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Perilymph fistulas secondary to gunshot wound. Localization with a radioactive pharmaceutical.

The symptoms, clinical evaluation, routes of formation, and repair of perilymph fistulas are reviewed. We report a case with oval and round window perilymph fistulas secondary to a gunshot wound. A radioactive pharmaceutical was injected into the subarachnoid space in the lumbar area. Subsequent radioactivity was detected at the sites of the fistulas. We discuss the implications and a suggested technique for fistula detection.

Adult↗

A non-radioactive assay system for screening for inhibitors of RNA-dependent reverse transcriptase activity; an analysis using aurintricarboxylic acid and plant flavonoids.

Ongoing and extensive screening for potentially selective and potent inhibitors of RNA-dependent DNA polymerases (reverse transcriptases) is important for the discovery of therapeutic agents active against retroviruses. Traditional systems for assaying the activity of reverse transcriptase enzymes have relied upon the use of radioactive nucleotides for monitoring complementary DNA (cDNA) synthesis. Moreover, such assays have also typically been programmed by the addition of synthetic template-primers. The recent trend, however, is towards the use of more natural nucleic acid template-primer-directed systems, which provides for a more realistic estimation of the activity of potential therapeutic anti-reverse transcriptase agents. A novel agarose gel-electrophoretic method originally developed to evaluate DNA polymerase activity during enzyme purification was adapted for screening purposes. The system was modified so that one can detect the synthesis of non-radioactively labeled cDNA synthesized in both DNA and RNA template-directed reverse transcription reactions. Such assays are programmed with either M13mp9 single-stranded DNA or rabbit globin mRNA as appropriate templates. Following incubation of reaction mixtures, agarose gels are used to determine the activity of the DNA-dependent DNA polymerase and RNA-dependent DNA polymerase activities of Maloney murine leukemia virus. This is done through a detection of the amount of double-stranded nucleic acid molecules (either DNA:DNA or RNA:DNA hybrids) generated by the enzyme in the presence and absence of various known enzyme inhibitors. The assay systems that have been developed will be useful in initial, general, rapid, inexpensive and safer screening programs for potential inhibitors of both the DNA- and RNA-dependent DNA-polymerase functions of reverse transcriptase enzymes. The system was tested and evaluated with potent inhibitors of reverse transcription, such as aurintricarboxylic acid and with several plant flavonoids known to be moderately potent inhibitors of reverse transcriptases. The details concerning the RNA-dependent DNA polymerizing system and some of our results are presented.

Animals↗

Nonabsorbable radioactive material in the treatment of carcinomas by local injections.

Nonabsorbable radioactive material was used in treating malignant lesions by local injection. One hundred thirty-five Lewis Wistar rats were inoculated subcutaneously with tumor cells. After 7 days, the rats grew tumors of approximately 1 X 1 X 1 cm at the injection site. The rats then were divided into two groups; 50% were kept as a control group while the other 50% received single injections of 1 mCi yttrium 90 (90Y) microspheres directly into the center of the tumor. This study shows a significant reduction of the tumor growth rate in treated animals. Tumor sizes after 4 days averaged 1.31 ml in the treated rats and 9.74 ml in the control group. Excluding animals that had complete regression of the tumor, treated rats lived an average of 30.8 days from the day of treatment compared with the control rats, which lived an average of 17.4 days. Pathology examinations showed no effects from injected radioactive materials in the liver, bone marrow or the kidney. Examination of the injected area after 6 months showed that microspheres remained locally and that the tumor was replaced with collagen tissue.

Animals↗

A novel platinum-iridium, potentially gamma radioactive stent: evaluation in a porcine model.

In-stent restenosis (ISR) is a major problem within stented arteries. Surface treatment of stents with platinum and gold were found to have the maximum charge with least neointima formation (NF). This study was designed to evaluate platinum (maximum electrical charge) as a material to make stents to reduce NF. Iridium was added to make an alloy suitable for stent manufacture, with the potential to make the stent radioactive. We implanted the novel platinum-iridium (PI) stent in 10 porcine coronaries and compared to the Palmaz-Schatz (PS) stent implanted in 8 coronary arteries. Six weeks after implantation, angiography of the stented vessel was performed before sacrifice. The coronaries were perfusion-fixed and stained, and vessel parameters were analyzed by computer-aided histomorphometry. The thrombus formation and the inflammatory response was less in the PI stent (0.04 +/- 0.1 vs. 0.24 +/- 0.2, P = 0.005; and 1.1 +/- 0.5 vs. 2.4 +/- 0.3, P < 0.001). The NF from PI-stented arteries was smaller in size than the PS controls (1.9 +/- 0.6 mm(2) vs. 2.4 +/- 0.4 mm(2), P = 0.06). However, PI stents presented with higher recoil than the PS stent (16% vs. 5%, P < 0.001). Platinum-iridium is a highly biocompatible material with high performance, low inflammatory response with small NF. This stent does not lead to thrombus formation and has the potential (due to the presence of iridium) to be irradiated to form a gamma radioactive stent. Cathet. Cardiovasc. Intervent. 51:364-368, 2000.

