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PubMed1983

Vitamin E--a selective inhibitor of the NADPH oxidoreductase enzyme system in human granulocytes.

The cellular sites of H2O2 formation in phagocytizing granulocytes have been identified with cerium chloride. A precipitate was visible in phagosomes and on plasma membranes from intact normal cells in the presence of either 0.71 mM NADH or NADPH. X-ray microanalysis permitted identification of cerium deposition within the phagosomes even in the absence of reduced pyridine nucleotides. Catalase ablated the formation of the reaction product. Intact granulocytes obtained from subjects receiving 1600 units of vitamin E daily for 2 weeks exhibited reaction product in the presence of NADH but not NADPH. Intact cells from subjects treated with vitamin E demonstrated diminished numbers of phagocytic vesicles containing reaction product. During phagocytosis the granulocytes treated with vitamin E consumed oxygen but exhibited significantly reduced rates of hydrogen-peroxide-dependent glucose-1-14C oxidation to 14CO2. Isolated phagocytic vesicles obtained from granulocytes after ingestion of opsonized lipopolysaccharide-paraffin oil droplets contained reaction product when exposed to 0.71 mM NADPH. No reaction product was evident at 0.71 mM NADH but was evident at 2.0 mM NADH. Isolated phagocytic vesicles from the granulocytes of subjects receiving vitamin E exhibited reaction product only in the presence of NADH. These observations suggest that vitamin E interferes with the electron transport chain apparently required for the oxidation of NADPH to form H2O2 in the phagocytizing granulocyte.

Cytochrome Reductases↗
PubMed1985

Stimulus-permeability coupling in rat pulmonary macrophages challenged by Pseudomonas aeruginosa. An X-ray microanalysis study.

Electron probe X-ray microanalysis (XRMA) of freeze-dried ultrathin sections provides the capability of measuring intracellular elemental content. This methodology was used to investigate the stimulus-permeability coupling responses associated with phagocytosis of Pseudomonas aeruginosa by cultured pulmonary alveolar macrophages (PAMs) of rats. PAMs were challenged with P. aeruginosa suspended in Gey's buffer at a bacteria to PAM ratio of 50:1 for 1 h at 37 degrees C. A 1-mm3 pellet of the unchallenged control PAMs, challenged PAMs and P. aeruginosa alone was quench-frozen in nitrogen-cooled, liquid propane, and 0.1-micron cryosections were cut at -100 degrees C. X-ray spectra were collected for nucleus and cytoplasm of 39 control PAMs, 36 challenged PAMs and 40 P. aeruginosa. Concentrations (mmole/kg dry weight) were obtained for Na, Cl, K, Ca, Mg, P, S for each cell. In the control PAMs, the content was similar to other mammalian cells. Moreover, there were no differences in elemental content between nucleus an cytoplasm. In the challenged PAMs, Na concentration was 4 times that of control PAMs (p less than 0.001) whereas Cl was double (p less than 0.001), K was 29% lower (p less than 0.001), and Ca was 4 times higher (p less than 0.05). The elemental concentration profile in the P. aeruginosa was distinctly different from that of the PAMs: higher Na, Ca, Mg, but lower Cl and K values. These results demonstrated elemental content changes in cultured PAMs challenged with P. aeruginosa that indicate a stimulus-permeability response by membranes associated with the phagocytic process.

Animals↗
PubMed1987

Subcellular calcium and magnesium mobilization in rat liver stimulated in vivo with vasopressin and glucagon.

The total Ca2+ content of the endoplasmic reticulum and the total Ca2+ and Mg2+ content of mitochondria were determined by electron probe microanalysis of rat liver rapidly frozen in vivo following brief (5-15 s) stimulation with vasopressin or prolonged (10-12 min) stimulation with vasopressin + glucagon. Brief vasopressin injection into the anterior mesenteric vein released 1.8 +/- 0.3 (S.D.) mmol of Ca2+/kg dry weight, from the rough endoplasmic reticulum (p less than 0.01), reducing Ca2+ content of the endoplasmic reticulum from 4.4 +/- 0.2 (S.E.) (controls) to 2.6 +/- 0.2 mmol of Ca2+/kg dry weight. Following vasopressin injection, endoplasmic reticulum Ca2+ was also significantly (p less than 0.025) lower than that in brief sham injected animals (3.5 +/- 0.2 mmol/kg dry weight). Mitochondrial Ca2+ was between 1.0 and 2.3 (+/-0.2) mmol/kg dry weight of mitochondrion, under all conditions studied, and no significant differences were observed. Both hormonal and brief sham injection into the anterior mesenteric vein increased mitochondrial Mg2+ from 42 (+/-0.8) to 49 (+/-1.8) mmol/kg dry weight (p less than 0.05). Hormonal stimulation of Mg2+ uptake was further confirmed by injection of vasopressin + glucagon into the jugular vein (to avoid any stimulation of the liver by the anterior mesenteric vein injection itself); mitochondrial Mg2+ increased from 43 (+/-0.9) (10-min sham) to 57 (+/-1.3) mmol/kg dry weight, with 10-min vasopressin + glucagon injection (p less than 0.01). These results demonstrate that hormones can release Ca2+ from the endoplasmic reticulum and modulate mitochondrial Mg2+ content in vivo without causing detectable changes in mitochondrial Ca2+.

