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Problems caused by high concentration of ATP on activation of the P2X7 receptor in bone marrow cells loaded with the Ca2+ fluorophore fura-2.

Fura-2 is one of the most used fluorophore for measuring intracellular calcium concentration ([Ca2+]i). In mouse bone marrow cell suspensions ATP produces a biphasic effect: till 1 mM, ATP produces increases in [Ca2+]i; from 1 mM on an increase is observed, that is followed by the decrease in the 340/380 nm ratio (R340/380). At high ATP (4 mM) concentration fura-2 leaked from loaded bone marrow cell suspensions. We observed that ATP decreases fluorescence in the absorption and excitation spectra of fura-2, consequently the emitted one is decreased including the isobestic point (360 nm). ATP analogs: BzATP, ATPyS and UTP, but not alphabetaATP, ADP or AMP, promote decrease of fluorescence in the isobestic point of fura-2. The physical/chemical process that reduces the absorption and excitation of fura-2 by ATP is unknown. The P2X7 inhibitors, Mg2+ (5 mM), OxATP (300 microM) and Brilliant Blue (100 nM), blocked the efflux of fura-2 and ATP-induced R340/380 decrease. The J774 cell line and mononuclear cells with a higher expression of P2X7 receptors show the same decrease in R340/380 as that induced by ATP. In the HL-60 cell line, myeloid cells and erythroblasts extracted from bone marrow, such effect does not occur. It is concluded that the use of the fluorescent Ca2+ indicator fura-2 does not allow the correct measurement of [Ca2+]i in these cells in the presence of a higher concentration of ATP which activated the P2X7 receptor.

Adenosine Triphosphate↗

Coomassie blue protein dye-binding assays measure formation of an insoluble protein-dye complex.

The solubility of the protein-Coomassie brilliant blue (CBB) complex formed upon Bradford (Anal. Biochem. 72, 248-254, 1976) or Sedmak and Grossberg (Anal. Biochem. 79, 544-552, 1977) protein assay has been investigated by centrifugation or filtration of the assay mix within 10 min of adding dye reagent. The results show complete loss of color yield in the respective supernates and filtrates. This indicates that the protein-CBB complexes are insoluble at the time of absorbance measurement. Protein solubility in the dye reagent may dictate the relative response of the assay to an individual protein and the requirement for macromolecular structure.

Animals↗

Photooxidative N-de-ethylation of anionic triarylmethane dye (sulfan blue) in titanium dioxide dispersions under UV irradiation.

The TiO(2)-mediated photocatalysis process has been successfully applied to degradation of dye pollutants. Our results indicate that the TiO(2) surface is negatively charged, and the sulfan blue (SB) adsorbs onto the TiO(2) surface through the positive di-ethylamino groups while the TiO(2) surface is positively charged and the SB adsorbs onto the TiO(2) surface through the negative sulfonyl groups. In order to obtain a better understanding of the mechanistic details of this TiO(2)-assisted photodegradation of the SB dye with UV irradiation, five intermediates of the processes were separated, identified, and characterized by the HPLC-ESI-MS technique, which included a positive- and negative-ion mode. The results indicated that the N-de-ethylation process continues until the N-de-ethylated SB dye is completely formed. The probable photodegradation pathways were proposed and discussed. The reaction mechanisms of UV/TiO(2) proposed in this study should be useful for future applications of the technology to the decolorization of dyes.

Adsorption↗

Nonlinear studies of Pararosanilin dye in liquid and solid media.