Animals↗

Use of the indirect platelet radioactive antiglobulin test with anti-IgG and anti-C3 in immune and nonimmune thrombocytopenias.

Indirect platelet radioactive antiglobulin tests using anti-IgG and anti-C3 were performed in 483 individuals. The subjects' diagnoses and clinical states were correlated with sensitizing protein (IgG and/or C3). Sensitivity of the technique was increased by increasing the serum to cell ratio and there was good correlation between direct and indirect antiglobulin tests in 56 patients. Seven percent of normal healthy non-thrombocytopenic untransfused males and 88% of patients in whom autoimmune thrombocytopenia was clinically suspected showed a positive test result. In the indirect antiglobulin test, mean +/- SD fg anti-C3d/platelet was 1.75 +/- 0.28 (N = 58) with serum from normal subjects, 3.91 +/- 1.86 (N = 123) with serum from patients with idiopathic thrombocytopenic purpura and systemic lupus erythematosus, and 2.78 +/- 0.91 (N = 52) with serum from patients with lymphoproliferative disease. The differences were significant with P less than 0.001. Levels of bound anti-IgG were in general accord with those of other reports; there was no significant relationship with serum immunoglobulin levels. When the indirect antiglobulin tests were positive for both IgG and C3, there was a significant correlation between IgG and C3 bound to normal platelets (r = 0.671; P less than 0.01). Although sera from 39% of patients whose thrombocytopenia was not initially suspected to be on an autoimmune basis gave a positive test result, indirect platelet radioactive antiglobulin tests using anti-IgG and anti-C3 may be convenient tools in the evaluation and follow-up of immune thrombocytopenias.

Adult↗

Estimation of metabolic flux rates in liver purine catabolism of tumour-bearing mice by computer simulation of radioactive tracer experiments.

Mouse hepatocytes from healthy control mice and from Ehrlich ascites tumour-bearing mice were used for tracer-kinetic studies of purine catabolism of liver cells during different periods of tumour growth. The dynamics of the radioactive tracers were modelled mathematically by a system of differential equations. Computer simulations, i.e. direct fitting of numerical solutions of these equations to the observed time-courses of metabolites and specific radioactivities, enables one to estimate unknown kinetic parameters of a simplified model of pathways of hepatic purine catabolism in tumour-bearing mice. There occurred great differences of metabolic flux rates between control hepatocytes, hepatocytes of mice during the proliferating period of tumour growth (6th day after inoculation of the tumour) and hepatocytes of mice during the resting period of tumour growth (12th day after inoculation of the tumour). The final purine degradation of hepatocytes prepared during the proliferating period was lower in comparison with that of control hepatocytes, but it was markedly higher in hepatocytes prepared during the resting period of tumour growth. The changes in hepatocyte purine catabolism during the proliferating period of tumour growth argue for transitions which aim at the maintenance of high purine nucleotide levels in the liver itself rather than for an increased nucleoside and nucleobase supply for the tumour. This suggestion is in accordance with the increased ATP level of the liver during the proliferating phase of tumour growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Emergency localization of radioactive seeds lost during intracoronary brachytherapy.

Recently, it has been reported that brachytherapy catheters ruptured in vivo. Localization of lost beta-radiation-emitting seeds is a problem because no appropriate technique is available that is rapid and precise. We developed a technique to localize beta-emitting seeds utilizing the effect that beta-radiation induces bremsstrahlung. The loss of a single radioactive source was simulated in an Alderson Phantom representing a human body. The beta-induced bremsstrahlung could be detected selectively by a gamma-camera. The position of the radioactive seed could be located within 5 min with an accuracy of +/- 0.5 cm. The result of this study suggests that in an emergency case of loss of a brachytherapy source, a commercially available gamma-camera can be a valuable tool to detect lost beta-radiation-emitting seeds rapidly and precisely. In addition, the technique minimizes the patient's as well as the surgeon's exposure to radiation and reduces the extent of surgical trauma.

