Amorphous Electron-dense Material Research Articles

Effect of sodium butyrate on actin distribution in rat 3Y1 fibroblasts in monolayer culture.

We studied the effect of sodium butyrate, a potent G1/G2-arresting agent, on actin distribution in rat 3Y1 fibroblasts in monolayer culture by fluorescence microscopy of cells stained with 7-nitrobenz-2-oxa-1, 3-diazole phallacidine (NBD-Ph). When randomly proliferating cells were arrested mainly in G1 phase with butyrate, a reversible overaccumulation of cellular net protein occurred. In the G1-arrested cells, actin markedly accumulated at the margin of cells, and a network structure of actin stress fibers appeared. When density-arrested cells were replated sparsely and rearrested in the G1, early S, and G2 phases with butyrate or hydroxyurea, the actin network was observed extensively in the cells arrested in the G1 and G2 phases with butyrate. These results agree with our previous results indicating the existence of some physiological similarity between cells in the G1 and G2 phases and suggest that actin distribution somehow depends on the phases of the cell cycle. The actin profiles observed by the NBD-Ph staining were confirmed by transmission electronmicroscopy (TEM) of negatively stained whole cells. TEM further revealed that electron-dense amorphous materials were present at crossing points in the network but rarely present on interconnecting microfilament bundles.

Flagellar morphology in stumpy-flagella mutants of Chlamydomonas reinhardtii.

Sixteen new mutants of the biflagellate green alga Chlamydomonas reinhardtii with either stumpy-flagella or no flagella at all were examined by electron microscopy. Four of the mutants were found to carry short bulbous flagella containing amorphous electron-dense material which may represent unassembled flagellar protein. Basal bodies of normal ultrastructure were present in all mutants. Dikaryon dominance tests indicated that the stumpy mutations were recessive to wild-type in all cases tested. Stumpy mutations also conferred a measure of detergent resistance to Chlamydomonas, apparently by affecting the detergent-solubility of the flagellar membrane.

Structure of the radially asymmetrical uncalcified region of the teeth of the red-backed salamander, Plethodon cinereus (Amphibia, Plethodontidae).

The teeth of the adult plethodontid salamander, Plethodon cinereus, were examined by light and electron microscopy with emphasis on the ringlike zone of uncalcified dentin that divides the calcified portion of each tooth into a proximal pedestal and a distal apex. The uncalcified region displays radial asymmetry, forming an integral part of the posterior wall of the tooth but bulging into the pulp cavity anteriorly, thus forming a hingelike structure. All portions of the dentin, including the uncalcified region, are composed predominantly of collagenous fibers but lack elastin. In scanning electron micrographs of teeth from which the oral mucosa has been removed, the location of the anterior uncalcified hinge is marked externally by a notch-like articulation of the apex and pedestal. Sites of transition between calcified and uncalcified areas of the dentin show no special modifications in transmission electron micrographs, but collagenous fibers in calcified portions are associated with more electron-dense amorphous material than are those in the uncalcified region. Odontoblasts associated with the uncalcified region possess ultrastructural features closely resembling those of odontoblasts found in calcified areas. The uncalcified region seems to afford the teeth a certain degree of flexibility, and the asymmetry of the region appears to allow the teeth to flex only in a posterior direction, thus facilitating the entry of living prey but hindering its escape. The uncalcified region also seems to permit the apex of a tooth to break away from its pedestal without damage to underlying bone.

Insights into the evolution of heliozoa (Protozoa, Sarcodina) as provided by ultrastructural studies on a new species of flagellate from the genus Pteridomonas

The fine structure of Pteridomonas danica, a new species of helioflagellate (protozoa), is described. The organism has a single apical flagellum which bears two rows of tripartite tubular hairs. There are two kinetosomes which are bound to each other and to the surface of the single nucleus by amorphous electron-dense material. The flagellum is surrounded by a wreath of fine arms, which are supported internally by triads of microtubules. The triads terminate on the nucleus. The mitochondria have tubular cristae which contain wisps of intracristal material. The dictyosomes are located in the posterior part of the cell, being clustered around the internal element of the contractile stalk. Extrusomes are present. Pteridomonas has substantial similarities with the colourless flagellate Ciliophrys and with the pedinellid algal flagellates. As Ciliophrys resembles the actinophryid heliozoa in several important respects, the evidence supports the contention that these heliozoa have a common ancestry with the heterokont algae and have evolved along a line involving the pedinellids and some colourless helioflagellates. If the perceived relationships are to be reflected in their taxonomy, then a substantial reclassification of these organisms is required.

