Health symptoms among adults living near a coal-burning power plant.

Coal ash is a waste product generated when coal is burned for energy. The purpose of this study was to assess health symptoms in adults living near a coal-burning power plant and compare the symptoms to a non-exposed population. A community-based mixed methods study was conducted with four neighborhoods adjacent to a coal-burning power plant. The comparison population was not exposed to coal ash and did not live near a coal-burning power plant. Adults who lived near the coal-burning power plant were significantly more likely to suffer from respiratory (AOR = 5.27, 95% CI = 2.16-12.0), gingiva (AOR = 2.46, 95% CI = 1.46-4.15), and skin symptoms (AOR = 3.37, 95% CI = 2.09-5.43). Results suggest that health symptoms may develop in people living near coal-burning power plants.

Effects of amiloride treatment on U-118 MG and U-251 MG human glioma and HT-29 human colon carcinoma cells.

Human glioma (U-118 MG, U-251 MG) and human colon carcinoma (HT-29) spheroids and monolayers were continuously exposed to amiloride under physiological Na+ and HCO3- conditions. Amiloride in concentrations of 0.1-0.2 mM inhibited growth, while 0.5 mM or higher induced disintegration of the glioma spheroids within 4-6 days. Growth retardation of the HT-29 spheroids was achieved at concentrations of 0.4-0.5 mM and total growth inhibition and disintegration were achieved at 1.0 mM. Monolayer cultures of glioma cells were also more sensitive to amiloride than those of colon carcinoma cells. The higher amiloride concentrations induced pyknotic nuclei mainly in the central areas of the spheroids where the extracellular pH (pHe) was low. The amiloride-sensitive glioma spheroids had lower pHe than the colon carcinoma spheroids. The intracellular pH (pHi), measured in monolayers, was higher (7.11-7.18) in glioma cells than in colon carcinoma cells (6.94). High concentrations of amiloride, 1.0 mM for 1 h in combination with low Na+ concentrations, caused a strong pHi decrease in glioma cells but only a slight decrease in the colon carcinoma cells. The pHi measurements in glioma monolayers were carried out after 2-6 days of continuous exposure to 0.1 mM amiloride at physiological levels of Na+ and HCO3- to simulate the conditions during growth inhibition. After several days this caused, when growth already was inhibited, an acidification of pHi. Parallel measurements with X-ray microanalysis showed an increase of intracellular sodium and a decrease of intracellular potassium in the gliomas, while no such changes were seen in the colon carcinoma cells under identical conditions. It is concluded that the two glioma cell lines were more sensitive to amiloride, both as monolayers and spheroids, than the corresponding cultures of the colon carcinoma cell line. The inhibition of proliferation by amiloride seemed not to have a clear connection to pHi regulation.

Magnesium in newly formed dentin mineral of rat incisor.

Small amounts of magnesium are always detectable in addition to calcium and phosphorus in mineralized tissues such as dentin or bone. Magnesium has been considered to influence the mineralization process, especially crystal growth. The present study reports on the location and enrichment of magnesium in the newly mineralized dentin by using the high lateral resolution of energy dispersive X-ray microanalysis combined with scanning transmission electron microscopy. To this end, we have used the continuously growing rat incisor as a model for a collagenous mineralizing system. Dental tissue was dissected free and cryofixed in liquid nitrogen-cooled propane. The distribution of elements was measured in freeze-dried ultrathin cryosections. The magnesium distribution of the newly formed dentin area near the predentin area was found to be inhomogeneous. In certain small dentin areas, characteristical magnesium enrichments were observed. Further, high magnesium-to-phosphate molar ratios were found in these areas, and these were correlated with low calcium-to-phosphate molar ratios. Our results support the theory that magnesium is involved in the process of biological apatite crystal formation.

Calcium-sequestering organelles of Dictyostelium discoideum: changes in element content during early development as measured by electron probe X-ray microanalysis.

Starving Dictyostelium discoideum amoebae aggregate within a few hours by chemotaxis towards the attractant cAMP to form a multicellular organism. The differentiating cells possess rapid and efficient calcium buffering and sequestration systems which enable them to restrict changes in the cytosolic free calcium concentration temporally and spatially during their chemotactic reaction and allow the continuous accumulation of Ca2+ during development. In order to identify and to characterize calcium storage compartments, we analyzed the element content of amoebae at three consecutive stages of differentiation. Determination of the element distribution was done using energy-dispersive X-ray microanalysis of freeze-dried cryosections of rapid-frozen cells. Amoebae were frozen in the vegetative and aggregation-competent state and after formation of aggregates. Aggregation-competent as well as aggregated cells contained mass dense granules with large amounts of calcium together with phosphorous and either potassium or magnesium: in aggregation-competent cells calcium was colocalized with potassium, whereas in aggregated cells the mass dense granules contained calcium and magnesium. Although mass dense granules were also present in undifferentiated, vegetative cells, they contained only low amounts of phosphorous and potassium together with little Ca and Mg. We conclude that during their differentiation D. discoideum cells use an intracellular storage compartment to sequester Ca and other cations constantly throughout development.

