The Influence of Calcium or Manganese on the Resistance to EDTA, Polymyxin B or Cold Shock, and the Composition of Pseudomonas aeruginosa Grown in Glucose- or Magnesium-depleted Batch Cultures

Cultures were batch grown in simple salts media in which growth was limited either by depletion of glucose and magnesium (C/Mg-dep) or by glucose alone (C-dep). Cultures were also grown in these media supplemented by calcium and/or manganese.
All cultures grown in the C-dep media were sensitive to ethylenediaminetetraacetic acid (EDTA), polymyxin and also to cold shock but were relatively resistant to ethyleneglycol-bis(2-aminoethyl ether)-N, N-tetraacetic acid (EGTA). Inclusion of calcium or manganese in the growth medium enhanced lysis by EDTA. Cultures grown in the basic C/Mg-dep media were resistant to EDTA, EGTA, polymyxin and to cold shock. Sensitivity to these agents was retained by cultures grown in C/Mg-dep media supplemented with Ca²⁺ and/or Mn²⁺. Cells grown in C/Mg-dep media with added Mn²⁺ were more sensitive to EDTA and polymyxin than those from the unsupplemented C/Mg-dep media but still resistant compared with C-dep cultures. All cultures from supplemented C/Mg-dep media were more sensitive to EGTA than those from any of the C-dep media.
Whole cells and cell walls from these various media had differing amounts of cell wall, phosphorus, amino sugar, carbohydrates, readily extractable lipid (REL), total phospholipid (PL), and especially differences in cell wall divalent metal cation content.
The differences in PL, REL and amino sugars and carbohydrate did not correlate with the response of C-dep and C/Mg-dep bacteria to EDTA, EGTA or polymyxin. The results are discussed in relation to the hypothesis that the sensitivity of Pseudomonas aeruginosa to polymyxin and EDTA is more dependent on outer membrane cation content rather than on other components, e.g. PL and lipopolysaccharide.     

Cost‐effective media for the rapid and high resolution of small DNA fragments using polyacrylamide‐based electrophoresis

Current Tris‐based solutions for DNA electrophoresis produce a positive feedback loop between current and temperature at high voltage, resulting in long running times for the separation of even small DNA fragments. We optimized the separation of small DNA fragments (90–300 bp) in polyacrylamide‐based electrophoresis at high voltages (200volts/cm) by substituting Tris with low concentration alkali salts (e.g. 1 mm LiCl and CsCl). These media reduced the heat produced during electrophoresis, enhanced the DNA fragment resolution, and allowed gels to be run at higher voltages, reducing gel running times by 25%. In addition, the elimination of Tris and EDTA from the buffer reduced material costs approximately 10‐fold.     

Reduction of Fe(II)EDTA-NO by a newly isolated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain DN-2 in NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> scrubber solution

Biological reduction of nitric oxide (NO) chelated by ferrous ethylenediaminetetraacetate (Fe(II)EDTA) to N₂ is one of the core processes in a chemical absorption–biological reduction integrated technique for nitrogen oxide (NO _x ) removal from flue gases. A new isolate, identified as Pseudomonas sp. DN-2 by 16S rRNA sequence analysis, was able to reduce Fe(II)EDTA-NO. The specific reduction capacity as measured by NO was up to 4.17 mmol g DCW⁻¹ h⁻¹. Strain DN-2 can simultaneously use glucose and Fe(II)EDTA as electron donors for Fe(II)EDTA-NO reduction. Fe(III)EDTA, the oxidation of Fe(II)EDTA by oxygen, can also serve as electron acceptor by strain DN-2. The interdependency between various chemical species, e.g., Fe(II)EDTA-NO, Fe(II)EDTA, or Fe (III)EDTA, was investigated. Though each complex, e.g., Fe(II)EDTA-NO or Fe(III)EDTA, can be reduced by its own dedicated bacterial strain, strain DN-2 capable of reducing Fe(III)EDTA can enhance the regeneration of Fe(II)EDTA, hence can enlarge NO elimination capacity. Additionally, the inhibition of Fe(II)EDTA-NO on the Fe(III)EDTA reduction has been explored previously. Strain DN-2 is probably one of the major contributors for the continual removal of NO _x due to the high Fe(II)EDTA-NO reduction rate and the ability of Fe(III)EDTA reduction.     

Effect of lysozyme and sodium EDTA on shrimp microflora

Summary The influence of lysozyme and salts on the growth of the microflora of shrimp was investigated. It was found that lysozyme at concentrations up to 150 g/ml could retard microbial growth in nutrient broth at 28°C. Growth of shrimp microflora was not affected much at low concentrations (0.05% and 0.1%) of EDTA but was totally inhibited in the presence of 0.5% Na₂EDTA. No growth was discernible using concentrations of 50 g/ml lysozyme and 0.02% Na₂EDTA, either in nutrient broth or in 2% shrimp homogenate.     

