Transfer RNA methylase activity in normal and dystrophic chicken muscle

Adult-dystrophic chicken muscle had 30% higher tRNA methylase activity and 42% higher tRNA methylating capacity than normal-adult chicken muscle. Eighty percent of the tRNA methylase activity of the dystrophic muscle resulted in the synthesis of N²-methylguanine, and 9% in the formation of N²,N²-dimethylguanine. From adult-normal muscle extracts, 33% of the tRNA methylase activity was due to the synthesis of N²-methylguanine, and 45% to the formation of N²,N²-dimethylguanine. Eight other methylated bases accounted for 5–15% of the enzyme activity in both tissues. Dialyzed and nondialyzed adult-normal muscle extracts had equivalent tRNA methylase activity. However, the dialyzed extracts synthesized 22% more N²-methylguanine and 18% less N²,N²-dimethylguanine than the nondialyzed extracts. Dialysis had no effect on the tRNA methylase activity or tRNA methylation pattern produced by adult-dystrophic muscle.     

The developmental pattern of homologous and heterologous tRNA methylation in rat brain differential effect of spermidine

UsingS-adenosyl-L-[Me-¹⁴C] methionine, rat cerebral cortex methyltransferase activity was determined during the early postnatal period in the absence of addedEscherichia coli tRNA and in its presence. [Me-¹⁴C] tRNA was purified from both systems and its [Me-¹⁴C] base composition determined. The endogenous formation of [Me-¹⁴C] tRNA (homologous tRNA methylation) was totally abolished in the presence of 2.5 mM spermidine, whereasE. coli B tRNA methylation (heterologous methylation) was markedly stimulated. Only [Me-¹⁴C] 1-methyl guanine and [Me-¹⁴C]N ²-methyl guanine were formed by homologous methylation, there being an inverse shift in their relative proportions with age. Heterologous tRNA methylation led, additionally, to the formation of [Me-¹⁴C]N ₂ ² -dimethyl guanine, 5-methyl cytosine, 1-methyl adenine, 5-methyl uracil, 2-methyl adenine, and 1-methyl hypoxanthine. A comparison of heterologous tRNA methylation between the whole brain cortex (containing nerve and glial cells) and bulk-isolated nerve cell bodies revealed markedly lower proportions of [Me-¹⁴C]N ₂-methyl andN ₂ ² -dimethyl guanine and significantly higher proportions of [Me-¹⁴C] 1-methyl adenine in the neurons. The present findings suggest (1) that homologous tRNA methylation may provide developing brain cells with continuously changing populations of tRNA and (2) that neurons are enriched in adenine residue-specific tRNA methyltransferases that are highly sensitive to spermidine.This research was supported by grant NS-06294 of the United States Public Health Service.     

A high-yield route to synthesize the P-glycoprotein radioligand [11C]N-desmethyl-loperamide and its parent radioligand [11C]loperamide

N-Desmethyl-loperamide and loperamide were synthesized from α,α-diphenyl-γ-butyrolactone and 4-(4-chlorophenyl)-4-hydroxypiperidine in five and four steps with 8% and 16% overall yield, respectively. The amide precursor was synthesized from 4-bromo-2,2-diphenylbutyronitrile and 4-(4-chlorophenyl)-4-hydroxypiperidine in 2 steps with 21–57% overall yield. [¹¹C]N-Desmethyl-loperamide and [¹¹C]loperamide were prepared from their corresponding amide precursor and N-desmethyl-loperamide with [¹¹C]CH₃OTf through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 20–30% and 10–15% radiochemical yields, respectively, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB), with 370–740 GBq/μmol specific activity at EOB.     

