Concentration dependence of sodium permeation and sodium ion interactions in the cyclic AMP-gated channels of mammalian olfactory receptor neurons

The dependence of currents through the cyclic nucleotide-gated (CNG) channels of mammalian olfactory receptor neurons (ORNs) on the concentration of NaCl was studied in excised inside-out patches from their dendritic knobs using the patch-clamp technique. With a saturating concentration (100 mu M) of adenosine 3', 5'-cyclic monophosphate (cAMP), the changes in the reversal potential of macroscopic currents were studied at NaCl concentrations from 25 to 300 mM. In symmetrical NaCl solutions without the addition of divalent cations, the current-voltage relations were almost linear, reversing close to O mV. When the external NaCl concentration was maintained at 150 mM and the internal concentrations were varied, the reversal potentials of the cAMP-activated currents closely followed the Na+ equilibrium potential indicating that P-Cl/P-Na approximate to 0. However, at low external NaCl concentrations (less than or equal to 100 mM) there was some significant chloride permeability. Our results further indicated that Na+ currents through these channels: (i) did not obey the independence principle; (ii) showed saturation kinetics with K(m)s in the range of 100-150 mM and (iii) displayed a lack of voltage dependence of conductance in asymmetric solutions that suggested that ion-binding sites were situated midway along the channel. Together, these characteristics indicate that the permeation properties of the olfactory CNG channels are significantly different from those of photoreceptor CNG channels.

Voltage gating and permeation in a gap junction hemichannel.

Gap junction channels are formed by members of the connexin gene family and mediate direct intercellular communication through linked hemichannels (connexons) from each of two adjacent cells. While for most connexins, the hemichannels appear to require an apposing hemichannel to open, macroscopic currents obtained from Xenopus oocytes expressing rat Cx46 suggested that some hemichannels can be readily opened by membrane depolarization [Paul, D. L., Ebihara, L., Takemoto, L. J., Swenson, K. I. & Goodenough, D. A. (1991), J. Cell Biol. 115, 1077-1089]. Here we demonstrate by single channel recording that hemichannels comprised of rat Cx46 exhibit complex voltage gating consistent with there being two distinct gating mechanisms. One mechanism partially closes Cx46 hemichannels from a fully open state, gammaopen, to a substate, gammasub, about one-third of the conductance of gammaopen; these transitions occur when the cell is depolarized to inside positive voltages, consistent with gating by transjunctional voltage in Cx46 gap junctions. The other gating mechanism closes Cx46 hemichannels to a fully closed state, gammaclosed, on hyperpolarization to inside negative voltages and has unusual characteristics; transitions between gammaclosed and gammaopen appear slow (10-20 ms), often involving several transient substates distinct from gammasub. The polarity of activation and kinetics of this latter form of gating indicate that it is the mechanism by which these hemichannels open in the cell surface membrane when unapposed by another hemichannel. Cx46 hemichannels display a substantial preference for cations over anions, yet have a large unitary conductance (approximately 300 pS) and a relatively large pore as inferred from permeability to tetraethylammonium (approximately 8.5 angstroms diameter). These hemichannels open at physiological voltages and could induce substantial cation fluxes in cells expressing Cx46.

Cation size effects-modified phase and PTCR development in Er3+ and Ca2+ co-doped BaTiO3 ceramics during sintering

Development of the positive temperature coefficient of resistivity (PTCR) in Er3+ and Ca2+ co-doped ferroelectric BaTiO3 was studied in this work, with Er3+ being used to act as a donor doping. Irrespective of all the materials showing high densities after sintering at 1200 to 1300 ºC, these revealed insulator at the lowest sintering temperature, changing to semiconducting and PTCR-type materials only when the sintering temperature was further increased. Observations from X-ray diffraction help correlating this effect with phase development in this formulated (Ba,Ca,Er)TiO3 system, considering the formation of initially two separated major (Ba,Ca)TiO3- and minor (Ca,Er)TiO3-based compounds, as a consequence of cation size-induced stress energy effects. Thus, appearance and enhancement here of the semiconducting and PTCR responses towards higher sintering temperatures particularly involve the incorporation of Er3+ into the major phase, rendering finally possible the generation and "percolative-like" migration of electrons throughout the whole material.

