Determination of pyrazinamide and its main metabolites in rat urine by high-performance liquid chromatography

A new high-performance liquid chromatograhic procedure for simultaneous determination of pyrazinamide (PZA) and its three metabolites 5-hydroxypyrazinamide (5-OH-PZA), pyrazinoic acid (PA), and 5-hydroxypyrazinoic acid (5-OH-PA), in rat urine was developed. 5-OH-PZA and 5-OH-PA standards were obtained by enzymatic synthesis (xanthine oxidase) and checked by HPLC and GC–MS. Chromatographic separation was achieved in 0.01 M KH₂PO₄ (pH 5.2), circulating at 0.9 ml/min, on a C₁₈ silica column, at 22°C. The limits of detection were 300 μg/l for PZA, 125 μg/l for PA, 90 μg/l for 5-OH-PZA and 70 μg/l for 5-OH-PA. Good linearity (r²>0.99) was observed within the calibration ranges studied: 0.375–7.50 mg/l for PZA, 0.416–3.33 mg/l for PA, 0.830–6.64 mg/l for 5-OH-PZA and 2.83–22.6 mg/l for 5-OHPA. Accuracy was always lower than ±10.8%. Precision was in the range 0.33–5.7%. The method will constitute a useful tool for studies on the influence of drug interactions in tuberculosis treatment.     

Use of activated carbon as a buffer in biofiltration of waste gases with fluctuating concentrations of toluene

Fluctuations in contaminant concentrations often adversely influence the effectiveness of bioreactors for waste gas treatment. Application of an adsorbent to minimize such fluctuations could improve the overall process. Therefore the buffer capacity of a number of activated carbons and other adsorbents was tested. The buffer capacity of the adsorbents depends on the desired concentration range of the contaminants entering the bioreactor and on the time available for desorption. When fluctuations between 0 and 1000 mg toluene m^–3 were applied to a biofilter this resulted in significant concentrations of toluene leaving the biofilter. Using one selected type of activated carbon it was demonstrated that these fluctuations could be decreased to a value of about 300 mg m^–3, which subsequently completely degraded in the biofilter.     

Comparison of soil nitrogen mineralization and nitrification in a mixed grassland and forested ecosystem in central Taiwan

We used the buried bag incubation method to study temporal patterns of net N mineralization and net nitrification in soils at Ta-Ta-Chia forest in central Taiwan. The site included a grassland zone, (dominant vegetation consists of Yushania niitakayamensis and Miscanthus transmorrisonensis Hayata) and a forest zone (Tsuga chinensis var. formosana and Yushania niitakamensis). In the grassland, soil concentration NH₄ ⁺ in the organic horizon (0.1–0.2 m) ranged from 1.0 to 12.4 mg N kg^–1 soil and that of NO₃ ^– varied from 0.2 to 2.1 mg N kg^–1 soil. In the forest zone, NH₄ ⁺ concentration was between 2.8 and 25.0 mg N kg^–1 soil and NO₃ ^–varied from 0.2 to 1.3 mg N kg^–1 soil. There were lower soil NH₄ ⁺ concentrations during the summer than other seasons. Net N mineralization was higher during the summer while net nitrification rates did not show a distinct seasonal pattern. In the grassland, net N mineralization and net nitrification rates were between –0.1 and 0.24 and from –0.04 to 0.04 mg N kg^–1 soil day^–1, respectively. In the forest zone, net N mineralization rates were between –0.03 and 0.45 mg N kg^–1 soil day^–1 and net nitrification rates were between –0.01 and 0.03 mg N kg^–1 soil day^–1. These differences likely result from differing vegetation communities (C₃ versus C₄ plant type) and soil characteristics.     

Prothrombin Concentration in the Cerebrospinal Fluid Is Not Altered in Alzheimer's Disease

