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Use of the sodium borohydride reduction technique to identify a gamma-glutamyl phosphate intermediary in the Escherichia coli glutamine synthetase reaction.

Incubation of unadenylylated Escherichia coli glutamine synthetase with ATP, L-[14C]glutamate and metal ion results in the formation of gamma-glutamyl-P which can under appropriate conditions be reduced by sodium borohydride. The acyl-P compound is formed catalytically as judged by the quantity of radioactive alpha-amino-delta-hydroxyvalerate produced compared to the concentration of enzyme subunits. Formation of the glutamyl-P compound occurs in the presence of magnesium or manganous ions, and the relation of this apparent lack of metal ion specificity with regard to the highly specific Mg2+-supported biosynthetic activity of the unadenylylated form is discussed.

Adenosine Triphosphate↗

Design, synthesis, and evaluation of [188Re]organorhenium-labeled antibody fragments with renal enzyme-cleavable linkage for low renal radioactivity levels.

Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. We have reported that Fab fragments labeled with 3'-[131I]iodohippuryl Nepsilon-maleoyl-lysine (HML) showed markedly low renal radioactivity levels even shortly after injection, due to a rapid and selective release of m-[131I]iodohippuric acid by the action of brush border enzymes. To estimate the applicability of the molecular design to metallic radionuclides, [188Re]tricarbonyl(cyclopentadienylcarbonate)rhenium ([188Re]CpTR-COOH) was conjugated with Nepsilon-tert-butoxycarbonyl-glycyl-lysine or Nepsilon-maleoyl-glycyl-lysine to prepare [188Re]CpTR-GK-Boc or [188Re]CpTR-GK. The cleavage of the glycyl-lysine linkage of the two compounds generates a glycine conjugate of [188Re]CpTR-COOH ([188Re]CpTR-Gly), which possesses in vivo behaviors similar to those of m-iodohippuric acid. The hydrolysis rate of the peptide bond in [188Re]CpTR-GK-Boc was compared with that in 3'-[125I]iodohippuryl Nepsilon-Boc-lysine ([125I]HL-Boc) using brush border membrane vesicles (BBMVs) prepared from rat kidneys. [188Re]CpTR-GK was conjugated to thiolated Fab fragments to prepare [188Re]CpTR-GK-Fab. The biodistribution of radioactivity after injection of [188Re]CpTR-GK-Fab was compared with that of [125I]HML-Fab and [188Re]CpTR-Fab prepared by conjugating N-hydroxysuccinimidyl ester of [188Re]CpTR-COOH with antibody fragments. While [188Re]CpTR-GK-Boc liberated [188Re]CpTR-Gly in BBMVs, [125I]HL-Boc liberated m-[125I]iodohippuric acid at a much faster rate. In addition, although [125I]HL-Boc was hydrolyzed by both metalloenzymes and nonmetalloenzymes, metalloenzymes were responsible for the cleavage of the peptide linkage in [188Re]CpTR-GK-Boc. In biodistribution studies, [188Re]CpTR-GK-Fab exhibited significantly lower renal radioactivity levels than did [188Re]CpTR-Fab. However, the renal radioactivity levels of [188Re]CpTR-GK-Fab were slightly higher than those of [125I]HML-Fab. The analysis of urine samples collected for 6 h postinjection of [188Re]CpTR-GK-Fab showed that [188Re]CpTR-Gly was the major radiometabolite. In tumor-bearing mice, [188Re]CpTR-GK-Fab significantly reduced renal radioactivity levels without impairing the radioactivity levels in tumor. These findings indicate that the molecular design of HML can be applied to metallic radionuclides by using a radiometal chelate of high inertness and by designing a radiometabolite of high urinary excretion when released from antibody fragments following cleavage of a glycyl-lysine linkage. This study also indicates that a change in chemical structure of a radiolabel attached to a glycyl-lysine linkage significantly affected enzymes involved in the hydrolysis reaction. Since there are many kinds of enzymes that cleave a variety of peptide linkages on the renal brush border membrane, selection of a peptide linkage optimal to a radiometal chelate of interest may provide radiolabeled antibody fragments that exhibit renal radioactivity levels similar to those of [131I]HML-labeled ones. The in vitro system using BBMVs might be useful for selecting an appropriate peptide linkage.

