Number Of Inclusion Bodies Research Articles

Fundamental insights in early-stage inclusion body formation.

Early-stage inclusion body formation is still mysterious. Literature is ambiguous about the existence of rod-shaped protein aggregates, a potential sponge-like inclusion body scaffold as well as the number of inclusion bodies per Escherichia coli cell. In this study, we verified the existence of rod-shaped inclusion bodies, confirmed their porous morphology, the presence of multiple protein aggregates per cell and modelled inclusion body formation as function of the number of generations.

C57BL/6 Background Attenuates mHTT Toxicity in the Striatum of YAC128 Mice.

Mouse models are frequently used to study Huntington’s disease (HD). The onset and severity of neuronal and behavioral pathologies vary greatly between HD mouse models, which results from different huntingtin expression levels and different CAG repeat length. HD pathology appears to depend also on the strain background of mouse models. Thus, behavioral deficits of HD mice are more severe in the FVB than in the C57BL/6 background. Alterations in medium spiny neuron (MSN) morphology and function have been well documented in young YAC128 mice in the FVB background. Here, we tested the relevance of strain background for mutant huntingtin (mHTT) toxicity on the cellular level by investigating HD pathologies in YAC128 mice in the C57BL/6 background (YAC128/BL6). Morphology, spine density, synapse function and membrane properties were not or only subtly altered in MSNs of 12-month-old YAC128/BL6 mice. Despite the mild cellular phenotype, YAC128/BL6 mice showed deficits in motor performance. More pronounced alterations in MSN function were found in the HdhQ150 mouse model in the C57BL/6 background (HdhQ150/BL6). Consistent with the differences in HD pathology, the number of inclusion bodies was considerably lower in YAC128/BL6 mice than HdhQ150/BL6 mice. This study highlights the relevance of strain background for mHTT toxicity in HD mouse models.

Degradation of Mutant Protein Aggregates within the Endoplasmic Reticulum of Vasopressin Neurons.

SummaryMisfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER.

Ulcerative Colitis and Cytomegalovirus Infection: From A to Z

Despite multiple studies, the role of cytomegalovirus [CMV] infection in exacerbating the severity of inflammation in ulcerative colitis [UC], and its response to treatment, remain debatable. Additionally, the optimal diagnostic tests for CMV infection in the setting of UC relapse, and timing of antiviral treatment initiation, remain unclear. The challenge faced by gastroenterologists is to differentiate between an acute UC flare and true CMV colitis. It seems that the presence of CMV colitis, as defined by the presence of intranuclear or intracellular inclusion bodies on haematoxylin and eosin [H&E] staining and/or positive immunohistochemistry [IHC] assay on histology, is associated with more severe colitis. Patients with CMV infection and acute severe colitis are more resistant to treatment with corticosteroids than non-infected patients. This refractoriness to steroids is related to colonic tissue CMV viral load and number of inclusion bodies [high-grade CMV infection] which may have a pronounced effect on clinical outcomes and colectomy rates. Whereas many studies showed no effect for antiviral treatment on colectomy rates in CMV-infected UC patients, there was a significant difference in colectomy rates of patients with high-grade infection who received anti-viral therapy compared with those who did not receive treatment. It was therefore proposed that high-grade CMV disease indicates that the virus is acting as a pathogen, whereas in those with low-grade CMV disease, the severity of IBD itself is more likely to influence outcome. The different algorithms that have been put forward for the management of patients with UC and concomitant CMV infection are discussed.

Construction and identification of the recombinant M13-IN5 phage and its effect on Chlamydia trachomatis

