Uranyl Precipitation by Pseudomonas aeruginosa via Controlled Polyphosphate Metabolism

The polyphosphate kinase gene from Pseudomonas aeruginosa was overexpressed in its native host, resulting in the accumulation of 100 times the polyphosphate seen with control strains. Degradation of this polyphosphate was induced by carbon starvation conditions, resulting in phosphate release into the medium. The mechanism of polyphosphate degradation is not clearly understood, but it appears to be associated with glycogen degradation. Upon suspension of the cells in 1 mM uranyl nitrate, nearly all polyphosphate that had accumulated was degraded within 48 h, resulting in the removal of nearly 80% of the uranyl ion and >95% of lesser-concentrated solutions. Electron microscopy, energy-dispersive X-ray spectroscopy, and time-resolved laser-induced fluorescence spectroscopy (TRLFS) suggest that this removal was due to the precipitation of uranyl phosphate at the cell membrane. TRLFS also indicated that uranyl was initially sorbed to the cell as uranyl hydroxide and was then precipitated as uranyl phosphate as phosphate was released from the cell. Lethal doses of radiation did not halt phosphate secretion from polyphosphate-filled cells under carbon starvation conditions.     

Uranyl precipitation by Pseudomonas aeruginosa via controlled polyphosphate metabolism

The polyphosphate kinase gene from Pseudomonas aeruginosa was overexpressed in its native host, resulting in the accumulation of 100 times the polyphosphate seen with control strains. Degradation of this polyphosphate was induced by carbon starvation conditions, resulting in phosphate release into the medium. The mechanism of polyphosphate degradation is not clearly understood, but it appears to be associated with glycogen degradation. Upon suspension of the cells in 1 mM uranyl nitrate, nearly all polyphosphate that had accumulated was degraded within 48 h, resulting in the removal of nearly 80% of the uranyl ion and >95% of lesser-concentrated solutions. Electron microscopy, energy-dispersive X-ray spectroscopy, and time-resolved laser-induced fluorescence spectroscopy (TRLFS) suggest that this removal was due to the precipitation of uranyl phosphate at the cell membrane. TRLFS also indicated that uranyl was initially sorbed to the cell as uranyl hydroxide and was then precipitated as uranyl phosphate as phosphate was released from the cell. Lethal doses of radiation did not halt phosphate secretion from polyphosphate-filled cells under carbon starvation conditions.     

A Stopped-Flow Method for the Determination of Ascorbic Acid in Orange Juice Containing Triose Reductone

The adsorption of uranium by chitin phosphate and chitosan phosphate was investigated to obtain information on uranium recovery from aqueous systems, especially sea water and uranium mine waste water. The adsorption of uranium by chitin phosphate and chitosan phosphate was much greater than copper, cadmium, manganese, zinc, cobalt, nickel, magnesium and calcium. The adsorption of uranium was very rapid during the first 10 min and was affected by pH of the solution, temperature, granule radius and the co-existence of carbonate ion. The amounts of uranium adsorbed on the adsorbents increased linearly as the external uranium concentration increased. Uranium adsorbed on chitin phosphate easily desorbed with diluted sodium carbonate solution. On the other hand, uranyl and cobalt ions were separated from each other by using chitin phosphate.     

Uranyl precipitation by biomass from an enhanced biological phosphorus removal reactor

Heavy metal and radionuclide contamination presents a significant environmental problem worldwide. Precipitation of heavy metals on membranes of cells that secrete phosphate has been shown to be an effective method of reducing the volume of these wastes, thus reducing the cost of disposal. A consortium of organisms, some of which secrete large quantities of phosphate, was enriched in a laboratory-scale sequencing batch reactor performing Enhanced Biological Phosphorus Removal, a treatment process widely used for removing phosphorus. Organisms collected after the aerobic phase of this process secreted phosphate and precipitated greater than 98% of the uranyl from a 1.5 mM uranyl nitrate solution when supplemented with an organic acid as a carbon source under anaerobic conditions. Transmission electron microscopy, energy dispersive x-ray spectroscopy, and fluorescence spectroscopy were used to identify the precipitate as membrane-associated uranyl phosphate, UO₂HPO₄.     

The Polarographic Determination of Phosphate

A method is described for estimating polarographically the amountof phosphate in small volumes of liquid, where the phosphoruscontent lies between 0.5 and 10.5 µg. P/ml. with an errorof ± 0.2 µg. P/ml. even when chloride, nitrate,and sulphate are present in excess. The method is based on precipitationof uranyl phosphate from uranyl acetate and estimation of theuranyl ion left in solution. A comparison is made with the colorimetric method of Berenblumand Chain.     

