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PubMed1999

Identification and characterization of a Chlamydia trachomatis early operon encoding four novel inclusion membrane proteins.

Chlamydia trachomatis is a bacterial obligate intracellular parasite that replicates within a vacuole, termed an inclusion, that does not fuse with lysosomes. Within 2 h after internalization, the C. trachomatis inclusion ceases to interact with the endocytic pathway and, instead, becomes fusogenic with exocytic vesicles containing exogenously synthesized NBD-sphingomyelin. Both fusion of exocytic vesicles and long-term avoidance of lysosomal fusion require early chlamydial gene expression. Modification of the chlamydial inclusion probably occurs through the expression and insertion of chlamydial protein(s) into the inclusion membrane. To identify candidate inclusion membrane proteins, antisera were raised against a total membrane fraction purified from C. trachomatis-infected HeLa cells. By indirect immunofluorescence, this antisera recognized the inclusion membrane and, by immunoblot analysis, recognized three chlamydial-specific antigens of approximate molecular weights 15, 18 and 21 kDa. IncG, encoding an 18 kDa and 21 kDa doublet chlamydial antigen, was identified by screening a C. trachomatis, serovar L2, genomic expression library. Three additional genes, incD, incE and incF, were co-transcribed with incG. Monospecific antisera against each of the four genes of this operon demonstrated that the gene products were localized to the chlamydial inclusion membrane. Immediately downstream from the operon containing incD-G was the C. trachomatis homologue of incA. Like IncD, E, F and G, C. trachomatis IncA is also localized to the inclusion membrane. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that IncD-G, but not incA, are transcribed within the first 2 h after internalization, making them candidates for chlamydial factors required for the modification of the nascent chlamydial inclusion.

Antigens, Bacterial↗
PubMed2004

alpha-synuclein is required for the fibrillar nature of ubiquitinated inclusions induced by proteasomal inhibition in primary neurons.

Proteasomal dysfunction may underlie certain neuro-degenerative conditions such as Parkinson disease. We have shown that pharmacological inhibition of the proteasome in cultured neuronal cells leads to apoptotic death and formation of cytoplasmic ubiquitinated inclusions. These inclusions stain for alpha-synuclein and assume a fibrillar structure, as assessed by thioflavine S staining, and therefore resemble Lewy bodies. alpha-Synuclein is thought to be a central component of Lewy bodies. Whether alpha-synuclein is required for inclusion formation or apoptotic death has not been formally assessed. The present study examines whether alpha-synuclein deficiency in neurons alters their sensitivity to proteasomal inhibition-induced apoptosis or inclusion formation. Cortical neurons derived from alpha-synuclein-null mice showed a similar sensitivity to death induced by the proteasomal inhibitor lactacystin compared with neurons derived from wild-type mice. Furthermore, the absence of alpha-synuclein did not influence the percentage of lactacystin-treated neurons harboring cytoplasmic ubiquitinated inclusions or alter the solubility of such inclusions. In contrast, however, ubiquitinated inclusions in alpha-synuclein-deficient neurons lacked amyloid-like fibrillization, as determined by thioflavine S staining. This indicates that although alpha-synuclein deficiency does not affect the formation of ubiquitinated inclusions, it does significantly alter their structure. The lack of effect on survival in alpha-synuclein knock-out cultures further suggests that the fibrillar nature of the inclusions does not contribute to neuronal degeneration in this model.

Acetylcysteine↗
PubMed2002

Fluid inclusion studies of chemosynthetic carbonates: strategy for seeking life on Mars.

