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PubMed1989

Radioactive-electrophoretic assay of adenosine 5'-triphosphate sulfurylase activity in crude extracts with sulfate or selenate as a substrate.

An assay method for ATP sulfurylase is presented which employs Na2(35)SO4 as a substrate and measures the production of labeled adenosine 5'-phosphosulfate and 3'-phosphoadenosine 5'-phosphosulfate by low-voltage, hanging paper strip electrophoresis. The method is applicable to crude bacterial or mammalian extracts and accurately measures picomole amounts of product(s). Na2(75SeO4 can also be employed as a substrate, if the unstable radioactive product, adenosine 5'-phosphoselenate, is converted to elemental 75Se degrees by inclusion of reduced glutathione in the reaction mixture. The same paper strip electrophoretic technique can then be used to separate 75Se degrees from the radiolabeled substrate. The method also has utility for measuring any direct reduction by crude microbial extracts of radioactive selenate to selenite, independent of ATP sulfurylase.

Adenosine Phosphosulfate↗
PubMed1990

Versatile stable isotope technique for the measurement of amino acids and keto acids: comparison with radioactive isotope and its use in measuring in vivo disposal rates.

Tracer methods using both carbon-13 and -14 have been utilized for determination of ovine fetal amino acid disposal and the results compared in seven animals. We infused [1-13C]leucine simultaneously with [1-14C]leucine into the fetal circulation of pregnant sheep chronically catheterized during late gestation. Radioactive and stable isotope enrichments of leucine (Leu) and stable isotope enrichments of ketoisocaproic acid (KIC) in the umbilical artery and vein and the maternal artery and uterine vein were measured. Stable isotope enrichments and concentrations of both Leu and KIC were determined from a single 0.2-ml sample by the use of internal standards and electron ionization GC/MS analysis after a simple isolation and derivatization procedure. The KIC/Leu enrichment ratio was measured for the first time in fetal arterial plasma and was 0.66 +/- 0.05 (SE). Fetal leucine disposal rate was 9.0 +/- 0.5 (SE) micron/min/kg. Disposal rates determined by stable isotopes were not different from those determined by radioactive isotopes. The GC/MS stable isotope method provided higher precision in both leucine concentration and enrichment measurements and has been shown to be a general method for the determination of concentration and isotopic enrichment of other amino acids and their corresponding keto acids. Furthermore, this method is ideally suited to clinical studies where large numbers of samples of rather small volume can easily be studied with a short turnaround time.

Amino Acids↗
PubMed1991

A method for quantifying radioactivity associated with protein in silver-stained polyacrylamide gels.

A method is described in which individual proteins labeled with weak beta-emitting radionuclides, separated by polyacrylamide gel electrophoresis, and stained with silver are released from the gel by the use of the periodate soluble cross-linking agent N,N'-dialyltartardiamide. The radioactivity can then be quantitated using liquid scintillation counting. The method is shown to be insensitive to reasonable variations in the intensity of staining as well as the gel volume over a practical range of gel slices. Recovery from the gel is extremely good with 93% of the counts associated with 14C-labeled proteins of known radioactive concentration being recovered. Analysis of a complex mixture of 3H-labeled proteins indicates resolution similar to that obtainable by autoradiography without the problems associated with quenching of autoradiographic signal by the staining procedure. The method is used to determine the amount of fucose and mannose incorporated into a putative cell adhesion protein during development of the cellular slime mold Dictyostelium purpureum.

Autoradiography↗
PubMed1993

Novel radioactive phospholipid probes as a tool for measurement of phospholipid translocation across biomembranes.

