Comparative ultrastructure of nonwounded Mexican lime and Yuzu leaves infected with the citrus canker bacterium Xanthomonas citri pv. citri

Ultrastructural aspects of citrus canker development were investigated in nonwounded leaves of citrus species by transmission electron microscopy (TEM). A susceptible species Mexican lime and a resistant species Yuzu were spray-inoculated with a virulent strain of Xanthomonas citri pv. citri. Initial symptoms occurred on Mexican lime ∼9 days after inoculation, whereas they appeared on Yuzu mostly 11 days after inoculation. In Mexican lime leaves, the bacterial invasion was usually accompanied by host cell wall dissolution and cellular disruption. Fibrillar materials from degenerated cell walls were usually found in intercellular spaces. Damaged host cells with necrotic cytoplasm showed the localized separation of plasma membrane from the cell wall. Bacterial multiplication and electron-transparent capsule-like structures around bacteria were commonly observed. Meanwhile, cell wall protuberances were prominent outside host cell walls in response to bacterial invasion in Yuzu leaves. Occlusion of intercellular spaces was also formed by the fusion of two or more individual cell wall protuberances originated from two adjacent host cells. Papillae-like materials accumulated locally within host cells in close proximity to bacteria. Some bacteria were found to be undergoing degeneration in xylem vessels. Also, the shrunken, inactive bacteria were surrounded by electron-translucent fibrillar materials in intercellular spaces, implying bacterial immobilization. These cellular responses are thought to be the consequences of defense responses of Yuzu leaves to invading bacteria. In both citrus species, X. citri pv. citri contained polyphosphate bodies showing electron-dense and elliptical structures in cytoplasm. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Plant regeneration after long-term callus culture in clones of Asparagus officinalis L.

Callus growth and plant regeneration from long-term callus cultures were studied in two elite clones of Asparagus officinalis cv. Argenteuil, to establish a suitable protocol for a prospective in vitro selection program. Callus initiation and growth was evaluated on MS medium with 3% sucrose, 0.9% agar, 1 mg x l(-1) kinetin, and three levels of 2,4-D. The highest callus relative growth was obtained on medium with 1.5 mg x l(-1) 2,4-D and 1 mg x l(-1) kinetin. Shoot primordia (SP) induction from > 18-months-old calluses was evaluated on several media; the highest percentage of SP induction (89%) and average number of SP per callus (8.6) were obtained with clone "265" on MS medium with 5 mg x l(-1) 2iP, 1 mg x l(-1) IAA, 3% sucrose and 0.9% agar. The highest percentage of root induction (100%) was achieved with clone '265' on MS medium with 0.1 mg x l(-1) kinetin, 0.1 mg x l(-1) NAA, 1.32 mg x l(-1) ancymidol, 7% glucose and 0.8% agar. Important medium x genotype interactions were detected, pointing to the need of adjusting this and other in vitro protocols for specific asparagus genotypes.     

Refractometry of fungi

Immersion refractometry is based on matching the refractive index of the medium to that of the cytoplasm of the cell, in which case the latter will have minimum contrast in the phase contrast microscope and will practically disappear. The cytoplasm is then said to be at the match point with the medium. Since the refractive index of a solution is related to its concentration by the formula n - n_o =αC, the values for the refractive indices can be expressed in terms of the concentration of solids (g/100 ml) which in fact has been done throughout this work. The properties of a suitable immersion medium, its preparation and interpretation of numerical results have been discussed. Refractometric measurements were carried out on fungal hyphae and spores. The solid concentration in hyphae varies according to their age, physiological state and function. In general the highest values are found at sites of physiological activity, such as growth and reproduction, and may range from 11% to 30% of solid concentration. The solid concentration of mature, resting spores was found to be in the range of 45–54%, with the exception of a powdery mildew spores in which the concentration of solids was 30–32%. The germination of spores is preceded by a fall in the concentration of solids, and subsequent swelling before the emergence of the germ tube. When the solid concentration in pregerminating spore has fallen to about 20–30%, metabolic activity can take place and germination can occur.     

