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At least 1,279 records · Page 71Linked to original sources
PubMed1994

A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay.

A one-step non-radioactive assay to determine the proliferation of murine lymphocytes, lymphoid tumor cells and hybridoma cells is described. This assay requires the addition of Alamar Blue dye to cell cultures and the degree of change in its color, which is reflective of the extent of cellular proliferation, can be determined by an ELISA plate reader. Alamar Blue must be added during the initial phase of cell culture. The pattern of concanavalin A (ConA) or anti-CD3 antibody-induced proliferative response of murine lymphocytes as assessed by Alamar Blue was similar to that of a [3H]thymidine assay. Similarly, the spontaneous proliferation curve of anti-CD3 antibody secreting cell line (YCD3-1), monocytic macrophage cell lines (PU5-1.8, P388D1, J774.1) and myeloma cells (Sp2/0) as determined by Alamar Blue closely resembled that of the [3H]thymidine assay. The minimum detectable number of proliferating cells was comparable in Alamar Blue and [3H]thymidine assays. Since cell lysis/extraction and washing procedures are not involved in the Alamar Blue assay, this approach has several distinct advantages over currently available assays (eg. [3H]thymidine). First, it allows daily monitoring of proliferation without compromising the sterility of cultures. An indication of proliferation can be evaluated (spectrophotometrically or visually) as early as 24 h after ConA stimulation. Second, unlike previously reported assays, Alamar Blue permits further analysis of proliferating cells by other methods. Analysis of cells in culture with Alamar Blue for various surface antigens (CD44, CD45RB, CD4, heat stable antigen) by flow cytometry revealed that the fluorescent profile and relative percentage of cells in cultures with the Alamar Blue were comparable to those without this reagent. The salient advantages of Alamar Blue assay over the [3H]thymidine assay include: (i) non-radioactivity; (ii) simplicity; (iii) less costly; (iv) non-labor intensive; (v) rapidity of assessment of proliferation of large number of samples; (vi) non-toxicity; (vii) usefulness in determining the kinetics of cell growth of hybridomas; and (viii) non-interference of secretion of antibodies by a hybridoma cell line.

Animals↗
PubMed1995

The use of non-radioactive chromium as an alternative to 51Cr in NK assay.

A novel method to measure target cell cytolysis based on the use of 'cold', non-radioactive chromium and on the determination of metal release by graphite furnace atomic absorption spectroscopy (FAAS) is proposed. Natural killer (NK) assays were performed by labelling target cells with chromium as Na2CrO4, and results were compared with those obtained by conventional overnight labelling with 51Cr of targets killed by the same effectors. The cytotoxic capacity of peripheral blood lymphocytes from healthy subjects was evaluated, and NK activity measured with both methods showed a good agreement at each of the tested effector to target cell ratios (between 100:1 and 1:1), with a high and significant coefficient of correlation (r = 0.931, p < 0.0001). The selection of the appropriate Cr concentrations for labelling target cells took into account both the sensitivity of our instrumentation and the possible toxic effects of the metal. A study of the effects of Cr on the cell line (K562) which is usually employed as a target in NK tests showed that Cr could have a detrimental effect on cellular function, with significant numbers of cells with depolarised mitochondria and reduced DNA synthesis after 24 h incubation using Cr levels higher than 15 mumol/l (780 micrograms/l). The method proposed here has a number of advantages, including the use of a non-radioactive tracer, limited costs, high sensitivity and reproducibility, and the possibility of storing samples. In addition, the technique uses a fixed Cr concentration which is known to be non toxic.

Adult↗
PubMed1982

Biosynthesis, purification and receptor binding properties of high specific radioactivity 1 alpha, 24(R),25-trihydroxy-[26,27-methyl-3H]-vitamin D3.

A procedure for the biosynthesis and purification of 1 alpha, 24(R),25-trihydroxy[26,27-methyl-3H]-vitamin D3 (1,24,25-(OH)3[3H]D3) is reported. A kidney homogenate from chicks receiving a high calcium diet (3%) and oral supplements of 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) was used for C-24-hydroxylation of 1 alpha, 25-dihydroxy[26,27-methyl-3H]-vitamin D3 (1,25-(OH)2[3H]D3), in vitro. Extraction and purification of the homogenate lipid fraction by Sephadex LH-20 and high performance liquid chromatography yielded radiochemically pure 1,24-25-(OH)3[3H]D3 with a specific radioactivity equivalent to the initial substrate (166 Ci/mmol). The authenticity of the generated metabolite was assessed by co-migration with synthetic 1,24,25-(OH)3D3 on high performance liquid chromatography and by equimolar competition with authentic radioinert 1,24,25-(OH)3D3 for binding to a purified receptor protein from rat kidney. Binding studies indicate the trihydroxylated metabolite competes 40-50% as effectively as 1,25-(OH)2D3 for hormone binding sites. Further analysis of 1,24,25-(OH)3D3-receptor interaction reveals a high-affinity, saturable binding with an apparent K4 of 2.2 x 10(-9) M. These studies demonstrate that although slightly less active than 1,25-(OH)2D3, 1,24,25-(OH)3D3 is capable of hormone-like interactions, in vitro. The availability of this high specific radioactivity sterol should allow for clarification of its potential physiologic significance.

