Effects of two cationic triphenylmethane dyes on the healing of skin incisions. A tensiometric and histologic study in the rat.
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A mathematical model for the description of the Coomassie brilliant blue protein assay.
The Coomassie brilliant blue dye-binding method for protein assay has become important relatively recently. The basis of the assay method is the binding of dye to protein, with production of a dye-protein complex which absorbs light intensely at 620 nm, but the mechanism of the binding process is not well understood. In this paper, two mathematical models for the binding process are developed, one involving the binding of both protonated (green) and deprotonated (blue) forms of the dye. The second model allows only binding of the blue species to proteins. These models are tested for their ability to estimate number of dye-binding sites (n) and binding constants (Kd) from protein assay data. The models are also tested for their ability to reproduce the experimental assay curve using either known values or reasonable estimates of the equation parameters. The models are shown to be approximately equal in ability to reproduce experimental data related to the protein assay, which somewhat favors the simpler of the two models. In this paper, a method for estimating n and Kd from standard curve-fitting procedures is established. Hitherto, binding constants were only available from assay data taken under conditions of very large molar protein/dye ratios. The possibility of protonated forms of the dye binding to proteins was not ruled out by this study, but for many purposes the use of the simple dye-binding model, in which only the deprotonated dye species binds, is sufficient.
Indicators and Reagents↗
A Coomassie blue-binding assay for the microquantitation of immobilized proteins.
A sensitive assay procedure for the determination of microgram quantities of immobilized proteins is described. The procedure is based on the property of Coomassie blue G-250 to bind strongly yet reversibly to proteins. The assay involves incubation of the immobilized protein with a solution containing 0.1% Coomassie blue, 10% acetic acid, and 25% isopropyl alcohol in distilled water at room temperature followed by washing off of the unbound dye. The protein-bound dye is eluted with methanolic NaOH, acidified, and the absorbance is measured at 605 nm. The assay is highly reproducible and several proteins immobilized on various matrices could be conveniently assayed. Protein values determined by the dye-binding assay showed good agreement with those obtained by other procedures.
Amines↗
Inhibition of human glutathione S-transferases by basic triphenylmethane dyes.
Human glutathione S-transferases (GSTs) of the Alpha-, Mu- and Pi- classes were expressed in E. coli and isolated by affinity chromatography. They were tested for their susceptibility to inhibition by basic triphenylmethane dyes. hGSTA 1-1 was inhibited by Malachite Green with a Ki value of the order of 10 microM. The inhibitory species appeared to be the dye-GSH adduct. This isoenzyme was not inhibited by either Crystal Violet or Ethyl Violet at concentrations up to 50 microM. hGSTM 2-2 was weakly inhibited by all three dyes tested with Ki values being in the range 40-80 microM. For all dyes the inhibition was best characterised as non-competitive. hGSTP 1-1 was not inhibited by Crystal Violet or by Ethyl Violet but was strongly inhibited by Malachite Green (Ki = 0.3 microM). The mode of inhibition appeared to be non-competitive but it seems probable that the mechanism is complex. There is at present no evidence to show clearly whether the dominant inhibitory species is the free dye or the adduct.
Coloring Agents↗
Basic fuchsins and the Schiff-aldehyde reaction. I. Spectral absorption characteristics in solution.
Spectral absorption profiles at the UV and visible ranges were detected spectrophotometrically for solutions of various basic fuchsins, for the SCHIFF reagent prepared from these stains and for the end product from SCHIFF-formaldehyde reaction. The aim was to detect whether absorption spectra could vary as a function of the basic fuchsin origin up to the point of being suspected of affecting potentially the spectrum of FEULGEN-stained chromatin. The results indicate a constancy in the spectral absorption characteristics for the absorption profiles at the visible range, when one considers SCHIFF reagents prepared from various basic fuchsins added with aldehyde. However, it is possible that size, stereoarrangement and electron distribution of the aldehyde- (eventually, ketone-) bearing molecule involved in SCHIFF-carbonyl reactions would function as auxochroms, the spectral characteristics of which could be affected by dye content and contaminants of the basic fuchsin.
