Mechanism of eosin Y action of activity of solubilized Ca2+, Mg2+- ATPase from smooth muscle sarcolemma

The eosin Y inhibitory effect on the activity of smooth muscle plasma membrane Ca(2+)-transporting ATPase was studied: effect of this inhibitor on the maximal initial rate of ATP-hydrolase reaction, catalyzed by Ca2+, Mg(2+)-ATPase, on the affinity of enzyme for the reaction reagents (Ca2+, Mg2+, ATP). Dependence of eosin Y inhibitory effect on some physicochemical factors of incubation medium was studied too. It was determined that eosin Y inhibited reversibly and with high specificity purified Ca2+, Mg(2+)-ATPase solubilized from myometrial cell plasma membrane (Ki--0.8 microM), decreased the turnover rate of this enzyme determined both by Mg2+, ATP and Ca2+. This inhibitor had no effect on the enzyme affinity for Ca2+, increased affinity for Mg2+ and decreased affinity for ATP. It was determined that inhibition of Ca2+, Mg(2+)-ATPase by eosin Y depended on pH and dielectric permeability of the incubation medium: increasing of pH from 6.5 to 8.0 reduced the apparent Ki, decreasing of dielectric permeability from 74.07 to 71.19 increased the apparent Ki.     

Preparation and characterization of reactive and stable glucose oxidase-containing liposomes modulated with detergent

Glucose oxidase-containing liposomes (GOL) as well as detergent-modulated glucose oxidase-containing liposomes were prepared and characterized, focusing not only on the reactivity of the liposomes upon external addition of glucose but also on the leakage of the entrapped glucose oxidase (GO) from the liposomes with the aim of developing a reactive and stable liposomal GO system. The membranes of the GOL prepared were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and modulated with either Triton X-100 or cholate. In the absence of added detergent, no GO leakage from the GOL was observed while its enzymatic activity was very low (low glucose permeability). As detergent-modulated liposomes, mixed POPC/Triton X-100 and mixed POPC/cholate liposomes (abbreviated as TL and CL, respectively) were prepared at different effective detergent/POPC molar ratios (R(e)) ranging from R(e) = 0 to R(e) = R(e) (sat) (R(e) (sat) is the critical value of R(e) at which the liposome membrane is saturated with detergent). The reactivity of GO-loaded TL (abbreviated as GOTL) or GO-loaded CL (GOCL) increased drastically with increase in the respective detergent content in the liposomes. In the case of GOTL, at R(e) (sat) = 0.40, a high reactivity was measured with a simultaneous high extent of GO leakage, suggesting that the observed enzymatic reaction was catalyzed mainly by leaked GO, caused by the interaction of Triton X-100 with the POPC membrane. On the other hand, GOCL prepared at R(e) (sat) = 0.43 showed relatively high reactivity with only a small extent of GO leakage, suggesting that most of the enzyme reaction was limited by the glucose permeation across the bilayers of GOCL. The GO leakage from GOCL was found to occur mostly during the rearrangement of the liposomal membrane during the preparation of the GOCL (mixing the GOL and cholate). Fluorescence polarization measurements of membrane-associated DPH (1,6-diphenyl-1,3,5-hexatriene) indicated that CL prepared by modifying POPC with cholate did not lead to a drastic change in membrane fluidity, indicating that the interacting cholate molecules did not penetrate deeply into the POPC bilayers. In summary, it was clearly shown that the membrane permeability of GOL can be quite simply modulated by mixing it with a certain amount of cholate to form highly reactive and stable GOCL with minimal enzyme leakage.     

