Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII

N-glycosylation is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. In the present study, however, we demonstrate posttranslational N-glycosylation of recombinant human coagulation factor VII (FVII) in CHO-K1 and 293A cells. Human FVII has two N-glycosylation sites (N145 and N322). Pulse-chase labeled intracellular FVII migrated as two bands corresponding to FVII with one and two N-glycans, respectively. N-glycosidase treatment converted both of these band into a single band, which comigrated with mutated FVII without N-glycans. Immediately after pulse, most labeled intracellular FVII had one N-glycan, but during a 1-h chase, the vast majority was processed into FVII with two N-glycans, demonstrating posttranslational N-glycosylation of FVII. Pulse-chase analysis of N-glycosylation site knockout mutants demonstrated cotranslational glycosylation of N145 but primarily or exclusively posttranslational glycosylation of N322. The posttranslational N-glycosylation appeared to take place in the same time frame as the folding of nascent FVII into a secretion-competent conformation, indicating a link between the two processes. We propose that the cotranslational conformation(s) of FVII are unfavorable for glycosylation at N332, whereas a more favorable conformation is obtained during the posttranslational folding. This is the first documentation of posttranslational N-glycosylation of a non-modified protein in mammalian cells with an intact N-glycosylation machinery. Thus, the present study demonstrates that posttranslational N-glycosylation can be a part of the normal processing of glycoproteins.     

Trochophora larvae: cell-lineages, ciliary bands and body regions. 2. Other groups and general discussion

The embryology of sipunculans, entoprocts, nemertines, platyhelminths (excluding acoelomorphs), rotifers, ectoprocts, phoronids, brachiopods, echinoderms and enteropneusts is reviewed with special emphasis on cell-lineage and differentiation of ectodermal structures. A group Spiralia comprising the four first-mentioned phyla plus annelids and molluscs seems well defined through the presence of spiral cleavage with early blastomere specification, prototroch with characteristic cell-lineage, cerebral ganglia developing from cells of the first micromere quartet (i.e., the episphere) and a ventral nervous system developing from the hyposphere. The planktotrophic trochophore was probably the larval type of the ancestor of this group. Another group comprising phoronids, brachiopods, echinoderms and enteropneusts appears equally well delimited. It is characterized by radial cleavage with late blastomere specification, possibly by the presence of a neotroch consisting of monociliate cells, by the absence of cerebral ganglia and of a well-defined brain and paired longitudinal nerve cords developing in connection with the blastopore, and by coelomic organization. Its ancestral larval type was probably a dipleurula. Several characters link rotifers with the spiralians, although they do not show the spiral pattern in the cleavage. Ectoprocts are still a problematic group, but some characters indicate spiralian affinities.     

An annotated checklist of macrozoobenthic species in German waters of the North and Baltic Seas

The present compilation is the first attempt to generate a comprehensive list of all macrozoobenthic species recorded at least once in the German regions of the North Sea and Baltic Sea including non-indigenous species and freshwater species which occurred in brackish waters (estuaries, bays, fjords etc.). Based on the data of several research institutes and consultancies, the macrozoobenthic species inventory comprises a total of 1.866 species belonging to 16 phyla including 193 threatened species. The most common groups were: malacostracan crustaceans (21%), Polychaeta (19%), and Gastropoda (12%). Even though the two major marine regions are separated by only 50 km of land, the composition of the respective communities was different. The two seas shared only 36.6% of the recorded species which should have profound and far-reaching consequences for conservation purposes. Considering all macroinvertebrates listed 96 species, or the equivalent of 5.2%, were introduced mainly during the last two centuries. Both seas are heavily affected by human activities and are sensitive to climate change displayed by effects on the faunal compositions. The present checklist is an important step to document these changes scientifically and may act as a base for political and management decisions.     

