Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3′→5′ DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.     

Exercise-induced activation of cardiac sympathetic nerve triggers cardioprotection via redox-sensitive activation of eNOS and upregulation of iNOS

We investigated the mechanism of exercise-induced late cardioprotection against ischemia-reperfusion (I/R) injury. C57BL/6 mice received treadmill exercise (60 min/day) for 7 days at a work rate of 60-70% maximal oxygen uptake. Exercise transiently increased oxidative stress and activated endothelial isoform of nitric oxide synthase (eNOS) during exercise and increased expression of inducible isoform of NOS (iNOS) in the heart after 7 days of exercise. The mice were subjected to regional ischemia by 30 min of occlusion of the left coronary artery, followed by 2 h of reperfusion. Infarct size was significantly smaller in the exercised mice. Ablation of cardiac sympathetic nerve by topical application of phenol abolished oxidative stress, activation of eNOS, upregulation of iNOS, and cardioprotection mediated by exercise. Treatment with the antioxidant N-(2-mercaptopropionyl)-glycine during exercise also inhibited activation of eNOS, upregulation of iNOS, and cardioprotection. In eNOS(-/-) mice, exercise-induced oxidative stress was conserved, but upregulation of iNOS and cardioprotection was lost. Exercise did not confer cardioprotection when the iNOS selective inhibitor 1400W was administered just before coronary artery occlusion or when iNOS(-/-) mice were employed. These results suggest that exercise stimulates cardiac sympathetic nerves that provoke redox-sensitive activation of eNOS, leading to upregulation of iNOS, which acts as a mediator of late cardioprotection against I/R injury.     

Temporary blockade of contractility during reperfusion elicits a cardioprotective effect of the p38 MAP kinase inhibitor SB-203580

p38 MAP kinase activation is known to be deleterious not only to mitochondria but also to contractile function. Therefore, p38 MAP kinase inhibition therapy represents a promising approach in preventing reperfusion injury in the heart. However, reversal of p38 MAP kinase-mediated contractile dysfunction may disrupt the fragile sarcolemma of ischemic-reperfused myocytes. We, therefore, hypothesized that the beneficial effect of p38 MAP kinase inhibition during reperfusion can be enhanced when contractility is simultaneously blocked. Isolated and perfused rat hearts were paced at 330 rpm and subjected to 20 min of ischemia followed by reperfusion. p38 MAP kinase was activated after ischemia and early during reperfusion (<30 min). Treatment with the p38 MAP kinase inhibitor SB-203580 (10 microM) for 30 min during reperfusion, but not the c-Jun NH(2)-terminal kinase inhibitor SP-600125 (10 microM), improved contractility but increased creatine kinase release and infarct size. Cotreatment with SB-203580 and the contractile blocker 2,3-butanedione monoxime (BDM, 20 mM) or the ultra-short-acting beta-blocker esmorol (0.15 mM) for the first 30 min during reperfusion significantly reduced creatine kinase release and infarct size. In vitro mitochondrial ATP generation and myocardial ATP content were significantly increased in the heart cotreated with SB-203580 and BDM during reperfusion. Dystrophin was translocated from the sarcolemma during ischemia and reperfusion. SB-203580 increased accumulation of Evans blue dye in myocytes depleted of sarcolemmal dystrophin during reperfusion, whereas cotreatment with BDM facilitated restoration of sarcolemmal dystrophin and mitigated sarcolemmal damage after withdrawal of BDM. These results suggest that treatment with SB-203580 during reperfusion aggravates myocyte necrosis but concomitant blockade of contractile force unmasks cardioprotective effects of SB-203580.     

G protein-coupled receptor kinase 2 mediates endothelin-1-induced insulin resistance via the inhibition of both Galphaq/11 and insulin receptor substrate-1 pathways in 3T3-L1 adipocytes

