Non-fluorescent Form Research Articles

Investigation of 3D ORAM Materials for Radiation Dosimetry

Three-dimensional optical random access memories (3D ORAM) are bistable photochromic materials that can be converted from the stable non-fluorescent form to the quasi-stable fluorescent form via two-photon absorption using intersecting laser beams. To investigate heavy charged particle (HCP) radiation effects on these materials, thin films of spyrobenzopyran-doped polymethylmethacrylate (SP/PMMA) were exposed to 0.6-2.5 MeV protons, 2 MeV and 5 MeV alpha particles, and 6.5 MeV 12C ions at fluences ranging from 1010 to 1014 cm-2. Exposed films were optically sectioned with a confocal laser scanning microscope (CLSM) and radiation effects were evaluated by measuring fluorescent intensity verses depth. Radiation exposure of the non-fluorescent form of SP/PMMA resulted in a permanent change, leaving fluorescent tracks excitable by optical wavelengths. Depth profiles revealed a correlation between maximum fluorescent intensity and absorbed dose. Additionally, the full width at half maximum (FWHM) of the depth profiles could be related to the incident HCP energy per nucleon.

Observation of radiation effects on three-dimensional optical random-access-memory materials for use in radiation dosimetry

The first experimental investigation has been performed of radiation effects on three-dimensional optical random-access-memory materials. Thin films of poly(methyl methacrylate) doped with spirobenzopyran were irradiated with uniform fluxes of protons, α-particles and 12C +3 ions, at fixed energies per nucleon from 0.5 to 2.5 MeV and fluences from 10 10 to 10 14 cm −2. The exposed films were examined under a confocal laser scanning microscopy system which is capable of optically sectioning the materials. The irradiation resulted in a permanent change in the materials from a nonfluorescent form to a form which is fluorescent under both 488 and 514 nm excitation. Profiles were measured of fluorescent intensity versus depth, and of intensity versus dose. It was found that both the particle energy and the dose can be obtained from measuring the width of the depth profile and the fluorescent intensity. These properties are very promising for dosimetry applications since they allow calculation of an accurate dose equivalent.

Conformational dynamics of Calcium Green 1 by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy was used to characterise the conformational equilibrium in “Calcium Green 1’', a fluorescent indicator of calcium. In the calcium bound form, a triplet bunching time of 6.7 µs is seen. We show that, in the absence of calcium, 10% of the indicator is in a fluorescent conformation that inter-converts to a non-fluorescent form in 10 µs. This confirms the probing mechanism based on a conformation controlled PET proposed for Calcium Green 1.

E2P phosphoforms of Na,K-ATPase. II. Interaction of substrate and cation-binding sites in Pi phosphorylation of Na,K-ATPase.

In this investigation the effects of alkali cations on the transient kinetics of Na,K-ATPase phosphoenzyme formation from either ATP (E2P) or Pi (E'2P) were characterized by chemical quench methods as well as by stopped-flow RH421 fluorescence experiments. By combining the two methods it was possible to characterize the kinetics of Na, K-ATPase from two sources, shark rectal glands and pig kidney. The rate of the spontaneous dephosphorylation of E2P and E'2P was identical with a rate constant of about 1.1 s-1 at 20 degreesC. However, whereas dephosphorylation of E2P formed from ATP was strongly stimulated by K+, dephosphorylation of E'2P formed from Pi in the absence of alkali cations was K+-insensitive, although in pig renal enzyme K+ binding to E'2P could be demonstrated with RH421 fluorescence. It appears, therefore, that in pig kidney enzyme the rapid binding of K+ to E'2P was followed by a slow transition to a nonfluorescent form. For shark enzyme the K+-induced decrease of RH421 fluorescence of Pi phosphorylated enzyme was due to K+ binding to the dephosphoenzyme (E1), thus shifting the equilibrium away from E'2P. When Pi phosphorylation was performed with enzyme equilibrated with K+ or its congeners Tl+, Rb+, and Cs+ but not with Na+ or Li+, both the phosphorylation and the dephosphorylation rates were considerably increased. This indicates that binding of cations modifies the substrate site in a cation-specific way, suggesting an allosteric interaction between the conformation of the cation-binding sites and the phosphorylation site of the enzyme.

Modulation of cellular antioxidant defense activities by sodium arsenite in human fibroblasts.

