Use Of Radioactive Isotopes Research Articles

Adverse pregnancy outcomes in offspring of fathers working in biomedical research laboratories

Laboratory work may constitute a possible health hazard for workers as well as for their offspring, and involves a wide range of exposures, such as organic solvents, carcinogenic agents, ionizing radiation, and/or microbiological agents. Adverse pregnancy outcomes in the offspring of male employees in biomedical research laboratories are examined. Offspring to males employed 1970-1989 at four Swedish universities were identified via the Medical Birth Register (MBR), along with other pregnancy parameters. Offspring of fathers with laboratory work (n = 2,281) is considered exposed, and of non-laboratory employees unexposed (n = 1,909). Exposure data were obtained by questionnaires to research group leaders. Logistic regression analysis estimated odds ratios (ORs) and 95% confidence intervals (CIs). Paternal laboratory work in general showed no statistically significant increased ORs concerning birth weight and/or gestational age, but work specifically with radioactive isotopes gave OR 1.8 (CI 1.0-3.2) for high birth weight and a relative risk of 1.2 (CI 1.0-1.4) for sex ratio (male/female). There was no clear association between periconceptional paternal laboratory work and adverse reproductive outcomes, but use of radioactive isotopes showed increased OR for high birth weight in offspring.

A nonradioactive, cell-free method for measuring protein synthesis inhibition by Pseudomonas exotoxin

Pseudomonas exotoxin A (PE) inhibits protein synthesis by NAD-dependent ADP-ribosylation of eukaryotic elongation factor 2. Traditionally, toxin activity has been characterized, either in living cells or cell-free systems, using radioactive compounds for quantification. The increased costs of radioactive waste disposal together with heightened security concerns have made the use of radioactive isotopes less attractive for routine laboratory assays. We therefore adapted a cell-free rabbit reticulocyte in vitro transcription-translation system that utilizes a reporter (β-galactosidase) to measure toxin activity. The assay for PE is rapid, scalable, log-linear, NAD dependent and can be used to assess the neutralizing activity of anti-PE antibody preparations.

Coral calcification: Use of radioactive isotopes and metabolic inhibitors to study the interactions with photosynthesis and respiration

In order to characterize the process of calcification in scleractinian corals, a series of laboratory experiments were conducted using radioactive isotopes. Labelled calcium, bicarbonate and glucose were used and the fates of the labelled tracers were followed in the skeleton and the tissue fractions of the coral Galaxea fascicularis. In addition, a variety of metabolic inhibitors were used to test the effects of various enzymes and processes on the incorporation rates. The incorporation rate of 45Ca into the coral skeleton decreased to about one-fifth upon inhibition of metabolic respiration by the specific inhibitor NaCN suggesting a major role of metabolic respiration in coral calcification, and decreased to one-half upon inhibition of carbonic anhydrase by the specific inhibitor acetazolamide indicating a role of the enzyme in the process. The results obtained have also shown that corals are able to incorporate carbon from seawater bicarbonate and added glucose in both skeleton and tissue fractions. The process of incorporation was influenced by light conditions, carbonic anhydrase, respiration and photosynthesis. The incorporation rate of 14C-HCO was reduced to about one-tenth in the skeleton, and one-fifth in the tissue, upon inhibition of carbonic anhydrase suggesting a major influence of the enzyme in the incorporation process. The inhibition of photosynthesis had more influence on the tissue incorporation rate of the tracer than the skeleton suggesting that photosynthesis is the main process responsible for tissue use of seawater bicarbonate in the coral. 14C-glucose incorporation into the skeleton was mainly affected by NaCN addition and to a lesser extent by dichlorophenyldimethylurea (DCMU) addition, while the tissue fraction was mainly affected by NaCN addition. It was concluded that respiration and photosynthesis, in addition to the enzyme carbonic anhydrase, are limiting factors for the process of calcification in the coral, and that various forms and sources of carbon can be used in coral calcification and tissue growth.

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake.

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo.

Assessment of cell-mediated toxicity has traditionally been achieved by measuring the specific activity of enriched effector cell populations against antigen-loaded target cells labeled with radioactive isotopes in vitro. Fluorometric techniques are viewed as a promising alternative to the use of radioactive isotopes for these analyses. Direct assessment of cytotoxicity in vivo can be achieved by monitoring survival of injected fluorescent targets relative to a differentially labeled internal control population without specific antigen. We have developed this approach, incorporating the use of multiple target cell populations labeled with different dyes so that cytotoxicity can be assessed against titrated doses of a given antigen, or against a range of different antigens, simultaneously. We show that this assay, referred to as the VITAL assay, can be used to assess cytotoxic activity of CTL and iNKT cells in vivo and in vitro. CTL responses measured in vivo could be correlated with antigen doses used in immunization strategies, and also with the size of specific CTL populations enumerated in the blood with fluorescent MHC/peptide tetramers. The VITAL assay is, therefore, a sensitive technique allowing analysis of complex multi-epitope responses.

