Reconstruction and forecast of doses due to ingestion of <Superscript>137</Superscript>Cs and <Superscript>90</Superscript>Sr after the Chernobyl accident

The assessment doses due to ingestion of ¹³⁷Cs and ⁹⁰Sr for the population suffering from the Chernobyl accident was performed on the basis of the new mechanistic ecological model for assessment of radiological consequences of agricultural lands contamination (EMARC). The EMARC model allows estimation of internal doses based on ecological factors influencing the contamination of foodstuff, for the post-accidental years in the countries of the former Soviet Union. The EMARC model allows estimation of all quantities required in radiation hygiene practice. For example, the proposed analytical method may be used for both retrospective dose reconstruction and prospective estimates of annual dose and integrated “life-time” dose, for different age intervals. According to the EMARC model, estimated reference “life-time” doses for adults are between 7 and 269 μSv kBq⁻¹ m² for ¹³⁷Cs, and between 25 and 235 μSv kBq⁻¹ m² for ⁹⁰Sr. Maximal doses were estimated for persons who were 3, 9 and 11 years old, at the time of the accident and these doses exceed those for adults by a factors of 1, 5 for ⁹⁰Sr, and 1.4 for ¹³⁷Cs.     

A long-term study of catchment inputs of <Superscript>137</Superscript>Cs to a subalpine lake in the form of allochthonous terrestrial plant material

The input of allochthonous plant material, largely terrestrial in origin, during the spring spate is a major source of primary production for montane lake ecosystems such as Øvre Heimdalsvatn and is readily incorporated into the food chain leading to fish. The transport from the lake catchment of allochthonous plant material contaminated with Chernobyl ¹³⁷Cs in 1986 was investigated from 1989 to 2008. ¹³⁷Cs activity concentrations were determined for both total samples before sorting and for the separate components in the two main tributary streams, Brurskardbekken and Lektorbekken, as well as the outflow river, Hinøgla. The total samples showed a similar long-term decline at all sites, although ¹³⁷Cs activity concentrations were generally somewhat higher in Hinøgla compared to the tributary streams. For the total samples, ecological half-lives were in the region of 4–5 years. However, there were major differences between components. The ¹³⁷Cs activity content of most components, including mosses, leaves of willow and woody material, decreased significantly over time. Lichens and juniper declined, but the relationship was not significant, while leaves of mountain birch and dwarf birch showed no obvious trend over the 20-year period.     

Application of enriched <Superscript>137</Superscript>Ba tracer to mark juvenile Persian sturgeon (<Emphasis Type="Italic">Acipenser persicus</Emphasis>)

The present study evaluates the variations of ¹³⁷Ba abundance in pectoral fin spine of 1-month-old juvenile Persian sturgeon (Acipenser persicus) upon marking using the stable isotope approach. The marking of the fish was achieved by incorporation of ¹³⁷Ba²⁺ in the calcified lattice of the pectoral fin spine through substitution with structural Ca²⁺. This process was carried out by rearing juveniles in treatment tanks containing elevated concentrations of ¹³⁷Ba for 1, 3 and 5 days. The marked fish were then retained in natural abundance fresh and brackish waters, to evaluate the trend of exchange of ¹³⁷Ba from the fin spines. The abundance of ¹³⁷Ba in fin spines during marking and post-marking experiments were detected by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that a significant isotope mark can be obtained with no mortality and 100% marking rate on the first day of exposure to the isotope. The marked juveniles maintained their isotopic signature for at least 25 days. Statistical analysis of the obtained ¹³⁸Ba/¹³⁷Ba ratios demonstrated that the successful incorporation of ¹³⁷Ba²⁺ in pectoral fin spines provides an effective marking method for Persian sturgeon restocking programs.     

Behavior of <Superscript>137</Superscript>Cs in the soil-rhizosphere-plant system (by the example of the Yenisei River floodplain)

¹³⁷Cs speciation and distribution in the components of the biogeocenosis of the Yenisei River floodplain such as soils, rhizosphere, and plants were studied. The study was carried out near zones of influence by the Krasnoyarsk Mining and Chemical Combine (MCC). Variation in the specific activity of radionuclides with increasing distance from the pollution source and its ratio in the soil and rhizosphere and distribution in the plant body were studied. A variety of occurrence forms of ¹³⁷Cs, including mobile ones, was revealed. The amount of ¹³⁷Cs leached from plants during flooding was calculated from the results of laboratory experiments.     