Prothrombin, known to be expressed in brain and to possess growth modulating properties, has been suggested to be involved in the pathogenesis of Alzheimer's disease (AD). We studied prothrombin concentration in lumbar CSF (L-CSF) in patients with AD (n = 25), neurologic disease controls (NDC; n = 33) covering a wide range of neurologic disorders, and subjects with Guillain-Barré syndrome (GBS; n = 4) as well as in samples of non-pathological ventricular CSF (V-CSF; n = 4). The results were evaluated with respect to CSF flow rate, as indicated by the albumin quotient (Q_Alb). The concentrations of prothrombin in L-CSF in NDC (mean: 0.46 mg/l, range: 0.21–0.96), and AD (mean: 0.6 mg/l, range: 0.19–1.2) were in the normal range reported previously. Expectedly, prothrombin concentration in L-CSF of GBS was increased (mean: 6.3 mg/l, range: 2.3–9.7) corresponding to the increased Q_Alb in this group (mean 54.6 × 10^–3, range: 17–88.1). The concentrations of both prothrombin and albumin were 5.5-fold higher in L-CSF than in V-CSF (mean Q_Alb : 1.1 × 10^–3, mean concentration of prothrombin: 0.088 mg/l). In conclusion, CSF prothrombin in all conditions evaluated here is exclusively derived from blood.     

Phenol degradation by a psychrotrophic strain of Pseudomonas putida

Summary Cell growth and phenol degradation kinetics were studied at 10°C for a psychrotrophic bacterium, Pseudomonas putida Q5. The batch studies were conducted for initial phenol concentrations, S_o, ranging from 14 to 1000 mg/1. The experimental data for 14<=S_o<=200 mg/1 were fitted by non-linear regression to the integrated Haldane substrate inhibition growth rate model. The values of the kinetic parameters were found to be: _m=0.119 h^–1, K _S=5.27 mg/1 and K _I=377 mg/1. The yield factor of dry biomass from substrate consumed was Y=0.55. Compared to mesophilic pseudomonads previously studied, the psychrotrophic strain grows on and degrades phenol at rates that are ca. 65–80% lower. However, use of the psychrotrophic microorganism may still be economically advantageous for waste-water treatment processes installed in cold climatic regions, and in cases where influent waste-water temperatures exhibit seasonal variation in the range 10–30°C.Nomenclature K _S saturation constant (mg/l) - K _I substrate inhibition constant (mg/l) - specific growth rate (h^–1) - _m maximum specific growth rate without substrate inhibition (h^–1) - _max maximum achievable specific growth rate with substrate inhibition (h^–1) - S substrate (phenol) concentration (mg/l) - S_o initial substrate concentration (mg/l) - S_max substrate concentration corresponding to _max (mg/l) - t time (h) - X cell concentration, dry basis (mg DW/l) - X_f final cell concentration, dry basis (mg DW/l) - X_o initial cell concentration, dry basis (mg DW/l) - Y yield factor (mg DW cell produced/mg substrate consumed)     

Augmentation of H2 photoproduction in Rhodopseudomonas palustris by N-heterocyclic aromatic compounds

Increases of 23- (5.6 mmol acetylene reduced mg dry wt^–1) and 16- (4 mmol acetylene reduced mg dry wt^–1) fold in nitrogenase activity and 12- (671 l H₂ mg dry wt^–1 h^–1) and 6- (349 l mg dry wt^–1 h^–1) fold in H₂ photoproduction in Rhodopseudomonas palustris JA1 over 24 h were achieved with pyrazine 2-carboxylate (3 mM) and 3-picoline (3 mM), respectively, and were higher than earlier reports of enhancement (1.5 to 5- fold) in biological H₂ production using various alternative methods.     

Effects of a clearcut on the net rates of nitrification and N mineralization in a northern hardwood forest,Catskill Mountains,New York,USA

The Catskill Mountains of southeastern New York receive among the highest rates of atmospheric nitrogen (N) deposition in eastern North America, and ecosystems in the region may be sensitive to human disturbances that affect the N cycle. We studied the effects of a clearcut in a northern hardwood forest within a 24-ha Catskill watershed on the net rates of N mineralization and nitrification in soil plots during 6 years (1994–1999) that encompassed 3-year pre- and post-harvesting periods. Despite stream NO₃^– concentrations that increased by more than 1400 mol l^–1 within 5 months after the clearcut, and three measures of NO₃^– availability in soil that increased 6- to 8-fold during the 1^st year after harvest, the net rates of N mineralization and nitrification as measured by in situ incubation in the soil remained unchanged. The net N-mineralization rate in O-horizon soil was 1– 2 mg N kg^–1 day^–1 and the net nitrification rate was about 1 mg N kg^–1 day^–1, and rates in B-horizon soil were only one-fifth to one-tenth those of the O-horizon. These rates were obtained in single 625 m² plots in the clearcut watershed and reference area, and were confirmed by rate measurements at 6 plots in 1999 that showed little difference in N-mineralization and nitrification rates between the treatment and reference areas. Soil temperature increased 1 ± 0.8 °C in a clearcut study plot relative to a reference plot during the post-harvest period, and soil moisture in the clearcut plot was indistinguishable from that in the reference plot. These results are contrary to the initial hypothesis that the clearcut would cause net rates of these N-cycling processes to increase sharply. The in situ incubation method used in this study isolated the samples from ambient roots and thereby prevented plant N uptake; therefore, the increases in stream NO₃^– concentrations and export following harvest largely reflect diminished uptake. Changes in temperature and moisture after the clearcut were insufficient to measurably affect the net rates of N mineralization and nitrification in the absence of plant uptake. Soil acidification resulting from the harvest may have acted in part to inhibit the rates of these processes. The US Governments right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Anaerobic degradation of phenol in batch and continuous cultures by a denitrifying bacterial consortium

