Ultrastructural electron probe X-ray microanalytical reaction product identification of three different enzymes in the same mouse resident peritoneal macrophage

Simultaneous cytochemical enzyme localization procedures for peroxidase (PO) plus acid phosphatase (AcP-ase) and/or aryl sulphatase (AS) have been investigated at the ultrastructural (EM) level. Electron probe X-ray microanalysis (EPMA) will identify and differentiate the reaction products. Dual reaction product localization of PO plus AcP-ase or alternatively PO plus AS have been obtained in the same mouse resident peritoneal macrophage. This has been acquired by first performing a PO-reaction followed by AcP-ase or followed by AS. In both cases PO-related reaction products (PODAB/Os or PODAB/Pt) were localized in nuclear envelope (NE) and rough endoplasmic reticulum (RER). Cells were identified by this reaction product as resident macrophages. Reaction products from the AcP-ase related cerium (AcP-aseCe), localized in lysosomes have been identified and differentiated from the PO-related osmium containing products. Similarly AS related barium (ASBa), localized in lysosomal structures and (R)ER was identified and differentiated. Triple reaction product localization of PO followed by AcP-ase plus AS could also be obtained. In this case, PO-related platinum containing reaction products (PODAB/Pt or PODAB/Os) in NE and RER has been identified and differentiated from the AcP-ase related lysosomal cerium (AcP-aseCe) and the AS related barium localized in lysosomal and (R)ER structures. Reversing the sequences in both dual cytochemical procedures: AcP-aseCe or ASBa followed by PODAB/Os (or PODAB/Pt) resulted in AcP-aseCe or ASBa activity related reaction products only. Reversing the sequence in the triple reaction procedures (ASBa followed by AcP-aseCe) resulted in the absence of the barium containing reaction products. By application of OsO4 postfixation with aminotriazole (ATR) additives the detrimental effects upon the various precipitates have been confirmed. In LM studies, using rat intestine and non-metal identification reactions for two of the enzymes (pararosaniline for AcP-ase, DAB for peroxidase), the influences of the metal ions used in EM were tested on the appearance of the coloured reaction products. Cerium ions used in EM for detection of AcP-aseCe activity have been shown to influence the PODAB visibility in LM and EM experiments. From the AS reaction media components neither barium ions nor p-nitro catachol sulphate influenced the LM visibility of the PO reaction.     

Benzidine dihydrochloride as a chromogen for single- and double-label light and electron microscopic immunocytochemical studies

Although very sensitive chromogens have been adapted for localization of horseradish peroxidase in anterograde and retrograde tracing studies, they have not been successfully applied in immunocytochemical studies. This report describes a protocol which uses benzidine dihydrochloride (BDHC) as the chromogen for light (LM) and electron microscopic (EM) immunocytochemical studies. The protocol is comparable to that used for tetramethylbenzidine, except that the pH of the reaction is above 6.0. At the LM level, the BDHC reaction product is bluish-green and crystalline. Both the color and form of the product are readily distinguished from the reddish-brown DAB reaction product. LM double-labeling studies are therefore feasible. The use of BDHC also increases significantly the sensitivity of the immunoreaction. Higher fixative concentrations can be used, less detergent is necessary, and higher primary antibody dilutions are possible. By osmicating at 45 degrees C in an s-collidine buffer it is possible to preserve the soluble BDHC reaction product for EM analysis. Immunoreactive cells are particularly well labeled with this new protocol. The BDHC crystals are easily detected at the EM level and can be distinguished from flocculent DAB reaction product. This feature makes EM double-labeling studies possible.     

Enterococcus faecalis Endocarditis Severity in Rabbits Is Reduced by IgG Fabs Interfering with Aggregation Substance

Background

Enterococcus faecalis is a significant cause of infective endocarditis, an infection of the heart endothelium leading to vegetation formation (microbes, fibrin, platelets, and host cells attached to underlying endothelial tissue). Our previous research determined that enterococcal aggregation substance (AS) is an important virulence factor in causation of endocarditis, although endocarditis may occur in the absence of AS production. Production of AS by E. faecalis causes the organism to form aggregates through AS binding to enterococcal binding substance. In this study, we assessed the ability of IgGs and IgG Fabs against AS to provide protection against AS⁺ E. faecalis endocarditis.