Fluid inclusions in minerals hold the potential to provide important data on the chemistry of the ambient fluids during mineral precipitation. Especially interesting to astrobiologists are inclusions in low-temperature minerals that may have been precipitated in the presence of microorganisms. We demonstrate that it is possible to obtain data from inclusions in chemosynthetic carbonates that precipitated by the oxidation of organic carbon around methane-bearing seepages. Chemosynthetic carbonates have been identified as a target rock for astrobiological exploration. Other surficial rock types identified as targets for astrobiological exploration include hydrothermal deposits, speleothems, stromatolites, tufas, and evaporites, each of which can contain fluid inclusions. Fracture systems below impact craters would also contain precipitates of minerals with fluid inclusions. As fluid inclusions are sealed microchambers, they preserve fluids in regions where water is now absent, such as regions of the martian surface. Although most inclusions are < 5 microns, the possibility to obtain data from the fluids, including biosignatures and physical remains of life, underscores the advantages of technological advances in the study of fluid inclusions. The crushing of bulk samples could release inclusion waters for analysis, which could be undertaken in situ on Mars.

Carbonates↗
PubMed2006

The GTPase Rab4 interacts with Chlamydia trachomatis inclusion membrane protein CT229.

Chlamydiae, which are obligate intracellular bacteria, replicate in a nonlysosomal vacuole, termed an inclusion. Although neither the host nor the chlamydial proteins that mediate the intracellular trafficking of the inclusion have been clearly identified, several enhanced green fluorescent protein (GFP)-tagged Rab GTPases, including Rab4A, are recruited to chlamydial inclusions. GFP-Rab4A associates with inclusions in a species-independent fashion by 2 h postinfection by mechanisms that have not yet been elucidated. To test whether chlamydial inclusion membrane proteins (Incs) recruit Rab4 to the inclusion, we screened a collection of chlamydial Incs for their ability to interact with Rab4A by using a yeast two-hybrid assay. From our analysis, we identified a specific interaction between Rab4A and Chlamydia trachomatis Inc CT229, which is expressed during the initial stages of infection. CT229 interacts with only wild-type Rab4A and the constitutively active GTPase-deficient Rab4AQ67L but not with the dominant-negative GDP-restricted Rab4AS22N mutant. To confirm the interaction between CT229 and Rab4A, we demonstrated that DsRed-CT229 colocalized with GFP-Rab4A in HeLa cells and more importantly wild-type and constitutively active GFP-Rab4A colocalized with CT229 at the inclusion membrane in C. trachomatis serovar L2-infected HeLa cells. Taken together, these data suggest that CT229 interacts with and recruits Rab4A to the inclusion membrane and therefore may play a role in regulating the intracellular trafficking or fusogenicity of the chlamydial inclusion.

Bacterial Proteins↗
PubMed1997

Ehrlichia chaffeensis inclusions are early endosomes which selectively accumulate transferrin receptor.

Ehrlichia chaffeensis is an obligatory intracellular bacterium which infects macrophages and monocytes. Double immunofluorescence labeling was used to characterize the nature of E. chaffeensis inclusion in the human promyelocytic leukemia cell line THP-1. E. chaffeensis was labeled with dog anti-E. chaffeensis serum and fluorescein isothiocyanate-conjugated anti-dog immunoglobulin G (IgG). Lissamine rhodamine-conjugated anti-mouse IgG was used to label various mouse monoclonal antibodies. Ehrlichial inclusions did not fuse with lysosomes, since they were not labeled with anti-CD63 or anti-LAMP-1. The ehrlichial inclusions were slightly acidic, since they weakly accumulated 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and stained weakly positive for vacuolar type H+ ATPase. Some ehrlichial inclusions were labeled positive with antibodies against HLA-DR, HLA-ABC, and beta2 microglobulin, while other inclusions in the same cell were labeled negative. The inclusions were labeled strongly positive for transferrin receptors (TfRs) and negative for the clathrin heavy chain. Time course labeling for TfRs showed that up to 3 h postinfection, most of the ehrlichial inclusions were negative for TfRs. After 6 h postinfection, 100% of the ehrlichial inclusions became TfR positive and the intensity of labeling was increased during the subsequent 3 days. Reverse transcription-PCR showed a gradual increase in the level of TfR mRNA postinfection, which reached a peak at 24 h postinfection. These results suggest that ehrlichial inclusions are early endosomes which selectively accumulate TfRs and that the ehrlichiae up-regulate TfR mRNA expression.

