Two tertiary branched tetraamines, N4-aminopropylnorspermidine and N4-aminopropylsperimidine, are the major polyamines in thermophilic eubacteria, Thermoleophilum

Abstract Two novel polyamines were found as major polyamines of Thermoleophilum album and Thermoleophilum minutum , which are Gram-negative eubacteria obligate for thermophily and n -alkane substrates. They were identified as tertiary branched tetraamines, N⁴-aminopropylnorspermidine ( tris (3-aminopropyl)amine) [NH₂(CH₂)₃N-((CH₂)₃NH₂)(CH₂)₃NH₂ or N(CH₂CH₂CH₂NH₂)₃] and N⁴-aminopropylspermidine [NH₂(CH₂)₃N((CH₂)₃NH₂)(CH₂)₄NH₂] by high-performance liquid chromatography and gas chromatography-mass spectrometry.     

Formation of aminopropylhomospermidine from homospermidine in yeasts and thermophilic bacilli

Abstract When the yeasts Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe and the thermophilic bacteria Bacillus stearothermophilus and Bacillus acidocaldarius were cultured in the presence of homospermidine, a new compound accumulated in the cells within a few days. This compound was identified as aminopropylhomospermidine [NH₂(CH₂)₃NH (CH₂)₄NH(CH₂)₄NH₂] by gas chromatographymass spectrometry (GC-MS) and by the enzymatic cleavage method developed in our laboratories. This polyamine was not produced from homospermidine in Escherichia coli, Bacillus subtilis, Bacillus alkalophilus , or a eukaryotic protozoon, Tetrahymena pyriformis , none of which usually contains appreciable amounts of spermine. These findings suggest that the synthesis of aminopropylhomospermidine from homospermidine is mediated by spermine synthase.     

Identification of N1-acetylnorspermidine in Vibrio parahaemolyticus and an enzyme activity responsible for its formation

Abstract N ¹-Acetylnorspermidine [CH₃CONH(CH₂)₃NH(CH₂)₃NH₃] was identified in Vibrio parahaemolyticus , which contains norspermidine as a major polyamine. This is the first example for the natural occurence of monoacetylated unusual polyamine. The N ¹-acetylnorspermidine content was the highest 4 h after inoculation. Incubation of norspermidine and acetyl CoA with a cell extract from V. parahaemolyticus produced N ¹-acetylnorspermidine. A remarkable increase in specific activity of the acetyltransferase was observed at the exponential phase of growth. Spermidine also served as a substrate for the enzyme, with the formation of two isomers of the acetylspermidines ( N ¹-acetylspermidine was predominant), but the reaction rate was less than 50% of that with norspermidine. These results suggest that norspermidine in V. parahaemolyticus may be associated with the cell growth and its role may be controlled through acetylation, as reported for spermidine in Escherichia coli .     

Utilization of nitrogen from storage and current-year uptake in walnut spurs during the spring flush of growth

[¹⁵N]-depleted (NH₄)₂SO₄ applied to the soil in 1985 resulted in residual labeling of about 16% of the storage nitrogen (N) pool of mature walnut ( Juglans regia L. cv. Serr) trees in 1987. Application of [¹⁵N]-depleted (NH₄)₂SO₄ fertilizer to a different set of mature walnut trees in 1987 allowed monitoring of the kinetics and utilization of N from current year uptake in 1987 and resulted in >20% labeling of fruit N following completion of leaf expansion. Redistribution of storage N to the new growth predominated during the spring flush of growth although N derived from the soil during current-year uptake contributed increasingly during leaf expansion. Labeled N from current year uptake accumulated preferentially in the leaves as compared with reproductive organs during leaf expansion but subsequent to leaf expansion, fruit were more highly labeled with N derived from current-year uptake than leaves. Pistillate flower abortion was coincident with an apparent competition for N among developing vegetative and reproductive organs and preceded the period of significant N contribution from current-year uptake.     

