Nonisotopic Method Research Articles

Biochar increases 15N fertilizer retention and indigenous soil N uptake in a cotton-barley rotation system

Biochar amendments can modify fertilizer nitrogen (N) availability in soil and crop N uptake. However, how biochar addition affects crop N uptake and fertilizer N recovery under various N levels is not yet well understood. To address this question, we conducted a two-season [cotton (Gossypium hirsutum L.)-barley (Hordeum vulgare L.) rotation] pot experiment that included four N fertilizer rates (0, 75, 150, and 300 kg N ha−1, supplied as urea-15N) combined with two straw-biochar rates (0 and 15 t ha−1). Soil properties, plant root morphology, N uptake, and biomass yield were studied. Biochar addition decreased soil inorganic N content but increased urea-N retention at cotton harvest, leading to 32% of the applied urea-N accumulating in soil compared with 27% without biochar, averaged across fertilizer N rates. Use of 15N fertilizer showed that biochar increased plant uptake of indigenous soil N, not fertilizer N. An obvious decrease in urea-15N recovery induced by biochar was observed at 75 kg N ha−1, but not at 150 or 300 kg N ha−1. The efficiency of urea-15N recovery by plants (15NRE, 34–45%), measured using the tracer method, was much lower than that measured using the traditional non-isotope method (NRE, 67–96%). At barley harvest, 2–5% of the urea-N, applied in the first season, was taken up by plants, and 12–19% remained in soils receiving biochar. We concluded that straw-biochar addition increased soil 15N fertilizer retention and plant N uptake of indigenous soil N, not fertilizer N, and the increased indigenous soil N uptake persisted into the second cropping season.

1844-P: Long-Term Remission of Type 2 Diabetes after Weight Loss Requires Effective Clearance of Hepatic VLDL-TG and Maintenance of Low Intrapancreatic Fat

The Diabetes Remission Clinical Trial (DiRECT) demonstrated initial remission of type 2 diabetes in about half of a large primary care population, and this was associated with decrease in intrapancreatic fat content and return of beta cell function. We have carried out detailed metabolic studies on a subgroup at 2 years, comparing those who remained in remission (HbA1c <6.5%; n=20, 7F/13M, 54.6±7.3 years, BMI 34.5±4.9kg/m2), and those who failed to maintain the initial remission (n=13, 6F/7M, 52.6±8.3 years, BMI 34.9±3.7kg/m2). Pancreas and liver fat were quantified by 3-point Dixon MRI. Hepatic VLDL-triglyceride (VLDL1-TG) production was measured in vivo using a non-isotopic competitive blocking method. Remission of diabetes was associated with major decreases in liver fat, pancreas fat, and hepatic VLDL-TG production during the weight loss phase between baseline to 5 months in both groups. However, those who failed to maintain remission were characterized by more weight regain (11. 3±1.9 vs. 6.6±1.0 kg, p=0.036), and greater increases in both liver (6.2±1.1 vs. 3.6±0.9%, p=0.05), and pancreas fat (+1.0±0.39 vs. -0.48±0.25%, p=0.005) between 5 months and 24 months. Hepatic VLDL-TG pool level remained stable under remission conditions (1046.4± 204.6 to 1638.6± 372.11 mg, p=0.35), whereas there was a significant increase when remission was lost (1328.0± 271.8 to 2865.6±692.4mg, p=0.04). In those who failed to maintain remission, fasting VLDL-TG increased (0.46±0.10 to 0.84±0.17 mmol/l, p<0.05), and there was a positive correlation between the rise in liver fat and intrapancreatic fat (r=0.64, p=0.018). These data emphasize the link between hepatic VLDL-TG-metabolism and accumulation of fat within the liver and pancreas in the pathogenesis of type 2 diabetes, provide further evidence for the role of fat in its pathophysiology, and show that longer term remission of type 2 diabetes is dependent upon avoidance of weight regain. Disclosure A. Al-Mrabeh: None. S.V. Zhyzhneuskaya: None. A.C. Barnes: None. K.G. Hollingsworth: None. N. Sattar: Advisory Panel; Self; Amgen Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Novo Nordisk A/S. Consultant; Self; NAPP Pharmaceuticals Limited. Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Speaker's Bureau; Self; Amgen Inc., Eli Lilly and Company, Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Roche Diagnostics France, Sanofi. M.E. Lean: Consultant; Self; Counterweight Ltd., Novo Nordisk A/S. Research Support; Self; Novo Nordisk A/S. R. Taylor: Advisory Panel; Self; Wilmington Healthcare. Speaker's Bureau; Self; Lilly Diabetes, Novartis AG. Funding Diabetes UK

