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PubMed2004

A role for SlyD in the Escherichia coli hydrogenase biosynthetic pathway.

The [NiFe] centers at the active sites of the Escherichia coli hydrogenase enzymes are assembled by a team of accessory proteins that includes the products of the hyp genes. To determine whether any other proteins are involved in this process, the sequential peptide affinity system was used. The analysis of the proteins in a complex with HypB revealed the peptidyl-prolyl cis/trans-isomerase SlyD, a metal-binding protein that has not been previously linked to the hydrogenase biosynthetic pathway. The association between HypB and SlyD was confirmed by chemical cross-linking of purified proteins. Deletion of the slyD gene resulted in a marked reduction of the hydrogenase activity in cell extracts prepared from anaerobic cultures, and an in-gel assay was used to demonstrate diminished activities of both hydrogenase 1 and 2. Western analysis revealed a decrease in the final proteolytic processing of the hydrogenase 3 HycE protein, indicating that the metal center was not assembled properly. These deficiencies were all rescued by growth in medium containing excess nickel, but zinc did not have any phenotypic effect. Experiments with radioactive nickel demonstrated that less nickel accumulated in DeltaslyD cells compared with wild type, and overexpression of SlyD from an inducible promoter doubled the level of cellular nickel. These experiments demonstrate that SlyD has a role in the nickel insertion step of the hydrogenase maturation pathway, and the possible functions of SlyD are discussed.

Amino Acid Sequence↗
PubMed1978

Occurrence and fate of organic and inorganic contaminants in marine animals.

Most chemical contaminants occur in highest concentrations in coastal waters, often maximized in very localized areas. In general, this situation represents the exposure pattern for marine animals. However, the availability of contaminant to an organism depends not only on its concentration but also on its chemical nature, its physical state, and whether the source of exposure is the surrounding seawater or the diet. Depending on the type of exposure, uptake occurs across absorptive surfaces, such as those of the respiratory apparatus or gastrointestinal tract, where selectivity may occur, even among neighboring homologs. Deposition in tissues, accumulation, degradation, or depuration depends on tissue type, metabolic processes, detoxification mechanisms, and the adaptive status of a particular animal. This hypothesis is examined briefly for hydrocarbons, pesticides, other miscellaneous organic contaminants, heavy metals, and radionuclides. It is concluded that most data relate to occurrence and distribution. Considerably less information is available on the underlying biochemical processes.

Animals↗
PubMed1991

Chernobyl fallout in southern and central Finland.

To study the levels and distributions of radionuclides released in the Chernobyl accident, we sampled surface peat from 62 sites in Southern and Central Finland and measured 131I, 134Cs, 137Cs, 132Te, 140Ba, 103Ru, 90Sr, 141Ce, and 95Zr. The distribution of fallout activities was highly uneven, depending on movement of the contaminated air mass and rainfall distribution during the critical days. The highest values observed were 420 kBq m-2 of 131I and 70 kBq m-2 of 137Cs. The nuclide ratios showed wide and partly unexpected variations. The high-boiling-point, or nonvolatile, elements Ce and Zr were spread mostly on a 200-km-wide zone extending across Finland from southwest to northeast. The more volatile elements, I, Ce, and Te, showed quite a different, more widespread, fallout distribution, while an intermediate behavior was observed for Ba, Ru, and possibly Sr. These results can be explained by assuming that pulverized nuclear fuel material released in the reactor explosion on 26 April reached Finland via Poland and the Baltic Sea and traversed the country along the above-mentioned narrow zone, while volatile material, evaporated in the reactor fire from 26 April to 5 May, arrived in several waves and was consequently more widely and evenly spread. From their elemental melting and boiling points, Ru and Mo would appear to belong to the nonvolatile group and Sr to the volatile. Yet, their actual behaviors were opposite; Ru in particular was found in the nonvolatile as well as the volatile fallout, possibly because Ru activities were present in the fuel partly in the metallic state and partly as volatile oxides.

Accidents↗
PubMed1993

Maturational changes in dermal absorption of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Fischer 344 rats.

