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In situ identification of nocardioform actinomycetes in activated sludge using fluorescent rRNA-targeted oligonucleotide probes.

Hitherto, few environmental samples have been investigated by a 'full cycle rRNA analysis'. Here the results of in situ hybridization experiments with specific rRNA-targeted oligonucleotide probes developed on the basis of new sequences derived from a previously described comparative 16S rRNA analysis of nocardioform actinomycetes in activated sludge are reported. Application of the specific probes enabled identification and discrimination of the distinct populations of nocardioform actinomycetes in activated sludge. One of the specific probes (DLP) detected rod-shaped bacteria which were found in 13 of the 16 investigated sludge samples from various wastewater treatment plants, suggesting their importance in the wastewater treatment process. Another probe (GLP2) hybridized with typically branched filaments of nocardioforms mainly found in samples from enhanced biological phosphorus removal plants, suggesting that these bacteria are involved in sludge foaming. The combination of in situ hybridization with fluorescently labelled rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy improved the detection of nocardioform actinomycetes, which often showed only weak signals inside the activated-sludge flocs.

Actinomycetales↗

Clinical effects of glass ionomer restorations on residual carious dentin in primary molars.

PURPOSE: To evaluate the mineralization and morphology of residual (infected and affected) carious dentin following the restoration of vital primary molars with viscous glass-ionomer cement (GIC). METHODS: Encapsulated Fuji IX GP and Ketac-Molar Aplicap GICs were placed in cavities that were prepared using the atraumatic restorative treatment (ART) approach. Four suitable exfoliated teeth with intact restorations were sectioned and then examined by electron probe microanalysis (EPMA) and scanning electron microscopy (SEM). RESULTS: EPMA demonstrated the presence of fluorine and strontium that had penetrated into the underlying residual carious dentin from the adjacent GIC. The concentrations of these two elements, and those of calcium and phosphorous, varied with distance from the GIC/dentin interface. SEM showed varying degrees of dentin tubule destruction and intratubular (peritubular) dentin present immediately subjacent to the GIC/dentin interface. Incomplete removal of carious dentin was observed in all specimens, and GIC remained adherent to the tissue.

Analysis of Variance↗

Does Puccinelia tenuiflora have the ability of salt exudation?

The leaves surface of Puccinelia tenuiflora seedling under stress of different concentration of Na(2)CO(3) was observed with scanning electron microscopy and X-ray electron probe micro analyzer. All the results indicated that varied salts crystalline distributing in stomatal apparatus on P. tenuiflora leaves surface could be observed by means of frozen-dried sampling. In the case of no stress, these leaves had many kinds of sediments such as Na, K, Ca, Mg, Zn, Cu, Cl, P, S, Si on their surface. When there was stress of Na(2)CO(3), the percentage content of each sediment would have an non-linear relationship to stress. As the concentration of Na(2)CO(3) increased, the percentage contents of Na, Cu, Zn, P, S generally decreased while that of K, Ca, Mg, Si generally increased. The ratio of K/Na also changed the same way as the later. From these results, we concluded, under stress of alkali and salts, P. tenuiflora leaves could exude salts through their stomata or together with wax secretion and these ways might participate many regulation process of P. tenuiflora leaves cells, for example in ion balancing, osmosis regulating and water metabolizing.

Calcium↗

Ultrastructure and chemical composition of calcite urinary calculi in Chinese swamp buffalo.

Four urinary calculi, derived from Chinese swamp buffalo, were studied by using qualitative chemical analysis, X-ray diffraction, scanning electron microscopy and qualitative energy dispersive (electron probe) microanalysis. Qualitative chemical analysis showed that the predominant ions were calcium and carbonate with small amounts of magnesium and ammonium. X-ray diffraction confirmed that the calculi were primarily composed of calcium carbonate (calcite). On ultrastructural examination, three apparently distinct structural regions were identified in the calculi: outer large laminations; cavities containing variable numbers of small spheres and rods; and large spheres. There did not appear to be material that acted as a nidus and all regions, on qualitative electron probe analysis, contained primarily calcium with trace amounts of magnesium, phosphorus, potassium and chloride. It was concluded that calcite calculi in Chinese swamp buffalo are probably formed through a process of asynchronous layering and that nidus formation may not be necessary. Moreover, the ultrastructure of the calcite calculi is similar to that reported for siliceous calculi in ruminants and this suggests that similar factors may be involved in their formation.

