Calmodulin Inhibitors from Aspergillus stromatoides

An organic extract was prepared from the culture medium and mycelia of the marine fungus Aspergillus stromatoides Raper & Fennell . The extract was fractionated via column chromatography, and the resulting fractions were tested for their abilities to quench the fluorescence of the calmodulin (CaM) biosensor hCaM M124C‐mBBr. From the active fraction, emodin ( 1 ) and ω‐hydroxyemodin ( 2 ) were isolated as CaM inhibitors. Anthraquinones 1 and 2 quenched the fluorescence of the hCaM M124C‐mBBr biosensor in a concentration‐dependent manner with K_d values of 0.33 and 0.76 μM , respectively. The results were compared with those of chlorpromazine (CPZ), a classical inhibitor of CaM, with a K_d value of 1.25 μM . Docking analysis revealed that 1 and 2 bind to the same pocket of CPZ. The CaM inhibitor properties of 1 and 2 were correlated with some of their reported biological properties. Citrinin ( 3 ), methyl 8‐hydroxy‐6‐methyl‐9‐oxo‐9H‐xanthene‐1‐carboxylate ( 4 ), and coniochaetone A ( 5 ) were also isolated in the present study. The X‐ray structure of 5 is reported for the first time.     

Jatropha curcas L. (Euphorbiaceae) exhibits a mixed mating system, high correlated mating and apomixis

The hierarchical mating system among and within fruits of Jatropha curcas was investigated in a base population using five microsatellite loci, employing mixed mating and correlated mating models. Open-pollinated fruits were collected from 15 randomly selected seed trees, sampling seven fruits per tree for a total of 21 seeds from each tree. We detected multilocus genotypes identical to the mother tree in 13 % of offspring, implying the occurrence of apomixis in J. curcas. The presumed apomictic individuals were excluded from the analysis of the remaining results. Evidence of substantial selfing was provided by the average multilocus outcrossing rate (t _m?=?0.683), showing that the species exhibits a mixed mating system. The outcrossing rate showed a large variation among seed trees, ranging from 0.21 to 1.0, indicating that the species is not self-incompatible. Significant differences were detected between the multilocus and the single locus outcrossing rates (t _m???t _s?=?0.347) that suggested mating among related individuals, possibly because of the presence of individuals from the same progeny (sibs) in the base population. The multilocus paternity correlation was extremely high for the population (r _p(m)?=?0.999), indicating that the progenies were manly composed of full-sibs. As a consequence of selfing and a high paternity correlation, the co-ancestry coefficient within the progeny was higher (Θ?=?0.369) than expected for panmictic populations. Our results indicated that J. curcas produces seeds asexually by apomixis and sexually by a mixed mating system, combining selfing and outcrossing.     

From the cradle of grapevine domestication: molecular overview and description of Georgian grapevine (Vitis vinifera L.) germplasm

Historical information and archaeological and palaeobotanical findings point Georgia, in the South Caucasus, as a cradle for grapevine (Vitis vinifera L.) domestication from its wild form (V. vinifera silvestris Beck.) and subsequent selection and development of varieties with characters suitable for human consumption. The hypothesis of Georgia being a center of domestication, combined with its distance from western countries and the importance of its viticulture and wine production, make Georgian grape germplasm particularly interesting to be investigated under the genetic point of view. Twenty nuclear microsatellite loci were used to genotype 112 Georgian grapevine accessions (V. vinifera sativa Beck.) from germplasm collections and 18 from spontaneous growing plants (V. vinifera silvestris Beck.) found in wild conditions and to compare them to a large international cultivar collection in France. Data analysis shows that Georgian grapevine germplasm has maintained distinctive traits despite arrival of international, foreign varieties and still conserve characteristics of local breeding linked to traditional wine production regions of the country. Results have identified alleles, overall loci, well represented in the Georgian germplasm (cultivated and wild) and absent or poorly represented in other countries, highlighting uniqueness and originality of traits of this viticulture. Moreover, the search for relationships between Georgian and foreign viticulture has evidenced few interesting cases linking the Georgian varieties with Western European ones and with neighboring Caucasian countries, helping to identify the real place of origin in some doubtful cases. In addition, populations or sparse individuals of wild grapevine still preserved in the Georgian natural environments present smaller genetic distances with local cultivars than in other European regions. Principal component analysis (PCA) has also identified special overlapping of the wild compartment with some cultivated varieties. This work provides a highly significant new contribution to applied aspects of Georgian grapevine genetic resources management and use. Uniqueness of the Georgian cultivated grapevine gene pool together with its close relatedness with the wild compartment makes this country a good candidate to address questions regarding domestication and grapevine genetic resource conservation.     

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: Its role in arsenic detoxification

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20 mM and 15 mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram + bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a k_cat/K_M value of 1.2 × 10⁴ M^− 1 s^− 1. It also exhibited weak phosphatase activity with a k_cat/K_M value of 2.7 × 10^− 4 M^− 1 s^− 1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase.     