A fluorescent compound for glucose uptake measurements in isolated rat cardiomyocytes.

A focus of current diabetes research is the development of insulinomimetic compounds for oral treatment of diabetes and its associated cardiac complications. Screening compounds for their potential insulinomimetic effects usually involves the use of radioactive isotopes. The focus of this study was to investigate a nonradioactive fluorescent compound for its use in screening insulinomimetic compounds. The indicator 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) has been used by some workers to measure glucose uptake in Escherichia coli and Candida albicans. We propose that 2-NBDG will also be a suitable indicator for mammalian cell lines, in particular rat cardiomyocytes. We found that the indicator could give a reliable reproducible standard curve following appropriate dilution and is taken up by isolated cardiomyocytes. The insulinomimetic compounds vanadyl sulfate and sodium molybdate showed rates of glucose uptake similar to that of insulin. Furthermore, the rate of uptake measured for insulin using this technique (0.04 +/- 0.003 nmol x min(-1) x 10(6) cells(-1) is comparable with previous literature using 2-deoxyglucose uptake measurements on isolated myocytes (0.040 nmol x min(-1) x 10(6) cells(-1), demonstrating the validity of this fluorescent compound for glucose uptake studies.

STRAIN IDENTIFICATION OF SPODOPTERA FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE) INSECTS AND CELL LINE: PCR-RFLP OF CYTOCHROME OXIDASE C SUBUNIT I GENE

A simple method was developed to analyze the two morphologically indistinguishable host-associated strains of the fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith). Total DNA extracted from the FAW corn and rice strains, as well as from the S. frugiperda cell line (SF9) was used to PCR amplify a 569 base pairs region of the mitochondrial gene cytochrome oxidase subunit I (COI). The amplified DNA from the Spodoptera corn strain and the SF9 cell line contained a restriction fragment length polymorphism (RFLP) marker, the MspI recognition site that was not present in the rice strain. This PCR-RFLP method does not require purification of mitochondrial DNA (mtDNA) or the use of radioactive isotopes, and differs from previous methods in that only a few nanograms of total DNA are needed to yield clear and accurate strain identification of individual insects.

A method for estimating the solids circulation rate in a closed-loop circulating fluidized bed

We propose a simple method for accurately estimating the solids circulation rate in a closed-loop liquid–solid circulating fluidized bed. The technique is based on the use of radioactive isotopes for estimating the solids velocity and volume fraction distribution in a chosen section of the circulating fluidized bed loop. The method is demonstrated for a liquid–solid circulating fluidized bed, and is readily applicable in other systems such as gas–solids circulating fluidized beds or closed-loop pneumatic and hydraulic transport lines. With minor modifications, the method may be used for industrial systems as well.

Cardiovascular Surgery Without Cardiopulmonary Bypass in Patients with Heparin-Induced Thrombocytopenia Type II Using Anticoagulation with Recombinant Hirudin

Cardiovascular Surgery Without Cardiopulmonary Bypass in Patients with Heparin-Induced Thrombocytopenia Type II Using Anticoagulation with Recombinant Hirudin

Determination of Kinetic Isotope Effects for Nucleoside Hydrolases using Gas Chromatography/Mass Spectrometry

Kinetic isotope effects are widely used to determine the transition state of chemical and enzymatic reactions. Radioactive isotopes are used most often to determine these kinetic isotope effects. However, stable isotopes offer a number of advantages over the use of radioactive isotopes. These advantages include ease of handling and disposal along with increased safety in the laboratory. [1′-13C]Inosine and [1′-2H]inosine kinetic isotope effects were determined using a gas chromatograph in conjunction with a mass selective detector for nucleoside hydrolase, a purine-metabolizing enzyme. Three ion pairs were used to determine kinetic isotope effects. These ion pairs were 158/159, 187/188, and 217/218. The average isotope effects for all ion pairs were 1.021 ± 0.006 for [1′-13C]inosine and 1.113 ± 0.008 for [1′-2H]inosine. The transition state consistent with these isotope effects is also consistent with the transition state proposed by Schramm and Horenstein using radioactive substrates.