Animals↗

Chemical exchange between glass-ionomer restorations and residual carious dentine in permanent molars: an in vivo study.

OBJECTIVE: To evaluate the remineralization of carious dentine following the restoration of an extensive lesion in a permanent molar with a high strength glass-ionomer cement (GIC). MATERIALS AND METHODS: Thirteen first permanent molars, which were scheduled for extraction because of the presence of extensive caries lesions, were selected for this study. They were first restored, according to the ART technique, using encapsulated Fuji IX(GP), which contains a strontium glass rather than the traditional calcium glass. The cavities were prepared with a clean enamel margin and minimal removal of the carious dentine around the walls. After a period of 1-3 months they were harvested and subsequently sectioned and examined using an electron probe microanalysis (EPMA) and scanning electron microscopy (SEM). RESULTS: EPMA demonstrated that both fluorine and strontium ions had penetrated deep into the underlying demineralized dentine. The only possible source of these ions was the GIC restoration. CONCLUSION: The pattern of penetration of the fluorine and strontium ions into the dentine was consistent with a remineralization process.

Adolescent↗

Direct visualization of phosphorylase-phosphorylase kinase complexes by scanning tunneling and atomic force microscopy.

In skeletal muscle the activation of phosphorylase b is catalyzed by phosphorylase kinase. Both enzymes occur in vivo as part of a multienzyme complex. The two enzymes have been imaged by atomic force microscopy and the results compared to those previously found by scanning tunneling microscopy. Scanning tunneling microscopy and atomic force microscopy have been used to view complexes between the activating enzyme phosphorylase kinase and its substrate phosphorylase b. Changes in the size and shape of phosphorylase kinase were observed when it bound phosphorylase b.

Animals↗

Microcapsules made of weak polyelectrolytes: templating and stimuli-responsive properties.

Hollow microcapsules composed of the weak polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(methacrylic acid) (PMA) are templated on silicon oxide particles using the layer-by-layer adsorption. The colloidal template is removed with a buffer system of hydrofluoric acid and ammonium fluoride. With this buffer system, the template can be dissolved in mild pH conditions, where the polymeric layers are still stable. The morphology and the thickness of the resulting capsules are investigated with atomic force microscopy. The resulting hollow capsules show pH-dependent properties. The shells are stable over a broad pH range and swell and immediately dissolve for pH values below 2.3 and above 11. If the molecular weight of the poly(methacrylic acid) is increased, the enhanced entanglement of the polymers results in a reversible swelling of the capsules at low and at high pH. The swelling degree is probed with confocal laser scanning microscopy. In addition to the pH-dependent size variations, the different ionization degree of poly(methacrylic acid) as a function of pH is used for the selective binding of calcium ions.

Binding Sites↗

Molecular evidence for association between the sphingobacterium-like organism "Candidatus comitans" and the myxobacterium Chondromyces crocatus.

Seven strains of the myxobacterium Chondromyces crocatus, isolated from widely separated geographic regions, were investigated for the presence of an associate gram-negative, rod-shaped companion bacterium that is phylogenetically related to the genus Sphingobacterium and has been named "Candidatus comitans" (C. A. Jacobi, E. Stackebrandt, H. Reichenbach, and B. J. Tindall, Int. J. Syst. Bacteriol. 46:119-122, 1996). Five of the Chondromyces strains were found to be associated with a companion bacterium, and one strain lost its companion during the study. A 16S ribosomal DNA (16S rDNA) clone library was generated for each Chondromyces culture. Sequence similarity was > 99.1% for all but one strain of C. crocatus and all but one strain of "Candidatus comitans". The three analyzed 16S rDNA clone sequences of the companion of Cm c7 indicated that this companion strain is slightly less related to the other companion strains. The association between the companion and the myxobacterium including the sporangioles was determined by in situ hybridization with fluorescently labeled rRNA probes and scanning confocal laser microscopy. Based on these results, there are indications that the companion strains may survive environmental stress by inclusion in the aggregates and in the sporangioles of the myxobacterium.

Bacteria↗

Observation of recording pits on phase-change film using a scanning probe microscope.

A phase-change film is a key material for optical data storage media such as rewritable compact disks (CD-RW) and digital versatile disk random access memory (DVD-RAM). Data pits are recorded as differences in crystal state (crystallized state vs. amorphous state) on phase-change film. It is very important to distinguish the crystal state difference in a very small area for material research of phase-change film. Measuring size and shape of recorded data pits is also very important for the development to achieve good data reliability and high data density of optical data storage media. The crystal state difference in very small areas of phase-change film is successfully observed by Kelvin probe force microscopy (KFM) and scanning near-field optical/atomic force microscopy (SNOAM). The advantage of KFM and SNOAM for measuring physical property differences in a very small area is demonstrated.

