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PubMed1983

In vitro transposition of bacteriophage Mu: a biochemical approach to a novel replication reaction.

The transposition-replication reaction of phage Mu has been reproduced in a cell-free reaction system. Two assay methods were used for the detection of transposition products. The first method uses lambda DNA as the target of transposition and a plasmid containing the ends of Mu DNA and an ampicillin-resistance gene as the donor; after the reaction, in vitro lambda packaging allows the scoring of ampr transducing phages generated by transposition. In the second method, the products made in the presence of a radioactive precursor for DNA synthesis are directly analyzed by gel electrophoresis and unique product species are identified. The reaction requires a donor DNA carrying the two Mu ends in their proper relative orientation, extracts containing the A and B gene products of Mu, and host factor(s). RNA synthesis by E. coli RNA polymerase is not required for the reaction. The products include both cointegrates and simple inserts. Both types of products show incorporation of radioactive DNA precursors; however, simple inserts do not seem to undergo a full round of DNA replication.

Bacteriophage mu↗

PubMed1987

Rare-earth elements in the NBS standard reference materials spinach, orchard leaves, pine needles and bovine liver.

A highly sensitive radiochemical neutron activation method for the determination of all rare-earth elements (REE) in NBS biological reference materials is described. The materials are irradiated, dissolved in HF/HCl solutions, mixed with scandium and REE carriers (except La, Pr, Nd, Dy, Er), and the resulting solutions evaporated to dryness. The residues are dissolved in HCl and the REE precipitated as fluorides on addition of HF/NH4F solutions. The REE fluorides were collected, dissolved in a nitric/boric acid solution and the radioactivity of the resulting solutions determined by gamma spectrometry. The concentrations of REE in the NBS SRM Spinach, Orchard Leaves, Pine Needles, and Bovine Liver were found to be in the ng/g to microgram/g range. The relative standard deviations are approximately 8%. The results agreed, within experimental errors, with literature values. The distribution patterns of REE in the NBS materials relative to chondritic meteorites resemble the patterns for geological materials.

Animals↗

PubMed1990

Transcapillary permeability and subendothelial distribution of endothelial and amniotic fluid insulin-like growth factor binding proteins in the rat heart.

Insulin-like growth factor (IGF) binding proteins (IGFBP) were purified from conditioned media of cultured bovine endothelial cells (ECBP) and from human amniotic fluid (IGFBP-1), and then labeled by radioiodination. 125I-ECBP and 125I-IGFBP-1 were perfused through isolated beating rat hearts for 1 and 5 min, and the hearts fixed and analyzed for 125I-BP content and distribution. One to 4% of the perfused 125I-ECBP and 125I-IGFBP-1 crossed the capillary boundary. The ECBPs predominantly localized as intact 125I-BP in connective tissue elements of the heart with less 125I-BP in cardiac muscle. The ratio of 125I-ECBP in connective tissue: muscle (normalized to percent vol of these compartments) was greater than or equal to 10:1. In contrast, the IGFBP-1 had a greater affinity for cardiac muscle with ratios of 125I-IGFBP-1 in connective tissue:muscle of approximately 1:2. When 125I-IGF-I, in the absence of any BPs, was perfused through the hearts approximately 3-5% left the microcirculation and was found in subendothelial tissues. 125I-IGF-I localized primarily to cardiac muscle with a distribution of connective tissue:cardiac muscle of about 1:3. The findings in the isolated perfused heart were confirmed in intact animals. After 125I-IGFBP-1 was injected into anesthetized rats and allowed to circulate for 5 min, substantial radioactivity was associated with the heart. As in the isolated heart, the IGFBP-1 preferentially localized to cardiac muscle with a connective tissue:cardiac muscle ratio of 1:3. We conclude that IGFBPs produced by endothelial cells and the IGFBP-1 contained in amniotic fluid can cross the capillary boundaries of the rat heart, and that the ECBPs preferentially localize in connective tissue elements of the myocardium, whereas IGFBP-1 predominantly localizes in cardiac muscle.

