Schistosoma mansoni: Electron probe X-ray microanalysis of the elemental composition of the tegument and subtegumental tissues of adult worms

The elementary composition [Na, Mg, P, S, Cl, K, Ca and Fe] of the tegument, tegumental spines, and subtegumental tissues of adult male and female Schistosoma mansoni have been determined by electron probe X-ray microanalysis of unfixed, freeze-dried cryosections. Statistical analysis of the results suggests that there are distinct differences in the elemental composition of the tissues both between and within individual male and female worms, and between male and female worms in general. In particular, there were significant variations in the elemental contents of the tissues between individual male and female worms, which may reflect differences in the physiology and/or metabolic state of the worms. Significant differences in the elemental composition of the various tissues examined within individual worms were also found. In general, in both male and female worms, there were significantly higher elemental levels in the tegument, as opposed to the subtegumental tissues. The elemental composition of the tegumental spines in both male and female worms differed from that of the tegumental cytoplasm, although the differences in the elemental composition between spines from male and female worms reflected the differences in the elemental content between the teguments themselves. Differences in the elemental composition of the tissues between male and female worms were also found, with the female tegument containing significantly higher elemental levels (with the exception of Cl) than the male tegument. In particular, the tegument of female worms contained higher levels of calcium and, in relatively small areas, isolated calcium-containing granules. This higher tegumental calcium level in female worms may reflect a higher calcium demand by sexually mature female worms due to the presence, within the mature vitelline cells, of calcium-containing corpuscles and the production of large numbers of eggs.     

Calcium content and concretions of pineal glands of young and old rats

Summary Calcium content and pineal concretions were studied in young (2–3 months) and old (28 months) Wistar rats. Samples, deep-frozen by liquid propane/isopentane and freeze-dried were analysed by means of X-ray microanalysis in a scanning electron microscope. Total semi-quantitative measurements revealed that pineals of old rats showed a marked increase of calcium compared with the pineals of young rats. It is thus suggested that a calcium-rich environment is responsible for the growth of pineal concretions, which only appear in old rats. Pineal calcifications in rats could thus be an indicator of aging and/or of a degenerating state.     

Calcium concretions in the pineal gland of aged rats: an ultrastructural and microanalytical study of their biogenesis

The genesis of calcium concretions in aged rats was studied by means of transmission and scanning electron microscopy. The potassium pyroantimonate method, combined with X-ray microanalysis, allowed us to study the distribution of cations and calcium. Notable accumulations of calcium (associated with phosphorus) were localized in vesicles, vacuoles, lipid droplets, lipopigments, and mitochondria of dark pinealocytes. The results obtained in the present investigation suggest that these organelles are involved in the genesis of the concretions. The presence of sulfur indicates the existence of an organic matrix. We propose that genesis takes place in dark pinealocytes, which contain more calcium than light pinealocytes. Mineralization foci are some-times associated with cellular debris and enlarge by further apposition of material. Two types of concretions, as determined by electron microscopy and confirmed by electron diffraction, could be observed: the amorphous type with concentric layers and the crystalline type with needle-shaped crystals. Once formed, the concretions reach the extracellular space and the cell breaks down. Possible extracellular calcification is suggested in the extracellular calcium-rich floculent material. The mineralization process is interpreted as being an age-related phenomenon and mainly a consequence of the degeneration of pinealocytes.     

