Estimation of 99Mo production rates from natural molybdenum in research reactors.

Molybdenum-99 is one of the most important radionuclides for medical diagnostics. In 2015, the International Atomic Energy Agency organized a round-robin exercise where the participants measured and calculated specific saturation activities achievable for the 98Mo(n,γ)99Mo reaction. This reaction is of interest as a means to locally, and on a small scale, produce 99Mo from natural molybdenum. The current paper summarises a set of experimental results and reviews the methodology for calculating the corresponding saturation activities. Activation by epithermal neutrons and also epithermal neutron self-shielding are found to be of high importance in this case.

Impact of axial root growth angles on nitrogen acquisition in maize depends on environmental conditions.

BACKGROUNDS AND AIMS: Crops with reduced requirement for nitrogen (N) fertilizer would have substantial benefits in developed nations, while improving food security in developing nations. This study employs the functional structural plant model SimRoot to test the hypothesis that variation in the growth angles of axial roots of maize (Zea mays L.) is an important determinant of N capture. METHODS: Six phenotypes contrasting in axial root growth angles were modelled for 42 d at seven soil nitrate levels from 10 to 250 kg ha(-1) in a sand and a silt loam, and five precipitation regimes ranging from 0·5× to 1·5× of an ambient rainfall pattern. Model results were compared with soil N measurements of field sites with silt loam and loamy sand textures. KEY RESULTS: For optimal nitrate uptake, root foraging must coincide with nitrate availability in the soil profile, which depends on soil type and precipitation regime. The benefit of specific root architectures for efficient N uptake increases with decreasing soil N content, while the effect of soil type increases with increasing soil N level. Extreme root architectures are beneficial under extreme environmental conditions. Extremely shallow root systems perform well under reduced precipitation, but perform poorly with ambient and greater precipitation. Dimorphic phenotypes with normal or shallow seminal and very steep nodal roots performed well in all scenarios, and consistently outperformed the steep phenotypes. Nitrate uptake increased under reduced leaching conditions in the silt loam and with low precipitation. CONCLUSIONS: Results support the hypothesis that root growth angles are primary determinants of N acquisition in maize. With decreasing soil N status, optimal angles resulted in 15-50 % greater N acquisition over 42 d. Optimal root phenotypes for N capture varied with soil and precipitation regimes, suggesting that genetic selection for root phenotypes could be tailored to specific environments.

The nutritional status of Dutch elderly patients with Parkinson's disease.

OBJECTIVES: To assess the prevalence of (risk of) undernutrition in Dutch elder Parkinson's disease patients as well as it's risk factors. DESIGN: Observational cross-sectional study. SETTING: An outpatient clinic at the department Neurology of Medical Centre Leeuwarden, a large teaching hospital. PARTICIPANTS: 102 outpatients with Parkinson's disease aged 65 years and older were recruited. MEASUREMENTS: Data regarding various aspects of undernutrition including socio-demographic aspect, disease characterisitics, nutritional status, appetite and overall-physical and psychological functioning were collected. RESULTS: Undernutrition was diagnosed in 2.0% and 20.5% of the patients were categorized as being at risk of undernutrition. Care dependency and appetite were the two risk factors with the highest predictive value for an unfavorable nutritional status. CONCLUSION: Of Dutch elderly patients with Parkinson's Disease 22.5% had an unfavourable nutritional status. Dependency and appetite were the two risk factors with the highest predictive value fort his outcome. Because undernutrition can be regarded as a geriatric syndrome a comprehensive nutritional assessment should be done followed by nutritional interventions next to interventions focused on the risk factors. Further studies are needed to evaluate these interventions.

Reproducibility of neutron activated Sm-153 oral dose formulations intended for human administration.

Neutron activation of Sm-152 offers a method of radiolabeling for the in vivo study of oral dose formulations by gamma scintigraphy. Reproducibility measurements are needed to ensure the robustness of clinical studies. 204 enteric-coated guaifenesin core tablets (10mg of Sm(2)O(3)) were irradiated by thermal neutrons to achieve 1 MBq at 48 h. Administered activities were 0.86±0.03 MBq. Good reproducibility (CV=3.5%) was observed over 24 weeks ensuring that volunteer doses were within the dose reference level of 0.8 mSv.

No evidence for antineutrinos significantly influencing exponential ß+ decay.

