Influence of nerve fiber K+ depolarization and altered membrane protein conformation on the state of myelin

Using Raman spectrometry and fluorescence microscopy, we studied the rearrangement of carotenoid molecules and membrane-bound Ca _mb ²⁺ in myelinated nerve fibers after K⁺ depolarization, K⁺-channel blocking, and altering the membrane protein conformation. We observed a decrease in Ca _mb ²⁺ and an increase of microviscosity in myelin after depolarization. Changes in Ca _mb ²⁺ and microviscosity were registered after blocking the K⁺ channels and modifying proteins with PCMB. Our results suggest an interconnection between the condition of nerve fiber membrane proteins, Ca _mb ²⁺ distribution, and myelin microviscosity.     

Competitive blockage of the sodium channel by intracellular magnesium ions in central mammalian neurones

The aim of this study was to determine from macroscopic current analysis how intracellular magnesium ions, Mg _i ²⁺ , interfere with sodium channels of mammalian neurones. It is reported here that permeation across the sodium channel is voltage- and concentration-dependently reduced by Mg _i ²⁺ . This results in a general reduction of sodium membrane conductance and an outward sodium peak current at large positive potentials. 30 mM Mg _i ²⁺ leads to a negative shift of voltage dependence of sodium channel gating parameters, probably due to the surface potential change of the membrane. This shift alone is, however, insufficient to explain the reduction of outward sodium currents. The blockage by Mg _i ²⁺ is decreased upon increasing intracellular or extracellular Na⁺ concentration, which suggests that Mg?' interferes with sodium permeation by competitively occupying sodium channels. Using a kinetic model to describe the sodium permeation, the dissociation constant (at zero membrane potential) of Mg _i ²⁺ for the sodium channel has been calculated to be 8.65 ± 1.51 mM, with its binding site located at 0.26 ± 0.05 electrical distance from the inner membrane. This dissociation constant is smaller than that of Na _i ⁺, which is 83.76 ± 7.60 mM with its binding site located at 0.75 ± 0.23. The low dissociation constant of Mg _i ²⁺ reflects its high affinity for the sodium channel.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

Effects of nitric oxide on protein-lipid interactions in the membranes of the myelinated nerve fiber

The influence of nitric oxide (NO) on the myelinated nerve fiber and the impact of modification of SHgroups of axon and myelin membrane proteins on the amplitude and propagation velocity of action potential (AP), amount of the membrane-bound calcium (Ca _mb ²⁺ , viscosity of the axon membrane, and saturation factor of phospholipid fatty acids (Sf) of myelin have been investigated. We established that the decrease in the number of extracellular SH-groups in membrane proteins induced by p-chloromercuribenzoate (pCMB, 10^?4 M), led to a decrease in the AP amplitude and a reversible desorption of Ca _mb ²⁺ but did not affect the axolemma viscosity and Sf. Nitric oxide (NO) caused a decrease in the AP amplitude and propagation velocity, an increase in the axolemma viscosity and a decrease in Sf of myelin; it also induced a reversible desorption of Ca _mb ²⁺ . Pretreatment of the nerve fiber with pCMB weakened the NO-induced desorption of Pretreatment of the nerve fiber with K⁺-channel blocker tetraethylammonium (10^?2 M) completely abolished the NO-induced change in the amount of Ca _mb ²⁺ . We suppose that NO-mediated changes in axolemma viscosity, Sf of myelin and desorption of Ca _mb ²⁺ affect protein-lipid interactions in axolemma and myelin, which in their turn influence the propagation of AP.     

