Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1

Summary The proteins P10 and P12 have been shown to be gene products of a new stability system, ParD, of plasmid R1. It is now shown that an R1 miniplasmid, pAB112, carrying a trans-complementable amber mutation in the gene of the P10 protein, is lethal for the host in the absence of suppression. This lethal effect is suppressed in a supF background and also by deletions in pAB112 that affect the gene of the P12 protein. These data indicate that the P12 protein has a lethal effect on the host and that this effect is neutralized by the P10 protein. The possibility that the stabilization conferred by the ParD system could be due to a counterselection, mediated by P12, of cells that lose the plasmid at cell division, is discussed.     

Nitrate inhibition of nodulation can be overcome by the ethylene inhibitor aminoethoxyvinylglycine

Previously, we reported (a) a positive correlation between the nitrate concentrations in growth medium and ethylene evolved from uninoculated and inoculated alfalfa (Medicago sativa) roots and (b) a negative correlation between ethylene evolution and nodulation. Here, we report that the inhibitory effect of NO₃⁻ on nodulation of alfalfa can be eliminated by the ethylene inhibitor aminoethoxyvinylglycine (AVG). This effect was probably related to the strong inhibition (90%) of ethylene biosynthesis caused by AVG in these inoculated and NO₃⁻-treated roots. These results support our hypothesis that the inhibitory effect of NO₃⁻ is mediated through the phytohormone ethylene. A possible role of endogenous ethylene in the autoregulation of nodulation also is discussed. AVG at 10 micromolar significantly (P < 0.05) increased total nitrogenase activity (acetylene reduction) in 2.5 and 5 millimolar NO₃⁻-fed plants probably as a result of the very high stimulation of nodulation.     

Distribution of nitrate reductase activity in Vicia faba: Effect of nitrate and plant genotype

The short term effect of NO₃⁻ (12 mM) on nitrate reductase (NR. EC 1.6.6.1) activity has been studied in the roots, nodules and leaves of different genotypes of Vicia faba L. at the end of vegetative growth. Root and leaf NR activity responded positively to NO₃⁻ while nodule activity, where detected, proved to he strongly inhibited. The withdraw of this NO₃⁻ from the solution consistently reduced activity in the roots and leaves but surprising, promoted a significant increase in nodule activity, which matched or surpassed that of control plants On the other hand, nodules developed in the presence of 8 mM NO₃⁻ expressed an on average 141% higher level of NR activity than did controls. This effect was observed even in nodules with negligible control activity. In any case, a naturally occurring mutant (VF17) lacking root and nodule NR activity is described. The results indicate that in V. faba. the effects of NO₃⁻ and plant genotype on NR activity depended on plant organ and time of NO₃⁻ application, hut the distribution of NO₃⁻ reduction through the plain was mainly dependent on plant genotype, and to a lesser extent on NO: supply and plant age.     

Nitrate-induced ethylene biosynthesis and the control of nodulation in alfalfa

We previously reported that inhibition of ethylene biosynthesis with aminoethoxyvinylglycine (AVG) eliminated the inhibitory effect of NO₃^– on nodulation of alfalfa (Medicago sativa L. cv. Aragon) plants grown aeroponically. In this work, the effect of Ag⁺, as an inhibitor of ethylene action, has been studied in plants growing aeroponically or in darkened tubes with vermiculite, and low-nitrate or high-nitrate solution. Vermiculite-grown plants developed up to 3 times as many nodules as did those growing aeroponically. Nodule formation was mirrored by dry-matter accumulation. High (10 mol m^–3) NO₃^– applied from planting inhibited nodulation to an equal extent (c. 50%) in the two growth conditions. In contrast, Ag⁺ treatment increased nodule formation at all NO₃^– concentrations assayed under the two growth conditions, with the stimulation being higher in plants grown aeroponically. Finally, no effect of Ag⁺ (10 mmol m^–3) on plant growth was observed in either of the growth conditions. The effectiveness of NO₃^– as a nodulation inhibitor and enhancer of ethylene biosynthesis in roots of alfalfa was also studied. Within 24 h after inoculation, 10 mol m^–3 NO₃^– exerted most of its inhibitory effect on nodulation. At the same time, both 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity and ethylene evolution rates markedly increased in inoculated and uninoculated alfalfa roots treated with NO₃^–. Support for a role of endogenous ethylene in the control of nodule formation in legumes is discussed.     

