Cellular and subcellular distribution of metals in molluscs

The cellular processes involved in metal metabolism in molluscs are reviewed, with emphasis on the contribution of microscopy (AMG, ARG, EPMA, and SIMS) to both basic research of metal cell biology and applied environmental research. In molluscs, metal uptake may occur by facilitated diffusion, active transport, or endocytosis, and can be enhanced by MT synthesis or formation of mineralized granules. In aquatic molluscs, gills constitute a key interface for dissolved metal uptake, where metals are bound to MT, incorporated into lysosomes, and released basally towards the blood plasma and circulating hemocytes. However, particulate metal uptake is mainly achieved via the digestive tract by endocytosis; further metals are transferred first to lysosomes and then to residual bodies, especially in the digestive cells of the digestive gland. Additionally, metals can be accumulated selectively in specific cell types. As ligands pools differ from cell to cell, different metals may be retained in different cell types. Class "a" metals are localized in cells with granules composed of carbonate, oxalate, phosphate, and sulfate (oxygen donors), whereas "b" metals are associated with those cell types rich in sulfur and nitrogen ligands (sulfur donors). In molluscs, oxygen donors occur in connective tissue calcium cells and basophilic cells, whereas sulfur donors are present in digestive cells, podocytes, nephrocytes, and rhogocytes. Hemocytes, which constitute the most relevant system for metal transport between tissues, move around the body and may penetrate tissues and remove metals from the inner medium to be accumulated in lysosomes as nondigested products. Rhogocytes also participate in metal mobilization, accumulation, and release. The assessment of metal levels in target cells of sentinel molluscs by microscopic techniques provides an early-warning measure, with promising applications as an exposure biomarker for environmental monitoring programs.     

Dynamic structural changes of synaptic contacts in the visual system of insects

The visual system of insects provides an excellent model to study processes of transduction and transmission of photic information, synaptogenesis, synaptic plasticity, and wiring between photoreceptors and their visual interneurons in the optic lobe. This review describes synaptic contacts between photoreceptors and other neurons in the visual system of insects, especially in the fly's first optic neuropile (the lamina), and summarizes changes observed in the synapses of visual cells that have been reported both in phylogeny and ontogeny, and also examples of synaptic plasticity in adult insects that have been evoked by intrinsic and extrinsic factors. Plasticity observed in synapses of the insect's visual system seems to exemplify not only synaptic contacts in insects but, given that similar examples of plasticity have been found in other animal groups, may also be a general phenomenon in the nervous system.     

Localization of metals in cells of saprophagous soil arthropods (Isopoda, Diplopoda, Collembola)

This review summarizes results on the intracellular distribution of metals in cells of woodlice (Isopoda), millipedes (Diplopoda), and springtails (Collembola), which are three major groups of saprophagous arthropods contributing to the turnover of soil organic matter. Although the impact of metals and also metal pollution has inevitably been shown at levels of higher biological organization than subcellular mechanisms in these animal groups, the aim of this review is to focus exclusively on storage sites and aspects of intracellular metal metabolism. Thus, methodologically, results obtained by microscopical techniques such as histochemistry, X-ray microanalysis, energy filter transmission electron microscopy, or laser microprobe mass spectrometry were given preference. Results from atomic absorption spectrophotometry of cellular fractions were kept to a minimum. In all three taxa, the main intracellular metal storage sites are various types of "granules" which are widely distributed throughout cell types associated with the digestive system.     

Real-time gene delivery vector tracking in the endo-lysosomal pathway of live cells

Using live-cell confocal microscopy and particle tracking technology, the simultaneous transport of intracellular vesicles of the endo-lysosomal pathway and nonviral polyethylenimine (PEI)/DNA nanocomplexes was investigated. Due to potential problems associated with the use of acid-sensitive probes in combination with a gene vector that is hypothesized to buffer the pH of intracellular vesicles, the biological location of PEI/DNA gene vectors was revealed by probing their trafficking in cells expressing fluorescent versions of either early endosome antigen 1, a protein that localizes to early endosomes, or Niemann Pick C1, a protein that localizes to late endosomes and lysosomes. Studies directly show that PEI/DNA nanoparticles are actively transported within both early and late endosomes, and display similar overall transport rates in each. Additionally, gene vector transfer between endosomes is observed. Over time post-transfection, gene vectors accumulate in late endosomes/lysosomes; however, real-time escape of vectors from membrane-bound vesicles is not observed.     