Ammonium nutrition increases water absorption in rice seedlings (Oryza sativa L.) under water stress

Water stress is a primary limitation on plant growth. In previous studies, it has been found that ammonium enhances the tolerance of rice plants to water stress, but how water is related to nitrogen form and water stress remains unknown. To study the effects of nitrogen form (NH ₄ ⁺ , NO ₃ ^? , and a mixture of NH ₄ ⁺ and NO ₃ ^? ) on the growth and water absorption of rice (Oryza sativa L.) seedlings, a hydroponic experiment with water stress, simulated by the addition of polyethylene glycol (PEG, 10% w/v, MW 6000), was conducted in a greenhouse. The results showed that, compared with non-water stress, under water stress, the fresh weight of rice seedlings increased by 14% with NH ₄ ⁺ nutrition, whereas it had decreased by about 20% with either NO ₃ ^? or mixed nitrogen nutrition. No significant difference was found in the transpiration rate of excised shoots or in xylem exudation of excised roots in NH ₄ ⁺ supply between the two water situations, whereas xylem flow decreased by 57% and 24% under water stress in NO ₃ ^? and mixed nutrition, and root hydraulic conductivity decreased by 29% and 54% in plants in NH ₄ ⁺ and NO ₃ ^? nutrition conditions, respectively. Although water absorption ability decreased in both NH ₄ ⁺ and NO ₃ ^? nutrition, aquaporin activity was higher in NH ₄ ⁺ than in NO ₃ ^? nutrition, regardless of water stress. We conclude that NH ₄ ⁺ nutrition can improve water handling in rice seedlings and subsequently enhance their resistance to drought.     

The effect of transpiration on selenium uptake and mobility in durum wheat and spring canola

Aims

The objective of this study was to determine the relative importance of transpirational pull, Se speciation, sulfate and species on Se accumulation by plants, in order to determine which of these factors must be considered in the future development of models to predict Se accumulation by plants.

Methods

Seedlings of durum wheat (Triticum turgidum L. var durum cv ‘Kyle’) and spring canola (Brassica napus L. var Hyola 401) were grown hydroponically and exposed to SeO ₄ ^2- (selenate) with or without SO ₄ ^2- (sulfate), or to HSeO ₃ ^- (biselenite) under different transpiration regimes altered through ‘low’ (~50%) or ‘high’ (~78%) relative humidity (RH). Plants were harvested after 0, 8, 16, or 24?h exposures, digested, and analyzed for Se by GFAAS.

Results

Accumulation and distribution of Se by plants is dependent on plant species, Se speciation in the nutrient solution, SO ₄ ^2- competition, and transpiration regimes. Canola accumulated and translocated more Se than wheat. In wheat and canola, the greatest accumulation and translocation of Se occurred when plants were exposed to SeO ₄ ^2- without SO ₄ ^2- compared to solutions of SeO ₄ ^2- with SO ₄ ^2- or HSeO ₃ ^2- . Wheat plants exposed to SeO ₄ ^2- and SO ₄ ^2- had an increased Se accumulation and translocation under increased transpiration rates than when exposed to SeO ₄ ^2- without SO ₄ ^2- or HSeO ₃ ^2- . On the other hand, increases in transpiration increased the translocation of Se to canola shoots when exposed to HSeO ₃ ^- more than any other treatments.

Conclusions

Overall, our results suggest that plant species is the most important factor influencing Se accumulation and translocation, but that these endpoints can be modified by climate and specific soil Se or S content. Models to predict accumulation of Se by plants must consider all of these factors to accurately calculate the mechanisms of uptake and translocation.     

Characterization of ammonium and nitrate uptake and assimilation in roots of tea plants

