RIG-I mediates the co-induction of tumor necrosis factor and type I interferon elicited by myxoma virus in primary human macrophages

The sensing of pathogen infection and subsequent triggering of innate immunity are key to controlling zoonotic infections. Myxoma virus (MV) is a cytoplasmic DNA poxvirus that in nature infects only rabbits. Our previous studies have shown that MV infection of primary mouse cells is restricted by virus-induced type I interferon (IFN). However, little is known about the innate sensor(s) involved in activating signaling pathways leading to cellular defense responses in primary human immune cells. Here, we show that the complete restriction of MV infection in the primary human fibroblasts requires both tumor necrosis factor (TNF) and type I IFN. We also demonstrate that MV infection of primary human macrophages (pHMs) activates the cytoplasmic RNA sensor called retinoic acid inducible gene I (RIG-I), which coordinately induces the production of both TNF and type I IFN. Of note, RIG-I sensing of MV infection in pHMs initiates a sustained TNF induction through the sequential involvement of the downstream IFN-regulatory factors 3 and 7 (IRF3 and IRF7). Thus, RIG-I-mediated co-induction of TNF and type I IFN by virus-infected pHMs represents a novel innate defense mechanism to restrict viral infection in human cells. These results also reveal a new regulatory mechanism for TNF induction following viral infection.     

Synthesis of 3'-deoxy-3'-C-methyl nucleoside derivatives

2',3'-Dideoxy-3'-C-methyl nucleosides bearing the five naturally occurring nucleic acid bases were synthesized. Additionally, the 3'-deoxy-3'-C-methyl nucleoside analogues bearing 5-aminoimidazole-4-carboxamide as well as 1,2,4-triazole-3-carboxamide moieties were prepared. The synthesis of the corresponding 2',3'-dideoxy-3'-C-methyl triazole derivative was also accomplished. The dideoxynucleoside derivatives were prepared by radical deoxygenation from their 3'-deoxy-3'-C-methyl parent ribonucleosides. When evaluated for their antiviral activity in cell culture experiments, none of these compounds showed any significant antiviral activity.     

Design of new potent and selective secretory phospholipase A2 inhibitors. Part 5: synthesis and biological activity of 1-alkyl-4-[4,5-dihydro-1,2,4-[4H]-oxadiazol-5-one-3-ylmethylbenz-4'-yl(oyl)] piperazines

Among the different PLA(2)s identified to date, the group IIA secretory PLA(2) (sPLA(2) GIIA) is implied in diverse pathological conditions. In this work we describe the synthesis, inhibitory activities, and structure-activity relationships (SAR) of a new class of substituted piperazine derivatives. The in vitro fluorimetric assay using two groups of enzymes, GIB and GIIA, revealed several compounds as highly potent inhibitors (IC(50)=0.1 microM). The in vivo activity assessed by ip or per os administration in a carrageenan-induced edema test in rats showed that two compounds proved to be as potent as indomethacin (10 mg/kg).     

2-N-Methyl modifications and SAR studies of manzamine A

Quaternary carbolinium salts have been reported to show improved antimalarial activity and reduced cytotoxicity as compared to electronically neutral beta-carbolines. In this study, mono- and di-methylated quaternary carbolinium cations of manzamine A were synthesized and evaluated for their in vitro antimalarial and antimicrobial activity, cytotoxicity, and also their potential for glycogen synthase kinase (GSK-3beta) inhibition using molecular docking studies. Among the analogs, 2-N-methylmanzamine A (2) exhibited antimalarial activity (IC(50) 0.7-1.0microM) but was less potent than manzamine A. However the compound was significantly less cytotoxic to mammalian kidney fibroblasts and the selectivity index was in the same range as manzamine A.     

Activity of native vs. synthetic promoters in Brucella

Brucellosis caused by Brucella species is reportedly the most common zoonotic infection worldwide. The bacterial pathogen is also classified by the Centers for Disease Control and Prevention as a category (B) pathogen that has the potential for development as a bioweapon. Although eight genomes of Brucella have been sequenced, little information is available regarding the regulation of gene expression and promoter activity in Brucella spp. We therefore constructed a set of broad-host-range vectors expressing the lacZ reporter gene from various promoters. Four groups of promoters (Brucella native, antibiotic resistant, bacteriophage and synthetic promoters) were tested in vivo and in vitro in Brucella suis. The highest level of heterologous gene expression was achieved with synthetic hybrid trc promoter carrying the adenine-rich upstream element. Furthermore, this demonstrates the usefulness of synthetic promoters for enhanced level of gene expression in Brucella spp.     

Agrobacterium-mediated transformation of apricot (Prunus armeniaca L.) leaf explants

A protocol for Agrobacterium-mediated stable transformation for scored, whole leaf explants of the apricot (Prunus armeniaca) cultivar Helena was developed. Regenerated shoots were selected using a two-step increased concentrations of paromomycin sulphate. Different factors affecting survival of transformed buds, including possible toxicity of green fluorescent protein (GFP) and time of exposure to high cytokine concentration in the regeneration medium, were examined. Transformation efficiency, based on PCR analysis of individual putative transformed shoots from independent lines was 5.6%, when optimal conditions for bud survival were provided. Southern blot analysis on four randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene. This is the first time that stable transformation of an apricot cultivar is reported and constitutes also one of the few reports on the transformation of Prunus cultivars.     

