Requirements for Glucose, Nitrate, and Phosphate and Their Variations in the Analysis of the Mixed Culture of Pseudomonas aeruginosa Variants

The calibration data and sensitivity analysis for the variational model of Pseudomonas aeruginosa mixed culture containing R, S, and M phenotypic variants are presented. The model with calibrated parameters (requirements of the variants for glucose, nitrates, and phosphates) adequately describes the experiment. The sensitivity indices (the species abundances and total community size) changed least under the influence of the parameter variations in the one-factor limitation areas. The variant proportions are more sensitive to variation of the requirements than the community size.     

Synthesis of Extracellular Proteinase by Pseudomonas fluorescens Under Conditions of Limiting Carbon, Nitrogen, and Phosphate

The influence of carbon, nitrogen, and phosphate concentrations on growth and proteinase production by Pseudomonas fluorescens 32A was examined. In mineral salts medium containing dialyzed skim milk supernatant as an inducer, maximum growth was obtained at 1.0 and 2.5 mM orthophosphate at 20 and 5°C, respectively. At both temperatures, 5 mM orthophosphate was required for maximum proteinase production, whereas significant inhibition was found at 10 mM. Orthophosphate was the only phosphate compound able to support growth. With sodium pyruvate as the carbon source, maximum enzyme synthesis was at 100 mM carbon at both temperatures. At both 20 and 5°C maximum growth and enzyme production was found with 10 mM NH₄Cl. A bioassay for available phosphate based on the growth of P. fluorescens 32A in phosphate-limited mineral salts medium showed that skim milk and skim milk supernatant contained 50 and 10 mM orthophosphate, respectively. Proteinase production in skim milk was 2.6- and 12-fold greater than that in optimal mineral salts medium at 20 and 5°C, respectively. These results suggest that proteinase production in milk does not occur as a result of nutrient limitation and may be regulated in part by milk phosphates.     

Association Studies of Calcium-Sensing Receptor (CaSR) Polymorphisms with Serum Concentrations of Glucose and Phosphate,and Vascular Calcification in Renal Transplant Recipients

Background

Cardiovascular disease is the major cause of death in renal transplant recipients (RTRs) and linked to arterial calcification. The calcium-sensing receptor (CaSR), a G-protein coupled receptor, plays a pivotal role in extracellular calcium homeostasis and is expressed in the intimal and medial layers of the arterial wall. We investigated whether common CASR gene variants are predictors for aortic and coronary artery calcification or influence risk factors such as serum calcium, phosphate and glucose concentrations in RTRs.

Methods

Two hundred and eighty four RTRs were investigated for associations between three CASR promoter region single nucleotide polymorphisms (SNPs) (rs115759455, rs7652589, rs1501899), three non-synonymous CASR coding region SNPs (A986S, R990G, Q1011E), and aortic and coronary artery calcium mass scores, cardiovascular outcomes and calcification risk factors that included serum phosphate, calcium, total cholesterol and glucose concentrations.

Results

Multivariate analysis revealed that RTRs homozygous for the minor allele (SS) of the A986S SNP, when compared to those homozygous for the major allele (AA), had raised serum glucose concentrations (8.7±5.4 vs. 5.7±2.1 mmol/L, P<0.05). In addition, RTRs who were heterozygous (CT) at the rs115759455 SNP, when compared to those homozygous for the major allele (CC), had higher serum phosphate concentrations (1.1±0.3 vs. 1.0±0.2 mmol/L, P<0.05). CASR SNPs were not significant determinants for aortic or coronary artery calcification, and were not associated with cardiovascular outcomes or mortality in this RTR cohort.

Conclusions

Common CASR SNPs may be independent predictors of serum glucose and phosphate concentrations, but are not determinants of vascular calcification or cardiovascular outcomes.     

