The Formation of Intracellular Crystals in Midgut Glands of Limnoria lignorum

Certain morphological features of intracellular crystal formation within the midgut glands of Limnoria lignorum (Rathke) have been studied with the electron microscope and cytochemical methods. A correlation has been established between Golgi membranes and formation of the crystals. The Prussian blue reaction reveals quantities of iron localized in the intracellular crystals and in small granular structures seen in the apical region of the cells. These granules can be identified as accumulations of Golgi membranes, with which iron-containing particles are associated. When these membrane configurations are studied with the electron microscope, they can be classified and arranged in an assumed sequence which is thought to represent successive stages in the development of crystals. As the membrane systems become progressively specialized, increasing accumulations of dense granular material appear within their interstices. This material is rich in iron and probably represents the component responsible for the positive Prussian blue reaction. This material also appears to be a precursor substance for iron-containing protein molecules which are synthesized and arranged to make up the crystals. These iron-containing molecules are first deposited in orderly array as double rows of dense particles on certain internal membranes of the specialized Golgi complexes. The membranes later disappear and the particles form definitive crystals by rearrangement into a hexagonal close-packed pattern.     

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires "cryofixation" of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 °C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at ≤-160 °C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 °C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their "characteristic" peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.     

猫肾离体保存中亚细胞水平上Ca^2＋浓度的X—射线微区分析

应用定量Ｘ－射线微区分析技术结合细胞化学技术,分析测定用单纯冷冻法保存离体猫肾脏过程中肾脏细胞的胞浆、线粒体、内质网、细胞核等细胞器内的Ｃａ２＋浓度变化,并探索钙通道阻滞剂对这种变化的影响。保存３６小时及７２小时后,线粒体与胞浆中Ｃａ２＋的峰背比极显著地提高,内质网、细胞核中钙颗粒减少。添加Ｖｅｒａｐａｍｉｌ后,保存过程中细胞器内Ｃａ２＋无显著变化。线粒体中的Ｃａ２＋峰背比与胞浆中的呈强的正相关,ｒ＝０９９０。实验结果显示：保存过程中,Ｃａ２＋由钙库（内质网等）进入胞浆中,线粒体在胞浆Ｃａ２＋浓度高时摄取Ｃａ２＋,而钙通道阻滞剂可抑制该过程。     

Salt Glands in the Zoysieae

Salt glands were found in two species of the genus Zoysia ofthe tribe Zoysieae, sub-family Chloridoideae (Poaceae). Glandsprotrude from and are recumbent to the leaf epidermis, and consistof two cells, a basal cell and upper cap cell. Glands were betterdeveloped on the adaxial surfaces. Those on the abaxial surfaceappear to be non-functional. Zoysia matrella, the more salt-tolerantspecies, had a higher density of larger glands, and secretedmore sodium per unit leaf mass, resulting in much lower leafsap osmolalities than those of the more salt-sensitive Z. japonica.The finding of salt glands in the tribe Zoysieae confirms itsrelation to the four other tribes within the sub-family Chloridoideaein which salt glands have previously been reported. Salt glands, Zoysieae, Poaceae, Japanese lawngrass, Zoysia japonica, Manilagrass, Zoysia matrella, sodium chloride, salt tolerance, secretion     

The Ultrastructure of Frankenia Salt Glands

The ultrastructure of the salt gland of Frankenia has been investigated.Glands were examined after treatment with distilled water andafter 4 h, 15 h, and 36 h of salt treatment. Characteristiccytoplasmic features can be correlated with each of these treatments.The results suggest that the acquisition and maintenance ofthe secreting state involves the vesiculation of endoplasmicreticulum.     

Functional Aspects of the Salt Glands of the Plumbaginaceae

X-ray microanalysis and diffuse reflectance infrared Fouriertransform spectrometry were used to determine the presence andratios of elements in salt secretions from salt glands of greenhouseand experimentally-manipulated leaves of five species of thePlumbaginaceae Sodium, magnesium, silica, sulphur, phosphorus,chloride, potassium, calcium and carbonate were detected insecretions of greenhouse-grown plants. The salt glands of excisedleaves challenged by solutions of KI, KCl, NaCl, Na₂SO₄, MgCl₂and MgSO₄ secreted principally the ions of the challenging solutions. Key words: Ion transport, secretion     