<Superscript>137</Superscript>Cs, <Superscript>40</Superscript>K, <Superscript>238</Superscript>Pu, <Superscript>239+240</Superscript>Pu and <Superscript>90</Superscript>Sr in biological samples from King George Island (Southern Shetlands) in Antarctica

There are few data reported on radionuclide contamination in Antarctica. The aim of this paper is to report ¹³⁷Cs, ⁹⁰Sr and ^238,239+240Pu and ⁴⁰K activity concentrations measured in biological samples collected from King George Island (Southern Shetlands, Antarctica), mostly during 2001–2002. The samples included: bones, eggshells and feathers of penguin Pygoscelis papua, bones and feathers of petrel Daption capense, bones and fur of seal Mirounga leonina, algae Himantothallus grandifolius, Desmarestia anceps and Cystosphaera jacquinotii, fish Notothenia corriceps, sea invertebrates Amphipoda, shells of limpet Nacella concina, lichen Usnea aurantiaco-atra, vascular plants Deschampsia antarctica and Colobanthus quitensis, fungi Omphalina pyxidata, moss Sanionia uncinata and soil. The results show a large variation in some activity concentrations. Samples from the marine environment had lower contamination levels than those from terrestrial ecosystems. The highest activity concentrations for all radionuclides were found in lichen and, to a lesser extent, in mosses, probably because lichens take up atmospheric pollutants and retain them. The only significant correlation (except for that expected between ²³⁸Pu and ^239+240Pu) was noted for moss and lichen samples between plutonium and ⁹⁰Sr. A tendency to a slow decrease with time seems to be occurring. Analyses of the activity ratios show varying fractionation between various radionuclides in different organisms. Algae were relatively more highly contaminated with plutonium and radiostrontium, and depleted with radiocesium. Feathers had the lowest plutonium concentrations. Radiostrontium and, to a lesser extent, Pu accumulated in bones. The present low intensity of fallout in Antarctic has a lower ²³⁸Pu/^239+240Pu activity ratio than that expected for global fallout.     

mFISH analysis of chromosomal damage in bone marrow cells collected from CBA/CaJ mice following whole body exposure to heavy ions (<Superscript>56</Superscript>Fe ions)

To date, there is scant information on in vivo induction of chromosomal damage by heavy ions found in space (i.e. ⁵⁶Fe ions). For radiation-induced response to be useful for risk assessment, it must be established in in vivo systems especially in cells that are known to be at risk for health problems associated with radiation exposure (such as hematopoietic cells, the known target tissue for radiation-induced leukemia). In this study, the whole genome multicolor fluorescence in situ hybridization (mFISH) technique was used to examine the in vivo induction of chromosomal damage in hematopoietic tissues, i.e. bone marrow cells. These cells were collected from CBA/CaJ mice at day 7 following whole-body exposure to different doses of 1 GeV/amu ⁵⁶Fe ions (0, 0.1, 0.5 and 1.0 Gy) or ¹³⁷Cs γ rays as the reference radiation (0, 0.5, 1.0 and 3.0 Gy, at the dose rate of 0.72 Gy/min using a GammaCell40). These radiation doses were the average total-body doses. For each radiation type, there were four mice per dose. Several types of aberrations in bone marrow cells collected from mice exposed to either type of radiation were found. These were exchanges and breaks (both chromatid- and chromosome-types). Chromosomal exchanges included translocations (Robertsonian or centric fusion, reciprocal and incomplete types), and dicentrics. No evidence of a non-random involvement of specific chromosomes in any type of aberrations observed in mice exposed to ⁵⁶Fe ions or ¹³⁷Cs γ rays was found. At the radiation dose range used in our in vivo study, the majority of exchanges were simple. Complex exchanges were detected in bone marrow cells collected from mice exposed to 1 Gy of ⁵⁶Fe ions or 3 Gy of ¹³⁷Cs γ rays only, but their frequencies were low. Overall, our in vivo data indicate that the frequency of complex chromosome exchanges was not significantly different between bone marrow cells collected from mice exposed to ⁵⁶Fe ions or ¹³⁷Cs γ rays. Each type of radiation induced significant dose-dependent increases (ANOVA, P < 0.01) in the frequencies of chromosomal damage, including the numbers of abnormal cells. Based upon the linear-terms of dose-response curves, ⁵⁶Fe ions were 1.6 (all types of exchanges), 4.3 (abnormal cells) and 4.2 (breaks, both chromatid- and chromosome-types) times more effective than ¹³⁷Cs γ rays in inducing chromosomal damage.     

