Symmetrie und Asymmetrie bei bilateralen Thieren

Ohne Zusammenfassung     

Dbf4 and Cdc7 Proteins Promote DNA Replication through Interactions with Distinct Mcm2–7 Protein Subunits

The essential cell cycle target of the Dbf4/Cdc7 kinase (DDK) is the Mcm2–7 helicase complex. Although Mcm4 has been identified as the critical DDK phosphorylation target for DNA replication, it is not well understood which of the six Mcm2–7 subunits actually mediate(s) docking of this kinase complex. We systematically examined the interaction between each Mcm2–7 subunit with Dbf4 and Cdc7 through two-hybrid and co-immunoprecipitation analyses. Strikingly different binding patterns were observed, as Dbf4 interacted most strongly with Mcm2, whereas Cdc7 displayed association with both Mcm4 and Mcm5. We identified an N-terminal Mcm2 region required for interaction with Dbf4. Cells expressing either an Mcm2 mutant lacking this docking domain (Mcm2ΔDDD) or an Mcm4 mutant lacking a previously identified DDK docking domain (Mcm4ΔDDD) displayed modest DNA replication and growth defects. In contrast, combining these two mutations resulted in synthetic lethality, suggesting that Mcm2 and Mcm4 play overlapping roles in the association of DDK with MCM rings at replication origins. Consistent with this model, growth inhibition could be induced in Mcm4ΔDDD cells through Mcm2 overexpression as a means of titrating the Dbf4-MCM ring interaction. This growth inhibition was exacerbated by exposing the cells to either hydroxyurea or methyl methanesulfonate, lending support for a DDK role in stabilizing or restarting replication forks under S phase checkpoint conditions. Finally, constitutive overexpression of each individual MCM subunit was examined, and genotoxic sensitivity was found to be specific to Mcm2 or Mcm4 overexpression, further pointing to the importance of the DDK-MCM ring interaction.     

Altered purinergic signaling in uridine adenosine tetraphosphate-induced coronary relaxation in swine with metabolic derangement

We previously demonstrated that uridine adenosine tetraphosphate (Up₄A) induces potent and partially endothelium-dependent relaxation in the healthy porcine coronary microvasculature. We subsequently showed that Up₄A-induced porcine coronary relaxation was impaired via downregulation of P1 receptors after myocardial infarction. In view of the deleterious effect of metabolic derangement on vascular function, we hypothesized that the coronary vasodilator response to Up₄A is impaired in metabolic derangement, and that the involvement of purinergic receptor subtypes and endothelium-derived vasoactive factors (EDVFs) is altered. Coronary small arteries, dissected from the apex of healthy swine and swine 6 months after induction of diabetes with streptozotocin and fed a high-fat diet, were mounted on wire myographs. Up₄A (10^?9–10^?5 M)-induced coronary relaxation was maintained in swine with metabolic derangement compared to normal swine, despite impaired endothelium-dependent relaxation to bradykinin and despite blunted P2X₇ receptor and NO-mediated vasodilator influences of Up₄A. Moreover, a thromboxane-mediated vasoconstrictor influence was unmasked. In contrast, an increased Up₄A-mediated vasodilator influence via P2Y₁ receptors was observed, while, in response to Up₄A, cytochrome P₄₅₀ 2C9 switched from producing vasoconstrictor to vasodilator metabolites in swine with metabolic derangement. Coronary vascular expression of A_2A and P2X₇ receptors as well as eNOS, as assessed with real-time PCR, was reduced in swine with metabolic derangement. In conclusion, although the overall coronary vasodilator response to Up₄A was maintained in swine with metabolic derangement, the involvement of purinergic receptor subtypes and EDVF was markedly altered, revealing compensatory mechanisms among signaling pathways in Up₄A-mediated coronary vasomotor influence in the early phase of metabolic derangement. Future studies are warranted to investigate the effects of severe metabolic derangement on coronary responses to Up₄A.     

Alterations of filtration coefficients in pulmonary edema of different pathogenesis.

