Dense Inclusions Research Articles

Sporogony, development and ultrastructure of extrinsic stages of Plasmodium mexicanum

Klein T. A., Akin D. C., Young D. G., Telford S. R., Jr. and Butler J. F. 1988. Sporogony, development and ultrastructure of extrinsic stages of Plasmodium mexicanum. International Journal for Parasitology 18: 463–476. The sporogonic stages of Plasmodium mexicanum in the midgut and salivary glands of Lutwmyia vexalor were studied by light and electron microscopy. The sporogonic cycle of P. mexicanum maintained at 27 °C was completed by 6.5 days after an infective bloodmeal. Sporozoites were relatively short (4–6 μm) when compared to most other Plasmodium sporozoites. The nucleus, with a distinct nucleolus, was relatively large in the early non-vacuolated oocyst. Oocyst differentiation was initiated by the formation of subcapsular spaces and internal vacuolization which eventually coalesced and formed cytoplasmic clefts (sporoblastoids). Sporozoite formation was initiated early in the differentiated oocyst by the formation of a pair of convex electron dense membranes. A moderately electron dense inclusion body. a nucleus, and a single spherical mitochondrian were observed migrating into the evaginating sporozoite. Rhoptries and micronemes appeared to arise de novo from the moderately dense inclusion bodies which degenerated and were not observed in mature Sporozoites. After their release from oocysts, Sporozoites appeared in the salivary glands of L. vexator. Sporozoites were constricted at the parasite-host cell junction during entry into the salivary gland and were enclosed within a parasitophorous vacuole formed by the host cell membrane after entering the salivary gland cell. Sporozoites became extracellular as they continued to migrate into the lumen of the salivary gland, in similar manner to that seen when entering the salivary gland cell. The arrangement of the subpellicular microtubules (asymmetrically, with 2 3 of the microtubules occupying 1 2 of the sporozoite circumference, and 1 3 in the remaining half) was different from that observed in mammalian malarias, where all but one microtubule lie in two-thirds of the sporozoite circumference, but was similar to Plasmodium agamae, a saurian malaria. Except for the arrangement of the subpellicular microtubules, the structure and arrangement of organelles was similar to that observed for other Plasmodium species.

High-level expression of a functional human fibrinogen gamma chain in Escherichia coli

Human fibrinogen γ chain has been expressed intact at high levels in Escherichia coli The construction of the expression plasmid, p253, is described. Synthesis of the recombinant protein is isopropyl-β- d-thiogalactopyranoside-dependent and is driven by the tac promoter. Western analysis of E. coli lysates demonstrates a novel protein of approx. 45 kDa which cross-reacts with antisera to human fibrinogen γ chains. The protein is not soluble in common E. coli lysis buffers and becomes soluble in 6 M guanidine · HC1 or 6 M urea. Initial insolubility is due to interchain disulfide bond formation and to noncovalent interactions. Induced cells examined by phase-contrast microscopy contain dense inclusion bodies. A known function of the γ chains of human fibrinogen is the clumping of Staphylococcus aureus Newman D 2C cells [Hawiger et al., Biochemistry (1982) 1407–1413]. We demonstrate that suspensions of recombinant γ chains retain the ability to clump cells from this strain of S. aureus.

Aldose reductase and retinal capillary basement membrane thickening

Weanling male Sprague-Dawley rats were given a 50% galactose diet with or without an aldose reductase inhibitor (0.04% tolrestat, Ayerst) for 88 weeks. Quantitative computer planimetry on electron micrographs of perpendicularly transected retinal capillaries showed mean basement membrane thicknesses to be 263 +/- 50 nm in galactose-fed rats, 153 +/- 10 nm in galactose-fed rats given tolrestat, and 114 +/- 16 nm in rats given control feed. The retinal capillary basement membrane thickening in galactose-fed rats was accompanied by other ultrastructural alterations mimicking changes observed in diabetic microangiopathy, such as multi-lamination and the formation of vacuoles and dense inclusions. The results extended previously reported (44-week) findings, showed greater galactose-induced changes in capillary basement membranes, and demonstrated the preventative effect of an aldose reductase inhibitor.