Photoinactivation of acetylcholinesterase by erythrosin B and related compounds

Acetylcholinesterase was rapidly inactivated when exposed to light in the presence of xanthene dyes. Photosensitizing efficiency paralleled the dye triplet state quantum yields, increasing in the order fluorescein less than eosin B less than eosin Y less than erythrosin B less than rose bengal. The observed first-order rate constants of photoinactivation increased hyperbolically with dye concentration. Evidence for the formation of a dye-enzyme complex prior to inactivation was obtained from spectrophotometric and protein fluorescence quenching methods. The latter technique allowed estimates of the dye-enzyme dissociation constants for rose bengal (20 microM) and erythrosin B (30 microM). After photoinactivation, a portion of the dye became covalently bound to the enzyme. The photoinactivation reaction occurs in both aerobic (air saturated) and anaerobic (argon saturated) solution, with the rates of photoinactivation being about three to five times greater under the latter conditions. The aerobic reaction exhibits a large deuterium isotope enhancement effect and is largely (but not completely) quenched by 10(-2) M azide. The anaerobic reaction is unaffected by azide and exhibits only a small deuterium isotope effect. These results indicate that the photoinactivation reaction proceeds mainly by a type II (singlet oxygen mediated) pathway under aerobic conditions and by a type I (radical) pathway under anaerobic conditions. The enzyme was protected from inactivation by edrophonium, a competitive inhibitor, but not by d-tubocurarine, a peripheral-site ligand, indicating that destruction of a crucial residue at or near the catalytic site is an important component of the inactivation process. Extensive destruction of tryptophan undoubtedly occurs, at least under aerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Central Asian cobra venom cytotoxins-induced aggregation, permeability and fusion of liposomes

The processes of membrane aggregation, permeability and fusion induced by cytotoxins from Central Asian cobra venom were investigated by studying optical density of liposome samples, permeability of liposome membranes for ferricyanide anions and exchange of lipid material between the membranes of adjacent liposomes. Cytotoxins Vc5 and Vc1 were found to induce aggregation of PC + CL and PC + PS liposomes. Cytotoxin Vc5 increased also the permeability of the liposomes for K3[Fe(CN)6] and enhanced their fusion. Cytotoxin Vc1 increased membrane permeability and enhanced fusion of PC + CL samples only. The changes in membrane permeability and fusion were found to occur within a single value of cytotoxin concentrations. The fusogenic properties of the cytotoxins studied are supposed to be due to the ability to dehydrate membrane surface and to destabilize the lipid bilayer structure. Fusion probability is largely defined by the phospholipid composition of the membranes. A model of interaction of cytotoxins with cardiolipin-containing membranes is offered.     

Photosensitizing dyes and fluorochromes as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) chromosome technique

Using Allium cepa chromosomes after 5-bromo, 2'-deoxyuridine (BrdU) incorporation, we studied several acid and basic dyes and fluorochromes for their potential as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) technique. All of the dyes and fluorochromes investigated showed a photosensitizing capacity which was slightly lower than 33258 Hoechst in the cases of daunomycin, phloxin, fluorescein, thioflavine T and nuclear fast red, and somewhat higher in the case of eosin Y. Observation and cytophotometric analysis of differentially Giemsa-stained sister chromatids when eosin Y was used as the photosensitizing agent revealed the unsubstituted chromatid to be reddish violet in colour (absorption maximum, 550 nm), while the BrdU-substituted chromatid was blue or pale violet blue (absorption maximum, 580 nm). These results indicate that eosin Y is a useful photosensitizing dye which could be used as a substitute for 33258 Hoechst in the FPG staining technique.     

Permeability of amino acids into liposomes.

1. A simple and rapid assay for the measurement of permeability of amino acids into liposome membrane was carried out by using the liposomes trapping D-amino acid oxidase (D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) inside the membrane. 2. Permeability of amino acids into liposomes depended on the lipid composition of the membrane. Permeability of amino acids into phosphatidylcholine-cholesterol liposomes depended critically on temperature. 3. Permeability also depended on the structure of amino acids. The order of permeability was norvaline greater than isoleucine greater than leucine greater than phenylalanine greater than tryptophan greater than methionine greater than tyrosine, valine greater than threonine greater than serine greater than alanine greater than glycine.     

Eosin interaction of alpha-synuclein leading to protein self-oligomerization

Among various dyes including congo red, thioflavin S, thioflavin T, eosin, rhodamine 6G, and phenol red, the eosin was the only dye that induced self-oligomerization of alpha-synuclein in the presence of a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline. To analyze chemical nature of the eosin interaction with alpha-synuclein, the phenomenon of self-oligomerization was further examined with eosin congeners such as ethyl eosin, eosin B, phloxine B, erythrosin B, and rose bengal. The followings are the conclusions we have reached. First of all, intactness of the benzoate moiety of eosin and the negative charge on the carboxylic group of the dye are important factors leading to the specific interaction with alpha-synuclein. Secondly, the localized negative charge on the xanthene moiety of eosin is another critical factor for the interaction. As far as substituting halides are concerned, bromides and iodides on the xanthene moiety of the dyes do not make any difference on the alpha-synuclein interaction because both eosin and erythrosin B have induced the common phenomenon of self-oligomerization. The binding curve between eosin and alpha-synuclein was sigmoidal as the dye concentrations were increased. A double reciprocal plot of the saturation curve showed that the maximum number of eosin binding sites on alpha-synuclein was 1.85 with a dissociation constant of 390 microM. The dye binding to the protein appeared to occur via a positive cooperativity. The eosin binding site(s) was suggested to be located predominantly on the NAC region and partly related to the acidic C-terminus of alpha-synuclein. It has been, therefore, expected that this information might be useful to develop alpha-synuclein interactive molecules, which could provide eventual preventive or possible therapeutic means against various alpha-synuclein related disorders including Parkinson's disease.     