Solid-state dye-doped polymers are attractive alternative to the conventional liquid dye solutions. In this paper, nonlinear properties of the dye Pararosanilin has been studied. The third-order nonlinear optical properties of Pararosanilin dye in 1-butanol and dye-doped polymer film were measured by the Z-scan technique using 532 nm diode pumped Nd:Yag laser. This material exhibits negative optical nonlinearity. The dye at 0.4 mM concentration exhibited nonlinear refractive coefficient (n(2) = -6.8 x 10(-8) and -7.11 x 10(-8) (cm(2)/W) in liquid and solid media, respectively), nonlinear absorption coefficient (beta = -7.7 x 10(-4) and -7.93 x 10(-4)cm/W in liquid and solid media, respectively) and susceptibility (chi((3))=3.38 x 10(-6) and 3.53 x 10(-5)esu in liquid and solid media, respectively). These results show that Pararosanilin dye has potential applications in nonlinear optics.

1-Butanol↗

Detection of low concentrations of malachite green and crystal violet in water.

A simple procedure for the detection of low concentrations of malachite green and crystal violet in water is presented. The dyes were preconcentrated from 1,000 ml of water samples with magnetic solid phase extraction using magnetic affinity adsorbent (magnetite with immobilized copper phthalocyanine dye). Due to the magnetic properties of the adsorbent the preconcentration process can also be performed in water samples containing suspended solids. After elution of the captured dyes, their presence in eluates was detected spectrophotometrically. Concentrations of both dyes in the range 0.5-1.0 microgl(-1) of water could be reproducibly detected. The dyes can be detected not only in potable water, but also in river ones.

Adsorption↗

Optimal number of sentinel nodes after intradermal injection isotope and blue dye.

BACKGROUND: Sentinel node (SN) biopsy for breast cancer is becoming more common owing to its lower morbidity when compared with full axillary dissection. However, the optimal method of finding and the number of SN to be dissected are still subject to conjecture. The aim of this study was to determine the optimal number of SN required to accurately stage an axilla after the i.d. injection of isotope and blue dye. METHOD: Prospective data from all patients undergoing SN biopsy from April 2000 to September 2004 were analysed. For positive SN, the order in which they became positive was then tabulated. RESULTS: During the 4 years, 113 patients who fulfilled the selection criteria had undergone SN biopsy with 216 SN harvested. Of these, 33 patients had positive SN results. If only the first SN was analysed, 87.9% of those positive biopsies would have been discovered. Two SN raised the predictive value to 97.0%. CONCLUSION: Two SN would seem to be the optimal number to harvest after i.d. injection of both isotope and blue dye.

Adult↗

[Effect of dye quality and the method of preparing a Schiff reagent on the staining intensity and DNA-fuchsin absorption spectra in performing the Feulgen reaction].

Dependence of the staining and absorption spectra of DNA-fuchsin on the dye quality and modes of preparation of the Schiff-reagent was studied. It is shown that many dyes supplied by different producers do not suit the purposes of quantitative cytochemical investigations. Before use the absorption spectra of the DNA-fuchsin must be taken using the standard object. The use of dyes possessing a multi-peak absorption curve should be avoided.

Absorption↗

[Protein assay for cellular receptors of steroids: a critical study of the technic with Coomassie-blue].

The Coomassie-Blue method for protein determination is the EORTC uniformed technique for steroid receptor assay in tumor tissues. Many quality control trials have shown unacceptable interlaboratory variation coefficients for protein determination in steroid receptor assay. The objective of this study is to check the critical parameters of the method. The authors propose some suggestions on stability of dye-protein complex, ionic strength and temperature influence to improve the accuracy of the method.

Animals↗

[The blue tests. Current data concerning its indications and interpretation (author's transl)].

The blue test is designed to reveal oedema of total or partial lymphatic origin. It consists of the subcutaneous or intradermal injection in oedematous areas of a very small amount of violet patent blue and study of its resorption. The diagnosis is immediate and although reliability is not absolute it is nevertheless highly constant. Intolerance to the dye injected is rare, though sometimes severe, and users should be aware of the possibility. The Blue test remains of great interest in angeiology.

Anaphylaxis↗

Quantitation of absolute Pneumocystis carinii nuclear DNA content. Trophic and cystic forms isolated from infected rat lungs are haploid organisms.

