Dense Inclusions Research Articles

Nuclear alterations induced in chick embryo fibroblasts by frog virus 3 (FV3) infection at permissive and non-permissive temperatures

In chick embryo fibroblasts, FV3 infection induced early alterations of the shape of the nucleus and clumping of chromatin; later in infection, the nucleoli displayed a segregation between their fibrillar and granular components and a dilatation of their fibrillar centres; some dense spherical inclusions, microtubules and exceptionally microfilaments appeared in the nucleoplasm. Chromatin condensation, nucleolar segregation and dilatation of fibrillar centres could be explained by the inhibition of host cell DNA and RNA synthesis by FV3 infection. Such a correlation is more difficult to establish concerning the presence of dense spherules and microtubules in the nucleus. These nuclear alterations were observed both in permissive infections at 29°C and in non-permissive infections at 37°C, where FV3 exerts mainly a cytotoxic effect. Thus these nuclear impairments seem to be a consequence of FV3 toxicity and not of viral multiplication.

Mucopolysaccharidoses: ultrastructure of leukocyte inclusions.

Ultrastructural study of leukocytes from 7 patients with mucopolysaccharidosis (MPS) I, II, or III demonstrated a variety of membrane-bound inclusions in 34 to 68% of lymphocytes and larger mononuclear cells. The most common inclusions were membrane-bound vacuoles containing faintly osmiophilic material, but a variety of clear vacuoles, dense granular inclusions and myelin figures were also seen. Fingerprint profiles were present in small percentages in MPS II and III, indicating that they are not as specific as previously considered. Granulocytes, lymphocytes, and larger mononuclear cells from a patient with MPS VII showed many small and large membrane-bound vacuolated inclusions, some of which contained large dense granules. The presence of alterations in a readily accessible tissue such as blood indicates that ultrastructural study of leukocytes may be a useful adjunct in guiding further biochemical studies in MPS, especially in early or obscure cases.

The histochemistry and infrastructure of the coelomocytes of species of Lumbricillus, and observations on certain other enchyfraeid genera (Oligochaeta: Annelida)

The main coelomocyte type–the granular mucocyte–of Lumbricillus mirabilis, L. reynold‐soni and L. rivalis has inclusions consisting of a mucopolysaccharide‐protein complex with very low lipid levels. Both neutral and carboxylated acid mucopolysaccharides occur, the latter predominating in the granules of L. rivalis in which protein levels are lower than in the other two species.The Golgi‐derived granules of the mucocytes of L. mirabilis and L. reynoldsoni are electron dense when mature and are separated from each other by cytoplasm housing, generally, a single endoplasmic reticulum profile. The mucocytes arise from the peritoneum. In L. rivalis the inclusions are of finely fibrillar material typical of acid mucopolysaccharides. Studies on species of other genera show the species of littoral origin to possess mucocytes with an ultrastructure similar to that of the littoral L. mirabilis and L. reynoldsoni; those of non‐littoral origin are similar to the non‐littoral L. rivalis. The differences therefore appear to be eco‐physiological rather than taxonomic.Amoebocytic coelomocytes occur and two types are described for L. mirabilis and L. reynoldsoni. Type I is finely pseudopodous and contains numerous small, electron dense inclusions. The origin of Type I from the peritoneum is demonstrated. Type II is characterized by fewer pseudopodia and large, electron dense inclusions similar to chloragosomes. Pinocytotic vesicles occur at the cell periphery. No intermediates between Type I and II were observed, and Type II was not seen in L. rivalis. Both types occur in Enchytraeus albidus, but only Type I in Fridericia bulbosa and no amoebocytes were observed in Mesen‐chytraeus sp.

Electron microscopy of the tumulus and origin of associated structures within the tegument of Eubothrium salvelini Schrank, 1790 (Cestoidea: Pseudophyllidea)

Tedesco J. L. and Coggins J. R. 1979. Electron microscopy of the tumulus and origin of associated structures within the tegument of Eubothrium salvelini Schrank, 1790 (Cestoidea: Pseudophyllidea). International Journal for Parasitology 10: 275–280. Validity of the tumulus, a new organ recently described on the tegument of Eubothrium salvelini, is confirmed. Spherical, dense inclusions approximately 0.23–0.29 μm were associated with the tumulus and with subtegumental cell bodies. Origin of these inclusions within subtegumental cell bodies and their transport via ducts to the tumulus is described. Inclusions are synthesized within granular endoplasmic reticulum and packaged by Golgi apparatus prior to transport. Inclusions were observed only in association with the tumulus within the tegumental distal cytoplasm.