It has been pointed out that tea (Camellia sinensis (L.) O. Kuntze) prefers ammonium (NH ₄ ⁺ ) over nitrate (NO ₃ ^? ) as an inorganic nitrogen (N) source. ¹⁵N studies were conducted using hydroponically grown tea plants to clarify the characteristics of uptake and assimilation of NH ₄ ⁺ and NO ₃ ^? by tea roots. The total ¹⁵N was detected, and kinetic parameters were calculated after feeding ¹⁵NH ₄ ⁺ or ¹⁵NO ₃ ^? to tea plants. The process of N assimilation was studied by monitoring the dynamic ¹⁵N abundance in the free amino acids of tea plant roots by GC-MS. Tea plants supplied with ¹⁵NH ₄ ⁺ absorbed significantly more ¹⁵N than those supplied with ¹⁵NO ₃ ^? . The kinetics of ¹⁵NH ₄ ⁺ and ¹⁵NO ₃ ^? influx into tea plants followed a classic biphasic pattern, demonstrating the action of a high affinity transport system (HATS) and a low affinity transport system (LATS). The V _max value for NH ₄ ⁺ uptake was 54.5 nmol/(g dry wt min), which was higher than that observed for NO ₃ ^? (39.3 nmol/(g dry wt min)). K_M estimates were approximately 0.06 mM for NH ₄ ⁺ and 0.16 mM for NO ₃ ^? , indicating a higher rate of NH ₄ ⁺ absorption by tea plant roots. Tea plants fed with ¹⁵NH ₄ ⁺ accumulated larger amounts of assimilated N, especially glutamine (Gln), compared with those fed with ¹⁵NO ₃ ^? . Gln, Glu, theanine (Thea), Ser, and Asp were the main free amino acids that were labeled with ¹⁵N under both conditions. The rate of N assimilation into Thea in the roots of NO ₃ ^? -supplied tea plants was quicker than in NH ₄ ⁺ -supplied tea plants. NO ₃ ^? uptake by roots, rather than reduction or transport within the plant, seems to be the main factor limiting the growth of tea plants supplied with NO ₃ ^? as the sole N source. The NH ₄ ⁺ absorbed by tea plants directly, as well as that produced by NO ₃ ^? reduction, was assimilated through the glutamine synthetase-glutamine oxoglutarate aminotransferase pathway in tea plant roots. The ¹⁵N labeling experiments showed that there was no direct relationship between the Thea synthesis and the preference of tea plants for NH ₄ ⁺ .     

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors

Effect of cations K⁺, Na⁺, Mg²⁺, and Ca²⁺ and anions Cl^?, SO ₄ ^2? , HCO ₃ ^? , and CO ₃ ^2? on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ecolum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC₅₀, decreased in the series Ca²⁺ > Na⁺ > Mg²⁺ > K⁺. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.     

Spatial and temporal variation of seepage water chemistry after femel and small scale clear-cutting in a N-saturated Norway spruce stand

The chemistry of seepage water was studied before and after small scale clear-cutting and femel cutting (removing 20% of the trees) between 1999 and 2002 at the H?glwald site in southern Bavaria. The interventions were performed in February 2000 on mature, N-saturated Norway spruce (Picea abies (L.) Karst.) stands with high NO ₃ ⁻ concentrations before felling. Seepage water was collected with suction cups at 40 cm soil depth in the following treatments: (I) a mature stand (control), (II) a femel-cut, and (III) a clear-cut. In the femel cut subvariants were created with suction cups (plots) at varying distances from pre-selected spruce, which were later removed. The femel treatment was replanted with beech (Fagus sylvatica L.) saplings. On the clear-cut, subvariants of planted beech (close to the stem, interstem area), planted spruce (interstem), or natural spruce regeneration were investigated. Clear-cutting caused high NO ₃ ⁻ peaks (average values up to 2750 μM) during 2000 and 2001 in all planted tree subvariants during times of comparatively low water fluxes. In contrast to peak concentrations, flux weighted yearly average concentrations showed different trends. In 2000, flux weighted yearly average NO ₃ ⁻ concentrations were significantly elevated, but only on the subvariants of the interstem area, which covered in the clear-cut plot ca. 62% of the area. However, the subvariant close to the stem (31% of clear-cut area), or the natural spruce regeneration subvariant (6% of clear-cut area) exhibited no significant felling effect. With respect to the whole treatment area, this resulted in no significant felling effect as compared with the control. In the next year (2001), flux weighted yearly average NO ₃ ⁻ concentrations were not significantly affected by clear-cutting, while the concentrations were even reduced for all of the clear-cut subvariants in 2002. On the subvariant natural spruce regeneration, NO ₃ ⁻ concentrations remained below the European limit of drinking water (806 μM) during almost the whole investigation period. Selective cutting resulted in slightly reduced NO ₃ ⁻ concentrations in 2000 and 2001 on the femel treatment. However, no significant effect could be detected for any subvariant in the femel-cut, even not for the subvariant with suction cups closest to the felled spruce. In contrast to many other investigations, clear-cutting did not increase the NO ₃ ⁻ problem on the treatment to a relevant extend. Quite contrary, a decline in NO ₃ ⁻ concentrations to values below the EU level for drinking water and levels below the control and femel treatment just 2 years after cutting were observed. Al³⁺ concentrations showed nearly the same trend as NO ₃ ⁻ , while Ca²⁺, Mg²⁺, and K⁺ concentrations were affected to a lesser degree. Only in 2002 was Ca²⁺ significantly lower on the clear-cut as compared to the femel treatment, but not compared to the control. Mg²⁺ increased in 2000 on the clear-cut subvariants in the interstem area, but decreased in the years 2001 and 2002. Changes could be observed for K⁺ only periodically on some subvariants, but not for the whole treatment area. Concentrations of SO ₄ ²⁻ , Na⁺, and Cl⁻ were reduced after clear-cutting and remained nearly unchanged after femel cutting.     

