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Sample records for bacterial inclusion body

  1. Bacterial inclusion body purification.

    PubMed

    Seras-Franzoso, Joaquin; Peternel, Spela; Cano-Garrido, Olivia; Villaverde, Antonio; García-Fruitós, Elena

    2015-01-01

    Purification of bacterial inclusion bodies (IBs) is gaining importance due to the raising of novel applications for this type of submicron particulate protein clusters, with potential uses in the biomedical field among others. Here, we present two optimized methods to purify IBs adapting classical procedures to the material nature as well as the requirements of its final application.

  2. Bacterial Inclusion Bodies: Discovering Their Better Half.

    PubMed

    Rinas, Ursula; Garcia-Fruitós, Elena; Corchero, José Luis; Vázquez, Esther; Seras-Franzoso, Joaquin; Villaverde, Antonio

    2017-02-26

    Bacterial inclusion bodies (IBs) are functional, non-toxic amyloids occurring in recombinant bacteria showing analogies with secretory granules of the mammalian endocrine system. The scientific interest in these mesoscale protein aggregates has been historically masked by their status as a hurdle in recombinant protein production. However, progressive understanding of how the cell handles the quality of recombinant polypeptides and the main features of their intriguing molecular organization has stimulated the interest in inclusion bodies and spurred their use in diverse technological fields. The engineering and tailoring of IBs as functional protein particles for materials science and biomedicine is a good example of how formerly undesired bacterial byproducts can be rediscovered as promising functional materials for a broad spectrum of applications.

  3. Towards revealing the structure of bacterial inclusion bodies

    PubMed Central

    2009-01-01

    Protein aggregation is a widely observed phenomenon in human diseases, biopharmaceutical production, and biological research. Protein aggregates are generally classified as highly ordered, such as amyloid fibrils, or amorphous, such as bacterial inclusion bodies. Amyloid fibrils are elongated filaments with diameters of 6–12 nm, they are comprised of residue-specific cross-β structure, and display characteristic properties, such as binding with amyloid-specific dyes. Amyloid fibrils are associated with dozens of human pathological conditions, including Alzheimer disease and prion diseases. Distinguished from amyloid fibrils, bacterial inclusion bodies display apparent amorphous morphology. Inclusion bodies are formed during high-level recombinant protein production, and formation of inclusion bodies is a major concern in biotechnology. Despite of the distinctive morphological difference, bacterial inclusion bodies have been found to have some amyloid-like properties, suggesting that they might contain structures similar to amyloid-like fibrils. Recent structural data further support this hypothesis, and this review summarizes the latest progress towards revealing the structural details of bacterial inclusion bodies. PMID:19806034

  4. Towards revealing the structure of bacterial inclusion bodies.

    PubMed

    Wang, Lei

    2009-01-01

    Protein aggregation is a widely observed phenomenon in human diseases, biopharmaceutical production, and biological research. Protein aggregates are generally classified as highly ordered, such as amyloid fibrils, or amorphous, such as bacterial inclusion bodies. Amyloid fibrils are elongated filaments with diameters of 6-12 nm, they are comprised of residue-specific cross-beta structure, and display characteristic properties, such as binding with amyloid-specific dyes. Amyloid fibrils are associated with dozens of human pathological conditions, including Alzheimer disease and prion diseases. Distinguished from amyloid fibrils, bacterial inclusion bodies display apparent amorphous morphology. Inclusion bodies are formed during high-level recombinant protein production, and formation of inclusion bodies is a major concern in biotechnology. Despite of the distinctive morphological difference, bacterial inclusion bodies have been found to have some amyloid-like properties, suggesting that they might contain structures similar to amyloid-like fibrils. Recent structural data further support this hypothesis, and this review summarizes the latest progress towards revealing the structural details of bacterial inclusion bodies.

  5. In situ protein folding and activation in bacterial inclusion bodies.

    PubMed

    Gonzalez-Montalban, Nuria; Natalello, Antonino; García-Fruitós, Elena; Villaverde, Antonio; Doglia, Silvia Maria

    2008-07-01

    Recent observations indicate that bacterial inclusion bodies formed in absence of the main chaperone DnaK result largely enriched in functional, properly folded recombinant proteins. Unfortunately, the molecular basis of this intriguing fact, with obvious biotechnological interest, remains unsolved. We have explored here two non-excluding physiological mechanisms that could account for this observation, namely selective removal of inactive polypeptides from inclusion bodies or in situ functional activation of the embedded proteins. By combining structural and functional analysis, we have not observed any preferential selection of inactive and misfolded protein species by the dissagregating machinery during inclusion body disintegration. Instead, our data strongly support that folding intermediates aggregated as inclusion bodies could complete their natural folding process once deposited in protein clusters, which conduces to significant functional activation. In addition, in situ folding and protein activation in inclusion bodies is negatively regulated by the chaperone DnaK.

  6. Isolation of cell-free bacterial inclusion bodies.

    PubMed

    Rodríguez-Carmona, Escarlata; Cano-Garrido, Olivia; Seras-Franzoso, Joaquin; Villaverde, Antonio; García-Fruitós, Elena

    2010-09-17

    Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces. Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10⁻¹ cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.

  7. Learning about protein solubility from bacterial inclusion bodies

    PubMed Central

    Martínez-Alonso, Mónica; González-Montalbán, Nuria; García-Fruitós, Elena; Villaverde, Antonio

    2009-01-01

    The progressive solving of the conformation of aggregated proteins and the conceptual understanding of the biology of inclusion bodies in recombinant bacteria is providing exciting insights on protein folding and quality. Interestingly, newest data also show an unexpected functional and structural complexity of soluble recombinant protein species and picture the whole bacterial cell factory scenario as more intricate than formerly believed. PMID:19133126

  8. Engineering the bacterial shapes for enhanced inclusion bodies accumulation.

    PubMed

    Jiang, Xiao-Ran; Wang, Huan; Shen, Rui; Chen, Guo-Qiang

    2015-05-01

    Many bacteria can accumulate inclusion bodies such as sulfur, polyphosphate, glycogen, proteins or polyhydroxyalkanoates. To exploit bacteria as factories for effective production of inclusion bodies, a larger intracellular space is needed for more inclusion body accumulation. In this study, polyhydroxybutyrate (PHB) was investigated as an inclusion bodies representative to be accumulated by Escherichia coli JM109SG. Various approaches were taken to increase the bacterial cell sizes including deletion on actin-like protein gene mreB, weak expression of mreB in mreB deletion mutant, and weak expression of mreB in mreB deletion mutant under inducible expression of SulA, the inhibitor of division ring protein FtsZ. All of the methods resulted in different levels of increases in bacterial sizes and PHB granules accumulation. Remarkably, an increase of over 100% PHB accumulation was observed in recombinant E. coli overexpressing mreB in an mreB deletion mutant under inducible expression of FtsZ inhibiting protein SulA. The molecular mechanism of enlarged bacterial size was found to be directly relate to weakened cytoskeleton which was the result of broken skeleton helix. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  9. Bacterial Inclusion Bodies Contain Amyloid-Like Structure

    PubMed Central

    Wang, Lei; Maji, Samir K; Sawaya, Michael R; Eisenberg, David; Riek, Roland

    2008-01-01

    Protein aggregation is a process in which identical proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as amorphous, lacking any long-range order, or highly ordered fibrils. Protein fibrils can be composed of native globular molecules, such as the hemoglobin molecules in sickle-cell fibrils, or can be reorganized β-sheet–rich aggregates, termed amyloid-like fibrils. Amyloid fibrils are associated with several pathological conditions in humans, including Alzheimer disease and diabetes type II. We studied the structure of bacterial inclusion bodies, which have been believed to belong to the amorphous class of aggregates. We demonstrate that all three in vivo-derived inclusion bodies studied are amyloid-like and comprised of amino-acid sequence-specific cross-β structure. These findings suggest that inclusion bodies are structured, that amyloid formation is an omnipresent process both in eukaryotes and prokaryotes, and that amino acid sequences evolve to avoid the amyloid conformation. PMID:18684013

  10. Amyloid-linked cellular toxicity triggered by bacterial inclusion bodies

    SciTech Connect

    Gonzalez-Montalban, Nuria; Villaverde, Antonio; Aris, Anna; E-mail: Anna.Aris@irta.es

    2007-04-13

    The aggregation of proteins in the form of amyloid fibrils and plaques is the characteristic feature of some pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. The mechanisms by which the aggregation processes result in cell damage are under intense investigation but recent data indicate that prefibrillar aggregates are the most proximate mediators of toxicity rather than mature fibrils. Since it has been shown that prefibrillar forms of the nondisease-related misfolded proteins are highly toxic to cultured mammalian cells we have studied the cytoxicity associated to bacterial inclusion bodies that have been recently described as protein deposits presenting amyloid-like structures. We have proved that bacterial inclusion bodies composed by a misfolding-prone {beta}-galactosidase fusion protein are clearly toxic for mammalian cells but the {beta}-galactosidase wild type enzyme forming more structured thermal aggregates does not impair cell viability, despite it also binds and enter into the cells. These results are in the line that the most cytotoxic aggregates are early prefibrilar assemblies but discard the hypothesis that the membrane destabilization is Key event to subsequent disruption of cellular processes, such as ion balance, oxidative state and the eventually cell death.

  11. Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells

    PubMed Central

    2010-01-01

    Background In recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry. To prepare large quantities of a high quality product, the whole bioprocess has to be optimised. This includes not only the cultivation of the bacterial culture, but also the isolation step itself, which can be of critical importance for the production process. To determine the most appropriate method for the isolation of biologically active nanoparticles, three methods for bacterial cell disruption were analyzed. Results In this study, enzymatic lysis and two mechanical methods, high-pressure homogenization and sonication, were compared. During enzymatic lysis the enzyme lysozyme was found to attach to the surface of IBs, and it could not be removed by simple washing. As this represents an additional impurity in the engineered nanoparticles, we concluded that enzymatic lysis is not the most suitable method for IBs isolation. During sonication proteins are released (lost) from the surface of IBs and thus the surface of IBs appears more porous when compared to the other two methods. We also found that the acoustic output power needed to isolate the IBs from bacterial cells actually damages proteins structures, thereby causing a reduction in biological activity. High-pressure homogenization also caused some damage to IBs, however the protein loss from the IBs was negligible. Furthermore, homogenization had no side-effects on protein biological activity. Conclusions The study shows that among the three methods tested, homogenization is the most appropriate method for the isolation of active nanoparticles from bacterial cells. PMID:20831775

  12. Packaging protein drugs as bacterial inclusion bodies for therapeutic applications

    PubMed Central

    2012-01-01

    A growing number of insights on the biology of bacterial inclusion bodies (IBs) have revealed intriguing utilities of these protein particles. Since they combine mechanical stability and protein functionality, IBs have been already exploited in biocatalysis and explored for bottom-up topographical modification in tissue engineering. Being fully biocompatible and with tuneable bio-physical properties, IBs are currently emerging as agents for protein delivery into mammalian cells in protein-replacement cell therapies. So far, IBs formed by chaperones (heat shock protein 70, Hsp70), enzymes (catalase and dihydrofolate reductase), grow factors (leukemia inhibitory factor, LIF) and structural proteins (the cytoskeleton keratin 14) have been shown to rescue exposed cells from a spectrum of stresses and restore cell functions in absence of cytotoxicity. The natural penetrability of IBs into mammalian cells (reaching both cytoplasm and nucleus) empowers them as an unexpected platform for the controlled delivery of essentially any therapeutic polypeptide. Production of protein drugs by biopharma has been traditionally challenged by IB formation. However, a time might have arrived in which recombinant bacteria are to be engineered for the controlled packaging of therapeutic proteins as nanoparticulate materials (nanopills), for their extra- or intra-cellular release in medicine and cosmetics. PMID:22686540

  13. Chemical Assistance in Refolding of Bacterial Inclusion Bodies

    PubMed Central

    Alibolandi, Mona; Mirzahoseini, Hasan

    2011-01-01

    Escherichia coli is one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins in E. coli. In vitro refolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes. PMID:21822494

  14. Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors

    PubMed Central

    2012-01-01

    Background The amyloid-β peptide (Aβ42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Aβ42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders. Results Here we describe a rapid and high-throughput screening method to identify molecules able to modulate amyloid aggregation. The approach exploits the inclusion bodies (IBs) formed by Aβ42 when expressed in bacteria. We have shown previously that these aggregates retain amyloid structural and functional properties. In the present work, we demonstrate that their in vitro refolding is selectively sensitive to the presence of aggregation-promoting metal ions, allowing the detection of inhibitors of metal-promoted amyloid aggregation with potential therapeutic interest. Conclusions Because IBs can be produced at high levels and easily purified, the method overcomes one of the main limitations in screens to detect amyloid modulators: the use of expensive and usually highly insoluble synthetic peptides. PMID:22553999

  15. Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent.

    PubMed

    Upadhyay, Vaibhav; Singh, Anupam; Jha, Divya; Singh, Akansha; Panda, Amulya K

    2016-06-08

    Formation of inclusion bodies poses a major hurdle in recovery of bioactive recombinant protein from Escherichia coli. Urea and guanidine hydrochloride have routinely been used to solubilize inclusion body proteins, but many times result in poor recovery of bioactive protein. High pH buffers, detergents and organic solvents like n-propanol have been successfully used as mild solubilization agents for high throughput recovery of bioactive protein from bacterial inclusion bodies. These mild solubilization agents preserve native-like secondary structures of proteins in inclusion body aggregates and result in improved recovery of bioactive protein as compared to conventional solubilization agents. Here we demonstrate solubilization of human growth hormone inclusion body aggregates using 30% trifluoroethanol in presence of 3 M urea and its refolding into bioactive form. Human growth hormone was expressed in E. coli M15 (pREP) cells in the form of inclusion bodies. Different concentrations of trifluoroethanol with or without addition of low concentration (3 M) of urea were used for solubilization of inclusion body aggregates. Thirty percent trifluoroethanol in combination with 3 M urea was found to be suitable for efficient solubilization of human growth hormone inclusion bodies. Solubilized protein was refolded by dilution and purified by anion exchange and size exclusion chromatography. Purified protein was analyzed for secondary and tertiary structure using different spectroscopic tools and was found to be bioactive by cell proliferation assay. To understand the mechanism of action of trifluoroethanol, secondary and tertiary structure of human growth hormone in trifluoroethanol was compared to that in presence of other denaturants like urea and guanidine hydrochloride. Trifluoroethanol was found to be stabilizing the secondary structure and destabilizing the tertiary structure of protein. Finally, it was observed that trifluoroethanol can be used to solubilize

  16. Tunable geometry of bacterial inclusion bodies as substrate materials for tissue engineering

    NASA Astrophysics Data System (ADS)

    García-Fruitós, Elena; Seras-Franzoso, Joaquín; Vazquez, Esther; Villaverde, Antonio

    2010-05-01

    A spectrum of materials for biomedical applications is produced in bacteria, and some of them, such as metals or polyhydroxyalkanoates, are straightforwardly obtained as particulate entities. We have explored the biofabrication process of bacterial inclusion bodies, particulate proteinaceous materials (ranging from 50 to 500 nm in diameter) recently recognized as suitable for surface topographical modification and tissue engineering. Inclusion bodies have been widely described as spherical or pseudo-spherical particles with only minor morphological variability, mostly restricted to their size. Here we have identified a cellular gene in Escherichia coli (clpP) that controls the in vivo fabrication process of inclusion bodies. In the absence of the encoded protease, the dynamics of protein deposition is perturbed, resulting in unusual tear-shaped particles with enhanced surface-volume ratios. This fact modifies the ability of inclusion bodies to promote mammalian cell attachment and differentiation upon surface decoration. The implications of the genetic control of inclusion body geometry are discussed in the context of their biological fabrication and regarding the biomedical potential of these protein clusters in regenerative medicine.

  17. Concepts and tools to exploit the potential of bacterial inclusion bodies in protein science and biotechnology.

    PubMed

    Gatti-Lafranconi, Pietro; Natalello, Antonino; Ami, Diletta; Doglia, Silvia Maria; Lotti, Marina

    2011-07-01

    Cells have evolved complex and overlapping mechanisms to protect their proteins from aggregation. However, several reasons can cause the failure of such defences, among them mutations, stress conditions and high rates of protein synthesis, all common consequences of heterologous protein production. As a result, in the bacterial cytoplasm several recombinant proteins aggregate as insoluble inclusion bodies. The recent discovery that aggregated proteins can retain native-like conformation and biological activity has opened the way for a dramatic change in the means by which intracellular aggregation is approached and exploited. This paper summarizes recent studies towards the direct use of inclusion bodies in biotechnology and for the detection of bottlenecks in the folding pathways of specific proteins. We also review the major biophysical methods available for revealing fine structural details of aggregated proteins and which information can be obtained through these techniques. © 2011 The Authors Journal compilation © 2011 FEBS.

  18. Nucleotide binding to human uncoupling protein-2 refolded from bacterial inclusion bodies.

    PubMed

    Jekabsons, Mika B; Echtay, Karim S; Brand, Martin D

    2002-09-01

    Experiments were performed to test the hypothesis that recombinant human uncoupling protein-2 (UCP2) ectopically expressed in bacterial inclusion bodies binds nucleotides in a manner identical with the nucleotide-inhibited uncoupling that is observed in kidney mitochondria. For this, sarkosyl-solubilized UCP2 inclusion bodies were treated with the polyoxyethylene ether detergent C12E9 and hydroxyapatite. Protein recovered from hydroxyapatite chromatography was approx. 90% pure UCP2, as judged by Coomassie Blue and silver staining of polyacrylamide gels. Using fluorescence resonance energy transfer, N-methylanthraniloyl-tagged purine nucleoside di- and tri-phosphates exhibited enhanced fluorescence with purified UCP2. Dissociation constants determined by least-squares non-linear regression indicated that the affinity of UCP2 for these fluorescently tagged nucleotides was 3-5 microM or perhaps an order of magnitude stronger, depending on the model used. Competition experiments with [8-14C]ATP demonstrated that UCP2 binds unmodified purine and pyrimidine nucleoside triphosphates with 2-5 microM affinity. Affinities for ADP and GDP were approx. 10-fold lower. These data indicate that: UCP2 (a) is at least partially refolded from sarkosyl-solubilized bacterial inclusion bodies by a two-step treatment with C12E9 detergent and hydroxyapatite; (b) binds purine and pyrimidine nucleoside triphosphates with low micromolar affinity; (c) binds GDP with the same affinity as GDP inhibits superoxide-stimulated uncoupling by kidney mitochondria; and (d) exhibits a different nucleotide preference than kidney mitochondria.

  19. Molecular properties of purified human uncoupling protein 2 refolded from bacterial inclusion bodies.

    PubMed

    Jekabsons, Mika B; Echtay, Karim S; Arechaga, Ignacio; Brand, Martin D

    2003-10-01

    One way to study low-abundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies. Problems encountered with this approach include protein refolding, homogeneity, and stability. In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2). N-methylanthraniloyl-tagged ATP (Mant-ATP) experiments indicated two independent inclusion body UCP2 binding sites with dissociation constants (Kd) of 0.3-0.5 and 23-92 microM. Dimethylanthranilate, the fluorescent tag without nucleotide, bound with a Kd of greater than 100 microM, suggesting that the low affinity site reflected binding of the tag. By direct titration, UCP2 bound [8-(14)C] ATP and [8-(14)C] ADP with Kds of 4-5 and 16-18 microM, respectively. Mg2+ (2 mM) reduced the apparent ATP affinity to 53 microM, an effect entirely explained by chelation of ATP; with Mg2+, Kd using calculated free ATP was 3 microM. A combination of gel filtration, Cu2+-phenanthroline cross-linking, and ultracentrifugation indicated that 75-80% of UCP2 was in a monodisperse, 197 kDa form while the remainder was aggregated. We conclude that (a) Mant-tagged nucleotides are useful fluorescent probes with isolated UCP2 when used with dimethylanthranilate controls; (b) UCP2 binds Mg2+-free nucleotides: the Kd for ATP is about 3-5 microM and for Mant-ATP it is about 10 times lower; and (c) in C12E9 detergent, the monodisperse protein may be in dimeric form.

  20. Bacterial inclusion bodies as potential synthetic devices for pathogen recognition and a therapeutic substance release

    PubMed Central

    2013-01-01

    Background Adhesins of pathogens recognise the glycans on the host cell and mediate adherence. They are also crucial for determining the tissue preferences of pathogens. Currently, glyco-nanomaterials provide potential tool for antimicrobial therapy. We demonstrate that properly glyco-tailored inclusion bodies can specifically bind pathogen adhesins and release therapeutic substances. Results In this paper, we describe the preparation of tailored inclusion bodies via the conjugation of indicator protein aggregated to form inclusion bodies with soluble proteins. Whereas the indicator protein represents a remedy, the soluble proteins play a role in pathogen recognition. For conjugation, glutaraldehyde was used as linker. The treatment of conjugates with polar lysine, which was used to inactivate the residual glutaraldehyde, inhibited unwanted hydrophobic interactions between inclusion bodies. The tailored inclusion bodies specifically interacted with the SabA adhesin from Helicobacter pylori aggregated to form inclusion bodies that were bound to the sialic acids decorating the surface of human erythrocytes. We also tested the release of indicator proteins from the inclusion bodies using sortase A and Ssp DNAB intein self-cleaving modules, respectively. Sortase A released proteins in a relatively short period of time, whereas the intein cleavage took several weeks. Conclusions The tailored inclusion bodies are promising “nanopills” for biomedical applications. They are able to specifically target the pathogen, while a self-cleaving module releases a soluble remedy. Various self-cleaving modules can be enabled to achieve the diverse pace of remedy release. PMID:23391325

  1. Bacterial inclusion bodies as potential synthetic devices for pathogen recognition and a therapeutic substance release.

    PubMed

    Talafová, Klaudia; Hrabárová, Eva; Chorvát, Dušan; Nahálka, Jozef

    2013-02-07

    Adhesins of pathogens recognise the glycans on the host cell and mediate adherence. They are also crucial for determining the tissue preferences of pathogens. Currently, glyco-nanomaterials provide potential tool for antimicrobial therapy. We demonstrate that properly glyco-tailored inclusion bodies can specifically bind pathogen adhesins and release therapeutic substances. In this paper, we describe the preparation of tailored inclusion bodies via the conjugation of indicator protein aggregated to form inclusion bodies with soluble proteins. Whereas the indicator protein represents a remedy, the soluble proteins play a role in pathogen recognition. For conjugation, glutaraldehyde was used as linker. The treatment of conjugates with polar lysine, which was used to inactivate the residual glutaraldehyde, inhibited unwanted hydrophobic interactions between inclusion bodies. The tailored inclusion bodies specifically interacted with the SabA adhesin from Helicobacter pylori aggregated to form inclusion bodies that were bound to the sialic acids decorating the surface of human erythrocytes. We also tested the release of indicator proteins from the inclusion bodies using sortase A and Ssp DNAB intein self-cleaving modules, respectively. Sortase A released proteins in a relatively short period of time, whereas the intein cleavage took several weeks. The tailored inclusion bodies are promising "nanopills" for biomedical applications. They are able to specifically target the pathogen, while a self-cleaving module releases a soluble remedy. Various self-cleaving modules can be enabled to achieve the diverse pace of remedy release.

  2. A mathematical approach to molecular organization and proteolytic disintegration of bacterial inclusion bodies.

    PubMed

    Cubarsi, R; Carrió, M M; Villaverde, A

    2005-09-01

    The in vivo proteolytic digestion of bacterial inclusion bodies (IBs) and the kinetic analysis of the resulting protein fragments is an interesting approach to investigate the molecular organization of these unconventional protein aggregates. In this work, we describe a set of mathematical instruments useful for such analysis and interpretation of observed data. These methods combine numerical estimation of digestion rate and approximation of its high-order derivatives, modelling of fragmentation events from a mixture of Poisson processes associated with differentiated protein species, differential equations techniques in order to estimate the mixture parameters, an iterative predictor-corrector algorithm for describing the flow diagram along the cascade process, as well as least squares procedures with minimum variance estimates. The models are formulated and compared with data, and successively refined to better match experimental observations. By applying such procedures as well as newer improved algorithms of formerly developed equations, it has been possible to model, for two kinds of bacterially produced aggregation prone recombinant proteins, their cascade digestion process that has revealed intriguing features of the IB-forming polypeptides.

  3. Biological role of bacterial inclusion bodies: a model for amyloid aggregation.

    PubMed

    García-Fruitós, Elena; Sabate, Raimon; de Groot, Natalia S; Villaverde, Antonio; Ventura, Salvador

    2011-07-01

    Inclusion bodies are insoluble protein aggregates usually found in recombinant bacteria when they are forced to produce heterologous protein species. These particles are formed by polypeptides that cross-interact through sterospecific contacts and that are steadily deposited in either the cell's cytoplasm or the periplasm. An important fraction of eukaryotic proteins form inclusion bodies in bacteria, which has posed major problems in the development of the biotechnology industry. Over the last decade, the fine dissection of the quality control system in bacteria and the recognition of the amyloid-like architecture of inclusion bodies have provided dramatic insights on the dynamic biology of these aggregates. We discuss here the relevant aspects, in the interface between cell physiology and structural biology, which make inclusion bodies unique models for the study of protein aggregation, amyloid formation and prion biology in a physiologically relevant background. © 2011 The Authors Journal compilation © 2011 FEBS.

  4. Insoluble protein applications: the use of bacterial inclusion bodies as biocatalysts.

    PubMed

    Hrabárová, Eva; Achbergerová, Lucia; Nahálka, Jozef

    2015-01-01

    Biocatalysis and biotransformations have a broad application in industrial synthetic chemistry. In addition to the whole cell catalysis, purified recombinant enzymes are successfully used for biocatalysis of specific chemical reactions. In this contribution, we report characterization, immobilization, and application of several model target enzymes (D-amino acid oxidase, sialic acid aldolase, maltodextrin phosphorylase, polyphosphate kinase) physiologically aggregated within inclusion bodies (IBs) retaining their biological activity as immobilized biocatalysts.

  5. Investigation of the phase morphology of bacterial PHA inclusion bodies by contrast variation SANS

    NASA Astrophysics Data System (ADS)

    Russell, R. A.; Holden, P. J.; Garvey, C. J.; Wilde, K. L.; Hammerton, K. M.; Foster, L. J.

    2006-11-01

    Under growth-limiting conditions, many bacteria are able to metabolise excess organic acids into polyhydroxyalkanoates (PHA) and store these polymers as intracellular inclusions until the return of favourable conditions. Various models have been proposed for the macromolecular organisation of the boundary layer surrounding the polymer, and contrast-variation small-angle neutron scattering (SANS) was used to study its organisation. Inclusions formed by Pseudomonas oleovorans under hydrogenating conditions showed lowest scattering intensity at ca. 20% D 2O. The inclusions consist of protein and membrane lipids in the boundary layer and polyhydroxyoctanoate (lipid) in the inclusion body. At 20% D 2O the contributions of lipids were contrast matched with the solvent, indicating that lipids contributed the bulk of the scattering intensity observed at other D 2O/H 2O ratios. These results are inconsistent with a model of the boundary layer which proposed outer and inner layers of crystalline protein lattice sandwiching a membrane lipid membrane layer [E.S. Stuart, R.W. Lenz, R.C. Fuller, Can J Microbiol 41(Suppl 1) (1995) 84-93], and is more consistent with a model consisting of a lipid monolayer containing embedded proteins [U. Pieper-furst, M.H. Madkour, F. Mayer, A. Steinbuchel, J. Bacteriol. 176 (1994) 4328-4337.] By altering the H/D content of the precursors, we were able to collect SANS data from preparations of both deuterated and H/D copolymer inclusions, where initial PHA produced was hydrogenated followed by deuteration. Deuterated inclusions showed minimum intensity above 90% D 2O/H 2O whereas the sequentially produced copolymer (assumed to be in a core/shell arrangement) displayed minimum scattering some 20% lower, which is consistent with the increased hydrogenation of the boundary layer expected from its synthesis during supply of hydrogenated followed by deuterated precursors.

  6. L-arginine mediated renaturation enhances yield of human, α6 Type IV collagen non-collagenous domain from bacterial inclusion bodies.

    PubMed

    Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Sudhakar, Yakkanti Akul

    2012-10-01

    The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.

  7. L-arginine mediated renaturation enhances yield of human, α6 type IV collagen non-collagenous domain from bacterial inclusion bodies

    PubMed Central

    Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Akul Sudhakar, Yakkanti

    2012-01-01

    The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated. PMID:22512648

  8. Supramolecular organization of protein-releasing functional amyloids solved in bacterial inclusion bodies.

    PubMed

    Cano-Garrido, Olivia; Rodríguez-Carmona, Escarlata; Díez-Gil, César; Vázquez, Esther; Elizondo, Elisa; Cubarsi, Rafael; Seras-Franzoso, Joaquin; Corchero, José Luis; Rinas, Ursula; Ratera, Imma; Ventosa, Nora; Veciana, Jaume; Villaverde, Antonio; García-Fruitós, Elena

    2013-04-01

    Slow protein release from amyloidal materials is a molecular platform used by nature to control protein hormone secretion in the endocrine system. The molecular mechanics of the sustained protein release from amyloids remains essentially unexplored. Inclusion bodies (IBs) are natural amyloids that occur as discrete protein nanoparticles in recombinant bacteria. These protein clusters have been recently explored as protein-based functional biomaterials with diverse biomedical applications, and adapted as nanopills to deliver recombinant protein drugs into mammalian cells. Interestingly, the slow protein release from IBs does not significantly affect the particulate organization and morphology of the material, suggesting the occurrence of a tight scaffold. Here, we have determined, by using a combined set of analytical approaches, a sponge-like supramolecular organization of IBs combining differently folded protein versions (amyloid and native-like), which supports both mechanical stability and sustained protein delivery. Apart from offering structural clues about how amyloid materials release their monomeric protein components, these findings open exciting possibilities for the tailored development of smart biofunctional materials, adapted to mimic the functions of amyloid-based secretory glands of higher organisms.

  9. Oxidative refolding from inclusion bodies.

    PubMed

    Nelson, Christopher A; Lee, Chung A; Fremont, Daved H

    2014-01-01

    This protocol describes the growth and purification of bacterial inclusion body proteins with an option to selenomethionine label the targeted protein through feedback inhibition of methionine biosynthesis in common (non-auxotrophic) strains of E. coli. The method includes solubilization of inclusion body proteins by chemical denaturation and disulfide reduction, renaturation of the solubilized material through rapid dilution by pulsed injection into refolding buffer containing arginine and a mixture of oxidized and reduced glutathione, recovery of the recombinant protein using a stirred cell concentrator, and removal of the aggregated or misfolded fraction by passage over size-exclusion chromatography. The quality of the resulting protein can be assessed by SDS-PAGE.

  10. Inclusion Body Myositis

    PubMed Central

    Dimachkie, Mazen M.; Barohn, Richard J.

    2012-01-01

    The idiopathic inflammatory myopathies are a group of rare disorders that share many similarities. These include dermatomyositis (DM), polymyositis (PM), necrotizing myopathy (NM), and sporadic inclusion body myositis (IBM). Inclusion body myositis is the most common idiopathic inflammatory myopathy after age 50 and it presents with chronic proximal leg and distal arm asymmetric mucle weakness. Despite similarities with PM, it is likely that IBM is primarily a degenerative disorder rather than an inflammatory muscle disease. Inclusion body myositis is associated with a modest degree of creatine kinase (CK) elevation and an abnormal electromyogram demonstrating an irritative myopathy with some chronicity. The muscle histopathology demonstrates inflammatory exudates surrounding and invading nonnecrotic muscle fibers often times accompanied by rimmed vacuoles. In this chapter, we review sporadic IBM. We also examine past, essentially negative, clinical trials in IBM and review ongoing clinical trials. For further details on DM, PM, and NM, the reader is referred to the idiopathic inflammatory myopathies chapter. PMID:23117948

  11. Improving protein delivery of fibroblast growth factor-2 from bacterial inclusion bodies used as cell culture substrates.

    PubMed

    Seras-Franzoso, Joaquin; Peebo, Karl; García-Fruitós, Elena; Vázquez, Esther; Rinas, Ursula; Villaverde, Antonio

    2014-03-01

    Bacterial inclusion bodies (IBs) have recently been used to generate biocompatible cell culture interfaces, with diverse effects on cultured cells such as cell adhesion enhancement, stimulation of cell growth or induction of mesenchymal stem cell differentiation. Additionally, novel applications of IBs as sustained protein delivery systems with potential applications in regenerative medicine have been successfully explored. In this scenario, with IBs gaining significance in the biomedical field, the fine tuning of this functional biomaterial is crucial. In this work, the effect of temperature on fibroblast growth factor-2 (FGF-2) IB production and performance has been evaluated. FGF-2 was overexpressed in Escherichia coli at 25 and 37 °C, producing IBs with differences in size, particle structure and biological activity. Cell culture topographies made with FGF-2 IBs biofabricated at 25 °C showed higher levels of biological activity as well as a looser supramolecular structure, enabling a higher protein release from the particles. In addition, the controlled use of FGF-2 protein particles enabled the generation of functional topographies with multiple biological activities being effective on diverse cell types. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  12. Inclusion bodies in Plesiomonas shigelloides.

    PubMed Central

    Pastian, M R; Bromel, M C

    1984-01-01

    Inclusion bodies were discovered in seven environmental isolates of Plesiomonas shigelloides and the P. shigelloides control (ATCC 14029). Differential staining indicated that the inclusion bodies may be composed of polyphosphates, and developmental stages of the bodies may occur. The inclusion bodies may be useful for rapid presumptive identification of this organism. Images PMID:6320723

  13. One-pot refolding of core histones from bacterial inclusion bodies allows rapid reconstitution of histone octamer.

    PubMed

    Lee, Young-Tae; Gibbons, Garrett; Lee, Shirley Y; Nikolovska-Coleska, Zaneta; Dou, Yali

    2015-06-01

    We report an optimized method to purify and reconstitute histone octamer, which utilizes high expression of histones in inclusion bodies but eliminates the time consuming steps of individual histone purification. In the newly modified protocol, Xenopus laevis H2A, H2B, H3, and H4 are expressed individually into inclusion bodies of bacteria, which are subsequently mixed together and denatured in 8M guanidine hydrochloride. Histones are refolded and reconstituted into soluble octamer by dialysis against 2M NaCl, and metal-affinity purified through an N-terminal polyhistidine-tag added on the H2A. After cleavage of the polyhistidine-tag, histone octamer is further purified by size exclusion chromatography. We show that the nucleosomes reconstituted using the purified histone octamer above are fully functional. They serve as effective substrates for the histone methyltransferases DOT1L and MLL1. Small angle X-ray scattering further confirms that the reconstituted nucleosomes have correct structural integration of histone octamer and DNA as observed in the X-ray crystal structure. Our new protocol enables rapid reconstitution of histone octamer with an optimal yield. We expect this simplified approach to facilitate research using recombinant nucleosomes in vitro. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Inclusion bodies: not that bad…

    PubMed Central

    Ramón, Ana; Señorale-Pose, Mario; Marín, Mónica

    2014-01-01

    The formation of inclusion bodies (IBs) constitute a frequent event during the production of heterologous proteins in bacterial hosts. Although the mechanisms leading to their formation are not completely understood, empirical data have been exploited trying to predict the aggregation propensity of specific proteins while a great number of strategies have been developed to avoid the generation of IBs. However, in many cases, the formation of such aggregates can be considered an advantage for basic research as for protein production. In this review, we focus on this positive side of IBs formation in bacteria. We present a compilation on recent advances on the understanding of IBs formation and their utilization as a model to understand protein aggregation and to explore strategies to control this process. We include recent information about their composition and structure, their use as an attractive approach to produce low cost proteins and other promising applications in Biomedicine. PMID:24592259

  15. Inclusion bodies: formation and utilisation.

    PubMed

    Fahnert, Beatrix; Lilie, Hauke; Neubauer, Peter

    2004-01-01

    The efficient in vivo folding of many heterologous proteins is a major bottleneck of high level production in bacterial hosts and simple optimisation protocols have not been available yet. Therefore, inclusion body (IB) based processes play a major role as a potential strategy for the production of complex recombinant proteins. These processes combine the advantages of a high accumulation of the target protein in well-characterised bacteria such as Escherichia coli, efficient strategies for IB isolation, purification and in vitro protein refolding without the need of complicated coexpression systems. Recent advances in the molecular physiology of IB formation and resolubilisation allow straight-forward optimisation of fermentation processes to obtain a high-quality product. In addition, simple strategies have been developed to optimise the purification and renaturation of disulfide bond containing proteins making a fast transfer of such processes into the industrial production scale realistic.

  16. Inclusion Body Myositis

    PubMed Central

    Barohn, Richard J.

    2014-01-01

    The idiopathic inflammatory myopathies (IIM) are a heterogenous group of rare disorders that share many similarities. In addition to sporadic inclusion body myositis (IBM), these include dematomyositis (DM), polymyositis (PM), and autoimmune necrotizing myopathy (NM). For discussion of later three disorders, the reader is referred to the IIM review in this issue. IBM is the most common IIM after age 50. It typically presents with chronic insidious proximal leg and/or distal arm asymmetric muscle weakness leading to recurrent falls and loss of dexterity. Creatine kinase (CK) is up to 15 times elevated in IBM and needle electromyograhy (EMG) mostly shows a chronic irritative myopathy. Muscle histopathology demonstrates endomysial inflammatory exudates surrounding and invading non-necrotic muscle fibers often times accompanied by rimmed vacuoles and protein deposits. Despite inflammatory muscle pathology suggesting similarity with PM, it likely that IBM is has a prominent degenerative component as supported by refractoriness to immunosuppressive therapy. We review the evolution of our knowledge in IBM with emphasis on recent developments in the field and discuss ongoing clinical trials. PMID:25037082

  17. Inclusion Body Myositis.

    PubMed

    Greenberg, Steven A

    2016-12-01

    Inclusion body myositis (IBM) is an enigmatic progressive disease of skeletal muscle. This review provides a summary of the clinical and pathophysiologic aspects of IBM. The development of diagnostic blood testing for IBM followed from the discovery of a B-cell pathway in IBM muscle and circulating autoantibodies against NT5C1A, further establishing IBM's status as an autoimmune disease. The key role of cytotoxic T cells in IBM is further supported by the identification of a link between IBM and T-cell large granular lymphocytic leukemia. The testing of research diagnostic criteria in patients is improving its accuracy. Increases in estimated prevalences may be due to a combination of true increases and improved recognition of disease. IBM has high unmet medical need. Advances in the mechanistic understanding of IBM as an autoimmune disease will drive effective therapeutic approaches. The identification of a B-cell pathway has resulted in the first identification of an IBM autoantigen and emphasized its status as an autoimmune disease. The recognition that large granular lymphocyte CD8+ T-cell expansions are present in both blood and muscle provides additional biomarkers for IBM and suggests a mechanistic relationship to the neoplastic disease T-cell large granular lymphocytic leukemia.

  18. Thioflavin-S staining of bacterial inclusion bodies for the fast, simple, and inexpensive screening of amyloid aggregation inhibitors.

    PubMed

    Pouplana, S; Espargaro, A; Galdeano, C; Viayna, E; Sola, I; Ventura, S; Muñoz-Torrero, D; Sabate, R

    2014-01-01

    Amyloid aggregation is linked to a large number of human disorders, from neurodegenerative diseases as Alzheimer's disease (AD) or spongiform encephalopathies to non-neuropathic localized diseases as type II diabetes and cataracts. Because the formation of insoluble inclusion bodies (IBs) during recombinant protein production in bacteria has been recently shown to share mechanistic features with amyloid self-assembly, bacteria have emerged as a tool to study amyloid aggregation. Herein we present a fast, simple, inexpensive and quantitative method for the screening of potential anti-aggregating drugs. This method is based on monitoring the changes in the binding of thioflavin-S to intracellular IBs in intact Eschericchia coli cells in the presence of small chemical compounds. This in vivo technique fairly recapitulates previous in vitro data. Here we mainly use the Alzheimer's related β-amyloid peptide as a model system, but the technique can be easily implemented for screening inhibitors relevant for other conformational diseases simply by changing the recombinant amyloid protein target. Indeed, we show that this methodology can be also applied to the evaluation of inhibitors of the aggregation of tau protein, another amyloidogenic protein with a key role in AD.

  19. Inclusion-Body Myositis: Diagnosis

    MedlinePlus

    ... Make a Donation Matching Gifts Legacy Gifts Product Donations Partner Become an MDA Partner Meet our Partners How to Get Involved Donate Inclusion-Body Myositis (IBM) Share print email share facebook twitter ...

  20. Genetics Home Reference: inclusion body myopathy 2

    MedlinePlus

    ... Conditions inclusion body myopathy 2 inclusion body myopathy 2 Enable Javascript to view the expand/collapse boxes. ... Open All Close All Description Inclusion body myopathy 2 is a condition that primarily affects skeletal muscles , ...

  1. Bacterial body plans

    PubMed Central

    Rieger, Tomáš; Neubauer, Zdeněk; Blahůšková, Anna; Cvrčková, Fatima

    2008-01-01

    The bacterium Serratia marcescens produces a plethora of multicellular shapes of different colorations on solid substrates, allowing immediate visual detection of varieties. Such a plasticity allows studies on multicellular community scale spanning two extremes, from well-elaborated individual colonies to undifferentiated cell mass. For a single strain and medium, we obtained a range of different multicellular bodies, depending on the layout of initial plating. Four principal factors affecting the morphogenetic pathways of such bodies can be distinguished: (1) amount, density and distribution pattern of founder cells; (2) the configuration of surrounding free medium; (3) the presence and character of other bacterial bodies sharing the same niche; and (4) self-perception, resulting in delimitation towards other bodies. The last feature results in an ability of well-formed multicellular individuals to maintain their identity upon a close mutual contact, as well as in spontaneous separation of cell masses in experimental chimeras. We propose an “embryo-like” colony model where multicellular bacterial bodies develop along genuine ontogenetic pathways inherent to the given species (clone), while external shaping forces (like nutrient gradients, pH, etc.,) exert not formative, but only regulative roles in the process. PMID:19513204

  2. One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system

    PubMed Central

    Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

    2008-01-01

    The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Jun × 4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems. PMID:18329890

  3. One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system.

    PubMed

    Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

    2008-05-01

    The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Junx4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems.

  4. Biomarkers of inclusion body myositis.

    PubMed

    Greenberg, Steven A

    2013-11-01

    Inclusion body myositis (IBM) is a poorly understood autoimmune and degenerative disorder of skeletal muscle. Here, pathophysiological and diagnostic biomarkers of IBM are reviewed. Muscle histopathological biomarkers have been successful in stimulating the study of IBM pathophysiology for over three decades. Their use as diagnostic biomarkers, in contrast, has significant limitations. A blood biomarker, autoantibodies against a 43-kDa muscle protein reported in 2011, has now been identified as targeting cytoplasmic 5' nucleotidase (cN1A; NT5C1A), a protein involved in nucleic acid metabolism. Diagnostic testing for these autoantibodies is of high diagnostic performance for IBM. Muscle biomarkers have suggested that IBM pathophysiology is linked to myonuclear degeneration and disordered nucleic acid metabolism. A blood biomarker has high diagnostic performance for IBM, and through identification of its target links, IBM autoimmunity and degeneration together, supporting the view that IBM pathophysiology includes abnormal nucleic acid metabolism.

  5. Update in inclusion body myositis

    PubMed Central

    Machado, Pedro; Brady, Stefen; Hanna, Michael G.

    2013-01-01

    Purpose of review The purpose of this study is to review recent scientific advances relating to the natural history, cause, treatment and serum and imaging biomarkers of inclusion body myositis (IBM). Recent findings Several theories regarding the aetiopathogenesis of IBM are being explored and new therapeutic approaches are being investigated. New diagnostic criteria have been proposed, reflecting the knowledge that the diagnostic pathological findings may be absent in patients with clinically typical IBM. The role of MRI in IBM is expanding and knowledge about pathological biomarkers is increasing. The recent description of autoantibodies to cytosolic 5′ nucleotidase 1A in patients with IBM is a potentially important advance that may aid early diagnosis and provides new evidence regarding the role of autoimmunity in IBM. Summary IBM remains an enigmatic and often misdiagnosed disease. The pathogenesis of the disease is still not fully understood. To date, pharmacological treatment trials have failed to show clear efficacy. Future research should continue to focus on improving understanding of the pathophysiological mechanisms of the disease and on the identification of reliable and sensitive outcome measures for clinical trials. IBM is a rare disease and international multicentre collaboration for trials is important to translate research advances into improved patient outcomes. PMID:24067381

  6. Update in inclusion body myositis.

    PubMed

    Machado, Pedro; Brady, Stefen; Hanna, Michael G

    2013-11-01

    The purpose of this study is to review recent scientific advances relating to the natural history, cause, treatment and serum and imaging biomarkers of inclusion body myositis (IBM). Several theories regarding the aetiopathogenesis of IBM are being explored and new therapeutic approaches are being investigated. New diagnostic criteria have been proposed, reflecting the knowledge that the diagnostic pathological findings may be absent in patients with clinically typical IBM. The role of MRI in IBM is expanding and knowledge about pathological biomarkers is increasing. The recent description of autoantibodies to cytosolic 5' nucleotidase 1A in patients with IBM is a potentially important advance that may aid early diagnosis and provides new evidence regarding the role of autoimmunity in IBM. IBM remains an enigmatic and often misdiagnosed disease. The pathogenesis of the disease is still not fully understood. To date, pharmacological treatment trials have failed to show clear efficacy. Future research should continue to focus on improving understanding of the pathophysiological mechanisms of the disease and on the identification of reliable and sensitive outcome measures for clinical trials. IBM is a rare disease and international multicentre collaboration for trials is important to translate research advances into improved patient outcomes.

  7. Purification of recombinant ovalbumin from inclusion bodies of Escherichia coli.

    PubMed

    Upadhyay, Vaibhav; Singh, Anupam; Panda, Amulya K

    2016-01-01

    Recombinant ovalbumin expressed in bacterial host is essentially free from post-translational modifications and can be useful in understanding the structure-function relationship of the protein. In this study, ovalbumin was expressed in Escherichia coli in the form of inclusion bodies. Ovalbumin inclusion bodies were solubilized using urea and refolded by decreasing the urea concentration by dilution. Refolded protein was purified by anion exchange chromatography. Overall recovery of purified recombinant ovalbumin from inclusion bodies was about 30% with 98% purity. Purified recombinant ovalbumin was characterized by mass spectrometry, circular dichroism and fluorescence spectroscopy. Recombinant ovalbumin was shown to be resistant to trypsin using protease resistance assay. This indicated proper refolding of ovalbumin from inclusion bodies of E. coli. This method provides a simple way of producing ovalbumin free of post-translational modifications. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Inclusion body hepatitis in kestrels (Falco sparverius)

    USGS Publications Warehouse

    Sileo, L.; Franson, J.C.; Graham, D.L.; Domermuth, C.H.; Rattner, B.A.; Pattee, O.H.

    1982-01-01

    Inclusion body disease of suspected adenovirus etiology was the apparent cause of death of 9 captive kestrels (Falco sparverius). Cloacal hemorrhage was the only prominent gross lesion; disseminated hepatocellular necrosis and intranuclear inclusion bodies were evident microscopically. Attempts to reproduce the disease, and to propagate and serologically characterize the agent were unsuccessful.

  9. Lagophthalmos and Ptosis in Inclusion Body Myositis.

    PubMed

    Shams, Fatemeh; Cauchi, Paul

    Sporadic inclusion body myositis is the most commonly acquired type of idiopathic inflammatory myopathy in people aged 50 and above. There is early weakness and atrophy of forearms and quadriceps and a third of patients also have mild facial weakness. Extraocular muscles are not affected and ptosis is rarely seen. The authors describe a unique case in which inclusion body myositis presented with early mid face weakness and atrophy resulting in unilateral lagophthalmus, and ptosis, which have not been documented before. This case is not only unique in its presentation but also emphasizes the importance of considering differential diagnoses and conservative measures before contemplating surgery.

  10. Familial Encephalopathy with Neuroserpin Inclusion Bodies

    PubMed Central

    Davis, Richard L.; Holohan, Peter D.; Shrimpton, Antony E.; Tatum, Arthur H.; Daucher, John; Collins, George H.; Todd, Robert; Bradshaw, Charles; Kent, Paul; Feiglin, David; Rosenbaum, Arthur; Yerby, Mark S.; Shaw, Cheng-Mei; Lacbawan, Felicitas; Lawrence, Daniel A.

    1999-01-01

    We report on a new familial neurodegenerative disease with associated dementia that has presented clinically in the fifth decade, in both genders, and in each of several generations of a large family from New York State—a pattern of inheritance consistent with an autosomal dominant mode of transmission. A key pathological finding is the presence of neuronal inclusion bodies distributed throughout the gray matter of the cerebral cortex and in certain subcortical nuclei. These inclusions are distinct from any described previously and henceforth are identified as Collins bodies. The Collins bodies can be isolated by simple biochemical procedures and have a surprisingly simple composition; neuroserpin (a serine protease inhibitor) is their predominant component. An affinity-purified antibody against neuroserpin specifically labels the Collins bodies, confirming their chemical composition. Therefore, we propose a new disease entity—familial encephalopathy with neuroserpin inclusion bodies (FENIB). The conclusion that FENIB is a previously unrecognized neurodegenerative disease is supported by finding Collins bodies in a small kindred from Oregon with familial dementia who are unrelated to the New York family. The autosomal dominant inheritance strongly suggests that FENIB is caused by mutations in the neuroserpin gene, resulting in intracellular accumulation of the mutant protein. PMID:10595921

  11. Familial encephalopathy with neuroserpin inclusion bodies.

    PubMed

    Davis, R L; Holohan, P D; Shrimpton, A E; Tatum, A H; Daucher, J; Collins, G H; Todd, R; Bradshaw, C; Kent, P; Feiglin, D; Rosenbaum, A; Yerby, M S; Shaw, C M; Lacbawan, F; Lawrence, D A

    1999-12-01

    We report on a new familial neurodegenerative disease with associated dementia that has presented clinically in the fifth decade, in both genders, and in each of several generations of a large family from New York State-a pattern of inheritance consistent with an autosomal dominant mode of transmission. A key pathological finding is the presence of neuronal inclusion bodies distributed throughout the gray matter of the cerebral cortex and in certain subcortical nuclei. These inclusions are distinct from any described previously and henceforth are identified as Collins bodies. The Collins bodies can be isolated by simple biochemical procedures and have a surprisingly simple composition; neuroserpin (a serine protease inhibitor) is their predominant component. An affinity-purified antibody against neuroserpin specifically labels the Collins bodies, confirming their chemical composition. Therefore, we propose a new disease entity-familial encephalopathy with neuroserpin inclusion bodies (FENIB). The conclusion that FENIB is a previously unrecognized neurodegenerative disease is supported by finding Collins bodies in a small kindred from Oregon with familial dementia who are unrelated to the New York family. The autosomal dominant inheritance strongly suggests that FENIB is caused by mutations in the neuroserpin gene, resulting in intracellular accumulation of the mutant protein.

  12. Dilated cardiomyopathy and inclusion body myositis.

    PubMed

    Ballo, Piercarlo; Chiodi, Leandro; Cameli, Matteo; Malandrini, Alessandro; Federico, Antonio; Mondillo, Sergio; Zuppiroli, Alfredo

    2012-04-01

    Inclusion body myositis (IBM) is the most common inflammatory myopathy after 50 years of age. In contrast to polymyositis and dermatomyositis, in which cardiac involvement is relatively common, current evidences indicate that IBM is not associated with cardiac disease. We report the case of a patient with biopsy-proven IBM who developed heart failure and major ventricular arrhythmias secondary to dilated cardiomyopathy few months after the clinical onset of IBM, and in whom no pathophysiologic causes explaining cardiac enlargement and dysfunction were found by laboratory and instrumental investigations. The hypothesis of a pathophysiologic association between the two conditions is discussed.

  13. Inclusion bodies and hepatopathies in psittacines.

    PubMed

    Pass, D A

    1987-01-01

    Intranuclear inclusion bodies in hepatic cells of seven Peach-faced Lovebirds (Agapornis roseicollis), two Black-masked Love birds (A. personata) and two galahs (Cacatua roseicapilla) were examined electron microscopically. Thin sections were prepared from formalin-fixed Epon-embedded tissue or from haematoxylin and eosin stained sections that were subsequently embedded in Epon. Discrete and diffuse eosinophilic inclusions in hepatocytes of eight birds were composed of filaments and virus particles were not demonstrated within them. Virus-like particles of at least three types were found in diffuse, lightly basophilic inclusions of similar appearance in hepatocytes and Kupffer cells or endothelial cells in four birds. Particles, 66 nm in diameter which were probably adenovirus were demonstrated in hepatocytes in one A. roseicollis. Particles 26 nm to 39 nm in diameter which resembled papovaviruses were seen in one A. personata and one C. roseicapilla. Particles of 40 nm to 50 nm in diameter were seen in one A. personata but because of the method of preparation it was not possible to state whether they were adenovirus or papovavirus.

  14. Inclusion of solid particles in bacterial cellulose.

    PubMed

    Serafica, G; Mormino, R; Bungay, H

    2002-05-01

    Depending upon the strain and the method of cultivation, bacterial cellulose can be reticulated filaments, pellets, or a dense, tough gel called a pellicle. The pellicular form is commonly made by surface culture, but a rotating disk bioreactor is more efficient and reduces the time of a run to about 3.5 days instead of the usual 12-20 days. Particles added to the medium as the gel is forming are trapped to form a new class of composite materials. Particles enter the films that are forming on the disks at rates depending on the size and geometry of the particle, as well as the rotational speed and concentration of the suspension.

  15. Inclusion body myositis: old and new concepts.

    PubMed

    Amato, A A; Barohn, R J

    2009-11-01

    Inclusion body myositis (IBM) is the most common idiopathic inflammatory myopathy occurring in patients over the age of 50 years and probably accounts for about 30% of all inflammatory myopathies. Muscle biopsy characteristically reveals endomysial inflammation, small groups of atrophic fibres, eosinophilic cytoplasmic inclusions and muscle fibres with one or more rimmed vacuoles. However, any given biopsy may lack these histopathological abnormalities; the clinical examination is often the key to diagnosis. Early and often asymmetrical weakness and atrophy of the quadriceps and flexor forearm muscles (ie, wrist and finger flexors) are the clinical hallmarks of IBM. The pathogenesis of IBM is unknown. It may be autoimmune inflammatory myopathy or a primary degenerative myopathy with a secondary inflammatory. A prevailing theory is that there is an overproduction of beta-amyloid precursor protein in muscle fibres that is somehow cleaved into abnormal beta-amyloid, and the accumulation of the latter is somehow toxic to muscle fibres. However, there are many problems with this theory and more work needs to be done. Unfortunately, IBM is generally refractory to therapy. Further research into the pathogenesis, along with both preliminary small pilot trials and larger double blind, placebo controlled efficacy trials, are needed to make progress in our understanding and therapeutic approach for this disorder.

  16. [Inclusion body myositis (IBM) -- a review].

    PubMed

    Czaplinski, A; Renaud, S; Fuhr, P

    2003-04-02

    Inclusion body myositis (IBM) is characterized by the insidious onset of slowly progressive proximal and distal weakness. The clinical hallmark of IBM are atrophy and weakness of the quadriceps and the wrist and finger flexors. Although frequently misdiagnosed, IBM is the most common inflammatory myopathy in patients over the age of 50 years. The diagnosis of IBM can be made, even in absence of a typical clinical history, exclusive on the basis of muscle biopsy when all of the characteristic histopathological findings are present (inflammation, rimmed vacuoles, protein deposits, and 15-18 nm tubofilaments). Apart from sporadic IBM there is a group of heterogeneous inherited myopathies with histopathologic similarities to sporadic IBM. Although some immunomodulating therapies may exert transient and mild benefits, there is no effective treatment for IBM.

  17. Ongoing developments in sporadic inclusion body myositis.

    PubMed

    Machado, Pedro M; Ahmed, Mhoriam; Brady, Stefen; Gang, Qiang; Healy, Estelle; Morrow, Jasper M; Wallace, Amanda C; Dewar, Liz; Ramdharry, Gita; Parton, Matthew; Holton, Janice L; Houlden, Henry; Greensmith, Linda; Hanna, Michael G

    2014-12-01

    Sporadic inclusion body myositis (IBM) is an acquired muscle disorder associated with ageing, for which there is no effective treatment. Ongoing developments include: genetic studies that may provide insights regarding the pathogenesis of IBM, improved histopathological markers, the description of a new IBM autoantibody, scrutiny of the diagnostic utility of clinical features and biomarkers, the refinement of diagnostic criteria, the emerging use of MRI as a diagnostic and monitoring tool, and new pathogenic insights that have led to novel therapeutic approaches being trialled for IBM, including treatments with the objective of restoring protein homeostasis and myostatin blockers. The effect of exercise in IBM continues to be investigated. However, despite these ongoing developments, the aetiopathogenesis of IBM remains uncertain. A translational and multidisciplinary collaborative approach is critical to improve the diagnosis, treatment, and care of patients with IBM.

  18. Inclusion body myositis with granuloma formation in muscle tissue.

    PubMed

    Sakai, Kenji; Ikeda, Yoshihisa; Ishida, Chiho; Matsumoto, Yasuko; Ono, Kenjiro; Iwasa, Kazuo; Yamada, Masahito

    2015-09-01

    Inclusion body myositis is a form of inflammatory myopathy. We identified 4 cases of inclusion body myositis showing granuloma formation in muscle tissue and aimed to assess the features of this atypical form of inclusion body myositis. We retrospectively reviewed consecutive patients who satisfied European Neuromuscular Centre IBM Research Diagnostic Criteria 2011. Then, we assessed clinical profiles and pathological findings in patients with inclusion body myositis with granuloma and compared these findings with those of typical inclusion body myositis without granuloma. We identified 15 patients with inclusion body myositis. Four patients showed granuloma formation in muscle tissue in addition to typical pathological features of inclusion body myositis. Granulomas comprised a mixture of inflammatory cells, such as macrophages, epithelioid histiocytic cells, and lymphocytes. One patient was found to have mediastinal granulomatous lymphadenopathy; however, the evidence in other patients was insufficient for a diagnosis of systemic sarcoidosis. There were no significant differences between groups with and without granuloma regarding clinical manifestations, laboratory findings, response to immunomodulating therapies, or myopathological profiles. We established a new form of inclusion body myositis showing granuloma formation in muscle tissue. Inclusion body myositis and granuloma formation could have identical pathomechanisms concerning dysregulation of autophagy.

  19. Proteomic analysis of inclusion body myositis.

    PubMed

    Li, Jie; Yin, Chunyue; Okamoto, Hiroaki; Jaffe, Howard; Oldfield, Edward H; Zhuang, Zhengping; Vortmeyer, Alexander O; Rushing, Elisabeth J

    2006-08-01

    Sporadic inclusion body myositis (IBM) is the most frequently acquired inflammatory myopathy of late adult life, yet its diagnostic criteria and pathogenesis remain poorly defined. Because effective treatment is lacking, research efforts have intensified to identify specific markers for this debilitating disorder. In this study, proteomic analysis of 4 cases of sporadic IBM was compared with 5 cases of inflammatory myopathy without clinicopathologic features of IBM to distinguish the IBM-specific proteome. Proteins were separated by 2-dimensional polyacrylamide gel electrophoresis and profiled by mass spectrometric sequencing. Expression of most proteins remained unchanged; however, 16 proteins were upregulated and 6 proteins were downregulated in IBM compared with cases of non-IBM inflammatory myopathy. These IBM-specific proteins included apolipoprotein A-I, amyloid beta precursor protein, and transthyretin, which have been associated with amyloidosis; superoxide dismutase, enolase, and various molecular chaperones indicate perturbations in detoxification, energy metabolism, and protein folding, respectively. The IBM-downregulated proteins mainly serve as carriers for muscle contraction and other normal muscle functions. We further applied Western blot and immunohistochemistry to verify the proteomic findings. This study validates proteomics as a powerful tool in the study of muscle disease and indicates a unique pattern of protein expression in IBM.

  20. Renal pathophysiologic role of cortical tubular inclusion bodies.

    PubMed

    Radi, Zaher A; Stewart, Zachary S; Grzemski, Felicity A; Bobrowski, Walter F

    2013-01-01

    Renal tubular inclusion bodies are rarely associated with drug administration. The authors describe the finding of renal cortical tubular intranuclear and intracytoplasmic inclusion bodies associated with the oral administration of a norepinephrine/serotonin reuptake inhibitor (NSRI) test article in Sprague-Dawley (SD) rats. Rats were given an NSRI daily for 4 weeks, and kidney histopathologic, ultrastructural pathology, and immunohistochemical examinations were performed. Round eosinophilic intranuclear inclusion bodies were observed histologically in the tubular epithelial cells of the renal cortex in male and female SD rats given the NSRI compound. No evidence of degeneration or necrosis was noted in the inclusion-containing renal cells. By ultrastructural pathology, inclusion bodies consisted of finely granular, amorphous, and uniformly stained nonmembrane-bound material. By immunohistochemistry, inclusion bodies stained positive for d-amino acid oxidase (DAO) protein. In addition, similar inclusion bodies were noted in the cytoplasmic tubular epithelial compartment by ultrastructural and immunohistochemical examination.  This is the first description of these renal inclusion bodies after an NSRI test article administration in SD rats. Such drug-induced renal inclusion bodies are rat-specific, do not represent an expression of nephrotoxicity, represent altered metabolism of d-amino acids, and are not relevant to human safety risk assessment.

  1. Secondary structure characterization of beta-lactamase inclusion bodies.

    PubMed

    Przybycien, T M; Dunn, J P; Valax, P; Georgiou, G

    1994-01-01

    The secondary structure of proteins in E. coli inclusion bodies was investigated via Raman spectroscopy. Inclusion bodies were purified from cells expressing different forms of RTEM beta-lactamase and grown at either 37 or 42 degrees C. All of the solid phase inclusion body samples examined gave amide I band spectra that were perturbed from that of the native, purified protein in both solution and powder forms; secondary structure estimates indicated significant decreases in alpha-helix and increases in beta-sheet contents in the inclusion body samples. The structure estimates for inclusion bodies isolated from 37 degrees C cultures were similar, regardless of aggregate localization in the E. coli cytoplasmic or periplasmic spaces or beta-lactamase precursor content. Inclusion bodies obtained from 42 degrees C cells exhibited a further reduction of alpha-helix and augmentation of beta-sheet contents relative to those from 37 degrees C cultures. These results are consistent with the paradigm for inclusion body formation via the self-association of intra-cellular folding intermediates having extensive secondary structure content. Further, the overall secondary structure content of inclusion bodies is not significantly affected by subcellular compartmentalization, but may be altered at increased temperatures.

  2. Inclusion bodies are a site of ebolavirus replication.

    PubMed

    Hoenen, Thomas; Shabman, Reed S; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F; Becker, Stephan; Feldmann, Heinz

    2012-11-01

    Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.

  3. Inclusion Bodies Are a Site of Ebolavirus Replication

    PubMed Central

    Hoenen, Thomas; Shabman, Reed S.; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F.; Becker, Stephan

    2012-01-01

    Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place. PMID:22915810

  4. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process.

    PubMed

    Singh, Anupam; Upadhyay, Vaibhav; Upadhyay, Arun Kumar; Singh, Surinder Mohan; Panda, Amulya Kumar

    2015-03-25

    Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by optimizing the individual steps of the process, especially solubilization of the inclusion bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in inclusion body aggregates have native-like structure. These methods solubilize the inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from inclusion bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.

  5. Refolding of proteins from inclusion bodies: rational design and recipes.

    PubMed

    Basu, Anindya; Li, Xiang; Leong, Susanna Su Jan

    2011-10-01

    The need to develop protein biomanufacturing platforms that can deliver proteins quickly and cost-effectively is ever more pressing. The rapid rate at which genomes can now be sequenced demands efficient protein production platforms for gene function identification. There is a continued need for the biotech industry to deliver new and more effective protein-based drugs to address new diseases. Bacterial production platforms have the advantage of high expression yields, but insoluble expression of many proteins necessitates the development of diverse and optimised refolding-based processes. Strategies employed to eliminate insoluble expression are reviewed, where it is concluded that inclusion bodies are difficult to eliminate for various reasons. Rational design of refolding systems and recipes are therefore needed to expedite production of recombinant proteins. This review article discusses efforts towards rational design of refolding systems and recipes, which can be guided by the development of refolding screening platforms that yield both qualitative and quantitative information on the progression of a given refolding process. The new opportunities presented by light scattering technologies for developing rational protein refolding buffer systems which in turn can be used to develop new process designs armed with better monitoring and controlling functionalities are discussed. The coupling of dynamic and static light scattering methodologies for incorporation into future bioprocess designs to ensure delivery of high-quality refolded proteins at faster rates is also discussed.

  6. Yeast prions form infectious amyloid inclusion bodies in bacteria

    PubMed Central

    2012-01-01

    Background Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. Results Here we show that both the prion domain of Sup35 (Sup35-NM) and the Ure2 protein (Ure2p) form inclusion bodies (IBs) displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. Conclusions An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity. PMID:22731490

  7. Thermolabile folding intermediates: inclusion body precursors and chaperonin substrates

    PubMed Central

    KING, JONATHAN; HAASE-PETTINGELL, CAMERON; ROBINSON, ANNE SKAJA; SPEED, MARGARET; MITRAKI, ANNA

    2007-01-01

    An unexpected aspect of the expression of cloned genes is the frequent failure of newly synthesized polypeptide chains to reach their native state, accumulating instead as insoluble inclusion bodies. Amyloid deposits represent a related state associated with a variety of human diseases. The critical folding intermediates at the juncture of productive folding and the off-pathway aggregation reaction have been identified for the phage P22 tailspike and coat proteins. Though the parallel β coil tailspike is thermostable, an early intracellular folding intermediate is thermolabile. As the temperature of intracellular folding is increased, this species partitions to inclusion bodies, a kinetic trap within the cell. The earliest intermediates along the in vitro aggregation pathway, sequential multimers of the thermolabile folding intermediates, have been directly identified by native gel electrophoresis. Temperature-sensitive folding (tsf) mutations identify sites in the β coil domain, which direct the junctional intermediate down the productive pathway. Global suppressors of tsf mutants inhibit the pathway to inclusion bodies, rescuing the mutant chains. These mutants identify sites important for avoiding aggregation. Coat folding intermediates also partition to inclusion bodies as temperature is increased. Coat tsf mutants are suppressed by overexpression of the GroE chaperonin, indicating that the thermolabile intermediate is a physiological substrate for GroE. We suggest that many proteins are likely to have thermolabile intermediates in their intracellular folding pathways, which will be precursors to inclusion body formation at elevated temperatures and therefore substrates for heat shock chaperonins. PMID:8566549

  8. Inclusion bodies in loggerhead erythrocytes are associated with unstable hemoglobin and resemble human Heinz bodies.

    PubMed

    Basile, Filomena; Di Santi, Annalisa; Caldora, Mercedes; Ferretti, Luigi; Bentivegna, Flegra; Pica, Alessandra

    2011-08-01

    The aim of this study was to clarify the role of the erythrocyte inclusions found during the hematological screening of loggerhead population of the Mediterranean Sea. We studied the erythrocyte inclusions in blood specimens collected from six juvenile and nine adult specimens of the loggerhead turtle, Caretta caretta, from the Adriatic and Tyrrhenian Seas. Our study indicates that the percentage of mature erythrocytes containing inclusions ranged from 3 to 82%. Each erythrocyte contained only one round inclusion body. Inclusion bodies stained with May Grünwald-Giemsa show that their cytochemical and ultrastructure characteristics are identical to those of human Heinz bodies. Because Heinz bodies originate from the precipitation of unstable hemoglobin (Hb) and cause globular osmotic resistance to increase, we analyzed loggerhead Hb using electrophoresis and high-performance liquid chromatography to detect and quantitate Hb fractions. We also tested the resistance of Hb to alkaline pH, heat, isopropanol denaturation, and globular osmosis. Our hemogram results excluded the occurrence of any infection, which could be associated with an inclusion body, in all the specimens. Negative Feulgen staining indicated that the inclusion bodies are not derived from DNA fragmentation. We hypothesize that amino acid substitutions could explain why loggerhead Hb precipitates under normal physiologic conditions, forming Heinz bodies. The identification of inclusion bodies in loggerhead erythrocytes allow us to better understand the haematological characteristics and the physiology of these ancient reptiles, thus aiding efforts to conserve such an endangered species. Copyright © 2011 Wiley-Liss, Inc., A Wiley Company.

