Distribution of Elements in Rat Peripheral Axons and Nerve Cell Bodies Determined by X-Ray Microprobe Analysis

X-ray microprobe analysis was used to determine concentrations (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in cellular compartments of frozen, unfixed sections of rat sciatic and tibial nerves and dorsal root ganglion (DRG). Five compartments were examined in peripheral nerve (axoplasm, mitochondria, myelin, extraaxonal space, and Schwann cell cytoplasm), and four were analyzed in DRG nerve cell bodies (cytoplasm, mitochondria, nucleus, and nucleolus). Each morphological compartment exhibited characteristic concentrations of elements. The extraaxonal space contained high concentrations of Na, Cl, and Ca, whereas intraaxonal compartments exhibited lower concentrations of these elements but relatively high K contents. Nerve axoplasm and axonal mitochondria had similar elemental profiles, and both compartments displayed proximodistal gradients of decreasing levels of K, Cl, and, to some extent, Na. Myelin had a selectively high P concentration with low levels of other elements. The elemental concentrations of Schwann cell cytoplasm and DRG were similar, but both were different from that of axoplasm, in that K and Cl were markedly lower whereas P was higher. DRG cell nuclei contained substantially higher K levels than cytoplasm. The subcellular distribution of elements was clearly shown by color-coded images generated by computer-directed digital x-ray imaging. The results of this study demonstrate characteristic elemental distributions for each anatomical compartment, which doubtless reflect nerve cell structure and function.     

Effects of Axotomy on Distribution and Concentration of Elements in Rat Sciatic Nerve

X-ray microprobe analysis was used to determine the effects of axotomy on distribution and concentration (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in frozen, unfixed sections of rat sciatic nerve. Elemental concentrations were measured in axoplasm, mitochondria, and myelin at 8, 16, and 48 h after transection in small-, medium-, and large-diameter fibers. In addition, elemental composition was determined in extraaxonal space (EAS) and Schwann cell cytoplasm. During the initial 16 h following transection, axoplasm of small fibers exhibited a decrease in dry weight concentrations of K and Cl, whereas Na and P increased compared to control values. Similar changes were observed in mitochondria of small axons, except for an early, large increase in Ca content. In contrast, intraaxonal compartments of larger fibers showed increased dry weight levels of K and P, with no changes in Na or Ca concentrations. Both Schwann cell cytoplasm and EAS at 8 and 16 h after injury had significant increases in Na, K, and Cl dry weight concentrations, whereas no changes, other than an increase in Ca, were observed in myelin. Regardless of fiber size, 48 h after transection, axoplasm and mitochondria displayed marked increases in Na, Cl, and Ca concentrations associated with decreased K. Also at 48 h, both Schwann cell cytoplasm and EAS had increased dry weight concentrations of Na, Cl, and K. The results of this study indicate that, in response to nerve transection, elemental content and distribution are altered according to a specific temporal pattern. This sequence of change, which occurs first in small axons, precedes the onset of Wallerian degeneration in transected nerves.     

Changes in the elemental composition of Asterionella formosa during the diatom spring bloom

Changes in the elemental composition of the diatom Asterionellaformosa within mixed phytoplankton samples were determined overthe spring bloom period using energy dispersive X-ray microanalysis.Using a 10 kV electron microprobe, X-ray information from thetop 1–2 µm of the cell revealed overall mean concentrationsof: Si (4636 mmol kg^–1 dry wt), P (82), S (54), Cl (71)K (94) and Ca (90). Concentrations of all elements showed widevariation within each date sample, with unimodal frequency distributionsapproximating to a Normal distribution. Correlation of the entiredata set demonstrated clear statistical associations betweenelements, including Si, P and K. Uptake of Si during the courseof the diatom bloom led to a major fall in lake water concentration(1.0 to 0.07 mg l^–1), which correlated with a decreasein the mean cell Si concentration (6735 to 3107 mmol kg^–1dry wt) during the final phase of the bloom. Mean cell concentrationsof P and K also showed marked decreases with time, in closeparallel with cell Si. These changes in P and K were attributedto the high level of internal correlation with Si and not toany significant decrease in P and K availability in the environment.     

