Characterization of the interaction of influenza virus NS1 with Akt

Avian influenza viruses belong to the genus influenza A virus of the family Orthomyxoviridae. The influenza virus consists of eight segmented minus stranded RNA that encode 11 known proteins. Among the 11 viral proteins, NS1 (non-structural protein 1, encoded on segment 8) has been implicated in the regulation of several important intra-cellular functions.In this report, we investigated the functional interaction of NS1 with serine threonine kinase Akt, a core intra-cellular survival regulator. In co-immunoprecipitation assays and GST pull-down assays, NS1 directly interacted with Akt. The interaction was mediated primarily through the Akt-PH (Pleckstrin Homology) domain and the RNA-binding domain of NS1. NS1 preferentially interacted with phosphorylated Akt, but not with non-phosphorylated Akt. Functionally, the NS1-Akt interaction enhanced Akt activity both in the intra-cellular context and in in vitro Akt kinase assays. Confocal microscopic analysis revealed that phosphorylated Akt interacted with NS1 during the interphase of the cell cycle predominantly within the nucleus. Finally, mass spectrometric analysis demonstrated the position at Thr215 of NS1 protein is primary phosphorylation target site through Akt activation. The results together supported the functional importance of influenza virus NS1 with Akt, a core intra-cellular survival regulator.     

Evidence that PAR2-triggered prostaglandin E2 (PGE2) formation involves the ERK-cytosolic phospholipase A2-COX-1-microsomal PGE synthase-1 cascade in human lung epithelial cells

We investigated possible involvement of three isozymes of prostaglandin E synthase (PGES), microsomal PGES-1 (mPGES-1), mPGES-2 and cytosolic PGES (cPGES) in COX-2-dependent prostaglandin E(2) (PGE(2)) formation following proteinase-activated receptor-2 (PAR2) stimulation in human lung epithelial cells. PAR2 stimulation up-regulated mPGES-1 as well as COX-2, but not mPGES-2 or cPGES, leading to PGE(2) formation. The PAR2-triggered up-regulation of mPGES-1 was suppressed by inhibitors of COX-1, cytosolic phospholipase A(2) (cPLA(2)) and MEK, but not COX-2. Finally, a selective inhibitor of mPGES-1 strongly suppressed the PAR2-evoked PGE(2) formation. PAR2 thus appears to trigger specific up-regulation of mPGES-1 that is dependent on prostanoids formed via the MEK/ERK/cPLA(2)/COX-1 pathway, being critical for PGE(2) formation.     

Oxygen uptake,heart rate,perceived exertion,and integrated electromyogram of the lower and upper extremities during level and Nordic walking on a treadmill

The purpose of this study was to characterize responses in oxygen uptake ( V·O2), heart rate (HR), perceived exertion (OMNI scale) and integrated electromyogram (iEMG) readings during incremental Nordic walking (NW) and level walking (LW) on a treadmill. Ten healthy adults (four men, six women), who regularly engaged in physical activity in their daily lives, were enrolled in the study. All subjects were familiar with NW. Each subject began walking at 60 m/min for 3 minutes, with incremental increases of 10 m/min every 2 minutes up to 120 m/min V·O2 , V·E and HR were measured every 30 seconds, and the OMNI scale was used during the final 15 seconds of each exercise. EMG readings were recorded from the triceps brachii, vastus lateralis, biceps femoris, gastrocnemius, and tibialis anterior muscles. V·O2 was significantly higher during NW than during LW, with the exception of the speed of 70 m/min (P < 0.01). V·E and HR were higher during NW than LW at all walking speeds (P < 0.05 to 0.001). OMNI scale of the upper extremities was significantly higher during NW than during LW at all speeds (P < 0.05). Furthermore, the iEMG reading for the VL was lower during NW than during LW at all walking speeds, while the iEMG reading for the BF and GA muscles were significantly lower during NW than LW at some speeds. These data suggest that the use of poles in NW attenuates muscle activity in the lower extremities during the stance and push-off phases, and decreases that of the lower extremities and increase energy expenditure of the upper body and respiratory system at certain walking speeds.     

Suppressive effects of 4-phenylbutyrate on the aggregation of Pael receptors and endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is defined as an accumulation of unfolded proteins in the endoplasmic reticulum. 4-phenylbutyrate (4-PBA) has been demonstrated to promote the normal trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutant from the ER to the plasma membrane and to restore activity. We have reported that 4-PBA protected against cerebral ischemic injury and ER stress-induced neuronal cell death. In this study, we revealed that 4-PBA possesses chemical chaperone activity in vitro, which prevents the aggregation of denatured alpha-lactalbumin and bovine serum albumin (BSA). Furthermore, we investigated the effects of 4-PBA on the accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R) pathologically relevant to the loss of dopaminergic neurons in autosomal recessive juvenile parkinsonism (AR-JP). Interestingly, 4-PBA restored the normal expression of Pael-R protein and suppressed ER stress induced by the overexpression of Pael-R. In addition, we showed that 4-PBA attenuated the activation of ER stress-induced signal transduction pathways and subsequent neuronal cell death. Moreover, 4-PBA restored the viability of yeasts that fail to induce an ER stress response under ER stress conditions. These results suggest that 4-PBA suppresses ER stress by directly reducing the amount of misfolded protein, including Pael-R accumulated in the ER.     

