Lupin (Lupinus spp.) seeds exert anthelmintic activity associated with their alkaloid content.

The growing range of drug resistant parasitic nematode populations threatens the sustainability of ruminant farming worldwide. In this context, nutraceuticals, animal feed that provides necessary dietary requirements while ensuring parasite control, could contribute to increase farming sustainability in developed and low resource settings. In this study, we evaluated the anthelmintic potential of lupin seed extracts against the major ruminant trichostrongylids, Haemonchus contortus and Teladorsagia circumcincta. In vitro observations showed that seed extracts from commercially available lupin varieties could significantly but moderately inhibit larval migration. This anthelmintic effect was mediated by the seed alkaloid content and was potent against both fully susceptible and multidrug resistant H. contortus isolates as well as a susceptible T. circumcincta isolate. Analytical chemistry revealed a set of four lupanine and sparteine-derivatives with anthelmintic activity, and electrophysiology assays on recombinant nematode acetylcholine receptors suggested an antagonistic mode of action for lupin alkaloids. An in vivo trial in H. contortus infected lupin-fed ewes and goats failed to demonstrate any direct anthelmintic effect of crude lupin seeds but infected lupin-fed goats suffered significantly less parasite-mediated blood losses. Altogether, our findings suggest that the anthelmintic potential of lupin remains limited. However, the potent alkaloids identified could lead to the development of novel drugs or may be used in combination with current anthelmintics to improve their efficacy.

Increased leukocyte survival and accelerated onset of lymphoma in the absence of MCL-1 S159-phosphorylation.

The antiapoptotic BCL-2 protein MCL-1, which opposes mitochondrial outer membrane permeabilization, was shown to have a crucial role in the survival of hematopoietic cells. We have previously shown that, upon loss of phosphatidylinositol 3-kinase signaling, S159 of MCL-1 is phosphorylated by glycogen synthase kinase-3 (GSK-3), earmarking MCL-1 for enhanced ubiquitylation and degradation. In this study, we introduced MCL-1(wt) or the phosphorylation-deficient mutant MCL-1(S159A) in mouse BM cells, followed by adoptive transfer to recipient mice. Mice expressing MCL-1(S159A) exhibited significantly elevated white blood cell and lymphocyte counts, whereas no effect was observed on the distribution of T and B lymphocyte subsets or the numbers of monocytes, red blood cells or platelets. Expression of MCL-1(S159A) in Eµ-Myc transgenic bone marrow significantly accelerated the onset of disease, and these mice displayed increased spleen weights compared with Eµ-Myc/MCL-1(wt) mice. Our data demonstrate that the absence of MCL-1 S159 phosphorylation provides a survival advantage for hematopoietic cells in vivo and facilitates oncogenesis.

Emodepside and SL0-1 potassium channels: a review.

Nematode parasites infect humans and domestic animals; treatment and prophylaxis require anthelmintic drugs because vaccination and sanitation is limited. Emodepside is a more recently introduced cyclooctadepsipeptide drug that has actions against GI nematodes, lungworm, and microfilaria. It has a novel mode of action which breaks resistance to the classical anthelmintics (benzimidazoles, macrocyclic lactones and cholinergic agonists). Here we review studies on its mode of action which suggest that it acts to inhibit neuronal and muscle activity of nematodes by increasing the opening of calcium-activated potassium (SLO-1) channels.

Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance.

BACKGROUND AND PURPOSE: The cholinergic agonist levamisole is widely used to treat parasitic nematode infestations. This anthelmintic drug paralyses worms by activating a class of levamisole-sensitive acetylcholine receptors (L-AChRs) expressed in nematode muscle cells. However, levamisole efficacy has been compromised by the emergence of drug-resistant parasites, especially in gastrointestinal nematodes such as Haemonchus contortus. We report here the first functional reconstitution and pharmacological characterization of H. contortus L-AChRs in a heterologous expression system. EXPERIMENTAL APPROACH: In the free-living nematode Caenorhabditis elegans, five AChR subunit and three ancillary protein genes are necessary in vivo and in vitro to synthesize L-AChRs. We have cloned the H. contortus orthologues of these genes and expressed them in Xenopus oocytes. We reconstituted two types of H. contortus L-AChRs with distinct pharmacologies by combining different receptor subunits. KEY RESULTS: The Hco-ACR-8 subunit plays a pivotal role in selective sensitivity to levamisole. As observed with C. elegans L-AChRs, expression of H. contortus receptors requires the ancillary proteins Hco-RIC-3, Hco-UNC-50 and Hco-UNC-74. Using this experimental system, we demonstrated that a truncated Hco-UNC-63 L-AChR subunit, which was specifically detected in a levamisole-resistant H. contortus isolate, but not in levamisole-sensitive strains, hampers the normal function of L-AChRs, when co-expressed with its full-length counterpart. CONCLUSIONS AND IMPLICATIONS: We provide the first functional evidence for a putative molecular mechanism involved in levamisole resistance in any parasitic nematode. This expression system will provide a means to analyse molecular polymorphisms associated with drug resistance at the electrophysiological level.

On the mode of action of emodepside: slow effects on membrane potential and voltage-activated currents in Ascaris suum.