Diabetes Remission Clinical Trial (DiRECT)—Changes in Hepatic VLDL1-TG Production and Intrapancreatic Fat during Weight Maintenance Phase

Type 2 diabetes is characterized by excess hepatic and intrapancreatic fat deposition. Liver-derived Very Low Density Lipoprotein-Triglyceride (VLDL1-TG) delivers fat to all peripheral tissues. Relative changes in VLDL1-TG production and intrapancreatic fat were investigated in a sub-group of the prospective, randomized Diabetes Remission Clinical Trial (DiRECT). Individuals with complete datasets at 12 months (n=45) were included. Detailed metabolic tests were carried out at baseline, 4 months, and 12 months after low calorie diet (825-853 kcal/day). Intra-organ fat was quantified using 3-point Dixon MRI, VLDL1-TG production was quantified using a competitive blocking non-isotopic method, and insulin secretion was measured by the Stepped Insulin Secretion Test with Arginine (SISTA). Weight loss induced major changes in liver and intrapancreatic fat with 69% remission of diabetes (responders, defined as HbA1c <6.5%). This was followed by weight regain between 4-12 months in responders (82.3±2.8 to 85.4±3.2kg, p=0.001) and non-responders (84.5±3.2 to 89.6±3.3kg, p<0.0001). However, liver fat, VLDL1-TG production, and intrapancreatic fat remained stable in responders (2.5 ±1.9 to 2.9±0.6%, p=0.20; 386.7±30.2 to 428.5±21.2 mg/kg/day, p=0.14; and 8.0±0.5 to 7.7 ±0.4%, p=0.26, respectively). In contrast, these parameters increased in non-responders (2.7±0.5 to 6.2 ±1.8%, p=0.02; 460.2±39.8 to 596.2±36.6 mg/kg/day, p=0.001; and 6.7±0.3 to 7.0±0.4%, p=0.18, respectively). The recovered first phase insulin secretion in responders continued to improve between 4-12 months (0.13±0.02 to 0.17±0.04 nmol/min/m2, p=0.17). There was no change in the non-responders (0.03±0.01 to 0.03±0.01 nmol/min/m2, p=0.98). These data consistent with VLDL1-TG being the link between liver fat and intrapancreatic fat, and a major modulator determining remission of type 2 diabetes. Disclosure A. Al-Mrabeh: None. S.V. Zhyzhneuskaya: None. C. Peters: None. A.C. Barnes: None. B. Aribisala: None. K.G. Hollingsworth: None. N. Sattar: Advisory Panel; Self; Boehringer Ingelheim GmbH. Speaker's Bureau; Self; Boehringer Ingelheim GmbH, Janssen Pharmaceuticals, Inc.. Advisory Panel; Self; Novo Nordisk A/S. Speaker's Bureau; Self; Novo Nordisk A/S, Eli Lilly and Company. Research Support; Self; Boehringer Ingelheim GmbH. Advisory Panel; Self; Amgen Inc.. Speaker's Bureau; Self; Amgen Inc., AstraZeneca, Mitsubishi Tanabe Pharma Corporation, Medscape, Sanofi-Aventis Deutschland GmbH. M.E. Lean: Advisory Panel; Self; Novo Nordisk Inc., Orexigen Therapeutics, Inc.. Research Support; Self; Cambridge Weight Plan. Consultant; Self; Counterweight Ltd. Stock/Shareholder; Self; Eat Balanced. R. Taylor: None.

GoldCLIP: Gel-omitted Ligation-dependent CLIP

Protein–RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins (RBPs) by the UV-crosslinking and immunoprecipitation (CLIP) represents one of the most powerful methods to map protein–RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method (GoldCLIP) that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein (PTB), a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.

