Yield and nitrogen uptake of rapessed (Brassica campestris L.) with ammonium and nitrate

Water culture, growth chamber, greenhouse and field experiments were conducted to compare the effect of NH₄−N and NO₃−N on yield and N uptake of rapeseed (Brassica campestris L.). In water culture, the yields of 28-day old rapeseed plants grown at 14 μg N ml⁻¹ were double with NO₃ compared to NH₄, but N uptake was little affected. There was no such effect when concentration was reduced to 3.5 or 7 μg N ml⁻¹. The yield and N uptake of 26-day old rapeseed grown on six soils (pH 4.6 to 6.5) in pots in a growth chamber were much greater with NO₃ than with NH₄, although N concentration was more in the NH₄- than the NO₃-grown plants. In a greenhouse experiment with rapeseed grown on 12 potted soils, the N uptake of applied N was greater with NO₃ than with NH₄ on all soils. Averages were 63% with NH₄ and 78% with NO₃. However, NH₄-fixation capacities of the soils were only weakly correlated with yield from the two sources of N (r=0.48) and the relation was similar with N uptake. In contrast to the behavior of water culture, growth chamber and greenhouse experiments, the 33 field experiments did not show consistent difference in seed yield with NH₄ and NO₃ applied at time of seeding. In nine field experiments where band application was used for Ca(NO₃)₂, (NH₄)₂ SO₄, NH₄ NO₃, yield tended to be greatest for (NH₄)₂SO₄. However, in 19 experiments on acid soils with and without lime, yields in most cases were similar with (NH₄)₂SO₄ and NH₄ NO₃. Nitrification inhibitors were added to spring banded NH₄-based fertilizers in five experiments, but the yields were not influenced. Scientific Paper No. 558, Lacombe Research Station, Agriculture Canada.     

Control of nitrification of fertilizer nitrogen: Effect of inhibitors,banding and nesting

Laboratory incubation and field experiments were conducted to evaluate thiourea, ATC (4-amino-1, 2, 4 triazole hydrochloride) and N-Serve 24 E (2-chloro-6-trichloromethyl-pyridine) as inhibitors of nitrification of fertilizer N. In the incubation experiment, most of the added aqueous NH₃ or urea was nitrified at 14 days on both soils, but addition of the inhibitors to fertilizer N decreased the conversion of NH₄−N to NO₃−N markedly. There was less nitrification for ATC and thiourea but not for N-Serve 24 E when the fertilizers and the inhibitors were placed at a point as opposed to when mixed into soil. After 28 days, ATC and N-Serve 24 E were more effective in inhibiting nitrification than thiourea. ATC and N-Serve 24 E also inhibited release of mineral N (NH₄−N+NO₃−N) from native soil N. In the uncropped field experiment, which received N fertilizers in the fall, nitrification of fall-applied N placed in the 15-cm bands was almost complete by early May in the Malmo soil, but not in the Breton soil. When ATC or thiourea had been applied with urea, nitrification of fall-applied N was depressed by May and the recovery of applied N as NH₄−N was greater with increasing band spacing to 60 cm or placing N fertilizer in nests (a method of application where urea prills were placed at a point in the soil in the center of 60×60 cm area). In late June, the percentage recovery of fall-applied N in soil as NH₄−N or mineral N increased with wide band spacing, or nest placement, or by adding ATC to fertilizer N on both soils. These results indicate that placing ammonium-based N fertilizers in widely-spaced bands or in nests with low rates of inhibitors slows nitrification enough to prevent much of the losses from fall-applied N. Scientific Paper No. 552, Lacombe Research Station, Research Branch, Agric, Can.     

Proteinase inhibitors I and II in fruit of wild tomato species: Transient components of a mechanism for defense and seed dispersal

The juice of unripe fruit from a wild species of tomato, Lycopersicon peruvianum (L.) Mill., LA 107, contains over 50% of its soluble proteins as the sum of two proteinase inhibitors. These are the highest levels of proteinase inhibitors and highest percentage of soluble proteins as proteinase inhibitors of any plant or animal tissue found to date. Fruit of the modern tomato, L. esculentum Mill., contains only negligible quantities of the two inhibitors. The two proteinase inhibitors in the fruit of L. peruvianum are members of the Inhibitor I and II families previously found in potato tubers and in leaves of wounded potato and tomato plants. The levels of the two inhibitors in the unripe fruit decrease significantly during ripening. Unripe fruit from other wild Lycopersicon species such as L. parviflorum Rick, Kesicki, Fobes et Holle, L. hirsutum Humb. et Bonpe., L. pimpinellifolium Mill., and other lines of L. peruvianum contain moderate levels of the inhibitors that also decrease during ripening. Another wild tomato species, L. pennellii Corr., is similar to L. esculentum in not containing the two proteinase inhibitors in either unripe or ripe fruit. The transient levels of the inhibitors in fruit of wild species indicate that they are present in unripe fruit as defensive chemicals against insects, birds or small mammals and their disappearance during ripening may render them edible to facilitate seed dispersal. High levels of mRNAs coding for Inhibitors I and II in unripe fruit of L. peruvianum, LA 107, indicate that strong promoters may regulate the developmentally expressed proteinase-inhibitor genes in tomato fruit that may have a substantial potential for use in genetic-engineering experiments to enhance the production of large quantities of proteinase inhibitors or other proteins in field tomatoes.Abbreviations poly(A)⁺ mRNA polyadenylated mRNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresis Project 1791, College of Agriculture and Home Economics Research Center, Washington, State University     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

X-ray and model-building studies on the specificity of the active site of proteinase K

Proteinase K, the extracellular serine endopeptidase (E.C. 3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy-Ala-Phechloromethyl Ketone to the active site of proteinase K was the first determined from a Fourier synthesis based on synchrotron X-ray diffraction data between 1.8 Å and 5.0 Å resolution. The protein inhibitor complexes was refined by restrained least-squares minimization with the data between 10.0 and 1.8 Å. The final R factor was 19.1% and the model contained 2,018 protein atoms, 28 inhibitors atoms, 125 water molecules, and two Ca²⁺ ions. The peptides portion of the inhibitor is bound to the active center of proteinase K by means of a three-stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.     

