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At least 577 records · Page 32Linked to original sources
PubMed2001

Alpha-synuclein-enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons.

Alpha-synuclein accumulates in the brains of sporadic Parkinson's disease patients as a major component of Lewy bodies, and mutations in alpha-synuclein are associated with familial forms of Parkinson's disease. The pathogenic mechanisms that precede and promote the aggregation of alpha-synuclein into Lewy bodies in neurons remain to be determined. Here, we constructed a series of alpha-synuclein-enhanced green fluorescent protein (alpha-synucleinEGFP, SynEGFP) fusion proteins to address whether the Parkinson's disease-associated mutations alter the subcellular distribution of alpha-synuclein, and to use as a tool for experimental manipulations to induce aggregate formation. When transfected into mouse cultured primary neurons, the 49-kDa alpha-synucleinEGFP fusion proteins are partially truncated to a approximately 27-kDa form. This non-fluorescent carboxy-terminally modified fusion protein spontaneously forms inclusions in the neuronal cytoplasm. A marked increase in the accumulation of inclusions is detected following treatment with each of three proteasome inhibitors, n-acetyl-leu-leu-norleucinal, lactacystin and MG132. Interestingly, Ala30Pro alpha-synucleinEGFP does not form the cytoplasmic inclusions that are characteristic of wild-type and Ala53Thr alpha-synucleinEGFP, supporting the idea that the Ala30Pro alpha-synuclein protein conformation differs from wild-type alpha-synuclein. Similar inclusions are formed if alpha-synuclein carboxy-terminus is modified by the addition of a V5/6xHistidine epitope tag. By contrast, overexpression of unmodified alpha-synuclein does not lead to aggregate formation. Furthermore, synphilin-1, an alpha-synuclein interacting protein also found in Lewy bodies, colocalizes with the carboxy-terminally truncated alpha-synuclein fusion protein in discrete cytoplasmic inclusions.Our finding that manipulations of the carboxy-terminus of alpha-synuclein lead to inclusion formation may provide a model for studies of the pathogenic mechanisms of alpha-synuclein aggregation in Lewy bodies.

Animals↗
PubMed2001

Macrophagic myofasciitis associated with inclusion body myositis: a report of three cases.

We describe three patients with macrophagic myofasciitis and inclusion body myositis. All patients fulfilled diagnostic criteria for inclusion body myositis and myopathologic criteria for macrophagic myofasciitis. In the three cases macrophagic myofasciitis complicated the evolution of a known and painless inclusion body myositis and was diagnosed in a repeated deltoid biopsy because of the appearance of myalgia during the course of inclusion body myositis in all cases. The unexpected appearance of myalgia during the course of painless inclusion body myositis must arouse the suspicion of an association of another inflammatory muscle disease, macrophagic myofasciitis.

Adult↗
PubMed2003

A novel homozygous missense mutation in the GNE gene of a patient with quadriceps-sparing hereditary inclusion body myopathy associated with muscle inflammation.

An adult-onset hereditary inclusion body myopathy with sparing of the quadriceps muscle was originally described in Iranian Jews and assigned to a locus on chromosome 9p12-p13. Recently, mutations of the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) gene were reported to cause hereditary inclusion body myopathy and one type of distal myopathy in a world-wide distribution. Importantly, the lack of muscle inflammation was used to distinguish hereditary inclusion body myopathy from the sporadic form of inclusion body myopathy. We report a case of a quadriceps-sparing myopathy in a non-Jewish, Iranian patient with a high degree of muscle inflammation. A novel homozygous G-to-A mutation (128933G-->A) in exon 7 changing a valine to isoleucine (V367I) in the epimerase domain of the GNE gene was found. We conclude that muscle inflammation is not sufficient to exclude the diagnosis of hereditary inclusion body myopathy.

Adult↗
PubMedDate not supplied

Direct refolding of inclusion bodies using reversed micelles.

The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT). Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies. The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles. To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system. The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies. The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h. The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies. The addition of cold acetone to the reversed micellar solution allowed over 90% recovery of the renatured RNase A.

Animals↗
PubMedDate not supplied

Optimized production of active alpha-glucosidase by recombinant Escherichia coli. evaluation of processes using in vivo reactivation from inclusion bodies.