Objective To construct active phages against Chlamydia trachomatis, and to evaluate its effect on Chlamydia trachomatis. Methods The M13 phage was recombined with the IN5 sequences encoding the capsid protein VP1 of chlamydiophage phiCPG1, and then the recombinant M13-IN5 phage was obtained. PCR amplification, enzyme digestion and sequencing were performed to verify whether the target fragment was inserted into the phage successfully. The viability of the phage was evaluated by plaque formation assay. Cell counting kit-8 (CCK8) assay was conducted to evaluate the effect of M13 phage and recombinant M13-IN5 phage at the titer of 1011 plaque-forming units (PFU) /ml on the proliferation of Hela cells, and Hela cells uninfected with chlamydia served as the blank control group. Western blot analysis was performed to determine the expression of the IN5 loop protein in the recombinant M13-IN5 phage, M13 phage and Escherichia coli ER2738 at exponential growth phase. Cultured standard Chlamydia trachomatis serovar E strain was treated with M13 phage and recombinant M13-IN5 phage at the titer of 1011 PFU/ml separately, and chlamydia control group without the treatment with phages was set up. After 36-hour infection, confocal microscopy was performed to detect the location of the M13 phage and the recombinant M13-IN5 phage. Moreover, iodine staining was conducted to count inclusion bodies at 36, 48, 60 and 72 hours separately after infection. Statistical analysis was carried out by a two-sample t-test for comparisons between two groups, one-way analysis of variance (ANOVA) for intergroup comparison, and Bonferroni test for multiple comparisons. Results The bioactive recombinant M13 phage containing the IN5 loop gene was constructed successfully, and Western blot analysis confirmed that the recombinant phage expressed IN5 loop/pⅢ fusion protein with a high titer of 3.05 × 1011 PFU/ml. As CCK8 assay showed, there was no significant difference in proliferation of Hela cells among the blank control group, M13 phage group and recombinant M13-IN5 phage group (A450 values: 3.63 ± 0.01, 3.55 ± 0.02, 3.70 ± 0.01, respectively, F = 12.0, P > 0.05) . Confocal microscopy showed overlap between the phage fluorescence and chlamydial inclusion body fluorescence. The M13-IN5 phage group and M13 phage group both showed significantly decreased number of inclusion bodies compared with the control group (both P 0.05) . After 48, and 60 hours of chlamydial infection, the number of inclusion bodies did not differ among the M13 phage group, M13-IN5 phage group and control group (both P > 0.05) . Conclusions The recombinant M13-IN5 phage was bioactive and could successfully express the IN5 loop protein. In the in vitro experiments, the recombinant phage could enter into chlamydia inclusion bodies, and markedly inhibited the infection of Chlamydia trachomatis. Key words: Chlamydia trachomatis; Bacteriophage M13; DNA, recombinant; Inclusion bodies; IN5 loop

Roles of DegP in prevention of protein misfolding in the periplasm upon overexpression of penicillin acylase in Escherichia coli.

Enhancement of the production of soluble recombinant penicillin acylase in Escherichia coli via coexpression of a periplasmic protease/chaperone, DegP, was demonstrated. Coexpression of DegP resulted in a shift of in vivo penicillin acylase (PAC) synthesis flux from the nonproductive pathway to the productive one when pac was overexpressed. The number of inclusion bodies, which consist primarily of protein aggregates of PAC precursors in the periplasm, was highly reduced, and the specific PAC activity was highly increased. DegP was a heat shock protein induced in response to pac overexpression, suggesting that the protein could possibly suppress the physiological toxicity caused by pac overexpression. Coexpression of DegP(S210A), a DegP mutant without protease activity but retaining chaperone activity, could not suppress the physiological toxicity, suggesting that DegP protease activity was primarily responsible for the suppression, possibly by degradation of abnormal proteins when pac was overexpressed. However, a shortage of periplasmic protease activity was not the only reason for the deterioration in culture performance upon pac overexpression because coexpression of a DegP-homologous periplasmic protease, DegQ or DegS, could not suppress the physiological toxicity. The chaperone activity of DegP is proposed to be another possible factor contributing to the suppression.

Estrogen induces cytokeratin aggregation in primary cultures of Armenian hamster hepatocytes

The effect of estrogen administration to cultured Armenian hamster was studied. Isolated Armenian hamster hepatocytes were cultured in RPMI medium supplemented with beta-estradiol (E2). Beta-estradiol treatment for 24-48 hr induced cytoplasmic inclusion bodies which by immunocytochemistry were positive for cytokeratin (CK) 8, CK 18, and ubiquitin but negative for CK 7 and CK 19. These inclusion bodies appeared as filamentous tangles or amorphous aggregates when observed by electron microscopy. F-actin, tubulin, and desmosomes were not influenced by the presence of the inclusion bodies. Addition of ethanol to culture medium increased the incidence of the inclusion formation. In combination with 0.5% ethanol 1 microM of E2 induced five to six times more inclusion bodies, while the number of inclusion bodies decreased when epidermal growth factor (EGF) was added to the medium in combination with E2. This reduction effect was nullified by treatment with anti-EGF receptor antibody. These findings suggest that E2 treatment to Armenian hamster hepatocytes in vitro induces Mallory body-like inclusions whose incidence can be influenced by addition of ethanol or EGF to the culture medium.

