System analysis shows distinct mechanisms and common principles of nuclear envelope protein dynamics

The nuclear envelope contains >100 transmembrane proteins that continuously exchange with the endoplasmic reticulum and move within the nuclear membranes. To better understand the organization and dynamics of this system, we compared the trafficking of 15 integral nuclear envelope proteins using FRAP. A surprising 30-fold range of mobilities was observed. The dynamic behavior of several of these proteins was also analyzed after depletion of ATP and/or Ran, two functions implicated in endoplasmic reticulum-inner nuclear membrane translocation. This revealed that ATP- and Ran-dependent translocation mechanisms are distinct and not used by all inner nuclear membrane proteins. The Ran-dependent mechanism requires the phenylalanine-glycine (FG)-nucleoporin Nup35, which is consistent with use of the nuclear pore complex peripheral channels. Intriguingly, the addition of FGs to membrane proteins reduces FRAP recovery times, and this also depends on Nup35. Modeling of three proteins that were unaffected by either ATP or Ran depletion indicates that the wide range in mobilities could be explained by differences in binding affinities in the inner nuclear membrane.     

Bioactivity,Chemical Profiling,and 16S rRNA-Based Phylogeny of <Emphasis Type="BoldItalic">Pseudoalteromonas</Emphasis> Strains Collected on a Global Research Cruise

One hundred one antibacterial Pseudoalteromonas strains that inhibited growth of a Vibrio anguillarum test strain were collected on a global research cruise (Galathea 3), and 51 of the strains repeatedly demonstrated antibacterial activity. Here, we profile secondary metabolites of these strains to determine if particular compounds serve as strain or species markers and to determine if the secondary metabolite profile of one strain represents the bioactivity of the entire species. 16S rRNA gene similarity divided the strains into two primary groups: One group (51 strains) consisted of bacteria which retained antibacterial activity, 48 of which were pigmented, and another group (50 strains) of bacteria which lost antibacterial activity upon sub-culturing, two of which were pigmented. The group that retained antibacterial activity consisted of six clusters in which strains were identified as Pseudoalteromonas luteoviolacea, Pseudoalteromonas aurantia, Pseudoalteromonas phenolica, Pseudoalteromonas ruthenica, Pseudoalteromonas rubra, and Pseudoalteromonas piscicida. HPLC-UV/VIS analyses identified key peaks, such as violacein in P. luteoviolacea. Some compounds, such as a novel bromoalterochromide, were detected in several species. HPLC-UV/VIS detected systematic intra-species differences for some groups, and testing several strains of a species was required to determine these differences. The majority of non-antibacterial, non-pigmented strains were identified as Pseudoalteromonas agarivorans, and HPLC-UV/VIS did not further differentiate this group. Pseudoalteromonas retaining antibacterial were more likely to originate from biotic or abiotic surfaces in contrast to planktonic strains. Hence, the pigmented, antibacterial Pseudoalteromonas have a niche specificity, and sampling from marine biofilm environments is a strategy for isolating novel marine bacteria that produce antibacterial compounds.     

Wide distribution of closely related, antibiotic-producing Arthrobacter strains throughout the Arctic Ocean

We isolated 16 antibiotic-producing bacterial strains throughout the central Arctic Ocean, including seven Arthrobacter spp. with almost identical 16S rRNA gene sequences. These strains were numerically rare, as revealed using 454 pyrosequencing libraries. Arthrobacter spp. produced arthrobacilins A to C under different culture conditions, but other, unidentified compounds likely contributed to their antibiotic activity.     

Thrombelastographic haemostatic status and antiplatelet therapy after coronary artery bypass surgery (TEG-CABG trial): assessing and monitoring the antithrombotic effect of clopidogrel and aspirin versus aspirin alone in hypercoagulable patients: study protocol for a randomized controlled trial

