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PubMed2000

Use of a microplate scintillation counter as a radioactivity detector for miniaturized separation techniques in drug metabolism.

In miniaturized separation techniques, such as capillary electrophoresis (CE) or capillary liquid chromatography (LC), conventional on-line radioactivity detection of labeled compounds is restricted, because of insufficient sensitivity. It will be shown that a microplate scintillation counter for 96-well plates (TopCount) can be used as a sensitive and easy-to-handle radioactivity detector for capillary LC and CE. The attractive combination of capillary LC, eluent fractionation, and subsequent off-line counting is described. The new method is applied for rapid and sensitive separation and detection of 3H-labeled parent drug and its metabolites at levels between 25 and 700 cpm in rat urine. The advantages of capillary LC coupled to the TopCount, and combined with LC-MS data, can be of benefit in many analytical areas, including the characterization of metabolites at low concentration within complex biological fluids. With the same setup, the fractionation with subsequent off-line counting is equally applicable to CE. This is demonstrated with electrophoretically separated 14C-labeled impurities, nicely resolved from a negatively charged main compound, at low levels.

Animals↗
PubMedDate not supplied

Rapid routes of synthesis of chemically reactive and highly radioactively labeled alpha- and beta-oligonucleotide derivatives for in vivo studies.

Development of the antisense oligonucleotide strategy for the regulation of gene expression in vivo poses several problems: the stability of oligonucleotides toward intracellular nucleases, labeling of oligonucleotides with high specific radioactivity, improvements of penetration of oligonucleotides into living cells, and enhancement of antisense action by coupling of chemically active groups. In the present paper synthesis of highly radioactively labeled [32P]- and [35S]oligonucleotide derivatives is described starting from both natural (beta) and nuclease-resistant (alpha) anomers of oligonucleotides. Conditions for preparative phosphorylation and thiophosphorylation suitable for oligonucleotides of various lengths, base composition, and anomeric forms were established. The stability of the phosphoramide bond under in vivo experimental conditions was checked. The methods of terminal phosphate chemical activation and terminal thiophosphate alkylation were applied to synthesize oligonucleotides equipped with hydrophobic, intercalating, alkylating, and photoactivatable groups. In the case of porphyrin-oligonucleotide conjugates, a series of new monofunctional porphyrin derivatives bearing a free aliphatic amino group was developed.

Acridines↗
PubMed1978

Length dependence in reassociation kinetics of radioactive tracer DNA.

The reassociation kinetics have been measured for radioactive Escherichia coli DNAs (tracers) of various average single-strand lengths reassociated alone and in the presence of excess unlabeled DNA (driver) of two different average lengths. Hydroxylapatite binding was used to follow the reaction time course. The length-dependence of the rate constant determined in the tracer self-reassociation reactions is in agreement with the square-root dependence previously determined (Wetmur, J. G., & Davisond, N. (1968) J. Mol. Biol. 31, 349-370) using optical methods to follow the time course. However, for the driver-tracer reactions, where the radioactive DNA reassociates largely with DNA of a different average length, the dependence of the rate constant upon average tracer length is increased and approaches an L to the first power dependence. In 0.18 M Na+, the variation of the rate constant for tracer reassociation with the lengths of the reassociating strands has been shown to fit the simple equation k = (9.0077).(L T 0.55 + 1/L D 0.55), where k is the observed rate constant in L mol-1 s-1 and L(T)and L(D) are the weight average tracer and driver lengths, respectively, in nucleotides. This dependence suggests that the rate of nucleation of two free strands is proportional to the sum of the reciprocals of the hydrodynamic radii of the two strands.

Base Sequence↗
PubMed1999

Combined use of radioimagers and radioactive 3'OH DNA nick end labelling to quantify apoptosis in cell lines and tissue sections: applications to virus-induced apoptosis.

DNA fragmentation is a key feature of the degradation phase of apoptosis. In this work we have developed an assay, based on radioimager (beta-IMAGER and micro-IMAGER) quantification of radioactive nick end labelling (RANEL), which is quantitative, rapid and sensitive to study in vitro and in vivo induced apoptosis. To establish the technique, in vitro apoptosis of T cell lines was induced by stimulation of the Fas receptor; cells were labelled using TdT-mediated [alpha-33P] dCTP nick end labelling, after which then radioactivity was quantified using a beta-IMAGER. We have also shown that the RANEL method can be applied to the quantification and visualisation, by micro-IMAGER analysis, of liver tissue sections from mouse Fas-induced fulminant hepatitis or from Dengue-1 virus infected individuals. Finally, this system has also been used to detect apoptosis induced by rabies virus in Jurkat T cells. These data have established a large field of application for the RANEL assay.

