Assimilation of 13NH 4 + by Anthoceros grown with and without symbiotic Nostoc

The pathways of assimilation of ammonium by pure cultures of symbiont-free Anthoceros punctatus L. and the reconstituted Anthoceros-Nostoc symbiotic association were determined from time-course (5–300 s) and inhibitor experiments using ¹³NH ₄ ⁺ . The major product of assimilation after all incubation times was glutamine, whether the tissues were cultured with excess ammonium or no combined nitrogen. The ¹³N in glutamine was predominantly in the amide-nitrogen position. Formation of glutamine and glutamate by Anthoceros-Nostoc was strongly inhibited by either 1mM methionine sulfoximine (MSX) or 1 mM exogenous ammonium. These data are consistent with the assimilation of ¹³NH ₄ ⁺ and formation of glutamate by the glutamine synthetase (EC 6.3.1.2)-glutamate synthase (EC 1.4.7.1) pathway in dinitrogen-grown Anthoceros-Nostoc. However, in symbiont-free Anthoceros, grown with 2.5 mM ammonium, formation of glutamine, but not glutamate, was decreased by either MSX or exogenous ammonium. These results indicate that during short incubation times ammonium is assimilated in nitrogenreplete Anthoceros by the activities of both glutamine synthetase and glutamate dehydrogenase (EC 1.4.1.2). In-vitro activities of glutamine synthetase were similar in nitrogen-replete Anthoceros and Anthoceros-Nostoc, indicating that the differences in the routes of glutamate formation were not based upon regulation of synthesis of the initial enzyme of the glutamine synthetase-glutamate synthase pathway. When symbiont-free Anthoceros was cultured for 2 d in the absence of combined nitrogen, total ¹³NH ₄ ⁺ assimilation, and glutamine and glutamate formation in the presence of inhibitors, were similar to dinitrogen-grown Anthoceros-Nostoc. The routes of immediate (within 2 min) glutamate formation and ammonium assimilation in Anthoceros were apparently determined by the intracellular levels of ammonium; at low levels the glutamine synthetase-glutamate synthase pathway was predominant, while at high levels independent activities of both glutamine synthetase and glutamate dehydrogenase were expressed.     

The ribosomal protein P0 of soybean (Glycine max L. Merr.) has antigenic cross-reactivity to soybean seed lectin

Soybean (Glycine max L. Merr.) mutants lacking the ability to produce the lectin normally found in soybean seeds (SBL) are designated Le-. A protein of higher molecular weight that cross-reacts with antibodies raised to SBL was found at nearly equivalent levels in roots, hypocotyls, and leaves, and at lower levels in cotyledons and dry seeds of both Le+ and Le- soybean cultivars. Earlier work suggested that this protein was a novel lectin. Clones isolated from a Le- soybean root cDNA library produced a cross-reacting protein of the same size in Escherichia coli. Sequence analysis of these clones revealed a high degree of similarity to the ribosomal protein P0. The cross-reacting protein co-purified with ribosomes, and a monoclonal antibody raised to purified brine shrimp P0 cross-reacted to the same protein. The protein showed no lectin activity in a hemagglutination assay, nor did it bind to an N-acetyl-D-galactosamine affinity column. On the basis of this evidence, we conclude that the SBL-cross-reacting protein is not a lectin but a homologue of the ribosomal protein P0. Consequently, Le- soybeans must produce a lectin that is dissimilar to SBL at both the DNA and amino acid levels and we suggest that it is this lectin which is involved in nodulation.     

Regulation of Expression of Nitrate and Dinitrogen Assimilation by Anabaena Species

