Sources of nitrate in rivers draining sixteen watersheds in the northeastern U.S.: Isotopic constraints

The feasibility of using nitrogen and oxygenisotope ratios of nitrate (NO₃ ^–) forelucidating sources and transformations ofriverine nitrate was evaluated in a comparativestudy of 16 watersheds in the northeastern U.S.A. Stream water was sampled repeatedly at theoutlets of the watersheds between January andDecember 1999 for determining concentrations,¹⁵N values, and ¹⁸Ovalues of riverine nitrate.In conjunction with information about land useand nitrogen fluxes,¹⁵N_nitrate and¹⁸O_nitrate values providedmainly information about sources of riverinenitrate. In predominantly forested watersheds,riverine nitrate had mean concentrations ofless than 0.4 mg NO₃ ^–-N L^–1,¹⁵N_nitrate values of lessthan +5, and ¹⁸O_nitratevalues between +12 and +19. This indicatesthat riverine nitrate was almost exclusivelyderived from soil nitrification processes withpotentially minor nitrate contributions fromatmospheric deposition in some catchments. Inwatersheds with significant agricultural andurban land use, concentrations of riverinenitrate were as high as 2.6 mg NO₃ ^–-NL^–1 with ¹⁵N_nitratevalues between +5 and +8 and¹⁸O_nitrate values generallybelow +15. Correlations between nitrateconcentrations, ¹⁵N_nitratevalues, and N fluxes suggest that nitrate inwaste water constituted a major, and nitrate inmanure a minor additional source of riverinenitrate. Atmospheric nitrate deposition ornitrate-containing fertilizers were not asignificant source of riverine nitrate inwatersheds with significant agricultural andurban land use. Although complementary studiesindicate that in-stream denitrification wassignificant in all rivers, the isotopiccomposition of riverine nitrate sampled at theoutlet of the 16 watersheds did not provideevidence for denitrification in the form ofelevated ¹⁵N_nitrate and¹⁸O_nitrate values. Relativelylow isotopic enrichment factors for nitrogenand oxygen during in-stream denitrification andcontinuous admixture of nitrate from theabove-described sources are thought to beresponsible for this finding.     

Subcellular Localization of "Peripheral-Type" Binding Sites for Benzodiazepines in Rat Brain

The binding of [3H]Ro 5-4864, a specific ligand for "peripheral-type" benzodiazepine binding sites and [3H]Ro 15-1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of "peripheral-type" benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1 and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding.     

Assessment of genetic variability for N2 fixation between and within provenances of Leucaena leucocephala and Acacia albida estimated by 15N labelling techniques

Nitrogen fixed in 13 provenances of Acacia albida and 11 isolines of Leucaena leucocephala inoculated with effective Rhizobium strains was measured by ¹⁵N techniques and the total N difference method. In the test soil, on the average, L. leucocephala derived about 65% of its total N from atmospheric N₂ fixation compared to about 20% by A. albida. Significant differences in the percentage of N derived from atmospheric N₂ (% Ndfa) occurred, between provenances or isolines within species. The % Ndfa ranged from 37 to 74% within L. leucocephala and from 6 to 37 within A. albida; (equivalent to 20–50 mg N plant^–1 and 4–37 mg N plant^–1 for the two species over three months, respectively) and was correlated with the nodule mass (r=0.91). The time course of N₂ fixation of three selected provenances (low, intermediate and good fixers) was followed at 12 weekly intervals over a 36 week period. The % Ndfa of all provenances and isolines increased with time; and except for one of the L. leucocephala provenances, % Ndfa was similar within species at the 36 weeks harvest. There was a significant correlation between % Ndfa and the amount of N₂ fixed (r=0.96). Significant interactions occurred between provenances and N treatments and often growth of uninoculated but N fertilized plants was less variable than for inoculated unfertilized plants.     

