Association of polyphosphate with protein in freeze-substituted sclerotia ofSclerotinia minor

Summary In freeze-substituted sclerotia stained with aqueous toluidine blue O, metachromatic material was found throughout the cytoplasm in discrete granules. It was also distributed evenly throughout spherical and elongate protein bodies. This material stained at low pH and was extracted by cold acid, indicating that it was polyphosphate. Retention of metachromatic material was much greater than previously reported in chemically fixed, conventionally processed sclerotia. X-ray microanalysis of dry-cut, unstained sections of freeze-substituted sclerotia confirmed that phosphorus was distributed evenly throughout the protein bodies and was not localised in discrete granules but phosphorus levels in the cytoplasm were very low. It is concluded that polyphosphate is lost during conventional preparation procedures but retained in dry-cut, unstained sections of freeze-substituted material. However, when freeze-substituted sections were stained with toluidine blue O, water soluble polyphosphate was extracted and subsequently precipitated in the cytoplasm as polyphosphate granules. Therefore it is considered that polyphosphate granules are an artefact, and that protein bodies are the major site for storage of phosphorus in this fungus.Abbreviations STEM scanning transmission electron microscope - ER endoplasmic reticulum     

Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate

Poly-beta-hydroxybutyrate granules exhibited a strong orange fluorescence when stained with Nile blue A. Heat-fixed cells were treated with 1% Nile blue A for 10 min and were observed at an excitation wavelength of 460 nm. Glycogen and polyphosphate did not stain. Nile blue A appears to be a more specific stain for poly-beta-hydroxybutyrate than Sudan black B.     

Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate.

Poly-beta-hydroxybutyrate granules exhibited a strong orange fluorescence when stained with Nile blue A. Heat-fixed cells were treated with 1% Nile blue A for 10 min and were observed at an excitation wavelength of 460 nm. Glycogen and polyphosphate did not stain. Nile blue A appears to be a more specific stain for poly-beta-hydroxybutyrate than Sudan black B.     

Dispersed polyphosphate in fungal vacuoles in Eucalyptus pilularis/Pisolithus tinctorius ectomycorrhizas.

Ectomycorrhizas produced between Pisolithus tinctorius and Eucalyptus pilularis under axenic conditions were rapidly frozen, freeze-substituted in tetrahydrofuran and embedded anhydrously, and dry-sectioned for X-ray microanalysis. The vacuoles of the sheath and Hartig net hyphae were rich in phosphorus and potassium. They also contained sulfur and variable amounts of chlorine. In anhydrously processed freeze-substituted mycorrhizas, dispersed electron-opaque material filled the fungal vacuoles. X-ray maps indicated that P was distributed evenly throughout the entire vacuole profile and was not concentrated in spherical bodies or subregions of the vacuole. There were no electron-opaque granules surrounded by electron-lucent areas, such as are commonly seen in chemically fixed material. The fungal vacuoles were also rich in K, which similarly gave a signal from the entire vacuolar profile. Such P-rich vacuoles occurred in both the mycorrhizal sheath and Hartig net hyphae. Stained sections of ether-acrolein freeze-substituted mycorrhizas also showed only dispersed material in the fungal vacuoles as, in most cases, did acetone-osmium freeze-substituted material. Precipitation of metachromatic granules by ethanol suggested that large amounts of polyphosphate are stored in these regions under the conditions of our experiments, as well as in the tips of actively growing hyphae of the same fungus. The higher plant vacuoles of ectomycorrhizas gave a much lower signal for K, and P was barely detectable. Much more K was located in the vacuoles of the root exodermal cells than in epidermal cells. The analysis of element distribution between the vacuole and cytoplasm in root cells agrees well with that found for other plant species using other techniques. We conclude that polyphosphate is indeed present in the vacuoles of the fungal cells of these ectomycorrhizas, but that in vivo it is in a dispersed form, not in granules.     

Changes in polyphosphate composition and localization in Propionibacterium acnes after near-ultraviolet irradiation.

Electron microscopy showed that electron-dense granules accumulated in Propionibacterium acnes in larger amounts when the bacteria were grown on a phosphate-rich medium. X-ray microanalysis demonstrated that the granules contained mostly phosphorus and potassium, indicating that the cells contained polyphosphate granules. When cells were grown on a complex Bacto-agar medium, the amount and the size of the polyphosphate granules were reduced. Polyphosphate was also detected with 31P nuclear magnetic resonance (31P-NMR). Of the polyphosphates observed with 31P-NMR, 20% seemed to be located outside the cell membrane. Broad-band near-ultraviolet irradiation (emission maximum 366 nm) corresponding to doses that killed 37% of the cells increased the amount of polyphosphate in cells grown on the phosphate-rich medium. The fluorescent chromophore 4',6-diamidino-2-phenylindole (DAPI) shifted the fluorescence emission from 478 to 538 nm when bound to polyphosphate and excited at 340 nm. DAPI was used to detect polyphosphates generated after near-ultraviolet irradiation of the cells. Nonirradiated cells showed no increased fluorescence at 538 nm, indicating no polyphosphate is presented in the cells. We conclude that DAPI did not have "access" to the intracellular polyphosphate as long as the cells were not light damaged. This observation is important for the interpretation of near-UV damage to cells.     

