Use of the fluorescent dye YOYO-1 to quantify oligonucleotides immobilized on plastic plates.
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Benzoxazoles↗
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Cytotoxicity of dopamine-derived 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines.
The in vivo effects of dopamine-derived alkaloids, 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines, salsolinols, and their N-methylated derivatives on a dopaminergic cell model, clonal rat pheochromocytoma PC12h cells, were examined by culture in the presence of various concentrations of the agents. The effects were evaluated in comparison with those by 1,2,3,4-tetrahydroisoquinoline and its N-methylated derivatives. Among 1,2,3,4-tetrahydroisoquinolines, only N-methylisoquinolinium ion had cytotoxic effect on PC12h cells. In general, 6,7-dihydroxyisoquinolines had more potent cytotoxic effect than N-methylisoquinolinium ion, and they reduced protein amounts of PC12h cells at 100 microM and 1 mM concentration. The specific activity of tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis, decreased with these isoquinolines at concentrations lower than those required to reduce the protein amount. The toxicity of N-methylated derivatives seems to be more potent than non-methylated isoquinolines. Salsolinols were proved to be accumulated in the mitochondrial fraction of the cells after 3 days in culture. N-methyl-1,2,3,4-tetrahydroisoquinoline depleted ATP from PC12h cells and it was prevented by preincubation with an inhibitor of type-A monoamine oxidase, clorgyline. These results indicate that N-methylated and oxidized derivatives of dopamine-derived alkaloids may be potent dopaminergic neurotoxins similar to 1-methyl-4-phenylpyridinium ion in the human brain and may induce Parkinson's disease after long years of accumulation.
Animals↗
Enhanced detection of PCR products through use of TOTO and YOYO intercalating dyes with laser induced fluorescence--capillary electrophoresis.
Recent developments in the chemical synthesis of DNA-binding dyes have enhanced detection of polymerase chain reaction (PCR) products by capillary electrophoresis. These dyes are dimers of thiazole orange (TOTO) or oxazole orange (YOYO) and have a very high binding affinity for DNA (Haugland, 1992). These dyes show enhanced fluorescence signals when they bind to double-stranded DNA and their fluorescence in the unbound state is almost zero, making them extremely useful in detecting minute (fg) quantities of DNA. We report here the utility of these dyes in DNA typing applications using a laser-induced fluorescence detector in conjunction with a capillary electrophoresis system.
Benzoxazoles↗
[Treatment of inflammatory diseases of the mouth and throat with Larypront in ENT practice (author's transl)].
Treatment of inflammations of the oral and pharyngeal cavities (glossitis, pharyngitis, pharyngolaryngitis, tonsillitis) in 60 patients with Larypront Throat Tablets alone, without administration of systemic antibiotics, gave very good results in 49 cases (81.6%) with complete regression of the complaints in 3-4 days. In 11 patients with acute pharyngitis and tonsillitis without demonstrable effect, systemic antibiotic therapy had to be added because of radiologically demonstrable acute sinusitis or increasing antibacterial infection. Side effects were not seen with the use of Larypront in any case.
Adolescent↗
Endotoxin-induced pulmonary leukostasis in the rat: role of platelet-activating factor and tumor necrosis factor.
Injection of bacterial lipopolysaccharide (LPS) into experimental animals induces septic shock associated with the release of tumor necrosis factor (TNF) and platelet-activating factor (PAF). Because both TNF and PAF stimulate neutrophil adhesion to endothelial cells in vitro, and because neutrophils are important effector cells in sepsis-induced lung vascular injury, the role of TNF and PAF in LPS-induced lung neutrophil sequestration was investigated. Lung myeloperoxidase (MPO) activity was measured as a quantitative assessment of pulmonary leukostasis. Injection of Salmonella enteritidis LPS into rats caused dose-dependent increases in lung MPO that peaked at 2 hours and persisted for up to 24 hours. Injection of purified human recombinant TNF (2 to 200 micrograms/kg i.v.) mimicked the effect of LPS on lung MPO activity. Injection of synthetic PAF increased lung MPO only at the highest and lethal dose (10 micrograms/kg). Lower doses (0.1 and 1 microgram/kg) of PAF had no effect on lung MPO by itself and did not enhance LPS- or TNF-induced lung neutrophil sequestration. Furthermore, pretreatment of the rats with two different PAF receptor-antagonists, WEB 2086 (10 mg/kg IP) and SRI 63-441 (10 mg/kg IP), failed to block LPS-induced (1 mg/kg) increase in lung MPO. These data suggest that TNF, not PAF, mediates LPS-induced pulmonary neutrophil sequestration in the intact rat.
