Dissipation of pH Gradients in Tonoplast Vesicles and Liposomes by Mixtures of Acridine Orange and Anions: Implications for the Use of Acridine Orange as a pH Probe

Acridine orange altered the response to anions of both ATP and in-organic pyrophosphate-dependent pH gradient formation in tonoplast vesicles isolated from oat (Avena sativa L.) roots and red beet (Beta vulgaris L.) storage tissue. When used as a fluorescent pH probe in the presence of I⁻, ClO₃⁻, NO₃⁻, Br⁻, or SCN⁻, acridine orange reported lower pH gradients than either quinacrine or [¹⁴C]methylamine. Acridine orange, but not quinacrine, reduced [¹⁴C]methylamine accumulation when NO₃⁻ was present indicating that the effect was due to a real decrease in the size of the pH gradient, not a misreporting of the gradient by acridine orange. Other experiments indicated that acridine orange and NO₃⁻ increased the rate of pH gradient collapse both in tonoplast vesicles and in liposomes of phosphatidylcholine and that the effect in tonoplast vesicles was greater at 24°C than at 12°C. It is suggested that acridine orange and certain anions increase the permeability of membranes to H⁺, possibly because protonated acridine orange and the anions form a lipophilic ion pair within the vesicle which diffuses across the membrane thus discharging the pH gradient. The results are discussed in relation to the use of acridine orange as a pH probe. It is concluded that the recently published evidence for a NO₃⁻/H⁺ symport involved in the export of NO₃⁻ from the vacuole is probably an artefact caused by acridine orange.     

Probing the Acceptor Active Site Organization of the Human Recombinant β1,4-Galactosyltransferase 7 and Design of Xyloside-based Inhibitors

Among glycosaminoglycan (GAG) biosynthetic enzymes, the human β1,4-galactosyltransferase 7 (hβ4GalT7) is characterized by its unique capacity to take over xyloside derivatives linked to a hydrophobic aglycone as substrates and/or inhibitors. This glycosyltransferase is thus a prime target for the development of regulators of GAG synthesis in therapeutics. Here, we report the structure-guided design of hβ4GalT7 inhibitors. By combining molecular modeling, in vitro mutagenesis, and kinetic measurements, and in cellulo analysis of GAG anabolism and decorin glycosylation, we mapped the organization of the acceptor binding pocket, in complex with 4-methylumbelliferone-xylopyranoside as prototype substrate. We show that its organization is governed, on one side, by three tyrosine residues, Tyr¹⁹⁴, Tyr¹⁹⁶, and Tyr¹⁹⁹, which create a hydrophobic environment and provide stacking interactions with both xylopyranoside and aglycone rings. On the opposite side, a hydrogen-bond network is established between the charged amino acids Asp²²⁸, Asp²²⁹, and Arg²²⁶, and the hydroxyl groups of xylose. We identified two key structural features, i.e. the strategic position of Tyr¹⁹⁴ forming stacking interactions with the aglycone, and the hydrogen bond between the His¹⁹⁵ nitrogen backbone and the carbonyl group of the coumarinyl molecule to develop a tight binder of hβ4GalT7. This led to the synthesis of 4-deoxy-4-fluoroxylose linked to 4-methylumbelliferone that inhibited hβ4GalT7 activity in vitro with a K_i 10 times lower than the K_m value and efficiently impaired GAG synthesis in a cell assay. This study provides a valuable probe for the investigation of GAG biology and opens avenues toward the development of bioactive compounds to correct GAG synthesis disorders implicated in different types of malignancies.     

Phenotype expression of human bone cells cultured on implant substrates

Bone cells derived from the human jaw were cultured on titanium, titanium coated with hydroxyapatite (THA) or with plasma spray (TPS) to study the behaviour of the cells anchored to implant substrates. Bone cells were cultured in MEM with the addition of [³H]-thymidine to evaluate cellular proliferation, and [³H]-glucosamine to evaluate GAG synthesis and accumulation in the extra-cellular matrix (ECM). Moreover, to study the degradation of GAG bone cells were cultured in the presence of NH₄Cl, an amine known to inhibit lysosomal activity. Our results show that TPS is the substrate that favours both cellular proliferation and the accumulation of GAG in the ECM. © 1997 John Wiley & Sons, Ltd.     