  9. Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies.

    PubMed

    Schipper-Krom, Sabine; Juenemann, Katrin; Jansen, Anne H; Wiemhoefer, Anne; van den Nieuwendijk, Rianne; Smith, Donna L; Hink, Mark A; Bates, Gillian P; Overkleeft, Hermen; Ovaa, Huib; Reits, Eric

    2014-01-03

    Neurodegenerative disorders such as Huntington's disease are hallmarked by neuronal intracellular inclusion body formation. Whether proteasomes are irreversibly recruited into inclusion bodies in these protein misfolding disorders is a controversial subject. In addition, it has been proposed that the proteasomes may become clogged by the aggregated protein fragments, leading to impairment of the ubiquitin-proteasome system. Here, we show by fluorescence pulse-chase experiments in living cells that proteasomes are dynamically and reversibly recruited into inclusion bodies. As these recruited proteasomes remain catalytically active and accessible to substrates, our results challenge the concept of proteasome sequestration and impairment in Huntington's disease, and support the reported absence of proteasome impairment in mouse models of Huntington's disease. Copyright © 2013 Federation of European Biochemical Societies. All rights reserved.

  10. Monitoring the centrifugal recovery of recombinant protein inclusion bodies.

    PubMed

    Middelberg, A P; O'Neill, B K

    1991-04-01

    The industrial processing of proteins expressed as insoluble inclusion bodies employs a reasonably standard sequence of unit operations. One of these is centrifugation, which serves to concentrate the inclusion bodies after disruption of the host microorganism, and also separates the inclusion bodies from other cellular debris. Monitoring the performance of the centrifuge is essential if excessive product and hence financial loss is to be avoided and a reasonable separation obtained. The analytical disc centrifuge may be used to monitor the centrifugation. This instrument returns the sample size distribution with high resolution and without fouling. By obtaining size distributions of the centrifuge feed, supernatant and concentrate, the fractional collection efficiency of the centrifuge may be determined as a function of the Stokes diameter, and a mass balance constructed.

  11. Inclusion body myositis – pathomechanism and lessons from genetics

    PubMed Central

    Murnyák, Balázs; Bodoki, Levente; Vincze, Melinda; Griger, Zoltán; Csonka, Tamás; Szepesi, Rita; Kurucz, Andrea; Dankó, Katalin

    2015-01-01

    Inclusion body myositis is a rare, late-onset myopathy. Both inflammatory and myodegenerative features play an important role in their pathogenesis. Overlapping clinicopathological entities are the familial inclusion body myopathies with or without dementia. These myopathies share several clinical and pathological features with the sporadic inflammatory disease. Therefore, better understanding of the genetic basis and pathomechanism of these rare familial cases may advance our knowledge and enable more effective treatment options in sporadic IBM, which is currently considered a relentlessly progressive incurable disease. PMID:28352694

  12. [Solubilization Specificities Interferon beta-1b from Inclusion Bodies].

    PubMed

    Zhuravko, A S; Kononova, N V; Bobruskin, A I

    2015-01-01

    A new solubilization method of recombinant interferon beta-1b (IFNβ-1b) from the inclusion bodies was developed. This method allows to extract the target protein selectively in the solutions of different alcohols, such as ethanol, propanol and isopropanol. It was shown that the more effective IFNβ-1b solubilization was achieved in the 55% propanol solution. This method allowed to extract the target protein from inclusion bodies around 85-90%, and significantly reduced Escherichia coli content in the solubilizate, in comparison with standard methods.

  13. Functional inclusion bodies produced in the yeast Pichia pastoris.

    PubMed

    Rueda, Fabián; Gasser, Brigitte; Sánchez-Chardi, Alejandro; Roldán, Mònica; Villegas, Sandra; Puxbaum, Verena; Ferrer-Miralles, Neus; Unzueta, Ugutz; Vázquez, Esther; Garcia-Fruitós, Elena; Mattanovich, Diethard; Villaverde, Antonio

    2016-10-01

    Bacterial inclusion bodies (IBs) are non-toxic protein aggregates commonly produced in recombinant bacteria. They are formed by a mixture of highly stable amyloid-like fibrils and releasable protein species with a significant extent of secondary structure, and are often functional. As nano structured materials, they are gaining biomedical interest because of the combination of submicron size, mechanical stability and biological activity, together with their ability to interact with mammalian cell membranes for subsequent cell penetration in absence of toxicity. Since essentially any protein species can be obtained as IBs, these entities, as well as related protein clusters (e.g., aggresomes), are being explored in biocatalysis and in biomedicine as mechanically stable sources of functional protein. One of the major bottlenecks for uses of IBs in biological interfaces is their potential contamination with endotoxins from producing bacteria. To overcome this hurdle, we have explored here the controlled production of functional IBs in the yeast Pichia pastoris (Komagataella spp.), an endotoxin-free host system for recombinant protein production, and determined the main physicochemical and biological traits of these materials. Quantitative and qualitative approaches clearly indicate the formation of IBs inside yeast, similar in morphology, size and biological activity to those produced in E. coli, that once purified, interact with mammalian cell membranes and penetrate cultured mammalian cells in absence of toxicity. Structurally and functionally similar from those produced in E. coli, the controlled production of IBs in P. pastoris demonstrates that yeasts can be used as convenient platforms for the biological fabrication of self-organizing protein materials in absence of potential endotoxin contamination and with additional advantages regarding, among others, post-translational modifications often required for protein functionality.

  14. Flexible queers, serious bodies: transgender inclusion in queer spaces.

    PubMed

    Stone, Amy L

    2013-01-01

    Queer spaces are significant for understanding transgender inclusion as "queer spaces were places where individuals were expected to be attentive to or aware of alternative possibilities for being, including non-normative formulations of bodies, genders, desires and practices" ( Nash, 2011 , p. 203). Indeed, in this interview study of members of a queer leather group called the Club, members described a flexible "sexual landscape" that easily includes transgender members. However, these same queer spaces have been criticized for the way they regulate queer bodies and organize queer subjectivities. In this study, queer members of the Club also contrasted playful queer flexibility with serious transgender bodies. This article argues that, although there is a reiterative relation between transgender inclusion and queer spaces, the idealization of flexibility within queer spaces can also serve to marginalize and regulate transgender bodies.

  15. Circovirus inclusion bodies in intestinal muscle cells of a canary.

    PubMed

    Rampin, T; Manarolla, G; Pisoni, G; Recordati, C; Sironi, G

    2006-08-01

    Multiple cytoplasmic inclusion bodies were observed in the intestinal smooth muscle cells of an adult canary from an aviary with a history of high mortality (50%) both in adult and young birds. Grossly, a mild enteritis was the only lesion appreciable. Smears of the proventricular contents contained a few megabacteria (Macrorhabdus ornithogaster). The intestinal inclusions were found in very high numbers in all parts of the tract examined. They appeared round to oval, amphophilic and hyaline in sections stained with haematoxylin and eosin, and magenta with Feulgen stain. Inclusions of the same type were occasionally detectable in the wall of a few splenic and pancreatic arteries. No inclusions or lesions were seen in the other organs examined. Transmission electron microscopy of the intestinal wall revealed circovirus-like particles either in paracrystalline arrays or loose arrangements, mostly within the cytoplasm of the intestinal muscule cells. Polymerase chain reaction amplification and sequence analysis confirmed infection with canary circovirus.

  16. High throughput inclusion body sizing: Nano particle tracking analysis.

    PubMed

    Reichelt, Wieland N; Kaineder, Andreas; Brillmann, Markus; Neutsch, Lukas; Taschauer, Alexander; Lohninger, Hans; Herwig, Christoph

    2017-06-01

    The expression of pharmaceutical relevant proteins in Escherichia coli frequently triggers inclusion body (IB) formation caused by protein aggregation. In the scientific literature, substantial effort has been devoted to the quantification of IB size. However, particle-based methods used up to this point to analyze the physical properties of representative numbers of IBs lack sensitivity and/or orthogonal verification. Using high pressure freezing and automated freeze substitution for transmission electron microscopy (TEM) the cytosolic inclusion body structure was preserved within the cells. TEM imaging in combination with manual grey scale image segmentation allowed the quantification of relative areas covered by the inclusion body within the cytosol. As a high throughput method nano particle tracking analysis (NTA) enables one to derive the diameter of inclusion bodies in cell homogenate based on a measurement of the Brownian motion. The NTA analysis of fixated (glutaraldehyde) and non-fixated IBs suggests that high pressure homogenization annihilates the native physiological shape of IBs. Nevertheless, the ratio of particle counts of non-fixated and fixated samples could potentially serve as factor for particle stickiness. In this contribution, we establish image segmentation of TEM pictures as an orthogonal method to size biologic particles in the cytosol of cells. More importantly, NTA has been established as a particle-based, fast and high throughput method (1000-3000 particles), thus constituting a much more accurate and representative analysis than currently available methods. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Insights into muscle degeneration from heritable inclusion body myopathies.

    PubMed

    Krause, Sabine

    2015-01-01

    Muscle mass and function are gradually lost in age-related, degenerative neuromuscular disorders, which also reflect the clinical hallmarks of sarcopenia. The consensus definition of sarcopenia includes a condition of age-related loss of muscle mass, quality, and strength. The most common acquired muscle disease affecting adults aged over 50 years is sporadic inclusion body myositis (sIBM). Besides inflammatory effects and immune-mediated muscle injury, degenerative myofiber changes are characteristic features of the disease. Although the earliest triggering events in sIBM remain elusive, a plethora of downstream mechanisms are implicated in the pathophysiology of muscle wasting. Although it remains controversial whether hereditary forms of inclusion body myopathy (IBM) may be considered as degenerative sIBM disease models, partial pathophysiological aspects can mimic the much more frequent sporadic condition, in particular the occurrence of inclusion bodies in skeletal muscle. Various clinical aspects in genetically determined skeletal muscle disorders reflect age-related alterations observed in sarcopenia. Several intriguing clues from monogenic defects in heritable IBMs contributing to the molecular basis of muscle loss will be discussed with special emphasis on inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) and GNE myopathy. Finally, also the recently identified dominant multisystem proteinopathy will be considered, which may rarely present as IBM.

  18. In Vivo Enzyme Immobilization by Inclusion Body Display▿ †

    PubMed Central

    Steinmann, Björn; Christmann, Andreas; Heiseler, Tim; Fritz, Janine; Kolmar, Harald

    2010-01-01

    A novel strategy for in vivo immobilization of enzymes on the surfaces of inclusion bodies has been established. It relies on expression in Escherichia coli of the polyhydroxybutyrate synthase PhaC from Cupriavidus necator, which carries at its amino terminus an engineered negatively charged α-helical coil (Ecoil) and forms inclusion bodies upon high-level expression. Coexpression in the same cell of galactose oxidase (GOase) from Fusarium spp. carrying a carboxy-terminal positively charged coil (lysine-rich coil [Kcoil]) sequence results in heterodimeric coiled-coil formation in vivo and in the capture of the enzyme in active form on the surface of the inclusion body particle. These round-shaped enzyme-decorated microparticles, with sizes of approximately 0.7 μm, can be isolated from lysed cells simply by centrifugation. The cost-effective one-step generation and isolation of enzymes immobilized on inclusion body particles may become useful for various applications in bioprocessing and biotransformation. PMID:20581198

  19. [Inclusion Bodies are Formed in SFTSV-infected Human Macrophages].

    PubMed

    Jin, Cong; Song, Jingdong; Han, Ying; Li, Chuan; Qiu, Peihong; Liang, Mifang

    2016-01-01

    The severe fever with thrombocytopenia syndrome virus (SFTSV) is a new member in the genus Phlebovirus of the family Bunyaviridae identified in China. The SFTSV is also the causative pathogen of an emerging infectious disease: severe fever with thrombocytopenia syndrome. Using immunofluorescent staining and confocal microscopy, the intracellular distribution of nucleocapsid protein (NP) in SFTSV-infected THP-1 cells was investigated with serial doses of SFTSV at different times after infection. Transmission electron microscopy was used to observe the ultrafine intracellular structure of SFTSV-infected THP-1 cells at different times after infection. SFTSV NP could form intracellular inclusion bodies in infected THP-1 cells. The association between NP-formed inclusion bodies and virus production was analyzed: the size of the inclusion body formed 3 days after infection was correlated with the viral load in supernatants collected 7 days after infection. These findings suggest that the inclusion bodies formed in SFTSV-infected THP-1 cells could be where the SFTSV uses host-cell proteins and intracellular organelles to produce new viral particles.

  20. In vivo enzyme immobilization by inclusion body display.

    PubMed

    Steinmann, Björn; Christmann, Andreas; Heiseler, Tim; Fritz, Janine; Kolmar, Harald

    2010-08-01

    A novel strategy for in vivo immobilization of enzymes on the surfaces of inclusion bodies has been established. It relies on expression in Escherichia coli of the polyhydroxybutyrate synthase PhaC from Cupriavidus necator, which carries at its amino terminus an engineered negatively charged alpha-helical coil (Ecoil) and forms inclusion bodies upon high-level expression. Coexpression in the same cell of galactose oxidase (GOase) from Fusarium spp. carrying a carboxy-terminal positively charged coil (lysine-rich coil [Kcoil]) sequence results in heterodimeric coiled-coil formation in vivo and in the capture of the enzyme in active form on the surface of the inclusion body particle. These round-shaped enzyme-decorated microparticles, with sizes of approximately 0.7 mum, can be isolated from lysed cells simply by centrifugation. The cost-effective one-step generation and isolation of enzymes immobilized on inclusion body particles may become useful for various applications in bioprocessing and biotransformation.

  1. Role of the disaggregase ClpB in processing of proteins aggregated as inclusion bodies.

    PubMed

    Zblewska, Kamila; Krajewska, Joanna; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

    2014-08-01

    Overproduction of heterologous proteins in bacterial systems often results in the formation of insoluble inclusion bodies (IBs), which is a major impediment in biochemical research and biotechnology. In principle, the activity of molecular chaperones could be employed to gain control over the IB formation and to improve the recombinant protein yields, but the potential of each of the major bacterial chaperones (DnaK/J, GroEL/ES, and ClpB) to process IBs has not been fully established yet. We investigated the formation of inclusion bodies (IBs) of two aggregation-prone proteins, VP1LAC and VP1GFP, overproduced in Escherichiacoli in the presence and absence of the chaperone ClpB. We found that both ClpB isoforms, ClpB95 and ClpB80 accumulated in E. coli cells during the production of IBs. The amount of IB proteins increased in the absence of ClpB. ClpB supported the resolubilization and reactivation of the aggregated VP1LAC and VP1GFP in E. coli cells. The IB disaggregation was optimal in the presence of both ClpB95 and ClpB80. Our results indicate an essential role of ClpB in controlling protein aggregation and inclusion body formation in bacteria. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. NEDD8 protein is involved in ubiquitinated inclusion bodies.

    PubMed

    Dil Kuazi, Afroz; Kito, Katsumi; Abe, Yasuhito; Shin, Ryong-Woon; Kamitani, Tetsu; Ueda, Norifumi

    2003-02-01

    Proteolysis by the ubiquitin-proteasome system is considered to play a pathological role in several degenerative diseases that involve ubiquitinated inclusion bodies. In recent years, several ubiquitin-like proteins have been isolated, but it is uncertain whether their roles are associated with protein degradation through the ubiquitin-proteasome system. NEDD8 (neural precursor cell-expressed and developmentally down-regulated gene), which consists of 81 amino acid residues, possesses the highest sequence similarity to ubiquitin. Recent studies have indicated that NEDD8 is covalently ligated to cullin family proteins, which are components of certain ubiquitin E3 ligases, by a pathway analogous to that of ubiquitin. Thus, by focusing on the structural and functional association between NEDD8 and ubiquitin, it would be of interest to know whether the NEDD8 system is involved in pathological disorders of the ubiquitin-proteasome system. This study has examined the immunohistochemical distribution of NEDD8 protein by using a highly purified antibody in normal tissues and in tissues known to contain ubiquitinated inclusions. NEDD8 protein expression was widely observed in most types of tissues. Furthermore, accumulation of the NEDD8 protein was commonly observed in ubiquitinated inclusion bodies, including Lewy bodies in Parkinson's disease, Mallory bodies in alcoholic liver disease, and Rosenthal fibres in astrocytoma. Two of ten cases of neurofibrillary tangles and senile plaques from patients with Alzheimer's disease showed intense staining for NEDD8 as well as for ubiquitin. These findings suggest the possibility that the NEDD8 system is involved in the metabolism of these inclusion bodies via the ubiquitin-proteasome system.

  3. Fast quantification of recombinant protein inclusion bodies within intact cells by FT-IR spectroscopy.

    PubMed

    Gross-Selbeck, Sven; Margreiter, Gerd; Obinger, Christian; Bayer, Karl

    2007-01-01

    The accomplishment of the quantification of the recombinant protein content of whole bacterial cells by FT-IR spectroscopy by application of chemometrics is shown. Recombinant Escherichia coli cells expressing an inclusion body forming fusion protein were dried on a 96-well silicon plate for the analysis in a high-throughput FT-IR spectrometer. Acquired spectra of additionally conventionally quantified samples were used to establish a multivariate calibration. The obtained method was tested by predicting inclusion body contents of samples not used for the multivariate model. Results from FT-IR spectra coincided well with the data of universalized electrophoresis analysis. Hence FT-IR spectroscopy could prove as a fast and simple alternative to conventional quantification methods.

  4. Refolding strategies from inclusion bodies in a structural genomics project.

    PubMed

    Trésaugues, Lionel; Collinet, Bruno; Minard, Philippe; Henckes, Gilles; Aufrère, Robert; Blondeau, Karine; Liger, Dominique; Zhou, Cong-Zhao; Janin, Joël; Van Tilbeurgh, Herman; Quevillon-Cheruel, Sophie

    2004-01-01

    The South-Paris Yeast Structural Genomics Project aims at systematically expressing, purifying and determining the structure of S. cerevisiae proteins with no detectable homology to proteins of known structure. We brought 250 yeast ORFs to expression in E. coli, but 37% of them form inclusion bodies. This important fraction of proteins that are well expressed but lost for structural studies prompted us to test methodologies to recover these proteins. Three different strategies were explored in parallel on a set of 20 proteins: (1) refolding from solubilized inclusion bodies using an original and fast 96-well plates screening test, (2) co-expression of the targets in E. coli with DnaK-DnaJ-GrpE and GroEL-GroES chaperones, and (3) use of the cell-free expression system. Most of the tested proteins (17/20) could be resolubilized at least by one approach, but the subsequent purification proved to be difficult for most of them.

  5. Soni-removal of nucleic acids from inclusion bodies.

    PubMed

    Neerathilingam, Muniasamy; Mysore, Sumukh; Gandham, Sai Hari A

    2014-05-23

    Inclusion bodies (IBs) are commonly formed in Escherichia coli due to over expression of recombinant proteins in non-native state. Isolation, denaturation and refolding of these IBs is generally performed to obtain functional protein. However, during this process IBs tend to form non-specific interactions with sheared nucleic acids from the genome, thus getting carried over into downstream processes. This may hinder the refolding of IBs into their native state. To circumvent this, we demonstrate a methodology termed soni-removal which involves disruption of nucleic acid-inclusion body interaction using sonication; followed by solvent based separation. As opposed to conventional techniques that use enzymes and column-based separations, soni-removal is a cost effective alternative for complete elimination of buried and/or strongly bound short nucleic acid contaminants from IBs. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Solubilization and refolding of inclusion body proteins in reverse micelles.

    PubMed

    Vinogradov, Alexei A; Kudryashova, Elena V; Levashov, Andrei V; van Dongen, Walter M A M

    2003-09-15

    Today, many valuable proteins can be obtained in sufficient amounts using recombinant DNA techniques. However, frequently the expression of recombinant proteins results in the accumulation of the product in dense amorphous deposits inside the cells, called inclusion bodies. The challenge then is to transform these inactive and misfolded protein aggregates into soluble bioactive forms. Although a number of general guidelines have been proposed, the search for proper reconstitution conditions can be very laborious and time consuming. Here, we suggest a new versatile approach for solubilization and refolding of inclusion body proteins using a water-sodium bis-2-ethylhexyl sulfosuccinate-isooctane reverse micellar system. Instead of amorphous aggregates, a transparent solution is obtained, where refolded protein is entrapped inside the micelles. The entrapped enzyme has native-like secondary structure and catalytic activity. This approach has been implemented with Fusarium galactose oxidase and Stigmatella aurantiaca putative galactose oxidase.

  7. New properties of inclusion bodies with implications for biotechnology.

    PubMed

    Peternel, Spela; Jevsevar, Simona; Bele, Marjan; Gaberc-Porekar, Vladka; Menart, Viktor

    2008-04-01

    Human G-CSF (granulocyte colony-stimulating factor) is a well-known biopharmaceutical drug being mostly produced by overexpression in Escherichia coli, where it appears in the form of IBs (inclusion bodies). Following our initial findings that properties of inclusion bodies strongly depend on the growth conditions used, especially growth temperature, we compared the characteristics of the G-CSF inclusion bodies prepared at two different temperatures, namely 42 and 25 degrees C. IBs formed at higher growth temperatures have properties similar to the usually described IBs, containing mainly denatured recombinant protein and being almost completely insoluble in aqueous solutions containing mild detergents or low concentrations of denaturants. In contrast, when produced at lower growth temperature of 25 degrees C, IBs show significantly different properties. Such IBs contain a significant proportion of G-CSF that is easily and directly extractable in the biologically active form, using non-denaturing solutions, which can be exploited for environmentally friendly biotechnological production. Irrespective of the production temperature, a significant decrease in IB volume was observed when transferring IBs from neutral to acidic (around 4) pH. Irreversible contraction of IBs at low pH was documented at the macro- and micro-scopic level using electron microscopy as a characterization tool. Together with volume decrease, a higher density, and thus decreased solubility, of IBs was observed at low pH, resulting in slower and less efficient extractability of the target protein.

  8. [In vitro renaturation of proteins from inclusion bodies].

    PubMed

    Porowińska, Dorota; Marszałek, Ewelina; Wardęcka, Paulina; Komoszyński, Michał

    2012-06-11

    Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic expression systems. However, a high expression level of foreign protein in bacteria cells results in formation of inactive and insoluble aggregates--inclusion bodies. Reactivation of aggregated proteins is a complex and time-consuming process. Every protein requires experimental optimization of the process conditions. The choice of the refolding method depends on the type of recombinant protein and its physical, chemical and biological properties. Recovery of the activity of proteins accumulated in inclusion bodies can be divided into 4 steps: 1) inclusion bodies isolation, 2) solubilization of aggregates, 3) renaturation, 4) purification of catalytically active molecules. Efficiency of the refolding process depends on many physical factors and chemical and biological agents. The above parameters determine the time of the folding and prevent protein aggregation. They also assist the folding and have an influence on the solubility and stability of native molecules. To date, dilution, dialysis and chromatography are the most often used methods for protein refolding.

  9. Dark proteins: effect of inclusion body formation on quantification of protein expression.

    PubMed

    Iafolla, Marco A J; Mazumder, Mostafizur; Sardana, Vandit; Velauthapillai, Tharsan; Pannu, Karanbir; McMillen, David R

    2008-09-01

    Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used

  10. Luminescent Conjugated Oligothiophenes for Sensitive Fluorescent Assignment of Protein Inclusion Bodies

    PubMed Central

    Klingstedt, Therése; Blechschmidt, Cristiane; Nogalska, Anna; Prokop, Stefan; Häggqvist, Bo; Danielsson, Olof; Engel, W King; Askanas, Valerie; Heppner, Frank L; Nilsson, K Peter R

    2013-01-01

    Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands. PMID:23450708

  11. Distribution of inclusion bodies in tissues from 100 dogs infected with canine distemper virus.

    PubMed

    Kubo, Takuya; Kagawa, Yumiko; Taniyama, Hiroyuki; Hasegawa, Atsuhiko

    2007-05-01

    One hundred dogs that were positive for canine distemper virus antigen and inclusion bodies in the tonsils were examined for the distribution of inclusion bodies in various tissues. Inclusion bodies were found in the lungs (70 dogs), brains (20 dogs), urinary bladders (73 dogs), stomachs (78 dogs), spleens (77 dogs), and lymph nodes (81 dogs) of the dogs. Based on these results, the tonsils may be the most suitable tissue for detection of inclusion bodies in canine distemper.

  12. Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

    PubMed

    Maggi, Maristella; Scotti, Claudia

    2017-08-01

    Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Treatment of sporadic inclusion body myositis with bimagrumab.

    PubMed

    Amato, Anthony A; Sivakumar, Kumaraswamy; Goyal, Namita; David, William S; Salajegheh, Mohammad; Praestgaard, Jens; Lach-Trifilieff, Estelle; Trendelenburg, Anne-Ulrike; Laurent, Didier; Glass, David J; Roubenoff, Ronenn; Tseng, Brian S; Greenberg, Steven A

    2014-12-09

    To study activin signaling and its blockade in sporadic inclusion body myositis (sIBM) through translational studies and a randomized controlled trial. We measured transforming growth factor β signaling by SMAD2/3 phosphorylation in muscle biopsies of 50 patients with neuromuscular disease (17 with sIBM). We tested inhibition of activin receptors IIA and IIB (ActRII) in 14 patients with sIBM using one dose of bimagrumab (n = 11) or placebo (n = 3). The primary outcome was the change in right thigh muscle volume by MRI at 8 weeks. Lean body mass, strength, and function were secondary outcomes. Twelve of the patients (10 bimagrumab, 2 placebo) participated in a subsequent 16-week observation phase. Muscle SMAD2/3 phosphorylation was higher in sIBM than in other muscle diseases studied (p = 0.003). Eight weeks after dosing, the bimagrumab-treated patients increased thigh muscle volume (right leg +6.5% compared with placebo, p = 0.024; left leg +7.6%, p = 0.009) and lean body mass (+5.7% compared with placebo, p = 0.014). Subsequently, bimagrumab-treated patients had improved 6-minute walking distance, which peaked at 16 weeks (+14.6%, p = 0.008) compared with placebo. There were no serious adverse events; the main adverse events with bimagrumab were mild acne and transient involuntary muscle contractions. Transforming growth factor β superfamily signaling, at least through ActRII, is implicated in the pathophysiology of sIBM. Inhibition of ActRII increased muscle mass and function in this pilot trial, offering a potential novel treatment of sIBM. This study provides Class I evidence that for patients with inclusion body myositis, bimagrumab increases thigh muscle volume at 8 weeks. © 2014 American Academy of Neurology.

  14. Targeting Protein Homeostasis in Sporadic Inclusion Body Myositis

    PubMed Central

    Ahmed, Mhoriam; Machado, Pedro M.; Miller, Adrian; Spicer, Charlotte; Herbelin, Laura; He, Jianghua; Noel, Janelle; Wang, Yunxia; McVey, April L.; Pasnoor, Mamatha; Gallagher, Philip; Statland, Jeffrey; Lu, Ching-Hua; Kalmar, Bernadett; Brady, Stefen; Sethi, Huma; Samandouras, George; Parton, Matt; Holton, Janice L.; Weston, Anne; Collinson, Lucy; Taylor, J. Paul; Schiavo, Giampietro; Hanna, Michael G.; Barohn, Richard J.; Dimachkie, Mazen M.; Greensmith, Linda

    2016-01-01

    Sporadic inclusion body myositis (sIBM) is the commonest severe myopathy in patients over age 50. Previous therapeutic trials have targeted the inflammatory features of sIBM, but all have failed. Since protein dyshomeostasis may also play a role in sIBM, we tested the effects of targeting this feature of the disease. Using rat myoblast cultures, we found that up-regulation of the heat shock response with Arimoclomol reduced key pathological markers of sIBM in vitro. Furthermore, in mutant valosin-containing protein VCP mice, which develop an inclusion body myopathy (IBM), treatment with Arimoclomol ameliorated disease pathology and improved muscle function. We therefore evaluated the safety and tolerability of Arimoclomol in an investigator-lead, randomised, double-blind, placebo-controlled, proof-of-concept patient trial and gathered exploratory efficacy data which showed that Arimoclomol was safe and well tolerated. Although Arimoclomol improved some IBM-like pathology in vitro and in vivo in the mutant VCP mouse, we did not see statistically significant evidence of efficacy in this proof of concept patient trial. PMID:27009270

  15. Formation of distinct inclusion bodies by inhibition of ubiquitin-proteasome and autophagy-lysosome pathways

    SciTech Connect

    Lee, Junho; Yang, Kyu-Hwan; Joe, Cheol O.; Kang, Seok-Seong

    2011-01-14

    Research highlights: {yields} Distinct inclusion bodies are developed by inhibition of UPP and ALP. {yields} The inclusion bodies differ in morphology, localization and formation process. {yields} The inclusion bodies are distinguishable by the localization of TSC2. {yields} Inhibition of both UPP and ALP simultaneously induces those inclusion bodies. -- Abstract: Accumulation of misfolded proteins is caused by the impairment of protein quality control systems, such as ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway (ALP). In this study, the formation of inclusion bodies was examined after the blockade of UPP and/or ALP in A549 cells. UPP inhibition induced a single and large inclusion body localized in microtubule-organizing center. Interestingly, however, ALP inhibition generated dispersed small inclusion bodies in the cytoplasm. Tuberous sclerosis complex 2 was selectively accumulated in the inclusion bodies of UPP-inhibited cells, but not those of ALP-inhibited cells. Blockade of transcription and translation entirely inhibited the formation of inclusion body induced by UPP inhibition, but partially by ALP inhibition. Moreover, the simultaneous inhibition of two protein catabolic pathways independently developed two distinct inclusion bodies within a single cell. These findings clearly demonstrated that dysfunction of each catabolic pathway induced formation and accumulation of unique inclusion bodies on the basis of morphology, localization and formation process in A549 cells.

  16. Activation of the Unfolded Protein Response in Sporadic Inclusion Body Myositis But Not in Hereditary GNE Inclusion Body Myopathy

    PubMed Central

    Nogalska, Anna; D’Agostino, Carla; Engel, W. King; Cacciottolo, Mafalda; Asada, Shinichi; Mori, Kazutoshi; Askanas, Valerie

    2015-01-01

    Muscle fibers in patients with sporadic inclusion-body myositis (s-IBM), the most common age-associated myopathy, are characterized by autophagic vacuoles and accumulation of ubiquitinated and congophilic multiprotein aggregates that contain amyloid-β and phosphorylated tau. Muscle fibers of autosomal-recessive hereditary inclusion-body myopathy due to the GNE mutation (GNE-h-IBM) display similar pathologic features, except with less pronounced congophilia. Accumulation of unfolded/misfolded proteins inside the ER lumen leads to ER stress, which elicits the unfolded protein response (UPR) as a protective mechanism. Here we demonstrate for the first time that UPR is activated in s-IBM muscle biopsies, since there was a) increased ATF4 protein and increased mRNA of its target CHOP, b) cleavage of the ATF6 and increased mRNA of its target GRP78, and c) an increase of the spliced form of XBP-1 and increased mRNA of EDEM, target of heterodimer of cleaved ATF6 and spliced XBP-1. In contrast, we did not find similar evidence of the UPR induction in GNE-h-IBM patient muscle, suggesting that different intracellular mechanisms might lead to the similar pathological phenotypes. Interestingly, cultured GNE-h-IBM muscle fibers had a robust UPR response to experimental ER stress stimuli, suggesting that the GNE mutation per se is not responsible for the lack of UPR in GNE-h-IBM biopsied muscle. PMID:25978849

  17. Bacterial communities in the fruit bodies of ground basidiomycetes

    NASA Astrophysics Data System (ADS)

    Zagryadskaya, Yu. A.; Lysak, L. V.; Chernov, I. Yu.

    2015-06-01

    Fruit bodies of basidiomycetes at different stages of decomposition serve as specific habitats in forest biocenoses for bacteria and differ significantly with respect to the total bacterial population and abundance of particular bacterial genera. A significant increase in the total bacterial population estimated by the direct microscopic method with acridine orange staining and in the population of saprotrophic bacteria (inoculation of glucose peptone yeast agar) in fruit bodies of basidiomycetes Armillaria mellea and Coprinus comatus was recorded at the final stage of their decomposition in comparison with the initial stage. Gramnegative bacteria predominated in the tissues of fruit bodies at all the stages of decomposition and were represented at the final stage by the Aeromonas, Vibrio, and Pseudomonas genera (for fruit bodies of A. mellea) the Pseudomonas genus (for fruit bodies of C. comatus). The potential influence of bacterial communities in the fruit bodies of soil basidiomycetes on the formation of bacterial communities in the upper soil horizons in forest biocenoses is discussed. The loci connected with the development and decomposition of fruit bodies of basidiomycetes on the soil surface are promising for targeted search of Gram-negative bacteria, the important objects of biotechnology.

  18. Inclusion Body Myositis: What Most Impacts Patients' Lives.

    PubMed

    Gibson, Cynthia; Johnson, Nicholas E; Eastwood, Eileen; Heatwole, Chad

    2016-12-01

    Inclusion body myositis (IBM) is the most common form of idiopathic inflammatory myopathy in adults older than 50 years. Few studies have focused on the functional, physical, and social limitations of this disease. This study identifies pertinent symptoms that impact the health and daily function of patients with IBM. We used semistructured interviews with 10 biopsy-confirmed adults with IBM to identify the psychological, physical, and functional limitations that have the greatest impact on the lives of patients with IBM. Participants with IBM provided 644 direct quotes identifying issues that have the greatest effect on their lives. Two hundred nine individual symptoms and 17 symptomatic themes were identified by patients as having a significant impact on their lives. The symptomatic themes mentioned most frequently involved mobility and ambulation, emotional distress, and activity impairment. Identifying critical issues to patients with IBM is potentially useful for clinicians whose aim is to provide optimal care to patients with IBM.

  19. Prediction of inclusion body solubilization from shaken to stirred reactors.

    PubMed

    Walther, Cornelia; Mayer, Sabrina; Trefilov, Alexandru; Sekot, Gerhard; Hahn, Rainer; Jungbauer, Alois; Dürauer, Astrid

    2014-01-01

    Inclusion bodies (IBs) were solubilized in a µ-scale system using shaking microtiter plates or a stirred tank reactor in a laboratory setting. Characteristic dimensionless numbers for mixing, the Phase number Ph and Reynolds number Re did not correlate with the kinetics and equilibrium of protein solubilization. The solubilization kinetics was independent of the mixing system, stirring or shaking rate, shaking diameter, and energy input. Good agreement was observed between the solubilization kinetics and yield on the µ-scale and laboratory setting. We show that the IB solubilization process is controlled predominantly by pore diffusion. Thus, for the process it is sufficient to keep the IBs homogeneously suspended, and additional power input will not improve the process. The high-throughput system developed on the µ-scale can predict solubilization in stirred reactors up to a factor of 500 and can therefore be used to determine optimal solubilization conditions on laboratory and industrial scale.

  20. [Pathogenesis of inclusion body myositis: autoimmune or degenerative disease?].

    PubMed

    Uruha, Akinori; Nishino, Ichizo

    2013-11-01

    While the pathogenesis of inclusion body myositis (IBM) remains undetermined, there are two major hypotheses: the autoimmune hypothesis and the degeneration hypothesis. Herein, we review these hypotheses as well as potential therapeutic approaches. Evidence in favor of a primary autoimmune etiology includes the frequent complication of other autoimmune diseases in patients with IBM and the presence of autoantibodies against cytosolic 5'-nucleotidase 1A. Interleukin (IL)-1β reportedly leads to accumulation of amyloid β via nitric oxide stress in vitro. The degeneration hypothesis addresses the following aspects of IBM: accumulation of amyloid β and other abnormal proteins that are observed in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease; relation to aging; and poor response to immunotherapy. Overexpression of IL-1β in skeletal muscles of patients with IBM and its secretion from skeletal muscle cells suggests an important role for IL-1β in the pathogenesis of IBM. Thus, IL-1β is a potential treatment target.

  1. Seborrheic inclusion cyst of the skin positive for cytoplasmic inclusion bodies and HPV antigen.

    PubMed

    Terada, Tadashi

    2012-01-01

    Seborrheic inclusion cyst (SIC) is a very rare variant of epidermal cyst of the skin. SIC shows seborrheic keratosis (SK)-like lesion in epidermal cyst. SIC is extremely rare; only 6 case reports have been published in the English literature. However, no immunohistochemical study of SIC has been reported. A 41-year-old Japanese man noticed a subcutaneous tumor in the neck. Physical examination showed slightly mobile tumor in the subcutaneous tissue, and total excision was performed. Grossly, the tumor (1 x 1 x 0.8 cm) was cyst containing atheromatous keratin. Microscopically, the lesion is a cyst containing keratins. About one half of the cyst showed features of epidermal cyst consisting of mature squamous epithelium with granular layers. The other one half showed SK-like epidermal proliferation. The SK-like area showed basaloid cell proliferation with pseudohorn cysts. No significant atypia was noted. Many eosinophilic cytoplasmic inclusion bodies were noted in the SK-like area. Immunohistochemically, the SK-like area was positive for pancytokeratin AE1/3, pancytokeratin CAM5.2, p63, and Ki-67 (labeling=8%) and HPV, but negative for p53. The pathological diagnosis was SIC.

  2. Interconnected Cavernous Structure of Bacterial Fruiting Bodies

    DOE PAGES

    Harvey, Cameron W.; Du, Huijing; Xu, Zhiliang; ...

    2012-12-27

    The formation of spore-filled fruiting bodies by myxobacteria is a fascinating case of multicelular self-organization by bacteria. The organization of Myxococcus xanthus into fruiting bodies has long been studied not only as an important example of collective motion of bacteria, but also as a simplified model for developmental morphogenesis. Sporulation within the nascent fruiting body requires signaling between moving cells in order that the rod-shaped self-propelled cells differentiate into spores at the appropriate time. Probing the three-dimensional structure of myxobacteria fruiting bodies has previously presented a challenge due to Imitations at different imaging methods. A new technique using Infrared Opticalmore » Coherence Tomography (OCT) revealed previously unknown details of the Internal structure of M. xanthus fruiting bodies consisting of interconnected pockets of relative nigh and low spore density regions. Here, to make sense of the experimentally observed structure, modeling and computer simulations were used to test a hypothesized mechanism that could produce high density pockets of spores. The mechanism consists of self-propelled cells aligning with each other and signaling by end-to-end contact to coordinate the process of differentiation resulting in a pattern of clusters observed in the experiment. The Integration of novel OCT experimental techniques with computational simulations can provide new insight Into the mechanisms that can give rise to the pattern formation seen In other biological systems such as dlctyostelids, social amoeba known to form multicellular aggregates observed as slugs under starvation conditions.« less

  3. Interconnected Cavernous Structure of Bacterial Fruiting Bodies

    PubMed Central

    Harvey, Cameron W.; Du, Huijing; Xu, Zhiliang; Kaiser, Dale; Aranson, Igor; Alber, Mark

    2012-01-01

    The formation of spore-filled fruiting bodies by myxobacteria is a fascinating case of multicellular self-organization by bacteria. The organization of Myxococcus xanthus into fruiting bodies has long been studied not only as an important example of collective motion of bacteria, but also as a simplified model for developmental morphogenesis. Sporulation within the nascent fruiting body requires signaling between moving cells in order that the rod-shaped self-propelled cells differentiate into spores at the appropriate time. Probing the three-dimensional structure of myxobacteria fruiting bodies has previously presented a challenge due to limitations of different imaging methods. A new technique using Infrared Optical Coherence Tomography (OCT) revealed previously unknown details of the internal structure of M. xanthus fruiting bodies consisting of interconnected pockets of relative high and low spore density regions. To make sense of the experimentally observed structure, modeling and computer simulations were used to test a hypothesized mechanism that could produce high-density pockets of spores. The mechanism consists of self-propelled cells aligning with each other and signaling by end-to-end contact to coordinate the process of differentiation resulting in a pattern of clusters observed in the experiment. The integration of novel OCT experimental techniques with computational simulations can provide new insight into the mechanisms that can give rise to the pattern formation seen in other biological systems such as dictyostelids, social amoeba known to form multicellular aggregates observed as slugs under starvation conditions. PMID:23300427

  4. [An easy way to purify the inclusion body protein with high purity from prokaryotic expression cells].

    PubMed

    Liu, Rong; Zhong, Qin-Ping; Jiang, Ming-Sen; Dong, Hui-Fen

    2011-10-01

    To clone partial ORF of SjBMP and to construct the recombinant SjBMP-pET-28a(+) plasmids, and then to transform them into the competent cells E. coli BL21 (DE3), finally a positive clone was used to be induced by IPTG. The bacterial aggregates with target protein expressed as inclusion bodies were purified by the methods of Ni(2+)-NTA affinity purification under denaturation condition and SDS-PAGE gel extraction. The purified protein was used to immune rabbits and make antiserum against the SjBMP, and the antiserum were then used to identify the rSjBMP by Western blotting. The target protein obtained by Ni(2+)-NTA Agarose affinity purification was not pure with unspecific proteins, but the protein further purified by SDS-PAGE gel extraction and the dialysis bag horizontal electrophoresis was quite pure, and the recovery rate was more than 11.0%. Meanwhile, Western blotting was used to identify the recombinant SjBMP protein by antiserum, only a specific single strip appeared, which suggested the protein purified by this method kept its antigenicity, and could be used for common immunological studies. Therefore, the SDS-PAGE gel extraction combining with electroosmosis and dialysis recycling are good and easy to purify the inclusion body proteins.

  5. Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies.

    PubMed

    Njengele, Zikhona; Kleynhans, Ronel; Sayed, Yasien; Mosebi, Salerwe

    2016-12-01

    Vpu is one of four accessory proteins encoded by human immunodeficiency virus type I (HIV-1). Vpu modulates the expression of several cellular restriction factors within the HIV-1 infected cell including CD4, CD74, the bone marrow stromal antigen 2 (BST-2) and NK-T-and-B antigen. The interaction of HIV-1 Vpu with these proteins interferes with the innate immune response directed against HIV-1; thereby promoting viral persistence. The involvement of HIV-1 Vpu in manipulating the cellular environment in ways that favor viral replication makes it an attractive target for anti-HIV drug intervention. This paper describes the over-expression and purification of a soluble HIV-1 Vpu from inclusion bodies by ion-exchange chromatography, allowing production of 6 mg of highly purified protein (>95% purity) per 10 mg of pelleted cells obtained from 1 L of bacterial culture. Far-UV circular dichroism showed that the recombinant protein is folded and retained its secondary structure. Moreover, using ELISA, known HIV-1 Vpu binding partners, BST-2 and CD74, showed that the refolded purified protein is functional or at least assumes a conformation that is capable of binding these putative binding partners. To our knowledge, this is the first report of the purification and successful solubilization of full-length, wild-type HIV-1 Vpu from inclusion bodies in Escherichia coli. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Catalytically-active inclusion bodies-Carrier-free protein immobilizates for application in biotechnology and biomedicine.

    PubMed

    Krauss, Ulrich; Jäger, Vera D; Diener, Martin; Pohl, Martina; Jaeger, Karl-Erich

    2017-09-20

    Bacterial inclusion bodies (IBs) consist of unfolded protein aggregates and represent inactive waste products often accumulating during heterologous overexpression of recombinant genes in Escherichia coli. This general misconception has been challenged in recent years by the discovery that IBs, apart from misfolded polypeptides, can also contain substantial amounts of active and thus correctly or native-like folded protein. The corresponding catalytically-active inclusion bodies (CatIBs) can be regarded as a biologically-active sub-micrometer sized biomaterial or naturally-produced carrier-free protein immobilizate. Fusion of polypeptide (protein) tags can induce CatIB formation paving the way towards the wider application of CatIBs in synthetic chemistry, biocatalysis and biomedicine. In the present review we summarize the history of CatIBs, present the molecular-biological tools that are available to induce CatIB formation, and highlight potential lines of application. In the second part findings regarding the formation, architecture, and structure of (Cat)IBs are summarized. Finally, an overview is presented about the available bioinformatic tools that potentially allow for the prediction of aggregation and thus (Cat)IB formation. This review aims at demonstrating the potential of CatIBs for biotechnology and hopefully contributes to a wider acceptance of this promising, yet not widely utilized, protein preparation. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. ALS-associated protein FIG4 is localized in Pick and Lewy bodies, and also neuronal nuclear inclusions, in polyglutamine and intranuclear inclusion body diseases.

    PubMed

    Kon, Tomoya; Mori, Fumiaki; Tanji, Kunikazu; Miki, Yasuo; Toyoshima, Yasuko; Yoshida, Mari; Sasaki, Hidenao; Kakita, Akiyoshi; Takahashi, Hitoshi; Wakabayashi, Koichi

    2014-02-01

    FIG4 is a phosphatase that regulates intracellular vesicle trafficking along the endosomal-lysosomal pathway. Mutations of FIG4 lead to the development of Charcot-Marie-Tooth disease type 4J and amyotrophic lateral sclerosis (ALS). Moreover, ALS-associated proteins (transactivation response DNA protein 43 (TDP-43), fused in sarcoma (FUS), optineurin, ubiquilin-2, charged mutivesicular body protein 2b (CHMP2B) and valosin-containing protein) are involved in inclusion body formation in several neurodegenerative diseases. Using immunohistochemistry, we examined the brains and spinal cords of patients with various neurodegenerative diseases, including sporadic TDP-43 proteinopathy (ALS and frontotemporal lobar degeneration). TDP-43 proteinopathy demonstrated no FIG4 immunoreactivity in neuronal inclusions. However, FIG4 immunoreactivity was present in Pick bodies in Pick's disease, Lewy bodies in Parkinson's disease and dementia with Lewy bodies, neuronal nuclear inclusions in polyglutamine and intranuclear inclusion body diseases, and Marinesco and Hirano bodies in aged control subjects. These findings suggest that FIG4 is not incorporated in TDP-43 inclusions and that it may have a common role in the formation or degradation of neuronal cytoplasmic and nuclear inclusions in several neurodegenerative diseases. © 2013 Japanese Society of Neuropathology.

  8. Acid-fast intranuclear inclusion bodies in the kidneys of mallards fed lead shot

    USGS Publications Warehouse

    Locke, L.N.; Bagley, George E.; Irby, H.D.

    1966-01-01

    Acid-fast intranuclear inclusion bodies were found in the cells of the proximal convoluted tubules of the kidneys of mallards fed one, two, three or eight number 6 lead shot and maintained on cracked or whole corn and on grain-duck pellet diets. No acid-fast inclusion bodies were found in mallards fed one or three lead shot but maintained on a duck pellet ration. Dietary factors may be responsible for the failure of mallards fed a duck pellet ration to develop lead Inclusion bodies when treated with one or three lead shot. The authors suggest these inclusion bodies can be used as presumptive evidence for lead intoxication in mallards.

  9. Evaluation and construction of diagnostic criteria for inclusion body myositis

    PubMed Central

    Mammen, Andrew L.; Amato, Anthony A.; Weiss, Michael D.; Needham, Merrilee

    2014-01-01

    Objective: To use patient data to evaluate and construct diagnostic criteria for inclusion body myositis (IBM), a progressive disease of skeletal muscle. Methods: The literature was reviewed to identify all previously proposed IBM diagnostic criteria. These criteria were applied through medical records review to 200 patients diagnosed as having IBM and 171 patients diagnosed as having a muscle disease other than IBM by neuromuscular specialists at 2 institutions, and to a validating set of 66 additional patients with IBM from 2 other institutions. Machine learning techniques were used for unbiased construction of diagnostic criteria. Results: Twenty-four previously proposed IBM diagnostic categories were identified. Twelve categories all performed with high (≥97%) specificity but varied substantially in their sensitivities (11%–84%). The best performing category was European Neuromuscular Centre 2013 probable (sensitivity of 84%). Specialized pathologic features and newly introduced strength criteria (comparative knee extension/hip flexion strength) performed poorly. Unbiased data-directed analysis of 20 features in 371 patients resulted in construction of higher-performing data-derived diagnostic criteria (90% sensitivity and 96% specificity). Conclusions: Published expert consensus–derived IBM diagnostic categories have uniformly high specificity but wide-ranging sensitivities. High-performing IBM diagnostic category criteria can be developed directly from principled unbiased analysis of patient data. Classification of evidence: This study provides Class II evidence that published expert consensus–derived IBM diagnostic categories accurately distinguish IBM from other muscle disease with high specificity but wide-ranging sensitivities. PMID:24975859

  10. Evaluation and construction of diagnostic criteria for inclusion body myositis.

    PubMed

    Lloyd, Thomas E; Mammen, Andrew L; Amato, Anthony A; Weiss, Michael D; Needham, Merrilee; Greenberg, Steven A

    2014-07-29

    To use patient data to evaluate and construct diagnostic criteria for inclusion body myositis (IBM), a progressive disease of skeletal muscle. The literature was reviewed to identify all previously proposed IBM diagnostic criteria. These criteria were applied through medical records review to 200 patients diagnosed as having IBM and 171 patients diagnosed as having a muscle disease other than IBM by neuromuscular specialists at 2 institutions, and to a validating set of 66 additional patients with IBM from 2 other institutions. Machine learning techniques were used for unbiased construction of diagnostic criteria. Twenty-four previously proposed IBM diagnostic categories were identified. Twelve categories all performed with high (≥97%) specificity but varied substantially in their sensitivities (11%-84%). The best performing category was European Neuromuscular Centre 2013 probable (sensitivity of 84%). Specialized pathologic features and newly introduced strength criteria (comparative knee extension/hip flexion strength) performed poorly. Unbiased data-directed analysis of 20 features in 371 patients resulted in construction of higher-performing data-derived diagnostic criteria (90% sensitivity and 96% specificity). Published expert consensus-derived IBM diagnostic categories have uniformly high specificity but wide-ranging sensitivities. High-performing IBM diagnostic category criteria can be developed directly from principled unbiased analysis of patient data. This study provides Class II evidence that published expert consensus-derived IBM diagnostic categories accurately distinguish IBM from other muscle disease with high specificity but wide-ranging sensitivities. © 2014 American Academy of Neurology.

  11. Sporadic Inclusion Body Myositis: new insights and potential therapy

    PubMed Central

    Machado, Pedro; Dimachkie, Mazen M.; Barohn, Richard J.

    2014-01-01

    Purpose of the review To describe new insights and developments in the pathogenesis, diagnosis and treatment of sporadic inclusion body myositis (IBM). Recent findings Various hypothesis about the pathogenesis of IBM continue to be investigated, including autoimmune factors, mitochondrial dysfunction, protein dyshomeostasis, altered nucleic acid metabolism, myonuclear degeneration and the role of the myostatin pathway. Serum autoantibodies against cytosolic 5′ nucleotidase 1A have been identified in IBM showing moderate diagnostic performance. The differential diagnostic value of histopathological features, including different protein aggregates, continues to be evaluated. MRI may also be of monitoring value in IBM. New therapeutic strategies are being tested in IBM patients, namely the up-regulation of the heat shock response and the antagonism of myostatin. Summary Recent important advances have occurred in IBM. These advances, including recent and ongoing clinical trials, may lead to earlier diagnosis and improved understanding and treatment of the disease. Despite improved knowledge, IBM continues to be a puzzling disease and the pathogenesis remains to be clarified. An interdisciplinary, bench to bedside translational research approach is crucial for the successful identification of novel treatments for this debilitating, currently untreatable disorder. PMID:25159931

  12. Protein misfolding specifies recruitment to cytoplasmic inclusion bodies.

    PubMed

    Bersuker, Kirill; Brandeis, Michael; Kopito, Ron R

    2016-04-25

    Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington's disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs.

  13. High-throughput automated refolding screening of inclusion bodies.

    PubMed

    Vincentelli, Renaud; Canaan, Stéphane; Campanacci, Valérie; Valencia, Christel; Maurin, Damien; Frassinetti, Frédéric; Scappucini-Calvo, Loréna; Bourne, Yves; Cambillau, Christian; Bignon, Christophe

    2004-10-01

    One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no "universal" refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high-throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96-well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large-scale purification tests, and five gave crystals.

  14. Flotillin-1 (Reggie-2) Contributes to Chlamydia pneumoniae Growth and Is Associated with Bacterial Inclusion

    PubMed Central

    Korhonen, Juha T.; Puolakkainen, Mirja; Häivälä, Reetta; Penttilä, Tuula; Haveri, Anu; Markkula, Eveliina

    2012-01-01

    Chlamydiae are obligate intracellular pathogens replicating only inside the eukaryotic host. Here, we studied the effect of human flotillin-1 protein on Chlamydia pneumoniae growth in human line (HL) and A549 epithelial cell lines. RNA interference was applied to disrupt flotillin-1-mediated endocytosis. Host-associated bacteria were detected by quantitative PCR, and C. pneumoniae growth was evaluated by inclusion counts. C. pneumoniae attachment to host cells was unaffected, but bacterial intracellular growth was attenuated in the flotillin-1-silenced cells. By using confocal microscopy, we detected flotillin-1 colocalized with the inclusion membrane protein A (IncA) in the C. pneumoniae inclusion membranes. In addition, flotillin-1 was associated with IncA in detergent-resistant membrane microdomains (DRMs) in biochemical fractioning. These results suggest that flotillin-1 localizes to the C. pneumoniae inclusion membrane and plays an important role for intracellular growth of C. pneumoniae. PMID:22215737

  15. Functional relevance of mitochondrial abnormalities in sporadic inclusion body myositis.

    PubMed

    Joshi, Pushpa Raj; Vetterke, Mirjam; Hauburger, Anja; Tacik, Pawel; Stoltenburg, Gisela; Hanisch, Frank

    2014-11-01

    Cytochrome c oxidase (COX)-deficient fibers and multiple mitochondrial DNA (mtDNA) deletions are frequent findings in sporadic inclusion body myositis (s-IBM). However, the functional impact of these defects is not known. We investigated oxygen desaturation during exercise using the forearm exercise test, accumulation of lactate during exercise using a cycle ergometry test and mitochondrial changes (COX-deficient fibers, biochemical activities of respiratory chain complexes, multiple mtDNA deletions by long-range polymerase chain reaction) in 10 patients with s-IBM and compared the findings with age and sex-matched normal and diseased controls (without mitochondrial disorders) as well as patients with mitochondrial disorder due to nuclear gene defects resulting in multiple mtDNA deletions (MITO group). The mean age of the s-IBM patients was 68.2 ± 5.7 years (range: 56-75). Patients with s-IBM had statistically significantly reduced oxygen desaturation (ΔsO2) during the handgrip exercise (p<0.05) and elevated peak serum lactate levels during cycle ergometry compared to normal controls (p<0.05). The percentage of COX-deficient fibers in s-IBM and MITO patients was significantly increased compared to normal controls (p<0.01). Five out of nine s-IBM patients had multiple mtDNA deletions. Thirty-three percent of s-IBM patients showed an increased citrate synthase content and decreased activities of complex IV (COX). The biochemical pattern of respiratory chain complexes in patients with s-IBM and MITO was similar. Histopathological analysis showed similar changes in s-IBM and MITO due to nuclear gene defects. Functional tests reflecting mitochondrial impairment suggest a contribution of mitochondrial defects to disease-related symptoms such as fatigue and exertion-induced symptoms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Hereditary inclusion body myopathy: the Middle Eastern genetic cluster.

    PubMed

    Argov, Z; Eisenberg, I; Grabov-Nardini, G; Sadeh, M; Wirguin, I; Soffer, D; Mitrani-Rosenbaum, S

    2003-05-13

    Recessively inherited hereditary inclusion body myopathy (HIBM) with quadriceps sparing was initially described only in Jews originating from the region of Persia. The recent identification of the gene responsible for this myopathy and the common "Persian Jewish mutation" (M712T) enabled the re-evaluation of atypical phenotypes and the epidemiology of HIBM in various communities in the Middle East. To test for the M712T mutation in the DNA from HIBM patients in the Middle East. DNA from all suspected HIBM patients was tested for the M712T mutation. Unaffected members of families with genetically proven HIBM were studied too. In the majority of families, haplotype construction with markers spanning the 700-kb region of the HIBM gene was performed. One hundred twenty-nine HIBM patients of 55 families (Middle Eastern Jews, Karaites, and Arab Muslims of Palestinian and Bedouin origin) were homozygous for the M712T mutation, and all carried the same haplotype. Five clinically unaffected subjects were also homozygous for the common mutation and haplotype, including two older adults (ages 50 and 68 years). Atypical features with this same mutation were marked quadriceps weakness in five patients, proximal weakness only in two patients, facial weakness in three patients, and a muscle biopsy showing perivascular inflammation in one patient. The phenotypic spectrum of recessive HIBM is wider than previously described, and the diagnostic criteria for this myopathy must be changed. The Middle Eastern cluster is the result of a founder mutation, with incomplete penetrance, that is approximately 1,300 years old and is not limited to Jews.

  17. Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence

    ERIC Educational Resources Information Center

    Peterson, Megan J.; Snyder, W. Kalani; Westerman, Shelley; McFarland, Benjamin J.

    2011-01-01

    We describe how to produce and purify proteins from "Escherichia coli" inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This 7-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies,…

  18. Luminescent conjugated oligothiophenes for sensitive fluorescent assignment of protein inclusion bodies.

    PubMed

    Klingstedt, Therése; Blechschmidt, Cristiane; Nogalska, Anna; Prokop, Stefan; Häggqvist, Bo; Danielsson, Olof; Engel, W King; Askanas, Valerie; Heppner, Frank L; Nilsson, K Peter R

    2013-03-18

    Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Ultrastructure of inclusion bodies in annulus cells in the degenerating human intervertebral disc.

    PubMed

    Gruber, H E; Hanley, E N

    2009-06-01

    The rough endoplasmic reticulum (rER) of the cell has an architectural editing function that checks whether protein structure and three-dimensional assembly have occurred properly prior to export of newly synthesized material out of the cell. If these have been faulty, the material is retained within the rER as an inclusion body. Inclusion bodies have been identified previously in chondrocytes and osteoblasts in chondrodysplasias and osteogenesis imperfecta. Inclusion bodies in intervertebral disc cells, however, have only recently been recognized. Our objectives were to use transmission electron microscopy to analyze more fully inclusion bodies in the annulus pulposus and to study the extracellular matrix (ECM) surrounding cells containing inclusion bodies. ECM frequently encapsulated cells with inclusion bodies, and commonly contained prominent banded aggregates of Type VI collagen. Inclusion body material had several morphologies, including relatively smooth, homogeneous material, or a rougher, less homogeneous feature. Such findings expand our knowledge of the fine structure of the human disc cell and ECM during disc degeneration, and indicate the potential utility of ultrastructural identification of discs with intracellular inclusion bodies as a screening method for molecular studies directed toward identification of defective gene products in degenerating discs.

  20. Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli.

    PubMed Central

    Strandberg, L; Enfors, S O

    1991-01-01

    Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation. PMID:1908208

  1. Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence

    ERIC Educational Resources Information Center

    Peterson, Megan J.; Snyder, W. Kalani; Westerman, Shelley; McFarland, Benjamin J.

    2011-01-01

    We describe how to produce and purify proteins from "Escherichia coli" inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This 7-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies,…

  2. Distinctive eosinophilic cytoplasmic inclusion bodies in melanocytic nevi: an immunohistochemical and ultrastructural study.

    PubMed

    Shon, Wonwoo; Wada, David A; Gibson, Lawrence E; Flotte, Thomas J; Scheithauer, Bernd W

    2011-11-01

    We sought to further determine the histochemical, immunohistochemical and ultrastructural properties of eosinophilic cytoplasmic inclusion bodies in melanocytic nevi. Skin specimens from four patients with a known diagnosis of conventional melanocytic nevus (3) or Spitz nevus (1) and containing intracytoplasmic eosinophilic inclusion bodies were selected. In addition, melanomas (25), Spitz nevi (10) and blue nevi (4) were examined to determine the frequency of the inclusions. Inclusions tended to be located in multinucleated melanocytes with abundant vacuolated cytoplasm. In conventional (hematoxylin and eosin-stained) sections, the degree of density and eosinophilia of intracytoplasmic inclusions varied with size. Periodic acid-Schiff, Fontana and Congo red stains showed no reactivity. All bodies were immunoreactive for ubiquitin but negative for tyrosinase, keratin and vimentin. Ultrastructurally, inclusion bodies were non-membrane bound, ranged from 4 to 7 µm, and were comprised of radiating filamentous structures with or without an electron-dense core. Electron probe x-ray microanalysis revealed no significant peaks. None of additional melanomas, Spitz nevi and blue nevi that were evaluated showed similar inclusions. The inclusion bodies described herein bear no resemblance to other cytoplasmic inclusion bodies previously described in melanocytic lesions. There is no discernible relationship to melanosomes by ultrastructural analysis. We postulate a relationship with dysfunction of ubiquitin-mediated protein degradation occurring in melanocytes. Copyright © 2011 John Wiley & Sons A/S.

  3. [Inclusion.

    ERIC Educational Resources Information Center

    Luzader, Carolyn, Ed.

    1995-01-01

    This special newsletter issue on inclusion of students with disabilities includes the following articles: "Inclusion: A Responsible Approach" (Harley A. Tomey, III); "Research Base Limited on Effects of Inclusion" (Thomas P. Lombardi); "Research Supports Inclusion for Physically Disabled--Vocational Ed Prevents Dropping…

  4. Formation of distinct inclusion bodies by inhibition of ubiquitin-proteasome and autophagy-lysosome pathways.

    PubMed

    Lee, Junho; Yang, Kyu-Hwan; Joe, Cheol O; Kang, Seok-Seong

    2011-01-14

    Accumulation of misfolded proteins is caused by the impairment of protein quality control systems, such as ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway (ALP). In this study, the formation of inclusion bodies was examined after the blockade of UPP and/or ALP in A549 cells. UPP inhibition induced a single and large inclusion body localized in microtubule-organizing center. Interestingly, however, ALP inhibition generated dispersed small inclusion bodies in the cytoplasm. Tuberous sclerosis complex 2 was selectively accumulated in the inclusion bodies of UPP-inhibited cells, but not those of ALP-inhibited cells. Blockade of transcription and translation entirely inhibited the formation of inclusion body induced by UPP inhibition, but partially by ALP inhibition. Moreover, the simultaneous inhibition of two protein catabolic pathways independently developed two distinct inclusion bodies within a single cell. These findings clearly demonstrated that dysfunction of each catabolic pathway induced formation and accumulation of unique inclusion bodies on the basis of morphology, localization and formation process in A549 cells. Copyright © 2010 Elsevier Inc. All rights reserved.

  5. TDP-43 Inclusion Bodies Formed in Bacteria Are Structurally Amorphous, Non-Amyloid and Inherently Toxic to Neuroblastoma Cells

    PubMed Central

    Capitini, Claudia; Conti, Simona; Perni, Michele; Guidi, Francesca; Cascella, Roberta; De Poli, Angela; Penco, Amanda; Relini, Annalisa; Cecchi, Cristina; Chiti, Fabrizio

    2014-01-01

    Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein. PMID:24497973

  6. TDP-43 inclusion bodies formed in bacteria are structurally amorphous, non-amyloid and inherently toxic to neuroblastoma cells.

    PubMed

    Capitini, Claudia; Conti, Simona; Perni, Michele; Guidi, Francesca; Cascella, Roberta; De Poli, Angela; Penco, Amanda; Relini, Annalisa; Cecchi, Cristina; Chiti, Fabrizio

    2014-01-01

    Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.

  7. Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study.

    PubMed

    Kateja, Nikhil; Agarwal, Harshit; Hebbi, Vishwanath; Rathore, Anurag S

    2016-12-15

    Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end-to-end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three-column periodic counter-current chromatography for capture of the product followed by a three-column con-current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end-to-end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2017.

  8. Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies.

    PubMed

    Ahlqvist, Josefin; Kumar, Ashok; Sundström, Heléne; Ledung, Erika; Hörnsten, E Gunnar; Enfors, Sven-Olof; Mattiasson, Bo

    2006-03-23

    A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B., 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.

  9. Monitoring the production of inclusion bodies during fermentation and enzyme-linked immunosorbent assay analysis of intact inclusion bodies using cryogel minicolumn plates.

    PubMed

    Ahlqvist, Josefin; Dainiak, Maria B; Kumar, Ashok; Hörnsten, E Gunnar; Galaev, Igor Yu; Mattiasson, Bo

    2006-07-15

    A novel minicolumn chromatographic method to monitor the production of inclusion bodies during fermentation and an enzyme-linked immunosorbent assay (ELISA) system allowing direct analysis of the particles with surface-displayed antigens are described. A 33-kDa protein containing 306 amino acids with three sulfur bridges produced as inclusion bodies was labeled with polyclonal antibodies against 15 amino acid (anti-A15) and 17 amino acid (anti-B17) residues at the N- and C-terminal ends of the protein, respectively. Labeled particles were bound to macroporous monolithic protein A-cryogel adsorbents inserted into the open-ended wells of a 96-well plate (referred to as protein A-cryogel minicolumn plate). The concept behind this application is that the binding degree of inclusion bodies from lysed fermentation broth to the cryogel minicolumns increases with an increase in their concentration during fermentation. The technique allowed us to monitor the increase in the production levels of the inclusion bodies as the fermentation process progressed. The system also has a built-in quality parameter to ensure that the target protein has been fully expressed. Alternatively, inclusion bodies immobilized on phenyl-cryogel minicolumn plate were used in indirect ELISA based on anti-A15 and anti-B17 antibodies against terminal amino acid residues displayed on the surface of inclusion bodies. Drainage-protected properties of the cryogel minicolumns allow performance of successive reactions with tested immunoglobulin G (IgG) samples and enzyme-conjugated secondary IgG and of enzymatic reaction within the adsorbent.

  10. The surface layer protein of Bacillus thuringiensis CTC forms unique intracellular parasporal inclusion body.

    PubMed

    Zhu, Chenguang; Yu, Ziniu

    2008-08-01

    Bacillus thuringiensis subsp. finitimus strain CTC forms round parasporal inclusion body. The inclusion body protein gene ctc has been cloned and characterized. Sequence homology analysis reveals that the amino acid sequence of CTC protein shows 87% identity with the surface layer (S-layer) protein Sap (GenBank Z36946) in B. anthracis. In this report, transmission electron microscope observation showed that CTC formed intracellular parasporal inclusion body and sheet structure of S-layer-like protein at the spore phase. Furthermore, the ctc gene was transformed into an acrystalliferous B. thuringiensis strain BMB171. The resulting transformant could form parasporal body which had the same shape and molecular weight of protein with that of B. thuringiensis CTC. These results, together with the sequence homology analysis of ctc gene, confirmed that the unique intracellular parasporal inclusion body of B. thuringiensis was comprised of S-layer protein.

  11. Creating Inclusive Physical Activity Spaces: The Case of Body-Positive Yoga.

    PubMed

    Pickett, Andrew C; Cunningham, George B

    2017-09-01

    Within the modern cultural climate, those in larger bodies face high levels of weight stigma, particularly in sport and physical activity spaces, which serves as a strong barrier to their participation. However, given the strong link between physical activity and general health and well-being for participants, it is important to explore strategies that encourage participation of these individuals. Thus, the current research examined strategies that physical activity instructors use to develop inclusive exercise spaces for all body sizes. This study employed a series of semistructured qualitative interviews (n = 9) with instructors of body-inclusive yoga classes to explore the ways in which they encourage participation for those in larger bodies. Emergent themes from the current study suggested support for 6 factors for creating body-inclusive physical activity spaces: authentic leadership, a culture of inclusion, a focus on health, inclusive language, leader social activism, and a sense of community. This study revealed that leaders must intentionally cultivate inclusion in their spaces to encourage those in nonconforming bodies to participate. These findings have important health and management implications for the sport and physical activity context and provide a basic outline of practical strategies that practitioners can use to foster inclusion in their spaces.

