Contribution of Position α4S336 on Functional Expression and Up-regulation of α4β2 Neuronal Nicotinic Receptors

Phosphorylation of the nicotinic acetylcholine receptor (nAChR) is believed to play a critical role in its nicotine-induced desensitization and up-regulation. We examined the contribution of a consensus PKC site in the α4 M3/M4 intracellular loop (α4S336) on the desensitization and up-regulation of α4β2 nAChRs expressed in oocytes. Position α4S336 was replaced with either alanine to abolish potential phosphorylation at this site or with aspartic acid to mimic phosphorylation at this same site. Mutations α4S336A and α4S336D displayed a threefold increase in the ACh-induced response and an increase in ACh EC₅₀. Epibatidine binding revealed a three and sevenfold increase in surface expression for the α4S336A and α4S336D mutations, respectively, relative to wild-type, therefore, both mutations enhanced expression of the α4β2 nAChR. Interestingly, the EC₅₀’s and peak currents for nicotine activation remained unaffected in both mutants. Both mutations abolished the nicotine-induced up-regulation that is normally observed in the wild-type. The present data suggest that adding or removing a negative charge at this phosphorylation site cannot be explained by a simple straightforward on-and-off mechanism; rather a more complex mechanism(s) may govern the functional expression of the α4β2 nAChR. Along the same line, our data support the idea that phosphorylation at multiple consensus sites in the α4 subunit could play a remarkable role on the regulation of the functional expression of the α4β2 nAChR.     

Distinct male reproductive strategies in two closely related oak species

Reproductive strategies of closely related species distributed along successional gradients should differ as a consequence of the trade‐off between competition and colonization abilities. We compared male reproductive strategies of Quercus robur and Q. petraea, two partly interfertile European oak species with different successional status. In the studied even‐aged stand, trees of the late‐successional species (Q. petraea) grew faster and suffered less from intertree competition than trees of the early‐successional species (Q. robur). A large‐scale paternity study and a spatially explicit individual‐based mating model were used to estimate parameters of pollen production and dispersal as well as sexual barriers between species. Male fecundity was found to be dependent both on a tree's circumference and on its environment, particularly so for Q. petraea. Pollen dispersal was greater and more isotropic in Q. robur than in Q. petraea. Premating barriers to hybridization were strong in both species, but more so in Q. petraea than in Q. robur. Hence, predictions based on the competition–colonization trade‐off are well supported, whereas the sexual barriers themselves seem to be shaped by colonization dynamics.     

Antibacterial activity against β- lactamase producing Methicillin and Ampicillin-resistants Staphylococcus aureus: fractional Inhibitory Concentration Index (FICI) determination

Background

The present study reports the antibacterial capacity of alkaloid compounds in combination with Methicillin and Ampicillin-resistants bacteria isolated from clinical samples. The resistance of different bacteria strains to the current antibacterial agents, their toxicity and the cost of the treatment have led to the development of natural products against the bacteria resistant infections when applied in combination with conventional antimicrobial drugs.

Method

The antibacterial assays in this study were performed by using inhibition zone diameters, MIC, MBC methods, the time-kill assay and the Fractional Inhibitory Concentration Index (FICI) determination. On the whole, fifteen Gram-positive bacterial strains (MRSA/ARSA) were used. Negative control was prepared using discs impregnated with 10 % DMSO in water and commercially available Methicillin and Ampicillin from Alkom Laboratories LTD were used as positive reference standards for all bacterial strains.

Results

We noticed that the highest activities were founded with the combination of alkaloid compounds and conventional antibiotics against all bacteria strains. Then, results showed that after 7 h exposition there was no viable microorganism in the initial inoculums.

Conclusion

The results of this study showed that alkaloid compounds in combination with conventional antibiotics (Methicillin, Ampicillin) exhibited antimicrobial effects against microorganisms tested. These results validate the ethno-botanical use of Cienfuegosia digitata Cav. (Malvaceae) in Burkina Faso. Moreover, this study demonstrates the potential of this herbaceous as a source of antibacterial agent that could be effectively used for future health care purposes.     

