Identifizierung, Expression und Charakterisierung des Nanos-verwandten Proteins aus dem marinen Schwamm Suberites domuncula

Im Rahmen dieser Arbeit konnte in dem marinen Schwamm Suberites domuncula ein Gen identifiziert werden, dessen C-terminale konservierte Domäne eine hohe Sequenzähnlichkeit zu den Zinkfingerdomänen der Nanos-Proteine aufweist. Weiter konnte ein N-terminales Sequenzmotiv identifiziert werden, das eine hohe Sequenzidentität zu den konservierten NIM Motiven (CNOT1-interagierendes-Motiv) von Nanos zeigt. Nach der Klonierung der cDNA erfolgte die Expression des als Sd_nrp bezeichneten Proteins in E. coli Bakterien, für dessen 231 Aminosäuren umfassende Polypeptidkette eine theoretische Molekülmasse von 25.8 kDa berechnet wurde. Anschließend gelang ein Nachweis des Proteins mithilfe eines polyklonalen, gegen Sd_nrp gerichteten Antikörpers in drei Gewebetypen, dem Pinacoderm, den Primmorphen (3D-Zellaggregate) und den Gemmulae (Dauerstadien der Schwämme). Dabei konnte die höchste Expression von Sd_nrp in den als totipotent geltenden Stammzellen der Schwämme, den Archaeocyten innerhalb der Gemmulae beobachtet werden. Die Identifizierung der Zellstrukturen, erfolgte dabei aufgrund morphologischer Vergleiche. Speziell die Merkmale der Zellen in den Gemmulae, der große Nukleus, die amöboide Form sowie die granulären Reservesubstanzen, entsprechen den typischen morphologischen Eigenschaften der Archaeocyten, und bestätigen die Interpretation der Ergebnisse. Weiter konnte mit Hilfe des Anti-Sd_nrp Antikörpers das native Protein in Proteinextrakten aus Gewebe adulter Tiere nachgewiesen werden. Die vergleichende Sequenzanalyse von Sd_nrp mit dem Nanos-verwandten Protein der Schwammspezies Ephydatia fluviatilis und die phylogenetische Stammbaum-Analyse mit Nanos-Homologen unterschiedlicher Invertebraten und Vertebraten lässt die Schlussfolgerung zu, dass es sich bei dem hier identifizierten Protein Sd_nrp um ein Nanos-verwandtes Protein handelt. Darüber hinaus konnte anhand eines Homologiemodells bestätigt werden, dass es sich bei der konservierten C-terminalen Domäne des Proteins Sd_nrp um die für Nanos-Proteine spezifische Zinkfingerstruktur mit dem konservierten Sequenzmotiv CCHC handelt. Dieses Ergebnis konnte auch bei einem Vergleich der Zinkfingerdomäne von Sd_nrp mit den Zinkfingerdomänen der Nanos-Homologen unterschiedlicher Invertebraten- und Vertebratenspezies bestätigt werden.

Temperature effects on larval growth and survival in five species of Caribbean Echinoids

Under the global change scenario, the possible effects of ocean warming were investigated on the larvae of five species of Caribbean Echinoids: Echinometra lucunter, Echinometra viridis, Clypeaster rosaceus, Tripneustes ventricosus and Lytechinus williamsi. Their thermal tolerance was evaluated rearing them for six days under different temperature regimes (26, 28, 30, 32, 34, 36°C). The larval sensitivity to the treatments was evaluated on the base of survival and growth. The rearing at higher temperatures has revealed a great suffering state of the larvae by inducing both reduction of live larvae and abnormality in their development. The extent of impact of the treatments varied from species to species, evidencing different levels of thermal tolerance. Anyway, higher temperature treatments have shown a general lethal threshold at about 34°C for most of the species. As an exception, the lethal threshold of Echinometra species was 36°C, few larvae of which being still capable of survive at the temperature of 34°C. The studies have also analyzed the effect of water warming on the larvae growth in terms of size and symmetry. The results put in evidence the presence of a critical upper temperature (about 32°C) at which the larvae of all species reveal a great suffering state that translates in the reduction of size (i.e., of body, stomach and postero-dorsal arm) and abnormalities (i.e., strong difference in the lengths of the two postero-dorsal arms). As sea surface temperatures are predicted to increase of 4-5°C by 2100, the high percentage of abnormal larvae and their scarce survival observed at 32- 34°C treatments indicate that the early stages of these species could be affected by future global warming.

