Increased 5-HT3-mediated signalling in pelvic afferent neurons from mice deficient in P2X2 and/or P2X3 receptor subunits

Extracellular ATP and 5-hydroxytryptamine (5-HT) are both involved in visceral sensory pathways by interacting with P2X and 5-HT₃ receptors, respectively. We have investigated the changes in P2X and 5-HT₃-mediated signalling in pelvic afferent neurons in mice deficient in P2X₂ and/or P2X₃ subunits by whole-cell recording of L₆–S₂ dorsal root ganglion (DRG) neurons and by multi-unit recording of pelvic afferents of the colorectum. In wildtype DRG neurons, ATP evoked transient, sustained or mixed (biphasic) inward currents. Transient currents were absent in P2X₃ ^−/− neurons, whereas sustained currents were absent in P2X₂ ^−/− DRG neurons. Neither transient nor sustained currents were observed following application of ATP or α,β-methylene ATP (α,β-meATP) in P2X₂/P2X₃ ^Dbl−/− DRG neurons. 5-HT was found to induce a fast inward current in 63% of DRG neurons from wildtype mice, which was blocked by tropisetron, a 5-HT₃ receptor antagonist. The percentage of DRG neurons responding to 5-HT was significantly increased in P2X ₂ ^−/−, P2X₃ ^−/− and P2X₂/P2X₃ ^Dbl−/− mice, and the amplitude of 5-HT response was significantly increased in P2X₂/P2X₃ ^Dbl−/− mice. The pelvic afferent response to colorectal distension was attenuated in P2X₂/P2X₃ ^Dbl−/− mice, but the response to serosal application of 5-HT was enhanced. Furthermore, tropisetron resulted in a greater reduction in pelvic afferent responses to colorectal distension in the P2X₂/P2X₃ ^Dbl−/− preparations. These data suggest that P2X receptors containing the P2X₂ and/or P2X₃ subunits mediate purinergic activation of colorectal afferents and that 5-HT signalling in pelvic afferent neurons is up-regulated in mice lacking P2X₂ or P2X₃ receptor genes. This effect is more pronounced when both subunits are absent.     

Clorgyline and other propargylamine derivatives as inhibitors of succinate-dependent H<Subscript>2</Subscript>O<Subscript>2</Subscript> release at NADH:UBIQUINONE oxidoreductase (Complex I) in brain mitochondria

Complex I is the main O₂ ⁻ producer of the mitochondrial respiratory chain. O₂ ⁻ release is low with NAD-linked substrates and increases strongly during succinate oxidation, which increases the QH₂/Q ratio and is rotenone sensitive. We show that the succinate dependent O₂ ⁻ production (measured as H₂O₂ release) is inhibited by propargylamine containing compounds (clorgyline, CGP 3466B, rasagiline and TVP-1012). The inhibition does not affect membrane potential and is unaffected by ΔpH modifications. Mitochondrial respiration is similarly unaffected. The propargylamines inhibition of O₂ ⁻/H₂O₂ production is monitored also in the presence of the Parkinson's disease toxin dopaminochrome which stimulates O₂ ⁻ release. Propargylamine-containing compounds are the first pharmacological inhibitors described for O₂ ⁻ release at Complex I.     

Transport measurements across Caco-2 monolayers of different organic and inorganic selenium

