Changes in elemental content during apoptotic cell death studied by electron probe X-ray microanalysis

Recent data suggest that changes in ionic content, primarily potassium, play a pivotal role in the progression of apoptosis. However, the changes in total element content, i.e., sodium (Na), magnesium (Mg), phosphorous (P), chlorine (Cl), potassium (K), and calcium (Ca), during apoptosis have not been evaluated. Electron probe X-ray microanalysis (EPXMA) was used to measure total element content in U937 cells before and after the induction of apoptosis. As an experimental model we used U937 cells irradiated with ultraviolet (UV) light. Apoptosis was evaluated with phase-contrast microscopy, with scanning and transmission electron microscopy, and with the fluorescent dye bisbenzimide (Hoechst 33342). Plasma membrane permeability as a measure of cell death was determined by trypan blue dye exclusion. To investigate element content with EPXMA, cells were cryoprepared, i.e., cryofixed and freeze-dried, and analyzed as whole cells using a scanning electron microscope. We found that the UV irradiation induced rapid (within 2 h) morphological changes associated with apoptosis, such as plasma membrane blebbing, condensation of the chromatin, and the formation of membrane-bound apoptotic bodies. At this time, 95% of the apoptotic cells excluded trypan blue dye. EPXMA results demonstrated that UV light-irradiated apoptotic cells (cells with membrane-bound apoptotic bodies) had a lower Cl content (P < 0.001) and K content (P < 0.001) and a higher Na content (P < 0.001) in comparison with nonirradiated control cells. Also, P and Ca content was higher in apoptotic cells than in control cells, but this difference did not reach statistical significance. No differences were found in Mg. These data indicated that morphological changes characteristic of apoptotic cell death are related with significant changes in sodium, chlorine, and potassium content. In addition, we demonstrated that these changes in elemental composition were not associated with loss of cell membrane integrity.     

Ultrastructure and X-Ray Microanalysis of Blood Cells of Ascidia malaca

Ultrastructural studies of Ascidia malaca blood reveal particular cell types, which are characterized by a polymorphism in the organization of the electron dense cytoplasmic material. A new pathway of morula cell differentiation is suggested. X-ray microanalysis shows that vanadium is localized in vacuolated, granular and morular cells. Iron, which is accumulated by this species to a lesser degree than vanadium, is found in vacuolated amebocytes and, together with vanadium, in granular cells. Our results are discussed in the light of the relations between selective metal absorption in blood cells and their specialization and differentiation.     

The effects of the adrenoreceptor agonists phenylephrine and isoproterenol on the intracellular ion concentrations of branchial epithelial cells of brown trout (Salmo trutta L.)

Brown trout were fitted with indwelling, intraperitoneal catheters and injected with 4–6 mol · kg^-1 of the -receptor agonist phenylephrine or the -receptor agonist isoproterenol. The intracellular concentrations of sodium, chlorine, potassium and phosphorus in the pavement epithelial cells and the mitochondria-rich cells of the branchial epithelium were measured by X-ray microanalysis 1 h after the injection of the adrenoreceptor agonists. Injection with phenylephrine resulted in a significant increase in intracellular chlorine and potassium in mitochondria-rich cells and a significant but relatively smaller increase in chlorine in pavement epithelial cells. Injection with isoproterenol resulted in a significant increase in sodium and chlorine concentration in pavement epithelial cells and a significant decrease in potassium concentration. The only significant effect of isoproterenol injection on mitochondria-rich cells was a decrease in intracellular chlorine concentration. The results suggest that these adrenoreceptor agonists have a direct effect on the influx of Na⁺ and Cl^- across the branchial epithelium. These effects may be a mechanism for acid-base regulation during the severe stress conditions that elicit catecholamine release in vivo. These results corroborate previous studies using X-ray microanalysis which suggested that pavement epithelial cells are the sites of Na⁺ uptake in freshwater fish whilst Cl^- uptake occurs via mitochondria-rich cells.Abbreviations LTSEM low-temperature scanning electron microscope - MR cells mitochondria-rich cells - PE cells pavement epithelial cells - XRMA X-ray microanalysis     

Analysis of the surface morphology of recovered silicone mammary prostheses

The surfaces of 11 silicone mammary prostheses were examined by scanning electron microscopy (SEM) and energy dispersive x-ray spectrography (XES). Three of the prostheses were recovered from clinical cases and eight from guinea pig models. The surfaces of the prostheses were covered with an investing film and by 2- to 5-microns spheres aggregated in 40- to 200-microns-wide patches. The embeddedded spheres were blebbed and surrounded by pits. X-ray energy spectrography showed high levels of sodium, chlorine, and potassium. It is concluded from this evidence that phagocytic cells embed themselves within the envelope, perhaps by an immune-mediated phagocytic process.     