  12. Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies

    PubMed Central

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2014-01-01

    Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies. PMID:24970214

  13. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.

    PubMed

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2014-02-20

    Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  14. Valosin-containing protein immunoreactivity in tauopathies, synucleinopathies, polyglutamine diseases and intranuclear inclusion body disease.

    PubMed

    Mori, Fumiaki; Tanji, Kunikazu; Toyoshima, Yasuko; Sasaki, Hidenao; Yoshida, Mari; Kakita, Akiyoshi; Takahashi, Hitoshi; Wakabayashi, Koichi

    2013-12-01

    Valosin-containing protein (VCP) is associated with multiple cellular functions, including ubiquitin-dependent protein degradation. Mutations in VCP are known to cause inclusion body myopathy with Paget's disease and frontotemporal dementia and familial amyotrophic lateral sclerosis (fALS; ALS14), both of which are characterized by trans-activation response DNA protein 43 (TDP-43)-positive neuronal cytoplasmic and nuclear inclusions. Recently, immunoreactivity for fALS-associated proteins (TDP-43, fused in sarcoma (FUS), optineurin and ubiquilin-2) were reported to be present in cytoplasmic and nuclear inclusions in various neurodegenerative diseases. However, the extent and frequency of VCP-immunoreactive structures in these neurodegenerative diseases are uncertain. We immunohistochemically examined the brains of 72 cases with neurodegenerative diseases and five control cases. VCP immunoreactivity was present in Lewy bodies in Parkinson's disease and dementia with Lewy bodies, and neuronal nuclear inclusions in five polyglutamine diseases and intranuclear inclusion body disease, as well as in Marinesco bodies in aged control subjects. However, other neuronal and glial cytoplasmic inclusions in tauopathies and TDP-43 proteinopathies were unstained. These findings suggest that VCP may have common mechanisms in the formation or degradation of cytoplasmic and nuclear inclusions of neurons, but not of glial cells, in several neurodegenerative conditions.

  15. Adenovirus-like inclusion body hepatitis in a flock of broiler chickens in Kermanshah province, Iran

    PubMed Central

    Rahimi, Morad; Minoosh Siavosh Haghighi, Zahra

    2015-01-01

    Inclusion body hepatitis (IBH) has been reported in many countries in the world. The IBH or similar cases characterized by hepatitis and presence of intra-nuclear inclusion bodies in hepatocytes have not been reported in broiler chickens in Iran. This is the first report on outbreak of adenovirus-like inclusion body hepatitis in Iran. On October 2012, an onset of high acute mortality in a flock of 2 day-old broiler chickens was reported to the Veterinary Clinic, Faculty of Veterinary Medicine, Razi University, Kermanshah, Iran. The birds showed lethargy, huddling, ruffled feathers, and inappetence. At necropsy the livers were the primary organ affected which were enlarged, pale yellow with necrotic foci and multiple petechial hemorrhages. Tissue samples of liver, kidneys and heart were fixed in 10% buffered formalin. They were processed and stained with hematoxylin and eosin for histopathological studies. Significant microscopic lesions were seen in the livers. Large eosinophilic intra-nuclear inclusion bodies were seen in hepatocytes. Based on the acute high mortality, age of the broilers, gross lesions and histopathological findings (especially intra-nuclear inclusion bodies), the condition was diagnosed as adenovirus-like inclusion body hepatitis. PMID:25992259

  16. Inclusion body myositis and sarcoid myopathy: coincidental occurrence or associated diseases.

    PubMed

    Sanmaneechai, Oranee; Swenson, Andrea; Gerke, Alicia K; Moore, Steven A; Shy, Michael E

    2015-04-01

    Inclusion body myositis (IBM) is a slowly progressive inflammatory myopathy characterized by selective weakness of finger flexors and quadriceps muscles commonly refractory to treatment. Another chronic inflammatory disorder, sarcoidosis, commonly involves muscle. The comorbidity of inclusion body myositis and sarcoid myopathy is rare. We describe clinical and muscle biopsy findings of a patient with sarcoidosis and inclusion body myositis. A 66-year-old man presented with a 6-year history of progressive, asymmetrical and selective weakness of the quadriceps, biceps and finger flexor muscles; he had a remote history of pulmonary sarcoidosis. A quadriceps muscle biopsy revealed a chronic inflammatory myopathy with ubiquitinated inclusion bodies, rimmed vacuoles, expression of major histocompatibility complex class I, numerous COX-negative fibers and TDP-43 cytoplasmic aggregates (features of IBM) and multiple non-necrotizing granulomata (feature of sarcoidosis). Clinical and histopathologic features of the current illness suggested the patient had sarcoidosis with inclusion body myositis overlap. This patient may represent the coincidental occurrence of both idiopathic inflammatory disorders. Alternatively, sarcoidoisis may promote the development of inclusion body myositis by a similar immune-mediated pathophysiologic process. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. p62-enriched inclusion bodies in macrophages protect against atherosclerosis

    PubMed Central

    Sergin, Ismail; Bhattacharya, Somashubhra; Emanuel, Roy; Esen, Emel; Stokes, Carl J.; Evans, Trent D.; Arif, Batool; Curci, John A.; Razani, Babak

    2016-01-01

    Autophagy is a catabolic cellular mechanism that degrades dysfunctional proteins and organelles. Atherosclerotic plaque formation is enhanced in mice with macrophages that cannot undergo autophagy because of a deficiency of an autophagy component such as ATG5. We showed that exposure of macrophages to atherogenic lipids led to an increase in the abundance of the autophagy chaperone p62, which colocalized with polyubiquitinated proteins in cytoplasmic inclusions. p62 accumulation was increased in ATG5-null macrophages, which had large cytoplasmic ubiquitin-positive p62 inclusions. Aortas from atherosclerotic mice and plaques from human endarterectomy samples showed increased abundance of p62 and polyubiquitinated proteins that co-localized with plaque macrophages, suggesting that p62-enriched protein aggregates were characteristic of atherosclerosis. The formation of the cytoplasmic inclusions depended on p62 because lipid-loaded p62-null macrophages accumulated polyubiquitinated proteins in a diffuse cytoplasmic pattern. The failure of these aggregates to form was associated with increased secretion of IL-1β and enhanced macrophage apoptosis, which depended on the p62 ubiquitin-binding domain and at least partly involved p62-mediated clearance of NLRP3 inflammasomes. Consistent with our in vitro observations, p62-deficient mice formed greater numbers of more complex atherosclerotic plaques, and p62 deficiency further increased atherosclerotic plaque burden in mice with a macrophage-specific ablation of ATG5. Together, these data suggested that sequestration of cytotoxic ubiquitinated proteins by p62 protects against atherogenesis, a condition in which the clearance of protein aggregates is disrupted. PMID:26732762

  18. Limit for the Survivability from Potassium Decay of Bacterial Spores in Halite Fluid Inclusions

    NASA Astrophysics Data System (ADS)

    Kminek, G.; Bada, J. L.

    2001-12-01

    Vreeland et al.1 recently claimed to have isolated and cultured a viable spore forming halotolerant bacterium from a 250 million year old brine inclusion present in a salt crystal from the Salado formation. An earlier report suggested that viable bacterial spores could be revived from samples obtained from insects entombed in 25-40 million year old Dominican amber2. On the bases of these reports, Parkes3 raised the question of whether bacterial spores under some conditions might be effectively immortal. Sporulation, induced by an adverse change in the environmental conditions, is able to stabilize the DNA primarily against hydrolytic depurination for extended periods of time4. However, the organism is still exposed to ionizing radiation from the environment. Dormant spores have a reduced sensitivity to ionizing radiation per se, but unlike active organisms are unable to repair DNA damage encountered during long-term exposure to ionizing radiation. The accumulated damage may overwhelm any repair mechanism that starts in the early stage of spore germination5. The main radionuclide in a halite fluid inclusion is 40K, which accounts for 0.0117% of natural potassium. 40K decays via beta decay to 40Ca and via electron capture to 40Ar, releasing a primary gamma-ray. About 83.3 % of the beta's emitted are in the energy range of 0.3-1.3 MeV. We assume 7 g/l for an average concentration of natural potassium in a halite fluid inclusion, which means that the amount of 40K in a 10 μ l fluid inclusion is 8.19 ng. We have chosen a 10 μ l because this volume is typical of that used to obtain chemical data and in the attempts to extract bacteria. Less than a percent of the gamma decay energy is absorbed in a fluid inclusion of 10 μ l. Thus, we will not take the gamma decay energy into account for the further discussion. Almost all the beta energy is absorbed in the fluid inclusion. The total decay energy absorbed in a time period of 250 million years is about 87 kGy. The most

  19. Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin.

    PubMed

    Mackin, Robert B

    2014-01-01

    The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix.

  20. Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin

    PubMed Central

    Mackin, Robert B.

    2014-01-01

    The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix. PMID:26150942

  1. Renaturation and one step purification of the chicken GIIA secreted phospholipase A2 from inclusion bodies.

    PubMed

    Karray, Aida; Amara, Sawsan; Carrière, Frédéric; Gargouri, Youssef; Bezzine, Sofiane

    2014-06-01

    The cDNA coding for a mature protein of 123 amino acids, containing all of the structural features of catalytically active group IIA sPLA2, has been amplified from chicken intestine. The gene has been cloned into the bacterial expression vector pET-21a(+), which allows protein over-expression as inclusion bodies and enables about 3mg/l of pure refolded fully active enzyme to be obtained. Recombinant expression of chicken intestinal sPLA2-IIA (ChPLA2-IIA) in Escherichia coli shows that the enzyme is Ca(2+) dependent, maximally active at pH 8-9, and hydrolyses phosphatidylglycerol versus phosphatidylcholine with a 10-fold preference. Indeed, we report in this work, a comparative kinetic study between the wild type and the recombinant ChPLA2-IIA, on zwitterionic head group phospholipids (DDPC) and negatively charged phospholipids (POPG) using the monomolecular film technique. The ability to express reasonably large amounts of the sPLA2 Group IIA, compared to that obtained with the classical purification will provide a basis for future site directed mutagenesis studies of this important enzyme. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Inclusion.

    ERIC Educational Resources Information Center

    Nathanson, Jeanne H., Ed.

    1992-01-01

    This theme journal issue focuses on current activities of the Office of Special Education and Rehabilitative Services which stress inclusion of students with disabilities in the mainstream. It begins with a message from the Assistant Secretary, Robert R. Davila which examines the full meaning of an "inclusive" education. Next, Barbara…

  3. Inclusion.

    ERIC Educational Resources Information Center

    Nathanson, Jeanne H., Ed.

    1992-01-01

    This theme journal issue focuses on current activities of the Office of Special Education and Rehabilitative Services which stress inclusion of students with disabilities in the mainstream. It begins with a message from the Assistant Secretary, Robert R. Davila which examines the full meaning of an "inclusive" education. Next, Barbara…

  4. Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli.

    PubMed

    Patra, A K; Mukhopadhyay, R; Mukhija, R; Krishnan, A; Garg, L C; Panda, A K

    2000-03-01

    Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.

  5. Huntingtin inclusion bodies are iron-dependent centers of oxidative events.

    PubMed

    Firdaus, Wance J J; Wyttenbach, Andreas; Giuliano, Paola; Kretz-Remy, Carole; Currie, R William; Arrigo, André-Patrick

    2006-12-01

    Recently, we reported that the transient expression of huntingtin exon1 polypeptide containing polyglutamine tracts of various sizes (httEx1-polyQ) in cell models of Huntington disease generated an oxidative stress whose intensity was CAG repeat expansion-dependent. Here, we have analyzed the intracellular localization of the oxidative events generated by the httEx1-polyQ polypeptides. Analysis of live COS-7 cells as well as neuronal SK-N-SH and PC12 cells incubated with hydroethidine or dichlorofluorescein diacetate revealed oxidation of these probes at the level of the inclusion bodies formed by httEx1-polyQ polypeptides. The intensity and frequency of the oxidative events among the inclusions were CAG repeat expansion-dependent. Electron microscopic analysis of cell sections revealed the presence of oxidation-dependent morphologic alterations in the vicinity of httEx1-polyQ inclusion bodies. Moreover, a high level of oxidized proteins was recovered in partially purified inclusions. We also report that the iron chelator deferroxamine altered the structure, localization and oxidative potential of httEx1-polyQ inclusion bodies. Hence, despite the fact that the formation of inclusion bodies may represent a defense reaction of the cell to eliminate httEx1 mutant polypeptide, this phenomenon appears inherent to the generation of iron-dependent oxidative events that can be deleterious to the cell.

  6. Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.

    PubMed

    Okegawa, Yuki; Koshino, Masanori; Okushima, Teruya; Motohashi, Ken

    2016-02-01

    Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams). Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli.

    PubMed

    Upadhyay, Arun K; Murmu, Aruna; Singh, Anupam; Panda, Amulya K

    2012-01-01

    The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2-3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.

  8. Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

    PubMed Central

    Upadhyay, Arun K.; Murmu, Aruna; Singh, Anupam; Panda, Amulya K.

    2012-01-01

    The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2–3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies. PMID:22479486

  9. Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli.

    PubMed

    Palmer, Ira; Wingfield, Paul T

    2012-11-01

    High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; however, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates by low-speed centrifugation. Following pre-extaction (or washing), protein is extracted from washed pellets using guanidine⋅HCl. The solubilized and unfolded protein is either directly folded or further purified by gel filtration in the presence of guanidine⋅HCl as described in this unit. A support protocol describes the removal of guanidine⋅HCl from column fractions so they can be monitored by SDS-PAGE.

  10. Microfluidic chips with multi-junctions: an advanced tool in recovering proteins from inclusion bodies

    PubMed Central

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2015-01-01

    Active recombinant proteins are used for studying the biological functions of genes and for the development of therapeutic drugs. Overexpression of recombinant proteins in bacteria often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. Protein refolding is an important process for obtaining active recombinant proteins from inclusion bodies. However, the conventional refolding method of dialysis or dilution is time-consuming and recovered active protein yields are often low, and a cumbersome trial-and-error process is required to achieve success. To circumvent these difficulties, we used controllable diffusion through laminar flow in microchannels to regulate the denaturant concentration. This method largely aims at reducing protein aggregation during the refolding procedure. This Commentary introduces the principles of the protein refolding method using microfluidic chips and the advantage of our results as a tool for rapid and efficient recovery of active recombinant proteins from inclusion bodies. PMID:25531187

  11. Microfluidic chips with multi-junctions: an advanced tool in recovering proteins from inclusion bodies.

    PubMed

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2015-01-01

    Active recombinant proteins are used for studying the biological functions of genes and for the development of therapeutic drugs. Overexpression of recombinant proteins in bacteria often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. Protein refolding is an important process for obtaining active recombinant proteins from inclusion bodies. However, the conventional refolding method of dialysis or dilution is time-consuming and recovered active protein yields are often low, and a cumbersome trial-and-error process is required to achieve success. To circumvent these difficulties, we used controllable diffusion through laminar flow in microchannels to regulate the denaturant concentration. This method largely aims at reducing protein aggregation during the refolding procedure. This Commentary introduces the principles of the protein refolding method using microfluidic chips and the advantage of our results as a tool for rapid and efficient recovery of active recombinant proteins from inclusion bodies.

  12. Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies

    PubMed Central

    Raghava, Smita; Barua, Bipasha; Singh, Pradeep K.; Das, Mili; Madan, Lalima; Bhattacharyya, Sanchari; Bajaj, Kanika; Gopal, B.; Varadarajan, Raghavan; Gupta, Munishwar N.

    2008-01-01

    Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of three-phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies. PMID:18780821

  13. A Rare Case of Sporadic Inclusion Body Myositis (s-IBM).

    PubMed

    Acharya, Sourya; Shukla, Samarth; Kitey, Pritum; Khan, Shameem; Mahajan, S N

    2016-01-01

    Sporadic inclusion body myositis (s-IBM) is an acquired inflammatory myopathy. Clinical presentation is variable. The usual presentation is progressive weakness and atrophy of the arms and leg muscles, especially of the quadriceps femoris which is invariably affected. It is classified under inflammatory myopathies, along with polymyositis and dermatomyositis. We present a case of s-IBM who presented with gradually progressive quadriparesis with characteristic quadriceps wasting and weakness in neck flexors. Electromyography revealed myopathic potential and muscle biopsy revealed features of inclusion body myositis.

  14. Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli

    PubMed Central

    Palmer, Ira; Wingfield, Paul T.

    2013-01-01

    High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; however, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates by low speed centrifugation. Following preextaction (or washing) protein is extracted from washed pellets using guanidine·HCl. The solubilized and unfolded protein is either directly folded as described in UNIT 6.1 or further purified by gel filtration in the presence of guanidine·HCl as described here. A support protocol describes the removal of guanidine·HCl from column fractions so they can be monitored by SDS-PAGE. High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies (UNITS 5.1 & 6.1). Inclusion bodies are normally formed in the cytoplasm; alternatively, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies are not restricted to E. coli; they can also form in yeast, mammalian, and insect cells. Inclusion bodies recovered from cell lysates by low-speed centrifugation are heavily contaminated with E. coli cell wall and outer membrane components. The latter are largely removed by selective extraction with detergents and low concentrations of either urea or guanidine·HCl to produce so-called washed pellets. These basic steps result in a significant purification of the recombinant protein, which usually makes up ~60% of the washed pellet protein. The challenge, therefore, is not to purify the recombinant-derived protein, but to solubilize it and then fold it into native and biologically active protein. Basic Protocol 1 describes preparation of washed pellets and solubilization of the protein using guanidine·HCl. The extracted protein, which is unfolded, is either directly

  15. Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli

    PubMed Central

    Palmer, Ira; Wingfield, Paul T.

    2012-01-01

    High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; however, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates by low speed centrifugation. Following preextaction (or washing) protein is extracted from washed pellets using guanidine·HCl. The solubilized and unfolded protein is either directly folded as described in UNIT 6.1 or further purified by gel filtration in the presence of guanidine·HCl as described here. A support protocol describes the removal of guanidine·HCl from column fractions so they can be monitored by SDS-PAGE. High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies (UNITS 5.1 & 6.1). Inclusion bodies are normally formed in the cytoplasm; alternatively, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies are not restricted to E. coli; they can also form in yeast, mammalian, and insect cells. Inclusion bodies recovered from cell lysates by low-speed centrifugation are heavily contaminated with E. coli cell wall and outer membrane components. The latter are largely removed by selective extraction with detergents and low concentrations of either urea or guanidine·HCl to produce so-called washed pellets. These basic steps result in a significant purification of the recombinant protein, which usually makes up ~60% of the washed pellet protein. The challenge, therefore, is not to purify the recombinant-derived protein, but to solubilize it and then fold it into native and biologically active protein. Basic Protocol 1 describes preparation of washed pellets and solubilization of the protein using guanidine·HCl. The extracted protein, which is unfolded, is either directly

  16. Expression and purification of biologically active recombinant human paraoxonase 1 from inclusion bodies of Escherichia coli.

    PubMed

    Bajaj, Priyanka; Tripathy, Rajan K; Aggarwal, Geetika; Pande, Abhay H

    2015-11-01

    Human PON1 (h-PON1) is a Ca(2+)-dependent serum enzyme and can hydrolyze (and inactivate) a wide range of substrates. It is a multifaceted enzyme and exhibit anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial, and organophosphate (OP)-detoxifying properties. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against these conditions in humans. Insufficient hydrolyzing activity of native h-PON1 against desirable substrate affirms the urgent need to develop improved variant(s) of h-PON1 having enhanced activity. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop such variant(s). However, generation of rh-PON1 using E. coli expression system has been elusive until now because of the aggregation of over-expressed rh-PON1 protein in inactive form as inclusion bodies (IBs) in the bacterial cells. In this study, we have over-expressed rh-PON1(wt) and rh-PON1(H115W;R192K) proteins as IBs in E. coli, and refolded the inactive enzymes present in the IBs to their active form using in vitro refolding. The active enzymes were isolated from the refolding mixture by ion-exchange chromatography. The catalytic properties of the refolded enzymes were similar to their soluble counterparts. Our results show that the pure and the active variant of rh-PON1 enzyme having enhanced hydrolyzing activity can be produced in large quantities using E. coli expression system. This method can be used for the industrial scale production of rh-PON1 enzymes and will aid in developing h-PON1 as a therapeutic candidate. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Direct Conversion of an Enzyme from Native-like to Amyloid-like Aggregates within Inclusion Bodies.

    PubMed

    Elia, Francesco; Cantini, Francesca; Chiti, Fabrizio; Dobson, Christopher Martin; Bemporad, Francesco

    2017-06-20

    The acylphosphatase from Sulfolobus solfataricus (Sso AcP) is a globular protein able to aggregate in vitro from a native-like conformational ensemble without the need for a transition across the major unfolding energy barrier. This process leads to the formation of assemblies in which the protein retains its native-like structure, which subsequently convert into amyloid-like aggregates. Here, we investigate the mechanism by which Sso AcP aggregates in vivo to form bacterial inclusion bodies after expression in E. coli. Shortly after the initiation of expression, Sso AcP is incorporated into inclusion bodies as a native-like protein, still exhibiting small but significant enzymatic activity. Additional experiments revealed that this overall process of aggregation is enhanced by the presence of the unfolded N-terminal region of the sequence and by destabilization of the globular segment of the protein. At later times, the Sso AcP molecules in the inclusion bodies lose their native-like properties and convert into β-sheet-rich amyloid-like structures, as indicated by their ability to bind thioflavin T and Congo red. These results show that the aggregation behavior of this protein is similar in vivo to that observed in vitro, and that, at least for a predominant part of the protein population, the transition from a native to an amyloid-like structure occurs within the aggregate state. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  18. Accumulation of Mutant Neuroserpin Precedes Development of Clinical Symptoms in Familial Encephalopathy with Neuroserpin Inclusion Bodies

    PubMed Central

    Galliciotti, Giovanna; Glatzel, Markus; Kinter, Jochen; Kozlov, Serguei V.; Cinelli, Paolo; Rülicke, Thomas; Sonderegger, Peter

    2007-01-01

    Intracellular protein deposition due to aggregation caused by conformational alteration is the hallmark of a number of neurodegenerative disorders, including Parkinson’s disease, tauopathies, Huntington’s disease, and familial encephalopathy with neuroserpin inclusion bodies. The latter is an autosomal dominant disorder caused by point mutations in neuroserpin resulting in its destabilization. Mutant neuroserpin polymerizes and forms intracellular aggregates that eventually lead to neurodegeneration. We generated genetically modified mice expressing the late-onset S49P-Syracuse or the early-onset S52R-Portland mutation of neuroserpin in central nervous system neurons. Mice exhibited morphological, biochemical, and clinical features resembling those found in the human disease. Analysis of brains revealed large intraneuronal inclusions composed exclusively of mutant neuroserpin, accumulating long before the development of clinical symptoms in a time-dependent manner. Clinical symptoms and amount of neuroserpin inclusions correlated with the predicted instability of the protein. The presence of inclusion bodies in subclinical mice indicates that in humans the prevalence of the disease could be higher than anticipated. In addition to shedding light on the pathophysiology of the human disorder, these mice provide an excellent model to study mechanisms of neurodegeneration or establish novel therapies for familial encephalopathy with neuroserpin inclusion bodies and other neurodegenerative diseases with intracellular protein deposition. PMID:17392169

  19. Semantic dementia with ubiquitin-positive tau-negative inclusion bodies.

    PubMed

    Rossor, M N; Revesz, T; Lantos, P L; Warrington, E K

    2000-02-01

    Three cases are reported with dementia and ubiquitin-positive but tau-negative inclusion bodies. All patients had a semantic dementia and the clinical details of two of these have been published as the first description of a selective semantic memory impairment. The original diagnosis had been of Pick's disease based on frontotemporal atrophy, but re-examination has revealed ubiquitin-positive but tau-negative inclusions as well as neurites in the frontotemporal cortices and ubiquitin-positive, intracytoplasmic inclusions in the granule cells of the dentate fascia. These inclusions are identical to those reported in association with amyotrophic lateral sclerosis (motor neuron disease), but none were seen in brainstem or spinal cord motor neurons.

  20. Incipient intranuclear inclusion body disease in a 78-year-old woman.

    PubMed

    Mori, Fumiaki; Miki, Yasuo; Tanji, Kunikazu; Ogura, Eriko; Yagihashi, Norito; Jensen, Poul H; Wakabayashi, Koichi

    2011-04-01

    We report an incipient case of intranuclear inclusion body disease (INIBD) in a 78-year-old woman. No apparent neurological symptoms were noticed during the clinical course. Post mortem examination revealed widespread occurrence of eosinophilic intranuclear inclusions in neuronal and glial cells of the central and peripheral nervous systems, as well as in parenchymal cells of the visceral organs. The inclusions were observed more frequently in glial cells than in neuronal cells. Ultrastructurally, the inclusions consisted of granular and filamentous material. Immunohistochemically, the inclusions were positive for ubiquitin, ubiquitin-related proteins (NEDD8 ultimate buster 1, small ubiquitin modifier-1, small ubiquitin modifier-2 and p62), promyelocytic leukemia protein and abnormally expanded polyglutamine. Consistent with previous studies, the vast majority of inclusion-bearing glial cells were astrocytes. Furthermore, p25α-positive oligodendrocytes rarely contained intranuclear inclusions. These findings suggest that INIBD may occur in non-demented elderly individuals and that oligodendrocyte is also involved in the disease process of INIBD.

  1. A constitutive model for micro-cracked bodies with growing inclusions

    NASA Astrophysics Data System (ADS)

    Bongué Boma, Malika; Alaoui, Amina

    2012-01-01

    A model of micro-cracked bodies having rigid inclusions growing in their pores is proposed, based on the theories of generalized continua. We first use the balance equations of an existing model of micro-cracked bodies, and we then perform a multiscale description in order to determine constitutive laws that account for the growth of the inclusions. We call macroscopic, the description in which the material is considered as a continuum with microstructure, whereas we refer to microscopic scale when one crack is observed at a closer view. We finally use equivalences between both descriptions in order to write the constitutive laws in terms of variables that are characteristic of (i) the geometry of the crack field and (ii) the growth of the inclusions. Such an approach can find, for instance, application in the modeling of expansion due to delayed ettringite formation: we perform numerical simulations using mechanical and geometrical parameters that are characteristic of high strength sulfoaluminate concrete.

  2. [Inclusion Body Disease (IBD of Boids)--a haematological, histological and electron microscopical study].

    PubMed

    Keilwerth, Melanie; Bühler, Ilina; Hoffmann, Rudolf; Soliman, Hatem; El-Matbouli, Mansour

    2012-01-01

    Our objective was to evaluate diagnostic tools for the detection of Inclusion Body Disease (IBD) in bold snakes. The aetiology of IBD is unknown, and the disease has non-specific clinical signs, hence there is a need for a clinically-applicable, specific diagnostic method. We examined blood smears and liver biopsies from 26 bold snakes (17 boas and nine pythons; some of which were suspected of having IBD) for the presence of characteristic inclusion bodies. We used haematology, histology and electron microscopy to characterise samples as IBD-positive or -negative. Our results indicate that examination of a simple blood smear is sufficient to diagnose IBD in boas. Inclusion bodies in lymphocytes, erythrocytes and thrombocytes were observed. In both, boas and pythons, we detected inclusion bodies within hepatocytes. We demonstrated also that IBD was more common in boas than in pythons: only samples from two Ball Pythons (Python regius) tested positive, whereas no other Pythonidae were positive. We consider that blood smears represents a rapid, non-invasive technique for detection of IBD.

  3. Replication and inclusion body characteristics of two Lymantria dispar nuclear polyhedrosis virus plaque variants

    Treesearch

    James M. Slavicek; Carita Lanner-Herrera; Nancy Hayes-Plazolles; Mary Ellen Kelly; Martha Fikes

    1991-01-01

    Propagation of Autographa californica nuclear polyhedrosis virus in cell culture results in the generation of a mutant virus, termed few polyhedra. This plaque variant is characterized by a high budded virus titer, the formation of few polyhedral inclusion bodies (PIBs), and the production of PIBs exhibiting a low potency against its natural host....

  4. Analysis of magnetite crystals and inclusion bodies inside magnetotactic bacteria from different environmental locations

    NASA Astrophysics Data System (ADS)

    Oestreicher, Z.; Lower, B.; Lower, S.; Bazylinski, D. A.

    2011-12-01

    Biomineralization occurs throughout the living world; a few common examples include iron oxide in chiton teeth, calcium carbonate in mollusk shells, calcium phosphate in animal bones and teeth, silica in diatom shells, and magnetite crystals inside the cells of magnetotactic bacteria. Biologically controlled mineralization is characterized by biominerals that have species-specific properties such as: preferential crystallographic orientation, consistent particle size, highly ordered spatial locations, and well-defined composition and structure. It is well known that magnetotactic bacteria synthesize crystals of magnetite inside of their cells, but how they mineralize the magnetite is poorly understood. Magnetosomes have a species-specific morphology that is due to specific proteins involved in the mineralization process. In addition to magnetite crystals, magnetotactic bacteria also produce inclusion bodies or granules that contain different elements, such as phosphorus, calcium, and sulfur. In this study we used the transmission electron microscope to analyze the structure of magnetite crystals and inclusion bodies from different species of magnetotactic bacteria in order to determine the composition of the inclusion bodies and to ascertain whether or not the magnetite crystals contain elements other than iron and oxygen. Using energy dispersive spectroscopy we found that different bacteria from different environments possess inclusion bodies that contain different elements such as phosphorus, calcium, barium, magnesium, and sulfur. These differences may reflect the conditions of the environment in which the bacteria inhabit.

  5. Asymptotics of eigenfrequencies of an elastic body with a heavy and hard peak-shaped inclusion*

    NASA Astrophysics Data System (ADS)

    Nazarov, Sergey A.

    2007-12-01

    A heavy and hard peak-shaped inclusion in an elastic body provokes to concentration of eigenvalues in the low-frequency range of the spectrum and localization of the corresponding eigenmodes near the peak tip. To cite this article: S.A. Nazarov, C. R. Mecanique 335 (2007).

  6. Refolding from denatured inclusion bodies, purification to homogeneity and simplified assay of MGDG synthases from land plants.

    PubMed

    Nishiyama, Yoshitaka; Hardré-Liénard, Hélène; Miras, Stéphane; Miège, Christine; Block, Maryse A; Revah, Frédéric; Joyard, Jacques; Maréchal, Eric

    2003-09-01

    In plant cells, the synthesis of monogalactosyldiacylglycerol (MGDG) is catalyzed within plastid envelope membranes by MGD proteins. MGDG synthesis was also reported in apicomplexan parasites, a phylum of protists harbouring a plastid that proved essential for the parasite survival. MGD activity is therefore a potent target for herbicidal and anti-parasitic molecules. In this study, we describe a detailed in vitro refolding protocol for denatured recombinant MGD accumulated in inclusion bodies from transformed Escherichia coli. The refolding process was dependent on CHAPS detergent and lipids, such as diacylglycerol and phosphatidylglycerol, as well as bivalent metals. Owing to this refolding procedure, the recombinant MGD protein from spinach was purified to homogeneity, allowing a definite characterization of its non-processivity and an investigation of its dimerization using cross-linking reagents. Additionally, using the portion of recombinant enzyme that accumulates in an active form in bacterial membranes, we developed a miniature assay for high-throughput screening for inhibitors.

  7. Expression of nattokinase in Escherichia coli and renaturation of its inclusion body.

    PubMed

    Ni, He; Guo, Peng-Cheng; Jiang, Wei-Ling; Fan, Xiao-Min; Luo, Xiang-Yu; Li, Hai-Hang

    2016-08-10

    Nattokinase is an important fibrinolytic enzyme with therapeutic applications for cardiovascular diseases. The full-length and mature nattokinase genes were cloned from Bacillus subtilis var. natto and expressed in pQE30 vector in Escherichia coli. The full-length gene expressed low nattokinase activity in the intracellular soluble and the medium fractions. The mature gene expressed low soluble nattokinase activity and large amount insoluble protein in inclusion bodies without enzyme activity. Large amount of refolding solutions (RSs) at different pH values were screening and RS-10 and RS-11 at pH 9 were selected to refold nattokinase inclusion bodies. The recombinant cells were lysed with 0.1mg/mL lysozyme and ultrasonic treatment. After centrifugation, the pellete was washed twice with 20mM Tris-HCl buffer (pH 7.5) containing 1% Triton X-100 to purify the inclusion bodies. The inclusion bodies were dissolved in water at pH 12.0 and refolded with RS-10. The refolded proteins showed 42.8IU/mg and 79.3IU/mg fibrinolytic activity by the traditional dilution method (20-fold dilution into RS-10) and the directly mixing the protein solution with equal volume RS-10, respectively, compared to the 52.0IU/mg of total water-soluble proteins from B. subtilis var. natto. This work demonstrated that the inclusion body of recombinant nattokinase expressed in E. coli could be simply refolded to the natural enzyme activity level by directly mixing the protein solution with equal volume refolding solution.

  8. Spinal motor neuron protein supersaturation patterns are associated with inclusion body formation in ALS

    PubMed Central

    Ciryam, Prajwal; Lambert-Smith, Isabella A.; Bean, Daniel M.; Freer, Rosie; Cid, Fernando; Tartaglia, Gian Gaetano; Saunders, Darren N.; Wilson, Mark R.; Morimoto, Richard I.; Dobson, Christopher M.; Vendruscolo, Michele; Favrin, Giorgio; Yerbury, Justin J.

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a heterogeneous degenerative motor neuron disease linked to numerous genetic mutations in apparently unrelated proteins. These proteins, including SOD1, TDP-43, and FUS, are highly aggregation-prone and form a variety of intracellular inclusion bodies that are characteristic of different neuropathological subtypes of the disease. Contained within these inclusions are a variety of proteins that do not share obvious characteristics other than coaggregation. However, recent evidence from other neurodegenerative disorders suggests that disease-affected biochemical pathways can be characterized by the presence of proteins that are supersaturated, with cellular concentrations significantly greater than their solubilities. Here, we show that the proteins that form inclusions of mutant SOD1, TDP-43, and FUS are not merely a subset of the native interaction partners of these three proteins, which are themselves supersaturated. To explain the presence of coaggregating proteins in inclusions in the brain and spinal cord, we observe that they have an average supersaturation even greater than the average supersaturation of the native interaction partners in motor neurons, but not when scores are generated from an average of other human tissues. These results suggest that inclusion bodies in various forms of ALS result from a set of proteins that are metastable in motor neurons, and thus prone to aggregation upon a disease-related progressive collapse of protein homeostasis in this specific setting. PMID:28396410

  9. Spinal motor neuron protein supersaturation patterns are associated with inclusion body formation in ALS.

    PubMed

    Ciryam, Prajwal; Lambert-Smith, Isabella A; Bean, Daniel M; Freer, Rosie; Cid, Fernando; Tartaglia, Gian Gaetano; Saunders, Darren N; Wilson, Mark R; Oliver, Stephen G; Morimoto, Richard I; Dobson, Christopher M; Vendruscolo, Michele; Favrin, Giorgio; Yerbury, Justin J

    2017-05-16

    Amyotrophic lateral sclerosis (ALS) is a heterogeneous degenerative motor neuron disease linked to numerous genetic mutations in apparently unrelated proteins. These proteins, including SOD1, TDP-43, and FUS, are highly aggregation-prone and form a variety of intracellular inclusion bodies that are characteristic of different neuropathological subtypes of the disease. Contained within these inclusions are a variety of proteins that do not share obvious characteristics other than coaggregation. However, recent evidence from other neurodegenerative disorders suggests that disease-affected biochemical pathways can be characterized by the presence of proteins that are supersaturated, with cellular concentrations significantly greater than their solubilities. Here, we show that the proteins that form inclusions of mutant SOD1, TDP-43, and FUS are not merely a subset of the native interaction partners of these three proteins, which are themselves supersaturated. To explain the presence of coaggregating proteins in inclusions in the brain and spinal cord, we observe that they have an average supersaturation even greater than the average supersaturation of the native interaction partners in motor neurons, but not when scores are generated from an average of other human tissues. These results suggest that inclusion bodies in various forms of ALS result from a set of proteins that are metastable in motor neurons, and thus prone to aggregation upon a disease-related progressive collapse of protein homeostasis in this specific setting.

  10. Adenomatoid mesothelioma with intranuclear inclusion bodies: a case report with cytological and histological findings.

    PubMed

    Kawai, Toshiaki; Kawashima, Katsuhiko; Serizawa, Hiromi; Miura, Hiroyuki; Kyeongil, Kim

    2014-05-01

    We report a very unusual cytologic feature, intranuclear inclusion bodies, in mesothelioma of a predominantly adenomatoid type. The patient, a 57-year-old woman, was presented with dyspnea and right pleural effusion. Pleural aspiration cytology revealed many cohesive ball-like clusters, with a tubular pattern, composed of small atypical cells displaying a high-nuclear-cytoplasmic ratio. They had a nuclear groove and irregular intranuclear inclusion bodies. Right lung partial resection with thoracoscopy revealed that a white tumor had proliferated along the pleural surface at S(8) . Histology revealed nodular tumor cells forming dilated structures mixed with small tubular or glandular structures similar to those seen in benign adenomatoid tumors. These tumor cells had invaded peripheral lung tissues. Such inclusion bodies have not been reported earlier in mesothelioma. On the basis of this observation, we propose that the adenomatoid type of malignant mesothelioma be added to the differential diagnosis of malignant effusions when tumor cells with nuclear grooves and intranuclear inclusions are found in pleural aspiration cytology. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

  11. Intracytoplasmic inclusion bodies in the chondrocytes of type I lethal achondrogenesis.

    PubMed

    Yang, S S; Heidelberger, K P; Bernstein, J

    1976-11-01

    Lethal achondrogenesis in the past has been frequently confused with achondroplasia. Clinical and radiologic advances in the last decade have led to clear differentiation of this condition from other types of bone dysplasia. It is further separated into two types on the basis of radiographic and pathologic findings. Re-evaluation of the histologic features has led to the recognition of heretofore unrecognized intracytoplasmic inclusion bodies in the chondrocytes of type 1 achondrogenesis. The finding of inclusions strengthens the differentiation of the two types and may have implications for the pathogenesis of the form of chondrodystrophy.

  12. Inclusion bodies in cerebral cortical astrocytes: a new change of astrocytes.

    PubMed

    Minagawa, M; Shioda, K; Shimizu, Y; Isshiki, T

    1992-01-01

    A unique pathological finding of astrocytes was observed in the brain of a 20-year-old man who had severe physical and mental retardation. The brain was malformed showing micropolygyria in several cortical areas. A large number of hypertrophic astrocytes with eosinophilic granular substances in their cytoplasm were found throughout the cerebral cortex. Several staining procedures and electron microscopical examinations were carried out on these intracytoplasmic inclusion. It was found that the appearance and staining character of these inclusions were different from other astrocytic changes, especially the Rosenthal fiber, described so far. The authors consider that these inclusion bodies in cerebral cortical astrocytes represent new pathological changes of astrocytes that appear to be associated with malformation of the brain.

  13. Singular invariant integrals for elastic bodies with thin elastic inclusions and cracks

    NASA Astrophysics Data System (ADS)

    Khludnev, A. M.; Shcherbakov, V. V.

    2016-12-01

    Equilibrium problems for an elastic body with partially delaminated thin elastic inclusions are considered. The inclusions are modeled within the framework of the Euler-Bernoulli and Timoshenko models of elastic beams. The presence of delamination means the existence of a crack between the inclusion and the elastic matrix. Displacements of the opposite crack faces are constrained with nonpenetration conditions. Formulas of the Griffith type giving the first derivatives of energy functionals with respect to the crack length are established. It is shown that the formulas for the derivatives admit representation in the form of invariant integrals independent of the smooth closed curve surrounding the crack tip. The obtained invariant integrals consist of the sum of regular and singular parts and are analogues of the classical Eshelby-Cherepanov-Rice J-integral.

  14. Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence.

    PubMed

    Peterson, Megan J; Snyder, W Kalani; Westerman, Shelley; McFarland, Benjamin J

    2011-07-01

    We describe how to produce and purify proteins from E. coli inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This seven-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies, which are collected, washed, then solubilized in urea. Stepwise dialysis to dilute urea over the course of a week produces refolded protein. Column chromatography is used to purify protein into fractions, which are then analyzed with gel electrophoresis and concentration assays. Students culminate the project by designing crystallization trials in sitting-drop trays. Student evaluation of the experience has been positive, listing 5-12 new techniques learned, which are transferrable to graduate research in academia and industry.

  15. Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence

    PubMed Central

    Peterson, Megan J.; Snyder, W. Kalani; Westerman, Shelley; McFarland, Benjamin J.

    2011-01-01

    We describe how to produce and purify proteins from E. coli inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This seven-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies, which are collected, washed, then solubilized in urea. Stepwise dialysis to dilute urea over the course of a week produces refolded protein. Column chromatography is used to purify protein into fractions, which are then analyzed with gel electrophoresis and concentration assays. Students culminate the project by designing crystallization trials in sitting-drop trays. Student evaluation of the experience has been positive, listing 5–12 new techniques learned, which are transferrable to graduate research in academia and industry. PMID:21691428

  16. Falcon Herpesvirus, the Etiologic Agent of Inclusion Body Disease of Falcons

    PubMed Central

    Maré, C. J.; Graham, D. L.

    1973-01-01

    A viral agent has been isolated from five fatal cases of naturally occurring inclusion body disease in three different falcon species, namely, the prairie falcon (Falco mexicanus), the red-headed falcon (F. chiquera), and the peregrine falcon (F. peregrinus). The virus has been shown to possess the physical, chemical, and biological properties of a herpesvirus and has been used to reproduce inclusion body disease in the prairie falcon, merlin (F. columbarius), and American kestrel (F. sparverius). A similar disease was also produced with this virus in the great horned owl (Bubo virginianus), screech owl (Otus asio), and ring-necked turtle dove (Streptopelia risoria). Serological comparison of the falcon herpesvirus with other known avian herpesviruses revealed that the virus is antigenically closely related to a pigeon herpesvirus and an owl herpesvirus while differing from the former in host range. No antigenic relationship to infectious laryngotracheitis virus, duck virus enteritis, or Marek's disease virus could be demonstrated. Images PMID:4352453

  17. Falcon Herpesvirus, the etiologic agent of inclusion body disease of falcons.

    PubMed

    Maré, C J; Graham, D L

    1973-07-01

    A viral agent has been isolated from five fatal cases of naturally occurring inclusion body disease in three different falcon species, namely, the prairie falcon (Falco mexicanus), the red-headed falcon (F. chiquera), and the peregrine falcon (F. peregrinus). The virus has been shown to possess the physical, chemical, and biological properties of a herpesvirus and has been used to reproduce inclusion body disease in the prairie falcon, merlin (F. columbarius), and American kestrel (F. sparverius). A similar disease was also produced with this virus in the great horned owl (Bubo virginianus), screech owl (Otus asio), and ring-necked turtle dove (Streptopelia risoria). Serological comparison of the falcon herpesvirus with other known avian herpesviruses revealed that the virus is antigenically closely related to a pigeon herpesvirus and an owl herpesvirus while differing from the former in host range. No antigenic relationship to infectious laryngotracheitis virus, duck virus enteritis, or Marek's disease virus could be demonstrated.

  18. Isolation of a herpesvirus from an American kestrel with inclusion body disease.

    PubMed

    Potgieter, L N; Kocan, A A; Kocan, K M

    1979-01-01

    A herpesvirus was isolated from the liver of a captive-bred American kestrel (Falco sparverius) which had died of inclusion body disease. Initial isolation was achieved in chicken embryo fibroblasts after three blind passages. Cell-adapted virus produced a distinct rounding of CEF cells within 24 to 48 h. Biologic and serologic tests suggested that the kestrel virus is similar to falcon herpesvirus and pigeon herpesvirus and is at least partially related to owl herpesvirus. However, serologic tests indicated that the kestrel herpesvirus is neither related to infectious laryngotracheitis virus nor to a herpesvirus from a psittacine bird; (Eupsittula canicularis) with Pacheco's parrot disease. This is the first report on the recovery of a herpesvirus similar to falcon herpesvirus from an American kestrel with naturally-occurring inclusion body disease, and on the serologic comparison between falcon herpesvirus and a psittacine herpesvirus.

  19. Standardization of stain used for diagnosing erythrocytic inclusion body syndrome (EIBS)

    USGS Publications Warehouse

    1987-01-01

    Erythrocytic inclusion body syndrome (EIBS), a viral erythrocytic necrosis (VEN)-like disease, has been observed in several areas in the Northwest. This virus disease is clinically diagnosed by microscopic examination of blood smears for intracytoplasmic erythrocytic inclusion bodies. Fish biologists involved in EIBS diagnostic work have been using several types of hematological stains. It became apparent that standardization of the staining procedure was needed. Comparative tests were conducted on blood smears and kidney imprints with the following commonly used blood stains: (1) Leishman-Giesma, (2) Pinacyanol chloride, (3) Powell 's Giemsa, (4) Harleco's Giemsa, (5) Diff Quik differential stain, (6) Wright's.Pinacyanol chloride stain was found to be the most consistent. The following staining procedure is recommended.

  20. Inclusion body myopathy and Paget disease is linked to a novel mutation in the VCP gene.

    PubMed

    Haubenberger, D; Bittner, R E; Rauch-Shorny, S; Zimprich, F; Mannhalter, C; Wagner, L; Mineva, I; Vass, K; Auff, E; Zimprich, A

    2005-10-25

    Mutations in the valosin-containing protein (VCP) on chromosome 9p13-p12 were recently found to be associated with hereditary inclusion body myopathy, Paget disease of the bone, and frontotemporal dementia (IBMPFD). We identified a novel missense mutation in the VCP gene (R159H; 688G>A) segregating with this disease in an Austrian family of four affected siblings, who exhibited progressive proximal myopathy and Paget disease of the bone but without clinical signs of dementia.

  1. Chromatographic methods for the isolation of, and refolding of proteins from, Escherichia coli inclusion bodies.

    PubMed

    Gu, Zhenyu; Weidenhaupt, Marianne; Ivanova, Natalia; Pavlov, Michail; Xu, Bingze; Su, Zhi-Guo; Janson, Jan-Christer

    2002-06-01

    New methods for the chromatographic isolation of inclusion bodies directly from crude Escherichia coli homogenates and for the refolding of denatured protein are presented. The traditional method of differential centrifugation for the isolation of purified inclusion bodies is replaced by a single gel-filtration step. The principle is that the exclusion limit of the gel particles is chosen such that only the inclusion bodies are excluded, i.e., all other components of the crude homogenate penetrate the gel under the conditions selected. In the novel column refolding process, a decreasing gradient of denaturant (urea or Gu-HCl), combined with an increasing pH gradient, is introduced into a gel-filtration column packed with a gel medium that has an exclusion limit lower than the molecular mass of the protein to be refolded. A limited sample volume of the protein, dissolved in the highest denaturant concentration at the lowest pH of the selected gradient combination, is applied to the column. During the course of elution, the zone of denatured protein moves down the column at a speed approximately threefold higher than that of the denaturant. This means that the protein sample will gradually pass through areas of increasingly lower denaturant concentrations and higher pH, which promotes refolding into the native conformation. The shape and slope of the gradients, as well as the flow rate, will influence the refolding rate and can be adjusted for different protein samples. The principle is illustrated using a denatured recombinant scFv fusion protein obtained from E. coli inclusion bodies.

  2. Arenavirus Coinfections Are Common in Snakes with Boid Inclusion Body Disease

    PubMed Central

    Salmenperä, P.; Sironen, T.; Hetzel, U.; Korzyukov, Y.; Kipar, A.; Vapalahti, O.

    2015-01-01

    Recently, novel arenaviruses were found in snakes with boid inclusion body disease (BIBD); these form the new genus Reptarenavirus within the family Arenaviridae. We used next-generation sequencing and de novo sequence assembly to investigate reptarenavirus isolates from our previous study. Four of the six isolates and all of the samples from snakes with BIBD contained at least two reptarenavirus species. The viruses sequenced comprise four novel reptarenavirus species and a representative of a new arenavirus genus. PMID:26041290

  3. Cellular inclusion bodies of mutant huntingtin exon 1 obscure small fibrillar aggregate species.

    PubMed

    Sahl, Steffen J; Weiss, Lucien E; Duim, Whitney C; Frydman, Judith; Moerner, W E

    2012-01-01

    The identities of toxic aggregate species in Huntington's disease pathogenesis remain ambiguous. While polyQ-expanded huntingtin (Htt) is known to accumulate in compact inclusion bodies inside neurons, this is widely thought to be a protective coping response that sequesters misfolded conformations or aggregated states of the mutated protein. To define the spatial distributions of fluorescently-labeled Htt-exon1 species in the cell model PC12m, we employed highly sensitive single-molecule super-resolution fluorescence imaging. In addition to inclusion bodies and the diffuse pool of monomers and oligomers, fibrillar aggregates -100 nm in diameter and up to -1-2 µm in length were observed for pathogenic polyQ tracts (46 and 97 repeats) after targeted photo-bleaching of the inclusion bodies. These short structures bear a striking resemblance to fibers described in vitro. Definition of the diverse Htt structures in cells will provide an avenue to link the impact of therapeutic agents to aggregate populations and morphologies.

  4. A simple strategy for the purification of native recombinant full-length human RPL10 protein from inclusion bodies.

    PubMed

    Pereira, Larissa M; Silva, Luana R; Alves, Joseane F; Marin, Nélida; Silva, Flavio Sousa; Morganti, Ligia; Silva, Ismael D C G; Affonso, Regina

    2014-09-01

    The L10 ribosomal protein (RPL10) plays a role in the binding of the 60 S and 40 S ribosomal subunits and in mRNA translation. The evidence indicates that RPL10 also has multiple extra-ribosomal functions, including tumor suppression. Recently, the presence of RPL10 in prostate and ovarian cancers was evaluated, and it was demonstrated to be associated with autistic disorders and premature ovarian failure. In the present work, we successfully cloned and expressed full-length human RPL10 (hRPL10) protein and isolated inclusion bodies containing this protein that had formed under mild growth conditions. The culture produced 376mg of hRPL10 protein per liter of induced bacterial culture, of which 102.4mg was present in the soluble fraction, and 25.6mg was recovered at approximately 94% purity. These results were obtained using a two-step process of non-denaturing protein extraction from pelleted inclusion bodies. We studied the characteristics of this protein using circular dichroism spectroscopy and by monitoring the changes induced by the presence or absence of zinc ions using fluorescence spectrometry. The results demonstrated that the protein obtained using these non-conventional methods retained its secondary and tertiary structure. The conformational changes induced by the incorporation of zinc suggested that this protein could interact with Jun or the SH3 domain of c-yes. The results suggested that the strategy used to obtain hRPL10 is simple and could be applied to obtaining other proteins that are susceptible to degradation. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Columbid herpesvirus-1 in two Cooper's hawks (Accipiter cooperii) with fatal inclusion body disease.

    PubMed

    Pinkerton, Marie E; Wellehan, James F X; Johnson, April J; Childress, April L; Fitzgerald, Scott D; Kinsel, Michael J

    2008-07-01

    We report two separate naturally occurring cases of fatal herpesviral disease in Cooper's Hawks (Accipiter cooperii). Gross lesions included splenomegaly and hepatomegaly, with diffuse pale mottling or scattered small white foci. Histologic lesions included splenic and hepatic necrosis associated with eosinophilic intranuclear inclusion bodies characteristic of herpesvirus. In one case, necrosis and inclusions were also noted in bone marrow, thymus, bursa of Fabricius, thyroid gland, parathyroid gland, ceca, and the enteric system. Transmission electron microscopy demonstrated viral particles typical of herpesvirus within hepatocyte nuclei and budding from the nuclear membrane. Herpesviral DNA was amplified via polymerase chain reaction (PCR) of paraffin-embedded liver and spleen, and sequence data were consistent with columbid herpesvirus-1, an alphaherpesvirus of Rock Pigeons (Columba livia). PCR results provide evidence that this disease is transmitted to raptors via Rock Pigeons, most likely through ingestion of Rock Pigeons as prey.

  6. Singular path-independent energy integrals for elastic bodies with thin elastic inclusions

    NASA Astrophysics Data System (ADS)

    Shcherbakov, V. V.

    2016-06-01

    An equilibrium problem for a two-dimensional homogeneous linear elastic body containing a thin elastic inclusion and an interfacial crack is considered. The thin inclusion is modeled within the framework of Euler-Bernoulli beam theory. An explicit formula for the first derivative of the energy functional with respect to the crack perturbation along the interface is presented. It is shown that the formulas for the derivative associated with translation and self-similar expansion of the crack are represented as path-independent integrals along smooth contour surrounding one or both crack tips. These path-independent integrals consist of regular and singular terms and are analogs of the well-known Eshelby-Cherepanov-Rice J-integral and Knowles-Sternberg M-integral.

  7. Actin Recruitment to the Chlamydia Inclusion Is Spatiotemporally Regulated by a Mechanism That Requires Host and Bacterial Factors

    PubMed Central

    Chin, Elizabeth; Kirker, Kelly; Zuck, Meghan; James, Garth; Hybiske, Kevin

    2012-01-01

    The ability to exit host cells at the end of their developmental growth is a critical step for the intracellular bacterium Chlamydia. One exit strategy, extrusion, is mediated by host signaling pathways involved with actin polymerization. Here, we show that actin is recruited to the chlamydial inclusion as a late event, occurring after 20 hours post-infection (hpi) and only within a subpopulation of cells. This event increases significantly in prevalence and extent from 20 to 68 hpi, and actin coats strongly correlated with extrusions. In contrast to what has been reported for other intracellular pathogens, actin nucleation on Chlamydia inclusions did not ‘flash’, but rather exhibited moderate depolymerization dynamics. By using small molecule agents to selectively disrupt host signaling pathways involved with actin nucleation, modulate actin polymerization dynamics and also to disable the synthesis and secretion of chlamydial proteins, we further show that host and bacterial proteins are required for actin coat formation. Transient disruption of either host or bacterial signaling pathways resulted in rapid loss of coats in all infected cells and a reduction in extrusion formation. Inhibition of Chlamydia type III secretion also resulted in rapid loss of actin association on inclusions, thus implicating chlamydial effector proteins(s) as being central factors for engaging with host actin nucleating factors, such as formins. In conclusion, our data illuminate the host and bacterial driven process by which a dense actin matrix is dynamically nucleated and maintained on the Chlamydia inclusion. This late stage event is not ubiquitous for all infected cells in a population, and escalates in prevalence and extent throughout the developmental cycle of Chlamydia, culminating with their exit from the host cell by extrusion. The initiation of actin recruitment by Chlamydia appears to be novel, and may serve as an upstream determinant of the extrusion mechanism. PMID

  8. Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars

    PubMed Central

    Kminek, Gerhard; Bada, Jeffrey L.; Pogliano, Kit; Ward, John F.

    2014-01-01

    Kminek, G., Bada, J. L., Pogliano, K. and Ward, J. F. Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars. Radiat. Res. 159, 722–729 (2003). When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed. We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B. subtilis and S. marismortui spores in solution as well as for dry spores of B. subtilis and B. thuringiensis. S. marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old (Vreeland et al., Nature 407, 897–900, 2000). B. thuringiensis is a soil bacterium that is genetically similar to the human pathogens B. anthracis and B. cereus (Helgason et al., Appl. Environ. Microbiol. 66, 2627–2630, 2000). To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time. Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods. In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the uppermost meter of Mars. PMID:12751954

  9. Isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body disease.

    PubMed

    Hetzel, Udo; Sironen, Tarja; Laurinmäki, Pasi; Liljeroos, Lassi; Patjas, Aino; Henttonen, Heikki; Vaheri, Antti; Artelt, Annette; Kipar, Anja; Butcher, Sarah J; Vapalahti, Olli; Hepojoki, Jussi

    2013-10-01

    Boid inclusion body disease (BIBD) is a progressive, usually fatal disease of constrictor snakes, characterized by cytoplasmic inclusion bodies (IB) in a wide range of cell types. To identify the causative agent of the disease, we established cell cultures from BIBD-positive and -negative boa constrictors. The IB phenotype was maintained in cultured cells of affected animals, and supernatants from these cultures caused the phenotype in cultures originating from BIBD-negative snakes. Viruses were purified from the supernatants by ultracentrifugation and subsequently identified as arenaviruses. Purified virus also induced the IB phenotype in naive cells, which fulfilled Koch's postulates in vitro. One isolate, tentatively designated University of Helsinki virus (UHV), was studied in depth. Sequencing confirmed that UHV is a novel arenavirus species that is distinct from other known arenaviruses including those recently identified in snakes with BIBD. The morphology of UHV was established by cryoelectron tomography and subtomographic averaging, revealing the trimeric arenavirus spike structure at 3.2-nm resolution. Immunofluorescence, immunohistochemistry, and immunoblotting with a polyclonal rabbit antiserum against UHV and reverse transcription-PCR (RT-PCR) revealed the presence of genetically diverse arenaviruses in a large cohort of snakes with BIBD, confirming the causative role of arenaviruses. Some snakes were also found to carry arenavirus antibodies. Furthermore, mammalian cells (Vero E6) were productively infected with UHV, demonstrating the potential of arenaviruses to cross species barriers. In conclusion, we propose the newly identified lineage of arenaviruses associated with BIBD as a novel taxonomic entity, boid inclusion body disease-associated arenaviruses (BIBDAV), in the family Arenaviridae.

  10. Influence of production process design on inclusion bodies protein: the case of an Antarctic flavohemoglobin

    PubMed Central

    2010-01-01

    Background Protein over-production in Escherichia coli often results in formation of inclusion bodies (IBs). Some recent reports have shown that the aggregation into IBs does not necessarily mean that the target protein is inactivated and that IBs may contain a high proportion of correctly folded protein. This proportion is variable depending on the protein itself, the genetic background of the producing cells and the expression temperature. In this paper we have evaluated the influence of other production process parameters on the quality of an inclusion bodies protein. Results The present paper describes the recombinant production in Escherichia coli of the flavohemoglobin from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125. Flavohemoglobins are multidomain proteins requiring FAD and heme cofactors. The production was carried out in several different experimental setups differing in bioreactor geometry, oxygen supply and the presence of a nitrosating compound. In all production processes, the recombinant protein accumulates in IBs, from which it was solubilized in non-denaturing conditions. Comparing structural properties of the solubilized flavohemoglobins, i.e. deriving from the different process designs, our data demonstrated that the protein preparations differ significantly in the presence of cofactors (heme and FAD) and as far as their secondary and tertiary structure content is concerned. Conclusions Data reported in this paper demonstrate that other production process parameters, besides growth temperature, can influence the structure of a recombinant product that accumulates in IBs. To the best of our knowledge, this is the first reported example in which the structural properties of a protein solubilized from inclusion bodies have been correlated to the production process design. PMID:20334669

  11. Isolation, Identification, and Characterization of Novel Arenaviruses, the Etiological Agents of Boid Inclusion Body Disease

    PubMed Central

    Hetzel, Udo; Sironen, Tarja; Laurinmäki, Pasi; Liljeroos, Lassi; Patjas, Aino; Henttonen, Heikki; Vaheri, Antti; Artelt, Annette; Kipar, Anja; Butcher, Sarah J.; Vapalahti, Olli

    2013-01-01

    Boid inclusion body disease (BIBD) is a progressive, usually fatal disease of constrictor snakes, characterized by cytoplasmic inclusion bodies (IB) in a wide range of cell types. To identify the causative agent of the disease, we established cell cultures from BIBD-positive and -negative boa constrictors. The IB phenotype was maintained in cultured cells of affected animals, and supernatants from these cultures caused the phenotype in cultures originating from BIBD-negative snakes. Viruses were purified from the supernatants by ultracentrifugation and subsequently identified as arenaviruses. Purified virus also induced the IB phenotype in naive cells, which fulfilled Koch's postulates in vitro. One isolate, tentatively designated University of Helsinki virus (UHV), was studied in depth. Sequencing confirmed that UHV is a novel arenavirus species that is distinct from other known arenaviruses including those recently identified in snakes with BIBD. The morphology of UHV was established by cryoelectron tomography and subtomographic averaging, revealing the trimeric arenavirus spike structure at 3.2-nm resolution. Immunofluorescence, immunohistochemistry, and immunoblotting with a polyclonal rabbit antiserum against UHV and reverse transcription-PCR (RT-PCR) revealed the presence of genetically diverse arenaviruses in a large cohort of snakes with BIBD, confirming the causative role of arenaviruses. Some snakes were also found to carry arenavirus antibodies. Furthermore, mammalian cells (Vero E6) were productively infected with UHV, demonstrating the potential of arenaviruses to cross species barriers. In conclusion, we propose the newly identified lineage of arenaviruses associated with BIBD as a novel taxonomic entity, boid inclusion body disease-associated arenaviruses (BIBDAV), in the family Arenaviridae. PMID:23926354

  12. Fusion tags and chaperone co-expression modulate both the solubility and the inclusion body features of the recombinant CLIPB14 serine protease.

    PubMed

    Schrödel, Andrea; Volz, Jennifer; de Marco, Ario

    2005-10-17

    Chaperone co-expression and the fusion to different tags were used to modify the aggregation pattern of the putative serine protease CLIPB14 precipitated in Escherichia coli inclusion bodies. A set of common tags used in expression vectors has been selected, as well as two bacterial strains over-expressing the chaperones GroELS and ibpA/B, respectively. The presence of the fused tags resulted in an improved solubility of CLIPB14 but also in a higher presence of contaminants in the inclusion bodies, while chaperone co-expression promoted the binding of all the chaperone machinery involved into the disaggregation to the CLIPB14. Furthermore, each tag influenced in a specific manner the re-aggregation of the denatured CLIPB14 constructs during urea dilution and the preliminary trials indicated that the CLIPB14 fusions with higher homogeneity and lower re-aggregation rate were the optimal candidates for refolding assays. In conclusion, it is possible to tune the quality of the inclusion bodies by choosing the suitable combination of tag and chaperone co-expression that minimize the non-productive side reactions during refolding.

  13. Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars

    NASA Technical Reports Server (NTRS)

    Kminek, Gerhard; Bada, Jeffrey L.; Pogliano, Kit; Ward, John F.

    2003-01-01

    When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed. We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B. subtilis and s. marismortui spores in solution as well as for dry spores of B. subtilis and B. thuringiensis. S. marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old, B. thuringiensis is a soil bacterium that is genetically similar to the human pathogens B. anthracis and B. cereus. To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time. Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods. In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the upper-most meter of Mars.

  14. Shear-stress-mediated refolding of proteins from aggregates and inclusion bodies.

    PubMed

    Yuan, Tom Z; Ormonde, Callum F G; Kudlacek, Stephan T; Kunche, Sameeran; Smith, Joshua N; Brown, William A; Pugliese, Kaitlin M; Olsen, Tivoli J; Iftikhar, Mariam; Raston, Colin L; Weiss, Gregory A

    2015-02-09

    Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer-wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin-1. Furthermore, the approach allowed refolding of a much larger protein, cAMP-dependent protein kinase A (PKA). The reported methods require only minutes, which is more than 100 times faster than conventional overnight dialysis. This rapid refolding technique could significantly shorten times, lower costs, and reduce waste streams associated with protein expression for a wide range of industrial and research applications.

  15. Processing of refractory meteorite inclusions (CAIs) in parent-body atmospheres

    NASA Technical Reports Server (NTRS)

    Podolak, Morris; Bunch, T. E.; Cassen, Pat; Reynolds, Ray T.; Chang, S.

    1990-01-01

    Ca-Al-rich inclusions (CAIs) in refractory meteorites are shown to have been subject to partial melting during a suitably high gas density/small scale height regime arising during gasdynamic deceleration in a temporary atmosphere around an accreting parent body. The presence of dust in such an atmosphere would have increased the pressure gradient with height, lowering the boiloff rate, and permitting dust particles to become trapped in the partially melted material. CAIs may therefore be studied as probes of a primitive atmosphere.

  16. Ubiquilin/Dsk2 promotes inclusion body formation and vacuole (lysosome)-mediated disposal of mutated huntingtin.

    PubMed

    Chuang, Kun-Han; Liang, Fengshan; Higgins, Ryan; Wang, Yanchang

    2016-07-01

    Ubiquilin proteins contain a ubiquitin-like domain (UBL) and ubiquitin-associated domain(s) that interact with the proteasome and ubiquitinated substrates, respectively. Previous work established the link between ubiquilin mutations and neurodegenerative diseases, but the function of ubiquilin proteins remains elusive. Here we used a misfolded huntingtin exon I containing a 103-polyglutamine expansion (Htt103QP) as a model substrate for the functional study of ubiquilin proteins. We found that yeast ubiquilin mutant (dsk2Δ) is sensitive to Htt103QP overexpression and has a defect in the formation of Htt103QP inclusion bodies. Our evidence further suggests that the UBL domain of Dsk2 is critical for inclusion body formation. Of interest, Dsk2 is dispensable for Htt103QP degradation when Htt103QP is induced for a short time before noticeable inclusion body formation. However, when the inclusion body forms after a long Htt103QP induction, Dsk2 is required for efficient Htt103QP clearance, as well as for autophagy-dependent delivery of Htt103QP into vacuoles (lysosomes). Therefore our data indicate that Dsk2 facilitates vacuole-mediated clearance of misfolded proteins by promoting inclusion body formation. Of importance, the defect of inclusion body formation in dsk2 mutants can be rescued by human ubiquilin 1 or 2, suggesting functional conservation of ubiquilin proteins.

  17. Valosin-containing protein and the pathogenesis of frontotemporal dementia associated with inclusion body myopathy.

    PubMed

    Guinto, Jake B; Ritson, Gillian P; Taylor, J Paul; Forman, Mark S

    2007-07-01

    Frontotemporal dementia with inclusion body myopathy and Paget's disease of bone (IBMPFD) is a rare, autosomal dominant disorder caused by mutations in the gene valosin-containing protein (VCP). The CNS pathology is characterized by a novel pattern of ubiquitin pathology distinct from sporadic and familial frontotemporal lobar degeneration with ubiquitin-positive inclusions without VCP mutations. Yet, the ubiquitin-positive inclusions in IBMPFD also stain for TAR DNA binding protein, a feature that links this rare disease with the pathology associated with the majority of sporadic FTD as well as disease resulting from different genetic alterations. VCP, a member of the AAA-ATPase gene family, associates with a plethora of protein adaptors to perform a variety of cellular processes including Golgi assembly/disassembly and regulation of the ubiquitin-proteasome system. However, the mechanism whereby mutations in VCP lead to CNS, muscle, and bone disease is largely unknown. In this report, we review current literature on IBMPFD, focusing on the pathology of the disease and the biology of VCP with respect to IBMPFD.

  18. Energy release rates for interfacial cracks in elastic bodies with thin semirigid inclusions

    NASA Astrophysics Data System (ADS)

    Shcherbakov, Viktor

    2017-02-01

    In this paper, we present some rigorous results for an equilibrium problem arising from the study of fiber-reinforced composites. We consider a two-dimensional homogeneous anisotropic linear elastic body containing a thin semirigid inclusion. The semirigid inclusion is an anisotropic thin structure that stretches along one direction and moves like a rigid body possessing both rotational and translatory motion along the perpendicular direction. A pre-existing interfacial crack is subject to nonlinear conditions that do not allow the opposite crack faces to penetrate each other. We focus on a variational approach to modelling the physical phenomenon of equilibrium and to demonstrate that the energy release rate associated with perturbation of the crack along the interface is well defined. A higher regularity result for the displacement field is formulated and proved. Then, taking into account this result, we deduce representations for the energy release rates associated with local translation and self-similar expansion of the crack by means of path-independent energy integrals along smooth contour surrounding one or both crack tips. Finally, some relations between the integrals obtained are discussed briefly.