Kit ligand has a critical role in mouse yolk sac and aorta–gonad–mesonephros hematopoiesis

Few studies report on the in vivo requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta–gonad–mesonephros (AGM) niche. In‐depth analysis of loss‐of‐function and transgenic reporter mouse models show that Kitl‐deficient embryos harbor decreased numbers of yolk sac erythro‐myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence. This EMP defect causes a dramatic decrease in fetal liver erythroid cells prior to the onset of hematopoietic stem cell (HSC)‐derived erythropoiesis, and a reduction in tissue‐resident macrophages. Pre‐HSCs in the AGM require Kitl for survival and maturation, but not proliferation. Although Kitl is expressed widely in all embryonic hematopoietic niches, conditional deletion in endothelial cells recapitulates germline loss‐of‐function phenotypes in AGM and yolk sac, with phenotypic HSCs but not EMPs remaining dependent on endothelial Kitl upon migration to the fetal liver. In conclusion, our data establish Kitl as a critical regulator in the in vivoAGM and yolk sac endothelial niche.     

Sechsj?hrige Untersuchungen an einer Population des Rauhfu?kauzes,Aegolius funereus (L.)

Zusammenfassung Von 1963–1968 wurden in einem Versuchsgebiet von 10 000 ha auf der Schwäbischen Alb Beobachtungen an einer Population des Rauhfußkauzes durchgeführt. Das Gebiet liegt 630 bis 690 m über NN.Nach den hier gemachten Erfahrungen ist der Rauhfußkauz keineswegs auf Nadelwälder angewiesen. Seine weitere Verbreitung wurde durch das Aufhängen von Nisthöhlen unterstützt. Es erwies sich als unumgänglich, sowohl künstliche Niststätten als auch Schwarzspechthöhlen mit zylindrisch um den Stamm gelegten Blechmanschetten von 50 cm Breite (ober- und unterhalb der Höhle) gegen Marder zu schützen. Vor allemMartes martes wurde als häufigster Vernichter von Bruten festgestellt. Nach der Durchführung der Schutzmaßnahmen fielen Brutstörungen durch Marder weg. Ein vonAegolius, das in einer Schwarzspechthöhle Junge huderte, wurde vom Kleiber(Sitta europaea) eingemauert. Die Gelegegröße und der Bruterfolg waren sehr stark vom Angebot an kleinen Wühlmäusen abhängig. In einem Mäusejahr betrug die durchschnittliche Gelegegröße 5,7 Eier und die Zahl der ausgeflogenen Jungen im Mittelwert 4. In einem Jahr mit Nahrungsknappheit wurden 2,7 Eier gelegt und 2,3 Junge flügge. Die Siedlungsdichte war in einem bestimmten Gebiet recht hoch, während weite Waldflächen keine Rauhfußkäuze beherbergten. Eine Erfassung des Bestandes durch Hochrechnung von in Repräsentativgebieten ermittelten Bestandszahlen erscheint daher bei dieser Art sehr fragwürdig.Die Ernährung zeigt, daß der Rauhfußkauz mit Vorliebe kleine Säugetiere, besonders Feld-, Erd- und Rötelmäuse erbeutet, d. h. sich in ähnlicher Weise ernährt wie die Waldohreule(Asio otus). Selbst in Jahren mit Mäuseknappheit überwogen Kleinsäuger mit 82,5 % gegenüber 12,5 % Kleinvögeln und 5 % Käfern. Bemerkenswert ist der Fund eines Haussperlings als Beutetier in einem vom Rauhfußkauz besetzten Nistkasten.