The transport and uptake of the most common Se compounds, selenate (SeO ₄ ²⁻ ), selenite (SeO ₃ ²⁻ ), selenomethionine, and selenocystine, were investigated using confluent monolayers of Caco-2 cells, a human carcinoma cell line. Comparative measurements were performed in the absorptive (apical to basolateral side) and exsorptive (basolateral to apical side) directions. Apparent permeability coefficients (P _app), calculated from transport experiments in the absorptive direction, showed increasing values in the following rank order: about 1×10⁻⁶ cm/s ≤ mannitol ≤ SeO ₃ ²⁻ ≤ selenocystine < selenomethionine < SeO ₄ ²⁻ ≤ about 16×10⁻⁶ cm/s. The ratios of the P _app measured in the absorptive versus exsorptive directions indicated that only the organic forms presented a net polarized transport (P _app ratio ≫1), suggesting the presence of a transcellular pathway. No significant excretion was observed. The transport of selenomethionine was inhibited by its sulfur analog, methionine, suggesting a common transport mechanism. In contrast, an inhibition of the transport of selenocystine by cysteine was not observed. From the two substrates tested, sulfate and thiosulfate, only thiosulfate inhibited the transport of SeO ₄ ²⁻ . This effect was also observed for SeO ₃ ²⁻ (i.e., was unspecific), which questioned the assertion of a common transport for sulfate and SeO ₄ ²⁻ and may confirm the paracellular pathway of SeO ₄ ²⁻ suggested by the P _app ratio of about 1. The addition of glutathione (GSH) in large excess had no consequence on the passage of SeO ₃ ²⁻ but strongly increased the uptake (about fourfold). The liquid chromatography — mass spectrometry (LC-MS) data showed that, in the ionic condition of incubation medium, GSH promptly reduced SeO ₃ ²⁻ (≤2 min) in its elemental form Se⁰, which cannot ascribe to selenodiglutathione a direct role in the effect of GSH.     

CO2 exchange of the endolithic lichen Verrucaria baldensis from karst habitats in northern Italy

CO₂ exchange of the endolithic lichen Verrucaria baldensis was measured in the laboratory under different conditions of water content, temperature, light, and CO₂ concentration. The species had low CO₂ exchange rates (maximum net photosynthesis: c. 0.45 μmol CO₂ m⁻² s⁻¹; maximum dark respiration: c. 0.3 μmol CO₂ m⁻² s⁻¹) and a very low light compensation point (7 μmol photons m⁻² s⁻¹ at 8°C). The net photosynthesis/respiration quotient reached a maximum at 9–15°C. Photosynthetic activity was affected only after very severe desiccation, when high resaturation respiratory rates were measured. Microclimatic data were recorded under different weather conditions in an abyss of the Trieste Karst (northeast Italy), where the species was particularly abundant. Low photosynthetically active radiation (normally below 40 μmol photons m⁻² s⁻¹), very high humidities (over 80%), and low, constant temperatures were measured. Thallus water contents sufficient for CO₂ assimilation were often measured in the absence of condensation phenomena. Received: 22 September 1996 / Accepted: 26 April 1997     

Photosystem 2 activity of <Emphasis Type="Italic">Citrus volkameriana</Emphasis> (L.) leaves as affected by Mn nutrition and irradiance

Citrus volkameriana (L.) plants were grown for 43 d in nutrient solutions containing 0, 2, 14, 98, or 686 μM Mn (Mn₀, Mn₂, Mn₁₄, Mn₉₈, and Mn₆₈₆, respectively). To adequately investigate the combined effects of Mn nutrition and irradiance on photosystem 2 (PS2) activity, irradiance response curves for electron transport rate (ETR), nonphotochemical quenching (q_N), photochemical quenching (q_P), and real photochemical efficiency of PS2 (Φ_PS2) were recorded under 10 different irradiances (66, 96, 136, 226, 336, 536, 811, 1 211, 1 911, and 3 111 μmol m⁻² s⁻¹, I₆₆ to I₃₁₁₁, respectively) generated with the PAM-2000 fluorometer. Leaf chlorophyll content was significantly lower under Mn excess (Mn₆₈₆) compared to Mn₀; its highest values were recorded in the treatments Mn₂-Mn₉₈. However, ETR and Φ_PS2 values were significantly lower under Mn₀ compared to the other Mn treatments, when plants were exposed to irradiances ≥96 μmol m⁻² s⁻¹. Furthermore, Mn₀ plants had significantly higher values of q_N and lower values of q_P at irradiances ≤226 and ≥336 μmol m⁻² s⁻¹, respectively, than those grown under Mn₂-Mn₆₈₆. Irrespective of Mn treatment, the values of Φ_PS2 and q_N decreased, while those of q_P increased progressively by increasing irradiance from I₁₃₆ to I₃₁₁₁. Finally, Mn₂-Mn₉₈ plants were less sensitive to photoinhibition of photosynthesis (≥811 μmol m⁻² s⁻¹) than the Mn₆₈₆ (≥536 μmol m⁻² s⁻¹) and Mn₀ (≥336 μmol m⁻² s⁻¹) ones.     

