Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs

The catalytic subunit of human cytomegalovirus (HCMV) DNA polymerase pUL54 is a 1242-amino-acid protein, whose function, stimulated by the processivity factor, phosphoprotein UL44 (ppUL44), is essential for viral replication. The C-terminal residues (amino acids 1220-1242) of pUL54 have been reported to be sufficient for ppUL44 binding in vitro. Although believed to be important for functioning in the nuclei of infected cells, no data are available on either the interaction of pUL54 with ppUL44 in living mammalian cells or the mechanism of pUL54 nuclear transport and its relationship with that of ppUL44. The present study examines for the first time the nuclear import pathway of pUL54 and its interaction with ppUL44 using dual color, quantitative confocal laser scanning microscopy on live transfected cells and quantitative gel mobility shift assays. We showed that of two nuclear localization signals (NLSs) located at amino acids 1153-1159 (NLSA) and 1222-1227 (NLSB), NLSA is sufficient to confer nuclear localization on green fluorescent protein (GFP) by mediating interaction with importin alpha/beta. We also showed that pUL54 residues 1213-1242 are sufficient to confer ppUL44 binding abilities on GFP and that pUL54 and ppUL44 can be transported to the nucleus as a complex. Our work thus identified distinct sites within the HCMV DNA polymerase, which represent potential therapeutic targets and establishes the molecular basis of UL54 nuclear import.     

A novel natural product compound enhances cAMP-regulated chloride conductance of cells expressing CFTR[delta]F508

BACKGROUND: Cystic fibrosis (CF) results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel localized at the plasma membrane of diverse epithelia. The most common mutation leading to CF, Delta F508, occurs in the first nucleotide-binding domain (NBD1) of CFTR. The Delta F508 mutation disrupts protein processing, leading to a decreased level of mutant channels at the plasma membrane and reduced transepithelial chloride permeability. Partial correction of the Delta F508 molecular defect in vitro is achieved by incubation of cells with several classes of chemical chaperones, indicating that further investigation of novel small molecules is warranted as a means for producing new therapies for CF. MATERIALS AND METHODS: The yeast two-hybrid assay was used to study the effect of CF-causing mutations on the ability of NBD1 to self-associate and form dimers. A yeast strain demonstrating defective growth as a result of impaired NBD1 dimerization due to Delta F508 was used as a drug discovery bioassay for the identification of plant natural product compounds restoring mutant NBD1 interaction. Active compounds were purified and the chemical structures determined. The purified compounds were tested in epithelial cells expressing CFTR Delta F508 and the resulting effect on transepithelial chloride permeability was assessed using short-circuit chloride current measurements. RESULTS: Wild-type NBD1 of CFTR forms homodimers in a yeast two-hybrid assay. CF-causing mutations within NBD1 that result in defective processing of CFTR (Delta F508, Delta I507, and S549R) disrupted NBD1 interaction in yeast. In contrast, a CF-causing mutation that does not impair CFTR processing (G551D) had no effect on NBD1 dimerization. Using the yeast-based assay, we identified a novel limonoid compound (TS3) that corrected the Delta F508 NBD1 dimerization defect in yeast and also increased the chloride permeability of Fisher Rat Thyroid (FRT) cells stably expressing CFTR Delta F508. CONCLUSION: The establishment of a phenotype for the Delta F508 mutation in the yeast two-hybrid system yielded a simple assay for the identification of small molecules that interact with the mutant NBD1 and restore dimerization. The natural product compound identified using the system (TS3) was found to increase chloride conductance in epithelial cells to an extent comparable to genistein, a known CFTR activator. The yeast system will thus be useful for further identification of compounds with potential for CF drug therapy.     

Three labdane diterpenoids from Aframomum sceptrum (Zingiberaceae)

Three labdane diterpenoids, 8beta,17-epoxy-3beta,7beta-dihydroxy-12(E)-labden-16,15-olide (1), methyl 8beta,17-epoxy-3beta,7beta,15-trihydroxy-12(E)-labden-16-oate (2) and 3beta,7beta,8beta,12zeta,17-pentahydroxylabdan-16,15-olide (3) have been isolated from the seeds of Afromomum sceptrum K. Schum (Zingiberaceae) and their structures assigned on the basis of their spectroscopic properties. Nerolidol, and the known flavonoids 3-acetoxy-4',5,7-trihydroxyflavanone, and 3,4',5,7-tetrahydroxyflavanone were also obtained.     

Impact of iron and vitamin C-containing supplements on preterm human milk: in vitro

Stress due to reactive oxygen species (ROS) may lead to neonatal diseases, such as necrotizing enterocolitis and respiratory distress. Enteral supplements for premature infants (PREM) added to human milk (HM) to increase nutrient content may induce lipid oxidation due to free radical formation via Fenton chemistry. We hypothesized that ferrous iron and vitamin C-containing supplements added to HM in vitro cause oxidation of milk fats, affect intracellular redox balance, and induce DNA damage. Lipid peroxidation in HM was measured by FOX-2 and TBARS assays; fatty acid composition of supplemented HM was measured by gas chromatography. Two cell culture bioassays were used for assessing either intracellular oxidative stress or DNA damage: the former involved Caco-2BBe cells, a secondary differentiated cell line, and the latter utilized FHS-74 Int cells, a primary fetal small intestinal culture. Lipid oxidation products of HM increased after the addition of iron alone, iron and vitamin C, or iron and a vitamin C-containing supplement (Trivisol, TVS). A reduced content of mono and polyunsaturated fatty acids in HM was also observed. Iron, not iron+vitamin C, but iron+TVS induced significant intracellular oxidative stress in FHS-74 Int cells. In contrast, iron, either alone or in combination with TVS or vitamin C, increased DNA damage in Caco-2BBE cells. Iron supplementation may increase oxidative stress in PREM infants and should be given separately from vitamin C-containing supplements.     

EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers

The involvement of the MET oncogene in de novo and acquired resistance of non-small cell lung cancers (NSCLCs) to tyrosine kinase inhibitors (TKIs) has previously been reported, but the precise mechanism by which MET overexpression contributes to TKI-resistant NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression, and their dysregulation has been implicated in tumorigenesis. To understand their role in TKI-resistant NSCLCs, we examined changes in miRNA that are mediated by tyrosine kinase receptors. Here we report that miR-30b, miR-30c, miR-221 and miR-222 are modulated by both epidermal growth factor (EGF) and MET receptors, whereas miR-103 and miR-203 are controlled only by MET. We showed that these miRNAs have important roles in gefitinib-induced apoptosis and epithelial-mesenchymal transition of NSCLC cells in vitro and in vivo by inhibiting the expression of the genes encoding BCL2-like 11 (BIM), apoptotic peptidase activating factor 1 (APAF-1), protein kinase C ? (PKC-?) and sarcoma viral oncogene homolog (SRC). These findings suggest that modulation of specific miRNAs may provide a therapeutic approach for the treatment of NSCLCs.     

Influence of lung oxidant and antioxidant status on alveolarization: role of light-exposed total parenteral nutrition

Parenteral multivitamins (MVP) are linked to the generation of peroxides, which cause oxidant injury in lungs associated with alveolar remodelling linked to lung disease of prematurity. This study was to investigate the relationship between alveolar development and lung oxidant-antioxidant status as modulated by the mode of administration of multivitamins with total parenteral nutrition (TPN). Four groups of guinea pig pups received parenteral nutrition differing by 1) mode of MVP admixture: with amino acid solution (AA-MVP) or lipid emulsion (LIP-MVP); 2) light exposure: TPN exposed (LE) or shielded from light (LP). After 2 or 4 days of TPN, vitamins C and E, 8-isoprostaneF(2alpha) and alveolarization index were determined in lungs and GSSG/GSH in lungs and blood. Exposure to light and the mode of MVP admixture did not influence vitamin E and isoprostane levels. Blood glutathione redox potential was more oxidized in LE and LIP-MVP groups after 4-day infusions, whereas lung redox potential was more reduced in LE groups. LP and LIP-MVP had a beneficial effect, with higher number of alveoli. Globally, results indicate that in this model, alveolarization and modifications in lung redox potential are two independent events induced by light exposed TPN.     

Fhit Delocalizes Annexin A4 from Plasma Membrane to Cytosol and Sensitizes Lung Cancer Cells to Paclitaxel

Fhit protein is lost or reduced in a large fraction of human tumors, and its restoration triggers apoptosis and suppresses tumor formation or progression in preclinical models. Here, we describe the identification of candidate Fhit-interacting proteins with cytosolic and plasma membrane localization. Among these, Annexin 4 (ANXA4) was validated by co-immunoprecipitation and confocal microscopy as a partner of this novel Fhit protein complex. Here we report that overexpression of Fhit prevents Annexin A4 translocation from cytosol to plasma membrane in A549 lung cancer cells treated with paclitaxel. Moreover, paclitaxel administration in combination with AdFHIT acts synergistically to increase the apoptotic rate of tumor cells both in vitro and in vivo experiments.     

Terpenoids constituents of Euphorbia sapinii

The investigation of an acetone extract of whole Euphorbia sapinii led to the isolation of eighteen terpenoids among which lanost-24-en-20-ol-3-tetradecanoate (1), euphorbol-7-one (2) are new while spectroscopic data of 30-nor-hopan-3β-ol-22-one (3) are reported for the first time. The structures of all compounds were determined using 1D, 2D NMR and MS techniques.     

Diterpenoids from Neoboutonia glabrescens (Euphorbiaceae)

Glabrescin, a daphnane diterpenoid, neoboutonin, a degraded diterpenoid with a novel skeleton, and neoglabrescins A and B, two rhamnofolane derivatives, have been isolated from the stem bark of Neoboutonia glabrescens Prain (Euphorbiaceae), together with the known tigliane derivative, baliospermin, and the known daphnane, montanin. Other constituents include squalene, 3-acetylaleuritolic acid, oleanolic acid and sitosterol, and the phenolic compounds 9-methoxy-1,7-dimethylphenanthrene and 2,3,8-tri-O-methylellagic acid. The structures were assigned on the basis of spectral studies and comparison with published literature data. The structures of neoglabrescins A and B were derived for their acetylated derivatives and, in the case of neoglabrescin A, confirmed by X-ray crystallographic analysis.