Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

PubMed Central

Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

2015-01-01

ABSTRACT Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high PrPC expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrPC-binding partners. Ten different putative PrPC ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrPC with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrPC-Stub1 interaction are under investigation. The PrPC-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrPC is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

The chemistry of prions: small molecules, protein conformers and mass spectrometry

USDA-ARS?s Scientific Manuscript database

Background/Introduction. Prions propagate by converting a normal cellular isoform (PrPC) into the prion isoform (PrPSc) in a template-driven process. The lysines in PrPC are highly conserved and strongly influence prion propagation, based on studies using natural polymorphisms of PrPC and transg...

Release of Full-Length PrPC from Cultured Neurons Following Neurotoxic Challenge

PubMed Central

Wang, Kevin K. W.; Zoltewicz, J. Susie; Chiu, Allen; Zhang, Zhiqun; Rubenstein, Richard

2012-01-01

The susceptibility of the normal cellular prion protein isoform, cellular prion protein (PrPC), to proteolytic digestion has been well documented. In addition, a link between PrPC and the cytosolic protease, calpain, has been reported although the specifics of the interaction remain unclear. We performed in vitro and in cell-based studies to examine this relationship. We observed that human recombinant PrP (HrPrP) was readily cleaved by calpain-1 and -2, and we have identified and defined the targeted cleavage sites. In contrast, HrPrP was resistant to caspase-3 digestion. Unexpectedly, when brain lysates from PrPC-expressing mice were treated with calpain, no appreciable loss of the intact PrPC, nor the appearance of PrPC breakdown products (BDPs) were observed, even though alpha II-spectrin was converted to its signature calpain-induced BDPs. In addition, when rat cerebrocortical neuronal cultures (RtCNC) were subjected to the two neurotoxins at subacute levels, maitotoxin (MTX) and N-methyl-d-aspartate (NMDA), PrPC-BDPs were also not detectable. However, a novel finding from these cell-based studies is that apparently full-length, mature PrPC is released into culture media from RtCNC challenged with subacute doses of MTX and NMDA. Calpain inhibitor SNJ-1945 and caspase inhibitor IDN-6556 did not attenuate the release of PrPC. Similarly, the lysosomal protease inhibitor, NH4Cl, and the proteasome inhibitor, lactacystin, did not significantly alter the integrity of PrPC or its release from the RtCNC. In conclusion, rat neuronal PrPC is not a significant target for proteolytic modifications during MTX and NMDA neurotoxic challenges. However, the robust neurotoxin-mediated release of full-length PrPC into the cell culture media suggests an unidentified neuroprotective mechanism for PrPC. PMID:23093947

Expression of cellular prion protein in the frontal and occipital lobe in Alzheimer's disease, diffuse Lewy body disease, and in normal brain: an immunohistochemical study.

PubMed

Rezaie, Payam; Pontikis, Charlie C; Hudson, Lance; Cairns, Nigel J; Lantos, Peter L

2005-08-01

Cellular prion protein (PrP(c)) is a glycoprotein expressed at low to moderate levels within the nervous system. Recent studies suggest that PrP(c) may possess neuroprotective functions and that its expression is upregulated in certain neurodegenerative disorders. We investigated whether PrP(c) expression is altered in the frontal and occipital cortex in two well-characterized neurodegenerative disorders--Alzheimer's disease (AD) and diffuse Lewy body disease (DLBD)--compared with that in normal human brain using immunohistochemistry and computerized image analysis. The distribution of PrP(c) was further tested for correlation with glial reactivity. We found that PrP(c) was localized mainly in the gray matter (predominantly in neurons) and expressed at higher levels within the occipital cortex in the normal human brain. Image analysis revealed no significant variability in PrP(c) expression between DLBD and control cases. However, blood vessels within the white matter of DLBD cases showed immunoreactivity to PrP(c). By contrast, this protein was differentially expressed in the frontal and occipital cortex of AD cases; it was markedly overexpressed in the former and significantly reduced in the latter. Epitope specificity of antibodies appeared important when detecting PrP(c). The distribution of PrP(c) did not correlate with glial immunoreactivity. In conclusion, this study supports the proposal that regional changes in expression of PrP(c) may occur in certain neurodegenerative disorders such as AD, but not in other disorders such as DLBD.

Alzheimer's Disease and Prion Protein

PubMed Central

Zhou, Jiayi; Liu, Bingqian

2013-01-01

Summary Alzheimer's disease (AD) is a devastating neurodegenerative disease with progressive loss of memory and cognitive function, pathologically hallmarked by aggregates of the amyloid-beta (Aβ) peptide and hyperphosphorylated tau in the brain. Aggregation of Aβ under the form of amyloid fibrils has long been considered central to the pathogenesis of AD. However, recent evidence has indicated that soluble Aβ oligomers, rather than insoluble fibrils, are the main neurotoxic species in AD. The cellular prion protein (PrPC) has newly been identified as a cell surface receptor for Aβ oligomers. PrPC is a cell surface glycoprotein that plays a key role in the propagation of prions, proteinaceous infectious agents that replicate by imposing their abnormal conformation to PrPC molecules. In AD, PrPC acts to transduce the neurotoxic signals arising from Aβ oligomers, leading to synaptic failure and cognitive impairment. Interestingly, accumulating evidence has also shown that aggregated Aβ or tau possesses prion-like activity, a property that would allow them to spread throughout the brain. In this article, we review recent findings regarding the function of PrPC and its role in AD, and discuss potential therapeutic implications of PrPC-based approaches in the treatment of AD. PMID:25343100

Does methionine oxidation influence the progression of classical or atypical scrapie

USDA-ARS?s Scientific Manuscript database

Introduction Prions are pathological proteins that propagate by converting a normal cellular prion protein (PrPC) into a prion (PrPSc). PrPC and PrPSc possess identical covalent structures and only differ in their conformations. The conversion of the PrPC conformation to the PrPSc one is template ...

PrP(C) regulates epidermal growth factor receptor function and cell shape dynamics in Neuro2a cells.

PubMed

Llorens, Franc; Carulla, Patricia; Villa, Ana; Torres, Juan M; Fortes, Puri; Ferrer, Isidre; del Río, José A

2013-10-01

The prion protein (PrP) plays a key role in prion disease pathogenesis. Although the misfolded and pathologic variant of this protein (PrP(SC)) has been studied in depth, the physiological role of PrP(C) remains elusive and controversial. PrP(C) is a cell-surface glycoprotein involved in multiple cellular functions at the plasma membrane, where it interacts with a myriad of partners and regulates several intracellular signal transduction cascades. However, little is known about the gene expression changes modulated by PrP(C) in animals and in cellular models. In this article, we present PrP(C)-dependent gene expression signature in N2a cells and its implication in the most overrepresented functions: cell cycle, cell growth and proliferation, and maintenance of cell shape. PrP(C) over-expression enhances cell proliferation and cell cycle re-entrance after serum stimulation, while PrP(C) silencing slows down cell cycle progression. In addition, MAP kinase and protein kinase B (AKT) pathway activation are under the regulation of PrP(C) in asynchronous cells and following mitogenic stimulation. These effects are due in part to the modulation of epidermal growth factor receptor (EGFR) by PrP(C) in the plasma membrane, where the two proteins interact in a multimeric complex. We also describe how PrP(C) over-expression modulates filopodia formation by Rho GTPase regulation mainly in an AKT-Cdc42-N-WASP-dependent pathway. © 2013 International Society for Neurochemistry.

Discovery of Novel Anti-prion Compounds Using In Silico and In Vitro Approaches

PubMed Central

Hyeon, Jae Wook; Choi, Jiwon; Kim, Su Yeon; Govindaraj, Rajiv Gandhi; Jam Hwang, Kyu; Lee, Yeong Seon; An, Seong Soo A.; Lee, Myung Koo; Joung, Jong Young; No, Kyoung Tai; Lee, Jeongmin

2015-01-01

Prion diseases are associated with the conformational conversion of the physiological form of cellular prion protein (PrPC) to the pathogenic form, PrPSc. Compounds that inhibit this process by blocking conversion to the PrPSc could provide useful anti-prion therapies. However, no suitable drugs have been identified to date. To identify novel anti-prion compounds, we developed a combined structure- and ligand-based virtual screening system in silico. Virtual screening of a 700,000-compound database, followed by cluster analysis, identified 37 compounds with strong interactions with essential hotspot PrP residues identified in a previous study of PrPC interaction with a known anti-prion compound (GN8). These compounds were tested in vitro using a multimer detection system, cell-based assays, and surface plasmon resonance. Some compounds effectively reduced PrPSc levels and one of these compounds also showed a high binding affinity for PrPC. These results provide a promising starting point for the development of anti-prion compounds. PMID:26449325

Behavioral abnormalities in prion protein knockout mice and the potential relevance of PrPc for the cytoskeleton

USDA-ARS?s Scientific Manuscript database

The cellular prion protein (PrPC) is a highly conserved protein, which is anchored to the outer surface of the plasma membrane. Even though its physiological function has already been investigated in different cell or mouse models where PrPC expression is either up-regulated or depleted, its exact p...

Therapeutic Molecules and Endogenous Ligands Regulate the Interaction between Brain Cellular Prion Protein (PrPC) and Metabotropic Glutamate Receptor 5 (mGluR5)*

PubMed Central

Haas, Laura T.; Kostylev, Mikhail A.; Strittmatter, Stephen M.

2014-01-01

Soluble Amyloid-β oligomers (Aβo) can trigger Alzheimer disease (AD) pathophysiology by binding to cell surface cellular prion protein (PrPC). PrPC interacts physically with metabotropic glutamate receptor 5 (mGluR5), and this interaction controls the transmission of neurotoxic signals to intracellular substrates. Because the interruption of the signal transduction from PrPC to mGluR5 has therapeutic potential for AD, we developed assays to explore the effect of endogenous ligands, agonists/antagonists, and antibodies on the interaction between PrPC and mGluR5 in cell lines and mouse brain. We show that the PrPC segment of amino acids 91–153 mediates the interaction with mGluR5. Agonists of mGluR5 increase the mGluR5-PrPC interaction, whereas mGluR5 antagonists suppress protein association. Synthetic Aβo promotes the protein interaction in mouse brain and transfected HEK-293 cell membrane preparations. The interaction of PrPC and mGluR5 is enhanced dramatically in the brains of familial AD transgenic model mice. In brain homogenates with Aβo, the interaction of PrPC and mGluR5 is reversed by mGluR5-directed antagonists or antibodies directed against the PrPC segment of amino acids 91–153. Silent allosteric modulators of mGluR5 do not alter Glu or basal mGluR5 activity, but they disrupt the Aβo-induced interaction of mGluR5 with PrPC. The assays described here have the potential to identify and develop new compounds that inhibit the interaction of PrPC and mGluR5, which plays a pivotal role in the pathogenesis of Alzheimer disease by transmitting the signal from extracellular Aβo into the cytosol. PMID:25148681

Alzheimer's amyloid-β oligomers rescue cellular prion protein induced tau reduction via the Fyn pathway.

PubMed

Chen, Rong-Jie; Chang, Wei-Wei; Lin, Yu-Chun; Cheng, Pei-Lin; Chen, Yun-Ru

2013-09-18

Amyloid-β (Aβ) and tau are the pathogenic hallmarks in Alzheimer's disease (AD). Aβ oligomers are considered the actual toxic entities, and the toxicity relies on the presence of tau. Recently, Aβ oligomers have been shown to specifically interact with cellular prion protein (PrP(C)) where the role of PrP(C) in AD is still not fully understood. To investigate the downstream mechanism of PrP(C) and Aβ oligomer interaction and their possible relationships to tau, we examined tau expression in human neuroblastoma BE(2)-C cells transfected with murine PrP(C) and studied the effect under Aβ oligomer treatment. By Western blotting, we found that PrP(C) overexpression down-regulated tau protein and Aβ oligomer binding alleviated the tau reduction induced by wild type but not M128V PrP(C), the high AD risk polymorphic allele in human prion gene. PrP(C) lacking the Aβ oligomer binding site was incapable of rescuing the level of tau reduction. Quantitative RT-PCR showed the PrP(C) effect was attributed to tau reduction at the transcription level. Treatment with Fyn pathway inhibitors, Fyn kinase inhibitor PP2 and MEK inhibitor U0126, reversed the PrP(C)-induced tau reduction and Aβ oligomer treatment modulated Fyn kinase activity. The results suggested Fyn pathway regulated Aβ-PrP(C)-tau signaling. Overall, our results demonstrated that PrP(C) down-regulated tau via the Fyn pathway and the effect can be regulated by Aβ oligomers. Our study facilitated the understanding of molecular mechanisms among PrP(C), tau, and Aβ oligomers.

Cellular Prion Protein and Caveolin-1 Interaction in a Neuronal Cell Line Precedes Fyn/Erk 1/2 Signal Transduction

PubMed Central

Toni, Mattia; Spisni, Enzo; Griffoni, Cristiana; Santi, Spartaco; Riccio, Massimo; Lenaz, Patrizia; Tomasi, Vittorio

2006-01-01

It has been reported that cellular prion protein (PrPc) is enriched in caveolae or caveolae-like domains with caveolin-1 (Cav-1) participating to signal transduction events by Fyn kinase recruitment. By using the Glutathione-S-transferase (GST)-fusion proteins assay, we observed that PrPc strongly interacts in vitro with Cav-1. Thus, we ascertained the PrPc caveolar localization in a hypothalamic neuronal cell line (GN11), by confocal microscopy analysis, flotation on density gradient, and coimmunoprecipitation experiments. Following the anti-PrPc antibody-mediated stimulation of live GN11 cells, we observed that PrPc clustered on plasma membrane domains rich in Cav-1 in which Fyn kinase converged to be activated. After these events, a signaling cascade through p42/44 MAP kinase (Erk 1/2) was triggered, suggesting that following translocations from rafts to caveolae or caveolaelike domains PrPc could interact with Cav-1 and induce signal transduction events. PMID:17489019

Cellular prion protein protects from inflammatory and neuropathic pain

PubMed Central

2011-01-01

Cellular prion protein (PrPC) inhibits N-Methyl-D-Aspartate (NMDA) receptors. Since NMDA receptors play an important role in the transmission of pain signals in the dorsal horn of spinal cord, we thus wanted to determine if PrPC null mice show a reduced threshold for various pain behaviours. We compared nociceptive thresholds between wild type and PrPC null mice in models of inflammatory and neuropathic pain, in the presence and the absence of a NMDA receptor antagonist. 2-3 months old male PrPC null mice exhibited an MK-801 sensitive decrease in the paw withdrawal threshold in response both mechanical and thermal stimuli. PrPC null mice also exhibited significantly longer licking/biting time during both the first and second phases of formalin-induced inflammation of the paw, which was again prevented by treatment of the mice with MK-801, and responded more strongly to glutamate injection into the paw. Compared to wild type animals, PrPC null mice also exhibited a significantly greater nociceptive response (licking/biting) after intrathecal injection of NMDA. Sciatic nerve ligation resulted in MK-801 sensitive neuropathic pain in wild-type mice, but did not further augment the basal increase in pain behaviour observed in the null mice, suggesting that mice lacking PrPC may already be in a state of tonic central sensitization. Altogether, our data indicate that PrPC exerts a critical role in modulating nociceptive transmission at the spinal cord level, and fit with the concept of NMDA receptor hyperfunction in the absence of PrPC. PMID:21843375

Signal transduction in neurons: effects of cellular prion protein on fyn kinase and ERK1/2 kinase.

PubMed

Tomasi, Vittorio

2010-12-16

It has been reported that cellular prion protein (PrPc) co-localizes with caveolin-1 and participates to signal transduction events by recruiting Fyn kinase. As PrPc is a secreted protein anchored to the outer surface membrane through a glycosylphosphatidylinositol (GPI) anchor (secPrP) and caveolin-1 is located in the inner leaflet of plasma membrane, there is a problem of how the two proteins can physically interact each other and transduce signals. By using the GST-fusion proteins system we observed that PrPc strongly interacts with caveolin-1 scaffolding domain and with a caveolin-1 hydrophilic C-terminal region, but not with the caveolin-1 N-terminal region. In vitro binding experiments were also performed to define the site(s) of PrPc interacting with cav-1. The results are consistent with a participation of PrPc octapeptide repeats motif in the binding to caveolin-1 scaffolding domain. The caveolar localization of PrPc was ascertained by co-immunoprecipitation, by co-localization after flotation in density gradients and by confocal microscopy analysis of PrPc and caveolin-1 distributions in a neuronal cell line (GN11) expressing caveolin-1 at high levels. We observed that, after antibody-mediated cross-linking or copper treatment, PrPc was internalized probably into caveolae. We propose that following translocation from rafts to caveolae or caveolae-like domains, secPrP could interact with caveolin-1 and induce signal transduction events.

Potential approaches for heterologous prion protein treatment of prion diseases

PubMed Central

Seelig, Davis M.; Goodman, Patricia A.; Skinner, Pamela J.

2016-01-01

ABSTRACT Prion diseases, or transmissible spongiform encephalopathies (TSEs) are progressive, fatal neurodegenerative diseases with no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPres). The efficiency of this conversion is predicated upon a number of factors, most notably a strong homology between cellular PrPC and PrPres. In our recently published study, we infected mice with the RML-Chandler strain of scrapie and treated them with heterologous hamster prion proteins. This treatment was seen to reduce clinical signs of prion disease, to delay the onset of clinical symptoms and to prolong survival. In this current article we discuss potential mechanisms of action of treatment with heterologous prion proteins. We also discuss potential extensions of these studies using a heterologous rabbit PrP-based treatment strategy or a peptide based strategy, and improvement of treatment delivery including a lentiviral-based system. PMID:26636482

Production of cattle lacking prion protein

PubMed Central

Richt, Jürgen A; Kasinathan, Poothappillai; Hamir, Amir N; Castilla, Joaquin; Sathiyaseelan, Thillai; Vargas, Francisco; Sathiyaseelan, Janaki; Wu, Hua; Matsushita, Hiroaki; Koster, Julie; Kato, Shinichiro; Ishida, Isao; Soto, Claudio; Robl, James M; Kuroiwa, Yoshimi

2010-01-01

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrPC, such as PrPBSE in bovine spongiform encephalopathy (BSE) in cattle and PrPCJD in Creutzfeldt-Jakob disease (CJD) in humans1. Disruption of PrPC expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities2–5. However, the impact of ablating PrPC function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrPC-deficient cattle produced by a sequential gene-targeting system6. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification7. PrPC-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins. PMID:17195841

Using mass spectrometry and small molecule reagents to detect distinctive structural features of different prion conformations (strains)

USDA-ARS?s Scientific Manuscript database

A prion (PrPSc) is a conformer of a normal cellular prion protein (PrPC). Although they are isosequential, PrPSc is an infectious protein able to convert PrPC into the prion conformation and thereby propagate an infection. PrPC is monomeric while PrPSc is a multimer. PrPSc can adopt more than one co...

Prion Protein Regulates Iron Transport by Functioning as a Ferrireductase

PubMed Central

Singh, Ajay; Haldar, Swati; Horback, Katharine; Tom, Cynthia; Zhou, Lan; Meyerson, Howard; Singh, Neena

2017-01-01

Prion protein (PrPC) is implicated in the pathogenesis of prion disorders, but its normal function is unclear. We demonstrate that PrPC is a ferrireductase (FR), and its absence causes systemic iron deficiency in PrP knock-out mice (PrP−/−). When exposed to non-transferrin-bound (NTB) radioactive-iron (59FeCl3) by gastric-gavage, PrP−/− mice absorb significantly more 59Fe from the intestinal lumen relative to controls, indicating appropriate systemic response to the iron deficiency. Chronic exposure to excess dietary iron corrects this deficiency, but unlike wild-type (PrP+/+) controls that remain iron over-loaded, PrP−/− mice revert back to the iron deficient phenotype after 5 months of chase on normal diet. Bone marrow (BM) preparations of PrP−/− mice on normal diet show relatively less stainable iron, and this phenotype is only partially corrected by intraperitoneal administration of excess iron-dextran. Cultured PrP−/− BM-macrophages incorporate significantly less NTB-59Fe in the absence or presence of excess extracellular iron, indicating reduced uptake and/or storage of available iron in the absence of PrPC. When expressed in neuroblastoma cells, PrPC exhibits NAD(P)H-dependent cell-surface and intracellular FR activity that requires the copper-binding octa-peptide-repeat region and linkage to the plasma membrane for optimal function. Incorporation of NTB-59Fe by neuroblastoma cells correlates with FR activity of PrPC, implicating PrPC in cellular iron uptake and metabolism. These observations explain the correlation between PrPC expression and cellular iron levels, and the cause of iron imbalance in sporadic-Creutzfeldt-Jakob-disease brains where PrPC accumulates as insoluble aggregates. PMID:23478311

PrP(C) homodimerization stimulates the production of PrPC cleaved fragments PrPN1 and PrPC1.

PubMed

Béland, Maxime; Motard, Julie; Barbarin, Alice; Roucou, Xavier

2012-09-19

An endoproteolytic cleavage termed α-cleavage between residues 111/112 is a characteristic feature of the cellular prion protein (PrP(C)). This cleavage generates a soluble N-terminal fragment (PrPN1) and a glycosylphosphatidylinositol-anchored C-terminal fragment (PrPC1). Independent studies demonstrate that modulating PrP(C) α-cleavage represents a potential therapeutic strategy in prion diseases. The regulation of PrP(C) α-cleavage is unclear. The only known domain that is essential for the α-cleavage to occur is a hydrophobic domain (HD). Importantly, the HD is also essential for the formation of PrP(C) homodimers. To explore the role of PrP(C) homodimerization on the α-cleavage, we used a well described inducible dimerization strategy whereby a chimeric PrP(C) composed of a modified FK506-binding protein (Fv) fused with PrP(C) and termed Fv-PrP is incubated in the presence of a dimerizer AP20187 ligand. We show that homodimerization leads to a considerable increase of PrP(C) α-cleavage in cultured cells and release of PrPN1 and PrPC1. Interestingly, enforced homodimerization increased PrP(C) levels at the plasma membrane, and preventing PrP(C) trafficking to the cell surface inhibited dimerization-induced α-cleavage. These observations were confirmed in primary hippocampal neurons from transgenic mice expressing Fv-PrP. The proteases responsible for the α-cleavage are still elusive, and in contrast to initial studies we confirm more recent investigations that neither ADAM10 nor ADAM17 are involved. Importantly, PrPN1 produced after PrP(C) homodimerization protects against toxic amyloid-β (Aβ) oligomers. Thus, our results show that PrP(C) homodimerization is an important regulator of PrP(C) α-cleavage and may represent a potential therapeutic avenue against Aβ toxicity in Alzheimer's disease.

Time-course study of retinal pathology in C57BL/6 mice infected with RML scrapie

USDA-ARS?s Scientific Manuscript database

Prions are proteinaceous pathogens that cause transmissible spongiform encephalopathies (TSEs). These diseases develop slowly as the misfolded and protease-resistant prion protein, PrP**Sc, interacts with the normal cellular form, PrP**C, a cell-surface protein found throughout the nervous system. T...

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

USDA-ARS?s Scientific Manuscript database

Classical scrapie is a form of transmissible spongiform encephalopathies (TSE) affecting domestic goats and sheep and disease is characterized by the accumulation of abnormal conformational isoform (PrP-Sc) of normal cellular prion protein (PrP-C) in the central nervous system and, in most cases, ly...

Detection of BSE prions by RT-QuIC in cattle with subclinical BSE

USDA-ARS?s Scientific Manuscript database

Bovine spongiform encephalopathy (BSE) belongs to a group of fatal prion diseases that result from the misfolding of the cellular prion protein (PrPC) into a pathogenic form (PrPSc) that accumulates in the brain and some lymphoid tissues. In vitro assays such as serial protein misfolding amplificati...

Spectroscopic and Theoretical Study of CuI Binding to His111 in the Human Prion Protein Fragment 106–115

PubMed Central

2016-01-01

The ability of the cellular prion protein (PrPC) to bind copper in vivo points to a physiological role for PrPC in copper transport. Six copper binding sites have been identified in the nonstructured N-terminal region of human PrPC. Among these sites, the His111 site is unique in that it contains a MKHM motif that would confer interesting CuI and CuII binding properties. We have evaluated CuI coordination to the PrP(106–115) fragment of the human PrP protein, using NMR and X-ray absorption spectroscopies and electronic structure calculations. We find that Met109 and Met112 play an important role in anchoring this metal ion. CuI coordination to His111 is pH-dependent: at pH >8, 2N1O1S species are formed with one Met ligand; in the range of pH 5–8, both methionine (Met) residues bind to CuI, forming a 1N1O2S species, where N is from His111 and O is from a backbone carbonyl or a water molecule; at pH <5, only the two Met residues remain coordinated. Thus, even upon drastic changes in the chemical environment, such as those occurring during endocytosis of PrPC (decreased pH and a reducing potential), the two Met residues in the MKHM motif enable PrPC to maintain the bound CuI ions, consistent with a copper transport function for this protein. We also find that the physiologically relevant CuI-1N1O2S species activates dioxygen via an inner-sphere mechanism, likely involving the formation of a copper(II) superoxide complex. In this process, the Met residues are partially oxidized to sulfoxide; this ability to scavenge superoxide may play a role in the proposed antioxidant properties of PrPC. This study provides further insight into the CuI coordination properties of His111 in human PrPC and the molecular mechanism of oxygen activation by this site. PMID:26930130

Using small molecule reagents to help distinguish among prion structural models

USDA-ARS?s Scientific Manuscript database

The only demonstrated difference between infectious prions (PrPSc) and the isosequential normal cellular prion protein (PrPC) is conformation. The structure of PrPC has been determined by a variety of instrumental techniques. The structure of prions remains uncertain. Recent instrumental analysis h...

Clinical features in prion protein-deficient and wild-type cattle inoculated with transmissible mink encephalopathy (TME)

USDA-ARS?s Scientific Manuscript database

Background: Transmissible spongiform encephalopathies (TSEs) or prion diseases are caused by the propagation of a misfolded form (PrP**d) of the normal cellular prion protein, PrP**c. Recently, we have reported the generation and characterization of PrP**C-deficient cattle (PrP-/-) produced by a seq...

PrPc Does Not Mediate Internalization of PrPSc but Is Required at an Early Stage for De Novo Prion Infection of Rov Cells▿

PubMed Central

Paquet, Sophie; Daude, Nathalie; Courageot, Marie-Pierre; Chapuis, Jérôme; Laude, Hubert; Vilette, Didier

2007-01-01

We have studied the interactions of exogenous prions with an epithelial cell line inducibly expressing PrPc protein and permissive to infection by a sheep scrapie agent. We demonstrate that abnormal PrP (PrPSc) and prion infectivity are efficiently internalized in Rov cells, whether or not PrPc is expressed. At odds with earlier studies implicating cellular heparan sulfates in PrPSc internalization, we failed to find any involvement of such molecules in Rov cells, indicating that prions can enter target cells by several routes. We further show that PrPSc taken up in the absence of PrPc was unable to promote efficient prion multiplication once PrPc expression was restored in the cells. This observation argues that interaction of PrPSc with PrPc has to occur early, in a specific subcellular compartment(s), and is consistent with the view that the first prion multiplication events may occur at the cell surface. PMID:17626095

Pressure-assisted dissociation and degradation of "proteinase K-resistant" fibrils prepared by seeding with scrapie-infected hamster prion protein.

PubMed

Akasaka, Kazuyuki; Maeno, Akihiro; Murayama, Taichi; Tachibana, Hideki; Fujita, Yuzo; Yamanaka, Hitoki; Nishida, Noriyuki; Atarashi, Ryuichiro

2014-01-01

The crucial step for the fatal neurodegenerative prion diseases involves the conversion of a normal cellular protein, PrP(C), into a fibrous pathogenic form, PrP(Sc), which has an unusual stability against heat and resistance against proteinase K digestion. A successful challenge to reverse the reaction from PrP(Sc) into PrP(C) is considered valuable, as it would give a key to dissolving the complex molecular events into thermodynamic and kinetic analyses and may also provide a means to prevent the formation of PrP(Sc) from PrP(C) eventually in vivo. Here we show that, by applying pressures at kbar range, the "proteinase K-resistant" fibrils (rHaPrP(res)) prepared from hamster prion protein (rHaPrP [23-231]) by seeding with brain homogenate of scrapie-infected hamster, becomes easily digestible. The result is consistent with the notion that rHaPrP(res) fibrils are dissociated into rHaPrP monomers under pressure and that the formation of PrP(Sc) from PrP(C) is thermodynamically controlled. Moreover, the efficient degradation of prion fibrils under pressure provides a novel means of eliminating infectious PrP(Sc) from various systems of pathogenic concern.

Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

NASA Astrophysics Data System (ADS)

Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

2000-05-01

The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells.

PubMed

Corsaro, Alessandro; Bajetto, Adriana; Thellung, Stefano; Begani, Giulia; Villa, Valentina; Nizzari, Mario; Pattarozzi, Alessandra; Solari, Agnese; Gatti, Monica; Pagano, Aldo; Würth, Roberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2016-06-21

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype.

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells

PubMed Central

Corsaro, Alessandro; Bajetto, Adriana; Thellung, Stefano; Begani, Giulia; Villa, Valentina; Nizzari, Mario; Pattarozzi, Alessandra; Solari, Agnese; Gatti, Monica; Pagano, Aldo; Würth, Roberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2016-01-01

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype. PMID:27229535

Quantifying the relative amounts of PrP polymorphisms present in prions isolated from heterozygous prion-infected animals

USDA-ARS?s Scientific Manuscript database

Prions cause protein misfolding diseases, such as transmissible spongiform encephalopathy. They propagate infections by converting a normal cellular prion protein into a prion (PrPSc). PrPC and PrPSc are isosequential and differ only in their respective conformations. PrPC is monomeric and sensit...

The α-Secretase-derived N-terminal Product of Cellular Prion, N1, Displays Neuroprotective Function in Vitro and in Vivo*

PubMed Central

Guillot-Sestier, Marie-Victoire; Sunyach, Claire; Druon, Charlotte; Scarzello, Sabine; Checler, Frédéric

2009-01-01

Cellular prion protein (PrPc) undergoes a disintegrin-mediated physiological cleavage, generating a soluble amino-terminal fragment (N1), the function of which remained unknown. Recombinant N1 inhibits staurosporine-induced caspase-3 activation by modulating p53 transcription and activity, whereas the PrPc-derived pathological fragment (N2) remains biologically inert. Furthermore, N1 protects retinal ganglion cells from hypoxia-induced apoptosis, reduces the number of terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling-positive and p53-immunoreactive neurons in a pressure-induced ischemia model of the rat retina and triggers a partial recovery of b-waves but not a-waves of rat electroretinograms. Our work is the first demonstration that the α-secretase-derived PrPc fragment N1, but not N2, displays in vivo and in vitro neuroprotective function by modulating p53 pathway. It further demonstrates that distinct N-terminal cleavage products of PrPc harbor different biological activities underlying the various phenotypes linking PrPc to cell survival. PMID:19850936

Spontaneous Generation of Infectious Prion Disease in Transgenic Mice

PubMed Central

Castilla, Joaquín; Pintado, Belén; Gutiérrez-Adan, Alfonso; Andréoletti, Olivier; Aguilar-Calvo, Patricia; Arroba, Ana-Isabel; Parra-Arrondo, Beatriz; Ferrer, Isidro; Manzanares, Jorge; Espinosa, Juan-Carlos

2013-01-01

We generated transgenic mice expressing bovine cellular prion protein (PrPC) with a leucine substitution at codon 113 (113L). This protein is homologous to human protein with mutation 102L, and its genetic link with Gerstmann–Sträussler–Scheinker syndrome has been established. This mutation in bovine PrPC causes a fully penetrant, lethal, spongiform encephalopathy. This genetic disease was transmitted by intracerebral inoculation of brain homogenate from ill mice expressing mutant bovine PrP to mice expressing wild-type bovine PrP, which indicated de novo generation of infectious prions. Our findings demonstrate that a single amino acid change in the PrPC sequence can induce spontaneous generation of an infectious prion disease that differs from all others identified in hosts expressing the same PrPC sequence. These observations support the view that a variety of infectious prion strains might spontaneously emerge in hosts displaying random genetic PrPC mutations. PMID:24274622

Orally administered indomethacin acutely reduces cellular prion protein in the small intestine and modestly increases survival of mice exposed to infectious prions.

PubMed

Martin, Gary R; Sharkey, Keith A; Jirik, Frank R

2015-05-01

The oral uptake of infectious prions represents a common way to acquire a prion disease; thus, host factors, such as gut inflammation and intestinal "leakiness", have the potential to influence infectivity. For example, the ingestion of nonsteroidal anti-inflammatory drugs (NSAIDs) is known to induce intestinal inflammation and increase intestinal permeability. Previously, we reported that normal cellular prion protein (PrP(C)) expression was increased in experimental colitis, and since the level of PrP(C) expressed is a determinant of prion disease propagation, we hypothesized that NSAID administration prior to the oral inoculation of mice with infectious prions would increase intestinal PrP(C) expression and accelerate the onset of neurological disease. In the long-term experiments, one group of mice was gavaged with indomethacin, followed by a second gavage with brain homogenate containing mouse-adapted scrapie (ME7). Control mice received ME7 brain homogenate alone. Brain and splenic tissues were harvested at several time points for immunoblotting, including at the onset of clinical signs of disease. In a second series of experiments, mice were gavaged with indomethacin to assess the acute effects of this treatment on intestinal PrP(C) expression. Acutely, NSAID treatment reduced intestinal PrP(C) expression, and chronically, there was a modest delay in the onset of neurological disease. In contrast to our hypothesis, brief exposure to an NSAID decreased intestinal PrP(C) expression and led to a modest survival advantage following oral ingestion of infectious prions.

Lack of Prion Accumulation in Lymphoid Tissues of Scrapie-affected Sheep with the AA136, QR171 Prion Protein Genotype

USDA-ARS?s Scientific Manuscript database

Background: Sheep scrapie is a transmissible spongiform encephalopathy which can be transmitted horizontally through the shedding of an infectious conformer (PrP**Sc) of the normal cellular prion protein (PrP**c). Genetics profoundly influence the susceptibility of sheep to scrapie. PrP**c amino-aci...

Covalent surface modification of prions: a mass spectrometry-based means of detecting distinctive structural features of prion strains

USDA-ARS?s Scientific Manuscript database

Prions (PrPSc) are molecular pathogens that are able to convert the isosequential normal cellular prion protein (PrPC) into a prion. The only demonstrated differences between PrPC and PrPSc is conformational, they are isoforms. A given host can be infected by more than one kind or strain of prion. F...

Novel epitopes identified by Anti-PrP monoclonal antibodies produced following immunization of Prnp0/0 Balb/cJ mice with purified scrapie prions

USDA-ARS?s Scientific Manuscript database

Prions, or infectious proteins, cause a class of uniformly fatal neurodegenerative diseases. Prions are composed solely of an aberrantly folded isoform(PrPSc)of a normal cellular protein (PrPC). Shared sequence identity of PrPSc with PrPC has limited the detection sensitivity of immunochemical assay...

Isolation of Novel Synthetic Prion Strains by Amplification in Transgenic Mice Coexpressing Wild-Type and Anchorless Prion Proteins

PubMed Central

Raymond, Gregory J.; Race, Brent; Hollister, Jason R.; Offerdahl, Danielle K.; Moore, Roger A.; Kodali, Ravindra; Raymond, Lynne D.; Hughson, Andrew G.; Rosenke, Rebecca; Long, Dan; Dorward, David W.

2012-01-01

Mammalian prions are thought to consist of misfolded aggregates (protease-resistant isoform of the prion protein [PrPres]) of the cellular prion protein (PrPC). Transmissible spongiform encephalopathy (TSE) can be induced in animals inoculated with recombinant PrP (rPrP) amyloid fibrils lacking mammalian posttranslational modifications, but this induction is inefficient in hamsters or transgenic mice overexpressing glycosylphosphatidylinositol (GPI)-anchored PrPC. Here we show that TSE can be initiated by inoculation of misfolded rPrP into mice that express wild-type (wt) levels of PrPC and that synthetic prion strain propagation and selection can be affected by GPI anchoring of the host's PrPC. To create prions de novo, we fibrillized mouse rPrP in the absence of molecular cofactors, generating fibrils with a PrPres-like protease-resistant banding profile. These fibrils induced the formation of PrPres deposits in transgenic mice coexpressing wt and GPI-anchorless PrPC (wt/GPI−) at a combined level comparable to that of PrPC expression in wt mice. Secondary passage into mice expressing wt, GPI−, or wt plus GPI− PrPC induced TSE disease with novel clinical, histopathological, and biochemical phenotypes. Contrary to laboratory-adapted mouse scrapie strains, the synthetic prion agents exhibited a preference for conversion of GPI− PrPC and, in one case, caused disease only in GPI− mice. Our data show that novel TSE agents can be generated de novo solely from purified mouse rPrP after amplification in mice coexpressing normal levels of wt and anchorless PrPC. These observations provide insight into the minimal elements required to create prions in vitro and suggest that the PrPC GPI anchor can modulate the propagation of synthetic TSE strains. PMID:22915801

PrP(C) signalling in neurons: from basics to clinical challenges.

PubMed

Hirsch, Théo Z; Hernandez-Rapp, Julia; Martin-Lannerée, Séverine; Launay, Jean-Marie; Mouillet-Richard, Sophie

2014-09-01

The cellular prion protein PrP(C) was identified over twenty-five years ago as the normal counterpart of the scrapie prion protein PrP(Sc), itself the main if not the sole component of the infectious agent at the root of Transmissible Spongiform Encephalopathies (TSEs). PrP(C) is a ubiquitous cell surface protein, abundantly expressed in neurons, which constitute the targets of PrP(Sc)-mediated toxicity. Converging evidence have highlighted that neuronal, GPI-anchored PrP(C) is absolutely required for prion-induced neuropathogenesis, which warrants investigating into the normal function exerted by PrP(C) in a neuronal context. It is now well-established that PrP(C) can serve as a cell signalling molecule, able to mobilize transduction cascades in response to interactions with partners. This function endows PrP(C) with the capacity to participate in multiple neuronal processes, ranging from survival to synaptic plasticity. A diverse array of data have allowed to shed light on how this function is corrupted by PrP(Sc). Recently, amyloid Aβ oligomers, whose accumulation is associated with Alzheimer's disease (AD), were shown to similarly instigate toxic events by deviating PrP(C)-mediated signalling. Here, we provide an overview of the various signal transduction cascades ascribed to PrP(C) in neurons, summarize how their subversion by PrP(Sc) or Aβ oligomers contributes to TSE or AD neuropathogenesis and discuss the ensuing clinical implications. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Prion replication without host adaptation during interspecies transmissions

PubMed Central

Bian, Jifeng; Khaychuk, Vadim; Angers, Rachel C.; Fernández-Borges, Natalia; Meyerett-Reid, Crystal; Kim, Sehun; Calvi, Carla L.; Bartz, Jason C.; Hoover, Edward A.; Agrimi, Umberto; Richt, Jürgen A.; Castilla, Joaquín; Telling, Glenn C.

2017-01-01

Adaptation of prions to new species is thought to reflect the capacity of the host-encoded cellular form of the prion protein (PrPC) to selectively propagate optimized prion conformations from larger ensembles generated in the species of origin. Here we describe an alternate replicative process, termed nonadaptive prion amplification (NAPA), in which dominant conformers bypass this requirement during particular interspecies transmissions. To model susceptibility of horses to prions, we produced transgenic (Tg) mice expressing cognate PrPC. Although disease transmission to only a subset of infected TgEq indicated a significant barrier to EqPrPC conversion, the resulting horse prions unexpectedly failed to cause disease upon further passage to TgEq. TgD expressing deer PrPC was similarly refractory to deer prions from diseased TgD infected with mink prions. In both cases, the resulting prions transmitted to mice expressing PrPC from the species of prion origin, demonstrating that transmission barrier eradication of the originating prions was ephemeral and adaptation superficial in TgEq and TgD. Horse prions produced in vitro by protein misfolding cyclic amplification of mouse prions using horse PrPC also failed to infect TgEq but retained tropism for wild-type mice. Concordant patterns of neuropathology and prion deposition in susceptible mice infected with NAPA prions and the corresponding prion of origin confirmed preservation of strain properties. The comparable responses of both prion types to guanidine hydrochloride denaturation indicated this occurs because NAPA precludes selection of novel prion conformations. Our findings provide insights into mechanisms regulating interspecies prion transmission and a framework to reconcile puzzling epidemiological features of certain prion disorders. PMID:28096357

Hematological shift in goat kids naturally devoid of prion protein.

PubMed

Reiten, Malin R; Bakkebø, Maren K; Brun-Hansen, Hege; Lewandowska-Sabat, Anna M; Olsaker, Ingrid; Tranulis, Michael A; Espenes, Arild; Boysen, Preben

2015-01-01

The physiological role of the cellular prion protein (PrP(C)) is incompletely understood. The expression of PrP(C) in hematopoietic stem cells and immune cells suggests a role in the development of these cells, and in PrP(C) knockout animals altered immune cell proliferation and phagocytic function have been observed. Recently, a spontaneous nonsense mutation at codon 32 in the PRNP gene in goats of the Norwegian Dairy breed was discovered, rendering homozygous animals devoid of PrP(C). Here we report hematological and immunological analyses of homozygous goat kids lacking PrP(C) (PRNP(Ter/Ter) ) compared to heterozygous (PRNP (+/Ter)) and normal (PRNP (+/+)) kids. Levels of cell surface PrP(C) and PRNP mRNA in peripheral blood mononuclear cells (PBMCs) correlated well and were very low in PRNP (Ter/Ter), intermediate in PRNP (+/Ter) and high in PRNP (+/+) kids. The PRNP (Ter/Ter) animals had a shift in blood cell composition with an elevated number of red blood cells (RBCs) and a tendency toward a smaller mean RBC volume (P = 0.08) and an increased number of neutrophils (P = 0.068), all values within the reference ranges. Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities. Studies of relative composition of PBMCs, phagocytic ability of monocytes and T-cell proliferation revealed no significant differences between the genotypes. Our data suggest that PrP(C) has a role in bone marrow physiology and warrant further studies of PrP(C) in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions. Altogether, this genetically unmanipulated PrP(C)-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrP(C).

Proteasome inhibitors promote the sequestration of PrPSc into aggresomes within the cytosol of prion-infected CAD neuronal cells.

PubMed

Dron, Michel; Dandoy-Dron, Françoise; Farooq Salamat, Muhammad Khalid; Laude, Hubert

2009-08-01

Dysfunction of the endoplasmic reticulum associated protein degradation/proteasome system is believed to contribute to the initiation or aggravation of neurodegenerative disorders associated with protein misfolding, and there is some evidence to suggest that proteasome dysfunctions might be implicated in prion disease. This study investigated the effect of proteasome inhibitors on the biogenesis of both the cellular (PrP(C)) and abnormal (PrP(Sc)) forms of prion protein in CAD neuronal cells, a newly introduced prion cell system. In uninfected cells, proteasome impairment altered the intracellular distribution of PrP(C), leading to a strong accumulation in the Golgi apparatus. Moreover, a detergent-insoluble and weakly protease-resistant PrP species of 26 kDa, termed PrP(26K), accumulated in the cells, whether they were prion-infected or not. However, no evidence was found that, in infected cells, this PrP(26K) species converts into the highly proteinase K-resistant PrP(Sc). In the infected cultures, proteasome inhibition caused an increased intracellular aggregation of PrP(Sc) that was deposited into large aggresomes. These findings strengthen the view that, in neuronal cells expressing wild-type PrP(C) from the natural promoter, proteasomal impairment may affect both the process of PrP(C) biosynthesis and the subcellular sites of PrP(Sc) accumulation, despite the fact that these two effects could essentially be disconnected.

Interactome analyses identify ties of PrP and its mammalian paralogs to oligomannosidic N-glycans and endoplasmic reticulum-derived chaperones.

PubMed

Watts, Joel C; Huo, Hairu; Bai, Yu; Ehsani, Sepehr; Jeon, Amy Hye Won; Won, Amy Hye; Shi, Tujin; Daude, Nathalie; Lau, Agnes; Young, Rebecca; Xu, Lei; Carlson, George A; Williams, David; Westaway, David; Schmitt-Ulms, Gerold

2009-10-01

The physiological environment which hosts the conformational conversion of the cellular prion protein (PrP(C)) to disease-associated isoforms has remained enigmatic. A quantitative investigation of the PrP(C) interactome was conducted in a cell culture model permissive to prion replication. To facilitate recognition of relevant interactors, the study was extended to Doppel (Prnd) and Shadoo (Sprn), two mammalian PrP(C) paralogs. Interestingly, this work not only established a similar physiological environment for the three prion protein family members in neuroblastoma cells, but also suggested direct interactions amongst them. Furthermore, multiple interactions between PrP(C) and the neural cell adhesion molecule, the laminin receptor precursor, Na/K ATPases and protein disulfide isomerases (PDI) were confirmed, thereby reconciling previously separate findings. Subsequent validation experiments established that interactions of PrP(C) with PDIs may extend beyond the endoplasmic reticulum and may play a hitherto unrecognized role in the accumulation of PrP(Sc). A simple hypothesis is presented which accounts for the majority of interactions observed in uninfected cells and suggests that PrP(C) organizes its molecular environment on account of its ability to bind to adhesion molecules harboring immunoglobulin-like domains, which in turn recognize oligomannose-bearing membrane proteins.

Cholesterol Balance in Prion Diseases and Alzheimer’s Disease

PubMed Central

Hannaoui, Samia; Shim, Su Yeon; Cheng, Yo Ching; Corda, Erica; Gilch, Sabine

2014-01-01

Prion diseases are transmissible and fatal neurodegenerative disorders of humans and animals. They are characterized by the accumulation of PrPSc, an aberrantly folded isoform of the cellular prion protein PrPC, in the brains of affected individuals. PrPC is a cell surface glycoprotein attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidyl-inositol (GPI) anchor. Specifically, it is associated with lipid rafts, membrane microdomains enriched in cholesterol and sphinoglipids. It has been established that inhibition of endogenous cholesterol synthesis disturbs lipid raft association of PrPC and prevents PrPSc accumulation in neuronal cells. Additionally, prion conversion is reduced upon interference with cellular cholesterol uptake, endosomal export, or complexation at the plasma membrane. Altogether, these results demonstrate on the one hand the importance of cholesterol for prion propagation. On the other hand, growing evidence suggests that prion infection modulates neuronal cholesterol metabolism. Similar results were reported in Alzheimer’s disease (AD): whereas amyloid β peptide formation is influenced by cellular cholesterol, levels of cholesterol in the brains of affected individuals increase during the clinical course of the disease. In this review, we summarize commonalities of alterations in cholesterol homeostasis and discuss consequences for neuronal function and therapy of prion diseases and AD. PMID:25419621

Human FDC express PrPc in vivo and in vitro

PubMed Central

Thielen, Caroline; Antoine, Nadine; Mélot, France; Cesbron, Jean-Yves; Heinen, Ernst

2001-01-01

Prion diseases are fatal neurodegenerative disorders caused by accumulation of abnormal prion protein (protease-resistant prion, PrPres). PrPres accumulation is also detected in lymphoid organs after peripheral infection. Several studies suggest that follicular dendritic cells (FDC) could be the site of PrPres retention and amplification. Here we show that human follicular dendritic cells can express normal cellular prion protein (PrPc) both in situ and in vitro. When tonsillar cryosections were treated with anti-PrP antibody, the label was found on some very delicate cell extensions inside the lymphoid follicles, especially in the germinal centres. These extensions react with DRC1 antibody, used frequently to label FDC. Other structures labelled with anti-PrP antibody were the keratinocytes. To confirm the ability of FDC to synthesise PrPc, we isolated FDC by a non-enzymatic procedure and cultured them. By cytochemistry and flow cytometry it was clearly shown that FDC do produce PrPc. PMID:11785675

The Prion Protein Modulates A-type K+ Currents Mediated by Kv4.2 Complexes through Dipeptidyl Aminopeptidase-like Protein 6*

PubMed Central

Mercer, Robert C. C.; Ma, Li; Watts, Joel C.; Strome, Robert; Wohlgemuth, Serene; Yang, Jing; Cashman, Neil R.; Coulthart, Michael B.; Schmitt-Ulms, Gerold; Jhamandas, Jack H.; Westaway, David

2013-01-01

Widely expressed in the adult central nervous system, the cellular prion protein (PrPC) is implicated in a variety of processes, including neuronal excitability. Dipeptidyl aminopeptidase-like protein 6 (DPP6) was first identified as a PrPC interactor using in vivo formaldehyde cross-linking of wild type (WT) mouse brain. This finding was confirmed in three cell lines and, because DPP6 directs the functional assembly of K+ channels, we assessed the impact of WT and mutant PrPC upon Kv4.2-based cell surface macromolecular complexes. Whereas a Gerstmann-Sträussler-Scheinker disease version of PrP with eight extra octarepeats was a loss of function both for complex formation and for modulation of Kv4.2 channels, WT PrPC, in a DPP6-dependent manner, modulated Kv4.2 channel properties, causing an increase in peak amplitude, a rightward shift of the voltage-dependent steady-state inactivation curve, a slower inactivation, and a faster recovery from steady-state inactivation. Thus, the net impact of wt PrPC was one of enhancement, which plays a critical role in the down-regulation of neuronal membrane excitability and is associated with a decreased susceptibility to seizures. Insofar as previous work has established a requirement for WT PrPC in the Aβ-dependent modulation of excitability in cholinergic basal forebrain neurons, our findings implicate PrPC regulation of Kv4.2 channels as a mechanism contributing to the effects of oligomeric Aβ upon neuronal excitability and viability. PMID:24225951

Cell-surface prion protein interacts with glycosaminoglycans.

PubMed Central

Pan, Tao; Wong, Boon-Seng; Liu, Tong; Li, Ruliang; Petersen, Robert B; Sy, Man-Sun

2002-01-01

We used ELISA and flow cytometry to study the binding of prion protein PrP to glycosaminoglycans (GAGs). We found that recombinant human PrP (rPrP) binds GAGs including chondroitin sulphate A, chondroitin sulphate B, hyaluronic acid, and heparin. rPrP binding to GAGs occurs via the N-terminus, a region known to bind divalent cations. Additionally, rPrP binding to GAGs is enhanced in the presence of Cu2+ and Zn2+, but not Ca2+ and Mn2+. rPrP binds heparin strongest, and the binding is inhibited by certain heparin analogues, including heparin disaccharide and sulphate-containing monosaccharides, but not by acetylated heparin. Full-length normal cellular prion protein (PrPC), but not N-terminally truncated PrPC species, from human brain bind GAGs in a similar Cu2+/Zn2+-enhanced fashion. We found that GAGs specifically bind to a synthetic peptide corresponding to amino acid residues 23-35 in the N-terminus of rPrP. We further demonstrated that while both wild-type PrPC and an octapeptide-repeat-deleted mutant PrP produced by transfected cells bound heparin at the cell surface, the PrP N-terminal deletion mutant and non-transfectant control failed to bind heparin. Binding of heparin to wild-type PrPC on the cell surface results in a reduction of the level of cell-surface PrPC. These results provide strong evidence that PrPC is a surface receptor for GAGs. PMID:12186633

Disruption of prion protein-HOP engagement impairs glioblastoma growth and cognitive decline and improves overall survival.

PubMed

Lopes, M H; Santos, T G; Rodrigues, B R; Queiroz-Hazarbassanov, N; Cunha, I W; Wasilewska-Sampaio, A P; Costa-Silva, B; Marchi, F A; Bleggi-Torres, L F; Sanematsu, P I; Suzuki, S H; Oba-Shinjo, S M; Marie, S K N; Toulmin, E; Hill, A F; Martins, V R

2015-06-01

Glioblastomas (GBMs) are resistant to current therapy protocols and identification of molecules that target these tumors is crucial. Interaction of secreted heat-shock protein 70 (Hsp70)-Hsp90-organizing protein (HOP) with cellular prion protein (PrP(C)) triggers a large number of trophic effects in the nervous system. We found that both PrP(C) and HOP are highly expressed in human GBM samples relative to non-tumoral tissue or astrocytoma grades I-III. High levels of PrP(C) and HOP were associated with greater GBM proliferation and lower patient survival. HOP-PrP(C) binding increased GBM proliferation in vitro via phosphatidylinositide 3-kinase and extracellular-signal-regulated kinase pathways, and a HOP peptide mimicking the PrP(C) binding site (HOP230-245) abrogates this effect. PrP(C) knockdown impaired tumor growth and increased survival of mice with tumors. In mice, intratumor delivery of HOP230-245 peptide impaired proliferation and promoted apoptosis of GBM cells. In addition, treatment with HOP230-245 peptide inhibited tumor growth, maintained cognitive performance and improved survival. Thus, together, the present results indicate that interfering with PrP(C)-HOP engagement is a promising approach for GBM therapy.

Primary Blast-Induced Traumatic Brain Injury in Rats Leads to Increased Prion Protein in Plasma: A Potential Biomarker for Blast-Induced Traumatic Brain Injury

PubMed Central

Pham, Nam; Sawyer, Thomas W.; Wang, Yushan; Jazii, Ferdous Rastgar; Vair, Cory

2015-01-01

Abstract Traumatic brain injury (TBI) is deemed the “signature injury” of recent military conflicts in Afghanistan and Iraq, largely because of increased blast exposure. Injuries to the brain can often be misdiagnosed, leading to further complications in the future. Therefore, the use of protein biomarkers for the screening and diagnosis of TBI is urgently needed. In the present study, we have investigated the plasma levels of soluble cellular prion protein (PrPC) as a novel biomarker for the diagnosis of primary blast-induced TBI (bTBI). We hypothesize that the primary blast wave can disrupt the brain and dislodge extracellular localized PrPC, leading to a rise in concentration within the systemic circulation. Adult male Sprague–Dawley rats were exposed to single pulse shockwave overpressures of varying intensities (15-30 psi or 103.4–206.8 kPa] using an advanced blast simulator. Blood plasma was collected 24 h after insult, and PrPC concentration was determined with a modified commercial enzyme-linked immunosorbent assay (ELISA) specific for PrPC. We provide the first report that mean PrPC concentration in primary blast exposed rats (3.97 ng/mL±0.13 SE) is significantly increased compared with controls (2.46 ng/mL±0.14 SE; two tailed test p<0.0001). Furthermore, we report a mild positive rank correlation between PrPC concentration and increasing blast intensity (psi) reflecting a plateaued response at higher pressure magnitudes, which may have implications for all military service members exposed to blast events. In conclusion, it appears that plasma levels of PrPC may be a novel biomarker for the detection of primary bTBI. PMID:25058115

Normal Cellular Prion Protein Protects against Manganese-induced Oxidative Stress and Apoptotic Cell Death

PubMed Central

Choi, Christopher J.; Anantharam, Vellareddy; Saetveit, Nathan J.; Houk, Robert. S.; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

2012-01-01

The normal prion protein is abundantly expressed in the CNS, but its biological function remains unclear. The prion protein has octapeptide repeat regions that bind to several divalent metals, suggesting that the prion proteins may alter the toxic effect of environmental neurotoxic metals. In the present study, we systematically examined whether prion protein modifies the neurotoxicity of manganese (Mn) by comparing the effect of Mn on mouse neural cells expressing prion protein (PrPC -cells) and prion-knockout (PrPKO -cells). Exposure to Mn (10 μM-1 mM) for 24 hr produced a dose-dependent cytotoxic response in both PrPC -cells and PrPKO -cells. Interestingly, PrPC -cells (EC50 117.6μM) were more resistant to Mn-induced cytotoxicity, as compared to PrPKO -cells (EC50 59.9μM), suggesting a protective role for PrPC against Mn neurotoxicity. Analysis of intracellular Mn levels showed less Mn accumulation in PrPC -cells as compared to PrPKO -cells. Furthermore, Mn-induced mitochondrial depolarization and ROS generation were significantly attenuated in PrPC -cells as compared to PrPKO -cells. Measurement of antioxidant status revealed similar basal levels of glutathione (GSH) in PrPC -cells and PrPKO -cells; however, Mn treatment caused greater depletion of GSH in PrPKO -cells. Mn-induced mitochondrial depolarization and ROS production were followed by time- and dose-dependent activation of the apoptotic cell death cascade involving caspase-9 and -3. Notably, DNA fragmentation induced by both Mn treatment and oxidative stress-inducer hydrogen peroxide (100μM) was significantly suppressed in PrPC -cells as compared to PrPKO -cells. Together, these results demonstrate that prion protein interferes with divalent metal Mn uptake and protects against Mn-induced oxidative stress and apoptotic cell death. PMID:17483122

Recombinant human prion protein inhibits prion propagation in vitro.

PubMed

Yuan, Jue; Zhan, Yi-An; Abskharon, Romany; Xiao, Xiangzhu; Martinez, Manuel Camacho; Zhou, Xiaochen; Kneale, Geoff; Mikol, Jacqueline; Lehmann, Sylvain; Surewicz, Witold K; Castilla, Joaquín; Steyaert, Jan; Zhang, Shulin; Kong, Qingzhong; Petersen, Robert B; Wohlkonig, Alexandre; Zou, Wen-Quan

2013-10-09

Prion diseases are associated with the conformational conversion of the cellular prion protein (PrP(C)) into the pathological scrapie isoform (PrP(Sc)) in the brain. Both the in vivo and in vitro conversion of PrP(C) into PrP(Sc) is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrP(Sc), but not PrP(C), suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrP(C) with PrP(Sc). Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrP(Sc) propagation without inducing immune response side effects.

Correlation of cellular factors and differential scrapie prion permissiveness in ovine microglia

USDA-ARS?s Scientific Manuscript database

Prion diseases are fatal neurodegenerative disorders by which the native cellular prion protein (PrP-C) is misfolded into an accumulating, disease-associated isoform (PrP-D). To improve the understanding of prion pathogenesis and develop effective treatments, it is essential to elucidate factors con...

Enhanced susceptibility of Prnp-deficient mice to kainate-induced seizures, neuronal apoptosis, and death: Role of AMPA/kainate receptors.

PubMed

Rangel, Alejandra; Burgaya, Ferran; Gavín, Rosalina; Soriano, Eduardo; Aguzzi, Adriano; Del Río, José A

2007-09-01

Normal physiologic functions of the cellular prion protein (PrPc) are still elusive. This GPI-anchored protein exerts many functions, including roles in neuron proliferation, neuroprotection or redox homeostasis. There are, however, conflicting data concerning its role in synaptic transmission. Although several studies report that PrPc participates in NMDA-mediated neurotransmission, parallel studies describe normal behavior of PrPc-mutant mice. Abnormal axon connections have been described in the dentate gyrus of the hippocampi of PrPc-deficient mice similar to those observed in epilepsy. A study indicates increased susceptibility to kainate (KA) in these mutant mice. We extend the observation of these studies by means of several histologic and biochemical analyses of KA-treated mice. PrPc-deficient mice showed increased sensitivity to KA-induced seizures in vivo and in vitro in organotypic slices. In addition, we show that this sensitivity is cell-specific because interference experiments to abolish PrPc expression increased susceptibility to KA in PrPc-expressing cells. We indicate a correlation of susceptibility to KA in cells lacking PrPc with the differential expression of GluR6 and GluR7 KA receptor subunits using real-time RT-PCR methods. These results indicate that PrPc exerts a neuroprotective role against KA-induced neurotoxicity, probably by regulating the expression of KA receptor subunits. (c) 2007 Wiley-Liss, Inc.

PrPC expression and prion seeding activity in the alimentary tract and lymphoid tissue of deer

PubMed Central

Davenport, Kristen A.; Hoover, Clare E.; Bian, Jifeng; Telling, Glenn C.; Mathiason, Candace K.; Hoover, Edward A.

2017-01-01

The agent responsible for prion diseases is a misfolded form of a normal protein (PrPC). The prion hypothesis stipulates that PrPC must be present for the disease to manifest. Cervid populations across the world are infected with chronic wasting disease, a horizontally-transmissible prion disease that is likely spread via oral exposure to infectious prions (PrPCWD). Though PrPCWD has been identified in many tissues, there has been little effort to characterize the overall PrPC expression in cervids and its relationship to PrPCWD accumulation. We used immunohistochemistry (IHC), western blot and enzyme-linked immunosorbent assay to describe PrPC expression in naïve white-tailed deer. We used real-time, quaking-induced conversion (RT-QuIC) to detect prion seeding activity in CWD-infected deer. We assessed tissues comprising the alimentary tract, alimentary-associated lymphoid tissue and systemic lymphoid tissue from 5 naïve deer. PrPC was expressed in all tissues, though expression was often very low compared to the level in the CNS. IHC identified specific cell types wherein PrPC expression is very high. To compare the distribution of PrPC to PrPCWD, we examined 5 deer with advanced CWD infection. Using RT-QuIC, we detected prion seeding activity in all 21 tissues. In 3 subclinical deer sacrificed 4 months post-inoculation, we detected PrPCWD consistently in alimentary-associated lymphoid tissue, irregularly in alimentary tract tissues, and not at all in the brain. Contrary to our hypothesis that PrPC levels dictate prion accumulation, PrPC expression was higher in the lower gastrointestinal tissues than in the alimentary-associated lymphoid system and was higher in salivary glands than in the oropharyngeal lymphoid tissue. These data suggest that PrPC expression is not the sole driver of prion accumulation and that alimentary tract tissues accumulate prions before centrifugal spread from the brain occurs. PMID:28880938

The CNS glycoprotein Shadoo has PrPC-like protective properties and displays reduced levels in prion infections

PubMed Central

Watts, Joel C; Drisaldi, Bettina; Ng, Vivian; Yang, Jing; Strome, Bob; Horne, Patrick; Sy, Man-Sun; Yoong, Larry; Young, Rebecca; Mastrangelo, Peter; Bergeron, Catherine; Fraser, Paul E; Carlson, George A; Mount, Howard T J; Schmitt-Ulms, Gerold; Westaway, David

2007-01-01

The cellular prion protein, PrPC, is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP (‘ΔPrP'). This paradigm has facilitated mapping of activity determinants in PrPC and implicated a cryptic PrPC-like protein, ‘π'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrPC, but is low in cerebellar granular neurons (CGNs) containing PrPC and high in PrPC-deficient dendritic processes. In Prnp0/0 CGNs, Sho transgenes were PrPC-like in their ability to counteract neurotoxic effects of either Doppel or ΔPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for π, and since it engenders a PrPC-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology. PMID:17703189

Prion Protein-mediated Toxicity of Amyloid-β Oligomers Requires Lipid Rafts and the Transmembrane LRP1*

PubMed Central

Rushworth, Jo V.; Griffiths, Heledd H.; Watt, Nicole T.; Hooper, Nigel M.

2013-01-01

Soluble oligomers of the amyloid-β (Aβ) peptide cause neurotoxicity, synaptic dysfunction, and memory impairments that underlie Alzheimer disease (AD). The cellular prion protein (PrPC) was recently identified as a high affinity neuronal receptor for Aβ oligomers. We report that fibrillar Aβ oligomers recognized by the OC antibody, which have been shown to correlate with the onset and severity of AD, bind preferentially to cells and neurons expressing PrPC. The binding of Aβ oligomers to cell surface PrPC, as well as their downstream activation of Fyn kinase, was dependent on the integrity of cholesterol-rich lipid rafts. In SH-SY5Y cells, fluorescence microscopy and co-localization with subcellular markers revealed that the Aβ oligomers co-internalized with PrPC, accumulated in endosomes, and subsequently trafficked to lysosomes. The cell surface binding, internalization, and downstream toxicity of Aβ oligomers was dependent on the transmembrane low density lipoprotein receptor-related protein-1 (LRP1). The binding of Aβ oligomers to cell surface PrPC impaired its ability to inhibit the activity of the β-secretase BACE1, which cleaves the amyloid precursor protein to produce Aβ. The green tea polyphenol (−)-epigallocatechin gallate and the red wine extract resveratrol both remodeled the fibrillar conformation of Aβ oligomers. The resulting nonfibrillar oligomers displayed significantly reduced binding to PrPC-expressing cells and were no longer cytotoxic. These data indicate that soluble, fibrillar Aβ oligomers bind to PrPC in a conformation-dependent manner and require the integrity of lipid rafts and the transmembrane LRP1 for their cytotoxicity, thus revealing potential targets to alleviate the neurotoxic properties of Aβ oligomers in AD. PMID:23386614

Brain immune interactions and air pollution: macrophage inhibitory factor (MIF), prion cellular protein (PrP(C)), Interleukin-6 (IL-6), interleukin 1 receptor antagonist (IL-1Ra), and interleukin-2 (IL-2) in cerebrospinal fluid and MIF in serum differentiate urban children exposed to severe vs. low air pollution.

PubMed

Calderón-Garcidueñas, Lilian; Cross, Janet V; Franco-Lira, Maricela; Aragón-Flores, Mariana; Kavanaugh, Michael; Torres-Jardón, Ricardo; Chao, Chih-Kai; Thompson, Charles; Chang, Jing; Zhu, Hongtu; D'Angiulli, Amedeo

2013-01-01

Mexico City Metropolitan Area children chronically exposed to high concentrations of air pollutants exhibit an early brain imbalance in genes involved in oxidative stress, inflammation, innate and adaptive immune responses along with accumulation of misfolded proteins observed in the early stages of Alzheimer and Parkinson's diseases. A complex modulation of serum cytokines and chemokines influences children's brain structural and gray/white matter volumetric responses to air pollution. The search for biomarkers associating systemic and CNS inflammation to brain growth and cognitive deficits in the short term and neurodegeneration in the long-term is our principal aim. We explored and compared a profile of cytokines, chemokines (Multiplexing LASER Bead Technology) and Cellular prion protein (PrP(C)) in normal cerebro-spinal-fluid (CSF) of urban children with high vs. low air pollution exposures. PrP(C) and macrophage inhibitory factor (MIF) were also measured in serum. Samples from 139 children ages 11.91 ± 4.2 years were measured. Highly exposed children exhibited significant increases in CSF MIF (p = 0.002), IL6 (p = 0.006), IL1ra (p = 0.014), IL-2 (p = 0.04), and PrP(C) (p = 0.039) vs. controls. MIF serum concentrations were higher in exposed children (p = 0.009). Our results suggest CSF as a MIF, IL6, IL1Ra, IL-2, and PrP(C) compartment that can possibly differentiate air pollution exposures in children. MIF, a key neuro-immune mediator, is a potential biomarker bridge to identify children with CNS inflammation. Fine tuning of immune-to-brain communication is crucial to neural networks appropriate functioning, thus the short and long term effects of systemic inflammation and dysregulated neural immune responses are of deep concern for millions of exposed children. Defining the linkage and the health consequences of the brain / immune system interactions in the developing brain chronically exposed to air pollutants ought to be of pressing importance for public health.

Cryo-immunogold electron microscopy for prions: toward identification of a conversion site.

PubMed

Godsave, Susan F; Wille, Holger; Kujala, Pekka; Latawiec, Diane; DeArmond, Stephen J; Serban, Ana; Prusiner, Stanley B; Peters, Peter J

2008-11-19

Prion diseases are caused by accumulation of an abnormally folded isoform (PrP(Sc)) of the cellular prion protein (PrP(C)). The subcellular distribution of PrP(Sc) and the site of its formation in brain are still unclear. We performed quantitative cryo-immunogold electron microscopy on hippocampal sections from mice infected with the Rocky Mountain Laboratory strain of prions. Two antibodies were used: R2, which recognizes both PrP(C) and PrP(Sc); and F4-31, which only detects PrP(C) in undenatured sections. At a late subclinical stage of prion infection, both PrP(C) and PrP(Sc) were detected principally on neuronal plasma membranes and on vesicles resembling early endocytic or recycling vesicles in the neuropil. The R2 labeling was approximately six times higher in the infected than the uninfected hippocampus and gold clusters were only evident in infected tissue. The biggest increase in labeling density (24-fold) was found on the early/recycling endosome-like vesicles of small-diameter neurites, suggesting these as possible sites of conversion. Trypsin digestion of infected hippocampal sections resulted in a reduction in R2 labeling of >85%, which suggests that a high proportion of PrP(Sc) may be oligomeric, protease-sensitive PrP(Sc).