Substrate specificity of a recombinant chicken <Emphasis Type="Italic">β</Emphasis>-carotene 15,15′-monooxygenase that converts <Emphasis Type="Italic">β</Emphasis>-carotene into retinal

The recombinant β-carotene 15,15′-monooxygenase from chicken liver was purified as a single 60 kDa band by His-Trap HP and Resource Q chromatography. It had a molecular mass of 240 kDa by gel filtration indicating the native form to be tetramer. The enzyme converted β-carotene under maximal conditions (pH 8.0 and 37°C) with a k _cat of 1.65 min⁻¹ and a K _m of 26 μM and its conversion yield of β-carotene to retinal was 120% (mol mol⁻¹). The enzyme displayed catalytic efficiency and conversion yield for β-carotene, β-cryptoxanthin, β-apo-8′-carotenal, β-apo-4′-carotenal, α-carotene and γ-carotene in decreasing order but not for zeaxanthin, lutein, β-apo-12′-carotenal and lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₀ seems to be essential for enzyme activity.     

Crankshaft motions of the polypeptide backbone in molecular dynamics simulations of human type-α transforming growth factor

Summary Order parameters for the backbone N–H and C–H bond vectors have been calculated from a 150 ps molecular dynamics (MD) simulation of human type- transforming growth factor in H₂O solvent. Two kinds of crankshaft motions of the polypeptide backbone are observed in this MD trajectory. The first involves small-amplitude rocking of the rigid peptide bond due to correlated changes in the backbone dihedral angles _i–1 and _i. These high-frequency librational crankshaft motions are correlated with systematically smaller values of motional order parameters for backbone N–H bond vectors compared to C–H bond vectors. In addition, infrequent crankshaft flips of the peptide bond from one local minimum to another are observed for several amino acid residues. These MD simulations demonstrate that comparisons of N–H and C–H order parameters provide a useful approach for identifying crank-shaft librational motions in proteins.     

Utilization of organic nitrogen at two different substrate pH by different ectomycorrhizal fungi growing in symbiosis with Pinus sylvestris seedlings

The aim of this study was to test the potential of four isolates of ectomycorrhizal (EM) fungi to utilize organic nitrogen (N) at two different substrate pHs. The organic N source (¹⁵N labelled lyophilised fungal mycelium) was mixed with either untreated peat/sand mixture (pH 4.9) or peat/sand mixture limed to a pH of 5.9 and put in cylindrical containers added to each pot. The content of the containers was separated from the roots of Pinus sylvestris seedlings by a nylon mesh and a 2 mm air gap to reduce diffusion of labelled N to the roots. The mycorrhizal plants (except those colonized by Suillus variegatus 2) took up significantly more ¹⁵N from the labelled mycelium than uncolonized seedlings. Liming significantly reduced the uptake of ¹⁵N by one of the EM fungi (unidentified) but not the other tested species (Paxillus involutus and two isolates of S. variegatus). The EM fungal isolates differed in their influence on the bacterial activity of the soil. This was reduced with P. involutus at both pH levels and increased with one of the two S. variegatus isolates at the high pH and with the other S. variegatus isolate at the low pH level. Liming the soil generally increased bacterial activity. The influence of liming on the proportion of organic N uptake in relation to inorganic N uptake by ectomycorrhizal trees is discussed.     

The eps15 homology (EH) domain-based interaction between eps15 and hrb connects the molecular machinery of endocytosis to that of nucleocytosolic transport

The Eps15 homology (EH) module is a protein-protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15-Hrb complex in regulating the stability of Rev.     