Animals↗
PubMed1998

Changes in magnesium content and subcellular distribution during retinoic acid-induced differentiation of HL60 cells.

Magnesium (Mg) is required for cellular proliferation; however, the differences in subcellular regulation of Mg between proliferating and differentiated cells has not been determined. We used electron probe microanalysis (EPMA) to investigate the subcellular distribution of Mg in HL60 cells (a promyelocytic leukemia cell line) before and after retinoic acid (RA)-induced differentiation. Most intracellular Mg is bound to ATP and the Mg-ATP complex regulates several metabolic enzymes. We also compared alterations in Mg content following differentiation with the changes in ATP and ADP levels. Using atomic absorption spectrophotometry, we observed a significant decrease (-20%) in cellular Mg content in RA-differentiated HL60 cells. To investigate which intracellular compartments were involved in these changes, we analyzed subcellular elemental composition in freeze-dried cryosections of rapidly frozen undifferentiated and differentiated HL60 cells by EPMA. Following differentiation of HL60 cells, we observed an 18% decrease in Mg content in both the cytoplasm (regions of the cell excluding mitochondria and nuclei) and mitochondria. There was also a significant (40%) decrease in cytoplasmic Ca content after RA-induced differentiation. Nuclear Mg concentration was not significantly different between differentiated and undifferentiated HL60 cells, although differentiation was accompanied by a 30% decrease in the nuclear K/Na ratio. After differentiation, cellular ATP and ADP content decreased by 31 and 40%, respectively. We conclude that during exit from the cell cycle, Mg redistributes within cells and that the decrease in cytoplasmic and mitochondrial Mg is accompanied by a decrease in ATP and ADP content.

Adenosine Diphosphate↗
PubMed1993

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↗
PubMed1981

Observations on electron probe x-ray microanalysis compared to other methods for measuring intracellular elemental concentration.

Electron probe X-ray microanalysis provides the capability of accurately measuring the concentration of a number of elements at the subcellular level in tissues which are appropriately prepared using cryofixative procedures. This preview compares quantitative data obtained by the authors, using X-ray microanalysis, with quantitative data obtained in other laboratories using routine chemical procedures such as flame photometry, atomic absorption spectrophotometry, titrimetry, and ion-selective electrodes. Results are compared for cells in whole tissue, for cells in suspension (erythrocytes), and for subcellular analysis. Subcellular elemental data obtained after cell fractionation and isolation and after cryomicrodissection are considered. Agreements and differences between the results obtained by the different methods of analysis as compared to microprobe are discussed and the sources of the differences are explored. The biological significance of microprobe concentration data itself is limited without additional information regarding the state of the elements, such as oxidation state, ionic activity, degree of binding, etc. In this regard, the supplementation of subcellular elemental concentration with ionic activity and flux data for the large amphibian oocyte is presented as an example of how measurement of multiple parameters can be used to explain the maintenance of subcellular concentration gradients.

Animals↗
PubMed1998

Morphology and pathogenesis of Liesegang rings in cyst aspirates: report of two cases with ancillary studies.

Liesegang rings are laminated ring-like structures occasionally found in benign cysts and abscesses. They have been confused with parasites (especially eggs), algae, calcifications, and psammoma bodies. We examined Liesegang rings from fine-needle aspiration (FNA) biopsies of a benign cyst of the breast and a complex renal cyst. Liesegang rings were best observed with Papanicolaou, hematoxylin-eosin, Masson's trichrome, acid-fast (AFB), and Gram stains, which accentuate the concentrically laminated morphology. An amorphous electron-dense core and fibrillary lucent concentric rings were seen with transmission electron microscopy with no significant energy peaks by electron probe microanalysis or distinct diffraction patterns by X-ray diffraction. This investigation indicates that Liesegang rings are composed of organic substances most likely formed by periodic precipitation from a supersaturated solution within cystic fluid. Awareness of the Liesegang phenomenon within cystic lesions will decrease the possibility of erroneous misdiagnosis as another type of pathologic process.

