Goodbye or welcome Gondwana? – insights into the phylogenetic biogeography of the leafy liverwort Plagiochila with a description of Proskauera, gen. nov. (Plagiochilaceae, Jungermanniales)

Molecular phylogenies based on chloroplast gene rps4 sequences and nuclear ribosomal ITS sequences have been generated to investigate relationships among species and putative segregates in Plagiochila (Plagiochilaceae), the largest genus of leafy liverworts. About a fourth of the ca. 450 accepted binomials of Plagiochilaceae are included in these phylogenetic analyses, several represented by multiple accessions. A clade with Chiastocaulon, Pedinophyllum, and Plagiochilion is placed sister to a clade with numerous accessions of Plagiochila. Plagiochila pleurata and P. fruticella are resolved sister to the remainder of Plagiochilaceae and transferred to the new Australasian genus Proskauera which differs from all other Plagiochilaceae by the occurrence of spherical leaf papillae. The historical biogeography of Plagiochilaceae is explored based on the reconstructions of the phylogeny, biogeographic patterns and diversification time estimates. The results indicate that the current distribution of Plagiochilaceae cannot be explained exclusively by Gondwanan vicariance. A more feasible explanation of the range is a combination of short distance dispersal, rare long distance dispersal events, extinction, recolonization and diversification.     

Conformational change of bacteriorhodopsin quantitatively monitored by microcantilever sensors

Bacteriorhodopsin proteoliposomes were used as a model system to explore the applicability of micromechanical cantilever arrays to detect conformational changes in membrane protein patches. The three main results of our study concern: 1), reliable functionalization of micromechanical cantilever arrays with proteoliposomes using ink jet spotting; 2), successful detection of the prosthetic retinal removal (bleaching) from the bacteriorhodopsin protein by measuring the induced nanomechanical surface stress change; and 3), the quantitative response thereof, which depends linearly on the amount of removed retinal. Our results show this technique to be a potential tool to measure membrane protein-based receptor-ligand interactions and conformational changes.     

Singlet oxygen generation by UVA light exposure of endogenous photosensitizers

UVA light (320-400 nm) has been shown to produce deleterious biological effects in tissue due to the generation of singlet oxygen by substances like flavins or urocanic acid. Riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), beta-nicotinamide adenine dinucleotide (NAD), and beta-nicotinamide adenine dinucleotide phosphate (NADP), urocanic acid, or cholesterol in solution were excited at 355 nm. Singlet oxygen was directly detected by time-resolved measurement of its luminescence at 1270 nm. NAD, NADP, and cholesterol showed no luminescence signal possibly due to the very low absorption coefficient at 355 nm. Singlet oxygen luminescence of urocanic acid was clearly detected but the signal was too weak to quantify a quantum yield. The quantum yield of singlet oxygen was precisely determined for riboflavin (PhiDelta = 0.54 +/- 0.07), FMN (PhiDelta = 0.51 +/- 0.07), and FAD (PhiDelta = 0.07 +/- 0.02). In aerated solution, riboflavin and FMN generate more singlet oxygen than exogenous photosensitizers such as Photofrin, which are applied in photodynamic therapy to kill cancer cells. With decreasing oxygen concentration, the quantum yield of singlet oxygen generation decreased, which must be considered when assessing the role of singlet oxygen at low oxygen concentrations (inside tissue).     

Allosteric activation of the protein kinase PDK1 with low molecular weight compounds

Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF-pocket and have the ability to allosterically activate phosphoinositide-dependent protein kinase 1 (PDK1) by modulating the phosphorylation-dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogs, interaction-displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode of action and may inspire future rational development of compounds with the ability to modulate phosphorylation-dependent conformational transitions in other proteins.     

Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin

Dodecamerization and insertion of the outer membrane secretin PulD is entirely determined by the C-terminal half of the polypeptide (PulD-CS). In the absence of its cognate chaperone PulS, PulD-CS and PulD mislocalize to the inner membrane, from which they are extractable with detergents but not urea. Electron microscopy of PulD-CS purified from the inner membrane revealed apparently normal dodecameric complexes. Electron microscopy of PulD-CS and PulD in inner membrane vesicles revealed inserted secretin complexes. Mislocalization of PulD or PulD-CS to this membrane induces the phage shock response, probably as a result of a decreased membrane electrochemical potential. Production of PulD in the absence of the phage shock response protein PspA and PulS caused a substantial drop in membrane potential and was lethal. Thus, PulD-CS and PulD assemble in the inner membrane if they do not associate with PulS. We propose that PulS prevents premature multimerization of PulD and accompanies it through the periplasm to the outer membrane. PulD is the first bacterial outer membrane protein with demonstrated ability to insert efficiently into the inner membrane.     

Lack of p53 function promotes radiation-induced mitotic catastrophe in mouse embryonic fibroblast cells

Background  

We have demonstrated that in some human cancer cells both chronic mild heat and ionizing radiation exposures induce a transient block in S and G2 phases of the cell cycle. During this delay, cyclin B1 protein accumulates to supranormal levels, cyclin B1-dependent kinase is activated, and abrogation of the G2/M checkpoint control occurs resulting in mitotic catastrophe (MC).     

