SSRs transferability and genetic diversity of Tunisian Festuca arundinacea and Lolium perenne

SSR primers specific to Lolium perenne generated a total of 96 alleles and 124 genotypes within Festuca arundinacea and Lolium perenne accessions. Their highly transferability (100 %) across genera was evidenced. Six alleles specific to loci H01F02, H02C11 and K01A03 and only 5/96 common alleles between both species (60, 140, 144, 190 and 192) expressed the differentiation between species. Besides, based on the Wrights fixation indices, the genetic variation within each species was attributable to differences within populations with a significant deficiency of heterozygous. The unweighted pair group method with arithmetic averaging dendrogram based on the Nei’s distances and the principal coordinate analysis based on Jaccard coefficient similarity distinguished each genus independently of the geographical origin. However, typically continuous genetic diversity and a low level of gene flow (N_m: 0.29–2.47) expressed the relatively closely relationships of both genera and suggest a possible hybridization in nature.     

Lebectin, a Macrovipera lebetina venom-derived C-type lectin, inhibits angiogenesis both in vitro and in vivo

Integrins play an essential role in endothelial cell motility processes during angiogenesis and thus present interesting targets for the development of new anti-angiogenic agents. Snake venoms naturally contain a variety of proteins that can affect integrin-ligand interactions. Recently, the C-type lectin proteins (CLPs) have been characterized as efficient modulators of integrin functions. In this study, we investigated the anti-angiogenic activity of lebectin, a newly discovered CLP from Macrovipera lebetina venom. Human brain microvascular endothelial cells (HBMEC), used as an in vitro model, express alphavbeta3, alphavbeta5, and alpha5beta1 integrins, as well as the alpha2, alpha3, alpha6, and beta4 subunits. Our data show that lebectin acts as a very potent inhibitor (IC(50) approximately 0.5 nM) of HBMEC adhesion and migration on fibronectin by blocking the adhesive functions of both the alpha5beta1 and alphaV integrins. In addition, lebectin strongly inhibits both HBMEC in vitro tubulogenesis on Matrigel trade mark (IC(50) = 0.4 nM) and proliferation. Finally, using both a chicken CAM assay and a Matrigel trade mark Plug assay in nude mice, our results show that lebectin displays potent anti-angiogenic activity in vivo. Lebectin thus represents a new C-type lectin with anti-angiogenic properties with great potential for the treatment of angiogenesis-related diseases.     

Novel missense mutations in gidB gene associated with streptomycin resistance in Mycobacterium tuberculosis: insights from molecular dynamics

Streptomycin was the first antibiotic used for the treatment of tuberculosis by inhibiting translational proof reading. Point mutation in gidB gene encoding S-adenosyl methionine (SAM)-dependent 7-methylguanosine (m7G) methyltransferase required for methylation of 16S rRNA confers streptomycin resistance. As there was no structural substantiation experimentally, gidB protein model was built by threading algorithm. In this work, molecular dynamics (MD) simulations coupled with binding free energy calculations were performed to outline the mechanism underlying high-level streptomycin resistance associated with three novel missense mutants including S70R, T146M, and R187M. Results from dynamics analyses suggested that the structure distortion in the binding pocket of gidB mutants modulate SAM binding affinity. At the structural level, these conformational changes bring substantial decrease in the number of residues involved in hydrogen bonding and dramatically reduce thermodynamic stability of mutant gidB–SAM complexes. The outcome of comparative analysis of the MD simulation trajectories revealed lower conformational stability associated with higher flexibility in mutants relative to the wild-type, turns to be major factor driving the emergence of drug resistance toward antibiotic. This study will pave way toward design and development of resistant defiant gidB inhibitors as potent anti-TB agents.     