Amniotic Fluid↗

PubMed1998

HIV-1 rev nuclear export signal binding peptides isolated by phage display.

The human immunodeficiency virus type 1 (HIV-1) Rev protein is absolutely essential in the viral replication cycle, where it induces the production of viral structural proteins. Rev functions in part by inducing the nuclear export of incompletely spliced mRNA species specified by the presence of an RNA element, the Rev response element (RRE). Several proteins implicated in RNA processing and nucleo-cytoplasmic transport have been shown to interact with Rev, however, their exact roles remain unknown. To map potential protein recognition sites within the Rev structure, we have screened a phage library, displaying random 15-mer peptides, and isolated clones exhibiting similar sequences that specifically interact with Rev. The binding sites on Rev of the corresponding synthetic peptides were characterised by protein footprinting, involving partial proteolysis of radioactively end-labelled Rev protein. Two of the peptides produced a significant footprint within the nuclear export signal of Rev, raising the possibility that they mimic the binding of cellular protein factors implicated in nuclear export.

Amino Acid Sequence↗

PubMed1996

Short-time neutron activation gamma-ray spectroscopy.

Short-time instrumental neutron activation analysis, with high throughput, sensitivity and accuracy without matrix interferences, can be achieved in spite of the initial high count rate from both short and long-lived nuclide activation, if the experimental conditions are optimized by the combination of techniques. Thus the initial usually high counting rate can be faced by a loss-free counting system to avoid blocking or distortion of the system. The rapid radioactive decay and the consequent low counting statistics of short lived nuclides can be compensated by source-detector distance variation during the counting period and by cyclic and cumulative activation. Matrix spectral interferences can be reduced by ion exchange before activation for selective element preseparation and by neutron spectrum optimization such as epithermal neutron activation for selective element peak enhancement.

Journal Article↗

PubMed1980

Relationships between iron and vanadium metabolism: the association of vanadium with bovine lactoferrin.

Bovine milk was incubated simultaneously with 48VO2+ and 59Fe3+ ions. The lactoferrin was separated by ion exchange gel chromatography on CM Sephadex C-50 resin. Sephacryl 200 gel filtration of the lactoferrin-containing fractions, from ion exchange chromatography was carried out to dertermine the proportions of 48V and 59Fe radioactivities associated with macromolecules of the size of lactoferrin. It was found that 48V was incorporated into lactoferrin, the milk protein which contains iron. This suggests that lactoferrin may play a role in the bioavailability of vanadium during lactation, as vanadium is an essential element for the growth of some animal species. More generally, the vanadium binding properties of lactoferrin further support previous findings on the possible biochemical role of Fe-containing non hemoproteins in the metabolism of vanadium.

Chromatography, Ion Exchange↗

PubMed2005

Radionuclides and trace metals in Canadian moose near uranium mines: comparison of radiation doses and food chain transfer with cattle and caribou.

Tissues from 45 moose and 4 cattle were collected to assess the health of country foods near uranium mines in northern Saskatchewan. Bone, liver, kidney, muscle and rumen contents were analyzed for uranium, radium-226 (226Ra), lead-210 (210Pb), and polonium-210 (210Po). Cesium-137 (137Cs), potassium-40 (40K), and 27 trace metals were also measured in some tissues. Within the most active mining area, Po in liver and muscle declined significantly with distance from tailings, possibly influenced by nearby natural uranium outcrops. Moose from this area had significantly higher 226Ra, 210Pb, 210Po, and 137Cs in some edible soft tissues vs. one control area. However, soil type and diet may influence concentrations as much as uranium mining activities, given that a) liver levels of uranium, 226Ra, and 210Po were similar to a second positive control area with mineral-rich shale hills and b) 210Po was higher in cattle kidneys than in all moose. Enhanced food chain transfer from rumen contents to liver was found for selenium in the main mining area and for copper, molybdenum and cadmium in moose vs. cattle. Although radiological doses to moose in the main mining area were 2.6 times higher than doses to control moose or cattle, low moose intakes yielded low human doses (0.0068 mSv y(-1)), a mere 0.3% of the dose from intake of caribou (2.4 mSv y(-1)), the dietary staple in the area.