Cyst Fluid↗
PubMed2004

Ti and Fe speciation by X-ray photoelectron spectroscopy(XPS) and Mössbauer spectroscopy for a full crystal chemical characterisation of Ti-garnets from Colli Albani (Italy).

Different analytical and structural methods (Electron Probe Micro-Analysis, Single Crystal X-ray diffraction, X-ray Photoelectron Spectroscopy, Mössbauer spectroscopy) were combined to fully characterise the crystal chemistry of natural Ti-bearing garnets from Colli Albani (Lazio, Italy). The study of the relevant complex crystal chemistry ( large number of cation substitutions affecting the three independent X(8-fold), Y(6-fold) and Z(4-fold) crystallographic sites and Fe and Ti transition elements exhibiting several oxidation states and coordination environments) benefited from the multi-technique approach. Electron probe microanalysis provided elemental composition of the analysed samples, which have low Ti-content (TiO2 in the range 1.99 - 3.48 wt %) and slightly different Fe/Al ratios. For all samples, two doublets were fitted to room temperature Mössbauer spectra and assigned to Fe3+(Y) (approximately 95%) and Fe2+(X) (approximately 5%). Up to three doublets (Ti3+(Y), Ti4+(Y), Ti4+(Z)) were fitted to XPS spectra and yielded direct evaluation of Ti site population. XPS technique confirmed its potential for the study of speciation of Ti in minerals.

Crystallization↗
PubMed2005

Cement lines of secondary osteons in human bone are not mineral-deficient: new data in a historical perspective.

Using qualitative backscattered electron (BSE) imaging and quantitative energy dispersive X-ray (EDX) spectroscopy, some investigators have concluded that cement (reversal) lines located at the periphery of secondary osteons are poorly mineralized viscous interfaces with respect to surrounding bone. This conclusion contradicts historical observations of apparent highly mineralized (or collagen-deficient) cement lines in microradiographs. Such conclusions, however, may stem from unrecognized artifacts that can occur during scanning electron microscopy. These include specimen degradation due to high-energy beams and the sampling of electron interaction volumes that extend beyond target locations during EDX analysis. This study used quantitative BSE imaging and EDX analysis, each with relatively lower-energy beams, to test the hypothesis that cement lines are poorly mineralized. Undemineralized adult human femoral diaphyses (n = 8) and radial diaphyses (n = 5) were sectioned transversely, embedded in polymethyl methacrylate, and imaged in a scanning electron microscope for BSE and EDX analyses. Unembedded samples were also evaluated. Additional thin embedded samples were stained and evaluated with light microscopy and correlated BSE imaging. BSE analyses showed the consistent presence of a bright line (higher atomic number) coincident with the classical location and description of the cement line. This may represent relative hypermineralization or, alternatively, collagen deficiency with respect to surrounding bone. EDX analyses of cement lines showed either higher Ca content or equivalent Ca content when compared to distant osteonal and interstitial bone. These data reject the hypothesis that cement lines of secondary osteons are poorly mineralized.

Adult↗
PubMed2006

Characterization and redox properties of cytochrome c552 from Thermus thermophilus adsorbed on different self-assembled thiol monolayers, used to model the chemical environment of the redox partner.

The structure of cytochrome c552 (Cyt-c552) from Thermus thermophilus shows many differences to other c-type cytochromes. The rich lysine domain close to the heme does not exist in this cytochrome, allowing us to postulate that the interaction with its redox partner must be different to the cytochrome c/cytochrome c oxidase interaction. We report a study of Cyt-c552 adsorbed on self-assembled monolayers (SAMs) of functionalized alkanethiols used to mimic the chemical properties of its redox partner (ba3-oxydase). Hydrophilic (-COOH), polar (-OH), hydrophobic (-CH3), and mixed (-OH/-CH3) SAMs grafted on roughened silver electrodes were characterized by X-ray photoelectron spectroscopy. Surface enhanced resonance Raman spectroscopy (SERRS) was employed to determine the structure and the redox properties (E degrees and number of transferred electron) of the heme of Cyt-c552 adsorbed on roughened silver electrodes coated by the different SAMs. The surface that most closely models the environment of the ba3-oxidase is a mixed SAM formed by 50% polar [Ag-(CH2)5-CH2OH] and 50% hydrophobic [Ag-(CH2)5-CH3] alkanethiols. Only the native form B1(6cLS) of Cyt-c552 is detected by SERRS when the protein is adsorbed on such a surface that promotes a protein orientation favorable for the electron transfer (number of transferred electron = 1). We shall discuss the differences and similarities of the electron-transfer mechanism of Cyt-c552 compared to cyt-c.

