Characterization of Nitrate Reductase from Corn Leaves (Zea mays cv W64A x W182E) : Two Molecular Forms of the Enzyme

The primary leaves from corn seedlings grown for 6 days were harvested, frozen with liquid N₂ and extracted in a Tris buffer (pH 8.5, 250 millimolar) containing 1 millimolar dithiothreitol, 10 millimolar cysteine, 1 millimolar EDTA, 20 micromolar flavin adenine dinucleotide and 10% (v/v) glycerol. Nitrate reductase (NR) in the crude extract was stable for several days at 0°C and for several months at −80°C. The enzyme was purified using (NH₄)₂SO₄ fractionation, brushite-hydroxyl-apatite chromatography and blue-sepharose affinity chromatography. The enzyme was eluted from the blue-sepharose column with a linear gradient of NADH (0-100 micromolar) or with 0.3 molar KNO₃. About 10% of the original activity was recovered with NADH (NADH-NR). It had a specific activity of about 60 to 70 units (micromoles NO₂⁻ per minute per milligram protein). A sequential elution with NADH followed by KNO₃ (0.3 molar) or KCl (0.3 molar) yielded 2 peaks. Rechromatography of each peak gave two peaks again. These results indicate that we are dealing with two forms of the same enzyme rather than two different NR proteins. The two NRs had different molecular weights as judged by chromatography on Toyopearl. The NADH-NR was more sensitive than the NO₃⁻-NR to antibody prepared against barley leaf NR. In Ouchterlony assays a single precipitin line, with completely fused boundaries, was observed.     

A Proteinase from Germinated Barley : II. Hydrolytic Specificity of a 30 Kilodalton Cysteine Proteinase From Green Malt

The hydrolytic specificity of a 30 kilodalton cysteine proteinase purified from germinated barley (Hordeum vulgare L. cv Morex) was investigated using high performance liquid chromatography to characterize its hydrolysis of two small barley seed proteins, the α- and β-hordothionins. The reduced and pyridylethylated thionins were rapidly cleaved, resulting in the production of a limited number of peptides. Peptide bonds Gly9-Arg10, Cys 16-Arg17, Cys25-Ala26, and Thr34-Ser35 were most susceptible to hydrolysis, the peptide bonds Arg5-Ser6, Arg19-Gly20 in both thionins and Lys38-Cys39 in β-hordothionin and Cys29-Arg30 of α-hordothionin being broken at much slower rates. The hydrolysis patterns were highly reproducible from assay to assay and with various enzyme preparations. The specificity was apparently defined by the amino acids in the P₂ position, not those immediately adjacent to the susceptible bonds. The P₂ amino acid residues of the released peptides were always either leucine, valine, tyrosine, or pyridylethylcysteine. From these observations and from the rates of release of the various peptides, it appears that the barley 30 kilodalton endoproteinase has an S2 subsite that preferentially binds the leucine side chain: i.e. for hydrolyzing the peptide bond P₁-P₁′ in the general sequence NH₂—P₂-P₁-P₁′—COOH, the enzyme is selective for leucine and, to a lesser extent, valine and tyrosine at position P₂. The barley proteinase thus resembles two other cysteine proteinases, papain and Streptococcal proteinase, in its specificity.     

Regulation of nitrate reductase in suspension culture of Silene alba Immunochemical approach

Silene alba cells grown on nitrate, usually develop NADH-nitrate reductase activity only at the beginning of their growth cycle. Immunodiffusion assays, with a specific nitrate reductase antiserum, revealed the presence of cross-reacting material in cells harvested at any time during their culture. Cells grown on ammonium lacked NADH-nitrate reductase activity but contained cross-reacting material. It is suggested that S. alba cells contain an enzymically inactive, antigenic form of nitrate reductase regardless of the nitrogen source.     

