Proteasome inhibition induces inclusion bodies associated with intermediate filaments and fragmentation of the Golgi apparatus

The ubiquitin-proteasome system is involved in a variety of biological processes. Inclusion bodies associated with intermediate filaments (IFs) and ubiquitin are observed in various diseases; however, the precise mechanisms of formation and the pathological significance of inclusion bodies have not been fully understood. We examined the effect of proteasome inhibitors on the structure of IF using anti-cytokeratin antibodies or transfection of green fluorescent protein-fused cytokeratin 18 in a hepatoma cell line, Huh7. Intracellular organelles were visualized by immunofluorescent and electron microscopies. Proteasome inhibitors induced IF inclusions associated with ubiquitin. Electron microscopic examination revealed inclusion bodies surrounded by filamentous structures. Autophagic vacuoles and lysosomes were frequently observed, and the organization of the Golgi apparatus was disrupted in these cells. After the removal of the proteasome inhibitors, the IF network and organization of the Golgi apparatus were restored. The IF inclusions could be induced by inhibition of the proteasome function. IF inclusions induced fragmentation of the Golgi apparatus and might inhibit the function of this important station of membrane traffic. The IF inclusions disappeared by restoring proteasome function, and autophagy and lysosomal degradation might be, at least in part, associated with the elimination of inclusion bodies.     

Intraepithelial inclusions resembling human Biondi bodies in the choroid plexus of an aged chimpanzee

Summary Complex intracellular inclusion bodies of the Biondi type were observed in the choroidal epithelium (choroid plexus of the lateral ventricle) of a 43-year-old male chimpanzee. The specific components of these inclusions are bundles of filaments 8–15 nm in diameter, which are associated with lipid droplets and a wide variety of unidentified inclusions of differing electron density. Biondi bodies are characteristic inclusions of the choroid plexus of aged humans but have been claimed to be absent from the choroidal epithelium of senescent animals including nonhuman primates. The present finding of Biondi body-like inclusions in an aged chimpanzee underscores the usefulness of nonhuman primates as models for studies of aging, seeking to gain a better understanding of gerontological aspects of the human brain.     

Inclusion bodies induced by Bean rugose mosaic comovirus seen under light microscopy

Two types of inclusion bodies were consistently observed under light microscopy in bean (Phaseolus vulgaris) leaf tissue infected with Bean rugose mosaic virus (BRMV), a species of the genus Comovirus, family Comoviridae. One type consisted of vacuolated inclusions found mainly in the cytoplasm of epidermal cells. The other type consisted of abundant crystalloid inclusions of different sizes and shapes found consistently in glandular hairs, guard cells, phloem tissue, xylem elements and occasionally in epidermal and mesophyll tissues. The two types of inclusion bodies stained with Azure A and Luxol Brilliant Green Bl-Calcomine Orange 2RS (O-G), and were similar to those seen to be caused by other species of comoviruses.     

Intramitochondrial rodlet-like inclusions in human leukemic lymphocytes.

Some mitochondria in leukemic cells in two of nine investigated patients with chronic lymphocytic leukemia and in one patient with morphologically less usual form of this disease contained unusual rodlet-like inclusion bodies. These structures were always present in the matrix of frequently dilatated intercristal spaces. The width of rodlet-like inclusions ranged between 10 and 20 nm, their maximal length was 114 nm. All observed rodlet-like inclusion bodies appeared to be composed of fine filaments 2...3 nm in width and resembled the DNA containing structures in mitochondria of few other cell types. In mitochondria of leukemic cells, however, these inclusions represent a further structural abnormality.     

Characterization of intracytoplasmic prokaryote infections in Dreissena sp. (Bivalvia: Dreissenidae)

