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Sample records for intracellular domain aicd

  1. Insulin-degrading enzyme rapidly removes the beta-amyloid precursor protein intracellular domain (AICD).

    PubMed

    Edbauer, Dieter; Willem, Michael; Lammich, Sven; Steiner, Harald; Haass, Christian

    2002-04-19

    The intramembranous gamma-secretase cleavage of the beta-amyloid precursor protein (APP) is dependent on biologically active presenilins (PS). Notch also undergoes a similar PS-dependent gamma-secretase-like cleavage, resulting in the liberation of the Notch intracellular domain (NICD), which is critically required for developmental signal transduction. gamma-Secretase processing of APP results in the production of a similar fragment called AICD (APP intracellular domain), which may function in nuclear signaling as well. AICD, like NICD, is rapidly removed. By using a battery of protease inhibitors we demonstrate that AICD, in contrast to NICD, is degraded by a cytoplasmic metalloprotease. In vitro degradation of AICD can be reconstituted with cytoplasmic fractions obtained from neuronal and non-neuronal cells. Taking into account the inhibition profile and the cytoplasmic localization, we identified three candidate enzymes (neurolysin, thimet oligopeptidase, and insulin-degrading enzyme (IDE), also known as insulysin), which all are involved in the degradation of bioactive peptides in the brain. When insulin, a well characterized substrate of IDE, was added to the in vitro degradation assay, removal of AICD was efficiently blocked. Moreover, overexpression of IDE resulted in enhanced degradation of AICD, whereas overexpression of the inactive IDE E111Q mutant did not affect AICD degradation. Finally, immunodepletion of IDE significantly reduced the AICD degrading activity. Therefore our data demonstrate that IDE, which is one of the proteases implicated in the removal of extracellular Abeta, also removes the cytoplasmic product of gamma-secretase cleaved APP.

  2. Phosphorylation of APP-CTF-AICD domains and interaction with adaptor proteins: signal transduction and/or transcriptional role--relevance for Alzheimer pathology.

    PubMed

    Schettini, Gennaro; Govoni, Stefano; Racchi, Marco; Rodriguez, Guido

    2010-12-01

    In recent decades, the study of the amyloid precursor protein (APP) and of its proteolytic products carboxy terminal fragment (CTF), APP intracellular C-terminal domain (AICD) and amyloid beta has been mostly focussed on the role of APP as a producer of the toxic amyloid beta peptide. Here, we reconsider the role of APP suggesting, in a provocative way, the protein as a central player in a putative signalling pathway. We highlight the presence in the cytosolic tail of APP of the YENPTY motif which is typical of tyrosine kinase receptors, the phosphorylation of the tyrosine, serine and threonine residues, the kinases involved and the interaction with intracellular adaptor proteins. In particular, we examine the interaction with Shc and Grb2 regulators, which through the activation of Ras proteins elicit downstream signalling events such as the MAPK pathway. The review also addresses the interaction of APP, CTFs and AICD with other adaptor proteins and in particular with Fe65 for nuclear transcriptional activity and the importance of phosphorylation for sorting the secretases involved in the amyloidogenic or non-amyloidogenic pathways. We provide a novel perspective on Alzheimer's disease pathogenesis, focussing on the perturbation of the physiological activities of APP-CTFs and AICD as an alternative perspective from that which normally focuses on the accumulation of neurotoxic proteolytic fragments.

  3. Regulation of Notch1 signaling by the APP intracellular domain facilitates degradation of the Notch1 intracellular domain and RBP-Jk.

    PubMed

    Kim, Mi-Yeon; Mo, Jung-Soon; Ann, Eun-Jung; Yoon, Ji-Hye; Jung, Jane; Choi, Yun-Hee; Kim, Su-Man; Kim, Hwa-Young; Ahn, Ji-Seon; Kim, Hangun; Kim, Kwonseop; Hoe, Hyang-Sook; Park, Hee-Sae

    2011-06-01

    The Notch1 receptor is a crucial controller of cell fate decisions, and is also a key regulator of cell growth and differentiation in a variety of contexts. In this study, we have demonstrated that the APP intracellular domain (AICD) attenuates Notch1 signaling by accelerated degradation of the Notch1 intracellular domain (Notch1-IC) and RBP-Jk, through different degradation pathways. AICD suppresses Notch1 transcriptional activity by the dissociation of the Notch1-IC-RBP-Jk complex after processing by γ-secretase. Notch1-IC is capable of forming a trimeric complex with Fbw7 and AICD, and AICD enhances the protein degradation of Notch1-IC through an Fbw7-dependent proteasomal pathway. AICD downregulates the levels of RBP-Jk protein through the lysosomal pathway. AICD-mediated degradation is involved in the preferential degradation of non-phosphorylated RBP-Jk. Collectively, our results demonstrate that AICD functions as a negative regulator in Notch1 signaling through the promotion of Notch1-IC and RBP-Jk protein degradation.

  4. Fe65 does not stabilize AICD during activation of transcription in a luciferase assay

    SciTech Connect

    Huysseune, Sandra; Kienlen-Campard, Pascal; Octave, Jean-Noel . E-mail: octave@nchm.ucl.ac.be

    2007-09-21

    The APP intracellular domain (AICD) could be involved in signaling via interaction with the adaptor protein Fe65, and with the histone acetyl transferase Tip60. However, the real function of AICD and Fe65 in regulation of transcription remains controversial. In this study, the human APPGal4 fusion protein was expressed in CHO cells and the transcriptional activity of AICDGal4 was measured in a luciferase-based reporter assay. AICDGal4 was stabilized by expression of Fe65 and levels of AICDGal4 controlled luciferase activity. On the contrary, when human APP was expressed in CHO cells, coexpression of Fe65 increased luciferase activity without affecting the amount of AICD fragment. AICD produced from APP was protected from degradation by orthophenanthroline, but not by lactacystine, indicating that AICD is not a substrate of the chymotryptic activity of the proteasome. It is concluded that Fe65 can control luciferase activity without stabilizing the labile AICD fragment.

  5. Increased AICD generation does not result in increased nuclear translocation or activation of target gene transcription

    SciTech Connect

    Waldron, Elaine; Isbert, Simone; Kern, Andreas; Jaeger, Sebastian; Martin, Anne M.; Hebert, Sebastien S.; Behl, Christian; Weggen, Sascha; De Strooper, Bart; Pietrzik, Claus U.

    2008-08-01

    A sequence of amyloid precursor protein (APP) cleavages culminates in the sequential release of the APP intracellular domain (AICD) and the amyloid {beta} peptide (A{beta}) and/or p3 fragment. One of the environmental factors favouring the accumulation of AICD appears to be a rise in intracellular pH. Here we further identified the metabolism and subcellular localization of artificially expressed constructs under such conditions. We also co-examined the mechanistic lead up to the AICD accumulation and explored possible significances for its increased expression. We found that most of the AICD generated under pH neutralized conditions is likely cleaved from C83. While the AICD surplus was unable to further activate transcription of a luciferase reporter via a Gal4-DNA-binding domain, it failed entirely via the endogenous promoter regions of proposed target genes, APP and KAI1. The lack of a specific transactivation potential was also demonstrated by the unchanged levels of target gene mRNA. However, rather than translocating to the nucleus, the AICD surplus remains membrane tethered or free in the cytosol where it interacts with Fe65. Therefore we provide strong evidence that an increase in AICD generation does not directly promote gene activation of previously proposed target 0011gen.

  6. Conformational landscape of an amyloid intra-cellular domain and Landau-Ginzburg-Wilson paradigm in protein dynamics.

    PubMed

    Dai, Jin; Niemi, Antti J; He, Jianfeng

    2016-07-28

    The Landau-Ginzburg-Wilson paradigm is proposed as a framework, to investigate the conformational landscape of intrinsically unstructured proteins. A universal Cα-trace Landau free energy is deduced from general symmetry considerations, with the ensuing all-atom structure modeled using publicly available reconstruction programs Pulchra and Scwrl. As an example, the conformational stability of an amyloid precursor protein intra-cellular domain (AICD) is inspected; the reference conformation is the crystallographic structure with code 3DXC in Protein Data Bank (PDB) that describes a heterodimer of AICD and a nuclear multi-domain adaptor protein Fe65. Those conformations of AICD that correspond to local or near-local minima of the Landau free energy are identified. For this, the response of the original 3DXC conformation to variations in the ambient temperature is investigated, using the Glauber algorithm. The conclusion is that in isolation the AICD conformation in 3DXC must be unstable. A family of degenerate conformations that minimise the Landau free energy is identified, and it is proposed that the native state of an isolated AICD is a superposition of these conformations. The results are fully in line with the presumed intrinsically unstructured character of isolated AICD and should provide a basis for a systematic analysis of AICD structure in future NMR experiments.

  7. Conformational landscape of an amyloid intra-cellular domain and Landau-Ginzburg-Wilson paradigm in protein dynamics

    NASA Astrophysics Data System (ADS)

    Dai, Jin; Niemi, Antti J.; He, Jianfeng

    2016-07-01

    The Landau-Ginzburg-Wilson paradigm is proposed as a framework, to investigate the conformational landscape of intrinsically unstructured proteins. A universal Cα-trace Landau free energy is deduced from general symmetry considerations, with the ensuing all-atom structure modeled using publicly available reconstruction programs Pulchra and Scwrl. As an example, the conformational stability of an amyloid precursor protein intra-cellular domain (AICD) is inspected; the reference conformation is the crystallographic structure with code 3DXC in Protein Data Bank (PDB) that describes a heterodimer of AICD and a nuclear multi-domain adaptor protein Fe65. Those conformations of AICD that correspond to local or near-local minima of the Landau free energy are identified. For this, the response of the original 3DXC conformation to variations in the ambient temperature is investigated, using the Glauber algorithm. The conclusion is that in isolation the AICD conformation in 3DXC must be unstable. A family of degenerate conformations that minimise the Landau free energy is identified, and it is proposed that the native state of an isolated AICD is a superposition of these conformations. The results are fully in line with the presumed intrinsically unstructured character of isolated AICD and should provide a basis for a systematic analysis of AICD structure in future NMR experiments.

  8. An AICD-based functional screen to identify APP metabolism regulators

    PubMed Central

    Zhang, Can; Khandelwal, Preeti J; Chakraborty, Ranjita; Cuellar, Trinna L; Sarangi, Srikant; Patel, Shyam A; Cosentino, Christopher P; O'Connor, Michael; Lee, Jeremy C; Tanzi, Rudolph E; Saunders, Aleister J

    2007-01-01

    Background A central event in Alzheimer's disease (AD) is the regulated intramembraneous proteolysis of the β-amyloid precursor protein (APP), to generate the β-amyloid (Aβ) peptide and the APP intracellular domain (AICD). Aβ is the major component of amyloid plaques and AICD displays transcriptional activation properties. We have taken advantage of AICD transactivation properties to develop a genetic screen to identify regulators of APP metabolism. This screen relies on an APP-Gal4 fusion protein, which upon normal proteolysis, produces AICD-Gal4. Production of AICD-Gal4 induces Gal4-UAS driven luciferase expression. Therefore, when regulators of APP metabolism are modulated, luciferase expression is altered. Results To validate this experimental approach we modulated α-, β-, and γ-secretase levels and activities. Changes in AICD-Gal4 levels as measured by Western blot analysis were strongly and significantly correlated to the observed changes in AICD-Gal4 mediated luciferase activity. To determine if a known regulator of APP trafficking/maturation and Presenilin1 endoproteolysis could be detected using the AICD-Gal4 mediated luciferase assay, we knocked-down Ubiquilin 1 and observed decreased luciferase activity. We confirmed that Ubiquilin 1 modulated AICD-Gal4 levels by Western blot analysis and also observed that Ubiquilin 1 modulated total APP levels, the ratio of mature to immature APP, as well as PS1 endoproteolysis. Conclusion Taken together, we have shown that this screen can identify known APP metabolism regulators that control proteolysis, intracellular trafficking, maturation and levels of APP and its proteolytic products. We demonstrate for the first time that Ubiquilin 1 regulates APP metabolism in the human neuroblastoma cell line, SH-SY5Y. PMID:17718916

  9. A cell-permeable tool for analysing APP intracellular domain function and manipulation of PIKfyve activity

    PubMed Central

    Guscott, Benjamin; Balklava, Zita; Safrany, Stephen T.; Wassmer, Thomas

    2016-01-01

    The mechanisms for regulating PIKfyve complex activity are currently emerging. The PIKfyve complex, consisting of the phosphoinositide kinase PIKfyve (also known as FAB1), VAC14 and FIG4, is required for the production of phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]. PIKfyve function is required for homoeostasis of the endo/lysosomal system and is crucially implicated in neuronal function and integrity, as loss of function mutations in the PIKfyve complex lead to neurodegeneration in mouse models and human patients. Our recent work has shown that the intracellular domain of the amyloid precursor protein (APP), a molecule central to the aetiology of Alzheimer's disease binds to VAC14 and enhances PIKfyve function. In the present study, we utilize this recent advance to create an easy-to-use tool for increasing PIKfyve activity in cells. We fused APP intracellular domain (AICD) to the HIV TAT domain, a cell-permeable peptide allowing proteins to penetrate cells. The resultant TAT–AICD fusion protein is cell permeable and triggers an increase in PI(3,5)P2. Using the PI(3,5)P2 specific GFP-ML1Nx2 probe, we show that cell-permeable AICD alters PI(3,5)P2 dynamics. TAT–AICD also provides partial protection from pharmacological inhibition of PIKfyve. All three lines of evidence show that the AICD activates the PIKfyve complex in cells, a finding that is important for our understanding of the mechanism of neurodegeneration in Alzheimer's disease. PMID:26934981

  10. APP intracellular domain derived from amyloidogenic β- and γ-secretase cleavage regulates neprilysin expression

    PubMed Central

    Grimm, Marcus O. W.; Mett, Janine; Stahlmann, Christoph P.; Grösgen, Sven; Haupenthal, Viola J.; Blümel, Tamara; Hundsdörfer, Benjamin; Zimmer, Valerie C.; Mylonas, Nadine T.; Tanila, Heikki; Müller, Ulrike; Grimm, Heike S.; Hartmann, Tobias

    2015-01-01

    Alzheimer's disease (AD) is characterized by an accumulation of Amyloid-β (Aβ), released by sequential proteolytic processing of the amyloid precursor protein (APP) by β - and γ-secretase. Aβ peptides can aggregate, leading to toxic Aβ oligomers and amyloid plaque formation. Aβ accumulation is not only dependent on de novo synthesis but also on Aβ degradation. Neprilysin (NEP) is one of the major enzymes involved in Aβ degradation. Here we investigate the molecular mechanism of NEP regulation, which is up to now controversially discussed to be affected by APP processing itself. We found that NEP expression is highly dependent on the APP intracellular domain (AICD), released by APP processing. Mouse embryonic fibroblasts devoid of APP processing, either by the lack of the catalytically active subunit of the γ-secretase complex [presenilin (PS) 1/2] or by the lack of APP and the APP-like protein 2 (APLP2), showed a decreased NEP expression, activity and protein level. Similar results were obtained by utilizing cells lacking a functional AICD domain (APPΔCT15) or expressing mutations in the genes encoding for PS1. AICD supplementation or retransfection with an AICD encoding plasmid could rescue the down-regulation of NEP further strengthening the link between AICD and transcriptional NEP regulation, in which Fe65 acts as an important adaptor protein. Especially AICD generated by the amyloidogenic pathway seems to be more involved in the regulation of NEP expression. In line, analysis of NEP gene expression in vivo in six transgenic AD mouse models (APP and APLP2 single knock-outs, APP/APLP2 double knock-out, APP-swedish, APP-swedish/PS1Δexon9, and APPΔCT15) confirmed the results obtained in cell culture. In summary, in the present study we clearly demonstrate an AICD-dependent regulation of the Aβ-degrading enzyme NEP in vitro and in vivo and elucidate the underlying mechanisms that might be beneficial to develop new therapeutic strategies for the

  11. APP intracellular domain derived from amyloidogenic β- and γ-secretase cleavage regulates neprilysin expression.

    PubMed

    Grimm, Marcus O W; Mett, Janine; Stahlmann, Christoph P; Grösgen, Sven; Haupenthal, Viola J; Blümel, Tamara; Hundsdörfer, Benjamin; Zimmer, Valerie C; Mylonas, Nadine T; Tanila, Heikki; Müller, Ulrike; Grimm, Heike S; Hartmann, Tobias

    2015-01-01

    Alzheimer's disease (AD) is characterized by an accumulation of Amyloid-β (Aβ), released by sequential proteolytic processing of the amyloid precursor protein (APP) by β - and γ-secretase. Aβ peptides can aggregate, leading to toxic Aβ oligomers and amyloid plaque formation. Aβ accumulation is not only dependent on de novo synthesis but also on Aβ degradation. Neprilysin (NEP) is one of the major enzymes involved in Aβ degradation. Here we investigate the molecular mechanism of NEP regulation, which is up to now controversially discussed to be affected by APP processing itself. We found that NEP expression is highly dependent on the APP intracellular domain (AICD), released by APP processing. Mouse embryonic fibroblasts devoid of APP processing, either by the lack of the catalytically active subunit of the γ-secretase complex [presenilin (PS) 1/2] or by the lack of APP and the APP-like protein 2 (APLP2), showed a decreased NEP expression, activity and protein level. Similar results were obtained by utilizing cells lacking a functional AICD domain (APPΔCT15) or expressing mutations in the genes encoding for PS1. AICD supplementation or retransfection with an AICD encoding plasmid could rescue the down-regulation of NEP further strengthening the link between AICD and transcriptional NEP regulation, in which Fe65 acts as an important adaptor protein. Especially AICD generated by the amyloidogenic pathway seems to be more involved in the regulation of NEP expression. In line, analysis of NEP gene expression in vivo in six transgenic AD mouse models (APP and APLP2 single knock-outs, APP/APLP2 double knock-out, APP-swedish, APP-swedish/PS1Δexon9, and APPΔCT15) confirmed the results obtained in cell culture. In summary, in the present study we clearly demonstrate an AICD-dependent regulation of the Aβ-degrading enzyme NEP in vitro and in vivo and elucidate the underlying mechanisms that might be beneficial to develop new therapeutic strategies for the

  12. Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain

    SciTech Connect

    Radzimanowski, Jens; Beyreuther, Konrad; Sinning, Irmgard; Wild, Klemens

    2008-05-01

    Alzheimer’s disease is characterized by proteolytic processing of the amyloid precursor protein (APP), which releases the aggregation-prone amyloid-β (Aβ) peptide and liberates the intracellular domain (AICD) that interacts with various adaptor proteins. The crystallized AICD–Fe65-PTB2 complex is of central importance for APP translocation, nuclear signalling, processing and Aβ generation. Alzheimer’s disease is associated with typical brain deposits (senile plaques) that mainly contain the neurotoxic amyloid β peptide. This peptide results from proteolytic processing of the type I transmembrane protein amyloid precursor protein (APP). During this proteolytic pathway the APP intracellular domain (AICD) is released into the cytosol, where it associates with various adaptor proteins. The interaction of the AICD with the C-terminal phosphotyrosine-binding domain of Fe65 (Fe65-PTB2) regulates APP translocation, signalling and processing. Human AICD and Fe65-PTB2 have been cloned, overproduced and purified in large amounts in Escherichia coli. A complex of Fe65-PTB2 with the C-terminal 32 amino acids of the AICD gave well diffracting hexagonal crystals and data have been collected to 2.1 Å resolution. Initial phases obtained by the molecular-replacement method are of good quality and revealed well defined electron density for the substrate peptide.

  13. Upregulation of PGC-1α expression by Alzheimer’s disease-associated pathway: presenilin 1/amyloid precursor protein (APP)/intracellular domain of APP

    PubMed Central

    Robinson, Ari; Grösgen, Sven; Mett, Janine; Zimmer, Valerie C; Haupenthal, Viola J; Hundsdörfer, Benjamin; P Stahlmann, Christoph; Slobodskoy, Yulia; Müller, Ulrike C; Hartmann, Tobias; Stein, Reuven; Grimm, Marcus O W

    2014-01-01

    Cleavage of amyloid precursor protein (APP) by β- and γ-secretase generates amyloid-β (Aβ) and APP intracellular domain (AICD) peptides. Presenilin (PS) 1 or 2 is the catalytic component of the γ-secretase complex. Mitochondrial dysfunction is an established phenomenon in Alzheimer’s disease (AD), but the causes and role of PS1, APP, and APP’s cleavage products in this process are largely unknown. We studied the effect of these AD-associated molecules on mitochondrial features. Using cells deficient in PSs expression, expressing human wild-type PS1, or PS1 familial AD (FAD) mutants, we found that PS1 affects mitochondrial energy metabolism (ATP levels and oxygen consumption) and expression of mitochondrial proteins. These effects were associated with enhanced expression of the mitochondrial master transcriptional coactivator PGC-1α and its target genes. Importantly, PS1-FAD mutations decreased PS1’s ability to enhance PGC-1α mRNA levels. Analyzing the effect of APP and its γ-secretase-derived cleavage products Aβ and AICD on PGC-1α expression showed that APP and AICD increase PGC-1α expression. Accordingly, PGC-1α mRNA levels in cells deficient in APP/APLP2 or expressing APP lacking its last 15 amino acids were lower than in control cells, and treatment with AICD, but not with Aβ, enhanced PGC-1α mRNA levels in these and PSs-deficient cells. In addition, knockdown of the AICD-binding partner Fe65 reduced PGC-1α mRNA levels. Importantly, APP/AICD increases PGC-1α expression also in the mice brain. Our results therefore suggest that APP processing regulates mitochondrial function and that impairments in the newly discovered PS1/APP/AICD/PGC-1α pathway may lead to mitochondrial dysfunction and neurodegeneration. PMID:24304563

  14. Hypoxia Affects Neprilysin Expression Through Caspase Activation and an APP Intracellular Domain-dependent Mechanism

    PubMed Central

    Kerridge, Caroline; Kozlova, Daria I.; Nalivaeva, Natalia N.; Turner, Anthony J.

    2015-01-01

    While gene mutations in the amyloid precursor protein (APP) and the presenilins lead to an accumulation of the amyloid β-peptide (Aβ) in the brain causing neurodegeneration and familial Alzheimer's disease (AD), over 95% of all AD cases are sporadic. Despite the pathologies being indistinguishable, relatively little is known about the mechanisms affecting generation of Aβ in the sporadic cases. Vascular disorders such as ischaemia and stroke are well established risk factors for the development of neurodegenerative diseases and systemic hypoxic episodes have been shown to increase Aβ production and accumulation. We have previously shown that hypoxia causes a significant decrease in the expression of the major Aβ-degrading enzyme neprilysin (NEP) which might deregulate Aβ clearance. Aβ itself is derived from the transmembrane APP along with several other biologically active metabolites including the C-terminal fragment (CTF) termed the APP intracellular domain (AICD), which regulates the expression of NEP and some other genes in neuronal cells. Here we show that in hypoxia there is a significantly increased expression of caspase-3, 8, and 9 in human neuroblastoma NB7 cells, which can degrade AICD. Using chromatin immunoprecipitation we have revealed that there was also a reduction of AICD bound to the NEP promoter region which underlies the decreased expression and activity of the enzyme under hypoxic conditions. Incubation of the cells with a caspase-3 inhibitor Z-DEVD-FMK could rescue the effect of hypoxia on NEP activity protecting the levels of AICD capable of binding the NEP promoter. These data suggest that activation of caspases might play an important role in regulation of NEP levels in the brain under pathological conditions such as hypoxia and ischaemia leading to a deficit of Aβ clearance and increasing the risk of development of AD. PMID:26617481

  15. RNAi for contactin 2 inhibits proliferation of U87-glioma stem cells by downregulating AICD, EGFR, and HES1

    PubMed Central

    Guo, Yang; Zhang, Peidong; Zhang, Hongtian; Zhang, Peng; Xu, Ruxiang

    2017-01-01

    Glioblastoma is the most common form of malignant brain tumors and has a poor prognosis. Glioma stem cells (GSCs) are thought to be responsible for the aberrant proliferation and invasion. Targeting the signaling pathways that promote proliferation in GSCs is one of the strategies for glioma treatment. In this study, we found increased expression of contactin 2 (CNTN2) and amyloid β precursor protein (APP) in U87-derived GSCs (U87-GSCs). RNA interference (RNAi) for CNTN2 downregulated the expression of APP intracellular domain (AICD), which is the proteolytic product of APP. Treatment with CNTN2 RNAi inhibited the proliferation of U87-GSCs. CNTN2 RNAi decreased the expression of epidermal growth factor receptor and HES1, which are potential targets of AICD. In summary, inhibition of the CNTN2/APP signaling pathway may repress the proliferation in U87-GSCs via downregulating the expression of HES1 and epidermal growth factor receptor. CNTN2/APP/AICD signaling pathway plays an important role in U87 glial tumorigenesis. Further studies are warranted to elucidate the role of these signaling pathways in other sources of GSCs. Depending on their role in proliferation in other sources of GSCs, members of the CNTN2/APP/AICD signaling pathway may provide novel targets for the development of therapy for glioblastomas. PMID:28243115

  16. Amyloidogenic Processing but not AICD Production Requires a Precisely Oriented APP Dimer Assembled by Transmembrane GXXXG Motifs

    PubMed Central

    Kienlen-Campard, Pascal; Tasiaux, Bernadette; Van Hees, Joanne; Li, Mingli; Huysseune, Sandra; Sato, Takeshi; Fei, Jeffrey Z.; Aimoto, Saburo; Courtoy, Pierre J.; Smith, Steven O.; Constantinescu, Stefan N.; Octave, Jean-Noël

    2009-01-01

    The β-amyloid peptide (Aβ) is the major constituent of the amyloid core of senile plaques found in the brain of patients with Alzheimer's disease (AD). Aβ is produced by the sequential cleavage of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Cleavage of APP by γ-secretase also generates the APP Intracellular C-terminal Domain (AICD) peptide, which might be involved in regulation of gene transcription. APP contains three glycine-xxx-glycine (GxxxG) motifs in its juxtamembrane and transmembrane (TM) regions. Such motifs are known to promote dimerization via close apposition of TM sequences. We demonstrate that pairwise replacement of glycines by leucines or isoleucines, but not alanines, in a GxxxG motif led to a drastic reduction of Aβ40 and Aβ42 secretion. β-Cleavage of mutant APP was not inhibited, and reduction of Aβ secretion resulted from inhibition of γ-cleavage. It was anticipated that decreased γ-cleavage of mutant APP would result from inhibition of its dimerization. Surprisingly, mutations of the GxxxG motif actually enhanced dimerization of the APP C-terminal fragments, possibly via a different TM α-helical interface. Increased dimerization of the TM APP C-terminal domain did not affect AICD production. These results clearly demonstrate that both orientation and dimerization of the APP TM domain differently affect Aβ and AICD production. PMID:18201969

  17. Neprilysin and Aβ Clearance: Impact of the APP Intracellular Domain in NEP Regulation and Implications in Alzheimer’s Disease

    PubMed Central

    Grimm, Marcus O. W.; Mett, Janine; Stahlmann, Christoph P.; Haupenthal, Viola J.; Zimmer, Valerie C.; Hartmann, Tobias

    2013-01-01

    One of the characteristic hallmarks of Alzheimer’s disease (AD) is an accumulation of amyloid β (Aβ) leading to plaque formation and toxic oligomeric Aβ complexes. Besides the de novo synthesis of Aβ caused by amyloidogenic processing of the amyloid precursor protein (APP), Aβ levels are also highly dependent on Aβ degradation. Several enzymes are described to cleave Aβ. In this review we focus on one of the most prominent Aβ degrading enzymes, the zinc-metalloprotease Neprilysin (NEP). In the first part of the review we discuss beside the general role of NEP in Aβ degradation the alterations of the enzyme observed during normal aging and the progression of AD. In vivo and cell culture experiments reveal that a decreased NEP level results in an increased Aβ level and vice versa. In a pathological situation like AD, it has been reported that NEP levels and activity are decreased and it has been suggested that certain polymorphisms in the NEP gene result in an increased risk for AD. Conversely, increasing NEP activity in AD mouse models revealed an improvement in some behavioral tests. Therefore it has been suggested that increasing NEP might be an interesting potential target to treat or to be protective for AD making it indispensable to understand the regulation of NEP. Interestingly, it is discussed that the APP intracellular domain (AICD), one of the cleavage products of APP processing, which has high similarities to Notch receptor processing, might be involved in the transcriptional regulation of NEP. However, the mechanisms of NEP regulation by AICD, which might be helpful to develop new therapeutic strategies, are up to now controversially discussed and summarized in the second part of this review. In addition, we review the impact of AICD not only in the transcriptional regulation of NEP but also of further genes. PMID:24391587

  18. A physiologic signaling role for the γ-secretase-derived intracellular fragment of APP

    PubMed Central

    Leissring, Malcolm A.; Murphy, M. Paul; Mead, Tonya R.; Akbari, Yama; Sugarman, Michael C.; Jannatipour, Mehrdad; Anliker, Brigitte; Müller, Ulrike; Saftig, Paul; De Strooper, Bart; Wolfe, Michael S.; Golde, Todd E.; LaFerla, Frank M.

    2002-01-01

    Presenilins mediate an unusual intramembranous proteolytic activity known as γ-secretase, two substrates of which are the Notch receptor (Notch) and the β-amyloid precursor protein (APP). γ-Secretase-mediated cleavage of APP, like that of Notch, yields an intracellular fragment [APP intracellular domain (AICD)] that forms a transcriptively active complex. We now demonstrate a functional role for AICD in regulating phosphoinositide-mediated calcium signaling. Genetic ablation of the presenilins or pharmacological inhibition of γ-secretase activity (and thereby AICD production) attenuated calcium signaling in a dose-dependent and reversible manner through a mechanism involving the modulation of endoplasmic reticulum calcium stores. Cells lacking APP (and hence AICD) exhibited similar calcium signaling deficits, and—notably—these disturbances could be reversed by transfection with APP constructs containing an intact AICD, but not by constructs lacking this domain. Our findings indicate that the AICD regulates phosphoinositide-mediated calcium signaling through a γ-secretase-dependent signaling pathway, suggesting that the intramembranous proteolysis of APP may play a signaling role analogous to that of Notch. PMID:11917117

  19. Structural rearrangement of the intracellular domains during AMPA receptor activation

    PubMed Central

    Zachariassen, Linda G.; Katchan, Ljudmila; Jensen, Anna G.; Pickering, Darryl S.; Plested, Andrew J. R.

    2016-01-01

    α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact. PMID:27313205

  20. Targeting of passenger protein domains to multiple intracellular membranes.

    PubMed Central

    Janiak, F; Glover, J R; Leber, B; Rachubinski, R A; Andrews, D W

    1994-01-01

    The role of passenger domains in protein targeting was examined by fusing previously characterized targeting motifs to different protein sequences. To compare the targeting requirements for a variety of subcellular compartments, targeting of the fusion proteins was examined for endoplasmic reticulum, mitochondria and peroxisomes in vitro and in yeast. Although most passenger domains were only partially passive to translocation, motif-dependent targeting via motifs positioned at either end of one passenger domain (gPA) was demonstrated for all of the subcellular compartments tested. The data presented extend earlier suggestions that translocation competence is an intrinsic property of the passenger protein. However, the properties that determine protein targeting are not mutually exclusive for the compartments tested. Therefore, although the primary determinant of specificity is the targeting motif, our results suggest that translocation competence of the targeted protein augments the fidelity of transport. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8198533

  1. Membrane-Tethered Intracellular Domain of Amphiregulin Promotes Keratinocyte Proliferation

    PubMed Central

    Stoll, Stefan W.; Stuart, Philip E.; Lambert, Sylviane; Gandarillas, Alberto; Rittié, Laure; Johnston, Andrew; Elder, James T.

    2016-01-01

    The EGF receptor (EGFR) and its ligands are essential regulators of epithelial biology, which are often amplified in cancer cells. We have previously shown that shRNA-mediated silencing of one of these ligands, amphiregulin (AREG), results in keratinocyte growth arrest that cannot be rescued by soluble extracellular EGFR ligands. To further explore the functional importance of specific AREG domains, we stably transduced keratinocytes expressing tetracycline-inducible AREG-targeted shRNA with lentiviruses expressing silencing-proof, membrane-tethered AREG cytoplasmic and extracellular domains (AREG-CTD and AREG-ECD), as well as full-length AREG precursor (proAREG). Here we show that growth arrest of AREG-silenced keratinocytes occurs in G2/M and is significantly restored by proAREG and AREG-CTD, but not by AREG-ECD. Moreover, the AREG-CTD was sufficient to normalize cell cycle distribution profiles and expression of mitosis-related genes. Our findings uncover an important role of the AREG-CTD in regulating cell division, which may be relevant to tumor resistance to EGFR-directed therapies. PMID:26802239

  2. Exploring functional roles of TRPV1 intracellular domains with unstructured peptide-insertion screening

    PubMed Central

    Ma, Linlin; Yang, Fan; Vu, Simon; Zheng, Jie

    2016-01-01

    TRPV1 is a polymodal nociceptor for diverse physical and chemical stimuli that interact with different parts of the channel protein. Recent cryo-EM studies revealed detailed channel structures, opening the door for mapping structural elements mediating activation by each stimulus. Towards this goal, here we have combined unstructured peptide-insertion screening (UPS) with electrophysiological and fluorescence recordings to explore structural and functional roles of the intracellular regions of TRPV1 in mediating various activation stimuli. We found that most of the tightly packed protein regions did not tolerate structural perturbation by UPS when tested, indicating that structural integrity of the intracellular region is critical. In agreement with previous reports, Ca2+-dependent desensitization is strongly dependent on both intracellular N- and C-terminal domains; insertions of an unstructured peptide between these domains and the transmembrane core domain nearly eliminated Ca2+-dependent desensitization. In contrast, channel activations by capsaicin, low pH, divalent cations, and even heat are mostly intact in mutant channels containing the same insertions. These observations suggest that the transmembrane core domain of TRPV1, but not the intracellular domains, is responsible for sensing these stimuli. PMID:27666400

  3. The intracellular domains of Notch1 and Notch2 are functionally equivalent during development and carcinogenesis.

    PubMed

    Liu, Zhenyi; Brunskill, Eric; Varnum-Finney, Barbara; Zhang, Chi; Zhang, Andrew; Jay, Patrick Y; Bernstein, Irv; Morimoto, Mitsuru; Kopan, Raphael

    2015-07-15

    Although Notch1 and Notch2 are closely related paralogs and function through the same canonical signaling pathway, they contribute to different outcomes in some cell and disease contexts. To understand the basis for these differences, we examined in detail mice in which the Notch intracellular domains (N1ICD and N2ICD) were swapped. Our data indicate that strength (defined here as the ultimate number of intracellular domain molecules reaching the nucleus, integrating ligand-mediated release and nuclear translocation) and duration (half-life of NICD-RBPjk-MAML-DNA complexes, integrating cooperativity and stability dependent on shared sequence elements) are the factors that underlie many of the differences between Notch1 and Notch2 in all the contexts we examined, including T-cell development, skin differentiation and carcinogenesis, the inner ear, the lung and the retina. We were able to show that phenotypes in the heart, endothelium, and marginal zone B cells are attributed to haploinsufficiency but not to intracellular domain composition. Tissue-specific differences in NICD stability were most likely caused by alternative scissile bond choices by tissue-specific γ-secretase complexes following the intracellular domain swap. Reinterpretation of clinical findings based on our analyses suggests that differences in outcome segregating with Notch1 or Notch2 are likely to reflect outcomes dependent on the overall strength of Notch signals.

  4. Functional genomics of intracellular peptide recognition domains with combinatorial biology methods.

    PubMed

    Sidhu, Sachdev S; Bader, Gary D; Boone, Charles

    2003-02-01

    Phage-displayed peptide libraries have been used to identify specific ligands for peptide-binding domains that mediate intracellular protein-protein interactions. These studies have provided significant insights into the specificities of particular domains. For PDZ domains that recognize C-terminal sequences, the information has proven useful in identifying natural binding partners from genomic databases. For SH3 domains that recognize internal proline-rich motifs, the results of database searches with phage-derived ligands have been compared with the results of yeast-two-hybrid experiments to produce overlap networks that reliably predict natural protein-protein interactions. In addition, libraries of phage-displayed PDZ and SH3 domains have been used to identify the residues responsible for ligand recognition, and also to engineer domains with altered specificities.

  5. NMR Dynamics of Transmembrane and Intracellular Domains of p75NTR in Lipid-Protein Nanodiscs

    PubMed Central

    Mineev, Konstantin S.; Goncharuk, Sergey A.; Kuzmichev, Pavel K.; Vilar, Marçal; Arseniev, Alexander S.

    2015-01-01

    P75NTR is a type I integral membrane protein that plays a key role in neurotrophin signaling. However, structural data for the receptor in various functional states are sparse and controversial. In this work, we studied the spatial structure and mobility of the transmembrane and intracellular parts of p75NTR, incorporated into lipid-protein nanodiscs of various sizes and compositions, by solution NMR spectroscopy. Our data reveal a high level of flexibility and disorder in the juxtamembrane chopper domain of p75NTR, which results in the motions of the receptor death domain being uncoupled from the motions of the transmembrane helix. Moreover, none of the intracellular domains of p75NTR demonstrated a propensity to interact with the membrane or to self-associate under the experimental conditions. The obtained data are discussed in the context of the receptor activation mechanism. PMID:26287629

  6. Intracellular Domain Fragment of CD44 Alters CD44 Function in Chondrocytes*

    PubMed Central

    Mellor, Liliana; Knudson, Cheryl B.; Hida, Daisuke; Askew, Emily B.; Knudson, Warren

    2013-01-01

    The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44. PMID:23884413

  7. The Notch intracellular domain integrates signals from Wnt, Hedgehog, TGFβ/BMP and hypoxia pathways.

    PubMed

    Borggrefe, Tilman; Lauth, Matthias; Zwijsen, An; Huylebroeck, Danny; Oswald, Franz; Giaimo, Benedetto Daniele

    2016-02-01

    Notch signaling is a highly conserved signal transduction pathway that regulates stem cell maintenance and differentiation in several organ systems. Upon activation, the Notch receptor is proteolytically processed, its intracellular domain (NICD) translocates into the nucleus and activates expression of target genes. Output, strength and duration of the signal are tightly regulated by post-translational modifications. Here we review the intracellular post-translational regulation of Notch that fine-tunes the outcome of the Notch response. We also describe how crosstalk with other conserved signaling pathways like the Wnt, Hedgehog, hypoxia and TGFβ/BMP pathways can affect Notch signaling output. This regulation can happen by regulation of ligand, receptor or transcription factor expression, regulation of protein stability of intracellular key components, usage of the same cofactors or coregulation of the same key target genes. Since carcinogenesis is often dependent on at least two of these pathways, a better understanding of their molecular crosstalk is pivotal.

  8. Domain reorientation and rotation of an intracellular assembly regulate conduction in Kir potassium channels.

    PubMed

    Clarke, Oliver B; Caputo, Alessandro T; Hill, Adam P; Vandenberg, Jamie I; Smith, Brian J; Gulbis, Jacqueline M

    2010-06-11

    Potassium channels embedded in cell membranes employ gates to regulate K+ current. While a specific constriction in the permeation pathway has historically been implicated in gating, recent reports suggest that the signature ion selectivity filter located in the outer membrane leaflet may be equally important. Inwardly rectifying K+ channels also control the directionality of flow, using intracellular polyamines to stem ion efflux by a valve-like action. This study presents crystallographic evidence of interdependent gates in the conduction pathway and reveals the mechanism of polyamine block. Reorientation of the intracellular domains, concomitant with activation, instigates polyamine release from intracellular binding sites to block the permeation pathway. Conformational adjustments of the slide helices, achieved by rotation of the cytoplasmic assembly relative to the pore, are directly correlated to the ion configuration in the selectivity filter. Ion redistribution occurs irrespective of the constriction, suggesting a more expansive role of the selectivity filter in gating than previously appreciated.

  9. Impact of intracellular domain flexibility upon properties of activated human 5-HT3 receptors*

    PubMed Central

    Kozuska, J L; Paulsen, I M; Belfield, W J; Martin, I L; Cole, D J; Holt, A; Dunn, S M J

    2014-01-01

    Background and Purpose It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance. Experimental Approach Mutations were introduced into the portal region of the human 5-HT3A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors. Key Results Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain. Conclusions and Implications These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy. Linked Article This article is commented on by Vardy and Kenakin, pp. 1614–1616 of volume 171 issue 7. To view this commentary

  10. TARP modulation of synaptic AMPA receptor trafficking and gating depends on multiple intracellular domains.

    PubMed

    Milstein, Aaron D; Nicoll, Roger A

    2009-07-07

    Previous work has established stargazin and its related family of transmembrane AMPA receptor regulatory proteins (TARPs) as auxiliary subunits of AMPA receptors (AMPARs) that control synaptic strength both by targeting AMPARs to synapses through an intracellular PDZ-binding motif and by modulating their gating through an extracellular domain. However, TARPs gamma-2 and gamma-8 differentially regulate the synaptic targeting of AMPARs, despite having identical PDZ-binding motifs. Here, we investigate the structural elements that contribute to this functional difference between TARP subtypes by using domain transplantation and truncation. We identify a component of synaptic AMPAR trafficking that is independent of the TARP C-terminal PDZ-binding motif, and we establish previously uncharacterized roles for the TARP intracellular N terminus, loop, and C terminus in modulating both the trafficking and gating of synaptic AMPARs.

  11. Membrane domain formation—a key factor for targeted intracellular drug delivery

    PubMed Central

    Popov-Čeleketić, Dušan; van Bergen en Henegouwen, Paul M. P.

    2014-01-01

    Protein molecules, toxins and viruses internalize into the cell via receptor-mediated endocytosis (RME) using specific proteins and lipids in the plasma membrane. The plasma membrane is a barrier for many pharmaceutical agents to enter into the cytoplasm of target cells. In the case of cancer cells, tissue-specific biomarkers in the plasma membrane, like cancer-specific growth factor receptors, could be excellent candidates for RME-dependent drug delivery. Recent data suggest that agent binding to these receptors at the cell surface, resulting in membrane domain formation by receptor clustering, can be used for the initiation of RME. As a result, these pharmaceutical agents are internalized into the cells and follow different routes until they reach their final intracellular targets like lysosomes or Golgi. We propose that clustering induced formation of plasma membrane microdomains enriched in receptors, sphingolipids, and inositol lipids, leads to membrane bending which functions as the onset of RME. In this review we will focus on the role of domain formation in RME and discuss potential applications for targeted intracellular drug delivery. PMID:25520666

  12. Localization of the Intracellular Activity Domain of Pasteurella multocida Toxin to the N Terminus

    PubMed Central

    Wilson, Brenda A.; Ponferrada, Virgilio G.; Vallance, Jefferson E.; Ho, Mengfei

    1999-01-01

    We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqα protein that is coupled to phosphatidylinositol-specific phospholipase Cβ1 in Xenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268–1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using the Xenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entire toxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein in Escherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells. PMID:9864199

  13. ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation

    SciTech Connect

    Shi, Fumin; Telesco, Shannon E.; Liu, Yingting; Radhakrishnan, Ravi; Lemmon, Mark A.

    2010-06-21

    ErbB3/HER3 is one of four members of the human epidermal growth factor receptor (EGFR/HER) or ErbB receptor tyrosine kinase family. ErbB3 binds neuregulins via its extracellular region and signals primarily by heterodimerizing with ErbB2/HER2/Neu. A recently appreciated role for ErbB3 in resistance of tumor cells to EGFR/ErbB2-targeted therapeutics has made it a focus of attention. However, efforts to inactivate ErbB3 therapeutically in parallel with other ErbB receptors are challenging because its intracellular kinase domain is thought to be an inactive pseudokinase that lacks several key conserved (and catalytically important) residues - including the catalytic base aspartate. We report here that, despite these sequence alterations, ErbB3 retains sufficient kinase activity to robustly trans-autophosphorylate its intracellular region - although it is substantially less active than EGFR and does not phosphorylate exogenous peptides. The ErbB3 kinase domain binds ATP with a K{sub d} of approximately 1.1 {micro}M. We describe a crystal structure of ErbB3 kinase bound to an ATP analogue, which resembles the inactive EGFR and ErbB4 kinase domains (but with a shortened {alpha}C-helix). Whereas mutations that destabilize this configuration activate EGFR and ErbB4 (and promote EGFR-dependent lung cancers), a similar mutation conversely inactivates ErbB3. Using quantum mechanics/molecular mechanics simulations, we delineate a reaction pathway for ErbB3-catalyzed phosphoryl transfer that does not require the conserved catalytic base and can be catalyzed by the 'inactive-like'configuration observed crystallographically. These findings suggest that ErbB3 kinase activity within receptor dimers may be crucial for signaling and could represent an important therapeutic target.

  14. Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses

    PubMed Central

    Nguyen, Quynh-Anh; Horn, Meryl E; Nicoll, Roger A

    2016-01-01

    Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that interact trans-synaptically with neurexins to mediate synapse development and function. NLGN2 is only at inhibitory synapses while NLGN3 is at both excitatory and inhibitory synapses. We found that NLGN3 function at inhibitory synapses in rat CA1 depends on the presence of NLGN2 and identified a domain in the extracellular region that accounted for this functional difference between NLGN2 and 3 specifically at inhibitory synapses. We further show that the presence of a cytoplasmic tail (c-tail) is indispensible, and identified two domains in the c-tail that are necessary for NLGN function at inhibitory synapses. These domains point to a gephyrin-dependent mechanism that is disrupted by an autism-associated mutation at R705 and a gephyrin-independent mechanism reliant on a putative phosphorylation site at S714. Our work highlights unique and separate roles for the extracellular and intracellular regions in specifying and carrying out NLGN function respectively. DOI: http://dx.doi.org/10.7554/eLife.19236.001 PMID:27805570

  15. Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses.

    PubMed

    Nguyen, Quynh-Anh; Horn, Meryl E; Nicoll, Roger A

    2016-11-02

    Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that interact trans-synaptically with neurexins to mediate synapse development and function. NLGN2 is only at inhibitory synapses while NLGN3 is at both excitatory and inhibitory synapses. We found that NLGN3 function at inhibitory synapses in rat CA1 depends on the presence of NLGN2 and identified a domain in the extracellular region that accounted for this functional difference between NLGN2 and 3 specifically at inhibitory synapses. We further show that the presence of a cytoplasmic tail (c-tail) is indispensible, and identified two domains in the c-tail that are necessary for NLGN function at inhibitory synapses. These domains point to a gephyrin-dependent mechanism that is disrupted by an autism-associated mutation at R705 and a gephyrin-independent mechanism reliant on a putative phosphorylation site at S714. Our work highlights unique and separate roles for the extracellular and intracellular regions in specifying and carrying out NLGN function respectively.

  16. Modulation of microtubule dynamics by a TIR domain protein from the intracellular pathogen Brucella melitensis.

    PubMed

    Radhakrishnan, Girish K; Harms, Jerome S; Splitter, Gary A

    2011-10-01

    TIR (Toll/interleukin-1 receptor) domain-containing proteins play a crucial role in innate immunity in eukaryotes. Brucella is a highly infectious intracellular bacterium that encodes a TIR domain protein (TcpB) to subvert host innate immune responses to establish a beneficial niche for pathogenesis. TcpB inhibits NF-κB (nuclear factor κB) activation and pro-inflammatory cytokine secretions mediated by TLR (Toll-like receptor) 2 and TLR4. In the present study, we have demonstrated that TcpB modulates microtubule dynamics by acting as a stabilization factor. TcpB increased the rate of nucleation as well as the polymerization phases of microtubule formation in a similar manner to paclitaxel. TcpB could efficiently inhibit nocodazole- or cold-induced microtubule disassembly. Microtubule stabilization by TcpB is attributed to the BB-loop region of the TIR domain, and a point mutation affected the microtubule stabilization as well as the TLR-suppression properties of TcpB.

  17. Intracellular domains interactions and gated motions of IKS potassium channel subunits

    PubMed Central

    Haitin, Yoni; Wiener, Reuven; Shaham, Dana; Peretz, Asher; Cohen, Enbal Ben-Tal; Shamgar, Liora; Pongs, Olaf; Hirsch, Joel A; Attali, Bernard

    2009-01-01

    Voltage-gated K+ channels co-assemble with auxiliary β subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore-forming subunits with KCNE1 β subunits generates the repolarizing K+ current IKS. However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life-threatening long or short QT syndromes. Here, we studied the interactions and voltage-dependent motions of IKS channel intracellular domains, using fluorescence resonance energy transfer combined with voltage-clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C-terminus interacts with the coiled-coil helix C of the Kv7.1 tetramerization domain. This association is important for IKS channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant-negative C-terminal domain. On channel opening, the C-termini of Kv7.1 and KCNE1 come close together. Co-expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K+ currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C-termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation. PMID:19521339

  18. Intracellular domains interactions and gated motions of I(KS) potassium channel subunits.

    PubMed

    Haitin, Yoni; Wiener, Reuven; Shaham, Dana; Peretz, Asher; Cohen, Enbal Ben-Tal; Shamgar, Liora; Pongs, Olaf; Hirsch, Joel A; Attali, Bernard

    2009-07-22

    Voltage-gated K(+) channels co-assemble with auxiliary beta subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore-forming subunits with KCNE1 beta subunits generates the repolarizing K(+) current I(KS). However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life-threatening long or short QT syndromes. Here, we studied the interactions and voltage-dependent motions of I(KS) channel intracellular domains, using fluorescence resonance energy transfer combined with voltage-clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C-terminus interacts with the coiled-coil helix C of the Kv7.1 tetramerization domain. This association is important for I(KS) channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant-negative C-terminal domain. On channel opening, the C-termini of Kv7.1 and KCNE1 come close together. Co-expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K(+) currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C-termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation.

