Elemental composition of bacterial metachromatic inclusions determined by electron microprobe X-ray analysis.

Electron microscopy and microprobe X-ray analysis were used to study metachromatic inclusions of Spirillum itersonii , Corynebacterium diphtheriae, and Micrococcus luteus. In situ metachromatic inclusions were electron dense and contained phosphorus and divalent cations. Metachromatic inclusions isolated by anion-exchange column chromatography and by isoosmolar Metrizamide density gradient centrifugation were similar in composition to in situ inclusions.     

Direct analysis of human blood (mothers and newborns) by energy dispersive X-ray fluorescence

This work is an application of energy dispersive X-ray fluorescence (EDXRF) as analytical technique for trace element determination in human tissues. Potassium (K), calcium (Ca), iron (Fe), copper (Cu), zinc (Zn), bromine (Br), rubidium (Rb) and lead (Pb) were determined directly in blood samples from 66 mothers at delivery after full-term pregnancies. The corresponding 66 cord-blood samples of the newborns were also analysed, in order to find element correlations between maternal and newborn blood at birth. The studied samples were obtained from mothers aged between 15 and 39 years old, the gestational age being between 35 and 41 weeks and the newborns' weight between 2.310 and 4.310 kg. Samples were lyophilised and analysed without any chemical treatment. Very low levels of Pb were found both in maternal and fetal cord blood samples. Cu values ranged from 3 to 13 microg g-1, both for mothers and children. A correlation between Cu and Fe concentrations in maternal and fetal cord blood was found. Zn is considered as one of the key elements in newborn health. Concentrations between 10 and 40 microg g-1 were measured. A positive correlation between Br levels in mothers and children was observed. Positive correlations for mothers were observed between Zn and Rb as well as K and Fe. The corresponding correlations in fetal cord blood samples were not observed, however positive correlations were found between Ca and K; Cu and Fe. The mean concentrations for each element were similar in maternal and in fetal cord blood, except for Cu and Zn, being higher in maternal samples. No correlations between element concentrations and pathologies of the mothers were observed.     

A study of methods for in situ X-ray energy dispersive analysis of polyphosphate bodies in Plectonema boryanum

A variety of preparative methods for in situ X-ray energy dispersive analysis were tested to determine their effects on the elemental composition of polyphosphate bodies in P. boryanum. The bodies were found to contain large amounts of P and K and small amounts of Ca and Mg. Air drying, freeze-drying and freeze-drying from a liquid nitrogen slush all gave similar results. Fixation of the cells in glutaraldehyde and/or OsO₄ resulted in loss of the K and enhancement of the Ca peak. Magnesium was lost during embedding in epoxy.     

Mitochondrial matrix granules in soft tissues : I. Elemental composition by X-ray microanalysis

The elemental composition of individual matrix granules in mitochondria of rat brown fat, mouse gall bladder and guinea pig kidney has been examined by X-ray microanalysis. The matrix granules showed a similar elemental composition that was strongly dependent upon the method of sample preparation. Low-temperature oxygen plasma microincineration or wavelength-dispersive X-ray spectrometers were used to demonstrate the presence of phosphorus in matrix granules of osmium tetroxide-fixed specimens. Matrix granule osmiophilia was retained in glutaraldehyde-fixed brown fat only if exposure to polar organic solvents was avoided during subsequent steps, e.g. by cryosectioning. As normally prepared, matrix granules lack detectable calcium but had bound this at detectable levels after fixation in osmium tetroxide, but not glutaraldehyde, supplemented with 5 mM Ca²⁺. The results demonstrate that mitochondrial matrix granules of normal soft tissues are not calcium phosphate deposits and contain phospholipids, apparently as a major constituent. Thus they provide evidence against the hypothesis that matrix granules are primarily involved in mitochondrial calcium sequestration and, indirectly, for the hypothesis that the granules may be related to inner membrane assembly.     

Accumulation of lead by Anabaena cylindrica : mathematical modeling and an energy dispersive X-ray study

The ability of microorganisms to selectively adsorb various heavy metal ions has been recognized for over a decade. We have investigated the biosorption of lead by an active culture of the cyanobacterium Anabaena cylindrica. Energy dispersive X-ray microanalysis was used to evaluate the different uptake mechanisms in the various subcellular regions. Three were identified: a very fast adsorption mechanism in the cell envelope; a time-dependent deposition reaction on the cell surface; and an adsorption mechanism, also time dependent, on the polyphosphate body inside the cell. Atomic absorption spectrometry was then used to quantify the changes with time of bulk fluid concentrations of lead solutions exposed to cyanobacteria. A mass transfer kinetic model was developed which quantitatively predicts the concentration of lead in cells of Anabaena cylindrica as a function of spatial dimensions and time. The model predictions are consistent with a pattern, documented in literature and confirmed by our own experimental evidence, of a very fast uptake in the cell envelope and then a longer uptake period inside the cell. Our experimental evidence also revealed a time-dependent uptake mechanism on the surface of the cells, which is included in the model. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 408-418, 1997.     

