Macrocyclic Lactones Differ in Interaction with Recombinant P-Glycoprotein 9 of the Parasitic Nematode Cylicocylus elongatus and Ketoconazole in a Yeast Growth Assay

Macrocyclic lactones (MLs) are widely used parasiticides against nematodes and arthropods, but resistance is frequently observed in parasitic nematodes of horses and livestock. Reports claiming resistance or decreased susceptibility in human nematodes are increasing. Since no target site directed ML resistance mechanisms have been identified, non-specific mechanisms were frequently implicated in ML resistance, including P-glycoproteins (Pgps, designated ABCB1 in vertebrates). Nematode genomes encode many different Pgps (e.g. 10 in the sheep parasite Haemonchus contortus). ML transport was shown for mammalian Pgps, Pgps on nematode egg shells, and very recently for Pgp-2 of H. contortus. Here, Pgp-9 from the equine parasite Cylicocyclus elongatus (Cyathostominae) was expressed in a Saccharomyces cerevisiae strain lacking seven endogenous efflux transporters. Pgp was detected on these yeasts by flow cytometry and chemiluminescence using the monoclonal antibody UIC2, which is specific for the active Pgp conformation. In a growth assay, Pgp-9 increased resistance to the fungicides ketoconazole, actinomycin D, valinomycin and daunorubicin, but not to the anthelmintic fungicide thiabendazole. Since no fungicidal activity has been described for MLs, their interaction with Pgp-9 was investigated in an assay involving two drugs: Yeasts were incubated with the highest ketoconazole concentration not affecting growth plus increasing concentrations of MLs to determine competition between or modulation of transport of both drugs. Already equimolar concentrations of ivermectin and eprinomectin inhibited growth, and at fourfold higher ML concentrations growth was virtually abolished. Selamectin and doramectin did not increase susceptibility to ketoconazole at all, although doramectin has been shown previously to strongly interact with human and canine Pgp. An intermediate interaction was observed for moxidectin. This was substantiated by increased binding of UIC2 antibodies in the presence of ivermectin, moxidectin, daunorubicin and ketoconazole but not selamectin. These results demonstrate direct effects of MLs on a recombinant nematode Pgp in an ML-specific manner.     

Human Polyhomeotic Homolog 3 (PHC3) Sterile Alpha Motif (SAM) Linker Allows Open-Ended Polymerization of PHC3 SAM

Sterile alpha motifs (SAMs) are frequently found in eukaryotic genomes. An intriguing property of many SAMs is their ability to self-associate, forming an open-ended polymer structure whose formation has been shown to be essential for the function of the protein. What remains largely unresolved is how polymerization is controlled. Previously, we had determined that the stretch of unstructured residues N-terminal to the SAM of a Drosophila protein called polyhomeotic (Ph), a member of the polycomb group (PcG) of gene silencers, plays a key role in controlling Ph SAM polymerization. Ph SAM with its native linker created shorter polymers compared to Ph SAM attached to either a random linker or no linker. Here, we show that the SAM linker for the human Ph ortholog, polyhomeotic homolog 3 (PHC3), also controls PHC3 SAM polymerization but does so in the opposite fashion. PHC3 SAM with its native linker allows longer polymers to form compared to when attached to a random linker. Attaching the PHC3 SAM linker to Ph SAM also resulted in extending Ph SAM polymerization. Moreover, in the context of full-length Ph protein, replacing the SAM linker with PHC3 SAM linker, intended to create longer polymers, resulted in greater repressive ability for the chimera compared to wild-type Ph. These findings show that polymeric SAM linkers evolved to modulate a wide dynamic range of SAM polymerization abilities and suggest that rationally manipulating the function of SAM containing proteins through controlling their SAM polymerization may be possible.     