Summary The anaerobic degradation of phenol under denitrifying conditions by a bacterial consortium was studied both in batch and continuous cultures. Anaerobic degradation was dependent on NO_f3 ^p– and concentrations up to 4 mm phenol were degraded within 2–5 days. During continuous growth in a fermenter, steady states could be maintained at eight dilution rates (D) corresponding to residence times between 12.5 and 50 h. Culture wash-out occurred at D=0.084 h^–1. The kinetic parameters obtained for anaerobic degradation of phenol under denitrifying conditions by the consortium were: maximam specific growth rate = 0.091 h^–1; saturation constant = 4.91 mg phenol/l; true growth yield = 0.57 mg dry wt/mg phenol; maintenance coefficient = 0.013 mg phenol/mg dry wt per hour. The Haldane model inhibition constant was estimated from batch culture data giving a value of 101 mg/l. The requirement of CO₂ for the anaerobic degradation of phenol with NO_f3 ^p– indicates that phenol carboxylation to 4-hydroxybenzoate was the first step of phenol degradation by this culture. 4-Hydroxybenzoate, proposed as an intermediate of phenol carboxylation under these conditions, was detected only in continuous cultures at very low growth rates (D=0.02 h^–1), but was never detected as a free intermediary metabolite either in batch or in continuous cultures. Correspondence to: N. Khoury     

Nitrite reduction by a mixed culture under conditions relevant to shortcut biological nitrogen removal

Dissimilative reduction of nitrite by nitrite-acclimated cellswas investigated in a batch reactor under various environmental conditions that can beencountered in shortcut biological nitrogen removal (SBNR: ammonia to nitrite andnitrite to nitrogen gas). The maximum specific nitrite reduction rate was as much as 4.3 times faster than the rate of nitrate reduction when individually tested, but the reaction was inhibited in the presence of nitrate when the initial nitrate concentration was greater than approximately 25 mg-N/l or the initialNO ₃ ^- N/NO ₂ ^- N ratio was larger than 0.5. Nitrite reduction was also inhibited by nitrite itself when theconcentration was higher than that to which the cells had been acclimated. Therefore, it was desirable to avoid excessively high nitrite and nitrate concentrations in a denitrification reactor. Nitrite reduction, however, was not affected by an alkaline pH (in the range of 7–9) or a high concentration of FA (in the range of 16–39 mg/l), which can be common in SBNR processes. The chemical oxygen demand (COD) requirement for nitrite reduction was approximately 22–38% lower than that for nitrate reduction, demonstrating that the SBNR process can be economical. The specific consumption,measured as the ratio of COD consumed to nitrogen removed, was affected by the availability of COD and the physiological state of the cells. The ratio increased when the cells grew rapidly and were storing carbon and electrons.     

Comparison of N losses (NO − 3, N 2O, NO) from surface applied, injected or amended (DCD) pig slurry of an irrigated soil in a Mediterranean climate