Synthesis and luminescence characterization of a new yellowish‐orange phosphor: Ba2B10O17:Sm3+

A new yellowish‐orange emitting phosphor, Ba₂B₁₀O₁₇:Sm³⁺ for use as a white light‐emitting diode (W‐LED) was synthesized by a solid‐state reaction method. The X‐ray diffraction results indicated that a pure Ba₂B₁₀O₁₇ material was obtained. As a potential yellowish‐orange luminescent material for W‐LEDs, the Ba₂B₁₀O₁₇:Sm³⁺ phosphor could be excited effectively by near‐ultraviolet (n‐UV) light and exhibited yellowish‐orange emission centered at 560 nm corresponding to the ⁴G_5/2 → ⁶H_5/2 transition of Sm³⁺ ions. The optimum concentration of Sm³⁺ ions in Ba₂B₁₀O₁₇, critical transfer distance (Ra) and concentration quenching mechanism of the presented phosphor were investigated. Moreover, CIE chromaticity coordinates and color purity performance of the Ba₂B₁₀O₁₇:Sm³⁺ phosphor were also discussed. The present work suggests that the Ba₂B₁₀O₁₇:Sm³⁺ phosphor has potential as a type of yellowish‐orange emitting phosphor. Copyright © 2016 John Wiley & Sons, Ltd.     

Association of glycosaminoglycan biosynthesis with the nuclear and microsomal cell apparatuses

The fraction composition of rat liver polysaccharides was characterized. Anabolism of glycosaminoglycans (GAG) was studied with ¹⁴C₁-glucose used as their precursor. It was found that synthesis of GAG polysaccharide fragments starts in structures associated with the cell nucleus and is completed in the microsomal fraction of the cell.     

Acridine orange staining of the mammalian fibroblast cell coat

Acridine orange selectively binds to glycos- and galactosaminoglycan (GAG) compounds in the presence of Na+ in low concentrations. We have worked out a cytochemical method, which is suitable for the specific demonstration of surface GAG components. The method consists of a glutaraldehyde prefixation, an acridine orange block-staining for 48 h and an OsO4 postfixation for some hours. The specificity of the staining was verified with the help of fluorimetric--in vitro--measurements and enzymatic digestions, respectively.     

Organ culture of human articular cartilage: Studies on sulphated glycosaminoglycan synthesis

Summary An organ culture system is described for adult human articular cartilage obtained from joints afterfemoral head replacement operations. Cartilage slices maintain maximal viability for 2 days in culture as assessed by uptake of [³H]uridine and [³H]leucine into whole tissue, and³⁵SO₄ into sulphated glycosaminoglycans (GAGs). Since GAGs are the components of cartilage matrix, the depletion of which is associated with osteoarthrosis, a method for measuring sulphated GAG synthesis in culture has been investigated.     

ATP-dependent and ionophore-induced proton translocation in pea stem microsomal vesicles

The presence of an electrogenic pump in pea stem microsomal vesicles has already been demonstrated, but no evidence on the nature of the electrogenic ion has been presented (Rasi-Caldogno, F., De Michelis, M.I. and Pugliarello, M.C. (1981) Biochim. Biophys. Acta 642, 37–45). In this work we tested the usefulness of the ΔpH probe Acridine orange to monitor both ATP-dependent and ionophore-induced H⁺ fluxes in pea stem microsomal vesicles. The H⁺/K⁺ exchanger nigericin causes a marked uptake of protons into the vesicles that can be followed, with similar results, both as Acridine orange absorbance changes and pH changes of the external medium. ATP induces an uptake of Acridine orange into the vesicles which is reversed by FCCP and abolished by the presence of Triton X-100 in the incubation medium, thus indicating an inward, ATP-driven, H⁺ translocation. The ATP-dependent acridine orange uptake is Mg²⁺-requiring and KCl-stimulated. Such activity is inhibited by two specific ATPase inhibitors, dicyclohexylcarbodiimide and diethylstilbestrol, while it is unaffected by oligomycin and Na₃VO₄. These results show that Acridine orange is a useful probe to measure pH gradients in our membrane system and are consistent with the hypothesis that an ATPase of plasmalemma may act as a proton pump.     