Intranuclear disease of uncertain etiology in larvae of the blackfly, Simulium vittatum

An intranuclear disease of uncertain etiology has been found in larvae of the blackfly, Simulium vittatum (Diptera: Simuliidae). The disease, which causes little mortality, is characterized by a lack of pigmentation in the cuticle and the occurrence of melanotic deposits in the cytoplasm of fat body and midgut epithelial cells. A single, dense lacunate inclusion develops within the nuclei of these cells, ranging from 20 to 30 μm in midgut epithelial cells, and averaging 18 μm in fat body cells. The disease has been reproduced in the laboratory by feeding first-instar blackfly larvae on homogenates of diseased larvae. However, attempts to isolate a causative agent have not been successful. The cytopathology of the disease resembles the cytopathology caused by densonucleosis viruses, but particles, either free or in aggregations, typical of the virions of this virus type have not been identified in infected nuclei.

Characterization of Potyvirus Isolates from West African Yams (Dioscorea spp.)

AbstractIn comparative studies on potyviruses from West African yams (Dioscorea spp.) the following isolates were used: Dioscorea greenbanding mosaic virus (DGMV) and a Nigerian yam virus (YV‐N), both isolated from Dioscorea rotundata, and a beet mosaic virus isolate from D. alata (BtMV‐Y) formerly designated Dioscorea alata ring mottle virus. Naturally infected D. alata containing very few particles of BtMV‐Y, contained primarily particles of a second potyvirus (Dioscorea alata virus, DaV) which could not be transmitted but which was included in these studies wherever possible.The normal lengths of DGMV, YV‐N, DaV, and BtMV‐Y were 754, 772, 805, and 812 nm, respectively. All viruses induced cytplasmic inclusions of the pinwheel type and laminated aggregates. In addition, the nucleoli of BtMV‐Y infected cells contained characteristic electron dense inclusions. The buoyant density of purified DGMV and BtMV‐Y in CsCl was 1.336 g/cm3 and 1.321 g/cm3, respectively. The sedimention velocities (Srel) of DGMV, YV‐N, and BtMV‐Y were 156, 158, and 162 Srespectively. In SDS‐polyacrylamide gel electrophoresis the coat protein of purified DGMV and YV‐N all migrated as a single band with an apparent molecular weight of 36 kd. Coat protein of purified DaV showed up to 5 bands with molecular weights of 36 to, 32 kd. Polypeptides of purified BtMV‐Y had an estimated molecular weight of 35 kd but those from infected plant extracts had a molecular weight of 36 kd. DGMV, YV‐N, and BtMV‐Y particles contained a single nucleic acid with an apparent molecular weightof 3.2, 3.2, and 3.1 Md, respectively. Using λ‐DNA digested with Hind III as a marker, the molecular weight of DGMV and BtMV‐Y nucleic acid was calculated to be 3.6 Md ± 10%. The nucleic acid was determined to be single‐stranded RNA by enzymatic digestion and by staining with acridine orange.In serological studies using immunoelectron microscopy (IEM), electro‐blot immunoassay (EBIA), and enzyme‐linked immunosorbent assay (ELISA), DGMV and YV‐N were closely related. Strong serological reactions were also obtained in IEM and EBIA when DGMV and YV‐N were tested with antiserum to yam mosaic virus (YMV). Antisera against DGMV, YV‐N, and YMV also reacted strongly with DaV antigen. Serological reactions between these viruses and BtMV‐Y were usually not found or were weak. A very close serological relationship could be detected between BtMV‐Y and beet mosaic virus isolated from beet (BtMV); both isolates were also very similar in host range, symptomatology, and cytopathology.