  19. Intracellular catalytic domain of symbiosis receptor kinase hyperactivates spontaneous nodulation in absence of rhizobia.

    PubMed

    Saha, Sudip; Dutta, Ayan; Bhattacharya, Avisek; DasGupta, Maitrayee

    2014-12-01

    Symbiosis Receptor Kinase (SYMRK), a member of the Nod factor signaling pathway, is indispensible for both nodule organogenesis and intracellular colonization of symbionts in rhizobia-legume symbiosis. Here, we show that the intracellular kinase domain of a SYMRK (SYMRK-kd) but not its inactive or full-length version leads to hyperactivation of the nodule organogenic program in Medicago truncatula TR25 (symrk knockout mutant) in the absence of rhizobia. Spontaneous nodulation in TR25/SYMRK-kd was 6-fold higher than rhizobia-induced nodulation in TR25/SYMRK roots. The merged clusters of spontaneous nodules indicated that TR25 roots in the presence of SYMRK-kd have overcome the control over both nodule numbers and their spatial position. In the presence of rhizobia, SYMRK-kd could rescue the epidermal infection processes in TR25, but colonization of symbionts in the nodule interior was significantly compromised. In summary, ligand-independent deregulated activation of SYMRK hyperactivates nodule organogenesis in the absence of rhizobia, but its ectodomain is required for proper symbiont colonization.

  20. Autophagy negatively regulates tumor cell proliferation through phosphorylation dependent degradation of the Notch1 intracellular domain

    PubMed Central

    Ahn, Ji-Seon; Ann, Eun-Jung; Kim, Mi-Yeon; Yoon, Ji-Hye; Lee, Hye-Jin; Jo, Eun-Hye; Lee, Keesook; Lee, Ji Shin; Park, Hee-Sae

    2016-01-01

    Autophagy is a highly conserved mechanism that degrades long-lived proteins and dysfunctional organelles, and contributes to cell fate. In this study, autophagy attenuates Notch1 signaling by degrading the Notch1 intracellular domain (Notch1-IC). Nutrient-deprivation promotes Notch1-IC phosphorylation by MEKK1 and phosphorylated Notch1-IC is recognized by Fbw7 E3 ligase. The ubiquitination of Notch1-IC by Fbw7 is essential for the interaction between Notch1-IC and p62 and for the formation of aggregates. Inhibition of Notch1 signaling prevents the transformation of breast cancer cells, tumor progression, and metastasis. The expression of Notch1 and p62 is inversely correlated with Beclin1 expression in human breast cancer patients. These results show that autophagy inhibits Notch1 signaling by promoting Notch1-IC degradation and therefore plays a role in tumor suppression. PMID:27806347

  1. Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein.

    PubMed

    Zhang, Yanfeng; Gao, Xiaoli; Michael Garavito, R

    2011-04-22

    The (pro)renin receptor (PRR) is an important component of the renin-angiotensin system (RAS), which regulates blood pressure and cardiovascular function. The integral membrane protein PRR contains a large extracellular domain (∼310 amino acids), a single transmembrane domain (∼20 amino acids) and an intracellular domain (∼19 amino acids). Although short, the intracellular (IC) domain of the PRR has functionally important roles in a number of signal transduction pathways activated by (pro)renin binding. Meanwhile, together with the transmembrane domain and a small portion of the extracellular domain (∼30 amino acids), the IC domain is also involved in assembly of V(0) portion of the vacuolar proton-translocating ATPase (V-ATPase). To better understand structural and multifunctional roles of the PRR-IC, we report the crystal structure of the PRR-IC domain as maltose-binding protein (MBP) fusion proteins at 2.0Å (maltose-free) and 2.15Å (maltose-bound). In the two separate crystal forms having significantly different unit-cell dimensions and molecular packing, MBP-PRR-IC fusion protein was found to be a dimer, which is different with the natural monomer of native MBP. The PRR-IC domain appears as a relatively flexible loop and is responsible for the dimerization of MBP fusion protein. Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermonomer interactions, suggesting a role for the PRR-IC domain in protein oligomerization.

  2. Emerin suppresses Notch signaling by restricting the Notch intracellular domain to the nuclear membrane.

    PubMed

    Lee, Byongsun; Lee, Tae-Hee; Shim, Jaekyung

    2017-02-01

    Emerin is an inner nuclear membrane protein that is involved in maintaining the mechanical integrity of the nuclear membrane. Increasing evidence supports the involvement of emerin in the regulation of gene expression; however, its precise function remains to be elucidated. Here, we show that emerin downregulated genes downstream of Notch signaling, which are activated exclusively by the Notch intracellular domain (NICD). Deletion mutant experiments revealed that the transmembrane domain of emerin is important for the inhibition of Notch signaling. Emerin interacted directly and colocalized with the NICD at the nuclear membrane. Emerin knockdown induced the phosphorylation of ERK and AKT, increased endogenous Notch signaling, and inhibited hydrogen peroxide-induced apoptosis in HeLa cells. Notably, the downregulation of barrier-to-autointegration factor (BAF) or lamin A/C increased Notch signaling by inducing the release of emerin into the cytosol, implying that nuclear membrane-bound emerin acts as an endogenous inhibitor of Notch signaling. Taken together, our results indicate that emerin negatively regulates Notch signaling by promoting the retention of the NICD at the nuclear membrane. This mechanism could constitute a new therapeutic target for the treatment of emerin-related diseases.

  3. The augmentation of intracellular delivery of peptide therapeutics by artificial protein transduction domains.

    PubMed

    Yoshikawa, Tomoaki; Sugita, Toshiki; Mukai, Yohei; Abe, Yasuhiro; Nakagawa, Shinsaku; Kamada, Haruhiko; Tsunoda, Shin-ichi; Tsutsumi, Yasuo

    2009-07-01

    Protein transduction domains (PTDs), such as HIV-derived Tat, have been successfully used as functional biomaterials for intracellular delivery of anti-cancer macromolecular drugs (protein, peptides, and oligonucleotides). Although there were therefore great expectations regarding the therapeutic potential of PTDs for the development of anti-cancer therapeutics, their clinical application so far has been extremely limited because of the relatively high concentrations required to mediate any effects on cancer cells in vitro or in vivo. In this context, improving the transduction efficiency of PTDs using phage display-based molecular evolution techniques may be useful for creating artificial PTDs with high efficiency and safety. Here, we report an evaluation of transduction efficiency and toxicity of such artificial PTDs (designated mT02 and mT03) compared with Tat. The internalization of mT02 was the most rapid and efficient by a mechanism different from the usual macropinocytosis. Furthermore, we found that artificial PTDs fused with survivin antagonistic peptide potentiate tumor cell-cytostatic activity. Thus, the results of this work provide new insights for designing new-generation peptide therapeutics for a wide variety of cancers as well as those expressing survivin.

  4. Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

    SciTech Connect

    Zhang, Yanfeng; Gao, Xiaoli; Michael Garavito, R.

    2011-04-22

    Highlights: {yields} Crystal structure of the intracellular domain of (pro)renin receptor (PRR-IC) as MBP fusion protein at 2.0 A (maltose-free) and 2.15 A (maltose-bound). {yields} MBP fusion protein is a dimer in crystals in the presence and absence of maltose. {yields} PRR-IC domain is responsible for the dimerization of the fusion protein. {yields} Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermolecular interactions, suggesting a role for the PRR-IC domain in PRR dimerization. -- Abstract: The (pro)renin receptor (PRR) is an important component of the renin-angiotensin system (RAS), which regulates blood pressure and cardiovascular function. The integral membrane protein PRR contains a large extracellular domain ({approx}310 amino acids), a single transmembrane domain ({approx}20 amino acids) and an intracellular domain ({approx}19 amino acids). Although short, the intracellular (IC) domain of the PRR has functionally important roles in a number of signal transduction pathways activated by (pro)renin binding. Meanwhile, together with the transmembrane domain and a small portion of the extracellular domain ({approx}30 amino acids), the IC domain is also involved in assembly of V{sub 0} portion of the vacuolar proton-translocating ATPase (V-ATPase). To better understand structural and multifunctional roles of the PRR-IC, we report the crystal structure of the PRR-IC domain as maltose-binding protein (MBP) fusion proteins at 2.0 A (maltose-free) and 2.15 A (maltose-bound). In the two separate crystal forms having significantly different unit-cell dimensions and molecular packing, MBP-PRR-IC fusion protein was found to be a dimer, which is different with the natural monomer of native MBP. The PRR-IC domain appears as a relatively flexible loop and is responsible for the dimerization of MBP fusion protein. Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermonomer interactions, suggesting a role for the PRR

  5. Intracellular Delivery of Peptidyl Ligands by Reversible Cyclization: Discovery of a PDZ Domain Inhibitor that Rescues CFTR Activity**

    PubMed Central

    Qian, Ziqing; Xu, Xiaohua; Amacher, Jeanine F.; Madden, Dean R.; Cormet-Boyaka, Estelle

    2015-01-01

    We report a general strategy for intracellular delivery of linear peptidyl ligands by fusing them with a cell-penetrating peptide and cyclizing the fusion peptides through a disulfide bond. The resulting cyclic peptides are cell permeable and have improved proteolytic stability. Once inside the cell, the disulfide bond is reduced to produce linear, biologically active peptides. This strategy was applied to generate a cell-permeable peptide substrate for real-time detection of intracellular caspase activities during apoptosis and a CAL-PDZ domain inhibitor for potential treatment of cystic fibrosis. PMID:25785567

  6. AICD -- Advanced Industrial Concepts Division Biological and Chemical Technologies Research Program. 1993 Annual summary report

    SciTech Connect

    Petersen, G.; Bair, K.; Ross, J.

    1994-03-01

    The annual summary report presents the fiscal year (FY) 1993 research activities and accomplishments for the United States Department of Energy (DOE) Biological and Chemical Technologies Research (BCTR) Program of the Advanced Industrial Concepts Division (AICD). This AICD program resides within the Office of Industrial Technologies (OIT) of the Office of Energy Efficiency and Renewable Energy (EE). The annual summary report for 1993 (ASR 93) contains the following: A program description (including BCTR program mission statement, historical background, relevance, goals and objectives), program structure and organization, selected technical and programmatic highlights for 1993, detailed descriptions of individual projects, a listing of program output, including a bibliography of published work, patents, and awards arising from work supported by BCTR.

  7. Structural fold, conservation and Fe(II) binding of the intracellular domain of prokaryote FeoB

    SciTech Connect

    Hung, Kuo-Wei; Chang, Yi-Wei; Eng, Edward T.; Chen, Jai-Hui; Chen, Yi-Chung; Sun, Yuh-Ju; Hsiao, Chwan-Deng; Dong, Gang; Spasov, Krasimir A.; Unger, Vinzenz M.; Huang, Tai-huang

    2010-09-17

    FeoB is a G-protein coupled membrane protein essential for Fe(II) uptake in prokaryotes. Here, we report the crystal structures of the intracellular domain of FeoB (NFeoB) from Klebsiella pneumoniae (KpNFeoB) and Pyrococcus furiosus (PfNFeoB) with and without bound ligands. In the structures, a canonical G-protein domain (G domain) is followed by a helical bundle domain (S-domain), which despite its lack of sequence similarity between species is structurally conserved. In the nucleotide-free state, the G-domain's two switch regions point away from the binding site. This gives rise to an open binding pocket whose shallowness is likely to be responsible for the low nucleotide-binding affinity. Nucleotide binding induced significant conformational changes in the G5 motif which in the case of GMPPNP binding was accompanied by destabilization of the switch I region. In addition to the structural data, we demonstrate that Fe(II)-induced foot printing cleaves the protein close to a putative Fe(II)-binding site at the tip of switch I, and we identify functionally important regions within the S-domain. Moreover, we show that NFeoB exists as a monomer in solution, and that its two constituent domains can undergo large conformational changes. The data show that the S-domain plays important roles in FeoB function.

  8. EndoU is a novel regulator of AICD during peripheral B cell selection

    PubMed Central

    Poe, Jonathan C.; Kountikov, Evgueni I.; Lykken, Jacquelyn M.; Natarajan, Abirami; Marchuk, Douglas A.

    2014-01-01

    Balanced transmembrane signals maintain a competent peripheral B cell pool limited in self-reactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22−/−[B6]) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag–specific B cells in IgTg hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance. PMID:24344237

  9. The Death Domain Superfamily in Intracellular Signaling of Apoptosis and Inflammation

    PubMed Central

    Park, Hyun Ho; Lo, Yu-Chih; Lin, Su-Chang; Wang, Liwei; Yang, Jin Kuk; Wu, Hao

    2010-01-01

    The death domain (DD) superfamily comprising the death domain (DD) subfamily, the death effector domain (DED) subfamily, the caspase recruitment domain (CARD) subfamily and the pyrin domains (PYD) subfamily is one of the largest domain superfamilies. By mediating homotypic interactions within each domain subfamily, these proteins play important roles in the assembly and activation of apoptotic and inflammatory complexes. In this article, we review the molecular complexes that are assembled by these proteins, the structural and biochemical features of these domains and the molecular interactions mediated by them. By analyzing the potential molecular basis for the function of these domains, we hope to provide a comprehensive understanding on the function, structure, interaction and evolution of this important family of domains. PMID:17201679

  10. Clinical significance of NOTCH1 intracellular cytoplasmic domain translocation into the nucleus in gastric cancer

    PubMed Central

    Saito, Shinichiro; Ishiguro, Hideyuki; Kimura, Masahiro; Ogawa, Ryo; Miyai, Hirotaka; Tanaka, Tatsuya; Mizoguchi, Koji; Takeyama, Hiromitsu

    2016-01-01

    Recent studies have shown constitutive activation of the Notch signaling pathway in various types of malignancies. However, it remains unclear whether this signaling pathway is activated in gastric cancer. In the present study, the aim was to investigate the role of Notch signaling in gastric cancer by investigating the subcellular localization of Notch-associated proteins in tissue samples from gastric cancer patients. Samples were obtained from 115 gastric cancer patients who had undergone surgery at the Department of Gastroenterological Surgery, Nagoya City University Graduate School of Medical Science without pre-operative chemotherapy or radiation. Subsequently the correlation between translocation of NOTCH1 intracellular cytoplasmic domain (NICD) into the nucleus (as measured by immunostaining) and survival in gastric cancer patients after surgery was investigated. The results were analyzed in reference to the patients' clinicopathological characteristics and the effects of these results on patient prognosis were determined. Significant correlations were observed between NICD nuclear localization and clinicopathological characteristics, such as tumor status (T factor), lymph node status (N factor), pathological stage and differentiation status. No significant correlations were observed between NICD nuclear localization and age, gender, tumor location, vein invasion or lymphatic invasion. Patients with >30% of cancer cell nuclei positively stained for NICD (as revealed by immunostaining) were associated with a significantly shorter survival following surgery than patients with <30% NICD-positive cancer cell nuclei (log-rank test, P=0.0194). Univariate analysis revealed that among the clinicopathological factors examined, T factor [risk rate (RR)=10.870; P=0.0016], N factor (RR=41.667; P=0.0003), lymphatic invasion (RR=13.158; P=0.0125), vein invasion (RR=25.000; P= 0.0019) and translocation of NICD to the nucleus (RR=3.937; P=0.0312) were all identified to be

  11. The intracellular domain of teneurin-1 interacts with MBD1 and CAP/ponsin resulting in subcellular codistribution and translocation to the nuclear matrix

    SciTech Connect

    Nunes, Samantha M.; Ferralli, Jacqueline; Choi, Karen; Brown-Luedi, Marianne; Minet, Ariane D.; Chiquet-Ehrismann, Ruth . E-mail: chiquet@fmi.ch

    2005-04-15

    Teneurin-1 is a type II transmembrane protein expressed in neurons of the developing and adult central nervous system. To investigate the intracellular signaling of teneurin-1, we searched for proteins interacting with its intracellular domain. One of the proteins identified is the c-Cbl-associated protein CAP/ponsin, an adaptor protein containing SH3 domains. This interaction results on one hand in the recruitment of the soluble intracellular domain of teneurin-1 to the cell membrane enriched in CAP/ponsin. On the other hand, it leads to the translocation of CAP/ponsin to the nucleus, the major site of accumulation of the intracellular domain of teneurin-1. The second interacting protein identified is the methyl-CpG binding protein MBD1. In the nucleus, the intracellular domain of teneurin-1 colocalizes with this transcriptional repressor in foci associated with the nuclear matrix. We propose that these interactions are part of a specific signaling pathway. Evidence for cleavage and nuclear translocation of the intracellular domain has been obtained by the detection of endogenous teneurin-1 immunoreactivity in nuclear speckles in chick embryo fibroblasts. Furthermore, in the nuclear matrix fraction of these cells as well as in cells expressing a hormone-inducible full-length teneurin-1 protein, a teneurin-1 fragment of identical size could be detected as in cells transfected with the intracellular domain alone.

  12. Long distance effect on ligand-gated ion channels extracellular domain may affect interactions with the intracellular machinery.

    PubMed

    Garret, Maurice; Boué-Grabot, Eric; Taly, Antoine

    2014-01-01

    Modulation of receptor trafficking is critical for controlling neurotransmission. A γ2(R43Q) point mutation on GABAA receptor subunit is linked to epilepsy in human. We recently analyzed the effect of this amino-acid substitution on GABAA receptor trafficking and showed that this mutation as well as agonist application, both affecting GABAA receptor extracellular domain, have an effect on receptor endocytosis. By comparing homology models based on ligand gated ion channels in their active and resting states, we reveal that the γ2R43 domain is located in a loop that is affected by motion resulting from receptor activation. Taken together, these results suggest that endocytosis of GABAA receptors is linked to agonist induced conformational changes. We propose that ligand or modulator binding is followed by a whole chain of interconnections, including the intracellular domain, that may influence ligand-gated channel trafficking.

  13. Long distance effect on ligand-gated ion channels extracellular domain may affect interactions with the intracellular machinery

    PubMed Central

    Garret, Maurice; Boué-Grabot, Eric; Taly, Antoine

    2014-01-01

    Modulation of receptor trafficking is critical for controlling neurotransmission. A γ2(R43Q) point mutation on GABAA receptor subunit is linked to epilepsy in human. We recently analyzed the effect of this amino-acid substitution on GABAA receptor trafficking and showed that this mutation as well as agonist application, both affecting GABAA receptor extracellular domain, have an effect on receptor endocytosis. By comparing homology models based on ligand gated ion channels in their active and resting states, we reveal that the γ2R43 domain is located in a loop that is affected by motion resulting from receptor activation. Taken together, these results suggest that endocytosis of GABAA receptors is linked to agonist induced conformational changes. We propose that ligand or modulator binding is followed by a whole chain of interconnections, including the intracellular domain, that may influence ligand-gated channel trafficking. PMID:25254078

  14. EpCAM Intracellular Domain Promotes Porcine Cell Reprogramming by Upregulation of Pluripotent Gene Expression via Beta-catenin Signaling

    PubMed Central

    Yu, Tong; Ma, Yangyang; Wang, Huayan

    2017-01-01

    Previous study showed that expression of epithelial cell adhesion molecule (EpCAM) was significantly upregulated in porcine induced pluripotent stem cells (piPSCs). However, the regulatory mechanism and the downstream target genes of EpCAM were not well investigated. In this study, we found that EpCAM was undetectable in fibroblasts, but highly expressed in piPSCs. Promoter of EpCAM was upregulated by zygotic activated factors LIN28, and ESRRB, but repressed by maternal factors OCT4 and SOX2. Knocking down EpCAM by shRNA significantly reduced the pluripotent gene expression. Conversely, overexpression of EpCAM significantly increased the number of alkaline phosphatase positive colonies and elevated the expression of endogenous pluripotent genes. As a key surface-to-nucleus factor, EpCAM releases its intercellular domain (EpICD) by a two-step proteolytic processing sequentially. Blocking the proteolytic processing by inhibitors TAPI-1 and DAPT could reduce the intracellular level of EpICD and lower expressions of OCT4, SOX2, LIN28, and ESRRB. We noticed that increasing intracellular EpICD only was unable to improve activity of EpCAM targeted genes, but by blocking GSK-3 signaling and stabilizing beta-catenin signaling, EpICD could then significantly stimulate the promoter activity. These results showed that EpCAM intracellular domain required beta-catenin signaling to enhance porcine cell reprogramming. PMID:28393933

  15. PRR repeats in the intracellular domain of KISS1R are important for its export to cell membrane.

    PubMed

    Chevrier, Lucie; de Brevern, Alexandre; Hernandez, Eva; Leprince, Jérome; Vaudry, Hubert; Guedj, Anne Marie; de Roux, Nicolas

    2013-06-01

    Inactivating mutations of KISS-1 receptor (KISS1R) have been recently described as a rare cause of isolated hypogonadotropic hypogonadism transmitted as a recessive trait. Few mutations have been described, and the structure-function relationship of KISS1R remains poorly understood. Here, we have taken advantage of the discovery of a novel mutation of KISS1R to characterize the structure and function of an uncommon protein motif composed of 3 proline-arginine-arginine (PRR) repeats located within the intracellular domain. A heterozygous insertion of 1 PRR repeat in-frame with 3 PRR repeats leading to synthesis of a receptor bearing 4 PRR repeats (PRR-KISS1R) was found in the index case. Functional analysis of PRR-KISS1R showed a decrease of the maximal response to kisspeptin stimulation, associated to a lower cell surface expression without modification of total expression. PRR-KISS1R exerts a dominant negative effect on the synthesis of the wild-type (WT)-KISS1R. This effect was due to the nature of inserted residues but also to the difference of the length of the intracellular domain between PRR-KISS1R and WT-KISS1R. A molecular dynamic analysis showed that the additional PRR constrained this arginine-rich region into a polyproline type II helix. Altogether, this study shows that a heterozygous insertion in KISS1R may lead to hypogonadotropic hypogonadism by a dominant negative effect on the WT receptor. An additional PRR repeat into a proline-arginine-rich motif can dramatically changed the conformation of the intracellular domain of KISS1R and its probable interaction with partner proteins.

  16. Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells

    PubMed Central

    Lu, Yinzhong; Wan, Jun; Yang, Zhifeng; Lei, Xiling; Niu, Qi; Jiang, Lanxin; Passtoors, Willemijn M.; Zang, Aiping; Fraering, Patrick C.; Wu, Fang

    2017-01-01

    Deregulation of the TAM (TYRO3, AXL, and MERTK) family of receptor tyrosine kinases (RTKs) has recently been demonstrated to predominately promote survival and chemoresistance of cancer cells. Intramembrane proteolysis mediated by presenilin/γ-secretase is known to regulate the homeostasis of some RTKs. In the present study, we demonstrate that AXL, but not TYRO3 or MERTK, is efficiently and sequentially cleaved by α- and γ-secretases in various types of cancer cell lines. Proteolytic processing of AXL redirected signaling toward a secretase-mediated pathway, away from the classic, well-known, ligand-dependent canonical RTK signaling pathway. The AXL intracellular domain cleavage product, but not full-length AXL, was further shown to translocate into the nucleus via a nuclear localization sequence that harbored a basic HRRKK motif. Of interest, we found that the γ-secretase–uncleavable AXL mutant caused an elevated chemoresistance in non–small-cell lung cancer cells. Altogether, our findings suggest that AXL can undergo sequential processing mediated by various proteases kept in a homeostatic balance. This newly discovered post-translational processing of AXL may provide an explanation for the diverse functions of AXL, especially in the context of drug resistance in cancer cells.—Lu, Y., Wan, J., Yang, Z., Lei, X., Niu, Q., Jiang, L., Passtoors, W. M., Zang, A., Fraering, P. C., Wu, F. Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells. PMID:28034848

  17. The γ-secretase-generated intracellular domain of β-amyloid precursor protein binds Numb and inhibits Notch signaling

    PubMed Central

    Roncarati, Roberta; Šestan, Nenad; Scheinfeld, Meir H.; Berechid, Bridget E.; Lopez, Peter A.; Meucci, Olimpia; McGlade, Jane C.; Rakic, Pasko; D'Adamio, Luciano

    2002-01-01

    The β-amyloid precursor protein (APP) and the Notch receptor undergo intramembranous proteolysis by the Presenilin-dependent γ-secretase. The cleavage of APP by γ-secretase releases amyloid-β peptides, which have been implicated in the pathogenesis of Alzheimer's disease, and the APP intracellular domain (AID), for which the function is not yet well understood. A similar γ-secretase-mediated cleavage of the Notch receptor liberates the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates the transcription of genes that regulate the generation, differentiation, and survival of neuronal cells. Hence, some of the effects of APP signaling and Alzheimer's disease pathology may be mediated by the interaction of APP and Notch. Here, we show that membrane-tethered APP binds to the cytosolic Notch inhibitors Numb and Numb-like in mouse brain lysates. AID also binds Numb and Numb-like, and represses Notch activity when released by APP. Thus, γ-secretase may have opposing effects on Notch signaling; positive by cleaving Notch and generating NICD, and negative by processing APP and generating AID, which inhibits the function of NICD. PMID:12011466

  18. Speckle fluctuation spectroscopy of intracellular motion in living tissue using coherence-domain digital holography

    NASA Astrophysics Data System (ADS)

    Jeong, Kwan; Turek, John J.; Nolte, David D.

    2010-05-01

    Dynamic speckle from 3-D coherence-gated optical sections provides a sensitive label-free measure of cellular activity up to 1 mm deep in living tissue. However, specificity to cellular functionality has not previously been demonstrated. In this work, we perform fluctuation spectroscopy on dynamic light scattering captured using coherence-domain digital holography to obtain the spectral response of tissue that is perturbed by temperature, osmolarity, and antimitotic cytoskeletal drugs. Different perturbations induce specific spectrogram response signatures that can show simultaneous enhancement and suppression in different spectral ranges.

  19. Secretin receptor oligomers form intracellularly during maturation through receptor core domains.

    PubMed

    Lisenbee, Cayle S; Miller, Laurence J

    2006-07-11

    Oligomerization of numerous G protein-coupled receptors has been documented, including the prototypic family B secretin receptor. The clinical significance of oligomerization of this receptor became clear with the recent observation that a misspliced form present in pancreatic cancer could associate with the wild-type receptor and act as a dominant negative inhibitor of its normal growth inhibitory function. Our goal was to explore the molecular mechanism of this interaction using bioluminescence (BRET) and fluorescence (FRET) resonance energy transfer and fluorescence microscopy with a variety of receptor constructs tagged with luciferase or cyan or yellow fluorescent proteins. BRET signals comparable to those obtained from cells coexpressing differentially tagged wild-type receptors were observed for similarly tagged secretin receptors in which all or part of the amino-terminal domain was deleted. As expected, neither of these constructs bound secretin, and only the partially truncated construct sorted to the plasma membrane. Receptors lacking the majority of the carboxyl-terminal domain, including that important for phosphorylation-mediated desensitization, also produced BRET signals above background. These findings suggested that the receptor's membrane-spanning core is responsible for secretin receptor oligomerization. Interestingly, alanine substitutions for a -GxxxG- helix interaction motif in transmembrane segment 7 created nonfunctional receptors that were capable of forming oligomers. Furthermore, treatment of receptor-expressing cells with brefeldin A did not eliminate the BRET signals, and morphologic FRET experiments confirmed the expected subcellular localizations of receptor oligomers. We conclude that secretin receptor oligomerization occurs through -GxxxG- motif-independent interactions of transmembrane segments during the maturation of nascent molecules.

  20. Allosteric Coupling between the Intracellular Coupling Helix 4 and Regulatory Sites of the First Nucleotide-binding Domain of CFTR

    PubMed Central

    Dawson, Jennifer E.; Farber, Patrick J.; Forman-Kay, Julie D.

    2013-01-01

    Cystic fibrosis is caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator), leading to folding and processing defects and to chloride channel gating misfunction. CFTR is regulated by ATP binding to its cytoplasmic nucleotide-binding domains, NBD1 and NBD2, and by phosphorylation of the NBD1 regulatory insert (RI) and the regulatory extension (RE)/R region. These regulatory effects are transmitted to the rest of the channel via NBD interactions with intracellular domain coupling helices (CL), particularly CL4. Using a sensitive method for detecting inter-residue correlations between chemical shift changes in NMR spectra, an allosteric network was revealed within NBD1, with a construct lacking RI. The CL4-binding site couples to the RI-deletion site and the C-terminal residues of NBD1 that precede the R region in full-length CFTR. Titration of CL4 peptide into NBD1 perturbs the conformational ensemble in these sites with similar titration patterns observed in F508del, the major CF-causing mutant, and in suppressor mutants F494N, V510D and Q637R NBD1, as well as in a CL4-NBD1 fusion construct. Reciprocally, the C-terminal mutation, Q637R, perturbs dynamics in these three sites. This allosteric network suggests a mechanism synthesizing diverse regulatory NBD1 interactions and provides biophysical evidence for the allosteric coupling required for CFTR function. PMID:24058550

  1. Intracellular Cleavage of the Cx43 C-Terminal Domain by Matrix-Metalloproteases: A Novel Contributor to Inflammation?

    PubMed Central

    De Bock, Marijke; Wang, Nan; Decrock, Elke; Bultynck, Geert; Leybaert, Luc

    2015-01-01

    The coordination of tissue function is mediated by gap junctions (GJs) that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexin (Cx) proteins of which Cx43 is most widespread in the human body. Beyond its role in direct intercellular communication, Cx43 also forms nonjunctional hemichannels (HCs) in the plasma membrane that mediate the release of paracrine signaling molecules in the extracellular environment. Both HC and GJ channel function are regulated by protein-protein interactions and posttranslational modifications that predominantly take place in the C-terminal domain of Cx43. Matrix metalloproteases (MMPs) are a major group of zinc-dependent proteases, known to regulate not only extracellular matrix remodeling, but also processing of intracellular proteins. Together with Cx43 channels, both GJs and HCs, MMPs contribute to acute inflammation and a small number of studies reports on an MMP-Cx43 link. Here, we build further on these reports and present a novel hypothesis that describes proteolytic cleavage of the Cx43 C-terminal domain by MMPs and explores possibilities of how such cleavage events may affect Cx43 channel function. Finally, we set out how aberrant channel function resulting from cleavage can contribute to the acute inflammatory response during tissue injury. PMID:26424967

  2. Differential functions of the Apoer2 intracellular domain in selenium uptake and cell signaling.

    PubMed

    Masiulis, Irene; Quill, Timothy A; Burk, Raymond F; Herz, Joachim

    2009-01-01

    Apolipoprotein E receptor 2 (Apoer2) is a multifunctional transport and signaling receptor that regulates the uptake of selenium into the mouse brain and testis through endocytosis of selenoprotein P (Sepp1). Mice deficient in Apoer2 or Sepp1 are infertile, with kinked and hypomotile spermatozoa. They also develop severe neurological defects on a low selenium diet, due to a profound impairment of selenium uptake. Little is known about the function of Apoer2 in the testis beyond its role as a Sepp1 receptor. By contrast, in the brain, Apoer2 is an essential component of the Reelin signaling pathway, which is required for proper neuronal organization and synapse function. Using knock-in mice, we have functionally dissociated the signaling motifs in the Apoer2 cytoplasmic domain from Sepp1 uptake. Selenium concentration of brain and testis was normal in the knock-in mutants, in contrast to Apoer2 knock-outs. Thus, the neurological defects in the signaling impaired knock-in mice are not caused by a selenium uptake defect, but instead are a direct consequence of a disruption of the Reelin signal. Reduced sperm motility was observed in some of the knock-in mice, indicating a novel signaling role for Apoer2 in sperm development and function that is independent of selenium uptake.

  3. Phase II Study of a HER-2/neu (HER2) Intracellular Domain (ICD) Peptide-Based Vaccine Administered to Stage IIIB and IV HER2 Positive Breast Cancer Patients Receiving Trastuzumab Monotherapy

    DTIC Science & Technology

    2008-05-01

    Intracellular Domain (ICD) Peptide - Based Vaccine Administered to Stage IIIB and IV HER2 Positive Breast Cancer Patients Receiving Trastuzumab...To) 27 APR 2007 - 26 APR 2008 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Phase II Study of a HER-2/neu (HER2) Intracellular Domain (ICD) Peptide ...intracellular domain (ICD) peptide -based vaccine while receiving maintenance trastuzumab. Patients enrolled will be HER2 overexpressing stage IIIB and IV

  4. Amino acids in the COOH-terminal region of the oxytocin receptor third intracellular domain are important for receptor function.

    PubMed

    Zhong, Miao; Parish, Bridgette; Murtazina, Dilyara A; Ku, Chun-Ying; Sanborn, Barbara M

    2007-04-01

    Previously, residue K6.30 in the COOH-terminal region of the third intracellular domain (3iC) of the oxytocin (OT) receptor (OTR) was identified as important for receptor function leading to phospholipase C activation in both OTR and the vasopressin V(2) receptor (V(2)R) chimera V(2)ROTR3iC. Substitution of either A6.28K or V6.30K in wild-type V(2)R did not recapitulate the increase in phosphatidylinositide (PI) turnover observed in V(2)ROTR3iC. Hence, the role of K6.30 may be context-specific. Deletion of two NH(2)-terminal OTR3iC segments in the V(2)ROTR3iC chimera did not diminish vasopressin-stimulated PI turnover, whereas deletion of RVSSVKL (residues 6.19-6.25) reduced receptor expression. Deletion of this sequence in wild-type OTR reduced expression by 50% without affecting affinity for [(3)H]OT. This OTR mutant was unable to activate PI turnover or extracellular signal-regulated kinase 1/2 phosphorylation. The effects of alanine substitution for individual residues in RVSSVKL indicated differential importance for OTR function. The R6.19A substitution lost high-affinity sites for [(3)H]OT and the ability to stimulate PI turnover. Affinity for [(3)H]OT and membrane expression was not affected by any other substitutions. OTR-V6.20A and OTR-K6.24A mutants functioned as well as wild-type OTR, whereas OTR S6.21A, S6.22A, and V6.23A mutants exhibited impaired abilities to activate PI turnover (20-40% of OTR), and the OTR-L6.25A mutant exhibited constitutive activity. In conclusion, specific amino acids in the RVSSVKL segment in the COOH-terminal region of the third intracellular domain of OTR influence the ability of OTR to activate G protein-mediated actions.

  5. Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis.

    PubMed

    Greenwood, Alexander I; Rogals, Monique J; De, Soumya; Lu, Kun Ping; Kovrigin, Evgenii L; Nicholson, Linda K

    2011-09-01

    The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, [Formula: see text] and apparent Michaelis constants, [Formula: see text]. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific (13)C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide (13)C-(1)H constant time HSQC spectra to determine [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.

  6. Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene in P. putida CA-3.

    PubMed

    O'Leary, Niall D; Mooney, Aisling; O'Mahony, Mark; Dobson, Alan Dw

    2014-01-01

    Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection and/or activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed.

  7. Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene in P. putida CA-3

    PubMed Central

    O'Leary, Niall D; Mooney, Aisling; O'Mahony, Mark; Dobson, Alan DW

    2014-01-01

    Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection and/or activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed. PMID:24637704

  8. Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain

    SciTech Connect

    Endsley, Mark A.; Somasunderam, Anoma D.; Li, Guangyu; Oezguen, Numan; Thiviyanathan, Varatharasa; Murray, James L.; Rubin, Donald H.; Hodge, Thomas W.; and others

    2014-04-15

    Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4{sup +} T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizes with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry. - Highlights: • Nuclear trafficking of the HIV-1 pre-integration complex depends on ADAM10. • ADAM10 associates with HIV-1 integrase in the pre-integration complex. • HIV-1 replication depends on the expression of ADAM15 and γ-secretase. • Silencing ADAM15 or γ-secretase expression inhibits nuclear import of viral cDNA. • ADAM10 is important for HIV-1 replication in human macrophages and CD4{sup +} T lymphocytes.

  9. EphB1 and EphB2 intracellular domains regulate the formation of the corpus callosum and anterior commissure.

    PubMed

    Robichaux, Michael A; Chenaux, George; Ho, Hsin-Yi Henry; Soskis, Michael J; Greenberg, Michael E; Henkemeyer, Mark; Cowan, Christopher W

    2016-04-01

    The two cortical hemispheres of the mammalian forebrain are interconnected by major white matter tracts, including the corpus callosum (CC) and the posterior branch of the anterior commissure (ACp), that bridge the telencephalic midline. We show here that the intracellular signaling domains of the EphB1 and EphB2 receptors are critical for formation of both the ACp and CC. We observe partial and complete agenesis of the corpus callosum, as well as highly penetrant ACp misprojection phenotypes in truncated EphB1/2 mice that lack intracellular signaling domains. Consistent with the roles for these receptors in formation of the CC and ACp, we detect expression of these receptors in multiple brain regions associated with the formation of these forebrain structures. Taken together, our findings suggest that a combination of forward and reverse EphB1/2 receptor-mediated signaling contribute to ACp and CC axon guidance.

  10. Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription

    PubMed Central

    Tuand, Krizia; Stijnen, Pieter; Volders, Karolien; Declercq, Jeroen; Nuytens, Kim; Meulemans, Sandra; Creemers, John

    2016-01-01

    Background Neurobeachin (NBEA) is an autism spectrum disorders (ASD) candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA)-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA), an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088) and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW). Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed. Methods Yeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO) enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression. Results Both domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated. Conclusion Defining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for

  11. Number and brightness analysis of sFRP4 domains in live cells demonstrates vesicle association signal of the NLD domain and dynamic intracellular responses to Wnt3a

    PubMed Central

    Perumal, Vanathi; Krishnan, Kannan; Gratton, Enrico; Dharmarajan, Arun M; Fox, Simon A

    2015-01-01

    The Wnts are secreted, lipidated glycoproteins that play a role in cellular processes of differentiation, proliferation, migration, survival, polarity and stem cell self-renewal. The majority of Wnts biological effects are through binding to specific frizzled (Fzd) receptor complexes leading to activation of downstream pathways. Secreted Frizzled-related proteins (sFRPs) were first identified as antagonists of Wnt signalling by binding directly to Wnts. They comprise two domains, a Fzd-like cysteine rich domain (CRD) and a netrin-like domain (NLD). Subsequently sFRPs have been shown to also interact with Fzd receptors and more diverse functions have been identified, including potentiation of Wnt signalling. Many aspects of the biology of this family remain to be elucidated. We used the number and brightness (N&B) method, a technique based on fluorescence fluctuation analysis, to characterise the intracellular aggregation and trafficking of sFRP4 domains. We expressed sFRP4 and its’ domains as EGFP fusions and then characterised the effect of endogenous Wnt3a by fluorescence confocal imaging. We observed vesicular trafficking of sFRP4 and that the NLD domain has a vesicular association signal. We found that sFRP4 and the CRD formed oligomeric aggregates in the perinuclear region while the NLD was distributed evenly throughout the cell with a larger proportion of aggregates. Most significantly we observed intracellular redistribution of sFRP4 in response to Wnt3a suggesting that Wnt3a can modulate intracellular localisation and secretion of sFRP4. Our results reveal a number of novel findings regarding sFRP4 which are likely to have relevance to this wider family. PMID:25805505

  12. Inhibition of insulin-stimulated phosphorylation of the intracellular domain of phospholemman decreases insulin-dependent GLUT4 translocation in streptolysin-O-permeabilized adipocytes.

    PubMed Central

    Walaas, O; Horn, R S; Walaas, S I

    1999-01-01

    A variety of studies indicate that protein kinase C might be involved in the insulin signalling cascade leading to translocation of the insulin-regulated glucose transporter GLUT4 from intracellular pools to the plasma membrane. Phospholemman is a plasma-membrane protein kinase C substrate whose phosphorylation is increased by insulin in intact muscle [Walaas, Czernik, Olstad, Sletten and Walaas (1994) Biochem. J. 304, 635-640]. The present study examined whether the inhibition of phospholemman phosphorylation modulates the effects of insulin on GLUT4 translocation. For this purpose, a synthetic peptide derived from the intracellular domain of phospholemman with the phosphorylatable serine residues replaced with alanine residues was prepared. This peptide was found to decrease the protein kinase C-catalysed phosphorylation of a synthetic phospholemman peptide in vitro. When introduced into streptolysin-O-permeabilized adipocytes, the peptide decreased the effects of insulin on both the phosphorylation of phospholemman and the recruitment of GLUT4 to the plasma membrane. Similarly, the internalization of phospholemman antibodies, which also decreased the protein kinase C-mediated phosphorylation of the synthetic phospholemman peptide in vitro, decreased the effect of insulin on GLUT4 translocation in the adipocytes. The results suggest that phosphorylation of the intracellular domain of phospholemman might be involved in modulating the insulin-induced translocation of GLUT4 to the plasma membrane. PMID:10493924

  13. The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

    PubMed

    Kim, Daehwan; Ko, Panseon; You, Eunae; Rhee, Sangmyung

    2014-12-01

    Discoidin domain receptors (DDRs) are unusual receptor tyrosine kinases (RTKs) that are activated by fibrillar collagens instead of soluble growth factors. DDRs play an important role in various cellular functions and disease processes, including malignant progression. Compared to other RTKs, DDRs have relatively long juxtamembrane domains, which are believed to contribute to receptor function. Despite this possibility, the function and mechanism of the juxtamembrane domain of DDRs have not yet been fully elucidated. In this study, we found that the cytoplasmic juxtamembrane 2 (JM2) region of DDR2 contributed to receptor dimerization, which is critical for receptor activation in response to collagen stimulation. A collagen-binding assay showed that JM2 was required for efficient binding of collagen to the discoidin (DS) domain. Immunohistochemical analysis of DDR2 expression using a tissue microarray demonstrated that DDR2 was overexpressed in several carcinoma tissues, including bladder, testis, lung, kidney, prostate and stomach. In H1299 cells, inhibition of DDR2 activity by overexpressing the juxtamembrane domain containing JM2 suppressed collagen-induced colony formation, cell proliferation and invasion via the inhibition of matrix metalloproteinase-2 and matrix metalloproteinase-9. Taken together, our results suggest that JM2-mediated dimerization is likely to be essential for DDR2 activation and cancer progression. Thus, inhibition of DDR2 function using a JM2-containing peptide might be a useful strategy for the treatment of DDR2-positive cancers.

  14. Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII

    PubMed Central

    Wei, Wei; Zheng, Chunlei; Zhu, Min; Zhu, Xiaofan; Yang, Renchi; Misra, Saurav; Zhang, Bin

    2017-01-01

    Missense mutation is the most common mutation type in hemophilia. However, the majority of missense mutations remain uncharacterized. Here we characterize how hemophilia mutations near the unused N-glycosylation site of the A2 domain (N582) of FVIII affect protein conformation and intracellular trafficking. N582 is located in the middle of a short 310-helical turn (D580-S584), in which most amino acids have multiple hemophilia mutations. All 14 missense mutations found in this 310-helix reduced secretion levels of the A2 domain and full-length FVIII. Secreted mutants have decreased activities relative to WT FVIII. Selected mutations also lead to partial glycosylation of N582, suggesting that rapid folding of local conformation prevents glycosylation of this site in wild-type FVIII. Protease sensitivity, stability and degradation of the A2 domain vary among mutants, and between non-glycosylated and glycosylated species of the same mutant. Most of the mutants interact with the ER chaperone BiP, while only mutants with aberrant glycosylation interact with calreticulin. Our results show that the short 310-helix from D580 to S584 is critical for proper biogenesis of the A2 domain and FVIII, and reveal a range of molecular mechanisms by which FVIII missense mutations lead to moderate to severe hemophilia A. PMID:28327546

  15. Chimeras of sperm PLCζ reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization

    PubMed Central

    Theodoridou, Maria; Nomikos, Michail; Parthimos, Dimitris; Gonzalez-Garcia, J. Raul; Elgmati, Khalil; Calver, Brian L.; Sideratou, Zili; Nounesis, George; Swann, Karl; Lai, F. Anthony

    2013-01-01

    Phospholipase C-zeta (PLCζ) is a sperm-specific protein believed to cause Ca2+ oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 isoform but is far more potent in mobilizing Ca2+ in eggs. To investigate how discrete protein domains contribute to Ca2+ release, we assessed the function of a series of PLCζ/PLCδ1 chimeras. We examined their ability to cause Ca2+ oscillations in mouse eggs, enzymatic properties using in vitro phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolyzed PIP2 with no major differences in Ca2+ sensitivity and enzyme kinetics. Insertion of a PH domain or replacement of the PLCζ EF hands domain had no deleterious effect on Ca2+ oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCζ completely abolished Ca2+ releasing activity. Notably, chimeras containing the PLCζ XY-linker bound to PIP2-containing liposomes, while chimeras containing the PLCζ C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca2+ sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XY-linker of PLCζ. To investigate the relationship between PLC enzymatic properties and Ca2+ oscillations in eggs, we have developed a mathematical model that incorporates Ca2+-dependent InsP3 generation by the PLC chimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency of Ca2+ oscillatory activity associated with specific PLC variants. PMID:24152875

  16. Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular c-terminal domain in complex with calmodulin.

    PubMed

    Sachyani, Dana; Dvir, Meidan; Strulovich, Roi; Tria, Giancarlo; Tobelaim, William; Peretz, Asher; Pongs, Olaf; Svergun, Dmitri; Attali, Bernard; Hirsch, Joel A

    2014-11-04

    Kv7 channels tune neuronal and cardiomyocyte excitability. In addition to the channel membrane domain, they also have a unique intracellular C-terminal (CT) domain, bound constitutively to calmodulin (CaM). This CT domain regulates gating and tetramerization. We investigated the structure of the membrane proximal CT module in complex with CaM by X-ray crystallography. The results show how the CaM intimately hugs a two-helical bundle, explaining many channelopathic mutations. Structure-based mutagenesis of this module in the context of concatemeric tetramer channels and functional analysis along with in vitro data lead us to propose that one CaM binds to one individual protomer, without crosslinking subunits and that this configuration is required for proper channel expression and function. Molecular modeling of the CT/CaM complex in conjunction with small-angle X-ray scattering suggests that the membrane proximal region, having a rigid lever arm, is a critical gating regulator.

  17. Synthesis of a pH-Sensitive Nitrilotriacetic Linker to Peptide Transduction Domains To Enable Intracellular Delivery of Histidine Imidazole Ring-Containing Macromolecules

    PubMed Central

    2010-01-01

    Intracellular delivery of functional macromolecules using peptide transduction domains (PTDs) is an exciting technology with both experimental and therapeutic applications. Recent data indicate that PTD-mediated transduction occurs via fluid-phase macropinocytosis involving an intracellular pH drop to ∼5. Nitrilotriacetic acid (NTA)-coordinated metals avidly bind hexahistidine-tagged macromolecules, including peptides and proteins. Histidine’s imidazole ring has a pKa of 6, making this an attractive target for the biological pH drop of PTD-mediated macropinocytotic delivery. The objective of this study was to develop a pH-sensitive PTD delivery peptide (NTA3-PTD). We demonstrate the in vitro function of this novel peptide by delivering fluorescently labeled peptides (1.6 kDa) and functional enzymes, β-galactosidase (119 kDa) and Cre recombinase (37 kDa). Furthermore, the NTA3-PTD peptide was able to deliver functional Cre recombinase in an in vivo mouse model. PMID:20681698

  18. Detection of novel intracellular agonist responsive pools of phosphatidylinositol 3,4-bisphosphate using the TAPP1 pleckstrin homology domain in immunoelectron microscopy.