Kinetics and mechanism for reduction of anticancer-active tetrachloroam(m)ine platinum(IV) compounds by glutathione

trans -[PtCl₄(NH₃)(thiazole)] (1), trans-[PtCl₄(cha)(NH₃)] (2), cis-[PtCl₄(cha)(NH₃)] (3) (cha =cyclohexylamine), and cis-[PtCl₄(NH₃)₂] (4) has been investigatedat 25 °C in a 1.0 M aqueous medium at pH 2.0–5.0 (1) and 4.5–6.8 (2–4) using stopped-flow spectrophotometry. The redox reactions follow the second-order rate law , where k is a pH-dependent rate constant and [GSH]_tot the total concentration of glutathione. The reduction takes place via parallel reactions between the platinum(IV) complexes and the various protolytic species of glutathione. The pH dependence of the redox kinetics is ascribed to displacement of these protolytic equilibria. The thiolate species GS⁻ is the major reductant under the reaction conditions used. The second-order rate constants for reduction of compounds 1–4 by GS⁻ are (1.43±0.01)×10⁷, (3.86±0.03)×10⁶, (1.83±0.01)×10⁶, and (1.18±0.01)×10⁶M⁻¹s⁻¹, respectively. Rate constants for reduction of 1 by the protonated species GSH are more than five orders of magnitude smaller. The mechanism for the reductive elimination reactions of the Pt(IV) compounds is proposed to involve an attack by glutathione on one of the mutually trans coordinated chloride ligands, leading to two-electron transfer via a chloride-bridged activated complex. The kinetics results together with literature data indicate that platinum(IV) complexes with a trans Cl-Pt-Cl axis are reduced rapidly by glutathione as well as by ascorbate. In agreement with this observation, cytotoxicity profiles for such complexes are very similar to those for the corresponding platinum(II) product complexes. The rapid reduction within 1 s of the platinum(IV) compounds with a trans Cl-Pt-Cl axis to their platinum(II) analogs does not seem to support the strategy of using kinetic inertness as a parameter to increase anticancer activity, at least for this class of compounds. Received: 8 December 1999 / Accepted: 15 February 2000     

Theoretical study of hydrogenation of the doubly aromatic B 7 − cluster

We have studied the influence of hydrogenation on the relative stability of the low-lying isomers of the anionic B₇⁻ cluster, computationally. It is known that the pure-boron B₇⁻ cluster has a doubly (σ- and π-) aromatic C_6v (³A₁) quasi-planar wheel-type triplet global minimum (structure 1), a low-lying σ-aromatic and π-antiaromatic quasi-planar singlet C_2v (¹A₁) isomer 2 (0.7 kcal mol⁻¹ above the global minimum), and a planar doubly (σ- and π-) antiaromatic C_2v (¹A₁) isomer 3 (7.8 kcal mol⁻¹ above the global minimum). However, upon hydrogenation, an inversion in the stability of the species occurs. The planar B₇H₂⁻ (C_2v, ¹A₁) isomer 4, originated from the addition of two hydrogen atoms to the doubly antiaromatic B₇⁻ isomer 3, becomes the global minimum structure. The second most stable B₇H₂⁻ isomer 5, originated from the quasi-planar triplet wheel isomer 1 of B₇⁻, was found to be 27 kcal mol⁻¹ higher in energy. The inversion in stability occurs due to the loss of the doubly aromatic character in the wheel-type global minimum isomer (C_6v, ³A₁) of B₇⁻ upon H₂−addition. In contrast, the planar isomer of B₇⁻ (C_2v, ¹A₁) gains aromatic character upon addition of two hydrogen atoms, which makes it more stable. Figure The B₇H₂-global minimum structure and its σ-aromatic and π-antiaromatic MOs Dedicated to Professor Dr. Paul von Ragué Schleyer on the occasion of his 75th birthday.     