Effects of acute temperature increase on performance and survival of Caribbean echinoids

Climate change is occurring at a faster rate than in the past, with an expected increase of mean sea surface temperatures up to 4.8°C by the end of this century. The actual capabilities of marine invertebrates to adapt to these rapid changes has still to be understood. Adult echinoids play a crucial role in the tropical ecosystems where they live. Despite their role, few studies about the effect of temperature increase on their viability have been reported in literature. This thesis work reports a first systematic study on several Caribbean echinoids about their tolerance to temperature rise in the context of global warming. The research - carried out at the Bocas del Toro Station of the Smithsonian Tropical Research Institute, in Panama - focalized on the 6 sea urchins Lytechinus variegatus, L. williamsi, Echinometra lucunter, E. viridis, Tripneustes ventricosus and Eucidaris tribuloides, and the 2 sand dollars Clypeaster rosaceus and C. subdepressus. Mortality and neuromuscular well-being indicators - such as righting response, covering behaviour, adhesion to the substrate, spine and tube feet movements - have been analysed in the temperature range 28-38°C. The righting time RT (i.e., the time necessary for the animal to right itself completely after inversion) measured in the 6 sea urchin species, demonstrated a clearly dependence on the water temperature. The experiments allowed to determine the “thermal safety margin” (TSM) of each species. Echinometra lucunter and E. viridis resulted the most tolerant species to high temperatures with a TSM of 5.5°C, while T. ventricosus was the most vulnerable with a TSM of only 3°C. The study assessed that all the species already live at temperatures close to their upper thermal limit. Their TSMs are comparable to the predicted temperature increase by 2100. In absence of acclimatization to such temperature change, these species could experience severe die-offs, with important consequences for tropical marine ecosystems.

Biomimetic coating of organic polymers with a protein-functionalized layer of calcium phosphate: the surface properties of the carrier influence neither the coating characteristics nor the incorporation mechanism or release kinetics of the protein

Polymers that are used in clinical practice as bone-defect-filling materials possess many essential qualities, such as moldability, mechanical strength and biodegradability, but they are neither osteoconductive nor osteoinductive. Osteoconductivity can be conferred by coating the material with a layer of calcium phosphate, which can be rendered osteoinductive by functionalizing it with an osteogenic agent. We wished to ascertain whether the morphological and physicochemical characteristics of unfunctionalized and bovine-serum-albumin (BSA)-functionalized calcium-phosphate coatings were influenced by the surface properties of polymeric carriers. The release kinetics of the protein were also investigated. Two sponge-like materials (Helistat® and Polyactive®) and two fibrous ones (Ethisorb and poly[lactic-co-glycolic acid]) were tested. The coating characteristics were evaluated using state-of-the-art methodologies. The release kinetics of BSA were monitored spectrophotometrically. The characteristics of the amorphous and the crystalline phases of the coatings were not influenced by either the surface chemistry or the surface geometry of the underlying polymer. The mechanism whereby BSA was incorporated into the crystalline layer and the rate of release of the truly incorporated depot were likewise unaffected by the nature of the polymeric carrier. Our biomimetic coating technique could be applied to either spongy or fibrous bone-defect-filling organic polymers, with a view to rendering them osteoconductive and osteoinductive.

Structure-activity relationship and mechanism of action studies of manzamine analogues for the control of neuroinflammation and cerebral infections

Structure-activity relationship studies were carried out by chemical modification of manzamine A (1), 8-hydroxymanzamine A (2), manzamine F (14), and ircinal isolated from the sponge Acanthostrongylophora. The derived analogues were evaluated for antimalarial, antimicrobial, and antineuroinflammatory activities. Several modified products exhibited potent and improved in vitro antineuroinflammatory, antimicrobial, and antimalarial activity. 1 showed improved activity against malaria compared to chloroquine in both multi- and single-dose in vivo experiments. The significant antimalarial potential was revealed by a 100% cure rate of malaria in mice with one administration of 100 mg/kg of 1. The potent antineuroinflammatory activity of the manzamines will provide great benefit for the prevention and treatment of cerebral infections (e.g., Cryptococcus and Plasmodium). In addition, 1 was shown to permeate across the blood-brain barrier (BBB) in an in vitro model using a MDR-MDCK monolayer. Docking studies support that 2 binds to the ATP-noncompetitive pocket of glycogen synthesis kinase-3beta (GSK-3beta), which is a putative target of manzamines. On the basis of the results presented here, it will be possible to initiate rational drug design efforts around this natural product scaffold for the treatment of several different diseases.