Regulation of GABAA and Glutamate Receptor Expression, Synaptic Facilitation and Long-Term Potentiation in the Hippocampus of Prion Mutant Mice

PubMed Central

Rangel, Alejandra; Madroñal, Noelia; Massó, Agnès Gruart i.; Gavín, Rosalina; Llorens, Franc; Sumoy, Lauro; Torres, Juan María; Delgado-García, José María; Río, José Antonio Del

2009-01-01

Background Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrPsc) of the natural cellular prion protein (PrPc) encoded by the Prnp gene. Although several roles have been attributed to PrPc, its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrPc studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. Methodology/Principal Findings Here we explore the role of PrPc expression in neurotransmission and neural excitability using wild-type, Prnp −/− and PrPc-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp −/− mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina™ microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp −/− and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABAA and AMPA-kainate receptors are co-regulated in both Prnp −/− and Tg20 mice. Conclusions/Significance Present results demonstrate that PrPc is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABAA and AMPA-Kainate neurotransmission. New PrPc functions have recently been described, which point to PrPc as a target for putative therapies in Alzheimer's disease. However, our results indicate that a “gain of function” strategy in Alzheimer's disease, or a “loss of function” in prionopathies, may impair PrPc function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies. PMID:19855845

Prion Protein Promotes Kidney Iron Uptake via Its Ferrireductase Activity*

PubMed Central

Haldar, Swati; Tripathi, Ajai; Qian, Juan; Beserra, Amber; Suda, Srinivas; McElwee, Matthew; Turner, Jerrold; Hopfer, Ulrich; Singh, Neena

2015-01-01

Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrPC) from its normal conformation to an aggregated, PrP-scrapie (PrPSc) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrPC in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrPC is lacking. Kidney provides a relevant model for this evaluation because PrPC is expressed in the kidney, and ∼370 μg of iron are reabsorbed daily from the glomerular filtrate by kidney proximal tubule cells (PT), requiring ferrireductase activity. Here, we report that PrPC promotes the uptake of transferrin (Tf) and non-Tf-bound iron (NTBI) by the kidney in vivo and mainly NTBI by PT cells in vitro. Thus, uptake of 59Fe administered by gastric gavage, intravenously, or intraperitoneally was significantly lower in PrP-knock-out (PrP−/−) mouse kidney relative to PrP+/+ controls. Selective in vivo radiolabeling of plasma NTBI with 59Fe revealed similar results. Expression of exogenous PrPC in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of 59Fe-NTBI and to a smaller extent 59Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrPΔ51–89) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrPC to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrPC promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism. PMID:25572394

Estimating chronic wasting disease susceptibility in cervids using real-time quaking-induced conversion

PubMed Central

Haley, Nicholas J.; Rielinger, Rachel; Davenport, Kristen A.; O'Rourke, Katherine; Mitchell, Gordon; Richt, Jürgen A.

2017-01-01

In mammals, susceptibility to prion infection is primarily modulated by the host’s cellular prion protein (PrPC) sequence. In the sheep scrapie model, a graded scale of susceptibility has been established both in vivo and in vitro based on PrPC amino acids 136, 154 and 171, leading to global breeding programmes to reduce the prevalence of scrapie in sheep. Chronic wasting disease (CWD) resistance in cervids is often characterized as decreased prevalence and/or protracted disease progression in individuals with specific alleles; at present, no PrPC allele conferring absolute resistance in cervids has been identified. To model the susceptibility of various naturally occurring and hypothetical cervid PrPC alleles in vitro, we compared the amplification rates and amyloid extension efficiencies of eight distinct CWD isolates in recombinant cervid PrPC substrates using real-time quaking-induced conversion. We hypothesized that the in vitro conversion characteristics of these isolates in cervid substrates would correlate to in vivo susceptibility – permitting susceptibility prediction for the rare alleles found in nature. We also predicted that hypothetical alleles with multiple resistance-associated codons would be more resistant to in vitro conversion than natural alleles with a single resistant codon. Our studies demonstrate that in vitro conversion metrics align with in vivo susceptibility, and that alleles with multiple amino acid substitutions, each influencing resistance independently, do not necessarily contribute additively to conversion resistance. Importantly, we found that the naturally occurring whitetail deer QGAK substrate exhibited the slowest amplification rate among those evaluated, suggesting that further investigation of this allele and its resistance in vivo is warranted. PMID:29058651

Differential effects of divalent cations on elk prion protein fibril formation and stability

USDA-ARS?s Scientific Manuscript database

Misfolding of the normally folded prion protein of mammals (PrPC) into infectious fibrils causes a variety of different diseases, from scrapie in sheep to bovine spongiform encephalopathy in cattle to chronic wasting disease (CWD) in deer and elk. The misfolded form of PrPC, termed PrPSc, or in this...

Octarepeat region flexibility impacts prion function, endoproteolysis and disease manifestation

PubMed Central

Lau, Agnes; McDonald, Alex; Daude, Nathalie; Mays, Charles E; Walter, Eric D; Aglietti, Robin; Mercer, Robert CC; Wohlgemuth, Serene; van der Merwe, Jacques; Yang, Jing; Gapeshina, Hristina; Kim, Chae; Grams, Jennifer; Shi, Beipei; Wille, Holger; Balachandran, Aru; Schmitt-Ulms, Gerold; Safar, Jiri G; Millhauser, Glenn L; Westaway, David

2015-01-01

The cellular prion protein (PrPC) comprises a natively unstructured N-terminal domain, including a metal-binding octarepeat region (OR) and a linker, followed by a C-terminal domain that misfolds to form PrPSc in Creutzfeldt-Jakob disease. PrPC β-endoproteolysis to the C2 fragment allows PrPSc formation, while α-endoproteolysis blocks production. To examine the OR, we used structure-directed design to make novel alleles, ‘S1’ and ‘S3’, locking this region in extended or compact conformations, respectively. S1 and S3 PrP resembled WT PrP in supporting peripheral nerve myelination. Prion-infected S1 and S3 transgenic mice both accumulated similar low levels of PrPSc and infectious prion particles, but differed in their clinical presentation. Unexpectedly, S3 PrP overproduced C2 fragment in the brain by a mechanism distinct from metal-catalysed hydrolysis reported previously. OR flexibility is concluded to impact diverse biological endpoints; it is a salient variable in infectious disease paradigms and modulates how the levels of PrPSc and infectivity can either uncouple or engage to drive the onset of clinical disease. PMID:25661904

Plasma soluble prion protein, a potential biomarker for sport-related concussions: a pilot study.

PubMed

Pham, Nam; Akonasu, Hungbo; Shishkin, Rhonda; Taghibiglou, Changiz

2015-01-01

Sport-related mild traumatic brain injury (mTBI) or concussion is a significant health concern to athletes with potential long-term consequences. The diagnosis of sport concussion and return to sport decision making is one of the greatest challenges facing health care clinicians working in sports. Blood biomarkers have recently demonstrated their potential in assisting the detection of brain injury particularly, in those cases with no obvious physical injury. We have recently discovered plasma soluble cellular prion protein (PrP(C)) as a potential reliable biomarker for blast induced TBI (bTBI) in a rodent animal model. In order to explore the application of this novel TBI biomarker to sport-related concussion, we conducted a pilot study at the University of Saskatchewan (U of S) by recruiting athlete and non-athlete 18 to 30 year-old students. Using a modified quantitative ELISA method, we first established normal values for the plasma soluble PrP(C) in male and female students. The measured plasma soluble PrP(C) in confirmed concussion cases demonstrated a significant elevation of this analyte in post-concussion samples. Data collected from our pilot study indicates that the plasma soluble PrP(C) is a potential biomarker for sport-related concussion, which may be further developed into a clinical diagnostic tool to assist clinicians in the assessment of sport concussion and return-to-play decision making.

Molecular architecture of human prion protein amyloid: a parallel, in-register beta-structure.

PubMed

Cobb, Nathan J; Sönnichsen, Frank D; McHaourab, Hassane; Surewicz, Witold K

2007-11-27

Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative diseases that are associated with conformational conversion of the normally monomeric and alpha-helical prion protein, PrP(C), to the beta-sheet-rich PrP(Sc). This latter conformer is believed to constitute the main component of the infectious TSE agent. In contrast to high-resolution data for the PrP(C) monomer, structures of the pathogenic PrP(Sc) or synthetic PrP(Sc)-like aggregates remain elusive. Here we have used site-directed spin labeling and EPR spectroscopy to probe the molecular architecture of the recombinant PrP amyloid, a misfolded form recently reported to induce transmissible disease in mice overexpressing an N-terminally truncated form of PrP(C). Our data show that, in contrast to earlier, largely theoretical models, the con formational conversion of PrP(C) involves major refolding of the C-terminal alpha-helical region. The core of the amyloid maps to C-terminal residues from approximately 160-220, and these residues form single-molecule layers that stack on top of one another with parallel, in-register alignment of beta-strands. This structural insight has important implications for understanding the molecular basis of prion propagation, as well as hereditary prion diseases, most of which are associated with point mutations in the region found to undergo a refolding to beta-structure.

Protease resistance of infectious prions is suppressed by removal of a single atom in the cellular prion protein.

PubMed

Leske, Henning; Hornemann, Simone; Herrmann, Uli Simon; Zhu, Caihong; Dametto, Paolo; Li, Bei; Laferriere, Florent; Polymenidou, Magdalini; Pelczar, Pawel; Reimann, Regina Rose; Schwarz, Petra; Rushing, Elisabeth Jane; Wüthrich, Kurt; Aguzzi, Adriano

2017-01-01

Resistance to proteolytic digestion has long been considered a defining trait of prions in tissues of organisms suffering from transmissible spongiform encephalopathies. Detection of proteinase K-resistant prion protein (PrPSc) still represents the diagnostic gold standard for prion diseases in humans, sheep and cattle. However, it has become increasingly apparent that the accumulation of PrPSc does not always accompany prion infections: high titers of prion infectivity can be reached also in the absence of protease resistant PrPSc. Here, we describe a structural basis for the phenomenon of protease-sensitive prion infectivity. We studied the effect on proteinase K (PK) resistance of the amino acid substitution Y169F, which removes a single oxygen atom from the β2-α2 loop of the cellular prion protein (PrPC). When infected with RML or the 263K strain of prions, transgenic mice lacking wild-type (wt) PrPC but expressing MoPrP169F generated prion infectivity at levels comparable to wt mice. The newly generated MoPrP169F prions were biologically indistinguishable from those recovered from prion-infected wt mice, and elicited similar pathologies in vivo. Surprisingly, MoPrP169F prions showed greatly reduced PK resistance and density gradient analyses showed a significant reduction in high-density aggregates. Passage of MoPrP169F prions into mice expressing wt MoPrP led to full recovery of protease resistance, indicating that no strain shift had taken place. We conclude that a subtle structural variation in the β2-α2 loop of PrPC affects the sensitivity of PrPSc to protease but does not impact prion replication and infectivity. With these findings a specific structural feature of PrPC can be linked to a physicochemical property of the corresponding PrPSc.

Prion protein functions as a ferrireductase partner for ZIP14 and DMT1

PubMed Central

Qian, Juan; Beserra, Amber; Suda, Srinivas; Singh, Ajay; Hopfer, Ulrich; Chen, Shu G.; Garrick, Michael D.; Turner, Jerrold R.; Knutson, Mitchell D.; Singh, Neena

2015-01-01

Excess circulating iron is stored in the liver, and requires reduction of non-Tf-bound-iron (NTBI) and transferrin (Tf)-iron at the plasma membrane and endosomes respectively by ferrireductase (FR) proteins for transport across biological membranes through divalent metal transporters. Here, we report that prion-protein (PrPC), a ubiquitously expressed glycoprotein most abundant on neuronal cells, functions as a FR partner for divalent-metal transporter-1 (DMT1) and ZIP14. Thus, absence of PrPC in PrP-knock-out (PrP−/−) mice resulted in markedly reduced liver iron stores, a deficiency that was not corrected by chronic or acute administration of iron by the oral or intra-peritoneal routes. Likewise, preferential radiolabeling of circulating NTBI with 59Fe revealed significantly reduced uptake and storage of NTBI by the liver of PrP−/− mice relative to matched PrP+/+ controls. However, uptake, storage, and utilization of ferritin-bound iron that does not require reduction for uptake was increased in PrP−/− mice, indicating a compensatory response to the iron-deficiency. Expression of exogenous PrPC in HepG2-cells increased uptake and storage of ferric-iron (Fe3+), not ferrous-iron (Fe2+) from the medium, supporting the function of PrPC as a plasma membrane FR. Co-expression of PrPC with ZIP14 and DMT1 in HepG2 cells increased uptake of Fe3+ significantly, and surprisingly, increased the ratio of N-terminally truncated PrPC forms lacking the FR domain relative to full-length PrPC. Together, these observations indicate that PrPC promotes, and possibly regulates the uptake of NTBI through DMT1 and Zip14 via its FR activity. Implications of these observations for neuronal iron homeostasis under physiological and pathological conditions are discussed. PMID:25862412

Determining the relative susceptibility of four prion protein genotypes to atypical scrapie

USDA-ARS?s Scientific Manuscript database

Atypical scrapie is a sheep prion (PrPSc) disease whose epidemiology is consistent with a sporadic origin and is associated with specific polymorphisms of the normal cellular prion protein (PrPC). We describe a mass spectrometry-based method of detecting and quantifying the polymorphisms of sheep P...

Molecular modeling of the conformational dynamics of the cellular prion protein

NASA Astrophysics Data System (ADS)

Nguyen, Charles; Colling, Ian; Bartz, Jason; Soto, Patricia

2014-03-01

Prions are infectious agents responsible for transmissible spongiform encephalopathies (TSEs), a type of fatal neurodegenerative disease in mammals. Prions propagate biological information by conversion of the non-pathological version of the prion protein to the infectious conformation, PrPSc. A wealth of knowledge has shed light on the nature and mechanism of prion protein conversion. In spite of the significance of this problem, we are far from fully understanding the conformational dynamics of the cellular isoform. To remedy this situation we employ multiple biomolecular modeling techniques such as docking and molecular dynamics simulations to map the free energy landscape and determine what specific regions of the prion protein are most conductive to binding. The overall goal is to characterize the conformational dynamics of the cell form of the prion protein, PrPc, to gain insight into inhibition pathways against misfolding. NE EPSCoR FIRST Award to Patricia Soto.

Pathogenic prions deviate PrPC signaling in neuronal cells and impair A-beta clearance

PubMed Central

Pradines, E; Hernandez-Rapp, J; Villa-Diaz, A; Dakowski, C; Ardila-Osorio, H; Haik, S; Schneider, B; Launay, J-M; Kellermann, O; Torres, J-M; Mouillet-Richard, S

2013-01-01

The subversion of the normal function exerted by the cellular prion protein (PrPC) in neurons by pathogenic prions is assumed to have a central role in the pathogenesis of transmissible spongiform encephalopathies. Using two murine models of prion infection, the 1C11 neuronal cell line and neurospheres, we document that prion infection is associated with the constitutive activation of signaling targets normally coupled with PrPC, including the Fyn kinase, the mitogen-associated protein kinases ERK1/2 and the CREB transcription factor. PrPC-dependent signaling overactivation in infected cells is associated with the recruitment of p38 and JNK stress-associated kinases. Downstream from CREB, prion-infected cells exhibit reduced activity of the matrix metalloprotease (MMP)-9. As MMP-9 catalyzes the degradation of the amyloid A-beta peptide, the decrease in MMP-9 activity in prion-infected cells causes a significant impairment of the clearance of A-beta, leading to its accumulation. By exploiting two 1C11-infected clones accumulating high or moderate levels of prions, we show that the prion-induced changes are correlated with the level of infectivity. Of note, a dose-dependent increase in A-beta levels was also found in the cerebrospinal fluid of mice inoculated with these infected clones. By demonstrating that pathogenic prions trigger increases in A-beta levels through the deviation of PrPC signaling, our data argue that A-beta may exacerbate prion-induced toxicity. PMID:23303130

Molecular approaches to detecting and discriminating among prions, a class of pathogenic molecules(Abstract)

USDA-ARS?s Scientific Manuscript database

Prions (PrPSc)are the pathogens that cause a set of fatal neurological diseases that include scrapie and chronic wasting disease (CWD). They are composed solely of protein and unlike viral, bacterial, or fungal pathogens, the information necessary to convert the normal cellular prion protein (PrPC) ...

Antiprion activity of DB772 and related monothiophene-and furan-based analogs in a persistently infected ovine microglia culture system

USDA-ARS?s Scientific Manuscript database

The transmissible spongiform encephalopathies are fatal neurodegenerative disorders characterized by the misfolding of the native cellular prion protein (PrP-C) into the accumulating, disease-associated isoform (PrP-Sc). Despite extensive research into the inhibition of prion accumulation, no effect...

Prion search and cellular prion protein expression in stranded dolphins.

PubMed

Di Guardo, G; Cocumelli, C; Meoli, R; Barbaro, K; Terracciano, G; Di Francesco, C E; Mazzariol, S; Eleni, C

2012-01-01

The recent description of a prion disease (PD) case in a free-ranging bottlenose dolphin (Tursiops truncatus) prompted us to carry out an extensive search for the disease-associated isoform (PrPSc) of the cellular prion protein (PrPC) in the brain and in a range of lymphoid tissues from 23 striped dolphins (Stenella coeruleoalba), 5 bottlenose dolphins and 2 Risso s dolphins (Grampus griseus) found stranded between 2007 and 2012 along the Italian coastline. Three striped dolphins and one bottlenose dolphin showed microscopic lesions of encephalitis, with no evidence of spongiform brain lesions being detected in any of the 30 free-ranging cetaceans investigated herein. Nevertheless, we could still observe a prominent PrPC immunoreactivity in the brain as well as in lymphoid tissues from these dolphins. Although immunohistochemical and Western blot investigations yielded negative results for PrPSc deposition in all tissues from the dolphins under study, the reported occurrence of a spontaneous PD case in a wild dolphin is an intriguing issue and a matter of concern for both prion biology and intra/inter-species transmissibility, as well as for cetacean conservation medicine.

Proximity of SCG10 and prion protein in membrane rafts.

PubMed

Iwamaru, Yoshifumi; Kitani, Hiroshi; Okada, Hiroyuki; Takenouchi, Takato; Shimizu, Yoshihisa; Imamura, Morikazu; Miyazawa, Kohtaro; Murayama, Yuichi; Hoover, Edward A; Yokoyama, Takashi

2015-12-10

The conversion of normal cellular prion protein (PrPC) into its pathogenic isoform (PrPSc) is an essential event in prion pathogenesis. In culture models, membrane rafts are suggested to play a critical role in PrPSc formation. To identify the candidate molecules capable of interacting with PrPC and facilitating PrPSc formation in membrane rafts, we applied a novel biochemical labelling method termed 'enzyme-mediated activation of radical sources (EMARS)'. EMARS was applied to the Lubrol WX insoluble detergent-resistant membrane fractions from mouse neuroblastoma (N2a) cells in which the surface PrPC was labeled with HRP-conjugated anti-PrP antibody. Two-dimensional Western blots of these preparations revealed biotinylated spots of approximately 20 kDa with an isoelectric point of 8.0-9.0. Liquid chromatography-tandem mass spectrometry analysis resulted in the identification of peptides containing SCG10, the neuron-specific microtubule regulator. Proximity of SCG10 and PrPC was confirmed using proximity ligation assay and co-immunoprecipitation assay. Transfection of persistently 22L prion infected N2a cells with SCG10 small interfering RNA reduced SCG10 expression but did not prevent PrPSc accumulation, indicating that SCG10 appears to be unrelated to PrPSc formation of 22L prion. Immunofluorescence and Western blot analyses showed reduced levels of SCG10 in the hippocampus of prion-infected mice, suggesting a possible association between SCG10 levels and the prion neuropathogenesis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Detecting and discriminating among pathogenic protein conformers(prions), using mass spectrometry-based and antibody-based approaches(Abstract)

USDA-ARS?s Scientific Manuscript database

A set of fatal neurological diseases that includes scrapie and chronic wasting disease (CWD) are caused by a pathological protein referred to as a prion (PrPSc). A prion propagates an infection by converting a normal cellular protein (PrPC) into a prion. Unlike viral, bacterial, or fungal pathogens,...

Relationships between PrPSc stability and incubation time for United States scrapie strains in a natural host system

USDA-ARS?s Scientific Manuscript database

Transmissible spongiform encephalopathies (TSEs), including scrapie in sheep (Ovis aries), are fatal neurodegenerative diseases caused by the misfolding of the cellular prion protein (PrP**C) into a beta-rich conformer (PrP**Sc) that accumulates into higher-order structures in the brain and other ti...

Quantitating PrP polymorphisms present in prions from heterozygous scrapie-infected sheep

USDA-ARS?s Scientific Manuscript database

Scrapie is a prion (PrPSc) disease of sheep. The incubation period of sheep scrapie is strongly influenced by polymorphisms at positions 136, 154, and 171 of a sheep’s normal cellular prion protein (PrPC). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic pept...

Prions amplify through degradation of the VPS10P sorting receptor sortilin.

PubMed

Uchiyama, Keiji; Tomita, Mitsuru; Yano, Masashi; Chida, Junji; Hara, Hideyuki; Das, Nandita Rani; Nykjaer, Anders; Sakaguchi, Suehiro

2017-06-01

Prion diseases are a group of fatal neurodegenerative disorders caused by prions, which consist mainly of the abnormally folded isoform of prion protein, PrPSc. A pivotal pathogenic event in prion disease is progressive accumulation of prions, or PrPSc, in brains through constitutive conformational conversion of the cellular prion protein, PrPC, into PrPSc. However, the cellular mechanism by which PrPSc is progressively accumulated in prion-infected neurons remains unknown. Here, we show that PrPSc is progressively accumulated in prion-infected cells through degradation of the VPS10P sorting receptor sortilin. We first show that sortilin interacts with PrPC and PrPSc and sorts them to lysosomes for degradation. Consistently, sortilin-knockdown increased PrPSc accumulation in prion-infected cells. In contrast, overexpression of sortilin reduced PrPSc accumulation in prion-infected cells. These results indicate that sortilin negatively regulates PrPSc accumulation in prion-infected cells. The negative role of sortilin in PrPSc accumulation was further confirmed in sortilin-knockout mice infected with prions. The infected mice had accelerated prion disease with early accumulation of PrPSc in their brains. Interestingly, sortilin was reduced in prion-infected cells and mouse brains. Treatment of prion-infected cells with lysosomal inhibitors, but not proteasomal inhibitors, increased the levels of sortilin. Moreover, sortilin was reduced following PrPSc becoming detectable in cells after infection with prions. These results indicate that PrPSc accumulation stimulates sortilin degradation in lysosomes. Taken together, these results show that PrPSc accumulation of itself could impair the sortilin-mediated sorting of PrPC and PrPSc to lysosomes for degradation by stimulating lysosomal degradation of sortilin, eventually leading to progressive accumulation of PrPSc in prion-infected cells.

Divergent prion strain evolution driven by PrPC expression level in transgenic mice

PubMed Central

Le Dur, Annick; Laï, Thanh Lan; Stinnakre, Marie-George; Laisné, Aude; Chenais, Nathalie; Rakotobe, Sabine; Passet, Bruno; Reine, Fabienne; Soulier, Solange; Herzog, Laetitia; Tilly, Gaëlle; Rézaei, Human; Béringue, Vincent; Vilotte, Jean-Luc; Laude, Hubert

2017-01-01

Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission. PMID:28112164

The multivesicular body is the major internal site of prion conversion

PubMed Central

Yim, Yang-In; Park, Bum-Chan; Yadavalli, Rajgopal; Zhao, Xiaohong; Eisenberg, Evan; Greene, Lois E.

2015-01-01

ABSTRACT The conversion of the properly folded prion protein, PrPc, to its misfolded amyloid form, PrPsc, occurs as the two proteins traffic along the endocytic pathway and PrPc is exposed to PrPsc. To determine the specific site of prion conversion, we knocked down various proteins in the endocytic pathway including Rab7a, Tsg101 and Hrs (also known as HGS). PrPsc was markedly reduced in two chronically infected cell lines by preventing the maturation of the multivesicular body, a process that begins in the early endosome and ends with the sorting of cargo to the lysosome. By contrast, knocking down proteins in the retromer complex, which diverts cargo away from the multivesicular body caused an increase in PrPsc levels. These results suggest that the multivesicular body is the major site for intracellular conversion of PrPc to PrPsc. PMID:25663703

Highly Efficient Protein Misfolding Cyclic Amplification

PubMed Central

Ostapchenko, Valeriy G.; Savtchenk, Regina; Alexeeva, Irina; Rohwer, Robert G.; Baskakov, Ilia V.

2011-01-01

Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrPC into PrPSc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrPC may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrPC into PrPSc from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrPSc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrPC susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrPSc in vitro. PMID:21347353

PrPC Undergoes Basal to Apical Transcytosis in Polarized Epithelial MDCK Cells

PubMed Central

Arkhipenko, Alexander; Syan, Sylvie; Victoria, Guiliana Soraya

2016-01-01

The Prion Protein (PrP) is an ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of prion disease, the cellular isoform (PrPC) is an enigmatic protein with unclear function. Of interest, PrP localization in polarized MDCK cells is controversial and its mechanism of trafficking is not clear. Here we investigated PrP traffic in MDCK cells polarized on filters and in three-dimensional MDCK cysts, a more physiological model of polarized epithelia. We found that, unlike other GPI-anchored proteins (GPI-APs), PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells. Following this event full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures. PMID:27389581

Use of an ELISA-based stability assay to examine host genotype, PrP**Sc stability, and incubation time relationships in U.S. livestock prion strains

USDA-ARS?s Scientific Manuscript database

Transmissible spongiform encephalopathies (TSEs) are caused by the misfolding of the cellular prion protein (PrP**C) into a disease-associated version (PrP**Sc) that accumulates in certain tissues, leading to pathological changes in the brain and eventual death. Different strains of TSEs have been d...

Prion Propagation in Cells Expressing PrP Glycosylation Mutants ▿

PubMed Central

Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-01-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides.

PubMed

Guichard, Cécile; Ivanyi-Nagy, Roland; Sharma, Kamal Kant; Gabus, Caroline; Marc, Daniel; Mély, Yves; Darlix, Jean-Luc

2011-10-01

Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrP(C)) into the aggregated misfolded scrapie isoform, named PrP(Sc). Recent studies on the physiological role of PrP(C) revealed that this protein has probably multiple functions, notably in cell-cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5'-GACACAAGCCGA-3' was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities.

Prion propagation in cells expressing PrP glycosylation mutants.

PubMed

Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-04-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

Methamphetamine increases Prion Protein and induces dopamine-dependent expression of protease resistant PrPsc.

PubMed

Ferrucci, M; Ryskalin, L; Biagioni, F; Gambardella, S; Busceti, C L; Falleni, A; Lazzeri, G; Fornai, F

2017-07-01

The cellular prion protein (PrPc) is physiologically expressed within selective brain areas of mammals. Alterations in the secondary structure of this protein lead to scrapie-like prion protein (PrPsc), which precipitates in the cell. PrPsc has been detected in infectious, inherited or sporadic neurodegenerative disorders. Prion protein metabolism is dependent on autophagy and ubiquitin proteasome. Despite not being fully elucidated, the physiological role of prion protein relates to chaperones which rescue cells under stressful conditions.Methamphetamine (METH) is a widely abused drug which produces oxidative stress in various brain areas causing mitochondrial alterations and protein misfolding. These effects produce a compensatory increase of chaperones while clogging cell clearing pathways. In the present study, we explored whether METH administration modifies the amount of PrPc. Since high levels of PrPc when the clearing systems are clogged may lead to its misfolding into PrPsc, we further tested whether METH exposure triggers the appearance of PrPsc. We analysed the effects of METH and dopamine administration in PC12 and striatal cells by using SDS-PAGE Coomassie blue, immune- histochemistry and immune-gold electron microscopy. To analyze whether METH administration produces PrPsc aggregates we used antibodies directed against PrP following exposure to proteinase K or sarkosyl which digest folded PrPc but misfolded PrPsc. We fond that METH triggers PrPsc aggregates in DA-containing cells while METH is not effective in primary striatal neurons which do not produce DA. In the latter cells exogenous DA is needed to trigger PrPsc accumulation similarly to what happens in DA containing cells under the effects of METH. The present findings, while fostering novel molecular mechanisms involving prion proteins, indicate that, cell pathology similar to prion disorders can be mimicked via a DA-dependent mechanism by a drug of abuse.

Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides

PubMed Central

Guichard, Cécile; Ivanyi-Nagy, Roland; Sharma, Kamal Kant; Gabus, Caroline; Marc, Daniel; Mély, Yves; Darlix, Jean-Luc

2011-01-01

Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrPC) into the aggregated misfolded scrapie isoform, named PrPSc. Recent studies on the physiological role of PrPC revealed that this protein has probably multiple functions, notably in cell–cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5′-GACACAAGCCGA-3′ was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities. PMID:21737432

Prion protein β2–α2 loop conformational landscape

PubMed Central

Caldarulo, Enrico; Wüthrich, Kurt; Parrinello, Michele

2017-01-01

In transmissible spongiform encephalopathies (TSEs), which are lethal neurodegenerative diseases that affect humans and a wide range of other mammalian species, the normal “cellular” prion protein (PrPC) is transformed into amyloid aggregates representing the “scrapie form” of the protein (PrPSc). Continued research on this system is of keen interest, since new information on the physiological function of PrPC in healthy organisms is emerging, as well as new data on the mechanism of the transformation of PrPC to PrPSc. In this paper we used two different approaches: a combination of the well-tempered ensemble (WTE) and parallel tempering (PT) schemes and metadynamics (MetaD) to characterize the conformational free-energy surface of PrPC. The focus of the data analysis was on an 11-residue polypeptide segment in mouse PrPC(121–231) that includes the β2–α2 loop of residues 167–170, for which a correlation between structure and susceptibility to prion disease has previously been described. This study includes wild-type mouse PrPC and a variant with the single-residue replacement Y169A. The resulting detailed conformational landscapes complement in an integrative manner the available experimental data on PrPC, providing quantitative insights into the nature of the structural transition-related function of the β2–α2 loop. PMID:28827331

Design of anti- and pro-aggregation variants to assess the effects of methionine oxidation in human prion protein

PubMed Central

Wolschner, Christina; Giese, Armin; Kretzschmar, Hans A.; Huber, Robert; Moroder, Luis; Budisa, Nediljko

2009-01-01

Prion disease is characterized by the α→β structural conversion of the cellular prion protein (PrPC) into the misfolded and aggregated “scrapie” (PrPSc) isoform. It has been speculated that methionine (Met) oxidation in PrPC may have a special role in this process, but has not been detailed and assigned individually to the 9 Met residues of full-length, recombinant human PrPC [rhPrPC(23-231)]. To better understand this oxidative event in PrP aggregation, the extent of periodate-induced Met oxidation was monitored by electrospray ionization-MS and correlated with aggregation propensity. Also, the Met residues were replaced with isosteric and chemically stable, nonoxidizable analogs, i.e., with the more hydrophobic norleucine (Nle) and the highly hydrophilic methoxinine (Mox). The Nle-rhPrPC variant is an α-helix rich protein (like Met-rhPrPC) resistant to oxidation that lacks the in vitro aggregation properties of the parent protein. Conversely, the Mox-rhPrPC variant is a β-sheet rich protein that features strong proaggregation behavior. In contrast to the parent Met-rhPrPC, the Nle/Mox-containing variants are not sensitive to periodate-induced in vitro aggregation. The experimental results fully support a direct correlation of the α→β secondary structure conversion in rhPrPC with the conformational preferences of Met/Nle/Mox residues. Accordingly, sporadic prion and other neurodegenerative diseases, as well as various aging processes, might also be caused by oxidative stress leading to Met oxidation. PMID:19416900

Olfactory behavior and physiology are disrupted in prion protein knockout mice.

PubMed

Le Pichon, Claire E; Valley, Matthew T; Polymenidou, Magdalini; Chesler, Alexander T; Sagdullaev, Botir T; Aguzzi, Adriano; Firestein, Stuart

2009-01-01

The prion protein PrP(C) is infamous for its role in disease, but its normal physiological function remains unknown. Here we found a previously unknown behavioral phenotype of Prnp(-/-) mice in an odor-guided task. This phenotype was manifest in three Prnp knockout lines on different genetic backgrounds, which provides strong evidence that the phenotype is caused by a lack of PrP(C) rather than by other genetic factors. Prnp(-/-) mice also showed altered behavior in a second olfactory task, suggesting that the phenotype is olfactory specific. Furthermore, PrP(C) deficiency affected oscillatory activity in the deep layers of the main olfactory bulb, as well as dendrodendritic synaptic transmission between olfactory bulb granule and mitral cells. Notably, both the behavioral and electrophysiological alterations found in Prnp(-/-) mice were rescued by transgenic neuronal-specific expression of PrP(C). These data suggest that PrP(C) is important in the normal processing of sensory information by the olfactory system.

Copper and the Prion Protein: Methods, Structures, Function, and Disease

NASA Astrophysics Data System (ADS)

Millhauser, Glenn L.

2007-05-01

The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrPC to PrPSc. Examples of the TSEs include mad cow disease, chronic wasting disease in deer and elk, scrapie in goats and sheep, and kuru and Creutzfeldt-Jakob disease in humans. Although the precise function of PrPC in healthy tissues is not known, recent research demonstrates that it binds Cu(II) in an unusual and highly conserved region of the protein termed the octarepeat domain. This review describes recent connections between copper and PrPC, with an emphasis on the electron paramagnetic resonance elucidation of the specific copper-binding sites, insights into PrPC function, and emerging connections between copper and prion disease.

Neuroinflammation, hyperphosphorylated tau, diffuse amyloid plaques, and down-regulation of the cellular prion protein in air pollution exposed children and young adults.

PubMed

Calderón-Garcidueñas, Lilian; Kavanaugh, Michael; Block, Michelle; D'Angiulli, Amedeo; Delgado-Chávez, Ricardo; Torres-Jardón, Ricardo; González-Maciel, Angelica; Reynoso-Robles, Rafael; Osnaya, Norma; Villarreal-Calderon, Rodolfo; Guo, Ruixin; Hua, Zhaowei; Zhu, Hongtu; Perry, George; Diaz, Philippe

2012-01-01

Air pollution exposures have been linked to neuroinflammation and neuropathology. Autopsy samples of the frontal cortex from control (n = 8) and pollution-exposed (n = 35) children and young adults were analyzed by RT-PCR (n = 43) and microarray analysis (n = 12) for gene expression changes in oxidative stress, DNA damage signaling, NFκB signaling, inflammation, and neurodegeneration pathways. The effect of apolipoprotein E (APOE) genotype on the presence of protein aggregates associated with Alzheimer's disease (AD) pathology was also explored. Exposed urbanites displayed differential (>2-fold) regulation of 134 genes. Forty percent exhibited tau hyperphosphorylation with pre-tangle material and 51% had amyloid-β (Aβ) diffuse plaques compared with 0% in controls. APOE4 carriers had greater hyperphosphorylated tau and diffuse Aβ plaques versus E3 carriers (Q = 7.82, p = 0.005). Upregulated gene network clusters included IL1, NFκB, TNF, IFN, and TLRs. A 15-fold frontal down-regulation of the prion-related protein (PrP(C)) was seen in highly exposed subjects. The down-regulation of the PrP(C) is critical given its important roles for neuroprotection, neurodegeneration, and mood disorder states. Elevation of indices of neuroinflammation and oxidative stress, down-regulation of the PrP(C) and AD-associated pathology are present in young megacity residents. The inducible regulation of gene expression suggests they are evolving different mechanisms in an attempt to cope with the constant state of inflammation and oxidative stress related to their environmental exposures. Together, these data support a role for air pollution in CNS damage and its impact upon the developing brain and the potential etiology of AD and mood disorders.

Infectious Prion Protein Alters Manganese Transport and Neurotoxicity in a Cell Culture Model of Prion Disease

PubMed Central

Martin, Dustin P.; Anantharam, Vellareddy; Jin, Huajun; Witte, Travis; Houk, Robert; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

2011-01-01

Protein misfolding and aggregation are considered key features of many neurodegenerative diseases, but biochemical mechanisms underlying protein misfolding and the propagation of protein aggregates are not well understood. Prion disease is a classical neurodegenerative disorder resulting from the misfolding of endogenously expressed normal cellular prion protein (PrPC). Although the exact function of PrPC has not been fully elucidated, studies have suggested that it can function as a metal binding protein. Interestingly, increased brain manganese (Mn) levels have been reported in various prion diseases indicating divalent metals also may play a role in the disease process. Recently, we reported that PrPC protects against Mn-induced cytotoxicity in a neural cell culture model. To further understand the role of Mn in prion diseases, we examined Mn neurotoxicity in an infectious cell culture model of prion disease. Our results show CAD5 scrapie-infected cells were more resistant to Mn neurotoxicity as compared to uninfected cells (EC50 = 428.8 μM for CAD5 infected cells vs. 211.6 μM for uninfected cells). Additionally, treatment with 300 μM Mn in persistently infected CAD5 cells showed a reduction in mitochondrial impairment, caspase-3 activation, and DNA fragmentation when compared to uninfected cells. Scrapie-infected cells also showed significantly reduced Mn uptake as measured by inductively coupled plasma-mass spectrometry (ICP-MS), and altered expression of metal transporting proteins DMT1 and transferrin. Together, our data indicate that conversion of PrP to the pathogenic isoform enhances its ability to regulate Mn homeostasis, and suggest that understanding the interaction of metals with disease-specific proteins may provide further insight to protein aggregation in neurodegenerative diseases. PMID:21871919

A visual dual-aptamer logic gate for sensitive discrimination of prion diseases-associated isoform with reusable magnetic microparticles and fluorescence quantum dots.

PubMed

Xiao, Sai Jin; Hu, Ping Ping; Chen, Li Qiang; Zhen, Shu Jun; Peng, Li; Li, Yuan Fang; Huang, Cheng Zhi

2013-01-01

Molecular logic gates, which have attracted increasing research interest and are crucial for the development of molecular-scale computers, simplify the results of measurements and detections, leaving the diagnosis of disease either "yes" or "no". Prion diseases are a group of fatal neurodegenerative disorders that happen in human and animals. The main problem with a diagnosis of prion diseases is how to sensitively and selectively discriminate and detection of the minute amount of PrP(Res) in biological samples. Our previous work had demonstrated that dual-aptamer strategy could achieve highly sensitive and selective discrimination and detection of prion protein (cellular prion protein, PrP(C), and the diseases associated isoform, PrP(Res)) in serum and brain. Inspired by the advantages of molecular logic gate, we further conceived a new concept for dual-aptamer logic gate that responds to two chemical input signals (PrP(C) or PrP(Res) and Gdn-HCl) and generates a change in fluorescence intensity as the output signal. It was found that PrP(Res) performs the "OR" logic operation while PrP(C) performs "XOR" logic operation when they get through the gate consisted of aptamer modified reusable magnetic microparticles (MMPs-Apt1) and quantum dots (QDs-Apt2). The dual-aptamer logic gate simplifies the discrimination results of PrP(Res), leaving the detection of PrP(Res) either "yes" or "no". The development of OR logic gate based on dual-aptamer strategy and two chemical input signals (PrP(Res) and Gdn-HCl) is an important step toward the design of prion diseases diagnosis and therapy systems.

Methods and Protocols for Developing Prion Vaccines.

PubMed

Marciniuk, Kristen; Taschuk, Ryan; Napper, Scott

2016-01-01

Prion diseases denote a distinct form of infectivity that is based in the misfolding of a self-protein (PrP(C)) into a pathological, infectious conformation (PrP(Sc)). Efforts to develop vaccines for prion diseases have been complicated by the potential dangers that are associated with induction of immune responses against a self-protein. As a consequence, there is considerable appeal for vaccines that specifically target the misfolded prion conformation. Such conformation-specific immunotherapy is made possible through the identification of vaccine targets (epitopes) that are exclusively presented as a consequence of misfolding. An immune response directed against these targets, termed disease-specific epitopes (DSEs), has the potential to spare the function of the native form of the protein while clearing, or neutralizing, the infectious isomer. Although identification of DSEs represents a critical first step in the induction of conformation-specific immune responses, substantial efforts are required to translate these targets into functional vaccines. Due to the poor immunogenicity that is inherent to self-proteins, and that is often associated with short peptides, substantial efforts are required to overcome tolerance-to-self and maximize the resultant immune response following DSE-based immunization. This often includes optimization of target sequences in terms of immunogenicity and development of effective formulation and delivery strategies for the associated peptides. Further, these vaccines must satisfy additional criteria from perspectives of specificity (PrP(C) vs. PrP(Sc)) and safety (antibody-induced template-driven misfolding of PrP(C)). The emphasis of this report is on the steps required to translate DSEs into prion vaccines and subsequent evaluation of the resulting immune responses.

Semiautomated cell-free conversion of prion protein: applications for high-throughput screening of potential antiprion drugs.

PubMed

Breydo, Leonid; Bocharova, Olga V; Baskakov, Ilia V

2005-04-01

Transmissible spongiform encephalitis (TSE) is a lethal illness with no known treatment. Conversion of the cellular prion protein (PrP(C)) into the infectious isoform (PrP(Sc)) is believed to be the central event in the development of this disease. Recombinant PrP (rPrP) protein folded into the amyloid conformation was shown to cause the transmissible form of prion disease in transgenic mice and can be used as a surrogate model for PrP(Sc). Here, we introduced a semiautomated assay of in vitro conversion of rPrP protein to the amyloid conformation. We have examined the effect of known inhibitors of prion propagation on this conversion and found good correlation between their activity in this assay and that in other in vitro assays. We thus propose that the conversion of rPrP to the amyloid isoform can serve as a high-throughput screen for possible inhibitors of PrP(Sc) formation and potential anti-TSE drugs.

Stochastic Modeling Approach to the Incubation Time of Prionic Diseases

NASA Astrophysics Data System (ADS)

Ferreira, A. S.; da Silva, M. A.; Cressoni, J. C.

2003-05-01

Transmissible spongiform encephalopathies are neurodegenerative diseases for which prions are the attributed pathogenic agents. A widely accepted theory assumes that prion replication is due to a direct interaction between the pathologic (PrPSc) form and the host-encoded (PrPC) conformation, in a kind of autocatalytic process. Here we show that the overall features of the incubation time of prion diseases are readily obtained if the prion reaction is described by a simple mean-field model. An analytical expression for the incubation time distribution then follows by associating the rate constant to a stochastic variable log normally distributed. The incubation time distribution is then also shown to be log normal and fits the observed BSE (bovine spongiform encephalopathy) data very well. Computer simulation results also yield the correct BSE incubation time distribution at low PrPC densities.

Dominant-Negative Inhibition of Prion Formation Diminished by Deletion Mutagenesis of the Prion Protein

PubMed Central

Zulianello, Laurence; Kaneko, Kiyotoshi; Scott, Michael; Erpel, Susanne; Han, Dong; Cohen, Fred E.; Prusiner, Stanley B.

2000-01-01

Polymorphic basic residues near the C terminus of the prion protein (PrP) in humans and sheep appear to protect against prion disease. In heterozygotes, inhibition of prion formation appears to be dominant negative and has been simulated in cultured cells persistently infected with scrapie prions. The results of nuclear magnetic resonance and mutagenesis studies indicate that specific substitutions at the C-terminal residues 167, 171, 214, and 218 of PrPC act as dominant-negative, inhibitors of PrPSc formation (K. Kaneko et al., Proc. Natl. Acad. Sci. USA 94:10069–10074, 1997). Trafficking of substituted PrPC to caveaola-like domains or rafts by the glycolipid anchor was required for the dominant-negative phenotype; interestingly, amino acid replacements at multiple sites were less effective than single-residue substitutions. To elucidate which domains of PrPC are responsible for dominant-negative inhibition of PrPSc formation, we analyzed whether N-terminally truncated PrP(Q218K) molecules exhibited dominant-negative effects in the conversion of full-length PrPC to PrPSc. We found that the C-terminal domain of PrP is not sufficient to impede the conversion of the full-length PrPC molecule and that N-terminally truncated molecules (with residues 23 to 88 and 23 to 120 deleted) have reduced dominant-negative activity. Whether the N-terminal region of PrP acts by stabilizing the C-terminal domain of the molecule or by modulating the binding of PrPC to an auxiliary molecule that participates in PrPSc formation remains to be established. PMID:10756050

Role of Prion Replication in the Strain-dependent Brain Regional Distribution of Prions*

PubMed Central

Hu, Ping Ping; Morales, Rodrigo; Duran-Aniotz, Claudia; Moreno-Gonzalez, Ines; Khan, Uffaf; Soto, Claudio

2016-01-01

One intriguing feature of prion diseases is their strain variation. Prion strains are differentiated by the clinical consequences they generate in the host, their biochemical properties, and their potential to infect other animal species. The selective targeting of these agents to specific brain structures have been extensively used to characterize prion strains. However, the molecular basis dictating strain-specific neurotropism are still elusive. In this study, isolated brain structures from animals infected with four hamster prion strains (HY, DY, 139H, and SSLOW) were analyzed for their content of protease-resistant PrPSc. Our data show that these strains have different profiles of PrP deposition along the brain. These patterns of accumulation, which were independent of regional PrPC production, were not reproduced by in vitro replication when different brain regions were used as substrate for the misfolding-amplification reaction. On the contrary, our results show that in vitro replication efficiency depended exclusively on the amount of PrPC present in each part of the brain. Our results suggest that the variable regional distribution of PrPSc in distinct strains is not determined by differences on prion formation, but on other factors or cellular pathways. Our findings may contribute to understand the molecular mechanisms of prion pathogenesis and strain diversity. PMID:27056328

The roles of the conserved tyrosine in the β2-α2 loop of the prion protein.

PubMed

Huang, Danzhi; Caflisch, Amedeo

2015-01-01

Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP(C)) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP(Sc)) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP(C) proteins have provided evidence of a conformational equilibrium between a 3(10)-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165-175). In vitro cell-free experiments of the seeded conversion of PrP(C) indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3(10)-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3(10)-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.

Experimental approaches to the interaction of the prion protein with nucleic acids and glycosaminoglycans: Modulators of the pathogenic conversion.

PubMed

Silva, Jerson L; Vieira, Tuane C R G; Gomes, Mariana P B; Rangel, Luciana P; Scapin, Sandra M N; Cordeiro, Yraima

2011-03-01

The concept that transmissible spongiform encephalopathies (TSEs) are caused only by proteins has changed the traditional paradigm that disease transmission is due solely to an agent that carries genetic information. The central hypothesis for prion diseases proposes that the conversion of a cellular prion protein (PrP(C)) into a misfolded, β-sheet-rich isoform (PrP(Sc)) accounts for the development of (TSE). There is substantial evidence that the infectious material consists chiefly of a protein, PrP(Sc), with no genomic coding material, unlike a virus particle, which has both. However, prions seem to have other partners that chaperone their activities in converting the PrP(C) into the disease-causing isoform. Nucleic acids (NAs) and glycosaminoglycans (GAGs) are the most probable accomplices of prion conversion. Here, we review the recent experimental approaches that have been employed to characterize the interaction of prion proteins with nucleic acids and glycosaminoglycans. A PrP recognizes many nucleic acids and GAGs with high affinities, and this seems to be related to a pathophysiological role for this interaction. A PrP binds nucleic acids and GAGs with structural selectivity, and some PrP:NA complexes can become proteinase K-resistant, undergoing amyloid oligomerization and conversion to a β-sheet-rich structure. These results are consistent with the hypothesis that endogenous polyanions (such as NAs and GAGs) may accelerate the rate of prion disease progression by acting as scaffolds or lattices that mediate the interaction between PrP(C) and PrP(Sc) molecules. In addition to a still-possible hypothesis that nucleic acids and GAGs, especially those from the host, may modulate the conversion, the recent structural characterization of the complexes has raised the possibility of developing new diagnostic and therapeutic strategies. Copyright © 2010 Elsevier Inc. All rights reserved.

Amyloid-beta-sheet formation at the air-water interface.

PubMed Central

Schladitz, C; Vieira, E P; Hermel, H; Möhwald, H

1999-01-01

An amyloid(1-40) solution rich in coil, turn, and alpha-helix, but poor in beta-sheet, develops monolayers with a high beta-sheet content when spread at the air-water interface. These monolayers are resistant to repeated compression-dilatation cycles and interaction with trifluoroethanol. The secondary structure motifs were detected by circular dichroism (CD) in solution and with infrared reflection-absorption spectroscopy (IRRAS) at the interface. Hydrophobic influences are discussed for the structure conversion in an effort to understand the completely unknown reason for the natural change of the normal prion protein cellular (PrP(C)) into the abnormal prion protein scrapie (PrP(Sc)). PMID:10585952

3D local structure around copper site of rabbit prion-related protein: Quantitative determination by XANES spectroscopy combined with multiple-scattering calculations

NASA Astrophysics Data System (ADS)

Cui, P. X.; Lian, F. L.; Wang, Y.; Wen, Yi; Chu, W. S.; Zhao, H. F.; Zhang, S.; Li, J.; Lin, D. H.; Wu, Z. Y.

2014-02-01

Prion-related protein (PrP), a cell-surface copper-binding glycoprotein, is considered to be responsible for a number of transmissible spongiform encephalopathies (TSEs). The structural conversion of PrP from the normal cellular isoform (PrPC) to the post-translationally modified form (PrPSc) is thought to be relevant to Cu2+ binding to histidine residues. Rabbits are one of the few mammalian species that appear to be resistant to TSEs, because of the structural characteristics of the rabbit prion protein (RaPrPC) itself. Here we determined the three-dimensional local structure around the C-terminal high-affinity copper-binding sites using X-ray absorption near-edge structure combined with ab initio calculations in the framework of the multiple-scattering (MS) theory. Result shows that two amino acid resides, Gln97 and Met108, and two histidine residues, His95 and His110, are involved in binding this copper(II) ion. It might help us understand the roles of copper in prion conformation conversions, and the molecular mechanisms of prion-involved diseases.

Transmission Characteristics of Variably Protease-Sensitive Prionopathy

PubMed Central

Notari, Silvio; Xiao, Xiangzhu; Espinosa, Juan Carlos; Cohen, Yvonne; Qing, Liuting; Aguilar-Calvo, Patricia; Kofskey, Diane; Cali, Ignazio; Cracco, Laura; Kong, Qingzhong; Torres, Juan Maria

2014-01-01

Variably protease-sensitive prionopathy (VPSPr), a recently identified and seemingly sporadic human prion disease, is distinct from Creutzfeldt-Jakob disease (CJD) but shares features of Gerstmann-Sträussler-Scheinker disease (GSS). However, contrary to exclusively inherited GSS, no prion protein (PrP) gene variations have been detected in VPSPr, suggesting that VPSPr might be the long-sought sporadic form of GSS. The VPSPr atypical features raised the issue of transmissibility, a prototypical property of prion diseases. We inoculated VPSPr brain homogenate into transgenic mice expressing various levels of human PrP (PrPC). On first passage, 54% of challenged mice showed histopathologic lesions, and 34% harbored abnormal PrP similar to that of VPSPr. Surprisingly, no prion disease was detected on second passage. We concluded that VPSPr is transmissible; thus, it is an authentic prion disease. However, we speculate that normal human PrPC is not an efficient conversion substrate (or mouse brain not a favorable environment) and therefore cannot sustain replication beyond the first passage. PMID:25418590

Role of Prion Replication in the Strain-dependent Brain Regional Distribution of Prions.

PubMed

Hu, Ping Ping; Morales, Rodrigo; Duran-Aniotz, Claudia; Moreno-Gonzalez, Ines; Khan, Uffaf; Soto, Claudio

2016-06-10

One intriguing feature of prion diseases is their strain variation. Prion strains are differentiated by the clinical consequences they generate in the host, their biochemical properties, and their potential to infect other animal species. The selective targeting of these agents to specific brain structures have been extensively used to characterize prion strains. However, the molecular basis dictating strain-specific neurotropism are still elusive. In this study, isolated brain structures from animals infected with four hamster prion strains (HY, DY, 139H, and SSLOW) were analyzed for their content of protease-resistant PrP(Sc) Our data show that these strains have different profiles of PrP deposition along the brain. These patterns of accumulation, which were independent of regional PrP(C) production, were not reproduced by in vitro replication when different brain regions were used as substrate for the misfolding-amplification reaction. On the contrary, our results show that in vitro replication efficiency depended exclusively on the amount of PrP(C) present in each part of the brain. Our results suggest that the variable regional distribution of PrP(Sc) in distinct strains is not determined by differences on prion formation, but on other factors or cellular pathways. Our findings may contribute to understand the molecular mechanisms of prion pathogenesis and strain diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure and polymorphism of the mouse prion protein gene.

PubMed Central

Westaway, D; Cooper, C; Turner, S; Da Costa, M; Carlson, G A; Prusiner, S B

1994-01-01

Missense mutations in the prion protein (PrP) gene, overexpression of the cellular isoform of PrP (PrPC), and infection with prions containing the scrapie isoform of PrP (PrPSc) all cause neurodegenerative disease. To understand better the physiology and expression of PrPC, we retrieved mouse PrP gene (Prn-p) yeast artificial chromosome (YAC), cosmid, phage, and cDNA clones. Physical mapping positions Prn-p approximately 300 kb from ecotropic virus integration site number 4 (Evi-4), compatible with failure to detect recombination between Prn-p and Evi-4 in genetic crosses. The Prn-pa allele encompasses three exons, with exons 1 and 2 encoding the mRNA 5' untranslated region. Exon 2 has no equivalent in the Syrian hamster and human PrP genes. The Prn-pb gene shares this intron/exon structure but harbors an approximately 6-kb deletion within intron 2. While the Prn-pb open reading frame encodes two amino acid substitutions linked to prolonged scrapie incubation periods, a deletion of intron 2 sequences also characterizes inbred strains such as RIII/S and MOLF/Ei with shorter incubation periods, making a relationship between intron 2 size and scrapie pathogenesis unlikely. The promoter regions of a and b Prn-p alleles include consensus Sp1 and AP-1 sites, as well as other conserved motifs which may represent binding sites for as yet unidentified transcription factors. Images PMID:7912827

Platelet-Rich Plasma Preparation Types Show Impact on Chondrogenic Differentiation, Migration, and Proliferation of Human Subchondral Mesenchymal Progenitor Cells.

PubMed

Kreuz, Peter Cornelius; Krüger, Jan Philipp; Metzlaff, Sebastian; Freymann, Undine; Endres, Michaela; Pruss, Axel; Petersen, Wolf; Kaps, Christian

2015-10-01

To evaluate the chondrogenic potential of platelet concentrates on human subchondral mesenchymal progenitor cells (MPCs) as assessed by histomorphometric analysis of proteoglycans and type II collagen. Furthermore, the migratory and proliferative effect of platelet concentrates were assessed. Platelet-rich plasma (PRP) was prepared using preparation kits (Autologous Conditioned Plasma [ACP] Kit [Arthrex, Naples, FL]; Regen ACR-C Kit [Regen Lab, Le Mont-Sur-Lausanne, Switzerland]; and Dr.PRP Kit [Rmedica, Seoul, Republic of Korea]) by apheresis (PRP-A) and by centrifugation (PRP-C). In contrast to clinical application, freeze-and-thaw cycles were subsequently performed to activate platelets and to prevent medium coagulation by residual fibrinogen in vitro. MPCs were harvested from the cortico-spongious bone of femoral heads. Chondrogenic differentiation of MPCs was induced in high-density pellet cultures and evaluated by histochemical staining of typical cartilage matrix components. Migration of MPCs was assessed using a chemotaxis assay, and proliferation activity was measured by DNA content. MPCs cultured in the presence of 5% ACP, Regen, or Dr.PRP formed fibrous tissue, whereas MPCs stimulated with 5% PRP-A or PRP-C developed compact and dense cartilaginous tissue rich in type II collagen and proteoglycans. All platelet concentrates significantly (ACP, P = .00041; Regen, P = .00029; Dr.PRP, P = .00051; PRP-A, P < .0001; and PRP-C, P < .0001) stimulated migration of MPCs. All platelet concentrates but one (Dr.PRP, P = .63) showed a proliferative effect on MPCs, as shown by significant increases (ACP, P = .027; Regen, P = .0029; PRP-A, P = .00021; and PRP-C, P = .00069) in DNA content. Platelet concentrates obtained by different preparation methods exhibit different potentials to stimulate chondrogenic differentiation, migration, and proliferation of MPCs. Platelet concentrates obtained by commercially available preparation kits failed to induce chondrogenic differentiation of MPCs, whereas highly standardized PRP preparations did induce such differentiation. These findings suggest differing outcomes with PRP treatment in stem cell-based cartilage repair. Our findings may help to explain the variability of results in studies examining the use of PRP clinically. Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

PrP0\\0 mice show behavioral abnormalities that suggest PrPC has a role in maintaining the cytoskeleton.

USDA-ARS?s Scientific Manuscript database

Background/Introduction. PrPC is highly conserved among mammals, but its natural function is unclear. Prnp ablated mice (PrP0/0) appear to develop normally and are able to reproduce. These observations seem to indicate that the gene is not essential for viability, in spite of it being highly conse...

Preliminary results of human PrPC protein studied by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Nowakowski, Michał; Czapla-Masztafiak, Joanna; Kozak, Maciej; Zhukov, Igor; Zhukova, Lilia; Szlachetko, Jakub; Kwiatek, Wojciech M.

2017-11-01

Neurodegenerative diseases are one of the malfunctions of human nervous system, being a class of complex and prominent pathologies. The human prion Protease Resistant Protein (PrP) is protein regulating copper metabolism in mammalian cells through binding of Cu(II) ions to specific fragments. Nowadays misfolding of this protein is associated with development of prion diseases. Therefore, it is crucial to obtain structural information about coordination of Cu(II) by PrP protein. Herein, we report X-ray absorption spectroscopy (XAS) measurements, carried out on SuperXAS beamline (SLS, PSI Villigen) on PrPC-Cu(II) complexes. Obtained results were compared with theoretical predictions done by FEFF 9.6 software. Complementary to XAS data, Atomic Force Microscopy (AFM) measurements were conducted to obtain low resolution structural information about prepared sample that allow to develop protocol of fixing PrPC molecules on solid substrate used for further experiments. It has been established that folded C-terminal domain of PrPC protein has around 5 nm in diameter. Presented results showed that both XAS and AFM methods are useful tools in detailed examination of complexes of human PrPC either with Cu(II) or with other divalent metal ions.

Instability of buried hydration sites increases protein subdomains fluctuations in the human prion protein by the pathogenic mutation T188R

NASA Astrophysics Data System (ADS)

Tomobe, Katsufumi; Yamamoto, Eiji; Akimoto, Takuma; Yasui, Masato; Yasuoka, Kenji

2016-05-01

The conformational change from the cellular prion protein (PrPc) to scrapie prion protein (PrPsc) is a key process in prion diseases. The prion protein has buried water molecules which significantly contribute to the stability of the protein; however, there has been no report investigating the influence on the buried hydration sites by a pathogenic mutation not adjacent to the buried hydration sites. Here, we perform molecular dynamics simulations of wild type (WT) PrPc and pathogenic point mutant T188R to investigate conformational changes and the buried hydration sites. In WT-PrPc, four buried hydration sites are identified by residence time and rotational relaxation analysis. However, there are no stable buried hydration sites in one of T188R simulations, which indicates that T188R sometimes makes the buried hydration sites fragile. We also find that fluctuations of subdomains S1-H1-S2 and H1-H2 increase in T188R when the buried hydration sites become unstable. Since the side chain of arginine which is replaced from threonine in T188R is larger than of threonine, the side chain cannot be embedded in the protein, which is one of the causes of the instability of subdomains. These results show correlations between the buried hydration sites and the mutation which is far from them, and provide a possible explanation for the instability by mutation.

Bison PRNP genotyping and potential association with Brucella spp. seroprevalence

USGS Publications Warehouse

Seabury, C.M.; Halbert, N.D.; Gogan, P.J.P.; Templeton, J.W.; Derr, J.N.

2005-01-01

The implication that host cellular prion protein (PrPC) may function as a cell surface receptor and/or portal protein for Brucella abortus in mice prompted an evaluation of nucleotide and amino acid variation within exon 3 of the prion protein gene (PRNP) for six US bison populations. A non-synonymous single nucleotide polymorphism (T50C), resulting in the predicted amino acid replacement M17T (Met ??? Thr), was identified in each population. To date, no variation (T50: Met) has been detected at the corresponding exon 3 nucleotide and/or amino acid position for domestic cattle. Notably, 80% (20 of 25) of the Yellowstone National Park bison possessing the C/C genotype were Brucella spp. seropositive, representing a significant (P = 0.021) association between seropositivity and the C/C genotypic class. Moreover, significant differences in the distribution of PRNP exon 3 alleles and genotypes were detected between Yellowstone National Park bison and three bison populations that were either founded from seronegative stock or previously subjected to test-and-slaughter management to eradicate brucellosis. Unlike domestic cattle, no indel polymorphisms were detected within the corresponding regions of the putative bison PRNP promoter, intron 1, octapeptide repeat region or 3???-untranslated region for any population examined. This study provides the first evidence of a potential association between nucleotide variation within PRNP exon 3 and the presence of Brucella spp. antibodies in bison, implicating PrPC in the natural resistance of bison to brucellosis infection. ?? 2005 International Society for Animal Genetics.

Progress on low susceptibility mechanisms of transmissible spongiform encephalopathies

PubMed Central

QING, Li-Li; ZHAO, Hui; LIU, Lin-Lin

2014-01-01

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are a group of fatal neurodegenerative diseases detected in a wide range of mammalian species. The “protein-only” hypothesis of TSE suggests that prions are transmissible particles devoid of nucleic acid and the primary pathogenic event is thought to be the conversion of cellular prion protein (PrPC) into the disease-associated isoform (PrPSc). According to susceptibility to TSEs, animals can be classified into susceptible species and low susceptibility species. In this review we focus on several species with low susceptibility to TSEs: dogs, rabbits, horses and buffaloes. We summarize recent studies into the characteristics of low susceptibility regarding protein structure, and biochemical and genetic properties. PMID:25297084

The non-octarepeat copper binding site of the prion protein is a key regulator of prion conversion

NASA Astrophysics Data System (ADS)

Giachin, Gabriele; Mai, Phuong Thao; Tran, Thanh Hoa; Salzano, Giulia; Benetti, Federico; Migliorati, Valentina; Arcovito, Alessandro; Longa, Stefano Della; Mancini, Giordano; D'Angelo, Paola; Legname, Giuseppe

2015-10-01

The conversion of the prion protein (PrPC) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrPC interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrPC constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrPC coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation.

Molecular dynamics studies on the buffalo prion protein.

PubMed

Zhang, Jiapu; Wang, Feng; Chatterjee, Subhojyoti

2016-01-01

It was reported that buffalo is a low susceptibility species resisting to transmissible spongiform encephalopathies (TSEs) (same as rabbits, horses, and dogs). TSEs, also called prion diseases, are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of species (except for rabbits, dogs, horses, and buffalo), manifesting as scrapie in sheep and goats; bovine spongiform encephalopathy (BSE or "mad-cow" disease) in cattle; chronic wasting disease in deer and elk; and Creutzfeldt-Jakob diseases, Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia, and Kulu in humans etc. In molecular structures, these neurodegenerative diseases are caused by the conversion from a soluble normal cellular prion protein (PrP(C)), predominantly with α-helices, into insoluble abnormally folded infectious prions (PrP(Sc)), rich in β-sheets. In this article, we studied the molecular structure and structural dynamics of buffalo PrP(C) (BufPrP(C)), in order to understand the reason why buffalo is resistant to prion diseases. We first did molecular modeling of a homology structure constructed by one mutation at residue 143 from the NMR structure of bovine and cattle PrP(124-227); immediately we found that for BufPrP(C)(124-227), there are five hydrogen bonds (HBs) at Asn143, but at this position, bovine/cattle do not have such HBs. Same as that of rabbits, dogs, or horses, our molecular dynamics studies also revealed there is a strong salt bridge (SB) ASP178-ARG164 (O-N) keeping the β2-α2 loop linked in buffalo. We also found there is a very strong HB SER170-TYR218 linking this loop with the C-terminal end of α-helix H3. Other information, such as (i) there is a very strong SB HIS187-ARG156 (N-O) linking α-helices H2 and H1 (if mutation H187R is made at position 187, then the hydrophobic core of PrP(C) will be exposed (L.H. Zhong (2010). Exposure of hydrophobic core in human prion protein pathogenic mutant H187R. Journal of Biomolecular Structure and Dynamics 28(3), 355-361)), (ii) at D178, there is a HB Y169-D178 and a polar contact R164-D178 for BufPrP(C) instead of a polar contact Q168-D178 for bovine PrP(C) (C.J. Cheng, & V. Daggett. (2014). Molecular dynamics simulations capture the misfolding of the bovine prion protein at acidic pH. Biomolecules 4(1), 181-201), (iii) BufPrP(C) owns three 310 helices at 125-127, 152-156, and in the β2-α2 loop, respectively, and (iv) in the β2-α2 loop, there is a strong π-π stacking and a strong π-cation F175-Y169-R164.(N)NH2, has been discovered.

Chronic Lymphocytic Inflammation Specifies the Organ Tropism of Prions

NASA Astrophysics Data System (ADS)

Heikenwalder, Mathias; Zeller, Nicolas; Seeger, Harald; Prinz, Marco; Klöhn, Peter-Christian; Schwarz, Petra; Ruddle, Nancy H.; Weissmann, Charles; Aguzzi, Adriano

2005-02-01

Prions typically accumulate in nervous and lymphoid tissues. Because proinflammatory cytokines and immune cells are required for lymphoid prion replication, we tested whether inflammatory conditions affect prion pathogenesis. We administered prions to mice with five inflammatory diseases of the kidney, pancreas, or liver. In all cases, chronic lymphocytic inflammation enabled prion accumulation in otherwise prion-free organs. Inflammatory foci consistently correlated with lymphotoxin up-regulation and ectopic induction of FDC-M1+ cells expressing the normal cellular prion protein PrPC. By contrast, inflamed organs of mice lacking lymphotoxin-α or its receptor did not accumulate the abnormal isoform PrPSc, nor did they display infectivity upon prion inoculation. By expanding the tissue distribution of prions, chronic inflammatory conditions may act as modifiers of natural and iatrogenic prion transmission.

Prion propagation and toxicity occur in vitro with two-phase kinetics specific to strain and neuronal type.

PubMed

Hannaoui, Samia; Maatouk, Layal; Privat, Nicolas; Levavasseur, Etienne; Faucheux, Baptiste A; Haïk, Stéphane

2013-03-01

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that occur in humans and animals. The neuropathological hallmarks of TSEs are spongiosis, glial proliferation, and neuronal loss. The only known specific molecular marker of TSEs is the abnormal isoform (PrP(Sc)) of the host-encoded prion protein (PrP(C)), which accumulates in the brain of infected subjects and forms infectious prion particles. Although this transmissible agent lacks a specific nucleic acid component, several prion strains have been isolated. Prion strains are characterized by differences in disease outcome, PrP(Sc) distribution patterns, and brain lesion profiles at the terminal stage of the disease. The molecular factors and cellular mechanisms involved in strain-specific neuronal tropism and toxicity remain largely unknown. Currently, no cellular model exists to facilitate in vitro studies of these processes. A few cultured cell lines that maintain persistent scrapie infections have been developed, but only two of them have shown the cytotoxic effects associated with prion propagation. In this study, we have developed primary neuronal cultures to assess in vitro neuronal tropism and toxicity of different prion strains (scrapie strains 139A, ME7, and 22L). We have tested primary neuronal cultures enriched in cerebellar granular, striatal, or cortical neurons. Our results showed that (i) a strain-specific neuronal tropism operated in vitro; (ii) the cytotoxic effect varied among strains and neuronal cell types; (iii) prion propagation and toxicity occurred in two kinetic phases, a replicative phase followed by a toxic phase; and (iv) neurotoxicity peaked when abnormal PrP accumulation reached a plateau.