This study characterizes intracytoplasmic infections with prokaryote microorganisms in Dreissena sp. (near Dreissena polymorpha) from northeastern Greece and represents the first report of such infections in freshwater bivalves. Light microscope observations of stained tissues revealed basophilic, cytoplasmic inclusion bodies in 87.5% (28/32) of the mussels sectioned. Inclusions in epithelial cells and connective tissues were noted, respectively, in 34.4 and 71.9% of the sample, with 5 mussels (15.6%) having both tissue types infected. Epithelial cell infections were observed in histological sections only in digestive gland tubules and ducts; within tubules, inclusions were present more often in secretory than digestive cells. Connective tissue infections, however, were systemic; among the 32 mussels sectioned, inclusions were found in the gills (65.6%), foot (12.5%), mantle (9.4%), labial palps (6.3%), digestive gland (6.3%), stomach (6.3%), and gonads (3.1%). Cytoplasmic inclusions (maximum dimension, 138 microm) were prominent enough in the gills to be visible in 17.0% of the 247 mussels dissected. Ultrastructurally, prokaryote cells in gill connective tissues were clearly characteristic of Chlamydiales-like organisms, with each intracytoplasmic inclusion containing a loosely packed mixture of elementary, reticulate, intermediate bodies, and blebs. Prokaryote colonies in digestive gland epithelial cells exclusively contained 1 of 4 morphological cell types and were considered Rickettsiales-like. Hexagonal, virus-like particles were present in the cytoplasm of the largest of these Rickettsiales-like prokaryotes. Although host stress was evident from localized cell necrosis and dense hemocyte infiltration, overall infection was fairly benign, with no major, adverse impact on body condition evident among sectioned or dissected mussels. A possible negative effect was partial constriction of gill water tubes, but at the infection intensity observed (typical range 1 to 7 inclusion bodies per section), significant interference with respiration and other metabolic functions of the gills was highly unlikely.     

Observation of Potato Virus X in Tobacco Mosaic Virus (TMV)- Induced Local Lesions and Surrounding Zones in Leaves of Datura stramonium L. in Mixed Infection

In mixed infection by Tobacco Mosaic Virus (TMV) and Potato Virus X (PVX) of leaves of Datura stramonium L., PVX particles were observed in the developing local lesions in both the central part and on the periphery, in addition to TMV. PVX virions were found either separately or together with TMV. Sometimes in local lesions mainly in their periphery, PVX-specific laminar inclusion components were observed and, in certain cases, cylindrical bodies about 120—140 nm in diameter. In 2 mm surrounding zones from the edge of the lesions, TMV particles were not observed. However, in the majority of cells of these zones, PVX intensively accumulated, often forming large masses. In some cases, we observed parts of cells with relatively small amounts of dispersed PVX particles, associated with laminar inclusion bodies. In cell areas with large accumulations of PVX, laminar inclusions were not found.     

Fine Structure of Cellular Inclusions in Measles Virus Infections

Cells which are infected with measles virus have been known for some time to contain inclusion material that is distinguishable from normal cellular components by application of traditional staining methods and observation in the light microscope. The fine structure of the inclusion material contained in HeLa cells infected with Edmonston strain of measles virus has been examined in the electron microscope. Two steps have been found necessary in this study: (1) the recognition by phase-contrast microscopy of the living cell of bodies that are defined as inclusion material when the cells are classically stained; and (2) the recognition in the electron microscope of inclusion-body material that had previously been identified in the living cell. The fine structure of the nuclear and cytoplasmic inclusion material in osmium-treated cells was found to consist mainly of randomly arrayed filaments of low electron density. Dense, highly ordered arrays of filaments were found near the center of the nuclear inclusions, sometimes as a two-dimensional, nearly orthogonal arrangement. If the size of the measles virus is taken to be around 100 mµ in diameter, the strands seen in the inclusions cannot be fully formed virus.     

Identification of the sulfur inclusion body inBeggiatoa alba B18LD by energy-dispersive X-ray microanalysis

Dense inclusion bodies were observed under the STEM mode of a scanning electron microscope to occupy peripheral locations in air-dried filaments ofBeggiatoa alba B18LD, and they were determined by energy-dispersive x-ray microanalysis to consist almost entirely of sulfur. These inclusions conform in position and size (220–275 nm in diameter) to bodies seen in thin sections to be both membrane-bounded and enclosed within pockets penetrating the individual cell from the cytoplasmic membrane.     

Inclusion bodies in pinealocytes of the cotton rat (Sigmodon hispidus)

Pinealocytes of the cotton rat (Sigmodon hispidus) often contain large (2-6 micron diameter) intracytoplasmic inclusions, the function of which is not known. These inclusions may represent nucleolus-like bodies, mineral deposits, secretory products or viral inclusions. In this study these inclusions were classified as type A, B or C inclusions based on the amount of electron-dense material interspersed within the finely granular material comprising the bulk of these inclusions. Each type of inclusion was analyzed by X-ray microanalysis and enzymatic proteinaceous digestion. X-ray microanalysis of these inclusions differed both quantitatively and semiquantitatively from that of human or gerbil pineal concretions, the latter two of which are extracellular deposits. Pronase, a proteolytic enzyme, digested the electron-dense material only after longer times of tissue exposure to this enzyme in contrast to the easily digested, finely granular matrix-like material of these inclusions. Such intrapinealocytic inclusions have only been observed in the cotton rat. Their functional significance remains unknown.     

