peptides inhibit B- and natural killer-cell function." Immunology 108(4): 458-464.

            A synthetic hydrophobic peptide (core peptide; CP) containing two positively charged amino acids, lysine and arginine was derived from the transmembrane sequence of the T-cell receptor (TCR) alpha chain and has been shown to inhibit T-cell-mediated inflammation. In this study, we investigated the specificity of CP (10 &mgr;m) on lymphocyte function and found that it significantly inhibited interleukin-2 production in T cells and natural killer cytotoxicity by 46-58% compared to positive control. CP had no effects on B-cell proliferative responses when used at these concentrations; however, it suppressed B-cell proliferation at higher concentrations (50 &mgr;m). Inhibition by CP was not the result of membrane pore formation or cytotoxicity when examined by trypan blue, propidium iodide staining or transmission electron microscopy. CP analogues, with both lysine and arginine replaced by neutral or negatively charged amino acids, or by randomly distributing charges in the peptide sequence, had no effect on lymphocyte function. These results suggest that peptide inhibition is affected by its structure and charge interactions, and may involve common signalling molecules in T, B and natural killer cells. The potential of the immuno-inhibitory effects of CP as a novel anti-inflammatory peptide in therapy should be further explored.

Ikeda, M., M. Arai, et al. (2003). "TMPDB: a database of experimentally-characterized transmembrane topologies." Nucleic Acids Res 31(1): 406-9.

            TMPDB is a database of experimentally-characterized transmembrane (TM) topologies. TMPDB release 6.2 contains a total of 302 TM protein sequences, in which 276 are alpha-helical sequences, 17 beta-stranded, and 9 alpha-helical sequences with short pore-forming helices buried in the membrane. The TM topologies in TMPDB were determined experimentally by means of X-ray crystallography, NMR, gene fusion technique, substituted cysteine accessibility method, N-linked glycosylation experiment and other biochemical methods. TMPDB would be useful as a test and/or training dataset in improving the proposed TM topology prediction methods or developing novel methods with higher performance, and as a guide for both the bioinformaticians and biologists to better understand TM proteins. TMPDB and its subsets are freely available at the following web site: http://bioinfo.si.hirosaki-u.ac.jp/~TMPDB/.

Imreh, G., D. Maksel, et al. (2003). "ER retention may play a role in sorting of the nuclear pore membrane protein POM121." Exp Cell Res 284(2): 171-182.

            Integral membrane proteins of the nuclear envelope (NE) are synthesized on the rough endoplasmic reticulum (ER) and following free diffusion in the continuous ER/NE membrane system are targeted to their proper destinations due to interactions of specific domains with other components of the NE. By studying the intracellular distribution and dynamics of a deletion mutant of an integral membrane protein of the nuclear pores, POM121, which lacks the pore-targeting domain, we investigated if ER retention plays a role in sorting of integral membrane proteins to the nuclear envelope. A nascent membrane protein lacking sorting determinants is believed to diffuse laterally in the continuous ER/NE lipid bilayer and expected to follow vesicular traffic to the plasma membrane. The GFP-tagged deletion mutant, POM121(1-129)-GFP, specifically distributed within the ER membrane, but was completely absent from the Golgi compartment and the plasma membrane. Experiments using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that despite having very high mobility within the whole ER network (D = 0.41 +/- 0.11 &mgr;m(2)/s) POM121(1-129)-GFP was unable to exit the ER. It was also not detected in post-ER compartments of cells incubated at 15 degrees C. Taken together, these experiments show that amino acids 1-129 of POM121 are able to retain GFP in the ER membrane and suggest that this retention occurs by a direct mechanism rather than by a retrieval mechanism. Our data suggest that ER retention might be important for sorting of POM121 to the nuclear pores.

Inkeniene, A. M., R. Klimas, et al. (2003). "[Development of formulation of hipromellose eye drops (artificial tears)]." Medicina (Kaunas) 39(1): 77-82.

            The task of this research work was to develop a formulation of hipromellose ophthalmic preparation by determining optimal composition of active ingredients and excipients. Aqueous solution of hipromellose 1% and sorbitol 4.5% was prepared with addition of phosphate buffer (pH 7.05) and preservative cetrimide 0.01%. Sterile filtration was used for solution sterilization. Polimeric membrane prefilters of 1 microm, 0.45 microm and 0.22 microm pore size were employed as sterilizing filter. It is recommended to increase the temperature up to 35-38 degrees C for more efficient filtration of the solution. Quality parameters and analytical methods for quality control have been developed to control the quality and to evaluate the stability of hipromellose eye drops during their storage for 24 months. The obtained results confirmed the sterility and stability of hipromellose eye drops for the period of two years.

