Study of the mechanism of action of anoplin, a helical antimicrobial decapeptide with ion channel-like activity, and the role of the amidated C-terminus

Anoplin, an antimicrobial, helical decapeptide from wasp venom, looses its biological activities by mere deamidation of its C-terminus. Secondary structure determination, by circular dichroism spectroscopy in amphipathic environments, and lytic activity in zwitterionic and anionic vesicles showed quite similar results for the amidated and the carboxylated forms of the peptide. The deamidation of the C-terminus introduced a negative charge at an all-positive charged peptide, causing a loss of amphipathicity, as indicated by molecular dynamics simulations in TFE/water mixtures and this subtle modification in a peptide`s primary structure disturbed the interaction with bilayers and biological membranes. Although being poorly lytic, the amidated form, but not the carboxylated, presented ion channel-like activity on anionic bilayers with a well-defined conductance step; at approximately the same concentration it showed antimicrobial activity. The pores remain open at trans-negative potentials, preferentially conducting cations, and this situation is equivalent to the interaction of the peptide with bacterial membranes that also maintain a high negative potential inside. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Chaotic model and memory in single calcium-activated potassium channel kinetics

Ion channels are pores formed by proteins and responsible for carrying ion fluxes through cellular membranes. The ion channels can assume conformational states thereby controlling ion flow. Physically, the conformational transitions from one state to another are associated with energy barriers between them and are dependent on stimulus, such as, electrical field, ligands, second messengers, etc. Several models have been proposed to describe the kinetics of ion channels. The classical Markovian model assumes that a future transition is independent of the time that the ion channel stayed in a previous state. Others models as the fractal and the chaotic assume that the rate of transitions between the states depend on the time that the ionic channel stayed in a previous state. For the calcium activated potassium channels of Leydig cells the R/S Hurst analysis has indicated that the channels are long-term correlated with a Hurst coefficient H around 0.7, showing a persistent memory in this kinetic. Here, we applied the R/S analysis to the opening and closing dwell time series obtained from simulated data from a chaotic model proposed by L. Liebovitch and T. Toth [J. Theor. Biol. 148, 243 (1991)] and we show that this chaotic model or any model that treats the set of channel openings and closings as independent events is inadequate to describe the long-term correlation (memory) already described for the experimental data. (C) 2008 American Institute of Physics.

Interactions of mast cell degranulating peptides with model membranes: A comparative biophysical study

In the last decade, there has been renewed interest in biologically active peptides in fields like allergy, autoimmume diseases and antibiotic therapy. Mast cell degranulating peptides mimic G-protein receptors, showing different activity levels even among homologous peptides. Another important feature is their ability to interact directly with membrane phospholipids, in a fast and concentration-dependent way. The mechanism of action of peptide HR1 on model membranes was investigated comparatively to other mast cell degranulating peptides (Mastoparan, Eumenitin and Anoplin) to evidence the features that modulate their selectivity. Using vesicle leakage, single-channel recordings and zeta-potential measurements, we demonstrated that HR1 preferentially binds to anionic bilayers, accumulates, folds, and at very low concentrations, is able to insert and create membrane spanning ion-selective pores. We discuss the ion selectivity character of the pores based on the neutralization or screening of the peptides charges by the bilayer head group charges or dipoles. (C) 2009 Elsevier Inc. All rights reserved.

Mouse Leydig cells express multiple P2X receptor subunits

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X(1)-P2X(7)) and seven heteromeric receptors (P2X(1/2), P2X(1/4), P2X(1/5), P2X(2/3), P2X(2/6), P2X(4/6), P2X(4/7)) have been described. ATP treatment of Leydig cells leads to an increase in [Ca(2+)](i) and testosterone secretion, supporting the hypothesis that Ca(2+) signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X(2). In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X(2), P2X(4), P2X(6), and P2X(7) subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 mu M ivermectin induced an increase (131.2 +/- 5.9%) and 3 mu M ivermectin a decrease (64.2 +/- 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X(4) subunits. P2X(7) receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X(2/4/6), is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X(2) subunit.

