In vitro assessment of adsorbents aiming to prevent deoxynivalenol and zearalenone mycotoxicoses

The high prevalence of the Fusarium mycotoxins, deoxynivalenol (DON) and zearalenone (ZON) in animal feeds in mild climatic zones of Europe and North America results in considerable economic losses, as these toxins affect health and productivity particularly of pigs from all age groups. The use of mycotoxin adsorbents as feed additives is one of the most prominent approaches to reduce the risk for mycotoxicoses in farm animals, and to minimise carry-over of mycotoxins from contaminated feeds into foods of animal origin. Successful aflatoxin adsorption by means of different substances (phyllosilicate minerals, zeolites, activated charcoal, synthetic resins or yeast cell-wall-derived products) has been demonstrated in vivo and in vitro. However, attempts to adsorb DON and ZON have been less encouraging. Here we describe the adsorption capacity of a variety of potential binders, including compounds that have not been evaluated before, such as humic acids. All compounds were tested at realistic inclusion levels for their capacity to bind ZON and DON, using an in vitro method that resembles the different pH conditions in the gastro-intestinal tract of pigs. Mycotoxin adsorption was assessed by chemical methods and distinct bioassays, using specific markers of toxicity as endpoints of toxicity in cytological assays. Whereas none of the tested substances was able to bind DON in an appreciable percentage, some of the selected smectite clays, humic substances and yeast-wall derived products efficiently adsorbed ZON (>70%). Binding efficiency was indirectly confirmed by the reduction of toxicity in the in vitro bioassays. In conclusion, the presented test protocol allows the rapid screening of potential mycotoxin binders. Like other in vitro assays, the presented protocol combining chemical and biological assays cannot completely simulate the conditions of the gastro-intestinal tract, and hence in vivo experiments remain mandatory to assess the efficacy of mycotoxin binders under practical conditions.     

Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea

The adult mammalian cochlea receives dual afferent innervation: the inner sensory hair cells are innervated exclusively by type I spiral ganglion neurons (SGN), whereas the sensory outer hair cells are innervated by type II SGN. We have characterized the spatiotemporal reorganization of the dual afferent innervation pattern as it is established in the developing mouse cochlea. This reorganization occurs during the first postnatal week just before the onset of hearing. Our data reveal three distinct phases in the development of the afferent innervation of the organ of Corti: (1) neurite growth and extension of both classes of afferents to all hair cells (E18-P0); (2) neurite refinement, with formation of the outer spiral bundles innervating outer hair cells (P0-P3); (3) neurite retraction and synaptic pruning to eliminate type I SGN innervation of outer hair cells, while retaining their innervation of inner hair cells (P3-P6). The characterization of this developmental innervation pattern was made possible by the finding that tetramethylrhodamine-conjugated dextran (TMRD) specifically labeled type I SGN. Peripherin and choline-acetyltransferase immunofluorescence confirmed the type II and efferent innervation patterns, respectively, and verified the specificity of the type I SGN neurites labeled by TMRD. These findings define the precise spatiotemporal neurite reorganization of the two afferent nerve fiber populations in the cochlea, which is crucial for auditory neurotransmission. This reorganization also establishes the cochlea as a model system for studying CNS synapse development, plasticity and elimination.     

The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents

Engineered extremely thermostable variants of the thermolysin-like protease from Bacillus stearothermophilus possessing an introduced disulfide bond G8C/N60C (double mutant, DM) and six additional amino acid substitutions in the exposed loop region 56-69 (Boilysin, BLN) have been probed with respect to stability toward water-miscible organic solvents and detergents. The solvent concentrations where 50% of enzyme activity were irreversibly lost (C(50)) decreased in the order methanol > 2-propanol > dimethylsulfoxide > dioxane > acetonitrile > dimethylformamide > acetone. The C(50) values were remarkably higher for the thermostable variants than for the wild-type enzymes. Therefore, the stabilization of this loop region also protects the molecule from irreversible inactivation by solvents, and inactivation seems to follow principally the same mechanism as thermal inactivation. However, in contrast to thermal inactivation where the corresponding T(50) values of DM and BLN differed by 10 K, the differences of the C(50) values of DM and BLN were not significant. Detergents had great effects on proteolytic activities which were dependent on the individual detergent and its concentration, but mostly without significant differences between the enzyme variants. These effects were inactivating (SDS, sulfobetaine) or strongly activating (CTAB, CHAPS). Triton X-100 and Tween 20 were activating or inactivating at low and high concentrations, respectively. In all detergents, stabilities of the enzymes were strongly decreased. However, the more thermostable variants were affected by the detergents to the same extent as the wild-type enzymes suggesting that the mechanism of detergent inactivation is different from that of thermal inactivation.     