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Summary From 1963–1968 observations were made on a population ofAegolius funereus, living in a territory of 10 000 ha in the Schwäbische Alb — mountains, about 630 to 690 m over sealevel. After our observations Tengmalm's Owl is here not restricted to coniferous forests. Nesting-boxes, being attached in the territory helped to enlarge the population.All nesting-sites (boxes and woodpecker-holes ofDryocopus martius) must be protected against martens(Martes martes) by fixing cuffs of sheet-iron cylindrically below and above the breeding-place. The breadth of these cuffs was 50 cm. After the protection of the nesting-sites, no one more was plundered by the marten. A female ofAegolius, hatching youngs in a woodpecker-hole, was shut up by a Nuthatch(Sitta europaea), who closed the entrance with mud. Clutch-size and breeding-success depended from the aboundance of small voles(Microtidae). In typical voleyears the average clutch-size was 5,7 eggs, and the number of fledged youngs 4. In years, when voles and mice were scarce 2,7 eggs were laid and 2,3 young fledged per brood.In certain parts of the observed territory, the density of Tengmalm's Owls was rather high, while in other, also very convenient parts of the extended forests noAegolius existed. It seems therefore very doubtful, to assume the number of couples in that species by counting them in certain representive-squares of the territory and then multiplying these numbers.Tengmalm's Owls of the Schwäbische Alb fed principally on small mammals, especially on small voles like the lesser Horned Owl(Asio otus). Even in years with a small offer of voles and mice 82,5 % mammals and only 12,5 % passerine birds and 5 % beetles were found. A remarquable prey of Tengmalm's Owl is a male House-sparrow(Passer domesticus), found by us in a nesting-box occupied by a hatching female ofAegolius.

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Vortrag gehalten auf der 81. Jahresversammlung der DO-G 1968 in Innsbruck.     

Feinstrukturelle Untersuchungen an den Horizontalzellen der menschlichen Netzhaut

Zusammenfassung Die Horizontalzellen der menschlichen Netzhaut wurden elektronenmikroskopisch untersucht. Es wurden erstmals in den Tubulusaggregaten verschiedene Vesikelformen, u.a. auch granulierte Bläschen nachgewiesen. Die Horizontalzellen sind wahrscheinlich neuronaler und nicht gliöser Natur. Es wird die Vermutung geäußert, daß die Tubulusaggregate an der Synthese der granulierten Bläschen beteiligt sind. Da sich dense core vesicle-haltige Horizontalzell-Fortsätze nur in den Stäbchensynapsen finden, ist es denkbar, daß diese Zellen ein spezifisches Schaltsystem in der Netzhaut aufbauen. Eventuell ist hierin eine morphologische Grundlage für die Hemmung des skotopischen Apparates durch die Zapfenaktivität zu sehen.

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On the ultrastructure of the horizontal cells in the human retina

Summary The horizontal cells of the human retina were studied with the electron microscope. For the first time different forms of vesicles especially dense core vesicles are shown in the complex of tubuli in these cells. The horizontal cells are probably of neuronal nature. Possibly the tubular complex participates in the synthesis of dense core vesicles. These vesicles occur only in the dendrites of rod spherule synapses. Thus it seems possible that the horizontal cells are a component of a spezific intraretinal circuit system. Perhaps these are the morphological bases for an inhibition of the scotopic apparatus exerted by the activity of retinal cones.

     

Characterization of a xylitol dehydrogenase and a d-arabitol dehydrogenase from the thermo- and acidophilic red alga Galdieria sulphuraria

Galdieria sulphuraria (Galdieri) Merola can grow heterotrophically on at least ten different polyols. We investigated their metabolic path to glycolysis/gluconeogenesis and identified two NAD-dependent polyol dehydrogenases. Activity of other enzymes metabolizing mannitol or sorbitol could not be detected. The two dehydrogenases had a broad substrate specificity and were termed xylitol dehydrogenase (EC 1.1.1.14; substrate specificity: xylitol > d-sorbitol > d-mannitol > l-arabitol) and d-arabitol dehydrogenase (EC 1.1.1.11; substrate specificity: d-arabitol > l-fucitol > d-mannitol > d-threitol) according to the substrate with the lowest K _m value. The xylitol dehydrogenase was stable during purification. In contrast, the d-arabitol dehydrogenase was thermolabile and depended on divalent ions for stability and activity, preferentially Mn²⁺ and Ni²⁺. The molecular mass of the xylitol dehydrogenase was estimated to be 295 kDa by size-exclusion chromatography and 220 kDa by rate-sedimentation centrifugation. The d-arabitol dehydrogenase had a molecular mass of 105 kDa as determined by rate-sedimentation centrifugation. The specific activity of both enzymes increased about fourfold when cells were transferred from autotrophic to heterotrophic conditions regardless of whether sugars or polyols were supplied as substrates. The significance of polyol metabolism in Galdieria sulphuraria with regard to the natural habitat of the alga is discussed. Received: 15 January 1997 / Accepted: 12 February 1997     