OBSERVATIONS ON PLASTID FINE‐STRUCTURE IN THE HOLOPARASITIC ANGIOSPERM EPIFAGUS VIRGINIANA

The ultrastructure of plastids in cortex and phloem parenchyma cells of Epifagus virginiana (L.) Bart. is described. Based upon morphology and content, several distinct plastid types appear to exist. “Tubular” complexes, lipid globules and electron dense inclusions in different arrangements appear to account for the degree of plastid variability. When results obtained with Epifagus are compared with those obtained by others for a closely related genus, a striking parallel is shown to exist.

Rapid degeneration of developing rat Pacinian corpuscles after denervation

The effect of denervation upon the development of Pacinian corpuscles was investigated in the crural interosseous membrane of the rat during the early postnatal period. When the sciatic nerve was transected in 1-day-old rats, further development of immature Pacinian corpuscles,was arrested and their structure rapidly disintegrated. In the main cluster of corpuscles, Pacinian axon terminals degenerated within 12 h after the operation and were phagocytised by the inner core cells. Subsequently large dense inclusion bodies and vacuoles with dense debris appeared in the innercore and capsule, and cell autolysis and pyknosis ensued from the 2nd day onwards. The debris of degenerated cells was removed by macrophages and the disintegration of individual corpuscles was completed 2–5 days after nerve section. Normal interosseous membranes of 2–6-day-old rats contained54.9 ± 1.2 (±S.E.) Pacinian corpscles, as revealed by staining for cholinesterase. The total number of corpuscles decreased to about 70% of the control values during the first 2 days after denervation, dropped to 38% on day 3, decreased further to 15% on day 4 and reached zero values on day 5 after nerve section. The experiments demonstrate that the postnatal development and growth of the non-nervous components of Pacinian corpuscles is completely dependent upon the neuronal induction exerted by sensory axon terminals.

Ultrastructural aspects of a serious disease of hatchery reared larval oysters, Crassostrea gigas Thtinberg

Abstract. Ultrastructural observations on a possible aetiological agent and tissue changes in a previously described disease of hatchery reared larval oysters Crassostrea gigas Thünberg are reported. Spherical bodies, implicated in gastro‐intestinal impaction, show a strong resemblance to the marine fungus, Hyalochlorella marina. Similarities include a thick outer wall structure, plate‐like mitochondria and dense cytoplasm with small vacuoles.The dense fascicular masses, previously observed histologically at epithelial surfaces, consist of accumulations of irregular electron opaque bodies. Large accumulations of these bodies appear to exacerbate tissue damage and prevent normal velar function. In comparison with the reported virus‐like particles in this disease, these dense inclusions and associated lesions are more frequently observed and thus seem to be more important in the disease process.Notes on normal epithelial surfaces of larval Crassostrea spp. are included.

Laser Microprobe Analysis of Inclusions in Dunaliella salina

Summary Laser microprobe mass analysis (LAMMA) is described as a method for element analysis of inclusions from thin-sectioned plant material (Dunaliella salina). This method is compared with results of element analysis by dispersive X-ray microanalysis (EDAX). The presence of high amounts of silicon, phosphorus, sulphur and chloride is documented in electron dense inclusions of the halotolerant alga by both methods.

Effects of doxorubicin and its aglycone metabolite on calcium sequestration by rabbit heart, liver, and kidney mitochondria

Previous investigators have shown that following doxorubicin treatment heart mitochondria appear swollen and contain intramitochondrial dense inclusion bodies identified as calcium phosphate. In vitro studies have shown that similar morphological changes occur in mitochondria previously loaded with excess calcium. The present studies were performed to determine the effects of doxorubicin and its aglycone metabolite on 45Ca 2+ uptake by mitochondria isolated from the heart, liver, and kidney of the rabbit. Doxorubicin (100 μM) significantly inhibited the initial rate of 45Ca 2+ accumulated by mitochondria isolated from the three tissues. In contrast, the aglycone metabolite (100 μM) induced the reverse effect. In preloaded mitochondria the aglycone stimulated the release of calcium while doxorubicin was without effect. Mitochondria from the heart were significantly more sensitive to the effects of these anthracyclines than were mitochondria from the other two tissues. If these in vitro effects also occur in vitro, then the aglycone metabolite would be a more likely candidate in explaining the morphological changes in heart mitochondria previously described.