Emission of nitrous oxide (N2O) from transgenic tobacco expressing antisense NiR mRNA

The emission of N₂ and N₂O from intact transgenic tobacco (clone 271) expressing antisense nitrite reductase (NiR) mRNA, and wild-type plants grown aseptically, on NO₃^–, NO₂^– or NH₄⁺ -containing medium was investigated. ¹⁵N contents of gas sampled from gas-sealed pots, in which the plants were grown on ¹⁵N-containing medium, were analyzed by gas chromato- graphy and mass spectrometry (GC–MS). No emission of N₂ was detected in either of the gas samples from plant clone 271 or the wild-type grown on NO₃^–-containing medium. N₂O emission from clone 271 grown on NO₃^–-containing medium was detected, but not from the wild-type plants. The N₂O emission rate of clone 271 was 106 ng N₂O mg^–1 incorporated N week^–1 and the N₂O emission was inhibited by tungstate (a nitrate reductase inhibitor). No emission of N₂O was found from clone 271 or wild-type plants grown on medium containing NH₄⁺. Emission of N₂O also was detected from clone 271 grown on NO₂^–-containing medium and its emission rate increased with increasing NO₂^– levels in plants. We speculate that NO₃^– is reduced to NO₂^– and that a part of NO₂^– is metabolized to N₂O in clone 271.     

Dynamics of nitrous oxide in groundwater at the aquatic–terrestrial interface

Few data are available to validate the Intergovernmental Panel on Climate Change's (IPCC) emission factors for indirect emissions of nitrous oxide (N₂O). In particular the N₂O emissions resulting from nitrogen leaching and the associated groundwater and surface drainage (EF5-g) are particularly poorly characterized. In situ push–pull methods have been used to identify the fate of NO₃⁻ in the groundwater. In this study, we adapted a previously published in situ denitrification push–pull method to examine the fate of ¹⁵N₂O introduced into the subsoil–groundwater matrix. Enriched ¹⁵N₂O was manufactured, added to groundwater via a closed system in the laboratory, and then introduced into the groundwater–subsoil matrix in an upland-marsh transition zone of a salt marsh and a forested alluvial riparian zone. Conservative tracers (SF₆ and Br⁻) and ¹⁵N₂O were injected into the groundwater and left for 1–4 h after which the groundwater was sampled. Added ¹⁵N₂O behaved in a conservative manner at one site while the other site showed variability with some injections showing significant consumption (3–8 μg N₂O-¹⁵N kg⁻¹ soil day⁻¹) of ¹⁵N₂O. Our results show that the fate and dynamics of N₂O in groundwater are complex and variable and that these dynamics should be considered in the development of improved IPCC inventory calculations.     

The importance of denitrification for N2O emissions from an N-saturated forest in SW China: results from in situ 15N labeling experiments

Long-term elevated atmogenic deposition (~5 g m^?2 year^?1) of reactive nitrogen (N) causes N saturation in forests of subtropical China which may lead to high nitrous oxide (N₂O) emissions. Recently, we found high N₂O emission rates (up to 1,730 μg N₂O–N m^?2 h^?1) during summer on well-drained acidic acrisols (pH = 4.0) along a hill slope in the forested Tieshanping catchment, Chongqing, southwest China. Here, we present results from an in situ ¹⁵N–NO₃ ^? labeling experiment to assess the contribution of nitrification and denitrification to N₂O emissions in these soils. Two loads of 99 at.% K¹⁵NO₃ (equivalent to 0.2 and 1.0 g N m^?2) were applied as a single dose to replicated plots at two positions along the hill slope (at top and bottom, respectively) during monsoonal summer. During a 6-day period after label application, we found that 71–100 % of the emitted N₂O was derived from the labeled NO₃ ^? pool irrespective of slope position. Based on this, we assume that denitrification is the dominant process of N₂O formation in these forest soils. Within 6 days after label addition, the fraction of the added ¹⁵N–NO₃ ^? emitted as ¹⁵N–N₂O was highest at the low-N addition plots (0.2 g N m^?2), amounting to 1.3 % at the top position of the hill slope and to 3.2 % at the bottom position, respectively. Our data illustrate the large potential of acid forest soils in subtropical China to form N₂O from excess NO₃ ^? most likely through denitrification.     

