Current-dependent block of rabbit sino-atrial node I(f) channels by ivabradine

"Funny" (f-) channels have a key role in generation of spontaneous activity of pacemaker cells and mediate autonomic control of cardiac rate; f-channels and the related neuronal h-channels are composed of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel subunits. We have investigated the block of f-channels of rabbit cardiac sino-atrial node cells by ivabradine, a novel heart rate-reducing agent. Ivabradine is an open-channel blocker; however, block is exerted preferentially when channels deactivate on depolarization, and is relieved by long hyperpolarizing steps. These features give rise to use-dependent behavior. In this, the action of ivabradine on f-channels is similar to that reported of other rate-reducing agents such as UL-FS49 and ZD7288. However, other features of ivabradine-induced block are peculiar and do not comply with the hypothesis that the voltage-dependence of block is entirely attributable to either the sensitivity of ivabradine-charged molecules to the electrical field in the channel pore, or to differential affinity to different channel states, as has been proposed for UL-FS49 (DiFrancesco, D. 1994. Pflugers Arch. 427:64-70) and ZD7288 (Shin, S.K., B.S. Rotheberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91-101), respectively. Experiments where current flows through channels is modified without changing membrane voltage reveal that the ivabradine block depends on the current driving force, rather than voltage alone, a feature typical of block induced in inwardly rectifying K(+) channels by intracellular cations. Bound drug molecules do not detach from the binding site in the absence of inward current through channels, even if channels are open and the drug is therefore not "trapped" by closed gates. Our data suggest that permeation through f-channel pores occurs according to a multiion, single-file mechanism, and that block/unblock by ivabradine is coupled to ionic flow. The use-dependence resulting from specific features of I(f) block by ivabradine amplifies its rate-reducing ability at high spontaneous rates and may be useful to clinical applications.     

Cellulose morphology and enzymatic reactivity: A modified solute exclusion technique

An expeditious and accurate simplification of Stone and Scallan's solute exclusion technique was developed, thereby avoiding several sources of experimental error coupled with the determination of cellulose pore volume. Using this method, it is shown that cellulolytic enzymes do not enter into the micropores of five studied celluloses. These results suggestes that hydrolysis occurs initially at the external surface of the fibers. This surface area was calculated with the help of adsorption isotherms of bovine serum albumin. The obtained values for the different samples agree with the microscopically observed cellulose morphology. The correlation obtained by several authors relating cellulose porosity and its digestibility is explained as a consequence of the lower crystallinity and easier fragmentation of the more porous celluloses during hydrolysis. (c) 1994 John Wiley & Sons, Inc.     

Nuclear pore distribution and relation to adjacent cytoplasmic organelles in cotyledon cells of developing Vicia faba

Following a zinc iodine-osmium tetroxide fixation, nuclear pore distribution was studied in 0.3-m sections from cotyledons of developing Vicia faba L. Localised absence of nuclear pores was found to be associated with proximity of organelles to the nucleus. Golgi cisternae and mitochondria are associated with areas of pore absence while cisternal endoplasmic reticulum and tubular endoplasmic reticulum are linked with areas showing reduction in pore density. Pores were seen in the nuclear membrane adjacent to vacuoles. Pattern analysis of pore distribution indicated possible clustering within an overall regularity.Abbreviations ER endoplasmic reticulum - ZIO zinc iodine-osmium tetroxide     

The spatial distribution of soil hyphae in structured spruce-forest soils

The external mycelium forms the major part of the absorbing surface of mycorrhized tree roots. Because the macro pore space of acid forest soils is selectively depleted of mobile nutrient cations, it is ecologically important, whether soil hyphae grow into the soil aggregates or not. Seedlings of Norway Spruce (Picea abies (L.) Karst.) with defined mycorrhiza were grown in unsterilized soil cores taken from the A and B-horizon of a limed and an unlimed cambisol on triassic sandstone in the Northern Black Forest, Germany. Water-tension treatments were 10, 30, 160 and 900 hPa. On ground and polished vertical cuts stained with acridine orange, we identified and measured the location of hyphae and characterized their micropedological environment using an image analyzing system. Mean length density varied between 17 m/cm³ and 100 m/cm³ and was independent of aeration parameters. The percentage of hyphae completely embedded in the soil matrix varied between 30% and 8% and decreased significantly with increasing CO₂ concentration in the soil air. Of the hyphae in the soil matrix, 70% were located in a 50 m shell around the macro pores. Pair correlation functions show, that the majority of soil hyphae occur in clusters with diameters below 100 m. Between 60% and 80% of randomly chosen circles with 250 m diameters were completely devoid of hyphae. The inefficient opening up of the intra-aggregate space by soil hyphae is explained by the very slow oxygen diffusion between air-filled macro pores and the intra-aggregate space and mechanical restrictions for hyphae growth.     

Polar paths of diffusion across plant cuticles: new evidence for an old hypothesis

BACKGROUND: The plant cuticle is an extracellular lipophilic biopolymer covering leaf and fruit surfaces. Its main function is the protection of land-living plants from uncontrolled water loss. In the past, the permeability of the cuticle to water and to non-ionic lipophilic molecules (pesticides, herbicides and other xenobiotics) was studied intensively, whereas cuticular penetration of polar ionic compounds was rarely investigated. RECENT PROGRESS: Recent work measuring cuticular penetration of inorganic and organic ions is presented; the effects of molecular size of ions, temperature, wax extraction, humidity and plasticizers strongly support the conclusion that ions penetrate cuticles via water-filled pores. The cuticle covering stomata and trichomes forms the preferential site of ion penetration. This indicates that cuticles possess a pronounced lateral heterogeneity: the largest fraction of the cuticle surface is covered by the lipophilic domains of cutin and wax, but to a certain extent polar domains are also present in the cuticle, which form preferential sites of penetration for polar compounds. THE FUTURE: The chemical nature of these polar domains awaits detailed characterization, which will be of major importance in agriculture and green biotechnology, since polar paths of diffusion represent the most important transport routes for foliar-applied nutrients. Furthermore, many compounds acting as inducers of gene expression in transgenic plants are ionic and need to penetrate the cuticle via polar paths in order to be active.     

