Subcellular targeting and agonist-induced site-specific phosphorylation of endothelial nitric-oxide synthase

The endothelial isoform of nitric-oxide synthase (eNOS) undergoes a complex pattern of covalent modifications, including acylation with the fatty acids myristate and palmitate as well as phosphorylation on multiple sites. eNOS acylation is a key determinant for the reversible subcellular targeting of the enzyme to plasmalemmal caveolae. We transfected a series of hemagglutinin epitope-tagged eNOS mutant cDNAs deficient in palmitoylation (palm(-)) and/or myristoylation (myr(-)) into bovine aortic endothelial cells; after treatment with the eNOS agonists sphingosine 1-phosphate or vascular endothelial growth factor, the recombinant eNOS was immunoprecipitated using an antibody directed against the epitope tag, and patterns of eNOS phosphorylation were analyzed in immunoblots probed with phosphorylation state-specific eNOS antibodies. The wild-type eNOS underwent agonist-induced phosphorylation at serine 1179 (a putative site for phosphorylation by kinase Akt), but phosphorylation of the myr(-) eNOS at this residue was nearly abrogated; the palm(-) eNOS exhibited an intermediate phenotype. The addition of the CD8 transmembrane domain to the amino terminus of eNOS acylation-deficient mutants rescued the wild-type phenotype of robust agonist-induced serine 1179 phosphorylation. Thus, membrane targeting, but not necessarily acylation, is the critical determinant for agonist-promoted eNOS phosphorylation at serine 1179. In striking contrast to serine 1179, phosphorylation of eNOS at serine 116 was enhanced in the myr(-) eNOS mutant and was markedly attenuated in the CD8-eNOS membrane-targeted fusion protein. We conclude that eNOS targeting differentially affects eNOS phosphorylation at distinct sites in the protein and suggest that the inter-relationships of eNOS acylation and phosphorylation may modulate eNOS localization and activity and thereby influence NO signaling pathways in the vessel wall.     

15-Deoxy-delta 12,14-prostaglandins D2 and J2 are potent activators of human eosinophils

15-Deoxy-Delta(12,14)-PDJ(2) (15d-PGJ(2)) is a degradation product of PGD(2) that has been proposed as an anti-inflammatory compound because of its various inhibitory effects, some of which are mediated by peroxisome proliferator-activated receptor-gamma. In contrast to its reported inhibitory effects on macrophages and other cells, we found that this compound is a potent activator of eosinophils, inducing calcium mobilization, actin polymerization, and CD11b expression. It is selective for eosinophils, having little or no effect on neutrophils or monocytes. 15d-PGJ(2) has an EC(50) of approximately 10 nM, similar to that of its precursor, PGD(2). The concentrations of 15d-PGJ(2) required to activate eosinophils are thus much lower than those required for its anti-inflammatory effects (usually micromolar). 15-Deoxy-Delta(12,14)-prostaglandin D(2) (15d-PGD(2)) is also a potent activator of eosinophils, with an EC(50) about the same as that of PGD(2), whereas Delta(12)-PGJ(2) is slightly less potent. Eosinophils pretreated with PGD(2) no longer respond to 15d-PGJ(2), and vice versa, but in both cases the cells still respond to another eicosanoid proinflammatory mediator, 5-oxo-6,8,11,14-eicosatetraenoic acid. This indicates that the effects of 15d-PGJ(2) are mediated by the DP(2)/chemoattractant receptor-homologous molecule expressed on Th2 cells that has recently been identified in eosinophils. 15d-PGJ(2) is selective for the DP(2) receptor, in that it has no effect on DP(1) receptor-mediated adenylyl cyclase activity in platelets. We conclude that 15d-PGJ(2) and 15d-PGD(2) are selective DP(2) receptor agonists that activate human eosinophils with potencies at least 100 times greater than those for the proposed anti-inflammatory effects of 15d-PGJ(2) on other cells.     

Blazeispirane and protoblazeispirane derivatives from the cultured mycelia of the fungus Agaricus blazei

Two blazeispirane derivatives including blazeispirols G and I were isolated from the cultured mycelia of the fungus Agaricus blazei Murill and were established to be (20S, 22S, 23R, 24S)-14 beta,22: 22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9-triene-11 alpha,23-diol and (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9,11-tetraene-23,28-diol by comparison of extensive 1D and 2D NMR spectral data with that of blazeispirol A. Furthermore, four blazeispirol derivatives blazeispirols, U, V, V(1) and Z(1) were isolated form the same source described above. Their structures were determined to be (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-23-hydroxyergosta-4,6,8,11-tetraen-3-one, (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-6 alpha,7 alpha,23-trihydroxyergosta-4,8,11-trien-3-one, (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-6 beta,7 alpha,23-trihydroxyergosta-4,8,11-trien-3-one and (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-23-hydroxy-4,5-seco-ergosta-6,8-diene-3,5-dione by extensive 1 D and 2D NMR spectral data.     

