Kinetics of phosphorus absorption byCorynebacterium bovis

The initial rate of phosphorus uptake by phosphorus-limited cells ofCorynebacterium bovis grown in batch culture and in a chemostat was measured with [³²P] orthophosphate. It was dependent on the external phosphorus concentration and was inversely related to the amount of intracellular phosphorus. The relationship between the initial rate of uptake, intracellular phosphorus, and phosphorus concentration in the medium can be expressed in terms of Haldane's modification of the Michaelis-Menten equation.     

Whole-stream phosphorus release studies: variation in uptake length with initial phosphorus concentration

A new dual channel flow injection analyser that can simultaneously analyse soluble reactive phosphorus and bromide in the field, has been used in an experiment to test the hypothesis that the phosphorus uptake length in Myrtle Creek, a small forested stream in the Australian Highlands, is influenced by the initial phosphorus concentration used in whole-stream release studies. The phosphorus uptake length was found to decrease with decreasing initial phosphorus concentration added; the uptake length was 98 m when an initial P concentration of 51.0 µg 1^–1 was used, 90 m with 21.7 µg 1^–1 and 63 m with 12.7 µg 1^–1. The estimated errors in the uptake lengths were 6–8%. Approximately 32% of the added phosphorus was retained in the 32 m study reach, with almost all (ca. 93%) of this retained phosphorus taken up by the sediments (microbial uptake plus physico-chemical adsorption) and only a small amount retained in transient storage zones.     

Metabolic flux and robustness analysis of glycerol metabolism in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The knowledge of the mechanism of flux distribution will benefit understanding cell physiology and regulation of metabolism. In this study, the measured fluxes obtained under steady-state conditions were used to estimate intracellular fluxes and identify the robustness of branch points of the anaerobic glycerol metabolism in Klebsiella pneumoniae for the production of 1,3-propanediol by metabolic flux analysis. The biomass concentration increased as NADH₂/NAD⁺ decreased at low initial concentration and inversed at high initial glycerol concentration. The flux distribution revealed that the branch points of glycerol and dihydroxyacetonephosphate were rigid to the environmental conditions. However, the pyruvate and acetyl coenzyme A metabolisms gave cells the flexibility to regulate the energy and intermediate fluxes under various environmental conditions. Additionly, it was found that the formation rate of ethanol and the ratio of pyruvate dehydrogenase to pyruvate formate lyase appeared visible fluctuations at high glycerol uptake rate.     

Kinetic studies on sodium-dependent calcium uptake by myocardial cells and neuroblastoma cells in culture

Kinetic analyses were made on intracellular Na⁺-dependent Ca²⁺ uptake by myocardial cells and neuroblastoma cells (N-18 strain) in culture. Cells loaded with various concentrations of Na⁺ could be prepared by incubating them in Ca²⁺-free medium containing various concentrations of Na⁺. Cells pre-loaded with various concentrations of Na⁺ were incubated in medium containing Ca²⁺ and ⁴⁵Ca. The resulting ⁴⁵Ca uptake by the two types of cell depended greatly on the initial intracellular concentrations of Na⁺. Lineweaver-Burk plots of the initial rate of Ca²⁺ uptake against the external concentration of Ca²⁺ fitted well to straight lines obtained by linear regression (r > 0.95). This result shows that Ca²⁺ uptake by the two types of cell was achieved by a carrier-mediated transport system. This Na⁺-dependent Ca²⁺ uptake was accompanied by Na⁺ release and the ratio of Na⁺ release to Ca²⁺ uptake was close to 3 : 1. A comparison of the kinetic data between myocardial cells and N-18 cells suggested that N-18 cells possess a carrier showing the same properties as that of myocardial cells, i.e.: (1) a similar dependency on the intracellular concentration of Na⁺; (2) the coincidence of the apparent Michaelis constants for Ca²⁺ (0.1 mM); (3) the similarities of the K_i values for Co²⁺, Sr²⁺ and Mg²⁺ (Co²⁺ < Sr²⁺ < Mg²⁺) and (4) a similar dependency on pH. However, the maximal initial rate, V, of N-18 cells was about $\text{1}\text{100}$ that of myocardial cells. The rate of Na⁺-dependent Ca²⁺ uptake by non-excitable cells was much lower than that by myocardial cells.     