Animals↗
PubMed1983

The measurement of glucose turnover and oxidation using radioactive and stable isotopes.

Isotopes have become the best means for investigating glucose kinetics in vivo. With the recent greater availability of stable isotopes there has developed a need to understand how data may be obtained from the use of both radioactive and stable glucose tracers. Described for the nonexpert is the calculation of glucose appearance and disappearance, clearance and oxidation using both stable and radioactive glucose isotopes, administered both by bolus and primed constant infusion and under both steady and nonsteady state conditions. Other substrates may be studied using similar methodology. The use of stable substrate isotopes will be an expanding field of metabolic research in man.

Glucose↗
PubMed1983

Salivary gland dysfunction following radioactive iodine therapy.

Radioactive iodine is used extensively for the treatment of thyrotoxicosis and thyroid carcinoma. Iodine is actively taken up by the salivary glands and, following its use, salivary dysfunction may result as a consequence of radiation damage. The literature is reviewed and a case is reported in which a patient presented with a significant increase in caries rate attributed to salivary dysfunction following radioactive iodine therapy for a thyroid carcinoma.

Adult↗
PubMed1975

Identifications of radioactive steroid estrogen conjugates in blood plasma of laying hens after intramuscular injection of (4--14C)-estrone.

[4--14C] Estrone was injected intramuscularly into six laying hens. Fifty minutes later the hens were exsanguinated. The plasmas were examined for conjugates of radioactive phenolic steroids by recovery on columns of Amberlite XAD-2 or by extraction with tetrahydrofuran followed by chromatography on a column of DEAE-Sephadex A-25 in a gradient of NaCl. The biggest Sephadex chromatographic fraction (50,4% of total) contained about 42% of its radioactivity as estradiol-17alpha-3-sulfate and 18% as estradiol-17beta-3-sulfate and the remaining 40% was identified tentatively as estradiol-17alpha-17-sulfate plus a small proportion of estradiol-17beta-17-sulfate. The second biggest Sephadex chromatographic fraction (12.7% of total) was a mixture of conjugates not further identified. Minor fractions identified comprised estrone-beta-glucuronide (2.8%), estradiol-17alpha-3-beta-glucuronide (2.8%), estradiol-17beta-3-beta-glucuronide (2.3%) and estrone sulfate (6.0%). Evidence was obtained for the presence of small proportions of estradiol-17alpha disulfate and estradiol-17beta disulfate.

Animals↗
PubMed1977

Preparation of radioactive iodinated cholylhistamine for use in the radioimmunoassay of cholic acid.

A major handicap in the development of simple and accurate radioimmunoassay procedures for bile acids has been the lack of a radioactive standard of high specific activity. To provide such a compound, we first synthesized cholylhistamine using the carbodiimide reaction. The hypothesized structure was confirmed by elemental analysis, thin-layer chromatography, infrared and mass spectral analysis. The cholylhistamine was then iodinated with 125I, using the choloramine-T method. The 125I-cholylhistamine was bound by antisera raised against a cholic acid-bovine serum albumin conjugate. This procedure should prove useful in preparing radioactive conjugates for all of the bile acids.

Cholic Acids↗
PubMed1977

In vitro biosynthesis of radioactive estradiol-17 beta, 17-glucosiduronate by rhesus monkey liver.

A method for the preparation of radioactive estradiol-17 beta, 17-glucosiduronate by incubating 3H-estradiol with rhesus monkey liver microsomal preparation in the presence of uridine diphosphoglucuronic acid is described. Small but significant amounts of the conjugate were also obtained from the 150,00 pellet and cytosol fractions. The addition of NADPH to the incubation media increased the yield of radioactive-estradiol-17 beta, 17-glucosiduronate perhaps by preserving the integrity of the C-17-hydroxyl group. As expected, the effect of the reduced nucleotide was more pronounced in the fractions other than the microsome. The biosynthesized conjugate was isolated and purified by multiple column chromatography and the structure was confirmed by derivative formation, enzyme hydrolysis and crystallization of the aglycone.

Animals↗
PubMed1995

Use of non-radioactive labels for half-life measurement of sex hormone-binding globulin in the rabbit.

