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PubMed1983

[Changes in the parameters of equilibrium binding of acidic dyes by contractile proteins during their functional activity and heat denaturation].

A study was made of the equilibrium distribution of acidic dyes (Heliogen blue, Bromthymol blue, Bromphenol blue, Phenol red) between actomyosin threads (intact, contracted by ATP, or denaturated by heating) and the medium. The limits of dye sorption (A infinity) were shown to rise with the increase in their hydrophobia. The heat denaturation is accompanied with similar changes in all the dyes examined: i.e. with the increase in the limits of dye sorption and constant dissociation, and with the decrease in dye affinity to protein. The functional activity of contractile proteins acted upon with ATP is accompanied with different changes. For Heliogen blue these are like those during denaturation, whereas for Phenol red these are quite opposite: the limits of dye sorption and constant dissociation diminish, and the affinity to protein rises. Thus, during the heat denaturation the number of polar and non-polar groups accessible to dyes increases, whereas during functional activity only the number of non-polar bonds increases, and the quantity of polar bonds is seen reduced.

Absorption↗
PubMed1988

Quantification of albumin in cerebrospinal fluid.

A standard spectrophotometric method for the quantitation of urinary albumin using bromphenol blue is evaluated to determine whether this method could be used to quantify cerebrospinal fluid albumin. In the modified procedure, 200 microliters of sample is added to 3 ml of bromphenol blue solution and the absorbance is read spectrophotometrically at 610 nm. Using standards and controls, the results were compared with known values and found to be both precise and accurate. The bromphenol blue method was compared with an immunoturbidometric method and found to be more precise and accurate, easier to perform, and cost effective. When compared to other dye-binding methods the bromphenol blue method is unique in its extremely low linear range and limit of detection. A minor disadvantage was the increased sample size necessary to obtain the increased precision.

Albumins↗
PubMed1978

[Essay of proteins after aldehyde treatment in biological objects (author's transl)].

Following fixation with formalin or glutaraldehyde, the protein content of bovine serum albumin, lysozyme, non-disintegrated yeast cells and liver nuclei of mice is determined by means of a brom-phenol blue staining procedure and the widely used Lowry-technique. The bromphenol blue technique permits determinations of soluble as well as particulate proteins or protein mixtures fixed with up to 6% formalin or 2% glutaraldehyde. Recovery rates differ no more than 20% as related to unfixed controls. Using the bromphenol blue method it is not necessary to separate aldehyde or any interfering material by additional steps prior to determination. Factors for correcting protein content of biological material after aldehyde treatment are available. In this way, comparative biochemical as well as cyto- and histochemical investigations of enzymatic activities after aldehyde fixation are possible. Some advantages of the bromphenol blue technique with special reference to the analysis of particulate and/or aldehyde-fixed specimens are discussed.

Animals↗
PubMed2003

Delivery of several forms of DNA, DNA-RNA hybrids, and dyes across human sclera by electrical fields.

PURPOSE: Iontophoresis has been used for drug delivery across the cornea for many years. We sought to test whether small charged dyes and DNA can be transferred across human sclera by an electric field. METHODS: Full-thickness human scleral fragments were embedded vertically in an agarose gel and positioned to completely span individual gel lanes. The scleral fragments were located approximately 1 cm downstream from the gel wells. DNA or dyes were loaded into the wells and electrophoresis was carried out at about 3.3 V/cm for approximately 2 h per run. Movement of DNA and dyes through the agarose and sclera was measured with either digital time-lapse photography or through DNA extraction and purification from the gel. SYBR green stain was used as a sensitive method to detect DNA. RESULTS: Digital time-lapse photography of agarose gel electrophoresis revealed that two dyes, xylene cyanol and bromphenol blue, passed through the sclera in the presence of an electric field. Xylene cyanol was driven through the sclera virtually unimpeded except for some spreading of the dye. Bromphenol blue was slowed markedly by the sclera, but it too eventually passed through the tissue. Small DNAs, including a single stranded 51-mer and a double hairpin 68-mer oligonucleotide, passed through the sclera as detected by SYBR green staining. Linear double stranded DNAs ranging from 50 bp to 12,000 bp passed through the sclera. The larger the DNA, the slower the rate of passage through the sclera, and the greater the band spreading. pEGFP-1 (a 3 kb plasmid) passed through the sclera but was accompanied by a great amount of band spreading. Following completion of the initial electrophoresis run, the plasmid DNA was extracted from the smeared bands in the agarose distal to the sclera and re-run on a second gel without sclera. The initially smeared plasmid bands resolved into 2 distinct bands after extraction and purification and matched well with control plasmid bands. CONCLUSIONS: Charged molecules such as xylene cyanol, bromphenol blue, and DNAs ranging from 51 bp oligonucleotides to a 3 kb plasmid can be driven across human sclera by an electric field and directly detected. Passage of plasmids was efficient, but the plasmid bands were diffuse after transit. This technique offers promise as a noninvasive DNA delivery tool, where gene therapy can be accomplished by small RNA or DNA synthetic oligonucleotides, larger double stranded fragments, or even plasmids.