An earlier study indicated that percutaneous absorption of a 40-nmol dose of TCDD decreased with aging in rats, suggesting that the potential for systemic exposure following dermal contact would be decreased in older age groups. In this study, maturational changes in potential for systemic exposure to TCDD following dermal application of a low dose (200 pmol) of this chemical were examined in male Fischer 344 rats. Absorption, tissue distribution, and elimination of TCDD, measured as TCDD-derived radioactivity, were examined 72 hr after dermal application of 200 pmol [3H]TCDD (111 pmol/cm2 applied over 1.8 cm2) to the interscapular region of 3-, 5-, 8-, 10-, and 36-week-old rats. The dose was applied in 60 microliters acetone and the application site was covered with a perforated metal cap; animals were held in individual metabolism cages. Dermal absorption was greatest in 3-week-old rats (approximately 129 pmol; approximately 64% of the administered dose), decreasing to approximately 80 pmol (approximately 40%) in 5-, 8-, and 10-week-old rats and to 45 pmol (approximately 22%) in 36-week-old rats. In each age group, 70 to 80% of the radioactivity remaining at the application site 72 hr after dosing could be removed with acetone swabs. Major tissue depots of radioactivity were liver and fat; skin and muscle were minor depots. Changes in distribution of absorbed TCDD-derived radioactivity reflected changes in body mass of these depots; however, tissue concentration also varied. Whole body dissection was performed on rats to determine body mass of tissue depots. Adipose tissue content (Y) increased linearly with body weight (X), Y = 0.03X + 2.1 (r2 = 0.95). Elimination of absorbed TCDD-derived radioactivity was incomplete in all age groups with larger residues being recovered in the carcass. Results indicate that TCDD is absorbed to a greater degree through skin of very young animals and that a significant decrease in potential for systemic exposure may occur during maturation and again during aging.

Administration, Cutaneous↗
PubMed2006

EXAFS study of U(VI) uptake by calcium silicate hydrates.

Among the different cement minerals, calcium silicate hydrates (C-S-H) are the prime candidates for heavy metal binding because of their abundance and appropriate structure. Immobilization processes of heavy metals by cementitious materials, and in particular C-S-H phases, thus play an important role in multibarrier concepts developed worldwide for the safe disposal of hazardous and radioactive wastes. In this study, the uptake of U(VI) by C-S-H has been investigated using X-ray absorption fine structure (XAFS) spectroscopy. C-S-H phases were synthesized using two different procedures: One is based on the mixing of CaO and SiO2 solids ("direct reaction" method); for the other one starting solutions of Ca and Si are used ("solution reaction" method). XAFS investigations were carried out on samples doped with U(VI). U(VI) was either sorbed onto previously precipitated C-S-H phases (sorption samples) or added during C-S-H synthesis (coprecipitation samples). The coordination environment of U(VI) in the sorption samples was found to be independent of the procedure used for C-S-H synthesis. A split equatorial oxygen shell (Oeq1: R=2.23-2.27 A; Oeq2: R=2.36-2.45 A), neighboring silicon atoms at short (R=3.07-3.11 A) and long (R=3.71-3.77 A) distances, and neighboring Ca atoms (R=3.77-3.81 and 4.15-4.29 A) were observed for all the samples. The structural parameters resemble those reported for uranophane. The coordination environment of U(VI) in the coprecipitation samples depends on the method used for C-S-H synthesis, and further, the spectra differ from those determined for the sorption samples. UU backscattering contributions were observed in the samples prepared using the direct reaction method, whereas no split equatorial shell appeared in the samples prepared using the solution reaction method.

Absorptiometry, Photon↗
PubMed1996

Identification of the ATP binding domain of recombinant human 40-kDa 2',5'-oligoadenylate synthetase by photoaffinity labeling with 8-azido-[alpha-32P]ATP.

