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PubMed2000

Modified telomeric repeat amplification protocol: a quantitative radioactive assay for telomerase without using electrophoresis.

A polymerase chain reaction (PCR)-based radioactive telomerase assay was developed in our laboratory which is quantitative and does not require electrophoretic evaluation (designated as TP-TRAP; it utilizes two reverse primers). The main steps of the assay include (1) extension of a 20-mer oligonucleotide substrate (MTS) by telomerase, (2) amplification of the telomerase products in the presence of [(3)H]dTTP using the substrate oligonucleotide and two reverse primers (RPC3, 38 mer; RP, 20 mer), (3) isolation of the amplified radioactive dsDNA by precipitation and filtration, (4) determination of the radioactivity of the acid-insoluble DNA. The length of the telomerase products does not increase on amplification. This valuable feature of the assay is achieved by utilization of the two reverse primers and a highly specific PCR protocol. The assay is linear, accurate, and suitable for cell-biological studies where slight quantitative differences in telomerase activity must be detected. The assay is also suitable for screening and characterization of telomerase inhibitors, as shown with a chemically modified oligonucleotide reverse transcriptase inhibitor [(s(4)dU)(35)].

Cell Extracts↗
PubMed1995

Fluorescein as a label for non-radioactive in situ hybridization.

Non-radioactive techniques can be applied to many in situ hybridization (ISH) applications, and a number of non-radioactive labels for this process have been reported. However, these labels have some inherent problems in terms of both background and signal-to-noise values. We have sought to address these issues by searching for an alternative label that has the following features: efficient incorporation into probes, non-endogenous to biological systems, the availability of a high-affinity, high-specificity antibody. Fluorescein has been shown to meet these requirements. In addition, due to the fluorescent nature of the label, it has been possible to design a rapid, non-radioactive labelling assay and also to view in situ hybridization results by direct fluorescence in certain ISH applications. The hybridization kinetics have been investigated. Significant improvements have been made to the hybridization buffer leading to reduced background and increased rates of hybridization when compared to traditional hybridization buffers.

Animals↗
PubMed1992

Two-step tumour targetting in ovarian cancer patients using biotinylated monoclonal antibodies and radioactive streptavidin.

A new method for intraperitoneal tumour targetting in ovarian cancer using biotinylated monoclonal antibodies (MoAb) and radioactive streptavidin is described. Fifteen patients with histologically documented ovarian carcinoma were injected intraperitoneally with 2 mg of biotinylated MoAb MOv18, followed 3-5 days later by 100-150 micrograms of indium-111 streptavidin, at the specific activity of 280-370 MBq/mg in 500 ml of normal saline. No toxicity was observed. Tumours were imaged from 2 to 48 h after radioactivity injection by recording both planar and single photon emission tomography (SPET) data. All patients underwent surgery 1-8 days later (mean 3 days) after scanning. The resected tumour and normal tissue radioactivity were measured. On the day of surgery, the tumour to normal tissue ratio was 9:1 (range 3:1-30:1) and 45:1 (range 12:1-120:1) for intra- and extraperitoneal samples, respectively. The mean tumor to blood ratio was 14:1 (range 4:1-30:1). The injected dose (i.d.) per gram of tumour was 0.112 (range 0.01-0.3) for recurrences and 0.05 for primary tumour (range 0.005-0.2). Over 24-48 h 14% i.d. (range 8-18% i.d.) was found in the urine, 14% i.d. (range 6-29% i.d.) in the blood and 63% i.d. (range 56-70% i.d.) was still in the peritoneal cavity. These preliminary clinical data suggest that this two-step strategy may be superior to the conventional approach (radiolabelled antibodies) for intraperitoneal radioimmunolocalization and radioimmunotherapy of ovarian cancer.

Bacterial Proteins↗
PubMed1976

Non-diffusional distribution of radioactivity in the rat median eminence after intraventricular injection of 3H-LH-RH.

