Development of a new real-time TaqMan PCR assay for quantitative analyses of Candida albicans resistance genes expression

Candida albicans is an important opportunistic pathogen that can cause serious fungal diseases in immunocompromised patients including cancer patients, transplant patients, and patients receiving immunosuppressive therapy in general, those with human immunodeficiency virus infections and undergoing major surgery. Its emergence spectrum varies from mucosal to systemic infections and the first line treatment is still based on fluconazole, a triazole derivate with a potent antifungal activity against most of C. albicans strains. Nevertheless the emergence of fluconazole-resistant C. albicans strains can lead to treatment failures and thus become a clinical problem in the management of such infections. For that reason we consider it important to study mechanisms inducing azole resistance and the possibilities to influence this process. In this work we give a short report on a real-time PCR (TaqMan) assay, which can be used for quantitative analyses of gene expression levels of MDR1, CDR1 and ERG11, genes supposed to contribute to development of the resistance mechanisms. We show some results achieved with that assay in fluconazole susceptible and resistant strains that confirm results seen earlier in experiments using Northern blot hybridisation and prove that the comparative DeltaCt method is valid for our system.     

Solid-state NMR [13C,15N] resonance assignments of the nucleotide-binding domain of a bacterial cyclic nucleotide-gated channel

Channels regulated by cyclic nucleotides are key signalling proteins in several biological pathways. The regulatory aspect is conferred by a C-terminal cyclic nucleotide-binding domain (CNBD). We report resonance assignments of the CNBD of a bacterial mlCNG channel obtained using 2D and 3D solid-state NMR under Magic-angle Spinning conditions. A secondary chemical shift analysis of the 141 residue protein suggests a three-dimensional fold seen in earlier X-ray and solution-state NMR work and points to spectroscopic polymorphism for a selected set of resonances.     

The Arabidopsis mitogen-activated protein kinase kinase MKK3 is upstream of group C mitogen-activated protein kinases and participates in pathogen signaling

Although the Arabidopsis thaliana genome contains genes encoding 20 mitogen-activated protein kinases (MAPKs) and 10 MAPK kinases (MAPKKs), most of them are still functionally uncharacterized. In this work, we analyzed the function of the group B MAPK kinase, MKK3. Transgenic ProMKK3:GUS lines showed basal expression in vascular tissues that was strongly induced by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000) infection but not by abiotic stresses. The growth of virulent Pst DC3000 was increased in mkk3 knockout plants and decreased in MKK3-overexpressing plants. Moreover, MKK3 overexpression lines showed increased expression of several PR genes. By yeast two-hybrid analysis, coimmunoprecipitation, and protein kinase assays, MKK3 was revealed to be an upstream activator of the group C MAPKs MPK1, MPK2, MPK7, and MPK14. Flagellin-derived flg22 peptide strongly activated MPK6 but resulted in poor activation of MPK7. By contrast, MPK6 and MPK7 were both activated by H(2)O(2), but only MPK7 activation was enhanced by MKK3. In agreement with the notion that MKK3 regulates the expression of PR genes, ProPR1:GUS expression was strongly enhanced by coexpression of MKK3-MPK7. Our results reveal that the MKK3 pathway plays a role in pathogen defense and further underscore the importance and complexity of MAPK signaling in plant stress responses.     

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.     

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.