Poly(ADP-Ribose) polymerase inhibition improves endothelial dysfunction induced by hypochlorite

Reactive oxygen species, such as myeloperoxidase-derived hypochlorite, induce oxidative stress and DNA injury. The subsequent activation of the DNA-damage-poly(ADP-ribose) polymerase (PARP) pathway has been implicated in the pathogenesis of various diseases, including ischemia-reperfusion injury, circulatory shock, diabetic complications, and atherosclerosis. We investigated the effect of PARP inhibition on the impaired endothelium-dependent vasorelaxation induced by hypochlorite. In organ bath experiments for isometric tension, we investigated the endothelium-dependent and endothelium-independent vasorelaxation of isolated rat aortic rings using cumulative concentrations of acetylcholine and sodium nitro-prusside. Endothelial dysfunction was induced by exposing rings to hypochlorite (100-400 microM). In the treatment group, rings were preincubated with the PARP inhibitor INO-1001. DNA strand breaks were assessed by the TUNEL method. Immunohistochemistry was performed for 4-hydroxynonenal (a marker of lipid peroxidation), nitrotyrosine (a marker of nitrosative stress), and poly(ADP-ribose) (an enzymatic product of PARP). Exposure to hypochlorite resulted in a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings, which was significantly improved by PARP inhibition, whereas the endothelium-independent vasorelaxation remained unaffected. In the hypochlorite groups we found increased DNA breakage, lipidperoxidation, and enhanced nitrotyrosine formation. The hypochloride-induced activation of PARP was prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of hypochlorite-induced endothelial dysfunction, which can be prevented by PARP inhibitors.     

Individual vocal production in a sperm whale (Physeter macrocephalus) social unit

The vocal repertoires of group‐living animals may communicate individual or group identity. Female and juvenile sperm whales live in long‐term social units that can be assigned to vocal clans based on the pattern of clicks in coda vocalizations. An unusual set of circumstances allowed us to record the vocalizations of photo‐identified individuals within a single social unit over a 41 d period. Using click interpulse intervals, we were able to assign codas to individuals and investigate coda production at the individual level within a social unit for the first time. Adult females in the unit vocalized at approximately equal rates. A calf and juvenile, both male, vocalized less often than the adult females. Repertoires were indistinguishable for all unit members apart from a mother and her calf, which possessed significantly different repertoires—even from one another. We suggest that similarity among the coda repertoires of most unit members indicates a function in advertising unit identity. In contrast, the distinctive repertoires of the calf and its mother may facilitate reunions between these whales. We hypothesize that sperm whales may be able to vary their vocal repertoires as their reproductive status alters the trade‐off between the benefits of individual and group identification.     

Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to the growing cell tip (anterograde). Occasionally, EEs run up to 90 μm in one direction. The underlying MT array consists of unipolar MTs at both cell ends and antipolar bundles in the middle region of the cell. Cytoplasmic MT-organizing centers, labeled with a γ-tubulin ring complex protein, are distributed along the antipolar MTs but are absent from the unipolar regions. Dynein colocalizes with EEs for 10-20 μm after they have left the cell tip. Inactivation of temperature-sensitive dynein abolishes EE motility within the unipolar MT array, whereas long-range motility is not impaired. In contrast, kinesin-3 is continuously present, and its inactivation stops long-range EE motility. This indicates that both motors participate in EE motility, with dynein transporting the organelles through the unipolar MT array near the cell ends, and kinesin-3 taking over at the beginning of the medial antipolar MT array. The cooperation of both motors mediates EE movements over the length of the entire cell.     

Myosin-V, Kinesin-1, and Kinesin-3 cooperate in hyphal growth of the fungus Ustilago maydis

Long-distance transport is crucial for polar-growing cells, such as neurons and fungal hyphae. Kinesins and myosins participate in this process, but their functional interplay is poorly understood. Here, we investigate the role of kinesin motors in hyphal growth of the plant pathogen Ustilago maydis. Although the microtubule plus-ends are directed to the hyphal tip, of all 10 kinesins analyzed, only conventional kinesin (Kinesin-1) and Unc104/Kif1A-like kinesin (Kinesin-3) were up-regulated in hyphae and they are essential for extended hyphal growth. deltakin1 and deltakin3 mutant hyphae grew irregular and remained short, but they were still able to grow polarized. No additional phenotype was detected in deltakin1rkin3 double mutants, but polarity was lost in deltamyo5rkin1 and deltamyo5rkin3 mutant cells, suggesting that kinesins and class V myosin cooperate in hyphal growth. Consistent with such a role in secretion, fusion proteins of green fluorescent protein and Kinesin-1, Myosin-V, and Kinesin-3 accumulate in the apex of hyphae, a region where secretory vesicles cluster to form the fungal Spitzenk?rper. Quantitative assays revealed a role of Kin3 in secretion of acid phosphatase, whereas Kin1 was not involved. Our data demonstrate that just two kinesins and at least one myosin support hyphal growth.     

Structure-based design of protein tyrosine phosphatase-1B inhibitors

Using structure-based design, a new class of inhibitors of protein tyrosine phosphatase-1B (PTP1B) has been identified, which incorporate the 1,2,5-thiadiazolidin-3-one-1,1-dioxide template.     