  19. Longitudinal observational study of sporadic inclusion body myositis: implications for clinical trials.

    PubMed

    Cortese, A; Machado, P; Morrow, J; Dewar, L; Hiscock, A; Miller, A; Brady, S; Hilton-Jones, D; Parton, M; Hanna, M G

    2013-05-01

    Sporadic inclusion body myositis (IBM) is the most common acquired myopathy occurring in adults aged over 50 years. The aim of the study was to assess prospectively the clinical features and functional impact of sporadic inclusion body myositis (IBM). Clinical data, manual muscle testing (MMT), quantitative muscle testing (QMT) of quadriceps muscle and IBM functional rating scale (IBM-FRS) were collected according to a standardised protocol at baseline (n=51) and one-year follow-up (n=23). MMT, quadriceps QMT and IBM-FRS significantly declined after one year (by 5.2%, 27.9%, and 13.8%, respectively). QMT of the quadriceps muscle and IBM-FRS were the most sensitive measures of disease progression. After a median time of seven years of disease duration, 63% of patients had lost independent walking. Disease onset after 55 years of age, but not sex or treatment, is predictive of a shorter time to requirement of a walking stick. We detected no differences in disease presentation and progression between clinically and pathologically defined IBM patients. The study provides evidence that quadriceps QMT and IBM-FRS could prove helpful as outcome measures in future therapeutic trials in IBM. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Periplasmically-exported lupanine hydroxylase undergoes transition from soluble to functional inclusion bodies in Escherichia coli.

    PubMed

    Stampolidis, Pavlos; Kaderbhai, Naheed N; Kaderbhai, Mustak A

    2009-04-01

    Pseudomonas lupanine hydroxylase is a periplasmic-localised, two domain quinocytochrome c enzyme. It requires numerous post-translocation modifications involving signal peptide processing, disulphide bridge formation and, heme linkage in the carboxy-terminal cytochrome c domain to eventually generate a Ca(2+)-bound quino-c hemoprotein that hydroxylates the plant alkaloid, lupanine. An exported, functional recombinant enzyme was generated in Escherichia coli by co-expression with cytochrome c maturation factors. Increased growth temperatures ranging from 18 to 30 degrees C gradually raised the enzyme production to a peak together with its concomitant aggregation as red solid particles, readily activatable in a fully functional form by mild chaotropic treatment. Here, we demonstrate that the exported lupanine hydroxylase undergoes a cascade transition from a soluble to "non-classical" inclusion body form when build-up in the periplasm exceeded a basal threshold concentration. These periplasmic aggregates were distinct from the non-secreted, signal-sequenceless counterpart that occurred as misfolded, non-functional concatamers in the form of classical inclusion bodies. We discuss our findings in the light of current models of how aggregation of lupanine hydroxylase arises in the periplasmic space.

  1. [A rare complication of dysarthria in a patient with inclusion body myositis: a case report].

    PubMed

    Isayama, Reina; Shiga, Kensuke; Tanaka, Eijirou; Itsukage, Masahiro; Tokuda, Takahiko; Nakagawa, Masanori

    2010-10-01

    We reported a 71-year-old man with inclusion body myositis with clinically overt dysarthria. He had been suffering from gradual progression of weakness in the hand muscles and lower extremities as well as dysarthria three years before admission. His neurological examination revealed muscle atrophy and weakness in the tongue, the forearm flexors, and the vastus medialis muscles. He had dysarthria to a moderate degree, while he denied any dysphasia. A biopsy from vastus lateralis muscle showed variation in fiber size, infiltration of mononucleated cells, and numerous fibers with rimmed vacuoles, leading to the diagnosis of definite inclusion body myositis. The EMG findings of the tongue demonstrated low amplitude motor unit potentials during voluntary contraction, abundant fibrillation potentials at rest, and preserved interference pattern at maximal contraction, implying myogenic changes. We surmised the dysarthria seen in this patient, an atypical clinical feature in IBM, presumably caused by muscle involvement in the tongue muscle. Dysphasia is common symptom in IBM patient and has been much reported previously. But dysarthria in IBM patient has not been aware, for this reason this report should be the rare case.

  2. The 2-min walk test is sufficient for evaluating walking abilities in sporadic inclusion body myositis.

    PubMed

    Alfano, L N; Lowes, L P; Dvorchik, I; Yin, H; Maus, E G; Flanigan, K M; Mendell, J R

    2014-03-01

    Sporadic inclusion body myositis causes progressive functional loss due to declining muscle strength. Although the underlying cause is unknown, clinical trials are underway to improve strength and function. Selection of appropriate outcome measures is critical for the success of these trials. The 6-min walk test has been the de facto standard for assessing function in neuromuscular disease; however, the optimal walking test has not been determined in this disease. In this study, 67 individuals with sporadic inclusion body myositis completed a battery of quantitative strength and functional tests including timed walking tests, patient-reported outcomes, and other tasks. The 2-min and 6-min walk tests are highly correlated to each other (r=0.97, p<0.001) and to all lower extremity strength, patient-reported, and functional measures in this population. All subjects completed the 2-min walk test, but 7% of subjects were unable to walk the full 6-min of the 6-min walk test due to fatigue. The 2-min walk test demonstrates similar correlation to all outcomes compared to the 6-min walk test, is less fatiguing and better tolerated. Results suggest that the 2-min walk test is a better alternative to tests of longer duration. Further research is needed to determine longitudinal changes on this outcome. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Rapid refolding and polishing of single-chain antibodies from Escherichia coli inclusion bodies.

    PubMed

    Sinacola, Jessica R; Robinson, Anne S

    2002-11-01

    An inexpensive and fast-folding strategy for single-chain antibody (scFv) recovered from Escherichia coli inclusion bodies has been developed. Two anti-fluorescein single-chain antibodies, 4-4-20 and 4M5.3, were expressed as inclusion bodies in E. coli for use in a comparative refolding study. Active protein yields as well as degree of aggregation were evaluated for scFv produced by stepwise dialysis, redox dialysis, and a newly developed controlled dilution and filtration strategy. Although all three methods produced active protein for both 4-4-20 and 4M5.3, the extent of aggregation differed greatly among the methods. For 4-4-20, the controlled dilution and filtration strategy reduced aggregation by half, allowed batch processing times of 8h (an 18-fold improvement), and significantly reduced denaturant usage while increasing active yields by 150%. A hydroxyapatite resin polishing step was used to remove completely the aggregate species and inactive monomeric scFv from active scFv.

  4. Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli.

    PubMed

    Hwang, Peter M; Pan, Jonathan S; Sykes, Brian D

    2014-01-21

    Today, proteins are typically overexpressed using solubility-enhancing fusion tags that allow for affinity chromatographic purification and subsequent removal by site-specific protease cleavage. In this review, we present an alternative approach to protein production using fusion partners specifically designed to accumulate in insoluble inclusion bodies. The strategy is appropriate for the mass production of short peptides, intrinsically disordered proteins, and proteins that can be efficiently refolded in vitro. There are many fusion protein systems now available for insoluble expression: TrpLE, ketosteroid isomerase, PurF, and PagP, for example. The ideal fusion partner is effective at directing a wide variety of target proteins into inclusion bodies, accumulates in large quantities in a highly pure form, and is readily solubilized and purified in commonly used denaturants. Fusion partner removal under denaturing conditions is biochemically challenging, requiring harsh conditions (e.g., cyanogen bromide in 70% formic acid) that can result in unwanted protein modifications. Recent advances in metal ion-catalyzed peptide bond cleavage allow for more mild conditions, and some methods involving nickel or palladium will likely soon appear in more biological applications. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Expression, purification and renaturation of truncated human integrin β1 from inclusion bodies of Escherichia coli.

    PubMed

    Shi, Tonglin; Zhang, Lichao; Li, Zhuoyu; Newton, Ian P; Zhang, Quanbin

    2015-03-01

    Integrins are a family of transmembrane receptors and among their members, integrin β1 is one of the best known. It plays a very important role in cell adhesion/migration and in cancer metastasis. Preparation of integrin β1 has a great potential value especially in studies focused on its function. To this end, recombinant plasmids were constructed containing DNA segments representing 454 amino acids of the N-terminal of integrin β1. The recombinant plasmid was transformed into Escherichiacoli BL21 (DE3) cells and after induction by isopropyl-β-D-thiogalactopyranoside (IPTG), the recombinant protein (molecular weight: 53 kD) was expressed, mainly in the form of inclusion bodies. The inclusion bodies were solubilized by 8M urea solution then purified by nickel affinity chromatography. The recombinant protein was renatured by a stepwise dialysis and finally dissolved in phosphate buffered saline. The final yield was approximately 5.4 mg/L of culture and the purity of the renatured recombinant protein was greater than 98% as assessed by SDS-PAGE. The integrity of the protein was shown by Western blot using monoclonal antibodies against his-tag and integrin β1. Its secondary structure was verified as native by circular dichroism spectra and the bioactivity of the recombinant protein was displayed through the conformation switch under Mn(2+) stimulation. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Pulmonary delivery of tea tree oil-β-cyclodextrin inclusion complexes for the treatment of fungal and bacterial pneumonia.

    PubMed

    Li, Miao; Zhu, Lifei; Zhang, Tongtong; Liu, Boming; Du, Lina; Jin, Yiguang

    2017-08-15

    Bacterial pneumonia is a common cause of death worldwide. Tea tree oil (TTO) is a potent antimicrobial natural product, which is formulated in dry powder inhalers (DPIs) for the treatment of fungal and bacterial pneumonia. Tea tree oil-β-cyclodextrin inclusion complexes (TTO-β-CD) were prepared and characterized. Aerodynamic properties of TTO-β-CD powders were measured. The rat models of fungal (Candida albicans) and bacterial (Acinetobacter baumannii) pneumonia were prepared. Saline, TTO, TTO-β-CD and the positive drug (fluconazole or penicillin) were directly delivered to the rat lungs. Pathological and biological assays were conducted. Tea tree oil-β-CD powders had an appropriate aerodynamic diameter of 5.59 μm and the fine particle fraction of 51.22%, suitable for pulmonary delivery. TTO-β-CD showed higher and similar antipneumonic effects on the rat models than fluconazole and penicillin, respectively. The effects of TTO-β-CD were higher than TTO alone. The antipneumonic mechanisms involved blocking the recruitment of leucocytes and neutrophils, eliminating the microbes, downregulating pro-inflammatory cytokines (including tumour necrosis factor-α, interleukin-1β and interleukin-6), suppressing cyclooxygenase 2 expression, and further reducing lung injury. Inhaled TTO-β-CD powders have the advantages of portability, high stability, self-administration, high lung deposition and good antipneumonic effect. It is a promising DPI for the treatment of fungal and bacterial pneumonia. © 2017 Royal Pharmaceutical Society.

  7. Plumage bacterial assemblages in a breeding wild passerine: relationships with ecological factors and body condition.

    PubMed

    Saag, Pauli; Tilgar, Vallo; Mänd, Raivo; Kilgas, Priit; Mägi, Marko

    2011-05-01

    Microorganisms have been shown to play an important role in shaping the life histories of animals, and it has recently been suggested that feather-degrading bacteria influence the trade-off between parental effort and self-preening behavior in birds. We studied a wild breeding population of great tits (Parus major) to explore habitat-, seasonal-, and sex-related variation in feather-degrading and free-living bacteria inhabiting the birds' yellow ventral feathers and to investigate associations with body condition. The density and species richness of bacterial assemblages was studied using flow cytometry and ribosomal intergenic spacer analysis. The density of studied bacteria declined between the nest-building period and the first brood. The number of bacterial phylotypes per bird was higher in coniferous habitat, while bacterial densities were higher in deciduous habitat. Free-living bacterial density was positively correlated with female mass; conversely, there was a negative correlation between attached bacterial density and female mass during the period of peak reproductive effort. Bacterial species richness was sex dependent, with more diverse bacterial assemblages present on males than females. Thus, this study revealed that bacterial assemblages on the feathers of breeding birds are affected both by life history and ecological factors and are related to body condition.

  8. Detection of arenavirus in a peripheral odontogenic fibromyxoma in a red tail boa (Boa constrictor constrictor) with inclusion body disease.

    PubMed

    Hellebuyck, Tom; Pasmans, Frank; Ducatelle, Richard; Saey, Veronique; Martel, An

    2015-03-01

    A captive bred red tail boa (Boa constrictor constrictor) was presented with a large intraoral mass originating from the buccal gingiva, attached to the right dentary teeth row. Based on the clinical features and histological examination, the diagnosis of a peripheral odontogenic fibromyxoma was made. Sections of liver biopsies and circulating lymphocytes contained relatively few eosinophilic intracytoplasmic inclusion bodies, indistinguishable from those observed in inclusion body disease-affected snakes. Inclusion bodies were not observed in cells comprising the neoplastic mass. Using reverse transcription polymerase chain reaction (RT-PCR), arenavirus was detected in the neoplastic tissue. Two years after surgical removal of the mass, recurrence of the neoplastic lesion was observed. Numerous large inclusion body disease inclusions were abundantly present in the neoplastic cells of the recurrent fibromyxoma. Sections of liver biopsies and circulating lymphocytes contained relatively few intracytoplasmic inclusions. The RT-PCR revealed the presence of arenavirus in blood, a liver biopsy, and neoplastic tissue. The present case describes the co-occurrence of an arenavirus infection and an odontogenic fibromyxoma in a red tail boa.

  9. Potential harmonics expansion method for trapped interacting bosons: Inclusion of two-body correlation

    SciTech Connect

    Das, T.K.; Chakrabarti, B.

    2004-12-01

    We study a system of A identical interacting bosons trapped by an external field by solving ab initio the many-body Schroedinger equation. A complete solution by using, for example, the traditional hyperspherical harmonics (HH) basis develops serious practical problems due to the large degeneracy of HH basis. Symmetrization of the wave function, calculation of the matrix elements, etc., become an immensely formidable task as A increases. Instead of the HH basis, here we use a new basis, called 'potential harmonics' (PH) basis, which is a subset of HH basis. We assume that the contribution to the orbital and grand orbital [in 3(A-1)-dimensional space of the reduced motion] quantum numbers comes only from the interacting pair. This implies inclusion of two-body correlations only and disregard of all higher-body correlations. Such an assumption is ideally suited for the Bose-Einstein condensate (BEC), which is required, for experimental realization of BEC, to be extremely dilute. Hence three and higher-body collisions are almost totally absent. Unlike the (3A-4) hyperspherical variables in HH basis, the PH basis involves only three active variables, corresponding to three quantum numbers--the orbital l, azimuthal m, and the grand orbital 2K+l quantum numbers for any arbitrary A. It drastically reduces the number of coupled equations and calculation of the potential matrix becomes tremendously simplified, as it involves integrals over only three variables for any A. One can easily incorporate realistic atom-atom interactions in a straightforward manner. We study the ground and excited state properties of the condensate for both attractive and repulsive interactions for various particle number. The ground state properties are compared with those calculated from the Gross-Pitaevskii equation. We notice that our many-body results converge towards the mean field results as the particle number increases.

  10. Inclusion bodies enriched for p62 and polyubiquitinated proteins in macrophages protect against atherosclerosis.

    PubMed

    Sergin, Ismail; Bhattacharya, Somashubhra; Emanuel, Roy; Esen, Emel; Stokes, Carl J; Evans, Trent D; Arif, Batool; Curci, John A; Razani, Babak

    2016-01-05

    Autophagy is a catabolic cellular mechanism that degrades dysfunctional proteins and organelles. Atherosclerotic plaque formation is enhanced in mice with macrophages deficient for the critical autophagy protein ATG5. We showed that exposure of macrophages to lipids that promote atherosclerosis increased the abundance of the autophagy chaperone p62 and that p62 colocalized with polyubiquitinated proteins in cytoplasmic inclusions, which are characterized by insoluble protein aggregates. ATG5-null macrophages developed further p62 accumulation at the sites of large cytoplasmic ubiquitin-positive inclusion bodies. Aortas from atherosclerotic mice and plaques from human endarterectomy samples showed increased abundance of p62 and polyubiquitinated proteins that colocalized with plaque macrophages, suggesting that p62-enriched protein aggregates were characteristic of atherosclerosis. The formation of the cytoplasmic inclusions depended on p62 because lipid-loaded p62-null macrophages accumulated polyubiquitinated proteins in a diffuse cytoplasmic pattern. Lipid-loaded p62-null macrophages also exhibited increased secretion of interleukin-1β (IL-1β) and had an increased tendency to undergo apoptosis, which depended on the p62 ubiquitin-binding domain and at least partly involved p62-mediated clearance of NLRP3 inflammasomes. Consistent with our in vitro observations, p62-deficient mice formed greater numbers of more complex atherosclerotic plaques, and p62 deficiency further increased atherosclerotic plaque burden in mice with a macrophage-specific ablation of ATG5. Together, these data suggested that sequestration of cytotoxic ubiquitinated proteins by p62 protects against atherogenesis, a condition in which the clearance of protein aggregates is disrupted. Copyright © 2016, American Association for the Advancement of Science.

  11. Characterization of inclusion bodies with cytoprotective properties formed by seipinopathy-linked mutant seipin.

    PubMed

    Ito, Daisuke; Yagi, Takuya; Ikawa, Masahito; Suzuki, Norihiro

    2012-02-01

    Gain-of-toxic mutations in the N-glycosylation motif of the seipin/BSCL2 gene (namely, the N88S and S90L mutations) cause autosomal dominant motor neuron diseases, termed 'seipinopathy'. Expressed mutant seipin is improperly folded and accumulates in the endoplasmic reticulum (ER), leading to an unfolded protein response (UPR). Furthermore, cells expressing mutant seipin contain unique cytoplasmic inclusion bodies (IB) that form via a different mechanism from that of ubiquitinated inclusions, or aggresomes. Whether the formation of these IB is pathogenic or protective in neurodegenerative diseases remains unclear. Here, we determined that mutant seipin IB are negative for two well-established ER markers, immunoglobulin-heavy-chain-binding protein and calnexin, indicating a distinct compartmentalization from the main ER, and that mutant seipin IB are formed via a mechanism that is independent of major UPR transducers and ER chaperons. Electron microscopy and coexpression study with variant α1-antitrypsin cDNA showed that seipin IB are compatible with unique cytoplasmic vesicles known as ER-derived protective organelles (ERPO). We also obtained evidence that seipin IB exhibit a cytoprotective property via the attenuation of ER stress. These findings suggest that ERPO, such as seipin IB, are a novel adaptation machinery against the accumulation of unfolded proteins in the ER.

  12. Mechanics of swimming of multi-body bacterial swarmers using non-labeled cell tracking algorithm

    NASA Astrophysics Data System (ADS)

    Phuyal, Kiran; Kim, Min Jun

    2013-01-01

    To better understand the survival strategy of bacterial swarmers and the mechanical advantages offered by the linear chain (head-tail) attachment of the multiple bacterial bodies in an individual swarmer cell at low Reynolds number, a non-labeled cell tracking algorithm was used to quantify the mechanics of multi-body flagellated bacteria, Serratia marcescens, swimming in a motility buffer that originally exhibited the swarming motility. Swarming is a type of bacterial motility that is characterized by the collective coordinated motion of differentiated swarmer cells on a two-dimensional surface such as agar. In this study, the bacterial swarmers with multiple cell bodies (2, 3, and 4) were extracted from the swarm plate, and then tracked individually after resuspending in the motility medium. Their motion was investigated and compared with individual undifferentiated swimming bacterial cells. The swarmers when released into the motility buffer swam actively without tumbles. Their speeds, orientations, and the diffusive properties were studied by tracking the individual cell trajectories over a short distance in two-dimensional field when the cells are swimming at a constant depth in a bulk aqueous environment. At short time scales, the ballistic trajectory was dominant for both multi-body swarmers and undifferentiated cells.

  13. Research note: the isolation of a herpes virus from captive cranes with an inclusion body disease

    USGS Publications Warehouse

    Docherty, D.E.; Henning, D.J.

    1980-01-01

    A viral agent, identified as a herpesvirus and tentatively called 'inclusion body disease of cranes' (IBDC), was isolated from captive cranes involved in a die-off at the International Crane Foundation near Baraboo, Wisconsin. Preliminary animal susceptibility tests, based on experimental infections, suggested that White Pekin ducklings up to 17 days old and adult coots were susceptible to the IBDC virus whereas 16-day-old White Leghorn chicks and 64-day-old Muscovy ducks were not. No serum antibody to IBDC virus was detected in 95 wild sandhill cranes collected in Wisconsin or Indiana in 1976 and 1977. However, 9 of 11 captive cranes in the affected area at the ICF had antibody to this agent.

  14. Selective recovery of polyhydroxyalkanoate inclusion bodies from fermentation broth by dissolved-air flotation.

    PubMed

    van Hee, Pim; Elumbaring, Andrea C M R; van der Lans, Rob G J M; Van der Wielen, Luuk A M

    2006-05-15

    Selective dissolved-air flotation for the separation of medium-chain-length polyhydroxyalkanoate (PHA) inclusion bodies (IBs) from Pseudomonas putida cell debris is investigated. Measurements show that both P. putida cell debris and PHA IBs have an iso-electric point of approximately pH 3.5. Selective aggregation and as a result selective flotation of PHA IBs was observed near this pH. Qualitative prediction of the aggregation behaviour was possible on the basis of the Van der Waals, hydrophobic and electrostatic interactions. In some cases however, the stability of the suspension could not be explained with these forces alone. It was therefore suggested that additional interactions, such as steric/brush effects, play an important role in the aggregation process.

  15. A dysphagia study in patients with sporadic inclusion body myositis (s-IBM).

    PubMed

    Murata, Ken-Ya; Kouda, Ken; Tajima, Fumihiro; Kondo, Tomoyoshi

    2012-08-01

    The nature of the swallowing impairment in patients with sporadic inclusion body myositis (s-IBM) has not been well characterized. In this study, we examined ten consecutive s-IBM patients using videofluoroscopy (VF) and computed pharyngoesophageal manometry (CPM). The patients were divided into two groups: patients with complaint and without complaint of dysphagia. VF results indicated pharyngeal muscle propulsion (PP) at the hypopharyngeal and upper esophagus sphincter (UES) in all s-IBM patients. Patients without complaint of dysphagia showed a mild degree of PP, whereas a severe form of PP was observed in patients with complaint of dysphagia. CPM revealed that negative pressure during UES opening was not observed in the s-IBM patients with complaint of dysphagia. Incomplete opening and PP at the UES were observed in all s-IBM patients. These results indicate that the dysphagic processes occur subclinically in s-IBM patients who may not report swallowing impairments.

  16. A protocol to develop clinical guidelines for inclusion-body myositis.

    PubMed

    Jones, Katherine L; Sejersen, Thomas; Amato, Anthony A; Hilton-Jones, David; Schmidt, Jens; Wallace, Amanda C; Badrising, Umesh A; Rose, Michael R

    2016-04-01

    Inclusion-body myositis (IBM) is a late-onset idiopathic inflammatory myopathy associated with selective and progressive muscle weakness and atrophy. Current clinical management of IBM is largely supportive due to its uncertain etiology and lack of effective treatment. Establishing a consensus of opinion on questions relating to diagnosis and management of IBM is expected to help reduce inconsistencies in the care and resources allocated to those living with this condition. A protocol has been developed to produce best practice clinical guidelines for IBM based on a combination of published research and expert consensus. In this study we describe the proposed protocol for developing methods for producing robust and transparent clinical guidance on aspects of diagnosis, drug treatment, physical and practical management, respiration, nutrition and cardiac management, psychosocial management, and multidisciplinary care. © 2016 The Authors. Muscle & Nerve Published by Wiley Periodicals, Inc.

  17. Magnetic resonance imaging pattern recognition in sporadic inclusion-body myositis.

    PubMed

    Tasca, Giorgio; Monforte, Mauro; De Fino, Chiara; Kley, Rudolf A; Ricci, Enzo; Mirabella, Massimiliano

    2015-12-01

    In sporadic inclusion-body myositis (IBM), additional tools are needed to confirm the diagnosis, particularly in clinically atypical or pathologically unproven patients. The aims of this study were to define the pattern of muscle MRI in IBM and to assess its accuracy in differentiating IBM from other myopathies that overlap with it clinically or pathologically. Blind assessment was done on the scans of 17 definite IBM, 2 possible IBM, and 118 patients with other myopathies. The diagnostic accuracy to detect definite IBM was 95% for the typical pattern (with 100% specificity) and 97% for both typical and consistent patterns (with 97% specificity). Muscle MRI is an accurate tool for diagnostic work-up of suspected IBM patients and may be particularly helpful in patients with early disease or who lack the classical IBM pathology. © 2015 Wiley Periodicals, Inc.

  18. Hereditary inclusion-body myopathy: clues on pathogenesis and possible therapy.

    PubMed

    Broccolini, Aldobrando; Gidaro, Teresa; Morosetti, Roberta; Mirabella, Massimiliano

    2009-09-01

    Hereditary inclusion-body myopathy (h-IBM), or distal myopathy with rimmed vacuoles (DMRV), is an autosomal recessive disorder with onset in early adult life and a progressive course leading to severe disability. h-IBM/DMRV is due to mutations of a gene (GNE) that codes for a rate-limiting enzyme in the sialic acid biosynthetic pathway. Despite the identification of the causative gene defect, it has not been unambiguously clarified how GNE gene mutations impair muscle metabolism. Although numerous studies have indicated a key role of hyposialylation of glycoproteins in h-IBM/DMRV pathogenesis, others have demonstrated new and unpredicted functions of the GNE gene, outside the sialic acid biosynthetic pathway, that may also be relevant. This review illustrates the clinical and pathologic characteristics of h-IBM/DMRV and the main clues available to date concerning the possible pathogenic mechanisms and therapeutic perspectives of this disorder.

  19. Production strategies for active heme-containing peroxidases from E. coli inclusion bodies - a review.

    PubMed

    Eggenreich, Britta; Willim, Melissa; Wurm, David Johannes; Herwig, Christoph; Spadiut, Oliver

    2016-06-01

    Heme-containing peroxidases are frequently used in medical applications. However, these enzymes are still extracted from their native source, which leads to inadequate yields and a mixture of isoenzymes differing in glycosylation which limits subsequent enzyme applications. Thus, recombinant production of these enzymes in Escherichia coli is a reasonable alternative. Even though production yields are high, the product is frequently found as protein aggregates called inclusion bodies (IBs). These IBs have to be solubilized and laboriously refolded to obtain active enzyme. Unfortunately, refolding yields are still very low making the recombinant production of these enzymes in E. coli not competitive. Motivated by the high importance of that enzyme class, this review aims at providing a comprehensive summary of state-of-the-art strategies to obtain active peroxidases from IBs. Additionally, various refolding techniques, which have not yet been used for this enzyme class, are discussed to show alternative and potentially more efficient ways to obtain active peroxidases from E. coli.

  20. Size characterization of inclusion bodies by sedimentation field-flow fractionation

    PubMed Central

    Margreiter, Gerd; Messner, Paul; Caldwell, Karin D.; Bayer, Karl

    2015-01-01

    Sedimentation field-flow fractionation (sedFFF) was evaluated to characterize the size of Δ(4–23)TEM-β-lactamase inclusion bodies (IBs) overexpressed in fed-batch cultivations of Escherichia coli. Heterologous Δ(4–23)TEM-β-lactamase protein formed different sizes of IBs, depending upon the induction conditions. In the early phases of recombinant protein expression, induced with low concentrations of IPTG (isopropyl-β-d-thiogalactoside), IB masses were larger than expected and showed heterogeneous size distributions. During cultivation, IB sizes showed a Gaussian distribution and reached a broad range by the end of the fed-batch cultivations. The obtained result proved the aptitude of sedFFF to rapidly assess the size distribution of IBs in a culture. PMID:18760314

  1. Practical applications of hydrostatic pressure to refold proteins from inclusion bodies for NMR structural studies.

    PubMed

    Ogura, Kenji; Kobashigawa, Yoshihiro; Saio, Tomohide; Kumeta, Hiroyuki; Torikai, Shinnosuke; Inagaki, Fuyuhiko

    2013-06-01

    Recently, the hydrostatic pressure refolding method was reported as a practical tool for solubilizing and refolding proteins from inclusion bodies; however, there have been only a few applications for protein structural studies. Here, we report the successful applications of the hydrostatic pressure refolding method to refold proteins, including the MOE-2 tandem zinc-finger, the p62 PB1 domain, the GCN2 RWD domain, and the mTOR FRB domain. Moreover, the absence of aggregation and the correct folding of solubilized protein samples were evaluated with size exclusion chromatography and NMR experiments. The analyses of NMR spectra for MOE-2 tandem zinc-finger and GCN2 RWD further led to the determination of tertiary structures, which are consistent with those from soluble fractions. Overall, our results indicate that the hydrostatic pressure method is effective for preparing samples for NMR structural studies.

  2. Protein folding, misfolding, and aggregation. Formation of inclusion bodies and aggresomes.

    PubMed

    Markossian, K A; Kurganov, B I

    2004-09-01

    In this review the mechanisms of protein folding, misfolding, and aggregation as well as the mechanisms of cell defense against toxic protein aggregates are considered. Misfolded and aggregated proteins in cells are exposed to chaperone-mediated refolding and are degraded by proteasomes if refolding is impossible. Proteolysis-stable protein aggregates accumulate, forming inclusion bodies. In eucaryotic cells, protein aggregates form structures in the pericentrosomal area that have been termed "aggresomes". Formation of aggresomes in cells is a general cellular response to the presence of misfolded proteins when the degrading capacity of the cells is exceeded. The role of aggresomes in disturbance of the proteasomal system operation and in cellular death, particularly in the so-called "protein conformational diseases", is discussed.

  3. One-step extraction of functional recombinant aquaporin Z from inclusion bodies with optimal detergent.

    PubMed

    Wang, Lili; Zhou, Hu; Li, Zhengjun; Lim, Teck Kwang; Lim, Xin Shan; Lin, Qingsong

    2015-11-01

    Aquaporins are integral membrane channel proteins found in all kingdoms of life. The Escherichia coli aquaporin Z (AqpZ) has been shown to solely conduct water at high permeability. Functional AqpZ is generally purified from the membrane fraction. However, the quantity of the purified protein is limited. In this study, a new method is developed to achieve high yield of bioactive AqpZ protein. A mild detergent n-dodecyl-β-D-maltopyranoside (DDM) was used to solubilize the over-expressed insoluble AqpZ from inclusion bodies without a refolding process. The recovered AqpZ protein showed high water permeability comparable with AqpZ obtained from the membrane fraction. In this way, the total yield of bioactive AqpZ has been increased greatly, which will facilitate the structural and functional characterization and future applications of AqpZ. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Characterization of a parasporal inclusion body from sporulating, enterotoxin-positive Clostridium perfringens type A.

    PubMed Central

    Löffler, A; Labbé, R

    1986-01-01

    Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens NCTC 8239 and 8798 were isolated and characterized. IB were isolated by disruption of sporangia by sonication in the presence of tetrasodium EDTA and phenylmethylsulfonyl fluoride. Fractionation was carried out in a linear gradient of sodium bromide, sucrose, or diatrizoate sodium. Denaturing and reducing agents were necessary to solubilize the IB. An alkylating agent was required to prevent reaggregation of the subunits. Molecular weight, compositional, and serological analyses and peptide mapping revealed strong similarities between the IB subunits and the enterotoxin synthesized during sporulation by C. perfringens. IB appear to represent the structural component where overproduced enterotoxin accumulates intracellularly. Enterotoxin-like subunits in the IB appeared to be held together by noncovalent and disulfide bonds, which were generally resistant to the action of intracellular proteases of C. perfringens, trypsin, or trypsin plus bile salts. Images PMID:2867991

  5. Experimental infection of Boa constrictor with an orthoreovirus isolated from a snake with inclusion body disease.

    PubMed

    Darke, Sabina; Marschang, Rachel E; Hetzel, Udo; Reinacher, Manfred

    2014-06-01

    Orthoreoviruses have been associated with disease in reptiles, but have not previously been isolated from snakes with inclusion body disease (IBD). An orthoreovirus was isolated from a Boa constrictor diagnosed with IBD and then used to conduct a transmission study to determine the clinical importance of this virus. For the transmission study, 10 juvenile boas were experimentally infected with the isolated orthoreovirus and compared to 5 sham-infected control animals. Orthoreovirus was reisolated for a period of 18 wk after infection and weight gain was reduced in infected snakes. Histological examination showed a mild hepatitis in three of four virologically positive snakes up to 12 wk after infection. Results indicated that the orthoreovirus was moderately pathogenic, but, no evidence was found to indicate that it was the causal agent of IBD. In the light of the discovery of Arenaviruses in some snakes with IBD, it was proposed that orthoreoviruses may play a role in synergistic infection.

  6. Lipid Droplets Are Essential for Efficient Clearance of Cytosolic Inclusion Bodies.

    PubMed

    Moldavski, Ofer; Amen, Triana; Levin-Zaidman, Smadar; Eisenstein, Miriam; Rogachev, Ilana; Brandis, Alexander; Kaganovich, Daniel; Schuldiner, Maya

    2015-06-08

    Exposing cells to folding stress causes a subset of their proteins to misfold and accumulate in inclusion bodies (IBs). IB formation and clearance are both active processes, but little is known about their mechanism. To shed light on this issue, we performed a screen with over 4,000 fluorescently tagged yeast proteins for co-localization with a model misfolded protein that marks IBs during folding stress. We identified 13 proteins that co-localize to IBs. Remarkably, one of these IB proteins, the uncharacterized and conserved protein Iml2, exhibited strong physical interactions with lipid droplet (LD) proteins. Indeed, we here show that IBs and LDs are spatially and functionally linked. We further demonstrate a mechanism for IB clearance via a sterol-based metabolite emanating from LDs. Our findings therefore uncover a function for Iml2 and LDs in regulating a critical stage of cellular proteostasis.

  7. Detection of novel divergent arenaviruses in boid snakes with inclusion body disease in The Netherlands.

    PubMed

    Bodewes, R; Kik, M J L; Raj, V Stalin; Schapendonk, C M E; Haagmans, B L; Smits, S L; Osterhaus, A D M E

    2013-06-01

    Arenaviruses are bi-segmented negative-stranded RNA viruses, which were until recently only detected in rodents and humans. Now highly divergent arenaviruses have been identified in boid snakes with inclusion body disease (IBD). Here, we describe the identification of a new species and variants of the highly divergent arenaviruses, which were detected in tissues of captive boid snakes with IBD in The Netherlands by next-generation sequencing. Phylogenetic analysis of the complete sequence of the open reading frames of the four predicted proteins of one of the detected viruses revealed that this virus was most closely related to the recently identified Golden Gate virus, while considerable sequence differences were observed between the highly divergent arenaviruses detected in this study. These findings add to the recent identification of the highly divergent arenaviruses in boid snakes with IBD in the United States and indicate that these viruses also circulate among boid snakes in Europe.

  8. Herpesviral inclusion body disease in owls and falcons is caused by the pigeon herpesvirus (columbid herpesvirus 1).

    PubMed

    Gailbreath, Katherine L; Oaks, J Lindsay

    2008-04-01

    A herpesviral disease of Rock Pigeons (Columba livia), called "inclusion body disease" or "inclusion body hepatitis," was first described in the 1940s. The disease involves hepatic and splenic necrosis with associated intranuclear inclusion bodies and occurs primarily in young squabs. A similar herpesviral disease occurs in falcons and owls. Serologic and restriction endonuclease digestion studies indicate that herpesviruses from pigeons, falcons, and owls are very closely related and that most reported cases of disease in falcons and owls involve prior documented or possible ingestion of pigeons. These findings led to the hypothesis that an endemic herpesvirus of pigeons may be causing disease in falcons and owls. In order to test this hypothesis, we sequenced a fragment of the herpesviral DNA polymerase gene from naturally infected owls, falcons, and pigeons with inclusion body disease collected between 1991 and 2006. Sequences from all three sources were almost identical, and we therefore propose that the usual agent of inclusion body hepatitis in owls and falcons is columbid herpesvirus 1.

  9. Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli

    PubMed Central

    2013-01-01

    Background Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. Results We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. Conclusion A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification. PMID:23638724

  10. Inclusion bodies from recombinant bacteria as a novel system for delivery of vaccine antigen by the oral route.

    PubMed

    Kesik, Małgorzata; Saczyńska, Violetta; Szewczyk, Bogusław; Płucienniczak, Andrzej

    2004-02-15

    A fragment of non-glycosylated E2 antigen of classical swine fever virus (CSFV), lacking the trans-membrane anchor (TM-) of the native glycoprotein, was produced in recombinant Escherichia coli strain BL21(DE3) in the form of inclusion bodies. These inclusion bodies isolated from the bacteria cells were administrated orally to mice twice at either 10 or 50 microg per dose. Each mouse fed with inclusion bodies carrying the E2 antigen responded with plasma antibodies and/or fecal IgA at least once during the entire investigation. Our study showed the capacity of inclusion bodies to induce both systemic and mucosal responses as well as to evoke relatively-long mucosal memory when fed to mice at low-number vaccination schedule and without any adjuvant. We propose the use of inclusion bodies for oral vaccination as an alternative to artificial systems for delivery of recombinant antigens by the oral route. Very few steps are needed to obtain an antigen ready for use as a vaccine. The procedure is easy and inexpensive and can be used for development of vaccine against classical swine fever.

  11. Online analysis of protein inclusion bodies produced in E. coli by monitoring alterations in scattered and reflected light.

    PubMed

    Ude, Christian; Ben-Dov, Nadav; Jochums, André; Li, Zhaopeng; Segal, Ester; Scheper, Thomas; Beutel, Sascha

    2016-05-01

    The online monitoring of recombinant protein aggregate inclusion bodies during microbial cultivation is an immense challenge. Measurement of scattered and reflected light offers a versatile and non-invasive measurement technique. Therefore, we investigated two methods to detect the formation of inclusion bodies and monitor their production: (1) online 180° scattered light measurement (λ = 625 nm) using a sensor platform during cultivation in shake flask and (2) online measurement of the light reflective interference using a porous Si-based optical biosensor (SiPA). It could be shown that 180° scattered light measurement allows monitoring of alterations in the optical properties of Escherichia coli BL21 cells, associated with the formation of inclusion bodies during cultivation. A reproducible linear correlation between the inclusion body concentration of the non-fluorescent protein human leukemia inhibitory factor (hLIF) carrying a thioredoxin tag and the shift ("Δamp") in scattered light signal intensity was observed. This was also observed for the glutathione-S-transferase-tagged green fluorescent protein (GFP-GST). Continuous online monitoring of reflective interference spectra reveals a significant increase in the bacterium refractive index during hLIF production in comparison to a non-induced reference that coincide with the formation of inclusion bodies. These online monitoring techniques could be applied for fast and cost-effective screening of different protein expression systems.

  12. Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies.

    PubMed

    Mačinković, Igor S; Abughren, Mohamed; Mrkic, Ivan; Grozdanović, Milica M; Prodanović, Radivoje; Gavrović-Jankulović, Marija

    2013-12-01

    High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6-8M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4M urea. The activity of rGST was assayed in 2M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli.

    PubMed

    Pandey, Neeraj; Sachan, Annapurna; Chen, Qi; Ruebling-Jass, Kristin; Bhalla, Ritu; Panguluri, Kiran Kumar; Rouviere, Pierre E; Cheng, Qiong

    2013-05-02

    Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification.

  14. Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization.

    PubMed

    Tomisawa, Satoshi; Sato, Yuji; Kamiya, Masakatsu; Kumaki, Yasuhiro; Kikukawa, Takashi; Kawano, Keiichi; Demura, Makoto; Nakamura, Kiminori; Ayabe, Tokiyoshi; Aizawa, Tomoyasu

    2015-08-01

    Mammalian α-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of α-defensins, large amounts of α-defensins are essential. Although many expression systems for the production of recombinant α-defensins have been developed, attempts to obtain large amounts of α-defensins have been only moderately successful. Therefore, in this study, we applied a previously developed aggregation-prone protein coexpression method for the production of mouse α-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in Escherichia coli expression system. By using this method, we succeeded in obtaining a large amount of Crp4 in the form of inclusion bodies. Moreover, we attempted to refold Crp4 directly during the inclusion-body solubilization step under oxidative conditions. Surprisingly, even without any purification, Crp4 was efficiently refolded during the solubilization step of inclusion bodies, and the yield was better than that of the conventional refolding method. NMR spectra of purified Crp4 suggested that it was folded into its correct tertiary structure. Therefore, the method described in this study not only enhances the expression of α-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step.

  15. Production and purification of refolded recombinant Plasmodium falciparum beta-ketoacyl-ACP reductase from inclusion bodies.

    PubMed

    Karmodiya, Krishanpal; Srivastav, Ratnesh Kumar; Surolia, Namita

    2005-07-01

    A recombinant form of Plasmodium falciparum beta-ketoacyl-ACP reductase (PfFabG) was overexpressed in Escherichia coli BL-21 codon plus (DE3). The resulting insoluble inclusion bodies were separated from cellular debris by extensive washing with buffer containing 0.05% Tween 20 and solubilized by homogenization with 8 M urea. Attempts to refold PfFabG from solubilized inclusion bodies employing Rotofor (separation based on different pIs of proteins in a mixture) followed by Ni(2+) or cation exchange chromatography were not successful either by bringing down the urea concentration instantaneously, stepwise, or by dialysis. Denatured PfFabG was therefore initially purified by cation exchange chromatography and was then correctly refolded at a final concentration of 100-200 microg/ml in a 20 mM Na-acetate buffer, pH 5.3, with 300 mM NaCl, 10% glycerol, and 0.05% Tween 20. The protein was found to be properly folded only in the presence of the cofactor NADPH and salt at a concentration 300 mM by drop dilution method at 2-8 degrees C for 12 h. The purified final product was >98% pure by denaturing gel electrophoresis. The purified protein was biologically active in a standard enzymatic assay using acetoacetyl-CoA as a substrate. The enzyme was found to be stable up to fourth day of purification and glycerol was found to stabilize enzyme activity for several weeks, during storage. This effort paves the way for elucidation of the structure-function correlations for PfFabG as well as exploration of the enzyme for developing inhibitors against it for combating malaria.

  16. Inclusion body formation, macroautophagy, and the role of HDAC6 in neurodegeneration.

    PubMed

    Richter-Landsberg, Christiane; Leyk, Janina

    2013-12-01

    The failure to clear misfolded or aggregated proteins from the cytoplasm of nerve cells and glia is a common pathogenic event in a variety of neurodegenerative disorders. This might be causally related to defects in the major proteolytic systems, i.e., the ubiquitin-proteasomal system and the autophagic pathway. Large protein aggregates and defective organelles are excluded from the proteasome. They can be degraded only by macroautophagy, which is a highly selective process. It requires p62 to act as a bridge connecting ubiquitinated protein aggregates and autophagosomes, and the tubulin deacetylase histone deacetylase 6 (HDAC6). HDAC6 has recently been identified as a constituent in Lewy bodies of Parkinson disease and glial cytoplasmic inclusions of multiple system atrophy. It is considered a sensor of proteasomal inhibition and a cellular stress surveillance factor, and plays a central role in autophagy by controlling the fusion process of autophagosomes with lysosomes. Upon proteasomal inhibition, HDAC6 is relocated and recruited to polyubiquitin-positive aggresomes. Tubulin acetylation is a major consequence of HDAC6 inhibition, and HDAC6 inhibition restores microtubule (MT)-dependent transport mechanisms in neurons. This suggests the involvement of HDAC6 in neurodegenerative diseases. Furthermore, the protein tau seems to be a substrate for HDAC6. Tau acetylation impairs MT assembly and promotes tau fibrillization in vitro. It has been suggested that acetylation and phosphorylation of tau at multiples sites may act synergistically in the pathogenesis of tau fibrillization. In this review, we will survey the process of aggresome formation, macroautophagy and the role of autophagosomal proteins and HDAC6 in inclusion body formation.

  17. Renal cell carcinoma with rhabdoid-like features lack intracytoplasmic inclusion bodies and show aggressive behavior.

    PubMed

    Sugimoto, Masaaki; Kohashi, Kenichi; Kuroiwa, Kentaro; Abe, Tatsuro; Yamada, Yuichi; Shiota, Masaki; Imada, Kenjiro; Naito, Seiji; Oda, Yoshinao

    2016-03-01

    In renal cell carcinoma (RCC), tumor cells with rhabdoid features are characterized by eccentric nuclei, prominent nucleoli, and eosinophilic cytoplasm with intracytoplasmic inclusion bodies. In RCC, tumor cells have also been observed resembling rhabdomyoblasts or rhabdoid but without intracytoplasmic inclusion bodies, and here, we defined these rhabdoid-like features of these cells. To this end, we studied a series of clear cell RCC (ccRCC) with rhabdoid features and compared them with a series of ccRCC with rhabdoid-like features to clarify the differences in the immunohistochemical profile and biological behavior. From 695 cases of ccRCC (80.8 % of all RCCs), 18 cases with rhabdoid features (2.1 % of all RCCs) and 25 cases with rhabdoid-like features (2.9 % of all RCCs) were investigated. The 5-year survival rate for ccRCC with rhabdoid features was 44.7 % and for ccRCC with rhabdoid-like features 30.3 %. Although ccRCC with rhabdoid features showed immunohistochemical co-expression of epithelial markers and vimentin as seen in malignant rhabdoid tumors, ccRCC with rhabdoid-like features showed no such co-expression. Multivariate analyses of cancer-specific survival revealed that perinephric tissues invasion was an independent prognostic factor in ccRCC with rhabdoid features (p = 0.0253) but not in ccRCC with rhabdoid-like features. In summary, although their prognosis is similar, the marker profile and pattern of extension of ccRCC with rhabdoid-like is different from that of ccRCC with rhabdoid features. Therefore, ccRCC with rhabdoid-like features should be distinguished from ccRCC with rhabdoid features.

  18. Intracellular Bacterial Pathogens Trigger the Formation of U Small Nuclear RNA Bodies (U Bodies) through Metabolic Stress Induction.

    PubMed

    Tsalikis, Jessica; Tattoli, Ivan; Ling, Arthur; Sorbara, Matthew T; Croitoru, David O; Philpott, Dana J; Girardin, Stephen E

    2015-08-21

    Invasive bacterial pathogens induce an amino acid starvation (AAS) response in infected host cells that controls host defense in part by promoting autophagy. However, whether AAS has additional significant effects on the host response to intracellular bacteria remains poorly characterized. Here we showed that Shigella, Salmonella, and Listeria interfere with spliceosomal U snRNA maturation in the cytosol. Bacterial infection resulted in the rerouting of U snRNAs and their cytoplasmic escort, the survival motor neuron (SMN) complex, to processing bodies, thus forming U snRNA bodies (U bodies). This process likely contributes to the decline in the cytosolic levels of U snRNAs and of the SMN complex proteins SMN and DDX20 that we observed in infected cells. U body formation was triggered by membrane damage in infected cells and was associated with the induction of metabolic stresses, such as AAS or endoplasmic reticulum stress. Mechanistically, targeting of U snRNAs to U bodies was regulated by translation initiation inhibition and the ATF4/ATF3 pathway, and U bodies rapidly disappeared upon removal of the stress, suggesting that their accumulation represented an adaptive response to metabolic stress. Importantly, this process likely contributed to shape the host response to invasive bacteria because down-regulation of DDX20 expression using short hairpin RNA (shRNA) amplified ATF3- and NF-κB-dependent signaling. Together, these results identify a critical role for metabolic stress and invasive bacterial pathogens in U body formation and suggest that this process contributes to host defense.

  19. Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation.

    PubMed

    Waelter, S; Boeddrich, A; Lurz, R; Scherzinger, E; Lueder, G; Lehrach, H; Wanker, E E

    2001-05-01

    The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin-proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14-3-3, and alpha-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin-proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.

  20. Inclusion body disease of cranes: comparison of pathologic findings in cranes with acquired vs. experimentally induced disease

    USGS Publications Warehouse

    Schuh, J.C.; Sileo, L.; Siegfried, L.M.; Yuill, Thomas M.

    1986-01-01

    Inclusion body disease of cranes was the cause of death in 17 immature and mature cranes of 5 different species in Wisconsin. A herpesvirus of unknown origin was the apparent cause. An isolate of this herpesvirus was used to experimentally infect 3 species of cranes. Macroscopic and microscopic lesions associated with naturally acquired and experimentally induced disease were essentially identical. Multifocal hepatic and splenic necrosis was found in all cranes evaluated. Necrosis of the gastrointestinal tract, thymus, and bursa of Fabricius also was seen in some of the cranes. Eosinophilic intranuclear inclusion bodies often were commonly associated with hepatic lesions, sometimes with the splenic lesions, and rarely with the thymic or gastrointestinal tract lesions. The lesions of this inclusion body disease were similar to those reported for cranes in Austria from which a crane herpesvirus was isolated.

  1. Recovery of active N-acetyl-D-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers.

    PubMed

    Lu, Shih-Chin; Lin, Sung-Chyr

    2012-01-05

    Overexpression of recombinant N-acetyl-D-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coli led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08±0.02 U/mg, was similar to that of the native protein, 2.13±0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Tris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41±10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32±5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. An In Vitro Model for Lewy Body-Like Hyaline Inclusion/Astrocytic Hyaline Inclusion: Induction by ER Stress with an ALS-Linked SOD1 Mutation

    PubMed Central

    Taniguchi, Manabu; Hitomi, Junichi; Kato, Masaaki; Aoki, Masashi; Itoyama, Yasuto; Kato, Shinsuke; Tohyama, Masaya

    2007-01-01

    Neuronal Lewy body-like hyaline inclusions (LBHI) and astrocytic hyaline inclusions (Ast-HI) containing mutant Cu/Zn superoxide dismutase 1 (SOD1) are morphological hallmarks of familial amyotrophic lateral sclerosis (FALS) associated with mutant SOD1. However, the mechanisms by which mutant SOD1 contributes to formation of LBHI/Ast-HI in FALS remain poorly defined. Here, we report induction of LBHI/Ast-HI-like hyaline inclusions (LHIs) in vitro by ER stress in neuroblastoma cells. These LHI closely resemble LBHI/Ast-HI in patients with SOD1-linked FALS. LHI and LBHI/Ast-HI share the following features: 1) eosinophilic staining with a pale core, 2) SOD1, ubiquitin and ER resident protein (KDEL) positivity and 3) the presence of approximately 15–25 nm granule-coated fibrils, which are morphological hallmark of mutant SOD1-linked FALS. Moreover, in spinal cord neurons of L84V SOD1 transgenic mice at presymptomatic stage, we observed aberrant aggregation of ER and numerous free ribosomes associated with abnormal inclusion-like structures, presumably early stage neuronal LBHI. We conclude that the LBHI/Ast-HI seen in human patients with mutant SOD1-linked FALS may arise from ER dysfunction. PMID:17925878

  3. The Metallothionein-Null Phenotype Is Associated with Heightened Sensitivity to Lead Toxicity and an Inability to Form Inclusion Bodies

    PubMed Central

    Qu, Wei; Diwan, Bhalchandra A.; Liu, Jie; Goyer, Robert A.; Dawson, Tammy; Horton, John L.; Cherian, M. George; Waalkes, Michael P.

    2002-01-01

    Susceptibility to lead toxicity in MT-null mice and cells, lacking the major forms of the metallothionein (MT) gene, was compared to wild-type (WT) mice or cells. Male MT-null and WT mice received lead in the drinking water (0 to 4000 ppm) for 10 to 20 weeks. Lead did not alter body weight in any group. Unlike WT mice, lead-treated MT-null mice showed dose-related nephromegaly. In addition, after lead exposure renal function was significantly diminished in MT-null mice in comparison to WT mice. MT-null mice accumulated less renal lead than WT mice and did not form lead inclusion bodies, which were present in the kidneys of WT mice. In gene array analysis, renal glutathione S-transferases were up-regulated after lead in MT-null mice only. In vitro studies on fibroblast cell lines derived from MT-null and WT mice showed that MT-null cells were much more sensitive to lead cytotoxicity. MT-null cells accumulated less lead and formed no inclusion bodies. The MT-null phenotype seems to preclude lead-induced inclusion body formation and increases lead toxicity at the organ and cellular level despite reducing lead accumulation. This study reveals important roles for MT in chronic lead toxicity, lead accumulation, and inclusion body formation. PMID:11891201

  4. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all raw...

  5. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all raw...

  6. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all raw...

  7. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all raw...

  8. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all raw...

  9. Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.

    PubMed

    Chen, Huanhuan; Li, Ninghuan; Xie, Yueqing; Jiang, Hua; Yang, Xiaoyi; Cagliero, Cedric; Shi, Siwei; Zhu, Chencen; Luo, Han; Chen, Junsheng; Zhang, Lei; Zhao, Menglin; Feng, Lei; Lu, Huili; Zhu, Jianwei

    2017-07-01

    It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.

  10. Increased expression in vivo and in vitro of foreign genes directed by A-type inclusion body hybrid promoters in recombinant vaccinia viruses.

    PubMed Central

    Funahashi, S; Itamura, S; Iinuma, H; Nerome, K; Sugimoto, M; Shida, H

    1991-01-01

    We constructed A-type inclusion body (ATI) hybrid promoters, that is, late ATI promoters followed by tandemly repeated early regions of the promoter for the 7.5-kDa protein (the 7.5-kDa promoter). The repetition of the whole early promoter sequence of the 7.5-kDa gene, including the upstream consensus sequence and initiation region, efficiently increased the early expression of the bacterial chloramphenicol acetyltransferase gene in recombinant vaccinia virus. Recombinant vaccinia virus could express influenza virus hemagglutinin via the hybrid promoter more efficiently, induced higher levels of neutralizing antibody and cytotoxic T lymphocytes, and consequently protected mice more efficiently against challenge with influenza virus than did recombinant vaccinia virus containing the widely used 7.5-kDa promoter. Images PMID:1654453

  11. Polyethyleneimine-mediated chemical extraction of cytoplasmic His-tagged inclusion body proteins from Escherichia coli.

    PubMed

    Nian, Rui; Tan, Lihan; Choe, Woo-Seok

    2008-01-01

    The selectivity of polyethyleneimine (PEI) in DNA precipitation during chemical extraction was investigated. Chemical extraction was used to recover two His-tagged model proteins: gloshedobin, a thrombin-like enzyme from snake venom, and IbpA, a molecular chaperone, which were expressed mainly in the form of inclusion bodies. High DNA removal efficiency (more than 90%) was achieved at various cell densities (with OD600 ranging from 30 to 150) without affecting the solubility of host cell proteins. Compared to spermine-induced precipitation method reported elsewhere, PEI provided a higher DNA precipitation efficiency at a significantly lower cost. Moreover, PEI obviated the use of EDTA, which has been reported to be essential for the chemical extraction methods, hence exhibiting dual roles in replacing cost-prohibitive spermine and EDTA. The residual PEI in the post-extraction mixture was efficiently counteracted by addition of Mg2+, allowing the streamlined application of the extraction broth to immobilized metal affinity chromatography. Taken together, the PEI-mediated chemical extraction method provides a simpler and more economically viable processing route for the production of recombinant proteins whose expression is hampered by IB formation.

  12. Diagnostic value of markers of muscle degeneration in sporadic inclusion body myositis.

    PubMed

    Dubourg, O; Wanschitz, J; Maisonobe, T; Béhin, A; Allenbach, Y; Herson, S; Benveniste, O

    2011-10-01

    Sporadic inclusion body myositis (s-IBM) is characterized histologically by the association of concomitant inflammatory and degenerative processes. We evaluated the sensitivity and specificity of different markers of the degenerative process in order to refine the histological diagnosis. We performed an immunohistochemical study with antibodies directed against ubiquitin, amyloid-beta precursor protein (AbetaPP), amyloid-beta (Abeta), SMI-31, SMI-310, Tar-DNA binding protein-43 (TDP-43) and p62 on s-IBM and control muscle biopsies. Based on conventional stains 36 patients with characteristic clinical features of s-IBM were subclassified as presumed definite s-IBM (d s-IBM, n = 17) or possible s-IBM (p s-IBM, n = 19) according to the presence or absence of vacuolated muscle fibers. Immunohistochemically, TDP-43 and p62 were the most sensitive markers, accumulating in all d s-IBM and in 31% and 37%, respectively, of the p s-IBM cases and thus enabling reclassification of these cases as d s-IBM. We recommend using TDP-43 and p62 antibodies in the histological diagnosis workup of s-IBM. The specificity of these markers has to be further validated in prospective series.

  13. Hepatitis C virus infection in inclusion body myositis: A case-control study.

    PubMed

    Uruha, Akinori; Noguchi, Satoru; Hayashi, Yukiko K; Tsuburaya, Rie S; Yonekawa, Takahiro; Nonaka, Ikuya; Nishino, Ichizo

    2016-01-19

    To clarify whether there is any association between inclusion body myositis (IBM) and hepatitis C virus (HCV) infection. We assessed the prevalence of HCV infection in 114 patients with IBM whose muscle biopsies were analyzed pathologically for diagnostic purpose from 2002 to 2012 and in 44 age-matched patients with polymyositis diagnosed in the same period as a control by administering a questionnaire survey to the physicians in charge. We also compared clinicopathologic features including the duration from onset to development of representative symptoms of IBM and the extent of representative pathologic changes between patients with IBM with and without HCV infection. A significantly higher number of patients with IBM (28%) had anti-HCV antibodies as compared with patients with polymyositis (4.5%; odds ratio 8.2, 95% confidence interval 1.9-36) and the general Japanese population in their 60s (3.4%). Furthermore, between patients with IBM with and without HCV infection, we did not find any significant difference in the clinicopathologic features, indicating that the 2 groups have essentially the same disease regardless of HCV infection. Our results provide the statistical evidence for an association between IBM and HCV infection, suggesting a possible pathomechanistic link between the 2 conditions. © 2015 American Academy of Neurology.

  14. Anesthetic Considerations of Sporadic Inclusion Body Myositis in an Elderly Man With Orthopedic Trauma.

    PubMed

    Steck, Dominik T; Choi, Christine; Gollapudy, Suneeta; Pagel, Paul S

    2016-04-01

    Sporadic inclusion body myositis (IBM) is an inflammatory myopathy characterized by progressive asymmetric extremity weakness, oropharyngeal dysphagia, and the potential for exaggerated sensitivity to neuromuscular blockers and respiratory compromise. The authors describe their management of a patient with IBM undergoing urgent orthopedic surgery. An 81-year-old man with IBM suffered a left intertrochanteric femoral fracture after falling down stairs. His IBM caused progressive left proximal lower extremity, bilateral distal upper extremity weakness (left > right), and oropharyngeal dysphagia (solid food, pills). He denied dyspnea, exercise intolerance, and a history of aspiration. Because respiratory insufficiency resulting from diaphragmatic dysfunction and prolonged duration of action of neuromuscular blockers may occur in IBM, the authors avoided using a neuromuscular blocker. After applying cricoid pressure, anesthesia was induced using intravenous lidocaine, propofol, remifentanil followed by manual ventilation with inhaled sevoflurane in oxygen. Endotracheal intubation was accomplished without difficulty; anesthesia was then maintained using remifentanil and sevoflurane. The fracture was repaired with a trochanteric femoral nail. The patient was extubated without difficulty and made an uneventful recovery. In summary, there is a lack of consensus about the use of neuromuscular blockers in patients with IBM. The authors avoided these drugs and were able to easily secure the patient's airway and maintain adequate muscle relaxation using a balanced sevoflurane-remifentanil anesthetic. Clinical trials are necessary to define the pharmacology of neuromuscular blockers in patients with IBM and determine whether use of these drugs contributes to postoperative respiratory insufficiency in these vulnerable patients.

  15. Intramuscular dissociation of echogenicity in the triceps surae characterizes sporadic inclusion body myositis.

    PubMed

    Nodera, H; Takamatsu, N; Matsui, N; Mori, A; Terasawa, Y; Shimatani, Y; Osaki, Y; Maruyama, K; Izumi, Y; Kaji, R

    2016-03-01

    Differential diagnosis of sporadic inclusion body myositis (s-IBM) and polymyositis (PM)/dermatomyositis (DM) is difficult and can affect proper disease management. Detection of heterogeneous muscular involvement in s-IBM by muscle sonography could be a unique diagnostic feature. Sonography of the lower leg and forearm was performed in patients with s-IBM, PM/DM and control subjects (n = 11 each). Echo intensities (EIs) of the adjacent muscles [medial head of the gastrocnemius versus soleus and the flexor digitorum profundus (FDP) versus flexor carpi ulnaris (FCU)] were scored by three blinded raters. The mean EIs of these muscles were compared using computer-assisted histogram analysis. Both evaluation methods showed high echoic signals in the gastrocnemius of patients with s-IBM. EIs were significantly different between the gastrocnemius and soleus in patients with s-IBM, but not in those with DM/PM and the controls. In the forearm, although the EI of the FDP was higher in the s-IBM group than in the other groups, the EI differences between the FDP and FCU did not differ significantly between disease groups. The difference in area under the curves to differentiate between s-IBM and DM/PM was greatest between the gastrocnemius-soleus EIs (0.843; P = 0.006). High echoic signals in the medial gastrocnemius compared with those of the soleus are suggestive of s-IBM over PM/DM. © 2015 EAN.

  16. Investigation of splicing changes and post-translational processing of LMNA in sporadic inclusion body myositis

    PubMed Central

    Luo, Yue-Bei; Mitrpant, Chalermchai; Johnsen, Russell; Fabian, Vicki; Needham, Merrilee; Fletcher, Sue; Wilton, Steve D; Mastaglia, Frank L

    2013-01-01

    Some features of sporadic inclusion body myositis (s-IBM) suggest that there is acceleration of the normal ageing process in muscle tissue. LMNA encodes the nuclear lamina proteins lamin A/C through alternative splicing, and aberrant splicing of exon 11 leads to the premature ageing disease, Hutchinson-Gilford progeria syndrome. Progerin, the pathogenic isoform expressed in HGPS tissues, has also been detected at low levels in tissues of normal individuals with aging. We therefore investigated the alternative splicing of LMNA gene transcripts, and the post-translational processing of prelamin A, in s-IBM and control muscle samples. Age-related low level expression of the progerin transcript was detected in both s-IBM and control muscles, but was not increased in s-IBM and there was no increase in progerin protein or demonstrable accumulation of intermediate prelamin isoforms in the s-IBM muscles. However, an age-related shift in the balance of splicing towards lamin A-related transcripts, which was present in normal muscles, was not found in s-IBM. Our findings indicate that while there are changes in the patterns of LMNA splicing in s-IBM muscle which are probably secondary to the underlying pathological process, it is unlikely that aberrant splicing of exon 11 or defective post-translational processing of prelamin A are involved in the pathogenesis of the disease. PMID:24040437

  17. Expression of myogenic regulatory factors and myo-endothelial remodeling in sporadic inclusion body myositis.

    PubMed

    Wanschitz, Julia V; Dubourg, Odile; Lacene, Emmanuelle; Fischer, Michael B; Höftberger, Romana; Budka, Herbert; Romero, Norma B; Eymard, Bruno; Herson, Serge; Butler-Browne, Gillian S; Voit, Thomas; Benveniste, Olivier

    2013-01-01

    Muscle repair relies on coordinated activation and differentiation of satellite cells, a process that is unable to counterbalance progressive degeneration in sporadic inclusion body myositis (s-IBM). To explore features of myo regeneration, the expression of myogenic regulatory factors Pax7, MyoD and Myogenin and markers of regenerating fibers was analyzed by immunohistochemistry in s-IBM muscle compared with polymyositis, dermatomyositis, muscular dystrophy and age-matched controls. In addition, the capillary density and number of interstitial CD34(+) hematopoietic progenitor cells was determined by double-immunoflourescence staining. Satellite cells and regenerating fibers were significantly increased in s-IBM similar to other inflammatory myopathies and correlated with the intensity of inflammation (R>0.428). Expression of MyoD, visualizing activated satellite cells and proliferating myoblasts, was lower in s-IBM compared to polymyosits. In contrast, Myogenin a marker of myogenic cell differentiation was strongly up-regulated in s-IBM muscle. The microvascular architecture in s-IBM was distorted, although the capillary density was normal. Notably, CD34(+) hematopoietic cells were significantly increased in the interstitial compartment. Our findings indicate profound myo-endothelial remodeling of s-IBM muscle concomitant to inflammation. An altered expression of myogenic regulatory factors involved in satellite cell activation and differentiation, however, might reflect perturbations of muscle repair in s-IBM. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Stable supply of large amounts of human Fab from the inclusion bodies in E. coli.

    PubMed

    Fujii, Testuro; Ohkuri, Takatoshi; Onodera, Reiko; Ueda, Tadashi

    2007-05-01

    Recombinant human Fab-H chain and L chain were separately expressed as inclusion body using Escherichia coli. After solubilization of Fab-H chain and L chain by the reduction and S-alkyldisulphidation in 8 M urea, about 100 mg of purified Fab-H chain and about 160 mg of L chain could be obtained from 1 l of each culture by ion-exchange chromatogram in the presence of 8 M urea. Combination of the lyophilized Fab-H chain and L chain could be efficiently folded to native human Fab by using the stepwise dialysis method and the human Fab was purified with cation-exchange chromatogram. In the folding procedure, it was found that cysteamine and cystamine with positive charge were effective to improve the folding yield of human Fab. Moreover, from comparison of folding yield in the presence of ten kinds of additives, it was suggested that taurine was effective to improve the folding of human Fab. Consequently, we could obtain about 60 mg of folded human Fab from 1 l of each culture under the optimum conditions.

  19. Efficacy of immunosuppressive treatment in a systemic lupus erythematosus patient presenting with inclusion body myositis.

    PubMed

    Varela-Rosario, Noemí; Pérez-Berenguer, Juan L; Vilá, Luis M

    2016-04-05

    Inclusion body myositis (IBM) is an inflammatory myopathy that is generally unresponsive to immunosuppressive drugs. The coexistence of IBM with other autoimmune connective tissue diseases is rare. We present a case of a 76-year-old woman with systemic lupus erythematosus (SLE) who developed proximal muscle weakness of lower extremities and mild elevation of serum creatine kinase (CK) at 495 U/L. Muscle biopsy showed changes of endomysial inflammation and rimmed vacuoles consistent with IBM. She was treated with prednisone 40 mg daily and methotrexate 12.5 mg weekly. One month later, her physical examination showed minimal proximal weakness of lower extremities. CK levels decreased to 44 U/L. Prednisone dose was gradually decreased to 5.0 mg daily. She remained stable with normal CK levels during a follow-up period of 10 months. This case, together with other reports, suggests that IBM in the setting of SLE represents a different subtype that can benefit from immunosuppressive treatment.

  20. Myonuclear breakdown in sporadic inclusion body myositis is accompanied by DNA double strand breaks.

    PubMed

    Nishii, Makoto; Nakano, Satoshi; Nakamura, Seika; Wate, Reika; Shinde, Akiyo; Kaneko, Satoshi; Kusaka, Hirofumi

    2011-05-01

    Rimmed vacuoles in sporadic inclusion body myositis (s-IBM) contain nuclear remnants. We sought to determine if the nuclear degeneration seen in s-IBM is associated with DNA damage. In muscle biopsy specimens from ten patients with s-IBM and 50 controls, we immunolocalized 1) phosphorylated histone H2AX (γ-H2AX), which is a sensitive immunocytochemical marker of DNA double-strand breaks and 2) DNA-PK, which is an enzyme involved in double-strand break repair. In s-IBM, vacuolar peripheries often showed strong immunoreactivity to γ-H2AX and the three components of DNA-PK (DNA-PKcs, Ku70, and Ku80). A triple fluorescence study of Ku70, emerin, and DNA displayed nuclear breakdown and it suggested impaired nuclear incorporation of Ku70. The percentage of positive nuclei for γ-H2AX was significantly higher in vacuolated fibers than non-vacuolated fibers in s-IBM, or fibers in polymyosits. We hypothesize that a dysfunction of nuclear envelope may cause nuclear fragility, double-strand breaks and impaired nuclear transport in s-IBM. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Production and purification of recombinant human BLyS mutant from inclusion bodies.