Prion Propagation and Toxicity Occur In Vitro with Two-Phase Kinetics Specific to Strain and Neuronal Type

PubMed Central

Hannaoui, Samia; Maatouk, Layal; Privat, Nicolas; Levavasseur, Etienne; Faucheux, Baptiste A.

2013-01-01

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that occur in humans and animals. The neuropathological hallmarks of TSEs are spongiosis, glial proliferation, and neuronal loss. The only known specific molecular marker of TSEs is the abnormal isoform (PrPSc) of the host-encoded prion protein (PrPC), which accumulates in the brain of infected subjects and forms infectious prion particles. Although this transmissible agent lacks a specific nucleic acid component, several prion strains have been isolated. Prion strains are characterized by differences in disease outcome, PrPSc distribution patterns, and brain lesion profiles at the terminal stage of the disease. The molecular factors and cellular mechanisms involved in strain-specific neuronal tropism and toxicity remain largely unknown. Currently, no cellular model exists to facilitate in vitro studies of these processes. A few cultured cell lines that maintain persistent scrapie infections have been developed, but only two of them have shown the cytotoxic effects associated with prion propagation. In this study, we have developed primary neuronal cultures to assess in vitro neuronal tropism and toxicity of different prion strains (scrapie strains 139A, ME7, and 22L). We have tested primary neuronal cultures enriched in cerebellar granular, striatal, or cortical neurons. Our results showed that (i) a strain-specific neuronal tropism operated in vitro; (ii) the cytotoxic effect varied among strains and neuronal cell types; (iii) prion propagation and toxicity occurred in two kinetic phases, a replicative phase followed by a toxic phase; and (iv) neurotoxicity peaked when abnormal PrP accumulation reached a plateau. PMID:23255799

PrPC Governs Susceptibility to Prion Strains in Bank Vole, While Other Host Factors Modulate Strain Features

PubMed Central

Espinosa, J. C.; Nonno, R.; Di Bari, M.; Aguilar-Calvo, P.; Pirisinu, L.; Fernández-Borges, N.; Vanni, I.; Vaccari, G.; Marín-Moreno, A.; Frassanito, P.; Lorenzo, P.; Agrimi, U.

2016-01-01

ABSTRACT Bank vole is a rodent species that shows differential susceptibility to the experimental transmission of different prion strains. In this work, the transmission features of a panel of diverse prions with distinct origins were assayed both in bank vole expressing methionine at codon 109 (Bv109M) and in transgenic mice expressing physiological levels of bank vole PrPC (the BvPrP-Tg407 mouse line). This work is the first systematic comparison of the transmission features of a collection of prion isolates, representing a panel of diverse prion strains, in a transgenic-mouse model and in its natural counterpart. The results showed very similar transmission properties in both the natural species and the transgenic-mouse model, demonstrating the key role of the PrP amino acid sequence in prion transmission susceptibility. However, differences in the PrPSc types propagated by Bv109M and BvPrP-Tg407 suggest that host factors other than PrPC modulate prion strain features. IMPORTANCE The differential susceptibility of bank voles to prion strains can be modeled in transgenic mice, suggesting that this selective susceptibility is controlled by the vole PrP sequence alone rather than by other species-specific factors. Differences in the phenotypes observed after prion transmissions in bank voles and in the transgenic mice suggest that host factors other than the PrPC sequence may affect the selection of the substrain replicating in the animal model. PMID:27654300

Infrared Microspectroscopy: A Multiple-Screening Platform for Investigating Single-Cell Biochemical Perturbations upon Prion Infection

PubMed Central

2011-01-01

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrPSc) possesses the capability to convert the host-encoded cellular isoform of the prion protein, PrPC, into nascent PrPSc. The present work aims at providing novel insight into cellular response upon prion infection evidenced by synchrotron radiation infrared microspectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations undergone by prion infected mouse hypothalamic GT1-1 cells at the cellular and subcellular level. A decrement in total cellular protein content upon prion infection was identified by infrared (IR) whole-cell spectra and validated by bicinchoninic acid assay and single-cell volume analysis by atomic force microscopy (AFM). Hierarchical cluster analysis (HCA) of IR data discriminated between infected and uninfected cells and allowed to deduce an increment of lysosomal bodies within the cytoplasm of infected GT1-1 cells, a hypothesis further confirmed by SR-IRMS at subcellular spatial resolution and fluorescent microscopy. The purpose of this work, therefore, consists of proposing IRMS as a powerful multiscreening platform, drawing on the synergy with conventional biological assays and microscopy techniques in order to increase the accuracy of investigations performed at the single-cell level. PMID:22778865

Infrared microspectroscopy: a multiple-screening platform for investigating single-cell biochemical perturbations upon prion infection.

PubMed

Didonna, Alessandro; Vaccari, Lisa; Bek, Alpan; Legname, Giuseppe

2011-03-16

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrP(Sc)) possesses the capability to convert the host-encoded cellular isoform of the prion protein, PrP(C), into nascent PrP(Sc). The present work aims at providing novel insight into cellular response upon prion infection evidenced by synchrotron radiation infrared microspectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations undergone by prion infected mouse hypothalamic GT1-1 cells at the cellular and subcellular level. A decrement in total cellular protein content upon prion infection was identified by infrared (IR) whole-cell spectra and validated by bicinchoninic acid assay and single-cell volume analysis by atomic force microscopy (AFM). Hierarchical cluster analysis (HCA) of IR data discriminated between infected and uninfected cells and allowed to deduce an increment of lysosomal bodies within the cytoplasm of infected GT1-1 cells, a hypothesis further confirmed by SR-IRMS at subcellular spatial resolution and fluorescent microscopy. The purpose of this work, therefore, consists of proposing IRMS as a powerful multiscreening platform, drawing on the synergy with conventional biological assays and microscopy techniques in order to increase the accuracy of investigations performed at the single-cell level.

Prion pathogenesis and secondary lymphoid organs (SLO)

PubMed Central

Mabbott, Neil A.

2012-01-01

Prion diseases are subacute neurodegenerative diseases that affect humans and a range of domestic and free-ranging animal species. These diseases are characterized by the accumulation of PrPSc, an abnormally folded isoform of the cellular prion protein (PrPC), in affected tissues. The pathology during prion disease appears to occur almost exclusively within the central nervous system. The extensive neurodegeneration which occurs ultimately leads to the death of the host. An intriguing feature of the prion diseases, when compared with other protein-misfolding diseases, is their transmissibility. Following peripheral exposure, some prion diseases accumulate to high levels within lymphoid tissues. The replication of prions within lymphoid tissue has been shown to be important for the efficient spread of disease to the brain. This article describes recent progress in our understanding of the cellular mechanisms that influence the propagation of prions from peripheral sites of exposure (such as the lumen of the intestine) to the brain. A thorough understanding of these events will lead to the identification of important targets for therapeutic intervention, or alternatively, reveal additional processes that influence disease susceptibility to peripherally-acquired prion diseases. PMID:22895090

Current Research Therapeutic Strategies for Alzheimer's Disease Treatment

PubMed Central

Folch, Jaume; Petrov, Dmitry; Ettcheto, Miren; Abad, Sonia; Sánchez-López, Elena; García, M. Luisa; Olloquequi, Jordi; Beas-Zarate, Carlos; Auladell, Carme; Camins, Antoni

2016-01-01

Alzheimer's disease (AD) currently presents one of the biggest healthcare issues in the developed countries. There is no effective treatment capable of slowing down disease progression. In recent years the main focus of research on novel pharmacotherapies was based on the amyloidogenic hypothesis of AD, which posits that the beta amyloid (Aβ) peptide is chiefly responsible for cognitive impairment and neuronal death. The goal of such treatments is (a) to reduce Aβ production through the inhibition of β and γ secretase enzymes and (b) to promote dissolution of existing cerebral Aβ plaques. However, this approach has proven to be only modestly effective. Recent studies suggest an alternative strategy centred on the inhibition of the downstream Aβ signalling, particularly at the synapse. Aβ oligomers may cause aberrant N-methyl-D-aspartate receptor (NMDAR) activation postsynaptically by forming complexes with the cell-surface prion protein (PrPC). PrPC is enriched at the neuronal postsynaptic density, where it interacts with Fyn tyrosine kinase. Fyn activation occurs when Aβ is bound to PrPC-Fyn complex. Fyn causes tyrosine phosphorylation of the NR2B subunit of metabotropic glutamate receptor 5 (mGluR5). Fyn kinase blockers masitinib and saracatinib have proven to be efficacious in treating AD symptoms in experimental mouse models of the disease. PMID:26881137

Stimulating the Release of Exosomes Increases the Intercellular Transfer of Prions.

PubMed

Guo, Belinda B; Bellingham, Shayne A; Hill, Andrew F

2016-03-04

Exosomes are small extracellular vesicles released by cells and play important roles in intercellular communication and pathogen transfer. Exosomes have been implicated in several neurodegenerative diseases, including prion disease and Alzheimer disease. Prion disease arises upon misfolding of the normal cellular prion protein, PrP(C), into the disease-associated isoform, PrP(Sc). The disease has a unique transmissible etiology, and exosomes represent a novel and efficient method for prion transmission. The precise mechanism by which prions are transmitted from cell to cell remains to be fully elucidated, although three hypotheses have been proposed: direct cell-cell contact, tunneling nanotubes, and exosomes. Given the reported presence of exosomes in biological fluids and in the lipid and nucleic acid contents of exosomes, these vesicles represent an ideal mechanism for encapsulating prions and potential cofactors to facilitate prion transmission. This study investigates the relationship between exosome release and intercellular prion dissemination. Stimulation of exosome release through treatment with an ionophore, monensin, revealed a corresponding increase in intercellular transfer of prion infectivity. Conversely, inhibition of exosome release using GW4869 to target the neutral sphingomyelinase pathway induced a decrease in intercellular prion transmission. Further examination of the effect of monensin on PrP conversion revealed that monensin also alters the conformational stability of PrP(C), leading to increased generation of proteinase K-resistant prion protein. The findings presented here provide support for a positive relationship between exosome release and intercellular transfer of prion infectivity, highlighting an integral role for exosomes in facilitating the unique transmissible nature of prions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Conformation-dependent epitopes recognized by prion protein antibodies probed using mutational scanning and deep sequencing.

PubMed

Doolan, Kyle M; Colby, David W

2015-01-30

Prion diseases are caused by a structural rearrangement of the cellular prion protein, PrP(C), into a disease-associated conformation, PrP(Sc), which may be distinguished from one another using conformation-specific antibodies. We used mutational scanning by cell-surface display to screen 1341 PrP single point mutants for attenuated interaction with four anti-PrP antibodies, including several with conformational specificity. Single-molecule real-time gene sequencing was used to quantify enrichment of mutants, returning 26,000 high-quality full-length reads for each screened population on average. Relative enrichment of mutants correlated to the magnitude of the change in binding affinity. Mutations that diminished binding of the antibody ICSM18 represented the core of contact residues in the published crystal structure of its complex. A similarly located binding site was identified for D18, comprising discontinuous residues in helix 1 of PrP, brought into close proximity to one another only when the alpha helix is intact. The specificity of these antibodies for the normal form of PrP likely arises from loss of this conformational feature after conversion to the disease-associated form. Intriguingly, 6H4 binding was found to depend on interaction with the same residues, among others, suggesting that its ability to recognize both forms of PrP depends on a structural rearrangement of the antigen. The application of mutational scanning and deep sequencing provides residue-level resolution of positions in the protein-protein interaction interface that are critical for binding, as well as a quantitative measure of the impact of mutations on binding affinity. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Neutral Sphingomyelinase Pathway Regulates Packaging of the Prion Protein into Exosomes*

PubMed Central

Guo, Belinda B.; Bellingham, Shayne A.; Hill, Andrew F.

2015-01-01

Prion diseases are a group of transmissible, fatal neurodegenerative disorders associated with the misfolding of the host-encoded prion protein, PrPC, into a disease-associated form, PrPSc. The transmissible prion agent is principally formed of PrPSc itself and is associated with extracellular vesicles known as exosomes. Exosomes are released from cells both in vitro and in vivo, and have been proposed as a mechanism by which prions spread intercellularly. The biogenesis of exosomes occurs within the endosomal system, through formation of intraluminal vesicles (ILVs), which are subsequently released from cells as exosomes. ILV formation is known to be regulated by the endosomal sorting complexes required for transport (ESCRT) machinery, although an alternative neutral sphingomyelinase (nSMase) pathway has been suggested to also regulate this process. Here, we investigate a role for the nSMase pathway in exosome biogenesis and packaging of PrP into these vesicles. Inhibition of the nSMase pathway using GW4869 revealed a role for the nSMase pathway in both exosome formation and PrP packaging. In agreement, targeted knockdown of nSMase1 and nSMase2 in mouse neurons using lentivirus-mediated RNAi also decreases exosome release, demonstrating the nSMase pathway regulates the biogenesis and release of exosomes. We also demonstrate that PrPC packaging is dependent on nSMase2, whereas the packaging of disease-associated PrPSc into exosomes occurs independently of nSMase2. These findings provide further insight into prion transmission and identify a pathway which directly assists exosome-mediated transmission of prions. PMID:25505180

An overview of animal prion diseases

PubMed Central

2011-01-01

Prion diseases are transmissible neurodegenerative conditions affecting human and a wide range of animal species. The pathogenesis of prion diseases is associated with the accumulation of aggregates of misfolded conformers of host-encoded cellular prion protein (PrPC). Animal prion diseases include scrapie of sheep and goats, bovine spongiform encephalopathy (BSE) or mad cow disease, transmissible mink encephalopathy, feline spongiform encephalopathy, exotic ungulate spongiform encephalopathy, chronic wasting disease of cervids and spongiform encephalopathy of primates. Although some cases of sporadic atypical scrapie and BSE have also been reported, animal prion diseases have basically occurred via the acquisition of infection from contaminated feed or via the exposure to contaminated environment. Scrapie and chronic wasting disease are naturally sustaining epidemics. The transmission of BSE to human has caused more than 200 cases of variant Cruetzfeldt-Jacob disease and has raised serious public health concerns. The present review discusses the epidemiology, clinical neuropathology, transmissibility and genetics of animal prion diseases. PMID:22044871

Amino acid sequence of the Amur tiger prion protein.

PubMed

Wu, Changde; Pang, Wanyong; Zhao, Deming

2006-10-01

Prion diseases are fatal neurodegenerative disorders in human and animal associated with conformational conversion of a cellular prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)). Various data indicate that the polymorphisms within the open reading frame (ORF) of PrP are associated with the susceptibility and control the species barrier in prion diseases. In the present study, partial Prnp from 25 Amur tigers (tPrnp) were cloned and screened for polymorphisms. Four single nucleotide polymorphisms (T423C, A501G, C511A, A610G) were found; the C511A and A610G nucleotide substitutions resulted in the amino acid changes Lysine171Glutamine and Alanine204Threoine, respectively. The tPrnp amino acid sequence is similar to house cat (Felis catus ) and sheep, but differs significantly from other two cat Prnp sequences that were previously deposited in GenBank.

Close Vicinity of PrP Expressing Cells (FDC) with Noradrenergic Fibers in Healthy Sheep Spleen

PubMed Central

Lezmi, S.; Hunsmann, G.; Baron, T.

2001-01-01

In naturally and experimentally occurring scrapie in sheep, prions invade the immune system and replicate in lymphoid organs. Here we analysed immunohistochemically, in seven spleens of 6-month-old healthy sheep, the nature of the cells expressing prion protein (PrP) potentially supporting prion replication, as well as their relationship with autonomic innervation. PrP was identified using either RB1 rabbit antiserum or 4F2 monoclonal antibody directed against AA 108–123 portion of the bovine and AA 79–92 of human prion protein respectively. Using double labelling analysis, we demonstrated that PrPc is expressed by follicular dendritic cells using a specific monoclonal antibody (CNA42). We also showed the close vicinity of these PrP expressing cells with noradrenergic fibers, using a polyclonal tyrosine hydroxylase antibody. Our results may help the study of the cellular requirements for the possible neuroinvasion from the spleen. PMID:11785673

Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions

PubMed Central

Moulick, Roumita; Das, Ranabir; Udgaonkar, Jayant B.

2015-01-01

The susceptibility of the cellular prion protein (PrPC) to convert to an alternative misfolded conformation (PrPSc), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23–231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a β-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4. PMID:26306043

Exploring the cause of initially reactive bovine brains on rapid tests for BSE

PubMed Central

Dudas, Sandor; James, Jace; Anderson, Renee; Czub, Stefanie

2015-01-01

ABSTRACT Bovine spongiform encephalopathy (BSE) is an invariably fatal prion disease of cattle. The identification of the zoonotic potential of BSE prompted safety officials to initiate surveillance testing for this disease. In Canada, BSE surveillance is primarily focused on high risk cattle including animals which are dead, down and unable to rise, diseased or distressed. This targeted surveillance results in the submission of brain samples with a wide range of tissue autolysis and associated contaminants. These contaminants have the potential to interfere with important steps of surveillance tests resulting in initially positive test results requiring additional testing to confirm the disease status of the animal. The current tests used for BSE screening in Canada utilize the relative protease resistance of the prion protein gained when it misfolds from PrPC to PrPSc as part of the disease process. Proteinase K completely digests PrPC in normal brains, but leaves most of the PrPSc in BSE positive brains intact which is detected using anti-prion antibodies. These tests are highly reliable but occasionally give rise to initially reactive/false positive results. Test results for these reactive samples were close to the positive/negative cut-off on a sub set of test platforms. This is in contrast to all of the previous Canadian positive samples whose numeric values on these same test platforms were 10 to 100 fold greater than the test positive/negative cut-off. Here we explore the potential reason why a sample is repeatedly positive on a sub-set of rapid surveillance tests, but negative on other test platforms. In order to better understand and identify what might cause these initial reactions, we have conducted a variety of rapid and confirmatory assays as well as bacterial isolation and identification on BSE positive, negative and initially reactive samples. We observed high levels of viable bacterial contamination in initially reactive samples suggesting that the reactivity may be related to bacterial factors. Several bacteria isolated from the initially reactive samples have characteristics of biofilm forming bacteria and this extracellular matrix might play a role in preventing complete digestion of PrPC in these samples. PMID:26689488

α-Secretase-derived Fragment of Cellular Prion, N1, Protects against Monomeric and Oligomeric Amyloid β (Aβ)-associated Cell Death*

PubMed Central

Guillot-Sestier, Marie-Victoire; Sunyach, Claire; Ferreira, Sergio T.; Marzolo, Maria-Paz; Bauer, Charlotte; Thevenet, Aurélie; Checler, Frédéric

2012-01-01

In physiological conditions, both β-amyloid precursor protein (βAPP) and cellular prion (PrPc) undergo similar disintegrin-mediated α-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-α (sAPPα) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid β (Aβ)-mediated toxicity. We first show that both recombinant sAPPα and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated βAPP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated βAPP (APPLDN) that yields Aβ oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APPLDN-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with Aβ-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPPα deficit taking place at the early asymptomatic phase of Alzheimer disease. PMID:22184125

The neutral sphingomyelinase pathway regulates packaging of the prion protein into exosomes.

PubMed

Guo, Belinda B; Bellingham, Shayne A; Hill, Andrew F

2015-02-06

Prion diseases are a group of transmissible, fatal neurodegenerative disorders associated with the misfolding of the host-encoded prion protein, PrP(C), into a disease-associated form, PrP(Sc). The transmissible prion agent is principally formed of PrP(Sc) itself and is associated with extracellular vesicles known as exosomes. Exosomes are released from cells both in vitro and in vivo, and have been proposed as a mechanism by which prions spread intercellularly. The biogenesis of exosomes occurs within the endosomal system, through formation of intraluminal vesicles (ILVs), which are subsequently released from cells as exosomes. ILV formation is known to be regulated by the endosomal sorting complexes required for transport (ESCRT) machinery, although an alternative neutral sphingomyelinase (nSMase) pathway has been suggested to also regulate this process. Here, we investigate a role for the nSMase pathway in exosome biogenesis and packaging of PrP into these vesicles. Inhibition of the nSMase pathway using GW4869 revealed a role for the nSMase pathway in both exosome formation and PrP packaging. In agreement, targeted knockdown of nSMase1 and nSMase2 in mouse neurons using lentivirus-mediated RNAi also decreases exosome release, demonstrating the nSMase pathway regulates the biogenesis and release of exosomes. We also demonstrate that PrP(C) packaging is dependent on nSMase2, whereas the packaging of disease-associated PrP(Sc) into exosomes occurs independently of nSMase2. These findings provide further insight into prion transmission and identify a pathway which directly assists exosome-mediated transmission of prions. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mouse-hamster chimeric prion protein (PrP) devoid of N-terminal residues 23-88 restores susceptibility to 22L prions, but not to RML prions in PrP-knockout mice.

PubMed

Uchiyama, Keiji; Miyata, Hironori; Yano, Masashi; Yamaguchi, Yoshitaka; Imamura, Morikazu; Muramatsu, Naomi; Das, Nandita Rani; Chida, Junji; Hara, Hideyuki; Sakaguchi, Suehiro

2014-01-01

Prion infection induces conformational conversion of the normal prion protein PrPC, into the pathogenic isoform PrPSc, in prion diseases. It has been shown that PrP-knockout (Prnp0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp 0/0 mice, neither developed the disease nor accumulated MHM2ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice developed the disease with abundant accumulation of MHM2ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2ScΔ23-88, and that the co-expressing wild-type PrPC can stimulate the conversion of MHM2Δ23-88 to MHM2ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp 0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp 0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice. However, wild-type PrPSc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice, compared with RML- and 22L-inoculated Prnp 0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2ScΔ23-88 without the help of the trans-acting PrPC, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrPC stimulates the conversion of MHM2Δ23-88 into MHM2ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrPC into PrPSc.

Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-Terminal Residues 23-88 Restores Susceptibility to 22L Prions, but Not to RML Prions in PrP-Knockout Mice

PubMed Central

Yano, Masashi; Yamaguchi, Yoshitaka; Imamura, Morikazu; Muramatsu, Naomi; Das, Nandita Rani; Chida, Junji; Hara, Hideyuki; Sakaguchi, Suehiro

2014-01-01

Prion infection induces conformational conversion of the normal prion protein PrPC, into the pathogenic isoform PrPSc, in prion diseases. It has been shown that PrP-knockout (Prnp0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp0/0 mice, neither developed the disease nor accumulated MHM2ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp0/+ mice developed the disease with abundant accumulation of MHM2ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2ScΔ23-88, and that the co-expressing wild-type PrPC can stimulate the conversion of MHM2Δ23-88 to MHM2ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp0/+ mice. However, wild-type PrPSc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp0/+ mice, compared with RML- and 22L-inoculated Prnp0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2ScΔ23-88 without the help of the trans-acting PrPC, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrPC stimulates the conversion of MHM2Δ23-88 into MHM2ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrPC into PrPSc. PMID:25330286

Does an infrasonic acoustic shock wave resonance of the manganese 3+ loaded/copper depleted prion protein initiate the pathogenesis of TSE?

PubMed

Purdey, Mark

2003-06-01

Intensive exposures to natural and artificial sources of infrasonic acoustic shock (tectonic disturbances, supersonic aeroplanes, etc.) have been observed in ecosystems supporting mammalian populations that are blighted by clusters of traditional and new variant strains of transmissible spongiform encephalopathy (TSE). But TSEs will only emerge in those 'infrasound-rich' environments which are simultaneously influenced by eco-factors that induce a high manganese (Mn)/low copper (Cu)-zinc (Zn) ratio in brains of local mammalian populations. Since cellular prion protein (PrPc) is a cupro-protein expressed throughout the circadian mediated pathways of the body, it is proposed that PrP's Cu component performs a role in the conduction and distribution of endogenous electromagnetic energy; energy that has been transduced from incoming ultraviolet, acoustic, geomagnetic radiations. TSE pathogenesis is initiated once Mn substitutes at the vacant Cu domain on PrPc and forms a nonpathogenic, protease resistant, 'sleeping' prion. A second stage of pathogenesis comes into play once a low frequency wave of infrasonic shock metamorphoses the piezoelectric atomic structure of the Mn 3+ component of the prion, thereby 'priming' the sleeping prion into its fully fledged, pathogenic TSE isoform - where the paramagnetic status of the Mn 3+ atom is transformed into a stable ferrimagnetic lattice work, due to the strong electron-phonon coupling resulting from the dynamic 'Jahn-Teller' type distortions of the oxygen octahedra specific to the trivalent Mn species. The so called 'infectivity' of the prion is a misnomer and should be correctly defined as the contagious field inducing capacity of the ferrimagnetic Mn 3+ component of the prion; which remains pathogenic at all temperatures below the 'curie point'. A progressive domino-like 'metal to ligand to metal' ferrimagnetic corruption of the conduits of electromagnetic superexchange is initiated. The TSE diseased brain can be likened to a solar charged battery on continuous charge; where the Mn contaminated/Cu depleted circadian-auditory pathways absorb and pile up, rather than conduct the vital life force energies of incoming ultra violet, acoustic and geomagnetic radiation. Instead of harnessing these energies for the body's own bio-rhythmic requirements, an infrasonic shock induced metamorphosis of the Mn atom intervenes; initiating an explosive pathogenesis that perverts the healthy pathways of darkness and light; Cu prions are replaced by hyperpolarized Mn 3+ prions that seed self perpetuating 'cluster bombs' of free radical mediated neurodegeneration. TSE ensues.

The Amino-Terminal PrP Domain Is Crucial to Modulate Prion Misfolding and Aggregation

PubMed Central

Cordeiro, Yraima; Kraineva, Julia; Gomes, Mariana P. B.; Lopes, Marilene H.; Martins, Vilma R.; Lima, Luís M. T. R.; Foguel, Débora; Winter, Roland; Silva, Jerson L.

2005-01-01

The main hypothesis for prion diseases is that the cellular protein (PrPC) can be altered into a misfolded, β-sheet-rich isoform (PrPSc), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we investigate the effects of amino-terminal deletion mutations, rPrPΔ51–90 and rPrPΔ32–121, on the stability and the packing properties of recombinant murine PrP. The region lacking in rPrPΔ51–90 is involved physiologically in copper binding and the other construct lacks more amino-terminal residues (from 32 to 121). The pressure stability is dramatically reduced with decreasing N-domain length and the process is not reversible for rPrPΔ51–90 and rPrPΔ32–121, whereas it is completely reversible for the wild-type form. Decompression to atmospheric pressure triggers immediate aggregation for the mutants in contrast to a slow aggregation process for the wild-type, as observed by Fourier-transform infrared spectroscopy. The temperature-induced transition leads to aggregation of all rPrPs, but the unfolding temperature is lower for the rPrP amino-terminal deletion mutants. The higher susceptibility to pressure of the amino-terminal deletion mutants can be explained by a change in hydration and cavity distribution. Taken together, our results show that the amino-terminal region has a pivotal role on the development of prion misfolding and aggregation. PMID:16040743

A closer look at prion strains

PubMed Central

Solforosi, Laura; Milani, Michela; Mancini, Nicasio; Clementi, Massimo; Burioni, Roberto

2013-01-01

Prions are infectious proteins that are responsible for transmissible spongiform encephalopathies (TSEs) and consist primarily of scrapie prion protein (PrPSc), a pathogenic isoform of the host-encoded cellular prion protein (PrPC). The absence of nucleic acids as essential components of the infectious prions is the most striking feature associated to these diseases. Additionally, different prion strains have been isolated from animal diseases despite the lack of DNA or RNA molecules. Mounting evidence suggests that prion-strain-specific features segregate with different PrPSc conformational and aggregation states. Strains are of practical relevance in prion diseases as they can drastically differ in many aspects, such as incubation period, PrPSc biochemical profile (e.g., electrophoretic mobility and glycoform ratio) and distribution of brain lesions. Importantly, such different features are maintained after inoculation of a prion strain into genetically identical hosts and are relatively stable across serial passages. This review focuses on the characterization of prion strains and on the wide range of important implications that the study of prion strains involves. PMID:23357828

Insights into mechanisms of transmission and pathogenesis from transgenic mouse models of prion diseases

PubMed Central

Moreno, Julie A.; Telling, Glenn C.

2018-01-01

Prions represent a new paradigm of protein-mediated information transfer. In the case of mammals, prions are the cause of fatal, transmissible neurodegenerative diseases, sometimes referred to as transmissible spongiform encephalopathies (TSE’s), which frequently occur as epidemics. An increasing body of evidence indicates that the canonical mechanism of conformational corruption of cellular prion protein (PrPC) by the pathogenic isoform (PrPSc) that is the basis of prion formation in TSE’s, is common to a spectrum of proteins associated with various additional human neurodegenerative disorders, including the more common Alzheimer’s and Parkinson’s diseases. The peerless infectious properties of TSE prions, and the unparalleled tools for their study, therefore enable elucidation of mechanisms of template-mediated conformational propagation that are generally applicable to these related disease states. Many unresolved issues remain including the exact molecular nature of the prion, the detailed cellular and molecular mechanisms of prion propagation, and the means by which prion diseases can be both genetic and infectious. In addition, we know little about the mechanism by which neurons degenerate during prion diseases. Tied to this, the physiological role of the normal form of the prion protein remains unclear and it is uncertain whether or not loss of this function contributes to prion pathogenesis. The factors governing the transmission of prions between species remain unclear, in particular the means by which prion strains and PrP primary structure interact to affect inter-species prion transmission. Despite all these unknowns, advances in our understanding of prions have occurred because of their transmissibility to experimental animals and the development of transgenic (Tg) mouse models has done much to further our understanding about various aspects of prion biology. In this review we will focus on advances in our understanding of prion biology that occurred in the past eight years since our last review of this topic. PMID:28861793

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

PubMed Central

Noble, Geoffrey P.; Wang, Daphne W.; Walsh, Daniel J.; Barone, Justin R.; Miller, Michael B.; Nishina, Koren A.; Li, Sheng; Supattapone, Surachai

2015-01-01

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure. PMID:26125623

Recombinant human prion protein fragment 90-231, a useful model to study prion neurotoxicity.

PubMed

Corsaro, Alessandro; Thellung, Stefano; Villa, Valentina; Nizzari, Mario; Aceto, Antonio; Florio, Tullio

2012-01-01

Transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of fatal neurodegenerative disorders of animals and humans. Human diseases include Creutzfeldt-Jakob (CJD) and Gerstmann-Straussler-Scheinker (GSSD) diseases, fatal familial insomnia, and Kuru. Human and animal TSEs share a common histopathology with a pathognomonic triad: spongiform vacuolation of the grey matter, neuronal death, glial proliferation, and, more inconstantly, amyloid deposition. According to the "protein only" hypothesis, TSEs are caused by a unique post-translational conversion of normal, host-encoded, protease-sensitive prion protein (PrP(sen) or PrP(C)) to an abnormal disease-associated isoform (PrP(res) or PrP(Sc)). To investigate the molecular mechanism of neurotoxicity induced by PrP(Sc) we developed a protocol to obtain millimolar amounts of soluble recombinant polypeptide encompassing the amino acid sequence 90-231 of human PrP (hPrP90-231). This protein corresponds to the protease-resistant prion protein fragment that originates after amino-terminal truncation. Importantly, hPrP90-231 has a flexible backbone that, similar to PrP(C), can undergo to structural rearrangement. This peptide, structurally resembling PrP(C), can be converted in a PrP(Sc)-like conformation, and thus represents a valuable model to study prion neurotoxicity. In this article we summarized our experimental evidence on the molecular and structural mechanisms responsible of hPrP90-231 neurotoxicity on neuroectodermal cell line SHSY5Y and the effects of some PrP pathogen mutations identified in familial TSE.

Similar folds with different stabilization mechanisms: the cases of prion and doppel proteins

PubMed Central

Colacino, Stefano; Tiana, Guido; Colombo, Giorgio

2006-01-01

Background Protein misfolding is the main cause of a group of fatal neurodegenerative diseases in humans and animals. In particular, in Prion-related diseases the normal cellular form of the Prion Protein PrP (PrPC) is converted into the infectious PrPSc through a conformational process during which it acquires a high β-sheet content. Doppel is a protein that shares a similar native fold, but lacks the scrapie isoform. Understanding the molecular determinants of these different behaviours is important both for biomedical and biophysical research. Results In this paper, the dynamical and energetic properties of the two proteins in solution is comparatively analyzed by means of long time scale explicit solvent, all-atom molecular dynamics in different temperature conditions. The trajectories are analyzed by means of a recently introduced energy decomposition approach (Tiana et al, Prot. Sci. 2004) aimed at identifying the key residues for the stabilization and folding of the protein. Our analysis shows that Prion and Doppel have two different cores stabilizing the native state and that the relative contribution of the nucleus to the global stability of the protein for Doppel is sensitively higher than for PrP. Moreover, under misfolding conditions the Doppel core is conserved, while the energy stabilization network of PrP is disrupted. Conclusion These observations suggest that different sequences can share similar native topology with different stabilizing interactions and that the sequences of the Prion and Doppel proteins may have diverged under different evolutionary constraints resulting in different folding and stabilization mechanisms. PMID:16857062

Bile Acids Reduce Prion Conversion, Reduce Neuronal Loss, and Prolong Male Survival in Models of Prion Disease

PubMed Central

Cortez, Leonardo M.; Campeau, Jody; Norman, Grant; Kalayil, Marian; Van der Merwe, Jacques; McKenzie, Debbie

2015-01-01

ABSTRACT Prion diseases are fatal neurodegenerative disorders associated with the conversion of cellular prion protein (PrPC) into its aberrant infectious form (PrPSc). There is no treatment available for these diseases. The bile acids tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) have been recently shown to be neuroprotective in other protein misfolding disease models, including Parkinson's, Huntington's and Alzheimer's diseases, and also in humans with amyotrophic lateral sclerosis. Here, we studied the therapeutic efficacy of these compounds in prion disease. We demonstrated that TUDCA and UDCA substantially reduced PrP conversion in cell-free aggregation assays, as well as in chronically and acutely infected cell cultures. This effect was mediated through reduction of PrPSc seeding ability, rather than an effect on PrPC. We also demonstrated the ability of TUDCA and UDCA to reduce neuronal loss in prion-infected cerebellar slice cultures. UDCA treatment reduced astrocytosis and prolonged survival in RML prion-infected mice. Interestingly, these effects were limited to the males, implying a gender-specific difference in drug metabolism. Beyond effects on PrPSc, we found that levels of phosphorylated eIF2α were increased at early time points, with correlated reductions in postsynaptic density protein 95. As demonstrated for other neurodegenerative diseases, we now show that TUDCA and UDCA may have a therapeutic role in prion diseases, with effects on both prion conversion and neuroprotection. Our findings, together with the fact that these natural compounds are orally bioavailable, permeable to the blood-brain barrier, and U.S. Food and Drug Administration-approved for use in humans, make these compounds promising alternatives for the treatment of prion diseases. IMPORTANCE Prion diseases are fatal neurodegenerative diseases that are transmissible to humans and other mammals. There are no disease-modifying therapies available, despite decades of research. Treatment targets have included inhibition of protein accumulation, clearance of toxic aggregates, and prevention of downstream neurodegeneration. No one target may be sufficient; rather, compounds which have a multimodal mechanism, acting on different targets, would be ideal. TUDCA and UDCA are bile acids that may fulfill this dual role. Previous studies have demonstrated their neuroprotective effects in several neurodegenerative disease models, and we now demonstrate that this effect occurs in prion disease, with an added mechanistic target of upstream prion seeding. Importantly, these are natural compounds which are orally bioavailable, permeable to the blood-brain barrier, and U.S. Food and Drug Administration-approved for use in humans with primary biliary cirrhosis. They have recently been proven efficacious in human amyotrophic lateral sclerosis. Therefore, these compounds are promising options for the treatment of prion diseases. PMID:25972546

Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins

PubMed Central

Imamura, Morikazu; Kato, Nobuko; Okada, Hiroyuki; Yoshioka, Miyako; Iwamaru, Yoshifumi; Shimizu, Yoshihisa; Mohri, Shirou; Yokoyama, Takashi; Murayama, Yuichi

2013-01-01

The central event in prion infection is the conformational conversion of host-encoded cellular prion protein (PrPC) into the pathogenic isoform (PrPSc). Diverse mammalian species possess the cofactors required for in vitro replication of PrPSc by protein-misfolding cyclic amplification (PMCA), but lower organisms, such as bacteria, yeasts, and insects, reportedly lack the essential cofactors. Various cellular components, such as RNA, lipids, and other identified cofactor molecules, are commonly distributed in both eukaryotes and prokaryotes, but the reasons for the absence of cofactor activity in lower organisms remain to be elucidated. Previously, we reported that brain-derived factors were necessary for the in vitro replication of glycosylphosphatidylinositol-anchored baculovirus-derived recombinant PrP (Bac-PrP). Here, we demonstrate that following protease digestion and heat treatment, insect cell lysates had the functional cofactor activity required for Bac-PrP replication by PMCA. Mammalian PrPSc seeds and Bac-PrPSc generated by PMCA using Bac-PrP and insect cell-derived cofactors showed similar pathogenicity and produced very similar lesions in the brains of inoculated mice. These results suggested that the essential cofactors required for the high-fidelity replication of mammalian PrPSc were present in the insect cells but that the cofactor activity was masked or inhibited in the native state. We suggest that not only RNA, but also DNA, are the key components of PMCA, although other cellular factors were necessary for the expression of the cofactor activity of nucleic acids. PMCA using only insect cell-derived substances (iPMCA) was highly useful for the ultrasensitive detection of PrPSc of some prion strains. PMID:24367521

Metals in obex and retropharyngeal lymph nodes of Illinois white-tailed deer and their variations associated with CWD status.

PubMed

Rivera, Nelda A; Novakofski, Jan; Weng, Hsin-Yi; Kelly, Amy; Satterthwaite-Phillips, Damian; Ruiz, Marilyn O; Mateus-Pinilla, Nohra

2015-01-01

Prion proteins (PrP(C)) are cell membrane glycoproteins that can be found in many cell types, but specially in neurons. Many studies have suggested PrP(C)'s participation in metal transport and cellular protection against stress in the central nervous system (CNS). On the other hand PrP(Sc), the misfolded isoform of PrP(C) and the pathogenic agent in transmissible spongiform encephalopathies (TSE), has been associated with brain metal dyshomeostasis in prion diseases. Thus, changes in metal concentration associated with protein misfolding and aggregation have been reported for human and animal prion diseases, as well as for other neurodegenerative disorders, such as Parkinson's and Alzheimer's disease. The use of metal concentrations in tissues as surrogate markers for early detection of TSEs has been suggested. Studies on the accumulation of metals in free-ranging white-tailed deer have not been conducted. This study established concentrations of copper, iron, manganese, and magnesium in 2 diagnostic tissues used for CWD testing (obex and retropharyngeal lymph nodes (RLN)). We compared these concentrations between tissues and in relation to CWD status. We established reference intervals (RIs) for these metals and explored their ability to discriminate between CWD-positive and CWD-negative animals. Our results indicate that independent of CWD status, white-tailed deer accumulate higher concentrations of Fe, Mn and Mg in RLN than in obex. White-tailed deer infected with CWD accumulated significantly lower concentrations of Mn and Fe than CWD-negative deer. These patterns differed from other species infected with prion diseases. Overlapping values between CWD positive and negative groups indicate that evaluation of these metals in obex and RLN may not be appropriate as a diagnostic tool for CWD infection in white-tailed deer. Because the CWD-negative deer were included in constructing the RIs, high specificities were expected and should be interpreted with caution. Due to the low sensitivity derived from the RIs, we do not recommend using metal concentrations for disease discrimination.

A New Method for the Characterization of Strain-Specific Conformational Stability of Protease-Sensitive and Protease-Resistant PrPSc

PubMed Central

Pirisinu, Laura; Di Bari, Michele; Marcon, Stefano; Vaccari, Gabriele; D'Agostino, Claudia; Fazzi, Paola; Esposito, Elena; Galeno, Roberta; Langeveld, Jan; Agrimi, Umberto; Nonno, Romolo

2010-01-01

Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrPSc, a disease-associated isoform of the host-encoded cellular protein (PrPC). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrPSc. However, PrPSc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrPSc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrPC and PrPSc by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrPSc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl]1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl]1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrPSc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrPSc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrPSc conformational stabilities of protease-resistant and protease-sensitive PrPSc and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep. PMID:20856860

β-sheet-like formation during the mechanical unfolding of prion protein

NASA Astrophysics Data System (ADS)

Tao, Weiwei; Yoon, Gwonchan; Cao, Penghui; Eom, Kilho; Park, Harold S.

2015-09-01

Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrPC, whose misfolded form PrPSc can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

PubMed Central

Timmes, Andrew G.; Moore, Roger A.; Fischer, Elizabeth R.; Priola, Suzette A.

2013-01-01

During prion infection, the normal, protease-sensitive conformation of prion protein (PrPC) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrPSc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrPSc in prion-infected brain homogenates as an initiating seed to convert PrPC and trigger the self-propagation of PrPSc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrPSc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrPSc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrPSc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res. PMID:23936256

PrkC-mediated phosphorylation of overexpressed YvcK protein regulates PBP1 protein localization in Bacillus subtilis mreB mutant cells.

PubMed

Foulquier, Elodie; Pompeo, Frédérique; Freton, Céline; Cordier, Baptiste; Grangeasse, Christophe; Galinier, Anne

2014-08-22

The YvcK protein has been shown to be necessary for growth under gluconeogenic conditions in Bacillus subtilis. Amazingly, its overproduction rescues growth and morphology defects of the actin-like protein MreB deletion mutant by restoration of PBP1 localization. In this work, we observed that YvcK was phosphorylated at Thr-304 by the protein kinase PrkC and that phosphorylated YvcK was dephosphorylated by the cognate phosphatase PrpC. We show that neither substitution of this threonine with a constitutively phosphorylated mimicking glutamic acid residue or a phosphorylation-dead mimicking alanine residue nor deletion of prkC or prpC altered the ability of B. subtilis to grow under gluconeogenic conditions. However, we observed that a prpC mutant and a yvcK mutant were more sensitive to bacitracin compared with the WT strain. In addition, the bacitracin sensitivity of strains in which YvcK Thr-304 was replaced with either an alanine or a glutamic acid residue was also affected. We also analyzed rescue of the mreB mutant strain by overproduction of YvcK in which the phosphorylation site was substituted. We show that YvcK T304A overproduction did not rescue the mreB mutant aberrant morphology due to PBP1 mislocalization. The same observation was made in an mreB prkC double mutant overproducing YvcK. Altogether, these data show that YvcK may have two distinct functions: 1) in carbon source utilization independent of its phosphorylation level and 2) in cell wall biosynthesis and morphogenesis through its phosphorylation state. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Prion subcellular fractionation reveals infectivity spectrum, with a high titre-low PrPres level disparity

PubMed Central

2012-01-01

Background Prion disease transmission and pathogenesis are linked to misfolded, typically protease resistant (PrPres) conformers of the normal cellular prion protein (PrPC), with the former posited to be the principal constituent of the infectious 'prion'. Unexplained discrepancies observed between detectable PrPres and infectivity levels exemplify the complexity in deciphering the exact biophysical nature of prions and those host cell factors, if any, which contribute to transmission efficiency. In order to improve our understanding of these important issues, this study utilized a bioassay validated cell culture model of prion infection to investigate discordance between PrPres levels and infectivity titres at a subcellular resolution. Findings Subcellular fractions enriched in lipid rafts or endoplasmic reticulum/mitochondrial marker proteins were equally highly efficient at prion transmission, despite lipid raft fractions containing up to eight times the levels of detectable PrPres. Brain homogenate infectivity was not differentially enhanced by subcellular fraction-specific co-factors, and proteinase K pre-treatment of selected fractions modestly, but equally reduced infectivity. Only lipid raft associated infectivity was enhanced by sonication. Conclusions This study authenticates a subcellular disparity in PrPres and infectivity levels, and eliminates simultaneous divergence of prion strains as the explanation for this phenomenon. On balance, the results align best with the concept that transmission efficiency is influenced more by intrinsic characteristics of the infectious prion, rather than cellular microenvironment conditions or absolute PrPres levels. PMID:22534096

Computational Calculation Of The Ionization Energies Of The Human Prion Protein By The Coarse-grain Method

NASA Astrophysics Data System (ADS)

Lyu, Justin; Andrianarijaona, V. M.

2016-05-01

The causes of the misfolding of prion protein -i.e. the transformation of PrPC to PrPSc - have not been clearly elucidated. Many studies have focused on identifying possible chemical conditions, such as pH, temperature and chemical denaturation, that may trigger the pathological transformation of prion proteins (Weiwei Tao, Gwonchan Yoon, Penghui Cao, `` β-sheet-like formation during the mechanical unfolding of prion protein'', The Journal of Chemical Physics, 2015, 143, 125101). Here, we attempt to calculate the ionization energies of the prion protein, which will be able to shed light onto the possible causes of the misfolding. We plan on using the coarse-grain method which allows for a more feasible calculation time by means of approximation. We believe that by being able to approximate the ionization potential, particularly that of the regions known to form stable β-strands of the PrPSc form, the possible sources of denaturation, be it chemical or mechanical, may be narrowed down.

Oxidized methionine is not a prion-specific covalent modification

USDA-ARS?s Scientific Manuscript database

The oxidation of methionine residues in the '-helical region of PrPC has been proposed to be important for prion formation. This proposal has been supported by structural studies, model systems and antibody-based experimental evidence. We developed a sensitive mass spectrometry-based method to stu...

High-resolution structure of infectious prion protein: the final frontier

PubMed Central

Diaz-Espinoza, Rodrigo; Soto, Claudio

2014-01-01

Prions are the proteinaceous infectious agents responsible for the transmission of prion diseases. The main or sole component of prions is the misfolded prion protein (PrPSc), which is able to template the conversion of the host’s natively folded form of the protein (PrPC). The detailed mechanism of prion replication and the high-resolution structure of PrPSc are unknown. The currently available information on PrPSc structure comes mostly from low-resolution biophysical techniques, which have resulted in quite divergent models. Recent advances in the production of infectious prions, using very pure recombinant protein, offer new hope for PrPSc structural studies. This review highlights the importance of, challenges for and recent progress toward elucidating the elusive structure of PrPSc, arguably the major pending milestone to reach in understanding prions. PMID:22472622

A short purification process for quantitative isolation of PrPSc from naturally occurring and experimental transmissible spongiform encephalopathies

PubMed Central

Polymenidou, Magdalini; Verghese-Nikolakaki, Susan; Groschup, Martin; Chaplin, Melanie J; Stack, Mick J; Plaitakis, Andreas; Sklaviadis, Theodoros

2002-01-01

Background Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases affecting both humans and animals. They are associated with post-translational conversion of the normal cellular prion protein (PrPC) into a heat- and protease-resistant abnormal isoform (PrPSc). Detection of PrPSc in individuals is widely utilized for the diagnosis of prion diseases. Methods TSE brain tissue samples have been processed in order to quantitatively isolate PrPSc. The protocol includes an initial homogenization, digestion with proteinase K and salt precipitation. Results Here we show that over 97 percent of the PrPSc present can be precipitated from infected brain material using this simple salting-out procedure for proteins. No chemically harsh conditions are used during the process in order to conserve the native quality of the isolated protein. Conclusion The resulting PrPSc-enriched preparation should provide a suitable substrate for analyzing the structure of the prion agent and for scavenging for other molecules with which it may associate. In comparison with most methods that exist today, the one described in this study is rapid, cost-effective and does not demand expensive laboratory equipment. PMID:12370086

A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity

PubMed Central

Munoz-Montesino, Carola; Sizun, Christina; Moudjou, Mohammed; Herzog, Laetitia; Reine, Fabienne; Igel-Egalon, Angelique; Barbereau, Clément; Chapuis, Jérôme; Ciric, Danica; Laude, Hubert; Béringue, Vincent; Rezaei, Human; Dron, Michel

2017-01-01

ABSTRACT Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the α-helical domain of the correctly folded protein and turns into a β-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrPC structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrPC nor for the formation of a stable prion structure. PMID:28281924

Solution structure of Syrian hamster prion protein rPrP(90-231).

PubMed

Liu, H; Farr-Jones, S; Ulyanov, N B; Llinas, M; Marqusee, S; Groth, D; Cohen, F E; Prusiner, S B; James, T L

1999-04-27

NMR has been used to refine the structure of Syrian hamster (SHa) prion protein rPrP(90-231), which is commensurate with the infectious protease-resistant core of the scrapie prion protein PrPSc. The structure of rPrP(90-231), refolded to resemble the normal cellular isoform PrPC spectroscopically and immunologically, has been studied using multidimensional NMR; initial results were published [James et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10086-10091]. We now report refinement with better definition revealing important structural and dynamic features which can be related to biological observations pertinent to prion diseases. Structure refinement was based on 2778 unambiguously assigned nuclear Overhauser effect (NOE) connectivities, 297 ambiguous NOE restraints, and 63 scalar coupling constants (3JHNHa). The structure is represented by an ensemble of 25 best-scoring structures from 100 structures calculated using ARIA/X-PLOR and further refined with restrained molecular dynamics using the AMBER 4.1 force field with an explicit shell of water molecules. The rPrP(90-231) structure features a core domain (residues 125-228), with a backbone atomic root-mean-square deviation (RMSD) of 0.67 A, consisting of three alpha-helices (residues 144-154, 172-193, and 200-227) and two short antiparallel beta-strands (residues 129-131 and 161-163). The N-terminus (residues 90-119) is largely unstructured despite some sparse and weak medium-range NOEs implying the existence of bends or turns. The transition region between the core domain and flexible N-terminus, i.e., residues 113-128, consists of hydrophobic residues or glycines and does not adopt any regular secondary structure in aqueous solution. There are about 30 medium- and long-range NOEs within this hydrophobic cluster, so it clearly manifests structure. Multiple discrete conformations are evident, implying the possible existence of one or more metastable states, which may feature in conversion of PrPC to PrPSc. To obtain a more comprehensive picture of rPrP(90-231), dynamics have been studied using amide hydrogen-deuterium exchange and 15N NMR relaxation times (T1 and T2) and 15N{1H} NOE measurements. Comparison of the structure with previous reports suggests sequence-dependent features that may be reflected in a species barrier to prion disease transmission.

Probing structural differences between PrPC and PrPSc by surface nitration and acetylation: evidence of conformational change in the C-terminus

USDA-ARS?s Scientific Manuscript database

We used two chemical modifiers, tetranitromethane (TNM) and acetic anhydride, which specifically target accessible tyrosine and lysine residues, respectively, to modify Syrian hamster recombinant PrP(90-231) (rPrP) and PrP27-30, aiming at finding locations of conformational change. Modified proteins...

Prion-Protein-interacting Amyloid-β Oligomers of High Molecular Weight Are Tightly Correlated with Memory Impairment in Multiple Alzheimer Mouse Models*

PubMed Central

Kostylev, Mikhail A.; Kaufman, Adam C.; Nygaard, Haakon B.; Patel, Pujan; Haas, Laura T.; Gunther, Erik C.; Vortmeyer, Alexander; Strittmatter, Stephen M.

2015-01-01

Alzheimer disease (AD) is characterized by amyloid-β accumulation, with soluble oligomers (Aβo) being the most synaptotoxic. However, the multivalent and unstable nature of Aβo limits molecular characterization and hinders research reproducibility. Here, we characterized multiple Aβo forms throughout the life span of various AD mice and in post-mortem human brain. Aβo exists in several populations, where prion protein (PrPC)-interacting Aβo is a high molecular weight Aβ assembly present in multiple mice and humans with AD. Levels of PrPC-interacting Aβo match closely with mouse memory and are equal or superior to other Aβ measures in predicting behavioral impairment. However, Aβo metrics vary considerably between mouse strains. Deleting PrPC expression in mice with relatively low PrPC-interacting Aβo (Tg2576) results in partial rescue of cognitive performance as opposed to complete recovery in animals with a high percentage of PrPC-interacting Aβo (APP/PSEN1). These findings highlight the relative contributions and interplay of Aβo forms in AD. PMID:26018073

Cell surface expression of PrP-c and the presence of scrapie prions in the blood of goats

USDA-ARS?s Scientific Manuscript database

Classical scrapie is a naturally occurring fatal brain disease of goats and sheep which is caused by prions, a novel class of infectious agent, and is accompanied by the accumulation of abnormal isoforms of prion protein (PrP-Sc) in certain neural and lymphoid tissues. Although collection of a blood...

Polythiophenes Inhibit Prion Propagation by Stabilizing Prion Protein (PrP) Aggregates*

PubMed Central

Margalith, Ilan; Suter, Carlo; Ballmer, Boris; Schwarz, Petra; Tiberi, Cinzia; Sonati, Tiziana; Falsig, Jeppe; Nyström, Sofie; Hammarström, Per; Åslund, Andreas; Nilsson, K. Peter R.; Yam, Alice; Whitters, Eric; Hornemann, Simone; Aguzzi, Adriano

2012-01-01

Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrPC (PrPSc) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrPSc to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23–231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23–231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrPSc by stabilizing the conformation of PrPC or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrPSc deposits. PMID:22493452

Oligomers of Amyloid β Prevent Physiological Activation of the Cellular Prion Protein-Metabotropic Glutamate Receptor 5 Complex by Glutamate in Alzheimer Disease.

PubMed

Haas, Laura T; Strittmatter, Stephen M

2016-08-12

The dysfunction and loss of synapses in Alzheimer disease are central to dementia symptoms. We have recently demonstrated that pathological Amyloid β oligomer (Aβo) regulates the association between intracellular protein mediators and the synaptic receptor complex composed of cellular prion protein (PrP(C)) and metabotropic glutamate receptor 5 (mGluR5). Here we sought to determine whether Aβo alters the physiological signaling of the PrP(C)-mGluR5 complex upon glutamate activation. We provide evidence that acute exposure to Aβo as well as chronic expression of familial Alzheimer disease mutant transgenes in model mice prevents protein-protein interaction changes of the complex induced by the glutamate analog 3,5-dihydroxyphenylglycine. We further show that 3,5-dihydroxyphenylglycine triggers the phosphorylation and activation of protein-tyrosine kinase 2-β (PTK2B, also referred to as Pyk2) and of calcium/calmodulin-dependent protein kinase II in wild-type brain slices but not in Alzheimer disease transgenic brain slices or wild-type slices incubated with Aβo. This study further distinguishes two separate Aβo-dependent signaling cascades, one dependent on extracellular Ca(2+) and Fyn kinase activation and the other dependent on the release of Ca(2+) from intracellular stores. Thus, Aβo triggers multiple distinct PrP(C)-mGluR5-dependent events implicated in neurodegeneration and dementia. We propose that targeting the PrP(C)-mGluR5 complex will reverse aberrant Aβo-triggered states of the complex to allow physiological fluctuations of glutamate signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Implications of prion adaptation and evolution paradigm for human neurodegenerative diseases.

PubMed

Kabir, M Enamul; Safar, Jiri G

2014-01-01

There is a growing body of evidence indicating that number of human neurodegenerative diseases, including Alzheimer disease, Parkinson disease, fronto-temporal dementias, and amyotrophic lateral sclerosis, propagate in the brain via prion-like intercellular induction of protein misfolding. Prions cause lethal neurodegenerative diseases in humans, the most prevalent being sporadic Creutzfeldt-Jakob disease (sCJD); they self-replicate and spread by converting the cellular form of prion protein (PrP(C)) to a misfolded pathogenic conformer (PrP(Sc)). The extensive phenotypic heterogeneity of human prion diseases is determined by polymorphisms in the prion protein gene, and by prion strain-specific conformation of PrP(Sc). Remarkably, even though informative nucleic acid is absent, prions may undergo rapid adaptation and evolution in cloned cells and upon crossing the species barrier. In the course of our investigation of this process, we isolated distinct populations of PrP(Sc) particles that frequently co-exist in sCJD. The human prion particles replicate independently and undergo competitive selection of those with lower initial conformational stability. Exposed to mutant substrate, the winning PrP(Sc) conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to the lowest stability. Thus, the evolution and adaptation of human prions is enabled by a dynamic collection of distinct populations of particles, whose evolution is governed by the selection of progressively less stable, faster replicating PrP(Sc) conformers. This fundamental biological mechanism may explain the drug resistance that some prions gained after exposure to compounds targeting PrP(Sc). Whether the phenotypic heterogeneity of other neurodegenerative diseases caused by protein misfolding is determined by the spectrum of misfolded conformers (strains) remains to be established. However, the prospect that these conformers may evolve and adapt by a prion-like mechanism calls for the reevaluation of therapeutic strategies that target aggregates of misfolded proteins, and argues for new therapeutic approaches that will focus on prior pathogenetic steps.

The Role of Shed PrPc in the Neuropathogenesis of HIV Infection.

PubMed

Megra, Bezawit W; Eugenin, Eliseo A; Berman, Joan W

2017-07-01

HIV-1 enters the CNS soon after peripheral infection and causes chronic neuroinflammation and neuronal damage that leads to cognitive impairment in 40-70% of HIV-infected people. The nonpathogenic cellular isoform of the human prion protein (PrP c ) is an adhesion molecule constitutively expressed in the CNS. Previously, our laboratory showed that shed PrP c (sPrP c ) is increased in the cerebrospinal fluid of HIV-infected people with cognitive deficits as compared with infected people with no impairment. In this article, we demonstrate that CCL2 and TNF-α, inflammatory mediators that are elevated in the CNS of HIV-infected people, increase shedding of PrP c from human astrocytes by increasing the active form of the metalloprotease ADAM10. We show that the consequence of this shedding can be the production of inflammatory mediators, because treatment of astrocytes with rPrP c increased secretion of CCL2, CXCL-12, and IL-8. Supernatants from rPrP c -treated astrocytes containing factors produced in response to this treatment, but not rPrP c by itself, cause increased chemotaxis of both uninfected and HIV-infected human monocytes, suggesting a role for sPrP c in monocyte recruitment into the brain. Furthermore, we examined whether PrP c participates in glutamate uptake and found that rPrP c decreased uptake of this metabolite in astrocytes, which could lead to neurotoxicity and neuronal loss. Collectively, our data characterize mediators involved in PrP c shedding and the effect of this sPrP c on monocyte chemotaxis and glutamate uptake from astrocytes. We propose that shedding of PrP c could be a potential target for therapeutics to limit the cognitive impairment characteristic of neuroAIDS. Copyright © 2017 by The American Association of Immunologists, Inc.

Changes in Protein Structure and Distribution Observed at Pre-Clinical Stages of Scrapie Pathogenesis

PubMed Central

Kretlow, Ariane; Wang, Qi; Beekes, Michael; Naumann, Dieter; Miller, Lisa M.

2011-01-01

Scrapie is a neurodegenerative disorder that involves the misfolding, aggregation and accumulation of the prion protein (PrP). The normal cellular PrP (PrPC) is rich in α-helical secondary structure, whereas the disease-associated pathogenic form of the protein (PrPSc) has an anomalously high β-sheet content. In this study, protein structural changes were examined in situ in the dorsal root ganglia from perorally 263K scrapie-infected and mock-infected hamsters using synchrotron Fourier Transform InfraRed Microspectroscopy (FTIRM) at four time points over the course of the disease (preclinical, 100 & 130 days post-infection (dpi); first clinical signs (~145 dpi); and terminal (~170 dpi)). Results showed clear changes in the total protein content, structure, and distribution as the disease progressed. At pre-clinical time points, the scrapie-infected animals exhibited a significant increase in protein expression, but the β-sheet protein content was significantly lower than controls. Based on these findings, we suggest that the pre-clinical stages of scrapie are characterized by an overexpression of proteins low in β-sheet content. As the disease progressed, the β-sheet content increased significantly. Immunostaining with a PrP-specific antibody, 3F4, confirmed that this increase was partly – but not solely – due to the formation of PrPSc in the tissue and indicated that other proteins high in β-sheet were produced, either by overexpression or misfolding. Elevated β-sheet was observed near the cell membrane at pre-clinical time points and also in the cytoplasm of infected neurons at later stages of infection. At the terminal stage of the disease, the protein expression declined significantly, likely due to degeneration and death of neurons. These dramatic changes in protein content and structure, especially at pre-clinical time points, emphasize the possibility for identifying other proteins involved in early pathogenesis, which are important for further understanding the disease. PMID:18625306

The Expanding Universe of Prion Diseases

PubMed Central

Watts, Joel C; Balachandran, Aru; Westaway, David

2006-01-01

Prions cause fatal and transmissible neurodegenerative disease. These etiological infectious agents are formed in greater part from a misfolded cell-surface protein called PrPC. Several mammalian species are affected by the diseases, and in the case of “mad cow disease” (BSE) the agent has a tropism for humans, with negative consequences for agribusiness and public health. Unfortunately, the known universe of prion diseases is expanding. At least four novel prion diseases—including human diseases variant Creutzfeldt-Jakob disease (vCJD) and sporadic fatal insomnia (sFI), bovine amyloidotic spongiform encephalopathy (BASE), and Nor98 of sheep—have been identified in the last ten years, and chronic wasting disease (CWD) of North American deer (Odocoileus Specis) and Rocky Mountain elk (Cervus elaphus nelsoni) is undergoing a dramatic spread across North America. While amplification (BSE) and dissemination (CWD, commercial sourcing of cervids from the wild and movement of farmed elk) can be attributed to human activity, the origins of emergent prion diseases cannot always be laid at the door of humankind. Instead, the continued appearance of new outbreaks in the form of “sporadic” disease may be an inevitable outcome in a situation where the replicating pathogen is host-encoded. PMID:16609731

The expanding universe of prion diseases.

PubMed

Watts, Joel C; Balachandran, Aru; Westaway, David

2006-03-01

Prions cause fatal and transmissible neurodegenerative disease. These etiological infectious agents are formed in greater part from a misfolded cell-surface protein called PrP(C). Several mammalian species are affected by the diseases, and in the case of "mad cow disease" (BSE) the agent has a tropism for humans, with negative consequences for agribusiness and public health. Unfortunately, the known universe of prion diseases is expanding. At least four novel prion diseases--including human diseases variant Creutzfeldt-Jakob disease (vCJD) and sporadic fatal insomnia (sFI), bovine amyloidotic spongiform encephalopathy (BASE), and Nor98 of sheep--have been identified in the last ten years, and chronic wasting disease (CWD) of North American deer (Odocoileus Specis) and Rocky Mountain elk (Cervus elaphus nelsoni) is undergoing a dramatic spread across North America. While amplification (BSE) and dissemination (CWD, commercial sourcing of cervids from the wild and movement of farmed elk) can be attributed to human activity, the origins of emergent prion diseases cannot always be laid at the door of humankind. Instead, the continued appearance of new outbreaks in the form of "sporadic" disease may be an inevitable outcome in a situation where the replicating pathogen is host-encoded.

Dissociation of recombinant prion autocatalysis from infectivity.

PubMed

Noble, Geoffrey P; Supattapone, Surachai

2015-01-01

Within the mammalian prion field, the existence of recombinant prion protein (PrP) conformers with self-replicating (ie. autocatalytic) activity in vitro but little to no infectious activity in vivo challenges a key prediction of the protein-only hypothesis of prion replication--that autocatalytic PrP conformers should be infectious. To understand this dissociation of autocatalysis from infectivity, we recently performed a structural and functional comparison between a highly infectious and non-infectious pair of autocatalytic recombinant PrP conformers derived from the same initial prion strain. (1) We identified restricted, C-terminal structural differences between these 2 conformers and provided evidence that these relatively subtle differences prevent the non-infectious conformer from templating the conversion of native PrP(C) substrates containing a glycosylphosphatidylinositol (GPI) anchor. (1) In this article we discuss a model, consistent with these findings, in which recombinant PrP, lacking post-translational modifications and associated folding constraints, is capable of adopting a wide variety of autocatalytic conformations. Only a subset of these recombinant conformers can be adopted by post-translationally modified native PrP(C), and this subset represents the recombinant conformers with high specific infectivity. We examine this model's implications for the generation of highly infectious recombinant prions and the protein-only hypothesis of prion replication.

Mechanisms of triggering H1 helix in prion proteins unfolding revealed by molecular dynamic simulation

NASA Astrophysics Data System (ADS)

Tseng, Chih-Yuan; Lee, H. C.

2006-03-01

In template-assistance model, normal Prion protein (PrP^C), the pathogen to cause several prion diseases such as Creutzfeldt-Jakob (CJD) in human, Bovine Spongiform Encephalopathy (BSE) in cow, and scrapie in sheep, converts to infectious prion (PrP^Sc) through a transient interaction with PrP^Sc. Furthermore, conventional studies showed S1-H1-S2 region in PrP^C to be the template of S1-S2 β-sheet in PrP^Sc, and Prion protein's conformational conversion may involve an unfolding of H1 and refolding into β-sheet. Here we prepare several mouse prion peptides that contain S1-H1-S2 region with specific different structures, which are corresponding to specific interactions, to investigate possible mechanisms to trigger H1 α-helix unfolding process via molecular dynamic simulation. Three properties, conformational transition, salt-bridge in H1, and hydrophobic solvent accessible surface (SAS) are analyzed. From these studies, we found the interaction that triggers H1 unfolding to be the one that causes dihedral angle at residue Asn^143 changes. Whereas interactions that cause S1 segment's conformational changes play a minor in this process. These studies offers an additional evidence for template-assistance model.

Shadoo/PrP (Sprn0/0/Prnp0/0) double knockout mice

PubMed Central

Daude, Nathalie; Westaway, David

2012-01-01

Shadoo (Sho) is a brain glycoprotein with similarities to the unstructured region of PrPC. Frameshift alleles of the Sho gene, Sprn, are reported in variant Creutzfeldt-Jakob disease (vCJD) patients while Sprn mRNA knockdown in PrP-null (Prnp0/0) embryos produces lethality, advancing Sho as the hypothetical PrP-like “pi” protein. Also, Sho levels are reduced as misfolded PrP accumulates during prion infections. To penetrate these issues we created Sprn null alleles (Daude et al., Proc. Natl. Acad. Sci USA 2012; 109(23): 9035–40). Results from the challenge of Sprn null and TgSprn transgenic mice with rodent-adapted prions coalesce to define downregulation of Sho as a “tracer” for the formation of misfolded PrP. However, classical BSE and rodent-adapted BSE isolates may behave differently, as they do for other facets of the pathogenic process, and this intriguing variation warrants closer scrutiny. With regards to physiological function, double knockout mice (Sprn0/0/Prnp0/0) mice survived to over 600 d of age. This suggests that Sho is not pi, or, given the accumulating data for many activities for PrPC, that the pi hypothesis invoking a discrete signaling pathway to maintain neuronal viability is no longer tenable. PMID:22929230

Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

NASA Astrophysics Data System (ADS)

Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A.; Iyer, Pooja; Lane, Fiona M.; Graham, James F.; Goldmann, Wilfred; Pinheiro, Teresa J. T.; Gill, Andrew C.

2015-10-01

The β2-α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

The Structural Architecture of an Infectious Mammalian Prion Using Electron Cryomicroscopy.

PubMed

Vázquez-Fernández, Ester; Vos, Matthijn R; Afanasyev, Pavel; Cebey, Lino; Sevillano, Alejandro M; Vidal, Enric; Rosa, Isaac; Renault, Ludovic; Ramos, Adriana; Peters, Peter J; Fernández, José Jesús; van Heel, Marin; Young, Howard S; Requena, Jesús R; Wille, Holger

2016-09-01

The structure of the infectious prion protein (PrPSc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrPSc replicates by converting the non-infectious, cellular prion protein (PrPC) into the misfolded, infectious conformer through an unknown mechanism. PrPSc and its N-terminally truncated variant, PrP 27-30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27-30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 Å3, predicted the height of each PrP 27-30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.

Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

USGS Publications Warehouse

Johnson, Christopher J.; Carlson, Christina M.; Morawski, Aaron R.; Manthei, Alyson; Cashman, Neil R.