Synthesis of sulfated glycosaminoglycans by embryonic corneal epithelium

The primary corneal stroma is produced by the overlying epithelium. The endothelium appears between 4 and 5 days, fibroblasts at 6 days, and at 12 days the epithelium stratifies. We investigated the synthesis of glycosaminoglycan (GAG) by the epithelium during this developmentally significant period. The sulfated GAG synthesized by isolated 4–6-day-old corneal epithelia during the first 24 hr in vitro are entirely accountable for as chondroitin sulfates and heparan sulfates. Nearly 50% of the total sulfated GAG synthesized by epithelia on Millipore filters is lost to the medium, but only 30–40% is lost when frozen killed lens capsule or stroma is the substratum. Retention of isotope by the tissue is correlated with visible matrix polymerization. The relative amount of heparan sulfate synthesized by the developing epithelium 24 hr in vitro decreases from about 50% of the total sulfated GAG for 4-day-old epithelium to 12% for 12-day-old epithelium. A similar decrease in heparan sulfate synthesis occurs with time in culture. The relative amount of GAG identified as chondroitin sulfate and heparan sulfate is the same when ³H-glucosamine is used to label GAG as when ³⁵SO₄ is used. We conclude that the corneal epithelium produces only sulfated polysaccharides. Since hyaluronate is synthesized by whole 5-day-old corneas, it must be the product of the endothelium.     

Tunable emission,energy transfer and charge compensation in SrMoO4:Sm3+,Tb3+,Na+ phosphor

A series of SrMoO₄:Sm³⁺,Tb³⁺,Na⁺ phosphors was synthesized using a high‐temperature solid‐state reaction method in air. On excitation at 290 nm, SrMoO₄:Sm³⁺,Tb³⁺ phosphor emitted light that varied systematically from green to reddish‐orange on changing the Sm³⁺ and Tb³⁺ ion concentrations. The emission intensities of SrMoO₄:Sm³⁺ and SrMoO₄:Sm³⁺,Tb³⁺ phosphors were increased two to four times due to charge compensation when Na⁺ was added as a charge compensator. The luminescence mechanism and energy transfer could be explained using energy‐level diagrams of the MoO₄^2– group, Sm³⁺ and Tb³⁺ ions. SrMoO₄:Sm³⁺,Tb³⁺,Na⁺ could be used as reddish‐orange phosphor in white light‐emitting diodes (LEDs) based on an ~ 405 nm near‐UV LED chip. This research is helpful in adjusting and improving the luminescence properties of other phosphors. Copyright © 2015 John Wiley & Sons, Ltd.     

Apparent inhibition of Na+/H+ exchange by amiloride and harmaline in acridine orange studies

Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na⁺ gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na⁺ gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K⁺ gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I₅₀) of Na⁺/H⁺ exchange and K⁺ gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I₅₀ for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na⁺/H⁺ exchange by these drugs.     

Orange light‐emitting Ca3Mg3(PO4)4:Sm3+ phosphors

This work reports the photoluminescence properties of Ca₃Mg₃(PO₄)₄:Sm³⁺ phosphors that were synthesized by the combustion method. The phase formation and morphology of the prepared phosphors were analysed by X‐ray diffraction studies and scanning electron microscopy. Ca₃Mg₃(PO₄)₄:Sm³⁺ phosphors give orange light emission when excited by near‐ultraviolet (NUV) and blue light. The photoluminescence characteristics of the as‐prepared phosphors were investigated and their emission spectra showed three peaks due to ⁴G_5/2 → ⁶H_5/2, ⁴G_5/2 → ⁶H_7/2 and ⁴G_5/2 → ⁶H_9/2 transitions. The mechanism responsible for the concentration quenching of luminescence was found to be an electric dipole–dipole interaction. The CIE chromaticity coordinates suggested that the prepared phosphors are potential candidates for orange light‐emitting diodes (LEDs).     