Influence of different mineral nitrogen sources (NO 3 − -N vs. NH 4 + -N) on arbuscular mycorrhiza development and N transfer in a Glomus intraradices–cowpea symbiosis

Labeled nitrogen (¹⁵?N) was applied to a soil-based substrate in order to study the uptake of N by Glomus intraradices extraradical mycelium (ERM) from different mineral N (NO ₃ ^? vs. NH ₄ ⁺ ) sources and the subsequent transfer to cowpea plants. Fungal compartments (FCs) were placed within the plant growth substrate to simulate soil patches containing root-inaccessible, but mycorrhiza-accessible, N. The fungus was able to take up both N-forms, NO ₃ ^? and NH ₄ ⁺ . However, the amount of N transferred from the FC to the plant was higher when NO ₃ ^? was applied to the FC. In contrast, analysis of ERM harvested from the FC showed a higher ¹⁵?N enrichment when the FC was supplied with ¹⁵NH ₄ ⁺ compared with ¹⁵NO ₃ ^? . The ¹⁵?N shoot/root ratio of plants supplied with ¹⁵NO ₃ ^? was much higher than that of plants supplied with ¹⁵NH ₄ ⁺ , indicative of a faster transfer of ¹⁵NO ₃ ^? from the root to the shoot and a higher accumulation of ¹⁵NH ₄ ⁺ in the root and/or intraradical mycelium. It is concluded that hyphae of the arbuscular mycorrhizal fungus may absorb NH ₄ ⁺ preferentially over NO ₃ ^? but that export of N from the hyphae to the root and shoot may be greater following NO ₃ ^? uptake. The need for NH ₄ ⁺ to be assimilated into organically bound N prior to transport into the plant is discussed.     

Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

The effect of nitrogen starvation on the NO₃-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH ₄ ⁺ as the only nitrogen source (NH ₄ ⁺ -cells) were transferred into NO ₃ ^? medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO ₃ ^? , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m^?2 s^?1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m^?2 s^?1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO ₃ ^? -induced NR synthesis when the cells, previously grown in NH ₄ ⁺ medium, were transferred into NO ₃ ^? medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH ₄ ⁺ to NO ₃ ^? medium (at time 0 h), NO ₃ ^? induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO ₃ ^? to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO ₃ ^? medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO ₃ ^? uptake and NR induction due to the expression of NR and NO ₃ ^? -transporter mRNAs.     