    PubMed Central

    Watt, Stephen A; Kimber, Wendy A; Fleming, Ian N; Leslie, Nick R; Downes, C Peter; Lucocq, John M

    2004-01-01

    PtdIns(3,4) P (2), a breakdown product of the lipid second messenger PtdIns(3,4,5) P (3), is a key signalling molecule in pathways controlling various cellular events. Cellular levels of PtdIns(3,4) P (2) are elevated upon agonist stimulation, mediating downstream signalling pathways by recruiting proteins containing specialized lipid-binding modules, such as the pleckstrin homology (PH) domain. A recently identified protein, TAPP1 (tandem-PH-domain-containing protein 1), has been shown to interact in vitro with high affinity and specificity with PtdIns(3,4) P (2) through its C-terminal PH domain. In the present study, we have utilized this PH domain tagged with glutathione S-transferase (GST-TAPP1-PH) as a probe in an on-section immunoelectron microscopy labelling procedure, mapping the subcellular distribution of PtdIns(3,4) P (2). As expected, we found accumulation of PtdIns(3,4) P (2) at the plasma membrane in response to the agonists platelet-derived growth factor and hydrogen peroxide. Importantly, however, we also found agonist stimulated PtdIns(3,4) P (2) labelling of intracellular organelles, including the endoplasmic reticulum and multivesicular endosomes. Expression of the 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in PTEN-null U87MG cells revealed differential sensitivity of these lipid pools to the enzyme. These data suggest a role for PtdIns(3,4) P (2) in endomembrane function. PMID:14604433

  19. The Arginine/Lysine-Rich Element within the DNA-Binding Domain Is Essential for Nuclear Localization and Function of the Intracellular Pathogen Resistance 1

    PubMed Central

    Yao, Kezhen; Wu, Yongyan; Chen, Qi; Zhang, Zihan; Chen, Xin; Zhang, Yong

    2016-01-01

    The mouse intracellular pathogen resistance 1 (Ipr1) gene plays important roles in mediating host immunity and previous work showed that it enhances macrophage apoptosis upon mycobacterium infection. However, to date, little is known about the regulation pattern of Ipr1 action. Recent studies have investigated the protein-coding genes and microRNAs regulated by Ipr1 in mouse macrophages, but the structure and the functional motif of the Ipr1 protein have yet to be explored. In this study, we analyzed the domains and functional motif of the Ipr1 protein. The resulting data reveal that Ipr1 protein forms a homodimer and that the Sp100-like domain mediates the targeting of Ipr1 protein to nuclear dots (NDs). Moreover, we found that an Ipr1 mutant lacking the classic nuclear localization signal (cNLS) also translocated into the nuclei, suggesting that the cNLS is not the only factor that directs Ipr1 nuclear localization. Additionally, mechanistic studies revealed that an arginine/lysine-rich element within the DNA-binding domain (SAND domain) is critical for Ipr1 binding to the importin protein receptor NPI-1, demonstrating that this element plays an essential role in mediating the nuclear localization of Ipr1 protein. Furthermore, our results show that this arginine/lysine-rich element contributes to the transcriptional regulation and apoptotic activity of Ipr1. These findings highlight the structural foundations of Ipr1 action and provide new insights into the mechanism of Ipr1-mediated resistance to mycobacterium. PMID:27622275

  20. Functional analysis of a breast cancer-associated mutation in the intracellular domain of the metalloprotease ADAM12.

    PubMed

    Stautz, Dorte; Wewer, Ulla M; Kveiborg, Marie

    2012-01-01

    A recently identified breast cancer-associated mutation in the metalloprotease ADAM12 alters a potential dileucine trafficking signal, which could affect protein processing and cellular localization. ADAM12 belongs to the group of A Disintegrin And Metalloproteases (ADAMs), which are typically membrane-associated proteins involved in ectodomain shedding, cell-adhesion, and signaling. ADAM12 as well as several members of the ADAM family are over-expressed in various cancers, correlating with disease stage. Three breast cancer-associated somatic mutations were previously identified in ADAM12, and two of these, one in the metalloprotease domain and another in the disintegrin domain, were investigated and found to result in protein misfolding, retention in the secretory pathway, and failure of zymogen maturation. The third mutation, p.L792F in the ADAM12 cytoplasmic tail, was not investigated, but is potentially significant given its location within a di-leucine motif, which is recognized as a potential cellular trafficking signal. The present study was motivated both by the potential relevance of this documented mutation to cancer, as well as for determining the role of the di-leucine motif in ADAM12 trafficking. Expression of ADAM12 p.L792F in mammalian cells demonstrated quantitatively similar expression levels and zymogen maturation as wild-type (WT) ADAM12, as well as comparable cellular localizations. A cell surface biotinylation assay demonstrated that cell surface levels of ADAM12 WT and ADAM12 p.L792F were similar and that internalization of the mutant occurred at the same rate and extent as for ADAM12 WT. Moreover, functional analysis revealed no differences in cell proliferation or ectodomain shedding of epidermal growth factor (EGF), a known ADAM12 substrate between WT and mutant ADAM12. These data suggest that the ADAM12 p.L792F mutation is unlikely to be a driver (cancer causing)-mutation in breast cancer.

  1. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels

    PubMed Central

    Wei, Shipeng; Roessler, Bryan C.; Icyuz, Mert; Chauvet, Sylvain; Tao, Binli; Hartman, John L.; Kirk, Kevin L.

    2015-01-01

    The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.—Wei, S., Roessler, B. C., Icyuz, M., Chauvet, S., Tao, B., Hartman IV, J. L., Kirk, K. L. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels. PMID:26606940

  2. Developmental analysis of Lingo-1/Lern1 protein expression in the mouse brain: interaction of its intracellular domain with Myt1l.

    PubMed

    Llorens, Franc; Gil, Vanesa; Iraola, Susana; Carim-Todd, Laura; Martí, Eulàlia; Estivill, Xavier; Soriano, Eduardo; del Rio, José Antonio; Sumoy, Lauro

    2008-03-01

    Lingo-1 (also known as Lern1) is a component of the Nogo receptor complex that mediates intracellular signaling in response to myelin associated inhibitors (MAIs): NogoA, MAG, and Omgp. Signaling through Nogo receptor extends to more than its well known role in preventing axon regeneration after lesion in the CNS, being implicated in neuronal functional maturation. Using Lingo-1-deficient mice, it has been demonstrated that Lingo-1 plays relevant roles in oligodendrocyte differentiation during brain development, and that treatment with Lingo-1 antagonists can improve axon regeneration after lesion in adult mice by decreasing MAI mediated signaling. However, a detailed description of the pattern of expression of Lingo-1 protein in correlation with the other partners of Nogo receptor is missing. Here, we show that components of the Nogo receptor complex, Lingo-1, NgR1, p75, and TROY coexist in mouse brain in a defined time window only at later postnatal stages. We have also determined the Lingo-1 distribution showing expression in particular subsets of neurons, but not in myelinating mature oligodendrocytes. Surprisingly, Lingo-1 is expressed at early developmental stages without NgR1, which supports the notion that Lingo-1 may participate in other activities in developing neurons different from oligodendrocyte maturation or axon extension inhibition in the adult. Finally, we propose that the intracellular domain of Lingo-1 contributes to signaling and show that it interacts with the postmitotic neuronal specific zinc finger protein Myt1l, suggesting that Lingo-1 may regulate Myt1l transcription factor activity by affecting its subcellular localization.

  3. Protein inhibitor of activated STAT3 (PIAS3) protein promotes SUMOylation and nuclear sequestration of the intracellular domain of ErbB4 protein.

    PubMed

    Sundvall, Maria; Korhonen, Anna; Vaparanta, Katri; Anckar, Julius; Halkilahti, Kalle; Salah, Zaidoun; Aqeilan, Rami I; Palvimo, Jorma J; Sistonen, Lea; Elenius, Klaus

    2012-06-29

    ErbB4 is a receptor tyrosine kinase implicated in the development and homeostasis of the heart, central nervous system, and mammary gland. Cleavable isoforms of ErbB4 release a soluble intracellular domain (ICD) that can translocate to the nucleus and function as a transcriptional coregulator. In search of regulatory mechanisms of ErbB4 ICD function, we identified PIAS3 as a novel interaction partner of ErbB4 ICD. In keeping with the small ubiquitin-like modifier (SUMO) E3 ligase function of protein inhibitor of activated STAT (PIAS) proteins, we showed that the ErbB4 ICD is modified by SUMO, and that PIAS3 stimulates the SUMOylation. Upon overexpression of PIAS3, the ErbB4 ICD generated from the full-length receptor accumulated into the nucleus in a manner that was dependent on the functional nuclear localization signal of ErbB4. In the nucleus, ErbB4 colocalized with PIAS3 and SUMO-1 in promyelocytic leukemia nuclear bodies, nuclear domains involved in regulation of transcription. Accordingly, PIAS3 overexpression had an effect on the transcriptional coregulatory activity of ErbB4, repressing its ability to coactivate transcription with Yes-associated protein. Finally, knockdown of PIAS3 with siRNA partially rescued the inhibitory effect of the ErbB4 ICD on differentiation of MDA-MB-468 breast cancer and HC11 mammary epithelial cells. Our findings illustrate that PIAS3 is a novel regulator of ErbB4 receptor tyrosine kinase, controlling its nuclear sequestration and function.

  4. Protein Inhibitor of Activated STAT3 (PIAS3) Protein Promotes SUMOylation and Nuclear Sequestration of the Intracellular Domain of ErbB4 Protein*

    PubMed Central

    Sundvall, Maria; Korhonen, Anna; Vaparanta, Katri; Anckar, Julius; Halkilahti, Kalle; Salah, Zaidoun; Aqeilan, Rami I.; Palvimo, Jorma J.; Sistonen, Lea; Elenius, Klaus

    2012-01-01

    ErbB4 is a receptor tyrosine kinase implicated in the development and homeostasis of the heart, central nervous system, and mammary gland. Cleavable isoforms of ErbB4 release a soluble intracellular domain (ICD) that can translocate to the nucleus and function as a transcriptional coregulator. In search of regulatory mechanisms of ErbB4 ICD function, we identified PIAS3 as a novel interaction partner of ErbB4 ICD. In keeping with the small ubiquitin-like modifier (SUMO) E3 ligase function of protein inhibitor of activated STAT (PIAS) proteins, we showed that the ErbB4 ICD is modified by SUMO, and that PIAS3 stimulates the SUMOylation. Upon overexpression of PIAS3, the ErbB4 ICD generated from the full-length receptor accumulated into the nucleus in a manner that was dependent on the functional nuclear localization signal of ErbB4. In the nucleus, ErbB4 colocalized with PIAS3 and SUMO-1 in promyelocytic leukemia nuclear bodies, nuclear domains involved in regulation of transcription. Accordingly, PIAS3 overexpression had an effect on the transcriptional coregulatory activity of ErbB4, repressing its ability to coactivate transcription with Yes-associated protein. Finally, knockdown of PIAS3 with siRNA partially rescued the inhibitory effect of the ErbB4 ICD on differentiation of MDA-MB-468 breast cancer and HC11 mammary epithelial cells. Our findings illustrate that PIAS3 is a novel regulator of ErbB4 receptor tyrosine kinase, controlling its nuclear sequestration and function. PMID:22584572

  5. Sequential and γ-secretase-dependent processing of the betacellulin precursor generates a palmitoylated intracellular-domain fragment that inhibits cell growth

    PubMed Central

    Stoeck, Alexander; Shang, Li; Dempsey, Peter J.

    2010-01-01

    Betacellulin (BTC) belongs to the family of epidermal growth factor (EGF)-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release soluble mature ligands. BTC is a dual-specificity ligand for ErbB1 and ErbB4 receptors, and can activate unique signal-transduction pathways that are beneficial for the function, survival and regeneration of pancreatic β-cells. We have previously shown that BTC precursor (proBTC) is cleaved by ADAM10 to generate soluble ligand and a stable, transmembrane remnant (BTC-CTF). In this study, we analyzed the fate of the BTC-CTF in greater detail. We demonstrated that proBTC is cleaved by ADAM10 to produce BTC-CTF, which then undergoes intramembrane processing by presenilin-1- and/or presenilin-2-dependent γ-secretase to generate an intracellular-domain fragment (BTC-ICD). We found that the proBTC cytoplasmic domain is palmitoylated and that palmitoylation is not required for ADAM10-dependent cleavage but is necessary for the stability and γ-secretase-dependent processing of BTC-CTF to generate BTC-ICD. Additionally, palmitoylation is required for nuclear-membrane localization of BTC-ICD, as demonstrated by the redistribution of non-palmitoylated BTC-ICD mutant to the nucleoplasm. Importantly, a novel receptor-independent role for BTC-ICD signaling is suggested by the ability of BTC-ICD to inhibit cell growth in vitro. PMID:20530572

  6. Relative impact of residues at the intracellular and extracellular ends of the human GABAC rho1 receptor M2 domain on picrotoxinin activity.

    PubMed

    Carland, Jane E; Johnston, Graham A R; Chebib, Mary

    2008-02-02

    The relative impact on picrotoxinin activity of residues at the intracellular (2' and 6' residues) and extracellular (15' and 17' residues) ends of the second transmembrane (M2) domain of the human gamma-aminobutyric acid-C (GABA(C)) rho1 receptor was investigated. A series of GABA(C) rho1 subunits were produced containing either single or multiple mutations at the positions of interest. Wild-type and mutant subunits (containing one or more of the following mutations: P2'S, T6'M, I15'N, G17'H) were expressed in Xenopus oocytes and characterized using agonists, partial agonists and antagonists. Changes in agonist activity were observed for mutant receptors. Most notably, mutation at the 2' position resulted in decreased agonist potency, while mutation at the 15' and 17' residues increased agonist potency. The affinity of the competitive antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA) was unchanged compared to wild-type at all mutant receptors. Of the four residues studied, mutation of residues at the 2' and 6' positions had the greatest impact on picrotoxinin activity. Inclusion of the P2'S mutation typically produced receptors with increased picrotoxinin potency, while the T6'M mutation reduced picrotoxinin potency. Picrotoxinin is a mixed antagonist at wild-type and all mutant receptors, with the exception of the double mutant rho1P2'S/T6'M receptors at which the non-competitive component was isolated. It is proposed that the contribution of M2 domain residues to picrotoxinin activity is potentially two-fold: (1) their role as a potential picrotoxinin binding site within the pore; and (2) they are critical for receptor activation properties of the receptor, thus may alter the allosteric mechanism of picrotoxinin.

  7. Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

    PubMed

    Tamura, Tomokazu; Ruggli, Nicolas; Nagashima, Naofumi; Okamatsu, Masatoshi; Igarashi, Manabu; Mine, Junki; Hofmann, Martin A; Liniger, Matthias; Summerfield, Artur; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-09-01

    Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.

  8. Model Hirano bodies protect against tau-independent and tau-dependent cell death initiated by the amyloid precursor protein intracellular domain.

    PubMed

    Furgerson, Matthew; Fechheimer, Marcus; Furukawa, Ruth

    2012-01-01

    The main pathological hallmarks of Alzheimer's disease are amyloid-beta plaques and neurofibrillary tangles, which are primarily composed of amyloid precursor protein (APP) and tau, respectively. These proteins and their role in the mechanism of neurodegeneration have been extensively studied. Hirano bodies are a frequently occurring pathology in Alzheimer's disease as well as other neurodegenerative diseases. However, the physiological role of Hirano bodies in neurodegenerative diseases has yet to be determined. We have established cell culture models to study the role of Hirano bodies in amyloid precursor protein and tau-induced cell death mechanisms. Exogenous expression of APP and either of its c-terminal fragments c31 or Amyloid Precursor Protein Intracellular Domain c58 (AICDc58) enhance cell death. The presence of tau is not required for this enhanced cell death. However, the addition of a hyperphosphorylated tau mimic 352PHPtau significantly increases cell death in the presence of both APP and c31 or AICDc58 alone. The mechanism of cell death induced by APP and its c-terminal fragments and tau was investigated. Fe65, Tip60, p53, and caspases play a role in tau-independent and tau-dependent cell death. In addition, apoptosis was determined to contribute to cell death. The presence of model Hirano bodies protected against cell death, indicating Hirano bodies may play a protective role in neurodegeneration.

  9. Intracellular domains of amyloid precursor-like protein 2 interact with CP2 transcription factor in the nucleus and induce glycogen synthase kinase-3beta expression.

    PubMed

    Xu, Y; Kim, H-S; Joo, Y; Choi, Y; Chang, K-A; Park, C H; Shin, K-Y; Kim, S; Cheon, Y-H; Baik, T-K; Kim, J-H; Suh, Y-H

    2007-01-01

    Amyloid precursor protein (APP) is a member of a gene family that includes two APP-like proteins, APLP1 and 2. Recently, it has been reported that APLP1 and 2 undergo presenilin-dependent gamma-secretase cleavage, as does APP, resulting in the release of an approximately 6 kDa intracellular C-terminal domain (ICD), which can translocate into the nucleus. In this study, we demonstrate that the APLP2-ICDs interact with CP2/LSF/LBP1 (CP2) transcription factor in the nucleus and induce the expression of glycogen synthase kinase 3beta (GSK-3beta), which has broad-ranged substrates such as tau- and beta-catenin. The significance of this finding is substantiated by the in vivo evidence of the increase in the immunoreactivities for the nuclear C-terminal fragments of APLP2, and for GSK-3beta in the AD patients' brain. Taken together, these results suggest that APLP2-ICDs contribute to the AD pathogenesis, by inducing GSK-3beta expression through the interaction with CP2 transcription factor in the nucleus.

  10. Structural analysis of the human interferon gamma receptor: a small segment of the intracellular domain is specifically required for class I major histocompatibility complex antigen induction and antiviral activity.

    PubMed

    Cook, J R; Jung, V; Schwartz, B; Wang, P; Pestka, S

    1992-12-01

    Mutations of the human interferon gamma (IFN-gamma) receptor intracellular domain have permitted us to define a restricted region of that domain as necessary for both induction of class I major histocompatibility complex antigen by IFN-gamma and protection against encephalomyocarditis virus. This region consists of five amino acids (YDKPH), all of which are conserved in the human and murine receptors. Tyr-457 and His-461 are essential for activity. Approximately 80% of the amino acids of the intracellular domain of the receptor is not required for major histocompatibility complex class I antigen induction or for antiviral protection against encephalomyocarditis virus. The observation that there was no protection by IFN-gamma against vesiculostomatitis virus indicates that other factors, in addition to chromosome 21 accessory factor(s), are required to generate the full complement of transduction signals from the human IFN-gamma receptor.

  11. The positive is inside the negative: HER2-negative tumors can express the HER2 intracellular domain and present a HER2-positive phenotype.

    PubMed

    Panis, Carolina; Pizzatti, Luciana; Corrêa, Stephany; Binato, Renata; Lemos, Gabriela Ferreira; Herrera, Ana Cristina da Silva do Amaral; Seixas, Teresa Fernandes; Cecchini, Rubens; Abdelhay, Eliana

    2015-02-01

    Overexpression of human epithelial growth factor receptor 2 (HER2) is a poor prognostic factor in breast cancer. HER2 is a transmembrane receptor comprising an extracellular domain (ECD), a single transmembrane domain, and an intracellular domain (ICD) with tyrosine-kinase activity. Receptor dimerization triggers pivotal effector pathways in cancer, such as phosphatidylinositol 3-kinase (PI3K) signaling. Currently, screening of HER2 in breast tumors for prognostic and therapeutic purposes involves immunohistochemical (IHC) phenotyping for the ECD, in which tumors with IHC scores below 2+ are reported as HER2-negative. We used a label-free liquid chromatography-mass spectrometry (LC-MS) proteomic approach to compare plasma samples from patients with HER2-positive breast tumors and patients with HER2-negative tumors. Patients with HER2-negative tumors expressed higher circulating levels of calpain-10 than patients with HER2-positive tumors. Calpains cleave HER2, releasing its ECD and transforming phenotypically positive tumors into phenotypically negative tumors. Therefore, we investigated the expression of the ICD in HER2-negative samples that overexpressed calpain-10. We found that 16% of HER2-negative tumors were positive for HER2-ICD, which was associated with circulating HER2-ECD. HER2 gene amplification was also observed in some HER2-negative tumors. Positive staining for the PI3K pathway was observed in the HER2-negative, ICD-positive tumors, similar to the HER2-positive cohort. Microarray analysis revealed that HER2-negative, ICD-positive samples clustered between HER2-positive tumors and triple-negative tumors. Survival analysis revealed that outcome in women with HER2-negative, ICD-positive tumors was better than in women bearing HER2-negative, ICD-negative (triple negative) tumors but was quite similar to HER2-positive tumors and worse than women with luminal A tumors. Moreover, in vitro analyses revealed that MDA-MB 231, a triple negative cell line

  12. Reduction in membranous immunohistochemical staining for the intracellular domain of epithelial cell adhesion molecule correlates with poor patient outcome in primary colorectal adenocarcinoma

    PubMed Central

    Wang, A.; Ramjeesingh, R.; Chen, C.H.; Hurlbut, D.; Hammad, N.; Mulligan, L.M.; Nicol, C.; Feilotter, H.E.; Davey, S.

    2016-01-01

    Background Epithelial cell adhesion molecule (epcam) is a multifunctional transmembrane glycoprotein expressed on both normal epithelium and epithelial neoplasms such as gastric, breast, and renal carcinomas. Recent studies have proposed that the proteolytic cleavage of the intracellular domain of epcam (epcam-icd) can trigger signalling cascades leading to aggressive tumour behavior. The expression profile of epcam-icd has not been elucidated for primary colorectal carcinoma. In the present study, we examined epcam-icd immunohistochemical staining in a large cohort of patients with primary colorectal adenocarcinoma and assessed its performance as a potential prognostic marker. Methods Immunohistochemical staining for epcam-icd was assessed on tissue microarrays consisting of 137 primary colorectal adenocarcinoma samples. Intensity of staining for each core was scored by 3 independent pathologists. The membranous epcam-icd staining score was calculated as a weighted average from 3 core samples per tumour. Univariate analysis of the average scores and clinical outcome measures was performed. Results The level of membranous epcam-icd staining was positively associated with well-differentiated tumours (p = 0.01); low preoperative carcinoembryonic antigen (p = 0.001); and several measures of survival, including 2-year (p = 0.02) and 5-year survival (p = 0.05), and length of time post-diagnosis (p = 0.03). A number of other variables—including stage, grade, and lymph node status—showed correlations with epcam staining and markers of poor outcome, but did not reach statistical significance. Conclusions Low membranous epcam-icd staining might be a useful marker to identify tumours with aggressive clinical behavior and potential poor prognosis and might help to select candidates who could potentially benefit from treatment targeting epcam. PMID:27330354

  13. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling.

    PubMed

    Calver, A R; Robbins, M J; Cosio, C; Rice, S Q; Babbs, A J; Hirst, W D; Boyfield, I; Wood, M D; Russell, R B; Price, G W; Couve, A; Moss, S J; Pangalos, M N

    2001-02-15

    GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

  14. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  15. Intracellular localization of human ZBP1: Differential regulation by the Z-DNA binding domain, Z{alpha}, in splice variants

    SciTech Connect

    Hong Thanh Pham; Park, Mi-Young; Kim, Kyeong Kyu; Kim, Yang-Gyun; Ahn, Jin-Hyun . E-mail: jahn@med.skku.ac.kr

    2006-09-15

    We investigated the subcellular distribution of human ZBP1, which harbors the N-terminal Z-DNA binding domains, Z{alpha} and Z{beta}. ZBP1 was distributed primarily in the cytoplasm and occasionally as nuclear foci in interferon (IFN)-treated primary hepatocellular carcinoma cells, and in several other transfected cell types. In leptomycin B (LMB)-treated cells, endogenous ZBP1 efficiently accumulated in nuclear foci, which overlapped PML oncogenic domains (PODs) or nuclear bodies (NBs). In transfection assays, the unique C-terminal region of ZBP1 was necessary for its typical cytoplasmic localization. Interestingly, the Z{alpha}-deleted form displayed an increased association with PODs compared to wild-type and, unlike wild-type, perfectly accumulated in PODs in LMB-treated cells, implying that the presence of Z{alpha} domain also facilitates the cytoplasmic localization. Our results demonstrate that ZBP1 is localized primarily in the cytoplasm but also associated with nuclear PODs in IFN or LMB-treated cells. Given that about half of ZBP1 mRNA lacks exon 2 encoding the Z{alpha} domain, our data also suggest that the localization of ZBP1 may be differentially regulated by the Z-DNA binding domain, Z{alpha}, in splice variants.

  16. Interactions of the Cytoplasmic Domains of Human and Simian Retroviral Transmembrane Proteins with Components of the Clathrin Adaptor Complexes Modulate Intracellular and Cell Surface Expression of Envelope Glycoproteins

    PubMed Central

    Berlioz-Torrent, Clarisse; Shacklett, Barbara L.; Erdtmann, Lars; Delamarre, Lelia; Bouchaert, Isabelle; Sonigo, Pierre; Dokhelar, Marie Christine; Benarous, Richard

    1999-01-01

    The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXØ, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXØ motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXØ motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXØ motifs interact with the μ1 and μ2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the β2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXØ-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved. PMID:9882340

  17. A quantum mechanical study on phosphotyrosyl peptide binding to the SH2 domain of p56lck tyrosine kinase with insights into the biochemistry of intracellular signal transduction events.

    PubMed

    Pichierri, Fabio

    2004-05-01

    A study on the interaction between a phosphotyrosyl peptide with the SH2 domain of Lck kinase has been undertaken with the aid of semiempirical linear-scaling quantum mechanical methods. The structure of this complex has been solved at atomic resolution and, hence, it represents the ideal candidate for studying the charge deformation effects induced by the phosphopeptide on the binding site. Substantial changes in the charge of amino acid residues located in the binding pocket of the protein are observed upon ligand binding. More specifically, our quantum chemical calculations indicate that H-bonds involving charged side-chains are subject to consistent charge deformation effects whereas those forming salt bridges are unaffected by ligand binding. Furthermore, ligand binding has the effect of changing both the magnitude and direction of the protein's macrodipole, which rotates approximately 150 degrees with respect that of the unliganded protein. This suggests that a change in the polarization state of the protein might acts as a switch during the transmission of intracellular signals. The binding energy calculated with the aid of the COSMO solvation model corresponds to about -200 kcal/mol, most of which is attributed to the interaction of the phosphotyrosine head with the amino acid chains located in the binding site of the SH2 domain.

  18. Silencing of the Tandem Pore Domain Halothane-inhibited K+ Channel 2 (THIK2) Relies on Combined Intracellular Retention and Low Intrinsic Activity at the Plasma Membrane*

    PubMed Central

    Chatelain, Franck C.; Bichet, Delphine; Feliciangeli, Sylvain; Larroque, Marie-Madeleine; Braud, Véronique M.; Douguet, Dominique; Lesage, Florian

    2013-01-01

    The tandem pore domain halothane-inhibited K+ channel 1 (THIK1) produces background K+ currents. Despite 62% amino acid identity with THIK1, THIK2 is not active upon heterologous expression. Here, we show that this apparent lack of activity is due to a unique combination of retention in the endoplasmic reticulum and low intrinsic channel activity at the plasma membrane. A THIK2 mutant containing a proline residue (THIK2-A155P) in its second inner helix (M2) produces K+-selective currents with properties similar to THIK1, including inhibition by halothane and insensitivity to extracellular pH variations. Another mutation in the M2 helix (I158D) further increases channel activity and affects current kinetics. We also show that the cytoplasmic amino-terminal region of THIK2 (Nt-THIK2) contains an arginine-rich motif (RRSRRR) that acts as a retention/retrieval signal. Mutation of this motif in THIK2 induces a relocation of the channel to the plasma membrane, resulting in measurable currents, even in the absence of mutations in the M2 helix. Cell surface delivery of a Nt-THIK2-CD161 chimera is increased by mutating the arginines of the retention motif but also by converting the serine embedded in this motif to aspartate, suggesting a phosphorylation-dependent regulation of THIK2 trafficking. PMID:24163367

  19. A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity.

    PubMed

    McKell, Allison O; LaConte, Leslie E W; McDonald, Sarah M

    2017-04-01

    Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1L138P-GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P-GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase.IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP

  20. Genetic evidence for the adhesion protein IgSF9/Dasm1 to regulate inhibitory synapse development independent of its intracellular domain.

    PubMed

    Mishra, Archana; Traut, Matthias H; Becker, Lore; Klopstock, Thomas; Stein, Valentin; Klein, Rüdiger

    2014-03-19

    Normal brain function requires balanced development of excitatory and inhibitory synapses. An imbalance in synaptic transmission underlies many brain disorders such as epilepsy, schizophrenia, and autism. Compared with excitatory synapses, relatively little is known about the molecular control of inhibitory synapse development. We used a genetic approach in mice to identify the Ig superfamily member IgSF9/Dasm1 as a candidate homophilic synaptic adhesion protein that regulates inhibitory synapse development. IgSF9 is expressed in pyramidal cells and subsets of interneurons in the CA1 region of hippocampus. Electrophysiological recordings of acute hippocampal slices revealed that genetic inactivation of the IgSF9 gene resulted in fewer functional inhibitory synapses; however, the strength of the remaining synapses was unaltered. These physiological abnormalities were correlated with decreased expression of inhibitory synapse markers in IgSF9(-/-) mice, providing anatomical evidence for a reduction in inhibitory synapse numbers, whereas excitatory synapse development was normal. Surprisingly, knock-in mice expressing a mutant isoform of IgSF9 lacking the entire cytoplasmic domain (IgSF9(ΔC/ΔC) mice) had no defects in inhibitory synapse development, providing genetic evidence that IgSF9 regulates synapse development via ectodomain interactions rather than acting itself as a signaling receptor. Further, we found that IgSF9 mediated homotypic binding and cell aggregation, but failed to induce synapse formation, suggesting that IgSF9 acts as a cell adhesion molecule (CAM) to maintain synapses. Juvenile IgSF9(-/-) mice exhibited increased seizure susceptibility indicative of an imbalance in synaptic excitation and inhibition. These results provide genetic evidence for a specific role of IgSF9 in inhibitory synapse development/maintenance, presumably by its CAM-like activity.

  1. pH-sensitive Self-associations of the N-terminal Domain of NBCe1-A Suggest a Compact Conformation under Acidic Intracellular Conditions

    PubMed Central

    Gill, Harindarpal S

    2012-01-01

    NBCe1-A is an integral membrane protein that cotransports Na+ and HCO3- ions across the basolateral membrane of the proximal tubule. It is essential for maintaining a homeostatic balance of cellular and blood pH. In X-ray diffraction studies, we reported that the cytoplasmic, N-terminal domain of NBCe1-A (NtNBCe1-A) is a dimer. Here, biophysical measurements show that the dimer is in a concentration-dependent dynamic equilibrium among three additional states in solution that are characterized by its hydrodynamic properties, molar masses, emission spectra, binding properties, and stabilities as a function of pH. Under physiological conditions, dimers are in equilibrium with monomers that are pronounced at low concentration and clusters of molecular masses up to 3-5 times that of a dimer that are pronounced at high concentration. The equilibrium can be influenced so that individual dimers predominate in a taut conformation by lowering the pH. Conversely, dimers begin to relax and disassociate into an increasing population of monomers by elevating the pH. A mechanistic diagram for the inter-conversion of these states is given. The self-associations are further supported by surface plasmon resonance (SPR-Biacore) techniques that illustrate NtNBCe1-A molecules transiently bind with one another. Bicarbonate and bicarbonate-analog bisulfite appear to enhance dimerization and induce a small amount of tetramers. A model is proposed, where the Nt responds to pH or bicarbonate fluctuations inside the cell and plays a role in self-association of entire NBCe1-A molecules in the membrane. PMID:22316307

  2. Identification of Canonical Tyrosine-dependent and Non-canonical Tyrosine-independent STAT3 Activation Sites in the Intracellular Domain of the Interleukin 23 Receptor*

    PubMed Central

    Floss, Doreen M.; Mrotzek, Simone; Klöcker, Tobias; Schröder, Jutta; Grötzinger, Joachim; Rose-John, Stefan; Scheller, Jürgen

    2013-01-01

    Signaling of interleukin 23 (IL-23) via the IL-23 receptor (IL-23R) and the shared IL-12 receptor β1 (IL-12Rβ1) controls innate and adaptive immune responses and is involved in the differentiation and expansion of IL-17-producing CD4+ T helper (TH17) cells. Activation of signal transducer and activator of transcription 3 (STAT3) appears to be the major signaling pathway of IL-23, and STAT binding sites were predicted in the IL-23R but not in the IL-12Rβ1 chain. Using site-directed mutagenesis and deletion variants of the murine and human IL-23R, we showed that the predicted STAT binding sites (pYXXQ; including Tyr-504 and Tyr-626 in murine IL-23R and Tyr-484 and Tyr-611 in human IL-23R) mediated STAT3 activation. Furthermore, we identified two uncommon STAT3 binding/activation sites within the murine IL-23R. First, the murine IL-23R carried the Y542PNFQ sequence, which acts as an unusual Src homology 2 (SH2) domain-binding protein activation site of STAT3. Second, we identified a non-canonical, phosphotyrosine-independent STAT3 activation motif within the IL-23R. A third predicted site, Tyr-416 in murine and Tyr-397 in human IL-23R, is involved in the activation of PI3K/Akt and the MAPK pathway leading to STAT3-independent proliferation of Ba/F3 cells upon stimulation with IL-23. In contrast to IL-6-induced short term STAT3 phosphorylation, cellular activation by IL-23 resulted in a slower but long term STAT3 phosphorylation, indicating that the IL-23R might not be a major target of negative feedback inhibition by suppressor of cytokine signaling (SOCS) proteins. In summary, we characterized IL-23-dependent signal transduction with a focus on STAT3 phosphorylation and identified canonical tyrosine-dependent and non-canonical tyrosine-independent STAT3 activation sites in the IL-23R. PMID:23673666

  3. Functional genomics of intracellular bacteria.

    PubMed

    de Barsy, Marie; Greub, Gilbert

    2013-07-01

    During the genomic era, a large amount of whole-genome sequences accumulated, which identified many hypothetical proteins of unknown function. Rapidly, functional genomics, which is the research domain that assign a function to a given gene product, has thus been developed. Functional genomics of intracellular pathogenic bacteria exhibit specific peculiarities due to the fastidious growth of most of these intracellular micro-organisms, due to the close interaction with the host cell, due to the risk of contamination of experiments with host cell proteins and, for some strict intracellular bacteria such as Chlamydia, due to the absence of simple genetic system to manipulate the bacterial genome. To identify virulence factors of intracellular pathogenic bacteria, functional genomics often rely on bioinformatic analyses compared with model organisms such as Escherichia coli and Bacillus subtilis. The use of heterologous expression is another common approach. Given the intracellular lifestyle and the many effectors that are used by the intracellular bacteria to corrupt host cell functions, functional genomics is also often targeting the identification of new effectors such as those of the T4SS of Brucella and Legionella.

  4. Evolution of intracellular compartmentalization.

    PubMed

    Diekmann, Yoan; Pereira-Leal, José B

    2013-01-15

    Cells compartmentalize their biochemical functions in a variety of ways, notably by creating physical barriers that separate a compartment via membranes or proteins. Eukaryotes have a wide diversity of membrane-based compartments, many that are lineage- or tissue-specific. In recent years, it has become increasingly evident that membrane-based compartmentalization of the cytosolic space is observed in multiple prokaryotic lineages, giving rise to several types of distinct prokaryotic organelles. Endosymbionts, previously believed to be a hallmark of eukaryotes, have been described in several bacteria. Protein-based compartments, frequent in bacteria, are also found in eukaryotes. In the present review, we focus on selected intracellular compartments from each of these three categories, membrane-based, endosymbiotic and protein-based, in both prokaryotes and eukaryotes. We review their diversity and the current theories and controversies regarding the evolutionary origins. Furthermore, we discuss the evolutionary processes acting on the genetic basis of intracellular compartments and how those differ across the domains of life. We conclude that the distinction between eukaryotes and prokaryotes no longer lies in the existence of a compartmentalized cell plan, but rather in its complexity.

  5. Highly prolific Booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells.

    PubMed

    Wilson, T; Wu, X Y; Juengel, J L; Ross, I K; Lumsden, J M; Lord, E A; Dodds, K G; Walling, G A; McEwan, J C; O'Connell, A R; McNatty, K P; Montgomery, G W

    2001-04-01

    The Booroola fecundity gene (FecB) increases ovulation rate and litter size in sheep and is inherited as a single autosomal locus. The effect of FecB is additive for ovulation rate (increasing by about 1.6 corpora lutea per cycle for each copy) and has been mapped to sheep chromosome 6q23-31, which is syntenic to human chromosome 4q21-25. Bone morphogenetic protein IB (BMP-IB) receptor (also known as ALK-6), which binds members of the transforming growth factor-beta (TGF-beta) superfamily, is located in the region containing the FecB locus. Booroola sheep have a mutation (Q249R) in the highly conserved intracellular kinase signaling domain of the BMP-IB receptor. The mutation segregated with the FecB phenotype in the Booroola backcross and half-sib flocks of sheep with no recombinants. The mutation was not found in individuals from a number of sheep breeds not derived from the Booroola strain. BMPR-IB was expressed in the ovary and in situ hybridization revealed its specific location to the oocyte and the granulosa cell. Expression of mRNA encoding the BMP type II receptor was widespread throughout the ovary. The mutation in BMPR-IB found in Booroola sheep is the second reported defect in a gene from the TGF-beta pathway affecting fertility in sheep following the recent discovery of mutations in the growth factor, GDF9b/BMP15.

  6. Colocalization of β-catenin with Notch intracellular domain in colon cancer: a possible role of Notch1 signaling in activation of CyclinD1-mediated cell proliferation.

    PubMed

    Gopalakrishnan, Natarajan; Saravanakumar, Marimuthu; Madankumar, Perumal; Thiyagu, Mani; Devaraj, Halagowder

    2014-11-01

    The Wnt and Notch1 signaling pathways play major roles in intestinal development and tumorigenesis. Sub-cellular localization of β-catenin has been implicated in colorectal carcinogenesis. However, the β-catenin and Notch intracellular domain (NICD) interaction has to be addressed. Immunohistochemistries of β-catenin, NICD, and dual immunofluorescence of β-catenin and NICD were analyzed in colorectal tissues and HT29 cell line. Moreover, real-time PCR analysis of CyclinD1, Hes1 and MUC2 was done in HT29 cells upon N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment. Dual staining emphasized the strong interaction of β-catenin and NICD in adenoma and adenocarcinoma than in normal tissues. Hes1 transcript levels were decreased 1.5- and 7.1-fold in 12.5 and 25 µM DAPT-treated HT29 cells. CyclinD1 transcript levels decreased 1.2- and 1.6-fold, and MUC2 transcript level increased 4.3- and 7.5-fold in 12.5 and 25 µM DAPT-treated HT29 cells. The results of this study showed that the sub-cellular localization of β-catenin converges with NICD inducing proliferation through the activation of CyclinD1 and Hes1. Moreover, the inhibition of Notch1 signaling by DAPT leads to the arrest of cell proliferation and induces apoptosis leading to the upregulation of MUC2, a secretory cell lineage marker.

  7. Revisiting intracellular calcium signaling semantics.

    PubMed

    Haiech, Jacques; Audran, Emilie; Fève, Marie; Ranjeva, Raoul; Kilhoffer, Marie-Claude

    2011-12-01

    Cells use intracellular free calcium concentration changes for signaling. Signal encoding occurs through both spatial and temporal modulation of the free calcium concentration. The encoded message is detected by an ensemble of intracellular sensors forming the family of calcium-binding proteins (CaBPs) which must faithfully translate the message using a new syntax that is recognized by the cell. The cell is home to a significant although limited number of genes coding for proteins involved in the signal encoding and decoding processes. In a cell, only a subset of this ensemble of genes is expressed, leading to a genetic regulation of the calcium signal pathways. Calmodulin (CaM), the most ubiquitous expressed intracellular calcium-binding protein, plays a major role in calcium signal translation. Similar to a hub, it is central to a large and finely tuned network, receiving information, integrating it and dispatching the cognate response. In this review, we examine the different steps starting with an external stimulus up to a cellular response, with special emphasis on CaM and the mechanism by which it decodes calcium signals and translates it into exquisitely coordinated cellular events. By this means, we will revisit the calcium signaling semantics, hoping that we will ease communication between scientists dealing with calcium signals in different biological systems and different domains.

  8. Intracellular Parasite Invasion Strategies

    NASA Astrophysics Data System (ADS)

    Sibley, L. D.

    2004-04-01

    Intracellular parasites use various strategies to invade cells and to subvert cellular signaling pathways and, thus, to gain a foothold against host defenses. Efficient cell entry, ability to exploit intracellular niches, and persistence make these parasites treacherous pathogens. Most intracellular parasites gain entry via host-mediated processes, but apicomplexans use a system of adhesion-based motility called ``gliding'' to actively penetrate host cells. Actin polymerization-dependent motility facilitates parasite migration across cellular barriers, enables dissemination within tissues, and powers invasion of host cells. Efficient invasion has brought widespread success to this group, which includes Toxoplasma, Plasmodium, and Cryptosporidium.

  9. Intracellular targeting with engineered proteins

    PubMed Central

    Miersch, Shane; Sidhu, Sachdev S.

    2016-01-01

    If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action

  10. Nanovehicular Intracellular Delivery Systems

    PubMed Central

    PROKOP, ALES; DAVIDSON, JEFFREY M.

    2013-01-01

    This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood–brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list “elementary” phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach. PMID:18200527

  11. Intracellular Sterol Dynamics

    PubMed Central

    Mesmin, Bruno; Maxfield, Frederick R.

    2009-01-01

    We review the cellular mechanisms implicated in cholesterol trafficking and distribution. Recent studies have provided new information about the distribution of sterols within cells, including analysis of its transbilayer distribution. The cholesterol interaction with other lipids and its engagement in various trafficking processes will determine its proper level in a specific membrane; making the cholesterol distribution uneven among the various intracellular organelles. The cholesterol content is important since cholesterol plays an essential role in membranes by controlling their physicochemical properties as well as key cellular events such as signal transduction and protein trafficking. Cholesterol movement between cellular organelles is highly dynamic, and can be achieved by vesicular and non-vesicular processes. Various studies have analyzed the proteins that play a significant role in these processes, giving us new information about the relative importance of these two trafficking pathways in cholesterol transport. Although still poorly characterized in many trafficking routes, several potential sterol transport proteins have been described in detail; as a result, molecular mechanisms for sterol transport among membranes start to be appreciated. PMID:19286471

  12. Peptidoglycan hydrolase fusion to protein transduction domains kill intracellular staphylococci.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococci and streptococci are both human and agricultural pathogens that are demonstrating an increasing frequency of antibiotic resistant strains resulting in chronic infections. The rise in bacterial resistance to antibiotics world-wide has precipitated the search for alternatives to broad r...

  13. INTRACELLULAR SIGNALING AND DEVELOPMENTAL NEUROTOXICITY.

    EPA Science Inventory

    A book chapter in ?Molecular Toxicology: Transcriptional Targets? reviewed the role of intracellular signaling in the developmental neurotoxicity of environmental chemicals. This chapter covered a number of aspects including the development of the nervous system, role of intrace...

  14. Structure of intracellular mature vaccinia virus observed by cryoelectron microscopy.

    PubMed Central

    Dubochet, J; Adrian, M; Richter, K; Garces, J; Wittek, R

    1994-01-01

    Intracellular mature vaccinia virus, also called intracellular naked virus, and its core envelope have been observed in their native, unfixed, unstained, hydrated states by cryoelectron microscopy of vitrified samples. The virion appears as a smooth rounded rectangle of ca. 350 by 270 nm. The core seems homogeneous and is surrounded by a 30-nm-thick surface domain delimited by membranes. We show that surface tubules and most likely also the characteristic dumbbell-shaped core with the lateral bodies which are generally observed in negatively stained or conventionally embedded samples are preparation artifacts. Images PMID:8107253

  15. Ectdomain shedding and regulated intracellular proteolysis in the central nervous system.

    PubMed

    Montes de Oca-B, Pavel

    2010-12-01

    The term Ectodomain Shedding (ES) refers to extracellular domain proteolytic release from cell membrane molecules. This proteolysis is mediated mainly by matrix metalloproteases (MMP) or disintegrin and metalloproteases (ADAM), although some other proteases may mediate it. Virtually, all functional categories of cell membrane molecules are subject of this kind of proteolysis, for this reason ES is involved in different cellular processes such as proliferation, apoptosis, migration, differentiation or pathologies such as inflammation, cancer and degeneration among others. ES releases membrane molecule's extracellular domain (or ectodomain) to the extracellular milieu where it can play different biological functions. ES of transmembrane molecules also generates membrane attached terminal fragments comprising transmembrane and intracellular domains that enable their additional processing by intracellular proteases known as Regulated Intracellular Proteolysis (RIP). This second proteolytic cleavage delivers molecule's intracellular domain (ICD) that carry out intracellular functions. RIP is mediated by the group of intracellular cleaving proteases (i-CLiPs) that include presenilin from the γ-secretase complex. In the CNS the best well known ES is that of the Amyloid Precursor Protein, although many other membrane molecules expressed by cells of the CNS are also subject to ES and RIP. In this review, these molecules are summarized, and some meaningful examples are highlighted and described. In addition, ES and RIP implications in the context of cell biology are discussed. Finally, some considerations that rise from the study of ES and RIP are formulated in view of the unexpected roles of intracellular fragments.

  16. Small Peptide Recognition Sequence for Intracellular Sorting

    PubMed Central

    Pandey, Kailash N.

    2010-01-01

    Increasing evidence indicate that complex arrays of short signals and recognition peptide sequence ensure accurate trafficking and distribution of transmembrane receptors and/or proteins and their ligands into intracellular compartments. Internalization and subsequent trafficking of cell-surface receptors into the cell interior is mediated by specific short-sequence peptide signals within the cytoplasmic domains of these receptor proteins. The short signals usually consist of small linear amino acid sequences, which are recognized by adaptor coat proteins along the endocytic and sorting pathways. In recent years, much has been learned about the function and mechanisms of endocytic pathways responsible for the trafficking and molecular sorting of membrane receptors and their ligands into intracellular compartments, however, the significance and scope of the short sequence motifs in these cellular events is not well understood. Here a particular emphasis has been given to the functions of short-sequence signal motifs responsible for the itinerary and destination of membrane receptors and proteins moving into subcellular compartments. PMID:20817434

  17. The translational potential for target validation and therapy using intracellular antibodies in oncology.

    PubMed

    Weidle, Ulrich H; Maisel, Daniela; Brinkmann, Ulrich; Tiefenthaler, Georg

    2013-01-01

    Antibody-based molecules can be delivered into cells either by intracellular expression or through cellular uptake. We describe technologies for identification and expression of intracellular antibodies for target validation, intracellular immunization and tumor therapy, such as intracellular antibody capture technology, suicide or silencing technology, antigen-antibody interaction dependent apoptosis and their application for inhibition of oncogenic intracellular proteins and induction of apoptosis. These strategies have to be viewed in the context that inhibition of protein-protein interactions by small molecules is often limited due to their large interaction surface. We summarize antibodies with the ability to penetrate cells and strategies to induce uptake of antibodies after modification with protein transduction domains. Interference in oncogenic pathways is described for moieties based on antibody 3E10, which translocates into the nucleus after extracellular administration. Finally, we discuss examples of tumor immunotherapy and vaccination against intracellular antigens, and possible interactions mediating their mode of action.

  18. Intracellular Signalling in Retinal Ischemia

    DTIC Science & Technology

    1990-07-01

    36) However, vascularization of the RPE is not known to occur in human diseases of photoreceptor degeneration, such as retinitis pigmentosa ...A.C. (1986) Retinitis pigmentosa and retinal neovascularization. Ophthalmology 91, 1599- 1603. Figure la: Control rat retina, 8 weeks of age, central...TITLE (Include Security Classification) Intracellular Signalling in Retinal Ischemia 12. PERSONAL AUTHOR(S) Burns, Margaret Sue; Bellhorn, Roy William

  19. Direct Measurement of Intracellular Pressure

    PubMed Central

    Petrie, Ryan J.; Koo, Hyun

    2014-01-01

    A method to directly measure the intracellular pressure of adherent, migrating cells is described in the Basic Protocol. This approach is based on the servo-null method where a microelectrode is introduced into the cell to directly measure the physical pressure of the cytoplasm. We also describe the initial calibration of the microelectrode as well as the application of the method to cells migrating inside three-dimensional (3D) extracellular matrix (ECM). PMID:24894836

  20. Stochastic models of intracellular transport

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Newby, Jay M.

    2013-01-01

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures.

  1. Modulation of lipoprotein receptor functions by intracellular adaptor proteins.

    PubMed

    Stolt, Peggy C; Bock, Hans H

    2006-10-01

    Members of the low density lipoprotein (LDL) receptor gene family are critically involved in a wide range of physiological processes including lipid and vitamin homeostasis, cellular migration, neurodevelopment, and synaptic plasticity, to name a few. Lipoprotein receptors exert these diverse biological functions by acting as cellular uptake receptors or by inducing intracellular signaling cascades. It was discovered that a short sequence in the intracellular region of all lipoprotein receptors, Asn-Pro-X-Tyr (NPXY) is important for mediating either endocytosis or signal transduction events, and that this motif serves as a binding site for phosphotyrosine-binding (PTB) domain containing scaffold proteins. These molecular adaptors connect the transmembrane receptors with the endocytosis machinery and regulate cellular trafficking, or function as assembly sites for dynamic multi-protein signaling complexes. Whereas the LDL receptor represents the archetype of an endocytic lipoprotein receptor, the structurally closely related apolipoprotein E receptor 2 (apoER2) and very low density lipoprotein (VLDL) receptor activate a kinase-dependent intracellular signaling cascade after binding to the neuronal signaling molecule Reelin. This review focuses on two related PTB domain containing adaptor proteins that mediate these divergent lipoprotein receptor responses, ARH (autosomal recessive hypercholesterolemia protein) and Dab1 (disabled-1), and discusses the structural and molecular basis of this different behaviour.

  2. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  3. Review: Intracardiac intracellular angiotensin system in diabetes

    PubMed Central

    Kumar, Rajesh; Yong, Qian Chen; Thomas, Candice M.