Rapid Conversion of Added Nitrate to Nitrous Oxide and Dinitrogen in Northern Forest Soil

The aims of this study were to simulate wet deposition of atmospheric nitrate (NO₃^?) onto forest soils and trace its fate via conversion spatially and temporally into gaseous products nitrous oxide (N₂O) and dinitrogen (N₂). The most likely mechanism is microbial denitrification, but an intermediate product nitrite (NO₂^?) can fuel N₂O production via a chemical pathway involving reactions with iron and/or organic matter referred to as chemodenitrification. During summer months, we applied tracer amounts of ¹⁵N-labeled NO₃^? onto forest soils (pH ～ 4) at three sites in the White Mountain Region of New Hampshire, USA. We recovered ¹⁵N as N₂O in 210 of 504 measurements (42%) versus ¹⁵N as N₂ in 51 of 504 measurements (10%), suggesting partial microbial denitrification and/or chemodenitrification. When recovery occurred, the mean percent recovery of added ¹⁵N was just 1.1% as N₂O, although N₂ recovery was 33%. A site with old-growth trees had a larger percentage recovery as N₂ (48%), whereas a site that had burned 100 years ago had a small percentage recovery as N₂O (0.24%). The ¹⁵N composition of N₂O in ambient air, collected before addition of the label, was markedly enriched in ¹⁵N. Since flux measurements were made 2 h after the addition, the results suggest that denitrification enzymes and conditions for chemodenitrification are present throughout the summer months but account for small amounts of NO₃^? conversion into N₂O and N₂.     

Waterlogging and fire impacts on nitrogen availability and utilization in a subtropical wet heathland (wallum)

Protein, amino acids and ammonium were the main forms of soluble soil nitrogen in the soil solution of a subtropical heathland (wallum). After fire, soil ammonium and nitrate increased 90- and 60-fold, respectively. Despite this increase in nitrate availability after fire, wallum species exhibited uniformly low nitrate reductase activities and low leaf and xylem nitrate. During waterlogging soil amino acids increased, particularly γ-aminobutyric acid (GABA) which accounted for over 50% of amino nitrogen. Non-mycorrhizal wallum species were significantly (P < 0.05) ¹⁵N-enriched (0.3–4.3‰) compared to species with mycorrhizal associations (ericoid-type, ecto-, va-mycorrhizal) which were strongly depleted in ¹⁵N (-6.3 to -1.8‰). Lignotubers and roots had δ¹⁵N signatures similar to that of the leaves of respective species. The exceptions were fine roots of ecto-, ecto/va-, and ericoid type mycorrhizal species which were enriched in ¹⁵N (0.1–2.4‰). The 5¹⁵N signatures of δ¹⁵N_{total soil N} and δ¹⁵N_{soil NH4+} were in the range 3.7–4.5‰, whereas δ¹⁵N_{soil NO3?} was significantly (P < 0.05) more enriched in ¹⁵N (9.2–9.8‰). It is proposed that there is discrimination against ¹⁵N during transfer of nitrogen from fungal to plant partner. Roots of selected species incorporated nitrogen sources in the order of preference: ammonium > glycine > nitrate. The exception were proteoid roots of Hakea (Proteaceae) which incorporated equal amounts of glycine and ammonium.     

New Method of Denitrification Analysis of Bradyrhizobium Field Isolates by Gas Chromatographic Determination of 15N-Labeled N2

To evaluate the denitrification abilities of many Bradyrhizobium field isolates, we developed a new ¹⁵N-labeled N₂ detection methodology, which is free from interference from atmospheric N₂ contamination. ³⁰N₂ (¹⁵N¹⁵N) and ²⁹N₂ (¹⁵N¹⁴N) were detected as an apparent peak by a gas chromatograph equipped with a thermal conductivity detector with N₂ gas having natural abundance of ¹⁵N (0.366 atom%) as a carrier gas. The detection limit was 0.04% ³⁰N₂, and the linearity extended at least to 40% ³⁰N₂. When Bradyrhizobium japonicum USDA110 was grown in cultures anaerobically with ¹⁵NO₃⁻, denitrification product (³⁰N₂) was detected stoichiometrically. A total of 65 isolates of soybean bradyrhizobia from two field sites in Japan were assayed by this method. The denitrification abilities were partly correlated with filed sites, Bradyrhizobium species, and the hup genotype.     