Effects of the cationic antimicrobial peptide eumenitin from the venom of solitary wasp Eumenes rubronotatus in planar lipid bilayers: Surface charge and pore formation activity

Eumenitin, a novel cationic antimicrobial peptide from the venom of solitary wasp Eumenes rubronotatus, was characterized by its effects on black lipid membranes of negatively charged (azolectin) and zwitterionic (1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or DPhPC-cholesterol) phospholipids: surface potential changes, single-channel activity, ion selectivity, and pore size were studied. We found that eumenitin binds preferentially to charged lipid membranes as compared with zwitterionic ones. Eumenitin is able to form pores in azolectin (G(1) = 118.00 +/- 3.67 pS or G(2) = 160.00 +/- 7.07 pS) and DPhPC membranes (G = 61.13 +/- 7.57 pS). Moreover, cholesterol addition to zwitterionic DPhPC membranes inhibits pore formation activity but does not interfere with the binding of peptide. Open pores presented higher cation (K (+)) over anion (Cl-) selectivity. The pore diameter was estimated at between 8.5and 9.8 angstrom in azolectin membranes and about 4.3 angstrom in DPhPC membranes. The results are discussed based on the toroidal pore model for membrane pore-forming activity and ion selectivity. (c) 2007 Elsevier Ltd. All rights reserved.

Effect on the heavy-ion fusion and elastic scattering cross sections of common approximations assumed in coupled-channel calculations

We study the effects of several approximations commonly used in coupled-channel analyses of fusion and elastic scattering cross sections. Our calculations are performed considering couplings to inelastic states in the context of the frozen approximation, which is equivalent to the coupled-channel formalism when dealing with small excitation energies. Our findings indicate that, in some cases, the effect of the approximations on the theoretical cross sections can be larger than the precision of the experimental data.

rho(0) photoproduction in ultraperipheral relativistic heavy ion collisions at root s(NN)=200 GeV

Photoproduction reactions occur when the electromagnetic field of a relativistic heavy ion interacts with another heavy ion. The STAR Collaboration presents a measurement of rho(0) and direct pi(+)pi(-) photoproduction in ultraperipheral relativistic heavy ion collisions at root s(NN) = 200 GeV. We observe both exclusive photoproduction and photoproduction accompanied by mutual Coulomb excitation. We find a coherent cross section of sigma(AuAu -> Au*Au*rho(0)) = 530 +/- 19(stat.) +/- 57(syst.) mb, in accord with theoretical calculations based on a Glauber approach, but considerably below the predictions of a color dipole model. The rho 0 transverse momentum spectrum (p(T)(2)) is fit by a double exponential curve including both coherent and incoherent coupling to the target nucleus; we find sigma(inc)/sigma(coh) = 0.29 +/- 0.03 (stat.) +/- 0.08 (syst.). The ratio of direct pi(+)pi(-) to rho(0) production is comparable to that observed in gamma(p) collisions at HERA and appears to be independent of photon energy. Finally, the measured rho(0) spin helicity matrix elements agree within errors with the expected s-channel helicity conservation.

Upregulation of intermediate calcium-activated potassium channels counterbalance the impaired endothelium-dependent vasodilation in stroke-prone spontaneously hypertensive rats

Endothelial dysfunction has been linked to a decrease in nitric oxide (NO) bioavailability and attenuated endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation. The small (SK(Ca)) and intermediate (IK(Ca)) calcium-activated potassium channels play a key role in endothelium-dependent relaxation. Because the repressor element 1-silencing transcription factor (REST) negatively regulates IK(Ca) expression, we hypothesized that augmented REST and decreased IK(Ca) expression contributes to impaired endothelium-dependent vasodilation associated with hypertension. Acetylcholine (ACh) responses were slightly decreased in small mesenteric arteries from male stroke-prone spontaneously hypertensive rats (SHRSPs) versus arteries from Wistar Kyoto (WKY) rats. Incubation with N-nitro-L-arginine methyl ester (L-NAME; 100 mu mol/L) and indomethacin (100 mu mol/L) greatly impaired ACh responses in vessels from SHRSP. lberiotoxin (0.1 mu mol/L), which is a selective inhibitor of large-conductance K(Ca) (BK(Ca)) channels, did not modify EDHF-mediated vasodilation in SHRSP or WKY. UCL-1684 (0.1 mu mol/L.), which is a selective inhibitor of SKCa channels, almost abolished EDHF-mediated vasodilation in WKY and decreased relaxation in SHRSP. 1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole (TRAM-34; 10 mu mol/L) and charybdotoxin (0.1 mu mol/L), which are both IKCa inhibitors, produced a small decrease of EDHF relaxation in WKY but completely abrogated EDHF vasodilation in SHRSP. EDHF-mediated relaxant responses were completely abolished in both groups by simultaneous treatment with UCL-1684 and TRAM-34 or charybdotoxin. Relaxation to SK(Ca)/IK(Ca) channels agonist NS-309 was decreased in SHRSP arteries. The expression of SK(Ca) was decreased, whereas IK(Ca) was increased in SHRSP mesenteric arteries. REST expression was reduced in arteries from SHRSP. Vessels incubated with TRAM-34 (10 mu mol/L) for 24h displayed reduced REST expression and demonstrated no differences in IK(Ca). In conclusion, IK(Ca) channel upregulation, via decreased REST, seems to compensate deficient activity of SK(Ca) channels in the vasculature of spontaneously hypertensive rats. (Translational Research 2009; 154:183-193)