Biological decolorization of xanthene dyes by anaerobic granular biomass

Biodegradation of a xanthene dyes was investigated for the first time using anaerobic granular sludge. On a first screening, biomass was able to decolorize, at different extents, six azo dye solutions: acid orange 7, direct black 19, direct blue 71, mordant yellow 10, reactive red 2 and reactive red 120 and two xanthene dyes--Erythrosine B and Eosin Y. Biomass concentration, type of electron donor, induction of biomass with dye and mediation with activated carbon (AC) were variables studied for Erythrosine B (Ery) as model dye. Maximum color removal efficiency was achieved with 4.71 g VSS L?1, while the process rates were independent of the biomass concentration above 1.89 g VSS L?1. No considerable effects were observed when different substrates were used as electron donors (VFA, glucose or lactose). Addition of Ery in the incubation period of biomass led to a fivefold increase of the decolorization rate. The rate of Ery decolorization almost duplicated in the presence of commercial AC (0.1 g L?1 AC?). Using different modified AC samples (from the treatment of AC?), a threefold higher rate was obtained with the most basic one, AC(H?), as compared with non-mediated reaction. Higher rates were obtained at pH 6.0. Chemical reduction using Na?S confirmed the recalcitrant nature of this dye. The results attest that decolorization of Ery is essentially due to enzymatic and adsorption phenomena.     

Glucose oxidation catalyzed by liposomal glucose oxidase in the presence of catalase-containing liposomes

A catalase-containing liposome (CAL) was prepared and characterized in terms of stability during storage and catalysis of the decomposition of hydrogen peroxide (H2O2) that was initially added or produced in the oxidation of glucose catalyzed by the glucose oxidase-containing liposomes (GOL). The reactors used were a test tube and an external loop airlift bubble column as the static liquid and circulating liquid flow systems, respectively. The free catalase (CA) at low concentrations was unstable during storage at 4 degrees C as a result of dissociation of the tetrameric CA subunits. On the other hand, the deactivation of the CA activity in the CAL was depressed because of the high CA concentration in the CAL liposome. The CAL effectively catalyzed the repeated decompositions at 25 degrees C with 10 mM H2O2 added initially, whereas the free CA was significantly deactivated during the repeated reactions. The high stability of the CAL was attributed to the moderately depressed reactivity, which was essentially derived from the diffusion limitation of the CAL membrane to H2O2 in the liquid bulk. In the GOL-catalyzed prolonged oxidation of 10 mM glucose at 40 degrees C in the static liquid in a test tube, both the free CA and CAL could continuously catalyze the decomposition of H2O2 produced. This was because the glucose oxidation rate was small due to the limited reactivity of the GOL to glucose with its low permeability through the GOL membrane. In the glucose oxidation catalyzed by the GOL with the free CA or the CAL in the airlift, much larger oxidation rates were observed compared to those in the test tube because the permeability of the GOL membrane to glucose was increased in the gas-liquid two phase flow in the airlift. The GOL/CAL system in the airlift operated in an acidic condition, which was preferable to the GO activity, gave the largest oxidation rate with negligible accumulation of the H2O2 produced. On the other hand, the GOL/free CA system gave an oxidation rate smaller than that of the GOL/CAL system even under the acidic condition due to an unfavorable interaction of the free CA molecules with the GOL membranes leading to the decreased reactivity of the GOL.     

