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An immobilized metal ion affinity adsorption and scintillation proximity assay for receptor-stimulated phosphoinositide hydrolysis.

A novel approach to measuring receptor-stimulated phosphoinositide hydrolysis was developed based on the principles of immobilized metal ion affinity chromatography (IMAC) and scintillation proximity assay (SPA). Hard Lewis metal ions, such as Zr(4+), Ga(3+), Al(3+), Fe(3+), Lu(3+), and Sc(3+), were immobilized on SPA beads via metal chelate and utilized as affinity ligands to entrap inositol phosphates. [3H]Inositol phosphates bound to IMAC-SPA beads through the strong interaction of their phosphate group with the immobilized metal ions. The binding brought [3H]inositol phosphates in close proximity to the scintillant embedded in the SPA beads, thereby allowing the radioactivity to be quantified. Quantification of [3H]inositol phosphate production in cells preincubated with [3H]inositol provided a highly sensitive measurement of phosphoinositide hydrolysis. The utility of this approach was demonstrated in measuring the response mediated by the G-protein-coupled neurokinin NK1 receptor and the tyrosine kinase-linked platelet-derived growth factor (PDGF) receptor. Substance P stimulated phosphoinositide hydrolysis concentration-dependently in CHO cells expressing NK1 receptors with a maximal 12-fold increase in inositol phosphate production. Similarly, PDGF-BB stimulated a 5-fold increase in phosphoinositide hydrolysis in quiescent Swiss 3T3 cells. This new approach is highly sensitive, fast, simple, easily performed on 96-well plates, and amenable for high-throughput screening.

3T3 Cells↗

Using rare earth element tracers and neutron activation analysis to study rill erosion process.

Spatially averaged soil erosion data provide little information on the process of rill erosion. The dynamically varied data on the temporal and spatial distributions in the rill erosion process are needed to better understand the erosion process and reveal its innate characteristics. The objectives of this study were to examine the feasibility and effectiveness of rare earth element (REE) tracers and the neutron activation analysis (NAA) method on the study of the rill erosion process and to reveal quantitatively the relationships and characteristics of temporal and spatial distributions of sediment yield in rill erosion. Four REEs were used to study the changeable process of rill erosion at 4 slope positions. Four water inflow rates were applied to a 0.3 x 5 m soil bed at 3 slopes of 10.5%, 15.8% and 21.2% in scouring experiments. All of the runoff was collected in the experiment. Each sample was air-dried and well mixed. Then 20 g of each sample was sieved through 100-mesh and about a 50 mg sample was weighed for analysis of the four elemental compositions by NAA. Results indicate that the REE tracers and NAA method can be used to not only quantitatively determine soil erosion amounts on different slope segments, but also to reveal the changeable process of rill erosion amount. All of the relative errors of the experimental results were less than 25%, which is considered satisfactory on the study of rill erosion process.

Environmental Monitoring↗

Study on the behavior of the heavy metals Cu, Cr, Ni, Zn, Fe, Mn and 137Cs in an estuarine ecosystem using Mytilus galloprovincialis as a bioindicator species: the case of Thermaikos gulf, Greece.

Mussels are worldwide recognized as pollution bioindicators and used in Mussel Watch programs, because they accumulate pollutants in their tissues at elevated levels in relation to pollutant biological availability in the marine environment. The present study deals with the use of Mytilus galloprovincialis as a local bioindicator of heavy metal and (137)Cs contamination in an estuarine ecosystem (Thermaikos gulf, Greece in Eastern Mediterranean). M. galloprovincialis samples were collected monthly from two aquaculture farms during the period April to October 2000. Analyses for the heavy metals Cu, Cr, Ni, Zn, Fe, Mn and (137)Cs showed that the concentrations measured were low and similar to those from other non-polluted Mediterranean areas. In terms of the two sampling stations, there were no statistically significant differences between them. On the contrary, the seasonal evolution of either heavy metals or (137)Cs levels presented high variation. The levels were found to increase during the cold period of the year, especially for Cu, Zn, Mn and Cr which are essential for life. Stable metals were positively inter-related and moreover, metals more involved in biochemical activities seem to present more correlations than others with less significant role in the metabolism of the organisms.