Jambrina, E., R. Alonso, et al. (2003). "Calcium Influx through Receptor-operated Channel Induces Mitochondria-triggered Paraptotic Cell Death." J Biol Chem 278(16): 14134-14145.

            We address the specific role of cytoplasmic Ca(2+) overload as a cell death trigger by expressing a receptor-operated specific Ca(2+) channel, vanilloid receptor subtype 1 (VR1), in Jurkat cells. Ca(2+) uptake through the VR1 channel, but not capacitative Ca(2+) influx stimulated by the muscarinic type 1 receptor, induced sustained intracellular [Ca(2+)] rises, exposure of phosphatidylserine, and cell death. Ca(2+) influx was necessary and sufficient to induce mitochondrial damage, as assessed by opening of the permeability transition pore and collapse of the mitochondrial membrane potential. Ca(2+)-induced cell death was inhibited by ruthenium red, protonophore carbonyl cyanide m-chlorophenylhydrazone, or cyclosporin A treatment, as well as by Bcl-2 expression, indicating that this process requires mitochondrial calcium uptake and permeability transition pore opening. Cell death occurred without caspase activation, oligonucleosomal/50-kilobase pair DNA cleavage, or release of cytochrome c or apoptosis inducer factor from mitochondria, but it required oxidative/nitrative stress. Thus, Ca(2+) influx triggers a distinct program of mitochondrial dysfunction leading to paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis.

Jena, B. P. (2003). "Fusion pore or porosome: structure and dynamics." J Endocrinol 176(2): 169-74.

            Electrophysiological measurements on live secretory cells almost a decade ago suggested the presence of fusion pores at the cell plasma membrane. Membrane-bound secretory vesicles were hypothesized to dock and fuse at these sites, to release their contents. Our studies using atomic force microscopy on live exocrine and neuroendocrine cells demonstrate the presence of such plasma membrane pores, revealing their morphology and dynamics at near nm resolution and in real time.

Jena, B. P., S. J. Cho, et al. (2003). "Structure and composition of the fusion pore." Biophys J 84(2): 1337-43.

            Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. Fusion pores were stable structures at the cell plasma membrane where secretory vesicles dock and fuse to release vesicular contents. In the present study, transmission electron microscopy confirms the presence of fusion pores and reveals their detailed structure and association with membrane-bound secretory vesicles in pancreatic acinar cells. Immunochemical studies demonstrated that t-SNAREs, NSF, actin, vimentin, alpha-fodrin and the calcium channels alpha1c and beta3 are associated with the fusion complex. The localization and possible arrangement of SNAREs at the fusion pore are further demonstrated from combined AFM, immunoAFM, and electrophysiological measurements. These studies reveal the fusion pore or porosome to be a cup-shaped lipoprotein structure, the base of which has t-SNAREs and allows for docking and release of secretory products from membrane-bound vesicles.

Jeong, H. K., S. Nair, et al. (2003). "A highly crystalline layered silicate with three-dimensionally microporous layers." Nat Mater 2(1): 53-8.

            Layered silicates with three-dimensional microporosity within the layers have the potential to enable new applications in catalysis, adsorption and ion-exchange. Until now no such materials have been reported. However, here we present the synthesis and structure of AMH-3, a silicate with three-dimensionally microporous layers, obtained in high purity and crystallinity. AMH-3 is composed of silicate layers containing eight-membered rings in all three principal crystal directions, and spaced by strontium cations, sodium cations and water molecules. Because of its three-dimensional pore structure, acid and thermal stability, this layered material could find applications in polymer-silicate composites for membrane applications, for synthesis of combined microporous-mesoporous materials, and for the formation of new zeolites and microporous films. Its existence also opens new possibilities for the synthesis of other layered silicates with multidimensional microporous framework layers.

Karatekin, E., O. Sandre, et al. (2003). "Cascades of transient pores in giant vesicles: line tension and transport." Biophys J 84(3): 1734-49.