Characterization of Osmotin

Cultured tobacco (Nicotiana tabacum var Wisconsin 38) cells adapted to grow under osmotic stress synthesize and accumulate a 26 kilodalton protein (osmotin) which can constitute as much as 12% of total cellular protein. In cells adapted to NaCl, osmotin occurs in two forms: an aqueous soluble form (osmotin-I) and a detergent soluble form (osmotin II) in the approximate ratio of 2:3. Osmotin-I has been purified to electrophoretic homogeneity, and osmotin-II has been purified to 90% electrophoretic homogeneity. The N-terminal amino acid sequences of osmotins I and II are identical through position 22. Osmotin-II appears to be much more resistant to proteolysis than osmotin-I. However, it cross-reacts with polyclonal antibodies raised in rabbits against osmotin-I. Osmotin strongly resembles the sweet protein thaumatin in its molecular weight, amino acid composition, N-terminal sequence, and the presence of a signal peptide on the precursor protein. Thaumatin does not cross-react with antiosmotin. An osmotin solution could not be detected as sweet at a concentration at least 100 times that of thaumatin which could be detected as sweet. Immunocytochemical detection of osmotin revealed that osmotin is concentrated in dense inclusion bodies within the vacuole. Although antiosmotin did not label organelles, cell walls, or membranes, osmotin appeared sparsely distributed in the cytoplasm.

Cytological studies of the susceptible reaction of sugarbeet roots to Heterodera schachtii

The cellular alterations induced in sugarbeet by Heterodera schachtii were studied at 3 and 12 days after nematode inoculation. Three days after inoculation the procambial cells involved in the nematode feeding were already transformed into a syncytium with ultrastructural features which indicated high metabolic activity. Feeding tubes produced by interaction of nematode saliva with the cell contents were observed; in their vicinity the cytoplasm was devoid of cell organelles except for smooth endoplasmic reticulum. Spiny structures occurred in the parenchyma syncytial cells and they appeared to be related to endoplasmic reticulum and dictyosomes; the relation could be functional and it is possible that the spiny component is involved in protein synthesis. Nuclei were enlarged and amoeboid in form and sometimes they migrated through a space in the cell wall. Conspicuous elements of rough endoplasmic reticulum, arranged in concentric layers and actively synthesizing, and plastids with dense inclusions indicating bulk protein synthesis, were detected in parenchyma cells, adjacent to syncytia. Twelve days after nematode inoculation the ground cytoplasm of the syncytia was densely packed, but without vacuoles and the membrane system resembled endoplasmic reticulum. At this time the modification of the cytoplasm and cell wall appeared to be involved in the rapid solute uptake which would be essential for nematode development.

Correlated histochemical and electron microscopic studies of the esophageal epithelium in the salamander, Hynobius nebulosus.

The esophageal epithelium of the adult salamander, Hynobius nebulosus, was studied by histochemistry, including periodic acid-Shiff (PAS), alcian blue both at pH 2.5 and pH 1.0, and alcian blue (pH 2.5)-PAS with or without neuraminidase-digestion, and scanning and transmission electron microscopy. The epithelium was columnar in type, comprising partly pseudostratified and partly two-layered architecture; it consisted mainly of ciliated cells, goblet cells, and basal cells. The ciliated cells consisted of two types, light and dark: both types, especially the latter, frequently contained numerous small mucous granules in their apical portion. Ciliated-mucous cells were also occasionally present. Immature ciliated cells were frequently found. Goblet cells were divided into two types: Type I cells possessed electron lucent mucous granules, which frequently contained dense specific inclusions, and frequently bulged into the lumen; Type II cells had moderately electron-dense mucous granules with no inclusions and a conical apex which did not exceed the level of the lumen. The Type I cells were closely distributed throughout the esophagus, while the Type II were mainly dispersed in the cranial portion, remarkably decreasing in the caudal portion. Correlated histochemical and electron microscopic observations suggested that, in the Type I cell, mucous granules contain acid mucosubstances, while in the Type II, they possess neutral mucosubstances, and that in the Type I cell mucous granules consist of sialic acid-containing glycoproteins and their swollen portions are more highly sulfated than the non-swollen ones.

Giant mitochondria in the myocardium of aging and endurance-trained mice.

During the course of an ultrastructural study on the apex region of the left ventricular myocardium of experimental female C57BL/6J mice, aged between 6 and 27 months, and that had been subjected to long-term daily enforced endurance running schedules, we encountered some instances of unusual giant mitochondria. These mitochondria showed degenerative changes including the disruption, disorganization and loss of cristae and the development of a very electron-lucent matrix. In many instances, these giant mitochondria showed dense inclusions within the matrix. The giant mitochondria appear to have developed as a result of fusion between adjacent hypertrophic mitochondria followed by a sequence of progressive degenerative changes. These giant mitochondria were most common in the myocardium of mice that had followed endurance training schedules for 10 or 15 months and were not encountered in age-matched nonrunning control mice.