Acceptor Correlated Centers in II-VI Compounds

We have investigated the acceptor doping of CdTe and ZnSe to elucidate various aspects of self-compensation. Before doping, a well-defined deviation of the stoichiometry was adjusted by thermal pre-treatment of the bulk crystals. We used ion implantation for doping. Radiation damage could be largely removed by proper annealing. Besides stable elements, we implanted radioactive isotopes of acceptors decaying into inactive centers (111Ag → Cd, 73As → Ge). On the other hand, we implanted native or isoelectronic isotopes which transmute by radioactive decay into relevant dopants (107Cd → Ag, 85Sr → Rb). In this way it is possible to avoid self-compensation. The use of radioactive isotopes allows an unambiguous correlation of defect features occurring in electrical and photoluminescence measurements with the incorporated dopants. Under optimum pre- and post-implantation annealing conditions, we find an almost one-to-one doping efficiency with respect to the implanted dose.

13C octanoic acid breath test

Although scintigraphy, a non-invasive procedure, is usually considered to be the reference technique for measuring gastric emptying in humans,1 several drawbacks limit its application in routine practice. Expensive equipment is...

An enzyme based assay for the measurement of complement mediated binding of immune complexes to red blood cells

A new in vitro method is presented for measuring directly the ability of sera to induce binding of immune complexes (ICs) to erythrocytes (ICRB assay). The assay measures the binding of alkaline phosphatase (AP)–anti-alkaline phosphatase (anti-AP) complexes formed in the presence of the test sera to the complement receptor 1 (CR1) on normal human red blood cells (RBCs). By using a standard serum source, the assay can also be used to measure the IC binding ability of RBCs from different donors. As compared to the traditional CH50 method, the ICRB assay generally showed more pronounced abnormality in 10 individuals tested, of whom 5 had primary deficiency of classical pathway components. Seven out of ten individuals had systemic lupus erythematosus (SLE) and 2/10 had other rheumatic diseases without primary complement deficiency. The ICRB measured in samples from 9 other patients with SLE was significantly decreased when compared to values from 80 normal individuals. ICRB in serum samples from a C2 deficient SLE patient collected during plasma infusion treatment reflected closely the rising amount of C2 in the serum. Using RBCs from different donors ICRB activity correlated well with the numbers of CR1 as measured by a flow cytometric assay (FCA). These methods should be valuable for measuring the overall IC clearance capacity of the blood and have the advantage that the use of radioactive isotopes is avoided.

Molecular HLA typing--the brave new world.

Molecular HLA typing--the brave new world.

Analysis of Avian Lymphocyte Proliferation by a New, Simple, Nonradioactive Assay (Lympho-Pro)

An assessment of T-cell-mediated immune functions (i.e., lymphocyte proliferation assay) in the chicken, unlike the determination of antibody levels, is not routinely performed. This is primarily because of difficulties in the isolation of relatively pure populations of lymphocytes and the use of radioactive isotopes. To address these issues, the goals of our study were to optimize a method for isolating and enriching avian lymphocyte populations and to develop a nonradioactive lymphocyte proliferation assay. To accomplish these goals, we used a multiple slow-speed centrifugation technique combined with a "swirl" collection technique for lymphocyte isolation from chicken peripheral blood. After a fraction enriched with lymphocytes was obtained, a simple, rapid colorimetric and fluorometric assay (lympho-pro) to indirectly determine mitogen-induced proliferation was adapted and compared with the "Gold Standard" [3H]thymidine. Chickens of different ages and two genetic strains were used in this study. Lymphocytes were stimulated with various concentrations of concanavalin A (Con A, T-cell mitogen) or phorbol 12-myristate 13-acetate + ionomycin (pan lymphocyte mitogen). Our studies showed that the pattern of lymphocyte proliferation assessed by the Alamar blue-based lympho-pro assay was similar to the [3H]thymidine incorporation assay. Younger birds had higher levels of mitogen-induced proliferation when compared with adults of the same genetic strain. Because the lympho-pro assay, unlike [3H]thymidine, does not require lysis of cells to assess proliferation, cells that have undergone stimulation/proliferation can be subsequently characterized by staining with antibodies against cell surface antigens and analysis by flow cytometry. Another notable advantage of the lympho-pro assay is the rapidity of assessment and nontoxicity. In conclusion, this assay may be of value in assessing some aspects of T-cell-mediated immunity in both avian research and avian medicine diagnostic settings.