Accumulation of radionuclids in amphibians (<Emphasis Type="Italic">Pelophylax ridibundus</Emphasis> Pall.) in the Middle Urals

The accumulation levels of anthropogenic ⁹⁰Sr and ¹³⁴Cs and ¹³⁷Cs radionuclides in the marsh frog have been studied in the areas of the Beloyarskii water-storage reservoir (an industrial storm-water discharge channel of the nuclear power station) and the Verkhnii Tagil water-storage reservoir (the Tagil River down-stream of the dam). No significant distinction in the radionuclide accumulation (⁹⁰Sr and ¹³⁷Cs) depending on the amphibian sex and age is detected. Comparable levels of the accumulation of radionuclides in the marsh frog, when compared to the other representatives of the water ecosystem, are estimated. An assumption of the presence of some unidentified source of radioactive contamination of marsh frogs has been made; cesium-137 may be transferred from it to the Tagil River by the frogs.     

DFT modeling on the suitable crown ether architecture for complexation with Cs<Superscript>+</Superscript> and Sr<Superscript>2+</Superscript> metal ions

Crown ether architectures were explored for the inclusion of Cs⁺ and Sr²⁺ ions within nano-cavity of macrocyclic crown ethers using density functional theory (DFT) modeling. The modeling was undertaken to gain insight into the mechanism of the complexation of Cs⁺ and Sr²⁺ ion with this ligand experimentally. The selectivity of Cs⁺ and Sr²⁺ ions for a particular size of crown ether has been explained based on the fitting and binding interaction of the guest ions in the narrow cavity of crown ethers. Although, Di-Benzo-18-Crown-6 (DB18C6) and Di-Benzo-21-Crown-7 (DB21C7) provide suitable host architecture for Sr²⁺ and Cs⁺ ions respectively as the ion size match with the cavity of the host, but consideration of binding interaction along with the cavity matching both DB18C6 and DB21C7 prefers Sr²⁺ ion. The calculated values of binding enthalpy of Cs metal ion with the crown ethers were found to be in good agreement with the experimental results. The gas phase binding enthalpy for Sr²⁺ ion with crown ether was higher than Cs metal ion. The ion exchange reaction between Sr and Cs always favors the selection of Sr metal ion both in the gas and in micro-solvated systems. The gas phase selectivity remains unchanged in micro-solvated phase. We have demonstrated the effect of micro-solvation on the binding interaction between the metal ions (Cs⁺ and Sr²⁺) and the macrocyclic crown ethers by considering micro-solvated metal ions up to eight water molecules directly attached to the metal ion and also by considering two water molecules attached to metal-ion-crown ether complexes. A metal ion exchange reaction involving the replacement of strontium ion in metal ion-crown ether complexes with cesium ion contained within a metal ion-water cluster serves as the basis for modeling binding preferences in solution. The calculated O-H stretching frequency of H₂O molecule in micro-solvated metal ion-crown complexes is more red-shifted in comparison to hydrated metal ions. The calculated IR spectra can be compared with an experimental spectrum to determine the presence of micro-solvated metal ion–crown ether complexes in extractant phase.     