Thermal quenching of luminescence of LiSr4(BO3)3:Eu2+ orange‐emitting phosphor

An orange‐emitting phosphor, Eu²⁺‐activated LiSr₄(BO₃)₃, was synthesized using the conventional solid‐state reaction. The photoluminescence excitation and emission spectra, and temperature dependence of the luminescence intensity of the phosphor were investigated. The results showed that LiSr₄(BO₃)₃:Eu²⁺ could be efficiently excited by incident light of 250–450 nm, and emits a strong orange light. With increasing temperature, the emission bands of LiSr₄(BO₃)₃:Eu²⁺ show an abnormal blue‐shift with broadening bandwidth and decreasing emission intensity. Copyright © 2013 John Wiley & Sons, Ltd.     

A novel reddish‐orange phosphor,NaLi2PO4:Eu3+

A series of Eu³⁺‐activated NaLi₂PO₄ novel phosphors was synthesized by the solid‐state reaction method. The X‐ray diffraction (XRD) and photoluminescence (PL) properties of these phosphors were investigated at room temperature. The excitation spectra indicate that these phosphors can be effectively excited by near‐UV (370–410 nm) light. The emission spectra exhibit strong reddish‐orange performance, which is due to the ⁵D₀ → ⁷F_J transitions of Eu³⁺ ions. The orange emission from transition ⁵D₀ → ⁷F₁ is dominant over that of ⁵D₀ → ⁷F_2. The concentration quenching of Eu³⁺ was observed in NaLi₂PO₄:Eu³⁺ when the Eu concentration was at 1 mol%. The impact of doping Eu³⁺ and photoluminescence properties were investigated and we propose a feasible interpretation. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of Dy3+ or Eu2+ co‐activator on a BaCa(SO4)2:Ce3+ mixed alkaline earth sulfate phosphor

The individual emission and energy transfer between Ce³⁺ and Eu²⁺ or Dy³⁺ in BaCa(SO₄)₂ mixed alkaline earth sulfate phosphor prepared using a co‐precipitation method is described. The phosphor was characterized by X‐ray diffraction (XRD) and photoluminescence (PL) studies and doped by Ce;Eu and Dy rare earths. All phosphors showed excellent blue–orange emission on excitation with UV light. PL measurements reveal that the emission intensity of Eu²⁺ or Dy³⁺ dopants is greater than when they are co‐doped with Ce³⁺. An efficient Ce³⁺ → Eu²⁺ [²T_2g(4f⁶5d) → ⁸S_7/2(4f₇)] and Ce³⁺ → Dy³⁺ (⁴ F_9/2 → ⁶H_15/2 and ⁴ F_9/2 → ⁶H_13/2) energy transfer takes place in the BaCa(SO₄)₂ host. A strong blue emission peak was observed at 462 nm for Eu²⁺ ions and an orange emission peak at 574 nm for Dy³⁺ ions. Hence, this phosphor may be used as a lamp phosphor. Copyright © 2015 John Wiley & Sons, Ltd.     

Kinetic of orange pigment production from <Emphasis Type="Italic">Monascus ruber</Emphasis> on submerged fermentation

Pigments produced by species of Monascus have been used to coloring rice, meat, sauces, wines and beers in East Asian countries. Monascus can produce orange (precursor), yellow and red pigments. Orange pigments have low solubility in culture media and when react with amino groups they become red and largely soluble. The orange pigments are an alternative to industrial pigment production because the low solubility facilitates the downstream operations. The aim of this work was to study the kinetic on the production of orange pigments by Monascus ruber CCT 3802. The shaking frequency of 300 rpm was favorable to production, whereas higher shaking frequencies showed negative effect. Pigment production was partially associated with cell growth, the critical dissolved oxygen concentration was between 0.894 and 1.388 mgO₂ L^?1 at 30 °C, and limiting conditions of dissolved oxygen decreased the production of orange pigments. The maintenance coefficient (mo) and the conversion factor of oxygen in biomass (Yo) were 18.603 mgO₂ g _x ^?1  h^?1 and 3.133 g_x gO ₂ ^?1 and the consideration of these parameters in the oxygen balance to estimate the biomass concentration provided good fits to the experimental data.     