Structural Aspects of the Salt Glands of the Plumbaginaceae

Faraday, C. D. and Thomson, W. W. 1986. Structural aspects ofthe salt glands of the Plumbaginaceae.—J. exp. Bot. 37:461–470. The epidermal salt-secreting glands of 11 species from six differentgenera within the Plumbaginaceae were examined Gland ultrastructurewas considered with respect to species, secretory activity,and secretory product. All mature glands had a similar ultrastructure.Cytoplasmically dense secretory cells contained a full complementof organelles and structures which included numerous mitochondriaand few plastids. Reconstruction of serial paradermal sectionsthrough entire glands revealed that each gland cell generallycontained one or two vacuoles with a convoluted tonoplast inboth secreting and non-secreting states. The absence of numerousvacuoles and vesicles during secretory activity suggested thation secretion was by a transmembrane pathway rather than bya vesicle-mediated pathway. Key words: Salt glands, ultrastructure, Plumbaginaceae     

X-ray Microanalysis of the Mineral Contents of Some Protozoa

With the aid of energy-dispersive X-ray microanalysis, several protozoa were tested for content of cations within inorganic minerals. The skeleton of acantharia consists mainly of Sr with small quantities of Ca and Ba. Two Loxodes species contain nothing but Ba, while in some Remanella species Sr with small quantities of Ba were present. In one Geleia species, Ca with small quantities of Sr was found; in two Trachelocerca species from Sylt (Germany), Ba is there in addition. Another Trachelocerca species from northern Italy lacked Ba, but did possess Mn. In Prorodon only Ca was found.     

X-ray Microanalysis of Phosphorus in Scenedesmus obtusiusculus

Results from energy dispersive x-ray microanalysis indicate considerable variation in phosphorus as well as in other elements found in polyphosphate granules in cells of Scenedesmus obtusiusculus starved of phosphorus for 24, 36 and 48 h. Comparison is made with cells at a 3-h stage of synchronization and after 48 h cultivation in a phosphorus-containing medium. After 48 h with a very limited external source of phosphorus, phosphorus was not detectable in‘polyphosphate granules’ in Epon-Araldite sections unless the resin and other organic molecules were removed by plasma microincineration. In spite of the low amounts of elements (Mg. Al, Si, P and Ca) detected in these granules they are still dense to electrons in unstained sections, like the granules in cells cultivated in the presence of phosphorus. That suggests that the localized differences in the structural packing of elements or radicals in the polyphosphale polymer is more important For visualization of unstained polyphosphate granules than the absolute concentration of phosphorus or other elements. In contrast to previous assumptions, the abundance of morphologically-observable‘polyphosphate’ granules can no longer be taken as an index of mobilizable phosphate reserves.     

花粉蛋白诱导胞内钙离子信号波动的研究

钙离子是细胞生命活动中的一个重要的调节因子,在细胞对外界刺激产生反应以及细胞死亡过程中,它扮演着重要的角色。天花粉蛋白是一种抗肿瘤药物,对绒毛膜上皮癌细胞的杀伤力特别强。通过对绒毛膜上皮癌细胞内钙离子进行ｆｌｕｏ－３／ＡＭ荧光染色发现,天花粉蛋白的加入能诱导绒毛膜上皮癌细胞内钙离子浓度的增加。经天花粉蛋白作用２４小时后,被天花粉蛋白损伤的绒毛膜上皮癌细胞内钙离子的浓度比正常细胞要高得多。在开花粉蛋     

The Apoplastic Pathway of Transport to Salt Glands

The availability of the apoplastic route for ion transport tosalt glands was assessed by electron microscope examinationsand by the use of lanthanum as an electron-dense tracer to demarcatethe apoplast. The results show that this solute can reach thesalt glands via the apoplast and this observation is discussedin relation to current ideas concerning salt gland function.     

