Invasion by mobile aquatic consumers enhances secondary production and increases top-down control of lower trophic levels

Increased biological diversity due to invasion by non-indigenous species (NIS) is a global phenomenon with potential effects on trophic interactions and ecosystem processes in the invaded habitat. We assessed the effects of resource availability and invasion of three non-indigenous invertebrate grazers (two crustaceans and a snail) on secondary production, relative dominance of NIS grazers and resource depletion in experimental freshwater mesocosms. The relative dominance of NIS grazers increased with increasing initial resource availability, although the effect was largest for one of the three species. The effect was due to the fact that all the included non-indigenous grazers were able to expand their populations quickly in response to resource addition. For the most dominating species, the increased grazer diversity due to invasion in turn resulted in higher production of grazer biomass and a more efficient depletion of the periphyton resource. The effect was largest at high initial resource availability, where NIS dominance was most pronounced. Our results show that an invasion-induced increase in species diversity can increase resource depletion and consequently production, but that the effect depends on identity of the introduced species. The results also suggest that properties of the recipient system, such as resource availability, can modulate ecosystem effects of NIS by affecting invader success and dominance.     

Cytokine production using membrane adsorbers: Human basic fibroblast growth factor produced by Escherichia coli

Basic fibroblast growth factor (FGF‐2) is a multifunctional cytokine that regulates various cellular processes both in vitro and in vivo. FGF‐2 is extensively used in embryonic stem cell cultures since it can maintain the cells in an undifferentiated state. However, the high price of FGF‐2 has limited its application in stem cell research. Here we present a fast and efficient process for the purification of FGF‐2 from recombinant Escherichia coli cultures using reusable membrane adsorbers. A high expression level of FGF‐2 (42 mg/g dry cell) was achieved by fed‐batch cultivation of E. coli BL21(DE3). A new combination of cation exchange membrane chromatography and heparin‐sepharose affinity chromatography was used for the purification of the protein. A novel anion exchange membrane chromatography was used in the polishing step to remove endotoxins and DNA. In this new process, about 200 mg soluble FGF‐2 was yielded from 1.9 L culture broth with a purity of 98%. The purified protein was identified to be endotoxin‐free and bioactive. It was successfully tested to keep primate embryonic stem cell and human‐induced pluripotent stem cell pluripotent. Our approach, in which a controlled cultivation process is combined with an optimized fast and versatile downstreaming process, is suitable for low‐cost preparation of bioactive FGF‐2 at bench‐scale and may be beneficial to the effective production of other cytokines.     

Calpains mediate integrin attachment complex maintenance of adult muscle in Caenorhabditis elegans

Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line- or M-line-specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks satellite cells, this mechanism is intrinsic to the muscles and raises the question if such a mechanism also exists in higher metazoans.     

Identification of novel benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein (FLAP)

Pharmacological suppression of leukotriene biosynthesis by 5-lipoxygenase (5-LO)-activating protein (FLAP) inhibitors is a promising strategy to intervene with inflammatory, allergic and cardiovascular diseases. Virtual screening targeting FLAP based on a combined ligand- and structure-based pharmacophore model led to the identification of 1-(2-chlorobenzyl)-2-(1-(4-isobutylphenyl)ethyl)-1H-benzimidazole (7) as developable candidate. Compound 7 potently suppressed leukotriene formation in intact neutrophils (IC(50)=0.31 μM) but essentially failed to directly inhibit 5-LO suggesting that interaction with FLAP causes inhibition of leukotriene synthesis. For structural optimization, a series of 46 benzimidazole-based derivatives of 7 were synthesized leading to more potent analogues (70-72, 82) with IC(50)=0.12-0.19 μM in intact neutrophils. Together, our results disclose the benzimidazole scaffold bearing an ibuprofen fingerprint as a new chemotype for further development of anti-leukotriene agents.     

Genome-wide transcript profiling indicates induction of energy-generating pathways and an adaptive immune response in the liver of sows during lactation

The present study aimed to explore the lactation-induced changes in hepatic gene expression in sows (Sus scrofa) during lactation. Using a porcine whole-genome microarray a total of 632 differentially expressed genes in the liver of lactating compared to non-lactating sows could be identified. Enrichment analysis revealed that the differentially expressed genes were mainly involved in fatty acid metabolism, pyruvate metabolism, glutathione metabolism, glycine, serine and threonine metabolism, citrate cycle, glycerophospholipid metabolism, PPAR signaling, and focal adhesion. The most striking observation with respect to intermediary metabolism was that genes involved in fatty acid catabolism, the catabolism of gluconeogenic amino acids, the citrate cycle and the respiratory chain were up-regulated in the liver of sows during lactation. With respect to immune response, it could be demonstrated that genes encoding acute phase proteins and genes involved in tissue repair were up-regulated and genes encoding adhesion molecules were down-regulated in the liver of sows during lactation. The results indicate that energy-generating pathways and pathways involved in the delivery of gluconeogenic substrates are induced in sow liver during lactation. The alterations of expression of genes encoding proteins involved in immune response suggest that lactation in sows may cause an adaptive immune response that possibly counteracts hepatic inflammation.     