Beta Particles↗

Distribution of radioactive octacosanol in response to exercise in rats.

To study the mechanism of action of octacosanol in rats the biodistribution of radioactivity of octacosanol was investigated in response to exercise. The amount of voluntary exercise was significantly higher in octacosanol fed rats than in the control. After ingestion of 14C-octacosanol, the accumulation of radioactivity of octacosanol in the muscle of exercised group given octacosanol was significantly higher in comparison with that of the exercised control group given no octacosanol and also the non-exercised groups irrespective of whether they were given octacosanol or not. The muscle thus seemed to be able to store a considerable amount of octacosanol in response to exercise. Although the exact mechanism of increase in physical exercise caused by octacosanol is not known, it is possible that octacosanol increases the mobilization of free fatty acids from fat cells within muscle. The results indicate that octacosanol possesses an adipokinetic activity, which might affect the lipolysis process of muscle.

Animals↗

Studies of bone and soft-tissue tumours induced in rats with radioactive cerium chloride.

A colloidal suspension of radioactive cerium chloride was inoculated into the hind legs of Sprague Dawley rats. Bone and soft-tissue tumours were induced at the site of inoculation in 77% of the animals. All bone tumours were osteogenic osteosarcomas. Soft tissue tumours were mostly malignant and were of various histological types, predominantly fibrosarcomas, haemangiopericytomas, angiosarcomas and rhabdomyosarcomas. A kinetic study showed that the doubling time (DT) of tumours was closely correlated with the anatomical site of tumour development: bone tumours had a DT of 17.4 +/- 4.3 days and malignant tumours which developed in soft tissues had a DT ranging from 7.4 to 8.4 days with the exception of two haemangiosarcomas which had a long DT of 17 +/- 0.6 days. Pulmonary metastases were frequent for osteosarcomas and tumours of vascular origin. This model of induction of bone and soft-tissue tumours in rats by injection of a colloidal suspension of radioactive cerium chloride offers the possibility of more comprehensive physiopathological and kinetic studies of these tumours and may constitute a good model for their human counterparts.

Animals↗

In vivo labelling of biotinylated monoclonal antibodies by radioactive avidin: a strategy to increase tumor radiolocalization.

Several monoclonal antibodies (MAbs), reactive with tumor-associated antigens, selectively persist on tumor sites in vivo for many days. If biotinylated, such highly specific tags on tumor cells could become targets for radioactive avidin, administered after suitable intervals. The proposed strategy is based on a number of assumptions concerning the ability of avidin to preserve its biological properties in heterologous in vivo environments, on its lack of toxicity and on its biodistribution. A preliminary study has been carried out in rabbits, using biotinylated nitrocellulose and polystyrene targets. The results of this study indicate that in rabbits 1) avidin can be administered i.v. and i.p. without adverse reactions, 2) it does not show any preferential localization, 3) it is eliminated with a biological half-life of 24 hr, 4) its biological properties are not impaired by in vivo conditions, since it accumulates at biotinylated targets only, 5) CEA-bearing targets can be biotinylated in vivo by biotin-labelled anti-CEA MAbs and 6) the biotin-avidin chain can be further extended in vivo since bound avidin is still able to bind biotinylated radioactive proteins.

Animals↗

Simple radioactive assay for the estimation of DNA breaks.

The intactness of DNA is an important part of the normal cellular structure. Any change to the DNA in the form of breaks leads to a change in the integrity, which in turn leads to abnormality in the cellular activity. Many discrepancies have been reported among the various methods of detecting DNA damage. Here, a simple, sensitive and reproducible method has been developed for the detection of DNA breaks by radioactive labelling of 5' broken ends. The method was evaluated by studying chemically induced DNA damage by using both organochloride (2,4-dichlorophenoxyacetic acid and lindane) and organophosphorus (sevin and phosphamidon) compounds at different concentrations. Phosphamidon, one of the organophosphorus compounds studied, showed complete degradation of the DNA after treatment. Radioactive analysis of phosphamidon showed higher counts at the lowest concentration (20 microg) of the chemical when compared with the control (2752 scintillation counts per minute, scm). Studies on the chemically induced DNA breaks by radiolabelling revealed that the cumulative effect of the organophosphorus and organochloride compounds showed maximum counts in all the samples (the highest being 2904 scm) when compared with the organophosphorus and organochloride compounds studied separately (the highest being 1881 and 2260 scm, respectively). Radiolabelling studies on the blood samples of 23 pesticide workers by the newly developed assay showed a significant positive correlation (0.893) between the number of years of exposure and the scintillation counts. A maximum of 11,702 scm (for 18 years of exposure) and a minimum of 1682 scm (for 4 years of exposure) were recorded compared with 1253 scm for the negative control. This method can be used effectively for estimation of the DNA breaks, irrespective of its nature.