Calcium stores in differentiated Dictyostelium discoideum: prespore cells sequester calcium more efficiently than prestalk cells.

Dictyostelium discoideum pseudoplasmodia exhibit a gradient of the cytosolic free Ca2+-concentration ([Ca2+]i) along their anterior-posterior axis involved in cell-type specific differentiation. [Ca2+]i is high in prestalk and low in prespore cells. We determined the content and localization of calcium and other elements in cryosectioned cells of pseudoplasmodia and fruiting bodies by X-ray microanalysis. Granular stores rich in Ca, Mg and P were identified. Average Ca was higher in prespore than prestalk granules (225vs 111 mmol/kg dry weight). Total Ca stored in granules was also higher in prespore than prestalk cells. The amount of P and S in granules differed between the two cell types indicating different store composition. In spores mean granular Ca was 120 mmol/kg dry weight. Stalk cells had smaller granules with 360 mmol Ca/kg dry weight. Complementary to microanalysis, vesicular Ca2+-fluxes were studied in fractionated cell homogenates. The rate of Ca2+-uptake was higher in pellet fractions of prespore than prestalk amoebae (4.7 vs 3.4 nmol/min x mg). Ca2+-release was greater in supernatant fractions from prestalk than prespore cells (16.5vs 7.7 nmol/10(8)cells). In summary, prestalk and prespore cells possess qualitatively different, high-capacity stores containing distinct amounts of Ca and probably being involved in regulation of the anterior-posterior [Ca2+]i-gradient.

Mass dense vacuoles in Drosophila Malpighian tubules contain zinc, not sodium. A reinvestigation by X-ray microanalysis of cryosections.

The intracellular storage of zinc in Malpighian tubules of Drosophila hydei was studied by X-ray microanalysis of freeze-dried cryosections. Mass dense vacuoles in the proximal region of the anterior larval Malpighian tubule cells were found to accumulate zinc, not sodium. The zinc content was enhanced considerably after addition of zinc to the food of the larvae. Zinc-containing vacuoles were also found after pupation. After starvation of larvae in sea water, Na was detected in these vacuoles in addition to Zn. A small increase of Na and a remarkable increase of Zn was found in the vacuoles after injection of Ringer solution with ouabain into the larvae. Similar vacuoles in cells of untreated posterior tubules exhibit only low zinc levels.

Morphology and element distribution of magnesium-secreting epithelium: the proximal tubule segment PII of dogfish, Scyliorhinus caniculus (L.).

Proximal tubule segment PII cells of marine elasmobranch fish were studied by transmission electron microscopy of thin sections, and X-ray microanalysis was performed with freeze-dried cryosections. Epithelial cells of PII are characterized by high and dense brush border at the apical side, elaborate folding of the lateral cell membrane and large basal extracellular labyrinth confined by a system of meandering cell extensions. Basal cytoplasmic zone, apical cytoplasmic zone, nuclei, mitochondria and apical small vacuoles were accessible for X-ray microanalysis. Concentrations of Na, Mg, P, S, Cl and K were different in the cytoplasmic zones along the basal-apical axis of the cell and in the organelles. PII cells lacked an apical tubulovesicular apparatus, instead they displayed an apical zone of smooth clear vesicles and small apical vacuoles. After freeze-drying, the small apical vacuoles and the smooth clear vesicles contained flocculent mass-dense material. Small apical vacuoles showed high concentrations of Mg (229 mmol/kg water), Na (132 mmol/kg water) and Cl (148 mmol/kg water). Sequestration of Mg in vesicles and small apical vacuoles and subsequent exocytosis between the microvilli of the brush border are supposed to be important steps in the transepithelial transport (tubular secretion) of magnesium by PII cells of marine fish.

The importance of the Golgi complex for epithelial ion transport in Drosophila Malpighian tubules, studied by electron microscopy, cytochemistry and X-ray microanalysis.