Many studies have shown that oxygen radicals can be produced during arsenic metabolism. We report here that in human fibroblasts (HFW cells) sodium arsenite exposure caused increased formation of fluorescent dichlorofluorescein (DCF) by oxidation of the nonfluorescent form. The enhanced DCF fluorescence was inhibited by a radical scavenger, butylated hydroxytoluene. The effects of sodium arsenite treatment on cellular antioxidant activities were then examined. Treatment of HFW cells with sodium arsenite resulted in a significant increase in heme oxygenase activity and ferritin level. Sodium arsenite-enhanced heme oxygenase synthesis was inhibited by co-treatment of cells with the antioxidants sodium azide and dimethyl sulfoxide. Furthermore, sodium arsenite treatment did not apparently affect glucose-6-phosphate dehydrogenase activity, but resulted in significantly increased glutathione levels and superoxide dismutase activity, slightly decreased glutathione peroxidase activity, and significantly decreased catalase activity. Sodium arsenite toxicity was partly reduced by addition of catalase to the culture medium. These results imply that arsenite can enhance oxidative stress in HFW cells.

In vivo visualization of oxidative changes in microvessels during neutrophil activation.

This study was designed to characterize temporal changes of oxidative metabolism in microvascular endothelium in vivo associated with neutrophil adhesion and diapedesis. The rat mesentery was loaded with dichlorofluorescein (DCFH) diacetate, a hydroperoxide-sensitive fluorescent probe that is trapped within viable cells as a nonfluorescent form and is converted to fluorescent dichlorofluorescein (DCF) by hydroperoxides. The fluorescent light emission was recorded with digital microscopy and its camera response was calibrated by superfusion of the mesentery with known concentrations of hydroperoxides. After rinsing with a precursor-free solution, it was possible to observe both the neutrophil adhesion and fluorescence changes in venular endothelium during superfusion with platelet-activating factor (PAF, 100 nM) by alternately changing light sources. In the PAF-treated animals, DCF fluorescence in the venular wall was markedly elevated concurrent with an increase in the number of adherent neutrophils. An onset of a significant DCF fluorescence was observed as early as 10 min after PAF application. The venular oxidative changes were co-localized with neutrophils adhering to the endothelium or migrating into the interstitium. The oxidative impact in the venular wall superfused with 100 nM PAF for 40 min was equivalent to that induced by the 10-min superfusion of 0.8 mM of tert-butyl hydroperoxide. Pretreatment with dipyridyl, an iron chelator, or dimethylthiourea, a hydroxyl radical scavenger, significantly attenuated the PAF-induced oxidative changes in the venule but had little effect on neutrophil adhesion. These findings suggest that an endothelial cell-dependent mechanism involving an iron-catalyzed reaction may have a role in enhancement of neutrophil-mediated oxidative stress in venular endothelium.

Effects of Ca2+ agonists on cytosolic Ca2+ in isolated hepatocytes and on bile secretion in the isolated perfused rat liver

The effects of increases in cytosolic Ca2+ on hepatocyte bile secretion are unknown. A number of agents that alter levels of cytosolic Ca2+ in the hepatocyte also produce hepatic vasoconstriction and activate protein kinase C, which complicates interpretations of their effects on bile secretion. To better understand the role of cytosolic Ca2+ in bile secretion, we examined the effect of the Ca2+ ionophore A23187 (0.1 mumol/L), the Ca2+ agonist vasopressin (10 nmol/L) and the Ca(2+)-mobilizing agent, 2,5-di(tert-butyl)-1,4-benzohydroquinone (25 mumol/L) on cytosolic Ca2+ in isolated hepatocytes and on bile flow in the isolated perfused rat liver, using vasodilators and inhibitors of protein kinase C and Ca2+ influx. Single-pass perfused livers were used, and cytosolic Ca2+ was measured by luminescent photometry in isolated hepatocytes loaded with the Ca(2+)-sensitive photoprotein aequorin. After A23187 perfusion, a sustained 74% +/- 10% (mean +/- S.D.) decrease in bile flow and a sustained 271% +/- 50% increase in perfusion pressure was observed. Simultaneous pretreatment with the vasodilator papaverine (25 mumol/L) and the protein kinase C inhibitor H-7 (50 mumol/L) abolished the pressure increase but not the decrease in bile flow, whereas pretreatment with Ni2+ (25 mumol/L) to block the influx of extracellular Ca2+ markedly reduced both the pressure increase and the decrease in bile flow. Vasopressin produced a transient (mean = 6 min) 75% +/- 4% decrease in bile flow and a sustained 7% +/- 4% increase in perfusion pressure. Pretreatment with H-7 alone corrected the vasopressin-induced pressure increase but also failed to eliminate the decrease in bile flow, whereas pretreatment with Ni2+ decreased the magnitude of the decrease by two-thirds without affecting the increase in perfusion pressure, 2,5'-di(tert-butyl)-1,4-benzohydroquinone produced a transient 65% +/- 20% decrease in bile flow and a transient 56% +/- 15% increase in perfusion pressure. In isolated hepatocytes, bromo-A23187, the nonfluorescent form of the ionophore, produced a sustained 56% +/- 32% increase in the cytosolic Ca2+ signal, whereas vasopressin resulted in a transient 241% +/- 75% increase and 2,5-di(tert-butyl)-1,4-benzohydroquinone resulted in a sustained 149% +/- 66% increase. The ionophore-induced increase in Ca2+ was abolished completely by pretreatment of the hepatocytes with Ni2+, whereas the vasopressin-induced increase was reduced by 38%.(ABSTRACT TRUNCATED AT 400 WORDS)