A quantitative immunoperoxidase procedure employing energy dispersive x-ray analysis.

A method is described for substituting gold for osmium as a marker in the unlabeled antibody technique. The gold marker can be detected in the light or electron microscope. The gold-labeled reaction product can be detected in lower concentrations than osmium and can be used as the basis for quantitating antigen concentrations in cells and tissues with the scanning electron microscope and energy dispersive x-ray analysis.     

Localization of selenium in bacterial cells using TEM and energy dispersive X-ray analysis

Bacteria isolated from lake sediment samples reduced sodium selenite to elemental selenium. Finestructural observations were made on a number of different bacterial species cultured in the presence of sodium selenite. Examination of Escherichia coli and a Pseudomonas species revealed electron-dense deposits of irregular shape, composed of smaller units, within the cytoplasm but not on the cell wall and cell membrane. Cells of Aeromonas and Flavobacterium species exhibited conspicuous intranuclear fibrillary aggregates and different electron-dense inclusions. It appeared that the membrane structures were somewhat more easily stained in some bacterial cells after growth on agar plates containing sodium selenite. The deposits and fibrillary accumulations were interpreted to contain selenium on the basis of energy dispersive X-ray analysis. Control preparations and cells grown in the presence of sodium selenate were void of any fine-structural abnormalities. Alterations in fine structure are discussed in relation to the metabolism of selenium by bacterial cells and possible sites of inhibition.Abbreviations TEM transmission electron microscopy - EDX energy dispersive X-ray     

Investigations on the cuticle of the polychaete elytra using energy dispersive X-ray analysis

Energy dispersive X-ray analysis of elements with Z>11 in a SEM (scanning electron microscope) was used to investigate the elytra ofLagisca extenuata, Lepidonotus clava, andHarmothoe areolata from Naples (Italy) and Banyuls (France). High concentrations of halogens and a few other elements were found in certain papillae in samples from both locations. Additional TEM-examinations and X-ray analysis of thin sections revealed that the halogen concentration is inversely related to the collagen content of the matrix. The halogens are presumably bound to tyrosines, which occur in these structures. In addition, accumulation of Mn²⁺ and possibly Fe³⁺ in the papillae might depend on environmental conditions. The results show that valuable information about the chemical composition of biological structures can be obtained by energy dispersive X-ray analysis. Moreover, the results indicate that this method may be useful for environmental investigations.This work is part of a doctoral thesis, written at the Zoological Institute of the University at Hamburg.     

Proteome analysis at the level of subcellular structures.

The targeting of proteins to particular subcellular sites is an important principle of the functional organization of cells at the molecular level. In turn, knowledge about the subcellular localization of a protein is a characteristic that may provide a hint as to the function of the protein. The combination of classic biochemical fractionation techniques for the enrichment of particular subcellular structures with the large-scale identification of proteins by mass spectrometry and bioinformatics provides a powerful strategy that interfaces cell biology and proteomics, and thus is termed 'subcellular proteomics'. In addition to its exceptional power for the identification of previously unknown gene products, the analysis of proteins at the subcellular level is the basis for monitoring important aspects of dynamic changes in the proteome such as protein transloction. This review summarizes data from recent subcellular proteomics studies with an emphasis on the type of data that can retrieved from such studies depending on the design of the analytical strategy.     

A chemical signal possibly related to physiology in fossil cells detected by energy dispersive X-ray microanalysis

Energy dispersive X-ray microanalysis (EDXMA) is a widely used tool employed to detect elemental composition and its spatial distribution in a sample without causing damage. Charcoalified cytoplasm is a new type of fossil material that came to people's attention only recently. In this paper, EDXMA is used for the first time to detect the spatial elemental distribution in charcoalified cytoplasm of two fossil plants that are more than 100 million years old. The results demonstrate certain elemental distribution patterns within charcoalified cytoplasm and the surrounding cell walls. Based on the results from cytological studies of extant material, the heterogeneous spatial elemental distribution within the charcoalified cytoplasm has the potential to be related to the maturation of cells, the presence of certain organelles, and the physiology of these organelles. This is the first chemical signal detected in cytoplasm residue that can possibly be related to plant physiology. This paves the way for further research on fossil cytoplasm, which will better our understanding on the physiology of fossil plants.     

Elemental composition of the human atherosclerotic artery wall

Summary The elemental composition of the human atheroselerotic popliteal artery was examined using the proton-induced X-ray-emission (PIXE) method. The application of a narrow proton beam (3×10 m²) enabled us to determine not only the concentrations of Cl, K, Ca, Fe, Cu, Zn, Br and Pb, but also their localization in different artery-wall regions. The highest mean concentrations of Cl, K, Zn and Br were found in the tunica media. In the investigated sections the distribution of Ca and Fe varied: sometimes, these elements were prevalent in the tunica intima, whereas in other cases, the highest concentrations were observed in the tunica media or tunica adventitia. The concentration profiles of each element were characterized by many sharp, narrow peaks. The highest concentrations of Ca and Fe showed such high levels that only one explanation is possible, i.e. the presence of crystals. The correlation of Ca peaks with those of Zn and Fe is discussed. The usefulness of the micro-PIXE method for the investigation of biomedical materials is also considered.     

