Reactive phosphate ester of the carcinogen 2-(N-hydroxy)acetamidofluorene

1. Reaction of 2-(N-acetoxy)-acetamidofluorene with orthophosphate buffer at pH7 yielded a large quantity of water-soluble fluorene derivatives, which showed absorption peaks at 303, 290 and 280nm. Tris buffer under similar conditions gave negligible reaction. 2. Hydrolysis of polar material with acid or alkaline phosphatases liberated equimolar amounts of inorganic phosphate and an ether-extractable fluorene derivative. On the basis of its u.v. spectrum, R(F) values after paper chromatography, solubility in alkali and colour with spray reagents, the derivative was characterized tentatively as 2-acetamido-5-hydroxyfluorene. 3. Polar material also contained a reactive fluorene derivative which gave characteristic reaction products with methionine and guanosine. The reactive derivative was characterized as a phosphate ester of 2-(N-hydroxy)-acetamidofluorene. 4. It is suggested that such reactive phosphate esters may also be some of the ultimate carcinogenic metabolites of carcinogenic aromatic hydroxamic acids.     

Effect of hydrogen ion buffers on photosynthetic oxygen evolution in the blue-green alga, Agmenellum quadruplicatum.

The photosynthetic oxygen evolution capacity of Agmenelium quadruplication suspended in four hydrogen ion buffers (pH 7.4, 0.05 M) and its synthetic marine growth medium was measured with an oxygen electrode. High rates of oxygen evolution were obtained in the growth medium and N-tris(hydroxymethyl)-methylglycine (Tricine) buffer. Compared to oxygen evolution in the growth medium, rates in phosphate buffer and N-tris(hydroxymethyl)-2-aminoethanesulphonic acid (TES) buffer were sometimes reduced by up to 30% and rates in tris (hydroxymethyl) amino-methane (Tris) were consistently reduced by 50%. An incubation-rinsing procedure caused inhibition of oxygen evolution in TES, phosphate, and Tris by 50 to 100%. Oxygen evolution could be restored to cells rinsed in TES or phosphate by resuspension in growth medium or in buffer plus magnesium and calcium ions. Bezoquinone-supported oxygen evolution was not affected by rinsing with any buffer tested except Tris. Ferricyanide was photoreduced at a low rate by cells rinsed in Tes but at a high rate in TES plus magnesium and calcium ions. We interpreted our results to mean that, in Agmenellum quadruplicatum, inhibition of photosynthetic oxygen evolution by Tris occurs at the level of photosystem 2 while the effects of TES and phosphate are on electron-transport occurring after the rate-limiting reaction.     

Disruptive effects of tris and sodium lauroyl sarcosinate on the outer membrane of Pseudomonas cepacia shown by fluorescent probes

The disruptive effects of Tris buffer and sodium lauroyl sarcosinate (Sarkosyl) on the outer membrane (OM) of Pseudomonas cepacia were investigated with several fluorescent probes. Tris increased the permeability of the OM to 6-anilino-l-naphthalenesulphonic acid and 2-p-toluidinylnaphthalene-6-sulphonate. The degree of damage to the OM was enhanced when the pH was decreased 3-(N-morpholino)propanesulphonic acid buffer had a small but significant effect at acid pH, while citrate/phosphate buffer showed insignificant effects. Sarkosyl released 3,3'-dipentyloxacarbocyanine iodide (CC5) from CC5-labelled OM or whole cells and altered OM fluidity as studied by fluorescence polarization.     

Influence of inorganic phosphate and organic buffers on cephalosporin production by Streptomyces clavuligerus

A high concentration of potassium phosphate (75–100 mM) stabilized pH and supported extensive growth of Streptomyces clavuligerus in a chemically defined medium; such a concentration also inhibited cephalosporin production. Although Tris buffer was found to have detrimental effects on growth and antibiotic production, 3-(N-morpholino)-propane sulfonate (MOPS) or 2-(N-morpholino)-ethane sulfonate (MES) buffer provided a nontoxic buffering system. In the presence of MOPS buffer, cephalosporin production was optimal at 25 mM phosphate, whereas higher concentrations of phosphate progressively inhibited antibiotic production up to 85% without modifying the pH pattern. MOPS buffer can be used to conduct fermentations at a relatively constant pH value in shake flasks.List of Non-Common Abbreviations MOPS 3-(N-morpholino)propane sulfonic acid - MES 2-(N-morpholino)ethane sulfonic acid     