Biotransformation of coumarin derivatives. (2). Oxidative metabolism of 7-alkoxycoumarin by microsomal enzymes and a simple assay procedure for 7-alkoxycoumarin O-dealkylase.

The in vitro biotransformation of 7-alkoxycoumarin by rat liver microsomes was studied to develop a simple and accurate assay procedure for 7-alkoxycoumarin O-dealkylase. 7-Alkoxycoumarin was converted to the O-dealkylated metabolite, 7-hydroxycoumarin, by aerobic incubation of the parent compound with microsomes and NADPH, but the decreased amount of 7-alkoxycoumarin in the reaction mixture was several times higher than that of the 7-hydroxycoumarin produced during the incubation. The thin-layer chromatogram of the ether extractable metabolites in the reaction mixture showed the existence of several fluorescent metabolites including 7-hydroxycoumarin. Fluorescent properties of the parent compound, 7-alkoxycoumarin, and most of the metabolites differed from that of 7-hydroxycoumarin, but the reaction cofactor, NADPH, showed similar properties. Treatment of the reaction mixture with perchloric acid resulted in conversion of NADPH to the non-fluorescent form without any effect upon the fluorescent properties of 7-hydroxycoumarin and its related compounds. Based on these properties, an improved and simple in vitro fluorometric assay of the O-dealkylation of 7-alkoxycoumarin was developed. The method is applicable to routine determination of O-dealkylase activity in both isolated microsomes and whole homogenate. Species differences in the substrate specificity of the O-dealkylation reaction and in the responsiveness of animals to the inducer were observed even with use of the liver homogenate obtained from untreated and phenobarbital- or beta-naphthoflavone-pretreated animals, similar to what was observed with the microsomal system.

BIOTRANSFORMATION OF COUMARIN DERIVATIVES (2) OXIDATIVE METABOLISM OF 7-ALKOXYCOUMARIN BY MICROSOMAL ENZYMES AND A SIMPLE ASSAY PROCEDURE FOR 7-ALKOXYCOUMARIN O-DEALKYLASE

The in vitro biotransformation of 7-alkoxycoumarin by rat liver microsomes was studied to develop a simple and accurate assay procedure for 7-alkoxycoumarin O-dealkylase. 7-Alkoxycoumarin was converted to the O-dealkylated metabolite, 7-hydroxycoumarin, by aerobic incubation of the parent compound with microsomes and NADPH, but the decreased amount of 7-alkoxycoumarin in the reaction mixture was several times higher than that of the 7-hydroxycoumarin produced during the incubation. The thin-layer chromatogram of the ether extractable metabolites in the reaction mixture showed the existence of several fluorescent metabolites including 7-hydroxycoumarin. Fluorescent properties of the parent compound, 7-alkoxycoumarin, and most of the metabolites differed from that of 7-hydroxycoumarin, but the reaction cofactor, NADPH, showed similar properties. Treatment of the reaction mixture with perchloric acid resulted in conversion of NADPH to the non-fluorescent form without any effect upon the fluorescent properties of 7-hydroxycoumarin and its related compounds. Based on these properties, an improved and simple in vitro fluorometric assay of the O-dealkylation of 7-alkoxycoumarin was developed. The method is applicable to routine determination of O-dealkylase activity in both isolated microsomes and whole homogenate. Species differences in the substrate specificity of the O-dealkylation reaction and in the responsiveness of animals to the inducer were observed even with use of the liver homogenate obtained from untreated and phénobarbital- or β-naphthoflavone-pretreated animals, similar to what was observed with the microsomal system.