    PubMed

    Xue, Xiaochang; Wang, Zenglu; Yan, Zhen; Shi, Jihong; Han, Wei; Zhang, Yingqi

    2005-07-01

    B lymphocyte stimulator (BLyS), a member of the tumor necrosis factor superfamily, is an important regulator of B cell homeostasis. In BLyS-deficient mice, B cell development is severely perturbed. On the other hand, mice transgenic for BLyS developed autoimmune disorders, such as increased germinal center formation, production of autoantibodies, and Ig deposition in kidneys. The overexpression of BLyS was found in some human autoimmune diseases. These findings suggest that BLyS has a crucial role in the humoral immune response and may be a therapeutic target for some human autoimmune diseases. To construct and express the therapeutic vaccine BLyS, we coupled a foreign immunodominant T-helper epitope to the N terminus of BLyS (named recombinant BLyS mutant, rBLySM) and expressed rBLySM in Escherichia coli. We have developed a purification process of rBLySM from inclusion bodies. A step-down urea concentration strategy was applied to the rBLySM renaturation process. By this strategy, a stable yield of 4.5mg purified rBLySM per gram of cell paste could be obtained.

  2. Demographic and clinical features of inclusion body myositis in North America

    PubMed Central

    Paltiel, A. David; Ingvarsson, Einar; Lee, Donald K. K.; Leff, Richard L.; Nowak, Richard J.; Petschke, Kurt D.; Richards-Shubik, Seth; Zhou, Ange; Shubik, Martin; O’Connor, Kevin C.

    2016-01-01

    Objective Define the demographics, natural history, and clinical management of patients with inclusion body myositis (IBM). Background Few studies of the demographics, natural history, and clinical management of IBM have been performed in a large patient population. Methods A cross-sectional, self-reporting survey was conducted. Results The mean age of the 916 participants was 70.4 years, the male-to-female ratio was 2:1, and the majority reported difficulty with ambulation and activities of daily living. The earliest symptoms included impaired use and weakness of arms and legs. The mean time from first symptoms to diagnosis was 4.7 years. Half reported that IBM was their initial diagnosis. A composite functional index negatively associated with age, disease duration, and positively associated with participation in exercise. Conclusion These data are valuable for informing patients how IBM manifestations are expected to impair daily living and indicate that self-reporting could be used to establish outcome measures in clinical trials. PMID:25557419

  3. A strategic crossflow filtration methodology for the initial purification of promegapoietin from inclusion bodies.

    PubMed

    Ledung, Erika; Eriksson, Per-Olov; Oscarsson, Sven

    2009-04-20

    A novel crossflow filtration methodology is demonstrated for the initial purification of the therapeutic protein, promegapoietin-1a (PMP), produced as inclusion bodies (IBs) in a recombinant Escherichia coli bioprocess. Two strategic separation steps were performed by utilizing a filtration unit with a 1000 kDa polyethersulphone membrane. The first step, aiming for separation of soluble contaminants, resulted in a 50% reduction of the host cell proteins, quantified by total amino acid analysis and a 70% reduction of all DNA, quantified by fluorometry, when washing the particulate material with a 10mM EDTA in 50mM phosphate buffer, pH 8. The second step, aiming for separation of particulate contaminants from solubilized IBs, resulted in a 97-99.5% reduction of endotoxin, used as a marker for cell debris, and was quantified by the kinetic turbidimetric LAL endotoxin assay. The overall PMP yield was 58% and 33% respectively for the two solubilizations investigated, guanidine hydrochloride and arginine, as measured by RP-HPLC. The scope was also to investigate the physical characteristics of the intermediate product/s with regard to the choice of IB solvent. Preliminary results from circular dichroism spectroscopy measurements indicate that the protein secondary structure was restored when arginine was used in the second step.

  4. Production, characterization, and application of an organic solvent-tolerant lipase present in active inclusion bodies.

    PubMed

    Li, Suxia; Lin, Kang; Pang, Huaiyu; Wu, Yixin; Xu, Jianhe

    2013-01-01

    An organic solvent-tolerant lipase from Serratia marcescens ECU1010 (rSML) was overproduced in Escherichia coli in an insoluble form. High concentrations of both biomass (50 g cell wet weight/L culture broth) and inclusion bodies (10.5 g/L) were obtained by applying a high-cell-density cultivation procedure. Activity assays indicated that the enzymatic activity of rSML reached 600 U/L. After treatment with isopropyl ether for 12 h, the maximum lipase activity reached 6,000 U/L. Scanning electron microscopy and Fourier transform infrared microspectroscopy revealed the activation mechanism of rSML in the presence of organic solvents. rSML was stable in broad ranges of temperatures and pH values, as well as in a series of organic solvents. Besides, rSML showed the best enantioselectivity for the kinetic resolution of (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester. These features render the S. marcescens ECU1010 lipase attractive for biotechnological applications in the field of organic synthesis and pharmaceutical industry.

  5. Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies.

    PubMed

    Espinosa-de la Garza, Carlos E; Perdomo-Abúndez, Francisco C; Campos-García, Víctor R; Pérez, Néstor O; Flores-Ortiz, Luis F; Medina-Rivero, Emilio

    2013-09-01

    In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines.

  6. Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells.

    PubMed

    Liovic, Mirjana; Ozir, Mateja; Zavec, Apolonija Bedina; Peternel, Spela; Komel, Radovan; Zupancic, Tina

    2012-05-24

    We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments. K14 IBs were electroporated into SW13 cells grown in culture together with a "reporter" plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5. Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells.

  7. Integrated refolding techniques for Schistosoma japonicum MTH1 overexpressed as inclusion bodies in Escherichia coli.

    PubMed

    Feng, Yanye; Liu, Lu; Wang, Jipeng; Liu, Jian; Hu, Wei; Wang, Xiaoning; Yang, Zhong

    2012-08-01

    The full-length cDNA of MTH1in Schistosoma japonicum was previously isolated. However, insoluble protein expression in Escherichia coli is the biggest bottleneck limiting biological and biophysical studies. Protein aggregation could not be significantly prevented using solubilization or refolding techniques, and denatured MTH1 protein could not be refolded to the native monomer form. Hence, integrating several refolding techniques within the protein refolding process of MTH1, a large amount of active MTH1 was obtained for protein crystallization. We primarily utilized the two-step-denaturing and refolding method and the protein refolding screening technique, as well as the continuous dialysis method. First, we identified the refolding buffer composition that allowed for successful refolding to overcome protein precipitation. Next, we used the two-step-denaturing and refolding method and the continuous dialysis method to suppress protein aggregation. In the end, we obtained 15 mg of active MTH1 monomer with 95% purity from 0.5l medium. Integrated refolding techniques proved to be excellent for obtaining the native monomer of S. japonicum MTH1 from inclusion bodies, paving the way for future biological and biophysical studies. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  8. Inclusion bodies of fuculose-1-phosphate aldolase as stable and reusable biocatalysts.

    PubMed

    Sans, Cristina; García-Fruitós, Elena; Ferraz, Rosa M; González-Montalbán, Núria; Rinas, Ursula; López-Santín, Josep; Villaverde, Antonio; Álvaro, Gregorio

    2012-01-01

    Fuculose-1-phosphate aldolase (FucA) has been produced in Escherichia coli as active inclusion bodies (IBs) in batch cultures. The activity of insoluble FucA has been modulated by a proper selection of producing strain, culture media, and process conditions. In some cases, when an optimized defined medium was used, FucA IBs were more active (in terms of specific activity) than the soluble protein version obtained in the same process with a conventional defined medium, supporting the concept that solubility and conformational quality are independent protein parameters. FucA IBs have been tested as biocatalysts, either directly or immobilized into Lentikat beads, in an aldolic reaction between DHAP and (S)-Cbz-alaninal, obtaining product yields ranging from 65 to 76%. The production of an active aldolase as IBs, the possibility of tailoring IBs properties by both genetic and process approaches, and the reusability of IBs by further entrapment in appropriate matrices fully support the principle of using self-assembled enzymatic clusters as tunable mechanically stable and functional biocatalysts. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  9. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.

    PubMed

    Nguyen, Hong-Loan Thi; Nguyen, Thuy Thi; Vu, Quy Thi; Le, Hang Thi; Pham, Yen; Trinh, Phuong Le; Bui, Thuan Phuong; Phan, Tuan-Nghia

    2015-12-01

    Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells

    PubMed Central

    2012-01-01

    Background We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments. Results K14 IBs were electroporated into SW13 cells grown in culture together with a “reporter” plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5. Conclusions Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells. PMID:22624805

  11. Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies.

    PubMed

    Spieler, Valerie; Valldorf, Bernhard; Maaß, Franziska; Kleinschek, Alexander; Hüttenhain, Stefan H; Kolmar, Harald

    2016-07-01

    Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost-effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co-immobilized them on modified poly-p-hydroxybutyrate synthase (PhaC)-inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled-coil interaction. Enzyme-loaded particles could be easily purified by centrifugation. Total conversion of 4'-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol could be accomplished using enzyme-loaded particles, catalytic amounts of NAD(+) and formate as substrates for FDH. Chiral GC-MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost-effective alternative to coupled reactions using purified enzymes. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. In vitro refolding of heterodimeric CapZ expressed in E. coli as inclusion body protein.

    PubMed

    Remmert, K; Vullhorst, D; Hinssen, H

    2000-02-01

    CapZ is a heterodimeric Ca(2+)-independent actin binding protein which plays an important role in organizing the actin filament lattice of cross-striated muscle cells. It caps the barbed end of actin filaments and promotes nucleation of actin polymerization, thereby regulating actin filament length. Here we report the expression of the two muscle-specific isoforms alpha2 and beta1, from chicken in Escherichia coli as individual subunits using the pQE60 expression vector and the subsequent renaturation of the functional CapZ heterodimer from inclusion bodies. Optimal renaturation conditions were obtained both by simultaneous refolding of urea-solubilized subunits and by rapid dilution into a buffer containing 20% glycerol, 5 mM EGTA, 2 mM DTT, 1 mM PMSF, and 100 mM Tris, pH 7.4. The refolding mixture was incubated for 24 h at 15 degrees C and the protein was concentrated by ultrafiltration. Biochemical characterization of the recombinant heterodimer revealed actin binding activities indistinguishable from those of native CapZ as purified from chicken skeletal muscle. Using the same protocol, we were able to refold the beta1, but not the alpha2 isoform as a single polypeptide, indicating a role for beta1 as a molecular template for the folding of alpha2. The reported recombinant approach leads to high yields of active heterodimer and allows the renaturation and characterization of the beta subunit.

  13. Investigation of splicing changes and post-translational processing of LMNA in sporadic inclusion body myositis.

    PubMed

    Luo, Yue-Bei; Mitrpant, Chalermchai; Johnsen, Russell; Fabian, Vicki; Needham, Merrilee; Fletcher, Sue; Wilton, Steve D; Mastaglia, Frank L

    2013-01-01

    Some features of sporadic inclusion body myositis (s-IBM) suggest that there is acceleration of the normal ageing process in muscle tissue. LMNA encodes the nuclear lamina proteins lamin A/C through alternative splicing, and aberrant splicing of exon 11 leads to the premature ageing disease, Hutchinson-Gilford progeria syndrome. Progerin, the pathogenic isoform expressed in HGPS tissues, has also been detected at low levels in tissues of normal individuals with aging. We therefore investigated the alternative splicing of LMNA gene transcripts, and the post-translational processing of prelamin A, in s-IBM and control muscle samples. Age-related low level expression of the progerin transcript was detected in both s-IBM and control muscles, but was not increased in s-IBM and there was no increase in progerin protein or demonstrable accumulation of intermediate prelamin isoforms in the s-IBM muscles. However, an age-related shift in the balance of splicing towards lamin A-related transcripts, which was present in normal muscles, was not found in s-IBM. Our findings indicate that while there are changes in the patterns of LMNA splicing in s-IBM muscle which are probably secondary to the underlying pathological process, it is unlikely that aberrant splicing of exon 11 or defective post-translational processing of prelamin A are involved in the pathogenesis of the disease.

  14. Cytoplasmic 5'-nucleotidase autoantibodies in inclusion body myositis: Isotypes and diagnostic utility.

    PubMed

    Greenberg, Steven A

    2014-10-01

    Recent studies have identified circulating immunoglobulin (Ig) G autoantibodies against cytoplasmic 5'-nucleotidase 1A (cN1A; NT5C1A) in patients with inclusion body myositis (IBM), whose detection provides for an IBM blood diagnostic test. Whether or not anti-cN1A autoantibody isotypes other than IgG are present in IBM has not previously been reported. Plasma and serum samples from 205 patients (50 with and155 without IBM) were studied for the presence of IgM and IgA, in addition to IgG, anti-cN1A autoantibodies using immunoblots and enzyme-linked immunoassays (ELISAs). IgM, IgA, and IgG anti-cN1A autoantibodies were detected by ELISA with similar sensitivities (49-53%) and specificities (94-96%), but with differing patterns of autoantibody isotype presence. Combination assays of all 3 autoantibody levels improved diagnostic sensitivity to 76%. In addition to previously recognized IgG anti-cN1A autoantibodies, IBM patients have circulating IgM and IgA anti-cN1A autoantibodies. Differing patterns of these isotypes may be present and useful for diagnosis. Copyright © 2014 Wiley Periodicals, Inc.

  15. Basement membrane remodelling and segmental fibrosis in sporadic inclusion body myositis.

    PubMed

    Doppler, K; Mittelbronn, M; Lindner, A; Bornemann, A

    2009-06-01

    Sporadic inclusion body myositis (sIBM) is a debilitating idiopathic inflammatory myopathy. Little is known about the pathogenetic mechanisms that lead to myofiber degeneration. In the present study, we evaluated the integrity of the myofiber basement membrane in non-necrotic myofibers invaded by inflammatory infiltrates. We used 100 ten mum thick serial sections obtained from biopsies of 5 patients suffering from sIBM. Biopsies from 5 patients suffering from polymyositis served as controls. We performed sequential HE staining and immunolabeling using anti-CD68, -CD8, -merosin, -laminin alpha4 chain, and -collagen IV antibodies. In sIBM, we detected a total of 89 non-necrotic myofibers that were invaded by inflammatory cells. The invasive process and its sequelae were segmental in nature and included destruction of the myofiber basement membrane, and eventually, partial replacement by fibrosis of the invaded myofiber. In polymyositis, we found only two myofibers that were affected in this way. In sIBM, basement membrane remodelling and irreversible replacement by fibrosis of myofibers appear to represent the end result of a process in which the balance between injury and repair are disrupted.

  16. Dean vortex membrane microfiltration and diafiltration of rBDNF E. coli inclusion bodies.

    PubMed

    Schutyser, Maarten; Rupp, Randall; Wideman, Janusz; Belfort, Georges

    2002-01-01

    Cross-flow microfiltration (CMF) and diafiltration were used to concentrate and purify recombinant Brain-Derived Neutrophic Factor (rBDNF) inclusion bodies from an E. coli cell suspension and a homogenized E. coli cell suspension (homogenate/lysate). Although these processes have been tested industrially in pilot scale with conventional linear membrane microfiltration modules, their performances were severely limited due to membrane fouling. The purpose of this work was to determine whether Dean vortex microfiltration with controlled centrifugal instabilities (Dean vortices produced in helical flow) could be used to improve filtration performance over that observed with conventional linear cross-flow microfiltration (CMF). For the microfiltration experiments with the feeds containing cell and homogenate suspensions, improvements in flux of about 50 and 70%, respectively, were obtained with the helical module as compared with that obtained with the linear module. For diafiltration with the homogenate suspension as feed, solute transport (as measured by mass) was from 100 to 40% higher after 40 and 100 min, respectively, with the helical module as compared with that obtained with the linear module. In the presence of the neutral surfactant, Tween 20, solute transport for diafiltration was at least 25 times higher during the first 10 min of operation and 100% higher after 300 min with the helical module as compared with that obtained with the linear module. Clearly, improved filtration performance, a purer and more concentrated product, and substantial savings can be expected with the new Dean vortex filters.

  17. Characterization of hereditary inclusion body myopathy myoblasts: possible primary impairment of apoptotic events.

    PubMed

    Amsili, S; Shlomai, Z; Levitzki, R; Krause, S; Lochmuller, H; Ben-Bassat, H; Mitrani-Rosenbaum, S

    2007-11-01

    Hereditary inclusion body myopathy (HIBM) is a unique muscular disorder caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene. GNE encodes a bi-functional enzyme acting in the biosynthetic pathway of sialic acid. Since the underlying myopathological mechanism leading to the disease phenotype is poorly understood, we have established human myoblasts cultures, derived from HIBM satellite cells carrying the homozygous M712T mutation, and identified cellular and molecular characteristics of these cells. HIBM and control myoblasts showed similar heterogeneous patterns of proliferation and differentiation. Upon apoptosis induction, phosphatidylserine externalization was similar in HIBM and controls. In contrast, the active forms of caspase-3 and -9 were strongly enhanced in most HIBM cultures compared to controls, while pAkt, downregulated in controls, remained high in HIBM cells. These results could indicate impaired apoptotic signaling in HIBM cells. Since satellite cells enable partial regeneration of the post-mitotic muscle tissue, these altered processes could contribute to the muscle mass loss seen in patients. The identification of survival defects in HIBM affected muscle cells could disclose new functions for GNE in muscle cells.

  18. Fast-twitch sarcomeric and glycolytic enzyme protein loss in inclusion body myositis.

    PubMed

    Parker, Kenneth C; Kong, Sek Won; Walsh, Ronan J; Salajegheh, Mohammad; Moghadaszadeh, Behzad; Amato, Anthony A; Nazareno, Remedios; Lin, Yin Yin; Krastins, Bryan; Sarracino, David A; Beggs, Alan H; Pinkus, Jack L; Greenberg, Steven A

    2009-06-01

    Inclusion body myositis (IBM) is an inflammatory disease of skeletal muscle of unknown cause. To further understand the nature of the tissue injury in this disease, we developed methods for large-scale detection and quantitation of proteins in muscle biopsy samples and analyzed proteomic data produced by these methods together with histochemical, immunohistochemical, and microarray data. Twenty muscle biopsy samples from patients with inflammatory myopathies (n = 17) or elderly subjects without neuromuscular disease (n = 3) were profiled by proteomic studies using liquid chromatographic separation of peptides followed by mass spectrometry. Thirteen of the diseased samples additionally underwent microarray studies. Seventy muscle specimens from patients with a range of neuromuscular disorders were examined by ATPase histochemical methods. Smaller numbers of samples underwent immunohistochemical and immunoblot studies. Mass spectrometric studies identified and quantified approximately 300 total distinct proteins in each muscle sample. In IBM and to a lesser extent in polymyositis, proteomic studies confirmed by histochemical, immunohistochemical, and immunoblot studies showed loss of many fast-twitch specific structural proteins and glycolytic enzymes despite relative preservation of transcript levels. Increased abundance of a nuclear membrane protein, immunoglobulins, and two calpain-3 substrates were present. The atrophy present in IBM muscle is accompanied by preferential loss of fast-twitch structural proteins and glycolytic enzymes, particularly glycogen debranching enzyme, with relative preservation of the abundance of their respective transcripts. Although muscle atrophy has long been recognized in IBM, these studies are the first to report specific proteins which are reduced in quantity in IBM muscle.

  19. Rare variants in SQSTM1 and VCP genes and risk of sporadic inclusion body myositis.

    PubMed

    Gang, Qiang; Bettencourt, Conceição; Machado, Pedro M; Brady, Stefen; Holton, Janice L; Pittman, Alan M; Hughes, Deborah; Healy, Estelle; Parton, Matthew; Hilton-Jones, David; Shieh, Perry B; Needham, Merrilee; Liang, Christina; Zanoteli, Edmar; de Camargo, Leonardo Valente; De Paepe, Boel; De Bleecker, Jan; Shaibani, Aziz; Ripolone, Michela; Violano, Raffaella; Moggio, Maurizio; Barohn, Richard J; Dimachkie, Mazen M; Mora, Marina; Mantegazza, Renato; Zanotti, Simona; Singleton, Andrew B; Hanna, Michael G; Houlden, Henry

    2016-11-01

    Genetic factors have been suggested to be involved in the pathogenesis of sporadic inclusion body myositis (sIBM). Sequestosome 1 (SQSTM1) and valosin-containing protein (VCP) are 2 key genes associated with several neurodegenerative disorders but have yet to be thoroughly investigated in sIBM. A candidate gene analysis was conducted using whole-exome sequencing data from 181 sIBM patients, and whole-transcriptome expression analysis was performed in patients with genetic variants of interest. We identified 6 rare missense variants in the SQSTM1 and VCP in 7 sIBM patients (4.0%). Two variants, the SQSTM1 p.G194R and the VCP p.R159C, were significantly overrepresented in this sIBM cohort compared with controls. Five of these variants had been previously reported in patients with degenerative diseases. The messenger RNA levels of major histocompatibility complex genes were upregulated, this elevation being more pronounced in SQSTM1 patient group. We report for the first time potentially pathogenic SQSTM1 variants and expand the spectrum of VCP variants in sIBM. These data suggest that defects in neurodegenerative pathways may confer genetic susceptibility to sIBM and reinforce the mechanistic overlap in these neurodegenerative disorders.

  20. A monoclonal antibody to inclusion body disease of cranes virus enabling specific immunohistochemistry and competitive ELISA

    USGS Publications Warehouse

    Letchworth, G.J.; Fishel, J.R.; Hansen, W.R.

    1997-01-01

    Inclusion body disease of cranes (IBDC) herpesvirus kills some infected cranes and persists in convalescent animals. To enable further study and rapid identification of carrier animals, we developed a monoclonal antibody (MAb) to IBDC virus and used it in immunohistochemistry and a competitive enzyme-linked immunosorbent assay (ELISA). We used conventional techniques to make murine MAbs directed against IBDC virus purified from infected duck embryo cells. Hybridomas reacting in an ELISA with IBDC virus but not uninfected duck embryo cells were characterized by radioimmunoprecipitation, in situ immunohistochemistry, and competitive ELISA with neutralizing and nonneutralizing crane sera. MAb 2C11 immunoprecipitated 59-, 61-, and 110-kD proteins from IBDC virus-infected but not uninfected cells and stained glutaraldehyde-fixed IBDC virus plaques but not surrounding uninfected duck embryo cells in vitro. Antibody 2C11 did not react with duck embryo cells infected with falcon herpesvirus, psittacine herpesvirus, infectious laryngotracheitis, pigeon herpesvirus, or duck plague virus. A competitive ELISA using antibody 2C11 identified most sera that were positive in the neutralization test. This antibody will be useful in further characterizing IBDC virus, its pathogenesis, and its natural history.

  1. Partial characterization of retroviruses from boid snakes with inclusion body disease.

    PubMed

    Jacobson, E R; Orós, J; Tucker, S J; Pollock, D P; Kelley, K L; Munn, R J; Lock, B A; Mergia, A; Yamamoto, J K

    2001-02-01

    To characterize retroviruses isolated from boid snakes with inclusion body disease (IBD). 2 boa constrictors with IBD and 1 boa exposed to an affected snake. Snakes were euthanatized, and tissue specimens and blood samples were submitted for virus isolation. Tissue specimens were cultured with or without commercially available viper heart cells and examined by use of transmission electron microscopy (TEM) for evidence of viral replication. Reverse transcriptase activ ty was determined in sucrose gradient-purified virus. Western blotting was performed, using polyclonal antibodies against 1 of the isolated viruses. Specificity of the rabbit anti-virus antibody was evaluated, using an immunogold-labeling TEM technique. 3 viruses (RV-1, RV-2, and RV-3) were isolated. The isolates were morphologically comparable to members of the Retroviridae family. Reverse transcriptase activity was high in sucrose gradient fractions that were rich in virus. Polyclonal antibody against RV-1 reacted with proteins of similar relative mobility in RV-1 and RV-2. By use of immunogold labeling, this antibody also recognized virions of both RV-1 and RV-2. A retrovirus was isolated from boid snakes with IBD or exposed to IBD. Western blot analysis of viral proteins indicated that viruses isolated from the different snakes were similar. Whether this virus represents the causative agent of IBD is yet to be determined. The isolation of retroviruses from boid snakes with IBD is an important step n the process of identifying the causative agent of this disease.

  2. Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions.

    PubMed

    Steinbuchel, A; Aerts, K; Babel, W; Follner, C; Liebergesell, M; Madkour, M H; Mayer, F; Pieper-Furst, U; Pries, A; Valentin, H E

    1995-01-01

    Some mathematical calculations were done that provided information about the structure and biochemistry of polyhydroxyalkanoic acid (PHA) granules and about the amounts of the different constituents that contribute to the PHA granules. The data obtained from these calculations are compared with data from the literature, which show that PHA granules consist not only of the polyester but also of phospholipids and proteins. The latter are referred to as granule-associated proteins, and they are always located at the surface of the PHA granules. A concept is proposed that distinguishes four classes of structurally and functionally different granule-associated proteins: (i) class I comprises the PHA synthases, which catalyze the formation of ester linkages between the constituents; (ii) class II comprises the PHA depolymerases, which are responsible for the intracellular degradation of PHA, (iii) class III comprises a new type of protein, which is referred to as phasins and which has most probably a function analogous to that of oleosins in oilseed plants, and (iv) class IV comprises all other proteins, which have been found to be associated with the granules but do not belong to classes I-III. Particular emphasis is placed on the phasins, which constitute a significant fraction of the total cellular protein. Phasins are assumed to form a close protein layer at the surface of the granules, providing the interface between the hydrophilic cytoplasm and the much more hydrophobic core of the PHA inclusion.

  3. An amino acid of human parainfluenza virus type 3 nucleoprotein is critical for template function and cytoplasmic inclusion body formation.

    PubMed

    Zhang, Shengwei; Chen, Longyun; Zhang, Guangyuan; Yan, Qin; Yang, Xiaodan; Ding, Binbin; Tang, Qiaopeng; Sun, Shengjun; Hu, Zhulong; Chen, Mingzhou

    2013-11-01

    The nucleoprotein (N) and phosphoprotein (P) interaction of nonsegmented negative-strand RNA viruses is essential for viral replication; this includes N⁰-P (N⁰, free of RNA) interaction and the interaction of N-RNA with P. The precise site(s) within N that mediates the N-P interaction and the detailed regulating mechanism, however, are less clear. Using a human parainfluenza virus type 3 (HPIV3) minigenome assay, we found that an N mutant (N(L478A) did not support reporter gene expression. Using in vivo and in vitro coimmunoprecipitation, we found that N(L478A) maintains the ability to form N(L478A)⁰-P, to self-assemble, and to form N(L478A)-RNA but that N(L478A)-RNA does not interact with P. Using an immunofluorescence assay, we found that N-P interaction provides the minimal requirement for the formation of cytoplasmic inclusion bodies, which contain viral RNA, N, P, and polymerase in HPIV3-infected cells. N(L478A) was unable to form inclusion bodies when coexpressed with P, but the presence of N rescued the ability of N(L478A) to form inclusion bodies and the transcriptional function of N(L478A), thereby suggesting that hetero-oligomers formed by N and N(L478A) are functional and competent to form inclusion bodies. Furthermore, we found that N(L478A) is also defective in virus growth. To our knowledge, we are the first to use a paramyxovirus to identify a precise amino acid within N that is critical for N-RNA and P interaction but not for N(0)-P interaction for the formation of inclusion bodies, which appear to be bona fide sites of RNA synthesis.

  4. An Amino Acid of Human Parainfluenza Virus Type 3 Nucleoprotein Is Critical for Template Function and Cytoplasmic Inclusion Body Formation

    PubMed Central

    Zhang, Shengwei; Chen, Longyun; Zhang, Guangyuan; Yan, Qin; Yang, Xiaodan; Ding, Binbin; Tang, Qiaopeng; Sun, Shengjun; Hu, Zhulong

    2013-01-01

    The nucleoprotein (N) and phosphoprotein (P) interaction of nonsegmented negative-strand RNA viruses is essential for viral replication; this includes N0-P (N0, free of RNA) interaction and the interaction of N-RNA with P. The precise site(s) within N that mediates the N-P interaction and the detailed regulating mechanism, however, are less clear. Using a human parainfluenza virus type 3 (HPIV3) minigenome assay, we found that an N mutant (NL478A) did not support reporter gene expression. Using in vivo and in vitro coimmunoprecipitation, we found that NL478A maintains the ability to form NL478A0-P, to self-assemble, and to form NL478A-RNA but that NL478A-RNA does not interact with P. Using an immunofluorescence assay, we found that N-P interaction provides the minimal requirement for the formation of cytoplasmic inclusion bodies, which contain viral RNA, N, P, and polymerase in HPIV3-infected cells. NL478A was unable to form inclusion bodies when coexpressed with P, but the presence of N rescued the ability of NL478A to form inclusion bodies and the transcriptional function of NL478A, thereby suggesting that hetero-oligomers formed by N and NL478A are functional and competent to form inclusion bodies. Furthermore, we found that NL478A is also defective in virus growth. To our knowledge, we are the first to use a paramyxovirus to identify a precise amino acid within N that is critical for N-RNA and P interaction but not for N0-P interaction for the formation of inclusion bodies, which appear to be bona fide sites of RNA synthesis. PMID:24027324

  5. Nuclear inclusions mimicking poly(A)-binding protein nuclear 1 inclusions in a case of inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia with a novel mutation in the valosin-containing protein gene.

    PubMed

    Matsubara, Shiro; Shimizu, Toshio; Komori, Takashi; Mori-Yoshimura, Madoka; Minami, Narihiro; Hayashi, Yukiko K

    2016-07-01

    A middle-aged Japanese man presented with slowly progressive asymmetric weakness of legs and arm but had neither ptosis nor dysphagia. He had a family history of similar condition suggestive of autosomal dominant inheritance. A muscle biopsy showed mixture of neurogenic atrophy and myopathy with rimmed vacuoles. Furthermore we found intranuclear inclusions that had a fine structure mimicking that of inclusions reported in oculopharyngeal muscular dystrophy (OPMD). Immunohistochemical staining for polyadenylate-binding nuclear protein 1, which is identified within the nuclear inclusions of OPMD, demonstrated nuclear positivity in this case. However, OPMD was thought unlikely based on the clinical features and results of genetic analyses. Instead, a novel mutation in valosin-containing protein, c.376A>T (p.Ile126Phe), was revealed. A diagnosis of inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia was made. This is the first report of polyadenylate-binding nuclear protein 1-positive nuclear inclusions in the muscle of this condition. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Dynamic JUNQ inclusion bodies are asymmetrically inherited in mammalian cell lines through the asymmetric partitioning of vimentin.

    PubMed

    Ogrodnik, Mikołaj; Salmonowicz, Hanna; Brown, Rachel; Turkowska, Joanna; Średniawa, Władysław; Pattabiraman, Sundararaghavan; Amen, Triana; Abraham, Ayelet-chen; Eichler, Noam; Lyakhovetsky, Roman; Kaganovich, Daniel

    2014-06-03

    Aging is associated with the accumulation of several types of damage: in particular, damage to the proteome. Recent work points to a conserved replicative rejuvenation mechanism that works by preventing the inheritance of damaged and misfolded proteins by specific cells during division. Asymmetric inheritance of misfolded and aggregated proteins has been shown in bacteria and yeast, but relatively little evidence exists for a similar mechanism in mammalian cells. Here, we demonstrate, using long-term 4D imaging, that the vimentin intermediate filament establishes mitotic polarity in mammalian cell lines and mediates the asymmetric partitioning of damaged proteins. We show that mammalian JUNQ inclusion bodies containing soluble misfolded proteins are inherited asymmetrically, similarly to JUNQ quality-control inclusions observed in yeast. Mammalian IPOD-like inclusion bodies, meanwhile, are not always inherited by the same cell as the JUNQ. Our study suggests that the mammalian cytoskeleton and intermediate filaments provide the physical scaffold for asymmetric inheritance of dynamic quality-control JUNQ inclusions. Mammalian IPOD inclusions containing amyloidogenic proteins are not partitioned as effectively during mitosis as their counterparts in yeast. These findings provide a valuable mechanistic basis for studying the process of asymmetric inheritance in mammalian cells, including cells potentially undergoing polar divisions, such as differentiating stem cells and cancer cells.

  7. Expression of the inclusion body myopathy 3 mutation in Drosophila depresses myosin function and stability and recapitulates muscle inclusions and weakness

    PubMed Central

    Wang, Yang; Melkani, Girish C.; Suggs, Jennifer A.; Melkani, Anju; Kronert, William A.; Cammarato, Anthony; Bernstein, Sanford I.

    2012-01-01

    Hereditary myosin myopathies are characterized by variable clinical features. Inclusion body myopathy 3 (IBM-3) is an autosomal dominant disease associated with a missense mutation (E706K) in the myosin heavy chain IIa gene. Adult patients experience progressive muscle weakness. Biopsies reveal dystrophic changes, rimmed vacuoles with cytoplasmic inclusions, and focal disorganization of myofilaments. We constructed a transgene encoding E706K myosin and expressed it in Drosophila (E701K) indirect flight and jump muscles to establish a novel homozygous organism with homogeneous populations of fast IBM-3 myosin and muscle fibers. Flight and jump abilities were severely reduced in homozygotes. ATPase and actin sliding velocity of the mutant myosin were depressed >80% compared with wild-type myosin. Light scattering experiments and electron microscopy revealed that mutant myosin heads bear a dramatic propensity to collapse and aggregate. Thus E706K (E701K) myosin appears far more labile than wild-type myosin. Furthermore, mutant fly fibers exhibit ultrastructural hallmarks seen in patients, including cytoplasmic inclusions containing aberrant proteinaceous structures and disorganized muscle filaments. Our Drosophila model reveals the unambiguous consequences of the IBM-3 lesion on fast muscle myosin and fibers. The abnormalities observed in myosin function and muscle ultrastructure likely contribute to muscle weakness observed in our flies and patients. PMID:22496423

  8. Lymphoblastic lymphoma and leukemic blood profile in a red-tail boa (Boa constrictor constrictor) with concurrent inclusion body disease.

    PubMed

    Schilliger, Lionel; Selleri, Paolo; Frye, Fredric L

    2011-01-01

    An adult male wild-caught true red-tail boa (Boa constrictor constrictor), imported from Surinam, was presented for anorexia, extreme lethargy, and coelomic swelling in the cranial third of the body, in the anatomic location of the thymus. The snake died a few minutes after blood sampling via cardiocentesis. Hematology revealed anemia and extreme leukocytosis (820 × 10(3)/ml) characterized by a predominance (95%) of lymphocytes. Necropsy revealed enlargement of most of the visceral organs. Histology confirmed lymphoblastic lymphoma with a leukemic blood profile and diffuse infiltration of some of the heart, thymus, bone marrow, kidney, spleen, lung, and liver. Several large intracytoplasmic eosinophilic inclusion bodies surrounded by narrow clear "halos" were identified within gastric mucosal cells, proximal and distal convoluted tubule epithelial cells, and splenic cells. The final diagnosis was lymphoblast lymphoma with a leukemic blood profile and concurrent inclusion body disease.

  9. TWEAK in inclusion-body myositis muscle: possible pathogenic role of a cytokine inhibiting myogenesis.

    PubMed

    Morosetti, Roberta; Gliubizzi, Carla; Sancricca, Cristina; Broccolini, Aldobrando; Gidaro, Teresa; Lucchini, Matteo; Mirabella, Massimiliano

    2012-04-01

    Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14 exert pleiotropic effects, including regulation of myogenesis. Sporadic inclusion-body myositis (IBM) is the most common muscle disease of the elderly population and leads to severe disability. IBM mesoangioblasts, different from mesoangioblasts in other inflammatory myopathies, display a myogenic differentiation defect. The objective of the present study was to investigate TWEAK-Fn14 expression in IBM and other inflammatory myopathies and explore whether TWEAK modulation affects myogenesis in IBM mesoangioblasts. TWEAK, Fn14, and NF-κB expression was assessed by immunohistochemistry and Western blot in cell samples from both muscle biopsies and primary cultures. Mesoangioblasts isolated from samples of IBM, dermatomyositis, polymyositis, and control muscles were treated with recombinant human TWEAK, Fn14-Fc chimera, and anti-TWEAK antibody. TWEAK-RNA interference was performed in IBM and dermatomyositis mesoangioblasts. TWEAK levels in culture media were determined by enzyme-linked immunosorbent assay. In IBM muscle, we found increased TWEAK-Fn14 expression. Increased levels of TWEAK were found in differentiation medium from IBM mesoangioblasts. Moreover, TWEAK inhibited myogenic differentiation of mesoangioblasts. Consistent with this evidence, TWEAK inhibition by Fn14-Fc chimera or short interfering RNA induced myogenic differentiation of IBM mesoangioblasts. We provide evidence that TWEAK is a negative regulator of human mesoangioblast differentiation. Dysregulation of the TWEAK-Fn14 axis in IBM muscle may induce progressive muscle atrophy and reduce activation and differentiation of muscle precursor cells. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  10. Cytosolic 5'-nucleotidase 1A autoimmunity in sporadic inclusion body myositis.

    PubMed

    Larman, H Benjamin; Salajegheh, Mohammad; Nazareno, Remedios; Lam, Theresa; Sauld, John; Steen, Hanno; Kong, Sek Won; Pinkus, Jack L; Amato, Anthony A; Elledge, Stephen J; Greenberg, Steven A

    2013-03-01

    We previously identified a circulating autoantibody against a 43 kDa muscle autoantigen in sporadic inclusion body myositis (IBM) and demonstrated the feasibility of an IBM diagnostic blood test. Here, we sought to identify the molecular target of this IBM autoantibody, understand the relationship between IBM autoimmunity and muscle degeneration, and develop an IBM blood test with high diagnostic accuracy. IBM blood samples were screened using mass spectrometry and a synthetic human peptidome. Plasma and serum samples (N=200 patients) underwent immunoblotting assays, and results were correlated to clinical features. Muscle biopsy samples (n=30) were examined by immunohistochemistry and immunoblotting. Exome or whole genome sequencing was performed on DNA from 19 patients. Both mass spectrometry and screening of a 413,611 human peptide library spanning the entire human proteome identified cytosolic 5'-nucleotidase 1A (cN1A; NT5C1A) as the likely 43 kDa IBM autoantigen, which was then confirmed in dot blot and Western blot assays using recombinant cN1A protein. Moderate reactivity of anti-cN1A autoantibodies was 70% sensitive and 92% specific, and high reactivity was 34% sensitive and 98% specific for the diagnosis of IBM. One to 3 major cN1A immunodominant epitopes were identified. cN1A reactivity by immunohistochemistry accumulated in perinuclear regions and rimmed vacuoles in IBM muscle, localizing to areas of myonuclear degeneration. Autoantibodies against cN1A are common in and highly specific to IBM among muscle diseases, and may provide a link between IBM's dual processes of autoimmunity and myodegeneration. Blood diagnostic testing is feasible and should improve early and reliable diagnosis of IBM. Copyright © 2013 American Neurological Association.

  11. Calcium dysregulation, functional calpainopathy, and endoplasmic reticulum stress in sporadic inclusion body myositis.

    PubMed

    Amici, David R; Pinal-Fernandez, Iago; Mázala, Davi A G; Lloyd, Thomas E; Corse, Andrea M; Christopher-Stine, Lisa; Mammen, Andrew L; Chin, Eva R

    2017-03-22

    Sporadic inclusion body myositis (IBM) is the most common primary myopathy in the elderly, but its pathoetiology is still unclear. Perturbed myocellular calcium (Ca(2+)) homeostasis can exacerbate many of the factors proposed to mediate muscle degeneration in IBM, such as mitochondrial dysfunction, protein aggregation, and endoplasmic reticulum stress. Ca(2+) dysregulation may plausibly be initiated in IBM by immune-mediated membrane damage and/or abnormally accumulating proteins, but no studies to date have investigated Ca(2+) regulation in IBM patients. We first investigated protein expression via immunoblot in muscle biopsies from IBM, dermatomyositis, and non-myositis control patients, identifying several differentially expressed Ca(2+)-regulatory proteins in IBM. Next, we investigated the Ca(2+)-signaling transcriptome by RNA-seq, finding 54 of 183 (29.5%) genes from an unbiased list differentially expressed in IBM vs. controls. Using an established statistical approach to relate genes with causal transcription networks, Ca(2+) abundance was considered a significant upstream regulator of observed whole-transcriptome changes. Post-hoc analyses of Ca(2+)-regulatory mRNA and protein data indicated a lower protein to transcript ratio in IBM vs. controls, which we hypothesized may relate to increased Ca(2+)-dependent proteolysis and decreased protein translation. Supporting this hypothesis, we observed robust (4-fold) elevation in the autolytic activation of a Ca(2+)-activated protease, calpain-1, as well as increased signaling for translational attenuation (eIF2a phosphorylation) downstream of the unfolded protein response. Finally, in IBM samples we observed mRNA and protein under-expression of calpain-3, the skeletal muscle-specific calpain, which broadly supports proper Ca(2+) homeostasis. Together, these data provide novel insight into mechanisms by which intracellular Ca(2+) regulation is perturbed in IBM and offer evidence of pathological downstream effects.

  12. Aberrant cell cycle reentry in human and experimental inclusion body myositis and polymyositis

    PubMed Central

    Kwon, Bumsup; Kumar, Pravir; Lee, Han-Kyu; Zeng, Ling; Walsh, Kenneth; Fu, Qinghao; Barakat, Amey; Querfurth, Henry W.

    2014-01-01

    Inclusion body myositis (IBM), a degenerative and inflammatory disorder of skeletal muscle, and Alzheimer's disease share protein derangements and attrition of postmitotic cells. Overexpression of cyclins and proliferating cell nuclear antigen (PCNA) and evidence for DNA replication is reported in Alzheimer's disease brain, possibly contributing to neuronal death. It is unknown whether aberrant cell cycle reentry also occurs in IBM. We examined cell cycle markers in IBM compared with normal control, polymyositis (PM) and non-inflammatory dystrophy sample sets. Next, we tested for evidence of reentry and DNA synthesis in C2C12 myotubes induced to express β-amyloid (Aβ42). We observed increased levels of Ki-67, PCNA and cyclins E/D1 in IBM compared with normals and non-inflammatory conditions. Interestingly, PM samples displayed similar increases. Satellite cell markers did not correlate with Ki-67-affected myofiber nuclei. DNA synthesis and cell cycle markers were induced in Aβ-bearing myotubes. Cell cycle marker and cyclin protein expressions were also induced in an experimental allergic myositis-like model of PM in mice. Levels of p21 (Cip1/WAF1), a cyclin-dependent kinase inhibitor, were decreased in affected myotubes. However, overexpression of p21 did not rescue cells from Aβ-induced toxicity. This is the first report of cell cycle reentry in human myositis. The absence of rescue and evidence for reentry in separate models of myodegeneration and inflammation suggest that new DNA synthesis may be a reactive response to either or both stressors. PMID:24556217

  13. Synaptic defects associated with s-inclusion body myositis are prevented by copper.

    PubMed

    Aldunate, R; Minniti, A N; Rebolledo, D; Inestrosa, N C

    2012-08-01

    Sporadic-inclusion body myositis (s-IBM) is the most common skeletal muscle disorder to afflict the elderly, and is clinically characterized by skeletal muscle degeneration. Its progressive course leads to muscle weakness and wasting, resulting in severe disability. The exact pathogenesis of this disease is unknown and no effective treatment has yet been found. An intriguing aspect of s-IBM is that it shares several molecular abnormalities with Alzheimer's disease, including the accumulation of amyloid-β-peptide (Aβ). Both disorders affect homeostasis of the cytotoxic fragment Aβ(1-42) during aging, but they are clinically distinct diseases. The use of animals that mimic some characteristics of a disease has become important in the search to elucidate the molecular mechanisms underlying the pathogenesis. With the aim of analyzing Aβ-induced pathology and evaluating the consequences of modulating Aβ aggregation, we used Caenorhabditis elegans that express the Aβ human peptide in muscle cells as a model of s-IBM. Previous studies indicate that copper treatment increases the number and size of amyloid deposits in muscle cells, and is able to ameliorate the motility impairments in Aβ transgenic C. elegans. Our recent studies show that neuromuscular synaptic transmission is defective in animals that express the Aβ-peptide and suggest a specific defect at the nicotine acetylcholine receptors level. Biochemical analyses show that copper treatment increases the number of amyloid deposits but decreases Aβ-oligomers. Copper treatment improves motility, synaptic structure and function. Our results suggest that Aβ-oligomers are the toxic Aβ species that trigger neuromuscular junction dysfunction.

  14. Isolated inclusion body myopathy caused by a multisystem proteinopathy–linked hnRNPA1 mutation

    PubMed Central

    Izumi, Rumiko; Warita, Hitoshi; Niihori, Tetsuya; Takahashi, Toshiaki; Tateyama, Maki; Suzuki, Naoki; Nishiyama, Ayumi; Shirota, Matsuyuki; Funayama, Ryo; Nakayama, Keiko; Mitsuhashi, Satomi; Nishino, Ichizo; Aoki, Yoko

    2015-01-01

    Objective: To identify the genetic cause of isolated inclusion body myopathy (IBM) with autosomal dominant inheritance in 2 families. Methods: Genetic investigations were performed using whole-exome and Sanger sequencing of the heterogeneous nuclear ribonucleoprotein A1 gene (hnRNPA1). The clinical and pathologic features of patients in the 2 families were evaluated with neurologic examinations, muscle imaging, and muscle biopsy. Results: We identified a missense p.D314N mutation in hnRNPA1, which is also known to cause familial amyotrophic lateral sclerosis, in 2 families with IBM. The affected individuals developed muscle weakness in their 40s, which slowly progressed toward a limb-girdle pattern. Further evaluation of the affected individuals revealed no apparent motor neuron dysfunction, cognitive impairment, or bone abnormality. The muscle pathology was compatible with IBM, lacking apparent neurogenic change and inflammation. Multiple immunohistochemical analyses revealed the cytoplasmic aggregation of hnRNPA1 in close association with autophagosomes and myonuclei. Furthermore, the aberrant accumulation was characterized by coaggregation with ubiquitin, sequestome-1/p62, valosin-containing protein/p97, and a variety of RNA-binding proteins (RBPs). Conclusions: The present study expands the clinical phenotype of hnRNPA1-linked multisystem proteinopathy. Mutations in hnRNPA1, and possibly hnRNPA2B1, will be responsible for isolated IBM with a pure muscular phenotype. Although the mechanisms underlying the selective skeletal muscle involvement remain to be elucidated, the immunohistochemical results suggest a broad sequestration of RBPs by the mutated hnRNPA1. PMID:27066560

  15. Improving the refolding efficiency for proinsulin aspart inclusion body with optimized buffer compositions.

    PubMed

    Chen, Ying; Wang, Qi; Zhang, Chun; Li, Xiunan; Gao, Qiang; Dong, Changqing; Liu, Yongdong; Su, Zhiguo

    2016-06-01

    Successfully recovering proinsulin's native conformation from inclusion body is the crucial step to guarantee high efficiency for insulin's manufacture. Here, two by-products of disulfide-linked oligomers and disulfide-isomerized monomers were clearly identified during proinsulin aspart's refolding through multiple analytic methods. Arginine and urea are both used to assist in proinsulin refolding, however the efficacy and possible mechanism was found to be different. The oligomers formed with urea were of larger size than with arginine. With the urea concentrations increasing from 2 M to 4 M, the content of oligomers decreased greatly, but simultaneously the refolding yield at the protein concentration of 0.5 mg/mL decreased from 40% to 30% due to the increase of disulfide-isomerized monomers. In contrast, with arginine concentrations increasing up to 1 M, the refolding yield gradually increased to 50% although the content for oligomers also decreased. Moreover, it was demonstrated that not redox pairs but only oxidant was necessary to facilitate the native disulfide bonds formation for the reduced denatured proinsulin. An oxidative agent of selenocystamine could increase the yield up to 80% in the presence of 0.5 M arginine. Further study demonstrated that refolding with 2 M urea instead of 0.5 M arginine could achieve similar yield as protein concentration is slightly reduced to 0.3 mg/mL. In this case, refolded proinsulin was directly purified through one-step of anionic exchange chromatography, with a recovery of 32% and purity up to 95%. All the results could be easily adopted in insulin's industrial manufacture for improving the production efficiency. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Delayed emergence of subdiffractionsized mutant huntingtin fibrils following inclusion body formation

    PubMed Central

    Sahl, Steffen J.; Lau, Lana; Vonk, Willianne I. M.; Weiss, Lucien E.; Frydman, Judith; Moerner, W. E.

    2015-01-01

    Aberrant aggregation of improperly folded proteins is the hallmark of several human neurodegenerative disorders, including Huntington’s Disease (HD) with autosomal-dominant inheritance. In HD, expansion of the CAG-repeat-encoded polyglutamine (polyQ) stretch beyond ~40 glutamines in huntingtin (Htt) and its N-terminal fragments leads to the formation of large (up to several μm) globular neuronal inclusion bodies (IBs) over time. We report direct observations of aggregating Htt exon 1 in living and fixed cells at enhanced spatial resolution by stimulated emission depletion (STED) microscopy and single-molecule super-resolution optical imaging. Fibrils of Htt exon 1 arise abundantly across the cytosolic compartment and also in neuritic processes only after nucleation and aggregation into a fairly advanced stage of growth of the prominent IB have taken place. Structural characterizations of fibrils by STED show a distinct length cutoff at ~1·5 μm and reveal subsequent coalescence (bundling/piling). Cytosolic fibrils are observed even at late stages in the process, side-by-side with the mature IB. Htt sequestration into the IB, which in neurons has been argued to be a cell-protective phenomenon, thus appears to saturate and over-power the cellular degradation systems and leaves cells vulnerable to further aggregation producing much smaller, potentially toxic, conformational protein species of which the fibrils may be comprised. We further found that exogenous delivery of the apical domain of the chaperonin subunit CCT1 to the cells via the cell medium reduced the aggregation propensity of mutant Htt exon 1 in general, and strongly reduced the occurrence of such late-stage fibrils in particular. PMID:26350150

  17. Preparation of active recombinant cathepsin K expressed in bacteria as inclusion body.

    PubMed

    Hwang, Hyo-Sung; Chung, Hye-Shin

    2002-08-01

    Human cathepsin K (EC 3.4.22.38) is a member of the cysteine protease family with high primary sequence homology to cathepsins S, L, and B. It has been shown that cathepsin K plays a major role in the resorption of the bone matrix by osteoclasts. Cathepsin K has a potential as a drug target for the diseases related to bone matrix metabolism such as osteoporosis. We have expressed recombinant human procathepsin K in Escherichia coli as inclusion bodies. Purified procathepsin K had size of 38kDa which is in agreement with the predicted mass of the construct. Refolding was done by rapid dilution into 50mM Tris-HCl, pH 8.0 buffer containing 5mM EDTA, 10 mM GSH, 1mM GSSG, 0.7 M L-arginine, 0.5 M NaCl, and 1% CHAPS and further dialysis against 25 mM Tris-HCl, pH 8.0 containing 0.5 M NaCl. Mature active cathepsin K was prepared from refolded procathepsin K by incubating at 40 degrees C in pH 4.0 buffers with or without pepsin or cysteine. The presence of pepsin or cysteine in autocatalysis buffer did not have effect on the degree of conversion of nascent to mature cathepsin K, but reduced the autocatalysis time slightly. Proteolytic activity was confirmed using synthetic substrate, and Western blotting identified mature cathepsin K. Active cathepsin K had type I and II collagenolytic activities which could be inhibited by E-64.

  18. Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination.

    PubMed

    Tamaki, Yukihiro; Harakuni, Tetsuya; Yamaguchi, Rui; Miyata, Takeshi; Arakawa, Takeshi

    2016-03-04

    The cholera toxin B subunit (CTB) is secreted in its pentameric form from Escherichia coli if its leader peptide is replaced with one of E. coli origin. However, the secretion of the pentamer is generally severely impaired when the molecule is mutated or fused to a foreign peptide. Therefore, we attempted to regenerate pentameric CTB from the inclusion bodies (IBs) of E. coli. Stepwise dialysis of the IBs solubilized in guanidine hydrochloride predominantly generated soluble high-molecular-mass (HMM) aggregates and only a small fraction of pentamer. Three methods to reassemble homogeneous pentameric molecules were evaluated: (i) using a pentameric coiled-coil fusion partner, expecting it to function as an assembly core; (ii) optimizing the protein concentration during refolding; and (iii) eliminating contaminants before refolding. Coiled-coil fusion had some effect, but substantial amounts of HMM aggregates were still generated. Varying the protein concentration from 0.05 mg/mL to 5mg/mL had almost no effect. In contrast, eliminating the contaminants before refolding had a robust effect, and only the pentamer was regenerated, with no detectable HMM aggregates. Surprisingly, the protein concentration at refolding was up to 5mg/mL when the contaminants were removed, with no adverse effects on refolding. The regenerated pentamer was indistinguishable in its biochemical and immunological characteristics from CTB secreted from E. coli or choleragenoid from Vibrio cholerae. This study provides a simple but very efficient strategy for pentamerizing CTB with a highly homogeneous molecular conformation, with which it may be feasible to engineer CTB derivatives and CTB fusion antigens. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Application of an E. coli signal sequence as a versatile inclusion body tag.

    PubMed

    Jong, Wouter S P; Vikström, David; Houben, Diane; van den Berg van Saparoea, H Bart; de Gier, Jan-Willem; Luirink, Joen

    2017-03-21

    Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form. When screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins. We present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs.

  20. Intravenous immune globulin in hereditary inclusion body myopathy: a pilot study

    PubMed Central

    Sparks, Susan; Rakocevic, Goran; Joe, Galen; Manoli, Irini; Shrader, Joseph; Harris-Love, Michael; Sonies, Barbara; Ciccone, Carla; Dorward, Heidi; Krasnewich, Donna; Huizing, Marjan; Dalakas, Marinos C; Gahl, William A

    2007-01-01

    Background Hereditary Inclusion Body Myopathy (HIBM) is an autosomal recessive, adult onset, non-inflammatory neuromuscular disorder with no effective treatment. The causative gene, GNE, codes for UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, which catalyzes the first two reactions in the synthesis of sialic acid. Reduced sialylation of muscle glycoproteins, such as α-dystroglycan and neural cell adhesion molecule (NCAM), has been reported in HIBM. Methods We treated 4 HIBM patients with intravenous immune globulin (IVIG), in order to provide sialic acid, because IgG contains 8 μmol of sialic acid/g. IVIG was infused as a loading dose of 1 g/kg on two consecutive days followed by 3 doses of 400 mg/kg at weekly intervals. Results For all four patients, mean quadriceps strength improved from 19.0 kg at baseline to 23.2 kg (+22%) directly after IVIG loading to 25.6 kg (+35%) at the end of the study. Mean shoulder strength improved from 4.1 kg at baseline to 5.9 kg (+44%) directly after IVIG loading to 6.0 kg (+46%) at the end of the study. The composite improvement for 8 other muscle groups was 5% after the initial loading and 19% by the end of the study. Esophageal motility and lingual strength improved in the patients with abnormal barium swallows. Objective measures of functional improvement gave variable results, but the patients experienced improvements in daily activities that they considered clinically significant. Immunohistochemical staining and immunoblotting of muscle biopsies for α-dystroglycan and NCAM did not provide consistent evidence for increased sialylation after IVIG treatment. Side effects were limited to transient headaches and vomiting. Conclusion The mild benefits in muscle strength experienced by HIBM patients after IVIG treatment may be related to the provision of sialic acid supplied by IVIG. Other sources of sialic acid are being explored as treatment options for HIBM. PMID:17261181

  1. Lower extremity peak force and gait kinematics in individuals with inclusion body myositis

    PubMed Central

    Davenport, Todd E.; Benson, Kimberly; Baker, Stephanie; Gracey, Christopher; Rakocevic, Goran; McElroy, Beverly; Dalakas, Marinos; Shrader, Joseph A.; Harris-Love, Michael O.

    2015-01-01

    Objective To determine the relationship between peak isometric muscle force and temporal characteristics of gait in individuals with sporadic inclusion body myositis (s-IBM). Patients and Methods An observational study of 42 individuals with s-IBM (12 female; age: 61.6 ±7.3 years [mean ±standard deviation]; disease duration 8.9 ±4.3 years) was conducted at a Federal hospital. Peak isometric force measurements for lower extremity (LE) muscle groups were obtained using quantitative muscle testing. Temporal characteristics of gait during habitual and fast walking conditions were measured using a portable gait analysis system. Results All observed muscle force values were significantly lower than predicted values (p <.001). During habitual walking, subjects’ gait speed and cadence were < 83% of normative literature values. During the fast walking, total gait cycle time was 133% of normal, while gait speed and cadence were 58% and 78%, respectively, of normative literature values. Scaled LE peak muscle forces showed significant moderate correlations with the temporal gait variables. Weaker subjects demonstrated greater limitations in gait speed and cadence compared to stronger subjects (p <.05). Peak isometric force of the knee flexors and ankle plantar flexors, but not knee extensors, were significantly correlated with most temporal features of habitual gait. Conclusions Muscle weakness associated with s-IBM disease activity may contribute to diminished gait kinematics. Temporal features of gait are not substantially influenced by knee extensor weakness alone, as the knee flexors and ankle plantar flexors play a compensatory role in maintaining the walking ability of individuals with s-IBM. PMID:25201017

  2. Effects of dietary inclusion of silymarin on performance, intestinal morphology and ileal bacterial count in aflatoxin-challenged broiler chicks.

    PubMed

    Jahanian, E; Mahdavi, A H; Asgary, S; Jahanian, R

    2017-01-04

    This study was conducted to investigate the effect of dietary supplementation of silymarin on performance, jejunal morphology and ileal bacterial population in broiler chicks intoxicated with a mix of aflatoxins. A total of three hundred thirty six 7-day-old Ross broiler chicks were randomly distributed between seven experimental groups with four replicates of 12 birds each. Experimental treatments consisted of a control group (unchallenged), and a 2 × 3 factorial arrangement, including two aflatoxin levels (0.5 and 2 ppm) and three levels of silymarin (0, 500 and 1000 ppm). Birds were challenged with a mix of aflatoxins from 7 to 28 days of age. Results showed that increasing aflatoxin level resulted in decreased average daily feed intake (ADFI) and weight gain (ADWG), consequently impaired feed conversion ratio (FCR) throughout the trial period. Dietary supplementation of silymarin resulted in the marked increases in ADFI and ADWG, and improved FCR values in aflatoxin-challenged chicks. Ileal bacterial populations at days 28 and 42 of age were increased by incremental levels of aflatoxins. On the other hand, dietary silymarin supplementation suppressed ileal populations of Escherichia coli, Salmonella, Klebsiella and total negative bacteria in aflatoxicated birds. Increase in dietary aflatoxin level resulted in the decreased villi height, villi height-to-crypt depth ratio (VH:CD), villi surface area and apparent villi absorptive area, while it increased crypt depth, goblet cell count and lymphoid follicular diameter. Feeding silymarin at the level of 1000 ppm increased villi height and VH:CD in aflatoxicated birds. Present results indicate that dietary inclusion of silymarin could improve performance by suppressing ileal bacteria and enhancing absorptive surface area in aflatoxin-challenged broiler chicks.

  3. Early bacterial genome detection in body fluids from patients with severe sepsis: a pilot study.

    PubMed

    Dugard, Anthony; Chainier, Delphine; Barraud, Olivier; Garnier, Fabien; Ploy, Marie-Cécile; Vignon, Philippe; François, Bruno

    2012-08-01

    The purpose of this study is to evaluate the feasibility and interest of real-time polymerase chain reaction (RT-PCR) testing for bacterial genomes in body fluids other than blood in patients with acute severe sepsis. Twenty-six consecutive patients admitted for severe sepsis or septic shock were prospectively studied. Body fluids were sampled as clinically indicated and tested using standard microbiological methods and modified RT-PCR methods (universal PCR and specific PCRs). Results of standard microbiological tests were compared with those of PCR tests. Direct RT-PCR testing was successfully performed on all nonblood body fluids. Of 29 body fluids collected, 23 were positive for at least 1 microorganism with conventional tests. Of 18 microbiological tests positive for a single microorganism, 15 fully agreed with RT-PCR assays, and the remaining 3 samples were infected with bacteria not screened by PCR testing. Among the 5 polymicrobial results obtained with conventional tests, RT-PCR agreed in 4 patients. The RT-PCR tests allowed additional clinically relevant bacterial identification in 3 of 6 samples with negative microbiological culture. Our results indicate that direct PCR testing may improve the detection of bacteria in body fluids other than blood in patients with acute severe sepsis. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Evaluation of the inclusion of circular RNAs in mRNA profiling in forensic body fluid identification.

    PubMed

    Zhang, Yaqi; Liu, Baonian; Shao, Chengchen; Xu, Hongmei; Xue, Aimin; Zhao, Ziqin; Shen, Yiwen; Tang, Qiqun; Xie, Jianhui

    2017-09-25

    The use of messenger RNA (mRNA) profiling is considered a promising method in the identification of forensically relevant body fluids which can provide crucial information for reconstructing a potential crime. However, casework samples are usually of limited quantity or have been subjected to degradation, which requires improvement of body fluid identification. Circular RNAs (circRNAs), a class of products from the backsplicing of pre-mRNAs, are shown to have high abundance, remarkable stability, and cell type-specific expression in human cells. In this study, we investigated whether the inclusion of circRNAs in mRNA profiling improve the detection of biomarkers including δ-aminolevulinate synthase 2 (ALAS2) and matrix metallopeptidase 7 (MMP7) in body fluid identification. The major circRNAs of ALAS2 and MMP7 were first identified and primer sets for the simultaneous detection of linear and circular transcripts were developed. The inclusion of circRNAs in mRNA profiling showed improved detection sensitivity and stability of biomarkers revealed by using serial dilutions, mixed samples, and menstrual bloodstains as well as degraded and aged samples. Therefore, the inclusion of circRNAs in mRNA profiling should facilitate the detection of mRNA markers in forensic body fluid identification.

  5. AN OUTBREAK OF PSITTACOSIS IN PIGEONS, INVOLVING THE PRODUCTION OF INCLUSION BODIES, AND TRANSFER OF THE DISEASE TO MAN

    PubMed Central

    Smadel, Joseph E.; Wall, M. J.; Gregg, Alan

    1943-01-01

    An epizootic disease in pigeons associated with atypical pneumonia in two persons handling the birds has been studied. Most of the observations made during the work were consistent with the idea that we were dealing with an infection caused by a member of the psittacosis-lymphogranuloma venereum group of viruses. The outbreak was peculiar, however, in that tissues of the diseased pigeons contained many intranuclear inclusions and that the viruses isolated from these birds produced both intranuclear inclusions and elementary bodies in the cytoplasm of cells of chorio-allantoic membranes of the developing egg. Whether the pigeons were simultaneously infected with two viruses or whether the virus of pigeon psittacosis can produce intranuclear inclusions under certain conditions remains to be determined. PMID:19871321

  6. Prolyl-isomerase Pin1 accumulates in lewy bodies of parkinson disease and facilitates formation of alpha-synuclein inclusions.

    PubMed

    Ryo, Akihide; Togo, Takashi; Nakai, Toshiki; Hirai, Akiko; Nishi, Mayuko; Yamaguchi, Akira; Suzuki, Kyoko; Hirayasu, Yoshio; Kobayashi, Hideki; Perrem, Kilian; Liou, Yih-Cherng; Aoki, Ichiro

    2006-02-17

    Parkinson disease (PD) is a relatively common neurodegenerative disorder that is characterized by the loss of dopaminergic neurons and by the formation of Lewy bodies (LBs), which are cytoplasmic inclusions containing aggregates of alpha-synuclein. Although certain post-translational modifications of alpha-synuclein and its related proteins are implicated in the genesis of LBs, the specific molecular mechanisms that both regulate these processes and initiate subsequent inclusion body formation are not yet well understood. We demonstrate in our current study, however, that the prolyl-isomerase Pin1 localizes to the LBs in PD brain tissue and thereby enhances the formation of alpha-synuclein immunoreactive inclusions. Immunohistochemical analysis of brain tissue from PD patients revealed that Pin1 localizes to 50-60% of the LBs that show an intense halo pattern resembling that of alpha-synuclein. By utilizing a cellular model of alpha-synuclein aggregation, we also demonstrate that, whereas Pin1 overexpression facilitates the formation of alpha-synuclein inclusions, dominant-negative Pin1 expression significantly suppresses this process. Consistent with these observations, Pin1 overexpression enhances the protein half-life and insolubility of alpha-synuclein. Finally, we show that Pin1 binds synphilin-1, an alpha-synuclein partner, via its Ser-211-Pro and Ser-215-Pro motifs, and enhances its interaction with alpha-synuclein, thus likely facilitating the formation of alpha-synuclein inclusions. These results indicate that Pin1-mediated prolyl-isomerization plays a pivotal role in a post-translational modification pathway for alpha-synuclein aggregation and in the resultant Lewy body formations in PD.

  7. Cooperation between two ClpB isoforms enhances the recovery of the recombinant {beta}-galactosidase from inclusion bodies

    SciTech Connect

    Guenther, Izabela; Zolkiewski, Michal; Kedzierska-Mieszkowska, Sabina

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer An important role of synergistic cooperation between the two ClpB isoforms. Black-Right-Pointing-Pointer Both ClpB isoforms are associated with IBs of {beta}-galactosidase. Black-Right-Pointing-Pointer ClpB is a key chaperone in IB protein release. -- Abstract: Bacterial ClpB is a molecular chaperone that solubilizes and reactivates aggregated proteins in cooperation with the DnaK chaperone system. The mechanism of protein disaggregation mediated by ClpB is linked to translocation of substrates through the central channel within the ring-hexameric structure of ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and the truncated ClpB80 (ClpB{Delta}N), which does not contain the N-terminal domain. The functional specificity of the two ClpB isoforms and the biological role of the N-terminal domain are still not fully understood. Recently, it has been demonstrated that ClpB may achieve its full potential as an aggregate-reactivating chaperone through the functional interaction and synergistic cooperation of its two isoforms. It has been found that the most efficient resolubilization and reactivation of stress-aggregated proteins occurred in the presence of both ClpB95 and ClpB80. In this work, we asked if the two ClpB isoforms functionally cooperate in the solubilization and reactivation of proteins from insoluble inclusion bodies (IBs) in Escherichia coli cells. Using the model {beta}-galactosidase fusion protein (VP1LAC), we found that solubilization and reactivation of enzymes entrapped in IBs occurred more efficiently in the presence of ClpB95 with ClpB80 than with either ClpB95 or ClpB80 alone. The two isoforms of ClpB chaperone acting together enhanced the solubility and enzymatic activity of {beta}-galactosidase sequestered into IBs. Both ClpB isoforms were associated with IBs of {beta}-galactosidase, what demonstrates their affinity to this type of aggregates. These results demonstrate a synergistic

  8. [Clinical and histopathological studies of cases of lafora-like inclusion bodies].

    PubMed

    Yoshimura, T

    1977-01-01

    In case 1, 41-year-old male, developed progressive demetia, paretic gait disturbance and pyramidal signs with the duration of three years. The neuropathological study revealed systemic atrophy as type Pick-disease i.e., lobal atrophy in the frontal and the parieto-occipital regions, degenerative changes in the basal ganglia and in the thalamus, nerve cell loss in the substantia nigra and myelin pallor in the pyramidal tract. Lafora-like inclusions were found in the cerebral cortex and in the cochleal nucleus. In case 2, 45-year-old male, showed character change, cerebellar symptomes and mental deteriotation, and ulcers on the oral mucosa during about 15 years long period. Neuropathological examination showed chronic encephalitis in the brain stem, vacuolar change in the neuron in the olivary nucleus and Lafora-like inclusions in the cochlear nucleus. Though neither generalized conversion nor myoclonus were clinicaly observed in these cases, the inclusions showed histochemically strong similarity with that of the Lafora-disease. These Lafora-like inclusions were compared with those in the literatur, which were reported on various disease of CNS. Finally in respect of predilection of the inclusions, it is likely that the inclusions result from same metabolic disturbance in the cochlear neurons in the Lafora-disease as well as in the present cases.

