X-ray microanalysis of cockroach fat body in relation to ion and water regulation

X-Ray microanalysis of fat body from the cockroach (Periplaneta americana) showed that the crystals in the urate cells sequestered potassium and sodium during water deprivation. Postassium sequestration appeared to occur by the formation of additional crystals whereas sodium appeared to be taken up by pre-existing crystals of the “amorphous granule” type. Urate cells increased in cytoplasmic density i.e. mass per unit volume as a result of water deprivation and lost potassium from the cytoplasm but not from the nucleus. Trophocyte cells also showed an increase in density and a loss of potassium from the cytoplasm. The nucleus in trophocyte cells however, did not increase in density and showed an increase in chlorine concentration. A previously undescribed type of crystal in the trophocyte cell cytoplasm also increased markedly in chlorine concentration. The mycetocyte cells did not appear to increase in density but chlorine concentration was elevated in symbionts, cytoplasm and nucleus. An increase in density is considered to be a consequence of a reduction in volume due to water loss. It is confirmed that the fat body plays a major role in the maintenance of ion balance during water deprivation.     

Spore discharge in the red algae Bostrychia tenella and Caloglossa leprieurii from the Godavari estuary,India

Bostrychia tenella (Vohl.) J. Ag. and Caloglossa leprieurii (Mont.) J. Ag., two examples of species important for human consumption from estuaries in Asia, were studied with respect to spore formation and release with a long-term view to their mass cultivation. Plants were collected between January and December 1987 from three different regions of the Gautami Godavari estuary, India. Both species behaved rather similarly. Tetrasporophytic plants were present throughout the year, whereas carposporophytic plants were found only in certain months. Shedding of tetraspores was observed throughout the year, but with seasonal difference in their output. Carpospores were liberated from October to May when the material was available. Maximum shedding of carpospores and tetraspores was observed in December and January and the minimum number of tetraspores in August and carpospores in May. The maximum number of spores was liberated, when plants were submerged at 20% salinity.     

Mannitol metabolism of the epiphytic mangrove red alga Caloglossa leprieurii (Ceramiales): A long-term field study

The concentrations of mannitol and floridean starch were determined in a 1–year field study of the epiphytic red alga Caloglossa leprieurii (Montagne) J. Agardh from warm temperate waters of eastern Australia. Seasonal environmental data for air and water temperature, day length and rainfall were recorded. The mannitol content and the floridean starch content varied significantly between collections but no seasonal responses were observed, nor were the contents correlated with any of the abiotic factors. A possible function of the starch pool as a supply for respiratory substrates under emergent conditions is discussed. All data indicate that productivity and biomass of C. leprieurii are affected by short-term abiotic and/or biotic conditions rather than controlled directly by seasonally fluctuating environmental factors. In addition, the activity of three highly specific enzymes (mannitol-1–phosphate dehydroge-nase, mannitol-1–phosphatase, mannitol-dehydroge-nase) and one non-specific enzyme (hexokinase), all of which are involved in the mannitol cycle, were measured in cell-free extracts every 2 weeks. All enzymes showed marked changes in activity over the year, but again no clear patterns emerged, neither with season nor in relationship to one another. On the basis of the results here, C. leprieurii is considered to be a 'season responded rather than a ‘season anticipator’.     

Leaf photosynthesis,fluorescence response to salinity and the relevance to chloroplast salt compartmentation and anti-oxidative stress in two poplars