Permeation of Large Antineoplastics into Wild Strain of Saccharomyces cerevisiae

Saccharomyces cerevisiae has been used in genotoxicity and cytotoxicity assays for several years before the Ames Test approach. However the cell permeability of yeast has been considered a limitant factor to this kind of assay and many researchers have been introducing genetic modifications into wild strains to improve the sensitivity to chemical compounds. In our study, we used Saccharomyces cerevisiae ATCC 9763, well known and very common strain in antibiotic assays, and we evaluated the cytotoxicity of some antineoplastic agents (etoposide, epirubicin, carboplatin, cisplatin and mitoxantrone). Each culture was observed under the light of microscope and photographed. Neither genetic modification nor addition of permeation inducers, as dimethylsulfoxide (DMSO), were introduced during the assays and the cells presented good sensitivity to those compounds, demonstrating that other potential strains and characteristics of cells should be reconsidered to improve these assays apart from the cellular permeability.

In Vitro Ultrasound Influence on Cutaneous Permeation of Hyaluronidase

In vitro skin permeation of hyaluronidase enzyme and the ultrasound (US) effects on cutaneous permeation of this enzyme were investigated. Diffusion cell technique, porcine skins, buffer solution, and a hydrophilic gel+hyaluronidase (100 TUR/g), were used for study. The experimental groups were: gel+enzyme and gel+enzyme+US. The activity of permeated hyaluronidase was determined by turbydimetric method in spectrophotometer at 400nm. Gel+enzyme group permitted a diffusion of 26.89 +/- 3.82 TUR of hyaluronidase in the total time of 240 minutes. However, the other group did not produce significant differences (p0.05) in the same period (13.68 +/- 5.35 TUR). Hyaluronidase permeation through the skin was verified; however US did not cause increase of this enzyme permeation.

Comparative analysis of the gas-phase reactions of cylindrospermopsin and the difference in the alkali metal cation mobility

Cylindrospermopsin (CYN) belongs to a group of toxins produced by several strains of freshwater cyanobacteria. It is a compact zwitterionic molecule composed of a uracil section and a tricyclic guanidinium portion with a primarily hepatotoxic effect. Using low multi-stage and high-resolution mass spectrometry, the gas-phase reactions of this toxin have been investigated. Our data show that collision-induced dissociation (CID) spectra of CYN are dominated by neutral losses, and three major initial fragmentation pathways are clearly distinguishable. Interestingly, comparative analysis of protonated and cationizated molecules showed a significant difference in the balance of the SO(3) and terminal ring elimination. These data indicate that the differential ion mobility of H(+), Li(+), Na(+) and K(+) leads to different fragmentation pathways, giving rise to mass spectra with different profiles. Copyright (C) 2008 John Wiley & Sons, Ltd.

In vitro studies of cutaneous retention of magnetic nanoemulsion loaded with zinc phthalocyanine for synergic use in skin cancer treatment

In this study was developed a new nano drug delivery system (NDDS) based on association of biodegradable surfactants with biocompatible magnetic fluid of maguemita citrate derivative. This formulation consists in a magnetic emulsion with nanostructured colloidal particles. Preliminary in vitro experiments showed that the formulation presents a great potential for synergic application in the topical release of photosensitizer drug (PS) and excellent target tissue properties in the photodynamic therapy (PDT) combined with hyperthermia (HPT) protocols. The physical chemistry characterization and in vitro assays were carried out by Zn(II) Phtalocyanine (ZnPc) photosensitizer incorporated into NDDS in the absence and the presence of magnetic fluid, showed good results and high biocompatibility. In vitro experiments were accomplished by tape-stripping protocols for quanti. cation of drug association with different skin tissue layers. This technique is a classical method for analyses of drug release in stratum corneum and epidermis+ dermis skin layers. The NDDS formulations were applied directly in pig skin (tissue model) fixed in the cell`s Franz device with receptor medium container with a PBS/EtOH 20% solution (10mM, pH 7.4) at 37 degrees C. After 12 h of topical administration stratum corneum was removed from fifty tapes and the ZnPc retained was evaluated by solvent extraction in dimetil-sulphoxide under ultrasonic bath. These results indicated that magnetic nanoemulsion (MNE) increase the drug release on the deeper skin layers when compared with classical formulation in the absence of magnetic particles. This could be related with the increase of biocompatibility of NDDS due to the great affinity for the polar extracelullar matrix in the skin and also for the increase in the drug partition inside of corneocites wall. (C) 2008 Elsevier B.V. All rights reserved.