2015-01-01

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitroprion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Elucidation of Prion Protein Conformational Changes Associated with Infectivity by Fluorescence Spectroscopy

DTIC Science & Technology

2007-06-01

developed the β-spiral model for PrPSc structure by using molecular dynamics simulations with the sequence of human PrPC (residues 90-231) under...SUPPLEMENTARY NOTES exerpts from thesis of Jessica Gilbert 14. ABSTRACT Steady state and lifetime fluorescence measurements were made on 12...lieu of F. We tried both methods. See Ms. Jessica Gilbert’s MS thesis for details, which is included. 6 Figure 4. Acrylamide quenching of Trp

Emergence of two prion subtypes in ovine PrP transgenic mice infected with human MM2-cortical Creutzfeldt-Jakob disease prions.

PubMed

Chapuis, Jérôme; Moudjou, Mohammed; Reine, Fabienne; Herzog, Laetitia; Jaumain, Emilie; Chapuis, Céline; Quadrio, Isabelle; Boulliat, Jacques; Perret-Liaudet, Armand; Dron, Michel; Laude, Hubert; Rezaei, Human; Béringue, Vincent

2016-02-05

Mammalian prions are proteinaceous pathogens responsible for a broad range of fatal neurodegenerative diseases in humans and animals. These diseases can occur spontaneously, such as Creutzfeldt-Jakob disease (CJD) in humans, or be acquired or inherited. Prions are primarily formed of macromolecular assemblies of the disease-associated prion protein PrP(Sc), a misfolded isoform of the host-encoded prion protein PrP(C). Within defined host-species, prions can exist as conformational variants or strains. Based on both the M/V polymorphism at codon 129 of PrP and the electrophoretic signature of PrP(Sc) in the brain, sporadic CJD is classified in different subtypes, which may encode different strains. A transmission barrier, the mechanism of which remains unknown, limits prion cross-species propagation. To adapt to the new host, prions have the capacity to 'mutate' conformationally, leading to the emergence of a variant with new biological properties. Here, we transmitted experimentally one rare subtype of human CJD, designated cortical MM2 (129 MM with type 2 PrP(Sc)), to transgenic mice overexpressing either human or the VRQ allele of ovine PrP(C). In marked contrast with the reported absence of transmission to knock-in mice expressing physiological levels of human PrP, this subtype transmitted faithfully to mice overexpressing human PrP, and exhibited unique strain features. Onto the ovine PrP sequence, the cortical MM2 subtype abruptly evolved on second passage, thereby allowing emergence of a pair of strain variants with distinct PrP(Sc) biochemical characteristics and differing tropism for the central and lymphoid tissues. These two strain components exhibited remarkably distinct replicative properties in cell-free amplification assay, allowing the 'physical' cloning of the minor, lymphotropic component, and subsequent isolation in ovine PrP mice and RK13 cells. Here, we provide in-depth assessment of the transmissibility and evolution of one rare subtype of sporadic CJD upon homologous and heterologous transmission. The notion that the environment or matrix where replication is occurring is key to the selection and preferential amplification of prion substrain components raises new questions on the determinants of prion replication within and between species. These data also further interrogate on the interplay between animal and human prions.

The Antemortem Detection and Conformational Switches of Prion Proteins

DTIC Science & Technology

2006-07-01

distribution of di-, mono-, and unglycosylated PrPC molecules (Fig. 7B, lane 2). Subsequent elution of the column with N-acetyl glucosamine yielded...pellet was washed in 50 mM Tris, pH 8.0, 150 mM NaCl, and then solubilized in 6 M guanidinium hydrochloride , 50 mM Tris pH 8.0, 150 mM NaCl. The...and 150mM sodium chloride (TCl) containing 6M guanidinium hydrochloride . The supernatant was centrifuged for 30min at 18,000g. The protein was

Ab initio Study of Transition metal binding to the Prion Protein

NASA Astrophysics Data System (ADS)

Cox, Daniel L.; Singh, Rajiv R. P.; Pan, Jianping

2004-03-01

Fundamental understanding of the prion protein (PrP) is of critical public health importance in view of mad cow and chronic wasting diseases. In recent years, it has been shown that the normal form (PrP^c) binds copper^1), and the structure of the copper binding domain has been elaborated. Hypotheses about toxicity associated with binding of other metals (notably manganese) have been put forward, Accordingly, using the ab initio SIESTA density functional theory code^2), we calculated the binding energy E_B(M) of M-(PrP) complexes relative to initially uncomplexed M ions, with M=Cu,Ni,Zn,Mn and (PrP)^* the minimal binding domain. The binding energy trend is E_B(Ni)>E_B(Cu)>E_B(Zn)>E_B(Mn), consistent with recent experiments apart from the surprising stability of Ni. We will also present preliminary results for binding of initially complexed M ions. *-Supported by U.S. DOE, Office of Basic Energy Sciences, Division of Materials Research 1) G.S. Jackson et al., Proc. Nat. Acad. Sci. (USA) 98, 8531 (2001). 2) P. Ordejón, et al., Phys. Rev. B53, R10441 (1996); J.M. Soler et al., J. Phys. Cond. Matt. 14, 2745 (2002).

Mammalian prions

PubMed Central

Salamat, Muhammad Khalid; Munoz-Montesino, Carola; Moudjou, Mohammed; Rezaei, Human; Laude, Hubert; Béringue, Vincent; Dron, Michel

2013-01-01

Upon prion infection, abnormal prion protein (PrPSc) self-perpetuate by conformational conversion of α-helix-rich PrPC into β sheet enriched form, leading to formation and deposition of PrPSc aggregates in affected brains. However the process remains poorly understood at the molecular level and the regions of PrP critical for conversion are still debated. Minimal amino acid substitutions can impair prion replication at many places in PrP. Conversely, we recently showed that bona fide prions could be generated after introduction of eight and up to 16 additional amino acids in the H2-H3 inter-helix loop of PrP. Prion replication also accommodated the insertions of an octapeptide at different places in the last turns of H2. This reverse genetic approach reveals an unexpected tolerance of prions to substantial sequence changes in the protease-resistant part which is associated with infectivity. It also demonstrates that conversion does not require the presence of a specific sequence in the middle of the H2-H3 area. We discuss the implications of our findings according to different structural models proposed for PrPSc and questioned the postulated existence of an N- or C-terminal prion domain in the protease-resistant region. PMID:23232499

Identification of bilateral changes in TID1 expression in the 6-OHDA rat model of Parkinson's disease.

PubMed

Proft, Juliane; Faraji, Jamshid; Robbins, Jerrah C; Zucchi, Fabiola C R; Zhao, Xiaoxi; Metz, Gerlinde A; Braun, Janice E A

2011-01-01

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra and the aggregation of α-synuclein into Lewy bodies. Existing therapies address motor dysfunction but do not halt progression of the disease. A still unresolved question is the biochemical pathway that modulates the outcome of protein misfolding and aggregation processes in PD. The molecular chaperone network plays an important defensive role against cellular protein misfolding and has been identified as protective in experimental models of protein misfolding diseases like PD. Molecular mechanisms underlying chaperone-neuroprotection are actively under investigation. Current evidence implicates a number of molecular chaperones in PD including Hsp25, Hsp70 and Hsp90, however their precise involvement in the neurodegenerative cascade is unresolved. The J protein family (DnaJ or Hsp40 protein family) has long been known to be important in protein conformational processes.We assessed sensory and motor function of control and PD rats and then evaluated the brain region-specific expression levels of select J proteins by Western analysis. Surprisingly, we observed a widespread 26 kDa breakdown product of the J protein, TID1, (tumorous imaginal discs, mtHsp40 or DnaJ3) in a 6-hydroxydopamine (6-OHDA) rat model of PD in which food handling, gait symmetry and sensory performance were impaired. Greater behavioral deficits were associated with lower TID1 expression. Furthermore, direct application of either 6-OHDA or MPP+ (1-methyl-4-phenylpyridinum) to CAD (CNS-derived catecholinaminergic neuronal cell line) cell cultures, reduced TID1 expression levels.Our results suggest that changes in cellular TID1 are a factor in the pathogenesis of PD by impeding functional and structural compensation and exaggerating neurodegenerative processes. In contrast, no changes were observed in CSPα, Hsp40, Hsp70, Hsc70 and PrP(C) levels and no activation of caspase3 was observed. This study links TID1 to PD and provides a new target for therapeutics that halts the PD progression.

Melt-processed polymeric cellular dosage forms for immediate drug release.

PubMed

Blaesi, Aron H; Saka, Nannaji

2015-12-28

The present immediate-release solid dosage forms, such as the oral tablets and capsules, comprise granular matrices. While effective in releasing the drug rapidly, they are fraught with difficulties inherent in processing particulate matter. By contrast, liquid-based processes would be far more predictable; but the standard cast microstructures are unsuited for immediate-release because they resist fluid percolation and penetration. In this article, we introduce cellular dosage forms that can be readily prepared from polymeric melts by incorporating the nucleation, growth, and coalescence of microscopic gas bubbles in a molding process. We show that the cell topology and formulation of such cellular structures can be engineered to reduce the length-scale of the mass-transfer step, which determines the time of drug release, from as large as the dosage form itself to as small as the thickness of the cell wall. This allows the cellular dosage forms to achieve drug release rates over an order of magnitude faster compared with those of cast matrices, spanning the entire spectrum of immediate-release and beyond. The melt-processed polymeric cellular dosage forms enable predictive design of immediate-release solid dosage forms by tailoring microstructures, and could be manufactured efficiently in a single step.

Genetic predictions of prion disease susceptibility in carnivore species based on variability of the prion gene coding region.

PubMed

Stewart, Paula; Campbell, Lauren; Skogtvedt, Susan; Griffin, Karen A; Arnemo, Jon M; Tryland, Morten; Girling, Simon; Miller, Michael W; Tranulis, Michael A; Goldmann, Wilfred

2012-01-01

Mammalian species vary widely in their apparent susceptibility to prion diseases. For example, several felid species developed prion disease (feline spongiform encephalopathy or FSE) during the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, whereas no canine BSE cases were detected. Whether either of these or other groups of carnivore species can contract other prion diseases (e.g. chronic wasting disease or CWD) remains an open question. Variation in the host-encoded prion protein (PrP(C)) largely explains observed disease susceptibility patterns within ruminant species, and may explain interspecies differences in susceptibility as well. We sequenced and compared the open reading frame of the PRNP gene encoding PrP(C) protein from 609 animal samples comprising 29 species from 22 genera of the Order Carnivora; amongst these samples were 15 FSE cases. Our analysis revealed that FSE cases did not encode an identifiable disease-associated PrP polymorphism. However, all canid PrPs contained aspartic acid or glutamic acid at codon 163 which we propose provides a genetic basis for observed susceptibility differences between canids and felids. Among other carnivores studied, wolverine (Gulo gulo) and pine marten (Martes martes) were the only non-canid species to also express PrP-Asp163, which may impact on their prion diseases susceptibility. Populations of black bear (Ursus americanus) and mountain lion (Puma concolor) from Colorado showed little genetic variation in the PrP protein and no variants likely to be highly resistant to prions in general, suggesting that strain differences between BSE and CWD prions also may contribute to the limited apparent host range of the latter.

The Physical Relationship between Infectivity and Prion Protein Aggregates Is Strain-Dependent

PubMed Central

Tixador, Philippe; Herzog, Laëtitia; Reine, Fabienne; Jaumain, Emilie; Chapuis, Jérôme; Le Dur, Annick; Laude, Hubert; Béringue, Vincent

2010-01-01

Prions are unconventional infectious agents thought to be primarily composed of PrPSc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrPC). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrPSc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrPSc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrPSc aggregates from PrPC. The distribution of PrPSc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrPSc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12–30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrPSc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics. PMID:20419156

Genetic Predictions of Prion Disease Susceptibility in Carnivore Species Based on Variability of the Prion Gene Coding Region

PubMed Central

Stewart, Paula; Campbell, Lauren; Skogtvedt, Susan; Griffin, Karen A.; Arnemo, Jon M.; Tryland, Morten; Girling, Simon; Miller, Michael W.; Tranulis, Michael A.; Goldmann, Wilfred

2012-01-01

Mammalian species vary widely in their apparent susceptibility to prion diseases. For example, several felid species developed prion disease (feline spongiform encephalopathy or FSE) during the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, whereas no canine BSE cases were detected. Whether either of these or other groups of carnivore species can contract other prion diseases (e.g. chronic wasting disease or CWD) remains an open question. Variation in the host-encoded prion protein (PrPC) largely explains observed disease susceptibility patterns within ruminant species, and may explain interspecies differences in susceptibility as well. We sequenced and compared the open reading frame of the PRNP gene encoding PrPC protein from 609 animal samples comprising 29 species from 22 genera of the Order Carnivora; amongst these samples were 15 FSE cases. Our analysis revealed that FSE cases did not encode an identifiable disease-associated PrP polymorphism. However, all canid PrPs contained aspartic acid or glutamic acid at codon 163 which we propose provides a genetic basis for observed susceptibility differences between canids and felids. Among other carnivores studied, wolverine (Gulo gulo) and pine marten (Martes martes) were the only non-canid species to also express PrP-Asp163, which may impact on their prion diseases susceptibility. Populations of black bear (Ursus americanus) and mountain lion (Puma concolor) from Colorado showed little genetic variation in the PrP protein and no variants likely to be highly resistant to prions in general, suggesting that strain differences between BSE and CWD prions also may contribute to the limited apparent host range of the latter. PMID:23236380

Determination of the mechanical properties of solid and cellular polymeric dosage forms by diametral compression.

PubMed

Blaesi, Aron H; Saka, Nannaji

2016-07-25

At present, the immediate-release solid dosage forms, such as the oral tablets and capsules, are granular solids. They release drug rapidly and have adequate mechanical properties, but their manufacture is fraught with difficulties inherent in processing particulate matter. Such difficulties, however, could be overcome by liquid-based processing. Therefore, we have recently introduced polymeric cellular (i.e., highly porous) dosage forms prepared from a melt process. Experiments have shown that upon immersion in a dissolution medium, the cellular dosage forms with polyethylene glycol (PEG) as excipient and with predominantly open-cell topology disintegrate by exfoliation, thus enabling rapid drug release. If the volume fraction of voids of the open-cell structures is too large, however, their mechanical strength is adversely affected. At present, the common method for determining the tensile strength of brittle, solid dosage forms (such as select granular forms) is the diametral compression test. In this study, the theory of diametral compression is first refined to demonstrate that the relevant mechanical properties of ductile and cellular solids (i.e., the elastic modulus and the yield strength) can also be extracted from this test. Diametral compression experiments are then conducted on PEG-based solid and cellular dosage forms. It is found that the elastic modulus and yield strength of the open-cell structures are about an order of magnitude smaller than those of the non-porous solids, but still are substantially greater than the stiffness and strength requirements for handling the dosage forms manually. This work thus demonstrates that melt-processed polymeric cellular dosage forms that release drug rapidly can be designed and manufactured to have adequate mechanical properties. Copyright © 2016. Published by Elsevier B.V.

Effects of different transferrin forms on transferrin receptor expression, iron uptake, and cellular proliferation of human leukemic HL60 cells. Mechanisms responsible for the specific cytotoxicity of transferrin-gallium.

PubMed Central

Chitambar, C R; Seligman, P A

1986-01-01

We have previously shown that human leukemic cells proliferate normally in serum-free media containing various transferrin forms, but the addition of transferrin-gallium leads to inhibition of cellular proliferation. Because gallium has therapeutic potential, the effects of transferrin-gallium on leukemic cell proliferation, transferrin receptor expression, and cellular iron utilization were studied. The cytotoxicity of gallium is considerably enhanced by its binding to transferrin and cytotoxicity can be reversed by transferrin-iron but not by other transferrin forms. Exposure to transferrin-gallium leads to a marked increase in cell surface transferrin binding sites, but despite this, cellular 59Fe incorporation is inappropriately low. Although shunting of transferrin-gallium to another cellular compartment has not been ruled out, other studies suggest that transferrin-gallium impairs intracellular release of 59Fe from transferrin by interfering with processes responsible for intracellular acidification. These studies, taken together, demonstrate that inhibition of cellular iron incorporation by transferrin-gallium is a prerequisite for inhibition of cellular proliferation. PMID:3465751

Asymmetric cellular memory in bacteria exposed to antibiotics.

PubMed

Mathis, Roland; Ackermann, Martin

2017-03-09

The ability to form a cellular memory and use it for cellular decision-making could help bacteria to cope with recurrent stress conditions. We analyzed whether bacteria would form a cellular memory specifically if past events are predictive of future conditions. We worked with the asymmetrically dividing bacterium Caulobacter crescentus where past events are expected to only be informative for one of the two cells emerging from division, the sessile cell that remains in the same microenvironment and does not migrate. Time-resolved analysis of individual cells revealed that past exposure to low levels of antibiotics increases tolerance to future exposure for the sessile but not for the motile cell. Using computer simulations, we found that such an asymmetry in cellular memory could be an evolutionary response to situations where the two cells emerging from division will experience different future conditions. Our results raise the question whether bacteria can evolve the ability to form and use cellular memory conditionally in situations where it is beneficial.

Double-Edge Sword of Sustained ROCK Activation in Prion Diseases through Neuritogenesis Defects and Prion Accumulation

PubMed Central

Alleaume-Butaux, Aurélie; Nicot, Simon; Pietri, Mathéa; Baudry, Anne; Dakowski, Caroline; Tixador, Philippe; Ardila-Osorio, Hector; Haeberlé, Anne-Marie; Bailly, Yannick; Peyrin, Jean-Michel; Launay, Jean-Marie; Kellermann, Odile; Schneider, Benoit

2015-01-01

In prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrPSc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrPSc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrPSc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrPC by TACE α-secretase, which physiologically precludes PrPSc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc. In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrPSc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases. PMID:26241960

Phase separation and the formation of cellular bodies

NASA Astrophysics Data System (ADS)

Xu, Bin; Broedersz, Chase P.; Meir, Yigal; Wingreen, Ned S.

Cellular bodies in eukaryotic cells spontaneously assemble to form cellular compartments. Among other functions, these bodies carry out essential biochemical reactions. Cellular bodies form micron-sized structures, which, unlike canonical cell organelles, are not surrounded by membranes. A recent in vitro experiment has shown that phase separation of polymers in solution can explain the formation of cellular bodies. We constructed a lattice-polymer model to capture the essential mechanism leading to this phase separation. We used both analytical and numerical tools to predict the phase diagram of a system of two interacting polymers, including the concentration of each polymer type in the condensed and dilute phase.

Detection and Control of Prion Diseases in Food Animals

PubMed Central

Hedlin, Peter; Taschuk, Ryan; Potter, Andrew; Griebel, Philip; Napper, Scott

2012-01-01

Transmissible spongiform encephalopathies (TSEs), or prion diseases, represent a unique form of infectious disease based on misfolding of a self-protein (PrPC) into a pathological, infectious conformation (PrPSc). Prion diseases of food animals gained notoriety during the bovine spongiform encephalopathy (BSE) outbreak of the 1980s. In particular, disease transmission to humans, to the generation of a fatal, untreatable disease, elevated the perspective on livestock prion diseases from food production to food safety. While the immediate threat posed by BSE has been successfully addressed through surveillance and improved management practices, another prion disease is rapidly spreading. Chronic wasting disease (CWD), a prion disease of cervids, has been confirmed in wild and captive populations with devastating impact on the farmed cervid industries. Furthermore, the unabated spread of this disease through wild populations threatens a natural resource that is a source of considerable economic benefit and national pride. In a worst-case scenario, CWD may represent a zoonotic threat either through direct transmission via consumption of infected cervids or through a secondary food animal, such as cattle. This has energized efforts to understand prion diseases as well as to develop tools for disease detection, prevention, and management. Progress in each of these areas is discussed. PMID:23738120

Feeding behavior of Aplysia: a model system for comparing cellular mechanisms of classical and operant conditioning.

PubMed

Baxter, Douglas A; Byrne, John H

2006-01-01

Feeding behavior of Aplysia provides an excellent model system for analyzing and comparing mechanisms underlying appetitive classical conditioning and reward operant conditioning. Behavioral protocols have been developed for both forms of associative learning, both of which increase the occurrence of biting following training. Because the neural circuitry that mediates the behavior is well characterized and amenable to detailed cellular analyses, substantial progress has been made toward a comparative analysis of the cellular mechanisms underlying these two forms of associative learning. Both forms of associative learning use the same reinforcement pathway (the esophageal nerve, En) and the same reinforcement transmitter (dopamine, DA). In addition, at least one cellular locus of plasticity (cell B51) is modified by both forms of associative learning. However, the two forms of associative learning have opposite effects on B51. Classical conditioning decreases the excitability of B51, whereas operant conditioning increases the excitability of B51. Thus, the approach of using two forms of associative learning to modify a single behavior, which is mediated by an analytically tractable neural circuit, is revealing similarities and differences in the mechanisms that underlie classical and operant conditioning.

Microstructural effects in drug release by solid and cellular polymeric dosage forms: A comparative study.

PubMed

Blaesi, Aron H; Saka, Nannaji

2017-11-01

In recent studies, we have introduced melt-processed polymeric cellular dosage forms to achieve both immediate drug release and predictable manufacture. Dosage forms ranging from minimally-porous solids to highly porous, open-cell and thin-walled structures were prepared, and the drug release characteristics investigated as the volume fraction of cells and the excipient molecular weight were varied. In the present study, both minimally-porous solid and cellular dosage forms consisting of various weight fractions of Acetaminophen drug and polyethylene glycol (PEG) excipient are prepared and analyzed. Microstructures of the solid forms and the cell walls range from single-phase solid solutions of the excipient and a small amount of drug molecules to two-phase composites of the excipient and tightly packed drug particles. Results of dissolution experiments show that the minimally-porous solid forms disintegrate and release drug by slow surface erosion. The erosion rate decreases as the drug weight fraction is increased. By contrast, the open-cell structures disintegrate rapidly by viscous exfoliation, and the disintegration time is independent of drug weight fraction. Drug release models suggest that the solid forms erode by convective mass transfer of the faster-eroding excipient if the drug volume fraction is small. At larger drug volume fractions, however, the slower-eroding drug particles hinder access of the free-flowing fluid to the excipient, thus slowing down erosion of the composite. Conversely, the disintegration rate of the cellular forms is limited by diffusion of the dissolution fluid into the excipient phase of the thin cell walls. Because the wall thickness is of the order of the drug particle size, and the particles are enveloped by the excipient during melt-processing, the drug particles cannot hinder diffusion through the excipient across the walls. Thus the disintegration time of the cellular forms is mostly unaffected by the volume fraction of drug in the walls. Copyright © 2017 Elsevier B.V. All rights reserved.

Simulation of root forms using cellular automata model

NASA Astrophysics Data System (ADS)

Winarno, Nanang; Prima, Eka Cahya; Afifah, Ratih Mega Ayu

2016-02-01

This research aims to produce a simulation program for root forms using cellular automata model. Stephen Wolfram in his book entitled "A New Kind of Science" discusses the formation rules based on the statistical analysis. In accordance with Stephen Wolfram's investigation, the research will develop a basic idea of computer program using Delphi 7 programming language. To best of our knowledge, there is no previous research developing a simulation describing root forms using the cellular automata model compared to the natural root form with the presence of stone addition as the disturbance. The result shows that (1) the simulation used four rules comparing results of the program towards the natural photographs and each rule had shown different root forms; (2) the stone disturbances prevent the root growth and the multiplication of root forms had been successfully modeled. Therefore, this research had added some stones, which have size of 120 cells placed randomly in the soil. Like in nature, stones cannot be penetrated by plant roots. The result showed that it is very likely to further develop the program of simulating root forms by 50 variations.

Molecular Dynamics Simulation Study on the Binding and Stabilization Mechanism of Antiprion Compounds to the "Hot Spot" Region of PrPC.

PubMed

Zhou, Shuangyan; Liu, Xuewei; An, Xiaoli; Yao, Xiaojun; Liu, Huanxiang

2017-11-15

Structural transitions in the prion protein from the cellular form, PrP C , into the pathological isoform, PrP Sc , are regarded as the main cause of the transmissible spongiform encephalopathies, also known as prion diseases. Hence, discovering and designing effective antiprion drugs that can inhibit PrP C to PrP Sc conversion is regarded as a promising way to cure prion disease. Among several strategies to inhibit PrP C to PrP Sc conversion, stabilizing the native PrP C via specific binding is believed to be one of the valuable approaches and many antiprion compounds have been reported based on this strategy. However, the detailed mechanism to stabilize the native PrP C is still unknown. As such, to unravel the stabilizing mechanism of these compounds to PrP C is valuable for the further design and discovery of antiprion compounds. In this study, by molecular dynamics simulation method, we investigated the stabilizing mechanism of several antiprion compounds on PrP C that were previously reported to have specific binding to the "hot spot" region of PrP C . Our simulation results reveal that the stabilization mechanism of specific binding compounds can be summarized as (I) to stabilize both the flexible C-terminal of α2 and the hydrophobic core, such as BMD42-29 and GN8; (II) to stabilize the hydrophobic core, such as J1 and GJP49; (III) to stabilize the overall structure of PrP C by high binding affinity, as NPR-056. In addition, as indicated by the H-bond analysis and decomposition analysis of binding free energy, the residues N159 and Q160 play an important role in the specific binding of the studied compounds and all these compounds interact with PrP C in a similar way with the key interacting residues L130 in the β1 strand, P158, N159, Q160, etc. in the α1-β2 loop, and H187, T190, T191, etc. in the α2 C-terminus although the compounds have large structural difference. As a whole, our obtained results can provide some insights into the specific binding mechanism of main antiprion compounds to the "hot spot" region of PrP C at the molecular level and also provide guidance for effective antiprion drug design in the future.

The cellular source for APOBEC3G's incorporation into HIV-1

PubMed Central

2011-01-01

Background Human APOBEC3G (hA3G) has been identified as a cellular inhibitor of HIV-1 infectivity. Viral incorporation of hA3G is an essential step for its antiviral activity. Although the mechanism underlying hA3G virion encapsidation has been investigated extensively, the cellular source of viral hA3G remains unclear. Results Previous studies have shown that hA3G forms low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of the mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation. Conclusions Together these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G. PMID:21211018

Removal of cellular debris formed in the Disse space in patients with cholestasis.

PubMed

Dubuisson, L; Bioulac-Sage, P; Boussarie, L; Quinton, A; Saric, J; de Mascarel, A; Balabaud, C

1987-01-01

Using electron microscopy, we investigated how cellular debris, formed in the Disse space during cholestasis, was cleared. Ten patients with cholestasis of varied origin and severity were studied and compared with 10 controls without liver disease. In cholestatic patients, sinusoidal cells contained variable amounts of amylase PAS-positive material. In clean perfusion-fixed sinusoids the endothelial cells often appeared swollen and active, with few fenestrations. Hepatocyte blebs and cellular debris were sometimes seen in the Disse space. Two mechanisms were apparently involved in the clearing process: phagocytosis by macrophages either infiltrated into the Disse space, or forming the barrier; and the passage of debris from the Disse space into the sinusoidal lumen through the endothelial wall. Debris was either forced through enlarged pores or through the wall, with a progressive invagination followed by an outpouching in the lumen. The force, possibly provided by endothelial massage, may not be sufficient to push out cellular debris from the Disse space; morphological data seemed to indicate that endothelial damage may be a necessary factor. Debris present in the lumen was phagocytized by numerous active macrophages. Cellular debris was not observed in the Disse space of control patients.

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kelly, Catriona, E-mail: catriona.kelly@qub.ac.uk; Flatt, Peter R.; McClenaghan, Neville H.

2010-08-20

Research highlights: {yields} TGP52 cells display enhanced functionality in pseudoislet form. {yields} Somatostatin content was reduced, but secretion increased in high glucose conditions. {yields} Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mMmore » glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.« less

Simulation of root forms using cellular automata model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Winarno, Nanang, E-mail: nanang-winarno@upi.edu; Prima, Eka Cahya; Afifah, Ratih Mega Ayu

This research aims to produce a simulation program for root forms using cellular automata model. Stephen Wolfram in his book entitled “A New Kind of Science” discusses the formation rules based on the statistical analysis. In accordance with Stephen Wolfram’s investigation, the research will develop a basic idea of computer program using Delphi 7 programming language. To best of our knowledge, there is no previous research developing a simulation describing root forms using the cellular automata model compared to the natural root form with the presence of stone addition as the disturbance. The result shows that (1) the simulation usedmore » four rules comparing results of the program towards the natural photographs and each rule had shown different root forms; (2) the stone disturbances prevent the root growth and the multiplication of root forms had been successfully modeled. Therefore, this research had added some stones, which have size of 120 cells placed randomly in the soil. Like in nature, stones cannot be penetrated by plant roots. The result showed that it is very likely to further develop the program of simulating root forms by 50 variations.« less

Subtotal Ablation of Parietal Epithelial Cells Induces Crescent Formation

PubMed Central

Sicking, Eva-Maria; Fuss, Astrid; Uhlig, Sandra; Jirak, Peggy; Dijkman, Henry; Wetzels, Jack; Engel, Daniel R.; Urzynicok, Torsten; Heidenreich, Stefan; Kriz, Wilhelm; Kurts, Christian; Ostendorf, Tammo; Floege, Jürgen; Smeets, Bart

2012-01-01

Parietal epithelial cells (PECs) of the renal glomerulus contribute to the formation of both cellular crescents in rapidly progressive GN and sclerotic lesions in FSGS. Subtotal transgenic ablation of podocytes induces FSGS but the effect of specific ablation of PECs is unknown. Here, we established an inducible transgenic mouse to allow subtotal ablation of PECs. Proteinuria developed during doxycycline-induced cellular ablation but fully reversed 26 days after termination of doxycycline administration. The ablation of PECs was focal, with only 30% of glomeruli exhibiting histologic changes; however, the number of PECs was reduced up to 90% within affected glomeruli. Ultrastructural analysis revealed disruption of PEC plasma membranes with cytoplasm shedding into Bowman’s space. Podocytes showed focal foot process effacement, which was the most likely cause for transient proteinuria. After >9 days of cellular ablation, the remaining PECs formed cellular extensions to cover the denuded Bowman’s capsule and expressed the activation marker CD44 de novo. The induced proliferation of PECs persisted throughout the observation period, resulting in the formation of typical cellular crescents with periglomerular infiltrate, albeit without accompanying proteinuria. In summary, subtotal ablation of PECs leads the remaining PECs to react with cellular activation and proliferation, which ultimately forms cellular crescents. PMID:22282596

Epithelial stem cells are formed by small-particles released from particle-producing cells

PubMed Central

Kong, Wuyi; Zhu, Xiao Ping; Han, Xiu Juan; Nuo, Mu; Wang, Hong

2017-01-01

Recent spatiotemporal report demonstrated that epidermal stem cells have equal potential to divide or differentiate, with no asymmetric cell division observed. Therefore, how epithelial stem cells maintain lifelong stem-cell support still needs to be elucidated. In mouse blood and bone marrow, we found a group of large cells stained strongly for eosin and containing coiled-tubing-like structures. Many were tightly attached to each other to form large cellular clumps. After sectioning, these large cell-clumps were composed of not cells but numerous small particles, however with few small “naked” nuclei. The small particles were about 2 to 3 μm in diameter and stained dense red for eosin, so they may be rich in proteins. Besides the clumps composed of small particles, we identified clumps formed by fusion of the small particles and clumps of newly formed nucleated cells. These observations suggest that these small particles further fused and underwent cellularization. E-cadherin was expressed in particle-fusion areas, some “naked” nuclei and the newly formed nucleated cells, which suggests that these particles can form epithelial cells via fusion and nuclear remodeling. In addition, we observed similar-particle fusion before epithelial cellularization in mouse kidney ducts after kidney ischemia, which suggests that these particles can be released in the blood and carried to the target tissues for epithelial-cell regeneration. Oct4 and E-cadherin expressed in the cytoplasmic areas in cells that were rich in protein and mainly located in the center of the cellular clumps, suggesting that these newly formed cells have become tissue-specific epithelial stem cells. Our data provide evidence that these large particle-producing cells are the origin of epithelial stem cells. The epithelial stem cells are newly formed by particle fusion. PMID:28253358

Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice

PubMed Central

Eigenbrod, Sabina; Frick, Petra; Bertsch, Uwe; Mitteregger-Kretzschmar, Gerda; Mielke, Janina; Maringer, Marko; Piening, Niklas; Hepp, Alexander; Daude, Nathalie; Windl, Otto; Levin, Johannes; Giese, Armin; Sakthivelu, Vignesh; Tatzelt, Jörg

2017-01-01

Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1–4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain. PMID:29220360

Introducing a Rigid Loop Structure from Deer into Mouse Prion Protein Increases Its Propensity for Misfolding In Vitro

PubMed Central

Kyle, Leah M.; John, Theodore R.; Schätzl, Hermann M.; Lewis, Randolph V.

2013-01-01

Prion diseases are fatal neurodegenerative disorders characterized by misfolding of the cellular prion protein (PrPc) into the disease-associated isoform (PrPSc) that has increased β-sheet content and partial resistance to proteolytic digestion. Prion diseases from different mammalian species have varying propensities for transmission upon exposure of an uninfected host to the infectious agent. Chronic Wasting Disease (CWD) is a highly transmissible prion disease that affects free ranging and farmed populations of cervids including deer, elk and moose, as well as other mammals in experimental settings. The molecular mechanisms allowing CWD to maintain comparatively high transmission rates have not been determined. Previous work has identified a unique structural feature in cervid PrP, a rigid loop between β-sheet 2 and α-helix 2 on the surface of the protein. This study was designed to test the hypothesis that the rigid loop has a direct influence on the misfolding process. The rigid loop was introduced into murine PrP as the result of two amino acid substitutions: S170N and N174T. Wild-type and rigid loop murine PrP were expressed in E. coli and purified. Misfolding propensity was compared for the two proteins using biochemical techniques and cell free misfolding and conversion systems. Murine PrP with a rigid loop misfolded in cell free systems with greater propensity than wild type murine PrP. In a lipid-based conversion assay, rigid loop PrP converted to a PK resistant, aggregated isoform at lower concentrations than wild-type PrP. Using both proteins as substrates in real time quaking-induced conversion, rigid loop PrP adopted a misfolded isoform more readily than wild type PrP. Taken together, these findings may help explain the high transmission rates observed for CWD within cervids. PMID:23825561

Terminal addition in a cellular world.

PubMed

Torday, J S; Miller, William B

2018-07-01

Recent advances in our understanding of evolutionary development permit a reframed appraisal of Terminal Addition as a continuous historical process of cellular-environmental complementarity. Within this frame of reference, evolutionary terminal additions can be identified as environmental induction of episodic adjustments to cell-cell signaling patterns that yield the cellular-molecular pathways that lead to differing developmental forms. Phenotypes derive, thereby, through cellular mutualistic/competitive niche constructions in reciprocating responsiveness to environmental stresses and epigenetic impacts. In such terms, Terminal Addition flows according to a logic of cellular needs confronting environmental challenges over space-time. A reconciliation of evolutionary development and Terminal Addition can be achieved through a combined focus on cell-cell signaling, molecular phylogenies and a broader understanding of epigenetic phenomena among eukaryotic organisms. When understood in this manner, Terminal Addition has an important role in evolutionary development, and chronic disease might be considered as a form of 'reverse evolution' of the self-same processes. Copyright © 2017. Published by Elsevier Ltd.

Long-distance communication by specialized cellular projections during pigment pattern development and evolution

PubMed Central

Eom, Dae Seok; Bain, Emily J; Patterson, Larissa B; Grout, Megan E; Parichy, David M

2015-01-01

Changes in gene activity are essential for evolutionary diversification. Yet, elucidating the cellular behaviors that underlie modifications to adult form remains a profound challenge. We use neural crest-derived adult pigmentation of zebrafish and pearl danio to uncover cellular bases for alternative pattern states. We show that stripes in zebrafish require a novel class of thin, fast cellular projection to promote Delta-Notch signaling over long distances from cells of the xanthophore lineage to melanophores. Projections depended on microfilaments and microtubules, exhibited meandering trajectories, and stabilized on target cells to which they delivered membraneous vesicles. By contrast, the uniformly patterned pearl danio lacked such projections, concomitant with Colony stimulating factor 1-dependent changes in xanthophore differentiation that likely curtail signaling available to melanophores. Our study reveals a novel mechanism of cellular communication, roles for differentiation state heterogeneity in pigment cell interactions, and an unanticipated morphogenetic behavior contributing to a striking difference in adult form. DOI: http://dx.doi.org/10.7554/eLife.12401.001 PMID:26701906

Segmented ceramic liner for induction furnaces

DOEpatents

Gorin, Andrew H.; Holcombe, Cressie E.

1994-01-01

A non-fibrous ceramic liner for induction furnaces is provided by vertically stackable ring-shaped liner segments made of ceramic material in a light-weight cellular form. The liner segments can each be fabricated as a single unit or from a plurality of arcuate segments joined together by an interlocking mechanism. Also, the liner segments can be formed of a single ceramic material or can be constructed of multiple concentric layers with the layers being of different ceramic materials and/or cellular forms. Thermomechanically damaged liner segments are selectively replaceable in the furnace.

Segmented ceramic liner for induction furnaces

DOEpatents

Gorin, A.H.; Holcombe, C.E.

1994-07-26

A non-fibrous ceramic liner for induction furnaces is provided by vertically stackable ring-shaped liner segments made of ceramic material in a light-weight cellular form. The liner segments can each be fabricated as a single unit or from a plurality of arcuate segments joined together by an interlocking mechanism. Also, the liner segments can be formed of a single ceramic material or can be constructed of multiple concentric layers with the layers being of different ceramic materials and/or cellular forms. Thermomechanically damaged liner segments are selectively replaceable in the furnace. 5 figs.

Cadmium accumulation, sub-cellular distribution and chemical forms in rice seedling in the presence of sulfur.

PubMed

Zhang, Wen; Lin, Kuangfei; Zhou, Jian; Zhang, Wei; Liu, Lili; Zhang, Qianqian

2014-01-01

Changes in cadmium (Cd) accumulation, distribution, and chemical form in rice seedling in the joint presence of different concentrations of sulfur (S) remain almost unknown. Therefore, the indoor experiments were performed to determine the accumulation, sub-cellular distribution and chemical forms of Cd under three S levels in rice seedling for the first time. The result showed that Cd accumulation in rice roots was more than in shoots. Sub-cellular distribution of Cd in rice roots and shoots indicated that the largest proportion of Cd accumulated in cell walls and soluble fractions. As S supply increased, the proportion of Cd in cell walls reduced, while it increased in the soluble fractions. The majority of Cd existed in inorganic form, and then gradually changed to organic forms that included pectates and proteins with increased S supply. The results showed that S supply significantly influenced Cd accumulation, distribution, and chemical forms, suggesting that S might provide the material for the synthesis of sulfhydryl protein and thereby affect Cd stress on plants. These observations provided a basic understanding of potential ecotoxicological effects of joint Cd and S exposure in the environment. Copyright © 2013 Elsevier B.V. All rights reserved.

Four domains: The fundamental unicell and Post-Darwinian Cognition-Based Evolution.

PubMed

Miller, William B; Torday, John S

2018-04-13

Contemporary research supports the viewpoint that self-referential cognition is the proper definition of life. From that initiating platform, a cohesive alternative evolutionary narrative distinct from standard Neodarwinism can be presented. Cognition-Based Evolution contends that biological variation is a product of a self-reinforcing information cycle that derives from self-referential attachment to biological information space-time with its attendant ambiguities. That information cycle is embodied through obligatory linkages among energy, biological information, and communication. Successive reiterations of the information cycle enact the informational architectures of the basic unicellular forms. From that base, inter-domain and cell-cell communications enable genetic and cellular variations through self-referential natural informational engineering and cellular niche construction. Holobionts are the exclusive endpoints of that self-referential cellular engineering as obligatory multicellular combinations of the essential Four Domains: Prokaryota, Archaea, Eukaryota and the Virome. Therefore, it is advocated that these Four Domains represent the perpetual object of the living circumstance rather than the visible macroorganic forms. In consequence, biology and its evolutionary development can be appraised as the continual defense of instantiated cellular self-reference. As the survival of cells is as dependent upon limitations and boundaries as upon any freedom of action, it is proposed that selection represents only one of many forms of cellular constraint that sustain self-referential integrity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Quantitative interactome reveals that porcine reproductive and respiratory syndrome virus nonstructural protein 2 forms a complex with viral nucleocapsid protein and cellular vimentin.

PubMed

Song, Tao; Fang, Liurong; Wang, Dang; Zhang, Ruoxi; Zeng, Songlin; An, Kang; Chen, Huanchun; Xiao, Shaobo

2016-06-16

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has heavily impacted the global swine industry. The PRRSV nonstructural protein 2 (nsp2) plays crucial roles in viral replication and host immune regulation, most likely by interacting with viral or cellular proteins that have not yet been identified. In this study, a quantitative interactome approach based on immunoprecipitation and stable isotope labeling with amino acids in cell culture (SILAC) was performed to identify nsp2-interacting proteins in PRRSV-infected cells with an nsp2-specific monoclonal antibody. Nine viral proteins and 62 cellular proteins were identified as potential nsp2-interacting partners. Our data demonstrate that the PRRSV nsp1α, nsp1β, and nucleocapsid proteins all interact directly with nsp2. Nsp2-interacting cellular proteins were classified into different functional groups and an interactome network of nsp2 was generated. Interestingly, cellular vimentin, a known receptor for PRRSV, forms a complex with nsp2 by using viral nucleocapsid protein as an intermediate. Taken together, the nsp2 interactome under the condition of virus infection clarifies a role of nsp2 in PRRSV replication and immune evasion. Viral proteins must interact with other virus-encoded proteins and/or host cellular proteins to function, and interactome analysis is an ideal approach for identifying such interacting proteins. In this study, we used the quantitative interactome methodology to identify the viral and cellular proteins that potentially interact with the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) under virus infection conditions, thus providing a rich source of potential viral and cellular interaction partners for PRRSV nsp2. Based on the interactome data, we further demonstrated that PRRSV nsp2 and nucleocapsid protein together with cellular vimentin, form a complex that may be essential for viral attachment and replication, which partly explains the role of nsp2 in PRRSV replication and immune evasion. Copyright © 2016 Elsevier B.V. All rights reserved.

A virocentric perspective on the evolution of life

PubMed Central

Koonin, Eugene V.; Dolja, Valerian V.

2015-01-01

Viruses and/or virus-like selfish elements are associated with all cellular life forms and are the most abundant biological entities on Earth, with the number of virus particles in many environments exceeding the number of cells by one to two orders of magnitude. The genetic diversity of viruses is commensurately enormous and might substantially exceed the diversity of cellular organisms. Unlike cellular organisms with their uniform replication-expression scheme, viruses possess either RNA or DNA genomes and exploit all conceivable replication-expression strategies. Although viruses extensively exchange genes with their hosts, there exists a set of viral hallmark genes that are shared by extremely diverse groups of viruses to the exclusion of cellular life forms. Coevolution of viruses and host defense systems is a key aspect in the evolution of both viruses and cells, and viral genes are often recruited for cellular functions. Together with the fundamental inevitability of the emergence of genomic parasites in any evolving replicator system, these multiple lines of evidence reveal the central role of viruses in the entire evolution of life. PMID:23850169

Inter-Cellular Exchange of Cellular Components via VE-Cadherin-Dependent Trans-Endocytosis

PubMed Central

Sakurai, Takashi; Woolls, Melissa J.; Jin, Suk-Won

2014-01-01

Cell-cell communications typically involve receptor-mediated signaling initiated by soluble or cell-bound ligands. Here, we report a unique mode of endocytosis: proteins originating from cell-cell junctions and cytosolic cellular components from the neighboring cell are internalized, leading to direct exchange of cellular components between two adjacent endothelial cells. VE-cadherins form transcellular bridges between two endothelial cells that are the basis of adherence junctions. At such adherens junction sites, we observed the movement of the entire VE-cadherin molecule from one endothelial cell into the other with junctional and cytoplasmic components. This phenomenon, here termed trans-endocytosis, requires the establishment of a VE-cadherin homodimer in trans with internalization proceeding in a Rac1-, and actomyosin-dependent manner. Importantly, the trans-endocytosis is not dependent on any known endocytic pathway including clathrin-dependent endocytosis, macropinocytosis or phagocytosis. This novel form of cell-cell communications, leading to a direct exchange of cellular components, was observed in 2D and 3D-cultured endothelial cells as well as in the developing zebrafish vasculature. PMID:24603875

Design and testing of botanical thermotropic actuator mechanisms in thermally adaptive building coverings

NASA Astrophysics Data System (ADS)

Barrett, Ronald M.; Barrett, Ronald P.; Barrett, Cassandra M.

2017-09-01

This paper lays out the inspiration, operational principles, analytical modeling and coupon testing of a new class of thermally adaptive building coverings. The fundamental driving concepts for these coverings are derived from various families of thermotropic plant structures. Certain plant cellular structures like those in Mimosa pudica (Sensitive Plant), Rhododendron leaves or Albizia julibrissin (Mimosa Tree), exhibit actuation physiology which depends on changes in cellular turgor pressures to generate motion. This form of cellular action via turgor pressure manipulation is an inspiration for a new field of thermally adaptive building coverings which use various forms of cellular foam to aid or enable actuation much like plant cells are used to move leaves. When exposed to high solar loading, the structures use the inherent actuation capability of pockets of air trapped in closed cell foam as actuators to curve plates upwards and outwards. When cold, these same structures curve back towards the building forming large convex pockets of dead air to insulate the building. This paper describes basic classical laminated plate theory models comparing theory and experiment of such coupons containing closed-cell foam actuators. The study concludes with a global description of the effectiveness of this class of thermally adaptive building coverings.

Multicellular regulation of entropy, spatial order, and information

NASA Astrophysics Data System (ADS)

Youk, Hyun

Many multicellular systems such as tissues and microbial biofilms consist of cells that secrete and sense signalling molecules. Understanding how collective behaviours of secrete-and-sense cells is an important challenge. We combined experimental and theoretical approaches to understand multicellular coordination of gene expression and spatial pattern formation among secrete-and-sense cells. We engineered secrete-and-sense yeast cells to show that cells can collectively and permanently remember a past event by reminding each other with their secreted signalling molecule. If one cell ``forgets'' then another cell can remind it. Cell-cell communication ensures a long-term (permanent) memory by overcoming common limitations of intracellular memory. We also established a new theoretical framework inspired by statistical mechanics to understand how fields of secrete-and-sense cells form spatial patterns. We introduce new metrics - cellular entropy, cellular Hamiltonian, and spatial order index - for dynamics of cellular automata that form spatial patterns. Our theory predicts how fast any spatial patterns form, how ordered they are, and establishes cellular Hamiltonian that, like energy for non-living systems, monotonically decreases towards a minimum over time. ERC Starting Grant (MultiCellSysBio), NWO VIDI, NWO NanoFront.

Cellular interface morphologies in directional solidification. II - The effect of grain boundaries

NASA Technical Reports Server (NTRS)

Ungar, Lyle H.; Brown, Robert A.

1984-01-01

A singular perturbation analysis valid for small grain-boundary slopes is used with the one-sided model for solidification to show that grain boundaries introduce imperfections into the symmetry of the developing cellular interfaces which rupture the junction between the family of planar shapes and the bifurcating cellular families. Undulating interfaces are shown to develop first near grain boundaries, and to evolve with decreasing temperature gradient either by a smooth transition from the almost planar family or by a sudden jump to moderate-amplitude cellular forms, depending on the growth rate.

Morphology of uranium electrodeposits on cathode in electrorefining process: A phase-field simulation

NASA Astrophysics Data System (ADS)

Shibuta, Yasushi; Sato, Takumi; Suzuki, Toshio; Ohta, Hirokazu; Kurata, Masaki

2013-05-01

Morphology of uranium electrodeposits on cathode with respect to applied voltage, zirconium concentration in the molten salt and the size of primary deposit during pyroprocessing is systematically investigated by the phase-field simulation. It is found that there is a threshold zirconium concentration in the molten salt demarcating planar and cellular/needle-like electrodeposits, which agrees with experimental results. In addition, the effect of size of primary deposits on the morphology of electrodeposits is examined. It is then confirmed that cellular/needle-like electrodeposits are formed from large primary deposits at all applied voltages considered, whereas both the planar and cellular/needle-like electrodeposits are formed from the primary deposits of 10 μm and less.

Global functional analyses of cellular responses to pore-forming toxins.

PubMed

Kao, Cheng-Yuan; Los, Ferdinand C O; Huffman, Danielle L; Wachi, Shinichiro; Kloft, Nicole; Husmann, Matthias; Karabrahimi, Valbona; Schwartz, Jean-Louis; Bellier, Audrey; Ha, Christine; Sagong, Youn; Fan, Hui; Ghosh, Partho; Hsieh, Mindy; Hsu, Chih-Shen; Chen, Li; Aroian, Raffi V

2011-03-01

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

Untangling the origin of viruses and their impact on cellular evolution.

PubMed

Nasir, Arshan; Sun, Feng-Jie; Kim, Kyung Mo; Caetano-Anollés, Gustavo

2015-04-01

The origin and evolution of viruses remain mysterious. Here, we focus on the distribution of viral replicons in host organisms, their morphological features, and the evolution of highly conserved protein and nucleic acid structures. The apparent inability of RNA viral replicons to infect contemporary akaryotic species suggests an early origin of RNA viruses and their subsequent loss in akaryotes. A census of virion morphotypes reveals that advanced forms were unique to viruses infecting a specific supergroup, while simpler forms were observed in viruses infecting organisms in all forms of cellular life. Results hint toward an ancient origin of viruses from an ancestral virus harboring either filamentous or spherical virions. Finally, phylogenetic trees built from protein domain and tRNA structures in thousands of genomes suggest that viruses evolved via reductive evolution from ancient cells. The analysis presents a complete account of the evolutionary history of cells and viruses and identifies viruses as crucial agents influencing cellular evolution. © 2015 New York Academy of Sciences.

Method of forming a continuous polymeric skin on a cellular foam material

DOEpatents

Duchane, David V.; Barthell, Barry L.

1985-01-01

Hydrophobic cellular material is coated with a thin hydrophilic polymer skin which stretches tightly over the outer surface of the foam but which does not fill the cells of the foam, thus resulting in a polymer-coated foam structure having a smoothness which was not possible in the prior art. In particular, when the hydrophobic cellular material is a specially chosen hydrophobic polymer foam and is formed into arbitrarily chosen shapes prior to the coating with hydrophilic polymer, inertial confinement fusion (ICF) targets of arbitrary shapes can be produced by subsequently coating the shapes with metal or with any other suitable material. New articles of manufacture are produced, including improved ICF targets, improved integrated circuits, and improved solar reflectors and solar collectors. In the coating method, the cell size of the hydrophobic cellular material, the viscosity of the polymer solution used to coat, and the surface tensin of the polymer solution used to coat are all very important to the coating.

Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments

NASA Astrophysics Data System (ADS)

Huber, Matthias C.; Schreiber, Andreas; von Olshausen, Philipp; Varga, Balázs R.; Kretz, Oliver; Joch, Barbara; Barnert, Sabine; Schubert, Rolf; Eimer, Stefan; Kele, Péter; Schiller, Stefan M.

2015-01-01

Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally ‘program’ the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials.

Cations Form Sequence Selective Motifs within DNA Grooves via a Combination of Cation-Pi and Ion-Dipole/Hydrogen Bond Interactions

PubMed Central

Stewart, Mikaela; Dunlap, Tori; Dourlain, Elizabeth; Grant, Bryce; McFail-Isom, Lori

2013-01-01

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl+) and the polarized first hydration shell waters of divalent cations (Mg2+, Ca2+) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves. PMID:23940752

Cations form sequence selective motifs within DNA grooves via a combination of cation-pi and ion-dipole/hydrogen bond interactions.

PubMed

Stewart, Mikaela; Dunlap, Tori; Dourlain, Elizabeth; Grant, Bryce; McFail-Isom, Lori

2013-01-01

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl⁺) and the polarized first hydration shell waters of divalent cations (Mg²⁺, Ca²⁺) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves.

DNA Secondary Structure at Chromosomal Fragile Sites in Human Disease

PubMed Central

Thys, Ryan G; Lehman, Christine E; Pierce, Levi C. T; Wang, Yuh-Hwa

2015-01-01

DNA has the ability to form a variety of secondary structures that can interfere with normal cellular processes, and many of these structures have been associated with neurological diseases and cancer. Secondary structure-forming sequences are often found at chromosomal fragile sites, which are hotspots for sister chromatid exchange, chromosomal translocations, and deletions. Structures formed at fragile sites can lead to instability by disrupting normal cellular processes such as DNA replication and transcription. The instability caused by disruption of replication and transcription can lead to DNA breakage, resulting in gene rearrangements and deletions that cause disease. In this review, we discuss the role of DNA secondary structure at fragile sites in human disease. PMID:25937814

Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

PubMed

Morohashi, Kengo; Sahara, Hiroeki; Watashi, Koichi; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

2011-04-29

Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

Giant viruses coexisted with the cellular ancestors and represent a distinct supergroup along with superkingdoms Archaea, Bacteria and Eukarya

PubMed Central

2012-01-01

Background The discovery of giant viruses with genome and physical size comparable to cellular organisms, remnants of protein translation machinery and virus-specific parasites (virophages) have raised intriguing questions about their origin. Evidence advocates for their inclusion into global phylogenomic studies and their consideration as a distinct and ancient form of life. Results Here we reconstruct phylogenies describing the evolution of proteomes and protein domain structures of cellular organisms and double-stranded DNA viruses with medium-to-very-large proteomes (giant viruses). Trees of proteomes define viruses as a ‘fourth supergroup’ along with superkingdoms Archaea, Bacteria, and Eukarya. Trees of domains indicate they have evolved via massive and primordial reductive evolutionary processes. The distribution of domain structures suggests giant viruses harbor a significant number of protein domains including those with no cellular representation. The genomic and structural diversity embedded in the viral proteomes is comparable to the cellular proteomes of organisms with parasitic lifestyles. Since viral domains are widespread among cellular species, we propose that viruses mediate gene transfer between cells and crucially enhance biodiversity. Conclusions Results call for a change in the way viruses are perceived. They likely represent a distinct form of life that either predated or coexisted with the last universal common ancestor (LUCA) and constitute a very crucial part of our planet’s biosphere. PMID:22920653

Characterization of a rare Unverricht-Lundborg disease mutation.

PubMed

Duarte, Ana Joana; Ribeiro, Diogo; Chaves, João; Amaral, Olga

2015-09-01

Cystatin B (CSTB) gene mutations cause Unverricht-Lundborg disease (ULD), a rare form of myoclonic epilepsy. The previous identification of a Portuguese patient, homozygous for a unique splicing defect (c.66G > A; p.Q22Q), provided awareness regarding the existence of variant forms of ULD. In this work we aimed at the characterization of this mutation at the population level and at the cellular level. The cellular fractionation studies here carried out showed mislocalization of the protein and add to the knowledge on this disease.

The cellular distribution of lignans in Tsuga heterophylla wood

Treesearch

R.l. Krahmer; Richard W. Hemingway; W.E. Hillis

1970-01-01

Western hemlock heartwood contains patches of tracheids with large amounts of cellular inclusions. Microscopic and chemical examination of the wood showed several types of deposits containing the lignans matairesinol, hydroxymatairesinol and conidendrin. The deposits, which were often relatively pure individual lignans, frequently assumed different physical forms and...

In silico studies and fluorescence binding assays of potential anti-prion compounds reveal an important binding site for prion inhibition from PrP(C) to PrP(Sc).

PubMed

Pagadala, Nataraj S; Perez-Pineiro, Rolando; Wishart, David S; Tuszynski, Jack A

2015-02-16

To understand the pharmacophore properties of 2-aminothiazoles and design novel inhibitors against the prion protein, a highly predictive 3D quantitative structure-activity relationship (QSAR) has been developed by performing comparative molecular field analysis (CoMFA) and comparative similarity analysis (CoMSIA). Both CoMFA and CoMSIA maps reveal the presence of the oxymethyl groups in meta and para positions on the phenyl ring of compound 17 (N-[4-(3,4-dimethoxyphenyl)-1,3-thiazol-2-yl]quinolin-2-amine), is necessary for activity while electro-negative nitrogen of quinoline is highly favorable to enhance activity. The blind docking results for these compounds show that the compound with quinoline binds with higher affinity than isoquinoline and naphthalene groups. Out of 150 novel compounds retrieved using finger print analysis by pharmacophoric model predicted based on five test sets of compounds, five compounds with diverse scaffolds were selected for biological evaluation as possible PrP inhibitors. Molecular docking combined with fluorescence quenching studies show that these compounds bind to pocket-D of SHaPrP near Trp145. The new antiprion compounds 3 and 6, which bind with the interaction energies of -12.1 and -13.2 kcal/mol, respectively, show fluorescence quenching with binding constant (Kd) values of 15.5 and 44.14 μM, respectively. Further fluorescence binding assays with compound 5, which is similar to 2-aminothiazole as a positive control, also show that the molecule binds to the pocket-D with the binding constant (Kd) value of 84.7 μM. Finally, both molecular docking and a fluorescence binding assay of noscapine as a negative control reveals the same binding site on the surface of pocket-A near a rigid loop between β2 and α2 interacting with Arg164. This high level of correlation between molecular docking and fluorescence quenching studies confirm that these five compounds are likely to act as inhibitors for prion propagation while noscapine might act as a prion accelerator from PrP(C) to PrP(Sc). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Formalizing Knowledge in Multi-Scale Agent-Based Simulations

PubMed Central

Somogyi, Endre; Sluka, James P.; Glazier, James A.

2017-01-01

Multi-scale, agent-based simulations of cellular and tissue biology are increasingly common. These simulations combine and integrate a range of components from different domains. Simulations continuously create, destroy and reorganize constituent elements causing their interactions to dynamically change. For example, the multi-cellular tissue development process coordinates molecular, cellular and tissue scale objects with biochemical, biomechanical, spatial and behavioral processes to form a dynamic network. Different domain specific languages can describe these components in isolation, but cannot describe their interactions. No current programming language is designed to represent in human readable and reusable form the domain specific knowledge contained in these components and interactions. We present a new hybrid programming language paradigm that naturally expresses the complex multi-scale objects and dynamic interactions in a unified way and allows domain knowledge to be captured, searched, formalized, extracted and reused. PMID:29338063

Formalizing Knowledge in Multi-Scale Agent-Based Simulations.

PubMed

Somogyi, Endre; Sluka, James P; Glazier, James A

2016-10-01

Multi-scale, agent-based simulations of cellular and tissue biology are increasingly common. These simulations combine and integrate a range of components from different domains. Simulations continuously create, destroy and reorganize constituent elements causing their interactions to dynamically change. For example, the multi-cellular tissue development process coordinates molecular, cellular and tissue scale objects with biochemical, biomechanical, spatial and behavioral processes to form a dynamic network. Different domain specific languages can describe these components in isolation, but cannot describe their interactions. No current programming language is designed to represent in human readable and reusable form the domain specific knowledge contained in these components and interactions. We present a new hybrid programming language paradigm that naturally expresses the complex multi-scale objects and dynamic interactions in a unified way and allows domain knowledge to be captured, searched, formalized, extracted and reused.

Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins.

PubMed

Inobe, Tomonao; Nukina, Nobuyuki

2016-07-01

Most proteins form larger protein complexes and perform multiple functions in the cell. Thus, artificial regulation of protein complex formation controls the cellular functions that involve protein complexes. Although several artificial dimerization systems have already been used for numerous applications in biomedical research, cellular protein complexes form not only simple dimers but also larger oligomers. In this study, we showed that fusion proteins comprising the induced heterodimer formation proteins FRB and FKBP formed various oligomers upon addition of rapamycin. By adjusting the configuration of fusion proteins, we succeeded in generating an inducible tetramer formation system. Proteins of interest also formed tetramers by fusing to the inducible tetramer formation system, which exhibits its utility in a broad range of biological applications. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment.

PubMed

Eguchi, Takanori; Sogawa, Chiharu; Okusha, Yuka; Uchibe, Kenta; Iinuma, Ryosuke; Ono, Kisho; Nakano, Keisuke; Murakami, Jun; Itoh, Manabu; Arai, Kazuya; Fujiwara, Toshifumi; Namba, Yuri; Murata, Yoshiki; Ohyama, Kazumi; Shimomura, Manami; Okamura, Hirohiko; Takigawa, Masaharu; Nakatsura, Tetsuya; Kozaki, Ken-Ichi; Okamoto, Kuniaki; Calderwood, Stuart K

2018-01-01

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment

PubMed Central

Okusha, Yuka; Uchibe, Kenta; Iinuma, Ryosuke; Ono, Kisho; Nakano, Keisuke; Murakami, Jun; Itoh, Manabu; Arai, Kazuya; Fujiwara, Toshifumi; Namba, Yuri; Murata, Yoshiki; Ohyama, Kazumi; Shimomura, Manami; Okamura, Hirohiko; Takigawa, Masaharu; Nakatsura, Tetsuya; Kozaki, Ken-ichi; Okamoto, Kuniaki; Calderwood, Stuart K.

2018-01-01

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression. PMID:29415026

Binding affinity of amyloid oligomers to cellular membranes is a generic indicator of cellular dysfunction in protein misfolding diseases

PubMed Central

Evangelisti, Elisa; Cascella, Roberta; Becatti, Matteo; Marrazza, Giovanna; Dobson, Christopher M.; Chiti, Fabrizio; Stefani, Massimo; Cecchi, Cristina

2016-01-01

The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer’s disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca2+ across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane. PMID:27619987

HLH-30/TFEB-mediated autophagy functions in a cell-autonomous manner for epithelium intrinsic cellular defense against bacterial pore-forming toxin in C. elegans.

PubMed

Chen, Huan-Da; Kao, Cheng-Yuan; Liu, Bang-Yu; Huang, Shin-Whei; Kuo, Cheng-Ju; Ruan, Jhen-Wei; Lin, Yen-Hung; Huang, Cheng-Rung; Chen, Yu-Hung; Wang, Horng-Dar; Aroian, Raffi V; Chen, Chang-Shi

2017-02-01

Autophagy is an evolutionarily conserved intracellular system that maintains cellular homeostasis by degrading and recycling damaged cellular components. The transcription factor HLH-30/TFEB-mediated autophagy has been reported to regulate tolerance to bacterial infection, but less is known about the bona fide bacterial effector that activates HLH-30 and autophagy. Here, we reveal that bacterial membrane pore-forming toxin (PFT) induces autophagy in an HLH-30-dependent manner in Caenorhabditis elegans. Moreover, autophagy controls the susceptibility of animals to PFT toxicity through xenophagic degradation of PFT and repair of membrane-pore cell-autonomously in the PFT-targeted intestinal cells in C. elegans. These results demonstrate that autophagic pathways and autophagy are induced partly at the transcriptional level through HLH-30 activation and are required to protect metazoan upon PFT intoxication. Together, our data show a new and powerful connection between HLH-30-mediated autophagy and epithelium intrinsic cellular defense against the single most common mode of bacterial attack in vivo.

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase.

PubMed

Kim, Sun Yee; Park, Jeen-Woo

2003-03-01

Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP+-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP+-dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.

Cellular automata and integrodifferential equation models for cell renewal in mosaic tissues

PubMed Central

Bloomfield, J. M.; Sherratt, J. A.; Painter, K. J.; Landini, G.

2010-01-01

Mosaic tissues are composed of two or more genetically distinct cell types. They occur naturally, and are also a useful experimental method for exploring tissue growth and maintenance. By marking the different cell types, one can study the patterns formed by proliferation, renewal and migration. Here, we present mathematical modelling suggesting that small changes in the type of interaction that cells have with their local cellular environment can lead to very different outcomes for the composition of mosaics. In cell renewal, proliferation of each cell type may depend linearly or nonlinearly on the local proportion of cells of that type, and these two possibilities produce very different patterns. We study two variations of a cellular automaton model based on simple rules for renewal. We then propose an integrodifferential equation model, and again consider two different forms of cellular interaction. The results of the continuous and cellular automata models are qualitatively the same, and we observe that changes in local environment interaction affect the dynamics for both. Furthermore, we demonstrate that the models reproduce some of the patterns seen in actual mosaic tissues. In particular, our results suggest that the differing patterns seen in organ parenchymas may be driven purely by the process of cell replacement under different interaction scenarios. PMID:20375040

In search of cellular control: signal transduction in context

NASA Technical Reports Server (NTRS)

Ingber, D.

1998-01-01

The field of molecular cell biology has experienced enormous advances over the last century by reducing the complexity of living cells into simpler molecular components and binding interactions that are amenable to rigorous biochemical analysis. However, as our tools become more powerful, there is a tendency to define mechanisms by what we can measure. The field is currently dominated by efforts to identify the key molecules and sequences that mediate the function of critical receptors, signal transducers, and molecular switches. Unfortunately, these conventional experimental approaches ignore the importance of supramolecular control mechanisms that play a critical role in cellular regulation. Thus, the significance of individual molecular constituents cannot be fully understood when studied in isolation because their function may vary depending on their context within the structural complexity of the living cell. These higher-order regulatory mechanisms are based on the cell's use of a form of solid-state biochemistry in which molecular components that mediate biochemical processing and signal transduction are immobilized on insoluble cytoskeletal scaffolds in the cytoplasm and nucleus. Key to the understanding of this form of cellular regulation is the realization that chemistry is structure and hence, recognition of the the importance of architecture and mechanics for signal integration and biochemical control. Recent work that has unified chemical and mechanical signaling pathways provides a glimpse of how this form of higher-order cellular control may function and where paths may lie in the future.

Transparent metal model study of the use of a cellular growth front to form aligned monotectic composite materials

NASA Technical Reports Server (NTRS)

Kaukler, William F.

1988-01-01

The purpose of this work was to resolve a scientific controversy in the understanding of how second phase particles become aligned during unidirectional growth of a monotectic alloy. A second aspect was to make the first systematic observations of the solidification behavior of a monotectic alloy during cellular growth in-situ. This research provides the first systematic transparent model study of cellular solidification. An interface stability diagram was developed for the planar to cellular transition of the succinonitrile glycerol (SNG) system. A method was developed utilizing Fourier Transform Infrared Spectroscopy which allows quantitative compositional analysis of directionally solidified SNG along the growth axis. To determine the influence of cellular growth front on alignment for directionally solidified monotectic alloys, the planar and cellular growth morphology was observed in-situ for SNG between 8 and 17 percent glycerol and for a range of over two orders of magnitude G/R.

Metal Dyshomeostasis and Their Pathological Role in Prion and Prion-Like Diseases: The Basis for a Nutritional Approach

PubMed Central

Toni, Mattia; Massimino, Maria L.; De Mario, Agnese; Angiulli, Elisa; Spisni, Enzo

2017-01-01

Metal ions are key elements in organisms' life acting like cofactors of many enzymes but they can also be potentially dangerous for the cell participating in redox reactions that lead to the formation of reactive oxygen species (ROS). Any factor inducing or limiting a metal dyshomeostasis, ROS production and cell injury may contribute to the onset of neurodegenerative diseases or play a neuroprotective action. Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are a group of fatal neurodegenerative disorders affecting the central nervous system (CNS) of human and other mammalian species. The causative agent of TSEs is believed to be the scrapie prion protein PrPSc, the β sheet-rich pathogenic isoform produced by the conformational conversion of the α-helix-rich physiological isoform PrPC. The peculiarity of PrPSc is its ability to self-propagate in exponential fashion in cells and its tendency to precipitate in insoluble and protease-resistance amyloid aggregates leading to neuronal cell death. The expression “prion-like diseases” refers to a group of neurodegenerative diseases that share some neuropathological features with prion diseases such as the involvement of proteins (α-synuclein, amyloid β, and tau) able to precipitate producing amyloid deposits following conformational change. High social impact diseases such as Alzheimer's and Parkinson's belong to prion-like diseases. Accumulating evidence suggests that the exposure to environmental metals is a risk factor for the development of prion and prion-like diseases and that metal ions can directly bind to prion and prion-like proteins affecting the amount of amyloid aggregates. The diet, source of metal ions but also of natural antioxidant and chelating agents such as polyphenols, is an aspect to take into account in addressing the issue of neurodegeneration. Epidemiological data suggest that the Mediterranean diet, based on the abundant consumption of fresh vegetables and on low intake of meat, could play a preventive or delaying role in prion and prion-like neurodegenerative diseases. In this review, metal role in the onset of prion and prion-like diseases is dealt with from a nutritional, cellular, and molecular point of view. PMID:28154522

Geomorphological Mapping of Sputnik Planum on Pluto: Convection, Glacial Flow, Sublimation and Re-deposition of Nitrogen Ice

NASA Astrophysics Data System (ADS)

White, O. L.; Moore, J. M.; Stern, S. A.; Weaver, H. A., Jr.; Olkin, C.; Ennico Smith, K.; Young, L. A.; Cheng, A. F.

