Highly sensitive quantitation of methamphetamine by time-resolved fluoroimmunoassay using a new europium chelate as a label.
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A simple and highly sensitive time-resolved fluoroimmunoassay of methamphetamine (MA) using a new fluorescent europium chelate (BHHCT-Eu3+) as a label is described. Two variations of competitive immunoassay were attempted. In the first (one-step) assay, microtiter plates coated with anti-MA were used, and the new label was bound to a conjugate of bovine serum albumin and N-(4-aminobutyl)-MA (MA-BSA). In the second (two-step) assay, instead of the labeled MA-BSA, biotinylated MA-BSA and BHHCT-Eu3+-labeled streptavidin-BSA were used. The lowest measurable concentrations of MA for the one-step and the two-step methods were 1 ng/mL (25 pg/assay) and 1 pg/mL (25 fg/assay), respectively. These were 10 to 1000 times superior to the detection limits of MA in any other immunoassay. Intra-assay coefficient of variation was approximately 2-8% at eight different concentrations (n = 4). Analysis of 34 urine samples with the new method and conventional gas chromatography showed a good correlation (r = 0.954). The high detectability of the present assay also enabled segmental hair analysis with a few centimeters of a hair. (+info)
Oligonucleotide-europium complex conjugate designed to cleave the 5' cap structure of the ICAM-1 transcript potentiates antisense activity in cells.
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The 5' cap structure of mRNA is a N7 methylated guanosine residue that is linked by a 5'-5' triphosphate linkage to the 5'-terminus of cellular and viral RNAs synthesized by RNA polymerase II. This unique structure facilitates several processes of mRNA metabolism, including splicing, nucleocytoplasmic transport,initiation of translation, and degradation. Previous research has demonstrated that the lanthanide macrocycle complex, Eu(THED)3+, effectively cleaves the 5' cap structure of mRNA in solution by nucleophilic attack of the triphosphate linkage via the metal-activated hydroxyethyl group of the THED ligand. This report shows that attachment of a Eu(THED)3+analog to the 3'-terminus of an antisense oligonucleotide, which targets the 5'-terminus of the intercellular adhesion molecule 1 mRNA, potentiates the inhibitory activity of the antisense oligonucleotide in cytokine-treatedendothelial cells. (+info)
Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer.
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We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged alpha1-, beta2-, or gamma2-subunits of the hetero-oligomeric gamma-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the alpha1(c-Myc)beta2gamma2 and the alpha1beta2(c-Myc)gamma2 receptors was twice that obtained with alpha1beta2gamma2(c-Myc), suggesting that there are 2x alpha-, 2x beta-, and 1x gamma-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining alpha1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with alpha1(c-Myc)beta2gamma2 and alpha1beta2(c-Myc)gamma2, but no significant energy transfer was observed with alpha1beta2gamma2(c-Myc) receptors, indicating the absence of a second gamma-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, alpha1beta2gamma2, is composed of two copies each of the alpha- and beta-subunits and one copy of the gamma-subunit (i.e. (alpha1)2(beta2)2(gamma2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins. (+info)
Specific radioactivity of europium-152 in roof tiles exposed to atomic bomb radiation in Nagasaki.
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Specific radioactivities of residual europium (Eu)-152 were measured in six roof tile samples exposed to the Nagasaki atomic bomb at two locations. The ground distances of the two locations from the hypocenter are 1020 m and 1060 m. In order to obtain reliable data, Eu-enriched samples (from 207 to 855 mg) were prepared by separating Eu from each roof tile sample (from 1 to 2 kg). For the major aliquot of the Eu-enriched sample, residual radioactivity of 152Eu was measured using a low-energy photon spectrometer. For the minor aliquot of the Eu-enriched sample, Eu content was determined by neutron activation analysis. Results of the specific radioactivity (152Eu/Eu, Bq mg-1) corrected to the time of bombing were in a range from 0.080 to 0.446. Although the measured values showed some scattering, they are moderately consistent with the calculated values by the DS86 methodology, i.e. the average ratio of the calculated to measured values is 1.3 +/- 0.8. (+info)
The species specificity of the microimmunofluorescence antibody test and comparisons with a time resolved fluoroscopic immunoassay for measuring IgG antibodies against Chlamydia pneumoniae.