In a 16-day study, the effect of increasing soil NaCl on leaf photosynthesis, chlorophyll a fluorescence, chloroplast ion compartmentation, variations of SOD (superoxide dismutase) and POD (peroxidase) isoenzymes and the relevance to salt resistance were investigated in seedlings of Populus euphratica Oliv. (P. euphratica) (salt-resistant) and rooted cuttings of P. “popularis 35–44” (P. popularis) (salt-sensitive). Initial salinity caused a rapid decline of net photosynthetic rate (Pn) and unit transpiration rate (TRN) in P. euphratica, resulting from the NaCl-induced stomatal closure. In a longer-term of salinity, CO₂ assimilation in P. popularis was severely reduced whereas stressed P. euphratica maintained a relatively higher and constant level of Pn. Pn–Ci curves showed that salt stress (12 days) reduced CO₂ saturation point (CSP), CO₂ saturated Pn (CSP _n ), and carboxylation efficiency (CE), but increased CO₂ compensation point (CCP) in the two genotypes. Similarly, salinity lowered light saturation point (LSP), light saturated Pn (LSP _n ), and apparent quantum yield (AQY) in both genotypes but the inhibitory effect of NaCl on light reaction was more pronounced in P. popularis, as compared to P. euphratica. Chlorophyll a fluorescence data indicated that a longer-term of salt stress (12 days) exhibited a marked influence on fluorescence parameters of P. popularis in both dark- and light-adapted states: (a) NaCl inhibited the maximal efficiency of PSII photochemistry (Fv/Fm) due to the salt-induced increase of Fo (the minimal fluorescence) and the marked decline of Fm (the maximal fluorescence); (b) salinity decreased coefficient of photochemical quenching (qP) but markedly elevated coefficient of nonphotochemical quenching (qN) in the light-adapted state. In contrast, there were no corresponding changes of chlorophyll a fluorescence in salinised P. euphratica. X-ray microanalysis results showed that salinity caused salt accumulation in the chloroplasts of P. popularis in which Na⁺ and Cl⁻ increased up to 42 and 221 mmol dm⁻³, respectively. Great buildup of Na⁺ and Cl⁻ in chloroplasts of P. popularis may exhibit direct and indirect restrictions on dark and light reactions. The activity of SOD isoenzymes (CuZn-SOD I and CuZn-SOD II) and POD isoenzymes in P. popularis decreased with increasing exposure period, and leaf malondialdehyde (MDA) content and membrane permeability (MP) increased correspondingly. In contrast to P. popularis, stressed P. euphratica maintained activity of SOD and POD isoenzymes and there was no significant increase of MDA and MP during the period of salt stress. In conclusion, P. euphratica plants exhibited a higher capacity to maintain the activity of anti-oxidant enzymes and restrict salt accumulation in the chloroplasts, the photosynthesis processes were less restricted consequently.     

Response to salinity in the homoploid hybrid species Helianthus paradoxus and its progenitors H. annuus and H. petiolaris

To contribute to the understanding of ecological differentiation in speciation, we compared salinity responses of the halophytic diploid hybrid species Helianthus paradoxus and its glycophytic progenitors Helianthus annuus and Helianthus petiolaris. Plants of three populations of each species were subjected to a control (nonsaline) and three salinity treatments, including one simulating the ion composition in the habitat of H. paradoxus. Relative to the control, saline treatments led to a 17% biomass increase in H. paradoxus while its progenitors suffered 19-33% productivity reductions and only in H. paradoxus, leaf contents of potassium, calcium, and magnesium were strongly reduced. Under all treatments, H. paradoxus allocated more resources to roots, was more succulent, and had higher leaf contents of sodium (> 200 mmol l(-1) tissue water) and sulfur than its progenitor species. These results suggest that salt tolerance and thus speciation of H. paradoxus is related to sodium replacing potassium, calcium and magnesium as vacuolar osmotica. The evolutionary and genetic mechanisms likely to be involved are discussed.     

Study of maturation of membrane transport function in red blood cells by X-ray microanalysis