Energy-linked swelling of EDTA submitochondrial particles

The osmotic properties of EDTA submitochondrial particles have been studied by means of light-scattering measurements and radioisotopic determination of water distribution. It is shown that EDTA particles exhibit a respiration-linked swelling which: (i) requires oligomycin and NO₃^?; (ii) is promoted by nigericin and inhibited by valinomycin in the presence of K⁺ but not in the presence of Na⁺; and (iii) is reversed by FCCP. It is concluded that the energy-linked swelling of EDTA particles is caused by energy-linked influx of salts.     

Evidence that bacterial ABC-type transporter imports free EDTA for metabolism

EDTA, a common chelating agent, is becoming a major organic pollutant in the form of metal-EDTA complexes in surface waters, partly due to its recalcitrance to biodegradation. Even an EDTA-degrading bacterium, BNC1, does not degrade stable metal-EDTA complexes. In the present study, an ABC-type transporter was identified for possible uptake of EDTA because the transporter genes and the EDTA monooxygenase gene were expressed from a single operon in BNC1. The ABC-type transporter had a periplasmic-binding protein (EppA) that should confer the substrate specificity for the transporter; therefore, EppA was produced in Escherichia coli, purified, and characterized. EppA was shown to bind free EDTA with a dissociation constant as low as 25 nM by using isothermal titration calorimetry. When unstable metal-EDTA complexes, e.g., (Mg-EDTA)²⁻, were added to the EppA solution, binding was also observed. However, experimental data and theoretical analysis supported EppA binding only of free EDTA. When stable metal-EDTA complexes, e.g., (Cu-EDTA)²⁻, were titrated into the EppA solution, no binding was observed. Since EDTA monooxygenase in the cytoplasm uses some of the stable metal-EDTA complexes as substrates, we suggest that the lack of EppA binding and EDTA uptake are responsible for the failure of BNC1 cells to degrade the stable complexes.     

Mechanisms of suppression of physiological function in hypothermia and a way of their restoration without body warming

It was shown that in hypothermic rats (rectal temperature 25-22 degrees C) it was possible to stimulate responses that had been suppressed by cold (i. e. thermoregulation and breathing) with the aid of injecting a solution of ethylenediaminetetraacetic acid disodium salt (EDTA) in quantity 16.5 mg/100 g of body weight (0.0045 mmol/100 g) into the blood stream of the cooled animals. EDTA connects calcium ions in blood and forms complexes. It was shown that enhancement of cold shivering intensity and that of breathing (in 5 min after beginning the injection of EDTA) coincided with a 42-45 % reduction of [Ca2+] in the blood]. After 15 min following the beginning of injection of EDTA [Ca2+] into the blood stream, a return to the initial level was observed in cooled animals. Simultaneously we observed suppression of the cold shivering and breathing. The repeated injection of EDTA again caused similar fall of [Ca2+] in the blood and the following enhancement of cold shivering and breathing.     

Anaerobic treatment of low-strength synthetic TCF effluents and biomass adhesion in fixed-bed systems

Toxicity effects produced by kraft mill effluents are due to the productive process. New bleaching processes have been proposed (e.g. total chlorine free, TCF) to reduce the production of toxic chlorine compounds. In the TCF processes large amounts of chelating compounds like the ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DPTA) are used. The aim of this work is to research the feasibility of the degradation of low-strength synthetic TCF effluents in a anaerobic filter reactor (AF) and the biomass adhesion. The effects on the operation of the AF at different EDTA loading rates were tested in the range from 0.07 to 0.51 g EDTA l(-1) days(-1). The maximum EDTA removal percentage achieved was of 27%. Acute toxicity (measured as 24 h-LC(50)) with Daphnia magna was reduced from 14.23 to 54.53% before and after anaerobic treatment, respectively. Observations of biomass samples from the AF under the scanning microscope verified the attached biomass.     

Solubilization of muscarinic acetylcholine receptors of bovine brain

The effectiveness of several detergents and salts in solubilizing the muscarinic acetylcholine receptor (identified by its atropine-sensitive [³H]3-quinuclidinyl benzilate (QNB) binding) from bovine striatal membranes is reported. The highest density of receptor is obtained by extraction with 1% digitonin-0.1 mM EDTA. Although the total solubilized muscarinic receptors (sites/ml) are increased and the nonspecific binding is decreased when 1 M NaCl is included in this extraction medium, the receptor density (sites/mg protein) is lower. The solubilized receptors have the same specific QNB binding affinity, and sensitivity to a variety of drugs, as the membrane-bound muscarinic receptors.     