Production of synthetically created phospholipase A(2) variants with industrial impact

Phospholipases A(2) (PLA(2)) play an important role for the production of lysophospholipids. Presently they are mainly obtained from porcine or bovine pancreas but these mammalian sources are not accepted in several fields of application. To make accessible a non-mammalian PLA(2) to industrial application, synthetic genes encoding PLA(2) from honey bee (Apis mellifera) with modified N-termini were constructed and expressed in Escherichia coli. While expression of the gene with an N-terminal leader sequence to direct the protein into the periplasm failed, four variants with slightly modified N-termini (I1A-PLA(2), I1V-PLA(2), His(6)-tagged PLA(2) and PLA(2) still containing the start methionine) were successfully expressed. In all cases, the PLA(2) variants were produced as inclusion bodies. Their protein content amounted to 26-35% of total cell protein. The optimized renaturation procedure and subsequent purification by cation-exchange chromatography yielded pure active enzymes in yields of 4-11 mg L(-1). The recombinant PLA(2) variants showed activities, far-UV CD and fluorescence spectra similar to the glycosylated PLA(2) isolated from the venom glands of honey bee (bv-PLA(2)). The thermodynamic stabilities of the recombinant enzymes calculated from the transition curves of guanidine hydrochloride induced unfolding were also nearly identical to the stability of bv-PLA(2). For the variant I1A-PLA(2) high-cell density fermentation in 10 L-scale using mineral salt medium was shown to increase the volumetric enzyme yield considerably.     

Secretory phospholipase A2-alpha from Arabidopsis thaliana: functional parameters and substrate preference

The secretory phospholipase A2-alpha from Arabidopsis thaliana (AtsPLA2-alpha), being one of the first plant sPLA2s obtained in purified state, has been characterised with respect to substrate preference and optimum conditions of catalysis. The optima of pH, temperature, and calcium concentration were similar to the parameters of secretory PLA2s from animals. However, substrate preferences markedly differed. In contrast to pancreatic PLA2s, AtsPLA2-alpha preferred zwitterionic phospholipids, and showed lower activity toward anionic phospholipids. In substrates with two identical fatty acid chains, AtsPLA2-alpha showed optimum activity toward phospholipids with decanoyl groups. In substrates with palmitoyl groups in sn-1 position, acyl chains with higher degree of unsaturation in sn-2 position were preferred, excluding arachidonic acid, showing the evolutionary adaptation of the enzyme to substrate composition in plants. Km values for short chain phospholipids were comparable to sPLA2s from animals, whereas k cat values were much smaller and interfacial activation was less important.     

Signal reliability compromised by genotype-by-environment interaction and potential mechanisms for its preservation

Sexual selection based on signaling requires that signals used by females in mate choice are reliable indicators of a male's heritable total fitness. A signal and the preference for it are expected to be heritable, resulting in the maintenance of genetic covariance between these two traits. However, a recent article has proposed that signals may quickly become unreliable in the presence of both environmental variation and genotype-by-environment interaction (G x E) with crossing reaction norms, potentially compromising the mechanisms of sexual selection. Here we examine the heritability and plasticity of a male dominance advertisement in the bank vole, Clethrionomys glareolus, in stable and changing rearing environments from father to son. The bank vole is naturally exposed to considerable sources of spatial and temporal environmental variation and male reproductive success is determined by both intra- (male-male competition) and inter- (females prefer to mate with dominant males) sexual selection. Significant G x E for male dominance was found with crossing reaction norms. Plasma testosterone level (T), rather than condition, determined a male's dominance and T also showed a significant G x E. Dominance showed a considerable plasticity across environments, but was only heritable under stable conditions. We document a negative between-environments correlation of male dominance, suggesting that when the environment changes between father and son, the dominance signal is unreliable to females and sexual selection may be compromised. We discuss how G x E and environmental variation interacting with other mechanisms may preserve the reliability of signals and thus the mechanism of sexual selection itself.