Storage reserves and cellular water in mature seeds of Araucaria angustifolia

Seed tissues of Araucaria angustifolia (Bertol.) Kuntze were investigated using histochemistry, transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) analysis. Moisture content and water status in tissues were also evaluated. In the embryo, TEM studies revealed the presence of one to several central vacuoles and a peripheral layer of cytoplasm in cells from different tissues of the cotyledons and axis. In the cytoplasm, lipid bodies, starch grains, mitochondria and a nucleus are evident. In most tissues, vacuoles contain proteins, indicating that the storage proteins are highly hydrated. In cells of the root cap, proteins are stored in discrete protein bodies. Both protein storage vacuoles and discrete protein bodies have inclusions of crystal globoids. EDX analysis of globoids revealed the presence of P, K and Mg as the main constituents and traces of S, Ca and Fe. In the root and shoot meristems, deposits of phytoferritin are present in the stroma of proplastids. The gametophyte consists of cells characterized by relatively thin cell walls and one to several nuclei per cell. Protein and lipid bodies are present, although starch is the most conspicuous reserve. Immediately after shedding, moisture content is approximately 145% (dry weight) for the embryo and 95% (dry weight) for the gametophyte. Calorimetric studies reveal that axes and cotyledons have a very high content of freezable water, corresponding to types 5 and 4, i.e. dilute and concentrated (or capillary) solution, respectively. The results are discussed in relation to the behaviour of the species, which has been categorized as recalcitrant.  © 2002 The Linnean Society of London . Botanical Journal of the Linnean Society , 2002, 140 , 273−281.     

Nuclear-cytoplasmic distribution of ions in the hepatocyte

To determine if nuclear/cytoplasmic (N/C) gradients of ions exist in mammalian hepatocytes, as has been shown to exist in the amphibian oocyte, small pieces of mouse liver were cryofixed for energy dispersive X-ray microanalysis of Na, K and Cl in thin freeze-dried cryosections. Expressing the data on a dry weight basis showed small but significant N/C gradients for Na and K but not for Cl. When the data were expressed on a wet weight basis no significant N/C gradients of Na, K or Cl were revealed. Questions of data interpretation are discussed.     

The distribution of nuclear and cytoplasmic proteins in the blastoderm of the loach, Misgurnus fossilis L

The concentration of dry substance (protein) and the dry weight of nuclei, cytoplasm and cells from different blastoderm regions at the early blastula and midgastrula stages were determined by interferentional microscopy. It was shown that at the early blastula stage the dry weight of cells in the basal layer is higher than that in the outer layer. Although the protein concentration in the basal layer cells appears to be somewhat higher, differences in their dry weight are due primarily to the big volume of cytoplasm of the basal layer cells. By the midgastrula stage, the total (nucleus + cytoplasm) protein concentration increases (by 17% in the basal layer cells and by 9% in the outer layer cells) due to the increase of nuclear protein concentration. At the same time dry weight of these cells markedly decreases due to the decrease of their volumes in the process of cell divisions. At the midgastrula stage the epiblast cells have the highest dry weight due to the highest protein concentration in the cytoplasm and the biggest cell volume. The results obtained are discussed with respect to the data on the pattern of accumulation of newly synthesized protein in nuclei and cytoplasm with special reference to the duration of individual cell cycle phases.     

In vivo localization of manganese in the hyperaccumulator Gossia bidwillii (Benth.) N. Snow & Guymer (Myrtaceae) by cryo-SEM/EDAX

Gossia bidwillii (Myrtaceae) is a manganese (Mn)-hyperaccumulating tree native to subtropical eastern Australia. It typically contains foliar Mn levels in excess of 1% dry weight. However, in G. bidwillii and other Mn-hyperaccumulating species, the cellular and subcellular localization of Mn has not been measured. Quantitative in vivo cryo-scanning electron microscopy (SEM)/energy dispersive X-ray analysis (EDAX) was used to localize Mn and other elements in tissue collected from mature trees growing in a natural population. Cryo-SEM showed that the leaf mesophyll is differentiated as a double-layer palisade mesophyll above spongy mesophyll. Transmission electron microscopy (TEM) revealed that the palisade and epidermal cells are highly vacuolated. EDAX data were used to estimate in situ vacuolar Mn concentrations of all cell types in fresh cryo-fixed leaf tissues. The highest average vacuolar Mn concentration of over 500 mM was found in the upper-layer palisade mesophyll, while the lowest concentration of around 100 mM was found in the spongy mesophyll. Qualitative in vivo cryo-SEM/EDAX was employed to further investigate the spatial distribution of Mn in fresh leaf tissues and young bark tissue, which was also found to have a high Mn concentration. It is concluded that Mn distribution in G. bidwillii is quantitatively different to metal distribution in other hyperaccumulating species where the highest localized concentrations of these elements occur in non-photosynthmetic tissues such as epidermal cells and associated dermal structures including trichomes and leaf hairs.     