An in-depth look into a tropical lowland forest soil: nitrogen-addition effects on the contents of N2O, CO2 and CH4 and N2O isotopic signatures down to 2-m depth

Atmospheric nitrogen (N) deposition is rapidly increasing in tropical regions. We investigated how a decade of experimental N addition (125 kg N ha^?1 year^?1) to a seasonal lowland forest affected depth distribution and contents of soil nitrous oxide (N₂O), carbon dioxide (CO₂) and methane (CH₄), as well as natural abundance isotopic signatures of N₂O, nitrate (NO₃ ^?) and ammonium (NH₄ ⁺). In the control plots during dry season, we deduced limited N₂O production by denitrification in the topsoil (0.05–0.40 m) as indicated by: ambient N₂O concentrations and ambient ¹⁵N-N₂O signatures, low water-filled pore space (35–60%), and similar ¹⁵N signatures of N₂O and NO₃ ^?. In the subsoil (0.40–2.00 m), we detected evidence of N₂O reduction to N₂ during upward diffusion, indicating denitrification activity. During wet season, we found that N₂O at 0.05–2.00 m was mainly produced by denitrification with substantial further reduction to N₂, as indicated by: lighter ¹⁵N-N₂O than ¹⁵N-NO₃ ^? throughout the profile, and increasing N₂O concentrations with simultaneously decreasing ¹⁵N-N₂O enrichment with depth. These interpretations were supported by an isotopomer map and by a positive correlation between ¹⁸O-N₂O and ¹⁵N-N₂O site preferences. Long-term N addition did not affect dry-season soil N₂O-N contents, doubled wet-season soil N₂O-N contents, did not affect ¹⁵N signatures of NO₃ ^?, and reduced wet-season ¹⁵N signatures of N₂O compared to the control plots. These suggest that the increased NO₃ ^? concentrations have stimulated N₂O production and decreased N₂O-to-N₂ reduction. Soil CO₂-C contents did not differ between treatments, implying that N addition essentially did not influence soil C cycling. The pronounced seasonality in soil respiration was largely attributable to enhanced topsoil respiration as indicated by a wet-season increase in the topsoil CO₂-C contents. The N-addition plots showed reduced dry-season soil CH₄-C contents and threshold CH₄ concentrations were reached at a shallower depth compared to the control plots, revealing an N-induced stimulation of methanotrophic activity. However, the net soil CH₄ uptake rates remained similar between treatments possibly because diffusive CH₄ supply from the atmosphere largely limited CH₄ oxidation.     

Nitrogen fixation by groundnut and soyabean and residual nitrogen benefits to rice in farmers' fields in Northeast Thailand

Nitrogen fixation in groundnut and soyabean and the residual benefits of incoporated legume stover to subsequent rice crops were estimated in farmers' fields using¹⁵N-isotope methods. Three field experiments were conducted, two which examined N₂-fixation in groundnut by¹⁵N-isotope dilution using a non-nodulating groundnut as a reference crop and one in which N₂-fixation in two soyabean genotypes was compared using maize as the non-fixing reference crop. Groundnut fixed 72–77% of its N amounting to 150–200 kg N ha^-1 in 106–119 days and soyabean derived 66–68% of its N from N₂-fixation which amounted to 108–152 kg N ha^-1 under similar conditions. When legume stover was returned to the soil, there was a net contribution of N from N₂-fixing varieties of groundnut in all cases ranging from 13–100 kg N ha^-1, whilst due to the high % N harvest index in soyabean (87–88%) there was a net removal of N of 37–46 kg N ha^-1. In all cases if the legume stover was removed there was a net removal of N in the legume crop which ranged between 54 and 74 kg N ha^-1 in N₂-fixing varieties of groundnut and from 58 to 73 kg N ha^-1 in soyabean, whilst maize removed 66 kg N ha^-1 if its stover was returned and 101 kg N ha^-1 when the stover was removed. Growth of rice was improved in all cases where groundnut stover was returned resulting in increases in grain yield of 12–26% and increases in total dry matter production of 26–31%. Soyabean residues gave no increases in rice grain yield but increased total dry matter production by 12–20%. Rice accumulated more N in all cases where legume stover was returned to the soil, and N yields were larger in all cases after the N₂-fixing legumes than after the non-fixing reference crops. N difference estimates of the total residual N benefits from the N₂-fixing legumes ranged from 11–19 kg N ha^-1 after groundnut and 15–16 kg N ha^-1 after soyabean. The amounts of N estimated directly by application of¹⁵N-labelled stover amounted to 7.2–20.5 kg N ha^-1 with groundnut which represented recovery of 8–22% of the N added in the stover. In soyabean only 3.0–5.8 kg N ha^-1 was estimated to be recovered by¹⁵N-labelling which was 15–23% of the added N, whilst only 1.3 kg N ha^-1 (4% of the N added) was recovered by rice from the maize stover. An indirect¹⁵N-method based on addition of unlabelled stover to microplots where the soil had previously been labelled with¹⁵N gave extremely variable and often negative estimates of residual N benefits. Estimates of residual N from the added stover made by N difference calculations did not correspond with the estimates by direct¹⁵N-labelling in all cases and possible reasons for this are discussed.     