Preparation and Properties of Recombinant Rat and Human Procholecystokinin57 –95

Recombinant human progastrin_6–80 binds two ferric ions with an apparent dissociation constant of 2.2 ± 0.1 μM [Baldwin (2004) Protein J 23:65–70]. The aims of the present study were to express fragments of recombinant procholecystokinin and to determine whether or not they bound ferric ions. Recombinant rat and human procholecystokinin_57–95 were expressed as glutathione S-transferase fusion proteins in E. coli. The fusion proteins were bound to glutathione-agarose, cleaved with thrombin, and purified by reverse phase HPLC. Recombinant procholecystokinin_57–95 did not bind to either the CCK1 or CCK2 receptor with high affinity. No change in absorption spectrum was observed on addition of ferric ions, and analysis of the quenching of tryptophan fluorescence observed in the presence of ferric ions indicated that binding to procholecystokinin_57–95 was at least 40–fold weaker than the binding of ferric ions to progastrin_6–80.     

A high confidence, manually validated human blood plasma protein reference set

Background

The immense diagnostic potential of human plasma has prompted great interest and effort in cataloging its contents, exemplified by the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) pilot project. Due to challenges in obtaining a reliable blood plasma protein list, HUPO later re-analysed their own original dataset with a more stringent statistical treatment that resulted in a much reduced list of high confidence (at least 95%) proteins compared with their original findings. In order to facilitate the discovery of novel biomarkers in the future and to realize the full diagnostic potential of blood plasma, we feel that there is still a need for an ultra-high confidence reference list (at least 99% confidence) of blood plasma proteins.

Methods

To address the complexity and dynamic protein concentration range of the plasma proteome, we employed a linear ion-trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) for mass spectrometry (MS) analysis. Both instruments allow the measurement of peptide masses in the low ppm range. Furthermore, we employed a statistical score that allows database peptide identification searching using the products of two consecutive stages of tandem mass spectrometry (MS3). The combination of MS3 with very high mass accuracy in the parent peptide allows peptide identification with orders of magnitude more confidence than that typically achieved.

Results

Herein we established a high confidence set of 697 blood plasma proteins and achieved a high 'average sequence coverage' of more than 14 peptides per protein and a median of 6 peptides per protein. All proteins annotated as belonging to the immunoglobulin family as well as all hypothetical proteins whose peptides completely matched immunoglobulin sequences were excluded from this protein list. We also compared the results of using two high-end MS instruments as well as the use of various peptide and protein separation approaches. Furthermore, we characterized the plasma proteins using cellular localization information, as well as comparing our list of proteins to data from other sources, including the HUPO PPP dataset.

Conclusion

Superior instrumentation combined with rigorous validation criteria gave rise to a set of 697 plasma proteins in which we have very high confidence, demonstrated by an exceptionally low false peptide identification rate of 0.29%.     

Active transport of the ubiquitin ligase MID1 along the microtubules is regulated by protein phosphatase 2A

Mutations in the MID1 protein have been found in patients with Opitz BBB/G syndrome (OS), which is characterised by multiple malformations of the ventral midline. MID1 is a microtubule-associated protein that stabilizes microtubules and, in association with the regulatory subunit of protein phosphatase 2A (PP2A), alpha4, provides ubiquitin ligase activity for the ubiquitin-specific modification of PP2A. Using Fluorescence Recovery After Photobleaching (FRAP) technology, we show here that MID1 is actively and bi-directionally transported along the microtubules, and that this movement is directly linked to its MAP kinase and PP2A-mediated phosphorylation status. Intact transport depends on both kinesins and dyneins and is inhibited upon colcemide treatments. MID1 proteins carrying missense mutations in the alpha4 binding domain still bind the microtubules but cannot be actively transported. Likewise, knock-down of the alpha4 protein, inhibition of PP2A activity by okadaic acid and fostriecin or the simulation of permanent phosphorylation at Ser96 in MID1 stop the migration of MID1-GFP, while preserving its microtubule-association. In summary, our data uncover an unexpected and novel function for PP2A, its regulatory subunit alpha4 and PP2A/alpha4/mTOR signaling in the active transport of the MID1 ubiquitin ligase complex along the cytoskeleton. Furthermore, a failure in the microtubule directed transport of this protein complex would be an attractive mechanism underlying the pathogenesis of OS in patients with B-box1 mutations.