Enzyme-gold affinity labelling of cellulose

The enzyme-linked colloidal gold affinity labelling technique was tested as a method to localize cellulose on thin sections of plant cell walls and slime mold spores. Commercially available cellulase from cultures of Trichoderma reesei, the main components being cellobiohydrolase I and II (CBH I, CBH II) and endoglucanase (EG), was linked to colloidal gold by using standard techniques and applied as a dilute, buffered suspension to thin sections. After brief exposure, e.g., 15–30 minutes, cellulose exposed on the surface of sections was labelled with the enzyme-gold complex. Poststaining did not appear to have a deleterious effect on the labelled sections. The specificity of labelling was demonstrated by its complete inhibition when carboxymethylcellulose was incorporated in the labelling mixture, by lack of labelling of 1,4-β-mannans or 1,3-β-xylans in noncellulosic walls of marine algae, by lack of labelling of 1,4-β-glucans in chitin, by much lower labelling density when done at 4°C, and by lack of labelling when sections were predigested with cellulase. Labelling with the crude commercial cellulase was compared to labelling with purified CBH I-, CBH II-, and EG-linked colloidal gold, and the labelling pattern was similar. This method was found useful on conventionally fixed material and required no special preparation other than the use of inert (Ni or Au) grids and 0.5% gelatin to reduce nonspecific binding of the gold complex. Labelling was similar in the several embedding resins tested: LR White, Lowicryl K4M, Epon 812, and Spurr's. The cellulase-gold probe remained active for at least 4 weeks at 4°C and much longer when frozen at ?80°C in 20% glycerol. This technique should prove useful in studies of cellulose degradation and cellulose deposition and of the interaction of cellulose with other wall components.     

Seawater Mercury Analysis at PPB Levels

Abstract

A new method of mercury detection at ng/mL concentrations in seawater, by total reflection x‐ray fluorescence (TXRF), is presented. Membranes that have complexation affinity to the mercury ions were produced on quartz reflectors. They were immersed in various seawater solutions containing mercury at low concentration (1–50 ng/mL) and they were left to equilibrate for 24 hours; membranes were well fixed on the reflectors. After the equilibration stage, they were taken out of the solution and they were analysed by TXRF; the minimum detection limit was determined to be equal to 0.4 ng/mL. The main ingredient of the membranes was the complexing reagent dithizone. The existence of high salt concentration in seawater did not prevent mercury's complexation from the membrane.     

Examination of occlusions in xylem vessels of cut rose flowers,using cryoscanning electron microscopy and cryoultramilling cross-sectioning

The occlusions occurring in xylem vessels of cut rose flowers (Rosa hybrida L.) were examined using freeze fracturing and conventional SEM, or cryoultramilling cross-sectioning followed by cryo-SEM. The study provides an excellent example of the utility of the latter procedure. The stems contained bacteria surrounded by amorphous material. The amorphous material, which was likely bacterial slime, was more abundant in cryo-SEM. The ultramilling method resulted in smooth cross-sectioning of the xylem walls. The results show that bacteria do not degrade the xylem walls.     

Use of primary cell cultures and intact isolated glandular epithelia for X-ray microanalysis

Changes in the elemental composition of cells during isolation of glandular epithelia were studied by electron probe X-ray microanalysis. Fine chopping of rat submandibular gland followed by enzymatic treatment for 15 min caused marked increases in Na and Cl and a decrease in K concentrations in acinar cells. After enzymatic treatment for 50 min, Na, Cl and K concentrations returned to close to the control level. Mechanical disaggregation of the acinar clumps following enzymatic treatment resulted again in minor increases in Na and Cl and a marked decrease in K concentration. Exposure of isolated acini to cholinergic stimulation in vitro resulted in secretion of Cl and K from the acinar cells. Dissection of the sweat gland from human skin caused a decrease in the K/Na ratio. Incubation of the gland for 30–45 min with collagenase gave rise to a gradual decrease in the K/Na ratio. After mechanical separation of the gland into the secretory coil and reabsorptive duct, a further reduction of the K/Na ratio was seen. However, the duct cells had a much lower K/Na ratio and higher Ca concentration than the coil cells. In primary cultures, the K/Na ratios of the coil and duct cells returned to the in situ level. The elemental composition of sweat gland cells incubated in collagenase-containing medium was no different from that in cells incubated in collagenase-free medium. In the intact collagenase-isolated tissue, Cl^? secretion in the coil was elicited by carbachol but not by cAMP, whereas in the duct cells the reverse was the case. In primary cell cultures, Cl^? efflux in both coil and duct cells could be elicited by both carbachol and cAMP. In conclusion, although changes in elemental composition of gland cells during the isolation procedure occur, physiological responses can be detected. When primary cell cultures are used, it should be borne in mind that cultured cells may have physiological properties different from those of the intact tissue.     

