GABA receptor binding in bovine retina: effects of Triton X-100 and perchloric acid

In two synaptosomal fractions from bovine retina, Triton X-100 and sodium perchlorate specifically enhanced the high affinity binding of ³H-GABA to sites with pharmacological specificity similar to the GABA receptor. Maximal effects were noted at 0.05% Triton X-100 and 100 mM sodium perchlorate. In the fraction enriched in photoreceptor cell synaptosomes from the outer plexiform layer, Triton and perchlorate had similar effects in that two binding sites were observed: a higher affinity site (～20 nMK_D) and a lower affinity site (～200 nMK_D). However, in the fraction enriched in conventional sized synaptosomes primarily from the inner plexiform layer, the 20 nM site was virtually absent after Triton treatment, but was readily detectable in the presence of perchlorate. These results may suggest that ³H-GABA binding $\text{in vitro}$ is inhibited by an endogenous substance which is removed by Triton or perchlorate treatment. The difference in the sensitivity of the two fractions to Triton and perchlorate suggests that in retina this substance (whether it is a membrane peptide or GABA itself) is not uniformly distributed and/or uniformly sensitive to Triton and perchlorate.     

Modelling salinity inhibition effects during biodegradation of perchlorate

AIMS: To determine the mathematical kinetic rates and mechanisms of acclimated perchlorate (ClO)-reducing microbial cultures by incorporating a term to relate the inhibitory effect of high salinity during biological reduction of concentrated perchlorate solutions. METHODS AND RESULTS: Salt toxicity associated with the biodegradation of concentrated perchlorate (200, 500, 1100, 1700 and 2400 mg l(-1) as ClO) was investigated using two microbial cultures isolated from a domestic wastewater treatment plant [return activated sludge (RAS) and anaerobic digester sludge (ADS)]. Experiments were performed in wastewaters containing various sodium chloride concentrations, ranging from 0% to 4.0% (w/v) NaCl (ionic strength: 0.14-0.82 mol l(-1), total dissolved solids: 5.3-42.6 g l(-1)) at near-neutral values of pH (6.7-7.8). Perchlorate biodegradation was stimulated through stepwise acclimation to high salinity. The ADS culture was capable of reducing perchlorate at salinities up to 4% NaCl, while the RAS culture exhibited complete inhibition of perchlorate degradation at 4% NaCl, probably resulting from either a toxic effect or enzyme inactivation of the perchlorate-reducing microbes. Further, a kinetic growth model was developed based on experimental data in order to express an inhibition function to relate specific growth rate and salinity. CONCLUSIONS: Biological reduction of concentrated perchlorate wastewaters using either acclimated RAS or ADS cultures is feasible up to 3% or 4% NaCl, respectively. In addition, the kinetic model including a salinity inhibition term should be effective in many practical applications such as improving reactor design and management, furthering the understanding of high salinity inhibition, and enhancing bioremediation under high salinity loading conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Applications of these findings in water treatment practice where ion exchange or membrane technologies are used to remove perchlorate from water can have the potential to increase the overall attractiveness of these processes by eliminating the need to dispose of a concentrated perchlorate solution.     

The nature of the electrophoretically separable multiple forms of rat liver monoamine oxidase

1. Treatment of a partly purified preparation of rat liver monoamine oxidase with the chaotropic agent sodium perchlorate caused the enzyme to migrate as a single band of activity of polyacrylamide-gel electrophoresis, whereas the untreated enzyme separated into a number of bands. 2. Treatment with the chaotropic agent caused no loss of enzyme activity towards benzylamine, dopamine or tyramine. 3. The activities of the untreated preparation towards different substrates were inhibited to different extents by heat treatment and by some inhibitors. No such differences could be detected after the enzyme preparation had been treated with sodium perchlorate. 4. Lipid material, which could be separated by gel filtration, was liberated from the enzyme preparation by sodium perchlorate treatment. 5. The molecular weight of the treated enzyme was found to be 380000+/-38000. 6. Perchlorate treatment altered the solubility of the enzyme. 7. A continuous assay method for monoamine oxidase is described.     