Animals↗
PubMed1975

Cytology as a guide to the presence of chlamydial inclusions in Giemsa-stained conjunctival smears in severe endemic trachoma.

Microscopical examination of 927 Giemsa-stained conjunctival smears from children with chronic trachoma in southern Tunisia showed 93 (10 per cent.) with typical trachoma (chlamydial) inclusions in epithelial cells. The accompanying cytological features were a useful indicator for inclusions. Inclusions were found only in slides with numerous polymorphonuclear neutrophils (PMNs) and separation of the epithelial cells. When these two features alone were present, 3 per cent. of the smears were inclusion-positive; when many lymphocytes were present also, 25 per cent. were inclusion positive; when other cytological features (plasma cells, macrophages, blastoid, and stem cells) were present as well, 70 per cent. of the smears were inclusion-positive. The occurrence of these sets of cytological features can be a useful guide for selecting smears for intensive examination for chlamydial inclusions. Immunofluorescent (FA) staining and Giemsa staining of 527 pairs of matched smears detected trachoma agent in 67 (13 per cent.); in thirty by both methods, in thirteen by Giemsa staining alone, and in 24 by FA alone. The examination of Giemsa-stained smears for chlamydial inclusions is a useful technique for the diagnosis of trachoma or inclusion conjunctivitis by laboratories that do not have the specialized facilities for the identification of these chlamydial infections by the technically more complex procedures of immunofluorescent staining or isolation in embryonated eggs or tissue cultures.

Bacteria↗
PubMed2006

Specific inclusion mode of guest compounds in the amylose complex analyzed by solid state NMR spectroscopy.

The inclusion compound formation between linear amylose of molecular weight 102500 (AS100) and p-aminobenzoic acid (PA) during the sealed-heating process was investigated by powder X-ray diffractometry, infrared spectroscopy and solid state NMR spectroscopy. Sealed-heating of AS100 and PA at 100 degrees C for 6 h provided an inclusion compound with 6(1)-helix structure, while a 7(1)-helix structure was found when sealed-heating was carried out at 150 degrees C for 1 h. The formation of an inclusion compound was not observed when sealed-heating was performed at 50 degrees C for 6 h. The 7(1)-helix inclusion compound maintained its structure even during storage at high temperature while the 6(1)-helix inclusion compound decomposed and returned to the original V(a)-amylose upon heating to 180 degrees C. Quantitative determination revealed that one PA molecule could be included per one helical turn of AS100 for both 6(1)-helix and 7(1)-helix inclusion compounds. Solid state NMR spectroscopy suggested that PA molecules were included in the amylose helix core in the 7(1)-helix inclusion compound, while in the case of 6(1)-helix inclusion compound, PA molecules were accommodated in the interstices between amylose helices. Moreover, the inclusion compound formation by sealed-heating of AS100 was also observed when using PA analogues as guest compounds. The binding ratio of AS100 and PA analogues varied depending on the size of guest molecules.

4-Aminobenzoic Acid↗
PubMed2004

[Determination of constant for the inclusion complex of Yinqiao volatile oil with beta-cyclodextrin].

OBJECTIVE: To determine the constant for the inclusion complex of Yinqiao volatile oil with beta-cyclodextrin. METHOD: The stability constant for the inclusion complex of Yinqiao the volatile oil with beta-cyclodextrin was detemined under multiwavelength by ultraviolet spectroscopy, and spectroscopy feature for inclusion complex was studied. The phase solubility-curve showed the inclusion proportion. The inclusion complex of Yinqiao the volatile oil with beta-cyclodextrin was prepared by saturated aqueous solution. The DTA spectroscopy of inclusion complex was different from not only oil with beta-cyclodextrin but also the mixture of them. RESULT: The inclusion complex of Yinqiao the volatile oil with beta-cyclodextrin was formed. The experimental data of inclusion complex fitted well to the molar ratio of 1:1. There was good liner feature under multiwavelength of ultraviolet spectroscopy. CONCLUSION: Adopting multiwavelength method may determine the appearant stability constant of multicomponential traditional drug. The determination of the stability constant of the inclusion complex can be used in traditional drug.