Morphometric analysis and fine structure of the vestibular epithelium of aged C57BL/6NNia mice

The vestibular organs of young and very old C57BL/6NNia (B6) mice were compared by light and electron microscopy. Hair cell density decreased an average of 14% in the utricle, 19% in the saccule and posterior crista, 23% in the horizontal crista, and 24% in the anterior crista. Hair cell size remained the same throughout the mouse's life span as did the ratio of Type I to Type II hair cells. The most apparent sign of advanced age was dense inclusions found in sensory and supporting cells. Although small inclusions were present at five weeks, by 29 months, additional, larger forms appeared. An unusual melanin-like form was characteristic of old Type I hair cells. Synaptic morphology and synaptic bodies were well preserved even in very old B6 mice. Elongated bars were common in Type I hair cells and spheroid synaptic bodies were the most common form in Type II hair cells. Large clusters of synaptic bodies occurring in both young and old mice were seen only in Type I hair cells. Although the B6 strain suffers from genetically determined early cochlear degeneration, it does not experience early degeneration of the peripheral vestibular organs.

Further observations on the ultrastructure of the flank gland of the musk shrew,Suncus murinus viridescens (Blyth)

The ultrastructure of the flank gland tissue of both sexes of the Indian musk shrew,Suncus murinus viridescens was investigated. The undifferentiated and differentiated sebaceous secretory cells in the glands of both sexes possess 1–8 mitochondrial dense osmophilic inclusions. A few inclusions of medium electron density, and clear vacuoles were also seen in the mitochondria. The muscle fibres in the glandular complex were devoid of these characteristic mitochondrial inclusions. Secretory activity of the well differentiated sebaceous cells was characterised by the presence of a number of large secretory granules. Mitochondria and polyribosomes were found in close association with the secretory granules. The flank gland tubules of the shrew were highly vascularised.

Evaluation of the arsenic inclusion using CTEM, STEM, and HVEM

The electron dense arsenic inclusion was first reported to occur in the nucleus of hepatocytes of Lepomis cvanellus. a freshwater species of sunfish, following laboratory exposures of this species to solutions of arsenate. Markedly elevated levels of arsenic were reported in the livers of the green sunfish, apparently due to the enterohepatic redistribution of the metalloid following biliary excretion. A maximum of approximately 50 and 160 ppm arsenic was found in the liver and bile, respectively, following a 6-d exposure of these fish to 60 ppm arsenic at 20°C. Translocation of arsenic from a cytoplasmic to nuclear location occurred during a 6-d period, ultimately leading to an increase in the size and number of intranuclear inclusions in the parenchymal hepatocytes. Microprobe analysis data indicated that the inclusion was composed of arsenic in a one to six ratio with sulfur.

Abnormal neurol fold development in mouse trisomy 12 and trisomy 14. II. LM and TEM

LM and TEM observations of embryonic tissue during the period of cranial neurulation are described in trisomic mouse embryos known to develop exencephaly, and are compared with tissue from normal mouse embryos. The earliest regularly observed differences were visible from the late presomite stage onwards, in the extracellular matrix of the cranial region. These were local defects of the basement membrane of the neural epithelium and enlarged areas of mesenchymal extracellular matrix, with associated abnormalities of mesenchymal cell distribution, cell number and cell contacts, and deficiency of alcian blue staining. Apical neuroepithelial microfilament bundles were observed at later somite-stages in trisomic embryos c.f.=compared with controls, and development of the concave neuroepithelial curvature was correspondingly retarded. Apposition of the neural folds at the forebrain/midbrain junction was never made, even though late neural fold fusion occurred in the hindbrain and ventral forebrain. At later stages (9–20 somites) the neuroepithelial cells showed pyknotic nuclei and dense intracellular inclusions. These are interpreted as secondary effects.