In an attempt to develop a new method to measure transbilayer phospholipid translocation, with a higher sensitivity and higher temporal resolution, novel radioactive phospholipid probes (*C5-PC, *C5-PE, and *C5-PS) with a short acyl chain at the 2-position were synthesized. The *C5-PC probe was made by coupling lysophosphatidylcholine with [14C]pentanoic acid, using N,N-carbonyldiimidazole as a coupling agent (yield 37%), and *C5-PE and *C5-PS were synthesized by exchanging the choline moiety of *C5-PC for ethanolamine and L-serine, respectively, as catalyzed by phospholipase D. The usefulness of the probes was confirmed by measuring phospholipid translocation across the human erythrocyte plasma membrane, in which the presence of aminophospholipid translocase was revealed using EPR techniques (Zachowski, A., Farve, E., Cribier, S., Herve, P. and Devaux, P.F. (1986) Biochemistry 25, 2585-2590). Using the present probes, ATP-dependent and SH-reagent-inhibitable translocation of *C5-PS and *C5-PE from outer to inner leaflets, which is characteristic to the translocation mediated by aminophospholipid translocase, was detected with a higher sensitivity than seen with the EPR technique. These radioactive phospholipid probes will be useful to measure phospholipid translocation with a high sensitivity and have the potential for application in measurements of transbilayer lipid-translocation for a wide variety of membranes.

Biological Transport↗
PubMed1976

Incorporation of radioactive amino acids into protein in isolated rat hepatocytes.

The incorporation of radioactivity from a 14C-labelled amino acid mixture (algal protein hydrolysate) into protein in isolated rat hepatocytes has been studied. The incorporation rate declined with increasing cell concentration, an effect which could be explained partly by isotope consumption, partly (and largely) by isotope dilution due to the formation of non-labelled amino acids by the cells. At a high extracellular amino acid concentration, the rate of incorporation into protein became independent of cell concentration, because the isotope dilution effect was now quantitatively insignificant. The time course of protein labelling at various cells concentrations correlated better with the intracellular than with the extracellular amino acid specific activity, suggesting that amino acids for protein synthesis were taken from an intracellular pool. With increasing extracellular amino acid concentrations both the intracellular amino acid concentration, the intracellular radioactivity and the rate of incorporation into protein increased. Protein labelling exhibited a distinct time lag at high amino acid concentrations, presumably reflecting the time-dependent expansion of the intracellular amino acid pool. The gradual increase in the rate of protein labelling could be due either to an increased intracellular specific activity, or to a real stimulation of protein synthesis by amino acids, depending on whether the total intracellular amino acid pool or just the expandable compartment is the precursor pool for protein synthesis.

Amino Acids↗
PubMed1984

Rat brain acetylcholinesterase turnover in vivo: use of a radioactive methylphosphonothiate irreversible inhibitor.

A new organophosphorus compound was used in its non radioactive and tritiated forms in order to study rat brain acetylcholinesterase. We measured the activity recovery of the total enzyme and of its two main molecular forms (4 S and 10 S) as a function of time following the inhibition. The radioactive compound allowed us to study the disappearance of the inhibitor irreversibly bound to the enzyme in the main cholinergic areas. Both approaches gave similar results: acetylcholinesterase turn-over proceeds in two steps, a rapid one of about 30 mn and a slow one of about 2 days. Our results suggest an in vivo reactivation process concerning a fraction of the bound inhibitor.

Acetylcholinesterase↗
PubMed1985

Photoaffinity labeling of the tetracycline binding site of the Escherichia coli ribosome. The uses of a high intensity light source and of radioactive sancycline derivatives.

[3H]Tetracycline (TC) has been shown to photoincorporate into the Escherichia coli ribosome. However, the utility of this process for characterizing the TC binding site on the ribosome is diminished by competing side reactions which also lead to incorporation of radioactivity. In this work we first conducted a detailed study of the labeling processes occurring when ribosomes are irradiated in the presence of [3H]TC with a common, rather low intensity, lamp. On the basis of the results of this study we next explored the usefulness for photoaffinity labeling of the TC site of both irradiation with a high-intensity laser and radioactive, functional TC derivatives having different photochemical properties than TC itself. Labeling patterns determined by polyacrylamide gel electrophoretic analysis of ribosomal proteins extracted from photoaffinity-labeled 30S subunits provided strong evidence that these two approaches offer distinct advantages for characterizing the TC binding site.

Affinity Labels↗
PubMed1975

Transfer of radioactive material between electrically coupled neurons of the leech central nervous system.

