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PubMed1977

Purification and Some Biological Properties of Asparaginase from Azotobacter vinelandii.

Asparaginase was found in the soluble fraction of cells of Azotobacter vinelandii, and its activity remained the same during growth of the organism in a nitrogen-free medium. The specific activity and the yield of A. vinelandii increased twofold in the presence of ammonium sulfate. Within limits, the temperature (30 to 37 degrees C) and pH (6.5 to 8.0) of the medium showed little effect on the levels of enzyme activity. The enzyme was purified to near homogeneity by standard methods of enzyme purification, including affinity chromatography, and had optimum activity at pH 8.6 and 48 degrees C. The approximate molecular weight was 84,000. The apparent K(m) value for the substrate was 1.1 x 10 M. Metal ions or sulfhydryl reagents were not required for enzyme activity. Cu, Zn, and Hg showed concentration-dependent inhibition, whereas amino and keto acids had no effect on the enzyme activity. Asparaginase was stable when incubated with rat serum and ascites fluid. The enzyme had no effect on the membrane of sheep erythrocytes and did not inhibit the incorporation of radioactive precursors into deoxyribonucleic acid, ribonucleic acid, and protein in Yoshida ascites sarcoma cells. Asparaginase activity was not detected in the tumor cells.

Journal Article↗

PubMed2006

[A novel anti-DNA antibody modified coronary stent for site-specific plasmid DNA delivery].

OBJECTIVE: To explore the feasibility of using an endovascular metal stent as a highly efficient and site-specific gene delivery system. METHODS: Stents were formulated with a collagen coating. Anti-DNA monoclonal antibodies were covalently bound to the collagen surface by a cross linking reagent of N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Binding capacity and stability of antibody and plasmid DNA on stents were quantified by radioactive labeling. The gene transduction efficiency was evaluated in cell culture and in rabbits. RESULTS: The amount of antibodies binding on collagen matrix through SPDP reaction was 15 times higher than that of through physical absorption (P < 0.005). The binding stability of plasmid was significantly better than the control groups (P < 0.01). There was no harmful effect on cell growth with the anti-DNA antibody modified stents. The stents retrieved from cell culture after 72 hours of incubation in A10 cells showed numerous transducted cells only infiltrating the surface coating indicating a highly localized and efficient gene delivery pattern. Results of in vivo gene transfer by this modified stent revealed (2.8 +/- 0.7)% of total cells transduction and the higher transduction location was neointimal layer (about 7%). No distal spread of vector was detectable in the anti-DNA antibody modified stent implantation animals. CONCLUSIONS: Anti-DNA antibody modified stents represent a novel highly efficient and site-specific gene delivery system which can deliver various kinds of plasmid vectors. The release of plasmid DNA tethered on the stents could be controlled in some conditions. This novel system provided a novel platform for cardiovascular site-specific gene therapy.

Animals↗

PubMed2006

Analysis of novel soluble chromate and uranyl reductases and generation of an improved enzyme by directed evolution.

Most polluted sites contain mixed waste. This is especially true of the U.S. Department of Energy (DOE) waste sites which hold a complex mixture of heavy metals, radionuclides, and organic solvents. In such environments enzymes that can remediate multiple pollutants are advantageous. We report here evolution of an enzyme, ChrR6 (formerly referred to as Y6), which shows a markedly enhanced capacity for remediating two of the most serious and prevalent DOE contaminants, chromate and uranyl. ChrR6 is a soluble enzyme and reduces chromate and uranyl intracellularly. Thus, the reduced product is at least partially sequestered and nucleated, minimizing the chances of reoxidation. Only one amino acid change, (Tyr)128(Asn), was responsible for the observed improvement. We show here that ChrR6 makes Pseudomonas putida and Escherichia coli more efficient agents for bioremediation if the cellular permeability barrier to the metals is decreased.

Directed Molecular Evolution↗

PubMed1987

The uptake and intracellular distribution of [35S]trithiomolybdate in bovine liver in vivo.