Sensitive high-performance liquid chromatography method for the determination of low levels of perchlorate in biological samples

A rapid and sensitive high-performance liquid chromatography (HPLC) method was developed to detect perchlorate in tissues of male and female rats, both pregnant and lactating (including milk) after administration of perchlorate. Supernatants of ethanol precipitated rat fluids and tissues were evaporated to dryness under nitrogen and reconstituted in deionized water. Reconstituted samples were injected into HPLC system coupled with conductivity detection. Isocratic separation of perchlorate was achieved using an anion-exchange column with sodium hydroxide as mobile phase and a conductivity detector. In this method, perchlorate showed a linear response range from 5 to 100 ng/ml. The lower detection limits for perchlorate in fluids and tissues of rats were 3-6 ng/ml and 0.007-0.7 mg/kg, respectively. The described method has the unique advantage over the existing methods of determining low traces of perchlorate in different biological matrices without complex sample preparation.     

Thyroid-stimulating hormone increases active transport of perchlorate into thyroid cells

Perchlorate blocks thyroidal iodide transport in a dose-dependent manner. The human sodium/iodide symporter (NIS) has a 30-fold higher affinity for perchlorate than for iodide. However, active transport of perchlorate into thyroid cells has not previously been demonstrated by direct measurement techniques. To demonstrate intracellular perchlorate accumulation, we incubated NIS-expressing FRTL-5 rat thyroid cells in various concentrations of perchlorate, and we used a sensitive ion chromatography tandem mass spectrometry method to measure perchlorate accumulation in the cells. Perchlorate caused a dose-related inhibition of 125-iodide uptake at 1-10 microM. The perchlorate content from cell lysate was analyzed, showing a higher amount of perchlorate in cells that were incubated in medium with higher perchlorate concentration. Thyroid-stimulating hormone increased perchlorate uptake in a dose-related manner, thus supporting the hypothesis that perchlorate is actively transported into thyroid cells. Incubation with nonradiolabeled iodide led to a dose-related reduction of intracellular accumulation of perchlorate. To determine potential toxicity of perchlorate, the cells were incubated in 1 nM to 100 microM perchlorate and cell proliferation was measured. Even the highest concentration of perchlorate (100 microM) did not inhibit cell proliferation after 72 h of incubation. In conclusion, perchlorate is actively transported into thyroid cells and does not inhibit cell proliferation.     

Cloning,expression and mutagenesis of a subunit contact of rabbit muscle-specific (betabeta) enolase

The cDNA for rabbit muscle-specific (betabeta) enolase was cloned, sequenced and expressed in Escherichia coli. This betabeta-enolase differs at eight positions from that sequenced by Chin (17). Site-directed mutagenesis was used to change residue 414 from glutamate to leucine, thereby abolishing a salt bridge involved in subunit contacts. Recombinant wild-type and mutant enolase were purified from E. coli and compared to enolase isolated from rabbit muscle. Molecular weights were determined by mass spectrometry. All three betabeta-enolases had similar kinetics, and UV and circular dichroism (CD) spectra. The mutant enolase was far more sensitive to inactivation by pressure, by KCl or EDTA, and by sodium perchlorate. We confirmed, by analytical ultracentrifugation, that the sodium perchlorate inactivation was due to dissociation. DeltaG(o) for dissociation of enolase was decreased from 49.7 kJ/mol for the wild-type enzyme to 42.3 kJ/mol for the mutant. In contrast to the wild-type enzyme, perchlorate inactivation of E414L was accompanied by a small loss of secondary structure.     