Losses of inorganic carbon and nitrous oxide from a temperate freshwater wetland in relation to nitrate loading

We studied the export of inorganic carbon and nitrous oxide (N₂O) from a Danish freshwater wetland. The wetland is situated in an agricultural catchment area and is recharged by groundwater enriched with nitrate (NO₃ ^–) (1000 M). NO₃ ^– in recharging groundwater was reduced (57.5 mol NO₃ ^– m^–2 yr^–) within a narrow zone of the wetland. Congruently, the annual efflux of carbon dioxide (CO₂) from the sediment was 19.1 mol C m^–2 when estimated from monthly in situ measurements. In comparison the CO₂ efflux was 4.8 mol C m^–2 yr^–1 further out in the wetland, where no NO₃ ^– reduction occurred. Annual exports of inorganic carbon in groundwater and surface water was 78.4 mol C m^–2 and 6.1 mol C m^–2 at the two sites, respectively. N₂O efflux from the sedimenst was detectable on five out of twelve sampling dates and was significantly (P < 0.0001) higher in the NO₃ ^– reduction zone (0.35–9.40 mol m^–2 h^–1, range of monthly means) than in the zone without NO₃ ^– reduction (0.21–0.41 mol m^–2 h^–1). No loss of dissolved N₂O could be measured. Total annual export of N₂O was not estimated. The reduction of oxygen (O₂) in groundwater was minor throughout the wetland and did not exceed 0.2 mol 0₂ m^–2yr^–1. Sulfate (SO₄ ^––) was reduced in groundwater (2.1 mol SO₄ ^–– m^–2 yr^–1) in the zone without NO₃ ^– reduction. Although the NO₃ ^– in our wetland can be reduced along several pathways our results strongly suggest that NO₃ ^– loading of freshwater wetlands disturb the carbon balance of such areas, resulting in an accelerated loss of inorganic carbon in gaseous and dissolved forms.     

Structure and Function of Metazoan Ciliary Bands and Their Phylogenetic Significance

Ciliated epithelia, especially the ciliary bands used in swimming and filter feeding, of representatives of the following phyla have been investigated: Porifera, Cnidaria, Annelida, Mollusca, Sipuncula, Nemertini, Platyhelminthes, Entoprocta, Ectoprocta, Rotifera, Pterobranchia, Phoronida, Brachiopoda, Echinodermata and Enteropneusta. The trochaea theory predicts that Porifera and Cnidaria have only monociliate cells and lack ciliary bands used in filter-feeding, that the gastroneuralian larvae have downstream-collecting ciliary bands with prototroch and metatroch of compound cilia on multiciliate cells, and that notoneuralian larvae have an upstream-collecting neotroch on monociliate cells. The observations generally fit these predictions and the exceptions are discussed. In all the ciliated epithelia, except that of the sponge larva, each ciliated cell has an accessory centriole perpendicular to the basal body of the cilium and situated on its downstream side.     

Pyrrolizidine alkaloids from Neurolaena lobata

Neurolaena lobata was investigated for the occurrence of pyrrolizidine alkaloids. The untypical methyl ester alkaloids tussilagine, isotussilagine and their possible biosynthetic precursor 2-pyrrolidineacetic acid were found in the methanolic leaf extract. As previously found for Arnica species and Tussilago farfara, all methyl esters are artifacts derived from the corresponding acids during Soxhlet extraction. The occurrence of toxic necines, often found in the Senecioneae, could be ruled out.