New Buffers to Improve the Quantitative Real-Time Polymerase Chain Reaction

Real-time PCR is a potent technique for nucleic acid quantification for research and diagnostic purposes, the wide dynamic range being one of the advantages over other techniques like the microarray. Several additives and enhancers have been studied to expand the PCR dynamic range in order to be more efficient in quantifying low quantities of nucleic acids, increase the yield and improve reaction efficiency. Shown here is that a combination of new buffers with the regularly used Tris buffer makes it possible to expand the real-time PCR dynamic range and to improve the efficiency and correlation coefficient. Mixing HEPES, TEA or MOPS with Tris was more efficient than Tris alone. It was also found that, if the pH value of the Tris buffer was calibrated with phosphoric acid instead of hydrochloric acid, then the dynamic range was significantly improved and low quantities could be detected and quantified more efficiently. Mixing more than one compound with the Tris buffer was also effective for expanding the dynamic range and increasing the efficiency and correlation coefficient in quantitative real-time PCR.     

New buffers to improve the quantitative real-time polymerase chain reaction

Real-time PCR is a potent technique for nucleic acid quantification for research and diagnostic purposes, the wide dynamic range being one of the advantages over other techniques like the microarray. Several additives and enhancers have been studied to expand the PCR dynamic range in order to be more efficient in quantifying low quantities of nucleic acids, increase the yield and improve reaction efficiency. Shown here is that a combination of new buffers with the regularly used Tris buffer makes it possible to expand the real-time PCR dynamic range and to improve the efficiency and correlation coefficient. Mixing HEPES, TEA or MOPS with Tris was more efficient than Tris alone. It was also found that, if the pH value of the Tris buffer was calibrated with phosphoric acid instead of hydrochloric acid, then the dynamic range was significantly improved and low quantities could be detected and quantified more efficiently. Mixing more than one compound with the Tris buffer was also effective for expanding the dynamic range and increasing the efficiency and correlation coefficient in quantitative real-time PCR.     

Kinetics of the reconstituted dicarboxylate carrier from rat liver mitochondria

The dicarboxylate carrier from rat liver mitochondria was purified by the Amberlite/hydroxyapatite procedure and reconstituted in egg yolk phospholipid vesicles by removing the detergent with Amberlite. The efficiency of reconstitution was optimized with respect to the ratio of detergent/phospholipid, the concentration of phospholipid and the number of Amberlite column passages. In the reconstituted system the incorporated dicarboxylate carrier catalyzed a first-order reaction of malate/phosphate exchange. V of the reconstituted malate/phosphate exchange was determined to be 6000 mumol/min per g protein at 25 degrees C. This value was independent of the type of substrate present at the external or internal space of the liposomes (malate, phosphate or malonate). The half-saturation constant was 0.49 mM for malate, 0.54 mM for malonate and 1.41 mM for phosphate. The activation energy of the exchange reaction was determined to be 95.8 kJ/mol. The transport was independent of the external pH in the range between pH 6 and 8.     

Detection of Escherichia coli O157:H7 using immunomagnetic separation and absorbance measurement

An assay system for detection of Escherichia coli O157:H7 was developed based on immunomagnetic separation of the target pathogen from samples and absorbance measurement of p-nitrophenol at 400 nm from p-nitrophenyl phosphate hydrolysis by alkaline phosphatase (EC 3.1.3.1) on the "sandwich" structure complexes (antibodies coated onto micromagnetic beads--E. coli O157:H7-antibodies conjugated with the enzyme) formed on the microbead surface. The effects of immunoreaction time, phosphate buffer concentration, pH and temperature on the immunomagnetic separation of E. coli O157:H7 from samples were determined and the conditions used for the separation were 1-h reaction time, 1.0 x 10(-2) M PBS, pH 8.0 and 33 degrees C in this system. The effects of MgCl(2) concentration, Tris buffer concentration, pH and temperature on the activity of alkaline phosphatase conjugated on the immuno-"sandwich" structure complexes were investigated after immunomagnetic separation of the target pathogen and the conditions used for the enzymatic amplification were 1.0 x 10(-4) M MgCl(2), 1.0 M Tris buffer, pH 8.0, 28 degrees C and 30-min reaction time during the assay. The selectivity of the system was examined and no interference from the other pathogens including Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes was observed. Its working range was from 3.2 x 10(2) to 3.2 x 10(4) CFU/ml, and the relative standard deviation was 2.5-9.9%. The total detection time was less than 2 h.     