Animals↗
Alternative labeling techniques for automated fluorescence based analysis of PCR products.
We present here convenient and simple methods that utilize two new fluorescent tags, TOTO-1 and fluorescein-12-dUTP, in conjunction with analysis of PCR products on automated, fluorescence-based electrophoretic instruments. These methods should prove valuable in those applications wherein the effort or expense of covalently attaching a fluorescent tag onto one or both of the PCR primers is not justified. Advantages and disadvantages of the new labeling methods are then reviewed.
Deoxyuracil Nucleotides↗
Trypanosoma evansi: measurement of pyruvate production as an indicator of the drug sensitivity of isolates in vitro.
In a previous study, three in vitro methods for the assessment of drug sensitivity among Trypanosoma evansi isolates were compared--a direct counting method, pyruvate production method and uptake of radiolabelled hypoxanthine. The pyruvate assay system, which measures the amount of pyruvate in the supernatant of growing populations of trypanosomes by a spectrophotometric method, was selected for further investigation with regard to its suitability for field studies. The effect of initial seeding density and incubation time on the growth of three stocks of T. evansi--TREU 1840 and TREU 1981 (suramin sensitive) and TREU 2136 (suramin resistant)--and drug sensitivities revealed by the pyruvate assay and direct counting were examined to optimise assay conditions. Maximum densities and pyruvate production achieved were not affected by varying the initial seeding densities in the range of 5 x 10(4)-5 x 10(5)/ml and had been reached after 48 hours incubation with one exception: Pyruvate levels continued to increase up to 72 hours in the suramin resistant stock. However, inhibition curves were affected by initial seeding density and incubation period. Results suggested that an initial seeding density of 1 x 10(5)/ml and an incubation time of 48 hours are optimal for the assay. Using these assay conditions, the isolates were screened against suramin, quinapyramine sulphate and Cymelarsan, the trypanocides used most commonly against T. evansi. This assay proved to be a relatively simple and cheap technique applicable to screening large numbers of isolates of differing sensitivities to trypanocidal drugs.
Animals↗
[Treatment of tonsillitis with dequalinium chloride].
44 children with acute tonsillitis were treated without the usual penicillin therapy. 22 children received lozenges containing Dequalinium chloride, 22 children received void lozenges under conditions of a double blind study. The results show that the treatment with Dequalinium chloride did not influence conditions caused by haemolytic streptococci -- verified by bacteriological examinations of pharyngeal smears -- inspite of its efficiency in vitro. Also general symptoms like fever, leukocytosis and rapid erythrocyte sedimentation rate were not influenced by Pequalinium chloride. Local symptoms like sore throat, tonsillar redness and exsudation were influenced beneficially. It can therefore be assumed that in cases of acute tonsillitis penicillin therapy is not necessary except in cases of streptococci infections. These should be excluded by pharyngeal smear. The medium of Liebermeister and Braveny is a very simple indicator to differentiate between tonsillitis caused by streptococci and tonsillitis of other origin.
Child↗
Modified Feulgen staining of DNA with aqueous solution of pinacyanol.
The paper reports on the use of a quinoline dye, pinacyanol, towards staining of acid hydrolysed DNA. The dye as an aqueous solution can be used after treatment of mammalian tissue sections in concentrated phosphoric acid at 5 degrees C for 20 min followed by hydrolysis in 6N HCl at room temperature for 15 min, for staining DNA-aldehyde molecules. It has also been observed that staining of DNA-phosphate groups is also possible in sections treated with cold concentrated phosphoric acid after selective extraction of RNA. Both in situ absorption characteristics of stained nuclei as well as in vitro absorption data of an aqueous solution of the dye have been presented. It has been suggested that staining DNA-aldehydes with pinacyanol, without any primary amino group in its molecules is due to a modified Feulgen reaction.
Animals↗
Quantitative fluorescence measurements of chloride secretion in native airway epithelium from CF and non-CF subjects.