Photophysical studies and in vitro skin permeation/retention of Foscan (R)/nanoemulsion (NE) applicable to photodynamic therapy skin cancer treatment.

In this work we evaluated the photophysical and in vitro properties of Foscan (R), a second-generation photosensitizer drug (PS) widely used in systemic clinical protocols for cancer therapy based on Photodynamic Therapy (PDT). We employed biodegradable nanoemulsions (NE) as a colloidal vehicle of the oil/water (o/w) type focusing in topical administration of Foscan (R) and other photosensitizer drugs. This formulation was obtained and stabilized by the methodology described by Tabosa do Egito et al.,(30) based on the mixture of two phases: an aqueous solution and an organic medium consisting of nonionic surfactants and oil. The photodynamic potential of the drug incorporated into the NE was studied by steady-state and time-resolved spectroscopic techniques. We also analyzed the in vitro biological behavior carried out in mimetic biological environment protocols based on the animal model. After topical application in a skin animal model, we evaluated the Foscan (R)/NE diffusion flux into the skin layers (stratum corneum and epidermis + dermis) by classical procedures using Franz Diffusion cells. Our results showed that the photophysical properties of PS were maintained after its incorporation into the NE when compared with homogeneous organic medium. The in vitro assays enabled the determination of an adequate profile for the interaction of this system in the different skin layers, with an ideal time lag of 6 h after topical administration in the skin model. The Foscan (R) diffusion flux (J) was increased when this PS was incorporated into the NE, if compared with its flux in physiological medium. These parameters demonstrated that the NE can be potentially applied as a drug delivery system (DDS) for Foscan (R) in both in vitro and in vivo assays, as well as in future clinical applications involving topical skin cancer PDT.

Examination into the accuracy of exchangeable cation measurement in saline soils

Despite the increasing prevalence of salinity world-wide, the measurement of exchangeable cation concentrations in saline soils remains problematic. Two soil types (Mollisol and Vertisol) were equilibrated with a range of sodium adsorption ratio (SAR) solutions at various ionic strengths. The concentrations of exchangeable cations were then determined using several different types of methods, and the measured exchangeable cation concentrations compared to reference values. At low ionic strength (low salinity), the concentration of exchangeable cations can be accurately estimated from the total soil extractable cations. In saline soils, however, the presence of soluble salts in the soil solution precludes the use of this method. Leaching of the soil with a pre-wash solution (such as alcohol) was found to effectively remove the soluble salts from the soil, thus allowing the accurate measurement of the effective cation exchange capacity (ECEC). However, the dilution associated with this pre-washing increased the exchangeable Ca concentrations while simultaneously decreasing exchangeable Na. In contrast, when calculated as the difference between the total extractable cations and the soil solution cations, good correlations were found between the calculated exchangeable cation concentrations and the reference values for both Na (Mollisol: y=0.873x and Vertisol: y=0.960x) and Ca (Mollisol: y=0.901x and Vertisol: y=1.05x). Therefore, for soils with a soil solution ionic strength greater than 50 mM (electrical conductivity of 4 dS/m) (in which exchangeable cation concentrations are overestimated by the assumption they can be estimated as the total extractable cations), concentrations can be calculated as the difference between total extractable cations and soluble cations.

