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PubMed2006

Chemical and mineralogical evaluation of slag products derived from the pyrolysis/melting treatment of MSW.

This paper provides the results of studies on the characteristics of novel material derived from pyrolysis/melting treatment of municipal solid waste in Japan. Slag products from pyrolysis/melting plants were sampled for the purpose of detailed phase analysis and characterization of heavy metal-containing phases using optical microscopy, electron probe microanalysis (EPMA), XRF and XRD. The study revealed that the slag material contains glass (over 95%), oxide and silicate minerals (spinel, melilite, pseudowollastonite), as well as individual metallic inclusions as the major constituents. A distinct chemical diversity was discovered in the interstitial glass in terms of silica content defined as low and high silica glass end members. Elevated concentrations of Zn, Cr, Cu, Pb and Ba were recorded in the bulk composition. Cu, Pb and Ba behave as incompatible elements since they have been markedly characterized as part of polymetallic alloys and insignificantly sulfides in the form of spherical metallic inclusions associated with tracer amounts of other elements such as Sb, Sn, Ni, Zn, Al, P and Si. In contrast, an appreciable amount of Zn is retained by zinc-rich end members of spinel and partially by melilite and silica glass. Chromium exhibits similar behavior, and is considerably held by Cr-rich spinel. The intense incorporation of Zn and Cr into spinel indicates the very effective enrichment of these two elements into phases more environmentally resistant than glass. There was no evidence, however, that Cu and Pb enter into the structure of the crystalline silicates or oxides that may lead to their easier leachability upon exposure to the environment.

Electron Probe Microanalysis↗
PubMed1986

Electron probe analysis of calcium content and movements in sarcoplasmic reticulum, endoplasmic reticulum, mitochondria, and cytoplasm.

Electron probe microanalysis (EPMA) of a variety of rapidly frozen nonmuscle (e.g., liver and retinal rods) and muscle cell systems indicates that the endoplasmic reticulum (ER) [in muscle, the sarcoplasmic reticulum (SR)] is the major intracellular store of Ca. In vascular smooth muscle, Ca stored in the SR can be released and recycled, and it is sufficient to activate maximal contractions even in those smooth muscles in which the volume of the SR is relatively small. The Ca content of mitochondria in situ in vascular smooth and striated muscles, in liver, and in retinal rods is low, indicating that mitochondria do not function as physiological regulators of cytoplasmic Ca2+ in any of the muscle or nonmuscle cells critically examined with EPMA. Mitochondria themselves may be regulated metabolically by small fluctuations in matrix free Ca2+. Massive accumulation of mitochondrial Ca occurs under pathological conditions, when mitochondria are exposed to abnormally high free Ca2+. In frog skeletal muscle, the return of Ca to the SR is characterized by two processes: a fast one (25% of the Ca released) associated with relaxation due to pumping by the SR, and a slow process (0.4/s) that occurs after relaxation and appears to be rate-limited by the removal of Ca from parvalbumin. Illumination in retinal rods causes no detectable change in the low endogenous Ca content of the outer segment.

Animals↗
PubMed1985

Calcium and sodium distribution and movements in smooth muscle.

Electron probe microanalysis (EPMA) has been used to study the subcellular distribution of Ca, Na, K, Cl, and Mg in smooth muscle. The EPMA results indicate that the sarcoplasmic reticulum (SR) is the major intracellular source and sink of activator Ca: norepinephrine decreases the Ca content of the junctional SR in portal vein smooth muscle. Mitochondria do not play a significant role in regulating cytoplasmic free Ca2+, but mitochondrial Ca content can be altered to a degree compatible with suggestions that fluctuations in matrix Ca contribute to the control of mitochondrial metabolism. The rise in total cytoplasmic Ca during a maintained, maximal contraction is very much greater than the rise in free Ca2+, and is probably in excess of the known binding sites available on calmodulin and myosin. Cell Ca is not increased in normal cells that are Na-loaded. The non-Donnan distribution of Cl is not due to compartmentalization, but reflects high cytoplasmic Cl. Na-loading of smooth muscle in K-free solutions is temperature dependent, and may exhibit cellular heterogeneity undetected by conventional techniques. The total cell Mg is equivalent to approximately 12 mM, and less than 50% of it can be accounted for by binding to ATP and to actin. Mitochondrial monovalent cations in smooth muscle are relatively rapidly exchangeable.

