Computer simulation of uranyl uptake by the rough lipopolysaccharide membrane of Pseudomonas aeruginosa

Heavy metal environmental contaminants cannot be destroyed but require containment, preferably in concentrated form, in a solid or immobile form for recycling or final disposal. Microorganisms are able to take up and deposit high levels of contaminant metals, including radioactive metals such as uranium and plutonium, into their cell wall. Consequently, these microbial systems are of great interest as the basis for potential environmental bioremediation technologies. The outer membranes of Gram-negative microbes are highly nonsymmetric and exhibit a significant electrostatic potential gradient across the membrane. This gradient has a significant effect on the uptake and transport of charged and dipolar compounds. However, the effectiveness of microbial systems for environmental remediation will depend strongly on specific properties that determine the uptake of targeted contaminants by a particular cell wall. To aid in the design of microbial remediation technologies, knowledge of the factors that determine the affinity of a particular bacterial outer membrane for the most common ionic species found in contaminated soils and groundwater is of great importance. Using our previously developed model for the lipopolysaccharide (LPS) membrane of Pseudomonas aeruginosa, this work presents the potentials of mean force as the estimate of the free energy profile for uptake of sodium, calcium, chloride, uranyl ions, and a water molecule by the bacterial LPS membrane. A compatible classical parameter set for uranyl has been developed and validated. Results show that the uptake of uranyl is energetically a favorable process relative to the other ions studied. At neutral pH, this nuclide is shown to be retained on the surface of the LPS membrane through chelation with the carboxyl and hydroxyl groups located in the outer core.     

Relationship between T-wave amplitude and oxygen pulse in guinea pigs in hyperbaric helium and hydrogen

Diving isknown to induce a change in the amplitude of the T wave(A_Tw) ofelectrocardiograms, but it is unknown whether this is linked to achange in cardiovascular performance. We analyzed A_Tw in guinea pigs at 10-60atm and 25-36°C, breathing 2%O₂ in either helium (heliox;n = 10) or hydrogen (hydrox;n = 9) for 1 h at each pressure. Coretemperature and electrocardiograms were detected by using implantedradiotelemeters. O₂ consumption rate was measured by using gas chromatography. In a previous study (S. R. Kayar and E. C. Parker. J. Appl.Physiol. 82: 988-997, 1997), we analyzed theO₂ pulse, i.e., theO₂ consumption rate per heartbeat, in the same animals. By multivariate regression analysis, weidentified variables that were significant toO₂ pulse: body surface area,chamber temperature, core temperature, and pressure. In this study,inclusion of A_Tw made asignificantly better model with fewer variables. After normalizing forchamber temperature and pressure, theO₂ pulse increased with increasing A_Tw in heliox(P = 0.001) but with decreasingA_Tw in hydrox(P < 0.001). ThusA_Tw is associated with thedifferences in O₂ pulse foranimals breathing heliox vs. hydrox.

     

VEGFR2 and Src kinase inhibitors suppress Andes virus-induced endothelial cell permeability