Influence of Nitrate and Ammonium Nutrition on the Uptake, Assimilation, and Distribution of Nutrients in Ricinus communis

Ricinus communis L. plants were grown in nutrient solutions in which N was supplied as NO₃⁻ or NH₄⁺, the solutions being maintained at pH 5.5. In NO₃⁻-fed plants excess nutrient anion over cation uptake was equivalent to net OH⁻ efflux, and the total charge from NO₃⁻ and SO₄²⁻ reduction equated to the sum of organic anion accumulation plus net OH⁻ efflux. In NH₄⁺-fed plants a large H⁺ efflux was recorded in close agreement with excess cation over anion uptake. This H⁺ efflux equated to the sum of net cation (NH₄⁺ minus SO₄²⁻) assimilation plus organic anion accumulation. In vivo nitrate reductase assays revealed that the roots may have the capacity to reduce just under half of the total NO₃⁻ that is taken up and reduced in NO₃⁻-fed plants. Organic anion concentration in these plants was much higher in the shoots than in the roots. In NH₄⁺-fed plants absorbed NH₄⁺ was almost exclusively assimilated in the roots. These plants were considerably lower in organic anions than NO₃⁻-fed plants, but had equal concentrations in shoots and roots. Xylem and phloem saps were collected from plants exposed to both N sources and analyzed for all major contributing ionic and nitrogenous compounds. The results obtained were used to assist in interpreting the ion uptake, assimilation, and accumulation data in terms of shoot/root pH regulation and cycling of nutrients.     

New Culture Medium Containing Ionic Concentrations of Nutrients Similar to Concentrations Found in the Soil Solution

A new growth medium which closely approximates the composition of the soil solution is presented. This soil solution equivalent (SSE) medium contains the following components (millimolar): NO₃, 2.5; NH₄, 2.5; HPO₄, 0.005; Na, 2.5; Ca, 4.0; Mg, 2.0; K, 0.503; Cl, 4.0; SO₄, 5.0; ethylenediamine-di(o-hydroxyphenylacetic acid), 0.02; and MES [2-(N-morpholino)ethanesulfonic acid] (to maintain the pH at 6.0), 10, plus 0.1% arabinose. The advantages of the SSE medium are discussed.     

Steady-State Cerebral Glucose Concentrations and Transport in the Human Brain

Abstract: Understanding the mechanism of brain glucose transport across the blood-brain barrier is of importance to understanding brain energy metabolism. The specific kinetics of glucose transport have been generally described using standard Michaelis-Menten kinetics. These models predict that the steady-state glucose concentration approaches an upper limit in the human brain when the plasma glucose level is well above the Michaelis-Menten constant for half-maximal transport, K _t. In experiments where steady-state plasma glucose content was varied from 4 to 30 m M , the brain glucose level was a linear function of plasma glucose concentration. At plasma concentrations nearing 30 m M , the brain glucose level approached 9 m M , which was significantly higher than predicted from the previously reported K _t of ∼4 m M ( p < 0.05). The high brain glucose concentration measured in the human brain suggests that ablumenal brain glucose may compete with lumenal glucose for transport. We developed a model based on a reversible Michaelis-Menten kinetic formulation of unidirectional transport rates. Fitting this model to brain glucose level as a function of plasma glucose level gave a substantially lower K _t of 0.6 ± 2.0 m M , which was consistent with the previously reported millimolar K _m of GLUT-1 in erythrocyte model systems. Previously reported and reanalyzed quantification provided consistent kinetic parameters. We conclude that cerebral glucose transport is most consistently described when using reversible Michaelis-Menten kinetics.     

Phosphate Deficiency in Maize. IV. Changes in Amounts of Sucrose Phosphate Synthase during the Course of Phosphate Deprivation

With low-P treatment of maize, the level of sucrose phosphatesynthase (SPS) protein decreased to 15% of the control, whilethe "V_max" activity stayed relatively high (100–80% ofthe control) and the substrate limiting activity increased about2 fold in the leaves. These results suggest that leaf phosphatestatus has dual effects on the amount of SPS protein and theactivation state of SPS. (Received January 20, 1992; Accepted April 14, 1993)     

Effects of Dopamine on Antioxidation,Mineral Nutrients,and Fruit Quality in Cucumber Under Nitrate Stress

Journal of Plant Growth Regulation - In greenhouses, growth of plants is affected by accumulation of nitrate in the soil due to extended durations of nitrogen fertilizer application. Dopamine has...     