Agricultural Workers' Diseases↗

Kit formulation for the preparation of radioactive blue liposomes for sentinel node lymphoscintigraphy.

A radioactively labeled blue liposome formulation was developed for use in presurgical lymphoscintigraphy and intraoperative sentinel node localization to avoid the differing injection site clearance kinetics of the conjunctive use of separate formulations of low molecular weight blue dye and radioactively labeled macromolecular sulfur colloid. Blue liposomes containing glutathione in the internal aqueous phase were prepared from blue dyed lipids obtained by covalently binding Reactive Blue II to phosphatidylethanolamine (PE) fractions of phospholipid extracts. Four phospholipid extracts with differing PE fractions and a centrifugation technique were evaluated with the goal of maximizing the blue color intensity of the formulation. Stability of the formulations was evaluated by studying radiolabeling efficiency (using a membrane permeable lipophilic carrier of the commonly used diagnostic radionuclide, technetium-99m) and particle-size distribution over 30-60-day periods. Blue color was not altered by varying the PE content, while centrifugation was an effective and convenient method to maximize the blue color intensity of the final preparation. The particle size distribution of the prepared liposomes ranged from 200-300 nm (considered ideal for lymphoscintigraphy studies) and did not change significantly, while radiolabeling efficiency exceeded 80% for up to 1 month. The described kit formulation for the preparation of radiolabeled blue liposomes is suitable for commercial production allowing widespread clinical use. The combination of a means of visual identification and tracking of the liposomes through the lymphatic channels along with the ability to trace the preparation using standard radiation detection instrumentation provides the surgeon with an improved radiolabeled compound for lymphoscintigraphy and intraoperative sentinel lymph node identification.

Drug Evaluation, Preclinical↗

Absorption and distribution of radioactivity from suppositories containing 3H-benzocaine in rats.

The effects of the suppository vehicle, drug concentration, and nonionic surfactants on in vitro benzocaine dialysis through a cellulose membrane and on rectal absorption in rats of total radioactivity following administration of 3H-benzocaine were investigated. In vitro dialysis correlated quite well with in vivo absorption, and drug release was greater from water-soluble vehicles than from oleaginous vehicles. Inclusion of a nonionic hydrophilic or lipophilic surfactant in cocoa butter resulted in a statistically significant increase for in vitro drug release, while a lipophilic surfactant showed little effect in vivo and a hydrophilic surfactant depressed release in vivo. Both types of surfactant had small effects on release from polyethylene glycol. In vitro release of benzocaine from some commercially available suppositories was compared with experimental preparations. Variation in blood radioactivity following administration of the same concentration of 3H-benzocaine in the same dosage form in male and female rats is reported.

Animals↗

Radioactive iodine-125 implantation for cancer of the prostate.

Localized cancer of the prostate can be treated by radical prostatectomy, external beam irradiation, or radioactive implantation with similar survival results. Radical prostatectomy, however, almost universally results in impotency, although a new, nerve-sparing procedure may preserve potency in B1 patients. External beam irradiation radiates a large volume of tissue with significant rectal and bladder morbidity, 23-47% risk of impotency, and requires prolonged treatment (6-8 weeks). Radioactive implantation may be done suprapubically or transperineally using iodine-125, gold-198, or radon-222 permanent implantation techniques and iridium-192 or radium-226 removable implantation techniques. Interstitial iodine-125 implantation is frequently employed since it is a short procedure and limits the morbidity to a 7% incidence of impotency, 20% urinary complications, and 5% rectal complications. The overall 5-year survival of patients with iodine-125 is 79%, the survival rate decreasing with increasing T or N stage or increasing grade of tumor.

Brachytherapy↗

Radioactive end labeling to determine hydrolytic rates of nuclease mimics.

