Determining anomericity of the glycosidic bond in Zn(II)-diethylenetriamine-disaccharide complexes using MSn in a quadrupole ion trap.
(9/5815)
Zinc-diethylenetriamine (Zn-dien) N-glycoside complexes of four 1,4 and four 1,6 linked disaccharides are prepared. Each reaction mixture is ionized by electrospray and the resulting species [Zn(dien)(disaccharide)-H]+ is allowed to undergo collision-induced dissociation in a quadrupole ion trap. An MS3 analysis is used to differentiate alpha versus beta anomericity of the glycosidic bond in the disaccharide moiety. In addition, the MS2 and MS3 spectra can be used together to determine the linkage position of this glycosidic bond. (+info)
Contaminations occurring in fungal PCR assays.
(10/5815)
Successful in vitro amplification of fungal DNA in clinical specimens has been reported recently. In a collaboration among five European centers, the frequency and risk of contamination due to airborne spore inoculation or carryover contamination in fungal PCR were analyzed. The identities of all contaminants were specified by cycle sequencing and GenBank analysis. Twelve of 150 PCR assays that together included over 2,800 samples were found to be contaminated (3.3% of the negative controls were contaminated during the DNA extraction, and 4.7% of the PCR mixtures were contaminated during the amplification process). Contaminants were specified as Aspergillus fumigatus, Saccharomyces cerevisiae, and Acremonium spp. Further analysis showed that commercially available products like zymolyase powder or 10x PCR buffer may contain fungal DNA. In conclusion, the risk of contamination is not higher in fungal PCR assays than in other diagnostic PCR-based assays if general precautions are taken. (+info)
Pretargeting of bacterial endocarditis in rats with streptavidin and 111In-labeled biotin.
(11/5815)
A radioimaging approach for the detection of endocarditis has been investigated using two-step pretargeting with streptavidin and radiolabeled biotin. METHODS: Hemodynamic alterations within the rat heart were induced by placing an in-dwelling catheter into the left ventricle through the aortic valves. The animals were subsequently infected with Staphylococcus aureus through a tail vein. After an incubation period, rats were first injected with streptavidin and, 2 h later, with 111In-labeled ethylene-diaminetetraacetic acid-biotin. Whole-body gamma camera images were taken 4-5 h postinjection of the radiolabeled biotin. Control animals consisted of catheterized but uninfected, infected but uncatheterized and normal untreated rats. As a further control, the labeled biotin was administered to a study animal without the preadministration of streptavidin. RESULTS: Histology showed typical endocarditic changes in the hearts of study animals with massive deposition of gram-positive cocci. Catheterized but uninfected animals showed alterations corresponding to nonbacterial thrombotic endocarditis. Macroautoradiography showed accumulation of radiolabel in the endocarditic vegetations of study animals. Whole-body gamma camera images showed important cardiac uptake in 7 of 8 catheterized and infected animals and in 3 of 6 catheterized but uninfected animals. Normal rats and those infected but not catheterized showed negative results by histology, autoradiography and imaging. The percent uptake of the injected dose in the heart was 0.20 (SD = 0.13) in catheterized and infected animals, 0.12 (SD = 0.10) in catheterized but uninfected animals, 0.10 (SD = 0.04) in infected but uncatheterized animals and 0.04 (SD = 0.01) in normal control animals. CONCLUSION: The two-step pretargeting approach using streptavidin and 111In-labeled biotin was used successfully to detect S. aureus-induced bacterial endocarditis in rats. (+info)
A simple high-performance liquid chromatography assay for the major cisapride metabolite, norcisapride, in human urine.
(12/5815)
A simple high-performance liquid chromatography assay using fluorescence detection for the major metabolite of the gastric prokinetic drug cisapride, norcisapride, is presented. Analysis is performed using an Alltech Platinum EPS C8 column with a mobile phase made up of methanol and 0.02M sodium dihygrogen phosphate (45:55, v/v) containing triethylamine (1 g/L). Complete resolution is achieved among norcisapride, the internal standard (metoclopramide), and endogenous urinary components. The assay is linear over the range 50-2000 ng/mL with a mean recovery of 71.2% across the analytical range following solvent extraction with toluene-isoamyl alcohol (95:5, v/v). Intraday coefficients of variation (precision) determined at 200 and 1000 ng/mL are 6.0 and 9.8%, respectively, and interday coefficients of variation are 8.8 and 6.6%, respectively. Intra- and interassay accuracy (as mean relative error) determined at the same concentrations is within 10% in all cases. An analysis of urine samples from a healthy volunteer following the administration of a single 10-mg oral dose of cisapride is shown. (+info)
High-performance liquid chromatography column switching applied to the trace determination of herbicides in environmental and drinking water samples.
