Measurement Of Specific Activity Research Articles

Click Chemistry Mediated Eu-Tagging: Activity-Based Specific Quantification and Simultaneous Activity Evaluation of CYP3A4 Using 153Eu Species-Unspecific Isotope Dilution Inductively Coupled Plasma Mass Spectrometry

P450 3A4 (CYP3A4) is one of the most important isoforms in the human cytochrome P450 superfamily. It was used as an example in this proof-of-concept study in order to demonstrate an activity-based labeling and then click chemistry (CC) mediated element-tagging strategy for simultaneously specific quantification and activity measurement of an enzyme using species-unspecific isotope dilution inductively coupled plasma mass spectrometry (SUID ICPMS). A dual functional hexynylated 17α-ethynylestradiol activity-based probe was synthesized for specifically labeling CYP3A4 and then CC-mediated Eu-tagging with an azido-DOTA-Eu complex for CYP3A4 quantification and activity measurement in human liver microsome and serum samples using (153)Eu SUID ICPMS. The LOD (3σ) of CYP3A4 reached 20.3 fmol when monitoring (151/153)Eu ICPMS signals, in addition to the merits of specificity and simultaneous activity measurement achieved. We believe that this activity-based CC-mediated element-tagging strategy will liberate more potential advantages of ICPMS in bioanalysis.

Specific activity measurement of 64Cu: A comparison of methods

Effective specific activity of 64Cu (amount of radioactivity per µmol metal) is important in order to determine purity of a particular 64Cu lot and to assist in optimization of the purification process. Metal impurities can affect effective specific activity and therefore it is important to have a simple method that can measure trace amounts of metals. This work shows that ion chromatography (IC) yields similar results to ICP mass spectrometry for copper, nickel and iron contaminants in 64Cu production solutions.

Implementation of the indirect assessment method for 129I and 135Cs accumulation in the RBMK-1500 reactor coolant purification system

The transport of long-lived radionuclides 129I and 135Cs in technologic chains of the Ignalina NPP and their accumulation in by-pass purification system filtering materials was investigated by spectrometric measurements and mathematical modelling. The database of regular gamma spectrometric measurements performed at the NPP was analysed. Results of gamma spectrometric measurement of specific activities of 131I and other short-lived isotopes of iodine as well as of 134Cs and 137Cs isotopes have been analysed and parameters specific to the reactor operation have been estimated. Accumulated activities of 129I and 135Cs in spent filtering materials of the reactor by-pass purification system as well as specific activities are evaluated. Specific activities are compared with preliminary acceptance criteria for a near surface repository which is at the stage of design at the Ignalina NPP. Moreover, scaling factors for these radionuclides for the by-pass purification system radioactive waste are assessed.

SAT0295 Comparison of Composite Measures of Disease Activity in Psoriatic Arthritis Using Data from an Interventional Study with Golimumab.

BackgroundFour new disease specific composite disease activity measures have been developed for psoriatic arthritis (PsA). Further validation of these measures in interventional trial data is now required.ObjectivesThe objective of this...

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

BackgroundMammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs. MethodsReducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis. ResultsOxidation of the CP is fast (k+1>103M−1s−1), however the rate of reduction by GSH is slow (k′+2=12.6M−1s−1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+1>103M−1s−1), but not by Trx. By surface plasmon resonance analysis, a KD=5.2μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo. ConclusionsGPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates. General significanceIn the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.

Development of collimator for in-situ measurement of 90Sr specific activity by β-ray survey meter and Monte Carlo calculation

For the in-situ measurement of 90Sr contamination, the collimators to be combined with the β-ray survey meter were designed with MCNP-4C calculations. The designed collimators were manufactured and its characteristics were measured in accordance with the calculations. The calculated energy deposition in the survey meter agreed within 27% to its counting rate measured in terms of their dependencies on the collimator dimension. This supports the usability of the manufactured collimators and numerical correction of the sensitivity of the survey meter depending on the geometry.