Elemental concentrations in isolated rat thymocytes prepared for cryofixation in the presence of different media

Rat thymocytes were isolated in suspension and the effect of preparing the cells for cryofixation in the presence of different media on the elemental content was investigated using the technique of X-ray microanalysis. Cells prepared in the different media showed variation in the concentrations of Na, K and Cl. The isolated cells were incubated at 310 K for 1 h to allow recovery from isolation. There was a decrease in Na and Cl content after incubation. The thymocyte population was disturbed by suspension in medium containing dextran, and this resulted in a number of cells with high concentrations of Na and low concentrations of K. These cells did not take up vital dye. Thymocytes were also prepared for freezing by using high-speed centrifugation to concentrate the cells. Thymocytes prepared by this method showed values for concentrations of Na, K and Cl similar to published values for these cells using other methods of estimation. There were, however, consistent differences in Na content between the cells prepared in Hanks' balanced salt solution and those prepared in serum. Factors which affect the apparent concentration of Na and Cl in isolated cells are discussed.     

Targeted recombinant aequorins: tools for monitoring [Ca2+] in the various compartments of a living cell.

In the last decade, the study of Ca2+ homeostasis within organelles in living cells has been greatly enhanced by the utilisation of a recombinant Ca(2+)-sensitive photoprotein, aequorin. Aequorin is a Ca2+ sensitive photoprotein of a coelenterate that, in the past, was widely employed to measure Ca2+ concentration in living cells. In fact, the purified protein was widely used to monitor cytoplasmic [Ca2+] changes in invertebrate muscle cells after microinjection. However, due to the time-consuming and traumatic procedure of microinjection, the role of aequorin in the study of Ca2+ homeostasis remained confined to a limited number of cells (giant cells) susceptible to microinjection. Thus, in most instances, it was replaced by the fluorescent indicators developed by Roger Tsien and coworkers. The cloning of aequorin cDNA [Inouye et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3154-3158] and the explosive development of molecular biology offered new possibilities in the use of aequorin, as microinjection has been replaced by the simpler technique of cDNA transfection. As a polypeptide, aequorin allows the endogenous production of the photoprotein in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localise it within the cell by including defined targeting signals in the amino acid sequence. Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles. In this review, we will not discuss the procedure of aequorin microinjection and its use as purified protein but we will present the new advances provided by recombinant aequorin in the study of intracellular Ca2+ homeostasis, discussing in greater detail the advantages and disadvantages in the use of this probe.     

Agar standards for quantitative X-ray microanalysis of resin-embedded plant tissues

Calibration standards for quantitative X-ray microanalysis of resin-embedded plant tissue were prepared by adding 6600 mM KC1 to 5% agar. Agar blocks with an edge length of 1–2 mm were rapidly frozen, freeze-dried and embedded in styrene-methacrylate. Dry sections 1 μm thick were mounted on adhesive-coated grids. Apart from fine-scale inhomogeneities caused by ice crystal formation, the KC1 is evenly distributed in the agar blocks. The peak-to-continuum values of K and Cl were highly linearly correlated to the K and Cl contents over the whole concentration range.     

Podocyte electrophysiology,in vivo and in vitro

Podocytes are the most differentiated cell types in the glomerulus, which have been assumed to participate in the regulation of the ultrafiltration coefficient K(f). In podocytes in vivo and in vitro vasoactive agonists, such as angiotensin II and acetylcholine, increase the free cytosolic Ca(2+) concentration via a release of Ca(2+) from intracellular stores and an influx of Ca(2+) from the extracellular space. An increase of the cytosolic Ca(2+) in podocytes activates Cl(-) channels in podocytes in vivo and in vitro, resulting in a depolarization of podocytes. In vitro studies have shown that in addition to Ca(2+)-activated Cl(-) channels, cAMP-activated Cl(-) channels and Ca(2+)-activated K(+) channels are present in cultured podocytes. The characterization of the signaling pathways that regulate ion channels in podocytes may be important in the understanding of the regulation of the ultrafiltration coefficient K(f). This review summarizes the currently known electrophysiological properties of podocytes.     

