Copepod nauplii use phosphorus from bacteria, creating a short circuit in the microbial loop.

In subtropical oceans phytoplankton carbon: phosphorus (C : P) ratios are high, and these ratios are predicted to increase further with rising ocean temperatures and stratification. Prey stoichiometry may pose a problem for copepod zooplankton nauplii, which have high phosphorus demands due to rapid growth. We hypothesised that nauplii meet this demand by consuming bacteria. Naupliar bacterial and phytoplankton carbon and phosphorus ingestion, assimilation and incorporation were traced using 33 P and 14 C radioisotopes. Bacterial carbon was incorporated four times less efficiently into biomass than phytoplankton carbon. In contrast, bacterial and phytoplankton phosphorus were incorporated at similar efficiencies, and bacteria could meet a substantial amount of naupliar phosphorus requirements. As parts of the ocean become more oligotrophic, bacteria could help sustain naupliar growth and survival under suboptimal stoichiometric conditions. Thus, nauplii may be a shortcut for phosphorus from the microbial loop to the classical food web.

Quantitative imaging of 33P in plant materials using 14C polymer references.

Phosphorus (P) research still lacks techniques for rapid imaging of P use and allocation in different soil, sediment, and biological systems in a quantitative manner. In this study, we describe a time-saving and cost-efficient digital autoradiographic method for in situ quantitative imaging of 33P radioisotopes in plant materials. Our method combines autoradiography of the radiotracer applications with additions of commercially available 14C polymer references to obtain 33P activities in a quantitative manner up to 2000 Bq cm-2. Our data show that linear standard regressions for both radioisotopes are obtained, allowing the establishment of photostimulated luminescence equivalence between both radioisotopes with a factor of 9.73. Validating experiments revealed a good agreement between the calculated and applied 33P activity (R2 = 0.96). This finding was also valid for the co-exposure of 14C polymer references and 33P radioisotope specific activities in excised plant leaves for both maize (R2 = 0.99) and wheat (R2 = 0.99). The outlined autoradiographic quantification procedure retrieved 100% ± 12% of the 33P activity in the plant leaves, irrespective of plant tissue density. The simplicity of this methodology opens up new perspectives for fast quantitative imaging of 33P in biological systems and likely, thus, also for other environmental compartments.

Impact of raking and bioturbation-mediated ecological manipulation on sediment-water phosphorus diagenesis: a mesocosm study supported with radioactive signature.

The study examined the impact of raking and fish bioturbation on modulating phosphorus (P) concentrations in the water and sediment under different trophic conditions. An outdoor experiment was set to monitor physicochemical and microbiological parameters of water and sediment influencing P diagenesis. A pilot study with radioactive 32P was also performed under the agency of raking and bacteria (Bacillus sp.). Raking was more effective in release of P under unfertilized conditions by significantly enhancing orthophosphate (35%) and soluble reactive phosphate (31.8%) over respective controls. Bioturbation increased total and available P in sediments significantly as compared to control. The rates of increase were higher in the unfertilized conditions (17.6-28.4% for total P and 12.2 to 23.2% for available P) than the fertilized ones (6.5-12.4% for total P and 9.1 to 15% for available P). The combined effects of raking and bioturbation on orthophosphate and soluble reactive phosphate were also stronger under unfertilized state (54.5 and 81.8%) than fertilized ones (50 and 70%). The tracer signature showed that coupled action of introduced bacteria and repeated raking resulted in 59.2, 23 and 16% higher counts of radioactive P than the treatments receiving raking once, repeated raking and bacteria inoculation, respectively. Raking alone or in sync with bioturbation exerted pronounced impact on P diagenesis through induction of coupled mineralization and nutrient release. It has significant implication for performing regular raking of fish-farm sediments and manipulation of bottom-grazing fish to regulate mineralization of organic matter and release of obnoxious gases from the system. Further, they synergistically can enhance the buffering capacity against organic overload and help to maintain aquatic ecosystem health.

Hair 32P measurement for body dose mapping in non-fatal exposures to fast neutrons.

