A fluorescent assay to quantitatively measure in vitro acyl CoA:diacylglycerol acyltransferase activity

Triacylglycerols (TG) are the major storage form of energy in eukaryotic organisms and are synthesized primarily by acyl CoA:1,2-diacylglycerol acyltransferase (DGAT) enzymes. In vitro DGAT activity has previously been quantified by measuring the incorporation of either radiolabeled fatty acyl CoA or diacylglycerol (DG) into TG. We developed a modified acyltransferase assay using a fluorescent fatty acyl CoA substrate to accurately quantify in vitro DGAT activity. In the modified assay, radioactive fatty acyl CoA is replaced with fluorescent NBD-palmitoyl CoA, which is used as a substrate by DGAT with DG to produce NBD-TG. After extraction with organic solvents and separation by thin layer chromatography, NBD-TG formation can be detected and accurately quantified using a fluorescent imaging system. We demonstrate that this method can be adapted to detect other acyltransferase activities. Because NBD-palmitoyl CoA is commercially available at a much lower cost compared with radioactive acyl CoA substrates, it is a more economical alternative to radioactive tracers. In addition, the exposure of laboratory personnel to radioactivity is greatly reduced.     

Production of low‐Cs+ rice plants by inactivation of the K+ transporter OsHAK1 with the CRISPR‐Cas system

The occurrence of radiocesium in food has raised sharp health concerns after nuclear accidents. Despite being present at low concentrations in contaminated soils (below μm ), cesium (Cs⁺) can be taken up by crops and transported to their edible parts. This plant capacity to take up Cs⁺ from low concentrations has notably affected the production of rice (Oryza sativa L.) in Japan after the nuclear accident at Fukushima in 2011. Several strategies have been put into practice to reduce Cs⁺ content in this crop species such as contaminated soil removal or adaptation of agricultural practices, including dedicated fertilizer management, with limited impact or pernicious side‐effects. Conversely, the development of biotechnological approaches aimed at reducing Cs⁺ accumulation in rice remain challenging. Here, we show that inactivation of the Cs⁺‐permeable K⁺ transporter OsHAK1 with the CRISPR‐Cas system dramatically reduced Cs⁺ uptake by rice plants. Cs⁺ uptake in rice roots and in transformed yeast cells that expressed OsHAK1 displayed very similar kinetics parameters. In rice, Cs⁺ uptake is dependent on two functional properties of OsHAK1: (i) a poor capacity of this system to discriminate between Cs⁺ and K⁺; and (ii) a high capacity to transport Cs⁺ from very low external concentrations that is likely to involve an active transport mechanism. In an experiment with a Fukushima soil highly contaminated with ¹³⁷Cs⁺, plants lacking OsHAK1 function displayed strikingly reduced levels of ¹³⁷Cs⁺ in roots and shoots. These results open stimulating perspectives to smartly produce safe food in regions contaminated by nuclear accidents.     

黄土区土壤放射性的生物循环

研究了不同类型土壤中放射性物质的生物学循环和平衡问题。结果表明,土壤中放射性核素剖面分布的总趋势是沿发生层往下逐步减少。其生物积聚系数因不同核素而变动在1.06—1.44间。放射性物质在各类植物体内的含量顺序是:牧草>农业作物≥灌木≥果树>乔木。植物对α放射性的富集系数一般均小于0.10,而β放射性的富集系数大都处于0.5—1.0间.不同土地利用情况下土壤放射性可分为积累、消耗和中间3种平衡类型。     

Determination of the specific activity of NaB3H4

A method for the rapid determination of the specific activity of NaB3H4 is presented. NaB3H4 is used to reduce NAD+ to [3H]NADH, which is then isolated by anion exchange chromatography. The specific activity of the NaB3H4 is calculated from measurements of radioactivity and absorbance (340 nm) in the [3H]NADH fractions.     

A simplified method for the estimation of individual amino acid radioactivity in plasma samples

A method is presented for the quantitative estimation of the individual amino acid radioactivity in biological samples. The material is deproteinized with cold acetone, and, after acetone evaporation, is passed through a column containing 1 g of Amberlite XAD-2, then eluted with 10% ethanol. The samples are derivatized with Sanger's reagent (alkaline 1-fluoro-2,4-dinitrobenzene) and passed again through the Amberlite XAD-2 column; the 10% ethanol eluate is now discarded and the DNP-amino acids eluted with acetone. Aliquots are used for TLC chromatography on Silicagel plates; the spots are identified, cut away and their radioactivity estimated. The actual recovery of radioactivity in the spots is about 86-92% of the initial radioactivity. No contamination with radioactive glucose, lactate, pyruvate or glycerol has been observed.     