Journal Article↗
PubMed2004

Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays.

Kinases represent one of the largest enzyme families and key regulatory proteins in the cell. Only a small subset of these enzymes has been characterised so far. We have prepared different types of phosphopeptide and peptide microarrays displaying peptides deduced from annotated human phosphorylation sites and cytoplasmic domains of all annotated human membrane proteins. This approach was enabled by fully-automated high throughput micro-scale synthesis of peptides by the SPOT technology combined with chemo-selective immobilisation on modified glass slides. The phosphopeptide microarrays displaying 2923 peptides in total have been used for the characterisation of commercially available generic anti-phosphopeptide antibodies. This enabled us to detect Abl kinase activity on a microarray with anti-phosphotyrosine antibodies yielding results comparable to those obtained from a radioactive assay. More than 13 000 peptides deposited on six glass slides were used to profile casein kinase 2 (CK2) using a radioactive assay, since no generic antibody for the reliable detection of serine or threonine phosphorylation could be identified. All previously identified substrates were detected in the microarray experiment. In order to confirm whether substrates on the microarray are substrates in solution phase assays, more than 700 peptides were synthesised and tested with CK2 in a solution phase assay. All substrates identified in the solution phase assay were also detected on the microarray.

Amino Acid Sequence↗
PubMed2004

Samuel Ruben's Contributions to Research on Photosynthesis and Bacterial Metabolism with Radioactive Carbon.

The earliest experiments on the pathways of carbon in photosynthetic and heterotrophic metabolism using radioactive carbon, (11)C, as a tracer were performed by Samuel (Sam) Ruben, Martin Kamen, and their colleagues. The short half-life of (11)C (20 min), however, posed severe limitations on identification of metabolic intermediates, and this was a major stimulus to search for a radioactive carbon isotope of longer half-life. (14)C was discovered by Ruben and Kamen in 1940, but circumstances prevented continuation of their research using the long-lived isotope. Because of the untimely accidental death of Ruben in 1943, there are very few published accounts on the life and work of this extraordinary scientist. This paper summarizes highlights of Ruben's outstanding accomplishments.

Journal Article↗
PubMed1970

Further studies on the uptake of Synkavit and a radioactive analogue into tumour cells in tissue culture.

In a previous paper, the exact conditions under which the radioactive drug 2-methyl-6,7-ditritio-1,4-naphthaquinol bis disodium phosphate could be selectively incorporated into HEp/2 cells were reported. This work has now been extended and suggests that the selective property associated with two human tumour cell lines established in culture, HEp/2 and HeLa, and two forms of mouse ascites tumour cells propagated in vivo, is a metabolic conversion of the drug (priming stage) to a form which can probably be freely incorporated by all cell types. It is suggested that the observed variations in uptake of label with changes in pH, cell concentration and the inorganic phosphate concentration of the medium indicate that the "priming" stage is critically dependent on the conditions of the experiment.Work with the non-radioactive analogue, Synkavit, indicates that under conditions where the drug is incorporated selectively into cells, incubations in excess of 20 minutes cause a large percentage of the population to lose its reproductive integrity.

Animals↗
PubMed1979

Host treatments affecting artificial pulmonary metastases: interpretation of loss of radioactively labelled cells from lungs.

The effect has been examined of various host treatments (C. parvum injection, immunization, thoracic irradiation, cyclophosphamide injection, and anticoagulation) on both lung colony formation and clearance of radioactive cells from the lungs after i.v. injection of tumour cells. Two tumour-host models have been used: the non-immunogenic KHT tumour in C3H/Km mice, and the immunogenic EMT6 tumour in BALB/c/Ka mice. Even for the at most weakly immunogenic KHT tumour, the number of artificial pulmonary metastases could be modified by a factor of up to 10(4) by different host treatments before i.v. inoculation of tumour cells. For all pretreatments except immunization, the shape of the curve of loss of radioactivity from the lungs vs time was biphasic, with an initial steep portion representing intravascular death of the tumor cells, followed 1--2 days after tumour-cell injection by a shallow exponential curve. It was concluded that the shallow slope represented spontaneous death of tumour cells in the perivascular tissues. Essentially all the injected tumour cells lodged initially in the lungs, and this was unaffected by the different host treatments. Furthermore, except for specific immunization, cell death in the perivascular tissues was also unaffected by host treatment. However, the survival of the tumour cells during the 24 h after injection (before they became extravascular) was extremely dependent on the particular host pretreatment. It would appear from these studies that host treatments such as C. parvum injection or anticoagulation can markedly affect the number of blood-borne pulmonary metastases, but they will only be effective if given before the tumor cells arrive in the lung vasculature.