Animals↗

Radioisotope blood volume measurement in uncontrolled retroperitoneal haemorrhage induced by a transfemoral iliac artery puncture.

Standard-of-care, large volume crystalloid infusion, in the setting of uncontrolled bleeding, has been challenged and it is not known if fluid resuscitation increases retroperitoneal hemorrhage. We developed an experimental model of retroperitoneal haemorrhage to correlate haemodynamic and metabolic alterations with the blood volume loss. Anaesthetised, spontaneously breathing dogs (17.1+/-0.56 kg) were randomised to unilateral (UL, n=11) or bilateral (BL, n=11) iliac artery puncture, using a metallic device introduced through the femoral arteries and followed for 120 min. Initial and final blood volumes were determined using radioactive tracers, 99mTC and 51Cr, respectively. UL was associated with a stable arterial pressure and a moderate decrease in cardiac output and oxygen delivery. BL induced an abrupt and sustained decrease in mean arterial pressure, from 131.9+/-5.9 to 88.6+/-10.8 mmHg, and a much greater reduction in cardiac output, oxygen delivery and consumption than UL throughout the experiment. Total retroperitoneal blood loss after BL was 36.8+/-3.2 ml/kg, while after UL was 25.1+/-3.4 ml/kg (P=0.0262). We conclude that a transfemoral bilateral iliac artery puncture produces a clinically relevant model of uncontrolled retroperitoneal haemorrhage, with hypotension and low flow state, while a unilateral iliac artery lesion causes a compensated shock state.

Animals↗

Bioaccumulation of radionuclides in fertilized Canadian Shield lake basins.

Radionuclide tracers of heavy metals (59Fe, 60Co, 65Zn, 75Se, 85Sr, 134Cs and 203Hg) representing potential contamination from nuclear power plants, industry and agriculture were added to separate basins of Lake 226, Experimental Lakes Area, northwestern Ontario. The two basins were part of a eutrophication experiment and differed in their trophic status; the north basin (L226N) was eutrophic whereas the south basin (L226S) was mesotrophic. Our objective was to determine the uptake of the radionuclides by biota and the effect of lake trophic status on their bioaccumulation. The trophic status of the lakes did not appear to have a marked effect on the accumulation of radionuclides by the biota. This may have been because of a mid-summer leakage of nutrients between the basins which enhanced primary production in L226S, because there is a time lag between primary production and the availability of the radionuclides to the fishes or because trophic status does not affect the uptake of at least some of these radionuclides. However, there was a tendency for faster uptake of the radionuclides in L226N by fish than L226S, but the differences were not significant. Concentrations in the biota generally decreased in the order: fathead minnow > pearl dace > tadpoles > slimy sculpin > leeches. Concentrations in biota generally decreased in the order. 65Zn > 203Hg > 75Se > 134Cs > 60Co > 85Sr = 59Fe. Cobalt-60 concentrations in tadpoles were greater than in the other biota. Radionuclide concentrations in the tissues of lake whitefish indicated that uptake was predominantly from food. Radionuclide concentrations were usually higher in the posterior gut, liver and kidney than in other tissues, whereas body burdens were generally high in the muscle for 75Se, 134Cs and 203Hg; kidney and gut for 60Co; and bone for 65Zn and 75Se. Mercury-203 burdens were also high in the bone and gut.

Agriculture↗

Developments in terrestrial bacterial remediation of metals.

Recent advances in understanding the role and application of bacteria to the remediation of toxic metal and radionuclide contaminated terrestrial environments have come from several avenues. Novel species capable of mobilization and immobilization of metal ions have been discovered. Remediation of toxicity has been accelerated by nutrient amendment, the use of chelating agents and novel methods for phosphate amendment. Major advances in the use of natural and genetically engineered species for bioprotection and remediation of organic co-contaminants have been reported. Construction of wetland function continues to be developed for containment and decontamination of wastewaters.

