Influence of ferulic acid on the production of feruloyl esterases by Aspergillus niger

Extracellular feruloyl esterases from the filamentous fungus Aspergillus niger are induced by growth on oat spelt xylan (OSX), which contains no detectable esterified ferulic acid. FAE-III accounted for most of the feruloyl esterase activity. Addition of free ferulic acid to OSX at the start of the culture induced FAE-III secretion a further 2.3-fold, and also induced other feruloyl esterases which could not be ascribed to FAE-III. Wheat bran- (WB)-grown cultures, containing 1% (m/v) ester-linked ferulic acid, gave almost identical FAE-III and total feruloyl esterase activities as the cultures grown on OSX plus ferulic acid. De-esterification of WB yielded less total feruloyl esterase, and 2.4-fold less FAE-III, compared to untreated WB. A slightly modified form of FAE-III was produced on de-esterified WB. These results show that production of FAE-III does not absolutely require ferulic acid. However, production is stimulated by the presence of free ferulic acid through increased expression, and is reduced by the removal of esterified ferulic acid from the growth substrate.     

The inheritance and chromosomal localization of AFLP markers in a non-inbred potato offspring

AFLP^TM is a new technique to generate large numbers of molecular markers for genetic mapping. The method involves the selective amplification of a limited number of DNA restriction fragments out of complex plant genomic DNA digests using PCR. With six primer combinations 264 segregating AFLP amplification products were identified in a diploid backcross population from non-inbred potato parents. The identity of an AFLP marker was specified by the primer combination of the amplification product and its size estimated in bases. The segregating AFLP amplification products were mapped by using a mapping population with 217 already known RFLP, isozyme and morphological trait loci. In general, the AFLP markers were randomly distributed over the genome, although a few clusters were observed. No indications were found that AFLP markers are present in other parts of the genome than those already covered by RFLP markers. Locus specificity of AFLP markers was demonstrated because equally sized amplification products segregating from both parental clones generally mapped to indistinguishable maternal and paternal map positions. Locus specificity of AFLP amplification products will allow to establish the chromosomal identity of linkage groups in future mapping studies.Since AFLP technology is a multi-locus detection system, it was not possible to identify the AFLP alleles which belong to a single AFLP locus. The consequences of a genetic analysis based on single alleles, rather than on loci with two or more alleles on mapping studies using progenies of non-inbred parents are discussed.     

Phylogenetic relationships among the species of the genus Testudo (Testudines: Testudinidae) inferred from mitochondrial 12S rRNA gene sequences

To test phylogenetic relationships within the genus Testudo (Testudines: Testudinidae), we have sequenced a fragment of the mitochondrial (mt) 12S rRNA gene of 98 tortoise specimens belonging to the genera Testudo, Indotestudo, and Geochelone. Maximum likelihood and neighbor-joining methods identify two main clades of Mediterranean tortoises, one composed of the species Testudo graeca, Testudo marginata, and Testudo kleinmanni and a second of Testudo hermanni, Testudo horsfieldii, and Indotestudo elongata. The first clade, but not the second, was also supported by maximum parsimony analysis. Together with the genus Geochelone, a star-like radiation of these clades was suggested, as a sister-group relationship between the two Testudo clades could not be confirmed. The intraspecies genetic variation was examined by sequencing the mt 12S rRNA fragment from 28 specimens of T. graeca and 49 specimens of T. hermanni from various geographic locations. Haplotype diversity was found to be significantly larger in T. graeca compared with T. hermanni, suggestive of reduced genetic diversity in the latter species, perhaps due to Pleistocene glaciations affecting northern and middle Europe or other sources of lineage reduction. No ancient mt 12S rRNA gene haplotypes were identified in T. graeca and/or T. hermanni originating from islands in the Mediterranean Sea, suggesting that these islands harbor tortoise populations introduced from the European and African mainland.     