Hantaviruses predominantly infect human endothelial cells and, in the absence of cell lysis, cause two diseases resulting from increased vascular permeability. Andes virus (ANDV) causes a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). ANDV infection enhances the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF) by increasing signaling responses directed by the VEGFR2-Src-VE-cadherin pathway, which directs adherens junction (AJ) disassembly. Here we demonstrate that inhibiting pathway-specific VEGFR2 and Src family kinases (SFKs) blocks ANDV-induced endothelial cell permeability. Small interfering RNA (siRNA) knockdown of Src within ANDV-infected endothelial cells resulted in an ～70% decrease in endothelial cell permeability compared to that for siRNA controls. This finding suggested that existing FDA-approved small-molecule kinase inhibitors might similarly block ANDV-induced permeability. The VEGFR2 kinase inhibitor pazopanib as well as SFK inhibitors dasatinib, PP1, bosutinib, and Src inhibitor 1 dramatically inhibited ANDV-induced endothelial cell permeability. Consistent with their kinase-inhibitory concentrations, dasatinib, PP1, and pazopanib inhibited ANDV-induced permeability at 1, 10, and 100 nanomolar 50% inhibitory concentrations (IC(50)s), respectively. We further demonstrated that dasatinib and pazopanib blocked VE-cadherin dissociation from the AJs of ANDV-infected endothelial cells by >90%. These findings indicate that VEGFR2 and Src kinases are potential targets for therapeutically reducing ANDV-induced endothelial cell permeability and, as a result, capillary permeability during HPS. Since the functions of VEGFR2 and SFK inhibitors are already well defined and FDA approved for clinical use, these findings rationalize their therapeutic evaluation for efficacy in reducing HPS disease. Endothelial cell barrier functions are disrupted by a number of viruses that cause hemorrhagic, edematous, or neurologic disease, and as a result, our findings suggest that VEGFR2 and SFK inhibitors should be considered for regulating endothelial cell barrier functions altered by additional viral pathogens.     

Alpine species seed longevity in the soil in relation to seed size and shape – A 5-year burial experiment in the Central Alps

Long-term persistent seeds are generally expected to be small and spherically shaped. In contrast, transient seeds are often large, flattened and elongated. We hypothesized that alpine species follow these trends, and that most species of unstable alpine habitats have the potential to form long-term persistent seed banks. The latter would enable them to delay germination until favourable conditions are present in their environment thereby increasing the likelihood for establishment of seedlings. We selected nine alpine species of more or less unstable habitats (i.e. Achillea moschata, Artemisia genipi, Anthyllis vulneraria ssp. alpicola, Geum reptans, Linaria alpina, Oxyria digyna, Saxifraga aizoides, Saxifraga oppositifolia, and Trifolium pallescens) for this study. Seed longevity was estimated by a 5-year burial experiment in the field with seed excavations after 1 winter, 2 years and 5 years. Germination experiments in the growth chamber and viability tests were performed after each excavation. We detected longevity patterns ranging from transient to long-term persistent. Two species were classified as transient. All other species have the potential to form persistent seed banks, with almost half of the investigated species showing a trend for long-term persistence. Despite contradictory reports, long-term persistent seed banks are an important life history trait of species of unstable alpine habitats. We found that seed shape and hard-seededness are good predictors of seed persistence of alpine species. Seed size seems to be less important. The data from this study support the general hypotheses describing the relationship among seed size, seed shape and seed persistence in the soil.     

Blood chemistry in relation to nutrition and ectoparasite load in Wilson’s storm-petrels<Emphasis Type="Italic"> Oceanites oceanicus</Emphasis>

Haematology and plasma biochemistry values are useful tools for ecological research, providing information on the physiological state and adaptation of individuals to their habitat, changes in nutritional state of birds, body condition, the level of parasite infestation, etc. We studied the effect of stress factors on haematological and plasma biochemistry values in adult and nestling Wilsons storm petrels Oceanites oceanicus (Aves, Procellariiformes). We measured packed cell volume, triglyceride levels, plasma protein levels, plasma hue and plasma corticosterone of nestlings and breeding adults at King George Island, South Shetland Islands. We used a snow storm as a natural experiment to test what effect starvation has on the stress response of nestlings. In particular, we predicted that: (1) plasma protein concentrations and plasma hue reflect ectoparasite load, (2) triglycerides and hue reflect the nutritional state, and (3) corticosterone levels increase with ectoparasite load and starvation. In line with our predictions, plasma triglycerides were higher in nestlings than adults, indicating a resorptive nutritional state in nestlings, during which dietary fat is deposited in adipose tissues. In adults, plasma triglycerides were positively correlated with body mass. Corticosterone levels increased in response to handling in adults, while we did not find a stress response in nestlings in good condition. However, nestlings sampled after their nests had been blocked by a snow storm showed a stress response in excess of that of adults. In nestlings sampled after the snow storm, corticosterone peak levels were positively correlated with the infestation intensity of the ectoparasite Philoceanus robertsi (Phthiraptera: Ischnocera), suggesting that the stress response is increased when more stressors act at the same time. In adults, plasma hue and proteins decreased with increasing ectoparasite load.     