Animals↗
PubMed2003

Experimental evidence that potassium is a substantial radioactive heat source in planetary cores.

The hypothesis that (40)K may be a significant radioactive heat source in the Earth's core was proposed on theoretical grounds over three decades ago, but experiments have provided only ambiguous and contradictory evidence for the solubility of potassium in iron-rich alloys. The existence of such radioactive heat in the core would have important implications for our understanding of the thermal evolution of the Earth and global processes such as the generation of the geomagnetic field, the core-mantle boundary heat flux and the time of formation of the inner core. Here we provide experimental evidence to show that the ambiguous results obtained from earlier experiments are probably due to previously unrecognized experimental and analytical difficulties. The high-pressure, high-temperature data presented here show conclusively that potassium enters iron sulphide melts in a strongly temperature-dependent fashion and that (40)K can serve as a substantial heat source in the cores of the Earth and Mars.

Journal Article↗
PubMed1966

Radioactive iodoprotein in thyroid lymph and blood.

1. Samples of thyroid and non-thyroid lymph and of thyroid and peripheral venous blood were obtained from primates and cats that had previously been given radioactive iodine. The distribution of the organic radioiodine between the protein and non-protein fractions in these samples was determined. 2. The proportion of the organic radioiodine in the form of iodoprotein was assessed by paper chromatography, acid-ethanol precipitation, hot-butanol washing, column chromatography and separate estimation of iodotyrosines after enzymic hydrolysis. 3. In thyroid lymph the relative proportion of the organic radioiodine in the form of iodoprotein was 75-98%; in blood it was much lower, probably no more than 6-7%. The absolute concentration of iodoprotein radioactivity also was considerably greater in thyroid lymph than in blood. 4. Enzymic hydrolysis of the protein of the thyroid lymph yielded a pattern of iodoamino acids that corresponded closely with that obtained after hydrolysis of protein extracted from the thyroid gland itself. 5. It can be concluded that the iodoprotein in thyroid lymph consists mainly of thyroglobulin or a closely related compound.

Animals↗
PubMed1967

A method for the degradation of radioactive nicotinic acid.

A chemical degradation scheme is reported, which permits the measurement of the radioactivity of each carbon atom of nicotinic acid. Nicotinic acid is decarboxylated by heating with copper chromite to give carbon dioxide (C-7) and pyridine. The pyridine is converted into 4-nitropyridine 1-oxide, which is heated with aqueous calcium hypobromite to give tribromonitromethane. Combustion of the latter gives carbon dioxide derived from C-4 of the nicotinic acid. Nicotinic acid is also reduced to nipecotic acid, which is oxidized to succinic acid by acidic potassium permanganate. Stepwise degradation of the succinic acid by standard procedures gives two samples of carbon dioxide, which correspond to C-3, C-6 and C-4, C-5 of the nicotinic acid. Benzoylation of the nipecotic acid, followed by oxidation with permanganate at pH7, gives 5-amino-4-carboxyvaleric acid; this is converted into 2-methyleneglutaric acid by the action of nitrous acid. Hydrogenation of the 2-methyleneglutaric acid over rhodium in methanol gives 2-methylglutaric acid, which is oxidized with dilute chromic acid to acetic acid. Stepwise degradation of the acetic acid by standard procedures gives two samples of carbon dioxide, which correspond to C-2 and C-3 of the nicotinic acid. Thus the radioactivities of C-2, C-3, C-4 and C-7 are determined directly and those of C-5 and C-6 by difference. The method was shown to be isotopically valid for [2,3,7-(14)C]-, [4,6-(14)C(2)]- and [5-(14)C]-nicotinic acid.

Carbon Dioxide↗
PubMed1970

Computer simulation of the non-steady-state radioactive labelling of a system of metabolite pools with constant rates of influx and efflux.