High-precision measurements were conducted on the time evolution of gamma-ray count rates during reactor-on and reactor-off periods to investigate the possible influence of antineutrinos on nuclear decay. This experiment was triggered by a recent analysis (Jenkins et al., 2009) of long-term measurements suggesting a possible link to variations in nuclear decay rate and solar neutrino flux. The antineutrino flux during reactor-off periods is mainly due to geoneutrinos and four orders of magnitude lower than during reactor-on periods. No effects have been observed for the two branches in the decay of (152)Eu and the decay of (137)Cs, (54)Mn and (22)Na. The upper limit determined of the ratio Δλ/λ for (22)Na is (-1±2)×10(-4), and (54)Mn is (-1±4)×10(-4). In comparison to the interpretation of Jenkins et al. our measurements do not show any such effect to at least two orders of magnitude less. Hence either the hypothesis of Jenkins et al. is not true or else one of two rather unlikely possibilities must also be true: either the effect of neutrinos on ß(-) decay differs considerably from the effect of antineutrinos on ß(+) decay, or the effect of antineutrinos on ß(+) decay must be identical to their effect on ß(-) and electron-capture decay.

An attempt to date an antique Benin bronze using neutron resonance capture analysis.

Neutron resonance capture analysis was applied to a bronze commemorative plaque from the West-African country Benin. By comparison with recently published element compositions of Benin memorial heads, the alloy of the plaque could be dated to the period 1725-1897 AD. In the analysis procedure, the object was not damaged, cleaned or altered, and very little long-lived radioactivity was induced.

Within-trophic group interactions of bacterivorous nematode species and their effects on the bacterial community and nitrogen mineralization.

Knowledge of the interactions between organisms within trophic groups is important for an understanding of the role of biodiversity in ecosystem functioning. We hypothesised that interactions between bacterivorous nematodes of different life history strategies would affect nematode population development, bacterial community composition and activity, resulting in increased N mineralization. A microcosm experiment was conducted using three nematode species (Bursilla monhystera, Acrobeloides nanus and Plectus parvus). All the nematode species interacted with each other, but the nature and effects of these interactions depended on the specific species combination. The interaction between B. monhystera and A. nanus was asymmetrically competitive (0,-), whereas that between B. monhystera and P. parvus, and also A. nanus and P. parvus was contramensal (+, -). The interaction that affected microcosm properties the most was the interaction between B. monhystera and P. parvus. This interaction affected the bacterial community composition, increased the bacterial biomass and increased soil N mineralization. B. monhystera and P. parvus have the most different life history strategies, whereas A. nanus has a life history strategy intermediate to those of B. monhystera and P. parvus. We suggest that the difference in life history strategies between species of the same trophic group is of importance for their communal effect on soil ecosystem processes. Our results support the idiosyncrasy hypothesis on the role of biodiversity in ecosystem functioning.

Specific growth rate plays a critical role in hydrogen peroxide resistance of the marine oligotrophic ultramicrobacterium sphingomonas alaskensis strain RB2256.

The marine oligotrophic ultramicrobacterium Sphingomonas alaskensis RB2256 has a physiology that is distinctly different from that of typical copiotrophic marine bacteria, such as Vibrio angustum S14. This includes a high level of inherent stress resistance and the absence of starvation-induced stress resistance to hydrogen peroxide. In addition to periods of starvation in the ocean, slow, nutrient-limited growth is likely to be encountered by oligotrophic bacteria for substantial periods of time. In this study we examined the effects of growth rate on the resistance of S. alaskensis RB2256 to hydrogen peroxide under carbon or nitrogen limitation conditions in nutrient-limited chemostats. Glucose-limited cultures of S. alaskensis RB2256 at a specific growth rate of 0.02 to 0.13 h(-1) exhibited 10,000-fold-greater viability following 60 min of exposure to 25 mM hydrogen peroxide than cells growing at a rate of 0.14 h(-1) or higher. Growth rate control of stress resistance was found to be specific to carbon and energy limitation in this organism. In contrast, V. angustum S14 did not exhibit growth rate-dependent stress resistance. The dramatic switch in stress resistance that was observed under carbon and energy limitation conditions has not been described previously in bacteria and thus may be a characteristic of the oligotrophic ultramicrobacterium. Catalase activity varied marginally and did not correlate with the growth rate, indicating that hydrogen peroxide breakdown was not the primary mechanism of resistance. More than 1,000 spots were resolved on silver-stained protein gels for cultures growing at rates of 0.026, 0.076, and 0.18 h(-1). Twelve protein spots had intensities that varied by more than twofold between growth rates and hence are likely to be important for growth rate-dependent stress resistance. These studies demonstrated the crucial role that nutrient limitation plays in the physiology of S. alaskensis RB2256, especially under oxidative stress conditions.

Efficient control of gene expression by a tetracycline-dependent transactivator in single Dictyostelium discoideum cells.

