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.     

Resprouting in the Mediterranean‐type shrub Erica australis afffected by soil resource availability

Abstract. Soil resource availability may affect plant regeneration by resprouting in disturbed environments directly, by affecting plant growth rates, or indirectly by determining allocation to storage in the resprouting organs. Allocation to storage may be higher in stressful, low resource‐supply soils, but under such conditions plant growth rates may be lower. These factors could act in opposite directions leading to poorly known effects on resprouting. This paper analyses the role played by soil resources in the production and growth of resprouts after removal of above‐ground plant tissues in the Mediterranean shrub Erica australis. At 13 sites, differing in substrate, we cut the base of the stems of six plants of E. australis and allowed them to resprout and grow for two years. Soils were chemically analysed and plant water potential measured during the summer at all sites to characterize soil resource availability. We used stepwise regression analysis to determine the relationships between the resprouting response [mean site values of the number of resprouts (RN), maximum length (RML) and biomass (RB)] and soil nutrient content and plant water potential at each site. During the first two years of resprouting there were statistically significant differences among sites in the variables characterizing the resprouting response. RML was always different among sites and had little relationship with lignotuber area. RN was less different among sites and was always positively correlated with lignotuber area. RB was different among sites after the two years of growth. During the first months of resprouting, RN and RML were highly and positively related to the water status of the plant during summer. At later dates soil fertility variables came into play, explaining significant amounts of variance of the resprouting variables. Soil extractable cations content was the main variable accounting for RML and RB. Our results indicate that resprout growth of E. australis is positively affected by high water availability at the beginning of the resprouting response and negatively so by high soil extractable cation content at later periods. Some of these factors had previously shown to be related, with an opposite sign, to the development of a relatively larger lignotuber. Indeed, RML and RB measured in the second year of resprouting were significantly and negatively correlated with some indices of biomass allocation to the lignotuber at each site. This indicates that sites favouring allocation to the resprouting organ may not favour resprout growth.     

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.     

Selenoprotein P associates with endothelial cells in rat tissues

 Selenoprotein P is an extracellular heparin-binding protein that has been implicated in protecting the liver against oxidant injury. Its location in liver, kidney, and brain was determined by conventional immunohistochemistry and confocal microscopy using a polyclonal antiserum. Selenoprotein P is associated with endothelial cells in the liver and is more abundant in central regions than in portal regions. It is also present in kidney glomeruli associated with capillary endothelial cells. Staining of selenoprotein P in the brain is also confined to vascular endothelial cells. The heparin-binding properties of selenoprotein P could be the basis for its binding to tissue. Its localization to the vicinity of endothelial cells is potentially relevant to its oxidant defense function. Accepted: 6 March 1997     

Cholinesterase Levels in Blood Plasma and Erythrocytes from Calves,Normal Delivering Cows and Cows Suffering from Parturient Paresis

Plasma cholinesterase (pChE) levels and erythrocyte acetylcholinesterase (eAChE) levels were studied in 6 cows before, during and after parturition (Group I), their calves (Group II), 38 cows suffering from parturient paresis (Group III) and 14 newly delivered non-paretic cows (Group IV). The mean of the pChE level in Group I was 1.5 μkat/1 ± 0.20 before parturition and decreased significantly (P ≦ 0.05) to 1.2 ukat/1 ± 0.16 after parturition. The eAChE level was before parturition ≅ 140 ukat/1 and decreased to ≅ 130 μkat/1 4–5 weeks after parturition. At birth the pChE level was 12.8 ukat/1 ± 5.9 in Group II. After 4 weeks the level had decreased to 2.3 ukat/1 ±0.3. In the bull calves the pChE level started to increase when they were 6 weeks old and reached a level of 5.7 μkat/1 ± 0.6 before slaughter at 6 months of age. The heifers did not show this increase. They had a level of around 2 μkat/1 throughout the investigation. The eAChE level at birth was 119 μkat/1 and increased slowly to a level of 145 μkat/1 at 6 months. No differences between the sexes were found. The cows suffering from parturient paresis had a pChE level of 1.80 μkat/1 ± 0.30 before treatment with calcium (Ca). The level decreased significantly (P ≦ 0.001) after Ca-infusion to a level of 1.67 ukat/1 ±0.29. Group IV had a pChE level of 1.65 μkat/1 ± 0.42 at parturition. Two to 4 months later the cows that had recovered from milk fever had a level of 1.61 μkat/1 ± 0.31 and the control cows 1.66 ukat/1 ± 0.48. No differences between the groups were found for the eAChE level. The findings show that parturition influences the pChE level in cows and that sex influences the pChE level in calves between 6 weeks to at least 6 months of age. Furthermore the elevated pChE level found in the cows suffering from parturient paresis before Ca infusion may be a further sign of a disturbance in the cholinergic system with a special preference to the neuromuscular junctions.     