Intracellular application of tritiated precursors by means of microiontophoresis was performed on nerve cells in isolated segmental ganglia of the leech ventral nerve cord. Incorporation as well as intra- and interneuronal transport were studied by autoradiography after injection of fucose, glucosamine, glycine, leucine, orotic acid and uridine. With several minutes of intraneuronal injection the precursors were incorporated into macromolecules. Depending upon the tracer used, the radioactive material was distributed in a specific pattern over the cell somata and then released into the nerve processes. After application of orotic acid and uridine a transport of radioactive material, presumably RNA, could be observed in the processes of the injected neurons at a distance of about 200-500 mum. Fucose and glucosamine injection resulted in the most extended labeling of the nerve cell processes, indicating a transport rate of about 11 mm/day. When the radiochemicals were injected into one of the two electrically coupled giant nerve cells -- the so-called Retzius cells (Rc) -- a specific labeling not only of the injected Rc but also of the coupled but not injected Rc was found. Injection of protein or glycoprotein precursors into one Rc produced heavy labeling of both Rcs including their processes; a slight labeling of other ganglion compartments was only found after increasing the dosage of the amino acids glycine and leucine. With orotic acid and uridine this interneuronal transfer was confined to the electrically coupled Rc twin. Intracellular injection of one Rc with puromycin followed by injection of amino acids or fucose into the same Rc or into the coupled Rc resulted in an inhibition of precursor incorporation within the puromycin-injected Rc and an exclusive labeling of the coupled Rc, thus indicating that the precursors themselves were transferred. It is suggested that after microiontophoretic application an interneuronal transfer of relatively low molecular weight material takes place, probably across the low-resistance junction through which the Rcs are electrically coupled.

Animals↗
PubMed1978

Developmental changes of protein-bound radioactivity distribution in rat brain slices incubated with labelled leucine.

The distribution of protein-bound labelled leucine in cortical slices prepared from young rats was investigated by autoradiography and complemented with measurements of the rate of protein synthesis in slices. During the second and third postnatal weeks the rate of protein synthesis in cortical slices from rat brain declined rapidly. The distribution of protein-bound radioactivity was fairly homogenous within the neuronal cell population of cortical slices from 7-day-old rats and was not influenced by the level of labelled leucine in the incubation medium (0.01 and 1 mM). On the contrary, the gradient-like distribution of incorporated radioactivity appeared in slices from 14-day-old rats, at both trace and 1 mM levels of the precursor. The laminar analysis of neuronal cell labelling confirmed the preponderance of incorporation into the neurones localized near the slice surface and the considerable inhibition of neuronal protein synthesis in the slice centre. Therefore, these limitations on the use of cortical slices for the study of brain protein synthesis in the rat should be respected from the beginning of the second postnatal week.

Age Factors↗
PubMed1985

Dexamethasone affects radioactive choline uptake in rat diaphragm nerve endings and not in muscle fibres.

The initial rate of radioactive choline (Ch) uptake in the endplate-rich area (EPA) of both stimulated and unstimulated hemidiaphragms is significantly increased by 0.2 microM dexamethasone (Dex) in the presence of 10 microM Ch. In autoradiographs, the mean grain densities above the muscle fibres are not altered by Dex. The mean grain densities above the nerve endings are significantly increased in the presence of Dex in stimulated tissue, and slightly but not significantly increased in unstimulated tissue. There is a positive correlation between the initial rate of Ch uptake in the EPA and the amount of isotope in the nerve terminals, in the absence and presence of Dex. Without correcting for the large amount of diffusion which occurs, the ratio of the grain densities above the nerve terminals to that above the muscle fibres in the presence of Dex is 2.12 in stimulated tissue, and 1.40 in unstimulated tissue. The ratio in the stimulated tissue is significantly greater than the control ratio in the absence of Dex (1.66). Therefore, Dex affects radioactive Ch uptake in nerve endings and not in muscle fibres in the rat diaphragm. The stimulation-induced increase in the uptake of isotope into the nerve endings is abolished in a Na+-depleted medium, and in the absence of Ca2+. Dex has no effect on this abolition. We conclude that relatively low concentrations of Dex affect Ch transport in rat diaphragm nerve endings by a mechanism as yet to be defined.

Animals↗
PubMed1989

Static and dynamic computerised radioactive tracer studies, vital dye staining and theoretical mathematical calculations to ascertain the mode of survival of single cephalad channel venous island flaps.

