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.     

Modification of surfactant metabolizing cells in rat lung by clofibrate, a hypolipidemic peroxisome proliferating agent. Evidence to suggest that clofibrate influences pulmonary surfactant metabolism

The influence of clofibrate (ethyl-alpha-p-chlorophenoxy-isobutyrate), a hypolipidemic peroxisome proliferating agent, has been tested on the lungs of adult male rats. Drug administration for 7 days caused structural changes in two types of lung cells, both of which are involved in the metabolism of the pulmonary surfactant. By light microscopy the prominent features were the presence of enlarged type II alveolar epithelial cells and foamy intraalveolar macrophages. Compared with controls, type II cells in treated rats apparently contained more numerous surfactant-containing lamellar bodies, as visualized in semi-thin sections of Epon-embedded tissue. This difference was quantified morphometrically by light microscopy: the number of lamellar bodies was estimated as the profile number per individual type II alveolar cell, transsected at its nucleus. Clofibrate administration for 7 days resulted in a significant increase in the number of the lamellar inclusions. In contrast the number of type II alveolar cells per area of lung remained unchanged. There was no evidence of atelectasis or inflammatory infiltration in the drug-treated lungs, a finding confirmed in sections of perfusion-fixed, paraffin-embedded whole lung-lobes. By electron microscopy the lamellar inclusion bodies in the type II alveolar cells in treated rats, apart from being more numerous and sometimes smaller, were morphologically identical to those in controls. The vacuolated alveolar macrophages seen in treated rats also contained various lamellar phospholipid inclusions.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Reovirus core protein mu2 determines the filamentous morphology of viral inclusion bodies by interacting with and stabilizing microtubules

Cells infected with mammalian reoviruses often contain large perinuclear inclusion bodies, or "factories," where viral replication and assembly are thought to occur. Here, we report a viral strain difference in the morphology of these inclusions: filamentous inclusions formed in cells infected with reovirus type 1 Lang (T1L), whereas globular inclusions formed in cells infected with our laboratory's isolate of reovirus type 3 Dearing (T3D). Examination by immunofluorescence microscopy revealed the filamentous inclusions to be colinear with microtubules (MTs). The filamentous distribution was dependent on an intact MT network, as depolymerization of MTs early after infection caused globular inclusions to form. The inclusion phenotypes of T1L x T3D reassortant viruses identified the viral M1 genome segment as the primary genetic determinant of the strain difference in inclusion morphology. Filamentous inclusions were seen with 21 of 22 other reovirus strains, including an isolate of T3D obtained from another laboratory. When the mu2 proteins derived from T1L and the other laboratory's T3D isolate were expressed after transfection of their cloned M1 genes, they associated with filamentous structures that colocalized with MTs, whereas the mu2 protein derived from our laboratory's T3D isolate did not. MTs were stabilized in cells infected with the viruses that induced filamentous inclusions and after transfection with the M1 genes derived from those viruses. Evidence for MT stabilization included bundling and hyperacetylation of alpha-tubulin, changes characteristically seen when MT-associated proteins (MAPs) are overexpressed. Sequencing of the M1 segments from the different T1L and T3D isolates revealed that a single-amino-acid difference at position 208 correlated with the inclusion morphology. Two mutant forms of mu2 with the changes Pro-208 to Ser in a background of T1L mu2 and Ser-208 to Pro in a background of T3D mu2 had MT association phenotypes opposite to those of the respective wild-type proteins. We conclude that the mu2 protein of most reovirus strains is a viral MAP and that it plays a key role in the formation and structural organization of reovirus inclusion bodies.     

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.     

Comparative Ultrastructure of the Thiobacilli

The ultrastructure of seven Thiobacillus species was studied. The structure of their cell envelopes is similar, if not identical, to that found in other gram-negative bacteria. Obvious differences were noted in the middle layer of the cell envelope of the seven cultures. Polyhedral inclusion bodies were apparent in four of the organisms: T. thioparus, T. neapolitanus, T. intermedius, and T. thiooxidans. Lamellar bodies, similar to those present in certain photosynthetic bacteria were found in a few cells of T. thioparus. Structures resembling mesosomes were discovered in T. dinitrificans. A few cells of T. intermedius possessed paracrystalline bodies. Other inclusions, probably volutin and polysaccharide, were present in some of the cultures.     