Differential synthesis of an alkaline endonuclease in Physarum polycephalum

During the sclerotization of microplasmodia of Physarum polycephalum in non-nutrient salt medium or in salt medium supplemented by glucose, RNA or nucleotides a 6-fold increase in the specific activity of an alkaline endonuclease was found within 6 h after the induction. The increase was based on de novo synthesis of the enzyme and it was strongly correlated to the sharp drop in the level of cellular RNA in the first hours of the process of scerotization. The induction in exhausted growth medium or in salt medium supplemented by protein or mannitol showed a gradual 2-3-fold increase of the endonuclease in 30 h, parallel to the gradual decrease of the RNA. No changes in the specific activity of the endonuclease were found during logarithmic growth or under conditions of starvation without the induction to sclerotization.The alkaline, polyA-specific endonuclease could possibly regulate the turnover of RNA.     

Monitoring lysin motif-ligand interactions via tryptophan analog fluorescence spectroscopy

The lysin motif (LysM) is a peptidoglycan binding protein domain found in a wide range of prokaryotes and eukaryotes. Various techniques have been used to study the LysM-ligand interaction, but a sensitive spectroscopic method to directly monitor this interaction has not been reported. Here a tryptophan analog fluorescence spectroscopy approach is presented to monitor the LysM-ligand interaction using the LysM of the N-acetylglucosaminidase enzyme of Lactococcus lactis. A three-dimensional model of this LysM protein was built based on available structural information of a homolog. This model allowed choosing the amino acid positions to be labeled with a Trp analog. Four functional single-Trp LysM mutants and one double-Trp LysM mutant were constructed and biosynthetically labeled with 7-azatryptophan or 5-hydroxytryptophan. These Trp analogs feature red-shifted absorption spectra, enabling the monitoring of the LysM-ligand interaction in media with a Trp background. The emission intensities of four of the five LysM constructs were found to change markedly on exposure to either L. lactis bacterium-like particles or peptidoglycan as ligands. The method reported here is suitable to monitor LysM-ligand interactions at (sub)micromolar LysM concentrations and can be used for the detection of low levels of peptidoglycan or microbes in solutions.     

Methane oxidation in anoxic lake water stimulated by nitrate and sulfate addition

Methanotrophic bacteria play a key role in limiting methane emissions from lakes. It is generally assumed that methanotrophic bacteria are mostly active at the oxic-anoxic transition zone in stratified lakes, where they use oxygen to oxidize methane. Here, we describe a methanotroph of the genera Methylobacter that is performing high-rate (up to 72 μM day⁻¹) methane oxidation in the anoxic hypolimnion of the temperate Lacamas Lake (Washington, USA), stimulated by both nitrate and sulfate addition. Oxic and anoxic incubations both showed active methane oxidation by a Methylobacter species, with anoxic rates being threefold higher. In anoxic incubations, Methylobacter cell numbers increased almost two orders of magnitude within 3 days, suggesting that this specific Methylobacter species is a facultative anaerobe with a rapid response capability. Genomic analysis revealed adaptations to oxygen-limitation as well as pathways for mixed-acid fermentation and H₂ production. The denitrification pathway was incomplete, lacking the genes narG/napA and nosZ, allowing only for methane oxidation coupled to nitrite-reduction. Our data suggest that Methylobacter can be an important driver of the conversion of methane in oxygen-limited lake systems and potentially use alternative electron acceptors or fermentation to remain active under oxygen-depleted conditions.     