Experiments were performed to ascertain the perfusion and drainage of single cephalad channel saphenous venous flaps in dogs. In the first experiment a standard saphenous flap and a single cephalad channel saphenous venous island flap were dissected on contralateral thighs of dogs. A radioactive tracer, 99mTc, was injected into a foreleg vein and its appearance in both flaps was monitored with the use of a static and dynamic scanner. After this experiment, Evans blue, a vital dye, was injected into the foreleg vein of the dog. Five minutes later both flaps were excised and the dye in the flaps was assayed quantitatively. In the third experiment radioactive tracer was injected into the single cephalad channel venous flap subcutaneously and its decay in the flap as well as its uptake in the rest of the body were measured dynamically. The experiments show that the single cephalad channel venous island flap is perfused by and drains through its single cephalad vein. A mathematical model to justify the above conclusion was worked out independently, based on standard values involved in mammalian circulatory mechanisms as in dog and man.

Animals↗
PubMed1993

Non-radioactive coloured microsphere measurement of regional tissue blood flow for axial pattern flaps in rabbits.

We administered non-radioactive coloured microspheres (NRACM) to measure the regional tissue blood flow (RTBF) of eight axial pattern flaps and four kidneys in four rabbits using four repeated injections into each animal. As a control, we used radioactive microspheres (RAM) for six kidneys in three rabbits. There were no significant differences for RTBF values between NRACM and RAM, between the numbers of microspheres used (2 x 10(6) and 4 x 10(6)), or between the number of injections. These results showed that NRACM is a useful and safe laboratory method.

Animals↗
PubMed1976

Titration of human placental alkaline phosphatase with radioactive orthophosphate.

Human placental alkaline phosphatase incorporates radioactive phosphate specifically and covalently at acid pH. By titration of solutions of the purified enzyme with radioactive orthophosphate, the enzyme was shown to incorporate up to 2 phosphate groups per molecule. No evidence was found to suggest that the two sites had different affinities for phosphate. Similar titrations can be used to determine the molarity of solutions of non-placental alkaline phosphatases of unknown purity, if these also are assumed to possess 2 binding sites per molecule.

Alkaline Phosphatase↗
PubMed1990

A new method for determination of the rate constant for clearance of radioactive indicators in gamma camera hepatography. Clinical examples with 99mTc-diethyl-IDA and 99mTc-diethyl-iodo-IDA.

Application of Stewart-Hamilton's formula to the activity-time curve recorded over a left ventricular region of interest following an intravenous bolus injection of a radioactive indicator in gamma camera hepatography has resulted in a new method for determination of the clearance rate constant of the indicator (i.e. the ratio of the clearance to the plasma volume). In a group of 19 normal subjects the clearance rate constant was 0.116 +/- 0.036 min-1 (means +/- 1 S.D.) with 99mTc-diethyl-IDA (SOLCO HIDA) and 0.184 +/- 0.037 min-1 (means +/- 1 S.D.) with 99mTc-diethyl-iodo-IDA (SOLCO IODIDA) in 15 normal subjects. The clearance rate constant is assumed to represent mainly hepatic clearance of the radioactive indicator and to a much smaller extent renal clearance. The literature shows diverging results as to the clearance of 99mTc-diethyl-IDA and the reasons for this are discussed. The correlations between the clearance rate constant of 99mTc-diethyl-IDA and a number of laboratory variables with relation to liver function were moderate to non-existent in a group of 16 subjects with abnormal values of at least one of these variables. The rate constant for clearance as described in the paper represents a non-invasive method for rapid evaluation of the uptake capacity of the liver and, in particular, with regard to the polygonal cells of the liver parenchyma when IDA derivatives are employed. The clinical usefulness of the clearance rate constant is probably superior to the group of routine laboratory variables which also describe other functions than liver uptake.

Adult↗
PubMedDate not supplied

Exchange of plasma radioactive cholesterol with atheroma lipids in situ and implanted subcutaneously.

Physicochemical exchange of radioactive cholesterol was investigated in mildly hypercholesterolaemic rabbits. Such exchange was examined in erythrocytes and atheroma lipids in situ in the aortic wall and in such lipids when implanted subcutaneously. The implanted atheroma lipids gained radioactivity from intravenously injected [3H]cholesterol, even though cholesterol was being resorbed from the implants. Autoradiography showed exchange between plasma cholesterol and both intracellular and dispersed extracellular lipid in these implants. Cholesterol crystals in both implants and atheromatous lesions labelled only sluggishly. The results are consistent with the view that physicochemical exchange is a major factor in cholesterol movement in vivo.