Avocado shoot culture, plantlet development and net CO2 assimilation in an ambient and CO2 enhanced environment

Summary The proliferation and survival of avocado nodal cultures of juvenile origin were affected by the form and concentration of nitrogen. Optimum growth was achieved on modified Murashige and Skoog medium containing 67% KNO₃ and 33% NH₄NO₃ with total N of 40 mM supplemented with 100 mg l⁻¹ myo-inositol, 1 mg l⁻¹ thiamine HCl, 30 g l⁻¹ sucrose, and 4.44 μM BA with a 16-h photoperiod (120–150 μmol m⁻² s⁻¹). Proliferating shoots and plantlets were photosynthetically active. Better shoot growth and accumulation of higher biomass occurred in a CO₂-enriched environment than under ambient CO₂ conditions. CO₂ assimilation efficiency, however, was higher under the latter conditions than in a CO₂-enhanced environment, e.g., 31±7 and 17±2 μmol CO₂ m⁻² s⁻¹, respectively. The net CO₂ assimilation rates of in vitro grown plantlets were comparable to those of seedlings ex vitro.     

Cardio-vascular and ventilatory effects of prostaglandin E2 in the European eel Anguilla anguilla

Prostaglandin E₂ (PGE₂) injection caused an initial decrease in heart rate (HR) (55%), cardiac output (QC) (55%), ventral (VAP, 40%) and dorsal (DAP, 60%) aortic pressures, associated with a marked gill vasoconstriction (100%) and a systemic vasodilation (30%) while cardiac stroke volume (CSV) remained unchanged. It was followed by a progressive recovery of HR, DAP, and vascular resistances to control values while VAP, QC, and CSV significantly increased (20%, 40%, and 40%, respectively). Bradycardia was abolished either by atropine treatment or bilateral section of the cardiac vagus, suggesting that bradycardia could be the consequence of a vagal mechanism triggered by a central action of PGE₂. The increase in QC is due to the increase in CSV, which suggests a positive inotropic action of PGE₂. Infusion with PGE₂ (10⁻⁹ to 10⁻⁵ mol · l⁻¹) did not cause any significant effect on the branchial vasculature in vitro, but when the preparation was pre-treated with adrenaline (10⁻⁸ mol · l⁻⁴), a dose-related vasoconstrictory response to PGE₂ (10⁻⁹ to 10⁻⁵ mol · l⁻¹) occurred. A significant rise (25%) in gill vascular resistance was observed in vivo after treatment with the β-blocker propranolol (2.0 mg · kg⁻¹), suggesting the existence of a vasodilatory β-adrenergic control of the branchial vasculature. PGE₂ caused an apnea followed by a simultaneous tachypnea (50%) and decrease in ventilatory amplitude (40%). The resulting decrease in ventilatory water flow (Q_w) was accompanied by a fall in arterial O₂ tension (Pa O ₂). Under steady Q_w in curarized eels, an initial fall in Pa O ₂ was still observed during the PGE₂-induced gill vasoconstriction. It is concluded that PGE₂ could play a role in the regulation of gas exchanges and blood circulation in particular environmental conditions which require an impairment of gill respiration and a redistribution of blood to the benefit of the systemic vascular bed. Accepted: 3 June 1997     

N2O, CH4 and CO2 emissions from seasonal tropical rainforests and a rubber plantation in Southwest China