Analysis of the biomacromolecular architecture of eukaryotic and prokaryotic serine proteases

Summary We have been developing computational approaches to increase our ability to analyze the growing body of three-dimensional structural data with applications centered on the serine proteases and their natural inhibitors and substrates. It is essential that these approaches emphasize the comparison of these macromolecules at the separate levels of secondary, tertiary and quaternary structure. We assume in our analysis that in functionally related macromolecules (i.e., a family of evolutionarily related enzymes), regions of structural and/or physicochemical similarity will exhibit functional similarity; regions that are different in structure and/or physicochemical properties will function differently and, therefore, be the source of observed specificity. It is the intent of our research to encapsulate such knowledge in a form which is capable of observing patterns which may serve as generalizable rules for macrostructural analysis (Liebman, M.N. 1986. Enzyme 36: 150–163), and to serve as the essential tools for the rational design of modified serine proteases and/or their natural inhibitors by the methods available through genetic engineering.     

Respiratory oxidation of reduced sulfur compounds by intact cells of Thiobacillus tepidarius (type strain)

Thiobacillus tepidarius (type strain) was grown in microaerophilic conditions, on tetrathionate, thiosulfate or crystalline S^o. The rates of tetrathionate, thiosulfate, elemental sulfur (S^o) and sulfite oxidation of the different cultures were measured respirometrically, using exponentially growing cells, with an oxygen electrode. Cells growing on the three different sulfur compounds retain thiosulfate-, tetrathionate, and S^o-oxidizing activities (SOA), but lack respiratory sulfite-oxidizing activity. The SOA for all the cultures was almost totally inhibited by 50 M myxothiazol, an inhibitor of the quinone-cytochrome b region, and by 10 M of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Tetrathionate- and thiosulfate-oxidizing activities were moderately and weakly inhibited by 50 M totally inhibited (>95%) all respiratory activities. This study suggests that electrons released by S^o oxidation enter the respiratory chain in the quinone-cytochrome b region.Abbreviation SOA sulfur-oxidizing activity     

Preferential inhibition of acetylcholinesterase molecular forms in rat brain

The effect of eight different acetylcholinesterase inhibitors (AChEIs) on the activity of acetylcholinesterase (AChE) molecular forms was investigated. Aqueous-soluble and detergent-soluble AChE molecular forms were separated from rat brain homogenate by sucrose density sedimentation. The bulk of soluble AChE corresponds to globular tetrameric (G₄), and monomeric (G₁) forms. Heptylphysostigmine (HEP) and diisopropylfluorophosphate were more selective for the G₁ than for the G₄ form in aqueous-soluble extract. Neostigmine showed slightly more selectivity for the G₁ form both in aqueous- and detergent-soluble extracts. Other drugs such as physostigmine, echothiophate, BW284C51, tetrahydroaminoacridine, and metrifonate inhibited both aqueous- and detergent-soluble AChE molecular forms with similar potency. Inhibition of aqueous-soluble AChE by HEP was highly competitive with Triton X-100 in a gradient, indicating that HEP may bind to a detergent-sensitive non-catalytic site of AChE. These results suggest a differential sensitivity among AChE molecular forms to inhibition by drugs through an allosteric mechanism. The application of these properties in developing AChEIs for treatment of Alzheimer disease is considered.Special issue dedicated to Dr. Morris H. Aprison.     

The role of ethylene and reducing agents on anther culture response of tetraploid potato (Solanum tuberosum L.)

Summary The role of ethylene in embryogenesis of cultured potato anthers was studied indirectly by testing various substances known to affect ethylene formation. The reducing agents ascorbic acid and L-cysteine prevented browning of anther cultures and significantly stimulated embryogenesis. Embryogenesis was also promoted by the use of the ethylene inhibitors AgNO₃ and n-propyl-gallate and by the polyamines spermidine and putrescine. The use of the ethylene releasing compound ethrel significantly inhibited embryogenesis.Abbreviations MS Murashige & Skoog - PVP polyvinylpyrrolidone - MW molecular weight - ACC 1-aminocyclopropane-1-carboxylic acid - ethrel 2-chloroethylphosphonic acid (ethephon)     

Michaelis complexes of porcine pancreatic elastase with 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins: translational sampling of inhibitor position and kinetic measurements.

A step leading to the formation of the covalent complexes between porcine pancreatic elastase (PPE) and 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins (alkylHNCO-EICs) is the formation of the noncovalent Michaelis complex. No average structures are available for the Michaelis complexes of PPE with alkylHNCO-EICs. We present the results of an initial step in obtaining these structures and have determined kinetic constants as well. The kinetic results indicate that formation of the Michaelis complex is what differentiates the effectiveness of these inhibitors in inactivating PPE. The structural and kinetic results together suggest that the structure of the Michaelis complex is necessary for the design of potent alkylHNCO-EIC inhibitors of PPE. Two novel alkylHNCO-EICs are predicted to be the best inhibitors of this series. An alternate mechanism for serine protease inhibition is also proposed. Evidence for, and studies that may add support to, the hypothesized mechanism are discussed.