Salt excretion from the salt glands in Rhodes grass (Chloris gayana Kunth) as evidenced by low-vacuum scanning electron microscopy

Some salt-tolerant plants belonging to the Poaceae excrete salts to the leaf surfaces under salinity conditions, and the bicellular glands on their leaf surfaces have been postulated to excrete salt. However, clear evidence of the salt excretion from these bicellular salt glands has not been shown at the electron-microscope level because soluble attachments on the leaf surface are completely removed during specimen preparation for conventional electron microscopy. To determine whether the bicellular salt glands actually excrete salt, we examined the leaves of Rhodes grass (Chloris gayana Kunth), Poaceae, by scanning electron microscopy in a low-vacuum mode, which allows to observe specimens without preparation procedures. Unwashed and washed fresh leaf surfaces were examined, and excreted materials on the leaf surface were analyzed by energy dispersive X-ray spectrometry. On the unwashed leaf surfaces, globular materials were observed arranged along the same lines as the macrohairs of the leaf surface, but the salt glands were hardly observed. After leaf surfaces were washed, the globular materials disappeared, and the salt glands appeared localized at the same lines as the macrohairs. Density of the globular materials observed under unwashed conditions and the salt glands under washed conditions was equal. These findings indicate that the glands indeed excrete globular materials just above their cap cells. The excreted materials contained sodium, chlorine, and potassium, and the counts of sodium and chlorine was increased greatly with NaCl treatment of the plants. After removing the excreted materials, most of the cap cells of the salt glands were smooth globular, without ruptures in their cuticle. We conclude that the leaves of Rhodes grass indeed excrete salt from the bicellular salt glands, but without rupturing the cuticle on the cap cell.     

The element distribution in ultrathin cryosections of cultivated fibroblast cells

Summary A preparation method for measurements of the intracellular distribution of elements in tissue culture cells is described which is based on cryofixation, cryoultramicrotomy, cryotransfer and X-ray microanalysis in a scanning transmission electron microscope. Dry weight concentrations of phosphorus, sulfur, chlorine and potassium in the nucleus, the cytoplasm and the mitochondria of L929 fibroblast cells of the mouse are reported. The preparation and quantitation procedures are discussed with respect to present limitations and possible improvements of the method.The paper was presented partly as a poster at the joint meeting on clectron microscopy of the Deutsche Gesellschaft für Elektronenmikroskopie and the Belgische Vereniging voor Elektronenmikroskopie in Antwerpen, 11.–16. 09. 1983     

Coelomocyte Morphology in the Holothurians Apostichopus japonicus (Aspidochirota: Stichopodidae) and Cucumaria japonica (Dendrochirota: Cucumariidae)

The cellular composition of the coelomic fluid of the Far Eastern holothurinans Apostichopus japonicus and Cucumaria japonica was studied using light and transmission electron microscopy and histochemistry. In the coelomic fluid of A. japonicus, the following types of coelomocytes were distinguished: progenitor cells; amoebocytes; vacuolated cells; small (or young) morula cells; morula cells of type I, type II, and type III; crystal cells; and vibratile cells. In the coelomic fluid of C. japonicawere found progenitor cells, amoebocytes, vacuolated cells, morula cells of type I and type II, crystal cells, and hemocytes containing a respiratory pigment. The issue of stem cell type, which gives rise to coelomocytes, is discussed.     

SCANNING ELECTRON MICROSCOPY AND ENERGY DISPERSIVE X-RAY ANALYSIS OF CHALK SECRETING LEAF GLANDS OF OF PLUMBAGO CAPENSIS

Chalk secreting leaf glands of Plumbago capensis Thunb. were examined by polarized reflected light microscopy, scanning electron microscopy, x-ray diffraction, and energy dispersive x-ray analysis. There are about 23 glands per mm² on the lower leaf surface and none on the upper surface. Each gland measures 20–30 μm in diam and consists of four small secretory cells surrounded by four subsidiary cells. Secretion of chalk is apparently through a pore in the surface of each secretory cell. The secreted material was crystalline and x-ray diffraction confirmed the presence of two minerals: calcite (CaCO₃) and nesquehonite (MgCO₃ · 3H₂O). Energy dispersive x-ray analysis revealed the presence of magnesium, silicon, phosphorus, sulfur, chlorine, potassium, and calcium in the epidermal cells. However, only calcium and magnesium and traces of silicon were detected in the secreted material. A distribution analysis showed calcium and magnesium to be uniformly distributed through the secreted material.     

