Development of a rickettsia isolated from an aborted bovine fetus.

An obligate intracellular rickettsial organism isolated from an aborted bovine fetus was studied in bovine turbinate and mouse macrophage cell cultures with light and electron microscopy. Development of the organism was similar in both cell types. The organism replicated within cytoplasmic vacuoles in a developmental cycle that resembled that of both the ehrlichiae and chlamydiae. The inoculum contained only electron-dense forms, which infected cells within 2 h postinoculation by adhering to cell membranes at thickened areas that appeared to be coated pits and then being endocytosed. A striking feature occurred next as the organisms became surrounded by host cell mitochondria and, by light microscopy, appeared to have halos. During this intimate association with mitochondria, the electron-dense organisms changed into large reticulated forms that began to divide by binary fission. These large forms were often in direct contact with mitochondrial membranes. The organisms continued to divide by binary fission, and host cells contained large cytoplasmic inclusions of reticulated organisms. The reticulated organisms gradually changed into electron-dense forms that were released from degenerated host cells.     

Ultrastructure of crystalline inclusions in the thylakoids of dodder<Emphasis Type="Italic">(cuscuta japonica)</Emphasis> plastids

The plastids from seedlings of the parasitic angiospermCuscuta japonica were ultrastructurally investigated. In shoot subapical cells from 3-d-old seedlings grown in the dark, the etioplasts contained prolamellar bodies and amorphous and dense inclusions. In the shoot subapical cells obtained from 6-d-old seedlings grown under light conditions for the last 3 d, the underdeveloped chloroplasts contained phytoferritin within the stroma as well as amorphous and dense inclusions that were limited by the thylakoid membranes. In the developing chloroplasts, electron-dense materials were detected within the transversely sectioned thylakoid lumens. This dense material presented two different images, depending upon the sectional plane. When transversely prepared, the materials appeared as somewhat thick, linear structures, whereas longitudinally sectioned thylakoids revealed very large crystalline inclusions. In the developed chloroplasts, the amounts of electron-dense material or crystalline inclusions were remarkably reduced in the thylakoid lumens, which were electron-translucent. Far fewer crystalline inclusions were observed in the developed chloroplasts of seedlings than in the developing chloroplasts. These results suggest that the crystalline inclusions may be temporarily reserved within the thylakoid lumens of chloroplasts in the Gjaponica seedlings.     

A Fine Structural Study and Energy-dispersive X-ray Spectrometrical Analysis of the Metals Present in Ovarian and Other Tissues of Normal and Lead-poisoned Quail (Coturnix coturnix japonica)

Abstract The fine structure was investigated of body tissues from Japanese quails which had received lead acetate added to their diet. A double fixation using glutaraldehyde and postfixation in osmic acid was compared with a fixation in glutaraldehyde alone. Because of the lack of contrast obtained in the specimens from the latter fixation, the presence of metal residues within the tissues were readily observable under the electron microscope. The various metal residues found in storage bodies in ovarian tissues from lead-poisoned quails were analyzed by means of energy-dispersive X-ray spectrometrical analysis. Out of a total of eight electron-dense, metal-containing inclusions which were analyzed, two contained lead. Other elements found in the inclusions were chlorine, phosphorus, iron and calcium, the latter element not being present in detectable amounts in those inclusions which also contained lead. Some correlation was found between the morphology and the elemental composition of the metal residues.     

New magnet-sensitive structures in bacterial and archaeal cells

The objects of the investigation were: distribution of intracellular magnet-sensitive structures among different taxonomic groups of prokaryotes, localisation and organisation of the magnet-sensitive inclusions (MsI) in cells. The MsI were discovered in representatives of both prokaryotic domains (Bacteria and Archaea), 2 kingdoms and 7 orders of bacteria. They were some amorphous or non-crystalline globules with the electron-transparent centre surrounded with an electron-dense homogenous matrix. The magnetic nature of the structures was shown by attraction with an applied magnet both for the cell suspensions and for the MsI isolated and separated from the destroyed cells. The MsI were studied with transparent electron microscopy and with X-ray analyses. When the cells were grown in the iron-containing nutrient medium, the matrix was enriched with iron. It was shown also that some bacteria grown with cobalt or with chromium contained the cobalt- or chromium-enriched magnetic inclusions.     