NITRATE UPTAKE BY LAMINARIA LONGICRURIS (PHAEOPHYCEAE)1,2

Laminaria longicrucis De la Pylaie took up exogenous nitrate under both summer and winter conditions. During July and August no NO₃^- was detected in the ambient water or in algal tissues although it was present in both in February. Discs (2.3 cm diam.) of thin blade tissue were incubated with NO₃^- at four temperatures, with and without illumination. Similar values Jor NO₃^- uptake were found for both summer and winter collected plants when measured in light at 0 C. An apparent K of 4–6 μM was recorded for both types of plants; the V_max ranged from 7 to 10 μmol h^-1 g^-1 dry wt measured in ca. 1800 μW cm^-2 of cool-white fluorescent light. Uptake rates at 5 C were 66%, and at 0 C 30% of those for controls run at 15 C. The alga scavenged NO₃- from solutions <0.5 μM. Ammonia did not inhibit NO₃- uptake. Antibiotic pretreatment reduced NO₃- uptake by a maximum of 12%. Nitrite uptake was inhibited in proportion to the concentration of NO₃- in the medium.     

Methionine Sulfoximine In Vivo Modifies the tRNALys Pool of Developing Rat Brain

Abstract: tRNA was extracted from brains of 3-, 8-, and 18-day-old rats that were injected intracerebrally, 45 min before death, with [³H]methyl methionine or [8-³H]guanosine, and intraperitoneally, 3 h before death, with l -methionine-dl-sulfoximine (MSO), a methylation-activating convulsant agent. Although there was no effect of age or of MSO on the per gram yield of tRNA, its specific radioactivity (dpm/A₂₆₀) was highest at 3 days in both the control and the MSO groups. Age- and MSO-related changes in the tRNA^Lys content of the brain tRNA pool were investigated by means of benzoylated DEAE- cellulose (BDC) and reverse-phase chromatography (RPC). BDC chromatography revealed tRNA^Lys species in the brains of the MSO-treated animals that were absent in control brains. Of particular interest was the finding that differences in RPC-5 chromatographic mobility between control and MSO-tRNA^Lys species were abolished by conversion to lysyl-tRNA, suggesting that the MSO-elicited change(s) in tRNAL^Lys structure involved the binding site(s) for lysine. Two additional findings were made: (a) lysine acceptance by the [³H]methyl-labeled tRNA^Lys purified from brains of the MSO-treated animals was higher than that of controls at 18 days; and (b) omission of the BDC chromatographic step accentuated the differences in mobility on RPC-5 columns between tRNA^Lys species of control and MSO-treated brains. Lastly, we found that some tRNA^Lys species present in the MSO-treated brains contained significantly different proportions of N²-methyl guanine and 1-methyl adenine, relative to controls. These MSO-elicited changes in the methyl base content of tRNA^Lys of immature rat brain are the first evidence of an alteration of brain tRNA structure by a centrally acting excitatory agent.     