Functional assessment of the efficacy of CFTR gene transfer protocols in humans has previously involved measurement of in vivo potential difference. We have studied whether freshly obtained airway epithelial cells may provide suitable tissue for studies of in vivo gene transfer using fluorescent digital imaging microscopy. Nasal epithelial cells from non-cystic fibrosis subjects (n = 6) and from cystic fibrosis (CF) patients (delta F508: delta F508, n = 5) were obtained by brushing and loaded with 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Addition of the cAMP-agonists forskolin (20 microM) and 3-isobutyl-1-methylxanthine (IBMX, 100 microM) produced an increased efflux of iodide from the cells which was significantly (P < 0.05) greater in non-CF than in CF cells. Efflux following addition of the calcium ionophore, ionomycin (100 microM) was similar in both non-CF and CF cells. Liposome-mediated transfection of CF nasal epithelial cells in vitro with CFTR-cDNA restored the cAMP-stimulated efflux to non-CF values. Bronchial epithelial cells from non-CF subjects showed responses to forskolin and ionomycin that were not different to those in non-CF nasal epithelia. These data demonstrate that the assay provides a useful method for assessing correction of abnormal ion transport in non-cultured CF epithelium and is likely to provide a further assay for assessment of in vivo gene transfer efficiency in protocols of gene therapy for CF.
Adult↗
Photodynamic efficiency of macrophage activity using a photosensitizer, lumin, with near-IR laser light for photoimmunotherapy of a cancer.
This study was carried out to determine the efficacy of photoimmunotherapy using a photosensitizer, lumin, and near-IR laser light irradiation for cancer therapy. Lumin is a potent cell-activating agent and a photosensitizer characterized by typical absorption peaks at 670 and 770 nm. To clarify that ingestion activity is enhanced by lumin administration with different light irradiation, He-Ne laser light (632.8 nm) and near-IR laser light (780 nm) were used. The results showed that a small dose of lumin (5 ng/ml) with near-IR laser light dramatically enhanced the ingestion activity. A human lung cancer grafted onto nude mice was used in this study. After the tumor hypertrophied, lumin was injected into it six times. Then, it was exposed to near-IR laser light (2 mW, 1 min) every other day for 2 weeks. The whole tumor was strongly cicatrized by collagen fiber from the stroma. It was removed 15 weeks after the treatment. Immunological function was investigated using a two-color flow cytometric assay method based on immunofluorescence labeling with specific monoclonal antibodies of 10(4) cell samples harvested from mouse peripheral blood. At 40 weeks, the ratio of T cells to B cells in untreated mice was 0.23, whereas that in treated mice was 0.58. These results demonstrated that photoimmunotherapy with lumin administration and laser light irradiation is a reliable method of cancer therapy.
Adenocarcinoma↗
Fluorometric determination of total mRNA with oligo(dT) immobilized on microtiter plates.
We have developed a rapid and nonradioactive method of quantifying cytosolic mRNA from crude cell lysates by using plastic plates to which oligonucleotides containing poly(dT) sequences were previously immobilized. Captured mRNA on the plate was mixed with Yoyo-1 fluorescent indicator dye, and the resulting Yoyo-1 fluorescence of the mRNA-Yoyo-1 complex was measured in a fluorometer. Because Yoyo-1 signals were linearly increased in proportion to the amount of applied mRNA in the range 10-250 ng, the amount of mRNA in test samples can be determined by comparing their Yoyo-1 fluorescence with that of known concentrations of calibrator mRNA. Using this system, we found that the amount of cytosolic mRNA in undifferentiated U937 and HL-60 cells was 268.6 +/- 13.1 and 282.0 +/- 7.8 ng/10(6) cells, respectively, significantly (P < 0.01) more than that of phorbol ester-induced differentiated U937 and HL-60 cells (145.3 +/- 13.9 and 164.7 +/- 11.6), respectively. Therefore, the present system may be applicable to both medical molecular biology research and diagnostics.
Animals↗
A flow cytometric method for measurement of intracellular chloride concentration in lymphocytes using the halide-specific probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ).
A flow cytometry method using the halide-specific fluorescent dye, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), has been developed to measure intracellular chloride concentration in single cells. Collisions with chloride quench the fluorescence of SPQ, making it possible to relate the measured fluorescence intensity to chloride concentration with a Stern-Volmer equation. To demonstrate the method, porcine lymphocytes were loaded in vitro, using a hypotonic method, with 5 mM SPQ. Fluorescence excitation was provided by a UV laser and the fluorescence emission intensity at 485 nm was recorded. Calibration was performed by using 7 microM nigericin (a K/H antiporter) and 10 microM tributyltin (a Cl/OH antiporter) to equilibrate the concentrations of intracellular and extracellular chloride. Calibration measurements were made for chloride concentrations between 0 mM and 140 mM. The calibration produced a Stern-Volmer quenching constant of 16.2 M(-1) which was used to relate measured cell fluorescence to intracellular chloride concentration. The intracellular chloride concentration for fresh porcine lymphocytes was determined to be 56.2 +/- 3.3 mM. Stable loading of cells with 5 mM SPQ was accomplished in 15 minutes, leakage of SPQ from the cells was minimal, and over 95% of the cells remained viable after loading.