Electron probe X-ray microanalysis of cellular ions in the eccrine secretory coil cells during methacholine stimulation

Summary Intracellular concentrations of Na, K, Cl ([Na], [K] and [Cl], respectively) and other elements were determined in isolated monkey eccrine sweat secretory coil cells using quantitative electron probe X-ray microanalysis of freeze dried cryosections. The validity of the methodology was partially supported by qualitative agreement of the X-ray microanalysis data with those obtained by micro-titration with a helium glow spectrophotometer. [Na], [K] and [Cl] of the cytoplasm were the same as those in the nucleus in both clear and dark cells. [Na], [K], and [Cl] of the clear cells were also the same as those of the dark cells at rest and after stimulation with methacholine (MCh), suggesting that these two cell types behave like a functional syncytium. MCh stimulation induced a pharmacologically specific, dose-dependent decrease in [K] and [Cl] (as much as 65%), and a 3.7-fold increase in [Na]. In myoepithelial cells, a similar change in [Na] and [K] was noted after MCh stimulation although the decrease in [Cl] was only 20%. The MCh-induced change in [Na], [K] and [Cl] was almost completely inhibited by removal of Ca²⁺ from the medium. 10^–4 m bumetanide inhibited the MCh-induced increase in [Na], reduced the decrease in [K] by about 50%, but slightly augmented the MCh-induced decrease in [Cl]. 10^–4 m ouabain increased [Na] and decreased [K] as did MCh; however, unlike MCh, ouabain increased [Cl] by 56% after 30 min of incubation. Thus the data may be best interpreted to indicate that Ca-dependent K efflux and (perhaps also Ca-dependent) Cl efflux are the predominat initial ionic movement in muscarinic cholinergic stimulation of the eccrine sweat secretory coils and that the ouabain-sensitive Na pump plays an important role in maintenance of intracellular ions and sweat secretion.     

Effects of dexamethasone on expression and maintenance of cartilage in serum-containing cultures of calvaria cells

Summary The effects of dexamethasone on the ability of cells enzymatically isolated from 21-day fetal rat calvaria to produce cartilage in vitro has been investigated. Primary cultures of single-cell suspensions of rat calvaria were grown for up to 28 days in vitro in -minimal essential medium containing 15% fetal bovine serum, 50 /ml ascorbic acid, 10 mM Na -glycerophosphate and dexamethasone at concentrations of 1 M to 1 nM. Two types of nodules were present in dexamethasone-containing cultures. One has been characterized previously as bone (Bellows et al. 1986). The second morphologically resembled hyaline cartilage, possessed a strong Alcian blue-positive matrix and contained type-II, but not type-I, collagen. Both bone and cartilaginous nodules were spatially distinct and developed in isolation from each other. Cartilaginous nodules were found in the highest number at a dexamethasone concentration of 100 nM. Time-course experiments revealed that while the number of bone nodules increased continuously at least to day 28, the number of cartilaginous nodules remained constant after cultures had reached confluency. When cells were isolated separately from frontal and parietal bones and suturai regions, the greatest number of cartilaginous nodules developed from parietal bones. Since 21-day fetal rat calvaria contains 2 distinct patches of cartilage at the periphery of the parietal bones, it seems likely that this cartilaginous tissue is the origin of the cartilage cells. The results demonstrate that cultures of rat calvaria cells contain chondrocytes and possibly chondroprogenitor cells that are distinct from osteoprogenitors. Results support previous data that 100 nM dexamethasone permits the expression of and maintains the phenotype of chondrocytes in serum-containing cultures in vitro.     

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。     

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.     

FGF2 and dexamethasone increase the production of hyaluronan in two-dimensional culture of elastic cartilage-derived cells: in vitro analyses and in vivo cartilage formation