Dosimetry bioassay methods are the backbone of a personal dosimetry in criticality accidents. Although methods like hair dosimetry and the use of activation foils (e.g., (32)S) have been employed for decades, capabilities of different techniques, effects of hair type and neutron spectrum on the dose response, sensitivity and uncertainties of different techniques, etc., need more investigations. For this reason, the use of the (32)S(n,p)(32)P reaction and hair samples for estimating non-fatal doses from fast neutrons was studied. The experiments were carried out with the hair samples attached on a RANDO phantom in a Cf-252 neutron field, in the dose range of about 0.05-1.15 Gy. In addition, the adequate post-accident preparation for hair samples including optimum conditioning and timing were investigated. Experimental results prove the good sensitivity and merit of the method for neutron quantification in the mentioned dose range for which other bioassay methods are of poor resolution and sensitivity. A rough estimation of the dose-response curve for Iranian hair was also derived.

A microautoradiographic method for fresh-frozen sections to reveal the distribution of radionuclides at the cellular level in plants.

Microautoradiography (MAR) is a conventional imaging method based on the daguerreotype. The technique is used to visualize the distribution of radionuclide-labeled compounds within a tissue section. However, application of the classical MAR method to plant tissue sections is associated with several difficulties. In this study, we report an MAR method applicable to fresh-frozen plant sections. Our method had two features: (i) the sample was kept frozen from plant tissue collection to radioisotope detection, making it possible to fix solutes without solvent exchange; and (ii) 1.2 µm thick polyphenylene sulfide film was inserted between the fresh-frozen plant section and the photosensitive nuclear emulsion to separate the section from the emulsion before autoradiography was conducted, which significantly improved the quality of the section until microscopic detection, the quality of the MAR image and the success rate. Then, the passage of cadmium (Cd) through vegetative rice stem tissue after 24 h of (109)Cd absorption was described for the first time using the MAR method. MAR clearly revealed the distribution of (109)Cd at the tissue level with high resolution. The (109)Cd concentration in phloem cells was found to be particularly high, whereas the xylem cells contained only small amounts of (109)Cd. The MAR method was also applicable for detecting (109)Cd and [(33)P]phosphate in roots. The MAR method developed here is expected to provide distribution images for a variety of compounds and ions in plant tissue.

Measure post-bloodmeal dispersal of mosquitoes and duration of radioactivity by using the isotope ³²P.

The radioactive isotope (32)P-labeled disodium phosphate (Na2H(32)PO4) was injected via the jugular vein into a cow kept in a shed in Maozhuang Village, Cao Township of Shanxian County, China. Over the following 5 d, mosquitoes feeding on the cow were captured at distances up to 400 m to determine dispersal distance. The duration of radioactivity in the cow and marked mosquitoes was 10 d. The results showed that after blood feeding, Anopheles sinensis and Culex tritaeniorhynchus temporarily rested in the cattle shed and then flew outdoors. In contrast, Culex pipiens pallens remained in the cattle shed after feeding. These findings confirmed that local An. sinensis and Cx. tritaeniorhynchus were partially endophilic and tended to rest out of doors, whereas Cx. pipiens pallens was endophilic. For marked An. sinensis and Cx. tritaeniorhynchus, there was a significant tendency for dispersal to be in a northeast and east direction, probably because of the presence of heavy shading by an agricultural field, a small river for mosquito oviposition sites, and locations downwind from the blood source. The furthest flight distances for An. sinensis and Cx. tritaeniorhynchus were 210 and 240 m; therefore, control of these mosquitoes should include resting places indoors and outdoors within a radius of 250 m from confirmed cases.

(32)P measurment of urine samples and internal dose assessment for radiation workers in life science laboratories.