The main focus of this study was to evaluate the effects of soil moisture and temperature on temporal variation of N₂O, CO₂ and CH₄ soil-atmosphere exchange at a primary seasonal tropical rainforest (PF) site in Southwest China and to compare these fluxes with fluxes from a secondary forest (SF) and a rubber plantation (RP) site. Agroforestry systems, such as rubber plantations, are increasingly replacing primary and secondary forest systems in tropical Southwest China and thus effect the N₂O emission in these regions on a landscape level. The mean N₂O emission at site PF was 6.0 ± 0.1 SE μg N m⁻² h⁻¹. Fluxes of N₂O increased from <5 μg N m⁻² h⁻¹ during dry season conditions to up to 24.5 μg N m⁻² h⁻¹ with re-wetting of the soil by the onset of first rainfall events. Comparable fluxes of N₂O were measured in the SF and RP sites, where mean N₂O emissions were 7.3 ± 0.7 SE μg N m⁻² h⁻¹ and 4.1 ± 0.5 SE μg N m⁻² h⁻¹, respectively. The dependency of N₂O fluxes on soil moisture levels was demonstrated in a watering experiment, however, artificial rainfall only influenced the timing of N₂O emission peaks, not the total amount of N₂O emitted. For all sites, significant positive correlations existed between N₂O emissions and both soil moisture and soil temperature. Mean CH₄ uptake rates were highest at the PF site (−29.5 ± 0.3 SE μg C m⁻² h⁻¹), slightly lower at the SF site (−25.6 ± 1.3 SE μg C m⁻² h⁻¹) and lowest for the RP site (−5.7 ± 0.5 SE μg C m⁻² h⁻¹). At all sites, CH₄ uptake rates were negatively correlated with soil moisture, which was also reflected in the lower uptake rates measured in the watering experiment. In contrast to N₂O emissions, CH₄ uptake did not significantly correlate with soil temperature at the SF and RP sites, and only weakly correlated at the PF site. Over the 2 month measurement period, CO₂ emissions at the PF site increased significantly from 50 mg C m⁻² h⁻¹ up to 100 mg C m⁻² h⁻¹ (mean value 68.8 ± 0.8 SE mg C m⁻² h⁻¹), whereas CO₂ emissions at the SF and RP site where quite stable and varied only slightly around mean values of 38.0 ± 1.8 SE mg C m⁻² h⁻¹ (SF) and 34.9 ± 1.1 SE mg C m⁻² h⁻¹ (RP). A dependency of soil CO₂ emissions on changes in soil water content could be demonstrated for all sites, thus, the watering experiment revealed significantly higher CO₂ emissions as compared to control chambers. Correlation of CO₂ emissions with soil temperature was significant at the PF site, but weak at the SF and not evident at the RP site. Even though we demonstrated that N and C trace gas fluxes significantly varied on subdaily and daily scales, weekly measurements would be sufficient if only the sink/ source strength of non-managed tropical forest sites needs to be identified.     

Microplankton respiratory activity and CO2 production rates in the Otranto Strait (Mediterranean Sea)

Respiratory activity and metabolic CO₂production of the microplankton in the Otranto Strait (Mediterranean Sea) were determined by monitoring the Electron Transport System activity. Ten stations were repeatedly investigated during two oceanographic surveys in February–March and August 1994. Respiratory activity and CO₂ production, estimated from the surface to the bottom, were higher in the euphotic layers (0-200 m) during summer (mean values: Winter = 0.024 μg C h⁻¹ dm⁻³; Summer = 0.042 μg C h⁻¹ dm⁻³); in the aphotic zone (deeper than 200 m), the rates were similar throughout different seasons (0.013 and 0.014 μg C h⁻¹ dm⁻³, respectively). A comparison with data collected by other authors from the euphotic layers of the Mediterranean Sea was made. Respiratory activities decreased from Western to Eastern Mediterranean Basins. The values of CO₂ production, integrated between 200 and 1000 m in the Otranto Strait (mean value 237.7 mg C m⁻² d⁻¹), were compared with other data collected from the Mediterranean Sea as well as from the Pacific, Atlantic and Indian Oceans. The comparison showed the Otranto Strait to be a site of organic matter oxidation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Contribution of plants to N 2 O emissions in soil-winter wheat ecosystem: pot and field experiments

Outdoor pot and field experiments were conducted to assess the role of growing plants in agricultural ecosystem N₂O emissions. N₂O emissions from plants were quantified as the difference in soil-crop system N₂O emissions before and immediately after cutting plants during the main growth stages in 2001–02 and 2002–03 winter wheat seasons. Emissions of N₂O from plants depended on biomass within the same plant developmental status. Field results indicated that the seasonal contribution of N₂O emissions from plants to ecosystem fluxes averaged 25%, ranging from 10% at wheat tillering to 62% at the heading stage. The fluxes of N₂O emissions from plants varied between 0.3 and 3.9 mg N₂O-N m⁻² day⁻¹ and its seasonal amount was equivalent to 0.23% of plant N released as N₂O. A N₂O emission coefficient (N₂O_E, mg N₂O-N g⁻¹ C day⁻¹), defined as N₂O-N emission in milligrams from per gram carbon of plant dry matter within a day, was represented by a 5-fold variation ranging from 0.021 to 0.004 mg N₂O-N g C⁻¹ day⁻¹. A linear relationship (y=0.4611x+0.0015, r ²=0.9352, p < 0.001) between N₂O_E (y) and plant dark respiration rate (x, mg CO₂-C g C⁻¹ day⁻¹) suggested that in the absence of photosynthesis, some N₂O production in plant N assimilation was associated with plant respiration. Although this study could not show whether N₂O was produced or transferred by winter wheat plants, these results indicated an important role for higher plant in N₂O exchange. Identifying its potential contribution is critical for understanding agricultural ecosystem N₂O sources.     

