The Novel Candida albicans Transporter Dur31 Is a Multi-Stage Pathogenicity Factor

Candida albicans is the most frequent cause of oral fungal infections. However, the exact pathogenicity mechanisms that this fungus employs are largely unknown and many of the genes expressed during oral infection are uncharacterized. In this study we sought to functionally characterize 12 previously unknown function genes associated with oral candidiasis. We generated homozygous knockout mutants for all 12 genes and analyzed their interaction with human oral epithelium in vitro. Eleven mutants caused significantly less epithelial damage and, of these, deletion of orf19.6656 (DUR31) elicited the strongest reduction in pathogenicity. Interestingly, DUR31 was not only involved in oral epithelial damage, but in multiple stages of candidiasis, including surviving attack by human neutrophils, endothelial damage and virulence in vivo. In silico analysis indicated that DUR31 encodes a sodium/substrate symporter with 13 transmembrane domains and no human homologue. We provide evidence that Dur31 transports histatin 5. This is one of the very first examples of microbial driven import of this highly cytotoxic antimicrobial peptide. Also, in contrast to wild type C. albicans, dur31Δ/Δ was unable to actively increase local environmental pH, suggesting that Dur31 lies in the extracellular alkalinization hyphal auto-induction pathway; and, indeed, DUR31 was required for morphogenesis. In agreement with this observation, dur31Δ/Δ was unable to assimilate the polyamine spermidine.     

Covalent coupling of bilirubin to albumin.

A method for covalent coupling of bilirubin to albumin is described. Human serum albumin-bilirubin (1:1 complex) has been treated with water soluble carbodiimide in order to obtain covalent coupling of bilirubin to albumin. The reaction conditions have been varied with respect to pH, reaction time and concentration of reagent to obtain the optimal coupling. The prepared albumin-bilirubin compounds were investigated by spectrophotometry, gel filtration and gel electrophoresis to ascertain the covalent nature of the bond and to characterize the products further. Gel electrophoresis and gel filtration showed that a monomer fraction could be prepared, and this fraction was a suitable material for further studies.     

Post-stocking survival of 0+ year pike in ponds as a function of water transparency, habitat complexity, prey availability and size heterogeneity

Survival of 0+ year pike Esox lucius (20–31 mm L _T) was evaluated over the first week following stocking in ponds (16 m²) depending on water transparencies (low and high), habitat complexity (low and high) and food availability (zooplankton). A multiple regression analysis showed that survival, essentially as a result of cannibalism, decreased with increasing size heterogeneity of pike, and the decrease was steeper when no food was available. Survival was equal in clear water and water containing copper chlorophyllin (chlorophyll), and slightly higher in complex than in simple habitats, but only when alternative prey availability was low. Conversely, growth in water containing chlorophyll was slightly faster than in clear water. This suggests that initial size heterogeneity is the prevailing factor controlling mortality when stocking 0+ year pike almost irrespective of the stocking environment.     

Spatial variation in natural formation of chloroform in the soils of four coniferous forests

Natural chloroform in soil gas below four coniferous forest sites was studied. High concentrations were found within narrow areas??Hot Spots??varying from ~25 to >400 m² in size, with chloroform concentrations being typically 20?C100 times those in corresponding Low Spots.Attempts to localize Hot Spots by visual inspection with regard to type and density of vegetation failed. Possible differences between Hot and Low Spots could be emission, leaching or degradation of chloroform. However, emissions of chloroform from Hot Spots were ~10 times higher than from Low Spots and similarly the chloroform concentration in groundwater below a Hot Spot was ~10 times higher than below the corresponding Low Spot. No differences in chloroform mineralization rates were observed between sites and incubation of soil cores confirmed a larger net formation of chloroform in the Hot Spots. Various soil parameters were measured in order to compare the soil sampled from Hot and Low Spots. The halogenation degree of organic soil samples was in the same range, although slightly higher in the H-horizon of the Hot Spot. The chloroform formation potential of the soil organic matter showed differences between soil horizons but not between sites. The high levels of chloroform in the Hot Spots are probably best explained by differences in chloroform forming activity caused by an uneven distribution of yet unidentified microorganisms, since differences in soil organic matter quality and in emission, leaching and degradation of chloroform as well as a number of additional soil parameters could be completely ruled out.     

An online catalogue of AFLP markers covering the potato genome

An AFLP marker catalogue is presented for gene mapping within cultivated potato. The catalogue is comprised of AFLP fingerprint images of 733 chromosome-specific AFLP markers which are mapped relative to 220 RFLP loci, isozyme loci, morphological characteristics and disease resistance traits. Use of the catalogue is based on identification of common AFLP markers which are visually recognized on autoradiogram images as co-migrating bands in fingerprints generated from different genotypes. Images of AFLP fingerprints combined with detailed information on the genomic location of all AFLP markers are available at URL: http://www.spg.wau.nl/pv/aflp/catalog.htm. It is demonstrated that the comparison of autoradiogram images and subsequent identification of common AFLP markers solely are efficient means for alignment of linkage groups and mapping target genes.     

DNA flow cytometry in metastases and a recurrency of malignant melanomas. A comparison of results from fresh and paraffin embedded material

Single cell suspensions from 16 biopsies from 15 patients with metastatic or recurrent malignant melanoma were prepared according to the method described by Vindel?v (1977) and the nuclear DNA content was measured by a laboratory-built flow cytometer. The DNA histograms thus obtained were compared with those obtained from suspensions of single nuclei from the same biopsies after formalin fixation and paraffin embedding, according to the method of Hedley et al. (1983). Linear regression analysis of ploidy values from fresh material compared with those from paraffin blocks showed a strong correlation (R2 = 0.85), while that of the S-phase fraction was somewhat weaker (R2 = 0.66). It is concluded that archival wax preparations of malignant melanoma cell populations are suitable for FCM analysis of ploidy, and to a lesser extent for analysis of fraction of cells in various cell cycle phases.     

A novel D-leucine-containing Conus peptide: diverse conformational dynamics in the contryphan family.

A Conus peptide family (the contryphans) is noteworthy because of the presence of a post-translationally modified D-amino acid in all members of the family. A new contryphan peptide, Leu-contryphan-P, was isolated from the venom of Conus purpurascens; the sequence of this peptide is: Gly-Cys-Val-D-Leu-Leu-Pro-Trp-Cys-OH. This is the first known occurrence of D-leucine in a Conus peptide. The discovery of Leu-contryphan-P suggests that there may be branches of the contryphan peptide family that diverge much more in sequence than previously anticipated. Several natural contryphans provide dramatic examples of interconversion between multiple conformational states in small constrained peptides. The contryphans that have 4-trans-hydroxyproline and D-tryptophan in positions 3 and 4, respectively, exhibit two peaks under reverse-phase HPLC conditions, indicating interconversion between two discrete conformations. However, [L-Trp4]contryphan-Sm (with L-Trp substituted for D-Trp) exhibits a single, broad peak that elutes later than the natural peptide, suggesting that D-Trp stabilizes a conformation in which hydrophobic residues are buried. Leucontryphan-P which has valine and D-leucine instead of 4-trans-hydroxyproline and D-tryptophan shows only a single peak that elutes much later than the other contryphans.