    2012-01-01

    The renin-angiotensin system (RAS) has mainly been categorized as a circulating and a local tissue RAS. A new component of the local system, known as the intracellular RAS, has recently been described. The intracellular RAS is defined as synthesis and action of ANG II intracellularly. This RAS appears to differ from the circulating and the local RAS, in terms of components and the mechanism of action. These differences may alter treatment strategies that target the RAS in several pathological conditions. Recent work from our laboratory has demonstrated significant upregulation of the cardiac, intracellular RAS in diabetes, which is associated with cardiac dysfunction. Here, we have reviewed evidence supporting an intracellular RAS in different cell types, ANG II's actions in cardiac cells, and its mechanism of action, focusing on the intracellular cardiac RAS in diabetes. We have discussed the significance of an intracellular RAS in cardiac pathophysiology and implications for potential therapies. PMID:22170614

  4. Dual Readout BRET/FRET Sensors for Measuring Intracellular Zinc

    PubMed Central

    2016-01-01

    Genetically encoded FRET-based sensor proteins have significantly contributed to our current understanding of the intracellular functions of Zn2+. However, the external excitation required for these fluorescent sensors can give rise to photobleaching and phototoxicity during long-term imaging, limits applications that suffer from autofluorescence and light scattering, and is not compatible with light-sensitive cells. For these applications, sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) would provide an attractive alternative. In this work, we used the bright and stable luciferase NanoLuc to create the first genetically encoded BRET sensors for measuring intracellular Zn2+. Using a new sensor approach, the NanoLuc domain was fused to the Cerulean donor domain of two previously developed FRET sensors, eCALWY and eZinCh-2. In addition to preserving the excellent Zn2+ affinity and specificity of their predecessors, these newly developed sensors enable both BRET- and FRET-based detection. While the dynamic range of the BRET signal for the eCALWY-based BLCALWY-1 sensor was limited by the presence of two competing BRET pathways, BRET/FRET sensors based on the eZinCh-2 scaffold (BLZinCh-1 and -2) yielded robust 25–30% changes in BRET ratio. In addition, introduction of a chromophore-silencing mutation resulted in a BRET-only sensor (BLZinCh-3) with increased BRET response (50%) and an unexpected 10-fold increase in Zn2+ affinity. The combination of robust ratiometric response, physiologically relevant Zn2+ affinities, and stable and bright luminescence signal offered by the BLZinCh sensors allowed monitoring of intracellular Zn2+ in plate-based assays as well as intracellular BRET-based imaging in single living cells in real time. PMID:27547982

  5. Anomalous dynamics in intracellular transport

    NASA Astrophysics Data System (ADS)

    Dinner, Aaron

    2013-03-01

    This talk will describe quantitative analyses of particle tracking data for systems with cytoskeletally associated molecular motors to better understand the motions contributing to intracellular transport and, more generally, means for characterizing systems far from equilibrium. In particular, we have studied the motions of insulin-containing vesicles (granules) in a pancreatic beta cell line. We find subdiffusive behavior with correlations in both space and time. These data can be modeled by subordinating an ergodic random walk process to a non-ergodic one. We relate the dynamics to the underlying microtubule structure by imaging in the presence of the drug vinblastine. Our results provide a simple physical mechanism for how diverse pools of insulin granules and, in turn, biphasic secretion could arise. Time permitting, these dynamics will be compared with those of actomyosin assemblies.

  6. THE ALTERATION OF INTRACELLULAR ENZYMES

    PubMed Central

    Kaplan, J. Gordin

    1954-01-01

    1. The ability of homologous series of alcohols, ketones, and aldehydes to cause alteration of intracellular catalase increases approximately threefold for each methylene group added, thus following Traube's rule. Equiactive concentrations of alcohols (methanol to octanol) varied over a 4,000-fold range, yet the average corresponding surface tension was 42 ± 2 dynes/cm., that for ketones 43 ± 2, and for aldehydes (above C1) 41 ± 3. 2. Above C8 the altering activity of alcohols ceased to follow Traube's rule, and at C18 was nil. Yet the surface activities of alcohols from nonanol to dodecanol did follow Traube's rule. These two facts show that the interface which is being affected by these agents is not the cell surface, for if it were, altering activity should not fall off between C9 and C12 where surface activity is undiminished; they show also that micelle formation by short range association of hydrocarbon "tails," usually invoked to explain decrease in biological activity of compounds above C8, is not responsible for this effect in these experiments, in which permeability of the cell membrane probably is involved. 3. The most soluble alcohols and aldehydes (alcohols C1 to C8; aldehydes C1, C2), but not ketones, cause, above optimal concentration, an irreversible inhibition of yeast catalase. 4. The critical concentration of altering agent (i.e., that concentration just sufficient to cause doubling of the catalase activity of the yeast suspension) was independent of the concentration of the yeast cells. 5. Viability studies show that the number of yeast cells killed by the altering agents was not related to the degree of activation of the catalase produced. While all the cells were invariably killed by concentrations of altering agent which produced complete activation, all the cells had been killed by concentrations which were insufficient to cause more than 50 per cent maximal activation. Further, the evidence suggested that the catalase may be partially

  7. Adaptation of fast marching methods to intracellular signaling

    NASA Astrophysics Data System (ADS)

    Chikando, Aristide C.; Kinser, Jason M.

    2006-02-01

    Imaging of signaling phenomena within the intracellular domain is a well studied field. Signaling is the process by which all living cells communicate with their environment and with each other. In the case of signaling calcium waves, numerous computational models based on solving homogeneous reaction diffusion equations have been developed. Typically, the reaction diffusion approach consists of solving systems of partial differential equations at each update step. The traditional methods used to solve these reaction diffusion equations are very computationally expensive since they must employ small time steps in order to reduce the computational error. The presented research suggests the application of fast marching methods to imaging signaling calcium waves, more specifically fertilization calcium waves, in Xenopus laevis eggs. The fast marching approach provides fast and efficient means of tracking the evolution of monotonically advancing fronts. A model that employs biophysical properties of intracellular calcium signaling, and adapts fast marching methods to tracking the propagation of signaling calcium waves is presented. The developed model is used to reproduce simulation results obtained with reaction diffusion based model. Results obtained with our model agree with both the results obtained with reaction diffusion based models, and confocal microscopy observations during in vivo experiments. The adaptation of fast marching methods to intracellular protein or macromolecule trafficking is also briefly explored.

  8. Intracellular Organisms as Placental Invaders

    PubMed Central

    Vigliani, Marguerite B.; Bakardjiev, Anna I.

    2015-01-01

    In this article we present a novel model for how the human placenta might get infected via the hematogenous route. We present a list of diverse placental pathogens, like Listeria monocytogenes or Cytomegalovirus, which are familiar to most obstetricians, but others, like Salmonella typhi, have only been reported in case studies or small case series. Remarkably, all of these organisms on this list are either obligate or facultative intracellular organisms. These pathogens are able to enter and survive inside host immune cells for at least a portion of their life cycle. We suggest that many blood-borne pathogens might arrive at the placenta via transportation inside of maternal leukocytes that enter the decidua in early pregnancy. We discuss mechanisms by which extravillous trophoblasts could get infected in the decidua and spread infection to other layers in the placenta. We hope to raise awareness among OB/GYN clinicians that organisms not typically associated with the TORCH list might cause placental infections and pregnancy complications. PMID:27695204

  9. Secretome of obligate intracellular Rickettsia

    PubMed Central

    Gillespie, Joseph J.; Kaur, Simran J.; Rahman, M. Sayeedur; Rennoll-Bankert, Kristen; Sears, Khandra T.; Beier-Sexton, Magda; Azad, Abdu F.

    2014-01-01

    The genus Rickettsia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently, there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, although others (e.g. josamycin, ciprofloxacin, chloramphenicol, and azithromycin) are also effective. Much of the recent research geared toward understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial ‘life on the inside’. PMID:25168200

  10. Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

    PubMed Central

    Pandey, Gouri Shankar; Tseng, Sandra C.; Howard, Tom E.; Sauna, Zuben E.

    2013-01-01

    Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs). An indirect intracellular staining (ICS) method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI) values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels. PMID:23555096

  11. Hydrophilic fluorescent nanogel thermometer for intracellular thermometry.

    PubMed

    Gota, Chie; Okabe, Kohki; Funatsu, Takashi; Harada, Yoshie; Uchiyama, Seiichi

    2009-03-04

    The first methodology to measure intracellular temperature is described. A highly hydrophilic fluorescent nanogel thermometer developed for this purpose stays in the cytoplasm and emits stronger fluorescence at a higher temperature. Thus, intracellular temperature variations associated with biological processes can be monitored by this novel thermometer with a temperature resolution of better than 0.5 degrees C.

  12. Intracellular minerals and metal deposits in prokaryotes.

    PubMed

    Edwards, K J; Bazylinski, D A

    2008-06-01

    Thanks to the work of Terrance J. Beveridge and other pioneers in the field of metal-microbe interactions, prokaryotes are well known to sequester metals and other ions intracellularly in various forms. These forms range from poorly ordered deposits of metals to well-ordered mineral crystals. Studies on well-ordered crystalline structures have generally focused on intracellular organelles produced by magnetotactic bacteria that are ubiquitous in terrestrial and marine environments that precipitate Fe(3)O(4) or Fe(3)S(4), Fe-bearing minerals that have magnetic properties and are enclosed in intracellular membranes. In contrast, studies on less-well ordered minerals have focused on Fe-, As-, Mn-, Au-, Se- and Cd-precipitates that occur intracellularly. The biological and environmental function of these particles remains a matter of debate.

  13. Nanoparticles for intracellular-targeted drug delivery

    NASA Astrophysics Data System (ADS)

    Paulo, Cristiana S. O.; Pires das Neves, Ricardo; Ferreira, Lino S.

    2011-12-01

    Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

  14. Intracellular Protein Delivery for Treating Breast Cancer

    DTIC Science & Technology

    2014-08-01

    nanocapsules with specific cancer cell targeting ligands; Task 3. Preparing and testing of MMP activatable cell penetrating peptides (ACCPs)-coupled...AD_________________ Award Number: W81XWH-11-1-0371 TITLE: Intracellular Protein Delivery for Treating Breast Cancer PRINCIPAL INVESTIGATOR: Dr...SUBTITLE Intracellular Protein Delivery for Treating Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-11-1-0371 5c. PROGRAM ELEMENT NUMBER 6

  15. Cancer-Selective Apoptotic Effects of Extracellular and Intracellular Par-4

    PubMed Central

    Bhattarai, Tripti Shrestha; Rangnekar, Vivek M.

    2010-01-01

    Selectivity toward cancer cells is the most desirable element in cancer therapeutics. Par-4 is a cancer cell-selective pro-apoptotic protein that functions intracellularly in the cytoplasmic and nuclear compartments, as a tumor suppressor. Moreover, recent findings indicate that the Par-4 protein is secreted by cells, and extracellular Par-4 induces cancer cell-specific apoptosis by interaction with the cell-surface receptor GRP78. This review describes the mechanisms underlying the apoptotic effects of both extracellular and intracellular Par-4 acting via its effector domain SAC. PMID:20440265

  16. Cache Domains That are Homologous to, but Different from PAS Domains Comprise the Largest Superfamily of Extracellular Sensors in Prokaryotes.

    PubMed

    Upadhyay, Amit A; Fleetwood, Aaron D; Adebali, Ogun; Finn, Robert D; Zhulin, Igor B

    2016-04-01

    Cellular receptors usually contain a designated sensory domain that recognizes the signal. Per/Arnt/Sim (PAS) domains are ubiquitous sensors in thousands of species ranging from bacteria to humans. Although PAS domains were described as intracellular sensors, recent structural studies revealed PAS-like domains in extracytoplasmic regions in several transmembrane receptors. However, these structurally defined extracellular PAS-like domains do not match sequence-derived PAS domain models, and thus their distribution across the genomic landscape remains largely unknown. Here we show that structurally defined extracellular PAS-like domains belong to the Cache superfamily, which is homologous to, but distinct from the PAS superfamily. Our newly built computational models enabled identification of Cache domains in tens of thousands of signal transduction proteins including those from important pathogens and model organisms. Furthermore, we show that Cache domains comprise the dominant mode of extracellular sensing in prokaryotes.

  17. Cache domains that are homologous to, but different from PAS domains comprise the largest superfamily of extracellular sensors in prokaryotes

    DOE PAGES

    Upadhyay, Amit A.; Fleetwood, Aaron D.; Adebali, Ogun; ...

    2016-04-06

    Cellular receptors usually contain a designated sensory domain that recognizes the signal. Per/Arnt/Sim (PAS) domains are ubiquitous sensors in thousands of species ranging from bacteria to humans. Although PAS domains were described as intracellular sensors, recent structural studies revealed PAS-like domains in extracytoplasmic regions in several transmembrane receptors. However, these structurally defined extracellular PAS-like domains do not match sequence-derived PAS domain models, and thus their distribution across the genomic landscape remains largely unknown. Here we show that structurally defined extracellular PAS-like domains belong to the Cache superfamily, which is homologous to, but distinct from the PAS superfamily. Our newly builtmore » computational models enabled identification of Cache domains in tens of thousands of signal transduction proteins including those from important pathogens and model organisms.Moreover, we show that Cache domains comprise the dominant mode of extracellular sensing in prokaryotes.« less

  18. Cache domains that are homologous to, but different from PAS domains comprise the largest superfamily of extracellular sensors in prokaryotes

    SciTech Connect

    Upadhyay, Amit A.; Fleetwood, Aaron D.; Adebali, Ogun; Finn, Robert D.; Zhulin, Igor B.; Schlessinger, Avner

    2016-04-06

    Cellular receptors usually contain a designated sensory domain that recognizes the signal. Per/Arnt/Sim (PAS) domains are ubiquitous sensors in thousands of species ranging from bacteria to humans. Although PAS domains were described as intracellular sensors, recent structural studies revealed PAS-like domains in extracytoplasmic regions in several transmembrane receptors. However, these structurally defined extracellular PAS-like domains do not match sequence-derived PAS domain models, and thus their distribution across the genomic landscape remains largely unknown. Here we show that structurally defined extracellular PAS-like domains belong to the Cache superfamily, which is homologous to, but distinct from the PAS superfamily. Our newly built computational models enabled identification of Cache domains in tens of thousands of signal transduction proteins including those from important pathogens and model organisms.Moreover, we show that Cache domains comprise the dominant mode of extracellular sensing in prokaryotes.

  19. Cache Domains That are Homologous to, but Different from PAS Domains Comprise the Largest Superfamily of Extracellular Sensors in Prokaryotes

    PubMed Central

    Upadhyay, Amit A.; Fleetwood, Aaron D.; Adebali, Ogun; Finn, Robert D.; Zhulin, Igor B.

    2016-01-01

    Cellular receptors usually contain a designated sensory domain that recognizes the signal. Per/Arnt/Sim (PAS) domains are ubiquitous sensors in thousands of species ranging from bacteria to humans. Although PAS domains were described as intracellular sensors, recent structural studies revealed PAS-like domains in extracytoplasmic regions in several transmembrane receptors. However, these structurally defined extracellular PAS-like domains do not match sequence-derived PAS domain models, and thus their distribution across the genomic landscape remains largely unknown. Here we show that structurally defined extracellular PAS-like domains belong to the Cache superfamily, which is homologous to, but distinct from the PAS superfamily. Our newly built computational models enabled identification of Cache domains in tens of thousands of signal transduction proteins including those from important pathogens and model organisms. Furthermore, we show that Cache domains comprise the dominant mode of extracellular sensing in prokaryotes. PMID:27049771

  20. Umami changes intracellular Ca2+ levels using intracellular and extracellular sources in mouse taste receptor cells.

    PubMed

    Narukawa, Masataka; Mori, Tomohiko; Hayashi, Yukako

    2006-11-01

    Recently, candidates for umami receptors have been identified in taste cells, but the precise transduction mechanisms of the downstream receptor remain unknown. To investigate how intracellular Ca(2+) increases in the umami transduction pathway, we measured changes in intracellular Ca(2+) levels in response to umami stimuli monosodium glutamate (MSG), IMP, and MSG + IMP in mouse taste receptor cells (TRCs) by Ca(2+) imaging. Even when extracellular Ca(2+) was absent, 1/3 of umami-responsive TRCs exhibited increased intracellular Ca(2+) levels. When intracellular Ca(2+) was depleted, half of the TRCs retained their response to umami. These results suggest that umami-responsive TRCs increase their intracellular Ca(2+) levels through two pathways: by releasing Ca(2+) from intracellular stores and by an influx of Ca(2+) from extracellular sources. We conclude that the Ca(2+) influx from extracellular source might play an important role in the synergistic effect between MSG and IMP.

  1. Non-recessive Bt toxin resistance conferred by an intracellular cadherin mutation in field-selected populations of cotton bollworm.

    PubMed

    Zhang, Haonan; Wu, Shuwen; Yang, Yihua; Tabashnik, Bruce E; Wu, Yidong

    2012-01-01

    Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.

  2. Neuroligin1 drives synaptic and behavioral maturation through intracellular interactions

    PubMed Central

    Hoy, Jennifer L.; Haeger, Paola A.; Constable, John R. L.; Arias, Renee J.; McCallum, Raluca; Kyweriga, Michael; Davis, Lawrence; Schnell, Eric; Wehr, Michael; Castillo, Pablo E.; Washbourne, Philip

    2013-01-01

    In vitro studies suggest that the intracellular C-terminus of Neuroligin1 (NL1) could play a central role in the maturation of excitatory synapses. However, it is unknown how this activity affects synapses in vivo, and whether it may impact the development of complex behaviors. To determine how NL1 influences the state of glutamatergic synapses in vivo, we compared the synaptic and behavioral phenotypes of mice overexpressing a full length version of NL1 (NL1FL) with mice overexpressing a version missing part of the intracellular domain (NL1ΔC). We show that overexpression of full length NL1 yielded an increase in the proportion of synapses with mature characteristics and impaired learning and flexibility. In contrast, the overexpression of NL1ΔC increased the number of excitatory postsynaptic structures and led to enhanced flexibility in mnemonic and social behaviors. Transient overexpression of NL1FL revealed that elevated levels are not necessary to maintain synaptic and behavioral states altered earlier in development. In contrast, overexpression of NL1FL in the fully mature adult was able to impair normal learning behavior after one month of expression. These results provide the first evidence that NL1 significantly impacts key developmental processes that permanently shape circuit function and behavior, as well as the function of fully developed neural circuits. Overall, these manipulations of NL1 function illuminate the significance of NL1 intracellular signaling in vivo, and enhance our understanding of the factors that gate the maturation of glutamatergic synapses and complex behavior. This has significant implications for our ability to address disorders such as ASD. PMID:23719805

  3. Crystallographic study of FABP5 as an intracellular endocannabinoid transporter

    SciTech Connect

    Sanson, Benoît; Wang, Tao; Sun, Jing; Wang, Liqun; Kaczocha, Martin; Ojima, Iwao; Deutsch, Dale; Li, Huilin

    2014-02-01

    FABP5 was recently found to intracellularly transport endocannabinoid signaling lipids. The structures of FABP5 complexed with two endocannabinoids and an inhibitor were solved. Human FABP5 was found to dimerize via a domain-swapping mechanism. This work will help in the development of inhibitors to raise endocannabinoid levels. In addition to binding intracellular fatty acids, fatty-acid-binding proteins (FABPs) have recently been reported to also transport the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), arachidonic acid derivatives that function as neurotransmitters and mediate a diverse set of physiological and psychological processes. To understand how the endocannabinoids bind to FABPs, the crystal structures of FABP5 in complex with AEA, 2-AG and the inhibitor BMS-309403 were determined. These ligands are shown to interact primarily with the substrate-binding pocket via hydrophobic interactions as well as a common hydrogen bond to the Tyr131 residue. This work advances our understanding of FABP5–endocannabinoid interactions and may be useful for future efforts in the development of small-molecule inhibitors to raise endocannabinoid levels.

  4. Extraction of intracellular protein from Glaciozyma antarctica for proteomics analysis

    NASA Astrophysics Data System (ADS)

    Faizura, S. Nor; Farahayu, K.; Faizal, A. B. Mohd; Asmahani, A. A. S.; Amir, R.; Nazalan, N.; Diba, A. B. Farah; Muhammad, M. Nor; Munir, A. M. Abdul

    2013-11-01

    Two preparation methods of crude extracts of psychrophilic yeast Glaciozyma antarctica were compared in order to obtain a good recovery of intracellular proteins. Extraction with mechanical procedures using sonication was found to be more effective for obtaining good yield compare to alkaline treatment method. The procedure is simple, rapid, and produce better yield. A total of 52 proteins were identified by combining both extraction methods. Most of the proteins identified in this study involves in the metabolic process including glycolysis pathway, pentose phosphate pathway, pyruyate decarboxylation and also urea cyle. Several chaperons were identified including probable cpr1-cyclophilin (peptidylprolyl isomerase), macrolide-binding protein fkbp12 and heat shock proteins which were postulate to accelerate proper protein folding. Characteristic of the fundamental cellular processes inferred from the expressed-proteome highlight the evolutionary and functional complexity existing in this domain of life.

  5. Targeted intracellular delivery of therapeutics: an overview.

    PubMed

    Rawat, A; Vaidya, B; Khatri, K; Goyal, A K; Gupta, P N; Mahor, S; Paliwal, R; Rai, S; Vyas, S P

    2007-09-01

    During the last decade, intracellular drug delivery has become an emerging area of research in the medical and pharmaceutical field. Many therapeutic agents such as drugs and DNA/oligonucleotides can be delivered not just to the cell but also to a particular compartment of that cell to achieve better activity e.g. proapoptotic drugs to the mitochondria, antibiotics and enzymes to the lysosomes and various anticancer drugs and gene to the nucleus. The lipidic nature of biological membrans is the major obstacle to the intracellular delivery of macromolecular and ionic drugs. Additionally, after endocytosis, the lysosome, the major degradation compartment, needs to be avoided for better activity. To avoid these problems, various carriers have been investigated for efficient intracellular delivery, either by direct entry to cytoplasm or by escaping the endosomal compartment. These include cell penetrating peptides, and carrier systems such as liposomes, cationic lipids and polymers, polymeric nanoparticles, etc. Various properties of these carriers, including size, surface charge, composition and the presence of cell specific ligands, alter their efficacy and specificity towards particular cells. This review summarizes various aspects of targeted intracellular delivery of therapeutics including pathways, mechanisms and approaches. Various carrier constructs having potential for targeted intracellular delivery are also been discussed.

  6. Internal affairs: investigating the Brucella intracellular lifestyle.

    PubMed

    von Bargen, Kristine; Gorvel, Jean-Pierre; Salcedo, Suzana P

    2012-05-01

    Bacteria of the genus Brucella are Gram-negative pathogens of several animal species that cause a zoonotic disease in humans known as brucellosis or Malta fever. Within their hosts, brucellae reside within different cell types where they establish a replicative niche and remain protected from the immune response. The aim of this article is to discuss recent advances in the field in the specific context of the Brucella intracellular 'lifestyle'. We initially discuss the different host cell targets and their relevance during infection. As it represents the key to intracellular replication, the focus is then set on the maturation of the Brucella phagosome, with particular emphasis on the Brucella factors that are directly implicated in intracellular trafficking and modulation of host cell signalling pathways. Recent data on the role of the type IV secretion system are discussed, novel effector molecules identified and how some of them impact on trafficking events. Current knowledge on Brucella gene regulation and control of host cell death are summarized, as they directly affect intracellular persistence. Understanding how Brucella molecules interplay with their host cell targets to modulate cellular functions and establish the intracellular niche will help unravel how this pathogen causes disease.

  7. Cell-mediated immune responses in healthy children with a history of subclinical infection with Japanese encephalitis virus: analysis of CD4+ and CD8+ T cell target specificities by intracellular delivery of viral proteins using the human immunodeficiency virus Tat protein transduction domain.

    PubMed

    Kumar, Priti; Krishna, Venkatramana D; Sulochana, Paramadevanapalli; Nirmala, Gejjehalli; Haridattatreya, Maganti; Satchidanandam, Vijaya

    2004-02-01

    Japanese encephalitis virus (JEV), a single-stranded positive-sense RNA virus of the family Flaviviridae, is the major cause of paediatric encephalitis in Asia. The high incidence of subclinical infections in Japanese encephalitis-endemic areas and subsequent evasion of encephalitis points to the development of immune responses against JEV. Humoral responses play a central role in protection against JEV; however, cell-mediated immune responses contributing to this end are not fully understood. The structural envelope (E) protein, the major inducer of neutralizing antibodies, is a poor target for T cells in natural JEV infections. The extent to which JEV non-structural proteins are targeted by T cells in subclinically infected healthy children would help to elucidate the role of cell-mediated immunity in protection against JEV as well as other flaviviral infections. The property of the Tat peptide of Human immunodeficiency virus to transduce proteins across cell membranes, facilitating intracellular protein delivery following exogenous addition to cultured cells, prompted us to express the four largest proteins of JEV, comprising 71 % of the JEV genome coding sequence, as Tat fusions for enumerating the frequencies of virus-specific CD4(+) and CD8(+) T cells in JEV-immune donors. At least two epitopes recognized by distinct HLA alleles were found on each of the non-structural proteins, with dominant antiviral Th1 T cell responses to the NS3 protein in nearly 96 % of the cohort. The data presented here show that non-structural proteins are frequently targeted by T cells in natural JEV infections and may be efficacious supplements for the predominantly antibody-eliciting E-based JEV vaccines.

  8. A γ-Secretase-independent Mechanism of Signal Transduction by the Amyloid Precursor Protein*

    PubMed Central

    Hass, Matthew R.; Yankner, Bruce A.

    2006-01-01

    It has been proposed that γ-secretase-mediated release of the amyloid precursor protein (APP) intracellular domain (AICD) results in nuclear translocation and signaling through a complex with the adaptor protein Fe65 and the histone acetyltransferase Tip60. Here, we show that APP and Fe65 activate transcription through a Gal4-Tip60 reporter in presenilin-1/2-deficient cells lacking generation of AICD. APP and Fe65 also activated transcription in the presence of γ-secretase inhibitors that prevent amyloid β-peptide production in human embryonic kidney 293 and SH-SY5Y cells. In contrast to the transcriptionally active Notch intracellular domain, expression of AICD did not activate transcription. An alternative mechanism for APP signal transduction is suggested by the identification of essential cyclin-dependent kinase (CDK) phosphorylation sites in Tip60. Mutation of these Tip60 phosphorylation sites or treatment with the CDK inhibitor roscovitine blocked the ability of APP to signal through Tip60. Moreover, APP stabilized Tip60 through CDK-dependent phosphorylation. Subcellular fractionation and confocal immunofluorescence showed that APP recruited Tip60 to membrane compartments. Thus, APP may signal to the nucleus by a γ-secretase-independent mechanism that involves membrane sequestration and phosphorylation of Tip60. PMID:16103124

  9. Efficient intracellular delivery and improved biocompatibility of colloidal silver nanoparticles towards intracellular SERS immuno-sensing.

    PubMed

    Bhardwaj, Vinay; Srinivasan, Supriya; McGoron, Anthony J

    2015-06-21

    High throughput intracellular delivery strategies, electroporation, passive and TATHA2 facilitated diffusion of colloidal silver nanoparticles (AgNPs) are investigated for cellular toxicity and uptake using state-of-art analytical techniques. The TATHA2 facilitated approach efficiently delivered high payload with no toxicity, pre-requisites for intracellular applications of plasmonic metal nanoparticles (PMNPs) in sensing and therapeutics.

  10. BDI-modelling of complex intracellular dynamics.

    PubMed

    Jonker, C M; Snoep, J L; Treur, J; Westerhoff, H V; Wijngaards, W C A

    2008-03-07

    A BDI-based continuous-time modelling approach for intracellular dynamics is presented. It is shown how temporalized BDI-models make it possible to model intracellular biochemical processes as decision processes. By abstracting from some of the details of the biochemical pathways, the model achieves understanding in nearly intuitive terms, without losing veracity: classical intentional state properties such as beliefs, desires and intentions are founded in reality through precise biochemical relations. In an extensive example, the complex regulation of Escherichia coli vis-à-vis lactose, glucose and oxygen is simulated as a discrete-state, continuous-time temporal decision manager. Thus a bridge is introduced between two different scientific areas: the area of BDI-modelling and the area of intracellular dynamics.

  11. Inhibition of APP gamma-secretase restores Sonic Hedgehog signaling and neurogenesis in the Ts65Dn mouse model of Down syndrome.

    PubMed

    Giacomini, Andrea; Stagni, Fiorenza; Trazzi, Stefania; Guidi, Sandra; Emili, Marco; Brigham, Elizabeth; Ciani, Elisabetta; Bartesaghi, Renata

    2015-10-01

    Neurogenesis impairment starting from early developmental stages is a key determinant of intellectual disability in Down syndrome (DS). Previous evidence provided a causal relationship between neurogenesis impairment and malfunctioning of the mitogenic Sonic Hedgehog (Shh) pathway. In particular, excessive levels of AICD (amyloid precursor protein intracellular domain), a cleavage product of the trisomic gene APP (amyloid precursor protein) up-regulate transcription of Ptch1 (Patched1), the Shh receptor that keeps the pathway repressed. Since AICD results from APP cleavage by γ-secretase, the goal of the current study was to establish whether treatment with a γ-secretase inhibitor normalizes AICD levels and restores neurogenesis in trisomic neural precursor cells. We found that treatment with a selective γ-secretase inhibitor (ELND006; ELN) restores proliferation in neurospheres derived from the subventricular zone (SVZ) of the Ts65Dn mouse model of DS. This effect was accompanied by reduction of AICD and Ptch1 levels and was prevented by inhibition of the Shh pathway with cyclopamine. Treatment of Ts65Dn mice with ELN in the postnatal period P3-P15 restored neurogenesis in the SVZ and hippocampus, hippocampal granule cell number and synapse development, indicating a positive impact of treatment on brain development. In addition, in the hippocampus of treated Ts65Dn mice there was a reduction in the expression levels of various genes that are transcriptionally regulated by AICD, including APP, its origin substrate. Inhibitors of γ-secretase are currently envisaged as tools for the cure of Alzheimer's disease because they lower βamyloid levels. Current results provide novel evidence that γ-secretase inhibitors may represent a strategy for the rescue of neurogenesis defects in DS.

  12. Macrophage defense mechanisms against intracellular bacteria.

    PubMed

    Weiss, Günter; Schaible, Ulrich E

    2015-03-01

    Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics.

  13. The ocular albinism type 1 gene product, OA1, spans intracellular membranes 7 times.

    PubMed

    Sone, Michio; Orlow, Seth J

    2007-12-01

    OA1 (GPR143) is a pigment cell-specific intracellular glycoprotein consisting of 404 amino acid residues that is mutated in patients with ocular albinism type 1, the most common form of ocular albinism. While its cellular localization is suggested to be endolysosomal and melanosomal, the physiological function of OA1 is currently unclear. Recent reports predicted that OA1 functions as a G protein coupled receptor (GPCR) based on its weak amino acid sequence similarity to known GPCRs, and on demonstration of GPCR activity in OA1 mislocalized to the plasma membrane. Because mislocalization of proteins is often caused by or induces defects in their proper folding/assembly, the significance of these studies remains unclear. A characteristic feature of GPCRs is a seven transmembrane domain structure. We analyzed the membrane topology of OA1 properly localized to intracellular lysosomal organelles in COS-1 cells. To accomplish this analysis, we established experimental conditions that allowed selective permeabilization of the plasma membrane while leaving endolysosomal membranes intact. Domains were mapped by the insertion of a hemagglutinin (HA) tag into the predicted cytosolic/luminal regions of OA1 molecule and the accessibility of tag to HA antibody was determined by immunofluorescence. HA-tagged lysosome associated membrane protein 1 (LAMP1), a type I membrane protein, was employed as a reporter for selective permeabilization of the plasma membrane. Our results show experimentally that the C-terminus of OA1 is directed to the cytoplasm and that the protein spans the intracellular membrane 7 times. Thus, OA1, properly localized intracellularly, is a 7 transmembrane domain integral membrane protein consistent with its putative role as an intracellular GPCR.

  14. NMR measurements of intracellular ions in hypertension

    NASA Astrophysics Data System (ADS)

    Veniero, Joseph C.; Gupta, R. K.

    1993-08-01

    The NMR methods for the measurement of intracellular free Na+, K+, Mg2+, Ca2+, and H+ are introduced. The recent literature is then presented showing applications of these methods to cells and tissues from hypertensive animal model systems, and humans with essential hypertension. The results support the hypothesis of consistent derangement of the intracellular ionic environment in hypertension. The theory that this derangement may be a common link in the disease states of high blood pressure and abnormal insulin and glucose metabolism, which are often associated clinically, is discussed.

  15. GTPases in intracellular trafficking: an overview.

    PubMed

    Segev, Nava

    2011-02-01

    Small GTPases that belong to the ras sub-families of Rab, Arf, and Rho, and the large GTPase dynamin, regulate intracellular trafficking. This issue of Seminars of Cell and Developmental Biology highlights topics regarding mechanisms by which these GTPases regulate the different steps of vesicular transport: vesicle formation, scission, targeting and fusion. In addition, the emerging roles of GTPases in coordination of individual transport steps as well as coordination of intracellular trafficking with other cellular processes are reviewed. Finally, common structures and mechanisms underlying the function of the ras-like GTPases and the importance of their function to human health and disease are discussed.

  16. Swine TRIM21 restricts FMDV infection via an intracellular neutralization mechanism.

    PubMed

    Fan, Wenchun; Zhang, Dong; Qian, Ping; Qian, Suhong; Wu, Mengge; Chen, Huanchun; Li, Xiangmin

    2016-03-01

    The tripartite motif protein 21 (TRIM21) is a ubiquitously expressed E3 ubiquitin ligase and an intracellular antibody receptor. TRIM21 mediates antibody-dependent intracellular neutralization (ADIN) in cytosol and provides an intracellular immune response to protect host defense against pathogen infection. In this study, swine TRIM21 (sTRIM21) was cloned and its role in ADIN was investigated. The expression of sTRIM21 is induced by type I interferon in PK-15 cells. sTRIM21 restricts FMDV infection in the presence of FMDV specific antibodies. Furthermore, sTRIM21 interacts with Fc fragment of swine immunoglobulin G (sFc) fused VP1 of FMDV and thereby causing its degradation. Both the RING and SPRY domains are essential for sTRIM21 to degrade sFc-fused VP1. These results suggest that the intracellular neutralization features of FMDV contribute to the antiviral activity of sTRIM21. sTRIM21 provide another intracellular mechanism to inhibit FMDV infection in infected cells.

  17. Interrogation of Cellular Innate Immunity by Diamond-Nanoneedle-Assisted Intracellular Molecular Fishing.

    PubMed

    Wang, Zixun; Yang, Yang; Xu, Zhen; Wang, Ying; Zhang, Wenjun; Shi, Peng

    2015-10-14

    Understanding intracellular signaling cascades and network is one of the core topics in modern biology. Novel tools based on nanotechnologies have enabled probing and analyzing intracellular signaling with unprecedented sensitivity and specificity. In this study, we developed a minimally invasive method for in situ probing specific signaling components of cellular innate immunity in living cells. The technique was based on diamond-nanoneedle arrays functionalized with aptamer-based molecular sensors, which were inserted into cytoplasmic domain using a centrifugation controlled process to capture molecular targets. Simultaneously, these diamond-nanoneedles also facilitated the delivery of double-strand DNAs (dsDNA90) into cells to activate the pathway involving the stimulator of interferon genes (STING). We showed that the nanoneedle-based biosensors can be successfully utilized to isolate transcriptional factor, NF-κB, from intracellular regions without damaging the cells, upon STING activation. By using a reversible protocol and repeated probing in living cells, we were able to examine the singling dynamics of NF-κB, which was quickly translocated from cytoplasm to nucleus region within ∼40 min of intracellular introduction of dsDNA90 for both A549 and neuron cells. These results demonstrated a novel and versatile tool for targeted in situ dissection of intracellular signaling, providing the potential to resolve new sights into various cellular processes.

  18. A salt bridge in intracellular loop 2 is essential for folding of human p-glycoprotein.

    PubMed

    Loo, Tip W; Clarke, David M

    2013-05-14

    There is no high-resolution structure of the human P-glycoprotein (P-gp, ABCB1) drug pump. Homology models based on the crystal structures of mouse and Caenorhabditis elegans P-gps show extensive contacts between intracellular loop 2 (ICL2, in the first transmembrane domain) and the second nucleotide-binding domain. Human P-gp modeled on these P-gp structures yields different ICL2 structures. Only the model based on the C. elegans P-gp structure predicts the presence of a salt bridge. We show that the Glu256-Arg276 salt bridge was critical for P-gp folding.

  19. The Effect of Size and Species on Lens Intracellular Hydrostatic Pressure

    PubMed Central

    Gao, Junyuan; Sun, Xiurong; Moore, Leon C.; Brink, Peter R.; White, Thomas W.; Mathias, Richard T.

    2013-01-01

    Purpose. Previous experiments showed that mouse lenses have an intracellular hydrostatic pressure that varied from 335 mm Hg in central fibers to 0 mm Hg in surface cells. Model calculations predicted that in larger lenses, all else equal, pressure should increase as the lens radius squared. To test this prediction, lenses of different radii from different species were studied. Methods. All studies were done in intact lenses. Intracellular hydrostatic pressures were measured with a microelectrode-manometer–based system. Membrane conductances were measured by frequency domain impedance analysis. Intracellular Na+ concentrations were measured by injecting the Na+-sensitive dye sodium-binding benzofuran isophthalate. Results. Intracellular hydrostatic pressures were measured in lenses from mice, rats, rabbits, and dogs with radii (cm) 0.11, 0.22, 0.49, and 0.57, respectively. In each species, pressure varied from 335 ± 6 mm Hg in central fiber cells to 0 mm Hg in surface cells. Further characterization of transport in lenses from mice and rats showed that the density of fiber cell gap junction channels was approximately the same, intracellular Na+ concentrations varied from 17 mM in central fiber cells to 7 mM in surface cells, and intracellular voltages varied from −45 mV in central fiber cells to −60 mV in surface cells. Fiber cell membrane conductance was a factor of 2.7 times larger in mouse than in rat lenses. Conclusions. Intracellular hydrostatic pressure is an important physiological parameter that is regulated in lenses from these different species. The most likely mechanism of regulation is to reduce the density of open Na+-leak channels in fiber cells of larger lenses. PMID:23211824

  20. [Magnetic nanoparticles and intracellular delivery of biopolymers].

    PubMed

    Kornev, A A; Dubina, M V

    2014-03-01

    The basic methods of intracellular delivery of biopolymers are present in this review. The structure and synthesis of magnetic nanoparticles, their stabilizing surfactants are described. The examples of the interaction of nanoparticles with biopolymers such as nucleic acids and proteins are considered. The final part of the review is devoted to problems physiology and biocompatibility of magnetic nanoparticles.

  1. Activities of Antimicrobial Agents against Intracellular Pneumococci

    PubMed Central

    Mandell, Gerald L.; Coleman, Elizabeth J.

    2000-01-01

    Pneumococci can enter and survive inside human lung alveolar carcinoma cells. We examined the activity of azithromycin, gentamicin, levofloxacin, moxifloxacin, penicillin G, rifampin, telithromycin, and trovafloxacin against pneumococci inside and outside cells. We found that moxifloxacin, trovafloxacin, and telithromycin were the most active, but only telithromycin killed all intracellular organisms. PMID:10952618

  2. Histoplasma capsulatum surmounts obstacles to intracellular pathogenesis

    PubMed Central

    Garfoot, Andrew L.; Rappleye, Chad A.

    2016-01-01

    The fungal pathogen Histoplasma capsulatum causes respiratory and disseminated disease, even in immunocompetent hosts. In contrast to opportunistic pathogens, which are readily controlled by phagocytic cells, H. capsulatum yeasts are able to infect macrophages, survive antimicrobial defenses, and proliferate as an intracellular pathogen. In this review, we discuss some of the molecular mechanisms that enable H. capsulatum yeasts to overcome obstacles to intracellular pathogenesis. H. capsulatum yeasts gain refuge from extracellular obstacles such as antimicrobial lung surfactant proteins by engaging the β-integrin family of phagocytic receptors to promote entry into macrophages. In addition, H. capsulatum yeasts conceal immunostimulatory β-glucans to avoid triggering signaling receptors such as the β-glucan receptor Dectin-1. H. capsulatum yeasts counteract phagocyte-produced reactive oxygen species by expression of oxidative stress defense enzymes including an extracellular superoxide dismutase and an extracellular catalase. Within the phagosome, H. capsulatum yeasts block phagosome acidification, acquire essential metals such as iron and zinc, and utilize de novo biosynthesis pathways to overcome nutritional limitations. These mechanisms explain how H. capsulatum yeasts avoid and negate macrophage defense strategies and establish a hospitable intracellular niche, making H. capsulatum a successful intracellular pathogen of macrophages. PMID:26235362

  3. Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics

    PubMed Central

    Lönn, Peter; Kacsinta, Apollo D.; Cui, Xian-Shu; Hamil, Alexander S.; Kaulich, Manuel; Gogoi, Khirud; Dowdy, Steven F.

    2016-01-01

    Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells. PMID:27604151

  4. Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics.

    PubMed

    Lönn, Peter; Kacsinta, Apollo D; Cui, Xian-Shu; Hamil, Alexander S; Kaulich, Manuel; Gogoi, Khirud; Dowdy, Steven F

    2016-09-08

    Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells.

  5. Mitochondrial γ-secretase participates in the metabolism of mitochondria-associated amyloid precursor protein.

    PubMed

    Pavlov, Pavel F; Wiehager, Birgitta; Sakai, Jun; Frykman, Susanne; Behbahani, Homira; Winblad, Bengt; Ankarcrona, Maria

    2011-01-01

    Intracellular amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). Mitochondria were found to be the target both for amyloid precursor protein (APP) that accumulates in the mitochondrial import channels and for Aβ that interacts with several proteins inside mitochondria and leads to mitochondrial dysfunction. Here, we have studied the role of mitochondrial γ-secretase in processing different substrates. We found that a significant proportion of APP is associated with mitochondria in cultured cells and that γ-secretase cleaves the shedded C-terminal part of APP identified as C83 associated with the outer membrane of mitochondria (OMM). Moreover, we have established the topology of the C83 in the OMM and found the APP intracellular domain (AICD) to be located inside mitochondria. Our data show for the first time that APP is a substrate for the mitochondrial γ-secretase and that AICD is produced inside mitochondria. Thus, we provide a mechanistic view of the mitochondria-associated APP metabolism where AICD, P3 peptide and potentially Aβ are produced locally and may contribute to mitochondrial dysfunction in AD.

  6. Functional domains in tetraspanin proteins.

    PubMed

    Stipp, Christopher S; Kolesnikova, Tatiana V; Hemler, Martin E

    2003-02-01

    Exciting new findings have emerged about the structure, function and biochemistry of tetraspanin proteins. Five distinct tetraspanin regions have now been delineated linking structural features to specific functions. Within the large extracellular loop of tetraspanins, there is a variable region that mediates specific interactions with other proteins, as well as a more highly conserved region that has been suggested to mediate homodimerization. Within the transmembrane region, the four tetraspanin transmembrane domains are probable sites of both intra- and inter-molecular interactions that are crucial during biosynthesis and assembly of the network of tetraspanin-linked membrane proteins known as the 'tetraspanin web'. In the intracellular juxtamembrane region, palmitoylation of cysteine residues also contributes to tetraspanin web assembly, and the C-terminal cytoplasmic tail region could provide specific functional links to cytoskeletal or signaling proteins.

  7. Intracellular mechanisms of solar water disinfection

    PubMed Central

    Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

    2016-01-01

    Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection. PMID:27909341

  8. Intracellular mechanisms of solar water disinfection.

    PubMed

    Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

    2016-12-02

    Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection.

  9. Intracellular mechanisms of solar water disinfection

    NASA Astrophysics Data System (ADS)

    Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

    2016-12-01

    Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection.

  10. Modeling of spatially-restricted intracellular signaling.

    PubMed

    Neves, Susana R

    2012-01-01

    Understanding the signaling capabilities of a cell presents a major challenge, not only due to the number of molecules involved, but also because of the complex network connectivity of intracellular signaling. Recently, the proliferation of quantitative imaging techniques has led to the discovery of the vast spatial organization of intracellular signaling. Computational modeling has emerged as a powerful tool for understanding how inhomogeneous signaling originates and is maintained. This article covers the current imaging techniques used to obtain quantitative spatial data and the mathematical approaches used to model spatial cell biology. Modeling-derived hypotheses have been experimentally tested and the integration of modeling and imaging approaches has led to non-intuitive mechanistic insights.

  11. Dynamics of gradient formation by intracellular shuttling

    SciTech Connect

    Berezhkovskii, Alexander M.; Shvartsman, Stanislav Y.

    2015-08-21

    A number of important cellular functions rely on the formation of intracellular protein concentration gradients. Experimental studies discovered a number of mechanisms for the formation of such gradients. One of the mechanisms relies on the intracellular shuttling of a protein that interconverts between the two states with different diffusivities, under the action of two enzymes, one of which is localized to the plasma membrane, whereas the second is uniformly distributed in the cytoplasm. Recent work reported an analytical solution for the steady state gradient in this mechanism, obtained in the framework of a one-dimensional reaction-diffusion model. Here, we study the dynamics in this model and derive analytical expressions for the Laplace transforms of the time-dependent concentration profiles in terms of elementary transcendental functions. Inverting these transforms numerically, one can obtain time-dependent concentration profiles of the two forms of the protein.

  12. Toward Intracellular Targeted Delivery of Cancer Therapeutics

    PubMed Central

    Pandya, Hetal; Debinski, Waldemar

    2013-01-01

    A number of anti-cancer drugs have their targets localized to particular intracellular compartments. These drugs reach the targets mainly through diffusion, dependent on biophysical and biochemical forces that allow cell penetration. This means that both cancer cells and normal cells will be subjected to such diffusion; hence many of these drugs, like chemotherapeutics, are potentially toxic and the concentration achieved at the site of their action is often suboptimal. The same relates to radiation that indiscriminately affects normal and diseased cells. However, nature-designed systems enable compounds present in the extracellular environment to end up inside the cell and even travel to more specific intracellular compartments. For example, viruses and bacterial toxins can more or less specifically recognize eukaryotic cells, enter these cells, and direct some protein portions to designated intracellular areas. These phenomena have led to creative thinking, such as employing viruses or bacterial toxins for cargo delivery to cells and, more specifically, to cancer cells. Proteins can be genetically engineered in order to not only mimic what viruses and bacterial toxins can do, but also to add new functions, extending or changing the intracellular routes. It is possible to make conjugates or, more preferably, single-chain proteins that recognize cancer cells and deliver cargo inside the cells, even to the desired subcellular compartment. These findings offer new opportunities to deliver drugs/labels only to cancer cells and only to their site of action within the cells. The development of such dual-specificity vectors for targeting cancer cells is an attractive and potentially safer and more efficacious way of delivering drugs. We provide examples of this approach for delivering brain cancer therapeutics, using a specific biomarker on glioblastoma tumor cells. PMID:22671766

  13. Targeting caspases in intracellular protozoan infections.

    PubMed

    Guillermo, Landi V C; Pereira, Wânia F; De Meis, Juliana; Ribeiro-Gomes, Flavia L; Silva, Elisabeth M; Kroll-Palhares, Karina; Takiya, Christina M; Lopes, Marcela F

    2009-06-01

    Caspases are cysteine aspartases acting either as initiators (caspases 8, 9, and 10) or executioners (caspases 3, 6, and 7) to induce programmed cell death by apoptosis. Parasite infections by certain intracellular protozoans increase host cell life span by targeting caspase activation. Conversely, caspase activation, followed by apoptosis of lymphocytes and other cells, prevents effective immune responses to chronic parasite infection. Here we discuss how pharmacological inhibition of caspases might affect the immunity to protozoan infections, by either blocking or delaying apoptosis.

  14. Intracellular serpins, firewalls and tissue necrosis.

    PubMed

    Marciniak, Stefan J; Lomas, David A

    2008-02-01

    Luke and colleagues have recently attributed a new role to a member of the serpin superfamily of serine proteinase inhibitors. They have used Caenorhabditis elegans to show that an intracellular serpin is crucial for maintaining lysosomal integrity. We examine the role of this firewall in preventing necrosis and attempt to integrate this with current theories of stress-induced protein degradation. We discuss how mutant serpins cause disease either through polymerization or now, perhaps, by unleashing necrosis.

  15. Bioreducible Lipid-like Nanoparticles for Intracellular Protein Delivery

    NASA Astrophysics Data System (ADS)

    Arellano, Carlos Luis

    Protein-based therapy is one of the most direct ways to manipulate cell function and treat human disease. Although protein therapeutics has made its way to clinical practice, with five of the top fifteen global pharmaceuticals being peptide or protein-based drugs, one common limitation is that the effects of protein therapy are only achieved through the targeting of cell surface receptors and intracellular domains. Due to the impermeability of the cell membrane to most foreign materials, entire classes of potentially therapeutic proteins cannot thoroughly be studied without a safe and efficient method of transporting proteins into the cytosol. We report the use of a combinatorially-designed bioreducible lipid-like material (termed "lipidoid") - based protein delivery platform for the transfection of human cancer cell lines. Lipidoid nanoparticles are synthesized through a thin film dispersion method. The degradation of the bioreducible nanoparticles was observed when exposed to glutathione, a highly reductive compound present in the cytosol. We demonstrate that the nanoparticles are capable of transfecting a dose-dependent concentration of our model protein, beta-galactosidase into HeLa cells. Furthermore, formulations of the lipidoid containing the cytotoxic proteins saporin and RNase-A are both capable of inhibiting tumor cell proliferation as observed in in vitro treatment of different human cancer cell lines. There was no observed loss in protein activity after lyophilization and long--term storage, indicating the potential of pre-clinical applications. Overall, we demonstrate an effective approach to protein formulation and intracellular delivery. We believe that our formulations will lead to the study of a whole class of previously untapped therapeutics that may generate new solutions for previously untreatable diseases.