Involvement of tachykinin NK(1) and NK(2) receptors in changes in lung mechanics and airway microvascular leakage during the early phase of endotoxemia in Guinea pigs

We investigated the role of tachykinins in airway neurogenic responses occurring in the early phase of endotoxemia. Forty-eight anesthetized guinea pigs were evenly divided into six groups pretreated with either saline vehicle, CP-96,345 (a tachykinin NK(1) receptor antagonist), SR-48,968 (a tachykinin NK(2) receptor antagonist) or CP-96,345 and SR-48,968 in combination. Animals then received an intravenous injection of either saline (the vehicle for endotoxin) or endotoxin (30 mg/kg). Total lung resistance (R(L)) and dynamic lung compliance (C(dyn)) were continuously measured before and 30 min after administration of saline or endotoxin. Airway microvascular leakage was assessed at the end of the observation period. Endotoxin significantly increased R(L) and decreased C(dyn) 10 min after intravenous endotoxin injection. Plasma extravasation significantly increased in the trachea, main bronchi and intrapulmonary airways with endotoxin administration. These changes in lung mechanics were abolished by SR-48,968, but were unaffected by CP-96,345. The plasma extravasation was largely attenuated by CP-96,345 and/or SR-48,968. We conclude that (1) endogenous tachykinins play an important role in producing changes in lung mechanics and airway microvascular leakage during the early phase of endotoxemia and (2) activation of tachykinin NK(2) receptors is responsible for the former response, while activation of both tachykinin NK(1) and NK(2) receptors is involved in the latter response.     

Allatotropic activity in the suboesophageal ganglia and corpora cardiaca of the adult male loreyi leafworm, Mythimna loreyi

Allatotropic activity was found in the methanolic extract of the suboesophageal ganglia (SOG) and the corpora cardiaca (CC) of the Mythimna loreyi virgin males. No allatotropic activity was observed in the extract of brain or corpora allata (CA). Although CA can be activated by the SOG and CC extract, respectively, CC extract inhibited the response to the SOG extract. A significant in vitro allatotropic effect was exerted by the SOG and CC extract within 10 and 15 min, respectively, and this effect can be sustained for several hours even after transferring to fresh medium without extracts. The time course pattern of the CA activation ratio in both the SOG and CC extract-treated group is very similar to, but with significantly higher level than, that in the control group, suggesting the existence of an intrinsic pacemaker or an in vitro effect that controls the fluctuation of the CA biosynthetic activity. Synthetic Manduca sexta allatotropin had no significant effect on the M. loreyi CA. The results of treatment with the adenylate cyclase activator forskolin, the phosphodiesterase inhibitor IBMX, and the cAMP analogue dibutyryl-cAMP did not indicate that cAMP might be involved in the allatotropic control of CA. Arch.     

Wood smoke-induced airway hyperreactivity in guinea pigs: time course, and role of leukotrienes and hydroxyl radical

A prior airway exposure to wood smoke induces a tachykinin-dependent increase in airway responsiveness to the subsequent smoke inhalation in guinea pigs (Life Sci. 63: 1513, 1998). To further investigate the time course of, and the contribution of other chemical mediators to, this smoke-induced airway hyperresponsiveness (SIAHR), two smoke challenges (each 10 ml) separated by 30 min were delivered into the lungs of anesthetized guinea pigs by a respirator. In the control animals, the SIAHR was evidenced by the bronchoconstrictive response to the second smoke challenge (SM2) which was approximately 5.2-fold greater than that to the first challenge (SM1). This SIAHR was alleviated by shortening the elapsed time between SM1 and SM2 to 10 min or by extending it to 60 min, and was abolished by extending it to 120 min. This SIAHR was reduced by pretreatment with either MK-571 (a leukotriene D4-receptor antagonist) or dimethylthiourea (a hydroxyl radical scavenger), but was not affected by pretreatment with either pyrilamine (a histamine H1-receptor antagonist) or indomethacin (a cyclooxygenase inhibitor). The smoke-induced reduction in the neutral endopeptidase activity (a major enzyme for tachykinin degradation) measured in airway tissues excised 30 min post SM1 was largely prevented by pretreatment with dimethylthiourea. However, this reduction was not seen in airway tissues excised 120 min post SM1. These results suggest that 1) the SIAHR to inhaled wood smoke has a rapid onset time following smoke inhalation and lasts for less than two hours, 2) leukotrienes and hydroxyl radical may play contributory roles in the development of this SIAHR, and 3) hydroxyl radical is the major factor responsible for the smoke-induced inactivation of airway neutral endopeptidase, which may possibly participate in the development of this SIAHR.     

