Interaction between dyes and polyelectrolytes, 9. Effect of polyanions on alkaline fading reaction of methylene blue

The effect of polyanions poly(potassium vinyl sulfate) (PVS), poly(sodium acrylate) (PANa), and poly(potassium p-styrenesulfonate) (PSS), on the alkaline fading reaction of methylene blue ( 1 ) was investigated. The fading rate of 1 was extremely retarded on addition of polyanions. The binding type of 1 to polyanions was investigated spectrophotometrically by following the changes in the molar absorption coefficient (ε) at 664nm band. The relationship between the binding type and the fading rate of 1 in aqueous alkaline solution of polyanions is discussed.     

Interaction between dyes and polyelectrolytes, 5. Effect of organic and inorganic salts on metachromasy of methylene blue

The effect of alkali metal chlorides and 1-substituted 3-carbamoylpyridinium bromides ( 2a – d ) on the metachromatic behavior of methylene blue ( 1 ) induced by poly(potassium styrenesulfonate) (PSS) and poly(potassium vinyl sulfate) (PVS) was investigated spectrophotometrically. The size of hydrated ion and the hydrophobicity of substituent were significant factors for inorganic and organic salts, respectively. Effect of these salts on the metachromasy was characteristic of each polyanion used. A peculiar phenomenon that the metachromasy of the PSS- 1 system was enhanced on addition of small amounts of 1-dodecyl-3-carbamoylpyridinium bromide ( 2d ) was observed.     

Interaction between dyes and polyelectrolytes, 8. Effect of polyanions on the mixed dimer formation of cationic dyes

The effect of polyanions on the formation of mixed dimers of methylene blue ( 1 ) and trypaflavine ( 2 ), methylene blue ( 1 ) and phenosafranine ( 3 ), and methylene blue ( 1 ) and pyronine G ( 4 ) was investigated spectrophotometrically. The following polyanions were used: poly(potassium styrenesulfonate) (PSS), poly(potassium vinyl sulfate) (PVS), and poly(sodium acrylate) (PAA). On addition of polyanions, the formation of mixed dimers was enhanced largely. Thermodynamic parameters inferred that the enhancement of the formation of mixed dimers in the presence of polyanions resulted from an entropic factor.     

Interaction between dyes and polyelectrolytes, 7. Metachromatic behavior of methylene blue induced by poly(sodium acrylate) and its homologues

The structural effect of polyanions on the metachromatic behavior of methylene blue ( 1 ) was investigated spectrophotometrically. The following polyanions were used: poly(sodium acrylate) (PAA), conventional poly(sodium methacrylate) (a-PMA), isotactic poly(sodium methacrylate) (i-PMA), syndiotactic poly(sodium methacrylate) (s-PMA), and the copolymer poly(sodium maleate-covinyl alcohol) (1:1) (PMVA). The metachromatic behavior was followed by the changes in the molar absorption coefficient (ε) at 664 nm of methylene blue ( 1 ) and in the wave length (λ_m) of the metachromatic band. Important factors for metachromasy were found to be the flexibility and charge density of the polymer. Differences between ? OSO and ? COO^? as binding sites are discussed.     

Interaction between dyes and polyelectrolytes, 14. Triplet energy transfer in a cationic dye-polyanion system

Polymers with different fractions of azobenzene residues were prepared by radical copolymerization of methacrylic acid with 4′-phenylazomethacrylanilid. The triplet excitation energy transfer from methylene blue ( 1 ) to the azobenzene residues in the polymers was investigated by using the 1 -photosensitized cis → trans isomerization of the azobenzene residue. The efficiency of the triplet excitation energy transfer from 1 to 4-(4′-phenylazoanilinocarbonyl)butanoic acid ( 4 ) as a monomer model compound. The number of azobenzene residues per polymer molecule and the molecular weight of the polymer were important factors for the efficient triplet excitation energy transfer. On addition of NaCl the energy transfer efficiency was lowered, suggesting that the binding of 1 to the polymer by electrostatic forces plays an important role. Binding of 1 to the polymers was investigated spectrophotometrically. Correlation with the binding of 1 to the polymers and the efficiency of triplet excitation energy transfer between 1 and azobenzene residues in the polymers is discussed.     