Methane-dependent nitrate production by a microbial consortium enriched from a cultivated humisol

Abstract A consortium was enriched from a humisol incubated with 3.6 kPa CH₄ and NH₄⁺. This consortium oxidized NH₄⁺ to NO₂⁻ and NO₃⁻ (NO₃⁻/NO₂⁻ ratio about 20) with smaller amounts of N₂O. This oxidation stopped in the stationary phase after depletion of CH₄. CH₃OH or CO₂ did not support oxidation. Growth and resting cell experiments suggested that nitrification was associated with methanotrophic activity and that chemoautotrophic nitrifiers were absent.     

Effect of changes in nitrogen nutrition on the polyamine content of Alnus glutinosa

Abstract. The principal polyamines in Alnus glutinosa roots, nodules and root pressure sap, putrescine, spermidine and spermine, were quantified by reversed-phase, high-performance liquid chromatography with fluorescence detection following precolumn derivatization with 9-fluorenylmethyl chloroformate and 1-ada-mantanamine. Putrescine was the major component of all tissues and sap. It comprised 70% or more of the polyamine pool except in roots of KNO₃-fed plants, in which similar amounts of putrescine and spermidine were present at levels five-fold lower than plants fed (NH₄)₂SO₄. Polyamine levels in nodules were 50% greater than in roots. The polyamine content of roots and nodules was not altered significantly when the nitrogen nutrition was changed from sole reliance on nitrogen fixation to partial or complete utilization of (NH₄)₂SO₄. However, the polyamine content of root pressure sap from nodulated plants increased almost four-fold when they were fed with increasing concentrations of NH₄NO₃, although the total polyamine content remained low (5mmol m⁻³ sap). The polyamine content of the Alnus root system changed with plant age. In particular, the spermidine content of both roots and nodules was higher in 10- as compared to 16-week-old plants.     

Relationship between osmoprotection and the structure and intracellular accumulation of betaines by Escherichia coli

Abstract Naturally occuring betaines, especially glycine betaine and proline betaine, were accumulated by Escherichia coli from urine. In synthetic hyperosmotic medium, with an homologous series of added betaines, (CH₃)₃N⁺-(CH₂) _n -COO⁻, osmoprotective activity and intracellular accumulation decreased monotonically as n increased from 1 to 5. In contrast, α -substituted glycine betaines were accumulated in a similar manner to glycine betaine, but with different osmoprotective activities. Arsenobetaine, with a quaternary arsonium group, was also accumulated but amino acids which can become negatively charged in a chemically basic environment were not.     

Reduced Development of Grey Mould (Botrytis cinerea) in Bean and Tomato Plants by Calcium Nutrition

Fertilization of bean plants grown in perlite with 1 and 3 mM CaCl₂ or Ca(NO₃)₂ reduced severity of grey mould as compared with control plants or plants fertilized with 5 mM of the compounds. Fertilization with Ca(NO₃)₂ reduced severity leaf grey mould and fruit ghost spots of tomato plants grown in perlite by 70 and 45%, respectively. The rate of decrease varied with the position of the fruits on the plants. Leaves from plants treated with calcium or otherwise [KNO₃, (NH₄)₂SO₄] produced less ethylene than leaves of nontreated plants. Rate of growth of B. cinerea was lower on growth medium prepared from washings from leaves of calcium fertilized plants than from leaves from other treatments. The fertilizer combination Ca(H₂PO₄)₂+ CaSO₄ (1 and 3 g/kg soil) applied once to tomato plants grown in soil reduced severity of leaf grey mould by 80 % (significant at P = 0.05) but 1–3 g CaSO₄/kg soil only tended to reduce disease severity (30–40 %, not significant) as compared with the control. The compounds CaCl₂ and Ca(NO₃)₂ increased significantly ( P = 0.05) the growth of B. cinerea on synthetic medium when applied at rates of 1 0–10.0 mM whereas reduction of growth was observed with 0.1 mM of the compounds and of CaSO₄.     