  16. Detection of intracellular phosphatidylserine in living cells.

    PubMed

    Calderon, Frances; Kim, Hee-Yong

    2008-03-01

    To demonstrate the intracellular phosphatidylserine (PS) distribution in neuronal cells, neuroblastoma cells and hippocampal neurons expressing green fluorescence protein (GFP)-AnnexinV were stimulated with a calcium ionophore and localization of GFP-AnnexinV was monitored by fluorescence microscopy. Initially, GFP-AnnexinV distributed evenly in the cytosol and nucleus. Raising the intracellular calcium level with ionomycin-induced translocation of cytoplasmic GFP-AnnexinV to the plasma membrane but not to the nuclear membrane, indicating that PS distributes in the cytoplasmic side of the plasma membrane. Nuclear GFP-AnnexinV subsequently translocated to the nuclear membrane, indicating PS localization in the nuclear envelope. GFP-AnnexinV also localized in a juxtanuclear organelle that was identified as the recycling endosome. However, minimal fluorescence was detected in any other subcellular organelles including mitochondria, endoplasmic reticulum, Golgi complex, and lysosomes, strongly suggesting that PS distribution in the cytoplasmic face in these organelles is negligible. Similarly, in hippocampal primary neurons PS distributed in the inner leaflet of plasma membranes of cell body and dendrites, and in the nuclear envelope. To our knowledge, this is the first demonstration of intracellular PS localization in living cells, providing an insight for specific sites of PS interaction with soluble proteins involved in signaling processes.

  17. A bacteriophage endolysin that eliminates intracellular streptococci

    PubMed Central

    Shen, Yang; Barros, Marilia; Vennemann, Tarek; Gallagher, D Travis; Yin, Yizhou; Linden, Sara B; Heselpoth, Ryan D; Spencer, Dennis J; Donovan, David M; Moult, John; Fischetti, Vincent A; Heinrich, Frank; Lösche, Mathias; Nelson, Daniel C

    2016-01-01

    PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB–PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities. DOI: http://dx.doi.org/10.7554/eLife.13152.001 PMID:26978792

  18. Invasion and Intracellular Survival by Protozoan Parasites

    PubMed Central

    Sibley, L. David

    2013-01-01

    Summary Intracellular parasitism has arisen only a few times during the long ancestry of protozoan parasites including in diverse groups such as microsporidians, kinetoplastids, and apicomplexans. Strategies used to gain entry differ widely from injection (e.g. microsporidians), active penetration of the host cell (e.g. Toxoplasma), recruitment of lysosomes to a plasma membrane wound (e.g. Trypanosoma cruzi), to host cell-mediated phagocytosis (e.g. Leishmania). The resulting range of intracellular niches is equally diverse ranging from cytosolic (e.g. T. cruzi) to residing within a nonfusigenic vacuole (e.g. Toxoplasma, Encephalitizoon) or a modified phagolysosome (e.g. Leishmania). These lifestyle choices influence access to nutrients, interaction with host cell signaling pathways, and detection by pathogen recognition systems. As such, intracellular life requires a repertoire of adaptations to assure entry-exit from the cell, as well as to thwart innate immune mechanisms and prevent clearance. Elucidating these pathways at the cellular and molecular level may identify key steps that can be targeted to reduce parasite survival or augment immunological responses and thereby prevent disease. PMID:21349087

  19. Intracellular Pressure Dynamics in Blebbing Cells.

    PubMed

    Strychalski, Wanda; Guy, Robert D

    2016-03-08

    Blebs are pressure-driven protrusions that play an important role in cell migration, particularly in three-dimensional environments. A bleb is initiated when the cytoskeleton detaches from the cell membrane, resulting in the pressure-driven flow of cytosol toward the area of detachment and local expansion of the cell membrane. Recent experiments involving blebbing cells have led to conflicting hypotheses regarding the timescale of intracellular pressure propagation. The interpretation of one set of experiments supports a poroelastic model of the cytoplasm that leads to slow pressure equilibration when compared to the timescale of bleb expansion. A different study concludes that pressure equilibrates faster than the timescale of bleb expansion. To address this discrepancy, a dynamic computational model of the cell was developed that includes mechanics of and the interactions among the cytoplasm, the actin cortex, the cell membrane, and the cytoskeleton. The model results quantify the relationship among cytoplasmic rheology, pressure, and bleb expansion dynamics, and provide a more detailed picture of intracellular pressure dynamics. This study shows the elastic response of the cytoplasm relieves pressure and limits bleb size, and that both permeability and elasticity of the cytoplasm determine bleb expansion time. Our model with a poroelastic cytoplasm shows that pressure disturbances from bleb initiation propagate faster than the timescale of bleb expansion and that pressure equilibrates slower than the timescale of bleb expansion. The multiple timescales in intracellular pressure dynamics explain the apparent discrepancy in the interpretation of experimental results.

  20. The Intracellular Loop of the Glycine Receptor: It’s not all about the Size

    PubMed Central

    Langlhofer, Georg; Villmann, Carmen

    2016-01-01

    The family of Cys-loop receptors (CLRs) shares a high degree of homology and sequence identity. The overall structural elements are highly conserved with a large extracellular domain (ECD) harboring an α-helix and 10 β-sheets. Following the ECD, four transmembrane domains (TMD) are connected by intracellular and extracellular loop structures. Except the TM3–4 loop, their length comprises 7–14 residues. The TM3–4 loop forms the largest part of the intracellular domain (ICD) and exhibits the most variable region between all CLRs. The ICD is defined by the TM3–4 loop together with the TM1–2 loop preceding the ion channel pore. During the last decade, crystallization approaches were successful for some members of the CLR family. To allow crystallization, the intracellular loop was in most structures replaced by a short linker present in prokaryotic CLRs. Therefore, no structural information about the large TM3–4 loop of CLRs including the glycine receptors (GlyRs) is available except for some basic stretches close to TM3 and TM4. The intracellular loop has been intensively studied with regard to functional aspects including desensitization, modulation of channel physiology by pharmacological substances, posttranslational modifications, and motifs important for trafficking. Furthermore, the ICD interacts with scaffold proteins enabling inhibitory synapse formation. This review focuses on attempts to define structural and functional elements within the ICD of GlyRs discussed with the background of protein-protein interactions and functional channel formation in the absence of the TM3–4 loop. PMID:27330534

  1. The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

    SciTech Connect

    Papazyan, Romeo; Rozengurt, Enrique; Rey, Osvaldo . E-mail: orey@mednet.ucla.edu

    2006-04-14

    We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.

  2. Functional domains of the poliovirus receptor

    SciTech Connect

    Koike, Satoshi; Ise, Iku; Nomoto, Akio )

    1991-05-15

    A number of mutant cDNAs of the human poliovirus receptor were constructed to identify essential regions of the molecule as the receptor. All mutant cDNAs carrying the sequence coding for the entire N-terminal immunoglobulin-like domain (domain I) confer permissiveness for poliovirus to mouse L cells, but a mutant cDNA lacking the sequence for domain I does not. The transformants permissive for poliovirus were able to bind the virus and were also recognized by monoclonal antibody D171, which competes with poliovirus for the cellular receptor. These results strongly suggest that the poliovirus binding site resides in domain I of the receptor. Mutant cDNAs for the sequence encoding the intracellular peptide were also constructed and expressed in mouse L cells. Susceptibility of these cells to poliovirus revealed that the entire putative cytoplasmic domain is not essential for virus infection. Thus, the cytoplasmic domain of the molecule appears not to play a role in the penetration of poliovirus.

  3. The roles of amyloid precursor protein (APP) in neurogenesis: Implications to pathogenesis and therapy of Alzheimer disease.

    PubMed

    Zhou, Zhi-dong; Chan, Christine Hui-shan; Ma, Quan-hong; Xu, Xiao-hong; Xiao, Zhi-cheng; Tan, Eng-king

    2011-01-01

    The amyloid-beta (Aβ) peptide is the derivative of amyloid precursor protein (APP) generated through sequential proteolytic processing by β- and γ-secretases. Excessive accumulation of Aβ, the main constituent of amyloid plaques, has been implicated in the etiology of Alzheimer's disease (AD). It was found recently that the impairments of neurogenesis in brain were associated with the pathogenesis of AD. Furthermore recent findings implicated that APP could function to influence proliferation of neural progenitor cells (NPC) and might regulate transcriptional activity of various genes. Studies demonstrated that influence of neurogenesis by APP is conferred differently via its two separate domains, soluble secreted APPs (sAPPs, mainly sAPPα) and APP intracellular domain (AICD). The sAPPα was shown to be neuroprotective and important to neurogenesis, whereas AICD was found to negatively modulate neurogenesis. Furthermore, it was demonstrated recently that microRNA could function to regulate APP expression, APP processing, Aβ accumulation and subsequently influence neurotoxicity and neurogenesis related to APP, which was implicated to AD pathogenesis, especially for sporadic AD. Based on data accumulated, secretase balances were proposed. These secretase balances could influence the downstream balance related to regulation of neurogenesis by AICD and sAPPα as well as balance related to influence of neuron viability by Aβ and sAPPα. Disruption of these secretase balances could be culprits to AD onset.

  4. The first intracellular loop of GLUT4 contains a retention motif.

    PubMed

    Talantikite, Maya; Berenguer, Marion; Gonzalez, Teresa; Alessi, Marie Christine; Poggi, Marjorie; Peiretti, Franck; Govers, Roland

    2016-06-01

    Glucose transporter GLUT4 (also known as SLC2A4) plays a major role in glucose homeostasis and is efficiently retained intracellularly in adipocytes and myocytes. To simplify the analysis of its retention, here, various intracellular GLUT4 domains were fused individually to reporter molecules. Of the four short cytoplasmic loops of GLUT4, only the first nine-residue-long loop conferred intracellular retention of truncated forms of the transferrin receptor and CD4 in adipocytes. In contrast, the same loop of GLUT1 was without effect. The reporter molecules to which the first loop of GLUT4 was fused localized, unlike GLUT4, to the trans-Golgi network (TGN), possibly explaining why these molecules did not respond to insulin. The retention induced by the GLUT4 loop was specific to adipocytes as it did not induce retention in preadipocytes. Of the SQWLGRKRA sequence that constitutes this loop, mutation of either the tryptophan or lysine residue abrogated reporter retention. Mutation of these residues individually into alanine residues in the full-length GLUT4 molecule resulted in a decreased retention for GLUT4-W105A. We conclude that the first intracellular loop of GLUT4 contains the retention motif WLGRK, in which W105 plays a prominent role.

  5. Polarized exocyst-mediated vesicle fusion directs intracellular lumenogenesis within the C. elegans excretory cell.

    PubMed

    Armenti, Stephen T; Chan, Emily; Nance, Jeremy

    2014-10-01

    Lumenogenesis of small seamless tubes occurs through intracellular membrane growth and directed vesicle fusion events. Within the Caenorhabditis elegans excretory cell, which forms seamless intracellular tubes (canals) that mediate osmoregulation, lumens grow in length and diameter when vesicles fuse with the expanding lumenal surface. Here, we show that lumenal vesicle fusion depends on the small GTPase RAL-1, which localizes to vesicles and acts through the exocyst vesicle-tethering complex. Loss of either the exocyst or RAL-1 prevents excretory canal lumen extension. Within the excretory canal and other polarized cells, the exocyst co-localizes with the PAR polarity proteins PAR-3, PAR-6 and PKC-3. Using early embryonic cells to determine the functional relationships between the exocyst and PAR proteins, we show that RAL-1 recruits the exocyst to the membrane, while PAR proteins concentrate membrane-localized exocyst proteins to a polarized domain. These findings reveal that RAL-1 and the exocyst direct the polarized vesicle fusion events required for intracellular lumenogenesis of the excretory cell, suggesting mechanistic similarities in the formation of topologically distinct multicellular and intracellular lumens.

  6. Understanding the Public Domain.

    ERIC Educational Resources Information Center

    Russell, Carrie

    2003-01-01

    This overview of the public domain covers: defining the public domain; figuring out if a work is protected by copyright; being sure a work is in the public domain; asserting the copyright protection and term; the Creative Commons initiative; building the Information Commons; when permission is needed for using a public domain work; and special…

  7. Landmark discoveries in intracellular transport and secretion

    PubMed Central

    Paknikar, Kishore M

    2007-01-01

    Abstract Cellular protein transport and secretion is fundamental to the very existence of an organism, regulating important physiological functions such as reproduction, digestion, energy production, growth, neurotransmission, hormone release, water and ion transport, etc., all required for the survival and maintenance of homeostasis within an organism. Molecular understanding of transport and secretion of intracellular product has therefore been of paramount importance and aggressively investigated for over six decades. Only in the last 20 years, the general molecular mechanism of the process has come to light, following discovery of key proteins involved in ER-Golgi transport, and discovery of the ‘porosome’– the universal secretion machinery in cells. PMID:17635635

  8. The Intracellular Life of Cryptococcus neoformans

    PubMed Central

    Coelho, Carolina; Bocca, Anamelia L.; Casadevall, Arturo

    2016-01-01

    Cryptococcus neoformans is a fungal pathogen with worldwide distribution. Serological studies of human populations show a high prevalence of human infection, which rarely progresses to disease in immunocompetent hosts. However, decreased host immunity places individuals at high risk for cryptococcal disease. The disease can result from acute infection or reactivation of latent infection, in which yeasts within granulomas and host macrophages emerge to cause disease. In this review, we summarize what is known about the cellular recognition, ingestion, and killing of C. neoformans and discuss the unique and remarkable features of its intracellular life, including the proposed mechanisms for fungal persistence and killing in phagocytic cells. PMID:24050625

  9. Intracellular pH in sperm physiology.

    PubMed

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction.

  10. Anchors aweigh: protein localization and transport mediated by transmembrane domains.

    PubMed

    Cosson, Pierre; Perrin, Jackie; Bonifacino, Juan S

    2013-10-01

    The transmembrane domains (TMDs) of integral membrane proteins have emerged as major determinants of intracellular localization and transport in the secretory and endocytic pathways. Unlike sorting signals in cytosolic domains, TMD sorting determinants are not conserved amino acid sequences but physical properties such as the length and hydrophilicity of the transmembrane span. The underlying sorting machinery is still poorly characterized, but several mechanisms have been proposed, including TMD recognition by transmembrane sorting receptors and partitioning into membrane lipid domains. Here we review the nature of TMD sorting determinants and how they may dictate transmembrane protein localization and transport.

  11. Anchors Aweigh: Protein Traffic Mediated by Transmembrane Domains

    PubMed Central

    Cosson, Pierre; Perrin, Jackie; Bonifacino, Juan S.

    2013-01-01

    The transmembrane domains (TMDs) of integral membrane proteins have emerged as major determinants of intracellular localization and transport in the secretory and endocytic pathways. Unlike sorting signals in the cytosolic domains, TMD sorting determinants are not conserved amino-acid sequences but physical properties such as length and hydrophilicity of the transmembrane span. The underlying sorting machinery is still poorly characterized but several mechanisms have been proposed, including TMD recognition by transmembrane sorting receptors and partitioning into membrane lipid domains. Here we review the nature of TMD sorting determinants and how they may dictate transmembrane protein localization and transport. PMID:23806646

  12. Backscattered light confocal imaging of intracellular MTT-formazan crystals.

    PubMed

    Bernas, Tytus; Dobrucki, Jurek W

    2004-06-01

    Metabolically active animal and plant cells reduce MTT tetrazolium salt to a corresponding nonfluorescent formazan. Reduction of MTT by viable cells is exploited in a number of tests widely used in biological research. The aim of this study was to optimize a microscopy method of detecting small crystals of MTT-formazan formed in intact cells maintained in in vitro cultures. We examined scattering properties of small intracellular crystals of MTT formazan and found that the efficiency of light scattering was dependent on wavelength. Small (<3 microm) crystals of MTT-formazan, formed in viable cells, scattered red, but not blue, light. Large crystals, which are formed later at a stage when cells begin to lose viability, scattered both red and blue light. We conclude that optimal detection of early stages of crystallization of MTT-formazan in living cells is possible using confocal microscopy of red, but not blue, scattered light. High contrast and resolution of images can be achieved by filtering out interference effects in the frequency domain.

  13. IQGAP1: a regulator of intracellular spacetime relativity.

    PubMed

    Malarkannan, Subramaniam; Awasthi, Aradhana; Rajasekaran, Kamalakannan; Kumar, Pawan; Schuldt, Kristina M; Bartoszek, Allison; Manoharan, Niranjan; Goldner, Nicholas K; Umhoefer, Colleen M; Thakar, Monica S

    2012-03-01

    Activating and inhibiting receptors of lymphocytes collect valuable information about their mikròs kósmos. This information is essential to initiate or to turn off complex signaling pathways. Irrespective of these advances, our knowledge on how these intracellular activation cascades are coordinated in a spatiotemporal manner is far from complete. Among multiple explanations, the scaffolding proteins have emerged as a critical piece of this evolutionary tangram. Among many, IQGAP1 is one of the essential scaffolding proteins that coordinate multiple signaling pathways. IQGAP1 possesses multiple protein interaction motifs to achieve its scaffolding functions. Using these domains, IQGAP1 has been shown to regulate a number of essential cellular events. This includes actin polymerization, tubulin multimerization, microtubule organizing center formation, calcium/calmodulin signaling, Pak/Raf/Mek1/2-mediated Erk1/2 activation, formation of maestrosome, E-cadherin, and CD44-mediated signaling and glycogen synthase kinase-3/adenomatous polyposis coli-mediated β-catenin activation. In this review, we summarize the recent developments and exciting new findings of cellular functions of IQGAP1.

  14. IQGAP1: A Regulator of Intracellular Spacetime Relativity

    PubMed Central

    Malarkannan, Subramaniam; Awasthi, Aradhana; Kamalakannan, Rajasekaran; Kumar, Pawan; Schuldt, Kristina M; Bartoszek, Allison; Manoharan, Niranjan; Goldner, Nicholas K; Umhoefer, Colleen M; Thakar, Monica S

    2012-01-01

    Activating and inhibiting receptors of lymphocytes collect valuable information about their mikròs kósmos. This information is essential to initiate or to turn off complex signaling pathways. Irrespective of these advances, our knowledge on how these intracellular activation cascades are coordinated in a spatiotemporal manner is far from complete. Amongst multiple explanations, the scaffolding proteins have emerged as a critical piece of this evolutionary tangram. Amongst many, IQGAP1 is one of the essential scaffolding proteins that coordinate multiple signaling pathways. IQGAP1 possesses multiple protein interaction motifs to achieve its scaffolding functions. Using these domains, IQGAP1 has been shown to regulate a number of essential cellular events. This includes actin polymerization, tubulin multimerization, MTOC formation, calcium/calmodulin signaling, Pak/Raf/Mek1/2-mediated Erk1/2 activation, formation of maestrosome, E-cadherin and CD44-mediated signaling and GSK3/APC-mediated β-catenin activation. In this review we summarize the recent developments and exciting new findings of cellular functions of IQGAP1. PMID:22345702

  15. Biodegradable nanoparticles for intracellular delivery of antimicrobial agents.

    PubMed

    Xie, Shuyu; Tao, Yanfei; Pan, Yuanhu; Qu, Wei; Cheng, Guyue; Huang, Lingli; Chen, Dongmei; Wang, Xu; Liu, Zhenli; Yuan, Zonghui

    2014-08-10

    Biodegradable nanoparticles have emerged as a promising strategy for ferrying antimicrobial agents into specific cells due to their unique properties. This review discusses the current progress and challenges of biodegradable nanoparticles for intracellular antimicrobial delivery to understand design principles for the development of ideal nanocarriers. The intracellular delivery performances of biodegradable nanoparticles for diverse antimicrobial agents are first summarized. Second, the cellular internalization and intracellular trafficking, degradation and release kinetics of nanoparticles as well as their relation with intracellular delivery of encapsulated antimicrobial agents are provided. Third, the influences of nanoparticle properties on the cellular internalization and intracellular fate of nanoparticles and their payload antimicrobial agents are discussed. Finally, the challenges and perspectives of nanoparticles for intracellular delivery of antimicrobial agents are addressed. The review will be helpful to the scientists who are interested in searching for more efficient nanosystem strategies for intracellular delivery of antimicrobial agents.

  16. Mg2+ mediates interaction between the voltage sensor and cytosolic domain to activate BK channels.

    PubMed

    Yang, Huanghe; Hu, Lei; Shi, Jingyi; Delaloye, Kelli; Horrigan, Frank T; Cui, Jianmin

    2007-11-13

    The voltage-sensor domain (VSD) of voltage-dependent ion channels and enzymes is critical for cellular responses to membrane potential. The VSD can also be regulated by interaction with intracellular proteins and ligands, but how this occurs is poorly understood. Here, we show that the VSD of the BK-type K(+) channel is regulated by a state-dependent interaction with its own tethered cytosolic domain that depends on both intracellular Mg(2+) and the open state of the channel pore. Mg(2+) bound to the cytosolic RCK1 domain enhances VSD activation by electrostatic interaction with Arg-213 in transmembrane segment S4. Our results demonstrate that a cytosolic domain can come close enough to the VSD to regulate its activity electrostatically, thereby elucidating a mechanism of Mg(2+)-dependent activation in BK channels and suggesting a general pathway by which intracellular factors can modulate the function of voltage-dependent proteins.

  17. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics.

    PubMed

    Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay

    2015-10-20

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design.

  18. Intracellular Calcium Dysregulation: Implications for Alzheimer's Disease

    PubMed Central

    Magi, Simona; Castaldo, Pasqualina; Macrì, Maria Loredana; Maiolino, Marta; Matteucci, Alessandra; Bastioli, Guendalina; Gratteri, Santo; Lariccia, Vincenzo

    2016-01-01

    Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by progressive neuronal loss. AD is associated with aberrant processing of the amyloid precursor protein, which leads to the deposition of amyloid-β plaques within the brain. Together with plaques deposition, the hyperphosphorylation of the microtubules associated protein tau and the formation of intraneuronal neurofibrillary tangles are a typical neuropathological feature in AD brains. Cellular dysfunctions involving specific subcellular compartments, such as mitochondria and endoplasmic reticulum (ER), are emerging as crucial players in the pathogenesis of AD, as well as increased oxidative stress and dysregulation of calcium homeostasis. Specifically, dysregulation of intracellular calcium homeostasis has been suggested as a common proximal cause of neural dysfunction in AD. Aberrant calcium signaling has been considered a phenomenon mainly related to the dysfunction of intracellular calcium stores, which can occur in both neuronal and nonneuronal cells. This review reports the most recent findings on cellular mechanisms involved in the pathogenesis of AD, with main focus on the control of calcium homeostasis at both cytosolic and mitochondrial level. PMID:27340665

  19. NPC1, intracellular cholesterol trafficking and atherosclerosis.

    PubMed

    Yu, Xiao-Hua; Jiang, Na; Yao, Ping-Bo; Zheng, Xi-Long; Cayabyab, Francisco S; Tang, Chao-Ke

    2014-02-15

    Post-lysosomal cholesterol trafficking is an important, but poorly understood process that is essential to maintain lipid homeostasis. Niemann-Pick type C1 (NPC1), an integral membrane protein on the limiting membrane of late endosome/lysosome (LE/LY), is known to accept cholesterol from NPC2 and then mediate cholesterol transport from LE/LY to endoplasmic reticulum (ER) and plasma membrane in a vesicle- or oxysterol-binding protein (OSBP)-related protein 5 (ORP5)-dependent manner. Mutations in the NPC1 gene can be found in the majority of NPC patients, who accumulate massive amounts of cholesterol and other lipids in the LE/LY due to a defect in intracellular lipid trafficking. Liver X receptor (LXR) is the major positive regulator of NPC1 expression. Atherosclerosis is the pathological basis of coronary heart disease, one of the major causes of death worldwide. NPC1 has been shown to play a critical role in the atherosclerotic progression. In this review, we have summarized the role of NPC1 in regulating intracellular cholesterol trafficking and atherosclerosis.

  20. Mechanisms of cellular invasion by intracellular parasites.

    PubMed

    Walker, Dawn M; Oghumu, Steve; Gupta, Gaurav; McGwire, Bradford S; Drew, Mark E; Satoskar, Abhay R

    2014-04-01

    Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world's population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite-host cell interactions, forming the basis of the parasite's cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.

  1. Quantitative proteomics of intracellular Porphyromonas gingivalis

    PubMed Central

    Xia, Qiangwei; Wang, Tiansong; Taub, Fred; Park, Yoonsuk; Capestany, Cindy A.; Lamont, Richard J.; Hackett, Murray

    2009-01-01

    Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18 hours within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were over-expressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be under-expressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction. PMID:17979175

  2. Intracellularly Swollen Polypeptide Nanogel Assists Hepatoma Chemotherapy

    PubMed Central

    Shi, Bo; Huang, Kexin; Ding, Jianxun; Xu, Weiguo; Yang, Yu; Liu, Haiyan; Yan, Lesan; Chen, Xuesi

    2017-01-01

    Nowadays, chemotherapy is one of the principal modes of treatment for tumor patients. However, the traditional formulations of small molecule drugs show short circulation time, low tumor selectivity, and high toxicity to normal tissues. To address these problems, a facilely prepared, and pH and reduction dual-responsive polypeptide nanogel was prepared for selectively intracellular delivery of chemotherapy drug. As a model drug, doxorubicin (DOX) was loaded into the nanogel through a sequential dispersion and dialysis technique, resulting in a high drug loading efficiency (DLE) of 96.7 wt.%. The loading nanogel, defined as NG/DOX, exhibited a uniform spherical morphology with a mean hydrodynamic radius of 58.8 nm, pH and reduction dual-triggered DOX release, efficient cell uptake, and cell proliferation inhibition in vitro. Moreover, NG/DOX exhibited improved antitumor efficacy toward H22 hepatoma-bearing BALB/c mouse model compared with free DOX·HCl. Histopathological and immunohistochemical analyses were implemented to further confirm the tumor suppression activity of NG/DOX. Furthermore, the variations of body weight, histopathological morphology, bone marrow cell micronucleus rate, and white blood cell count verified that NG/DOX showed excellent safety in vivo. With these excellent properties in vitro and in vivo, the pH and reduction dual-responsive polypeptide nanogel exhibits great potential for on-demand intracellular delivery of antitumor drug, and holds good prospect for future clinical application. PMID:28255361

  3. Strategies for Intracellular Survival of Burkholderia pseudomallei

    PubMed Central

    Allwood, Elizabeth M.; Devenish, Rodney J.; Prescott, Mark; Adler, Ben; Boyce, John D.

    2011-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high mortality that is prevalent in tropical regions of the world. A key component of the pathogenesis of melioidosis is the ability of B. pseudomallei to enter, survive, and replicate within mammalian host cells. For non-phagocytic cells, bacterial adhesins have been identified both on the bacterial surface and associated with Type 4 pili. Cell invasion involves components of one or more of the three Type 3 Secretion System clusters, which also mediate, at least in part, the escape of bacteria from the endosome into the cytoplasm, where bacteria move by actin-based motility. The mechanism of actin-based motility is not clearly understood, but appears to differ from characterized mechanisms in other bacterial species. A small proportion of intracellular bacteria is targeted by host cell autophagy, involving direct recruitment of LC3 to endosomes rather than through uptake by canonical autophagosomes. However, the majority of bacterial cells are able to circumvent autophagy and other intracellular defense mechanisms such as the induction of inducible nitric oxide synthase, and then replicate in the cytoplasm and spread to adjacent cells via membrane fusion, resulting in the formation of multi-nucleated giant cells. A potential role for host cell ubiquitin in the autophagic response to bacterial infection has recently been proposed. PMID:22007185

  4. Intracellular accumulation of ethanol in yeast

    SciTech Connect

    Loueiro, V.; Ferreira, H.G.

    1983-09-01

    Ethanol produced in the course of a batch fermentation by Saccharomyces cerevisiae or added from the outside, affects adversely the specific rate of growth of the yeast population, its viability, its specific rate of fermentation, and the specific rates of the uptake of sugar and amino acids. The underlying mechanisms are many and include irreversible denaturation and hyperbolic noncompetitive inhibition of glycolytic enzymes, the exponential noncompetitive inhibition of glucose, maltose, and ammonium transport, the depression of the optimum and the maximum temperature for growth, the increase of the minimum temperature for growth, and the enhancement of thermal death and petite mutation. Nagodawithana and Steinkraus reported that added ethanol was less toxic for S. cerevisiae than ethanol produced by the yeast. The death rates were lower in the presence of added ethanol than those measured at similar external ethanol concentrations endogenously produced. They proposed that, due to an unbalance between the rates of production and the net outflux of ethanol, there would be an intracellular accumulation of ethanol which in turn would explain the apparently greater inhibitory potency of endogenously produced ethanol present in the medium. This hypothesis was supported by the findings of several authors who reported that the intracellular concentration of ethanol, in the course of batch fermentation, is much higher than its concentration in the extracellular medium. The present work is an attempt to clarify this matter. (Refs. 32).

  5. Intracellular trafficking of hybrid gene delivery vectors.

    PubMed

    Keswani, Rahul K; Lazebnik, Mihael; Pack, Daniel W

    2015-06-10

    Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles.

  6. Novel targets for treating heart and muscle disease: stabilizing ryanodine receptors and preventing intracellular calcium leak.

    PubMed

    Lehnart, Stephan E

    2007-04-01

    Ryanodine receptors (RyRs) function as intracellular Ca(2+) release channels on the endoplasmic and sarcoplasmic reticulum membranes. In striated muscles, Ca(2+) release through RyRs controls muscle excitation-contraction coupling. RyR channel function is regulated by a cytoplasmic scaffold domain that forms a macromolecular signaling complex including calstabin (formerly known as FK506-binding protein), calmodulin, phosphodiesterase, kinase and phosphatase proteins. An increasing number of genetic and acquired diseases has been associated with intracellular Ca(2+) leak. In heart failure, for instance, the RyR complex becomes altered, resulting in chronic channel dysfunction and chronic sarcoplasmic reticulum Ca(2+) leak. Recently, the efficacy of novel Ca(2+) release channel-stabilizing drugs has been demonstrated in cardiac and skeletal muscle disease models.

  7. Plant VAP27 proteins: domain characterization, intracellular localization and role in plant development.

    PubMed

    Wang, Pengwei; Richardson, Christine; Hawkins, Timothy J; Sparkes, Imogen; Hawes, Chris; Hussey, Patrick J

    2016-06-01

    The endoplasmic reticulum (ER) is connected to the plasma membrane (PM) through the plant-specific NETWORKED protein, NET3C, and phylogenetically conserved vesicle-associated membrane protein-associated proteins (VAPs). Ten VAP homologues (VAP27-1 to 27-10) can be identified in the Arabidopsis genome and can be divided into three clades. Representative members from each clade were tagged with fluorescent protein and expressed in Nicotiana benthamiana. Proteins from clades I and III localized to the ER as well as to ER/PM contact sites (EPCSs), whereas proteins from clade II were found only at the PM. Some of the VAP27-labelled EPCSs localized to plasmodesmata, and we show that the mobility of VAP27 at EPCSs is influenced by the cell wall. EPCSs closely associate with the cytoskeleton, but their structure is unaffected when the cytoskeleton is removed. VAP27-labelled EPCSs are found in most cell types in Arabidopsis, with the exception of cells in early trichome development. Arabidopsis plants expressing VAP27-GFP fusions exhibit pleiotropic phenotypes, including defects in root hair morphogenesis. A similar effect is also observed in plants expressing VAP27 RNAi. Taken together, these data indicate that VAP27 proteins used at EPCSs are essential for normal ER-cytoskeleton interaction and for plant development.

  8. Triple-acting Lytic Enzyme Treatment of Drug-Resistant and Intracellular Staphylococcus aureus.

    PubMed

    Becker, Stephen C; Roach, Dwayne R; Chauhan, Vinita S; Shen, Yang; Foster-Frey, Juli; Powell, Anne M; Bauchan, Gary; Lease, Richard A; Mohammadi, Homan; Harty, William J; Simmons, Chad; Schmelcher, Mathias; Camp, Mary; Dong, Shengli; Baker, John R; Sheen, Tamsin R; Doran, Kelly S; Pritchard, David G; Almeida, Raul A; Nelson, Daniel C; Marriott, Ian; Lee, Jean C; Donovan, David M

    2016-04-28

    Multi-drug resistant bacteria are a persistent problem in modern health care, food safety and animal health. There is a need for new antimicrobials to replace over used conventional antibiotics. Here we describe engineered triple-acting staphylolytic peptidoglycan hydrolases wherein three unique antimicrobial activities from two parental proteins are combined into a single fusion protein. This effectively reduces the incidence of resistant strain development. The fusion protein reduced colonization by Staphylococcus aureus in a rat nasal colonization model, surpassing the efficacy of either parental protein. Modification of a triple-acting lytic construct with a protein transduction domain significantly enhanced both biofilm eradication and the ability to kill intracellular S. aureus as demonstrated in cultured mammary epithelial cells and in a mouse model of staphylococcal mastitis. Interestingly, the protein transduction domain was not necessary for reducing the intracellular pathogens in cultured osteoblasts or in two mouse models of osteomyelitis, highlighting the vagaries of exactly how protein transduction domains facilitate protein uptake. Bacterial cell wall degrading enzyme antimicrobials can be engineered to enhance their value as potent therapeutics.

  9. Triple-acting Lytic Enzyme Treatment of Drug-Resistant and Intracellular Staphylococcus aureus

    PubMed Central

    Becker, Stephen C.; Roach, Dwayne R.; Chauhan, Vinita S.; Shen, Yang; Foster-Frey, Juli; Powell, Anne M.; Bauchan, Gary; Lease, Richard A.; Mohammadi, Homan; Harty, William J.; Simmons, Chad; Schmelcher, Mathias; Camp, Mary; Dong, Shengli; Baker, John R.; Sheen, Tamsin R.; Doran, Kelly S.; Pritchard, David G.; Almeida, Raul A.; Nelson, Daniel C.; Marriott, Ian; Lee, Jean C.; Donovan, David M.

    2016-01-01

    Multi-drug resistant bacteria are a persistent problem in modern health care, food safety and animal health. There is a need for new antimicrobials to replace over used conventional antibiotics. Here we describe engineered triple-acting staphylolytic peptidoglycan hydrolases wherein three unique antimicrobial activities from two parental proteins are combined into a single fusion protein. This effectively reduces the incidence of resistant strain development. The fusion protein reduced colonization by Staphylococcus aureus in a rat nasal colonization model, surpassing the efficacy of either parental protein. Modification of a triple-acting lytic construct with a protein transduction domain significantly enhanced both biofilm eradication and the ability to kill intracellular S. aureus as demonstrated in cultured mammary epithelial cells and in a mouse model of staphylococcal mastitis. Interestingly, the protein transduction domain was not necessary for reducing the intracellular pathogens in cultured osteoblasts or in two mouse models of osteomyelitis, highlighting the vagaries of exactly how protein transduction domains facilitate protein uptake. Bacterial cell wall degrading enzyme antimicrobials can be engineered to enhance their value as potent therapeutics. PMID:27121552

  10. Association of ActA to Peptidoglycan Revealed by Cell Wall Proteomics of Intracellular Listeria monocytogenes*

    PubMed Central

    García-del Portillo, Francisco; Calvo, Enrique; D'Orazio, Valentina; Pucciarelli, M. Graciela

    2011-01-01

    Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen that colonizes the cytosol of eukaryotic cells. Recent transcriptomic studies have revealed that intracellular L. monocytogenes alter expression of genes encoding envelope components. However, no comparative global analysis of this cell wall remodeling process is yet known at the protein level. Here, we used high resolution mass spectrometry to define the cell wall proteome of L. monocytogenes growing inside epithelial cells. When compared with extracellular bacteria growing in a nutrient-rich medium, a major difference found in the proteome was the presence of the actin assembly-inducing protein ActA in peptidoglycan purified from intracellular bacteria. ActA was also identified in the peptidoglycan of extracellular bacteria growing in a chemically defined minimal medium. In this condition, ActA maintains its membrane anchoring domain and promotes efficient bacterial entry into nonphagocytic host cells. Unexpectedly, Internalin-A, which mediates entry of extracellular L. monocytogenes into eukaryotic cells, was identified at late infection times (6 h) as an abundant protein in the cell wall of intracellular bacteria. Other surface proteins covalently bound to the peptidoglycan, as Lmo0514 and Lmo2085, were detected exclusively in intracellular and extracellular bacteria, respectively. Altogether, these data provide the first insights into the changes occurring at the protein level in the L. monocytogenes cell wall as the pathogen transits from the extracellular environment to an intracytosolic lifestyle inside eukaryotic cells. Some of these changes include alterations in the relative amount and the mode of association of certain surface proteins. PMID:21846725

  11. Myometrial oxytocin receptor expression and intracellular pathways.

    PubMed

    Yulia, A; Johnson, M R

    2014-06-01

    Oxytocin (OT) signalling plays a fundamental role in the mechanisms of parturition. OT is one of the most frequently used drugs in obstetrics, promoting uterine contractions for labor induction and augmentation and to prevent postpartum hemorrhage (PPH). Expression of the oxytocin receptor (OTR) in the human myometrium is tightly regulated during pregnancy and its levels have been shown to peak upon labour onset and to fall sharply in advanced labour and the postpartum period, when the uterus become refractive to OT. However, uterine sensitivity to OT varies between pregnant women, probably reflecting differences in their myometrial OTR expression. Control of OTR expression is mediated by a combination of steroid hormone stimulation, stretch, and inflammation. This review summarises current knowledge regarding the complex regulation of myometrial OTR expression and its associated intracellular signaling pathways.

  12. Intracellular pH in Sperm Physiology

    PubMed Central

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L.; Darszon, Alberto

    2014-01-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca2+ channel; Slo3, a K+ channel; the sperm-specific Na+/H+ exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. PMID:24887564

  13. [Measurement of intracellular pH].

    PubMed

    Hanaoka, K; Imai, M; Yoshitomi, K

    1992-09-01

    Since various cellular processes depend on changes in pH, the regulation of intracellular pH (pHi) is important both for the individual cell and for the organism. The mechanisms of the regulation of pHi can be investigated by monitoring pHi. In this report, we discuss the four major techniques available for measuring pHi, which are 1) Distribution of weak acids and bases, 2) pH-sensitive microelectrodes, 3) pH-sensitive dyes, and 4) Nuclear magnetic resonance. Among four techniques, the advantage of the microelectrode approach is that it can monitor membrane potential at the same time and be applied to a single cell. The dye technique is a relative new developing technique, which has lots of advantages. It is easy to use, and is capable of monitoring rapid pHi changes, and being applied to a smaller cell, or a single cell.

  14. Impaired intracellular trafficking defines early Parkinson's disease.

    PubMed

    Hunn, Benjamin H M; Cragg, Stephanie J; Bolam, J Paul; Spillantini, Maria-Grazia; Wade-Martins, Richard

    2015-03-01

    Parkinson's disease (PD) is an insidious and incurable neurodegenerative disease, and represents a significant cost to individuals, carers, and ageing societies. It is defined at post-mortem by the loss of dopamine neurons in the substantia nigra together with the presence of Lewy bodies and Lewy neurites. We examine here the role of α-synuclein and other cellular transport proteins implicated in PD and how their aberrant activity may be compounded by the unique anatomy of the dopaminergic neuron. This review uses multiple lines of evidence from genetic studies, human tissue, induced pluripotent stem cells, and refined animal models to argue that prodromal PD can be defined as a disease of impaired intracellular trafficking. Dysfunction of the dopaminergic synapse heralds trafficking impairment.

  15. Intracellular dynamics with the phase microscope Airyscan

    NASA Astrophysics Data System (ADS)

    Tychinsky, Vladimir P.; Perevedentseva, Elena V.; Vyshenskaia, Tatiana V.; Kufal, Georgy E.

    1997-12-01

    Investigation of intracellular dynamics of Allium cepa inner epidermal cells are described. The applicability of the method for quantitative estimation of spatio-temporal phase fluctuations and the effect due to external factors is discussed. The analysis of time-sampled series allows one to detect the regions of various motility in cytoplasm. The intense Fourier-spectra harmonics in 0.2 - 8 Hz interval were observed inside a cell wall and cytoplasm. Regularly spaced 2- to 4-s long batches of 100-ms pulses at cell-wall sites are recorded. The phase-fluctuation intensity decreased and the frequencies of certain harmonics were shifted with lowering temperature. The advantages and specific features of the method are discussed.

  16. Glycosaminoglycans: Sorting determinants in intracellular protein traffic.

    PubMed

    Mihov, Deyan; Spiess, Martin

    2015-11-01

    Intracellular transport of proteins to their appropriate destinations is crucial for the maintenance of cellular integrity and function. Sorting information is contained either directly in the amino acid sequence or in a protein's post-translational modifications. Glycosaminoglycans (GAGs) are characteristic modifications of proteoglycans. GAGs are long unbranched polysaccharide chains with unique structural and functional properties also contributing to protein sorting in various ways. By deletion or insertion of GAG attachment sites it has been shown that GAGs affect polarized sorting in epithelial cells, targeting to and storage in secretory granules, and endocytosis. Most recently, the role of GAGs as signals for rapid trans-Golgi-to-cell surface transport, dominant over the cytosolic sorting motifs in the core protein, was demonstrated. Here, we provide an overview on existing data on the roles of GAGs on protein and proteoglycan trafficking.

  17. An intracellular anion channel critical for pigmentation.

    PubMed

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-12-16

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation.

  18. Cell-cell and intracellular lactate shuttles.

    PubMed

    Brooks, George A

    2009-12-01

    Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilized continuously in diverse cells under fully aerobic conditions. 'Cell-cell' and 'intracellular lactate shuttle' concepts describe the roles of lactate in delivery of oxidative and gluconeogenic substrates as well as in cell signalling. Examples of the cell-cell shuttles include lactate exchanges between between white-glycolytic and red-oxidative fibres within a working muscle bed, and between working skeletal muscle and heart, brain, liver and kidneys. Examples of intracellular lactate shuttles include lactate uptake by mitochondria and pyruvate for lactate exchange in peroxisomes. Lactate for pyruvate exchanges affect cell redox state, and by itself lactate is a ROS generator. In vivo, lactate is a preferred substrate and high blood lactate levels down-regulate the use of glucose and free fatty acids (FFA). As well, lactate binding may affect metabolic regulation, for instance binding to G-protein receptors in adipocytes inhibiting lipolysis, and thus decreasing plasma FFA availability. In vitro lactate accumulation upregulates expression of MCT1 and genes coding for other components of the mitochondrial reticulum in skeletal muscle. The mitochondrial reticulum in muscle and mitochondrial networks in other aerobic tissues function to establish concentration and proton gradients necessary for cells with high mitochondrial densities to oxidize lactate. The presence of lactate shuttles gives rise to the realization that glycolytic and oxidative pathways should be viewed as linked, as opposed to alternative, processes, because lactate, the product of one pathway, is the substrate for the other.

  19. Cell–cell and intracellular lactate shuttles

    PubMed Central

    Brooks, George A

    2009-01-01

    Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilized continuously in diverse cells under fully aerobic conditions. ‘Cell–cell’ and ‘intracellular lactate shuttle’ concepts describe the roles of lactate in delivery of oxidative and gluconeogenic substrates as well as in cell signalling. Examples of the cell–cell shuttles include lactate exchanges between between white-glycolytic and red-oxidative fibres within a working muscle bed, and between working skeletal muscle and heart, brain, liver and kidneys. Examples of intracellular lactate shuttles include lactate uptake by mitochondria and pyruvate for lactate exchange in peroxisomes. Lactate for pyruvate exchanges affect cell redox state, and by itself lactate is a ROS generator. In vivo, lactate is a preferred substrate and high blood lactate levels down-regulate the use of glucose and free fatty acids (FFA). As well, lactate binding may affect metabolic regulation, for instance binding to G-protein receptors in adipocytes inhibiting lipolysis, and thus decreasing plasma FFA availability. In vitro lactate accumulation upregulates expression of MCT1 and genes coding for other components of the mitochondrial reticulum in skeletal muscle. The mitochondrial reticulum in muscle and mitochondrial networks in other aerobic tissues function to establish concentration and proton gradients necessary for cells with high mitochondrial densities to oxidize lactate. The presence of lactate shuttles gives rise to the realization that glycolytic and oxidative pathways should be viewed as linked, as opposed to alternative, processes, because lactate, the product of one pathway, is the substrate for the other. PMID:19805739

  20. Spatiotemporal intracellular calcium dynamics during cardiac alternans

    PubMed Central

    Restrepo, Juan G.; Karma, Alain

    2009-01-01

    Cellular calcium transient alternans are beat-to-beat alternations in the peak cytosolic calcium concentration exhibited by cardiac cells during rapid electrical stimulation or under pathological conditions. Calcium transient alternans promote action potential duration alternans, which have been linked to the onset of life-threatening ventricular arrhythmias. Here we use a recently developed physiologically detailed mathematical model of ventricular myocytes to investigate both stochastic and deterministic aspects of intracellular calcium dynamics during alternans. The model combines a spatially distributed description of intracellular calcium cycling, where a large number of calcium release units are spatially distributed throughout the cell, with a full set of ionic membrane currents. The results demonstrate that ion channel stochasticity at the level of single calcium release units can influence the whole-cell alternans dynamics by causing phase reversals over many beats during fixed frequency pacing close to the alternans bifurcation. They also demonstrate the existence of a wide range of dynamical states. Depending on the sign and magnitude of calcium-voltage coupling, calcium alternans can be spatially synchronized or desynchronized, in or out of phase with action potential duration alternans, and the node separating out-of-phase regions of calcium alternans can be expelled from or trapped inside the cell. This range of states is found to be larger than previously anticipated by including a robust global attractor where calcium alternans can be spatially synchronized but out of phase with action potential duration alternans. The results are explained by a combined theoretical analysis of alternans stability and node motion using general iterative maps of the beat-to-beat dynamics and amplitude equations. PMID:19792040

  1. CBS domains: structure, function, and pathology in human proteins.

    PubMed

    Ignoul, Sofie; Eggermont, Jan

    2005-12-01

    The cystathionine-beta-synthase (CBS) domain is an evolutionarily conserved protein domain that is present in the proteome of archaebacteria, prokaryotes, and eukaryotes. CBS domains usually come in tandem repeats and are found in cytosolic and membrane proteins performing different functions (metabolic enzymes, kinases, and channels). Crystallographic studies of bacterial CBS domains have shown that two CBS domains form an intramolecular dimeric structure (CBS pair). Several human hereditary diseases (homocystinuria, retinitis pigmentosa, hypertrophic cardiomyopathy, myotonia congenital, etc.) can be caused by mutations in CBS domains of, respectively, cystathionine-beta-synthase, inosine 5'-monophosphate dehydrogenase, AMP kinase, and chloride channels. Despite their clinical relevance, it remains to be established what the precise function of CBS domains is and how they affect the structural and/or functional properties of an enzyme, kinase, or channel. Depending on the protein in which they occur, CBS domains have been proposed to affect multimerization and sorting of proteins, channel gating, and ligand binding. However, recent experiments revealing that CBS domains can bind adenosine-containing ligands such ATP, AMP, or S-adenosylmethionine have led to the hypothesis that CBS domains function as sensors of intracellular metabolites.

  2. Conserved residues in the HAMP domain define a new family of proposed bipartite energy taxis receptors.

    PubMed

    Elliott, Kathryn T; Zhulin, Igor B; Stuckey, Jeanne A; DiRita, Victor J

    2009-01-01

    HAMP domains, found in many bacterial signal transduction proteins, generally transmit an intramolecular signal between an extracellular sensory domain and an intracellular signaling domain. Studies of HAMP domains in proteins where both the input and output signals occur intracellularly are limited to those of the Aer energy taxis receptor of Escherichia coli, which has both a HAMP domain and a sensory PAS domain. Campylobacter jejuni has an energy taxis system consisting of the domains of Aer divided between two proteins, CetA (HAMP domain containing) and CetB (PAS domain containing). In this study, we found that the CetA HAMP domain differs significantly from that of Aer in the predicted secondary structure. Using similarity searches, we identified 55 pairs of HAMP/PAS proteins encoded by adjacent genes in a diverse group of microorganisms. We propose that these HAMP/PAS pairs form a new family of bipartite energy taxis receptors. Within these proteins, we identified nine residues in the HAMP domain and proximal signaling domain that are highly conserved, at least three of which are required for CetA function. Additionally, we demonstrated that CetA contributes to the invasion of human epithelial cells by C. jejuni, while CetB does not. This finding supports the hypothesis that members of HAMP/PAS pairs possess the capacity to act independently of each other in cellular traits other than energy taxis.