Growth and metabolism characteristics of anaerobic ammonium-oxidizing bacteria aggregates

The present study reported the growth and metabolism characteristics of anaerobic ammonium-oxidizing (anammox) bacteria aggregates in an expanded granular sludge bed (EGSB). The results showed that the anammox bacteria aggregates presented starvation, growth, and inhibition phase along with the increase of substrate supply. The substrate conversion rates for survival were 0.05 kgNH ₄ ⁺ –N/(kgVSS·day), 0.07 kgNO ₂ ^? –N/(kgVSS·day), and 0.12 kgN/(kgVSS·day); the substrate conversion rates for maximum growth were 0.21 kgNH ₄ ⁺ –N/(kgVSS·day), 0.24 kgNH ₄ ⁺ –N/(kgVSS·day), and 0.45 kgNH ₄ ⁺ –N/(kgVSS·day), respectively. In the growth phase, the yield of anammox bacteria aggregates was 0.14 gVSS/(gNH ₄ ⁺ –N), 0.12 gVSS/(gNO ₂ ^? –N), and 0.70 gVSS/(gNO ₃ ^? –N); the yield of extracellular polymeric substances (EPS) was 0.11 gEPS/(gNH ₄ ⁺ –N), 0.09 gEPS/(gNO ₂ ^? –N), and 0.55 gEPS/(gNO ₃ ^? –N), respectively. The EPS contents in anammox bacteria aggregates were high compared to that in anaerobic granular sludge. Speculated from the cell yield, the energy for anammox bacteria growth was not only from nitrite oxidation, but also from anammox reaction.     

Relation between electron exchange and nuclear vibrations in the H 2 + +H 2 + → H 3 + +p exothermic ion-molecular reaction

The effective cross section for the H ₂ ⁺ +H ₂ ⁺ → H ₃ ⁺ +p reaction in the energy range 5.7–11.5 eV is measured by the split beam method. The maximum of the cross section at an energy of ～8 eV is related to the production of the H ₄ ⁺⁺ compound system. The reaction threshold W _thr≈5 eV provides evidence in favor of the classical model of the H ₂ ⁺ ion with the charge fixed on one of the nuclei throughout the collision event.     

Denitrification in soil amended with thermophile-fermented compost suppresses nitrate accumulation in plants

NO ₃ ^? is a major nitrogen source for plant nutrition, and plant cells store NO ₃ ^? in their vacuoles. Here, we report that a unique compost made from marine animal resources by thermophiles represses NO ₃ ^? accumulation in plants. A decrease in the leaf NO ₃ ^? content occurred in parallel with a decrease in the soil NO ₃ ^? level, and the degree of the soil NO ₃ ^? decrease was proportional to the compost concentration in the soil. The compost-induced reduction of the soil NO ₃ ^? level was blocked by incubation with chloramphenicol, indicating that the soil NO ₃ ^? was reduced by chloramphenicol-sensitive microbes. The compost-induced denitrification activity was assessed by the acetylene block method. To eliminate denitrification by the soil bacterial habitants, soil was sterilized with γ irradiation and then compost was amended. After the 24-h incubation, the N₂O level in the compost soil with presence of acetylene was approximately fourfold higher than that in the compost soil with absence of acetylene. These results indicate that the low NO ₃ ^? levels that are often found in the leaves of organic vegetables can be explained by compost-mediated denitrification in the soil.     

Production of H 3 + ions in low-energy collisions between hydrogen molecular ions

The effective cross section for the H ₂ ⁺ + H ₂ ⁺ → H ₃ ⁺ + p reaction in the energy range 5.7–11.5 eV is measured by the split beam method. The cross-section maximum at an energy of ～8 eV is related to the production of the H ₄ ⁺⁺ compound system. The reaction threshold W _thr ≈5 eV provides evidence in favor of the classical model with the H ₂ ⁺ ion charge fixed on one of the two nuclei during the entire collision event.     

Concentration-dependent modulations of potassium and calcium currents of rat osteoblastic cells by arachidonic acid

We show that the voltage-gated K⁺ and Ca²⁺ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 μm, AA reduces the amplitude and accelerates the inactivation of the K⁺ current activated by depolarization; at higher concentrations (≥5 μm), AA still blocks this K⁺ current, but also induces a very large noninactivating K⁺ current. At 2 or 3 μm, AA enhances the T-type Ca²⁺ current, close to its threshold of activation, whereas at 10 μm, it blocks that current. AA (1–10 μm) also blocks the dihydropyridine-sensitive L-type Ca²⁺ current. Thus, the effect of AA on Ca²⁺ entry through voltage-gated Ca²⁺ channels can change qualitatively with the AA concentration: at 2 or 3 μm, AA will favor Ca²⁺ entry through T channels, both by lowering the voltage-gated K⁺ conductance and by increasing the T current, whereas at 10 μm, AA will prevent Ca²⁺ entry through voltage-gated Ca²⁺ channels, both by inducing a K⁺ conductance and by blocking Ca²⁺ channels.     