Animals↗
PubMed1981

A radioactive antigen-binding assay for the measurement of antibody to Haemophilus influenzae type b capsular polysaccharide.

A new polyethylene glycol (PEG) radioimmunoprecipitation assay was developed for the detection of antibody to Haemophilus influenzae b capsular polysaccharide, polyribosylribitol phosphate (PRP). The radioactive antigen, [3H]PRP, with a high specific activity, was produced by growing the organism in the presence of [3H]ribose and was purified by hydroxylapatite and Sepharose 4B column chromatography. In the assay, PEG (12.5%) was used to separate antibody-bound [3H]PRP from free [3H]PRP. The assay covered the range of 0.5 and 20 ng antibody/assay at a maximum sensitivity of 0.5 approximately 1.0 ng antibody/assay. With various dilutions (1-20 ng antibody/assay) of S. Klein reference antiserum, the within-run coefficient of variation (CV) of 10 replicates ranged from 3.5 to 8.5%. Average CVs of 8.9% and 11.0% were obtained in the between-run and day-to-day reproducibility studies. The binding of [3H]PRP to S. Klein reference antiserum was severely inhibited by a minute amount of non-radioactive PRP; however, no significant interference was found in the presence of high concentrations of polysaccharides from Escherichia coli K100 and Streptococcus pneumoniae indicating that the RIA was highly specific for antibody to H. influenzae b PRP. The present RIA is a simple, specific, sensitive and reproducible procedure for the evaluation of antibody responses of young animals and infants to H. influenzae b vaccines and infections.

Antibodies, Bacterial↗
PubMed1994

Non-radioactive labelling and immunoprecipitation analysis of leukocyte surface proteins using different methods of protein biotinylation.

The biotinylation of surface proteins and the detection of immunoprecipitated protein(s) after transfer to nitrocellulose using chemiluminescence methods is a highly sensitive alternative to hazardous radioactive labelling procedures. Ligation of proteins using the common biotin-NHS-ester (N-hydroxysuccinimido-biotin) is often associated with a decrease in immunoreactivity. Here a new non-radioactive method of leukocyte surface glycoprotein labelling using biotin-LC-hydrazide is described. This technique is based on the labelling of glycoproteins after mild oxidation of carbohydrate hydroxyl groups to reactive aldehydes. Flow cytometric and immunoprecipitation analyses of selected leukocyte markers such as CD3, CD26 and CD65 indicated that the alteration in immunoreactivity achieved by NHS-mediated biotin ligation was different from that obtained with hydrazide-mediated biotin ligation. CD3 and CD26 immunoreactivity was diminished using NHS biotinylation but preserved by biotin-LC-hydrazide, whereas CD65 binding to monoclonal antibodies was completely abolished after treatment with biotin-hydrazide. However, the immunoreactivity of CD13 was found to be totally unaffected by both NHS and hydrazide biotinylation. The combination of different biotinylation methods for surface protein labelling offers a viable alternative to radioiodination in the biochemical analysis of membrane proteins.

Antibodies, Monoclonal↗
PubMed1994

Competitive RT-PCR ELISA: a rapid, sensitive and non-radioactive method to quantitate cytokine mRNA.

We have developed a non-radioactive method to quantitate precisely levels of gene expression. This method is based on RT-PCR (reverse transcriptase-polymerase chain reaction) with an RNA competitor, followed by the covalent capture of the amplified DNA onto the wells of microtiter plates, and the quantitation of the PCR product by oligonucleotide hybridization and ELISA (enzyme-linked immunosorbent assay). The assay can reproducibly detect 1 zeptomole mRNA. The assay was successfully used to quantitate mRNA levels of the T cell derived cytokines interleukin-2, interleukin-4 and interferon-gamma in resting and stimulated human lymphocytes. Because it is performed in a microtiter ELISA format, this rapid, sensitive and non-radioactive method should facilitate measurements of gene expression, particularly in large clinical studies.

Base Sequence↗