Bacteria↗

Fe(O)/lignitic coal: an efficient and mechanically stable reactive material for purification of water containing heavy metals, radionuclides, and nitroaromatics.

Mine water in the former uranium mine of Königstein (Saxony, Germany) contains high concentrations of acid, sulfate, iron, aluminum, various heavy metals, radionuclides, and nitroaromatics. Research has been conducted for several years to establish the extent to which reduction of pollutant concentrations can be positively influenced and accelerated by storage of reactive materials in mine cavities. Investigations were made at different scales to test and select materials with respect to maximum fixation of contaminants (underground column tests) to examine hydraulic effects (underground large-scale column tests) and to optimize material properties (laboratory tests). The investigations have shown that a mixture of Fe chips and lignitic coal is capable of efficiently cleaning acid and contaminant-containing mine water. The examined material is easily available and compatible with the environment. A large-scale application of such a reactive barrier is being considered for mine water treatment in the future and is deemed to be a reasonable conception for a safety component after conclusion of the flooding.

Coal↗

Determination of gold and platinum traces in biological materials as a part of a multi-element radiochemical activation analysis system.

For the analysis of human tissues for traces of gold and platinum--being used as constituents of therapeutic agents--a radiochemical neutron activation method has been developed. The radiochemical separation involves the selective removal of radioactive gold--formed by the reaction 197Au (n, gamma)198Au and the reaction 198Pt (n, gamma) 199Pt ---- 199Au --as small metallic nuggets . The determination of gold and platinum is carried out as a part of an automated multi-element radiochemical separation scheme, allowing the determination of about 20 additional trace elements, and thus giving the possibility to study interelement relations. The analytical characteristics of the determination are evaluated. Gold and platinum levels measured in Bowen's Kale, NBS Bovine Liver and NBS Orchard Leaves are presented. Values are shown for gold, platinum, and 20 other trace elements in various healthy and cancerous tissues from patients treated with cis-platin. (Cis-Diamminedichloroplatinum II).

Activation Analysis↗

Bremsstrahlung doses from natural uranium ingots.

In the past, some privately owned commercial facilities in the United States were involved in producing or processing radioactive materials used in the production of atomic weapons. Seven different geometrical objects, representative of the configurations of natural uranium metal potentially encountered by workers at these facilities, are modelled to determine gamma ray and bremsstrahlung dose rates. The dose rates are calculated using the MCNP5 code and also by using the MICROSHIELD point-kernel code. Both gamma ray and bremsstrahlung dose rates are calculated and combined to obtain a total dose rate. The two methods were found to be in good agreement despite differences in modelling assumptions and method differences. Computed total dose rates on the surface of these objects ranged from approximately 51-84 microSv h(-1) and 17-95 microSv h(-1) using the MCNP5 and the MICROSHIELD modeling, respectively. The partitioning of the computed dose rates between gamma rays and bremsstrahlung were the same order of magnitude for each object.

Computer Simulation↗

Proteolysis of sialoglycoprotein by Pasteurella haemolytica cytotoxic culture supernatant.

Proteolytic enzyme activity releasing sialo glycopeptides from 3H-labeled human erythrocyte ghosts was detected in cytotoxic (leukotoxic) culture supernatants from 9 of 12 Pasteurella haemolytica serotypes. Microcrystalline cellulose thin-layer chromatograms of radioactive water-soluble products showed the following two radioactive peaks: a high-mobility minor peak (Rf, 0.54 to 0.74), identified as sialic acid, and a low-mobility major peak (Rf, 0.18 to 0.21), partially characterized as a trichloroacetic acid-soluble, sialic acid-rich fragment with a molecular weight of greater than 3,500, not extractable by chloroform. The sialic acid content of this fragment after treatment with Clostridium perfringens neuraminidase was estimated to be 7.2 X 10(-2) mumol mg-1. The presence of neuraminidase as a separate activity in some culture supernatants was confirmed. It is considered to be responsible for the observed release of free sialic acid. Preliminary studies with the crude enzyme showed that it has a broad pH optimum around pH 7.0 and that activity is not affected by inhibitors of trypsin, chymotrypsin, thermolysin, thio and serine enzymes, nor by an inhibitor of neuraminidase, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. Activity was, however, inhibited by o-phenanthroline at a high concentration after prolonged treatment. The enzyme hydrolyzed glycophorin at a rate four times higher than the rate for casein. Free glycophorin inhibited the enzyme-induced release of radioactive products from 3H-labeled ghosts. It is speculated that the novel enzyme is a neutral protease, probably metal-dependent, with specificity for sialoglycopeptides. The possible relationship of this protease to the previously reported host species-specific leukotoxicity of P. haemolytica and its potential role in virulence is discussed.