The distribution of radioactivity in the rat hypothalamus after intraventricular injection of tritiated luteinizing hormone-releasing hormone (LH-RH) was studied by autoradiography and compared with that expected from Fick's second law which defines the character of ordinary diffusion. The results suggest that LH-RH penetrates through the ependyma and thin subependymal layer in the median eminence and into the suprachiasmatic area by ordinary diffusion. By contrast, concentration of radioactivity in outer layers of the median eminence is increased and, therefore, can not be explained by the diffusion law. In this region incorporation of radioactivity into cell bodies was demonstrated. In the subependymal layer of the median eminence a few rows of silver grains seemingly located in cellular processes were also observed. The diffusion coefficient in the periventricular zone was lower than that in the deeper tissue. It is suggested that the ependymal-subependymal complex of the third and lateral ventricles acts as passive membrane towards LH-RH contained in the cerebrospinal fluid. The significance of this mechanism for the intrahypothalamic transport of RH's is discussed.

Animals↗
PubMed1988

Localization and identification of nuclear radioactivity in the pituitary gland and genital tract after administering 3H-testosterone, 3H-dihydrotestosterone, or 3H-estradiol to male rhesus monkeys.

Target cells for testosterone, dihydrotestosterone, and estradiol in the pituitary gland and genital tract of the male primate were localized by thaw-mount autoradiography, and high performance liquid chromatography was used to identify the metabolites of these steroids in cell nuclei. Castrated rhesus monkeys were injected with 3H-testosterone, 3H-dihydrotestosterone, or 3H-estradiol and killed 60 min later. In the anterior pituitary gland, fewer cells were labeled and less radioactivity was taken up by cell nuclei following the administration of either 3H-testosterone (4% of pars distalis cells and 5 dpm/micrograms DNA) or 3H-dihydrotestosterone (5% of cells and 13 dpm/micrograms DNA) than following the administration of 3H-estradiol (43% of cells and 214 dpm/micrograms DNA). Most of the radioactivity in nuclei was in the form of the unmetabolized parent compound (78-94%). In prostate, seminal vesicles, and penis, 3H-dihydrotestosterone was the predominant form of nuclear radioactivity following both 3H-testosterone (67-90%) and 3H-dihydrostestosterone (94-97%) administration, and both androgens labeled epithelial and smooth muscle cells. In contrast, 3H-estradiol was taken up in unchanged form, by cell nuclei of the genital tract and it labeled connective tissue fibroblasts, but not epithelial cells. Thus, the distributions of target cells for androgens and estrogens were clearly different in all these tissues, and the uptake of testosterone resembled that of its androgenic rather than that of its estrogenic metabolite.

Animals↗
PubMed1976

Investigations on the turnover of adrenocortical mitochondria. V. An autoradiographic study of the radioactivity decay in the mitochondrial compartment from the adrenal cortex of 3H-thymidine-injected rats.

The radioactivity decay of the mitochondrial compartment of 3H-thymidine-injected rats was followed by high resolution autoradiography. The number of days in which the radioactivity of the mitochondrial compartment is reduced to a half was calculated from the semilogarithmic plots of radioactivity versus time. Since DNA is a very stable molecule, it was assumed that this parameter can be an estimate of the half-life of adrenocortical mitochondria. It was found that the half-lives of mitochondria from the zonae glomerulosa, fasciculata and reticularis are, respectively, 8.20, 11.51 and 8.16 days.

Adrenal Cortex↗
PubMed1993

Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies.

In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a 35S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascin-specific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.

Acrylic Resins↗
PubMed1982

Increased survival due to radioactive estradiol in mice with C3HBA or BW 10232 tumors.

The influence of progesterone and estradiol labeled with tritium was studied in mice inoculated with transplantable mammary adenocarcinomas C3HBA or BW 10232. Tumor size, tumor growth rate, and host survival were measured. Radioactive [3H]estradiol administration increased survival time and inhibited tumor growth in mice inoculated with these tumor lines. Tumor growth retardation depended on the amount of radioactivity injected and nonradioactive estradiol was without any salutary effect on tumor size or host survival. Neither survival times nor tumor growth rate were altered by radioactive [3H]progesterone. The underlying mechanism(s) is (are) referable to ionizing radiation by the specific carrier estradiol or to an isotope effect of [3H]estradiol.

Animals↗
PubMed1975

The radioactive microsphere method for the assessment of regional myocardial blood flow after coronary artery occlusion. Inaccuracies due to variations in the diameter distribution of the spheres.

