Identification of a Zds-like gene ZDS3 as a new mediator of stress resistance, capsule formation and virulence of the human pathogenic yeast Cryptococcus neoformans

The fungal Zds proteins are regulators of the serine/threonine phosphatase 2A (PP2A) and the protein kinase A. Here, we characterize a Zds-like gene ZDS3 that plays a broad range of roles in the basidiomycetous pathogenic yeast Cryptococcus neoformans. ZDS3 harbors the conserved activation domain ZDS_C of Zds proteins. By gene disruption, ZDS3 is shown to play roles in capsule production, cell wall integrity, growth at a high temperature, resistance to H(2)O(2) stress, osmotic pressures and glucose-dependent invasive growth on the agar. As a consequence, the disruption of ZDS3 resulted in complete loss of virulence in a mouse cryptococcosis model. The data suggest that ZDS3 is a novel mediator of the virulence of C. neoformans. Zds3 may serve as an antifungal drug target as no homologs are found in mammals.     

Mitochondrial Unselective Channels throughout the eukaryotic domain

Mitochondria from diverse species can undergo a massive permeability increase known as the permeability transition, a process first thought to be an artifact. It is currently accepted that in the inner mitochondrial membrane there is a Mitochondrial Unselective Channel (MUC), also known as the permeability transition pore. Regardless of the species, MUC opening leads to uncoupling of oxidative phosphorylation. In each species, MUC regulation appears to be different, probably as a result of the adaptation of each organism to its specific environment. To date, the components and the putative physiological role of MUCs are still a matter of debate. Current hypothesis suggests that proteins normally participating in diverse metabolic functions constitute MUCs. Among these proteins, the Adenine Nucleotide Translocase and the phosphate carrier have been proposed as putative MUC components in mammalian and yeast mitochondria. In this review, the characteristics of MUCs from different species and strains are discussed. The data from the literature reinforce the current notion that these channels are preserved through evolution albeit with different control factors. We emphasize the knowledge available of Mitochondrial Unselective Channels from different yeast species.     

Heterologous Expression of Human Cytochromes P450 2D6 and CYP3A4 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and Their Functional Characterization

This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17α sequence were inserted into a pCWori⁺ vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K_m and V_max values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.     

Novel terpenoids from Calocedrus macrolepis var. formosana

Calocetriol ( 1 ), diacetylcalocediol ( 2 ), and ferrugimenthenol ( 3 ) were isolated from the bark of Calocedrus macrolepis var. formosana. Among them, 1 and 2 are secoabietane‐type diterpenoids, and 3 , with a novel C₂₀ C₁₀ skeleton, is classified as a meroterpenoid. The structures of 1 – 3 were elucidated by spectroscopic analyses, and their biological activities were also evaluated. Compound 3 exhibited significant cytotoxic activity against human oral epidermoid carcinoma KB cells with an IC₅₀ value of 9.0±0.1 μM .     

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

We demonstrate the versatility of a collection of insertions of the transposon Minos-mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 integrase attP sites. MiMIC integrates almost at random in the genome to create sites for DNAmanipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase-mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system. Insertions in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the D. melanogaster toolkit.     

Factors influencing wolf <Emphasis Type="Italic">Canis lupus</Emphasis> roadkills in Northwest Spain

Roadkill is one of the most prominent causes of wildlife mortality. Much research effort has focussed on collisions with ungulates because of traffic safety. However, studies about large carnivore roadkills are scarce despite vehicles being a main cause of mortality. The absence of studies can be explained in part because of difficulties in obtaining sufficient sample sizes. We collected data from locations of 82 wolf roadkill sites in the Castilla y León Region, northwest Spain. We evaluated different models to characterise collision localities using logistic regressions with corrections for rare events. The best models included traffic and human disturbance parameters. Landscape variables did not improve predictive power. Fencing was a decisive key predictor; roadkill was proportionally higher along fenced highways than on similar major roads that lacked fences. Wolf–vehicle collisions were more common in agricultural areas, although wolf densities were lower in these zones. Both the higher density of important roads and a greater proportion of roaming wolves on the plateau may explain this pattern.     

New pregnane glycosides from Brucea javanica and their antifeedant activity

Three new pregnane glycosides, 3-O-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl-(20R)-pregn-5-ene-3β,20-diol (1), 3-O-α-L-arabinopyranosyl-(20R)-pregn-5-ene-3β,20-diol-20-O-β-D-glucopyranoside (2), 3-O-α-L-arabinopyranosyl-(20R)-pregn-5-ene-3β,20-diol-20-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside (3) were isolated along with four known compounds, 4-7, from the leaves and stems of Brucea javanica. Their structures were determined by detailed analyses of 1D- and 2D-NMR spectroscopic data. All of the compounds isolated from Brucea javanica were tested for the antifeedant activities against the larva of Pieris rapae. Compounds 1, 3, and 5 showed significant antifeedant activities after 72 h incubation.     

Essential oils from neotropical Myrtaceae: chemical diversity and biological properties

Myrtaceae family (121 genera, 3800-5800 spp.) is one of the most important families in tropical forests. They are aromatic trees or shrubs, which frequently produce edible fruits. In the neotropics, ca. 1000 species were found. Several members of this family are used in folk medicine, mainly as an antidiarrheal, antimicrobial, antioxidant, cleanser, antirheumatic, and anti-inflammatory agent and to decrease the blood cholesterol. In addition, some fruits are eaten fresh or used to make juices, liqueurs, and sweets very much appreciated by people. The flavor composition of some fruits belonging to the Myrtaceae family has been extensively studied due to their pleasant and intense aromas. Most of the essential oils of neotropical Myrtaceae analyzed so far are characterized by predominance of sesquiterpenes, some with important biological properties. In the present work, chemical and pharmacological studies carried out on neotropical Myrtaceae species are reviewed, based on original articles published since 1980. The uses in folk medicine and chemotaxonomic importance of secondary metabolites are also briefly discussed.