Adsorption↗
PubMed2002

Effects of enamel matrix derivative to titanium implantation in rat femurs.

The effects of enamel matrix derivative (EMD; Emdogain) on new trabecular bone induction after pure bioinert titanium (Ti) implantation in the rat femur were examined by means of routine light and transmission electron microscopy, immunohistochemistry, and backscattered electron image analysis. Newly designed mini-Ti implants (3.5 mm in length and 1.6 mm in diameter) were placed in the corticotrabecular area of the femur with either EMD or its carrier, propylene glycol alginate, as control. On post-implantation days 4, 7, 14, and 30, the dissected femur was examined in the transverse direction through Ti implants. In both control and EMD-applied femurs, trabecular bone formation was recognized over the implant surfaces and within medullary cavities even at 4 days post-implantation. These newly formed bone trabeculae around the Ti implants were immunoreactive for bone sialoproteins as a bone matrix marker, and osteoclastic bone resorption became evident in these bone trabeculae after 7 days post-implantation. Although trabecular bone area around the implants was markedly decreased at 30 days post-implantation compared with those at 14 days, the trabecular bone areas in EMD-applied femurs were significantly greater than those in propylene glycol alginate-applied femurs at both 14 and 30 days post-implantation. Our results suggest that EMD is an effective biological matrix for enhancing new trabecular bone induction and resulting attachment of orthopedic prostheses to the recipient bone.

Animals↗
PubMedDate not supplied

Influence of alloy composition on the hardening of silver-tin dental amalgam.

The objective of the investigation was to examine the reactions of mercury with silver-tin alloys with compositions spanning the phase fields beta, (beta + gamma), gamma, and (gamma + Sn). The experimental methods employed include the application of light microscopy, scanning electron microscopy, and electron probe microanalysis. These techniques were used to investigate the mechanisms of reaction and to identify the nature and morphology of the reaction products formed on bulk specimens of the alloys. The progress and characteristics of the reactions that occur during hardening of amalgams prepared from powders of these alloys were monitored using a high-sensitivity dilatometer. These results were correlated with direct observations on the development of the microstructures. The reaction of mercury with the beta-phase alloy occurred rapidly and resulted in a very marked and rapid expansion during the initial stages of hardening. gamma-Phase alloys, on the other hand, reacted more slowly and contracted markedly during hardening. The behavior of amalgams made from alloys with compositions lying between these two extremes appeared to be explicable in terms of the characteristics of the separate phases from which they were constituted.

Absorption↗
PubMed2006

Effects of in vitro bone formation on the mechanical properties of a trabeculated hydroxyapatite bone substitute.

This study was designed to test the hypothesis that the mechanical properties of a trabecular bone substitute can be enhanced through in vitro tissue formation. Our specific objectives were to (1) determine the effects of in vitro marrow stromal cell-mediated tissue deposition upon a trabeculated hydroxyapatite scaffold on the strength and toughness of the resulting bone substitute; and (2) identify and characterize regions of newly deposited matrix and mineral. This work provides a basis for future investigations aimed at transforming a brittle hydroxyapatite scaffold into an osteoinductive, biomechanically functional implant through in vitro bone deposition. As hypothesized, the mechanical properties of the trabecular bone substitutes were significantly enhanced by in vitro tissue formation. As a result of cell seeding and a 5 week culture protocol, mean strength increased by 85% (p = 0.008) and energy to fracture increased by 130% (p = 0.003). Accompanying the enhancement of mechanical properties was the deposition of significant amounts of bone matrix and mineral. Fluorescence imaging, scanning electron microscopy, electron probe microanalysis, and nanoindentation confirmed the presence of bonelike mineral with Ca/P ratio, modulus, and hardness similar to that within human and rat trabecular bone tissue. This new mineralization was found to exist within a newly deposited parallel-fibered matrix both encasing and bridging between scaffold trabeculae. Taken as a whole, our results establish the feasibility of the production of an osteoinductive hydroxyapatite-based trabecular bone substitute with mechanical properties enhanced through in vitro bone deposition.

Animals↗
PubMed2005

Elemental distributions and microtensile bond strength of the adhesive interface to normal and caries-affected dentin.