The distribution of potassium in the cells of Drosophila Malpighian tubules is not homogeneous. In the microvilli of the apical part of the cell the cytoplasmic potassium content was found to be 2 to 3 times higher than in the neighboring intermediate cytoplasm. Data obtained by electron microscopy, histochemistry and electron probe X-ray microanalysis indicate that glucosaminoglycans (GAGs), synthesized by the Golgi-ER complex, are responsible for potassium accumulation in the apical microvilli. Vesicles bud from the Golgi complex and then move to the apical cell region, where they discharge their contents into the cytoplasm or into the lumen. Budded vesicles also discharge their contents into the hemolymph space between the folds of the basal plasma membrane. GAGs, transformed to proteoglycans (PGs), were identified on the folds of the basal cell surface including basal lamina by reaction with alcian blue. Brefeldin A (BFA) was found to disintegrate Golgi-ER structures to vesicles, whereas budded vesicles vanished. Within the microvilli the K+-content decreased to 32%, the water content to 77%. These data provide evidence that the ER-Golgi complex is involved in the delivery of GAGs (and PGs) into the luminal space and the hemolymph. After disintegration of the Golgi complex, GAGs are missing as temporary ion stores from the vicinity of the membrane transporters.

Intracellular storage of sodium and magnesium in Drosophila Malpighian tubules. X-ray microanalysis of native cryosections.

Using electron probe X-ray microanalysis after cryofixation, cryosectioning and freeze-drying we investigated the content of electron-dark vacuoles in the intermediate cell region of the proximal segment of Malpighian tubules in Drosophila larvae. According to this method these vacuoles store sodium and magnesium in a high correlation (r = 0.98) of 5:1 ratio. Phosphorus, potassium and sulfur are also stored. In the intermediate groundplasm surrounding the vacuoles the element content is different from that in the vacuoles. The significance of vacuolar sodium and magnesium storage for the ionic metabolism is unknown. In addition to Na, Mg, P, K and S the vacuoles also contain 3-OH-kynurenine and other fluorochromes. With the pyroantimonate technique intravacuolar precipitates were demonstrated. X-ray microanalysis of the precipitates revealed sodium and calcium, although following cryofixation calcium was not detectable in the vacuoles by X-ray analysis.

X-ray microanalysis of organic thin sections in TEM using an UTW Si(Li) detector: comparison of quantification methods.

We compared Hall's peak to continuum ratio method with a peak ratio method in order to quantify light elements (C, N, and O) in organic specimens as a model for biological thin sections. X-ray spectra were recorded by an energy dispersive X-ray spectrometer equipped with an ultra thin window detector. Spectra were processed by means of a top-hat filter adapted to peak full-width half maximum. The peak intensities were measured by multiple least square fitting to reference spectra. For most elements of biological interest, theoretical and experimental k-factors were determined. Absorption correction was found to be important for quantitation of carbon, nitrogen, and oxygen. Boron was efficiently detected; however, quantitative analysis was not possible. We conclude from our experiments that the peak ratio method is more suitable for quantitation of elemental concentrations in biological thin sections than the peak to continuum method.

Element distribution in organelles of pancreatic acinar cells of rat, mouse, and pig investigated by energy-dispersive X-ray microanalysis.

The exocrine pancreas was long thought to be composed of identical subunits, the acinar cells that store the inactive forms of the digestive enzymes in zymogen granules (ZGs). These were generally seen as a homogeneous population of vesicles. This homogeneity was recently questioned: Digestive demands are answered by the release of specific enzymes and immunocytochemical labeling showed distinctive nonidentical populations of ZGs. We have aimed at finding concomitant differences in element contents. We analyzed by energy-dispersive x-ray microanalysis (EDX) the subcellular distribution of elements in acinar cells of resting and stimulated rat, resting mouse, and resting pig pancreas and compared the results with values from the literature. We found large variances in the concentrations of Na, Mg, P, S, Cl, K, and Ca in cytoplasm rich in endoplasmic reticulum (C/E), whereas the concentrations of P, Cl, K, and Ca in mitochondria and ZGs had surprisingly small variations. Na and Mg were detected in measurable amounts only in C/E and mitochondria and Ca was detectable only in ZGs. We could not find any other elements. We have not found clearly distinguishable populations of ZGs. We critically discuss our findings in comparison with the literature. Many discrepancies can be explained by the different preparation procedures. We show that it is questionable to present absolute values of concentration in biological specimens on the basis of EDX. The technique should, in our opinion, be used only for the study of relative concentrations.

Photoaffinity labeling of plasma membrane proteins involved in the transport of loop diuretics into hepatocytes.