The pathway of pepsin-catalysed transpeptidation. Evidence for the reactive species being the anion of the acceptor molecule

Several minor pepsinogens, present in extracts of bovine fundic mucosa obtained from the fourth stomach or abomasum, were separated from the main pepsinogen by chromatography on hydroxyapatite at pH7.3. The major pepsinogen and two of these minor pepsinogens were studied in detail. All three zymogens have N-terminal Ser-Val-, C-terminal -Val-Ala and not more than 1mol of glucose/mol of protein; no significant differences in amino acid composition were found. The pepsinogens differ in their organic phosphate content, which accounts for their chromatographic separation. By activation at 0 degrees C and pH2, a corresponding series of pepsins is formed. These enzymes were separated by hydroxyapatite chromatography at pH5.7. All the pepsins have N-terminal valine, C-terminal alanine and are free from carbohydrate. Again the only difference detected among them is in their organic phosphate content. The pepsins of high phosphate content are converted by an acid phosphatase in vitro into pepsins of low phosphate content.     

Quantitative evaluation of the role of organic acid exudation in the mobilization of rock phosphate by rape

Phosphorus-deficient rape plants appear to acidify part of their rhizosphere by exuding malic and citric acid. A simulation model was used to evaluate the effect of measured exudation rates on phosphate uptake from Mali rock phosphate. The model used was one on nutrient uptake, extended to include both the effect of ion uptake and exudation on rhizosphere pH and the effect of rhizosphere pH on the solubilization of rock phosphate. Only the youngest zones of the root system were assumed to exude organic acids. The transport of protons released by organic acids was described by mass flow and diffusion. An experimentally determined relation was used describing pH and phosphate concentration in the soil solution as a function of total soil acid concentration. Model parameters were determined in experiments on organic acid exudation and on the uptake of phosphate by rape from a mixture of quartz sand and rock phosphate. Results based on simulation calculations indicated that the exudation rates measured in rape plants deficient in phosphorus can provide the roots with more phosphate than is actually taken up. Presence of root hairs enhanced the effect of organic acid exudation on calculated phosphate uptake. However, increase of root hair length without exudation as an alternative strategy to increase phosphate uptake from rock phosphate appeared to be less effective than exudation of organic acids. It was concluded that organic acid exudation is a highly effective strategy to increase phosphate uptake from rock phosphate, and that it unlikely that other rhizosphere processes play an important role in rock phosphate mobilization by rape.     

Interchange reaction of disulfides and denaturation of oxytocin by copper(II)/ascorbic acid/O2 system

The interchange reaction of disulfides was caused by the copper(II)/ascorbic acid/O2 system. The incubation of two symmetric disulfides, L-cystinyl-bis-L-phenylalanine (PP) and L-cystinyl-bis-L-tyrosine (TT), with L-ascorbic acid and CuSO4 in potassium phosphate buffer (pH 7.2, 50 mM) resulted in the formation of an asymmetric disulfide, L-cystinyl-L-phenylalanine-L-tyrosine (PT), and the final ratio of PP:PT:TT was 1:2:1. As the reaction was inhibited by catalase and DMSO only at the initial time, hydroxyl radical generated by the copper(II)/ascorbic acid/O2 system seemed to be responsible for the initiation of the reaction. Oxytocin and insulin were denatured by this system, and catalase and DMSO similarly inhibited these denaturations. As the composition of amino acids was unchanged after the reaction, hydroxyl radical was thought to cause the cleavage and/or interchange reaction of disulfides to denature the peptides.     