The Cancer Mutation D83V Induces an α-Helix to β-Strand Conformation Switch in MEF2B

MEF2B is a major target of somatic mutations in non-Hodgkin lymphoma. Most of these mutations are non-synonymous substitutions of surface residues in the MADS-box/MEF2 domain. Among them, D83V is the most frequent mutation found in tumor cells. The link between this hotspot mutation and cancer is not well understood. Here we show that the D83V mutation induces a dramatic α-helix to β-strand switch in the MEF2 domain. Located in an α-helix region rich in β-branched residues, the D83V mutation not only removes the extensive helix stabilization interactions but also introduces an additional β-branched residue that further shifts the conformation equilibrium from α-helix to β-strand. Cross-database analyses of cancer mutations and chameleon sequences revealed a number of well-known cancer targets harboring β-strand favoring mutations in chameleon α-helices, suggesting a commonality of such conformational switch in certain cancers and a new factor to consider when stratifying the rapidly expanding cancer mutation data.     

A functional NQO1 609C>T polymorphism and risk of gastrointestinal cancers: a meta-analysis

Background

The functional polymorphism (rs1800566) in the NQO1 gene, a 609C>T substitution, leading to proline-to-serine amino-acid and enzyme activity changes, has been implicated in cancer risk, but individually published studies showed inconclusive results.

Methodology/Principal Findings

We performed a meta-analysis of 20 publications with a total of 5,491 cases and 5,917 controls, mainly on gastrointestinal (GI) cancers. We summarized the data on the association between the NQO1 609C>T polymorphism and risk of GI cancers and performed subgroup analyses by ethnicity, cancer site, and study quality. We found that the variant CT heterozygous and CT/TT genotypes of the NQO1 609 C>T polymorphism were associated with a modestly increased risk of GI cancers (CT vs. CC: OR = 1.10, 95% CI = 1.01 – 1.19, P _{heterogeneity} = 0.27, I ² = 0.15; CT/TT vs. CC: OR = 1.11, 95%CI = 1.02 – 1.20, P _{heterogeneity} = 0.14; I ² = 0.27). Following further stratified analyses, the increased risk was only observed in subgroups of Caucasians, colorectal cancer in Caucasians, and high quality studies.

Conclusions

This meta-analysis suggests that the NQO1 609T allele is a low-penetrance risk factor for GI cancers. Although the effect on GI cancers may be modified by ethnicity and cancer sites, small sample seizes of the subgroup analyses suggest that further larger studies are needed, especially for non-colorectal GI cancers in Caucasians and GI cancers in Asians.     

α-Lipoic acid ameliorates impaired glucose uptake in LYRM1 overexpressing 3T3-L1 adipocytes through the IRS-1/Akt signaling pathway

Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.     

Diploid hybrid origin of Hippophaë gyantsensis (Elaeagnaceae) in the western Qinghai–Tibet Plateau

Homoploid hybrid speciation, the origin of a hybrid species without change in chromosome number, is currently considered to be a rare form of speciation. In the present study, we examined the phylogenetic origin of Hippophaë gyantsensis, a diploid species occurring in the western Qinghai–Tibet Plateau. Some of its morphological and molecular traits suggest a close relationship to H. rhamnoides ssp. yunnanensis while others indicate H. neurocarpa. We conducted phylogenetic analyses of sequence data of two maternally inherited chloroplast (cp) DNA fragments and the bi‐parentally inherited nuclear ribosomal internal transcribed spacer (ITS) from 17 populations of H. gyantsensis, 15 populations of H. rhamnoides ssp. yunnanensis and 27 populations of H. neurocarpa across their distributional ranges, and modelled the niche differentiation of the three taxa. Multiple lines of evidence suggested that H. gyantsensis is a morphologically stable, genetically independent and ecologically distinct species. The inconsistent phylogenetic placements of the H. gyantsensis clade that comprised the dominant cpDNA haplotypes and ITS ribotypes suggested a probable diploid hybrid origin from multiple crosses between H. rhamnoides ssp. yunnanensis and H. neurocarpa. This tentative hypothesis is more parsimonious than alternative explanations according to the data available, although more evidence based on further testing is needed.