Three isoforms of the interferon-inducible 2',5'-oligoadenylate (2-5A) synthetase that require double-stranded RNA have been isolated and cloned. However, identification of the amino acid(s) of 2-5A synthetase directly interacting with ATP is crucial to the elucidation of the mechanism of the enzymatic conversion of ATP to 2',5'-oligoadenylates by 2-5A synthetase. Recombinant human 40-kDa 2-5A synthetase has been expressed as a glutathione S-transferase fusion protein in E. coli and purified to near homogeneity in milligram quantities. The azido photoprobe, 8-azido-[alpha-32P]ATP, has been used to identify the ATP binding domain of the recombinant human 40-kDa 2-5A synthetase. Specific covalent photoincorporation of 8-azido-[alpha-32P]ATP into the 2-5A synthetase, tryptic digestion of the covalently 32P-labeled enzyme, isolation of the photolabeled phosphopeptide by metal (Al3+) chelate chromatography, and high pressure liquid chromatography identified a 32P-pentapeptide, which has been assigned to the ATP binding domain of 2-5A synthetase. The radioactive pentapeptide has the sequence D196FLKQ200 in which the photoprobe, 8-azido-[alpha-32P]ATP, chemically modified the amino acid lysine 199. The catalytic importance of Lys199 was further established by mutation of lysine 199 to arginine 199 and histidine 199 using site-directed mutagenesis. The K199R and K199H recombinant human 40-kDa 2-5A synthetase mutants bind 8-azido-ATP and the allosteric activator, poly(I) poly(C) but are enzymatically inactive. These photoaffinity labeling and mutation data strongly suggest that lysine 199 is essential for the formation of a productive 2-5A synthetase-ATP-double-stranded RNA complex for the enzymatic conversion of ATP to 2-5A.

2',5'-Oligoadenylate Synthetase↗
PubMed1999

Technetium-99m complexes with steroid-2-aminooxyethyliminodiacetic acid conjugates.

Conjugates of 2-aminooxyethyliminodiacetic acid with estrone, testosterone, epiandrosterone, 17-alpha-hydroxy-progesterone and progesterone were synthesized and their complexes with Tc-99m were successfully prepared in good yields, which indicated the agent to be promising for metal-labeling of biomolecules and related substances containing one or more carbonyl groups. The biodistribution and scintigraphic studies in mice bearing Ehrlich tumor and mammary tumor showed that the radioactivity accumulated considerably in the tumor tissues, but the tumor images were somewhat obscured.

Animals↗
PubMed1992

[The effect of Ca-diethylenetriamine pentaacetate on the bio-behavior of tumor-affine metal complexes].

The complexon CaDTPA was injected into tumour-bearing mice in concentrations of 0.05, 0.1, 0.3, and 0.6 mole/l (pH:6) 30 min after the 169Yb-injection. 100 microliters of a 0.3 M CaDTPA solution were injected at different time points (simultaneously, 2, 5, 10, 20, 30, 40 and 50 min, 1, 1.25, 1.5, 2.5 and 10 h) after 169Yb-citrate injection. The animals were killed 24 h after radionuclide administration. A strong radioactivity decrease was observable 24 h p.i. not only in blood, liver, spleen, muscle and bone but also in the tumour if CaDTPA was administered within the first 2 h after ytterbium injection. Thereafter no change in radioactivity could be achieved by DTPA. A time phase in which the Yb could be eliminated from the tissues by means of DTPA (time intervals < 2 h) was distinguishable from a time phase in which it was not attainable for DTPA (time intervals > 5 h). This indicates that the incorporation of Yb into the cells is completed after 5 h and that the metals are intracellularly bound, probably to the lysosomes. Improvements of the scintigraphic tumour detection cannot be expected from the use of complexons.

Animals↗
PubMed2001

Significance of (111)In-DTPA chelate in renal radioactivity levels of (111)In-DTPA-conjugated peptides.

Metabolic studies of (111)In-DTPA-labeled polypeptides and peptides showed that the radiolabeled (poly)peptides generated (111)In-DTPA-adducts of amino acid that possess long residence times in the lysosomal compartment of the tissues where (poly)peptides accumulated. However, a recent study suggested that metal-chelate-methionine (Met) might possess in vivo behaviors different from metal-chelate adducts of other amino acids. In this study, to elucidate whether some biological characteristics of Met may accelerate the renal elimination rate of (111)In-DTPA-adduct of Met into urine, (111)In-DTPA-Met(1)-octreotide was synthesized and the renal handling of (111)In-DTPA-Met was investigated using (111)In-DTPA-L-Phe(1)-octreotide (Phe represents phenylalanine), which was reported previously, as a reference. Both (111)In-DTPA-conjugated octreotide analogs were stable against 3-h incubation in murine serum at 37 degrees C. Both (111)In-DTPA-octreotide analogs also showed rapid clearance of the radioactivity from the blood and similar accumulation of the radioactivity in the kidney. No significant differences were observed in the renal radioactivity levels from 10 min to 24 h postinjection between the two. Metabolic studies indicated that (111)In-DTPA-Met(1)-octreotide and (111)In-DTPA-L-Phe(1)-octreotide generated (111)In-DTPA-adducts of Met and Phe, respectively, as the final radiometabolites at similar rates. These findings suggested that the long residence times of the radioactivity in tissues after administration of (111)In-DTPA-labeled peptides and polypeptides would be attributed to inherent characteristics of (111)In-DTPA chelate.