Disease Activity Measures in Paediatric Rheumatic Diseases

Disease activity refers to potentially reversible aspects of a disease. Measurement of disease activity in paediatric rheumatic diseases is a critical component of patient care and clinical research. Disease activity measures are developed systematically, often involving consensus methods. To be useful, a disease activity measure must be feasible, valid, and interpretable. There are several challenges in quantifying disease activity in paediatric rheumatology; namely, the conditions are multidimensional, the level of activity must be valuated in the context of treatment being received, there is no gold standard for disease activity, and it is often difficult to incorporate the patient's perspective of their disease activity. To date, core sets of response variables are defined for juvenile idiopathic arthritis, juvenile systemic lupus erythematosus, and juvenile dermatomyositis, as well as definitions for improvement in response to therapy. Several specific absolute disease activity measures also exist for each condition. Further work is required to determine the optimal disease activity measures in paediatric rheumatology.

Granular biomass selection in a double-stage biogas collection UASB reactor: effects on SMA, abundance and diversity of the methanogenic population

The present work aimed at investigating biomass selection in a pilot-scale double-stage biogas collection (DSBC) upflow anaerobic sludge bed (USAB) reactor treating domestic wastewater. Specific methanogenic activity (SMA) measurements and FISH countings were applied to sludge samples collected during 102 days of operation of the DSBC-UASB and of a control reactor. Results showed that both reactors presented similar SMA values in early stages of operation however the UASB-DSBC reactor showed much higher SMA after day 45, when the biomass was in granular stage. In terms of archaeal abundance, no statistical difference was observed between the reactors. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) revealed a similar composition of the archaeal communities in the two reactors and during the operational period, mainly constituted by Methanosaeta concilii. The results suggest that cell activity rather than archaeal abundance or diversity drive the methane production in the UASB reactors.

Measurement of Specific Activity of Dihydrofolate Reductase (DHFR) Enzyme and Determination of Folate Content Extracted from UV-Exposed Stem- Derived Callus of Sunflower (Helianthus annuus L.)

Measurement of Specific Activity of Dihydrofolate Reductase (DHFR) Enzyme and Determination of Folate Content Extracted from UV-Exposed Stem- Derived Callus of Sunflower (Helianthus annuus L.)

Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency

Type II restriction endonucleases (REases) are one of the basic tools of recombinant DNA technology. They also serve as models for elucidation of mechanisms for both site-specific DNA recognition and cleavage by proteins. However, isolation of catalytically active mutants from their libraries is challenging due to the toxicity of REases in the absence of protecting methylation, and techniques explored so far had limited success. Here, we present an improved SOS induction-based approach for in vivo screening of active REases, which we used to isolate a set of active variants of the catalytic mutant, Cfr10IE204Q. Detailed characterization of plasmids from 64 colonies screened from the library of ∼200 000 transformants revealed 29 variants of cfr10IR gene at the level of nucleotide sequence and 15 variants at the level of amino acid sequence, all of which were able to induce SOS response. Specific activity measurements of affinity-purified mutants revealed >200-fold variance among them, ranging from 100% (wild-type isolates) to 0.5% (S188C mutant), suggesting that the technique is equally suited for screening of mutants possessing high or low activity and confirming that it may be applied for identification of residues playing a role in catalysis.

Health assessment of natural radioactivity and radon exhalation rate in granites used as buildingmaterials in Lebanon

Measurements of specific activities (Bq kg(-1)) of gamma-emissions from radioactive nuclides, (238)U, (226)Ra, (214)Bi, (232)Th, (212)Pb and (40)K, contained in 28 granite types, used as building materials in indoors in Lebanon, were performed on the powdered granites. The concentration of the nuclides, (226)Ra, (232)Th and (40)K, in the granites varied from below detection level (BDL) to 494 Bq kg(-1), BDL to 157.2 Bq kg(-1) and BDL to 1776 Bq kg(-1), respectively. (226)Ra concentration equivalents, C(Raeq), were obtained and ranged between 37 and 591 Bq kg(-1), with certain values above the allowed limit of 370 Bq kg(-1). Calculated annual gamma-absorbed dose in air, D(aR), varied from 17.7 to 274.5 (nGy h(-1)). Annual effective dose, E (mSv y(-1)), of gamma radiations related to the studied granites and absorbed by the inhabitants was evaluated. E (mSv y(-1)) ranged from 0.09 to 1.35 mSv y(-1). Some granite types produced E above the allowed limit of 1 mSv y(-1) set by ICRP. Values of (222)Rn mass exhalation rate, E(M) (mBq kg(-1)h(-1))(,) in granite powder were obtained using the CR-39 detector technique. Diffusion factors, f, in 23 granite types were calculated with f ranging between (0.1 ± 0.02)×10(-2) and (6.6 ± 1.01)×10(-2).