(32)P measurements of urine samples and internal dose assessments were conducted for workers in life science laboratories. A procedure for sample pre-treatment was established and validation was performed to exclude interference and to detect (32)P levels accurately. The detection conditions for Cherenkov radiation were evaluated and the accuracy of Cherenkov radiation measurements validated. The analytical and measurement procedures were applied to urine samples collected from 11 workers from life sciences laboratories. The results of the measurements generally indicated very low background radiation levels, but daily urine samples from two workers were above the minimum detectable activity. The (32)P concentrations for two of the workers were 29.3 ± 10.4 Bq•d(-1) and 24.1 ± 11.8 Bq•d(-1), respectively, at intake levels of 4.12 kBq and 2.61 kBq. The effective doses for these two workers were 4.6 µSv and 2.9 µSv. Overall, the results indicate very low levels of radioactivity, except for cases related to specific working conditions.

Direct and indirect influences of arbuscular mycorrhizal fungi on phosphorus uptake by two root hemiparasitic Pedicularis species: do the fungal partners matter at low colonization levels?

BACKGROUND AND AIMS: Because most parasitic plants do not form mycorrhizal associations, the nutritional roles of arbuscular mycorrhizal (AM) fungi in them have hardly been tested. Some facultative root hemiparasitic Pedicularis species form AM associations and hence are ideal for testing both direct and indirect effects of AM fungi on their nutrient acquisition. The aim of this study was to test the influence of AM inoculation on phosphorus (P) uptake by Pedicularis rex and P. tricolor. METHODS: (32)P labelling was used in compartmented pots to assess the contribution of the AM pathway and the influence of AM inoculation on P uptake from a host plant into the root hemiparasites. Laboratory isolates of fungal species (Glomus mosseae and G. intraradices) and the host species (Hordeum vulgare 'Fleet') to which the two Pedicularis species showed obvious responses in haustorium formation and growth in previous studies were used. KEY RESULTS: The AM colonization of both Pedicularis spp. was low (<15 % root length) and only a very small proportion of total plant P (<1 %) was delivered from the soil via the AM fungus. In a separate experiment, inoculation with AM fungi strongly interfered with P acquisition by both Pedicularis species from their host barley, almost certainly because the numbers of haustoria formed by the parasite were significantly reduced in AM plants. CONCLUSIONS: Roles of AM fungi in nutrient acquisition by root parasitic plants were quantitatively demonstrated for the first time. Evidence was obtained for a novel mechanism of preventing root parasitic plants from overexploiting host resources through AM fungal-induced suppression of the absorptive structures in the parasites.

Analysis of yeast protein kinases using protein chips.

We have developed a novel protein chip technology that allows the high-throughput analysis of biochemical activities, and used this approach to analyse nearly all of the protein kinases from Saccharomyces cerevisiae. Protein chips are disposable arrays of microwells in silicone elastomer sheets placed on top of microscope slides. The high density and small size of the wells allows for high-throughput batch processing and simultaneous analysis of many individual samples. Only small amounts of protein are required. Of 122 known and predicted yeast protein kinases, 119 were overexpressed and analysed using 17 different substrates and protein chips. We found many novel activities and that a large number of protein kinases are capable of phosphorylating tyrosine. The tyrosine phosphorylating enzymes often share common amino acid residues that lie near the catalytic region. Thus, our study identified a number of novel features of protein kinases and demonstrates that protein chip technology is useful for high-throughput screening of protein biochemical activity.

Sustained overexpression of CYP1A1 and 1B1 and steady accumulation of DNA adducts by low-dose, continuous exposure to benzo[a]pyrene by polymeric implants.