Hominoid dispersal patterns and human evolution

Recent advances in DNA and isotope analyses have allowed tentative reconstructions of dispersal strategies of Plio-Pleistocene hominins.(1,2) Comparing their findings to dispersal patterns of some extant apes and humans suggested groups of related males and unrelated females in Neandertals indicating patrilocality(2) and Pan-like male philopatry in australopiths.(1) Here we review the demographic, ethnographic, and genetic evidence of dispersal patterns in extant apes and humans and compare the results to the suggestions for Plio-Pleistocene hominins. We find that alternative dispersal patterns, for example among gorillas or gibbons, could explain the findings of related or natal males in a confined geographic area. Based on sexual size dimorphism, we speculate that gorillas might currently be the best model for reconstructing dispersal in robust australopiths. Given that the sexual size dimorphism in other australopiths is still hotly debated, the question of which hominoid model best matches their dispersal pattern must remain unanswered. Neandertal dispersal patterns have been compared to patrilocality of modern humans. However, the latter is related to the advent of food production. Consequently, hunter-gatherers exhibiting primarily multilocality appear to be the better comparison for Neandertals. Overall, human-like patrilocality and Pan-like male philopatry appear to be poor models for the reconstruction of dispersal patterns in Plio-Pleistocene hominins.     

New QTLs identified for plant water status, water-soluble carbohydrate and osmotic adjustment in a barley population grown in a growth-chamber under two water regimes

Quantitative trait locus (QTL) analysis was carried out with 167 recombinant inbred lines (RILs) of barley derived from a cross between Tadmor and Er/Apm to identify the genomic regions controlling traits related to plant water status and osmotic adjustment (OA). The experiment was conducted in a growth chamber using a random incomplete block design (nine blocks). Relative water content (RWC) and leaf osmotic potential (ψ_π) were measured at 100% and 14% of the field capacity on 105 RILs in each block. In addition, the water-soluble carbohydrate concentration (WSC) was measured in the four first-blocks. The leaf osmotic potential at full turgor (ψ_π100), the water-soluble carbohydrate concentration at full turgor (WSC100), and also OA, the accumulation of water-soluble carbohydrates (dWSC100), the contribution of a change in water content to OA (CWC) and of the net solute accumulation to OA (SA) have also been calculated. In a previous paper (Teulat et al. 1998), 12 QTLs were identified for RWC, ψ_π, ψ_π100 and OA with adjusted means (block effects and pot-within-block effects fixed) with an incomplete genetic map. In the present paper, a more-saturated and improved map is described. A new QTL analysis as been performed with adjusted means. The new QTLs identified for previous evaluated traits, as well as the QTLs for the new traits, are presented. Eight additional regions (22 QTLs) were identified which increased to 13 the total number of chromosomal regions (32 QTLs) controlling traits related to plant water status and/or osmotic adjustment in this barley genetic background. The results emphasise the value of the experimental design employed for the evaluation of traits difficult to assess in genetic studies. The putative target regions for drought-tolerance improvement are discussed combining arguments on the consistency of QTLs and, when possible, the physiological value of QTLs (trait relevance, syntenic relationships and clustering of QTLs). Received: 8 March 2000 / Accepted: 18 October 2000     

What Hanuman langur males know about female reproductive status

In species with a high risk of infanticide, a conflict of interest exists between the sexes over the amount of paternity information that is available to males. While females are expected to keep males unaware of their reproductive status in order to confuse paternity, selection should favor those male traits that enhance the males' assessment of female status and consequently of paternity probability. In Hanuman langurs (Semnopithecus entellus), a species that is extremely vulnerable to infanticide, females have been shown to successfully conceal the exact timing of ovulation from males--perhaps because they exhibit no sexual swelling and mate during all reproductive phases, including gestation. Nevertheless, it remains unclear whether males have hitherto unrecognized information about females' reproductive condition on a broader level that could still enhance male reproductive success. We investigated male assessment of female reproductive states in a population of wild Hanuman langurs as indicated by changes in male behavior, such as rates of copulations, anogenital inspections, and consortships, in relation to different female receptive periods (pregnant, fertile-nonconceptional, and conceptional). Our data indicate that males were able to discern qualitatively distinct reproductive states. Males were more interested in fertile than pregnant females, as indicated by higher copulation rates. Based on consortships, males distinguished fertile from nonfertile phases, as well as fertile, nonconceptional receptive periods from conceptional ones. Hanuman langur males are thus not as unaware of female reproductive condition as previously thought, supporting the idea of an ongoing battle of the sexes over paternity information. However, granting some knowledge while at the same time concealing the exact day of ovulation may also reflect a pure female strategy of balancing paternity concentration with paternity confusion, which is the most likely strategy in this system with high infanticide risk and male defense of infants.     