Methodology/Principal Findings

When challenged with AS⁺ E. faecalis, 10 rabbits actively immunized against AS⁺ E. faecalis developed more significant vegetations than 9 animals immunized against AS⁻ E. faecalis, and 9/10 succumbed compared to 2/9 (p<0.005), suggesting enhanced aggregation by IgG contributes significantly to disease. IgG antibodies against AS also enhanced enterococcal aggregation as tested in vitro. In contrast, Fab fragments of IgG from rabbits immunized against purified AS, when passively administered to rabbits (6/group) immediately before challenge with AS⁺ E. faecalis, reduced total vegetation (endocarditis lesion) microbial counts (7.9×10⁶ versus 2.0×10⁵, p = 0.02) and size (40 mg versus 10, p = 0.05). In vitro, the Fabs prevented enterococcal aggregation.

Conclusions/Significance

The data confirm the role of AS in infective endocarditis formation and suggest that use of Fabs against AS will provide partial protection from AS⁺ E. faecalis illness.     

Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10⁶ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 μM) is then added directly to the growing culture and AS-resistant (AS^r) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT^? clones. The AS^r phenotype is characterized both physiologically and biochemically. All AS^r clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.     

Optical and thermal properties of rare earth–doped K4Ca(PO4)2 phosphor

The luminescent properties and energy transfer (ET) mechanism in the Ln³⁺ pair of the RE³⁺ (RE = Eu³⁺, Ce³⁺, Dy³⁺ and Sm³⁺) doped K₄Ca(PO₄)₂ phosphor were successfully investigated using a conventional high-temperature solid-state reaction. In the near infrared (NIR) range, Ce³⁺-doped K₄Ca(PO₄)₂ phosphor exhibited a UV–Vis. emission band, whereas K₄Ca(PO₄)₂:Dy³⁺ exhibited characteristic emission bands centred at 481 and 576 nm in the near-ultraviolet excitation range. The possibility of ET from Ce³⁺ to Dy³⁺ in K₄Ca(PO₄)₂ phosphor was confirmed by a significant increase in the photoluminescence intensity of the Dy³⁺ ion based on the spectral overlap of acceptor and donor ions. X-ray diffraction, Fourier-transform infrared and thermogravimetric analysis/differential thermal analysis TGA/DTA were carried out to study phase purity, presence of functional groups and amount of weight loss under different temperature regimes. Therefore, the RE³⁺-doped K₄Ca(PO₄)₂ phosphor may be a stable phosphor host for light-emitting diode applications.     

Luminescence of the near‐ultraviolet blue‐emitting Ba9Al2Si6O24:Ce3+ phosphor

A near‐ultraviolet (NUV) blue‐emitting phosphor Ba₉Al₂Si₆O₂₄:Ce³⁺ (BAS:Ce³⁺) was synthesized using a high‐temperature solid‐state reaction. BAS:Ce³⁺ had an excitation band peak at about 328 nm and showed a blue emission band. The NUV‐blue emission band had a peak at about 386 nm with a band width of about 60 nm, attributed to the 5d–4f transition of Ce³⁺. Fluorescent decay showed an exponential model with a lifetime of 27.2 nsec. At 150°C, the luminescence intensity decreased to 68.7% compared with the intensity at room temperature.     