Summary Red blood cells of certain species of animals, such as dogs and cats, contain low potassium and high sodium, whereas the erythropoietic stem cells giving rise to these cells are of high potassium type. This paper examines the sequence of membrane transport changes during erythropoiesis by analyzing the K, Na and Fe in single bone marrow cells, reticulocytes and mature red blood cells with X-ray microanalysis. The relationship between K/Na ratios and Fe/(K+Na) ratios were examined by X-ray microanalysis. The K/Na ratios give a measure of the membrane cation transport function. The Fe/(K+Na), which is analogous to hemoglobin concentration, gives an index of maturation stage. The relationships between K/Na and Fe/(K+Na) in the marrow cells of normal adult dog and those of a phenylhydrazine-injected dog with accelerated erythropoiesis show that the modification of cation composition occurs after the initiation of hemoglobin synthesis but before its completion. Similar relationships in the reticulocytes obtained from phenylhydrazine-injected dogs as well as from newborn dogs show a consistent decrease in K/Na with increased Hb, indicating a drastic change in cation composition during the maturation of the reticulocytes. Therefore the modification in membrane transport function must have occurred before or during the formation of reticulocytes.     

Quantitative energy dispersive X-ray microanalysis of eight elements in pancreatic endocrine and exocrine cells after cryo-fixation

Quantitative X-ray microanalysis of 8 elements was performed on ultrathin, freeze-dried sections of islets and pancreas pieces from non-inbredob/ob-mice. Diffusion of elements was reduced to a minimum by rapidly freezing the tissue samples between nitrogen-cooled polished copper surfaces and avoiding the use of chemical fixatives and stains. The ultrastructural morphology was adequately maintained to allow measurements on secretory granules, mitochondria, cell nuclei, and cytoplasm free of these organelles. The distribution of the various elements between cellular compartments was similar in islet -cells and exocrine pancreas cells. However, the insulin secretory granules were outstanding in exhibiting the highest concentrations of zinc and calcium. In comparison with cytoplasm in the -cells, the insulin granules accumulated calcium 2-fold and zinc as much as 40-fold. As no correlation could be made for endoplasmic reticulum in the cytoplasmic measurements areas, the true accumulations above cytosol are likely to be even higher.     

X-ray microanalysis and chlorotetracycline staining of calcium vesicles in the green alga Mougeotia

Calcium ions have been proposed to play a key role in the sensory transduction of phytochrome-governed chloroplast movement in the green alga Mougeotia. To test this hypothesis, the intracellular pattern of calcium distribution was studied in this alga by two independent techniques, namely, X-ray microanalysis of fixed and of unfixed frozen-hydrated cells, as well as in vivo fluorescence by chlorotetracycline. Both methods of detection reveal a significant compartmentation of calcium in vesicles close to the chloroplast edge and, less frequently, in the cortical cytoplasm. Microfilaments, presumably actin, which could function in driving chloroplast movement, have been observed running between the chloroplast edge and the cortical cytoplasm (Wagner, G., Klein, K. (1978) Photochem. Photobiol. 27, 137). The vesicular calcium concentration is stable and decays only slowly in the absence of extracellular calcium much in the same way as the ability of the chloroplast to perform movements decreases. A functional relationship between vesicular calcium compartmentation and phytochrome-governed chloroplast movement in the green alga Mougeotia seems indicated.Abbreviation CTC chlorotetracycline     

Ion measurements by X-ray microanalysis in unfixed,frozen, hydrated plant cells of species differing in salt tolerance

A technique is described for X-ray microanalysis of unfixed, frozen, hydrated higher plant cells using a scanning electron microscope in conjunction with a cryostage. Freezing in liquid N₂ is the only preparative step required. Using this method, ion distribution was compared in the roots of Zea mays L. (termed a salt excluder) and Hordeum vulgare L. (which is rather more tolerant), both grown in the presence of NaCl. Distinct differences were observed between the two species in Na, K and Cl distribution. Evidence is presented to support the hypothesis that reabsorption of Na from the xylem sap in the mature regions of the root may occur in salt-sensitive glycophytes such as Z. mays.     