Animals↗
PubMed1987

Calcium and magnesium transport by in situ mitochondria: electron probe analysis of vascular smooth muscle.

The extent, time course, and reversibility of mitochondrial Ca2+ uptake secondary to cellular Ca2+ influx stimulated by massive Na+ efflux were evaluated by electron probe microanalysis of rabbit portal vein smooth muscle. Strips of portal vein were Na+ loaded for 3 hours at 37 degrees C in a K+-free 1 mM ouabain solution, after which rapid Na+ efflux was induced by washing with a Na+-free K+-Li+ solution (1 mM ouabain). Li+ washing Na+-loaded portal vein produced a large transient contraction accompanied by an increase (over 100-fold) in mitochondrial Ca2+ and also significant (p less than 0.05) increases in phosphorus and Mg2+. The Ca2+ loading of the mitochondria was reversed during prolonged Li+ wash, and by 2 hours, mitochondrial Ca2+, Mg2+, and phosphorus had returned to control levels. The maximal contractile response to stimulation remained normal, demonstrating that pathologic Ca2+ loading of mitochondria is reversible in situ and compatible with normal maximal force developed by the smooth muscle. Mitochondrial Ca2+ and phosphorus uptake were reduced but still significant when the Li+ wash contained 0.2 mM Ca2+ or when ouabain was omitted. The fact that mitochondrial Ca2+ loading accompanied submaximal contractions during 0.2 mM Ca2+-Li wash suggests "supranormal" affinity of mitochondria for Ca2+ and may be due, in part, to reverse operation of the mitochondrial Na+-Ca2+ exchanger. Mitochondrial Ca2+, Mg2+, and phosphorus uptake were eliminated when the Li+ wash was performed at 2 degrees C or when the wash contained no Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Progression of left ventricular hypertrophy does not change the sarcoplasmic reticulum calcium store in the spontaneously hypertensive rat heart.

The spontaneously hypertensive rat (SHR) is characterized by elevated blood pressure and the development of left ventricular hypertrophy. During compensatory hypertrophy in the SHR, (26 weeks) when baseline contractile function is normal or increased, the inotropic response to beta-adrenergic stimulation is impaired. We recently showed by electron probe microanalysis (EPMA) that the amount of Ca2+ stored in the sarcoplasmic reticulum (SR) following sympathetic stimulation is not decreased in the 26-week-old SHR heart. However, with disease progression, cardiac function declines further in the SHR and the response to beta-adrenergic stimulation is more impaired. To determine whether a decreased availability of SR Ca2+ is responsible for the severely depressed inotropic response in the older SHR, we used EPMA to measure directly the amount of Ca2+ stored in the SR following activation of the beta-adrenergic pathway in papillary muscles from 76-week-old SHR and Wistar-Kyoto (WKY) controls. In order to determine if there are other alterations in ion homeostasis, we also compared elemental content of A-band and mitochondria. Papillary muscles from 76-week-old SHR and WKY were stimulated by 10 microM isoproterenol and then rapidly frozen during relaxation. The elemental content of the junctional SR. A-band and mitochondria was measured by EPMA. We observed no significant difference in SR Ca2+ content between SHR and WKY. There was also no strain-dependent difference in mitochondrial or A-band Ca2+. Overall, these results indicate that the impaired response to beta-adrenergic stimulation in the SHR at 76 weeks is not due to altered availability of SR Ca2+.

Adrenergic beta-Agonists↗
PubMed2005

Physical properties of root cementum: Part 4. Quantitative analysis of the mineral composition of human premolar cementum.

