The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin

The influence of the hyaluronanbinding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The procofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the Nterminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5(H) and D6(H) are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system

Three-Dimensional Reconstruction and Segmentation of Intact Drosophila by Ultramicroscopy

Genetic mutants are invaluable for understanding the development, physiology and behaviour of Drosophila. Modern molecular genetic techniques enable the rapid generation of large numbers of different mutants. To phenotype these mutants sophisticated microscopy techniques are required, ideally allowing the 3D-reconstruction of the anatomy of an adult fly from a single scan. Ultramicroscopy enables up to cm fields of view, whilst providing micron resolution. In this paper, we present ultramicroscopy reconstructions of the flight musculature, the nervous system, and the digestive tract of entire, chemically cleared, drosophila in autofluorescent light. The 3D-reconstructions thus obtained verify that the anatomy of a whole fly, including the filigree spatial organization of the direct flight muscles, can be analysed from a single ultramicroscopy reconstruction. The recording procedure, including 3D-reconstruction using standard software, takes no longer than 30 min. Additionally, image segmentation, which would allow for further quantitative analysis, was performed

FLEXBAR: flexible barcode and adapter processing for next-generation sequencing platforms

Quantitative and systems biology approaches benefit from the unprecedented depth of next-generation sequencing. A typical experiment yields millions of short reads, which oftentimes carry particular sequence tags. These tags may be: (a) specific to the sequencing platform and library construction method (e.g., adapter sequences); (b) have been introduced by experimental design (e.g., sample barcodes); or (c) constitute some biological signal (e.g., splice leader sequences in nematodes). Our software FLEXBAR enables accurate recognition, sorting and trimming of sequence tags with maximal flexibility, based on exact overlap sequence alignment. The software supports data formats from all current sequencing platforms, including color-space reads. FLEXBAR maintains read pairings and processes separate barcode reads on demand. Our software facilitates the fine-grained adjustment of sequence tag detection parameters and search regions. FLEXBAR is a multi-threaded software and combines speed with precision. Even complex read processing scenarios might be executed with a single command line call. We demonstrate the utility of the software in terms of read mapping applications, library demultiplexing and splice leader detection. FLEXBAR and additional information is available for academic use from the website: http://sourceforge.net/projects/flexbar/

Fisheries long term monitoring program : Syngnathids in the East Coast Trawl Fishery: a review and trawl survey

The Department of Primary Industries and Fisheries (DPI&F), Queensland, manages the state’s fish, mollusc and crustacean species and the habitats in which they live. Inherent in this responsibility is a commitment to monitor the condition of, and trends in, fish populations and their associated habitats. This information is used to assess the effectiveness of fisheries management strategies and contributes to ensuring that the fisheries remain ecologically sustainable. The family Syngnathidae (seahorses, seadragons, pipefish and pipehorses) are harvested incidentally in the Queensland East Coast Trawl Fishery (ECTF) and as a target species in the Marine Aquarium Fish Fishery (MAFF), due to their value as curios, aquarium fish and traditional Asian medicines (Dunning et al., 2001; Vincent, 1996). There is very little information available on the biology or ecology of syngnathid species and as such many are listed as a worldwide conservation concern. The objectives of this project were to carry out a fishery-independent trawl survey to: • improve our understanding of the distribution and abundance of syngnathids in shallow water eastern king prawn (EKP) trawl grounds • collect basic biological information on the syngnathid species within the shallow water EKP trawl grounds • compare the patterns of syngnathid distribution and abundance using the fisheryindependent data collected from the trawl survey to the patterns using fishery-dependent commercial logbook data from the Commerical Fisheries Information System (CFISH) • investigate the relationship between syngnathid distribution and abundance, and assemblages and habitat characteristics. During this fishery-independent assessment, 87 trawl shots were undertaken over a 10 night stratified survey in the shallow water EKP trawl grounds. Information was collected on the syngnathids and the entire community composition collected from the trawls. The information collected was statistically compared against the current syngnathid monitoring information. Over 90% of the total syngnathid catch comprised of pipehorses, S. dunckeri and S. hardwickii. In addition, there were two species of seahorse and one pipefish collected in the survey area. The highest density of all syngnathids was collected in the ‘low effort, zero reported catch’ grid east of Noosa. The shallowest depth where pipehorses were caught in the survey area was 50 m, however no trawls were conducted deeper than 85 m and therefore no maximum depth limit was indicated. There was little correlation in terms of syngnathid abundance between the information gathered from the survey and the daily commercial fishery logbook data used to currently monitor syngnathids. This result indicates that the logbook data alone is not an accurate or comprehensive monitoring tool for fine scale areas of syngnathid distribution and abundance along the Queensland coast. The survey showed that the highest mean abundance of syngnathids occurs in the low trawl effort grids, signifying that syngnathids either prefer habitats not targeted by trawlers or that syngnathid populations in high effort trawl grounds have been reduced due to fishing mortality. Further investigation is required on the biology and preferred habitat of syngnathids to further support either of these potential causal mechanisms. The data presented in this report is limited to providing information on the first three objectives of the study. The final objective is to be assessed at a later date as the data become available, and will be the subject of a future report