Chemical Phenomena↗
Modification of malachite green by Fomes sclerodermeus and reduction of toxicity to Phanerochaete chrysosporium.
Malachite green (MG) is a triphenylmethane dye used as a fungicide but also possesses a high toxicity to mammalian cells. The toxicity of MG to Fomes sclerodermeus and Phanerochaete chrysosporium was assessed. P. chrysosporium was highly sensitive to the dye and it was unable to grow on solid media containing 64 microM of MG, lower concentrations caused a delay in growth. The radial growth of F. sclerodermeus was not affected at this concentration and up to 128 microM. In liquid media both fungi were more sensitive. F. sclerodermeus not only was able to grow in the presence of high concentrations of MG, but also it was able to decolorize and detoxify the dye. MG treated with supernatants containing high laccase activity in the presence or absence of 1-hydroxybenzotriazole (1-HBT) gave a colorless product (DMG) that was not toxic to P. chrysosporium and other white rot fungi tested. On the basis of the data of maximal absorbance, it is probable that the mechanism involved in the modification of the dye was different if 1-HBT was added to the reaction.
Culture Media↗
Anaphylaxis to isosulfan blue and cross-reactivity to patent blue V: case report and review of the nomenclature of vital blue dyes.
BACKGROUND: Blue dyes used for lymphatic mapping in sentinel lymph node biopsy cause intraoperative anaphylactic reactions in up to 2.7% of patients. With increasing implementation of this technique, the incidence of anaphylaxis to these dyes can be expected to increase. In the literature, the chemically often unrelated and inconsistently designated dyes have been confused, adding to other inconsistencies in the nomenclature. OBJECTIVE: To demonstrate the nomenclature, chemical and physiologic differences, and allergenicity of the various blue dyes used in a medical context. METHODS: We describe a patient with an intraoperative grade IV anaphylactic reaction to isosulfan blue. Immediate-type hypersensitivity was proved by positive skin test reactions and CD63 expression to isosulfan blue and cross-reactivity to patent blue V. RESULTS: A review of the literature clarified the exact nomenclature of the blue dyes and the possible pitfalls of confusing nomenclature in the context of structurally closely related dyes with different allergenic properties. For the detection of type I hypersensitivity, intracutaneous tests are valuable tools. An IgE-mediated mechanism has been shown recently. In most cases, sensitization exists without known previous exposure in a medical context. This may be due to the widespread use of such dyes in objects of everyday life. Preoperative antiallergic medication use does not prevent anaphylactic reactions but apparently reduces their severity. CONCLUSION: For better comparison and precision, the Chemical Abstracts Service number of the respective dye should always be given.
Aged↗
Use of aqueous solutions of two basic dyes for the demonstration of DNA.
This paper presents informations as to the ability of aqueous solutions of two basic dyes, such as Dahlia and Victoria blue, belonging to aminotriarylmethane group for the staining of DNA-aldehyde molecules as well as DNA-phosphate groups. It has been found that sections of rat tissues stained with aqueous solutions of these dyes after acid hydrolysis followed by drying between folds of filter paper and treatment in n-butanol for a minute and then by a very brief treatment in a mixture consisting of equal parts of n-butanol and absolute ethanol reveal well-stained nuclei. Tissue sections after acid hydrolysis when stained with aqueous solutions of these dyes and then treated with SO2 water do not reveal any colouration of the nuclei. Since both the dyes are without any primary amino group in their molecules, it has been concluded that the imino group of Dahlia and the tertiary amino group of Victoria blue with cold concentrated phosphoric acid and then stained with any of these dyes also exhibit well-stained nuclei. The absorption spectra of nuclei stained with these dyes for DNA-aldehyde molecules as well as DNA-phosphate groups reveal positions of the peaks of maximum absorption at the same wavelength, which, however, are different in the case of nuclei stained with the two dyes. The implications of these findings have been discussed.
Aldehydes↗
Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P.