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AIMS: To examine the species specificity of the microimmunofluorescence test (MIF) and assess a time resolved fluoroscopic immunoassay (TRIA) for measuring IgG antibodies to C pneumoniae. METHODS: Sera from 1020 subjects were tested by MIF for IgG, IgM, and IgA antibodies to C pneumoniae, C trachomatis, and C psittaci; 501 serum samples were also tested by TRIA for IgG antibodies to C pneumoniae. RESULTS: C pneumoniae antibody titres as measured by MIF were correlated with those for C psittaci and trachomatis. It was estimated that on average, one third of the twofold dilution steps that make up the final C pneumoniae antibody titre may be due to cross reacting genus specific antibody. The results of TRIA correlated well with those of MIF. In 75% of cases, the TRIA result predicted a three titre range within which the actual MIF result would fall. CONCLUSIONS: MIF does not appear to be as species specific as claimed. TRIA is unlikely to be as specific but as it is completely objective, easier to perform, amenable to automation, and gives reproducible results, it is a rapid and useful method for comparing populations. (+info)
DS86 neutron dose: Monte Carlo analysis for depth profile of 152Eu activity in a large stone sample.
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The depth profile of 152Eu activity induced in a large granite stone pillar by Hiroshima atomic bomb neutrons was calculated by a Monte Carlo N-Particle Transport Code (MCNP). The pillar was on the Motoyasu Bridge, located at a distance of 132 m (WSW) from the hypocenter. It was a square column with a horizontal sectional size of 82.5 cm x 82.5 cm and height of 179 cm. Twenty-one cells from the north to south surface at the central height of the column were specified for the calculation and 152Eu activities for each cell were calculated. The incident neutron spectrum was assumed to be the angular fluence data of the Dosimetry System 1986 (DS86). The angular dependence of the spectrum was taken into account by dividing the whole solid angle into twenty-six directions. The calculated depth profile of specific activity did not agree with the measured profile. A discrepancy was found in the absolute values at each depth with a mean multiplication factor of 0.58 and also in the shape of the relative profile. The results indicated that a reassessment of the neutron energy spectrum in DS86 is required for correct dose estimation. (+info)
Alamethicin-mediated fusion of lecithin vesicles.
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It was recently shown that alamethicin greatly facilitates the fusion of small, sonicated, lecithin bilayer vesicles. In the present work the details of this fusion process have been followed by monitoring the inner and outer choline methyl signals separately by proton magnetic resonance spectroscopy. It is shown that during the alamethicine-induced fusion some of the antibiotic molecules become translocated from the extravesicular aqueous medium into the enclosed intravesicular space, and these alamethicine molecules were found to affect the choline methyl signals from the inner half of the bilayer only. No evidence was obtained for transmembrane coupling of the two halves of the bilayer in the presence of alamethicin or for any effects that might be construed as due to incorporation of alamethicin molecules into the hydrophobic core of the bilayer. (+info)
Probing the binding of Tb(III) and Eu(III) to the hammerhead ribozyme using luminescence spectroscopy.
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BACKGROUND: Divalent metal ions serve as structural as well as catalytic cofactors in the hammerhead ribozyme reaction. The natural cofactor in these reactions is Mg(II), but its spectroscopic silence makes it difficult to study. We previously showed that a single Tb(III) ion inhibits the hammerhead ribozyme by site-specific competition for a Mg(II) ion and therefore can be used as a spectroscopic probe for the Mg(II) it replaces. RESULTS: Lanthanide luminescence spectroscopy was used to study the coordination environment around Tb(III) and Eu(III) ions bound to the structurally well-characterized site on the hammerhead ribozyme. Sensitized emission and direct excitation experiments show that a single lanthanide ion binds to the ribozyme under these conditions and that three waters of hydration are displaced from the Tb(III) upon binding the RNA. Furthermore, we show that these techniques allow the comparison of binding affinities for a series of ions to this site. The binding affinities for ions at the G5 site correlates linearly with the function Z(2)/r of the aqua ion (where Z is the charge and r is the radius of the ion). CONCLUSIONS: This study compares the crystallographic nature of the G5 metal-binding site with solution measurements and gives a clearer picture of the coordination environment of this ion. These results provide one of the best characterized metal-binding sites from a ribozyme, so we use this information to compare the RNA site with that of typical metalloproteins. (+info)