Dual emission of Ce3+,Mn2+‐coactivated Ca3YNa(PO4)3F via energy transfer: a single component white/yellow‐emitting phosphor

A series of Ce³⁺,Mn²⁺‐coactivated Ca₃YNa(PO₄)₃F phosphors were synthesized via a traditional solid‐state reaction under a reductive atmosphere. X‐Ray powder diffraction was used to confirm that the crystal structure and diffraction peaks of Ce³⁺/Mn²⁺‐doped samples matched well with the standard data. A spectral overlap between the emission band of Ce³⁺ and the excitation band of Mn²⁺ suggested the occurrence of energy transfer from Ce³⁺ to Mn²⁺. With increasing Mn²⁺ content, the emission intensities and lifetime values of the Ce³⁺ emission for Ca₃YNa(PO₄)₃F:Ce³⁺,Mn²⁺ phosphors linearly decrease, whereas the energy transfer efficiencies gradually increase to 89.35%. By adjusting the relative concentrations of Ce³⁺ and Mn²⁺, the emission hues are tuned from blue to white and eventually to yellow. These results suggest that Ca₃YNa(PO₄)₃F:Ce³⁺,Mn²⁺ phosphors have promising application as white‐emitting phosphors for near‐ultraviolet light‐emitting diodes.     

Synthesis and characterization of KCe(PO3)4 doped with some lanthanide activators

KCe(PO₃)₄ doped with Dy³⁺,Tb³⁺,Yb³⁺and Nd³⁺ phosphors were synthesized by a solid state diffusion method. The prepared samples were characterized by X‐ray diffraction and photoluminescence. KCe(PO₃)₄ exhibits emission in ultraviolet (UV) region which indicates weak Ce³⁺–Ce³⁺ interaction. The Ce³⁺–Ce³⁺energy transfer is not efficient. In light of this, energy transfer from Ce³⁺ to other lanthanides like Dy³⁺, Tb³⁺,Yb³⁺ and Nd³⁺ is rather surprising.     

Energy transfer in M5(PO4)3 F:Eu2+,Ce3+ (M = Ca and Ba) phosphors

M₅(PO₄)₃ F:Eu²⁺ (M = Ca and Ba) co‐doped with Ce³⁺ phosphors were successfully prepared by the combustion synthesis method. The introduction of co‐dopant (Ce³⁺) into the host enhanced the luminescent intensity of the M₅(PO₄)₃ F:Eu²⁺ (M = Ca and Ba) efficiently. Previously, we have reported the synthesis and photoluminescence properties of same phosphors. The aim of this article is to report energy transfer mechanism between Ce³⁺?Eu²⁺ ions in M₅(PO₄)₃ F:Eu²⁺ (M = Ca and Ba) phosphors, where Ce³⁺ ions act as sensitizers and Eu²⁺ ions act as activators. The M₅(PO₄)₃ F:Eu²⁺ (M = Ca and Ba) co‐doped with Ce³⁺ phosphor exhibits great potential for use in white ultraviolet (UV) light‐emitting diode applications to serve as a single‐phased phosphor that can be pumped with near‐UV or UV light‐emitting diodes. Copyright © 2013 John Wiley & Sons, Ltd.     

Optical properties and energy transfer of Eu2+/Ce3+ co‐activated Na3Ca6(PO4)5 phosphor

Ce³⁺/Eu²⁺ co‐doped Na₃Ca₆(PO₄)₅ phosphors were prepared using a combustion‐assisted synthesis method. X‐Ray powder diffraction (XRD) analysis confirmed the formation of a Na₃Ca₆(PO₄)₅ crystal phase. Na₃Ca₆(PO₄)₅:Eu²⁺ phosphors have an efficient bluish‐green emission band that peaks at 489 nm, whereas Ce³⁺‐doped Na₃Ca₆(PO₄)₅ showed a bright emission band at 391 nm. Analysis of the experimental results suggests that enhancement of the Eu²⁺ emission intensity in co‐doped Na₃Ca₆(PO₄)₅:Eu²⁺,Ce³⁺ phosphors is due to a resonance‐type energy transfer from Ce³⁺ to Eu²⁺ ions, which is predominantly governed by an exchange interaction mechanism. These results indicate that Ce³⁺/Eu²⁺ co‐doped Na₃Ca₆(PO₄)₅ is potentially useful as a highly efficient, bluish‐green emitting, UV‐convertible phosphor for white‐light‐emitting diodes. Copyright © 2014 John Wiley & Sons, Ltd.     