2016-12-01

The New Horizons flyby of Pluto provided extensive high-resolution coverage of its encounter hemisphere. The most prominent surface feature in this hemisphere is the high albedo region informally named Tombaugh Regio, the western portion of which is represented by the expansive nitrogen ice plains informally named Sputnik Planum. A large fraction of Sputnik Planum displays a distinct cellular pattern, with individual cells typically displaying ovoid planforms and shallow pitting on a scale of a few hundred meters. Troughs with medial ridges define the boundaries between cells. Prior studies have argued that this pattern is indicative of solid-state convection occurring within the nitrogen ice. The southern non-cellular plains are either featureless or display dense fields of often elongate and aligned pits typically reaching a few km across, interpreted to have formed via sublimation. The mapping that will be presented at AGU focuses on identifying the different plains units that compose Sputnik Planum and defining the boundaries between them, which aids in assessing their time sequencing and correlation to one another. The cellular plains are divided into bright and dark units, with the bright unit forming a continuous high albedo zone with the bright uplands of east Tombaugh Regio. We interpret the dark plains to represent the main body of convecting N2 ice that forms the cellular plains of Sputnik Planum, with the low albedo caused by a high concentration of entrained dark material (likely tholins). Preferential sublimation of N2 ice from these plains would leave the dark ice exposed, and re-deposition of the N2 ice on the eastern cellular plains and uplands of east Tombaugh Regio would create a thin veneer of pure, bright N2 ice covering these landscapes. The non-cellular plains are universally bright and display evidence for southwards flow of the N2 ice, based on the orientations of fields of elongate sublimation pits as well as the presence of `extinct cells' that appear to have migrated away from the zone of active convection. The larger pits that occur within the non-cellular plains imply that these plains are older than the cellular plains, where resurfacing via convection limits the maximum size attainable by sublimation pits.

Cyclosporin A Associated Helicase-Like Protein Facilitates the Association of Hepatitis C Virus RNA Polymerase with Its Cellular Cyclophilin B

PubMed Central

Sahara, Hiroeki; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

2011-01-01

Background Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Principal Findings Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. Conclusions We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology. PMID:21559518

Evolution of the new vertebrate head by co-option of an ancient chordate skeletal tissue.

PubMed

Jandzik, David; Garnett, Aaron T; Square, Tyler A; Cattell, Maria V; Yu, Jr-Kai; Medeiros, Daniel M

2015-02-26

A defining feature of vertebrates (craniates) is a pronounced head that is supported and protected by a robust cellular endoskeleton. In the first vertebrates, this skeleton probably consisted of collagenous cellular cartilage, which forms the embryonic skeleton of all vertebrates and the adult skeleton of modern jawless and cartilaginous fish. In the head, most cellular cartilage is derived from a migratory cell population called the neural crest, which arises from the edges of the central nervous system. Because collagenous cellular cartilage and neural crest cells have not been described in invertebrates, the appearance of cellular cartilage derived from neural crest cells is considered a turning point in vertebrate evolution. Here we show that a tissue with many of the defining features of vertebrate cellular cartilage transiently forms in the larvae of the invertebrate chordate Branchiostoma floridae (Florida amphioxus). We also present evidence that during evolution, a key regulator of vertebrate cartilage development, SoxE, gained new cis-regulatory sequences that subsequently directed its novel expression in neural crest cells. Together, these results suggest that the origin of the vertebrate head skeleton did not depend on the evolution of a new skeletal tissue, as is commonly thought, but on the spread of this tissue throughout the head. We further propose that the evolution of cis-regulatory elements near an ancient regulator of cartilage differentiation was a major factor in the evolution of the vertebrate head skeleton.

Oligodeoxynucleotide nanostructure formation in the presence of polypropyleneimine dendrimers and their uptake in breast cancer cells

NASA Astrophysics Data System (ADS)

Chen, Alex M.; Santhakumaran, Latha M.; Nair, Sandhya K.; Amenta, Peter S.; Thomas, Thresia; He, Huixin; Thomas, T. J.

2006-11-01

We studied the efficacy of five generations of polypropyleneimine (PPI) dendrimer to provoke nanostructure formation from a 21-nucleotide antisense oligodeoxynucleotide (ODN). Nanostructure formation was observed with all generations of dendrimer by light scattering and microscopic techniques. The efficacy of the dendrimers increased with generation number. Atomic force microscopy (AFM) was used to study the morphology of the structures at different condensation stages. Based on the observed nanostructures, we propose a zipping condensation mechanism, which is very different from the condensation pathways of high molecular weight DNA polymers. Electron microscopy showed the presence of toroidal nanoparticles. Confocal microscopic analysis showed that the nanostructures formed with G-4 and G-5 dendrimers could undergo facile cellular uptake in a breast cancer cell line, MDA-MB-231, whereas nanostructures formed with G-1 to G-3 dendrimers lacked this ability. Nanoparticles formed with G-1 to G-3 dendrimers showed significantly lower zeta potential (5.2-6.5 mV) than those (12-18 mV) of particles formed with G-4 and G-5 dendrimers. These results show that the structure and charge density of the dendrimers are important in ODN nanoparticle formation and cellular transport and that G-4 and G-5 dendrimers are useful in cellular delivery of antisense ODN.

Nitric Oxide Synthase and Cyclooxygenase Pathways: A Complex Interplay in Cellular Signaling.

PubMed

Sorokin, Andrey

2016-01-01

The cellular reaction to external challenges is a tightly regulated process consisting of integrated processes mediated by a variety of signaling molecules, generated as a result of modulation of corresponding biosynthetic systems. Both, nitric oxide synthase (NOS) and cyclooxygenase (COX) systems, consist of constitutive forms (NOS1, NOS3 and COX-1), which are mostly involved in housekeeping tasks, and inducible forms (NOS2 and COX-2), which shape the cellular response to stress and variety of bioactive agents. The complex interplay between NOS and COX pathways can be observed at least at three levels. Firstly, products of NOS and Cox systems can mediate the regulation and the expression of inducible forms (NOS2 and COX-2) in response of similar and dissimilar stimulus. Secondly, the reciprocal modulation of cyclooxygenase activity by nitric oxide and NOS activity by prostaglandins at the posttranslational level has been shown to occur. Mechanisms by which nitric oxide can modulate prostaglandin synthesis include direct S-nitrosylation of COX and inactivation of prostaglandin I synthase by peroxynitrite, product of superoxide reaction with nitric oxide. Prostaglandins, conversely, can promote an increased association of dynein light chain (DLC) (also known as protein inhibitor of neuronal nitric oxide synthase) with NOS1, thereby reducing its activity. The third level of interplay is provided by intracellular crosstalk of signaling pathways stimulated by products of NOS and COX which contributes significantly to the complexity of cellular signaling. Since modulation of COX and NOS pathways was shown to be principally involved in a variety of pathological conditions, the dissection of their complex relationship is needed for better understanding of possible therapeutic strategies. This review focuses on implications of interplay between NOS and COX for cellular function and signal integration.

Embryo as an active granular fluid: stress-coordinated cellular constriction chains

NASA Astrophysics Data System (ADS)

Holcomb, Michael; Gao, Guo-Jie; Thomas, Jeffrey; Blawzdziewicz, Jerzy

2016-11-01

Mechanical stress plays an intricate role in gene expression in individual cells and sculpting of developing tissues. Motivated by our observation of the cellular constriction chains (CCCs) during the initial phase of ventral furrow formation in the Drosophila melanogaster embryo, we propose an active granular fluid (AGF) model that provides valuable insights into cellular coordination in the apical constriction process. In our model, cells are treated as circular particles connected by a predefined force network, and they undergo a random constriction process in which the particle constriction probability P is a function of the stress exerted on the particle by its neighbors. We find that when P favors tensile stress, constricted particles tend to form chain-like structures. In contrast, constricted particles tend to form compact clusters when P favors compression. A remarkable similarity of constricted-particle chains and CCCs observed in vivo provides indirect evidence that tensile-stress feedback coordinates the apical constriction activity.

Exploiting the biomolecular corona: pre-coating of nanoparticles enables controlled cellular interactions.

PubMed

Simon, Johanna; Müller, Laura K; Kokkinopoulou, Maria; Lieberwirth, Ingo; Morsbach, Svenja; Landfester, Katharina; Mailänder, Volker

2018-06-14

Formation of the biomolecular corona ultimately determines the successful application of nanoparticles in vivo. Adsorption of biomolecules such as proteins is an inevitable process that takes place instantaneously upon contact with physiological fluid (e.g. blood). Therefore, strategies are needed to control this process in order to improve the properties of the nanoparticles and to allow targeted drug delivery. Here, we show that the design of the protein corona by a pre-formed protein corona with tailored properties enables targeted cellular interactions. Nanoparticles were pre-coated with immunoglobulin depleted plasma to create and design a protein corona that reduces cellular uptake by immune cells. It was proven that a pre-formed protein corona remains stable even after nanoparticles were re-introduced to plasma. This opens up the great potential to exploit protein corona formation, which will significantly influence the development of novel nanomaterials.

Impact of uranium (U) on the cellular glutathione pool and resultant consequences for the redox status of U.

PubMed

Viehweger, Katrin; Geipel, Gerhard; Bernhard, Gert

2011-12-01

Uranium (U) as a redox-active heavy metal can cause various redox imbalances in plant cells. Measurements of the cellular glutathione/glutathione disulfide (GSH/GSSG) by HPLC after cellular U contact revealed an interference with this essential redox couple. The GSH content remained unaffected by 10 μM U whereas the GSSG level immediately increased. In contrast, higher U concentrations (50 μM) drastically raised both forms. Using the Nernst equation, it was possible to calculate the half-cell reduction potential of 2GSH/GSSG. In case of lower U contents the cellular redox environment shifted towards more oxidizing conditions whereas the opposite effect was obtained by higher U contents. This indicates that U contact causes a consumption of reduced redox equivalents. Artificial depletion of GSH by chlorodinitrobenzene and measuring the cellular reducing capacity by tetrazolium salt reduction underlined the strong requirement of reduced redox equivalents. An additional element of cellular U detoxification mechanisms is the complex formation between the heavy metal and carboxylic functionalities of GSH. Because two GSH molecules catalyze electron transfers each with one electron forming a dimer (GSSG) two UO(2) (2+) are reduced to each UO(2) (+) by unbound redox sensitive sulfhydryl moieties. UO(2) (+) subsequently disproportionates to UO(2) (2+) and U(4+). This explains that in vitro experiments revealed a reduction to U(IV) of only around 33% of initial U(VI). Cellular U(IV) was transiently detected with the highest level after 2 h of U contact. Hence, it can be proposed that these reducing processes are an important element of defense reactions induced by this heavy metal.

The emergence of extracellular matrix mechanics and cell traction forces as important regulators of cellular self-organization.

PubMed

Checa, Sara; Rausch, Manuel K; Petersen, Ansgar; Kuhl, Ellen; Duda, Georg N

2015-01-01

Physical cues play a fundamental role in a wide range of biological processes, such as embryogenesis, wound healing, tumour invasion and connective tissue morphogenesis. Although it is well known that during these processes, cells continuously interact with the local extracellular matrix (ECM) through cell traction forces, the role of these mechanical interactions on large scale cellular and matrix organization remains largely unknown. In this study, we use a simple theoretical model to investigate cellular and matrix organization as a result of mechanical feedback signals between cells and the surrounding ECM. The model includes bi-directional coupling through cellular traction forces to deform the ECM and through matrix deformation to trigger cellular migration. In addition, we incorporate the mechanical contribution of matrix fibres and their reorganization by the cells. We show that a group of contractile cells will self-polarize at a large scale, even in homogeneous environments. In addition, our simulations mimic the experimentally observed alignment of cells in the direction of maximum stiffness and the building up of tension as a consequence of cell and fibre reorganization. Moreover, we demonstrate that cellular organization is tightly linked to the mechanical feedback loop between cells and matrix. Cells with a preference for stiff environments have a tendency to form chains, while cells with a tendency for soft environments tend to form clusters. The model presented here illustrates the potential of simple physical cues and their impact on cellular self-organization. It can be used in applications where cell-matrix interactions play a key role, such as in the design of tissue engineering scaffolds and to gain a basic understanding of pattern formation in organogenesis or tissue regeneration.

Differential polymer structure tunes mechanism of cellular uptake and transfection routes of poly(β-amino ester) polyplexes in human breast cancer cells.

PubMed

Kim, Jayoung; Sunshine, Joel C; Green, Jordan J

2014-01-15

Successful gene delivery with nonviral particles has several barriers, including cellular uptake, endosomal escape, and nuclear transport. Understanding the mechanisms behind these steps is critical to enhancing the effectiveness of gene delivery. Polyplexes formed with poly(β-amino ester)s (PBAEs) have been shown to effectively transfer DNA to various cell types, but the mechanism of their cellular uptake has not been identified. This is the first study to evaluate the uptake mechanism of PBAE polyplexes and the dependence of cellular uptake on the end group and molecular weight of the polymer. We synthesized three different analogues of PBAEs with the same base polymer poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (B4S4) but with small changes in the end group or molecular weight. We quantified the uptake and transfection efficiencies of the pDNA polyplexes formulated from these polymers in hard-to-transfect triple negative human breast cancer cells (MDA-MB 231). All polymers formed positively charged (10-17 mV) nanoparticles of ∼200 nm in size. Cellular internalization of all three formulations was inhibited the most (60-90% decrease in cellular uptake) by blocking caveolae-mediated endocytosis. Greater inhibition was shown with polymers that had a 1-(3-aminopropyl)-4-methylpiperazine end group (E7) than the others with a 2-(3-aminopropylamino)-ethanol end group (E6) or higher molecular weight. However, caveolae-mediated endocytosis was generally not as efficient as clathrin-mediated endocytosis in leading to transfection. These findings indicate that PBAE polyplexes can be used to transfect triple negative human breast cancer cells and that small changes to the same base polymer can modulate their cellular uptake and transfection routes.

On the exfoliating polymeric cellular dosage forms for immediate drug release.

PubMed

Blaesi, Aron H; Saka, Nannaji

2016-06-01

The most prevalent pharmaceutical dosage forms at present-the oral immediate-release tablets and capsules-are granular solids. Though effective in releasing drug rapidly, development and manufacture of such dosage forms are fraught with difficulties inherent to particulate processing. Predictable dosage form manufacture could be achieved by liquid-based processing, but cast solid dosage forms are not suitable for immediate drug release due to their resistance to fluid percolation. To overcome this limitation, we have recently introduced cellular dosage forms that can be readily prepared from polymeric melts. It has been shown that open-cell structures comprising polyethylene glycol 8000 (PEG 8k) excipient and a drug exfoliate upon immersion in a dissolution medium. The drug is then released rapidly due to the large specific surface area of the exfoliations. In this work, we vary the molecular weight of the PEG excipient and investigate its effect on the drug release kinetics of structures with predominantly open-cell topology. We demonstrate that the exfoliation rate decreases substantially if the excipient molecular weight is increased from 12 to 100kg/mol, which causes the drug dissolution time to increase by more than a factor of ten. A model is then developed to elucidate the exfoliation behavior of cellular structures. Diverse transport processes are considered: percolation due to capillarity, diffusion of dissolution medium through the cell walls, and viscous flow of the saturated excipient. It is found that the lower exfoliation rate and the longer dissolution time of the dosage forms with higher excipient molecular weight are primarily due to the greater viscosity of the cell walls after fluid penetration. Copyright © 2016 Elsevier B.V. All rights reserved.

Adaptable Hydrogel Networks with Reversible Linkages for Tissue Engineering

PubMed Central

Wang, Huiyuan

2015-01-01

Adaptable hydrogels have recently emerged as a promising platform for three-dimensional (3D) cell encapsulation and culture. In conventional, covalently crosslinked hydrogels, degradation is typically required to allow complex cellular functions to occur, leading to bulk material degradation. In contrast, adaptable hydrogels are formed by reversible crosslinks. Through breaking and re-forming of the reversible linkages, adaptable hydrogels can be locally modified to permit complex cellular functions while maintaining their long-term integrity. In addition, these adaptable materials can have biomimetic viscoelastic properties that make them well suited for several biotechnology and medical applications. In this review, adaptable hydrogel design considerations and linkage selections are overviewed, with a focus on various cell compatible crosslinking mechanisms that can be exploited to form adaptable hydrogels for tissue engineering. PMID:25989348

RRM2 induces NF-{kappa}B-dependent MMP-9 activation and enhances cellular invasiveness

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duxbury, Mark S.; Whang, Edward E.

2007-03-02

Ribonucleotide reductase is a dimeric enzyme that catalyzes conversion of ribonucleotide 5'-diphosphates to their 2'-deoxynucleotide forms, a rate-limiting step in the production of 2'-deoxyribonucleoside 5'-triphosphates required for DNA synthesis. The ribonucleotide reductase M2 subunit (RRM2) is a determinant of malignant cellular behavior in a range of human cancers. We examined the effect of RRM2 overexpression on pancreatic adenocarcinoma cellular invasiveness and nuclear factor-{kappa}B (NF-{kappa}B) transcription factor activity. RRM2 overexpression increases pancreatic adenocarcinoma cellular invasiveness and MMP-9 expression in a NF-{kappa}B-dependent manner. RNA interference (RNAi)-mediated silencing of RRM2 expression attenuates cellular invasiveness and NF-{kappa}B activity. NF-{kappa}B is a key mediator ofmore » the invasive phenotypic changes induced by RRM2 overexpression.« less

Microfluidic device to control interstitial flow-mediated homotypic and heterotypic cellular communication.

PubMed

Alonzo, Luis F; Moya, Monica L; Shirure, Venktesh S; George, Steven C

2015-09-07

Tissue engineering can potentially recreate in vivo cellular microenvironments in vitro for an array of applications such as biological inquiry and drug discovery. However, the majority of current in vitro systems still neglect many biological, chemical, and mechanical cues that are known to impact cellular functions such as proliferation, migration, and differentiation. To address this gap, we have developed a novel microfluidic device that precisely controls the spatial and temporal interactions between adjacent three-dimensional cellular environments. The device consists of four interconnected microtissue compartments (~0.1 mm(3)) arranged in a square. The top and bottom pairs of compartments can be sequentially loaded with discrete cellularized hydrogels creating the opportunity to investigate homotypic (left to right or x-direction) and heterotypic (top to bottom or y-direction) cell-cell communication. A controlled hydrostatic pressure difference across the tissue compartments in both x and y direction induces interstitial flow and modulates communication via soluble factors. To validate the biological significance of this novel platform, we examined the role of stromal cells in the process of vasculogenesis. Our device confirms previous observations that soluble mediators derived from normal human lung fibroblasts (NHLFs) are necessary to form a vascular network derived from endothelial colony forming cell-derived endothelial cells (ECFC-ECs). We conclude that this platform could be used to study important physiological and pathological processes that rely on homotypic and heterotypic cell-cell communication.

Effects of pH and aggregation in the human prion conversion into scrapie form: a study using molecular dynamics with excited normal modes.

PubMed

Lima, Angelica Nakagawa; de Oliveira, Ronaldo Junio; Braz, Antônio Sérgio Kimus; de Souza Costa, Maurício Garcia; Perahia, David; Scott, Luis Paulo Barbour

2018-03-15

There are two different prion conformations: (1) the cellular natural (PrP C ) and (2) the scrapie (PrP Sc ), an infectious form that tends to aggregate under specific conditions. PrP C and PrP Sc are widely different regarding secondary and tertiary structures. PrP Sc contains more and longer β-strands compared to PrP C . The lack of solved PrP Sc structures precludes a proper understanding of the mechanisms related to the transition between cellular and scrapie forms, as well as the aggregation process. In order to investigate the conformational transition between PrP C and PrP Sc , we applied MDeNM (molecular dynamics with excited normal modes), an enhanced sampling simulation technique that has been recently developed to probe large structural changes. These simulations yielded new structural rearrangements of the cellular prion that would have been difficult to obtain with standard MD simulations. We observed an increase in β-sheet formation under low pH (≤ 4) and upon oligomerization, whose relevance was discussed on the basis of the energy landscape theory for protein folding. The characterization of intermediate structures corresponding to transition states allowed us to propose a conversion model from the cellular to the scrapie prion, which possibly ignites the fibril formation. This model can assist the design of new drugs to prevent neurological disorders related to the prion aggregation mechanism.

Endogenous sterol biosynthesis is important for mitochondrial function and cell morphology in procyclic forms of Trypanosoma brucei.

PubMed

Pérez-Moreno, Guiomar; Sealey-Cardona, Marco; Rodrigues-Poveda, Carlos; Gelb, Michael H; Ruiz-Pérez, Luis Miguel; Castillo-Acosta, Víctor; Urbina, Julio A; González-Pacanowska, Dolores

2012-10-01

Sterol biosynthesis inhibitors are promising entities for the treatment of trypanosomal diseases. Insect forms of Trypanosoma brucei, the causative agent of sleeping sickness, synthesize ergosterol and other 24-alkylated sterols, yet also incorporate cholesterol from the medium. While sterol function has been investigated by pharmacological manipulation of sterol biosynthesis, molecular mechanisms by which endogenous sterols influence cellular processes remain largely unknown in trypanosomes. Here we analyse by RNA interference, the effects of a perturbation of three specific steps of endogenous sterol biosynthesis in order to dissect the role of specific intermediates in proliferation, mitochondrial function and cellular morphology in procyclic cells. A decrease in the levels of squalene synthase and squalene epoxidase resulted in a depletion of cellular sterol intermediates and end products, impaired cell growth and led to aberrant morphologies, DNA fragmentation and a profound modification of mitochondrial structure and function. In contrast, cells deficient in sterol methyl transferase, the enzyme involved in 24-alkylation, exhibited a normal growth phenotype in spite of a complete abolition of the synthesis and content of 24-alkyl sterols. Thus, the data provided indicates that while the depletion of squalene and post-squalene endogenous sterol metabolites results in profound cellular defects, bulk 24-alkyl sterols are not strictly required to support growth in insect forms of T. brucei in vitro. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Absolute quantification of prion protein (90-231) using stable isotope-labeled chymotryptic peptide standards in a LC-MRM AQUA workflow.

PubMed

Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M; Pedersen, Joel A; Li, Lingjun

2012-09-01

Substantial evidence indicates that the disease-associated conformer of the prion protein (PrP(TSE)) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrP(TSE) has the ability to convert the conformation of the normal prion protein (PrP(C)) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrP(TSE) at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrP(TSE), the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

Absolute quantification of prion protein (90-231) using stable isotope-labeled chymotryptic peptide standards in a LC-MRM AQUA workflow

PubMed Central

Sturm, Robert; Kreitinger, Gloria; Booth, Clarissa; Smith, Lloyd; Pedersen, Joel; Li, Lingjun

2012-01-01

Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiological agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at sub-femtomole levels while animal bioassays, cell culture, and in vitro conversion assays offer ultrasensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein’s signature peptide, often with sub-femtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors’ knowledge, this is the first report of the use of a non-tryptic peptide in a LC-MRM AQUA workflow. PMID:22714949

Absolute Quantification of Prion Protein (90-231) Using Stable Isotope-Labeled Chymotryptic Peptide Standards in a LC-MRM AQUA Workflow

NASA Astrophysics Data System (ADS)

Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M.; Pedersen, Joel A.; Li, Lingjun

2012-09-01

Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

Fate of prions in soil: trapped conformation of full-length ovine prion protein induced by adsorption on clays.

PubMed

Revault, M; Quiquampoix, H; Baron, M H; Noinville, S

2005-08-05

Studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. We consider the surface-induced conformational changes that the recombinant ovine prion protein (ovPrP) may undergo under different pH conditions when interacting with soil minerals of highly adsorptive capacities such as montmorillonite. The conformational states of the full-length ovine prion protein adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pD range 3.5-9, using FTIR spectroscopy. The in vitro pH-induced conversion of the alpha-helical monomer of ovPrP into oligomers of beta-like structure prone to self-aggregation does not occur when the protein is adsorbed on the clay surface. The conformation of the trapped ovPrP molecules on montmorillonite is pH-independent and looks like that of the ovPrP solvated state at pD higher than 7, suggesting the major role of Arg and Lys residues in the electrostatic origin of adsorption. The uneven distribution of positively and negatively charged residues of the ovPrP protein would promote a favored orientation of the protein towards the clay, so that not only the basic residues embedded in the N-terminal flexible part but also external basic residues in the globular part of the protein might participate to the attractive interaction. From these results, it appears unlikely that the interaction of normal prions (PrP(C)) with soil clay surfaces could induce a change of conformation leading to the pathogenic form of prions (PrP(Sc)).

Novel Inhibitors of Cholesterol Degradation in Mycobacterium tuberculosis Reveal How the Bacterium’s Metabolism Is Constrained by the Intracellular Environment

PubMed Central

VanderVen, Brian C.; Fahey, Ruth J.; Lee, Wonsik; Liu, Yancheng; Abramovitch, Robert B.; Memmott, Christine; Crowe, Adam M.; Eltis, Lindsay D.; Perola, Emanuele; Deininger, David D.; Wang, Tiansheng; Locher, Christopher P.; Russell, David G.

2015-01-01

Mycobacterium tuberculosis (Mtb) relies on a specialized set of metabolic pathways to support growth in macrophages. By conducting an extensive, unbiased chemical screen to identify small molecules that inhibit Mtb metabolism within macrophages, we identified a significant number of novel compounds that limit Mtb growth in macrophages and in medium containing cholesterol as the principle carbon source. Based on this observation, we developed a chemical-rescue strategy to identify compounds that target metabolic enzymes involved in cholesterol metabolism. This approach identified two compounds that inhibit the HsaAB enzyme complex, which is required for complete degradation of the cholesterol A/B rings. The strategy also identified an inhibitor of PrpC, the 2-methylcitrate synthase, which is required for assimilation of cholesterol-derived propionyl-CoA into the TCA cycle. These chemical probes represent new classes of inhibitors with novel modes of action, and target metabolic pathways required to support growth of Mtb in its host cell. The screen also revealed a structurally-diverse set of compounds that target additional stage(s) of cholesterol utilization. Mutants resistant to this class of compounds are defective in the bacterial adenylate cyclase Rv1625/Cya. These data implicate cyclic-AMP (cAMP) in regulating cholesterol utilization in Mtb, and are consistent with published reports indicating that propionate metabolism is regulated by cAMP levels. Intriguingly, reversal of the cholesterol-dependent growth inhibition caused by this subset of compounds could be achieved by supplementing the media with acetate, but not with glucose, indicating that Mtb is subject to a unique form of metabolic constraint induced by the presence of cholesterol. PMID:25675247

Protein Corona Analysis of Silver Nanoparticles Links to Their Cellular Effects.

PubMed

Juling, Sabine; Niedzwiecka, Alicia; Böhmert, Linda; Lichtenstein, Dajana; Selve, Sören; Braeuning, Albert; Thünemann, Andreas F; Krause, Eberhard; Lampen, Alfonso

2017-11-03

The breadth of applications of nanoparticles and the access to food-associated consumer products containing nanosized materials lead to oral human exposure to such particles. In biological fluids nanoparticles dynamically interact with biomolecules and form a protein corona. Knowledge about the protein corona is of great interest for understanding the molecular effects of particles as well as their fate inside the human body. We used a mass spectrometry-based toxicoproteomics approach to elucidate mechanisms of toxicity of silver nanoparticles and to comprehensively characterize the protein corona formed around silver nanoparticles in Caco-2 human intestinal epithelial cells. Results were compared with respect to the cellular function of proteins either affected by exposure to nanoparticles or present in the protein corona. A transcriptomic data set was included in the analyses in order to obtain a combined multiomics view of nanoparticle-affected cellular processes. A relationship between corona proteins and the proteomic or transcriptomic responses was revealed, showing that differentially regulated proteins or transcripts were engaged in the same cellular signaling pathways. Protein corona analyses of nanoparticles in cells might therefore help in obtaining information about the molecular consequences of nanoparticle treatment.

Embryo as an active granular fluid: stress-coordinated cellular constriction chains

NASA Astrophysics Data System (ADS)

Gao, Guo-Jie Jason; Holcomb, Michael C.; Thomas, Jeffrey H.; Blawzdziewicz, Jerzy

2016-10-01

Mechanical stress plays an intricate role in gene expression in individual cells and sculpting of developing tissues. However, systematic methods of studying how mechanical stress and feedback help to harmonize cellular activities within a tissue have yet to be developed. Motivated by our observation of the cellular constriction chains (CCCs) during the initial phase of ventral furrow formation in the Drosophila melanogaster embryo, we propose an active granular fluid (AGF) model that provides valuable insights into cellular coordination in the apical constriction process. In our model, cells are treated as circular particles connected by a predefined force network, and they undergo a random constriction process in which the particle constriction probability P is a function of the stress exerted on the particle by its neighbors. We find that when P favors tensile stress, constricted particles tend to form chain-like structures. In contrast, constricted particles tend to form compact clusters when P favors compression. A remarkable similarity of constricted-particle chains and CCCs observed in vivo provides indirect evidence that tensile-stress feedback coordinates the apical constriction activity. Our particle-based AGF model will be useful in analyzing mechanical feedback effects in a wide variety of morphogenesis and organogenesis phenomena.

Endoplasmic Reticulum-Golgi Intermediate Compartment Membranes and Vimentin Filaments Participate in Vaccinia Virus Assembly

PubMed Central

Risco, Cristina; Rodríguez, Juan R.; López-Iglesias, Carmen; Carrascosa, José L.; Esteban, Mariano; Rodríguez, Dolores

2002-01-01

Vaccinia virus (VV) has a complex morphogenetic pathway whose first steps are poorly characterized. We have studied the early phase of VV assembly, when viral factories and spherical immature viruses (IVs) form in the cytoplasm of the infected cell. After freeze-substitution numerous cellular elements are detected around assembling viruses: membranes, ribosomes, microtubules, filaments, and unidentified structures. A double membrane is clearly resolved in the VV envelope for the first time, and freeze fracture reveals groups of tubules interacting laterally on the surface of the viroplasm foci. These data strongly support the hypothesis of a cellular tubulovesicular compartment, related to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), as the origin of the first VV envelope. Moreover, the cytoskeletal vimentin intermediate filaments are found around viral factories and inside the viroplasm foci, where vimentin and the VV core protein p39 colocalize in the areas where crescents protrude. Confocal microscopy showed that ERGIC elements and vimentin filaments concentrate in the viral factories. We propose that modified cellular ERGIC membranes and vimentin intermediate filaments act coordinately in the construction of viral factories and the first VV form through a unique mechanism of viral morphogenesis from cellular elements. PMID:11799179

Evolutionary cell biology: functional insight from "endless forms most beautiful".

PubMed

Richardson, Elisabeth; Zerr, Kelly; Tsaousis, Anastasios; Dorrell, Richard G; Dacks, Joel B

2015-12-15

In animal and fungal model organisms, the complexities of cell biology have been analyzed in exquisite detail and much is known about how these organisms function at the cellular level. However, the model organisms cell biologists generally use include only a tiny fraction of the true diversity of eukaryotic cellular forms. The divergent cellular processes observed in these more distant lineages are still largely unknown in the general scientific community. Despite the relative obscurity of these organisms, comparative studies of them across eukaryotic diversity have had profound implications for our understanding of fundamental cell biology in all species and have revealed the evolution and origins of previously observed cellular processes. In this Perspective, we will discuss the complexity of cell biology found across the eukaryotic tree, and three specific examples of where studies of divergent cell biology have altered our understanding of key functional aspects of mitochondria, plastids, and membrane trafficking. © 2015 Richardson et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

[Homeostasis and Disorder of Musculoskeletal System.Cellular dynamics in musculoskeletal system visualized by intravital imaging techniques.

PubMed

Kikuta, Junichi; Ishii, Masaru

Bone is continually remodeled by bone-resorbing osteoclasts and bone-forming osteoblasts. Although it has long been believed that bone homeostasis is tightly regulated by communication between osteoclasts and osteoblasts, the fundamental process and dynamics have remained elusive. We originally established an advanced imaging system to visualize living bone tissues using intravital two-photon microscopy. By means of this system, we revealed the in vivo behavior of bone-resorbing osteoclasts and bone-forming osteoblasts in bone tissues. This approach facilitates investigation of cellular dynamics in the pathogenesis of musculoskeletal disorders, and would thus be useful for evaluating the efficacy of novel therapeutic agents.

Fabp4-CreER lineage tracing reveals two distinctive coronary vascular populations.

PubMed

He, Lingjuan; Tian, Xueying; Zhang, Hui; Wythe, Joshua D; Zhou, Bin

2014-11-01

Over the last two decades, genetic lineage tracing has allowed for the elucidation of the cellular origins and fates during both embryogenesis and in pathological settings in adults. Recent lineage tracing studies using Apln-CreER tool indicated that a large number of post-natal coronary vessels do not form from pre-existing vessels. Instead, they form de novo after birth, which represents a coronary vascular population (CVP) distinct from the pre-existing one. Herein, we present new coronary vasculature lineage tracing results using a novel tool, Fabp4-CreER. Our results confirm the distinct existence of two unique CVPs. The 1(st) CVP, which is labelled by Fabp4-CreER, arises through angiogenic sprouting of pre-existing vessels established during early embryogenesis. The 2(nd) CVP is not labelled by Fabp4, suggesting that these vessels form de novo, rather than through expansion of the 1(st) CVP. These results support the de novo formation of vessels in the post-natal heart, which has implications for studies in cardiovascular disease and heart regeneration. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

The Vitamin Nicotinamide: Translating Nutrition into Clinical Care

PubMed Central

Maiese, Kenneth; Chong, Zhao Zhong; Hou, Jinling; Shang, Yan Chen

2009-01-01

Nicotinamide, the amide form of vitamin B3 (niacin), is changed to its mononucleotide compound with the enzyme nicotinic acide/nicotinamide adenylyl-transferase, and participates in the cellular energy metabolism that directly impacts normal physiology. However, nicotinamide also influences oxidative stress and modulates multiple pathways tied to both cellular survival and death. During disorders that include immune system dysfunction, diabetes, and aging-related diseases, nicotinamide is a robust cytoprotectant that blocks cellular inflammatory cell activation, early apoptotic phosphatidylserine exposure, and late nuclear DNA degradation. Nicotinamide relies upon unique cellular pathways that involve forkhead transcription factors, sirtuins, protein kinase B (Akt), Bad, caspases, and poly (ADP-ribose) polymerase that may offer a fine line with determining cellular longevity, cell survival, and unwanted cancer progression. If one is cognizant of the these considerations, it becomes evident that nicotinamide holds great potential for multiple disease entities, but the development of new therapeutic strategies rests heavily upon the elucidation of the novel cellular pathways that nicotinamide closely governs. PMID:19783937

Domain 4 (D4) of Perfringolysin O to Visualize Cholesterol in Cellular Membranes-The Update.

PubMed

Maekawa, Masashi

2017-03-03

The cellular membrane of eukaryotes consists of phospholipids, sphingolipids, cholesterol and membrane proteins. Among them, cholesterol is crucial for various cellular events (e.g., signaling, viral/bacterial infection, and membrane trafficking) in addition to its essential role as an ingredient of steroid hormones, vitamin D, and bile acids. From a micro-perspective, at the plasma membrane, recent emerging evidence strongly suggests the existence of lipid nanodomains formed with cholesterol and phospholipids (e.g., sphingomyelin, phosphatidylserine). Thus, it is important to elucidate how cholesterol behaves in membranes and how the behavior of cholesterol is regulated at the molecular level. To elucidate the complexed characteristics of cholesterol in cellular membranes, a couple of useful biosensors that enable us to visualize cholesterol in cellular membranes have been recently developed by utilizing domain 4 (D4) of Perfringolysin O (PFO, theta toxin), a cholesterol-binding toxin. This review highlights the current progress on development of novel cholesterol biosensors that uncover new insights of cholesterol in cellular membranes.

Electromagnetic cellular interactions.

PubMed

Cifra, Michal; Fields, Jeremy Z; Farhadi, Ashkan

2011-05-01

Chemical and electrical interaction within and between cells is well established. Just the opposite is true about cellular interactions via other physical fields. The most probable candidate for an other form of cellular interaction is the electromagnetic field. We review theories and experiments on how cells can generate and detect electromagnetic fields generally, and if the cell-generated electromagnetic field can mediate cellular interactions. We do not limit here ourselves to specialized electro-excitable cells. Rather we describe physical processes that are of a more general nature and probably present in almost every type of living cell. The spectral range included is broad; from kHz to the visible part of the electromagnetic spectrum. We show that there is a rather large number of theories on how cells can generate and detect electromagnetic fields and discuss experimental evidence on electromagnetic cellular interactions in the modern scientific literature. Although small, it is continuously accumulating. Copyright © 2010 Elsevier Ltd. All rights reserved.

Growth and Decay in Life-Like Cellular Automata

NASA Astrophysics Data System (ADS)

Eppstein, David

Since the study of life began, many have asked: is it unique in the universe, or are there other interesting forms of life elsewhere? Before we can answer that question, we should ask others: What makes life special? If we happen across another system with life-like behavior, how would we be able to recognize it? We are speaking, of course, of the mathematical systems of cellular automata, of the fascinating patterns that have been discovered and engineered in Conway's Game of Life, and of the possible existence of other cellular automaton rules with equally complex behavior to that of Life.

Debio 0507 primarily forms diaminocyclohexane-Pt-d(GpG) and -d(ApG) DNA adducts in HCT116 cells

PubMed Central

King, C. L.; Ramachandran, S.; Collins, L.; Swenberg, J. A.; deKrafft, K. E.; Lin, W.; Cicurel, L.; Barbier, M.

2013-01-01

Purpose To characterize the cellular action mechanism of Debio 0507, we compared the major DNA adducts formed by Debio 0507- and oxaliplatin-treated HCT116 human colon carcinoma cells by a combination of inductively coupled plasma mass spectrometry (ICP-MS) and ultra-performance liquid chromatography mass spectrometry (UPLC-MS/MS). Methods HCT116 cells were treated with IC50 doses of Debio 0507 or oxaliplatin for 3 days. Total cellular Pt–DNA adducts were determined by ICP-MS. The DNA was digested, and the major Pt–DNA adducts formed by both drugs were characterized by UPLC/MS/MS essentially as described previously for cisplatin (Baskerville-Abraham et al. in Chem Res Toxicol 22:905–912, 2009). Results The Pt level/deoxynucleotide was 7.4/104 for DNA from Debio 0507-treated cells and 5.5/104 for oxaliplatin-treated cells following a 3-day treatment at the IC50 for each drug. UPLC-MS/MS in the positive ion mode confirmed the major Pt–DNA adducts formed by both drugs were dach-Pt-d(GpG) (904.2 m/z → 610 m/z and 904.2 m/z → 459 m/z) and dach-Pt-d(ApG) (888.2 m/z → 594 m/z and 888.2 m/z → 459 m/z). Conclusions These data show that the major DNA adducts formed by Debio 0507 are the dach-Pt-d(GpG) and dach-Pt-d(ApG) adducts and at equitoxic doses Debio 0507 and oxaliplatin form similar levels of dach-Pt-d(GpG) and dach-Pt-d(ApG) adducts. This suggests that the action mechanisms of Debio 0507 and oxaliplatin are similar at a cellular level. PMID:21968950

Patterned substrates and methods for nerve regeneration

DOEpatents

Mallapragada, Surya K.; Heath, Carole; Shanks, Howard; Miller, Cheryl A.; Jeftinija, Srdija

2004-01-13

Micropatterned substrates and methods for fabrication of artificial nerve regeneration conduits and methods for regenerating nerves are provided. Guidance compounds or cells are seeded in grooves formed on the patterned substrate. The substrates may also be provided with electrodes to provide electrical guidance cues to the regenerating nerve. The micropatterned substrates give physical, chemical, cellular and/or electrical guidance cues to promote nerve regeneration at the cellular level.

A Major Binding Protein for Leukemia Inhibitory Factor in Normal Mouse Serum: Identification as a Soluble Form of the Cellular Receptor

NASA Astrophysics Data System (ADS)

Layton, Meredith J.; Cross, Bronwyn A.; Metcalf, Donald; Ward, Larry D.; Simpson, Richard J.; Nicola, Nicos A.

1992-09-01

A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (K_d = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the α chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 μg/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.

Pulsating Hydrodynamic Instability in a Dynamic Model of Liquid-Propellant Combustion

NASA Technical Reports Server (NTRS)

Margolis, Stephen B.; Sacksteder, Kurt (Technical Monitor)

1999-01-01

Hydrodynamic (Landau) instability in combustion is typically associated with the onset of wrinkling of a flame surface, corresponding to the formation of steady cellular structures as the stability threshold is crossed. In the context of liquid-propellant combustion, such instability has recently been shown to occur for critical values of the pressure sensitivity of the burning rate and the disturbance wavenumber, significantly generalizing previous classical results for this problem that assumed a constant normal burning rate. Additionally, however, a pulsating form of hydrodynamic instability has been shown to occur as well, corresponding to the onset of temporal oscillations in the location of the liquid/gas interface. In the present work, we consider the realistic influence of a nonzero temperature sensitivity in the local burning rate on both types of stability thresholds. It is found that for sufficiently small values of this parameter, there exists a stable range of pressure sensitivities for steady, planar burning such that the classical cellular form of hydrodynamic instability and the more recent pulsating form of hydrodynamic instability can each occur as the corresponding stability threshold is crossed. For larger thermal sensitivities, however, the pulsating stability boundary evolves into a C-shaped curve in the disturbance-wavenumber/ pressure-sensitivity plane, indicating loss of stability to pulsating perturbations for all sufficiently large disturbance wavelengths. It is thus concluded, based on characteristic parameter values, that an equally likely form of hydrodynamic instability in liquid-propellant combustion is of a nonsteady, long-wave nature, distinct from the steady, cellular form originally predicted by Landau.

Pulsating Hydrodynamic Instability and Thermal Coupling in an Extended Landau/Levich Model of Liquid-Propellant Combustion. 1; Inviscid Analysis

NASA Technical Reports Server (NTRS)

Margolis, Stephen B.; Sacksteder, Kurt (Technical Monitor)

1999-01-01

Hydrodynamic (Landau) instability in combustion is typically associated with the onset of wrinkling of a flame surface, corresponding to the formation of steady cellular structures as the stability threshold is crossed. In the context of liquid-propellant combustion, such instability has recently been shown to occur for critical values of the pressure sensitivity of the burning rate and the disturbance wavenumber, significantly generalizing previous classical results for this problem that assumed a constant normal burning rate. Additionally, however, a pulsating form of hydrodynamic instability has been shown to occur as well, corresponding to the onset of temporal oscillations in the location of the liquid/gas interface. In the present work, we consider the realistic influence of a non-zero temperature sensitivity in the local burning rate on both types of stability thresholds. It is found that for sufficiently small values of this parameter, there exists a stable range of pressure sensitivities for steady, planar burning such that the classical cellular form of hydrodynamic instability and the more recent pulsating form of hydrodynamic instability can each occur as the corresponding stability threshold is crossed. For larger thermal sensitivities, however, the pulsating stability boundary evolves into a C-shaped curve in the (disturbance-wavenumber, pressure-sensitivity) plane, indicating loss of stability to pulsating perturbations for all sufficiently large disturbance wavelengths. It is thus concluded, based on characteristic parameter values, that an equally likely form of hydrodynamic instability in liquid-propellant combustion is of a non-steady, long-wave nature, distinct from the steady, cellular form originally predicted by Landau.

The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation.

PubMed

Halamoda-Kenzaoui, Blanka; Ceridono, Mara; Urbán, Patricia; Bogni, Alessia; Ponti, Jessica; Gioria, Sabrina; Kinsner-Ovaskainen, Agnieszka

2017-06-26

Significant progress of nanotechnology, including in particular biomedical and pharmaceutical applications, has resulted in a high number of studies describing the biological effects of nanomaterials. Moreover, a determination of so-called "critical quality attributes", that is specific physicochemical properties of nanomaterials triggering the observed biological response, has been recognised as crucial for the evaluation and design of novel safe and efficacious therapeutics. In the context of in vitro studies, a thorough physicochemical characterisation of nanoparticles (NPs), also in the biological medium, is necessary to allow a correlation with a cellular response. Following this concept, we examined whether the main and frequently reported characteristics of NPs such as size and the agglomeration state can influence the level and the mechanism of NP cellular internalization. We employed fluorescently-labelled 30 and 80 nm silicon dioxide NPs, both in agglomerated and non-agglomerated form. Using flow cytometry, transmission electron microscopy, the inhibitors of endocytosis and gene silencing we determined the most probable routes of cellular uptake for each form of tested silica NPs. We observed differences in cellular uptake depending on the size and the agglomeration state of NPs. Caveolae-mediated endocytosis was implicated particularly in the internalisation of well dispersed silica NPs but with an increase of the agglomeration state of NPs a combination of endocytic pathways with a predominant role of macropinocytosis was noted. We demonstrated that the agglomeration state of NPs is an important factor influencing the level of cell uptake and the mechanism of endocytosis of silica NPs.

Cellular distribution, purification and electrophoretic properties of malate dehydrogenase in Trichuris ovis and inhibition by benzimidazoles and pyrimidine derivatives.

PubMed

Sanchez-Moreno, M; Ortega, J E; Valero, A

1989-12-01

High levels of malate dehydrogenase were found in Trichuris ovis. Two molecular forms of the enzyme, of different cellular location and electrophoretic pattern, were isolated and purified. The activity of soluble malate dehydrogenase was greater than that of mitochondrial malate dehydrogenase. Both forms also displayed different electrophoretic profiles in comparison with purified extracts from goat (Capra hircus) liver. Substrate concentration directly affected enzyme activity. Host and parasite malate dehydrogenase activity were both inhibited by a series of benzimidazoles and pyrimidine-derived compounds, some of which markedly reduced parasite enzyme activity, but not host enzyme activity. Percentage inhibition by some pyrimidine derivatives was greater than that produced by benzimidazoles.

Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome.

PubMed

Rönnberg, Tuomas; Jääskeläinen, Kirsi; Blot, Guillaume; Parviainen, Ville; Vaheri, Antti; Renkonen, Risto; Bouloy, Michele; Plyusnin, Alexander

2012-01-01

Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

Identification of Modules in Protein-Protein Interaction Networks

NASA Astrophysics Data System (ADS)

Erten, Sinan; Koyutürk, Mehmet

In biological systems, most processes are carried out through orchestration of multiple interacting molecules. These interactions are often abstracted using network models. A key feature of cellular networks is their modularity, which contributes significantly to the robustness, as well as adaptability of biological systems. Therefore, modularization of cellular networks is likely to be useful in obtaining insights into the working principles of cellular systems, as well as building tractable models of cellular organization and dynamics. A common, high-throughput source of data on molecular interactions is in the form of physical interactions between proteins, which are organized into protein-protein interaction (PPI) networks. This chapter provides an overview on identification and analysis of functional modules in PPI networks, which has been an active area of research in the last decade.

Navigating novel mechanisms of cellular plasticity with the NAD+ precursor and nutrient nicotinamide.

PubMed

Li, Faqi; Chong, Zhao Zhong; Maiese, Kenneth

2004-09-01

Interest in neuroprotectants for the central nervous system continues to garner significant attention. Nicotinamide, the amide form of niacin (vitamin B3), is the precursor for the coenzyme beta-nicotinamide adenine dinucleotide (NAD+) and is considered to be necessary for cellular function and metabolism. However, recent work has focused on the development of nicotinamide as a novel agent that is critical for modulating cellular plasticity, longevity, and inflammatory microglial function. The ability of nicotinamide to preserve both neuronal and vascular cell populations in the brain during injury is intriguing, but further knowledge of the specific cellular mechanisms that determine protection by this agent is required. The capacity of nicotinamide to govern not only intrinsic cellular integrity, but also extrinsic cellular inflammation rests with the modulation of a host of cellular targets that involve protein kinase B, glycogen synthase kinase-3 beta (GSK-3 beta), Forkhead transcription factors, mitochondrial dysfunction, poly(ADP-ribose) polymerase, cysteine proteases, and microglial activation. Intimately tied to the cytoprotection of nicotinamide is the modulation of an early and late phase of apoptotic injury that is triggered by the loss of membrane asymmetry. Identifying robust cytoprotective agents as nicotinamide in conjunction with the elucidation of the cellular mechanisms responsible for cell survival will continue to solidify the development of therapeutic strategies against neurodegenerative diseases

Transient inter-cellular polymeric linker.

PubMed

Ong, Siew-Min; He, Lijuan; Thuy Linh, Nguyen Thi; Tee, Yee-Han; Arooz, Talha; Tang, Guping; Tan, Choon-Hong; Yu, Hanry

2007-09-01

Three-dimensional (3D) tissue-engineered constructs with bio-mimicry cell-cell and cell-matrix interactions are useful in regenerative medicine. In cell-dense and matrix-poor tissues of the internal organs, cells support one another via cell-cell interactions, supplemented by small amount of the extra-cellular matrices (ECM) secreted by the cells. Here we connect HepG2 cells directly but transiently with inter-cellular polymeric linker to facilitate cell-cell interaction and aggregation. The linker consists of a non-toxic low molecular-weight polyethyleneimine (PEI) backbone conjugated with multiple hydrazide groups that can aggregate cells within 30 min by reacting with the aldehyde handles on the chemically modified cell-surface glycoproteins. The cells in the cellular aggregates proliferated; and maintained the cortical actin distribution of the 3D cell morphology while non-aggregated cells died over 7 days of suspension culture. The aggregates lost distinguishable cell-cell boundaries within 3 days; and the ECM fibers became visible around cells from day 3 onwards while the inter-cellular polymeric linker disappeared from the cell surfaces over time. The transient inter-cellular polymeric linker can be useful for forming 3D cellular and tissue constructs without bulk biomaterials or extensive network of engineered ECM for various applications.

Mechanisms for Variation of Cellular P Stoichiometry: Diverse Cellular Phosphorus Allocation Strategies Across Microbial Groups from the Sargasso Sea

NASA Astrophysics Data System (ADS)

Popendorf, K.; Duhamel, S.

2016-02-01

Phosphorus is the least abundant of the three major macronutrients that define the canonical Redfield ratio, but its place in the backbone of nucleic acids and as an energy trafficking molecule lays a lower bound of cellular phosphorus content that is essential for all life. In addition to forming DNA, RNA, and adenosine triphosphate (ATP), significant amounts of cellular phosphorus may also be allocated to the production of phospholipids and polyphosphate. These latter two biochemicals in particular may occur in significant but highly variable amounts across different microbial groups, and the variation in cellular allocation to these biochemicals may be a contributing factor in defining the elemental stoichiometry of microbes. We investigated this variation in cellular phosphorus allocation across the most abundant microbial groups in the P-depleted Sargasso Sea: Prochlorococcus, Synechococcus, and heterotrophic bacteria. By coupling radioisotope tracing of phosphate and ATP with cell sorting flow cytometry and subsequent biochemical extractions, we made novel measurements of the P allocation to DNA, phospholipids, and polyphosphate in individual microbial groups from environmental populations. These results provide new insights into the cellular mechanisms of variation in stoichiometry and different microbial strategies for adaptation to low-P environments.

TWO INNEXINS OF Spodoptera litura INFLUENCES HEMICHANNEL AND GAP JUNCTION FUNCTIONS IN CELLULAR IMMUNE RESPONSES.

PubMed

Pang, Zunyu; Li, Ming; Yu, Dongshuai; Yan, Zhang; Liu, Xinyi; Ji, Xinglai; Yang, Yang; Hu, Jiansheng; Luo, Kaijun

2015-09-01

Insect cellular immune responses include encapsulation, nodule formation, and phagocytosis. Hemichannels and gap junctions are involved in these cellular actions. Innexins (Inxs: analogous to the vertebrate connexins) form hemichannels and gap junctions, but the molecular mechanisms underlying their biology is still unclear. In this article, we reported a steady-state level of Inxs (SpliInxs) in hemocytes of Spodoptera litura, which formed nonfunctional hemichannels on the cell surface to maintain normal metabolism. We also reported that two innnexins (SpliInx2 and SpliInx3) were expressed significantly higher in hemocytes compared to other tissues, suggesting that they play important roles in hemocytes. Amino acid analysis found that two cysteine residues in two extracellular loops provided the capability for SpliInx2 and SpliInx3 hemichannels to dock into gap junctions. Western blotting demonstrated that both extracellular and intracellular loops of SpliInx3 and the extracellular loops of SpliInx2 might undergo posttranslational modification during the formation of a steady-state hemichannel. During hemichannel formation, SpliInx2 presented as one isoform, while SpliInx3 presented as three isoforms. These results provide fundamental knowledge for further study of how steady-state levels of SpliInxs are dynamically adjusted to perform cellular immune responses under immune challenge. © 2015 Wiley Periodicals, Inc.

Cellular Chaperonin CCTγ Contributes to Rabies Virus Replication during Infection

PubMed Central

Zhang, Jinyang; Wu, Xiaopeng; Zan, Jie; Wu, Yongping; Ye, Chengjin; Ruan, Xizhen

2013-01-01

Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCTγ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit. PMID:23637400

Understanding of Leaf Development-the Science of Complexity.

PubMed

Malinowski, Robert

2013-06-25

The leaf is the major organ involved in light perception and conversion of solar energy into organic carbon. In order to adapt to different natural habitats, plants have developed a variety of leaf forms, ranging from simple to compound, with various forms of dissection. Due to the enormous cellular complexity of leaves, understanding the mechanisms regulating development of these organs is difficult. In recent years there has been a dramatic increase in the use of technically advanced imaging techniques and computational modeling in studies of leaf development. Additionally, molecular tools for manipulation of morphogenesis were successfully used for in planta verification of developmental models. Results of these interdisciplinary studies show that global growth patterns influencing final leaf form are generated by cooperative action of genetic, biochemical, and biomechanical inputs. This review summarizes recent progress in integrative studies on leaf development and illustrates how intrinsic features of leaves (including their cellular complexity) influence the choice of experimental approach.

Understanding of Leaf Development—the Science of Complexity

PubMed Central

Malinowski, Robert

2013-01-01

The leaf is the major organ involved in light perception and conversion of solar energy into organic carbon. In order to adapt to different natural habitats, plants have developed a variety of leaf forms, ranging from simple to compound, with various forms of dissection. Due to the enormous cellular complexity of leaves, understanding the mechanisms regulating development of these organs is difficult. In recent years there has been a dramatic increase in the use of technically advanced imaging techniques and computational modeling in studies of leaf development. Additionally, molecular tools for manipulation of morphogenesis were successfully used for in planta verification of developmental models. Results of these interdisciplinary studies show that global growth patterns influencing final leaf form are generated by cooperative action of genetic, biochemical, and biomechanical inputs. This review summarizes recent progress in integrative studies on leaf development and illustrates how intrinsic features of leaves (including their cellular complexity) influence the choice of experimental approach. PMID:27137383

Analytical formulation of cellular automata rules using data models

NASA Astrophysics Data System (ADS)

Jaenisch, Holger M.; Handley, James W.

2009-05-01

We present a unique method for converting traditional cellular automata (CA) rules into analytical function form. CA rules have been successfully used for morphological image processing and volumetric shape recognition and classification. Further, the use of CA rules as analog models to the physical and biological sciences can be significantly extended if analytical (as opposed to discrete) models could be formulated. We show that such transformations are possible. We use as our example John Horton Conway's famous "Game of Life" rule set. We show that using Data Modeling, we are able to derive both polynomial and bi-spectrum models of the IF-THEN rules that yield equivalent results. Further, we demonstrate that the "Game of Life" rule set can be modeled using the multi-fluxion, yielding a closed form nth order derivative and integral. All of the demonstrated analytical forms of the CA rule are general and applicable to real-time use.

Requirement for the Murine Zinc Finger Protein ZFR in Perigastrulation Growth and Survival

PubMed Central

Meagher, Madeleine J.; Braun, Robert E.

2001-01-01

The transition from preimplantation to postimplantation development leads to the initiation of complex cellular differentiation and morphogenetic movements, a dramatic decrease in cell cycle length, and a commensurate increase in the size of the embryo. Accompanying these changes is the need for the transfer of nutrients from the mother to the embryo and the elaboration of sophisticated genetic networks that monitor genomic integrity and the homeostatic control of cellular growth, differentiation, and programmed cell death. To determine the function of the murine zinc finger protein ZFR in these events, we generated mice carrying a null mutation in the gene encoding it. Homozygous mutant embryos form normal-appearing blastocysts that implant and initiate the process of gastrulation. Mutant embryos form mesoderm but they are delayed in their development and fail to form normal anterior embryonic structures. Loss of ZFR function leads to both an increase in programmed cell death and a decrease in mitotic index, especially in the region of the distal tip of the embryonic ectoderm. Mutant embryos also have an apparent reduction in apical vacuoles in the columnar visceral endoderm cells in the extraembryonic region. Together, these cellular phenotypes lead to a dramatic development delay and embryonic death by 8 to 9 days of gestation, which are independent of p53 function. PMID:11283266

Can mechanics control pattern formation in plants?

PubMed

Dumais, Jacques

2007-02-01

Development of the plant body entails many pattern forming events at scales ranging from the cellular level to the whole plant. Recent evidence suggests that mechanical forces play a role in establishing some of these patterns. The development of cellular configurations in glandular trichomes and the rippling of leaf surfaces are discussed in depth to illustrate how intricate patterns can emerge from simple and well-established molecular and cellular processes. The ability of plants to sense and transduce mechanical signals suggests that complex interactions between mechanics and chemistry are possible during plant development. The inclusion of mechanics alongside traditional molecular controls offers a more comprehensive view of developmental processes.

Understanding the Form, Function, and Logic of Clandestine Cellular Networks: The First Step in Effective Counternetwork Operations

DTIC Science & Technology

2009-04-01

REPORT ...PAGE Form ApprovedOMB No. 0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per...suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports

Differential growth of wrinkled biofilms

NASA Astrophysics Data System (ADS)

Espeso, D. R.; Carpio, A.; Einarsson, B.

2015-02-01

Biofilms are antibiotic-resistant bacterial aggregates that grow on moist surfaces and can trigger hospital-acquired infections. They provide a classical example in biology where the dynamics of cellular communities may be observed and studied. Gene expression regulates cell division and differentiation, which affect the biofilm architecture. Mechanical and chemical processes shape the resulting structure. We gain insight into the interplay between cellular and mechanical processes during biofilm development on air-agar interfaces by means of a hybrid model. Cellular behavior is governed by stochastic rules informed by a cascade of concentration fields for nutrients, waste, and autoinducers. Cellular differentiation and death alter the structure and the mechanical properties of the biofilm, which is deformed according to Föppl-Von Kármán equations informed by cellular processes and the interaction with the substratum. Stiffness gradients due to growth and swelling produce wrinkle branching. We are able to reproduce wrinkled structures often formed by biofilms on air-agar interfaces, as well as spatial distributions of differentiated cells commonly observed with B. subtilis.

The coming of age of chaperone-mediated autophagy.

PubMed

Kaushik, Susmita; Cuervo, Ana Maria

2018-06-01

Chaperone-mediated autophagy (CMA) was the first studied process that indicated that degradation of intracellular components by the lysosome can be selective - a concept that is now well accepted for other forms of autophagy. Lysosomes can degrade cellular cytosol in a nonspecific manner but can also discriminate what to target for degradation with the involvement of a degradation tag, a chaperone and a sophisticated mechanism to make the selected proteins cross the lysosomal membrane through a dedicated translocation complex. Recent studies modulating CMA activity in vivo using transgenic mouse models have demonstrated that selectivity confers on CMA the ability to participate in the regulation of multiple cellular functions. Timely degradation of specific cellular proteins by CMA modulates, for example, glucose and lipid metabolism, DNA repair, cellular reprograming and the cellular response to stress. These findings expand the physiological relevance of CMA beyond its originally identified role in protein quality control and reveal that CMA failure with age may aggravate diseases, such as ageing-associated neurodegeneration and cancer.

[THE SYSTEMIC IMMUNITY CELLULAR LINK REACTION IN PATIENTS WITH TRAUMATIC ILLNESS].

PubMed

Plehutsa, I M; Sydorchuk, R I; Plehutsa, O M

2015-01-01

The effect of trauma on parameters of cellular immunity changes is studied. The study includes 52 patients with various forms of traumatic illness, aged 18-69 years (37.91-4.28). The control group consisted of 16 patients who underwent routine surgery not related to the pathology of musculoskeletal system. All patients of the main group were divided into 3 groups according to severity of the condition. Analysis of parameters of cellular link of immune system was performed by defining subpopulations of T-lymphocytes in indirect immunofluorescence method using a panel of monoclonal antibodies for CD3, CD4, CD8, CD22 lymphocytes' receptors and calculation of integrated indicators. The highest expression (immune disorders of II-III grades) of changes of cellular immunity observed in patients with severe traumatic: illness (expand clinical picture). Surgical intervention, even without traumatic injury significantly impact cellular immunity, but in patients with traumatic illness immunity violation were significantly higher than in comparison groups patients except immunoregulatory index.

[Cell signaling pathways interaction in cellular proliferation: Potential target for therapeutic interventionism].

PubMed

Valdespino-Gómez, Víctor Manuel; Valdespino-Castillo, Patricia Margarita; Valdespino-Castillo, Víctor Edmundo

2015-01-01

Nowadays, cellular physiology is best understood by analysing their interacting molecular components. Proteins are the major components of the cells. Different proteins are organised in the form of functional clusters, pathways or networks. These molecules are ordered in clusters of receptor molecules of extracellular signals, transducers, sensors and biological response effectors. The identification of these intracellular signaling pathways in different cellular types has required a long journey of experimental work. More than 300 intracellular signaling pathways have been identified in human cells. They participate in cell homeostasis processes for structural and functional maintenance. Some of them participate simultaneously or in a nearly-consecutive progression to generate a cellular phenotypic change. In this review, an analysis is performed on the main intracellular signaling pathways that take part in the cellular proliferation process, and the potential use of some components of these pathways as target for therapeutic interventionism are also underlined. Copyright © 2015 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.

Discrimination of Self and Non-Self Ribonucleic Acids

PubMed Central

Gebhardt, Anna; Laudenbach, Beatrice T.

2017-01-01

Most virus infections are controlled through the innate and adaptive immune system. A surprisingly limited number of so-called pattern recognition receptors (PRRs) have the ability to sense a large variety of virus infections. The reason for the broad activity of PRRs lies in the ability to recognize viral nucleic acids. These nucleic acids lack signatures that are present in cytoplasmic cellular nucleic acids and thereby marking them as pathogen-derived. Accumulating evidence suggests that these signatures, which are predominantly sensed by a class of PRRs called retinoic acid-inducible gene I (RIG-I)-like receptors and other proteins, are not unique to viruses but rather resemble immature forms of cellular ribonucleic acids generated by cellular polymerases. RIG-I-like receptors, and other cellular antiviral proteins, may therefore have mainly evolved to sense nonprocessed nucleic acids typically generated by primitive organisms and pathogens. This capability has not only implications on induction of antiviral immunity but also on the function of cellular proteins to handle self-derived RNA with stimulatory potential. PMID:28475460

Experimental approaches to identify cellular G-quadruplex structures and functions.

PubMed

Di Antonio, Marco; Rodriguez, Raphaël; Balasubramanian, Shankar

2012-05-01

Guanine-rich nucleic acids can fold into non-canonical DNA secondary structures called G-quadruplexes. The formation of these structures can interfere with the biology that is crucial to sustain cellular homeostases and metabolism via mechanisms that include transcription, translation, splicing, telomere maintenance and DNA recombination. Thus, due to their implication in several biological processes and possible role promoting genomic instability, G-quadruplex forming sequences have emerged as potential therapeutic targets. There has been a growing interest in the development of synthetic molecules and biomolecules for sensing G-quadruplex structures in cellular DNA. In this review, we summarise and discuss recent methods developed for cellular imaging of G-quadruplexes, and the application of experimental genomic approaches to detect G-quadruplexes throughout genomic DNA. In particular, we will discuss the use of engineered small molecules and natural proteins to enable pull-down, ChIP-Seq, ChIP-chip and fluorescence imaging of G-quadruplex structures in cellular DNA. Copyright © 2012 Elsevier Inc. All rights reserved.

Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

PubMed

Stone, Matthew B; Shelby, Sarah A; Veatch, Sarah L

2017-06-14

Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

Tissue Engineering Strategies for Myocardial Regeneration: Acellular Versus Cellular Scaffolds?

PubMed

Domenech, Maribella; Polo-Corrales, Lilliana; Ramirez-Vick, Jaime E; Freytes, Donald O

2016-12-01

Heart disease remains one of the leading causes of death in industrialized nations with myocardial infarction (MI) contributing to at least one fifth of the reported deaths. The hypoxic environment eventually leads to cellular death and scar tissue formation. The scar tissue that forms is not mechanically functional and often leads to myocardial remodeling and eventual heart failure. Tissue engineering and regenerative medicine principles provide an alternative approach to restoring myocardial function by designing constructs that will restore the mechanical function of the heart. In this review, we will describe the cellular events that take place after an MI and describe current treatments. We will also describe how biomaterials, alone or in combination with a cellular component, have been used to engineer suitable myocardium replacement constructs and how new advanced culture systems will be required to achieve clinical success.

Cellular compartmentation follows rules: The Schnepf theorem, its consequences and exceptions: A biological membrane separates a plasmatic from a non-plasmatic phase.

PubMed

Moog, Daniel; Maier, Uwe G

2017-08-01

Is the spatial organization of membranes and compartments within cells subjected to any rules? Cellular compartmentation differs between prokaryotic and eukaryotic life, because it is present to a high degree only in eukaryotes. In 1964, Prof. Eberhard Schnepf formulated the compartmentation rule (Schnepf theorem), which posits that a biological membrane, the main physical structure responsible for cellular compartmentation, usually separates a plasmatic form a non-plasmatic phase. Here we review and re-investigate the Schnepf theorem by applying the theorem to different cellular structures, from bacterial cells to eukaryotes with their organelles and compartments. In conclusion, we can confirm the general correctness of the Schnepf theorem, noting explicit exceptions only in special cases such as endosymbiosis and parasitism. © 2017 WILEY Periodicals, Inc.

Method for determining gene knockouts

DOEpatents

Maranas, Costas D [Port Matilda, PA; Burgard, Anthony R [State College, PA; Pharkya, Priti [State College, PA

2011-09-27

A method for determining candidates for gene deletions and additions using a model of a metabolic network associated with an organism, the model includes a plurality of metabolic reactions defining metabolite relationships, the method includes selecting a bioengineering objective for the organism, selecting at least one cellular objective, forming an optimization problem that couples the at least one cellular objective with the bioengineering objective, and solving the optimization problem to yield at least one candidate.

Method for determining gene knockouts

DOEpatents

Maranas, Costa D; Burgard, Anthony R; Pharkya, Priti

2013-06-04

A method for determining candidates for gene deletions and additions using a model of a metabolic network associated with an organism, the model includes a plurality of metabolic reactions defining metabolite relationships, the method includes selecting a bioengineering objective for the organism, selecting at least one cellular objective, forming an optimization problem that couples the at least one cellular objective with the bioengineering objective, and solving the optimization problem to yield at least one candidate.

Distinct cellular distributions of Kv4 pore-forming and auxiliary subunits in rat dorsal root ganglion neurons.