Nitrogen losses in puddled soils as affected by timing of water deficit and nitrogen fertilization

Erratic rainfall in rainfed lowlands and inadequate water supply in irrigated lowlands can results in alternate soil drying and flooding during a rice (Oryza sativa L.) cropping period. Effects of alternate soil drying and flooding on N loss by nitrification-denitrification have been inconsistent in previous field research. To determine the effects of water deficit and urea timing on soil NO₃ and NH₄, floodwater NO₃, and N loss from added ¹⁵N-labeled urea, a field experiment was conducted for 2 yr on an Andaqueptic Haplaquoll in the Philippines. Water regimes were continuously flooded, not irrigated from 15 to 35 d after transplanting (DT), or not irrigated from 41 to 63 DT. The nitrogen treatments in factorial combination with water regimes were no applied N and 80 kg urea-N ha^–1, either applied half basally and half at 37 DT or half at 11 DT and half at 65 DT. Water deficit at 15 to 35 DT and 41 to 63 DT, compared with continuous soil flooding, significantly reduced extractable NH₄ in the top 30-cm soil layer and resulted in significant but small (<1.0 kg N ha^–1) soil NO₃ accumulations. Soil NO₃, which accumulated during the water deficit, rapidly disappeared after reflooding. Water deficit at 15 to 35 DT, unlike that at 41 to 63 DT, increased the gaseous loss of added urea N as determined from unrecovered ¹⁵N in ¹⁵N balances. The results indicate that application of urea to young rice in saturated or flooded soil results in large, rapid losses of N (mean = 35% of applied N), presumably by NH₃ volatilization. Subsequent soil drying and flooding during the vegetative growth phase can result in additional N loss (mean = 14% of applied N), presumably by nitrification-denitrification. This additional N loss due to soil drying and flooding decreases with increasing crop age, apparently because of increased competition by rice with soil microorganisms for NH₄ and NO₃.     

Serotonin Stimulates Both Cytosolic and Membrane-Bound Guanylate Cyclase in NG108–15 Cells

The cyclic GMP (cGMP) content was rapidly (greater than 30 s) increased by serotonin [5-hydroxytryptamine (5-HT)] (EC50 = 10 microM), and the increase lasted for greater than 10 min in NG108-15 cells. The 5-HT-induced elevation of cGMP level (EC50 = 10 microM) at 20 s ("fast" elevation) was inhibited by ICS 205-930 or MDL 72,222 and by Ca2+ deficiency in the reaction medium but not by organic Ca2+ antagonists. The 5-HT effect at 10 min ("slow" elevation) was not inhibited by several antagonists for 5-HT receptors of the 1A, 1B, 1C, 1D, 2, and 3 subtypes and was independent from external Ca2+ concentration. The fast and slow effects of 5-HT were similar to the effects of bradykinin and atrial natriuretic peptide (ANP), respectively, in aspects of both Ca2+ dependency and time course of the effects. Bradykinin transiently stimulated formation of inositol phosphates as well as accumulation of cGMP, a finding suggesting that intracellular Ca2+ is involved in bradykinin-induced cGMP accumulation as shown in the fast response to 5-HT. ANP, an activator of membrane-associated guanylate cyclase (mGC), slowly (approximately 60 s) increased the cGMP content (EC50 = 10 nM), a result lasting for greater than 10 min, and the effects were independent from external Ca2+, as shown in the slow response to 5-HT. 5-HT and ANP did not induce formation of inositol phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystalline 2-Ketogluconate Reductase from Acetobacter ascendens,the Second Instance of Crystalline Enzyme in Genus Acetobacter

Crystalline 2-ketogluconate reductase in genus Acetobacter was prepared from cell free extract of Acetobacter ascendens. Crystalline enzyme was purified 13,000-fold with a yield of 15%. Affinity chromatography on blue-dextran Sepharose 4B column successfully purified the enzyme. The enzyme was composed of three identical subunits with a molecular weight of 40,000. Substrate specificity of 2-ketogluconate reductase from two genera of acetic acid bacteria was compared using highly purified enzyme preparations, and it was confirmed that gluconate oxidation activity of the enzyme was intrinsically weak or absent in genus Acetobacter and intense in Gluconobacter. This fact must be a useful criterion for classification of acetic acid bacteria.