The composition of wall alterations and appositions (reaction material) and their role in the resistance of onion bulb scale epidermis to colonization by Botrytis allii

Ultrastructural examination of deposits of reaction material (RM) showed them to be composed of paramural granules (osmiophilic to varying degrees) lying within amorphous electron–dense material. Epidermal cell walls per se became increasingly electron–dense around the short infection hyphae produced by Botrytis allii. Fluorescence microscopy, histochemical tests and microautoradiography showed the accumulation of phenolic compounds at infection sites. Use of phenylalanine and cinnamate for E.M. autoradiography demonstrated the accumulation of phenolics within paramural granules, amorphous aggregates and within the penetrated cell wall, Paramural deposits were soluble in organic solvents and probably represented reservoirs of low MW phenolics which were progressively incorporated into insoluble polymers within the cell wall. Initial synthesis or accumulation of phenolics appeared to take place in rough endoplasmic reticulum during the first 2 h after cuticle penetration. Suppression of RM deposition and wall alterations was achieved by treatment of bulb scales with cycloheximide. Tissues treated with the protein synthesis inhibitor became susceptible to colonization by B. allii. Accumulation of phenolics at infection sites rendered the onion cell walls resistant to attack by a mixture of cell wall–degrading enzymes, Methanolic extracts of epidermal strips containing RM deposits contained very weakly active inhibitors of B. allii germ–tube growth. The role of RM deposits and wall alterations in the resistance of onion epidermis to colonization by Botrytis is discussed.

Light and electron microscopic observations on granulomatous lesions in pigs dosed with Mycobacterium intracellulare

The histology and ultrastructure of the granulomatous lesions were studied in 18 pigs dosed orally with Mycobacterium intracellulare serotype 8. The pigs were killed 2, 4, 6, 8 and 12 weeks after dosing. Histologically, initial ganulomatous lesions were seen in the tonsils 6 and 8 weeks after dosing. Initial or more advanced granulomatous lesions were observed in the tonsils and lymph nodes (mandibular, jejunal, ileocolic and superficial inguinal) 12 weeks after dosing. The initial granulomatous lesions appeared as slight proliferations of epithelioid cells and multinucleated giant cells. Advanced granulomatous lesions were nodular proliferations of epithelioid cells and multinucleated giant cells (epithelioid-cell nodule). In more advanced granulomatous lesions, the epithelioid-cell nodules increased in size and showed caseation necrosis, calcification and fibrosis. Ultrastructural examination revealed that the epithelioid-cell nodules consisted of mononuclear cells, epithelioid cells and multinucleated giant cells. In the caseous foci of the epithelioid-cell nodules, an electron-dense amorphous material with collagen fibres was deposited in the intercellular spaces. Although a small number of acid-fast organisms were observed histologically, they could not be detected ultrastructurally. The histological and ultrastructural findings of the present cases are compared with those of pigs infected with M. avium and with human lesions of tuberculosis and sarcoidosis.

Differentiation of raphide crystal idioblasts in isolated root cultures of Yucca torreyi (Agavaceae)

Excised primary roots of Yucca torreyi (L.) were cultured in various media, and development of cortical raphide crystal idioblasts was observed. Idioblasts were consistently produced, required 24 h to differentiate fully from initials in isolated root cultures, and developed normally relative to those cells in intact roots. In vacuoles of idioblast initials, we observed an electron-dense amorphous material associated with paracrystalline bodies and membranous crystal chambers. Crystallization occurs within and is apparently controlled by crystal chamber membranes. After crystal production, crystalloplastids differentiate lobed regions, and cables of tubules and mucilage form within the central vacuole. Many aspects of Yucca idioblast development are common to differentiation of similar cells in intact organs of other plants. Based on our results, we believe that isolated root cultures of Yucca will be useful for future physiological investigations of oxalate metabolism and control of idioblast cytodifferentiation.