The derivation is given of the general case of a differential equation describing the labelling of a single metabolite pool that is subjected to constant rates of influx of radioactive material when the pool size is itself changing at constant rate. A programme, written in ALGOL, is briefly described that solves a set of these differential equations, and that can be used to simulate the radioactive labelling of a system of metabolite pools. A copy of the programme, together with examples of input and output, has been deposited as Supplementary Publication no. SUP 50001 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1970), 116, 7.

Computers↗
PubMed1970

Disappearance of radioactivity from the various ribonucleic acid pools and acid-soluble fractions of mouse liver and kidney after a single injection of labelled orotic acid. The effect of castration.

1. An attempt was made to study the rate of synthesis as well as the distribution of RNA in the various cellular fractions in the livers and kidneys of normal and castrated mice. 2. The tissue was fractionated by the procedure of Blobel & Potter (1967). By using this method it was not possible to find any pronounced difference in the relative proportions of RNA in isolated subcellular fractions when kidneys from normal and castrated mice were compared. On the other hand there was an indication of a shift toward the bound ribosomes in livers from normal mice in comparison with livers from castrated mice. 3. Disappearance of the radioactivity followed the pattern of the first-order reaction. Comparing the half-lives of RNA in liver and kidneys it was found that in the latter in both groups of animals half-lives were shorter no matter which cellular fraction was studied. 4. The half-lives for total homogenate RNA, total ribosomal RNA and low-molecular-weight RNA from kidneys of castrated mice were approximately 20-25% longer than the half-lives for the corresponding fractions from normal mouse kidneys. 5. An explanation is put forward for the anomalous finding that RNA from the castrated-mouse kidneys has a higher specific radioactivity than that isolated from normal mice.

Animals↗
PubMed1970

The preparation of alpha-oxo acid derivatives suitable for specific-radioactivity determination.

1. Conditions favouring the condensation of o-phenylenediamine with alpha-oxo acids to form substituted quinoxalines have been investigated. The best yields (85-100%) are obtained by incubation of the reagents in 2m-hydrochloric acid. Substitution of acetic acid for hydrochloric acid, or of halogen-substituted 1,2-diaminobenzenes for phenylenediamine, increases the initial rate of reaction, but not the yield of quinoxalines. 2. The quinoxaline derivatives are insoluble in water and can be readily purified. They are very suitable for determination of the specific radioactivities of radioactively labelled alpha-oxo acids because they are colourless, soluble in dioxan-based scintillators, and their strong absorption of u.v. light facilitates their chemical determination. As little as 1 nmol can be accurately determined by means of the blue fluorescence produced by absorption of light at 340-360nm. 3. Optimum conditions for preparation, purification and separation by paper and thin-layer chromatography are described.

Acetates↗
PubMed1973

The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent.

1. A new method is described for labelling proteins to high specific radioactivities with (125)I. The protein is treated with a (125)I-labelled acylating agent, iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester, which reacts with free amino groups in the protein molecule to attach the (125)I-labelled groups by amide bonds. 2. Three protein hormones have been labelled by this method, human growth hormone, human thyroid-stimulating hormone and human luteinizing hormone. Specific radioactivities of up to 170, 120 and 55muCi/mug respectively have been obtained for these hormones. 3. The immunoreactivity of these labelled hormones has been investigated by using a radioimmunoassay system specific for each hormone. These preparations have also been compared with and found to be equal or superior to labelled hormones prepared by chemical substitution of (125)I into tyrosine residues of the proteins by using the chloramine-t-oxidation procedure. 4. With some antisera the immunoreactivity of the antigen was diminished by the introduction of a single I atom into the tyrosyl groups, whereas antigen containing a single (125)I-labelled 3-(4-hydroxyphenyl)propionamide group showed the same immunoreactivity as the unmodified antigen.

Acylation↗
PubMed1975

The preparation of a semisynthetic tritiated insulin with a specific radioactivity of up to 20 Curies per millimole.

1. This paper describes the semisynthesis of [3H]insulin of high specific radioactivity which is indistinguishable from native insulin in its hypoglycaemic activity towards mice. 2. The maximum specific radioactivity so far obtained is 20 Ci/mmol. At this value the recommended scale of preparation is between 1 mCi (30 mug of product) and 10mCi (300 mug of product). Reaction volumes are then of the order of 10 mul. 3. Special problems arise when the techniques of peptide synthesis are applied to the production of such small quantities of material. We describe these problems and discuss the means of overcoming them.

Animals↗