Cytotoxic effects of 2-arylbenzofuran phytoestrogens on human cancer cells: modulation by adrenal and gonadal steroids

Although 2-arylbenzofuran phytoalexins are known for decades, their anticancer activity has not been studied systematically. We have previously reported on the isolation and the estrogen receptor (ER) modulation properties of three new 2-arylbenzofurans from Onobrychis ebenoides, ebenfuran I [2-(2,4-dihydroxyphenyl)-5-hydroxy-6-methoxy-benzofuran], ebenfuran II [2-(2,4-dihydroxyphenyl)-3-formyl-4-hydroxy-6-methoxy-benzofuran] and ebenfuran III [2-(2,4-dihydroxyphenyl)-3-formyl-4-hydroxy-6-methoxy-5-(3-methyl-buten-2-yl)-benzofuran]. We now show that, while I and II could stimulate the proliferation of MCF-7 cells, III was inhibitory in a proliferation-dependent manner. III inhibited the growth of all human cancer cells examined, regardless of ER or multidrug resistance status. Estradiol rendered MCF-7 cells more sensitive to III, and this coincided with the ability of the hormone at concentrations ≥0.1 nM to bind to the ER of the cells and stimulate their proliferation in the presence of III. Cell proliferation stimulating concentrations of I and II also enhanced the effect of III on MCF-7 cells. However, dehydroepiandrosterone and dihydrotestosterone were ineffective in this respect. III-treated MCF-7 cells exhibited G1 phase arrest followed by detachment-induced cell death and/or apoptosis in the adherent fraction, pronounced induction of Bax and suppression of estradiol induction of Bcl-2. Our data indicate that the largely unexplored pool of benzofuran phytoalexins includes entities potentially suitable for chemoprevention and treatment of human cancer.     

ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y₁ receptor agonist, induced a large signal while UTPyS (P2Y₂ agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y₁. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.     

A semi-quantitative, synteny-based method to improve functional predictions for hypothetical and poorly annotated bacterial and archaeal genes

During microbial evolution, genome rearrangement increases with increasing sequence divergence. If the relationship between synteny and sequence divergence can be modeled, gene clusters in genomes of distantly related organisms exhibiting anomalous synteny can be identified and used to infer functional conservation. We applied the phylogenetic pairwise comparison method to establish and model a strong correlation between synteny and sequence divergence in all 634 available Archaeal and Bacterial genomes from the NCBI database and four newly assembled genomes of uncultivated Archaea from an acid mine drainage (AMD) community. In parallel, we established and modeled the trend between synteny and functional relatedness in the 118 genomes available in the STRING database. By combining these models, we developed a gene functional annotation method that weights evolutionary distance to estimate the probability of functional associations of syntenous proteins between genome pairs. The method was applied to the hypothetical proteins and poorly annotated genes in newly assembled acid mine drainage Archaeal genomes to add or improve gene annotations. This is the first method to assign possible functions to poorly annotated genes through quantification of the probability of gene functional relationships based on synteny at a significant evolutionary distance, and has the potential for broad application.     

SSU‐rRNA Gene Sequencing Survey of Benthic Microbial Eukaryotes from Guaymas Basin Hydrothermal Vent

Microbial eukaryotes have important roles in marine food webs, but their diversity and activities in hydrothermal vent ecosystems are poorly characterized. In this study, we analyzed microbial eukaryotic communities associated with bacterial (Beggiatoa) mats in the 2,000 m deep‐sea Guaymas Basin hydrothermal vent system using 18S rRNA gene high‐throughput sequencing of the V4 region. We detected 6,954 distinct Operational Taxonomic Units (OTUs) across various mat systems. Of the sequences that aligned with known protistan phylotypes, most were affiliated with alveolates (especially dinoflagellates and ciliates) and cercozoans. OTU richness and community structure differed among sediment habitats (e.g. different mat types and cold sediments away from mats). Additionally, full‐length 18S rRNA genes amplified and cloned from single cells revealed the identities of some of the most commonly encountered, active ciliates in this hydrothermal vent ecosystem. Observations and experiments were also conducted to demonstrate that ciliates were trophically active and ingesting fluorescent bacteria or Beggiatoa trichomes. Our work suggests that the active and diverse protistan community at the Guaymas Basin hydrothermal vent ecosystem likely consumes substantial amounts of bacterial biomass, and that the different habitats, often defined by distances of just a few 10s of cm, select for particular assemblages and levels of diversity.     

Effects of the entomopathogenic fungus Neozygites tanajoae and the predatory mite Typhlodromalus aripo on cassava green mite densities: screenhouse experiments

To improve biocontrol of the cassava green mite, Mononychellus tanajoa, a series of screenhouse experiments were conducted on cassava to determine the effects of single and combined releases of the predatory mite Typhlodromalus aripo and the entomopathogenic fungus Neozygites tanajoae on the suppression of M. tanajoa. We showed that, separately or in combination, T. aripo and N. tanajoae significantly reduced M. tanajoa populations. Moreover, combined release of T. aripo and N. tanajoae on the same cassava plants improved M. tanajoa control. However, our data suggest between T. aripo and N. tanajoae an asymmetric competition that significantly affected N. tanajoae only. This interference is indirect, probably mediated by their common prey or host mite, M. tanajoa since N. tanajoae is not pathogenic to T. aripo. We conclude that adding N. tanajoae to T. aripo populations in Africa would not negatively affect T. aripo and would further promote biocontrol of M. tanajoa.