Animals↗

Processes, dynamics and modelling of radiocaesium cycling in a chronosequence of Chernobyl-contaminated Scots pine (Pinus sylvestris L.) plantations.

In a large forested area affected by the Chernobyl radioactive fallout, especially in CIS, the lasting recycling of radiocaesium (137Cs) by the trees is a source of long-term contamination of woody products. The quantitative description of the 137Cs dynamics in contaminated forest is a prerequisite to predictive modelling and further management of such territories. Three even-aged mono-specific Scots pine stands (17, 37 and 57 years old) were selected in a contaminated woodland in southeastern Belarus to constitute an adequate chronosequence. We determined the potassium and radiocaesium annual fluxes involved in the biological cycling in each stand using a well-documented calculation methodology. Qualitatively, 137Cs was shown to be rapidly recycled in trees through the same pathways as K and to redistribute similarly between the tree components. Compared to K, a higher fraction of 137Cs, corresponding to about the half of the annual uptake, is immobilised in perennial organs. With tree development, trunk wood and bark become prevailing sinks for 137Cs since they represent an increasing pool of biomass. In the pine chronosequence, the current root absorption, respectively, mobilizes 0.53, 0.32 and 0.31% year(-1) of the total 137Cs pool in soil. Variations in the 137Cs uptake do not reflect differences in the 137Cs balance between stands. In the two older stands, 51 and 71% of the current tree contamination are related to earlier accumulation subsequent to the initial fallout interception and recycling. The soil is the dominant source of long-term tree contamination. A simple modelling based on the measured 137Cs fluxes indicates that, for young stands, radioactive decay-corrected contamination would stabilize after reaching a maximum of 25 years after the 137Cs deposition. Stemwood presents a maximum of 15 years after the deposition and decrease afterwards mainly through radioactive decay. In the older stands, the decontamination is constant without local maximum of 137Cs level in the wood. The 137Cs contamination of tree components is the result of different influential processes like root uptake, internal translocation and immobilisation. For more accurate predictions, the calibration of existing models would be benefited by comparing with the 137Cs annual fluxes instead of the simple transfer factor coefficients. In the perspective of other applications, there is a need of such data for other radionuclides as well as for heavy metals.

Calibration↗

[Isolation and investigation of natural yeast strains resistant to heavy metal salts and radionuclides].

The collection of yeasts (more than 2000 strains) from extreme natural environment of Kamchatka peninsular and Kuril Islands was created. 448 strains were selected for their resistance to salts of such heavy metals as Cu, Cd, Co and to high temperature (37-52 degrees C). 72% of strains appeared to be resistant to one or more selective factors. We obtained several strains able to grow on medium with 0.1 M/L nonradioactive strontium and (or) cesium. Four of this strains accumulated radioactive isotope 90Sr with 45-80% efficiency. Thus, we demonstrated that yeast strains from nature could be used for bioremediation of industrial waste solutions, polluted by radionuclides and salts of heavy metals.

Drug Resistance, Microbial↗

[Percutaneous absorption of metallic cations: uranium particulates].

Washing is usually used to reduce the risk of a skin contamination. Skin biopsies were used and contaminations were realised with 1) soluble radioactive calcium 2) insoluble particles of uranium. The result obtained with different wash liquids are presented here. Water and soap had no effect but hypertonic DTPA was the better therapy to remove uranium from skin.

Calcium Radioisotopes↗

Irradiation of choroidal melanoma with iodine 125 ophthalmic plaque.