BACKGROUND: The aim of this study was to perform a quantitative analysis of the calcium (Ca), phosphorus (P), and fluoride (F) concentrations in human first premolars. METHODS: This study was conducted on 18 maxillary or mandibular first premolars that were collected from 16 prospective orthodontic patients (10 male, 6 female), mean age 13.9 years (range, 11.7-16.1 years), requiring first premolar extractions. After extraction, the teeth were prepared for electron probe microanalysis. The Ca, P, and F concentrations were measured on the buccal and lingual surfaces at the midpoint of the cervical, middle, and apical thirds of the root from the outer to middle to inner third of the cementum. RESULTS AND CONCLUSIONS: In first premolar cementum, there was significant interindividual variation in the Ca, P, and F concentrations ( P = .024, .017, and .000, respectively). There was no significant difference in the Ca, P, and F concentrations of cementum between the buccal and lingual surfaces, except for a significantly higher F content at the cervical region on the buccal surface ( P = .000). There was a decreasing gradient in the Ca, P, and F concentrations from the cervical to the apical third of the root, which was highly significant from the cervical to middle third ( P = .000) and from the middle to apical third ( P = .000), except for F, for which there was no significant difference from the cervical to the middle third on the lingual surface ( P = .966). There was a significant increasing gradient in the Ca and P concentrations from the outer to inner third of cementum at the cervical ( P < .01) and middle ( P < .01) thirds of the root but no significant difference at the apical third of the root. For F, there was a significant decreasing gradient from the outer to the inner third of cementum at the cervical ( P < .01), middle ( P < .01) and apical ( P < .01) thirds of the root.

Adolescent↗
PubMed1995

Subcellular calcium pools of ischaemic and reperfused myocardium characterised by electron probe.

OBJECTIVE: The subcellular redistribution of calcium and other electrolytes was analysed in myocardium subjected to global ischaemia and reperfusion in order to establish possible causes of reperfusion injury. METHODS: Isolated ferret papillary muscles (maintained at 37 degrees C, 1.2 Hz stimulation) were exposed to ischaemic conditions for 1 h by isolating from room air, removing buffer, and exposing to a constant flow of water-saturated 95% N2/5% CO2 gas. Some muscles were "reperfused" for 5 min by exposing to control buffer. Electron probe microanalysis was used to measure subcellular electrolyte content. RESULTS: Ischaemia caused severe swelling and doubling of cell sodium [84(SEM 5) to 156(9) mmol.kg-1 dry weight (dw), P < 0.05]. Mitochondrial electrolyte concentrations were generally increased, most notably calcium [0.40(0.15) to 1.99(0.32) mmol.kg-1 dw, P < 0.05]. Electrolytes in other structures were more moderately affected. Reperfusion resulted in two general myocyte populations. Irreversibly injured cells exhibited contracture knots and an influx of extracellular fluid. Analysis was restricted to moderately injured cells, which had reduced swelling and varying numbers of vacuolated mitochondria. In the moderately injured cells, sodium remained high [167(11) mmol.kg-1 dw] and magnesium had increased from 39(2) to 52(2) mmol.kg-1 dw (P < 0.05). Mitochondrial calcium was near control levels [0.64(0.42) mmol.kg-1 dw]. Junctional sarcoplasmic reticular calcium significantly increased [6.56(0.59) to 18.53(1.64) mmol.kg-1 dw in control and reperfused cells, respectively, P < 0.05], while calcium had not changed significantly in T tubule lumen or "3rd compartment", composed of sarcoplasmic reticulum, T tubules, and free sarcoplasm. "3rd compartment" and junctional sarcoplasmic reticulum results suggest that sarcolemma bound calcium had decreased. CONCLUSIONS: Surviving myocytes had no change in total calcium, but showed a large degree of calcium redistribution. Mitochondria accumulated calcium during ischaemia, but released it upon reperfusion. Sarcoplasmic reticulum in reperfused cells appeared functional and may have helped maintain physiological [Ca2+]i by storing calcium released from mitochondria and the sarcolemma. Mitochondrial and sarcolemmal damage are proposed as critical factors in reperfusion injury.

