The influence of alkyl pyridinium sponge toxins on membrane properties, cytotoxicity, transfection and protein expression in mammalian cells

The ability of two alkyl pyridinium sponge toxin preparations (poly-APS and halitoxin) to form transient pores/lesions in cell membranes and allow transfection of plasmid cDNA have been investigated using HEK 293 cells. Poly-APS and halitoxin preparations caused a collapse in membrane potential, reductions in input resistance and increased Ca²⁺ permeability. At least partial recovery was observed after poly-APS application but recovery was more rarely seen with halitoxin. The transfection with plasmid cDNAs for an enhanced green fluorescent protein (EGFP) and human tumour necrosis factor receptor 2 (TNFR2) was assessed for both toxin preparations and compared with lipofectamine. Stable transfection was achieved with poly-APS although it was less efficient than lipofectamine. These results show that viable cells transfected with alien cDNA can be obtained using novel transient pore-forming alkyl pyridinium sponge toxins and a simple pre-incubation protocol. This provides the first proof of principle that pore-forming alkyl pyridinium compounds can be used to deliver cDNA to the intracellular environment without permanently compromising the plasma membrane.     

Cyclic GMP directly regulates a cation conductance in membranes of bovine rods by a cooperative mechanism

Cyclic GMP causes the release of endogenous Ca2+ from rod outer segments, whose plasma membrane has been made permeable, or from isolated discs. Approximately 11,000 Ca2+ ions are released per disc at saturating concentrations of cyclic GMP. The velocity and the amplitude of the release of Ca2+ are dependent on the concentration of cyclic GMP. The maximal rate of the Ca2+ efflux is approximately 7 X 10(4) Ca2+ ions s-1 rod-1. The Ca2+ release by cyclic GMP is independent of light. The activation of the efflux occurred within a narrow range of the cyclic GMP concentration (30-80 microM) and does not obey a simple Michaelis-Menten scheme. Instead, the kinetic analysis of the Ca2+ efflux suggests that a minimum number of 2 molecules of cyclic GMP activates the ion conductance in a cooperative fashion. The release of Ca2+ by cyclic GMP requires a gradient of Ca2+ ions across the disc membrane. If the endogenous Ca2+ gradient is dissipated by means of the ionophore A23187, the release of Ca2+ by cyclic GMP is abolished. Ca2+ is released by analogues of cyclic GMP which are either modified at the 8-carbon position of the imidazole ring or by the deaza-analogue of cyclic GMP. Congeners of cyclic GMP which are modified at the ribose, phosphodiester, or pyrimidine portion of the molecule are ineffective. The hydrolysis of cyclic GMP by the light-regulated phosphodiesterase of rod outer segments is not a necessary condition for the Ca2+ release because 8-bromo-cyclic GMP, a congener resistant to hydrolysis, is a more powerful activator of the release than cyclic GMP itself. Ca2+ release by cyclic GMP is inhibited by organic and inorganic blockers of Ca2+ channels. The l-stereoisomer of cis-diltiazem blocks the release of Ca2+ at micromolar concentrations, whereas the d-form is much less effective. These results suggest that disc membranes contain a cationic conductance which is permeable to Ca2+ ions and which is regulated through the cooperative binding of at least 2 molecules of cyclic GMP to regulatory sites of the transport protein. By this mechanism, subtle changes in the concentration of cyclic GMP could promote large changes in the flux of Ca2+ ions across the disc membrane.     

Elevated CO2 Levels do not Affect the Shell Structure of the Bivalve Arctica islandica from the Western Baltic

Shells of the bivalve Arctica islandica are used to reconstruct paleo-environmental conditions (e.g. temperature) via biogeochemical proxies, i.e. biogenic components that are related closely to environmental parameters at the time of shell formation. Several studies have shown that proxies like element and isotope-ratios can be affected by shell growth and microstructure. Thus it is essential to evaluate the impact of changing environmental parameters such as high pCO₂ and consequent changes in carbonate chemistry on shell properties to validate these biogeochemical proxies for a wider range of environmental conditions. Growth experiments with Arctica islandica from the Western Baltic Sea kept under different pCO₂ levels (from 380 to 1120 µatm) indicate no affect of elevated pCO₂ on shell growth or crystal microstructure, indicating that A. islandica shows an adaptation to a wider range of pCO₂ levels than reported for other species. Accordingly, proxy information derived from A. islandica shells of this region contains no pCO₂ related bias.     

Pulsed-field gel electrophoresis for genotypic comparison of Rhizobium bacteria that nodulate leguminous trees

Abstract Pulsed-field gel electrophoresis (PFGE) was applied to characterize Rhizobium bacteria isolated from the root nodules of Acacia senegal and Prosopis chilensis trees growing in Sudan and Keya. For the electrophoresis, the total DNA of 42 isolates, embedded in agarose, was digested by a rare-cutting restriction endonuclease, Xba I. The PFGE run resulted in good resolution of the DNA fragments and gave the strains distinctive fingerprint patterns. The patterns were analysed visually and using automated clustering analysis, which divided the strains into groups resembling the results generated by numerical taxonomy. However, several strains had unique banding patterns, which indicates that these strains are genetically very diverse.     

Über die proteolytische Aktivität von Aspergillus niger und ihre Beeinflussung durch Spurenelemente

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