Exendin-4 Induces Cell Adhesion and Differentiation and Counteracts the Invasive Potential of Human Neuroblastoma Cells

Exendin-4 is a molecule currently used, in its synthetic form exenatide, for the treatment of type 2 diabetes mellitus. Exendin-4 binds and activates the Glucagon-Like Peptide-1 Receptor (GLP-1R), thus inducing insulin release. More recently, additional biological properties have been associated to molecules that belong to the GLP-1 family. For instance, Peptide YY and Vasoactive Intestinal Peptide have been found to affect cell adhesion and migration and our previous data have shown a considerable actin cytoskeleton rearrangement after exendin-4 treatment. However, no data are currently available on the effects of exendin-4 on tumor cell motility. The aim of this study was to investigate the effects of this molecule on cell adhesion, differentiation and migration in two neuroblastoma cell lines, SH-SY5Y and SK-N-AS. We first demonstrated, by Extra Cellular Matrix cell adhesion arrays, that exendin-4 increased cell adhesion, in particular on a vitronectin substrate. Subsequently, we found that this molecule induced a more differentiated phenotype, as assessed by i) the evaluation of neurite-like protrusions in 3D cell cultures, ii) the analysis of the expression of neuronal markers and iii) electrophysiological studies. Furthermore, we demonstrated that exendin-4 reduced cell migration and counteracted anchorage-independent growth in neuroblastoma cells. Overall, these data indicate for the first time that exendin-4 may have anti-tumoral properties.     

Neuroimmunomodulation in human autoimmune liver disease

Bidirectional interaction between immune and nervous systems is considered an important biological process in health and disease. However, little is known about the mechanisms involved in their interaction in the human liver. This study examines the distribution of intrahepatic NPY, SP immunoreactive (IR) nerve fibers and their antomical relationship with immunocells containing tumor necrosis factor-α (TNF-α) and nuclear factor κB (NF-κB) in patients with autoimmune hepatitis. Liver specimens were obtained from control liver and autoimmune hepatitis patients. The immunoreactivity was determined by immunohisto- and immunocytochemistry and confocal laser microscopy. In hepatitis, the number of NPY-IR and SP-IR nerve fibers increased significantly. These IR nerve fibers were in very close contact with the lymphocytes. In healthy controls, no NPY-IR, SP-IR or NF-κB IR lymphocytes and only a few TNF-α positive cells, were observed. In hepatitis, some of the lymphocytes showed immunoreactivity for SP and NPY in the portal area. Fluorescent double-labeled immunostaining revealed that in these cells NPY did not colocalize with TNF-α or NF-κB. However, some of the SP fluorescence-positive immune cells exhibited immunostaining for p65 of NF-κB, where their labeling was detected in the nuclei. Under the electronmicroscope, these cells could be identified (lymphocytes, plasmacells and mast cells). The gap between the IR nerve fibers and immunocells was 1 μm or even less. Overexpression of SP in lymphocytes may amplify local inflammation, while NPY may contribute to liver homeostasis in hepatitis. Neural immunomodulation (SP antagonists and NPY) might be a novel therapeutic concept in the management of liver inflammation.     

Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids,viral vectors and cell lines

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.     

Multiscale simulation of flow-induced texture formation in polymer liquid crystals and carbonaceous mesophases

This paper presents theory and simulation of flow-induced structures in liquid crystalline materials, useful to the creation of synthetic material structures and to the biomimetics of natural fibers. A multiscale theory and simulation of hydrodynamic texture formation is presented; it provides fundamental principles for control and optimization of structures in liquid crystal polymers and carbonaceous mesophases. In thermotropic flow-aligning nematic polymers it is found that as the shear-rate increases, the pathway between an oriented non-planar state and an oriented planar state is through meso-texture formation and coarsening, with temperature and shear rate being efficient fields to control the grain size of the texture. For capillary flow of carbonaceous mesophases, the simulations predict the emergence of macroscopic ring patterns whose thickness and density can be controlled by the applied pressure drops. The results provide insight on microstructure formation and control in liquid crystalline materials.     

A new metriorhynchid crocodylomorph from the Lower Kimmeridge Clay Formation (Late Jurassic) of England,with implications for the evolution of dermatocranium ornamentation in Geosaurini

A new metriorhynchid crocodylomorph from the Lower Kimmeridge Clay Formation (Kimmeridgian, Upper Jurassic) of England is described. This specimen, a three‐dimensionally preserved skull and left mandibular ramus, is referred to a new species: T orvoneustes coryphaeus sp. nov. Within the genus Torvoneustes, T . coryphaeus sp. nov. is unique as it has a long anteromedial process of the frontal, ornamented dermatocranium, and the supraorbital notch forms a strongly acute angle. Our phylogenetic analysis confirms the placement of this specimen in Torvoneustes. The dentition of T . coryphaeus sp. nov. , like that of the type species, has a blunt apex, crown basal–mid regions with numerous tightly packed apicobasally aligned ridges, and apical region with an anastomosed pattern of ridges that interact with the carinae. Within Thalattosuchia these dental characteristics are only found in Torvoneustes and the teleosaurid Machimosaurus. The heavily ornamented dermatocranium of T . coryphaeus sp. nov. is in contrast to the unornamented (nasals and frontal)–lightly ornamented (maxillae and premaxillae) pattern seen in Torvoneustes carpenteri. Curiously, this pattern of reduction and loss of dermatocranium ornamentation is also observed in Metriorhynchus, Dakosaurus, and the subclade Rhacheosaurini. We hypothesize that the ‘smooth’ dermatocranium of Late Jurassic metriorhynchids evolved independently in each subclade (parallel evolution), and would have reduced drag, thereby making locomotion through water more energy efficient. © 2013 The Linnean Society of London