Animals↗
PubMed2003

Intracellular chloride accumulation and subcellular elemental distribution during atrial fibrillation.

BACKGROUND: Ion channel remodeling occurs during atrial fibrillation (AF); however, the extent of alteration in the subcellular distribution of elements (Na, K, Cl, Ca, Mg, P) is unknown. Electron probe microanalysis was used to determine the total (free+bound) in vivo subcellular concentration of these elements during AF. METHODS AND RESULTS: The left atrial appendage (LAA) was snap-frozen in situ after pacing (640 bpm) for 3 minutes (n=5 dogs), 30 minutes (n=3), or 48 hours (n=5). Dogs in sinus rhythm (n=3) served as controls. Whole-cell, cytosolic, and mitochondrial elemental concentrations were measured in cryosections. LAA effective refractory period (ERP) was measured before and after pacing. LAA ERP decreased significantly after 48 hours (116+/-3 to 88+/-10 ms, P=0.02). Whole-cell Cl increased by 9.0 mmol/L and 17 mmol/L after 3 and 30 minutes of pacing, respectively (P<0.0001), without a concomitant increase in Na. However, at 48 hours, whole-cell Na was reduced by 51% (P<0.01). Cytosolic Ca increased by 1.1 mmol/kg dry wt after 3 minutes (P<0.005), but mitochondrial Ca remained low and unchanged. Cell size measured in transverse cryosections increased after 3 minutes of pacing (75+/-5 to 109+/-11 microm2, P=0.007) but returned to baseline by 30 minutes (66+/-5 microm2). CONCLUSIONS: Intracellular Cl accumulation induced by rapid pacing is a novel finding and may play a role in AF pathogenesis by causing resting membrane depolarization and ERP reduction. There was no evidence of cellular or mitochondrial Ca overload despite the development of electrical remodeling and transient increase in cytoplasmic Ca.

Animals↗
PubMed1991

Calcium is released from the junctional sarcoplasmic reticulum during cardiac muscle contraction.

We have used electron-probe microanalysis (EPMA) to address the question of Ca2+ release by junctional sarcoplasmic reticulum (JSR) as well as Ca2+ regulation by mitochondria (MT) during cardiac muscle contraction. Hamster papillary muscles were rapidly frozen during relaxation or at the peak rate of tension rise (+dT/dt). Total Ca2+ content was measured by EPMA in the JSR, within a MT, over the A band, and in the whole cell, in nine cells per animal (five animals per group). JSR Ca2+ content was found to be significantly lower in muscles frozen at the peak of contraction [7.3 +/- 1.3 (mean +/- SE) mmol Ca2+/kg dry wt] than in those frozen during relaxation (12.5 +/- 1.9 mmol Ca2+/kg dry wt; P less than 0.01), suggesting that Ca2+ is released from this storage site during cardiac muscle contraction. In contrast, MT Ca2+ content did not change significantly during contraction (0.4 +/- 0.1 mmol/kg dry wt) compared with relaxation (0.1 +/- 0.2 mmol/kg dry wt). A third group of muscles was frozen during relaxation after pretreatment with 10(-7) M ryanodine. Ca2+ content of the JSR was significantly decreased (P less than 0.01) in this group of muscles, (6.4 +/- 1.8 mmol/kg dry wt) compared with those frozen during relaxation in the absence of the drug. This suggests that the intracellular storage site with a decreased Ca2+ content in muscles frozen at the peak of contraction is the ryanodine-releasable store. These results provide the first direct measurement of the Ca2+ content of both JSR and MT during a normal cardiac muscle contraction and demonstrate that Ca2+ is released from the JSR during muscle contraction.

Animals↗
PubMed1993

Changes in mitochondrial calcium concentration during the cardiac contraction cycle.