Autofluorescence of living cells

We have investigated the autofluorescence of viable mammalian cells (DU-145 and V79) with a confocal laser scanning microscope equipped with a UV laser. Our aim was to investigate the autofluorescence dependence on different treatments in mitochondria and lysosomes by using different reagents and to improve the confocal laser scanning microscope image quality by deconvolution. The following conclusions were drawn from the results: (1) not all of the autofluorescence comes from mitochondria; (2) one can significantly affect the signal which comes from the mitochondria; (3) the other organelles involved are probably lysosomes; (4) it is harder to affect the autofluorescence signal from the lysosomes than that from the mitochondria, and (5) deconvoluted autofluorescence images provide better information than undeconvoluted ones.     

Demonstration of lanthanum in liver cells by energy-dispersive X-ray spectroscopy, electron energy loss spectroscopy and high-resolution transmission electron microscopy

The appearance of lanthanum in liver cells as a result of the injection of lanthanum chloride into rats is investigated by advanced transmission electron microscopy techniques, including electron energy loss spectroscopy and high‐resolution transmission electron microscopy. It is demonstrated that the lysosomes contain large amounts of lanthanum appearing in a granular form with particle dimensions between 5 and 25 nm, whereas no lanthanum could be detected in other surrounding cellular components.     

Intracellular localization of samarium in the lactating mammary gland cells: ultrastructural and microanalytical study

The frequent use of some rare earths in the medical and industrial domains make us worry about their intracellular behavior into the body. Reason for which we have investigated the subcellular localization of one of these elements, the samarium, in the mammary gland of lactating female wistar rats using two very sensitive methods of observation and microanalysis, the transmission electron microscopy and the secondary ion mass spectrometry. The ultrastructural study showed the presence of electron dense deposits in the lactating mammary glandular epithelial cell lysosomes of the samarium-treated rats, but no loaded lysosomes were observed in those of control rats. The microanalytical study allowed both the identification of the chemical species present in those deposits as samarium isotopes ((152) Sm(+)) and the cartography of its distribution. Our results confirm the previous ones showing that lysosomes of the glandular epithelial cells are the site of the intracellular concentration of foreign elements such as gallium. The intralysosomal deposits observed in the mammary glandular cells of the samarium-treated rats are similar in their form and density to those observed with the same element in other varieties of cells, such as liver, bone marrow, and spleen cells. Our ultrastructural and microanalytical results and those obtained in previous studies allow deducing that the intralysosomal deposits are very probably composed of an insoluble samarium phosphate salt.     

Ultrastructural study of the intracellular behavior of four mineral elements in the lactating mammary gland cells: study using conventional transmission electron microscopy

The effects of parenteral injection of aluminum, indium, gadolinium, or terbium in rats have been previously studied in several organs such as the liver, the kidneys, etc., but never in mammary glands. In this work, we have attempted to study the subcellular localization of these elements after their intraperitoneal administration. Their subsequent effects in the lactating mammary gland cells have also been studied. Our results using conventional transmission electron microscopy have shown that the lysosomes of the mammary glandular epithelial cells are the intracellular site of accumulation of the studied elements. Our results have also show intracellular deteriorations such as an expanded ergastoplasm and altered mitochondria after intraperitoneal injection of aluminum and indium.     

