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At least 127 records · Page 7Linked to original sources
PubMed2005

Development of secondary inclusions in cells infected by Chlamydia trachomatis.

The chlamydiae are obligate intracellular bacteria that occupy a non-acidified vacuole (the inclusion) during their entire developmental cycle. These bacteria produce a set of proteins (Inc proteins) that localize to the surface of the inclusion within infected cells. Chlamydia trachomatis IncA is also commonly found in long fibers that extend away from the inclusion. We used standard and confocal immunofluorescence microscopy to demonstrate that these fibers extend to newly developed inclusions, termed secondary inclusions, within infected cells. Secondary inclusions observed at early time points postinfection were devoid of chlamydial reticulate bodies. Later in the developmental cycle, secondary inclusions containing variable numbers of reticulate bodies were common. Reticulate bodies were also observed within the IncA-laden fibers connecting primary and secondary inclusions. Quantitative differences in secondary inclusion formation were found among clinical isolates, and these differences were associated with serovar. Isolates of serovar G consistently produced secondary inclusions at the highest frequency (P < 0.0001). Similar quantitative studies demonstrated that secondary inclusion formation was associated with segregation of inclusions to daughter cells following cytokinesis. We conclude that the production of secondary inclusions via IncA-laden fibers allows chlamydiae to generate an expanded intracellular niche in which they can grow and may provide a means for continuous infection within progeny cells following cell division.

Bacterial Proteins↗
PubMed1999

Intranuclear inclusions of meningioma associated with abnormal cytoskeletal protein expression.

We describe a case of meningothelial meningioma with a large number of intranuclear inclusions. Morphologically, these are divided into cytoplasmic inclusions and nuclear vacuoles. The cytoplasmic inclusion has a limiting membrane with cell organelles and filaments. Inclusions of this type are generally eosinophilic, like the cytoplasm. However, there are many inclusions that are more eosinophilic than the cytoplasm or that have a ground-glass appearance. Some of them may contain fine or coarse granules. On the other hand, the nuclear vacuole lacks a limiting membrane and appears empty. In most of the inclusions of this type, there is a faintly basophilic substance in the margin. Generally, the cytoplasmic inclusions are as immunopositive as cytoplasm with vimentin, but some of these cytoplasmic inclusions are more reactive. Under the electron microscope, abnormal aggregation of intermediate filaments is recognized in the cytoplasmic inclusions. It is considered that a strong reaction of cytoplasmic inclusions with vimentin immunostaining is due to abnormal aggregation of intermediate filaments. The present study distinctly demonstrates abnormal localization of intermediate filaments in the cytoplasmic inclusions, and it is suggested that the cytoskeleton participates in the evolution of the cytoplasmic inclusions.

Aged↗
PubMed1982

Unusual cytoplasmic inclusion bodies in medullary carcinoma of the thyroid gland.

Medullary carcinoma of the thyroid from a 43-year-old patient showed 4 types of unusual cytoplasmic inclusion bodies ultrastructurally. Type 1 inclusion is composed of rod and doughnut-shaped bodies aggregated in the cytoplasma and not enclosed by a limiting membrane. Typical neurosecretory granules are intermingled with the rod and doughnut-shaped bodies. The doughnut-shaped bodies often have an electron dense core which is similar to the neurosecretory granules. The rod-shaped bodies are bounded by trilaminar unit membrane and have a slightly electron dense matrix. A centriole is often found associated with this type of inclusion. Type 2 inclusion is composed of small secretory granules and clear vesicles. These inclusions can be found either with or without surrounding membrane. Type 3 inclusion is a large osmiophilic body, or secondary lysosome, in which some degenerated organelles are discernible. Type 4 inclusion is a fragmented nucleus with condensation and margination of the chromatin, so-called karyorrhexis. Routine hematoxylin and eosin sections did not show each type of inclusion but semithin sections stained with Paragon suggested 4 different types of inclusions. Type 3 and 4 inclusions represent forms of degeneration and cell death in the tumor cells and probably represent a remote effect of radiation. Type 1 inclusion is not commonly associated with cell death and may represent a unique organellar aberration in these tumor cells. Type 2 inclusion is simply an aggregation of secretory granules and clear vesicles.