Elastic cartilage-derived cells cultured two-dimensionally with FGF2 and corticosteroid produce gel-type masses that become mature cartilage when injected into a subcutaneous pocket. This unique method has previously been clinically applied for treatments of nasal augmentation. However, the components of the gel-type mass and the mechanism of its synthesis remain unknown. Here, we have investigated the components of the gel-type mass produced by elastic cartilage-derived cells, and whether this gel-type mass can be produced by using other cell sources or other media. Human elastic cartilage-derived cells from auricular cartilage, hyaline cartilage-derived cells from articular cartilage, and mesenchymal stem cells from synovium were cultured in three media: “redifferentiation medium” containing FGF2 and dexamethasone; “chondrogenic medium” containing bone morphogenetic protein-2, transforming growth factor-β3, and dexamethasone specific for in vitro chondrogenesis of mesenchymal stem cells; control medium. The elastic cartilage-derived cells cultured in redifferentiation medium produced a gelatinous matrix positive for Alcian blue. During culture, the amount of chondroitin 4-sulfate, chondroitin 6-sulfate, and especially hyaluronan increased. However, the expression of RNAs for most chondrogenic genes did not increase. We also reproduced cartilage tissue formation by the injection of elastic cartilage-derived cells with the gelatinous mass into the subcutaneous space of the nude mouse. The synthesis of gelatinous matrix in vitro and the formation of cartilage tissue in vivo could be obtained only for the combination of elastic cartilage-derived cells with redifferentiation medium. This study was supported in part by grants from the “Japan Society for the Promotion of Science (19591752)” and “Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone in Tokyo Medical and Dental University” to Takeshi Muneta, and the “Japan Society for the Promotion of Science (18591657)” to Ichiro Sekiya.     

X-ray microanalysis of freeze-dried digestive mucus in Anguilla anguilla L: modifications of ion content during the early steps of secretion

Summary— Digestive mucus of sea-water adapted eels has been observed and analyzed by the scanning electron microscope (SEM) after rapid freezing at liquid nitrogen temperature followed by freeze-drying. No chemical procedures were used in this technique. This allowed the maintenance of the mucous coating. Preliminary X-ray microanalysis carried out on freeze-fractured and freeze-dried samples of the oesophagus showed a decrease of K⁺ and an increase of Ca²⁺ and Cl^? from the basal part of the mucous cell towards its the apical part. This technique has proven to be satisfactory for it prevents translocation and loss of diffusible elements in situ and allows X-ray microanalysis in the SEM.     

Synthesis of fibronectin in normal and osteoarthritic articular cartilage

The content and the biosynthesis of fibronectin was examined in disease-free articular cartilage and in articular cartilage from osteoarthritic canine joints. Fibronectin content was increased in extracts of cartilage from osteoarthritic joints. Incubation of cartilage in vitro with [³H]phenylalanine and subsequent isolation of [³H]fibronectin from a gelatin affinity column and characterization by SDS-polyacrylamide gel electrophoresis and by immunoprecipitation indicated that disease-free and osteoarthritic cartilage explants synthesized fibronectin. About 50% of the [³H]fibronectin was recovered in the incubation medium. The osteoarthritic cartilage synthesized and accumulated up to 5-fold more [³H]fibronectin than disease-free cartilage.     

The effect of TGF-beta1 and beta-estradiol on glycosaminoglycan and type II collagen distribution in articular chondrocyte cultures

Articular cartilage extracellular matrix (ECM) plays a crucial role in regulating chondrocyte functions via cell-matrix interaction, cytoskeletal organization and integrin-mediated signaling. Factors such as interleukins, basic fibroblast growth factor (bFGF), bone morphogenic proteins (BMPs) and insulin-like growth factor (IGF) have been shown to modulate the synthesis of extracellular matrix in vitro. However, the effects of TGF-beta1 and beta-estradiol in ECM regulation require further investigation, although there have been suggestions that these factors do play a positive role. To establish the role of these factors on chondrocytes derived from articular joints, a study was conducted to investigate the effects of TGF-beta1 and beta-estradiol on glycosaminoglycan secretion and type II collagen distribution (two major component of cartilage ECM in vivo). Thus, chondrocyte cultures initiated from rabbit articular cartilage were treated with 10ng/ml of TGF-beta1, 10nM of beta-estradiol or with a combination of both factors. Sulphated glycosaminoglycan (GAG) and type II collagen levels were then measured in both these culture systems. The results revealed that the synthesis of GAG and type II collagen was shown to be enhanced in the TGF-beta1 treated cultures. This increase was also noted when TGF-beta1 and beta-estradiol were both used as culture supplements. However, beta-estradiol alone did not appear to affect GAG or type II collagen deposition. There was also no difference between the amount of collagen type II and GAG being expressed when chondrocyte cultures were treated with TGF-beta1 when compared with cultures treated with combined factors. From this, we conclude that although TGF-beta1 appears to stimulate chondrocyte ECM synthesis, beta-estradiol fails to produce similar effects. The findings of this study confirm that contrary to previous claims, beta-estradiol has little or no effect on chondrocyte ECM synthesis. Furthermore, the use of TGF-beta1 may be useful in future studies looking into biological mechanisms by which ECM synthesis in chondrocyte cultures can be augmented, particularly for clinical application.     

