Experimental Infection of Rabbits with Newly Excysted Metacercariae of Japanese Fasciola Sp. and American Fasciola Hepatica by Portal Vein Route

日本産およびアメリカ産脱嚢幼肝蛭を家兎の門脈内に移入・感染させ,各種変化について検索した。幼肝蛭は感染後3時間で肝実質内に認められ,その後感染経過に伴い肝病変は漸次増大した。アメリカ産肝蛭では胆管への定着と虫卵排出も認められた。また,日本産肝蛭についても感染後虫体は漸次成長することが認められた。沈降抗体の出現時期は,感染後21-28日であった。幼肝蛭は血行によって肝臓に達してもよく発育し,感染が成立することが明らかとなった。 / Rabbits were experimentally infected with newly excysted metacercariae of the Japanese Fasciola sp. and the American Fasciola hepatica by portal vein route. At early stages of the infection, histopathological changes of the liver of rabbits infected with either F. sp. or F. hepatica were tract lesions characterized by haemorrhages, necrosis, cellular infiltration, and so on. The tract lesions grew larger and more numerous with the progress of the infection. At the 77th day of infection, increases of connective tissue around the intrahepatic bile ducts and in thickness of the wall of the bile ducts were noted. Precipitating antibodies were first detected at the 28th and 21st days of infection in sera of the rabbits infected with F. sp. and F. hepatica, respectively. Fluke eggs were first detected at the 63rd day in feces of the rabbits infected with F. hepatica, and were not detected even at the 77th day in the case of F. sp. Fluke eggs recovered from the infected rabbits were measured the maximum length of 15.9mm and 16.2mm, for F. sp. and F. hepatica, respectively. The infection of rabbits with F. sp. and F. hepatica by the vein route was successful

Isolation and characterization of Pas2p, a peroxisomal membrane protein essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris

The pas2 mutant of the methylotrophic yeast Pichia pastoris is characterized by a deficiency in peroxisome biogenesis. We have cloned the PpPAS2 gene by functional complementation and show that it encodes a protein of 455 amino acids with a molecular mass of 52 kDa. In a Pppas2 null mutant, import of both peroxisomal targeting signal 1 (PTS1)- and PTS2-containing proteins is impaired as shown by biochemical fractionation and fluorescence microscopy. No morphologically distinguishable peroxisomal structures could be detected by electron microscopy in Pppas2 null cells induced on methanol and oleate, suggesting that PpPas2p is involved in the early stages of peroxisome biogenesis. PpPas2p is a peroxisomal membrane protein (PMP) and is resistant to extraction by 1 M NaCl or alkaline sodium carbonate, suggesting that it is a peroxisomal integral membrane protein. Two hydrophobic domains can be distinguished which may be involved in anchoring PpPas2p to the peroxisomal membrane. PpPas2p is homologous to the Saccharomyces cerevisiae Pas3p. The first 40 amino acids of PpPas2p, devoid of the hydrophobic domains, are sufficient to target a soluble fluorescent reporter protein to the peroxisomal membrane, with which it associates tightly, A comparison with the membrane peroxisomal targeting signal of PMP47 of Candida boidinii revealed a stretch of positively charged amino acids common to both sequences. The role of peroxisomal membrane targeting signals and transmembrane domains in anchoring PMPs to the peroxisomal membrane is discussed.</p

The FLUXNET2015 dataset and the ONEFlux processing pipeline for eddy covariance data

The FLUXNET2015 dataset provides ecosystem-scale data on CO2, water, and energy exchange between the biosphere and the atmosphere, and other meteorological and biological measurements, from 212 sites around the globe (over 1500 site-years, up to and including year 2014). These sites, independently managed and operated, voluntarily contributed their data to create global datasets. Data were quality controlled and processed using uniform methods, to improve consistency and intercomparability across sites. The dataset is already being used in a number of applications, including ecophysiology studies, remote sensing studies, and development of ecosystem and Earth system models. FLUXNET2015 includes derived-data products, such as gap-filled time series, ecosystem respiration and photosynthetic uptake estimates, estimation of uncertainties, and metadata about the measurements, presented for the first time in this paper. In addition, 206 of these sites are for the first time distributed under a Creative Commons (CC-BY 4.0) license. This paper details this enhanced dataset and the processing methods, now made available as open-source codes, making the dataset more accessible, transparent, and reproducible.Peer reviewe