[35S] trithiomolybdate was administered intravenously to a group of four steer at two dose rates, 1 and 26 mg Mo per animal. Radioactivity appeared rapidly in the liver and was distributed in all the subcellular fractions examined. Examination by Sephadex G-100 gel-filtration of the cytosol fraction showed that distinct 35S-binding protein peaks were present. The protein-bound radioactivity was displaceable and was identified as [35S]thiomolybdates. No radioactivity eluted with metallothionein, but 35S was associated with the high molecular weight copper fraction, eluted in the void volume of the column, which increased transiently after the administration of the higher dose. It was suggested that the presence of protein-bound thiomolybdates in the liver gave rise to new ligands, which altered the equilibrium of copper between the different metal-binding proteins. This might be similar to the alteration in the copper-binding of albumin produced by the presence of thiomolybdates.

Animals↗

PubMed1978

Assay and properties of IgA protease of Streptococcus sanguis.

An assay procedure for streptococcal IgA protease is described which uses isotopically labelled human serum IgA as substrate. Enzyme activity was monitored by the radioactive counts in the Fab alpha product, which was separated from other components in the digestion mixture by electrophoresis. Cleavage of IgA was linear with respect to time using catalytic amounts of the enzyme. Km was calculated to be 5.5 X 10(-6)M, pH optimum 6.0-7.0 at 37 degrees C, and the enzyme was fully inactivated at low concentrations of the metal chelator ethylenediaminetetraacetic acid.

Edetic Acid↗

PubMed1979

Bioavailability of lead in rats fed "human" diets.

The bioavailability of lead was studied in rats fed various baby foods (Babymix-turkey, Babymix-vegetables, Frutolino-fruit, Frutamix-bananas, Babyron-S-26, Truefood), cow's milk, bread, liver and standard rat diet. Lead absorption was determined by measuring the whole body retention of 203Pb 6 days after a single oral application. Highest absorption values ranging from 17 to 20% were obtained in animals fed cow's milk and fruit foods. Rats on other "human" diets absorbed between 3 and 8% of the radioactive lead dose. Only in animals on rat diet lead absorption was below 1%. It is concluded that rats fed "human" diets show absorption values similar to those in humans. This might indicate that the bioavailability of lead is primarily dependent on dietary habits. This experimental model, if confirmed by further work, might be useful for obtaining preliminary data on the bioavailability of metals from various foods.

Animals↗

PubMed1984

Influence of dietary ingredients on the body retention of strontium, cadmium and mercury in suckling rats.

Six-day-old suckling rats were artificially fed over 8 h with cow's milk or a mixture of rat food ingredients (fish meal, sunflower meal, alfalfa, cane molasses and premix) labelled with 85Sr, 115mCd or 203Hg. Whole-body radioactivity was determined in a double crystal scintillation counter every 24 h over a 6-day period. Carcass, gut, liver and kidney retention were determined at the end of the experiment. Relative to pups that were fed on cow's milk, administration of the mixture of the solid dietary ingredients caused a significant reduction in whole body, carcass and organ retention of all metals and was highest for 203Hg. This indicates that high absorption of toxic metals in sucklings can be reduced by dietary means. Rat food ingredients deserve further consideration because they are non-toxic dietary constituents and might be useful for decreasing the body burden of some metals in conditions of increased environmental exposure.

Animal Population Groups↗

PubMed1986

Distribution of tungstate in pregnant mice and effects on embryonic cells in vitro.

Whole-body autoradiography and impulse counting experiments were used to study the distribution and retention of radioactivity in the pregnant mouse after administration of [185W]tungstate. A rapid uptake was found in a number of tissues--skeleton, red pulp of the spleen, adrenal, liver, thyroid, pituitary, and ovary--and in the intestine and kidneys, through which it was rapidly excreted. 185W was also readily transported from mother to fetus, although more in late than in early gestation. The largest metal retention was found in the maternal skeleton, kidneys, and spleen and in the visceral yolk sac epithelium and the skeleton of the fetus. Furthermore, in vitro cytotoxicity experiments showed inhibition by tungstate of cartilage production in limb bud mesenchymal cultures at concentrations similar to those found in vivo.

Animals↗

PubMed1989

The biology of amiloride-sensitive sodium channels.

