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PubMed1985

Modified rapid radiometric method for detection of Mycobacterium tuberculosis from sputum samples.

A modified, simple and radiometric method for early detection of M. tuberculosis from sputum samples has been developed using a biphasic vial system for detection of 14C-CO2 produced by the metabolism of 14C-U-acetate on glycerol-free Lowenstein-Jensen medium (LJM). Of the 84 smear positive sputum samples examined, 85.7% and 86.9% were scored positive by radiometric and visual methods respectively. The detection rates at 1st, 2nd and 3rd week of the test were 53.3%, 60.7% and 82.1% by radiometry and 1.2%, 11.9% and 54.8% by visual methods respectively. The mean detection time was 10.7 days by the radiometric and 21.0 days by the visual method. An average replication time of primary culture from 54 sputum samples was 25.58 +/- 6.92 h (range 10.0-39.1 h).

Acetates↗
PubMed1984

Analysis of transferrin recycling in mitotic and interphase HeLa cells by quantitative fluorescence microscopy.

Recent findings suggest that membrane vesicle transport during mitosis may be generally inhibited. To test this, we examined the kinetics of uptake and exocytosis of RITC-transferrin in mitotic and interphase HeLa cells. We used quantitative image-intensification fluorescence microscopy to analyze the content of ligands in single cells. This technique was validated by comparison of 3H or RITC-transferrin release from interphase cells determined by microscopy or radiometry. Both methods gave a t1/2 of release of 5-6 min. The uptake of RITC-transferrin was depressed in mitotics. More importantly, we monitored the exocytosis of label during mitosis. Labeled mitotics were obtained by the progression of interphase cells into mitosis during a 50 min incubation with RITC-transferrin. After 30 min chase with unlabeled transferrin, the intensities of interphase cells approached background, whereas those of mitotic cells remained nearly constant. Thus both exocytosis and endocytosis of transferrin were exocytosis and endocytosis of transferrin were blocked during mitosis.

Fluorescent Dyes↗
PubMed2004

Automated segmentation of human brain MR images using a multi-agent approach.

Image interpretation consists in finding a correspondence between radiometric information and symbolic labeling with respect to specific spatial constraints. It is intrinsically a distributed process in terms of goals to be reached, zones in the image to be processed and methods to be applied. To cope the the difficulty inherent in this process, several information processing steps are required to gradually extract information. In this paper we advocate the use of situated cooperative agents as a framework for managing such steps. Dedicated agent behaviors are dynamically adapted depending on their position in the image, of their topographic relationships and of the radiometric information available. The information collected by the agents is gathered, shared via qualitative maps, or used as soon as available by acquaintances. Incremental refinement of interpretation is obtained through a coarse to fine strategy. Our work is essentially focused on radiometry-based tissue interpretation where knowledge is introduced or extracted at several levels to estimate models for tissue-intensity distribution and to cope with noise, intensity non-uniformity and partial volume effect. Several experiments on phantom and real images were performed. A complete volume can be segmented in less than 5 min with about 0.84% accuracy of the segmented reference. Comparison with other techniques demonstrates the potential interest of our approach for magnetic resonance imaging (MRI) brain scan interpretation.

Artificial Intelligence↗
PubMed2004

Mass-tag technology for monitoring of protein kinase activity using mass spectrometry.

Monitoring of intracellular protein kinase activity is very important for fields involving diagnosis and drug screening. However, current methods, such as radiometry using (32)P, or ELISA, are laborious and time-consuming. We have developed high-throughput assay system of protein kinase activity using mass-tagged substrate peptide probes and mass spectrometry. This assay system can easily evaluate target kinase activity and will potentially be able to simultaneously profile many protein kinase activities.

Mass Spectrometry↗
PubMed1982

Multiple inverse isotope dilution assay for the stereospecific quantitative determination of R(-) and S(+)-oxaprotiline in biological fluids.

