Modification of flavin adenine dinucleotide in alcohol oxidase of the yeast Hansenula polymorpha.

Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch and continuous culture. The relative amount of mFAD in the enzyme varied from 5 to 95%, depending on the growth or storage conditions. The presence of mFAD led to a slight decrease in Vmax and a significant (about one order) decrease in the Km of alcohol oxidase with respect to methanol. The kinetics of modification measured in purified preparations of the enzyme obeyed first-order kinetics (k = 0.78 h-1). The modification process was strongly inhibited by methanol, formaldehyde or hydroxylamine. Modification observed in continuous culture under steady state conditions depended on the dilution rate and could also be described as a spontaneous first-order reaction (kapp = 0.27 h-1). FAD modification could only be detected in alcohol oxidase and not in other yeast peroxisomal flavoenzymes, such as D-amino acid oxidase from Candida boidinii.     

Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.     

Function of caspase-14 in trophoblast differentiation

Background  

Within the human placenta, the cytotrophoblast consists of a proliferative pool of progenitor cells which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast, which forms the barrier between the maternal and fetal tissues. Disruption to trophoblast differentiation and function may result in impaired fetal development and preeclampsia. Caspase-14 expression is limited to barrier forming tissues. It promotes keratinocyte differentiation by cleaving profilaggrin to stabilise keratin intermediate filaments, and indirectly providing hydration and UV protection. However its role in the trophoblast remains unexplored.     

Defining the healthy "core microbiome" of oral microbial communities

Background  

Most studies examining the commensal human oral microbiome are focused on disease or are limited in methodology. In order to diagnose and treat diseases at an early and reversible stage an in-depth definition of health is indispensible. The aim of this study therefore was to define the healthy oral microbiome using recent advances in sequencing technology (454 pyrosequencing).     

AFLP markers as a tool to reconstruct complex relationships: A case study in Rosa (Rosaceae)

The genus Rosa has a complex evolutionary history caused by several factors, often in conjunction: extensive hybridization, recent radiation, incomplete lineage sorting, and multiple events of polyploidy. We examined the applicability of AFLP markers for reconstructing (species) relationships in Rosa, using UPGMA clustering, Wagner parsimony, and Bayesian inference. All trees were well resolved, but many of the deeper branches were weakly supported. The cluster analysis showed that the rose cultivars can be separated into a European and an Oriental cluster, each being related to different wild species. The phylogenetic analyses showed that (1) two of the four subgenera (Hulthemia and Platyrhodon) do not deserve subgeneric status; (2) section Carolinae should be merged with sect. Cinnamomeae; (3) subsection Rubigineae is a monophyletic group within sect. Caninae, making sect. Caninae paraphyletic; and (4) there is little support for the distinction of the five other subsections within sect. Caninae. Comparison of the trees with morphological classifications and with previous molecular studies showed that all methods yielded reliable trees. Bayesian inference proved to be a useful alternative to parsimony analysis of AFLP data. Because of their genome-wide sampling, AFLPs are the markers of choice to reconstruct (species) relationships in evolutionary complex groups.     

Evolution of the Rhodococcus equi vap pathogenicity island seen through comparison of host-associated vapA and vapB virulence plasmids

The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB(+) plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA(+) plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA(+) plasmid but differed over an approximately 22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes (vapB and vapJ to -M, with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA(+) plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.     

Oestrogenic activity of CPRG (chlorophenol red-β-D-galactopyranoside), a β-galactosidase substrate commonly used in recombinant yeast oestrogenic assays

The finding that a variety of chemicals display oestrogenic activity has resulted in the development of in vitro and in vivo assays to assess oestrogenic activity. One such assay, the yeast oestrogen assay (YES) makes use of recombinant yeast cells that harbour an oestrogen receptor expression cassette and a reporter construct, coding for bgalactosidase. The induction mechanism starts with the binding of oestrogenic compounds to the oestrogen receptor. This complex activates the production of β-galactosidase. The β-galactosidase activity is thus a measure of the oestrogenic activity of chemical compounds. In the YES assay, the β-galactosidase activity may be quantified with the chromogenic substrate chlorophenol red-β-d-galactopyranoside (CPRG). In the present study it is reported that CPRG or its β-galactosidase degradation product chlorophenol red act in the YES as an oestrogenic compound itself. The implications of this finding are described. It is especially argued that chlorophenol red production after prolonged incubation of the assay might be misinterpreted as an oestrogenic effect of the test compound.     

Towards a new classification of the giant paraphyletic genus Cyperus (Cyperaceae): phylogenetic relationships and generic delimitation in C4 Cyperus

Maximum likelihood and Bayesian inference analyses of nuclear ribosomal DNA (ETS1f) and plastid DNA (rpl32‐trnL, trnH‐psbA) sequence data are presented for ‘C₄ Cyperus’ (Cyperaceae). The term ‘C₄ Cyperus’ encompasses all species of Cyperus s.l. that use C₄ photosynthesis linked with chlorocyperoid vegetative anatomy. Sampling comprises 107 specimens of 104 different taxa, including many of the subdivisions of C₄ Cyperus s.s. and all C₄ segregate genera (Alinula, Ascolepis, Kyllinga, Lipocarpha, Pycreus, Queenslandiella, Remirea, Sphaerocyperus and Volkiella). According to our results, C₄ Cyperus is a well‐supported monophyletic clade nested in C₃ Cyperus. Despite the lack of resolution along the backbone of the C₄ Cyperus clade and for some internal branches, several well‐supported clades can be distinguished. The first clade in C₄ Cyperus is formed by Cyperus cuspidatus and C. waterloti. Other recognizable and well‐supported clades correspond to segregate genera, i.e. Ascolepis, Lipocarpha including Volkiella, and Kyllinga. Species of C₄ Cyperus s.s. form a core grade in which the C₄ segregate genera are embedded. Pycreus, the largest segregate genus composed of c. 120 species, is not monophyletic as it includes several C₄ species of Cyperus s.s. This study establishes a phylogenetic framework for revising the classification and character evolution in Cyperus s.l. © 2013 The Linnean Society of London