The impact of the fourth disulfide bridge in scorpion toxins of the alpha-KTx6 subfamily

Animal toxins are highly reticulated and structured polypeptides that adopt a limited number of folds. In scorpion species, the most represented fold is the alpha/beta scaffold in which an helical structure is connected to an antiparallel beta-sheet by two disulfide bridges. The intimate relationship existing between peptide reticulation and folding remains poorly understood. Here, we investigated the role of disulfide bridging on the 3D structure of HsTx1, a scorpion toxin potently active on Kv1.1 and Kv1.3 channels. This toxin folds along the classical alpha/beta scaffold but belongs to a unique family of short-chain, four disulfide-bridged toxins. Removal of the fourth disulfide bridge of HsTx1 does not affect its helical structure, whereas its two-stranded beta-sheet is altered from a twisted to a nontwisted configuration. This structural change in HsTx1 is accompanied by a marked decrease in Kv1.1 and Kv1.3 current blockage, and by alterations in the toxin to channel molecular contacts. In contrast, a similar removal of the fourth disulfide bridge of Pi1, another scorpion toxin from the same structural family, has no impact on its 3D structure, pharmacology, or channel interaction. These data highlight the importance of disulfide bridging in reaching the correct bioactive conformation of some toxins.     

GROWTH STIMULATION OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) BY HUMIC SUBSTANCES FROM THE MANICOUAGAN RIVER (EASTERN CANADA)1

In the St. Lawrence Estuary, annual recurrent blooms of the toxic dinoflagellate Alexandrium tamarense L. Balech are associated with brackish waters. Riverine inputs are suspected to favor bloom development by increasing water column stability and/or by providing growth stimulants such as humic substances (HS). A 17‐day culture experiment was conducted to evaluate the importance of HS as growth factors for A. tamarense. Nonaxenic cultures were exposed to four HS extracts from three different sources: humic and fulvic acids isolated from the Manicouagan River, Quebec, Canada; humic acids from the Suwannee River, Georgia, United States; and a desalted alkaline soil extract. For each extract, four concentrations were tested as supplements to the artificial Keller medium, a nitrate‐rich algal culture medium. Additions of HS from all sources significantly enhanced the overall growth rates relative to the controls. Concentrations of HS, estimated by UV spectrophotometry, remained constant throughout the exponential growth phase, suggesting that the HS were acting mainly as growth promoters during our experiment. Dose–response curves indicated that HS could increase the growth rate of A. tamarense even at low concentrations, such as those encountered in the St. Lawrence Estuary. Our results support the hypothesis that HS from the Manicouagan River plume can stimulate the development of toxic dinoflagellate blooms.     

The endoproteolytic processing of alphavbeta5 integrin is involved in cytoskeleton remodelling and cell migration

We previously showed that the post-translational cleavage of alphav subunit is essential for integrin-dependent signalling and cell adhesion. Here, we report that blocking alphav subunit cleavage by expression of alpha1-PDX, a convertase inhibitor, modified the capacity of cells to change shape, via a remodelling of the actin cytoskeleton upon cell attachment. These changes are associated with cell scattering and with a dramatic increase in cell migration to vitronectin. The alphav subunit cleavage is thus essential for integrin function and has a considerable impact on integrin-dependent events, especially those leading to cell migration.     

Validation of Horne and Ostberg morningness-eveningness questionnaire in a middle-aged population of French workers