The effect of polyethylene glycol (PEG) on the isolation of Chlamydia trachomatis in H-1 HeLa cells

One hundred and ninety-six urethral and endocervical swabs were processed for isolation of C. trachomatis, using H-1 HeLa cells on shell vials, in the presence and absence of 7% PEG in the chlamydial overlay medium. The results were divided into three evaluable groups based on the number of inclusions per coverslip in the shell vials without PEG. The number of inclusions were compared with that of PEG-treated cultures using the paired t-test. The ranges for the three evaluable groups were one to ten, 11-100 and 101-1000 inclusions per coverslip. All three groups showed a significant increase (2.8- to 3.8-fold) in the number of inclusion bodies in the PEG-treated cultures compared to the untreated cultures.

Effects of cisplatin on testicular tissue and the Leydig cell-pituitary axis.

The mechanisms responsible for the rapid increases in levels of gonadotropin and steroid hormones upon administration of cisplatin to rats are not clearly understood. In the present study, the morphologic effects of cisplatin on the testis and the relationship of these changes to serum hormone levels were studied in adult rats. Upon administration of a single dose (5 mg/kg) of cisplatin, the levels of testosterone in the peripheral blood decreased and the level of luteinizing hormone (LH) increased. After 3 days, significant damage to the germinal epithelium became detectable by morphologic examination. In Leydig and Sertoli cells, dilatation of the endoplasmic reticulum, structural changes in mitochondria and an increase in the numbers of inclusion bodies were detected. Although blood-testis barriers between Sertoli cells were normal, the relationship between germinal cells and the relationship between germinal cells and Sertoli cells were distorted. It is concluded that damage to Leydig cells and withdrawal of the controlling effects of the germinal epithelium on the LH-Leydig cell axis could explain the changes in serum hormone levels.

Increased oxidant resistance of alveolar macrophages isolated from rats repeatedly exposed to cadmium aerosols

This study investigated potential mechanisms of oxidant resistance in alveolar macrophages (AM) isolated from Lewis rats exposed repeatedly to cadmium aerosols. Macrophages from Cd-adapted animals exhibited significantly greater resistance to oxidant-induced cytotoxicity than control cells when challenged with hydrogen peroxide in vitro. Elevations in glutathione peroxidase and glutathione reductase activities were associated with increased oxidant tolerance but catalase activity was unchanged. Metallothionein (MT) expression (protein and mRNA) was dramatically up-regulated in response to in vivo Cd exposure. A study using immunocytochemistry and in situ hybridization techniques revealed significant heterogeneity in the expression of metallothionein by AMs. The percentage of AMs positive for MT (protein and mRNA) and the degree of MT expression within individual cells increased in response to additional Cd exposures. A putative state of activation was suggested by the differences in size and number of inclusion bodies in macrophages from Cd-adapted animals and by secretion of a cytokine with interleukin-1-like characteristics. In summary, AMs from Cd-adapted animals are distinguished from control cells with respect to: (1) increased oxidant resistance, (2) secretion of cytokines, (3) elevations in enzymes associated with glutathione metabolism, and (4) up-regulation in metallothionein expression.

Comparison of Pharmacia Chlamydia EIA, IDEIA and Cell Culture in the Detection of Urogenital Chlamydial Infection

Two commercial test kits, Pharmacia Chlamydia EIA (PhEIA) and IDEIA Chlamydia Test, for the identification of Chlamydia trachomasis and McCoy cell culture were compared in urogenital specimens. The sediments of the transportation buffers of specimens with discordant results were investigated for elementary bodies (EB) with fluorescein-labelled antichlamydial antibodies. The prevalence of chlamydial infection among the men was 16% (48 of 293), 47 culture positive and one EB positive, and among the women 10% (10 of 97), 10 culture positive. In men, the sensitivity of PhEIA, IDEIA and culture was 71%, 40% and 98%, respectively. In women, irrespective of site, corresponding figures were 100%, 80% and 100%. The specificity and positive predictive values were 100% for both enzyme immunoassays in men and women. The low sensitivity of IDEIA could not be explained by the degree of infection as measured by the number of inclusion bodies in cell culture, the presence of antigen as measured by the number of EBs or the sampling order.

The fate of intramitochondrial paracrystalline inclusion bodies in germ line cells of water frogs (Amphibia, Anura)

Numerous intramitochondrial paracrystalline inclusion bodies (ICIB) were observed in the germinal plasm of a mid-blastula, and in primordial germ cells (PGCs) after their migration to the germinal ridges, in Rana ridibunda, R. lessonae and R. esculenta. In oogonia the number of ICIB decreases rapidly. Single ICIB are observed in the germ cells up to the leptotene stage; they have never been observed in pachytene oocytes. In diplotene oocytes that have reached a diameter of about 100 microns ICIB are visible again, and their number increases concomitantly with oocyte growth.