Metabolism of Specifically Labeled Glucose, Glucose 1-Phosphate, and Glucose 6-Phosphate via the Oxidative Pentose Phosphate Cycle in a Reconstituted Spinach Chloroplast System in Darkness and in the Light

Using isolated spinach (hybrid 424) chloroplasts deprived of their envelopes (reconstituted chloroplast system), the metabolism of glucose, glucose 1-phosphate, and glucose 6-phosphate via the oxidative pentose phosphate cycle was analyzed. The activity of oxidative pentose phosphate cycle was monitored by continuous sampling of the CO₂ released during the decarboxylation process of 6-phosphogluconate.     

The Effect of Inorganic Nutrients on the Hormonal Requirements of Normal, Habituated, and Crown-gall Tissue 4

The addition of Braun and Wood's inorganic supplements (845mg l^–1 KCl, 1800 mgl^–1 NaNO₃, 300 mg l^–1 NaH₂PO₄.2H₂O,790 mg l^–1 (NH₄)₂SO₄) to White's medium caused markedincreases in the growth of normal tissues of Helianthus annuus,Nicotiana rustica, Daucus carota, and Vinca rosea and crown-galltumour tissues of H. annuus. However, no evidence was obtainedwhich suggested that the presence of these extra salts markedlyinfluenced the essential requirements of normal callus for auxinsand kinetin. In contrast their presence significantly influencedthe hormonal requirements of certain habituated cultures ofH. annuus and V. rosea. These habituated cultures had specificauxin requirements on White's medium while either an auxin orkinetin was sufficient on high-salts medium. These results arediscussed in relation to previous reports which suggested thatthe biosyntheses of auxins and other growth factors in normaland crown-gall cultures are specifically activated by certaininorganic ions.     

The Requirements of Pseudomonas aeruginosa Dissociants for Carbon,Nitrogen, and Phosphorus

Quantitative data on the nutritional requirements of microorganisms are necessary to predict the behavior of bacterial populations and to control their cultivation. The requirements of the R, S, and M dissociants of P. aeruginosa for carbon, nitrogen, and phosphorus were derived from the results of 88 cultivation experiments. For each of the dissociants, we derived a coefficient that relates the optical density and the number of cells in the dissociant culture, determined the time when the cultures entered the stationary growth phase, studied cultural changes induced by transfer to the stationary phase, and determined what nutrients limit the growth of particular dissociants. The nutritional requirements of the dissociants are discussed in relation to our earlier data.     

Glucose Metabolism in Batch and Continuous Cultures of Gluconacetobacter diazotrophicus PAL 3

Periplasmic glucose oxidation (by way of a pyrrolo-quinoline-quinone [PQQ]–linked glucose dehydrogenase [GDH]) was observed in continuous cultures of Gluconacetobacter diazotrophicus regardless of the carbon source (glucose or gluconate) and the nitrogen source (N₂ or NH₃). Its synthesis was stimulated by conditions of high energetic demand (i.e., N₂-fixation) and/or C-limitation. Under C-excess conditions, PQQ-GDH synthesis increased with the glucose concentration in the culture medium. In batch cultures, PQQ-GDH was actively expressed in very early stages with higher activities under conditions of N₂-fixation. Hexokinase activity was almost absent under any culture condition. Cytoplasmic nicotinamide adenine dinucleotide (NAD)–linked glucose dehydrogenase (GDH) was expressed in continuous cultures under all tested conditions, and its synthesis increased with the glucose concentration. In contrast, low activities of this enzyme were detected in batch cultures. Periplasmic oxidation, by way of PQQ-GDH, seems to be the principal pathway for metabolism of glucose in G. Diazotrophicus, and NAD-GDH is an alternative route under certain environmental conditions.     