Intranuclear inclusion bodies within neurons of spinal and cranial ganglia in three cyprinid species

Summary A histological examination of 205 fish representing four cyprinid species from a site 2.5 miles north of Wheeling, West Virginia, on the Ohio River revealed large (2–4 m) cuboidal intranuclear inclusion bodies (NIB's) within neurons in the cranial and spinal ganglia of three species. Because the minnows had been caught during a yearly sampling of fish, an additional 63 minnows were taken the following year. Inclusions were again observed. The NIB's stain strongly with phloxine as well as with Mallory and Giemsa stains, appearing bright red or pink. Various histochemical tests indicated that the inclusions contain protein and lipid but no carbohydrates or nucleic acids. No heavy metals were detected by electron probe analysis. At the ultrastructural level the inclusions exhibit subunits resembling hexagons measuring 326–350 nm. Previously suggested causes for such inclusions include effects of viruses, aging, drugs, cellular transformation, and an altered metabolic state of affected cells.     

Disseminated cytomegalovirus disease in the guinea pig

Two guinea pigs (Cavia porcellus), which were housed conventionally in separate animal facilities and had not been experimentally manipulated, were found to have evidence of disseminated cytomegalovirus disease at necropsy. Microscopic examination revealed multiple focal areas of necrosis in numerous organs. The spleen, liver, kidney and lungs were affected particularly. These lesions contained numerous cells with large intranuclear and cytoplasmic inclusion bodies resembling cytomegalovirus inclusions. Characteristic cytomegalovirus virions were observed by electron microscopy in both cases. Antigens of guinea pig cytomegalovirus were detected in paraffin sections of lesions from both cases with an immunoperoxidase technique. Although subclinical infection is common, this is the only contemporary report of disseminated disease due to cytomegalovirus in guinea pigs.     

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.     

Soil-borne wheat mosaic virus inclusion bodies: structural, compositional and staining properties

Anatomy and cytochemistry of inclusion bodies induced by Soil‐borne wheat mosaic virus infection were studied in roots and leaves to learn more about the nature of inclusions and their roles in pathogenesis. Acid Fuchsin, Giemsa stain, Toluidine Blue and Trypan Blue stains facilitated visualization of inclusion bodies. Combined, simultaneous staining with Acid Fuchsin and Toluidine Blue clearly differentiated inclusion bodies from host nuclei. The overall anatomy, composition and structure of virus inclusions in leaves and roots were generally similar, as shown by phase contrast, differential interference contrast, epifluorescence, laser scanning confocal and transmission electron microscopy. Both were often closely associated with host nuclei; both were comprised of intertwined masses of tubular material, presumably endoplasmic reticulum, and in which varied numbers and sizes of vacuolar cavities occurred. Leaf inclusions, however, were typically larger and more vacuolate than those in roots. Lipids were found to be significant constituents of both the tubular and vacuolar components of inclusions, indicated by positive staining with Nile Red and Sudan Black. Inclusion bodies in both leaves and roots lost their structural and compositional integrity, eventually becoming disorganized and devoid of clearly identifiable components as host tissue aged and symptom expression advanced. Significant results of this study include the first published examination of virus inclusion bodies in root tissue, the degree of structural detail of inclusion body anatomy revealed by laser scanning confocal microscopy and the presence of an extensive lipid component in virus inclusion bodies.     

Inclusion bodies in loggerhead erythrocytes are associated with unstable hemoglobin and resemble human Heinz bodies

The aim of this study was to clarify the role of the erythrocyte inclusions found during the hematological screening of loggerhead population of the Mediterranean Sea. We studied the erythrocyte inclusions in blood specimens collected from six juvenile and nine adult specimens of the loggerhead turtle, Caretta caretta, from the Adriatic and Tyrrhenian Seas. Our study indicates that the percentage of mature erythrocytes containing inclusions ranged from 3 to 82%. Each erythrocyte contained only one round inclusion body. Inclusion bodies stained with May Grünwald-Giemsa show that their cytochemical and ultrastructure characteristics are identical to those of human Heinz bodies. Because Heinz bodies originate from the precipitation of unstable hemoglobin (Hb) and cause globular osmotic resistance to increase, we analyzed loggerhead Hb using electrophoresis and high-performance liquid chromatography to detect and quantitate Hb fractions. We also tested the resistance of Hb to alkaline pH, heat, isopropanol denaturation, and globular osmosis. Our hemogram results excluded the occurrence of any infection, which could be associated with an inclusion body, in all the specimens. Negative Feulgen staining indicated that the inclusion bodies are not derived from DNA fragmentation. We hypothesize that amino acid substitutions could explain why loggerhead Hb precipitates under normal physiologic conditions, forming Heinz bodies. The identification of inclusion bodies in loggerhead erythrocytes allow us to better understand the haematological characteristics and the physiology of these ancient reptiles, thus aiding efforts to conserve such an endangered species.     