Performance and Accuracy of Lightweight and Low-Cost GPS Data Loggers According to Antenna Positions,Fix Intervals,Habitats and Animal Movements

Recently developed low-cost Global Positioning System (GPS) data loggers are promising tools for wildlife research because of their affordability for low-budget projects and ability to simultaneously track a greater number of individuals compared with expensive built-in wildlife GPS. However, the reliability of these devices must be carefully examined because they were not developed to track wildlife. This study aimed to assess the performance and accuracy of commercially available GPS data loggers for the first time using the same methods applied to test built-in wildlife GPS. The effects of antenna position, fix interval and habitat on the fix-success rate (FSR) and location error (LE) of CatLog data loggers were investigated in stationary tests, whereas the effects of animal movements on these errors were investigated in motion tests. The units operated well and presented consistent performance and accuracy over time in stationary tests, and the FSR was good for all antenna positions and fix intervals. However, the LE was affected by the GPS antenna and fix interval. Furthermore, completely or partially obstructed habitats reduced the FSR by up to 80% in households and increased the LE. Movement across habitats had no effect on the FSR, whereas forest habitat influenced the LE. Finally, the mean FSR (0.90 ± 0.26) and LE (15.4 ± 10.1 m) values from low-cost GPS data loggers were comparable to those of built-in wildlife GPS collars (71.6% of fixes with LE < 10 m for motion tests), thus confirming their suitability for use in wildlife studies.     

When should a trophically and vertically transmitted parasite manipulate its intermediate host? The case of Toxoplasma gondii

Parasites with complex life cycles are expected to manipulate the behaviour of their intermediate hosts (IHs), which increase their predation rate and facilitate the transmission to definitive hosts (DHs). This ability, however, is a double-edged sword when the parasite can also be transmitted vertically in the IH. In this situation, as the manipulation of the IH behaviour increases the IH death rate, it conflicts with vertical transmission, which requires healthy and reproducing IHs. The protozoan Toxoplasma gondii, a widespread pathogen, combines both trophic and vertical transmission strategies. Is parasite manipulation of host behaviour still adaptive in this situation? We model the evolution of the IH manipulation by T. gondii to study the conflict between these two routes of transmission under different epidemiological situations. Model outputs show that manipulation is particularly advantageous for virulent strains and in epidemic situations, and that different levels of manipulation may evolve depending on the sex of the IH and the transmission routes considered. These results may help to understand the variability of strain characteristics encountered for T. gondii and may extend to other trophically transmitted parasites.     

Role of dog behaviour and environmental fecal contamination in transmission of Echinococcus multilocularis in Tibetan communities

On the Eastern Tibetan Plateau region (Sichuan province, China) dogs are regarded as important definitive hosts of Echinococcus multilocularis. We studied dog spatial behaviour in 4 Tibetan villages in order to determine the role of dogs in environmental contamination and their potential interactions with small mammal intermediate hosts. We identified definitive host species and Echinococcus spp. infection status of feces collected in the field by PCR methods and analysed the spatial distribution of canid feces. Nocturnal space utilization of GPS collared dogs in and around villages was also undertaken. E. multilocularis DNA was amplified in 23% of dog feces (n=142) and in 15% of fox feces (n=13) but this difference was not significant. However, dog feces were more frequently observed (78% of collected feces) than fox feces and are therefore assumed to largely contribute to human environment contamination. Feces were mainly distributed around houses of dog owners (0-200 m) where collared dogs spent the majority of their time. Inside villages, the contamination was aggregated in some micro-foci where groups of dogs defecated preferentially. Finally, small mammal densities increased from the dog core areas to grasslands at the periphery of villages occasionally used by dogs; male dogs moving significantly farther than females. This study constitutes a first attempt to quantify in a spatially explicit way the role of dogs in E. multilocularis peri-domestic cycles and to identify behavioural parameters required to model E. multilocularis transmission in this region.     

Landscape genetics highlights the role of bank vole metapopulation dynamics in the epidemiology of Puumala hantavirus

Rodent host dynamics and dispersal are thought to be critical for hantavirus epidemiology as they determine pathogen persistence and transmission within and between host populations. We used landscape genetics to investigate how the population dynamics of the bank vole Myodes glareolus, the host of Puumala hantavirus (PUUV), vary with forest fragmentation and influence PUUV epidemiology. We sampled vole populations within the Ardennes, a French PUUV endemic area. We inferred demographic features such as population size, isolation and migration with regard to landscape configuration. We next analysed the influence of M. glareolus population dynamics on PUUV spatial distribution. Our results revealed that the global metapopulation dynamics of bank voles were strongly shaped by landscape features, including suitable patch size and connectivity. Large effective size in forest might therefore contribute to the higher observed levels of PUUV prevalence. By contrast, populations from hedge networks highly suffered from genetic drift and appeared strongly isolated from all other populations. This might result in high probabilities of local extinction for both M. glareolus and PUUV. Besides, we detected signatures of asymmetric bank vole migration from forests to hedges. These movements were likely to sustain PUUV in fragmented landscapes. In conclusion, our study provided arguments in favour of source‐sink dynamics shaping PUUV persistence and spread in heterogeneous, Western European temperate landscapes. It illustrated the potential contribution of landscape genetics to the understanding of the epidemiological processes occurring at this local scale.     