Dosimetric comparison between realistic ocular model and other models for COMS plaque brachytherapy with <Superscript>103</Superscript>Pd, <Superscript>131</Superscript>Cs,and <Superscript>125</Superscript>I radioisotopes

Nowadays, Monte Carlo calculations are commonly used for the evaluation of dose distributions and dose volume histograms in eye brachytherapy. However, currently available eye models have simple geometries, and main substructures of the eye are either not defined in details or not distinguished at all. In this work absorbed doses of eye substructures have been estimated for eye plaque brachytherapy using the most realistic eye model available, and compared with absorbed doses obtained with other available eye models. For this, a medium-sized tumour on the left sides of the right eye was considered. Dosimetry calculations were performed for four different eye models developed based on a literature review, and using a 12 mm Collaborative Ocular Melanoma Study plaque containing ¹³¹Cs, ¹⁰³Pd, and ¹²⁵I sources. Obtained results illustrate that the estimated doses received by different eye substructures strongly depend on the model used to represent the eye. It is shown here that using a non-realistic eye model leads to a wrong estimation of doses for some eye substructures. For example, dose differences of up to 35% were observed between the models proposed by Nogueira and co-workers and Yoriyaz and co-workers, while doses obtained by use of the models proposed by Lesperance and co-workers, and Behrens and co-workers differed up to 100 and 63% as compared to the situation when a realistic model was used, respectively. Moreover, comparing different radionuclides showed that the most uniform dose distribution in the considered tumour region was that from ¹³¹Cs, with a coefficient of variation of 33%. In addition, considering the realistic eye model, it was found that the radiosensitive region of the lens received more than the threshold dose of cataract induction (0.5 Gy), for all investigated radionuclides.     

Salt tolerance of barley: Relations between expression of isoforms of vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter and <Superscript>22</Superscript>Na<Superscript>+</Superscript> accumulation

Two barley cultivars (Hordeum vulgare L., cvs. Elo and Belogorskii) differing in salt tolerance were used to study ²²Na⁺ uptake, expression of three isoforms of the Na⁺/H⁺ antiporter HvNHX1-3, and the cellular localization of these isoforms in the elongation zone of seedling roots. During short (1 h) incubation, seedling roots of both cultivars accumulated approximately equal quantities of ²²Na⁺. However, after 24-h incubation the content of ²²Na⁺ in roots of a salt-tolerant variety Elo was 40% lower than in roots of the susceptible variety Belogorskii. The content of ²²Na⁺ accumulated in shoots of cv. Elo after 24-h incubation was 6.5 times lower than in shoots of cv. Belogorskii and it was 4 times lower after the salt stress treatment. The cytochemical examination revealed that three proteins HvNHX1-3 are co-localized in the same cells of almost all root tissues; these proteins were present in the tonoplast and prevacuolar vesicles. Western blot analysis of HvNHX1-3 has shown that the content of isoforms in vacuolar membranes increased in response to salt stress in seedling roots and shoots of both cultivars, although the increase was more pronounced in the tolerant cultivar. The content of HvNHX1 in the seedlings increased in parallel with the enhanced expression of HvNHX1, whereas the increase in HvNHX2 and HvNHX3 protein content was accompanied by only slight changes in expression of respective genes. The results provide evidence that salt tolerance of barley depends on plant ability to restrict Na⁺ transport from the root to the shoot and relies on regulatory pathways of HvNHX1-3 expression in roots and shoots during salt stress.     

Relations between resuspension and the content of 137Cs in freshwater fish in some Swedish lakes

In many Swedish lakes, the fallout of ¹³⁷Cs from the Chernobyl accident in April 1986 has largely accumulated in the sediments. The availability and transfer of deposited ¹³⁷Cs to biota is influenced by factors such as resuspension. The frequency of resuspension and the ¹³⁷Cs-content of different fish species was studied in three shallow lakes in Uppland, central Sweden, and in one deeper lake in northern Sweden. Resuspension was measured by the use of sediment traps. Sedimentation rates measured from the traps in the shallower lakes were 5–10 times higher than normal for this type of lake, indicating that resuspension was an important factor. The decrease of the ¹³⁷Cs-content in muscle tissue of pike, perch and roach was slow in each of the shallow lakes. ¹³⁷Cs decreased by about 30% over a period of 2 years in the shallowest lake (maximum depth 4 m), whereas ¹³⁷Cs decreased by 50% in the deeper lakes (maximum depth 10 m). The slower rates of decline of ¹³⁷Cs in biota from the shallow lakes, are probably a function of sediment dynamics (mainly influenced by lake morphometry, wind direction and strength). They may be influenced, also, by bioavailability of resuspended sediment material. In the deepest northern lake, much of the ¹³⁷Cs-containing material collected in the sediment traps originated from the catchment area. Resuspension was minimal, and the high activity of ¹³⁷Cs in the sediment had no effect on content or decline of ¹³⁷Cs in lake fish.     