During fast production in recombinant Escherichia coli, the enzyme alpha-glucosidase from Saccharomyces cerevisiae accumulates partially as inclusion bodies. The inclusion bodies are reactivated inside the cell upon temperature downshift. This in vivo reactivation was most efficient on complex medium with inclusion body production at 42 degrees C and reactivation at 30 degrees C, yielding volumetric activities 85% higher than those of extended isothermal production at low temperature. On defined medium, however, in vivo reactivation was slow. In fed-batch cultivations, feeding controls the specific growth rate independent of the temperature. Here, high growth rates fostered inclusion body formation even at low temperature. Intermediate growth rates permitted accumulation of active alpha-glucosidase without affecting the total amount of alpha-glucosidase. Low growth rates yielded similar activities and additionally prevented inclusion body formation. Moreover, high growth rates during production forestalled subsequent in vivo reactivation. Accumulation of activity after temperature reduction was possible with intermediate growth rates. The best time for temperature shift was concomitant to induction. Thus, in fed-batch culture, isothermal production at 30 degrees C and with a set growth rate of 0.12 h(-)(1) controlled by feeding was most efficient for production of active alpha-glucosidase. Compared to production under optimal conditions on complex medium, the specific and volumetric activities obtained were 3 and 45 times higher, respectively.

Bioreactors↗
PubMedDate not supplied

Solubilization of recombinant ovine growth hormone with retention of native-like secondary structure and its refolding from the inclusion bodies of Escherichia coli.

Ovine growth hormone was expressed in Escherichia coli in the form of inclusion bodies using the pQE-30 expression vector. In a simple fed-batch fermentation, 800 mg/L of recombinant ovine growth hormone (r-oGH) was produced at a cell concentration of 12 g dry cell weight/L. Inclusion bodies were isolated from cells with >95% purity by extensive washing using detergent, and the r-oGH from the purified inclusion bodies was solubilized in 2 M Tris-HCl buffer at pH 12 containing 2 M urea. The r-oGH solubilized in the above conditions exhibited considerable secondary structure as determined by circular dichroism spectra and was immunologically active. Solubilization of the inclusion body protein with retention of native-like secondary structure gave higher yields during refolding. To suppress protein aggregation, refolding was carried out in gel filtration column. Refolding, buffer exchange, and the purification of monomeric r-oGH from aggregated complex was achieved in a single step using gel filtration chromatography. More than 60% of the initial inclusion body protein was refolded into a native-like conformation by the use of this procedure. The refolded protein was characterized by circular dichroism, fluorescence, SDS-PAGE, Western blotting, and radio receptor binding assay and found to be similar to native, pituitary-derived, ovine growth hormone.

Animals↗
PubMed2003

Protein aggregation in recombinant bacteria: biological role of inclusion bodies.

Protein aggregation is an ordinary consequence of thermal stress. In recombinant bacteria, the over-expression of plasmid-encoded genes triggers transcription of heat-shock genes and other stress responses and often results in the aggregation of the encoded protein as inclusion bodies. The formation of these deposits represents a major obstacle for the production of biologically active polypeptides and restricts the spectrum of protein products being available for the industrial-biomedical market. Inclusion body formation was formerly considered to occur passively by the irretrievable deposition of partially-folded intermediates. Increasing evidence, however, indicates that protein aggregation in bacteria occurs as a reversible process deeply integrated in the cell mechanisms for coping with thermal stress, and that inclusion bodies are structurally dynamic structures. Inclusion body formation might actually be supported by the cellular machinery that when operated under specific stress conditions, transiently stores misfolded polypeptides until they could be further processed: either refolded or proteolysed. A better understanding of protein aggregation in cell physiology could allow not only inclusion body formation to be minimized more efficiently for a higher soluble yield, but also to comprehend in detail the intricacy of cell mechanisms committed to handling the misfolding danger.

Bacteria↗
PubMed2005

Alpha-synuclein phosphorylation controls neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease.