Potential of the seaweed Gracilaria lemaneiformis for integrated multi-trophic aquaculture with scallop Chlamys farreri in North China

In this study the red alga, Gracilaria lemaneiformis, was cultivated with the scallop Chlamys farreri in an integrated multi-trophic aquaculture (IMTA) system for 3 weeks at the Marine Aquaculture Laboratory of the Institute of Oceanology, Chinese Academy of Sciences (IOCAS) in Qingdao, Shandong Province, North China. The nutrient uptake rate and nutrient reduction efficiency of ammonium and phosphorus from scallop excretion were determined. The experiment included four treatments each with three replicates, and three scallop monoculture systems served as the control. Scallop density (407.9 ± 2.84 g m⁻³) remained the same in all treatments while seaweed density differed. The seaweed density was set at four levels (treatments 1, 2, 3, 4) with thallus wet weight of 69.3 ± 3.21, 139.1 ± 3.80, 263.5 ± 6.83, and 347.6 ± 6.30 g m⁻³, respectively. There were no significant differences in the initial nitrogen and phosphorus concentration between each treatment and the control group (ANOVA, p > 0.05). The results showed that at the end of the experiment, the nitrogen concentration in the control group and treatment 1 was significantly higher than in the other treatments. There was also a significant difference in phosphorus concentration between the control group and the IMTA treatments (ANOVA, p < 0.05). Growth rate, C and N content of the thallus, and mortality of scallop was different between the IMTA treatments. The nutrient uptake rate and nutrient reduction efficiency of ammonium and phosphorus changed with different cultivation density and time. The maximum reduction efficiency of ammonium and phosphorus was 83.7% and 70.4%, respectively. The maximum uptake rate of ammonium and phosphorus was 6.3 and 3.3 μmol g⁻¹ DW h⁻¹. A bivalve/seaweed biomass ratio from 1:0.33 to 1:0.80 (treatments 2, 3, and 4) was preferable for efficient nutrient uptake and for maintaining lower nutrient levels. Results indicate that G. lemaneiformis can efficiently absorb the ammonium and phosphorus from scallop excretion and is a suitable candidate for IMTA.     

Uncoupling of Methanogenesis from Growth of Methanosarcina barkeri by Phosphate Limitation

Production of methane by Methanosarcina barkeri from H₂-CO₂ was studied in fed-batch culture under phosphate-limiting conditions. A transition in the kinetics of methanogenesis from an exponentially increasing rate to a constant rate was due to depletion of phosphate from the medium. The period of exponentially increasing rate of methanogenesis was extended by increasing the initial concentration of phosphate in the medium. Addition of phosphate during the constant period changed the kinetics to an exponentially increasing rate of methanogenesis, indicating the reversibility of phosphate depletion. The relation between methanogenesis and growth of M. barkeri was investigated by measuring the incorporation of phosphorus, supplied as KH₂³²PO₄, in the medium. At a low (1 μM) initial concentration of phosphate in the medium and during the constant period of methanogenesis, there was no net cell growth. At a higher (10 μM) initial concentration of phosphate, cell growth proceeded linearly with time after phosphate had been removed from the medium by uptake into cells.     

Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions

Microalgal starch is a potential feedstock for biofuel production. Nutrient stress is widely used to stimulate starch accumulation in microalgae. Cell growth and starch accumulation in the marine green microalga Tetraselmis subcordiformis were evaluated under extracellular phosphorus deprivation with initial cell densities (ICD) of 1.5, 3.0, 6.0, and 9.0?×?10⁶ cells mL^?1. The intracellular stored phosphorus supported cell growth when extracellular phosphorus was absent. The maximum starch content of 44.1 % was achieved in the lowest ICD culture, while the maximum biomass productivity of 0.71 g L^?1 day^?1, starch concentration of 1.6 g L^?1, and starch productivity of 0.30 g L^?1 day^?1 were all obtained in the culture with the ICD of 3.0?×?10⁶ cells mL^?1. Appropriate ICD could be used to regulate the intracellular phosphorus concentration and maintain adequate photosynthetic activity to achieve the highest starch productivity, along with biomass and starch concentration. The recovery of phosphorus-deprived T. subcordiformis in medium containing 0.5, 1.0, or 6.0 mM KH₂PO₄ was also tested. Cell growth and starch accumulation ability could be recovered completely. A phosphorus pool in T. subcordiformis was shown to manipulate its metabolic activity under different environmental phosphorus availability. Though lower starch productivity and starch content were achieved under phosphorus deprivation compared with nitrogen- or sulfur-deprived conditions, the higher biomass and starch concentration make T. subcordiformis a good candidate for biomass and starch production under extracellular phosphorus deprivation.     

Adaptation of potassium metabolism and restoration of mitosis during prolonged treatment of mouse lymphoblasts with ouabain

The effects of ouabain on the growth of murine lymphoblasts in vitro have been studied. Exposure of cells to ouabain (0.1 mM) initially inhibited ⁸⁶Rb⁺ uptake rate, reduced the intracellular potassium concentration, and decreased population growth rates. Continued exposure to the same ouabain concentration resulted in an increase of ⁸⁶Rb⁺ uptake rate, intracellular potassium content and population growth rates to control values (adaptation). When treated cells were resuspended in medium free of ouabain after 12 to 15 hours of ouabain treatment, ⁸⁶Rb⁺ uptake rates and intracellular potassium levels exceeded those of untreated cells. Adaptation was inhibited by cycloheximide (3 μg/ml) and by actinomycin D (0.05 μg/ml). Kinetic analysis of transport suggested that while the total capacity of the Na⁺, K⁺ transport system increased, the affinity for both the cation (⁸⁶Rb⁺) and ouabain decreased.     

Effect of nitrogen and phosphorus availability on the growth response of Eucalyptus grandis to high CO2

The response of Eucalyptus grandis seedlings to elevated atmospheric CO₂ concentrations was examined by growing seedlings at either 340 or 660 n mol CO₂ mol^-1 for 6 weeks. Graded increments of phosphorus and nitrogen fertilizers were added to a soil deficient in these nutrients to establish if the growth response to increasing nutrient availability was affected by CO₂ concentration. At 660 μmol CO₂ mol^-1, seedling dry weight was up to five times greater than at 340 μmol CO₂ mol^-1. The absolute response was largest when both nitrogen and phosphorus availability was high but the relative increase in dry weight was greatest at low phosphorus availability. At 340 μmol CO₂ mol^-1 and high nitrogen availability, growth was stimulated by addition of phosphorus up to 76 mg kg 1 soil. Further additions of phosphorus had little effect. However, at 660 μmol CO₂ mol^-1, growth only began to plateau at a phosphorus addition rate of 920mg kg^-1 soil. At 340 μmol CO₂ mol^-1 and high phosphorus availability, increasing nitrogen from 40 to 160mg kg^-1 soil had little effect on plant growth. At high CO₂, growth reached a maximum at between 80 and 160mg nitrogen kg^-1 soil. Total uptake of phosphorus was greater at high CO₂ concentration at all fertilizer addition rates, but nitrogen uptake was either lower or unchanged at high CO₂ concentration except at the highest nitrogen fertilizer rate. The shoot to root ratio was increased by CO₂ enrichment, primarily because the specific leaf weight was greater. The nitrogen and phosphorus concentration in the foliage was lower at elevated CO₂ concentration partly because of the higher specific leaf weight. These results indicate that critical foliar concentrations currently used to define nutritional status and fertilizer management may need to be reassessed as the atmospheric CO₂ concentration rises.     