OBJECTIVE: The aim was to examine whether mitochondrial Ca2+ fluxes are high enough to change mitochondrial and cytosolic calcium concentration during the contraction cycle. METHODS: Isolated guinea pig ventricular myocytes were stimulated with paired voltage clamp pulses until contractions were maximal (2 mM [Ca2+]o, 36 degrees C). At defined times of diastole or systole, the cells were shock frozen. Electron-probe microanalysis measured the concentration of total calcium in mitochondria (sigma Ca(mito)) and surrounding cytosol (sigma Cac). Other experiments were performed to evaluate DNP sensitive mitochondrial Ca2+ uptake from depolarisation induced [Ca2+]c transients (K5indo-1 fluorescence). RESULTS: At end of diastole, sigma Ca(mito) was 446 mumol.litre-1. During systole, sigma Ca(mito) increased with a 20 ms delay. A peak sigma Ca(mito) of 1050 mumol.litre-1 was measured 40 ms after start of systole, while 95 ms after start of systole sigma Ca(mito) had fallen to 530 mumol.litre-1. From the changes in sigma Ca(mito) the rates of net mitochondrial Ca2+ flux were estimated at 100 nmol.s-1 x mg-1 protein for Ca2+ influx and 36 nmol.s-1 x mg-1 protein for Ca2+ egress. Decay of sigma Ca(mito) was coupled to a rise in sigma Na(mito). sigma Cl(mito) and sigma K(mito) rose and fell in parallel with sigma Ca(mito), suggesting Ca2+ activation of mitochondrial anion and cation channels. Activation of the non-specific permeability can be excluded. Block of mitochondrial Ca2+ uptake with DNP (100 microM) or FCCP (10 microM) increased the amplitude of the [Ca2+]c transients for 1-3 min by about 50%; evaluation of mitochondrial Ca2+ uptake from DNP sensitive difference signals, however, was hampered by sequestration of mitochondrial Ca2+ into the sarcoplasmic reticulum. CONCLUSIONS: Mitochondrial calcium content changes during each individual contraction cycle; a substantial amount of calcium is taken up during the systole and released during later systole and diastole.

Animals↗
PubMed2006

Physical properties of root cementum: part 6. A comparative quantitative analysis of the mineral composition of human premolar cementum after the application of orthodontic forces.

INTRODUCTION: The aim of this study was to examine quantitatively with electron probe microanalysis (EPMA) the calcium (Ca), phosphorus (P), and fluoride (F) concentrations in human first premolar cementum after the application of light and heavy orthodontic forces. METHODS: Thirty-six maxillary and mandibular first premolars (18 experimental, 18 control) were extracted from 16 subjects (10 male, 6 female; mean age, 13.9 years; range, 11.7-16.1 years) who were randomly assigned to the light-force or the heavy-force group. In the light-force group, 25 g of buccally directed force was applied to the experimental premolar; in the heavy-force group, 225 g of buccally directed force was applied to the experimental premolar. The contralateral premolar served as the control. The experimental and control premolars were extracted 28 or 29 days after initial force application and prepared for EPMA. The Ca, P, and F concentrations were measured on the buccal and lingual surfaces at the midpoint of the cervical, middle, and apical thirds of the root from the outer to the middle to the inner third of the cementum. RESULTS: Little change was found in the mineral composition of cementum after the application of light forces; however, there was a trend toward an increase in the mineral composition (Ca, P, and F) of cementum at various areas of periodontal ligament compression. The application of heavy forces caused a significant (P = .000) decrease in the Ca concentration of cementum at certain areas of periodontal ligament tension. The application of both light and heavy orthodontic forces did not appear to influence the F concentrations in cementum. CONCLUSIONS: Heavy orthodontic forces cause alterations in the mineral content of cementum; light forces cause little change.

Adolescent↗
PubMed1999

Twitch-potentiation increases calcium in peripheral more than in central mitochondria of guinea-pig ventricular myocytes.