Evaluation of a commercial enzyme immunoassay versus culture for the detection ofChlamydia trachomatis

A commercial EIA (Chlamydiazyme) for detection of Chlamydia trachomatis was evaluated in comparison to culture using genital specimens from 472 men and 279 women. The sensitivity of the EIA compared with culture was 66.0% in men and 71.4% in women, while the specificity was 99.7% and 95.9% respectively. The EIA failed more often to detect chlamydial antigen when the number of inclusion bodies found in the corresponding cultures was less than or equal to 100/well. A direct test (MicroTrak) was performed on the EIA samples which showed discordant results compared to corresponding cultures. One of 17 EIA positive samples, and 12 of 36 EIA negative samples were positive in the direct test (p less than 0.05). A cut-off absorbance value of 0.1 is recommended by the manufacturer in the EIA. However, 84.2% of the EIA negative samples in the negative absorbance interval 0.05-0.099 corresponded with a positive culture. In view of variations in the sensitivity of the culture technique between laboratories and the low sensitivity of the EIA found in this study, it is recommended that each laboratory using the EIA compare it to culture. It is also recommended that an equivocal zone around the cut-off value be used in the EIA, the zone to be established by each laboratory using the test.

Quantitative changes of Holmes positive nucleolus-like inclusion bodies in the mouse locus coeruleus under various experimental conditions.

The author demonstrated earlier that the nucleolus-like inclusion bodies in the central nervous system observed under an electron microscope are characteristically stained by the modified Holmes silver impregnation method. These inclusion bodies were then counted in the entire mouse brain; they numbered 1,547 +/- 144 on the unilateral side of the locus coeruleus. In the present experiment, mice were subjected to various experimental conditions or given several medicaments that might induce changes in body weight and in the number of inclusion bodies. Following dehydration, fasting, dehydration with fasting, induction of stress condition, reserpine, alpha-methyl-p-tyrosine, cycloheximide, cephalexin and puromycin administrations, body weight was decreased along with a decrease in the number of inclusion bodies in the locus coeruleus. After streptomycin sulfate injection, however, body weight was increased and inclusion bodies showed a moderate increase in number. Actinomycin D and mitomycin C, inhibitors of nucleic acid synthesis, brought about a definite decrease in body weight, but little if any changes in the number of inclusion bodies. Furthermore, after reserpine injection several small inclusion bodies were observed in the cytoplasm of locus coeruleus cells in a recovery stage and after inclusion bodies regained their normal number and size. The results suggested that the inclusion bodies might have some relation to body weight, as well as to monoamine of the brain and protein synthesis.

Establishment of a persistent baculovirus infection in a lepidopteran cell line

The paper describes the first overt attempt to establish an insect cell line ( Spodoptera frugiperda), persistently infected with its homologous baculovirus. The persistently infected cells were morphologically different and grew to a higher density than the noninfected parent line. The parent line, however, had a shorter doubling time. Persistently infected cells were passaged 40 times over 10 months; they still continued to produce infectious virus and polyhedral inclusion bodies. However, the infectious viral titer was ca. 100 times lower in the persistently infected line than in the parent line; also, the number of inclusion bodies was reduced ca. 98%. Interference with both homologous and heterologous baculoviruses was demonstrated in the persistently infected cell line. Sevently percent of the persistently infected cells contained antigens for S. frugiperda nuclear polyhedrosis virus, ca. 1% of the cells showed infectious viral centers, and ca. 3% of the cells contained inclusion bodies. Although the inclusion bodies from the persistently infected cells were infectious for S. frugiperda larvae, they were about 3 times less infectious than the inclusion bodies produced in the parent line.

Dermosporidium penneri sp. n. from the Skin of the American Toad, Bufo americanus (Amphibia: Bufonidae)

A protozoan parasite, Dermosporidium penneri sp. n., was found in the skin of the American Toad, Bufo americanus, in Connecticut. The life cycle was not determined. Attempts to infect other B. americanus were unsuccessful. This parasite differed from the four other described species in sizes of the cyst and of the spore, and in the number of inclusion bodies. Other members of this genus are known from Brazil and Czechoslovakia. Cysts are subcutaneous, in the ventral skin, spherical in shape, 4 to 8 mm in diameter. The spores are 10 to 12 ,um in diameter and contain about 70 inclusion bodies, each 1.2 ,um in diameter. All the infected toads were found within a 1.6-km area along a single road in Mansfield, Connecticut, USA.