Binding of von Willebrand factor cleaving protease ADAMTS13 to Lys-plasmin(ogen)

The metalloprotease ADAMTS13 affects platelet adhesion and aggregation through depolymerization of von Willebrand factor (VWF) multimers. Identification of ADAMTS13-binding proteins would reveal the hitherto unrecognized mechanisms underlying microvascular thrombus. To identify ADAMTS13-binding proteins, we performed a yeast two-hybrid screen using the Cys-rich and spacer domains of ADAMTS13, the critical regions for the binding and cleavage of VWF, as a bait region. We identified Lys-plasminogen, an amino-terminal truncated form of plasminogen, as the binding protein to ADAMTS13. Intact Glu-plasminogen did not bind to ADAMTS13. Active-site blocked Lys-plasmin bound to ADAMTS13. Domain truncation of ADAMTS13 and elastase digest of plasminogen indicated that the Cys-rich and spacer domains of ADAMTS13 and the kringle 5 and protease domains of plasminogen served as the main binding sites. Biacore measurements revealed that Lys-plasminogen bound to ADAMTS13 with a K(d) of 1.9 ± 0.1 × 10(-7) M and Glu-plasminogen exhibited a significantly lower affinity to ADAMTS13. Specific activity measurements revealed that ADAMTS13 and Lys-plasmin were still active even after the binary complex was formed. The binding of ADAMTS13 to Lys-plasminogen may play an important role to localize these two proteases at sites of thrombus formation or vascular injury where the fibrinolytic system is activated.

238PU

The alpha spectrometry measurements of specific activity of 238Pu and 239Pu in urine from bioassay examinations of 1,013 workers employed at the radiochemical and plutonium production facilities of the Mayak Production Association and in autopsy specimens of lung, liver, and skeleton from 85 former nuclear workers who died between 1974-2009, are summarized.The accumulation fraction of 238Pu in the body and excreta has not changed with time in workers involved in production of weapons-grade plutonium production (e.g., the plutonium production facility and the former radiochemical facility). The accumulation fraction of 238Pu in individuals exposed to plutonium isotopes at the newer Spent Nuclear Fuel Reprocessing Plant ranged from 0.13% up to 27.5% based on the autopsy data. No statistically significant differences between 238Pu and 239Pu in distribution by the main organs of plutonium deposition were found in the Mayak workers. Based on the bioassay data,the fraction of 238Pu activity in urine is on average 38-69% of the total activity of 238Pu and 239Pu, which correlates with the isotopic composition in workplace air sampled at the Spent Nuclear Fuel Reprocessing Plant. In view of the higher specific activity of 238Pu, the contribution of 238Pu to the total internal dose, particularly in the skeleton and liver, might be expected to continue to increase, and continued surveillance is recommended.

Stable isotope liquid chromatography–tandem mass spectrometry assay for fatty acid amide hydrolase activity

Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for the hydrolysis of the endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) to arachidonic acid (AA) and ethanolamine (EA). Published FAAH activity assays mostly employ radiolabeled anandamide or synthetic fluorogenic substrates. We report a stable isotope liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for specific, sensitive, and high-throughput capable FAAH activity measurements. The assay uses AEA labeled with deuterium on the EA moiety (d4-AEA) as substrate and measures the specific reaction product tetradeutero-EA (d4-EA) and the internal standard 13C2-EA. Selected reaction monitoring of m/z 66→m/z 48 (d4-EA) and m/z 64→m/z 46 (13C2-EA) in the positive electrospray ionization mode after liquid chromatographic separation on a HILIC (hydrophilic interaction liquid chromatography) column is performed. The assay was developed and thoroughly validated using recombinant human FAAH (rhFAAH) and then was applied to human blood and dog liver samples. rhFAAH-catalyzed d4-AEA hydrolysis obeyed Michaelis–Menten kinetics (KM=12.3μM, Vmax=27.6nmol/minmg). Oleoyl oxazolopyridine (oloxa) was a potent, partial noncompetitive inhibitor of rhFAAH (IC50=24.3nM). Substrate specificity of other fatty acid ethanolamides decreased with decreasing length, number of double bonds, and lipophilicity of the fatty acid skeleton. In human whole blood, we detected FAAH activity that was inhibited by oloxa.

