NMR and HPLC-UV profiling of potatoes with genetic modifications to metabolic pathways

Metabolite profiling has been carried out to assess the compositional changes occurring in potato tubers after genetic modifications have been made to different metabolic pathways. Most major features in the (1)H NMR and HPLC-UV profiles of tuber extracts have been assigned. About 40 GM lines and controls belonging to 4 groups of samples (derived from cv. Record or cv. Desirée and modified in primary carbon metabolism, starch synthesis, glycoprotein processing, or polyamine/ethylene metabolism) were analyzed. Differences were assessed at the level of whole profiles (by PCA) or individual compounds (by ANOVA). The most obvious differences seen in both NMR and HPLC-UV profiles were between the two varieties. There were also significant differences between two of the four Desirée GM lines with modified polyamine metabolism and their controls. Compounds notably affected were proline, trigonelline, and numerous phenolics. However, that modification gave rise to a very abnormal phenotype. Certain lines from the other groups had several compounds present in significantly higher or lower amounts compared to the control, but the differences in mean values amounted to no more than a 2-3-fold change: in the context of variability in the whole data set, such changes did not appear to be important.     

Antioxidant properties and composition of aqueous extracts from Mentha species, hybrids, varieties, and cultivars

Water-soluble extracts from the Mentha species M. aquatica L. and M. haplocalyx Briq., the hybrids M. x dalmatica L. and M. x verticillata L., the varieties M. arvensis var. japanensis [M. arvensis L. var. piperascens Holmes ex Christ] and M. spicata L. var. crispa Benth, and M. x piperita L. "Frantsila", M. "Morocco", and M. "Native Wilmet" cultivars were screened for potential antioxidative properties. These properties included iron(III) reduction, iron(II) chelation, 1,1-diphenyl-2-picrylhydrazyl radical scavenging, and the ability to inhibit iron(III)-ascorbate-catalyzed hydroxyl radical-mediated brain phospholipid peroxidation. Total phenol content and qualitative and quantitative compositional analyses of each extract were also made. The extracts demonstrated varying degrees of efficacy in each assay, with the M. x piperita "Frantsila" extract being better than the other extracts, except for ferrous iron chelation. With the exception of iron chelation, it appeared that the level of activity identified was strongly associated with the phenolic content.     

Nitrate leaching in soil: Tracing the NO3 sources with the help of stable N and O isotopes

Legumes increase the plant-available N pool in soil, but might also increase NO₃⁻ leaching to groundwater. To minimize NO₃⁻ leaching, N-release processes and the contribution of legumes to NO₃⁻ concentrations in soil must be known. Our objectives were (1) to quantify NO₃⁻-N export to >0.3 m soil depth from three legume monocultures (Medicago x varia Martyn, Onobrychis viciifolia Scop., Lathyrus pratensis L.) and from three bare ground plots. Furthermore, we (2) tested if it is possible to apply a mixing model for NO₃⁻ in soil solution based on its dual isotope signals, and (3) estimated the contribution of legume mineralization to NO₃⁻ concentrations in soil solution under field conditions. We collected rainfall and soil solution at 0.3 m soil depth during 1 year, and determined NO₃⁻ concentrations and δ¹⁵N and δ¹⁸O of NO₃⁻ for >11.5 mg NO₃⁻-N l⁻¹. We incubated soil samples to assess potential N release by mineralization and determined δ¹⁵N and δ¹⁸O signals of NO₃⁻ derived from mineralization of non-leguminous and leguminous organic matter.Mean annual N export to >0.3 m soil depth was highest in bare ground plots (9.7 g NO₃⁻-N m⁻²; the SD reflects the spatial variation) followed by Medicago x varia monoculture (6.0 g NO₃⁻-N m⁻²). The O. viciifolia and L. pratensis monocultures had a much lower mean annual N export (0.5 and 0.3 g NO₃⁻-N m⁻²). The averaged NO₃⁻-N leaching during 70 days was not significantly different between field estimates and incubation for the Medicago x varia Martyn monoculture.The δ¹⁵N and δ¹⁸O values in NO₃⁻ of rainfall (δ¹⁵N: 3.3±0.8‰; δ¹⁸O: 30.8±4.7‰), mineralization of non-leguminous SOM (9.3±0.9‰; 6.7±0.8‰), and mineralization of leguminous SOM (1.5±0.6‰; 5.1±0.9‰) were markedly different. Applying a linear mixing model based on these three sources to δ¹⁵N and δ¹⁸O values in NO₃⁻ of soil solution during winter 2003, we calculated 18-41% to originate from rainfall, 38-57% from mineralization of non-leguminous SOM, and 18-40% from mineralization of leguminous SOM.Our results demonstrate that (1) even under legumes NO₃⁻-N leaching was reduced compared to bare ground, (2) the application of a three-end-member mixing model for NO₃⁻ based on its dual isotope signals produced plausible results and suggests that under particular circumstances such models can be used to estimate the contributions of different NO₃⁻ sources in soil solution, and (3) in the 2nd year after establishment of legumes, they contributed approximately one-fourth to NO₃⁻-N loss.     

Impact of Maillard reaction on immunoreactivity and allergenicity of the hazelnut allergen Cor a 11

Few studies exist on the influence of processing methods on structural changes and allergenic potential of hazelnut proteins. This study focused on the effect of glycation (Maillard reaction) on the immunoreactivity and degranulation capacity of the purified hazelnut 7S globulin, Cor a 11. After heating, the extent of the Maillard reaction, sensitivity to proteolysis, binding of human IgE or rabbit IgG, and degranulation capacity were analyzed. Changes in electrophoretic mobility, amount of free amino groups, and contents of bound sugar and fructosamine indicated that glycation of Cor a 11 occurred at all conditions. Glycation at 37 °C did not influence the specific IgG or IgE binding and was decreased after heating at 60 and 145 °C. Heating, with or without glucose, at 145 °C increased basophil degranulation capacity. The results suggest that glycation of Cor a 11 at 60 and 145 °C may decrease the IgE/IgG binding properties but not the degranulation capacity of basophils. This is possibly related to aggregation of the proteins as a result of the Maillard reaction.     

Cross-linking behavior and foaming properties of bovine α-lactalbumin after glycation with various saccharides

α-Lactalbumin was glycated via the Maillard reaction in the dry state using various mono- and oligosaccharides. The reaction resulted not only in coupling of the saccharides to α-lactalbumin but also in cross-linked proteins. The glycation rate and the extent of cross-link formation were highly dependent on the saccharide used. Glycation by arabinose and xylose led to a very fast protein cross-link formation, whereas glucose showed a relatively low protein cross-linking ability. The stability of foams, created using the various glycated protein samples, depended on the type of saccharide used, the extent of glycation, and possibly the amount of cross-linked protein. Compared to nonmodified α-lactalbumin, glycation with rhamnose and fucose improved foam stability, whereas application of glucose, galacturonic acid, and their oligosaccharides did not exert a clear effect. Mass spectrometric analysis revealed that dehydration of the Amadori products is an indicator of the formation of protein cross-links.     

Association between skin surface pH, temperature and Staphylococcus pseudintermedius in dogs with immunomodulatory-responsive lymphocytic-plasmacytic pododermatitis

Secondary bacterial infection is a frequent complication in lesional skin of dogs with immunomodulatory‐responsive lymphocytic–plasmacytic pododermatitis (ImR‐LPP). However, the influence of skin pH and temperature in determining the composition of the cutaneous microflora at lesional sites has not been investigated. The association between ImR‐LPP and pedal skin temperature, pH and Staphylococcus pseudintermedius isolates was thus evaluated. Temperature and pH were measured in 20 dogs with ImR‐LPP and in 30 clinically healthy control dogs, and S. pseudintermedius was cultured from interdigital and palmoplantar swabs in both groups and scored semi‐quantitatively for bacterial growth. In the ImR‐LPP group, mean skin pH was slightly, but significantly, higher at both interdigital and palmoplantar sites. Staphylococcus pseudintermedius was isolated more frequently, and scores for bacterial growth were also significantly higher. However, mean skin temperatures were not significantly different from those in the control group. The isolation of S. pseudintermedius was significantly associated with ImR‐LPP, with the single exception of isolates on Columbia blood agar from the palmoplantar region. However, pH and temperature were not significantly associated with the disease, and were not associated with the isolation of S. pseudintermedius at most sites sampled. Staphylococcus pseudintermedius was not isolated from all feet sampled in dogs with ImR‐LPP. Taken together, these data would suggest that S. pseudintermedius infection is most likely to be a secondary phenomenon in dogs with ImR‐LPP, and that changes in skin pH and temperature are not significant risk factors for this disease.     

Characterization of Escherichia coli isolates from peritonitis lesions in commercial laying hens

Five clinically normal chickens from three farms (farm A, farm B, and farm C), for a total of 15 clinically normal chickens, were examined bacteriologically. In a similar manner, five dead chickens with lesions of peritonitis from each of the same three commercial egg-laying operations were selected for bacterial culturing. Escherichia coli were isolated from the cloaca in 14 of 15 healthy chickens and from all 15 chickens with peritonitis. Oviducts of normal chickens did not contain E. coli (0/15) whereas oviducts from 13 of 15 hens with peritonitis were positive for this pathogen. No lesions and no E. coli (0/15) were found in the peritoneal cavity of healthy hens, but peritonitis lesions from 13 of 15 dead chickens yielded E. coli. On farm A and farm B, a flock consisted of all chickens within a single house and all chickens in each flock were of the same age and same genetic strain. In flock 1 from farm A, all five E. coli isolates from the oviduct and all five isolates from the peritoneal cavity were serogrouped as O78; contained the virulence genes iroN, sitA, iutA, tsh, and iss; and belonged to phylogenetic group A. In flock 2 from farm B, all four E. coli isolates from the oviduct and all four isolates from the peritoneal cavity were serogrouped as O111; contained virulence genes iroN, sitA, iutA, traT, iss, and ompT; and belonged to phylogenetic group D. These data suggest that all chickens with peritonitis in a single flock on farms A and B were likely infected by the same E. coli strain. Escherichia coli isolates from the magnum and peritoneum had the same serogroup, virulence genotype, and phylogenetic group, which is consistent with an ascending infection from the oviduct to the peritoneal cavity.     

Effects of the sodium and calcium concentrations on the in vitro growth of Atriplex halimus L. plantlets

The objective of this work is to analyse the sodium (Na⁺) and calcium (Ca⁺⁺) importance in the in vitro morphogenesis of a halophile species the Atriplex halimus. The experiments have been made on plantlets coming from a seed population collected in a natural area (Algeria). Twenty medium, all different by their sodium chloride (NaCl) and calcium chloride (CaCl₂) content, have been tested. For five Na⁺ concentrations, four [Na⁺]/[Ca⁺⁺] ratios have been tested. The [Na⁺]/[Ca⁺⁺] ratio has a significant effect on the studied criteria (stem length, longest root length and leaf number). The ratio equal to two is the one which gives the lowest values and the ratio equal to one (for the same [Na⁺] quantity and a double quantity of [Ca⁺⁺]) gives the strongest ones. A new doubling of the [Ca⁺⁺] (ratio equal to 0.5) induces a decrease of the values. These results are remarkable if we take into account the low cation concentrations used in the experiments. The morphogenetical events observed here will be explained when results about cation ratios inside the plant and the enzyme activity will have been obtained.     

Development of maternal and foetal immune responses in cattle following experimental challenge with Neospora caninum at day 210 of gestation

This study examined the immunological responses of pregnant cattle and their foetuses following an experimental challenge with live Neospora caninum tachyzoites at day 210 of gestation. Animals were bled prior to and weekly throughout the experiment and sacrificed at 14, 28, 42 and 56 days post inoculation (dpi). At post mortem examination, samples of lymph nodes and spleen were collected from both dam and foetus for immunological analysis. Subcutaneous (sc) inoculation over the left prefemoral (LPF) lymph node of pregnant cattle at day 210 of gestation, led to the vertical transmission of parasites by 14 dpi, however no foetal deaths were observed in the infected animals. Foetuses from infected dams mounted Neospora-specific humoral and cell-mediated immune (CMI) responses by 14 dpi. These responses involved anti-Neospora IgG, antigen-specific lymphocyte proliferation, and the production of the cytokines IFN–γ, interleukin (IL)-4 and IL-10. There was also evidence of innate immunity during the response against Neospora from infected dams, with statistically significant (p < 0.05) increases in mean expression of toll like receptors (TLR)-2 on 56 dpi in maternal spleen, LPF, right prefemoral (RPF), left uterine (LUL) and right uterine (RUL) lymph nodes and TLR-9 in retropharyngeal (RLN), LPF and RPF lymph nodes from 28 dpi. Statistically significant (p < 0.05) increases in mean TLR-9 were detected in spleen samples from foetuses of infected dams, compared to the foetuses from control animals. Our results show that vertical transmission of the parasite occurred in all infected dams, with their foetuses showing effective Neospora-specific cell mediated, humoral and innate immune responses.