AT1-receptor blockade by telmisartan upregulates GTP-cyclohydrolase I and protects eNOS in diabetic rats

Several enzymatic sources of reactive oxygen species (ROS) were described as potential reasons of eNOS uncoupling in diabetes mellitus. In the present study, we investigated the effects of AT(1)-receptor blockade with chronic telmisartan (25 mg/kg/day, 6.5 weeks) therapy on expression of the BH(4)-synthesizing enzyme GTP-cyclohydrolase I (GCH-I), eNOS uncoupling, and endothelial dysfunction in streptozotocin (STZ, 60 mg/kg iv, 7 weeks)-induced diabetes mellitus (type I). Telmisartan therapy did not modify blood glucose and body weight. Aortas from diabetic animals had vascular dysfunction as revealed by isometric tension studies (acetylcholine and nitroglycerin potency). Vascular and cardiac ROS produced by NADPH oxidase, mitochondria, eNOS, and xanthine oxidase were increased in the diabetic group as was the expression of NADPH oxidase subunits at the protein level. The expression of GCH-I and the phosphorylation of eNOS at Ser1177 was decreased by STZ treatment. Therapy with telmisartan normalized these parameters. The present study demonstrates for the first time that AT(1)-receptor blockade by telmisartan prevents downregulation of the BH(4) synthase GCH-I and thereby eNOS uncoupling in experimental diabetes. In addition, telmisartan inhibits activation of superoxide sources like NADPH oxidase, mitochondria, and xanthine oxidase. These effects may explain the beneficial effects of telmisartan on endothelial dysfunction in diabetes.     

Prevention of estrogen-DNA adduct formation in MCF-10F cells by resveratrol

Resveratrol (Resv), a natural occurring phytolexin present in grapes and other foods, possesses chemopreventive effects revealed by its striking modulation of diverse cellular events associated with tumor initiation, promotion, and progression. Catechol estrogens generated in the metabolism of estrogens are oxidized to catechol quinones that react with DNA to form predominantly depurinating estrogen-DNA adducts. This event can generate the mutations responsible for cancer initiation. In this regard, Resv acts as both an antioxidant and an inducer of the phase II enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1). In this report, we present the effects of Resv on the metabolism of estrogens in normal breast epithelial cells (MCF-10F) treated with 4-hydroxyestradiol (4-OHE(2)) or estradiol-3,4-quinone (E(2)-3,4-Q). Resv induced NQO1 in a dose- and time-dependent manner, but did not affect the expression of catechol-O-methyltransferase. Ultraperformance liquid chromatography/tandem mass spectrometry was used to determine the effects of Resv on estrogen metabolism. Preincubation of the cells with Resv for 48 h decreased the formation of depurinating estrogen-DNA adducts from 4-OHE(2) or E(2)-3,4-Q and increased formation of methoxycatechol estrogens. When Resv was also present with the 4-OHE(2) or E(2)-3,4-Q, even greater increases in methoxycatechol estrogens were observed, and the DNA adducts were undetectable. We conclude that Resv can protect breast cells from carcinogenic estrogen metabolites, suggesting that it could be used in breast cancer prevention.     

Historical colonization of the Mediterranean Sea by Atlantic fishes: do biological traits matter?

Since its opening 5.33 million years ago, the Gibraltar Strait has always contributed to the Mediterranean fauna and flora. Despite the increasing importance of the phenomenon, ecological determinants underlying colonization success of Atlantic fishes in the Mediterranean Sea have been poorly investigated. Here we reconstruct the recent historical colonization of the whole Mediterranean Sea by Atlantic fishes and we aim to determine where Atlantic fishes preferentially establish and whether some biological traits and ecological factors can be correlated to the colonization success (climate match, position in the water column, maximum body length, propagules, confamilial resistance, depth). A database on Atlantic fish species records from 1810 to 2006 was built and the colonization rate of each introduced species was estimated. Analysis of Variance, Chi squared test and logistic regression were used to investigate the relationships between ecological variables and colonization success. In addition, an index of asymmetry was used to analyse the relative colonization on the two sides of the Mediterranean Sea. Overall 48.33% of Atlantic species introduced in the Mediterranean Sea succeeded in colonizing eastwards. We found that habitat depth of Atlantic species is significantly related to their colonization success due to the obstacle of the shallow depth of the Gibraltar Strait (300 m). It also appears that despite the cyclonic general water circulation on the North Western basin, the northern side is more colonized than the southern one: 70.40% of the studied species colonize the northern side, while only 29.62% colonize the southern one. Two hypotheses may explain this trend: the bottom-up process that enhances the colonization success of Atlantic fishes along the Spanish coast of Alboran Sea owning to its high productivity and the intensiveness of the scientific explorations along the northern Mediterranean side. We conclude that crossing the Gibraltar Strait does not guarantee the colonization success and that species life-history and functional traits are poor predictors. Instead we suggest that environmental factors may determine favourable locations for invasive species installation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: J. Cambray.