Serum Proteins Promoting [3H]Thymidine Uptake by Trypanosoma (Schizotrypanum) cruzi (Chagas) in vitro

SYNOPSIS. Five proteins capable of stimulating [³H]thymidine uptake by Trypanosoma cruzi in vitro were isolated from fetal calf serum by (NH₄)₂SO₄ precipitation and ion exchange column chromatography. The proteins were partially characterized by immunodiffusion, immunoelectrophoresis, polyacrylamide gel disc electrophoresis, and SDS electrophoresis. As estimated by SDS electrophoresis, using 4 standards, the molecular weight of protein 1 was 100,000, that of protein 2 was 76,000. and that of proteins 3–5 was 68,000 daltons.     

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

NIDA522131, a new radioligand for imaging extrathalamic nicotinic acetylcholine receptors: in vitro and in vivo evaluation

A novel radioligand, 6-chloro-3-((2-( S )-azetidinyl)methoxy)-5-(2-fluoropyridin-4-yl)pyridine (NIDA522131), for imaging extrathalamic nicotinic acetylcholine receptors (nAChRs) was characterized in vitro and in vivo using positron emission tomography. The K_d and T_1/2 of dissociation of NIDA522131 binding measured at 37°C in vitro were 4.9 ± 0.4 pmol/L and 81 ± 5 min, respectively. The patterns of radioactivity distribution in monkey brain in vivo was similar to that of 2-[¹⁸F]fluoro-3-(2( S )-azetidinylmethoxy)pyridine (2FA), a radioligand that has been successfully used in humans, and matched the α₄β₂* nAChRs distribution. Comparison between [¹⁸F]NIDA522131 and 2FA demonstrated better in vivo binding properties of the new radioligand and substantially greater radioactivity accumulation in brain. Consistent with [¹⁸F]NIDA522131 elevated affinity for nAChRs and its increased lipophilicity, both, the total and non-displaceable distribution volumes were substantially higher than those of 2FA. Estimated binding potential values in different brain regions, characterizing the specificity of receptor binding, were 3–4 fold higher for [¹⁸F]NIDA522131 than those of 2FA. Pharmacological evaluation in mice demonstrated a toxicity that was comparable to 2FA and is in agreement with a 2300 fold higher affinity at α₄β₂* versus α₃β₄* nAChRs. These results suggest that [¹⁸F]NIDA522131 is a promising positron emission tomography radioligand for studying extrathalamic nAChR in humans.     

Associative cellulolysis and N2 fixation by co-cultures of Trichoderma harzianum and Clostridium butyricum: the effects of ammonium-N on these processes

Mixed cultures of the cellulolytic fungus Trichoderma harzianum with the anaerobic diazotroph Clostridium butyricum were shown to co-operatively degrade cellulose and utilize the degradation products for N₂ fixation. Cellulose degradation and N₂ fixation were stimulated by small (0.1 mg/ml) additions of (NH₄)₂SO₄. The (NH₄₂SO₄ stimulates cellulolysis thereby increasing the supply of cellulose degradation products to the diazotroph. In aerobic environments the anaerobe depends on the respiration of the aerobe to create anaerobic microsites. The N source increased O₂ uptake by the fungus increasing the number of sites suitable for the development of the anaerobe. Stimulation in the growth of T. harzianum by (NH₄₂SO₄ resulted in increased growth and N₂ fixation by Cl. butyricum.     