To identify proteins involved in the hepatocellular uptake of loop diuretics, [3H]bumetanide was photoactivated by light flash in the presence of either intact isolated rat hepatocytes, rat liver basolateral plasma membranes or integral membrane proteins extracted from the basolateral plasma membranes. Proteins of 52-54, 48, 33, 27, 25 and 23 kDa in sodium dodecyl sulfate (SDS) gel electrophoresis were radiolabeled on intact hepatocytes. On liver basolateral plasma membranes a 50-52 kDa protein was the most intensely labeled protein. After separation into integral and associated membrane proteins by extraction with Triton X-114, radioactive labeling was only found in integral membrane proteins with a molecular weight of 50-52 kDa. Photoactivated bumetanide irreversibly inhibited the hepatocellular uptake of cholate, taurocholate but not of serine. Binding proteins for photoactivated bumetanide were absent on AS 30-D ascites hepatoma cells. Labeling of all proteins was sodium dependent in intact hepatocytes but was sodium independent in plasma membranes. Labeling was prevented by non-labeled bumetanide and by the loop diuretics piretanide and furosemide. Labeling protection was further achieved with organic anions such as bromosulfophthalein, rifampicin, probenecid and by the bile acids taurocholate, deoxycholate and dehydrocholate. The radiolabeled proteins did not belong to the bumetanide-sensitive NaCl/KCl co-transport system which apparently does not occur in intact isolated rat hepatocytes.

Potassium is involved in apatite biomineralization.

The biogenetic formation of mineral crystals, one aspect of biomineralization, is a multistep process of apatite formation throughout the growth of dentin tissue. An important step is the transformation of the non-mineralized predentin matrix to mineralizing dentin matrix and its biological control. In this study, the high capacity of elemental mapping is combined with single x-ray point measurements to elucidate whether special elements are involved in initiation or regulation of mineral nucleation. Directly at the mineralization front, micro-areas with a strong co-enrichment of phosphorus (e.g., as phosphate) and potassium are found. During the beginning of the calcium enrichment and the subsequent apatite mineral formation in the characteristic micro-areas, the content of potassium decreases significantly. These findings indicate that potassium is involved in the process of dentin mineralization.

Summer work and injury among middle school students, aged 10-14 years.

BACKGROUND: Little information exists on injury and factors associated with injury in working youth aged 10-14 years. Most studies do not involve children younger than 15. METHODS: A cross-sectional anonymous survey was administered to middle school students in five school districts and one large urban school in October 2001. RESULTS: Of the 3189 working middle school students who responded to the survey, the majority were employed in informal job settings, such as working for someone in a home, newspaper delivery, and working on family farms or in family businesses. Overall, 18% of children reported being injured at work. Of those injured, 26% reported that their injury was severe enough to affect their activities for more than three days. Variables that were associated with injury included having a "near-miss" incident at work (AOR 6.61, 95% CI 4.92 to 8.89), having a co-worker injured (AOR 2.65, 95% CI 1.95 to 3.60), and being asked to do something dangerous (AOR 2.25, 95% CI 1.61 to 3.14). CONCLUSIONS: Children are working and being injured in jobs that are not covered by existing child labour laws. Injury rates in non-covered occupations are high, warranting review of current laws.

Preparation of biological cryosections for analytical electron microscopy.

A cooling chain for studies of ultrastructure and elemental composition of cryofixed biological cells and tissues is described. The technique is demonstrated for yeast cells. The preparation steps such as cryofixation, cryosectioning, transfer into the electron microscope as well as imaging and X-ray microanalysis in the STEM are discussed with respect to the present possibilities and problems. Whereas freeze-dried cryosections can be studied routinely, imaging of the ultrastructure and measurement of the elemental distribution in frozen-hydrated sections turn out to be limited.

A cooling chain for studies of cryofixed biological specimens by scanning transmission electron microscopy and X-ray microanalysis.

A cooling chain is described which enables the transfer of frozen hydrated biological specimens (ultrathin cryosections as well as about 1 micrometer thick cultured cells) from a cryoultramicrotome into a scanning transmission electron microscope with a field emission gun. Transfer is done at 118 K, specimen temperature in the microscope is 165 K. Sublimation processes are controlled visually and by mass spectrometry. Electron micrographs and X-ray microanalytical spectra of cryofixed unstained tissue culture cells and rat liver tissue sections are described and discussed. Contamination of the specimen is much reduced by use of the cold stage.

Heavy metal cytotoxicity studied by electron probe X-ray microanalysis of cultured rat hepatocytes.