Isolation and characterization of Dechlorospirillum anomalous strain JB116 from a sewage treatment plant

The dissimilatory perchlorate reducers mainly belong to two monophyletic groups, viz. Dechloromonas and Azospira in the beta subclass of Proteobacteria. The present study describes isolation and genetic characterization of Dechlorospirillum anomalous strain JB116 that belongs to alpha subclass of Proteobacteria. The strain JB116 was isolated under facultative anaerobic conditions on a growth medium containing sodium perchlorate and sodium acetate as electron (e(-)) acceptor and e(-) donor, respectively. The strain is a spirillum shaped, dissimilatory perchlorate and nitrate reducer that prefers nitrate to perchlorate. It grows heterotrophically with acetate at temperatures between 25-35 degrees C, NaCl concentrations between 0-0.5% and pH of 7-7.8. The strain JB116 is the second only representative strain within D. anomalous that shares 99% 16S rDNA sequence similarity with the type strain D. anomalous strain WD.     

Alpha-helix in the carboxy-terminal domains of histones H1 and H5.

Although the carboxy-terminal domains of histones H1 and H5 exist as random-coil in aqueous solution, secondary structure prediction suggests that this region has a high potential for alpha-helix formation. We have measured CD spectra in various conditions known to stabilize alpha-helices, to determine whether this potential can be realized in an appropriate environment. Trifluoroethanol increases the helix contents of H1, H5 and their carboxy-terminal fragments, presumably through promotion of axial hydrogen bonding. Sodium perchlorate is also effective and better than sodium chloride, suggesting stabilization by binding of bulky perchlorate ions rather than simple charge screening. Extrapolating from these measurements in solution, and taking into account the occurrence of proline residues throughout the carboxy-terminal domain, we propose that binding to DNA stabilizes helical segments in the carboxy-terminal domains of histones H1 and H5, and that it is this structured form of the domain that is functionally important in chromatin.     

Rapid isolation of RNA using proteinase K and sodium perchlorate.

A simple, efficient procedure for the isolation of cellular nucleic acids is described. It combines the use of sodium dodecyl sulfate, proteinase K, sodium perchlorate, and isopropanol precipitation. The yields and purity of RNA extracted from a variety of sources are comparable or superior to those obtained by phenol extraction. High molecular weight RNA (ribosomal as well as nonribosomal) is recovered intact and in high yield. Fibroin messenger RNA (M_r 5.8 × 10⁶) isolated by this procedure is biologically active.     

Effect of perchlorate on mitochondrial function

Administration of sodium perchlorate to rats for 45 days leads to decreased activities of citric acid cycle enzymes. The oxidation of succinate both in state 3 and state 4 conditions and endogenous ATP content of mitochondria decreased during perchlorate toxicity. The significant decrease in cytochrome aa3 in perchlorate-treated rats may be one of the prime factors involved in the decreased rate of respiration. The permeability of mitochondria of perchlorate-treated rats is altered as indicated by increased oxidation of NADH and low respiratory control ratio (RCR).     

Experimental characterization of poly(hydroxyalkyl-L-glutamine) conformations in aqueous calcium chloride and sodium perchlorate solutions

Poly(hydroxyalkyl-L -glutamine) (alkyl = ethyl, propyl, butyl) solutions have been studied by CD as functions of temperature and activity of calcium chloride and sodium perchlorate. Helical content is altered by changes in salt activity and temperature. The helicity of poly(hydroxybutyl-L -glutamine) and poly(hydroxypropyl-L -glutamine) falls to zero in a monotonic fashion with increasing calcium chloride activity. A nonzero helicity reappears at activities in excess of 5–50 mol kg^?1. Poly(hydroxypropyl-L -glutamine) is much more sensitive to calcium chloride than is poly(hydroxybutyl-L -glutamine), and both polypeptides are more sensitive to calcium chloride than are typical proteins. Markedly different behavior is observed with sodium perchlorate. This salt acts as a helix stabilizer at low activities but becomes a destabilizer at activities higher than 0.3–1.0 mol kg^?1. In this respect the effect of sodium perchlorate on nonionic poly(hydroxyalkyl-L -glutamines) resembles that seen with cationic poly(L -lysine) and poly(L -arginine). Helix stabilization at low sodium perchlorate activity is moderate for poly(hydroxybutyl-L -glutamine) and large for poly(hydroxypropyl-L -glutamine) and poly(hydroxyethyl-L -glutamine).     