Endocytosis is essential for pathogenic development in the corn smut fungus Ustilago maydis

It is well established that polarized exocytosis is essential for fungal virulence. By contrast, the contribution of endocytosis is unknown. We made use of a temperature-sensitive mutant in the endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptor Yup1 and demonstrate that endocytosis in Ustilago maydis is essential for the initial steps of pathogenic development, including pheromone perception and cell-cell fusion. Furthermore, spore formation and germination were drastically reduced, whereas colonization of the plant was only slightly inhibited. The function of endocytosis in the recognition of mating pheromone through the G protein-coupled pheromone receptor Pra1 was analyzed in greater detail. Biologically active Pra1-green fluorescent protein localizes to the plasma membrane and is constitutively endocytosed. Yup1(ts) mutants that are blocked in the fusion of endocytic transport vesicles with early endosomes are impaired in pheromone perception and conjugation hyphae formation. This is attributable to an accumulation of Pra1-carrying endocytic vesicles in the cytoplasm and the depletion of the receptor from the membrane. Consistently, strong Pra1 expression rescues the signaling defects in endocytosis mutants, but subsequent cell fusion is still impaired. Thus, we conclude that endocytosis is essential for recognition of the partner at the beginning of the pathogenic program but has additional roles in mating as well as spore formation and germination.     

Polar localizing class V myosin chitin synthases are essential during early plant infection in the plant pathogenic fungus Ustilago maydis

Fungal chitin synthases (CHSs) form fibers of the cell wall and are crucial for substrate invasion and pathogenicity. Filamentous fungi contain up to 10 CHSs, which might reflect redundant functions or the complex biology of these fungi. Here, we investigate the complete repertoire of eight CHSs in the dimorphic plant pathogen Ustilago maydis. We demonstrate that all CHSs are expressed in yeast cells and hyphae. Green fluorescent protein (GFP) fusions to all CHSs localize to septa, whereas Chs5-GFP, Chs6-GFP, Chs7-yellow fluorescent protein (YFP), and Myosin chitin synthase1 (Mcs1)-YFP were found at growth regions of yeast-like cells and hyphae, indicating that they participate in tip growth. However, only the class IV CHS genes chs7 and chs5 are crucial for shaping yeast cells and hyphae ex planta. Although most CHS mutants were attenuated in plant pathogenicity, Deltachs6, Deltachs7, and Deltamcs1 mutants were drastically reduced in virulence. Deltamcs1 showed no morphological defects in hyphae, but Mcs1 became essential during invasion of the plant epidermis. Deltamcs1 hyphae entered the plant but immediately lost growth polarity and formed large aggregates of spherical cells. Our data show that the polar class IV CHSs are essential for morphogenesis ex planta, whereas the class V myosin-CHS is essential during plant infection.     

The influence of storage temperature during machine perfusion on preservation quality of marginal donor livers

Background

Although non-heart-beating donors have the potential to increase the number of available organs, the livers are used very seldom because of the risk of primary non-function. There is evidence that machine perfusion is able to improve the preservation of marginal organs, and therefore we evaluated in our study the influence of the perfusate temperature during oxygenated machine perfusion on the graft quality.

Methods

Livers from male Wistar rats were harvested after 60-min warm ischemia induced by cardiac arrest. The portal vein was cannulated and the liver flushed with Lifor® (Lifeblood Medical, Inc.) organ preservation solution for oxygenated machine perfusion (MP) at 4, 12 or 21 °C. Other livers were flushed with HTK and stored at 4 °C by conventional cold storage (4 °C-CS). Furthermore two groups with either warm ischemic damage only or without any ischemic damage serve as control groups. After 6 h of either machine perfusion or cold storage all livers were normothermic reperfused with Krebs–Henseleit buffer, and functional as well as structural data were analyzed.

Results

Contrary to livers stored by static cold storage, machine perfused livers showed independently of the perfusate temperature a significantly decreased enzyme release of hepatic transaminases (ALT) during isolated reperfusion. Increasing the machine perfusion temperature to 21 °C resulted in a marked reduction of portal venous resistance and an increased bile production.

Conclusions

Oxygenated machine perfusion improves viability of livers after prolonged warm ischemic damage. Elevated perfusion temperature of 21 °C reconstitutes the hepatic functional capacity better than perfusion at 4 or 12 °C.     

Selective photostimulation of genetically chARGed neurons.

To permit direct functional analyses of neural circuits, we have developed a method for stimulating groups of genetically designated neurons optically. Coexpression of the Drosophila photoreceptor genes encoding arrestin-2, rhodopsin (formed by liganding opsin with retinal), and the alpha subunit of the cognate heterotrimeric G protein--an explosive combination we term "chARGe"--sensitizes generalist vertebrate neurons to light. Illumination of a mixed population of neurons elicits action potentials selectively and cell-autonomously in its genetically chARGed members. In contrast to bath-applied photostimulants or caged neurotransmitters, which act indiscriminately throughout the illuminated volume, chARGe localizes the responsiveness to light. Distributed activity may thus be fed directly into a circumscribed population of neurons in intact tissue, irrespective of the spatial arrangement of its elements.