Permeability changes induced by 130 GHz pulsed radiation on cationic liposomes loaded with carbonic anhydrase

The effects of pulsed 130 GHz radiations on lipid membrane permeability were investigated by using cationic liposomes containing dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and stearylamine. Carbonic anhydrase (CA) was loaded inside the liposomes and the substrate p-nitrophenyl acetate (p-NPA) added in the bulk aqueous phase. Upon permeation across the lipid bilayer, the trapped CA catalyzes the conversion of the p-NPA molecules into products. Because the self-diffusion rate of p-NPA across intact liposomes is very low the CA reaction rate, expressed as Delta A/min, is used to track membrane permeability changes. The effect of 130 GHz radiation pulse-modulated at low frequencies of 5, 7, or 10 Hz, and at time-averaged incident intensity (I(AV)) up to 17 mW/cm(2) was studied at room temperature (22 degrees C), below the phase transition temperature of DPPC liposomes. At all the tested values of I(AV) a significant enhancement of the enzyme reaction rate in CA-loaded liposomes occurred when the pulse repetition rate was 7 Hz. Typically, an increase from Delta A/min = 0.0026 +/- 0.0010 (n = 11) to Delta A/min = 0.0045 +/- 0.0013 (n = 12) (P < 0.0005) resulted at I(AV) = 7.7 mW/cm(2). The effect of 130 GHz pulse-modulated at 7 Hz was also observed on cationic liposomes formed with palmitoyloleoyl phosphatidylcholine (POPC), at room temperature (22 degrees C), above the phase transition temperature of POPC liposomes.     

Various properties of urease encapsulated into liposomes

The enzymic activity of plant urease encapsulated into liposomes from egg lecithin was studied. Liposomes contained 3-5% of the initial enzymic preparation. Incorporation of urease into liposomes increases the permeability of the lecithin membrane for urea. The liposome membrane provides protection of the incorporated material from the inhibitory action of heavy metal ions. Kinetics of the reactions catalyzed by the free enzyme and encapsulated one is different. Km for the encapsulated enzyme is 1 X 10(-3) M and for free urease--4 X 10(-4) M, that is related to limited substrate mass transfer rate and as a result of it due to inhomogeneity of the catalysis proceeding in liposomes.     

Traditional staining for routine diagnostic pathology including the role of tannic acid. 1. Value and limitations of the hematoxylin-eosin stain

The components of the hematoxylin and eosin (H & E) stain (i.e. hemalum and eosin Y), their contributions to the typical staining pattern, and the reasons why the H & E stains are the preferred oversight stains for routine diagnostic histopathology are discussed. The essential diagnostic significance of effective nuclear staining by hemalum, providing information on nuclear morphology and texture, is emphasized; as is the ironic advantage for routine diagnostic histopathology of the limited range of colors provided by H & E staining, that allows recognition of significant features under low microscopic magnifications. Standardization of hemalum is considered, along with probable reasons why users show resistance to such a concept. Counterstaining with anionic (acid) dyes is discussed, as is the important phenomenon of contrast. The particular advantages and disadvantages of eosin Y and phloxin B as counterstains to hemalum are outlined. The concept of an “ideal routine histological stain” is considered, and H & E is compared to such an ideal case. Finally, deficiencies of H & E staining are discussed, and a program to develop an improved oversight stain is introduced.     

Oxygen uptake induced by electron transfer from donors to the triplet state of methylene blue and xanthene dyes in air-saturated aqueous solution.

The effects of oxygen in the photolysis of rose bengal, eosin, erythrosin and methylene blue were studied in the presence of formate and electron donors, such as ascorbic acid, aromatic amino acids or aliphatic amines, e.g. triethylamine (TEA). The overall reaction is conversion of oxygen via the hydroperoxyl/superoxide ion radical into hydrogen peroxide. The quantum yield of oxygen uptake (Phi(-O2)) increases with the donor concentration. The photoinduced formation of H2O2 is initiated by quenching of the triplet state of the dye by the donor and subsequent reactions of both the dye and donor radicals with oxygen. For methylene blue and the xanthene dyes in the presence of 10 mM ascorbic acid or 0.1 M TEA Phi(-O2)=0.07-0.25. The spectral and kinetic properties of the specific dye transients, including the radicals involved and the pH and concentration dependences, are discussed.     