The purpose of this study was to investigate two methods for labeling rabbit sex hormone-binding globulin (rSHBG) with non-radioactive material, biotin (B) and europium (Eu3+), in order to obtain stable labeled SHBG and measure in vivo its metabolism and distribution. The obtained half-life values were compared with [125I]rSHBG half-lives. rSHBG was first isolated by immunoaffinity chromatography using an immobilized monoclonal anti-human SHBG (hSHBG) antibody that cross-reacts with rSHBG. This purified rSHBG was labeled by either biotin-X-N-hydroxysuccinimide ester (rSHBG-B), Eu3(+)-diethylenetriaminepentaacetic dianhydride, or Eu(3+)-isothiocyanatobenzyldiethylenetriamine-tetraacetic acid reagents (rSHBG-Eu3+) or by 125I using Bolton and Hunter reagent ([125I]rSHBG). The labeling procedure preserved the main properties of native SHBG: interaction with the lectine concanavaline A-Sepharose, recognition by anti-hSHBG monoclonal antibody, and, although lower than in native SHBG, the binding affinity for 5 alpha-dihydrotestosterone. These characteristics were the prerequisite for reliable measurement of the metabolism of labeled SHBG. Labeled rSHBG was injected into various rabbits with blood sampling at 2 min and at 1, 2, 4, 8, 12, 24, 48, 72, and 96 h after injection. rSHBG-B or desiaylated rSHBG-B and rSHBG-Eu3+ were captured from serum samples by tubes coated with anti-hSHBG antibody prior to the following detection procedure: biotin was detected by luminometry with the [streptavidin-alkaline phosphatase-dioxetane (AMPPD)] system and europium by time-resolved fluorimetry. [125I]rSHBG was detected by measurement of radioactivity either directly on serum or after fixation on concanavaline A-Sepharose.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

The radioactivity content of United Kingdom coal.

Twenty samples of coal representing each of the seven major regions of the National Coal Board have been analysed for their natural radioactivity content. A variety of methods have been used to verify the results, but the major technique used was radiation spectrometry of 3 kg samples. The results indicate a mean value for uranium and radium activity in British coals of 14.5 Bq/kg, for thorium 12.5 Bq/kg and for potassium 150 Bq/kg. These are significantly lower levels of actinides than have been previously reported and represent only two thirds of those previously used as source terms for assessment of the radiological impact of fossil fuel burning in the U.K.. The content of potassium in U.K. coal is twice the accepted global mean but the radiological significance of this element is negligible. A subsidiary finding is that uranium and its daughter radium are in secular radioactive equilibrium in coal within the experimental error of the analysis.

Coal↗
PubMed1985

Radioactivity connected with coal burning.

The enhanced environmental radioactivity resulting from the operation of a 72 MWe brown coal-fired power plant in central Italy is considered. A source-related control procedure is suggested. The calculated values for the atmospheric dispersion of radioactive effluents and the results of some measurements on brown coals, ash, environmental samples and gamma-exposure levels performed at representative points are reported.

Coal↗
PubMed1985

Effect of an enhanced natural radioactivity on mammal fertility.

Male and female mice of the BALB/c strain were placed on the floor of a hut built at a site of the Permian Basin of Lodève (Southwest France) where the dose rate of gamma radiation amounts to about 10 mrad/h. In previous experiments such high natural radioactivity was shown to induce point mutations in plant material as well as chromosome aberrations in somatic cells of experimental mammals. Controls were kept near the radioactive site under comparable conditions. After the exposure period, the animals were mated in our laboratory, with control mice of the same strain. Due to the high radiosensitivity of mouse oocytes, the fertility of the exposed females was drastically reduced in spite of the fact that the dose received did not exceed 13.8 rad. The fertility of the males exposed to 13 rad, 15 rad or to 45 rad was increased above the control values but was reduced for the animals receiving 63 rad. Analysis of the results show that the differences in fertility are mainly due to a decrease in the number of sterile pairs when males have been exposed to doses upto 45 rad and to an inverse effect when animals received 63 rad. Since histology and weight of the testes suggest that the germ cell population was normal, one has to conclude that the variations in fertility result from physiological effects.

Animals↗
PubMed1985

Limitation of exposure to natural radioactivity in Canada.

The jurisdictional responsibility for natural radioactivity in Canada is divided between the federal and provincial governments. The desire for uniform standards has resulted in the formation of a scientific group to advise governments concerning the desirability of standards for exposure to natural radioactivity. The various types of exposure are reviewed with emphasis on naturally occurring radon in homes. The scientific Committee could not agree either philosophically or practically on the control level for radon in homes. To resolve this impasse, it undertook a calculation designed to illustrate for Canada, the potential cancers saved each year compared to the actual lung cancer rate, with an estimate of the cost for each limit derived. A second calculation reviewed the cost of modifying the building code and estimated the long term effect by comparing avoided cancers and cost. The calculations are based on cross-Canada surveys, intensive long term investigation of smaller groups of homes and national housing statistics. The results are discussed and serve to illustrate development of the proposed policy.