Calorimetry, Differential Scanning↗
PubMed2006

[Inclusion of coenzyme Q10 with beta-cyclodextrin studied by polarography].

AIM: To investigate the inclusion of coenzyme Q10 with beta-cyclodextrin (beta-CD). METHODS: The inclusion of the electroactive guest molecule coenzyme Q10 with the host molecule beta-CD was studied by the polarography. The change of the reduction peak current of the inclusion complex with time and the change of the peak potential of the inclusion complex with beta-CD concentration were examined. In order to study the photostability, the change of the reduction peak current of both coenzyme Q10 and coenzyme Q10-beta-CD inclusion complex with time were also examined under light, separately. RESULTS: In 0.1 mol x L(-1) HAc/NaAc (pH 4.7) buffer-ethanol/water (60:40) medium, coenzyme Q10 was included with p-CD to form an 1:1 inclusion complex. The formation constant Kf was 1.26 x 10(4) L x mol(-1) the apparent formation rate constant was 6.64 x 10(-2) min(-1). The photodegradation apparent rate constant of coenzyme Q10 as 7.77 x 10(-3) min(-1) and that of the coenzyme Q10-beta-CD inclusion complex was 3.38 x 10(-3) min(-1). CONCLUSION: The inclusion of coenzyme Q10 with beta-CD took place. The stability of coenzyme Q10 to lights was improved in a certain degree due to the formation of the inclusion complex.

Coenzymes↗
PubMed1987

Nuclear inclusions in the neurons of the developing cerebral cortex of Gallus gallus.

In this work an ultrastructural analysis of the nuclear inclusions of neurons of chick (Gallus gallus) dorsomedial cerebral cortex during different perinatal stages has been made. Two types of nuclear inclusions could be identified: a) membranous nuclear inclusions and b) nuclear bodies. Into the type of membranous nuclear inclusions we distinguish compact and pleomorphic membranous inclusions. Compact membranous nuclear inclusions are located close to the nuclear envelope and they are circularly shaped; the largest inclusions of this type frequently contain a fine granular material. Pleomorphic membranous inclusions are irregularly shaped and do not contain any electrondense material; this type of membranous inclusions can be mainly seen in newborn and rarely at later stages. These features suggest a transport mechanism cytoplasm-nucleus and nucleus-cytoplasm which coincides with the hyperactivity of these neurons during these perinatal stages. The nuclear bodies can be exclusively seen in the newborn stage and they possibly have an important role related to the cellular hyperactivity of these stages of the development.

Aging↗
PubMed1979

Nuclear inclusions in alveolar epithelium of patients with fibrotic lung disorders.

Ultrastructural study of pulmonary biopsy specimens from patients with fibrotic lung disease disclosed the presence of nuclear inclusions in 1% or less of cuboidal alveolar epithelial cells in 9 of 19 patients, including 6 of 12 patients with idiopathic pulmonary fibrosis, 2 of 3 patients with collagen-vascular disease, and 1 of 3 patients with sarcoidosis. Nuclear inclusions were not observed by ultrastructural study in 5 control patients. The inclusions consisted of masses or aggregates of tubules which probably were derived from the inner nuclear membranes. These tubules were smooth-walled, showed branchings and bifurcations, were composed of single trilaminar membranes, usually had a clear content, and ranged from 500 to 1000 A in diameter. They resembled nuclear tubules which occur in other cell types under conditions of rapid growth or specific hormonal stimulation. Statistically significant differences between the groups of patients with and without nuclear inclusions in cuboidal alveolar epithelial cells were not found with respect to smoking history, degree of fibrosis in the lung biopsy specimen, or the degree of pulmonary physiologic impairment. However, the average age of the patients having nuclear inclusions was significantly greater than that of patients not having nuclear inclusions. In addition, the frequency of indentations in the nuclei of cuboidal alveolar epithelial cells was greater in patients with nuclear inclusions than in patients without nuclear inclusions. Highly significant correlations were observed between the presence of nuclear inclusions and the presence of a) anchoring fibrils and hemidesmosomes along the basal surfaces of alveolar epithelial cells and b) multilayering of the alveolar epithelium.