X-ray Microanalysis of Ion Distribution in Abutilon Nectary Hairs

Robards, A. W. and Oates, K. 1986. X-ray microanalysis of iondistribution in Abutilon nectaryhairs—J. exp. Bot. 37:940–946. X-ray microanalysis of frozen-hydrated Abutilon nectary hairsshows that potassium is present at easily detectable levelsin all cells between the phloem and the trichome tip cells.There is little overall variation in the amount of potassiumfrom cell to cell although it was found that: (i) some basalcells accumulate potassium to a significantly higher level thanthe hair cells; and (ii) there is more potassium in the cellstowards the distal end of the trichome. These findings wouldbe consistent with a model for secretion envisaging a trans-membraneloading of nectar into a ‘secretory reticulum’ inall trichome cells. This loading process would be selectiveand would exclude ions. A pronounced increase in the level ofchlorine from the basal cell along the hair was observed. Nocomplementary cation was detected but equivalent changes inthe level of Na would have been below the detection limit ofthe system. Key words: Abutilon, nectary hairs, X-ray microanalysis, ion distribution     

Isolation of Salt Glands from the Larval Brine Shrimp Artemia salina

Use of isolated cell preparations have been a powerful approachto understanding cellular and biochemical processes of a widevariety of tissues, including transporting epithelia. A methodfor isolating the extra-renal organ or salt gland from a larvalbrachiopod crustacean, the brine shrimp, has been developed.This preparation is unique as it is the first isolated epithelialcell preparation from a crustacean and is derived from an earlystage of development. As both the removal and purification protocolsdiffer markedly from previous cell isolation methods, detailsof their development have been described. This preparation hasallowed measurement of the Na$K AT Pase specific activity inthesalt gland and intestine for the first time. Use of the isolatedsalt glands will facilitate cellular and subcellular studiesof both the structure and function of this ion transportingepithelium.     

应用电子探针对植物根际和根内营养元素微区分布的探讨

用电子探针可检测出玉米、大豆根际和根内含有Na,Mg,Al,Si,P,S,Cl,K,Ca,Ti,Fe,Cu和Zn 13种元素。这些元素在根际土壤、粘液层和根组织内的含量分布有一定的规律性。除Si,Al,Ca,Fe在根际土壤中峰值较高外,Ti仅在土壤中达到可检测量;S,Fe和Zn富集在粘液层,Mg,P,Cl只在根组织内才有较明显的峰。这些规律可作为区分根—土界面的参考指标。K含量在根内明显高于根际土壤,并由表皮层到中柱径向增加;Ca则与K不同,且受植物种类的影响。     

Salt glands of recretohalophyte Tamarix under salinity: Their evolution and adaptation

Here, we studied the evolution of salt glands in 11 species of Tamarix and determined their role in adaptation to saline environments by measuring the effect of NaCl on plant growth and salt gland characteristics. Cluster analysis divided Tamarix species into three types (types I–III) according to salt‐gland characteristics. A phylogenetic tree based on ITS sequences indicated an evolutionary relationship consistent with the geographical distribution of Tamarix. We measured growth under different NaCl conditions (0, 100, 200, and 300 mM) for 40 days in three species (T. gallica, T. ramosissima, and T. laxa) representing the three Tamarix types. With increasing NaCl concentration, the biomass of all species was significantly reduced, especially that of T. gallica. Salt secretion ability and salt‐gland density showed similar trends in three types. The order of salt tolerance was type I > type II > type III. We conclude that during Tamarix adaptation to salinity, salt‐gland evolution followed two directions: one increasing salt‐gland density, and the other increasing salt secretion rate per salt‐gland. This study provides a basis for potential mechanisms of recretohalophyte adaptation to salinity.     

The Ultrastructure of Ion-Secreting and Non-Secreting Salt Glands of Limonium platyphyllum

The ultrastructure of salt glands in developing leaves of Limoniumplatyphyllum is described prior to exposure to 3% NaCl solution(with non-secreting glands) and after 4.5 and 18 h exposureto the salt solution. It is shown that in most glands, the transitionto active chloride transport was accompanied by the displacementof vacuoles toward the cell periphery and by the establishmentof plasmalemma contact sites with the tonoplast which appearedsimilar to gap junctions in animal epithelial cells. No evidencefor the exocytosis of vacuoles was found. It is suggested thatgland vacuoles may have a primary role in chloride secretionand that the tonoplast may be functionally asymmetrical, sothat the free part facing the hyaloplasm bears ion pumps, whereashighly permeable ion channels are active along the zone of contactwith the plasmalemma. It follows that the active step in chloridetransport in Limonium glands is the influx of ions into thevacuoles. Within the inner cup cells of the gland, vacuolescome into contact with the plasmalemma only at sites where thecell wall is adjacent to secretory and accessory cells. Suchan asymmetry appears to ensure the directed flux of ions intothis cell wall. Wall protuberances in the gland cells are rudimentaryand presumably not involved directly in NaCl secretion. Thenucleolus is activated during secretion and the frequency offree ribosomes is significantly increased, which is suggestiveof their involvement in the synthesis of membrane transportproteins. The ultrastructure of about one-third of the glandsremained unchanged in salt-treated leaves. Key words: Salt glands, ultrastructure, ion fluxes     

