Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin

The peptide antibiotic nisin is shown to disrupt valinomycin-induced potassium diffusion potentials imposed on intact cells of Staphylococcus cohnii 22. Membrane depolarization occurred rapidly at high diffusion potentials while at low potentials nisin-induced depolarization was slower suggesting that nisin requires a membrane potential for activity. This assumption was proven in experiments with planar lipid bilayers (black lipid membranes). Macroscopic conductivity measurements indicated a voltage-dependent action of nisin. The potential must have a trans-negative orientation with respect to the addition of nisin (added to the cis-side) and a sufficient magnitude (ca. -100 mV). With intact cells the threshold potential was lower (-50 to -80 mV at pH 7.5 and below -50 mV at pH 5.5). Single channel recordings resolved transient multistate pores, strongly resembling those introduced by melittin into artificial bilayers. The pores had diameters in the range of 0.2–1 nm, and lifetimes of few to several hundred milliseconds. The results indicate that nisin has to be regarded as a membrane-depolarizing agent which acts in a voltage-dependent fashion.Abbreviations BLM Black lipid membranes - CCCP carbonyl cyanide m-chlorophenylhydrazone - DOPC dioleoyl phosphatidylcholine - PS phosphatidylserine - TPP⁺ tetraphenylphosphonium cation     

Heat shock proteins induce pores in membranes

Human heat shock protein (hsp) 70 and bacterial protein groEL promote leakage of calcein from liposomes induced by human serum albumin signal peptide, byS. aureus toxin or by diphtheria toxin. Hsp 70 and groEL, as well as two mycobacterial homologues hsp 71 and hsp 65, induce ion conducting pores across planar lipid bilayers at low or neutral pH. It is concluded that hsp induce pores in membranes and that this may contribute to their action within cells.     

Identification of a nucleo-cytoplasmic ionic pathway by osmotic shock in isolated mouse liver nuclei

Summary The observation that the nuclear envelope outer mem brane contains ion channels raises the question of whether these conductances communicate between the cytosol and the nuclear envelope cisternae or between the cytosol and the cytoplasm. Failure to detect large, nonselective holes using the patch-clamp technique has led to the speculation that ion channels and nuclear pores are in fact the same. In this paper we present evidence that the ionic channel, recorded in isolated liver nuclei with the patch-clamp configura tion of “nucleus-attached,” spans the double membrane of the envelope, providing a direct contact between nucleoplasm and cytoplasm.     

A densitometrical method for the study of pattern formation in a ciliateChilodonella

Summary An easy and sensitive method is reported here for testing the similarities of individual patterns by photographically transforming maps of these patterns to given, deductively chosen conventions involving constant distances between selected reference points. A cumulative map is produced by loading all landmarks from a set of individual maps on to one sheet of paper. The use of various a priori conventions results in variable cumulative maps, which are then optically transformed on an analog digital converter, with additional input for optical picture processing. The densitometrical maps thus obtained may be compared as to the cumulative degree of areas of maximal and minimal density of landmarks. The best conventions are those that yield the map with the most contrast.Maps of spatial patterns of the sites of contractile vacuole pore (CVP) primordia in an early stage of divisional morphogenesis of the ciliateChilodonella steini were compared after four different transformations and adjustments of the same set of individual maps. The best focusing of the sites of CVP differentiation was achieved by use of the postoral axis, defined by the center of the oral apparatus and the posterior end of the cell as the scaling parameter. The composite domain map obtained by optical transformation of this cumulative map could distinguish the specific CVP territories observed in earlier work (Kaczanowska 1981). These results confirm earlier findings that indicated the site of the oral apparatus is an important reference point in CVP primordia positioning. They also strongly suggest the existence of an overriding scaling factor governing the positioning of sites of differentiation in both dimensions of the developmental field. The method of superposition and scaling of pattern maps is generally applicable to situations in which pattern elements appear at discrete points on a flat surface.     

Freeze-etching studies on ultrastructural changes of endothelial cells in the thyroid of normal,TSH-treated and Thyradin-treated mice

Summary Freeze-etching images of the capillary endothelium in the thyroid of normal, TSH-treated and Thyradin (powdered thyroid)-treated mice were examined. Numerous pores represent vesicular stomata or fenestrations. The number of the pores and their population density are increased in TSH-treated mice, and decreased in Thyradin-treated animals. In addition, the width of the parajunctional zone and of the flat ray free from endothelial pores is smaller in TSH-treated mice and larger in Thyradin-treated animals. These facts indicate that the number of endothelial pores changes according to the functional activity of the gland.Supported by a grant from the Japanese Educational Ministry     

On the Application of Widom's Test Particle Method to Homogeneous and Inhomogeneous Fluids