Activity and composition of ammonia oxidizing bacterial communities and emission dynamics of NH3 and N2O in a compost reactor treating organic household waste

Aims:  To monitor emissions of NH₃ and N₂O during composting and link these to ammonia oxidation rates and the community structure of ammonia oxidizing bacteria (AOB).
Methods and Results:  A laboratory-scale compost reactor treating organic household waste was run for 2 months. NH₃ emissions peaked when pH started to increase. Small amounts of N₂O and CH₄ were also produced. In total, 16% and less than 1% of the initial N was lost as NH₃-N and N₂O-N respectively. The potential ammonia oxidation rate, determined by a chlorate inhibition assay, increased fourfold during the first 9 days and then remained high. Initially, both Nitrosospira and Nitrosomonas populations were detected using DGGE analysis of AOB specific 16S rRNA fragments. Only Nitrosomonas europaea was detected under thermophilic conditions, but Nitrosospira populations re-established during the cooling phase.
Conclusions:  Thermophilic conditions favoured high potential ammonia oxidation rates, suggesting that ammonia oxidation contributed to reduced NH₃ emissions. Small but significant amounts of N₂O were emitted during the thermophilic phase. The significance of different AOBs detected in the compost for ammonia oxidation is not clear.
Significance and Impact of Study:  This study shows that ammonia oxidation occurs at high temperature composting and therefore most likely reduces NH₃ emissions.     

Nitrogen concentration, ammonium/nitrate ratio and NaCl interaction in vegetative and reproductive growth of peanuts

Peanuts ( Arachis hypogaea L. cv. Shulamit) grown with NO₃⁻ and saline water in hydroponics responded positively to addition of nitrogen (N) in their vegetative growth, but not in desert dune sand. In order to clarify these conflicting results, peanut plants were grown in a greenhouse pot experiment with fine calcareous sand. The nutrient solution contained 0 or 50 m M NaCl and 2 or 6 m M N in the form of Ca(NO₃)₂, NH₄NO₃ or (NH₄)₂SO₄. Three replicates were harvested after 48 days (beginning of reproductive stage) and three after 109 days (pod filling). In addition, gynophores were treated with 0, 50, 100, 150 or 200 m M NaCl outside the growth pot to check their sensitivity to salt. Shoot dry weight became greater with increasing NH₄⁺/NO₃⁻ ratio. Increasing the N concentration from 2 to 6 m M did not change shoot dry weight of the NH₄NO₃ or NH₄⁺-fed plants, but caused a reduction in shoot dry weight of NO₃⁻-fed plants. Shoot dry weight was not affected by increasing the NaCl concentration to 50 m M . Salt caused an increase in the number of gynophores per plant and a reduction of the mean pod weight. A NaCl concentration of 100 m M and above reduced gynophore vitality. It is concluded that the salt sensitivity of peanut plants resides mainly in the sensitivity of the reproductive organs.     

The role of ultraviolet radiation, photosensitizers, reactive oxygen species and ester groups in mechanisms of methane formation from pectin

Ultraviolet (UV) radiation has recently been demonstrated to drive an aerobic production of methane (CH₄) from plant tissues and pectins, as do agents that generate reactive oxygen species (ROS) in vivo independently of UV. As the major building-blocks of pectin do not absorb solar UV found at the earth's surface (i.e. >280 nm), we explored the hypothesis that UV radiation affects pectin indirectly via generation of ROS which themselves release CH₄ from pectin. Decreasing the UV absorbance of commercial pectin by ethanol washing diminished UV-dependent CH₄ production, and this was restored by the addition of the UV photosensitizer tryptophan. Certain ROS scavengers [mannitol, a hydroxyl radical (^•OH) scavenger; 1,4-diazabicyclo[2.2.2] octane; and iodide] strongly inhibited UV-induced CH₄ production from dry pectin. Furthermore, pectin solutions emitted CH₄ in darkness upon the addition of ^•OH, but not superoxide or H₂O₂. Model carbohydrates reacted similarly if they possessed —CH₃ groups [e.g. methyl esters or (more weakly) acetyl esters but not rhamnose]. We conclude that UV evokes CH₄ production from pectic methyl groups by interacting with UV photosensitizers to generate ^•OH. We suggest that diverse processes generating ^•OH could contribute to CH₄ emissions independently of UV irradiation, and that environmental stresses and constitutive physiological processes generating ROS require careful evaluation in studies of CH₄ formation from foliage.     