Animals↗
Antiproliferative gene BTG1 is highly expressed in apoptotic cells in macrophage-rich areas of advanced lesions in Watanabe heritable hyperlipidemic rabbit and human.
In the present study, the expression and potential role of the highly conserved antiproliferative gene BTG1 in the process of apoptosis as it occurs in atherosclerotic lesions was examined. In situ hybridization and immunodetection studies demonstrated that BTG1 localized to specific macrophage-rich regions of lesions in both Watanabe heritable hyperlipidemic rabbits and humans. In addition, the spatial distribution of BTG1 mRNA was shown to colocalize not only with macrophage-rich regions but also to cells that exhibited changes consistent with apoptosis, such as DNA fragmentation and nuclear condensation. In vitro studies demonstrated that forced overexpression of BTG1 induced a 3-fold increase in apoptosis in NIH/3T3 cells. Furthermore, significantly increased expression of BTG1 mRNA resulted from lipid loading of human monocyte-derived macrophages in vitro. The process of increasing lipid content in macrophages is frequently associated with decreased macrophage viability. Taken together, these data underscore a potentially important role for BTG1 in regulating the cellularity of advanced atherosclerotic lesions.
3T3 Cells↗
The use of two fluorescent dyes to identify sperm in a competitive binding assay to oocytes.
The relationship of most sperm laboratory assays to male fertility is inconsistent. Assays that measure traits required to fertilize oocytes are expected to have the most predictive value. A new assay that measures the competitive ability of two sperm samples to bind to oocytes was developed. Two populations of sperm were labeled using a pair of lipophilic dyes. A concentration of 75 microM of the two dyes, DiQ (4-[4-(dihexadecylamino)styryl]-N-methylquinolinium iodide; an orange-red dye) and DiOC16 (3,3'-dihexadecyloxacarbocyanine perchlorate; a yellow-green dye), intensely stained 66 and 73% of sperm, respectively, without affecting sperm motility or oocyte-binding ability. Because sperm could be stained with fluorescent dyes, sperm from two semen samples were mixed together in a droplet, and oocytes were added to allow sperm to bind oocytes competitively. Oocyte-bound sperm from each sample were counted. Binding was specific; nonspecific sperm binding was estimated by sperm bound to two-cell mouse embryos and averaged one to three sperm per embryo. Staining with DiQ or DiOC16 did not affect oocyte-binding ability since more than 80% of the sperm bound were stained with either dye. Furthermore, if different ratios of DiQ- or DiOC16-stained sperm from the same ejaculate were prepared in droplets and oocytes were added, the percentage of sperm bound to the oocytes reflected the percentage of sperm in the droplet; there was no differential effect of either dye. This assay used fixed oocytes because sperm bound equally to fixed and fresh bovine oocytes. This competitive oocyte-binding assay allows one to make a series of pairwise comparisons between a group of males or to include an internal control sample in sperm-oocyte binding assays. This assay may allow more accurate prediction of the oocyte-binding ability of sperm.
Animals↗
[Cytostatic effect of 1-methyl-2-(p-(bis-(beta-chlorethyl)-amino)-phenyliminomethyl)-quinolinium chloride (IMET 3106) on the growth of transplantable neoplasms].
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Animals↗
Pyridinium- and quinolinium-2-dithioacetic acid zwitterions: antiradiation and anticancer activities.
Pyridinium-, quinolinium-, and one pyrimidinium-2-dithioacetic acid zwitterions were prepared by condensation of the N-methyl heterocyclic anhydro bases with carbon disulfide. Reaction of the 2-methylpyridine methiodide anhydro base with carbon disulfide resulted in replacement of the 2-methyl group to give the 1-methylpyridinium-2-dithioacetic acid zwitterion. The 1,6-dimethylquinolinium-2-dithioacetic acid zwitterion showed appreciable anticancer activity against P-388 lymphocytic leukemia in mice, but the other zwitterions tested showed no activity. No antiradiation activity was found for 1-methyl-pyridinium-2-dithioacetic acid zwitterion.
Acetates↗
Quinolinium salt as a potent inhibitor of lymphocyte apoptosis.
The synthesis of several quinolinium salts and related compounds and their ability to inhibit glucocorticoid-induced apoptosis in murine thymocytes are described. Interestingly, 1-[2-methoxyimino-2-(4-pyrrolidin-1-yl-phenyl)ethyl]quinolinium bromide (11) showed a potent protective effect with an EC(50) of 0.013 microM, which was at least 300-fold more potent than the reference compound pifithrin-alpha.
Antineoplastic Agents↗