How can iron salts mediate the degradation of nucleos(t)ides by elliptinium acetate via free-radicals?

Elliptinium acetate (NSC 264137) is an antineoplastic agent currently used in anticancer chemotherapy. We report here the first evidence that this drug is able to modify DNA models via a redox process with iron salts. In presence of iron (III) salts, EDTA and H2O2, 9-OH-NME degrades deoxyguanosine. The two main products are guanine and 8-hydroxydeoxyguanosine which result from the formation of hydroxyl radicals. The biological implications of this phenomenon are briefly discussed.     

Bacterial strain characterizing by EDTA requirement

A novel strain of bacteria (LPM-4) was isolated that is characterized by a unique EDTA requirement for cell growth. Suspensions of washed cells of strain LPM-4 degrated EDTA complexes with Ba2+, Mg 2+, Ca2+, and Mn2+ at constant rates (0.310-0.486 mmol EDTA/(g h)) and Zn-EDTA at an initial rate of 0.137 +/- 0.016 mmol EDTA/(g h). The temperature optima for cell growth and EDTA degradation were determined under pH-auxostat cultivation. As compared with the known EDTA-degrating bacteria, strain LPM-4 exhibited a higher specific growth rate (0.095 h(-1)) and lower mass cell yield (0.219 g cells/g EDTA) that is promising for its practical applications for EDTA removal in wastewater treatment plants.     

Oxalate Extraction of Pb and Zn from Polluted Soils: Solubility Limitations

Oxalate (Ox) was used to extract Pb and Zn from industrially contaminated soils. Although Ox effectively releases metals bound by hydrous oxide soil components, it forms insoluble salts with some heavy metals unlike conventional extractants (e.g., EDTA). The insolubility of PbOx(s) (K_sp=2.74 × 10^?11) precluded the use of Ox as a single-step extractant even for soils mildly contaminated with Pb. The usefulness of Ox as a Zn extractant, however, depends on the level of soil contamination. A Zn solubility model, based on published equilibrium constants, was developed to assess Ox suitability as a function of system conditions. Precipitation of ZnOx(s) hindered Zn recovery under acidic conditions where formation of soluble oxalato complexes was small. For pH<3, the presence of 1?M Ox actually reduced Zn release compared to simple acid washing. Although Ox displaces oxide-bound metals and thus is potentially useful in soil washing, solubility limitations must be defined for effective remediation of metal-laden soils.     

Photoinduced oxygen uptake for 9,10-anthraquinone in air-saturated aqueous acetonitrile in the presence of formate, alcohols, ascorbic acid or amines.

The photolysis of 9,10-anthraquinone (AQ), 2-methyl- and 2,3-dimethyl-AQ was studied in air-saturated acetonitrile-water in the presence of various donors: formate, ascorbic acid, alcohols, e.g. 2-propanol or methanol, and amines, e.g. ethylenediaminetetraacetate (EDTA). The photoreaction is initiated by H-atom or electron transfer from the donor to the AQ triplet state. The conversion of oxygen into hydrogen peroxide occurs via the superoxide radical and its conjugate acid. The quantum yield of oxygen uptake (Phi(-O2)) increases with increasing donor concentration. Phi(-O2) = 0.3-0.6 in the presence of 1 M 2-propanol and 3-10 mM ascorbic acid or EDTA. The properties of the quinone and donor radicals involved and the pH and concentration dependences of Phi(-O2) are described.     

Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis

It is well established that salt enhances the interaction between solutes (e.g., proteins, displacers) and the weak hydrophobic ligands in hydrophobic interaction chromatography (HIC) and that various salts (e.g., kosmotropes, chaotropes, and neutral) have different effects on protein retention. In this article, the solute affinity in kosmotropic, chaotropic, and neutral mobile phases are compared and the selectivity of solutes in the presence of these salts is examined. Since solute binding in HIC systems is driven by the release of water molecules, the total number of released water molecules in the presence of various types of salts was calculated using the preferential interaction theory. Chromatographic retention times and selectivity reversals of both proteins and displacers were found to be consistent with the total number of released water molecules. Finally, the solute surface hydrophobicity was also found to have a significant effect on its retention in HIC systems.     

