Improved approach for LSC detection of 35S aiming at its application as tracer for short groundwater residence times.

The knowledge of groundwater residence times in (vulnerable) aquifers is essential for the sustainable management of the associated groundwater resources. A powerful tool for related investigations is the application of naturally occurring radioisotopes as water age indicators. However, due to the limited number of suitable (i.e. omnipresent, short-lived and easily detectable) radionuclides only few studies focus on groundwater ages below one year. A natural radionuclide that does have the potential to cover this time range is 35S (87.4 day half-life). 35S is continually produced in the upper atmosphere and transferred with the rain to the groundwater. Since no natural sources of 35S exist in the subsurface the decrease of the 35S activity concentration in such young groundwater can be used for the determination of its age. Still, 35S activities in precipitation (and hence even more in groundwater) are very low and necessitate appropriate analytical protocols based on liquid scintillation counting (LSC). This turns out to be challenging due to the required large sample volumes and due to potentially high SO42- loads of the samples, both limiting the range of possible applications of 35S as indicator for short groundwater residence times. In the paper we present an improved straightforward LSC based approach for the detection of 35S in natural water samples. We recommend using Insta-Gel Plus as scintillation cocktail for allowing a homogeneous suspension of 35S-containing BaSO4 in the cocktail. The recommended improvements in instrument setting concern the LSC (TriCarb 3170 Tr/SL) counting window, the pulse decay discriminator setting and the delay before burst setting. The settings allow measuring low activity concentrations of 35S, which was previously pre-concentrated from natural water samples, containing SO42- loads of up to 1500 mg with a reasonably high statistical reliability.

Stable isotopes reveal patterns of diet and mobility in the last Neandertals and first modern humans in Europe.

Correlating cultural, technological and ecological aspects of both Upper Pleistocene modern humans (UPMHs) and Neandertals provides a useful approach for achieving robust predictions about what makes us human. Here we present ecological information for a period of special relevance in human evolution, the time of replacement of Neandertals by modern humans during the Late Pleistocene in Europe. Using the stable isotopic approach, we shed light on aspects of diet and mobility of the late Neandertals and UPMHs from the cave sites of the Troisième caverne of Goyet and Spy in Belgium. We demonstrate that their diet was essentially similar, relying on the same terrestrial herbivores, whereas mobility strategies indicate considerable differences between Neandertal groups, as well as in comparison to UPMHs. Our results indicate that UPMHs exploited their environment to a greater extent than Neandertals and support the hypothesis that UPMHs had a substantial impact not only on the population dynamics of large mammals but also on the whole structure of the ecosystem since their initial arrival in Europe.

SDS-PAGE for 35S Immunoprecipitation and Immunoprecipitation Western Blotting.

This report discusses recent methods of sample preparation and gel electrophoresis for 35S immunoprecipitation (IP) and IP western blotting. In both methods, IP is used to obtain purified proteins, and sodium dodecyl sulfate polyacrylamide gel electrophoresis is used to separate the proteins on a gel. In 35S IP, the proteins are radiolabeled and visualized on film by fluorography; in IP blotting, proteins are transferred onto nitrocellulose paper, and antibodies are used to detect specific proteins. A similar IP and SDS-PAGE method can be used for both procedures, but IP blotting has the potential advantages of improvement in sensitivity for low-abundance proteins and enhanced specificity for identification of proteins from a mixture. Some of the technical adaptations discussed here to facilitate IP blotting and avoid loss of beads or purified proteins may also be useful for 35S IP.

DOSE EFFECT OF THE 33S(n,α) 30SI REACTION IN BNCT USING THE NEW n_TOF-CERN DATA.

33S is a stable isotope of sulphur which is being studied as a potential cooperative target for Boron Neutron Capture Therapy (BNCT) in accelerator-based neutron sources because of its large (n,α) cross section in the epithermal neutron energy range. Previous measurements resolved the resonances with a discrepant description of the lowest-lying and strongest one (at 13.5 keV). However, the evaluations of the major databases do not include resonances, except EAF-2010 which shows smaller values in this range than the experimental data. Furthermore, the glaring lack of data below 10 keV down to thermal (25.3 meV) has motivated a new measurement at n_TOF at CERN in order to cover the whole energy range. The inclusion of this new 33S(n,α) cross section in Monte Carlo simulations provides a more accurate estimation of the deposited kerma rate in tissue due to the presence of 33S. The results of those simulations represent the goal of this work.

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding.

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPγS), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTPγS binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the µ-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Sequential Immunoprecipitation of Secretory Vesicle Proteins from Biosynthetically Labelled Cells.