Alpha-synuclein is phosphorylated at serine 129 (Ser129) in intracellular protein aggregates called Lewy bodies. These inclusion bodies are the characteristic pathologic lesions of Parkinson disease. Here we define the role of phosphorylation of Ser129 in alpha-synuclein toxicity and inclusion formation using a Drosophila model of Parkinson disease. Mutation of Ser129 to alanine to prevent phosphorylation completely suppresses dopaminergic neuronal loss produced by expression of human alpha-synuclein. In contrast, altering Ser129 to the negatively charged residue aspartate, to mimic phosphorylation, significantly enhances alpha-synuclein toxicity. The G protein-coupled receptor kinase 2 (Gprk2) phosphorylates Ser129 in vivo and enhances alpha-synuclein toxicity. Blocking phosphorylation at Ser129 substantially increases aggregate formation. Thus Ser129 phosphorylation status is crucial in mediating alpha-synuclein neurotoxicity and inclusion formation. Because increased number of inclusion bodies correlates with reduced toxicity, inclusion bodies may protect neurons from alpha-synuclein toxicity.

Amino Acid Substitution↗
PubMed2001

Distribution and dynamic process of neuronal cytoplasmic inclusion (NCI) in MSA: correlation of the density of NCI and the degree of involvement of the pontine nuclei.

MSA is a sporadic degenerative disease that occurs in striatonigral degeneration (SND), SDS and most cases of sporadic OPCA. Oligodendroglial inclusion is a hallmark of MSA. Recently there have been a small number of reports of neuronal argyrophilic inclusions. To clarify the distribution and dynamic process of neuronal cytoplasmic inclusions (NCI), 31 cases of MSA were studied using histology, immunohistochemistry, and electron microscopy. The inclusions were exclusively found in the pontine nucleus and there was a correlation between the incidence of NCI and the severity of OPCA, but not of SND. NCI were increased to some extent in the cases with moderate OPCA and decreased in number in proportion to devastation of the pontine nuclei. Immunohistochemical and ultrastructural features of NCI were virtually identical to those of glial cytoplasmic inclusions (GCI), which gives some clues to the pathogenesis of MSA. It is tempting to interpret this as NCI playing a significant role in the degenerative changes of the neurons at least in the pons. Further systematic studies on NCI in the other brain regions are necessary to elucidate the pathogenesis of neuronal degeneration in MSA.

Adult↗
PubMed2004

Ubiquitylation of synphilin-1 and alpha-synuclein by SIAH and its presence in cellular inclusions and Lewy bodies imply a role in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterized by Lewy body formation and death of dopaminergic neurons. Mutations in alpha-synuclein and parkin cause familial forms of PD. Synphilin-1 was shown to interact with alpha-synuclein and to promote the formation of cytosolic inclusions. We now report that synphilin-1 interacts with the E3 ubiquitin-ligases SIAH-1 and SIAH-2. SIAH proteins ubiquitylate synphilin-1 both in vitro and in vivo, promoting its degradation by the ubiquitin-proteasome system. Inability of the proteasome to degrade synphilin-1/SIAH complex leads to a robust formation of ubiquitylated cytosolic inclusions. Ubiquitylation is required for inclusion formation, because a catalytically inactive mutant of SIAH-1, which still binds to synphilin-1, fails to promote inclusions. Like synphilin-1, alpha-synuclein associates with SIAH in intact cells, but the interaction with SIAH-2 was much stronger that with SIAH-1. In vitro experiments show that SIAH-2 monoubiquitylates alpha-synuclein. Further evidence that SIAH proteins may play a role in inclusion formation comes from the demonstration of SIAH immunoreactivity in Lewy bodies of PD patients.

Animals↗
PubMed2001

Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons.

Human apolipoprotein (apo) E4, a major risk factor for Alzheimer's disease (AD), occurs in amyloid plaques and neurofibrillary tangles (NFTs) in AD brains; however, its role in the pathogenesis of these lesions is unclear. Here we demonstrate that carboxyl-terminal-truncated forms of apoE, which occur in AD brains and cultured neurons, induce intracellular NFT-like inclusions in neurons. These cytosolic inclusions were composed of phosphorylated tau, phosphorylated neurofilaments of high molecular weight, and truncated apoE. Truncated apoE4, especially apoE4(Delta 272--299), induced inclusions in up to 75% of transfected neuronal cells, but not in transfected nonneuronal cells. ApoE4 was more susceptible to truncation than apoE3 and resulted in much greater intracellular inclusion formation. These results suggest that apoE4 preferentially undergoes intracellular processing, creating a bioactive fragment that interacts with cytoskeletal components and induces NFT-like inclusions containing phosphorylated tau and phosphorylated neurofilaments of high molecular weight in neurons.