INTRAMITOCHONDRIAL FIBERS WITH DNA CHARACTERISTICS : I. Fixation and Electron Staining Reactions

Chick embryo mitochondria, studied with the electron microscope, show crista-free areas of low electron opacity. These areas are observable after fixation with osmium tetroxide, calcium permanganate, potassium permanganate, formaldehyde, acrolein, acrolein followed by osmium tetroxide, uranyl acetate followed by calcium permanganate, and acetic acid-alcohol. Staining of sections with lead hydroxide or uranyl acetate, or with both, resulted in an increased density of a fibrous material within these areas. The appearance of the fibrous structures varied with the fixative employed; after fixation with osmium tetroxide the material was clumped and bar-like (up to 400 A in diameter), whereas after treatment of osmium tetroxide-fixed tissues with uranyl acetate before dehydration the fibrous structures could be visualized as 15 to 30 A fibrils. Treatment with ethylenediaminetetraacetate (EDTA) in place of uranyl acetate coarsened the mitochondrial fibrils. After fixation with calcium permanganate or potassium permanganate, or a double fixation by uranyl acetate followed by calcium permanganate, the fibers appeared to have a pattern and ultrastructure similar to that observed after the osmium tetroxide-uranyl acetate technique, except that some of them had a slightly greater diameter (up to 50 A). Other fixatives did not preserve the fibers so well. The fibers appeared strongly clumped by formaldehyde fixation, and were difficult to identify after fixation with acrolein or acetic acid-alcohol. The staining of nucleic acid-containing structures by uranyl acetate and lead hydroxide was improved by treatment of osmium tetroxide-fixed sections with hydrogen peroxide, and the mitochondrial fibers also had an increased density in the electron beam after this procedure. The staining characteristics suggest the fibrous material of chick embryo mitochondria to be a nucleic acid-containing structure, and its variable appearance after different fixations parallels that previously reported, or described in this paper, for the nucleoplasm of bacteria and blue-green algae. The results, in addition to those described in the accompanying communication, indicate that these mitochondria contain DNA.     

Electron Dense Artefactual Deposits in Tissue Sections: The Role of Ethanol, Uranyl Acetate and Phosphate Buffer

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.     

Electron dense artefactual deposits in tissue sections: the role of ethanol, uranyl acetate and phosphate buffer.

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.     

Phosphate release and heavy metal accumulation by biofilm-immobilized and chemically-coupled cells of a Citrobacter sp. pre-grown in continuous culture

A heavy metal-accumulating Citrobacter sp. was grown in carbon-limiting continuous culture in an air-lift fermentor containing raschig rings as support for biofilm development. Planktonic cells from the culture outflow were immobilized in parallel on raschig rings by chemical coupling (silanization), for quantitative comparison of phosphatase activity and uranyl uptake by both types of immobilized cell. The flow rate giving 50% conversion of substrate to product (phosphate) in flow-through reactors was higher, by 35-40%, for the biofilm-immobilized cells, possibly exploiting a pH-buffering effect of inorganic phosphate species within the extracellular polymeric material. Upon incorporation of uranyl ions (0.2 mM UO22+), both types of cell removed more than 90% of the input UO22+ at slow flow rates, but the chemically-coupled cells performed better at higher flow rates. The deposited material (HUO2PO4) subsequently removed Ni2+ from a second flow via intercalative ion exchange of Ni2+ into the crystalline HUO2PO4.4H2O lattice. This occurred irrespective of the method of coupling of the biomass to the support and suggested that uranyl phosphate accumulated by both types of cell has potential as a bio-inorganic ion exchanger-a potential use for the uranium recoved from primary waste treatment processes.     

Pentacopper(II) complexes of alpha-aminohydroxamic acids: uranyl-induced conversion of a 12-metallacrown-4 to a 15-metallacrown-5

Effects of uranyl on the pentacopper(II) complexes of alpha-leucinehydroxamic acid and alpha-tyrosinehydroxamic acid were studied in water and methanol by means of electrospray ionisation mass spectrometry (ES-MS), absorption spectrophotometry, circular dichroism spectroscopy and proton NMR spectroscopy. All the measurements were consistent with the complete conversion of a 12-metallacrown-4 to a 15-metallacrown-5 upon addition of one equivalent of the uranyl ion. The uranyl ion is accommodated in the cavity formed by five copper(II) ions and five alpha-aminohydroxamate ligands. The 15-metallacrown-5 inclusion complexes have a high affinity for the uranyl ion. Competition studies showed that even in the presence of a large excess of calcium(II), the 15-metallacrown-5 remained stable, and no exchange reactions between calcium(II) and uranyl were observed. Extraction of uranyl from the 15-metallacrown-5 was also not detected in the presence of a large excess of 18-crown-6. Trivalent lanthanide ions can be partially sequestered by the 15-metallacrown-5, however, even these trivalent ions are displaced by uranyl.     