Receptor Binding Affinities of Synthetic Cannabinoids Determined by Non-Isotopic Receptor Binding Assay

A major predictor of the efficacy of natural or synthetic cannabinoids is their binding affinity to the cannabinoid type I receptor (CB1) in the central nervous system, as the main psychological effects of cannabinoids are achieved via binding to this receptor. Conventionally, receptor binding assays have been performed using isotopes, which are inconvenient owing to the effects of radioactivity. In the present study, the binding affinities of five cannabinoids for purified CB1 were measured using a surface plasmon resonance (SPR) technique as a putative non-isotopic receptor binding assay. Results were compared with those of a radio-isotope-labeled receptor binding assay. The representative natural cannabinoid Δ9-tetrahydrocannabinol and four synthetic cannabinoids, JWH-015, JWH-210, RCS-4, and JWH-250, were assessed using both the SPR biosensor assay and the conventional isotopic receptor binding assay. The binding affinities of the test substances to CB1 were determined to be (from highest to lowest) 9.52 × 10−13 M (JWH-210), 6.54 × 10−12 M (JWH-250), 1.56 × 10−11 M (Δ9-tetrahydrocannabinol), 2.75 × 10−11 M (RCS-4), and 6.80 ×10−11 M (JWH-015) using the non-isotopic method. Using the conventional isotopic receptor binding assay, the same order of affinities was observed. In conclusion, our results support the use of kinetic analysis via SPR in place of the isotopic receptor binding assay. To replace the receptor binding affinity assay with SPR techniques in routine assays, further studies for method validation will be needed in the future.

IrCLIP platform for efficient characterization of protein-RNA interactions.

The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein-RNA interactions using far less material than standard protocols.

The yeast ζ-crystallin/NADPH:quinone oxidoreductase (Zta1p) is under nutritional control by the target of rapamycin pathway and is involved in the regulation of argininosuccinate lyase mRNA half-life.

The yeast ζ-crystallin (Zta1p) is a quinone oxidoreductase belonging to the ζ-crystallin family, with activity in the reduction of alkenal/alkenone compounds. Various biological functions have been ascribed to the members of this protein family, such as their ability to interact specifically with AU-rich sequences in mRNA, and thus they have been proposed to act as AU-rich element-binding proteins (AREBPs). In this study, we evaluated the specificity of Zta1p for RNA versus DNA by means of a novel nonisotopic method for the in vitro quantitative detection of protein · RNA complexes. Through comparative transcriptomic analysis, we found that the lack of Zta1p negatively affects the expression of a group of genes involved in amino acid biosynthesis, the argininosuccinate lyase (ARG4) gene being one of them. Here, we propose that Zta1p participates in the post-transcriptional regulation of ARG4 expression by increasing the ARG4 mRNA half-life. In addition, expression of the ζ-crystallin gene (ZTA1) is itself regulated by nutrient availability through the general amino acid control and target of rapamycin pathways. Our results shed new light on the ζ-crystallin family members from yeast to humans as stress response proteins with a bifunctional role in the detoxification of alkenal and alkenone compounds, and the regulation of gene expression.

Assessing the stability of soil organic matter by fractionation and 13C isotope techniques

Carbon pools of different stabilities have been separated from the soil organic matter of agrochernozem and agrogray soil samples. The work has been based on the studies of the natural abundance of the carbon isotope composition by C3-C4 transition using the biokinetic, size-density, and chemical fractionation (6 M HCl hydrolysis) methods. The most stable pools with the minimum content of new carbon have been identified by particle-size and chemical fractionation. The content of carbon in the fine fractions has been found to be close to that in the nonhydrolyzable residue. This pool makes up 65 and 48% of Corg in the agrochernozems and agrogray soils, respectively. The combination of the biokinetic approach with particle-size fractionation or 6 M HCl hydrolysis has allowed assessing the size of the medium-stable organic carbon pool with a turnover time of several years to several decades. The organic matter pool with this turnover rate is usually identified from the variation in the 13C abundance by C3-C4 transition. In the agrochernozems and agrogray soils, the medium-stable carbon pool makes up 35 and 46% of Corg, respectively. The isotope indication may be replaced by a nonisotope method to significantly expand the study of the inert and mediumstable organic matter pools in the geographical aspect, but this requires a comparative analysis of particle-size and chemical fractionation data for all Russian soils.