Elemental composition of the human atherosclerotic artery wall

The elemental composition of the human atherosclerotic popliteal artery was examined using the proton-induced X-ray-emission (PIXE) method. The application of a narrow proton beam (3 X 10 micron 2) enabled us to determine not only the concentrations of Cl, K, Ca, Fe, Cu, Zn, Br and Pb, but also their localization in different artery-wall regions. The highest mean concentrations of Cl, K, Zn and Br were found in the tunica media. In the investigated sections the distribution of Ca and Fe varied: sometimes, these elements were prevalent in the tunica intima, whereas in other cases, the highest concentrations were observed in the tunica media or tunica adventitia. The concentration profiles of each element were characterized by many sharp, narrow peaks. The highest concentrations of Ca and Fe showed such high levels that only one explanation is possible, i.e. the presence of crystals. The correlation of Ca peaks with those of Zn and Fe is discussed. The usefulness of the micro-PIXE method for the investigation of biomedical materials is also considered.     

Ultrastructural differentiation within the central nervous system between indolamines and catecholamines by energy dispersive X-ray analysis following paraformaldehyde pretreatment

Dense bodies containing high amounts of chrome were localized in the perikarya of substantia nigra and dorsal raphe neurons following the cytochemical reaction of endogenous dopamine and serotonin (respectively) with glutaraldehyde-dichromate (GDC). Energy dispersive X-ray analysis of these bodies revealed chrome levels two to four times higher than those recorded from the cytoplasmic background. Pretreatment with paraformaldehyde blocked the GDC reaction within the dense bodies in the substantia nigra (chrome levels similar to background), while the chrome levels in the dense bodies of the raphe neurons remained elevated. This demonstrates that pretreatment with paraformaldehyde allows selective localization of central nervous system serotonin stores by the GDC technique.     

A comparison of X-ray and NMR structures for human endothelin-1.

Direct comparisons between the recently solved X-ray and NMR structures of human endothelin-1 with respect to secondary structure, RMS deviations, surface accessibilities, and side-chain conformers indicate important differences in conformation, especially in the C-terminus, but also in the central loop region, that are important for defining the specificity of binding. These differences are larger than seen for other X-ray and NMR structures that have been compared. Comparisons between the X-ray structure and the NMR NOE constraints highlight the regions of flexibility and environment-induced diversity in the endothelin structures.     

Elemental analysis of growth plate cartilage by synchrotron-radiation-induced X-ray emission (SRIXE).

The elemental composition of growth plate cartilage from calf scapula has been studied by means of SRIXE. X-ray emission spectra were obtained from the resting, hypertrophic and calcified regions of cartilage; then, each element was mapped with a lateral definition of about 10 microns x 10 microns. Evidence was found for a homogeneous distribution of the elements in resting cartilage compared to changes in local concentration of some atoms in the hypertrophic-calcified tissue. In this zone Ca, Sr, Ni, Zn, S, reach the maximal concentration at the calcification front while Cu shows a uniform distribution. A Zn distribution similar to that of the Zn-containing enzyme alkaline phosphatase, the key enzyme of calcification, is found.     

Localization of alkaline phosphatase in subcellular structures of human osteogenic sarcomas.

Electron microscopic study of osteogenic sarcomas has revealed association of the product of the reaction for alkaline phosphatase with membranous structures. The structural and function polymorphism of osteogenic sarcoma cells is also shown.     

Screening for selected trace elements in single cell populations by energy dispersive X-ray fluorescence spectrometry

An adaptation of the thin film approach to energy dispersive X-ray fluorescence spectrometry (EDXRF) is described, which provides for a screening analysis for Fe, Ni, Cu, Zn, Se, and Cd in K-562 leukemic cells incubated in various metal-enhanced cultures. Valuable quantitative and semiquantitative data can be obtained on as few as 400,000 cells.     

Energy dispersive X-ray analysis of tissue gold after silver amplification by physical development

Rats were treated intraperitoneally with the gold-containing compounds sodium aurothiomalate (Myocrisin), sodium aurothiosulfate (Sanocrysin), and aurothioglucose. Using stem energy dispersive X-ray analysis, gold and silver were shown to be located at the same point in lysosomes of proximal tubular cells of the kidney, in hepatocytes and in macrophages of lymph glands, spleen and liver. This result indicates that, after exposure to ultraviolet radiation, chemically bound tissue gold is transformed to metallic gold that subsequently can catalyze the reduction of silver ions to silver when subjected to physical development, i.e. exposed to a photographic developer containing silver ions in addition to the reducing molecules.