Acclimation of the summer annual species, Lolium temulentum, to CO2 enrichment

Lolium temulentum L. Ba 3081 was grown hydroponically in air (350 μmol mol⁻¹ CO₂) and elevated CO₂ (700 μmol mol⁻¹ CO₂) at two irradiances (150 and 500 μmol m⁻² s⁻¹) for 35 days at which point the plants were harvested. Elevated CO₂ did not modify relative growth rate or biomass at either irradiance. Foliar carbon-to-nitrogen ratios were decreased at elevated CO₂ and plants had a greater number of shorter tillers, particularly at the lower growth irradiance. Both light-limited and light-saturated rates of photosynthesis were stimulated. The amount of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) protein was increased at elevated CO₂, but maximum extractable Rubisco activities were not significantly increased. A pronounced decrease in the Rubisco activation state was found with CO₂ enrichment, particularly at the higher growth irradiance. Elevated-CO₂-induced changes in leaf carbohydrate composition were small in comparison to those caused by changes in irradiance. No CO₂-dependent effects on fructan biosynthesis were observed. Leaf respiration rates were increased by 68% in plants grown with CO₂ enrichment and low light. We conclude that high CO₂ will only result in increased biomass if total light input favourably increases the photosynthesis-to-respiration ratio. At low irradiances, biomass is more limited by increased rates of respiration than by CO₂-induced enhancement of photosynthesis. Received: 23 February 1999 / Accepted: 15 June 1999     

Subsurface CO<Subscript>2</Subscript> Dynamics in Temperate Beech and Spruce Forest Stands

Rates of soil respiration (CO₂ effluxes), subsurface pore gas CO₂/O₂ concentrations, soil temperature and soil water content were measured for 15 months in two temperate and contrasting Danish forest ecosystems: beech (Fagus sylvatica L.) and Norway spruce (Picea abies [L.] Karst.). Soil CO₂ effluxes showed a distinct seasonal trend in the range of 0.48–3.3 μmol CO₂ m⁻² s⁻¹ for beech and 0.50–2.92 μmol CO₂ m⁻² s⁻¹ for spruce and were well-correlated with near-surface soil temperatures. The soil organic C-stock (upper 1 m including the O-horizon) was higher in the spruce stand (184±23 Mg C ha⁻¹) compared to the beech stand (93±19 Mg C ha⁻¹) and resulted in a faster turnover time as calculated by mass/flux in soil beneath the beech stand (28 years) compared to spruce stand (60 years). Observed soil CO₂ concentrations and effluxes were simulated using a Fickian diffusion-reaction model based on vertical CO₂ production rates and soil diffusivity. Temporal trends were simulated on the basis of observed trends in the distribution of soil water, temperature, and live roots as well as temperature and water content sensitivity functions. These functions were established based on controlled laboratory incubation experiments. The model was successfully validated against observed soil CO₂ effluxes and concentrations and revealed that temporal trends generally could be linked to variations in subsurface CO₂ production rates and diffusion over time and with depths. However, periods with exceptionally high CO₂ effluxes (> 20 μmol CO₂ m⁻² s⁻¹) were noted in March 2000 in relation to drying after heavy rain and after the removal of snow from collars. Both cases were considered non-steady state and could not be simulated.     