An application of EDXRF on the study of barley seedlings growth on sewage sludge

An energy dispersive X-ray fluorescence method for trace element analysis in plants (leaves and roots) is presented. The method is characterized by the use of a secondary target excitation, thin specimen, and microwave acid digestion. The accuracy is about 10% and the sensitivity is in the range 10–50 ng/cm². The analysis time (from dry sample to concentration data) is about 4×10³ s. The effects of Cr in sewage sludge on barley seedling growth is presented.     

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.     

An investigation of the mineral content of barley grains and seedlings

The Ca, Mg, K, and P content of dry barley (Hordeum vulgare) grains and seedlings was investigated using energy dispersive x-ray analysis and neutron activation analysis. Energy dispersive x-ray analysis of protein bodies in aleurone cells showed that these bodies contained very little Ca in relation to P, Mg, and K. Neutron activation analysis also showed that the endosperm contained very little Ca in relation to the other three elements. Surface sterilization and soaking treatments brought about slight loss of Ca but substantial loss of K from embryos. Over 6 days of growth the seedling plant gained minerals from the endosperm.     

Electron probe microanalysis of the subcellular compartments of bovine adrenal chromaffin cells. Comparison of chromaffin granules in situ and in vitro

The elemental and water content of cultured bovine adrenal chromaffin cells and their secretory chromaffin granules have been measured and compared with isolated chromaffin granules using quick freezing, ultracryomicrotomy, and electron microprobe analysis methods. In units of millimole/kilogram dry weight (+/- S.E.) granules in situ contained: P, 523 +/- 32; K+, 124 +/- 9; S, 82 +/- 3; Cl-, 74 +/- 9; Ca2+, 13 +/- 2; Mg2+, 6 +/- 2; and Na+, -2 +/- 2. Following routine isolation in isotonic sucrose buffer, granule K and Cl- had decreased while granule Na+ increased. Cl- exhibited a consistent decrease to 35-40 mmol/kg dry weight. Granule Na+ and K+ concentrations ranged from 43 to 12 mmol/kg and 28 to 60 mmol/kg dry weight, respectively, depending on the Na+ and K+ content of the buffer. Despite the redistribution of monovalent ions, granule Ca2+, granule P, being in the form of ATP, and granule S, being in the form of protein, were not significantly changed. The stability of these elements is consistent with the existence of a stable storage complex for Ca2+, ATP, and protein. Using the granule as an internal standard with a water content of 66%, the water contents of external space, nucleus, cytoplasm, and mitochondria were estimated to be 89, 88, 82, and 70%, respectively. Wet weight concentrations for each element were calculated for granules and cytoplasm from which the transgranular concentration gradients for K+, Cl-, and Na+ were determined. Cl-, a permeant anion, was 2-fold higher in the granule than in the cytoplasm while K+, a slightly permeant cation, had an opposite distribution ratio slightly less than two. Together, the K+ and Cl- data suggest the presence of an inside-positive granule membrane potential of approximately 10-16 mV. The surprising lack of Na+ from the granule matrix suggests a hugh inward gradient for Na+ even though the Na+ content of chromaffin cell cytoplasm is low at 5 mmol/kg water. The lack of an outward Na+ gradient is important in that it indicates that the previously described electroneutral Na+-Ca2+ exchange system, by which isolated granules accumulate Ca2+, does not operate in mature granules in situ. Consequently, if chromaffin granules regulate internal calcium during stimulus secretion coupling, a mechanism other that Na+-Ca2+ exchange is necessary.     