Ca2+ uptake by hyperpermeable mouse heart cells: Effects of inhibitors of mitochondrial function

The relative importance of heart mitochondria in regulating intracellular [Ca²⁺] in cardiac muscle is controversial. In a new approach to the question, we have measured the energy-linked ⁴⁵Ca uptake of an unusual myocardial tissue preparation in which the cells appear to be intact yet the sarcolemmae are highly permeable to exogenous solutes. Inhibitors of mitochondrial energy metabolism were used to estimate the mitochondrial contribution to rate and extent of total cell uptake. At 6.6μM Ca, which is close to the probable intracellular [Ca] range, inhibitors of mitochondrial energy metabolism did not diminish initial rates of ⁴⁵Ca uptake by myocardial fragments, if ATP was present to drive Ca²⁺ sequestration by the sarcoplasmic reticulum. The ultimate extent of uptake was reduced somewhat, however. Similar uptake profiles were obtained in the presence of carbonyl cyanide m-chlorophenyl-hydrazone, CN^?, and atractyloside, each of which acts at a different locus to inhibit mitochondrial Ca²⁺ transport. These data suggest that the mitochondria cannot control beat-to-beat [Ca²⁺] oscillations, because at μM Ca concentrations, the Ca²⁺ uptake rate of mitochondria $\text{in}$$\text{situ}$ is slow in comparison to the extra-mitochondrial (sarcoplasmic reticulum) uptake rate.     

Lactose uptake rate by kefir yeast using C-labelled lactose to explain kinetic aspects in its fermentation

Τhe present work shows the relation between kefir fermentation ability and carbohydrate uptake rate. This was examined in a model system containing kefir co-culture and lactose in order to study fermentation induced by yeasts and bacteria at the same time. Lactose uptake rate was recorded by using ¹⁴C-labelled lactose. The effect of lactose, cell concentration and pH on lactose fermentation was examined. Results have shown increase of lactose uptake rate at lower cell concentrations and specifically the maximum values of lactose uptake rate were obtained at 30 °C, 5.5 pH value and initial lactose and cell concentration 10% w/v and 16 g/L, respectively. Likewise, lighten that the increase of the fermentation rate by immobilized cells can be attributed also, in addition to other factors, to lower cell concentration on the surface of the support or of the promoter. Besides, it is shown that the effect of pH value on the biochemical reactions, carried out by intracellular enzymes can be attributed, except to the effect of pH on enzyme ability, in addition to the effect of pH on carbohydrate uptake rate.     

Mutational Engineering of the Cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis> for Resistance to Growth-Inhibitory Action of LiCl and NaCl

The effect of NaCl on two vital processes of cyanobacterial metabolism, viz. N₂ fixation and oxygenic photosynthesis, was studied in the cyanobacterium Nostoc muscorum grown diazotrophically. An increase in NaCl concentration suppressed the formation of heterocyst and adversely affected the nitrogenase activity in the parent, whereas in Li⁺-R and Na⁺-R mutants NaCl stress did not cause any adverse effect. The rate of photosynthetic O₂-evolution was also adversely affected by the NaCl stress, but the magnitude was less than that of nitrogenase activity. L-Proline, the well-known osmoprotectant, provided protection to the cyanobacterium against NaCl stress. The parent strain utilized L-proline as a nitrogen source and suppressed heterocyst formation and nitrogenase activity, while mutants showed normal heterocyst frequency and nitrogenase activity. Therefore, it may be that the proline metabolism is altered as a result of mutation. The intracellular levels of proline in the parent were enhanced about threefold in the medium containing 1 mol m⁻³ proline, while in mutants there was no significant increase in the intracellular level of proline. In the medium containing both NaCl and proline, the intracellular level of proline was enhanced in the parent as well as in both mutant strains. This suggests that the parent strain possessed both normal proline uptake and salt-induced proline uptake systems, whereas the mutant strains were defective in normal proline uptake and had only salt-induced proline uptake. The over-accumulation of proline in the presence of NaCl stress is due either to the loss of proline oxidase activity or to the accumulation of exogenous proline. Received: 10 July 2002 / Accepted: 13 August 2002     

Uptake of abscisic acid by isolated mesophyll protoplasts from Vicia Faba

Mesophyll protoplasts were isolated from Vicia faba (cv. exhibition longpod) leaf tissue and [³H] abscisic acid ([³H]ABA) uptake was measured as a function of time, concentration, pH and temperature. [³H] ABA uptake with time was linear for 30 s and then reached equilibrium. The uptake rate was a linear function of the external ABA concentration and had a pH optimum of 4–5. Various metabolic inhibitors did not effect the rate of uptake. The Q₁₀ value was less than 1.5. The results suggested that initial uptake was not a metabolically dependent or carrier mediated process but diffusive.     