Relationships between environmental factors and vegetation in nutrient-enriched fens at fishpond margins

Vegetation-environment relationships were investigated in fens of the T?eboň basin (Czech Republic), which are enriched by nutrients and calcium from intensively managed and limed fishponds to test the hypothesis of altered gradient structure after long-term nutrient enrichment in fens. Water-table depth, pH, conductivity, N-NH ₄ ⁺ , N-NO ₃ ^? , PO ₄ ^3? , total P, SO ₄ ^2? , K⁺, Ca²⁺, Mg²⁺ and Fe were measured four times in 30 vegetation plots of 16 m² during the 2004 vegetation season. Both constrained and unconstrained ordination (DCA, CCA) were used to relate environmental factors to the species composition of the vegetation. The relationships among particular factors were revealed using PCA. Four fen vegetation types obtained by TWINSPAN classification were compared with measured factors using repeated measures ANOVA. Vegetation types differed significantly in water-table depth, water pH and Ca²⁺, Mg²⁺, K⁺, and N-NO ₃ ^? content. The concentration of major nutrients fluctuated noticeably during vegetation season and displayed large variation within vegetation types. Temporarily the concentration of different nutrients reached extremely high values. However, high nutrient supply has not altered the gradient structure of the vegetation. Water pH and water-table depths were found to be two major determinants of species variation in fishpond-margin fens, as in the majority of other environments studied throughout the Northern Hemisphere. Species richness of both vascular plants and bryophytes was partly explained by pH. However, the extent of variation in total mineral richness and potassium concentration were the next two most important variables determining bryophyte species richness. Water in flooded poor-fen vegetation, directly affected by water from limed fishponds, had calcium and magnesium concentration similar to fishpond water. The calcium concentrations of about 20 mg l^?1 in typical poor-fen vegetation have no analogy in the ecological literature. High phosphorus level presumably buffers the effect of calcium by enhancing bryophyte biomass depositing superfluous calcium. In conclusion, extremely high long-term nutrient supply to fishpond-margin fens have not altered gradient structure, but shifted chemical limits of plant communities.     

Culture medium optimization for improved puerarin production by cell suspension cultures of Pueraria tuberosa (Roxb. ex Willd.) DC.

The effects of carbon, nitrogen, phosphate, and copper on cell growth and production of the isoflavone puerarin by suspension cultures of Pueraria tuberosa (Roxb. ex. Willd.) DC were investigated. Among the various sugars evaluated (glucose, galactose, fructose, maltose, and sucrose), use of sucrose in the medium led to the maximum accumulation of puerarin. A sucrose-feeding strategy in which additional sucrose was added to the flasks 15?d into the culture cycle stimulated both cell biomass and puerarin production. The maximum production of puerarin was obtained when a concentration balance of 20:60?mM NH ₄ ⁺ /NO ₃ ^? was used as the nitrogen source. Alteration in the concentration balance of nitrogen components (NH ₄ ⁺ /NO ₃ ^? 60:20?mM) or the use of either NH ₄ ⁺ or NO ₃ ^? alone decreased biomass production and puerarin accumulation compared with the control culture (NH ₄ ⁺ /NO ₃ ^? 20:20?mM). High amounts of phosphate (2.5 and 5?mM) in the medium inhibited puerarin production whereas 0.625?mM phosphate promoted puerarin production (68.3???g/g DW on day?25). An increase in Cu²⁺ concentration from 0.025 to 0.05?mg/l in the P. tuberosa cell culture medium resulted in a 2.2-fold increase in puerarin production (up to 141???g/g DW on day?25) but reduced cell culture biomass.