The Clinical Utility of Free Thyroxine in Oral Levothyroxine Absorption Testing

Original absorption studies for levothyroxine (LT4) were validated using total thyroxine (TT4) measurements. Free thyroxine (FT4) has largely supplanted TT4 in clinical practice. The objective of our study was to assess the clinical utility of FT4 in oral LT4 absorption testing. In this retrospective electronic health record analysis, we recorded data of patients who underwent LT4 oral absorption testing between November 2010 and January 2012 because of persistent hypothyroidism despite a greater than anticipated weight-based dose of LT4. Patients included had primary hypothyroidism and an absorption test with assessment of both TT4 and FT4 measured at times 0, 30, 60, 90, 120, 180, 240, 300, and 360 minutes. The test was conducted with 1 mg (five 200-μg tablets) of Synthroid® after an overnight fast by a standard nonisotopic method. A total of 10 patients (3 men/7 women) underwent absorption testing. Prior to testing, the median daily LT4 dose was 250 μg (range, 150 to 350 μg). Three patients were also on liothyronine (10, 20, or 50 μg daily). Based on the calculated amount absorbed, 1 patient demonstrated subnormal absorption, and 9 patients were normal. Median body mass index was 33 kg/m2 (range, 21 to 50 kg/m2). Median calculated absorption was 105% (range, 3.7 to 195.6%). The correlation comparing FT4 and TT4 was 0.88 (95% confidence interval, 0.56 to 0.97; P<.001), a significant correlation. FT4 and TT4 correlated highly, even in patients who were severely hypothyroid; FT4 may be used interchangeably with TT4 as a qualitative assessment of suspected malabsorption using an oral LT4 absorption test.

Performance of formulae based estimates of glomerular filtration rate for carboplatin dosing in stage 1 seminoma

BackgroundSingle cycle carboplatin, dosed by glomerular filtration rate (GFR), is standard adjuvant therapy for stage 1 seminoma. Accurate measurement of GFR is essential for correct dosing. Isotopic methods remain the gold standard for the determination of GFR. Formulae to estimate GFR have improved the assessment of renal function in non-oncological settings. We assessed the utility of these formulae for carboplatin dosing. MethodsWe studied consecutive subjects receiving adjuvant carboplatin for stage 1 seminoma at our institution between 2007 and 2012. Subjects underwent 51Cr-ethylene diamine tetra-acetic acid (EDTA) measurement of GFR with carboplatin dose calculated using the Calvert formula. Theoretical carboplatin doses were calculated from estimated GFR using Chronic Kidney Disease-Epidemiology (CKD-EPI), Management of Diet in Renal Disease (MDRD) and Cockcroft–Gault (CG) formulae with additional correction for actual body surface area (BSA). Carboplatin doses calculated by formulae were compared with dose calculated by isotopic GFR; a difference <10% was considered acceptable. Results115 patients were identified. Mean isotopic GFR was 96.9ml/min/1.73m2. CG and CKD-EPI tended to overestimate GFR whereas MDRD tended to underestimate GFR. The CKD-EPI formula had greatest accuracy. The CKD-EPI formula, corrected for actual BSA, performed best; 45.9% of patients received within 10% of correct carboplatin dose. Patients predicted as underdosed (13.5%) by CKD-EPI were more likely to be obese (p=0.013); there were no predictors of the 40.5% receiving an excess dose. ConclusionsOur data support further evaluation of the CKD-EPI formula in this patient population but clinically significant variances in carboplatin dosing occur using non-isotopic methods of GFR estimation. Isotopic determination of GFR should remain the recommended standard for carboplatin dosing when accuracy is essential.