Electron microscopic observation of photoreceptor cells in directly inserted anesthetized Drosophila into a high‐pressure freezing unit

The high‐pressure freezing (HPF) technique is known to cryofix water‐containing materials with little ice‐crystal formation in deep depths compared with other freezing techniques. In this study, HPF for anesthetized living Drosophila was performed by placing them directly on the carrier of the HPF unit and exposing them to light. Frozen Drosophila were freeze substituted, and their compound eyes were examined by transmission electron microscopy. The ultrastructures of ommatidia composed of photoreceptor cells were well preserved. The location of the cytoplasmic organelles inside the photoreceptor cells was observed. In some photoreceptor cells in ommatidia of the light‐exposed Drosphila, the cytoplasmic small granules were localized nearer the base of rhabdomeres, compared with those of the nonlight‐exposed Drosophila. Thus, HPF with the direct insertion of living Drosophila under light exposure into the HPF machine enabled us to examine changes to functional structures of photoreceptor cells that occur within seconds.     

电子探针测定镁橄榄石中元素含量的不确定度

本文基于氧化物的ZAF修正法对典型样品进行定量分析。在得出了定量分析结果之后,分析了样品测量不确定度的来源,得出电子探针定量分析的不确定度模型,测算了每个影响因素的具体数据,最后计算出了定量结果的扩展不确定度。结果表明ZAF修正法引入的不确定度对测试结果的不确定度影响非常小,主要影响结果不确定度的因素是测量重复性引入的不确定度造成的。利用不确定度提高测试和仪器校准的准确度,可以对电子探针定量分析的后续工作提供基础和保障。     

Localization of lead in Allium cepa L. cells by electron microscopy

The study of mechanisms by which plants tolerate lead requires ultrastructural observations of lead distribution in cells. However, the conventional technique used in electron microscopy brings up the problem of lead translocation from tissues during chemical processing. If most of the lead is washed out of tissues, then the method is not suitable for this type of study; if, however, it remains in the tissues, the method can be used. The amount of lead washed out of Allium cepa root tips during successive stages of fixing and dehydrating was determined in this study. Allium cepa plants were treated with Pb(NO₃)₂ in hydroponic cultures. The conventional fixing (GA, OsO₄) and dehydration (alcohol + propylene oxide or acetone) methods used in the preparation of tissues for electron microscopy were then applied to root tips. The lead content in tissues and in reagents was analysed in the successive stages of the procedure. It was shown that 96.2% of the lead taken up was retained in the tissues and was not washed out during fixing and dehydrating. Of the 3.8% of the lead lost in the chemical procedure, about half was washed out during fixing in osmium tetroxide. This reagent is thus the least suitable for this type of study. Our study showed that the conventional electron microscopy preparative technique is appropriate for studying the distribution of lead deposits in A. cepa root cells, owing to the way in which lead is compartmentalized/sequestered in plant cells.     

虚拟企业伙伴选择中的多目标决策问题

在竞争，合作，动态的市场环境中，虚拟企业被认为是21世纪最有竞争力的企业运行模式，但如何选择合适的伙伴，则是虚拟企业能否组建成功的关键。在应用整数规划描述虚拟企业伙伴选择多目标决策模型的基础上，给出了支持伙伴选择的多目标决策工具与算法，并以实例说明该工具能有效支持虚拟企业伙伴的选择。     

Processing of auditory information in insects

Insects exhibit an astonishing diversity in the design of their ears and the subsequent processing of information within their auditory pathways. The aim of this review is to summarize and compare the present concepts of auditory processing by relating behavioral performance to known neuronal mechanisms. We focus on three general aspects, that is frequency, directional, and temporal processing. The first part compares the capacity (in some insects high) for frequency analysis in the ear with the rather low specificity of tuning in interneurons by looking at Q10dB values and frequency dependent inhibition of interneurons. Since sharpening of frequency does not seem to be the prime task of a set of differently tuned receptors, alternative hypotheses are discussed. Moreover, the physiological correspondence between tonotopic projections of receptors and dendritic organization of interneurons is not in all cases strong. The second part is concerned with directional hearing and thus with the ability for angular resolution of insects. The present concepts, as derived from behavioral performances, for angular resolution versus lateralization and serial versus parallel processing of directional and pattern information can be traced to the thoracic level of neuronal processing. Contralateral inhibition, a mechanism for enhancing directional tuning, appears to be most effective in parallel pathways, whereas in serial processing it may have detrimental effects on pattern processing. The third part, after some considerations of signal analysis in the temporal domain, demonstrates that closely related species often use different combinations of temporal parameters in their recognition systems. On the thoracic level, analysis of temporal modulation functions and effects of inhibition on spiking patterns reveals relatively simple processing, whereas brain neurons may exhibit more complex properties.     

