Pharmacological postconditioning by bolus injection of phosphodiesterase-5 inhibitors vardenafil and sildenafil

Postconditioning enables cardioprotection against ischemia/reperfusion injury either by application of short, repetitive ischemic periods or by pharmacological intervention prior to reperfusion. Pharmacological postconditioning has been described for phosphodiesterase-5 inhibitors when the substances were applied as a permanent infusion. For clinical purposes, application of a bolus is more convenient. In a rat heart in situ model of ischemia reperfusion vardenafil or sildenafil were applied as a bolus prior to reperfusion. Cardioprotective effects were found over a broad dosage range. In accordance with current hypotheses on pharmacological postconditioning signaling, the protective effect was mediated by extracellular signal-regulated kinase and protein kinase C pathway. Interestingly, the extent of protection was independent of the concentration applied for both substances. Full protection comparable to ischemic postconditioning was reached with half-maximal human equivalence dose. In contrast, mean arterial pressure dropped upon bolus application in a dose-dependent manner. Taken together, the current study extends previous findings obtained in a permanent infusion model to bolus application. This is an important step toward clinical application of pharmacological postconditioning with sildenafil and vardenafil, especially because the beneficial effects were proven for concentrations with reduced hemodynamic side effects compared to the dosage applied for erectile dysfunction treatment.     

Acidic Cave-Wall Biofilms Located in the Frasassi Gorge,Italy

Acidic biofilms present on cave walls in the sulfidic region of the Frasassi Gorge, Italy, were investigated to determine their microbial composition and their potential role in cave formation and ecosystem functioning. All biofilm samples examined had pH values < 1.0. Scanning electron microscopy of the biofilms revealed the presence of various filaments and rods associated in large clusters with mineral crystals. Qualitative energy-dispersive x-ray analysis was used to determine that the crystals present on the cave walls, associated with the microbial biofilm, were composed of calcium and barium sulfate. Ribosomal RNA-based methods to determine the microbial composition of these biofilms revealed the presence of at least two strains of potential acidophilic, sulfur-oxidizing bacteria, belonging to the genera Thiobacillus and Sulfobacillus. An acid-producing strain of Thiobacillus sp. also was obtained in pure culture. Stable isotope ratio analysis of carbon and nitrogen showed that the wall biofilms are isotopically light, suggesting that in situ chemoautotrophic activity plays an important role in this subsurface ecosystem.     

Pandemic 2009 H1N1 Influenza A Virus Carrying a Q136K Mutation in the Neuraminidase Gene Is Resistant to Zanamivir but Exhibits Reduced Fitness in the Guinea Pig Transmission Model

Resistance of influenza A viruses to neuraminidase inhibitors can arise through mutations in the neuraminidase (NA) gene. We show here that a Q136K mutation in the NA of the 2009 pandemic H1N1 virus confers a high degree of resistance to zanamivir. Resistance is accompanied by reduced numbers of NA molecules in viral particles and reduced intrinsic enzymatic activity of mutant NA. Interestingly, the Q136K mutation strongly impairs viral fitness in the guinea pig transmission model.     

Bird attacks on a butterfly with marginal eyespots and the role of prey concealment against the background

Small eyespots on butterflies have long been thought to deflect attacks, and birds are the presumptive drivers selecting for these patterns; however, evidence of this function is still ambiguous. Marginal eyespots typically consist of a UV‐reflective white pupil, surrounded by one black and one yellowish ring. We have recently shown that Cyanistes caeruleus (blue tits) attack such eyespots, but only under low light intensities with accentuated UV levels: the increased salience of the eyespots relative to the rest of the butterfly probably explains this result. Possibly the background against which the butterfly is concealed may deceive birds to make similar errors. We therefore presented speckled wood butterflies decorated with eyespots (or controls without eyespots) to C. caeruleus against two backgrounds: oak and birch bark. Our results show that: (1) eyespots, independent of background, were effective in deflecting attacks; (2) the time elapsed between a bird landing and the attack was interactively dependent on the background and whether the butterfly bore an eyespot; and (3) the speed at which a butterfly was attacked predicted the outcome, with faster birds being more prone to errors than slower birds. This underscores a speed–accuracy trade‐off in the predators, and that background plays a role in the defensive qualities of marginal eyespots. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 290–297.     