Effect of the successive steps of a cryopreservation protocol on the structural integrity of Rubia akane Nakai hairy roots

In this work, we studied the impact of the successive steps of the droplet-vitrification protocol technique employed for cryopreservation of Rubia akane hairy roots on the features of cortical, pericycle and endoderm cells of apical and central root segments, using histology techniques and combining qualitative and quantitative observations. In apical segments, plasmolysis (22–71 %, depending on cell type) was observed only after the loading treatment and did not increase after the following steps of the protocol. By contrast, in central segments, plasmolysis (39–45 %) was already observed after the sucrose pretreatment; it increased to 54–68 %, depending on cell type, after the loading treatment, but no further changes were noted after treatment with the vitrification solution. After liquid nitrogen exposure and unloading treatment, deplasmolysis was more rapid in apical segments, with cortical and pericycle cells having retrieved their original features. In central segments, only cortical cells had retrieved their original features and endoderm and pericycle cells were still highly plasmolysed. Nuclei were more strongly impacted by the cryopreservation protocol in central segments, where they displayed a highly condensed nucleoplasm from the loading treatment onwards and had not retrieved their original aspect after the unloading treatment. By contrast, nuclei had a much less condensed nucleoplasm in cells of apical segments, and they had retrieved their original aspect after the unloading treatment.     

Penicillium verruculosum SG: a source of polyketide and bioactive compounds with varying cytotoxic activities against normal and cancer lines

A newly isolated fungus Penicillium verruculosum SG was evaluated for the production and characterization of bioactive colored secondary metabolites using solid-state fermentation along with their cytotoxic activities against normal and cancer cell lines. Logical fragmentation pattern following column chromatography, thin layer chromatography and liquid chromatography and mass spectrometry of crude culture filtrate of fungus revealed the presence of different polyketide pigments and other bioactive compounds. Cytotoxicity of the selected colored fractions of fungal filtrate containing different compounds revealed IC50 (μg/ml) values ranging from 5 to 100. It was significantly higher in case of orevactaene (5 + 0.44) and monascorubrine followed by pyripyropene (8 + 0.63) against cancer cell line KA3IT. Overall, these compounds considerably showed less toxicity toward normal cell lines NIH3T3, HSCT6, HEK293 and MDCK. XRD of a yellow crystalline compound (224.21 m/z) confirmed its 3-dimensional structure as phenazine 1 carboxylic acid (C13H8N2O2) (broad spectrum antibiotic), and it is first time reported in fungi.     

Determination of amlodipine using terbium‐sensitized luminescence in the presence of europium(III) as a co‐luminescence reagent

A sensitive time‐resolved luminescence method for the determination of amlodipine (AM) in methanol and in aqueous solution is described. The method is based on the luminescence sensitization of terbium (Tb³⁺) by formation of a ternary complex with AM in the presence of tri‐n‐octylphosphine oxide (TOPO) as co‐ligand, dodecylbenzenesulfate as surfactant and europium ion as a co‐luminescence reagent. The signal for Tb–AM–TOPO is monitored at λ_ex = 242 nm and λ_em = 550 nm. Optimum conditions for the formation of the complex in aqueous system were 0.015 m Tris (hydroxylmethyl) amino methane buffer, pH 9.0, TOPO (1.0 × 10^–4 m ), Eu³⁺ (2.0 × 10^–7 m ), dodecylbenzenesulfate (0.14%) and 6.0 × 10^–5 m of Tb³⁺, which allows the determination of 10–50 ppb of AM with a limit of detection of 1.2 ppb. The relative standard deviations of the method range between 0.1 and 0.2% indicated excellent reproducibility of the method. The proposed method was successfully applied for the assay of AM in pharmaceutical formulations and in plasma samples. Average recoveries of 98.5 ± 0.2% and 95.2 ± 0.2% were obtained for AM in tablet and plasma samples respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Spectrofluorimetric analysis of gemifloxacin mesylate in pharmaceutical formulations

A simple, rapid and highly sensitive spectrofluorimetric method was developed for determination of gemifloxacin mesylate (GFX) in tablets. The method is based on measuring the native fluorescence of GFX in isopropanol at 400 nm after excitation at 272 nm. The fluorescence–concentration plot was rectilinear over the range of 0.01–0.50 µg/mL with a lower detection limit of 1.19 ng/mL and quantification limit of 3.6 ng/mL. The method was fully validated and successfully applied to the determination of GFX tablets with an average percentage recovery of 99.65 ± 0.532. The method was extended to the stability study of GFX. The drug was exposed to acidic, alkaline, oxidative and photolytic degradation according to International Conference on Harmonization guidelines. The rate of GFX degradation was found at its highest in acidic conditions, and in its lowest in the neutral one. However, it was stable under dry heat and photolytic degradation conditions. Copyright © 2013 John Wiley & Sons, Ltd.