Spatial distribution of soil contamination by Toxoplasma gondii in relation to cat defecation behaviour in an urban area

In urban areas, there may be a high local risk of zoonosis due to high densities of stray cat populations. In this study, soil contamination by oocysts of Toxoplasma gondii was searched for, and its spatial distribution was analysed in relation to defecation behaviour of cats living in a high-density population present in one area of Lyon (France). Sixteen defecation sites were first identified. Cats were then repeatedly fed with marked food and the marked faeces were searched for in the defecation sites. Of 260 markers, 72 were recovered from 24 different cats. Defecation sites were frequented by up to 15 individuals. Soil samples were also examined in order to detect the presence of T. gondii using real-time PCR. The entire study area was then sampled according to cat density and vegetation cover type. Only three of 55 samples were positive and all came from defecation sites. In a second series of observations, 16 defecation sites were sampled. Eight of 62 samples tested positive, originating in five defecation sites. Laboratory experiments using experimental seeding of soil showed that the inoculated dose that can be detected in 50% of assays equals 100-1000oocysts/g, depending on the strain. This study shows that high concentrations of oocysts can be detected in soil samples using molecular methods and suggests that spatial distribution of contamination areas is highly heterogeneous. Positive samples were only found in some of the defecation sites, signifying that at-risk points for human and animal infection may be very localised.     

Dynamics of spatial relationships among members of a fox group (Vulpes vulpes: Mammalia: Carnivora)

The 'Resources Dispersion Hypothesis' (RDH, Macdonald, 1983) suggests that, for solitary foragers such as the red fox Vulpes vulpes , group formation is dependent on resource distribution heterogeneity. Our data are compatible with this hypothesis. Rodents, which constituted the main fox prey, were heterogeneously distributed in time and space. Six foxes (three males and three females) were radiotracked continuously from February 1989 to October 1990 (20 months) and we observed spatial sharing between one male and two or three females, considered as members of a 'spatial group'. Even though their home ranges overlapped between 30 and 100%, members of the group foraged alone and had very few contacts with conspecifics during the night. Furthermore, they partitioned the common home range so that each fox made exclusive use of foraging patches. In contrast, during the daytime, two to four members of the group were frequently in association in a communal resting place. Such associations were observed all year round; they were durable and dynamic. Their advantages were examined. We suggest that they play a role in the maintenance of social cohesion within the group in providing the opportunity for direct contact between foxes. They might also permit increased security through mutual vigilance during resting.     

Characterization of Nitrate Reductases from Corn Leaves (Zea mays cv W64AxW182E) and Chlorella vulgaris: Sensitivity to a Proteinase Extracted from Corn Roots

The sensitivity of the two forms of nitrate reductase, NR_I and NR_II, obtained from the primary leaf of corn, to a limited action corn root proteinase has been examined. The corn inactivating protein (CIP) inhibited the overall reaction (NADH-NR) and the two partial reactions, cytochrome c reductase and reduced methyl viologen NR (MV-NR) of both forms of NR. NADH-cytochrome c reductase was more sensitive to the protease than MV-NR. NR_II was less sensitive to inactivation than NR_I. When NR_I and NR_II were inactivated and then subjected to native gel electrophoresis the protein bands associated with MV-NR activity shifted from an R_m value of 0.32 to 0.61 for NR_I and from an R_m of 0.28 to 0.60 for NR_II. For Chlorella NR these values are 0.32 and 0.70. The initial cleavage of the 116 kilodalton subunit of NR_I yielded fragments of 84 and 80 kilodaltons after a 5 minute incubation with CIP. With longer incubation times smaller fragments were also identified. For the Chlorella NR the initial cleavage products are approximately 68 and 25 kilodaltons. Longer incubation times also led to smaller fragments. The products of hydrolysis by this limited action protease are quite different for the corn and Chlorella NRs.