Distribution of elements in the lily pollen tube tip,determined with the Oxford scanning proton microprobe

Summary Pollen tubes ofLilium longiflorum were fixed with glutaraldehyde and investigated unsectioned with the Oxford scanning proton microprobe (SPM). Two-dimensional maps which show the distribution and concentration of phosphorus, sulphur, chlorine, potassium, calcium, iron, copper, zinc and arsenic are presented. The maps show that, within the pollen tube tip region, calcium and zinc exhibit relatively steep longitudinal concentration gradients compared to the more flat distributions of phosphorus and sulphur. Chlorine, potassium, iron and copper appear equally distributed along the tube. All elements with the expception of arsenic show the highest concentration within the cell protoplasm and not in the cell wall. Additional signals of arsenic, chlorine and potassium originate from the remaining fixative dried around the tube, containing also the free ions of the cell. The arsenic signals originate exclusively from the buffer used during fixation. The different maps are compared and discussed in relation to their significance to the pollen tubes.Abbreviations EDAX energy dispersive analysis of X-rays - EIXE electron induced X-ray emission - PIXE proton induced X-ray emission - SPM scanning proton microprobe     

Effects of the cationic protein poly-L-arginine on airway epithelial cells in vitro

BACKGROUND: Allergic asthma is associated with an increased number of eosinophils in the airway wall. Eosinophils secrete cationic proteins, particularly major basic protein (MBP). AIM: To investigate the effect of synthetic cationic polypeptides such as poly-L-arginine, which can mimic the effect of MBP, on airway epithelial cells. METHODS: Cultured airway epithelial cells were exposed to poly-L-arginine, and effects were determined by light and electron microscopy. RESULTS: Poly-L-arginine induced apoptosis and necrosis. Transmission electron microscopy showed mitochondrial damage and changes in the nucleus. The tight junctions were damaged, as evidenced by penetration of lanthanum. Scanning electron microscopy showed a damaged cell membrane with many pores. Microanalysis showed a significant decrease in the cellular content of magnesium, phosphorus, sodium, potassium and chlorine, and an increase in calcium. Plakoglobin immunoreactivity in the cell membrane was decreased, indicating a decrease in the number of desmosomes CONCLUSIONS: The results point to poly-L-arginine induced membrane damage, resulting in increased permeability, loss of cell-cell contacts and generalized cell damage.     

Development of polyembryonic insects: a major departure from typical insect embryogenesis

 The parasitic wasp Copidosoma floridanum represents the most extreme form of polyembryonic development known, forming up to 2000 embryos from a single egg. To understand the mechanisms of embryonic patterning in polyembryonic wasps and the evolutionary changes that led to this form of development we have analyzed embryonic development at the cellular level using confocal and scanning electron microscopy. C. floridanum embryogenesis can be divided into three phases: (1) early cleavage that leads to formation of a primary morula, (2) a proliferative phase that involves partitioning of embryonic cells into thousands of morulae, and (3) morphogenesis whereby individual embryos develop into larvae. This developmental program represents a major departure from typical insect embryogenesis, and we describe several features of morphogenesis unusual for insects. The early development of polyembryonic wasps, which likely evolved in association with a shift in life history to endoparasitism, shows several analogies with mammalian embryogenesis, including early separation of extraembryonic and embryonic cell lineages, formation of a morula and embryonic compaction. However, the late morphogenesis of polyembryonic wasps proceeds in a fashion conserved in all insects. Collectively, this suggests a lack of developmental constraints in early development, but a strong conservation of the phylotypic stage. Received: 27 June 1997 / Accepted: 11 January 1998     

The assembly of keratins from higher plant cells

Summary In vitro assembly and morphological characteristics of purified 58 kDa, 52 kDa, 50 kDa, and 45 kDa polypeptides in the leaves and the cotyledons of the cabbage (Brassica pekinensis Rupt.) were investigated by electron microscopy and scanning tunneling microscopy. The three or four purified intermediate filament (IF) polypeptides can spontaneously assemble into intermediate filaments in vitro with a 23–24 nm axial repeat, which indicates that keratin IFs in higher plant cells have the same molecular arrangement as in animal cells. STM images suggest that the plant keratin filaments display a pronounced structural polymorphism, which can be composed of 3 nm, 4.5 nm, or 6 nm wide keratin protofilaments.Abbreviation IF intermediate filament - STM scanning tunneling microscopy - SDS sodium dodecyl sulfate - BCIP 5-bromo-4-chloro-3-indolyl phosphate-toluidine - NBC p-nitroblue tetrazolium chloride - PMSF phenylmethyl sulfonylfluoride - HOPG high oriented pyrolytic graphite     