The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis. The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry. Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA. It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P. carinii organisms, as well as the cell wall of S. cerevisiae, bound all fluorescent dyes tested to varying degrees. Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase. Two stains that appeared most specific for DNA, DB181 and 4',6-diamidino-2-phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181. Haploid wild type Saccharomyces cerevisiae and cdc-28 temperature-sensitive mutant cells, accumulated at the restrictive temperature (37 degrees C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P. carinii. Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities. The P. carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different. The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively). Since these would include 2C (G-2 phase) and S-phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181-stained nuclei. Almost all nuclei analyzed in all life cycle stages fell within this population. The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A-T binding of the dye and taking into account the G + C content of S. cerevisiae and P. carinii. A 4C (G-2-phase diploid nuclei) population was not detected in histograms of DB181- or DAPI-stained nuclei. The P. carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organism's chromosomes resolved by electrophoretic techniques. Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life-cycle stages demonstrate that P. carini carinii isolated from infected rat lungs are haploid organisms.

Animals↗

Cutaneous distribution and localization of dyes affected by vehicles all with different lipophilicity.

The penetration of topically applied substances into the stratum corneum (SC) depends on several factors, e.g., the physicochemical properties of the vehicle used for application. The penetration of highly hydrophilic and lipophilic dyes into the skin was studied using a pure oil (o) or water (w) for the application compared to an o/w emulsion. The penetration and localization of both dyes, the lipophilic curcumin and the hydrophilic Patent blue V, was investigated in vivo using the method of tape stripping and microscopy. In addition, histological sections of biopsies, removed from porcine ear skin were studied using microscopy. Differences in the distribution and the localization of both dyes within the SC were observed. These differences depend on the physicochemical properties of both the vehicles and the dyes. The vehicle appears to affect, in particular, the pathways of penetration.

Administration, Cutaneous↗

Lymphatic mapping and focused analysis of sentinel lymph nodes upstage gastrointestinal neoplasms.

BACKGROUND: Lymph node analysis is essential for staging gastrointestinal (GI) neoplasms. Intraoperative lymphatic mapping and sentinel lymphadenectomy were originally described for melanoma but have not yet been investigated for most GI neoplasms. HYPOTHESES: (1) Lymphatic mapping and sentinel lymphadenectomy is feasible in GI neoplasms, (2) the sentinel node (SN) status reflects the regional node status, and (3) focused analysis of the SN improves staging accuracy. DESIGN: Prospective patient series. PATIENTS AND METHODS: Lymphatic mapping was performed in 65 patients with GI neoplasms by injecting 0.5 to 1 mL of isosulfan blue dye around the periphery of the neoplasm. Blue-stained SNs were analyzed by hematoxylin-eosin staining, multiple sectioning, and cytokeratin immunohistochemistry. RESULTS: Lymphatic mapping identified at least 1 SN in 62 patients (95%). Of the 36 cases with nodal metastasis, 32 (89%) had at least 1 positive SN and 15 (42%) had nodal metastasis only in the SN. In 11 cases, tumor deposits were identified by multiple sectioning (n = 2) or immunohistochemistry (n = 9) only. In 5 cases (8%), lymphatic mapping identified aberrant lymphatic drainage that altered the extent of the lymphadenectomy. CONCLUSIONS: Lymphatic mapping and sentinel lymphadenectomy are feasible in GI neoplasms and identify aberrant lymphatic drainage. The SN status accurately reflects the regional node status. Focused analysis of the SN increases the detection of micrometastases and may improve selection of patients for adjuvant treatment.

Adult↗

Orange peel as an adsorbent in the removal of acid violet 17 (acid dye) from aqueous solutions.