Formalin-Induced Fluorescence Reveals Cell Shape and Morphology in Biological Tissue Samples

Ultramicroscopy is a powerful tool to reveal detailed three-dimensional structures of large microscopical objects. Using high magnification, we observed that formalin induces fluorescence more in extra-cellular space and stains cellular structures negatively, rendering cells as dark objects in front of a bright background. Here, we show this effect on a three-dimensional image stack of a hippocampus sample, focusing on the CA1 region. This method, called FIF-Ultramicroscopy, allows for the three-dimensional observation of cellular structures in various tissue types without complicated staining techniques

Aktivierung von Kaliumkanälen durch Transaktivierung von Rezeptortyrosinkinasen in Mäusefibroblasten

In der vorliegenden Arbeit konnte erstmals gezeigt werden, daß über Liganden-gesteuerte Rezeptortyrosinkinasen ein calciumabhängiger spannungsunabhängiger Kaliumstrom transient aktivieren kann. In NIH3T3-Mäusefibroblasten reichte hierzu eine Konzentration von 10 ng/ml Plättchen-Wachstumsfaktor (PDGF) aus. Die pharmakologische Charakterisierung zeigte eine vollständige Blockade des Kaliumstroms durch die Skorpiontoxine Charybdotoxin, Iberiotoxin und Margatoxin in nanomolaren Konzentrationen. Der selektive PDGF-Rezeptortyrosinkinase-Inhibitor AG 1295 verhinderte in NIH3T3-Zellen vollständig einen PDGF-induzierten Kalium-strom. Der epidermale Wachstumsfaktor (EGF) konnte in NIH3T3-Zellen in einer Konzentration bis 100 ng/ml keinen Kaliumstrom induzieren. In EGF-R Zellen, die EGF-Rezeptoren in hoher Dichte überexprimieren, waren sowohl PDGF als auch EGF in der Lage, in einer Konzentration von 10 ng/ml einen spannungsunabhängien, calciumabhängigen Kaliumstrom maximal zu aktivieren. Charakteristisch im Vergleich zur Kaliumstromaktivierung durch den G-Protein-gekoppelten LPA-Rezeptor ist bei den Rezeptortyrosinkinasen zum einen die lange Latenz zwischen Applikation des Wachstumsfaktors und Beginn der Kaliumstrom-aktivierung und zum anderen die langen Inaktivierungszeiten mit 200 s im Vergleich zu LPA mit 50 s. Es konnte gezeigt werden, daß die Wachstumsfaktor-Rezeptoren nach ihrer ersten Stimulation einem Internalisierungsprozeß unterliegen. Eine zweite Kaliumstromaktivierung ist durch EGF bzw. PDGF innerhalb von 30 min nicht möglich, im Gegensatz zum G-Protein-gekoppelten LPA-Rezeptor. Die Kaliumstrom-aktivierung konnte durch die entsprechenden selektiven EGF- bzw. PDGF-Rezeptortyrosinkinase-Inhibitoren AG 1478 und AG 1295 vollständig blockiert werden. Dies zeigt die wichtige Rolle der Rezeptortyrosinkinaseaktivität für die Signalweiterleitung von den Rezeptoren zum Kaliumkanal. Dies wird auch bestätigt durch Experimente an DEL-Zellen, deren EGF-Rezeptoren drei fehlende Auto-phosphorylierungsstellen und deshalb eine verminderte Tyrosinkinaseaktivität haben. Eine Blockade der EGF-Rezeptortyrosinkinase durch den selektiven EGF-Rezeptor-tyrosinkinase-Inhibitor AG 1478 führte auch zu einer Blockade der PDGF-vermittelten Kaliumstromaktivierung und umgekehrt. Die vorliegende Arbeit konnte damit auch erstmals zeigen, daß eine Transaktivierung bzw. ein „cross-talk“ zwischen Wachs-tumsfaktor-Rezeptoren auch hinsichtlich einer Kaliumkanalaktivierung stattfindet