Radioactive iodine 125 is a low-energy gamma isotope with physical characteristics suitable for irradiation of intraocular tumors. Metal ophthalmic applicators have been designed to shield vital ocular structures while allowing irradiation of the tumor. We compared the radiation effects of iodine 125 and cobalt 60. The Greene melanoma was transplanted into the suprachoroidal space of rabbits. The tumor then grew as an intraocular mass, was irradiated, and was followed up for two months before enucleation. Histopathologic examination defined the extent of the radiation damage to the tumor and other ocular structures from the iodine 125 and from the cobalt 60. The eye irradiated with iodine 125 suffered minimal radiation damage, whereas the tumor was sterilized. The eye irradiated with cobalt 60 showed substantial radiation damage, and the melanoma was incompletely treated. Our results support the use of iodine 125 in treating intraocular tumors. More research is needed as to optimum total dose and dose rate.

Animals↗

Preparation of homogeneous crystals of myo-inositol 1-phosphate synthase from rat testicles--further data on the chemical and catalytic properties of the enzyme (studies on the biosynthesis cyclitols, XXXIX).

Pre-purified preparations of myoinositol-1-phosphate synthase (E.C. 5.5.1.4) from rat testes can be purified to homogeneity by first crystallizing the enzyme according to JAKOBY and then recrystallizing it at a pH value close to the isoelectric point while slowly increasing the temperature from 0 to 15 degrees C. This method gives a much yield of homogeneous enzyme than the previously used purification by affinity chromatography. It was further found that the pure enzyme contains close to 2 mol NAD+ per mol enzyme; it does not contain any metal. At substrate saturation the enzyme binds close to 1 mol substrate per mol enzyme, as determined by using radioactively labelled substrate and binding it to the enzyme by reduction with NaBH4. The reaction catalyzed by the enzyme is irreversible.

Animals↗

The chemical activities of the Viking biology experiments and the arguments for the presence of superoxides, peroxides, gamma-Fe2O3 and carbon suboxide polymer in the Martian soil.

The evolution of N2, Ar, O2, and CO2 from Martian soil as a function of humidity in the Gas Exchange Experiment are correlated with the mean level of water vapor in the Martian atmosphere. All but O2 are associated with desorption. The evolution of oxygen is consistent with the presence of alkaline earth and alkali metal superoxides; and their peroxides and the gamma-Fe2O3 in the soil can account for the generation of radioactive gas in the Labeled Release Experiment. The slower evolution of CO2 from both the Gas Exchange Experiment and the Labeled Release Experiment are associated with the direct oxidation of organics by gamma-Fe2O3. The Pyrolytic Release Experiment's second peak may be carbon suboxide as demonstrated by laboratory experiments. A necessary condition is that the polymer exists in the Martian soil. We ascribe the activity of the surface samples to the reaction of Martian particulates with an anhydrous CO2 atmosphere activated by uv and ionizing radiations. The surface particles are ultimately altered by exposure to small but significant amounts of water at the sites. From the working model, we have predicted the peculiar nature of the chemical entities and demonstrated that the model is justified by laboratory data. The final confirmation of this model will entail a return to Mars, but the nature and implications of this chemistry for the Martian surface is predicted to reveal even more about Mars with further simulations in the laboratory.

Argon↗

Acetylation of S-substituted cysteines by a rat liver and kidney microsomal N-acetyltransferase.

1. An acetyl-CoA--S-substituted cysteine N-acetyltransferase in rat liver and kidney preparations was investigated, by using an assay involving incubations with S-benzyl-L-cysteine and [l-14C]acetyl-CoA and extraction of the radioactive product with ethyl acetate. 2. The enzyme was associated with the microsomal fraction and could not be solubilized. Metal ions, EDTA and detergents did not significantly affect the enzyme activity. p-Chloromercuribenzoate and N-ethylmaleimide inhibited the enzyme. 3. Other S-substituted cysteines were acetylated at about the same rate as S-benzyl-L-cysteine. Acetylation of cysteine itself and of methionine, ethionine and tryptophan could be detected but was much slower. Acetylation of aspartic acid, glycine, phenylalanine and serine could not be detected. Palmitoyl-CoA was not a substrate. 4. The enzyme is presumably responsible for the acetylation step of mercapturic acid synthesis; a more physiological function is not yet known, except that the enzyme may be involved in acetylation of those amino acids which occur in elevated amounts in some disorders of amino acid metabolism.