Different pathomechanisms in the development of pulmonary edema are being discussed. We investigated the effect of pathogenetically varying forms of edema on lung vascular barrier function in isolated cell-free perfused rabbit lungs. As an index of permeability, capillary filtration coefficients (Kfc) were determined from the slope of lung weight change over periods of stepwise venous pressure elevation (5, 7.5, and 10 mmHg) before (controls) and 60 min after edema induction. Edema was induced by venous congestion (n = 6), by application of arachidonic acid in the presence of diclofenac sodium (n = 6), and by elastase application (n = 6). Control values ranged from 0.28 to 0.51 ml.min-1 x mmHg-1 x 100 g-1. Kfc was significantly enhanced after edema induction up to 243% of control value in the hydrostatic edema, 357% in the arachidonic acid edema, and 594% in the elastase edema. When the alterations in capillary filtration due to the different types of edema were compared, Kfc was significantly higher in the proteinase edema, indicating an irreversibly damaged barrier function. These data exemplify different pathophysiological characteristics due to the pathogenesis of interstitial edema formation.     

The origin recognition complex protein family

Origin recognition complex (ORC) proteins were first discovered as a six-subunit assemblage in budding yeast that promotes the initiation of DNA replication. Orc1-5 appear to be present in all eukaryotes, and include both AAA+ and winged-helix motifs. A sixth protein, Orc6, shows no structural similarity to the other ORC proteins, and is poorly conserved between budding yeast and most other eukaryotic species. The replication factor Cdc6 has extensive sequence similarity with Orc1 and phylogenetic analysis suggests the genes that encode them may be paralogs. ORC proteins have also been found in the archaea, and the bacterial DnaA replication protein has ORC-like functional domains. In budding yeast, Orc1-6 are bound to origins of DNA replication throughout the cell cycle. Following association with Cdc6 in G1 phase, the sequential hydrolysis of Cdc6 - then ORC-bound ATP loads the Mcm2-7 helicase complex onto DNA. Localization of ORC subunits to the kinetochore and centrosome during mitosis and to the cleavage furrow during cytokinesis has been observed in metazoan cells and, along with phenotypes observed following knockdown with short interfering RNAs, point to additional roles at these cell-cycle stages. In addition, ORC proteins function in epigenetic gene silencing through interactions with heterochromatin factors such as Sir1 in budding yeast and HP1 in higher eukaryotes. Current avenues of research have identified roles for ORC proteins in the development of neuronal and muscle tissue, and are probing their relationship to genome integrity.     

Ultrastructural changes of lung capillary endothelium in response to botulinum C2 toxin

Ermert, L., H.-R. Duncker, H. Brückner, F. Grimminger,T. Hansen, R. Rössig, K. Aktories, and W. Seeger.Ultrastructural changes of lung capillary endothelium in responseto botulinum C₂ toxin.J. Appl. Physiol. 82(2): 382-388, 1997.The role of the endothelial cytoskeleton for the structuralintegrity of the pulmonary gas exchange area was probed with the use ofClostridium botulinumC₂ toxin. This agent causesselective loss of nonmuscle F-actin. In buffer-perfused rabbit lungs,vascular pressures were kept within physiological ranges. In differentgroups, low-dose [0.3(C_2,I)/0.6(C_2,II) ng/ml] andhigh-dose [10 (C_2,I)/20(C_2,II) ng/ml] toxin wereapplicated into the buffer fluid; experiments were terminated after atotal weight gain of either 1 or 7.5 g. Electron microscopy revealedextensive attenuations, undulations, and protrusions of the endotheliallayer, suggestive of "remodeling" and "flowing" of the cellmembrane in low C₂ toxin-treatedlungs accompanied by few disruptions of the endothelial layer and edema formation. In addition, endothelial cells displayed vesiculation andbleb formation. Lungs that were exposed to high-toxin doses displayedmarked attenuations of the endothelial layer in addition to largeendothelial cell disruptions, which did not include interendothelial junctions. Interestingly, type II epithelial cells displayed fusion oflamellar bodies. Collectively, these data suggest that the actinmicrofilament system is instrumental in supporting endothelial cellmembrane configuration and integrity and maintains the intimal barrierfunction of the lung microvasculature.