Adult↗
PubMed2003

Rab GTPases are recruited to chlamydial inclusions in both a species-dependent and species-independent manner.

Chlamydiae are obligate intracellular bacteria that replicate within an inclusion that is trafficked to the peri-Golgi region where it fuses with exocytic vesicles. The host and chlamydial proteins that regulate the trafficking of the inclusion have not been identified. Since Rab GTPases are key regulators of membrane trafficking, we examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydia-infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFP-Rab1, which functions in endoplasmic reticulum (ER)-to-Golgi trafficking, are recruited to Chlamydia trachomatis, Chlamydia muridarum, and Chlamydia pneumoniae inclusions, whereas GFP-Rab5, GFP-Rab7, and GFP-Rab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi-to-ER and endosome-to-Golgi trafficking, is associated with C. trachomatis inclusions but not with C. pneumoniae or C. muridarum inclusions, while the opposite was observed for the Golgi-localized GFP-Rab10. Colocalization studies between transferrin and GFP-Rab11 demonstrate that a portion of GFP-Rab11 that localizes to inclusions does not colocalize with transferrin, which suggests that GFP-Rab11's association with the inclusion is not mediated solely through Rab11's association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after disassembly of microtubules, which disperses recycling endosomes and the Golgi apparatus within the cytoplasm, suggesting a specific interaction with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with C. trachomatis IncG at the inclusion membrane. Therefore, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may function to regulate the trafficking or fusogenic properties of the inclusion.

Base Sequence↗
PubMed2002

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.

Animals↗
PubMed1975

Effect of dose of estradiol and age of animals on intranuclear inclusions in mammotrophs of the Mongolian gerbil.

Intranuclear inclusions have been examined in mammotrophs of the Mongolian gerbil. No inclusions were identified in nuclei of newborn females, although inclusions were seen in the pituitary gland of young mature females. The number of inclusions in retired breeders was similar to that in young females. Estradiol benzoate increased the number of inclusions, although 10 mug/day for 5 or 14 days induced more inclusions than 5 or 100 mug for 14 days; significantly fewer inclusions were seen with the higher dose (2.81 +/- 0.10 inclusions per field for 10 mug and 2.19 +/- 0.01 inclusions for 100 mug). Significantly fewer inclusions were present at 3 and 4 weeks. The reduced number of inclusions may be attributable at least in part to cellular hypertrophy of mammotrophs which was especially prominent. Vacuolar inclusions predominated at 3 and 4 weeks and there were fewer membranous types than at previous times.

Age Factors↗
PubMed2005

Different proportions of aneusomic cells in ovarian inclusion cysts associated with serous borderline tumours and serous high-grade carcinomas support different pathogenetic pathways.

Ovarian serous tumours may arise from the ovarian surface epithelium or from ovarian cortical epithelial inclusion cysts. However, little is known about the pathogenetic mechanisms involved in the progression from ovarian surface epithelium or inclusion cysts to neoplastic disease. In the present study, chromosomal aberrations typical of ovarian serous tumours were studied in ovarian surface epithelium and inclusion cysts. Ten ovaries with inclusion cysts obtained from patients without a gynaecological tumour, as well as 15 serous borderline tumours and 16 invasive high-grade serous carcinomas with inclusion cysts either in the ipsi- or in the contralateral ovary, were investigated by fluorescence in situ hybridization (FISH) using centromere enumeration probes directed against chromosomes 1, 6, 7, and X. The proportions of aneusomic cells were assessed. Trisomies 1 and 7 and monosomies 6 and X were present in the surface epithelium, inclusion cysts, and tumours, providing evidence for a link between the surface epithelium, and inclusion cysts, and serous neoplasia. Inclusion cysts generally harboured more aneusomic cells than the associated surface epithelium, suggesting an influence of the ovarian stroma on the development of chromosomal instability. Moreover, inclusion cysts associated with borderline tumours displayed a higher proportion of aneusomic cells than inclusion cysts associated with invasive high-grade carcinoma and than inclusion cysts in ovaries without neoplastic disease. These results suggest a genetic field defect of the inclusion cyst epithelium in serous borderline tumours. Invasive high-grade serous carcinomas, by contrast, may arise from single cell clones subject to a different set of genetic events.