1. The mitochondrial total calcium content ([Ca]mt) was studied with electron probe microanalysis (EPMA) in isolated guinea-pig ventricular myocytes in order to answer the question of whether electrical stimulation increases [Ca]mt in subsarcolemmal and central mitochondria to a different extent. 2. In unstimulated myocytes subsarcolemmal [Ca]mt was (mean +/- s.e.m.) 535 +/- 229 micromol (kg dry weight (DW))-1 and central [Ca]mt was 513 +/- 162 micromol (kg DW)-1. These values do not differ and correspond to approximately 180 micromol calcium per litre of mitochondria or 180 microM. 3. Contractions were potentiated to an optimum by stimulation with trains of 12 paired stimuli. After potentiation with 12 paired action potentials, cells were shock-frozen 120 ms after the start of the first action potential of the 13th pair. Subsarcolemmal [Ca]mt was 1.3 +/- 0.4 mmol (kg DW)-1 (433 microM) and central [Ca]mt was 227 +/- 104 micromol (kg DW)-1 (76 microM). The difference was significant. 4. After potentiation with 12 paired voltage-clamp pulses, cells were shock-frozen 120 ms after the start of the first pulse of the 13th pair. Subsarcolemmal [Ca]mt was 2.2 +/- 1.0 mmol (kg DW)-1 (733 microM) and central [Ca]mt was 630 +/- 180 micromol (kg DW)-1 (210 microM). After removal of extracellular K+, five paired voltage-clamp pulses increased subsarcolemmal [Ca]mt to 2.1 +/- 0.8 mmol (kg DW)-1 (700 microM), which was significantly higher than the central [Ca]mt of 389 +/- 88 micromol (kg DW) -1 or 130 microM. 5. In unstimulated cells, [Na] and [K] in subsarcolemmal and central mitochondria were not different. In potentiated myocytes, subsarcolemmal [Na]mt was 236 +/- 20 mmol (kg DW)-1 or 79 mM, which is significantly higher than the central [Na]mt of 50 +/- 5 mmol (kg DW)-1 or 16 mM. 6. The differences in [Ca]mt and [Na]mt are attributed to subsarcolemmal cytosolic microdomains of elevated [Ca2+] and [Na+] generated during contractile potentiation by transmembrane Ca2+ and Na+ fluxes.

Animals↗
PubMed1988

Uptake of a fluorinated bisphosphonate by cultured bones.

The uptake of bisphosphonates into bone was studied using 19-day-old fetal rat bones cultured with a new fluorinated bisphosphonate, difluoromethylidene bisphosphonate (F2MBP). F2MBP uptake was assessed by determining the weight percent of fluoride using electron probe microanalysis. By 30 min the weight percent of fluoride was significantly greater in the F2MBP-treated bones than in controls and continually increased throughout the duration of the experiment to reach a fluoride concentration 6-fold greater than controls after 120 h of incubation. When the peripheral cortical bone was analyzed separately from the interior trabecular bone in the F2MBP-treated bones, the fluoride concentration in the periphery increased until 24 h and then remained somewhat constant, while the interior, which is more actively remodeling, showed a continual increase. The uptake of F2MBP during the 1 to 6 h time intervals demonstrated no differences between vital and devitalized bone and, thus, is not cell-mediated. Because analysis of free fluoride in F2MBP media incubated with bones showed that the concentration of fluoride was less than 1% of the total amount of fluoride, the fluoride detected by the probe was most likely that of the intact molecule and not free fluoride. The rapid uptake of the F2MBP molecule was supported by assessing the effects of short-term F2MBP treatment on subsequent bone resorption, as determined by the release of 45Ca from prelabeled bones. Bones treated with F2MBP for only 5 min exhibited reductions in the percentage of 45Ca released during the remainder of the 120 h incubation period similar to that when F2MBP was continuously in the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

In vitro corrosion resistance of plasma source ion nitrided austenitic stainless steels.