Lateral sensitivity in electron probe microanalysis studied by Monte Carlo simulations involving fluorescence enhancements

In electron probe microanalysis, secondary fluorescence can occur leading to an increase of the volume analysed, degrading the lateral resolution of this technique. An adequate knowledge of the interaction volumes from where the different signals of interest are detected is determinant to estimate the minimum size of the zone that can be characterized. In this work, the size of the signal source volume is surveyed for a wide set of samples at different beam energies. To this aim, the PENELOPE software package was chosen to run Monte Carlo simulations for several experimental situations in order to produce the various lateral radiation distributions of interest. A comparison between the interaction volumes of the different signals was performed by taking into account the different fluorescence enhancement possibilities. An unexpected behaviour was found in the particular cases of aluminium and alumina, where the secondary photons signal exhibits a decreasing trend up to certain beam energy (～17 keV); this implies that lower beam energies may degrade the lateral resolution of the technique in these materials.     

The potentials of MS-based subproteomic approaches in medical science: the case of lysosomes and breast cancer

Because of the great number of women who are diagnosed with breast cancer each year, and though this disease presents the lowest mortality rate among cancers, breast cancer remains a major public health problem. As for any cancer, the tumorigenic and metastatic processes are still hardly understood, and the biochemical markers that allow either a precise monitoring of the disease or the classification of the numerous forms of breast cancer remain too scarce. Therefore, great hopes are put on the development of high-throughput genomic and proteomic technologies. Such comprehensive techniques should help in understanding the processes and in defining steps of the disease by depicting specific genes or protein profiles. Because techniques dedicated to the current proteomic challenges are continuously improving, the probability of the discovery of new potential protein biomarkers is rapidly increasing. In addition, the identification of such markers should be eased by lowering the sample complexity; e.g., by sample fractionation, either according to specific physico-chemical properties of the proteins, or by focusing on definite subcellular compartments. In particular, proteins of the lysosomal compartment have been shown to be prone to alterations in their localization, expression, or post-translational modifications (PTMs) during the cancer process. Some of them, such as the aspartic protease cathepsin D (CatD), have even been proven as participating actively in the disease progression. The present review aims at giving an overview of the implication of the lysosome in breast cancer, and at showing how subproteomics and the constantly refining MS-based proteomic techniques may help in making breast cancer research progress, and thus, hopefully, in improving disease treatment.     

汽车零部件铝合金轴瓦及其表面涂层的产品解析

轴承部件具有耐磨、耐蚀、一定温度区间的热化学稳定性以及优良的润滑特性。某进口铝合金轴瓦经过表面涂层处理后,具有良好的无油自润滑性能。借助电子探针显微分析仪器EPMA、能量色散X射线荧光光谱仪EDX、X射线光电子能谱仪XPS和傅立叶变换红外光谱仪FTIR,分析其基体和涂层信息,推断了其实现方案,为开发类似的产品工艺分析方案提供了一定的参考。     

Wavelength- and energy-dispersive Electron Probe Microanalysis (EPMA) measurements with non-perpendicular incidence of the electron beam

A database of 416 wavelength‐ and energy‐dispersive EPMA measurements on tilted specimens of NiAl, TiO₂ and Ti₃Al is presented. The analyses were performed between 10 and 30 kV and the tilt angles were varied between 0° and 60° in seven steps. The necessary hardware modifications for the specimen holder are discussed, as well as the various focusing techniques used in the measurements. A comparison between the experimental data, the calculations of our proza 96t program and the results of Monte Carlo simulations shows that up to 50° tilt the predictions of our software are more than satisfactory. At larger tilt angles some deviations become noticeable. The Monte Carlo simulations appear to produce deviations at a somewhat earlier stage already, for reasons as yet unknown.