Nitrous oxide (N ₂O), nitric oxide (NO), denitrification losses and NO^–₃ leaching from an irrigated sward were quantified under Mediterranean conditions. The effect of injected pig slurry (IPS) with and without the nitrification inhibitor dicyandiamide (DCD) was evaluated and also compared with that of a surface pig slurry application (SPS) and a control treatment (Control) without fertiliser. After application, fluxes of NO and N ₂O peaked from SPS (3.06 mg NO-N m ^–2 d ^–1 and 108 mg N ₂O-N m ^–2 d ^–1) and IPS (3.50 mg NO-N m ^–2 d ^–1 and 105 mg N ₂O-N m ^–2 d ^–1). However, when irrigation was applied, N ₂O and NO emissions declined. The total N ₂O and denitrification losses were slightly large from IPS than from SPS, although the differences were not significant (P < 0.05). Emission of NO was not affected by the method of pig slurry application. DCD inhibited nitrification during the first 20–30 days and reduced N ₂O and NO emissions from pig slurry by at least 46% and 37%, respectively. Considering the 215 days following pig slurry application, the emission factor of N ₂O based on N fertiliser was 1.60% (SPS), 2.95% (IPS), and 0.50% (IPS + DCD). The emission factor for NO was 0.14% (SPS), 0.12% (IPS), and 0.02% (IPS + DCD). Environmental conditions of the crop favoured the denitrification process as the most important source of N ₂O during the experimental period. The differences in the denitrification rate between treatments could be explained by the pattern of water soluble carbon (WSC), that was the highest value in injected pig slurry (with and without DCD). Due to low drainage (5% of water applied), leaching losses of NO₃^– were lower than those of denitrification from the upper soil layer (0–10 cm) in all treatments and especially with IPS + DCD, where the nitrification inhibitor was very efficient in reducing leaching losses.     

The effect of cropping and fertiliser nitrogen rates on nitrogen balance in soil

Summary Fertilizer/soil N balance of cropped and fallow soil has been studied in a pot experiment carried out with grey forest soil (southern part of Moscow region) at increasing rates of¹⁵N labelled ammonium sulfate (0; 8; 16; 32 mg N/100 g of soil). The fertilizer¹⁵N balance has been shown to depend upon its application rate and the presence of growing plants. Fertilizer N uptake efficiency was maximum (72.5%) and gaseous losses-minimum (12.5%) at the application rate of 16 mg N/100 g of soil. Fertilizer N losses from the fallow soil were 130–220% versus those from the cropped soil. At the application of fertilizer N the plant uptake of soil N was 170–240% and the amount of soil N as N–NH₄ exchangeable + N–NO₃ in fallow was 350–440% as compared to the control treatment without nitrogen (PK).After cropping without or with N fertilizer application at the rates of 8 and 32 mg N/100 g of soil, a positive nitrogen balance has been found which is likely due to nonsymbiotic (associative) N-fixation. It has been shown that biologically fixed nitrogen contributes to plant nutrition.     

Accession and cycling of elements in a coastal stand ofPinus radiata D. Don in New Zealand

Summary The accession and cycling of elements in a 14-year-old coastal stand ofPinus radiata D. Don was measured for one year. The element contents (mg m^–2 year^–1) of bulk precipitation and throughfall respectively were: NO₃–N 41, 12; NH₄–N 133, 154; organic-N 157, 396; Na 4420, 9700; K 387, 2900; Ca 351, 701; Mg 486, 1320. Of the increase in element content of rainwater beneath the forest canopy 20% (NH₄–N), 70% (organic-N), 3% (Na), 90% (K), 20% (Ca) and 30% (Mg) was attributed to leaching; the remainder to washing of aerosols filtered from the atmosphere by the vegetation. The canopy absorbed approximately 40 mg m^–2 year^–1 of NO₃–N. Litterfall was the major pathway for the above-ground biogeochemical cycle of N (93%), Ca (96%) and Mg (74%), and leaching was the major (73%) pathway for K.     

High-performance liquid chromatographic determination of a new oral thrombin inhibitor in the blood of rats and dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of a new oral thrombin inhibitor (compound I) in the blood of rats and dogs. The analyte was deproteinized with a 1.5 volume of methanol and a 0.5 volume of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a mixture of acetonitrile and 0.2% triethylamine of pH 2.3 (31:69, v/v) with a flow-rate of 1.0 ml/min at UV 231 nm. The retention time of compound I was approximately 9.3 min. The calibration curve was linear over the concentration range of 0.05–100 mg/l for rat blood (r²>0.9995, n=6) and dog blood (r²>0.9993, n=6). The limit of quantitation was 0.05 mg/l for both bloods using a 100-μl sample. For the 5 concentrations (0.05, 0.1, 1, 10, and 100 mg/l), the within-day recovery (n=4) and precision (n=4) were 98.1–104.1% and 1.5–6.8% for rat blood and 95.4–105.7% and 1.4–5.3% for dog blood, respectively. The between-day recovery (n=6) and precision (n=6) were 99.8–105.3% and 3.7–12.6% for rat blood and 87.5–107.1% and 2.9–15.3% for dog blood, respectively. The absolute recoveries were 82.4–93.3%. No interferences from endogenous substances were observed. In conclusion, the presented simple, sensitive, and reproducible HPLC method proved and was used successfully for the determination of compound I in the preclinical pharmacokinetics.     