4-N-Alkanoyl and 4-N-alkyl gemcitabine analogues with NOTA chelators for 68-gallium labelling

The conjugation of 4-N-(3-aminopropanyl)-2′-deoxy-2′,2′-difluorocytidine with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) ligand in 0.1?M Na₂CO₃ buffer (pH 11) at ambient temperature provided 4-N-alkylgemcitabine-NOTA chelator. Incubation of latter with excess of gallium(III) chloride (GaCl₃) (0.6?N AcONa/H₂O, pH?=?9.3) over 15?min gave gallium 4-N-alkylgemcitabine-NOTA complex which was characterized by HRMS. Analogous [⁶⁸Ga]-complexation of 4-N-alkylgemcitabine-NOTA conjugate proceeded with high labeling efficiency (94%–96%) with the radioligand almost exclusively found in the aqueous layer (～95%). The high polarity of the gallium 4-N-alkylgemctiabine-NOTA complex resulted in rapid renal clearance of the ⁶⁸Ga-labelled radioligand in BALB/c mice.     

C-A base pairs in transfer ribonucleic acids and codon-anticodon complexes

Two hundred and thirty three nucleotide sequences of tRNAs were investigated to elucidate the frequency of appearance of C-A (cytosine-adenine) pairs in their main two-stranded regions, in the positions 26–44 and 15–48². It was supposed that in the formation of C-A pairs on antiparallel polynucleotide chains the atomic groups -N₄H and -N₃ of cytosine make up Hbonds with the groups N₇- and HN₆- of adenine. On parallel chains, Hbonds, probably, form -N₆H and -N₁ groups of adenine with N₃- and HN₄- of cytosine. The calculation results predicted a significant energy of interaction between cytosine and adenine. By the investigation of the molecular models it was shown that the formation of Hbonded C-A pairs requires considerable changes of conformation in ribose-phosphate chains. In addition, a theoretical analysis revealed the possibility of formation of C-A pairs at the wobble-position of the codon-anticodon complex. The significance of this nucleotide pair in the processes of genetic coding proved to depend on the stability of the codon-anticodon complex, the modification of cytosine 34 and structural features of the distant regions of the tRNA.     

Denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Rhodopseudomonas sphaeroides f. denitrificans grown photosynthetically with NO ₃ ^- under anaerobic conditions accumulated NO ₂ ^- in the culture medium. In washed cells succinate, lactate, fumarate, citrate and malate, were effective electron donors for the reduction of NO ₃ ^- , NO ₂ ^- and N₂O to N₂ gas. Nitrate reductase was inhibited by amytal and potassium thocyanate. Nitrite reductase activity was severely restricted by potassium cyanide, sodium diethyldithiocarbamate, Amytal and 2-n-heptyl-4-hydroxyquinoline-N-oxide whereas N₂O reductase was inhibited by NaN₃, C₂H₂ and KCNS. Cells incubated with either K¹⁵NO₃ or K¹⁵NO₂ produced ¹⁵N₂O and ¹⁵N₂. A stoichiometry of 2:1 was recorded for the reduction of either NO ₃ ^- or NO ₂ ^- to N₂O and N₂ and for N₂O to N₂ it was 1:1.Abbreviations BVH reduced benzyl viologen - MVH reduced methyl viologen - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - CCCP carbonyl cyanide-m-chlorophenyl-hydrazone - DIECA diethyl dithiocarbamate - KCN potassium cyanide     

Measurement of N2 fixation in 30 cowpea (Vigna unguiculata L. Walp.) genotypes under field conditions in Ghana, using the15N natural abundance technique