Anabaena sp. strain 7120 appeared more responsive to nitrogen control than A. cylindrica. Growth in the presence of nitrate strongly repressed the differentiation of heterocysts and fixation of dinitrogen in Anabaena sp. strain 7120, but only weakly in A. cylindrica. Nitrate assimilation by ammonium-grown cultures was strongly repressed in Anabaena sp. strain 7120, but less so in A. cylindrica. The repressive effect of nitrate on dinitrogen assimilation in Anabaena sp. strain 7120, compared to A. cylindrica, did not correlate with a greater rate of nitrate transport, reduction to ammonium, assimilation into amino acids, or growth. Although both species grew at similar rates with dinitrogen, A. cylindrica grew faster with nitrate, incorporated more ¹³NO₃⁻ into amino acids, and assimilated (transported) nitrate at the same rate as Anabaena sp. strain 7120. Full expression of nitrate assimilation in the two species occurred within 2.5 h (10 to 14% of their generation times) after transfer to nitrate medium. The induction and continued expression of nitrate assimilation was dependent on protein synthesis. The half-saturation constants for nitrate assimilation and for nitrate and ammonium repression of dinitrogen assimilation have ecological significance with respect to nitrogen-dependent growth and competitiveness of the two Anabaena species.     

Oxygen regulation of a nodule-located carbonic anhydrase in alfalfa

Control of the permeability to oxygen is critical for the function of symbiotic nitrogen fixation in legume nodules. The inner cortex (IC) seems to be a primary site for this regulation. In alfalfa (Medicago sativa) nodules, expression of the Msca1 gene encoding a carbonic anhydrase (CA) was previously found to be restricted to the IC. We have now raised antibodies against recombinant Msca1 protein and used them, together with antibodies raised against potato leaf CA, to demonstrate the presence of two forms of CA in mature nodules. Each antibody recognizes a different CA isoform in nodule tissues. Immunolocalization revealed that leaf-related CAs were localized primarily in the nitrogen-fixing zone, whereas the Msca1 protein was restricted exclusively to the IC region, in indeterminate and determinate nodules. In alfalfa nodules grown at various O(2) concentrations, an inverse correlation was observed between the external oxygen pressure and Msca1 protein content in the IC, the site of the putative diffusion barrier. Thus Msca1 is a molecular target of physiological processes occurring in the IC cells involved in gas exchange in the nodule.     

Chlorate-resistant rose cells: influx, efflux and reduction of [36Cl]-chlorate

Clones of Rosa damascena Mill. cv. Gloire de Guillan, selected for growth in solid medium containing 56 m M NaClO₃, were studied to determine the reason for their resistance to this toxic salt. The cells grew on medium containing nitrate as the only nitrogen source, and they synthesized nitrate reductase (EC 1.6.6.2) in the presence of nitrate. The cells were resistant in the presence of nitrate. However, their resistance was greatly increased by the presence of glutamate in the medium. The cells took up [³⁶Cl]-ClO₃- and reduced it to ClO₂⁻, but the fraction of ClO₃⁻ that they reduced under our experimental conditions was less than that reduced by wild type. The slower production of ClO₂⁻ apparently accounted for the resistance of the cells to ClO₃⁻. We suggest several possible reasons for the low rate of reduction of ClO₃⁻.     

Chronic wasting disease management in ranched elk using rectal biopsy testing

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) affecting members of the cervid species, and is one of the few TSEs with an expanding geographic range. Diagnostic limitations, efficient transmission, and the movement of infected animals are important contributing factors in the ongoing spread of disease. Managing CWD in affected populations has proven difficult, relying on population reduction in the case of wild deer and elk, or quarantine and depopulation in farmed cervids. In the present study, we evaluated the effectiveness of managing endemic CWD in a closed elk herd using antemortem sampling combined with both conventional and experimental diagnostic testing, and selective, targeted culling of infected animals. We hypothesized that the real-time quaking-induced conversion (RT-QuIC) assay, a developing amplification assay, would offer greater detection capabilities over immunohistochemistry (IHC) in the identification of infected animals using recto-anal mucosa associated lymphoid tissue (RAMALT). We further sought to develop a better understanding of CWD epidemiology in elk with various PRNP alleles, and predicted that CWD prevalence would decrease with targeted culling. We found that RT-QuIC identified significantly more CWD-positive animals than IHC using RAMALT tissues (121 vs. 86, respectively, out of 553 unique animals), and that longstanding disease presence was associated with an increasing frequency of less susceptible PRNP alleles. Prevalence of CWD increased significantly over the first two years of the study, implying that refinements in our management strategy are necessary to reduce the prevalence of CWD in this herd.