Caseins↗

Impaired function of salvaged myocardium: two-dimensional echocardiographic quantification of regional wall thickening in the open-chest dog.

This study was designed to examine the functional properties of myocardium subjected to acute coronary occlusion but surviving the ischemic insult. Ten conscious mongrel dogs underwent mild-circumflex coronary occlusion and were treated for 6 hours with prostacyclin, 540 ng/kg/min, and ibuprofen, 110 micrograms/kg/min, or dipyridamole (7-9.7 micrograms/kg/min). At 7 days, each dog was anesthetized, the chest was opened, and cross-sectional two-dimensional echocardiograms were obtained through the middle of the occluded vascular bed. A computer-aided contouring system was used to assess percent systolic thickening in 16 equally spaced segments around the left ventricle. Metal markers sewn to the epicardium permitted precise regional correlation of histology, percent systolic thickening, and flow, as measured by radioactive microspheres. Necrosis was minimal, averaging only 2.2 +/- 0.8% (+/- SEM) of the left ventricular ring corresponding to the echocardiographic cross section. Percent systolic thickening was 28.6 +/- 4.7 in the nonischemic anterior wall, but was reduced to -4.5 +/- 3.1 in the occluded bed (p less than 0.01). In individual echo segments, percent systolic thickening correlated with local flow (r = 0.69, p less than 0.001), but was still depressed even when flow was normal. In six segments within the occluded bed that had normal histology and flow, percent systolic thickening was 52% less than that in the nonischemic region (p less than 0.02). Thus, coronary artery occlusion combined with drug treatment results in myocardium that, although histologically normal and supplied by normal myocardial blood flow, remains functionally abnormal 7 days after occlusion.

Animals↗

Fate and stability of 14C-labeled 2,4,6-trinitrotoluene in contaminated soil following microbial bioremediation processes.

Biological treatment of 2,4,6-trinitrotoluene (TNT) in soil rarely results in complete mineralization of the parent compound. More often, the largest proportion of the TNT carbon is incorporated into the soil organic matrix. Therefore, we evaluated the stability of nonextractable residues from various bioremediation processes of 14C-TNT in soils. The extractable amounts of the residual radioactivity varied between 7 and 33% and thus the nonextractable amount between 93 and 67% (3-15% in fulvic acids, 26-46% in humic acids, and 27-44% in the humin fraction). The residue-containing soils were analyzed for the release of radioactivity after treatment by physical (freeze and thaw, grinding of soil, and steam extraction), chemical (acid rain and addition of metal complexing agent), and biological methods (addition of compost, white rot fungi, radical-generating enzymes, and germination of plants). Freeze and thaw treatment and grinding of the soil did not alter the partitioning of the label significantly. Steam extraction and acid rain extraction increased the water extractability to 11 to 29% and to 51.6% in the native TNT-contaminated soil. The addition of ethylenediamine-tetraacetate (EDTA) increased the extractability from 7 to 12%. After biological treatment, only slightly increased extractability (<10%) was observed. No increase of extractable TNT or known metabolites was observed with any of the treatments. Thus, under the treatment conditions applied in this study, the residues formed during microbial transformation of TNT may be biogenic residues with low mobilization potential and low hazardous impact.