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.     

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.     

α-Synuclein Immunoisolation of Glial Inclusions from Multiple System Atrophy Brain Tissue Reveals Multiprotein Components

Immunohistochemical studies have shown that oligodendroglial inclusions in multiple system atrophy contain alpha-synuclein, a synaptic protein also found in Lewy bodies in Parkinson's disease. We have now used density gradient enrichment and an anti-alpha-synuclein immunomagnetic technique to isolate pure and morphologically intact oligodendroglial inclusions from brain white matter of patients dying with multiple system atrophy. Filamentous inclusion structures were obtained only from multiple system atrophy tissue, but not from normal brain tissues, or from multiple system atrophy tissue processed without anti-alpha-synuclein antibody. We confirmed the purity and morphology of isolated inclusions by electron microscopy. The inclusions comprised multiple protein bands after separation by polyacrylamide gel electrophoresis. Immunoblotting demonstrated that these proteins included alpha-synuclein, alphaB-crystallin, tubulins, ubiquitin, and prominent, possibly truncated alpha-synuclein species as high-molecular-weight aggregates. Our study provides the first biochemical evidence that oligodendroglial inclusion filaments consist of multiple protein components, suggesting that these inclusions may form as a result of multiprotein interactions with alpha-synuclein.     

A histochemical and ultrastructural study of yucca seed proteins

Embryos and nutritive tissues in ungerminated Yucca seeds of 4 species contain many spherical bodies which stain positively for proteins. Two distinct morphological types were observed at both the light and electron microscope levels. A meshwork-type consists of electron-dense and electron-transparent regions in which are embedded slightly birefringent inclusions. The second type, named the core-type, consists of a core surrounded by a matrix in which the inclusions are embedded. A single unit membrane surrounds each protein body. Both types are present in the embryo while only the core-type protein body appears in the surrounding nutritive tissue (perisperm). All regions in each of the two protein body types, except the inclusions, stain histochemically for proteins. Seeds were planted at 2-day intervals and allowed to germinate through 14 days. As germination commences (day 0) protein bodies in the embryo begin to break down. By day 4 the bodies are depleted in embryos of 3 of the 4 species. About day 4, protein bodies in perisperm surrounding the embryo begin to break down and this process continues outward to the seed coat until day 14 when all seed proteins have disappeared. During germination the protein bodies in the embryo and perisperm of 3 species coalesce and then undergo breakdown. In a fourth species, there is no appreciable increase in size of the bodies, but an erosion of the periphery and possibly internally as well takes place, followed by ultimate dissolution.     

Cytoplasmic bar-like structures of alveolar type II cells: an ultrastructural study in freshly isolated cells from rat lungs

Bar-like structures are tubular cytoplasmic inclusions found in situ in pulmonary epithelial type II cells of several animal species. The physiological significance and mode of formation of these inclusions are not fully established. In this paper, we describe bar-like structures as found in freshly isolated type II cells from rat lungs. Pulmonary cells were dissociated from the tissue with elastase and separated on a discontinuous density gradient of Percoll. The complete isolation procedure yielded 17 X 10(6) type II cells per animal (purity = 80%). Either from the crude cell suspensions or the purified preparations, only a small fraction of the type II cell population harbored the inclusions (less than 5%). It is shown that the bounding membranes of the bar-like structures can derive from the endoplasmic reticulum, the nuclear membrane, or the Golgi apparatus. Occasional connections with lamellar bodies were observed, and different levels of complexity in the bar-like structures were also found. The apparent rigid conformation and the orientation of the bar-like structures were taken as evidence for a role of the cytoskeleton in their formation. Because the inclusions do not appear to be new organelles or cellular structures performing a specific function, we propose that their formation may be a transient and limited cellular event in normal cells. However, the stabilization and the generation of the osmiophilic structures, as well as their overproduction, may reflect alterations of the normal physiology of the type II cells.     