A Dictyostelium cellobiohydrolase orthologue that affects developmental timing

Dictyostelium discoideum is a facultative multicellular amoebozoan with cellulose in the stalk and spore coat of its fruiting body as well as in the extracellular matrix of the migrating slug. The organism also harbors a number of cellulase genes. One of them, cbhA, was identified as a candidate cellobiohydrolase gene based on the strong homology of its predicted protein product to fungal cellobiohydrolase I (CBHI). Expression of the cbhA was developmentally regulated, with strong expression in the spores of the mature fruiting body. However, a weak but detectable level of expression was observed in the extracellular matrix at the mound — tipped finger stages, in prestalk O cells, and in the slime sheath of the migrating slug — late culminant stages. A null mutant of the cbhA showed almost normal morphology. However, the developmental timing of the mutant was delayed by 2–4 h. When a c-Myc epitope-tagged CbhA was expressed, it was secreted into the culture medium and was able to bind crystalline cellulose. The CbhA-myc protein was glycosylated, as demonstrated by its ability to bind succinyl concanavalin A-agarose. Moreover, conditioned medium from the cbhA-myc ^oe strain displayed 4-methylumbelliferyl β-d-cellobioside (4-MUC) digesting activity in Zymograms in which conditioned medium was examined via native-polyacrylamide gel electrophoresis or spotted on an agar plate containing 4-MUC, one of the substrates of cellobiohydrolase. Taken together, these findings indicate that Dictyostelium CbhA is an orthologue of CBH I that is required for a normal rate of development.     

Angiotensin II type 1 (AT1)-receptor blocker prevents impairment of endothelium-dependent cerebral vasodilation by acute cigarette smoking in rats

Our aim was to test for smoking-induced endothelial dysfunction in rat cerebral vessels, then to evaluate the effect of valsartan [angiotensin II type I (AT1)-receptor blocker] on that impairment. In pentobarbital-anesthetized, mechanically ventilated Sprague-Dawley rats, we used a cranial window preparation to measure changes in pial vessel diameters following topical applications of acetylcholine (Ach) (before and after smoking or intravenous nicotine infusion; n = 6 in each group), and adenosine (n = 6 for before and after smoking). Then, after intravenous valsartan pretreatment we reexamined the pial vasodilator response to topical Ach (before and after cigarette smoking). Under control conditions, cerebral arterioles were dilated by 6.9 +/- 4.2% and 13.6 +/- 4.8% by topical Ach (10(-6) M and 10(-5) M, respectively) and by 6.4 +/- 2.5% and 12.2 +/- 3.1% by topical adenosine (10(-5) M and 10(-4) M, respectively). One hour after a 1-min inhalation of mainstream smoke (1-mg nicotine cigarette), 10(-5) M Ach constricted cerebral arterioles (-4.4 +/- 4.1%), while 10(-4) M adenosine dilated them by 13.4 +/- 3.4%. One hour after a 1-min nicotine infusion (0.05 mg), 10(-5) M Ach dilated cerebral arterioles by 9.9 +/- 2.4%. Thus, vasodilator response to topical Ach was impaired after smoking, whereas that to adenosine was unaffected. However, the vasodilator response to Ach was unaffected by intravenous nicotine. Valsartan prevented smoking from impairing Ach-induced vasodilation. In conclusion, acute single-cigarette smoking causes a dysfunction of endothelium-dependent, but not endothelium-independent, vasodilation of rat cerebral vessels in vivo, and the effect was not mimicked by intravenous nicotine. AT1-receptor blockade prevented the above smoking-induced impairment of endothelium-dependent vasodilation.     

Antimalarial activity enhancement in hydroxymethylcarbonyl (HMC) isostere-based dipeptidomimetics targeting malarial aspartic protease plasmepsin

Plasmepsin (Plm) is a potential target for new antimalarial drugs, but most reported Plm inhibitors have relatively low antimalarial activities. We synthesized a series of dipeptide-type HIV protease inhibitors, which contain an allophenylnorstatine-dimethylthioproline scaffold to exhibit potent inhibitory activities against Plm II. Their activities against Plasmodium falciparum in the infected erythrocyte assay were largely different from those against the target enzyme. To improve the antimalarial activity of peptidomimetic Plm inhibitors, we attached substituents on a structure of the highly potent Plm inhibitor KNI-10006. Among the derivatives, we identified alkylamino compounds such as 44 (KNI-10283) and 47 (KNI-10538) with more than 15-fold enhanced antimalarial activity, to the sub-micromolar level, maintaining their potent Plm II inhibitory activity and low cytotoxicity. These results suggest that auxiliary substituents on a specific basic group contribute to deliver the inhibitors to the target Plm.