Many carcinogenesis and tumorigenesis studies reported in the past several decades have relied upon bolus dose(s) of test compounds to determine their DNA damage and carcinogenic potential. The high doses are far from the human scenario where exposure is almost always to low doses and for long duration. In this study, we report a novel polymeric implant system that provides continuous ("24/7") exposure to low doses using benzo[a]pyrene (BP) as a model carcinogen. Cylindrical implants (1 cm length, 3.2 mm diameter; 10 mg BP/100 mg implant) prepared from polycaprolactone:F68 (9:1) showed controlled release in vitro for long duration. To determine the rate of release and biochemical effects in vivo, groups of female Sprague-Dawley rats received either no treatment or subcutaneous sham or BP implants (1 cm, 10% load) and were euthanized after 6, 15, 30, and 180 days; the average dose of BP by the implant route was 16.7 ± 3 µg/rat. For comparison, rats were also treated with a single bolus dose of BP intraperitoneally (10 mg/rat) and euthanized at 6, 15, and 30 days. DNA adducts analyzed by (32)P-postlabeling in the lung and liver increased steadily with time with levels reaching 31 ± 3 and 17 ± 6 adducts/10(9) nucleotides, respectively, after 25 weeks; the adduct burden in the mammary tissue initially increased but then declined with time presumably due to high cell turn over. In contrast, the bolus dose treatment showed the highest DNA adduct levels after 6 days, followed by a steady decline. The steady accumulation of tissue DNA adducts in the implant groups corroborates the sustained overexpression of CYP1A1 and 1B1, the cytochrome P450s involved in the conversion of BP to its electrophilic metabolites. In contrast, the overexpression of CYP1A1 and 1B1 resulting from the bolus dose of BP lasted only for a few days. This is the first demonstration revealing that low-dose, continuous exposure to environmental polycyclic aromatic hydrocarbons such as BP can render sustained expression of CYPs and steady accumulation of tissue DNA adducts. On the basis of our recent study in which we showed the presence of 17ß-estradiol in the lung, the sustained overexpression of CYP1A1 and 1B1 due to continuous exposure to BP may increase the susceptibility to estrogen-mediated carcinogenicity.

Pharmacology of the phosphate binder, lanthanum carbonate.

Studies were conducted to compare the phosphate-binding efficacy of lanthanum carbonate directly with other clinically used phosphate binders and to evaluate any potential adverse pharmacology. To examine the phosphate-binding efficacy, rats with normal renal function and chronic renal failure received lanthanum carbonate, aluminum hydroxide, calcium carbonate, or sevelamer hydrochloride in several experimental models. Lanthanum carbonate and aluminum hydroxide markedly increased excretion of [(32)P]-phosphate in feces and reduced excretion in urine in rats with normal renal function (p < 0.05), indicating good dietary phosphate-binding efficacy. In rats with chronic renal failure, lanthanum carbonate and aluminum hydroxide reduced urinary phosphate excretion to a greater degree and more rapidly than calcium carbonate, which in turn was more effective than sevelamer hydrochloride. The potential to induce adverse pharmacological effects was assessed systematically in mice, rats, and dogs with normal renal function using standard in vivo models. There was no evidence of any adverse secondary pharmacological effects of lanthanum carbonate on the central nervous, cardiovascular, respiratory, or gastrointestinal systems. These studies indicate that lanthanum carbonate is the more potent of the currently available dietary phosphate binders. No adverse secondary pharmacological actions were observed in vivo in a systematic evaluation at high doses.

Abundance and diversity of heterotrophic bacterial cells assimilating phosphate in the subtropical North Atlantic Ocean.

Microorganisms play key roles in the cycles of carbon and nutrients in the ocean, and identifying the extent to which specific taxa contribute to these cycles will establish their ecological function. We examined the use of (33)P-phosphate to identify heterotrophic bacteria actively involved in the cycling of phosphate, an essential inorganic nutrient. Seawater from the sub-tropical North Atlantic Ocean was incubated with (33)P-phosphate and analysed by microautoradiography to determine the proportion and diversity of the bacterial community-assimilating phosphate. Complementary incubations using (3)H-leucine and (3)H-thymidine were also conducted. We found that a higher proportion of total heterotrophic bacterial cells in surface water samples assimilated phosphate compared with leucine or thymidine. Bacteria from all of the phylogenetic groups we identified by CARD-FISH were able to assimilate phosphate, although the abundances of cells within each group did not scale directly with the number found to assimilate phosphate. Furthermore, a significantly higher proportion of Alphaproteobacteria, Gammaproteobacteria and Cytophaga-like cells assimilated phosphate compared with leucine or thymidine. Our results suggest that a greater proportion of bacterial cells in surface waters are actively participating in the biogeochemical cycling of phosphorus, and possibly other elements, than is currently estimated through the use of (3)H-leucine or (3)H-thymidine.

Dose-rate distribution of 32P-glass microspheres for intra-arterial brachytherapy.