In 2005 and 2006, 30 and 15 cowpea genotypes were respectively evaluated for plant growth and symbiotic performance at Manga in Northern Ghana, in order to identify N₂-fixing potential of these cowpea genotypes as source of N for cropping systems. The results showed differences in biomass production by the 30 or 15 cowpea genotypes. In 2005, cultivars Fahari, Mchanganyiko, IT97K-499-39, IT93K-2045-29 and IT84S-2246 produced the most shoot biomass, while Apagbaala, Brown Eye, ITH98-46, Vita 7 and Iron Grey produced the least. Of the 15 genotypes tested in 2006, cv. TVu11424 produced the largest amount of biomass, and ITH98-46, the least. Isotopic analysis of¹⁵N in plant parts also revealed significant differences in δ¹⁵N of the cowpea genotypes studied. As a result, the percent N derived from fixation (% Ndfa) also differed among the cowpea genotypes tested in 2005, with only 5 out of the 30 cultivars obtaining over 50% of their N from symbiotic fixation. Whether expressed as mg N.plant^?1 or kg N.ha^?1, the levels of N₂ fixation by the cowpea genotypes varied considerably during 2005 and 2006, with values of N contribution ranging from 14.1 kg N.ha^?1 by cv. TVu1509 to 157.0 kg N.ha^?1 by IT84S-2246 in 2005. The amounts of N-fixed in 2006 ranged from 16.7 kg N.ha^?1 by cv. ITH98-46 to 171.2 kg N.ha^?1 by TVu11424, clearly indicating genotypic differences in symbiotic N yield. Re-evaluating 15 out of the 30 cowpea genotypes for N₂ fixation in 2006, revealed higher % Ndfa values (>50%) in all (15 cowpea genotypes) relative to those tested in 2005, indicating greater dependence on N₂ fixation for their N nutrition even though, the actual amounts of fixed-N were lower in 2006. This was due, in part, to reduced plant biomass as a result of very late sampling in 2006, close to physiological maturity (72 DAP in 2006 vs. 46 DAP in 2005) when considerable leaf matter was lost. The amount of N-fixed in 2006 can therefore be considered as being under-estimated.     

Incorporation of cytokinin N-benzyladenine into tobacco callus transfer ribonucleic Acid and ribosomal ribonucleic Acid preparations

The incorporation of the cytokinin N⁶-benzyladenine into tobacco (Nicotiana tabacum) callus tRNA and rRNA preparations isolated from tissue grown on medium containing either N⁶-benzyladenine-8-¹⁴C or N⁶-benzyladenine-8-¹⁴C: benzene-³H(G) has been examined. N⁶-benzyladenine was incorporated into both the tRNA and rRNA preparations as the intact base. Over 90% of the radioactive N⁶-benzyladenosine recovered from the RNA preparations was associated with the rRNA. Purification of the crude rRNA by either MAK chromatography or Sephadex G-200 gel filtration had no effect on the N⁶-benzyladenosine content of the RNA preparation. The distribution of N⁶-benzyladenosine moieties in tobacco callus tRNA fractionated by BD-cellulose chromatography did not correspond to the distribution of ribosylzeatin activity. N⁶-benzyladenosine was released from the rRNA preparation by treatment with venom phosphodiesterase and phosphatase, ribonuclease T₂ and phosphatase, or ribonuclease T₂ and a 3′-nucleotidase. N⁶-benzyladenosine was not released from the RNA preparation by treatment with either ribonuclease T₂ or phosphatase alone or by successive treatment with ribonuclease T₂ and a 5′-nucleotidase. Brief treatment of the rRNA preparation with ribonuclease T₁ and pancreatic ribonuclease converted the N⁶-benzyladenosine moieties into an ethyl alcohol soluble form. On the basis of these and earlier results, the N⁶-benzyladenosine recovered from the tobacco callus RNA preparations appears to be present as a constituent of RNA and not as a nonpolynucleotide contaminant.     

Deciphering the oxygen isotope composition of nitrous oxide produced by nitrification