We established a tetracycline-regulated gene expression system that tightly controls expression of genes in Dictyostelium discoideum. The control elements are contained in two plasmid vectors, one being an integrated plasmid encoding a chimeric tetracycline-controlled transcriptional activator protein (tTA(s)(*)). The second component is an extrachromosomal plasmid harboring the gene of interest preceded by an inducible promoter. This promoter contains a tetracycline-responsive element, which is the binding site for tTA(s)(*). Tetracycline prevents tTA(s)(*) from binding to the tetracycline-responsive element, rendering the promoter virtually silent. In the absence of tetracycline, tTA(s)(*) binds to its target sequence and strongly induces gene expression. The kinetics of activation and repression of the system were monitored using luciferase as a reporter. The results reveal efficient inhibition of gene expression by low concentrations of tetracycline and an induction of gene expression by several orders of magnitude within a few hours after removal of tetracycline. Green fluorescent protein (GFP) provided information about the effects of modulation of the tetracycline concentration on gene expression, at the single cell level, using fluorescence activated cell sorting (FACS). We also report that not all cells in a clonal population express the reporter gene.

Structural evidence for dimerization-regulated activation of an integral membrane phospholipase.

Dimerization is a biological regulatory mechanism employed by both soluble and membrane proteins. However, there are few structural data on the factors that govern dimerization of membrane proteins. Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme which participates in secretion of colicins in Escherichia coli. In Campilobacter and Helicobacter pylori strains, OMPLA is implied in virulence. Its activity is regulated by reversible dimerization. Here we report X-ray structures of monomeric and dimeric OMPLA from E. coli. Dimer interactions occur almost exclusively in the apolar membrane-embedded parts, with two hydrogen bonds within the hydrophobic membrane area being key interactions. Dimerization results in functional oxyanion holes and substrate-binding pockets, which are absent in monomeric OMPLA. These results provide a detailed view of activation by dimerization of a membrane protein.

Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins in Dictyostelium discoideum.

The soil amoeba Dictyostelium discoideum is a host cell that provides simple genetics in combination with complex protein synthesis. We show that the complex human heterodimeric gonadotropins can be produced and secreted by this organism. Furthermore, both follicle stimulation hormone and choriogonadotropin produced by D. dictyostelium bind to their human receptors and elicit a biological response comparable to the wild-type hormones. We also show that structure-function analysis using random mutagenesis and screening of recombinant glycoprotein hormones is feasible. Thus, expression of gonadotropins in D. dictyostelium opens the way to the engineering of potential new therapeutic analogues.

Expression of a bioactive, single-chain choriogonadotropin in Dictyostelium discoideum.

Human choriogonadotropin (hCG) is a highly complex glycoprotein consisting of two non-covalently associated subunits. We aimed for the expression of a single-chain hCG in the soil amoebae Dictyostelium discoideum, a host which, in principle, provides simple genetics in combination with complex protein synthesis. To limit anticipated problems in mRNA translation, the first 30 bases of the coding sequence were altered to conform to the Dictyostelium preferred codon usage. We show that, immunologically, active single-chain hCG is indeed produced by Dictyostelium. Furthermore, this single-chain hCG is able to bind to the human luteinizing hormone/CG receptor and elicit a biological response. Its receptor-binding affinity is comparable to single-chain hCG produced by mammalian cells. We conclude that Dictyostelium is able to express bioactive highly complex heterologous glycoproteins.

The accuracy of instrumental neutron activation analysis of kilogram-size inhomogeneous samples.

The feasibility of quantitative instrumental neutron activation analysis (INAA) of samples in the kilogram range without internal standardization has been demonstrated by Overwater et al. (Anal. Chem. 1996, 68, 341). In their studies, however, they demonstrated only the agreement between the "corrected" γ ray spectrum of homogeneous large samples and that of small samples of the same material. In this paper, the k(0) calibration of the IRI facilities for large samples is described, and, this time in terms of (trace) element concentrations, some of Overwater's results for homogeneous materials are presented again, as well as results obtained from inhomogeneous materials and subsamples thereof. It is concluded that large-sample INAA can be as accurate as ordinary INAA, even when applied to inhomogeneous materials.

Crystallization and preliminary X-ray analysis of outer membrane phospholipase A from Escherichia coli.

The outer membrane phospholipase A (OMPLA) of Escherichia coli is one of the few integral outer membrane proteins displaying enzymatic activity. It is encoded as a mature protein of 269 amino acids preceded by a signal sequence of 20 amino acids. There is no sequence homology with water-soluble lipases and phospholipases. Crystals of the mature enzyme were obtained at 22 degrees C from 24-28% (v/v) 2-methyl-2,4-pentanediol in Bis-Tris buffer, pH 5.9-6.0, with 1 mM calcium chloride and 1.5% (w/v) beta-octylglucoside. They have the symmetry of the trigonal spacegroup P3(1)21 (or P3(2)21) with cell dimensions of a = b = 79.6 A and c = 102.8 A (alpha = beta = 90 degrees, gamma = 120 degrees). Native crystals diffract to a resolution of 2.6 A.

