Über Arthropoden-Abwehrstoffe. LVIII Zur Feinstruktur der Pygidialdrüsen des Gelbrandkäfers (Dytiscus marginalis L., Dytiscidae,Coleoptera)

Zusammenfassung Die Pygidialdrüsen von Dytiscus sezernieren eine Emulsion, die p-Hydroxybenzaldehyd, p-Hydroxybenzoesäuremethylester, Benzoesäure und ein Glycoproteid enthält. Ihre Loben sind aus zwei verschiedenen Arten von Drüsenzellen aufgebaut, den Kanalzellen, die die Einzelkanäle und den Sammelkanal umgeben, und den Tracheoblasten. Die chitinigen Einzelkanäle enden mit einer traubigen oder blasigen Anschwellung, die aus einer massiven inneren und einer schwammigen äußeren Wandschicht besteht, in einer Höhle der sekretorischen Zellen. Die Zahl der Blasen - und Trauben-Zellen ist etwa gleich. Die zentrale Höhle ist von Mikrovilli umgeben, die durch Mikrofibrillen in hexagonaler Packung ausgesteift werden. Wie die Untersuchung nach Gefrierätzung zeigt, ist die konvexe Seite der Mikrovilli-Membran dichter mit Partikeln besetzt als die konkave Seite. Beide Zelltypen haben ein ausgedehntes tubuläres glattes endoplasmatisches Reticulum; freie Ribosomen und granuläre Zisternen sind selten. In den Traubenzellen ist der Golgi-Apparat besser als in den Blasenzellen entwickelt. Die zentrale Höhle der Traubenzellen enthält eine fein-flockige Substanz, die der Blasenzellen ein dichtes osmiophiles Material. Die Sekretionsmechanismen, die Beteiligung verschiedener Typen von Vesikeln und anderer Zellorganellen an der Sekretion und die Unterschiede zwischen den beiden Drüsenzelltypen werden diskutiert. Es wird angenommen, daß die Hauptkomponenten des Sekretes eccrin ausgeschieden werden.

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On defense substances of arthropods LVIII the fine structure of the pygidial glands of the water beetle, Dytiscus marginalis L.

Summary The pygidial glands of Dytiscus secrete an emulsion containing p-hydroxybenzaldehyde, p-hydroxybenzoate, benzoic acid, and a glycoproteid (Schildknecht, 1970). Their lobes are composed of two different types of secretory cells, channel cells (which surround a channel, draining the secretory cell, as well as the collecting channel), and of tracheoblasts. The chitinous draining channel ends in the form of either a racemous or a bulbous swelling consisting of a massive inner and a spongy outer wall layer in a cavity of the secretory cell. The number of racemous and bulbous cells is nearly the same. The central cavity is surrounded by microvilli which are stiffened by microfibrils in a hexagonal packing. After freeze etching the convex surface of the microvilli reveals more membrane particles than their concave surface. Both cell types have an extended smooth surfaced tubular endoplasmic reticulum; the amount of free ribosomes and of granular cisternae is low. In the racemous cells the Golgi apparatus is better developed than in the bulbous cells. In the racemous cells the central cavity contains a fine-fluffy substance, in the bulbous cells a dense osmiophilic material. The mode of secretion, the participation of various kinds of vesicles and other cell organelles in this process, and the differences between the two types of secretory cells are discussed. It is assumed that the main components of the secretion are released in the eccrine way.

Wir danken der Deutschen Forschungsgemeinschaft für Sachbeihilfen.     

Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES cell line

We characterized phenotype and function of a fetal human mesencephalic cell line (LUHMES, Lund human mesencephalic) as neuronal model system. Neurodevelopmental profiling of the proliferation stage (d0, day 0) of these conditionally-immortalized cells revealed neuronal features, expressed simultaneously with some early neuroblast and stem cell markers. An optimized 2-step differentiation procedure, triggered by shut-down of the myc transgene, resulted in uniformly post-mitotic neurons within 5 days (d5). This was associated with down-regulation of some precursor markers and further up-regulation of neuronal genes. Neurite network formation involved the outgrowth of 1-2, often > 500 μm long projections. They showed dynamic growth cone behavior, as evidenced by time-lapse imaging of stably GFP-over-expressing cells. Voltage-dependent sodium channels and spontaneous electrical activity of LUHMES continuously increased from d0 to d11, while levels of synaptic markers reached their maximum on d5. The developmental expression patterns of most genes and of the dopamine uptake- and release-machinery appeared to be intrinsically predetermined, as the differentiation proceeded similarly when external factors such as dibutyryl-cAMP and glial cell derived neurotrophic factor were omitted. Only tyrosine hydroxylase required the continuous presence of cAMP. In conclusion, LUHMES are a robust neuronal model with adaptable phenotype and high value for neurodevelopmental studies, disease modeling and neuropharmacology.     