Inhibition by light of CO2 evolution from dark respiration: Comparison of two gas exchange methods

Two approaches to determine the fraction (μ) of mitochondrial respiration sustained during illumination by measuring CO₂ gas exchange are compared. In single leaves, the respiration rate in the light (`day respiration' rate R<sub>d</sub>) is determined as the ordinate of the intersection point of A–c<sub>i</sub> curves at various photon flux densities and compared with the CO<sub>2</sub> evolution rate in darkness (`night respiration' rate R_n). Alternatively, using leaves with varying values of CO₂ compensation concentration (Γ), intracellular resistance (r_i) and R_n, an average number for μ can be derived from the linear regression between Γ and the product r_iċR_n. Both methods also result in a number c* for that intercellular CO₂ concentration at which net CO₂ uptake rate is equal to –R_d. c* is an approximate value of the photocompensation point Γ* (Γ in the absence of mitochondrial respiration), which is related to the CO₂/O₂ specificity factor of Rubisco S_c/o. The presuppositions and limitations for application of both approaches are discussed. In leaves of Nicotiana tabacum, at 22 °C, single leaf measurements resulted in mean values of μ = 0.71 and c_* = 34 μmol mol⁻¹. At the photosynthetically active photon flux density of 960 μmol quanta m⁻² s⁻¹, nearly the same numbers were derived from the linear relationship between Γ and r_iċR_n. c* and R_d determined by single leaf measurements varied between 31 and 41 μmol mol⁻¹ and between 0.37 and 1.22 μmol m⁻² s⁻¹, respectively. A highly significant negative correlation between c* and R_d was found. From the regression equation we obtained estimates for Γ* (39 μmol mol⁻¹), S_c/o (96.5 mol mol⁻¹) and the mesophyll CO₂ transfer resistance (7.0 mol⁻¹ m² s). This revised version was published online in June 2006 with corrections to the Cover Date.     

Density functional theory studies of model complexes for molybdenum-dependent nitrate reductase active sites

Molybdenum and tungsten complexes as models for the active sites of assimilatory or dissimilatory nitrate reductases (NR) were computed at the CPCM-B98/SDDp//B3LYP/Lanl2DZp* plus zero point energy level of density functional theory. The ligands were chosen on the basis of available experimental protein or small chemical model structures. A water molecule is found to bind to assimilatory NR models [(Me₂C₂S₂)MO(YMe)]⁻ (−11.5 kcal mol⁻¹ for M is Mo, Y is S) and may be replaced by nitrate (−4.5 kcal mol⁻¹) (but a hydroxy group may not). Nature’s choice of M is Mo and Y is S for NR has the largest activation energy for protein-free models (13.3 kcal mol⁻¹) and the least exothermic reaction energy for the nitrate reduction (−14.9 kcal mol⁻¹) compared with M is W and Y is O or Se alternatives. Water binding to dissimilatory NR model complexes [(Me₂C₂S₂)₂M(YR)]⁻ is considerably endothermic (10.3 kcal mol⁻¹); nitrate binding is only slightly so (1.5 kcal mol⁻¹ for RY⁻ is MeS⁻). The exchange of an oxo ligand (assimilatory NR) for a dithiolato ligand (dissimilatory NR model) reduces the exothermicity (−8.6 kcal mol⁻¹ relative to the fivefold-coordinate reduced complex) and raises the barrier for oxygen atom transfer (OAT) in the nitrate complex (19.2 kcal mol⁻¹). Not for the mono but only for the bisdithiolato complexes hydrogen bonding involving the coordinated substrate may significantly lower the OAT barrier as shown by explicitly adding water molecules. Substitution of tungsten for molybdenum generally lowers OAT activation energies and makes nitrate reduction reaction energies more negative. Bidentate carboxylato binding identified in Escherichia coli NarGHI is the preferred binding mode also for an acetato model. However, one dithiolato ligand folds when the Mo^VI center is bare of a good π-donor ligand, e.g., an oxo group. Computations on [(mnt)₂Mo^IV(YR)(PPh₃)]⁻ [mnt is (CN)₂C₂S₂ ²⁻] gave a smaller nitrate reduction activation energy for RY⁻ is Cl⁻, compared with RY⁻ is PhS⁻, although experimentally only the phenyl thiolato complex and not the chloro complex was found to be a functional NR model. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Photosynthetic parameters of young Brazil nut (<Emphasis Type="Italic">Bertholletia excelsa</Emphasis> H. B.) plants subjected to fertilization in a degraded area in Central Amazonia