Studies of the molecular nature of epithelial sodium channels are in their infancy and have largely involved experiments in which the interaction between amiloride and this transport process has been examined. Because of the inherent geometric complexity of epithelial tissues, these studies have in large measure been macroscopic in nature, with the molecular details of transport being deduced. In the past five years, however, the molecular biology of these critical ion channels has been studied directly. The development of radioactive high-affinity probes, the application of patch-clamp and reconstitution techniques, the generation of specific antibodies, and the formulation of epithelial cDNA expression libraries have propelled the field of epithelial ion channels into a new era. Now, for the first time, we can rigorously address questions concerning the molecular nature of the amiloride block, the channel's selectivity to alkali metal cations, and the modulation of ion transport through this channel by other ions (such as calcium), hormones (such as vasopressin, aldosterone, and atrial natriuretic factor), or intracellular second messengers (such as cAMP or cGMP). The complexity of the epithelial sodium channel's structure may reflect the constitutive and regulatory role this protein plays in sodium homeostasis. The epithelial sodium channel is continually operating, constantly changing its activity on a second-to-second basis. Hence, its tonic functions are probably modulated by a myriad of factors, most of which are unknown. With the application of molecular techniques, a much clearer understanding of the nature and regulation of epithelial sodium channel processes in health and disease will emerge in the years to come.

Aldosterone↗

PubMed2001

ICCVAM evaluation of the murine local lymph node assay. Conclusions and recommendations of an independent scientific peer review panel.

The validation status of the murine local lymph node assay (LLNA), a method for assessing the allergic contact dermatitis potential of chemicals, was evaluated by an independent peer review panel (Panel) convened by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). The LLNA measures lymphocyte proliferation using incorporation of radioactive thymidine or iododeoxyuridine into cells of the draining lymph nodes of mice topically exposed to a test article. The Panel concluded that the assay performed as well as currently accepted guinea pig methods [guinea pig maximization test (GPMT)/Buehler assay (BA)] for the hazard identification of strong to moderate chemical sensitizing agents, but that it might not correctly identify all weak sensitizers or metals (potential false negative response) or all strong irritants (potential false positive response). The Panel concluded also that the LLNA involves less pain and distress than conventional guinea pig methods. The Panel unanimously recommended the LLNA as a stand-alone alternative for contact sensitization hazard assessment, provided that certain protocol modifications were made. These included collection of individual, rather than pooled, animal response data; the inclusion of a concurrent positive control; and consideration of dose-response information and statistical analyses. A standardized LLNA protocol is provided.

Animals↗

PubMed1966

Aspartate carbamoyltransferase from rat liver.

1. Aspartate-carbamoyltransferase activity was concentrated from rat-liver preparations. Only l-aspartate, beta-benzyl-l-aspartate and beta-erythro-hydroxy-dl-aspartate were carbamoylated enzymically. The K(m) for l-aspartate and carbamoyl phosphate have been determined by three methods: colorimetric procedure, radioactive assay with [(14)C]aspartate and an assay with [(14)C]carbamoyl phosphate. 2. The K(m) for aspartate has been determined as a function of the pH; the pK of the functional group at the active site of the enzyme, pK(e), was at pH9.0. Enzymic activity was diminished in the presence of N-ethylmaleimide, p-hydroxymercuribenzoate and the heavy metals Ag(+), Hg(2+), or Zn(2+). The inhibitions could be prevented by mercaptoethanol. These findings suggested the association of a thiol group with the enzymic activity. 3. Enzymic activity was also decreased by sodium lauryl sulphate, urea and dioxan. Competitive inhibition (with l-aspartate) was manifested by maleate, succinate, oxaloacetate, beta-erythro-hydroxy-dl-aspartate and beta-benzyl-l-aspartate. The K(i) for most of these inhibitions has been determined. 4. The properties of the liver enzyme are compared with those of Escherichia coli aspartate carbamoyltransferase and the implications of the findings are discussed.

Animals↗

PubMed2002

High-throughput fluorescence polarization method for identification of FKBP12 ligands.