An isotope dilution assay for the determination of both oxaprotiline enantiomers in biological samples after administration of the racemic mixture has been developed. The enantiomers were reacted with synthetically prepared, optically pure N-trifluoroacetyl-S(-)-prolyl chloride, followed by high-performance liquid chromatographic separation of the diastereoisomers formed. Quantitation was performed by on-line UV detection at 260 nm and off-line radiometry by liquid scintillation counting. Endogenous compounds and metabolites do not interfere in the assay. Analysis of water and the blood and urine of rats spiked with [14C]oxaprotiline X HCl showed recoveries for S(+)-oxaprotiline X HCl (mean +/- coefficient of variation, n = 4-6) of 98.0 +/- 1.0% (water), 100.5 +/- 0.6% (blood) and 101.5 +/- 2.0% (urine), and for R(-)-oxaprotiline X HCl of 101.3 +/- 2.0% (water), 102.2 +/- 2.1% (blood) and 103.2 +/- 0.2% (urine). A pilot study to determine blood levels of the two enantiomers in two rats dosed with racemic [14C]oxaprotiline X HCl (10 mg/kg i.v.) was carried out to test the method. The results indicated stereoselective disposition of oxaprotiline enantiomers in the rat. The ratio of the areas under the blood concentration curves for R(-)-to S(+)-oxaprotiline X HCl was 1.14.

Animals↗
PubMed1985

Isolation and stereospecific determination of the enantiomers of oxindazac by direct liquid chromatographic resolution on triacetylcellulose.

The preparation of the optically pure enantiomers of the antiphlogistic trial drug oxindazac via liquid chromatographic resolution of the corresponding tert.-butyl or benzyl ester on triacetylcellulose is described. Cleavage of the optically pure enantiomeric esters to the acids proceeds without significant racemization. The methyl ester of oxindazac is also completely resolved on the same chiral phase. Whereas oxindazac racemizes easily upon derivatization to diastereomers, no racemization is observed upon methylation to the corresponding methyl ester with diazomethane. An inverse isotope dilution method has been developed to determine both enantiomers of the drug in biological fluids after administration of 14C-labelled oxindazac. The enantiomers are converted into their methyl esters and separated on triacetylcellulose. Quantitation is performed by on-line UV detection at 290 nm and off-line radiometry. In the analysis of plasma samples, endogenous compounds do not interfere. The recoveries of [14C]oxindazac from water, rat and human plasma were 99.6 +/- 1.8% for the (+/- and 96.0 +/- 1.4% for the (-)-enantiomer. The plasma concentrations and urinary excretion of the two enantiomers were determined in a human volunteer who had received 200 mg of racemic 14C-labelled oxindazac.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1983

Multiple inverse isotope dilution assay for cadralazine and four metabolites in biological fluids.

An inverse isotope dilution assay was developed for the specific determination of 14C-labelled cadralazine and four of its metabolites in biological samples. After addition of unlabelled carrier substances to the sample, metabolite IV was derivatized. The derivative and the unaltered compounds (I, II, III, V) were extracted and separated by high-performance liquid chromatography on silica gel. Quantitation was performed by on-line ultraviolet detection at 274 nm and off-line radiometry by liquid scintillation counting. Endogenous compounds and unknown metabolites did not interfere in the assay. The analysis of water, plasma and urine samples spiked with [14C]cadralazine showed mean recoveries between 98.4 and 101.3%. The lower limit of detection was 10 nmol/1 (3 ng/ml) for any of the compounds I-V. The method was used for the analysis of plasma and urine samples of rats dosed with [14C]cadralazine.

Animals↗
PubMed1983

Multiple inverse isotope dilution assay for oxprenolol and nine metabolites in biological fluids.

An isotope dilution assay for the specific determination of 14C-labelled oxprenolol and nine of its metabolites in the same biological sample is described. After addition of unlabelled carriers to the sample, oxprenolol and the metabolites were isolated by base- and acid-specific extraction and separated by normal-phase high-performance liquid chromatography using two different mobile phases. Quantitation of the various peaks was performed by on-line ultraviolet detection at 275 nm and off-line radiometry by liquid scintillation counting. Endogenous compounds and unknown metabolites did not interfere in the assay. The analysis of rat and dog blood, plasma and urine samples spiked with [14C]oxprenolol hydrochloride, showed mean recoveries between 98.7 and 99.8%. The assay was used to investigate the metabolic fate of [14C]oxprenolol in the dog. Analyses of blood and urine demonstrated the quantitative significance of the various metabolites in the biotransformation of oxprenolol.