Volume changes in the formation of internal complexes of flavinyltryptophan peptides.

The effect of pressure, up to 10 kbar, on the fluorescence yield and lifetime of two flavinyltryptophan peptides was investigated. These peptides differed only in the number of methylene groups, respectively three and five, separating the chromophores. At atmospheric pressure the closed nonfluorescent form predominated in both compounds constitutin 94% of the total in the short-linked peptide and 80% in the long-linked one. The fluorescence of both peptides decreased at high pressure and the volume change upon formation of the nonfluorescent complex in the short peptide (--1.8 mL/mol) was less than half of the change in the long peptide (--4.8 mL/mol) or the value for FAD (--4.3 mL/mol). The much smaller compressibility of the short peptides is attributed to the mechanical constraint to the approach of the interacting rings, imposed by the short link. Mechanical constraints of similar nature may be expected to be operative in proteins. Their importance in pressure denaturation is discussed.

The binding of polyamines and of ethidium bromide to tRNA.

The binding of spermidine and ethidium bromide to mixed tRNA and phenylalanine tRNA has been studied under equilibrium conditions. The numbers and classes of binding sites obtained have been compared to those found in complexes isolated by gel filtration a low ionic strength. The latter complexes contain 10-11 moles of either spermidine or ethidium per mole of tRNA; either cation is completely displaceable by the other. In ethidium complexes, the first 2-3 moles are bound in fluorescent binding sites; the remaining 7-8 molecules bind in non-fluorescent form. At least one of the binding sites for spermidine appears similar to a binding site for fluorescent ethidium. Similar results are found with E. coli formylmethionine tRNA. Spermine, in excess of 18-20 moles per mole tRNA, causes precipitation of the complex. Putrescine does not form isolable complexes with yeast tRNA and displaces ethidium less readily from preformed ethidium-tRNA complexes. Under equilibrium conditions, in the absence of Mg++, there are 16-17 moles of spermidine bound per mole of tRNA as determined by equilibrium dialysis. Of these, 2-3 bind with a Ksence of 9 mM Mg++, the total number of binding sites is decreased slightly and there appears to be only one class of sites with a Ka = 600 M(-1). Quantitatively similar results are obtained for the binding of spermidine to yeast phenylalanine tRNA. When the interaction between ethidium bromide and mixed tRNA is studied by equilibrium dialysis or spectrophotometric titration, two classes of binding sites are obtained: 2-3 molecules bind with an average Ka = 6.6 x 10(5) M(-1) and 14-15 molecules bind with an average Ka = 4.1 x 10(4) M(-1). Spermidine, spermine, and Mg++ compete effectively for both classes of ethidium sites and have the effect of reducing the apparent binding constants for ethidium. When the binding of ethidium is studied by fluorometry, there are 3-4 highly fluorescent sites per tRNA. These sites are also affected by spermidine, spermine and Mg++. Putrescine has little effect on any of the classes of binding sites. These data are consistent with those found under non-equilibrium conditions. They suggest that polyamines bind to fairly specific regions of tRNA and may be involved in the maintenance of certain structural features of tRNA.

Isolation and characterization of the fluorescent alkali product from diphosphopyridine nucleotide.