NITRATE AND PHOSPHATE UPTAKE INTERACTIONS IN A MARINE PRYMNESIOPHYTE1

The short- and long-term uptake of nitrate and phosphate ions, and their interactions, were studied as functions of the preconditioning of Pavlova lutheri (Droop) Green. Populations were preconditioned in continuous culture at a variety of growth rates and N:P supply ratios. The maximum uptake rates cell^?1 for nitrate and phosphate were of similar magnitudes, in spite of the forty-fold smaller requirement for phosphorus. Short-term phosphate uptake was independent of the nitrate concentration, but the short-term nitrate uptake rate was reduced in the presence of phosphate. The severity of inhibition of nitrate uptake by phosphate was positively correlated with the preconditioning N:P supply ratio and the preconditioning growth rate. In response to large additions of nutrients, P. lutheri was able to increase its phosphorus content sixty-fold, but was only able to take up enough nitrate to double its nitrogen content. The high rate of phosphate uptake relative to its requirement, the inhibition of nitrate uptake by phosphate, and the large capacity for phosphorus storage relative to its requirement, all of which were observed even under N limitation, may imply that even where nitrogen is limiting there can be interspecific competition for available phosphate.     

The role of intracellular pH in the regulation of cation exchanges in yeast

1. When yeast oxidizes propan-2-ol in the presence of KCl no uptake of K⁺ occurs. 2. When propionate is added to suspensions containing propan-2-ol, or if the suspensions are bubbled with CO₂, a considerable uptake of K⁺ occurs. 3. Maximum K⁺ uptake occurs at a propionate concentration of 2mm. 4. The addition of 20mm-propionate to the suspension lowers the intracellular pH of the yeast from a resting value in the region of 6.2 to approx. 5.6. 5. When K⁺ uptake is measured in the presence of 20mm-propionate, progressive changes in the rate of K⁺ uptake and intracellular pH occur. The optimum rate of K⁺ uptake occurs at an intracellular pH of 5.70. 6. The effect of both intra- and extra-cellular pH on K⁺–K⁺ exchange was studied and an optimum rate was found at an extracellular pH of 5.35, the corresponding intracellular pH being 6.44. 7. When a Na⁺-loaded yeast oxidizes propan-2-ol in the presence of KCl, a steady efflux of Na⁺ and influx of K⁺ occurs. The addition of 10mm-propionate to the suspension markedly inhibited the Na⁺ efflux but only slightly decreased the K⁺ influx. 8. The effect of both extra- and intra-cellular pH on Na⁺ efflux was studied with propan-2-ol and with glucose. The results can be best interpreted in terms of intracellular pH changes, and an optimum was obtained at approx. pH6.40.     

Studies on the uptake of fatty acids by Escherichia coli

Oleate uptake by Escherichia coli showed saturation kinetics with a K_m of 34 μm and an activation energy of 6.25 kcal/mole indicating that the rate limiting step in oleate uptake involves an enzyme-catalyzed step. The rate of oleate uptake was decreased by the respiratory poisons, arsenate and 4-pentenoate, which apparently is activated to pentenoyl CoA, thus reducing the intracellular concentration of free intracellular CoA. These data indicated that oleate uptake is dependent on cellular ATP and CoA. During short pulses with [1-¹⁴C]oleate, most of the radioactivity which was taken up was released as ¹⁴C0₂; cells accumulated radioactivity in phospholipids and compounds with the chromatographic mobility of Krebs cycle intermediates. Neither free fatty acid nor oleyl CoA were detectable in the cells. The results support the hypothesis that long-chain fatty acids are translocated by the long-chain fatty acyl CoA synthetase and that uptake is the rate limiting step in the utilization of exogenous fatty acid.     