Animals↗
PubMed1983

Multiple inverse isotope dilution assay for the stereospecific determination of R(+)- and S(-)-oxprenolol in biological fluids.

An isotope dilution assay has been developed for the determination of both oxprenolol enantiomers in biological samples after administration of the racemic 14C-labelled mixture. The enantiomers were reacted with optically pure S(-)-1-phenylethyl isocyanate and the diastereoisomeric urea derivatives formed were separated by normal-phase high-performance liquid chromatography. Quantitation was performed by on-line ultraviolet detection at 275 nm and off-line radiometry. Endogenous compounds and oxprenolol metabolites did not interfere with the assay. Analysis of water and blood, plasma and urine samples of rats and dogs spiked with [14C]oxprenolol hydrochloride showed mean recoveries for R(+)-oxprenolol hydrochloride of 99.2% (water), 99.3% (blood), 99.1% (plasma) and 97.9% (urine), and for S(-)-oxprenolol hydrochloride of 99.7% (water), 98.1% (blood), 98.6% (plasma) and 96.9% (urine). In a pilot study, the presented method was used to investigate the metabolic fate of the enantiomers in two dogs dosed orally with racemic [14C]oxprenolol hydrochloride (3 mg/kg). The results show that conjugation of R(+)-oxprenolol exceeded that of S(-)-oxprenolol.

Animals↗
PubMed1997

Temperature-controlled CO2 laser tissue welding of ocular tissues.

Lasers can be used for binding tissues by welding, but the clinical application of this method has been limited by the difficulties in defining and maintaining the optimal conditions. Fiberoptic radiometry allows accurate remote temperature measurements for control of laser tissue welding. We evaluated the use of a temperature-controlled tissue welding system to close corneal and corneoscleral wounds. Eighty ex vivo bovine eyes were used for the determination of welding parameters optimal for corneal wound closure. A 4 mm central corneal cut was closed with use of a CO2 laser (600 mw, 0.9 mm spot size), with tissue temperatures ranging from 45-70 degrees C and welding time ranging from 1-30 seconds. Wound strength was measured as burst pressure of the sealed wound. The welding parameters found to cause the strongest wound binding were used to weld a limbal incision of 4 mm in 10 adult albino rabbits. The fellow eye of each animal was used as a control, and the same wound was closed with one 10/0 mersilen suture. Two animals were killed immediately after the procedure, and the eyes were sent for histologic examination. Eight rabbits were followed for 1 month. Clinical examination and refraction were done 1 day, 1 week, 2 weeks, and 1 month after the procedure. Corneal topographic evaluations were done 1 week after the procedure. After 1 month the animals were killed and the eyes were examined histologically. The optimal results of wound binding by laser welding in the enucleated bovine eyes were achieved with 55-60 degrees C and at a welding time of 12-20 seconds. At these parameters the burst pressure of corneal wounds was 70 mm Hg. All laser-welded limbal wounds in the rabbits were tightly closed at the end of procedure and during the follow-up period. The refractive results after laser welding were equal to those of the controlled suture-closed wound. Laser tissue welding combined with tissue temperature monitoring can be used to close corneal wounds.

Animals↗
PubMedDate not supplied

Lipid peroxidation-coupled co-oxygenation of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol in human term placental microsomes.