Deglycosylation of individual flavonoids and flavonoid containing plant extracts by purified human intestinal lactase-phlorizin hydrolase (LPH)

Lactase-phlorizin hydrolase (LPH, EC 3.2.123/62) is an apically-sorted glycoprotein of the small intestine. One of the two catalytic sites of LPH is responsible for hydrolyzing lactose, the main carbohydrate in milk. The second active site exhibits a broad specificity against substrates like phlorizin and glycosyl-N-acylsphingosines [1]. Phlorizin, a dihydrochalcone, belongs to the group of flavonoids often present as β-glycosides in food and herbal medicinal plant products. Bioavailability of flavonoids may depend on intestinal hydrolysis before absorption and delivery to systemic circulation. The ability of purified LPH to hydrolyze various flavonoid glycosides was investigated. Isolation of human LPH from stably transfected CHO-cells by immunoprecipitation with monoclonal anti-LPH antibodies allows specific activity measurements with known substrates, individual flavonoids and plant extracts. Deglycosylation of approximately 20 putative substrates was tested by determination of the released products by a photometric method (glucose) and by HPLC-DAD (flavonoid aglyca). Flavonoid glycosides with terminal rhamnose (e.g. rutin, naringin and quercitrin) and isoflavone glycosides (e.g. sophoricoside and genistin) were not hydrolyzed by human LPH. A cleavage of flavonoid-mono-/di-β-glycosides with glucose residues in different positions (3, 4', 3', 7) of the aglycone could be observed (e.g. kaempferol-3-O-glucoside, quercetin-4'-O-glucoside, apigenin-7-O-glucoside and luteolin-3'-7-O-diglucoside). Hydrolysis of specific flavonoids could also be shown for plant extracts of onion and curly kale. As an example about 40% of quercetin-4'-O-glucoside, the main component of an onion extract, was deglycosylated by LPH demonstrating its importance in the metabolisation of biologically active compounds.

Are one early muscle pH and one early temperature measurement sufficient to detect PSE breast meat in turkeys?

1. Within a large flock of turkey toms (2000 BUT9 conventionally reared and slaughtered), early muscle pH measurements were randomly done to distinguish two groups of birds presenting low (fast glycolysing, GR) or normal (normal glycolysing, GN) values. 2. Subsequently, ultimate pH values and meat quality parameters were also recorded. Meat quality parameters from GR or GN samples differ more or less indicating more or less severe PSE conditions. Proteins extracted from the samples at 20 min post mortem were similar while they differed greatly at 24 h post mortem. Moreover, among the GR birds, a subgroup of animals (called AB) presented SDS-PAGE profiles largely different from other GR or GN birds. 3. All the subsequent analysis developed on meat quality parameters as well as for protein extractabilities also differed between AB and other animals indicating that they must be considered differently in term of PSE syndrome development. 4. Western blots against Myosin Heavy Chain and actin at 24 h post mortem indicate that myofibrillar protein alterations are different in AB and GR or GN samples. 5. At 20 min post mortem, glycogen content was lowest in AB samples while the glycolytic potential was similar in all samples at the time of death. Measurements of PFK enzyme specific activity did not indicate a different regulation of post mortem glycolysis in AB samples. 6. Our results suggest that a unique pH measurement at 20 min post is insufficient to detect animals more prone to developing a severe PSE syndrome in turkeys. In consequence, it is suggested that a more precise evaluation of the kinetics of pH and temperature decrease has to be conducted to understand the aetiology of meat quality parameter alterations in poultry.