Effects of potassium-free media and ouabain on epithelial cell composition in toad urinary bladder studied with X-ray microanalysis

Summary The technique of X-ray microanalysis was used to study the composition of toad urinary bladder epithelial cells incubated in Na Ringer's and K-free medium, with and without ouabain. Following incubation under short-circuit conditions, portions of tissue were coated with an external albumin standard and plunge-frozen. Cryosections were freeze-dried and analyzed. In Na Ringer's, granular and basal cells, and also the basal portion of the goblet cells, had similar water and ion compositions. In contrast, mitochondria-rich cells contained less Cl and Na. On average, the granular cells and a subpopulation of the basal cells lost K and gained Na after ouabain and in K-free medium alone. However, there was considerable variation from cell to cell in the responses, indicating differences between cells in the availabilities of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control. In contrast, the compositions of both the low Cl, mitochondria-rich cells and a sub-population of the basal cells were little affected by the different incubation conditions. This is consistent with a comparatively low Na permeability of these cells. The results also indicate that (i) much, if not all, of the K in the dominant cell type, the granular cells, is potentially exchangeable with serosal medium Na, and (ii) Na is accumulated from the serosal medium under K-free conditions. They also provide information about the role of the (Na–K)-ATPase in the maintenance of cellular K in K-free medium, being consistent with other evidence that removal of serosal medium K inhibits transepithelial Na transport by decreasing Na entry to the cells from the mucosal medium, rather than solely by inhibiting the basolateral membrane (Na–K)-ATPase.     

The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats

Recent studies of Parkinson's disease indicate that dorsal motor nucleus of nerve vagus is one of the earliest brain areas affected by alpha-synuclein and Lewy bodies pathology. The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats is investigated. Synchrotron radiation based X-ray fluorescence was applied to the elemental micro-imaging and quantification in thin tissue sections. It was found that elements such as P, S, Cl, K, Ca, Fe, Cu, Zn, Se, Br and Rb are present in motor cortex, corpus striatum, nucleus accumbens, substantia nigra, ventral tectal area, and dorsal motor nucleus of vagus. The topographic analysis shows that macro-elements like P, S, Cl and K are highly concentrated within the fiber bundles of corpus striatum. In contrast the levels of trace elements like Fe and Zn are the lowest in these structures. It was found that statistically significant differences between the animals with electrical stimulation of vagus nerve and the control are observed in the left side of corpus striatum for P (p = 0.04), S (p = 0.02), Cl (p = 0.05), K (p = 0.02), Fe (p = 0.04) and Zn (p = 0.02). The mass fractions of these elements are increased in the group for which the electrical stimulation of vagus nerve was performed. Moreover, the contents of Ca (p = 0.02), Zn (p = 0.07) and Rb (p = 0.04) in substantia nigra of right hemisphere are found to be significantly lower in the group with stimulation of vagus nerve than in the control rats.     