  3. Strategies of Intracellular Pathogens for Obtaining Iron from the Environment.

    PubMed

    Leon-Sicairos, Nidia; Reyes-Cortes, Ruth; Guadrón-Llanos, Alma M; Madueña-Molina, Jesús; Leon-Sicairos, Claudia; Canizalez-Román, Adrian

    2015-01-01

    Most microorganisms are destroyed by the host tissues through processes that usually involve phagocytosis and lysosomal disruption. However, some organisms, called intracellular pathogens, are capable of avoiding destruction by growing inside macrophages or other cells. During infection with intracellular pathogenic microorganisms, the element iron is required by both the host cell and the pathogen that inhabits the host cell. This minireview focuses on how intracellular pathogens use multiple strategies to obtain nutritional iron from the intracellular environment in order to use this element for replication. Additionally, the implications of these mechanisms for iron acquisition in the pathogen-host relationship are discussed.

  4. Strategies of Intracellular Pathogens for Obtaining Iron from the Environment

    PubMed Central

    Leon-Sicairos, Nidia; Reyes-Cortes, Ruth; Guadrón-Llanos, Alma M.; Madueña-Molina, Jesús; Leon-Sicairos, Claudia; Canizalez-Román, Adrian

    2015-01-01

    Most microorganisms are destroyed by the host tissues through processes that usually involve phagocytosis and lysosomal disruption. However, some organisms, called intracellular pathogens, are capable of avoiding destruction by growing inside macrophages or other cells. During infection with intracellular pathogenic microorganisms, the element iron is required by both the host cell and the pathogen that inhabits the host cell. This minireview focuses on how intracellular pathogens use multiple strategies to obtain nutritional iron from the intracellular environment in order to use this element for replication. Additionally, the implications of these mechanisms for iron acquisition in the pathogen-host relationship are discussed. PMID:26120582

  5. Misidentification of Mycobacterium leprae as Mycobacterium intracellulare by the COBAS AMPLICOR M. intracellulare Test

    PubMed Central

    Lefmann, M.; Moter, A.; Schweickert, B.; Göbel, U. B.

    2005-01-01

    Commercially available nucleic acid probe- and amplification-based systems for detection and differentiation of mycobacteria are widely used in clinical microbiology laboratories. Here we report two cases of human leprosy in which the COBAS AMPLICOR Mycobacterium intracellulare test led to false- positive results. Correct identification of Mycobacterium leprae was possible only by amplification and comparative sequence analysis of the 16S rRNA gene. PMID:15815021

  6. The intracellular localization of poliomyelitis virus.

    PubMed

    KAPLAN, A S; MELNICK, J L

    1953-01-01

    A study was made of the intracellular localization of Type 2 poliomyelitis virus, using the technique of Mirsky and Pollister (23) for cellular fractionation. After isotonic saline homogenization of central nervous system tissue from infected mice, and subsequent centrifugation of the suspension, the virus present in the supernatant fluid was held to be of cytoplasmic origin. Upon serial washings of the sediment with physiological saline, the resulting supernates contained progressively less virus until by the seventh washing, virtually none was present. At this point extraction of the washed sediment with molar NaCl, which lyses the nuclei, yielded substantial amounts of virus, and this was assumed to be from nuclear sources. The possibility has not been excluded however that the "nuclear" sediment was contaminated by cytoplasmic particles too large to remain in the supernate. Experiments on the increase of virus during the incubation and acute stages of infection have revealed that it was first detectable in the "cytoplasmic" fraction and subsequently in the "nuclear" fraction. Virus in the "nuclear" fraction from paralyzed mice sometimes reached titers almost as high as those found in the "cytoplasm." Adsorption experiments indicated that the "nuclear" fraction of CNS tissue from normal, uninoculated mice did not adsorb added Type 2 poliomyelitis virus, nor did such fractions adsorb virus procured from the "cytoplasm" or "nuclei" of infected cells. Although individual mice varied in their response after virus injection, the "cytoplasmic" fraction of paralytic mice was found to contain virus regularly, whereas little more than half of the non-paralytic mice yielded it. When virus was present in the "cytoplasm," it could be found in the "nuclear" fraction of paralytic mice with much greater regularity than in that of non-paralytic mice. A comparison between the lines of the MEF1 strain of poliomyelitis virus, "adapted" and "non-adapted" to newborn mice, and the

  7. Tollip, an intracellular trafficking protein, is a novel modulator of the transforming growth factor-β signaling pathway.

    PubMed

    Zhu, Lu; Wang, Lingdi; Luo, Xiaolin; Zhang, Yongxian; Ding, Qiurong; Jiang, Xiaomeng; Wang, Xiao; Pan, Yi; Chen, Yan

    2012-11-16

    Upon activation, TGF-β type I receptor (TβRI) undergoes active ubiquitination via recruitment of E3 ligases to the receptor complex by Smad7. However, how ubiquitination of TβRI is coupled to intracellular trafficking, and protein degradation remains unclear. We report here that Tollip, an adaptor protein that contains both ubiquitin-associated domains and endosome-targeting domain, plays an important role in modulating trafficking and degradation of TβRI. Tollip was previously demonstrated to possess a functional role in modulating the signaling of interleukin-1 and Toll-like receptors. We identify here that Tollip interacts with Smad7, a major modulatory protein involved in the negative regulation of TGF-β signaling. Overexpression of Tollip antagonizes TGF-β-stimulated transcriptional response, Smad2 phosphorylation, and epithelial-mesenchymal transition. Tollip also interacts with ubiquitinated TβRI, and such interaction requires ubiquitin-associated domains of Tollip. The interaction and intracellular colocalization of Tollip with TβRI is enhanced by Smad7. Overexpression of Tollip accelerates protein degradation of activated TβRI. In addition, Tollip alters subcellular compartmentalization and endosomal trafficking of activated TβRI. Collectively, our studies reveal that Tollip cooperates with Smad7 to modulate intracellular trafficking and degradation of ubiquitinated TβRI, whereby negatively regulates TGF-β signaling pathway.

  8. Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles

    PubMed Central

    Abraham, Ambily; Natraj, Usha; Karande, Anjali A.; Gulati, Ashutosh; Murthy, Mathur R. N.; Murugesan, Sathyabalan; Mukunda, Pavithra; Savithri, Handanahal S.

    2016-01-01

    The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the βH-βI loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies–D6F10 (targeting abrin), anti-α-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies. PMID:26905902

  9. Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells.

    PubMed

    Subramanian, Veedamali S; Marchant, Jonathan S; Ye, Dongmei; Ma, Thomas Y; Said, Hamid M

    2007-11-01

    Occludin, a transmembrane (TM)-spanning protein, is an integral component of the tight junctional (TJ) complexes that regulate epithelial integrity and paracellular barrier function. However, the molecular determinants that dictate occludin targeting and delivery to the TJs remain unclear. Here, using live cell imaging of yellow fluorescent protein-labeled occludin fragments, we resolved the intracellular trafficking of occludin-fusion proteins in polarized Madin-Darby canine kidney and Caco-2 cells to delineate the regions within the occludin polypeptide that are important for occludin targeting to the TJs. Live cell confocal imaging showed that complete or partial truncation of the COOH-terminal tail of the occludin polypeptide did not prevent occludin targeting to the TJs in epithelial cell lines. Progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression; after the removal of the regions proximal to the fourth transmembrane domain (TM4), the efficiency of expression increased. However, further deletions into the TM4 abolished TJ targeting, which resulted in constructs that were retained intracellularly within the endoplasmic reticulum. The full-length occludin polypeptide trafficked to the cell surface within a heterogenous population of intracellular vesicles that delivered occludin to the plasma membrane in a microtubule- and temperature-dependent manner. In contrast, the steady-state localization of occludin at the cell surface was dependent on intact microfilaments but not microtubules.

  10. Mapping intracellular diffusion distribution using single quantum dot tracking: compartmentalized diffusion defined by endoplasmic reticulum.

    PubMed

    Li, Hui; Dou, Shuo-Xing; Liu, Yu-Ru; Li, Wei; Xie, Ping; Wang, Wei-Chi; Wang, Peng-Ye

    2015-01-14

    The crowded intracellular environment influences the diffusion-mediated cellular processes, such as metabolism, signaling, and transport. The hindered diffusion of macromolecules in heterogeneous cytoplasm has been studied over years, but the detailed diffusion distribution and its origin still remain unclear. Here, we introduce a novel method to map rapidly the diffusion distribution in single cells based on single-particle tracking (SPT) of quantum dots (QDs). The diffusion map reveals the heterogeneous intracellular environment and, more importantly, an unreported compartmentalization of QD diffusions in cytoplasm. Simultaneous observations of QD motion and green fluorescent protein-tagged endoplasmic reticulum (ER) dynamics provide direct evidence that the compartmentalization results from micron-scale domains defined by ER tubules, and ER cisternae form perinuclear areas that restrict QDs to enter. The same phenomenon was observed using fluorescein isothiocyanate-dextrans, further confirming the compartmentalized diffusion. These results shed new light on the diffusive movements of macromolecules in the cell, and the mapping of intracellular diffusion distribution may be used to develop strategies for nanoparticle-based drug deliveries and therapeutics.

  11. Extracellular stimulation of VSIG4/complement receptor Ig suppresses intracellular bacterial infection by inducing autophagy.

    PubMed

    Kim, Kwang H; Choi, Beom K; Kim, Young H; Han, Chungyong; Oh, Ho S; Lee, Don G; Kwon, Byoung S

    2016-09-01

    VSIG4/CRIg (V-set and immunoglobulin domain containing 4) is a transmembrane receptor of the immunoglobulin superfamily that is expressed specifically on macrophages and mature dendritic cells. VSIG4 signaling accelerates phagocytosis of C3-opsonized bacteria, thereby efficiently clearing pathogens within macrophages. We found that VSIG4 signaling triggered by C3-opsonized Listeria (opLM) or by agonistic anti-VSIG4 monoclonal antibody (mAb) induced macrophages to form autophagosomes. VSIG4-induced autophagosomes were selectively colocalized with the intracellular LM while starvation-induced autophagosomes were not. Consistent with these results, the frequency of autophagosomes induced by infection with opLM was lower in VSIG4-deficient bone marrow-derived macrophages (BMDMs) than in WT BMDMs. Furthermore, when VSIG4 molecules were overexpressed in HeLa cells, which are non-macrophage cells, VSIG4 triggering led to efficient uptake of LM, autophagosome formation, and killing of the infected LM. These findings suggest that VSIG4 signaling not only promotes rapid phagocytosis and killing of C3-opsonized intracellular bacteria, as previously reported, but also induces autophagosome formation, eliminating the LM that have escaped from phagosomes. We conclude that VSIG4 signaling provides an anti-immune evasion mechanism that prevents the outgrowth of intracellular bacteria in macrophages.

  12. Ectodomain Shedding of Interleukin-2 Receptor β and Generation of an Intracellular Functional Fragment*

    PubMed Central

    de Oca B., Pavel Montes; Malardé, Valerie; Proust, Richard; Dautry-Varsat, Alice; Gesbert, Franck

    2010-01-01

    Interleukin-2 (IL-2) regulates different functions of various lymphoid cell subsets. These are mediated by its binding to the IL-2 receptor (IL-2R) composed of three subunits (IL2-Rα, -β, and -γc). IL-2Rβ is responsible for the activation of several signaling pathways. Ectodomain shedding of membrane receptors is thought to be an important mechanism for down-regulation of cell surface receptor abundance but is also emerging as a mechanism that cell membrane-associated molecules require for proper action in vivo. Here, we demonstrate that IL-2Rβ is cleaved in cell lines of different origin, including T cells, generating an intracellular 37-kDa fragment (37βic) that comprises the full intracellular C-terminal and transmembrane domains. Ectodomain shedding of IL-2Rβ decreases in a mutant deleted of the juxtamembrane region, where cleavage is predicted to occur, and is inhibited by tissue inhibitor of metalloproteases-3. 37βic is tyrosine-phosphorylated and associates with STAT-5, a canonic signal transducer of IL-2R. Finally, lymphoid cell transfection with a truncated form of IL-2Rβ mimicking 37βic increases their proliferation. These data indicate that IL-2Rβ is subject to ectodomain shedding generating an intracellular fragment biologically functional, because (i) it is phosphorylated, (ii) it associates with STAT5A, and (iii) it increases cell proliferation. PMID:20495002

  13. Emerging intracellular receptors for hemorrhagic fever viruses.

    PubMed

    Jae, Lucas T; Brummelkamp, Thijn R

    2015-07-01

    Ebola virus and Lassa virus belong to different virus families that can cause viral hemorrhagic fever, a life-threatening disease in humans with limited treatment options. To infect a target cell, Ebola and Lassa viruses engage receptors at the cell surface and are subsequently shuttled into the endosomal compartment. Upon arrival in late endosomes/lysosomes, the viruses trigger membrane fusion to release their genome into the cytoplasm. Although contact sites at the cell surface were recognized for Ebola virus and Lassa virus, it was postulated that Ebola virus requires a critical receptor inside the cell. Recent screens for host factors identified such internal receptors for both viruses: Niemann-Pick disease type C1 protein (NPC1) for Ebola virus and lysosome-associated membrane protein 1 (LAMP1) for Lassa virus. A cellular trigger is needed to permit binding of the viral envelope protein to these intracellular receptors. This 'receptor switch' represents a previously unnoticed step in virus entry with implications for host-pathogen interactions and viral tropism.

  14. Cardiac alternans and intracellular calcium cycling

    PubMed Central

    Edwards, Joshua N.; Blatter, Lothar A.

    2014-01-01

    Cardiac alternans refers to a condition in which there is a periodic beat-to-beat oscillation in electrical activity and the strength of cardiac muscle contraction at a constant heart rate. Clinically, cardiac alternans occurs in settings that are typical for cardiac arrhythmias and has been causally linked to these conditions. At the cellular level, alternans is defined as beat-to-beat alternations in contraction amplitude (mechanical alternans), action potential duration (APD; electrical or APD alternans), and Ca2+ transient amplitude (Ca2+ alternans). The cause of alternans is multifactorial, however alternans always originate from disturbances of the bi-directional coupling between membrane voltage (Vm) and intracellular calcium ([Ca2+]i). Bi-directional coupling refers to the fact that in cardiac cells, Vm depolarization and the generation of action potentials cause the elevation of [Ca2+]i that is required for contraction (a process referred to as excitation-contraction coupling), the changes of [Ca2+]i on the other hand control Vm because important membrane currents are Ca2+-dependent. Evidence is mounting that alternans is ultimately caused by disturbances of cellular Ca2+ signaling. Here we review how two key factors of cardiac cellular Ca2+ cycling - the release of Ca2+ from internal stores and the capability of clearing the cytosol from Ca2+ after each beat - determine the conditions under which alternans occurs. The contributions from key Ca2+ handling proteins - surface membrane channels, ion pumps and transporters, and internal Ca2+ release channels - are discussed. PMID:25040398

  15. Uncoupling Caveolae from Intracellular Signaling In Vivo

    PubMed Central

    Kraehling, Jan R.; Hao, Zhengrong; Lee, Monica Y.; Vinyard, David J.; Velazquez, Heino; Liu, X.; Stan, Radu V.; Brudvig, Gary W.; Sessa, William C.

    2015-01-01

    Rationale Caveolin-1 negatively regulates eNOS derived NO production and this has been mapped to several residues on Cav-1 including F92. Herein, we reasoned that endothelial expression of an F92ACav-1 transgene would let us decipher the mechanisms and relationships between caveolae structure and intracellular signaling. Objective This study was designed to separate caveolae formation from its downstream signaling effects. Methods and Results An endothelial-specific doxycycline-regulated mouse model for the expression of Cav-1-F92A was developed. Blood pressure by telemetry and nitric oxide bioavailability by electron paramagnetic resonance and phosphorylation of VASP were determined. Caveolae integrity in the presence of Cav-1-F92A was measured by stabilization of Cav-2, sucrose gradient and electron microscopy. Histological analysis of heart and lung, echocardiography and signaling were performed. Conclusions This study shows that mutant Cav-1-F92A forms caveolae structures similar to WT but leads to increases in NO bioavailability in vivo thereby demonstrating that caveolae formation and downstream signaling events occur through independent mechanisms. PMID:26602865

  16. Tumour suppressors hamartin and tuberin: intracellular signalling.

    PubMed

    Krymskaya, Vera P

    2003-08-01

    Tumour suppressors hamartin and tuberin, encoded by tuberous sclerosis complex 1(TSC1) and TSC2 genes, respectively, are critical regulators of cell growth and proliferation. Mutations in TSC1 and TSC2 genes are the cause of an autosomal dominant disorder known as tuberous sclerosis complex (TSC). Another genetic disorder, lymphangioleiomyomatosis (LAM), is also associated with mutations in the TSC2 gene. Hamartin and tuberin control cell growth by negatively regulating S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), potentially through their upstream modulator mammalian target of rapamycin (mTOR). Growth factors and insulin promote Akt/PKB-dependent phosphorylation of tuberin, which in turn, releases S6K1 from negative regulation by tuberin and results in the activation of S6K1. Although much has been written regarding the molecular genetics of TSC and LAM, which is associated with either the loss of or mutation in the TSC1 and TSC2 genes, few reviews have addressed the intracellular signalling pathways regulated by hamartin and tuberin. The current review will fill the gap in our understanding of their role in cellular signalling networks, and by improving this understanding, an integrated picture regarding the normal function of tuberin and hamartin is beginning to emerge.

  17. Stapled peptides for intracellular drug targets.

    PubMed

    Verdine, Gregory L; Hilinski, Gerard J

    2012-01-01

    Proteins that engage in intracellular interactions with other proteins are widely considered among the most biologically appealing yet chemically intractable targets for drug discovery. The critical interaction surfaces of these proteins typically lack the deep hydrophobic involutions that enable potent, selective targeting by small organic molecules, and their localization within the cell puts them beyond the reach of protein therapeutics. Considerable interest has therefore arisen in next-generation targeting molecules that combine the broad target recognition capabilities of protein therapeutics with the robust cell-penetrating ability of small molecules. One type that has shown promise in early-stage studies is hydrocarbon-stapled α-helical peptides, a novel class of synthetic miniproteins locked into their bioactive α-helical fold through the site-specific introduction of a chemical brace, an all-hydrocarbon staple. Stapling can greatly improve the pharmacologic performance of peptides, increasing their target affinity, proteolytic resistance, and serum half-life while conferring on them high levels of cell penetration through endocytic vesicle trafficking. Here, we discuss considerations crucial to the successful design and evaluation of potent stapled peptide interactions, our intention being to facilitate the broad application of this technology to intractable targets of both basic biologic interest and potential therapeutic value.

  18. On the Computing Potential of Intracellular Vesicles

    PubMed Central

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal ‘circuitry’ and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a ‘vesicle modification’ of the archetypal CBC ‘billiard ball model’ of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle ‘programming’ in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing. PMID:26431435

  19. Characterizations of intracellular arsenic in a bacterium

    NASA Astrophysics Data System (ADS)

    Wolfe-Simon, F.; Yannone, S. M.; Tainer, J. A.

    2011-12-01

    Life requires a key set of chemical elements to sustain growth. Yet, a growing body of literature suggests that microbes can alter their nutritional requirements based on the availability of these chemical elements. Under limiting conditions for one element microbes have been shown to utilize a variety of other elements to serve similar functions often (but not always) in similar molecular structures. Well-characterized elemental exchanges include manganese for iron, tungsten for molybdenum and sulfur for phosphorus or oxygen. These exchanges can be found in a wide variety of biomolecules ranging from protein to lipids and DNA. Recent evidence suggested that arsenic, as arsenate or As(V), was taken up and incorporated into the cellular material of the bacterium GFAJ-1. The evidence was interpreted to support As(V) acting in an analogous role to phosphate. We will therefore discuss our ongoing efforts to characterize intracellular arsenate and how it may partition among the cellular fractions of the microbial isolate GFAJ-1 when exposed to As(V) in the presence of various levels of phosphate. Under high As(V) conditions, cells express a dramatically different proteome than when grown given only phosphate. Ongoing studies on the diversity and potential role of proteins and metabolites produced in the presence of As(V) will be reported. These investigations promise to inform the role and additional metabolic potential for As in biology. Arsenic assimilation into biomolecules contributes to the expanding set of chemical elements utilized by microbes in unusual environmental niches.

  20. Intracellular sphingosine releases calcium from lysosomes

    PubMed Central

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. DOI: http://dx.doi.org/10.7554/eLife.10616.001 PMID:26613410

  1. Intracellular recording from a spider vibration receptor.

    PubMed

    Gingl, Ewald; Burger, Anna-M; Barth, Friedrich G

    2006-05-01

    The present study introduces a new preparation of a spider vibration receptor that allows intracellular recording of responses to natural mechanical or electrical stimulation of the associated mechanoreceptor cells. The spider vibration receptor is a lyriform slit sense organ made up of 21 cuticular slits located on the distal end of the metatarsus of each walking leg. The organ is stimulated when the tarsus receives substrate vibrations, which it transmits to the organ's cuticular structures, reducing the displacement to about one tenth due to geometrical reasons. Current clamp recording was used to record action potentials generated by electrical or mechanical stimuli. Square pulse stimulation identified two groups of sensory cells, the first being single-spike cells which generated only one or two action potentials and the second being multi-spike cells which produced bursts of action potentials. When the more natural mechanical sinusoidal stimulation was applied, differences in adaptation rate between the two cell types remained. In agreement with prior extracellular recordings, both cell types showed a decrease in the threshold tarsus deflection with increasing stimulus frequency. Off-responses to mechanical stimuli have also been seen in the metatarsal organ for the first time.

  2. On the Computing Potential of Intracellular Vesicles.

    PubMed

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal 'circuitry' and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a 'vesicle modification' of the archetypal CBC 'billiard ball model' of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle 'programming' in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing.

  3. A viral peptide for intracellular delivery

    NASA Astrophysics Data System (ADS)

    Falanga, Annarita; Tarallo, Rossella; Cantisani, Marco; Della Pepa, Maria Elena; Galdiero, Massimiliano; Galdiero, Stefania

    2012-10-01

    Biological membranes represent a critical hindrance for administering active molecules which are often unable to reach their designated intracellular target sites. In order to overcome this barrier-like behavior not easily circumvented by many pharmacologically-active molecules, synthetic transporters have been exploited to promote cellular uptake. Linking or complexing therapeutic molecules to peptides that can translocate through the cellular membranes could enhance their internal delivery, and consequently, a higher amount of active compound would reach the site of action. Use of cell penetrating peptides (CPPs) is one of the most promising strategy to efficiently translocate macromolecules through the plasma membrane, and have attracted a lot of attention. New translocating peptides are continuously described and in the present review, we will focus on viral derived peptides, and in particular a peptide (gH625) derived from the herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) that has proved to be a useful delivery vehicle due to its intrinsic properties of inducing membrane perturbation.

  4. Impact of photosensitizers activation on intracellular trafficking and viscosity.

    PubMed

    Aubertin, Kelly; Bonneau, Stéphanie; Silva, Amanda K A; Bacri, Jean-Claude; Gallet, François; Wilhelm, Claire

    2013-01-01

    The intracellular microenvironment is essential for the efficiency of photo-induced therapies, as short-lived reactive oxygen species generated must diffuse through their intracellular surrounding medium to reach their cellular target. Here, by combining measurements of local cytoplasmic dissipation and active trafficking, we found that photosensitizers activation induced small changes in surrounding viscosity but a massive decrease in diffusion. These effects are the signature of a return to thermodynamic equilibrium of the system after photo-activation and correlated with depolymerization of the microtubule network, as shown in a reconstituted system. These mechanical measurements were performed with two intracellular photosensitizing chlorins having similar quantum yield of singlet oxygen production but different intracellular localizations (cytoplasmic for mTHPC, endosomal for TPCS2a). These two agents demonstrated different intracellular impact.

  5. Impact of Photosensitizers Activation on Intracellular Trafficking and Viscosity

    PubMed Central

    Aubertin, Kelly; Bonneau, Stéphanie; Silva, Amanda K. A.; Bacri, Jean-Claude; Gallet, François; Wilhelm, Claire

    2013-01-01

    The intracellular microenvironment is essential for the efficiency of photo-induced therapies, as short-lived reactive oxygen species generated must diffuse through their intracellular surrounding medium to reach their cellular target. Here, by combining measurements of local cytoplasmic dissipation and active trafficking, we found that photosensitizers activation induced small changes in surrounding viscosity but a massive decrease in diffusion. These effects are the signature of a return to thermodynamic equilibrium of the system after photo-activation and correlated with depolymerization of the microtubule network, as shown in a reconstituted system. These mechanical measurements were performed with two intracellular photosensitizing chlorins having similar quantum yield of singlet oxygen production but different intracellular localizations (cytoplasmic for mTHPC, endosomal for TPCS2a). These two agents demonstrated different intracellular impact. PMID:24386423

  6. Control of Intracellular Calcium Signaling as a Neuroprotective Strategy

    PubMed Central

    Duncan, R. Scott; Goad, Daryl L.; Grillo, Michael A.; Kaja, Simon; Payne, Andrew J.; Koulen, Peter

    2010-01-01

    Both acute and chronic degenerative diseases of the nervous system reduce the viability and function of neurons through changes in intracellular calcium signaling. In particular, pathological increases in the intracellular calcium concentration promote such pathogenesis. Disease involvement of numerous regulators of intracellular calcium signaling located on the plasma membrane and intracellular organelles has been documented. Diverse groups of chemical compounds targeting ion channels, G-protein coupled receptors, pumps and enzymes have been identified as potential neuroprotectants. The present review summarizes the discovery, mechanisms and biological activity of neuroprotective molecules targeting proteins that control intracellular calcium signaling to preserve or restore structure and function of the nervous system. Disease relevance, clinical applications and new technologies for the identification of such molecules are being discussed. PMID:20335972

  7. The Rab1 GTPase of Sciaenops ocellatus modulates intracellular bacterial infection.

    PubMed

    Hu, Yong-hua; Deng, Tian; Sun, Li

    2011-12-01

    The Rab family proteins belong to the Ras-like GTPase superfamily and play important roles in intracellular membrane trafficking. To date no studies on fish Rab have been documented, though rab-like sequences have been found in a number of teleosts. In this study, we identified and analyzed a Rab homologue, SoRab1, from red drum, Sciaenops ocellatus. The cDNA of SoRab1 contains a 5'- untranslated region (UTR) of 358 bp, an open reading frame (ORF) of 612 bp, and a 3'-UTR of 265 bp. The ORF encodes a putative protein of 203 residues, which shares 92-99% overall sequence identities with the Rab1 from fish, human, and mouse. SoRab1 possesses a typical Rab1 GTPase domain with the conserved G box motifs and the switch I and switch II regions. Recombinant SoRab1 purified from Escherichia coli exhibits apparent GTPase activity. Quantitative real time RT-PCR analysis showed that SoRab1 expression was detected in a number of tissues, with the lowest expression found in blood and highest expression found in muscle. Bacterial and lipopolysaccharide challenges significantly upregulated SoRab1 expression in liver, kidney, and spleen in time-dependent manners. Transient overexpression of SoRab1 in primary hepatocytes reduced intracellular bacterial infection, whereas interference with SoRab1 expression by RNAi enhanced intracellular bacterial invasion. These results provide the first indication that a fish Rab1 GTPase, SoRab1, regulates intracellular bacterial infection and thus is likely to play a role in bacteria-induced host immune defense.

  8. Arrhythmogenic consequences of intracellular calcium waves.

    PubMed

    Xie, Lai-Hua; Weiss, James N

    2009-09-01

    Intracellular Ca(2+) (Ca(i)(2+)) waves are known to cause delayed afterdepolarizations (DADs), which have been associated with arrhythmias in cardiac disease states such as heart failure, catecholaminergic polymorphic ventricular tachycardia, and digitalis toxicity. Here we show that, in addition to DADs, Ca(i)(2+) waves also have other consequences relevant to arrhythmogenesis, including subcellular spatially discordant alternans (SDA, in which the amplitude of the local Ca(i)(2+) transient alternates out of phase in different regions of the same cell), sudden repolarization changes promoting the dispersion of refractoriness, and early afterdepolarizations (EADs). Ca(i)(2+) was imaged using a charge-coupled device-based system in fluo-4 AM-loaded isolated rabbit ventricular myocytes paced at constant or incrementally increasing rates, using either field stimulation, current clamp, or action potential (AP) clamp. Ca(i)(2+) waves were induced by Bay K 8644 (50 nM) + isoproterenol (100 nM), or low temperature. When pacing was initiated during a spontaneous Ca(i)(2+) wave, SDA occurred abruptly and persisted during pacing. Similarly, during rapid pacing, SDA typically arose suddenly from spatially concordant alternans, due to an abrupt phase reversal of the subcellular Ca(i)(2+) transient in a region of the myocyte. Ca(i)(2+) waves could be visualized interspersed with AP-triggered Ca(i)(2+) transients, producing a rich variety of subcellular Ca(i)(2+) transient patterns. With free-running APs, complex Ca(i)(2+) release patterns were associated with DADs, EADs, and sudden changes in AP duration. These findings link Ca(i)(2+) waves directly to a variety of arrhythmogenic phenomena relevant to the intact heart.

  9. Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

    NASA Astrophysics Data System (ADS)

    Payne, Christine

    2014-03-01

    Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.

  10. Visualizing domain wall and reverse domain superconductivity.

    PubMed

    Iavarone, M; Moore, S A; Fedor, J; Ciocys, S T; Karapetrov, G; Pearson, J; Novosad, V; Bader, S D

    2014-08-28

    In magnetically coupled, planar ferromagnet-superconductor (F/S) hybrid structures, magnetic domain walls can be used to spatially confine the superconductivity. In contrast to a superconductor in a uniform applied magnetic field, the nucleation of the superconducting order parameter in F/S structures is governed by the inhomogeneous magnetic field distribution. The interplay between the superconductivity localized at the domain walls and far from the walls leads to effects such as re-entrant superconductivity and reverse domain superconductivity with the critical temperature depending upon the location. Here we use scanning tunnelling spectroscopy to directly image the nucleation of superconductivity at the domain wall in F/S structures realized with Co-Pd multilayers and Pb thin films. Our results demonstrate that such F/S structures are attractive model systems that offer the possibility to control the strength and the location of the superconducting nucleus by applying an external magnetic field, potentially useful to guide vortices for computing application.

  11. Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel.

    PubMed

    Yamada, Toshiki; Krzeminski, Mickael; Bozoky, Zoltan; Forman-Kay, Julie D; Strange, Kevin

    2016-11-01

    Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple α-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-β-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane α-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structure-function relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways.

  12. Functionally active t1-t1 interfaces revealed by the accessibility of intracellular thiolate groups in kv4 channels.

    PubMed

    Wang, Guangyu; Shahidullah, Mohammad; Rocha, Carmen A; Strang, Candace; Pfaffinger, Paul J; Covarrubias, Manuel

    2005-07-01

    Gating of voltage-dependent K(+) channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn(2+) coordination. Also, added Zn(2+) or a potent Zn(2+) chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is approximately 200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact alpha-subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1--T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn(2+) binding.

  13. Functionally Active T1-T1 Interfaces Revealed by the Accessibility of Intracellular Thiolate Groups in Kv4 Channels

    PubMed Central

    Wang, Guangyu; Shahidullah, Mohammad; Rocha, Carmen A.; Strang, Candace; Pfaffinger, Paul J.; Covarrubias, Manuel

    2005-01-01

    Gating of voltage-dependent K+ channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn2+ coordination. Also, added Zn2+ or a potent Zn2+ chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is ∼200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact α-subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1–T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn2+ binding. PMID:15955876

  14. A Domain Analysis Bibliography

    DTIC Science & Technology

    1990-06-01

    Bauhaus , a prototype CASE workstation for D-SAPS development. [ARAN88A] Guillermo F. Arango. Domain Engineering for Software Reuse. PhD thesis...34 VITA90B: Domain Analysis within the ISEC Rapid Center 48 CMU/SEI-90-SR-3 Appendix III Alphabetical by Organization/Project BAUHAUS * ALLE87A

  15. Domain wall filters

    SciTech Connect

    Baer, Oliver; Narayanan, Rajamani; Neuberger, Herbert; Witzel, Oliver

    2007-03-15

    We propose using the extra dimension separating the domain walls carrying lattice quarks of opposite handedness to gradually filter out the ultraviolet fluctuations of the gauge fields that are felt by the fermionic excitations living in the bulk. This generalization of the homogeneous domain wall construction has some theoretical features that seem nontrivial.

  16. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  17. Causal Learning Across Domains

    ERIC Educational Resources Information Center

    Schulz, Laura E.; Gopnik, Alison

    2004-01-01

    Five studies investigated (a) children's ability to use the dependent and independent probabilities of events to make causal inferences and (b) the interaction between such inferences and domain-specific knowledge. In Experiment 1, preschoolers used patterns of dependence and independence to make accurate causal inferences in the domains of…

  18. Ultraviolet-irradiated monocytes efficiently inhibit the intracellular replication of Mycobacterium avium intracellulare.

    PubMed Central

    Mirando, W S; Shiratsuchi, H; Tubesing, K; Toba, H; Ellner, J J; Elmets, C A

    1992-01-01

    The purpose of this study was to evaluate the effect of ultraviolet (UV) radiation on the antimicrobial activities of monocytes for the intracellular pathogen Mycobacterium avium intracellulare (MAI). UV radiation augmented monocyte antimicrobial activity for MAI in a dose-dependent fashion. UVB doses of greater than or equal to 25 J/m2 resulted in a 50-100-fold reduction in MAI growth 7 d after initiation of culture. The increased monocyte antibacterial effect could be blocked by a plate glass filter, indicating that wavelengths within the UVB were responsible for the effect. UV radiation did not stimulate monocyte phagocytosis, and enhanced inhibition of MAI growth was observed in populations of adherent mononuclear cells that were devoid of T cells. This suggested that UV radiation acted directly to augment intrinsic monocyte antimicrobial activities. The administration of 8-methoxypsoralen plus UVA radiation to monocytes also augmented their antimicrobial activities against MAI. UV radiation thus may serve as a unique agent by which to evaluate the mechanisms by which mononuclear phagocytes control the growth of MAI. Images PMID:1556188

  19. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation.

    PubMed

    Chamaillard, Mathias; Girardin, Stephen E; Viala, Jérôme; Philpott, Dana J

    2003-09-01

    The innate immune system is the most ancestral and ubiquitous system of defence against microbial infection. The microbial sensing proteins involved in innate immunity recognize conserved and often structural components of microorganisms. One class of these pattern-recognition molecules, the Toll-like receptors (TLRs), are involved in detection of microbes in the extracellular compartment whereas a newly discovered family of proteins, the NBS-LRR proteins (for nucleotide-binding site and leucine-rich repeat), are involved in intracellular recognition of microbes and their products. NBS-LRR proteins are characterized by three structural domains: a C-terminal leucine-rich repeat (LRR) domain able to sense a microbial motif, an intermediary nucleotide binding site (NBS) essential for the oligomerization of the molecule that is necessary for the signal transduction induced by different N-terminal effector motifs, such as a pyrin domain (PYD), a caspase-activating and recruitment domain (CARD) or a baculovirus inhibitor of apoptosis protein repeat (BIR) domain. Two of these family members, Nod1 and Nod2, play a role in the regulation of pro-inflammatory pathways through NF-kappaB induced by bacterial ligands. Recently, it was shown that Nod2 recognizes a specific peptidoglycan motif from bacteria, muramyl dipeptide (MDP). A surprising number of human genetic disorders have been linked to NBS-LRR proteins. For example, mutations in Nod2, which render the molecule insensitive to MDP and unable to induce NF-kappaB activation when stimulated, are associated with susceptibility to a chronic intestinal inflammatory disorder, Crohn's disease. Conversely, mutations in the NBS region of Nod2 induce a constitutive activation of NF-kappaB and are responsible for Blau syndrome, another auto-inflammatory disease. Nalp3, which is an NBS-LRR protein with an N-terminal Pyrin domain, is also implicated in rare auto-inflammatory disorders. In conclusion, NBS-LRR molecules appear as a new

  20. Sac phosphatase domain proteins.

    PubMed Central

    Hughes, W E; Cooke, F T; Parker, P J

    2000-01-01

    Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids. One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins. Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p. The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p. These proteins therefore contain both Sac phosphatase and 5-phosphatase domains. This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae. PMID:10947947

  1. Structure-Function Analysis of DipA, a Francisella tularensis Virulence Factor Required for Intracellular Replication

    PubMed Central

    Chong, Audrey; Child, Robert; Wehrly, Tara D.; Rockx-Brouwer, Dedeke; Qin, Aiping; Mann, Barbara J.; Celli, Jean

    2013-01-01

    Francisella tularensis is a highly infectious bacterium whose virulence relies on its ability to rapidly reach the macrophage cytosol and extensively replicate in this compartment. We previously identified a novel Francisella virulence factor, DipA (FTT0369c), which is required for intramacrophage proliferation and survival, and virulence in mice. DipA is a 353 amino acid protein with a Sec-dependent signal peptide, four Sel1-like repeats (SLR), and a C-terminal coiled-coil (CC) domain. Here, we determined through biochemical and localization studies that DipA is a membrane-associated protein exposed on the surface of the prototypical F. tularensis subsp. tularensis strain SchuS4 during macrophage infection. Deletion and substitution mutagenesis showed that the CC domain, but not the SLR motifs, of DipA is required for surface exposure on SchuS4. Complementation of the dipA mutant with either DipA CC or SLR domain mutants did not restore intracellular growth of Francisella, indicating that proper localization and the SLR domains are required for DipA function. Co-immunoprecipitation studies revealed interactions with the Francisella outer membrane protein FopA, suggesting that DipA is part of a membrane-associated complex. Altogether, our findings indicate that DipA is positioned at the host–pathogen interface to influence the intracellular fate of this pathogen. PMID:23840797

  2. Intracellular acidosis enhances the excitability of working muscle.

    PubMed

    Pedersen, Thomas H; Nielsen, Ole B; Lamb, Graham D; Stephenson, D George

    2004-08-20

    Intracellular acidification of skeletal muscles is commonly thought to contribute to muscle fatigue. However, intracellular acidosis also acts to preserve muscle excitability when muscles become depolarized, which occurs with working muscles. Here, we show that this process may be mediated by decreased chloride permeability, which enables action potentials to still be propagated along the internal network of tubules in a muscle fiber (the T system) despite muscle depolarization. These results implicate chloride ion channels in muscle function and emphasize that intracellular acidosis of muscle has protective effects during muscle fatigue.

  3. Cross Domain Analogies for Learning Domain Theories

    DTIC Science & Technology

    2007-01-01

    Example Problem and Worked Solution All problems and worked solutions used in this work were taken from the same physics textbook ( Giancoli 1991...domain theory. We close with a discussion of related work and our plans for the future. Representations and Problem Solving Representing physics ...small compared to the 30,000+ concepts and 8,000+ predicates already defined in the KB. Thus, objects, relations, and events that appear in physics

  4. [Intracellular signals involved in glucose control].

    PubMed

    Cruz, M; Velasco, E; Kumate, J

    2001-01-01

    Many proteins are involved in glucose control. The first step for glucose uptake is insulin receptor-binding. Stimulation of the insulin receptor results in rapid autophosphorylation and conformational changes in the beta chain and the subsequent phosphorylation of the insulin receptor substrate. This results in the docking of several SH2 domain proteins, including PI 3-kinase and other adapters. The final event is glucose transporter (GLUT) translocation to the cell surface. GLUT is in the cytosol but after insulin stimulation, several proteins are activated either in the GLUT vesicles or in the inner membrane. The role of the cytoskeleton is not well known, but it apparently participates in membrane fusion and vesicle mobilization. After glucose uptake, several hexokines metabolize the glucose to generate energy, convert the glucose in glycogen and store it. Type 2 diabetes is characterized by high glucose levels and insulin resistance. The insulin receptor is diminished on the cell surface membrane, tyrosine phosphorylation is decreased, serine and threonine phosphorylation is augmented. Apparently, the main problem with GLUT protein is in its translocation to the cell surface. At present, we know the role of many proteins involved in glucose control. However, we do not understand the significance of insulin resistance at the molecular level with type 2 diabetes.

  5. Quantitating intracellular oxygen tension in vivo by phosphorescence lifetime measurement

    PubMed Central

    Hirakawa, Yosuke; Yoshihara, Toshitada; Kamiya, Mako; Mimura, Imari; Fujikura, Daichi; Masuda, Tsuyoshi; Kikuchi, Ryohei; Takahashi, Ippei; Urano, Yasuteru; Tobita, Seiji; Nangaku, Masaomi

    2015-01-01

    Hypoxia appears to have an important role in pathological conditions in many organs such as kidney; however, a method to quantify intracellular oxygen tension in vivo has not been well established. In this study, we established an optical method to quantify oxygen tension in mice kidneys using a cationic lipophilic phosphorescence probe, BTPDM1, which has an intracellular oxygen concentration-sensitive phosphorescence lifetime. Since this probe is distributed inside the tubular cells of the mice kidney, we succeeded in detecting acute renal hypoxic conditions and chronic kidney disease. This technique enabled us to estimate intracellular partial pressures of oxygen in vivo by extrapolating the calibration curve generated from cultured tubular cells. Since intracellular oxygen tension is directly related to cellular hypoxic reactions, such as the activation of hypoxia-inducible factors, our method will shed new light on hypoxia research in vivo. PMID:26644023

  6. Inhibition of intracellular growth of Listeria monocytogenes by antibiotics.

    PubMed Central

    Michelet, C; Avril, J L; Cartier, F; Berche, P

    1994-01-01

    We studied the activities of 15 antibiotics on the intracellular growth of Listeria monocytogenes in a HeLa cell line. After 24 h of contact with the infected cells, the antibiotics most effective against the intracellular growth of the 10 strains tested were amoxicillin, temafloxacin, and sparfloxacin, which nevertheless failed to totally eliminate the intracellular bacteria. Rifampin and co-trimoxazole had variable effects, depending on the isolates studied. The most active combinations were amoxicillin-sparfloxacin, co-trimoxazole-gentamicin, and sparfloxacin-co-trimoxazole. The results suggest the value of using a cell culture technique to study the activities of antibiotics against certain bacteria with intracellular sites of multiplication. PMID:8203836

  7. Acute disseminated encephalomyelitis associated with meningitis due to Mycobacterium intracellulare.

    PubMed

    Okada, Hiroshi; Yoshioka, Keiji

    2010-01-01

    A 73-year-old woman was admitted to our hospital because of persistent fever, headache and fatigue for several weeks. On admission, she was diagnosed as having meningitis due to Mycobacterium intracellulare (M. intracellulare) detected in her cerebrospinal fluid (CSF) by polymerase chain reaction. Even though anti-tuberculous therapy improved her CSF findings, her condition was not restored. Brain MRI showed multifocal and asymmetrical increases in T2 signals involving white matter and cortical gray-white junction of cerebral hemispheres, cerebellum and brainstem. Based on the progression of clinical symptoms and radiological features, we diagnosed her illness as acute disseminated encephalomyelitis (ADEM) associated with meningitis due to M. intracellulare. Steroid therapy dramatically improved her condition. This is the first report of ADEM following meningitis due to M. intracellulare in a non-immunocompromized host.

  8. EVIDENCE FOR THE MACROPHAGE INDUCING GENE IN MYCOBACTERIUM INTRACELLULARE

    EPA Science Inventory

    Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-des...

  9. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungalrelated parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and...

  10. Intracellular dynamics of hippocampal place cells during virtual navigation

    PubMed Central

    Harvey, Christopher D.; Collman, Forrest; Dombeck, Daniel A.; Tank, David W.

    2009-01-01

    Hippocampal place cells encode spatial information in rate and temporal codes. To examine the mechanisms underlying hippocampal coding, we measured the intracellular dynamics of place cells by combining in vivo whole cell recordings with a virtual reality system. Head-restrained mice, running on a spherical treadmill, interacted with a computer-generated visual environment to perform spatial behaviors. Robust place cell activity was present during movement along a virtual linear track. From whole cell recordings, we identified three subthreshold signatures of place fields: (1) an asymmetric ramp-like depolarization of the baseline membrane potential; (2) an increase in the amplitude of intracellular theta oscillations; and, (3) a phase precession of the intracellular theta oscillation relative to the extracellularly-recorded theta rhythm. These intracellular dynamics underlie the primary features of place cell rate and temporal codes. The virtual reality system developed here will enable new experimental approaches to study the neural circuits underlying navigation. PMID:19829374

  11. Predicting the morphology of sickle red blood cells using coarse-grained models of intracellular aligned hemoglobin polymers†

    PubMed Central

    Lei, Huan; Karniadakis, George Em

    2013-01-01

    Sickle red blood cells (SS-RBCs) exhibit heterogeneous cell morphologies (sickle, holly leaf, granular, etc.) in the deoxygenated state due to the polymerization of the sickle hemoglobin. Experimental evidence points to a close relationship between SS-RBC morphology and intracellular aligned hemoglobin polymers. Here, we develop a coarse-grained (CG) stochastic model to represent the growth of the intracellular aligned hemoglobin polymer domain. The CG model is calibrated based on the mechanical properties (Young’s modulus, bending rigidity) of the sickle hemoglobin fibers reported in experiments. The process of the cell membrane transition is simulated for physiologic aligned hemoglobin polymer configurations and mean corpuscular hemoglobin concentration. Typical SS-RBC morphologies observed in experiments can be obtained from the current model as a result of the intracellular aligned hemoglobin polymer development without introducing any further ad hoc assumptions. It is found that the final shape of SS-RBCs is primarily determined by the angular width of the aligned hemoglobin polymer domain, but it also depends, to a lesser degree, on the polymer growth rate and the cell membrane rigidity. Cell morphologies are quantified by structural shape factors, which agree well with experimental results from medical images. PMID:24307912

  12. Assessment of Methods for the Intracellular Blockade of GABAA Receptors

    PubMed Central

    Atherton, Laura A.; Burnell, Erica S.; Mellor, Jack R.

    2016-01-01

    Selective blockade of inhibitory synaptic transmission onto specific neurons is a useful tool for dissecting the excitatory and inhibitory synaptic components of ongoing network activity. To achieve this, intracellular recording with a patch solution capable of blocking GABAA receptors has advantages over other manipulations, such as pharmacological application of GABAergic antagonists or optogenetic inhibition of populations of interneurones, in that the majority of inhibitory transmission is unaffected and hence the remaining network activity preserved. Here, we assess three previously described methods to block inhibition: intracellular application of the molecules picrotoxin, 4,4’-dinitro-stilbene-2,2’-disulphonic acid (DNDS) and 4,4’-diisothiocyanostilbene-2,2’-disulphonic acid (DIDS). DNDS and picrotoxin were both found to be ineffective at blocking evoked, monosynaptic inhibitory postsynaptic currents (IPSCs) onto mouse CA1 pyramidal cells. An intracellular solution containing DIDS and caesium fluoride, but lacking nucleotides ATP and GTP, was effective at decreasing the amplitude of IPSCs. However, this effect was found to be independent of DIDS, and the absence of intracellular nucleotides, and was instead due to the presence of fluoride ions in this intracellular solution, which also blocked spontaneously occurring IPSCs during hippocampal sharp waves. Critically, intracellular fluoride ions also caused a decrease in both spontaneous and evoked excitatory synaptic currents and precluded the inclusion of nucleotides in the intracellular solution. Therefore, of the methods tested, only fluoride ions were effective for intracellular blockade of IPSCs but this approach has additional cellular effects reducing its selectivity and utility. PMID:27501143

  13. Study of neurotoxic intracellular calcium signalling triggered by amyloids.

    PubMed

    Villalobos, Carlos; Caballero, Erica; Sanz-Blasco, Sara; Núñez, Lucía

    2012-01-01

    Neurotoxicity in Alzheimer's disease (AD) is associated to dishomeostasis of intracellular Ca(2+) induced by amyloid β peptide (Aβ) species. Understanding of the effects of Aβ on intracellular Ca(2+) homeostasis requires preparation of the different Aβ assemblies including oligomers and fibrils and the testing of their effects on cytosolic and mitochondrial Ca(2+) in neurons. Procedures for cerebellar granule cell culture, preparation of Aβ species as well as fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca(2+) in neurons are described.