FKBP12 is best known as the target of the widely used immunosuppressive drug FK506 but may also play a role in neuronal survival. Nonimmunosuppressive ligands of FKBP12 have been shown to have neuroprotective and neuroregenerative activity both in vitro and in vivo, stimulating interest in the development of high-throughput screens to rapidly identify novel ligands. FKBP12 was expressed as a His(6)-fusion in bacteria and purified by metal ion affinity and gel filtration chromatography. A high-throughput fluorescence polarization assay was developed to identify novel ligands of FKBP12. Dissociation constant values of known FKBP12 ligands measured by the new method agreed closely with K(i) values obtained by assaying inhibition of the rotamase activity of the enzyme. The fluorescence polarization assay is rapid, robust, and inexpensive and does not generate radioactive waste. It is very well suited for high-throughput screening efforts.

Drug Evaluation, Preclinical↗

PubMed1990

[Mortality reduction in mice administered a single abundant dose of zinc, manganese or magnesium after irradiation by gamma-rays at sublethal doses].

Radioprotective effect has been observed in mice which have subcutaneously received a single dose of abundant inorganic zinc or manganese 24 hours before gamma-ray irradiation with a sublethal dose. Mechanism of the radioprotection has been suggested to be an abundant production of metallothionein (MT) induced by the metals. But process of the protection might be more complicated because immunological and hematopoietic functions are remarkably inhibited by the irradiation and many zinc binding-compounds with biological functions are known at present. Furthermore, the effect of these metals has been observed by the administration only before the irradiation. Radioprotective effect was shown in mice which were administered with not only zinc and manganese, but also magnesium. It seems that mechanism of the radioprotection of magnesium is different from that of zinc or manganese since magnesium is not known to induce MT production. It was observed that the relative concentration ratio of radioactivity in tissues and organs at 7th day (after injection of 65Zn) showed a marked difference between in irradiated and non-irradiated mice.

Animals↗

PubMed1991

[Phase I clinical study on 99mTc-MIBI].

A phase I clinical study on 99mTc-hexakis 2-methoxy isobutylisonitrile (99mTc-MIBI) was carried out in 6 normal volunteers. There was no significant change in vital signs and laboratory parameters attributing to the reagent other than complaint of slight and transient metallic taste immediately after the injection in 4 volunteers. The highest dosimetry was calculated as 1.1 mGy/37 MBq at lower large intestine, which was within the acceptable range. 99mTc-MIBI was rapidly cleared from the blood and accumulated in the heart immediately after the injection with 1.4% dose and 1.8% dose at 5 min at rest and at stress, respectively. The retention of radioactivity in the heart well continued for at least several hours. The heart-to-lung ratio was over 2.00 at 5 min and heart-to-liver ratio was over 1.00 at 60 min. Myocardial planar and SPECT images were obtained with high quality. In conclusion, 99mTc-MIBI is a useful myocardial perfusion imaging agent.

Adult↗

PubMed2005

Bioreduction of uranium: environmental implications of a pentavalent intermediate.

The release of uranium and other transuranics into the environment, and their subsequent mobility, are subjects of intense public concern. Uranium dominates the inventory of most medium- and low-level radioactive waste sites and under oxic conditions is highly mobile as U(VI), the soluble uranyl dioxocation (UO2)2+. Specialist anaerobic bacteria are, however, able to reduce U(VI)to insoluble U(IV), offering a strategy for the bioremediation of uranium-contaminated groundwater and a potential mechanism for the biodeposition of uranium ores. Despite the environmental importance of U(VI) bioreduction, there is little information on the mechanism of this transformation. In the course of this study we used X-ray absorption spectroscopy (XAS) to show that the subsurface metal-reducing bacterium Geobacter sulfurreducens reduces U(VI) by a one-electron reduction, forming an unstable (UO2)+ species. The final, insoluble U(IV) product could be formed either through further reduction of U(V) or through its disproportionation. When G. sulfurreducens was challenged with the chemically analogous (NpO2)+, which is stable with respect to disproportionation, it was not reduced, suggesting that it is disproportionation of U(V) which leads to the U(IV) product. This surprising discrimination between U and Np illustrates the need for mechanistic understanding and care in devising in situ bioremediation strategies for complex wastes containing other redox-active actinides, including plutonium.

Biodegradation, Environmental↗

PubMed2006

Determination of 210Po in environmental materials: a review of analytical methodology.