Pulse radiolabelling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labelled proteins can then be immunoprecipitated and analysed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labelling and immunoprecipitation of pancreatic islet proteins which are known to be affected in psychiatric disorders such as schizophrenia.

2D Gel Electrophoresis of Insulin Secretory Granule Proteins from Biosynthetically Labelled Pancreatic Islets.

Pulse radiolabelling of cells with radioactive amino acids such is a common method for investigating the biosynthetic rates of proteins. In this way, the abundance of newly synthesized proteins can be determined by several proteomic techniques including 2D gel electrophoresis (2DE). This chapter describes a protocol for labelling pancreatic islets with 35S-methionine in the presence of low and high concentrations of glucose, followed by subcellular fractionation enrichment of secretory granule proteins and analysis of the granule protein contents by 2DE. This demonstrated that the biosynthetic rates of most of the granule proteins are co-ordinately regulated in the presence of stimulatory glucose concentrations.

Improved efficacy by using the pTnT-rhtTG plasmid for the detection of celiac disease specific tissue transglutaminase autoantibodies in radioligand binding assays.

BACKGROUND: Tissue transglutaminase (tTG) autoantibodies are serological markers for celiac disease. The aim was to study the efficacy of the pTnT-rhtTG plasmid and subsequent diagnostic accuracy of tTG autoantibodies for childhood celiac disease using radioligand binding assays. METHODS: Coupled in vitro transcription and translation of tTG were performed by pTnT-rhtTG as well as by the pGEMt Easy-rhtTG vectors using the TNT SP6 Coupled Reticulocyte Lysate System in the presence of [³5S] methionine. Sera from 190 celiac disease children and 74 controls were measured for tTG autoantibodies in two separate radioligand binding assays using anti-human IgA agarose and protein A sepharose beads for the detection of IgA-tTG and IgG-tTG, respectively. RESULTS: Median incorporation of [³5S] methionine into the pTnT-rhtTG was 26% compared to 16% for the pGEMt Easy-rhtTG plasmid (p = 0.0016). Using pTnT-rhtTG (as compared to pGEMt Easy-rhtTG), sensitivities were IgA-tTG = 96.3% (95.7%) and IgG-tTG = 95.8% (97.3%) and specificities were IgA-tTG = 91.9% (90.5%) and IgG-tTG = 94.6% (98.4%). According to receiver operator characteristics for the pTnT (pGEMt Easy) assays, area under the curves were IgA-tTG = 98.4% (98.4%) and IgG-tTG = 97.7% (97.2%), respectively. CONCLUSION: The pTnT-rhtTG plasmid increased the efficacy of tTG antigen usage without reducing the diagnostic accuracy of IgA-tTG and IgG-tTG for childhood celiac disease. The pTnT-rhtTG plasmid is therefore recommended over the pGEMt Easy-rhtTG for the assessment of IgA-tTG and IgG-tTG using radioligand binding assays.

Evidence of neutron leakage at the Fukushima nuclear plant from measurements of radioactive 35S in California.

A recent earthquake and the subsequent tsunami have extensively damaged the Fukushima nuclear power plant, releasing harmful radiation into the environment. Despite the obvious implication for human health and the surrounding ecology, there are no quantitative estimates of the neutron flux leakage during the weeks following the earthquake. Here, using measurements of radioactive (35)S contained in sulfate aerosols and SO(2) gas at a coastal site in La Jolla, California, we show that nearly 4 × 10(11) neutrons per m(2) leaked at the Fukushima nuclear power plant before March 20, 2011. A significantly higher (35)SO(2-)(4) activity as measured on March 28 is in accord with neutrons escaping the reactor core and being absorbed by the coolant seawater (35)Cl to produce (35)S by a (n, p) reaction. Once produced, (35)S oxidizes to (35)SO(2) and (35)SO(2-)(4) and was then transported to Southern California due to the presence of strong prevailing westerly winds at this time. Based on a moving box model, we show that the observed activity enhancement in (35)SO(2-)(4) is compatible with long-range transport of the radiation plume from Fukushima. Our model predicts that (35)SO(2-)(4), the concentration in the marine boundary layer at Fukushima, was approximately 2 × 10(5) atoms per m(3), which is approximately 365 times above expected natural concentrations. These measurements and model calculations imply that approximately 0.7% of the total radioactive sulfate present at the marine boundary layer at Fukushima reached Southern California as a result of the trans-Pacific transport.

Validation of efficiency tracing and zero detection threshold techniques using liquid scintillation analyser TriCarb.