Agaricales↗

Preparation of tritium standard gas by the use of tritiated methane.

Tritium standard gas for calibrating radioactivity detectors was prepared by the use of tritiated methane synthesized by the reaction of tritiated water with aluminum carbide at ca. 470 degrees C. A long-path proportional counter was used to standardize the radioactivity of the prepared gas. Tritiated gas of less than 10(4) Bq could be measured by this counter with the overall error of 3.5% (3 sigma). Finally, two types of standard were prepared in a metal cylinder and a glass ampoule. The latter type could be used as a handy calibration source for detectors.

Aluminum↗

[Concentration of heavy elements and their compounds in the cells from BALF and pulmonary tumors in radiation-exposed subjects].

AIM: Measurement of heavy elements in alveolar macrophages (AM) of bronchoalveolar lavage (BAL) and pulmonary tumor tissue (PTT); investigation of spacial distribution of the heavy particles in the tissues. MATERIALS AND METHODS: Laser mass-spectrometry estimated quantitative and qualitative characteristics of heavy elements (Mn, Fe, Co, Cu, Zr, etc.) in diagnostic and therapeutic BAL of 6 patients exposed to radiation after the Chernobyl accident and in pulmonary tumor tissue of patients operated for central small-cell and peripheral bronchoalveolar cancer who also had been exposed to radiation after the Chernobyl. RESULTS: Heavy elements concentration in the secondary BAL was less than in the primary one. This shows effectiveness of the procedures aimed at removal of heavy particles from the respiratory organs. Heavy elements content was quite different in two histologically different tumors. CONCLUSION: Laser mass-spectrometry proved its usefulness in simultaneous study of the quantity and quality of heavy elements in AM of BAL and tumors in radiation-exposed subjects, in investigation of the elements distribution in the pulmonary tissues and changes of elements composition in the tissues in the course of tumors development.

Adenocarcinoma, Bronchiolo-Alveolar↗

Influence of metal (Cd and Zn) waterborne exposure on radionuclide (134Cs, 110Ag, and 57Co) bioaccumulation by rainbow trout (Oncorhynchus mykiss): a field and laboratory study.

Field and laboratory experiments were carried out to assess the influence of Cd and Zn on the contamination levels of 110Ag, 57Co, and 134Cs in rainbow trout. During a four-week prior exposure phase, two fish groups were held in tanks in the Lot River (France) at a reference (<0.05 microg Cd/L and 68 microg Zn/L) and at a polluted site (1.5 microg Cd/L and 152 microg Zn/L). During a subsequent phase, organisms were brought back to the laboratory, where the radionuclide accumulation and depuration were studied for 14 and 7 d, respectively. During this second phase, the water used in the experiments was brought back from the two sites on the Lot River in order to work under the same chemical conditions. The potential effect of chronic exposure to stable metals on several biomarkers has been explored: Plasma analysis indicated the disruption of certain variables linked to the energetic metabolism and to the maintenance of the ionic balance. In contrast, no significant disruption of the measured enzyme activities was observed. With regard to the bioaccumulation of radionuclides, concentrations in fish exposed to metals are much lower than those in fish from the control group. Various hypotheses are proposed to link fish metabolic profiles due to metal exposure to the radiocontamination of organisms.

Animals↗

Monitoring of cobalt(II) uptake and transformation in cells of the plant-associated soil bacterium Azospirillum brasilense using emission Mössbauer spectroscopy.