A study of the growth condition and solubilization of phosphate from hydroxyapatite byPantoea agglomerans

The growth conditions ofPantoea agglomerans, a phosphate solubilizing organism, were studied in our laboratory to determine the optimal conditions.Pantoea agglomerans showed the highest growth rate at 30°C, pH 7.0 and 2 vvm, after 50 h cultivation. A certain relationship between pH and phosphate concentration, was evident when the glucose concentration in the medium was changed. Increasing glucose concentration increased the pH buffer action of the broth. At glucose concentrations higher than the optimum concentration of 0.2 M, the cell growth was retarded.P. agglomerans consumed glucose as a substrate to produce organic acids which caused the pH decrease in the culture medium. The phosphate concentration in the medium was increased by the presence of the organic acids, which solubilized insoluble phosphates such as hydroxyapatite.     

pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I.

Human kynurenine aminotransferase I/glutamine transaminase K (hKAT-I) is an important multifunctional enzyme. This study systematically studies the substrates of hKAT-I and reassesses the effects of pH, Tris, amino acids and alpha-keto acids on the activity of the enzyme. The experiments were comprised of functional expression of the hKAT-I in an insect cell/baculovirus expression system, purification of its recombinant protein, and functional characterization of the purified enzyme. This study demonstrates that hKAT-I can catalyze kynurenine to kynurenic acid under physiological pH conditions, indicates indo-3-pyruvate and cysteine as efficient inhibitors for hKAT-I, and also provides biochemical information about the substrate specificity and cosubstrate inhibition of the enzyme. hKAT-I is inhibited by Tris under physiological pH conditions, which explains why it has been concluded that the enzyme could not efficiently catalyze kynurenine transamination. Our findings provide a biochemical basis towards understanding the overall physiological role of hKAT-I in vivo and insight into controlling the levels of endogenous kynurenic acid through modulation of the enzyme in the human brain.     

Identification of nutrient-dependent changes in extracellular pH and acid phosphatase secretion in Aspergillus nidulans

The present study was designed to identify nutrient-dependent changes in extracellular pH and acid phosphatase secretion in the biA1 palC4 mutant strain of Aspergillus nidulans. The palC4 mutant was selected as lacking alkaline phosphatase, but having substantially increased acid phosphatase activity when grown on solid minimal medium under phosphate starvation, pH 6.5. Gene palC was identified as a putative member of a conserved signaling cascade involved in ambient alkaline sensing whose sole function is to promote the proteolytic activation of PacC at alkaline pH. We showed that both poor growth and conidiation of the palC4 mutant strain on solid medium, alkaline pH, were relative to its hypersensitivity to Tris (hydroxymethyl) aminomethane buffer. Also, the secretion of acid phosphatase was repressed when both the wild-type and palC4 mutant strains were grown in low-phosphate yeast extract liquid medium, pH 5.0, indicating that the secretion of this enzyme is not necessary to regenerate inorganic phosphate from the organic phosphate pool present in yeast extract.     

Steroid sulfatase in the human ovary and placenta: enzyme kinetics and phosphate inhibition.

In 5 placental homogenates the Km of steroid sulfatase for DHEA sulfate increased from 15.4 in Tris buffer to 26.8 microM in phosphate (both buffers 0.1 M, pH 7.4), P less than 0.05. In 3 pooled ovarian preparations the Km increased from 14.3 microM in Tris to 33.0 microM in phosphate, P less than 0.01. There was no significant difference between the ovarian and placental values for Km in either Tris or phosphate (P greater than 0.5), and the increase in the Km produced by phosphate in ovarian tissue was not significantly different from that in the placenta (P greater than 0.5). In the placentas the Vmax in Tris was 1420 pmol/min/mg protein and this fell to 523 pmol/min/mg protein in phosphate (P less than 0.005). The Vmax was 50-fold higher in the placenta than in the ovary in either Tris or phosphate (both P less than 0.001). In the ovary, the Vmax was 27.6 pmol/min/mg protein in Tris and 11.0 pmol/min/mg protein in phosphate (P less than 0.05). The reduction of Vmax produced by phosphate in the ovary was not significantly different from that in the placenta (P greater than 0.5). The slope of the 1/v vs 1/S plot (Km/Vmax) increased 4.7-fold in the placentas and 5.8-fold in the ovaries in phosphate over that in Tris (both P less than 0.001); the increase in the placentas was not significantly different from that in the ovaries (P greater than 0.5). Phosphate ion acts as a mixed inhibitor of both placental and ovarian steroid sulfatase.     

Isolation of protoplasts and vacuoles from cell suspension cultures of Coleus blumei Benth.