PubMed

Matsuyoshi, Hiroko; Takimoto, Koichi; Yunoki, Takakazu; Erickson, Vickie L; Tyagi, Pradeep; Hirao, Yoshihiko; Wanaka, Akio; Yoshimura, Naoki

2012-09-17

Dorsal root ganglia contain heterogeneous populations of primary afferent neurons that transmit various sensory stimuli. This functional diversity may be correlated with differential expression of voltage-gated K(+) (Kv) channels. Here, we examine cellular distributions of Kv4 pore-forming and ancillary subunits that are responsible for fast-inactivating A-type K(+) current. Expression pattern of Kv α-subunit, β-subunit and auxiliary subunit was investigated using immunohistochemistry, in situ hybridization and RT-PCR technique. The two pore-forming subunits Kv4.1 and Kv4.3 show distinct cellular distributions: Kv4.3 is predominantly in small-sized C-fiber neurons, whereas Kv4.1 is seen in DRG neurons in various sizes. Furthermore, the two classes of Kv4 channel auxiliary subunits are also distributed in different-sized cells. KChIP3 is the only significantly expressed Ca(2+)-binding cytosolic ancillary subunit in DRGs and present in medium to large-sized neurons. The membrane-spanning auxiliary subunit DPP6 is seen in a large number of DRG neurons in various sizes, whereas DPP10 is restricted in small-sized neurons. Distinct combinations of Kv4 pore-forming and auxiliary subunits may constitute A-type channels in DRG neurons with different physiological roles. Kv4.1 subunit, in combination with KChIP3 and/or DPP6, form A-type K(+) channels in medium to large-sized A-fiber DRG neurons. In contrast, Kv4.3 and DPP10 may contribute to A-type K(+) current in non-peptidergic, C-fiber somatic afferent neurons. Copyright © 2012 Elsevier Inc. All rights reserved.

Blending of diblock and triblock copolypeptide amphiphiles yields cell penetrating vesicles with low toxicity.

PubMed

Rodriguez, April R; Choe, Uh-Joo; Kamei, Daniel T; Deming, Timothy J

2015-01-01

We prepared dual hydrophilic triblock copolypeptide vesicles that form both micron and nanometer scale vesicles in aqueous media. The incorporation of terminal homoarginine segments into methionine sulfoxide-based vesicles was found to significantly enhance their cellular uptake compared to a non-ionic control. We also demonstrated that diblock and triblock copolypeptides with similar hydrophobic domains were found to mix well and form vesicle populations with uniform compositions. Blending of amphiphiles in vesicle nanocarriers was found to impart these materials with many advantageous properties, including good cellular uptake while maintaining minimal toxicity, as well as biological responsiveness to promote vesicle disruption and release of encapsulated cargos. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

More Haste, Less Speed: Could Public–Private Partnerships Advance Cellular Immunotherapies?

PubMed Central

Bubela, Tania; Bonter, Katherine; Lachance, Silvy; Delisle, Jean-Sébastien; Gold, E. Richard

2017-01-01

Cellular immunotherapies promise to transform cancer care. However, they must overcome serious challenges, including: (1) the need to identify and characterize novel cancer antigens to expand the range of therapeutic targets; (2) the need to develop strategies to minimize serious adverse events, such as cytokine release syndrome and treatment-related toxicities; and (3) the need to develop efficient production/manufacturing processes to reduce costs. Here, we discuss whether these challenges might better be addressed through forms of public–private research collaborations, including public–private partnerships (PPPs), or whether these challenges are best addressed by way of standard market transactions. We reviewed 14 public–private relationships and 25 underlying agreements for the clinical development of cancer cellular immunotherapies in the US. Most were based on bilateral research agreements and pure market transactions in the form of service contracts and technology licenses, which is representative of the commercialization focus of the field. We make the strategic case that multiparty PPPs may better advance cancer antigen discovery and characterization and improved cell processing/manufacturing and related activities. In the rush toward the competitive end of the translational continuum for cancer cellular immunotherapy and the attendant focus on commercialization, many gaps have appeared in our understanding of cellular biology, immunology, and bioengineering. We conclude that the model of bilateral agreements between leading research institutions and the private sector may be inadequate to efficiently harness the interdisciplinary skills and knowledge of the public and private sectors to bring these promising therapies to the clinic for the benefit of cancer patients. PMID:28861415

More Haste, Less Speed: Could Public-Private Partnerships Advance Cellular Immunotherapies?

PubMed

Bubela, Tania; Bonter, Katherine; Lachance, Silvy; Delisle, Jean-Sébastien; Gold, E Richard

2017-01-01

Cellular immunotherapies promise to transform cancer care. However, they must overcome serious challenges, including: (1) the need to identify and characterize novel cancer antigens to expand the range of therapeutic targets; (2) the need to develop strategies to minimize serious adverse events, such as cytokine release syndrome and treatment-related toxicities; and (3) the need to develop efficient production/manufacturing processes to reduce costs. Here, we discuss whether these challenges might better be addressed through forms of public-private research collaborations, including public-private partnerships (PPPs), or whether these challenges are best addressed by way of standard market transactions. We reviewed 14 public-private relationships and 25 underlying agreements for the clinical development of cancer cellular immunotherapies in the US. Most were based on bilateral research agreements and pure market transactions in the form of service contracts and technology licenses, which is representative of the commercialization focus of the field. We make the strategic case that multiparty PPPs may better advance cancer antigen discovery and characterization and improved cell processing/manufacturing and related activities. In the rush toward the competitive end of the translational continuum for cancer cellular immunotherapy and the attendant focus on commercialization, many gaps have appeared in our understanding of cellular biology, immunology, and bioengineering. We conclude that the model of bilateral agreements between leading research institutions and the private sector may be inadequate to efficiently harness the interdisciplinary skills and knowledge of the public and private sectors to bring these promising therapies to the clinic for the benefit of cancer patients.

The cellular responses of the rat to an intraperitoneal inoculation of Nippostrongylus brasiliensis larvae

PubMed Central

Greenberg, Z.; Wertheim, Guta

1973-01-01

The cellular responses to intraperitoneal inoculation of infective (L3) or non-infective (L2) larvae of Nippostrongylus brasiliensis were studied in unprimed rats. Peritoneal macrophages adhered to the larvae immediately after inoculation and the coated larvae became attached to the omentum. As additional inflammatory cells, appearing in the peritoneal exudate, adhered to the larvae, nodules were formed which with time organized into granulomas. The initial response was not specific and consisted of an intense neutrophilia which developed in all rats a few hours after inoculation and lasted 24 hours. Thereafter the cellular responses were distinctly different in the case of each larval stage. In rats receiving L3 larvae an intense eosinophilia in the peritoneal exudate began to develop 7 days after inoculation, and islands of numerous pyroninophilic blast- and plasma cells were present at the periphery of the granuloma. The L3 larvae survived in the granulomas for 7–10 days. The granulomas formed around the L2 larvae consisted mainly of macrophages; the number of eosinophils did not rise above normal and there were no pyroninophilic cells. The L2 larvae survived in the granuloma for 3 days. In control rats, in which an intestinal infection was established by subcutaneous administration of larvae, no changes were detected in the cellular composition of the peritoneal exudate. The significance of these responses is discussed in relation to recent reports about the cellular composition of antigenic and non-antigenic granulomas. ImagesFIG. 3FIG. 4FIG. 5FIG. 6FIG. 7FIG. 8FIG. 9FIG. 10 PMID:4705618

Heterogeneous Human Periodontal Ligament-Committed Progenitor and Stem Cell Populations Exhibit a Unique Cementogenic Property Under In Vitro and In Vivo Conditions.

PubMed

Shinagawa-Ohama, Rei; Mochizuki, Mai; Tamaki, Yuichi; Suda, Naoto; Nakahara, Taka

2017-05-01

An undesirable complication that arises during dental treatments is external apical-root resorption, which causes root-cementum and root-dentin loss. To induce de novo cementogenesis, stem cell therapy is required. Cementum-forming cells (cementoblasts) are known to be differentiated from periodontal-lineage mesenchymal stem cells (MSCs), which are derived from the dental follicle (DF) in developing tissues and the periodontal ligament (PDL) in adult tissues, but the periodontal-lineage MSC type that is optimal for inducing de novo cementogenesis remains unidentified, as does the method to isolate these cells from harvested tissues. Thus, we investigated the cementogenic potential of DF- and PDL-derived MSCs that were isolated by using two widely used cell-isolation methods: enzymatic digestion and outgrowth (OG) methods. DF- and PDL-derived cells isolated by using both methods proliferated actively, and all four isolated cell types showed MSC gene/protein expression phenotype and ability to differentiate into adipogenic and chondrogenic lineages. Furthermore, cementogenic-potential analysis revealed that all cell types produced alizarin red S-positive mineralized materials in in vitro cultures. However, PDL-OG cells presented unique cementogenic features, such as nodular formation of mineralized deposits displaying a cellular intrinsic fiber cementum-like structure, as well as a higher expression of cementoblast-specific genes than in the other cell types. Moreover, in in vivo transplantation experiments, PDL-OG cells formed cellular cementum-like hard tissue containing embedded osteocalcin-positive cells, whereas the other cells formed acellular cementum-like materials. Given that the root-cementum defect is likely regenerated through cellular cementum deposition, PDL-OG cell-based therapies might potentially facilitate the de novo cellular cementogenesis required for regenerating the root defect.

Interdependence of free zinc changes and protein complex assembly - insights into zinc signal regulation.

PubMed

Kocyła, Anna; Adamczyk, Justyna; Krężel, Artur

2018-01-24

Cellular zinc (Zn(ii)) is bound with proteins that are part of the proteomes of all domains of life. It is mostly utilized as a catalytic or structural protein cofactor, which results in a vast number of binding architectures. The Zn(ii) ion is also important for the formation of transient protein complexes with a Zn(ii)-dependent quaternary structure that is formed upon cellular zinc signals. The mechanisms by which proteins associate with and dissociate from Zn(ii) and the connection with cellular Zn(ii) changes remain incompletely understood. In this study, we aimed to examine how zinc protein domains with various Zn(ii)-binding architectures are formed under free Zn(ii) concentration changes and how formation of the Zn(ii)-dependent assemblies is related to the protein concentration and reactivity. To accomplish these goals we chose four zinc domains with different Zn(ii)-to-protein binding stoichiometries: classical zinc finger (ZnP), LIM domain (Zn 2 P), zinc hook (ZnP 2 ) and zinc clasp (ZnP 1 P 2 ) folds. Our research demonstrated a lack of changes in the saturation level of intraprotein zinc binding sites, despite various peptide concentrations, while homo- and heterodimers indicated a concentration-dependent tendency. In other words, at a certain free Zn(ii) concentration, the fraction of a formed dimeric complex increases or decreases with subunit concentration changes. Secondly, even small or local changes in free Zn(ii) may significantly affect protein saturation depending on its architecture, function and subcellular concentration. In our paper, we indicate the importance of interdependence of free Zn(ii) availability and protein subunit concentrations for cellular zinc signal regulation.

Evaluation of cellular immunological responses in mono- and polymorphic clinical forms of post-kala-azar dermal leishmaniasis in India.

PubMed

Kaushal, H; Bras-Gonçalves, R; Avishek, K; Kumar Deep, D; Petitdidier, E; Lemesre, J-L; Papierok, G; Kumar, S; Ramesh, V; Salotra, P

2016-07-01

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic dermal complication that occurs usually after recovery from visceral leishmaniasis (VL). The disease manifests into macular, papular and/or nodular clinical types with mono- or polymorphic presentations. Here, we investigated differences in immunological response between these two distinct clinical forms in Indian PKDL patients. Peripheral blood mononuclear cells of PKDL and naive individuals were exposed in vitro to total soluble Leishmania antigen (TSLA). The proliferation index was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based lymphoproliferative assay. Cytokines and granzyme B levels were determined by cytometric bead array. Parasite load in tissue biopsy samples of PKDL was quantified by quantitative polymerase chain reaction (qPCR). The proportion of different lymphoid subsets in peripheral blood and the activated T cell population were estimated using flow cytometry. The study demonstrated heightened cellular immune responses in the polymorphic PKDL group compared to the naive group. The polymorphic group showed significantly higher lymphoproliferation, increased cytokines and granzyme B levels upon TSLA stimulation, and a raised proportion of circulating natural killer (NK) T cells against naive controls. Furthermore, the polymorphic group showed a significantly elevated proportion of activated CD4(+) and CD8(+) T cells upon in-vitro TSLA stimulation. Thus, the polymorphic variants showed pronounced cellular immunity while the monomorphic form demonstrated a comparatively lower cellular response. Additionally, the elevated level of both activated CD4(+) and CD8(+) T cells, coupled with high granzyme B secretion upon in-vitro TSLA stimulation, indicated the role of cytotoxic cells in resistance to L. donovani infection in polymorphic PKDL. © 2016 British Society for Immunology.

Transmembrane 4 L Six Family Member 5 (TM4SF5)-Mediated Epithelial-Mesenchymal Transition in Liver Diseases.

PubMed

Lee, Jung Weon

2015-01-01

The membrane protein TM4SF5, a member of the transmembrane 4L six family, forms a tetraspanin-enriched microdomain (TEM) on the cell surface, where many different membrane proteins and receptors form a massive protein-protein complex to regulate cellular functions including transdifferentiation, migration, and invasion. We recently reported that TM4SF5 causes epithelial-mesenchymal transition (EMT), eventually contributing to aberrant multilayer cellular growth, drug resistance, enhanced migration, invasion, its circulation in the blood, tumor initiation for successful metastasis, and muscle development in zebrafish. In this review, I summarize the information on the role of TM4SF5 in EMT-related functions at TM4SF5-enriched microdomain (T5EM) on cell surface, where proteins such as TM4SF5, CD151, CD44, integrins, and epidermal growth factor receptor (EGFR) can form numerous protein complexes. TM4SF5-mediated EMT contributes to diverse cellular functions, leading to fibrotic phenotypes and initiating and maintaining tumors in primary and/or metastatic regions, in addition to its role in muscle development in zebrafish. Anti-TM4SF5 strategies for addressing the protein networks can lead to regulation of the fibrotic, tumorigenic, and tumor-maintaining functions of TM4SF5-positive hepatic cells. This review is for us to (re)consider the antifibrotic or antitumorigenic (i.e., anti-EMT-related diseases) strategies of dealing with protein networks that would be involved in cross-talks to regulate various cellular functions during TM4SF5-dependent progression from fibrotic to cancerous hepatic cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Reciprocal Control of the Circadian Clock and Cellular Redox State - a Critical Appraisal.

PubMed

Putker, Marrit; O'Neill, John Stuart

2016-01-01

Redox signalling comprises the biology of molecular signal transduction mediated by reactive oxygen (or nitrogen) species. By specific and reversible oxidation of redox-sensitive cysteines, many biological processes sense and respond to signals from the intracellular redox environment. Redox signals are therefore important regulators of cellular homeostasis. Recently, it has become apparent that the cellular redox state oscillates in vivo and in vitro, with a period of about one day (circadian). Circadian time-keeping allows cells and organisms to adapt their biology to resonate with the 24-hour cycle of day/night. The importance of this innate biological time-keeping is illustrated by the association of clock disruption with the early onset of several diseases (e.g. type II diabetes, stroke and several forms of cancer). Circadian regulation of cellular redox balance suggests potentially two distinct roles for redox signalling in relation to the cellular clock: one where it is regulated by the clock, and one where it regulates the clock. Here, we introduce the concepts of redox signalling and cellular timekeeping, and then critically appraise the evidence for the reciprocal regulation between cellular redox state and the circadian clock. We conclude there is a substantial body of evidence supporting circadian regulation of cellular redox state, but that it would be premature to conclude that the converse is also true. We therefore propose some approaches that might yield more insight into redox control of cellular timekeeping.

Reciprocal Control of the Circadian Clock and Cellular Redox State - a Critical Appraisal

PubMed Central

Putker, Marrit; O’Neill, John Stuart

2016-01-01

Redox signalling comprises the biology of molecular signal transduction mediated by reactive oxygen (or nitrogen) species. By specific and reversible oxidation of redox-sensitive cysteines, many biological processes sense and respond to signals from the intracellular redox environment. Redox signals are therefore important regulators of cellular homeostasis. Recently, it has become apparent that the cellular redox state oscillates in vivo and in vitro, with a period of about one day (circadian). Circadian time-keeping allows cells and organisms to adapt their biology to resonate with the 24-hour cycle of day/night. The importance of this innate biological time-keeping is illustrated by the association of clock disruption with the early onset of several diseases (e.g. type II diabetes, stroke and several forms of cancer). Circadian regulation of cellular redox balance suggests potentially two distinct roles for redox signalling in relation to the cellular clock: one where it is regulated by the clock, and one where it regulates the clock. Here, we introduce the concepts of redox signalling and cellular timekeeping, and then critically appraise the evidence for the reciprocal regulation between cellular redox state and the circadian clock. We conclude there is a substantial body of evidence supporting circadian regulation of cellular redox state, but that it would be premature to conclude that the converse is also true. We therefore propose some approaches that might yield more insight into redox control of cellular timekeeping. PMID:26810072

Autophagic activity in BC3H1 cells exposed to yessotoxin.

PubMed

Korsnes, Mónica Suárez; Kolstad, Hilde; Kleiveland, Charlotte Ramstad; Korsnes, Reinert; Ørmen, Elin

2016-04-01

The marine toxin yessotoxin (YTX) can induce programmed cell death through both caspase-dependent and -independent pathways in various cellular systems. It appears to stimulate different forms of cellular stress causing instability among cell death mechanisms and making them overlap and cross-talk. Autophagy is one of the key pathways that can be stimulated by multiple forms of cellular stress which may determine cell survival or death. The present work evaluates a plausible link between ribotoxic stress and autophagic activity in BC3H1 cells treated with YTX. Such treatment produces massive cytoplasmic compartments as well as double-membrane vesicles termed autophagosomes which are typically observed in cells undergoing autophagy. The observed autophagosomes contain a large amount of ribosomes associated with the endoplasmic reticulum (ER). Western blotting analysis of Atg proteins and detection of the autophagic markers LC3-II and SQSTM1/p62 by flow cytometry and immunofluorescence verified autophagic activity during YTX-treatment. The present work supports the idea that autophagic activity upon YTX exposure may represent a response to ribotoxic stress. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Real-Time Monitoring of Cellular Bioenergetics with a Multi-Analyte Screen-Printed Electrode

PubMed Central

McKenzie, Jennifer R.; Cognata, Andrew C.; Davis, Anna N.; Wikswo, John P.; Cliffel, David E.

2016-01-01

Real-time monitoring of changes to cellular bioenergetics can provide new insights into mechanisms of action for disease and toxicity. This work describes the development of a multi-analyte screen-printed electrode for the detection of analytes central to cellular bioenergetics: glucose, lactate, oxygen, and pH. Platinum screen-printed electrodes were designed in-house and printed by Pine Research Instrumentation. Electrochemical plating techniques were used to form quasi-reference and pH electrodes. A Dimatix materials inkjet printer was used to deposit enzyme and polymer films to form sensors for glucose, lactate, and oxygen. These sensors were evaluated in bulk solution and microfluidic environments, and found to behave reproducibly and possess a lifetime of up to six weeks. Linear ranges and limits of detection for enzyme-based sensors were found to have an inverse relationship with enzyme loading, and iridium oxide pH sensors were found to have super-Nernstian responses. Preliminary measurements where the sensor was enclosed within a microfluidic channel with RAW 264.7 macrophages were performed to demonstrate the sensors’ capabilities for performing real-time microphysiometry measurements. PMID:26125545

Enhanced splicing correction effect by an oligo-aspartic acid-PNA conjugate and cationic carrier complexes.

PubMed

Bae, Yun Mi; Kim, Myung Hee; Yu, Gwang Sig; Um, Bong Ho; Park, Hee Kyung; Lee, Hyun-il; Lee, Kang Taek; Suh, Yung Doug; Choi, Joon Sig

2014-02-10

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA. They recognize specific cellular nucleic acid sequences and form stable complexes with complementary DNA or RNA. Here, we designed an oligo-aspartic acid-PNA conjugate and showed its enhanced delivery into cells with high gene correction efficiency using conventional cationic carriers, such as polyethylenimine (PEI) and Lipofectamine 2000. The negatively charged oligo-aspartic acid-PNA (Asp(n)-PNA) formed complexes with PEI and Lipofectamine, and the resulting Asp(n)-PNA/PEI and Asp(n)-PNA/Lipofectamine complexes were introduced into cells. We observed significantly enhanced cellular uptake of Asp(n)-PNA by cationic carriers and detected an active splicing correction effect even at nanomolar concentrations. We found that the splicing correction efficiency of the complex depended on the kind of the cationic carriers and on the number of repeating aspartic acid units. By enhancing the cellular uptake efficiency of PNAs, these results may provide a novel platform technology of PNAs as bioactive substances for their biological and therapeutic applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Haemophilus parasuis encodes two functional cytolethal distending toxins: CdtC contains an atypical cholesterol recognition/interaction region.

PubMed

Zhou, Mingguang; Zhang, Qiang; Zhao, Jianping; Jin, Meilin

2012-01-01

Haemophilus parasuis is the causative agent of Glässer's disease of pigs, a disease associated with fibrinous polyserositis, polyarthritis and meningitis. We report here H. parasuis encodes two copies of cytolethal distending toxins (Cdts), which these two Cdts showed the uniform toxin activity in vitro. We demonstrate that three Cdt peptides can form an active tripartite holotoxin that exhibits maximum cellular toxicity, and CdtA and CdtB form a more active toxin than CdtB and CdtC. Moreover, the cellular toxicity is associated with the binding of Cdt subunits to cells. Further analysis indicates that CdtC subunit contains an atypical cholesterol recognition/interaction amino acid consensus (CRAC) region. The mutation of CRAC site resulted in decreased cell toxicity. Finally, western blot analysis show all the 15 H. parasuis reference strains and 109 clinical isolates expressed CdtB subunit, indicating that Cdt is a conservative putative virulence factor for H. parasuis. This is the first report of the molecular and cellular basis of Cdt host interactions in H. parasuis.

ATP-driven and AMPK-independent autophagy in an early branching eukaryotic parasite.

PubMed

Li, Feng-Jun; Xu, Zhi-Shen; Soo, Andy D S; Lun, Zhao-Rong; He, Cynthia Y

2017-04-03

Autophagy is a catabolic cellular process required to maintain protein synthesis, energy production and other essential activities in starved cells. While the exact nutrient sensor(s) is yet to be identified, deprivation of amino acids, glucose, growth factor and other nutrients can serve as metabolic stimuli to initiate autophagy in higher eukaryotes. In the early-branching unicellular parasite Trypanosoma brucei, which can proliferate as procyclic form (PCF) in the tsetse fly or as bloodstream form (BSF) in animal hosts, autophagy is robustly triggered by amino acid deficiency but not by glucose depletion. Taking advantage of the clearly defined adenosine triphosphate (ATP) production pathways in T. brucei, we have shown that autophagic activity depends on the levels of cellular ATP production, using either glucose or proline as a carbon source. While autophagosome formation positively correlates with cellular ATP levels; perturbation of ATP production by removing carbon sources or genetic silencing of enzymes involved in ATP generation pathways, also inhibited autophagy. This obligate energy dependence and the lack of glucose starvation-induced autophagy in T. brucei may reflect an adaptation to its specialized, parasitic life style.

Synthesis of structurally diverse major groove DNA interstrand crosslinks using three different aldehyde precursors

PubMed Central

Mukherjee, Shivam; Guainazzi, Angelo; Schärer, Orlando D.

2014-01-01

DNA interstrand crosslinks (ICLs) are extremely cytotoxic lesions that block essential cellular processes, such as replication and transcription. Crosslinking agents are widely used in cancer chemotherapy and form an array of structurally diverse ICLs. Despite the clinical success of these agents, resistance of tumors to crosslinking agents, for example, through repair of these lesions by the cellular machinery remains a problem. We have previously reported the synthesis of site-specific ICLs mimicking those formed by nitrogen mustards to facilitate the studies of cellular responses to ICL formation. Here we extend these efforts and report the synthesis of structurally diverse major groove ICLs that induce severe, little or no distortion in the DNA. Our approach employs the incorporation of aldehyde precursors of different lengths into complementary strands and ICL formation using a double reductive amination with a variety of amines. Our studies provide insight into the structure and reactivity parameters of ICL formation by double reductive amination and yield a set of diverse ICLs that will be invaluable for exploring structure–activity relationships in ICL repair. PMID:24782532

Clay energetics in chemical evolution

NASA Technical Reports Server (NTRS)

Coyne, L. M.

1986-01-01

Clays have been implicated in the origin of terrestrial life since the 1950's. Originally they were considered agents which aid in selecting, concentrating and promoting oligomerization of the organic monomeric substituents of cellular life forms. However, more recently, it has been suggested that minerals, with particular emphasis on clays, may have played a yet more fundamental role. It has been suggested that clays are prototypic life forms in themselves and that they served as a template which directed the self-assembly of cellular life. If the clay-life theory is to have other than conceptual credibility, clays must be shown by experiment to execute the operations of cellular life, not only individually, but also in a sufficiently concerted manner as to produce some semblance of the functional attributes of living cells. Current studies are focussed on the ability of clays to absorb, store and transfer energy under plausible prebiotic conditions and to use this energy to drive chemistry of prebiotic relevance. Conclusions of the work are applicable to the role of clays either as substrates for organic chemistry, or in fueling their own life-mimetic processes.

Ultralife's polymer electrolyte rechargeable lithium-ion batteries for use in the mobile electronics industry

NASA Astrophysics Data System (ADS)

Cuellar, Edward A.; Manna, Michael E.; Wise, Ralph D.; Gavrilov, Alexei B.; Bastian, Matthew J.; Brey, Rufus M.; DeMatteis, Jeffrey

Ultralife Polymer™ brand batteries for cellular phones as made by Nokia Mobile Phones Incorporated were introduced in July 2000. Characteristics of the UBC443483 cell and UB750N battery are described and related to the power and battery requirements of these cellular phones and chargers. Current, power, and pulse capability are presented as functions of temperature, depth of discharge, and storage at the cell level. Safety protection devices and chargers are discussed at the battery pack level, as well as performance in cellular phones under various wireless communication protocols. Performance is competitive with liquid lithium-ion systems while offering opportunity for non-traditional form factors.

Survival and reversion of a stable L form in soil

NASA Technical Reports Server (NTRS)

Horwitz, A. H.; Casida, L. E., Jr.

1978-01-01

The stable L form of Agromyces ramosus reverted to a bacterial form when incubated in sterilized soil. The cellular and colonial morphology of this bacterial form resembled that of the original parent bacterial form. The two forms differed, however, in that the revertant maintained its bacterial form when transferred onto a low-salt (NaCl) medium but was virtually completely induced into the L-form state on a high-salt medium. The original parent bacterial form was not sensitive to salt. The possibility is discussed that an L-form - bacterial-form cycle for this bacterium might occur naturally in soil. This cycle would be mediated by fluctuations in local salt concentrations in the soil.

A simple and fast method for fixation of cultured cell lines that preserves cellular structures containing gamma-tubulin.

PubMed

Alvarado-Kristensson, Maria

2018-01-01

When using fluorescence microscope techniques to study cells, it is essential that the cell structure and contents are preserved after preparation of the samples, and that the preparation method employed does not create artefacts that can be perceived as cellular structure/components. γ-Tubulin forms filaments that in some cases are immunostained with an anti-γ-tubulin antibody, but this immunostaining is not reproducible [[1], [2

Heparin concentration is critical for cell culture with human platelet lysate.

PubMed

Hemeda, Hatim; Kalz, Jana; Walenda, Gudrun; Lohmann, Michael; Wagner, Wolfgang

2013-09-01

Culture media for mesenchymal stromal cells (MSCs) are generally supplemented with fetal bovine serum. Human platelet lysate (hPL) has been proven to be a very effective alternative without the risk of xenogeneic infections or immune reactions. In contrast to fetal bovine serum, hPL comprises plasma, and anticoagulants-usually unfractionated heparin (UFH)-need to be added to prevent gel formation. Cultures of MSCs in hPL media with various concentrations of UFH and enoxaparin, a low-molecular-weight heparin (LMWH), were systematically compared with regard to proliferation, fibroblastoid colony-forming unit frequency, immunophenotype and in vitro differentiation. At least 0.61 IU/mL UFH or 0.024 mg/mL LMWH was necessary for reliable prevention of coagulation of hPL pools used in this study. Higher concentrations impaired cellular proliferation in a dose-dependent manner even without benzyl alcohol, which is commonly added to heparins as a bacteriostatic agent. Colony-forming unit frequency was also reduced at higher heparin concentrations, particularly with LMWH, whereas no significant effect was observed on cellular morphology or immunophenotype. High concentrations of heparins reduced the in vitro differentiation toward adipogenic and osteogenic lineages. Heparin concentration is critical for culture of MSCs in hPL media; this is of particular relevance for cellular therapy where cell culture procedures need to be optimized and standardized. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Pore-forming subunits of K-ATP channels, Kir6.1 and Kir6.2, display prominent differences in regional and cellular distribution in the rat brain.

PubMed

Thomzig, Achim; Laube, Gregor; Prüss, Harald; Veh, Rüdiger W

2005-04-11

K-ATP channels consist of two structurally different subunits: a pore-forming subunit of the Kir6.0-family (Kir6.1 or Kir6.2) and a sulfonylurea receptor (SUR1, SUR2, SUR2A, SUR2B) with regulatory activity. The functional diversity of K-ATP channels in brain is broad and of fundamental importance for neuronal activity. Here, using immunocytochemistry with monospecific antibodies against the Kir6.1 and Kir6.2 subunits, we analyze the regional and cellular distribution of both proteins in the adult rat brain. We find Kir6.2 to be widely expressed in all brain regions, suggesting that the Kir6.2 subunit forms the pore of the K-ATP channels in most neurons, presumably protecting the cells during cellular stress conditions such as hypoglycemia or ischemia. Especially in hypothalamic nuclei, in particular the ventromedial and arcuate nucleus, neurons display Kir6.2 immunoreactivity only, suggesting that Kir6.2 is the pore-forming subunit of the K-ATP channels in the glucose-responsive neurons of the hypothalamus. In contrast, Kir6.1-like immunolabeling is restricted to astrocytes (Thomzig et al. [2001] Mol Cell Neurosci 18:671-690) in most areas of the rat brain and very weak or absent in neurons. Only in distinct nuclei or neuronal subpopulations is a moderate or even strong Kir6.1 staining detected. The biological functions of these K-ATP channels still need to be elucidated. Copyright 2005 Wiley-Liss, Inc.

Debio 0507 primarily forms diaminocyclohexane-Pt-d(GpG) and -d(ApG) DNA adducts in HCT116 cells.

PubMed

King, C L; Ramachandran, S; Chaney, S G; Collins, L; Swenberg, J A; DeKrafft, K E; Lin, W; Cicurel, L; Barbier, M

2012-03-01

To characterize the cellular action mechanism of Debio 0507, we compared the major DNA adducts formed by Debio 0507- and oxaliplatin-treated HCT116 human colon carcinoma cells by a combination of inductively coupled plasma mass spectrometry (ICP-MS) and ultraperformance liquid chromatography mass spectrometry (UPLC-MS/MS). HCT116 cells were treated with IC(50) doses of Debio 0507 or oxaliplatin for 3 days. Total cellular Pt-DNA adducts were determined by ICP-MS. The DNA was digested, and the major Pt-DNA adducts formed by both drugs were characterized by UPLC/MS/MS essentially as described previously for cisplatin (Baskerville-Abraham et al. in Chem Res Toxicol 22:905-912, 2009). The Pt level/deoxynucleotide was 7.4/10(4) for DNA from Debio 0507-treated cells and 5.5/10(4) for oxaliplatin-treated cells following a 3-day treatment at the IC(50) for each drug. UPLC-MS/MS in the positive ion mode confirmed the major Pt-DNA adducts formed by both drugs were dach-Pt-d(GpG) (904.2 m/z → 610 m/z and 904.2 m/z → 459 m/z) and dach-Pt-d(ApG) (888.2 m/z → 594 m/z and 888.2 m/z → 459 m/z). These data show that the major DNA adducts formed by Debio 0507 are the dach-Pt-d(GpG) and dach-Pt-d(ApG) adducts and at equitoxic doses Debio 0507 and oxaliplatin form similar levels of dach-Pt-d(GpG) and dach-Pt-d(ApG) adducts. This suggests that the action mechanisms of Debio 0507 and oxaliplatin are similar at a cellular level.

Smooth muscle architecture within cell-dense vascular tissues influences functional contractility.

PubMed

Win, Zaw; Vrla, Geoffrey D; Steucke, Kerianne E; Sevcik, Emily N; Hald, Eric S; Alford, Patrick W

2014-12-01

The role of vascular smooth muscle architecture in the function of healthy and dysfunctional vessels is poorly understood. We aimed at determining the relationship between vascular smooth muscle architecture and contractile output using engineered vascular tissues. We utilized microcontact printing and a microfluidic cell seeding technique to provide three different initial seeding conditions, with the aim of influencing the cellular architecture within the tissue. Cells seeded in each condition formed confluent and aligned tissues but within the tissues, the cellular architecture varied. Tissues with a more elongated cellular architecture had significantly elevated basal stress and produced more contractile stress in response to endothelin-1 stimulation. We also found a correlation between the contractile phenotype marker expression and the cellular architecture, contrary to our previous findings in non-confluent tissues. Taken with previous results, these data suggest that within cell-dense vascular tissues, smooth muscle contractility is strongly influenced by cell and tissue architectures.

Cellular complexity captured in durable silica biocomposites

PubMed Central

Kaehr, Bryan; Townson, Jason L.; Kalinich, Robin M.; Awad, Yasmine H.; Swartzentruber, B. S.; Dunphy, Darren R.; Brinker, C. Jeffrey

2012-01-01

Tissue-derived cultured cells exhibit a remarkable range of morphological features in vitro, depending on phenotypic expression and environmental interactions. Translation of these cellular architectures into inorganic materials would provide routes to generate hierarchical nanomaterials with stabilized structures and functions. Here, we describe the fabrication of cell/silica composites (CSCs) and their conversion to silica replicas using mammalian cells as scaffolds to direct complex structure formation. Under mildly acidic solution conditions, silica deposition is restricted to the molecularly crowded cellular template. Inter- and intracellular heterogeneity from the nano- to macroscale is captured and dimensionally preserved in CSCs following drying and subjection to extreme temperatures allowing, for instance, size and shape preserving pyrolysis of cellular architectures to form conductive carbon replicas. The structural and behavioral malleability of the starting material (cultured cells) provides opportunities to develop robust and economical biocomposites with programmed structures and functions. PMID:23045634

Non-transferrin bound iron: a key role in iron overload and iron toxicity.

PubMed

Brissot, Pierre; Ropert, Martine; Le Lan, Caroline; Loréal, Olivier

2012-03-01

Besides transferrin iron, which represents the normal form of circulating iron, non-transferrin bound iron (NTBI) has been identified in the plasma of patients with various pathological conditions in which transferrin saturation is significantly elevated. To show that: i) NTBI is present not only during chronic iron overload disorders (hemochromatosis, transfusional iron overload) but also in miscellaneous diseases which are not primarily iron overloaded conditions; ii) this iron species represents a potentially toxic iron form due to its high propensity to induce reactive oxygen species and is responsible for cellular damage not only at the plasma membrane level but also towards different intracellular organelles; iii) the NTBI concept may be expanded to include intracytosolic iron forms which are not linked to ferritin, the major storage protein which exerts, at the cellular level, the same type of protective effect towards the intracellular environment as transferrin in the plasma. Plasma NTBI and especially labile plasma iron determinations represent a new important biological tool since elimination of this toxic iron species is a major therapeutic goal. The NTBI approach represents an important mechanistic concept for explaining cellular iron excess and toxicity and provides new important biochemical diagnostic tools. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders. Copyright © 2011 Elsevier B.V. All rights reserved.

Familial CJD Associated PrP Mutants within Transmembrane Region Induced Ctm-PrP Retention in ER and Triggered Apoptosis by ER Stress in SH-SY5Y Cells

PubMed Central

Wang, Xin; Shi, Qi; Xu, Kun; Gao, Chen; Chen, Cao; Li, Xiao-Li; Wang, Gui-Rong; Tian, Chan; Han, Jun; Dong, Xiao-Ping

2011-01-01

Background Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrPC) to the pathogenic one (PrPSc). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease. Methodology/Principal Findings To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and capase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed. Conclusions/Significance The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants within the transmembrane region undergo an ER-stress mediated cell apoptosis. PMID:21298055

Identification of Major Signaling Pathways in Prion Disease Progression Using Network Analysis

PubMed Central

Newaz, Khalique; Sriram, K.; Bera, Debajyoti

2015-01-01

Prion diseases are transmissible neurodegenerative diseases that arise due to conformational change of normal, cellular prion protein (PrPC) to protease-resistant isofrom (rPrPSc). Deposition of misfolded PrpSc proteins leads to an alteration of many signaling pathways that includes immunological and apoptotic pathways. As a result, this culminates in the dysfunction and death of neuronal cells. Earlier works on transcriptomic studies have revealed some affected pathways, but it is not clear which is (are) the prime network pathway(s) that change during the disease progression and how these pathways are involved in crosstalks with each other from the time of incubation to clinical death. We perform network analysis on large-scale transcriptomic data of differentially expressed genes obtained from whole brain in six different mouse strain-prion strain combination models to determine the pathways involved in prion diseases, and to understand the role of crosstalks in disease propagation. We employ a notion of differential network centrality measures on protein interaction networks to identify the potential biological pathways involved. We also propose a crosstalk ranking method based on dynamic protein interaction networks to identify the core network elements involved in crosstalk with different pathways. We identify 148 DEGs (differentially expressed genes) potentially related to the prion disease progression. Functional association of the identified genes implicates a strong involvement of immunological pathways. We extract a bow-tie structure that is potentially dysregulated in prion disease. We also propose an ODE model for the bow-tie network. Predictions related to diseased condition suggests the downregulation of the core signaling elements (PI3Ks and AKTs) of the bow-tie network. In this work, we show using transcriptomic data that the neuronal dysfunction in prion disease is strongly related to the immunological pathways. We conclude that these immunological pathways occupy influential positions in the PFNs (protein functional networks) that are related to prion disease. Importantly, this functional network involvement is prevalent in all the five different mouse strain-prion strain combinations that we studied. We also conclude that the dysregulation of the core elements of the bow-tie structure, which belongs to PI3K-Akt signaling pathway, leads to dysregulation of the downstream components corresponding to other biological pathways. PMID:26646948

In Vitro Approach To Identify Key Amino Acids in Low Susceptibility of Rabbit Prion Protein to Misfolding

PubMed Central

Eraña, Hasier; Fernández-Borges, Natalia; Elezgarai, Saioa R.; Harrathi, Chafik; Charco, Jorge M.; Chianini, Francesca; Dagleish, Mark P.; Ortega, Gabriel; Millet, Óscar

2017-01-01

ABSTRACT Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a group of rare progressive neurodegenerative disorders caused by an abnormally folded prion protein (PrPSc). This is capable of transforming the normal cellular prion protein (PrPC) into new infectious PrPSc. Interspecies prion transmissibility studies performed by experimental challenge and the outbreak of bovine spongiform encephalopathy that occurred in the late 1980s and 1990s showed that while some species (sheep, mice, and cats) are readily susceptible to TSEs, others are apparently resistant (rabbits, dogs, and horses) to the same agent. To study the mechanisms of low susceptibility to TSEs of certain species, the mouse-rabbit transmission barrier was used as a model. To identify which specific amino acid residues determine high or low susceptibility to PrPSc propagation, protein misfolding cyclic amplification (PMCA), which mimics PrPC-to-PrPSc conversion with accelerated kinetics, was used. This allowed amino acid substitutions in rabbit PrP and accurate analysis of misfolding propensities. Wild-type rabbit recombinant PrP could not be misfolded into a protease-resistant self-propagating isoform in vitro despite seeding with at least 12 different infectious prions from diverse origins. Therefore, rabbit recombinant PrP mutants were designed to contain every single amino acid substitution that distinguishes rabbit recombinant PrP from mouse recombinant PrP. Key amino acid residue substitutions were identified that make rabbit recombinant PrP susceptible to misfolding, and using these, protease-resistant misfolded recombinant rabbit PrP was generated. Additional studies characterized the mechanisms by which these critical amino acid residue substitutions increased the misfolding susceptibility of rabbit PrP. IMPORTANCE Prion disorders are invariably fatal, untreatable diseases typically associated with long incubation periods and characteristic spongiform changes associated with neuronal loss in the brain. Development of any treatment or preventative measure is dependent upon a detailed understanding of the pathogenesis of these diseases, and understanding the mechanism by which certain species appear to be resistant to TSEs is critical. Rabbits are highly resistant to naturally acquired TSEs, and even under experimental conditions, induction of clinical disease is not easy. Using recombinant rabbit PrP as a model, this study describes critical molecular determinants that confer this high resistance to transmissible spongiform encephalopathies. PMID:28978705

The Bicomponent Pore-Forming Leucocidins of Staphylococcus aureus

PubMed Central

Alonzo, Francis

2014-01-01

SUMMARY The ability to produce water-soluble proteins with the capacity to oligomerize and form pores within cellular lipid bilayers is a trait conserved among nearly all forms of life, including humans, single-celled eukaryotes, and numerous bacterial species. In bacteria, some of the most notable pore-forming molecules are protein toxins that interact with mammalian cell membranes to promote lysis, deliver effectors, and modulate cellular homeostasis. Of the bacterial species capable of producing pore-forming toxic molecules, the Gram-positive pathogen Staphylococcus aureus is one of the most notorious. S. aureus can produce seven different pore-forming protein toxins, all of which are believed to play a unique role in promoting the ability of the organism to cause disease in humans and other mammals. The most diverse of these pore-forming toxins, in terms of both functional activity and global representation within S. aureus clinical isolates, are the bicomponent leucocidins. From the first description of their activity on host immune cells over 100 years ago to the detailed investigations of their biochemical function today, the leucocidins remain at the forefront of S. aureus pathogenesis research initiatives. Study of their mode of action is of immediate interest in the realm of therapeutic agent design as well as for studies of bacterial pathogenesis. This review provides an updated perspective on our understanding of the S. aureus leucocidins and their function, specificity, and potential as therapeutic targets. PMID:24847020

Resistance of Soil-Bound Prions to Rumen Digestion

PubMed Central

Saunders, Samuel E.; Bartelt-Hunt, Shannon L.; Bartz, Jason C.

2012-01-01

Before prion uptake and infection can occur in the lower gastrointestinal system, ingested prions are subjected to anaerobic digestion in the rumen of cervids and bovids. The susceptibility of soil-bound prions to rumen digestion has not been evaluated previously. In this study, prions from infectious brain homogenates as well as prions bound to a range of soils and soil minerals were subjected to in vitro rumen digestion, and changes in PrP levels were measured via western blot. Binding to clay appeared to protect noninfectious hamster PrPc from complete digestion, while both unbound and soil-bound infectious PrPSc proved highly resistant to rumen digestion. In addition, no change in intracerebral incubation period was observed following active rumen digestion of unbound hamster HY TME prions and HY TME prions bound to a silty clay loam soil. These results demonstrate that both unbound and soil-bound prions readily survive rumen digestion without a reduction in infectivity, further supporting the potential for soil-mediated transmission of chronic wasting disease (CWD) and scrapie in the environment. PMID:22937149

Role of Mitochondrial Ca2+ in the Regulation of Cellular Energetics

PubMed Central

Glancy, Brian; Balaban, Robert S.

2012-01-01

Calcium is an important signaling molecule involved in the regulation of many cellular functions. The large free energy in the Ca2+ ion membrane gradients make Ca2+ signaling inherently sensitive to the available cellular free energy, primarily in the form of ATP. In addition, Ca2+ regulates many cellular ATP consuming reactions such as muscle contraction, exocytosis, biosynthesis and neuronal signaling. Thus, Ca2+ becomes a logical candidate as a signaling molecule to modulate ATP hydrolysis and synthesis during changes in numerous forms of cellular work. Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca2+ gradient across their inner membrane providing a signaling potential for this molecule. The demonstrated link between cytosolic and mitochondrial [Ca2+], identification of transport mechanisms as well as proximity of mitochondria to Ca2+ release sites further supports the notion that Ca2+ can be an important signaling molecule in the energy metabolism interplay of the cytosol with the mitochondria. Here we review sites within the mitochondria where Ca2+ plays a role in the regulation of ATP generation and potentially contributes to the orchestration of the cellular metabolic homeostasis. Early work on isolated enzymes pointed to several matrix dehydrogenases that are stimulated by Ca2+, which were confirmed in the intact mitochondrion as well as cellular and in vivo systems. However, studies in these intact systems suggested a more expansive influence of Ca2+ on mitochondrial energy conversion. Numerous non-invasive approaches monitoring NADH, mitochondrial membrane potential, oxygen consumption and workloads suggest significant Ca2+ effects on other elements of NADH generation as well as downstream elements of oxidative phosphorylation including the F1FO-ATPase and the cytochrome chain. These other potential elements of Ca2+ modification of mitochondrial energy conversion will be the focus of this review. Though most of specific molecular mechanisms have yet to be elucidated, it is clear that Ca2+ provides a balanced activation of mitochondrial energy metabolism which exceeds the alteration of dehydrogenases alone. PMID:22443365

Ultraflexible nanoelectronic probes form reliable, glial scar–free neural integration

PubMed Central

Luan, Lan; Wei, Xiaoling; Zhao, Zhengtuo; Siegel, Jennifer J.; Potnis, Ojas; Tuppen, Catherine A; Lin, Shengqing; Kazmi, Shams; Fowler, Robert A.; Holloway, Stewart; Dunn, Andrew K.; Chitwood, Raymond A.; Xie, Chong

2017-01-01

Implanted brain electrodes construct the only means to electrically interface with individual neurons in vivo, but their recording efficacy and biocompatibility pose limitations on scientific and clinical applications. We showed that nanoelectronic thread (NET) electrodes with subcellular dimensions, ultraflexibility, and cellular surgical footprints form reliable, glial scar–free neural integration. We demonstrated that NET electrodes reliably detected and tracked individual units for months; their impedance, noise level, single-unit recording yield, and the signal amplitude remained stable during long-term implantation. In vivo two-photon imaging and postmortem histological analysis revealed seamless, subcellular integration of NET probes with the local cellular and vasculature networks, featuring fully recovered capillaries with an intact blood-brain barrier and complete absence of chronic neuronal degradation and glial scar. PMID:28246640

Tissue morphodynamics shaping the early mouse embryo.

PubMed

Sutherland, Ann E

2016-07-01

Generation of the elongated vertebrate body plan from the initially radially symmetrical embryo requires comprehensive changes to tissue form. These shape changes are generated by specific underlying cell behaviors, coordinated in time and space. Major principles and also specifics are emerging, from studies in many model systems, of the cell and physical biology of how region-specific cell behaviors produce regional tissue morphogenesis, and how these, in turn, are integrated at the level of the embryo. New technical approaches have made it possible more recently, to examine the morphogenesis of the mouse embryo in depth, and to elucidate the underlying cellular mechanisms. This review focuses on recent advances in understanding the cellular basis for the early fundamental events that establish the basic form of the embryo. Copyright © 2016 Elsevier Ltd. All rights reserved.

Vegetative Incompatibility and the Mating-Type Locus in the Cellular Slime Mold DICTYOSTELIUM DISCOIDEUM

PubMed Central

Robson, Gillian E.; Williams, Keith L.

1979-01-01

The genetic basis of vegetative incompatibility in the cellular slime mold, Dictyostelium discoideum, is elucidated. Vegetatively compatible haploid strains from parasexual diploids at a frequency of between 10-6 and 10-5, whereas "escaped" diploids are formed between vegetatively incompatible strains at a frequency of ∼10-8. There is probably only a single vegetative incompatibility site, which appears to be located at, or closely linked to, the mating-type locus. The nature of the vegetative incompatibility is deduced from parasexual diploid formation between wild isolates and tester strains of each mating type, examination of the frequency of formation of "escaped" diploids formed between vegetatively incompatible strains, and examination of the mating type and vegetative incompatibility of haploid segregants obtained from "escaped" diploids. PMID:17248984

Environmental Electrophiles: Protein Adducts, Modulation of Redox Signaling, and Interaction with Persulfides/Polysulfides.

PubMed

Kumagai, Yoshito; Abiko, Yumi

2017-01-17

Included among the many environmental electrophiles are aromatic hydrocarbon quinones formed during combustion of gasoline, crotonaldehyde in tobacco smoke, methylmercury accumulated in fish, cadmium contaminated in rice, and acrylamide in baked foods. These electrophiles can modify nucleophilic functions such as cysteine residues in proteins forming adducts and in the process activate cellular redox signal transduction pathways such as kinases and transcription factors. However, higher concentrations of electrophiles disrupt such signaling by nonselective covalent modification of cellular proteins. Persulfide/polysulfides produced by various enzymes appear to capture environmental electrophiles because of the formation of their sulfur adducts without electrophilicity. We therefore speculate that persulfide/polysulfides are candidates for the regulation of redox signal transduction pathways (e.g., cell survival, cell proliferation, and adaptive response) and toxicity during exposure to environmental electrophiles.

Deterministic Integration of Biological and Soft Materials onto 3D Microscale Cellular Frameworks

PubMed Central

McCracken, Joselle M.; Xu, Sheng; Badea, Adina; Jang, Kyung-In; Yan, Zheng; Wetzel, David J.; Nan, Kewang; Lin, Qing; Han, Mengdi; Anderson, Mikayla A.; Lee, Jung Woo; Wei, Zijun; Pharr, Matt; Wang, Renhan; Su, Jessica; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

2018-01-01

Complex 3D organizations of materials represent ubiquitous structural motifs found in the most sophisticated forms of matter, the most notable of which are in life-sustaining hierarchical structures found in biology, but where simpler examples also exist as dense multilayered constructs in high-performance electronics. Each class of system evinces specific enabling forms of assembly to establish their functional organization at length scales not dissimilar to tissue-level constructs. This study describes materials and means of assembly that extend and join these disparate systems—schemes for the functional integration of soft and biological materials with synthetic 3D microscale, open frameworks that can leverage the most advanced forms of multilayer electronic technologies, including device-grade semiconductors such as monocrystalline silicon. Cellular migration behaviors, temporal dependencies of their growth, and contact guidance cues provided by the nonplanarity of these frameworks illustrate design criteria useful for their functional integration with living matter (e.g., NIH 3T3 fibroblast and primary rat dorsal root ganglion cell cultures). PMID:29552634

[A Cellular Automata Model for a Community Comprising Two Plant Species of Different Growth Forms].

PubMed

Frolov, P V; Zubkova, E V; Komarov, A S

2015-01-01

A cellular automata computer model for the interactions between two plant species of different growth forms--the lime hairgrass Deschampsia caespitosa (L.) P. Beauv., a sod cereal, and the moneywort Lysimachia nummularia L., a ground creeping perennial herb--is considered. Computer experiments on the self-maintenance of the populations of each species against the background of a gradual increase in the share of randomly eliminated individuals, coexistence of the populations of two species, and the effect of the phytogenous field have been conducted. As has been shown, all the studied factors determine the number of individuals and self-sustainability of the simulated populations by the degree of their impact. The limits of action have been determined for individual factors; within these limits, the specific features in plant reproduction and dispersal provide sustainable coexistence of the simulated populations. It has been demonstrated that the constructed model allows for studying the long-term developmental dynamics of the plants belonging to the selected growth forms.

Effects of multiple enzyme-substrate interactions in basic units of cellular signal processing

NASA Astrophysics Data System (ADS)

Seaton, D. D.; Krishnan, J.

2012-08-01

Covalent modification cycles are a ubiquitous feature of cellular signalling networks. In these systems, the interaction of an active enzyme with the unmodified form of its substrate is essential for signalling to occur. However, this interaction is not necessarily the only enzyme-substrate interaction possible. In this paper, we analyse the behaviour of a basic model of signalling in which additional, non-essential enzyme-substrate interactions are possible. These interactions include those between the inactive form of an enzyme and its substrate, and between the active form of an enzyme and its product. We find that these additional interactions can result in increased sensitivity and biphasic responses, respectively. The dynamics of the responses are also significantly altered by the presence of additional interactions. Finally, we evaluate the consequences of these interactions in two variations of our basic model, involving double modification of substrate and scaffold-mediated signalling, respectively. We conclude that the molecular details of protein-protein interactions are important in determining the signalling properties of enzymatic signalling pathways.

Synthesis, characterisation, and in vitro cellular uptake kinetics of nanoprecipitated poly(2-methacryloyloxyethyl phosphorylcholine)- b-poly(2-(diisopropylamino)ethyl methacrylate) (MPC-DPA) polymeric nanoparticle micelles for nanomedicine applications

NASA Astrophysics Data System (ADS)

Salvage, Jonathan P.; Smith, Tia; Lu, Tao; Sanghera, Amendeep; Standen, Guy; Tang, Yiqing; Lewis, Andrew L.

2016-10-01

Nanoscience offers the potential for great advances in medical technology and therapies in the form of nanomedicine. As such, developing controllable, predictable, and effective, nanoparticle-based therapeutic systems remains a significant challenge. Many polymer-based nanoparticle systems have been reported to date, but few harness materials with accepted biocompatibility. Phosphorylcholine (PC) based biomimetic materials have a long history of successful translation into effective commercial medical technologies. This study investigated the synthesis, characterisation, nanoprecipitation, and in vitro cellular uptake kinetics of PC-based polymeric nanoparticle micelles (PNM) formed by the biocompatible and pH responsive block copolymer poly(2-methacryloyloxyethyl phosphorylcholine)- b-poly(2-(diisopropylamino)ethyl methacrylate) (MPC-DPA). Atom transfer radical polymerisation (ATRP), and gel permeation chromatography (GPC) were used to synthesise and characterise the well-defined MPC100-DPA100 polymer, revealing organic GPC, using evaporative light scatter detection, to be more accurate than aqueous GPC for this application. Subsequent nanoprecipitation investigations utilising photon correlation spectroscopy (PCS) revealed PNM size increased with polymer concentration, and conferred Cryo-stability. PNM diameters ranged from circa 64-69 nm, and increased upon hydrophobic compound loading, circa 65-71 nm, with loading efficiencies of circa 60 % achieved, whilst remaining monodisperse. In vitro studies demonstrated that the PNM were of low cellular toxicity, with colony formation and MTT assays, utilising V79 and 3T3 cells, yielding comparable results. Investigation of the in vitro cellular uptake kinetics revealed rapid, 1 h, cellular uptake of MPC100-DPA100 PNM delivered fluorescent probes, with fluorescence persistence for 48 h. This paper presents the first report of these novel findings, which highlight the potential of the system for nanomedicine application development.

Irradiation With Carbon Ion Beams Induces Apoptosis, Autophagy, and Cellular Senescence in a Human Glioma-Derived Cell Line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jinno-Oue, Atsushi; Shimizu, Nobuaki; 21st Century Center of Excellence Program for Biomedical Research Using Accelerator Technology, Maebashi, Gunma

2010-01-15

Purpose: We examined biological responses of human glioma cells to irradiation with carbon ion beams (C-ions). Methods and Materials: A human glioma-derived cell line, NP-2, was irradiated with C-ions. Apoptotic cell nuclei were stained with Hoechst 33342. Induction of autophagy was examined either by staining cells with monodansylcadaverine (MDC) or by Western blotting to detect conversion of microtuble-associated protein light chain 3 (MAP-LC3) (LC3-I) to the membrane-bound form (LC3-II). Cellular senescence markers including induction of senescence-associated beta-galactosidase (SA-beta-gal) were examined. The mean telomere length of irradiated cells was determined by Southern blot hybridization. Expression of tumor suppressor p53 and cyclin/cyclin-dependentmore » kinase inhibitor p21{sup WAF1/CIP1} in the irradiated cells was analyzed by Western blotting. Results: When NP-2 cells were irradiated with C-ions at 6 Gy, the major population of the cells died of apoptosis and autophagy. The residual fraction of attached cells (<1% of initially irradiated cells) could not form a colony: however, they showed a morphological phenotype consistent with cellular senescence, that is, enlarged and flattened appearance. The senescent nature of these attached cells was further indicated by staining for SA-beta-gal. The mean telomere length was not changed after irradiation with C-ions. Phosphorylation of p53 at serine 15 as well as the expression of p21{sup WAF1/CIP1} was induced in NP-2 cells after irradiation. Furthermore, we found that irradiation with C-ions induced cellular senescence in a human glioma cell line lacking functional p53. Conclusions: Irradiation with C-ions induced apoptosis, autophagy, and cellular senescence in human glioma cells.« less

Life's Still Lifes

NASA Astrophysics Data System (ADS)

McIntosh, Harold V.

The de Bruijn diagram describing those decompositions of the neighborhoods of a one dimensional cellular automaton which conform to predetermined requirements of periodicity and translational symmetry shows how to construct extended configurations satisfying the same requirements. Similar diagrams, formed by stages, describe higher dimensional automata, although they become more laborious to compute with increasing neighborhood size. The procedure is illustrated by computing some still lifes for Conway's game of Life, a widely known two dimensional cellular automaton. This paper is written in September 10, 1988.

Multiscale modelling of Flow-Induced Blood Cell Damage

NASA Astrophysics Data System (ADS)

Liu, Yaling; Sohrabi, Salman

2017-11-01

We study red blood cell (RBC) damage and hemolysis at cellular level. Under high shear rates, pores form on RBC membranes through which hemoglobin (Hb) leaks out and increases free Hb content of plasma leading to hemolysis. By coupling lattice Boltzmann and spring connected network models through immersed boundary method, we estimate hemolysis of a single RBC under various shear rates. The developed cellular damage model can be used as a predictive tool for hydrodynamic and hematologic design optimization of blood-wetting medical devices.

The Transcription Factor EB Links Cellular Stress to the Immune Response



PubMed Central

Nabar, Neel R.; Kehrl, John H.

2017-01-01

The transcription factor EB (TFEB) is the master transcriptional regulator of autophagy and lysosome biogenesis. Recent advances have led to a paradigm shift in our understanding of lysosomes from a housekeeping cellular waste bin to a dynamically regulated pathway that is efficiently turned up or down based on cellular needs. TFEB coordinates the cellular response to nutrient deprivation and other forms of cell stress through the lysosome system, and regulates a myriad of cellular processes associated with this system including endocytosis, phagocytosis, autophagy, and lysosomal exocytosis. Autophagy and the endolysosomal system are critical to both the innate and adaptive arms of the immune system, with functions in effector cell priming and direct pathogen clearance. Recent studies have linked TFEB to the regulation of the immune response through the endolysosmal pathway and by direct transcriptional activation of immune related genes. In this review, we discuss the current understanding of TFEB’s function and the molecular mechanisms behind TFEB activation. Finally, we discuss recent advances linking TFEB to the immune response that positions lysosomal signaling as a potential target for immune modulation. PMID:28656016

The Transcription Factor EB Links Cellular Stress to the Immune Response

.

PubMed

Nabar, Neel R; Kehrl, John H

2017-06-01

The transcription factor EB (TFEB) is the master transcriptional regulator of autophagy and lysosome biogenesis. Recent advances have led to a paradigm shift in our understanding of lysosomes from a housekeeping cellular waste bin to a dynamically regulated pathway that is efficiently turned up or down based on cellular needs. TFEB coordinates the cellular response to nutrient deprivation and other forms of cell stress through the lysosome system, and regulates a myriad of cellular processes associated with this system including endocytosis, phagocytosis, autophagy, and lysosomal exocytosis. Autophagy and the endolysosomal system are critical to both the innate and adaptive arms of the immune system, with functions in effector cell priming and direct pathogen clearance. Recent studies have linked TFEB to the regulation of the immune response through the endolysosmal pathway and by direct transcriptional activation of immune related genes. In this review, we discuss the current understanding of TFEB's function and the molecular mechanisms behind TFEB activation. Finally, we discuss recent advances linking TFEB to the immune response that positions lysosomal signaling as a potential target for immune modulation.

Biofilms formed by the archaeon Haloferax volcanii exhibit cellular differentiation and social motility, and facilitate horizontal gene transfer.

PubMed

Chimileski, Scott; Franklin, Michael J; Papke, R Thane

2014-08-14

Archaea share a similar microbial lifestyle with bacteria, and not surprisingly then, also exist within matrix-enclosed communities known as biofilms. Advances in biofilm biology have been made over decades for model bacterial species, and include characterizations of social behaviors and cellular differentiation during biofilm development. Like bacteria, archaea impact ecological and biogeochemical systems. However, the biology of archaeal biofilms is only now being explored. Here, we investigated the development, composition and dynamics of biofilms formed by the haloarchaeon Haloferax volcanii DS2. Biofilms were cultured in static liquid and visualized with fluorescent cell membrane dyes and by engineering cells to express green fluorescent protein (GFP). Analysis by confocal scanning laser microscopy showed that H. volcanii cells formed microcolonies within 24 h, which developed into larger clusters by 48 h and matured into flake-like towers often greater than 100 μm in height after 7 days. To visualize the extracellular matrix, biofilms formed by GFP-expressing cells were stained with concanavalin A, DAPI, Congo red and thioflavin T. Stains colocalized with larger cellular structures and indicated that the extracellular matrix may contain a combination of polysaccharides, extracellular DNA and amyloid protein. Following a switch to biofilm growth conditions, a sub-population of cells differentiated into chains of long rods sometimes exceeding 25 μm in length, compared to their planktonic disk-shaped morphology. Time-lapse photography of static liquid biofilms also revealed wave-like social motility. Finally, we quantified gene exchange between biofilm cells, and found that it was equivalent to the mating frequency of a classic filter-based experimental method. The developmental processes, functional properties and dynamics of H. volcanii biofilms provide insight on how haloarchaeal species might persist, interact and exchange DNA in natural communities. H. volcanii demonstrates some biofilm phenotypes similar to bacterial biofilms, but also has interesting phenotypes that may be unique to this organism or to this class of organisms, including changes in cellular morphology and an unusual form of social motility. Because H. volcanii has one of the most advanced genetic systems for any archaeon, the phenotypes reported here may promote the study of genetic and developmental processes in archaeal biofilms.

ISG15 in the tumorigenesis and treatment of cancer: An emerging role in malignancies of the digestive system

PubMed Central

Zuo, Chaohui; Sheng, Xinyi; Ma, Min; Xia, Man; Ouyang, Linda

2016-01-01

The interferon-stimulated gene 15 ubiquitin-like modifier (ISG15) encodes an IFN-inducible, ubiquitin-like protein. The ISG15 protein forms conjugates with numerous cellular proteins that are involved in a multitude of cellular functions, including interferon-induced immune responses and the regulation of cellular protein turnover. The expression of ISG15 and ISG15-mediated conjugation has been implicated in a wide range of human tumors and cancer cell lines, but the roles of ISG15 in tumorigenesis and responses to anticancer treatments remain largely unknown. In this review, we discuss the findings of recent studies with regard to the role of ISG15 pathways in cancers of the digestive system. PMID:27626310

Micro-thermocouple probe for measurement of cellular thermal responses.

PubMed

Watanabe, M; Kakuta, N; Mabuchi, K; Yamada, Y

2005-01-01

We have produced micro-thermocouple probes for the measurement of cellular thermal responses. Cells generate heat with their metabolisms and more heat with reactions to a certain physical or chemical stimulation. Therefore, the analysis of the cellular thermal responses would provide new physiological information. However, a real-time thermal measurement technique on a target of a single cell has not been established. In this study, glass micropipettes, which are widely used in bioengineering and medicine, are used for the base of the thermocouple probes. Using microfabrication techniques, the junction of two different metal films is formed at the micropipette tip with a diameter of 1 μm. This probe can inject a chemical substance into a cell and to detect its subsequent temperature changes simultaneously.

Discontinuous precipitation at the deformation band in copper alloy

NASA Astrophysics Data System (ADS)

Han, Seung Zeon; Ahn, Jee Hyuk; You, Young Soo; Lee, Jehyun; Goto, Masahiro; Kim, Kwangho; Kim, Sangshik

2018-01-01

The Cu-Ni-Si alloy is known as a precipitation hardening alloy, where the Ni2Si intermetallic compound is precipitated in the matrix during aging. There are two types of precipitation of Ni2Si: continuous and discontinuous cellular. The discontinuous cellular precipitation is generally initiated at interfaces especially grain boundaries in the matrix. To observe the grain boundary effect on the discontinuous precipitation, a large-grained Cu-Ni-Si-Ti alloy was intentionally fabricated by unidirectional solidification and plastically deformed by groove rolling. While discontinuous cellular precipitation has been generally known to occur only at the high angled grain boundaries in the alloys, we found that it was also generated inside the grains, at the deformation bands formed by plastic deformation.

ISG15 in the tumorigenesis and treatment of cancer: An emerging role in malignancies of the digestive system.

PubMed

Zuo, Chaohui; Sheng, Xinyi; Ma, Min; Xia, Man; Ouyang, Linda

2016-11-08

The interferon-stimulated gene 15 ubiquitin-like modifier (ISG15) encodes an IFN-inducible, ubiquitin-like protein. The ISG15 protein forms conjugates with numerous cellular proteins that are involved in a multitude of cellular functions, including interferon-induced immune responses and the regulation of cellular protein turnover. The expression of ISG15 and ISG15-mediated conjugation has been implicated in a wide range of human tumors and cancer cell lines, but the roles of ISG15 in tumorigenesis and responses to anticancer treatments remain largely unknown. In this review, we discuss the findings of recent studies with regard to the role of ISG15 pathways in cancers of the digestive system.

Mechanism of protection from primary bovine viral diarrhea virus infection. I. The effects of dexamethasone.

PubMed Central

Shope, R E; Muscoplat, C C; Chen, A W; Johnson, D W

1976-01-01

A series of investigations was designed to study the role of cellular immunity and passive antibody in protecting neonatal calves from primary bovine viral diarrhea virus infection. Administration of corticosteroids (dexamethasone) in doses capable of suppressing cellular immunity markedly potentiated systemic bovine viral diarrhea virus infection in calves which lacked bovine viral diarrhea passive neutralizing antibody. Immunosuppressed calves did not form neutralizing antibody to bovine viral diarrhea virus and developed a fatal viremia. Calves with high levels of passive bovine viral diarrhea neutralizing antibodies were protected from the effect of corticosteroids. The results suggest an essential role for humoral passive antibody, but not for cellular immunity, in protection from primary systemic bovine viral diarrhea virus infection in calves. PMID:187303

Support grid for fuel elements in a nuclear reactor

DOEpatents

Finch, Lester M.

1977-01-01

A support grid is provided for holding nuclear fuel rods in a rectangular array. Intersecting sheet metal strips are interconnected using opposing slots in the strips to form a rectangular cellular grid structure for engaging the sides of a multiplicity of fuel rods. Spring and dimple supports for engaging fuel and guide rods extending through each cell in the support grid are formed in the metal strips with the springs thus formed being characterized by nonlinear spring rates.

3D-micro-patterned fibrous dosage forms for immediate drug release.

PubMed

Blaesi, Aron H; Saka, Nannaji

2018-03-01

At present, the most prevalent pharmaceutical dosage forms, the orally-delivered immediate-release tablets and capsules, are porous, granular solids. They disintegrate into their constituent particulates upon ingestion to release drug rapidly. The design, development, and manufacture of such granular solids, however, is inefficient due to difficulties associated with the unpredictable inter-particle interactions. Therefore, to achieve more predictable dosage form properties and processing, we have recently introduced melt-processed polymeric cellular dosage forms. The cellular forms disintegrated and released drug rapidly if the cells were predominantly interconnected. Preparation of interconnected cells, however, relies on the coalescence of gas bubbles in the melt, which is unpredictable. In the present work, therefore, new melt-processed fibrous dosage forms with contiguous void space are presented. The dosage forms are prepared by melt extrusion of the drug-excipient mixture followed by patterning the fibrous extrudate on a moving surface. It is demonstrated that the resulting fibrous structures are fully predictable by the extruder nozzle diameter and the motion of the surface. Furthermore, drug release experiments show that the disintegration time of the fibrous forms prepared in this work is of the order of that of the corresponding single fibers. The thin fibers of polyethylene glycol (excipient) and acetaminophen (drug) in turn disintegrate in a time proportional to the fiber radius and well within immediate-release specification. Finally, models of dosage form disintegration and drug release by single fibers and fibrous dosage forms are developed. It is found that drug release from fibrous forms is predictable by the physico-chemical properties of the excipient and such microstructural parameters as the fiber radius, the inter-fiber spacing, and the volume fraction of water-soluble excipient in the fibers. Copyright © 2017 Elsevier B.V. All rights reserved.