Aneuploidy↗
PubMed1987

Ultrastructural cytochemical analysis of intranuclear arsenic inclusions.

To establish the chemical composition of the arsenic inclusion, freshly isolated preparations of inclusions and epon-embedded thin sections of inclusions were subjected to ultrastructural cytochemical analysis. Intranuclear inclusions are composed of amorphous, arsenic-containing subunits aligned linearly to form a coiled complex. Lipase, ribonuclease, deoxyribonuclease, trypsin, pepsin, protease, amylase, or ethylenediaminetetraacetic acid (EDTA) was used to digest or chelate these inclusions. Following enzymatic digestion or chelation, the electron opacity of inclusions was compared with that of control sections exposed for equal times to equivalent solutions lacking the enzymes. Exposure to amylase caused a consistent reduction in the electron opacity of thin sections of inclusions and almost complete digestion of the freshly isolated preparations of inclusions. This was indicative of the presence of a carbohydrate moiety within arsenic inclusions. Incubation of inclusions with EDTA resulted in solubilization of freshly isolated and thin-sectioned embedded material. These data indicated that the intranuclear arsenic inclusion is composed of both metallic and carbohydrate moieties, confirming earlier studies which identified arsenic within inclusions using instrumental neutron activation analysis and X-ray microprobe analysis.

Amylases↗
PubMed1996

The late chlamydial inclusion membrane is not derived from the endocytic pathway and is relatively deficient in host proteins.

Chlamydiae are obligate intracellular parasites which multiply within infected cells in a membrane-bound structure termed an inclusion. Newly internalized bacteria are surrounded by host plasma membrane; however, the source of membrane for the expansion of the inclusion is unknown. To determine if the membrane for the mature inclusion was derived by fusion with cellular organelles, we stained infected cells with fluorescent or electron-dense markers specific for organelles and examined inclusions for those markers. We observed no evidence for the presence of endoplasmic reticulum, Golgi, late endosomal, or lysosomal proteins in the inclusion. These data suggest that the expansion of the inclusion membrane, beginning 24 h postinoculation, does not occur by the addition of host proteins resulting from either de novo host synthesis or by fusion with preexisting membranes. To determine the source of the expanding inclusion membrane, antibodies were produced against isolated membranes from Chlamydia-infected mouse cells. The antibodies were demonstrated to be solely against Chlamydia-specified proteins by both immunoprecipitation of [35S]methionine-labeled extracts and Western blotting (immunoblotting). Techniques were used to semipermeabilize Chlamydia-infected cells without disrupting the permeability of the inclusion, allowing antibodies access to the outer surface of the inclusion membrane. Immunofluorescent staining demonstrated a ring-like fluorescence around inclusions in semipermeabilized cells, whereas Triton X-100-permeabilized cells showed staining throughout the inclusion. These studies demonstrate that the inclusion membrane is made up, in part, of Chlamydia-specified proteins and not of existing host membrane proteins.

3T3 Cells↗
PubMed1996

Inclusion body myositis: clinical and pathological boundaries.

Inclusion body myositis, polymyositis, and dermatomyositis are three distinct categories of inflammatory myopathy. Some authorities commented on the selective early weakness of the volar forearm muscles, quadriceps, and ankle dorsiflexors in inclusion body myositis. The most important feature distinguishing inclusion body myositis from the other two inflammatory myopathies is the lack of responsiveness to immunosuppressive treatment. Although most patients with inclusion body myositis have characteristic muscle biopsy findings, some cannot be distinguished histologically early from polymyositis. Predicting responsiveness to immunosuppressive medications, independent of muscle histology, would be valuable to clinicians. We retrospectively reviewed the pattern of weakness and other clinical features of 46 patients newly diagnosed with either inclusion body myositis, polymyositis, or dermatomyositis. Asymmetrical muscle weakness with prominent wrist flexor, finger flexor, and knee extensor involvement was specific for inclusion body myositis and unresponsive polymyositis. Male sex, lower creatine kinase levels, slower rate of progression, and peripheral neuropathy were also more common in inclusion body myositis and unresponsive polymyositis than in responsive polymyositis and dermatomyositis patients. Repeat muscle biopsy in 2 patients in the unresponsive polymyositis group demonstrated histological features of inclusion body myositis. We suspect that patients with clinical features of inclusion body myositis but lacking histological confirmation may nonetheless have inclusion body myositis. Our study supports the recently proposed criteria for definite and possible inclusion body myositis.