Efficiency tracing with unquenched 14C and zero detection threshold with unquenched 3H as tracers are practical and simple techniques which have been implemented to quantify the activity of various beta emitters using liquid scintillation analyser. These techniques are used to study the influence of quench level on activity quantification and the activity levels up to which these techniques are applicable. The results indicate that, for an activity level of 166.67 Bq, both the techniques are in good agreement with the reference activity with a relative discrepancy of ≤4.6 %. The relative discrepancy of ~10 % is observed for extreme quench values of ~111. For all the radionuclides with the activity level of 1.67 Bq, the uncertainty in activity quantification raises to ~8 % and for the activity level from 8.33 to 100 Bq, the uncertainty reduces to 1 %.

33S nuclear magnetic resonance spectroscopy of biological samples obtained with a laboratory model 33S cryogenic probe.

(33)S nuclear magnetic resonance (NMR) spectroscopy is limited by inherently low NMR sensitivity because of the quadrupolar moment and low gyromagnetic ratio of the (33)S nucleus. We have developed a 10 mm (33)S cryogenic NMR probe, which is operated at 9-26 K with a cold preamplifier and a cold rf switch operated at 60 K. The (33)S NMR sensitivity of the cryogenic probe is as large as 9.8 times that of a conventional 5 mm broadband NMR probe. The (33)S cryogenic probe was applied to biological samples such as human urine, bile, chondroitin sulfate, and scallop tissue. We demonstrated that the system can detect and determine sulfur compounds having SO(4)(2-) anions and -SO(3)(-) groups using the (33)S cryogenic probe, as the (33)S nuclei in these groups are in highly symmetric environments. The NMR signals for other common sulfur compounds such as cysteine are still undetectable by the (33)S cryogenic probe, as the (33)S nuclei in these compounds are in asymmetric environments. If we shorten the rf pulse width or decrease the rf coil diameter, we should be able to detect the NMR signals for these compounds.

Sulphur isotopes and the search for life: strategies for identifying sulphur metabolisms in the rock record and beyond.

The search for life can only be as successful as our understanding of the tools we use to search for it. Here we present new sulphur isotope data (32S, 33S, 34S, 36S) from a variety of modern marine environments and use these observations, along with previously published work, to contribute to this search. Specifically, we use these new data to gain a sense of life's influences on the sulphur isotope record and to distinguish these biologically influenced signatures from their non-biological counterparts. This treatment extends sulphur isotope analyses beyond traditional (34S/32S) measures and employs trace isotope relationships (33S/32S, 36S/32S), as the inclusion of these isotopes provides unique information about biology and its role in the sulphur cycle through time. In the current study we compare and contrast isotope effects produced by sulphur-utilizing microorganisms (experimental), modern and ancient sedimentary records (observational) and non-biological reactions (theoretical). With our collective search for life now extending to neighbouring planets, we present this study as a first step towards more fully understanding the capability of the sulphur isotope system as a viable tool for life detection, both on Earth and beyond.

Glucuronidation and sulfonation, in vitro, of the major endocrine-active metabolites of methoxychlor in the channel catfish, Ictalurus punctatus, and induction following treatment with 3-methylcholanthrene.

The organochlorine pesticide, methoxychlor (MXC), is metabolized in animals to phenolic mono- and bis-demethylated metabolites (OH-MXC and HPTE, respectively) that interact with estrogen receptors and may be endocrine disruptors. The phase II detoxication of these compounds will influence the duration of action of the estrogenic metabolites, but has not been investigated extensively. In this study, the glucuronidation and sulfonation of OH-MXC and HPTE were investigated in subcellular fractions of liver and intestine from untreated, MXC-treated and 3-methylcholanthrene (3-MC)-treated channel catfish, Ictalurus punctatus. MXC-treated fish were given i.p. injections of 2mg MXC/kg daily for 6 days and sacrificed 24h after the last dose. The 3-MC treatment was a single 10mg/kg i.p. dose 5 days prior to sacrifice. In hepatic microsomes from control fish, the V(max) value (mean+/-S.D., n=4) for glucuronidation of OH-MXC was 270+/-50pmol/min/mg protein, higher than found for HPTE (110+/-20pmol/min/mg protein). For each substrate, the V(max) values observed in intestinal microsomes were approximately twice those found in the liver. The K(m) values for OH-MXC and HPTE glucuronidation in control liver were not significantly different and were 0.32+/-0.04mM for OH-MXC and 0.26+/-0.06mM for HPTE. The K(m) for the co-substrate, UDPGA, was higher in liver (0.28+/-0.09mM) than intestine (0.04+/-0.02mM). Treatment with 3-MC but not MXC increased the V(max) for glucuronidation in liver and intestine. Glucuronidation was a more efficient pathway than sulfonation for both substrates, in both tissues. The V(max) values for sulfonation of OH-MXC and HPTE, respectively, in liver cytosol were 7+/-3 and 17+/-4pmol/min/mg protein and in intestinal cytosol were 13+/-3 and 30+/-5pmol/min/mg protein. Treatment with 3-MC but not MXC increased rates of sulfonation of OH-MXC and HPTE and the model substrate, 3-hydroxy-benzo(a)pyrene in both intestine and liver. Comparison of the kinetics of the conjugation pathways with those published for the demethylation of MXC showed that formation of the endocrine-active metabolites was more efficient than either conjugation pathway. Residues of OH-MXC and HPTE were detected in extracts of liver microsomes from MXC-treated fish. This work showed that although OH-MXC and HPTE could be eliminated by glucuronidation and sulfonation, the phase II pathways were less efficient than the phase I pathway leading to formation of these endocrine-active metabolites.