PURPOSE: The intra-arterial administration of radioactive glass microspheres is an alternative therapy option for treating primary hepatocellular carcinoma, the main cause of liver cancer death, and metastatic liver cancer, another important kind of cancer induced in the liver. The technique involves the administration of radioactive microspheres in the hepatic artery, which are trapped preferentially in the tumor. METHODS: In this work the GEANT4 toolkit was used to calculate the radial dose-rate distributions in water from 32P-loaded glass microspheres and also from 90Y-loaded glass microspheres. To validate the toolkit for this application, the authors compared the dose-rate distribution of 32P and 90Y point sources in water with data from the International Commission on Radiation Units and Measurements report 72. RESULTS: Tables of radial dose-rate distributions are provided for practical use in brachytherapy planning with these microspheres. CONCLUSIONS: The simulations with the microspheres show that the shape of the beta ray energy spectra with respect to the 32P and 90Y sources is significantly modified by the glass matrix.

The role of ionic release from NovaMin (calcium sodium phosphosilicate) in tubule occlusion: an exploratory in vitro study using radio-labeled isotopes.

OBJECTIVE: The primary objective of this work was to develop a method of quantifying the levels and source of calcium and phosphate deposited on dental hard tissue from a novel calcium phosphosilicate (NovaMin) material using neutron activation analysis (NAA). A second objective was to explore the utility of radiotracing to determine dentin porosity following exposure to calcium phosphosilicate. METHODS: Neutron activation was used to create isotopes of Ca and P in the calcium phosphosilicate particles. Gamma radiation emitted from these isotopes was used to identify and measure their uptake (concentration) onto dental hard tissue. Three experiments were conducted to explore calcium and phosphate uptake to dental hard tissue: 1) a dose response to quantify the relative levels of calcium and phosphate deposited on dental hard tissue as a function of calcium phosphosilicate dose; 2) the effect of calcium phosphosilicate particle size on the relative levels of calcium and phosphate uptake; and 3) the permeability of calcium phosphosilicate-treated dentin by employing the radiotracer technetium. For all experiments, extracted bovine incisors were employed as the test substrate. RESULTS: The results indicate there is a strong dose relationship between the wt% and particle size of calcium phosphosilicate in the dentifrice formulation and new Ca and P deposition. At above 5.0 wt% calcium phosphosilicate, there appears to be an exponential increase in the number of counts from the tooth surface. Finer particle size calcium phosphosilicate appears to deposit much higher levels of Ca and P than the larger range of particle sizes. The results from the technetium study show that when treated with the dentifrice slurry containing calcium phosphosilicate, dentin shows only a slight amount of technetium infiltration, indicating a lowering of dentin permeability. CONCLUSION: This exploratory study has demonstrated that NAA and the use of radio isotopes have utility in monitoring the uptake of Ca and P into both dentin and enamel tooth structure. The data generated from these studies have shown that there is a dose dependence and particle size effect for calcium phosphosilicate on the deposition of calcium and phosphate to dental hard tissue.

Autoradiography and Phosphorimaging.

Many of the commonly used techniques in molecular cloning depend on methods to map accurately the distribution of radioactive atoms on two-dimensional (2D) surfaces. Without this ability, methods such as Southern blotting, northern hybridizations, radiolabeled DNA sequencing, and library screening would not have been possible. In the 1970s and 1980s-the pioneering days of molecular cloning-imaging of 2D surfaces was obtained using autoradiography. In this technique, ß-particles emitted by radioactive specimens were recorded on X-ray film, producing a latent image that can be converted to a true image by developing and fixing the film. Autoradiography was a lot of fun, but it was also messy. In the impatient excitement of wanting to see how an experiment had turned out, people used to hold the newly developed X-ray films in their metal frames up to the darkroom light. Drips of the final wash would run down their arms, clothes would be stained, and shoes ruined. It is hardly surprising that autoradiography was quickly abandoned when sensitive phosphorimagers came onto the market at the end of the 1990s.

NEUTRON DOSE ASSESSMENT USING SAMPLES OF HUMAN BLOOD AND HAIR.