细胞壁矿质元素含量在细胞生长中的动态变化

在组织水平上已经描述了许多植物通气组织的形成过程,但对其发育过程的调控仍然知道得很少.利用CSEM-EDX微量分析技术,定点测量慈姑叶柄通气组织不唰发育时期的细胞壁矿质元素的组成.这些元素除了C,O以外,还包括Mg,Ca,Cu,Zn,P等必需的矿质元素.结果发现,在叶柄发育的早期,通气组织细胞壁的K和Cl含量很高,分别高达36%和4.3%细胞壁干重.Mg的含量在第二阶段最高,达到细胞壁干重的0.86%.只有在第三和第四阶段监测到Cu和Zn元素,最高含量分别为2.5%和1.5%细胞干重.仅在第四和第五阶段才能检测到Ca,其最高含量为1.3%细胞壁干重.通气组织横膈膜细胞和圆柱体腔壁细胞的元素构成变化有相似的趋势,说明这种变化与组织的发育阶段关系密切.细胞壁的一些元素间呈现较高的相关性,其中K和Cl及Cu和Zn之间成较高的正相关.在不同发育阶段,细胞壁的元素含量呈现动态变化,说明细胞壁(质外体物质)的元素构成有很大的变动范围.早期的通气组织细胞壁大量积累K和Cl,暗示早期的气体空间充满液体(组织液);Mg可能参与细胞伸展的调控;伸展中细胞的细胞壁积累高浓度的Cu和zn,并不影响细胞的正常功能;而Ca的出现使细胞比硬度增加,将终止细胞伸展.Cu和Zn在细胞壁中的积累呈高度的直线关系,回归分析显示,二者呈现定量关系,推测它们可能有共同的或者类似的转运和吸收机制.     

Identification of a new glucosinolate-rich cell type in Arabidopsis flower stalk

Distribution of K, Ca, Cl, S, and P in freeze-dried sections of Arabidopsis flower stalk was analyzed by energy dispersive x-ray imaging. Concentrations of these elements in different cell types were quantified by microanalysis of single-cell samples and phloem exudates. Results showed a differential pattern of distribution for all five elements. K concentration was found to be highest in the parenchymatous tissue around vascular bundles. Ca and Cl were present mainly in the central part of the flower stalk. P was largely located in the bundles and in the parenchyma surrounding them. S signal was extraordinary high in groups of cells (S-cells) situated between the phloem of every vascular bundle and the endodermis. Enzymatic hydrolysis by thioglucosidase of cell sap collected from S-cells using a glass microcapillary resulted in the release of glucose, indicating that these cells contain glucosinolates at high (> 100 mM) concentration, which is consistent with the concentration of S (> 200 mM) estimated by x-ray analysis of cell sap samples. Since their position outside of the phloem is ideally suited for protecting the long-distance transport system from feeding insects, the possible roles of these cells as components of a plant defense system are discussed.     

Isolation and immunochemical characteristics of the immunodepressive substance from Escherichia coli.

A substance, inhibiting the production of haemolysins against sheep erythrocytes in mice was isolated from two non-pathogenic strains of E. coli, 020:K4 and M-17, by the methods of differential centrifugation and gel filtration. Spectrophotometric studies and chemical analysis have shown the isolated substance to be glycolipid. The immunodepressive substance is localized in the cytoplasm of the microbial cell. The isolated and partly purified immunodepressive substance did not contain any admixture of O-antigen (endotoxin) of the cell wall or of antigens giving cross reactions with sheep erythrocytes. The isolated substance exhibited weak antigenic properties and was not toxic for mice when administered in a dose of 2 mg (dry weight).     

Subcellular flux of potassium and rubidium in amphibian oocytes

To determine the net Rb+ influx and K+ efflux at a subcellular level, fully grown and a Ringer's solution where K+ was substituted for by Rb+ on a molar bases. For 40 hrs serial samples of the oocytes were cryofixed and cryosectioned for elemental analysis in mM per kg dry weight using electron probe x-ray microanalysis. Oocyte volume remained constant. Net Rb+ influx showed a slow exponential increase into the nucleus, the yolk-free cytoplasm and the yolk platelets. There was significant K+ efflux from the nucleus but not from the yolk-free cytoplasm or the yolk. The Na+ concentration remained unchanged in all compartments during the course of the experiment. There was however a slow but significant increase in the concentration of Cl- in each of the subcellular compartments but this increase was not sufficient to balance the observed increase in the sum of K+ plus Rb+. Thus Rb+ accumulates selectively in all three subcellular compartments indicating that Rb+ is not a good K+ surrogate in the oocytes. That K+ demonstrates efflux from the nucleus but not from the cytoplasmic compartments is interpreted to suggest that some of the nuclear K+ is lost in exchange for Rb+ but that essentially none of the cytoplasmic K+ is-lost in exchange for Rb+. The findings provide strong evidence for adsorption of Rb+ in the cell.     