  9. Efficient inclusion body processing using chemical extraction and high gradient magnetic fishing.

    PubMed

    Heebøll-Nielsen, Anders; Choe, Woo-Seok; Middelberg, Anton P J; Thomas, Owen R T

    2003-01-01

    In this study we introduce a radical new approach for the recovery of proteins expressed in the form of inclusion bodies, involving (i) chemical extraction from the host cells, (ii) adsorptive capture of the target protein onto small magnetic adsorbents, and (iii) subsequent rapid collection of the product-loaded supports with the aid of high gradient magnetic fields. The manufacture and testing of two types of micron-sized nonporous superparamagnetic metal chelator particles derivatized with iminodiacetic acid is described. In small-scale adsorption studies conducted with a hexahistidine tagged form of the L1 coat protein of human papillomavirus type 16 dissolved in 8 M urea-phosphate buffer, the best binding performance (Q(max) = 58 mg g(-1) and K(d) approximately 0.08 microM) was exhibited by Cu(2+)-charged type II support materials. Equilibrium adsorption of L1 to these nonporous supports was achieved very rapidly (<300 s), and approximately 90% of the tightly bound L1 could be desorbed in just one elution step by including >100 mM imidazole in the equilibration buffer. The influence of feedstock complexity on L1 adsorption to the Cu(2+)-charged type II magnetic chelators was studied using various dilutions of four crude chemical E. coli cell extracts containing denatured L1 protein. Undiminished L1 adsorption to these adsorbents (relative to the 8 M urea-phosphate buffer case) was observed with the least complex of these feed materials, i.e., a partially clarified (12 g dry weight L(-1)) and spermine-treated chemical cell extract (feedstock B). Efficient recovery of L1 from feed B was demonstrated at a 60-fold increased scale using the high gradient magnetic fishing (HGMF) system to collect loaded Cu(2+)-chelator particles following batch adsorption of L1. Over 70% of the initial L1 present was recovered within the HGMF rig in a highly clarified form in two batch elution cycles with an overall purification factor of approximately 10.

  10. Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8.

    PubMed

    Wang, Xu; Zhou, Bihong; Hu, Weike; Zhao, Qing; Lin, Zhanglin

    2015-06-16

    In the last few decades, several groups have observed that proteins expressed as inclusion bodies (IBs) in bacteria could still be biologically active when terminally fused to an appropriate aggregation-prone partner such as pyruvate oxidase from Paenibacillus polymyxa (PoxB). More recently, we have demonstrated that three amphipathic self-assembling peptides, an alpha helical peptide 18A, a beta-strand peptide ELK16, and a surfactant-like peptide L6KD, have properties that induce target proteins into active IBs. We have developed an efficient protein expression and purification approach for these active IBs by introducing a self-cleavable intein molecule. In this study, the self-assembling peptide GFIL8 (GFILGFIL) with only hydrophobic residues was analyzed, and this peptide effectively induced the formation of cytoplasmic IBs in Escherichia coli when terminally attached to lipase A and amadoriase II. The protein aggregates in cells were confirmed by transmission electron microscopy analysis and retained ~50% of their specific activities relative to the native counterparts. We constructed an expression and separation coupled tag (ESCT) by incorporating an intein molecule, the Mxe GyrA intein. Soluble target proteins were successfully released from active IBs upon cleavage of the intein between the GFIL8 tag and the target protein, which was mediated by dithiothreitol. A variant of GFIL8, GFIL16 (GFILGFILGFILGFIL), improved the ESCT scheme by efficiently eliminating interference from the soluble intein-GFIL8 molecule. The yields of target proteins at the laboratory scale were 3.0-7.5 μg/mg wet cell pellet, which is comparable to the yields from similar ESCT constructs using 18A, ELK16, or the elastin-like peptide tag scheme. The all-hydrophobic self-assembling peptide GFIL8 induced the formation of active IBs in E. coli when terminally attached to target proteins. GFIL8 and its variant GFIL16 can act as a "pull-down" tag to produce purified soluble proteins with

  11. Sleep disordered breathing in a cohort of patients with sporadic inclusion body myositis.

    PubMed

    Della Marca, Giacomo; Sancricca, Cristina; Losurdo, Anna; Di Blasi, Chiara; De Fino, Chiara; Morosetti, Roberta; Broccolini, Aldobrando; Testani, Elisa; Scarano, Emanuele; Servidei, Serenella; Mirabella, Massimiliano

    2013-08-01

    The aims of the study were: (1) to evaluate subjective sleep quality and daytime sleepiness in patients affected by sporadic inclusion-body myositis (IBM); (2) to define the sleep and sleep-related respiratory pattern in IBM patients. Thirteen consecutive adult patients affected by definite IBM were enrolled, six women and seven men, mean age 66.2 ± 11.1 years (range: 50-80). Diagnosis was based on clinical and muscle biopsy studies. All patients underwent subjective sleep evaluation (Pittsburgh Sleep Quality Index, PSQI and Epworth Sleepiness Scale, ESS), oro-pharingo-esophageal scintigraphy, pulmonary function tests, psychometric measures, anatomic evaluation of upper airways, and laboratory-based polysomnography. Findings in IBM patients were compared to those obtained from a control group of 25 healthy subjects (13 men and 12 women, mean age 61.9 ± 8.6 years). Disease duration was >10 years in all. Mean IBM severity score was 28.8 ± 5.4 (range 18-36). Dysphagia was present in 10 patients. Nine patients had PSQI scores ≥ 5; patients had higher mean PSQI score (IBM: 7.2 ± 4.7, CONTROLS: 2.76 ± 1.45, p=0.005); one patient (and no controls) had EES>9. Polysomnography showed that IBM patients, compared to controls, had lower sleep efficiency (IBM: 78.8 ± 12.0%, 94.0 ± 4.5%, p<0.001), more awakenings (IBM: 11.9 ± 11.0, CONTROLS: 5.2 ± 7.5, p=0.009) and increased nocturnal time awake (IBM: 121.2 ± 82.0 min., 46.12 ± 28.8 min., p=0.001). Seven Patients (and no controls) had polysomnographic findings consistent with sleep disordered breathing (SDB). Data suggest that sleep disruption, and in particular SDB, might be highly prevalent in IBM. Data indicate that IBM patients have poor sleep and high prevalence of SDB. Copyright © 2013 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

  12. Unfolding story of inclusion-body myositis and myopathies: role of misfolded proteins, amyloid-beta, cholesterol, and aging.

    PubMed

    Askanas, Valerie; Engel, W King

    2003-03-01

    Sporadic inclusion-body myositis and hereditary inclusion-body myopathies are progressive muscle diseases leading to severe disability. We briefly summarize their clinical pictures and pathologic diagnostic criteria and discuss the latest advances in illuminating their pathogenic mechanism(s). We emphasize how different etiologies might lead to the strikingly similar pathology and possibly similar pathogenic cascade. On the basis of our research, several processes seem to be important in relation to the still speculative pathogenesis, including (a) increased transcription and accumulation of amyloid-beta precursor protein and accumulation of its proteolytic fragment amyloid-beta; (b) abnormal accumulation of components related to lipid metabolism, for example, cholesterol, accumulation of which is possibly owing to its abnormal trafficking; (c) oxidative stress; (d) accumulations of other Alzheimer's disease-related proteins; and (e) a milieu of muscle cellular aging in which these changes occur. We discuss a potentially very important role of unfolded and/or misfolded proteins as a possible mechanism in the formations of the inclusion bodies and other abnormalities.

  13. Iron- and 4-hydroxy-2-alkylquinoline-containing periplasmic inclusion bodies of Pseudomonas aeruginosa: A chemical analysis

    USGS Publications Warehouse

    Royt, P.W.; Honeychuck, R.V.; Pant, R.R.; Rogers, M.L.; Asher, L.V.; Lloyd, J.R.; Carlos, W.E.; Belkin, H.E.; Patwardhan, S.

    2007-01-01

    Dark aggregated particles were seen on pellets of iron-rich, mid-logarithmic phase Pseudomonas aeruginosa. Transmission electron microscopy of these cells showed inclusion bodies in periplasmic vacuoles. Aggregated particles isolated from the spent medium of these cells contained iron as indicated by atomic absorption spectroscopy and by electron paramagnetic resonance spectroscopy that revealed Fe3+. Scanning electron microscopy/energy dispersive X-ray analysis of whole cells revealed the presence of iron-containing particles beneath the surface of the cell, indicating that the isolated aggregates were the intracellular inclusion bodies. Collectively, mass spectroscopy and nuclear magnetic resonance spectroscopy of the isolated inclusion bodies revealed the presence of 3,4-dihydroxy-2-heptylquinoline which is the Pseudomonas quinolone signaling compound (PQS) and an iron chelator; 4-hydroxy-2-heptylquinoline (pseudan VII), which is an iron chelator, antibacterial compound and precursor of PQS; 4-hydroxy-2-nonylquinoline (pseudan IX) which is an iron chelator and antibacterial compound; 4-hydroxy-2-methylquinoline (pseudan I), and 4-hydroxy-2-nonylquinoline N-oxide. ?? 2006 Elsevier Inc. All rights reserved.

  14. Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3).

    PubMed

    Kumar, Sandeep; Jain, Kavish Kumar; Singh, Anupam; Panda, Amulya K; Kuhad, Ramesh Chander

    2015-06-01

    Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with specific activity ∼3194IU/mg and ∼1493IU/mg were obtained from soluble and inclusion bodies (IBs) fraction with recovery of 56% and 74% in terms of activity, respectively. The recombinant enzyme was moderately thermostable (t1/2 60min at 50°C) and optimally active in wider alkaline pH range (7.0-10.5). Interaction of protein with its cofactor CaCl2 was found to stimulate the change in tertiary structure as revealed by near UV CD spectra. Intrinsic tryptophan fluorescence spectra indicated that tryptophan is involved in substrate binding and there might be independent binding of Ca(2+) and polygalacturonic acid to the active site. The recombinant enzyme was found to be capable of degrading pectin and polygalacturonic acid. The work reports novel conditions for refolding to obtain active recombinant pectate lyase from inclusion bodies and elucidates the effect of ligand and substrate binding on protein conformation by circular dichroism (CD) and fluorescence spectrofluorometry. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Repeated-batch operation of immobilized β-galactosidase inclusion bodies-containing Escherichia coli cell reactor for lactose hydrolysis.

    PubMed

    Yeon, Ji-Hyeon; Jung, Kyung-Hwan

    2011-09-01

    In this study, we investigated the performance of an immobilized β-galactosidase inclusion bodies-containing Escherichia coli cell reactor, where the cells were immobilized in alginate beads, which were then used in repeated-batch operations for the hydrolysis of o-nitrophenyl-β-D-galactoside or lactose over the long-term. In particular, in the Tris buffer system, disintegration of the alginate beads was not observed during the operation, which was observed for the phosphate buffer system. The o-nitrophenyl-β-D-galactoside hydrolysis was operated successfully up to about 80 h, and the runs were successfully repeated at least eight times. In addition, hydrolysis of lactose was successfully carried out up to 240 h. Using Western blotting analyses, it was verified that the beta-galactosidase inclusion bodies were sustained in the alginate beads during the repeated-batch operations. Consequently, we experimentally verified that β-galactosidase inclusion bodies-containing Escherichia coli cells could be used in a repeated-batch reactor as a biocatalyst for the hydrolysis of o-nitrophenyl-β-D-galactoside or lactose. It is probable that this approach can be applied to enzymatic synthesis reactions for other biotechnology applications, particularly reactions that require long-term and stable operation.

  16. Highly Efficient Production of Soluble Proteins from Insoluble Inclusion Bodies by a Two-Step-Denaturing and Refolding Method

    PubMed Central

    Zhang, Yan; Zhang, Ting; Feng, Yanye; Lu, Xiuxiu; Lan, Wenxian; Wang, Jufang; Wu, Houming; Cao, Chunyang; Wang, Xiaoning

    2011-01-01

    The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This “two-step-denaturing and refolding” (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes. PMID:21829569

  17. Bacterial community variation in human body habitats across space and time.

    PubMed

    Costello, Elizabeth K; Lauber, Christian L; Hamady, Micah; Fierer, Noah; Gordon, Jeffrey I; Knight, Rob

    2009-12-18

    Elucidating the biogeography of bacterial communities on the human body is critical for establishing healthy baselines from which to detect differences associated with diseases. To obtain an integrated view of the spatial and temporal distribution of the human microbiota, we surveyed bacteria from up to 27 sites in seven to nine healthy adults on four occasions. We found that community composition was determined primarily by body habitat. Within habitats, interpersonal variability was high, whereas individuals exhibited minimal temporal variability. Several skin locations harbored more diverse communities than the gut and mouth, and skin locations differed in their community assembly patterns. These results indicate that our microbiota, although personalized, varies systematically across body habitats and time; such trends may ultimately reveal how microbiome changes cause or prevent disease.

  18. Comparison of haemoglobin H inclusion bodies with embryonic zeta globin in screening for alpha thalassaemia.

    PubMed

    Chan, L C; So, J C; Chui, D H

    1995-09-01

    To compare the haemoglobin (Hb) H inclusion test with immunocytochemical detection of embryonic zeta chains in screening for alpha thalassaemia. Blood samples from 115 patients with relevant clinical history and hypochromic microcytic indexes were screened using the HbH inclusion test and the Variant Hemoglobin Testing System (BioRad, Hercules, CA, USA). The HbH inclusion test was positive in 61 of 115 cases, three of whom had HbH disease confirmed by electrophoresis. The remaining 58 had alpha thalassaemia 1. All three HbH cases and 56 of 58 cases of alpha thalassaemia 1 expressed embryonic zeta chains, giving a specificity of 96.7%. Fifty four of 115 cases had a negative HbH inclusion test, of whom 50 had beta thalassaemia trait and three had iron deficiency. No diagnosis was reached for the remaining patient. The immunocytochemical test is as sensitive as the HbH inclusion test in screening for alpha thalassaemia. The presence of zeta chains is highly specific for alpha thalassaemia 1 incorporating the (--/SEA) deletion. The specificity and simplicity of the immunocytochemical test make it the test of choice in screening for alpha thalassaemia.

  19. Comparison of haemoglobin H inclusion bodies with embryonic zeta globin in screening for alpha thalassaemia.

    PubMed Central

    Chan, L C; So, J C; Chui, D H

    1995-01-01

    AIMS--To compare the haemoglobin (Hb) H inclusion test with immunocytochemical detection of embryonic zeta chains in screening for alpha thalassaemia. METHODS--Blood samples from 115 patients with relevant clinical history and hypochromic microcytic indexes were screened using the HbH inclusion test and the Variant Hemoglobin Testing System (BioRad, Hercules, CA, USA). RESULTS--The HbH inclusion test was positive in 61 of 115 cases, three of whom had HbH disease confirmed by electrophoresis. The remaining 58 had alpha thalassaemia 1. All three HbH cases and 56 of 58 cases of alpha thalassaemia 1 expressed embryonic zeta chains, giving a specificity of 96.7%. Fifty four of 115 cases had a negative HbH inclusion test, of whom 50 had beta thalassaemia trait and three had iron deficiency. No diagnosis was reached for the remaining patient. CONCLUSION--The immunocytochemical test is as sensitive as the HbH inclusion test in screening for alpha thalassaemia. The presence of zeta chains is highly specific for alpha thalassaemia 1 incorporating the (--/SEA) deletion. The specificity and simplicity of the immunocytochemical test make it the test of choice in screening for alpha thalassaemia. Images PMID:7490322

  20. Lack of Accuracy of Body Temperature for Detecting Serious Bacterial Infection in Febrile Episodes.

    PubMed

    De, Sukanya; Williams, Gabrielle J; Teixeira-Pinto, Armando; Macaskill, Petra; McCaskill, Mary; Isaacs, David; Craig, Jonathan C

    2015-09-01

    Body temperature is a time-honored marker of serious bacterial infection, but there are few studies of its test performance. The aim of our study was to determine the accuracy of temperature measured on presentation to medical care for detecting serious bacterial infection. Febrile children 0-5 years of age presenting to the emergency department of a tertiary care pediatric hospital were sampled consecutively. The accuracy of the axillary temperature measured at presentation was evaluated using logistic regression models to generate receiver operating characteristic curves. Reference standard tests for serious bacterial infection were standard microbiologic/radiologic tests and clinical follow-up. Age, clinicians' impression of appearance of the child (well versus unwell) and duration of illness were assessed as possible effect modifiers. Of 15,781 illness episodes 1120 (7.1%) had serious bacterial infection. The area under the receiver operating characteristic curve for temperature was 0.60 [95% confidence intervals (CI): 0.58-0.62]. A threshold of ≥ 38°C had a sensitivity of 0.67 (95% CI: 0.64-0.70), specificity of 0.45 (95% CI: 0.44-0.46), positive likelihood ratio of 1.2 (95% CI: 1.2-1.3) and negative likelihood ratio of 0.7 (95% CI: 0.7-0.8). Age and illness duration had a small but significant effect on the accuracy of temperature increasing its "rule-in" potential. Measured temperature at presentation to hospital is not an accurate marker of serious bacterial infection in febrile children. Younger age and longer duration of illness increase the rule-in potential of temperature but without substantial overall change in its test accuracy.

  1. Late onset glycogen storage disease type II with reducing body-like inclusions.

    PubMed

    Gayathri, N; Yasha, T C; Vani, S; Taly, A B; Nalini, A; Shankar, S K

    2010-01-01

    Skeletal muscle tissue from 3 patients with clinical diagnosis of limb girdle muscular dystrophy revealed a vacuolar myopathy with glycogen storage and lysosomal activity. A diagnosis of late onset GSD Type II was considered. An interesting finding was the presence of round to oval eosinophilic inclusions which reduced on menadione linked a-glycerophosphate dehydrogenase (MAG). There are only two reports in the literature describing similar inclusions in late onset GSD II. We report morphological findings of this rare disorder and compare the findings with earlier two reports.

  2. Titration of Adenovirus by Counting Cells Containing Virus-Induced Inclusion Bodies

    PubMed Central

    Weber, Joseph

    1972-01-01

    A new method for the titration of adenovirus types 2 and 12 based on the enumeration of viral inclusions in infected cells was devised and evaluated. The technique gave virus titers comparable to those obtained by the plaque assay procedure. Images PMID:4113255

  3. Detection of occupational and environmental exposures by bacterial mutagenesis assays of human body fluids.

    PubMed

    Everson, R B

    1986-08-01

    Assays of human body fluids provide a means to document human exposure to mutagens in the environment. In contrast to measurements of ambient levels, these assays demonstrate absorption of mutagens and provide estimates of minimal systemic doses. For most studies reviewed here, specimens of urine were concentrated by adsorption to columns of XAD-2 resin or by liquid partition extraction prior to the mutagenesis assays. The resulting extracts most commonly were analyzed for mutagenicity using the Salmonella/mammalian microsomal plate assay. Less frequently used assays included bacterial fluctuation tests instead of the plate assay and assays for the induction of sister chromatid exchanges in cultured mammalian cells. In addition to reviewing literature reports where body fluids were tested, the advantages, disadvantages, and potential role of this approach will be briefly discussed and compared with other approaches to the identification of mutagenic hazards in the workplace.

  4. Detection of occupational and environmental exposures by bacterial mutagenesis assays of human body fluids

    SciTech Connect

    Everson, R.B.

    1986-08-01

    Assays of human body fluids provide a means to document human exposure to mutagens in the environment. In contrast to measurements of ambient levels, these assays demonstrate absorption of mutagens and provide estimates of minimal systemic doses. For most studies reviewed here, specimens of urine were concentrated by adsorption to columns of XAD-2 resin or by liquid partition extraction prior to the mutagenesis assays. The resulting extracts most commonly were analyzed for mutagenicity using the Salmonella/mammalian microsomal plate assay. Less frequently used assays included bacterial fluctuation tests instead of the plate assay and assays for the induction of sister chromatid exchanges in cultured mammalian cells. In addition to reviewing literature reports where body fluids were tested, the advantages, disadvantages, and potential role of this approach will be briefly discussed and compared with other approaches to the identification of mutagenic hazards in the workplace.

  5. Dorfin Localizes to the Ubiquitylated Inclusions in Parkinson’s Disease, Dementia with Lewy Bodies, Multiple System Atrophy, and Amyotrophic Lateral Sclerosis

    PubMed Central

    Hishikawa, Nozomi; Niwa, Jun-ichi; Doyu, Manabu; Ito, Takashi; Ishigaki, Shinsuke; Hashizume, Yoshio; Sobue, Gen

    2003-01-01

    In many neurodegenerative diseases, the cytopathological hallmark is the presence of ubiquitylated inclusions consisting of insoluble protein aggregates. Lewy bodies in Parkinson’s disease and dementia with Lewy bodies disease, glial cell inclusions in multiple system atrophy, and hyaline inclusions in amyotrophic lateral sclerosis (ALS) are representative of these inclusions. The elucidation of the components of these inclusions and the mechanisms underlying inclusion formation is important in uncovering the pathogenesis of these disorders. We hypothesized that Dorfin, a perinuclearly located E3 ubiquitin ligase, participates in the formation of ubiquitylated inclusions in a wide range of neurodegenerative diseases. Here, we report that affinity-purified anti-Dorfin antibody labeled ubiquitylated inclusions of Parkinson’s disease, dementia with Lewy bodies disease, multiple system atrophy, and sporadic and familial ALS. A double-immunofluorescence study revealed that Dorfin shows a distribution pattern parallel to that of ubiquitin. Furthermore, by a filter trap assay, we detected that Dorfin is present in the ubiquitylated high-molecular weight structures derived from these diseases. These results suggest that Dorfin plays a crucial role in the formation of ubiquitylated inclusions of α-synucleinopathy and ALS. However, because we failed to show the direct binding of α-synuclein with Dorfin, future investigations into the binding partner(s) of Dorfin will be needed to deepen our understanding of the pathophysiology of α-synucleinopathy and ALS. PMID:12875980

  6. Endosomal sorting related protein CHMP2B is localized in Lewy bodies and glial cytoplasmic inclusions in α-synucleinopathy.

    PubMed

    Tanikawa, Satoshi; Mori, Fumiaki; Tanji, Kunikazu; Kakita, Akiyoshi; Takahashi, Hitoshi; Wakabayashi, Koichi

    2012-10-03

    Charged multivesicular body protein 2B (CHMP2B) is a component of the endosomal sorting complex required for transport-III, which is involved in the degradation of proteins in the endocytic and autophagic pathways. Mutations in the CHMP2B gene cause frontotemporal dementia and amyotrophic lateral sclerosis characterized by accumulation of ubiquitinated protein aggregates. Recent studies have shown that autophagosomal proteins are present in α-synuclein aggregates in neurons and glial cells in Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We therefore immunohistochemically examined the brains of various neurodegenerative diseases using CHMP2B-specific antibody. CHMP2B immunoreactivity was present in intracytoplasmic and axonal Lewy bodies in PD and DLB as well as in neuronal and glial cytoplasmic inclusions in MSA. No CHMP2B immunoreactivity was found in a variety of other neuronal and glial inclusions in TDP-43 proteinopathy and tauopathy. These findings suggest that endosomal and autophagic pathway is associated with degradation or formation of α-synuclein aggregates in α-synucleinopathy. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  7. Polyglucosan inclusions (Lafora bodies) in a gray-headed flying fox (Pteropus poliocephalus).

    PubMed

    Gabor, Les J; Srivastava, Mukesh

    2010-03-01

    Polyglucosan bodies (Lafora bodies) were identified in a juvenile gray-headed flying fox (Pteropus poliocephalus) with neurological signs. The structures were only noted in the brain stem, and no associated degenerative changes were present. These structures have not been previously identified in any species in the order Chiroptera.

  8. Folding and aggregation of TEM beta-lactamase: analogies with the formation of inclusion bodies in Escherichia coli.

    PubMed Central

    Georgiou, G.; Valax, P.; Ostermeier, M.; Horowitz, P. M.

    1994-01-01

    The enzyme TEM beta-lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM beta-lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0-1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis-1-anilino-8-naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM beta-lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0-1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM beta-lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM beta-lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131-136). Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo. PMID:7703842

  9. Solubilization of growth hormone and other recombinant proteins from Escherichia coli inclusion bodies by using a cationic surfactant.

    PubMed Central

    Puri, N K; Crivelli, E; Cardamone, M; Fiddes, R; Bertolini, J; Ninham, B; Brandon, M R

    1992-01-01

    Recombinant pig growth hormone (rPGH) was solubilized from inclusion bodies by using the cationic surfactant cetyltrimethylammonium chloride (CTAC). The solubilizing action of CTAC appeared to be dependent on the presence of a positively charged head group, as a non-charged variant was inactive. Relatively low concentrations of CTAC were required for rapid solubilization, and protein-bound CTAC was easily removed by ion-exchange chromatography. Compared with solubilization and recovery of rPGH from inclusion bodies with 7.5 M-urea and 6 M-guanidinium chloride, the relative efficiency of solubilization was lower with CTAC. However, superior refolding efficiency resulted in final yields of purified rPGH being in the order of CTAC greater than urea greater than or equal to guanidinium chloride. Detailed comparison of the different rPGH preparations as well as pituitary-derived growth hormone by h.p.l.c., native PAGE, c.d. spectral analysis and radioreceptor-binding assay showed that the CTAC-derived rPGH was essentially indistinguishable from the urea and guanidinium chloride preparations. The CTAC-derived rPGH was of greater biopotency than pituitary-derived growth hormone. The advantages of CTAC over urea and guanidinium chloride for increasing recovery of monomeric rPGH by minimizing aggregation during refolding in vitro were also found with recombinant sheep interleukin-I beta and a sheep insulin-like growth factor II fusion protein. In addition, the bioactivity of the CTAC-derived recombinant interleukin-1 beta was approximately ten-fold greater than that of an equivalent amount obtained from urea and guanidinium chloride preparations. It is concluded that CTAC represents, in general, an excellent additional approach or a superior alternative to urea and in particular guanidinium chloride for solubilization and recovery of bioactive recombinant proteins from inclusion bodies. Images Fig. 1. Fig. 3. Fig. 5. Fig. 9. PMID:1497625

  10. Detection and prevalence of boid inclusion body disease in collections of boas and pythons using immunological assays.

    PubMed

    Chang, L; Fu, D; Stenglein, M D; Hernandez, J A; DeRisi, J L; Jacobson, E R

    2016-12-01

    Inclusion body disease (IBD) of boas and pythons is characterized by the intracytoplasmic accumulation of an antigenic 68 kDa viral protein IBDP, more recently known as the nucleoprotein (NP) of the reptarenaviruses. Blood samples of 131 captive boas and pythons (53 boa constrictors, Boa constrictor; 35 rainbow boas, Epicrates cenchria; 22 ball pythons, Python regius; 5 carpet pythons, Morelia spilota; 6 Burmese pythons, Python bivittatus; 4 Jamaican boas, Epicrates subflavus; 5 anacondas, Eunectes spp.; and 1 green tree python, Morelia viridis) were obtained from 28 collections in the USA. Diagnosis of IBD was initially made by the identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin (HE) stained blood films and isolated peripheral white blood cells (PWBC). The overall prevalence of IBD in study snakes was 25/131 or 19% (95% CI = 12.4%, 25.8%) with boa constrictors being more commonly infected (22/53 or 41.5%; 95% CI = 28.2%, 54.8%) than other species in this study. Of the 22 IBD positive boa constrictors, 87% were clinically healthy, 13% had various signs of chronic illness, and none showed signs of central nervous system disease. Using a validated monoclonal anti-NP antibody, NP was confirmed within the isolated PWBC by immunohistochemical staining and Western blots. The presence of reptarenaviruses within blood samples of 27 boa constrictors and three rainbow boas was also assessed by PCR. Among boa constrictors, very good agreements were shown between the observation of inclusion bodies (by HE stain) and the presence of NP (by immunohistochemistry, kappa = 0.92; and Western blots, kappa = 0.89), or the presence of reptarenaviruses (by PCR; kappa = 0.92). Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Replication of boid inclusion body disease-associated arenaviruses is temperature sensitive in both boid and mammalian cells.

    PubMed

    Hepojoki, Jussi; Kipar, Anja; Korzyukov, Yegor; Bell-Sakyi, Lesley; Vapalahti, Olli; Hetzel, Udo

    2015-01-15

    Boid inclusion body disease (BIDB) is a fatal disease of boid snakes, the etiology of which has only recently been revealed following the identification of several novel arenaviruses in diseased snakes. BIBD-associated arenaviruses (BIBDAV) are genetically divergent from the classical Old and New World arenaviruses and also differ substantially from each other. Even though there is convincing evidence that BIBDAV are indeed the etiological agent of BIBD, the BIBDAV reservoir hosts--if any exist besides boid snakes themselves--are not yet known. In this report, we use University of Helsinki virus (UHV; a virus that we isolated from a Boa constrictor with BIBD) to show that BIBDAV can also replicate effectively in mammalian cells, including human cells, provided they are cultured at 30°C. The infection induces the formation of cytoplasmic inclusion bodies (IB), comprised mainly of viral nucleoprotein (NP), similar to those observed in BIBD and in boid cell cultures. Transferring infected cells from 30°C to 37°C ambient temperature resulted in progressive declines in IB formation and in the amounts of viral NP and RNA, suggesting that BIBDAV growth is limited at 37°C. These observations indirectly indicate that IB formation is linked to viral replication. In addition to mammalian and reptilian cells, UHV infected arthropod (tick) cells when grown at 30°C. Even though our findings suggest that BIBDAV have a high potential to cross the species barrier, their inefficient growth at mammalian body temperatures indicates that the reservoir hosts of BIBDAV are likely species with a lower body temperature, such as snakes. The newly discovered boid inclusion body disease-associated arenaviruses (BIBDAV) of reptiles have drastically altered the phylogeny of the family Arenavirus. Prior to their discovery, known arenaviruses were considered mainly rodent-borne viruses, with each arenavirus species having its own reservoir host. BIBDAV have so far been demonstrated in

  12. Replication of Boid Inclusion Body Disease-Associated Arenaviruses Is Temperature Sensitive in both Boid and Mammalian Cells

    PubMed Central

    Kipar, Anja; Korzyukov, Yegor; Bell-Sakyi, Lesley; Vapalahti, Olli; Hetzel, Udo

    2014-01-01

    ABSTRACT Boid inclusion body disease (BIDB) is a fatal disease of boid snakes, the etiology of which has only recently been revealed following the identification of several novel arenaviruses in diseased snakes. BIBD-associated arenaviruses (BIBDAV) are genetically divergent from the classical Old and New World arenaviruses and also differ substantially from each other. Even though there is convincing evidence that BIBDAV are indeed the etiological agent of BIBD, the BIBDAV reservoir hosts—if any exist besides boid snakes themselves—are not yet known. In this report, we use University of Helsinki virus (UHV; a virus that we isolated from a Boa constrictor with BIBD) to show that BIBDAV can also replicate effectively in mammalian cells, including human cells, provided they are cultured at 30°C. The infection induces the formation of cytoplasmic inclusion bodies (IB), comprised mainly of viral nucleoprotein (NP), similar to those observed in BIBD and in boid cell cultures. Transferring infected cells from 30°C to 37°C ambient temperature resulted in progressive declines in IB formation and in the amounts of viral NP and RNA, suggesting that BIBDAV growth is limited at 37°C. These observations indirectly indicate that IB formation is linked to viral replication. In addition to mammalian and reptilian cells, UHV infected arthropod (tick) cells when grown at 30°C. Even though our findings suggest that BIBDAV have a high potential to cross the species barrier, their inefficient growth at mammalian body temperatures indicates that the reservoir hosts of BIBDAV are likely species with a lower body temperature, such as snakes. IMPORTANCE The newly discovered boid inclusion body disease-associated arenaviruses (BIBDAV) of reptiles have drastically altered the phylogeny of the family Arenavirus. Prior to their discovery, known arenaviruses were considered mainly rodent-borne viruses, with each arenavirus species having its own reservoir host. BIBDAV have so far been

  13. Mechanical ventilation weaning in inclusion body myositis: feasibility of isokinetic inspiratory muscle training as an adjunct therapy.

    PubMed

    Cordeiro de Souza, Leonardo; Campos, Josué Felipe; Daher, Leandro Possidente; Furtado da Silva, Priscila; Ventura, Alex; do Prado, Pollyana Zamborlini; Brasil, Daniele; Mendonça, Debora; Lugon, Jocemir Ronaldo

    2014-01-01

    Inclusion body myositis is a rare myopathy associated with a high rate of respiratory complications. This condition usually requires prolonged mechanical ventilation and prolonged intensive care stay. The unsuccessful weaning is mainly related to respiratory muscle weakness that does not promptly respond to immunosuppressive therapy. We are reporting a case of a patient in whom the use of an inspiratory muscle-training program which started after a two-week period of mechanical ventilation was associated with a successful weaning in one week and hospital discharge after 2 subsequent weeks.

  14. Mechanical Ventilation Weaning in Inclusion Body Myositis: Feasibility of Isokinetic Inspiratory Muscle Training as an Adjunct Therapy

    PubMed Central

    Campos, Josué Felipe; Daher, Leandro Possidente; Ventura, Alex; do Prado, Pollyana Zamborlini; Brasil, Daniele; Mendonça, Debora; Lugon, Jocemir Ronaldo

    2014-01-01

    Inclusion body myositis is a rare myopathy associated with a high rate of respiratory complications. This condition usually requires prolonged mechanical ventilation and prolonged intensive care stay. The unsuccessful weaning is mainly related to respiratory muscle weakness that does not promptly respond to immunosuppressive therapy. We are reporting a case of a patient in whom the use of an inspiratory muscle-training program which started after a two-week period of mechanical ventilation was associated with a successful weaning in one week and hospital discharge after 2 subsequent weeks. PMID:25147743

  15. Presence of BACE1 and BACE2 in muscle fibres of patients with sporadic inclusion-body myositis.

    PubMed

    Vattemi, G; Engel, W K; McFerrin, J; Buxbaum, J D; Pastorino, L; Askanas, V

    2001-12-08

    Sporadic inclusion-body myositis (IBM) is the most common, progressive muscle disease of older individuals. We investigated the presence of BACE1 and BACE2-two beta secretases that cleave amyloid-beta-precursor protein-in muscle-biopsy samples from patients with IBM and from controls. On immunofluorescence, BACE1 and BACE2 co-localised with amyloid beta in IBM vacuolated muscle fibres, but were not found in controls. Immunoblotting showed increased BACE2 but not BACE1 in patients with IBM compared with controls. Our study suggests that both of these proteases might participate in processing of amyloid-beta-precursor protein in IBM muscle fibres.

  16. Sporadic Inclusion Body Myositis Manifesting as Isolated Muscle Weakness of the Finger Flexors Three Years after Disease Onset

    PubMed Central

    Suwa, Yuichi; Suzuki, Naoki; Soga, Temma; Harada, Ryuhei; Shibui, Aya; Kuroda, Hiroshi; Izumi, Rumiko; Tateyama, Maki; Nakashima, Ichiro; Sonoo, Masahiro; Aoki, Masashi

    2016-01-01

    Sporadic inclusion body myositis (sIBM) is a chronic progressive myopathy characterized by muscle weakness of both the quadriceps femoris and finger flexors. We herein present the case of a typical male patient with sIBM, which manifested as the isolated weakness of the finger flexors three years after the disease onset. We have identified several patients with sIBM in our cohort with muscle weakness of the flexors but not the quadriceps femoris. Examination of the flexor digitorum profundus muscle is important for the early and proper diagnosis of sIBM, even if a patient only presents with isolated finger flexor muscle weakness. PMID:27904121

  17. High and compact formation of baculoviral polyhedrin-induced inclusion body by co-expression of baculoviral FP25 in Escherichia coli.

    PubMed

    Li, Lin; Kim, Young Soo; Hwang, Dong Soo; Seo, Jeong Hyun; Jung, Hee Jung; Du, Juan; Cha, Hyung Joon

    2007-04-15

    Previously, we found that baculoviral polyhedrin (Polh) can successfully be used in Escherichia coli as a fusion partner for the expression of special foreign proteins as inclusion bodies, and the resulting, easily isolatable Polh-induced fusion inclusion bodies had almost the same characteristics as the native Polh. Here, we investigated the effects of co-expression of baculoviral FP25 protein on Polh-induced inclusion-body production in an E. coli expression system, as FP25 is known to be involved specifically in polyhedra formation. Using several analytical tools, including SDS-PAGE, pronase proteolysis, solubilization under alkaline conditions, and electron microscopy, we found that co-expressed FP25 was associated with Polh-induced inclusion bodies and that its co-expression led to formation of compact inclusion bodies as well as high production levels. We confirmed that FP25 co-expression induced higher production levels of other heterologous protein, antimicrobial peptide Hal18, fused with aggregation-prone Polh. Therefore, co-expression of baculoviral FP25 can be promisingly used to increase the levels of baculoviral Polh-fused foreign proteins, especially harmful proteins, expressed as inclusion bodies in an E. coli expression system.

  18. Studies of a nuclear matrix protein restricted to normal brain cells and lead-induced intranuclear inclusion bodies of kidney

    SciTech Connect

    Shelton, K.; Egle, P.; Redford, K.; Bigbee, J.

    1986-05-01

    A nuclear matrix protein, p32/6.3, with an unusual tissue distribution, has been identified. Protein from 21 tissues was surveyed by immunoprobing Western blots. In normal adult rats p32/6.3 is found only in grey matter from the cerebrum and the cerebellum, occurring in both neurons and astrocytes. Other brain cell types have not been examined. The protein appears to be developmentally regulated. It is detectable in the brain within a few days after birth and reaches adult levels within one to two weeks. Brain p32/6.3 has been found in all animals tested including rat, mouse, dog, cow, pig, chicken and human. This conservation indicates a fundamental role for p32/6.3 in the nucleus of brain cells. Possible functions for p32/6.3 may be indicated by a second novel occurrence. Chronic lead poisoning characteristically induces intranuclear inclusion bodies in the cells lining kidney proximal tubules. p32/6.3 is a major constituent of these inclusion bodies. They are also rich in lead and other metals including calcium, iron, zinc, copper and cadmium. These diverse observations suggest that p32/6.3 may have a role in metal homeostasis in the brain of normal animals.

  19. Hereditary inclusion-body myopathy with sparing of the quadriceps: the many tiles of an incomplete puzzle.

    PubMed

    Broccolini, A; Gidaro, T; Morosetti, R; Sancricca, C; Mirabella, M

    2011-10-01

    The hereditary inclusion-body myopathies encompass several syndromes with autosomal recessive or dominant inheritance. Despite a different clinical presentation they all have a progressive course leading to severe disability and share similar pathologic findings at the muscle biopsy. Quadriceps-sparing autosomal recessive hereditary inclusion-body myopathy (h-IBM) is the commonest form and is tied to mutations of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) that codes for a rate-limiting enzyme in the sialic acid biosynthetic pathway. Despite the identification of the causative gene defect, it has not been clarified how mutations of the GNE gene impair muscle homeostasis. Although several lines of evidence argue in favor of an abnormal sialylation of muscle glycoproteins playing a key role in h-IBM pathogenesis, others studies have demonstrated new functions of the GNE gene, outside the sialic acid biosynthetic pathway, that may also be relevant. This review illustrates the clinical and pathologic characteristics of h-IBM and the main clues available to date concerning the possible pathogenic mechanisms of this disorder. Understanding the molecular mechanism underlying h-IBM pathology is a fundamental requisite to plan a future attempt to therapy.

  20. Highly Effective Renaturation of a Streptokinase from Streptococcus pyogenes DT7 as Inclusion Bodies Overexpressed in Escherichia coli

    PubMed Central

    Nguyen, Sy Le Thanh; Quyen, Dinh Thi; Vu, Hong Diep

    2014-01-01

    The streptokinase (SK) is emerging as an important thrombolytic therapy agent in the treatment of patients suffering from cardiovascular diseases. We reported highly effective renaturation of a SK from S. pyogeness DT7 overexpressed in E. coli, purification, and biochemical characterization. A gene coding for the SK was cloned from S. pyogeness DT7. Because accumulation of active SK is toxic to the host cells, we have expressed it in the form of inclusion bodies. The mature protein was overexpressed in E. coli BL21 DE3/pESK under the control of the strong promoter tac induced by IPTG with a level of 60% of the total cell proteins. The activity of the rSK, renatured in phosphate buffer supplemented with Triton X-100 and glycerol, was covered with up to 41 folds of its initial activity. The purified of protein was identified with MALDI-TOF mass spectrometry through four peptide fragments, which showed 100% identification to the corresponding peptides of the putative SK from GenBank. Due to overexpression and highly effective renaturation of large amounts of inclusion bodies, the recombinant E. coli BL21 DE3/pESK system could be potentially applied for large-scale production of SK used in the therapy of acute myocardial infarction. PMID:24883307

  1. A new derivatizing agent, trimethylammoniopropyl methanethiosulphonate, is efficient for preparation of recombinant brain-derived neurotrophic factor from inclusion bodies.

    PubMed

    Inoue, M; Akimaru, J; Nishikawa, T; Seki, N; Yamada, H

    1998-12-01

    Derivatization with trimethylammoniopropyl methanethiosulphonate (TAPS-sulphonate) enabled brain-derived neurotrophic factor (BDNF) to be prepared efficiently from Escherichia coli inclusion bodies. Reduced BDNF obtained from inclusion bodies solubilized by urea and reduced by dithiothreitol was suggested to form a complex with itself or with other compounds such as lipids. It could hardly be adsorbed on to cation-exchange resin for partial purification prior to a refolding reaction. Reversible derivatization of cysteine residues was tested as a method of dissociating BDNF from such complexes. However, even if a methyl or aminoethyl group was introduced, BDNF could not be dissociated readily. Derivatization with TAPS-sulphonate brought about good dissociation of BDNF, and more than 50% adsorbed on to the cation-exchange resin. BDNF derivatized with TAPS-sulphonate refolded well, and the refolded samples showed the same biological activity as purified BDNF. Derivatization with TAPS-sulphonate would increase the intermolecular repulsion of BDNF, due to the positively charged character of the quaternized amine, and inhibit complex formation. Thus, TAPS-sulphonate is effective for the preparation of BDNF under denatured conditions.

  2. Interferon-alpha 2b quantification in inclusion bodies using reversed phase-ultra performance liquid chromatography (RP-UPLC).

    PubMed

    Cueto-Rojas, H F; Pérez, N O; Pérez-Sánchez, G; Ocampo-Juárez, I; Medina-Rivero, E

    2010-04-15

    Interferon-alpha 2b (IFN-alpha 2b) is a recombinant therapeutic cytokine produced as inclusion bodies using a strain of Escherichia coli as expression system. After fermentation and recovery, it is necessary to know the amount of recombinant IFN-alpha 2b, in order to determine the yield and the load for solubilization, and chromatographic protein purification steps. The present work details the validation of a new short run-time and fast sample-preparation method to quantify IFN-alpha 2b in inclusion bodies using Reversed Phase-Ultra Performance Liquid Chromatography (RP-UPLC). The developed method demonstrated an accuracy of 100.28%; the relative standard deviations for method precision, repeatability and inter-day precision tests were found to be 0.57%, 1.54% and 1.83%, respectively. Linearity of the method was assessed in the range of concentrations from 0.05 mg/mL to 0.5 mg/mL, the curve obtained had a determination coefficient (r(2)) of 0.9989. Detection and quantification limits were found to be 0.008 mg/mL and 0.025 mg/mL, respectively. The method also demonstrated robustness for changes in column temperature, and specificity against host proteins and other recombinant protein expressed in the same E. coli strain.

  3. Data on enhanced expression and purification of camelid single domain antibodies from Escherichia coli classical inclusion bodies.

    PubMed

    Maggi, Maristella; Scotti, Claudia

    2017-06-01

    Heterologous expression of high amounts of recombinant proteins is a milestone for research and industrial purposes. Single domain antibodies (sdAbs) are heavy-chain only antibody fragments with applications in the biotechnological, medical and industrial fields. The simple nature and small size of sdAbs allows for efficient expression of the soluble molecule in different hosts. However, in some cases, it results in low functional protein yield. To overcome this limitation, expression of a 6xHistag sdAb was attempted in different conditions in Escherichia coli BL21(DE3) cells. Data showed that high amount of sdAb can be expressed in E. coli classical inclusion bodies, efficiently extracted by urea in a short-time, and properly purified by metal ion affinity chromatography. These data originate from the research article "Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies" Maggi and Scotti (2017) [1] (DOI: http://dx.doi.org/10.1016/j.pep.2017.02.007).

  4. Integrated continuous dissolution, refolding and tag removal of fusion proteins from inclusion bodies in a tubular reactor.

    PubMed

    Pan, Siqi; Zelger, Monika; Jungbauer, Alois; Hahn, Rainer

    2014-09-20

    An integrated continuous tubular reactor system was developed for processing an autoprotease expressed as inclusion bodies. The inclusion bodies were suspended and fed into the tubular reactor system for continuous dissolving, refolding and precipitation. During refolding, the dissolved autoprotease cleaves itself, separating the fusion tag from the target peptide. Subsequently, the cleaved fusion tag and any uncleaved autoprotease were precipitated out in the precipitation step. The processed exiting solution results in the purified soluble target peptide. Refolding and precipitation yields performed in the tubular reactor were similar to batch reactor and process was stable for at least 20 h. The authenticity of purified peptide was also verified by mass spectroscopy. Productivity (in mg/l/h and mg/h) calculated in the tubular process was twice and 1.5 times of the batch process, respectively. Although it is more complex to setup a tubular than a batch reactor, it offers faster mixing, higher productivity and better integration to other bioprocessing steps. With increasing interest of integrated continuous biomanufacturing, the use of tubular reactors in industrial settings offers clear advantages. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Interaction of cellular proteins with BCL-xL targeted to cytoplasmic inclusion bodies in adenovirus infected cells.

    PubMed

    Subramanian, T; Vijayalingam, S; Kuppuswamy, M; Chinnadurai, G

    2015-09-01

    Adenovirus-mediated apoptosis was suppressed when cellular anti-apoptosis proteins (BCL-2 and BCL-xL) were substituted for the viral E1B-19K. For unbiased proteomic analysis of proteins targeted by BCL-xL in adenovirus-infected cells and to visualize the interactions with target proteins, BCL-xL was targeted to cytosolic inclusion bodies utilizing the orthoreovirus µNS protein sequences. The chimeric protein was localized in non-canonical cytosolic factory-like sites and promoted survival of virus-infected cells. The BCL-xL-associated proteins were isolated from the cytosolic inclusion bodies in adenovirus-infected cells and analyzed by LC-MS. These proteins included BAX, BAK, BID, BIK and BIM as well as mitochondrial proteins such as prohibitin 2, ATP synthase and DNA-PKcs. Our studies suggested that in addition to the interaction with various pro-apoptotic proteins, the association with certain mitochondrial proteins such as DNA-PKcs and prohibitins might augment the survival function of BCL-xL in virus infected cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein-folding liquid-chromatography columns.

    PubMed

    Yuan, Jie; Zhou, Huifang; Yang, Yicong; Li, Weimin; Wan, Yi; Wang, Lili

    2015-05-01

    Protein-folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high-performance size-exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low-urea gradient-elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS-18% PAGE, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and the biological activity assay by HP-RPLC. Copyright © 2014 John Wiley & Sons, Ltd.

  7. Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.

    PubMed

    Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidic, Jasmina

    2015-12-19

    The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

  8. Highly effective renaturation of a streptokinase from Streptococcus pyogenes DT7 as inclusion bodies overexpressed in Escherichia coli.

    PubMed

    Nguyen, Sy Le Thanh; Quyen, Dinh Thi; Vu, Hong Diep

    2014-01-01

    The streptokinase (SK) is emerging as an important thrombolytic therapy agent in the treatment of patients suffering from cardiovascular diseases. We reported highly effective renaturation of a SK from S. pyogeness DT7 overexpressed in E. coli, purification, and biochemical characterization. A gene coding for the SK was cloned from S. pyogeness DT7. Because accumulation of active SK is toxic to the host cells, we have expressed it in the form of inclusion bodies. The mature protein was overexpressed in E. coli BL21 DE3/pESK under the control of the strong promoter tac induced by IPTG with a level of 60% of the total cell proteins. The activity of the rSK, renatured in phosphate buffer supplemented with Triton X-100 and glycerol, was covered with up to 41 folds of its initial activity. The purified of protein was identified with MALDI-TOF mass spectrometry through four peptide fragments, which showed 100% identification to the corresponding peptides of the putative SK from GenBank. Due to overexpression and highly effective renaturation of large amounts of inclusion bodies, the recombinant E. coli BL21 DE3/pESK system could be potentially applied for large-scale production of SK used in the therapy of acute myocardial infarction.

  9. The receptor for advanced glycation end products promotes bacterial growth at distant body sites in Staphylococcus aureus skin infection.

    PubMed

    Achouiti, Ahmed; Van't Veer, Cornelis; de Vos, Alex F; van der Poll, Tom

    2015-09-01

    The receptor for advanced glycation endproducts (RAGE) has been implicated in the regulation of skin inflammation. We here sought to study the role of RAGE in host defense during skin infection caused by Staphylococcus (S.) aureus, the most common pathogen in this condition. Wild-type (Wt) and RAGE deficient (rage(-/-)) mice were infected subcutaneously with S. aureus and bacterial loads and local inflammation were quantified at regular intervals up to 8 days after infection. While bacterial burdens were similar in both mouse strains at the primary site of infection, rage(-/-) mice had lower bacterial counts in lungs and liver. Skin cytokine and chemokine levels did not differ between groups. In accordance with the skin model, direct intravenous infection with S. aureus was associated with lower bacterial loads in lungs and liver of rage(-/-) mice. Together these data suggest that RAGE does not impact local host defense during S. aureus skin infection, but facilitates bacterial growth at distant body sites.

  10. Mitochondrial inclusion bodies (intracytoplasmic acidophilic droplets) in neurons of chicken spinal cords increase with age.

    PubMed

    Watanabe, O; Maruo, Y

    2012-03-01

    We studied the pathologic features of neurons that contain intracytoplasmic acidophilic droplets (IADs) in chicken spinal cords. The IADs were lustrous spheroid bodies scattered in the cytoplasm of neurons, variable in size, and protein-rich bodies stained eosinophilic with hematoxylin-eosin, acidophilic with Azan, blue indigo with phosphotungstic acid hematoxylin, and yellow-green with Elastica van Gieson stain histopathologically. Ultrastructurally, almost all IADs were observed as homogeneous highly electron-dense spheroid bodies enclosed by double-limited membranes. Small IADs were observed in mitochondria. Anatomically, IAD-CNs were observed only in the ventral horn of the spinal cord between the fourth sacral and third lumbal vertebrae, and they were particularly frequent in the third sacral vertebrae. Their appearance and accumulative amount were likely to increase with age, while the clinical and pathologic significances of IAD-CNs remain unclear.

  11. Body Temperature at the Emergency Department as a Predictor of Mortality in Patients With Bacterial Infection.

    PubMed

    Yamamoto, Shungo; Yamazaki, Shin; Shimizu, Tsunehiro; Takeshima, Taro; Fukuma, Shingo; Yamamoto, Yosuke; Tochitani, Kentaro; Tsuchido, Yasuhiro; Shinohara, Koh; Fukuhara, Shunichi

    2016-05-01

    Hypothermia is a risk factor for death in intensive care unit (ICU) patients with severe sepsis and septic shock. In the present study, we investigated the association between body temperature (BT) on arrival at the emergency department (ED) and mortality in patients with bacterial infection.We conducted a retrospective cohort study in consecutive ED patients over 15 years of age with bacterial infection who were admitted to an urban teaching hospital in Japan between 2010 and 2012. The main outcome measure was 30-day in-hospital mortality. Each patient was assigned to 1 of 6 categories based on BT at ED admission. We conducted multivariable logistic regression analysis to adjust for predictors of death.A total of 913 patients were enrolled in the study. The BT categories were <36, 36 to 36.9, 37 to 37.9, 38 to 38.9, 39 to 39.9, and ≥40 °C, with respective mortalities of 32.5%, 14.1%, 8.7%, 8.2%, 5.7%, and 5.3%. Multivariable analysis showed that the risk of death was significantly low in patients with BT 37 to 37.9 °C (adjusted odds ratio [AOR]: 0.2; 95% confidence interval [CI] 0.1-0.6, P = 0.003), 38-38.9 °C (AOR: 0.2; 95% CI 0.1-0.6, P = 0.002), 39-39.9 °C (AOR: 0.2; 95% CI 0.1-0.5, P = 0.001), and ≥40 °C (AOR: 0.1; 95% CI 0.02-0.4, P = 0.001), compared with hypothermic patients (BT <36 °C).The higher BT on arrival at ED, the better the outcomes observed in patients with bacterial infection were.

  12. Neutral weak-current two-body contributions in inclusive scattering from {sup 12}C

    SciTech Connect

    Lovato, Alessandro; Gandolfi, Stefano; Carlson, Joseph; Pieper, S. C.; Schiavilla, Rocco

    2014-05-01

    An {\\it ab initio} calculation of the sum rules of the neutral weak response functions in $^{12}$C is reported, based on a realistic Hamiltonian, including two- and three-nucleon potentials, and on realistic currents, consisting of one- and two-body terms. We find that the sum rules of the response functions associated with the longitudinal and transverse components of the (space-like) neutral current are largest and that a significant portion ($\\simeq 30$\\%) of the calculated strength is due to two-body terms. This fact may have implications for the MiniBooNE and other neutrino quasi-elastic scattering data on nuclei.

  13. The bacterial communities associated with fecal types and body weight of rex rabbits.

    PubMed

    Zeng, Bo; Han, Shushu; Wang, Ping; Wen, Bin; Jian, Wensu; Guo, Wei; Yu, Zhiju; Du, Dan; Fu, Xiangchao; Kong, Fanli; Yang, Mingyao; Si, Xiaohui; Zhao, Jiangchao; Li, Ying

    2015-03-20

    Rex rabbit is an important small herbivore for fur and meat production. However, little is known about the gut microbiota in rex rabbit, especially regarding their relationship with different fecal types and growth of the hosts. We characterized the microbiota of both hard and soft feces from rex rabbits with high and low body weight by using the Illumina MiSeq platform targeting the V4 region of the 16S rDNA. High weight rex rabbits possess distinctive microbiota in hard feces, but not in soft feces, from the low weight group. We detected the overrepresentation of several genera such as YS2/Cyanobacteria, and Bacteroidales and underrepresentation of genera such as Anaeroplasma spp. and Clostridiaceae in high weight hard feces. Between fecal types, several bacterial taxa such as Ruminococcaceae, and Akkermansia spp. were enriched in soft feces. PICRUSt analysis revealed that metabolic pathways such as "stilbenoid, diarylheptanoid, gingerol biosynthesis" were enriched in high weight rabbits, and pathways related to "xenobiotics biodegradation" and "various types of N-glycan biosynthesis" were overrepresented in rabbit soft feces. Our study provides foundation to generate hypothesis aiming to test the roles that different bacterial taxa play in the growth and caecotrophy of rex rabbits.

  14. The bacterial communities associated with fecal types and body weight of rex rabbits

    PubMed Central

    Zeng, Bo; Han, Shushu; Wang, Ping; Wen, Bin; Jian, Wensu; Guo, Wei; Yu, Zhiju; Du, Dan; Fu, Xiangchao; Kong, Fanli; Yang, Mingyao; Si, Xiaohui; Zhao, Jiangchao; Li, Ying

    2015-01-01

    Rex rabbit is an important small herbivore for fur and meat production. However, little is known about the gut microbiota in rex rabbit, especially regarding their relationship with different fecal types and growth of the hosts. We characterized the microbiota of both hard and soft feces from rex rabbits with high and low body weight by using the Illumina MiSeq platform targeting the V4 region of the 16S rDNA. High weight rex rabbits possess distinctive microbiota in hard feces, but not in soft feces, from the low weight group. We detected the overrepresentation of several genera such as YS2/Cyanobacteria, and Bacteroidales and underrepresentation of genera such as Anaeroplasma spp. and Clostridiaceae in high weight hard feces. Between fecal types, several bacterial taxa such as Ruminococcaceae, and Akkermansia spp. were enriched in soft feces. PICRUSt analysis revealed that metabolic pathways such as “stilbenoid, diarylheptanoid, gingerol biosynthesis” were enriched in high weight rabbits, and pathways related to “xenobiotics biodegradation” and “various types of N-glycan biosynthesis” were overrepresented in rabbit soft feces. Our study provides foundation to generate hypothesis aiming to test the roles that different bacterial taxa play in the growth and caecotrophy of rex rabbits. PMID:25791609

  15. Creating Inclusive Physical Activity Spaces: The Case of Body-Positive Yoga

    ERIC Educational Resources Information Center

    Pickett, Andrew C.; Cunningham, George B.

    2017-01-01

    Purpose: Within the modern cultural climate, those in larger bodies face high levels of weight stigma, particularly in sport and physical activity spaces, which serves as a strong barrier to their participation. However, given the strong link between physical activity and general health and well-being for participants, it is important to explore…

  16. Observations on glial inclusion bodies in a case of acute disseminated sclerosis

    PubMed Central

    Field, E. J.; Miller, Henry; Russell, Dorothy S.

    1962-01-01

    An unusual rod-like structure enclosed within a vacuole is described as occurring in enlarged glial cells associated with the lesions encountered in an uncommonly acute case of multiple sclerosis apparently heralded by an attack of `viral encephalitis'. Similar bodies were not found in a variety of other enlarged glial cells. An encapsulated `grape-fruit' like structure was also seen. Images PMID:13892761

  17. Benchmark Calculation of Inclusive Electromagnetic Responses in the Four-Body Nuclear System

    SciTech Connect

    Stetcu, I; Quaglioni, S; Bacca, S; Barrett, B R; Johnson, C W; Navratil, P; Barnea, N; Leidemann, W; Orlandini, G

    2006-05-09

    Both the no-core shell model and the effective interaction hyperspherical harmonic approaches are applied to the calculation of different response functions to external electromagnetic probes, using the Lorentz integral transform method. The test is performed on the four-body nuclear system, within a simple potential model. The quality of the agreement in the various cases is discussed, together with the perspectives for rigorous ab initio calculations of cross sections of heavier nuclei.

  18. Systematic Significance of Cell Inclusions in Haemodoraceae and Allied Families: Silica Bodies and Tapetal Raphides

    PubMed Central

    PRYCHID, CHRISTINA J.; FURNESS, CAROL A.; RUDALL, PAULA J.

    2003-01-01

    This paper presents the first record of silica deposits in tissues of Haemodoraceae and adds new records of tapetal raphides in this family. Within the order Commelinales, silica is present in leaves of three families (Hanguanacaeae, Haemodoraceae and Commelinaceae), but entirely absent from the other two (Pontederiaceae and Philydraceae). Presence or absence of characteristic cell inclusions may have systematic potential in commelinid monocotyledons, although the existing topology indicates de novo gains and losses in individual families. Silica sand was observed in leaves of five out of nine genera examined of Haemodoraceae, predominantly in vascular bundle sheath cells and epidermal cells. Within Haemodoraceae, silica is limited to subfamily Conostylidoideae. The occurrence of silica in Phlebocarya supports an earlier transfer of this genus from Haemodoroideae to Conostylidoideae. The presence of raphides (calcium oxalate crystals) in the anther tapetum represents a rare character, only reported in a few monocot families of the order Commelinales, and possibly representing a mechanism for regulation of cytoplasmic free calcium levels. Tapetal raphides were observed here in Anigozanthus and Conostylis (both Haemodoraceae), and Tradescantia (Commelinaceae), thus supplementing two earlier records in Haemodoraceae, Philydraceae and Commelinaceae. PMID:14507742

  19. Comparison of bacterial and archaeal communities in depth-resolved zones in an LNAPL body.

    PubMed

    Irianni-Renno, Maria; Akhbari, Daria; Olson, Mitchell R; Byrne, Adam P; Lefèvre, Emilie; Zimbron, Julio; Lyverse, Mark; Sale, Thomas C; De Long, Susan K

    2016-04-01

    Advances in our understanding of the microbial ecology at sites impacted by light non-aqueous phase liquids (LNAPLs) are needed to drive development of optimized bioremediation technologies, support longevity models, and develop culture-independent molecular tools. In this study, depth-resolved characterization of geochemical parameters and microbial communities was conducted for a shallow hydrocarbon-impacted aquifer. Four distinct zones were identified based on microbial community structure and geochemical data: (i) an aerobic, low-contaminant mass zone at the top of the vadose zone; (ii) a moderate to high-contaminant mass, low-oxygen to anaerobic transition zone in the middle of the vadose zone; (iii) an anaerobic, high-contaminant mass zone spanning the bottom of the vadose zone and saturated zone; and (iv) an anaerobic, low-contaminant mass zone below the LNAPL body. Evidence suggested that hydrocarbon degradation is mediated by syntrophic fermenters and methanogens in zone III. Upward flux of methane likely contributes to promoting anaerobic conditions in zone II by limiting downward flux of oxygen as methane and oxygen fronts converge at the top of this zone. Observed sulfate gradients and microbial communities suggested that sulfate reduction and methanogenesis both contribute to hydrocarbon degradation in zone IV. Pyrosequencing revealed that Syntrophus- and Methanosaeta-related species dominate bacterial and archaeal communities, respectively, in the LNAPL body below the water table. Observed phylotypes were linked with in situ anaerobic hydrocarbon degradation in LNAPL-impacted soils.

  20. Foreign Body Infection Models to Study Host-Pathogen Response and Antimicrobial Tolerance of Bacterial Biofilm

    PubMed Central

    Nowakowska, Justyna; Landmann, Regine; Khanna, Nina

    2014-01-01

    The number of implanted medical devices is steadily increasing and has become an effective intervention improving life quality, but still carries the risk of infection. These infections are mainly caused by biofilm-forming staphylococci that are difficult to treat due to the decreased susceptibility to both antibiotics and host defense mechanisms. To understand the particular pathogenesis and treatment tolerance of implant-associated infection (IAI) animal models that closely resemble human disease are needed. Applications of the tissue cage and catheter abscess foreign body infection models in the mouse will be discussed herein. Both models allow the investigation of biofilm and virulence of various bacterial species and a comprehensive insight into the host response at the same time. They have also been proven to serve as very suitable tools to study the anti-adhesive and anti-infective efficacy of different biomaterial coatings. The tissue cage model can additionally be used to determine pharmacokinetics, efficacy and cytotoxicity of antimicrobial compounds as the tissue cage fluid can be aspirated repeatedly without the need to sacrifice the animal. Moreover, with the advance in innovative imaging systems in rodents, these models may offer new diagnostic measures of infection. In summary, animal foreign body infection models are important tools in the development of new antimicrobials against IAI and can help to elucidate the complex interactions between bacteria, the host immune system, and prosthetic materials. PMID:27025752

  1. Stanley Fahn Lecture 2005: The staging procedure for the inclusion body pathology associated with sporadic Parkinson's disease reconsidered.

    PubMed

    Braak, Heiko; Bohl, Jürgen R; Müller, Christian M; Rüb, Udo; de Vos, Rob A I; Del Tredici, Kelly

    2006-12-01

    The synucleinopathy known as sporadic Parkinson's disease (PD) is a multisystem disorder that severely damages predisposed nerve cell types in circumscribed regions of the human nervous system. A recent staging procedure for the inclusion body pathology associated with PD proposes that, in the brain, the pathological process (formation of proteinaceous intraneuronal Lewy bodies and Lewy neurites) begins at two sites and continues in a topographically predictable sequence in six stages, during which components of the olfactory, autonomic, limbic, and somatomotor systems become progressively involved. In stages 1 to 2, the Lewy body pathology is confined to the medulla oblongata/pontine tegmentum and anterior olfactory structures. In stages 3 to 4, the substantia nigra and other nuclei of the basal mid- and forebrain become the focus of initially subtle and, then, severe changes. During this phase, the illness probably becomes clinically manifest. In the final stages 5 to 6, the lesions appear in the neocortex. This cross-sectional study originally was performed on 168 autopsy cases using material from 69 incidental cases and 41 clinically diagnosed PD patients as well as 58 age- and gender-matched controls. Here, the staging hypothesis is critically reconsidered and discussed.

  2. Improving the In-Medium Similarity Renormalization Group via approximate inclusion of three-body effects

    NASA Astrophysics Data System (ADS)

    Morris, Titus; Bogner, Scott

    2016-09-01

    The In-Medium Similarity Renormalization Group (IM-SRG) has been applied successfully to the ground state of closed shell finite nuclei. Recent work has extended its ability to target excited states of these closed shell systems via equation of motion methods, and also complete spectra of the whole SD shell via effective shell model interactions. A recent alternative method for solving of the IM-SRG equations, based on the Magnus expansion, not only provides a computationally feasible route to producing observables, but also allows for approximate handling of induced three-body forces. Promising results for several systems, including finite nuclei, will be presented and discussed.

  3. Improving the In-Medium Similarity Renormalization Group via approximate inclusion of three-body effects

    NASA Astrophysics Data System (ADS)

    Morris, Titus; Bogner, Scott

    2015-10-01

    The In-Medium Similarity Renormalization Group (IM-SRG) has been applied successfully not only to several closed shell finite nuclei, but has recently been used to produce effective shell model interactions that are competitive with phenomenological interactions in the SD shell. A recent alternative method for solving of the IM-SRG equations, called the Magnus expansion, not only provides a computationally feasible route to producing observables, but also allows for approximate handling of induced three-body forces. Promising results for several systems, including finite nuclei, will be presented and discussed.

  4. Novel demonstration of amyloid-β oligomers in sporadic inclusion-body myositis muscle fibers.

    PubMed

    Nogalska, Anna; D'Agostino, Carla; Engel, W King; Klein, William L; Askanas, Valerie

    2010-11-01

    Accumulation of amyloid-β (Aβ) within muscle fibers has been considered an upstream step in the development of the s-IBM pathologic phenotype. Aβ42, which is considered more cytotoxic than Aβ40 and has a higher propensity to oligomerize, is preferentially increased in s-IBM muscle fibers. In Alzheimer disease (AD), low-molecular weight Aβ oligomers and toxic oligomers, also referred to as "Aβ-Derived Diffusible Ligands" (ADDLs), are considered strongly cytotoxic and proposed to play an important pathogenic role. ADDLs have been shown to be increased in AD brain. We now report for the first time that in s-IBM muscle biopsies Aβ-dimer, -trimer, and -tetramer are identifiable by immunoblots. While all the s-IBM samples we studied had Aβ-oligomers, their molecular weights and intensity varied between the patient samples. None of the control muscle biopsies had Aβ oligomers. Dot-immunoblots using highly specific anti-ADDL monoclonal antibodies also showed highly increased ADDLs in all s-IBM biopsies studied, while controls were negative. By immunofluorescence, in some of the abnormal s-IBM muscle fibers ADDLs were accumulated in the form of plaque-like inclusions, and were often increased diffusely in very small fibers. Normal and disease-controls were negative. By gold-immuno-electron microscopy, ADDL-immunoreactivities were in close proximity to 6-10 nm amyloid-like fibrils, and also were immunodecorating amorphous and floccular material. In cultured human muscle fibers, we found that inhibition of autophagy led to the accumulation of Aβ oligomers. This novel demonstration of Aβ42 oligomers in s-IBM muscle biopsy provides additional evidence that intra-muscle fiber accumulation of Aβ42 oligomers in s-IBM may contribute importantly to s-IBM pathogenic cascade.

  5. Iron and aluminum deposition in the meninges of the lamprey: identification of an aluminum-ferritin inclusion body

    SciTech Connect

    Youson, J.H.; Sargent, P.A.; Pearce, G.W.

    1989-01-01

    The meningeal tissue of the brain and spinal cord of larval and juvenile adults of lampreys (Petromyzon marinus) was examined by routine electron microscopy, electron microscopic histochemistry, and electron-probe x-ray microanalysis to locate sites of iron deposition. A magnetometer was used for identification of ferromagnetic iron. Ferritin particles, representing ferric iron, are present in abundance within the cytoplasmic matrices and in dense bodies of meningeal cells of both the brain and spinal cord of larvae and juveniles. These round cells of the meninges also contain abundant glycogen and lipid. Small quantities of ferrous iron are associated to the latter inclusion. Aluminum deposits are present within an electron-dense material of many ferritin-containing inclusions of meningeal cells of the larval brain. Ferromagnetic material was not detected in larval and upstream-migrant lampreys. The deposition of iron and aluminum in the meninges of lampreys may be related to physiological and environmental factors, respectively, and/or to an important interaction between the two metals.

  6. Sporadic inclusion body myositis: A review of recent clinical advances and current approaches to diagnosis and treatment.