  14. Regulation of Intracellular Free Calcium in Neuronal Cells by Opioids

    DTIC Science & Technology

    1995-06-19

    APPROVAL SHEET Title of Dissertation: "Regulation ofIntracellular Free Calcium in Neuronal Cells by Opioids" Name of Candidate: Tianlai Tang Doctor...Calcium in Neuronal Cells by Opioids" beyond brief excerpts is with the pennission of the copyright owner, and will save and hold harmless the...Intracellular Free Calcium in Neuronal Cells by Opioids Doctor of Philosophy, 1995 Brian M. Cox, Professor, Department of Pharmacology The

  15. Intracellular delivery of peptides and siRNAs using microbubble enhanced focused ultrasound

    NASA Astrophysics Data System (ADS)

    Kinoshita, Manabu; Hynynen, Kullervo

    2006-05-01

    Bioactive substances such as peptides and nucleic acid based agents have attracted great attention for the next generation drug for various diseases. However, the greatest challenge for using these bioactive substances is the development of their delivery system, especially the method for delivering these substances through the cell membrane. With the advancement of ultrasound and ultrasound contrast agent technology, it has become possible to transiently change the permeability of the cell membrane. Moreover, using a focused ultrasound transducer, it is possible to narrow and focus the ultrasound energy within a small target, avoiding damage to the surrounding tissue. In this research we have searched the possibility of delivering the Bak BH3 peptide, the death domain of the Bc1-2 family of proteins, or the short interfering RNA (siRNA) targeting the enhanced green fluorescent protein (EGFP) using microbubble-enhanced focused ultrasound in an in vitro setting. Using a 1.696 MHz focused ultrasound and a microbubble ultrasound contrast agent OPTISON®, we first tested the stability of BH3 peptide under microbubble-enhanced focused ultrasound exposure and proved that the peptide is stable under these circumstances. Next, we have tested the cell-killing effect of the intracellularly delivered Bak BH3 peptide in HeLa and BJAB cell line and observed a statistically enhanced cell death in BJAB cells but not in HeLa cells, leading to the conclusion that intracellularly delivered BH3 peptide by microbubble-enhanced ultrasound can exert its cell killing effect in some cells. We also investigated if we can silence the EGFP expression in the cell by delivering siRNA targeting the EGFP in both transient and stable EGFP expression cell line. Using a 1.653 MHz focused ultrasound and OPTISON®, in both cases, intracellularly delivered siRNA by microbubble-enhanced ultrasound was able to knock down the EGFP expression, which demonstrates the feasibility of using this novel method

  16. Is membrane occupation and recognition nexus domain functional in plant phosphatidylinositol phosphate kinases?

    PubMed

    Mikami, Koji; Saavedra, Laura; Sommarin, Marianne

    2010-10-01

    Phosphatidylinositol phosphate kinase (PIPK) catalyzes a key step controlling cellular contents of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], a critical intracellular messenger involved in vesicle trafficking and modulation of actin cytoskeleton and also a substrate of phospholipase C to produce the two intracellular messengers, diacylglycerol and inositol-1,4,5-trisphosphate. In addition to the conserved C-terminal PIPK catalytic domain, plant PIPKs contain a unique structural feature consisting of a repeat of membrane occupation and recognition nexus (MORN) motifs, called the MORN domain, in the N-terminal half. The MORN domain has previously been proposed to regulate plasma membrane localization and phosphatidic acid (PA)-inducible activation. Recently, the importance of the catalytic domain, but not the MORN domain, in these aspects was demonstrated. These conflicting data raise the question about the function of the MORN domain in plant PIPKs. We therefore performed analyses of PpPIPK1 from the moss Physcomitrella patens to elucidate the importance of the MORN domain in the control of enzymatic activity; however, we found no effect on either enzymatic activity or activation by PA. Taken together with our previous findings of lack of function in plasma membrane localization, there is no positive evidence indicating roles of the MORN domain in enzymatic and functional regulations of PpPIPK1. Therefore, further biochemical and reverse genetic analyses are necessary to understand the biological significance of the MORN domain in plant PIPKs.

  17. Chaperone receptors: guiding proteins to intracellular compartments.

    PubMed

    Kriechbaumer, Verena; von Löffelholz, Ottilie; Abell, Ben M

    2012-01-01

    Despite mitochondria and chloroplasts having their own genome, 99% of mitochondrial proteins (Rehling et al., Nat Rev Mol Cell Biol 5:519-530, 2004) and more than 95% of chloroplast proteins (Soll, Curr Opin Plant Biol 5:529-535, 2002) are encoded by nuclear DNA, synthesised in the cytosol and imported post-translationally. Protein targeting to these organelles depends on cytosolic targeting factors, which bind to the precursor, and then interact with membrane receptors to deliver the precursor into a translocase. The molecular chaperones Hsp70 and Hsp90 have been widely implicated in protein targeting to mitochondria and chloroplasts, and receptors capable of recognising these chaperones have been identified at the surface of both these organelles (Schlegel et al., Mol Biol Evol 24:2763-2774, 2007). The role of these chaperone receptors is not fully understood, but they have been shown to increase the efficiency of protein targeting (Young et al., Cell 112:41-50, 2003; Qbadou et al., EMBO J 25:1836-1847, 2006). Whether these receptors contribute to the specificity of targeting is less clear. A class of chaperone receptors bearing tetratricopeptide repeat domains is able to specifically bind the highly conserved C terminus of Hsp70 and/or Hsp90. Interestingly, at least of one these chaperone receptors can be found on each organelle (Schlegel et al., Mol Biol Evol 24:2763-2774, 2007), which suggests a universal role in protein targeting for these chaperone receptors. This review will investigate the role that chaperone receptors play in targeting efficiency and specificity, as well as examining recent in silico approaches to find novel chaperone receptors.

  18. Intracellular signalling involved in volume regulatory decrease.

    PubMed

    Hoffmann, E K

    2000-01-01

    The following volume-sensitive channels are characterized in Ehrlich ascites tumor cells (EATC), (i) a tamoxifen- and AA acid sensitive, outwardly rectifying small anion channel (I(Cl,vol)) with low field anion selectivity (I(-)>Cl(-)) and moderate depolarisation-induced inactivation, (ii) a separate DIDS- and niflumic acid-sensitive organic osmolyte/anion channel (OOC) transporting predominantly taurine, and (iii) a clofilium- and Ba(2+)-sensitive, voltage- and Ca(2+)-insensitive 5 pS K(+) channel (I(K,vol)), resistant to a range of K(+) channel inhibitors including ChTX, clotrimazole, apamin, kaliotoxin, margatoxin, and TEA, and with a pH(o) dependence reminiscent of the two-pore domain background K(+) channels TASK. Cell swelling leads to an immediate and transient 3.3 fold increase in the rate of AA release resulting from activation of cPLA(2)alpha, which is found to be translocated to the nucleus upon cell swelling (probably to the inner nuclear membrane), where it is phosphorylated and activated by a G-protein coupled process. AA is a precursor for LTC(4), which is transported out of the cell, where it is converted to LTD(4), which activates I(K,vol), and OOC, whereas I(Cl,vol) is activated via a different pathway. In the absence of an increase in [Ca(2+)](i), the unitary conductance, kinetics, and pharmacological profile are similar for I(K,vol) and the K(+)-channels activated by LTD(4). Tyrosine phosphorylations are involved in the volume regulatory pathways and in defining the volume set-point. Tyrosin kinases appear to be involved in the signalling sequence leading to opening of the channels, and tyrosin phosphatases seem to be involved in closing of the channels. Finally a significant de-polymerization of F-actin is observed after cell swelling, the potential role of which in the volume regulatory mechanisms is under investigation.

  19. Invasion of the Central Nervous System by Intracellular Bacteria

    PubMed Central

    Drevets, Douglas A.; Leenen, Pieter J. M.; Greenfield, Ronald A.

    2004-01-01

    Infection of the central nervous system (CNS) is a severe and frequently fatal event during the course of many diseases caused by microbes with predominantly intracellular life cycles. Examples of these include the facultative intracellular bacteria Listeria monocytogenes, Mycobacterium tuberculosis, and Brucella and Salmonella spp. and obligate intracellular microbes of the Rickettsiaceae family and Tropheryma whipplei. Unfortunately, the mechanisms used by intracellular bacterial pathogens to enter the CNS are less well known than those used by bacterial pathogens with an extracellular life cycle. The goal of this review is to elaborate on the means by which intracellular bacterial pathogens establish infection within the CNS. This review encompasses the clinical and pathological findings that pertain to the CNS infection in humans and includes experimental data from animal models that illuminate how these microbes enter the CNS. Recent experimental data showing that L. monocytogenes can invade the CNS by more than one mechanism make it a useful model for discussing the various routes for neuroinvasion used by intracellular bacterial pathogens. PMID:15084504

  20. INTRA-CELLULAR STAPHYLOCOCCUS AUREUS ALONE CAUSES INFECTION IN VIVO#

    PubMed Central

    Hamza, Therwa; Dietz, Matthew; Pham, Danh; Clovis, Nina; Danley, Suzanne; Li, Bingyun

    2013-01-01

    Chronic and recurrent bone infections occur frequently but have not been explained. Staphylococcus aureus (S. aureus) is often found among chronic and recurrent infections and may be responsible for such infections. One possible reason is that S. aureus can internalize and survive within host cells and by doing so, S. aureus can evade both host defense mechanisms and most conventional antibiotic treatments. In this study, we hypothesized that intra-cellular S. aureus could induce infections in vivo. Osteoblasts were infected with S. aureus and, after eliminating extra-cellular S. aureus, inoculated into an open fracture rat model. Bacterial cultures and radiographic observations at post-operative day 21 confirmed local bone infections in animals inoculated with intra-cellular S. aureus within osteoblasts alone. We present direct in vivo evidence that intra-cellular S. aureus could be sufficient to induce bone infection in animals; we found that intra-cellular S. aureus inoculation of as low as 102 colony forming units could induce severe bone infections. Our data may suggest that intra-cellular S. aureus can “hide” in host cells during symptom-free periods and, under certain conditions, they may escape and lead to infection recurrence. Intra-cellular S. aureus therefore could play an important role in the pathogenesis of S. aureus infections, especially those chronic and recurrent infections in which disease episodes may be separated by weeks, months, or even years. PMID:23832687

  1. Intra-cellular Staphylococcus aureus alone causes infection in vivo.

    PubMed

    Hamza, T; Dietz, M; Pham, D; Clovis, N; Danley, S; Li, B

    2013-07-08

    Chronic and recurrent bone infections occur frequently but have not been explained. Staphylococcus aureus (S. aureus) is often found among chronic and recurrent infections and may be responsible for such infections. One possible reason is that S. aureus can internalize and survive within host cells and by doing so, S. aureus can evade both host defense mechanisms and most conventional antibiotic treatments. In this study, we hypothesized that intra-cellular S. aureus could induce infections in vivo. Osteoblasts were infected with S. aureus and, after eliminating extra-cellular S. aureus, inoculated into an open fracture rat model. Bacterial cultures and radiographic observations at post-operative day 21 confirmed local bone infections in animals inoculated with intra-cellular S. aureus within osteoblasts alone. We present direct in vivo evidence that intra-cellular S. aureus could be sufficient to induce bone infection in animals; we found that intra-cellular S. aureus inoculation of as low as 102 colony forming units could induce severe bone infections. Our data may suggest that intra-cellular S. aureus can "hide" in host cells during symptom-free periods and, under certain conditions, they may escape and lead to infection recurrence. Intra-cellular S. aureus therefore could play an important role in the pathogenesis of S. aureus infections, especially those chronic and recurrent infections in which disease episodes may be separated by weeks, months, or even years.

  2. Uptake and intracellular activity of fluconazole in human polymorphonuclear leukocytes.

    PubMed Central

    Pascual, A; García, I; Conejo, C; Perea, E J

    1993-01-01

    The penetration of fluconazole into human polymorphonuclear leukocytes (PMNs) and tissue culture epithelial cells (McCoy) was evaluated. At different extracellular concentrations (0.5 to 10 mg/liter), fluconazole reached cell-associated concentrations greater than the extracellular ones in either human PMNs (intracellular concentration to extracellular concentration ratio, > or = 2.2) or McCoy cells (intracellular concentration to extracellular concentration ratio, > or = 1.3). The uptake of fluconazole by PMNs was rapid and reversible but was not energy dependent. The intracellular penetration of fluconazole was not affected by environmental pH or temperature. Ingestion of opsonized zymosan and opsonized Candida albicans did not significantly increase the amount of PMN-associated fluconazole. At therapeutic extracellular concentrations, the intracellular activity of fluconazole against C. albicans in PMNs was significantly lower than that of amphotericin B. It was concluded that fluconazole reaches high intracellular concentrations within PMNs but shows moderate activity against intracellular C. albicans in vitro. PMID:8452347

  3. Cell adhesion and intracellular calcium signaling in neurons

    PubMed Central

    2013-01-01

    Cell adhesion molecules (CAMs) play indispensable roles in the developing and mature brain by regulating neuronal migration and differentiation, neurite outgrowth, axonal fasciculation, synapse formation and synaptic plasticity. CAM-mediated changes in neuronal behavior depend on a number of intracellular signaling cascades including changes in various second messengers, among which CAM-dependent changes in intracellular Ca2+ levels play a prominent role. Ca2+ is an essential secondary intracellular signaling molecule that regulates fundamental cellular functions in various cell types, including neurons. We present a systematic review of the studies reporting changes in intracellular Ca2+ levels in response to activation of the immunoglobulin superfamily CAMs, cadherins and integrins in neurons. We also analyze current experimental evidence on the Ca2+ sources and channels involved in intracellular Ca2+ increases mediated by CAMs of these families, and systematically review the role of the voltage-dependent Ca2+ channels (VDCCs) in neurite outgrowth induced by activation of these CAMs. Molecular mechanisms linking CAMs to VDCCs and intracellular Ca2+ stores in neurons are discussed. PMID:24330678

  4. Visualizing Knowledge Domains.

    ERIC Educational Resources Information Center

    Borner, Katy; Chen, Chaomei; Boyack, Kevin W.

    2003-01-01

    Reviews visualization techniques for scientific disciplines and information retrieval and classification. Highlights include historical background of scientometrics, bibliometrics, and citation analysis; map generation; process flow of visualizing knowledge domains; measures and similarity calculations; vector space model; factor analysis;…

  5. Two apextrin-like proteins mediate extracellular and intracellular bacterial recognition in amphioxus.

    PubMed

    Huang, Guangrui; Huang, Shengfeng; Yan, Xinyu; Yang, Ping; Li, Jun; Xu, Weiya; Zhang, Lingling; Wang, Ruihua; Yu, Yingcai; Yuan, Shaochun; Chen, Shangwu; Luo, Guangbin; Xu, Anlong

    2014-09-16

    Animals exploit different germ-line-encoded proteins with various domain structures to detect the signature molecules of pathogenic microbes. These molecules are known as pathogen-associated molecular patterns (PAMPs), and the host proteins that react with PAMPs are called pattern recognition proteins (PRPs). Here, we present a novel type of protein domain structure capable of binding to bacterial peptidoglycan (PGN) and the minimal PGN motif muramyl dipeptide (MDP). This domain is designated as apextrin C-terminal domain (ApeC), and its presence was confirmed in several invertebrate phyla and subphyla. Two apextrin-like proteins (ALP1 and ALP2) were identified in a basal chordate, the Japanese amphioxus Branchiostoma japonicum (bj). bjALP1 is a mucosal effector secreted into the gut lumen to agglutinate the Gram-positive bacterium Staphylococcus aureus via PGN binding. Neutralization of secreted bjALP1 by anti-bjALP1 monoclonal antibodies caused serious damage to the gut epithelium and rapid death of the animals after bacterial infection. bjALP2 is an intracellular PGN sensor that binds to TNF receptor-associated factor 6 (TRAF6) and prevents TRAF6 from self-ubiquitination and hence from NF-κB activation. MDP was found to compete with TRAF6 for bjALP2, which released TRAF6 to activate the NF-κB pathway. BjALP1 and bjALP2 therefore play distinct and complementary functions in amphioxus gut mucosal immunity. In conclusion, discovery of the ApeC domain and the functional analyses of amphioxus ALP1 and ALP2 allowed us to define a previously undocumented type of PRP that is represented across different animal phyla.

  6. Software architecture design domain

    SciTech Connect

    White, S.A.

    1996-12-31

    Software architectures can provide a basis for the capture and subsequent reuse of design knowledge. The goal of software architecture is to allow the design of a system to take place at a higher level of abstraction; a level concerned with components, connections, constraints, rationale. This architectural view of software adds a new layer of abstraction to the traditional design phase of software development. It has resulted in a flurry of activity towards techniques, tools, and architectural design languages developed specifically to assist with this activity. An analysis of architectural descriptions, even though they differ in notation, shows a common set of key constructs that are present across widely varying domains. These common aspects form a core set of constructs that should belong to any ADL in order to for the language to offer the ability to specify software systems at the architectural level. This analysis also revealed a second set of constructs which served to expand the first set thereby improving the syntax and semantics. These constructs are classified according to whether they provide representation and analysis support for architectures belonging to many varying application domains (domain-independent construct class) or to a particular application domain (domain-dependent constructs). This paper presents the constructs of these two classes, their placement in the architecture design domain and shows how they may be used to classify, select, and analyze proclaimed architectural design languages (ADLs).

  7. Regulation of the processivity and intracellular localization of Saccharomyces cerevisiae dynein by dynactin

    PubMed Central

    Kardon, Julia R.; Reck-Peterson, Samara L.; Vale, Ronald D.

    2009-01-01

    Dynactin, a large multisubunit complex, is required for intracellular transport by dynein; however, its cellular functions and mechanism of action are not clear. Prior studies suggested that dynactin increases dynein processivity by tethering the motor to the microtubule through its own microtubule binding domains. However, this hypothesis could not be tested without a recombinant source of dynactin. Here, we have produced recombinant dynactin and dynein in Saccharomyces cerevisiae, and examined the effect of dynactin on dynein in single-molecule motility assays. We show that dynactin increases the run length of single dynein motors, but does not alter the directionality of dynein movement. Enhancement of dynein processivity by dynactin does not require the microtubule (MT) binding domains of Nip100 (the yeast p150Glued homolog). Dynactin lacking these MT binding domains also supports the proper localization and function of dynein during nuclear segregation in vivo. Instead, a segment of the coiled-coil of Nip100 is required for these activities. Our results directly demonstrate that dynactin increases the processivity of dynein through a mechanism independent of microtubule tethering. PMID:19293377

  8. Positive and negative regulation of a SNARE protein by control of intracellular localization.

    PubMed

    Nakanishi, Hideki; de los Santos, Pablo; Neiman, Aaron M

    2004-04-01

    In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores. Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain. We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or prospore membrane localization to green fluorescent protein. In vitro, this helix binds to acidic phospholipids, and mutations that reduce or eliminate phospholipid binding in vitro inactivate Spo20p in vivo. Genetic manipulation of phospholipid pools indicates that the likely in vivo ligand of this domain is phosphatidic acid. The inhibitory activity is a nuclear targeting signal, which confers nuclear localization in vegetative cells and in cells entering meiosis. However, as cells initiate spore formation, fusions containing the inhibitory domain exit the nucleus and localize to the nascent prospore membrane. Thus, the SNARE Spo20p is both positively and negatively regulated by control of its intracellular localization.

  9. Domains in Ferroelectric Nanostructures

    NASA Astrophysics Data System (ADS)

    Gregg, Marty

    2010-03-01

    Ferroelectric materials have great potential in influencing the future of small scale electronics. At a basic level, this is because ferroelectric surfaces are charged, and so interact strongly with charge-carrying metals and semiconductors - the building blocks for all electronic systems. Since the electrical polarity of the ferroelectric can be reversed, surfaces can both attract and repel charges in nearby materials, and can thereby exert complete control over both charge distribution and movement. It should be no surprise, therefore, that microelectronics industries have already looked very seriously at harnessing ferroelectric materials in a variety of applications, from solid state memory chips (FeRAMs) to field effect transistors (FeFETs). In all such applications, switching the direction of the polarity of the ferroelectric is a key aspect of functional behavior. The mechanism for switching involves the field-induced nucleation and growth of domains. Domain coarsening, through domain wall propagation, eventually causes the entire ferroelectric to switch its polar direction. It is thus the existence and behavior of domains that determine the switching response, and ultimately the performance of the ferroelectric device. A major issue, associated with the integration of ferroelectrics into microelectronic devices, has been that the fundamental properties associated with ferroelectrics, when in bulk form, appear to change quite dramatically and unpredictably when at the nanoscale: new modes of behaviour, and different functional characteristics from those seen in bulk appear. For domains, in particular, the proximity of surfaces and boundaries have a dramatic effect: surface tension and depolarizing fields both serve to increase the equilibrium density of domains, such that minor changes in scale or morphology can have major ramifications for domain redistribution. Given the importance of domains in dictating the overall switching characteristics of a device

  10. Characterization of intracellular pteroylpolyglutamate hydrolase (PPH) from human intestinal mucosa

    SciTech Connect

    Wang, T.T.Y.; Chandler, C.J.; Halsted, C.H.

    1986-03-01

    There are two forms of pteroylpolyglutamate hydrolase (PPH) in the human intestinal mucosa, one in the brush border membrane and the other intracellular; brush border PPH is an exopeptidase with optimal activity at pH 6.5 and a requirement for zinc. The presence study characterized human intracellular PPH and compared its properties to those of brush border PPH. Intracellular PPH was purified 30-fold. The enzyme had a MW of 75,000 by gel filtration, was optimally active at pH 4.5, and had an isoelectric point at pH 8.0. In contrast to brush border PPH, intracellular PPH was unstable at increasing temperatures, was unaffected by dialysis against chelating agents and showed no requirement for Zn/sup 2 +/. Using PteGlu/sub 2/(/sup 14/C)Glu as substrate, they demonstrated a K/sub m/ of 1.2 ..mu..M and increasing affinity for folates with longer glutamate chains. Intracellular PPH required the complete folic acid (PteGlu) moiety and a ..gamma..-glutamyl linkage for activity. Using ion exchange chromatography and an HPLC method to determine the hydrolytic products of the reaction, they found intracellular PPH could cleave both internal and terminal ..gamma..-glutamyl linkages, with PteGlu as an end product. After subcellular fractionation of the mucosa, PPH was found in the lysosomes. In summary, the distinct characteristics of brush border and intracellular PPH suggest that the two hydrolases serve different roles in folate metabolism.

  11. Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins.

    PubMed

    Kim, Minsoo; Carman, Christopher V; Springer, Timothy A

    2003-09-19

    Although critical for development, immunity, wound healing, and metastasis, integrins represent one of the few classes of plasma membrane receptors for which the basic signaling mechanism remains a mystery. We investigated cytoplasmic conformational changes in the integrin LFA-1 (alphaLbeta2) in living cells by measuring fluorescence resonance energy transfer between cyan fluorescent protein-fused and yellow fluorescent protein-fused alphaL and beta2 cytoplasmic domains. In the resting state these domains were close to each other, but underwent significant spatial separation upon either intracellular activation of integrin adhesiveness (inside-out signaling) or ligand binding (outside-in signaling). Thus, bidirectional integrin signaling is accomplished by coupling extracellular conformational changes to an unclasping and separation of the alpha and beta cytoplasmic domains, a distinctive mechanism for transmitting information across the plasma membrane.

  12. Modulation of the slow/common gating of CLC channels by intracellular cadmium

    PubMed Central

    Yu, Yawei; Tsai, Ming-Feng; Yu, Wei-Ping

    2015-01-01

    Members of the CLC family of Cl− channels and transporters are homodimeric integral membrane proteins. Two gating mechanisms control the opening and closing of Cl− channels in this family: fast gating, which regulates opening and closing of the individual pores in each subunit, and slow (or common) gating, which simultaneously controls gating of both subunits. Here, we found that intracellularly applied Cd2+ reduces the current of CLC-0 because of its inhibition on the slow gating. We identified CLC-0 residues C229 and H231, located at the intracellular end of the transmembrane domain near the dimer interface, as the Cd2+-coordinating residues. The inhibition of the current of CLC-0 by Cd2+ was greatly enhanced by mutation of I225W and V490W at the dimer interface. Biochemical experiments revealed that formation of a disulfide bond within this Cd2+-binding site is also affected by mutation of I225W and V490W, indicating that these two mutations alter the structure of the Cd2+-binding site. Kinetic studies showed that Cd2+ inhibition appears to be state dependent, suggesting that structural rearrangements may occur in the CLC dimer interface during Cd2+ modulation. Mutations of I290 and I556 of CLC-1, which correspond to I225 and V490 of CLC-0, respectively, have been shown previously to cause malfunction of CLC-1 Cl− channel by altering the common gating. Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0. The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd2+ likely depends on their alteration of subunit interactions. PMID:26621774

  13. Discovery and functional characterization of a novel small molecule inhibitor of the intracellular phosphatase, SHIP2

    PubMed Central

    Suwa, A; Yamamoto, T; Sawada, A; Minoura, K; Hosogai, N; Tahara, A; Kurama, T; Shimokawa, T; Aramori, I

    2009-01-01

    Background and purpose: The lipid phosphatase known as SH2 domain-containing inositol 5′-phosphatase 2 (SHIP2) plays an important role in the regulation of the intracellular insulin signalling pathway. Recent studies have suggested that inhibition of SHIP2 could produce significant benefits in treatment of type 2 diabetes. However, there were no small molecule SHIP2 inhibitors and we, therefore, aimed to identify this type of compound. Experimental approach: The phosphatase assay with malachite green was used for high-throughput screening. The pharmacological profiles of suitable compounds were further characterized in phosphatase assays, cellular assays and oral administration in normal and diabetic (db/db) mice. Key results: During high-throughput screening, AS1949490 was identified as a potent SHIP2 inhibitor (IC50= 0.62 µM for SHIP2). This compound was also selective for SHIP2 relative to other intracellular phosphatases. In L6 myotubes, AS1949490 increased the phosphorylation of Akt, glucose consumption and glucose uptake. In FAO hepatocytes, AS1949490 suppressed gluconeogenesis. Acute administration of AS1949490 inhibited the expression of gluconeogenic genes in the livers of normal mice. Chronic treatment of diabetic db/db mice with AS1949490 significantly lowered the plasma glucose level and improved glucose intolerance. These in vivo effects were based in part on the activation of intracellular insulin signalling pathways in the liver. Conclusions and implications: This is the first report of a small molecule inhibitor of SHIP2. This compound will help to elucidate the physiological functions of SHIP2 and its involvement in various diseases, such as type 2 diabetes. PMID:19694723

  14. Modulation of the slow/common gating of CLC channels by intracellular cadmium.

    PubMed

    Yu, Yawei; Tsai, Ming-Feng; Yu, Wei-Ping; Chen, Tsung-Yu

    2015-12-01

    Members of the CLC family of Cl(-) channels and transporters are homodimeric integral membrane proteins. Two gating mechanisms control the opening and closing of Cl(-) channels in this family: fast gating, which regulates opening and closing of the individual pores in each subunit, and slow (or common) gating, which simultaneously controls gating of both subunits. Here, we found that intracellularly applied Cd(2+) reduces the current of CLC-0 because of its inhibition on the slow gating. We identified CLC-0 residues C229 and H231, located at the intracellular end of the transmembrane domain near the dimer interface, as the Cd(2+)-coordinating residues. The inhibition of the current of CLC-0 by Cd(2+) was greatly enhanced by mutation of I225W and V490W at the dimer interface. Biochemical experiments revealed that formation of a disulfide bond within this Cd(2+)-binding site is also affected by mutation of I225W and V490W, indicating that these two mutations alter the structure of the Cd(2+)-binding site. Kinetic studies showed that Cd(2+) inhibition appears to be state dependent, suggesting that structural rearrangements may occur in the CLC dimer interface during Cd(2+) modulation. Mutations of I290 and I556 of CLC-1, which correspond to I225 and V490 of CLC-0, respectively, have been shown previously to cause malfunction of CLC-1 Cl(-) channel by altering the common gating. Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0. The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd(2+) likely depends on their alteration of subunit interactions.

  15. Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

    PubMed

    Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, C L; Kaul, M; Singh, K K

    2014-09-01

    Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons.

  16. Bioinformatic Characterization of the Trimeric Intracellular Cation-Specific Channel Protein Family

    PubMed Central

    Silverio, Abe L. F.

    2014-01-01

    Trimeric intracellular cation-specific (TRIC) channels are integral to muscle excitation–contraction coupling. TRIC channels provide counter-ionic flux when calcium is rapidly transported from intracellular stores to the cell cytoplasm. Until recently, knowledge of the presence of these proteins was limited to animals. We analyzed the TRIC family and identified a profusion of prokaryotic family members with topologies and motifs similar to those of their eukaryotic counterparts. Prokaryotic members far outnumber eukaryotic members, and although none has been functionally characterized, the evidence suggests that they function as secondary carriers. The presence of fused N- or C-terminal domains of known biochemical functions as well as genomic context analyses provide clues about the functions of these prokaryotic homologs. They are proposed to function in metabolite (e.g., amino acid/ nucleotide) efflux. Phylogenetic analysis revealed that TRIC channel homologs diverged relatively early during evolutionary history and that horizontal gene transfer was frequent in prokaryotes but not in eukaryotes. Topological analyses of TRIC channels revealed that these proteins possess seven putative transmembrane segments (TMSs), which arose by intragenic duplication of a three-TMS polypeptide-encoding genetic element followed by addition of a seventh TMS at the C terminus to give the precursor of all current TRIC family homologs. We propose that this family arose in prokaryotes. PMID:21519847

  17. From surface to intracellular non-invasive nanoscale study of living cells impairments

    NASA Astrophysics Data System (ADS)

    Ewald, M.; Tetard, L.; Elie-Caille, C.; Nicod, L.; Passian, A.; Bourillot, E.; Lesniewska, E.

    2014-07-01

    Among the enduring challenges in nanoscience, subsurface characterization of living cells holds major stakes. Developments in nanometrology for soft matter thriving on the sensitivity and high resolution benefits of atomic force microscopy have enabled detection of subsurface structures at the nanoscale. However, measurements in liquid environments remain complex, in particular in the subsurface domain. Here we introduce liquid-mode synthesizing atomic force microscopy (l-MSAFM) to study both the inner structures and the chemically induced intracellular impairments of living cells. Specifically, we visualize the intracellular stress effects of glyphosate on living keratinocytes skin cells. This new approach, l-MSAFM, for nanoscale imaging of living cell in their physiological environment or in presence of a chemical stress agent could resolve the loss of inner structures induced by glyphosate, the main component of a well-known pesticide (RoundUp™). This firsthand ability to monitor the cell’s inner response to external stimuli non-destructively and in liquid, has the potential to unveil critical nanoscale mechanisms of life science.

  18. ROP18 Is a Rhoptry Kinase Controlling the Intracellular Proliferation of Toxoplasma gondii

    PubMed Central

    El Hajj, Hiba; Lebrun, Maryse; Arold, Stefan T; Vial, Henri; Labesse, Gilles; Dubremetz, Jean François

    2007-01-01

    Toxoplasma gondii is an obligate intracellular parasite for which the discharge of apical organelles named rhoptries is a key event in host cell invasion. Among rhoptry proteins, ROP2, which is the prototype of a large protein family, is translocated in the parasitophorous vacuole membrane during invasion. The ROP2 family members are related to protein-kinases, but only some of them are predicted to be catalytically active, and none of the latter has been characterized so far. We show here that ROP18, a member of the ROP2 family, is located in the rhoptries and re-localises at the parasitophorous vacuole membrane during invasion. We demonstrate that a recombinant ROP18 catalytic domain (amino acids 243–539) possesses a protein-kinase activity and phosphorylate parasitic substrates, especially a 70-kDa protein of tachyzoites. Furthermore, we show that overexpression of ROP18 in transgenic parasites causes a dramatic increase in intra-vacuolar parasite multiplication rate, which is correlated with kinase activity. Therefore, we demonstrate, to our knowledge for the first time, that rhoptries can discharge active protein-kinases upon host cell invasion, which can exert a long-lasting effect on intracellular parasite development and virulence. PMID:17305424

  19. From surface to intracellular non-invasive nanoscale study of living cells impairments

    SciTech Connect

    Ewald, Dr. Maxime; Tetard, Laurene; Elie-Caille, Dr. Cecile; Nicod, Laurence; Passian, Ali; Bourillot, Dr. Eric; Lesniewska, Prof. Eric

    2014-01-01

    Among the enduring challenges in nanoscience, subsurface characterization of live cells holds major stakes. Developments in nanometrology for soft matter thriving on the sensitivity and high resolution benefits of atomic force microscopy have enabled detection of subsurface structures at the nanoscale (1,2,3). However, measurements in liquid environments remain complex (4,5,6,7), in particular in the subsurface domain. Here we introduce liquid-Mode Synthesizing Atomic Force Microscopy (l-MSAFM) to study both the inner structures and the chemically induced intracellular impairments of living cells. Specifically, we visualize the intracellular stress effects of glyphosate on living keratinocytes skin cells. This new approach for living cell nanoscale imaging, l-MSAFM, in their physiological environment or in presence of a chemical stress agent confirmed the loss of inner structures induced by glyphosate. The ability to monitor the cell's inner response to external stimuli, non-destructively and in real time, has the potential to unveil critical nanoscale mechanisms of life science.

  20. Intracellular Mannose Binding Lectin Mediates Subcellular Trafficking of HIV-1 gp120 in Neurons

    PubMed Central

    Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, CL; Kaul, M; Singh, KK

    2014-01-01

    Human immunodeficiency virus -1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. PMID:24825317

  1. Role of caveolae in Leishmania chagasi phagocytosis and intracellular survival in macrophages.

    PubMed

    Rodríguez, Nilda E; Gaur, Upasna; Wilson, Mary E

    2006-07-01

    Caveolae are membrane microdomains enriched in cholesterol, ganglioside M1 (GM1) and caveolin-1. We explored whether caveolae facilitate the entry of Leishmania chagasi into murine macrophages. Transient depletion of macrophage membrane cholesterol by 1 h exposure to methyl-beta-cyclodextrin (MbetaCD) impaired the phagocytosis of non-opsonized and serum-opsonized virulent L. chagasi. In contrast, MbetaCD did not affect the phagocytosis of opsonized attenuated L. chagasi. As early as 5 min after phagocytosis, virulent L. chagasi colocalized with the caveolae markers GM1 and caveolin-1, and colocalization continued for over 48 h. We explored the kinetics of lysosome fusion. Whereas fluorescent-labelled dextran entered macrophage lysosomes by 30 min after addition, localization of L. chagasi in lysosomes was delayed for 24-48 h after phagocytosis. However, after transient depletion of cholesterol from macrophage membrane with MbetaCD, the proportion of L. chagasi-containing phagosomes that fused with lysosomes increased significantly. Furthermore, intracellular replication was impaired in parasites entering after transient cholesterol depletion, even though lipid microdomains were restored by 4 h after treatment. These observations suggest that virulent L. chagasi localize in caveolae during phagocytosis by host macrophages, and that cholesterol-containing macrophage membrane domains, such as caveolae, target parasites to a pathway that promotes delay of lysosome fusion and intracellular survival.

  2. A Carboxyl Ester Lipase (CEL) Mutant Causes Chronic Pancreatitis by Forming Intracellular Aggregates That Activate Apoptosis.

    PubMed

    Xiao, Xunjun; Jones, Gabrielle; Sevilla, Wednesday A; Stolz, Donna B; Magee, Kelsey E; Haughney, Margaret; Mukherjee, Amitava; Wang, Yan; Lowe, Mark E

    2016-10-28

    Patients with chronic pancreatitis (CP) frequently have genetic risk factors for disease. Many of the identified genes have been connected to trypsinogen activation or trypsin inactivation. The description of CP in patients with mutations in the variable number of tandem repeat (VNTR) domain of carboxyl ester lipase (CEL) presents an opportunity to study the pathogenesis of CP independently of trypsin pathways. We tested the hypothesis that a deletion and frameshift mutation (C563fsX673) in the CEL VNTR causes CP through proteotoxic gain-of-function activation of maladaptive cell signaling pathways including cell death pathways. HEK293 or AR42J cells were transfected with constructs expressing CEL with 14 repeats in the VNTR (CEL14R) or C563fsX673 CEL (CEL maturity onset diabetes of youth with a deletion mutation in the VNTR (MODY)). In both cell types, CEL MODY formed intracellular aggregates. Secretion of CEL MODY was decreased compared with that of CEL14R. Expression of CEL MODY increased endoplasmic reticulum stress, activated the unfolded protein response, and caused cell death by apoptosis. Our results demonstrate that disorders of protein homeostasis can lead to CP and suggest that novel therapies to decrease the intracellular accumulation of misfolded protein may be successful in some patients with CP.

  3. Active site structure and catalytic mechanism of phosphodiesterase for degradation of intracellular second messengers

    NASA Astrophysics Data System (ADS)

    Zhan, Chang-Guo

    2002-03-01

    Phosphodiesterases are clinical targets for a variety of biological disorders, because this superfamily of enzymes regulate intracellular concentration of cyclic nucleotides that serve as the second messengers playing a critical role in a variety of physiological processes. Understanding structure and mechanism of a phosphodiesterase will provide a solid basis for rational design of the more efficient therapeutics. Although a three-dimensional X-ray crystal structure of the catalytic domain of human phosphodiesterase 4B2B was recently reported, it was uncertain whether a critical bridging ligand in the active site is a water molecule or a hydroxide ion. The identity of this bridging ligand has been determined by performing first-principles quantum chemical calculations on models of the active site. All the results obtained indicate that this critical bridging ligand in the active site of the reported X-ray crystal structure is a hydroxide ion, rather than a water molecule, expected to serve as the nucleophile to initialize the catalytic degradation of the intracellular second messengers.

  4. Coupling mechanical forces to electrical signaling: molecular motors and the intracellular transport of ion channels.

    PubMed

    Barry, Joshua; Gu, Chen

    2013-04-01

    Proper localization of various ion channels is fundamental to neuronal functions, including postsynaptic potential plasticity, dendritic integration, action potential initiation and propagation, and neurotransmitter release. Microtubule-based forward transport mediated by kinesin motors plays a key role in placing ion channel proteins to correct subcellular compartments. PDZ- and coiled-coil-domain proteins function as adaptor proteins linking ionotropic glutamate and GABA receptors to various kinesin motors, respectively. Recent studies show that several voltage-gated ion channel/transporter proteins directly bind to kinesins during forward transport. Three major regulatory mechanisms underlying intracellular transport of ion channels are also revealed. These studies contribute to understanding how mechanical forces are coupled to electrical signaling and illuminating pathogenic mechanisms in neurodegenerative diseases.

  5. Structure and intracellular targeting of the SARS-coronavirus Orf7a accessory protein.

    PubMed

    Nelson, Christopher A; Pekosz, Andrew; Lee, Chung A; Diamond, Michael S; Fremont, Daved H

    2005-01-01

    The open reading frame (ORF) 7a of the SARS-associated coronavirus (SARS-CoV) encodes a unique type I transmembrane protein of unknown function. We have determined the 1.8 A resolution crystal structure of the N-terminal ectodomain of orf7a, revealing a compact seven-stranded beta sandwich unexpectedly similar in fold and topology to members of the Ig superfamily. We also demonstrate that, in SARS-CoV- infected cells, the orf7a protein is expressed and retained intracellularly. Confocal microscopy studies using orf7a and orf7a/CD4 chimeras implicate the short cytoplasmic tail and transmembrane domain in trafficking of the protein within the endoplasmic reticulum and Golgi network. Taken together, our findings provide a structural and cellular framework in which to explore the role of orf7a in SARS-CoV pathogenesis.

  6. Structural Basis of Intracellular TGF-β Signaling: Receptors and Smads.

    PubMed

    Chaikuad, Apirat; Bullock, Alex N

    2016-11-01

    Stimulation of the transforming growth factor β (TGF-β) family receptors activates an intracellular phosphorylation-dependent signaling cascade that culminates in Smad transcriptional activation and turnover. Structural studies have identified a number of allosteric mechanisms that control the localization, conformation, and oligomeric state of the receptors and Smads. Such mechanisms dictate the ordered binding of substrate and adaptor proteins that determine the directionality of the signaling process. Activation of the pathway has been illustrated by the various structures of the receptor-activated Smads (R-Smads) with SARA, Smad4, and YAP, respectively, whereas mechanisms of down-regulation have been elucidated by the structural complexes of FKBP12, Ski, and Smurf1. Interesting parallels have emerged between the R-Smads and the Forkhead-associated (FHA) and interferon regulatory factor (IRF)-associated domains, as well as the Hippo pathway. However, important questions remain as to the mechanism of Smad-independent signaling.

  7. Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria

    PubMed Central

    Benzerara, Karim; Skouri-Panet, Feriel; Li, Jinhua; Férard, Céline; Gugger, Muriel; Laurent, Thierry; Couradeau, Estelle; Ragon, Marie; Cosmidis, Julie; Menguy, Nicolas; Margaret-Oliver, Isabel; Tavera, Rosaluz; López-García, Purificación; Moreira, David

    2014-01-01

    Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Ca-carbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra- and extracellular Ca-carbonate biomineralization by cyanobacteria. PMID:25009182

  8. Intracellular glasses and seed survival in the dry state.

    PubMed

    Buitink, Julia; Leprince, Olivier

    2008-10-01

    So-called orthodox seeds can resist complete desiccation and survive the dry state for extended periods of time. During drying, the cellular viscosity increases dramatically and in the dry state, the cytoplasm transforms into a glassy state. The formation of intracellular glasses is indispensable to survive the dry state. Indeed, the storage stability of seeds is related to the packing density and molecular mobility of the intracellular glass, suggesting that the physico-chemical properties of intracellular glasses provide stability for long-term survival. Whereas seeds contain large amounts of soluble non-reducing sugars, which are known to be good glass formers, detailed in vivo measurements using techniques such as FTIR and EPR spectroscopy reveal that these intracellular glasses have properties that are quite different from those of simple sugar glasses. Intracellular glasses exhibit slow molecular mobility and a high molecular packing, resembling glasses made of mixtures of sugars with proteins, which potentially interact with additional cytoplasmic components such as salts, organic acids and amino acids. Above the glass transition temperature, the cytoplasm of biological systems still exhibits a low molecular mobility and a high stability, which serves as an ecological advantage, keeping the seeds stable under adverse conditions of temperature or water content that bring the tissues out of the glassy state.

  9. Relevance of intracellular polarity to accuracy of eukaryotic chemotaxis

    NASA Astrophysics Data System (ADS)

    Hiraiwa, Tetsuya; Nagamatsu, Akihiro; Akuzawa, Naohiro; Nishikawa, Masatoshi; Shibata, Tatsuo

    2014-10-01

    Eukaryotic chemotaxis is usually mediated by intracellular signals that tend to localize at the front or back of the cell. Such intracellular polarities frequently require no extracellular guidance cues, indicating that spontaneous polarization occurs in the signal network. Spontaneous polarization activity is considered relevant to the persistent motions in random cell migrations and chemotaxis. In this study, we propose a theoretical model that connects spontaneous intracellular polarity and motile ability in a chemoattractant solution. We demonstrate that the intracellular polarity can enhance the accuracy of chemotaxis. Chemotactic accuracy should also depend on chemoattractant concentration through the concentration-dependent correlation time in the polarity direction. Both the polarity correlation time and the chemotactic accuracy depend on the degree of responsiveness to the chemical gradient. We show that optimally accurate chemotaxis occurs at an intermediate responsiveness of intracellular polarity. Experimentally, we find that the persistence time of randomly migrating Dictyostelium cells depends on the chemoattractant concentration, as predicted by our theory. At the optimum responsiveness, this ameboid cell can enhance its chemotactic accuracy tenfold.

  10. Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria.

    PubMed

    Benzerara, Karim; Skouri-Panet, Feriel; Li, Jinhua; Férard, Céline; Gugger, Muriel; Laurent, Thierry; Couradeau, Estelle; Ragon, Marie; Cosmidis, Julie; Menguy, Nicolas; Margaret-Oliver, Isabel; Tavera, Rosaluz; López-García, Purificación; Moreira, David

    2014-07-29

    Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Ca-carbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra- and extracellular Ca-carbonate biomineralization by cyanobacteria.

  11. Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria

    NASA Astrophysics Data System (ADS)

    Benzerara, Karim; Skouri-Panet, Feriel; Li, Jinhua; Férard, Céline; Gugger, Muriel; Laurent, Thierry; Couradeau, Estelle; Ragon, Marie; Cosmidis, Julie; Menguy, Nicolas; Margaret-Oliver, Isabel; Tavera, Rosaluz; López-García, Purificación; Moreira, David

    2014-07-01

    Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Ca-carbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra- and extracellular Ca-carbonate biomineralization by cyanobacteria.

  12. High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila

    PubMed Central

    Chiaraviglio, Lucius

    2015-01-01

    Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche. PMID:26392509

  13. Exploring Anti-Bacterial Compounds against Intracellular Legionella

    PubMed Central

    Harrison, Christopher F.; Kicka, Sébastien; Trofimov, Valentin; Berschl, Kathrin; Ouertatani-Sakouhi, Hajer; Ackermann, Nikolaus; Hedberg, Christian; Cosson, Pierre; Soldati, Thierry; Hilbi, Hubert

    2013-01-01

    Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an ‘accidental’ human pathogen and cause a severe pneumonia known as Legionnaires’ disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target. PMID:24058631

  14. Intracellular Neural Recording with Pure Carbon Nanotube Probes

    PubMed Central

    Yoon, Inho; Hamaguchi, Kosuke; Borzenets, Ivan V.; Finkelstein, Gleb; Mooney, Richard; Donald, Bruce R.

    2013-01-01

    The computational complexity of the brain depends in part on a neuron’s capacity to integrate electrochemical information from vast numbers of synaptic inputs. The measurements of synaptic activity that are crucial for mechanistic understanding of brain function are also challenging, because they require intracellular recording methods to detect and resolve millivolt- scale synaptic potentials. Although glass electrodes are widely used for intracellular recordings, novel electrodes with superior mechanical and electrical properties are desirable, because they could extend intracellular recording methods to challenging environments, including long term recordings in freely behaving animals. Carbon nanotubes (CNTs) can theoretically deliver this advance, but the difficulty of assembling CNTs has limited their application to a coating layer or assembly on a planar substrate, resulting in electrodes that are more suitable for in vivo extracellular recording or extracellular recording from isolated cells. Here we show that a novel, yet remarkably simple, millimeter-long electrode with a sub-micron tip, fabricated from self-entangled pure CNTs can be used to obtain intracellular and extracellular recordings from vertebrate neurons in vitro and in vivo. This fabrication technology provides a new method for assembling intracellular electrodes from CNTs, affording a promising opportunity to harness nanotechnology for neuroscience applications. PMID:23840357

  15. Nanoparticles and intracellular applications of surface-enhanced Raman spectroscopy

    PubMed Central

    Taylor, Jack; Huefner, Anna; Li, Li; Wingfield, Jonathan

    2016-01-01

    Surface-enhanced Raman spectrocopy (SERS) offers ultrasensitive vibrational fingerprinting at the nanoscale. Its non-destructive nature affords an ideal tool for interrogation of the intracellular environment, detecting the localisation of biomolecules, delivery and monitoring of therapeutics and for characterisation of complex cellular processes at the molecular level. Innovations in nanotechnology have produced a wide selection of novel, purpose-built plasmonic nanostructures capable of high SERS enhancement for intracellular probing while microfluidic technologies are being utilised to reproducibly synthesise nanoparticle (NP) probes at large scale and in high throughput. Sophisticated multivariate analysis techniques unlock the wealth of previously unattainable biomolecular information contained within large and multidimensional SERS datasets. Thus, with suitable combination of experimental techniques and analytics, SERS boasts enormous potential for cell based assays and to expand our understanding of the intracellular environment. In this review we trace the pathway to utilisation of nanomaterials for intracellular SERS. Thus we review and assess nanoparticle synthesis methods, their toxicity and cell interactions before presenting significant developments in intracellular SERS methodologies and how identified challenges can be addressed. PMID:27479539

  16. Intracellular transport of fat-soluble vitamins A and E.

    PubMed

    Kono, Nozomu; Arai, Hiroyuki

    2015-01-01

    Vitamins are compounds that are essential for the normal growth, reproduction and functioning of the human body. Of the 13 known vitamins, vitamins A, D, E and K are lipophilic compounds and are therefore called fat-soluble vitamins. Because of their lipophilicity, fat-soluble vitamins are solubilized and transported by intracellular carrier proteins to exert their actions and to be metabolized properly. Vitamin A and its derivatives, collectively called retinoids, are solubilized by intracellular retinoid-binding proteins such as cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP) and cellular retinal-binding protein (CRALBP). These proteins act as chaperones that regulate the metabolism, signaling and transport of retinoids. CRALBP-mediated intracellular retinoid transport is essential for vision in human. α-Tocopherol, the main form of vitamin E found in the body, is transported by α-tocopherol transfer protein (α-TTP) in hepatic cells. Defects of α-TTP cause vitamin E deficiency and neurological disorders in humans. Recently, it has been shown that the interaction of α-TTP with phosphoinositides plays a critical role in the intracellular transport of α-tocopherol and is associated with familial vitamin E deficiency. In this review, we summarize the mechanisms and biological significance of the intracellular transport of vitamins A and E.

  17. Relevance of intracellular polarity to accuracy of eukaryotic chemotaxis.

    PubMed

    Hiraiwa, Tetsuya; Nagamatsu, Akihiro; Akuzawa, Naohiro; Nishikawa, Masatoshi; Shibata, Tatsuo

    2014-08-14

    Eukaryotic chemotaxis is usually mediated by intracellular signals that tend to localize at the front or back of the cell. Such intracellular polarities frequently require no extracellular guidance cues, indicating that spontaneous polarization occurs in the signal network. Spontaneous polarization activity is considered relevant to the persistent motions in random cell migrations and chemotaxis. In this study, we propose a theoretical model that connects spontaneous intracellular polarity and motile ability in a chemoattractant solution. We demonstrate that the intracellular polarity can enhance the accuracy of chemotaxis. Chemotactic accuracy should also depend on chemoattractant concentration through the concentration-dependent correlation time in the polarity direction. Both the polarity correlation time and the chemotactic accuracy depend on the degree of responsiveness to the chemical gradient. We show that optimally accurate chemotaxis occurs at an intermediate responsiveness of intracellular polarity. Experimentally, we find that the persistence time of randomly migrating Dictyostelium cells depends on the chemoattractant concentration, as predicted by our theory. At the optimum responsiveness, this ameboid cell can enhance its chemotactic accuracy tenfold.