Interaction of cobalt(II) at micromolar concentrations with live cells of the plant-growth-promoting rhizobacterium Azospirillum brasilense (strain Sp245) and further transformations of the metal cation were monitored using 57Co emission Mössbauer spectroscopy (EMS). Cell suspensions of the bacterial culture (2.4 x 10(8) cells ml(-1)) were doped with radioactive 57CoCl2 (1 mCi; final concentration 2 x 10(-6) M 57Co2+), kept under physiological conditions for various periods of time (from 2 min up to 1 hour) and then rapidly frozen in liquid nitrogen. Analysis of emission Mössbauer spectra of the frozen aqueous suspensions of the bacterial cell samples shows that the primary absorption of cobalt(II) at micromolar concentrations by the bacterial cells is rapid and virtually complete, giving at least two major forms of cobalt(II) species bound to the cells. Within an hour, the metal is involved in further metabolic transformations reflected by changes occurring in the spectra. The Mössbauer parameters calculated from the EMS data by statistical treatment were different for suspensions of live and dead (thermally killed) bacterial cells that had been in contact with 57Co2+ for 1 h, as well as for the cell-free culture medium containing the same concentration of 57Co2+. Chemical after-effects of the nuclear transition (57Co --> 5 7Fe), which provide additional information on the chemical environment of metal ions, are also considered. The data presented demonstrate that EMS is a valuable tool for monitoring the chemical state of cobalt species in biological matter providing information at the atomic level in the course of its uptake and/or metabolic transformations.

Azospirillum brasilense↗

Separation of critical radioactive and non-radioactive species from aqueous waste streams.

The separation of radioactive and non-radioactive species from the simulated DOE neutralized current acid waste (NCAW) stream was studied. Cation and anion species were referred to their possible basic compounds, and divided into seven groups (nitrate, phosphate, sulfate, fluoride, nitrite, carbonate, and hydroxide). The nitrate group (the major anion in the DOE waste streams) contains several cations species, while the rest of the groups are only in the form of sodium. The precipitation measurements were conducted in three experimental stages. In the first stage, the precipitation of sodium sulfate, sodium phosphate, sodium-sulfate-phosphate, and aluminum nitrate systems were studied using isopropylamine (IPA) as a precipitation solvent. The objectives of this stage were to evaluate the precipitation ability of IPA in precipitating these compounds individually, and to validate the consistency of the analytical instruments and the employed experimental procedure. Tests performed on the acquired data indicated a high level of experimental consistency. The removal of phosphate, sulfate and aluminum were very high. In the second stage, the precipitation studies were conducted on the: (1) nitrate group alone; (2) binary groups containing the groups of nitrate-phosphate, nitrate-sulfate, nitrate-fluoride, nitrate-nitrite, and nitrate-carbonate; (3) combined nitrate, phosphate, sulfate, and fluoride groups and (4) combined nitrate, phosphate, sulfate, fluoride, nitrite, and carbonate groups. IPA was used as a precipitation solvent. The objectives of this stage were to evaluate the interactions of these groups in the absence of the hydroxide group (e.g. DOE acid-dissolved sludge and acidified supernate streams), and the influence of such interactions on the individual removal of the targeted species. The removal of the aluminum, phosphate, fluoride, and alkaline cations were significantly high (reached 99.9%). The removal of sulfate were moderately high (reached 87%), and the removal of nitrate and alkali cations including cesium were to some extent low (reached about 50%). In the third stage, the precipitation of inorganic species from the simulated NCAW stream was studied using IPA and ethylamine (EA). The precipitation process is very feasible for reducing the radioactivity contents of alkaline cations. However, the process is less effective in separating alkali cations including cesium. The removal of polyvalent transition metals such as aluminum ion is negatively influenced by the significant presence of hydroxide. While the process is effectively capable of separating phosphate, fluoride, and sulfate, it is significantly less effective in separating nitrate and nitrite. A previously derived thermodynamics framework was used to model the precipitation measurements. The framework provided two predictive equations (the 2-Suffix and 3-Suffix equations). Both equations were reasonably adequate for predicting the solubility phase behavior of tested inorganic species in a mixed-solvents mixture as well as for estimating optimum interaction parameters. However, the 3-Suffix equation was better than the 2-Suffix equation. The parameters were useful for estimating the: (1) precipitation fractions (%P) of the studied species, for instance, at different concentration levels of similar targeted species, or in different waste streams with similar or approximate abundance of species, or at different solvents volume ratio (V(r)) where no experimental data are available.

Chemical Precipitation↗