The aim of this study was to evaluate the microtensile bond strength (microTBS) and the elemental contents of the adhesive interface created to normal versus caries-affected dentin. Extracted human molars with coronal carious lesions were used in this study. A self-etching primer/adhesive system (Clearfil Protect Bond) was applied to flat dentin surfaces with normal and caries-affected dentin according to the manufacturer's instructions. After 24 h water storage, the bonded specimens were cross-sectioned and subjected to a microTBS test and electron probe microanalysis for the elemental distributions [calcium (Ca), phosphorus (P), magnesium (Mg), and nitrogen (N)] of the resin-dentin interface after gold sputter-coating. The microTBS to caries-affected dentin was lower than that of normal dentin. The demineralized zone of the caries-affected dentin-resin interface was thicker than that of normal dentin (approximately 3 microm thick in normal dentin; 8 microm thick in caries-affected dentin), and Ca and P in both types of dentin gradually increased from the interface to the underlying dentin. The caries-affected dentin had lost most of its Mg content. The distributions of the minerals, Ca, P, and Mg, at the adhesive interface to caries-affected dentin were different from normal dentin. Moreover, a N peak, which was considered to be the collagen-rich zone resulting from incomplete resin infiltration of exposed collagen, was observed to be thicker within the demineralized zone of caries-affected dentin compared with normal dentin.

Dental Caries↗
PubMed1980

Intracellular analysis of sodium, potassium, and chloride in mouse erythrocytes.

Recently, Cameron et al. ('79) published measurements of intracellular solute amounts (expressed as mmoles per kilogram dry cell solids) obtained by energy dispersive electron probe microanalysis in different rodent tissues. In this communication, I wish to compare Cameron et al.'s ('79) erythrocyte values of Na, K, and Cl with those I have made using more conventional techniques of elemental analysis. This comparison is necessitated by Cameron et al.'s ('79) observation of extremely high intracellular sodium levels. If their findings are accurate, the possibility of a polymorphism with respect to intracellular Na levels therefore presents itself. If a polymorphism between different inbred strains of mice exists, and if, as in ruminants, this trait has a genetic basis, an examination of the genetic aspects of the control, differentiation, and the ultimate expression of the ion transport mechanisms responsible would undoubtedly provide insight into the molecular basis as well as the adaptive dynamics of transport systems in general.

Animals↗
PubMed1992

Dynamic calcium requirements for activation of human ventricular muscle calculated from tension-independent heat.

The heat and tension generated by strips of human left ventricle taken from nonfailing hearts were measured at 30 C before and after partial inhibition of ATP splitting by the contractile proteins. We used 2, 3-butanedione monoxime (BDM) (4mM) as the chemical inhibition agent and alterations in solution calcium concentration and stimulus frequency to estimate the heat associated with calcium cycling for a wide range of activation levels. Tension-independent heat (TIH) was used to calculate the total calcium cycled per twitch by assuming that two-thirds of TIH was due to ATP splitting by the sarcoplasmic reticulum CA2+ ATPase with a coupling ratio of 2 Ca2+/ATP split and that one-third of TIH was due to ATP splitting by the sarcolemmal Na+ -K+ ATPase supporting the Na+ -Ca2+ exchanger (1 Ca2+/ATP). The enthalpy of creatine phosphate hydrolysis buffering ATP was taken as -34 KJ/mol. There was a highly positive correlation between TIH and mechanical activation during steady-state and nonsteady-state stimulation. The estimated total calcium turnover per twitch at 39% activation (0.3 Hz pacing rate and 2.5 mM Calcium) was approximately 0.17 nmol/g wet weight. This estimate is less than that calculated from biochemical data describing the cellular content and Ca2+ affinity of major Ca2+ buffers, but is similar to values calculated from recent electron probe microanalysis experiments.

Adenosine Triphosphate↗
PubMed1986

On the interaction between anthralin and mitochondria: a revision.

Anthralin is an inhibitor of oxidative phosphorylation at concentrations found in vivo. ADP-stimulated oxygen consumption is diminished. Consequently, the rate of ATP synthesis is reduced and mitochondrial ATP content declines. Neither the isolated ATPase (F1F0-ATPase), nor the mitochondrial membrane-bound ATPase are influenced by the drug. Respiration under resting conditions is not affected. The experimental data unequivocally indicate that anthralin is not an uncoupler of oxidative phosphorylation, as previously stated. Furthermore, the interpretation that respiratory deficiency induced in yeast strains by anthralin is a consequence of petite mutations has to be reconsidered. Under in vivo conditions, anthralin inhibits respiration per se. Our experiments, including the electron spin resonance spectroscopy, reveal that anthralin alters mitochondrial membrane structure and function simultaneously. A redox or free-radical mediated step may be involved. In consequence, inhibition of ATP production occurs which may become the limiting factor for increased cellular metabolism in psoriasis.

Adenine Nucleotides↗