Eag1 potassium channel immunohistochemistry in the CNS of adult rat and selected regions of human brain

Eag1 (K(v)10.1) is the founding member of an evolutionarily conserved superfamily of voltage-gated K+ channels. In rats and humans Eag1 is preferentially expressed in adult brain but its regional distribution has only been studied at mRNA level and only in the rat at high resolution. The main aim of the present study is to describe the distribution of Eag1 protein in adult rat brain in comparison to selected regions of the human adult brain. The distribution of Eag1 protein was assessed using alkaline-phosphatase based immunohistochemistry. Eag1 immunoreactivity was widespread, although selective, throughout rat brain, especially noticeable in the perinuclear space of cells and proximal regions of the extensions, both in rat and human brain. To relate the results to the relative abundance of Eag1 transcripts in different regions of rat brain a reverse-transcription coupled to quantitative polymerase chain reaction (real time PCR) was performed. This real time PCR analysis showed high Eag1 expression in the olfactory bulb, cerebral cortex, hippocampus, hypothalamus, and cerebellum. The results indicate that Eag1 protein expression greatly overlaps with mRNA distribution in rats and humans. The physiological relevance of potassium channels in the different regions expressing Eag1 protein is discussed. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

An imaginary potential with universal normalization for dissipative processes in heavy-ion reactions

In this work we present new coupled channel calculations with the Sao Paulo potential (SPP) as the bare interaction, and an imaginary potential with system and energy independent normalization that has been developed to take into account dissipative processes in heavy-ion reactions. This imaginary potential is based on high-energy nucleon interaction in nuclear medium. Our theoretical predictions for energies up to approximate to 100 MeV/nucleon agree very well with the experimental data for the p, n + nucleus, (16)O + (27)Al, (16)O + (60)Ni, (58)Ni + (124)Sn, and weakly bound projectile (7)Li + (120)Sn systems. (C) 2008 Elsevier B.V. All rights reserved.

Potassium Current in Mature Bovine Spermatozoa

Ion channels have been assigned a pivotal importance in various sperm functions and are therefore promising targets for contraceptive development. The lack of data on channel functionality and pharmacology has hampered this goal. This is a consequence of technical problems of applying electrophysiological techniques to spermatozoa due to their small size and form. By using a laminin coating to increase adherence of spermatozoa and nystatin in the patch pipette for pore formation, we have adapted the whole-cell recording technique to study currents in mature uncapacitated bovine spermatozoa. Employing these conditions, in the head region, patched spermatozoa could be transferred into the whole-cell configuration. For the first time we document an outward rectifying current in mature bovine spermatozoa was blocked by tetraethyl ammonium (TEA) chloride. The observation of a shift in the reversal potential as a response to changes in the extracellular concentration of K+ ions allowed us to identify this current as K+ selective. This result shows that K+ channels in the head region of mature uncapacitated bovine spermatozoa can be suitably investigated using the whole-cell recording patch-clamp technique.

Mitochondrial ion transport pathways: Role in metabolic diseases

Mitochondria are the central coordinators of energy metabolism and alterations in their function and number have long been associated with metabolic disorders such as obesity, diabetes and hyperlipidemias. Since oxidative phosphorylation requires an electrochemical gradient across the inner mitochondrial membrane, ion channels in this membrane certainly must play an important role in the regulation of energy metabolism. However, in many experimental settings, the relationship between the activity of mitochondrial ion transport and metabolic disorders is still poorly understood. This review briefly summarizes some aspects of mitochondrial H(+) transport (promoted by uncoupling proteins, UCPs). Ca(2+) and K(+) uniporters which may be determinant in metabolic disorders. (C) 2009 Elsevier B.V. All rights reserved.