Friction and wear behaviors of a (Ca, Mg)-sialon/SAE 52100 steel pair under the lubrication of various polyols as water-based lubricating additives

The friction and wear behaviors of (Ca, Mg)-sialon/SAE 52100 steel pair under the lubrication of water or various polyol aqueous solutions were investigated with an SRV friction and wear tester in a ball-on-disc configuration. This was conducted to simulate the effect of polyols as aqueous additive in machining sialon ceramic. The morphologies of and elemental distributions in the worn surfaces of the lubricated sialon ceramics and counterpart steel were observed and determined with scanning electron microscopy (SEM) and electron probe microanalysis (EPMA). All solutions of the tested polyols decreased the friction coefficient of (Ca, Mg)-sialon/SAE 52100 steel effectively and increased the wear volume loss of (Ca, Mg)-sialon to some extent as compared with dry sliding. The friction coefficients under the lubrication of distilled water and various polyols aqueous solutions of polyols showed almost no difference, and propanetriol was found to be the most effective for machining (Ca, Mg)-sialon with the concentration of polyols in water fixed as 5 wt%. The friction coefficients under the lubrication of propanetriol aqueous solutions in varied concentrations are closely related with the concentration, which came to the lowest value of 0.04 at a concentration of 75%. The friction-reducing performance of the polyols as additives in water was roughly correlated with their wetting behaviors on the sialon ceramic surface. In other words, the higher the wetting ability is, the lower the friction coefficients will be. Moreover, the wear volume losses of (Ca, Mg)-sialon also varied with the variation in the concentration of propanetriol in water. Accounting for the friction-reduction and wear behavior, 20% concentration of propanetriol in water could be recommended for machining (Ca, Mg)-sialon. Electron microscopic analysis indicates that polyols as additives in water enhanced the corrosive wear of sialon ceramic, which could be beneficial for increasing the machining efficiency. There existed interactions among water, polyols and sialon surfaces, which were dependent on the compositions of the lubricant solution. This accounts for the variations in the friction and wear behaviors with the concentration of polyols in water.     

EDX measurements and SEM examination of surface of some imported woods inoculated by three mold fungi

Wood samples of Pinus rigida, Juglans nigra, and Fagus sylvatica were superficially inoculated with three mold fungi (Penicillium selerotigenum, Paecilomyces variotii, and Aspergillus niger). The changes in wood surfaces and the elemental composition were observed by scan electron microscope (SEM) and the electron dispersive X-ray spectroscopy (EDX), respectively. Wood elemental composition was an indication of the growth of mold fungi, in which P. selerotigenum, and A. niger have consumed high amount of carbon (C) of F. sylvatica wood compared to control treatment. Also, P. selerotigenum consumed high amount of C of J. nigra wood compared to the control. While, the three mold fungi showed little changes in C content in P. rigida wood. The nitrogen element in the studied woods was completely consumed by the three mold fungi. Cl element increased in the three woods as inoculated by the three fungi compared to untreated woods. Other elements such as S, Si, Ca, Zn, Cu, and Fe varied in their content in accordance to the interaction among wood species and fungi. The growth of the three mold fungi showed distribution and structures of spores and hyphae covering deteriorated wood surfaces. The study suggests that the three mold fungi metabolize the carbon-rich constituents of the investigated wood species in a proportional manner and produce large fruiting structures that release vast numbers of spores in nature.     

Quantitative X-ray microanalysis of solutes in individual plant cells: a comparison of microdroplet and in situ frozen-hydrated data

Two different approaches to X-ray microanalysis were tested and compared. These were the analysis of sap droplets extracted from individual cells (plants grown and analysed in Bangor, U.K.), and the analysis of cells in situ in frozen tissue (plants grown and analysed in Hannover, Germany). The data suggest that both these methods can produce quantitative data accurately reflecting in vivo concentrations in cereal leaf epidermal cells. The relative merits of the two procedures are discussed with reference to possible sources of error and their application to other cell types. Bulk wheat leaf tissue concentrations of K and Cl did not differ significantly between the two locations, but Ca concentration was significantly higher in the plants grown in Hannover.
Microdroplet analysis invariably yielded linear responses in the range of concentrations found in plant tissue ( r ² for Ca > 0.97, r ² for K, Cl > 0.99), and interference of other components of the sap was minimal. The calibration curves for the frozen-hydrated material were typically linear in the same range of concentrations ( r ² for K, Ca, Cl > 0.95), and the results for K and Cl concentration in these samples were highly consistent with those measured in the microdroplet experiments. In wheat, for example, the cellular Cl concentration varied between 12 m M and 119 m M , but no significant differences were found between the two techniques of measurement. The results for cellular Ca differed in a manner which might be predicted from the results of the bulk tissue analyses.     