Calcium Oxalate Crystals: An Integral Component of the Sclerotinia sclerotiorum/Brassica carinata Pathosystem

Oxalic acid is an important virulence factor for disease caused by the fungal necrotrophic pathogen Sclerotinia sclerotiorum, yet calcium oxalate (CaOx) crystals have not been widely reported. B. carinata stems were infected with S. sclerotiorum and observed using light microscopy. Six hours post inoculation (hpi), CaOx crystals were evident on 46% of stem sections and by 72 hpi on 100%, demonstrating that the secretion of oxalic acid by S. sclerotiorum commences before hyphal penetration. This is the first time CaOx crystals have been reported on B. carinata infected with S. sclerotiorum. The shape of crystals varied as infection progressed. Long tetragonal rods were dominant 12 hpi (68% of crystal-containing samples), but by 72 hpi, 50% of stems displayed bipyramidal crystals, and only 23% had long rods. Scanning electron microscopy from 24 hpi revealed CaOx crystals in all samples, ranging from tiny irregular crystals (< 0.5 μm) to large (up to 40 μm) highly organized arrangements. Crystal morphology encompassed various forms, including tetragonal prisms, oval plates, crystal sand, and druses. Large conglomerates of CaOx crystals were observed in the hyphal mass 72 hpi and these are proposed as a strategy of the fungus to hold and detoxify Ca²⁺ions. The range of crystal morphologies suggests that S. sclerotiorum growth and infection controls the form taken by CaOx crystals.     

Microbeam X-ray Diffraction Study of Granular Crystals Formed in Water-in-Oil Emulsion

Margarine and fat spread contain typical water-in-oil emulsions, including semi-solid fats, as continuous oil phases. The application of palm oil, one of the most promising trans-fat alternatives, for semi-solid fats is increasing. However, granular crystals often occur in palm-oil-based solid fats and cause deterioration. In this study, we carried out differential scanning calorimetry (DSC), optical microscopy, and X-ray diffraction (XRD) experiments on granular crystals in margarine. A conventional laboratory-scale technique was applied to examine thermal properties of high-melting fat fractions in granular crystals. In addition, microstructures of the granular crystal were precisely observed with a synchrotron radiation small-angle XRD (SR-μ-SAXD) technique by using a microbeam having a cross section of 5 × 5 μm². The following results were obtained. (1) DSC indicated that granular crystals are composed of high-melting fractions having a melting temperature of 23 °C. (2) Conventional XRD of granular crystals indicated that β-fat crystals of a triple chain length structure (β-3) melted below the melting of β′-fat of a double chain length structure (β′-2). (3) SR-μ-SAXD indicated that the fat crystals in normal margarine were β′-2. However, the fat crystals of the double and triple chain length structures were simultaneously observed at the center region of a granular crystal, whereas only the fat crystals of β-3 were observed at the outer region of a granular crystal. We analyzed the microstructures and formation processes of granular crystals in relation to the fractional crystallization of the β form of 1,3-dipalmitoyl-2-oleoyl-sn-glycerol promoted by crystallization and transformation of tripalmitin and tristearin fractions.     

细胞内贮存钙释放的机制

细胞内贮存钙的释放主要由１,４,５－三磷酸肌醇（ＩＰ３）受体系统和ｒｙａｎｏｄｉｎｅ受体系统调控。前通过ＩＰ３与其受体结合后,诱发细胞内钙释放;后通过复杂的机制调节环腺苷二磷酸核糖含量,由ｃＡＤＰＲ直接或间接作用于ｒｙａｎｏｄｉｎｅ受体,进而启动由Ｃａ＾２＋诱发的Ｃａ＾２＋释放机制。上述两系统之间相互作用,共同调节细胞内贮存钙的释放。