Acetyl Coenzyme A↗

Ruthenium hexaammine trichloride chemography for aggrecan mapping in cartilage is a sensitive indicator of matrix degradation.

We developed a new quantitative histochemical method for mapping aggrecan content in articular cartilage and applied it to models of cartilage degradation. Ruthenium hexaammine trichloride (RHT) forms co-precipitates with aggrecan, the main proteoglycan component of cartilage, and was previously found to be a good fixative in aiding the maintenance of chondrocyte morphology. We show that these RHT-aggrecan precipitates generate a positive chemographic signal on autoradiographic emulsions, in the absence of any radioactivity in the tissue section, via a process similar to the autometallographic process used previously for localization of trace metals ions in tissues. By exploiting the inherent depth-dependence of aggrecan concentration in adult articular cartilage, we demonstrated that the density of silver grains produced by RHT-derived chemography on autoradiographic emulsions correlated with locally measured aggrecan concentration as determined by the dimethylmethylene blue assay of microdissected tissue from these different depths of cartilage. To explore the benefits of this new method in monitoring tissue degradation, cartilage explants were degraded during culture using interleukin-1 (IL-1) or digested after culture using chondroitinase and keratinase. The RHT chemographic signal derived from these samples, compared to controls, showed sensitivity to loss of aggrecan and distinguished cell-mediated loss (IL-1) from degradation due to addition of exogenous enzymes. The RHT-derived chemographic grain density represents an interesting new quantitative tool for histological analysis of cartilage in physiology and in arthritis.

Aggrecans↗

Characterization of the TE-NORM waste associated with oil and natural gas production in Abu Rudeis, Egypt.

The present study was conducted to characterize the Technically Enhanced Naturally Occurring Radioactive Materials (TE-NORM) waste generated from oil and gas production. The waste was characterized by means of dry screening solid fractionation, X-ray analysis (XRF and XRD) and gamma-ray spectrometry. Sediment of the TE-NORM waste was fractionated into ten fractions with particle sizes varying from less than 100 microm to more than 3 mm. The results showed that the TE-NORM waste contains mainly radionuclides of the 238U, 235U and 232Th series. The mean activity concentrations of 226Ra (of U-series), 228Ra (of Th-series) and 40K in the waste samples before fractionation (i.e. 3 mm) were found to amount to 68.9, 24 and 1.3 Bq/g (dry weight), respectively. After dry fractionation, the activity concentrations were widely distributed and enriched in certain fractions. This represented a 1.48 and 1.82-fold enrichment of 226Ra and 228Ra, respectively, in fraction F8 (2.0-2.5 mm) over those in bulk TE-NORM waste samples. The activity ratios of 238U/226Ra, 210Pb/226Ra, 223Ra/226Ra and 228Ra/224Ra were calculated and evaluated. Activity of the most hazardous radionuclide 226Ra was found to be higher than the exemption levels established by IAEA [International Atomic Energy Agency, 1994. International Basic Safety Standards for the Protection against Ionizing Radiation and for the Safety of Radiation Sources. GOV/2715/94, Vienna]. The radium equivalent activity (Ra-eq), radon (222Rn) emanation coefficient (EC) and absorbed dose rate (Dgammar) were estimated and these are further discussed.

Egypt↗

Calcineurin immunoprecipitated from bovine brain extract contains no detectable Ni2+ or Mn2+.

Purified calcineurin phosphatase is converted upon incubation in millimolar Ni2+ or Mn2+ to an active form by association with these metal activators. The bound metal ion is not dissociable from calcineurin by dialysis or gel filtration, but can be released upon prolonged incubation of the enzyme with Ca2+/calmodulin or chelating agents (Pallen, C.J., and Wang, J.H. (1986) J. Biol. Chem. 261, 16115-16120). The present study has been undertaken to test the possibility that calcineurin in brain may contain tightly bound Ni2+ or Mn2+. A monoclonal antibody (VA1) immunoaffinity matrix was prepared and shown to affect specific precipitation of calcineurin from crude bovine brain extract. Using [3H]-, [63Ni2+]-, and [54Mn2+]calcineurin added to the extract as radioactive tracer, it was found that up to 80% of the calcineurin could be immunoprecipitated, and that more than 50% of the originally bound metal ions could be detected in the immunoprecipitate. When samples of calcineurin immunoprecipitated from brain extracts were analyzed by atomic absorption spectroscopy, Ni2+ and Mn2+ were not detected, whereas, Zn2+, a constitutive metal of calcineurin (King, M. M., and Huang, C. Y. (1984) J. Biol. Chem. 259, 8847-8856) was found in the expected amount. The result suggests that calcineurin in brain does not contain tightly associated Ni2+ or Mn2+.