Pathology of rabies: a light- and electron-microscopical study with particular reference to the changes in cases with prolonged survival.

Light- and electron-microscopical studies were conducted on necropsy material from six cases of rabies encephalitis including three with the unusual feature of surviving for over 14 days as a result of intensive medical care. This included administration of antiviral agents and interferon inducers and prevention of hypoxia by intermittent positive pressure ventilation. In all these cases, typical Negri bodies were demonstrated. Inflammatory reaction was absent or minimal. Unlike the cases with short survival where Negri bodies were infrequently seen and restricted mostly to the hippocampus, in cases with prolonged survival, they were present in large number, widely distributed throughout the grey matter of the brain. The associated inflammatory reaction in these cases, however, did not keep pace with the increase in number of inclusion bodies. Peripheral neuritis was observed in two of these cases, which also showed myelitis involving the cervical region and inflammation of dorsal root ganglia. One of them showed necrosis and severe inflammation of the lower cervical sympathetic ganglion. An electron-microscopical study conducted in four cases showed three forms of the inclusion body in the cytoplasm of neurons.

Ultrastructural expression of virus-like particles in human leukaemic cells growing in vitro

The ultrastructure of newly discovered virus-like particles in human leukaemic myeloblasts and lymphoblasts growing in vitro has been studied. Three EBNA-negative cultures contained cytoplasmic inclusion bodies which consist probably of viroplasms. After addition of 5-bromo-2-deoxyuridine (BUDR) or dimethyl sulphoxide (Me 2SO) to the cultured cells there was an approximately 50-fold increase in the number of inclusion bodies, supporting the notion that these bodies are the site of synthesis of viral structural proteins. A suggested sequence of development of inclusion bodies into virus-like particles is described and illustrated. Similar inclusions have been reported to date only in fresh human leukaemic cells but not in cell cultures. The availability of these particles for further studies will help to determine whether they play a role in the aetiology of human leukaemia. EBNA-positive human haemic cell lines studied did not contain similar intracytoplasmic inclusion bodies, and treatment of two of them with BUDR did not produce inclusion bodies.

Ultrastructural investigation of various membrane configurations in Nicotiana tabacum

Lamellar and tubular inclusion bodies in the cell vacuole became more numerous as the severity of plant stunting was increased. In normal-appearing greenhouse plants, only a few of these inclusions were seen. In vacuoles of the severely stunted plants, much larger quantities of inclusion bodies were observed. In severely stunted plants, lamellar inclusions occurred that contained osmiophilic globules along the lamellar strands. These types of lamellar inclusions were also seen accumulating in the starch cavities of chloroplasts. From these observations, we postulated that a cell, with a given capacity to produce membrane material, produces membranous material in anticipation of growth. When the cell does not grow, owing to stunting, this excess of membrane material forms the large number of inclusion bodies seen in the vacuoles of stunted cells.

Electron microscopy of African cereal streak diseased plants

The African cereal streak disease (ACS) was further characterized as to transmissibility and cytological changes produced in cells of affected plants. Virus-like particles were regularly associated with diseased plants grown under field conditions, but no similar particles could be found in diseased plants of wheat, triticale, rice, Harpachne schimperi, Chloris pycnothrix, or Eragrostis tenuifolia grown in a growth cabinet set on an 18-h–6-h light–dark cycle and a corresponding 24 °C–17.5 °C temperature cycle. The efficiency of continuous transmission by Toya catilina Fenn. under growth-cabinet conditions was low, at 5%, 3%, and 5% for females, males, and nymphs, respectively.Except for the absence of recognizable virus-like particles in infected plants grown in the growth cabinet, the symptoms and cytological features of diseased plants grown under growth-cabinet and field conditions were similar. The main cytological features of diseased plants were as follows. (1) A dense, fine ground substance which filled many of the phloem cells was developed. (2) Large numbers of inclusion bodies, which were of globular and hexagonal-elongated forms, were formed. These forms are probably related. Virus-like particles were occasionally associated with the inclusions in diseased field-grown plants (3) Non-specific changes, such as may be induced by various types of pathogens, or are associated with senescence, e.g. phloem cell necrosis, abnormal callose-like cell wall deposits, and degenerative changes in the chloroplasts, were observed.