A small molecule that disrupts S. Typhimurium membrane voltage without cell lysis reduces bacterial colonization of mice

As pathogenic bacteria become increasingly resistant to antibiotics, antimicrobials with mechanisms of action distinct from current clinical antibiotics are needed. Gram-negative bacteria pose a particular problem because they defend themselves against chemicals with a minimally permeable outer membrane and with efflux pumps. During infection, innate immune defense molecules increase bacterial vulnerability to chemicals by permeabilizing the outer membrane and occupying efflux pumps. Therefore, screens for compounds that reduce bacterial colonization of mammalian cells have the potential to reveal unexplored therapeutic avenues. Here we describe a new small molecule, D66, that prevents the survival of a human Gram-negative pathogen in macrophages. D66 inhibits bacterial growth under conditions wherein the bacterial outer membrane or efflux pumps are compromised, but not in standard microbiological media. The compound disrupts voltage across the bacterial inner membrane at concentrations that do not permeabilize the inner membrane or lyse cells. Selection for bacterial clones resistant to D66 activity suggested that outer membrane integrity and efflux are the two major bacterial defense mechanisms against this compound. Treatment of mammalian cells with D66 does not permeabilize the mammalian cell membrane but does cause stress, as revealed by hyperpolarization of mitochondrial membranes. Nevertheless, the compound is tolerated in mice and reduces bacterial tissue load. These data suggest that the inner membrane could be a viable target for anti-Gram-negative antimicrobials, and that disruption of bacterial membrane voltage without lysis is sufficient to enable clearance from the host.     

Biological soil crusts in ecological restoration: emerging research and perspectives

Drylands encompass over 40% of terrestrial ecosystems and face significant anthropogenic degradation causing a loss of ecosystem integrity, services, and deterioration of social‐ecological systems. To combat this degradation, some dryland restoration efforts have focused on the use of biological soil crusts (biocrusts): complex communities of cyanobacteria, algae, lichens, bryophytes, and other organisms living in association with the top millimeters of soil. Biocrusts are common in many ecosystems and especially drylands. They perform a suite of ecosystem functions: stabilizing soil surfaces to prevent erosion, contributing carbon through photosynthesis, fixing nitrogen, and mediating the hydrological cycle in drylands. Biocrusts have emerged as a potential tool in restoration; developing methods to implement effective biocrust restoration has the potential to return many ecosystem functions and services. Although culture‐based approaches have allowed researchers to learn about the biology, physiology, and cultivation of biocrusts, transferring this knowledge to field implementation has been more challenging. A large amount of research has amassed to improve our understanding of biocrust restoration, leaving us at an opportune time to learn from one another and to join approaches for maximum efficacy. The articles in this special issue improve the state of our current knowledge in biocrust restoration, highlighting efforts to effectively restore biocrusts through a variety of different ecosystems, across scales and utilizing a variety of lab and field methods. This collective work provides a useful resource for the scientific community as well as land managers.