In an experimental site for reforestation of degraded area, three-year-old plants of Bertholletia excelsa Humb. & Bonpl. were subjected to different fertilization treatments: T0 = unfertilized control, T1 = green fertilization (branches and leaves) and T2 = chemical fertilization. Higher net photosynthetic rates (P _N) were observed in T1 [13.2±1.0 μmol(CO₂) m⁻² s⁻¹] compared to T2 [8.0±1.8 μmol(CO₂) m⁻² s⁻¹] and T0 [4.8±1.3 μmol(CO₂) m⁻² s⁻¹]. Stomatal conductance (g _s), transpiration rate (E) and water use efficiency (WUE) of individuals of T1 and T2 did not differ significantly, however, they were by 88, 55 and 63%, respectively, higher in T1 than in the control. The mean values of variable fluorescence (F_v), performance index (P.I.) and total chlorophyll [Chl (a+b)] were higher in T1. Our results indicate that green fertilization improves photosynthetic structure and function in plants of B. excelsa in young phase.     

Short-term inhibition of legume N2 fixation by nitrate

The hypothesis of NO ₂ ⁻ toxicity as the causative factor of NO ₃ ⁻ inhibition of nitrogenase (N₂ase; EC 1.18.6.1) activity has been evaluated using a short-term exposure (3 d) of several legumes. Treatment of plants with 10 mM NO ₃ ⁻ induced nitrate reductase (NR) from bacteroids (EC 1.7.99.4) and nodule cytosol (EC 1.6.6.1) in most species. Regardless of the levels of both enzymes, significant accumulation of NO ₂ ⁻ did not occur in nodules. Dissection of nodules into cortical and infected regions, and subsequent NO ₂ ⁻ assays in conditions that suppressed enzyme activities, indicated that, in the short-term, bacteroid NR does not generate NO ₂ ⁻ in vivo. This is probably because NO ₃ ⁻ access is restricted to the nodule cortex. Accumulation of NO ₂ ⁻ at levels that are damaging for N₂ase and leghaemoglobin were only observed when a delay occurred between dissection and assaying of nodules. It is concluded that NO ₂ ⁻ is not responsible for the initial NO ₃ ⁻ -induced decline of N₂ase activity, and that toxic amounts of NO ₂ ⁻ only build up in nodules following longer exposures to NO ₃ ⁻ , when this anion is actively reduced by bacteroid and cytosol enzymes.     

Kinetics of urea uptake by Melosira italica (Ehr.) Kütz at different luminosity conditions

The kinetic parameters K_m and V_max for urea uptake by Melosira italica were determined at 160 μeinsteins m⁻² s⁻¹ and in the dark. The transport systems showed an affinity for the substrate and a storing capacity in the dark (K_m = 65.07 μM; V_max = 2.18 nmoles 10⁵ cells ⁻¹ h⁻¹) greater than under 160 μE m⁻² s ⁻¹ (K_m = 111.2 μM; V_max = 1.11 nmoles 105 cells⁻¹ h⁻¹). Similarly, a reduction in consumption rate of urea under increasing photon flux densities was observed. The use of an inhibitor (potassium cyanide) indicated that the uptake process requires metabolic energy. That urea transport is more important in darkness, may constitute a survival strategy in which this compound is utilized by cells mainly during heterotrophic growth.     

Nitrite utilization by Chaetoceros muelleri under elevated CO2 concentration

Chaetoceros muelleri (Lemn.) was cultured with nitrite (NO₂⁻) or nitrate (NO₃⁻) as the sole nitrogen source and aerated with air or with CO₂-enriched air. Cells of C. muelleri excreted into the medium nitrite produced by reduction of nitrate when grown with 100 μM NaNO₃ as nitrogen source. Accordingly, NO₂⁻ concentration reached 10.4 μM after 95 h at the low CO₂ condition (aerated with air); while the maximum NO₂⁻ concentration was only around 2.0 μM at the high CO₂ condition (aerated with 5% CO₂ in air), furthermore, after 30 h it decreased to no more than 1.0 μM. NO₂⁻ was almost assimilated in 80 h when C. muelleri was cultured at the high CO₂ condition with 100 μM NaNO₂ as sole nitrogen source. At the high CO₂ condition, after 3 h the activity of nitrite reductase was as much as 50% higher than that at the low CO₂ condition. It was indicated that enriched CO₂ concentration could inhibit nitrite excretion and enhance nitrite assimilation by cells. Therefore, aeration with enriched CO₂ might be an effective way to control nitrite content in aquaculture systems.