Ein neues In-Vitro-Modell zur Untersuchung der Degradation von Peroxisomen

Peroxisomen sind Zellorganellen, die in fast allen eukaryonten Zellen vorkommen. Sie erfüllen wichtige Funktionen im Lipidstoffwechsel und beim Metabolismus von reaktiven Sauerstoffverbindungen. Bei Veränderungen der Stoffwechselsituationen werden sie in ihrer Enzymausstattung flexibel an neue Bedingungen angepasst, wobei es zum Umbau und Abbau der Organellen kommt. In höheren Eukaryonten ist bisher nur wenig bekannt über die Mechanismen der Degradation von Peroxisomen. Es bestehen jedoch zwei unterschiedliche Modellvorstellungen, nach denen die Peroxisomen entweder komplett auf dem Wege der Autophagie abgebaut werden oder alternativ durch eine Lipoxygenase derart geschädigt werden, dass peroxisomale Proteine dann im Proteasom abgebaut werden können. Um zu klären, welcher Abbauweg tatsächlich genutzt wird, wurde im Rahmen dieser Arbeit ein neues Modell zur Untersuchung der Degradation von Peroxisomen etabliert. Aus Voruntersuchungen war bekannt, dass ein Fusionsprotein aus dem peroxisomalen Membranprotein Pxmp2 mit dem grün fluoreszierenden Protein (GFP) einen toxischen Effekt auf die Peroxisomen ausübt. Ausgehend von diesen Beobachungen wurde eine stabile Zelllinie (BGL231) etabliert, in der die Expression des Pxmp2-GFP-Fusionsproteins unter der Kontrolle des Ecdyson-Promotorsystems induzierbar war. In diesen Zellen konnte die Degradation von Peroxisomen in einer synchronisierten Weise in allen Zellen induziert werden und war dadurch einer systematischen Untersuchung zugänglich. Im Verlauf der induzierten Peroxisomendegradation kam es zu charakteristischen morphologischen Veränderungen des zellulären GFP-Fluoreszenzmusters, so dass vier Stadien dieses Prozesses unterschieden werden konnten. Nach Expression reicherte sich das Pxmp2-GFP-Fusionsprotein zunächst in den Peroxisomen an, die dann nach ca. 24 Stunden zu großen Konglomeraten aggregierten und nach weiteren 24 Stunden der Induktion nicht mehr nach-weisbar waren. Das Pxmp2-GFP-Fusionsprotein war dann in diesen Peroxisomendefizienten Zellen in der mitochondrialen Membran lokalisiert. Der charakteristische Ablauf der indu-zierten Peroxisomendegradation konnte mit etablierten Induktoren und Inhibitoren der Autophagie beschleunigt oder in bestimmten Stadien arretiert werden. Eine Hemmung der Lipoxygenase hatte hingegen keinen Effekt auf den Degradationsprozess. Die Pxmp2-GFP-induzierte Peroxisomendegradation in höheren Eukaryonten verläuft daher in Analogie zur Autophagie und wird nicht durch die Lipoxygenase vermittelt. Während des gesamten Prozesses konnte keine Kolokalisationen von Lysosomen und Peroxisomen gezeigt werden, was die Vermutung zulässt, dass für die Peroxisomen eine von der regulären Autophagie abweichende spezifische Endstrecke der Degradation dieser Organellen existiert. Die hier etablierte Zelllinie stellt das erste Modellsystem zur Untersuchung der synchronisierten Peroxisomendegradation in höheren Eukaryonten dar. In dieser Studie ist es gelungen, damit das grundlegende Degradationsmuster für Peroxisomen aufzuklären. Mit Hilfe dieses Modellsystems sollen nun auch die molekularen Mechanismen dieses Prozesses in höheren Eukaryonten systematisch untersucht werden

Structural characterisation of peroxisomal import receptor complexes

Peroxisomes are dynamic eukaryotic organelles that require import of their membrane and matrix proteins by soluble receptors in the cytosol. Their biogenesis, protein import, and proliferation are regulated by a distinct set of proteins, collectively called peroxins.Two peroxins, Pex3 and Pex19, are involved in the insertion of various Peroxisomal Membrane Proteins (PMPs) in the peroxisomal membrane and are thus crucial for its formation. Pex3 is a PMP that acts as a docking receptor for Pex19. The role of Pex19, in turn, is to bind, stabilise, and guide PMPs to the membrane-docked Pex3, where they are inserted into the peroxisomal membrane by an unknown mechanism. Pex14 is a PMP protein that is a key component of the peroxisomal import pore. Its interaction with Pex19 is established, yet little structural information is available about their full-length complex. In this thesis we investigated the conformation of the full-length human PEX19 in complex with the cytosolic domain of PEX3. A hybrid structural and biochemical approach was employed in order to characterize this interaction. Furthermore, the role of PEX19-PEX14 binding was addressed and the full-length complex was structurally characterised, providing insight into the stoichiometry, binding and shape of the never-before-described full-length assembly, using a variety of biochemical, biophysical and structural methods.The second part of this thesis aims to shed light on the peroxisomal protein import mechanism process. Matrix proteins (cargoes) can be imported into the peroxisomal lumen using peroxisomal targeting signal 1 (PTS1), or peroxisomal targeting signal 2 (PTS2) import pathways. The PTS1 pathway is the most common, utilising peroxin Pex5p. Pex5p is a soluble receptor, cycling between a free cytoplasmic state -where it recognises and binds peroxisomal matrix proteins- and a membrane bound state, as part of the transient PTS1 pore. The interaction between Pex5p and cargo proteins occurs via the TPR domains (tetratricopeptide repeats) of Pex5p and the PTS1 sequence at the C-terminus of the cargo proteins. Two peroxisomal cargoes, Pcs60 and MIF1, were studied in this part of the PhD project. Pcs60 is a yeast peroxisomal oxalyl-CoA synthetase, while MIF1 is a plant peroxisomal cargo associated with stress response, both of which contain a PTS1 recognition signal peptide. These proteins were crystallised and structurally characterised by means of X-ray crystallography. Further characterisation of Pcs60 and its complex with Pex5p was performed by biophysical methods, small angle X-ray scattering (SAXS), and negative stain electron microscopy. Elucidation of the structure of the Pex5p-Pcs60 complex will lead to enhancing our understanding of peroxisomal protein import