Androgen receptor in human skin fibroblasts. Characterization of a specific 17beta-hydroxy-5alpha-androstan-3-one-protein complex in cell sonicates and nuclei.

Cultured human skin fibroblasts were shown to contain an androgen binding activity (receptor) which was heat-labile and destroyed by trypsin. Specific binding was seen after incubations of these cells with 1,2-3-H-testosterone, 1,2-3-H17beta-hydroxy-5alpha-androstan-3-one (dihydrotestosterone, DHT) and 1,2-3-H-5alpha-androstane-3alpha, 17beta-diol. This receptor had a high affinity (Kd=0,2-1.6 nM) and a high degree of specificity for DHT. It was measured as a 3-H-DHT-protein complex by gel filtration chromatography using a method which distinguishes specific from nonspecific binding. Receptor activity was distributed about equally between nuclear and extranuclear components at all times studied and was present in both compartments when cell incubations were carried out at 4 degrees and 37 degrees. Saturation analysis indicated that there were 1250-18,600 binding sites per whole cell. By sucrose gradient centrifugation the receptor had a sedimentation coefficient (S20,w) of about 4. Cells grown for 8 days without serum in the medium maintained the same levels of 3-H-DHT binding. Within 15 hours puromycin (20 mug/ml) in serum-free medium caused a 40-60 percent decrease in binding for the same cell lines. Although the highest levels of 3-H-DHT binding were observed in fibroblasts from newborn foreskin, appreciable cytosol and nuclear binding were seen in cells from forearm, neck and abdominal skin. Receptor activity was stable during prolonged culture. Fibroblasts from several skin sites from patients with the androgen insensitivity syndrome (testicular feminization) had no detectable specific DHT binding. In this study it was demonstrated that skin fibroblasts can rapidly convert testosterone to its active form, DHT, bind DHT to a specific receptor protein and transport this complex to their nuclei. Therefore this may prove to be a convenient system for studying androgen action in vitro.     

Natural radioactivity concentrations of 226Ra, 232Th,and 40K in commercial building materials and their lifetime cancer risk assessment in dwellers

Elevated radioactivity levels of ²²⁶Ra, ²³²Th, and ⁴⁰K in building materials were measured using gamma-ray spectrometry and their associated lifetime cancer risks were also determined. The mean activity concentrations of ²²⁶Ra, ²³²Th, and ⁴⁰K are 45.72 ± 0.55, 65.90 ± 8.89, and 487.32 ± 15.20 Bq kg^?1, respectively. Statistically, the principal component (PC) analysis indicates that higher loadings were recorded in Principal Component One (PC1) with large contribution from ²³²Th and ⁴⁰K. The leverage studies indicate that BN Ceramics (BNC) contributes more to the loadings in PC1 followed by Golden Crown Ceramic (GCC) sample. The mean values of 0.399 mSv y^?1 do not surpass the world average value of 0.7 mSv y^?1. The mean gamma index from the measured samples is 0.644, whereas a mean value of 0.271 for alpha index is noted in the samples. The activity utilization index (AUI) from the samples satisfied the AUI <2, which corresponded with the annual effective dose of <0.3 mSv y^?1, except interlock Site 2 and Gomez Spain tiles. Significantly, the mean value of excess lifetime cancer risk of 0.0014 is slightly lower than the world average value of 0.29 × 10^?3.     

Modified protocols for rapid carrot genomic DNA extraction and AFLP™ analysis using silver stain or radioisotopes

AFLP^™ is one of the most frequently used techniques for identification of molecular markers. We have modified the procedures for genomic DNA extraction, AFLP product generation and silver staining in order to speed up analyses and screen large numbers of plant samples. Using this protocol, we were able to achieve an 82% reduction of costs without compromising the reliability and quality of data gathered.     

Radon in dwellings: The importance of ventilation

Summary People who live in industrial countries receive a radioactive dose of 2.4 mSv/y: the most important contribution to this dose is given by radon decay products. Radon is a noble gas generated from the disintegration of radium, which is found in soils and building materials. Owing to a bad air circulation, radon and its daughters may accumulate in a house. By using a mathematical model it may be shown that radon concentration is inversely proportional to the ventilation rate. Measurements of radon concentration through the method of activated carbon canisters, show that in a room with a double-pane window, kept continuously closed, the mean radon concentration can exceed the concentration of a similar room with a single-pane window of 190%. However, the radon concentration inside the energy-saving room may be decreased up to values slightly higher than those measured in the conventional window room, by simply opening the double-pane window a few minutes a day. A set of measurements carried out in a group of house in Reggio Emilia validates the effectiveness of that practice: radon concentrations in rooms with double-pane window closely approximated to those of rooms with ordinary window.