Glycosomes (protein-glycogen complex) in the canine heart. Ultrastructure, histochemistry and changes induced by acidic treatment.

Cardiac conducting fibers were selected from two dogs defined as A and B. The specimens differed in the reaction of their electron dense granules, commonly referred to as glycogen, to the treatment en bloc with uranyl acetate. Material was fixed in glutaraldehyde and OsO4. Blocks were processed either conventionally or immersed in uranyl acetate before dehydration. Sections were examined unsatined, stained with U and/or Pb or with a histochemical technique (PA-TSC-SP) specific for glycogen. Electron dense granules have affinity to Os, U and Pb which suggests ionic reactions specific for protein but improbable for glycogen. Large granules in A turned into pale ghosts and small granules in B disappeared after treatment en bloc with uranyl acetate. PA-TSC-SP in conventional samples showed glycogen particles arranged into aggregates corresponding in size to the electron dense granules. Treatment en bloc slightly affected glycogen aggregates in A and resulted in a formation of large clumps of glycogen particles in B. It was concluded that the electron dense granules represented protein bound to glycogen in the organelles called glycosomes. Acidic action of uranyl acetate removed protein from glycosomes. The degree of this removal depended on the amount of protein present in glycosomes in the moment of fixation.     

The interaction of uranyl ions with inorganic pyrophosphatase from baker's yeast.

The interaction of uranyl ions with inorganic pyrophosphatase from baker's yeast was investigated by measurement of their effect on the protein fluorescence. Fluorescence titrations of the native enzyme with uranyl nitrate show that there is a specific binding of uranyl ions to the enzyme. It was deduced that each subunit of the enzyme binds one uranyl ion. The binding constant was estimated to be in the order of 10(7) M-1. The enzyme which contains a small number of chemically modified carboxyl groups was not able to bind uranyl ions specifically. The modification of carboxyl groups was carried out by use of a water soluble carbodiimide and the nucleophilic reagent N-(2,4-dinitro-phenyl)-hexamethylenediamine. The substrate analogue calcium pyrophosphate displaced the uranyl ions from their binding sites at the enzyme. From the results it is concluded that carboxyl groups of the active site are the ligands for the binding of uranyl ions.     

Zum chemischen Status der Deoxyribonucleinsäure im Bakteriennucleoid

Zusammenfassung Der in einer früheren Mitteilung beschriebene Effekt der Uranylsalze auf die Struktur des Bakteriennucleoids tritt bereits bei wesentlich niedrigeren Uranylionenkonzentrationen ein, wenn die Zellen bereits mit Uranylacetat und ÄDTE (successiv) vorbehandelt wurden. ÄDTE allein verursacht eine geringere Senkung der Grenzkonzentration. Durch Anwendung eines Gemisches von Calcium-und Magnesiumionen und Polyaminen wird diese Erscheinung größtenteil wieder rückgängig gemacht.Außerdem wurde versucht, die Bindungsverhältnisse der Uranylionen an DNS in vitro genauer zu charakterisieren.Alle Befunde sprechen dafür, daß die Uranylionen natürliche Liganden von den Phosphatgruppen der DNS zu verdrängen vermögen. Diese Liganden sind, soweit sie mit Hilfe von ÄDTE allein ausgetauscht werden können, Metallionen, im übrigen sind sie organischer Natur. Die Experimente sind als erster Schritt zu einer indirekten Charakterisierung dieser Liganden aufzufassen.Der Struktureffekt der Uranylionen am Nucleoid tritt ein, wenn etwa die Hälfte der DNS-Phosphatgruppen durch Uranylionen neutralisiert ist.