Cytotoxicity of the heavy metals gold, mercury, thallium and lead was studied by measuring the intracellular element distribution of cultured rat hepatocytes by energy dispersive electron probe X-ray microanalysis of freeze-dried cryosections in a scanning transmission electron microscope. Exposure of the cells to aqueous solutions containing heavy metal ions in concentrations reaching a critical concentration caused increase of intracellular sodium and chloride content accompanied or followed by decrease of intracellular potassium content. Thus, the intracellular potassium/sodium ratio drastically decreased from control values of approximately 10 to values below 1 before changes of cell morphology became visible. In experiments with gold or mercury the decrease of the potassium/sodium ratio was preceded by transient cytoplasmic increase of sulfur and phosphorus. Heavy metal concentrations exceeding the critical concentration also caused an increase of cytoplasmic calcium concentration and finally decay of the cell structure. Cytotoxicity of heavy metals was found to increase in the order Pb, Au, Tl, Hg. Cytotoxic effects by Au, Tl or Hg in moderate concentrations were reduced by simultaneous addition of Zn or Pb to the culture medium. The results obtained prove electron probe X-ray microanalysis of cryosections as a sensitive probe of cell viability.

Effects of the ionophores valinomycin, ionomycin and gramicidin A on the element compartmentation in cultured rat hepatocytes.

The element compartmentation in cultured rat hepatocytes was studied by electron probe X-ray microanalysis of freeze-dried cryosections after exposure of the cells to the ionophores valinomycin, ionomycin or gramicidin A. The most striking effect of these ionophores is the decrease of the intracellular potassium/sodium ratio from values of approximately 10 under control conditions to values below 1 after application of the ionophores. Changes of sodium, potassium and chloride are similar in cytoplasm and nucleus. However, elemental changes are delayed or impeded in mitochondria with respect to the surrounding cytoplasm. The water portion of cytoplasm and mitochondria slightly increases. Besides that, each ionophore has specific effects on the intracellular ion distribution. As compared to gramicidin A and ionomycin, valinomycin does not change the intracellular chloride content. Ionomycin induces calcium accumulation in mitochondria. The cytotoxic effects of the studied ionophores on the intracellular element distribution are more complex than supposed from their ion selective properties in membranes.

X-ray microanalysis of elements present in the matrix of cnidarian nematocysts.

The composition and concentration of elements, in particular those of metallic cations, present in the intracapsular matrix and the wall of nematocysts of various cnidarian species have been recorded by means of X-ray microanalysis performed on 100nm thick cryosections. The predominant cation detected in the nematocyst matrix of the hydrozoan Podocoryne carnea (medusa), the scyphozoan Aurelia aurita (scyphopolyp) and the anthozoan Calliactis parasitica (tentacles and acontia) is K(+). Mg(2+) prevails in tentacular cysts of Anthopleura elegantissima, Actinia equina and Anemonia viridis, whereas, the acrorhagial cysts of A. elegantissima and A. equina contain Ca(2+) instead of Mg(2+). The acrorhagial cysts of A. viridis contain Mg(2+) like those of the tentacles. In the tentacular nematocysts of Podocoryne carnea polyps (Hydrozoa) on the other hand ambiguous element contents were found indicating that the cysts of this species has no preference for a particular cation. The high values of sulfur recorded in the matrix and particularly the wall of all the cysts are reflecting the presence of numerous protein disulfide bonds within the structural components (wall, shaft, tubule) of the nematocysts.

X-ray microanalysis of ion contents in vacuoles and cytoplasm of the growing tips of a hydroid polyp as related to osmotic changes and growth pulsations.

Growth pulsations (GP) in hydroid polyps are associated with changes in vacuolar patterns which can be imitated by altering external osmolarity. With the use of X-ray spectroscopy we measured the elemental contents in the vacuoles and cytoplasm of the growing tips of a hydroid polyp, Podocoryne carnea, under various tonicity conditions. Under hypertonic condition which arrested the samples at the retraction phase of normal GP, the elemental content within the vacuolar compartment appeared to be similar to that of the external medium, confirming our previous conclusion about the dehermetization of the vacuolar compartment under these conditions. Under hypotonical condition which arrested samples at the extension GP phase (vacuoles isolated) element ratio data displayed an obvious bimodality. At least one of the data groups could be characterized by a significant increase in the concentrations of sodium and potassium, as related to Cl, Ca and Mg, and in comparison to the same ratios in hypotonical samples and those in the external medium. We suggest that under hypotonical conditions the isolated vacuolar compartment is formed by influx of sodium and potassium ions. These cations are accompanied by anions other than chloride. Potassium appears to be transferred into the vacuoles from the cytoplasm while the sodium derives from the external environment.