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The cationic dye auramine O forms a fluorescent complex with Ca(2+)-liganded calmodulin. One moderately strong binding site is present, as well as one or more weaker sites. The binding site for auramine O is different from those for toluidinyl-naphthalene sulfonate. The dependence of binding upon electrolyte concentration suggests a substantial electrostatic component of the free energy of binding. The splitting of the bond between residues 77 and 78 by trypsin digestion abolishes auramine O binding; the N- and C-terminal half-molecules have virtually no binding capacity. This suggests that the primary binding site is located near the midpoint of the connecting strand and includes elements of both half-molecules. Thrombin digestion, which splits calmodulin between residues 106 and 107, also substantially reduces auramine O binding; this may be interpreted in terms of the stabilization of the structure of the connecting strand by interaction with residues within binding domain IV. The binding affinity at pH 5.0, where the helical organization of the connecting strand may be intact, is greater than at neutral pH.
The purpose of this study was to evaluate the clinical usefulness of a commercial ligase-based gene amplification method (LCx Mycobacterium tuberculosis test; Abbott Laboratories, USA) for detection of Mycobacterium tuberculosis. The tuberculosis infection rate among clinical samples was 10.6%. The sensitivity, specificity, and positive and negative predictive values were 23.5%, 100%, 100%, and 91.7%, respectively, with the fluorochrome auramine stain; 32.4%, 100%, 100%, and 92.6%, respectively, with culture; and 76.5%, 95.8%, 68.4% and 97.2%, respectively, with the gene amplification method. When only samples from patients without current or previous treatment were studied, the sensitivity was 36.4% with the auramine stain, 63.6% with culture, and 100% with the gene amplification assay. The mean treatment time for culture-negative and assay-negative samples was greater than that of culture-negative and assay-positive samples. The LCx Mycobacterium tuberculosis test is a sensitive method for detection and identification of Mycobacterium tuberculosis. It produces few false-positive results. However, as it can remain positive after the culture becomes negative, it is not recommended for evaluation of treatment efficiency.
A study was carried out on the presence of oocysts of Cryptosporidium spp. in water for human consumption in the Province of Salamanca, Spain, using four different staining techniques: Ziehl-Neelsen, auramine, indirect immunofluorescence with monoclonal antibodies (Meridian, USA) and direct immunofluorescence with monoclonal antibodies (Pasteur, France). The presence of oocysts was detected in the water used for the washing of sand filters in the four water purifying plants and in the drinking water of two of the six geographical areas studied. The Ziehl-Neelsen and auramine staining techniques were found to have excellent diagnostic correlation. Immunofluorescence stainings with monoclonal antibodies proved to be less sensitive.
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Acridine orange staining for the detection of mycobacteria was compared with staining by auramine O and with mycobacterial culture in a series of 1071 clinical specimens. A total of 78 (7%) specimens were positive by staining. No false positive or negative findings were recorded by the acridine orange method. The two fluorochromes proved equal in their ability to detect mycobacteria in specimens from culture positive cases of tuberculosis. In the rapid bacteriological diagnosis of tuberculosis, acridine orange offers a good alternative to auramine O which is considered carcinogenic.
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The blue product of the Lowry et al. (1951, J. Biol. Chem. 193, 265-275) reaction interacts with malachite green (MG), inducing a change in the visible light spectrum. At A690 nm the absorbance of malachite green solutions increases 10-fold in the presence of Lowry blue (LB). Under the optimum conditions, 0.01 A700 nm unit of Lowry blue produces a change in A690 nm unit of malachite green of 0.5 and the delta A690 nm is a linear function of Lowry blue concentration. Conditions under which this 50-fold amplification can be exploited to detect less than 100 ng of protein (or 4 micrograms X ml-1) are described. A number of chemicals including sodium dodecyl sulfate can interfere with the assay but a strategy has been devised to overcome these problems. Amplification of the Lowry assay appears to involve a cooperative interaction between malachite green and the Lowry blue product such that about 23 molecules of malachite green undergo a spectral shift per molecule of a model reactant such as tyrosine. Malachite green can be used to amplify the molybdenum blue signal obtained in other assays. Less than 10 pmol of tyrosine can be detected using this procedure. Lowry blue also interacts with auramine O, giving a large increase in A500 nm and a 40-fold amplification of the LB signal. As with malachite green, there is a cooperative interaction between auramine O and LB. About 72 molecules of auramine O undergo a spectral shift per molecule of tyrosine. The product of this reaction is also fluorescent and could be exploited in a protein assay.(ABSTRACT TRUNCATED AT 250 WORDS)
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A cationic fluorescent dye, auramine O (AO), exhibited an intense increase in fluorescence after binding to human alpha 1-acid glycoprotein (alpha 1-AG). The interaction between AO and the protein was studied by fluorescence spectroscopy and by equilibrium dialysis. AO binds to the protein via a single site with a dissociation constant of 24 microM. Various basic drugs such as chlorpromazine, imipramine, desipramine, quinidine, propranolol and lidocaine, which are known to bind to the protein, competitively inhibited the AO binding to the protein. The dissociation constants of these basic drugs obtained from such inhibitory experiments were comparable to those obtained with other methods (equilibrium dialysis, quenching of protein intrinsic fluorescence, and the difference spectrophotometric method) and from the literature. It is concluded that AO may be a useful fluorescent probe that binds to a single basic drug binding site on alpha 1-AG. In addition, a simple fluorometric method for the determination of alpha 1-AG in serum was developed using AO, and the validity of this method was confirmed by comparing it with the conventional radial immunodiffusion method.