Calcium containing particles in mitochondria of heart muscle cells as shown by cryo-ultramicrotomy and X-ray microanalysis

Summary Mitochondria of normal myocardial cells of the sand rat and the mouse as well as of the left ventricle of man, have been examined for their content of calcium. Ultrahistochemistry and X-ray microanalysis revealed two basically different inclusions: Osmiophilic mitochondrial granules and Spherical mitochondrial particles. Osmiophilic mitochondrial granules were found in conventionally fixed and plastic embedded tissues as well as in cryosections of chemically fixed and sucrose infused tissues. Such granules lacked inert electron density and probably consisted mainly of unsaturated lipids. X-ray spectra obtained from these tissues revealed no peaks for calcium. Spherical mitochondrial particles were present in dry-cut cryo-sections of N₂-frozen tissues not treated by fixatives and/or cryoprotectants. These particles were deeply electron dense in unstained, freeze-dried cryo-sections. They usually measured from 600Å–900Å in diameter in the normal myocardium of the sand rat and the mouse and from 250 Å–400Å in diameter in the left ventricular myocardium of man. Significant calcium peaks could be identified in the X-ray spectra of these particles, whereas none occurred in the analyses of other tissue regions. Potassium was detected with about equal frequency in the particles and in other parts of the tissue. On the basis of the inert electron density of the particles and their absence in chemically fixed tissues as well as of the results of the X-ray analysis, it is concluded that they contain precipitates of extremely labile ions of mitochondrial calcium.We should like to thank Mrs. Trine Jensen for skillful technical assistance. This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from the Norwegian Research Council for Science and the Humanities     

Programmed cell death. Cytochemical evidence for accumulation of calcium in mitochondria and its translocation into lysosomes: X-ray microanalysis in metamorphosing insect muscles

Summary The intersegmental muscles in the metamorphosing silkmothAntheraea polyphemus were examined by two electron cytochemical procedures for demonstration of calcium compartmentation during the two-day period of degeneration after emergence. Muscle fibres were treated with either oxalate—pyroantimonate, or phosphate—pyroantimonate procedures. The elemental composition of the reaction product arising from the oxalate procedure was determined with electron probe X-ray microanalysis of unstained thin sections by energy dispersive spectrometry and wavelength dispersive spectrometry. The wavelength dispersive data revealed high peaks of calcium and antimony in the electron-dense precipitates. No reaction was obtained in muscles after treatment with the phosphate—pyroantimonate method.Shortly after the emergence of the moth, very few calcium deposits were found in the mitochondria, which also contained amorphous matrix densities. During the rapid lytic phase (17 and 30 h after ecdysis), the mitochondria, autophagic vacuoles sequestering mitochondria, and lysosomal dense bodies issuing from the latter were highly reactive in each muscle fibre.These results demonstrate that the collapse of tracheae (hypoxic conditions) is correlated with the calcium overload of mitochondria when the cell calcium homeostasis is apparently lost. Such calcium overload of the mitochondria appears to cause irreversible damage to these organelles which are then sequestered in autophagic vacuoles. This mitochondrial autophagic process leads to calcium translocation into a lysosomal compartment. We suggest that the calcium lysosomal stores may have a transient function of cell detoxification and stimulation of calcium-dependent degradative processes prior to the final muscle collapse.     

Fine structure of the parotid and mandibular glands of the cotton rat (Sigmodon hispidus).

The parotid and mandibular glands of the cotton rat were examined by light and transmission electron microscopy. Parotid gland: Acinar cells were serous in nature, and contained electron-dense granules. Intercalated duct cells contained electron-dense granules. Striated duct cells had small granules of moderate and high electron densities. Mandibular gland: Acinar cells were seromucous in nature, and contained granules of low and moderate electron densities. Intercalated duct cells contained granules of moderate and high electron densities. Striated ducts were comprised of two portions - a secretory portion and a striated portion without granules. The secretory portion had many electron-dense granules. A sexual dimorphism was obserbed in these granules, which were smaller and fewer in females than in males.     

Morphological and immunological confirmation of the presence of chlamydiae in the gut tissues of the chesapeake bay clam,Mercenaria mercenaria

The original electron microscopic identification by other investigators in 1977 of chlamydiae in the gut tissues of the Chesapeake Bay hard clam (Mercenaria mercenaria) is corroborated and further supported by evidence ofChlamydia-specific immunofluorescence (IF). Our electron microscopy demonstrated that gut tissue cells were heavily infected with chlamydiae in all stages of development but the intrachlamydial phage-like particles reported in 1977 were not seen. Tissue sections stained with IF reagents were strongly positive, and IF was blocked in varying degrees with chlamydial antisera produced in a goat and a turkey. The IF-positive tissue sections contained intracytoplasmic inclusions that stained darkly with Lugol's iodine (indicating the presence of glycogen) while IF-negative tissues had little if any iodinestaining material. Furthermore, electron micrographs of chlamydiae-containing phagosomes showed numerous rosettes of electron-dense particles typical of glycogen. The presence of iodine-positive phagosomes with electron dense rosettes suggests that the organisms are glycogen-producing chlamydiae biochemically related toChlamydia trachomatis. Repeated attempts to cultivate chlamydiae from the clam tissues in cell cultures and laboratory animals failed.     