In this study, we have tried to determine the magnitude of the inaccuracy of the radioactive microsphere method - due to variations in the diameter distribution of the spheres - for measuring regional myocardial blood flow after coronary artery occlusion. In 5 mongrel dogs, three types of 15 mum microspheres, labelled with 125I, 141Ce or 85Sr, were injected simultaneously after the descending branch of the left coronary artery had been ligated. Myocardial samples wert taken from the left ventricle and divided into four groups according to the number of spheres per sample. The radioactivity of the various isotopes per gram tissue was expressed as percentage of their activity per milliliter of the reference sample. The diameter distribution of microspheres, labelled with each of the isotopes, was determined light-microscopically in suspensions belonging to three different batches. The relative error, as determined from the difference in relative radioactivity of the various types of microspheres in the tissue samples, was higher than the theoretical error for each of the number of spheres per sample. It is very likely that this discrepancy is caused by the differences in diameter distribution of the various types of microspheres, resulting in non-random error. The smaller spheres tended to go to low flow areas and the larger ones to high flow areas. Because of the non-randomness, the error due to diameter variations in the spheres can be diminished by randomizing the order of injection of the various isotopes. The present study indicates that the relatively high degree of accuracy of the microsphere method for the determination of blood flow to large parts of the myocardium with an unimpeded coronary circulation, as was described in literature, cannot be extrapolated to the determination of regional myocardial blood flow after coronary artery occlusion, when the combination of small tissue samples, variations in the diameter distribution of the spheres and an unevenly distributed myocardial blood flow unfavourably affect the accuracy of the method.

Animals↗
PubMed1981

Differential diagnosis of maldigestion and malabsorption of fat. I. Comparison of the radioactivity of triglyceride and non-esterified fatty acid in fecal fat using purified 131I-triolein.

To differentiate maldigestion from malabsorption of fat, we compared the radioactivity of triglyceride with non-esterified fatty acid in fecal fat after ingestion of radiochemically purified 131I-triolein. A large amount of radioactivity was present in the triglyceride fraction from patients with pancreatic insufficiency, while such was found only in non-esterified fatty acid fraction in patients with short bowel syndrome, as determined by thin-layer chromatogram scanning. Thus, the "Digestion-Absorption Index" was calculated as a ratio of the radioactivity of triglyceride to that of non-esterified fatty acid. Mean Digestion-Absorption Index was 0.946 in the maldigestive group, and 0.045 in the malabsorptive group, and there was a statistical significance. However, an overlap in a few cases was confirmed in the area of 0.10 +/- 0.02, when this index was plotted on a table of semilogarithms. We assumed that this overlap was chiefly the result of maldigestion plus malasorption of fat.

Adult↗
PubMed1999

Direct preparation of radioactive fullerenes as a tracer for applications.

The C60 and C70 fullerenes were irradiated by high-energy gamma-rays and charged particles. The irradiated samples were dissolved in CS2 and/or toluene and filtered to remove insoluble by-products. Finally, radioactive fullerenes and products labeled with 11C or 13N were isolated and detected in the liquid phase by radiochromatography. It was found that (1) not only 11C or 13N radioactive monomer fullerenes but also their dimers (trimers and, possibly, tetramers) were produced by recoil implantation process following nuclear reaction and (2) the radioactive fullerene labeled with 11C yields has led to high yields.

Carbon↗
PubMed2001

In-vitro uptake of radioactive lipiodol I-131 and I-125 by hepatoblastoma: implications for targeted radiotherapy.

Lipiodol, a stable iodine addition product of fatty ethyl esters derived from poppyseed oil, has been used as a vehicle for targeted cytotoxic or radiotherapeutic treatment in adults with unresectable hepatocellular carcinoma and in a few children with advanced hepatoblastoma (HB). Prolonged retention of lipiodol by the cancer cells might enable more effective targeted therapy to advanced, multifocal, or metastatic HB. To investigate the uptake and cytotoxic efficacy of beta and Auger electron-emitting radioconjugates on HB cells, monolayers of HB (C3a, Hep Tow1) and normal human hepatocyte cell lines were exposed to lipiodol131 (L131), lipiodol125 (L125), or a cocktail of both isotopes. Uptake of radioactivity was assessed autoradiographically using phosphoimages and the cytotoxicity assessed by trypan blue exclusion and clonogenic assay. The uptake of the different radioconjugates was identical in both HB cell lines. The "cocktail" of both radioactive lipiodols caused the greatest cytotoxicity to HB C3a, and HepTow1. L125 alone had a similar level of uptake and cytotoxicity as L131. Radioactive iodine alone did not show any cytotoxicity on any of the liver cell lines in culture for up to 72 h. These experimental results provide support for a clinical strategy of combinations ("cocktails") of radioconjugates in targeted radiotherapy in patients with HB.

