The biological and toxicological importance of molybdenum in the environment and in the nutrition of plants, animals and man. Part 1: Molybdenum in plants

In 1930, Bortels showed that molybdenum is necessary for nitrogen fixation in Acetobacter, and in 1939 Arnon and Stout reported that molybdenum is essential for life in higher plants. Nitrogenase is the nitrogen-fixing enzyme complex, while nitrate reductase requires molybdenum for its activity. Molybdenum occurs in the earth crust with an abundance of 1.0-1.4 mg/kg. The molybdenum content of the vegetation is determined by the amount of this element in the soil and its pH-value. The weathering soils of granite, porphyry, gneiss and Rotliegendes produce a molybdenum-rich vegetation. Significantly poorer in Mo is the vegetation on loess, diluvial sands, alluvial riverside soils and especially on Keuper and Muschelkalk weathering soils, which produce legumes and, e.g. cauliflower with molybdenum deficiency symptoms. The molybdenum content of the flora decreases with increasing age. Legumes store the highest molybdenum levels in the bulbs of their roots; on average, they accumulate more molybdenum than herbs and grasses do. The danger of molybdenum toxicity in plants is small.     

Reduced sialylation status in UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE)-deficient mice

Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. They are involved in a variety of cellular functions, such as cell adhesion or signal recognition. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. Previously, we have shown that inactivation of the GNE by gene targeting causes early embryonic lethality in mice, whereas heterozygous GNE-deficient mice are vital. In this study we compared the amount of membrane-bound sialic acids of wildtype mice with those of heterozygous GNE-deficient mice. For that we quantified membrane-bound sialic acid concentration in various organs of wildtype- and heterozygous GNE-deficient mice. We found an organ-specific reduction of membrane-bound sialic acids in heterozygous GNE-deficient mice. The overall reduction was 25%. Additionally, we analyzed transferrin and polysialylated neural cell adhesion molecule (NCAM) by one- or two-dimensional gel electrophoresis. Transferrin-expression was unchanged in heterozygous GNE-deficient mice; however the isoelectric point of transferrin was shifted towards basic pH, indicating a reduced sialylation. Furthermore, the expression of polysialic acids on NCAM was reduced in GNE-deficient mice. Daniel Gagiannis and André Orthmann have contributed equally to this work.     

Formation of giant spicules in the deep-sea hexactinellid <Emphasis Type="Italic">Monorhaphis chuni</Emphasis> (Schulze 1904): electron-microscopic and biochemical studies

The siliceous sponge Monorhaphis chuni (Hexactinellida) synthesizes the largest biosilica structures on earth (3 m). Scanning electron microscopy has shown that these spicules are regularly composed of concentrically arranged lamellae (width: 3–10 μm). Between 400 and 600 lamellae have been counted in one giant basal spicule. An axial canal (diameter: ~2 μm) is located in the center of the spicules; it harbors the axial filament and is surrounded by an axial cylinder (100–150 μm) of electron-dense homogeneous silica. During dissolution of the spicules with hydrofluoric acid, the axial filament is first released followed by the release of a proteinaceous tubule. Two major proteins (150 kDa and 35 kDa) have been visualized, together with a 24-kDa protein that cross-reacts with antibodies against silicatein. The spicules are surrounded by a collagen net, and the existence of a hexactinellidan collagen gene has been demonstrated by cloning it from Aphrocallistes vastus. During the axial growth of the spicules, silicatein or the silicatein-related protein is proposed to become associated with the surface of the spicules and to be finally internalized through the apical opening to associate with the axial filament. Based on the data gathered here, we suggest that, in the Hexactinellida, the growth of the spicules is mediated by silicatein or by a silicatein-related protein, with the orientation of biosilica deposition being controlled by lectin and collagen. Carsten Eckert was previously with the Museum für Naturkunde, Invalidenstrasse 43, 10115 Berlin, Germany. The collagen sequence from Aphrocallistes vastus reported here, viz., [COL_APHRO] APHVACOL (accession number AM411124), has been deposited in the EMBL/GenBank data base. This work was supported by grants from the European Commission, the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung Germany (project: Center of Excellence BIOTECmarin), the National Natural Science Foundation of China (grant no. 50402023), and the International Human Frontier Science Program.     

Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2 to facilitate biochemical studies using thiol-specific modifying reagents. Of 10 endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site saturation mutagenesis scheme based on the Stratagene Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in Saccharomyces cerevisiae cells auxotrophic for purines, several highly functional noncysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position.     

Conflicts between humans over wildlife management: on the diversity of stakeholder attitudes and implications for conflict management

Conflicts involving wildlife are, in essence, often conflicts between human parties with differing wildlife management objectives. However, the study and management of wildlife conflicts often focuses on the ecological context without addressing disagreements between people over these objectives. This research uses quantitative approaches to examine actors’ views on a complex wildlife-related conflict: a raptor of conservation concern that impacts on game-bird management. Four dominant elements of the debate emerged from initial semi-structured interviews: perceptions of conflict related issues; perceptions of each other; perceived barriers to consensus within the debate; and assessment of proposed practical management solutions. A quantitative survey that built on these elements demonstrates the degree to which perceptions differ between groups and how local variation in these elements may be obscured in a regional or national level debate. The findings emphasise the importance of understanding the social issues involved in wildlife related conflicts if management aims are to be agreed and achieved.     

N9-benzyl-substituted 1,3-dimethyl- and 1,3-dipropyl-pyrimido[2,1-f]purinediones: synthesis and structure-activity relationships at adenosine A1 and A2A receptors

Synthesis and physicochemical properties of N-benzyl pyrimido[2,1-f]purinediones are described. These derivatives were synthesized by the cyclization of 7-chloropropylo-8-bromo-1,3-dimethyl- or 1,3-dipropyl xanthine derivatives with corresponding (un)substituted benzylamines. Dipropyl derivatives were obtained under microwave irradiation conditions either. The obtained compounds (1-20) were evaluated for their affinity to adenosine A1 and A2A receptors, selected compounds were additionally investigated for affinity to the A3 receptor subtype. The results of the radioligand binding assays to A1 and A2A adenosine receptors showed that most of the 1,3-dimethyl-9-benzylpyrimidopurinediones exhibited selective affinity to A2A receptors at micromolar or submicromolar concentrations (for example, derivative 9 with o-methoxy substituent displayed a Ki value of 0.699 microM at rat A2A receptor with more than 36-fold selectivity). Contrary to previously described arylpyrimido[2,1-f]purinediones dipropyl derivatives (compounds 15-20) showed affinity to both kinds of receptors increased, however A1 affinity increased to a larger extent, with the result that A2A selectivity was abolished. The best adenosine A1 receptor ligand was m-chlorobenzyl derivative 18 (Ki=0.089 microM and 5-fold A1 selectivity). Structure-activity relationships were discussed with the analysis of lipophilic and spatial properties of the investigated compounds. Pharmacophore model of adenosine A1 receptor antagonist was adopted for this purpose.     

Soybean oil-degrading bacterial cultures as a potential for control of green peach aphids (Myzus persicae)

Microorganisms capable of degrading crude oil were isolated and grown in soybean oil as a sole carbon source. The microbial cultures were used to control green peach aphids in vitro. Approximately 60% mortality of aphids was observed when the cultures were applied alone onto aphids. To examine the cultures as a pesticide formulation mixture, the cultures were combined with a low dose of the insecticide imidacloprid (one-fourth dose of recommended field-application rate) and applied onto aphids. The cultures enhanced significantly the insecticidal effectiveness of imidacloprid, which was higher than imidacloprid alone applied at the low dose. The isolated microorganisms exhibited high emulsifying index values and decreased surface tension values after being grown in soybean oil media. GC/MS analyses showed that microorganisms degraded soybean oil to fatty acids. The cultures were suggested to play the roles of wetting, spreading, and sticking agents to improve the effectiveness of imidacloprid. This is the first report on the control of aphids by using oil-degrading microbial cultures.     