Non-ohmic proton conductance of mitochondria and liposomes

Direct measurements of the proton/hydroxyl ion flux across rat liver mitochondria and liposome membranes are reported. H+/OH- fluxes driven by membrane potential (delta psi) showed nonlinear dependence on delta psi both in mitochondria and in liposomes whereas delta pH-driven H+/OH- flux shows linear dependence on delta pH in liposomes. In the presence of low concentrations of a protonophore the H+/OH- flux was linearly dependent on delta psi and showed complex dependence on delta pH. The nonlinearity of H+/OH- permeability without protonophore is described by an integrated Nernst- Plank equation with trapezoidal energy barrier. Permeability coefficients depended on the driving force but were in the range 10(-3) cm/s for mitochondria and 10(-4)-10(-6) cm/s for liposomes. The nonlinear dependence of H+/OH- flux on delta psi explains the nonlinear dependence of electrochemical proton gradient on the rate of electron transport in energy coupling systems.     

Traditional staining for routine diagnostic pathology including the role of tannic acid. 1. Value and limitations of the hematoxylin-eosin stain

The components of the hematoxylin and eosin (H & E) stain (i.e. hemalum and eosin Y), their contributions to the typical staining pattern, and the reasons why the H & E stains are the preferred oversight stains for routine diagnostic histopathology are discussed. The essential diagnostic significance of effective nuclear staining by hemalum, providing information on nuclear morphology and texture, is emphasized; as is the ironic advantage for routine diagnostic histopathology of the limited range of colors provided by H & E staining, that allows recognition of significant features under low microscopic magnifications. Standardization of hemalum is considered, along with probable reasons why users show resistance to such a concept. Counterstaining with anionic (acid) dyes is discussed, as is the important phenomenon of contrast. The particular advantages and disadvantages of eosin Y and phloxin B as counterstains to hemalum are outlined. The concept of an “ideal routine histological stain” is considered, and H & E is compared to such an ideal case. Finally, deficiencies of H & E staining are discussed, and a program to develop an improved oversight stain is introduced.     

Membrane fusion activity of influenza virus. Effects of gangliosides and negatively charged phospholipids in target liposomes

Fusion of influenza virus with liposomes composed of negatively charged phospholipids differs from fusion with biological membranes or zwitterionic liposomes with ganglioside receptors [Stegmann, T., Hoekstra, D., Scherphof, G., & Wilschut, J. (1986) J. Biol. Chem. 261, 10966-10969]. In this study, we investigated how the kinetics and extent of fusion of influenza virus, monitored with a fluorescence resonance energy-transfer assay, are influenced by the surface charge and the presence of receptors on liposomal membranes. The results were analyzed in terms of mass action kinetic model, providing separate rate constants for the initial virus-liposome adhesion, or aggregation, and for the actual fusion reaction. Incorporation of increasing amounts of cardiolipin (CL) or phosphatidylserine (PS) into otherwise zwitterionic phosphatidylcholine (PC)/phosphatidylethanolamine (PE) vesicles results in a gradual shift of the pH threshold of fusion to neutral, relative to the pH threshold obtained with PC/PE vesicles containing the ganglioside GD1a, while also the rate of fusion increases. This indicates the emergence of a fusion mechanism not involving the well-documented conformational change in the viral hemagglutinin (HA). However, only with pure CL liposomes this nonphysiological fusion reaction dominates the overall fusion process; with pure PS or with zwitterionic vesicles containing CL or PS, the contribution of the nonphysiological fusion reaction is small. Accordingly, preincubation of the virus alone at low pH results in a rapid inactivation of the viral fusion capacity toward all liposome compositions studied, except pure CL liposomes. The results of the kinetic analyses show that with pure CL liposomes the rates of both virus-liposome adhesion and fusion are considerably higher than with all other liposome compositions studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes: energy transfer to nonfluorescent dimers