The link between lipid peroxidation and benzo(a)pyrene activation was studied in microsomes isolated from human term placenta. Lipid peroxidation was initiated in the presence of NADPH by partially chelated iron. Covalently bound and free metabolites of benzo(a)pyrene or benzo(a)pyrene-7,8-diol were quantitated by radiometry and/or HPLC. Peroxidative conditions increased the amounts of benzo(a)pyrene-trans-anti-tetrol produced from benzo(a)pyrene-7,8-diol, benzo(a)pyrene-diones from benzo(a)pyrene, and protein bound metabolites from both. A reactive oxo-iron complex is proposed as an ultimate species initiating hydrogen abstraction and lipid peroxidation. It is suggested that partially chelated iron catalyzes co-oxygenation of benzo(a)pyrene and benzo(a)pyrene-7,8-diol by peroxyl radical in placental microsomes. This peroxidative reaction may be crucial for bioactivation of benzo(a)pyrene in human term placenta.

Benzo(a)pyrene↗
PubMed2000

Relationships between acid-soluble thiol peptides and accumulated Pb in the green alga Stichococcus bacillaris.

Stichococcus bacillaris, an ubiquitous green microalga accumulated inorganic lead (Pb) from aqueous solutions extra- and intracellularly. In response to Pb uptake acid-soluble thiol peptides (glutathione - GSH and phytochelatins - PC) were synthesized. The proportion of the intracellular Pb uptake by algal cells was low and comprised only 3-6% of the total metal sorption. The intracellular uptake was dependent on external Pb concentration, time of metal exposure and cell metabolism. Pb accumulation in alga was determined by means of 210Pb radiometry. Reduced GSH and PC were determined in algal cells using HPLC with the post-column derivatization with Ellman's reagent. Within the studied concentration range 0.1-20 µM, inorganic lead caused a significant production of induced thiol peptides: PC (n=2-4) and some other unidentified oligopeptides, probably (GluCys)n. The time of appearance and the concentration of individual oligomers of phytochelatins were dependent on the external Pb concentration and time of metal exposure. In algal cells exposed to Pb, significant changes in the GSH level accompanying the formation of the induced thiol peptides were also observed. The GSH level decreased in the cells exposed to the lower (up to 10 µM) studied Pb concentrations or increased in the cells treated with higher (20 µM) Pb concentrations. The thiol groups originated from induced peptides (mainly phytochelatins) followed a stoichiometric relationship 2:1 to the intracellular Pb amounts, however, only at the lowest studied external concentration (0.1 µM). At higher concentrations (up to 2.5 µM), intracellular Pb concentration was equal or even exceeded (at Pb>2.5 µM) two to three times the level of induced thiols. S. bacillaris accumulated intracellularly by 46% more Pb in light than in dark and the level of induced thiol peptides was significantly higher in the cells exposed to Pb under illumination. The rapid formation of these peptides in S. bacillaris in response to Pb, and their elimination (by about 90%) when algae were placed into the Pb-free solution reveal a tight regulation of GSH and phytochelatin pools in the algal cells exposed to toxic metals. The obtained results suggest that both PCs and GSH are the primary line of defence against the Pb toxicity. Additionally, the induced thiol peptides in S. bacillaris could be a good indicator of intracellular Pb availability and stress at the metal concentrations found in polluted fresh waters.

Journal Article↗
PubMed1996

Application of molluscs for radioecological monitoring of the Chernobyl outburst.

Accumulation of radionuclides was studied in molluscs from the territories polluted by the Chernobyl outburst. In addition to radiochemical evaluation of (90)Sr, a simple method of beta-radiometry of shells was applied which allowed processing of extensive samples and mapping of contamination of large territories: the Dnieper drainage area and the Kiev administrative region. Pre-Hiroshima and pre-Chernobyl radioactivity was investigated in museum collections. Differences in (90)Sr accumulation in molluscs of different genera were demonstrated for freshwater and terrestrial snails, an especially high accumulation factor was found in Helix. Indices of relative accumulation were calculated treating Lymnaea stagnalis as a standard. All the measurements were recalculated to this standard in order to provide comparisons between sampling sites disregarding collected species and to reduce variance before mapping. Based on shell beta-activity measurements, the average accumulation factor for (90)Sr in Lymnaea compared to its concentration in the river water was about 5000, its transfer factor compared to the soil contamination was about 0.1 m(2) kg(-1), figures for (137)Cs were smaller by an order of magnitude. Inverse dependence between free calcium content in the environment and the transfer factor was demonstrated, as well as a difference in (90)Sr/(137)Cs ratio in molluscs collected on different tracks of Chernobyl pollution. Monitoring the biologically accessible and biologically active fraction of (90)Sr contamination, the shell beta-activity above 15 kBq kg(-1) indicates a dangerous level of contamination for the human population.