The role of co-operativity in determining the binding behaviour of hetero-charged polyanions with metachromatic dyes

The binding of acridine orange to carboxyl and sulfate containing polyanions has been investigated by fluorescence spectroscopy. Particular attention has been given to solutions containing both carboxyl and sulfate groups in two different situations, first where both types of site are present attached to the same polymer backbone in a regular repeating structure (heparin), and secondly where individual polymer chains carry exclusively one type of site (hyaluronic acid/K-carrageenan mixtures). Greatly differing behaviour of the binding profiles as the concentrations of components were reduced have been explained in terms of the strong co-operativity of the dye-binding process. The carboxyl and sulfate sites of heparin appear to be equivalent in binding strength, while those of the polycarboxylate polysulfate mixture are no longer equivalent, binding profiles being dominated by the strong binding sulfate group.     

The effect of toluidine blue and methylene blue in immunochemical reactions in vitro

Our observation that the effect of a patient's autoantibody with a potent antithrombin activity could be inhibited by toluidine blue and methylene blue led us to investigate the effect of these dyes in several immunochemical reactions in vitro. Both dyes reduced the titer or prevented the detection of IgG-specific single- and double-stranded DNA-binding antibodies and rheumatoid factors but did not reduce the titer of antinuclear factor and had no effect on detection of microsomal and thyroglobulin antibodies. These dyes did not aggregate or precipitate the immunoglobulins and their inhibitory effect in serum could be removed by dialysis of serum. The possible mechanism of action of these dyes in immunochemical reactions is discussed.     

Aqueous gel permeation chromatography of electrolytes and polyelectrolytes, 2. Donnan salt exclusion effect

The salt exclusion of electrolytes and polyelectrolytes in aqueous GPC was investigated theoretically and experimentally, in order to elucidate the feasibility of GPC as a technique for measuring the Donnan salt exclusion parameter, Γ¯, of polyelectrolytes. Three simple salts different in the valency of the anion, i.e., sodium chloride, sodium sulfate and naphthalene-1,3,6-trisulfonic acid trisodium salt, were eluted with aqueous NaNO₃ solution on a Sephadex G-10 gel column. The experimental results were analyzed in terms of the Donnan equilibrium established on the gel, and the Γ¯ value of these simple salts was evaluated. It was found that the Γ¯ value from GPC was in good agreement with the theoretical value of 1/2 for an ideal case, showing that the colligative properties of electrolytes and polyelectrolytes such as Γ¯ can be accurately investigated by means of GPC. GPC experiments on the Donnan salt exclusion were further carried out for the heparin-NaCl system by using a Sephadex G-25 gel column. From the experimental data, the Γ¯ value of heparin was estimated.     

Photodynamic inactivation of pseudorabies virus with methylene blue dye, light, and electricity.

Pseudorabies virus was photoinactivated with a combination of methylene blue dye, light, and electricity. Viral suspensions were mixed with variable amounts of methylene blue dye and then were placed in a current source apparatus. Total inactivation of pseudorabies virus (1.7 X 10(6) 50% tissue culture infective doses per ml) was achieved with constant mixing, a methylene blue dye concentration of 10(-4) M, and an electrical current of 12 microA for 12 min.     

Comparison of acridine orange, methylene blue, and Gram stains for blood cultures.

Direct microscopic screening of blood cultures by Gram stain or methylene blue stain is time consuming and frequently insensitive. Therefore, we evaluated a fluorescent-staining procedure that uses acridine orange (AO) at pH 3.5 and compared it with the methylene blue and Gram stain procedures. All smears were prepared within 24 h of receiving the culture, fixed with methanol, and examined without the results of the companion smears being known. AO-stained smears were examined with incident-light fluorescence at 600 x magnification and confirmed at 1,500x magnification. All bottles macroscopically positive within 24 h were excluded from the study. Of 2,946 cultures entered into the study, 204 (6.9%) were positive within 3 days. The sensitivity and specificity of AO based on these culture results were 52 and 98%, respectively, compared with 38% sensitivity and 99% specificity by methylene blue and Gram stains. The AO staining procedure is a simple, sensitive, screening technique for the early detection of positive blood cultures.     