Animals↗

Heavy metal pollution of some Danube Delta lacustrine sediments studied by neutron activation analysis.

Using neutron activation analysis and radiometric measurements, the vertical distribution of six possible pollutant elements (Zn, Cr, Co, As, Sb and Br), two trace elements of natural origin (Sc and Hf) and radioactive 137Cs were determined in three lakes, Furtuna, Lung and Mesteru, located in an active sedimentary zone of the Danube Delta. The accumulation rate of recent sediments was estimated from radiocesium profiles. Zn, As, Sb and Br were identified as possible pollutants. The vertical profiles of their concentrations reflect recent historic inputs, showing a gradual increase until around 1990, followed by a continuous decrease.

Cesium Radioisotopes↗

The use of a hydroxylapatite-filter steroid receptor assay method in the study of the modulation of androgen receptor interaction.

Receptors for androgen, estrogen, and glucocorticoid can be assayed by hydroxylapatite adsorption of the radioactive steroid-receptor complex and washing of the adducts on membrane filters mounted on a multiple filter holder. The method is economical, very rapid and sensitive. This new receptor assay method was used to study the modulation of androgen receptor of rat ventral prostate by metal ions, thiols, and ligand structure. The interaction of androgen with the naked receptor is inhibited by 10 microM ZnCl2, CdSO4, or CuSO4 but this inhibition is competed by androgen and is reversed by DTT. The androgen-receptor complex is less sensitive to divalent metal ions but Zn2+, at 3 mM, appears to alter the conformation of the receptor and promote the release of androgen. Certain phenanthrene derivatives exhibited striking structural specificities in their ability to compete with radioactive androgen for binding to the prostate receptor. The results suggest that the receptor has binding preference toward individual ring structure in the steroid.

Animals↗

Monte Carlo modeling of the transverse-axis dose distribution of the model 200 103Pd interstitial brachytherapy source.

This study presents the first theoretical analysis of the absolute dose-rate distribution about the Model 200 103Pd interstitial brachytherapy source. Monte Carlo photon-transport (MCPT) simulation techniques have been used to evaluate the transverse-axis dose-rate distribution of the Model 200 source as a function of thickness of the Pd metal coating (containing the 103Pd) plated onto the surfaces of right cylindrical graphite pellets contained within the seed. The dose-rate constant, A, was realistically estimated by simulating the wide-angle, free-air chamber (WAFAC) calibration geometry. The WAFAC is the experimental realization of NIST's (National Institute of Standards and Technology) recently implemented primary standard of air-kerma strength (S(K)). Our results show that polar angle- and distance-dependent oblique filtration and shielding effects induce significant and unexpected photon fluence anisotropy near the transverse-axis and inverse square law deviations at typical calibration distances. Any source consisting of radioactivity deposited on a highly attenuating surface with sharp edges may exhibit such effects. In the case of the Model 200 seed, the Pd metal thickness does not significantly influence the relative dose distribution in water at distances less than 5 cm, but does make A sensitive to the S(K) measurement geometry. Fortunately, the WAFAC averages fluence over a sufficiently large aperture that the resultant A, 0.68 +/- 0.02 cGy x h(-1) x U(-1), is almost independent of Pd metal layer thickness and in close agreement with recent measurements and calculations. This value is 20% higher than that of the renormalized Task Group 43 A value.

Brachytherapy↗

The relationships between urinary elimination, airborne concentration, and radioactive hand contamination for workers exposed to uranium.

