Development and application of a brain-specific cDNA microarray for effect evaluation of neuro-active pharmaceuticals in zebrafish (Danio rerio)

The environmental fate and ecotoxicological effect of pharmaceuticals are poorly understood, and standardized tests to detect and evaluate their potential effects in the environment are not available. We developed a zebrafish brain-specific microarray containing 682 neurologically relevant cDNA-fragments. To investigate the applicability of this microarray for studying neurotoxic modes-of-action and impact assessment of neuro-active pharmaceuticals in zebrafish, chlorpromazine was used as a model compound. After exposure to chlorpromazine (75 μg/L) for 2, 4, 14 and 28 days or control treatment RNA was extracted from brains of males and females. Fluorescently labeled cDNA was prepared and hybridized to the custom microarray. In total, 56 genes were differentially expressed in brains of male and/or female zebrafish, of which most genes were down-regulated. A clear difference in response to chlorpromazine exposure between males and females was observed with exposure time as well as in functional classes of affected genes. The presented study is one of the first reports on molecular effects of human neuro-active pharmaceuticals in aquatic non-target organisms. This new genomic tool successfully detected gene expression effects of exposure to chlorpromazine in the brain of zebrafish. Reported gene expression effects are found to be consistent with literature data for other laboratory animals.     

Defining a holoprosencephaly locus on human chromosome 14q13 and characterization of potential candidate genes

Holoprosencephaly (HPE) is the most common developmental field defect in patterning of the human prosencephalon and associated craniofacial structures. The genetics is complex, with 12 loci defined on 11 chromosomes. We defined a locus for HPE (HPE8) on human chromosome 14q13 between markers D14S49 and AFM205XG5, by mapping deletion intervals of affected subjects with proximal chromosome 14q interstitial cytogenetic deletions. A 35-BAC contig was built by chromosome walking. By annotation of the 2.82-Mb minimal critical region, we identified 28 possible genes. Seven genes were expressed in human fetal brain: NPAS3, SNX6, C14ORF11, C14ORF10, PAX9, NKX2.1, and C14ORF19, the last an apparent gene fragment. Molecular embryology, animal modeling, and human mutation studies were reported elsewhere for PAX9 and NKX2.1. We focused on three genes, SNX6, NPAS3, and C14ORF11, as potential candidates for HPE. Genomic structure, human expression patterns, protein cellular localization, and embryonic expression patterns of orthologous murine genes were determined, showing that the three genes have properties similar to those of known HPE genes.     

Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions

In a forward screen for genes affecting neurotransmission in Drosophila, we identified mutations in dynamin-related protein (drp1). DRP1 is required for proper cellular distribution of mitochondria, and in mutant neurons, mitochondria are largely absent from synapses, thus providing a genetic tool to assess the role of mitochondria at synapses. Although resting Ca2+ is elevated at drp1 NMJs, basal synaptic properties are barely affected. However, during intense stimulation, mutants fail to maintain normal neurotransmission. Surprisingly, FM1-43 labeling indicates normal exo- and endocytosis, but a specific inability to mobilize reserve pool vesicles, which is partially rescued by exogenous ATP. Using a variety of drugs, we provide evidence that reserve pool recruitment depends on mitochondrial ATP production downstream of PKA signaling and that mitochondrial ATP limits myosin-propelled mobilization of reserve pool vesicles. Our data suggest a specific role for mitochondria in regulating synaptic strength.     

Incorporation of galactomannans in the diet of newly weaned piglets: effect on bacteriological and some morphological characteristics of the small intestine

In search of substances replacing antibiotics as growth promoters for farm animals, non-digestible oligosaccharides (NDO) or non-starch polysaccharides (NSP) have been proposed as possible alternatives. In this context, the influence of galactomannans on bacteriological and morphological aspects of the gastrointestinal tract in weanling pigs was investigated. Four groups of five newly weaned piglets received one of the following diets: control feed (C), C supplemented with guar gum (1%), C supplemented with locust bean gum (1%) and C supplemented with 10% of carob tree seeds meal as source of locust bean gum. The animals were euthanized after 11-12 days and digesta were sampled in stomach, jejunum (proximal and distal) and caecum, while mucosal scrapings and ring shaped tissue samples were taken of proximal and distal jejunum. On these samples bacteriological, biochemical and morphological determinations were carried out. Total count of bacteria in digesta and mucosal scrapings was not influenced by the different diets, with the exception of the proximal jejunum where a small decrease (0.5 log10 CFU) was noted with the guar gum and carob tree seeds diet. The number of E. coli increased by feeding both gums and carob tree seeds. With the latter diet, higher counts of streptococci were observed. In agreement with the lower concentration of lactic acid in jejunal contents, guar gum decreased the number of lactobacilli. Locust bean gum decreased the molar proportion of acetate in caecal contents while butyrate and valerate were augmented. Feeding the carob tree seeds resulted in shorter villi and a lower villus height/crypt depth ratio in the jejunum mucosa, which was an indication for a faster renewal rate of the epithelium. Both locust bean gum feeds significantly lowered the mitotic index in the crypts of the small intestine. Only with the carob tree seeds diet, viscosity of jejunal contents was increased. In conclusion, the effects of the addition of 1% of pure guar gum or locust bean gum were inconsistent and not very outspoken, whereas 10% of carob tree seeds meal in the diet resulted in influences on intestinal characteristics at the bacteriological and morphological level.     