Aerenchyma tissue development and gas-pathway structure in root of Avicennia marina (Forsk.) Vierh.

The aerenchyma differentiation in cable roots, pneumatophores, anchor roots, and feeding roots of the mangrove plant, Avicennia marina (Verbenaceae) was analyzed using a light microscope and scanning electron microscope. In all types, cortex cells were arranged in longitudinal columns extending from the endodermis to the epidermis. No cells in the cortex had intercellular spaces at the root tip (0–150 m), and aerenchyma started developing at 200 m from the root apex. The aerenchyma formation was due to cell separation (schizogeny) rather than cell lysis. The cell separation occurred between the longitudinal cell columns, forming long intercellular spaces along the root axis. During aerenchyma formation, the cortex cells enlarged longitudinally by 1.8–3.9 times and widened horizontally by 2.2–2.9 times. As a result, the aerenchyma had a pronounced tubular structure that was radially long, elliptical or oval in cross section and that ran parallel to the root axis. The tube had tapering ends, as did vessel elements, although there were no perforated plates. The interconnection between neighboring tubes was made by abundant small pores or canals that were schizogenous intercellular spaces between the wall cells. All aerenchyma tubes in the root were interconnected by these small pores serving as a gas pathway.     

Molecular Simulations of Phase Transitions in Pores

Abstract

Methods for simulating phase transitions in narrow pores are reviewed, and the advantages and disadvantages of different techniques are discussed. Examples are given of applications to vapor-liquid, liquid-liquid, melting and freezing, solid-solid and layering transitions. While there has been a considerable body of simulation work on vapor-liquid, wetting and layering transitions for simple fluids and pore geometries, much remains to be done on more complex geometries and network effects, on heterogeneous surfaces, and on liquid-liquid, melting and solid-solid transitions in pores.     

Uni-molecular detection and quantification of selected β-lactam antibiotics with a hybrid α-hemolysin protein pore

Single-nanopores have recently been used to electrically detect a wide range of analytes. Similarly, using electrophysiology, we demonstrate how a system comprised of an ion channel formed by α-hemolysin (α-HL) and single-cyclic γ-cyclodextrin (γ-CD) molecule permits the detection of, and differentiation between three different antibiotics from the β-lactam family. Specifically, histograms of the time between the successive binding events, and the residence time distributions of the antibiotic in the γ-CD molecular adapter vary with the antibiotic type. The results show that the association times of amoxicillin, azlocillin, and ampicillin are τ(on) = 2.1 ± 0.2, 2.2 ± 0.3, and 3.1 ± 0.4 ms, respectively. Interestingly, we found that the residence time of the bulkier and negatively charged azlocillin (τ(off) = 0.008 ± 0.0005 ms) is much less than that of ampicillin (τ(off) = 0.07 ± 0.005 ms) and amoxicillin (τ(off) = 0.1 ± 0.02 ms), even though the γ-CD-α-HL complex is anionic selective. The data were also used to estimate the standard free energy of binding between ampicillin to γ-CDs binding (-12 kJ mol(-1) [corrected]). The difference in association times might be due to γ-CDs-imposed steric hindrance or an energetically more expensive desolvation step for the antibiotics to gain access to the binding site in the CD. We suggest that this technique may be used to detect other analytes used in pharmaceutical applications.     

Melittin: a Membrane-active Peptide with Diverse Functions

Melittin is the principal toxic component in the venom of the European honey bee Apis mellifera and is a cationic, hemolytic peptide. It is a small linear peptide composed of 26 amino acid residues in which the amino-terminal region is predominantly hydrophobic whereas the carboxy-terminal region is hydrophilic due to the presence of a stretch of positively charged amino acids. This amphiphilic property of melittin has resulted in melittin being used as a suitable model peptide for monitoring lipid–protein interactions in membranes. In this review, the solution and membrane properties of melittin are highlighted, with an emphasis on melittin–membrane interaction using biophysical approaches. The recent applications of melittin in various cellular processes are discussed.     

Ion channels of various types induced in lipid membranes by gramicidin a derivatives carrying a cationic sequence at their C-termini

The channel-forming activity of gramicidin A derivatives carrying positively charged amino acid sequences at their C-termini was studied on planar bilayer lipid membranes and liposomes. We showed previously (FEBS Lett., 2005, vol. 579, pp. 5247–5252) that, at low concentrations, these peptides form classical cation-selective pores typical of gramicidin A, whereas, at high concentrations, they form large nonselective pores. The ability of the peptides to form nonselective pores, which was determined by the efflux of carboxyfluorescein, an organic dye, from liposomes, decreased substantially as the length of the gramicidin fragment in the series of cationic analogues was truncated. CD spectra showed that large pores are formed by peptides having both β^6.3 single-stranded and β^5.6 double-stranded helical conformations of the gramicidin fragment, with the C-terminal cationic sequence being extended. The dimerization of the peptides by the oxidation of the terminal cysteine promoted the formation of nonselective pores. It was shown that nonselective pores are not formed in membranes of erythrocytes, which may indicate a dependence of the channel-forming ability on the membrane type. The results may be of interest for the directed synthesis of peptides with antibacterial activity.