Aging↗
PubMed1978

Ultrastructure of anaplasmal inclusions (Pawhuska isolate) and their appendages in intact and hemolyzed erythrocytes and in complement-fixation antigen.

Hemolysis of parasitized erythrocytes augmented visualization of the anaplasmal inclusion, including its initial bodies, inclusion membrane, and inclusion appendage ("tail" or "band"). A dense attachment complex joined the appendage to the inclusion membrane (wall of the inclusion vacuole). The inclusion appendage consisted of tightly packed, interconnected laminae and assumed loop, dumbbell, and comet configurations described by other workers. The erythrocytic plasmalemma and the inclusion membrane had a thickness of 9.0 +/- 0.8 nm and similar structures. The initial bodies were covered by a thin inner organismic membrane (7.0 +/- 0.7 nm thick) attached to the organismic chromatin, an intermembranous matrix, and by an outer membranous sheath or pellicle (12.5 +/- 1.2 nm thick). Dense granular aggregates (24 to 40 nm in diameter) within chromatin clumps were the only structures in the initial body remotely similar to ribosomes, yet they were too large, were never free of chromatin, and appeared to disappear upon hemolysis. Complement-fixation antigen prepared by fractionation contained initial bodies, inclusion appendages, a few mitochondria, vesicularized membranes, and stromal debris. The preparatory treatment caused segregation of the organismic chromatin into independent dense particles 103 +/- 12 nm in diameter still bound by inner organismic membrane. Similar particles were seen also in the plasma and inclusion vacuoles of hemolyzed erythrocytes.

Anaplasma↗
PubMed1998

Pleomorphic mitochondrial and different filamentous inclusions in inflammatory myopathies associated with mtDNA deletions.

Mitochondrial changes are frequently observed in muscle fibers of patients with inclusion body myositis (IBM) and polymyositis (PM), suggesting that mitochondrial function may be especially impaired in these forms of inflammatory myopathies. Intranuclear and cytoplasmic tubulofilamentous inclusions are characteristic, although not totally specific for IBM. In the present cases, the inclusions were strikingly pleomorphic when chloroquine had been given for long periods. The nuclear inclusions were always tubular, whereas the cytoplasmic filaments had either a tubular, a helical, or a cross-striated structure with different diameters and arrangements in association with myelin-like figures, and vacuoles. Abnormal mitochondria containing paracrystalline, globoid, and other inclusions, noted in IBM, were occasionally also seen in PM or vasculitis. By contrast, in the latter, no intranuclear or cytoplasmic tubulofilamentous inclusions were apparent in muscle fibers. This study reports for the first time the presence of membrane-bound crystalloid inclusions in a muscle fiber with numerous abnormal mitochondria; similar structures have thus far only been observed in macrophages. The identity and function of these inclusions remains unknown. Using PCR analysis we detected different mtDNA deletions not only in IBM, but also in PM and vasculitis, indicating at least some degree of association between the structural mitochondrial abnormalities and the mtDNA mutations. There was no topographical correlation between the presence of tubular or helical filaments and the mitochondrial abnormalities. As already noted by others, the mitochondrial changes in IBM were more frequent than expected in this age group. It is suggested that the presence of the mtDNA deletions in IBM and PM are not primary, but rather the result of the underlying, presumably immunological disorder causing nuclear and secondary or simultaneous mitochondrial changes.