The application of optical microscopy and electron-probe microanalysis to a mineral processing topic—the high-temperature reduction of ilmenite

A combination of optical microscopy and electron-probe microanalysis (EPMA) has been used to study the phases which occur during reduction of the mineral ilmenite to metallic iron and titanium oxides. Quantitative EPMA of natural and chemically-processed minerals frequently suffers from variable specimen conductivity caused by the porosity and varying texture of the mineral grains. It has been found, however, that the element concentration ratios are largely independent of porosity and texture and can be used to identify the phases with precision. The microprobe has revealed some hitherto unsuspected effects arising from small concentrations of magnesium and manganese dissolved in the ilmenite lattice. The morphology and composition of the reduced product are in fact controlled by the way in which these impurity solutes segregate within the mineral grains during reduction.     

The adhesion mechanism of the built-up edge and the layer on the rake face of a cutting tool

When machining carbon steel a built-up edge forms on the rake face of the cutting tool during low speed cutting and a layer is deposited on the tool during high speed cutting. Differences in the mechanism of adhesion between the built-up edge and the layer on the tool were investigated by electron probe microanalysis (EPMA) and electron diffraction. EPMA was used to detect element transfer at the boundary between the deposit and the tool. Electron diffraction was used to determine the identity of the characteristic materials at the boundary. Element diffusion occurred in the built-up edge on the tool from a depth about 40 μm below the tool surface to the surface of the tool. Many carbides were found at the boundary between the built-edge and the tool. In the layer deposited on the tool, alloying elements diffused from the tool to the chip, which resulted in a reduction of their concentration at the tool surface. Many oxides were present at the boundary between the layer and the tool, and the layer strongly adhered to the tool.     

武钢三硅钢水处理中大型石灰乳制备装置的研发

在钢厂冷轧生产过程产生的废水中,含有大量的酸,需要通过投加碱性的CaO或Ca(OH)2乳液进行中和。针对已有的石灰乳制备装置存在的问题,研制开发了机电一体化大型石灰乳制备装置,并成功应用于武钢三硅钢冷轧水处理项目。本文主要叙述了该大型石灰乳制备装置结构及工艺流程、设备的技术参数说明及与国外同类技术产品的比较。     

Biological calcium absorption edge imaging using monochromatic synchrotron radiation.

Soft X-ray contact absorption edge images of unfixed, unstained biological specimens were made using monochromatic synchrotron radiation. X-ray contact replicas of unfixed, hydrated biological specimens at the nitrogen absorption edge and above and below the CaLIII absorption edge were compared to comparative conventional morphological and elemental high-resolution imaging methods (scanning and transmission electron microscopy, TEM-histochemistry and TEM-X-ray microanalysis). Soft X-ray absorption edge images made above the calcium absorption edge clearly revealed morphological detail and identified regions ladened with calcium as verified by TEM histochemistry of identical spores. Similarly, nitrogen absorption edge images identified residual nitrogenous material in the spore resuspension medium, and non-viable spores with nitrogen loss due to protoplast disaggregation.     

Mechanism by which an elevation of extracellular glucide concentration induces pigment aggregation in medaka melanophores

An increase in glucide concentration induces pigment aggregation in melanophores in the skin on scales isolated from the medaka, Oryzias latipes. In this study, hexoses (including the common D-isomers of glucose, galactose, fructose, and mannitol) were examined. Denervated melanophores were refractory to such stimuli. An alpha-adrenolytic agent, phentolamine, effectively blocked the responses of normally innervated melanophores. The pigment-aggregating action of glucide was inhibited by withdrawal of Ca(2+) and Mg(2+) ions from the medium. A specific blocker of voltage-dependent N-type Ca(2+) channels, the omega-conotoxin GVIA, also inhibited the glucide action. The conclusion derived is that an elevation of glucide levels acts to open Ca(2+) channels of presynaptic membranes of sympathetic postganglionic fibers, and the consequently released adrenergic transmitter acts on the effector cells to induce the aggregation of their pigmentary organelles.     

FEA for damping of structures having elastic bodies,viscoelastic bodies,porous media and gas

A numerical method is proposed to calculate damping properties for soundproof structures involving solid bodies, porous media and air in two-dimensional regions. Both effective density and bulk modulus have complex quantity to represent damped sound fields in the porous media. Particle displacements in the media are discretized using finite element method. For damped solid bodies, displacements are formulated using conventional finite elements including complex modulus of elasticity. Displacement vectors as common unknown variables are solved under coupled condition between solid bodies, porous media and gas. Further, by applying asymptotic method to complex eigenvalue problem, explicit expressions of modal loss factor for the mixed structures are derived. The proposed methods yield appropriate results for some typical problems and this method diminish computational time for large-scaled finite element models concerning the mixed structure. Moreover, it is found that damping can be coupled in the mixed structures between solid bodies, porous media and air.