Welwitschia mirabilis: Fine Structure of the Germinating Seed IV. The Imbibed Gametophyte

Summary During the first two days after imbibition, in the cells of the gametophyte of Welwitschia, the volume of the nucleolus increases and membranes become clearly defined. Microbodies increase strikingly up to day 3, but decline in number after day 4 as lipid reserves are consumed. Microbodies, often containing spherical inclusions within an amorphous electron dense region near the enclosing membrane, and lipid bodies are sequestered into protein bodies. Digestion of protein body reserves begins from within the protein-carbohydrate deposits, giving the body a fenestrated appearance. Small, spherical, electron dense crystalline inclusions, presumably globoids, are found not only in the protein-carbohydrate deposits of the protein body, but also in contact with the outer protein body membrane as well as in the cytoplasm. This suggests a cytoplasmic origin for the globoid. Mitochondria, ER and ribosomes are numerous by day 4; amyloplasts, dictyosomes and myelin figures appear by day 5. By day 6 protein bodies fuse to form aqueous vacuoles, lipid reserves are depleted and amyloplasts are numerous. Although nuclear inclusions disappear upon imbibition nucleoli tend to remain in a semi-quiescent state; this, coupled with the pattern of protein body degradation, suggests an embryo-controlled mobilization of food reserves.

The corpus luteum of the guinea pig. IV. Fine structure of macrophages during pregnancy and postpartum luteolysis, and the phagocytosis of luteal cells

Little information is available on the ultrastructure of macrophages in the corpus luteum or their importance in the regression of luteal tissue. In the present study, the fine structure of activated luteal macrophages during pregnancy and the postpartum period was examined by electron microscopy of guinea pig ovaries fixed by vascular perfusion. In these corpora lutea, macrophages can readily be distinguished from luteal cells. Activated macrophages typically display three prominent inclusions in their cytoplasm: (1) heterophagic vacuoles, (2) distinctive large dense inclusions, and (3) large and small electron-lucent vacuoles. In addition, they contain numerous smaller lysosome-like dense bodies. Activated macrophages in corpora lutea also characteristically show many surface protrusions, such as processes, folds or pseudopodia, which often occur in close contact with nearby luteal cells. Generally, nuclei of macrophages are irregular in shape and display a dense border of heterochromatin, thus differing from those of luteal cells. Macrophages seem to be most abundant in regressing corpora lutea, where they commonly display heterophagic vacuoles containing recognizable luteal cell fragments, evidence that these phagocytes ingest senescent luteal cells. The digestion of luteal cell components in heterophagic vacuoles presumably gives rise to the distinctive large dense inclusions typically seen in macrophages. The findings of this study indicate that macrophages play a central role in luteolysis by phagocytizing luteal cells or their remnants. They therefore appear to bring about the reduction in volume of the corpus luteum that occurs as this tissue regresses. These results taken together with those previously published (Paavola, '78) further indicate that breakdown of the corpus luteum during postpartum luteolysis in guinea pigs involves both autophagy and heterophagy.

Cellular Development of the American Lobster Heart

The heart of the American lobster, Homarus americanus , represents a relatively simple nerve-muscle system. A nine-celled cardiac ganglion functions to initiate the contractions of the transversely striated myocardium. To describe the development of this nerve-muscle system, hearts of lobster embryos in different stages of development have been examined with an electron microscope. The heart wall consists of an outer adventitial layer of fibroblast cells and an inner layer of transversely striated myocardial cells. Adjacent fibroblast cells form fasciae adherent and gap junctions. Intercalated discs and membrane regions of close apposition (4 nm) occur between adjacent myocardial cells. A cardiac ganglion that contains nine neurons and synaptic neuropil is present. Neuromuscular synapses occur in the myocardium. Cells that we have termed storage cells contain dense cytoplasmic inclusions that resemble yolk material and form gap junctions with myocardial cells. The heart at six weeks is about 200 μm long and 160 μm wide; at six months it is 300 and 250 /m respectively. The myocardium grows by an increase in the size and in the number of myocardial cells. The myocardium contains pre-myoblast, myoblast and well-differentiated myocardial cells. Sarcomere formation occurs first adjacent to the inner surface of the sarcolemma where the myofilaments align and anchor. Distinct A and I bands appear first, followed by the Z lines. The myocardium is sparsely innervated when the initial heartbeats occur; a preliminary quantitative analysis has shown that three days later, the density of innervation has increased six-fold.