Conditional bistability, a generic cellular mnemonic mechanism for robust and flexible working memory computations.

PubMed

Rodriguez, Guillaume; Sarazin, Matthieu; Clemente, Alexandra; Holden, Stephanie; Paz, Jeanne T; Delord, Bruno

2018-04-30

Persistent neural activity, the substrate of working memory, is thought to emerge from synaptic reverberation within recurrent networks. However, reverberation models do not robustly explain fundamental dynamics of persistent activity, including high-spiking irregularity, large intertrial variability, and state transitions. While cellular bistability may contribute to persistent activity, its rigidity appears incompatible with persistent activity labile characteristics. Here, we unravel in a cellular model a form of spike-mediated conditional bistability that is robust, generic and provides a rich repertoire of mnemonic computations. Under asynchronous synaptic inputs of the awakened state, conditional bistability generates spiking/bursting episodes, accounting for the irregularity, variability and state transitions characterizing persistent activity. This mechanism has likely been overlooked because of the sub-threshold input it requires and we predict how to assess it experimentally. Our results suggest a reexamination of the role of intrinsic properties in the collective network dynamics responsible for flexible working memory. SIGNIFICANCE STATEMENT This study unravels a novel form of intrinsic neuronal property, i.e. conditional bistability. We show that, thanks of its conditional character, conditional bistability favors the emergence of flexible and robust forms of persistent activity in PFC neural networks, in opposition to previously studied classical forms of absolute bistability. Specifically, we demonstrate for the first time that conditional bistability 1) is a generic biophysical spike-dependent mechanism of layer V pyramidal neurons in the PFC and that 2) it accounts for essential neurodynamical features for the organisation and flexibility of PFC persistent activity (the large irregularity and intertrial variability of the discharge and its organization under discrete stable states), which remain unexplained in a robust fashion by current models. Copyright © 2018 the authors.

Dynamic positional fate map of the primary heart-forming region.

PubMed

Cui, Cheng; Cheuvront, Tracey J; Lansford, Rusty D; Moreno-Rodriguez, Ricardo A; Schultheiss, Thomas M; Rongish, Brenda J

2009-08-15

Here we show the temporal-spatial orchestration of early heart morphogenesis at cellular level resolution, in vivo, and reconcile conflicting positional fate mapping data regarding the primary heart-forming field(s). We determined the positional fates of precardiac cells using a precision electroporation approach in combination with wide-field time-lapse microscopy in the quail embryo, a warm-blooded vertebrate (HH Stages 4 through 10). Contrary to previous studies, the results demonstrate the existence of a "continuous" circle-shaped heart field that spans the midline, appearing at HH Stage 4, which then expands to form a wide arc of progenitors at HH Stages 5-7. Our time-resolved image data show that a subset of these cardiac progenitor cells do not overlap with the expression of common cardiogenic factors, Nkx-2.5 and Bmp-2, until HH Stage 10, when a tubular heart has formed, calling into question when cardiac fate is specified and by which key factors. Sub-groups and anatomical bands (cohorts) of heart precursor cells dramatically change their relative positions in a process largely driven by endodermal folding and other large-scale tissue deformations. Thus, our novel dynamic positional fate maps resolve the origin of cardiac progenitor cells in amniotes. The data also establish the concept that tissue motion contributes significantly to cellular position fate - i.e., much of the cellular displacement that occurs during assembly of a midline heart tube (HH Stage 9) is NOT due to "migration" (autonomous motility), a commonly held belief. Computational analysis of our time-resolved data lays the foundation for more precise analyses of how cardiac gene regulatory networks correlate with early heart tissue morphogenesis in birds and mammals.

The role of hydrodynamic stress on the phenotypic characteristics of single and binary biofilms of Pseudomonas fluorescens.

PubMed

Simões, M; Pereira, M O; Vieira, M J

2007-01-01

This study investigates the phenotype of turbulent (Re = 5,200) and laminar (Re = 2,000) flow-generated Pseudomonas fluorescens biofilms. Three P. fluorescens strains, the type strain ATCC 13525 and two strains isolated from an industrial processing plant, D3-348 and D3-350, were used throughout this study. The isolated strains were used to form single and binary biofilms. The biofilm physiology (metabolic activity, cellular density, mass, extracellular polymeric substances, structural characteristics and outer membrane proteins [OMP] expression) was compared. The results indicate that, for every situation, turbulent flow-generated biofilms were more active (p < 0.05), had more mass per cm(2) (p < 0.05), a higher cellular density (p < 0.05), distinct morphology, similar matrix proteins (p > 0.1) and identical (isolated strains -single and binary biofilms) and higher (type strain) matrix polysaccharides contents (p < 0.05) than laminar flow-generated biofilms. Flow-generated biofilms formed by the type strain revealed a considerably higher cellular density and amount of matrix polysaccharides than single and binary biofilms formed by the isolated strains (p < 0.05). Similar OMP expression was detected for the several single strains and for the binary situation, not dependent on the hydrodynamic conditions. Binary biofilms revealed an equal coexistence of the isolated strains with apparent neutral interactions. In summary, the biofilms formed by the type strain represent, apparently, the worst situation in a context of control. The results obtained clearly illustrate the importance of considering strain variation and hydrodynamics in biofilm development, and complement previous studies which have focused on physical aspects of structural and density differences.

The Cellular Form of Human Fibronectin as an Adhesion Target for the S Fimbriae of Meningitis-Associated Escherichia coli

PubMed Central

Sarén, Anne; Virkola, Ritva; Hacker, Jörg; Korhonen, Timo K.

1999-01-01

The adhesion of the S fimbriae of meningitis-associated Escherichia coli O18ac:K1:H7 to the cellular and the plasma forms of human fibronectin was studied. E. coli HB101(pAZZ50) expressing the complete S-fimbria II gene cluster of E. coli O18 adhered to cellular fibronectin (cFn) on glass but not to plasma fibronectin (pFn). Adhesion to cFn was specifically inhibited by neuraminidase treatment of cFn as well as by incubation of the bacteria with sialyl-α2-3-lactose, a receptor analog of the S fimbriae. No significant adhesion to cFn or pFn was detected with E. coli HB101(pAZZ50-67) expressing S fimbriae lacking the SfaS lectin subunit. Strain HB101(pAZZ50) also adhered to a human fibroblast cell culture known to be rich in cFn, and the adhesion was specifically inhibited in the presence of polyclonal antibodies to cFn. The results show that the SfaS lectin of the S fimbriae mediates the adherence of meningitis-associated E. coli to sialyl oligosaccharide chains of cFn. PMID:10225941

Prohibitin( PHB) roles in granulosa cell physiology.

PubMed

Chowdhury, Indrajit; Thomas, Kelwyn; Thompson, Winston E

2016-01-01

Ovarian granulosa cells (GC) play an important role in the growth and development of the follicle in the process known as folliculogenesis. In the present review, we focus on recent developments in prohibitin (PHB) research in relation to GC physiological functions. PHB is a member of a highly conserved eukaryotic protein family containing the repressor of estrogen activity (REA)/stomatin/PHB/flotillin/HflK/C (SPFH) domain (also known as the PHB domain) found in diverse species from prokaryotes to eukaryotes. PHB is ubiquitously expressed in a circulating free form or is present in multiple cellular compartments including mitochondria, nucleus and plasma membrane. In mitochondria, PHB is anchored to the mitochondrial inner membrane and forms complexes with the ATPases associated with proteases having diverse cellular activities. PHB continuously shuttles between the mitochondria, cytosol and nucleus. In the nucleus, PHB interacts with various transcription factors and modulates transcriptional activity directly or through interactions with chromatin remodeling proteins. Many functions have been attributed to the mitochondrial and nuclear PHB complexes such as cellular differentiation, anti-proliferation, morphogenesis and maintenance of the functional integrity of the mitochondria. However, to date, the regulation of PHB expression patterns and GC physiological functions are not completely understood.

Prohibitin (PHB) roles in granulosa cell physiology

PubMed Central

Chowdhury, Indrajit; Thomas, Kelwyn; Thompson, Winston E.

2015-01-01

Ovarian granulosa cells (GC) play an important role in the growth and development of the follicle in the process known as folliculogenesis. In the present review, we focus on the recent developments in prohibitin (PHB) research in relation to GC physiological functions. PHB is a member of highly conserved eukaryotic protein family containing the repressor of estrogen activity (REA)/stomatin/prohibitin/flotillin/HflK/C (SPFH) domain [also known as the PHB domain] found in divergent species from prokaryotes to eukaryotes. PHB is ubiquitously expressed either in circulating free form or is present in multiple cellular compartments including mitochondria, nucleus and plasma membrane. In mitochondria, PHB is anchored to the mitochondrial inner membrane (IMM), and form complexes with the ATPases Associated with diverse cellular Activities (m-AAA) proteases. PHB continuously shuttles between the mitochondria, cytosol and nucleus. In the nucleus, PHB interacts with various transcription factors and modulate transcriptional activity directly or through interactions with chromatin remodeling proteins. Multiple functions have been attributed to the mitochondrial and nuclear prohibitin complexes such as cellular differentiation, anti-proliferation, morphogenesis and maintaining the functional integrity of the mitochondria. However, to date, the regulation of PHB expression patterns and GC physiological functions are not completely understood. PMID:26496733

THE CELLULAR ORIGIN OF HUMAN IMMUNOGLOBULINS (γ2, γ1M, γ1A)

PubMed Central

Mellors, Robert C.; Korngold, Leonhard

1963-01-01

A study was made of the cellular origin of human immunoglobulins (γ2, γ1M, γ1A). The results indicated that two closely related families of cells form immunoglobulins in human lymphoid tissue: germinal (reticular) centers and plasma cells. Thus their cellular origin in addition to their known antigenic relations further justifies placing the immunoglobulins in one family of proteins. Immunoglobulins were also formed to a small extent in primitive reticular cells which resembled those of germinal centers but were separated from them. Possibly such cells were undergoing transition to the much more numerous plasma cells with which they were commonly associated. The mantles of small lymphocytes which surrounded germinal centers did not contain detectable quantities of immunoglobulins. While in general only one type of immunoglobulin was present in an individual cell or germinal center, γ2- and γ1M-globulin were identified on occasion in the same plasma cell and germinal center. A peculiarity of the fetal thymus gland was the presence of immunoglobulin, mainly γ1M, in a small number of cells of small and intermediate size and primitive reticular appearance and in Hassall's corpuscles. PMID:14077999

Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging

PubMed Central

Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

2015-01-01

Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction. PMID:25762114

Molecular and Cellular Aspects of Calcific Aortic Valve Disease

PubMed Central

Towler, Dwight A.

2014-01-01

Calcific aortic valve disease (CAVD) increasingly afflicts our aging population. One-third of our elderly have echocardiographic or radiological evidence of aortic valve sclerosis (CAVS), an early and subclinical form of CAVD. Age, gender, tobacco use, hypercholesterolemia, hypertension, and type II diabetes all contribute to the risk of disease that has worldwide distribution. Upon progression to its most severe form --- calcific aortic stenosis (CAS) --- CAVD becomes debilitating and devastating, and 2% of individuals over age 60 suffer from CAS to the extent that surgical intervention is required. No effective pharmacotherapies exist for treating those at risk for clinical progression. It is becoming increasingly apparent that a diverse spectrum of cellular and molecular mechanisms converge to regulate valvular calcium load; this is evidenced not only in histopathologic heterogeneity of CAVD but also from the multiplicity of cell types that can participate in valve biomineralization. In this review, we highlight our current understanding of CAVD disease biology, emphasizing molecular and cellular aspects of its regulation. We end by pointing to important biological and clinical questions that must be answered to enable sophisticated disease staging and the development of new strategies to medically treat CAVD. PMID:23833294

Enhanced Cellular Uptake and Pharmacokinetic Characteristics of Doxorubicin-Valine Amide Prodrug.

PubMed

Park, Yohan; Park, Ju-Hwan; Park, Suryeon; Lee, Song Yi; Cho, Kwan Hyung; Kim, Dae-Duk; Shim, Won-Sik; Yoon, In-Soo; Cho, Hyun-Jong; Maeng, Han-Joo

2016-09-22

In this study, we synthesized the valine (Val)-conjugated amide prodrug of doxorubicin (DOX) by the formation of amide bonds between DOX and Val. The synthesis of the DOX-Val prodrug was identified by a proton nuclear magnetic resonance (¹H-NMR) assay. In the MCF-7 cells (human breast adenocarcinoma cell; amino acid transporter-positive cell), the cellular accumulation efficiency of DOX-Val was higher than that of DOX according to the flow cytometry analysis data. Using confocal laser scanning microscopy (CLSM) imaging, it was confirmed that DOX-Val as well as DOX was mainly distributed in the nucleus of cancer cells. DOX-Val was intravenously administered to rats at a dose of 4 mg/kg, and the plasma concentrations of DOX-Val (prodrug) and DOX (formed metabolite) were quantitatively determined. Based on the systemic exposure (represented as area under the curve (AUC) values) of DOX-Val (prodrug) and DOX (formed metabolite), approximately half of DOX-Val seemed to be metabolized into DOX. However, it is expected that the remaining DOX-Val may exert improved cellular uptake efficiency in cancer cells after its delivery to the cancer region.

Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium.

PubMed

Miyoshi, Hiroyuki; VanDussen, Kelli L; Malvin, Nicole P; Ryu, Stacy H; Wang, Yi; Sonnek, Naomi M; Lai, Chin-Wen; Stappenbeck, Thaddeus S

2017-01-04

Adaptive cellular responses are often required during wound repair. Following disruption of the intestinal epithelium, wound-associated epithelial (WAE) cells form the initial barrier over the wound. Our goal was to determine the critical factor that promotes WAE cell differentiation. Using an adaptation of our in vitro primary epithelial cell culture system, we found that prostaglandin E2 (PGE 2 ) signaling through one of its receptors, Ptger4, was sufficient to drive a differentiation state morphologically and transcriptionally similar to in vivo WAE cells. WAE cell differentiation was a permanent state and dominant over enterocyte differentiation in plasticity experiments. WAE cell differentiation was triggered by nuclear β-catenin signaling independent of canonical Wnt signaling. Creation of WAE cells via the PGE 2 -Ptger4 pathway was required in vivo, as mice with loss of Ptger4 in the intestinal epithelium did not produce WAE cells and exhibited impaired wound repair. Our results demonstrate a mechanism by which WAE cells are formed by PGE 2 and suggest a process of adaptive cellular reprogramming of the intestinal epithelium that occurs to ensure proper repair to injury. © 2016 The Authors.

Model of polar auxin transport coupled to mechanical forces retrieves robust morphogenesis along the Arabidopsis root

NASA Astrophysics Data System (ADS)

Romero-Arias, J. Roberto; Hernández-Hernández, Valeria; Benítez, Mariana; Alvarez-Buylla, Elena R.; Barrio, Rafael A.

2017-03-01

Stem cells are identical in many scales, they share the same molecular composition, DNA, genes, and genetic networks, yet they should acquire different properties to form a functional tissue. Therefore, they must interact and get some external information from their environment, either spatial (dynamical fields) or temporal (lineage). In this paper we test to what extent coupled chemical and physical fields can underlie the cell's positional information during development. We choose the root apical meristem of Arabidopsis thaliana to model the emergence of cellular patterns. We built a model to study the dynamics and interactions between the cell divisions, the local auxin concentration, and physical elastic fields. Our model recovers important aspects of the self-organized and resilient behavior of the observed cellular patterns in the Arabidopsis root, in particular, the reverse fountain pattern observed in the auxin transport, the PIN-FORMED (protein family of auxin transporters) polarization pattern and the accumulation of auxin near the region of maximum curvature in a bent root. Our model may be extended to predict altered cellular patterns that are expected under various applied auxin treatments or modified physical growth conditions.

Cytotoxicity and Physiological Effects of Silver Nanoparticles on Marine Invertebrates.

PubMed

Magesky, Adriano; Pelletier, Émilien

2018-01-01

Silver nanoparticles (AgNPs) incorporation in commercial products is increasing due to their remarkable physical and chemical properties and their low cost on the market. Silver has been known for a long time to be highly toxic to bacterial communities, aquatic organisms, and particularly to marine biota. Strong chloro-complexes dominate Ag speciation in seawater and facilitate its persistence in dissolved form. It has a great impact on marine organisms because low concentration of silver can lead to strong bioaccumulation, partly because the neutral silver chloro complex (AgCl 0 ) is highly bioavailable. Owing to the fact that estuaries and coastal areas are considered as the ultimate fate for AgNPs, the study of their toxic effects on marine invertebrates can reveal some environmental risks related to nanosilver exposure. In an attempt to reach this goal, many invertebrate taxa including mollusks, crustaceans, echinoderms and polychaetes have been used as biological models. The main findings related to AgNP toxicity and marine invertebrates are summarized hereafter. Some cellular mechanisms involving nano-internalization (cellular uptake, distribution and elimination), DNA damaging, antioxidant cellular defenses and protein expression are discussed. Physiological effects on early stage development, silver metabolic speciation, immune response, tissue damaging, anti-oxidant effects and nano-depuration are also described. Finally, we paid attention to some recent interesting findings using sea urchin developmental stages and their cells as models for nanotoxicity investigation. Cellular and physiological processes characterizing sea urchin development revealed new and multiple toxicity mechanisms of both soluble and nano forms of silver.

Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.

PubMed

Moeller, Marcus J; Soofi, Abdulsalaam; Hartmann, Inge; Le Hir, Michel; Wiggins, Roger; Kriz, Wilhelm; Holzman, Lawrence B

2004-01-01

Cellular crescents are a defining histologic finding in many forms of inflammatory glomerulonephritis. Despite numerous studies, the origin of glomerular crescents remains unresolved. A genetic cell lineage-mapping study with a novel transgenic mouse model was performed to investigate whether visceral glomerular epithelial cells, termed podocytes, are precursors of cells that populate cellular crescents. The podocyte-specific 2.5P-Cre mouse line was crossed with the ROSA26 reporter line, resulting in irreversible constitutive expression of beta-galactosidase in doubly transgenic 2.5P-Cre/ROSA26 mice. In these mice, crescentic glomerulonephritis was induced with a previously described rabbit anti-glomerular basement membrane antiserum nephritis approach. Interestingly, beta-galactosidase-positive cells derived from podocytes adhered to the parietal basement membrane and populated glomerular crescents during the early phases of cellular crescent formation, accounting for at least one-fourth of the total cell mass. In cellular crescents, the proliferation marker Ki-67 was expressed in beta-galactosidase-positive and beta-galactosidase-negative cells, indicating that both cell types contributed to the formation of cellular crescents through proliferation in situ. Podocyte-specific antigens, including WT-1, synaptopodin, nephrin, and podocin, were not expressed by any cells in glomerular crescents, suggesting that podocytes underwent profound phenotypic changes in this nephritis model.

Tetraspanin CD37 contributes to the initiation of cellular immunity by promoting dendritic cell migration.

PubMed

Gartlan, Kate H; Wee, Janet L; Demaria, Maria C; Nastovska, Roza; Chang, Tsz Man; Jones, Eleanor L; Apostolopoulos, Vasso; Pietersz, Geoffrey A; Hickey, Michael J; van Spriel, Annemiek B; Wright, Mark D

2013-05-01

Previous studies on the role of the tetraspanin CD37 in cellular immunity appear contradictory. In vitro approaches indicate a negative regulatory role, whereas in vivo studies suggest that CD37 is necessary for optimal cellular responses. To resolve this discrepancy, we studied the adaptive cellular immune responses of CD37(-/-) mice to intradermal challenge with either tumors or model antigens and found that CD37 is essential for optimal cell-mediated immunity. We provide evidence that an increased susceptibility to tumors observed in CD37(-/-) mice coincides with a striking failure to induce antigen-specific IFN-γ-secreting T cells. We also show that CD37 ablation impairs several aspects of DC function including: in vivo migration from skin to draining lymph nodes; chemo-tactic migration; integrin-mediated adhesion under flow; the ability to spread and form actin protrusions and in vivo priming of adoptively transferred naïve T cells. In addition, multiphoton microscopy-based assessment of dermal DC migration demonstrated a reduced rate of migration and increased randomness of DC migration in CD37(-/-) mice. Together, these studies are consistent with a model in which the cellular defect that underlies poor cellular immune induction in CD37(-/-) mice is impaired DC migration. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

At a glance: cellular biology for engineers.

PubMed

Khoshmanesh, K; Kouzani, A Z; Nahavandi, S; Baratchi, S; Kanwar, J R

2008-10-01

Engineering contributions have played an important role in the rise and evolution of cellular biology. Engineering technologies have helped biologists to explore the living organisms at cellular and molecular levels, and have created new opportunities to tackle the unsolved biological problems. There is now a growing demand to further expand the role of engineering in cellular biology research. For an engineer to play an effective role in cellular biology, the first essential step is to understand the cells and their components. However, the stumbling block of this step is to comprehend the information given in the cellular biology literature because it best suits the readers with a biological background. This paper aims to overcome this bottleneck by describing the human cell components as micro-plants that form cells as micro-bio-factories. This concept can accelerate the engineers' comprehension of the subject. In this paper, first the structure and function of different cell components are described. In addition, the engineering attempts to mimic various cell components through numerical modelling or physical implementation are highlighted. Next, the interaction of different cell components that facilitate complicated chemical processes, such as energy generation and protein synthesis, are described. These complex interactions are translated into simple flow diagrams, generally used by engineers to represent multi-component processes.

Alginic acid cell entrapment: a novel method for measuring in vivo macrophage cholesterol homeostasis

PubMed Central

Sontag, Timothy J.; Chellan, Bijoy; Bhanvadia, Clarissa V.; Getz, Godfrey S.; Reardon, Catherine A.

2015-01-01

Macrophage conversion to atherosclerotic foam cells is partly due to the balance of uptake and efflux of cholesterol. Cholesterol efflux from cells by HDL and its apoproteins for subsequent hepatic elimination is known as reverse cholesterol transport. Numerous methods have been developed to measure in vivo macrophage cholesterol efflux. Most methods do not allow for macrophage recovery for analysis of changes in cellular cholesterol status. We describe a novel method for measuring cellular cholesterol balance using the in vivo entrapment of macrophages in alginate, which retains incorporated cells while being permeable to lipoproteins. Recipient mice were injected subcutaneously with CaCl2 forming a bubble into which a macrophage/alginate suspension was injected, entrapping the macrophages. Cells were recovered after 24 h. Cellular free and esterified cholesterol mass were determined enzymatically and normalized to cellular protein. Both normal and cholesterol loaded macrophages undergo measureable changes in cell cholesterol when injected into WT and apoA-I-, LDL-receptor-, or apoE-deficient mice. Cellular cholesterol balance is dependent on initial cellular cholesterol status, macrophage cholesterol transporter expression, and apolipoprotein deficiency. Alginate entrapment allows for the in vivo measurement of macrophage cholesterol homeostasis and is a novel platform for investigating the role of genetics and therapeutic interventions in atherogenesis. PMID:25465389

Experimental investigation of the limits of ethanol combustion in the boundary layer behind an obstacle

NASA Astrophysics Data System (ADS)

Boyarshinov, B. F.

2018-01-01

Experimental data on the flow structure and mass transfer near the boundaries of the region existence of the laminar and turbulent boundary layers with combustion are considered. These data include the results of in-vestigation on reacting flow stability at mixed convection, mass transfer during ethanol evaporation "on the floor" and "on the ceiling", when the flame surface curves to form the large-scale cellular structures. It is shown with the help of the PIV equipment that when Rayleigh-Taylor instability manifests, the mushroom-like structures are formed, where the motion from the flame front to the wall and back alternates. The cellular flame exists in a narrow range of velocities from 0.55 to 0.65 m/s, and mass transfer is three times higher than its level in the standard laminar boundary layer.

The pathobiology and mechanisms of infection of HPV.

PubMed

Wood, N H; Khammissa, R A G; Chikte, U M E; Meyerov, R; Lemmer, J; Feller, L

2010-04-01

There are more than 120 types of low-risk and high-risk human papillomaviruses, all of which are epitheliotropic. HPV infection may be latent, or active in a subclinical form or a symptomatic form, the latter manifesting as benign or malignant neoplasms. In basal cells with non-productive HPV infection some early HPV proteins are expressed independently of cell maturation: the productive cycle of HPV replication depends upon specific cellular factors of the maturation of the infected keratinocytes. In HPV-mediated oncogenesis, the combined pathobiological effects of E6 and E7 oncoproteins of high-risk HPV culminate in cellular genomic instability and transformation of persistently infected cells, that progress to the development of a malignant phenotype. In this article we provide insights into the stages of HPV infection, and into the viral genomic organization and replicative cycle.

Computational Modeling of Proteins based on Cellular Automata: A Method of HP Folding Approximation.

PubMed

Madain, Alia; Abu Dalhoum, Abdel Latif; Sleit, Azzam

2018-06-01

The design of a protein folding approximation algorithm is not straightforward even when a simplified model is used. The folding problem is a combinatorial problem, where approximation and heuristic algorithms are usually used to find near optimal folds of proteins primary structures. Approximation algorithms provide guarantees on the distance to the optimal solution. The folding approximation approach proposed here depends on two-dimensional cellular automata to fold proteins presented in a well-studied simplified model called the hydrophobic-hydrophilic model. Cellular automata are discrete computational models that rely on local rules to produce some overall global behavior. One-third and one-fourth approximation algorithms choose a subset of the hydrophobic amino acids to form H-H contacts. Those algorithms start with finding a point to fold the protein sequence into two sides where one side ignores H's at even positions and the other side ignores H's at odd positions. In addition, blocks or groups of amino acids fold the same way according to a predefined normal form. We intend to improve approximation algorithms by considering all hydrophobic amino acids and folding based on the local neighborhood instead of using normal forms. The CA does not assume a fixed folding point. The proposed approach guarantees one half approximation minus the H-H endpoints. This lower bound guaranteed applies to short sequences only. This is proved as the core and the folds of the protein will have two identical sides for all short sequences.

GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

PubMed

Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

2015-01-01

Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

HSP90 Inhibition and Cellular Stress Elicits Phenotypic Plasticity in Hematopoietic Differentiation

PubMed Central

Lawag, Abdalla A.; Napper, Jennifer M.; Hunter, Caroline A.; Bacon, Nickolas A.; Deskins, Seth; El-hamdani, Manaf; Govender, Sarah-Leigh; Koc, Emine C.

2017-01-01

Abstract Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%–90% of the cells die when placed in medium where the major growth factor is granulocyte–macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer. PMID:28910138

HSP90 Inhibition and Cellular Stress Elicits Phenotypic Plasticity in Hematopoietic Differentiation.

PubMed

Lawag, Abdalla A; Napper, Jennifer M; Hunter, Caroline A; Bacon, Nickolas A; Deskins, Seth; El-Hamdani, Manaf; Govender, Sarah-Leigh; Koc, Emine C; Sollars, Vincent E

2017-10-01

Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%-90% of the cells die when placed in medium where the major growth factor is granulocyte-macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer.

Engineering cellular fibers for musculoskeletal soft tissues using directed self-assembly.

PubMed

Schiele, Nathan R; Koppes, Ryan A; Chrisey, Douglas B; Corr, David T

2013-05-01

Engineering strategies guided by developmental biology may enhance and accelerate in vitro tissue formation for tissue engineering and regenerative medicine applications. In this study, we looked toward embryonic tendon development as a model system to guide our soft tissue engineering approach. To direct cellular self-assembly, we utilized laser micromachined, differentially adherent growth channels lined with fibronectin. The micromachined growth channels directed human dermal fibroblast cells to form single cellular fibers, without the need for a provisional three-dimensional extracellular matrix or scaffold to establish a fiber structure. Therefore, the resulting tissue structure and mechanical characteristics were determined solely by the cells. Due to the self-assembly nature of this approach, the growing fibers exhibit some key aspects of embryonic tendon development, such as high cellularity, the rapid formation (within 24 h) of a highly organized and aligned cellular structure, and the expression of cadherin-11 (indicating direct cell-to-cell adhesions). To provide a dynamic mechanical environment, we have also developed and characterized a method to apply precise cyclic tensile strain to the cellular fibers as they develop. After an initial period of cellular fiber formation (24 h postseeding), cyclic strain was applied for 48 h, in 8-h intervals, with tensile strain increasing from 0.7% to 1.0%, and at a frequency of 0.5 Hz. Dynamic loading dramatically increased cellular fiber mechanical properties with a nearly twofold increase in both the linear region stiffness and maximum load at failure, thereby demonstrating a mechanism for enhancing cellular fiber formation and mechanical properties. Tissue engineering strategies, designed to capture key aspects of embryonic development, may provide unique insight into accelerated maturation of engineered replacement tissue, and offer significant advances for regenerative medicine applications in tendon, ligament, and other fibrous soft tissues.

In vitro effects of platinum compounds on renal cellular respiration in mice.

PubMed

Almarzooqi, Saeeda-S; Alfazari, Ali-S; Abdul-Kader, Hidaya-M; Saraswathiamma, Dhanya; Albawardi, Alia-S; Souid, Abdul-Kader

2015-01-01

Cisplatin, carboplatin and oxaliplatin are structurally-related compounds, which are commonly used in cancer therapy. Cisplatin (Platinol(®)) has Boxed Warning stating: "Cumulative renal toxicity associated with PLATINOL is severe", while carboplatin and oxaliplatin are less nephrotoxic. These drugs form platinum adducts with cellular DNA. Their bindings to cellular thiols (e.g., glutathione and metallothionein) are known to contribute to drug resistance while thiol depletion augments platinum toxicity. Using phosphorescence oxygen analyzer, this study investigated the effects of platinum drugs on renal cellular respiration (mitochondrial O2 consumption) in the presence and absence of the thiol blocking agent N-ethylmaleimide (used here as a model for thiol depletion). Renal cellular ATP was also determined. Kidney fragments from C57BL/6 mice were incubated at 37 °C in Krebs-Henseleit buffer (gassed with 95% O2:5% CO2) with and without 100 μM platinum drug in the presence and absence of 100 μM N-ethylmaleimide for ≤ 6 h. Platinum drugs alone had no effects on cellular respiration (P ≥ 0.143) or ATP (P ≥ 0.161). N-ethylmaleimide lowered cellular respiration (P ≤ 0.114) and ATP (P = 0.008). The combination of platinum drug and N-ethylmaleimide significantly lowered both cellular respiration (P ≤ 0.006) and ATP (P ≤ 0.003). Incubations with N-ethylmaleimide alone were associated with moderate-to-severe tubular necrosis. Incubations with cisplatin+N-ethylmaleimide vs. cisplatin alone produced similar severities of tubular necrosis. Tubular derangements were more prominent in carboplatin+N-ethylmaleimide vs. carboplatin alone and in oxaliplatin+N-ethylmaleimide vs. oxaliplatin alone. These results demonstrate the adverse events of thiol depletion on platinum-induced nephrotoxicities. The results suggest cellular bioenergetics is a useful surrogate biomarker for assessing drug-induced nephrotoxicities.

Neural correlates of social odor recognition and the representation of individual distinctive social odors within entorhinal cortex and ventral subiculum.

PubMed

Petrulis, A; Alvarez, P; Eichenbaum, H

2005-01-01

Recognition of individual conspecifics is important for social behavior and requires the formation of memories for individually distinctive social signals. Individual recognition is often mediated by olfactory cues in mammals, especially nocturnal rodents such as golden hamsters. In hamsters, this form of recognition requires main olfactory system input to the lateral entorhinal cortex (LEnt). Here, we tested whether neurons in LEnt and the nearby ventral subiculum (VS) would show cellular correlates of this natural form of recognition memory. Two hundred ninety single neurons were recorded from both superficial (SE) and deep layers of LEnt (DE) and VS while male hamsters investigated volatile odorants from female vaginal secretions. Many neurons encoded differences between female's odors with many discriminating between odors from different individual females but not between different odor samples from the same female. Other neurons discriminated between odor samples from one female and generalized across collections from other females. LEnt and VS neurons showed enhanced or suppressed cellular activity during investigation of previously presented odors and in response to novel odors. A majority of SE neurons decreased firing to odor repetition and increased activity to novel odors. In contrast, DE neurons often showed suppressed activity in response to novel odors. Thus, neurons in LEnt and VS of male hamsters encode information that is critical for the identification and recognition of individual females by odor cues. This study reveals cellular mechanisms in LEnt and VS that may mediate a natural form of recognition memory in hamsters. These neuronal responses were similar to those observed in rats and monkeys during performance in standard recognition memory tasks. Consequently, the present data extend our understanding of the cellular basis for recognition memory and suggest that individual recognition requires similar neural mechanisms as those employed in laboratory tests of recognition memory.

Cellular Auxin Homeostasis under High Temperature Is Regulated through a SORTING NEXIN1–Dependent Endosomal Trafficking Pathway[C][W

PubMed Central

Hanzawa, Taiki; Shibasaki, Kyohei; Numata, Takahiro; Kawamura, Yukio; Gaude, Thierry; Rahman, Abidur

2013-01-01

High-temperature-mediated adaptation in plant architecture is linked to the increased synthesis of the phytohormone auxin, which alters cellular auxin homeostasis. The auxin gradient, modulated by cellular auxin homeostasis, plays an important role in regulating the developmental fate of plant organs. Although the signaling mechanism that integrates auxin and high temperature is relatively well understood, the cellular auxin homeostasis mechanism under high temperature is largely unknown. Using the Arabidopsis thaliana root as a model, we demonstrate that under high temperature, roots counterbalance the elevated level of intracellular auxin by promoting shootward auxin efflux in a PIN-FORMED2 (PIN2)-dependent manner. Further analyses revealed that high temperature selectively promotes the retrieval of PIN2 from late endosomes and sorts them to the plasma membrane through an endosomal trafficking pathway dependent on SORTING NEXIN1. Our results demonstrate that recycling endosomal pathway plays an important role in facilitating plants adaptation to increased temperature. PMID:24003052

Therapeutic intervention at cellular quality control systems in Alzheimer's and Parkinson's diseases.

PubMed

Arduino, Daniela M; Esteves, A Raquel; Silva, Diana F F; Martins-Branco, Diogo; Santos, Daniel; Pimentel, Diana F Gomes; Cardoso, Sandra M

2011-01-01

Cellular homeostasis relies on quality control systems so that damaged biologic structures are either repaired or degraded and entirely replaced by newly formed proteins or even organelles. The clearance of dysfunctional cellular structures in long-lived postmitotic cells, like neurons, is essential to eliminate, per example, defective mitochondria, lipofuscin-loaded lysosomes and oxidized proteins. Short-lived proteins are degraded mainly by proteases and proteasomes whether most long-lived proteins and all organelles are digested by autophagy in the lysosomes. Recently, it an interplay was established between the ubiquitin-proteasome system and macroautophagy, so that both degradative mechanisms compensate for each other. In this article we describe each of these clearance systems and their contribution to neuronal quality control. We will highlight some of the findings that provide evidence for the dysfunction of these systems in Alzheimer's and Parkinson's diseases. Ultimately, we provide an outline on potential therapeutic interventions based on the modulation of cellular degradative systems.

High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection

PubMed Central

Choudhry, Priya

2016-01-01

Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays. PMID:26848849

Cellular proteostasis: degradation of misfolded proteins by lysosomes

PubMed Central

Jackson, Matthew P.

2016-01-01

Proteostasis refers to the regulation of the cellular concentration, folding, interactions and localization of each of the proteins that comprise the proteome. One essential element of proteostasis is the disposal of misfolded proteins by the cellular pathways of protein degradation. Lysosomes are an important site for the degradation of misfolded proteins, which are trafficked to this organelle by the pathways of macroautophagy, chaperone-mediated autophagy and endocytosis. Conversely, amyloid diseases represent a failure in proteostasis, in which proteins misfold, forming amyloid deposits that are not degraded effectively by cells. Amyloid may then exacerbate this failure by disrupting autophagy and lysosomal proteolysis. However, targeting the pathways that regulate autophagy and the biogenesis of lysosomes may present approaches that can rescue cells from the deleterious effects of amyloidogenic proteins. PMID:27744333

Cellular and multicellular form and function.

PubMed

Liu, Wendy F; Chen, Christopher S

2007-11-10

Engineering artificial tissue constructs requires the appropriate spatial arrangement of cells within scaffolds. The introduction of microengineering tools to the biological community has provided a valuable set of techniques to manipulate the cellular environment, and to examine how cell structure affects cellular function. Using micropatterning techniques, investigators have found that the geometric presentation of cell-matrix adhesions are important regulators of various cell behaviors including cell growth, proliferation, differentiation, polarity and migration. Furthermore, the presence of neighboring cells in multicellular aggregates has a significant impact on the proliferative and differentiated state of cells. Using microengineering tools, it will now be possible to manipulate the various environmental factors for practical applications such as engineering tissue constructs with greater control over the physical structure and spatial arrangement of cells within their surrounding microenvironment.

Cellular interface morphologies in directional solidification. III - The effects of heat transfer and solid diffusivity

NASA Technical Reports Server (NTRS)

Ungar, Lyle H.; Bennett, Mark J.; Brown, Robert A.

1985-01-01

The shape and stability of two-dimensional finite-amplitude cellular interfaces arising during directional solidification are compared for several solidification models that account differently for latent heat released at the interface, unequal thermal conductivities of melt and solid, and solute diffusivity in the solid. Finite-element analysis and computer-implemented perturbation methods are used to analyze the families of steadily growing cellular forms that evolve from the planar state. In all models a secondary bifurcation between different families of finite-amplitude cells exists that halves the spatial wavelength of the stable interface. The quantitative location of this transition is very dependent on the details of the model. Large amounts of solute diffusion in the solid retard the growth of large-amplitude cells.

Failover in Cellular Automata

NASA Astrophysics Data System (ADS)

Kumar, Shailesh; Rao, Shrisha

This paper studies a phenomenon called failover, and shows that this phenomenon (in particular, stateless failover) can be modeled by Game of Life cellular automata. This is the first time that this sophisticated real-life system behavior has been modeled in abstract terms. A cellular automata (CA) configuration is constructed that exhibits emergent failover. The configuration is based on standard Game of Life rules. Gliders and glider-guns form the core messaging structure in the configuration. The blinker is represented as the basic computational unit, and it is shown how it can be recreated in case of a failure. Stateless failover using the primary-backup mechanism is demonstrated. The details of the CA components used in the configuration and its working are described, and a simulation of the complete configuration is also presented.

Stress-induced O-GlcNAcylation: an adaptive process of injured cells.

PubMed

Martinez, Marissa R; Dias, Thiago Braido; Natov, Peter S; Zachara, Natasha E

2017-02-08

In the 30 years, since the discovery of nucleocytoplasmic glycosylation, O -GlcNAc has been implicated in regulating cellular processes as diverse as protein folding, localization, degradation, activity, post-translational modifications, and interactions. The cell co-ordinates these molecular events, on thousands of cellular proteins, in concert with environmental and physiological cues to fine-tune epigenetics, transcription, translation, signal transduction, cell cycle, and metabolism. The cellular stress response is no exception: diverse forms of injury result in dynamic changes to the O -GlcNAc subproteome that promote survival. In this review, we discuss the biosynthesis of O -GlcNAc, the mechanisms by which O -GlcNAc promotes cytoprotection, and the clinical significance of these data. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

75 FR 31791 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-04

... reveal the genetic processes occurring in ccRCC tissues that may contribute to advanced disease. Positive... transformed into blood brain barrier permeable forms by the coupling of an Inter-Cellular Adhesion Molecule-1...

Converging cellular themes for the hereditary spastic paraplegias.

PubMed

Blackstone, Craig

2018-05-10

Hereditary spastic paraplegias (HSPs) are neurologic disorders characterized by prominent lower-extremity spasticity, resulting from a length-dependent axonopathy of corticospinal upper motor neurons. They are among the most genetically-diverse neurologic disorders, with >80 distinct genetic loci and over 60 identified genes. Studies investigating the molecular pathogenesis underlying HSPs have emphasized the importance of converging cellular pathogenic themes in the most common forms of HSP, providing compelling targets for therapy. Most notably, these include organelle shaping and biogenesis as well as membrane and cargo trafficking. Published by Elsevier Ltd.

A study for bank effect on ship traffic in narrow water channels using cellular automata

NASA Astrophysics Data System (ADS)

Sun, Zhuo; Cong, Shuang; Pan, Junnan; Zheng, Jianfeng

2017-12-01

In narrow water channels, bank might affect nearby ships due to hydrodynamic forces (bank effect). To avoid accidents, different sailing rules (i.e., lane-changing, speed control) are required. In this paper, a two-lane cellular automata model is proposed to evaluate such phenomena. Numerical experiments show that ships will form a “slow-moving chunk” in the bank area, which will significantly block the flux. As further study demonstrated to alleviate bank effect, ship speed and bank length should be controlled.

Method of encapsulating polyaminopolycarboxylic acid chelating agents in liposomes

DOEpatents

Rahman, Yueh Erh

1977-11-10

A method is provided for transferring a polyaminopolycarboxylic acid chelating agent across a cellular membrane by encapsulating the charged chelating agent within liposomes, which liposomes will be taken up by the cells, thereby transferring the chelating agent across the cellular membrane. The chelating agent is encapsulated within liposomes by drying a lipid mixture to form a thin film and wetting the lipid film with a solution containing the chelating agent. Mixing then results in the formation of a suspension of liposomes encapsulating the chelating agent, which liposomes can then be separated.

Wallerian demyelination: chronicle of a cellular cataclysm.

PubMed

Tricaud, Nicolas; Park, Hwan Tae

2017-11-01

Wallerian demyelination is characteristic of peripheral nerve degeneration after traumatic injury. After axonal degeneration, the myelinated Schwann cell undergoes a stereotypical cellular program that results in the disintegration of the myelin sheath, a process termed demyelination. In this review, we chronologically describe this program starting from the late and visible features of myelin destruction and going backward to the initial molecular steps that trigger the nuclear reprogramming few hours after injury. Wallerian demyelination is a wonderful model for myelin degeneration occurring in the diverse forms of demyelinating peripheral neuropathies that plague human beings.

Use of Lightweight Cellular Mats to Reduce the Settlement of Structure on Soft Soil

NASA Astrophysics Data System (ADS)

Ganasan, R.; Lim, A. J. M. S.; Wijeyesekera, D. C.

2016-07-01

Construction of structures on soft soils gives rise to some difficulties in Malaysia and other country especially in settlement both in short and long term. The focus of this research is to minimize the differential and non-uniform settlement on peat soil with the use of an innovative cellular mat. The behaviour and performance of the lightweight geo-material (in block form) is critically investigated and in particular the use as a fill in embankment on soft ground. Hemic peat soil, sponge and innovative cellular mat will be used as the main material in this study. The monitoring in settlement behavior from this part of research will be done as laboratory testing only. The uneven settlement in this problem was uniquely monitored photographically using spot markers. In the end of the research, it is seen that the innovative cellular mat has reduce the excessive and differential settlement up to 50% compare to flexible and rigid foundations. This had improve the stiffness of soils as well as the porous contain in cellular structure which help in allowing water/moisture to flow through in or out thus resulting in prevent the condition of floating.

Welcome to pandoraviruses at the ‘Fourth TRUC’ club

PubMed Central

Sharma, Vikas; Colson, Philippe; Chabrol, Olivier; Scheid, Patrick; Pontarotti, Pierre; Raoult, Didier

2015-01-01

Nucleocytoplasmic large DNA viruses, or representatives of the proposed order Megavirales, belong to families of giant viruses that infect a broad range of eukaryotic hosts. Megaviruses have been previously described to comprise a fourth monophylogenetic TRUC (things resisting uncompleted classification) together with cellular domains in the universal tree of life. Recently described pandoraviruses have large (1.9–2.5 MB) and highly divergent genomes. In the present study, we updated the classification of pandoraviruses and other reported giant viruses. Phylogenetic trees were constructed based on six informational genes. Hierarchical clustering was performed based on a set of informational genes from Megavirales members and cellular organisms. Homologous sequences were selected from cellular organisms using TimeTree software, comprising comprehensive, and representative sets of members from Bacteria, Archaea, and Eukarya. Phylogenetic analyses based on three conserved core genes clustered pandoraviruses with phycodnaviruses, exhibiting their close relatedness. Additionally, hierarchical clustering analyses based on informational genes grouped pandoraviruses with Megavirales members as a super group distinct from cellular organisms. Thus, the analyses based on core conserved genes revealed that pandoraviruses are new genuine members of the ‘Fourth TRUC’ club, encompassing distinct life forms compared with cellular organisms. PMID:26042093

Interface Pattern Selection in Directional Solidification

NASA Technical Reports Server (NTRS)

Trivedi, Rohit; Tewari, Surendra N.

2001-01-01

The central focus of this research is to establish key scientific concepts that govern the selection of cellular and dendritic patterns during the directional solidification of alloys. Ground-based studies have established that the conditions under which cellular and dendritic microstructures form are precisely where convection effects are dominant in bulk samples. Thus, experimental data can not be obtained terrestrially under pure diffusive regime. Furthermore, reliable theoretical models are not yet possible which can quantitatively incorporate fluid flow in the pattern selection criterion. Consequently, microgravity experiments on cellular and dendritic growth are designed to obtain benchmark data under diffusive growth conditions that can be quantitatively analyzed and compared with the rigorous theoretical model to establish the fundamental principles that govern the selection of specific microstructure and its length scales. In the cellular structure, different cells in an array are strongly coupled so that the cellular pattern evolution is controlled by complex interactions between thermal diffusion, solute diffusion and interface effects. These interactions give infinity of solutions, and the system selects only a narrow band of solutions. The aim of this investigation is to obtain benchmark data and develop a rigorous theoretical model that will allow us to quantitatively establish the physics of this selection process.

An insight into morphometric descriptors of cell shape that pertain to regenerative medicine.

PubMed

Lobo, Joana; See, Eugene Yong-Shun; Biggs, Manus; Pandit, Abhay

2016-07-01

Cellular morphology has recently been indicated as a powerful indicator of cellular function. The analysis of cell shape has evolved from rudimentary forms of microscopic visual inspection to more advanced methodologies that utilize high-resolution microscopy coupled with sophisticated computer hardware and software for data analysis. Despite this progress, there is still a lack of standardization in quantification of morphometric parameters. In addition, uncertainty remains as to which methodologies and parameters of cell morphology will yield meaningful data, which methods should be utilized to categorize cell shape, and the extent of reliability of measurements and the interpretation of the resulting analysis. A large range of descriptors has been employed to objectively assess the cellular morphology in two-dimensional and three-dimensional domains. Intuitively, simple and applicable morphometric descriptors are preferable and standardized protocols for cell shape analysis can be achieved with the help of computerized tools. In this review, cellular morphology is discussed as a descriptor of cellular function and the current morphometric parameters that are used quantitatively in two- and three-dimensional environments are described. Furthermore, the current problems associated with these morphometric measurements are addressed. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Welcome to pandoraviruses at the 'Fourth TRUC' club.

PubMed

Sharma, Vikas; Colson, Philippe; Chabrol, Olivier; Scheid, Patrick; Pontarotti, Pierre; Raoult, Didier

2015-01-01

Nucleocytoplasmic large DNA viruses, or representatives of the proposed order Megavirales, belong to families of giant viruses that infect a broad range of eukaryotic hosts. Megaviruses have been previously described to comprise a fourth monophylogenetic TRUC (things resisting uncompleted classification) together with cellular domains in the universal tree of life. Recently described pandoraviruses have large (1.9-2.5 MB) and highly divergent genomes. In the present study, we updated the classification of pandoraviruses and other reported giant viruses. Phylogenetic trees were constructed based on six informational genes. Hierarchical clustering was performed based on a set of informational genes from Megavirales members and cellular organisms. Homologous sequences were selected from cellular organisms using TimeTree software, comprising comprehensive, and representative sets of members from Bacteria, Archaea, and Eukarya. Phylogenetic analyses based on three conserved core genes clustered pandoraviruses with phycodnaviruses, exhibiting their close relatedness. Additionally, hierarchical clustering analyses based on informational genes grouped pandoraviruses with Megavirales members as a super group distinct from cellular organisms. Thus, the analyses based on core conserved genes revealed that pandoraviruses are new genuine members of the 'Fourth TRUC' club, encompassing distinct life forms compared with cellular organisms.

Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

PubMed

Zou, Wei; Wang, Zekun; Xiong, Min; Chen, Aaron Yun; Xu, Peng; Ganaie, Safder S; Badawi, Yomna; Kleiboeker, Steve; Nishimune, Hiroshi; Ye, Shui Qing; Qiu, Jianming

2018-03-01

Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication. IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome and played a critical role in viral DNA replication. In contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication. Copyright © 2018 American Society for Microbiology.

Where else might be life in the Solar system?

NASA Astrophysics Data System (ADS)

Vidmachenko, A. P.; Steklov, A. F.

2017-05-01

We know that there is life on Earth. But some bacteria live in nearly boiling liquid of an extinct volcano, which is saturated by acids, alkalis and salts in various combinations. The main problem of the modern theory of the origin of life is the emergence from the initial chaotic mixture of chemical elements and simple compounds of polymer systems that can to organize themselves, and their subsequent evolution. The main forms of life on Earth are organisms of cellular structure. Exceptions are viruses, that are non-cellular life forms. If we find somewhere life in the Solar system, most likely, it will be microscopic cells. The most likely candidates for this honorable role are: Jupiter's moon Io, Jupiter's moon Europa, Saturn's moons Titan and Enceladus, Neptune's satellite Triton; on the surface of Pluto also found two cryovolcanoes, spacecraft "Dawn" discovered the vast reserves of water on dwarf planet Ceres; also, on its surface was found a large cryovolcano. The most likely candidate for the presence of life is Mars. These bodies are possible objects in the Solar system, where one can search for life of different forms.

Assessment of cellular materials generated by co-cultured ‘inflamed’ and healthy periodontal ligament stem cells from patient-matched groups

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Hao-Ning; Department of Stomatology, The First Affiliated Hospital of the Chinese PLA General Hospital, Beijing 100048; Xia, Yu

Recently, stem cells derived from the'inflamed’ periodontal ligament (PDL) tissue of periodontally diseased teeth (I-PDLSCs) have been increasingly suggested as a more readily accessible source of cells for regenerative therapies than those derived from healthy PDL tissue (H-PDLSCs). However, substantial evidence indicates that I-PDLSCs exhibit impaired functionalities compared with H-PDLSCs. In this study, patient-matched I-PDLSCs and H-PDLSCs were co-cultured at various ratios. Cellular materials derived from these cultures were investigated regarding their osteogenic potential in vitro and capacity to form new bone following in vivo transplantation. While patient-matched I-PDLSCs and H-PDLSCs could co-exist in co-culture systems, the proportion of I-PDLSCsmore » tended to increase during in vitro incubation. Compared with H-PDLSC monoculture, the presence of I-PDLSCs in the co-cultures appeared to enhance the overall cell proliferation. Although not completely rescued, the osteogenic and regenerative potentials of the cellular materials generated by co-cultured I-PDLSCs and H-PDLSCs were significantly improved compared with those derived from I-PDLSC monocultures. Notably, cells in co-cultures containing either 50% I-PDLSCs plus 50% H-PDLSCs or 25% I-PDLSCs plus 75% H-PDLSCs expressed osteogenesis-related proteins and genes at levels similar to those expressed in H-PDLSC monocultures (P>0.05). Irrespective of the percentage of I-PDLSCs, robust cellular materials were obtained from co-cultures with 50% or more H-PDLSCs, which exhibited equivalent potential to form new bone in vivo compared with sheets generated by H-PDLSC monocultures. These data suggest that the co-culture of I-PDLSCs with patient-matched H-PDLSCs is a practical and effective method for increasing the overall osteogenic and regenerative potentials of resultant cellular materials. - Highlights: • Co-culturing H-PDLSCs with I-PDLSCs led to rapid cell expansion. • H-PDLSCs and I-PDLSCs co-cultured at proper ratios retained cell differentiation. • Co-culturing proper ratios of H-PDLSCs and I-PDLSCs produced robust cell sheets. • I-PDLSCs can be used as an adjuvant to H-PDLSCs for yielding cellular materials.« less

Systems Modeling in Developmental Toxicity

EPA Science Inventory

An individual starts off as a single cell, the progeny of which form complex structures that are themselves integrated into progressively larger systems. Developmental biology is concerned with how this cellular complexity and patterning arises through orchestration of cell divi...

Detection of Protein SUMOylation In Situ by Proximity Ligation Assays.

PubMed

Sahin, Umut; Jollivet, Florence; Berthier, Caroline; de Thé, Hugues; Lallemand-Breitenbach, Valérie

2016-01-01

Sumoylation is a posttranslational process essential for life and concerns a growing number of crucial proteins. Understanding the influence of this phenomenon on individual proteins or on cellular pathways in which they function has become an intense area of research. A critical step in studying protein sumoylation is to detect sumoylated forms of a particular protein. This has proven to be a challenging task for a number of reasons, especially in the case of endogenous proteins and in vivo studies or when studying rare cells such as stem cells. Proximity ligation assays that allow detection of closely interacting protein partners can be adapted for initial detection of endogenous sumoylation or ubiquitination in a rapid, ultrasensitive, and cheap manner. In addition, modified forms of a given protein can be detected in situ in various cellular compartments. Finally, the flexibility of this technique may allow rapid screening of drugs and stress signals that may modulate protein sumoylation.

[Arterial media calcification in patients with type 2 diabetes mellitus].

PubMed

Belovici, Maria Isabela; Pandele, G I

2008-01-01

Arterial calcification was previously viewed as an inevitable, passive, and degenerative process that occurred at the end stages of atherosclerosis. Recent studies, however, have demonstrated that calcification of arteries is a complex and regulated process. It may occur in conjunction with atherosclerosis or in an isolated form that is commonly associated with diabetes and renal failure. Higher artery calcium scores are associated with increased cardiovascular events, and some aspects of arterial calcification are similar to the biology of forming bone. Arterial calcification can thus be viewed as a distinct inflammatory arteriopathy, much like atherosclerosis and aneurysms, with its own contribution to cardiovascular morbidity and mortality. Current research involves efforts to define the complex interactions between cellular and molecular mediators of arterial calcification and, in particular, the role of endogenous calcification inhibitors. This review discusses the clinical relevance, cellular events, and suspected molecular pathways that control arterial calcification.

Membrane interactions of ionic liquids and imidazolium salts.

PubMed

Wang, Da; Galla, Hans-Joachim; Drücker, Patrick

2018-06-01

Room-temperature ionic liquids (RTILs) have attracted considerable attention in recent years due to their versatile properties such as negligible volatility, inflammability, high extractive selectivity and thermal stability. In general, RTILs are organic salts with a melting point below ~100 °C determined by the asymmetry of at least one of their ions. Due to their amphiphilic character, strong interactions with biological materials can be expected. However, rising attention has appeared towards their similarity and interaction with biomolecules. By employing structural modifications, the biochemical properties of RTILs can be designed to mimic lipid structures and to tune their hydrophobicity towards a lipophilic behavior. This is evident for the interaction with lipid-membranes where some of these compounds present membrane-disturbing effects or cellular toxicity. Moreover, they can form micelles or lipid-like bilayer structures by themselves. Both aspects, cellular effects and membrane-forming capacities, of a novel class of lipophilic imidazolium salts will be discussed.

Hereditary spastic paraplegia.

PubMed

Blackstone, Craig

2018-01-01

The hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurologic disorders with the common feature of prominent lower-extremity spasticity, resulting from a length-dependent axonopathy of corticospinal upper motor neurons. The HSPs exist not only in "pure" forms but also in "complex" forms that are associated with additional neurologic and extraneurologic features. The HSPs are among the most genetically diverse neurologic disorders, with well over 70 distinct genetic loci, for which about 60 mutated genes have already been identified. Numerous studies elucidating the molecular pathogenesis underlying HSPs have highlighted the importance of basic cellular functions - especially membrane trafficking, mitochondrial function, organelle shaping and biogenesis, axon transport, and lipid/cholesterol metabolism - in axon development and maintenance. An encouragingly small number of converging cellular pathogenic themes have been identified for the most common HSPs, and some of these pathways present compelling targets for future therapies. Copyright © 2018 Elsevier B.V. All rights reserved.

Hemichannel composition and electrical synaptic transmission: molecular diversity and its implications for electrical rectification

PubMed Central

Palacios-Prado, Nicolás; Huetteroth, Wolf; Pereda, Alberto E.

2014-01-01

Unapposed hemichannels (HCs) formed by hexamers of gap junction proteins are now known to be involved in various cellular processes under both physiological and pathological conditions. On the other hand, less is known regarding how differences in the molecular composition of HCs impact electrical synaptic transmission between neurons when they form intercellular heterotypic gap junctions (GJs). Here we review data indicating that molecular differences between apposed HCs at electrical synapses are generally associated with rectification of electrical transmission. Furthermore, this association has been observed at both innexin and connexin (Cx) based electrical synapses. We discuss the possible molecular mechanisms underlying electrical rectification, as well as the potential contribution of intracellular soluble factors to this phenomenon. We conclude that asymmetries in molecular composition and sensitivity to cellular factors of each contributing hemichannel can profoundly influence the transmission of electrical signals, endowing electrical synapses with more complex functional properties. PMID:25360082

Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase.

PubMed

Mohr, S; Hallak, H; de Boitte, A; Lapetina, E G; Brüne, B

1999-04-02

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.

Compact seaweed growth of peritectic phase on confined, flat properitectic dendrites

NASA Astrophysics Data System (ADS)

Ludwig, A.; Mogeritsch, J.

2016-12-01

Peritectic alloys form a variety of different solidification morphologies at low growth rates. An alloy with a concentration that corresponds to the hyper-peritectic limit should show a cellular/dendritic solidification of the peritectic phase for growth velocities above the corresponding constitutional undercooling limit. However, due to nucleation retardation of the peritectic phase we observed growth of properitectic dendrites before cellular growth of the peritectic could established. The transition happened via an overgrowth of dendrites with a thin layer of peritectic phase. The observations were made using a transparent, metal-like solidifying peritectic system that was solidified directionally in thin samples. In the gap between the flat dendrites and the tubing walls, the peritectic phase grew with a compact seaweed morphology, whereas in the interdendritic spacing it formed small-curved bumps. At same distance behind the tip region, more and more polycrystalline-like objects appeared at the elongated traces of the compact seaweed morphology.

BK channels are required for multisensory plasticity in the oculomotor system

PubMed Central

Nelson, Alexandra; Faulstich, Michael; Moghadam, Setareh; Onori, Kimberly; Meredith, Andrea; du Lac, Sascha

2017-01-01

SUMMARY Neural circuits are endowed with several forms of intrinsic and synaptic plasticity that could contribute to adaptive changes in behavior, but circuit complexities have hindered linking specific cellular mechanisms with their behavioral consequences. Eye movements generated by simple brainstem circuits provide a means for relating cellular plasticity to behavioral gain control. Here we show that firing rate potentiation, a form of intrinsic plasticity mediated by reductions in BK-type calcium activated potassium currents in spontaneously firing neurons, is engaged during optokinetic reflex compensation for inner ear dysfunction. Vestibular loss triggers transient increases in postsynaptic excitability, occlusion of firing rate potentiation, and reductions in BK currents in vestibular nucleus neurons. Concurrently, adaptive increases in visually-evoked eye movements rapidly restore oculomotor function in wildtype mice but are profoundly impaired in BK channel null mice. Activity-dependent regulation of intrinsic excitability may be a general mechanism for adaptive control of behavioral output in multisensory circuits. PMID:27989457

The 29-kDa proteins phosphorylated ion thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mendelsohn, M.E.; Yan Zhu; O'Neill, S.

Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. The authors have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, they isolatedmore » and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the estrogen receptor-related protein is HSP27, and the three major 20-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.« less

RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC

PubMed Central

Majumder, Mrinmoyee; House, Reniqua; Palanisamy, Nallasivam; Qie, Shuo; Day, Terrence A.; Neskey, David; Diehl, J. Alan

2016-01-01

RNA-binding proteins (RBP) regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1), plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4) RNA structure within) both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1) and the non-coding RNA Telomerase RNA Component (TERC), and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells. PMID:27606879

Organic-inorganic interface-induced multi-fluorescence of MgO nanocrystal clusters and their applications in cellular imaging.

PubMed

Xie, Shuifen; Bao, Shixiong; Ouyang, Junjie; Zhou, Xi; Kuang, Qin; Xie, Zhaoxiong; Zheng, Lansun

2014-04-25

Surface functionalization of inorganic nanomaterials through chemical binding of organic ligands on the surface unsaturated atoms, forming unique organic-inorganic interfaces, is a powerful approach for creating special functions for inorganic nanomaterials. Herein, we report the synthesis of hierarchical MgO nanocrystal clusters (NCs) with an organic-inorganic interface induced multi-fluorescence and their application as new alternative labels for cellular imaging. The synthetic method was established by a dissolution and regrowth process with the assistance of carboxylic acid, in which the as-prepared MgO NCs were modified with carboxylic groups at the coordinatively unsaturated atoms of the surface. By introducing acetic acid to partially replace oleic acid in the reaction, the optical absorption of the produced MgO NCs was progressively engineered from the UV to the visible region. Importantly, with wider and continuous absorption profile, those MgO NCs presented bright and tunable multicolor emissions from blue-violet to green and yellow, with the highest absolute quantum yield up to (33±1) %. The overlap for the energy levels of the inorganic-organic interface and low-coordinated states stimulated a unique fluorescence resonance energy transfer phenomenon. Considering the potential application in cellular imaging, such multi-fluorescent MgO NCs were further encapsulated with a silica shell to improve the water solubility and stability. As expected, the as-formed MgO@SiO2 NCs possessed great biocompatibility and high performance in cellular imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

PubMed

Brumm, Andrew J; Nunez, Stefanie; Doroudchi, Mehdi M; Kawaguchi, Riki; Duan, Jinhzu; Pellegrini, Matteo; Lam, Larry; Carmichael, S Thomas; Deb, Arjun; Hinman, Jason D

2017-08-01

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein

PubMed Central

Fitzgerald, Kerry D.; Semler, Bert L.

2013-01-01

Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. PMID:23830997

Toxicity evaluations of nanoclays and thermally degraded byproducts through spectroscopical and microscopical approaches

PubMed Central

Wagner, Alixandra; Eldawud, Reem; White, Andrew; Agarwal, Sushant; Stueckle, Todd A.; Sierros, Konstantinos A.; Rojanasakul, Yon; Gupta, Rakesh K.; Dinu, Cerasela Zoica

2016-01-01

Background Montmorillonite is a type of nanoclay that originates from the clay fraction of the soil and is incorporated into polymers to form nanocomposites with enhanced mechanical strength, barrier, and flammability properties used for food packaging, automotive, and medical devices. However, with implementation in such consumer applications, the interaction of montmorillonite-based composites or derived byproducts with biological systems needs to be investigated. Methods Herein we examined the potential of Cloisite Na+ (pristine) and Cloisite 30B (organically modified montmorillonite nanoclay) and their thermally degraded byproducts’ to induce toxicity in model human lung epithelial cells. The experimental set-up mimicked biological exposure in manufacturing and disposal areas and employed cellular treatments with occupationally relevant doses of nanoclays previously characterized using spectroscopical and microscopical approaches. For nanoclay-cellular interactions and for cellular analyses respectively, biosensorial-based analytical platforms were used, with induced cellular changes being confirmed via live cell counts, viability assays, and cell imaging. Results Our analysis of byproducts’ chemical and physical properties revealed both structural and functional changes. Real-time high throughput analyses of exposed cellular systems confirmed that nanoclay induced significant toxic effects, with Cloisite 30B showing time-dependent decreases in live cell count and cellular viability relative to control and pristine nanoclay, respectively. Byproducts produced less toxic effects; all treatments caused alterations in the cell morphology upon exposure. Conclusions Our morphological, behavioral, and viability cellular changes show that nanoclays have the potential to produce toxic effects when used both in manufacturing or disposal environments. General significance The reported toxicological mechanisms prove the extensibility of a biosensorial-based platform for cellular behavior analysis upon treatment with a variety of nanomaterials. PMID:27612663

Cellular Automata Generalized To An Inferential System

NASA Astrophysics Data System (ADS)

Blower, David J.

2007-11-01

Stephen Wolfram popularized elementary one-dimensional cellular automata in his book, A New Kind of Science. Among many remarkable things, he proved that one of these cellular automata was a Universal Turing Machine. Such cellular automata can be interpreted in a different way by viewing them within the context of the formal manipulation rules from probability theory. Bayes's Theorem is the most famous of such formal rules. As a prelude, we recapitulate Jaynes's presentation of how probability theory generalizes classical logic using modus ponens as the canonical example. We emphasize the important conceptual standing of Boolean Algebra for the formal rules of probability manipulation and give an alternative demonstration augmenting and complementing Jaynes's derivation. We show the complementary roles played in arguments of this kind by Bayes's Theorem and joint probability tables. A good explanation for all of this is afforded by the expansion of any particular logic function via the disjunctive normal form (DNF). The DNF expansion is a useful heuristic emphasized in this exposition because such expansions point out where relevant 0s should be placed in the joint probability tables for logic functions involving any number of variables. It then becomes a straightforward exercise to rely on Boolean Algebra, Bayes's Theorem, and joint probability tables in extrapolating to Wolfram's cellular automata. Cellular automata are seen as purely deductive systems, just like classical logic, which probability theory is then able to generalize. Thus, any uncertainties which we might like to introduce into the discussion about cellular automata are handled with ease via the familiar inferential path. Most importantly, the difficult problem of predicting what cellular automata will do in the far future is treated like any inferential prediction problem.

Restructuring of Enterococcus faecalis biofilm architecture in response to antibiotic-induced stress

DOE PAGES

Dale, Jennifer L.; Nilson, Jennifer L.; Barnes, Aaron M. T.; ...

2017-06-30

Bacterial biofilms are intrinsically resistant to antimicrobial treatment, which contributes to microbial persistence in clinical infections. Enterococcus faecalis is an opportunistic pathogen that readily forms biofilms and is the most prevalent enterococcal species identified in healthcare-associated infections. Since intrinsic resistance to multiple antibiotics is common for enterococci, and antibiotic resistance is elevated in biofilm populations, it is imperative to understand the mechanisms involved. Previously, we identified two glycosyltransferase genes whose disruption resulted in impaired nascent biofilm formation in the presence of antibiotic concentrations subinhibitory for parent growth and biofilm formation. The glycosyltransferases are involved in synthesis of the cell-wall-associated rhamnopolysaccharidemore » Epa. Here we examined the effect of epa mutations on the temporal development of E. faecalis biofilms, and on the effects of antibiotics on pre-formed biofilms using scanning electron microscopy. We show that ΔepaOX mutant cells arrange into complex multidimensional biofilms independent of antibiotic exposure, while parent cells form biofilms that are monolayers in the absence of antibiotics. Remarkably, upon exposure to antibiotics parent biofilm cells restructure into complex three-dimensional biofilms resembling those of the ΔepaOX mutant without antibiotics. All biofilms exhibiting complex cellular architectures were less structurally stable than monolayer biofilms, with the biofilm cells exhibiting increased detachment. Our results indicate that E. faecalis biofilms restructure in response to cellular stress whether induced by antibiotics in the case of parent cells, or by deficiencies in Epa composition for the ΔepaOX strain. The data demonstrate a link between cellular architecture and antibiotic resistance of E. faecalis biofilms.« less

Cellular changes in tears associated with keratoconjunctival responses induced by nasal allergy.