G protein-coupled receptor kinases (GRKs) regulate seven-transmembrane receptors (7TMRs) by phosphorylating agonist-activated 7TMRs. Recently, we have reported that GRK2 can function as a negative regulator of insulin action by interfering with G protein-q/11 alpha-subunit (Galphaq/11) signaling, causing decreased glucose transporter 4 (GLUT4) translocation. We have also reported that chronic endothelin-1 (ET-1) treatment leads to heterologous desensitization of insulin signaling with decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and Galphaq/11, and decreased insulin-stimulated glucose transport in 3T3-L1 adipocytes. In the current study, we have investigated the role of GRK2 in chronic ET-1-induced insulin resistance. Insulin-induced GLUT4 translocation was inhibited by pretreatment with ET-1 for 24 h, and we found that this inhibitory effect was rescued by microinjection of anti-GRK2 antibody or GRK2 short interfering RNA. We further found that GRK2 mediates the inhibitory effects of ET-1 by two distinct mechanisms. Firstly, adenovirus-mediated overexpression of either wild-type (WT)- or kinase-deficient (KD)-GRK2 inhibited Galphaq/11 signaling, including tyrosine phosphorylation of Galphaq/11 and cdc42-associated phosphatidylinositol 3-kinase activity. Secondly, ET-1 treatment caused Ser/Thr phosphorylation of IRS-1 and IRS-1 protein degradation. Overexpression of KD-GRK2, but not WT-GRK2, inhibited ET-1-induced serine 612 phosphorylation of IRS-1 and restored activation of this pathway. Taken together, these results suggest that GRK2 mediates ET-1-induced insulin resistance by 1) inhibition of Galphaq/11 activation, and this effect is independent of GRK2 kinase activity, and 2) GRK2 kinase activity-mediated IRS-1 serine phosphorylation and degradation.     

Thyrotropin releasing hormone stimulation of GABA-gated but not basal chloride ion influx in rat cerebellum

Potential interaction between gamma-amino butyric acid (GABA) and thyrotropin-releasing hormone (TRH) in eliciting a variety of central nervous system (CNS)-related biologic activities is well known; however, the mechanism underlying this interaction is not clearly defined. To gain further insight into this interaction, we examined the effects of TRH and two of its central nervous system selective analogs, DN 1417 and TA 0910, on basal and GABA-mediated chloride ion influx into rat cerebellar neurosynaptosomes. The results of these studies show that TRH may facilitate GABA action by augmenting chloride ion influx and hyperpolarization of cerebellar neurons.     

Conversion of gastric mucosa to intestinal metaplasia in Cdx2-expressing transgenic mice

Gastric intestinal metaplasia occurs as a pathological condition in the gastric mucosa. To clarify how an intestine-specific homeobox gene, Cdx2, affects the morphogenesis of gastric mucosa, we generated transgenic mice expressing Cdx2 in parietal cells. Until Day 18 after birth, the number of parietal cells inthegastric mucosa of transgenic mice was the same as for their normal littermates. However, at Day 19, we detected several glands in which parietal cells disappeared and the proliferating zone moved from the isthmus to the base of the glands. Thereafter, parietal cells decreased gradually and disappeared at Day 37. All of the gastric mucosal cells, except for enterochromaffin-like (ECL) cells, were completely replaced by intestinal metaplasia, consisting of goblet cells, enteroendocrine cells, and absorptive cells expressing alkaline phosphatase. Pseudopyloric gland metaplasia was also formed. The transgenic mouse is a very useful model for clarifying physiological differentiation of gastric and intestinal cell lineages and analyzing the molecular events from intestinal metaplasia to adenocarcinoma.     

Compilation and characterization of histidine-containing phosphotransmitters implicated in His-to-Asp phosphorelay in plants: AHP signal transducers of Arabidopsis thaliana

Histidine (His)-to-Aspartate (Asp) phosphorelay signal transduction systems are generally made up of a "sensor histidine (His)-kinase", a "response regulator", and a "histidine-containing phosphotransmitter (HPt)". In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that the His-to-Asp phosphorelay mechanism is at least partly responsible for propagation of environmental stimuli, such as phytohormones (e.g. ethylene and cytokinin). Here we compiled the members of the HPt family of phosphotransmitters in Arabidopsis thaliana (AHP-series, Arabidopsis HPt phosphotransmitters), based on both database and experimental analyses, in order to provide a comprehensive basis at the molecular level for understanding the function of the AHP phosphotransmitters that are implicated in the His-to-Asp phosphorelay of higher plants.     

An antibiotic from Penicillium sp. covering the cocoon of the leaf-rolling moth, Dactylioglypha tonica

An antibiotic-producing Penicillium sp. strain was isolated from cocoons of the leaf-rolling moth, Dactylioglypha tonica. An antibacterial compound was isolated from the cultured broth, and the chemical structure of the principle was determined by spectroscopic data to be a derivative of isocoumarincarboxylate.     

Structure-activity relationships of trans-3,5-disubstituted pyrrolidinylthio-1beta-methylcarbapenems. Part 2: J-111,225, J-114,870, J-114,871 and related compounds

Through further derivatization of J-111,347 (1a), a trans-3,5-disubstituted pyrrolidinylthio-1beta-methylcarbapenem, undesired epileptogenicity in a rat intracerebroventricular assay (200 microg/rat) could be eliminated to afford J-111,225 (2a), J-114,870 (3a) and J-114,871 (3b) which preserved comparable broad antimicrobial activity.