Journal Article↗
PubMed2004

Influence of vasopressin in the pathogenesis of oligohydramnios-polyhydramnios in monochorionic twins.

OBJECTIVES: The pathophysiology of oligohydramnios-polyhydramnios in monochorionic (MC) twins complicated by chronic twin-twin transfusion syndrome (TTTS) is poorly understood. We hypothesise that oliguria and oligohydramnios in the donor twin of chronic TTTS, occurs due to antidiuretic and vasoconstrictive activity of vasopressin (AVP). METHODS: We measured AVP levels in maternal, fetal and amniotic fluid samples obtained in utero and at birth from 44 MC twins with (n=27) or without chronic TTTS (n=17). Concentrations of AVP in pg/ml were determined by immuno-radiometry assay. RESULTS: In donor fetuses, plasma and amniotic fluid AVP levels were higher than those of the recipient twins in utero (P<0.001) and at birth (P<0.001). No such differences were found between the non-TTTS twins. The plasma AVP concentrations were higher in the recipient fetuses with severe hydrops than those without hydrops (2.8+/-0.7 pg/ml versus 0.3+/-0.3 pg/ml; P<0.05). Maternal AVP levels were comparable between the TTTS and non-TTTS groups. In the non-TTTS twins, both plasma and amniotic fluid AVP levels were higher than those of the recipient twins (P<0.001) but lower than those of the donor twins (P<0.001). There was a significant association between amniotic fluid and plasma AVP levels both in the TTTS (r=0.78; P<0.001) and non-TTTS (r=0.70; P<0.01) infants. CONCLUSIONS: Vasopressin concentrations in the donor twins were three times higher than their co-twins which suggests that oligohydramnios may occur as a consequence of AVP mediated reduction in fetal urine output.

Adult↗
PubMed2000

Nuclear scintigraphic assessment of intestinal dysfunction after combined treatment with 9-amino-20(S)-camptothecin (9-AC) and irradiation.

PURPOSE: The camptothecins (CPTs) are potent radiosensitizers of malignant tumors in vivo. The extent of normal tissue damage after combined CPT and radiation treatment is unknown. In this article, a jejunal absorption assay with (99m)Tc- pertechnetate (Na[(99m)TcO(4)]) was used to assess C3H/Kam mice given total body irradiation (TBI) of 4 Gy, 6 Gy, and 8 Gy, 2 mg/kg single intramuscular injection of 9-AC or a combination of 2 mg/kg 9-AC + 4 Gy TBI. We also correlated the absorption data with morphologic changes in the jejunal mucosa. MATERIALS AND METHODS: ((99m)TcO(4))(-) absorption from the intestinal lumen into the circulation was studied with dynamic gamma-scintigraphy combined with a multichannel analyzer to record the radiometry data in a time-dependent fashion. Jejunal cross sections were scored for the number of cells per villus and the percentage of apoptotic and mitotic cells in the crypt compartment. The jejunal microcolony assay was used to quantify jejunal crypt survival. RESULTS: A dose-dependent decrease in the absorption function was observed 3.5 days following TBI. The mean absorption rate was reduced to 89 +/- 16% of control in response to a sublethal 4 Gy TBI and dropped to 47. 5 (9.8% in response to 8 Gy TBI. The mean rate of intestinal absorption was delayed by single sublethal 2 mg/kg 9-AC injection to 62 (11% in comparison with control values. The combination of a single 4 Gy TBI with a 9-AC treatment decreased the ((99m)TcO(4))(-) jejunal absorption in an additive fashion producing absorption lifetime values more than twofold longer than controls. The decrease in ((99m)TcO(4))(-) absorption at 3.5 days after irradiation, 9-AC treatment or the combination of the two agents correlates with the number of cells per villus and the percentage of apoptotic cells in the crypt compartment. CONCLUSION: Dynamic enteroscintigraphy with (99m)Tc-pertechnetate is a sensitive functional assay for rapid evaluation of radiation and chemotherapy induced intestinal damage. Reduced intestinal absorptive function has a cellular basis and correlates directly with the numbers of cells lost per villus in a treatment-dependent manner.