Endotoxin elicits nitric oxide release in rat but prostacyclin synthesis in human and bovine vascular smooth muscle cells

Lipopolysaccharide (LPS) exposure to cells and tissues can mimic the biochemical events leading to septic shock. Previous data demonstrated a massive upregulation of prostaglandin endoperoxide H2 synthase (PGHS-2), but not NO synthase-2 (NOS-2) in bovine smooth muscle cells (SMC) between 2 and 12 h of LPS exposure. This caused an abundant release of prostacyclin (PGI2) by constitutive PGI2-synthase as a counterregulation to a dysfunctional endothelium. We here report that human as well as bovine SMC mainly respond by the induction of PGHS-2 and the subsequent release of PGI2, whereas rat SMC exhibited a distinct induction of NOS-2 and released significantly higher amounts of *NO compared with cattle and human. The induction of either PGHS-2 or NOS-2 in the three different species investigated seems to be mutually exclusive in the time window of 2-24 h. This finding should be considered in the setup of experimental models for the investigation of septic shock.     

Hydralazine is a powerful inhibitor of peroxynitrite formation as a possible explanation for its beneficial effects on prognosis in patients with congestive heart failure

The hemodynamic and anti-ischemic effects of nitroglycerin (GTN) are rapidly blunted as a result of the development of nitrate tolerance. Hydralazine has been shown to prevent tolerance in experimental and clinical studies, all of which may be at least in part secondary to antioxidant properties of this compound. The antioxidant effects of hydralazine were tested in cell free systems, cultured smooth muscle cells, isolated mitochondria, and isolated vessels. Inhibitory effects on the formation of superoxide and/or peroxynitrite formation were tested using lucigenin and L-012 enhanced chemiluminescence as well as DHE-fluorescence. The peroxynitrite scavenging properties were also assessed by inhibition of nitration of phenol. Prevention of impairment of NO downstream signaling and GTN bioactivation was determined by measurement of P-VASP (surrogate parameter for the activity of the cGMP-dependent kinase-I, cGK-I) and mitochondrial aldehyde dehydrogenase (ALDH-2) activity. Hydralazine dose-dependently decreased the chemiluminescence signal induced by peroxynitrite from SIN-1 and by superoxide from HX/XO in a cell free system, by superoxide in smooth muscle cells and mitochondria acutely challenged with GTN. Moreover, hydralazine inhibited the peroxynitrite-mediated nitration of phenols as well as proteins in smooth muscle cells in a dose-dependent fashion. Finally, hydralazine normalized impaired cGK-I activity as well as impaired vascular ALDH-2 activity. Our results indicate that hydralazine is a highly potent radical scavenger. Thus, the combination with isosorbide dinitrate (ISDN) will favorably influence the nitroso-redox balance in the cardiovascular system in patients with congestive heart failure and may explain at least in part the improvement of prognosis in patients with chronic congestive heart failure.     

B cell activation state-governed formation of germinal centers following viral infection

Germinal centers are structures that promote humoral memory cell formation and affinity maturation, but the triggers for their development are not entirely clear. Activated extrafollicular B cells can form IgM-producing plasmablasts or enter a germinal center reaction and differentiate into memory or plasma cells, mostly of the IgG isotype. Vesicular stomatitis virus (VSV) induces both types of response, allowing events that promote each of these pathways to be studied. In this work, extrafollicular vs germinal center responses were examined at a cellular level, analyzing VSV-specific B cells in infected mice. We show that VSV-specific germinal centers are transiently formed when insufficient proportions of specific T cell help is available and that strong B cell activation in cells expressing high levels of the VSV-specific BCR promoted their differentiation into early blasts, whereas moderate stimulation of B cells or interaction with Th cells restricted extrafollicular responses and promoted germinal center formation.     

Role of reduced lipoic acid in the redox regulation of mitochondrial aldehyde dehydrogenase (ALDH-2) activity. Implications for mitochondrial oxidative stress and nitrate tolerance

Chronic therapy with nitroglycerin results in a rapid development of nitrate tolerance, which is associated with an increased production of reactive oxygen species. We have recently shown that mitochondria are an important source of nitroglycerin-induced oxidants and that the nitroglycerin-bioactivating mitochondrial aldehyde dehydrogenase is oxidatively inactivated in the setting of tolerance. Here we investigated the effect of various oxidants on aldehyde dehydrogenase activity and its restoration by dihydrolipoic acid. In vivo tolerance in Wistar rats was induced by infusion of nitroglycerin (6.6 microg/kg/min, 4 days). Vascular reactivity was measured by isometric tension studies of isolated aortic rings in response to nitroglycerin. Chronic nitroglycerin infusion lead to impaired vascular responses to nitroglycerin and decreased dehydrogenase activity, which was corrected by dihydrolipoic acid co-incubation. Superoxide, peroxynitrite, and nitroglycerin itself were highly efficient in inhibiting mitochondrial and yeast aldehyde dehydrogenase activity, which was restored by dithiol compounds such as dihydrolipoic acid and dithiothreitol. Hydrogen peroxide and nitric oxide were rather insensitive inhibitors. Our observations indicate that mitochondrial oxidative stress (especially superoxide and peroxynitrite) in response to organic nitrate treatment may inactivate aldehyde dehydrogenase thereby leading to nitrate tolerance. Glutathionylation obviously amplifies oxidative inactivation of the enzyme providing another regulatory pathway. Furthermore, the present data demonstrate that the mitochondrial dithiol compound dihydrolipoic acid restores mitochondrial aldehyde dehydrogenase activity via reduction of a disulfide at the active site and thereby improves nitrate tolerance.     