Synaptic fine structure and the termination of corticospinal fibers in the lateral basal region of the cat spinal cord

The lateral basal region (LBR) of the spinal cord gray matter (Rexed's laminae IV-VII) by physiologic and anatomic criteria is the major terminal zone for the corticospinal (CS) tract in the cat. The neurons in this area are medium-sized with abundant spines on their dendrites. Axon terminals on the dendrites and somata of these neurons form synapses easily classified as asymmetric with spheroid vesicles and symmetric with flattened vesicles. There are rare exceptions to this. In a systematic count of terminals, 82% have spheroid and 18% flattened vesicles. The majority of all terminals are on dendrites (84.9%) and a minority on somata (14.1%). Less than 1% are axoaxonic. Degeneration of the corticospinal tract was produced by transecting one hemisphere of our experimental cats. Its termination in the lower cervical cord was studied for 17 hours to 7 days after surgery. Vesicle depletion and clumping and dense polymembranous inclusions were the most common forms of degeneration. Filamentous proliferation in the terminals was also prominent; dark degeneration, however, was infrequent. The percent of degenerating CS terminals in the LBR was the following: 17 hours - 2.2%, 36 hours - 4.19%, 2 days - 10.3%, 3 days - 8.4%, 4 days - 6.9%, 7 days - 10.25%; 84.8% of degenerating CS terminals were axodendritic and 15.2% axosomatic.

Ultrastructural Concomitants of Sodium Butyrate-Enhanced Ectopic Production of Chorionic Gonadotropin and Its Alpha Subunit in Human Bronchogenic Carcinoma (ChaGo) Cells

Sodium butyrate treatment of cultures of ChaGo (human lung cancer) cells resulted in increased production of human chorionic gonadotropin (hCG) and its alpha subunit (hCG-alpha) and induced a variety of morphologic changes. Elongation and flattening of cells were seen by light microscopy. Immunocytochemistry with antisera against hCG and against hCG-alpha showed an increase in cells containing stainable hCG-alpha. Scanning electron microscopy demonstrated enhanced adhesion of cells to glass cover slips, with elongation, flattening, and decreased cytoplasmic blebs. Ultrastructural changes were examined by transmission electron microscopy and evaluated quantitatively by an unbiased observer. Significant findings included increases in perinuclear tonofilaments, smooth endoplasmic reticulum vesicles, dense mitochondrial inclusions, and lipid granules, as well as decreases in intercellular desmosomes, free polyribosomes, mitochondrial dense granules, and Golgi complexes. The most notable change, a marked decrease in condensed chromatin clumps, may have reflected a butyrate-induced biochemical modification of chromatin leading to enhanced accessibility of certain genes for transcription.

Host-dependent restriction of influenza virus maturation

Host-dependent restriction of influenza virus replication in nonpermissive HeLa cells was studied under single cycle conditions using as inoculum bovine kidney (MDBK)-grown virus which was relatively free of defective interfering particles. Biochemical and ultrastructural changes in infected HeLa cells were compared to MDBK cells, which are permissive for influenza virus replication [ Choppin and Pons (1970), Virology, 42, 603–610]. Two subclasses of virus-specific nucleoproteins separated in renografin density gradients were present in the nucleus and cytoplasm of both MDBK and HeLa cells; however, the assembly of the 1.26 g/ml virus-specific RNPs occurred at a faster rate in HeLa cells than in MDBK cells. The overall patterns of synthesis of polypeptides in HeLa cells were similar to those seen in MDBK cells, and the polypeptide compositions of virus released from HeLa and MDBK cells were also similar. Electron microscopy showed that elongated virus particles accumulated in tightly packed arrays on the plasma membrane of HeLa cells; however, only a small number of spherical virions were observed budding from the plasma membrane of MDBK cells. Large, intracytoplasmic vesicles filled with budding virus particles were numerous in HeLa cells but were not observed in MDBK cells. Many finely striated inclusions and dense granular inclusions were observed in the cytoplasm of HeLa cells within 24 hr after infection. In contrast, MDBK cells showed much less cytopathic effect at 24 hr after infection, and virus-induced inclusions occurred much less frequently than in HeLa cells. These results suggest that. in HeLa cells the final stages of maturation of virus particles at the plasma membrane may be,blocked, allowing the accumulation of viral products within the cytoplasm and budding virus particles on. the surface of nonpermissive cells. Thus, in the absence of defective interfering particles in the inoculum, the host-dependent restriction in influenza virus replication in HeLa cells does not occur early in the virus growth cycle, but appears to be due to a defect in a late event involving the participation of the plasma membrane. in virus maturation.