The effect of extracellular ATP on the intracellular calcium concentration ([Ca2+]i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1-30 microM concentration range and a further increase at concentrations higher than 100 microM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATP tau S blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca2+]i in response to 100 microM carbachol. By itself, substance P (100 pM-1 microM) increased the [Ca2+]i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 microM and substance P (100 pM-1 microM) increased the release of inositol trisphosphate (IP3) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 microM carbachol (-50%) and by 100 nM substance P (-60% at 1 nM substance P and -40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 microM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P2z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca2+]i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca2+]i.
Adenosine Triphosphate↗
Neurons projecting to the retrocerebral complex of the adult blow fly, Protophormia terraenovae.
Anatomical study of neurons projecting to the retrocerebral complex of the adult blow fly, Protophormia terraenovae, was done by NiCl2 filling and immunocytochemistry. Retrograde filling through the cardiac-recurrent nerve labeled three groups of neurons in the brain/subesophageal ganglion: (1) paramedial clusters of the pars intercerebralis, (2) neurons in each pars lateralis, and (3) neurons in the subesophageal ganglion. The pars intercerebralis neurons send prominent axons into the median bundle and exit from the brain via the contralateral nervus corporis cardiaci. Based on the projection pattern, two types of the pars lateralis neurons can be distinguished: the most lateral pairs of neurons contralaterally extend through the posterior lateral tract and the remainder ipsilaterally extend through the posterior lateral tract. The neurons in the subesophageal ganglion run through the contralateral nervus corporis cardiaci. The dendritic arborization of the pars intercerebralis and pars lateralis neurons is restricted to the superior protocerebral neuropil and to the anterior neuropil of the subesophageal ganglion where the neurons in the subesophageal ganglion also project. Retrograde filling from the corpus allatum indicated that the pars lateralis neurons and a few pars intercerebralis neurons project to the corpus allatum, but that the neurons in the subesophageal ganglion do not. Orthograde filling from the pars intercerebralis and staining by paraldehyde-thionin/paraldehyde-fuchsin indicated that the pars intercerebralis neurons project primarily to the corpus cardiacum/hypocerebral ganglion complex. Immunostaining with a polyclonal antiserum against diapause hormone, a member of the FXPRLamide family, suggests that some of the subesophageal ganglion neurons contain FXPRLamide-like peptides.
Age Factors↗
Induction of apoptosis in vivo in the rabbit brain with focused ultrasound and Optison.
Histologic effects of focused ultrasound (FUS) exposures combined with an ultrasound contrast agent (Optison) were investigated to examine whether the lesions were dominated by apoptosis or necrosis. The rabbit brains (n = 17) were sonicated (1.5 MHz, peak rarefactional pressure amplitude: 1.4 to 8.8 MPa) after Optison was injected intravenously (IV). MRI and light microscopy were used to examine tissue effects. To detect apoptosis, TUNEL staining based on labeling of DNA strand breaks was used. The average number of apoptotic and necrotic cells in 300 x 220 microm microscopic fields were counted in 18 representative lesions. Lesions in the rabbit brains were created at lowered acoustic power levels when FUS was combined with Optison. In histology, the lesions exhibited red blood cell extravasations and destruction of blood vessels. At 4 h after sonication, the lesions lost many cells, and the remaining cells exhibited both necrotic and apoptotic features. Overall, apoptosis dominated; there were, on average, 32.3 +/- 13.2 apoptotic cells per microscopic field compared with only 5.1 +/- 3.4 necrotic cells per field. In conclusion, FUS combined with Optison could produce lesions that are dominated by apoptosis, presumably induced primarily via ischemia after cavitation-produced damage to the brain vasculature.
Albumins↗
Regulation by purinergic agonists of zinc uptake by rat submandibular glands.
The zinc uptake in rat submandibular cells was measured using fura2 as a fluorescent probe. Basal zinc uptake was observed in a 100 microM - 1 mM concentration range. Carbachol and isoproterenol had no effect but ATP4- dose-dependently increased the basal zinc uptake (half-maximal concentration: 250 microM). The purinergic agonist shifted the concentration curve for zinc to the left by one order of magnitude. The response to ATP was not reproduced by adenosine or ADP and was blocked by Coomassie blue. Calcium, nickel or lanthanum were inhibitors of zinc uptake, while the substitution of extracellular sodium by potassium or lithium increased the basal zinc uptake. We conclude that in submandibular cells zinc can permeate through the non-specific cation channel coupled to ATP-sensitive purinergic receptors.