            Under ordinary circumstances, the membrane tension of a giant unilamellar vesicle is essentially nil. Using visible light, we stretch the vesicles, increasing the membrane tension until the membrane responds by the sudden opening of a large pore (several micrometers in size). Only a single pore is observed at a time in a given vesicle. However, a cascade of transient pores appear, up to 30-40 in succession, in the same vesicle. These pores are transient: they reseal within a few seconds as the inner liquid leaks out. The membrane tension, which is the driving force for pore opening, is relaxed with the opening of a pore and the leakage of the inner liquid; the line tension of the pore's edge is then able to drive the closure of a pore. We use fluorescent membrane probes and real-time videomicroscopy to study the dynamics of the pores. These can be visualized only if the vesicles are prepared in a viscous solution to slow down the leakout of the internal liquid. From measurements of the closure velocity of the pores, we are able to infer the line tension,. We have studied the effect of the shape of inclusion molecules on. Cholesterol, which can be modeled as an inverted cone-shaped molecule, increases the line tension when incorporated into the bilayers. Conversely, addition of cone-shaped detergents reduces. The effect of some detergents can be dramatic, reducing by two orders of magnitude, and increasing pore lifetimes up to several minutes. We give some examples of transport through transient pores and present a rough measurement of the leakout velocity of the inner liquid through a pore. We discuss how our results can be extended to less viscous aqueous solutions which are more relevant for biological systems and biotechnological applications.

Kargol, M. and A. Kargol (2003). "Mechanistic equations for membrane substance transport and their identity with Kedem-Katchalsky equations." Biophys Chem 103(2): 117-27.

            Since the physical interpretation of practical Kedem-Katchalsky (KK) equations is not clear, we consider an alternative, mechanistic approach to membrane transport generated by osmotic and hydraulic pressure. We study a porous membrane with randomly distributed pore sizes (radii). We postulate that reflection coefficient (sigma(p)) of a single pore may equal 1 or 0. From this postulate we derive new (mechanistic) transport equations. Their advantage is in clear physical interpretation and since we show they are equivalent to the KK equations, the interpretation of the latter became clearer as well. Hence the equations allow clearer and more detailed interpretation of results concerning membrane substances transport.

Kerschbaum, H. H., J. A. Kozak, et al. (2003). "Polyvalent Cations as Permeant Probes of MIC and TRPM7 Pores." Biophys J 84(4): 2293-305.

            Recent studies in Jurkat T cells and in rat basophilic leukemia cells revealed an Mg(2+)-inhibited cation (MIC) channel that has electrophysiological properties similar to TRPM7 Eyring rate model expressed exogenously in mammalian cells. Here we compare the characteristics of several polyvalent cations and Mg(2+) to block monovalent MIC current from the outside. Putrescine, spermidine, spermine, PhTX-343 (a derivative of the naturally occurring polyamine toxin philanthotoxin), and Mg(2+) each blocked in a dose- and voltage-dependent manner, indicating a blocking site within the electric field of the ion channel. Spermine and the relatively bulky PhTX-343 exhibited voltage dependence steeper than that expected for the number of charges on the molecule. Polyamines and Mg(2+) are permeant blockers, as judged by relief of block at strongly negative membrane potentials. Intracellular dialysis with spermine (300 micro M) had no effect, indicating an asymmetrical pore. At the single-channel level, spermine and Mg(2+) induced flickery block of 40-pS single channels. I/V characteristics and polyamine block are similar in expressed TRPM7 and in native MIC currents, consistent with the conclusion that native MIC channels are composed of TRPM7 subunits. An Eyring rate model is developed to account for I/V characteristics and block of MIC channels by polyvalent cations from the outside.

Kindler, C. H., M. Paul, et al. (2003). "Amide Local Anesthetics Potently Inhibit the Human Tandem Pore Domain Background K+ Channel TASK-2 (KCNK5)." J Pharmacol Exp Ther.

            Blockade of voltage-gated sodium (Na(+)) channels by local anesthetics represents the main mechanism for inhibition of impulse propagation. Local anesthetic induced potassium (K(+)) channel inhibition is also known to influence transmission of sensory impulses and to potentiate inhibition. K(+) channels involved in this mechanism may belong to the emerging family of background tandem pore domain K(+) channels (2P K(+) channels). Oocytes of Xenopus laevis were injected with cRNAs encoding the acid-sensitive 2P K(+) channels TASK-2 (KCNK5), TASK-1 (KCNK3) and chimeric TASK-1/TASK-2 channels in order to study the effects of local anesthetics on heterologously expressed channels using two-electrode voltage-clamp techniques. TASK-2 cDNA-transfected HEK 293 cells were used for single channel recordings. Local anesthetic inhibition of TASK-2 was dose-dependent, agent-specific, and stereoselective. The IC50s for R(+)-bupivacaine and S(-)-bupivacaine were 1