Four stage liquid chromatographic selection of methionyl peptides for peptide-centric proteome analysis: the proteome of human multipotent adult progenitor cells

Serial application of strong cation-exchange and diagonal reversed-phase chromatography selecting methionyl peptides by stepwise shifting them from their reduced to their sulfoxide and sulfone forms generates a four-stage fractionation system, allowing high coverage analysis of complex proteome digests by LC-MALDI-MS/MS. Application to the proteome of a human multipotent adult progenitor cell line (MAPC) identified 2151 proteins with high confidence as on average four MS/MS-spectra were linked to each protein. Our dataset contains several novel, potential marker proteins that may be evaluated as affinity-anchors for isolating different adult stem cells in further studies. Furthermore, at least 2 tyrosine kinases that were previously linked to the self-renewal potential of stem cells were identified, validating the stemness of the analyzed cells. We also present data hinting at possible involvement of the ubiquitin/proteasome machinery in steering proliferation and/or differentiation of MAPC. Finally, following comparison of the MAPC proteome with proteomes of four human differentiated cell lines reveals differential usage of chromosomal information: compared to differentiated cells, MAPC do not appear to hold any preference for expressing genes located on specific chromosomes.     

Genetic diversity in Australian ancient asexual <Emphasis Type="Italic">Vestalenula</Emphasis> (Ostracoda,Darwinulidae): little variability down under

Darwinulid ostracods are putative ancient asexuals, and are thus assumed to be unable to purge deleterious mutations from their genomes. Some darwinulids species can be found both above (epigeic) and below ground (hypogeic). We hypothesize that surface populations carry more mutations than their below-ground counterparts, which are buffered from mutagens such as UV-B. Given the age of the investigated area, the Pilbara in Western Australia, we also expect geographic patterning of observed haplotypes. We have used DNA sequence data from the nuclear ITS and the mitochondrial COI region to investigate a (limited) data set on two Australian species, the endemic Vestalenula matildae and V. marmonieri from the Pilbara region. We do not find differences in genetic variability between specimens from subterranean habitats as compared to those from habitats above ground. There was also no congruence between hydrological basins and distribution patterns of the haplotypes identified. Although our data indicate that the two species may have split from each other ca. 70 myr ago, this has not resulted in any clear phylogeographic patterns among the analysed specimens across the regions of the Pilbara.     

Variation in ostracod (Crustacea,Ostracoda) communities in the alluvial valley of the upper Paraná River (Brazil) in relation to substrate

Large river floodplains are convenient model systems to test for variation in animal and plant community structure, as they have a variety of habitats and substrates and are generally dynamic systems through the occurrence of flood pulses with varying intensity. South American floodplain systems furthermore have unique types of substrates, in the form of root systems of floating macrophytes. Here, we investigate the variation in ostracod (small, bivalved crustaceans) communities in relation to substrates and related environmental variables. Sampling was effected in 2004 in the alluvial valley of the upper Paraná River, Brazil, in the wet and dry seasons. Five different substrates, including littoral sediment and four macrophyte species root and leaf systems, in four hydrological systems and a variety of habitat types, were sampled. Fifty-four species of Ostracoda were found. Variation partitioning analysis (RDA) showed that ostracod communities significantly differed between different substrates, mainly between the littoral and plants with small root systems (Eichhornia azurea) on the one hand, and plants with large and complex root systems on the other hand (Eichhornia crassipes and Pistia stratiotes). RDA analyses indicated that the pleuston (biotic communities associated with root systems of floating plants) of E. crassipes comprised more non-swimming species than the pleuston of the smaller roots of P. stratiotes, but species-level Kruskal–Wallis analyses could not detect significant differences between both macrophyte species. Also habitat type and hydrological systems contributed to variation amongst ostracod communities, but less so than the factor substrate. Abiotic factors also contributed to variation, but the ranges of all measured water chemistry variables were narrow. This uniformity in abiotic factors, which might be owing to the occurrence of large flooding events, unites all water bodies, even those that are generally separated.     

Quantification of right ventricular afterload in patients with and without pulmonary hypertension

Right ventricular (RV) afterload is commonly defined as pulmonary vascular resistance, but this does not reflect the afterload to pulsatile flow. The purpose of this study was to quantify RV afterload more completely in patients with and without pulmonary hypertension (PH) using a three-element windkessel model. The model consists of peripheral resistance (R), pulmonary arterial compliance (C), and characteristic impedance (Z). Using pulmonary artery pressure from right-heart catheterization and pulmonary artery flow from MRI velocity quantification, we estimated the windkessel parameters in patients with chronic thromboembolic PH (CTEPH; n = 10) and idiopathic pulmonary arterial hypertension (IPAH; n = 9). Patients suspected of PH but in whom PH was not found served as controls (NONPH; n = 10). R and Z were significantly lower and C significantly higher in the NONPH group than in both the CTEPH and IPAH groups (P < 0.001). R and Z were significantly lower in the CTEPH group than in the IPAH group (P < 0.05). The parameters R and C of all patients obeyed the relationship C = 0.75/R (R(2) = 0.77), equivalent to a similar RC time in all patients. Mean pulmonary artery pressure P and C fitted well to C = 69.7/P (i.e., similar pressure dependence in all patients). Our results show that differences in RV afterload among groups with different forms of PH can be quantified with a windkessel model. Furthermore, the data suggest that the RC time and the elastic properties of the large pulmonary arteries remain unchanged in PH.