Canada↗
PubMed1992

Subcellular localization of radioactively labelled defibrotide in cultured endothelial cells.

Defibrotide is a new antithrombotic and fibrinolytic drug which is obtained by controlled depolymerization of mammalian DNA. In various models of arterial and venous thrombosis, it has been shown that it induces tissue plasminogen activator [tPA] and prostacyclin [PGI2] release from the vessel wall. We have previously shown the presence of specific binding sites with a Kd of 4.2 micrograms/ml for radioactively labelled defibrotide. The present study was undertaken to identify the location of the binding site. Confluent cultures of endothelial cells from human umbilical vein were incubated with media containing 3H-acetyl-defibrotide for various intervals of time. Cells were then washed and harvested nonenzymatically. Subcellular location of 3H-defibrotide was investigated by fractionating cells on discontinuous sucrose gradient and measuring the distribution of radioactivity. 5'-nucleotidase enzyme activity was also measured to ensure the location of membrane fraction. Our results suggest that the major location of 3H-defibrotide in endothelial cells is the plasma membrane. On the other hand, nuclei also contain a considerable amount of the drug which suggests a mechanism where binding to a membrane protein is followed by internalization.

Binding Sites↗
PubMed1985

Stereotaxic biopsy and radioactive implantation for interstitial therapy of tumors of the pineal region.

Stereotaxic biopsy was performed in 10 patients with tumors of the pineal region. On the basis of intraoperative tissue diagnosis, low-energy radioactive sources (125I) were implanted in seven patients for interstitial irradiation during the same stereotaxic procedure. Results were good in five cases. This therapeutic modality appears to be indicated in cases of proven low-grade malignancy, inasmuch as the implantation of radioactive sources in a highly malignant lesion carries the risk of severe, often irreversible, damage to the surrounding brain, because of possible seed migration.

Adolescent↗
PubMedDate not supplied

Radioautographic localization of radioactivity in rat brain after intraventricular or intracarotid injection of 3H-L-prolyl-L-leucyl glycinamide.

Tritiated L-prolyl-L-leucyl-glycinamide was injected into the carotid artery or lateral ventricle of rats and the radioactivity localized by radioautography. After intracarotid administration, the radioactivity labeled the meninges, ependymal cells bordering the ventricles, and cells of the choroid plexus, as well as the median eminence and subfornical organ and thus may have reached the cerebrospinal fluid. After intraventricular injection, globus pallidus, indusium griseum, hippocampus, corpus callosum, and meninges. The results after intracarotid injection suggest that L-prolyl-L-leucyl-glycinamide may cross the blood brain barrier and the results after intraventricular injection suggest sites of localization which might be correlated with the previously reported effects of the tripeptide on the brain.

Animals↗
PubMed1982

Flow measurement of blood radioactivity in animals.

In order to permit flow measurement of blood radioactivity in an animal after establishing extracorporeal circulation, counting cells were constructed of various plastic scintillator materials. The cell was placed in a liquid scintillation spectrometer replacing the sample vial. Stable and reproducible counting rates were obtained only with cells made of blue Altustipe plastic. The counting efficiency was 1.8%. Using this detection system, continuous records were produced of blood radioactivity in miniature pigs after administration of 14C-urethane and [14C]methyl-piperazinyl pyrazino benzoxazepine maleate.

Animals↗
PubMed1978

Tumor promoting phorbol diesters stimulate release of radioactivity from [3H]-arachidonic acid labeled- but not [14C]linoleic acid labeled-cells. Indomethacin inhibits the stimulated release from [3H] arachidonate labeled cells.

The tumor promoting phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate, stimulates MDCK cells to deacylate cellular phospholipids and to produce prostaglandins when measured as the release of arachidonic acid and its metabolites into the culture fluid. Indomethacin, at levels of 2.8 x 10(-8) to 2.8 x 10(-6) M, inhibits the release of radioactivity from [3H]arachidonate labeled cells stimulated by 12-O-tetradecanoyl-phorbol-13-acetate treatment in a concentration dependent manner. At these concentrations, the conversion of released [3H]arachidonic acid into prostaglandins E2 and F2alpha and the production of PGE2 measured serologically also is suppressed in a concentration dependent manner. Indomethacin, at these levels, has no effect on the acylation of [3H]arachidonic acid into cellular lipids. The tumor promoting phorbol diester does not stimulate the release of radioactive materials from MDCK cells labeled with [14C]linoleic acid, although prostaglandin production by these cells is stimulated.

Animals↗