Animals↗
PubMed1997

An enzyme inhibition assay for the quantitative determination of the new bisphosphonate zoledronate in plasma.

Zoledronate, 2-(imidazole-1-yl)-1-hydroxyethane-1,1-bisphosphonic acid is a new 3rd generation bisphosphonate. A specific enzyme inhibition assay was developed for the determination of zoledronate in plasma of animals and man. The multistep synthesis of cholesterol and some of its precursors (lanosterol, squalene) from 14C-labeled mevalonic acid lactone is catalyzed by the S12 fraction of homogenized rat liver in the presence of ATP, NADH and Mg2+. After hydrolysis of the reaction mixture with KOH, lipophilic reaction products were extracted with hexane and the overall yield determined by radiometry. Addition of zoledronate inhibited the formation of cholesterol and its precursors in a dose dependent manner. The described method is suitable to specifically and quantitatively measure concentrations of zoledronate down to 25 ng ml-1 in human and animal (dog, rat) plasma with acceptable reproducibility and accuracy.

Animals↗
PubMed2006

The determination of the efficiency of energy dispersive X-ray spectrometers by a new reference material.

A calibration procedure for the detection efficiency of energy dispersive X-ray spectrometers (EDS) used in combination with scanning electron microscopy (SEM) for standardless electron probe microanalysis (EPMA) is presented. The procedure is based on the comparison of X-ray spectra from a reference material (RM) measured with the EDS to be calibrated and a reference EDS. The RM is certified by the line intensities in the X-ray spectrum recorded with a reference EDS and by its composition. The calibration of the reference EDS is performed using synchrotron radiation at the radiometry laboratory of the Physikalisch-Technische Bundesanstalt. Measurement of RM spectra and comparison of the specified line intensities enables a rapid efficiency calibration on most SEMs. The article reports on studies to prepare such a RM and on EDS calibration and proposes a methodology that could be implemented in current spectrometer software to enable the calibration with a minimum of operator assistance.

Journal Article↗
PubMed2002

Effects of soil pH and soil water content on prosulfuron dissipation.

The sulfonylurea herbicide prosulfuron, 1-(4-methoxy-6-methyltriazin-2-yl)-3-[2-(3,3,3-trifluoropropyl)phenylsulfonyl]urea, is used for the selective control of broadleaf weeds in corn, sorghum, and cereal grains. To investigate its fate in soils, this study examined the effects of soil pH and water content on the rates of dissipation processes and the products formed under aerobic conditions. Radiometry and chromatography analyses were used to quantify the degradation products and bound residues formed in incubations of 10 different soils. The pH-dependent hydrolysis of the sulfonylurea bridge to form phenyl sulfonamide was the primary transformation process. Significant microbial degradation of prosulfuron occurred in 2 of the 10 soils, yielding (14)CO(2) and desmethyl prosulfuron among the major products. The time required for 50% dissipation of the herbicide (DT(50)) was determined for each soil and water content treatment. At equivalent water contents, prosulfuron DT(50) values were positively correlated with soil pH (P < 0.0001), varying from 6.5 days at pH 5.4 to 122.9 days at pH 7.9. Soil pH and water content strongly influence the fate of sulfonylurea herbicides in agricultural fields. Differences in the effect of soil water content on dissipation kinetics in a comparison of two soils were attributed to differences in soil pH, texture, and the ability of indigenous microorganisms to transform the herbicide.

Biodegradation, Environmental↗