Adhesive properties of sea cucumber coelomoyctes

Summary— The adhesive properties of the coelomocytes of the sea cucumber, Holothuria polii, have been investigated by studying their ability to attach to glass coverslips in vitro, and their morphology examined by scanning electron microscopy. Both amoeboyctes and spherule cells in cell suspensions attached themselves to glass coverslips, but spreading activity was restricted to an amoebocyte subset which assumed an extremely flattened morphology. Coelomocyte adhesion was a time- and temperature-dependent phenomenon and required cations for attachment to the glass surface. Mg²⁺ ions were more effective than Ca²⁺ in facilitating cell binding. The addition of potassium cyanide or sodium azide to the cell suspension did not inhibit amoebocyte attachment but vinblastine did. Cytochalasin B had no effect. Cell adhesiveness was greatly enhanced with both coelomic fluid and purified 200-kDa coelomocyte-aggregating factor.     

Vanadium inhibits DNA-protein cross-links and ameliorates surface level changes of aberrant crypt foci during 1,2-dimethylhydrazine induced rat colon carcinogenesis

The trace mineral vanadium inhibits cancer development in a variety of experimental animal models. The present study was to gain insight into a putative anticancer effect of vanadium in a rat model of colon carcinogenesis. The in vivo study was intended to clarify the effect of vanadium on DNA-protein cross-links (DPC), surface level changes of aberrant crypt foci (ACF) and biotransformation status during 1,2-dimethylhydrazine (1,2-DMH) induced preneoplastic rat colon carcinogenesis. The comet assay showed statistically higher mean base values of DNA-protein mass (p<0.01) and mean frequencies of tailed cells (p<0.001) in the carcinogen-induced group after treatment with proteinase K. Treatment with vanadium in the form of ammonium monovanadate supplemented ad libitum in drinking water for the entire experimental period caused a significant (p<0.02) reduction (40%) in DNA-protein cross-links in colon cells. Further, the biotransformation status of vanadium was ascertained measuring the drug metabolising enzymes, glutathione S-transferase (GST) and cytochrome P-450 (Cyt P-450). Significantly, there was an increase in glutathione S-transferase and cytochrome P-450 levels (p<0.01 and p<0.02, respectively) in rats supplemented with vanadium as compared to their carcinogen controls. As an endpoint marker, we also evaluated the effect of vanadium on surface level changes of aberrant crypt foci induced by 1,2-DMH by scanning electron microscopy. Animals induced with 1,2-DMH and supplemented with vanadium showed a marked improvement in colonic architecture with less number of aberrant crypt foci in contrast to the animals induced with 1,2-DMH alone, thereby exhibiting its anticarcinogenicity by modulating the markers studied herein.     

Ionic regulation in the Antarctic nematode Panagrolaimus davidi,measured using electron probe X-ray microanalysis

The element composition of the pseudocoelomic fluid of the Antarctic nematode Panagrolaimus davidi was analysed by electron probe X-ray microanalysis after absorbing the fluid into Sephadex G-25 beads, and after producing calibration curves by analysing various concentrations of elements of interest absorbed into beads. The nematodes maintain higher concentrations of sodium and potassium in their pseudocoelomic fluid than in the external medium but lower concentrations of magnesium and calcium. When external concentrations of specific ions were elevated there was evidence for the regulation of internal concentrations of sodium, potassium, magnesium and chlorine. The time course of changes in response to exposure to elevated levels of KCl shows an increase in internal concentrations of potassium and chlorine up to 2 h after exposure, followed by a decline. This is consistent with a model of ionic regulation proposed for Caenorhabditis elegans which suggests that high concentrations of ionic osmolytes are replaced by compatible organic osmolytes.     

X-ray microanalysis of cockroach fat body in relation to ion and water regulation

X-Ray microanalysis of fat body from the cockroach (Periplaneta americana) showed that the crystals in the urate cells sequestered potassium and sodium during water deprivation. Postassium sequestration appeared to occur by the formation of additional crystals whereas sodium appeared to be taken up by pre-existing crystals of the “amorphous granule” type. Urate cells increased in cytoplasmic density i.e. mass per unit volume as a result of water deprivation and lost potassium from the cytoplasm but not from the nucleus. Trophocyte cells also showed an increase in density and a loss of potassium from the cytoplasm. The nucleus in trophocyte cells however, did not increase in density and showed an increase in chlorine concentration. A previously undescribed type of crystal in the trophocyte cell cytoplasm also increased markedly in chlorine concentration. The mycetocyte cells did not appear to increase in density but chlorine concentration was elevated in symbionts, cytoplasm and nucleus. An increase in density is considered to be a consequence of a reduction in volume due to water loss. It is confirmed that the fat body plays a major role in the maintenance of ion balance during water deprivation.     