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Summary The limiting concentration of uranyl ions which is necessary to induce a fibrillar appearance in bacterial nucleoids as observed in ultrathin section, was decreased markedly by pretreatment of the cells with solutions of uranyl salt and subsequent removal of uranyl ions by EDTA, in a lesser extent by pretreatment with EDTA alone. If the pretreatment was followed by application of a mixture of calcium and magnesium ions and polyamines, its effect was reversed to a great extent.In in vitro experiments the association of uranyl ions to DNA was studied in some detail.All observations are in accordance with the view that uranyl ions displace ligands from the phosphate groups of intracellular DNA which are partly (as far as they are removed by EDTA alone) divalent metal ions, the remainder being of organic nature and possibly of low molecular weight.The experiments may be considered as a first step to an indirect characterization of these ligands.The effect of uranylions on nucleoid structure is induced as soon as about 50% of the phosphate groups of DNA are neutralized by uranyl ions.

     

Ultrastructure of the cell wall and cytoplasmic membrane of gram-negative bacteria with different fixation techniques

The ultrastructure of the cytoplasmic membrane and cell wall of two strains of Escherichia coli, Proteus morganii, P. vulgaris, Acinetobacter anitratum, Moraxella lacunata, Erwinia amylovora, Acinetobacter sp., and of a plant pathogen, unclassified gram-negative, fixed by the Ryter-Kellenberger procedure, was found to be significantly affected by the use or omission of the uranyl postfixation included in that procedure, and by the presence or absence of calcium in the OsO(4) fixative. The omission of the uranyl treatment results in a less clear profile of both the outer membrane of the cell wall and of the cytoplasmic membrane. The observation of these two membranes is further limited when both uranyl and calcium are omitted. The R-layer and the material covering the surface of the cell wall appear more distinct when the uranyl postfixation is not used. Evidence is given suggesting that the influence of uranyl and calcium ions on the appearance of the outer and cytoplasmic membranes would be primarily due to their action as fixatives, whereas the influence of uranyl on the appearance of the R-layer would be due to a direct action on the peptidoglycan component of this layer. When uranyl acetate is used as a section stain after the embedding in plastic, it improves the observation of the R-layer. In this case, a well contrasted R-layer is consistently observed in all strains studied, provided that the postfixation has been omitted. The frequent difficulty in clearly observing the R-layer in many published micrographs probably results from the common use of uranyl postfixation.     

The correlation between bismuth and uranyl staining and phosphorus content of intracellular structures as determined by electron spectroscopic imaging

Four groups of intracellular structures can be recognized according to bismuth and uranyl staining and phosphorus content. (1) Those which contain phosphorus and stain strongly with uranyl acetate but not with bismuth (ribosomes, heterochromatin and mature ribosomal precursor granules), presumably because of their nucleic acid content. (2) Those which contain phosphorus and stain with uranyl acetate and bismuth (interchromatin granules, immature ribosomal precursor granules and mitochondrial granules), presumably because at least some of their phosphate is available to react with bismuth. (3) Those which contain little phosphorus but which stain strongly with bismuth and weakly with uranyl acetate (Golgi complex beads), perhaps because some ligand in addition to phosphate reacts with bismuth, and (4) those which do not contain phosphorus and stain with neither uranyl acetate nor bismuth (portasomes). Uranyl staining correlates strongly with the phosphorus content of nucleic acids, proteins and inorganic deposits. Bismuth will stain some phosphorylated molecules but not all. Thus only some phosphates stain with bismuth.     

Uranium(VI) bioprecipitation mediated by a phosphate solubilizing Acinetobacter sp. YU-SS-SB-29 isolated from a high natural background radiation site

Bioprecipitation of uranium (U) into uranyl phosphate (U-P) mediated by soluble ortho-phosphate is an attractive proposition for U bioremediation. As an alternative to the microbial phosphatase, we have investigated the dissolution of phosphate by the organic acids produced by bacteria to aid in U precipitation. The bacterium Acinetobacter sp. YU-SS-SB-29, isolated from monazite sand of natural background radiation site solubilized 952.0 ± 46.7 mg L⁻¹ phosphate from tri-calcium phosphate (TCP) in the Pikovskaya's medium and showed tolerance to 120 ppm U(VI). U(VI) bioprecipitation was investigated by adding different concentrations of U(VI) to a cell-free culture supernatant containing ortho-phosphate released from TCP by the bacterium. A yellow precipitate was immediately formed following which there was a reduction in U(VI) concentration. A strong positive correlation (R² = 0.98) was observed between % decrease in phosphate and U(VI) concentration (up to 750 ppm U) added. FTIR and EDX spectra of the yellow precipitate demonstrated the involvement of phosphate groups in U(VI) binding. Furthermore, the XRD pattern of the precipitate agrees well with that of chernikovite, a uranyl phosphate mineral. The results from this study demonstrate the potential of the U tolerant, phosphate solubilizing bacterium Acinetobacter sp. YU-SS-SB-29 for non-reductive in situ bioprecipitation of uranium.     