As suggested by the authors, the Horne and Ostberg morning/evening questionnaire (MEQ) has never been adapted to evaluate a nonstudent population. The purpose of this study was to validate this MEQ in a sample of middle-aged workers by modifying only the cutoffs. It was administered in 566 non-shift-workers aged 51.2 to 3.2 years who presented no sleep disorders. According to the Home and Ostberg classification, the sample consisted of 62.1% morning type, 36.6% neither type, and 2.2% evening type. Multiple correspondence analysis, which determines the principal components, was performed on all MEQ items. Then an ascending hierarchical classification was applied to determine 3 clusters from these principal components. On the basis of these 3 clusters, new cutoffs were determined: evening types were considered as scoring under 53 and morning types above 64, thus giving 28.1% morning type, 51.7% neither type, and 20.2% evening type. As an external validation, eveningness was associated with later bedtime and waking-up time (more pronounced at the weekend), greater need for sleep, larger daily sleep debt, greater morning sleepiness, and ease of returning to sleep in the early morning. A positive correlation between age and morningness was again found. This study confirms that "owls" are not rare in a middle-aged sample. We conclude that this adapted MEQ could be useful when investigating age-related changes in sleep.     

Two Antiproliferative Triterpene Saponins from Nematostylis anthophylla from the Highlands of Central Madagascar

Investigation of the endemic Madagascan plant Nematostylis anthophylla (Rubiaceae) for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of the known triterpene saponin randianin ( 1 ), and the two new bioactive triterpene saponins 2″‐O‐acetylrandianin ( 2 ) and 6″‐O‐acetylrandianin ( 3 ). The structures of the two new compounds were elucidated based on analysis of their 1D‐ and 2D‐NMR spectra, and mass spectrometric data. The three isolated triterpene saponins displayed moderate but selective antiproliferative activities, with IC₅₀ values of 1.2, 1.7, and 2.2 μM , respectively, against the A2780 ovarian cancer, but only weak inhibitions of the proliferation of A2058 melanoma and the H522 lung cancer cell lines.     

Human impacts on minimum subsets of species critical for maintaining ecosystem structure

Humans have indirectly influenced species at lower trophic levels by driving losses of apex consumers. Furthermore, humans have indirectly influenced species at higher trophic levels by driving losses of primary producers. Beyond these broad classes of apex consumers and primary producers, it remains challenging to identify minimum subsets of species that are particularly important for maintaining ecosystem structure and functioning. Here we use a novel method at the intersection of control theory and network theory to identify a minimum set of driver node species upon which ecosystem structure strongly depends. Specifically, humans could unintentionally completely restructure ecosystems (i.e., change species abundances from any initial values to any final values, including zero) by altering the abundances of these few critical driver node species. We then quantify the proportion of these driver nodes that are influenced by humans, top predators, and primary producers in several marine food webs. We find that humans could unintentionally completely restructure marine food webs while only directly influencing less than one in four species. Additionally, humans directly influence: (1) most or all of the species necessary to completely restructure each network, (2) more driver nodes than top predators, and at least as many driver nodes as primary producers, and (3) an increasing proportion of driver nodes over time in the Adriatic Sea. We conclude that humans have potentially huge impacts on marine ecosystems while directly influencing only the relatively small subset of species that are currently fished. It may be possible to reduce unintentional and undesirable cascading human influences by decreasing human impacts on driver node species in these and other food webs.     

In vitro activity of novel dual action MDR anthranilamide modulators with inhibitory activity at CYP-450

Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their inhibition potency of the P-glycoprotein (P-gp) transporter. The aromatic spacer group between nitrogen atoms (N1 and N2) in the known inhibitor XR9576 was replaced with a flexible alkyl chain of 2 to 6 carbon atoms in length. 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline and their open-chain N-methylhomoveratrylamine counterparts were shown to be potent P-gp inhibitors. The maximal inhibition was obtained when using an ethyl or propyl spacer. Several compounds were more potent than verapamil and intrinsically less cytotoxic than XR9576. In addition, in vitro metabolism studies of 23a with a subset of human CYP-450 isoforms revealed that, unlike XR9576, 23a inhibited CYP3A4, an enzyme that colocalizes with P-gp in the intestine and contributes to tumor cell chemoresistance by enhancing the biodisposition of anticancer drugs such as paclitaxel toward metabolism. In this context, 22a might be a suitable candidate for further drug development.     

Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep

Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.