Structure of the Hsp110:Hsc70 nucleotide exchange machine

Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family. We report the structure of an Hsp110:Hsc70 nucleotide exchange complex. The complex is characterized by extensive protein:protein interactions and symmetric bridging interactions between the nucleotides bound in each partner protein's NBD. An electropositive pore allows nucleotides to enter and exit the complex. The role of nucleotides in complex formation and dissociation, and the effects of the protein:protein interactions on nucleotide exchange, can be understood in terms of the coupled effects of the nucleotides and protein:protein interactions on the open-closed isomerization of the NBDs. The symmetrical interactions in the complex may model other Hsp70 family heterodimers in which two Hsp70s reciprocally act as NEFs.     

Paternity analysis of alternative male reproductive routes among the langurs (Semnopithecus entellus) of Ramnagar

Because primate males usually invest very little in offspring, male reproductive success will mainly be determined by access to fertile females with differences in access leading to differential male reproductive success. To determine the outcome of alternative male reproductive routes, we investigated a wild population of Hanuman langurs at Ramnagar, South Nepal, where groups were either one-male or multimale. Paternity was established by DNA analysis from faeces for 42 infants in five groups. In one-male groups all infants were sired by the only resident male in the group. In multimale groups the alpha male fathered significantly more infants (57%) than all other resident males. Nonresident males sired at least 21% of all infants born into multimale groups. Because of the lower mean number of infants sired by alpha males during their whole tenure as alpha compared with males in one-male groups (2.3 versus 6.8) and the higher maximum value (8.9 versus 6.0), the alpha male route is considered the riskier option. Based on demographic data we suggest that the considerable variance in short-term reproductive success might not translate to the same degree into differences in long-term reproductive success. Copyright 2001 The Association for the Study of Animal Behaviour.     

Analytical sedimentation of the IIAChb and IIBChb proteins of the Escherichia coli N,N'-diacetylchitobiose phosphotransferase system. Demonstration of a model phosphotransfer transition state complex

The phosphoenolpyruvate:glycose transferase system (PTS) is a prototypic signaling system responsible for the vectorial uptake and phosphorylation of carbohydrate substrates. The accompanying papers describe the proteins and product of the Escherichia coli N, N-diacetylchitobiose ((GlcNAc)(2)) PTS-mediated permease. Unlike most PTS transporters, the Chb system is composed of two soluble proteins, IIA(Chb) and IIB(Chb), and one transmembrane receptor (IIC(Chb)). The oligomeric states of PTS permease proteins and phosphoproteins have been difficult to determine. Using analytical ultracentrifugation, both dephospho and phosphorylated IIA(Chb) are shown to exist as stable dimers, whereas IIB(Chb), phospho-IIB(Chb) and the mutant Cys10SerIIB(Chb) are monomers. The mutant protein Cys10SerIIB(Chb) is unable to accept phosphate from phospho-IIA(Chb) but forms a stable higher order complex with phospho-IIA(Chb) (but not with dephospho-IIA(Chb)). The stoichiometry of proteins in the purified complex was determined to be 1:1, indicating that two molecules of Cys10SerIIB(Chb) are associated with one phospho-IIA(Chb) dimer in the complex. The complex appears to be a transition state analogue in the phosphotransfer reaction between the proteins. A model is presented that describes the concerted assembly and disassembly of IIA(Chb)-IIB(Chb) complexes contingent on phosphorylation-dependent conformational changes, especially of IIA(Chb).