Polonium-210 ((210)Po) is analysed for a variety of purposes, including for radiological impact assessment, as a tracer of environmental processes, and as an indirect measure of its progenitor lead-210 ((210)Pb). Losses of polonium may occur at temperatures above 100 degrees C, depending on conditions, requiring particular care in sample preparation and treatment. In spite of this problem, the analysis of (210)Po is relatively straightforward, due to the ease of source preparation through spontaneous auto-deposition onto metal surfaces and the uncomplicated alpha spectrum. Although several optimisation studies have been carried out, published source preparation methods remain remarkably diverse. Some areas where further study would be useful are identified.

Air Pollutants, Radioactive↗

PubMed2006

Physicochemical and mineralogical characterization of soil-saprolite cores from a field research site, Tennessee.

Site characterization is an essential initial step in determining the feasibility of remedial alternatives at hazardous waste sites. Physicochemical and mineralogical characterization of U-contaminated soils in deeply weathered saprolite at Area 2 of the DOE Field Research Center (FRC) site, Oak Ridge, TN, was accomplished to examine the feasibility of bioremediation. Concentrations of U in soil-saprolite (up to 291 mg kg(-1) in oxalate-extractable U(o)) were closely related to low pH (ca. 4-5), high effective cation exchange capacity without Ca (64.7-83.2 cmol(c) kg(-1)), amorphous Mn content (up to 9910 mg kg(-1)), and the decreased presence of relative clay mineral contents in the bulk samples (i.e., illite 2.5-12 wt. %, average 32 wt. %). The pH of the fill material ranged from 7.0 to 10.5, whereas the pH of the saprolite ranged from 4.5 to 8. Uranium concentration was highest (about 300 mg kg(-1)) at around 6 m below land surface near the saprolite-fill interface. The pH of ground water at Area 2 tended to be between 6 and 7 with U concentrations of about 0.9 to 1.7 mg L(-1). These site specific characteristics of Area 2, which has lower U and nitrate contamination levels and more neutral ground water pH compared with FRC Areas 1 and 3 (ca. 5.5 and <4, respectively), indicate that with appropriate addition of electron donors and nutrients bioremediation of U by metal reducing microorganisms may be possible.

Biodegradation, Environmental↗

PubMed2006

Rhenium(V) oxocomplexes with novel pyrazolyl-based N4- and N3S-donor chelators.

The novel pyrazolyl-based ligands 3,5-Me2pz(CH2)2NH(CH2)2NH(CH2)2NH2 and pz*(CH2)2NH-Gly-CH2STrit (pz*=pz, 3,5-Me2pz, 4-(EtOOC)CH(2)-3,5-Me2pz) were synthesized, and their suitability to stabilize Re(V) oxocomplexes was evaluated using different starting materials, namely (NBu4)[ReOCl4], [ReOCl3(PPh3)2] and trans-[ReO2(py)4]Cl. Compound reacts with trans-[ReO2(py)4]Cl yielding the cationic compound [ReO(OMe){3,5-Me2pz(CH2)2N(CH2)2NH(CH2)2NH2}](BPh4) in a low isolated yield. In contrast, the neutral complexes [ReO{pz*(CH2)2NH-Gly-CH2S}] (pz*=pz, 3,5-Me2pz, 4-(EtOOCCH2)-3,5-Me2pz) were synthesized almost quantitatively by reacting [ReOCl3(PPh3)2] or (NBu4)[ReOCl4] with the trityl-protected chelators. The X-ray diffraction analysis of and confirmed the tetradentate coordination mode of the respective ancillary ligands. In the monoanionic chelator coordinates to the metal through four nitrogen atoms, while in the chelator is trianionic, coordinating to the metal through three nitrogens and one sulfur atom. Solution NMR studies of , including two-dimensional NMR techniques (1H COSY and 1H/13C HSQC), confirmed that the N3S coordination mode of the chelators is retained in solution. Unlike , complexes may be considered relevant in the development of radiopharmaceuticals, as further corroborated by the synthesis of the congener [99mTcO{pz(CH2)2-NH-Gly-CH2S}]. This radioactive compound was obtained from 99mTcO4- in aqueous medium, in almost quantitative yield and with high specific activity and radiochemical purity.

Chelating Agents↗