In order to study the accumulation and transport of rosmarinic acid in suspension cells of Coleus blumei we established an efficient method to isolate protoplasts and vacuoles. Protoplasts were disrupted by an osmotic shock in a medium with basic pH containing ethylenediamine tetraacetic acid. The resulting vacuoles were purified on a two-step Ficoll gradient. The comparison of the rosmarinic acid contents of cells, protoplasts and vacuoles showed that the depside is localized in the vacuole. Data concerning the yield and purity of the vacuoles are presented. In addition we show that at the physiological pH of the cytoplasm rosmarinic acid is present almost exclusively as an anion and cannot pass a membrane by simple diffusion. We therefore propose a carrier system for the transport of rosmarinic acid into the vacuole.Abbreviations EDTA ethylenediamine tetraacetic acid - HEPES 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethane sulfonic acid - HPLC high performance liquid chromatography - MES morpholinoethane sulfonic acid - NADP⁺ ß-nicotinamide adenine dinucleotide phosphate - PEG polyethylene glycol - RA rosmarinic acid - Tris Tris(hydroxymethyl)aminomethane     

Solubilization of inorganic phosphate byRhizobium

A large number of strains ofRhizobium were able to solubilize the insoluble phosphate compound, hydroxy-apatite, in liquid culture. Solubilization of hydroxyapatite byRhizobium was not mediated by an enzyme but acidity developed in the cultures was involved in the process. An inverse relationship between the level of soluble phosphate and medium pH was evident. The ability to solubilize hydroxyapatite varied among the strains. In a medium without NH ₄ ⁺ , some of the strains showed better activity than when NH ₄ ⁺ was present, suggesting involvement of different mechanisms for phosphate solubilization.R. meliloti SU 47 produced 2-ketogluconic acid along with an unidentified acid in the medium containing NH ₄ ⁺ . 2-Ketogluconic acid was identified as the major factor in inorganic phosphate solubilization. Initial presence of soluble phosphate in the medium had no discernible influence on the extent of hydroxyapatite solubilization. Initial presence of calcium reduced solubilization of phosphate and addition of EDTA to stationary phase cultures caused an increase in the level of soluble phosphate.     

Characterization by high performance liquid chromatography (HPLC) of the solubilization of phosphorus in iron ore by a fungus

Summary The value of iron ore is adversely affected by phosphorus in concentrations over 0.03% by weight. The present research concerns the use of metabolic products of aPenicillium-like fungus to leach insoluble phosphates (hydroxyapatite) from ores. Ion chromatography was used to measure metabolism of glucose into acidic fragments. The rate and products of glucose degradation depended on both the chemical composition of the growth medium (buffered or not) and incubation conditions (shaken or quiescent). The principal products were identified as oxalic acid and isomers of propylene dicarboxylic acid, mainly itaconic acid. Continued, slow metabolism of itaconic acid generates more oxalic acid. Aliphatic acids were not detected. Both iron ore phosphate and calcium phosphate were partially solubilized by either the spent broth or aqueous oxalic acid. Solubilization of ore phosphorus was greatly assisted by hydrochloric acid added to the spent broth in small increments. The data suggest biological alternatives to costly leaching procedures that use only mineral acids.     

A STUDY OF SOIL BACTERIA DISSOLVING CERTAIN MINERAL PHOSPHATE FERTILIZERS AND RELATED COMPOUNDS

SUMMARY: Over a hundred isolates which produced haloes around their colonies on dilution plates containing calcium carbonate or dicalcium phosphate have been obtained in pure culture from the root region of the oat plant. Of these, more than 50% were pleomorphic, and this group had the highest proportion of isolates which could produce clear zones on agar media containing either calcium carbonate, dicalcium phosphate, tricalcium phosphate, freshly precipitated hydroxyapatite or basic slag. None of the isolates showed dissolving ability on agar media containing gafsa rock phosphate, variscite, strengite or taranakite. However, when an analytical method was used, 82% of the isolates tested proved able to release phosphate from gafsa rock phosphate, though to a much lesser extent than from dicalcium phosphate. None of the isolates tested by this method released phosphate from variscite, strengite or taranakite.
The nature of the organic acids produced from glucose by 26 of the isolates was also investigated. The majority produced mainly lactic acid, but a few also gave an acid with chromatographic properties similar to those of 2-keto-gluconic acid.