Nitrogen regulation of erythromycin formation in Streptomyces erythreus

Abstract Erythromycin formation decreased in Streptomyces erythreus as a function of the ammonium concentration present in the medium. Total inhibition of synthesis was obtained with 100 mM NH₄Cl but medium pH and culture growth were not significantly affected. A similar effect was obtained with NH₄NO₃ or (NH₄)₂SO₄ indicating that ammonium ion probably repressed formation of antibiotic.     

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans

Aims:  To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans .
Methods and Results:  In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0·13 g l⁻¹ N) initial NaNO₃ or (NH₄)₂SO₄ levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0·78 g l⁻¹ N) initial NaNO₃ levels. EPS produced by CSTR grown cultures with high (NH₄)₂SO₄ levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO₃ levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units.
Conclusions:  While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology.
Significance and Impact of the Study:  These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.     

Morphological changes in the gills of Lophiosilurus alexandri exposed to un-ionized ammonia

The average lethal concentration of un-ionized ammonia (48-h LC₅₀NH₃) has been determined by the static assay for larvae (0.48 mg l⁻¹) and alevins (0.92 mg l⁻¹) of 'pacamã' Lophiosilurus alexandri. Studies by light and scanning electron microscopes at the greatest concentration of NH₃ (0.99 mg l⁻¹ for larvae and 1.5 mg l⁻¹ for alevins) have shown that the changes in the cells and branchial tissue were more intense in the alevins.     

The synthesis and structures of tantalum complexes that contain a triamido or a diamidoamine ligand

Addition of (Me₃SiNHCH₂CH₂)₂NH (H₃[N₃(TMS)]) or (Me₃SiNH-o-C₆H₄)₂NH (H₃[ArN₃(TMS)]) to a solution of TaMe₅ yields [N₃(TMS)]TaMe₂ or [ArN₃(TMS)]TaMe₂, respectively. An X-ray study of [ArN₃(TMS)]TaMe₂ showed it to have an approximate trigonal bipyramidal structure in which the two methyl groups are in equatorial positions and the triamido ligand is approximately planar. Addition of (C₆F₅NHCH₂CH₂)₂NH (H₃[N₃(C₆F₅)]) to TaMe₅ yields first [(C₆F₅NCH₂CH₂)₂NH]TaMe₃, which then decomposes to [(C₆F₅NCH₂CH₂)₂N]TaMe₂. An X-ray study of [(C₆F₅NCH₂CH₂)₂N]TaMe₂ shows it to be approximately a trigonal bipyramid, but the C₆F₅ rings are oriented so that they lie approximately in the TaN₃ plane and two ortho fluorines interact weakly with the metal. Trimethylaluminum attacks the central nitrogen atom in [N₃(TMS)]TaMe₂ to give [(Me₃SiNCH₂CH₂)₂NAlMe₃]TaMe₂, an X-ray study of which shows it to be a trigonal bipyramidal species similar to the first two structures, except that the C-Ta-C bond angle is approximately 30° smaller (106.6(12)°). Addition of B(C₆F₅)₃ to [(C₆F₅NCH₂CH₂)₂NH]TaMe₃ yields {[(C₆F₅NCH₂CH₂)₂NH]TaMe₂}⁺ {B(C₆F₅)₃Me}⁻, the structure of which most closely resembles that of [(Me₃SiNCH₂CH₂)₂NAlMe₃]TaMe₂ in that the C-Ta-C angle is 102.0(6)°. The C₆F₅ rings in {[(C₆F₅NCH₂CH₂)₂NH]TaMe₂}⁺ are turned roughly perpendicular to the TaN₃ plane, i.e. ortho fluorines do not interact with the metal in this molecule.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.