Carcinoma, Hepatocellular↗
PubMedDate not supplied

Detection of mRNAs encoding peroxisomal proteins by non-radioactive in situ hybridization with digoxigenin-labelled cRNAs.

We have used a non-radioactive in situ hybridization (ISH) protocol for the detection of mRNAs encoding proteins localized in peroxisomes. In this presentation the literature on detection of "peroxisomal mRNAs" is reviewed and the results obtained by application of the non-radioactive method are compared with those obtained by hybridization with radioactive probes. Moreover, the special processing conditions and the application of the method for the specific visualization of mRNAs coding for several peroxisomal proteins with different abundance levels and distinct tissue distributions are presented. The combination of the following technical details in the ISH procedure were found to be essential for obtaining optimal sensitivity and good histological quality of the preparations: (a) perfusion-fixation with a fixative containing 4% depolymerized paraformaldehyde/0.05% glutaraldehyde, (b) the use of paraffin embedding instead of frozen sections, (c) specific proteinase K-digestion time for each tissue, and (d) the use of digoxigenin-labelled cRNA probes (hydrolyzed to a length of about 200 bases) for detection. By using this technique, we were able to localize several peroxisome-specific mRNAs with different degrees of abundance: (1) high-level (catalase and urate oxidase) and (2) low-level (all beta-oxidation enzymes and the 70-kDa peroxisomal membrane protein) in rat liver and kidney. The specificity of the method was confirmed by the negative results obtained with corresponding sense controls and the distinct positive staining patterns obtained for albumin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs. All transcripts for mRNAs encoding peroxisomal proteins were localized to the cytoplasm of hepatocytes, with all nuclei as well as epithelial cells of bile ducts and sinusoidal cells remaining negative. In rat kidney, the catalase transcripts were confined to proximal tubular epithelial cells, which is consistent with the high abundance of peroxisomes in this part of the nephron. In contrast, no transcripts for urate oxidase were present in the kidney, corresponding to the absence of that protein in this organ. The transcripts for GAPDH on the other hand were localized in proximal and distal tubular epithelial cells as well as in collecting ducts. The application of this technique to the rat adrenal gland and testis in recent unpublished studies have revealed exclusive localization of catalase transcripts to the adrenal cortex and to interstitial cells of Leydig, which are known to be rich in microperoxisomes. These observations demonstrate the suitability of this technique for accurate localization of mRNAs encoding peroxisomal proteins and for the analysis of alterations in the expression of the corresponding genes under different experimental conditions.

Animals↗
PubMedDate not supplied

Guidelines for the safe use of radioactive materials during localization and resection of the sentinel lymph node.

BACKGROUND: Several reports have demonstrated accurate prediction of nodal metastasis with radiolocalization and selective resection of the radiolocalized sentinel lymph node (SLN) in patients with breast cancer and melanoma. As reliance on this technique grows, its use by those without experience in radiation safety will increase. METHODS: Tissue obtained during radioguided SLN biopsies was examined for residual radioactivity. Specimens with a specific activity greater than the radiologic control level (RCL) of 0.002 microCi/g were considered radioactive. Radiation exposure to the surgical team was measured. RESULTS: A total of 24 primary tissue specimens and 318 lymph nodes were obtained during 57 operations (37 for breast cancer, 20 for melanoma). All 24 (100%) of the specimens injected with radiopharmaceutical and 89 of 98 (91%) of the localized nodes were radioactive after surgery. Activity fell below the RCL 71+/-3.6 hours in primary tissue specimens, 46+/-1.7 hours in nodes from melanoma patients, and 33+/-3.5 hours in nodes from breast cancer patients (P = .037). The hands of the surgical team (n = 22 cases) were exposed to 9.4+/-3.6 mrem/case. CONCLUSION: Although low levels of radiation exposure are associated with radiolocalization and resection of the SLN, the presented guidelines ensure conformity to existing regulations and allow timely pathologic analysis.