Effects of a high fat diet on liver mitochondria: increased ATP-sensitive K(+) channel activity and reactive oxygen species generation

High fat diets are extensively associated with health complications within the spectrum of the metabolic syndrome. Some of the most prevalent of these pathologies, often observed early in the development of high-fat dietary complications, are non-alcoholic fatty liver diseases. Mitochondrial bioenergetics and redox state changes are also widely associated with alterations within the metabolic syndrome. We investigated the mitochondrial effects of a high fat diet leading to non-alcoholic fatty liver disease in mice. We found that the diet does not substantially alter respiratory rates, ADP/O ratios or membrane potentials of isolated liver mitochondria. However, H(2)O(2) release using different substrates and ATP-sensitive K(+) transport activities are increased in mitochondria from animals on high fat diets. The increase in H(2)O(2) release rates was observed with different respiratory substrates and was not altered by modulators of mitochondrial ATP-sensitive K(+) channels, indicating it was not related to an observed increase in K(+) transport. Altogether, we demonstrate that mitochondria from animals with diet-induced steatosis do not present significant bioenergetic changes, but display altered ion transport and increased oxidant generation. This is the first evidence, to our knowledge, that ATP-sensitive K(+) transport in mitochondria can be modulated by diet.

Short-Term effect of COX-2 selective inhibitor as an adjunct for the treatment of periodontal disease: a clinical double-blind study in humans

Adjunctive therapeutic strategies that modulate the inflammatory mediators can play a significant role in periodontal therapy. In this double-blind, placebo-controlled study, 60 subjects diagnosed as periodontitis patients were evaluated for 28 days after periodontal treatment combined with selective cyclooxygenase-2 (COX-2) inhibitor. The experimental group received scaling and root planning (SRP) combined with the Loxoprofen antiinflammatory drug (SRP+Loxoprofen). The control group received SRP combined with placebo (SRP+placebo). Plaque index (PI), probing pocket depth (PD) and bleeding on probing (BOP) were monitored with an electronic probe at baseline and after 14 and 28 days. Both groups displayed clinical improvement in PD, PI and BOP. They also showed statistically similar values (p>0.05) of PD reduction on day 14 (0.4 mm) and on day 28 (0.6 mm). At the baseline, few deeper sites (>7 mm) from SRP+Loxoprofen group were responsible and most PD reduction was observed after 14 days (p<0.05). The percentage of remaining deep pockets (>7 mm) after 14 days in the SRP+Loxoprofen group was significantly lower (p<0.05) than in the SRP+placebo group. Loxoprofen presents potential effect as an adjunct of periodontal disease treatment, but long-term clinical trials are necessary to confirm its efficacy.

Effect of different cleansers on the weight and ion release of removable partial denture: an in vitro study

OBJECTIVE: Removable partial dentures (RPD) require different hygiene care, and association of brushing and chemical cleansing is the most recommended to control biofilm formation. However, the effect of cleansers has not been evaluated in RPD metallic components. The aim of this study was to evaluate in vitro the effect of different denture cleansers on the weight and ion release of RPD. MATERIAL AND METHODS: Five specimens (12x3 mm metallic disc positioned in a 38x18x4 mm mould filled with resin), 7 cleanser agents [Periogard (PE), Cepacol (CE), Corega Tabs (CT), Medical Interporous (MI), Polident (PO), 0.05% sodium hypochlorite (NaOCl), and distilled water (DW) (control)] and 2 cobalt-chromium alloys [DeguDent (DD), and VeraPDI (VPDI)] were used for each experimental situation. One hundred and eighty immersions were performed and the weight was analyzed with a high precision analytic balance. Data were recorded before and after the immersions. The ion release was analyzed using mass spectrometry with inductively coupled plasma. Data were analyzed by two-way ANOVA and Tukey HSD post hoc test at 5% significance level. RESULTS: Statistical analysis showed that CT and MI had higher values of weight loss with higher change in VPDI alloy compared to DD. The solutions that caused more ion release were NaOCl and MI. CONCLUSIONS: It may be concluded that 0.05% NaOCl and Medical Interporous tablets are not suitable as auxiliary chemical solutions for RPD care.