Evaluation of the immune response in visceral leishmaniasis

Detection of parasites in culture or by microscopy is still necessary to make diagnosis of visceral leishmaniasis (VL). Serological methods still need assessment, as they are quick but not very sensitive, especially in immunosuppressed subjects. This paper compares the results obtained with three serological methods (indirect immunofluorescence test (IFAT), direct agglutination test (DAT), and enzyme-linked immunosorbent assay (ELISA) and the specific cell-mediated immune response, evaluated as proliferation and IFN-γ production by peripheral blood lymphocytes (PBL) following stimulation with heat-killed L. infantum promastigotes. PBL and sera were obtained from 10 healthy donors, 3 VL patients in acute phase, and 3 patients recovering after two glucantim treatment courses. No false positive results were observed with the serological methods. IFAT can be considered the most sensitive and best suited for follow-up, as it allowed a good discrimination between the acute and remission phase. DAT did not discriminate between healthy donors and remission-phase patients, whereas ELISA is unsuited for follow-up, as it did not show any significant difference between remission- and acutephase patients. Assessment of the cellular response is not recommended for making a diagnosis, because false positive results are frequent. However, a strong cellular response in a patient stands for a successful treatment. IFN-γ titration is preferable to the proliferation test, because it gives earlier results and does not require the use of radioactive isotopes.

Enhanced ignition of cold cathode gauges through the use of radioactive isotopes

The Relativistic Heavy Ion Collider (RHIC) at Brookhaven National Laboratory comprises vacuum systems that range in pressure from 10−4 to 10−11 Torr. For cost savings associated with commonality, it is desirable to use one ultrahigh vacuum (UHV)-compatible high vacuum gauge throughout the accelerator. Work was done with radioactive isotopes to enhance ignition of inverted magnetron gauges at very low pressures (i.e., <10−10 Torr). Tests were initially conducted using cobalt-coated copper washers with a nickel flash to prevent oxidation. Solid nickel washers were also activated and tested. Problems with these sources will be discussed. Finally, an americium source was tested. The UHV-compatible americium sources yielded average ignition times of about 10 min at 4×10−11 Torr. The addition of the americium source produced a gauge suitable for operation at UHV pressures, and as a consequence for all of the RHIC.

Use of fluorochromes in the determination of chemotaxis and haptotaxis of granulocytes by micropore filter assays

Most conventional assays for the in vitro measurement of polymorphonuclear leukocyte (PMN) migration are modifications of the Boyden chamber technique, which requires quantification of migrated cells on micropore filters. This quantification is accompanied by several disadvantages, comprising errors in direct cell counting, low sensitivity of turbidimetric methods, the loss of marker enzymes by activation of PMNs, or the use of radioactive isotopes. We set up an improved fluorometric method to measure chemotactic and haptotactic migration of PMNs using polycarbonate filter-bearing Transwell culture plate inserts. This improved fluorometric method allows the evaluation of effects of fluorescent-dye labeling on chemotaxis and haptotaxis - defined as cell migration due to cell surface- or matrix-bound gradients of chemoattractants - by collecting data in an automated system. Results were compared with data obtained from direct microscopic cell counting in Transwell experiments as well as in conventional 48-multiwell Boyden chamber assays. Calcein-AM and BCECF-AM proved to be fluorochromes with minimal effect on both types of PMN migration. We conclude that the fluorochromes are powerful tools for the analysis of PMN migration and allow modifications of chemotaxis/haptotaxis assays for automated quantification.

A new luminometer for sensitive quantification by chemiluminescence of specific proteins in microtitre plates and on blot membranes

Generally applicable technologies are described, which depend on the use of chemiluminescence and a new type of a versatile luminometer, that can measure also weak light in microtitre plates (microplates) and on blotting membranes that is especially useful for dot blot immunoassay. Applications are described using alkaline phosphatase conjugated antibodies against IgG and IgE and the reagent Lumi-Phos 530. This chemiluminescence offers advantages over the use of radioactive isotopes, densitometry and light reflection measurement on membranes and also ELISA, for sensitive quantification of e.g. specific proteins. Special procedures are described for the first time that with the mentioned reagent in agarose gel allows specific and very sensitive quantification of proteins on dot blot membranes. The luminometer, which has temperature control, is very sensitive, precise and allows efficient protocols for various assays. It thus fulfils many of the requirements for good quantification, is time-saving and in addition brings significant improvements due to very low detection limits and large linear concentration ranges that can be measured with excellent regression coefficients r 2 often about 0.999).

Radioisotopes and Their Use in the Diagnosis and Management of Thyroid Disease

The use of radioactive isotopes in thyroid evaluation and therapy gives important information in the endocrine assessment of the aged. Thyroid uptake imaging can be used to establish a hypothyroid or hyperthyroid state and can determine the functional status of a palpable nodule. Investigational agents, such as T1 201m DMSA, MIBG, FDG, and Gallium are reviewed in relation to the evaluation of neoplasia. A detailed discussion of thyroid carcinoma ablation and subsequent radioiodine therapy is described, as are radiation safety procedures as they relate to the elderly.