Radioisotope Counting Research Articles

Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA

The activation of plasminogen by t-PA was measured in the presence and absence of fibrin stimulation, using natural human plasminogen (nPlg) and rPlg-Ala740, a recombinant plasminogen with the active site Ser740 mutagenized to Ala. Recombinant wild type t-PA (rt-PA) was used as well as rt-PA-Glu275, a recombinant single chain t-PA in which the Arg of the plasmin sensitive Arg275-Ile276 peptide bond was substituted with Glu. Conversion of 125I-labeled single chain plasminogen to two-chain plasmin by wild-type or mutant t-PA, was quantitated by SDS gel electrophoresis and radioisotope counting of gel slices, and expressed as initial activation rates (v0 in pM s-1) per 1 nM enzyme. In the absence of fibrin stimulation, the v0 for the activation of nPlg and rPlg-Ala740 with the single chain forms of both t-PAs were comparable (0.6 to 2.7 pM s-1) but were lower than with the corresponding two-chain forms (5.3 to 23 pM s-1). In the presence of 1 microM soluble fibrin monomer (desAAfibrin), the v0 for nPlg and rPlg-Ala740 by single chain rt-PA was also comparable (24 and 33 pM s-1 respectively), whereas with 1 microM CNBr-digested fibrinogen, the v0 for nPlg with single chain rt-PA was about 20-fold higher than that of rPlg-Ala740 (135 and 7.5 pM s-1 respectively). In contrast, the v0 for nPlg and rPlg-Ala740 by single chain rt-PA-Glu275, two-chain rt-PA-Glu275 or two-chain rt-PA were comparable in the presence of either desAAfibrin or CNBr-digested fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

On the molecular interactions between fibrin, tissue-type plasminogen activator and plasminogen

The molecular interactions involved in the fibrin-mediated stimulation of plasminogen activation by tissue-type plasminogen activator (t-PA) were studied using natural human plasminogen (nPlg) and rPlg-Ala 740, a recombinant human plasminogen in which the catalytic site is destroyed by mutagenesis of the active site Ser 740 to Ala. Using this rPlg-Ala 740 moiety, the dissociation constant of the interaction between plasminogen and CNBr-digested fibrinogen was determined to be 0.40 μM. In addition, conversion of 125I-labeled single chain plasminogen to two chain plasmin by single chain recombinant t-PA (rt-PA) in the absence or the presence of CNBr-digested fibrinogen was quantitated on reduced SDS-gel electrophoresis, combined with autoradiography and radioisotope counting of gel bands. In the absence of fibrin, the activation rate of nPlg and rPlg-Ala 740 by single-chain rt-PA was comparable. In the presence of fibrin, however, the activation rate of rPlg-Ala 740 was about 20-fold lower than that of nPlg. These results with rPlg-Ala 740 may be explained by an impaired formation of the stable cyclic ternary complex between plasminogen, t-PA and fibrin, which mediates the fibrin stimulation of plasminogen activation by t-PA or, alternatively, by impaired conversion of single chain rt-PA to two chain rt-PA at the fibrin surface.

Direct observation of dopamine compartmentation in striatal nerve terminal by ‘in vivo’ measurement of the specific activity of released dopamine

Rats were anesthetized with fluothane and implanted in the caudate nucleus with a push-pull cannula supplied with artificial CSF containing the tritiated precursor of dopamine (DA), [ 3H]tyrosine. Total DA and dihydroxyphenyladetic acid (DOPAC) were measured in successive 20 min fractions using high performance liquid chromatography and electrochemical detection. Radioisotopic counting of the peaks permitted the calculation of the specific activity of both DA and DOPAC released into the extracellular space. Local applications of potassium (8, 16 and 32 mM) induced a dose-dependent increase of DA with a decrease of its specific activity as evidence of the involvement of a stored DA pool. Base release of DOPAC was increased by repeating potassium applications with a temporary decrease during the applications. Superfusion withα-methyl- p-tyrosine produced a decrease of both the [ 3H]DA and total DA with a simultaneous decrease of its specific activity. This decrease was considered to be an indicator of the involvement by synthesis inhibition of the stored amine, but the simultaneous decrease of the specific activity of DOPAC suggests that this release was intraterminal. These results constitute the first direct observation that DA terminals act with two separate pools (stored and releasable) and suggest that the stored amine is preferentially released intraterminally. Systemic injection of reserpine induced a decrease of the release of DA and DOPAC without alteration of DA-specific activity when the specific activity of DOPAC was lowered. From these results it is concluded that the releasable compartment of the amine is located, in part, in vesicles different in nature from the vesicles containing the stored amine. A functional model of a DA terminal is proposed using 4 intraterminal compartments, two of which are vesicular and two cytosolic. The measurement of the DOPAC release and the calculation of its specific activity appeared as an effective tool to evidence the intraterminal transfers occurring between the various intraterminal compartments.