Steps to Take to Enhance Gait Stability: The Effect of Stride Frequency,Stride Length,and Walking Speed on Local Dynamic Stability and Margins of Stability

The purpose of the current study was to investigate whether adaptations of stride length, stride frequency, and walking speed, independently influence local dynamic stability and the size of the medio-lateral and backward margins of stability during walking. Nine healthy subjects walked 25 trials on a treadmill at different combinations of stride frequency, stride length, and consequently at different walking speeds. Visual feedback about the required and the actual combination of stride frequency and stride length was given during the trials. Generalized Estimating Equations were used to investigate the independent contribution of stride length, stride frequency, and walking speed on the measures of gait stability. Increasing stride frequency was found to enhance medio-lateral margins of stability. Backward margins of stability became larger as stride length decreased or walking speed increased. For local dynamic stability no significant effects of stride frequency, stride length or walking speed were found. We conclude that adaptations in stride frequency, stride length and/or walking speed can result in an increase of the medio-lateral and backward margins of stability, while these adaptations do not seem to affect local dynamic stability. Gait training focusing on the observed stepping strategies to enhance margins of stability might be a useful contribution to programs aimed at fall prevention.     

Is slow walking more stable?

Several efforts have been made to study gait stability using measures derived from nonlinear time-series analysis. The maximum finite time Lyapunov exponent (λ_max) quantifies how a system responds to an infinitesimally small perturbation. Recent studies suggested that slow walking leads to lower λ_max values, and thus is more stable than fast walking, but these studies suffer from methodological limitations. We studied the effects of walking speed on the amount of kinematic variability and stability in human walking. Trunk motions of 15 healthy volunteers were recorded in 3D during 2 min of treadmill walking at different speeds. From those time series, maximum Lyapunov exponents, indicating short-term and long-term divergence (λ_S-stride and λ_L-stride), and mean standard deviation (MeanSD) were calculated. λ_S-stride showed a linear decrease with increasing speed for forward–backward (AP) movements and quadratic effects (inverted U-shaped) for medio-lateral (ML) and up–down (VT) movements. λ_L-stride showed a quadratic effect (inverted U-shaped) of walking speed for AP movements, a linear decrease for ML movements, and a linear increase for VT movements. Moreover, positive correlations between λ_S and MeanSD were found for all directions, while λ_L-stride and MeanSD were correlated negatively in the AP direction. The different effects of walking speed on λ_S-stride and λ_L-stride for the different planes suggest that slow walking is not necessarily more stable than fast walking. The absence of a consistent pattern of correlations between λ_L-stride and MeanSD over the three directions suggests that variability and stability reflect, at least to a degree, different properties of the dynamics of walking.     

Association of earthworm-denitrifier interactions with increased emission of nitrous oxide from soil mesocosms amended with crop residue

Earthworm activity is known to increase emissions of nitrous oxide (N(2)O) from arable soils. Earthworm gut, casts, and burrows have exhibited higher denitrification activities than the bulk soil, implicating priming of denitrifying organisms as a possible mechanism for this effect. Furthermore, the earthworm feeding strategy may drive N(2)O emissions, as it determines access to fresh organic matter for denitrification. Here, we determined whether interactions between earthworm feeding strategy and the soil denitrifier community can predict N(2)O emissions from the soil. We set up a 90-day mesocosm experiment in which (15)N-labeled maize (Zea mays L.) was either mixed in or applied on top of the soil in the presence or absence of the epigeic earthworm Lumbricus rubellus and/or the endogeic earthworm Aporrectodea caliginosa. We measured N(2)O fluxes and tested the bulk soil for denitrification enzyme activity and the abundance of 16S rRNA and denitrifier genes nirS and nosZ through real-time quantitative PCR. Compared to the control, L. rubellus increased denitrification enzyme activity and N(2)O emissions on days 21 and 90 (day 21, P = 0.034 and P = 0.002, respectively; day 90, P = 0.001 and P = 0.007, respectively), as well as cumulative N(2)O emissions (76%; P = 0.014). A. caliginosa activity led to a transient increase of N(2)O emissions on days 8 to 18 of the experiment. Abundance of nosZ was significantly increased (100%) on day 90 in the treatment mixture containing L. rubellus alone. We conclude that L. rubellus increased cumulative N(2)O emissions by affecting denitrifier community activity via incorporation of fresh residue into the soil and supplying a steady, labile carbon source.