    PubMed

    Needham, Merrilee; Mastaglia, Frank L

    2016-03-01

    Sporadic inclusion body myositis is the most frequent acquired myopathy of middle and later life and is distinguished from other inflammatory myopathies by its selective pattern of muscle involvement and slowly progressive course, and by the combination of inflammatory and degenerative muscle pathology and multi-protein deposits in muscle tissue. This review summarises the findings of recent studies that provide a more complete picture of the clinical phenotype and natural history of the disease and its global prevalence and genetic predisposition. Current diagnostic criteria, including the role of electrophysiological and muscle imaging studies and the recently identified anti-5'-nucleotidase (anti-cN1A) antibody in diagnosis are also discussed as well as current trends in the treatment of the disease. Copyright © 2015 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

  7. Anti-aggregatory effect of cyclodextrins in the refolding process of recombinant growth hormones from Escherichia coli inclusion bodies.

    PubMed

    Bajorunaite, Egle; Cirkovas, Andrejus; Radzevicius, Kostas; Larsen, Kim Lambertsen; Sereikaite, Jolanta; Bumelis, Vladas-Algirdas

    2009-06-01

    Cyclodextrins with different ring size and ring substituents were tested for recombinant mink and porcine growth hormones aggregation suppression in the refolding process from Escherichia coli inclusion bodies. Methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin show a positive effect on the aggregation suppression of both proteins. The influence of different methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin concentrations on the renaturation yield of both growth hormones was investigated. Moreover, methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin suppress not only folding-related, but also temperature-related aggregates formation of both proteins. Circular dichroism experiments (monitoring of protein solution turbidity by registering high tension voltage) showed that the onset temperature of aggregation of both growth hormones increased with increasing 2-hydroxypropyl-beta-cyclodextrin concentration. In conclusion, cyclodextrins have perspectives in biotechnology of veterinary growth hormones not only for protein production, but also for its storage.

  8. Overexpression and refolding of thioredoxin/TRAIL fusion from inclusion bodies and further purification of TRAIL after cleavage by enteropeptidase.

    PubMed

    Gasparian, Marine E; Ostapchenko, Valeriy G; Yagolovich, Anne V; Tsygannik, Igor N; Chernyak, Boris V; Dolgikh, Dmitry A; Kirpichnikov, Mikhail P

    2007-10-01

    The human TRAIL gene (encoding residues 114-281) was synthesized by PCR and cloned into plasmid pET-32a. High level expression (1.5 g l(-1)) of thioredoxin/TRAIL fusion was achieved in Escherichia coli strain BL21(DE3), mainly as inclusion bodies. Refolded fusion thioredoxin/TRAIL was cleaved by enteropeptidase and TRAIL was separated from thioredoxin on Ni-NTA agarose. High yield (400 mg l(-1)) of TRAIL without N-terminal methionine and His tag was obtained. Sedimentation coefficient demonstrated that 98% of TRAIL formed trimers. TRAIL formed crystals of space group P3 (1) with unit-cell dimensions a = b = 72.5 A, c = 141.5 A. Apoptosis induced in HeLa cells by purified TRAIL was 5-fold enhanced by emetine.

  9. Approaches for the generation of active papain-like cysteine proteases from inclusion bodies of Escherichia coli.

    PubMed

    Ling, Chunfang; Zhang, Junyan; Lin, Deqiu; Tao, Ailin

    2015-05-01

    Papain-like cysteine proteases are widely expressed, fulfill specific functions in extracellular matrix turnover, antigen presentation and processing events, and may represent viable drug targets for major diseases. In depth and rigorous studies of the potential for these proteins to be targets for drug development require sufficient amounts of protease protein that can be used for both experimental and therapeutic purposes. Escherichia coli was widely used to express papain-like cysteine proteases, but most of those proteases are produced in insoluble inclusion bodies that need solubilizing, refolding, purifying and activating. Refolding is the most critical step in the process of generating active cysteine proteases and the current approaches to refolding include dialysis, dilution and chromatography. Purification is mainly achieved by various column chromatography. Finally, the attained refolded proteases are examined regarding their protease structures and activities.

  10. Immunohistochemical Detection of a Unique Protein within Cells of Snakes Having Inclusion Body Disease, a World-Wide Disease Seen in Members of the Families Boidae and Pythonidae

    PubMed Central

    Chang, Li-Wen; Fu, Ann; Wozniak, Edward; Chow, Marjorie; Duke, Diane G.; Green, Linda; Kelley, Karen; Hernandez, Jorge A.; Jacobson, Elliott R.

    2013-01-01

    Inclusion body disease (IBD) is a worldwide disease in captive boa constrictors (boa constrictor) and occasionally in other snakes of the families Boidae and Pythonidae. The exact causative agent(s) and pathogenesis are not yet fully understood. Currently, diagnosis of IBD is based on the light microscopic identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin stained tissues or blood smears. An antigenically unique 68 KDa protein was identified within the IBD inclusion bodies, called IBD protein. A validated immuno-based ante-mortem diagnostic test is needed for screening snakes that are at risk of having IBD. In this study, despite difficulties in solubilizing semi-purified inclusion bodies, utilizing hybridoma technology a mouse anti-IBD protein monoclonal antibody (MAB) was produced. The antigenic specificity of the antibody was confirmed and validated by western blots, enzyme-linked immunosorbent assay, immuno-transmission electron microscopy, and immunohistochemical staining. Paraffin embedded tissues of IBD positive and negative boa constrictors (n=94) collected from 1990 to 2011 were tested with immunohistochemical staining. In boa constrictors, the anti-IBDP MAB had a sensitivity of 83% and specificity of 100% in detecting IBD. The antibody also cross-reacted with IBD inclusion bodies in carpet pythons (Morelia spilota) and a ball python (python regius). This validated antibody can serve as a tool for the development of ante-mortem immunodiagnostic tests for IBD. PMID:24340066

  11. Immunohistochemical detection of a unique protein within cells of snakes having inclusion body disease, a world-wide disease seen in members of the families Boidae and Pythonidae.

    PubMed

    Chang, Li-Wen; Fu, Ann; Wozniak, Edward; Chow, Marjorie; Duke, Diane G; Green, Linda; Kelley, Karen; Hernandez, Jorge A; Jacobson, Elliott R

    2013-01-01

    Inclusion body disease (IBD) is a worldwide disease in captive boa constrictors (boa constrictor) and occasionally in other snakes of the families Boidae and Pythonidae. The exact causative agent(s) and pathogenesis are not yet fully understood. Currently, diagnosis of IBD is based on the light microscopic identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin stained tissues or blood smears. An antigenically unique 68 KDa protein was identified within the IBD inclusion bodies, called IBD protein. A validated immuno-based ante-mortem diagnostic test is needed for screening snakes that are at risk of having IBD. In this study, despite difficulties in solubilizing semi-purified inclusion bodies, utilizing hybridoma technology a mouse anti-IBD protein monoclonal antibody (MAB) was produced. The antigenic specificity of the antibody was confirmed and validated by western blots, enzyme-linked immunosorbent assay, immuno-transmission electron microscopy, and immunohistochemical staining. Paraffin embedded tissues of IBD positive and negative boa constrictors (n=94) collected from 1990 to 2011 were tested with immunohistochemical staining. In boa constrictors, the anti-IBDP MAB had a sensitivity of 83% and specificity of 100% in detecting IBD. The antibody also cross-reacted with IBD inclusion bodies in carpet pythons (Morelia spilota) and a ball python (python regius). This validated antibody can serve as a tool for the development of ante-mortem immunodiagnostic tests for IBD.

  12. The small heat shock protein, HSP30, is associated with aggresome-like inclusion bodies in proteasomal inhibitor-, arsenite-, and cadmium-treated Xenopus kidney cells.

    PubMed

    Khan, Saad; Khamis, Imran; Heikkila, John J

    2015-11-01

    In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with the proteasomal inhibitor, MG132, or the environmental toxicants, sodium arsenite or cadmium chloride, induced the accumulation of the small heat shock protein, HSP30, in total and in both soluble and insoluble protein fractions. Immunocytochemical analysis revealed the presence of relatively large HSP30 structures primarily in the perinuclear region of the cytoplasm. All three of the stressors promoted the formation of aggresome-like inclusion bodies as determined by immunocytochemistry and laser scanning confocal microscopy using a ProteoStat aggresome dye and additional aggresomal markers, namely, anti-γ-tubulin and anti-vimentin antibodies. Further analysis revealed that HSP30 co-localized with these aggresome-like inclusion bodies. In most cells, HSP30 was found to envelope or occur within these structures. Finally, we show that treatment of cells with withaferin A, a steroidal lactone with anti-inflammatory, anti-tumor, and proteasomal inhibitor properties, also induced HSP30 accumulation that co-localized with aggresome-like inclusion bodies. It is possible that proteasomal inhibitor or metal/metalloid-induced formation of aggresome-like inclusion bodies may sequester toxic protein aggregates until they can be degraded. While the role of HSP30 in these aggresome-like structures is not known, it is possible that they may be involved in various aspects of aggresome-like inclusion body formation or transport. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed

    Khan, R H; Rao, K B; Eshwari, A N; Totey, S M; Panda, A K

    1998-01-01

    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.

  14. Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.

    PubMed

    Maseko, Sibusiso B; Natarajan, Satheesh; Sharma, Vikas; Bhattacharyya, Neelakshi; Govender, Thavendran; Sayed, Yasien; Maguire, Glenn E M; Lin, Johnson; Kruger, Hendrik G

    2016-06-01

    Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 μmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Advanced glycation end products induce in vitro cross-linking of alpha-synuclein and accelerate the process of intracellular inclusion body formation.

    PubMed

    Shaikh, Shamim; Nicholson, Louise F B

    2008-07-01

    Cross-linking of alpha-synuclein and Lewy body formation have been implicated in the dopaminergic neuronal cell death observed in Parkinson's disease (PD); the mechanisms responsible, however, are not clear. Reactive oxygen species and advanced glycation end products (AGEs) have been found in the intracellular, alpha-synuclein-positive Lewy bodies in the brains of both PD as well as incidental Lewy body disease patients, suggesting a role for AGEs in alpha-synuclein cross-linking and Lewy body formation. The aims of the present study were to determine 1) whether AGEs can induce cross-linking of alpha-synuclein peptides, 2) the progressive and time-dependent intracellular accumulation of AGEs and inclusion body formation, and 3) the effects of extracellular or exogenous AGEs on intracellular inclusion formation. We first investigated the time-dependent cross-linking of recombinant human alpha-synuclein in the presence of AGEs in vitro, then used a cell culture model based on chronic rotenone treatment of human dopaminergic neuroblastoma cells (SH-SY5Y) over a period of 1-4 weeks, in the presence of different doses of AGEs. Cells (grown on coverslips) and cell lysates, collected at the end of every week, were analyzed for the presence of intracellular reactive oxygen species, AGEs, alpha-synuclein proteins, and intracellular alpha-synuclein- and AGE-positive inclusion bodies by using immunocytochemical, biochemical, and Western blot techniques. Our results show that AGEs promote in vitro cross-linking of alpha-synuclein, that intracellular accumulation of AGEs precedes alpha-synuclein-positive inclusion body formation, and that extracellular AGEs accelerate the process of intracellular alpha-synuclein-positive inclusion body formation.

  16. Characterization of the 4,6-α-glucanotransferase GTFB enzyme of Lactobacillus reuteri 121 isolated from inclusion bodies.

    PubMed

    Bai, Yuxiang; van der Kaaij, Rachel Maria; Woortman, Albert Jan Jacob; Jin, Zhengyu; Dijkhuizen, Lubbert

    2015-06-09

    The GTFB enzyme of the probiotic bacterium Lactobacillus reuteri 121 is a 4,6-α-glucanotransferase of glycoside hydrolase family 70 (GH70; http://www.cazy.org ). Contrary to the glucansucrases in GH70, GTFB is unable to use sucrose as substrate, but instead converts malto-oligosaccharides and starch into isomalto-/malto- polymers that may find application as prebiotics and dietary fibers. The GTFB enzyme expresses well in Escherichia coli BL21 Star (DE3), but mostly accumulates in inclusion bodies (IBs) which generally contain wrongly folded protein and inactive enzyme. Denaturation followed by refolding, as well as ncIB preparation were used for isolation of active GTFB protein from inclusion bodies. Soluble, refolded and ncIB GTFB were compared using activity assays, secondary structure analysis by FT-IR, and product analyses by NMR, HPAEC and SEC. Expression of GTFB in E. coli yielded > 100 mg/l relatively pure and active but mostly insoluble GTFB protein in IBs, regardless of the expression conditions used. Following denaturing, refolding of GTFB protein was most efficient in double distilled H2O. Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein. When expressed as units of activity obtained per liter E. coli culture, the total amount of ncIB GTFB expressed possessed around 180 % hydrolysis activity and 100 % transferase activity compared to the amount of soluble GTFB enzyme obtained from one liter culture. The product profiles obtained for the three GTFB enzyme preparations were similar when analyzed by HPAEC and NMR. SEC investigation also showed that these 3 enzyme preparations yielded products with similar size distributions. FT-IR analysis revealed extended β-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs. The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB. In view of their relatively high yield

  17. Cauliflower mosaic virus major inclusion body protein interacts with the aphid transmission factor, the virion-associated protein, and gene VII product.

    PubMed

    Lutz, Lindy; Raikhy, Gaurav; Leisner, Scott M

    2012-12-01

    The Cauliflower mosaic virus (CaMV) gene VI product (P6) is a multifunctional protein essential for viral infection. In order to perform its various tasks, P6 interacts with both viral and host factors, as well as forming electron-dense cytoplasmic inclusion bodies. Here we investigate the interactions of P6 with three CaMV proteins: P2 (aphid transmission factor), P3 (virion-associated protein), and P7 (protein of unknown function). Based on yeast two-hybrid and maltose-binding protein pull-down experiments, P6 interacted with all three of these CaMV proteins. P2 helps to stabilize P6 inclusion bodies. Although the P2s from two CaMV isolates (W260 and CM1841) differ in the ability to stabilize inclusion bodies, both interacted similarly with P6. This suggests that inclusion body stability may not be dependent on the efficiency of P2-P6 interaction. However, neither P2 nor P3 interacted with P7 in yeast two-hybrid assays.

  18. High pH solubilization and chromatography-based renaturation and purification of recombinant human granulocyte colony-stimulating factor from inclusion bodies.

    PubMed

    Li, Ming; Fan, Hua; Liu, Jiahua; Wang, Minhong; Wang, Lili; Wang, Chaozhan

    2012-03-01

    Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a very efficient therapeutic protein drug which has been widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was solubilized from inclusion bodies by using a high-pH solution containing low concentration of urea. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the buffer pH employed; alkalic pH significantly favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured with simultaneous purification by using weak anion exchange, strong anion exchange, and hydrophobic interaction chromatography, separately. The results indicated that the rhG-CSF solubilized by the high-pH solution containing low concentration of urea had much higher mass recovery than the one solubilized by 8 M urea when using anyone of the three refolding methods employed in this work. In the case of weak anion exchange chromatography, the high pH solubilized rhG-CSF could get a mass recovery of 73%. The strategy of combining solubilization of inclusion bodies at high pH with refolding of protein using liquid chromatography may become a routine method for protein production from inclusion bodies.

  19. Silver stainings distinguish Lewy bodies and glial cytoplasmic inclusions: comparison between Gallyas-Braak and Campbell-Switzer methods.

    PubMed

    Uchihara, Toshiki; Nakamura, Ayako; Mochizuki, Yoko; Hayashi, Masaharu; Orimo, Satoshi; Isozaki, Eiji; Mizutani, Toshio

    2005-09-01

    Lewy bodies (LBs) of idiopathic Parkinson's disease and glial cytoplasmic inclusions (GCIs) of multiple system atrophy are pathological deposits both composed of phosphorylated alpha-synuclein woven into different filaments. Although both LBs and GCIs are considered to be hallmarks for each independent synucleinopathy, until now they could not be clearly distinguished on the basis of their biochemical or immunohistochemical features. We have examined possible differences in their argyrophilic features and their relation to synuclein-like or ubiquitin-like immunoreactivity (IR). Pairs of mirror sections from different brain areas were triple-fluorolabeled with an anti-alpha-synuclein antibody, an anti-ubiquitin antibody and thiazin red (TR), a fluorochrome that labels fibrillary structures such as Lewy bodies or neurofibrillary tangles. One of the paired sections was subsequently stained using the Campbell-Switzer method (CS), and the other by the Gallyas-Braak method (GB). By comparing of the same microscopic field on the paired fluorolabeled sections, subsequently silver-stained with either CS or GB, five different profiles of each structure could be determined: alpha-synuclein-like IR, ubiquitin-like IR, affinity to TR, argyrophilia with CS or GB. GCIs exhibited argyrophilia with both CS and GB but lacked affinity to TR. In contrast, LBs exhibited argyrophilia with CS but not with GB and some affinity to TR. These disease-specific profiles of argyrophilia were consistent, and were not influenced by areas or cases examined. Although immunohistochemical features of LBs and GCIs were similar in exhibiting IR for alpha-synuclein and ubiquitin, the contrast in their argyrophilic profiles may indicate possible differences in the molecular composition or conformation of alpha-synuclein. Even though these empirical differences still remain to be explained, awareness of this clear distinction is potentially of diagnostic and pathological relevance.

  20. Autoantibodies produced at the site of tissue damage provide evidence of humoral autoimmunity in inclusion body myositis.

    PubMed

    Ray, Arundhati; Amato, Anthony A; Bradshaw, Elizabeth M; Felice, Kevin J; DiCapua, Daniel B; Goldstein, Jonathan M; Lundberg, Ingrid E; Nowak, Richard J; Ploegh, Hidde L; Spooner, Eric; Wu, Qian; Willis, Simon N; O'Connor, Kevin C

    2012-01-01

    Inclusion body myositis (IBM) belongs to a group of muscle diseases known as the inflammatory myopathies. The presence of antibody-secreting plasma cells in IBM muscle implicates the humoral immune response in this disease. However, whether the humoral immune response actively contributes to IBM pathology has not been established. We sought to investigate whether the humoral immune response in IBM both in the periphery and at the site of tissue damage was directed towards self-antigens. Peripheral autoantibodies present in IBM serum but not control serum recognized self-antigens in both muscle tissue and human-derived cell lines. To study the humoral immune response at the site of tissue damage in IBM patients, we isolated single plasma cells directly from IBM-derived muscle tissue sections and from these cells, reconstructed a series of recombinant immunoglobulins (rIgG). These rIgG, each representing a single muscle-associated plasma cell, were examined for reactivity to self-antigens. Both, flow cytometry and immunoblotting revealed that these rIgG recognized antigens expressed by cell lines and in muscle tissue homogenates. Using a mass spectrometry-based approach, Desmin, a major intermediate filament protein, expressed abundantly in muscle tissue, was identified as the target of one IBM muscle-derived rIgG. Collectively, these data support the view that IBM includes a humoral immune response in both the periphery and at the site of tissue damage that is directed towards self-antigens.

  1. Autoantibodies Produced at the Site of Tissue Damage Provide Evidence of Humoral Autoimmunity in Inclusion Body Myositis

    PubMed Central

    Ray, Arundhati; Amato, Anthony A.; Bradshaw, Elizabeth M.; Felice, Kevin J.; DiCapua, Daniel B.; Goldstein, Jonathan M.; Lundberg, Ingrid E.; Nowak, Richard J.; Ploegh, Hidde L.; Spooner, Eric; Wu, Qian

    2012-01-01

    Inclusion body myositis (IBM) belongs to a group of muscle diseases known as the inflammatory myopathies. The presence of antibody-secreting plasma cells in IBM muscle implicates the humoral immune response in this disease. However, whether the humoral immune response actively contributes to IBM pathology has not been established. We sought to investigate whether the humoral immune response in IBM both in the periphery and at the site of tissue damage was directed towards self-antigens. Peripheral autoantibodies present in IBM serum but not control serum recognized self-antigens in both muscle tissue and human-derived cell lines. To study the humoral immune response at the site of tissue damage in IBM patients, we isolated single plasma cells directly from IBM-derived muscle tissue sections and from these cells, reconstructed a series of recombinant immunoglobulins (rIgG). These rIgG, each representing a single muscle-associated plasma cell, were examined for reactivity to self-antigens. Both, flow cytometry and immunoblotting revealed that these rIgG recognized antigens expressed by cell lines and in muscle tissue homogenates. Using a mass spectrometry-based approach, Desmin, a major intermediate filament protein, expressed abundantly in muscle tissue, was identified as the target of one IBM muscle-derived rIgG. Collectively, these data support the view that IBM includes a humoral immune response in both the periphery and at the site of tissue damage that is directed towards self-antigens. PMID:23071619

  2. Atypical sporadic CJD-MM phenotype with white matter kuru plaques associated with intranuclear inclusion body and argyrophilic grain disease.

    PubMed

    Berghoff, Anna S; Trummert, Anita; Unterberger, Ursula; Ströbel, Thomas; Hortobágyi, Tibor; Kovacs, Gabor G

    2015-08-01

    We describe an atypical neuropathological phenotype of sporadic Creutzfeldt-Jakob disease in a 76-year-old man. The clinical symptoms were characterized by progressive dementia, gait ataxia, rigidity and urinary incontinence. The disease duration was 6 weeks. MRI did not show prominent atrophy or hyperintensities in cortical areas, striatum or thalamus. Biomarker examination of the cerebrospinal fluid deviated from that seen in pure Alzheimer's disease. Triphasic waves in the EEG were detected only later in the disease course, while 14-3-3 assay was positive. PRNP genotyping revealed methionine homozygosity (MM) at codon 129. Neuropathology showed classical CJD changes corresponding to the MM type 1 cases. However, a striking feature was the presence of abundant kuru-type plaques in the white matter. This rare morphology was associated with neuropathological signs of intranuclear inclusion body disease and advanced stage of argyrophilic grain disease. These alterations did not show correlation with each other, thus seemed to develop independently. This case further highlights the complexity of neuropathological alterations in the ageing brain. © 2015 Japanese Society of Neuropathology.

  3. Inclusion body myopathy, Paget's disease of the bone and fronto-temporal dementia: a disorder of autophagy.

    PubMed

    Ju, Jeong-Sun; Weihl, Conrad C

    2010-04-15

    Inclusion body myopathy associated with Paget's disease of the bone and fronto-temporal dementia (IBMPFD) is a progressive autosomal dominant disorder caused by mutations in p97/VCP (valosin-containing protein). p97/VCP is a member of the AAA+ (ATPase associated with a variety of activities) protein family and participates in multiple cellular processes. One particularly important role for p97/VCP is facilitating intracellular protein degradation. p97/VCP has traditionally been thought to mediate the ubiquitin-proteasome degradation of proteins; however, recent studies challenge this dogma. p97/VCP clearly participates in the degradation of aggregate-prone proteins, a process principally mediated by autophagy. In addition, IBMPFD mutations in p97/VCP lead to accumulation of autophagic structures in patient and transgenic animal tissue. This is likely due to a defect in p97/VCP-mediated autophagosome maturation. The following review will discuss the evidence for p97/VCP in autophagy and how a disruption in this process contributes to IBMPFD pathogenesis.

  4. Evasion of Antiviral Immunity through Sequestering of TBK1/IKKε/IRF3 into Viral Inclusion Bodies

    PubMed Central

    Wu, Xiaodong; Qi, Xian; Qu, Bingqian; Zhang, Zerui; Liang, Mifang; Li, Chuan; Cardona, Carol J.; Li, Dexin

    2014-01-01

    Cells are equipped with pattern recognition receptors (PRRs) such as the Toll-like and RIG-I-like receptors that mount innate defenses against viruses. However, viruses have evolved multiple strategies to evade or thwart host antiviral responses. Viral inclusion bodies (IBs), which are accumulated aggregates of viral proteins, are commonly formed during the replication of some viruses in infected cells, but their role in viral immune evasion has rarely been explored. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging febrile illness caused by a novel phlebovirus in the Bunyaviridae. The SFTS viral nonstructural protein NSs can suppress host beta interferon (IFN-β) responses. NSs can form IBs in infected and transfected cells. Through interaction with tank-binding kinase 1 (TBK1), viral NSs was able to sequester the IKK complex, including IKKε and IRF3, into IBs, although NSs did not interact with IKKε or IRF3 directly. When cells were infected with influenza A virus, IRF3 was phosphorylated and active phosphorylated IRF3 (p-IRF3) was translocated into the nucleus. In the presence of NSs, IRF3 could still be phosphorylated, but p-IRF3 was trapped in cytoplasmic IBs, resulting in reduced IFN-β induction and enhanced viral replication. Sequestration of the IKK complex and active IRF3 into viral IBs through the interaction of NSs and TBK1 is a novel mechanism for viral evasion of innate immunity. PMID:24335286

  5. A Brazilian family with hereditary inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia.

    PubMed

    Fanganiello, R D; Kimonis, V E; Côrte, C C; Nitrini, R; Passos-Bueno, M R

    2011-04-01

    Inclusion body myopathy associated with Paget disease and frontotemporal dementia (IBMPFD) is a progressive and usually misdiagnosed autosomal dominant disorder. It is clinically characterized by a triad of features: proximal and distal myopathy, early onset Paget disease of bone (PDB), and frontotemporal dementia (FTD). It is caused by missense mutations in the valosin-containing protein (VCP) gene. We describe here the clinical and molecular findings of the first Brazilian family identified with IBMPFD. Progressive myopathy affecting the limb girdles was detected by clinical examination followed by muscle biopsy and creatine kinase measurement. PDB was suggested after anatomopathological bone examination and FTD was diagnosed by clinical, neuropsychological and language evaluations. Brain magnetic resonance revealed severe atrophy of the anterior temporal lobes, including the hippocampi. A R93C mutation in VCP was detected by direct sequencing screening in subject W (age 62) and in his mother. Four more individuals diagnosed with "dementia" were reported in this family. We also present a comprehensive genotype-phenotype correlation analysis of mutations in VCP in 182 patients from 29 families described in the literature and show that while IBM is a conspicuously penetrant symptom, PDB has a lower penetrance when associated with mutations in the AAAD1 domain and FTD has a lower penetrance when associated with mutations in the Junction (L1-D1) domain. Furthermore, the R93C mutation is likely to be associated with the penetrance of all the clinical symptoms of the triad.

  6. The presence of OMP inclusion bodies in a Escherichia coli K-12 mutated strain is not related to lipopolysaccharide structure.

    PubMed

    Corsaro, M Michela; Parrilli, Ermenegilda; Lanzetta, Rosa; Naldi, Teresa; Pieretti, Giuseppina; Lindner, Buko; Carpentieri, Andrea; Parrilli, Michelangelo; Tutino, M Luisa

    2009-08-01

    The role of lipopolysaccharides (LPSs) in the biogenesis of outer membrane proteins have been investigated in several studies. Some of these analyses showed that LPS is required for correct and efficient folding of outer membrane proteins; other studies support the idea of independence of outer membrane proteins biogenesis from LPS structure. In this article, we investigated the involvement of LPS structure in the anomalous aggregation of outer membrane proteins in a E. coli mutant strain (S17-1(lambdapir)). To achieve this aim, the LPS structure of the mutant strain was carefully determined and compared with the E. coli K-12 one. It turned out that LPS of these two strains differs in the inner core for the absence of a heptose residue (HepIII). We demonstrated that this difference is due to a mutation in waaQ, a gene encoding the transferase for the branch heptose HepIII residue. The mutation was complemented to find out if the restoration of LPS structure influenced the observed outer membrane proteins aggregation. Data reported in this work demonstrated that, in E. coli S17-1(lambdapir) there is no influence of LPS structure on the outer membrane proteins inclusion bodies formation.

  7. Psychological Impact of Predictive Genetic Testing in VCP Inclusion Body Myopathy, Paget Disease of Bone and Frontotemporal Dementia.

    PubMed

    Surampalli, Abhilasha; Khare, Manaswitha; Kubrussi, Georgette; Wencel, Marie; Tanaja, Jasmin; Donkervoort, Sandra; Osann, Kathryn; Simon, Mariella; Wallace, Douglas; Smith, Charles; M McInerney-Leo, Aideen; Kimonis, Virginia

    2015-10-01

    Inclusion Body Myopathy associated with Paget's disease of bone and Fronto-temporal Dementia, also known as multisystem proteinopathy is an autosomal dominant, late onset neurodegenerative disorder caused by mutations in Valosin containing protein (VCP) gene. This study aimed to assess uptake and decision making for predictive genetic testing and the impact on psychological well-being. Individuals who had participated in the gene discovery study with a 50 % a priori risk of inheriting VCP disease were sent a letter of invitation offering genetic counseling and testing and were also invited to participate in this psychosocial study. A total of 102 individuals received an invitation and 33 individuals participated in genetic counseling and testing (32.3 %) with 29 completing baseline questionnaires. Twenty completed the follow-up post-test Hospital Anxiety and Depression Scale questionnaire including 13 of the 18 who had tested positive. Mean risk perception at baseline was 50.1 %. Reasons for testing included planning for the future, relieving uncertainty, informing children and satisfying curiosity. At baseline, one quarter of the participants had high levels of anxiety. However, scores were normal one year following testing. In this small cohort, one third of individuals at 50 % risk chose pre-symptomatic testing. Although one quarter of those choosing testing had high anxiety at baseline, this was not evident at follow-up.

  8. Impact of different cultivation and induction regimes on the structure of cytosolic inclusion bodies of TEM1-beta-lactamase.

    PubMed

    Margreiter, Gerd; Schwanninger, Manfred; Bayer, Karl; Obinger, Christian

    2008-10-01

    The enzyme TEM1-beta-lactamase has been used as a model to study the impact of different cultivation and induction regimes on the structure of cytosolic inclusion bodies (IBs). The protein has been heterologously expressed in Escherichia coli in fed-batch cultivations at different temperatures (30, 37, and 40 degrees C) as well as induction regimes that guaranteed distinct product formation rates and ratios of soluble to aggregated protein. Additionally, shake flask cultivations at 20, 30, and 37 degrees C were performed. IBs were sampled during the whole bioprocess and structural analysis was performed by attenuated total reflectance Fourier transform infrared (ATR-FT-IR) spectroscopy. This work clearly demonstrates that the tested production regimes and rates had no impact on the IB structure, which was characterized by decreased alpha-helical and increased and modified beta-sheet contents compared to the native protein. Moreover, aggregates formed during refolding of IBs by solubilization and simple dilution showed very similar FT-IR spectra suggesting (i) the existence of only one critical folding step from which either aggregation (IB formation) or native folding branches off, and (ii) underlining the important role of the specific amino acid sequence in aggregation. The findings are discussed with respect to the known structure of TEM1-beta-lactamase and the reported kinetics of its (un)folding as well as contradictory data on the effect of cultivation regimes on IB structure(s) of other proteins.

  9. Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein

    PubMed Central

    Upadhyay, Arun K.; Singh, Anupam; Mukherjee, K. J.; Panda, Amulya K.

    2014-01-01

    A tetrameric protein of therapeutic importance, Escherichia coli L-asparaginase-II was expressed in Escherichia coli as inclusion bodies (IBs). Asparaginase IBs were solubilized using low concentration of urea and refolded into active tetrameric protein using pulsatile dilution method. Refolded asparaginase was purified in two steps by ion-exchange and gel filtration chromatographic techniques. The recovery of bioactive asparaginase from IBs was around 50%. The melting temperature (Tm) of the purified asparaginase was found to be 64°C. The specific activity of refolded, purified asparaginase was found to be comparable to the commercial asparaginase (190 IU/mg). Enzymatic activity of the refolded asparaginase was high even at four molar urea solutions, where the IB aggregates are completely solubilized. From the comparison of chemical denaturation data and activity at different concentrations of guanidine hydrochloride, it was observed that dissociation of monomeric units precedes the complete loss of helical secondary structures. Protection of the existing native-like protein structure during solubilization of IB aggregates with 4 M urea improved the propensity of monomer units to form oligomeric structure. Our mild solubilization technique retaining native-like structures, improved recovery of asparaginase in bioactive tetrameric form. PMID:25309524

  10. Sporadic inclusion-body myositis: A degenerative muscle disease associated with aging, impaired muscle protein homeostasis and abnormal mitophagy.

    PubMed

    Askanas, Valerie; Engel, W King; Nogalska, Anna

    2015-04-01

    Sporadic inclusion-body myositis (s-IBM) is the most common degenerative muscle disease in which aging appears to be a key risk factor. In this review we focus on several cellular molecular mechanisms responsible for multiprotein aggregation and accumulations within s-IBM muscle fibers, and their possible consequences. Those include mechanisms leading to: a) accumulation in the form of aggregates within the muscle fibers, of several proteins, including amyloid-β42 and its oligomers, and phosphorylated tau in the form of paired helical filaments, and we consider their putative detrimental influence; and b) protein misfolding and aggregation, including evidence of abnormal myoproteostasis, such as increased protein transcription, inadequate protein disposal, and abnormal posttranslational modifications of proteins. Pathogenic importance of our recently demonstrated abnormal mitophagy is also discussed. The intriguing phenotypic similarities between s-IBM muscle fibers and the brains of Alzheimer and Parkinson's disease patients, the two most common neurodegenerative diseases associated with aging, are also discussed. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.

  11. Aldehyde dehydrogenase, Ald4p, is a major component of mitochondrial fluorescent inclusion bodies in the yeast Saccharomyces cerevisiae

    PubMed Central

    Misonou, Yoshiko; Kikuchi, Maiko; Sato, Hiroshi; Inai, Tomomi; Kuroiwa, Tsuneyoshi; Tanaka, Kenji; Miyakawa, Isamu

    2014-01-01

    ABSTRACT When Saccharomyces cerevisiae strain 3626 was cultured to the stationary phase in a medium that contained glucose, needle-like structures that emitted autofluorescence were observed in almost all cells by fluorescence microscopy under UV excitation. The needle-like structures completely overlapped with the profile of straight elongated mitochondria. Therefore, these structures were designated as mitochondrial fluorescent inclusion bodies (MFIBs). The MFIB-enriched mitochondrial fractions were successfully isolated and 2D-gel electrophoresis revealed that a protein of 54 kDa was only highly concentrated in the fractions. Determination of the N-terminal amino acid sequence of the 54-kDa protein identified it as a mitochondrial aldehyde dehydrogenase, Ald4p. Immunofluorescence microscopy showed that anti-Ald4p antibody specifically stained MFIBs. Freeze-substitution electron microscopy demonstrated that cells that retained MFIBs had electron-dense filamentous structures with a diameter of 10 nm in straight elongated mitochondria. Immunoelectron microscopy showed that Ald4p was localized to the electron-dense filamentous structures in mitochondria. These results together showed that a major component of MFIBs is Ald4p. In addition, we demonstrate that MFIBs are common features that appear in mitochondria of many species of yeast. PMID:24771619

  12. Mutant LRRK2 Toxicity in Neurons Depends on LRRK2 Levels and Synuclein But Not Kinase Activity or Inclusion Bodies

    PubMed Central

    Skibinski, Gaia; Nakamura, Ken; Cookson, Mark R.

    2014-01-01

    By combining experimental neuron models and mathematical tools, we developed a “systems” approach to deconvolve cellular mechanisms of neurodegeneration underlying the most common known cause of Parkinson's disease (PD), mutations in leucine-rich repeat kinase 2 (LRRK2). Neurons ectopically expressing mutant LRRK2 formed inclusion bodies (IBs), retracted neurites, accumulated synuclein, and died prematurely, recapitulating key features of PD. Degeneration was predicted from the levels of diffuse mutant LRRK2 that each neuron contained, but IB formation was neither necessary nor sufficient for death. Genetic or pharmacological blockade of its kinase activity destabilized LRRK2 and lowered its levels enough to account for the moderate reduction in LRRK2 toxicity that ensued. By contrast, targeting synuclein, including neurons made from PD patient-derived induced pluripotent cells, dramatically reduced LRRK2-dependent neurodegeneration and LRRK2 levels. These findings suggest that LRRK2 levels are more important than kinase activity per se in predicting toxicity and implicate synuclein as a major mediator of LRRK2-induced neurodegeneration. PMID:24403142

  13. Mutant LRRK2 toxicity in neurons depends on LRRK2 levels and synuclein but not kinase activity or inclusion bodies.

    PubMed

    Skibinski, Gaia; Nakamura, Ken; Cookson, Mark R; Finkbeiner, Steven

    2014-01-08

    By combining experimental neuron models and mathematical tools, we developed a "systems" approach to deconvolve cellular mechanisms of neurodegeneration underlying the most common known cause of Parkinson's disease (PD), mutations in leucine-rich repeat kinase 2 (LRRK2). Neurons ectopically expressing mutant LRRK2 formed inclusion bodies (IBs), retracted neurites, accumulated synuclein, and died prematurely, recapitulating key features of PD. Degeneration was predicted from the levels of diffuse mutant LRRK2 that each neuron contained, but IB formation was neither necessary nor sufficient for death. Genetic or pharmacological blockade of its kinase activity destabilized LRRK2 and lowered its levels enough to account for the moderate reduction in LRRK2 toxicity that ensued. By contrast, targeting synuclein, including neurons made from PD patient-derived induced pluripotent cells, dramatically reduced LRRK2-dependent neurodegeneration and LRRK2 levels. These findings suggest that LRRK2 levels are more important than kinase activity per se in predicting toxicity and implicate synuclein as a major mediator of LRRK2-induced neurodegeneration.

  14. Cloning and expression of phosphoenolpyruvate carboxykinase from a cestode parasite and its solubilization from inclusion bodies using l-arginine.

    PubMed

    Dutta, Asim K; Ramnath; Dkhar, Barilin; Tandon, Veena; Das, Bidyadhar

    2016-09-01

    Phosphoenolpyruvate carboxykinase is an essential regulatory enzyme of glycolysis in the cestode parasite, Raillietina echinobothrida, and is considered a potential target for anthelmintic action because of its differential activity from that of its avian host. However, due to the unavailability of its structure, the mechanism of regulation of PEPCK from R. echinobothrida (rePEPCK) and its interaction with possible modulators remain unclear. Hence, in this study, the rePEPCK gene was cloned into pGEX-4T-3 and overexpressed for its characterization. On being induced by IPTG, the recombinant rePEPCK was expressed as inclusion bodies (IBs); hence, various agents, like different inducer concentrations, temperature, time, host cell types, culture media, pH, and additives, were used to bring the protein to soluble form. Finally, a significant amount (∼46%) of rePEPCK was solubilized from IBs by adding 2M l-arginine. Near-UV circular dichroism spectra analysis indicated that l-arginine (2M) had no effect on the conformation of the protein. In this study, we have reported a yield of ∼73mg of purified rePEPCK per 1L of culture. The purified rePEPCK retained its biological activity, and Km of the enzyme for its substrate was determined and discussed. The availability of recombinant rePEPCK may help in biochemical- and biophysical-studies to explore its molecular mechanisms and regulations. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Copper reduces Aβ oligomeric species and ameliorates neuromuscular synaptic defects in a C. elegans model of inclusion body myositis.

    PubMed

    Rebolledo, Daniela L; Aldunate, Rebeca; Kohn, Rebecca; Neira, Iván; Minniti, Alicia N; Inestrosa, Nibaldo C

    2011-07-13

    Alzheimer's disease and inclusion body myositis (IBM) are disorders frequently found in the elderly and characterized by the presence of amyloid-β peptide (Aβ) aggregates. We used Caenorhabditis elegans that express Aβ in muscle cells as a model of IBM, with the aim of analyzing Aβ-induced muscle pathology and evaluating the consequences of modulating Aβ aggregation. First, we tested whether the altered motility we observed in the Aβ transgenic strain could be the result of a compromised neuromuscular synapse. Our pharmacological analyses show that synaptic transmission is defective in our model and suggest a specific defect on nicotine-sensitive acetylcholine receptors (AChRs). Through GFP-coupled protein visualization, we found that synaptic dysfunction correlates with mislocalization of ACR-16, the AChR subunit essential for nicotine-triggered currents. Histological and biochemical analysis allowed us to determine that copper treatment increases the amyloid deposits and decreases Aβ oligomers in this model. Furthermore, copper treatment improves motility, ACR-16 localization, and synaptic function and delays Aβ-induced paralysis. Our results indicate that copper modulates Aβ-induced pathology and suggest that Aβ oligomers are triggering neuromuscular dysfunction. Our findings emphasize the importance of neuromuscular synaptic dysfunction and the relevance of modulating the amyloidogenic component as an alternative therapeutic approach for this debilitating disease.

  16. Sequestration of mutated alpha1-antitrypsin into inclusion bodies is a cell-protective mechanism to maintain endoplasmic reticulum function.

    PubMed

    Granell, Susana; Baldini, Giovanna; Mohammad, Sameer; Nicolin, Vanessa; Narducci, Paola; Storrie, Brian; Baldini, Giulia

    2008-02-01

    A variant alpha1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers, and it is associated with liver disease. In the hepatocytes of individuals carrying the mutation, alpha1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or whether they are protective to the cell. We found that in hepatoma cells, mutated alpha1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, and contained several ER components, but not calnexin. Mutated alpha1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulfide isomerase, inhibited formation of IBs and lead to retention of mutated alpha1-antitrypsin in the ER. In hepatoma cells, shift of mutated alpha1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress, and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated alpha1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway.

  17. Sequestration of Mutated α1-Antitrypsin into Inclusion Bodies Is a Cell-protective Mechanism to Maintain Endoplasmic Reticulum Function

    PubMed Central

    Granell, Susana; Baldini, Giovanna; Mohammad, Sameer; Nicolin, Vanessa; Narducci, Paola; Storrie, Brian

    2008-01-01

    A variant α1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers, and it is associated with liver disease. In the hepatocytes of individuals carrying the mutation, α1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or whether they are protective to the cell. We found that in hepatoma cells, mutated α1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, and contained several ER components, but not calnexin. Mutated α1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulfide isomerase, inhibited formation of IBs and lead to retention of mutated α1-antitrypsin in the ER. In hepatoma cells, shift of mutated α1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress, and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated α1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway. PMID:18045994

  18. Transcriptional profiling identifies differential expression of long non-coding RNAs in Jo-1 associated and inclusion body myositis.

    PubMed

    Hamann, Philip D; Roux, Benoit T; Heward, James A; Love, Seth; McHugh, Neil J; Jones, Simon W; Lindsay, Mark A

    2017-08-14

    Myositis is characterised by muscle inflammation and weakness. Although generally thought to be driven by a systemic autoimmune response, increasing evidence suggests that intrinsic changes in the muscle might also contribute to the pathogenesis. Long non-coding RNAs (lncRNAs) are a family of novel genes that regulate gene transcription and translation. To determine the potential role of lncRNAs, we employed next generation sequencing to examine the transcriptome in muscle biopsies obtained from two histologically distinct patient populations, inclusion body myositis (IBM) and anti-Jo-1-associated myositis (Jo-1). 1287 mRNAs and 1068 mRNAs were differentially expressed in the muscle from Jo-1 and IBM patients, respectively. Pathway analysis showed the top canonical pathway in both Jo-1 and IBM was oxidative phosphorylation and mitochondrial dysfunction. We identified 731 known and 325 novel lncRNAs in the muscles biopsies. Comparison with controls showed 55 and 46 lncRNAs were differentially expressed in IBM and Jo-1 myositis, respectively. Of these, 16 lncRNAs were differentially expressed in both IBM and Jo-1 myositis and included upregulated H19, lncMyoD and MALAT1. Given that these are known to regulate muscle proliferation and differentiation, we speculate that changes in lncRNAs might contribute to the phenotypic changes in Jo-1 and IBM myositis.

  19. The effects of an intronic polymorphism in TOMM40 and APOE genotypes in sporadic inclusion body myositis.

    PubMed

    Gang, Qiang; Bettencourt, Conceicao; Machado, Pedro M; Fox, Zoe; Brady, Stefen; Healy, Estelle; Parton, Matt; Holton, Janice L; Hilton-Jones, David; Shieh, Perry B; Zanoteli, Edmar; De Paepe, Boel; De Bleecker, Jan; Shaibani, Aziz; Ripolone, Michela; Violano, Raffaella; Moggio, Maurizio; Barohn, Richard J; Dimachkie, Mazen M; Mora, Marina; Mantegazza, Renato; Zanotti, Simona; Hanna, Michael G; Houlden, Henry

    2015-04-01

    A previous study showed that, in carriers of the apolipoprotein E (APOE) genotype ε3/ε3 or ε3/ε4, the presence of a very long (VL) polyT repeat allele in "translocase of outer mitochondrial membrane 40" (TOMM40) was less frequent in patients with sporadic inclusion body myositis (sIBM) compared with controls and associated with a later age of sIBM symptom onset, suggesting a protective effect of this haplotype. To further investigate the influence of these genetic factors in sIBM, we analyzed a large sIBM cohort of 158 cases as part of an International sIBM Genetics Study. No significant association was found between APOE or TOMM40 genotypes and the risk of developing sIBM. We found that the presence of at least 1 VL polyT repeat allele in TOMM40 was significantly associated with about 4 years later onset of sIBM symptoms. The age of onset was delayed by 5 years when the patients were also carriers of the APOE genotype ε3/ε3. In addition, males were likely to have a later age of onset than females. Therefore, the TOMM40 VL polyT repeat, although not influencing disease susceptibility, has a disease-modifying effect on sIBM, which can be enhanced by the APOE genotype ε3/ε3.

  20. Molecular characterisation of fowl adenovirus type 7 isolated from poultry associated with inclusion body hepatitis in Poland.

    PubMed

    Niczyporuk, Jowita Samanta

    2017-02-03

    The fowl adenovirus field strain FAdV-JSN-5/10j (GenBank accession number KP879219) was isolated from the intestine of a 7-week-old chicken diagnosed with inclusion body hepatitis and simultaneously with Marek's disease, and for that reason, it was chosen for molecular study. It was identified as fowl adenovirus genotype 7 (species Fowl aviadenovirus E) based on nucleotide sequence analysis of the loop L1 region of the hexon gene. Nucleotide sequence alignment of this strain, FAdV-7 reference strains B-3A ATCC VR-832 (AF339922) and YR36 (AF508955), and eight additional FAdV-7 field strains confirmed its classification as FAdV-JS-5/10j and showed that these viruses are very similar to each other. Additionally, we described mutations and their influence on the amino acid sequence, nucleotide composition, and relative synonymous codon usage. Immunofluorescence of cell cultures infected with 10(4.5) TCID 50 per 0.1-ml dose of the FAdV-JSN-5/10j strain demonstrated the presence of a cytopathic effect. Infection of fowl with adenoviruses raises concerns for poultry production, and thus, the efficient detection of adenovirus infection is crucial. This is the first attempt to describe the molecular characteristics of FadV-7 strains isolated in Poland.

  1. Aqueous two-phase systems as an alternative process route for the fractionation of small inclusion bodies.

    PubMed

    Walker, S G; Lyddiatt, A

    1998-06-26

    Aqueous two-phase protocols have been established which successfully generate highly purified preparations of small inclusion bodies (IBs) from whole cell homogenates. Particle size analysis of disruptates confirmed that intense disruption (concomitant with maximal product release) was compromised by the corelease of contaminating solutes and the micronisation of cell debris yielding a similar particle size range to the IBs (100-200 nm). PEG 300-phosphate systems enabled partial recovery of IBs in the top phase of ATPS. In contrast, PEG 8000-phosphate systems partitioned IBs more efficiently as a discrete sediment within the lower phase, whilst the majority of micronised debris remained in the interphase. The alpha-glucosidase IB yield and purity in ATPS was bettered only by analytical sucrose density gradient centrifugation, which is not readily scaleable for application in process operations. The successful recovery of such small IBs from complex homogenates highlights a generic role that ATPS techniques might play in the recovery and purification of new bioparticulate products (viral and plasmid gene therapy vectors, particulate protein vaccines etc.).

  2. Inclusion Body Myositis

    MedlinePlus

    ... Rigor & Reproducibility Scientific Resources Animal Models Cell/Tissue/DNA Clinical and Translational Resources Gene Expression Research Reagents ... Rigor & Reproducibility Scientific Resources Animal Models Cell/Tissue/DNA Clinical and Translational Resources Gene Expression Research Reagents ...

  3. The ND10 Component Promyelocytic Leukemia Protein Relocates to Human Papillomavirus Type 1 E4 Intranuclear Inclusion Bodies in Cultured Keratinocytes and in Warts

    PubMed Central

    Roberts, Sally; Hillman, Michele L.; Knight, Gillian L.; Gallimore, Phillip H.

    2003-01-01

    Human papillomavirus type 1 (HPV1) E4 protein is associated with cytoplasmic and nuclear inclusions in productively infected keratinocytes. Here we have used transient expression of HPV1 E4 (also known as E1^E4) protein in keratinocytes to reproduce formation of E4 inclusions. Immunofluorescence analysis showed that progressive formation of inclusions correlated with diminished colocalization between E4 and keratin intermediate filaments (IFs). Our results support a model in which the HPV1 E4-keratin IF association is transient, occurring only at an early stage of inclusion formation. We also demonstrate that E4 induces relocation of the promyelocytic leukemia protein (PML) from multiple intranuclear speckles (ND10 bodies) to the periphery of nuclear E4 inclusions and that this activity is specific to full-length E4 protein. Analysis of HPV1-induced warts demonstrated that nuclear PML-E4 inclusions were present in productively infected keratinocytes, indicating that reorganization of PML occurs during the virus's replication cycle. It has been suggested that ND10 bodies are the sites for papillomavirus genome replication and virion assembly. Our finding that E4 induces reorganization of ND10 bodies in vitro and in vivo is further strong evidence that these domains play an important role in the papillomavirus life cycle. This study indicates that HPV1 is analogous to other DNA viruses that disrupt or reorganize ND10 domains, possibly to increase efficiency of virus infection. We hypothesize that HPV1 E4-induced reorganization of PML is necessary for efficient replication of the virus during the virus-producing phase. PMID:12477870

  4. Selected clinical chemistry analytes correlate with the pathogenesis of inclusion body hepatitis experimentally induced by fowl aviadenoviruses.

    PubMed

    Matos, Miguel; Grafl, Beatrice; Liebhart, Dieter; Schwendenwein, Ilse; Hess, Michael

    2016-10-01

    In the present study, clinical chemistry was applied to assess the pathogenesis and progression of experimentally induced inclusion body hepatitis (IBH). For this, five fowl aviadenovirus (FAdV) strains from recent IBH field outbreaks were used to orally inoculate different groups of day-old specific pathogen-free chickens, which were weighed, sampled and examined during necropsy by sequential killing. Mortalities of 50% and 30% were recorded in two groups between 6 and 9 days post-infection (dpi), along with a decreased weight of 23% and 20%, respectively, compared to the control group. Macroscopical changes were seen in the liver and kidney between 6 and 10 dpi, with no lesions being observed in the other organs. Histological lesions were observed in the liver and pancreas during the same period. Plasma was collected from killed birds of each group at each time point and the following clinical chemistry analytes were investigated: aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), bile acids, total protein, albumin, uric acid and lipase. Plasma protein profile, AST and GLDH, together with bile acids values paralleled the macroscopical and histopathological lesions in the liver, while plasma lipase activity levels coincided with lesions observed in pancreas. In agreement with the histology and clinical chemistry, viral load in the target organs, liver and pancreas, was highest at 7 dpi. Thus, clinical chemistry was found to be a valuable tool in evaluating and monitoring the progression of IBH in experimentally infected birds, providing a deeper knowledge of the underlying pathophysiological mechanisms of a FAdV infection in chickens.

  5. A retrospective cohort study identifying the principal pathological features useful in the diagnosis of inclusion body myositis.

    PubMed

    Brady, Stefen; Squier, Waney; Sewry, Caroline; Hanna, Michael; Hilton-Jones, David; Holton, Janice L

    2014-04-28

    The current pathological diagnostic criteria for sporadic inclusion body myositis (IBM) lack sensitivity. Using immunohistochemical techniques abnormal protein aggregates have been identified in IBM, including some associated with neurodegenerative disorders. Our objective was to investigate the diagnostic utility of a number of markers of protein aggregates together with mitochondrial and inflammatory changes in IBM. Retrospective cohort study. The sensitivity of pathological features was evaluated in cases of Griggs definite IBM. The diagnostic potential of the most reliable features was then assessed in clinically typical IBM with rimmed vacuoles (n=15), clinically typical IBM without rimmed vacuoles (n=9) and IBM mimics-protein accumulation myopathies containing rimmed vacuoles (n=7) and steroid-responsive inflammatory myopathies (n=11). Specialist muscle services at the John Radcliffe Hospital, Oxford and the National Hospital for Neurology and Neurosurgery, London. Individual pathological features, in isolation, lacked sensitivity and specificity. However, the morphology and distribution of p62 aggregates in IBM were characteristic and in a myopathy with rimmed vacuoles, the combination of characteristic p62 aggregates and increased sarcolemmal and internal major histocompatibility complex class I expression or endomysial T cells were diagnostic for IBM with a sensitivity of 93% and specificity of 100%. In an inflammatory myopathy lacking rimmed vacuoles, the presence of mitochondrial changes was 100% sensitive and 73% specific for IBM; characteristic p62 aggregates were specific (91%), but lacked sensitivity (44%). We propose an easily applied diagnostic algorithm for the pathological diagnosis of IBM. Additionally our findings support the hypothesis that many of the pathological features considered typical of IBM develop later in the disease, explaining their poor sensitivity at disease presentation and emphasising the need for revised pathological criteria

  6. Abnormalities of NBR1, a novel autophagy-associated protein, in muscle fibers of sporadic inclusion-body myositis.

    PubMed

    D'Agostino, Carla; Nogalska, Anna; Cacciottolo, Mafalda; Engel, W King; Askanas, Valerie

    2011-11-01

    Intra-muscle fiber accumulation of ubiquitinated protein aggregates containing several conformationally modified proteins, including amyloid-β and phosphorylated tau, is characteristic of the pathologic phenotype of sporadic inclusion-body myositis (s-IBM), the most common progressive degenerative myopathy of older persons. Abnormalities of protein-degradation, involving both the 26S proteasome and autophagic-lysosomal pathways, were previously demonstrated in s-IBM muscle. NBR1 is a ubiquitin-binding scaffold protein importantly participating in autophagic degradation of ubiquitinated proteins. Whereas abnormalities of p62, a ubiquitin-binding protein, were previously described in s-IBM, abnormalities of NBR1 have not been reported in s-IBM. We have now identified in s-IBM muscle biopsies that NBR1, by: (a) immunohistochemistry, was strongly accumulated within s-IBM muscle-fiber aggregates, where it closely co-localized with p62, ubiquitin, and phosphorylated tau; (b) immunoblots, was increased threefold (p < 0.001); and (c) immunoprecipitation, was associated with p62 and LC3. By real-time PCR, NBR1 mRNA was increased twofold (p < 0.01). None of the various disease- and normal-control muscle biopsies had any NBR1 abnormality. In cultured human muscle fibers, NBR1 also physically associated with both p62 and LC3, and experimental inhibition of either the 26S proteasome or the lysosomal activity resulted in NBR1 increase. Our demonstration of NBR1 abnormalities in s-IBM provides further evidence that altered protein degradation pathways may be critically involved in the s-IBM pathogenesis. Accordingly, attempts to unblock defective protein degradation might be a therapeutic strategy for s-IBM patients.

  7. Impaired Autophagy in Sporadic Inclusion-Body Myositis and in Endoplasmic Reticulum Stress-Provoked Cultured Human Muscle Fibers

    PubMed Central

    Nogalska, Anna; D'Agostino, Carla; Terracciano, Chiara; Engel, W. King; Askanas, Valerie

    2010-01-01

    The hallmark pathologies of sporadic inclusion-body myositis (s-IBM) muscle fibers are autophagic vacuoles and accumulation of ubiquitin-positive multiprotein aggregates that contain amyloid-β or phosphorylated tau in a β-pleated sheet amyloid configuration. Endoplasmic reticulum stress (ERS) and 26S proteasome inhibition, also associated with s-IBM, putatively aggrandize the accumulation of misfolded proteins. However, autophagosomal-lysosomal pathway formation and function, indicated by autophagosome maturation, have not been previously analyzed in this system. Here we studied the autophagosomal-lysosomal pathway using 14 s-IBM and 30 disease control and normal control muscle biopsy samples and our cultured human muscle fibers in a microenvironment modified to resemble aspects of s-IBM pathology. We report for the first time that in s-IBM, lysosomal enzyme activities of cathepsin D and B were decreased 60% (P < 0.01) and 40% (P < 0.05), respectively. We also detected two indicators of increased autophagosome maturation, the presence of LC3-II and decreased mammalian target of rapamycin-mediated phosphorylation of p70S6 kinase. Moreover, in cultured human muscle fibers, ERS induction significantly decreased activities of cathepsins D and B, increased levels of LC3-II, decreased phosphorylation of p70S6 kinase, and decreased expression of VMA21, a chaperone for assembly of lysosomal V-ATPase. We conclude that in s-IBM muscle, decreased lysosomal proteolytic activity might enhance accumulation of misfolded proteins, despite increased maturation of autophagosomes, and that ERS is a possible cause of s-IBM-impaired lysosomal function. Thus, unblocking protein degradation in s-IBM muscle fibers may be a desirable therapeutic strategy. PMID:20616343

  8. A retrospective cohort study identifying the principal pathological features useful in the diagnosis of inclusion body myositis

    PubMed Central

    Brady, Stefen; Squier, Waney; Sewry, Caroline; Hanna, Michael; Hilton-Jones, David; Holton, Janice L

    2014-01-01

    Objective The current pathological diagnostic criteria for sporadic inclusion body myositis (IBM) lack sensitivity. Using immunohistochemical techniques abnormal protein aggregates have been identified in IBM, including some associated with neurodegenerative disorders. Our objective was to investigate the diagnostic utility of a number of markers of protein aggregates together with mitochondrial and inflammatory changes in IBM. Design Retrospective cohort study. The sensitivity of pathological features was evaluated in cases of Griggs definite IBM. The diagnostic potential of the most reliable features was then assessed in clinically typical IBM with rimmed vacuoles (n=15), clinically typical IBM without rimmed vacuoles (n=9) and IBM mimics—protein accumulation myopathies containing rimmed vacuoles (n=7) and steroid-responsive inflammatory myopathies (n=11). Setting Specialist muscle services at the John Radcliffe Hospital, Oxford and the National Hospital for Neurology and Neurosurgery, London. Results Individual pathological features, in isolation, lacked sensitivity and specificity. However, the morphology and distribution of p62 aggregates in IBM were characteristic and in a myopathy with rimmed vacuoles, the combination of characteristic p62 aggregates and increased sarcolemmal and internal major histocompatibility complex class I expression or endomysial T cells were diagnostic for IBM with a sensitivity of 93% and specificity of 100%. In an inflammatory myopathy lacking rimmed vacuoles, the presence of mitochondrial changes was 100% sensitive and 73% specific for IBM; characteristic p62 aggregates were specific (91%), but lacked sensitivity (44%). Conclusions We propose an easily applied diagnostic algorithm for the pathological diagnosis of IBM. Additionally our findings support the hypothesis that many of the pathological features considered typical of IBM develop later in the disease, explaining their poor sensitivity at disease presentation and

  9. A Systematic Review and Meta-Analysis of Prevalence Studies of Sporadic Inclusion Body Myositis.

    PubMed

    Callan, Aoife; Capkun, Gorana; Vasanthaprasad, Vijayalakshmi; Freitas, Rita; Needham, Merrilee

    2017-01-01

    Sporadic Inclusion Body Myositis (sIBM) is a rare and slowly progressive debilitating muscle disease with symptoms generally developing≥50 years of age. To conduct a systematic review and meta-analysis of the prevalence of sIBM literature, including a methodological quality assessment of the selected papers. A systematic search of Medline, Embase, Cochrane Database of Systematic Reviews and major Myositis and Neurological conferences was conducted. Articles reporting prevalence and published in English up to March 2017 were assessed for methodology quality using the Loney quality assessment, Downs & Black score, and the Methodological Evaluation of Observational Research checklists. Meta-analyses using random effects were completed on both general population and≥50 years prevalence estimates. 315 articles were retrieved and data were extracted from 10 relevant studies. One study was subsequently excluded due to methodological issues. The meta-prevalence estimate from 9 papers was 24.8/1,000,000 (95% CI: 20.0-29.6). The methodological quality results were consistent across assessment tools with four articles scoring 4 or 5 out of 8 in the Loney assessment. The meta-prevalence of these four articles was 45.6/ 1,000,000 (95% CI: 35.9-55.2). There was high variability in reported sIBM prevalence estimates and the quality of the studies conducted. Existing evidence suggests an increase of prevalence estimates over time, which may be explained by growing disease awareness, improvements in diagnostic criteria and study methodologies. Further high quality studies are needed to understand if prevalence varies across geographies or ethnicities.

  10. Disease specificity of autoantibodies to cytosolic 5'-nucleotidase 1A in sporadic inclusion body myositis versus known autoimmune diseases.

    PubMed

    Herbert, Megan K; Stammen-Vogelzangs, Judith; Verbeek, Marcel M; Rietveld, Anke; Lundberg, Ingrid E; Chinoy, Hector; Lamb, Janine A; Cooper, Robert G; Roberts, Mark; Badrising, Umesh A; De Bleecker, Jan L; Machado, Pedro M; Hanna, Michael G; Plestilova, Lenka; Vencovsky, Jiri; van Engelen, Baziel G; Pruijn, Ger J M

    2016-04-01

    The diagnosis of inclusion body myositis (IBM) can be challenging as it can be difficult to clinically distinguish from other forms of myositis, particularly polymyositis (PM). Recent studies have shown frequent presence of autoantibodies directed against cytosolic 5'-nucleotidase 1A (cN-1A) in patients with IBM. We therefore, examined the autoantigenicity and disease specificity of major epitopes of cN-1A in patients with sporadic IBM compared with healthy and disease controls. Serum samples obtained from patients with IBM (n=238), PM and dermatomyositis (DM) (n=185), other autoimmune diseases (n=246), other neuromuscular diseases (n=93) and healthy controls (n=35) were analysed for the presence of autoantibodies using immunodominant cN-1A peptide ELISAs. Autoantibodies directed against major epitopes of cN-1A were frequent in patients with IBM (37%) but not in PM, DM or non-autoimmune neuromuscular diseases (<5%). Anti-cN-1A reactivity was also observed in some other autoimmune diseases, particularly Sjögren's syndrome (SjS; 36%) and systemic lupus erythematosus (SLE; 20%). In summary, we found frequent anti-cN-1A autoantibodies in sera from patients with IBM. Heterogeneity in reactivity with the three immunodominant epitopes indicates that serological assays should not be limited to a distinct epitope region. The similar reactivities observed for SjS and SLE demonstrate the need to further investigate whether distinct IBM-specific epitopes exist. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  11. Kinetics of viral load and erythrocytic inclusion body formation in Pacific herring artificially infected with erythrocytic necrosis virus.

    PubMed

    Glenn, Jolene A; Emmenegger, Eveline J; Grady, Courtney A; Roon, Sean R; Gregg, Jacob L; Conway, Carla M; Winton, James R; Hershberger, Paul K

    2012-09-01

    Viral erythrocytic necrosis (VEN) is a condition that affects marine and anadromous fish species, including herrings and salmonids, in the Atlantic and Pacific oceans. Infection is frequently associated with severe anemia and causes episodic mortality among wild and hatchery fish when accompanied by additional stressors; VEN can be presumptively diagnosed by (1) light microscopic identification of a single characteristic-a round, magenta-colored, 0.8-μm-diameter inclusion body (IB) within the cytoplasm of erythrocytes and their precursors on Giemsa-stained blood films; or (2) observation (via transmission electron microscopy [TEM]) of the causative iridovirus, erythrocytic necrosis virus (ENV), within erythrocytes or their precursors. To better understand the kinetics of VEN, specific-pathogen-free Pacific herring Clupea pallasii were infected with ENV by intraperitoneal injection. At 1, 4, 7, 10, 14, 21, and 28 d postexposure, samples of blood, spleen, and kidney were collected and assessed (1) via light microscopy for the number of intracytoplasmic IBs in blood smears and (2) via TEM for the number of virions within erythrocytes. The mean prevalence of intracytoplasmic IBs in the blood cells increased from 0% at 0-4 d postexposure to 94% at 28 d postexposure. Viral load within circulating red blood cells peaked at 7 d postexposure, fell slightly, and then reached a plateau. However, blood cells observed within the kidney and spleen tissues demonstrated high levels of ENV between 14 and 28 d postexposure. The results indicate that the viral load within erythrocytes does not correlate well with IB prevalence and that the virus can persist in infected fish for more than 28 d.

  12. Sporadic inclusion body myositis: phenotypic variability and influence of HLA-DR3 in a cohort of 57 Australian cases.

    PubMed

    Needham, M; James, I; Corbett, A; Day, T; Christiansen, F; Phillips, B; Mastaglia, F L

    2008-09-01

    There have been few studies of the variability in the clinical phenotype in sporadic inclusion body myositis (sIBM) and it is not known whether the human leucocyte antigen (HLA) haplotype influences the phenotype and course of the disease. We studied a large cohort of patients with sIBM in order to determine the degree of phenotypic variability and different modes of presentation, as well as the influence of HLA haplotypes. A cross-sectional study of 57 biopsy-proven sIBM cases from three Australian centres was performed. Patients were interviewed and examined by a single investigator, and had HLA typing and autoantibody studies. Although the initial symptoms in the majority of cases were attributable to quadriceps weakness (79%), a proportion of patients presented due to finger weakness (12%), foot drop (7%) or dysphagia (1.8%). Although the majority had the classic combination of quadriceps and forearm muscle involvement, some patients had predominantly forearm weakness with sparing of the quadriceps, or severe involvement of the anterior tibial muscles. Asymmetrical involvement was common (82%), particularly of the forearm muscles, with the non-dominant side being more severely affected in most cases. Carriage of the HLA-DRB1*0301 (DR3) allele was associated with lower quadriceps muscle strength and a more rapid decline in strength. The findings emphasise the variability in the mode of presentation, patterns of muscle involvement and clinical course of sIBM in this population, and indicate that the HLA-DRB1*0301 (DR3) allele may influence the rate of progression as well as susceptibility to the disease.

  13. High hydrostatic pressure enables almost 100% refolding of recombinant human ciliary neurotrophic factor from inclusion bodies at high concentration.

    PubMed

    Wang, Qi; Liu, Yongdong; Zhang, Chun; Guo, Fangxia; Feng, Cui; Li, Xiunan; Shi, Hong; Su, Zhiguo

    2017-05-01

    Protein refolding from inclusion bodies (IBs) often encounters a problem of low recovery at high protein concentration. In this study, we demonstrated that high hydrostatic pressure (HHP) could simultaneously achieve high refolding concentration and high refolding yield for IBs of recombinant human ciliary neurotrophic factor (rhCNTF), a potential therapeutic for neurodegenerative diseases. The use of dilution refolding obtained 18% recovery at 3 mg/mL, even in the presence of 4 M urea. In contrast, HHP refolding could efficiently increase the recovery up to almost 100% even at 4 mg/mL. It was found that in the dilution, hydrophobic aggregates were the off-path products and their amount increased with the protein concentration. However, HHP could effectively minimize the formation of hydrophobic aggregates, leading to almost complete conversion of the rhCNTF IBs to the correct configuration. The stable operation range of concentration is 0.5-4.0 mg/mL, in which the refolding yield was almost 100%. Compared with the literatures where HHP failed to increase the refolding yield beyond 90%, the reason could be attributed to the structural difference that rhCNTF has no disulfide bond and is a monomeric protein. After purification by one-step of anionic chromatography, the purity of rhCNTF reached 95% with total process recovery of 54.1%. The purified rhCNTF showed similar structure and in vitro bioactivity to the native species. The whole process featured integration of solubilization/refolding, a high refolding yield of 100%, a high concentration of 4 mg/mL, and a simple chromatography to ensure a high productivity. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Formation of orthopoxvirus cytoplasmic A-type inclusion bodies and embedding of virions are dynamic processes requiring microtubules.