Aged↗
PubMed1993

Comparative light and electron microscopic analyses of tenuivirus major noncapsid protein (NCP) inclusion bodies in infected plants, and of the NCP in vitro.

Tenuivirus infections are associated with the formation of abundant inclusion bodies and with the accumulation of large quantities of a viral noncapsid protein (NCP) in infected plants. Examination of maize stripe virus and rice hoja blanca virus-infected plant tissues using light, immunofluorescent, and electron microscopy showed that the inclusion bodies induced by the two viruses were very similar. Light microscopy revealed that both induced arrays of ring-like, figure-eight-like, and amorphous inclusions, frequently with a substructure of needle-shaped crystals. Immunofluorescent staining showed that all types of inclusion bodies contained the viral NCP but not the viral N protein, associated to the viral RNA. Electron microscopy revealed abundant amorphous semi-electron-opaque inclusion bodies; these had a fibrillar appearance but also occurred as compact, more electron-dense structures. Filamentous electron-opaque inclusion bodies were also detected. Immunogold labeling of ultrathin sections confirmed that all inclusion bodies included NCP and that none included viral N protein. Examination of purified NCP showed that it can form similar amorphous and crystalline arrays in vitro to the inclusion bodies observed in vivo. We propose that the common presence of NCP in all inclusion bodies implies the existence of a single type of intracellular inclusion body, the different developmental stages of which have previously been considered to be distinct inclusion bodies.

Inclusion Bodies, Viral↗
PubMed2003

Bioprocessing of therapeutic proteins from the inclusion bodies of Escherichia coli.

Escherichia coli has been most extensively used for the large-scale production of therapeutic proteins, which do not require complex glycosylation for bioactivity. In recent years tremendous progress has been made on the molecular biology, fermentation process development and protein refolding from inclusion bodies for efficient production of therapeutic proteins using E. coli. High cell density fermentation and high throughput purification of the recombinant protein from inclusion bodies of E. coli are the two major bottle necks for the cost effective production of therapeutic proteins. The aim of this review is to summarize the developments both in high cell density, high productive fermentation and inclusion body protein refolding processes using E. coli as an expression system. The first section deals with the problems of high cell density fermentation with an aim to high volumetric productivity of recombinant protein. Process engineering parameters during the expression of ovine growth hormone as inclusion body in E. coli were analyzed. Ovine growth hormone yield was improved from 60 mg L(-1) to 3.2 g L(-1) using fed-batch culture. Similar high volumetric yields were also achieved for human growth hormone and for recombinant bonnet monkey zona pellucida glycoprotein expressed as inclusion bodies in E. coli. The second section deals with purification and refolding of recombinant proteins from the inclusion bodies of E. coli. The nature of inclusion body protein, its characterization and isolation from E. coli has been discussed in detail. Different solubilization and refolding methods, which have been used to recover bioactive protein from inclusion bodies of E. coli have also been discussed. A novel inclusion body protein solubilization method, while retaining the existing native-like secondary structure of the protein and its subsequent refolding in to bioactive form, has been discussed. This inclusion body solubilization and refolding method has been applied to recover bioactive recombinant ovine growth hormone, recombinant human growth hormone and bonnet monkey zona pellucida glycoprotein from the inclusion bodies of E. coli.

Bacterial Proteins↗
PubMed1996

Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630.

An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a variety of substrates and to produce large amounts of three different types of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large numbers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morphology occurred in small amounts. The electron-transparent inclusions of alkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major component of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in gluconate- or olive-oil-grown cells, respectively. The fatty acid composition of the inclusions depended on the substrate used for cultivation. In cells cultivated on n-alkanes, the composition of the fatty acids was related to the substrate, and intermediates of the beta-oxidation pathway, such as hexadecanoic or pentadecanoic acid, were among the acylglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-grown cells. This indicated that strain PD630 utilized beta-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydrocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, and phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have definite structures, probably with membranes at their surfaces, which allow them to maintain their shape, and with some associated proteins, probably involved in the inclusion formation.