The effect of monensin on gametogenesis and zoosporogenesis in the aquatic fungus,Allomyces macrogynus

Cytoplasmic cleavage in the gametangia and zoosporangia ofA. macrogynus was studied using monensin, an ionophore known to disrupt several endomembrane functions in plant and animal cells. Monensin interfered with normal gamete and zoospore formation in a dose dependent manner such that at a 20 μM concentration very abnormal cells were released from the reproductive structures. It was evident that monensin's effect was most pronounced during the first 25 minutes of gametogenesis and parallels in time the onset and continuation of the cytoplasmic cleavage events. Observations using fluorescence and differential interference contrast microscopy indicated that the ionophore inhibited normal cytoplasmic cleavage resulting in the production of multinucleate cells, many of which had either no flagella or multiple flagella. Transmission electron microscopy showed that the monensin-treated gametangia had many large vacuoles which contained amorphous electron-opaque material. X-ray microprobe analysis demonstrated that the elemental composition of the large vacuoles was identical to that of the dense globular inclusions seen in untreated gametangia, and morphological analysis confirmed the relationship between these endomembrane structures. Thus this swollen endomembrane component probably is not involved in the cleavage process. Single endomembrane cisternae which were very common in untreated gametangia were seldom seen in monensin-treated preparations. Instead, many smaller electron-transparent vacuoles were observed. These swollen cisternae may both represent monensin-modified Golgi apparatus equivalents and/or play a critical role during the process of gametogenesis and zoosporogenesis inA. macrogynus.

Heterogeneity of calcium compartmentation: electron probe analysis of renal tubules.

The objective of this study has been to determine the intracellular localization of calcium in cryofixed, cryosectioned suspensions of kidney proximal tubules using quantitative electron probe X-ray microanalysis. Two populations of cells have been identified: 1) "Viable" cells, representing the majority of cells probed, are defined by their relatively normal K/Na concentration ratio of approximately 4:1. Their measured Ca content is 4.1 +/- 1.4 (SEM) mmol/kg dry wt in the cytoplasm and 3.1 +/- 1.1 mmol/kg dry wt in the mitochondria, or an average cell calcium content of approximately 3.8 mmol/kg dry wt. 2) "Nonviable" cells, defined by the presence of dense inclusions in their mitochondria and a K/Na concentration ratio of approximately 1. The Ca content is 15 +/- 2 mmol/kg dry wt in the cytoplasm and 685 +/- 139 mmol/kg dry wt in the mitochondria of such cells. Assuming 25 to 30% of the cell volume is mitochondrial, the overall calcium content of such nonviable cells is approximately 210 mmol/kg dry wt. The presence of these inclusions in 4 to 5% of the cells would account for the average total Ca content measured in perchloric acid extracts of isolated proximal tubule suspensions (approximately equal to 18 nmol/mg protein or 12.6 mmol/kg dry wt). Whole kidney tissues display a large variability in total Ca content (4.5 to 18 nmol/mg protein, or 3.4 to 13.5 mmol/kg dry wt), which could be accounted for by inclusions in 0 to 4% of the cells. The electron probe X-ray microanalysis (EPXMA) data conclusively demonstrate that the in situ mitochondrial Ca content of viable cells from the kidney proximal tubule is low and support the idea that mitochondrial Ca may regulate dehydrogenase activity but probably does not normally control cytosolic free Ca.

Qualitative and quantitative effects of acid misting and two air pollutants on foliar structures of Liriodendron tulipifera

One-year-old seedlings of Liriodendron tulipifera (yellow poplar) were exposed to a daily misting regime at pH levels of 5.6, 4.6, 3.6, or 2.6 for a period of 8 weeks. Identical misting conditions supplemented with either a 0.1 ppm fumigation of ozone or NO2 were established for additional treatments. Leaf samples were prepared for light and transmission electron microscopy and light micrographs were used for quantitative analyses of transectional cellular and air-space areas. Leaves exposed to acid misting alone exhibited a cellular collapse at pH 2.6. With increasing acidity, a steady decline in mesophyll air space, an increase in dense inclusion bodies, and a decline in plastid starch content were observed. Plants that received supplemental ozone fumigation during misting exhibited cellular swelling at all pH levels and an increase in total mesophyll area from pH 5.6 to 3.6. While mesophyll cell transectional areas were virtually unchanged from pH 3.6 to 2.6, the air space was significantly reduced. Distinctive differences in apparent cell wall thickness were noted between palisade and spongy cells at pH 3.6 and 2.6. Leaves exposed to NO2 fumigation plus mist macroscopically appeared vigorous and generally showed increased blade thickness with increasing acidity primarily because of changes in mesophyll cell content. Chloroplasts showed prominent stroma protrusions into the cytoplasm. Thus, this study reveals distinctively different quantitative and morphological foliar responses to the misting and fumigation regimes.