The Synthesis and characteristic study of transferrin-conjugated liposomes carrying brain-derived neurotrophic factor

This study aimed to establish a novel non-viral liposome vector delivering brain derived neurotrophic factor (BDNF) through the blood brain barrier. For this purpose, different water-oil ratios were tested to create liposomes for packaging the prophase synthesized plasmids encoding the BDNF proteins. In order to increase the targeted and peripheral circulation time, we connected the liposomes with transferrin (Tf) and a polyethylene glycol (PEG) long chain. The non-isotope method was used to measure the liposome envelopment ratio and ligand-binding ratio, and also to detect molecular biological features, such as particle size and stability. Tf-conjugated liposomes could be synthesized satisfactorily under the following conditions: the ratio of phospholipid to cholesterol was 1:1; the ratio of enter to plasmid was 100:1; oil phase was dichloromethane; the oil to water ratio was 4:1; the rotary evaporation temperature was 30 °C; the ultrasonic temperature was 10 °C; the ultrasonic time was 10 min; and 10% trehalose was in the presence. Generated liposomes had a uniform circular shape and particle size distribution. In this experiment, we successfully established a new type of Tf-conjugated liposomes carrying the gene of BDNF and the study provides an experimental basis for the future.

Serum 7-Alpha-Hydroxy-4-Cholesten-3-One as a Marker for Bile Acid Loss in Children

To establish age-related reference values for 7-alpha-hydroxy-4-cholesten-3-one (C4) in a pediatric population and to investigate bile acid malabsorption in children with short bowel syndrome (SBS). Serum was obtained between 8:00 a.m. and 11:00 a.m. from 100 healthy children (52% males, 9 months to 18 years of age) after 10 hours of fasting. Pediatric patients with SBS served as disease controls (n = 12). Following solid-phase extraction and purification, C4 was determined by high-performance liquid chromatography using a ultraviolet detector at a wavelength of 241 nm. The upper limit of normal for C4 concentrations was defined as the mean plus 2 SD of the log-normal distribution. The mean concentration and SD of C4 in healthy children was 22.8 ± 15.8 ng/mL with no relation to age or sex and an upper limit of normal of 66.5 ng/mL. Normal C4 values were found in 97 of 100 healthy children, and all 12 patients with SBS had C4 concentrations above 100 ng/mL (mean 299.6 ± 167.8 ng/mL; range 105.7-562.1 ng/mL, P < .0001 compared with controls). The determined upper limit of normal for C4 concentration in healthy children corresponds to previously published levels in healthy adults and is independent of age and sex. The consistently elevated C4 concentrations in our patients with SBS confirm the reliability of this noninvasive, nonisotopic method to assess bile acid malabsorption in children.

Can estimated glomerular filtration rate (eGFR) replace isotopic measurement of GFR for carboplatin dosing in stage 1 seminoma?

2589 Background: Accurate determination of GFR is essential for correct dosing of carboplatin, the standard adjuvant therapy for stage 1 seminoma. Isotopic methods (e.g. 51Cr-EDTA) remain the gold standard for determination of GFR. However, the use of eGFR could reduce the need for such isotope studies. As novel formulae to estimate GFR such as CKD-EPI and MDRD4 have improved the assessment of renal function in non-oncological settings, we investigated their utility for carboplatin dosing. Methods: 115 patients (pts) (mean age 40.3, std dev. 10.1) who received adjuvant carboplatin for stage 1 seminoma at our institution between 2007-2012 were identified. All pts underwent 51Cr-EDTA measurement of GFR with carboplatin dose calculated using the Calvert formula, based on GFR uncorrected for body surface area (BSA). Theoretical carboplatin doses were then calculated using eGFR values obtained using the CKD-EPI and MDRD4 formulae with additional calculation to uncorrect for BSA. Creatinine clearance was calculated by Cockcroft-Gault (CG) formula. For each pt the carboplatin doses calculated by eGFR were compared with the actual dose calculated by the gold-standard method; a difference of less than 10% was considered acceptable. Results: The Table shows the percentage of pts who would have received an equivalent carboplatin dose using each eGFR formula compared to the dose calculated using 51Cr-EDTA. The CKD-EPI formula performed best with 58.9% of pts receiving within 10% of the correct dose. Pts predicted to be underdosed by CKD-EPI eGFR were more likely to be obese (BMI &gt;30) (p=0.01); there were no predictors of the 18.8% who would have received an excess dose. Conclusions: Our data support further evaluation of the CKD-EPI formula but highlight the clinically significant variances in carboplatin dosing when using non-isotopic methods of GFR estimation. [Table: see text]

Absolute quantification of NAD(P)H:quinone oxidoreductase 1 in human tumor cell lines and tissues by liquid chromatography–mass spectrometry/mass spectrometry using both isotopic and non-isotopic internal standards

NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase) is a prognostic biomarker and a potential therapeutic target for various tumors. Therefore, it is of significance to develop a robust method for the absolute quantification of NQO1. This study aimed to develop and validate a LC–MS/MS based method and to test the appropriateness of using non-isotopic analog peptide as the internal standard (IS) by comparing with a stable isotope labeled (SIL) peptide. The chromatographic performance and mass spectra between the selected signature peptide of NQO1 and the non-isotopic peptide were observed to be very similar. The use of the two internal standards was validated appropriate for the absolute quantification of NQO1, as evidenced by satisfactory validation results over a concentration range of 1.62–162fmolμL−1. This method has been successfully applied to the absolute quantification of NQO1 expression in various tumor cell lines and tissues. NQO1 expression in human tumor tissues is much higher than that in the neighboring normal tissues in both the cases of lung and colon cancer. The quantitative results obtained from the isotopic and non-isotopic methods are quite similar, further supporting that the use of non-isotopic analog peptide as internal standard is appropriate and feasible for the quantification of NQO1. By comparing with a classical isotopic IS, the present study indicates that the use of a non-isotopic peptide analog to the proteotypic peptide as the internal standard can get equal accuracy and preciseness in measuring NQO1. The universal applicability of the non-isotopic IS approach for the quantification of proteins warrants further research.

Effects of mutations on herpes simplex virus 1 thymidine kinase functionality: An in vitro assay based on detection of monophosphate forms of acyclovir and thymidine using HPLC/DAD

Discrimination between the mutations responsible for drug resistance and those of UL23 TK gene polymorphism can be difficult. A non-isotopic method has been developed to assess TK functionality by measuring monophosphate forms of both acyclovir (ACV) and thymidine using HPLC/DAD. Phenotypes of TKs could thus be characterized as TK altered (P84L, A189V, L227F), TK deficient (G200S, L291P) or TK partial (R163H). A reliable link between HSV UL23 TK mutations and ACV resistance is necessary for developing a powerful genotyping tool to detect ACV resistance quickly in clinical samples.

Use of Stable Isotopes Deuterium and Oxygen-18 to Derive Evaporation from Flood Irrigation on the Basis of Pan Evaporation Techniques

The loss of water to the atmosphere during flood irrigation occurs through evaporation and transpiration. Whereas transpiration can be estimated through the FAO56 methodology, actual evaporation is difficult to quantify in water balance studies. In this study, two analytical models, previously developed to quantify evaporation from lakes on the basis of stable isotopes, were applied to determine evaporation losses from four flood irrigation sites of varied characteristics. Evaporation losses were determined by empirical relationships derived between heavy isotope enrichment and percentage of water loss in evaporation pan experiments. Validation of the two isotopic models in this setting was achieved by comparison with conventional nonisotopic methods, carried out in parallel. Results showed that heavy isotope enrichment of applied irrigation waters varied among each of the study sites. Isotope enrichment was notably different among irrigation bays that drained rapidly [+0.05 to +0.18 per mil (‰) for δO18 ...

A method for detecting genetic toxicity using the RNA synthesis response to DNA damage

To date, biological risk assessment studies of chemicals that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication but also with transcription. Therefore, detecting the damaging effects of DNA lesions during transcription might be important for estimating the safety of chemical mutagens and carcinogens. However, methods to address these effects have not been developed. Here, we report a simple, non-isotopic method for determining the toxicity of chemical agents by visualizing transcription in a mammalian cell system. The method is based on the measurement of the incorporation of bromouridine (as the uridine analogue) into the nascent RNA during RNA synthesis inhibition (RSI) induced by the stalling of RNA polymerases at DNA lesions on the transcribed DNA strand, which triggers transcription-coupled nucleotide excision repair (TC-NER). When we tested chemical agents (camptothecin, etoposide, 4-nitroquinoline-1-oxide, mitomycin C, methyl methanesulfonate, and cisplatin) in HeLa cells by the method, RSI indicative of genomic toxicity was observed in the nucleoli of the tested cells. This procedure provides the following advantages: 1) it uses common, affordable mammalian cells (HeLa cells, WI38VA13 cells, human dermal fibroblasts, or Chinese hamster ovary cells) rather than genetically modified microorganisms; 2) it can be completed within approximately 8 hr after the cells are prepared because RNA polymerase responses during TC-NER are faster than other DNA damage responses (replication, recombination, and apoptosis); and 3) it is safe because it uses non-radioactive bromouridine and antibodies to detect RNA synthesis on undamaged transcribed DNA strands.