Fatty Acids↗
PubMed2006

The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, and provides an anchor to target other proteins.

In Ralstonia eutropha, the H16 phasin PhaP1 represents the major phasin that binds to the surface of polyhydroxyalkanoate (PHA) inclusions. In this study, C-terminal fusions of PhaP1 with enhanced green fluorescent protein (eGFP) and with Escherichia coli beta-galactosidase (LacZ) were expressed separately in the triacylglycerol (TAG)-accumulating actinomycetes Rhodococcus opacus PD630 and Mycobacterium smegmatis mc(2)155, employing the M. smegmatis acetamidase (ace) promoter of the Escherichia-Mycobacterium/Rhodococcus shuttle plasmid pJAM2. PhaP1 and the PhaP1 fusion proteins were expressed stably in the recombinant strains. Western blot analysis of cell fractions of Rh. opacus revealed that PhaP1 and the PhaP1-eGFP fusion protein were associated with the TAG inclusions, whereas no phasin or phasin fusion protein was detected in the soluble and membrane fractions. Additional electron microscopy/immunocytochemistry studies demonstrated that PhaP1 was mainly located on the surface of intracellular TAG inclusions; in addition, some PhaP1 also occurred at the plasma membrane. Fluorescence microscopic investigations of the subcellular distribution of the PhaP1-eGFP fusion protein in vivo and on isolated TAG inclusions revealed that the fusion protein was bound to TAG inclusions at all stages of their formation, and to some extent at the cytoplasmic membrane. The PhaP1-LacZ fusion protein also bound to the TAG inclusions, and could be separated together with the inclusions from Rh. opacus crude extracts, thus demonstrating the immobilization of beta-galactosidase activity on the inclusions. This is believed to be the first report demonstrating the ability of PhaP1 to bind to lipid inclusions in addition to PHA inclusions. Furthermore, it was demonstrated that this non-specificity of PhaP1 can be utilized to anchor enzymically active fusion proteins to a matrix of bacterial TAG inclusions.

Bacterial Proteins↗
PubMed2002

Inhibition of fusion of Chlamydia trachomatis inclusions at 32 degrees C correlates with restricted export of IncA.

Chlamydia trachomatis is an obligate intracellular bacterium that develops within a parasitophorous vacuole termed an inclusion. The inclusion is nonfusogenic with lysosomes but intercepts lipids from a host cell exocytic pathway. Initiation of chlamydial development is concurrent with modification of the inclusion membrane by a set of C. trachomatis-encoded proteins collectively designated Incs. One of these Incs, IncA, is functionally associated with the homotypic fusion of inclusions. Inclusions also do not fuse when cultures are multiply infected with C. trachomatis and cultivated at 32 degrees C. We obtained evidence linking these experimental observations by characterizing IncA localization in 32 degrees C cultures. Analysis of inclusions by light and transmission electron microscopy confirmed that HeLa cells infected with multiple C. trachomatis elementary bodies and cultivated at 32 degrees C for 24 h contained multiple, independent inclusions. Reverse transcriptase PCR and immunoblot analyses of C. trachomatis-infected HeLa cells demonstrated the presence of IncA at 24 h in 32 degrees C cultures. When parallel cultures were probed with IncA-specific antibodies in indirect immunofluorescence assays, IncA was detectable in intracellular chlamydiae but not within the inclusion membrane. In addition, analysis of purified reticulate bodies from 37 and 32 degrees C cultures showed that bacterium-associated pools of IncA are enriched in cultures grown at 32 degrees C. Microscopic observation of infected cells revealed that some vacuoles had fused by 48 h postinfection, and this finding was correlated with the detection of IncA in inclusion membranes by immunofluorescence microscopy. The data are consistent with a requirement for IncA in fusions of C. trachomatis inclusions and suggest that the effect of incubation at 32 degrees C is manifested by restricted export of IncA to the inclusion membrane.

Bacterial Proteins↗
PubMed1973

A paracrystalline inclusion formed during sporulation of enterotoxin-producing strains of Clostridium perfringens type A.