Respective role of the 42- and 51-kDa components of the Bacillus sphaericus toxin overexpressed in Bacillus thuringiensis

Abstract The 42- and 51-kDa protein genes of Bacillus sphaericus 1593 have been subcloned independently downstream from the cytA gene promoter of Bacillus thuringiensis serovar israelensis and introduced into a non-mosquitocidal strain of Bacillus thuringiensis . Consequently, each protein was overproduced and accumulated as inclusion bodies which were purified. For the first time, the 42-kDa protein inclusions alone were found to be toxic to Culex pipiens larvae (LC₅₀ at 48 h 300 ng ml⁻¹); in contrast, the 51-kDa protein inclusions were not. Moreover, a synergistic effect between these two components was observed.     

Polymorphism of nuclear inclusions in carcinoma of the cervix uteri

Frequency, polymorphism and ultrastructural characteristics of the nuclear inclusion bodies encountered in cancers of the uterine cervix are reported and briefly discussed. The nuclear inclusions are grouped in three distinct types: a) nuclear bodies (comprising type I and II inclusions according to Bouteille et al.'s classification), b) inclusion of cytoplasmic origin, and c) particles of chromatic material. The ultrastructural aspects of the chromatic particles suggest an early structuration of viral chromatin into core material. There appears to be a direct relation between the frequency of chromatic particles and raised antiherpetic antibodies in the patient's sera.     

Viral basophilic inclusions in the digestive gland of razor clams Ensis arcuatus (Pharidae) in Galicia (NW Spain)

During a histological survey of razor clam Ensis arcuatus (Jeffreys, 1865) from Galicia (NW Spain), basophilic inclusion bodies were observed in epithelial cells of the digestive gland. Transmission electron microscopy revealed the intranuclear position of these inclusions containing viral particles with icosahedral symmetry. Size and symmetry of these unenveloped virus particles suggest similarities to the families Papillomaviridae and Polyomaviridae which have been described as causing a viral gametocytic hypertrophy in oysters Crassostrea virginica and C. gigas. This is the first report of viral particles in E. arcuatus.     

Origins and formation of intracellular inclusions associated with two leguminous virus diseases

Summary Two hitherto undescribed viruses were isolated from naturally-infected white clover plants.One induced both cytoplasmic and intranuclear inclusions, and the other caused cytoplasmic inclusions of a new type, which we have called Corner inclusion bodies.All three kinds of inclusion were found in the same cells.Vital observations showed that the two kinds of cytoplasmic inclusion bodies developed principally from the plastids.The intranuclear inclusions were shown to develop from the nucleoli.Cytochemical tests showed that the cytoplasmic inclusions contained protein and ribonucleic acid.On the basis of these findings, the possibility of virus synthesis in plastids and nucleoli is discussed.     

Intranuclear inclusions in urinary cytology

Twenty-one urinary cytology specimens from 16 patients showed red intranuclear inclusion bodies. The literature pertinent to these inclusions mainly suggests that lead poisoning is a cause for such inclusions. Histories were reviewed for possible causes, particularly lead poisoning, associated medications, associated illness or relationship to neoplasia. The only consistent associations found were that all the patients were women over the age of 50 and that all inclusions were found in voided urine specimens. Four slides containing inclusions were stained by acid-fast methods, with negative results. The origin of these inclusions remains unknown.     

Intranuclear filamentous inclusions in the gastro-entero-pancreatic (GEP) endocrine cells of birds

Summary Intranuclear filamentous inclusions were found in the normal endocrine cells of the avian stomach and pancreas. These inclusions were composed of a bundle of closely packed filaments (6–8 nm in diameter), being ultrastructurally similar to those found in the nucleus of various neurons. Most of them appeared as single rod- or spindle-shaped bodies; aggregations of two or more inclusions were rarely seen within a single nucleus. Cells with an intranuclear inclusion often contained a cytoplasmic fibrillar bundle similar to the intranuclear inclusion.