Hypoxia and regulation of messenger RNA translation.

Poor oxygenation (hypoxia) influences important physiological and pathological conditions, including development, ischemia, stroke, and cancer. The influence of hypoxia is due in large part to changes in gene expression, which occur through changes in transcription and translation. In response to hypoxic conditions, cells reduce their overall rate of messenger RNA (mRNA) translation. However, individual mRNA species are effected to highly varying degrees, with some even translationally stimulated under these conditions. Regulation of translation in response to hypoxia thereby gives rise to differential gene expression. The ability of cells to regulate translation during hypoxia is important for their survival. In the first part of this chapter, we review the effects of hypoxia on overall and gene-specific mRNA translation efficiencies and summarize the molecular pathways activated by hypoxia that regulate mRNA translation. In the second part, we describe the methods employed to investigate overall and gene-specific translation, including radioactive metabolic labeling, polysome fractionation, and reporter assays. We have emphasized the qualitative and quantitative analysis of polysome profiles, which can yield interesting information regarding the mechanistic basis for (gene-specific) translational regulation.

Measurements of short-lived cosmic-ray-produced radionuclides in rainwater.

Cosmic-ray-produced (CP) nuclides with half-lives shorter than 21h were measured in rainwater by ultra-low-background gamma spectrometry at Ogoya Underground Laboratory. As levels of CP nuclides are extremely low and their half-lives are very short, quick sampling methods for a large volume of rainwater and rapid chemical separations by ion exchange method were developed. The nuclides measured were short-lived (24)Na, (28)Mg, (38)S, (38)Cl, (39)Cl, as well as nuclides with longer half-lives (7)Be and (22)Na. The number of atoms of CP nuclides in rainwater were evaluated to range from 30 to 1500L(-1) for (24)Na (n=16, mean; 520 [6.7mBqL(-1)]), 80 to 600L(-1) for (28)Mg (n=13, mean; 260 [2.4mBqL(-1)]), 400 to 1900L(-1) for (39)Cl (n=6, mean; 1200 [250mBqL(-1)]), 1x10(6) to 4x10(7)L(-1) for (7)Be (n=16, mean; 7x10(6) [1.05BqL(-1)]) and 2x10(3) to 1x10(5)L(-1) for (22)Na (n=9, mean; 2x10(4) [0.2mBqL(-1)]).

Effects of sodium hyaluronate and methylprednisolone acetate on proteoglycan metabolism in equine articular chondrocytes treated with interleukin-1.

OBJECTIVE: To determine the effects of sodium hyaluronate (HA) in combination with methylprednisolone acetate (MPA) on interleukin-1 (IL-1)-induced inflammation in equine articular cartilage pellets. SAMPLE POPULATION: Chondrocytes collected from 7 horses euthanatized for problems unrelated to the musculoskeletal system. PROCEDURES: Chondrocyte pellets were treated with medium (negative control); medium containing IL-1 (positive control); or medium containing IL-1 with MPA only (0.05 or 0.5 mg/mL), HA only (0.2 or 2 mg/mL), or MPA (0.05 or 0.5 mg/mL) and HA (0.2 or 2 mg/mL) in combination. Proteoglycan (PG) synthesis was determined by incorporation of sulfur 35-labeled sodium sulfate into PGs. Glycosaminoglycan (GAG) content of the media and the pellets and total pellet DNA content were determined. RESULTS: Methylprednisolone acetate at 0.5 mg/mL caused an increase in PG synthesis, whereas HA had no effect alone. The combination of MPA, both 0.05 mg/mL and 0.5 mg/mL, with HA at 2 mg/mL increased PG synthesis, compared with IL-1-treated control. All treatment groups containing the high concentration of MPA (0.5 mg/mL) and the high concentration of HA (2.0 mg/mL) had pellets with increased GAG content. The addition of HA caused an increase in total GAG content in the media, regardless of MPA treatment. Cyclooxygenase-2 mRNA and aggrecan mRNA expression was significantly reduced with MPA treatment. Total pellet DNA content was unchanged by any treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that MPA in combination with HA has beneficial effects on PG metabolism of IL-1-treated equine chondrocytes.