Detection by bismuth staining of highly phosphorylated nucleo-proteins in plants. Determination of its specificity by X-ray microanalysis,SDS-PAGE and immunological analysis

Summary— Staining with bismuth salts after glutaraldehyde fixation is a very useful technique for preferential detection of phosphorylated nucleoproteins in mammalians and insects. In the present work we report an adaptation of this method for plant nuclei: staining with bismuth salts either in tissue blocks before embedding, or on thin sections of acrylic resin. Both procedures are highly reproducible and give the same pattern of staining in the nuclei in situ or isolated at the electron microscope. The specificity of bismuth binding to the dense nucleolar fibrillar component and interchromatin granules is proven by X-ray microanalysis. The nuclear proteins which bind bismuth have been identified by bismuth and immunostains of blots from total nuclear proteins. This technique is a very useful and specific cytochemical tool for studying nuclear organization and functions in plants.     

The volume and ionic composition of cells in incubated slices of rat renal cortex, medulla and papilla

The apparent extracellular space in incubated slices of rat renal cortex, medulla and papilla has been measured using three differently sized marker molecules, mannitol, sucrose and inulin. Cellular volumes have been estimated by following the efflux of from equilibrated slices. Sucrose appears to be the most accurate extracellular marker in each of the regions examined, in that the sum of its volume of distribution plus cellular volume approximates most closely to the total slice fluid volume. Inulin has the same volume of distribution as sucrose in cortical slices, but under-penetrates medullary and papillary tissue. Mannitol overestimates the extracellular space in all three regions, although its larger volume of distribution, relative to that of sucrose, was not statistically significant in papillary slices. When cell volume and composition are estimated (a) using sucrose as extracellular marker and (b) making appropriate allowance for the presence of bound tissue electrolytes, it is found that cells in each region have low Na⁺ and high K⁺ concentrations and contents. When papillary slices are incubated in medium of very high osmolality (NaCl plus urea, 2000 mosmol/kg H₂O) there is a moderate (approx. 23%) decrease in cell volume and an increase in cell fluid Na⁺ and Cl⁻ concentrations equal to approx. 50% of the increase in the extracellular concentrations. Cell K⁺ concentrations remain unchanged. The results show that cells in renal slices are able to maintain high K⁺-to-Na⁺ ratios when incubated in isosmotic (cortex) or moderately hyperosmotic media (medulla and papilla), and suggest that regulation of papillary cell volume following hyperosmotic shock can only partly be ascribed to uptake of extracellular electrolytes.     

Effects of Increased Extracellular K on the Elemental Composition and Water Content of Neuron and Glial Cells in Leech CNS

Elemental (Na, Cl, K) and water contents of leech (Macrobdella decora) neurons and glial cells were determined under steady-state exposure to 4, 10, and 20 mM KCl concentrations (bathing media) using x-ray microanalysis for quantitative digital imaging of frozen hydrated and dried cryosections. Effects of furosemide, 5-hydroxytryptamine (5-HT), and ouabain on elemental distribution changes, induced by exposure to 20 mM K, were also determined. Results demonstrated that packet glial cells and neurons accumulated substantial amounts of K that appeared evenly distributed throughout the cytoplasm. Cell water content also increased as a function of increased cytoplasmic K so that the net effect was an unchanged wet-weight K concentration (expressed as millimoles per kilogram wet weight). Dry-weight Na and Cl concentration (expressed as millimoles per kilogram dry weight) increased slightly in glial cells; however, because cell water increased, both Na and Cl (wet-weight) concentrations decreased. Neurons, in contrast, had no significant change in either Na or K on a wet-weight basis, so a relatively constant Na/K ratio was maintained despite a small, but significant, increase in K (dry weight) and cell water. These increases, like those in packet glia, were a function of exposure to different concentrations of extracellular space K. These changes were completely abolished by 10(-4) M ouabain. Neither furosemide nor 5-HT appeared to affect neuronal or glial K wet-weight concentrations. These data show that both glial cells and neurons can act as substantial reservoirs for K while maintaining stable K concentrations (by altering cell water content and elemental composition). This process appears to depend on a functioning Na+, K+-ATPase system.     