The effectiveness of orange peel in adsorbing Acid violet 17 from aqueous solutions has been studied as a function of agitation time, adsorbent dosage, initial dye concentration and pH. The adsorption follows both Langmuir and Freundlich isotherms. The adsorption capacity Q0 was 19.88 mg/g at initial pH 6.3. The equilibrium time was found to be 80 min for 10, 20, 30 and 40 mg/L, dye concentration respectively. A maximum removal of 87% was obtained at pH 2.0 for an adsorbent dose of 600 mg/50 ml of 10 mg/L dye concentration. Adsorption increases with increase in pH. Maximum desorption of 60% was achieved in water medium at pH 10.0.

Adsorption↗

On some characteristic properties of ATR liquid light conductors.

Electronic spectra of aqueous solutions of the malachite green dye have been measured by liquid and solid spectroscopy. Particular characteristics of the light conductor itself have been shown to have no considerable effect on the results of the experiment.

Rosaniline Dyes↗

MTS interferon assay: a simplified cellular dehydrogenase assay for interferon activity using a water-soluble tetrazolium salt.

MTS, a tetrazolium dye, is reduced by hydrogenases in living cells to a water-soluble formazan. When it is added to the medium at the end of a cytopathic effects (CPE) inhibition interferon assay, the formazan formed diffuses into the medium; the resultant optical density directly and quantitatively measures how much cellular damage has been produced by the challenge virus in the presence of different amounts of interferon. The use of MTS has considerable advantages in that after it is added, no further steps, such as washing of the cells, extraction of dye, or other manipulations, are needed.

Cell Line↗

Inactivation of myelin-associated glycoprotein enhances optic nerve regeneration.

CNS regeneration in higher vertebrates is a long sought after goal in neuroscience. The lack of regeneration is attributable in part to inhibitory factors found in myelin (Caroni and Schwab, 1988a). Myelin-associated glycoprotein (MAG) is an abundant myelin protein that inhibits neurite outgrowth in vitro (McKerracher et al., 1994; Mukhopadhyay et al., 1994), but its role in regeneration remains controversial. To address this role, we performed nerve crush on embryonic day 15 chick retina-optic nerve explants and then acutely eliminated MAG function along the nerve using chromophore-assisted laser inactivation (CALI). CALI of MAG permitted significant regrowth of retinal axons past the site of lesion containing CNS myelin in contrast to various control treatments. Electron microscopy of the site of nerve crush shows abundant regenerating axons crossing the gap. When crushed optic nerve was retrogradely labeled at the nerve stump, no labeling of retinal neurons was observed. In contrast, labeling of CALI of MAG-treated crushed optic nerve showed significant retinal labeling (89 +/- 16 cells per square millimeter), a value indistinguishable from that seen with non-crushed nerve (98 +/- 13 cells per square millimeter). These findings implicate MAG as an important component of the myelin-derived inhibition of nerve regeneration. The acute loss of MAG function can promote significant axon growth across a site of CNS nerve damage.

Animals↗

The analysis and interpretation of DNA distributions measured by flow cytometry.

A principal use of flow cytometers is for the measurement of fluorescence distributions of cells stained with DNA specific dyes. A large amount of effort has been and is being expended currently in the analysis of these distributions for the fractions of cells in the G1, S, and G2 + M phases. Several methods of analysis have been proposed and are being used; new methods continue to be introduced. Many, if not most, of these methods differ only in the mathematical function used to represent the phases of the cell cycle and represent attempts to fit exactly distributions with known phase fractions or unusual shapes. In this paper we show that these refinements probably are not necessary because of cell staining and sampling variability. This hypothesis was tested by measuring fluorescence distributions for Chinese hamster ovary and KHT mouse sarcoma cells stained with Hoechst-33258, chromomycin A3, propidium iodide, and acriflavine. Our results show that: a) single measurements can result in phase fraction estimates that are in error by as much as 40% for G2 + M phase and 15-20% for G1 and S phases; b) different dyes can yield phase fraction estimates that differ by as much as 40% due to differences in DNA specificity; c) the shapes of fluorescence distributions and their interpretation are very dependent on the dye being used and on its binding mechanism.

Acriflavine↗