Journal Article↗

Mechanisms by which intracellular calcium induces susceptibility to secretory phospholipase A2 in human erythrocytes.

Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A(2) (sPLA(2)). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA(2). Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A(2) was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA(2) is a consequence of increased order of membrane lipids.

Biological Transport↗

Backscattered electron imaging of cultured cells: application to electron probe X-ray microanalysis using a scanning electron microscope.

We report a simple method to study the elemental content in cultured human adherent cells by electron probe X-ray microanalysis with scanning electron microscopy. Cells were adapted to grow on polycarbonate tissue culture cell inserts, washed with distilled water, plunge-frozen with liquid nitrogen and freeze-dried. Unstained, freeze-dried cultured cells were visualized in the secondary and backscattered electron imaging modes of scanning electron microscopy. With backscattered electron imaging it was possible to identify unequivocally major subcellular compartments, i.e. the nucleus, nucleoli and cytoplasm. X-ray microanalysis was used simultaneously to determine the elemental content in cultured cells at the cellular level. In addition, we propose some improvements to optimize backscattered electron and X-ray signal collection. Our findings demonstrate that backscattered electron imaging offers a powerful method to examine whole, freeze-dried cultured cells for scanning electron probe X-ray microanalysis.

Animals↗

The crystalline pattern of calcium in different topographical regions of the otoconial membrane. An electron probe and spectroscopic diffraction study.

Quantitative electron probe microanalysis and electron spectroscopic diffraction analysis was used to determine the gradient of distribution of calcium and its crystalline pattern at different levels (lower gelatinous membrane, upper gelatinous membrane and otoliths) in the otoconial membrane of adult OF1 mice. Our quantitative electron probe microanalytical data, obtained with scanning-transmission electron microscopy, indicated that there was a gradient in calcium concentration which increased from the vestibular surface towards the otoliths. Differences between the three regions of the otoconial membrane were statistically significant in both the utricle and saccule. Our results with electron spectroscopic diffraction revealed an increasing crystalline development from the lower gelatinous membrane towards the otoliths. Our findings with both techniques suggest that the gelatinous membrane is involved in the maturation and crystallization of the otoliths.

Animals↗

[Fluorescence quantification of intracellular calcium of rat smooth muscle cells].

OBJECTIVE: To explore the kinetic changes of calcium in rat smooth muscle cells and establish a method for quantification of intracellular calcium. METHODS: Rat mesenteric arteriolar smooth muscle cells (ASMCs) were isolated and the kinetic changes of calcium were measured using highly sensitive Ca2+ fluorescent probe indo-1 with laser scanning confocal microscopy (LSCM). The dissociation constant values (Kd) of the fluorescent probe indo-1 was measured at 37 degrees Celsius, and according to the conversion formula from fluorescence intensity to concentration, the concentration of Ca2+ was calculated. RESULTS: Analysis of the fluorescent images using LSCM showed that [Ca2+]i in the ASMCs were significantly elevated in response to stimulation with dexamethasone, and spontaneous calcium waves as well as intracellular calcium overloading were observed occasionally. CONCLUSION: Fluorescence quantification with LSCM is applicable for detecting the kinetic changes of intracellular [Ca2+]i.

Animals↗

Preparation of silica encapsulated quantum dot encoded beads for multiplex assay and its properties.

Novel -COOH modified polystyrene beads were prepared by sulfonation grafting, and the surface area and pore volume are greatly improved in comparison with the swelling-treated beads. The optimization coating time is 4 h, and the corresponding -COOH content is approximately 2.1 mmol/g. The scanning electron microscope results show that the silica particles deposited on the beads and formed a silica shell that decreases the leakage of quantum dots (QDs) preferably and improves the bar code stability greatly. The anti-photobleaching of silica-coated beads was studied systemically, and the results show that the half-decay time (t1/2) of the coated beads increases to 537 s--seven times longer than that of the uncoated ones. Further DNA probe hybridization experiments indicated that the coding signal and target signal can be detected simultaneously and that the assays based on these probe-conjugated silica/QD/polystyrene beads have good specificity and sensitivity that can detect a concentration as low as 0.01 microg/ml target DNA in denatured calf thymus DNA solution, indicating that it is feasible to use this kind of bead for multiplex analysis.