Aged↗
PubMed2003

The chaperone environment at the cytoplasmic face of the endoplasmic reticulum can modulate rhodopsin processing and inclusion formation.

The human DnaJ (Hsp40) proteins HSJ1a and HSJ1b are type II DnaJ proteins with different C termini generated by alternate splicing. Both protein isoforms can regulate the ATPase activity and substrate binding of Hsp70. In this study, we have confirmed the neuronal expression of HSJ1a and HSJ1b proteins and localized their expression in human neural retina using isoform-specific antisera. HSJ1a and HSJ1b were enriched in photoreceptors, particularly the inner segments, but had different intracellular localization due to the prenylation of HSJ1b by a geranylgeranyl moiety. Because of their enrichment at the site of rhodopsin production, we investigated the effect of HSJ1 isoforms on the cellular processing of wild-type and mutant rhodopsin apoprotein in SK-N-SH cells. The expression of HSJ1b, but not HSJ1a, inhibited the normal cellular processing of wild-type rhodopsin-GFP, which co-localized with HSJ1b at the ER. HSJ1b expression also increased the incidence of inclusion formation by the wild-type protein. Both isoforms were recruited to mutant P23H rhodopsin inclusions, but only HSJ1b enhanced inclusion formation. Investigation of a prenylation-null mutant showed that the modulation of rhodopsin processing and inclusion formation was dependent on the correct subcellular targeting of HSJ1b to the cytosolic face of the ER. An Hsp70 interaction-null mutant of HSJ1b had the same effect as HSJ1b, suggesting that these phenomena were independent of Hsp70 and, furthermore, overexpression of Hsp70 with HSJ1b did not modulate the HSJ1b effect on inclusion formation, showing that Hsp70 was not limiting. The data provide evidence that cytoplasmic chaperones when targeted to the ER can influence the folding and processing of a GPCR and show that DnaJ protein function can be further specialized by alternative splicing and post-translational modification.

Adenosine Triphosphatases↗
PubMed2003

Association of the cytoskeletal GTP-binding protein Sept4/H5 with cytoplasmic inclusions found in Parkinson's disease and other synucleinopathies.

alpha-Synuclein-positive cytoplasmic inclusions are a pathological hallmark of several neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Here we report that Sept4, a member of the septin protein family, is consistently found in these inclusions, whereas five other septins (Sept2, Sept5, Sept6, Sept7, and Sept8) are not found in these inclusions. Sept4 and alpha-synuclein can also be co-immunoprecipitated from normal human brain lysates. When co-expressed in cultured cells, FLAG-tagged Sept4 and Myc-tagged alpha-synuclein formed detergent-insoluble complex, and upon treatment with a proteasome inhibitor, they formed Lewy body-like cytoplasmic inclusions. The tagged Sept4 and alpha-synuclein synergistically accelerated cell death induced by the proteasome inhibitor, and this effect was further enhanced by expression of another Lewy body-associated protein, synphilin-1, tagged with the V5 epitope. Moreover, co-expression of the three proteins (tagged Sept4, alpha-synuclein, and synphilin-1) was sufficient to induce cell death. These data raise the possibility that Sept4 is involved in the formation of cytoplasmic inclusions as well as induction of cell death in alpha-synuclein-associated neurodegenerative disorders.

Aged↗
PubMed1986

Cytoplasmic inclusions in Giardia: an electron microscopy study.

Ultrastructural observations of Giardia trophozoites showed two different types of cytoplasmic inclusions. In Giardia muris from hamsters, the inclusions scattered in the cytoplasm were round, about 0.2 micron in diameter, limited by a double membrane, and with some electron-dense contents. In G. duodenalis from domestic rats, the inclusions were polyhedral, about 0.2 micron in diameter, limited by a double membrane, and with less electron dense contents. These inclusions have an unknown nature, and evidences show interaction between the inclusions and the Giardia trophozoites.

Animals↗
PubMed1995

Basal cell carcinoma with hyaline inclusions.