Accumulation of procollagen in the degenerative articular cartilage of dogs with osteoarthritis

Collagen metabolism was studied in degenerative articular cartilage of dogs with spontaneous, early onset osteoarthritis. A fraction of collagen which represented about 1.5.% of the total was extracted from cartilage samples with dilute phosphate buffer (pH 7.4) containing 0.2% sodium dodecyl sulfate. Agarose gel filtration in the presence of sodium dodecul sulfate revealed that extracts of degenerative cartilage had about 24% procollagen whereas extracts of normal samples had only 3%. The isolated procollagen fraction was rechromatographed on agarose columns in the presence of mercaptoethanol. This resulted in the identification of a collagen species which migrated between marker β and α collagen chains. The molecular weight of this collagen was estimated to be 150000. Based on incorporation of [¹⁴C]proline, its ratio of hydroxy[¹⁴C]proline to total ¹⁴C was 0.32. Procollagen was not found after limited pepsin digestion (pH 3,4°C, 16 h) of degenerative cartilage samples.Since the total collagen content (μg hydroxyproline/mg cartilage), hydroxy[¹⁴C]proline/mg cartilage, specific radioactivity of hydroxy[¹⁴C]proline (cpm/μg), in the whole cartilage, and the specific radioactivity of hydroxyproline in the extractable collagen fraction were similar for normal and degenerative cartilage we propose that procollagen accumulated in the degenerative cartilage due to a partial defect in conversion of procollagen to collagen.     

Atrial-specific granules in the hearts of normal and water-deprived rats

Summary The morphology of atrial-specific granules, which contain atrial natriuretic polypeptide (ANP), was studied in the cardiac tissue of untreated controls and water-deprived rats by means of conventional and immunoelectron microscopy. Immature secretory vesicles or granules appeared to become buded off from the Golgi cisternae and then fused to form specific A-granules. An electron-dense plate with a fuzzy coat was frequently found on the limiting membrane at the end of such fusion. Pale specific B-granules, which were less electron-dense, larger, and more granular than A-granules, were found in small numbers in the left atrial cardiocytes, but rarely in the right ones. Very pale granules with a less granular matrix, considered to be B-type granules which had lost their electron-density, and which had less immunoreactivity for ANP, were numerous in the cardiac tissue after water deprivation. This morphological change, which is interpreted as an indication of granule degradation, was in agreement with the noted increase of natriuretic activity in the atrial tissue of water-deprived specimens.     

Quantitative X-ray microanalysis of the morula cell of the blood of the ascidian Halocynthia papillosa (Stolidobranchiata)

Summary The elemental composition of the morula cell of Halocynthia papillosa blood was studied by X-ray microanalysis with respect to the possible iron accumulation in this cell type. We found various amounts of Na, Mg, P, S, Cl, K, Ca, Fe and Br in the cytoplasm, nucleus and vacuoles. With the exception of a few cells, Ca, Fe and Br were not detected. Thus, the morula cells of the studied species are not iron-rich cells.     

The effect of primycin on the intracellular monovalent ion and water contents of rat hepatocytes as revealed by energy dispersive X-ray microanalysis and interference microscopy

Using energy-dispersive X-ray microanalytic and interference microscopic techniques, the intracellular concentration of the monovalent ions (Na+, K+, Cl+) as well as the intracytoplasmic and intracellular water contents were studied in normal and adrenalectomized rat hepatocytes with and without primycin treatment. Although primycin influenced significantly only the intracellular potassium content of the adrenalectomized group, it exerted a marked influence on the intranuclear water content in both the normal and adrenalectomized rats. The intranuclear water content increased significantly in the primycin-treated animals. The conclusion is drawn that the increased level of hydration of the nuclear substances reflects a 'decondensation' of the chromatin which on the other hand, may represent the basis for the various effects of primycin on the induction of certain hepatic enzymes.