Local strain distribution in real three-dimensional alveolar geometries

Mechanical ventilation is not only a life saving treatment but can also cause negative side effects. One of the main complications is inflammation caused by overstretching of the alveolar tissue. Previously, studies investigated either global strains or looked into which states lead to inflammatory reactions in cell cultures. However, the connection between the global deformation, of a tissue strip or the whole organ, and the strains reaching the single cells lining the alveolar walls is unknown and respective studies are still missing. The main reason for this is most likely the complex, sponge-like alveolar geometry, whose three-dimensional details have been unknown until recently. Utilizing synchrotron-based X-ray tomographic microscopy, we were able to generate real and detailed three-dimensional alveolar geometries on which we have performed finite-element simulations. This allowed us to determine, for the first time, a three-dimensional strain state within the alveolar wall. Briefly, precision-cut lung slices, prepared from isolated rat lungs, were scanned and segmented to provide a three-dimensional geometry. This was then discretized using newly developed tetrahedral elements. The main conclusions of this study are that the local strain in the alveolar wall can reach a multiple of the value of the global strain, for our simulations up to four times as high and that thin structures obviously cause hotspots that are especially at risk of overstretching.

Impact of Holocene climate changes on alpine and treeline vegetation at Sanetsch Pass, Bernese Alps, Switzerland

In order to infer reactions of treeline and alpine vegetation to climatic change, past vegetation changes are reconstructed on the basis of pollen, macrofossil and charcoal analysis. The sampled sediment cores originate from the small pond Emines, located at the Sanetsch Pass (connecting the Valais and Bern, Switzerland) at an altitude of 2288 m a.s.l. Today's treeline is at ca. 2200 m a.s.l. in the area, though due to special pass (saddle) conditions it is locally depressed to ca. 2060 m a.s.l. Our results reveal that the area around Emines was covered by treeless alpine vegetation during most of the past 12,000 years. Single individuals of Betula, Larix decidua and possibly Pinus cembra occurred during the Holocene. Major centennial to millennial-scale responses of treeline vegetation to climatic changes are evident. However, alpine vegetation composition remained rather stable between 11,500 and 6000 cal. BP, showing that Holocene climatic changes of +/− 1 °C hardly influenced the local vegetation at Emines. The rapid warming of 3–4 °C at the Late Glacial/Holocene transition (11,600 cal. BP) caused significant altitudinal displacements of alpine species that were additionally affected by the rapid upward movement of trees and shrubs. Since the beginning of the Neolithic, vegetation changes at Sanetsch Pass resulted from a combination of climate change and human impact. Anthropogenic fire increase and land-use change combined with a natural change from subcontinental to more oceanic climate during the second half of the Holocene led to the disappearance of P. cembra in the study area, but favoured the occurrence of Picea abies and Alnus viridis. The mid- to late-Holocene decline of Abies alba was primarily a consequence of human impact, since this mesic species should have benefitted from a shift to more oceanic conditions. Future alpine vegetation changes will be a function of the amplitude and rapidity of global warming as well as human land use. Our results imply that alpine vegetation at our treeline pass site was never replaced by forests since the last ice-age. This may change in the future if anticipated climate change will induce upslope migration of trees. The results of this study emphasise the necessity of climate change mitigation in order to prevent biodiversity losses as a consequence of unprecedented community and species displacement in response to climatic change.

Mandibular reconstruction using a combination graft of rhBMP-2 with bone marrow cells expanded in vitro

BACKGROUND: The aim of this study was to evaluate the efficacy of a combination graft, using recombinant human bone morphogenetic protein-2 (rhBMP-2) and culture-expanded cells derived from bone marrow, for bone regeneration in a nonhuman primate mandible. METHODS: Five Japanese monkeys were used. Three milliliters of bone marrow was obtained from the tibia and plated into culture flasks. Adherent cells were cultured until near confluence; then, the proliferated cells were transferred to a three-dimensional culture system using collagen beads as the cell carrier. The medium was supplemented with ascorbic acid, beta-glycerophosphate, and dexamethasone to promote osteoblastic differentiation. After further proliferation on beads, the cells were mixed with a collagen sponge that was impregnated with rhBMP-2 and grafted into surgically created segmental bone defects of the mandibles. Three animals received this treatment, and either culture-expanded cells alone or collagen beads without cells were implanted into the remaining two monkeys as controls. The animals were killed 24 weeks after surgery, and the results were assessed by radiographic and histologic evaluation. RESULTS: The combination graft of culture-expanded bone marrow cells with rhBMP-2 in a collagen sponge regenerated the mandibular bone completely. By contrast, the graft of culture-expanded cells alone resulted in only a small amount of bone formation, and the implantation of collagen beads alone led to no bone formation. CONCLUSION: The combination graft of rhBMP-2 and culture-expanded cells, which requires only a small amount of bone marrow, is a reliable method for the reconstruction of segmental bone defects of the mandible.