Nutritional Requirements of Plasmodium falciparum in Culture. III. Further Observations on Essential Nutrients and Antimetabolites1

In a semi-defined minimal medium for cultivation of Plasmodium falciparum, ribose, mannose, fructose, galactose, and maltose could not replace glucose. Hypoxanthine was the preferred purine source for the parasite over adenine, guanine, inosine, adenosine and guanosine although all supported growth equally. Inhibitors of nucleoside uptake had low potency in killing the parasites but depressed incorporation of [³H]adenosine more than [³H]hypoxanthine. Glutamate could not be replaced by 5-oxoproline, indicating that the γ-glutamyl transferase pathway for amino acid uptake is probably not found in this organism. Adenine, nicotinamide, and orotic acid could not supplement glutamine-deficient medium. The pyridoxine antagonists isoniazid and 4-deoxypyridoxine were reversed by amino acid supplementation, suggesting that transaminases may be targets of these drugs. Orotic acid, but not glutathione or its amino acid components, partially reversed the effects of 8-methylamino-8-desmethyl riboflavin. Thus, the flavin enzyme, dihydroorotic acid dehydrogenase, but not glutathione reductase, appears to be a target of this riboflavin antagonist. Five biotin antagonists had no significant activity. The choline antagonist 2-(tert-butylamino)ethanol and thiamin uptake inhibitors had nonspecific inhibitory effects, which were not reversed by the respective target vitamin. Buthionine sulfoximine and methionine sulfoximine, inhibitors of glutathione synthesis, had significant oxygen-dependent toxicity. Six sulfonamides showed marked variation in potency and efficacy. Sulfathiazole and sulfadoxine were reversed differentially by p-aminobenzoic acid, folic acid, and folinic acid. Folinic acid was more effective than folic acid at reversing the toxicity of the dihydrofolate reductase inhibitors aminopterin and pyrimethamine; p-aminobenzoic acid had no effect.     

Phosphate transport in Pseudomonas aeruginosa. Involvement of a periplasmic phosphate-binding protein

A binding protein for inorganic phosphate was purified to apparent homogeneity from the shock fluids of phosphate-limited Pseudomonas aeruginosa. The purified protein bound one molecule of phosphate per molecule of binding protein with an average Kd of 0.34 microM. Arsenate, pyrophosphate and polyphosphates up to 15 units long could inhibit the binding of phosphate to the binding protein, although organic phosphates, such as glucose 6-phosphate, glycerol 3-phosphate and adenosine 5'-monophosphate could not. Mutants lacking the phosphate-binding protein were isolated and shown to be deficient in phosphate transport compared with wild-type cells. Two kinetically distinct systems for phosphate uptake could be observed in wild-type cells, with apparent Km values of 0.46 +/- 0.10 microM (high affinity) and 12.0 +/- 1.6 microM (low affinity). In contrast, only a single low-affinity transport system was observable in mutants lacking the binding protein (Km apparent = 19.3 +/- 1.4 microM Pi), suggesting the involvement of the binding protein in the inducible high-affinity phosphate-uptake system of P. aeruginosa.     

An Integrated View of the Kinetics of Glucose and Phosphate Transport, and of Glucose 6-Phosphate Transport and Hydrolysis in Intact Rat Liver Microsomes

The dynamics of the glucose 6-phosphatase system were investigated in intact rat liver microsomes using a fast-sampling, rapid-filtration apparatus. Glucose and phosphate transport followed single exponential kinetics, appeared to be homogeneous, was unaffected by unlabeled substrate concentrations up to 100 mm, proved insensitive to various potential inhibitors, and demonstrated similarly low energies of activation. The extent of tracer accumulation from glucose 6-phosphate depended on which of the glucose or phosphate moieties was the labeled species in the parent molecule. The rates of tracer equilibration reflected those of glucose or phosphate transport but similar initial rates of uptake were observed for the glucose and phosphate products of hydrolysis. However, the latter accounted for only 12–13% of the steady-state rate of total glucose production. It is concluded that tracer uptake cannot represent substrate transport, that labeled glucose 6-phosphate at best represents a tiny fraction of the intramicrosomal glucose or phosphate pools, and that glucose 6-phosphate transport is not an obligatory prerequisite to its hydrolysis. The latter conclusion invalidates a major postulate of the substrate transport-catalytic unit concept but proves compatible with a conformational model whereby glucose 6-phosphate transport and hydrolysis are tightly coupled processes while glucose and phosphate share, along with water and a variety of other organic and inorganic solutes, a common porelike structure for their transport through the microsomal membrane. Received: 26 May 2000/Revised: 16 October 2000     

Glucose utilization in heart, diaphragm and skeletal muscle during the fed-to-starved transition.