The ability to use δ¹⁸O values of nitrous oxide (N₂O) to apportion environmental emissions is currently hindered by a poor understanding of the controls on δ¹⁸O–N₂O from nitrification (hydroxylamine oxidation to N₂O and nitrite reduction to N₂O). In this study fertilized agricultural soils and unfertilized temperate forest soils were aerobically incubated with different ¹⁸O/¹⁶O waters, and conceptual and mathematical models were developed to systematically explain the δ¹⁸O–N₂O formed by nitrification. Modeling exercises used a set of defined input parameters to emulate the measured soil δ¹⁸O–N₂O data (Monte Carlo approach). The Monte Carlo simulations implied that abiotic oxygen (O) exchange between nitrite (NO₂^?) and H₂O is important in all soils, but that biological, enzyme‐controlled O‐exchange does not occur during the reduction of NO₂^? to N₂O (nitrifier‐denitrification). Similarly, the results of the model simulations indicated that N₂O consumption is not characteristic of aerobic N₂O formation. The results of this study and a synthesis of the published literature data indicate that δ¹⁸O–N₂O formed in aerobic environments is constrained between +13‰ and +35‰ relative to Vienna Standard Mean Ocean Water (VSMOW). N₂O formed via hydroxylamine oxidation and nitrifier‐denitrification cannot be separated using δ¹⁸O unless ¹⁸O tracers are employed. The natural range of nitrifier δ¹⁸O–N₂O is discussed and explained in terms of our conceptual model, and the major and minor controls that define aerobically produced δ¹⁸O–N₂O are identified. Despite the highly complex nature of δ¹⁸O–N₂O produced by nitrification this δ¹⁸O range is narrow. As a result, in many situations δ¹⁸O values may be used in conjunction with δ¹⁵N–N₂O data to apportion nitrifier‐ and denitrifier‐derived N₂O. However, when biological O‐exchange during denitrification is high and N₂O consumption is low, there may be too much overlap in δ¹⁸O values to distinguish N₂O formed by these pathways.     

Differential Effects of the Alkylating Agent N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline on Brain α2-Adrenoceptors and I2-Imidazoline Sites In Vitro and In Vivo

Abstract— The alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is a peptide-coupling agent that is being used to inactivate irreversibly α₂-adrenoceptors and other receptors. The aim of the present study was to assess the in vitro and in vivo effects of EEDQ on the newly discovered brain l₂-imidazoline sites, located mainly in mitochondria. Preincubation of rat cortical membranes with EEDQ (10^?8-10^?5M) markedly decreased (20–90%) the specific binding of the selective antagonist [³H]R821002 to α₂-adrenoceptors without affecting that of [³H]idazoxan (in the presence of adrenaline) to l₂-imidazoline sites. In EEDQ-pretreated membranes (10^?5M, 30 min at 25°c), the density of l₂-imidazoline sites (B_max= 80 ± 4 fmol/mg of protein) was not different from that determined in untreated membranes in the presence of 10^?6M (-)-adrenaline (B_max= 83 ± 4 fmol/mg of protein), and both densities were lower (24%, p < 0.05) than the total native density of [³H]idazoxan binding sites (B_max= 107 ± 6 fmol/mg of protein) (l₂-imidazoline sites plus a₂-adrenoceptors). Treatment of rats with an optimal dose of EEDQ (1.6 mg/kg, i.p., for 2 h to 30 days) reduced maximally at 6 h (by 95 ± 1%) the specific binding of [³H]-R821002 to α₂-adrenoceptors, but also the binding of [³H]idazoxan to l₂-imidazoline sites (by 44 ± 5%). Pretreatment with yohimbine (10 mg/kg, i.p.) fully protected against EEDQ-induced α₂-adrenoceptor inactivation. In contrast, pretreatment with cirazoline (1 mg/kg, i.p.), did not protect against EEDQ-induced inactivation of l₂-imidazoline sites. Treatment with EEDQ (1.6 mg/kg, i.p., for 6 h) did not alter the density of brain monoamine oxidase-A sites labeled by [³H]Ro 41–1049 or that of monoamine oxidase-B sites labeled by [³H]Ro 19–6327 (lazabemide), two relevant mitochondrial markers. Competition experiments with cirazoline against the specific binding of [³H]idazoxan to l₂-imidazoline sites demonstrated the presence of the expected two affinity states for the drug in EEDQ-pretreated membranes as well as in rats treated with EEDQ. The results indicate that EEDQ in vitro is a useful tool for quantitating l₂-imidazoline sites when using [³H]-imidazoline ligands that also recognize α₂-adrenoceptors. In vivo, however, EEDQ is also able to inactivate partially brain l₂-imidazoline sites probably by an indirect mechanism. Key Words: Brain l₂-imidazoline sites—[³H]-Idazoxan—α₂-Adrenoceptors—[³H] R821002—N -Ethoxycarbonyl-2-ethoxy-li2-dihydroquinoline—Monoamine oxidase-A—[³H]Ro 41–1049—Monoamine oxidase-B—[³H]Ro 19–6327.