Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors

The multidrug exporter AcrB is the inner membrane component of the AcrAB-TolC drug efflux system in Escherichia coli and is responsible for the resistance of this organism to a wide range of drugs. Here we describe the crystal structure of the trimeric AcrB in complex with a designed ankyrin-repeat protein (DARPin) inhibitor at 2.5-Å resolution. The three subunits of AcrB are locked in different conformations revealing distinct channels in each subunit. There seems to be remote conformational coupling between the channel access, exit, and the putative proton-translocation site, explaining how the proton motive force is used for drug export. Thus our structure suggests a transport pathway not through the central pore but through the identified channels in the individual subunits, which greatly advances our understanding of the multidrug export mechanism.     

Condition and coalition formation by brood-rearing common eider females

Partner choice is important in nature, and partnerships or coalitionswithin which reproduction is shared are the subject of growinginterest. However, little attention has been given to questionsof which individuals are suitable partners and why. Common eider(Somateria mollissima) females sometimes pool their broods andshare brood-rearing duties, and body condition affects caredecisions. We constructed a model in which females, based ontheir body condition and the structure of the joint brood, assessthe fitness consequences of joining a coalition versus tendingfor young alone. We tested the model's predictions by comparingdata on the condition of females in enduring and transient coalitions.Our model showed that the range of acceptable brood arrays ina female coalition decreases with increasing condition of thefemale, so females tending alone should be in better conditionthan multifemale tenders. This prediction is in agreement withprevious data. The model also predicts that females in goodcondition should join coalitions with females in poor conditionand not with other females in good condition. This predictionwas also supported by data: in enduring two-female coalitions,the positive correlation between the better female's conditionand the difference in condition between the two females wasstronger than would be expected by random grouping of females.In contrast, in transient coalitions of females, this correlationdid not differ from the correlation expected under random grouping.Model assumptions seem to fit with eider natural history, andthe model may prove to be a useful way to study brood amalgamationbehavior of waterfowl in general.     

Evolutionary relationships in the Asteraceae tribe Inuleae (incl. Plucheeae) evidenced by DNA sequences of ndhF; with notes on the systematic positions of some aberrant genera

The phylogenetic relationships between the tribes Inuleae sensu stricto and Plucheeae are investigated by analysis of sequence data from the cpDNA gene ndhF. The delimitation between the two tribes is elucidated, and the systematic positions of a number of genera associated with these groups, i.e. genera with either aberrant morphological characters or a debated systematic position, are clarified. Together, the Inuleae and Plucheeae form a monophyletic group in which the majority of genera of Inuleae s.str. form one clade, and all the taxa from the Plucheeae together with the genera Antiphiona, Calostephane, Geigeria, Ondetia, Pechuel-loeschea, Pegolettia, and Iphionopsis from Inuleae s.str. form another. Members of the Plucheeae are nested with genera of the Inuleae s.str., and support for the Plucheeae clade is weak. Consequently, the latter cannot be maintained and the two groups are treated as one tribe, Inuleae, with the two subtribes Inulinae and Plucheinae. The genera Asteriscus, Chrysophthalmum, Inula, Laggera, Pentanema, Pluchea, and Pulicaria are demonstrated to be non-monophyletic. Cratystylis and Iphionopsis are found to belong to the same clade as the taxa of the former Plucheeae. Caesulia is shown to be a close relative of Duhaldea and Blumea of the Inuleae-Inulinae. The genera Callilepis and Zoutpansbergia belong to the major clade of the family that includes the tribes Heliantheae sensu lato and Inuleae (incl. Plucheeae), but their exact position remains unresolved. The genus Gymnarrhena is not part of the Inuleae, but is either part of the unresolved basal complex of the paraphyletic Cichorioideae, or sister to the entire Asteroideae.     

Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion

Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion.     

Helix packing moments reveal diversity and conservation in membrane protein structure

Helical membrane proteins are more tightly packed and the packing interactions are more diverse than those found in helical soluble proteins. Based on a linear correlation between amino acid packing values and interhelical propensity, we propose the concept of a helix packing moment to predict the orientation of helices in helical membrane proteins and membrane protein complexes. We show that the helix packing moment correlates with the helix interfaces of helix dimers of single pass membrane proteins of known structure. Helix packing moments are also shown to help identify the packing interfaces in membrane proteins with multiple transmembrane helices, where a single helix can have multiple contact surfaces. Analyses are described on class A G protein-coupled receptors (GPCRs) with seven transmembrane helices. We show that the helix packing moments are conserved across the class A family of GPCRs and correspond to key structural contacts in rhodopsin. These contacts are distinct from the highly conserved signature motifs of GPCRs and have not previously been recognized. The specific amino acid types involved in these contacts, however, are not necessarily conserved between subfamilies of GPCRs, indicating that the same protein architecture can be supported by a diverse set of interactions. In GPCRs, as well as membrane channels and transporters, amino acid residues with small side-chains (Gly, Ala, Ser, Cys) allow tight helix packing by mediating strong van der Waals interactions between helices. Closely packed helices, in turn, facilitate interhelical hydrogen bonding of both weakly polar (Ser, Thr, Cys) and strongly polar (Asn, Gln, Glu, Asp, His, Arg, Lys) amino acid residues. We propose the use of the helix packing moment as a complementary tool to the helical hydrophobic moment in the analysis of transmembrane sequences.     

Retrospective study of the parental origin of the extra chromosome in trisomy 18 (Edwards syndrome)

The parental origin of the extra chromosome in trisomy 18 was traced in 30 informative families using highly polymorphic (CA) repeats mapped on the long arm of chromosome 18. Proband DNA was recovered from slides of chromosome preparations in 28 cases and from paraffin-embedded tissues in two cases. The extra chromosome was found to be of maternal origin in 26 cases (86.7%), and paternal origin in 4 cases (13.3%).     

A key role for E-cadherin in intestinal homeostasis and Paneth cell maturation

Background

E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn''s disease.

Methods and Findings

To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen.

Conclusion

These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells.