N2 fixation and nitrogen allocation to above and below ground plant parts in red clover-grasslands

Leys, used for grazing or production of forage to be conserved as silage or hay, are very important crops in northern areas. In order to measure the N₂ fixation in leys of varying ages and during different parts of the season, detailed measurements were taken of yield, N₂ fixation and the amounts of N remaining in the field after harvesting red clover (Trifolium pratense L.)-grass leys at a site in northern Sweden, where they are generally harvested twice per growing season. Entire plants, including stubble and roots, were sampled at the time of first and second harvest and, in addition, at the end of the growing season in three neighbouring fields, carrying a first, a second and a third year ley, respectively. N₂ fixation was measured by both ¹⁵N isotope dilution (ID) and ¹⁵N natural abundance (NA) methods. The proportion of clover dry matter (DM) in the stands increased from the first to the second harvest, but the grasses dominated throughout the entire season, especially below ground. The N concentrations, in both herbage and whole plants, were about twice as high in the clover as in the grasses. Seasonal variations in N concentrations were minor, and total N contents followed the same trends as DM. The clover acquired nearly all of its N from N₂ fixation: the proportion of N in clover herbage derived from N₂ fixation was often >0.8 throughout the season. The variations in the amounts of N₂ fixed during the course of the season corresponded well to the seasonal changes in clover biomass. Amounts of fixed N₂ allocated to clover herbage during the whole season were in the range 4 to 6 g N m⁻² in this unusually rainy year. Calculations of daily N allocation rates to herbage showed that N uptake rates were similar, and high, in grasses during May–June and July–August, while N₂ fixation rates in clover were about 10-fold as high in July–August as in May–June, reflecting the need for N in clover growth. The proportion of N remaining in clover stubble and roots after the first and second harvests was about 60 and 25%, respectively, while about 60% of the N in grasses remained in stubble and roots after both harvests. The considerable amounts of biomass and N that were left in field after harvesting red clover-grass leys are important for re-growth of the plants and provide substantial N fertilization for the next crop in the crop rotation.     

Dopamine depletion and subsequent treatment with L-DOPA, but not the long-lived dopamine agonist pergolide, enhances activity of the Akt pathway in the rat striatum

Dysregulation of signaling pathways is believed to contribute to Parkinson's disease pathology and l-DOPA-induced motor complications. Long-lived dopamine (DA) agonists are less likely to cause motor complications by virtue of continuous stimulation of DA receptors. In this study, we compared the effects of the unilateral 6-hydroxydopamine lesion and subsequent treatment with l-DOPA and DA agonist pergolide on signaling pathways in rats. Pergolide caused less pronounced behavioral sensitization than l-DOPA (25 mg/kg, i.p., 10 days), particularly at lower dose (0.5 and 0.25 mg/kg, i.p.). Pergolide, but not l-DOPA, reversed lesion-induced up-regulation of preproenkephalin and did not up-regulate preprodynorphine or DA D3 receptor in the lesioned hemisphere. Pergolide was as effective as l-DOPA in reversing the lesion-induced elevation of ERK2 phosphorylation in response to acute apomorphine administration (0.05 mg/kg, s.c.). Chronic l-DOPA significantly elevated the level of Akt phosphorylation at both Thr(308) and Ser(473) and concentration of phosphorylated GSK3alpha, whereas pergolide suppressed the lesion- and/or challenge-induced supersensitive Akt responses. The data indicate that l-DOPA, unlike pergolide, exacerbates imbalances in the Akt pathway caused by the loss of DA. The results support the hypothesis that the Akt pathway is involved in long-term actions of l-DOPA and may be linked to l-DOPA-induced dyskinesia.     

Affinities of Shiga toxins 1 and 2 for univalent and oligovalent Pk-trisaccharide analogs measured by electrospray ionization mass spectrometry

The binding stoichiometry and affinities of the Shiga toxins, Stx1 and Stx2, for a series of uni- and oligovalent analogs of the Pk-trisaccharide were measured using the direct electrospray ionization mass spectrometry (ES-MS) assay. Importantly, it is shown that, for a given ligand, Stx1 and Stx2 exhibit similar affinities. The binding data suggest a high degree of similarity in the spatial arrangement and structural characteristics of the Pk binding sites in Stx1 and Stx2. The results confirm that both toxins recognize the alpha-D-Galp(1-->4)-beta-D-Galp(1-->4)-beta-D-Glcp carbohydrate motif of the cell surface glycolipid Gb3. This, taken together with the results of the chemical mapping study, suggests that the nature of the Pk binding interactions with Stx1 and Stx2 are similar. The affinities of Stx1-B(5) and Stx2 for the multivalent ligands reveals that site 2 of Stx2, which shares the same spatial arrangement as site 2 in Stx1, is the primary Pk binding site and that site 1 of Stx1 and of Stx2 can also participate in Pk binding.     

A laboratory exercise to illustrate increased salivary cortisol in response to three stressful conditions using competitive ELISA

Perceived stress activates the hypothalamus-pituitary-adrenal axis, resulting in the release of glucocorticoids into the systemic circulation. Glucocorticoids cause the elevation of blood glucose, providing the necessary energy for the organism to cope with stress. Here, we outline a laboratory exercise that uses a competitive ELISA kit to illustrate the response of salivary cortisol concentrations to three stressful conditions. Twelve undergraduate students in the General and Comparative Endocrinology course at Iowa State University were subjected to presentation stress, fasting stress, and competition stress to determine their effect on salivary cortisol concentrations. Students had elevated salivary cortisol in response to each of these stresses compared with basal conditions. These results reiterate the importance of glucocorticoids as mediators of the stress response. This study also incorporates the use of the ELISA technique, a modern laboratory tool used to determine the amount of endogenous antigens in plasma or saliva. This laboratory exercise can easily be adapted to fit into already existing physiology and endocrinology curriculums.     

Toxic effects of zinc on anaerobic microbiota from Zimapán Reservoir (Mexico)

The toxic effects of heavy metals have been extensively documented in different organisms. Nevertheless, a lack of information exists with regard to this topic in the case of autochthonous microorganism communities. The aim of this study was to evaluate the toxic effects of zinc on the anaerobic microorganisms present in the sediment and anoxic water of Zimapán Reservoir (Mexico), with particular focus on dissimilatory sulphate reducing bacteria. In the laboratory, a system of enrichment microcosms was set up with sediment and water from the reservoir. ATP, protein, carbohydrates and lactate and alcohol dehydrogenase activity were determined. The physicochemical parameters of the reservoir were evaluated over the course of one year. Sulphate reduction occurred in the reservoir throughout the year, but was most pronounced at the end of the wet season and during winter. In the field, increases in the rate of sulphate reduction coincided with the lowest levels of total phosphorus and hydrosoluble organic carbon. Zinc enrichment was observed to modify protein and carbohydrate content as well as to affect lactate and alcohol dehydrogenase activity. All responses followed a zinc concentration-response relationship and were dependent on reservoir physicochemical parameters. ATP content was used as a biomarker to evaluate the sublethal toxic effects of zinc. The acceptable threshold concentration of zinc in the aquatic and sediment enrichment microcosms was determined to be 0.06mgZn/L and 711.1mgZn/kg, respectively.     

Mouse translation elongation factor eEF1A-2 interacts with Prdx-I to protect cells against apoptotic death induced by oxidative stress

eEF1A-1 and eEF1A-2 are two isoforms of translation elongation factor eEF1A. In adult mammalian tissues, isoform eEF1A-1 is present in all tissues except neurons, cardiomyocytes, and myotubes, where its isoform, eEF1A-2, is the only form expressed. Both forms of eEF1A have been characterized to function in the protein elongation step of translation, and eEF1A-1 is shown to possess additional non-canonical roles in actin binding/bundling, microtubule bundling/severing, and cellular transformation processes. To study whether eEF1A-2 has similar non-canonical functions, we carried out a yeast two-hybrid screening using a full sequence of mouse eEF1A-2 as bait. A total of 78 hits, representing 23 proteins, were identified and validated to be true positives. We have focused on the protein with the highest frequency of hits, peroxiredoxin I (Prdx-I), for in-depth study of its functional implication for eEF1A-2. Here we show that Prdx-I coimmunoprecipitates with eEF1A-2 from extracts of both cultured cells and mouse tissues expressing this protein, but it does not do so with its isoform, eEF1A-1, even though the latter is abundantly present. We also report that an eEF1A-2 and Prdx-I double transfectant increases resistance to peroxide-induced cell death as high as 1 mM peroxide treatment, significantly higher than do single transfectants with either gene alone; this protection is correlated with reduced activation of caspases 3 and 8, and with increased expression of pro-survival factor Akt. Thus, our results suggest that eEF1A-2 interacts with Prdx-I to functionally provide cells with extraordinary resistance to oxidative stress-induced cell death.     

Profiling the Bordetella pertussis proteome during iron starvation

Regulation of gene expression in response to local iron concentration is commonly observed in bacterial pathogens that face this nutrient limitation during host infection. In this study, a proteomic approach was used to analyze the differential protein expression of Bordetella pertussis under iron limitation. Whole cell lysates (WCL) and outer membrane fractions of bacteria grown either under iron-starvation or iron-excess conditions were analyzed by two-dimensional (2-D) gel electrophoresis. Statistical analysis revealed 36 proteins displaying differential expression, 9 with higher expression under iron-excess and 27 with increased expression under iron-starvation. These proteins were subjected to tryptic digestion and MALDI-TOF MS. Apart from those previously reported, we identified new low-iron-induced proteins that might help to explain the increased virulence of this phenotype. Additionally, we found evidence that at least one of the identified proteins, solely expressed under iron starvation, is highly immunogenic in infected individuals.