PubMed

Pelikan, Z

2014-04-01

Allergic keratoconjunctivitis occurs in a primary form, caused by an allergic reaction localized in the conjunctiva, and in a secondary form, induced by an allergic reaction originating in the nasal mucosa. Various hypersensitivity mechanisms involved in the keratoconjunctivitis forms result in different keratoconjunctival response types. To investigate the cytologic changes in tears during the secondary immediate (SIKCR), late (SLKCR), and delayed (SDYKCR) keratoconjunctival responses. In 61 patients, comprising 20 SIKCRs, 23 SLKCRs, and 18 SDYKCRs, nasal provocation tests (NPTs) with allergens and 61 phosphate-buffered control challenges were repeated and supplemented with cell counting in the tears. The SIKCR (P<0.01), appearing 10-120 min after the NPT, was associated with increased eosinophil and mast cell counts in tears. The SLKCR (P<0.01), appearing 5-12 h after the NPT, was accompanied by increased counts of eosinophils, neutrophils, basophils, and conjunctival epithelial and goblet cells. The SDYKCR (P<0.05), appearing 24-48 h after NPT, was associated with increased counts of lymphocytes, neutrophils, monocytes, basophils, conjunctival epithelial, corneal epithelial and goblet cells. The SIKCR, SLKCR, and SDYKCR, induced by nasal allergy, were associated with different cellular profiles in the tears. The cells, except mast, epithelial and goblet cells, displaying no intracellular changes, migrated probably from the conjunctival capillaries, in response to the factors released during the primary allergic reaction in the nasal mucosa and subsequently penetrating into the conjunctiva. These results demonstrate a causal role of nasal allergy and diagnostic value of NPT combined with recording of ocular features and cellular profiles in tears in some keratoconjunctivitis patients.

Microtubule self-organisation by reaction-diffusion processes causes collective transport and organisation of cellular particles

PubMed Central

Glade, Nicolas; Demongeot, Jacques; Tabony, James

2004-01-01

Background The transport of intra-cellular particles by microtubules is a major biological function. Under appropriate in vitro conditions, microtubule preparations behave as a 'complex' system and show 'emergent' phenomena. In particular, they form dissipative structures that self-organise over macroscopic distances by a combination of reaction and diffusion. Results Here, we show that self-organisation also gives rise to a collective transport of colloidal particles along a specific direction. Particles, such as polystyrene beads, chromosomes, nuclei, and vesicles are carried at speeds of several microns per minute. The process also results in the macroscopic self-organisation of these particles. After self-organisation is completed, they show the same pattern of organisation as the microtubules. Numerical simulations of a population of growing and shrinking microtubules, incorporating experimentally realistic reaction dynamics, predict self-organisation. They forecast that during self-organisation, macroscopic parallel arrays of oriented microtubules form which cross the reaction space in successive waves. Such travelling waves are capable of transporting colloidal particles. The fact that in the simulations, the aligned arrays move along the same direction and at the same speed as the particles move, suggest that this process forms the underlying mechanism for the observed transport properties. Conclusions This process constitutes a novel physical chemical mechanism by which chemical energy is converted into collective transport of colloidal particles along a given direction. Self-organisation of this type provides a new mechanism by which intra cellular particles such as chromosomes and vesicles can be displaced and simultaneously organised by microtubules. It is plausible that processes of this type occur in vivo. PMID:15176973

Restructuring of Enterococcus faecalis biofilm architecture in response to antibiotic-induced stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dale, Jennifer L.; Nilson, Jennifer L.; Barnes, Aaron M. T.

Bacterial biofilms are intrinsically resistant to antimicrobial treatment, which contributes to microbial persistence in clinical infections. Enterococcus faecalis is an opportunistic pathogen that readily forms biofilms and is the most prevalent enterococcal species identified in healthcare-associated infections. Since intrinsic resistance to multiple antibiotics is common for enterococci, and antibiotic resistance is elevated in biofilm populations, it is imperative to understand the mechanisms involved. Previously, we identified two glycosyltransferase genes whose disruption resulted in impaired nascent biofilm formation in the presence of antibiotic concentrations subinhibitory for parent growth and biofilm formation. The glycosyltransferases are involved in synthesis of the cell-wall-associated rhamnopolysaccharidemore » Epa. Here we examined the effect of epa mutations on the temporal development of E. faecalis biofilms, and on the effects of antibiotics on pre-formed biofilms using scanning electron microscopy. We show that ΔepaOX mutant cells arrange into complex multidimensional biofilms independent of antibiotic exposure, while parent cells form biofilms that are monolayers in the absence of antibiotics. Remarkably, upon exposure to antibiotics parent biofilm cells restructure into complex three-dimensional biofilms resembling those of the ΔepaOX mutant without antibiotics. All biofilms exhibiting complex cellular architectures were less structurally stable than monolayer biofilms, with the biofilm cells exhibiting increased detachment. Our results indicate that E. faecalis biofilms restructure in response to cellular stress whether induced by antibiotics in the case of parent cells, or by deficiencies in Epa composition for the ΔepaOX strain. The data demonstrate a link between cellular architecture and antibiotic resistance of E. faecalis biofilms.« less

Protein Corona in Response to Flow: Effect on Protein Concentration and Structure.

PubMed

Jayaram, Dhanya T; Pustulka, Samantha M; Mannino, Robert G; Lam, Wilbur A; Payne, Christine K

2018-04-09

Nanoparticles used in cellular applications encounter free serum proteins that adsorb onto the surface of the nanoparticle, forming a protein corona. This protein layer controls the interaction of nanoparticles with cells. For nanomedicine applications, it is important to consider how intravenous injection and the subsequent shear flow will affect the protein corona. Our goal was to determine if shear flow changed the composition of the protein corona and if these changes affected cellular binding. Colorimetric assays of protein concentration and gel electrophoresis demonstrate that polystyrene nanoparticles subjected to flow have a greater concentration of serum proteins adsorbed on the surface, especially plasminogen. Plasminogen, in the absence of nanoparticles, undergoes changes in structure in response to flow, characterized by fluorescence and circular dichroism spectroscopy. The protein-nanoparticle complexes formed from fetal bovine serum after flow had decreased cellular binding, as measured with flow cytometry. In addition to the relevance for nanomedicine, these results also highlight the technical challenges of protein corona studies. The composition of the protein corona was highly dependent on the initial mixing step: rocking, vortexing, or flow. Overall, these results reaffirm the importance of the protein corona in nanoparticle-cell interactions and point toward the challenges of predicting corona composition based on nanoparticle properties. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A Programmable Cellular-Automata Polarized Dirac Vacuum

NASA Astrophysics Data System (ADS)

Osoroma, Drahcir S.

2013-09-01

We explore properties of a `Least Cosmological Unit' (LCU) as an inherent spacetime raster tiling or tessellating the unique backcloth of Holographic Anthropic Multiverse (HAM) cosmology as an array of programmable cellular automata. The HAM vacuum is a scale-invariant HD extension of a covariant polarized Dirac vacuum with `bumps' and `holes' typically described by extended electromagnetic theory corresponding to an Einstein energy-dependent spacetime metric admitting a periodic photon mass. The new cosmology incorporates a unique form of M-Theoretic Calabi-Yau-Poincaré Dodecadedral-AdS5-DS5space (PDS) with mirror symmetry best described by an HD extension of Cramer's Transactional Interpretation when integrated also with an HD extension of the de Broglie-Bohm-Vigier causal interpretation of quantum theory. We incorporate a unique form of large-scale additional dimensionality (LSXD) bearing some similarity to that conceived by Randall and Sundrum; and extend the fundamental basis of our model to the Unified Field, UF. A Sagnac Effect rf-pulsed incursive resonance hierarchy is utilized to manipulate and ballistically program the geometric-topological properties of this putative LSXD space-spacetime network. The model is empirically testable; and it is proposed that a variety of new technologies will arise from ballistic programming of tessellated LCU vacuum cellular automata.

Perfringolysin O Theta Toxin as a Tool to Monitor the Distribution and Inhomogeneity of Cholesterol in Cellular Membranes

PubMed Central

Maekawa, Masashi; Yang, Yanbo; Fairn, Gregory D.

2016-01-01

Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has crucial functions in membrane trafficking. Furthermore, cellular cholesterol homeostasis and regulation of de novo synthesis rely on transport via both vesicular and non-vesicular pathways. Thus, the ability to visualize and detect intracellular cholesterol, especially in the plasma membrane, is critical to understanding the complex biology associated with cholesterol and the nanodomains. Perfringolysin O (PFO) theta toxin is one of the toxins secreted by the anaerobic bacteria Clostridium perfringens and this toxin forms pores in the plasma membrane that causes cell lysis. It is well understood that PFO recognizes and binds to cholesterol in the exofacial leaflets of the plasma membrane, and domain 4 of PFO (D4) is sufficient for the binding of cholesterol. Recent studies have taken advantage of this high-affinity cholesterol-binding domain to create a variety of cholesterol biosensors by using a non-toxic PFO or the D4 in isolation. This review highlights the characteristics and usefulness of, and the principal findings related to, these PFO-derived cholesterol biosensors. PMID:27005662

Perfringolysin O Theta Toxin as a Tool to Monitor the Distribution and Inhomogeneity of Cholesterol in Cellular Membranes.

PubMed

Maekawa, Masashi; Yang, Yanbo; Fairn, Gregory D

2016-03-08

Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has crucial functions in membrane trafficking. Furthermore, cellular cholesterol homeostasis and regulation of de novo synthesis rely on transport via both vesicular and non-vesicular pathways. Thus, the ability to visualize and detect intracellular cholesterol, especially in the plasma membrane, is critical to understanding the complex biology associated with cholesterol and the nanodomains. Perfringolysin O (PFO) theta toxin is one of the toxins secreted by the anaerobic bacteria Clostridium perfringens and this toxin forms pores in the plasma membrane that causes cell lysis. It is well understood that PFO recognizes and binds to cholesterol in the exofacial leaflets of the plasma membrane, and domain 4 of PFO (D4) is sufficient for the binding of cholesterol. Recent studies have taken advantage of this high-affinity cholesterol-binding domain to create a variety of cholesterol biosensors by using a non-toxic PFO or the D4 in isolation. This review highlights the characteristics and usefulness of, and the principal findings related to, these PFO-derived cholesterol biosensors.

Aging and differentiation in yeast populations: elders with different properties and functions.

PubMed

Palková, Zdena; Wilkinson, Derek; Váchová, Libuše

2014-02-01

Over the past decade, it has become evident that similarly to cells forming metazoan tissues, yeast cells have the ability to differentiate and form specialized cell types. Examples of yeast cellular differentiation have been identified both in yeast liquid cultures and within multicellular structures occupying solid surfaces. Most current knowledge on different cell types comes from studies of the spatiotemporal internal architecture of colonies developing on various media. With a few exceptions, yeast cell differentiation often concerns nongrowing, stationary-phase cells and leads to the formation of cell subpopulations differing in stress resistance, cell metabolism, respiration, ROS production, and others. These differences can affect longevity of particular subpopulations. In contrast to liquid cultures, where various cell types are dispersed within stationary-phase populations, cellular differentiation depends on the specific position of particular cells within multicellular colonies. Differentiated colonies, thus, resemble primitive multicellular organisms, in which the gradients of certain compounds and the position of cells within the structure affect cellular differentiation. In this review, we summarize and compare the properties of diverse types of differentiated chronologically aging yeast cells that have been identified in colonies growing on different media, as well as of those found in liquid cultures. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Experimental verification of using nanostructured ceramic implants and osteograft

NASA Astrophysics Data System (ADS)

Rerikh, V. V.; Lastevskiy, A. D.; Sadovoy, M. A.; Zaidman, A. M.; Bataev, A. V.; Predein, Yu. A.; Avetisyan, A. R.; Romanenko, V. V.; Mamonova, E. V.; Nikulina, A. A.; Semantsova, E. S.; Smirnov, A. I.

2017-09-01

Ventral interbody fusion was carried out in 8 mini pigs in order to determine the effectiveness of anterior stabilization of the vertebral unit with implants made of nanostructured alumina ceramics using cellular technologies to form a bone block. A ceramic cage with a through cylindrical orifice in the center was implanted into the interbody gap; either cellular osteograft (group 1) or cellular autograft (group 2) was placed in it. The adjacent vertebrae were fixed anteriorly with a ceramic plate containing 4 screws. Bone block formation was studied radiographically, morphologically, and by MSCT. The signs of osteointegration of ceramic implants were observed in both groups after 90 days. MSCT and morphological analysis revealed the formation of the osteoceramic block and completed osteogenesis in the bone-graft contact region in group 1 compared to the control group (p < 0.05).

DNA Knots: Theory and Experiments

NASA Astrophysics Data System (ADS)

Sumners, D. W.

Cellular DNA is a long, thread-like molecule with remarkably complex topology. Enzymes that manipulate the geometry and topology of cellular DNA perform many vital cellular processes (including segregation of daughter chromosomes, gene regulation, DNA repair, and generation of antibody diversity). Some enzymes pass DNA through itself via enzyme-bridged transient breaks in the DNA; other enzymes break the DNA apart and reconnect it to different ends. In the topological approach to enzymology, circular DNA is incubated with an enzyme, producing an enzyme signature in the form of DNA knots and links. By observing the changes in DNA geometry (supercoiling) and topology (knotting and linking) due to enzyme action, the enzyme binding and mechanism can often be characterized. This paper will discuss some personal research history, and the tangle model for the analysis of site-specific recombination experiments on circular DNA.

Texturing Silicon Nanowires for Highly Localized Optical Modulation of Cellular Dynamics.

PubMed

Fang, Yin; Jiang, Yuanwen; Acaron Ledesma, Hector; Yi, Jaeseok; Gao, Xiang; Weiss, Dara E; Shi, Fengyuan; Tian, Bozhi

2018-06-18

Engineered silicon-based materials can display photoelectric and photothermal responses under light illumination, which may lead to further innovations at the silicon-biology interfaces. Silicon nanowires have small radial dimensions, promising as highly localized cellular modulators, however the single crystalline form typically has limited photothermal efficacy due to the poor light absorption and fast heat dissipation. In this work, we report strategies to improve the photothermal response from silicon nanowires by introducing nanoscale textures on the surface and in the bulk. We next demonstrate high-resolution extracellular modulation of calcium dynamics in a number of mammalian cells including glial cells, neurons, and cancer cells. The new materials may be broadly used in probing and modulating electrical and chemical signals at the subcellular length scale, which is currently a challenge in the field of electrophysiology or cellular engineering.

Process-morphology scaling relations quantify self-organization in capillary densified nanofiber arrays.

PubMed

Kaiser, Ashley L; Stein, Itai Y; Cui, Kehang; Wardle, Brian L

2018-02-07

Capillary-mediated densification is an inexpensive and versatile approach to tune the application-specific properties and packing morphology of bulk nanofiber (NF) arrays, such as aligned carbon nanotubes. While NF length governs elasto-capillary self-assembly, the geometry of cellular patterns formed by capillary densified NFs cannot be precisely predicted by existing theories. This originates from the recently quantified orders of magnitude lower than expected NF array effective axial elastic modulus (E), and here we show via parametric experimentation and modeling that E determines the width, area, and wall thickness of the resulting cellular pattern. Both experiments and models show that further tuning of the cellular pattern is possible by altering the NF-substrate adhesion strength, which could enable the broad use of this facile approach to predictably pattern NF arrays for high value applications.

Method and Apparatus for the Collection Storage and Real Time Analysis of Blood and Other Bodily Fluids

NASA Technical Reports Server (NTRS)

Whitson, Peggy A. (Inventor); Clift, Vaughan L. (Inventor)

1997-01-01

The present invention provides an apparatus for separating a relatively large volume of blood into cellular and acellular fractions without the need for centrifugation. The apparatus comprises a housing divided by a fibrous filter into a blood sample collection chamber having a volume of at least about 1 milliliter and a serum sample collection chamber. The fibrous filter has a pore size of less than about 3 microns, and is coated with a mixture of mannitol and plasma fraction protein (or an animal or vegetable equivalent thereof). The coating causes the cellular fraction to be trapped by the small pores, leaving the cellular fraction intact on the fibrous filter while the acellular fraction passes through the filter for collection in unaltered form from the serum sample collection chamber.

Heat shock cognate 71 (HSC71) regulates cellular antiviral response by impairing formation of VISA aggregates.

PubMed

Liu, Zhigang; Wu, Shu-Wen; Lei, Cao-Qi; Zhou, Qian; Li, Shu; Shu, Hong-Bing; Wang, Yan-Yi

2013-05-01

In response to viral infection, RIG-I-like RNA helicases detect viral RNA and signal through the mitochondrial adapter protein VISA. VISA activation leads to rapid activation of transcription factors IRF3 and NF-κB, which collaborate to induce transcription of type I interferon (IFN) genes and cellular antiviral response. It has been demonstrated that VISA is activated by forming prion-like aggregates. However, how this process is regulated remains unknown. Here we show that overexpression of HSC71 resulted in potent inhibition of virus-triggered transcription of IFNB1 gene and cellular antiviral response. Consistently, knockdown of HSC71 had opposite effects. HSC71 interacted with VISA, and negatively regulated virus-triggered VISA aggregation. These findings suggest that HSC71 functions as a check against VISA-mediated antiviral response.

Chemo-enzymatic synthesis of isotopically labeled nicotinamide riboside.

PubMed

Tran, Ai; Yokose, Ryota; Cen, Yana

2018-05-15

As a cofactor for numerous reactions, NAD+ is found widely dispersed across many maps of cellular metabolism. This core redox role alone makes the biosynthesis of NAD+ of great interest. Recent studies have revealed new biological roles for NAD+ as a substrate for diverse enzymes that regulate a broad spectrum of key cellular tasks. These NAD+-consuming enzymes further highlight the importance of understanding NAD+ biosynthetic pathways. In this study, we developed a chemo-enzymatic synthesis of isotopically labeled NAD+ precursor, nicotinamide riboside (NR). The synthesis of NR isotopomers allowed us to unambiguously determine that NR is efficiently converted to NAD+ in the cellular environment independent of degradation to nicotinamide, and it is incorporated into NAD+ in its intact form. The versatile synthetic method along with the isotopically labeled NRs will provide powerful tools to further decipher the important yet complicated NAD+ metabolism.

Propulsion and navigation within the advancing monolayer sheet

PubMed Central

Kim, Jae Hun; Serra-Picamal, Xavier; Tambe, Dhananjay T.; Zhou, Enhua H.; Park, Chan Young; Sadati, Monirosadat; Park, Jin-Ah; Krishnan, Ramaswamy; Gweon, Bomi; Millet, Emil; Butler, James P.; Trepat, Xavier; Fredberg, Jeffrey J.

2013-01-01

As a wound heals, or a body plan forms, or a tumor invades, observed cellular motions within the advancing cell swarm are thought to stem from yet to be observed physical stresses that act in some direct and causal mechanical fashion. Here we show that such a relationship between motion and stress is far from direct. Using monolayer stress microscopy, we probed migration velocities, cellular tractions and intercellular stresses in an epithelial cell sheet advancing towards an island on which cells cannot adhere. We found that cells located near the island exert tractions that pull systematically towards this island regardless of whether the cells approach the island, migrate tangentially along its edge or, paradoxically, recede from it. This unanticipated cell-patterning motif, which we call kenotaxis, represents the robust and systematic mechanical drive of the cellular collective to fill unfilled space. PMID:23793160

Impaired activity of CCA-adding enzyme TRNT1 impacts OXPHOS complexes and cellular respiration in SIFD patient-derived fibroblasts.

PubMed

Liwak-Muir, Urszula; Mamady, Hapsatou; Naas, Turaya; Wylie, Quinlan; McBride, Skye; Lines, Matthew; Michaud, Jean; Baird, Stephen D; Chakraborty, Pranesh K; Holcik, Martin

2016-06-18

SIFD (Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay) is a novel form of congenital sideroblastic anemia associated with B-cell immunodeficiency, periodic fevers, and developmental delay caused by mutations in the CCA-adding enzyme TRNT1, but the precise molecular pathophysiology is not known. We show that the disease causing mutations in patient-derived fibroblasts do not affect subcellular localization of TRNT1 and show no gross morphological differences when compared to control cells. Analysis of cellular respiration and oxidative phosphorylation (OXPHOS) complexes demonstrates that both basal and maximal respiration rates are decreased in patient cells, which may be attributed to an observed decrease in the abundance of select proteins of the OXPHOS complexes. Our data provides further insight into cellular pathophysiology of SIFD.

Assessment of cellular materials generated by co-cultured 'inflamed' and healthy periodontal ligament stem cells from patient-matched groups.

PubMed

Tang, Hao-Ning; Xia, Yu; Xu, Jie; Tian, Bei-Min; Zhang, Xi-Yu; Chen, Fa-Ming

2016-08-01

Recently, stem cells derived from the'inflamed' periodontal ligament (PDL) tissue of periodontally diseased teeth (I-PDLSCs) have been increasingly suggested as a more readily accessible source of cells for regenerative therapies than those derived from healthy PDL tissue (H-PDLSCs). However, substantial evidence indicates that I-PDLSCs exhibit impaired functionalities compared with H-PDLSCs. In this study, patient-matched I-PDLSCs and H-PDLSCs were co-cultured at various ratios. Cellular materials derived from these cultures were investigated regarding their osteogenic potential in vitro and capacity to form new bone following in vivo transplantation. While patient-matched I-PDLSCs and H-PDLSCs could co-exist in co-culture systems, the proportion of I-PDLSCs tended to increase during in vitro incubation. Compared with H-PDLSC monoculture, the presence of I-PDLSCs in the co-cultures appeared to enhance the overall cell proliferation. Although not completely rescued, the osteogenic and regenerative potentials of the cellular materials generated by co-cultured I-PDLSCs and H-PDLSCs were significantly improved compared with those derived from I-PDLSC monocultures. Notably, cells in co-cultures containing either 50% I-PDLSCs plus 50% H-PDLSCs or 25% I-PDLSCs plus 75% H-PDLSCs expressed osteogenesis-related proteins and genes at levels similar to those expressed in H-PDLSC monocultures (P>0.05). Irrespective of the percentage of I-PDLSCs, robust cellular materials were obtained from co-cultures with 50% or more H-PDLSCs, which exhibited equivalent potential to form new bone in vivo compared with sheets generated by H-PDLSC monocultures. These data suggest that the co-culture of I-PDLSCs with patient-matched H-PDLSCs is a practical and effective method for increasing the overall osteogenic and regenerative potentials of resultant cellular materials. Copyright © 2016 Elsevier Inc. All rights reserved.

Methylation-based classification of benign and malignant peripheral nerve sheath tumors.

PubMed

Röhrich, Manuel; Koelsche, Christian; Schrimpf, Daniel; Capper, David; Sahm, Felix; Kratz, Annekathrin; Reuss, Jana; Hovestadt, Volker; Jones, David T W; Bewerunge-Hudler, Melanie; Becker, Albert; Weis, Joachim; Mawrin, Christian; Mittelbronn, Michel; Perry, Arie; Mautner, Victor-Felix; Mechtersheimer, Gunhild; Hartmann, Christian; Okuducu, Ali Fuat; Arp, Mirko; Seiz-Rosenhagen, Marcel; Hänggi, Daniel; Heim, Stefanie; Paulus, Werner; Schittenhelm, Jens; Ahmadi, Rezvan; Herold-Mende, Christel; Unterberg, Andreas; Pfister, Stefan M; von Deimling, Andreas; Reuss, David E

2016-06-01

The vast majority of peripheral nerve sheath tumors derive from the Schwann cell lineage and comprise diverse histological entities ranging from benign schwannomas and neurofibromas to high-grade malignant peripheral nerve sheath tumors (MPNST), each with several variants. There is increasing evidence for methylation profiling being able to delineate biologically relevant tumor groups even within the same cellular lineage. Therefore, we used DNA methylation arrays for methylome- and chromosomal profile-based characterization of 171 peripheral nerve sheath tumors. We analyzed 28 conventional high-grade MPNST, three malignant Triton tumors, six low-grade MPNST, four epithelioid MPNST, 33 neurofibromas (15 dermal, 8 intraneural, 10 plexiform), six atypical neurofibromas, 43 schwannomas (including 5 NF2 and 5 schwannomatosis associated cases), 11 cellular schwannomas, 10 melanotic schwannomas, 7 neurofibroma/schwannoma hybrid tumors, 10 nerve sheath myxomas and 10 ganglioneuromas. Schwannomas formed different epigenomic subgroups including a vestibular schwannoma subgroup. Cellular schwannomas were not distinct from conventional schwannomas. Nerve sheath myxomas and neurofibroma/schwannoma hybrid tumors were most similar to schwannomas. Dermal, intraneural and plexiform neurofibromas as well as ganglioneuromas all showed distinct methylation profiles. Atypical neurofibromas and low-grade MPNST were indistinguishable with a common methylation profile and frequent losses of CDKN2A. Epigenomic analysis finds two groups of conventional high-grade MPNST sharing a frequent loss of neurofibromin. The larger of the two groups shows an additional loss of trimethylation of histone H3 at lysine 27 (H3K27me3). The smaller one retains H3K27me3 and is found in spinal locations. Sporadic MPNST with retained neurofibromin expression did not form an epigenetic group and most cases could be reclassified as cellular schwannomas or soft tissue sarcomas. Widespread immunohistochemical loss of H3K27me3 was exclusively seen in MPNST of the main methylation cluster, which defines it as an additional useful marker for the differentiation of cellular schwannoma and MPNST.

The small GTPase Arf6 regulates sea urchin morphogenesis

PubMed Central

Stepicheva, Nadezda A.; Dumas, Megan; Kobi, Priscilla; Donaldson, Julie G.; Song, Jia L.

2017-01-01

The small GTPase Arf6 is a conserved protein that is expressed in all metazoans. Arf6 remodels cytoskeletal actin and mediates membrane protein trafficking between the plasma membrane in its active form and endosomal compartments in its inactive form. While a rich knowledge exists for the cellular functions of Arf6, relatively little is known about its physiological role in development. This study examines the function of Arf6 in mediating cellular morphogenesis in early development. We dissect the function of Arf6 with a loss-of-function morpholino and constitutively active Arf6-Q67L construct. We focus on the two cell types that undergo active directed migration: the primary mesenchyme cells (PMCs) that give rise to the sea urchin skeleton and endodermal cells that form the gut. Our results indicate that Arf6 plays an important role in skeleton formation and PMC migration, in part due to its ability to remodel actin. We also found that embryos injected with Arf6 morpholino have gastrulation defects and embryos injected with constitutively active Arf6 have endodermal cells detached from the gut epithelium with decreased junctional cadherin staining, indicating that Arf6 may mediate the recycling of cadherin. Thus, Arf6 impacts cells that undergo coordinated movement to form embryonic structures in the developing embryo. PMID:28188999

Yeast as a model system to study metabolic impact of selenium compounds

PubMed Central

Herrero, Enrique; Wellinger, Ralf E.

2015-01-01

Inorganic Se forms such as selenate or selenite (the two more abundant forms in nature) can be toxic in Saccharomyces cerevisiae cells, which constitute an adequate model to study such toxicity at the molecular level and the functions participating in protection against Se compounds. Those Se forms enter the yeast cell through other oxyanion transporters. Once inside the cell, inorganic Se forms may be converted into selenide through a reductive pathway that in physiological conditions involves reduced glutathione with its consequent oxidation into diglutathione and alteration of the cellular redox buffering capacity. Selenide can subsequently be converted by molecular oxygen into elemental Se, with production of superoxide anions and other reactive oxygen species. Overall, these events result in DNA damage and dose-dependent reversible or irreversible protein oxidation, although additional oxidation of other cellular macromolecules cannot be discarded. Stress-adaptation pathways are essential for efficient Se detoxification, while activation of DNA damage checkpoint and repair pathways protects against Se-mediated genotoxicity. We propose that yeast may be used to improve our knowledge on the impact of Se on metal homeostasis, the identification of Se-targets at the DNA and protein levels, and to gain more insights into the mechanism of Se-mediated apoptosis. PMID:28357286

Genetics Home Reference: lysinuric protein intolerance

MedlinePlus

... abnormally large amount of these amino acids in urine. A shortage of lysine, arginine, and ornithine disrupts many vital functions. Arginine and ornithine are involved in a cellular process called the urea cycle, which processes excess nitrogen (in the form ...

Up to Four Distinct Polypeptides Are Produced from the γ34.5 Open Reading Frame of Herpes Simplex Virus 2

PubMed Central

Korom, Maria; Davis, Katie L.

2014-01-01

ABSTRACT The herpes simplex virus 1 (HSV-1) ICP34.5 protein strongly influences neurovirulence and regulates several cellular antiviral responses. Despite the clinical importance of HSV-2, relatively little is known about its ICP34.5 ortholog. We found that HSV-2 produces up to four distinct forms of ICP34.5 in infected cells: a full-length protein, one shorter form sharing the N terminus, and two shorter forms sharing the C terminus. These forms appeared with similar kinetics and accumulated in cells over much of the replication cycle. We confirmed that the N-terminal form is translated from the primary unspliced transcript to a stop codon within the intron unique to HSV-2 γ34.5. We found that the N-terminal form was produced in a variety of cell types and by 9 of 10 clinical isolates. ICP27 influenced but was not required for expression of the N-terminal form. Western blotting and reverse transcription-PCR indicated the C-terminal forms did not contain the N terminus and were not products of alternative splicing or internal transcript initiation. Expression plasmids encoding methionine at amino acids 56 and 70 generated products that comigrated in SDS-PAGE with the C1 and C2 forms, respectively, and mutation of these sites abolished C1 and C2. Using a recombinant HSV-2 encoding hemagglutinin (HA)-tagged ICP34.5, we demonstrated that the C-terminal forms were also produced during infection of many human and mouse cell types but were not detectable in mouse primary neurons. The protein diversity generated from the HSV-2 γ34.5 open reading frame implies additional layers of cellular regulation through potential independent activities associated with the various forms of ICP34.5. IMPORTANCE The herpes simplex virus 1 (HSV-1) protein ICP34.5, encoded by the γ34.5 gene, interferes with several host defense mechanisms by binding cellular proteins that would otherwise stimulate the cell's autophagic, translational-arrest, and type I interferon responses to virus infection. ICP34.5 also plays a crucial role in determining the severity of nervous system infections with HSV-1 and HSV-2. The HSV-2 γ34.5 gene contains an intron not present in HSV-1 γ34.5. A shorter N-terminal form of HSV-2 ICP34.5 can be translated from the unspliced γ34.5 mRNA. Here, we show that two additional forms consisting of the C-terminal portion of ICP34.5 are generated in infected cells. Production of these N- and C-terminal forms is highly conserved among HSV-2 strains, including many clinical isolates, and they are broadly expressed in several cell types, but not mouse primary neurons. Multiple ICP34.5 polypeptides add additional complexity to potential functional interactions influencing HSV-2 neurovirulence. PMID:25031346

Impact of Environmental Conditions on the Form and Function of Candida albicans Biofilms

PubMed Central

Daniels, Karla J.; Park, Yang-Nim; Srikantha, Thyagarajan; Pujol, Claude

2013-01-01

Candida albicans, like other pathogens, can form complex biofilms on a variety of substrates. However, as the number of studies of gene regulation, architecture, and pathogenic traits of C. albicans biofilms has increased, so have differences in results. This suggests that depending upon the conditions employed, biofilms may vary widely, thus hampering attempts at a uniform description. Gene expression studies suggest that this may be the case. To explore this hypothesis further, we compared the architectures and traits of biofilms formed in RPMI 1640 and Spider media at 37°C in air. Biofilms formed by a/α cells in the two media differed to various degrees in cellular architecture, matrix deposition, penetrability by leukocytes, fluconazole susceptibility, and the facilitation of mating. Similar comparisons of a/a cells in the two media, however, were made difficult given that in air, although a/a cells form traditional biofilms in RPMI medium, they form polylayers composed primarily of yeast cells in Spider medium. These polylayers lack an upper hyphal/matrix region, are readily penetrated by leukocytes, are highly fluconazole susceptible, and do not facilitate mating. If, however, air is replaced with 20% CO2, a/a cells make a biofilm in Spider medium similar architecturally to that of a/α cells, which facilitates mating. A second, more cursory comparison is made between the disparate cellular architectures of a/a biofilms formed in air in RPMI and Lee's media. The results demonstrate that C. albicans forms very different types of biofilms depending upon the composition of the medium, level of CO2 in the atmosphere, and configuration of the MTL locus. PMID:23954841

Application of “omics” to Prion Biomarker Discovery

PubMed Central

Huzarewich, Rhiannon L. C. H.; Siemens, Christine G.; Booth, Stephanie A.

2010-01-01

The advent of genomics and proteomics has been a catalyst for the discovery of biomarkers able to discriminate biological processes such as the pathogenesis of complex diseases. Prompt detection of prion diseases is particularly desirable given their transmissibility, which is responsible for a number of human health risks stemming from exogenous sources of prion protein. Diagnosis relies on the ability to detect the biomarker PrPSc, a pathological isoform of the host protein PrPC, which is an essential component of the infectious prion. Immunochemical detection of PrPSc is specific and sensitive enough for antemortem testing of brain tissue, however, this is not the case in accessible biological fluids or for the detection of recently identified novel prions with unique biochemical properties. A complementary approach to the detection of PrPSc itself is to identify alternative, “surrogate” gene or protein biomarkers indicative of disease. Biomarkers are also useful to track the progress of disease, especially important in the assessment of therapies, or to identify individuals “at risk”. In this review we provide perspective on current progress and pitfalls in the use of “omics” technologies to screen body fluids and tissues for biomarker discovery in prion diseases. PMID:20224650

Sex effects in mouse prion disease incubation time.

PubMed

Akhtar, Shaheen; Wenborn, Adam; Brandner, Sebastian; Collinge, John; Lloyd, Sarah E

2011-01-01

Prion disease incubation time in mice is determined by many factors including PrP expression level, Prnp alleles, genetic background, prion strain and route of inoculation. Sex differences have been described in age of onset for vCJD and in disease duration for both vCJD and sporadic CJD and have also been shown in experimental models. The sex effects reported for mouse incubation times are often contradictory and detail only one strain of mice or prions, resulting in broad generalisations and a confusing picture. To clarify the effect of sex on prion disease incubation time in mice we have compared male and female transmission data from twelve different inbred lines of mice inoculated with at least two prion strains, representing both mouse-adapted scrapie and BSE. Our data show that sex can have a highly significant difference on incubation time. However, this is limited to particular mouse and prion strain combinations. No sex differences were seen in endogenous PrP(C) levels nor in the neuropathological markers of prion disease: PrP(Sc) distribution, spongiosis, neuronal loss and gliosis. These data suggest that when comparing incubation times between experimental groups, such as testing the effects of modifier genes or therapeutics, single sex groups should be used.

Live imaging of prions reveals nascent PrPSc in cell-surface, raft-associated amyloid strings and webs

PubMed Central

Rouvinski, Alexander; Karniely, Sharon; Kounin, Maria; Moussa, Sanaa; Goldberg, Miri D.; Warburg, Gabriela; Lyakhovetsky, Roman; Papy-Garcia, Dulce; Kutzsche, Janine; Korth, Carsten; Carlson, George A.; Godsave, Susan F.; Peters, Peter J.; Luhr, Katarina; Kristensson, Krister

2014-01-01

Mammalian prions refold host glycosylphosphatidylinositol-anchored PrPC into β-sheet–rich PrPSc. PrPSc is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrPSc rather than on its truncated PrP27-30 product. We show that N-terminal PrPSc epitopes are exposed in their physiological context and visualize, for the first time, PrPSc in living cells. PrPSc resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrPSc amyloids. PMID:24493590

A crucial role for B cells in neuroinvasive scrapie.

PubMed

Brandner, S; Klein, M A; Aguzzi, A

1999-02-01

Although prions are most efficiently propagated via intracerebral inoculation, peripheral administration has caused kuru [Gajdusek et al, 1966], iatrogenic Creutzfeldt-Jakob disease (CJD) [Gibbs et al, 1997], bovine spongiform encephalitis (BSE), and new variant CJD [Hill et al, 1997; Bruce et al, 1997]. Neurological disease after peripheral inoculation depends on prion expansion within cells of the lymphoreticular system (LRS) [Lasmezas et al. 1996; Wilesmith et al, 1992]. In order to identify the nature of the latter cells, we inoculated a panel of immune deficient mice with prions intraperitoneally. While defects affecting only T lymphocytes had no apparent effect, all mutations affecting differentiation and responses of B lymphocytes prevented development of clinical scrapie. Since absence of B cells and of antibodies correlates with severe defects in follicular dendritic cells (FDCs), the lack of any of these three components may prevent clinical scrapie. Yet, mice expressing immunoglobulins exclusively of the M subclass without detectable specificity for PrPc, and mice with differentiated B cells but lacking functional FDCs, developed scrapie after peripheral inoculation: therefore, differentiated B cells appear to play a crucial role in neuroinvasion of scrapie regardless of B-cell receptor specificity.

Unique Cellular Organization in the Oldest Root Meristem.

PubMed

Hetherington, Alexander J; Dubrovsky, Joseph G; Dolan, Liam

2016-06-20

Roots and shoots of plant bodies develop from meristems-cell populations that self-renew and produce cells that undergo differentiation-located at the apices of axes [1].The oldest preserved root apices in which cellular anatomy can be imaged are found in nodules of permineralized fossil soils called coal balls [2], which formed in the Carboniferous coal swamp forests over 300 million years ago [3-9]. However, no fossil root apices described to date were actively growing at the time of preservation [3-10]. Because the cellular organization of meristems changes when root growth stops, it has been impossible to compare cellular dynamics as stem cells transition to differentiated cells in extinct and extant taxa [11]. We predicted that meristems of actively growing roots would be preserved in coal balls. Here we report the discovery of the first fossilized remains of an actively growing root meristem from permineralized Carboniferous soil with detail of the stem cells and differentiating cells preserved. The cellular organization of the meristem is unique. The position of the Körper-Kappe boundary, discrete root cap, and presence of many anticlinal cell divisions within a broad promeristem distinguish it from all other known root meristems. This discovery is important because it demonstrates that the same general cellular dynamics are conserved between the oldest extinct and extant root meristems. However, its unique cellular organization demonstrates that extant root meristem organization and development represents only a subset of the diversity that has existed since roots first evolved. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Piezoelectricity and ferroelectricity of cellular polypropylene electrets films characterized by piezoresponse force microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miao, Hongchen; Sun, Yao; Zhou, Xilong

Cellular electrets polymer is a new ferroelectret material exhibiting large piezoelectricity and has attracted considerable attentions in researches and industries. Property characterization is very important for this material and current investigations are mostly on macroscopic properties. In this work, we conduct nanoscale piezoelectric and ferroelectric characterizations of cellular polypropylene (PP) films using piezoresponse force microscopy (PFM). First, both the single-frequency PFM and dual-frequency resonance-tracking PFM testings were conducted on the cellular PP film. The localized piezoelectric constant d{sub 33} is estimated to be 7–11pC/N by correcting the resonance magnification with quality factor and it is about one order lower thanmore » the macroscopic value. Next, using the switching spectroscopy PFM (SS-PFM), we studied polarization switching behavior of the cellular PP films. Results show that it exhibits the typical ferroelectric-like phase hysteresis loops and butterfly-shaped amplitude loops, which is similar to that of a poly(vinylidene fluoride) (PVDF) ferroelectric polymer film. However, both the phase and amplitude loops of the PP film are intensively asymmetric, which is thought to be caused by the nonzero remnant polarization after poling. Then, the D-E hysteresis loops of both the cellular PP film and PVDF film were measured by using the same wave form as that used in the SS-PFM, and the results show significant differences. Finally, we suggest that the ferroelectric-like behavior of cellular electrets films should be distinguished from that of typical ferroelectrics, both macroscopically and microscopically.« less

Intelligent call admission control for multi-class services in mobile cellular networks

NASA Astrophysics Data System (ADS)

Ma, Yufeng; Hu, Xiulin; Zhang, Yunyu

2005-11-01

Scarcity of the spectrum resource and mobility of users make quality of service (QoS) provision a critical issue in mobile cellular networks. This paper presents a fuzzy call admission control scheme to meet the requirement of the QoS. A performance measure is formed as a weighted linear function of new call and handoff call blocking probabilities of each service class. Simulation compares the proposed fuzzy scheme with complete sharing and guard channel policies. Simulation results show that fuzzy scheme has a better robust performance in terms of average blocking criterion.

Investigation of Molecular Mechanism of JC virus Viroporin Activity.

PubMed

Suzuki, Tadaki

2015-01-01

Viroporins are small and hydrophobic viral proteins that form pores on host cell membranes, and their expression can increase the permeability of cellular membranes and the production of progeny virus particles. JC virus (JCV) is the causative agent of progressive multifocal leukoenchephalopathy (PML). We demonstrate that JCV Agno, which is the small and hydrophobic protein, andincreases the plasma membrane permeability and virion release, acts as a viroporin. We also demonstrate that an interaction of Agno with a host cellular protein regulates the viroporin activity of Agno. These findings indicate a new paradigm in virus-host interactions regulating viroporin activity and viral replication.

Environmental phototoxicity: Solar ultraviolet radiation affects the toxicity of natural and man-made chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larson, R.A.; Berenbaum, M.R.

1988-04-01

Ultraviolet radiation appears to be toxic to all forms of unpigmented living cells, including bacteria, protozoa, nematodes, arthropods, fish, birds, and mammals. In addition to the direct absorption of solar energy by cellular constituents, toxicity may occur because of the absorption of sunlight by xenobiotics (or by naturally occurring compounds outside the target cell); these may be converted by light or by subsequent light-promoted reactions that induce cellular damage. This article describes the phototoxicity of photodynamic dyes, light-activated synthetic herbicides, petroleum and its constituents, and naturally occurring chemicals from plants. Detoxification mechanisms are also discussed.

Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina.

PubMed

Muranyi, Walter; Haas, Jürgen; Wagner, Markus; Krohne, Georg; Koszinowski, Ulrich H

2002-08-02

The passage of large-sized herpesviral capsids through the nuclear lamina and the inner nuclear membrane to leave the nucleus requires a dissolution of the nuclear lamina. Here, we report on the functions of M50/p35, a beta-herpesviral protein of murine cytomegalovirus. M50/p35 inserts into the inner nuclear membrane and is aggregated by a second viral protein, M53/p38, to form the capsid docking site. M50/p35 recruits the cellular protein kinase C for phosphorylation and dissolution of the nuclear lamina, suggesting that herpesviruses target a critical element of nuclear architecture.

A Conspectus of Cellular Mechanisms of Nitrosothiol Formation from Nitric Oxide

PubMed Central

Li, Qian; Lancaster, Jack R.

2013-01-01

Although chemical mechanisms for the formation of nitrosothiol from •NO have been studied extensively “in the test tube”, surprisingly little is known regarding the mechanism(s) of how nitrosothiols are formed in vivo. This lack of understanding has hampered more general acceptance of the concept of cysteine nitrosothiol formation as a generally applicable, regulated, and functionally significant protein posttranslational modification (as opposed to multiple other •NO-induced thiol modifications). Here we provide a brief overview/summary of the cellular formation of nitrosothiols from •NO via two possible mechanisms involving oxygen or transition metals. PMID:23503678

The Synaptic Function of α-Synuclein

PubMed Central

Burré, Jacqueline

2015-01-01

α-Synuclein is an abundant neuronal protein which localizes predominantly to presynaptic terminals, and is strongly linked genetically and pathologically to Parkinson’s disease and other neurodegenerative diseases. While the accumulation of α-synuclein in the form of misfolded oligomers and large aggregates defines multiple neurodegenerative diseases called “synucleinopathies”, its cellular function has remained largely unclear, and is the subject of intense investigation. In this review, I focus on the structural characteristics of α-synuclein, its cellular and subcellular localization, and discuss how this relates to its function in neurons, in particular at the neuronal synapse. PMID:26407041

A Fork in the Road: The Effects of Different Cellular Pathways on Melanoma | Center for Cancer Research

Cancer.gov

Malignant melanoma is one of the most deadly forms of cancer because of its high capacity to metastasize and because there are few treatments effective in stopping its progression. The extensive body of research on melanoma has identified several important protein mutations that contribute to development of the disease. One of these proteins, Ras, is mutated in 25 percent of cutaneous malignant melanomas. These mutations disrupt Ras regulation, switching the protein permanently "on," leading to constant signaling of cellular messengers and stimulating growth without normal checks and balances.

Spatiotemporal dynamics of oscillatory cellular patterns in three-dimensional directional solidification.

PubMed

Bergeon, N; Tourret, D; Chen, L; Debierre, J-M; Guérin, R; Ramirez, A; Billia, B; Karma, A; Trivedi, R

2013-05-31

We report results of directional solidification experiments conducted on board the International Space Station and quantitative phase-field modeling of those experiments. The experiments image for the first time in situ the spatially extended dynamics of three-dimensional cellular array patterns formed under microgravity conditions where fluid flow is suppressed. Experiments and phase-field simulations reveal the existence of oscillatory breathing modes with time periods of several 10's of minutes. Oscillating cells are usually noncoherent due to array disorder, with the exception of small areas where the array structure is regular and stable.

Effects of endoplasmic reticulum stressors on maturation and signaling of hemizygous and heterozygous wild-type and mutant forms of KIT.

PubMed

Brahimi-Adouane, Sabrina; Bachet, Jean-Baptiste; Tabone-Eglinger, Séverine; Subra, Frédéric; Capron, Claude; Blay, Jean-Yves; Emile, Jean-François

2013-06-01

Gain of function mutations of KIT are frequent in some human tumors, and are sensible to tyrosine kinase inhibitors. In most tumors, oncogenic mutations are heterozygous, however most in vitro data of KIT activation have been obtained with hemizygous mutation. This study aimed to investigate the maturation and activation of wild-type (WT) and mutant (M) forms of KIT in hemizygous and heterozygous conditions. WT and two types of exon 11 deletions M forms of human KIT were expressed in NIH3T3 cell lines. Membrane expression of KIT was quantified by flow cytometry. Quantification of glycosylated forms of KIT and phosphorylated forms of AKT and ERK were performed by western blot. Simultaneous activation of WT KIT and treatment with endoplasmic reticulum (ER) inhibitors, tunicamycin or brefeldin A induced a complete inhibition of membrane expression of the 145 kDa form of KIT. By contrast activation or ER inhibitors alone, only partly inhibited this form. ER inhibitors also inhibited KIT activation-dependent phosphorylation of AKT and ERK1/2. Brefeldin A induced a complete down regulation of the 145 kDa form in hemizygous M, and induced an intra-cellular accumulation of the 125 kDa form in WT but not in hemizygous M. Heterozygous cells had glycosylation and response to ER inhibitors patterns more similar to WT than to hemizygous M. Phosphorylated AKT was reduced in hemizygous cells in comparison to WT KIT cells and heterozygous cells, and in the presence of brefeldin A in all cell lines. Effects of ER inhibitors are significantly different in hemizygous and heterozygous mutants. Differences in intra-cellular trafficking of KIT forms result in differences in downstream signaling pathways, and activation of PI3K/AKT pathway appears to be tied to the presence of the mature 145 kDa form of KIT at the membrane surface. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Disorders of lysosomal acidification - the emerging role of v-ATPase in aging and neurodegenerative disease

PubMed Central

Colacurcio, Daniel J.; Nixon, Ralph A.

2016-01-01

Autophagy and endocytosis deliver unneeded cellular materials to lysosomes for degradation. Beyond processing cellular waste, lysosomes release metabolites and ions that serve signaling and nutrient sensing roles, linking the functions of the lysosome to various pathways for intracellular metabolism and nutrient homeostasis. Each of these lysosomal behaviors is influenced by the intraluminal pH of the lysosome, which is maintained in the low acidic range by a proton pump, the vacuolar ATPase (v-ATPase). New reports implicate altered v-ATPase activity and lysosomal pH dysregulation in cellular aging, longevity, and adult-onset neurodegenerative diseases, including forms of Parkinson Disease and Alzheimer Disease. Genetic defects of subunits composing the v-ATPase or v-ATPase-related proteins occur in an increasingly recognized group of familial neurodegenerative diseases. Here, we review the expanding roles of the v-ATPase complex as a platform regulating lysosomal proteolysis and cellular homeostasis. We discuss the unique vulnerability of neurons to persistent low level lysosomal dysfunction and review recent clinical and experimental studies that link dysfunction of the v-ATPase complex to neurodegenerative diseases across the age spectrum. PMID:27197071

Targeting Virus-host Interactions of HIV Replication.

PubMed

Weydert, Caroline; De Rijck, Jan; Christ, Frauke; Debyser, Zeger

2016-01-01

Cellular proteins that are hijacked by HIV in order to complete its replication cycle, form attractive new targets for antiretroviral therapy. In particular, the protein-protein interactions between these cellular proteins (cofactors) and viral proteins are of great interest to develop new therapies. Research efforts have led to the validation of different cofactors and some successes in therapeutic applications. Maraviroc, the first cofactor inhibitor approved for human medicinal use, provided a proof of concept. Furthermore, compounds developed as Integrase-LEDGF/p75 interaction inhibitors (LEDGINs) have advanced to early clinical trials. Other compounds targeting cofactors and cofactor-viral protein interactions are currently under development. Likewise, interactions between cellular restriction factors and their counteracting HIV protein might serve as interesting targets in order to impair HIV replication. In this respect, compounds targeting the Vif-APOBEC3G interaction have been described. In this review, we focus on compounds targeting the Integrase- LEDGF/p75 interaction, the Tat-P-TEFb interaction and the Vif-APOBEC3G interaction. Additionally we give an overview of currently discovered compounds presumably targeting cellular cofactor-HIV protein interactions.

Identification and characterization of a novel ISG15-ubiquitin mixed chain and its role in regulating protein homeostasis

PubMed Central

Fan, Jun-Bao; Arimoto, Kei-lchiro; Motamedchaboki, Khatereh; Yan, Ming; Wolf, Dieter A.; Zhang, Dong-Er

2015-01-01

As a ubiquitin-like modifier, ISG15 is conjugated to many cellular proteins in a process termed protein ISGylation. However, the crosstalk between protein ISGylation and the ubiquitin proteasome system is not fully understood. Here, we report that cellular ubiquitin is a substrate of ISG15 and Lys 29 on ubiquitin is the major ISG15 acceptor site. Using a model substrate, we demonstrate that ISG15 can modify ubiquitin, which is immobilized on its substrate, to form ISG15-ubiquitin mixed chains. Furthermore, our results indicate that ISG15-ubiquitin mixed chains do not serve as degradation signals for a ubiquitin fusion degradation substrate. Accordingly, an ISG15-ubiquitin fusion protein, which mimics an ISG15-ubiquitin mixed chain, negatively regulates cellular turnover of ubiquitylated proteins. In addition, ISG15-ubiquitin mixed chains, which are detectable on endogenously ubiquitylated proteins, dampen cellular turnover of these proteins. Thus, our studies unveil an unanticipated interplay between two protein modification systems and highlight its role in coordinating protein homeostasis. PMID:26226047

Effect of alternate energy substrates on mammalian brain metabolism during ischemic events.

PubMed

Koppaka, S S; Puchowicz; LaManna, J C; Gatica, J E

2008-01-01

Regulation of brain metabolism and cerebral blood flow involves complex control systems with several interacting variables at both cellular and organ levels. Quantitative understanding of the spatially and temporally heterogeneous brain control mechanisms during internal and external stimuli requires the development and validation of a computational (mathematical) model of metabolic processes in brain. This paper describes a computational model of cellular metabolism in blood-perfused brain tissue, which considers the astrocyte-neuron lactate-shuttle (ANLS) hypothesis. The model structure consists of neurons, astrocytes, extra-cellular space, and a surrounding capillary network. Each cell is further compartmentalized into cytosol and mitochondria. Inter-compartment interaction is accounted in the form of passive and carrier-mediated transport. Our model was validated against experimental data reported by Crumrine and LaManna, who studied the effect of ischemia and its recovery on various intra-cellular tissue substrates under standard diet conditions. The effect of ketone bodies on brain metabolism was also examined under ischemic conditions following cardiac resuscitation through our model simulations. The influence of ketone bodies on lactate dynamics on mammalian brain following ischemia is studied incorporating experimental data.

Adverse outcome pathway (AOP) development and evaluation

EPA Science Inventory

The Adverse Outcome Pathway provides a construct for assembling mechanistic information at different levels of biological organization in a form designed to support regulatory decision making. In particular, it frames the link between molecular and cellular events that can be mea...

Sewage sludge dewatering using flowing liquid metals

DOEpatents

Carlson, L.W.

1985-08-30

This invention relates generally to the dewatering of sludge, and more particularly to the dewatering of a sewage sludge having a moisture content of about 50 to 80% in the form of small cellular micro-organism bodies having internally confined water.

The ubiquitin conjugating enzyme UbcH7, controls cell migration

USDA-ARS?s Scientific Manuscript database

Post translational modification by ubiquitination can target proteins for degradation, allow the interaction of proteins to form complexes or direct relocalization of proteins to different subcellular compartments. As such, ubiquitin controls a variety of essential cellular processes. Previously we ...

Evolution of Microbial Quorum Sensing to Human Global Quorum Sensing: An Insight into How Gap Junctional Intercellular Communication Might Be Linked to the Global Metabolic Disease Crisis.

PubMed

Trosko, James E

2016-06-15

The first anaerobic organism extracted energy for survival and reproduction from its source of nutrients, with the genetic means to ensure protection of its individual genome but also its species survival. While it had a means to communicate with its community via simple secreted molecules ("quorum sensing"), the eventual shift to an aerobic environment led to multi-cellular metazoan organisms, with evolutionary-selected genes to form extracellular matrices, stem cells, stem cell niches, and a family of gap junction or "connexin" genes. These germinal and somatic stem cells responded to extracellular signals that triggered intra-cellular signaling to regulate specific genes out of the total genome. These extra-cellular induced intra-cellular signals also modulated gap junctional intercellular communication (GJIC) in order to regulate the new cellular functions of symmetrical and asymmetrical cell division, cell differentiation, modes of cell death, and senescence. Within the hierarchical and cybernetic concepts, differentiated by neurons organized in the brain of the Homo sapiens, the conscious mind led to language, abstract ideas, technology, myth-making, scientific reasoning, and moral decision-making, i.e., the creation of culture. Over thousands of years, this has created the current collision between biological and cultural evolution, leading to the global "metabolic disease" crisis.

Enhancing proliferation and optimizing the culture condition for human bone marrow stromal cells using hypoxia and fibroblast growth factor-2.

PubMed

Lee, Jung-Seok; Kim, Seul Ki; Jung, Byung-Joo; Choi, Seong-Bok; Choi, Eun-Young; Kim, Chang-Sung

2018-04-01

This study aimed to determine the cellular characteristics and behaviors of human bone marrow stromal cells (hBMSCs) expanded in media in a hypoxic or normoxic condition and with or without fibroblast growth factor-2 (FGF-2) treatment. hBMSCs isolated from the vertebral body and expanded in these four groups were evaluated for cellular proliferation/migration, colony-forming units, cell-surface characterization, in vitro differentiation, in vivo transplantation, and gene expression. Culturing hBMSCs using a particular environmental factor (hypoxia) and with the addition of FGF-2 increased the cellular proliferation rate while enhancing the regenerative potential, modulated the multipotency-related processes (enhanced chondrogenesis-related processes/osteogenesis, but reduced adipogenesis), and increased cellular migration and collagen formation. The gene expression levels in the experimental samples showed activation of the hypoxia-inducible factor-1 pathway and glycolysis in the hypoxic condition, with this not being affected by the addition of FGF-2. The concurrent application of hypoxia and FGF-2 could provide a favorable condition for culturing hBMSCs to be used in clinical applications associated with bone tissue engineering, due to the enhancement of cellular proliferation and regenerative potential. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Computer Modeling of the Earliest Cellular Structures and Functions

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Chipot, Christophe; Schweighofer, Karl

2000-01-01

In the absence of extinct or extant record of protocells (the earliest ancestors of contemporary cells). the most direct way to test our understanding of the origin of cellular life is to construct laboratory models of protocells. Such efforts are currently underway in the NASA Astrobiology Program. They are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures and developing designs for molecules that perform proto-cellular functions. Many of these functions, such as import of nutrients, capture and storage of energy. and response to changes in the environment are carried out by proteins bound to membrane< We will discuss a series of large-scale, molecular-level computer simulations which demonstrate (a) how small proteins (peptides) organize themselves into ordered structures at water-membrane interfaces and insert into membranes, (b) how these peptides aggregate to form membrane-spanning structures (eg. channels), and (c) by what mechanisms such aggregates perform essential proto-cellular functions, such as proton transport of protons across cell walls, a key step in cellular bioenergetics. The simulations were performed using the molecular dynamics method, in which Newton's equations of motion for each item in the system are solved iteratively. The problems of interest required simulations on multi-nanosecond time scales, which corresponded to 10(exp 6)-10(exp 8) time steps.

A Continuum Damage Mechanics Model for the Static and Cyclic Fatigue of Cellular Composites

PubMed Central

Huber, Otto

2017-01-01

The fatigue behavior of a cellular composite with an epoxy matrix and glass foam granules is analyzed and modeled by means of continuum damage mechanics. The investigated cellular composite is a particular type of composite foam, and is very similar to syntactic foams. In contrast to conventional syntactic foams constituted by hollow spherical particles (balloons), cellular glass, mineral, or metal place holders are combined with the matrix material (metal or polymer) in the case of cellular composites. A microstructural investigation of the damage behavior is performed using scanning electron microscopy. For the modeling of the fatigue behavior, the damage is separated into pure static and pure cyclic damage and described in terms of the stiffness loss of the material using damage models for cyclic and creep damage. Both models incorporate nonlinear accumulation and interaction of damage. A cycle jumping procedure is developed, which allows for a fast and accurate calculation of the damage evolution for constant load frequencies. The damage model is applied to examine the mean stress effect for cyclic fatigue and to investigate the frequency effect and the influence of the signal form in the case of static and cyclic damage interaction. The calculated lifetimes are in very good agreement with experimental results. PMID:28809806

The AAA+ ATPase p97, a cellular multitool

PubMed Central

Stach, Lasse

2017-01-01

The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy. PMID:28819009

Stress Management in Cyst-Forming Free-Living Protists: Programmed Cell Death and/or Encystment

PubMed Central

Khan, Naveed Ahmed; Iqbal, Junaid

2015-01-01

In the face of harsh conditions and given a choice, a cell may (i) undergo programmed cell death, (ii) transform into a cancer cell, or (iii) enclose itself into a cyst form. In metazoans, the available evidence suggests that cellular machinery exists only to execute or avoid programmed cell death, while the ability to form a cyst was either lost or never developed. For cyst-forming free-living protists, here we pose the question whether the ability to encyst was gained at the expense of the programmed cell death or both functions coexist to counter unfavorable environmental conditions with mutually exclusive phenotypes. PMID:25648302

Matrix description of the complete topology of three-dimensional cells

PubMed Central

Xue, Weihua; Wang, Hao; Liu, Guoquan; Meng, Li; Xiang, Song; Ma, Guang; Li, Wenwen

2016-01-01

A new, efficient method based on a series of matrices is introduced to completely describe the detailed topology of individual domains and their topology evolution in three-dimensional cellular structures. With this approach, we found a lot of new topological grain forms which are never reported before, i.e., there are total 8 and 32 topological forms for 7- and 8-faced grains respectively, other than the reported 7 and 27. This method is proved to be a practical tool to predict all possible grain forms efficiently. Moreover, a connectivity index of grain forms serves as a new convenient differentiator of different multicellular structures. PMID:27160500

Apparatus for forming a continuous lightweight multicell material

NASA Technical Reports Server (NTRS)

Wang, Taylor G. (Inventor); Elleman, Daniel D. (Inventor); Kendall, James M., Jr. (Inventor)

1984-01-01

An apparatus is described for producing a lightweight structural material (12), by forming gas-filled shells (38) of molten material from a matrix of nozzles (22) that form shells of very uniform size at very uniform rates. The matrix of molten shells coalesce into a multi-cell material of controlled cellular structure. The shells can be of two different sizes (38, 44) that are interspersed, to form a multicell material that has a regular cell pattern but which avoids planes of weakness and localized voids. The gas (50) in the shells can be under a high pressure, and can be a fire extinguishing gas.

Non-Mendelian determinants of morphology in fungi.

PubMed

Malagnac, Fabienne; Silar, Philippe

2003-12-01

Morphological plasticity is a hallmark of eumycetes. In addition to genes and environment, epigenetic factors control cell, colony and thallus forms in many species, by creating reversible switches. Current knowledge indicates that the different shapes are due to structural or regulatory heritable states of cytoplasmic components. Cellular physiology differs in the various forms, permitting adaptation to fluctuation in the environment. These switches are part of the adaptation repertoire that fungi exhibit to colonize most niches.

Inner ear development: building a spiral ganglion and an organ of Corti out of unspecified ectoderm.

PubMed

Fritzsch, Bernd; Pan, Ning; Jahan, Israt; Elliott, Karen L

2015-07-01

The mammalian inner ear develops from a placodal thickening into a complex labyrinth of ducts with five sensory organs specialized to detect position and movement in space. The mammalian ear also develops a spiraled cochlear duct containing the auditory organ, the organ of Corti (OC), specialized to translate sound into hearing. Development of the OC from a uniform sheet of ectoderm requires unparalleled precision in the topological developmental engineering of four different general cell types, namely sensory neurons, hair cells, supporting cells, and general otic epithelium, into a mosaic of ten distinctly recognizable cell types in and around the OC, each with a unique distribution. Moreover, the OC receives unique innervation by ear-derived spiral ganglion afferents and brainstem-derived motor neurons as efferents and requires neural-crest-derived Schwann cells to form myelin and neural-crest-derived cells to induce the stria vascularis. This transformation of a sheet of cells into a complicated interdigitating set of cells necessitates the orchestrated expression of multiple transcription factors that enable the cellular transformation from ectoderm into neurosensory cells forming the spiral ganglion neurons (SGNs), while simultaneously transforming the flat epithelium into a tube, the cochlear duct, housing the OC. In addition to the cellular and conformational changes forming the cochlear duct with the OC, changes in the surrounding periotic mesenchyme form passageways for sound to stimulate the OC. We review molecular developmental data, generated predominantly in mice, in order to integrate the well-described expression changes of transcription factors and their actions, as revealed in mutants, in the formation of SGNs and OC in the correct position and orientation with suitable innervation. Understanding the molecular basis of these developmental changes leading to the formation of the mammalian OC and highlighting the gaps in our knowledge might guide in vivo attempts to regenerate this most complicated cellular mosaic of the mammalian body for the reconstitution of hearing in a rapidly growing population of aging people suffering from hearing loss.

Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thayanithy, Venugopal; Babatunde, Victor; Dickson, Elizabeth L.

Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24–48 h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formationmore » (1.3–1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells. - Highlights: • Exosomes derived from malignant cells can stimulate an increased rate in the formation of tunneling nanotubes. • Tunneling nanotubes can serve as conduits for intercellular transfer of these exosomes. • Most notably, exosomes derived from benign mesothelial cells had no effect on nanotube formation. • Cells forming nanotubes were enriched in lipid rafts at a greater number compared with cells not forming nanotubes. • Our findings suggest causal and potentially synergistic association of exosomes and tunneling nanotubes in cancer.« less

Smooth muscle-like tissue constructs with circumferentially oriented cells formed by the cell fiber technology.

PubMed

Hsiao, Amy Y; Okitsu, Teru; Onoe, Hiroaki; Kiyosawa, Mahiro; Teramae, Hiroki; Iwanaga, Shintaroh; Kazama, Tomohiko; Matsumoto, Taro; Takeuchi, Shoji

2015-01-01

The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments.

Smooth Muscle-Like Tissue Constructs with Circumferentially Oriented Cells Formed by the Cell Fiber Technology

PubMed Central

Hsiao, Amy Y.; Okitsu, Teru; Onoe, Hiroaki; Kiyosawa, Mahiro; Teramae, Hiroki; Iwanaga, Shintaroh; Kazama, Tomohiko; Matsumoto, Taro; Takeuchi, Shoji

2015-01-01

The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments. PMID:25734774

Mössbauer spectroscopic study of 57Fe metabolic transformations in the rhizobacterium Azospirillum brasilense Sp245

NASA Astrophysics Data System (ADS)

Kamnev, Alexander A.; Tugarova, Anna V.; Kovács, Krisztina; Biró, Borbála; Homonnay, Zoltán; Kuzmann, Ernő

2014-04-01

Preliminary 57Fe transmission Mössbauer spectroscopic data were obtained for the first time for live cells of the plant-growth-promoting rhizobacterium Azospirillum brasilense (wild-type strain Sp245) grown aerobically with 57FeIII-nitrilotriacetate (NTA) complex as a sole source of iron. The results obtained have shown that live cells actively reduce part of the assimilated iron(III) to iron(II), the latter amounting up to 33 % of total cellular iron after 18 h of growth, and 48 % after additional 3 days of storage of the dense wet cell suspension in nutrient-free saline solution in air at room temperature (measured at 80 K). The cellular iron(II) was found to be represented by two quadrupole doublets of different high-spin forms, while the parameters of the cellular iron(III) were close to those typical for bacterioferritins.

Genetic networks and soft computing.

PubMed

Mitra, Sushmita; Das, Ranajit; Hayashi, Yoichi

2011-01-01

The analysis of gene regulatory networks provides enormous information on various fundamental cellular processes involving growth, development, hormone secretion, and cellular communication. Their extraction from available gene expression profiles is a challenging problem. Such reverse engineering of genetic networks offers insight into cellular activity toward prediction of adverse effects of new drugs or possible identification of new drug targets. Tasks such as classification, clustering, and feature selection enable efficient mining of knowledge about gene interactions in the form of networks. It is known that biological data is prone to different kinds of noise and ambiguity. Soft computing tools, such as fuzzy sets, evolutionary strategies, and neurocomputing, have been found to be helpful in providing low-cost, acceptable solutions in the presence of various types of uncertainties. In this paper, we survey the role of these soft methodologies and their hybridizations, for the purpose of generating genetic networks.

Proteomic methods for analysis of S-nitrosation⋄

PubMed Central

Kettenhofen, Nicholas; Broniowska, Katarzyna; Keszler, Agnes; Zhang, Yanhong; Hogg, Neil

2007-01-01

This review discusses proteomic methods to detect and identify S-nitrosated proteins. Protein S-nitrosation, the post-translational modification of thiol residues to form S-nitrosothiols, has been suggested to be a mechanism of cellular redox signaling by which nitric oxide can alter cellular function through modification of protein thiol residues. It has become apparent that methods that will detect and identify low levels of S-nitrosated protein in complex protein mixtures are required in order to fully appreciate the range, extent and selectivity of this modification in both physiological and pathological conditions. While many advances have been made in the detection of either total cellular S-nitrosation or individual S-nitrosothiols, proteomic methods for the detection of S-nitrosation are in relative infancy. This review will discuss the major methods that have been used for the proteomic analysis of protein S-nitrosation and discuss the pros and cons of this methodology. PMID:17360249

Coated foams, preparation, uses and articles

DOEpatents

Duchane, D.V.; Barthell, B.L.

1982-10-21

Hydrophobic cellular material is coated with a thin hydrophilic polymer skin which stretches tightly over the foam but which does not fill the cells of the foam, thus resulting in a polymer-coated foam structure having a smoothness which was not possible in the prior art. In particular, when the hydrophobic cellular material is a specially chosen hydrophobic polymer foam and is formed into arbitrarily chosen shapes prior to the coating with hydrophilic polymer, inertial confinement fusion (ICF) targets of arbitrary shapes can be produced by subsequently coating the shapes with metal or with any other suitable material. New articles of manufacture are produced, including improved ICF targets, improved integrated circuits, and improved solar reflectors and solar collectors. In the coating method, the cell size of the hydrophobic cellular material, the viscosity of the polymer solution used to coat, and the surface tension of the polymer solution used to coat are all very important to the coating.