The Effects of Zinc Sulphate, Manganese Sulphate, and Iron Sulphate on the Lead Storage Cell.

The lead storage battery as it is used today is made up of the pasted type plates of lead dioxide, the anode, and sponge lead, the cathode, and wooden or hard rubber separators, which serve to insulate these from one another. In manufacturing these, it is desirable to keep them free from impurities.

Apparatus and Method for Removing Mercury Vapor from a Gas Stream

A metallic filter effectively removes vapor from gas streams. The filter captures the mercury which then can be released and collected as a product. The metallic filter is a copper mesh sponge plated with a six micrometer thickness of gold. The filter removes up to 90% of mercury vapor from a mercury contaminated gas stream.

A 700-year paleoecological record of boreal ecosystem responses to climatic variation from Alaska

Recent observations and model simulations have highlighted the sensitivity of the forest - tundra ecotone to climatic forcing. In contrast, paleoecological studies have not provided evidence of tree-line fluctuations in response to Holocene climatic changes in Alaska, suggesting that the forest - tundra boundary in certain areas may be relatively stable at multicentennial to millennial time scales. We conducted a multiproxy study of sediment cores from an Alaskan lake near the altitudinal limits of key boreal-forest species. Paleoecological data were compared with independent climatic reconstructions to assess ecosystem responses of the forest - tundra boundary to Little Ice Age (LIA) climatic. uctuations. Pollen, diatom, charcoal, macrofossil, and magnetic analyses provide the first continuous record of vegetation -. re - climate interactions at decadal to centennial time scales during the past 700 years from southern Alaska. Boreal-forest diebacks characterized by declines of Picea mariana, P. glauca, and tree Betula occurred during the LIA ( AD 1500 - 1800), whereas shrubs ( Alnus viridis, Betula glandulosa/nana) and herbaceous taxa (Epilobium, Aconitum) expanded. Marked increases in charcoal abundance and changes in magnetic properties suggest increases in. re importance and soil erosion during the same period. In addition, the conspicuous reduction or disappearance of certain aquatic ( e. g., Isoetes, Nuphar, Pediastrum) and wetland ( Sphagnum) plants and major shifts in diatom assemblages suggest pronounced lake-level. uctuations and rapid ecosystem reorganization in response to LIA climatic deterioration. Our results imply that temperature shifts of 1 - 2 degrees C, when accompanied by major changes in moisture balance, can greatly alter high-altitudinal terrestrial, wetland, and aquatic ecosystems, including conversion between boreal-forest tree line and tundra. The climatic and ecosystem variations in our study area appear to be coherent with changes in solar irradiance, suggesting that changes in solar activity contributed to the environmental instability of the past 700 years.

Climatic and human impacts on mountain vegetation at Lauenensee (Bernese Alps, Switzerland) during the last 14,000 years

Lake sediments from Lauenensee (1381 m a.s.l.), a small lake in the Bernese Alps, were analysed to reconstruct the vegetation and fire history. The chronology is based on 11 calibrated radiocarbon dates on terrestrial plant macrofossils suggesting a basal age of 14,200 cal. BP. Pollen and macrofossil data imply that treeline never reached the lake catchment during the Bølling–Allerød interstadial. Treeline north of the Alps was depressed by c. 300 altitudinal meters, if compared with southern locations. We attribute this difference to colder temperatures and to unbuffered cold air excursions from the ice masses in northern Europe. Afforestation started after the Younger Dryas at 11,600 cal. BP. Early-Holocene tree-Betula and Pinus sylvestris forests were replaced by Abies alba forests around 7500 cal. BP. Continuous high-resolution pollen and macrofossil series allow quantitative assessments of vegetation dynamics at 5900–5200 cal. BP (first expansion of Picea abies, decline of Abies alba) and 4100–2900 cal. BP (first collapse of Abies alba). The first signs of human activity became noticeable during the late Neolithic c. 5700–5200 cal. BP. Cross-correlation analysis shows that the expansion of Alnus viridis and the replacement of Abies alba by Picea abies after c. 5500 cal. BP was most likely a consequence of human disturbance. Abies alba responded very sensitively to a combination of fire and grazing disturbance. Our results imply that the current dominance of Picea abies in the upper montane and subalpine belts is a consequence of anthropogenic activities through the millennia.