An evaluation of two newly formulated calcium hydroxide cements: A leakage study

One-hundred extracted human single-rooted teethwere biomechanically instrumented. Sixty teeth were obturated with gutta-percha and three types of root canal cements, which were Roth Root Canal Cement, Calciobiotic Root Canal Sealer, and Sealapex. None of the remaining 40 teeth (controls) were obturated; however, 20 had their apical foramina sealed and the other 20 had patent foramina. Five microliters of [3H]uridine were deposited into the prepared root canal of each tooth, and radioisotope counts were obtained at various intervals postoperatively (2 to 60 days). The results showed that there was no statistical difference in leakage between the obturated groups and the sealed control group; however, when compared with the control group with patent foramina there was a significant difference. One-hundred extracted human single-rooted teethwere biomechanically instrumented. Sixty teeth were obturated with gutta-percha and three types of root canal cements, which were Roth Root Canal Cement, Calciobiotic Root Canal Sealer, and Sealapex. None of the remaining 40 teeth (controls) were obturated; however, 20 had their apical foramina sealed and the other 20 had patent foramina. Five microliters of [3H]uridine were deposited into the prepared root canal of each tooth, and radioisotope counts were obtained at various intervals postoperatively (2 to 60 days). The results showed that there was no statistical difference in leakage between the obturated groups and the sealed control group; however, when compared with the control group with patent foramina there was a significant difference.

Reduced adherence of micro-organisms to human mucosal epithelial cells following treatment with Taurolin, a novel antimicrobial agent.

Taurolin, a non-antibiotic antimicrobial agent, significantly reduced the adherence of buccal and vaginal strains of Candida albicans blastospores and urine isolates of Escherichia coli and Staphylococcus saprophyticus to epithelial cells. Light microscopy and radio-isotopic counting methods were used to quantify the adherence of the micro-organisms to either uroepithelial or buccal epithelial cells. A maximum reduction in adherence of approximately 65% was obtained. The anti-adherence capacity was time-dependent, requiring a contact time of 30 min to achieve maximum effect. Taurolin at sub-minimum inhibitory concentrations (MIC) significantly reduced the adherence of Candida and E. coli. A concentration slightly higher than the MIC was required for Staph. saprophyticus. Treatment of either epithelial cells or micro-organisms with Taurolin resulted in reduced adherence of microorganisms.

Uptake of Inorganic Phosphate by Plastids Purified from the Roots ofPisum sativumL.

Uptake of Inorganic Phosphate by Plastids Purified from the Roots of<i>Pisum sativum</i>L.

Metabolic fate and elimination in milk, urine and bile of deoxynivalenol following administration to lactating sheep.

Using a combination of radioisotopic counting and chromatographic detection techniques, the kinetics and metabolic fate of deoxynivalenol (DON) in plasma, urine and bile were studied in lactating sheep, as was the transmission of residues to milk. Following intravenous administration, the plasma clearance of 14C-DON-derived radioactivity was rapid and followed a tri-phasic decay curve comprised of a bi-exponential decrease in DON (rapid distribution phase, t1/2 alpha = 16.2 min; slower elimination phase t1/2 beta = 66.5 min) and the formation and elimination (t1/2 beta = 188.0 min) of its major plasma metabolite, DON-glucuronide conjugate, which accounted for 13% of the plasma radioactivity levels. DON was rapidly cleared from the body by metabolism to 7 possible metabolites, which were excreted essentially in the urine (91%) and to a lesser extent in the bile (6%). Most (67%) of the recovered radioactivity was in the form of the glucuronide conjugates of DON (54%) and the de-epoxide metabolite, DOM-1 (13%). Excretion of unmetabolized DON accounted for 11%. The remaining recovered dose (18%) comprised of minor amounts of DOM-1 (6%), DON-sulfate conjugate (2%) and 3 unidentified radioactive components (10%). Studies on the presence of DON-derived residues in milk indicated that, relative to the dose, only trace amounts were transmitted following either oral or iv administration of the toxin.