Adolescent↗
PubMed1986

New artificial electron donors for in vitro assay of nitrate reductase isolated from cultured tobacco cells and other organisms.

The capacity of bromphenol blue and its analogs to act as electron donors for measurement of in vitro nitrate reductase activity from tobacco cells (Nicotiana tabacum var Techné SP 25 strain) was determined. Competitive inhibition was demonstrated to occur between NADH, the natural electron donor, and bromphenol blue, the artificial electron donor, suggesting that both donors bind to a similar active site on the enzyme. NADH-dependent or bromphenol blue-dependent nitrate reductase activity was carried out by a similar molecular weight protein exhibiting similar antigenic sites. Following ammonium sulfate precipitation, sucrose density gradient and two chromatographic steps, nitrate reductase activity from tobacco cells was purified near homogeneity using bromphenol blue as an electron donor in the absence of measurable NADH-dependent activity. The enzyme is composed of two identical subunits of 83 kilodaltons < Momega < 94 kilodaltons.

Journal Article↗
PubMed1978

[Comparative evaluation of modern intraoperative diagnostic methods in malignant gliomas of the brain].

The following methods were employed: Intravital staining with bromphenol blue (70 patients), radiometry with P 32 (127 patients), echo encephalography (12 patients,) puncture biopsy (18 patients), rheometry (96 patients). Bromphenol blue is injected into the a. cerebri media or the supraclinoidal a. carotis interna and stains the periphery of malignant tumours and the entire tissue of benign tumours. Radiometry also serves for the intraoperative finding of tumour remainders which must be located at least 20 to 22 mm below the cortex of the brain. Tumour biopsy was carried out with a sharp cannula to avoid deformation of the tissue cylinder. Rheometry proved to be very useful when avoiding the mixing with blood, liquor, and cyst fluid at the measuring tip.

Biopsy, Needle↗
PubMed1990

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia.

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.

Journal Article↗
PubMed2002

Acid diffusion through rat duodenal mucosal cap.

Acid induced duodenal injury occurs immediately following exposure to strong acid but does not worsen despite continuous perfusion with the acid. A thick gelatinous cap is found overlying the duodenum when examined histologically. The purpose of this study was to quantitate the resistance to acid diffusion by native duodenal mucus and the cap material. Rat duodenum was perfused with acid. The mucus from control and acid-perfused animals was harvested and the diffusion coefficient measured. Mucus from the duodenums of anesthetized rats was mixed with bromphenol blue and placed into capillary tubes. The tubes were submersed in 0.15 N HCI at 37 degrees C. The color change from blue to yellow was measured with time as the HCl migrated through the tube. From the distance and time data, the diffusion coefficient could be calculated. Controls of bromphenol blue in water were studied. The diffusion coefficient for HCl in water was 30.1+/-0.9 x 10(-6) cm2/sec, nearly identical to the standard value reported in the literature. For the duodenal mucus adherent to the epithelial surface, it was 1.1+/-0.1 x 10(-6) cm2/sec. Following perfusion with 0.15 N HCl, a 279.6+/-44-microm-thick mucus cap formed over the duodenum that had a diffusion coefficient of 1.8+/-.3 x 10(-8) cm2/sec. In response to injury, the duodenum protects against continued damage by producing a mucoid cap that it highly resistant to acid diffusion.

Acids↗
PubMed1995

Modeling of the gastric gel mucus layer: application to the measured pH gradient.

The gastric gel mucus layer plays an important role in the defense against acid-induced injury. Previous investigations have modeled the interaction between the mucus, bicarbonate, acid moving towards the epithelial surface, and water carrying ions away from the epithelium. We measured the diffusion coefficient of protons through gastric mucus and applied this parameter to the model. Using the measured diffusion coefficient, we applied the model to previously published gastric mucus gel pH gradients to assess the relative contribution of bicarbonate or water fluxes in maintaining the gradient in the face of luminal acid. Mucus was harvested from the stomachs of anesthetized rats, mixed with bromphenol blue, and placed in capillary tubes. The tubes were submerged in 0.15 N HCl at 37 degrees C. The color change from blue to yellow was measured with time as the HCl migrated through the tube. From the distance and time data, the diffusion coefficient could be calculated. Controls of bromphenol blue in water were studied. The diffusion coefficient for water was 30.2 +/- 0.8 x 10(-6) cm2 s-1, almost identical to the standard value reported in the literature. The HCl diffusion coefficient through saline was 67.8 +/- 0.7 x 10(-6) cm2 s-1. For gastric mucus it was 5.4 +/- 1.3 x 10(-6) cm2 s-1. Application of this diffusion coefficient to the model and subsequently to observed pH gradients suggested that the most potent defense mechanism against concentrations of luminal acid is the bulk movement of water away from the epithelial surface through the mucus gel layer.