Die Vegetationsgeschichte der letzten 14 000 Jahre am Lauenensee

Sedimente des Lauenensees (Berner Oberland, 7381 m ü.M.) wurden untersucht, um mittels Pollen-, Pflanzenrest-und Holzkohleanalyse die regionale Vegetations-und Feuergeschichte zu rekonstruieren. Die Chronologie basiert auf elf kalibrierten Radiokarbondaten von terrestrischen Pflanzenresten und ergab ein Basisalter von 14 200 kal. Jahren vor heute (12 250 v. Chr.). Während des Bölling-Allerød lnterstadials war das Einzugsgebiet des Lauenensees noch nicht bewaldet. Somit lag die Waldgrenze in den Nordalpen tiefer als in den Zentral-und Südalpen. Die Wieder-bewaldung startete nach der Jüngeren Dryas vor 11 600 kal. Jahren (9650 v. Chr.). Diese frühen Birken-und Föhrenwälder (Betula und Pinus sylvestris) wurden vor rund 7500 kal. Jahren (5550 v. Chr.) durch Weisstannenwälder(Abies alba) ersetzt. Der erste menschliche Einfluss ist ab dem späten Neolithikum belegt (5700 bis 5200 kal. Jahre vor heute, 3750-3250 v. Chr.). Mit zwei hochaufgelösten Sequenzen wurde die Ausbreitung der Fichte (Picea abies) im Neolithikum (5700 bis 5200 kal. Jahre vor heute, 3750-3250 v. Chr.) und der Kollaps der Weisstanne in der Bronzezeit (4100 bis 2900 kal. Jahre vor heute, 2150-950 v. Chr.) untersucht. Die Resultate der Kreuzkorrelationen zeigen, dass die Ausbreitung der Grünerle (Ainus viridis) sowie der Wechsel von Weisstannen-zu Fichtenwäldern durch den Menschen ausgelöst wurde. Die Weisstanne reagiert nachweislich sehr sensibel auf Beweidung und Feuer, weshalb sich die heutige Fichtendominanz im oberen montanen und subalpinen Vegetationsgürtel aus der menschlichen Nutzung über die Jahrtausende ergeben haben muss.

Holocene climate, fire and vegetation dynamics at the treeline in the Northwestern Swiss Alps

Treelines are expected to rise to higher elevations with climate warming; the rate and extent however are still largely unknown. Here we present the first multi-proxy palaeoecological study from the treeline in the Northwestern Swiss Alps that covers the entire Holocene. We reconstructed climate, fire and vegetation dynamics at Iffigsee, an alpine lake at 2,065 m a.s.l., by using seismic sedimentary surveys, loss on ignition, visible spectrum reflectance spectroscopy, pollen, spore, macrofossil and charcoal analyses. Afforestation with Larix decidua and tree Betula (probably B. pendula) started at ~9,800 cal. b.p., more than 1,000 years later than at similar elevations in the Central and Southern Alps, indicating cooler temperatures and/or a high seasonality. Highest biomass production and forest position of ~2,100–2,300 m a.s.l. are inferred during the Holocene Thermal Maximum from 7,000 to 5,000 cal. b.p. With the onset of pastoralism and transhumance at 6,800–6,500 cal. b.p., human impact became an important factor in the vegetation dynamics at Iffigsee. This early evidence of pastoralism is documented by the presence of grazing indicators (pollen, spores), as well as a wealth of archaeological finds at the nearby mountain pass of Schnidejoch. Human and fire impact during the Neolithic and Bronze Ages led to the establishment of pastures and facilitated the expansion of Picea abies and Alnus viridis. We expect that in mountain areas with land abandonment, the treeline will react quickly to future climate warming by shifting to higher elevations, causing drastic changes in species distribution and composition as well as severe biodiversity losses.

Cladorhiza corona sp. nov. (Porifera : Demospongiae : Cladorhizidae) from the Aleutian Islands (Alaska)

A new species of Cladorhizidac, front the Aleutian Islands is described and compared with all known species of Cladorhizza worldwide. Cladorhiza corona sp. now has a unique growth form with two planes of differently shaped appendages. Appendages are Inserted directly at the stalk; a spherical or conical body at the stalk is lacking. It is the only species reported where different spicule types occur in three morphologically different areas of the sponge. The spiculation of the basal plate is characterized by the occurrence of short, thick anisoxcas and the lack of anisochelae. Anisochelac arc found in the stalk and the basal appendages only. Flattened sigmancistras and (sub-)tylostyles are restricted to the crown. The arrangement of spicules is different in the basal plate, the stalk with the basal appendages, and in the distal append ages. The dimensions and combination of spicule types separate C. corona sp. nov. from all known members of the genus.