In vitro cartilage formation: effects of 6-diazo-5-oxo-L-norleucine (DON) on glycosaminoglycan and collagen synthesis.

To determine the relationships between glycosaminoglycan (GAG) synthesis and type-specific collagen synthesis, we have investigated mouse limbs cultured in the presence of antiglutamine DON (6-diazo-5-oxo-l-norleucine). When compared to control limbs, ultrastructural examination of the DON-treated limbs shows that newly formed cartilage lacks matrix granules and the collagen fibrils have an altered morphology. Using [³⁵S]sulfate as a precursor, we have found that DON (5 μg/ml) suppresses chondroitin sulfate synthesis to less than 15% of the control level. We have also examined the collagen synthesized in equivalent limbs labeled with [³H]proline. The α-chain patterns from CM-cellulose chromatography were very similar for control and experimental limbs (α1:α2 ～ 7), suggesting that both (α1)₃- and (α1)₂α2-type molecules were being produced. The (α1)₃ molecules in both cases were identified as type II collagen by fractional salt separation and cyanogen bromide peptide mapping on CM-cellulose columns. We conclude that (1) the synthesis of type II collagen can be dissociated from the production of GAG, and (2) environmental influences can be involved in controlling the fibrillogenesis of collagen.     

Biodegradation of 14C-Labeled Model and Cornstalk Lignins, Phenols, Model Phenolase Humic Polymers, and Fungal Melanins as Influenced by a Readily Available Carbon Source and Soil

After 6 months of incubation in a fertile neutral sandy loam, about 48% of the ring carbons and 2-carbons and 60% of the OCH₃ carbons of specifically labeled coniferyl alcohol had evolved as CO₂. After 1 year, corresponding values were 55 and 65%. When coniferyl alcohol units were linked into model and cornstalk lignins, about 23% of the ring carbons and 2-carbons and 39% of the OCH₃ carbons had evolved as CO₂ after 6 months. After 1 year, corresponding values were about 28 and 46%. The addition of orange leaves (0.5%, wt/wt) after 6 months did not significantly increase the evolution of ¹⁴CO₂. Addition of orange leaves (0.5%, wt/wt) with specifically ¹⁴C-labeled pyrocatechol, coumaryl alcohol, model lignins, humic acid-type phenolic polymers and of uniformly ¹⁴C-labeled fungal melanins did not increase labeled C losses or C losses from the orange leaves. Decomposition of protein and pyrocatechol linked into model humic acid polymers, coniferyl alcohol C in model lignins, and Eurotium echinulatum melanin in six soils varied from 2 to 14%. Significant differences in C losses were related to soils and were not influenced by orange leaf applications.     

Optical properties of silver(I) perrhenate: luminescence from a metal-to-metal charge transfer state

The salt Ag⁺ReO₄ ⁻ shows an orange luminescence (λ_max=580 nm), which originates from a Ag^I → Re^VII MMCT triplet.     

Sr2ZnWO6:Eu3+,Bi3+,Li+: a potential white‐emitting phosphor for near‐ultraviolet white light‐emitting diodes

A series of Sr₂ZnWO₆ phosphors co‐doped with Eu³⁺, Bi³⁺ and Li⁺ were prepared using the Pechini method. The samples were tested using X‐ray diffraction and luminescence spectroscopy. The results show that the samples can be effectively excited by near‐ultraviolet (UV) and UV light. The introduction of Bi³⁺ and Li⁺ significantly enhances the fluorescence emission of Sr₂ZnWO₆:Eu³⁺ and changes the light emitted by the phosphors from bluish‐green to white. When excited at 371 nm, Sr_2–x–zZn_1–yWO₆:xEu³⁺,yBi³⁺,zLi⁺ (x = 0.05, y = 0.05, z = 0.05, 0.1 and 0.15) samples emit high‐performance white light. Intense red–orange emission is also observed when excited by UV light. The obtained phosphor is a potential white‐emitting phosphor that could meet the needs of excitation sources with near‐UV chips. In addition, this phosphor might have promising application as a red–orange emitting phosphor for white light‐emitting diodes based on UV light‐emitting diodes. Copyright © 2015 John Wiley & Sons, Ltd.