Animals↗
PubMed1972

Observations on a new family showing bisalbuminaemia of the slow type.

A new family with an electrophoretically more slowly moving albumin variant is described. The pattern of variance was studied using three different electrophoretic techniques, and compared with certain other known fast and slow variants. Features of penicillin and bromphenol blue binding by normal and variant albumins were also studied. Both normal human albumin and the albumin variant treated with penicillin broadened and moved faster. Native albumin A, prestained with bromphenol blue, binds approximately twice as much of the dye as the slow-moving variant. On the other hand, staining of denatured proteins on paper showed that both bands take up equal amounts of dye.

Adult↗
PubMed1979

[Coelomic gonadal epithelia during the period of human embryonic morphologic sex differentiation].

The course of divergent differentiation of coelomic epithelium in the indifferent gonad has been traced up to the stage when morphological sex signs are distinctly seen. Eleven human embryos fixed in Carnoy and Bouin's fluid have been studied from 6 to 10 weeks of gestation. Paraffin sections were stained with hematoxylin, ferric hematoxylin after Heidenhain. Basal membrane fibres were revealed by silver impregnation after Karupu. PAS-reaction after McManus and additional staining with hematoxylin and light green were applied, as well as the reaction for total proteins with bromphenol blue after Miguel--Calvo. Sex differentiation of coelomic epithelium in the gonad developing according to the male type begins a little later than in the ovary. In the epithelium of the forming testis a great amount of mitotic figures is observed that might be connected with the presence of Y-chromosome stimulating cells for extra mitotic cycles. The increasing presence of Y-chromosome stimulating cells for extra mitotic cycles. The increasing number of mitotic figures in the epithelium should be considered as a sign demonstrating that differentiation according to the male type has started. Superficial epithelium of the embryonic ovary has a peculiar false pseudostratified structure. This secures cellular reserve for the ovarian cortex formation from the external epithelium. The apical surface of the cells in the external epithelium has a border which is evidently formed by microvilli and revealed by PAS-reaction and bromphenol blue. Ovarian follicular cells and Sertoli's cells (sustentocytes) in testis have the common origin in the human fetus--from coelomic epithelium of the gonad germ.

Epithelial Cells↗
PubMed2003

[Examination of drugs and poisons in institutions not equipped with an analyzer of them and poisons--focusing on the simple qualitative test of surfactants].

We examined the simple qualitative test of surfactants, and discussed its possible usefulness in clinical emergency medicine and the analysis system in institutions not equipped with an analyzer of drugs and poisons. The procedures for the cobalt thiocyanate ammonium test, methylene blue chloroform test, and bromphenol blue test are easy, if reagents are prepared beforehand. If concomitantly used, these tests enable analysis of ion properties of surfactants. These tests thus seem useful for the screening of drugs and poisons in emergency medicine. We think that it is possible for institutions not equipped with an analyzer of drugs and poisons to establish a system to support clinical practice by giving priority to the establishment of drugs and poisons that frequently cause intoxication as well as of items for the simple qualitative test that are specified in "proposals for the guidelines for the analysis of drugs and poisons" advanced by the Japanese Society for Clinical Toxicology.

Chemistry Techniques, Analytical↗
PubMed1990

Resolution and kinetic characterization of glutathione S-transferases from human jejunal mucosa.

Cytosolic glutathione S-transferases were purified from human jejunal mucosa by affinity chromatography on S-hexylglutathione-Sepharose 4B. Chromatofocusing in the pH range 7-4 yielded peaks with apparent pI's of 7.2 (peak 1), 5.2 (peak 2), and 4.4 (peak 3). Each enzymatic fraction was shown to have a homodimeric structure, with subunit mass of 24.9 +/- 0.5 (P1), 27.9 +/- 0.9 (P2), and 23.4 +/- 0.8 (P3) kDa, as determined by SDS-PAGE. The substrate specificity of each peak was tested using discriminating substrates for basic, near-neutral, and acidic GSTs. With cumene hydroperoxide, the diagnostic substrate for the alpha (basic) class of GSTs, P1 showed 8- to 36-fold higher activity than P2 and P3. Ethacrynic acid, the selective substrate for the acidic enzyme (pi), gave highest activity with P3. The inhibitory potentials of sulfobromophthalein, cibacron blue, tributyltin acetate, triphenyltin chloride, and bromphenol blue were also tested. A qualitative resemblance between P1 and alpha, and P3 and pi GSTs was noted. The substrate specificity and inhibiton parameters of P2 corresponded most closely to those of mu-GST. The relative abundances of P1, P2, and P3 (based on CDNB-conjugating activity) were 35, 5, and 60%, respectively.

Adult↗