Adult↗
PubMed1999

Diagnostic significance of papillary structures and intranuclear inclusions in Hurthle-cell neoplasms of the thyroid.

Papillary structures and intranuclear inclusions, features which are not ordinarily associated with Hurthle-cell lesions, are occasionally noted within Hurthle-cell-rich aspirates. The diagnostic significance of these features in this setting, if any, is unknown. The purpose of this study was to determine whether these features are diagnostically useful. Specifically, since fine-needle aspiration cytology cannot distinguish between Hurthle-cell adenoma and carcinoma, we wanted to know whether the presence of either feature might aid in preoperatively assessing the likelihood of carcinoma vs. adenoma. We identified 12 cases in which a diagnosis of Hurthle-cell lesion was made by thyroid fine-needle aspiration biopsy. All cases had subsequent surgery. For every case, all cytology slides were reviewed for the presence or absence of papillary structures and intranuclear inclusions, after which the findings were correlated with the final surgical pathological diagnosis. Seven of the 12 cases had identifiable papillary structures, ranging from cases where papillary structures comprised the predominant architectural pattern to cases where rare papillary structures were found amidst a predominance of sheets and single Hurthle cells. Four of the 12 cases had intranuclear inclusions. In all cases, intranuclear inclusions were uncommon. Of 6 cases that proved to be Hurthle-cell carcinoma, 5 had papillary structures on the antecedent fine-needle aspirate cytology, and 2 had intranuclear inclusions. Of 6 cases that proved to be Hurthle-cell adenoma, 2 had papillary structures, and 2 had intranuclear inclusions. Papillary structures had a sensitivity of 83% for the presence of carcinoma, a specificity of 66%, a positive predictive value of 71%, and a negative predictive value of 80% (P = 0.2). Intranuclear inclusions had a sensitivity of 33%, a specificity of 66%, and positive and negative predictive values of 50%. Papillary structures are more common in Hurthle-cell aspirates than previously recognized. Their presence is not diagnostic of carcinoma, but neither does their absence rule out carcinoma. However, when found, the likelihood of an ultimate diagnosis of Hurthle-cell carcinoma is increased. On the other hand, intranuclear inclusions, when rare, appear to have no diagnostic value.

Adenocarcinoma↗
PubMed2004

Relation between cell disruption conditions, cell debris particle size, and inclusion body release.

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric peptidoglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity.

Biotechnology↗
PubMed2025

Fluorescence Loss After Photoactivation (FLAPh): A Pulse-Chase Cellular Assay for Understanding Kinetics and Dynamics of Viral Inclusions.

Influenza A virus (IAV) relies on host cellular machinery for replication. Upon infection, the eight genomic segments, independently packed as viral ribonucleoproteins (vRNPs), are released into the cytosol before nuclear import for viral replication. After nucleocytoplasmic transport, the resulting progeny vRNPs reach the cytosol, accumulating in highly mobile and dynamic viral inclusions that display liquid properties. Being sites postulated to support IAV genome assembly, the biophysical properties of IAV inclusions may be critical for function. In agreement, imposing liquid-to-solid transitions was demonstrated to impact viral replication negatively. Therefore, screening for host factors or compounds able to alter the material properties may provide the molecular basis for how influenza genomic complex forms as well as identify novel antivirals. Conventional techniques employed to investigate biomolecular condensates' material properties include fluorescence correlation spectroscopy, raster image correlation spectroscopy, single molecule or microrheology particle tracking, and Fluorescence Recovery After Photobleaching (FRAP). These approaches allow measuring molecular dynamics in systems that do not move very much. However, the analysis of highly mobile intracellular condensates, such as IAV inclusions, poses significant challenges as these structures not only constantly move within the cell but also exchange material, fusing, and dividing, rendering the quantitation of internal rearrangements and diffusion coefficients of molecules within condensates inaccurate. As an alternative, we opted for measuring the kinetics and the exchange of material between IAV inclusions using the Fluorescence Loss After Photoactivation (FLAPh) technique. It involves pulse photoactivation of individual or pools of viral inclusions in the cell, and chasing over time in photoactivated and non-photoactivated regions. This approach is suitable for quantifying the movement and spatial distribution of components within inclusions over time, enabling the determination of both the distance and speed from a specific cellular location. As a result, this method allows the quantification of decay profiles, half-lives, decay constant rate, and mobile and immobile fractions in viral inclusions. It, therefore, enables high throughput screenings for compounds or host factors that affect this dynamism and indirectly allows assessing the material properties of IAV inclusions.