ULTRASTRUCTURE OF THE MATURE UNGERMINATED RICE (ORYZA SATIVA) CARYOPSIS. THE GERM

An in-depth transmission electron microscope study of the ungerminated, unimbibed rice germ was conducted. All tissues in the germ were examined. Plastids were similar in all cells; many contained osmiophilic globules and phytoferritin, some displayed a limited thylakoid system, and all contained cytoplasmic tubular and vesicular inclusions formed by invaginations of the outer plastid membranes. Filament bundles were found in cells of the coleoptile, plumule, mesocotyl, radicle, and epiblast. Three general categories of cells could be identified in the germ based on protein body characteristics and on lipid body distribution: 1) Cells having inclusions in protein bodies and having numerous lipid bodies scattered throughout the cytoplasm; 2) Cells having or lacking protein body inclusions and possessing peripheral lipid bodies and/or having electron-dense deposits in the lipid bodies; and 3) Cells lacking protein bodies and having peripheral lipid bodies.     

Polyhydroxybutyrate particles in Synechocystis sp. PCC 6803: facts and fiction

Transmission electron microscopy has been used to identify poly-3-hydroxybutyrate (PHB) granules in cyanobacteria for over 40 years. Spherical inclusions inside the cell that are electron-transparent and/or slightly electron-dense and that are found in transmission electron micrographs of cyanobacteria are generally assumed to be PHB granules. The aim of this study was to test this assumption in different strains of the cyanobacterium Synechocystis sp. PCC 6803. Inclusions that resemble PHB granules were present in strains lacking a pair of genes essential for PHB synthesis and in wild-type cells under conditions that no PHB granules could be detected by fluorescence staining of PHB. Indeed, in these cells PHB could not be demonstrated chemically by GC/MS either. Based on the results gathered, it is concluded that not all the slightly electron-dense spherical inclusions are PHB granules in Synechocystis sp. PCC 6803. This result is potentially applicable to other cyanobacteria. Alternate assignments for these inclusions are discussed.     

Morphometric and ultrastructural features of the mare oviduct epithelium during oestrus

Morphometric, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) investigations have displayed regional differences in the mare oviductal epithelium. The entire mucosa of the oviduct was lined with a pseudostratified epithelium, which consisted of two distinct cell types, ciliated and non-ciliated. Ciliated cells were predominant in the three different segments of the oviduct and their percentage increased from fimbriae to ampulla and significantly decreased in the isthmus. SEM revealed in the infundibulum finger-like mucosal folds, some of them interconnected, in the ampulla numerous and elaborated branched folds of the mucosa, whereas the isthmus displayed a narrow lumen, short and non-branched mucosal folds. In the ampulla and isthmus the majority of non-ciliated cells showed apical blebs provided or not of short microvilli. TEM displayed different ultrastructural features of ciliated and non-ciliated cells along the oviduct. Isthmus ciliated cells presented a more electron-dense cytoplasm than in infundibulum and ampulla cells and its cilia were enclosed in an amorphous matrix. The non-ciliated cells of infundibulum did not contain secretory granules but some apical endocytic vesicles and microvilli coated by a well developed glycocalyx. Non-ciliated cells of ampulla and isthmus contained secretory granules. Apical protrusions of ampulla displayed two types of secretory granules as well as occasional electron-lucent vesicles. Isthmus non-ciliated cells showed either electron-lucent or electron-dense cytoplasm and not all contained apical protrusions. The electron-dense non-ciliated cells displayed microvilli coated with a well developed glycocalyx. Three types of granules were observed in the isthmus non-ciliated cells. The regional differences observed along the epithelium lining the mare oviduct suggest that the epithelium of the each segment is involved in the production of a distinctive microenvironment with a unique biochemical milieu related to its functional role.     