Prevention of basement membrane thickening in retinal capillaries by a novel inhibitor of aldose reductase, tolrestat.

A diabetic-like thickening of retinal capillary basement membranes induced in rats fed for 207 consecutive days a diet containing 50% galactose was prevented by the addition to the diet of tolrestat, a potent, structurally novel inhibitor of aldose reductase. Analysis of electron micrographs (X 25,000) of capillaries from the outer plexiform layer of the retina by computer planimetry showed that the basement membranes were approximately twofold thicker in rats fed galactose than in those fed either a standard diet or a diet containing galactose and tolrestat in doses of 43 or 57 mg/kg/day. The thickening of basement membranes in galactose-fed rats was accompanied by other ultrastructural alterations mimicking changes typical of diabetic microangiopathy, such as multilamination and the formation of vacuoles and dense inclusions. Therefore, the galactosemic rat represents a useful model for studying basement membrane-related complications of diabetes and their possible prevention by aldose reductase inhibitors.

Fine structure and cytochemistry of the endothelial cells and rodlet cells in the bulbus arteriosus in species of Cichlidae (Teleostei)

The ultrastructure of endothelial cells and rodlet cells in the bulbus arteriosus of specimens representing six genera of Cichlidae is described. The former are very closely packed by membrane–bound and mainly electron–dense inclusion bodies (0.3–0.7μm).In Apistogramma ramirezi I observed numerous subendothelial rodlet cells throughout the entire length of the bulbus arteriosus. These cells penetrate the endothelium and connect to the latter by desmosomes and tight junctions. The luminal part of the cell contains numerous vesicles and tubules (width 50–100 nm), whereas the basal part is occupied by a number of membrane–bound, club–like inclusions (length ≤ 5 μm). Between these two layers there occurs a layer of small, elongated mitochondria. Peripherally, these cells consist of a filamentous wall, except in the apical area.The endothelial and rodlet cell inclusion bodies do not react with phosphotungstic acid (pH 1) or Sudan black B stain. The endothelial cells react strongly with periodic acid–Schiff (PAS) stain, whereas the rodlet cells are only moderately coloured by this stain.The present results are discussed and compared with those reported previously for endothelial/ endocardial cells and rodlet cells in bony fish.

Fine structural analysis of lipofuscin in various tissues of rats of different ages

Comparative fine structural analysis of intracellular dense inclusions in various tissues of rats of different ages (2, 11 and 29.5 months old) was made. The majority of dense inclusions observed in 2-month-old rats were of the granular type. With advancing age, granular type inclusions gradually decreased in frequency, while compound type inclusions, such as granular-homogeneous and granular-lamellated type increased except for spleen. In the bronchial epithelia, granular-lamellated inclusions appeared at 2 months of age. On the other hand, in the heart, liver, spleen, and skeletal muscle, lamellated or granular-lamellated inclusions were not observed at any of the three age levels. Dense inclusions seemed to have various patterns of distribution, frequency of appearance, and source of organelles in each tissue and cell. They also did not show the maturity of pigment in the process of pigment formation. These findings suggested that these inclusions were not merely age-related granules, but seemed to be inlfuenced by their relationship to the cell metabolism and other functions.

An electron microscopic study of astroglia and oligodendroglia in the lateral geniculate nucleus of aged rats

The ultrastructural features of glial cells in the lateral geniculate nucleus of aged rats have been studied. Abundant filaments as well as heterogeneous dense bodies are observed in the majority of astrocytes. They frequently surround both axons and nerve terminals showing signs of degeneration. In addition, some degenerating myelinated axons are seen in phase suggestive of engulfment by astrocyte processes. Oligodendrocytes display broad processes containing an organelle-rich cytoplasm and a continuity between their plasma membrane and the outer myelin lamellae which partially ensheath the adjacent axons. Multivesicular bodies and pleomorphic dense inclusions, composed of amorphous material as well as laminated structures, are also present in oligodendrocytes. The significance of these morphological features is discussed in relation to process of normal ageing.