New immunoenzymatic assay for human thyrotropin compared with two radioimmunoassays.

We have evaluated a new immunoenzymatic assay for human thyrotropin involving three monoclonal antibodies (Abbott HTSH EIA) and compared the results with those of two conventional nonequilibrium double-antibody radioimmunoassay (RIA) methods: Clinical Assays' RIA and a research RIA (J Clin Endocrinol Metab 1975; 41:676). Mean values for thyrotropin in 100 euthyroid serum samples were similar in the Abbott and Clinical Assays methods, but both sets were significantly higher than those by the research RIA. By all methods, values for hypothyroid patients were clearly higher than values for euthyroid subjects. Results for hyperthyroid and euthyroid subjects were resolved slightly better with the research RIA than with the Abbott kit. The new Abbott assay was far more sensitive than either the Clinical Assays RIA or our research RIA. The correlation of results of the Abbott assay with those of the Clinical Assays and the research RIA exceeded 90% for samples from hypothyroid patients. The Abbott assay replaces radioisotope counting with spectrophotometric detection.

Cellular sodium transport in arterial wall - summary of studies by digital computer simulation

The distribution of electrolytes between extracellular and intracellular compartments is of greater functional significance in biological tissues than the total amount. A method commonly employed for the study of a substance distribution among different spaces is the continuous outflow and radioisotope counting of a fragment of tissue previously incubated with a radioactive tracet (here 22Na +). The experimental data can be analyzed within the frame of a compartmental analysis system which is defined by transport rate constants (k ij) and solved when all k ij are numerically determined. In practical applications the direct solution requires measurements in different compartments or knowledge of the initial condition for each compartment, but this is not possible in most biological tissues where the experimentally observable data represent the summation of tracer in all compartments at each point in time. The availability of the SAAM (Simulation, Analysis And Modelling) computer program allows for a powerful simulation procedure to arrive at values for k ij . Experimental data and specifications for a chosen model are entered in the SAAM format. A simulated ‘inflow’ experiment is mde by the computer by assigning to k ij some estimate values. All initial conditions are now zero, since at the beginning there is no radioactive tracer in any compartment. After a time equivalent in the experimental realm to achieving constant specific activity, a ‘time change’ programmed into the computer takes place so that the outflow part of the experiment is developed with the same k ij as for the inflow part, the final conditions for the inflow before the time change being the initial conditions for the outflow. An iterative procedure modifies the initial estimates of k ij to seek the best fit. By coupling a powerful simulation procedure to an advanced radionuclide tracer biological methodology, we have been able to find effects of the hormone aldosterone and of experimental hypertension on Na + distribution in arterial wall years before such effects were demonstrated by purely conventional experimental techniques. This computer simulation procedure may be suited to other experimental situations in which outflow data are the only available.

Treatment of osteoid osteoma of the proximal phalanx by use of cryosurgery

A case report of an osteoid osteoma of the proximal phalanx is presented in which radioisotope counting was employed as an intraoperative diagnostic aid and cryosurgery was combined with resection for tumor control.