Histochemical, ultrastructural and X-ray microprobe analytical studies of localization of calcium in the mucous lining of the rat duodenum

Localization of calcium in a rapid frozen and freeze substituted duodenum of normal, starved or calcium-repleted rat was examined using either of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, a sensitive histochemical calcium stain or electron microscopy. In normally-fed rats, a majority of absorptive cells of the duodenum showed numerous discrete red granular GBHA reactions, approximately 1 micron or less in diameter, located primarily along their lateral plasma membranes and within intercellular spaces. Electron microscopy also revealed electron-dense granules, 30-100 nm in diameter, showing a similar distribution as the GBHA granules in the respective absorptive cells, and confirmed their absence in mitochondria and other intracellular compartments. Some of the absorptive cells located exclusively at the tip of each villus contained highly GBHA-reactive tubulo-vesicular structures extending throughout the cytoplasm. However, they displayed virtually no granular GBHA reaction. In these cells, electron microscopy revealed numerous electron-dense granules in the nucleus, mitochondria and in other unidentified organelles. X-ray microprobe analyses of ultrathin sections confirmed the presence of calcium within electron-dense granules associated with both types of absorptive cells. The number and intensity of all GBHA reactions fluctuated according to luminal calcium concentration. In calcium-repleted rats, strong GBHA reactions appeared in a narrow zone of lamina propria at the tip of the villus, overlaid, predominantly, with absorptive cells showing tubulo-vesicular GBHA reactions. These results suggest the existence of distinct types of absorptive epithelial cells in the rat duodenum, with respect to patterns of calcium localization which they display.     

Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.     

Histochemical,ultrastructural and X-ray microprobe analytical studies of localization of calcium in the mucous lining of the rat duodenum

Summary Localization of calcium in a rapid frozen and freeze substituted duodenum of normal, starved or calciumrepleted rat was examined using either of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, a sensitive histochemical calcium stain or electron microscopy. In normallyfed rats, a majority of absorptive cells of the duodenum showed numerous discrete red granular GBHA reactions, approximately 1 m or less in diameter, located primarily along their lateral plasma membranes and within intercellular spaces. Electron microscopy also revealed electron-dense granules, 30–100 nm in diameter, showing a similar distribution as the GBHA granules in the respective absorptive cells, and confirmed their absence in mitochondria and other intracellular compartments. Some of the absorptive cells located exclusively at the tip of each villus contained highly GBHA-reactive tubulo-vesicular structures extending throughout the cytoplasm. However, they displayed virtually no granular GBHA reaction. In these cells, electron microscopy revealed numerous electron-dense granules in the nucleus, mitochondria and in other unidentified organelles. X-ray microprobe analyses of ultrathin sections confirmed the presence of calcium within electron-dense granules associated with both types of absorptive cells. The number and intensity of all GBHA reactions fluctuated according to luminal calcium concentration. In calcium-repleted rats, strong GBHA reactions appeared in a narrow zone of lamina propria at the tip of the villus, overlaid, predominantly, with absorptive cells showing tubulo-vesicular GBHA reactions. these results suggest the existence of distinct types of absorptive epithelial cells in the rat duodenum, with respect to patterns of calcium localization which they display.     

Inhibition of growth of Chlamydia trachomatis by the calcium antagonist verapamil

Treatment of BGM (African Green Monkey kidney) cells with the calcium antagonist Verapamil resulted in a reduced yield of chlamydial infectious particles. The inhibitory effect was concentration-dependent, the maximal effect being achieved at 200 microM-Verapamil, which produced a 99.99% reduction of infectious particle yield. Electron microscopy showed that control Chlamydia trachomatis-infected BGM cells contained typical large inclusions in which most of the particles were elementary bodies, whereas Verapamil-treated infected cells contained small inclusions consisting predominantly of reticulate bodies. The findings indicate a possible therapeutic use of this calcium antagonist as an anti-chlamydial drug.     

Formation of "ghost" bodies and calcification in experimental atherosclerosis in nonhuman primates. An ultrastructural study

Aortic lesions from African green monkeys fed a cholesterol diet for up to 24 months were studied by electron microscopy. The lesions, grossly classified as fatty dots and fatty streaks consisted of foam cells, increased amounts of interstitial connective tissues and osmiophilic lipid material. In addition, in the interstitial spaces, there were membrane-bound detached cytoplasmic fragments and deeply osmiophilic calcium spherules. The smooth muscle cells had a frayed appearance and bulbous cytoplasmic pseudopodlike processes. Figures suggestive of transition between these cell processes and the detached cytoplasmic fragments were observed. The detached cytoplasmic fragments or ghost bodies often contained lipid droplets, myelin-like figures and calcific material. The process of budding off cytoplasmic fragments was interpreted as a form of clasmatosis enabling smooth muscle cells to eliminate substances which could not be degraded intracellularly. It is proposed that material presented within the ghost bodies may become a nucleation site for calcium salts deposition. Cell necrosis was not a feature observed in this material.     