Water and heat balance during flight in the rose-colored starling (Sturnus roseus)

Water imbalance during flight is considered to be a potentially limiting factor for flight ranges in migrating birds, but empirical data are scarce. We studied flights under controlled ambient conditions with rose-colored starlings in a wind tunnel. In one experiment, we measured water fluxes with stable isotopes at a range of flight speeds (9-14 m s(-1)) at constant temperature (15 degrees C). In a second experiment, we measured evaporation rates at variable ambient temperatures (Ta = 5 deg -27 deg C) but constant speed (12 m s(-1)). During all flights, the birds experienced a net water loss. On average, water influx was 0.98 g h(-1) (SD = 0.16; n = 8), and water efflux was 1.29 g h(-1) (SD = 0.14; n = 8), irrespective of flight speed. Evaporation was related to temperature in a biphasic pattern. At temperatures below 18.2 degrees C, net evaporation was constant at 0.36 g h(-1) (SD = 0.18; n = 10), rising at higher temperatures with a slope of 0.11 per degree to about 1.5 g h(-1) at 27 degrees C. We calculated the relative proportion of dry and evaporative heat loss during flight. Evaporative heat loss at Ta < 18.2 deg C was 14% of total heat production during flight, and dry heat loss accounted for 84%. At higher temperatures, evaporative heat loss increased linearly with T(a) to about 25% at 27 degrees C. Our data suggest that for prolonged flights, rose-colored starlings should adopt behavioral water-saving strategies and that they cannot complete their annual migration without stopovers to replenish their water reserves.     

Relationship of taurine and other amino acids in plasma and in neutrophils of septic trauma patients

Summary. Recently, an interdependency of plasma taurine and other amino acids as well as metabolic and clinical variables implicating therapeutic options was reported. This result may be an indication that plasma taurine levels are directly related to intracellular levels. Therefore, the aim of this study was to analyse the possible relationship between taurine levels in plasma and in neutrophils, the relationship to other amino acids, and variables quantifying metabolic impairment and severity of sepsis in multiple trauma patients developing sepsis. After multiple trauma taurine decreased significantly in plasma in thirty-two patients as well as within the neutrophil and does not recover in sepsis. Lower individual levels in the neutrophil did not follow lower individual levels in plasma and no correlation of taurine in plasma and in the neutrophils could be observed. In sepsis, only plasma showed an interdependency of taurine, aspartate, and glutamate. No association between taurine plasma or intracellular levels and SOFA score as indicator for severity of sepsis or metabolic variables was observed. After multiple trauma and in sepsis, taurine uptake in cells (which is regulated in different ways), and intracellular taurine (which serves e.g. as an osmolyte) can be influenced. Therefore a prediction of the neutrophil taurine pool seems not fully possible from taurine plasma levels. Intracellular taurine has some unique properties explaining the missing interdependency despite some similarities in osmoregulation and metabolic interactions to other amino acids. The association of taurine, aspartate, and glutamate in plasma cannot be simply transferred to the neutrophils intracellular level. The clinical meaning of the plasma correlation remains unclear. A dependency of plasma and neutrophil taurine to severity of sepsis and to metabolic variables seems not possible because of the multifactorial pathophysiology of sepsis.     

Bacteriorhodopsin chimeras containing the third cytoplasmic loop of bovine rhodopsin activate transducin for GTP/GDP exchange

The mechanisms by which G-protein-coupled receptors (GPCRs) activate G-proteins are not well understood due to the lack of atomic structures of GPCRs in an active form or in GPCR/G-protein complexes. For study of GPCR/G-protein interactions, we have generated a series of chimeras by replacing the third cytoplasmic loop of a scaffold protein bacteriorhodopsin (bR) with various lengths of cytoplasmic loop 3 of bovine rhodopsin (Rh), and one such chimera containing loop 3 of the human beta2-adrenergic receptor. The chimeras expressed in the archaeon Halobacterium salinarum formed purple membrane lattices thus facilitating robust protein purification. Retinal was correctly incorporated into the chimeras, as determined by spectrophotometry. A 2D crystal (lattice) was evidenced by circular dichroism analysis, and proper organization of homotrimers formed by the bR/Rh loop 3 chimera Rh3C was clearly illustrated by atomic force microscopy. Most interestingly, Rh3C (and Rh3G to a lesser extent) was functional in activation of GTPgamma35S/GDP exchange of the transducin alpha subunit (Galphat) at a level 3.5-fold higher than the basal exchange. This activation was inhibited by GDP and by a high-affinity peptide analog of the Galphat C terminus, indicating specificity in the exchange reaction. Furthermore, a specific physical interaction between the chimera Rh3C loop 3 and the Galphat C terminus was demonstrated by cocentrifugation of transducin with Rh3C. This Galphat-activating bR/Rh chimera is highly likely to be a useful tool for studying GPCR/G-protein interactions.