A review of the use of the basic cation saturation ratio and the "ideal" soil

The use of 'balanced' Ca, Mg, and K ratios, as prescribed by the basic cation saturation ratio (BCSR) concept, is still used by some private soil-testing laboratories for the interpretation of soil analytical data. This review aims to examine the suitability of the BCSR concept as a method for the interpretation of soil analytical data. According to the BCSR concept, maximum plant growth will be achieved only when the soil’s exchangeable Ca, Mg, and K concentrations are approximately 65 % Ca, 10 % Mg, and 5 % K (termed the ‘ideal soil’). This ‘ideal soil’ was originally proposed by Firman Bear and co-workers in New Jersey (USA) during the 1940s as a method of reducing luxury K uptake by alfalfa (Medicago sativa L.). At about the same time, William Albrecht, working in Missouri (USA), concluded through his own investigations that plants require a soil with a high Ca saturation for optimal growth. Whilst it now appears that several of Albrecht’s experiments were fundamentally flawed, the BCSR (‘balanced soil’) concept has been widely promoted, suggesting that the prescribed cationic ratios provide optimum chemical, physical, and biological soil properties. Our examination of data from numerous studies (particularly those of Albrecht and Bear, themselves) would suggest that, within the ranges commonly found in soils, the chemical, physical, and biological fertility of a soil is generally not influenced by the ratios of Ca, Mg, and K. The data do not support the claims of the BCSR, and continued promotion of the BCSR will result in the inefficient use of resources in agriculture and horticulture.

Nitric oxide inhibition of the rat olfactory cyclic nucleotide-gated cation channel

The effects of nitric oxide (NO) and other cysteine modifying agents were examined on cyclic nucleotide-gated (CNG) cation channels from rat olfactory receptor neurons. The NO compounds, S-nitroso-cysteine (SNC) and 3-morpholino-sydnonomine (SIN-1), did not activate the channels when applied for up to 10 min. The cysteine alkylating agent, N-ethylmaleimide (NEM), and the oxidising agent, dithionitrobensoate (DTNB), were also without agonist efficacy. Neither SNC nor DTNB altered the cAMP sensitivity of the channels. However, 2-min applications of SIN-1, SNC and DTNB inhibited the cAMP-gated current to approximately 50% of the control current level. This inhibition showed no spontaneous reversal for 5 min but was completely reversed by a 2-min exposure to DTT. The presence of cAMP protected the channels against NO-induced inhibition. These results indicate that inhibition is caused by S-nitrosylation of neighboring sulfhydryl groups leading to sulfhydryl bond formation. This reaction is favored in the closed channel state. Since recombinantly expressed rat olfactory alpha and beta CNG channel homomers and alpha/beta heteromers are activated and not inhibited by cysteine modification, the results of this study imply the existence of a novel subunit or tightly bound factor which dominates the effect of cysteine modification in the native channels. As CNG channels provide a pathway for calcum influx, the results may also have important implications for the physiological role of NO in mammalian olfactory receptor neurons.

Pore structure tailoring of pillared clays with cation doping techniques

Techniques and mechanism of doping controlled amounts of various cations into pillared clays without causing precipitation or damages to the pillared layered structures are reviewed and discussed. Transition metals of great interest in catalysis can be doped in the micropores of pillared clay in ionic forms by a two-step process. The micropore structures and surface nature of pillared clays are altered by the introduced cations, and this results in a significant improvement in adsorption properties of the clays. Adsorption of water, air components and organic vapors on cation-doped pillared clays were studied. The effects of the amount and species of cations on the pore structure and adsorption behavior are discussed. It is demonstrated that the presence of doped Ca2+ ions can effectively aides the control of modification of the pillared clays of large pore openings. Controlled cation doping is a simple and powerful tool for improving the adsorption properties of pillared clay.

Thermal stability of mixed-cation alpha-sialon ceramics

A series of alpha-sialon (alpha') compositions containing mixed stabilising cations were prepared, by introducing additional CaO to a basic Sm alpha-sialon compositions. The thermal stability of these Sm-Ca-containing alpha-sialon phases was investigated using XRD, SEM and EDXS techniques. It was found that the addition of calcium into the Sm alpha-sialon systems greatly improved the stability of the alpha-sialon phases. Calcium was found to be incorporated into the alpha-sialon structure, coexistent with the samarium, and partitioning of the calcium and samarium was observed between the alpha' phase and grain boundary phases. This indicates a technique which may be used to improve the thermal stability of the alpha' phase while maintaining good refractory phases at the gialon grain boundaries. (C) 2003 Elsevier Science B.V. All rights reserved.

Cyclopropyl fatty acids implicate a radical but not a cation as an intermediate in P450(BM3)-catalysed hydroxylations

Novel cyclopropyl containing fatty acids are good substrates for P450(BM3) catalysed hydroxylation and analysis of their oxidation products indicates the presence of a radical intermediate (maximum rebound rate 2.6x10(10) s(-1)) and the absence of any cationic intermediate.