    PubMed

    Howard, Amanda R; Moss, Bernard

    2012-05-01

    In cells infected with some orthopoxviruses, numerous mature virions (MVs) become embedded within large, cytoplasmic A-type inclusions (ATIs) that can protect infectivity after cell lysis. ATIs are composed of an abundant viral protein called ATIp, which is truncated in orthopoxviruses such as vaccinia virus (VACV) that do not form ATIs. To study ATI formation and occlusion of MVs within ATIs, we used recombinant VACVs that express the cowpox full-length ATIp or we transfected plasmids encoding ATIp into cells infected with VACV, enabling ATI formation. ATI enlargement and MV embedment required continued protein synthesis and an intact microtubular network. For live imaging of ATIs and MVs, plasmids expressing mCherry fluorescent protein fused to ATIp were transfected into cells infected with VACV expressing the viral core protein A4 fused to yellow fluorescent protein. ATIs appeared as dynamic, mobile bodies that enlarged by multiple coalescence events, which could be prevented by disrupting microtubules. Coalescence of ATIs was confirmed in cells infected with cowpox virus. MVs were predominantly at the periphery of ATIs early in infection. We determined that coalescence contributed to the distribution of MVs within ATIs and that microtubule-disrupting drugs abrogated coalescence-mediated MV embedment. In addition, MVs were shown to move from viral factories at speeds consistent with microtubular transport to the peripheries of ATIs, whereas disruption of microtubules prevented such trafficking. The data indicate an important role for microtubules in the coalescence of ATIs into larger structures, transport of MVs to ATIs, and embedment of MVs within the ATI matrix.

  15. Human Respiratory Syncytial Virus Nucleoprotein and Inclusion Bodies Antagonize the Innate Immune Response Mediated by MDA5 and MAVS

    PubMed Central

    Lifland, Aaron W.; Jung, Jeenah; Alonas, Eric; Zurla, Chiara; Crowe, James E.

    2012-01-01

    Currently, the spatial distribution of human respiratory syncytial virus (hRSV) proteins and RNAs in infected cells is still under investigation, with many unanswered questions regarding the interaction of virus-induced structures and the innate immune system. Very few studies of hRSV have used subcellular imaging as a means to explore the changes in localization of retinoic-acid-inducible gene-I (RIG-I)-like receptors or the mitochondrial antiviral signaling (MAVS) protein, in response to the infection and formation of viral structures. In this investigation, we found that both RIG-I and melanoma differentiation-associated gene 5 (MDA5) colocalized with viral genomic RNA and the nucleoprotein (N) as early as 6 h postinfection (hpi). By 12 hpi, MDA5 and MAVS were observed within large viral inclusion bodies (IB). We used a proximity ligation assay (PLA) and determined that the N protein was in close proximity to MDA5 and MAVS in IBs throughout the course of the infection. Similar results were found with the transient coexpression of N and the phosphoprotein (P). Additionally, we demonstrated that the localization of MDA5 and MAVS in IBs inhibited the expression of interferon β mRNA 27-fold following Newcastle disease virus infection. From these data, we concluded that the N likely interacts with MDA5, is in close proximity to MAVS, and localizes these molecules within IBs in order to attenuate the interferon response. To our knowledge, this is the first report of a specific function for hRSV IBs and of the hRSV N protein as a modulator of the innate immune response. PMID:22623778

  16. Production of soluble truncated spike protein of porcine epidemic diarrhea virus from inclusion bodies of Escherichia coli through refolding.

    PubMed

    Piao, Da-Chuan; Lee, Yoon-Seok; Bok, Jin-Duck; Cho, Chong-Su; Hong, Zhong-Shan; Kang, Sang-Kee; Choi, Yun-Jaie

    2016-10-01

    The emergence of highly pathogenic variant porcine epidemic diarrhea virus (PEDV) strains, from 2013 to 2014, in North American and Asian countries have greatly threatened global swine industry. Therefore, development of effective vaccines against PEDV variant strains is urgently needed. Recently, it has been reported that the N-terminal domain (NTD) of S1 domain of PEDV spike protein is responsible for binding to the 5-N-acetylneuraminic acid (Neu5Ac), a possible sugar co-receptor. Therefore, the NTD of S1 domain could be an attractive target for the development of subunit vaccines. In this study, the NTD spanning amino acid residues 25-229 (S25-229) of S1 domain of PEDV variant strain was expressed in Escherichia coli BL21 (DE3) in the form of inclusion bodies (IBs). S25-229 IBs were solubilized in 20 mM sodium acetate (pH 4.5) buffer containing 8 M urea and 1 mM dithiothreitol with 95% yield. Solubilized S25-229 IBs were refolded by 10-fold flash dilution and purified by one-step cation exchange chromatography with >95% purity and 20% yield. The CD spectrum of S25-229 showed the characteristic pattern of alpha helical structure. In an indirect ELISA, purified S25-229 showed strong reactivity with mouse anti-PEDV sera. In addition, immunization of mice with 20 μg of purified S25-229 elicited highly potent serum IgG titers. Finally, mouse antisera against S25-229 showed immune reactivity with native PEDV S protein in an immunofluorescence assay. These results suggest that purified S25-229 may have potential to be used as a subunit vaccine against PEDV variant strains. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by MDA5 and MAVS.

    PubMed

    Lifland, Aaron W; Jung, Jeenah; Alonas, Eric; Zurla, Chiara; Crowe, James E; Santangelo, Philip J

    2012-08-01

    Currently, the spatial distribution of human respiratory syncytial virus (hRSV) proteins and RNAs in infected cells is still under investigation, with many unanswered questions regarding the interaction of virus-induced structures and the innate immune system. Very few studies of hRSV have used subcellular imaging as a means to explore the changes in localization of retinoic-acid-inducible gene-I (RIG-I)-like receptors or the mitochondrial antiviral signaling (MAVS) protein, in response to the infection and formation of viral structures. In this investigation, we found that both RIG-I and melanoma differentiation-associated gene 5 (MDA5) colocalized with viral genomic RNA and the nucleoprotein (N) as early as 6 h postinfection (hpi). By 12 hpi, MDA5 and MAVS were observed within large viral inclusion bodies (IB). We used a proximity ligation assay (PLA) and determined that the N protein was in close proximity to MDA5 and MAVS in IBs throughout the course of the infection. Similar results were found with the transient coexpression of N and the phosphoprotein (P). Additionally, we demonstrated that the localization of MDA5 and MAVS in IBs inhibited the expression of interferon β mRNA 27-fold following Newcastle disease virus infection. From these data, we concluded that the N likely interacts with MDA5, is in close proximity to MAVS, and localizes these molecules within IBs in order to attenuate the interferon response. To our knowledge, this is the first report of a specific function for hRSV IBs and of the hRSV N protein as a modulator of the innate immune response.

  18. Molecular events linking cholesterol to Alzheimer’s disease and inclusion body myositis in a rabbit model

    PubMed Central

    Liu, Qing Yan; Koukiekolo, Roger; Zhang, Dong Ling; Smith, Brandon; Ly, Dao; Lei, Joy X; Ghribi, Othman

    2016-01-01

    Alzheimer’s disease (AD) is the most common neurodegenerative disorder, characterized by cognitive impairment and dementia, resulting from progressive synaptic dysfunction, loss and neuronal cell death. Inclusion body myositis (IBM) is a skeletal muscle degenerative disease, displaying progressive proximal and distal muscle weakness, in association with muscle fiber atrophy, degeneration and death. Studies have shown that the late onset version of AD (LOAD) and sporadic IBM (sIBM) in muscle share many pathological features, including the presence of extracellular plaques of β-amyloid peptides and intracellular tangles of hyperphosphorylated tau proteins. High blood cholesterol is suggested to be a risk factor for LOAD. Many neuropathological changes of LOAD can be reproduced by feeding rabbits a 2% enriched cholesterol diet for 12 weeks. The cholesterol fed rabbit model also simultaneously develops sIBM like pathology, which makes it an ideal model to study the molecular mechanisms common to the development of both diseases. In the present study, we determined the changes of gene expression in rabbit brain and muscle during the progression of LOAD and sIBM pathology using a custom rabbit nucleotide microarray, followed by qRT-PCR analyses. Out of 869 unique transcripts screened, 47 genes showed differential expression between the control and the cholesterol-treated group during the 12 week period and 19 changed transcripts appeared to be common to LOAD and sIBM. The most notable changes are the upregulation of the hemoglobin gene family and the downregulation of the genes required for mitochondrial oxidative phosphorylation in both brain and muscle tissues throughout the time course. The significant overlap on the changes of gene expression in the brain and muscle of rabbits fed with cholesterol-enriched diet supports the notion that LOAD and sIBM may share a common etiology. PMID:27073745

  19. Kinetics of viral load and erythrocytic inclusion body formation in pacific herring artificially infected with erythrocytic necrosis virus

    USGS Publications Warehouse

    Glenn, Jolene A.; Emmenegger, Eveline J.; Grady, Courtney A.; Roon, Sean R.; Gregg, Jacob L.; Conway, Carla M.; Winton, James R.; Hershberger, Paul K.

    2012-01-01

    Viral erythrocytic necrosis (VEN) is a condition that affects marine and anadromous fish species, including herrings and salmonids, in the Atlantic and Pacific oceans. Infection is frequently associated with severe anemia and causes episodic mortality among wild and hatchery fish when accompanied by additional stressors; VEN can be presumptively diagnosed by (1) light microscopic identification of a single characteristic—a round, magenta-colored, 0.8-μm-diameter inclusion body (IB) within the cytoplasm of erythrocytes and their precursors on Giemsa-stained blood films; or (2) observation (via transmission electron microscopy [TEM]) of the causative iridovirus, erythrocytic necrosis virus (ENV), within erythrocytes or their precursors. To better understand the kinetics of VEN, specific-pathogen-free Pacific herring Clupea pallasii were infected with ENV by intraperitoneal injection. At 1, 4, 7, 10, 14, 21, and 28 d postexposure, samples of blood, spleen, and kidney were collected and assessed (1) via light microscopy for the number of intracytoplasmic IBs in blood smears and (2) via TEM for the number of virions within erythrocytes. The mean prevalence of intracytoplasmic IBs in the blood cells increased from 0% at 0–4 d postexposure to 94% at 28 d postexposure. Viral load within circulating red blood cells peaked at 7 d postexposure, fell slightly, and then reached a plateau. However, blood cells observed within the kidney and spleen tissues demonstrated high levels of ENV between 14 and 28 d postexposure. The results indicate that the viral load within erythrocytes does not correlate well with IB prevalence and that the virus can persist in infected fish for more than 28 d.

  20. Solubilization and purification of recombinant modified C-reactive protein from inclusion bodies using reversible anhydride modification.

    PubMed

    Potempa, Lawrence A; Yao, Zhen-Yu; Ji, Shang-Rong; Filep, János G; Wu, Yi

    The precise function of C-reactive protein (CRP) as a regulator of inflammation in health and disease continues to evolve. The true understanding of its role in host defense responses has been hampered by numerous reports of comparable systems with contradictory interpretations of CRP as a stimulator, suppressor, or benign contributor to such processes. These discrepancies may be explained in part by the existence of a naturally occurring CRP isoform, termed modified CRP (i.e., mCRP), that is expressed when CRP subunits are dissociated into monomeric structures. The free mCRP subunit undergoes a non-proteolytic conformational change that has unique solubility, antigenicity, and bioactivity compared to the subunits that remain associated in the native, pentameric CRP molecule (i.e., pCRP). As specific reagents have been developed to identify and quantify mCRP, it has become apparent that this isoform can be formed spontaneously in calcium-free solutions. Furthermore, mCRP can be expressed on perturbed cell membranes with as little as 24-48 h incubation in tissue culture. Because mCRP has the same size as pCRP subunits as evaluated by SDS-PAGE, its presence in a pCRP reagent would not be apparent using this technique to evaluate purity. Finally, because many antibody reagents purported to be specific for "CRP" contains some, or substantial specificity to mCRP, antigen-detection techniques using such reagents may fail to distinguish the specific CRP isoform detected. All these caveats concerning CRP structures and measurements suggest that the aforementioned contradictory studies may reflect to some extent on distinctive bioactivities of mCRP rather than on pCRP. To provide a reliable, abundant supply of mCRP for separate and comparable studies, a recombinant protein was engineered and expressed in E. coli (i.e., recombinant mCRP or rmCRP). Synthesized protein was produced as inclusion bodies which proved difficult to solubilize for purification and characterization

  1. Exploring the Medicinal Potential of the Fruit Bodies of Oyster Mushroom, Pleurotus ostreatus (Agaricomycetes), against Multidrug-Resistant Bacterial Isolates.

    PubMed

    Skariyachan, Sinosh; Prasanna, Apoorva; Manjunath, Sirisha P; Karanth, Soujanya S; Nazre, Ambika

    2016-01-01

    Bacterial resistance to present-generation antibiotics is increasing drastically, which has become a major public health concern. The present study focuses on demonstrating the antimicrobial potential of fruit bodies of the culinary/medicinal oyster mushroom Pleurotus ostreatus against clinical pathogens. Five bacterial isolates were collected from Sagar Hospital in Bangalore, India. The collected strains were grown on selective and differential media and antibiotic susceptibility testing was applied using 48 antibiotics by disc diffusion assay. The antibacterial efficiency of the mushroom extract against clinical pathogens, which were found to be multidrug resistant (MDR) to most of the tested antibiotics, was studied. The yield of cultivated mushrooms was evident at moist, cooler, and humid conditions. The clinical isolates of Staphylococcus aureus, Salmonella typhi, Acinetobacter sp., Proteus mirabilis, and Proteus spp. were found to be MDR to β-lactam, fluoroquinolones, sulfonamides, third- and fourth-generation cephalosporins, aminoglycosides, macrolides, tetracyclines, and carbapenems. The methanolic extracts of mushroom fruit bodies were found to be more effective than present-generation antibiotics against methicillin- and vancomycin- resistant S. aureus, S. typhi, Acinetobacter sp., and P. mirabilis at a concentration ranging from 50 to 100 µg/disc or 50 to 100 µL/well. The current study suggests that the methanolic extract of P. ostreatus can be used as a promising antibacterial agent against MDR bacterial pathogens.

  2. Marburg virus inclusions: A virus-induced microcompartment and interface to multivesicular bodies and the late endosomal compartment.

    PubMed

    Dolnik, Olga; Stevermann, Lea; Kolesnikova, Larissa; Becker, Stephan

    2015-01-01

    Filovirus infection of target cells leads to the formation of virally induced cytoplasmic inclusions that contain viral nucleocapsids at different stages of maturation. While the role of the inclusions has been unclear since the identification of Marburg and Ebola viruses, it recently became clear that the inclusions are the sites of viral replication, nucleocapsid formation and maturation. Live cell imaging analyses revealed that mature nucleocapsids are transported from inclusions to the filopodia, which represent the major budding sites. Moreover, inclusions recruit cellular proteins that have been shown to support the transport of nucleocapsids. For example, the tumor susceptibility gene 101 protein (Tsg101) interacts with a late domain motif in the nucleocapsid protein NP and recruits the actin-nucleation factor IQGAP1. Complexes of nucleocapsids together with Tsg101 and IQGAP1 are then co-transported along actin filaments. We detected additional proteins (Alix, Nedd4 and the AAA-type ATPase VPS4) of the endosomal sorting complex required for transport (ESCRT) that are recruited into inclusions. Together, the results suggest that nucleocapsids recruit the machinery that enhances viral budding at the plasma membrane. Furthermore, we identified Lamp1 as a marker of the late endosomal compartment in inclusions, while ER, Golgi, TGN and early endosomal markers were absent. In addition, we observed that LC3, a marker of autophagosomal membranes, was present in inclusions. The 3D structures of inclusions show an intricate structure that seems to accommodate an intimate cooperation between cellular and viral components with the intention to support viral transport and budding.

  3. Correlation of blood metabolite concentrations and body condition scores with persistent postpartum uterine bacterial infection in dairy cows

    PubMed Central

    GHANEM, Mohamed Elshabrawy; TEZUKA, Erisa; SASAKI, Kouya; TAKAHASHI, Masahiro; YAMAGISHI, Norio; IZAIKE, Yoshiaki; OSAWA, Takeshi

    2016-01-01

    To analyze the relationship of blood metabolite concentrations and body condition score (BCS) with persistent bacterial uterine infection, specifically that caused by Trueperella pyogenes and anaerobic bacteria, uterine bacteriological swabs (n = 128) were collected from 64 Holstein cows at 5 (W5) and 7 (W7) weeks postpartum, and the percentage of neutrophils in the endometrium was evaluated. Blood glucose, total cholesterol (T-cho), blood urea nitrogen (BUN), non-esterified fatty acid (NEFA), and β-hydroxybutyric acid concentrations were analyzed at 3 weeks (W-3) and 1 week (W-1) prepartum and W3, W5, and W7 postpartum. BCS were evaluated at W-3, W3, and W7. Blood glucose concentrations at W-3 and W-1 in cows with persistent bacterial infection were lower (P = 0.05) than in the rest of the cows. Total BUN concentrations in cows with persistent bacterial infection were lower (P < 0.01) than those in other cows, although the association between the pre or postpartum time and status of infection was not significant. Total NEFA concentrations in cows with persistent bacterial infection were similar to those in uninfected cows and cows positive for infection at W5 but not W7. Total BCS in cows with persistent bacterial infection were lower (P < 0.01) than those in cows positive for infection at both W5 but not W7 and W7 but not W5; however, the association between the pre or postpartum time and status of infection was not significant. Glucose concentrations at W-3 and W-1 negatively correlated with persistent bacterial infection at W5 and W7 (P < 0.01). BUN concentrations at W3 (P < 0.01), W5 (P < 0.05), and W7 (P < 0.05) and BCS at W3 (P < 0.01) negatively correlated with persistent postpartum bacterial infection. Decreased prepartum blood glucose concentrations might be an important risk factor for persistent postpartum bacterial uterine infection in dairy cows. PMID:27349443

  4. Autophagic adapter protein NBR1 is localized in Lewy bodies and glial cytoplasmic inclusions and is involved in aggregate formation in α-synucleinopathy.

    PubMed

    Odagiri, Saori; Tanji, Kunikazu; Mori, Fumiaki; Kakita, Akiyoshi; Takahashi, Hitoshi; Wakabayashi, Koichi

    2012-08-01

    Macroautophagy is a dynamic process whereby cytoplasmic components are initially sequestered within autophagosomes. Recent studies have shown that the autophagosome membrane can selectively recognize ubiquitinated proteins and organelles through interaction with adapter proteins such as p62 and NBR1. Both proteins are structurally similar at the amino acid level, and bind with ubiquitin and ubiquitinated proteins. Although p62 is incorporated into a wide spectrum of pathological inclusions in various neurodegenerative diseases, abnormalities of NBR1 have not been reported in these diseases. Our immunohistochemical examination revealed that the vast majority of Lewy bodies (LBs) in Parkinson's disease and dementia with LBs (DLB) as well as of glial cytoplasmic inclusions in multiple system atrophy (MSA) were positive for NBR1. Neuronal and glial inclusions in tauopathies and TAR DNA-binding protein of 43 kDa proteinopathies were rarely immunolabeled, or were unstained. Using cultured cells bearing LB-like inclusions, formation of α-synuclein aggregates was repressed in cells with NBR1 knockdown. Immunoblot analysis showed that the level of NBR1 was significantly increased by 2.5-fold in MSA, but not in DLB. These findings suggest that NBR1 is involved in the formation of cytoplasmic inclusions in α-synucleinopathy.

  5. A simple and effective strategy for solving the problem of inclusion bodies in recombinant protein technology: His-tag deletions enhance soluble expression.

    PubMed

    Zhu, Shaozhou; Gong, Cuiyu; Ren, Lu; Li, Xingzhou; Song, Dawei; Zheng, Guojun

    2013-01-01

    The formation of inclusion bodies (IBs) in recombinant protein biotechnology has become one of the most frequent undesirable occurrences in both research and industrial applications. So far, the pET System is the most powerful system developed for the production of recombinant proteins when Escherichia coli is used as the microbial cell factory. Also, using fusion tags to facilitate detection and purification of the target protein is a commonly used tactic. However, there is still a large fraction of proteins that cannot be produced in E. coli in a soluble (and hence functional) form. Intensive research efforts have tried to address this issue, and numerous parameters have been modulated to avoid the formation of inclusion bodies. However, hardly anyone has noticed that adding fusion tags to the recombinant protein to facilitate purification is a key factor that affects the formation of inclusion bodies. To test this idea, the industrial biocatalysts uridine phosphorylase from Aeropyrum pernix K1 and (+)-γ-lactamase and (-)-γ-lactamase from Bradyrhizobium japonicum USDA 6 were expressed in E. coli by using the pET System and then examined. We found that using a histidine tag as a fusion partner for protein expression did affect the formation of inclusion bodies in these examples, suggesting that removing the fusion tag can promote the solubility of heterologous proteins. The production of soluble and highly active uridine phosphorylase, (+)-γ-lactamase, and (-)-γ-lactamase in our results shows that the traditional process needs to be reconsidered. Accordingly, a simple and efficient structure-based strategy for the production of valuable soluble recombinant proteins in E. coli is proposed.

  6. Epidemiological and pathological investigation of fowl aviadenovirus serotypes 8b and 11 isolated from chickens with inclusion body hepatitis in Spain (2011-2013).

    PubMed

    Oliver-Ferrando, S; Dolz, R; Calderón, C; Valle, R; Rivas, R; Pérez, M; Biarnés, M; Blanco, A; Bertran, K; Ramis, A; Busquets, N; Majó, N

    2017-04-01

    Inclusion body hepatitis caused by different fowl aviadenovirus (FAdV) serotypes has been described in several countries in recent years. In Spain, from the spring of 2011 to 2013, an increased number of outbreaks in broiler and broiler breeder flocks from different regions occurred. The objectives of the present work were to carry out the molecular characterization of FAdV strains from Spanish inclusion body hepatitis cases and to study the pathogenicity and viral dynamics of these strains in specific pathogen-free (SPF) chickens. A total of 52 inclusion body hepatitis clinical cases, including 45 from broiler farms and seven from broiler breeder farms, were analysed by conventional polymerase chain reaction and sequencing targeting the FAdV hexon gene. From these, 37 strains were classified as FAdV type 8b, while the remaining 15 were classified as FAdV types 11 (n = 10), 2 (n = 4) and 8a (n = 1). In addition, two different FAdVs belonging to the genotypes 8b and 11 were used for experimental infection. Specific pathogen-free five-day-old birds were inoculated intramuscularly with a high (10(6.5) tissue culture infective dose (TCID)50/ml) or low (10(4) TCID50/ml) dose of the above-mentioned FAdVs. No mortality was observed in any of the experimental groups, and only one bird showed evident clinical signs. However, macroscopic and microscopic hepatic lesions, as well as viral DNA, were detected in birds from all infection groups. Inclusion bodies and viral DNA were also detected in the pancreas and in the small and the large intestine in some birds. Long-lasting shedding and transmission to contact birds were confirmed in all infected groups.

  7. Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture.

    PubMed

    Chung, Wen-Jen; Huang, Chi-Lung; Gong, Hong-Yi; Ou, Tsung-Yin; Hsu, Jue-Liang; Hu, Shao-Yang

    2015-06-01

    Growth hormone (GH) performs important roles in regulating somatic growth, reproduction, osmoregulation, metabolism and immunity in teleosts, and thus, it has attracted substantial attention in the field of aquaculture application. Herein, giant grouper GH (ggGH) cDNA was cloned into the pET28a vector and expressed in Shuffle® T7 Competent Escherichia coli. Recombinant N-terminal 6× His-tagged ggGH was produced mainly in insoluble inclusion bodies; the recombinant ggGH content reached 20% of total protein. For large-scale ggGH production, high-cell density E. coli culture was achieved via fed-batch culture with pH-stat. After 30h of cultivation, a cell concentration of 41.1g/l dry cell weight with over 95% plasmid stability was reached. Maximal ggGH production (4.0g/l; 22% total protein) was achieved via mid-log phase induction. Various centrifugal forces, buffer pHs and urea concentrations were optimized for isolation and solubilization of ggGH from inclusion bodies. Hydrophobic interactions and ionic interactions were the major forces in ggGH inclusion body formation. Complete ggGH inclusion body solubilization was obtained in PBS buffer at pH 12 containing 3M urea. Through a simple purification process including Ni-NTA affinity chromatography and refolding, 5.7mg of ggGH was obtained from 10ml of fed-batch culture (45% recovery). The sequence and secondary structure of the purified ggGH were confirmed by LC-MS/MS mass spectrometry and circular dichroism analysis. The cell proliferation-promoting activity was confirmed in HepG2, ZFL and GF-1 cells with the WST-1 colorimetric bioassay. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. The fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen.

    PubMed

    Arai, Shinpei; Ogiwara, Naoko; Mukai, Saki; Takezawa, Yuka; Sugano, Mitsutoshi; Honda, Takayuki; Okumura, Nobuo

    2017-06-01

    Fibrinogen storage disease (FSD) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. Six mutations in the γC domain (γG284R, γT314P, γD316N, the deletion of γG346-Q350, γG366S, and γR375W) have been identified for FSD. Our group previously established γ375W fibrinogen-producing Chinese hamster ovary (CHO) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. The aim of this study was to investigate whether fibrous intracellular inclusion bodies are specific to FSD-inducible variant fibrinogen. Thirteen expression vectors encoding the variant γ-chain were stably or transiently transfected into CHO cells expressing normal fibrinogen Aα- and Bβ-chains or HuH-7 cells, which were then immunofluorescently stained. Six CHO and HuH-7 cell lines that transiently produced FSD-inducible variant fibrinogen presented the fibrous (3.2-22.7 and 2.1-24.5%, respectively) and large granular (5.4-25.5 and 7.7-23.9%) forms of intracellular inclusion bodies. Seven CHO and HuH-7 cell lines that transiently produced FSD-non-inducible variant fibrinogen only exhibit the large granular form. These results demonstrate that transiently transfected variant fibrinogen-producing CHO cells and inclusion bodies of the fibrous form may be useful in non-invasive screening for FSD risk factors for FSD before its onset.

  9. The effect of protein acetylation on the formation and processing of inclusion bodies and endogenous protein aggregates in Escherichia coli cells.

    PubMed

    Kuczyńska-Wiśnik, Dorota; Moruno-Algara, María; Stojowska-Swędrzyńska, Karolina; Laskowska, Ewa

    2016-11-10

    Acetylation of lysine residues is a reversible post-translational modification conserved from bacteria to humans. Several recent studies have revealed hundreds of lysine-acetylated proteins in various bacteria; however, the physiological role of these modifications remains largely unknown. Since lysine acetylation changes the size and charge of proteins and thereby may affect their conformation, we assumed that lysine acetylation can stimulate aggregation of proteins, especially for overproduced recombinant proteins that form inclusion bodies. To verify this assumption, we used Escherichia coli strains that overproduce aggregation-prone VP1GFP protein. We found that in ΔackA-pta cells, which display diminished protein acetylation, inclusion bodies were formed with a delay and processed faster than in the wild-type cells. Moreover, in ΔackA-pta cells, inclusion bodies exhibited significantly increased specific GFP fluorescence. In CobB deacetylase-deficient cells, in which protein acetylation was enhanced, the formation of inclusion bodies was increased and their processing was significantly inhibited. Similar results were obtained with regard to endogenous protein aggregates formed during the late stationary phase in ΔackA-pta and ΔcobB cells. Our studies revealed that protein acetylation affected the aggregation of endogenous E. coli proteins and the yield, solubility, and biological activity of a model recombinant protein. In general, decreased lysine acetylation inhibited the formation of protein aggregates, whereas increased lysine acetylation stabilized protein aggregates. These findings should be considered during the designing of efficient strategies for the production of recombinant proteins in E. coli cells.

  10. Mineralogy and Petrology of Amoeboid Olivine Inclusions in CO3 Chondrites: Relationship to Parent-Body Aqueous Alteration

    NASA Technical Reports Server (NTRS)

    Chizmadia, Lysa J.; Rubin, Alan E.; Wasson, John T.

    2003-01-01

    Petrographic and mineralogic studies of amoeboid olivine inclusions (AOIs) in CO3 carbonaceous chondrites reveal that they are sensitive indicators of parent-body aqueous and thermal alteration. As the petrologic subtype increases from 3.0 to 3.8, forsteritic olivine (Fa(sub 0-1)) is systematically converted into ferroan olivine (Fa(sub 60-75)). We infer that the Fe, Si and O entered the assemblage along grain boundaries, forming ferroan olivine that filled fractures and voids. As temperatures increased, Fe(+2) from the new olivine exchanged with Mg(+2) from the original AOI to form diffusive haloes around low-FeO cores. Cations of Mn(+2), Ca(+2) and Cr(+3) were also mobilized. The systematic changes in AOI textures and olivine compositional distributions can be used to refine the classification of CO3 chondrites into subtypes. In subtype 3.0, olivine occurs as small forsterite grains (Fa(sub 0-1)), free of ferroan olivine. In petrologic subtype 3.2, narrow veins of FeO-rich olivine have formed at forsterite grain boundaries. With increasing alteration, these veins thicken to form zones of ferroan olivine at the outside AOI margin and within the AOI interior. By subtype 3.7, there is a fairly broad olivine compositional distribution in the range Fa(sub 63-70), and by subtype 3.8, no forsterite remains and the high-Fa peak has narrowed, Fa(sub 64-67). Even at this stage, there is incomplete equilibration in the chondrite as a whole (e.g., data for coarse olivine grains in Isna (CO3.8) chondrules and lithic clasts show a peak at Fa(sub39)). We infer that the mineral changes in A01 identified in the low petrologic types required aqueous or hydrothermal fluids whereas those in subtypes greater than or equal to 3.3 largely reflect diffusive exchange within and between mineral grains without the aid of fluids.

  11. Active inclusion bodies of acid phosphatase PhoC: aggregation induced by GFP fusion and activities modulated by linker flexibility

    PubMed Central

    2013-01-01

    Background Biologically active inclusion bodies (IBs) have gained much attention in recent years. Fusion with IB-inducing partner has been shown to be an efficient strategy for generating active IBs. To make full use of the advantages of active IBs, one of the key issues will be to improve the activity yield of IBs when expressed in cells, which would need more choices on IB-inducing fusion partners and approaches for engineering IBs. Green fluorescent protein (GFP) has been reported to aggregate when overexpressed, but GFP fusion has not been considered as an IB-inducing approach for these fusion proteins so far. In addition, the role of linker in fusion proteins has been shown to be important for protein characteristics, yet impact of linker on active IBs has never been reported. Results Here we report that by fusing GFP and acid phosphatase PhoC via a linker region, the resultant PhoC-GFPs were expressed largely as IBs. These IBs show high levels of specific fluorescence and specific PhoC activities (phosphatase and phosphotransferase), and can account for up to over 80% of the total PhoC activities in the cells. We further demonstrated that the aggregation of GFP moiety in the fusion protein plays an essential role in the formation of PhoC-GFP IBs. In addition, PhoC-GFP IBs with linkers of different flexibility were found to exhibit different levels of activities and ratios in the cells, suggesting that the linker region can be utilized to manipulate the characteristics of active IBs. Conclusions Our results show that active IBs of PhoC can be generated by GFP fusion, demonstrating for the first time the potential of GFP fusion to induce active IB formation of another soluble protein. We also show that the linker sequence in PhoC-GFP fusion proteins plays an important role on the regulation of IB characteristics, providing an alternative and important approach for engineering of active IBs with the goal of obtaining high activity yield of IBs. PMID:23497261

  12. MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies.

    PubMed

    Kuo, Mario Meng-Chiang; Nguyen, Phuong Hong; Jeon, Yun-Hui; Kim, Subin; Yoon, So-Mi; Choe, Senyon

    2014-02-24

    The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 - Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of aggregation suppressors and the

  13. MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies

    PubMed Central

    2014-01-01

    Background The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Results Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 – Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. Conclusion MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of

  14. Molecular differentiation and pathogenicity of Aviadenoviruses isolated during an outbreak of inclusion body hepatitis in South Africa.

    PubMed

    Joubert, Hilda W; Aitchison, Henry; Maartens, Louis H; Venter, Estelle H

    2014-11-05

    Fowl adenovirus (FAdV) is a member of the genus Aviadenovirus and causes a number of economically important poultry diseases. One of these diseases, inclusion body hepatitis (IBH), has a worldwide distribution and is characterised by acute mortality (5% - 20%) in production chickens. The disease was first described in the United States of America in 1963 and has also been reported in Canada, the United Kingdom, Australia, France and Ireland, but until now, not in South Africa. Adenoviruses isolated from the first outbreak of IBH in South Africa were able to reproduce the disease in chicken embryo livers. The aim of the present study was to characterise the viruses and determine the pathogenicity of the FAdV strains responsible for the first reported case of IBH in South Africa. Polymerase chain reaction (PCR) amplification of the L1 loop region of the fowl adenovirus hexon gene using degenerate primer pair hexon A/B was used to identify the viruses that were isolated. Restriction fragment length polymorphism (RFLP) of the amplification products was used for the differentiation of 14 isolates of fowl adenovirus. Sequencing of the PCR products followed by amino acid comparison and phylogenetic analysis using the L1 loop region of the hexon protein was done to determine the identity of the isolates. Amino acid sequences of the hexon genes of all the South African isolates were compared with those of reference strains representing FAdV species. Amino acid comparison of 12 South Africa field isolates to FAdV reference strains revealed a high sequence identity (> 93.33%) with reference strains T8-A and 764. Two of the isolates had high sequence identity (93.40%) with reference strains P7-A, C2B and SR48. Phylogenetic analysis of the L1 loop region of the hexon protein of all 14 South African isolates was consistent with their RFLP clusters. The mortality rates of embryos challenged with 106 egg infective doses (EID50) FAdV 2 were 80% - 87% and mortality rates for

  15. An aromatase-associated cytoplasmic inclusion, the "stigmoid body," in the rat brain: II. Ultrastructure (with a review of its history and nomenclature).

    PubMed

    Shinoda, K; Nagano, M; Osawa, Y

    1993-03-01

    The ultrastructure of aromatase-associated "stigmoid (dot-like) structures," which were detected in a previous study using light-microscopic immunohistochemistry (Shinoda et al.: J. Comp. Neurol. 322:360-376, '92), were examined in the rat medial preoptic region, bed nucleus of the stria terminalis, medial amygdaloid nucleus, and arcuate nucleus by pre- and post-embedding marking with a polyclonal antibody against human placental antigen X-P2 (hPAX-P2) for immuno-electron microscopic analysis. The immunoreactive stigmoid structure was identified as a distinct, non-membrane-bounded cytoplasmic inclusion (approximately 1-3 microns in diameter), which has a granulo-fuzzy texture with moderate-to-low electron density in non-immunostained preparations. It consists of at least four distinct granular and three distinct fibrillo-tubular elements forming a granulo-fibrillar conglomerate. This type of inclusions was formally termed the "stigmoid body" under the electron microscope. The stigmoid body is composed of the outer granulo-fibrillar and inner hyaloplasmic compartments. The immunoreactivity for hPAX-P2 is mainly localized to the former, especially to the low density granulo-fuzzy materials associated with the fibrillo-tubular elements. Identification of the ultrastructure of stigmoid body clarified their prevalence not only in the limbic and hypothalamic regions, but also in sex-steroid-sensitive peripheral tissues (e.g., peripheral sensory ganglia, ovary, testis) by consulting earlier electron-microscopic studies. Reviewing the history and nomenclature of this inclusion body, we reorganized the terminology of related neuronal cytoplasmic inclusions, the terms of which have often been confused, and discussed its functional significance on the basis of the present and previously accumulated data. In conclusion, we emphasized the importance of the stigmoid bodies in the sex-steroid-sensitive neural system because of their large size, high frequency, specific

  16. VCP Associated Inclusion Body Myopathy and Paget Disease of Bone Knock-In Mouse Model Exhibits Tissue Pathology Typical of Human Disease

    PubMed Central

    Kitazawa, Masashi; Su, Hailing; Tanaja, Jasmin; Dec, Eric; Wallace, Douglas C.; Mukherjee, Jogeshwar; Caiozzo, Vincent; Warman, Matthew; Kimonis, Virginia E.

    2010-01-01

    Dominant mutations in the valosin containing protein (VCP) gene cause inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD). We have generated a knock-in mouse model with the common R155H mutation. Mice demonstrate progressive muscle weakness starting approximately at the age of 6 months. Histology of mutant muscle showed progressive vacuolization of myofibrils and centrally located nuclei, and immunostaining shows progressive cytoplasmic accumulation of TDP-43 and ubiquitin-positive inclusion bodies in quadriceps myofibrils and brain. Increased LC3-II staining of muscle sections representing increased number of autophagosomes suggested impaired autophagy. Increased apoptosis was demonstrated by elevated caspase-3 activity and increased TUNEL-positive nuclei. X-ray microtomography (uCT) images show radiolucency of distal femurs and proximal tibiae in knock-in mice and uCT morphometrics shows decreased trabecular pattern and increased cortical wall thickness. Bone histology and bone marrow derived macrophage cultures in these mice revealed increased osteoclastogenesis observed by TRAP staining suggestive of Paget bone disease. The VCPR155H/+ knock-in mice replicate the muscle, bone and brain pathology of inclusion body myopathy, thus representing a useful model for preclinical studies. PMID:20957154

  17. L-cysteine-enhanced renaturation of bioactive soluble tumor necrosis factor ligand family member LIGHT from inclusion bodies in Escherichia coli.

    PubMed

    Tsuji, Isamu; Mastui, Hideki; Ito, Tatsuo; Kurokawa, Tomofumi; Shintani, Yasushi

    2011-12-01

    LIGHT is a membrane-bound protein that belongs to the tumor necrosis factor (TNF) superfamily ligands. In this study, we established an effective strategy for producing a bioactive soluble form of LIGHT (sLIGHT), an extracellular region (Ile⁸⁴-Val²⁴⁰) of human LIGHT. Because sLIGHT was expressed as inclusion bodies in Escherichia coli, we investigated reagents that enhance the renaturation of sLIGHT from the inclusion bodies. Interestingly, L-cysteine in the denaturation buffer containing 3.5 M guanidine hydrochloride significantly improved the renaturation efficiency of sLIGHT. The effect of L-cysteine was synergistically enhanced by L-arginine in the refolding buffer. The optimal concentrations of L-cysteine and L-arginine in the denaturation and refolding buffers were 8 mM and 0.8 M, respectively. With these buffers, approximately 90 mg of sLIGHT was purified from 200 g of frozen E. coli cells. sLIGHT thus obtained significantly induced apoptosis in the WiDr human colon adenocarcinoma cell line at nanomolar concentrations, the same amount of sLIGHT that was produced by Sf9 insect cells. These results suggest that L-cysteine in the denaturation buffer enhances the renaturation of recombinant proteins from inclusion bodies in E. coli.

  18. Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system.

    PubMed

    Moricoli, Diego; Muller, William Anthony; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Watson, Richard; Fiori, Valentina; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

    2014-06-01

    Migration of leukocytes into site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells, inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies and the absence of toxic reagents utilized for solubilization and refolding step of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting, we herein describe an efficient and large scale production of the antibody fragments expressed in E. coli as periplasmic insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signaling. This protocol can be useful for the successful purification of other monomeric scFvs which are expressed as periplasmic inclusion bodies in bacterial systems. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication-associated inclusion bodies.

    PubMed

    Sun, Liying; Andika, Ida Bagus; Shen, Jiangfeng; Yang, Di; Chen, Jianping

    2014-06-01

    Viruses commonly modify host endomembranes to facilitate biological processes in the viral life cycle. Infection by viruses belonging to the genus Bymovirus (family Potyviridae) has long been known to induce the formation of large membranous inclusion bodies in host cells, but their assembly and biological roles are still unclear. Immunoelectron microscopy of cells infected with the bymovirus Wheat yellow mosaic virus (WYMV) showed that P1, P2 and P3 are the major viral protein constituents of the membranous inclusions, whereas NIa-Pro (nuclear inclusion-a protease) and VPg (viral protein genome-linked) are probable minor components. P1, P2 and P3 associated with the endoplasmic reticulum (ER), but only P2 was able to rearrange ER and form large aggregate structures. Bioinformatic analyses and chemical experiments showed that P2 is an integral membrane protein and depends on the active secretory pathway to form aggregates of ER membranes. In planta and in vitro assays demonstrated that P2 interacts with P1, P3, NIa-Pro or VPg and recruits these proteins into the aggregates. In vivo RNA labelling using WYMV-infected wheat protoplasts showed that the synthesis of viral RNAs occurs in the P2-associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house the genomic replication of WYMV. © 2013 BSPP AND JOHN WILEY & SONS LTD.

  20. Bodies Displayed on Walls: Are Children's Bodies Represented in an Inclusive Way in the Pictures on the Walls in Their Early Childhood Educational Environments?

    ERIC Educational Resources Information Center

    Martínez-Bello, Vladimir E.; Martínez-Bello, Judith T.

    2017-01-01

    The messages conveyed by visual representations in the early childhood education (ECE) environment are critical to ensuring the success of inclusive practices. Given that anti-bias education permeates and affects everything which takes place in ECE institutions, the challenge for early childhood educators is to think creatively about how classroom…

  1. The Mrj co-chaperone mediates keratin turnover and prevents the formation of toxic inclusion bodies in trophoblast cells of the placenta.

    PubMed

    Watson, Erica D; Geary-Joo, Colleen; Hughes, Martha; Cross, James C

    2007-05-01

    Defects in protein-folding and -degradation machinery have been identified as a major cause of intracellular protein aggregation and of aggregation-associated diseases. In general, it remains unclear how these aggregates are harmful to normal cellular function. We demonstrate here that, in the developing placenta of the mouse, the absence of the Mrj (Dnajb6) co-chaperone prevents proteasome degradation of keratin 18 (K18; Krt18) intermediate filaments, resulting in the formation of keratin inclusion bodies. These inclusions in chorionic trophoblast cells prevent chorioallantoic attachment during placental development. We show further that keratin-deficient embryos undergo chorioallantoic attachment and that, by genetically reducing keratin expression in Mrj(-/-) conceptuses, chorioallantoic attachment was rescued. Therefore, the chorioallantoic attachment phenotype in Mrj mutants is not due to a deficiency of the normal keratin cytoskeleton, but rather is cytotoxicity caused by keratin aggregates that disrupt chorion trophoblast cell organization and function.

  2. Pasteurization Procedures for Donor Human Milk Affect Body Growth, Intestinal Structure, and Resistance against Bacterial Infections in Preterm Pigs.

    PubMed

    Li, Yanqi; Nguyen, Duc Ninh; de Waard, Marita; Christensen, Lars; Zhou, Ping; Jiang, Pingping; Sun, Jing; Bojesen, Anders Miki; Lauridsen, Charlotte; Lykkesfeldt, Jens; Dalsgaard, Trine Kastrup; Bering, Stine Brandt; Sangild, Per Torp

    2017-06-01

    Background: Holder pasteurization (HP) destroys multiple bioactive factors in donor human milk (DM), and UV-C irradiation (UVC) is potentially a gentler method for pasteurizing DM for preterm infants.Objective: We investigated whether UVC-treated DM improves gut maturation and resistance toward bacterial infections relative to HP-treated DM.Methods: Bacteria, selected bioactive components, and markers of antioxidant capacity were measured in unpasteurized donor milk (UP), HP-treated milk, and UVC-treated milk (all from the same DM pool). Fifty-seven cesarean-delivered preterm pigs (91% gestation; ratio of males to females, 30:27) received decreasing volumes of parental nutrition (average 69 mL · kg(-1) · d(-1)) and increasing volumes of the 3 DM diets (n = 19 each, average 89 mL · kg(-1) · d(-1)) for 8-9 d. Body growth, gut structure and function, and systemic bacterial infection were evaluated.Results: A high bacterial load in the UP (6×10(5) colony forming units/mL) was eliminated similarly by HP and UVC treatments. Relative to HP-treated milk, both UVC-treated milk and UP showed greater activities of lipase and alkaline phosphatase and concentrations of lactoferrin, secretory immunoglobulin A, xanthine dehydrogenase, and some antioxidant markers (all P < 0.05). The pigs fed UVC-treated milk and pigs fed UP showed higher relative weight gain than pigs fed HP-treated milk (5.4% and 3.5%), and fewer pigs fed UVC-treated milk had positive bacterial cultures in the bone marrow (28%) than pigs fed HP-treated milk (68%) (P < 0.05). Intestinal health was also improved in pigs fed UVC-treated milk compared with those fed HP-treated milk as indicated by a higher plasma citrulline concentration (36%) and villus height (38%) (P < 0.05) and a tendency for higher aminopeptidase N (48%) and claudin-4 (26%) concentrations in the distal intestine (P < 0.08). The gut microbiota composition was similar among groups except for greater proportions of Enterococcus in pigs fed

  3. NIH Human Microbiome Project defines normal bacterial makeup of the body

    Cancer.gov

    Microbes inhabit just about every part of the human body, living on the skin, in the gut, and up the nose. Sometimes they cause sickness, but most of the time, microorganisms live in harmony with their human hosts, providing vital functions essential for

  4. Human body morphology, prevalence of nasopharyngeal potential bacterial pathogens, and immunocompetence handicap principal.

    PubMed

    Pawlowski, Boguslaw; Nowak, Judyta; Borkowska, Barbara; Drulis-Kawa, Zuzanna

    2014-01-01

    Body height, body mass index (BMI) and waist-to-hip ratio (WHR) are the main traits characterizing human body morphology. Studies show that these traits are related to attractiveness and, therefore, according to an evolutionary point of view, are supposed to be honest signals of biological quality. If the immunocompetence handicap principal (IHP) is true, people with more attractive values of these traits should be more immunologically competent. To test this, we analyzed whether nasal and throat colonization with potentially pathogenic bacteria is related to body height and BMI in both sexes and to WHR in females. 103 healthy females and 90 healthy males (with the mean age of 21.4 and 22.8, respectively) participated in the study. The heights and weights were self-reported and waist and hip circumferences measured. Six potentially pathogenic species (with the most common Staphylococcus aureus) isolated from nasal and throat swabs were identified by colony morphology, standard biochemical assays, and latex tests. To compare carrier and noncarrier individuals, Kruskal-Wallis test was used. Colonized males had higher BMI than non-colonized males (no difference for females) and colonized females had lower WHR's. Body height was not related to colonization in either sex. We confirmed our hypothesis only for BMI in males. This result and a higher WHR in non-colonized females indicate higher immunocompetence of those who bear the costs of higher levels of testosterone, which according to previous studies is correlated negatively to BMI in males and positively to WHR in females. Copyright © 2014 Wiley Periodicals, Inc.

  5. Inclusion in Political and Public Life: The Experiences of People with Intellectual Disability on Government Disability Advisory Bodies in Australia

    ERIC Educational Resources Information Center

    Frawley, Patsie; Bigby, Christine

    2011-01-01

    Background: Civil and political participation lies at the core of citizenship. Increasingly, people with intellectual disability are members of disability advisory bodies. This study investigated the political orientations of advisory body members with intellectual disability, their participatory experiences, and the types of support they…

  6. Importin-α7 Is Involved in the Formation of Ebola Virus Inclusion Bodies but Is Not Essential for Pathogenicity in Mice

    PubMed Central

    Gabriel, Gülsah; Feldmann, Friederike; Reimer, Rudolph; Thiele, Swantje; Fischer, Meike; Hartmann, Enno; Bader, Michael; Ebihara, Hideki; Hoenen, Thomas; Feldmann, Heinz

    2015-01-01

    Ebola virus (EBOV) protein 24 antagonizes the host interferon (IFN) response by hijacking select nuclear importin-α isoforms. Thereby, it blocks STAT1-mediated IFN-α/β and IFN-γ synthesis. However, owing to the lack of importin-α knockout animal models in the past, their role in EBOV pathogenesis remained largely unknown. Here, we demonstrate that importin-α7 is involved in the formation of EBOV inclusion bodies and replication. However, deletion of the gene encoding importin-α7 was not sufficient to increase survival rates among mice infected with EBOV. PMID:26185094

  7. Human body temperature and new approaches to constructing temperature-sensitive bacterial vaccines.

    PubMed

    White, Matthew D; Bosio, Catharine M; Duplantis, Barry N; Nano, Francis E

    2011-09-01

    Many of the live human and animal vaccines that are currently in use are attenuated by virtue of their temperature-sensitive (TS) replication. These vaccines are able to function because they can take advantage of sites in mammalian bodies that are cooler than the core temperature, where TS vaccines fail to replicate. In this article, we discuss the distribution of temperature in the human body, and relate how the temperature differential can be exploited for designing and using TS vaccines. We also examine how one of the coolest organs of the body, the skin, contains antigen-processing cells that can be targeted to provoke the desired immune response from a TS vaccine. We describe traditional approaches to making TS vaccines, and highlight new information and technologies that are being used to create a new generation of engineered TS vaccines. We pay particular attention to the recently described technology of substituting essential genes from Arctic bacteria for their homologues in mammalian pathogens as a way of creating TS vaccines.

  8. Human body temperature and new approaches to constructing temperature-sensitive bacterial vaccines

    PubMed Central

    White, Matthew D.; Bosio, Catharine M.; Duplantis, Barry N.

    2012-01-01

    Many of the live human and animal vaccines that are currently in use are attenuated by virtue of their temperature-sensitive (TS) replication. These vaccines are able to function because they can take advantage of sites in mammalian bodies that are cooler than the core temperature, where TS vaccines fail to replicate. In this article, we discuss the distribution of temperature in the human body, and relate how the temperature differential can be exploited for designing and using TS vaccines. We also examine how one of the coolest organs of the body, the skin, contains antigen-processing cells that can be targeted to provoke the desired immune response from a TS vaccine. We describe traditional approaches to making TS vaccines, and highlight new information and technologies that are being used to create a new generation of engineered TS vaccines. We pay particular attention to the recently described technology of substituting essential genes from Arctic bacteria for their homologues in mammalian pathogens as a way of creating TS vaccines. PMID:21626408

  9. Computational biomechanics of human brain with and without the inclusion of the body under different blast orientation.

    PubMed

    Salimi Jazi, Mehdi; Rezaei, Asghar; Azarmi, Fardad; Ziejewski, Mariusz; Karami, Ghodrat

    2016-01-01

    Three different human head models in a free space are exposed to blast waves coming from four different directions. The four head-neck-body models composed of model a, with the neck free in space; model b, with neck fixed at the bottom; and model c, with the neck attached to the body. The results show that the effect of the body can be ignored for the first milliseconds of the head-blast wave interactions. Also one can see that although most biomechanical responses of the brain have similar patterns in all models, the shear stresses are heavily increased after a few milliseconds in model b in which the head motion is obstructed by the fixed-neck boundary conditions. The free-floating head model results are closer to the attached-body model.

  10. Identification, Characterization, and In Vitro Culture of Highly Divergent Arenaviruses from Boa Constrictors and Annulated Tree Boas: Candidate Etiological Agents for Snake Inclusion Body Disease

    PubMed Central

    Stenglein, Mark D.; Sanders, Chris; Kistler, Amy L.; Ruby, J. Graham; Franco, Jessica Y.; Reavill, Drury R.; Dunker, Freeland; DeRisi, Joseph L.

    2012-01-01

    ABSTRACT Inclusion body disease (IBD) is an infectious fatal disease of snakes typified by behavioral abnormalities, wasting, and secondary infections. At a histopathological level, the disease is identified by the presence of large eosinophilic cytoplasmic inclusions in multiple tissues. To date, no virus or other pathogen has been definitively characterized or associated with the disease. Using a metagenomic approach to search for candidate etiologic agents in snakes with confirmed IBD, we identified and de novo assembled the complete genomic sequences of two viruses related to arenaviruses, and a third arenavirus-like sequence was discovered by screening an additional set of samples. A continuous boa constrictor cell line was established and used to propagate and isolate one of the viruses in culture. Viral nucleoprotein was localized and concentrated within large cytoplasmic inclusions in infected cells in culture and tissues from diseased snakes. In total, viral RNA was detected in 6/8 confirmed IBD cases and 0/18 controls. These viruses have a typical arenavirus genome organization but are highly divergent, belonging to a lineage separate from that of the Old and New World arenaviruses. Furthermore, these viruses encode envelope glycoproteins that are more similar to those of filoviruses than to those of other arenaviruses. These findings implicate these viruses as candidate etiologic agents of IBD. The presence of arenaviruses outside mammals reveals that these viruses infect an unexpectedly broad range of species and represent a new reservoir of potential human pathogens. PMID:22893382

  11. Inclusion in political and public life: the experiences of people with intellectual disability on government disability advisory bodies in Australia.

    PubMed

    Frawley, Patsie; Bigby, Christine

    2011-03-01

    Civil and political participation lies at the core of citizenship. Increasingly, people with intellectual disability are members of disability advisory bodies. This study investigated the political orientations of advisory body members with intellectual disability, their participatory experiences, and the types of support they received. The 9 people with intellectual disability who in 2005 were members of disability advisory bodies at a state, national, and Victorian local government level were interviewed, together with 12 other members or secretariat staff of these bodies. Observations were also conducted of advisory body meetings. The political perspective of members with intellectual disability varied, but all had a background in self-advocacy. They found the work hard but rewarding and encountered both practical and intangible obstacles to participation. Members received varying types of practical support, but a supportive collegial milieu was characteristic among members who felt most confident about their participation. The milieu, structures, and processes of advisory bodies must all be adjusted to accommodate people with intellectual disability if they are to participate meaningfully.

  12. Quantitative relationships between huntingtin levels, polyglutamine length, inclusion body formation, and neuronal death provide novel insight into Huntington’s disease molecular pathogenesis

    PubMed Central

    Miller, Jason; Arrasate, Montserrat; Shaby, Benjamin A.; Mitra, Siddhartha; Masliah, Eliezer; Finkbeiner, Steven

    2010-01-01

    An expanded polyglutamine (polyQ) stretch in the protein huntingtin (htt) induces self-aggregation into inclusion bodies (IBs) and causes Huntington’s disease (HD). Defining precise relationships between early observable variables and neuronal death at the molecular and cellular levels should improve our understanding of HD pathogenesis. Here, we utilized an automated microscope that can track thousands of neurons individually over their entire lifetime to quantify interconnected relationships between early variables, such as htt levels, polyQ length, and IB formation, and neuronal death in a primary striatal model of HD. The resulting model revealed that: mutant htt increases the risk of death by tonically interfering with homeostatic coping mechanisms rather than producing accumulated damage to the neuron; htt toxicity is saturable; the rate limiting steps for inclusion body formation and death can be traced to different conformational changes in monomeric htt; and IB formation reduces the impact of a neuron’s starting levels of htt on its risk of death. Finally, the model that emerges from our quantitative measurements places critical limits on the potential mechanisms by which mutant htt might induce neurodegeneration, which should help direct future research. PMID:20685997

  13. Enhancement of solubility, purification and inclusion-bodies-refolding of an active pectin lyase from Penicillium occitanis expressed in Escherichia coli.

    PubMed

    Hadj Sassi, Azza; Trigui-Lahiani, Hèla; Abdeljalil, Salma; Gargouri, Ali

    2017-02-01

    Pectin lyase (pnl) is the only pectinase able to hydrolyze directly the highly methylated pectin without liberating the toxic methanol and without disturbing ester content responsible for specific aroma of juices. The cDNA of Penicillium occitanis pnl (mature form) was cloned into pET-21a as expression vector and over-expressed into Esherichia coli. Most of recombinant pnl was expressed as inclusion bodies. Pnl activity was confirmed by colorimetric assay. To enhance the solubility yield of the expressed pnl, the effects of induction temperature, host strain and expression level were optimized. Maximal production of functional pnl was obtained after induction by 0.4mM IPTG at 30°C and 150rpm for 16h. Interestingly, the use of Origami host strain, having an oxidized cytoplasm favoring disulfide bonds formation required for the active conformation of the enzyme, has significantly improved the yield of the soluble active form of recombinant pnl. This pnl was successfully purified through a single step purification using His-Trap affinity column chromatography. This work is the first to report pnl expression into Origami strain. Alternatively, the inclusion bodies were isolated, denatured by high concentration of urea and gradually refolded by successive dialysis, leading to their transformation into soluble and active form.

  14. Establishment of mouse model of MYH9 disorders: heterozygous R702C mutation provokes macrothrombocytopenia with leukocyte inclusion bodies, renal glomerulosclerosis and hearing disability.

    PubMed

    Suzuki, Nobuaki; Kunishima, Shinji; Ikejiri, Makoto; Maruyama, Shoichi; Sone, Michihiko; Takagi, Akira; Ikawa, Masahito; Okabe, Masaru; Kojima, Tetsuhito; Saito, Hidehiko; Naoe, Tomoki; Matsushita, Tadashi

    2013-01-01

    Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. MYH9 disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation. We generated knock-in mice expressing the Myh9 R702C mutation. R702C knock-in hetero mice (R702C+/- mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/- mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May-Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm. In other organs, R702C+/- mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response. These findings indicate that Myh9 R702C knock-in mice mirror features of human MYH9 disorders arising from the R702C mutation.

  15. Continuous processing of recombinant proteins: Integration of inclusion body solubilization and refolding using simulated moving bed size exclusion chromatography with buffer recycling.

    PubMed

    Wellhoefer, Martin; Sprinzl, Wolfgang; Hahn, Rainer; Jungbauer, Alois

    2013-12-06

    An integrated process which combines continuous inclusion body dissolution with NaOH and continuous matrix-assisted refolding based on closed-loop simulated moving bed size exclusion chromatography was designed and experimentally evaluated at laboratory scale. Inclusion bodies from N(pro) fusion pep6His and N(pro) fusion MCP1 from high cell density fermentation were continuously dissolved with NaOH, filtered and mixed with concentrated refolding buffer prior to refolding by size exclusion chromatography (SEC). This process enabled an isocratic operation of the simulated moving bed (SMB) system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling by concentrating the raffinate using tangential flow filtration. With this continuous refolding process, we increased the refolding and cleavage yield of both model proteins by 10% compared to batch dilution refolding. Furthermore, more than 99% of the refolding buffer of the raffinate could be recycled which reduced the buffer consumption significantly. Based on the actual refolding data, we compared throughput, productivity, and buffer consumption between two batch dilution refolding processes - one using urea for IB dissolution, the other one using NaOH for IB dissolution - and our continuous refolding process. The higher complexity of the continuous refolding process was rewarded with higher throughput and productivity as well as significantly lower buffer consumption compared to the batch dilution refolding processes. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Further research on the biological function of inclusion bodies of Anomala cuprea entomopoxvirus, with special reference to the effect on the insecticidal activity of a Bacillus thuringiensis formulation.

    PubMed

    Mitsuhashi, Wataru; Asano, Shoji; Miyamoto, Kazuhisa; Wada, Sanae

    2014-01-01

    Entomopoxviruses (EVs) form two types of inclusion body: spheroids, which contain virions, and spindles, which do not. The authors tested whether the spindles from a coleopteran EV, Anomala cuprea EV (ACEV), enhanced the insecticidal activity of a commercial Bacillus thuringiensis (Bt) formulation and the susceptibility of scarabaeid pest species in Japan to the virus's spheroids, to assess whether ACEV inclusion bodies are potential biological control agents for pest insects. Peroral inoculation with both ACEV spindles and the Bt toxin only or the complete Bt formulation shortened the survival and increased the mortality of treated insects compared with those of insects inoculated with Bt without the spindles (8-38 h of decrease in LT50 values among assays). ACEV showed high infectivity to a major scarabaeid pest species in Japanese sugar cane fields. The results suggest that spindles or the constituent protein fusolin can be used as a coagent with Bt formulations, and that fusolin coexpression with a Bt toxin in crops might improve the insecticidal efficacy. In addition, the spheroids are potential biocontrol agents for some scarabaeid pests that are not easy to control because of their underground habitation. © 2013 Society of Chemical Industry.

  17. Development of a rapid high-efficiency scalable process for acetylated Sus scrofa cationic trypsin production from Escherichia coli inclusion bodies.

    PubMed

    Zhao, Mingzhi; Wu, Feilin; Xu, Ping

    2015-12-01

    Trypsin is one of the most important enzymatic tools in proteomics and biopharmaceutical studies. Here, we describe the complete recombinant expression and purification from a trypsinogen expression vector construct. The Sus scrofa cationic trypsin gene with a propeptide sequence was optimized according to Escherichia coli codon-usage bias and chemically synthesized. The gene was inserted into pET-11c plasmid to yield an expression vector. Using high-density E. coli fed-batch fermentation, trypsinogen was expressed in inclusion bodies at 1.47 g/L. The inclusion body was refolded with a high yield of 36%. The purified trypsinogen was then activated to produce trypsin. To address stability problems, the trypsin thus produced was acetylated. The final product was generated upon gel filtration. The final yield of acetylated trypsin was 182 mg/L from a 5-L fermenter. Our acetylated trypsin product demonstrated higher BAEE activity (30,100 BAEE unit/mg) than a commercial product (9500 BAEE unit/mg, Promega). It also demonstrated resistance to autolysis. This is the first report of production of acetylated recombinant trypsin that is stable and suitable for scale-up. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Expression, characterization of a novel nitrilase PpL19 from Pseudomonas psychrotolerans with S-selectivity toward mandelonitrile present in active inclusion bodies.

    PubMed

    Sun, Huihui; Gao, Wenyuan; Wang, Hualei; Wei, Dongzhi

    2016-03-01

    To identify a novel nitrilase with S-selectivity toward mandelonitrile that can produce (S)-mandelic acid in one step. A novel nitrilase PpL19 from Pseudomonas psychrotolerans L19 was discovered by genome mining. It showed S-selectivity with an enantiomeric excess of 52.7 % when used to hydrolyse (R, S)-mandelonitrile. No byproduct was observed. PpL19 was overexpressed in Escherichia coli BL21 (DE3) and formed inclusion bodies that were active toward mandelonitrile and stable across a broad range of temperature and pH. In addition, PpL19 hydrolysed nitriles with diverse structures; arylacetonitriles were the optimal substrates. Homology modelling and docking studies of both enantiomers of mandelonitrile in the active site of nitrilase PpL19 shed light on the enantioselectivity. A novel nitrilase PpL19 from P. psychrotolerans L19 was mined and distinguished from other nitrilases as it was expressed as an active inclusion body and showed S-selectivity toward mandelonitrile.

  19. Establishment of Mouse Model of MYH9 Disorders: Heterozygous R702C Mutation Provokes Macrothrombocytopenia with Leukocyte Inclusion Bodies, Renal Glomerulosclerosis and Hearing Disability

    PubMed Central

    Suzuki, Nobuaki; Kunishima, Shinji; Ikejiri, Makoto; Maruyama, Shoichi; Sone, Michihiko; Takagi, Akira; Ikawa, Masahito; Okabe, Masaru; Kojima, Tetsuhito; Saito, Hidehiko; Naoe, Tomoki; Matsushita, Tadashi

    2013-01-01

    Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. MYH9 disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation. We generated knock-in mice expressing the Myh9 R702C mutation. R702C knock-in hetero mice (R702C+/− mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/− mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May–Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm. In other organs, R702C+/− mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response. These findings indicate that Myh9 R702C knock-in mice mirror features of human MYH9 disorders arising from the R702C mutation. PMID:23976996

  20. Mammalian G-protein-coupled receptor expression in Escherichia coli: I. High-throughput large-scale production as inclusion bodies.

    PubMed

    Michalke, Kerstin; Gravière, Marie-Eve; Huyghe, Céline; Vincentelli, Renaud; Wagner, Renaud; Pattus, Franc; Schroeder, Kathrin; Oschmann, Jan; Rudolph, Rainer; Cambillau, Christian; Desmyter, Aline

    2009-03-15

    G-protein-coupled receptors (GPCRs) represent approximately 3% of human proteome and the most prominent class of pharmacological targets. Despite their important role in many functions, only the X-ray structures of rhodopsin, and more recently of the beta(1)- and beta(2)-adrenergic receptors, have been resolved. Structural studies of GPCRs require that several tedious preliminary steps be fulfilled before setting up the first crystallization experiments: protein expression, detergent solubilization, purification, and stabilization. Here we report on screening expression conditions of approximately 100 GPCRs in Escherichia coli with a view to obtain large amounts of inclusion bodies, a prerequisite to the subsequent refolding step. A set of optimal conditions, including appropriate vectors (Gateway pDEST17oi), strain (C43), and fermentation at high optical density, define the best first instance choice. Beyond this minimal setting, however, the rate of success increases significantly with the number of conditions tested. In contrast with experiments based on a single GPCR expression, our approach provides statistically significant results and indicates that up to 40% of GPCRs can be expressed as inclusion bodies in quantities sufficient for subsequent refolding, solubilization, and purification.

  1. The Inclusive Classroom: How Inclusive Is Inclusion?

    ERIC Educational Resources Information Center

    Reid, Claudette M.

    2010-01-01

    This paper presents the position that inclusion is limited; inclusion does not go far enough. The inclusive classroom has been assessed to be of benefit both to the teacher and student. There are, however, limits set on inclusion. In most classrooms only children with learning disability are included omitting those with severe disabilities,…

  2. Protein-protein interaction network prediction by using rigid-body docking tools: application to bacterial chemotaxis.

    PubMed

    Matsuzaki, Yuri; Ohue, Masahito; Uchikoga, Nobuyuki; Akiyama, Yutaka

    2014-01-01

    Core elements of cell regulation are made up of protein-protein interaction (PPI) networks. However, many parts of the cell regulatory systems include unknown PPIs. To approach this problem, we have developed a computational method of high-throughput PPI network prediction based on all-to-all rigid-body docking of protein tertiary structures. The prediction system accepts a set of data comprising protein tertiary structures as input and generates a list of possible interacting pairs from all the combinations as output. A crucial advantage of this docking based method is in providing predictions of protein pairs that increases our understanding of biological pathways by analyzing the structures of candidate complex structures, which gives insight into novel interaction mechanisms. Although such exhaustive docking calculation requires massive computational resources, recent advancements in the computational sciences have made such large-scale calculations feasible. In this study we applied our prediction method to a pathway reconstruction problem of bacterial chemotaxis by using two different rigid-body docking tools with different scoring models. We found that the predicted interactions were different between the results from the two tools. When the positive predictions from both of the docking tools were combined, all the core signaling interactions were correctly predicted with the exception of interactions activated by protein phosphorylation. Large-scale PPI prediction using tertiary structures is an effective approach that has a wide range of potential applications. This method is especially useful for identifying novel PPIs of new pathways that control cellular behavior.

  3. Bacterial meningoencephalitis and ventriculitis due to migrating plant foreign bodies in three dogs.

    PubMed

    Dennis, M M; Pearce, L K; Norrdin, R W; Ehrhart, E J

    2005-11-01

    Regional suppurative meningoencephalitis and ventriculitis of variable chronicity was diagnosed in three young dogs residing in Colorado. Grass awns were grossly identified in the right occipital cortex of one dog and in the right lateral ventricle of another. Intralesional plant material was microscopically evident in the dura mater overlying the right occipital cortex of the third dog. One grass awn was identified as a floret of Hordeum jabatum. In each case, aerobic culture of brain tissue identified multiple isolates of bacteria. The dogs presented with clinically variable, rapidly progressive neurologic dysfunction, including tetraplegia, depressed mentation, and episodic extensor rigidity, ataxia, circling, stupor, vocalization, and head-pressing. Encephalitis due to bacteria introduced from migrating plant foreign material is a potential sequela of intranasal, periocular, or pharyngeal foreign bodies.

  4. Inclusion of Body-Bias Effect in SPICE Modeling of 4H-SiC Integrated Circuit Resistors

    NASA Technical Reports Server (NTRS)

    Neudeck, Philip G.

    2017-01-01

    The DC electrical behavior of n-type 4H-SiC resistors used for realizing 500 C durable integrated circuits (ICs) is studied as a function of substrate bias and temperature. Improved fidelity electrical simulation is described using SPICE NMOS model to simulate resistor substrate body bias effect that is absent from the SPICE semiconductor resistor model.

  5. Identification, characterization, and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas: candidate etiological agents for snake inclusion body disease.

    PubMed

    Stenglein, Mark D; Sanders, Chris; Kistler, Amy L; Ruby, J Graham; Franco, Jessica Y; Reavill, Drury R; Dunker, Freeland; Derisi, Joseph L

    2012-01-01

    Inclusion body disease (IBD) is an infec