1. 1,4-Dideoxy-1,4-imino-L-threitol was synthesized and the synthesis of 1,4-dideoxy-1,4-imino-L-arabinitol was improved. 2. Both compounds are competitive inhibitors of Monilinia fructigena alpha-L-arabinofuranosidase III, the additional hydroxymethyl group in the arabinitol contributing about 17.8 kj/mol (4.25 kcal/mol) to the Gibbs free energy of binding. 3. The affinities (1/Ki) of both compounds vary with pH in a classical bell-shaped way, the pKa value being that of the acid-catalytic group on the enzyme [5.9; Selwood & Sinnott (1988) Biochem. J. 254, 899-901] and the pKb values being those of the free inhibitors, 7.6 and 7.8 respectively. 4. On the basis of these and literature data we suggest that efficient inhibition of a glycosidase at its pH optimum by an appropriate iminoalditol will be found when the pKa of the iminoalditol is below that of the acid-catalytic group of the target enzyme.     

Apoplastic Solute Concentrations of Organic Acids and Mineral Nutrients in the Leaves of Several Fagaceae

Ion chromatographic methods determined organic acids and mainnutrient minerals in the apoplastic solution from leaves ofseveral Fagaceae (Quercus ilex L., Quercus cerris L., Quercusvirgiliana (Ten.) Ten, and Fagus sylvatica L.). The anions oforganic acids found in high amounts (250 to 650 µM) werequinate, malate, and oxalate. Lactate, pyruvate, formate andacetate were detected in relatively low amounts with concentrationsbetween 20 and 200 µM. The total concentration of organicacids in the apoplastic sap ranged between 1.5 and 2 mM. Thetotal concentration of inorganic cations (K⁺, Mg²⁺, NH₄⁺, Ca²⁺,Na⁺) and anions (C1^–, NO₃^–, SO^2–₄ and PO^3–₄)in the apoplastic sap varied between 5 and 10 mM, and 0.35 and1.8 mM, respectively. We conclude that the concentration oforganic acid ions in the leaf apoplast depends mainly on theexchange with the leaf cells and is influenced by the electrochemicalgradient between the symplast and the apoplast in relation tothe water potential of the leaf. The determination of formateand acetate in the apoplastic compartment of leaves lend weightto the argument that the production of these acids by treesis a important emission source to the atmosphere. (Received June 9, 1998; Accepted April 8, 1999)     

Heat Production by the Denitrifying Bacterium Pseudomonas fluorescens and the Dissimilatory Ammonium-Producing Bacterium Pseudomonas putrefaciens during Anaerobic Growth with Nitrate as the Electron Acceptor

The heat production rate and the simultaneous nitrate consumption and production and consumption of nitrite and nitrous oxide were monitored during the anaerobic growth of two types of dissimilatory nitrate reducers. Pseudomonas fluorescens, a denitrifier, consumed nitrate and accumulated small amounts of nitrite or nitrous oxide. The heat production rate increased steadily during the course of nitrate consumption and decreased rapidly concomitant with the depletion of the electron acceptors. A mean experimental enthalpy change value of −800 kJ/mol of nitrate and a mean growth yield value of 33 g (dry weight)/mol of nitrate consumed were obtained for different concentrations of nitrate. For Pseudomonas putrefaciens, a dissimilatory ammonium producer, the nitrate consumption resulted in an accumulation of nitrite and nitrous oxide. Nitrite consumption commenced after depletion of the nitrate; consequently, two phases were noted in the heat production rate curve during growth. A mean experimental enthalpy change value of −810 kJ/mol of nitrate was obtained for different concentrations of nitrate.