Crystallization and preliminary X-ray analysis of a lipase from Staphylococcus hyicus.

Single cyrstals of a lipase from Staphylococcus hyicus have been obtained using a combination of 18 to 24% dimethylsulfoxide and 10% isopropanol as a precipitant. The crystals grow at 4 degrees C in 2-3 months. They belong to the orthorhombic space group P212121 with a = 73.31 A, b = 77.96 A, and c = 169.81 A, with two protein molecules per asymmetrical unit. The crystals diffract to at least 2.8 A resolution and are suitable for an X-ray structure analysis.

Crystallization and preliminary X-ray analysis of a lipase from Bacillus subtilis.

Single crystals of the lipase from Bacillus subtilis have been obtained using a mixture of polyethylene glycol 4000 and sodium sulphate solution as the precipitant. The crystals grow at room temperature in two to three weeks in the presence of n-octyl-beta-D-glucoside. They belong to the monoclinic space group C2 with a = 121.20 A, b = 93.19 A, c = 80.96 A, and beta = 110.67 degrees, with four protein molecules per asymmetric unit. The crystals diffract to at least 2.5 A resolution and are suitable for an X-ray structure analysis.

The structure-function relationship of the lipases from Pseudomonas aeruginosa and Bacillus subtilis.

Within the BRIDGE T-project on lipases we investigate the structure-function relationships of the lipases from Bacillus subtilis and Pseudomonas aeruginosa. Construction of an overproducing Bacillus strain allowed the purification of > 100 mg lipase from 30 l culture supernatant. After testing a large variety of crystallization conditions, the Bacillus lipase gave crystals of reasonable quality in PEG-4000 (38-45%), Na2SO4 and octyl-beta-glucoside at 22 degrees C, pH 9.0. A 2.5 A dataset has been obtained which is complete from 15 to 2.5 A resolution. P.aeruginosa wild-type strain PAC1R was fermented using conditions of maximum lipase production. More than 90% of the lipase was cell bound and could be solubilized by treatment of the cells with Triton X-100. This permitted the purification of approximately 50 mg lipase. So far, no crystals of sufficient quality were obtained. Comparison of the model we built for the Pseudomonas lipase, on the basis of sequences and structures of various hydrolases which were found to possess a common folding pattern (alpha/beta hydrolase fold), with the X-ray structure of the P.glumae lipase revealed that it is possible to correctly build the structure of the core of a protein even in the absence of obvious sequence homology with a protein of known 3-D structure.

Molecular analysis of the cytosolic and glycosomal glyceraldehyde-3-phosphate dehydrogenase in Leishmania mexicana.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in two cell compartments of Leishmania mexicana promastigotes. These activities could be attributed to two different isoenzymes, one residing in glycosomes, the other in the cytosol. We have cloned and sequenced the genes for both isoenzymes. The glycosomal enzyme is encoded by two tandemly linked genes of identical sequence and contains features frequently found in glycosomal enzymes: the presence of peptide insertions, a small carboxy-terminal extension with a potential glycosomal targeting signal (-SKM) and an excess of positively charged residues (net charge +7). Only one open reading frame was detected for the cytosolic enzyme. The amino acid sequences of the two proteins are only 55% identical. We discuss some evolutionary aspects of the observed organization of the GAPDH genes in the Trypanosomatidae and the role of the two isoenzymes in the metabolism of these organisms. The possibility to develop GAPDH-specific inhibitors that will be effective against the enzyme of various parasitic members of this family is explored.

Mutations in the FAD-binding fold of alcohol oxidase from Hansenula polymorpha.

Alcohol oxidase of methylotrophic yeast is an FAD-containing enzyme. When in its active form, the enzyme is an octamer and located in the peroxisomes. To study the importance of FAD-binding on the activity, octamerization and intracellular localization of the enzyme, alcohol oxidase of Hansenula polymorpha was mutated in its presumed nucleotide-binding domain, which is formed by the N-terminal sequence. Whereas mutations of a glutamic acid residue (E42) reduced the stability of the octamer, it hardly affected enzyme activity and expression. However, replacements of three conserved glycines (G13, G15 and G18) and a conserved glutamic acid (E39) within the fold had severe effects. The mutations not only resulted in loss of enzyme activity but in reduced protein levels as well, probably due to decreased stability of the mutant alcohol oxidase. However, octamerization of the protein still occurred. The existence of inactive octameric proteins provides information about the formation pathway of this octameric flavoprotein.