Direct measurement of perchlorate exposure biomarkers in a highly exposed population: a pilot study

Exposure to perchlorate is ubiquitous in the United States and has been found to be widespread in food and drinking water. People living in the lower Colorado River region may have perchlorate exposure because of perchlorate in ground water and locally-grown produce. Relatively high doses of perchlorate can inhibit iodine uptake and impair thyroid function, and thus could impair neurological development in utero. We examined human exposures to perchlorate in the Imperial Valley among individuals consuming locally grown produce and compared perchlorate exposure doses to state and federal reference doses. We collected 24-hour urine specimen from a convenience sample of 31 individuals and measured urinary excretion rates of perchlorate, thiocyanate, nitrate, and iodide. In addition, drinking water and local produce were also sampled for perchlorate. All but two of the water samples tested negative for perchlorate. Perchlorate levels in 79 produce samples ranged from non-detect to 1816 ppb. Estimated perchlorate doses ranged from 0.02 to 0.51 μg/kg of body weight/day. Perchlorate dose increased with the number of servings of dairy products consumed and with estimated perchlorate levels in produce consumed. The geometric mean perchlorate dose was 70% higher than for the NHANES reference population. Our sample of 31 Imperial Valley residents had higher perchlorate dose levels compared with national reference ranges. Although none of our exposure estimates exceeded the U. S. EPA reference dose, three participants exceeded the acceptable daily dose as defined by bench mark dose methods used by the California Office of Environmental Health Hazard Assessment.     

Factors affecting the extraction of intact ribonucleic Acid from plant tissues containing interfering phenolic compounds

Using conventional methods it is impossible to extract RNA as uncomplexed intact molecules from the leaves of grapevines (Vitis vinifera L.) and from a number of woody perennial species that contain high levels of reactive phenolic compounds. A procedure involving the use of high concentrations of the chaotropic agent sodium perchlorate prevents the binding of phenolic compounds to RNA during extraction. Analyses of the phenolics present in plant tissues used in these experiments indicate that there is a poor correlation between the total phenolic content and the complexing of RNA. However, qualitative analyses suggest that proanthocyanidins are involved in the tanning of RNA during conventional extractions.     

Characterization of a microbial community capable of reducing perchlorate and nitrate in high salinity

Denitrifying up-flow packed-bed bioreactors fed with perchlorate and nitrate allowed for the examination of the impact of a variety of salt conditions (up to 10% w/v NaCl) on the complete perchlorate and nitrate removal capacity of the reactor using activated sludge taken from a municipal wastewater treatment plant. Based on the evaluation of the microbial community in the bioreactor by cloning analysis, Clostridium sp. and a Rhodocyclaceae bacteria were identified as the dominant clones. This suggests that they may be tolerant to high salt and can reduce both nitrate and perchlorate in such conditions.     