The cellular changes in primary and recurrent infection with herpes simplex virus type 2 in an in vitro model

The cellular changes of primary and recurrent herpes simplex virus (HSV) infection were investigated in in vitro models. In the primary infection model, nuclear changes were characterized by clumping and margination of the nuclear chromatin, a homogeneous ground-glass nuclear appearance, multinucleation and the appearance of two different types of intranuclear inclusions. One of the two types of inclusions appeared as early as six hours postinfection, reached a maximum at nine hours postinfection and gradually decreased thereafter. This early inclusion, relatively small in size as compared to the other type of inclusion, gave a basophilic staining when the Papanicolaou staining method was used. The other type of inclusion was the typical Cowdry type A inclusion, which appeared as early as 12 hours postinfection. Both types of inclusions contained clear perinuclear halo. In the recurrent infection model, the appearance of all the nuclear changes was delayed, the appearance of early inclusions was infrequent, and the Cowdry type A inclusions were observed more frequently than in the primary infection model. These results may indicate that the early inclusion is a sign of rapid virus replication while the Cowdry type A inclusion is one form of the remains of an HSV infection.     

Annulate lamellae and crystalline inclusions in granular endoplasmic reticulum of the islet organ and associated tissues of a cyclostome,Myxine glutinosa

Cytoplasmic annylate lamellae were found in the islet organ of a cyclostome, the hagfish (Myxine glutinosa), predominantly in cells interpreted as young proliferating beta-cells, and also in endocrine cells and enterocytes of the bile duct and gut and in the endothelial cells of small blood vessels. A close association was observed annulate lamellae and granular endoplasmic reticulum. Both in cells with and in those without annulate lamellae, crystalline inclusions of proteinaceous nature were seen in granular endoplasmic reticulum. These inclusions were occasionally closely associated to annulate lamellae, and a direct continuity could be seen between granular endoplasmic reticulum and the outer nuclear membrane surrounding an inclusion partially situated in the perinuclear cisterna. Rod-shaped structures and rounded electron dense bodies were seen in the nuclei of some islet parenchymal cells. The presence of annulate lamellae in the islet organ and associated tissues of Myxine glutinosa is believed to be related to the very high phylogenetic age of this species. The close association observed between annulate lamellae, granular endoplasmic reticulum, crystalline inclusions, and sometimes also nuclear membranes, may be of functional significance.     

Intranuclear Inclusions in the Ameboid Cells of Protostelium zonatum*

SYNOPSIS. Two morphologically distinct types of intranuclear inclusions are found in ameboid cells of the protostelid mycetozoan Protostelium zonatum. One type of inclusion is a coiled tubular structure which in cross section appears as cisternae and oval to elliptical vesicles 40–60 nm in diameter. These tubular and vesicular structures are formed by a unit membrane that is connected directly with the inner nuclear membrane. The other type of inclusion is a membrane-bound structure that contains amorphous and/or fibrous material. These inclusions usually are present at several locations in a nucleus. No similar structures occur in the cytoplasm.     

Protein inclusions produced by the entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus.

The entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus produces two types of intracellular inclusion bodies during in vitro culture. Large cigar-shaped inclusions (designated type 1) and smaller ovoid inclusions (designated type 2) were purified from cell lysates, using differential centrifugation in discontinuous glycerol gradients and isopycnic density gradient centrifugation in sodium diatrizoate. The inclusions, composed almost exclusively of protein, are readily soluble at high and low pH values and in the presence of cation chelators such as EDTA, anionic detergents (sodium dodecyl sulfate), or protein denaturants (urea, NaBr). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inclusions revealed a single 26-kilodalton protein (IP-1) in type 1 inclusions and a 22-kilodalton protein (IP-2) in type 2 inclusions. Analysis of these proteins by isoelectric focusing in the presence of 8 M urea showed that IP-1 is acidic and IP-2 is neutral. Furthermore, each protein occurred in multiple forms differing slightly in isoelectric point. Other variations in peptides released by trypsin digestion, immunological properties, and amino acid composition revealed significant structural differences between IP-1 and IP-2. Kinetic studies using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting procedures showed that inclusion protein synthesis occurs only during the second half of exponential culture growth. Synthesis of inclusion proteins and their aggregation to form inclusions occurred concurrently. Possible functions for these abundant proteins are discussed.     