Air mass origin and its influence on radionuclide activities ( 7Be and 210Pb) in aerosol particles at a coastal site in the western Mediterranean

Studies of radionuclide activities in aerosol particles provide a means for evaluating the integrated effects of transport and meteorology on the atmospheric loadings of substances with different sources. Measurements of aerosol mass concentration and specific activities of 7Be and 210Pb in aerosols at Málaga (36° 43′ 40″ N; 4° 28′ 8″ W) for the period 2000–2006 were used to obtain the relationships between radionuclide activities and airflow patterns by comparing the data grouped by air mass trajectory clusters. The average concentration values of 7Be and 210Pb over the 7 year period have been found to be 4.6 and 0.58 mBq m −3, respectively, with mean aerosol mass concentration of 53.6 μg m −3. The identified air flow types arriving at Málaga reflect the transitional location of the Iberian Peninsula and show significant differences in radionuclide activities. Air concentrations of both nuclides and the aerosol mass concentration are controlled predominantly by the synoptic scenarios leading to the entrance of dust-laden continental flows from northern Africa and the arrival of polar maritime air masses, as implied by the strong correlations found between the monthly frequencies of the different air masses and the specific activities of both radionuclides. Correlations between activity concentrations and precipitation are significant though lower than with air masses.

Development of the radiosynthesis of high-specific-activity 123I-NKJ64

(123)I-NKJ64, a reboxetine analogue, is currently under development as a potential novel single photon emission computed tomography radiotracer for imaging the noradrenaline transporter in brain. This study describes the development of the radiosynthesis of (123)I-NKJ64, highlighting the advantages and disadvantages, pitfalls and solutions encountered while developing the final radiolabelling methodology. The synthesis of (123)I-NKJ64 was evaluated using an electrophilic iododestannylation method, where a Boc-protected trimethylstannyl precursor was radioiodinated using peracetic acid as an oxidant and deprotection was investigated using either trifluoroacetic acid (TFA) or 2 M hydrochloric acid (HCl). Radioiodination of the Boc-protected trimethylstannyl precursor was achieved with an incorporation yield of 92±6%. Deprotection with 2 M HCl produced (123)I-NKJ64 with the highest radiochemical yield of 98.05±1.63% compared with 83.95±13.24% with TFA. However, the specific activity of the obtained (123)I-NKJ64 was lower when measured after using 2 M HCl (0.15±0.23 Ci/μmol) as the deprotecting agent in comparison to TFA (1.76±0.60 Ci/μmol). Further investigation of the 2 M HCl methodology found a by-product, identified as the deprotected proto-destannylated precursor, which co-eluted with (123)I-NKJ64 during the high-performance liquid chromatography (HPLC) purification. The radiosynthesis of (123)I-NKJ64 was achieved with good isolated radiochemical yield of 68% and a high specific activity of 1.8 Ci/μmol. TFA was found to be the most suitable deprotecting agent, since 2 M HCl generated a by-product that could not be fully separated from (123)I-NKJ64 using the HPLC methodology investigated. This study highlights the importance of HPLC purification and accurate measurement of specific activity while developing new radiosynthesis methodologies.

The ins and outs of cholesterol in the vertebrate retina

The vertebrate retina has multiple demands for utilization of cholesterol and must meet those demands either by synthesizing its own supply of cholesterol or by importing cholesterol from extraretinal sources, or both. Unlike the blood-brain barrier, the blood-retina barrier allows uptake of cholesterol from the circulation via a lipoprotein-based/receptor-mediated mechanism. Under normal conditions, cholesterol homeostasis is tightly regulated; also, cholesterol exists in the neural retina overwhelmingly in unesterified form, and sterol intermediates are present in minimal to negligible quantities. However, under certain pathological conditions, either due to an inborn error in cholesterol biosynthesis or as a consequence of exposure to selective inhibitors of enzymes in the cholesterol pathway, the ratio of sterol intermediates to cholesterol in the retina can rise dramatically and persist, in some cases resulting in progressive degeneration that significantly compromises the structure and function of the retina. Although the relative contributions of de novo synthesis versus extraretinal uptake are not yet known, herein we review what is known about these processes and the dynamics of cholesterol in the vertebrate retina and indicate some future avenues of research in this area.

Study of properties of catalysts for the oxidation of carbon black prepared by “ceramic” synthesis and by pyrolysis of polymeric salt compositions

Samples of catalysts of the composition La1−xCsxVO4−y were obtained by the standard “ceramic” technology and by pyrolysis of polymeric salt compositions. Comparative measurements of specific surface area and catalytic activity of the samples in reactions of carbon black and carbon oxide oxidation were carried out.