Locally resolved membrane binding affinity of the N-terminus of α-synuclein

α-Synuclein is abundantly present in Lewy bodies, characteristic of Parkinson's disease. Its exact physiological role has yet to be determined, but mitochondrial membrane binding is suspected to be a key aspect of its function. Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling allowed for a locally resolved analysis of the protein-membrane binding affinity for artificial phospholipid membranes, supported by a study of binding to isolated mitochondria. The data reveal that the binding affinity of the N-terminus is nonuniform.     

Über Arthropoden-Abwehrstoffe XLI. Zur Explosionschemie der Bombardierkäfer (Coleoptera,Carabidae)

Zusammenfassung Die Drüsenzellen in den Loben der Wehrdrüsen vonBrachynus crepitans bilden eine Mischung aus H₂O₂ und Hydrochinonen. Diese Drüsenzellen ähneln in ihrer Struktur, trotz ihrer außergewöhnlichen Sekrete, einigen Hautdrüsen anderer Insekten. Sie enthalten ein gut entwickeltes endoplasmatisches Reticulum, einige Dictyosomen und viele lysosomenähnliche Organellen; die Zahl der Mitochondrien ist unerwartet niedrig. Eine große Höhle im mittleren Teil der Zelle steht durch einen cuticularen Kanal mit dem Hauptkanal des Lobenastes in offener Verbindung. Das Zentrum dieser Höhle wird von einem Sack aus feinen Fibrillen eingenommen. Zwischen diesem Sack und dem Zellkörper befinden sich zahlreiche Mikrovilli. An der Basis dieser Mikrovilli bildet das Plasmalemm Invaginationen, in denen es mit einem beiderseitigen Belag versehen ist. Die Menge dieser coated membranes und die Zahl der Mikrovilli hängt vom Funktionszustand der Zelle ab. — Verschiedene Möglichkeiten des Sekretionsmechanismus werden diskutiert.

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On defense substances of arthropods XLI. The chemistry of the bombardier beetle (Coleoptera, Carabidae)IV. The fine structure of the defensive glands of Brachynus crepitans

Summary The gland cells of the lobular defensive glands ofBrachynus crepitans produce a highly concentrated mixture of H₂O₂ and hydroquinones. The fine structure of these cells is comparable to that of some other insect glandular cells despite of their extraordinary products. Their cytoplasm contains a well developed endoplasmic reticulum, some dictyosomes and many lysosome-like organelles of different kinds; the number of mitochondria is lower than expected. A large cavity in the center of the cell is drained by an efferent cuticular tubule which is a branch of the central lobular channel. The center of the cavity is occupied by a sac-like structure consisting of a meshwork of fibrils. Between this sac and the cell body there are many crowded microvilli. At the base of the microvilli the plasmalemma forms invaginations which are coated on both surfaces. The number of these coated invaginations and the amount of the microvilli depends on the functional state of the cell. — Several possibilities of the mechanism of the secretory process are discussed.

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XL. Mitteilung:H. Schildknecht: Angew. Chem.81 (im Druck) (1969).

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III. Mitteilung:H. Schildknecht, E. Maschwitz u.U. Maschwitz: Z. Naturforsch.23b, 1213–1218 (1968).

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Wir danken Frl.G. Deichgräber für ihre wertvolle Mitarbeit, den Herren Dr.U. Maschwitz und Dr.H. G. Heumann für wichtige Hinweise und anregende Diskussionen, Herrn Prof. Dr.J. Knappe für die Diskussion der biochemischen Probleme, der Deutschen Forschungs gemeinschaft für Sachbeihilfen.     

Simultaneous Statistical Inference for Epigenetic Data

Epigenetic research leads to complex data structures. Since parametric model assumptions for the distribution of epigenetic data are hard to verify we introduce in the present work a nonparametric statistical framework for two-group comparisons. Furthermore, epigenetic analyses are often performed at various genetic loci simultaneously. Hence, in order to be able to draw valid conclusions for specific loci, an appropriate multiple testing correction is necessary. Finally, with technologies available for the simultaneous assessment of many interrelated biological parameters (such as gene arrays), statistical approaches also need to deal with a possibly unknown dependency structure in the data. Our statistical approach to the nonparametric comparison of two samples with independent multivariate observables is based on recently developed multivariate multiple permutation tests. We adapt their theory in order to cope with families of hypotheses regarding relative effects. Our results indicate that the multivariate multiple permutation test keeps the pre-assigned type I error level for the global null hypothesis. In combination with the closure principle, the family-wise error rate for the simultaneous test of the corresponding locus/parameter-specific null hypotheses can be controlled. In applications we demonstrate that group differences in epigenetic data can be detected reliably with our methodology.