Cas3p belongs to a seven-member family of capsule structure designer proteins

The polysaccharide capsule is the main virulence factor of the basidiomycetous yeast Cryptococcus neoformans. Four genes (CAP10, CAP59, CAP60, and CAP64) essential for capsule formation have been previously identified, although their roles in the biosynthetic pathway remain unclear. A genetic and bioinformatics approach allowed the identification of six CAP64-homologous genes, named CAS3, CAS31, CAS32, CAS33, CAS34, and CAS35, in the C. neoformans genome. This gene family is apparently specific in a subclass of the basidiomycete fungi. Single as well as double deletions of these genes in all possible combinations demonstrated that none of the CAP64-homologous genes were essential for capsule formation, although the cas35Delta strains displayed a hypocapsular phenotype. The chemical structure of the glucuronomannan (GXM) produced by the CAS family deletants revealed that these genes determined the position and the linkage of the xylose and/or O-acetyl residues on the mannose backbone. Hence, these genes are all involved in assembly of the GXM structure in C. neoformans.     

Genetic identification of AChE as a positive modulator of addiction to the psychostimulant D-amphetamine in zebrafish

Addiction is a complex maladaptive behavior involving alterations in several neurotransmitter networks. In mammals, psychostimulants trigger elevated extracellular levels of dopamine, which can be modulated by central cholinergic transmission. Which elements of the cholinergic system might be targeted for drug addiction therapies remains unknown. The rewarding properties of drugs of abuse are central for the development of addictive behavior and are most commonly measured by means of the conditioned place preference (CPP) paradigm. We demonstrate here that adult zebrafish show robust CPP induced by the psychostimulant D-amphetamine. We further show that this behavior is dramatically reduced upon genetic impairment of acetylcholinesterase (AChE) function in ache/+ mutants, without involvement of concomitant defects in exploratory activity, learning, and visual performance. Our observations demonstrate that the cholinergic system modulates drug-induced reward in zebrafish, and identify genetically AChE as a promising target for systemic therapies against addiction to psychostimulants. More generally, they validate the zebrafish model to study the effect of developmental mutations on the molecular neurobiology of addiction in vertebrates.     

Mistletoe viscotoxins induce membrane permeabilization and spore death in phytopathogenic fungi

Viscotoxins (Vts) are basic peptides expressed in mistletoe leaves, seeds and stems which have been shown to be cytotoxic to mammalian cells. The aim of this study was to analyse whether Vts were able to control and/or inhibit the growth of phytopathogenic fungi to obtain a clue to their biological function. Incubation of two Vt isoforms, VtA₃ and VtB, at a final concentration of 10 µ M resulted in a complete blockage of the germination of spores from three different pathogenic fungi. It was also shown that lower concentrations than 10 µ M of VtA₃ and VtB inhibit their mycelial growth in a dose-dependent manner. The protein dose required to inhibit the growth of Fusarium solani and Sclerotinia sclerotiorum to a 50% was between 1.5 and 3.75 µ M , which represents a potent activity. No significant differences in the antifungal potency for each Vt isoform, either VtA₃ and VtB, were observed, although they have been shown to exert differential cytotoxicity on mammalian cells. It was also demonstrated that Vts act as fungicidal compounds. To explore the basis of the antifungal activity the ability of VtA₃ to induce changes in membrane permeability and on the oxidative status of F. solani spores was analysed. By using a specific fluorescent probe on intact spores, it was demonstrated that VtA₃ produces rapid changes in fungal membrane permeability. It also induces H₂O₂ production verified by a histochemical staining. The data presented in this study support a direct role of Vts in the plant defence determined by their lethal effect on fungal pathogens.     

Recognition of unknown conserved alternatively spliced exons

The split structure of most mammalian protein-coding genes allows for the potential to produce multiple different mRNA and protein isoforms from a single gene locus through the process of alternative splicing (AS). We propose a computational approach called UNCOVER based on a pair hidden Markov model to discover conserved coding exonic sequences subject to AS that have so far gone undetected. Applying UNCOVER to orthologous introns of known human and mouse genes predicts skipped exons or retained introns present in both species, while discriminating them from conserved noncoding sequences. The accuracy of the model is evaluated on a curated set of genes with known conserved AS events. The prediction of skipped exons in the approximately 1% of the human genome represented by the ENCODE regions leads to more than 50 new exon candidates. Five novel predicted AS exons were validated by RT-PCR and sequencing analysis of 15 introns with strong UNCOVER predictions and lacking EST evidence. These results imply that a considerable number of conserved exonic sequences and associated isoforms are still completely missing from the current annotation of known genes. UNCOVER also identifies a small number of candidates for conserved intron retention.     