(13/5815)
A selective and sensitive coupled-column high-performance liquid chromatographic method is developed for the simultaneous determination of 5 phenylurea herbicides (monuron, linuron, isoproturon, monolinuron, and diuron) in environmental and drinking water samples. Sample clean-up is performed automatically by means of a column switching technique. Using 2 octadecyl silica columns connected via two programmable 6-port valves and ultraviolet detection at 244 nm, the aforementioned compounds can be determined at the low concentration levels required for pesticide residue analysis in water samples. A mobile phase consisting of a mixture of methanol-water (55:45, v/v) is pumped at 1 mL/min. For the 5 phenylureas, high recoveries ranging from 94.9 to 101.6%, good reproducibility with relative standard deviations lower than 5%, and wide linear ranges up to 20 micrograms/L are observed with determination limits of 0.05 microgram/L. The method is successfully applied to the screening of different environmental water samples such as surface, ground, rain, and drinking water. (+info)
Histology and tissue chemistry of tidemark separation in hamsters.
(14/5815)
Adult articular cartilage is divided by the tidemark into a deep calcified layer and a more superficial uncalcified layer. Histologic examination of articular cartilage from the knee joint of golden Syrian hamsters 123 days of age or older revealed defects at the tidemark in the tibia. Defects ranged from small separations of the calcified and uncalcified layers along the tidemark to progressively larger defects apparently formed by dissolution. These larger defects appeared as cavities in the noncalcified cartilage, had smooth rather than rough edges, frequently contained coalesced debris, and often resulted in a bulge in the articular surface. Occasionally, these large defects broke through the articular surface. Defects were not observed in tibial cartilage of younger (<90 days old) hamsters or in femoral cartilage from hamsters of any age. Exercise neither protected against nor increased the severity of the defects. Collagen cross-linking by pyridinoline was examined as a function of age and increased from 1,090 to 3,062 micromoles of pyridinoline/mole of hydroxyproline over the period of 1-9 months of age but was not correlated with defect formation. With increasing age, these focal tidemark defects could lead to osteoarthrosis-like cartilage lesions. (+info)
Inhibitory effects of catecholamines and anti-oxidants on the fluorescence reaction of 4,5-diaminofluorescein, DAF-2, a novel indicator of nitric oxide.
(15/5815)
4,5-Diaminofluorescein (DAF-2) is a newly developed indicator of nitric oxide (NO). Two amino groups of DAF-2 are oxidized by NO. We investigated the effects of reducers on the NO-induced oxidation of DAF-2. NOC-5 (0.1-10 microM), a NO-donor, concentration-dependently elicited fluorescence with 10 microM DAF-2. The rate of the fluorescence reaction was dependent on the width of the excitation band path. The presence of catecholamines (1 microM), but not tyrosine or phenylephrine, attenuated the fluorescence induced by NOC-5. Ascorbate and other reducers like dithiothreitol, 2-mercaptoethanol, or glutathione (all 1 mM) abolished the fluorescence. These results suggest that reducers attenuate the NO-induced fluorescence of DAF-2 mainly through an anti-oxidative action. (+info)
LY303366 exhibits rapid and potent fungicidal activity in flow cytometric assays of yeast viability.
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LY303366 is a semisynthetic analog of the antifungal lipopeptide echinocandin B that inhibits (1,3)-beta-D-glucan synthase and exhibits efficacy in animal models of human fungal infections. In this study, we utilized flow cytometric analysis of propidium iodide uptake, single-cell sorting, and standard microbiological plating methods to study the antifungal effect of LY303366 on Saccharomyces cerevisiae and Candida albicans. Our data indicate that an initial 5-min pulse treatment with LY303366 caused yeasts to take up propidium iodide and lose their ability to grow. Amphotericin B and cilofungin required longer exposure periods (30 and 180 min, respectively) and higher concentrations to elicit these fungicidal effects. These two measurements of fungicidal activity by LY303366 were highly correlated (r > 0.99) in concentration response and time course experiments. As further validation, LY303366-treated yeasts that stained with propidium iodide were unable to grow in single-cell-sorted cultures. Our data indicate that LY303366 is potent and rapidly fungicidal for actively growing yeasts. The potency and rapid action of this new fungicidal compound suggest that LY303366 may be useful for antifungal therapy. (+info)