Assessment of glucose disposal with the hyperglycaemic clamp technique during low intensity exercise in Warmblood horses

The quantity of glucose disposal during exercise (walk and trot) compared to rest by use of the hyperglycaemic clamp technique has not been reported previously and has relevance to nutritional requirements. Exercise (walk and trot) significantly increases glucose disposal compared to rest. Seven healthy Dutch Warmblood mares, all in dioestrus, mean ± s.d. age 11.6 ± 2.4 years and weighing 569 ± 40 kg were fasted for 12 h prior to a hyperglycaemic clamp at rest (maintaining a steady state of the blood glucose concentration during 30 min), walk (10 min, 1.5 m/s), trot (20 min, 4.4 m/s), walk (10 min, 1.5 m/s) and rest again (maintaining a steady state during 30 min). Plasma glucose concentrations were measured every 5 min. The mean rate of glucose disposal was calculated by corrections for glucose loss via the glucose space and urine. A one-way ANOVA with post hoc Bonferroni was performed. The mean ± s.d. rate of glucose disposal was 15.0 ± 2.1 at first rest, 25.1 ± 6.2 at first walk, 37.4 ± 9.1 at trot, 33.0 ± 13.1 at second walk and 18.7 ± 4.6 µmol/kg bwt/min at second rest. Values at trot and at second walk differed significantly from values at first rest, whereas values at both rests were similar as well as at first rest and at first walk. Mean rate of glucose disposal of Warmblood horses increased 2.5 times during trot compared to basal. The hyperglycaemic clamp technique is an attractive nonisotope method to assess the rate of glucose disposal in exercising horses.

Real-time monitoring of DNAzyme cleavage process using fluorescent assay

Detection of deoxyribozyme (DNAzyme) cleavage process usually needs complex and time-consuming radial labeling, gel electrophoresis and autoradiography. This paper reported an approach to detect DNAzyme cleavage process in real time using a fluorescence probe. The probe was employed as DNAzyme substrate to convert directly the cleavage information into fluorescence signal in real time. Compared with traditional approach, this non-isotope method not only brought a convenient means to monitor the DNAzyme cleavage reaction, but also offered abundant dynamic data for choosing potential gene therapeutic agents. It provides a new tool for DNAzyme research, as well as a new insight into research on human disease diagnosis. Based on this method, 8–17deoxyribozyme (8–17DNAzyme) against hepatitis C virus RNA (HCV-RNA) was designed and the cleavage process was studied in real time.

Development of an isoenzyme-specific colorimetric assay for tissue transglutaminase 2 cross-linking activity

Tissue transglutaminase (TGase 2) belongs to the multigene transglutaminase family of Ca 2+-dependent protein cross-linking enzymes. Based on the transamidation activity of TGase 2, a novel colorimetric assay has been developed using covalently coupled spermine to carboxy-substituted polystyrene plates and biotinylated pepT26, an excellent acyl-donor substrate, highly specific for TGase 2. The assay is based on the incorporation of the gamma-carboxamide of glutamine of pepT26 into the immobilized spermine. The amount of biotinylated pepT26 bound to the plate, as measured by the activity of streptavidin-peroxidase, is directly proportional to the TGase activity. The colorimetric procedure showed a good correlation ( r = 0.995) with the commonly used radiometric filter paper method for TGase2, and provides linear dose–response curves over a wide range of hrTGase2 concentrations (2.5–40 μU/ml). In addition, the assay shows higher sensitivity when compared with our previous TG-colorimetric test (more than 50-fold increase) and other existing assays. PepT26 displays strong reactivity with TGase 2, and no reactivity with TGases 1, 3, and FXIII. The procedure constitutes a rapid, TG2-specific, sensitive, and nonisotopic method for the measurement of TGase 2 activity in as low as 4 ng of hrTGase 2 and purified guinea pig liver transglutaminase, and 1.25 μg of guinea pig liver extracts.