Philosophy of Biology: Naturalistic or Transcendental?

The aim of this article is to clarify the meaning of a naturalistic position within philosophy of biology, against the background of an alternative view, founded on the basic insights of transcendental philosophy. It is argued that the apparently minimal and neutral constraints naturalism imposes on philosophy of science turn out to involve a quite heavily constraining metaphysics, due to the naturalism's fundamental neglect of its own perspective. Because of its intrinsic sensitivity to perspectivity and historicity, transcendental philosophy can avoid this type of hidden metaphysics.     

Molecular dissection of the interactions of an antitumor interleukin‐2‐derived mutein on a phage display‐based platform

A mutein with stronger antitumor activity and lower toxicity than wild‐type human interleukin‐2 (IL‐2) has been recently described. The rationale behind its design was to reinforce the immunostimulatory potential through the introduction of four mutations that would selectively disrupt the interaction with the IL‐2 receptor alpha chain (thought to be critical for both IL‐2‐driven expansion of T regulatory cells and IL‐2‐mediated toxic effects). Despite the successful results of the mutein in several tumor models, characterization of its interactions was still to be performed. The current work, based on phage display of IL‐2‐derived variants, showed the individual contribution of each mutation to the impairment of alpha chain binding. A more sensitive assay, based on the ability of phage‐displayed IL‐2 variants to induce proliferation of the IL‐2‐dependent CTLL‐2 cell line, revealed differences between the mutated variants. The results validated the mutein design, highlighting the importance of the combined effects of the four mutations. The developed phage display‐based platform is robust and sensitive, allows a fast comparative evaluation of multiple variants, and could be broadly used to engineer IL‐2 and related cytokines, accelerating the development of cytokine‐derived therapeutics. Copyright © 2015 John Wiley & Sons, Ltd.     

Glial calcium signaling and neuron-glia communication

The existence of bidirectional signaling between astrocytes and neurons has revealed an important active role of astrocytes in the physiology of the nervous system. As a consequence, there is a new concept of the synaptic physiology-"the tripartite synapse", where astrocytes exchange information with the pre- and postsynaptic elements and participate as dynamic regulatory elements in neurotransmission. The control of the Ca2+ excitability in astrocytes is a key element in this loop of information exchange. The ability of astrocytes to respond to neuronal activity and discriminate between the activity of different synapses, the modulation of the astrocytic cellular excitability by the synaptic activity, and the expression of cellular intrinsic properties indicate that astrocytes are endowed with cellular computational characteristics that process synaptic information. Therefore, we propose that astrocytes can be considered as cellular elements involved in the information processing by the nervous system.     

Efficient construction of a highly useful phage-displayed human antibody repertoire

We have constructed a highly useful phage-displayed human antibody repertoire with limited cloning efforts. Our strategy was to maximize diversity during the first steps of library construction through the use of various lymphoid sources from several donors, inclusion of different immunoglobulin isotypes, and performance of multiple separate amplification reactions with all possible combinations within a complex primer set. The resulting variable region collections were cloned to form a moderate size library, composed by 4.25x10(8) single chain antibody fragments. This repertoire was successfully used to retrieve binders to seven model antigens: six proteins and one 12 aa peptide. Binding affinities reached nanomolar and even subnanomolar range. Sequence diversity and V-gene usage variability among binders were proven. Our approach was not focused on absolute library size, but on a high quality sampling of variable regions from the human antibody repertoire.