Animals↗
PubMed2002

Diffusion studies in non-oxide ceramics: analytical aspects of the use of ion implanted stable tracers and SIMS.

The physical and chemical properties of complex non-oxide ceramic materials require advanced methods of diffusivity determination. In this study, we present a method based on the high-dose ion implantation of stable tracers in combination with secondary ion mass spectroscopy for depth profiling. The analytical basics, advantages and problems of the method are discussed for two examples of complex materials, the Si-B-C-N precursor ceramics and the Ti-based transition metal diborides. We demonstrate that is possible to measure the temperature dependence of diffusivities, especially for ceramic systems with low diffusivities, for systems that contain elements for which no suitable radioactive tracers exist for extended measurements.

Ceramics↗
PubMed1976

Metabolism of 109Cd in rats fed normal and low-calcium diets.

Growing male rats were fed purified diets that contained either 0.6% or 0.1% calcium to investigate the relationship of calcium intake to the uptake, tissue distribution, and excretion of 109Cd. An equal number of rats were fed either the 0.6 or 0.1% calcium diets for 4 wk before they were used for experiments. In the first experiment 11 rats from each dietary group were administered 5 muCi 109Cd by stomach tube and were then maintained in metabolism cages for 72 hr. Animals fed the low-calcium diet took up more 109Cd, as significantly higher levels of radioactivity were found in the intestinal mucosa, serum, lungs, liver, kidneys, and urine and a significantly lower level was found in the feces. Higher levels of 109Cd, associated with low-molecular-weight proteins that may be related to the absorption process, were found in the intestinal mucosa of the low-calcium group. In the second experiment 10 rats from each dietary group were administered 5 muCi 109Cd by subcutaneous injection and then maintained in a metabolism cage for 72 hr. No significant differences were found in the distribution or excretion of 109Cd except for the lungs where radioactivity was greater in the low-calcium group. The results of the study indicate that the enhanced cadmium toxicity observed in calcium-deficient animals exposed to the heavy metal is the result of an increased uptake from the small intestine.

Animals↗
PubMed1976

GAMMA-Glutamyl transpeptidase of sheep-kidney cortex. Isolation, catalytic properties and dissociation into two polypeptide chains.

Gamma-Glutamyl transpeptidase was isolated from sheep kidney cortex as an apparently homogeneous, highly active protein. At optimal pH and in the absence of acceptors, the enzyme catalyzes the release of about 510 mumol of p-nitroaniline per mg protein per min from the model substrate L-gamma-glutamyl-p-nitroanilide. Polyacrylamide gel electrophoresis in a sodium dodecylsulfate buffer system showed the presence of a large (Mr approximately 65000) and a small (Mr approximately 27000) polypeptide chain. Dissociation into two polypeptide chains was also achieved in 8 M urea. Amidination with dimethylsuberimidate produced a crosslinked protein of molecular weight approximately 90000. In the course of this work a convenient procedure was developed for the determination of gamma-glutamyl transpeptidase activity using L[glycine-2-3H]glutathione as the substrate. In this procedure the release of cysteinyl-[2-3H]glycine from glutathione is followed, after separation of the radioactive di-peptide from unreacted glutathione on a small Dowex-1 acetate column. The reactions with gamma-glutamyl-p-nitroanilide and glutathione are both strongly activated by several metal ions (Ca2+, Mg2+, Na+ and K+) and by a number of amino acids and peptide acceptors. The products of the reaction with glutathione were identified as cysteinylglycine, gamma-glutamylglutathione and glutamate. The formation of these products is consistent with the function of gamma-glutamyl transpeptidase in both the gamma-glutamyl transfer reaction and in the hydrolysis of the gamma-glutamyl bond. The activating effect of metal ions in the reaction with glutathione was shown to be dependent on the acceleration of the transfer reaction; the rate of hydrolysis of the gamma-glutamyl bond remaining unchanged.