Diphosphopyridine nucleotide undergoes a ringshown to be identical with one another and to have the same opening reaction in strongly alkaline solutions. This has been structure as synthetic 2-hydroxynicotinaldehyde by comshown by isolation of the same final fluorescent alkaline prodparison of their infrared, mass, nuclear magnetic resonance, uct from both DPN and 1-(N,N-dimethylcarbamoy1)nicoultraviolet, and fluorescence spectra. The yield of 2-hydroxytinamide chloride, which is known to undergo ring-opening at nicotinaldehyde from DPN increases with increasing hydroxpH above 7. The final alkaline products from DPN and 1ide ion concentration and reaches a maximal value of 80% (N,N-dimethycarbamoy1)nicotinamide chloride have been at 7.5 N NaOH. T he action of strong base on DPN produces a transient 370-mp-absorbing intermediate which rapidly decomposes to a 340-mp-absorbing species (Johnson and Morrison, 1970a). This 340-mp absorbing intermediate decays to a final product absorbing at 360 mp in its basic fluorescent form and at 340 my in its acidic nonfluorescent form (Kaplan et al., 1951). A pK, of 9.6 has been measured both spectrally (Johnson and Morrison, 1970b) and by acid quenching of fluorescence (Kaplan et al., 1951) for this 360-my-absorbing material. Production of fluorescence has been developed as an analytical method for DPN (Lowry et al., 1957). A ring-opening reaction of l-(N,Ndimethylcarbamoy1)nicotinamide chloride, I, where R = CONH2, at pH values above 7 produces a product with the identical spectral, fluorescent, chromatographic, and pK, properties as the 360-mp-absorbing product from DPN (Johnson and Morrison, 1970b). The isolation and characterization of these two products are the subject of this paper.

Absorption spectra of chlorophyll a in aqueous carbitol solvents

The absorption spectrum of chlorophyll a in aqueous methyl and butyl carbitol has been measured. The red-absorption maximum of chlorophyll dissolved in methyl carbitol (665 mμ) shifts to the red as the solvent is made increasingly aqueous (672 mμ in 30% methyl carbitol). Concurrently, the extinction coefficient decreases and fluorescence is lost. It has also been shown that only the non-fluorescent form of chlorophyll can form a colloid absorbing at 685 mμ in the presence of 6% dioxane. Butyl carbitol is more effective than methyl carbitol in keeping chlorophyll in a fluorescent form at high water concentrations, some fluorescence persisting when the solvent is 70–80% aqueous. Dioxane colloids prepared in 10% butyl carbitol exhibit an absorption maximum at 690 mμ, with a subsidiary maximum at 695 mμ. The 695-mμ absorption is considerably strengthened on standing. Thus, in these two solvent systems it is possible to prepare colloidal states of chlorophyll absorbing maximally at 672, 685, 690, and 695 mμ.

Studies on dissociation in Pasteurella multocida

Summary Little correlation exists between the morphologic cultural characteristics and virulence of strains of P. multocida . There is a relation between the type of fluorescence and virulence of a strain. Since the variants isolated from the blood and organs of mice dead of experimental infection are always identical to those injected, it cannot be proved that the seruin of susceptible animals necessarily contains a selective factor for a given variant as occurs in the case of the Brucella species. A fluorescent, virulent swine strain of P. multocida and the fluorescent and the non-fluorescent variants of a Chinese water buffalo strain ( P. bubaliseptica 4606) of P. multocida possess common antigens. By agglutination adsorption it was shown that each of the strains possesses additional specific antigens not shared by the other. The fluorescent virulent variant of the water buffalo strain could not completely adsorb agglutinins from an antiserum against the non-fluorescent avirulent variant. Either the non-fluorescent variants possess an antigen which is absent in the fluorescent variant or failure to adsorb was due to quantitative differences in the distribution of the same antigens. Normal horse serum and extract broth possessed a selective factor for the growth of the virulent fluorescent type of P. multocida . Continuous transfer in infusion broth promoted dissociation to the non-fluorescent form despite the presence of serum. Extract broth stippressed development of non-fluorescent variants even on rapid transfer. Dissociation of the fluorescent type to the non-fluorescent occurred in decreasing order in infusion broth, serum infusion broth, nutrient broth and serum nutrient broth when cultures were stored at 31°C.

Excretion of stilbamidine

1. 1. There was a marked contrast between a therapeutic dose level (1 mg./kg.) and a toxic level (10 mg./kg.) in the percentage urinary excretion of stilbamidine in rats. 2. 2. Considerably more (in one series of experiments about twenty times) amidine was determined by the glyoxal than by the fluorescence method. 3. 3. By the glyoxal method the daily excretion reached a maximum after about five daily injections of a therapeutic dose. 4. 4. A large proportion of stilbamidine was presumably metabolized and excreted in a non-fluorescent form. The preparation of 2-amino stilbamidine is described by Ashley and Harris (to be published in the Journal of the Chemical Society).