Adenosine↗
Benzidine stain for the histochemical detection of hemoglobin in splinter hemorrhage (subungual hematoma) and black heel.
Minor nail trauma may cause bluish discoloration of the nail, while tangential skin trauma on the heel can result in a so-called black heel. To rule out melanoma in such clinical situations, a biopsy is needed to reveal homogeneous eosinophilic masses deposited under the nail plate or within it (transepidermal elimination). Most dermatopathologists attempt to demonstrate the presence of hemoglobin in these eosinophilic masses with Prussian blue stain, which typically remains negative. In our experience, these traumatically induced blood deposits are always situated in avascular spaces, devoid of degrading phagocytes. Consequently, a histochemical stain for these deposits should be directed specifically toward hemoglobin, not hemosiderin. In the dermatopathologic literature, the various techniques to detect hemoglobin deposits in tissue sections are not well-known. We would like to emphasize benzidine stain, a highly selective and efficient method to demonstrate the presence of hemoglobin deposits in histologic sections. To date, benzidine stain has not been utilized to characterize splinter hemorrhage (subungual hematoma). Of concern, the use of benzidine in histopathology laboratories is restricted because this agent is a known carcinogen, while the non-mutagenic derivative, 3,3',5,5'-tetramethylbenzidine, does not stain histologic sections. Patent blue V, a completely different and less specific agent, stains hemoglobin an intense blue-green.
Benzidines↗
Staining of endotracheal tube with isosulfan blue dye after sentinel node mapping: a case report.
Sentinel node mapping is successfully performed for the detection of cancer and nodal metastasis. There is a reported complication rate of 1.5%, most being mild allergic reactions, with a few rare reported cases of anaphylaxis. Isosulfan blue has been found to routinely create a modest artifactual desaturation via pulse oximetry, as well as a transient skin discoloration that may last for several hours to weeks. It causes discoloration of urine and has been detected in other body secretions, none of which have been found to be of clinical significance. This is a case report involving a 61-year-old woman who underwent sentinel node mapping and axillary dissection for breast adenocarcinoma. On postoperative extubation, the endotracheal tube was noted to be stained dark blue with isosulfan dye. This unusual complication is unreported to date and is likely the result of systemic absorption of the dye.
Absorption↗
Oxidative degradation of malachite green by Fenton generated hydroxyl radicals in aqueous acidic media.
Fenton-generated hydroxyl radicals removes the color of the malachite green, a basic dye with triphenylmethane group, almost (approximately 98%) completely in weakly acidic aqueous media possibly through oxidative degradation pathways as evidenced from a remarkable lowering in the COD value of the product mixture in comparison to the title dye under identical conditions and almost full quenching of the reaction in presence of hydroxyl radical scavengers. The dye can most effectively be degraded at dye:Fe2+:H2O2 molar ratio of 1:3.3:81.7 for 1.08 x 10(-5) mol dm(-3) dye at pH 2.5-2.8 and at 299K. The rate law of the dye degradation process appears to be: -a[dye]/dt = k[dye] [Fe2+]0.79 [H2O2]0.12, where k = (33 +/- 5) (dm3 mol(-1))0.91 s(-1) at 299K. Salts like NaCl or NaBr retard the degradation rate markedly whereas SO4(2-) or ClO4- are rather innocent. In presence of Cl-, the radical reaction: ClOH*- + Fe2+ --> Cl- + HO- + Fe3+ may account for the gross lowering of degradation rate. The results may be helpful for designing the treatment plants of wastewater containing dyes with triphenylmethane group.
Coloring Agents↗
An improvement of the Coomassie Blue dye binding method allowing an equal sensitivity to various proteins: application to cerebrospinal fluid.
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Albumins↗
The use of vital dyes to predict survival after experimental coronary artery ligation.
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Angiocardiography↗
Interference of patent blue V dye with pulse oximetry and co-oximetry.
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Adolescent↗