Fine structural localization of alkaline phosphatase in the granular pneumonocytes of hamster lung.

The fine structural localization of nonspecific alkaline phosphatase was studied in the granular pneumonocytes (type II alveolar epithelial cells) of hamster lung by incubating sections of glutaraldehyde-fixed tissues in a medium containing lead ions and sodium beta-glycerophosphate or alpha-naphthyl acid phosphate. The specificity of the reaction was tested by exposing the sections to inhibitors of alkaline phosphatase. The results showed that alkaline phosphatase activity was present in the inclusion bodies of granular pneumonocytes. The enzyme reaction was strong in the membrane lining the inclusion bodies and a weaker reaction was generally detectable in the inclusion contents. Although only a proportion of the inclusion bodies showed enzyme activity, there was no obvious correlation between the reactivity of the inclusions and their intracellular position or size. The other organelles were unreactive. The finding of alkaline phosphatase activity within the inclusion bodies of granular pneumonocytes is an enigma as these organelles are generally considered to be lyosomes.     

Enhanced lysosomal pathology caused by beta-synuclein mutants linked to dementia with Lewy bodies

Two missense mutations (P123H and V70M) of beta-synuclein (beta-syn), the homologue of alpha-syn, have been recently identified in dementia with Lewy bodies. However, the mechanism through which these mutations influence the pathogenesis of dementia with Lewy bodies is unclear. To investigate the role of the beta-syn mutations in neurodegeneration, each mutant was stably transfected into B103 neuroblastoma cells. Cells overexpressing mutated beta-syn had eosinophilic cytoplasmic inclusion bodies immunopositive for mutant beta-syn, and electron microscopy revealed that these cells were abundant in various cytoplasmic membranous inclusions resembling the histopathology of lysosomal storage disease. Consistent with these findings, the inclusion bodies were immunopositive for lysosomal markers, including cathepsin B, LAMP-2, GM2 ganglioside, and ATP13A2, which has recently been linked to PARK9. Notably, formation of these lysosomal inclusions was greatly stimulated by co-expression of alpha-syn, was dependent on the phosphorylation of alpha-syn at Ser-129, and was more efficient with the A53T familial mutant of alpha-syn compared with wild type. Furthermore, the inclusion formation in cells overexpressing mutant beta-syn and transfected with alpha-syn was significantly suppressed by treatment with autophagy-lysosomal inhibitors, which were associated with impaired clearance of syn proteins and enhanced apoptosis, indicating that formation of lysosomal inclusions might be protective. Collectively, the results demonstrated unambiguously that overexpression of beta-syn mutants (P123H and V70M) in neuroblastoma cells results in an enhanced lysosomal pathology. We suggest that these missense mutations of beta-syn might play a causative role in stimulating neurodegeneration.     

Ultrastructural characterization of core structures and paracrystalline inclusion bodies in L-form cells of streptomycetes

Protoplast type L-form cells of Streptomyces hygroscopicus and S. griseus contain different types of inclusion bodies. Cytoplasmic cores and paracrystalline structures are peculiar inclusions which could not be observed in normal parent bacteria. The cytoplasmic cores are 1-4 micron long and 0.05-0.25 micron broad straight and stiff non-tubular structures consisting of homogeneous mode-rate electron opaque material. Paracrystalline inclusions have side-lengths between 0.2 and 0.5 micron and show a characteristic pattern of 15-20 nm thick straight dark lines and electron lucent intervening spaces of 20-30 nm. Both cytoplasmic cores and paracrystalline inclusions are apparently proteins. Their occurrence in L-form cells indicates an altered synthesis of one or several proteins in these cell types.