ABSTRACT: BACKGROUND: Hypercoagulability, assessed by the thrombelastography (TEG) assay, has in several observational studies been associated with an increased risk of post-procedural thromboembolic complications. We hypothesize that intensified antiplatelet therapy with clopidogrel and aspirin, as compared to aspirin alone, will improve saphenous vein graft patency in preoperatively TEG-Hypercoagulable coronary artery bypass surgery (CABG) patients and reduce their risk for thromboembolic complications and death postoperatively. METHODS: This is a prospective randomized clinical trial, with an open-label design with blinded evaluation of graft patency. TEG-Hypercoagulability is defined as a TEG maximum amplitude above 69 mm. Two hundred and fifty TEG-Hypercoagulable patients will be randomized to either an interventional group receiving clopidogrel 75 mg daily for three months (after initial oral bolus of 300 mg) together with aspirin 75 mg or a control group receiving aspirin 75 mg daily alone. Monitoring of antiplatelet efficacy and on-treatment platelet reactivity to clopidogrel and aspirin will be conducted with Multiplate aggregometry. Graft patency will be assessed with Multislice computed tomography (MSCT) at three months after surgery. CONCLUSIONS: The present trial is the first randomized clinical trial to evaluate whether TEG-Hypercoagulable CABG patients will benefit from intensified antiplatelet therapy after surgery. Monitoring of platelet inhibition from instituted antithrombotic therapy will elucidate platelet resistance patterns after CABG surgery. The results could be helpful in redefining how clinicians can evaluate patients preoperatively for their postoperative thromboembolic risk and tailor individualized postoperative antiplatelet therapy. Trial registration Clinicaltrials.gov Identifier NCT01046942.     

A BRET assay for monitoring insulin receptor interactions and ligand pharmacology

The insulin receptor (IR) belongs to the receptor tyrosine kinase super family and plays an important role in glucose homeostasis. The receptor interacts with several large docking proteins that mediate signaling from the receptor, including the insulin receptor substrate (IRS) family and Src homology-2-containing proteins (Src). Here, we applied the bioluminescence resonance energy transfer 2 (BRET2) technique to study the IR signaling pathways. The interaction between the IR and the substrates IRS1, IRS4 and Shc was examined in response to ligands with different signaling properties. The association between IR and the interacting partners could successfully be monitored when co-expressing green fluorescent protein 2 (GFP2) tagged substrates with Renilla reniformis luciferase 8 (Rluc8) tagged IR. Through additional optimization steps, we developed a stable and flexible BRET2 assay for monitoring the interactions between the IR and its substrates. Furthermore, the insulin analogue X10 was characterized in the BRET2 assay and was found to be 10 times more potent with respect to IRS1, IRS4 and Shc recruitment compared to human insulin. This study demonstrates that the BRET2 technique can be applied to study IR signaling pathways, and that this assay can be used as a platform for screening and characterization of IR ligands.     

Variable responses of waterfowl breeding populations to long-term removal of introduced American mink

It is suspected that feral American mink, an introduced predator in Europe, have seriously affected local densities of birds breeding in archipelagos and coastal areas. We studied the effects of mink removal on breeding densities of waterfowl in two manipulation and two control areas in the outer archipelago of SW Finland, Baltic Sea. The study was conducted in two phases: during 1992–2001 a total of 98 mink was removed from 60 islands and islets (total area 72 km²) whereas on 37 islands and islets (35 km²) mink was not removed. Additional mink removal and control areas were established during 1998–2001 to replicate the experiment. The breeding densities of the shelduck, tufted duck and the velvet scoter increased as a response to mink removal, while in the control areas their populations remained unchanged. The breeding densities of mallards increased during the first 7 yr of mink removal, but a steep decrease in the last study year resulted in a statistically non-significant overall increase. The species with low breeding densities (the gadwall, northern shoveler, pintail and the red-breasted merganser) increased as well. In contrast, the populations of large waterfowl species, the mute swan, greylag goose, common eider and the goosander, did not show obvious increases in breeding densities after mink removal. We conclude that feral mink may locally limit the breeding densities of some smaller waterfowl species and thus reduce the diversity of the waterfowl community in the outer archipelago.     

Human GLTP and mutant forms of ACD11 suppress cell death in the Arabidopsis acd11 mutant

The Arabidopsis acd11 mutant exhibits runaway, programmed cell death due to the loss of a putative sphingosine transfer protein (ACD11) with homology to mammalian GLTP. We demonstrate that transgenic expression in Arabidopsis thaliana of human GLTP partially suppressed the phenotype of the acd11 null mutant, resulting in delayed programmed cell death development and plant survival. Surprisingly, a GLTP mutant form impaired in glycolipid transfer activity also complemented the acd11 mutants. To understand the relationship between functional complementarity and transfer activity, we generated site-specific mutants in ACD11 based on homologous GLTP residues required for glycolipid transfer. We show that these ACD11 mutant forms are impaired in their in vitro transfer activity of sphingolipids. However, transgenic expression of these mutant forms fully complemented acd11 mutant cell death, and transgenic plants showed normal induction of hypersensitive cell death upon infection with avirulent strains of Pseudomonas syringae. The significance of these findings with respect to the function(s) of ACD11 in sphingolipid transport and cell death regulation is discussed.