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The tetrameric molecule of pig skeletal muscle lactate dehydrogenase binds a cationic fluorescent probe, auramine O, at four equal non-interacting sites with a dissociation constant of (1.25 +/- 0.2) X 10(-4) M. Fluorescence of the dye/enzyme mixture is strongly pH-dependent, with a maximum at pH 6.3-6.8. Auramine O-binding sites are located outside the active center of the enzyme. The microenvironment of the bound dye changes upon interaction of lactate dehydrogenase with NAD+, NADH, ADP and pyruvate. The binding of specific ligands induces an increase in fluorescence of auramine O-enzyme complex. This effect was used to determine the dissociation constants of the complexes of lactate dehydrogenase with specific ligands. Pyruvate was demonstrated to bind to the apoenzyme-auramine O complex with a dissociation constant of 5.2 X 10(-4) M. With the use of auramine O, it became possible to reveal subunit interactions within the tetrameric molecule of lactate dehydrogenase. They are manifested in the changes of the microenvironment of a dye-binding site located on one of the subunits induced by the binding of ligands in the active center of a neighboring subunit.
A case of Isospora belli in a homosexual male is described. The procedures used for recovery and staining the cyst stages of Isospora are included.
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A large fraction of carcinogens score negative in short-term genotoxicity assays such as the Salmonella reverse mutation (Ames) assay. More information is needed about the mechanism of action of such Salmonella-negative carcinogens. Many Salmonella-negative carcinogens induce deletions due to intrachromosomal recombination in Saccharomyces cerevisiae with an apparent threshold. We have previously shown that the Salmonella-negative carcinogens cadmium, aniline, chloroform and carbon tetrachloride generate free radical species in S. cerevisiae. We have further investigated the possible generation of intracellular free radical species by the diverse Salmonella-negative carcinogens benzene, urethane, thiourea and auramine O. The toxicity and recombinagenicity of thiourea and auramine O was reduced in the presence of the free radical scavenger N-acetyl cysteine. N-acetyl cysteine did not protect against toxicity or recombination induced by the Salmonella-positive carcinogens ethyl methane sulfonate, methyl methane sulfonate or nitroquinoline-N-oxide. A strain deficient in the enzyme superoxide dismutase, which catalyses the dismutation of superoxide anion radical, was hypersensitive to killing by benzene, urethane and thiourea. The sod- strain was only slightly more sensitive to the Salmonella-positive carcinogens. Intracellular oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate was increased in yeast cultures exposed to benzene, urethane and auramine O; again, the Salmonella mutagens had no effect on oxidation of the dye. These data show that free radical species are produced in Saccharomyces cerevisiae following exposure to benzene, urethane, thiourea and auramine O, and suggest a possible role for oxidative stress is recombination induced by these carcinogens.
Nontuberculous (i.e., atypical) mycobacterial infections are increasing among pediatric and immunosuppressed patients, who commonly present with subcutaneous inflammatory masses or adenitis, which is often surgically excised. Since the most frequently isolated species also grow slowly in culture, early diagnosis may depend on histologic detection of a mycobacterial organism in the biopsy specimen. However, the histologic methods used for this purpose are of uncertain value in the diagnosis of these infections. Biopsy specimens from 22 patients with clinical histories highly consistent with nontuberculous mycobacteriosis in which part of the tissue was cultured were selected for study. Coded tissue blocks and control specimens were stained by the Ziehl-Neelsen (ZN) or auramine-O (A0) fluorescent technique and examined blindly for the presence of characteristic organisms. Results of these studies were compared with the culture results, and predictive values were calculated. This experience showed that the AO technique is technically simpler, allowing faster screening at lower power and showing greater sensitivity and predictive value of a negative result although less specificity than the ZN technique. The lower specificity of AO may be factitious and due to the detection in the tissue of organisms that did not grow in culture. Previous observations that nontuberculous mycobacterial infections may elicit tissue reactions that simulate cat-scratch disease, sarcoidosis, and nonspecific chronic inflammation were also confirmed.
The effect of DNA hydrolysis, staining time, dye concentration and some other important factors was evaluated on measurement of nuclear DNA content in plant cells by cytofluorometry. Based on the results optimal conditions for making permanent Auramine O--SO2 stained squash preparations can be chosen.