The unique feature of nuclear accidents with neutron exposure is the induced radioactivity in body tissues. For dosimetry purposes, the most important stable isotopes occurring in human body, which can be activated by neutrons, are 23 Na and 32 S. The respective activation reactions are as follows:23Na(n,γ)24Na and32S(n,p)32P. While sodium occurs in human blood, sulfur is present in human hair. In order to verify the practical feasibility of this dosimetry technique in conditions of our laboratory, samples of human blood and hair were irradiated in a channel of a training reactor VR-1.24Na activity was measured by gamma-ray spectrometry.32P activity in hair was measured by means of a proportional counter. Based on neutron-spectrum calculation, relationships between neutron dose and induced activity were derived for both blood and hair.

NEUTRON FIELD MEASUREMENT OF P(35)+Be SOURCE USING THE MULTI-FOIL ACTIVATION METHOD.

Neutron field from the p+Be interaction was investigated at the NPI CAS for a proton beam energy of 35 MeV and thick beryllium target. Broad neutron spectra at close source-to-sample distances were determined using the multi-foil activation technique. Two large sets of dosimetry foils containing the Ni, Co, Au, In, Ti, Al, Y, Lu, Nb and Fe were irradiated at a distance of 74 mm at direct neutron beam axis and at a distance of 34 mm from beam axis. Supporting Monte-Carlo MCNPX calculations of the irradiation system were performed as well. From measured reaction rates, the neutron energy spectra at both positions were reconstructed employing the modified version of the SAND-II unfolding code and activation cross-section data from the EAF-2010 library. At the position of irradiated samples, the total fast neutron flux reaches the value up to 1010 cm-2 s-1, and the neutron field is utilizable for radiation hardness study and integral benchmark experiments within the International Fusion Material Irradiation Facility (IFMIF) program.

Verification of Safety Compliance of Delivering Radionuclides at Vanderbilt University.

Various radionuclides are transported at Vanderbilt University and Vanderbilt University Medical Center on a daily basis, to provide the necessities for diagnostic, therapeutic, and research applications. The delivery of the radionuclides takes various pathways where the general public may receive radiation doses. The Tennessee Department of Environment and Health Radiological Division regulates the dose limits for members of the public to be less than 1 mSv per year and 20 µSv in any hour. We designed a project to verify that potential doses received by the general public meet state regulations. Before the departure of the delivery, dose rates from three directions at a distance of 30 cm with respect to the transport vehicle, were measured using a tissue equivalent survey meter. During the shipment, times were recorded and the number of persons encountered along the path was estimated. Annual and hourly doses were calculated, conservatively assuming that a member of general public would follow the shipment at a distance of 30 cm, for the entire duration of the delivery. Calculated dose rates for each delivery and various combinations of radionuclides were found to be below state regulation limits.

Assaying Protein Kinase Activity with Radiolabeled ATP.

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [γ-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Non-radioisotope detection of pol sequences of HTLV-1 proviral DNA: Standardisation and sensitivity analysis.

Proviral DNA amplification methods may be used for identification of HTLV-1 infection or in basic virology research. Published standardised methods in this regard usually depend on hybridisation of PCR products with radioisotope-labelled probes. However, this procedure has limited use in routine testing, due to environmental and health risks. The aim was to assess the feasibility of routine use and the accuracy of an alternative detection system that employs an HTLV-1-specific enzyme-labelled probe. For this purpose DNA was extracted from MT-2 cells, quantified and submitted to serial dilution (1:10), starting from 1.2 microg of genomic DNA. Primary and nested PCR amplifications of pol sequences of the HTLV-1 genome were carried out with standardised primers (SK110/111 and POL1.1/3.1). After Southern blotting, two different detection systems were compared, consisting of hybridisation with either 32P- or alkaline phosphatase-labelled SK112 probes. Both detection systems yielded similar results, detecting PCR products generated from 120 pg of DNA (genomic DNA amount equivalent to 20 diploid human cells) after primary and nested PCR. The alkaline phosphatase-labelled detection technique was feasible for the diagnosis of HTLV-1 with the advantage of precluding the handling of radioisotopes.