Ultrastructural observations on the erythrocytes and thrombocytes of the tuatara, Sphenodon punctatus (gray)

The erythrocytes of Sphenodon punctatus (Gray) are nucleated, ellipsoidal and flattened, and contain 55–65 microtubules in their marginal band. The thrombocytes are also flattened, ellipsoidal, nucleated cells and in electron-microscopic preparations occurred in aggregrates. The thrombocytes appeared to be ‘activated’ and possessed many pseudopodia which were devoid of organelles. The latter were concentrated in the perinuclear region and were encircled by a ring of microtubules. The organelles included ribosomes, mitochondria, membrane-bound dense material and numerous actin-like microfilaments. Cytoplasmic vacuoles contained a moderately dense, filamentous material and/or spheroidal electron-dense inclusions. β-glycogen particles were scattered in the general cytoplasm and were most concentrated in the pseudopodia. The erythrocytes and thrombocytes of S. punctatus are compared with those in other vertebrates.

Intracellular calcium deposition in brain following electrical stimulation.

The effects of electrical stimulation of the cat cerebral cortex have been evaluated by light and electron microscopy following a wide variety of stimulation parameters (QD/ph of 10 - 300 muC/cm2/ph). Platinum or rhodium disc electrode arrays were bilaterally implanted subdurally on the parietal cortex and subjected to 36-hour stimulations (9 hrs./day for 4 days). Prominent among the degenerative changes shown by electron microscopy were dense crystalline inclusions that were identified as calcium hydroxyapatite (CHA) crystals by electron diffraction and energy dispersive X-ray analysis. The appearance of intracellular calcification generally paralleled the onset of other degenerative changes in stimulated tissue, including gliosis, mitochondrial swelling, lipid inclusions, degenerating cells, neuronal loss, and phagocytic activity. A preferential deposition of calcium was noted in mitochondria of several cell types and in postsynaptic dendrites. The mechanism of the apparently electroresponsive calcium deposition is obscure; however, a plausible explanation is that increased cyclic AMP levels, known to occur with electrical stimulation of nervous tissue, result in enhanced calcium plasmalemmal permeability.

Correlations between epidermal cell structure and endogenous hormone titers during the fifth larval instar of the tobacco hornworm, Manduca sexta

Epidermal cell morphology and cuticle production in Manduca sexta are directly influenced by both ecdysterone and juvenile hormone. Up to day 6 of the last larval instar, post-molt endocuticle is continuously deposited even though cells undergo a partial and temporary separation from the overlying cuticle at the time when a small ecdysteroid peak is detected (~ day 3.5). At about days 6–7 when another, larger ecdysteroid peak is present, apolysis occurs accompanied by the appearance of ecdysial droplets. Following apolysis, layers of pupal cuticle are deposited. Increased quantities of rough endoplasmic reticulum characterize the epidermis at times of peak endocuticle deposition (day 3, larval cuticle; day 9, pupal cuticle). Dense pigment inclusions are found in epidermis from the day of ecdysis to the last larval instar until they are eliminated 5 days later. These dense bodies migrate from cell apex to base in the absence of juvenile hormone (or in the presence of a negligible amount of juvenile hormone) and probably contain insecticyanin.

Electron microscopy of malachite green--glutaraldehyde fixed bacteria.

Malachite green combined with glutaraldehyde has been used recently as a fixative for preserving and revaling lipid complexes in thin sections of eukaryotic cells examined by electron microscopy. When bacteria were prefixed with the above mixture granular electron dense inclusions were revealed in all cultures tested. These inclusions were replaced by electron transparent areas in cells fixed with glutaraldehyde alone. The structures were frequently located near to or within the nucleoid and adjacent to the cell membrane in Gram-negative bacteria and were associated with the nucleoid and mesosomes in Gram-positive species. Polyhydroxybutyrate granules, generally poorly preserved in thin sections of Aquaspirillum serpens, were well preserved by the malachite green-glutaraldehyde fixative. Malachite green complexes were observed outside of the cells in all preparations. Capsules were neither preserved nor stained.

A novel method for the isolation and study of a magnetotactic bacterium

The magnetococcus, a magnetotactic bacterium, has been grown in a complex simulated natural environment. Sufficiently pure samples of cells were obtained magnetically making axenic cultures unnecessary for many purposes. The magnetococcus is a Gram-negative coccus, 1.6 micron in diameter and readily distinguished by highly refractile inclusions and its magnetotactic behavior. This organism is actively motile by means of two bundles of flagella. Electron dense ferromagnetic inclusions were localized between the flagellar bundles. Collections of magnetococci were morphologically homogeneous and negligibly contaminated by extraneous bacteria. DNA extracted from pooled collections of cells was homogeneous by analytical CsC1 centrifugation. The guanine-cytosine content was 61.7%. Total iron by percent cellular dry weight was 3.8%. Comparisons with a previously described magnetotactic marine coccus were made.