The pharmacokinetics and metabolism of ethinyl estradiol and its three sulfates in the baboon

Ethinyl estradiol sulfates are major circulating metabolites of ethinyl estradiol (EE2); this is a relationship analogous to that of endogenous estrone and estrone sulfate. Because of the wide use of contraceptives containing EE2, the pharmacokinetics of its sulfate conjugates are of some importance. In previous studies of the intermediate metabolism of ethinyl estrogens we have shown that the baboon is an appropriate animal model. Accordingly, oral and/or intravenous doses of EE2 or each of its three sulfates were administered to castrate female baboons, and plasma levels of EE2 and its sulfates were studied by specific radioimmunoassay or radioisotope counting. After intravenous administration of EE2, the 3-sulfate and the 3,17-disulfate are the major circulating metabolites. After oral dosage administration, the 3-glucuronide and, in some cases, the 3,17-diglucuronide also become important. After intravenous administration, about twice as much of the drug exists in the sulfate as in the free form, as reflected by the areas under the plasma level curves. The bioavailability of orally administered EE2 was about 60%, confirming the presence of a substantial first-pass effect. Hydrolysis at the 17-position occurs when EE2-17-sulfate is administered orally but appears not to occur with intravenous administration. EE2 and the three sulfates, given intravenously, exhibited two-compartment open-model kinetics. The elimination phase half-lives of all four compounds were similar, ranging from 8.8 to 11.2 hours. The area under the plasma level curve of EE2 resulting from the intravenous administration of the 3-sulfate was approximately 8% of the total area under the plasma level curve of both EE2 and EE2 sulfates. The ratio of the area under the plasma level curve of sulfates resulting from 3-sulfate administration compared to the other two sulfates was approximately 0.3, reflecting the existence of other metabolic pathways for its disposition.

シンチカメラによる甲状腺ヨード摂取率

The use of scinticamera for thyroid iodine uptake was evaluated by a method which allows simultaneously imaging of thyroid scintigram. It was found that thyroid uptake measurement made with scinticamera was reliable and it was not necessary to use scintillation counter for kinetic study. ^<123>Iodine capsule as well as ^<131>Iodine were useful. Thyroid uptake measured by with scinticamera methods, (1) and (2), here under were very similar to that of with scintillation counter. (1) : Thyroid uptake with a parallel collimator was measured under following conditions : neck to collimator distance was 10 cm ; radio-isotope counting included the counts of 9 cm×9 cm on the neck and subtraction of natural counts as background ; plane phantom was necessary as a standard source. (2) : When pinhole collimator was used, the measuring distance was 3 cm, the counting was in the whole area of the imaging screen without natural counts and the ORINS phantom was used as a standard source.

Study of infrasonoric bladder vibrations, rise in blood pressure and headache in tetraplegic patients. Spectral analysis of radioisotope counts during radioisotope renogram.

The object of this work is the study of the relation of a full bladder and the development of headache and rise in blood pressure in tetraplegic patients. Tetraplegic patients are known to develop a m

Pulmonary Antibacterial Defense Mechanisms Are Depressed by Halothane

Pulmonary antibacterial activity was determined by challenging halothane-exposed and control mice to radiolabeled (32P) Staphylococcus aureus produced by an aerosol generator and by determining the relative changes in viable bacterial and radioisotope counts in ground lung immediately after exposure and 4 hours later. Mice that were anesthetized for 4 hours with 1 or 2 MAC halothane and allowed to recover for 1 hour showed higher 4-hour bacterial counts when compared to controls, while radioisotope counts were the same in both groups. These data indicate that 1 and 2 MAC halothane anesthesia is associated with depressed lung bactericidal activity but that physical clearance mechanisms are not depressed.

Cyclic nucleotide metabolism and vasodilation in canine mesenteric artery

The uptake and extracellular and intracellular metabolism of radioisotopically labeled cyclic 3',5'-adenosine monophosphate (cAMP) and dibutyryl cAMP (DBcAMP) was determined in canine mesenteric arteries incubated in vitro. Intracellular tissue uptake was measured by radioisotope counting and labeled metabolites separated by thin-layer chromatography. Extracellularly, cAMP was extensively metabolized to AMP, adenosine, and Pi. DBcAMP was metabolized to monobutyryl cAMP (MBcAMP) intracellularly. Vasodilation of the mesenteric circulation in vivo was produced by cAMP, its metabolites and DBcAMP. DBcAMP caused greater vasodilation than cAMP but had a response time to its peak effect of 12 min versus 90 s for cAMP. The vasodilator properties of cAMP and DBcAMP were related to their metabolism. It was concluded that the vasodilation caused by cAMP was due to cAMP metabolites produced by extracellular metabolism.