Animals↗
PubMed1995

Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zinc.

Glutathione disulfide, the major cellular disulfide, releases zinc from metallothionein (MT) [W. Maret (1994) Oxidative metal release from metallothionein via zinc-thiol/disulfide interchange, Proc. natn. Acad. Sci. U.S.A. 91, 237-241]. Here, the interaction of rabbit liver MT-II with other selected biological disulfides (coenzyme A/glutathione mixed disulfide, coenzyme A disulfide, and cystamine) was investigated by measuring concomitant release of radioactive 65-zinc from MT. These disulfides react more rapidly than glutathione disulfide, thus underscoring the reactivity of zinc sulfur bonds in the clusters of MT and the importance of the MT/disulfide interaction as a chemical mechanism for mobilizing zinc from a thermodynamically stable zinc complex. Two implications of these in vitro findings are discussed. (i) Apparently, in the case of zinc which is redox inert, Nature has availed itself of the redox activity of the cysteine ligand to mobilize the metal, and, presumably to permit redox-control of cellular zinc distribution. The mobilization of zinc from MT suggests a possible function of MT as a physiological zinc donor. (ii) A shift of the glutathione redox balance under conditions of oxidative stress will accelerate metal release from MT. Such a disturbance of metal metabolism has important consequences for the progression of diseases such as Alzheimer's and Parkinson's disease where oxidative stress occurs in affected brain tissue.

Animals↗
PubMed2004

A simplified method to create quantitative, "fixed" uranyl-contaminated metal coupons.

A method was developed and validated to quantitatively apply and "fix" uranyl contamination onto a metal surface (steel). Simple approaches are needed to create test surfaces in order to quantify contaminant removal or "decon" methods. We used steel discs sized to allow direct and accurate alpha counting in a Ludlum scanner from radioactive contaminants. A typical 3.8-cm-diameter coupon had a depleted uranyl loading of about 0.1 mg U cm with a count of 980 dpm. The resulting alpha radiation was measured with a precision of >97% for the same coupon. The alpha concentration on replicate coupons differed by as much as 9% (standard deviation). This method, based on earlier methods, required a uranyl solution to be dried but lowers the baking temperature to less than 100 degrees C to increase safety in a typical radiological laboratory. A dike was used to provide a uniform coating of the uranyl solution.

Calibration↗
PubMed2001

Characterization of a multi-functional metal-mediated nuclease by MALDI-TOF mass spectrometry.

Mass spectrometric analysis of reaction products allows simultaneous characterization of activities mediated by multifunctional enzymes. By use of MALDI-TOF mass spectrometry, the relative influence of magnesium and manganese promoted exonuclease and phosphatase activities of Esherichia coli exonuclease III have been quantitatively measured, offering a rapid and sensitive alternative to radioactivity quantification and gel electrophoresis procedures for determination of reaction rate constants. Manganese is found to promote higher levels of exonuclease activity, which could be a source of mutagenic effects if this ion were selected as the natural cofactor. Several potential applications of these methods to quantitative studies of DNA repair chemistry are also described.

Base Sequence↗
PubMed2006

International standards related to the classification and deregulation of radioactive waste.

Although solid radioactive waste management is mainly a national concern, there are some aspects that have international implications. One important example is the decommissioning of nuclear facilities, which results in the release of materials that could be reused and recycled. It is possible that these materials could enter international trade, especially if the material is a metal. It is clearly desirable, therefore, to have appropriate international standards to help regulate trade. This paper describes recent international developments relating to the establishment of radiological criteria for the release of materials from regulatory control (clearance). There have already been some experiences of clearance and the transfer of recycled materials within Europe, and this paper reviews that experience. It also discusses recent developments in relation to the international classification of radioactive waste.

Decision Making↗
PubMed2006

Ranges of 40 keV ions in polycrystalline tungsten and gold.

Integral range profiles for various 40keV radioactive ions from (7)Be to (204)Tl were measured in tungsten and gold using sectioning techniques based on anodizing and stripping. Measured range distributions exhibited long tails extending into the metals. Median range values for a given ion were the same in tungsten as in gold, but were about 1.7 times greater than calculations of projected ranges based on the SRIM computer code.

Journal Article↗