Aberrant spindle dynamics and cytokinesis in Dictyostelium discoideum cells that lack glycogen synthase kinase 3

Eukaryotic cell division requires the co-ordinated assembly and disassembly of the mitotic spindle, accurate chromosome segregation and temporal control of cytokinesis to generate two daughter cells. While the absolute details of these processes differ between organisms, there are evolutionarily conserved core components common to all eukaryotic cells, whose identification will reveal the key processes that control cell division. Glycogen synthase kinase 3 (GSK-3) is a major protein kinase found throughout the eukaryotes and regulates many processes, including cell differentiation, growth, motility and apoptosis. In animals, GSK-3 associates with mitotic spindles and its inhibition causes mis-regulation of chromosome segregation. Two suppressor screens in yeast point to a more general effect of GSK-3 on cell division, however the direct role of GSK-3 in control of mitosis has not been explored outside the animal kingdom. Here we report that the Dictyostelium discoideum GSK-3 orthologue, GskA, associates with the mitotic spindle during cell division, as seen for its mammalian counterparts. Dictyostelium possesses only a single GSK-3 gene that can be deleted to eliminate all GSK-3 activity. We found that gskA-null mutants failed to elongate their mitotic spindle and were unable to divide in shaking culture, but have no chromosome segregation defect. These results suggest further conservation for the role of GSK-3 in the regulation of spindle dynamics during mitosis, but also reveal differences in the mechanisms ensuring accurate chromosome segregation.     

Sulfur mustard induced nuclear translocation of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)

Sulfur Mustard (SM) is a vesicant chemical warfare agent, which is acutely toxic to a variety of organ systems including skin, eyes, respiratory system and bone marrow. The underlying molecular pathomechanism was mainly attributed to the alkylating properties of SM. However, recent studies have revealed that cellular responses to SM exposure are of more complex nature and include increased protein expression and protein modifications that can be used as biomarkers. In order to confirm already known biomarkers, to detect potential new ones and to further elucidate the pathomechanism of SM, we conducted large-scale proteomic experiments based on a human keratinocyte cell line (HaCaT) exposed to SM. Surprisingly, our analysis identified glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as one of the up-regulated proteins after exposure of HaCaT cells to SM. In this paper we demonstrate the sulfur mustard induced nuclear translocation of GAPDH in HaCaT cells by 2D gel-electrophoresis (2D GE), immunocytochemistry (ICC), Western Blot (WB) and a combination thereof. 2D GE in combination with MALDI-TOF MS/MS analysis identified GAPDH as an up-regulated protein after SM exposure. Immunocytochemistry revealed a distinct nuclear translocation of GAPDH after exposure to 300 μM SM. This finding was confirmed by fractionated WB analysis. 2D GE and subsequent immunoblot staining of GAPDH demonstrated two different spot locations of GAPH (pI 7.0 and pI 8.5) that are related to cytosolic or nuclear GAPDH respectively. After exposure to 300 μM SM a significant increase of nuclear GAPDH at pI 8.5 occurred. Nuclear GAPDH has been associated with apoptosis, detection of structural DNA alterations, DNA repair and regulation of genomic integrity and telomere structure. The results of our study add new aspects to the pathophysiology of sulfur mustard toxicity, yet further studies will be necessary to reveal the specific function of nuclear GAPDH in the pathomechanism of sulfur mustard.     

The Shaft of the Type 1 Fimbriae Regulates an External Force to Match the FimH Catch Bond

Type 1 fimbriae mediate adhesion of uropathogenic Escherichia coli to host cells. It has been hypothesized that due to their ability to uncoil under exposure to force, fimbriae can reduce fluid shear stress on the adhesin-receptor interaction by which the bacterium adheres to the surface. In this work, we develop a model that describes how the force on the adhesin-receptor interaction of a type 1 fimbria varies as a bacterium is affected by a time-dependent fluid flow mimicking in vivo conditions. The model combines in vivo hydrodynamic conditions with previously assessed biomechanical properties of the fimbriae. Numerical methods are used to solve for the motion and adhesion force under the presence of time-dependent fluid profiles. It is found that a bacterium tethered with a type 1 pilus will experience significantly reduced shear stress for moderate to high flow velocities and that the maximum stress the adhesin will experience is limited to ∼120 pN, which is sufficient to activate the conformational change of the FimH adhesin into its stronger state but also lower than the force required for breaking it under rapid loading. Our model thus supports the assumption that the type 1 fimbria shaft and the FimH adhesin-receptor interaction are optimized to each other, and that they give piliated bacteria significant advantages in rapidly changing fluidic environments.