Fusiogenic endogenous-retroviral syncytin-1 exerts anti-apoptotic functions in staurosporine-challenged CHO cells

Fusiogenic glycoprotein syncytin-1, expressed in human placenta, is a promising candidate for acquiring a basic knowledge of placental syncytialization. However, its cellular mode of action is unidentified. We investigated whether syncytin-1 may exert influence on apoptotic processes. Therefore, we incubated CHO cells after stable transfection with syncytin-1 (CHO-52) in the presence or absence of staurosporine (STS), a kinase inhibitor well characterized to induce apoptosis. When testing the phenotype of CHO-52 cells, we could demonstrate that the induction of apoptosis by STS was delayed over a period of up to 24 h. Furthermore, the cell death rate was decreased by approx 75% following transfection of syncytin-1 in CHO-52 compared to mock-treated cells. In detail, after 18h of incubation with 500 nM STS, 64 ± 2% of CHO-52 cells were viable compared to 16 ± 1% of CHO-mocks, after 24 h 43 ± 3% vs 5 ± 2%, respectively. CHO-52 cells exhibited a lower expression of active caspase 3 and anti-apoptotic Bcl-2 was found to be increased in CHO-52 cells at baseline and following STS treatment. Our study provides first evidence that syncytin-1 serves anti-apoptotic function under certain conditions. A lessened activation of caspase 3 and an increased expression of Bcl-2 are possible mechanisms.     

Erythropoietin in Cerebrospinal Fluid: Age-related Reference Values and Relevance in Neurological Disease

We aimed to establish age-related reference values for Erythropoietin (EPO) in cerebrospinal fluid (CSF) and to evaluate concentrations in neurological diseases. CSF and serum EPO was measured in controls with tension-type headache (CTTH), in patients with ALS, dementia and depression using ELISA technique. Stability experiments showed CSF EPO to be stable for two and a half months and over two thaw/freeze cycles. A positive correlation of CSF EPO with age was found (P < 0.01). We found a CSF/serum EPO concentration ratio of 0.126, pointing towards an intrathecal synthesis of EPO. The ALS group showed significantly lowered CSF EPO compared to age-matched CTTH (P < 0.012), whereas the dementia and depression group showed no significant differences compared to CTTH. The establishment of age-related reference values in a large cohort of controls will improve the interpretation of future CSF EPO evaluations in neurological diseases. The authors have not reported any conflicts of interest.     

Biodegradation of chlorobenzene under hypoxic and mixed hypoxic-denitrifying conditions

Pseudomonas veronii strain UFZ B549, Acidovorax facilis strain UFZ B530, and a community of indigenous groundwater bacteria, adapted to oxygen limitation, were cultivated on chlorobenzene and its metabolites 2-chloro-cis,cis-muconate and acetate/succinate under hypoxic and denitrifying conditions. Highly sensitive approaches were used to maintain defined low oxygen partial pressures in an oxygen-re-supplying headspace. With low amounts of oxygen available all cultures converted chlorobenzene, though the pure strains accumulated 3-chlorocatechol and 2-chloro-cis,cis-muconate as intermediates. Under strictly anoxic conditions no chlorobenzene transformation was observed, while 2-chloro-cis,cis-muconate, the fission product of oxidative ring cleavage, was readily degraded by the investigated chlorobenzene-degrading cultures at the expense of nitrate as terminal electron acceptor. Hence, we conclude that oxygen is an obligatory reactant for initial activation of chlorobenzene and fission of the aromatic ring, but it can be partially replaced by nitrate in respiration. The tendency to denitrify in the presence of oxygen during growth on chlorobenzene appeared to depend on the oxygen availability and the efficiency to metabolize chlorobenzene under oxygen limitation, which is largely regulated by the activity of the intradiol ring fission dioxygenase. Permanent cultivation of a groundwater consortium under reduced oxygen levels resulted in enrichment of a community almost exclusively composed of members of the β-Proteobacteria and Bacteroidetes. Thus, it is deduced that these strains can still maintain high activities of oxygen-requiring enzymes that allow for efficient CB transformation under hypoxic conditions.