A large paracrystalline inclusion is formed by certain strains of Clostridium perfringens type A during spore morphogenesis. In most cell thin sections, the inclusion appeared rod-shaped when sectioned at an angle perpendicular to its longer axis, and circular or oval-shaped when sectioned at an angle parallel to its longer axis. Measurements performed on electron micrographs of inclusions sectioned to reveal the rod shape indicated a fairly consistent thickness (width) of 192 +/- 23 nm. The length of the inclusions varied considerably with a maximum of approximately 2,120 nm being observed. Ultrastructurally, the inclusion was composed of closely packed, periodically spaced, parallel layers. Usually a single inclusion was randomly located in the cytoplasm of the cell. Two inclusions per cell were rarely observed. The inclusion was formed only by ent(+) strains of C. perfringens. Mutants of the ent(+) strain NCTC 8798 that were altered in their sporulating and enterotoxin-producing capacities and revertants of these mutants were tested for inclusion formation. The results indicate that, as with the ent(+) trait, a direct relationship exists between inclusion formation and spore formation. The synthesis of enterotoxin, formation of a morphologically distinct inclusion, and the initial deposition of discontinuous coat fragments around the forespore appear to be events closely related in time during spore morphogenesis.

Clostridium↗
PubMed2005

Solubilization and refolding of bacterial inclusion body proteins.

Inclusion bodies produced in Escherichia coli are composed of densely packed denatured protein molecules in the form of particles. Refolding of inclusion body proteins into bioactive forms is cumbersome, results in poor recovery and accounts for the major cost in production of recombinant proteins from E. coli. With new information available on the structure and function of protein aggregates in bacterial inclusion bodies, it has been possible to develop improved solubilization and refolding procedures for higher recovery of bioactive protein. Inclusion bodies are formed from partially folded protein intermediates and are composed of aggregates of mostly single types of polypeptide. This helps to isolate and purify the protein aggregates to homogeneity before solubilization and refolding. Proteins inside inclusion body aggregates have native-like secondary structures. It is assumed that restoration of this native-like secondary structure using mild solubilization conditions will help in improved recovery of bioactive protein in comparison to solubilization using a high concentration of chaotropic agent. Analysis of the dominant forces causing aggregation during inclusion body formation provides information to develop suitable mild solubilization procedures for inclusion body proteins. Refolding from such solubilized protein will be very high due to restoration of native-like secondary structure. Human growth hormone inclusion bodies were purified to homogeneity from E. coli cells before solubilization and refolding. Pure inclusion bodies were solubilized at alkaline pH in the presence of 2 M urea solution. The solubilized proteins were refolded using a pulsatile renaturation process and subsequently purified using chromatographic procedures. More than 40% of the inclusion body proteins could be refolded back to the bioactive native conformation. Mild solubilization is thus the key for high recovery of bioactive protein from inclusion bodies.

Cell Fractionation↗
PubMed2006

Light and electron microscopic observation of intracytoplasmic inclusion bodies in the locus coeruleus of the hamster.

The authors previously demonstrated that intracytoplasmic inclusion bodies (1-3 microm) in the mouse locus coeruleus under light and electron microscopy are characteristically stained using the Holmes modified method. We reported that one inclusion body existed in almost all neurons of the locus coeruleus. The present study examined whether similar inclusion bodies are present in the Syrian hamster (weight, about 60 g). Paraffin sections stained with the modified Holmes' method dis played numerous small inclusion bodies in the cytoplasm of cells in the locus coeruleus. Epon sections (1 microm thick) stained using toluidine blue were observed under light microscopy, and numerous small inclusion bodies were again observed. Under electron microscopy observation, inclusion bodies (<1 microm in diameter) predominantly comprised small granular materials, similar to those described by previous investigators. Although inclusion bodies were devoid of a limiting membrane, the relation ship to cytoplasmic organelles was unclear. However, free and polyribosomes were occasionally noted in close proximity to inclusion bodies. Inclusion bodies may thus be formed from ribosomes. Intracytoplasmic inclusion bodies in the hamster locus coeruleus differed in appearance compared with inclusion bodies in the mouse locus coeruleus.

Animals↗