<Superscript>13</Superscript>C-<Superscript>13</Superscript>C NOESY: A constructive use of <Superscript>13</Superscript>C-<Superscript>13</Superscript>C spin-diffusion

¹³C-¹³C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, ¹⁵N, ^13C and 70% ²H labeled). ¹³C-¹³C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time _m of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C and C. The interpretation was confirmed by simulations. This approach broadens the range of applicability of ¹³C-¹³C NOESY spectroscopy.     

Dynamics of <Superscript>18</Superscript>O Incorporation from H <Subscript>2</Subscript><Superscript>18</Superscript>O into Soil Microbial DNA

Heavy water (H₂¹⁸O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the ¹⁸O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% ¹⁸O for DNA extracted from soil incubated with the addition of H₂¹⁸O. Four experiments were conducted to test how the atom% ¹⁸O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% ¹⁸O of added H₂O. In the time series experiment, the atom% ¹⁸O of DNA increased linearly (R ² = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R ² = 0.991, p = 0.006) between the log₁₀ of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log₁₀ of the atom% ¹⁸O of DNA. For the experiment where soil moisture was manipulated, the atom% ¹⁸O of DNA increased with higher soil moisture until soil moisture reached 30%, above which ¹⁸O enrichment of DNA declined as soils became more saturated. When the atom% ¹⁸O for H₂O added was varied, there was a positive linear relationship between the atom% ¹⁸O of the added water and the atom% ¹⁸O of the DNA. Results indicate that quantification of ¹⁸O incorporated into DNA from H₂¹⁸O has potential to be used as a proxy for microbial growth in soil.     

Transfer of 137Cs from water to fish is not linear in two northern lakes

Empirical data on the behavior of radionuclides in the biosphere are needed for validating radioecological models. In this study, data collected from two lakes in Northern Finland were used to investigate the transfer of ¹³⁷Cs from lake water into fish during a 20-year period after the Chernobyl nuclear power plant accident. The results indicated that transfer of ¹³⁷Cs in the food chains investigated is nonlinear: the water-to-fish concentration ratios (CRs) decreased with increasing ¹³⁷Cs concentration in water. Major deviation from linearity (constant CR) commonly assumed in radioecological modeling was observed only at low ¹³⁷Cs concentrations in water. Other findings of potential importance for radioecological models were that ¹³⁷Cs concentrations were threefold higher in piscivores than in non-piscivores and that no differences in ¹³⁷Cs uptake were found between the two piscivorous species (pike and perch) studied or between three non-piscivorous species (vendace, white fish, and roach) representing different feeding habits (planktivore, benthivore, and mixed diet).     

Comparison of <Superscript>13</Superscript>CH<Subscript>3</Subscript>, <Superscript>13</Superscript>CH<Subscript>2</Subscript>D,and <Superscript>13</Superscript>CHD<Subscript>2</Subscript> methyl labeling strategies in proteins

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using ¹³CH₃,¹³CH₂D, and ¹³CHD₂ methyl isotopomers, is presented. For each labeling scheme, ¹H–¹³C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with ¹³CH₃,¹³CH₂D, and ¹³CHD₂ labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for ¹³CH₃-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (¹³CH₃ HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the ¹³CH₃ HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both ¹³C and ¹H spins. The HSQC and HMQC spectra of ¹³CH₂D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, ¹³CH₂D TROSY, that exploits cross-correlation effects to record ¹H–¹³C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of ¹³CHD₂-labeled methyl groups can be recorded with HSQC or HMQC; a new ¹³CHD₂ HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the ¹³CH₃ labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either ¹³CH₂D or ¹³CHD₂ labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.     