Design,synthesis and luminescence properties of Ba2YB2O6Cl‐ and Ba2YB2O6F‐based phosphors

Using a high‐temperature solid‐state reaction, the chlorine in Ba₂YB₂O₆Cl is gradually replaced by F, and a new compound with the nominal chemical formula Ba₂YB₂O₆F and two phosphors doped with Ce³⁺ and Eu³⁺, respectively, are obtained. X‐Ray diffraction and photoluminescence spectroscopy are used to characterize the as‐synthesized samples. The as‐synthesized Ba₂YB₂O₆Cl exhibits bright blue emission in the spectral range ~ 330–410 nm with a maximum around 363 nm under X‐ray or UV excitation. Ba₂YB₂O₆F:0.01Ce³⁺ exhibits blue emission in the range ~ 340–570 nm with a maximum around 383 nm. Ba₂YB₂O₆F:0.01Eu³⁺ exhibits a predominantly ⁵D₀–⁷ F₂ emission (~610 nm) and the relative intensities of the ⁵D₀–⁷ F_0,1,2 emissions are tunable under different wavelength UV excitation. The luminescence behaviors of the two phosphors are explained simply in terms of the host composition and site occupancy probability of Ce³⁺ and Eu³⁺, respectively. The results indicate that these phosphors have potential application as a blue phosphor or as a red phosphor. Copyright © 2014 John Wiley & Sons, Ltd.     

5d–4f transition in new phosphate‐based phosphors

The preparation of Ce³⁺‐doped Sr₆AlP₅O₂₀ and Ba₆AlP₅O₂₀ by a combustion method is described. Formation of compounds was confirmed by X‐ray diffraction (XRD) analysis. The photoluminescence (PL) emission spectra were observed at 355 nm when excited at 307 nm for the various concentrations. The PL emission spectra of phosphors showed strong Ce³⁺ emission due to the 5 d → 4f transition of Ce³⁺ ions. The Ce³⁺ emission intensity in Sr₆AlP₅O₂₀:Ce phosphor was higher than that in Ba₆AlP₅O₂₀:Ce and it may be useful for scintillation applications. Copyright © 2011 John Wiley & Sons, Ltd.     

Stimulation of the insulin secretory mechanism following barium accumulation in pancreatic β-cells

Electrothermal atomic absorption spectroscopy was employed for measuring barium in β-cell-rich pancreatic islets microdissected from ob/ob-mice. Both the uptake and efflux of barium displayed two distinct phases. There was a 4-fold accumulation of barium into intracellular stores when its extracellular concentration was 0.26 mM. Unlike divalent cations with more extensive intracellular accumulation, the washout of Ba²⁺ was not inhibited by d-glucose. Ba²⁺ served as a substitute for Ca²⁺ both in maintaining the glucose metabolism after removal of extracellular Ca²⁺ and making it possible for glucose to stimulate insulin release. Furthermore, Ba²⁺ elicited insulin release in the absence of glucose and other secretagogues. The latter effect was reversible and was markedly potentiated under conditions known to increase the β-cell content of cyclic AMP. It is likely that the observed actions of Ba²⁺ are mediated by Ca²⁺, since Ca²⁺-dependent regulatory proteins, such as calmodulin, apparently cannot bind Ba²⁺ specifically.     

Detection of fragmented DNA in apoptotic cells embedded in LR white: A combined histochemical (LM) and ultrastructural (EM) study.

We developed an improved method for the detection of double-strand DNA breaks in apoptotic cells at both the light (LM) and electron microscopic (EM) levels using a modification of the TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labeling (TUNEL) technique. Cultured rat cerebellar granule cells were exposed to low potassium conditions to induce apoptosis. Twenty-four hr after treatment, one group of cells was fixed in situ with 4% paraformaldehyde and labeled for DNA fragmentation characteristic of apoptosis. Apoptotic cells were visualized with diaminobenzidine (DAB) and viewed by LM. The second group of cells was detached from the culture dish, pelleted, fixed with a 4% paraformaldehyde and 0. 2% glutaraldehyde mixture, and embedded in LR White. For LM, the modified TUNEL technique was performed on 1.5-microm LR White sections and apoptotic cells were visualized using an enzymatic reaction to generate a blue precipitate. For EM, thin sections (94 nm) were processed and DNA fragmentation was identified using modified TUNEL with streptavidin-conjugated gold in conjunction with in-depth ultrastructural detail. Alternate sections of cells embedded in LR White can therefore be used for LM and EM TUNEL-based detection of apoptosis. The present findings suggest that the modified TUNEL technique on LR White semithin and consecutive thin sections has useful application for studying the fundamental mechanism of cell death. (J Histochem Cytochem 47:561-568, 1999)     