Pigmented Conjunctival Cysts Following Tetracycline/Minocycline Therapy: Histochemical and Electron Microscopic Observations

Unpigmented and pigmented cystic epithelial inclusions were found bilaterally within the lower palpebral conjunctiva temporally of a 31-year-old man. He had a history of tetracycline/minocycline therapy for 14 years because of acne vulgaris. The cysts were studied by light and electron microscopy, histochemistry, and ultraviolet light. By light microscopy the unpigmented cysts contained faintly eosinophilic globular material that disclosed yellowish-green autofluorescence indicating the presence of tetracycline/minocycline. The pigmented cysts revealed laminated eosinophilic to brownish concretions that also showed yellowish-green autofluorescence. Autofluorescence, however, decreased with increasing brown pigmentation of the concretions. Histochemically, the pigment, which failed to show the staining characteristics of lipofuscin, melanin or iron, probably represented an oxidation product of tetracycline. By electron microscopy the cysts contained moderately electron dense amorphous material as well as degenerating epithelial cells. The epithelial cells lining the conjunctival cysts, did not contain membrane-bound, large, pigment granules within their cytoplasm as has been demonstrated in the colloid and follicular epithelium of the thyroid following minocycline therapy.

Morphogenesis of a cytomegalovirus from an American bison affected with malignant catarrhal fever.

A herpesvirus isolated from several organs of an American bison affected with malignant catarrhal fever was cultured in bovine foetal spleen cells and studied by electron microscopy. The fine structural features of the mature virion and the mode of virus morphogenesis were found to be similar to herpesviruses classified in the subgroup cytomegalovirus. The capsids were granular, hexagonal in shape and contained pleomorphic cores in thin sections. Envelopment of the capsids occurred primarily by budding on cytoplasmic membranes which appeared to be formed as extended vesicles of the Golgi apparatus; budding on nuclear membranes was only rarely observed. Cytoplasmic inclusions consisting of granular threads and amorphous electron-dense material were found in association with virions during the late stages of infection. The formation of cytoplasmic inclusions, the morphogenesis and ultrastructure of the virus are all consistent with classification of this virus as a cytomegalovirus.

A light and electron microscopical investigation of Ochromonas sphaerocystis Matvienko (Chrysophyceae): the statospore, vegetative cell and its peripheral vesicles

Abstract Vegetative cells of Ochromonas sphaerocystis Matvienko are examined by light and electron microscopy. The ultrastructure is generally typical for the Chrysophyceae although the nucleus has a sheet-like evagination which penetrates between the two chloroplast lobes and which is surrounded by a pyrenoid. There are numerous vesicles, approximately 1 μm diam. at the periphery of the cell. These stain with Sudan Black B, but not with methylene blue, brilliant cresyl blue or Sudans III and IV. An electron dense amorphous material appears adjacent to the membrane when fixed with osmium tetroxide. Statospore formation involves retraction of extracystic cytoplasm, including the chloroplast. The fully formed statospore has a thick neck with a recurved collar. Ochromonas sphaerocystis is compared with O. crenata Klebs and is concluded to be a separate species.

An ultrastructural study of recurrent disc herniation: a preliminary report.

One early and two late recurrent herniations located at the same interspace as the original prolapse were studied by light and electron microscopy. In the early recurrent herniation, most chondrocytes appeared to be actively engaged in protein synthesis, and the intercellular matrix contained densely packed collagen fibers, numerous matrix vesicles, and abundant electron-dense amorphous material. In late recurrent herniations, chondrocytes had scant endoplasmic reticulum and extensive cytoplasmic filaments, and the intercellular matrix showed intermediate features between fibrocartilage and fibrous connective tissue. On the basis of these morphologic characteristics, it seems reasonable to assume that the newly formed disc tissue arose mainly from remnants of the original herniation in the early recurrent prolapse and from the annulus fibrous and the granulation tissue invading the disc space after discectomy in the late recurrent herniations.

Membrane distribution in dividing endosperm cells of Haemanthus.

Membranes in cell-wall-free dividing endosperm cells of Haemanthus were examined after postfixation with osmium tetroxide-potassium ferrocyanide. We found that preservation and staining of membranes in metaphase cells was highly variable. Even adjacent cells often showed different degrees of preservation of membrane. However, this method does reveal a much more extensive membrane system in the mitotic spindle of Haemanthus than has been revealed previously using glutaraldehyde-osmium fixation. At prometaphase a system of membranes becomes associated with the kinetochore bundles. By metaphase, membranes constitute a prominent feature of kinetochore bundles, terminating near the kinetichores. Minipoles, identified by converging microtubules and associated membranes, are distributed in a zone extending laterally across the polar regions of the cell. The microtubules appear to terminate at the minipoles, whereas the membrane system becomes oriented generally perpendicular to the spindle axis and interfaces distally with a region of amorphous electron-dense material, helical polyribosomes, and cell organelles. The role of this extensive membrane system, if any, in chromosome movement is unknown. However, its distribution is coincident with the distribution of calcium-rich membranes and kinetochore fibers at metaphase in these cells (Wolniak, S. M., P. K. Hepler, and W. T. Jackson, 1981, Eur. J. Cell Biol., 25:171-174). Thus, these membranes may function in creating calcium domains that, in turn, may play a regulatory role in chromosome movement.