Nitrogen fixation by Rhizobium sp. 32H1. A morphological and ultrastructural comparison of asymbiotic and symbiotic nitrogen-fixing forms.

The induction of nitrogenase (C2H2) activity in asymbiotically cultured Rhizobium sp. 32H1 was found to be associated with morphological changes in the cells which were more pronounced than those seen in bacteroids. Polyphosphate granules were found in both bacteroids and cultured cells, but poly-beta-hydroxybutyrate vesicles were almost absent in bacteroids but were present in cultured cells. Freeze-etching techniques revealed no differences between the asymbiotically cultured nitrogen-fixing forms and bacteroids in that both the cell wall and cytoplasmic membrane cleavage planes were normal for gram-negative bacteria.     

A one-step dual-labeling method for antigen detection in mast cells

This paper describes a one-step light microscopy method for demonstrating the antigen contents of unequivocally identified mast cells. It is based on the differential metachromatic properties of proteoglycans, mostly heparin and chondroitin sulfate, and 1-naphthol in the presence of toluidine blue in an acidic medium. Proteoglycans occur in all mast cells and 1-naphthol is used to demonstrate the peroxidase activity of the sections treated by the horseradish peroxidase-labeled avidin–biotin complex method for antigen detection. Granules containing proteoglycans present the classical metachromatic reaction by appearing purplish-red, while granules containing antigen appear a brilliant green. When both types of granules are distinct inside the cell, single- and double-stained cells can be accurately separated and counted. We hope that this new procedure will contribute to a further identification of mast cell mediator contents and to a better understanding of the physiology of this cellular population.     

Structural changes occurring in interrenal tissue of the duck (Anas platyrhynchos) following adenohypophysectomy and treatment in vivo and in vitro with corticotropin

Adrenal glands from ACTH-treated intact ducks and chronically adenohypophysectomized ducks showed clear zonation into a subcapsular zone (SCZ) and an inner zone (IZ). Adenohypophysectomy caused ultrastructural changes in the IZ but not in the SCZ cells. These included increases in lipid droplets, changes in mitochondrial cristae from tubular to shelf-like, and changes in the shape of the nuclei from spherical to crenated. These changes were reversed by treatment with ACTH. Also, cells of the IZ, but not the SCZ, of adrenals from intact birds given ACTH showed more SER, more dense bodies, fewer lipid droplets and more prominent Golgi complexes. IZ cells incubated in buffer containing no ACTH developed mitochondria with shelf-like cristae and numerous opaque granules in the matrix. Exposure to buffer containing ACTH caused the mitochondrial cristae to become tubular and the matrix granules either decreased in number or disappeared. The granules could be extracted by incubating sections with chelating agents. The mitochondria in SCZ cells did not respond structurally to the presence of ACTH in the incubation medium but the matrix granules, like those in IZ cells, responded to the presence of chelating agents.     

A Neoplastic Connective Tissue Mast Cell Capable of Continuous Growth In Tissue Culture

A neoplastic connective tissue mast cell from a dog mast cell sarcoma has been grown in tissue culture for 50 passages over a period of 2 years. The cells were grown as monolayer cultures in glass bottles, using Eagle's basal medium fortified with calf serum. The cultures were contaminated with an Alkaligenes sp. for 10 months but finally were sterilized bacteriologically by treatment with specific antiserum combined with antibiotics. The cells grow in a fibroblastic pattern, and contain mitochondria, mast cell granules, and lipid granules or droplets. The mast cell granules stain basophilic with Giemsa's stain and metachromatically with azure A or toluidine blue. They also stain with Sudan black B and with periodic acid-Schiff stain. The interphase nuclei are vesicular, contain from 1 to 20 nucleoli, and frequently show bizarre outlines. Multinucleate cells are often seen, as are mitotic figures. Extracellular fibrous material occurs in all cultures and apparently originates from the cell surface. This material does not have the structure of connective tissue fibers and has not been identified. The cells develop an increased number of metachromatic granules when grown in medium containing heparin and an increased number of sudanophilic granules when grown in medium containing stearic acid. Only small amounts of histamine were present in the tumor from which this cell line was derived and in the cells grown in tissue culture.     

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.     

Atypical polyphosphate accumulation by the denitrifying bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-beta-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

Cellular incorporation of poly-beta-hydroxybutyrate into plasma membranes of Escherichia coli and Azotobacter vinelandii alters native membrane structure

Under growth-limiting conditions or conditions which mediate genetic transformation, Escherichia coli and Azotobacter vinelandii incorporate poly-beta-hydroxybutyrate into their plasma membranes. Genetic transformation competence of both bacteria increased in proportion to the concentration of membrane poly-beta-hydroxybutyrate. The effects of this lipid polymer on membrane structure were investigated by freeze-fracture electron microscopy. Before poly-beta-hydroxybutyrate incorporation, freeze-fracture revealed a typical mosaic of particles and pits on both concave and convex surfaces of the plasma membrane. As the cells incorporated the lipid polymer into the membrane, transformability developed and small semiregular plaques which possessed shallow particles were seen. These plaques grew in size and frequency as the membrane poly-beta-hydroxybutyrate concentrations and transformability increased.     

Localization of divalent cations in phosphate-rich cytoplasmic granules in yeast

Phosphate-rich yeast cells (Saccharomyces cerevisiae) take up more calcium and strontium than phosphate-deficient cells. The divalent cations appear to be tightly bound in phosphate-rich cells. Electron microscopical investigations, combined with energy-dispersive X-ray microanalysis, showed that divalent cations were sequestered in cytoplasmic granules, together with a large amount of phosphorus. The major part of the divalent cations is retrieved in the polyphosphate fraction, a minor part is bound to lipids. The results suggest that polyphosphate granules may serve as an important store for divalent cations.     

REJUVENATION OF MELOSIRA GRANULATA (BACILLARIOPHYCEAE) RESTING CELLS FROM THE ANOXIC SEDIMENTS OF DOUGLAS LAKE,MICHIGAN. I. LIGHT MIGROSCOPY AND 14C UPTAKE 1

Resting cells of Melosira granulate (Ehr.) Ralfs were collected from the anoxic sediments of Douglas Lake, Michigan. Sediment containing M. granulata was inoculated into distilled water and incubated in a growth chamber for one week during which observations were made on the cytological differentiation process. Cells classified as “condensed,” i.e. containing a dark brown cytoplasmic mass were identified as resting cells. The differentiation process consisted of a series of gradual cytological changes that included elongation of the cytoplasmic mass and recognition of definable organelles to the point where the cells were non-distinguishable from water column vegetative cells. Differentiating cells accumulated large polyphosphate and lipid granules. However, these granules disappeared just prior to cell division. The complete differentiation or rejuvenation sequence occurred in some cells in less than 24 h. However, not all dormant cells rejuvenated at the same time and it was observed that the lag period for rejuvenation increased with resting cell age (depth of burial in sediments). In the ¹⁴C uptake studies, label was initially observed in condensed state cells. The label gradually progressed to the more differentiated forms. Total carbon uptake during the rejuvenation process was initially lower in the rejuvenating cells, but roughly equal to water column populations after 8 h, indicating a period of high metabolic activity in the rejuvenating cells between 1 and 8 h.     

Differences in the behavior of cytoplasmic granules and lipid bodies during human lung mast cell degranulation

We used a morphometric and autoradiographic approach to analyze changes in specific cytoplasmic granules and cytoplasmic lipid bodies associated with human lung mast cell degranulation. Mast cells were dissociated from lung tissue by enzymatic digestion and were then enriched to purities of up to 99% by countercurrent centrifugation elutriation and recovery from columns containing specific antigen bound to Sepharose 6 MB. Degranulation was induced by goat anti-IgE. At various intervals after stimulation, parallel aliquots of cells were recovered for determination of histamine release or were fixed for transmission electron microscopy. We found that lipid bodies, electron- dense structures that lack unit membranes, were present in both control and stimulated mast cells. Autoradiographic analysis showed that lipid bodies represented the major repository of 3H-label derived from [3H]arachidonic acid taken up from the external milieu. By contrast, the specific cytoplasmic granules contained no detectable 3H-label. In addition, lipid bodies occurred in intimate association with degranulation channels during mast cell activation, but the total volume of lipid bodies did not change during the 20 min after stimulation with anti-IgE. This result stands in striking contrast to the behavior of specific cytoplasmic granules, the great majority of which (77% according to aggregate volume) exhibited ultrastructural alterations during the first 20 min of mast cell activation. These observations establish that mast cell cytoplasmic granules and cytoplasmic lipid bodies are distinct organelles that differ in ultrastructure, biochemistry, and behavior during mast cell activation.     

Formation of volutin granules in Corynebacterium glutamicum

Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted.     