Ethylene Removal by a Biofilter with Immobilized Bacteria

A biofilter which eliminated ethylene (C₂H₄) from the high parts-per-million range to levels near the limit for plant hormonal activity (0.01 to 0.1 ppm) was developed. Isolated ethylene-oxidizing bacteria were immobilized on peat-soil in a biofilter (687 cm³) and subjected to an atmospheric gas flow (73.3 ml min⁻¹) with 2 or 117 ppm of C₂H₄. Ethylene was eliminated to a minimum level of 0.017 ppm after operation with 2.05 ppm of C₂H₄ for 16 days. Also, the inlet C₂H₄ concentration of 117 ppm was reduced to <0.04 ppm. During operation with 2 and 117 ppm of C₂H₄, an increase in the C₂H₄ removal rate was observed, which was attributed to proliferation of the immobilized bacteria, notably in the first 0- to 5-cm segment of the biofilter. The maximal C₂H₄ elimination capacity of the biofilter was 21 g of C₂H₄ m⁻³ day⁻¹ during operation with 117 ppm of C₂H₄ in the inlet gas. However, for the first 0- to 5-cm segment of the biofilter, an elimination capacity of 146 g of C₂H₄ m⁻³ day⁻¹ was calculated. Transition of the biofilter temperature from 21 to 10°C caused a 1.6-fold reduction in the C₂H₄ removal rate, which was reversed during operation for 18 days. Batch experiments with inoculated peat-soil demonstrated that C₂H₄ removal still occurred after storage at 2, 8, and 20°C for 2, 3, and 4 weeks. However, the C₂H₄ removal rate decreased with increasing storage time and was reduced by ca. 50% after storage for 2 weeks at all three temperatures. The biofilter could be a suitable tool for C₂H₄ removal in, e.g., horticultural storage facilities, since it (i) removed C₂H₄ to 0.017 ppm, (ii) had a good operational stability, and (iii) operated efficiently at 10°C.     

The influence of nitrogen concentration and ammonium/nitrate ratio on N-uptake,mineral composition and yield of citrus

In short-term water culture experiments with different ¹⁵N labeled ammonium or nitrate concentrations, citrus seedlings absorbed NH₄ ⁺ at a higher rate than NO₃ ^–. Maximum NO₃ ^– uptake by the whole plant occurred at 120 mg L^–1 NO₃ ^–-N, whereas NH₄ ⁺ absorption was saturated at 240 mg L^–1 NH₄ ⁺-N. ¹⁵NH₄ ⁺ accumulated in roots and to a lesser degree in both leaves and stems. However, ¹⁵NO₃ ^– was mostly partitioned between leaves and roots.Adding increasing amounts of unlabeled NH₄ ⁺ (15–60 mg L^–1 N) to nutrient solutions containing 120 mg L^–1 N as ¹⁵N labeled nitrate reduced ¹⁵NO₃ ^– uptake. Maximum inhibition of ¹⁵NO₃ ^– uptake was about 55% at 2.14 mM NH₄ ⁺ (30 mg L^–1 NH₄ ⁺-N) and it did not increase any further at higher NH₄ ⁺ proportions.In a long-term experiment, the effects of concentration and source of added N (NO₃ ^– or NH₄ ⁺) on nutrient concentrations in leaves from plants grown in sand were evaluated. Leaf concentration of N, P, Mg, Fe and Cu were increased by NH₄ ⁺ versus NO₃ ^– nutrition, whereas the reverse was true for Ca, K, Zn and Mn.The effects of different NO₃ ^–-N:NH₄ ⁺-N ratios (100:0, 75:25, 50:50, 25:75 and 0:100) at 120 mg L^–1 total N on leaf nutrient concentrations, fruit yield and fruit characteristics were investigated in another long-term experiment with plants grown in sand cultures. Nitrogen concentrations in leaves were highest when plants were provided with either NO₃ ^– or NH₄ ⁺ as a sole source of N. Lowest N concentration in leaves was found with a 75:25 NO₃ ^–-N/NH₄ ⁺-N ratio. With increasing proportions of NH₄ ⁺ in the N supply, leaf nutrients such as P, Mg, Fe and Cu increased, whereas Ca, K, Mn and Zn decreased. Yield in number of fruits per tree was increased significantly by supplying all N as NH₄ ⁺, although fruit weight was reduced. The number of fruits per tree was lowest with the 75:25 NO₃ ^–-N:NH₄ ⁺-N ratio, but in this treatment fruits reached their highest weight. Rind thickness, juice acidity, and colour index of fruits decreased with increasing NH₄ ⁺ in the N supply, whereas the % pulp and maturity index increased. Percent of juice in fruits and total soluble solids were only slightly affected by NO₃ ^–:NH₄ ⁺ ratio.     