Radioactive end labeling can be used to determine the hydrolytic rates of nuclease mimics on moderate to long lengths of RNA or DNA. However, the reliability of end labeling as an assay can vary depending on how well the unincorporated label is removed from the labeled RNA or DNA products. Therefore, gel filtration, acid precipitation, membrane diafiltration, and paper chromatography were tested to determine which technique was the most effective at such separation. The results in order of decreasing contamination by [gamma-32P]ATP were gel filtration (40%), acid precipitation (5%), diafiltration (2%), and paper chromatography (1%); and, in order of decreasing loss of RNA, were acid precipitation (30%), diafiltration (11%), gel filtration (10%), and paper chromatography (1%). In order for the resultant radioactive counts to be linearly proportional to the number of cleavage sites, the total ATP in the end-labeling reaction should be in excess of 5'-hydroxyl ends by a factor of 10 or more. Interference by nuclease mimics in the end-labeling reaction should be accounted for by including the mimics when developing a standard curve based on known concentrations of 5'-hydroxyl ends.

Chromatography, Gel↗

Quantification of free sphingosine in cultured cells by acylation with radioactive acetic anhydride.

A simple and sensitive method for quantification of sphingosine in cellular lipid extracts was developed. The assay is based on quantitative conversion of sphingosine to N-[3H]acetylated sphingosine ([3H]C2-ceramide) by N-acylation with [3H]acetic anhydride under certain conditions. Sphingosine was extracted from cultured cells with chloroform and methanol and then treated with base to remove interfering glycerolipids having reactive amino groups (e.g., phosphatidylethanolamine or phosphatidylserine). Sphingosine was acylated with [3H]acetic anhydride in the presence of 0.004 N NaOH. Acylation was complete in 1 h at 37 degrees C when sphingosine was present in the picomole range. After the acylation, samples were treated with NaOH to reduce background radioactivity by removing the remaining [3H]acetic anhydride and hydrolyzing any ester linkages formed during the acylation and resolved by thin-layer chromatography. [3H]C2-ceramide converted from sphingosine with the acylation was detected with radioautography and quantitated by scraping the corresponding band and counting its radioactivity with a scintillation counter. [3H]C2-ceramide formed was quantitatively measured. This assay allows quantification of sphingosine over a range of 10 to 1500 pmol. The amount of sphingosine in lipid extracts from cultured cells was proportional to the number of cells. Sphingosine levels in human gastric cancer KATO III cells, human promyelocytic leukemic HL60 cells, and human monoblastic U937 cells, determined by this method, were 26.6 +/- 2.2, 6.3 +/- 0.4, and 6.8 +/- 0.6 pmol per 10(6) cells, respectively. Our new procedure allows quantification of sphingosine levels present in the low picomole range in lipid extracts from biological samples.

Acetic Anhydrides↗

Development of a quantitative polyacrylamide gel electrophoresis analysis using a multichannel radioactivity counter for the evaluation of oligonucleotide-bound drug carrier.

A quantitative polyacrylamide gel electrophoresis (PAGE) analysis using a multichannel radioactivity counter was designed for the evaluation of 33P-labeled antisense oligonucleotide associated with polymeric drug carrier (nanoparticles). The proposed analytical method was first validated. The criteria of specificity, linearity, reliability, detection and quantification limits, and resolution power were determined. Results were compared to those obtained using liquid scintillation counting of crude samples or after solubilization of gel slices. The proposed method gave a better linearity and reliability than liquid scintillation counting of solubilized gel slices. In comparison with the liquid scintillation counting of crude samples, the method presented the advantage of being able to directly separate oligonucleotides differing by only one nucleotide in length. This method was applied for the separation of free oligonucleotides and oligonucleotides bound onto nanoparticles, allowing quantification of the amount of free and bound oligonucleotides without any further separation steps. Thus, because it is easy and rapid, the quantitative PAGE analysis using a multichannel radioactivity counter offers interesting possibilities for the characterization of oligonucleotide nanoparticles.

Cyanoacrylates↗

PCR product quantification by non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells.

Oligonucleotides derived from IS6110, an insertion sequence from Mycobacterium tuberculosis, have been covalently immobilized on polystyrene Covalink NH microwells to develop a sandwich and a competitive non-radioactive hybridization assay for the quantitative determination of the DNA fragments obtained by polymerase chain reaction (PCR). Using the appropriate standard DNA, the method can be employed for the quantitative analysis of PCR fragments. The sandwich assay can detect as little as 3 fmol of target DNA per well and the standard curve may be used with quantities ranging from 3 to 300 fmol per well. The competitive hybridization assay is less sensitive since it is quantitative between 100 and 8000 fmol per well. We show here that both kinds of assays can be used to identify M. tuberculosis strains isolated from clinical samples. The non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells involve few and simple operations, and are thus suitable for routine diagnosis. Moreover, when stored at 5 degrees C, precoated strips can still be used for hybridization up to at least 10 months.

Base Sequence↗