Integrative taxonomy provides evidence for the species status of the Ibero‐Maghrebian grass snake Natrix astreptophora

The grass snake (Natrix natrix) is Europe's most widely distributed and, in many regions, most common snake species, with many morphologically defined subspecies. Yet, the taxonomy of grass snakes is relatively little studied and recent work has shown major conflicts between morphologically defined subspecies and phylogeographical differentiation. Using external morphology, osteological characters, and information from 13 microsatellite loci and two mitochondrial markers, we examine differentiation of the subspecies N. n. astreptophora from the North African Maghreb region, the Iberian Peninsula and neighbouring France. According to previous studies, N. n. astreptophora corresponds to a deeply divergent mitochondrial clade and constitutes the sister taxon of all remaining grass snakes. In the French Pyrenees region, there is a contact zone of N. n. astreptophora with another subspecies, N. n. helvetica. Our analyses of microsatellites and mitochondrial DNA reveal that the distribution ranges of the two taxa abut there, but both hybridize only exceptionally. Even though many morphological characters are highly variable and homoplastic in grass snakes, N. n. astreptophora differs consistently from all other grass snakes by its reddish iris coloration and in having significantly fewer ventral scales and another skull morphology. Considering further the virtual absence of gene flow between N. n. astreptophora and N. n. helvetica, and acknowledging the morphological distinctiveness of N. n. astreptophora and its sister group relationship to all remaining subspecies of grass snakes, we conclude that Natrix astreptophora (Seoane, 1884) should be recognized as a distinct species. Further research is needed to explore whether N. astreptophora is polytypic because a single sample of N. astreptophora from Tunisia turned out to be genetically highly distinct from its European conspecifics.     

Increase of methane formation by ethanol addition during continuous fermentation of biogas sludge

Very recently, it was shown that the addition of acetate or ethanol led to enhanced biogas formation rates during an observation period of 24 h. To determine if increased methane production rates due to ethanol addition can be maintained over longer time periods, continuous reactors filled with biogas sludge were developed which were fed with the same substrates as the full-scale reactor from which the sludge was derived. These reactors are well reflected conditions of a full-scale biogas plant during a period of 14 days. When the fermenters were pulsed with 50–100 mM ethanol, biomethanation increased by 50–150 %, depending on the composition of the biogas sludge. It was also possible to increase methane formation significantly when 10–20 mM pure ethanol or ethanolic solutions (e.g. beer) were added daily. In summary, the experiments revealed that “normal” methane production continued to take place, but ethanol led to production of additional methane.     

<Emphasis Type="Italic">Mycosphaerella podagrariae</Emphasis>—a necrotrophic phytopathogen forming a special cellular interaction with its host <Emphasis Type="Italic">Aegopodium podagraria</Emphasis>

We present a new kind of cellular interaction found between Mycosphaerella podagrariae and Aegopodium podagraria, which is remarkably different to the interaction type of the obligate biotrophic fungus Cymadothea trifolii, another member of the Mycosphaerellaceae (Capnodiales, Dothideomycetes, Ascomycota) which we have described earlier. Observations are based on both conventional and cryofixed material and show that some features of this particular interaction are better discernable after chemical fixation. We were also able to generate sequences for nuclear ribosomal DNA (complete SSU, 5.8 S and flanking ITS-regions, D1–D3 region of the LSU) confirming the position of M. podagrariae within Mycosphaerellaceae.     

Role of mass spectrometry in the purification of peptides and proteins

Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV-Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used.     

Maternal-embryonic relationships in the goodeid teleost,Xenoophorus captivus

In goodeid teleosts, prolonged embryonic development takes place within the ovarian cavity. Apposed maternal and embryonic epithelia interface via a nutritive liquid (embryotrophe) and facilitate aplacental matrotrophy. The role of the internal ovarian epithelium (IOE) in providing proteins for the embryotrophe has been studied using transmission electron-microscopic examinations of both the resting and the active ovarian lining, and isoelectric focusing of embryotrophe and maternal blood serum. The simple IOE is apparently composed of only one, filament-containing cell-type. In the non-gravid ovary these cells are cuboidal to columnar in shape, and are either compact and electron-dense or oedematous and light. During gestation, swelling of the ovarian connective tissue gives rise to dovetailing of the IOE with the subjacent capillary plexus. Part of the IOE overlying the capillaries becomes stretched, resulting in a thin endothelium-like demarcation. The nuclei and the bulk of the cytoplasm are usually recessed between the meshes of the protruding capillary network. The blood-embryotrophe pathway is thus reduced in places, to less than one m. The active form of the IOE contains a well-developed vacuolar apparatus composed of small vesicles, vacuoles, multivesicular bodies, and a few lysosomes. Elements of the RER are sparsely distributed throughout the cytoplasm. Endocytotic activity is observable at the apical and basolateral plasma membrane. Isoelectric focusing of both serum and embryotrophe produces numerous bands each between pI 4–8, which reveal many homologies. The intensity of corresponding bands varies considerably. It is concluded that the cells of the IOE provide a transport pathway for serum-derived macromolecular substances rather than produce proteinaceous secretions.