Rapid fluorometric micromethod for in situ quantitation of lipids on thin-layer chromatograms

A simple, rapid method is described that is capable of measuring directly and reliably nanomole amounts of triglycerides, free fatty acids, fatty acid esters, free cholesterol, and cholesterol esters. The technique involves ( a) chromatography of as little as 120 μg of total extracted lipid on silica gel thin-layer plates incorporating rhodamine 6G, ( b) development of the chromatogram in 2 solvent systems in sequence and ( c) monitoring the intensity of emitted fluorescence by fluorometry. Standard curves for each of the 5 neutral lipid classes are reproducible over a large range of concentrations. Phospholipid values can be obtained as differences from the total lipid extracted. This microtechnique reduces sample size by 95% from that needed for chemical tests and compares favorably with densitometric methods. The method does not destroy the sample and lends itself easily to other kinds of measurement, such as radioisotope counting and autoradiography, on the same samples which have been quantitated.

Measurement of Oral Calcium Absorption from the Gut by External Isotope Counting

AbstractAmong the factors which regulate normal gastro-intestinal calcium absorption, current thought recognises calcium intake, vitamin D and parathyroid hormone as playing an important part. It is well documented that gastro-intestinal calcium absorption is reduced in states of malabsorption, hypoparathyroidism and in patients with chronic renal failure, and raised in states of hyperparathyroidism, urolithiasis and idiopathic hypercalcuria.Difficulties inherent in classic calcium-balance techniques have prompted a search for more practical and reliable methods of measuring the degree of impairment of calcium absorption. This paper describes a study of intestinal calcium absorption in 10 normal subjects and four patients, with various disorders of calcium metabolism, using an external radio-isotope counting technique.

A computer program for immunoassay of protein hormones, with special reference to insulin and growth hormone.

A Fortran language program suitable for use in computer processing of two-antibody radioimmunoassay data has been developed. Radioisotope counts transferred to punch-cards are automatically converted to a semilog plot of standard values from which assay values are derived and “printed out” as microunits of insulin and/or milli-micrograms of growth hormone per milliliter of serum or plasma.

CONCENTRATIVE AND REVERSIBLE CHARACTER OF INTESTINAL AMINO ACID TRANSPORT.

Either α-aminoisobutyric acid-1-C14 or 1-aminocyclopentanecarboxylic acid-1-C14 was injected into a rat and, after at least 24 hr, a suitable dilution of the same solution of the amino acid was introduced into an intestinal loop in situ and the change in its distribution observed by radioisotope counting. The two amino acids could be concentrated from the small intestine to establish steady-state distribution ratios of from 12 to 100, for the plasma level with respect to the level in the lumen. These values are far higher than the distribution ratios produced across excised intestine. These steady-state values were approached from either above or below, by absorption or release of the amino acid. The release was accelerated by the presence of the same or another amino acid of high transport affinity in fluid perfusing the intestinal loop, an action that tends to identify the outward migration from lumen to plasma with a transport process. The test amino acids were released into the colon also, although at slower rates, the steady-state distribution ratio lying in the neighborhood of unity. This release appears also to be a specific process, and to account for the slow fecal excretion of these difficultly metabolized amino acids.

A study of the intranasal challenge assay of pertussis vaccine using iodine-131.

The present study was undertaken to determine the cause of the wide variation in the response of mice following intranasal challenge with virulent H. pertussis. Mice, both normal and immunized, were inoculated intranasally with killed H. pertussis organisms labelled with iodine-131. The course of the tagged antigens, in the lungs and other organs of these animals, was followed by radioisotope counting. The lung uptake varied from 31.6% to 77.5% of the challenge dose in immunized animals and from 25.0% to 83.7% in non-immunized animals. It was considered that this variation was large enough to contribute substantially to the wide discrepancies found in animal assays. To date the cultures employed for intranasal challenge have all been of low virulence when administered by this route. A considerably more virulent strain of challenge organism than those in use now would be necessary to overcome the variations in the assay caused by uneven lung uptake of challenge culture. It was also found that the lungs of immunized mice were cleared more rapidly than the lungs of non-immunized mice in the 24-hr. period following the intranasal inoculation.