Characterization of multi-layered membrane generated by rabies virus

Electron microscopy revealed multi-layered membranes within the cytoplasmic inclusion (accumulation of nucleocapsids) produced by rabies virus. When infected BHK cells were maintained at 31 C, an enhancement in production of these membranes occurred in approximately 60% of inclusion-containing cells. Multi-layered membranes were composed of an alternate array of two different layers; an electron-dense, thin membrane and a less dense layer which was thicker. SDS-polyacrylamide gel electrophoresis and immune electron microscopy of isolated multi-layered membrane preparations demonstrated that the structures contained viral G and M2 polypeptides. Our observations suggest that these membranous structures are not a degenerative product of rabies virus infection but rather are related to the replication of viral envelope constituents, although they represent themselves to be an abortive form of viral assembly.     

Alcohol oxidase and catalase in peroxisomes of methanol-grown Candida boidinii.

Microbodies, designated as peroxisomes because of their enzyme complement, have been isolated from methanol-grown cells of Candida boidinii. Spheroplast lysates were separated on non-continuous Ficoll density gradients, resulting in a mitochondrial fraction and a peroxisome fraction. Estimates of purity using the mitochondrial enzyme markers suggested that the contamination of mitochondria in the peroxisome fraction was about 2-3%. As shown by electron microscopy the peroxisomes were 0.4-0.6 mum in diameter and contained crystalloid inclusions. Alcohol oxidase and catalase, which catalyse the oxidation of methanol to formaldehyde in Candida boidinii, could be localized within the peroxisomes. Gel-electrophoretic studies of the peroxisome fraction demonstrated that it contained only two predominant protein bands consistent with alcohol oxidase and catalase. No alcohol oxidase and catalase activity was found in mitochondria.     

An umbraviral protein,involved in long-distance RNA movement,binds viral RNA and forms unique,protective ribonucleoprotein complexes

Umbraviruses are different from most other viruses in that they do not encode a conventional capsid protein (CP); therefore, no recognizable virus particles are formed in infected plants. Their lack of a CP is compensated for by the ORF3 protein, which fulfils functions that are provided by the CPs of other viruses, such as protection and long-distance movement of viral RNA. When the Groundnut rosette virus (GRV) ORF3 protein was expressed from Tobacco mosaic virus (TMV) in place of the TMV CP [TMV(ORF3)], in infected cells it interacted with the TMV RNA to form filamentous ribonucleoprotein (RNP) particles that had elements of helical structure but were not as uniform as classical virions. These RNP particles were observed in amorphous inclusions in the cytoplasm, where they were embedded within an electron-dense matrix material. The inclusions were detected in all types of cells and were abundant in phloem-associated cells, in particular companion cells and immature sieve elements. RNP-containing complexes similar in appearance to the inclusions were isolated from plants infected with TMV(ORF3) or with GRV itself. In vitro, the ORF3 protein formed oligomers and bound RNA in a manner consistent with its role in the formation of RNP complexes. It is suggested that the cytoplasmic RNP complexes formed by the ORF3 protein serve to protect viral RNA and may be the form in which it moves through the phloem. Thus, the RNP particles detected here represent a novel structure which may be used by umbraviruses as an alternative to classical virions.     

Secretory products of the haptoral reservoirs and peduncular glands in two species of Bravohollisia (Monogenea: Ancyrocephalidae)

Abstract. Light and electron microscopy were used to characterize the structure of secretory cells and their products involved in attachment of two monogenean parasites of fish, in order to understand their role in the attachment process. In Bravohollisia rosetta and Bravohollisia gussevi , peduncular gland cells with two nuclei, granular endoplasmic reticulum, and Golgi bodies produce dual electron-dense (DED) secretory bodies with a homogenous electron-dense rind and a less electron-dense fibrillar core (oval and concave in B. rosetta and oval in B. gussevi ). The DED secretory bodies are altered as they migrate from the gland cell to the haptoral reservoir, the superficial anchor grooves, and into the gill tissues. The contents of the DED secretory bodies are exocytosed into the reservoirs, fibrillar cores persisting in the matrix, some of which condense, forming highly electron-dense spherical bodies. Small, oval, electron-dense bodies occur in the grooves, while no inclusions are visible in the homogenous exudate within the gill tissues. The single tubular extension of the reservoir enters a bifurcate channel within the anchor via a concealed, crevice-like opening on one side of the anchor. The channel directs secretions into the left and the right grooves via concealed apertures. The secretions, introduced into the tissues by the anchors, probably assist in attachment. The secretions are manifested externally as net-like structures and observed in some cases to be still attached to the point of exudation, on anchors detached from the gill tissues. This suggests that despite having the anchors detached, the worms can still remain anchored to the gill tissues via these net-like structures. Based on this, it is postulated that the net-like secretions probably function as a safety line to anchor the worm during the onset of locomotion and in doing so reduce the risk of tearing host tissues.