Experimentally elevated testosterone increases status signalling in male Greylag geese (<Emphasis Type="Italic">Anser anser</Emphasis>)

Testosterone modulates male vertebrates sexual and social behaviour. We experimentally investigated the testosterone-sensitive behaviours in male greylag geese (Anser anser) by implanting silastic tubes containing crystalline testosterone during the mating season (February; 5 implanted and 5 control males) and in the early winter (November; 7 and 7). Focal animals were part of a semi-tame, unrestrained flock with fully intact social relationships. Excreted testosterone and corticosterone immunoreactive metabolites (TM, BM) were determined by enzyme immunoassay. Individual faecal samples and behavioural protocols were collected daily over a period of 5 weeks, including 1 control week before implantation. In February, no significant behavioural effects of the supplemental testosterone were observed, which may be due to the naturally occurring high systemic androgen levels in spring. In November, however, implanted males had higher TM excretion rates and performed status signalling behaviour (beak up) more frequently than control males. No differences between implanted and control males were found with respect to BM, agonistic interactions or vigilance behaviour. Furthermore, during the second week after implantation, TM positively correlated with the frequency of beak up of implanted males, whilst their female partners were attacked with lower latency by other members of the flock than the females of control males. Hence, status signalling in greylag ganders seems to be testosterone-sensitive year-long and inappropriate status signalling of males may draw attacks towards their females.     

Cell volume and the regulation of apoptotic cell death

Apoptosis is a physiological mechanism allowing for the removal of abundant or potentially harmful cells. The hallmarks of apoptosis include degradation of cellular DNA, exposure of phosphatidylserine at the outer leaflet of the cell membrane and cell shrinkage. Phosphatidylserine exposure favours adhesion to macrophages with subsequent phagocytosis of the shrunken apoptotic particles. The interaction of cell volume regulatory mechanisms and apoptosis is illustrated in two different model systems, i.e. (a) lymphocyte apoptosis following stimulation of CD95 receptor and (b) erythrocyte apoptosis upon cell shrinkage. (a) Triggering of CD95 in Jurkat T lymphocytes is paralleled by activation of cell volume regulatory Cl- channels, inhibition of the Na+/H+ exchanger and osmolyte release. The latter coincides with cell shrinkage, DNA fragmentation and phosphatidylserine exposure. CD95 stimulation leads to early inhibition of the voltage gated K+ channel Kv1.3, which may contribute to the inhibition of the Ca2+ release activated Ca2+ channel I(CRAC). (b) Osmotic shock of erythrocytes activates a cell volume regulatory cation conductance allowing the entry not only of Na+ but of Ca2+ as well. Increased cytosolic Ca2+ stimulates a scramblase which disrupts the phosphatidylserine asymmetry of the cell membrane, leading to phosphatidylserine exposure. The cation conductance is further activated by oxidative stress and energy depletion and inhibited by Cl-. Shrinkage of erythrocytes stimulates in addition a sphingomyelinase with subsequent formation of ceramide which potentiates the effect of cytosolic Ca2+ on phosphatidylserine. In conclusion, cell volume-sensitive mechanisms participate in the triggering of apoptosis following receptor stimulation or cell injury.     

Discovery of novel aspartyl ketone dipeptides as potent and selective caspase-3 inhibitors

The discovery of a series of potent, selective and reversible dipeptidyl caspase-3 inhibitors are reported. The iterative discovery process of using combinatorial chemistry, parallel synthesis, moleculare modelling and structural biology will be discussed.