Animals↗

PubMed2006

Rubidium-rich asymptotic giant branch stars.

A long-debated issue concerning the nucleosynthesis of neutron-rich elements in asymptotic giant branch (AGB) stars is the identification of the neutron source. We report intermediate-mass (4 to 8 solar masses) AGB stars in our Galaxy that are rubidium-rich as a result of overproduction of the long-lived radioactive isotope (87)Rb, as predicted theoretically 40 years ago. This finding represents direct observational evidence that the (22)Ne(alpha,n)(25)Mg reaction must be the dominant neutron source in these stars. These stars challenge our understanding of the late stages of the evolution of intermediate-mass stars and would have promoted a highly variable Rb/Sr environment in the early solar nebula.

Journal Article↗

PubMed2003

Detection of the content and activity of the transcription factor AP-1 in a multistage skin carcinogenesis model.

Investigation of transcription factor activity in animal tissue during the early stage of cancer development can be difficult because of a low number of affected cells in a background of a large number of normal cells. We have used a well-established multistage skin carcinogenesis model to study the effect of manganese superoxide dismutase on the activity of AP-1 during an early stage of mouse skin carcinogenesis. The DNA binding activity of AP-1 proteins to a concensus DNA regulatory binding element known as TRE (TPA Responsive Element) is used as an in vitro assay for AP-1 activity in the nucleus of skin cells. This activity is detected by electrophoretic mobility shift assay (EMSA) which is based on the ability of specific proteins to bind and retard the migration of radioactive labeled oligonucleotides on a native gel. The presence of a specific protein in the complex can be identified by adding to EMSA an antibody specific for that protein, which will result in supershift complexes on the gel. The presence of the proteins in question can be further verified by standard Western analysis of the nuclear proteins.

Animals↗

PubMed1989

Trifluorothymidine localization in the rabbit cornea by secondary ion mass spectrometry imaging microanalysis.

Imaging microanalysis by secondary ion mass spectrometry is a sensitive surface analytical technique that allows the detection and localization of elements and compounds in biological tissues. We report the detection by this approach of intracorneal trifluorothymidine following topical administration in rabbit with normal cornea. The presence of trifluorothymidine is revealed using 19F as a marker, allowing the acquisition of ultrastructural microanalytical images without using radioactive tracers.

Animals↗

PubMed1985

The statistical analysis of distribution of blood deposits in arterial prostheses after short-term exposure to reduced blood flow in dogs: a computerized study using radioisotopes.

This study was undertaken to analyse the distribution pattern of blood elements deposits in 15 cases of vascular prostheses submitted, ex vivo, to reduced blood flow in a canine model. Autologous blood platelets labeled with Indium-111 and human 125 Iodinated fibrinogen were injected into the animals. Ten minutes later, the blood flow in the aorta was reduced to 50 ml/min and the prostheses (Woven DeBakey, 30 cm long and 6 mm internal diameter), were implanted as substitutes for the infrarenal abdominal aorta. The blood flow was then reestablished to 50 ml/min and controlled throughout the entire experiment. When a thrombosis occurred, or after 4 hours of low blood circulation, the prosthesis was collected, weighted and chemically fixed, and the radioactivity was measured by means of a two-channel gamma scintillation counter. These data were then formatted and processed by a special computer program. Final values were processed for graphical representation and analysed statistically. Results show a lack of correlation between weight of thrombotic matrix, platelet accumulation, and fibrin deposits retained from one animal to the other. There is, though, an appearance of a complex interrelationship between the mean pattern of longitudinal distribution of these elements, which shall be qualified as contagious distal accumulation of the deposits. The influence of each animals' blood characteristics, as evaluated by means of regression analysis, account for 40 to 60% of the interindividual variance in blood deposits. Data corrections following evidence of undesirable influences due to uncontrolled parameters are presented. This experimental method permits simultaneous graphical representation of the distribution of several elements in the thrombotic matrix, and at the same time an elaborate numerical analysis of their longitudinal distribution and total accumulation. The method is easy to use, does not require sophisticated equipment, and is less expensive than scintigraphy.