X-ray microanalysis of non-aldehyde-fixed glycogen contrast stained with OsVIIIO4, OsVIIIFeIII, or OsVIFeII complex in vitro

Summary Fibrin-enrobed, commercially produced glycogen was treated, without prior glutaraldehyde fixation, to a form of post-fixation with solutions of Os^VIIIO₄ or with a mixture of either Os^VIIIO₄ plus K₃Fe^III(CN)₆ or K₂Os^VIO₄ plus K₄Fe^II(CN)₆.Only the last mixture gave constrast staining of the glycogen in unstained ultrathin sections. The first mixture rendered the glycogen just barely visible but the glycogen contrast was increased by lead staining. The glycogen treated with the Os^VIIIO₄ solution was not contrast stained and was just observable after lead staining.Qualitative X-ray microanalysis of the glycogen in the ultrathin sections confirmed the presence of osmium and iron in the glycogen treated with both mixtures. The glycogen treated with Os^VIIIO₄ alone was difficult to analyse.Quantitative X-ray microanalysis showed that, in the glycogen treated with the Os^VIIIO₄ mixture plus K₃Fe^III(CN)₆, the mean atomic osmium to iron ratio was 15. In the glycogen treated with K₂Os^VIO₄ plus K₄Fe^II(CN)₆ this ratio was 117. However, the mean net osmium intensity in the latter case was 15 times higher than in the former case and for the iron even 40 times higher.The Unit for Analytical Electron Microscopy was established by collaboration between the Erasmus University of Rotterdam (W. C. de Bruijn), the University of Leiden and the Organization for Health Research TNO. The analytical microscope was purchased with funds from the Netherlands Organization for Pure Scientific Research (ZWO).     

Metal cluster topology. 1. Osmium carbonyl clusters

Important theoretical approaches to metal cluster bonding including the Wade-Mingos skeletal electron pair method, the Teo topological electron count, the King-Rouvray graph theory derived method, and Lauher's extended Hückel calculations are shown to agree in their apparent skeletal electron counts for the most prevalent metal cluster polyhedra including the tetrahedron, the trigonal bipyramid (both ordinary and elongated), square pyramid, octahedron, bicapped tetrahedron, pentagonal bipyramid, and capped octahedron. The graph theory derived method is used to treat osmium carbonyl clusters containing from five to eleven osmium atoms. In this connection most osmium carbonyl clusters can be classified into the following types: (1) Clusters exhibiting edge- localized bonding containing multiple tetrahedral chambers (e.g., Os₅(CO)₁₆, Os₆(CO)₁₈, H₂Os₇(CO)₂₀ and HOs₈(CO)₂₂⁻); (2) Capped octahedral clusters derived from osmium carbonyl fragments of the type Os_6+p(CO)_19+2p (p = 0, 1, 2, and 4) (e.g., Os₆- (CO)₁₈²⁻, Os₇(CO)₂₁, Os₈(CO)₂₂²⁻, and H₄Os₁₀- (CO)₂₄²⁻). Other more unusual osmium carbonyl clusters such as the planar Os₆(CO)₁₇ [P(OCH₃)₃]₄, the Os₉ cluster [Os₉(CO)₂₁C₃H₂R]⁻, and the Os₁₁ cluster Os₁₁C(CO)₂₇²⁻ can also be treated satisfactorily by these methods. The importance of the number of ligands around isoelectronic Os_n systems in determining the cluster polyhedron is illustrated by the different cluster polyhedra found for each member of the following isoelectronic pairs: HOs₆- (CO)₁₈⁻/H₂Os₆(CO)₁₈. Os₇(CO)₂₁/H₂Os₇(CO)₂₀, Os₈(CO)₂₂²⁻/HOs₈(CO)₂₂⁻. The tendency for osmium carbonyl clusters frequently to form polyhedra exhibiting edge-localized rather than globally delocalized bonding relates to the facility for osmium carbonyl vertices to contribute more than three internal orbitals to the cluster bonding. In this way Wade's well-known analogy between boron hydride clusters and metal clusters, which assumes exactly three internal orbitals for each vertex atom, is frequently no longer followed in the case of osmium carbonyl clusters.     