Metal X-ray microanalysis in the olfactory system of rainbow trout exposed to low level of copper

Summary— It has recently been shown that a chronic copper exposure induces specific degeneration of olfactory receptor cells in rainbow trout; however, the exact mechanism of action of the metal is not yet known. Using X-ray microanalysis in transmission electron microscopy, we have studied the distribution of metal in the olfactory system of fish exposed for 15, 30 and 60 days to 20 μg/l of copper. This was done in order to determine if it was accumulated in receptor cells and transported into the central nervous system via the olfactory nerve. No copper accumulation was detected either in the olfactory epithelium, in the olfactory nerve or in the olfactory bulb. The heavy metal was exclusively found within melanosomes of melanophores located in the lamina propria. After 60 days of exposure, the copper content in melanosomes was about two-fold higher than that in controls. As far as some morphological recovery took place in the olfactory organ during the metal exposure, which lets us suppose that some detoxication mechanism occurs, it could be suggested that melanophores might be somehow involved in such a mechanism.     

Ultrastructural localization of calcium, by electron-probe X-ray microanalysis, in the small intestine of suckling rats

The subcellular distribution of calcium has been investigated in samples, from the intestinal mucosa of 10-day rats, prepared for X-ray microanalysis by various techniques designed to minimize the loss of this element. Calcium retention and its threshold of detection was most satisfactory in freeze-dried frozen thin sections. In resin-embedded samples the best retention of calcium was found in specimens fixed in absolute ethanol, embedded without osmication, and sectioned onto glycerol. The results of this investigation indicate the presence of calcium in the supranuclear vacuole of enterocytes in the distal intestine of the neonatal rat. This calcium is probably taken up during the endocytosis of material from the intestinal lumen. The same mechanism may also be important in the uptake of other metals by suckling animals.     

Chondroitin sulphate at the endothelial lumen of pig aorta: Assay by radiolabelling and by X-ray microanalysis

Trypsin-releasable glycosaminoglycans from the luminal surface of intact pig aorta were measured following metabolic labelling with³⁵S]sulphate. Chondroitin sulphate was found to be present at a surface density equal to that already established for heparan sulphate (5×10¹¹ chains per cm²). This result was confirmed by X-ray microanalysis of the luminal sulphur content before and after treatment with specific glycosaminoglycan-degrading enzymes. This result implies that approximately half of the luminal surface is occupied by sulphated glycosaminoglycans.     

Calcium pathway through a mineralizing epithelium in the crustacean Orchestia in pre-molt: Ultrastructural cytochemistry and X-ray microanalysis

During the pre-exuvial period of the terrestrial crustacean Orchestia, the calcium of the old cuticle is almost entirely reabsorbed and stored as calcareous concretions in the lumen of the midgut posterior caeca. The elaboration of these concretions is due to transport by the caecal epithelium. With ultrastructural cytochemistry controlled by X-ray microanalysis, it can be demonstrated that the main sites of ionized or ionizable calcium are the apical microvilli and an extracellular (lateral and basal) network of channels. Direct precipitating cytochemical methods, using potassium pyroantimonate or pyrophosphate, potassium oxalate or oxalic acid, sodium fluoride, sodium tungstate, and indirect substitution methods, using lead acetate or nitrate and cobalt nitrate were comparatively used. The results are interpreted in favour of the hypothesis of an extracellular transport pathway for calcium through the lateral smooth septate junctions, in conjunction with a more classical apical transport through the microvilli.     

盐胁迫下白杨无性系苗木体内离子分配及比较

以白杨杂种无性系及其亲本当年生苗木为材料 ,分析了盐胁迫下无性系的离子含量 ,及 Na 、K 、Ca 在植物体内运输和分配特点 ,并对无性系间差异进行了比较。研究表明 ,随盐浓度提高 ,植物体内Na 含量迅速提高 ,K 、Ca 含量降低 ;盐分胁迫下 ,根部 Na 含量较高 ,叶片 Na 中含量最低 ,K 、Ca 含量则相反 ,特别是 Ca ,其分布顺序为叶 >茎 >根。杂种无性系 B430及其亲本新疆杨对 K 和 Ca 运输的选择性比毛白杨高 ,而对 Na 运输的选择性则比毛白杨低 ,从而导致根部存留的 Na 较多 ,叶片分配的 Na 数量较少 ,从而减轻 Na 对叶片的伤害。综合分析表明 B430和新疆杨耐盐能力最强 ,毛新杨其次 ,毛白杨最差。     