Abstract

Chemical potentials of a homogeneous and an inhomogeneous Lennard-Jones fluid have been determined by molecular dynamics simulations on the vector computer CYBER 205 by applying essentially the fictitious test particle method of Widom. For the homogeneous fluid we find, contrary to the previous result of Guillot and Guissani, that the simulated chemical potential is independent of the particle number. The crucial point, however, is a sufficiently large cut-off radius in the evaluation of the Boltzmann factor. Comparing with our WCA-type perturbation theory, we get agreement in the chemical potentials within 0.1 kT up to the density n[sgrave]³ = 0.80 and a difference of 0.2 kT at n[sgrave]³ = 0.85. For the inhomogeneous case we consider a fluid in a cylindrical pore and integrate Widom's equation over a certain probe volume as suggested earlier by us. Chemical potentials are then calculated independently in five different probe volumes, which are cylindrical shells. The results agree well from the second to the fourth shell. Inaccuracies in the innermost cylinder can be easily explained by bad statistics. In the shell close to the wall the extremely high local density is responsible for the inaccuracies. Extending the probe volume over all cylindrical shells besides the one closest to the wall is thought to yield rather reliable results for the chemical potential. As a by-product of the simulations we also obtained diffusion coefficients, which are given in an appendix.     

Editorial

Abstract

Grand canonical molecular dynamics (GCMD) simulations are used to study the adsorption and desorption of Lennard-Jones nitrogen in three slit pore junction models of microporous graphite. These networks consist of two narrow pores separated by a wider (cavity) pore. We report results for cases where the narrow pore has a width of only two or three molecular diameters. Using the GCMD technique, a novel freezing transition is observed which results in pore blocking in the narrow pores of the network, which are less than 1 nm wide. This freezing results from the adsorption energy barrier at the junction between the narrow and wider pores. This type of pore blocking could account for the apparent increase in pore volume with increasing temperature that has been experimentally observed in microporous graphite systems. For networks in which the narrower pores are somewhat larger, with a width of 1.28 nm, this pore blocking effect is much reduced, and adsorbate molecules enter and fill the central cavity. In such cases, however, desorption is incomplete, some residual adsorbate remaining in the central cavity even at the lowest pressures.     

Depletion of nucleoporins from HeLa nuclear pore complexes to facilitate the production of ghost pores for in vitro reconstitution

During cell division, Nuclear Pore Complexes (NPCs) are broken down into protein subcomplexes that are the basis for reassembly in daughter cells. This is the driving force for the establishment of an in vitro reconstitution system to study aspects of NPC reassembly. In this study, nuclear envelope (NE) was isolated from HeLa cells. NE was treated with increasing concentrations of heparin to extract nucleoporins (Nups) for the production of “ghost pores” which are pores severely deficient in Nups, while still containing Pore Membrane proteins (POM) needed to anchor the NPC. Ghost pores have been subjected to incubation with previously stripped Nups and some re-binding has been shown to occur by western blot analysis. This in vitro assay provides a powerful tool to investigate the protein–protein interactions of NPC reassembly from a human cell line. Through a better understanding of the process of NPC reassembly, we can continue to piece together the puzzle of this macromolecular structure. It is most advantageous to establish a straightforward reconstitution procedure at the mammalian level.     

Comparison of three formal methods used to estimate the functional axis of rotation: an extensive in-vivo analysis performed on the knee joint

Estimating the main axis of rotation (AoR) of a human joint represents an important issue in biomechanics. This study compared three formal methods used to estimate functional AoR, namely a cylindrical fitting method, a mean helical axis transformation, and a symmetrical axis approach. These methods were tested on 106 subjects undergoing navigated total knee arthroplasty. AoR orientation in 3D and in the frontal and coronal planes provided by each method was compared to the transepicondylar axis direction. Although all the methods resulted effective, significant differences were identified among them, relatively to the orientation in 3D and in the frontal plane projection. This was probably due to the presence of secondary rotations during the first degrees of knee flexion.     

Differential effect of dopamine catabolism and uptake inhibition on dopamine-induced calcium dysregulation and viability loss

The present study was aimed at evaluating of the effects of dopamine (DA) toxicity on PC12 cells' calcium homeostasis, cellular viability, and free radical levels. Moreover, the effect of receptor inhibition, and DA metabolism and reuptake antagonism on all parameters was also evaluated. Acute treatment with DA impaired the ability of PC12 cells to buffer excess calcium after K+-depolarization, decreased cellular viability by approximately 35%, and increased free radical levels by about 10% in a dose dependent manner. Pretreatment with both active and inactive pargyl monoamine oxidase inhibitors (MAOi) protected PC12 cells from DA toxicity on cellular viability and free radical levels, regardless of the presence or absence of their target enzymes in PC12 cells. These results suggest a lack of specific involvement of DA metabolism by MAO in dopamine's effects on cellular viability and production of free radicals. However, DA-induced dysregulation of calcium homeostasis seems to be more specifically mediated by DA metabolism by MAO. Results indicate that, in order for toxicity to occur the DA must be taken up into the cells. DA receptors do not mediate dopamine cytoxicity, and the D2 receptor plays a modest role in DA-induced calcium dysregulation and generation of free radicals. Moreover, DA-induced cell viability loss is not mediated by calcium, nor by caspase-3 enzyme, but is prevented by inhibition of mitochondrial permeability transition pores.