Luminescent properties of single‐phase Ba2‐x‐1.5y‐1.5zP2O7:xEu2+,yCe3+, zTb3+ phosphors for white light‐emitting diode

A series of Ba₂P₂O₇:xEu²⁺,yCe³⁺,zTb³⁺ phosphors was synthesized via a co‐precipitation method, then their crystal structure, quantum efficiency and luminescent properties were analyzed by XRD and FL, respectively. The results showed that these phosphors not only presented the excitation characteristics of Ba₂P₂O₇:xEu²⁺,zTb³⁺, but also exhibited that of the Ba₂P₂O₇:yCe³⁺,zTb³⁺ phosphor. Meanwhile, the tri‐doped phosphor showed a stronger absorption around 320 nm in contrast with the Eu²⁺/Ce³⁺:Tb³⁺ co‐doped phosphor. Not only can energy transfer from Ce³⁺→Eu²⁺ be observed; the energy transfer mechanism from Eu²⁺ to Tb³⁺ is discussed in the tri‐doped system. Ce³⁺ affects the luminescence properties of Ba₂P₂O₇:xEu²⁺,yCe³⁺,zTb³⁺ phosphors just as the sensitizer whereas Eu²⁺ is considered both as the sensitizer and the activator. The chromaticity coordinates of tri‐doped phosphors excited at 320 nm stayed steadily in the bluish‐white light region,and the emitted color and color temperature (CCT) of these phosphors could be tuned by adjusting the relative contents of Eu²⁺, Ce³⁺ and Tb³⁺. Hence, the single phase Ba₂P₂O₇:xEu²⁺,yCe³⁺,zTb³⁺ phosphors may be considered as potential candidates for white light‐emitting diodes.     

The Study of Impedance and Conductivity of New Modified Polystyrene Used as an Effective Adsorbent of Ba(II) Ions

In the research described here we prepared a novel, modified polystyrene (PS) with iminoether as the complexing agent for Ba²⁺. Most heavy metals cause environmental, atmospheric pollution.^[1–2] They cause consequence for humans health and aquatic life due to their toxicity. They become strongly toxic by mixing with different environmental elements and their removal from contaminated water is very important. The structure of all modified polystyrene such as nitrated polystyrene (PS−NO₂), aminated polystyrene (PS−NH₂), aminated polystyrene with imidate group (PS−NH−Im) and the complex with barium metal (PS−NH−Im/Ba²⁺) were analyzed by Fourier transform infrared spectroscopy (FT-IR), and the formation of N-2-Benzimidazolyl iminoether grafted PS was proved. The thermal stability and structure of the polystyrene and modified polystyrene were studied by differential thermal analysis (DTA) and X-ray diffractometry (XRD), respectively. The elemental analysis was used for the determination of the chemical composition of the modified PS. The grafted polystyrene was used in order to adsorb barium from wastewater with an acceptable cost before the wastewater distribution in the environment. The impedance analysis of the polystyrene complex PS−NH−Im/Ba²⁺ indicated an activated thermal conduction mechanism.The conductivity analysis of the complex with barium metal PS−NH−Im/Ba²⁺ was studied; the activation energy was deduced from an Arrhenius plot and corresponded to, , suggesting PS−NH−Im/Ba²⁺ was a proton-type of semiconductor.