1-<Superscript>13</Superscript>C amino acid selective labeling in a <Superscript>2</Superscript>H<Superscript>15</Superscript>N background for NMR studies of large proteins

Isotope labeling by residue type (LBRT) has long been an important tool for resonance assignments at the limit where other approaches, such as triple-resonance experiments or NOESY methods do not succeed in yielding complete assignments. While LBRT has become less important for small proteins it can be the method of last resort for completing assignments of the most challenging protein systems. Here we present an approach where LBRT is achieved by adding protonated ¹⁴N amino acids that are ¹³C labeled at the carbonyl position to a medium for uniform deuteration and ¹⁵N labeling. This has three important benefits over conventional ¹⁵N LBRT in a deuterated back ground: (1) selective TROSY-HNCO cross peaks can be observed with high sensitivity for amino-acid pairs connected by the labeling, and the amide proton of the residue following the ¹³C labeled amino acid is very sharp since its alpha position is deuterated, (2) the ¹³C label at the carbonyl position is less prone to scrambling than the ¹⁵N at the α-amino position, and (3) the peaks for the 1-¹³C labeled amino acids can be identified easily from the large intensity reduction in the ¹H-¹⁵N TROSY-HSQC spectrum for some residues that do not significantly scramble nitrogens, such as alanine and tyrosine. This approach is cost effective and has been successfully applied to proteins larger than 40 kDa. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Superscript>18</Superscript> O-rich Oxygen from Land Photosynthesis

ATMOSPHERIC oxygen is not in equilibrium with sea water with respect to the isotope exchange illustration but has an ¹⁸O excess of about 22‰ compared to sea water¹. This could be due to isotope fractionation during respiration². Another large contribution to the effect has been overlooked up to now. Photosynthesis on land takes place in transpiring leaves, where the difference in the vapour pressure of ¹⁶OH₂ and ¹⁸OH₂ concentrates the heavy molecules in their stationary water content. Since the free oxygen stems from the water in which photosynthesis takes place^3–8 (with only a very small shift in isotopic composition⁹), photosynthesis on land is an ¹⁸O source for atmospheric O₂. We have begun to study this effect quantitatively.

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<Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C chemical shift assignments of the regulatory domain of human calcineurin

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM–CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete ¹³C, ¹⁵N and ¹H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of ¹³C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A ¹⁵N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.     

<Superscript>65</Superscript>Zn<Superscript>2+</Superscript> transport by lobster hepato-pancreatic baso-lateral membrane vesicles

The lobster (Homarus americanus) hepato-pancreatic epithelial baso-lateral cell membrane possesses three transport proteins that transfer calcium between the cytoplasm and hemolymph: an ATP-dependent calcium ATPase, a sodium-calcium exchanger, and a verapamil-sensitive cation channel. We used standard centrifugation methods to prepare purified hepato-pancreatic baso-lateral membrane vesicles and a rapid filtration procedure to investigate whether ⁶⁵Zn²⁺ transfer across this epithelial cell border occurs by any of these previously described transporters for calcium. Baso-lateral membrane vesicles were osmotically reactive and exhibited a time course of uptake that was linear for 10–15 s and approached equilibrium by 120 s. In the absence of sodium, ⁶⁵Zn²⁺ influx was a hyperbolic function of external zinc concentration and followed the Michaelis-Menten equation for carrier transport. This carrier transport was stimulated by the addition of 150 M ATP (increase in K_m and J_max) and inhibited by the simultaneous presence of 150 mol l^–1 ATP+250 mol l^–1 vanadate (decrease in both K_m and J_max). In the absence of ATP, ⁶⁵Zn²⁺ influx was a sigmoidal function of preloaded vesicular sodium concentration (0, 5, 10, 20, 30, 45, and 75 mmol l^–1) and exhibited a Hill Coefficient of 4.03±1.14, consistent with the exchange of 3 Na⁺/1Zn²⁺. Using Dixon analysis, calcium was shown to be a competitive inhibitor of baso-lateral membrane vesicle ⁶⁵Zn²⁺ influx by both the ATP-dependent (K_i=205 nmol l^–1 Ca²⁺) and sodium-dependent (K_i=2.47 mol l^–1 Ca²⁺) transport processes. These results suggest that zinc transport across the lobster hepato-pancreatic baso-lateral membrane largely occurred by the ATP-dependent calcium ATPase and sodium-calcium exchanger carrier proteins.Communicated by: I.D. Hume