Humans↗
PubMed1991

Argyrophilic ubiquitinated cytoplasmic inclusions of Leu-7-positive glial cells in olivopontocerebellar atrophy (multiple system atrophy).

We described cytoplasmic inclusions in glial cells in 18 patients with olivopontocerebellar atrophy (OPCA) (multiple system atrophy, MSA). These glial inclusions showed intense argyrophilia with modified Bielschowsky's and Bodian's silver impregnation techniques, and were observed in the pons, cerebellar white matter, midbrain, medulla oblongata and basal ganglia, as well as cerebral white matter and spinal cord. None of the 54 control cases had glial argyrophilic inclusions. Immunohistochemically, these inclusions were intensely labeled by anti-ubiquitin antibody. Some of them reacted with an antibody to Rosenthal fiber (RF) protein. The cytoplasm of ubiquitinated inclusion-bearing glial cells was immunostained by anti-Leu-7 antibody, but not by anti-GFAP antibody. Ultrastructurally, the glial inclusions were composed primarily of approximately 24- to 40-nm fibrils, which were coated with osmiophilic granular material along their length in longitudinal section. These fibrils appeared as annuli in cross section. Often, a central granule approximately 5 nm in diameter was seen in the lucent lumen of a cross-sectioned fibril. The granule-coated fibrils were not seen in the glial filament-containing astrocytes. Electron microscopic examination of silver-impregnated specimens revealed that the granule-coated fibrils had strong affinity for silver. Immunoelectron microscopy using the indirect immunoperoxidase techniques with antibodies to ubiquitin and RF protein revealed that the electron-dense reaction products respective to both were located on constituents of glial inclusions. Our observation that Leu-7-positive glial cells, mainly oligodendroglial cells, had argyrophilic ubiquitinated inclusions may be of significance for the evaluation of the pathology of OPCA(MSA).

Adult↗
PubMed1989

An electron microscopic study of inclusion bodies in synaptic terminals of scrapie-infected animals.

Inclusion bodies consisting of vesicles of about 25 nm diameter and occurring in the synaptic terminals of scrapie-infected animals have been described by a number of people. In the present study these inclusion bodies were looked for in the neocortex, hippocampus and corpus callosum in a variety of strains of mice (C3H, LM, RIII, IM, VL) infected with different strains of scrapie agent (22C, 79A, ME7, 87V) after intracerebral inoculation. In plaque-bearing models of scrapie, terminals containing synaptic inclusion bodies were frequently found surrounding the amyloid plaque cores in the neocortex but not in the corpus callosum. In non-plaque-bearing models, terminals containing synaptic inclusion bodies were found in the neuropil of the neocortex and hippocampus. For all models, these bodies were either presynaptic or postsynaptic but were not, as a rule, found on both sides of the same synapse. Fibrillary material was frequently seen in the postsynaptic terminals containing the inclusion bodies in both the plaque- and non-plaque-bearing models. On one occasion fibrillary material was seen, together with the inclusion bodies, in a neuron cell body. Inclusion bodies were also seen in the neocortex of hamsters infected with the 263K strain of scrapie agent and a Cheviot sheep infected with the ME7 strain of agent. The inclusion bodies and the fibrillary material were thought to be derived from the breakdown of neurotubules.

Animals↗