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.     

Antimalarial efficacy of methylene blue and menadione and their effect on glutathione metabolism of Plasmodium yoelii-infected albino mice

Plasmodium yoelii infection caused significant decline in the hepatic and splenic glutathione content and the activities of the key enzymes, that is, glutamate cysteine ligase (EC 6.3.2.2) and glutathione reductase (EC 1.8.1.7) of their murine host, that is, Swiss albino mice. Methylene blue as well as menadione were found to restore these constituents when given to P. yoelii-infected mice at the dose levels of 2.5 and 100 mg/kg, respectively, compared to mefloquine which does the same at 5.0 mg/kg dose. Methylene blue, like mefloquine also caused a rapid decline in percent parasitaemia, whereas menadione caused a delay in maturation of the infection, but could not cure the mice.     

Early detection of Chlamydia trachomatis using fluorescent, DNA binding dyes.

HeLa 229 cells were infected with genital tract strains of Chlamydia trachomatis. After incubation for varying times the infected cells were fixed and stained with the fluorescent DNA binding dyes Hoechst 33258 or DAPI for comparison with conventional Giemsa stain. Fluorochrome-treated preparations were examined by incident ultraviolet fluorescence microscopy and the Giemsa-stained preparations by dark-ground light microscopy. Chlamydial inclusion bodies could be identified unambiguously as early as 18 hours after infection of HeLa 229 cells using either Hoechst 33258 or DAPI but not until some 48 hours in Giemsa-stained preparations. The DNA rich chlamydial elementary bodies in infected egg yolk suspension were readily detected using Hoechst 33258. The fluorescent dye technique was simpler and more rapid than Giemsa staining. Using Hoechst 33258 it is possible to speed up the identification of chlamydial isolates growing in tissue culture.     

Human lipoprotein binding to schistosomula of schistosoma mansoni. Displacement by polyanions, parasite antigen masking, and persistence in young larvae.

It was previously shown by the authors that the binding of human low-density lipoprotein (LDL) to the surface of schistosomula inhibits the binding of human anti-schistosomal antibodies and is inhibited by suramin. Here, three questions were considered. 1) Are LDLs bound to schistosomula displaced from the membrane by polyanions? 2) Does bound LDL mask or hide antigens recognized by human anti-schistosomal antibodies? 3) Is LDL, binding capability present when the larvae enter the blood stream? The first question was tested by measuring the percentage of the schistosomular surface membrane covered by LDL after exposure to LDL with or without dextran sulfate or suramin. The bound LDL was visualized with polyclonal goat anti-human apolipoprotein B (anti-apo B) antibodies and peroxidase-conjugated secondary antibodies. After overnight culture in 20 micrograms/300 microliters LDL, 84.0% +/- 0.3% of the parasite surface was covered by LDL reaction product. When the polyanions suramin or dextran sulfate were added to the cultures for 30 minutes, only 59.7% +/- 4.9% of the surface was covered by reaction product, demonstrating that the LDL was partially displaced from the membrane by these compounds. The second question was tested by measuring the binding of human and mouse monoclonal anti-schistosomal antibodies before and after exposure to LDL, with or without partial removal of the bound LDL by suramin. LDL partially inhibited antibody binding in a reversible fashion. The LDL clearly masked parasite antigens, most probably by steric hindrance. However, there may be competitive inhibition of antibody binding by the LDL as well, because human anti-schistosomal antibodies inhibited LDL binding to worms and both human anti-schistosomal antibody and LDL binding to schistosomula were inhibited by suramin. Finally, the third question was tested by quantitative immunofluorescence. The LDL binding capability persisted and nearly doubled by 72 hours after transformation from cercariae. These experiments demonstrated that LDL bound to the surface of schistosomula through the time they enter the blood stream. LDL bound to the parasite surface may help the parasite to evade antibody-dependent cytotoxic reactions by masking parasite antigens.