Nuclear membrane cholesterol can modulate nuclear nucleoside triphosphatase activity

Previous work has suggested that changes in nuclear membrane cholesterol may induce a stimulation in nuclear nucleoside triphosphatase (NTPase) activity. The purpose of the present study was to directly investigate if nuclear membrane cholesterol can stimulate nuclear NTPase activity. The cholesterol content of nuclei was altered with a liposomal methodology. The cholesterol content of nuclei isolated from hepatic tissue was relatively low in comparison to that typically exhibited by other membrane fractions. Because of this, it was difficult to further deplete the nuclear membrane of cholesterol, but we could successfully increase the cholesterol content after exposure to cholesterol-enriched liposomes. Nuclear NTPase activity was potently stimulated (∼︁ 150–200% of control) by an increase in the nuclear membrane cholesterol content. The Vmax of the NTPase activity in the presence of ATP or GTP was significantly increased after cholesterol enrichment without altering the affinity of the enzyme for these moieties. Mg²⁺ dependency of NTPase activity was also altered by cholesterol incorporation into the nuclear membrane. Cholesterol enrichment of the nuclear membrane also left the nuclei more susceptible to damage by salt-induced lysis than control nuclei. Our results clearly demonstrate that the cholesterol content of the nuclear membrane will have significant, direct effects on nuclear integrity and NTPase activity. © 1996 Wiley-Liss, Inc.     

Differential range and activity of various forms of the Hedgehog protein

Background  

The Hedgehog (Hh) family of secreted proteins act as extra cellular messengers to control and coordinate growth and differentiation. The mechanism by which Hh protein travels across a field of cells, and results in a range of specific effects relating to the distance from the source, has been the subject of much debate. It has been suggested that the range and activity of the pathway can be linked to modifications of the Hh protein, specifically the addition of lipid groups at N- and C-terminal sites.     

Neutralization of CD95 ligand promotes regeneration and functional recovery after spinal cord injury

The clinical outcome of spinal cord injury (SCI) depends in part on the extent of secondary damage, to which apoptosis contributes. The CD95 and tumor necrosis factor (TNF) ligand/receptor systems play an essential role in various apoptotic mechanisms. To determine the involvement of these ligands in SCI-induced damage, we neutralized the activity of CD95 ligand (CD95L) and/or TNF in spinal cord-injured mice. Therapeutic neutralization of CD95L, but not of TNF, significantly decreased apoptotic cell death after SCI. Mice treated with CD95L-specific antibodies were capable of initiating active hind-limb movements several weeks after injury. The improvement in locomotor performance was mirrored by an increase in regenerating fibers and upregulation of growth-associated protein-43 (GAP-43). Thus, neutralization of CD95L promoted axonal regeneration and functional improvement in injured adult animals. This therapeutic strategy may constitute a potent future treatment for human spinal injury.     

Endosymbiont Dominated Bacterial Communities in a Dwarf Spider

The microbial community of spiders is little known, with previous studies focussing primarily on the medical importance of spiders as vectors of pathogenic bacteria and on the screening of known cytoplasmic endosymbiont bacteria. These screening studies have been performed by means of specific primers that only amplify a selective set of endosymbionts, hampering the detection of unreported species in spiders. In order to have a more complete overview of the bacterial species that can be present in spiders, we applied a combination of a cloning assay, DGGE profiling and high-throughput sequencing on multiple individuals of the dwarf spider Oedothorax gibbosus. This revealed a co-infection of at least three known (Wolbachia, Rickettsia and Cardinium) and the detection of a previously unreported endosymbiont bacterium (Rhabdochlamydia) in spiders. 16S rRNA gene sequences of Rhabdochlamydia matched closely with those of Candidatus R. porcellionis, which is currently only reported as a pathogen from a woodlouse and with Candidatus R. crassificans reported from a cockroach. Remarkably, this bacterium appears to present in very high proportions in one of the two populations only, with all investigated females being infected. We also recovered Acinetobacter in high abundance in one individual. In total, more than 99% of approximately 4.5M high-throughput sequencing reads were restricted to these five bacterial species. In contrast to previously reported screening studies of terrestrial arthropods, our results suggest that the bacterial communities in this spider species are dominated by, or even restricted to endosymbiont bacteria. Given the high prevalence of endosymbiont species in spiders, this bacterial community pattern could be widespread in the Araneae order.     