Peptide biomarkers as evidence of perchlorate biodegradation

Perchlorate is a known health hazard for humans, fish, and other species. Therefore, it is important to assess the response of an ecosystem exposed to perchlorate contamination. The data reported here show that a liquid chromatography-mass spectrometry-based proteomics approach for the detection of perchlorate-reducing enzymes can be used to measure the ability of microorganisms to degrade perchlorate, including determining the current perchlorate degradation status. Signature peptides derived from chlorite dismutase (CD) and perchlorate reductase can be used as biomarkers of perchlorate presence and biodegradation. Four peptides each derived from CD and perchlorate reductase subunit A (PcrA) and seven peptides derived from perchlorate reductase subunit B (PcrB) were identified as signature biomarkers for perchlorate degradation, as these sequences are conserved in the majority of the pure and mixed perchlorate-degrading microbial cultures examined. However, chlorite dismutase signature biomarker peptides from Dechloromonas agitata CKB were found to be different from those in other cultures used and should also be included with selected CD biomarkers. The combination of these peptides derived from the two enzymes represents a promising perchlorate presence/biodegradation biomarker system. The biomarker peptides were detected at perchlorate concentrations as low as 0.1 mM and at different time points both in pure cultures and within perchlorate-reducing environmental enrichment consortia. The peptide biomarkers were also detected in the simultaneous presence of perchlorate and an alternate electron acceptor, nitrate. We believe that this technique can be useful for monitoring bioremediation processes for other anthropogenic environmental contaminants with known metabolic pathways.     

Equilibrium unfolding of dimeric and engineered monomeric forms of lambda Cro (F58W) repressor and the effect of added salts: evidence for the formation of folded monomer induced by sodium perchlorate

The equilibrium unfolding transitions of Cro repressor variants, dimeric variant Cro F58W and monomer Cro K56[DGEVK]F58W, have been studied by urea and guanidine hydrochloride to probe the folding mechanism. The unfolding transitions of a dimeric variant are well described by a two state process involving native dimer and unfolded monomer with a free energy of unfolding, DeltaG(0,un)(0), of approximately 10-11 kcal/mol. The midpoint of transition curves is dependent on total protein concentration and DeltaG(0,un)(0) is independent of protein concentration, as expected for this model. Unfolding of Cro monomer is well described by the standard two state model. The stability of both forms of protein increases in the presence of salt but decreases with the decrease in pH. Because of the suggested importance of a N2<-->2F dimerization process in DNA binding, we have also studied the effect of sodium perchlorate, containing the chaotropic perchlorate anion, on the conformational transition of Cro dimer by CD, fluorescence and NMR (in addition to urea and guanidine hydrochloride) in an attempt both to characterize the thermodynamics of the process and to identify conditions that lead to an increase in the population of the folded monomers. Data suggest that sodium perchlorate stabilizes the protein at low concentration (<1.5 M) and destabilizes the protein at higher perchlorate concentration with the formation of a "significantly folded" monomer. The tryptophan residue in the "significantly folded" monomer induced by perchlorate is more exposed to the solvent than in native dimer.     

Treatment of amiodarone induced hyperthyroidism with potassium perchlorate and methimazole during amiodarone treatment.

To exploit the antiarrhythmic effect of amiodarone when patients develop the side effect of thyrotoxicosis three patients with hyperthyroidism induced by amiodarone were given simultaneously 1 g potassium perchlorate a day for 40 days and a starting dose of 40 mg methimazole a day while they continued to take amiodarone. As hyperthyroidism might have recurred after potassium perchlorate treatment was stopped the dose of methimazole was not reduced until biochemical hypothyroidism (raised thyroid stimulating hormone concentrations) was achieved. The patients became euthyroid (free triiodothyronine concentration returned to normal values) in two to five weeks and hypothyroid in 10 to 14 weeks. One patient became euthyroid while taking 5 mg methimazole a day and 600 mg amiodarone weekly; the two others required substitution treatment with thyroxine sodium while taking 5 mg methimazole or 50 mg propylthiouracil (because of an allergic reaction to methimazole) and 2100 or 1400 mg amiodarone weekly. Hyperthyroidism induced by amiodarone may be treated with potassium perchlorate and methimazole given simultaneously while treatment with amiodarone is continued.     