The purposes of this study were to estimate the proportional contribution of uranium dust exposure to urinary excretion of the metal, and to examine the influence of hand contamination on the elimination of uranium from the urine of workers who performed different jobs. The study was based on records taken from a gaseous diffusion plant in 1956. Regression techniques were used to investigate the relationship between uranium concentration in urine and exposure variables, including the concentration of uranium dust and the incidence of hand contamination. The proportional contribution of airborne to urinary uranium was estimated by the slope of this relationship. It is concluded that the workers eliminated uranium at relatively constant rates per unit air concentration (0.67 to 1.27 micrograms/L per micrograms/m3). In addition to airborne uranium, hand contamination significantly contributed to the elimination among maintenance crew, but not among production workers. Furthermore, the production index was shown to be a useful surrogate for airborne and urinary uranium.

Air Pollutants, Occupational↗

An approach for immunoradiometric assay with metallic radionuclides: gallium-67-deferoxamine-dialdehyde starch-IgG.

Radiogallium (Ga) labeling of an immunoglobulin-G-deferoxamine conjugate (DF-IgG) to a high-specific radioactivity was performed to allow the development of a radiometallic immunoradiometric assay (IRMA) system. To increase the specific radioactivity of Ga-DF-IgG, we used dialdehyde starch (DAS) as a multi-site spacer for the binding of DF to IgG. Six DF molecules bound to each IgG molecule after DAS conjugation. DF-DAS-IgG was then labeled with the previously reported 67Ga labeling solution, producing labeled IgG with a specific radioactivity of 11,766 MBq/mg IgG. Using this method, we labeled an anti-CA125 tumor-associated antigen monoclonal antibody (130-22), allowing the first application of 67Ga-DF-DAS-IgG to an IRMA system. With this system, a higher sensitivity could be obtained than with 125I IRMA. In addition, a very high correlation (r = 0.995) was obtained between serum CA125 levels as determined by 67Ga IRMA and 125I IRMA. Gallium-67-labeled antibodies with a high-specific radioactivity appear to hold promise for use in highly sensitive radioassay systems.

Antigens, Tumor-Associated, Carbohydrate↗

Metal-dependent nucleotide binding to the Escherichia coli rotamase SlyD.

Upon expression and purification of the first catalytic domain of mammalian adenylate cyclase type 1 (IC1), a 27 kDa contaminant was observed, which was labelled by three radioactive ATP analogues (8-azido-ATP, 3'-O-(4-benzoyl)benzoyl-ATP and 2',3'-dialdehyde-ATP); the protein was purified separately and identified as Escherichia coli SlyD by N-terminal amino acid sequence determination. SlyD is the host protein required for lysis of E. coli upon infection with bacteriophage PhiX174 and has recently been shown to display rotamase (peptidylproline cis-trans-isomerase) activity. The covalent incorporation of ATP analogues into SlyD was promoted by bivalent transition metal ions (Zn(2+)>/=Ni(2+)>Co(2+)>Cu(2+)) but not by Mg(2+) or Ca(2+); this is consistent with the known metal ion specificity of SlyD. ATP, ADP, GTP and UTP suppressed labelling of SlyD with comparable potencies. Similarly, SlyD bound 2',3'-O-(-2,4, 6-trinitrophenyl)-ATP with an affinity in the range of 10 microM, as determined by fluorescence enhancement. This interaction was further augmented in the presence of Zn(2+) (K(d)= approximately 2 microM at saturating Zn(2+)) but not of Mg(2+). Irrespective of the assay conditions, hydrolysis of nucleotides by SlyD was not detected. Upon gel filtration on a Superose HR12 column, SlyD (predicted molecular mass=21 kDa) migrated with an apparent molecular mass of 44 kDa, indicating that the protein was a dimer. However, the migration of SlyD was not affected by the presence of Zn(2+) or of Zn(2+) and ATP. Thus we concluded that SlyD binds nucleotides in the presence of metal ions. These findings suggest that SlyD serves a physiological role that goes beyond that accounted for by its intrinsic rotamase activity, which is observed in the absence of metal ions.

Adenosine Diphosphate↗