Demonstration of the display of components of the endoplasmic reticulum system by indirect immunofluorescence microscopy using antibodies against cytochrome b(5) from rat liver microsomes.

Antibodies against the purified fragment of cytochrome b(5) released by trypsin treatment from rat liver microsomes were raised in rabbits and used for the demonstration of membranes rich in cytochrome b(5), in particular the endoplasmic reticulum (ER) system, by indirect immunofluorescence microscopy. The method allowed the demonstration of ER not only in frozen sections of various tissues, including liver and lactating mammary gland from different species, but also in cultured cells of a diversity of species and cell types. In the cultured cells the structures most prominently decorated with the antibodies against cytochrome b(5) were the nuclear envelope and the ER system which in many cells could be recognized as a system of smoothly bending, branching threads, extending from the perinuclear cytoplasm toward the cell periphery. Changes in the display of such elements during mitosis and cell plating and possible influences of the specific fixations used are discussed.     

The X-ray microanalysis method for investigation of chemical composition of normal and pathologic human peripheral blood lymphocytes

The X-ray microanalysis method for investigation of chemical composition, mainly trace elements, of normal and pathologic human peripheral blood lymphocytes has been presented. The method in question consists of two stages. During the first stage the scanning electron microscope with the energy dispersive X-ray spectrometer has been used. The second stage of the method consisted of employing the above microscope with the microwave dispersive spectrometer. The method enables the short-term determination of the various elements content within the lymphocytes with particular insight into the content of elements in a single cell. The preliminary investigations showed significant differences in the content of chlorine, sulfur and lead in normal human lymphocytes and that from patients with cancer of the larynx.     

ELEMENTAL COMPOSITION,CORRELATIONS, AND RATIOS WITHIN A POPULATION OF STAURASTRUM PLANCTONICUM (ZYGNEMATALES): AN X-RAY MICROANALYTICAL STUDY1

Individual cells of Staurastrum planctonicum (Teil.) within a mixed freshwater phytoplankton sample were analyzed by scanning electron microscope X-ray microanalysis to determine their elemental composition. X-ray emission spectra routinely showed clear peaks of P, S, and Cl, plus monovalent (Na, K, and divalent (Mg, Ca) cations. Si and Cu were also present in lower quantities. Concentrations of individual elements (expressed as mmol.kg⁻¹ dry weight) varied widely among cells, with values over the sample population approximating to a normal distribution. Although intracellular anion and cation levels varied considerably, significant correlations occurred between concentrations of monovalent and divalent cations (mean ratio 1.4), major diffusible anions and cations (mean ratio 1,2), and total levels of electropositive and electronegative elements (mean ratio 1.2). The monovalent cations of K and Na occurred at a mean ratio of 1.2 and were not significantly correlated. Concentrations of individual elements (except Si) showed clear positive correlations within the analyses, with 12 highly significant (99% probability) correlations out of 36 possible combinations. Principal factor analysis showed that elemental correlations were determined by two major factors, with two resulting groups of elements—(Na, S, Cl, Ca, Cu) and (Mg, P, K). Statistical relationships between elements followed a clear correlation pattern, which retained its characteristics even when elemental concentrations were expressed per unit P rather than per unit dry weight. Elemental concentrations were closely similar in matching, but not nonmatching, smicells. The statistical pattern of elemental associations noted in Staurastrum parallels that seen in X-ray micro-analytical studies of other algae but differs in detail. This pattern of statistical associations has biological implications in terms of cell compartmentation, characterization of different cell types, and cell interactions with their environment.     

Localization of cadmium in the root cells of <Emphasis Type="Italic">Allium cepa</Emphasis> by energy dispersive x-ray analysis

Allium cepa L. roots were exposed to 0.1 and 1.0 mM Cd for 6, 24 and 48 h and the localization of Cd in the root tissue was investigated. Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDXA) were performed on frozen-dried tissues of roots. No Cd was detected in the roots treated with only 0.1 mM Cd, while after exposure to higher Cd concentration (1.0 mM) Cd was observed in cell wall and in cytoplasm in the epidermis, cortex and vascular tissues in the roots.