DNA↗

The surface oxidation potential of human neuromelanin reveals a spherical architecture with a pheomelanin core and a eumelanin surface.

Neuromelanin (NM) isolated from the substantia nigra region of the human brain was studied by scanning probe and photoelectron emission microscopies. Atomic force microscopy reveals that NM granules are comprised of spherical structures with a diameter of approximately 30 nm, similar to that observed for Sepia cuttlefish, bovine eye, and human eye and hair melanosomes. Photoelectron microscopy images were collected at specific wavelengths of UV light between 248 and 413 nm, using the spontaneous-emission output from the Duke OK-4 free electron laser. Analysis of the data establishes a threshold photoionization potential for NM of 4.5 +/- 0.2 eV, which corresponds to an oxidation potential of -0.1 +/- 0.2 V vs. the normal hydrogen electrode (NHE). The oxidation potential of NM is within experimental error of the oxidation potential measured for human eumelanosomes (-0.2 +/- 0.2 V vs. NHE), despite the presence of a significant fraction of the red pigment, pheomelanin, which is characterized by a higher oxidation potential (+0.5 +/- 0.2 V vs. NHE). Published kinetic studies on the early chemical steps of melanogenesis show that in the case of pigments containing a mixture of pheomelanin and eumelanin, of which NM is an example, pheomelanin formation occurs first with eumelanin formation predominantly occurring only after cysteine levels are depleted. Such a kinetic model would predict a structural motif with pheomelanin at the core and eumelanin at the surface, which is consistent with the measured surface oxidation potential of the approximately 30-nm constituents of NM granules.

Animals↗

The effect of a tannin-fluoride mixture on human dental enamel.

In this study, the effect of a tannin-fluoride mixture (Ta-F, 0.5% tannic acid, 450 ppm fluoride, pH 5.9) on dental enamel has been investigated by using scanning electron microscopy (SEM), electron probe microanalysis (EPMA) and X-ray diffraction (XRD), compared with the effect of acidulated phosphate fluoride (APF, 0.015 M phosphoric acid, 450 ppm fluoride, pH 5.3). Under the SEM, a large number of spherical globules (1-5 microns in diameter) were observed on the enamel surface treated with Ta-F. On a fractured cross section, these large globules showed a columnar appearance, measuring 2.5-5 microns each in height. They had a good range and formed a single coating layer on the enamel, whereas on the APF-treated enamel only very small spherical globules 0.1-0.5 micron in diameter were seen. Moreover, three types of connective patterns were observed between the basal ends of these columnar deposits and the enamel surface: (a) a pattern loosely attached to the enamel surface, (b) a pattern partially connected with the crystals of enamel, and (c) a pattern inserted into the pores of enamel. These columnar deposits also showed very strong resistance either to acid decalcification or to water washing. By EPMA and XRD examinations, a remarkable elevation of the fluoride profile accompanied by a high elevation of calcium was observed and CaF2 peaks were detected on the enamel surface. These results suggest that the columnar deposits might contain the CaF2-like substances and possess unique morphological and qualitative features which are quite different from the deposits found after APF or NaF treatment as described in previous studies.

Acid Etching, Dental↗

Calcium phosphate formation on the phosphorylated dental bonding agent in electrolyte solution.

The aim of the present study was to study the mineral formation on a phosphorylated dental bonding agent using a mineralization inductive solution. Clearfil Photobond, which contained phosphate monomer, was cured by photo-irradiation and heat treated, and was then immersed in Hanks' balanced salt solution (HBSS) with pH = 7.4 for 1, 3, 5, 7, 14, and 28 days at 37 degrees C. The white substances were deposited on the phosphorylated polymer, i.e. cured Photobond disk, after the immersion in HBSS. The white substances become visible after 3 days immersion. After 7 days immersion, surface of the phosphorylated polymer disk was almost covered with white substance layers. The measurement of white substances by means of X-ray diffraction, Fourier-transform infrared and electron probe microanalysis revealed that their main component was carbonate-containing hydroxyapatite. Scanning electron microscopy pictures showed that a large number of globules of hydroxyapatite were fused together, and that each globule was composed of a group of numerous thin-film form flakes uniting and/or clustering together. The results obtained in this study concluded that the presence of phosphonic acid and phosphate group of phosphorylated dental bonding agent enhanced the nucleation and growth of hydroxyapatite crystals on its surface.

Biocompatible Materials↗