中国桫椤科植物叶、孢子和孢子囊环带元素的X-射线微区成分分析

用扫描电镜和X ray微区分析方法测定和分析了中国产的13种及变种的桫椤科植物的叶、孢子和孢子囊环带中元素组成和平均质量分数。结果表明这13种及变种植物在叶、孢子和孢子囊环带的元素组成上彼此均有一定的差异;在元素的平均质量分数上,差异更加明显;同一植物的叶、孢子和孢子囊环带元素组成大多不完全相同,在叶中的元素种类均多于孢子和孢子囊环带的元素;并且同一植物的叶、孢子和孢子囊环带中元素的平均质量分数差异也较大;13种及变种植物叶、孢子和孢子囊环带元素的的平均质量分数最高的大多为K元素,而孢子中质量分数最高的为Si元素。13种及变种的桫椤科植物在元素组成和平均质量分数上的差异可能与其结构、生理和生态环境有关。     

Purification and Characterization of Mannitol-l-Phosphatase in the Red Alga Caloglossa continua (Ceramiales, Rhodophyta)

Purification of mannitol-l-phosphatase, an enzyme catalyzing the final step of mannitol biosynthesis, was first achieved in the mannitol-accumulating red alga Caloglossa continua (Okamura) King et Puttock. The enzyme was shown to be a monomer, since gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis gave close values of apparent molecular weights of 28,500 and 30,200, respectively. The protein exhibited an isoelectric point of 4.8. The substrate specificity for mannitol-l-phosphate (MIP) was very high, and that for K _m(MIP) was 0.41 mM. The catalytic activity was optimal at pH 7.4. The enzyme was activated by Mg²⁺, but was strongly inhibited by Ca²⁺, NaF, N-ethylmaleimide, and p-hydroxymercuribenzoic acid. Seawater levels of NaCl and physiological levels of mannitol also inhibited the activity by 50% or more. Changes in the concentrations of those ions and metabolites may regulate the biosynthesis of mannitol as an osmoregulant in vivo. Received May 7, 2001; accepted June 15, 2001.     

Posthodiplostomum minimum: Examination of cyst wall and metacercaria containing calcarious concretions with scanning electron microscopy and X-ray microanalysis

Scanning electron micrographs show a smoothly fibrous structure of the cyst wall of the trematode Posthodiplostomum minimum, whereas the enclosed metacercaria posses a porous and papillate surface with occasional spines. Numerous excretory concretions fill the body of the metacercaria. The concretions appear layered from a central core and show an abundance of calcium, and, in some cases, magnesium.     

Immunolocalization of alkaloids and X-ray microanalysis of elements in lupin seeds

Summary Immunolocalization of alkaloids in lupin seeds (Lupinus spp.) has been performed by cryofixation and conventional methods. Alkaloids were localized in the protein bodies of the cotyledon cells. Some immunogold particles in the walls of these cells were also observed. There were no differences in the sites of localization between the two mentioned methods. X-ray microanalysis of elements showed the presence of P, Mg, S, and K in the protein bodies of cotyledon cells in lupin seeds. The role of K⁺ in alkaloids transport is discussed.     

Intracellular water and dry mass content as measured in bulk specimens by energy-dispersive X-ray microanalysis

Intracellular water content (IWC) was measured in freeze-fractured biological bulk specimens by means of energy-dispersive X-ray microanalysis. The method is based on the concentration differences of certain elements (potassium and phosphorus) between frozen-hydrated and frozen-dried states of the tissues as applied formerly to sectioned material by others. A new mathematical formula has been derived giving rather precise figures for IWC. No elemental standards are necessary for the measurement: one has to obtain only the peak to background ratios in wet and dry states of the cells. The method is sensitive enough to reveal age-dependent as well as drug-induced changes of IWC in liver and brain cells. The values obtained are quite comparable with the theoretically expected ones. Technical problems of the application of this method are discussed in detail.