A novel functional assay for fungal histidine kinases group III reveals the role of HAMP domains for fungicide sensitivity

Signal transduction systems comprising histidine kinases are suggested as new molecular targets of antibiotics. The important human fungal pathogen Candida albicans possesses three histidine kinases, one of which is the type III histidine kinase CaNik1, which activates the MAP kinase Hog1. We established a screening system for inhibitors of this class of histidine kinases by functional expression of the CaNIK1 gene in S. cerevisiae. This transformant was susceptible to fungicides to which the wild type strain was resistant, such as fludioxonil and ambruticin. Growth inhibition correlated with phosphorylation of Hog1 and was dependent on an intact Hog1 pathway. At the N-terminus the histidine kinase CaNik1 has four amino acid repeats of 92 amino acids each and one truncated repeat of 72 amino acids. Within these repeats we identified 9 HAMP domains with a paired structure. We constructed mutants in which one or two pairs of these domains were deleted. S. cerevisiae transformants expressing the full-length CaNIK1 showed the highest sensitivity to the fungicides, any truncation reduced the susceptibility of the transformants to the fungicides. This indicates that the HAMP domains are decisive for the mode of action of the antifungal compounds.     

Preparation and characterization of a stratum corneum substitute for in vitro percutaneous penetration studies

The intercellular stratum corneum (SC) lipids form the main barrier for diffusion of substances through the skin. A porous substrate covered with synthetic SC lipids would be an attractive model to study percutaneous penetration, hereby replacing native human SC. Prerequisite is that this stratum corneum substitute (SCS) is prepared with a uniform lipid composition and layer thickness. Furthermore, the lipid organization and orientation should resemble that in SC. The objective of this study was to investigate the utility of an airbrush spraying device to prepare a SCS composed of cholesterol, ceramides and free fatty acids on a polycarbonate filter. The results demonstrate that a proper choice of solvent mixture and lipid concentration is crucial to achieve a uniform distribution of the applied lipids over the filter surface. A smooth and tightly packed lipid layer is only obtained when the equilibration conditions are appropriately chosen. The SCS possesses two crystalline lamellar phases with periodicities similar to those present in native SC. The orientation of these lamellae is mainly parallel to the surface of the polycarbonate filter, which resembles the orientation of the intercellular SC lipids. In conclusion, the airbrush technique enables generation of a homogeneous SCS, which ultimately may function as a predictive in vitro percutaneous penetration model.     

Structure of the type III secretion recognition protein YscU from Yersinia enterocolitica

The inner-membrane protein YscU has an important role during the assembly of the Yersinia enterocolitica type III secretion injectisome. Its cytoplasmic domain (YscU^C) recognizes translocators as individual substrates in the export hierarchy. Activation of YscU entails autocleavage at a conserved NPTH motif. Modification of this motif markedly changes the properties of YscU, including translocator export cessation and production of longer injectisome needles. We determined the crystal structures of the uncleaved variants N263A and N263D of YscU^C at 2.05 Å and 1.55 Å resolution, respectively. The globular domain is found to consist of a central, mixed β-sheet surrounded by α-helices. The NPTH motif forms a type II β-turn connecting two β-strands. NMR analysis of cleaved and uncleaved YscU^C indicates that the global structure of the protein is retained in cleaved YscU^C. The structure of YscU^C variant N263D reveals that wild type YscU^C is poised for cleavage due to an optimal reaction geometry for nucleophilic attack of the scissile bond by the side chain of Asn263. In vivo analysis of N263Q and H266A/R314A YscU variants showed a phenotype that combines the absence of translocator secretion with normal needle-length control. Comparing the structure of YscU to those of related proteins reveals that the linker domain between the N-terminal transmembrane domain and the autocleavage domain can switch from an extended to a largely α-helical conformation, allowing for optimal positioning of the autocleavage domain during injectisome assembly.     

Dissociative Symptoms and Reported Trauma Among Patients with Spirit Possession and Matched Healthy Controls in Uganda

Spirit possession is a common, worldwide phenomenon with dissociative features. Studies in Europe and the United States have revealed associations among psychoform and somatoform dissociation and (reported) potential traumatic events. The aim of this study was to explore the relationships among spirit possession, dissociative symptoms and reported potentially traumatizing events in Uganda. One hundred nineteen persons with spirit possession, diagnosed by traditional healers, were compared to a matched control group of 71 nonpossessed persons. Assessments included demographic items and measures of dissociation and potentially traumatizing events. Compared to the nonpossessed group, the possessed group reported more severe psychoform dissociation and somatoform dissociation and more potentially traumatizing events. The associations between these events and both types of dissociation were significant. Yet, consistent with the cultural perception of dissociative symptoms, the participants subjectively did not associate dissociative symptoms with potentially traumatizing events. In conclusion, spirit possession deserves more interest as a possible idiom of distress and a culture-specific expression of dissociation related to potential traumatizing events.     

Continuous-time modeling of cell fate determination in Arabidopsis flowers

Background  

The genetic control of floral organ specification is currently being investigated by various approaches, both experimentally and through modeling. Models and simulations have mostly involved boolean or related methods, and so far a quantitative, continuous-time approach has not been explored.     

Effect of size and charge on endocytosis of lysozyme derivatives by sinusoidal rat liver cells in vivo

Hen egg-white lysozyme has been modified by intermolecular cross-linking with dimethyl suberimidate or by acylation with acetic or succinic anhydride. Retention of the native conformation of the modified enzyme was checked by measuring enzyme activity, resistance of disulfide bridges to reduction by thiols, and susceptibility to proteases.Unmodified lysozyme and its derivatives (labelled with ¹²⁵I) were intravenously injected into nephrectomized rats, and plasma clearance and uptake by liver cells were determined. Under these conditions, about 6% of the unmodified lysozyme was taken up by liver 15 min after injection. Cross-linking led to a greatly increased uptake (up to 89% of the dose in 15 min), whereas acylation reduced the uptake to 3–4%. Cell isolations showed that the unmodified enzyme and the cross-linked derivatives were taken up by sinusoidal cells. Differential fractionation of liver homogenates indicated that the unmodified enzyme was taken up in lysosomes. The cross-linked derivatives were concentrated in the nuclear and microsomal fractions as well as in the lysosomal fraction, suggesting adsorption on plasma membranes besides uptake in lysosomes.The experiments described in this paper, together with previous results on ribonuclease and lactate dehydrogenase, indicate that endocytosis of some proteins by sinusoidal liver cells is positively correlated with size and positive charge of the molecules.     

Evaluation of the extent of among-family variation in inbreeding depression in the perennial herb Scabiosa columbaria (Dipsacaceae)

Significantly different maternal line responses to inbreeding provide a mechanism for the invasion of a selfing variant into a population. The goal of this study was to examine the extent of family-level variation in inbreeding depression in the mixed-mating, perennial herb Scabiosa columbaria. Plants from one population were raised, and hand-pollinated to produce selfed and outcrossed progeny, and the effects of inbreeding depression on life-cycle traits were analyzed. Inbreeding depression significantly affected early life cycle traits. The pollination treatment by family interaction was significant for almost all traits, indicating a high family-level variation in inbreeding depression. The correlations between inbreeding depression values (e.g., percentage germination and flowering date, and flowering date and aboveground biomass) exhibited alternate signs, illustrating the type of association between inbreeding depression loci for different traits across the life cycle. Overall, it is concluded that the extent of among-family variation in inbreeding depression might allow a selfing variant of S. columbaria to invade an outcrossing population, though the pattern of correlations between inbreeding depression values might prevent effective purging of the deleterious genetic load.