Evidence of remodeling in dura mater cardiac valves

Subcellular changes in 12 dura mater cardiac valves (in the mitral or aortic position) surgically removed after 23 to 108 months of implantation owing to calcification, rupture, or endocarditis show signs of a remodeling process. Significant morphologic changes in the connective tissue fiber matrices and cell populations were noted in the recovered valvular leaflets. Macrophages were found within electronlucent (cleared-out) areas, and they seemed to play an essential role in the remodeling process by ingesting and digesting selected connective tissue components. Fibroblasts found within these "rebuilding" areas in the dura mater tissue possessed small cytoplasmic vesicles (65 nm in diameter) being extruded from the cell. Evidence of early collagen formation was also found in association with both peripheral filaments and peripheral condensations, as well as within the connective tissue matrices surrounding the cellular elements, where electron dense amorphous material was observed. In conclusion, the long-term durability of dura mater bioprosthetic cardiac valves may be directly related to (1) glycerin stabilization and preservation of the collagen fibers, (2) the viability of the fibroblasts and macrophages within the implanted valves, and (3) the unique morphology and fine structure of the double-layered dura mater encephali. We hypothesize that the fibroblasts or myofibroblast-like cells found within the implanted leaflets, no matter what their origin, are capable of giving form and organization to the early developing connective tissue.

Epidermolysis bullosa acquisita and inflammatory bowel disease

Immunologic studies on lesional and nonlesional "normal" -appearing skin of a 32-year-old man with epidermolysis bullosa acquisita (EBA) revealed the following: (1) Noninflammatory subepidermal bullae formation in the periodic acid-Schiff (PAS)-positive basement membrane; (2) linear deposits of polyclonal IgG, Clq, C4, C3, properdin, and factor B at the dermoepidermal junction; IgA and IgM were not found; (3) broad subbasal lamina electron-dense amorphous material (EDAM) lacking amyloid features by electron microscopy; EDAM was not associated with anchoring fibers; (4) cleavage plane below the EDAM. Gastrointestinal endoscopy showed inflammatory erosions in the duodenum. Microscopy of the involved duodenum showed a mixed inflammatory infiltrate, subepithelial edema, microvesiculation, and granular deposits of IgG and C3, but no EDAM. However, EDAM and immune reactants were found in the esophagus. EBA is a distinct entity characterized by the presence and location of EDAM ultrastructurally, and it may represent a gastrointestinal-cutaneous syndrome with an immune pathogenesis.

CRYSTALLINE INCULSION IN MYXOID LIPOSARCOMA

An electron-microscopic study of a myxoid liposarcoma is presented in which amorphous electron-dense material and crystalline inclusions in the granular endoplasmic reticulum were observed. These inclusions showed transversal striations and were associated with round electrodense bodies. The previous finding of similar structures in normal cells from vertebrates and plants without relationship to adipose tissue discards the direct association of these inclusions with this particular tumor and suggests a possible functional origin.

Morphological diagnosis of Niemann-Pick disease type C by skin and conjunctival biopsies.

Skin and conjunctival biopsies were performed in six children with mental deterioration, incoordination, involuntary movements and discrete visceromegaly. Two main types of inclusions were observed: 1) intracytoplasmic bodies containing membranous profiles loosely disposed in concentric or parallel arrays, lying in clear cells of eccrine sweat glands, hair follicles and cells of the basal layer of skin and 2) pleomorphous membrane bound bodies with a multivesicular aspect which are formed by vacuoles, stacks of lamellae and amorphous electron dense material. Subsequent biochemical studies showing a slight increase of sphingomyelin in liver and normal sphingomyelinase in leukocytes supported the morphological diagnosis of Niemann-Pick disease type C. The qualitative regional diversity of storage is stressed and the interest of both skin and conjunctival biopsies in the diagnosis of this affection is discussed.

Further studies of the central nervous system in canine giant axonal neuropathy.