The fine structure of blood cells in the ascidian Perophora viridis

The fine structure of each of the blood cell types of Perophora viridis has been characterized and strong evidence for localization of vanadium in two of these types is given. There are eight cell types; phagocytes which may contain completely engulfed cells, lymphocytes with a prominant nucleolus and scanty cytoplasm packed with clustered ribosomes, and six other cell types each with distinctive granules. Morula cells contain a central nucleus and cytoplasm filled by wedged bodies, about five of which are seen in section. These bodies contain regularly spaced electron dense foci. Green cells have the same organization but contain bodies which are electron dense throughout. Granular amoebocytes contain many smaller lightly staining oval bodies and much glycogen. Another cell type (probably orange cells of light microscopy) contains numerous granular rounded bodies. Compartment cells have vacuoles containing electron dense particles and signet ring cells have usually one large vacuole which is electron dense lined and may contain electron dense particles. Developmental stages of these cell types show involvement of endoplasmic reticulum and Golgi bodies in granule formation. After glutaraldehyde fixation alone the only extremely electron dense components are particles in the compartment cells and signet ring cells implicating these as sites of vanadium localization, although not excluding other cell types.     

Changes in the ionic composition and shape of erythrocytes in response to cholera toxin

Blood of 10 inbred rats has been investigated after effect of cholera toxin for 1 h by means of roentgenospectral energetic spectrometer Revex-5100 (USA). Simultaneously, erythrocytes have been studied by means of the scanning electron microscope. Contents of sodium, phosphorus, chlorine, serum, potassium, and ferrum ions have been estimated. The cholera toxin changes the contents of the ions mentioned in erythrocytes and contributes to certain changes of their form. A suggestion is made that the cholera toxin changes permeability of the erythrocytes membranes without participation of the cell cyclase systems.     

The element distribution in ultrathin cryosections of cultivated fibroblast cells

A preparation method for measurements of the intracellular distribution of elements in tissue culture cells is described which is based on cryofixation, cryoultramicrotomy, cryotransfer and X-ray microanalysis in a scanning transmission electron microscope. Dry weight concentrations of phosphorus, sulfur, chlorine and potassium in the nucleus, the cytoplasm and the mitochondria of L929 fibroblast cells of the mouse are reported. The preparation and quantitation procedures are discussed with respect to present limitations and possible improvements of the method.     

Stimulatory (insulin-mimetic) and inhibitory (ouabain-like) action of vanadate on potassium uptake and cellular sodium and potassium in heart cells in culture

(1) The influence of vanadate (Na3VO4) on sodium and potassium uptake as well as on cellular ion contents of sodium and potassium has been studied in heart muscle and non-muscle cells obtained from various species. An ouabain-like inhibition of potassium uptake (up to 50%), combined with a decrease of cellular potassium (up to 20%) has been observed by vanadate (10(-4)-10(-3) M) in heart non-muscle cells obtained from neonatal guinea pigs and chick embryos. In heart muscle and non-muscle cells prepared from neonatal rats, as well as in Girardi human heart cells, a vanadate-induced stimulation of potassium uptake (up to 100%), combined with a rise in cellular potassium (up to 20%) and without significant alteration of cellular sodium, has been found. A slight increase of 22Na+ influx can be measured in rat heart muscle cells and in Girardi human heart cells in the presence of vanadate (10(-4)--10(-3) M). (2) In beating rat heart muscle cells in culture, detrimental effects of serum deprivation--concerning beating properties, potassium uptake and cellular potassium--can at least in part be overcome by addition of vanadate. Furthermore, this compound prevents ouabain-induced signs of toxicity (contractures) in these cells. (3) The stimulatory effects of vanadate on potassium can be mimicked by insulin (1-10 mU/ml). Furthermore, vanadate produces an insulin-like stimulation of 2-deoxy-D-glucose uptake in rat heart muscle and non-muscle cells as well as in Girardi human heart cells. (4) The experimental data demonstrate an ouabain-like inhibition as well as an insulin-mimetic stimulation of potassium-uptake in various heart cells. The reason for this antagonistic mode of action may be due to the different capabilities of the heart cell types to reduce vanadium in the V-valence state of vanadium in the IV-valence state, thereby favouring either ouabain-like inhibition (vanadium V) or insulin-mimetic stimulation (vanadium IV) of potassium transport.