Animals↗

PubMed1978

In vitro labeling of platelets with stable rubidium compounds.

It is a common practice in diagnostic hematology to mark blood cells with radioactive tracers, such as 51Cr, 55Fe, 75Fe, etc., to determine their life-span and study their metabolism. A program has been started to verify the possibility of marking blood cells with stable indicators, assayed by radioisotope-induced X-ray fluorescence analysis. All elements may be considered as indicators aggregating to blood cells, and for in vivo measurements, which are not toxic in the quantities injected, calculated on the basis of the limit of detection of the technique. In this study platelets were marked with a very small quantity of stable compounds of rubidium, a potassium analog. Measurements were carried out in vitro on platelets of normal subjects and in vivo on platelets of rats and rabbits. Several survival curves were deduced and life-span values obtained in accord with previously reported values.

Animals↗

PubMed1991

Production of no carrier added 80mBr for investigation of Auger electron toxicity.

80mBr (half-life = 4.43 h) is an Auger electron emitting nuclide with convenient properties for investigating Auger electron cytotoxicity and with potential for labeling in vivo radiotherapeutic agents. We have investigated three cyclotron target systems capable of generating 80mBr of sufficiently high specific radioactivity (no carrier added) for biomedical experiments. A 83Kr gas target irradiated with 21.5 MeV deuterons made 80mBr at a production yield of 1.6 +/- 0.2 mCi/muAh at saturation. A five-fold increase in 80mBr yield was obtained from 15 MeV proton irradiation of thin elemental Se enriched in 80Se targets although technical improvements are expected to further raise this production yield. This route is therefore superior for current medical cyclotrons. Irradiation of a reusable 80Se copper selenide target also yielded multi-millicurie amounts of 80mBr, and recovery of radiobromine by dry distillation is faster and more convenient than in the elemental Se target, but an optimum copper selenide target for 80mBr production has not yet been built.

Bromine Radioisotopes↗

PubMed1969

The isolation, identification and synthesis of two metabolites of guanethidine formed in pig and rabbit liver homogenates.

1. Two metabolites of radioactively labelled guanethidine were isolated from rabbit and pig liver homogenates by ion-exchange chromatography on a sulphonic acid resin. 2. One of the metabolites was eluted from the column with ammonia and identified as 2-(6-carboxyhexylamino)ethylguanidine on the basis of the elemental analysis, i.r. spectrum and pH titration curve of the pure compound, and the observed partial loss of tritium for ring-labelled guanethidine during the formation of this metabolite. 3. This identification was confirmed by synthesis. 4. 2-(6-Carboxyhexylamino)ethylguanidine underwent ring-closure in hot alkaline solution to 1-(6-carboxyhexyl)-2-iminoimidazolidine. 5. The other metabolite of guanethidine was eluted from the ion-exchange column with 6m-hydrochloric acid along with the unchanged drug. It was purified by countercurrent distribution and shown to be identical with synthetic guanethidine N-oxide. 6. The two metabolites and the product of ring-closure had less than one-tenth of the antihypertensive activity of guanethidine in the renal-hypertensive rat and are unlikely to contribute to the pharmacological properties of the drug.

Animals↗

PubMed1995

Intermolecular binding sites of human immunodeficiency virus type 1 Rev protein determined by protein footprinting.