BACKGROUND AND PURPOSE: Anthelmintics are required for treatment and prophylaxis of nematode parasites of humans and domestic animals. Emodepside, a cyclooctadepsipeptide, is a modern anthelmintic that has a novel mode of action involving a Ca-activated K channel (SLO-1) in Caenorhabditis elegans, sometimes mediated by a latrophilin (LAT) receptor. We examined mechanisms of action of emodepside in a parasitic nematode, Ascaris suum. EXPERIMENTAL APPROACH: RT-PCR was used to investigate expression of slo-1 and lat-1 in A. suum muscle flaps, and two-micropipette current-clamp and voltage-clamp techniques were used to record electrophysiological effects of emodepside. KEY RESULTS: Expression of slo-1 and lat-1 were detected. Emodepside produced a slow time-dependent (20 min), 4-aminopyridine sensitive, concentration-dependent hyperpolarization and increase in voltage-activated K currents. Sodium nitroprusside increased the hyperpolarizations and K currents. N-nitro-L-arginine inhibited the hyperpolarizations and K currents. Phorbol-12-myristate-13 acetate increased the K currents, while staurosporine inhibited the hyperpolarizations and K currents. Iberiotoxin reduced these emodepside K currents. The effect of emodepside was reduced in Ca-free solutions. Emodepside had no effect on voltage-activated Ca currents. CONCLUSIONS AND IMPLICATIONS: Asu-slo-1 and Asu-lat-1 are expressed in adult A. suum muscle flaps and emodepside produces slow activation of voltage-activated Ca-dependent SLO-1-like K channels. The effect of emodepside was enhanced by stimulation of protein kinase C and NO pathways. The data are consistent with a model in which NO, PKC and emodepside signalling pathways are separate and converge on the K channels, or in which emodepside activates NO and PKC signalling pathways to increase opening of the K channels.

Identification of levamisole resistance markers in the parasitic nematode Haemonchus contortus using a cDNA-AFLP approach.

cDNA-AFLP (cDNA-Amplified Fragment Length Polymorphism)-based strategy has been used to identify levamisole (LEV) resistance markers in the nematode Haemonchus contortus. Transcript profiles of adult nematodes from two LEV-resistant and two susceptible isolates were compared. Among the 17 280 transcript-derived fragments (TDFs) amplified, 26 presented a polymorphic pattern between resistant and susceptible nematodes: 11 TDFs were present in both resistant isolates and absent from both susceptible isolates whereas 15 TDFs were present in both susceptible isolates and absent from both resistant isolates. 8 TDFs specifically present in resistant isolates were cloned and sequenced. Some of these TDFs could represent novel genes, as their sequences presented no homologies in databases. Interestingly, specific expression of one candidate (HA17) in resistant nematodes from different isolates was confirmed by RT-PCR experiments. The finding that HA17 expression correlates with LEV resistance in three H. contortus isolates vs five susceptible isolates strongly suggest that we identified a new potential marker of LEV resistance. This differential approach at the transcriptome level could be of great interest for the identification of the molecular mechanism involved in this phenotype.

Vav2 activates c-fos serum response element and CD69 expression but negatively regulates nuclear factor of activated T cells and interleukin-2 gene activation in T lymphocyte.

Vav1 and Vav2 are members of the Dbl family of guanine nucleotide exchange factors for the Rho family of small GTPases. Although the role of Vav1 during lymphocyte development and activation is well characterized, the function of Vav2 is still unclear. In this study, we compared the signaling pathways regulated by Vav1 and Vav2 following engagement of the T cell receptor (TCR). We show that Vav2 is tyrosine-phosphorylated upon TCR stimulation and by co-expressed Src and Syk family kinases. Using glutathione S-transferase fusion proteins, we observed that the Src homology 2 domain of Vav2 binds tyrosine-phosphorylated proteins from TCR-stimulated Jurkat T cell lysates, including c-Cbl and SLP-76. Like Vav1, Vav2 cooperated with TCR stimulation to increase extracellular signal-regulated kinase activation and to promote c-fos serum response element transcriptional activity. Moreover, both proteins displayed a similar action in increasing the expression of the early activation marker CD69 in Jurkat T cells. However, in contrast to Vav1, Vav2 dramatically suppressed TCR signals leading to nuclear factor of activated T cells (NF-AT)-dependent transcription and induction of the interleukin-2 promoter. Vav2 appears to act upstream of the phosphatase calcineurin because a constitutively active form of calcineurin rescued the effect of Vav2 by restoring TCR-induced NF-AT activation. Interestingly, the Dbl homology and Src homology 2 domains of Vav2 were necessary for its inhibitory effect on NF-AT activation and for induction of serum response element transcriptional activity. Taken together, our results indicate that Vav1 and Vav2 exert overlapping but nonidentical functions in T cells. The negative regulatory pathway elicited by Vav2 might play an important role in regulating lymphocyte activation processes.

[The free union and marriage: assessment of work in demography]. / L'union libre et le mariage: un bilan des travaux en demographie.

PIP: Begun in the middle of the 19th century, the demographic transition will have soon reached almost all of the world¿s countries. Such transition also involves social and economic changes. Now, during a second wave of transformation, the Western world is going through a sort of revolution of its familial system linked to changes in the systems of reproduction. Marriage, long considered to be a permanent union, is increasingly threatened as couples declare the failure of their unions. During the mid-1970s, one began to see a greater degree of cohabitation outside of marriage, while legal marriage diminished in importance. These events challenged the definition of family, after which there was an increase in the number of free unions of varying duration, and either fecund or not. Marriage, coresidence of the couple, then childbearing became less common. Rather, that pattern is often replaced by unions other than marriage, where partners progress through unions with new and different partners, and where certain individuals go through rather long periods of single parenthood. The authors note works undertaken on marriage and free union. After exploring the theoretical explanations proposed by demographers, the authors review studies on the progression from free union, the dynamics of free union creation and dissolution, the role of conception or child birth in the establishment of an union or its evolution into marriage, and the links between cohabitation with marriage and fertility.^ieng