Removal of toluene in waste gases using a biological trickling filter

The removal of toluene from waste gas was studied in a trickling biofilter. A high level of water recirculation (4.7 m h^–1) was maintained in order to keep the liquid phase concentration constant and to achieve a high degree of wetting. For loads in the range from 6 to 150 g m^–3 h^–1 the maximum volumetric removal rate (elimination capacity) was 35±10 g m^–3 h^–1, corresponding to a zero order removal rate of 0.11±0.03 g m^–2 h^–1 per unit of nominal surface area. The surface removal was zero order above the liquid phase concentrations of approximately 1.0 g m^–3, corresponding to inlet gas concentrations above 0.7–0.8 g m^–3. Below this concentration the surface removal was roughly of first order. The magnitude of the first order surface removal rate constant, k_1A , was estimated to be 0.08–0.27 m h^–1 (k_1A a=24–86 h^–1). Near-equilibrium conditions existed in the gas effluent, so mass transfer from gas to liquid was obviously relatively fast compared to the biological degradation. An analytical model based on a constant liquid phase concentration through the trickling filter column predicts the effluent gas concentration and the liquid phase concentration for a first and a zero order surface removal. The experimental results were in reasonable agreement with a very simple model valid for conditions with an overall removal governed by the biological degradation and independent of the gas/liquid mass transfer. The overall liquid mass transfer coefficient, K_La, was found to be a factor 6 higher in the system with biofilm compared to the system without. The difference may be explained by: 1. Difference in the wetting of the packing material, 2. Mass transfer occurring directly from the gas phase to the biofilm, and 3. Enlarged contact area between the gas phase and the biofilm due to a rough biofilm surface.     

Microbial biomass and nitrogen cycling responses to fertilization and litter removal in young northern hardwood forests

The influence of site fertility on soil microbial biomass and activity is not well understood but is likely to be complex because of interactions with plant responses to nutrient availability. We examined the effects of long-term (8 yr) fertilization and litter removal on forest floor microbial biomass and N and C transformations to test the hypothesis that higher soil resource availability stimulates microbial activity. Microbial biomass and respiration decreased by 20–30 % in response to fertilization. Microbial C averaged 3.8 mg C/g soil in fertilized, 5.8 mg C/g in control, and 5.5 mg C/g in litter removal plots. Microbial respiration was 200 µg CO₂-C g^–1 d^–1 in fertilized plots, compared to 270 µg CO₂-C g^–1 d^–1 in controls. Gross N mineralization and N immobilization did not differ among treatments, despite higher litter nutrient concentrations in fertilized plots and the removal of substantial quantities of C and N in litter removal plots. Net N mineralization was significantly reduced by fertilization. Gross nitrification and NO₃ ^– immobilization both were increased by fertilization. Nitrate thus became a more important part of microbial N cycling in fertilized plots even though NH₄ ⁺ availability was not stimulated by fertilization.Soil microorganisms did not mineralize more C or N in response to fertilization and higher litter quality; instead, results suggest a difference in the physiological status of microbial biomass in fertilized plots that influenced N transformations. Respiration quotients (qCO₂, respiration per unit biomass) were higher in fertilized plots (56 µg CO₂-C mg C^–1 d^–1) than control (48 µg CO₂-C mg C^–1 d ^–1) or litter removal (45 µg CO₂-C mg C^–1 d^–1), corresponding to higher microbial growth efficiency, higher proportions of gross mineralization immobilized, and lower net N mineralization in fertilized plots. While microbial biomass is an important labile nutrient pool, patterns of microbial growth and turnover were distinct from this pool and were more important to microbial function in nitrogen cycling.     