Human immunodeficiency virus encodes the regulatory protein Rev, which is required for expression of viral structural proteins. It binds to an RNA element (RRE) in the viral transcript and up-regulates the cytoplasmic appearance of unspliced and singly spliced viral mRNA. We have studied the structure of Rev alone and complexed with the RRE and two monoclonal antibodies, using a protein footprinting approach. The method involves radioactive labeling at the C-terminal end of Rev fusion protein followed by limited proteolysis under native conditions, using 10 different proteinases. Rev protein was mainly cleaved within the basic domain and in the C-terminal part. The periodicity of the proteolytic cleavages within the basic domain strongly suggests that it forms an alpha-helical structure with one side facing the solvent. In the presence of RRE, these cleavages became significantly reduced. In addition, strong protection was observed at position 66 outside the basic domain. As a control for the specificity of the footprinting reaction, we confirmed the position of the epitopes for two monoclonal antibodies. This protein footprinting methodology is generally applicable to other proteins for which terminal modifications are acceptable, and provides a useful tool for mapping structure, substrate binding, and conformational changes.

Base Sequence↗

PubMed1978

Baclofen (beta-p-chlorophenyl-gamma-aminobutyric acid) enhances [3H]gamma-aminobutyric acid (3H-GABA) release from rat globus pallidus in vitro.

The rat globus pallidus has been investigated as a possible model in which to study pre-synaptic GABA mechanisms in vitro. (+/-)-Baclofen (300 micrometer-1 mM) significantly enhanced the release of radioactivity from superfused slices of rat globus pallidus prelabelled with 3H-GABA in vitro. This releasing action was specific to the (+)-isomer of baclofen: neither the (-)-isomer nor another neuronal depressant dl-alpha-epsilon-diaminopimelic acid had any significant effect. The releasing effect of baclofen appeared unrelated to the phenethylamine moiety of its structure as neither beta-phenethylamine nor dopamine evoked release of 3H-GABA from pallidal slices. Baclofen increased the efflux of radioactivity from pallidal slices prelabelled with either [3H]-beta-alanine or [3H]diaminobutyric acid in vitro. The use of specific glial and neuronal GABA uptake blocking compounds (beta-alanine and (+/-)-cis-1,3-amino-cyclohexanecarboxylic acid) did not permit resolution of the elements from which baclofen was evoking [3H]GABA release. Baclofen also inhibited uptake of [3H]GABA into pallidal slices with an IC50 value of 6 x 10(-4) m. The GABA-like properties of baclofen may be related to the (+)-isomer while non-specific neuronal depressant actions are an effect of the (-)-isomer. The potential of the (+)-isomer as an antipsychotic agent while (-)-baclofen remains the effective antispastic drug free from unwanted side-effects, is discussed.

Alanine↗

PubMed1980

Chronic airways obstruction in pulmonary sarcoidosis: its poor response to bronchodilators.

Emphysema, chronic bronchitis, asthma, and cystic fibrosis are often cited as examples of chronic airways obstruction, while sarcoidosis is typically restrictive. Approximately 15 percent of sarcoidosis patients, however, have airways obstruction clinically characterized by wheezing with granulomatous involvement of airways. Since the majority have Stage IV disease by chest radiograph, their lungs usually have honey-combining with pulmonary fibrosis, adhesions, cavities, and mediastinal distortion.Patients had a mixed ventilatory defect, but obstruction of large airways was present as shown by decreased specific airways conductances. Small airways obstruction was also present as shown by low instantaneous flows at the terminal portion of the maximum expiratory flow-volume curve and diminished helium response of this curve. The closing volume, however, was not very sensitive. Radioactive xenon washout from ventilation lung scans and N2 washout from the lungs were prolonged in patients with worse disease.The authors conclude that the obstructive type of physiologic pattern is more frequent than recognized in sarcoidosis, which like that of cystic fibrosis has some restrictive element and is characterized by poor reversibility to bronchodilators. A trial period of beclomethasone dipropionate aerosol was not helpful in two patients. Relief of this distressing airways obstruction continues to pose a challenging problem in management.

Adult↗

PubMedDate not supplied

[Basic principles of radioimmunoanalysis].

The authors review the principal characteristics of radioimmunoassay (R.I.A.), in grounds of the so-called "saturation analysis" techniques, which are of increasing interest among the chemical laboratory procedures. They study each element of R.I.A. separatedly, and finally they make a brief display of the different applications of radioimmunoassay stressing its significance in the endocrinological field.

Antibodies↗