Mast cells are present in epithelial layers of different tissues of the mollusc <Emphasis Type="Italic">Anomalocardia brasiliana</Emphasis>. <Emphasis Type="Italic">In situ</Emphasis> characterization of heparin and a correlation of heparin and histamine concentration

Heparin, other sulphated glycosaminoglycans and histamine were extracted from various dissected organs of Anomalocardia brasiliana, a mollusc from the South Atlantic, and quantified. A good correlation between heparin and histamine content was found in the labial palp, intestine, ctenidium, mantle and foot tissues. The tissue location of metachromatic cells, putatively containing heparin, was identified histologically with Alcian Blue, Toluidine Blue, Masson trichrome, Haematoxylin–Eosin and PAS. Except for the foot, cells containing metachromatic granules were found in the epithelium surfaces of all the organs analysed. An in situ identification of heparin using nitrous acid and heparinase degradation has established unequivocally the presence of this compound in the metachromatic cells. The location of 'mast-like' cells at the epithelium surface of mollusc tissues exposed to the environment are very similar to the distribution of mammalian and other vertebrate mast cells and gives support to the suggestion for a role of mast cells in defense mechanisms.     

Metabolic and ultrastructural response to glucose of two eurytrophic bacteria isolated from seawater at different enriching concentrations.

Two marine bacteria, an Acinetobacter sp. (strain GO1) and a vibrio sp. (strain G1), were isolated by extinction dilution and maintained in natural seawater supplemented with nitrogen, phosphorus, and glucose at 0.01 and 10 mg of glucose carbon per liter above ambient monosaccharide concentrations, respectively. After 3 days in unsupplemented natural seawater, growth in batch culture with glucose supplements was determined by changes in cell numbers and glucose concentration. The exponential growth of the Acinetobacter strain with added glucose was indistinguishable from that in natural seawater alone, whereas that of the Vibrio strain was more rapid in the presence of glucose supplements, suggesting that the Acinetobacter strain preferred the natural organic matter in seawater as a carbon source. The ultrastructure for both isolates was unaffected by glucose supplements during exponential growth, but there were marked changes in stationary-phase cells. The Vibrio strain formed polyphosphate at 10 mg of glucose carbon per liter, whereas poly-beta-hydroxybutyrate formation occurred at 100 mg and became excessive at 1,000 mg, disrupting the cells. In contrast, the Acinetobacter strain elongated at 100 and 1,000 mg of glucose carbon per liter but failed to show poly-beta-hydroxybutyrate formation. The diversity of responses shown here would not have been detected with a single concentration of substrate, often used in the literature to characterize both pure and natural populations of marine bacteria.     

Cytochemical study of developing anthers of Amomum villosum Lour.

We analyzed anther development in Amomum villosum Lour. (Zingiberaceae) using the periodic acid-Schiff's technique and Sudan black staining to test for the presence of starch and lipids, respectively. Our analyses showed that microspore mother cells of A. villosum lack typical callose walls, and numerous lipid granules appear in the cells early in development. Some starch granules are present in anther wall cells, but not in tapetal cells. After meiosis, numerous lipid granules remain unchanged in the microspores. During microspore development, some small starch granules first appear in the central cell region, and then the starch granules increase in size. After microspore division, the bicellular pollen grains become filled with starch and lipids, and remain in this state until the pollen grains reach maturity. At anthesis, the anther wall of A. villosum consists of several layers of endothecium cells with an evidently thickened radial wall, and some layers of parenchyma cells containing numerous starch granules.     

Metabolic and ultrastructural response to glucose of two eurytrophic bacteria isolated from seawater at different enriching concentrations

Two marine bacteria, an Acinetobacter sp. (strain GO1) and a vibrio sp. (strain G1), were isolated by extinction dilution and maintained in natural seawater supplemented with nitrogen, phosphorus, and glucose at 0.01 and 10 mg of glucose carbon per liter above ambient monosaccharide concentrations, respectively. After 3 days in unsupplemented natural seawater, growth in batch culture with glucose supplements was determined by changes in cell numbers and glucose concentration. The exponential growth of the Acinetobacter strain with added glucose was indistinguishable from that in natural seawater alone, whereas that of the Vibrio strain was more rapid in the presence of glucose supplements, suggesting that the Acinetobacter strain preferred the natural organic matter in seawater as a carbon source. The ultrastructure for both isolates was unaffected by glucose supplements during exponential growth, but there were marked changes in stationary-phase cells. The Vibrio strain formed polyphosphate at 10 mg of glucose carbon per liter, whereas poly-beta-hydroxybutyrate formation occurred at 100 mg and became excessive at 1,000 mg, disrupting the cells. In contrast, the Acinetobacter strain elongated at 100 and 1,000 mg of glucose carbon per liter but failed to show poly-beta-hydroxybutyrate formation. The diversity of responses shown here would not have been detected with a single concentration of substrate, often used in the literature to characterize both pure and natural populations of marine bacteria.