Synthesis, biophysical and biological evaluation of 3,6-bis-amidoacridines with extended 9-anilino substituents as potent G-quadruplex-binding telomerase inhibitors

Telomerase and telomere maintenance are emerging targets for the treatment of human cancers. We report here on the targeting of the telomere-telomerase complex with a series of small molecules based on an acridine platform. A series of 3,6-bisamidoacridines with extended 9-anilino sidechains were designed and synthesised as potential telomeric G-quadruplex DNA (G4) interacting compounds. G4-stabilisation was assessed using a high-throughput FRET (fluorescence resonance energy transfer) assay and telomerase inhibition quantified by a modified TRAP (telomerase repeat amplification protocol) method. Within the series, the compounds showed significant G4-stabilising ability (Delta T(m) values of 25-36 degrees C at 1 microM concentration) and telomerase inhibition in the nanomolar region ((tel)EC(50) values of 80-318 nM). Furthermore, a direct correlation between the FRET and TRAP assays was observed, supporting the use of the rapid screening FRET assay for early assessment of potential G4-stabilising telomerase inhibitors.     

N2-benzyl-N1-(1-(1-naphthyl)ethyl)-3-phenylpropane-1,2-diamines and conformationally restrained indole analogues: development of calindol as a new calcimimetic acting at the calcium sensing receptor

The synthesis and calcimimetic activities of two new families of compounds are described. The most active derivatives of the first family, N(2)-(2-chloro-(or 4-fluoro-)benzyl)-N(1)-(1-(1-naphthyl)ethyl)-3-phenylpropane-1,2-diamine (4b and 4d, respectively, tested at 10 microM) produced 98+/-6% and 95+/-4%, respectively, of the maximal stimulation of [(3)H]inositol phosphates production obtained by 10mM Ca(2+) in CHO cells expressing the rat calcium sensing receptor (CaSR). The second family of calcimimetics was obtained by conformationally restraining the compounds of type 4 to provide the 2-aminomethyl derivatives 5. One of these compounds, (R)-2-[N-(1-(1-naphthyl)ethyl)aminomethyl]indole ((R)-5a, calindol), displayed improved calcimimetic activity compared to 4b and 4d as well as stereoselectivity. In the presence of 2mM Ca(2+), calindol stimulated [(3)H]inositol phosphates accumulation with an EC(50) of 1.0+/-0.1 or 0.31+/-0.05 microM in cells expressing the rat or the human CaSR, respectively. The calcimimetic activities of these novel compounds were shown to be due to a specific interaction with the CaSR.     

Vesicular stomatitis virus glycoprotein: a transducing coat for SFV-based RNA vectors

BACKGROUND: Semliki Forest virus (SFV) vectors have a great potential for the induction of protective immunity in a large number of clinical conditions including cancer. Such a potential accounts for the huge efforts made to improve the in vivo expression from SFV vectors. It is noteworthy that efficient in vivo expression strongly relies on the ability to deliver high-titre vectors. To achieve this, the generation of recombinant SFV particles, using independent expression systems for structural SFV genes, has been proposed. However, despite several modifications in the production process, a risk of contamination with replication-competent, or partially recombined, virus has remained. METHODS: Here, we exploit the ability of the vesicular stomatitis virus glycoprotein (VSV-G), expressed in trans, to hijack full-length genomic SFV RNA into secreted virus-like particles (VLPs). To allow SFV vector mobilisation, we designed a CMV driven SFV vector in which the internal 26S promoter has been extensively mutated. With this vector, mobilisation events were monitored using the Green Fluorescent Protein (GFP). The production procedure involves a sequential transfection protocol, of plasmids expressing the VSV-G and the SFV vector respectively. RESULTS: We show that the VLPs are effective for cellular delivery of SFV vectors in a broad range of human and non-human cellular targets. Furthermore, production of VLPs is easy and allows, through concentration, the harvest of high-titre vector. CONCLUSIONS: The present paper describes a convenient process aimed at mobilising full length SFV vectors. A major issue to consider, while developing clinically relevant gene transfer vectors, is the risk of undesirable generation of replication competent by-products. Importantly, as the VSV-G gene shares no homology with the SFV genome, our VLPs offer a strong guarantee of biosafety.     

Possible apoptotic mechanism in epidermal cell acantholysis induced by pemphigus vulgaris autoimmunoglobulins

Through a still unclear mechanism, pemphigus vulgaris autoantibodies (PV-IgG) induce intra-epidermal acantholytic lesions responsible for severe to fatal skin wounding. We present evidence that PV lesions contain apoptotic keratinocytes, and that cell death is induced in the lesional tissue apparently before cell separation. These data suggest that apoptosis could be the cause of the acantholytic phenomenon. We show that PV-IgG and an antibody against Fas receptor (anti-FasR) induce lesions in vitro in a similar way, causing: (1) secretion of soluble FasL; (2) elevated cellular amounts of FasR, FasL (soluble and membranal), Bax and p53 proteins; (3) reduction in levels of cellular Bcl-2; (4) enrichment in caspase 8, and activation of caspases 1 and 3; (5) co-aggregation of FasL and FasR with caspase 8 in membranal death-inducing signaling complex (DISC). Hence, the Fas-mediated death signaling pathway seems to be involved in lesion formation. Moreover, we have shown that in skin organ cultures and in keratinocyte cultures, PV-IgG can induce caspase activation and DNA fragmentation, and caspase inhibitors can prevent the formation of PV-IgG-induced epidermal lesions. Altogether, these results suggest that PV-IgG-induced acantholysis may proceed through the death-signaling pathway. They highlight new perspectives on mechanisms of tissue damage in autoimmune diseases.     

Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese

AIMS: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great significance for the dairy industry to improve food safety. METHODS AND RESULTS: Six-hundred strains of LAB isolated from 'rigouta', a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identified as Lactococcus lactis and produced nisin Z as demonstrated by mass spectrometry analysis of the purified antibacterial compound. Polymerase chain reaction experiments using nisin gene-specific primers confirmed the presence of nisin operon. Plasmid profiles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identified, at molecular level, as Enterococcus faecalis. The purified bacteriocin produced by this strain showed a molecular mass of 10 201.33 +/- 0.85 Da. This new member of class III bacteriocins was termed enterocin MMT05. CONCLUSIONS: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or beneficial role in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety.     

Functional significance of a periplasmic Mn-superoxide dismutase from Aeromonas hydrophila

AIMS: A better understanding of the role of superoxide dismutases (SODs) from Aeromonas hydrophila and particularly the Mn-SOD which shares a peculiar localization within the bacterial periplasm and is only detected during the stationary phase of growth. METHODS AND RESULTS: A. hydrophila ATCC 7966 can express two distinct SODs: an Fe-SOD and an Mn-SOD. Using insertional mutagenesis, an Mn-SOD-deficient mutant was isolated. After growth of this mutant under conditions leading to the expression of an Mn-SOD, only the Fe-SOD could be detected in nondenaturing PAGE. Study of its response to the oxidative stress showed that the Mn-SOD was not implicated in the protection against intracellular superoxide but defended the bacterial cells against environmental superoxide. CONCLUSIONS: By protecting the bacteria against external superoxide, the role of the Mn-SOD from A. hydrophila is equivalent to that of the Cu/Zn-SOD from the well-studied Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The function of this Mn-SOD is in agreement with its periplasmic localization and may confer an advantage on the bacteria such as a virulence factor in cases of pathogenicity.     

Inhibition of the p53-hdm2 interaction with low molecular weight compounds

The hdm2 protein, upon binding to p53, inhibits its tumor suppressor activity. The inhibition of the p53-hdm2 interaction represents therefore a new therapeutic strategy to activate wild type p53 in tumors. Potent low molecular weight compounds inhibiting this protein-protein interaction, which are active in vivo, have just been identified. This offers new perspectives and hopes in this research area.     

SdFFF monitoring of cellular apoptosis induction by diosgenin and different inducers in the human 1547 osteosarcoma cell line

Apoptosis is one of the most important phenomena of cellular biology. Sedimentation field flow fractionation (SdFFF) has been described as an effective tool for cell separation, respecting integrity and viability. Because SdFFF takes advantage of intrinsic properties of eluted cells (size, density, shape or rigidity), we investigated the capacity of SdFFF in monitoring the early and specific biophysical modifications which occurred during cellular apoptosis induction. Then, we used, as an in vitro cellular apoptosis model, the association between human 1547 osteosarcoma cells and diosgenin, a plant steroid known to induce apoptosis. Four other molecules were studied: hecogenin, tigogenin, staurosporine and MG132. Our results demonstrated a correlation between SdFFF elution profile changes (peak shape modification and retention ratio evolution) and effective apoptosis induction. For the first time, we demonstrated that SdFFF could be used to monitor apoptosis induction as early as 6 h incubation, suggesting different applications such as screening series of molecules to evaluate their ability to induce apoptosis, or sorting apoptotic cells to study apoptosis pathway.     

Proteomic analysis of aneuploid lines in the homeologous group 1 of the hexaploid wheat cultivar Courtot

Three monosomic lines (MSLs) and three nullisomic lines (NSLs) of the homeologous group 1 and one euploid line of the bread wheat Triticum aestivum cultivar Courtot were used in a proteomic approach to investigate the effects of zero, one or two doses of chromosomes 1A, 1B and 1D on the amount of endosperm proteins. Polypeptides whose amounts changed significantly between each aneuploid line and the euploid line were identified using image analyses of two-dimensional gel electrophoresis patterns resulting from specific endosperm protein extractions. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry were also used for protein identification. Removing one chromosome or a chromosome pair allowed varying responses to be observed for the remaining endosperm protein genes. Compensation phenomena for the high molecular weight glutenin subunits (HMW-GS) were detected only in the MSLs. Subunits Bx7, By8 and Dy12 were the only HMW-GS overexpressed (from 152-737%) when chromosomes 1A or 1B or 1D were at hemizygous state. Thirteen new protein spots were detected only in the NSL1D, and seven were identified as HMW-GS analogs. These seven new spots may result from the expression of inactive genes. The HMW-GS were of significantly higher volume in MSLs, whereas the low molecular weight glutenin subunits and the gamma-gliadins were of lower volume in aneuploid lines. Most of the down-regulated proteins in the MSLs were storage proteins encoded at loci located on another chromosome pair. Complex regulations between chromosomes and loci of the homeologous groups 1 and 6 in bread wheat are discussed.