Effect of vaccinations with recombinant fusion proteins on Ancylostoma caninum habitat selection in the canine intestine

Laboratory dogs were vaccinated subcutaneously with 3 different recombinant fusion proteins, each precipitated with alum or calcium phosphate. The vaccinated dogs were then challenged orally with 400 third-stage infective larvae (L3) of the canine hookworm, Ancylostoma caninum. The 3 A. caninum antigens selected were Ac-TMP, an adult-specific secreted tissue inhibitor of metalloproteases; Ac-AP, an adult-specific secreted factor Xa serine protease inhibitor anticoagulant; and Ac-ARR-1, a cathepsin D-like aspartic protease. Each of the 3 groups comprised 6 male beagles (8 +/- 1 wk of age). A fourth group comprised control dogs injected with alum. All of the dogs vaccinated with Ac-TMP or Ac-APR-1 exhibited a vigorous antigen-specific antibody response, whereas only a single dog vaccinated with Ac-AP developed an antibody response. Dogs with circulating antibody responses exhibited 4.5-18% reduction in the numbers of adult hookworms recovered from the small intestines at necropsy, relative to alum-injected dogs. In contrast, there was a concomitant increase in the number of adult hookworms recovered from the colon. The increase in colonic hookworms was as high as 500%, relative to alum-injected dogs. Female adult hookworms were more likely to migrate into the colon than were males. Anti-enzyme and anti-enzyme inhibitor antibodies correlated with an alteration in adult hookworm habitat selection in the canine gastroinntestinal tract.     

A micromethod for the determination of an unbound radioactive iodine in radiolabeled glycoproteins by means of uranyl acetate

A method for determination of an unbound radioactive iodine in radiolabeled glycoproteins is based on coprecipitation of these macromolecules with uranyl and phosphate ions. Under these conditions unbound 125I is not precipitated. The method is simple, rapid, and very sensitive.     

Ectopic Osteoid and Bone Formation by Three Calcium-Phosphate Ceramics in Rats,Rabbits and Dogs

Calcium phosphate ceramics with specific physicochemical properties have been shown to induce de novo bone formation upon ectopic implantation in a number of animal models. In this study we explored the influence of physicochemical properties as well as the animal species on material-induced ectopic bone formation. Three bioceramics were used for the study: phase-pure hydroxyapatite (HA) sintered at 1200°C and two biphasic calcium phosphate (BCP) ceramics, consisting of 60 wt.% HA and 40 wt.% TCP (β-Tricalcium phosphate), sintered at either 1100°C or 1200°C. 108 samples of each ceramic were intramuscularly implanted in dogs, rabbits, and rats for 6, 12, and 24 weeks respectively. Histological and histomorphometrical analyses illustrated that ectopic bone and/or osteoid tissue formation was most pronounced in BCP sintered at 1100°C and most limited in HA, independent of the animal model. Concerning the effect of animal species, ectopic bone formation reproducibly occurred in dogs, while in rabbits and rats, new tissue formation was mainly limited to osteoid. The results of this study confirmed that the incidence and the extent of material-induced bone formation are related to both the physicochemical properties of calcium phosphate ceramics and the animal model.     

Calcium precipitation in acute and chronic brain diseases.

In rat brain, calcification associated with excitotoxicity has been proposed to play a protective role, whereas in human brain, nonartherosclerotic calcification is present in several pathological conditions without any clear significance. To determine if calcification can be viewed as a protective step of calcium homeostasis during chronic and acute neuronal suffering, cerebral cortex and hippocampus of patients with Alzheimer's disease, vascular dementia and neonatal hypoxia-ischemia were investigated. To investigate the human specificity, these two areas were also studied in dogs with established cognitive deficits. In all groups, calcium precipitates were observed in the cerebral parenchyma associated with neuronal damage. The cerebral cortex presented a higher degree of calcification than the hippocampus. The neonatal hypoxia-ischemia group was characterised by a higher degree of calcification, whereas the groups with lowest calcification were the Alzheimer's patients and dogs. As shown by X-ray microanalysis, in the precipitates, calcium is mainly associated with phosphorus in a form that resembles hydroxyapatites. Thus, intracellular calcium concentration associated with neuronal suffering may reduce the energy extrusion. We propose that, to help overcome excitotoxicity, calcium precipitation acts in CNS of vertebrates as a new compartment of the calcium homeostasis in which free cytoplasmic calcium ions are inactivated by phosphate ones.