The Potential of the Ni-Resistant TCE-Degrading Pseudomonas putida W619-TCE to Reduce Phytotoxicity and Improve Phytoremediation Efficiency of Poplar Cuttings on A Ni-TCE Co-Contamination

To examine the potential of Pseudomonas putida W619-TCE to improve phytoremediation of Ni-TCE co-contamination, the effects of inoculation of a Ni-resistant, TCE-degrading root endophyte on Ni-TCE phytotoxicity, Ni uptake and trichloroethylene (TCE) degradation of Ni-TCE-exposed poplar cuttings are evaluated.

After inoculation with P. putida W619-TCE, root weight of non-exposed poplar cuttings significantly increased. Further, inoculation induced a mitigation of the Ni-TCE phytotoxicity, which was illustrated by a diminished exposure-induced increase in activity of antioxidative enzymes. Considering phytoremediation efficiency, inoculation with P. putida W619-TCE resulted in a 45% increased Ni uptake in roots as well as a slightly significant reduction in TCE concentration in leaves and TCE evapotranspiration to the atmosphere.

These results indicate that endophytes equipped with the appropriate characteristics can assist their host plant to deal with co-contamination of toxic metals and organic contaminants during phytoremediation. Furthermore, as poplar is an excellent plant for biomass production as well as for phytoremediation, the obtained results can be exploited to produce biomass for energy and industrial feedstock applications in a highly productive manner on contaminated land that is not suited for normal agriculture. Exploiting this land for biomass production could contribute to diminish the conflict between food and bioenergy production.     

Frequency-based haplotype reconstruction from deep sequencing data of bacterial populations

Clonal populations accumulate mutations over time, resulting in different haplotypes. Deep sequencing of such a population in principle provides information to reconstruct these haplotypes and the frequency at which the haplotypes occur. However, this reconstruction is technically not trivial, especially not in clonal systems with a relatively low mutation frequency. The low number of segregating sites in those systems adds ambiguity to the haplotype phasing and thus obviates the reconstruction of genome-wide haplotypes based on sequence overlap information.Therefore, we present EVORhA, a haplotype reconstruction method that complements phasing information in the non-empty read overlap with the frequency estimations of inferred local haplotypes. As was shown with simulated data, as soon as read lengths and/or mutation rates become restrictive for state-of-the-art methods, the use of this additional frequency information allows EVORhA to still reliably reconstruct genome-wide haplotypes. On real data, we show the applicability of the method in reconstructing the population composition of evolved bacterial populations and in decomposing mixed bacterial infections from clinical samples.     

Fitness trade‐offs explain low levels of persister cells in the opportunistic pathogen Pseudomonas aeruginosa

Microbial populations often contain a fraction of slow‐growing persister cells that withstand antibiotics and other stress factors. Current theoretical models predict that persistence levels should reflect a stable state in which the survival advantage of persisters under adverse conditions is balanced with the direct growth cost impaired under favourable growth conditions, caused by the nonreplication of persister cells. Based on this direct growth cost alone, however, it remains challenging to explain the observed low levels of persistence (<<1%) seen in the populations of many species. Here, we present data from the opportunistic human pathogen Pseudomonas aeruginosa that can explain this discrepancy by revealing various previously unknown costs of persistence. In particular, we show that in the absence of antibiotic stress, increased persistence is traded off against a lengthened lag phase as well as a reduced survival ability during stationary phase. We argue that these pleiotropic costs contribute to the very low proportions of persister cells observed among natural P. aeruginosa isolates (3 × 10^?8–3 × 10^?4) and that they can explain why strains with higher proportions of persister cells lose out very quickly in competition assays under favourable growth conditions, despite a negligible difference in maximal growth rate. We discuss how incorporating these trade‐offs could lead to models that can better explain the evolution of persistence in nature and facilitate the rational design of alternative therapeutic strategies for treating infectious diseases.