When 5(6)-carboxyfluorescein (6CF) is encapsulated in liposomes at 0.2 M, 97-98% of the fluorescence is quenched. We have studied the mechanism of this effect. The dye-liposome system is a special case of concentration quenching of dyes, a phenomenon recognized for 100 years. Absorption spectra of encapsulated dye show that 6CF dimerizes, and the dimer is nonfluorescent. The dimerization constant was estimated, and it was concluded that dimerization can account for only part of the quenching. In 6CF solutions, the fluorescence lifetime decreased drastically as concentration was changed over the narrow range 0.02-0.05 M, a finding which was attributed to energy transfer to dimers. Inhibition of dimerization by propylene glycol also inhibited the shortening of lifetime. F?rster critical transfer distances were calculated to be 51 and 57 A for monomer-monomer and monomer-dimer transfer, respectively. Monomer-monomer transfer was demonstrated directly by steady-state or time-resolved anisotropy experiments, while transfer to dimer was modeled by using sulforhodamine B, which has a critical transfer distance like that for the dimer and also quenches 6CF emission. No direct evidence for collisional self-quenching of 6CF could be found, although a model compound, salicylate, did quench weakly. For xanthene dyes, the rate of energy transfer is much faster than that for quenching collisions, implying that collisional quenching in the usual 6CF-liposome system is insignificant. The reason why 6CF is not 100% quenched in liposomes is attributed to dye interaction with lipid as evidenced by (i) multiexponential decay of 6CF in liposomes with a long component of 3-4 ns, (ii) inhibition of dimerization in liposomes, (iii) partial protection of dye from quenching by KI, (iv) differing amounts of dimerization in liposomes made from different kinds of phospholipid, and (v) enhancement of fluorescence lifetime in the presence of Triton X-100.     

Fusion of planar bilayer phospholipid membranes with liposomes]

Lecithine-cholesterol liposomes containing amphotericin B ionoforic marker were used to study the interaction between liposomes and planar phospholipid membranes. The liposomes were shown to increase the permeability of the planar membrane, which may be explained in terms of membrane fusion. Bivalent cations (Mg2+ and particularly Ca2+), dicetylphosphate producing negatively charged groups on the membrane surface and the n-decane suspension in water promote the fusion, whereas the increase of the cholesterol content in the liposomes prevents it.     

Reconstitution of a porcine submaxillary gland beta-D-galactoside alpha 2----3 sialyltransferase into liposomes

Form A of the beta-D-galactoside alpha 2----3 sialyltransferase from porcine submaxillary glands was incorporated into liposomes. Incorporation was achieved by gel filtration of the enzyme in the presence of octylglucoside-phospholipid micelles. As detergent was removed during gel filtration, liposomes (average diameter, 370 A) with bound enzyme were formed and emerged unretarded from the column. The recovery of enzyme activity in the liposomes was about 40% of the initial activity starting with as little as 9 micrograms of transferase. Chromatography on Sepharose CL6B and sucrose density gradient centrifugation confirmed the association of enzyme with liposomes. In contrast to Form A, Form B of the sialyltransferase, which lacks the proposed lipid-binding domain of Form A, cannot be incorporated into liposomes. Form A of the transferase was also incorporated into liposomes composed of phosphatidylcholine, cholesterol, and a mixture of phospholipids from the membranes of the Golgi apparatus from porcine submaxillary glands. Although the transferase was distributed about equally on the internal and external surface of the phosphatidylcholine liposomes, most of the transferase was on the external surface in liposomes containing cholesterol (72%) or in liposomes containing Golgi apparatus phospholipids (88%). The enzyme bound to phosphatidylcholine liposomes was shown by kinetic analysis to have the same activity as that found in the presence of activity-stimulating detergents such as Triton X-100. Enzyme incorporated into cholesterol-containing liposomes had the same activity. In contrast, enzyme bound to liposomes formed from the Golgi apparatus mixed phospholipids had a lower activity, but one similar to that of the transferase in Golgi apparatus membranes. These studies suggest that the composition of a biological membrane may well influence the orientation of the transferase in the membrane as well as modulate its enzymic activity.     

Photosensitizing dyes and fluorochromes as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) chromosome technique

Summary UsingAllium cepa chromosomes after 5-bromo, 2-deoxyuridine (BrdU) incorporation, we studied several acid and basic dyes and fluorochromes for their potential as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) technique. All of the dyes and fluorochromes investigated showed a photosensitizing capacity which was slightly lower than 33258 Hoechst in the cases of daunomycin, phloxin, fluorescein, thioflavine T and nuclear fast red, and somewhat higher in the case of eosin Y. Observation and cytophotometric analysis of differentially Giemsa-stained sister chromatids when eosin Y was used as the photosensitizing agent revealed the unsubstituted chromatid to be reddish violet in colour (absorption maximum, 550 nm), while the BrdU-substituted chromatid was blue or pale violet blue (absorption maximum, 580 nm). These results indicate that eosin Y is a useful photosensitizing dye which could be used as a substitute for 33258 Hocchst in the FPG staining technique