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.     

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.     

Evidence for genomic regulation of the telomeric activity in Drosophila melanogaster

The structural integrity of TART elements has been used as reporter of instability at chromosomal ends in numerous Drosophila stocks and over time in an unstable stock. The results show that telomeric activity is a regulated process that may differ between the stocks as well as over time within a stock. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of phosphorylation sites in protein kinase A substrates using artificial neural networks and mass spectrometry

Protein phosphorylation plays a key role in cell regulation and identification of phosphorylation sites is important for understanding their functional significance. Here, we present an artificial neural network algorithm: NetPhosK (http://www.cbs.dtu.dk/services/NetPhosK/) that predicts protein kinase A (PKA) phosphorylation sites. The neural network was trained with a positive set of 258 experimentally verified PKA phosphorylation sites. The predictions by NetPhosK were validated using four novel PKA substrates: Necdin, RFX5, En-2, and Wee 1. The four proteins were phosphorylated by PKA in vitro and 13 PKA phosphorylation sites were identified by mass spectrometry. NetPhosK was 100% sensitive and 41% specific in predicting PKA sites in the four proteins. These results demonstrate the potential of using integrated computational and experimental methods for detailed investigations of the phosphoproteome.     

Structural basis for a direct interaction between FGFR1 and NCAM and evidence for a regulatory role of ATP

The neural cell adhesion molecule (NCAM) promotes axonal outgrowth, presumably through an interaction with the fibroblast growth factor receptor (FGFR). NCAM also has a little-understood ATPase activity. We here demonstrate for the first time a direct interaction between NCAM (fibronectin type III [F3] modules 1 and 2) and FGFR1 (Ig modules 2 and 3) by surface plasmon resonance (SPR) analysis. The structure of the NCAM F3 module 2 was determined by NMR and the module was shown by NMR to interact with the FGFR1 Ig module 3 and ATP. The NCAM sites binding to FGFR and ATP were found to overlap and ATP was shown by SPR to inhibit the NCAM-FGFR binding, indicating that ATP probably regulates the NCAM-FGFR interaction. Furthermore, we demonstrate that the NCAM module was able to induce activation (phosphorylation) of FGFR and to stimulate neurite outgrowth. In contrast, ATP inhibited neurite outgrowth induced by the module.     

Effects of PYY1-36 and PYY3-36 on appetite, energy intake, energy expenditure, glucose and fat metabolism in obese and lean subjects

Nitric oxide (NO) and 5'-AMP-activated protein kinase (AMPK) are involved in glucose transport and mitochondrial biogenesis in skeletal muscle. Here, we examined whether NO regulates the expression of the major glucose transporter in muscle (GLUT4) and whether it influences AMPK-induced upregulation of GLUT4. At low levels, the NO donor S-nitroso-N-penicillamine (SNAP, 1 and 10 microM) significantly increased GLUT4 mRNA ( approximately 3-fold; P < 0.05) in L6 myotubes, and cotreatment with the AMPK inhibitor compound C ablated this effect. The cGMP analog 8-bromo-cGMP (8-Br-cGMP, 2 mM) increased GLUT4 mRNA by approximately 50% (P < 0.05). GLUT4 protein expression was elevated 40% by 2 days treatment with 8-Br-cGMP, whereas 6 days treatment with 10 microM SNAP increased GLUT4 expression by 65%. Cotreatment of cultures with the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one prevented the SNAP-induced increase in GLUT4 protein. SNAP (10 microM) also induced significant phosphorylation of alpha-AMPK and acetyl-CoA carboxylase and translocation of phosphorylated alpha-AMPK to the nucleus. Furthermore, L6 myotubes exposed to 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) for 16 h presented an approximately ninefold increase in GLUT4 mRNA, whereas cotreatment with the non-isoform-specific NOS inhibitor N(G)-nitro-l-arginine methyl ester, prevented approximately 70% of this effect. In vivo, GLUT4 mRNA was increased 1.8-fold in the rat plantaris muscle 12 h after AICAR injection, and this induction was reduced by approximately 50% in animals cotreated with the neuronal and inducible nitric oxide synthases selective inhibitor 1-(2-trifluoromethyl-phenyl)-imidazole. We conclude that, in skeletal muscle, NO increases GLUT4 expression via a cGMP- and AMPK-dependent mechanism. The data are consistent with a role for NO in the regulation of AMPK, possibly via control of cellular activity of AMPK kinases and/or AMPK phosphatases.