Electrophoretic mobility shift assays to study protein binding to damaged DNA.

The electrophoretic mobility shift assay (EMSA) can be used to identify proteins that bind specifically to damaged DNA. EMSAs detect the presence of key DNA repair proteins, such as ultraviolet (UV)-damaged DNA binding protein, which is involved in nucleotide excision repair, and Ku and DNA-PKcs, which are involved in double-strand break repair. This chapter describes EMSA protocols for detecting proteins that bind to UV-damaged DNA, cisplatin-damaged DNA, and DNA ends. The chapter also describes variations of the EMSA that can be used to obtain additional information about these important proteins. The variations include the reverse EMSA, which can detect binding of 35S-labeled protein to damaged DNA, and the antibody supershift assay, which can define the composition of protein-DNA complexes.

Measurement of specific radioactivity in proteins separated by two-dimensional gel electrophoresis.

We report a method to quantify the specific radioactivity of proteins that have been separated by 2-DE. Gels are stained with SyproRuby, and protein spots are excised. The SyproRuby dye is extracted from each spot using DMSO, and the fluorescence is quantified automatically using a plate reader. The extracted gel piece is then dissolved in hydrogen peroxide and radioactivity is quantified by liquid scintillation counting. Gentle agitation with DMSO for 24 h was found to extract all the SyproRuby dye from gel fragments. The fluorescence of the extract was linearly related to the amount of BSA loaded onto a series of 1-D gels. When rat muscle samples were run on 2-DE gels, the fluorescence extracted from 54 protein spots showed a good correlation (r = 0.79, p < 0.001) with the corresponding spot intensity measured by conventional scanning and image analysis. DMSO extraction was found not to affect the amount of radioactive protein left in the gel. When a series of BSA solutions of known specific radioactivity were run on 2-DE gels, the specific radioactivity measured by the new method showed a good correlation (r = 0.98, p < 0.01, n = 5) with the specific radioactivity measured directly before loading. Reproducibility of the method was measured in a series of 2-DE gels containing proteins from the livers of rats and mice that had been injected with [35S]methionine. Variability tended to increase when the amount of radioactivity in the protein spot was low, but for samples containing at least 10 dpm above background the CV was around 30%, which is comparable to that obtained when measuring protein expression by conventional image analysis of SyproRuby-stained 2-DE gels. Similar results were obtained whether spots were excised manually or using a spot excision robot. This method offers a high-throughput, cost-effective and reliable method of quantifying the specific radioactivity of proteins from metabolic labelling experiments carried out in vivo, so long as sufficient quantities of radioactive tracer are used.

Modelling the fate of sulphur-35 in crops. 2. Development and validation of the CROPS-35 model.

Gas-cooled nuclear power plants in the UK release sulphur-35 during their routine operation, which can be readily assimilated by vegetation. It is therefore necessary to be able to model the uptake of such releases in order to quantify any potential contamination of the food chain. A model is described which predicts the concentration of (35)S in crop components following an aerial gaseous release. Following deposition the allocation to crop components is determined by an export function from a labile pool, the leaves, to those components growing most actively post exposure. The growth rates are determined by crop growth data, which is also used to determine the concentration. The loss of activity is controlled by radioactive decay only. The paper describes the calibration and the validation of the model. To improve the model, further experimental work is required particularly on the export kinetics of (35)S. It may be possible to adapt such a modelling approach to the prediction of crop content for gaseous releases of (3)H and (14)C from nuclear facilities.

Analysis of triple-label samples by liquid scintillation spectrometry.

The simultaneous analysis of three radionuclides present in a sample is demonstrated. The approach requires fitting a series of counting data acquired over a period of time to a triple exponential mathematical expression. The use and advantages of the method are illustrated for the case of the standardisation of 32P in the presence of 33P and 35S by 4pi-beta liquid scintillation spectrometry. The effects of sample preparation, chemical quenching and different activity ratios were studied.