The CNS of three further cases of canine giant axonal neuropathy (GAN) were examined. The axonal swellings were present in the distal portions of the spinal long tracts and their terminations in the cerebellar vermis; in the distal optic pathways; the nuclei of the habenulo-interpeduncular tract; certain thalamic relay nuclei and the cerebral cortex. The swellings were present both paranodally and internodally with the myelin sheath being attenuated or even absent. Excessive numbers of disordered 10 nm neurofilaments were the main constituent although mitochondria, membranous bodies, glycogen bodies and amorphous electron dense material also accumulated, particularly in the fasciculus gracilis. The neurotubules tended to form small subaxolemmal or intra-axonal islands. Complex interdigitations of oligodendroglia and axolemma were found in the affected areas. A small number of fibres in the rostral fasciculus gracilis showed marked proliferation of the smooth endoplasmic reticulum which may represent abortive attempts at regeneration. A small proportion of astrocytic processes were markedly enlarged with excessive whorling of the glial filaments. The accumulation of these various organelles in the non-terminal axon in the absence of mechanical obstruction suggests a defect in axoplasmic transport which may result from an energy failure as suggested in toxic neuropathies. The significance and pathogenesis of the glial filamentous changes and those reported in other cells in human GAN is at present uncertain.

Zostera capensis Setchell II. Fine structure of the cavities in the wall of leaf blade epidermal cells

Summary The ultrastructural morphology of cavities in the outer walls of leaf blade epidermal cells of the marine angiosperm Zostera capensis is described. The cavities usually occur beneath the cuticle and are generally spherical to ovoid in shape. They are not bounded by a membrane although their margins are occasionally more electron-dense than the wall that surrounds them. In young epidermal cells the subcuticular cavities penetrate partially into the overlying cuticle. In more mature cells, flask-shaped extensions of the cavities penetrate through the cuticle. The actual opening through the cuticle appears to be slit-like. It is extremely small and is filled with an electron-dense material. While the cavities in the youngest epidermal cells examined generally lack contents, those of more mature cells contain mainly fibrillar material. In addition to fibrillar material, electron-dense amorphous or crystalline-like material maybe present within the cavities. The occurrence of the latter types of material on the cuticle surface, as well as within the wall below the cavities, suggests that these materials are probably excreted from the epidermal cells via the cavities. Structural similarities between the subcuticular cavities and the subcuticular spaces in the outermost secretory cells of salt glands on the leaves of certain halophytes are pointed out, and some suggestions are offered as to the nature of the material apparently being excreted from the cavities.

Primary epithelial cell culture of adult rat kidney, enhancement of cell growth by ammonium acetate

Abstract Primary monolayer cultures of epithelial-like cells from adult Lewis rat kidney demonstrated enhanced growth and survival over a 3-week period when cultured in medium supplemented with 10 mM ammonium acetate. In addition to enhancing the growth and survival of epithelial-like cells, 10 mM ammonium acetate also suppressed the growth of a subpopulation of cells having a fibroblast-like morphology. Both epithelial-like and fibroblast-like cells reacted positively in histochemical reactions for gamma glutamyl transpeptidase (GGT) and 5′-nucleotidase, enzymes found in high amounts in kidney tubular epithelium. Despite the enhanced survival of cells in the presence of ammonium acetate, most cells in primary culture eventually died out. Electron microscopic examination of dying epithelial-like cells revealed the presence of numerous large lysosomes filled with amorphous electron-dense material. These findings substantiate the prediction of Seglen (Exp cell res 107 (1977) 207) that ammonia might cause increased growth of primary cells in culture, presumably through its nitrogen-preserving action.

Fibrogenesis and Biosynthesis of Elastin in Cartilage

This study presents direct evidence that dissociated chondroblasts from rabbit ear cartilage grown in vitro are capable of synthesizing insoluble elastin. Ultrastructural examination indicated that at an early stage of tissue development, elastogenesis is initiated producing a form of primary fibrils which later condense into an electron dense amorphous material which, unlike other elastin-containing tissues, is heavily stained by metal cations and lacks peripheral microfibrils. Native elastic fibrils and the mature elastic fiber bundles are both susceptible to elastase digestion. Transmission electronmicroscopy demonstrated the presence of many intracellular filaments all showing a substructural organization and localized in close proximity to the nucleus. Their possible contractile nature is discussed. Amino acid analysis of cartilage elastin and of the elastin synthesized in vitro revealed a close chemical similarity between the two molecules. Ultrastructural analysis of the in vitro elastin demonstrated a substructural organization quite similar to that of the elastin observed in an in vivo system.