Intracellular pH and the kinetics of Rb+ uptake by yeast non-carrier versus mobile carrier-mediated uptake

The effect of changes in the intracellular pH upon the concentration dependence of the Rb⁺ uptake by yeast is investigated. It is shown, that the uptake of Rb⁺ can be described by a mechanism in which the total concentration of primary binding sites at the outer side of the membrane is independent of the intracellular ligand composition and of the membrane potential, and the influx rate constants depend upon the intracellular pH and/or upon the membrane potential. It is argued that the involvement of a mobile carrier mechanism is not likely.     

Aluminum Effects on Uptake and Metabolism of Phosphorus by the Cyanobacterium Anabaena cylindrica

Aluminum severely affects the growth of the cyanobacterium Anabaena cylindrica and induces symptoms indicating phosphorus starvation. Preor post-treating the cells with high (90 micromolar) phosphorus reduces the toxicity of aluminum compared to cells receiving a lower orthophosphate concentration. In this study aluminum (ranging from 9 to 36 micromolar) and phosphorus concentrations were chosen so that the precipitation of insoluble AIPO₄ never exceeded 10% of the total phosphate concentration. The uptake of ³²P-phosphorus is not disturbed by aluminum either at high (100 micromolar) or low (10 micromolar) concentrations of phosphate. Also, the rapid accumulation of polyphosphate granules in cells exposed to aluminum indicates that the incorporation of phosphate is not disturbed. However, a significant decrease in the mobilization of the polyphosphates is observed, as is a lowered activity of the enzyme acid phosphatase, in aluminum treated cells. We conclude that aluminum acts on the intracellular metabolism of phosphate, which eventually leads to phosphorus starvation rather than on its uptake in the cyanobacterium A. cylindrica.     

Calcium channels and cation transport ATPases in cardiac hypertrophy induced by aortic constriction in newborn rats

In order to evaluate the contribution of pinocytosis to basal (no agonist) and lanthanide-insensitive store-activated Ca²⁺ inflow in freshly-isolated rat hepatocytes, the uptake of extracellular fluid by pinocytosis was measured at 20°C and used to predict the amount of extracellular Ca²⁺ taken up by pinocytosis. This was compared with the measured rate of Ca²⁺ uptake in the basal state, and with the measured lanthanide-insensitive component of divalent cation uptake stimulated by 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the smooth endoplasmic reticulum (Ca²⁺ + Mg²⁺)ATP-ase. Fluid uptake by pinocytosis was measured using [¹⁴C]sucrose. In hepatocytes incubated at 20°C, DBHQ increased the initial rate of sucrose uptake by about 35%. The data for sucrose uptake were used to calculate the volume of extracellular fluid taken up by pinocytosis which, in turn, was used to predict the amount of extracellular Ca ²⁺ taken up through pinocytosis in the basal and DBHQ-stimulated states. Rates of divalent cation inflow in the basal state were determined at 20°C by measuring the uptake of ⁴⁵Ca²⁺. The degree of stimulation of Ca²⁺ inflow by DBHQ and the lanthanide-insensitive component of DBHQ-stimulated divalent cation inflow were determined by measuring the rate of Mn²⁺-induced quenching of intracellular quin-2 in the absence of an agonist, and in the presence of DBHQ or DBHQ plus Gd³⁺. It was calculated that the process of pinocytosis accounts for at least 15% of Ca²⁺ uptake in the basal (no agonist) state, and for about 10% of DBHQ-stimulated lanthanide-insensitive Ca²⁺ uptake. It is concluded that in isolated hepatocytes (i) the release of Ca²⁺ from intracellular stores stimulates pinocytosis and (ii) the process of pinocytosis can account for a substantial proportion of basal Ca²⁺ inflow and a small proportion of DBHQ-stimulated lanthanide-insensitive Ca²⁺ inflow.Abbreviations RACC receptor-activated Ca²⁺ channel - DBHQ 2,5-di-tert-butylhydroquinone - [Ca²⁺] intracellular free Ca²⁺ concentration