Preparation of a highly active ATPase of the mesophilic cyanobacterium Spirulina maxima

In this work, we report new studies on the ATPase attached to the photosynthetic membranes of the mesophilic cyanobacterium Spirulina maxima. This enzyme does not display persistent latency as had been previously reported for the ATPase of Spirulina platensis. The enzyme is readily activated by the careful application of methods currently used to activate chloroplast CF₁. Photosynthetic membranes of Spirulina maxima show a Mg²⁺-dependent ATPase activity of 195±25 mol Pi (mg chl)^–1 h^–1 after a light plus dithiothreitol (DDT) treatment. Methanol treatment of these membranes elicits Mg²⁺-dependent ATPase activity of 222±18 mol Pi (mg chl)^–1 h^–1.Here, we also describe the purification of the soluble coupling factor AF₁ of Spirulina maxima. This enzyme is unique among mesophilic cyanobacterial F₁ preparations in regard to its high specific Ca²⁺-dependent ATPase activity after heat treatment (14.75±1.91 mol Pi (mg prot)^–1 min^–1) and its room temperature stability.Abbreviations AF₁ cyanobacterial coupling factor - DTT dithiothreitol - DEAE cellulose diethylaminoethyl cellulose - DCCD N,N'-dicyclohexylcarbodiimide - EDTA ethylenediamine tetracetic, sodium salt - PAGE polyacrylamide gel electrophoresis - PMS phenazine methosulfate - PMSF phenylmethylsulfonyl fluoride - MV methylviologen - SDS sodium dodecylsulfate - TPSnCl triphenyltin chloride - Tris tris (hydroxymethyl) aminomethane - tricine N Tris (hydroxymethyl) methylglycine - BAEE N-benzoyl-L-arginine ethyl ester     

Somatic embryogenesis and plant regeneration from protoplasts of asparagus (Asparagus officinalis L.)

Protoplasts were isolated from embryogenic calli of Asparagus officinalis L. cv. Mary Washington and cultured in 1/2 MS medium with 1 mg/l NAA, 0.5 mg/l zeatin, 1 g/l L-glutamine, 0.6 M glucose and 0.1% Gellan Gum. Protoplasts started to divide after 3–4 d of culture and formed visible colonies after 30 d of culture. The percentage of colony formation (plating efficiency) was 7.2%. The colonies were then transferred onto Gellan Gum-solidified MS medium containing 1 mg/l 2,4-D and 3% sucrose for further growth. Somatic embryos were induced from all colonies of 0.5–1.0 mm size after transferring to 1/2 MS medium lacking growth regulators. After treating these somatic embryos (1–3 mm) in distilled water for a week, 30–40% of them germinated normally and grew into plantlets 20–30 d after transplanting on 1/2 MS medium containing 1 mg/l IBA, 1 mg/l GA₃ and 1% sucrose. These protoplast-derived plants were diploid with 20 chromosomes.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962)     

Foliar N application reduces soil NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>-N leaching loss in apple orchards

A comparison of the effects of foliar and soil N application was made in field-grown mature fruiting Gala/M9 apple trees (Malus domestica Borkh) in 2001 and 2002 growing seasons under Pacific Northwest growing conditions in southern British Columbia, Canada. The trees, six years old at the start of the experiment, were treated: (1) with 5 g/l urea sprays supplied every two weeks (7 times) from mid May to mid August (total about 50 g N/tree/year), (2) with the same amount of N applied to the soil with the same timing and quantity as for the foliar treatment, and (3) with no N (control). Leaf color (as SPAD readings) and N concentrations (mg/g), and soil NH₄⁺-N and NO₃^–-N were measured periodically throughout the two seasons. Leached NO₃^–-N was monitored monthly via an anion exchange probe from June to October in 2001 and from May to November in 2002. Shoot length was measured in October and N concentration of one-year-old wood and roots was determined in December of each growing s eason. Soil N application significantly increased shoot length relative to control or foliar N application. Leaf color, leaf N, and N concentration of one-year-old wood and roots were similarly increased relative to control by both soil and foliar N application. These treatments also increased fruit yield relative to control. There was no significant difference in yield and fruit quality between soil and foliar N applications. Soil N application increased soil NH₄⁺-N and NO₃^–-N content in the root zone, and also increased the NO₃^– leaching loss below the root zone especially late in the growing season. Our results suggested that tree N status and yield and fruit quality could be maintained by multiple urea sprays during the growing season in apple orchards, and foliar N application will reduce the risk of soil NO₃^–-N leaching.