A 69 yr old man had a 4 mm basal cell carcinoma completely excised from the chin. Numerous hyaline cytoplasmic inclusions were contained within the tumor cells. The inclusions stained intensely red with Masson's trichrome, and immunocytochemically there was prominent rim labelling for keratins (bovine, callus and AE1/3) and muscle-specific actin, the latter more faintly decorating the centre of some inclusions. The inclusions were negative for antibodies to cytokeratin Cam5.2, epithelial membrane antigen (EMA), vimentin, S100, neurofilaments, glial fibrillary acidic protein (GFAP) and carcinoembryonic antigen (CEA) and there was no post Congo red apple green birefringence to indicate amyloid. Ultrastructure indicated the inclusions were composed of proteinaceous material surrounded by a defined rim of tonofilaments in cells showing no degenerative features. The findings suggested aberrant tumor cell keratinization. Familiarization with this rare variant of a common cutaneous carcinoma will alleviate diagnostic difficulties that may arise, particularly in superficial tumor curettage.

Aged↗
PubMed1998

Atypical cervical polyp with intracytoplasmic inclusions.

Benign polyps containing atypical stromal cells are described at many anatomical sites and some such lesions have been shown to contain intracytoplasmic actin-rich inclusions, believed to represent deranged filament metabolism in proliferating myofibroblastic cells. We present a case of an atypical cervical polyp with intracytoplasmic inclusions, occurring in a 23 year old female, and provide support for the proposal that these inclusions are composed of actin filaments, identical to those initially reported in infantile digital fibromatosis. This report emphasises the need to recognise the benign nature of such stromal proliferations and expands the range of myofibroblastic lesions in which actin inclusions may occur. Characterisation of the inclusions will provide further insight into the complexities of actin metabolism.

Actins↗
PubMedDate not supplied

Peculiar cytoplasmic inclusions in oncocytic adrenal cortical tumors: an electron microscopic observation.

Two cases of an oncocytic adrenal cortical tumor that contained peculiar cytoplasmic crystalline inclusions in the tumor cells are presented. The patients were 49- and 72-year-old females without clinical and biochemical evidence of adrenal cortical or medullary dysfunction. The adrenal tumors weighed 80 and 200 g each. These crystalline inclusions were present in groups of longitudinal profiles or clusters of crossly cut aggregates. They appeared in clusters of membrane-bound columns. On longitudinal sections, they appeared as rigid rods of homogenous density measuring 36 nm in width, but when they were cut transversely their paracrystalline nature became apparent. They were composed of closely packed microtubules in rectangular blocks. The microtubules measured 12.5 nm with a hollow center measuring 4.2 nm. The inclusions were within the membrane-bound cisterna of rough-surfaced endoplasmic reticulum. The significance of these inclusions is not clearly understood; however, they have been seen only in adrenal cortical tumors and their presence may be helpful in the differential diagnosis of adrenal oncocytic tumors. One patient presented with a tumor in which gross and microscopic appearance was compatible with a pheochromocytoma. This case exhibited an oncocytic appearance and pronounced cellular pleomorphism. Ultrastructural studies were necessary to recognize the tumor cells as cortical cells. The tumor cells contained abundant mitochondria with tubular cristae, paranuclear parallel stacks of granular endoplasmic reticulum, and relatively prominent smooth endoplasmic reticulum. These features are typical of adrenocortical cells. In addition, frequent tumor cells contained the peculiar cytoplasmic inclusions herein described.

Adenoma, Oxyphilic↗
PubMedDate not supplied

Actin inclusions in stromal cells of fibroepithelial tumor of breast: immunohistochemical and ultrastructural studies.

An uncommon occurrence of actin inclusions in the stromal cells of a benign fibroepithelial tumor of breast is reported. Histologically, many of the stromal cells contained round and eosinophilic intracytoplasmic inclusions identical to those seen in inclusion body fibromatosis. Ultrastructurally, these inclusions represented dense spherical clumps of microfilaments derived from rough endoplasmic reticulum. The literature was reviewed and follow-up data showed that the clinical course of these morphologically distinctive benign fibroepithelial tumors was relatively indolent if completely excised, in contrast to inclusion body fibromatosis, which commonly recurs. The pathogenesis may be related to abnormal production of truncated actin filaments or alteration in microenvironment.

Actins↗