Biopsy↗
PubMed1982

Metabolic fate of radioactive acyclovir in humans.

The metabolic fate and the kinetics of elimination of [8-14C]acyclovir in plasma and blood was investigated in five cancer patients. Doses of 0.5 and 2.5 mg/kg were administered by one-hour intravenous infusion. Radioactivity was distributed nearly equally in blood and plasma. The plasma and blood concentration-time data were defined by a two-compartment open pharmacokinetic model. The overall mean acyclovir plasma half-life and total body clearance +/- SD were 2.1 +/- 0.5 hours and 297 +/- 53 ml/min/1.73 m2. Binding of acyclovir to plasma proteins was 15.4 +/- 4.4 percent. The radioactive dose was excreted predominantly in the urine (71 to 99 percent) with less than 2 percent excretion in the feces and only trace amounts of radioactivity in the expired air. Reverse-phase high-performance liquid chromatography indicated that 9-carboxymethoxymethylguanine was the only significant urinary metabolite of acyclovir accounting for 8.5 to 14.1 percent of the dose. A minor metabolite (less than 0.2 percent of dose) had the retention time of 8-hydroxy-9-(2-hydroxyethoxymethyl)guanine. Unchanged urinary acyclovir ranged from 62 to 91 percent of the dose. There was no indication of acyclovir cleavage to guanine. The renal clearances of acyclovir were three times higher than the corresponding creatinine clearances.

Acyclovir↗
PubMed1975

Use of radioactive microspheres for studies of tubal ovum transport.

Experiments were designed to ascertain the fidelity with which radioactive plastic ovum models imitated the time-course of transport of natural ova through the rabbit oviduct. After location of the radioactive microspheres within the intact oviducts, the genital tracts were removed and cleared in benzyl benzoate. Natural ova and ovum surrogates were then visualized in situ. The radioactive microspheres were reasonably good models of transport of natural ova and their sojourn in the oviduct was shortened by progesterone and prolonged by estrogen. Thus they should be useful tools for future studies of own transport under various conditions.

Animals↗
PubMed1977

Comparing radioactive and trephine-disk bioassays of dicloxacillin and gentamicin in ocular tissues in vitro.

Trephined disks of tissue (6 mm diameter) from fresh rabbit eyes were immersed overnight in solutions of radioactive carbon (14C)-dicloxacillin or gentamicin, then assayed by one of two methods: agar-diffusion bioassay, using filter-paper disks immersed in known concentrations of antibiotic as standards, or radioactive assay. The bioassay underestimated the concentrations of antibiotic in sclera and iris by 38 to 72%, and slightly overestimated those in the cornea. A corrected bioassay was calculated, based on the differences in fluid mass between tissue specimens and filter-paper disks; this value was within 20% of the radioactive assay in 18 of 24 experiments with dicloxacillin, and in 14 of 24 with gentamicin. There was evidence of pronounced binding of gentamicin to ocular tissues (iris greater than sclera greater than cornea); this appeared to be a saturable and reversible phenomenon. Less marked binding of dicloxacillin to iris and sclera was noted. The results indicated that, depending on the degree of accuracy required, the corrected trephine-disk agar-diffusion bioassay provides a reasonable estimate of the antibiotic content of ocular tissues.

Animals↗
PubMed1985

The determination of specific radioactivity of proteins eluted intact from polyacrylamide gels, utilizing a fluorescamine assay.

The methodology described permits the measurement of the specific radioactivity of diverse proteins resolvable by separatory techniques using cylindrical polyacrylamide gels. Following separation, the proteins are electroeluted; eluted protein is quantitated in the microgram range using a fluorescamine assay, while the major portion of the recovered sample is used for radioactivity measurement. These procedures have been adapted for use in tracer studies of protein metabolism. Their utility in kinetic investigations is demonstrated with data on the time course of changing specific radioactivities of human plasma albumin and apolipoprotein B labeled in vivo with a [3H]leucine tracer.

Apolipoproteins B↗