Plasma source ion nitriding has emerged as a low-temperature, low-pressure nitriding approach for low-energy implanting nitrogen ions and then diffusing them into steel and alloy. In this work, a single high nitrogen face-centered-cubic (f.c.c.) phase (gammaN) formed on the 1Cr18Ni9Ti and AISI 316L austenitic stainless steels with a high nitrogen concentration of about 32 at % was characterized using Auger electron spectroscopy, electron probe microanalysis, glancing angle X-ray diffraction, and transmission electron microscopy. The corrosion resistance of the gammaN-phase layer was studied by the electrochemical cyclic polarization measurement in Ringer's solutions buffered to pH from 3.5 to 7.2 at a temperature of 37 degrees C. No pitting corrosion in the Ringer's solutions with pH = 7.2 and 5.5 was detected for the gammaN-phase layers on the two stainless steels. The high pitting potential for the gammaN-phase layers is higher, about 500 and 600 mV, above that of the two original stainless steels, respectively, in the Ringer's solution with pH = 3.5. The corroded surface morphologies of the gammaN-phase layers observed by scanning electron microscopy are consistent with the results of the electrochemical polarization measurement.

Biocompatible Materials↗
PubMed2001

Phase identification of microfeatures using EPMA methods, especially high-resolution X-ray spectroscopy

Methods of electron-probe microanalysis (EPMA), with some input from scanning and transmission electron microscopy (SEM/TEM), are applied for the identification of micro-scale constituents in a solid matrix. The subject of the study is a magnesium alloy composite, which contains silicon carbide-based fibres made by a liquid metal infiltration process. Backscattered electron imaging of the composite in the SEM showed that during composite manufacture, fibres were chemically attacked by the metal, many of the fibres exhibiting three distinct grey levels, indicative of different reaction zones, and others appearing uniformly black. EPMA measurements showed that each region contained approximately 12wt.% oxygen and that penetration of the fibre by magnesium was accompanied by a reduction in the concentration of silicon and carbon. From studying the position and shape of specific X-ray lines it was shown that magnesium penetration involved a chemical reaction with silicon oxycarbide, established in earlier EPMA studies as one of the fibre constituents. Also, in the outermost region, aluminium in the alloy reacted with free carbon in the fibre to form aluminium carbide and with magnesium to produce a Mg-Al intermetallic. The composition of black fibres was quite different from the grey ones, with negligible silicon and only a small amount of aluminium. Oxygen levels in black fibres were consistent with complete oxidation, indicating these fibres were subjected, locally, to severe oxidising conditions during composite manufacture. In the metal matrix itself, particles of a mixed magnesium/aluminium oxide, silicon carbide and magnesium silicide were observed, the latter two phases forming as silicon and carbon were ejected from fibres.

Journal Article↗
PubMed1999

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy.

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g, washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 mM ammonium acetate, 300 mM sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10-11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.

Cell Compartmentation↗
PubMed1977

Vanadate is a potent (Na,K)-ATPase inhibitor found in ATP derived from muscle.

A potent (Na,K)-ATPase inhibitor purified from "Sigma Grade* ATP has been identified as vanadium using electron probe microanalysis and confirmed by microwave-induced emission spectroscopy and electron paramagnetic resonance spectroscopy. Sodium orthovanadate (Na3 VO4) is identical with the purified inhibitor with respect to ultraviolet absorbance, migration on thin layer chromatography, and inhibition of (Na,K)-ATPase. The (Na,K)-ATPase is in-inhibited 50% by 40 nM Na3 VO4 under optimal conditions (28 mM Mg2+) and the inhibition is 100% reversible by millimolar concentrations of norepinephrine. The physiological significance of this inhibition is discussed in relation to vanadium concentrations in vivo.

Adenosine Triphosphatases↗
PubMed1991

Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system.

Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes. Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis. Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta-glycerophosphate. Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio. In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20-fold. Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease. In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy. Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course. Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise. Type IX synthesis remained at undetectable levels throughout the time course. The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis. Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo. Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan. Numerous vesicular structures could be detected. Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils. There was no clear evidence of mineral association with extracellular vesicles. The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction. In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaline Phosphatase↗