Using a Wetland Bioreactor to Remediate Ground Water Contaminated with Nitrate (mg/L) and Perchlorate (μg/L)

Wetland plants and their rhizospheric microorganisms were used to remove perchlorate from nitrate-contaminated ground water. A wetland bioreactor was constructed with the wetland plants Typha spp., Scirpus spp., and Cyperus spp. These plants provided a habitat and a food source for the indigenous denitrifying microorganisms presumed responsible for the perchlorate and nitrate removal. Ground water contaminated with nitrate (68 mg/L) was collected from a well and injected with concentrations of potassium perchlorate similar to those found in several local wells (50 and 100 μg/L). The water flowed continuously through the wetland bioreactor with hydraulic retention times (HRT) ranging from 0.5 to 400 h. Because microbial denitrification depends, in part, on the nature and amount of carbon available to the microorganisms, four carbon sources (dried leaf matter, dried algae, milk replacement starter, and sodium acetic acid) were evaluated. Analyses for perchlorate

This document was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor the University of California nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial products, process or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or the University of California, and shall not be used for advertising or product endorsement purposes.

Work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract W-7405-Eng-18. concentration in ground water were performed using negative electrospray mass spectrometry/mass spectrometry. Of the carbon sources tested, sodium acetic acid was the most efficient at accelerating the nitrate and perchlorate removal rates. The HRT required to remove perchlorate (100 μg/L) to below the detection limit, without carbon source amendment, was 4 d. With the addition of sodium acetic acid (0.26 g/L) the required HRT decreased to 0.5 d. These results suggest that denitrifying microorganisms, supported in a wetland bioreactor amended with a carbon source, remove perchlorate from nitrate contaminated ground water.     

Mechanistic changes during phytoremediation of perchlorate under different root-zone conditions

Two types of hydroponic bioreactors were used to investigate the mechanisnistic changes during phytoremediation of perchlorate under different root-zone conditions. The bioreactors included: (1) an aerobic ebb-and-flow system planted with six willow trees, and (2) individual willow trees grown in sealed root-zone bioreactors. Rhizosphere probes were used to monitor for the first time during phytoremediation of perchlorate, diurnal swings in oxidation-reduction potential (E(H)), dissolved oxygen (DO), and pH. Radiolabeled (36Cl-labeled) perchlorate was used as a tracer in a subset of the sealed bioreactor experiments to quantify the contribution of phytodegradation and rhizodegradation mechanisms. Rhizodegradation accounted for the removal of 96.1 +/- 4.5% (+/-95% CI) of the initial perchlorate dose in experiments conducted in sealed hydroponic bioreactors with low DO and little or no nitrate N. Meanwhile, the contribution of rhizodegradation decreased to 76 +/- 14% (+/-95% CI) when nitrate (a competing terminal electron acceptor) was provided as the nitrogen source. Slower rates of phytoremediation by uptake and phytodegradation were observed under high nitrate concentrations and aerobic conditions, which allowed perchlorate to persist in solution and resulted in a higher fraction uptake by the plant. Specifically, the rate of removal of perchlorate from bulk solution ranged from 5.4 +/- 0.54 to 37.1 +/- 2.25 mg/L/d (+/-SE) in the absence of nitrate to 1.78 +/- 0.27 to 0.46 +/- 0.02 mg/L/d (+/-SE) at high nitrate concentration. The results of this study indicate that the root-zone environment of plants can be manipulated to optimize rhizodegradation and to minimize undesirable processes such as uptake, temporal phytoaccumulation, and slow phytodegradation during phytoremediation of perchlorate. Rhizodegradation is desired because contaminants resident in plant tissue may remain an ecological risk until completely phytodegraded.     

Design of a test system based on magnetic particles with immobilized monoclonal antibodies for selective <Emphasis Type="Italic">Bacillus anthracis</Emphasis> spore concentration

Magnetic immunosorbents (MIS) have been designed using antianthrax monoclonal antibodies immobilized on aluminum silicate—iron oxide matrix activated by sodium perchlorate. MIS allows us to concentrate the analyzed microorganism and to decontaminate culture from concomitant microflora. Diagnostic test systems constructed on the basis of MIS were tested on pure Bacillus anthracis cultures and in the model experiments. The results testify to the high specificity and sensitivity (10²–10³ spore/ml) of the designed test systems.