Clastogenicity in vitro of the Na, K, Ca and Mg salts of saccharin; and of magnesium chloride; consideration of significance

The sodium, potassium, calcium and magnesium salts of saccharin, and magnesium chloride, have been shown to be clastogenic to Chinese hamster lung (CHL) fibroblasts in vitro, but only at elevated dose levels (8-16 mg/ml). Saccharin acid was inactive to the limits of its solubility (4 mg/ml). When the data are expressed in terms of ionic concentration, each salt showed a similar clastogenic potency. This suggests that ionic effects induced by these salts in the assay medium may be the critical determinant of the clastogenic effects seen, rather than that the saccharin moiety presents a genotoxic insult to the chromosomes of the cells. The metal-chelating agents EDTA and EGTA were non-clastogenic, but the disodium salt of EDTA showed weak activity prior to toxicity at 0.5 mg/ml. The absence of a clastogenic response for the salts of saccharin at dose levels lower than 4 mg/ml is discussed within the context of the threshold-dependent tumour-promoting activity of high dose levels of sodium saccharin to the bladder of male rats. The doubtful value of conducting in vitro clastogenicity studies at dose levels greater than 10(-2) M is discussed.     

Relationship between alkaline phosphatase and neomycin formation in Streptomyces fradiae

Studies on phosphatase activity of Streptomyces fradiae 3535 grown in three different media indicate that neomycin formation varies directly with enzyme activity, sodium nitrate-maltose-mineral salts medium giving the highest yields of alkaline phosphatase and neomycin. S. fradiae contains more than one alkaline phosphatase and the phosphatase responsible for hydrolysis of neomycin phosphate appears to be substrate specific. The same enzyme apparently hydrolyses both the N-P and P-O-P bonds of neomycin pyrophosphate. The enzyme is stimulated by Ca(2+), is inactive at a pH below 7 and is inhibited by EDTA. Enzymic activity increases when mycelia are incubated in mineral salts medium, but decreases when phosphate or glucose is included in the medium, although the latter is more effective. The inhibitory effect of EDTA on neomycin formation by resting mycelia is completely reversed by Ca(2+).     

Effect of copper speciation on whole-cell soluble methane monooxygenase activity in Methylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) activity in Methylosinus trichosporium OB3b was found to be more strongly affected as copper-to-biomass ratios changed in a newly developed medium, M2M, which uses pyrophosphate for metal chelation, than in nitrate mineral salts (NMS), which uses EDTA. When M2M medium was amended with EDTA, sMMO activity was similar to that in NMS medium, indicating that EDTA-bound copper had lower bioavailability than pyrophosphate-bound copper. EDTA did not limit the association of copper with the cells; rather, copper was sequestered in a form which did not affect sMMO activity.     

Effect of Copper Speciation on Whole-Cell Soluble Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) activity in Methylosinus trichosporium OB3b was found to be more strongly affected as copper-to-biomass ratios changed in a newly developed medium, M2M, which uses pyrophosphate for metal chelation, than in nitrate mineral salts (NMS), which uses EDTA. When M2M medium was amended with EDTA, sMMO activity was similar to that in NMS medium, indicating that EDTA-bound copper had lower bioavailability than pyrophosphate-bound copper. EDTA did not limit the association of copper with the cells; rather, copper was sequestered in a form which did not affect sMMO activity.     

Ferrous ion-EDTA-stimulated phospholipid peroxidation. A reaction changing from alkoxyl-radical- to hydroxyl-radical-dependent initiation.

The stimulatory effect of ferrous salts on the peroxidation of phospholipids can be enhanced by EDTA when the concentration of Fe2+ in the reaction is greater than that of EDTA. Hydroxyl-radical scavengers do not inhibit peroxidation until the concentrations of Fe2+ and EDTA in the reaction are equal. Lipid peroxidation is then substantially initiated by hydroxyl radicals derived from a Fenton-type reaction requiring hydrogen peroxide. Superoxide radicals appear to play some role in the formation of initiating species.     

Bacterial Strain Characterizing by EDTA Requirement

A novel strain of bacteria (LPM-4) characterized by a unique EDTA requirement for cell growth was isolated. Suspensions of washed cells of strain LPM-4 degraded EDTA complexes with Ba²⁺, Mg²⁺, Ca²⁺, and Mn²⁺ at constant rates ( 0.310 ± 0.486 mmol EDTA/(g h)) and Zn-EDTA at an initial rate of 0.137 ± 0.016 mmol EDTA/(g h). The temperature optima for cell growth and EDTA degradation were determined under pH-auxostat cultivation. As compared with the known EDTA-degrading bacteria, strain LPM-4 exhibited a higher specific growth rate (0.095^{? 1}) and lower mass cell yield (0.219 g cells/g EDTA), which is promising for its practical applications for EDTA removal in wastewater treatment plants.