  18. Quantitative intracellular magnetic nanoparticle uptake measured by live cell magnetophoresis

    PubMed Central

    Jing, Ying; Mal, Niladri; Williams, P. Stephen; Mayorga, Maritza; Penn, Marc S.; Chalmers, Jeffrey J.; Zborowski, Maciej

    2008-01-01

    Superparamagnetic iron oxide (SPIO) particles have been used successfully as an intracellular contrast agent for nuclear MRI cell tracking in vivo. We present a method of detecting intracellular SPIO colloid uptake in live cells using cell magnetophoresis, with potential applications in measuring intracellular MRI contrast uptake. The method was evaluated by measuring shifts in mean and distribution of the cell magnetophoretic mobility, and the concomitant changes in population frequency of the magnetically positive cells when compared to the unmanipulated negative control. Seven different transfection agent (TA) -SPIO complexes based on dendrimer, lipid, and polyethylenimine compounds were used as test standards, in combination with 3 different cell types: mesenchymal stem cells, cardiac fibroblasts, and cultured KG-1a hematopoietic stem cells. Transfectol (TRA) -SPIO incubation resulted in the highest frequency of magnetically positive cells (>90%), and Fugene 6 (FUG) -SPIO incubation the lowest, below that when using SPIO alone. A highly regular process of cell magnetophoresis was amenable to intracellular iron mass calculations. The results were consistent in all the cell types studied and with other reports. The cell magnetophoresis depends on the presence of high-spin iron species and is therefore expected to be directly related to the cell MRI contrast level.—Jing, Y., Mal, N., Williams, P. S., Mayorga, M., Penn, M. S., Chalmers, J. J., Zborowski, M. Quantitative intracellular magnetic nanoparticle uptake measured by live cell magnetophoresis. PMID:18725459

  19. Intracellular calcium levels can regulate Importin-dependent nuclear import

    SciTech Connect

    Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A.

    2014-07-18

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca{sup 2+} on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

  20. Innate Immunity to Intracellular Pathogens: Lessons Learned from Legionella pneumophila.

    PubMed

    Shin, Sunny

    2012-01-01

    Intracellular bacterial pathogens have the remarkable ability to manipulate host cell processes in order to establish a replicative niche within the host cell. In response, the host can initiate immune defenses that lead to the eventual restriction and clearance of intracellular infection. The bacterial pathogen Legionella pneumophila has evolved elaborate virulence mechanisms that allow for its survival inside protozoa within a specialized membrane-bound organelle. These strategies also enable L. pneumophila to survive and replicate within alveolar macrophages, and can result in the severe pneumonia Legionnaires' disease. Essential to L. pneumophila's intracellular lifestyle is a specialized type IV secretion system, termed Dot/Icm, that translocates bacterial effector proteins into host cells. The ease with which L. pneumophila can be genetically manipulated has facilitated the comparison of host responses to virulent and isogenic avirulent mutants lacking a functional Dot/Icm system. This has made L. pneumophila an excellent model for understanding how the host discriminates between pathogenic and nonpathogenic bacteria and for systematically dissecting host defense mechanisms against intracellular pathogens. In this chapter, I discuss a few examples demonstrating how the study of immune responses triggered specifically by the L. pneumophila type IV secretion system has provided unique insight into our understanding of host immunity against intracellular bacterial pathogens.

  1. Axion domain wall baryogenesis

    SciTech Connect

    Daido, Ryuji; Kitajima, Naoya; Takahashi, Fuminobu

    2015-07-28

    We propose a new scenario of baryogenesis, in which annihilation of axion domain walls generates a sizable baryon asymmetry. Successful baryogenesis is possible for a wide range of the axion mass and decay constant, m≃10{sup 8}–10{sup 13} GeV and f≃10{sup 13}–10{sup 16} GeV. Baryonic isocurvature perturbations are significantly suppressed in our model, in contrast to various spontaneous baryogenesis scenarios in the slow-roll regime. In particular, the axion domain wall baryogenesis is consistent with high-scale inflation which generates a large tensor-to-scalar ratio within the reach of future CMB B-mode experiments. We also discuss the gravitational waves produced by the domain wall annihilation and its implications for the future gravitational wave experiments.

  2. The intracellular citrus huanglongbing bacterium, 'Candidatus Liberibacter asiaticus' encodes two novel autotransporters.

    PubMed

    Hao, Guixia; Boyle, Michael; Zhou, Lijuan; Duan, Yongping

    2013-01-01

    Proteins secreted by the type V secretion system (T5SS), known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. In this study, we characterized two novel autotransporter proteins of 'Candidatus Liberibacter asiaticus' (Las), and redesignated them as LasAI and LasAII in lieu of the previous names HyvI and HyvII. As a phloem-limited, intracellular bacterial pathogen, Las has a significantly reduced genome and causes huanglongbing (HLB), a devastating disease of citrus worldwide. Bioinformatic analyses revealed that LasAI and LasAII share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasAI C-terminus and the full length LasAII were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of a typical signal peptide, LasAI was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasAI protein. Its surface localization was also confirmed by the removal of the LasAI antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasAI translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria when expressed in the plants. These findings may lead to a better understanding of the pathogenesis of this intracellular bacterium.

  3. Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment

    PubMed Central

    Jura, Natalia; Endres, Nicholas F.; Engel, Kate; Deindl, Sebastian; Das, Rahul; Lamers, Meindert H.; Wemmer, David E.; Zhang, Xuewu; Kuriyan, John

    2009-01-01

    Signaling by the epidermal growth factor receptor requires an allosteric interaction between the kinase domains of two receptors, whereby one activates the other. We show that the intracellular juxtamembrane segment of the receptor, known to potentiate kinase activity, is able to dimerize the kinase domains. The C-terminal half of the juxtamembrane segment latches the activated kinase domain to the activator, and the N-terminal half of this segment further potentiates dimerization, most likely by forming an antiparallel helical dimer that engages the transmembrane helices of the activated receptor. Our data are consistent with a mechanism in which the extracellular domains block the intrinsic ability of the transmembrane and cytoplasmic domains to dimerize and activate, with ligand binding releasing this block. The formation of the activating juxtamembrane latch is prevented by the C-terminal tails in a new structure of an inactive kinase domain dimer, suggesting how alternative dimers can prevent ligand-independent activation. PMID:19563760

  4. Optimal domain decomposition strategies

    NASA Technical Reports Server (NTRS)

    Yoon, Yonghyun; Soni, Bharat K.

    1995-01-01

    The primary interest of the authors is in the area of grid generation, in particular, optimal domain decomposition about realistic configurations. A grid generation procedure with optimal blocking strategies has been developed to generate multi-block grids for a circular-to-rectangular transition duct. The focus of this study is the domain decomposition which optimizes solution algorithm/block compatibility based on geometrical complexities as well as the physical characteristics of flow field. The progress realized in this study is summarized in this paper.

  5. Quantifying intracellular hydrogen peroxide perturbations in terms of concentration

    PubMed Central

    Huang, Beijing K.; Sikes, Hadley D.

    2014-01-01

    Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS) in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies. PMID:25460730

  6. An intracellularly activatable, fluorogenic probe for cancer imaging.

    PubMed

    Tian, Ruisong; Li, Mingjie; Wang, Jin; Yu, Min; Kong, Xiuqi; Feng, Yupeng; Chen, Zeming; Li, Yuxi; Huang, Weiqiang; Wu, Wenjie; Hong, Zhangyong

    2014-08-07

    A newly designed, dual-functional probe based on intracellular activation has been successfully developed for the detection of cancer cells. The probe is nearly non-fluorescent in buffer due to its highly efficient FRET quenching, but it can be specifically activated with dramatic fluorescence enhancement upon intracellular cathepsin B cleavage in target cancer cells after selective internalization via folate receptor-dependent endocytosis. Therefore, this probe enables "turn-on" visualization of cancer cells with desirable specificity and contrast enhancement. This targeted, intracellularly activatable probe exhibits low fluorescence-quenched background when compared with "always-on" probes and avoids non-specific activation by non-specifically expressed enzymes in normal tissue, which normally occurs when using common "turn on" probe design strategies. Therefore, this probe can be potentially applied in intraoperative inspection during clinical cancer surgery with higher contrast and sensitivity.

  7. Neuronal Recordings with Solid-Conductor Intracellular Nanoelectrodes (SCINEs)

    PubMed Central

    Angle, Matthew R.; Schaefer, Andreas T.

    2012-01-01

    Direct electrical recording of the neuronal transmembrane potential has been crucial to our understanding of the biophysical mechanisms subserving neuronal computation. Existing intracellular recording techniques, however, limit the accuracy and duration of such measurements by changing intracellular biochemistry and/or by damaging the plasma membrane. Here we demonstrate that nanoengineered electrodes can be used to record neuronal transmembrane potentials in brain tissue without causing these physiological perturbations. Using focused ion beam milling, we have fabricated Solid-Conductor Intracellular NanoElectrodes (SCINEs), from conventional tungsten microelectrodes. SCINEs have tips that are <300 nm in diameter for several micrometers, but can be easily handled and can be inserted into brain tissue. Performing simultaneous whole-cell patch recordings, we show that SCINEs can record action potentials (APs) as well as slower, subthreshold neuronal potentials without altering cellular properties. These results show a key role for nanotechnology in the development of new electrical recording techniques in neuroscience. PMID:22905231

  8. Direct Determination of the Intracellular Oxidation State of Plutonium

    PubMed Central

    Gorman-Lewis, Drew; Aryal, Baikuntha P.; Paunesku, Tatjana; Vogt, Stefan; Lai, Barry; Woloschak, Gayle E.; Jensen, Mark P.

    2013-01-01

    Microprobe X-ray absorption near edge structure (μ-XANES) measurements were used to determine directly, for the first time, the oxidation state of intracellular plutonium in individual 0.1 μm2 areas within single rat pheochromocytoma cells (PC12). The living cells were incubated in vitro for 3 hours in the presence of Pu added to the media in different oxidation states (Pu(III), Pu(IV), and Pu(VI)) and in different chemical forms. Regardless of the initial oxidation state or chemical form of Pu presented to the cells, the XANES spectra of the intracellular Pu deposits was always consistent with tetravalent Pu even though the intracellular milieu is generally reducing. PMID:21755934

  9. Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.

    PubMed

    Lehar, Sophie M; Pillow, Thomas; Xu, Min; Staben, Leanna; Kajihara, Kimberly K; Vandlen, Richard; DePalatis, Laura; Raab, Helga; Hazenbos, Wouter L; Morisaki, J Hiroshi; Kim, Janice; Park, Summer; Darwish, Martine; Lee, Byoung-Chul; Hernandez, Hilda; Loyet, Kelly M; Lupardus, Patrick; Fong, Rina; Yan, Donghong; Chalouni, Cecile; Luis, Elizabeth; Khalfin, Yana; Plise, Emile; Cheong, Jonathan; Lyssikatos, Joseph P; Strandh, Magnus; Koefoed, Klaus; Andersen, Peter S; Flygare, John A; Wah Tan, Man; Brown, Eric J; Mariathasan, Sanjeev

    2015-11-19

    Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

  10. Intracellular activity of azithromycin against bacterial enteric pathogens.

    PubMed Central

    Rakita, R M; Jacques-Palaz, K; Murray, B E

    1994-01-01

    Azithromycin, a new azalide antibiotic, is active in vitro against a variety of enteric bacterial pathogens. Since it is concentrated inside human neutrophils and other cells, it might be particularly useful in the treatment of infections caused by enteropathogens that invade host tissues. The intracellular activity of azithromycin against several enteric pathogens that had been phagocytosed by neutrophils was determined. Azithromycin was effective in reducing the intracellular viabilities of almost all strains tested, including representative strains of Salmonella, Shigella, and enteroinvasive, enteropathogenic, enterotoxigenic, and enterohemorrhagic Escherichia coli. Erythromycin was also effective in this model system, although azithromycin was generally more effective than erythromycin against strains of invasive enteric pathogens. Cefotaxime reduced intracellular bacterial viability to a lesser extent than either azithromycin or erythromycin. The presence of neutrophils did not significantly affect the activity of azithromycin in this system. Azithromycin may be a useful agent for the treatment of bacterial diarrhea, and clinical trials should be considered. PMID:7810998

  11. Role of the Extracellular and Intracellular Loops of Follicle-Stimulating Hormone Receptor in Its Function

    PubMed Central

    Banerjee, Antara A.; Mahale, Smita D.

    2015-01-01

    Follicle-stimulating hormone receptor (FSHR) is a leucine-rich repeat containing class A G-protein coupled receptor belonging to the subfamily of glycoprotein hormone receptors (GPHRs), which includes luteinizing hormone/choriogonadotropin receptor (LH/CGR) and thyroid-stimulating hormone receptor. Its cognate ligand, follicle-stimulating hormone binds to, and activates FSHR expressed on the surface of granulosa cells of the ovary, in females, and Sertoli cells of the testis, in males, to bring about folliculogenesis and spermatogenesis, respectively. FSHR contains a large extracellular domain (ECD) consisting of leucine-rich repeats at the N-terminal end and a hinge region at the C-terminus that connects the ECD to the membrane spanning transmembrane domain (TMD). The TMD consists of seven α-helices that are connected to each other by means of three extracellular loops (ELs) and three intracellular loops (ILs) and ends in a short-cytoplasmic tail. It is well established that the ECD is the primary hormone binding domain, whereas the TMD is the signal transducing domain. However, several studies on the ELs and ILs employing site directed mutagenesis, generation of chimeric receptors and in vitro characterization of naturally occurring mutations have proven their indispensable role in FSHR function. Their role in every phase of the life cycle of the receptor like post translational modifications, cell surface trafficking, hormone binding, activation of downstream signaling, receptor phosphorylation, hormone–receptor internalization, and recycling of hormone–receptor complex have been documented. Mutations in the loops causing dysregulation of these processes lead to pathophysiological conditions. In other GPHRs as well, the loops have been convincingly shown to contribute to various aspects of receptor function. This review article attempts to summarize the extensive contributions of FSHR loops and C-terminal tail to its function. PMID:26236283

  12. Regulatory Conformational Coupling between CLC Anion Channel Membrane and Cytoplasmic Domains.

    PubMed

    Yamada, Toshiki; Strange, Kevin

    2016-11-01

    CLC anion channels are homodimeric proteins. Each subunit is comprised of 18 α-helices designated "A-R" and an intracellular carboxy-terminus containing two cystathionine-β-synthase (CBS1 and CBS2) domains. Conformational coupling between membrane and intracellular domains via poorly understood mechanisms is required for CLC regulation. The activity of the C. elegans CLC channel CLH-3b is reduced by phosphorylation of a carboxy-terminus "activation domain," which disrupts its interaction with CBS domains. CBS2 interfaces with a short intracellular loop, the H-I loop, connecting membrane helices H and I. Alanine mutation of a conserved H-I loop tyrosine residue, Y232, prevents regulation demonstrating that the loop functions to couple phosphorylation-dependent CBS domain conformational changes to channel membrane domains. To gain further insight into the mechanisms of this coupling, we mutated conserved amino acid residues in membrane helices H and I. Only mutation of the H-helix valine residue V228 to leucine prevented phosphorylation-dependent channel regulation. Structural and functional studies of other CLC proteins suggest that V228 may interact with Y529, a conserved R-helix tyrosine residue that forms part of the CLC ion conduction pathway. Mutation of Y529 to alanine also prevented CLH-3b regulation. Intracellular application of the sulfhydryl reactive reagent MTSET using CLH-3b channels engineered with single-cysteine residues in CBS2 indicate that V228L, Y529A, and Y232A disrupt putative regulatory intracellular conformational changes. Extracellular Zn(2+) inhibits CLH-3b and alters the effects of intracellular MTSET on channel activity. The effects of Zn(2+) are disrupted by V228L, Y529A, and Y232A. Collectively, our findings indicate that there is conformational coupling between CBS domains and the H and R membrane helices mediated by the H-I loop. We propose a simple model by which conformational changes in H and R helices mediate CLH-3b regulation

  13. Phenotypic lentivirus screens to identify functional single domain antibodies

    PubMed Central

    Schmidt, Florian I.; Hanke, Leo; Morin, Benjamin; Brewer, Rebeccah; Brusic, Vesna; Whelan, Sean P.J.; Ploegh, Hidde L.

    2016-01-01

    Manipulation of proteins is key in assessing their in vivo function. While genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway. PMID:27573105

  14. Danger signals, inflammasomes, and the intricate intracellular lives of chlamydiae.

    PubMed

    Pettengill, Matthew A; Abdul-Sater, Ali; Coutinho-Silva, Robson; Ojcius, David M

    2016-10-01

    Chlamydiae are obligate intracellular bacterial pathogens, and as such are sensitive to alterations in the cellular physiology of their hosts. Chlamydial infections often cause pathologic consequences due to prolonged localized inflammation. Considerable advances have been made in the last few years regarding our understanding of how two key inflammation-associated signaling pathways influence the biology of Chlamydia infections: inflammation regulating purinergic signaling pathways significantly impact intracellular chlamydial development, and inflammasome activation modulates both chlamydial growth and infection mediated pro-inflammatory cytokine production. We review here elements of both pathways, presenting the latest developments contributing to our understanding of how chlamydial infections are influenced by inflammasomes and purinergic signaling.

  15. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms

    PubMed Central

    Mansilla Pareja, Maria Eugenia; Colombo, Maria I.

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance. PMID:24137567

  16. In vitro and ex vivo strategies for intracellular delivery

    NASA Astrophysics Data System (ADS)

    Stewart, Martin P.; Sharei, Armon; Ding, Xiaoyun; Sahay, Gaurav; Langer, Robert; Jensen, Klavs F.

    2016-10-01

    Intracellular delivery of materials has become a critical component of genome-editing approaches, ex vivo cell-based therapies, and a diversity of fundamental research applications. Limitations of current technologies motivate development of next-generation systems that can deliver a broad variety of cargo to diverse cell types. Here we review in vitro and ex vivo intracellular delivery approaches with a focus on mechanisms, challenges and opportunities. In particular, we emphasize membrane-disruption-based delivery methods and the transformative role of nanotechnology, microfluidics and laboratory-on-chip technology in advancing the field.

  17. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms.

    PubMed

    Pareja, Maria Eugenia Mansilla; Colombo, Maria I

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance.

  18. Cellulose binding domain proteins

    SciTech Connect

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  19. The Domains of TESOL.

    ERIC Educational Resources Information Center

    Robinett, Betty Wallace

    The domains of Teachers of English to Speakers of Other Languages (TESOL) are those spheres of concern involving persons who speak languages other than English or dialects of English other than the standard. This clientele has been classified traditionally in terms of programs in English as a foreign language, English as a second language, English…

  20. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  1. Domain Validity and Generalizability

    ERIC Educational Resources Information Center

    Kaiser, Henry F.; Michael, William B.

    1975-01-01

    An alternative derivation of Tryon's basic formula for the coefficient of domain validity or the coefficient of generalizability developed by Cronbach, Rajaratnam, and Glaser is provided. This derivation, which is also the generalized Kuder-Richardson coefficient, requires a relatively minimal number of assumptions compared with that in previously…

  2. BAR domain proteins regulate Rho GTPase signaling

    PubMed Central

    Aspenström, Pontus

    2014-01-01

    BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis. PMID:25483303

  3. Remote homology between Munc13 MUN domain and vesicle tethering complexes

    PubMed Central

    Pei, Jimin; Ma, Cong; Rizo, Josep; Grishin, Nick V.

    2011-01-01

    Most core components of the neurotransmitter release machinery have homologues in other types of intracellular membrane traffic, likely underlying a universal mechanism of intracellular membrane fusion. However, no clear similarity between Munc13s and protein families generally involved in membrane traffic has been reported, despite the essential nature of Munc13s for neurotransmitter release. This crucial function was ascribed to a minimal Munc13 region called the MUN domain, which likely participates in SNARE complex assembly and is also found in CAPS. We have now used comparative sequence and structural analyses to study the structure and evolutionary origin of the MUN domain. We found weak, yet significant sequence similarities between the MUN domain and a set of protein subunits from several related vesicle tethering complexes, such as Sec6 from the exocyst complex and Vps53 from the GARP complex. Such an evolutionary relationship allows structure prediction of the MUN domain and suggests functional similarities between MUN domain-containing proteins and multisubunit tethering complexes such as exocyst, COG, GARP and Dsl1p. These findings further unify the mechanism of neurotransmitter release with those of other types of intracellular membrane traffic, and in turn support a role for tethering complexes in SNARE complex assembly. PMID:19563813

  4. Magnetic domain and domain wall in Co/Pt multilayer

    NASA Astrophysics Data System (ADS)

    Talapatra, A.; Mohanty, J.

    2016-05-01

    We report systematic micromagnetic investigation of formation of magnetic domains in perpendicularly magnetized Co/Pt multilayer with the variation in magnetic anisotropy and stack thickness. The lowering of anisotropy makes the domain wall broader and domain formation less efficient. Domain sizeincreases with increasing thickness of the stack to minimize the stray field energy.The minimization of energy of the system due to domain formation makes the M-H loop narrower whereas, lower stack thickness results in a wider loop. The magnetization reversalin this system occurs due tothe nucleation and growth of reverse domains.

  5. Cholesterol and the interaction of proteins with membrane domains.

    PubMed

    Epand, Richard M

    2006-07-01

    Cholesterol is not uniformly distributed in biological membranes. One of the factors influencing the formation of cholesterol-rich domains in membranes is the unequal lateral distribution of proteins in membranes. Certain proteins are found in cholesterol-rich domains. In some of these cases, it is as a consequence of the proteins interacting directly with cholesterol. There are several structural features of a protein that result in the protein preferentially associating with cholesterol-rich domains. One of the best documented of these is certain types of lipidations. In addition, however, there are segments of a protein that can preferentially sequester cholesterol. We discuss two examples of these cholesterol-recognition elements: the cholesterol recognition/interaction amino acid consensus (CRAC) domain and the sterol-sensing domain (SSD). The requirements for a CRAC motif are quite flexible and predict that a large number of sequences could recognize cholesterol. There are, however, certain proteins that are known to interact with cholesterol-rich domains of cell membranes that have CRAC motifs, and synthetic peptides corresponding to these segments also promote the formation of cholesterol-rich domains. Modeling studies have provided a rationale for certain requirements of the CRAC motif. The SSD is a larger protein segment comprising five transmembrane domains. The amino acid sequence YIYF is found in several SSD and in certain other proteins for which there is evidence that they interact with cholesterol-rich domains. The CRAC sequences as well as YIYF are generally found adjacent to a transmembrane helical segment. These regions appear to have a strong influence of the localization of certain proteins into domains in biological membranes. In addition to the SSD, there is also a domain found in soluble proteins, the START domain, that binds lipids. Certain proteins with START domains specifically bind cholesterol and are believed to function in

  6. Imaging atrial arrhythmic intracellular calcium in intact heart

    PubMed Central

    Xie, Wenjun; Santulli, Gaetano; Guo, Xiaoxiao; Gao, Melanie; Chen, Bi-Xing; Marks, Andrew R.

    2014-01-01

    Abnormalities in intracellular Ca2+ signaling have been proposed to play an essential role in the pathophysiology of atrial arrhythmias. However, a direct observation of intracellular Ca2+ in atrial myocytes during atrial arrhythmias is lacking. Here, we have developed an ex vivo model of simultaneous Ca2+ imaging and electrocardiographic recording in cardiac atria. Using this system we were able to record atrial arrhythmic intracellular Ca2+ activities. Our results indicate that atrial arrhythmias can be tightly linked to intracellular Ca2+ waves and Ca2+ alternans. Moreover, we applied this strategy to analyze Ca2+ signals in the hearts of WT and knock-in mice harboring a ‘leaky’ type 2 ryanodine receptor (RyR2-R2474S). We showed that sarcoplasmic reticulum (SR) Ca2+ leak increases the susceptibility to Ca2+ alternans and Ca2+ waves increasing the incidence of atrial arrhythmias. Reduction of SR Ca2+ leak via RyR2 by acute treatment with S107 reduced both Ca2+ alternans and Ca2+ waves, and prevented atrial arrhythmias. PMID:24041536

  7. Controls of Intracellular Communication Mediated by Gap Junctions

    NASA Technical Reports Server (NTRS)

    Bennett, M. V. L.

    1983-01-01

    Experiments were done using aequorin and no increase in aequorin luminescence during acidification adequate to uncouple cells was seen. The pH sensitivity of the conductance of the perfused membrane was essentially the same as that observed with intracellular pH microelectrodes.

  8. Monitoring intracellular oxidative events using dynamic spectral unmixing microscopy

    EPA Science Inventory

    There is increasing interest in using live-cell imaging to monitor not just individual intracellular endpoints, but to investigate the interplay between multiple molecular events as they unfold in real time within the cell. A major impediment to simultaneous acquisition of multip...

  9. Activity of 10 antimicrobial agents against intracellular Rhodococcus equi.

    PubMed

    Giguère, Steeve; Berghaus, Londa J; Lee, Elise A

    2015-08-05

    Studies with facultative intracellular bacterial pathogens have shown that evaluation of the bactericidal activity of antimicrobial agents against intracellular bacteria is more closely associated with in vivo efficacy than traditional in vitro susceptibility testing. The objective of this study was to determine the relative activity of 10 antimicrobial agents against intracellular Rhodococcus equi. Equine monocyte-derived macrophages were infected with virulent R. equi and exposed to erythromycin, clarithromycin, azithromycin, rifampin, ceftiofur, gentamicin, enrofloxacin, vancomycin, imipenem, or doxycycline at concentrations achievable in plasma at clinically recommended dosages in foals. The number of intracellular R. equi was determined 48h after infection by counting colony forming units (CFUs). The number of R. equi CFUs in untreated control wells were significantly higher than those of monolayers treated with antimicrobial agents. Numbers of R. equi were significantly lower in monolayers treated with enrofloxacin followed by those treated with gentamicin, and vancomycin, when compared to monolayers treated with other antimicrobial agents. Numbers of R. equi in monolayers treated with doxycycline were significantly higher than those of monolayers treated with other antimicrobial agents. Differences in R. equi CFUs between monolayers treated with other antimicrobial agents were not statistically significant. Enrofloxacin, gentamicin, and vancomycin are the most active drugs in equine monocyte-derived macrophages infected with R. equi. Additional studies will be needed to determine if these findings correlate with in vivo efficacy.

  10. Nutrient salvaging and metabolism by the intracellular pathogen Legionella pneumophila.

    PubMed

    Fonseca, Maris V; Swanson, Michele S

    2014-01-01

    The Gram-negative bacterium Legionella pneumophila is ubiquitous in freshwater environments as a free-swimming organism, resident of biofilms, or parasite of protozoa. If the bacterium is aerosolized and inhaled by a susceptible human host, it can infect alveolar macrophages and cause a severe pneumonia known as Legionnaires' disease. A sophisticated cell differentiation program equips L. pneumophila to persist in both extracellular and intracellular niches. During its life cycle, L. pneumophila alternates between at least two distinct forms: a transmissive form equipped to infect host cells and evade lysosomal degradation, and a replicative form that multiplies within a phagosomal compartment that it has retooled to its advantage. The efficient changeover between transmissive and replicative states is fundamental to L. pneumophila's fitness as an intracellular pathogen. The transmission and replication programs of L. pneumophila are governed by a number of metabolic cues that signal whether conditions are favorable for replication or instead trigger escape from a spent host. Several lines of experimental evidence gathered over the past decade establish strong links between metabolism, cellular differentiation, and virulence of L. pneumophila. Herein, we focus on current knowledge of the metabolic components employed by intracellular L. pneumophila for cell differentiation, nutrient salvaging and utilization of host factors. Specifically, we highlight the metabolic cues that are coupled to bacterial differentiation, nutrient acquisition systems, and the strategies utilized by L. pneumophila to exploit host metabolites for intracellular replication.

  11. Intracellular GTP level determines cell's fate toward differentiation and apoptosis

    SciTech Connect

    Meshkini, Azadeh; Yazdanparast, Razieh Nouri, Kazem

    2011-06-15

    Since the adequate supply of guanine nucleotides is vital for cellular activities, limitation of their syntheses would certainly result in modulation of cellular fate toward differentiation and apoptosis. The aim of this study was to set a correlation between the intracellular level of GTP and the induction of relevant signaling pathways involved in the cell's fate toward life or death. In that regard, we measured the GTP level among human leukemia K562 cells exposed to mycophenolic acid (MPA) or 3-hydrogenkwadaphnin (3-HK) as two potent inosine monophosphate dehydrogenase inhibitors. Our results supported the maturation of the cells when the intracellular GTP level was reduced by almost 30-40%. Under these conditions, 3-HK and/or MPA caused up-regulation of PKC{alpha} and PI3K/AKT pathways. Furthermore, co-treatment of cells with hypoxanthine plus 3-HK or MPA, which caused a reduction of about 60% in the intracellular GTP levels, led to apoptosis and activation of mitochondrial pathways through inverse regulation of Bcl-2/Bax expression and activation of caspase-3. Moreover, our results demonstrated that attenuation of GTP by almost 60% augmented the intracellular ROS and nuclear localization of p21 and subsequently led to cell death. These results suggest that two different threshold levels of GTP are needed for induction of differentiation and/or ROS-associated apoptosis. - Graphical abstract: Display Omitted

  12. Dynamic Reorganization of Metabolic Enzymes into Intracellular Bodies

    PubMed Central

    O’Connell, Jeremy D.; Zhao, Alice; Ellington, Andrew D.; Marcotte, Edward M.

    2013-01-01

    Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci—such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis—to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality—and dysfunctionality—of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable. PMID:23057741

  13. Imaging atrial arrhythmic intracellular calcium in intact heart.

    PubMed

    Xie, Wenjun; Santulli, Gaetano; Guo, Xiaoxiao; Gao, Melanie; Chen, Bi-Xing; Marks, Andrew R

    2013-11-01

    Abnormalities in intracellular Ca(2+) signaling have been proposed to play an essential role in the pathophysiology of atrial arrhythmias. However, a direct observation of intracellular Ca(2+) in atrial myocytes during atrial arrhythmias is lacking. Here, we have developed an ex vivo model of simultaneous Ca(2+) imaging and electrocardiographic recording in cardiac atria. Using this system we were able to record atrial arrhythmic intracellular Ca(2+) activities. Our results indicate that atrial arrhythmias can be tightly linked to intracellular Ca(2+) waves and Ca(2+) alternans. Moreover, we applied this strategy to analyze Ca(2+) signals in the hearts of WT and knock-in mice harboring a 'leaky' type 2 ryanodine receptor (RyR2-R2474S). We showed that sarcoplasmic reticulum (SR) Ca(2+) leak increases the susceptibility to Ca(2+) alternans and Ca(2+) waves increasing the incidence of atrial arrhythmias. Reduction of SR Ca(2+) leak via RyR2 by acute treatment with S107 reduced both Ca(2+) alternans and Ca(2+) waves, and prevented atrial arrhythmias.

  14. Novel Waddlia Intracellular Bacterium in Artibeus intermedius Fruit Bats, Mexico

    PubMed Central

    Pierlé, Sebastián Aguilar; Morales, Cirani Obregón; Martínez, Leonardo Perea; Ceballos, Nidia Aréchiga; Rivero, Juan José Pérez; Díaz, Osvaldo López; Brayton, Kelly A.

    2015-01-01

    An intracellular bacterium was isolated from fruit bats (Artibeus intermedius) in Cocoyoc, Mexico. The bacterium caused severe lesions in the lungs and spleens of bats and intracytoplasmic vacuoles in cell cultures. Sequence analyses showed it is related to Waddlia spp. (order Chlamydiales). We propose to call this bacterium Waddlia cocoyoc. PMID:26583968

  15. Poly-arginine conjugated triarylmethyl radical as intracellular spin label.

    PubMed

    Driesschaert, Benoit; Bobko, Andrey A; Eubank, Timothy D; Samouilov, Alexandre; Khramtsov, Valery V; Zweier, Jay L

    2016-04-01

    Stable triarylmethyl radicals are ideal spin labels used for biomedical electron paramagnetic resonance applications. Previously reported structures exhibit polar charged functions for water solubilization preventing them from crossing the cell membrane. We report the synthesis of a triarylmethyl radical conjugated to poly-arginine peptide allowing intracellular delivery of the paramagnetic label.

  16. Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

    PubMed Central

    Abe, Tomoko; Kobayashi, Teruyuki; Saito, Terumi

    2005-01-01

    A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases. PMID:16199568

  17. Properties of a novel intracellular poly(3-hydroxybutyrate) depolymerase with high specific activity (PhaZd) in Wautersia eutropha H16.

    PubMed

    Abe, Tomoko; Kobayashi, Teruyuki; Saito, Terumi

    2005-10-01

    A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.

  18. Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells*

    PubMed Central

    Traenkle, Bjoern; Emele, Felix; Anton, Roman; Poetz, Oliver; Haeussler, Ragna S.; Maier, Julia; Kaiser, Philipp D.; Scholz, Armin M.; Nueske, Stefan; Buchfellner, Andrea; Romer, Tina; Rothbauer, Ulrich

    2015-01-01

    β-catenin is the key component of the canonical Wnt pathway and plays a crucial role in a multitude of developmental and homeostatic processes. The different tasks of β-catenin are orchestrated by its subcellular localization and participation in multiprotein complexes. To gain a better understanding of β-catenin's role in living cells we have generated a new set of single domain antibodies, referred to as nanobodies, derived from heavy chain antibodies of camelids. We selected nanobodies recognizing the N-terminal, core or C-terminal domain of β-catenin and applied these new high-affinity binders as capture molecules in sandwich immunoassays and co-immunoprecipitations of endogenous β-catenin complexes. In addition, we engineered intracellularly functional anti-β-catenin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. For the first time, we were able to visualize the subcellular localization and nuclear translocation of endogenous β-catenin in living cells using these chromobodies. Moreover, the chromobody signal allowed us to trace the accumulation of diffusible, hypo-phosphorylated β-catenin in response to compound treatment in real time using High Content Imaging. The anti-β-catenin nanobodies and chromobodies characterized in this study are versatile tools that enable a novel and unique approach to monitor the dynamics of subcellular β-catenin in biochemical and cell biological assays. PMID:25595278

  19. Assembly and Intracellular Targeting of the βγ Subunits of Heterotrimeric G Proteins

    PubMed Central

    Rehm, Armin; Ploegh, Hidde L.

    1997-01-01

    The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent α, β, and γ subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact. Monoclonal anti-β antibodies (ARC5 and ARC9), raised against immunoaffinity purified βγ complexes, recognize β subunits when not associated with γ and can thus be used to monitor assembly of βγ complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of βγ complexes with the plasma membrane fraction starts between 15–30 min of chase. Three pools of β subunits can be distinguished based on their association with γ subunits, their localization, and their detergent solubility. Association of β and α subunits with detergent-insoluble domains occurs within 1 min of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase. Brefeldin A treatment does not interfere with delivery of βγ subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane. PMID:9128244

  20. Molecular insights into Adgra2/Gpr124 and Reck intracellular trafficking

    PubMed Central

    Bostaille, Naguissa; Gauquier, Anne; Twyffels, Laure

    2016-01-01

    ABSTRACT Adgra2, formerly known as Gpr124, is a key regulator of cerebrovascular development in vertebrates. Together with the GPI-anchored glycoprotein Reck, this adhesion GPCR (aGPCR) stimulates Wnt7-dependent Wnt/β-catenin signaling to promote brain vascular invasion in an endothelial cell-autonomous manner. Adgra2 and Reck have been proposed to assemble a receptor complex at the plasma membrane, but the molecular modalities of their functional synergy remain to be investigated. In particular, as typically found in aGPCRs, the ectodomain of Adgra2 is rich in protein-protein interaction motifs whose contributions to receptor function are unknown. In opposition to the severe ADGRA2 genetic lesions found in previously generated zebrafish and mouse models, the zebrafish ouchless allele encodes an aberrantly-spliced and inactive receptor lacking a single leucine-rich repeat (LRR) unit within its N-terminus. By characterizing this allele we uncover that, in contrast to all other extracellular domains, the precise composition of the LRR domain determines proper receptor trafficking to the plasma membrane. Using CRISPR/Cas9 engineered cells, we further show that Adgra2 trafficking occurs in a Reck-independent manner and that, similarly, Reck reaches the plasma membrane irrespective of Adgra2 expression or localization, suggesting that the partners meet at the plasma membrane after independent intracellular trafficking events. PMID:27979830

  1. IMPDH2 Is an Intracellular Target of the Cyclophilin A and Sanglifehrin A Complex.

    PubMed

    Pua, Khian Hong; Stiles, Dylan T; Sowa, Mathew E; Verdine, Gregory L

    2017-01-10

    Natural products have demonstrated utility in the clinic and can also act as probes to understand complex cellular pathways. Sanglifehrin A (SFA) is a mixed polyketide and non-ribosomal peptide synthase natural product with sub-nano-molar affinity for its receptor cyclophilin A (PPIA). It has been shown to behave in vitro as an immune suppressant. Here, we identify inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) as an intracellular target of the PPIA-SFA binary complex. The formation of this ternary complex does not inhibit the enzymatic activity of IMPDH2. Rather, ternary complex formation modulates cell growth through interaction with the cystathionine-β-synthase (CBS) domain of IMPDH2. We further demonstrate that the SFA complex is highly isoform selective for IMPDH2 (versus IMPDH1). This work reveals a role for the CBS domains of IMPDH2 in cellular proliferation, suggesting a more complex role than previously suspected for IMPDH2 in T cell activation and proliferation.

  2. Intracellular trafficking of VP22 in bovine herpesvirus-1 infected cells

    SciTech Connect

    Lobanov, Vladislav A.; Babiuk, Lorne A.; Drunen Littel-van den Hurk, Sylvia van

    2010-01-20

    The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion proteins expressed by bovine herpesvirus-1 (BHV-1) recombinants was examined by live-cell imaging. Our results demonstrate that (i) the fusion of EYFP to the C terminus of VP22 does not alter the trafficking of the protein in infected cells, (ii) VP22 expressed during BHV-1 infection translocates to the nucleus through three different pathways, namely early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage, (iii) the addition of the SV40 large-T-antigen nuclear localization signal (NLS) to VP22-EYFP abrogates its early cytoplasmic stage, and (iv) the VP22 {sup 131}PRPR{sup 134} NLS is not required for the late massive nuclear translocation of the protein, but this motif is essential for the targeting of VP22 to discrete dot-like nuclear domains during the early cytoplasmic stage. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection and suggest that the early pathways of VP22 nuclear accumulation may be more relevant to the infection process as the late massive nuclear influx starts when most of the viral progeny has already emerged from the cell.

  3. Single-cell intracellular nano-pH probes.

    PubMed

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

  4. Maintenance of skeletal muscle intracellular glutamine during standard surgical trauma.

    PubMed

    Kapadia, C R; Colpoys, M F; Jiang, Z M; Johnson, D J; Smith, R J; Wilmore, D W

    1985-01-01

    Skeletal muscle glutamine (GLN) concentration falls following injury and infection. In an attempt to prevent this decline and to characterize its influence on the efflux of amino acid (AA) from skeletal muscle, we administered varying quantities of AA (0,2, and 4 g/kg X day) as saline or AA solutions with or without GLN enrichment to 22 postoperative dogs. Plasma and muscle AA were determined before and 24 hr after standard laparotomy. Hindquarter AA efflux was measured at 6 and 24 hr. Skeletal muscle nitrogen declined in saline controls (69.8 +/- 8.5 vs 52.8 +/- 8.4 mmol/liter; p less than 0.01), largely due to the fall in intracellular GLN (21.48 +/- 3.21 vs 15.86 +/- 3.80; p less than 0.05). Similar alterations were seen in the animals receiving 2 g/kg. However, both intracellular nitrogen and GLN were maintained in animals receiving 4 g/kg, whether the AA solutions contained GLN or not (skeletal muscle nitrogen before 64.3 +/- 8.6 mmol/l vs 65.4 +/- 7.0 after, GLN 19.2 +/- 3.4 vs 19.9 +/- 3.0). Hindquarter AA efflux was reduced in those animals at 6 hr compared with saline-treated animals (-6.52 +/- 1.8 and -7.70 +/- 5.90 vs -19.05 +/- 4.06 mumol/kg X min; p less than 0.05). Intracellular GLN can be maintained during operative stress with adequate nitrogen infusion. Replacing 50% of the balanced AA solution with GLN resulted in equally effective maintenance of intracellular GLN levels and a comparable reduction in skeletal muscle AA efflux. Preservation of normal intracellular GLN levels with adequate AA nutrition may be essential for the conservation of muscle protein.

  5. Versatile Roles of Intracellularly Located TRPV1 Channel.

    PubMed

    Zhao, Rui; Tsang, Suk Ying

    2016-11-27

    The ubiquitous expression in many organs throughout the body and the ability to respond to a wide variety of physical and chemical stimuli have brought transient receptor potential (TRP) channels to the vanguards of our sensory systems. TRP vanilloid-1 (TRPV1) is the founding member of the TRPV subfamily. TRPV1 can be activated by noxious heat, protons, and vanilloids. Previous studies have shown that TRPV1 is located on the plasma membrane, serving to non-selectively permeate calcium ion from the extracellular region to the cytoplasm. Interestingly, increasing evidence suggests that TRPV1 is also located intracellularly in various cell types such as neurons, myocytes, and numerous cancer cells. By immunocytochemistry and/or fractionation followed by Western blotting, TRPV1 was found to express on the endoplasmic reticulum/sarcoplasmic reticulum and the mitochondria. By using various pharmacological and molecular tools, intracellular TRPV1 was also found to functionally express to control calcium level both inside the organelles and in the cytoplasm. Recent studies have shown that intracellularly located TRPV1 serves versatile functions in various physiological and pathological conditions (e.g., exercise endurance and hypertrophy). In this review, we not only have summarized the well-characterized roles of TRPV1, but also have highlighted the increasing importance of intracellular TRPV1-mediated pathways. Lastly, we have pointed out future research direction for answering several important questions that have remained unanswered. Vigorous investigation of the emerging roles of intracellular TRPV1 can allow a better understanding of how TRPV1 controls the cellular calcium homeostasis and its role in various physiological and pathophysiological conditions. J. Cell. Physiol. 9999: 1-9, 2016. © 2016 Wiley Periodicals, Inc.

  6. Microsporidia Are Natural Intracellular Parasites of the Nematode Caenorhabditis elegans

    PubMed Central

    Troemel, Emily R; Félix, Marie-Anne; Whiteman, Noah K; Barrière, Antoine; Ausubel, Frederick M

    2008-01-01

    For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes. PMID:19071962

  7. Intracellular singlet oxygen photosensitizers: on the road to solving the problems of sensitizer degradation, bleaching and relocalization.

    PubMed

    da Silva, Elsa F F; Pimenta, Frederico M; Pedersen, Brian W; Blaikie, Frances H; Bosio, Gabriela N; Breitenbach, Thomas; Westberg, Michael; Bregnhøj, Mikkel; Etzerodt, Michael; Arnaut, Luis G; Ogilby, Peter R

    2016-02-01

    Selected singlet oxygen photosensitizers have been examined from the perspective of obtaining a molecule that is sufficiently stable under conditions currently employed to study singlet oxygen behavior in single mammalian cells. Reasonable predictions about intracellular sensitizer stability can be made based on solution phase experiments that approximate the intracellular environment (e.g., solutions containing proteins). Nevertheless, attempts to construct a stable sensitizer based solely on the expected reactivity of a given functional group with singlet oxygen are generally not sufficient for experiments in cells; it is difficult to construct a suitable chromophore that is impervious to all of the secondary and/or competing degradative processes that are present in the intracellular environment. On the other hand, prospects are reasonably positive when one considers the use of a sensitizer encapsulated in a specific protein; the local environment of the chromophore is controlled, degradation as a consequence of bimolecular reactions can be mitigated, and genetic engineering can be used to localize the encapsulated sensitizer in a given cellular domain. Also, the option of directly exciting oxygen in sensitizer-free experiments provides a useful complementary tool. These latter systems bode well with respect to obtaining more accurate control of the "dose" of singlet oxygen used to perturb a cell; a parameter that currently limits mechanistic studies of singlet-oxygen-mediated cell signaling.

  8. Modularity and determinants of a (bi-)polarization control system from free-living and obligate intracellular bacteria

    PubMed Central

    Bergé, Matthieu; Campagne, Sébastien; Mignolet, Johann; Holden, Seamus; Théraulaz, Laurence; Manley, Suliana; Allain, Frédéric H-T; Viollier, Patrick H

    2016-01-01

    Although free-living and obligate intracellular bacteria are both polarized it is unclear whether the underlying polarization mechanisms and effector proteins are conserved. Here we dissect at the cytological, functional and structural level a conserved polarization module from the free living α-proteobacterium Caulobacter crescentus and an orthologous system from an obligate intracellular (rickettsial) pathogen. The NMR solution structure of the zinc-finger (ZnR) domain from the bifunctional and bipolar ZitP pilus assembly/motility regulator revealed conserved interaction determinants for PopZ, a bipolar matrix protein that anchors the ParB centromere-binding protein and other regulatory factors at the poles. We show that ZitP regulates cytokinesis and the localization of ParB and PopZ, targeting PopZ independently of the previously known binding sites for its client proteins. Through heterologous localization assays with rickettsial ZitP and PopZ orthologs, we document the shared ancestries, activities and structural determinants of a (bi-)polarization system encoded in free-living and obligate intracellular α-proteobacteria. DOI: http://dx.doi.org/10.7554/eLife.20640.001 PMID:28008852

  9. Domain architecture evolution of pattern-recognition receptors

    PubMed Central

    Zhang, Qing; Zmasek, Christian M.

    2010-01-01

    In animals, the innate immune system is the first line of defense against invading microorganisms, and the pattern-recognition receptors (PRRs) are the key components of this system, detecting microbial invasion and initiating innate immune defenses. Two families of PRRs, the intracellular NOD-like receptors (NLRs) and the transmembrane Toll-like receptors (TLRs), are of particular interest because of their roles in a number of diseases. Understanding the evolutionary history of these families and their pattern of evolutionary changes may lead to new insights into the functioning of this critical system. We found that the evolution of both NLR and TLR families included massive species-specific expansions and domain shuffling in various lineages, which resulted in the same domain architectures evolving independently within different lineages in a process that fits the definition of parallel evolution. This observation illustrates both the dynamics of the innate immune system and the effects of “combinatorially constrained” evolution, where existence of the limited numbers of functionally relevant domains constrains the choices of domain architectures for new members in the family, resulting in the emergence of independently evolved proteins with identical domain architectures, often mistaken for orthologs. Electronic supplementary material The online version of this article (doi:10.1007/s00251-010-0428-1) contains supplementary material, which is available to authorized users. PMID:20195594

  10. Solution Structure of the cGMP Binding GAF Domain from Phosphodiesterase 5: Insights into Nucleotide Specificity, Dimerization, and cGMP-Dependent Conformational Change

    SciTech Connect

    Heikaus, Clemens C.; Stout, Joseph R.; Sekharan, Monica R.; Eakin, Catherine M.; Rajagopal, Ponni; Brzovic, Peter S.; Beavo, Joseph A.; Klevit, Rachel E.

    2008-08-15

    Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular NOEs.

  11. Supramolecular nanoreactors for intracellular singlet-oxygen sensitization

    NASA Astrophysics Data System (ADS)

    Swaminathan, Subramani; Fowley, Colin; Thapaliya, Ek Raj; McCaughan, Bridgeen; Tang, Sicheng; Fraix, Aurore; Burjor, Captain; Sortino, Salvatore; Callan, John F.; Raymo, Françisco M.

    2015-08-01

    An amphiphilic polymer with multiple decyl and oligo(ethylene glycol) chains attached to a common poly(methacrylate) backbone assembles into nanoscaled particles in aqueous environments. Hydrophobic anthracene and borondipyrromethene (BODIPY) chromophores can be co-encapsulated within the self-assembling nanoparticles and transported across hydrophilic media. The reversible character of the noncovalent bonds, holding the supramolecular containers together, permits the exchange of their components with fast kinetics in aqueous solution. Incubation of cervical cancer (HeLA) cells with a mixture of two sets of nanoparticles, pre-loaded independently with anthracene or BODIPY chromophores, results in guest scrambling first and then transport of co-entrapped species to the intracellular space. Alternatively, incubation of cells with the two sets of nanocarriers in consecutive steps permits the sequential transport of the anthracene and BODIPY chromophores across the plasma membrane and only then allows their co-encapsulation within the same supramolecular containers. Both mechanisms position the two sets of chromophores with complementary spectral overlap in close proximity to enable the efficient transfer of energy intracellularly from the anthracene donors to the BODIPY acceptors. In the presence of iodine substituents on the BODIPY platform, intersystem crossing follows energy transfer. The resulting triplet state can transfer energy further to molecular oxygen with the concomitant production of singlet oxygen to induce cell mortality. Furthermore, the donor can be excited with two near-infrared photons simultaneously to permit the photoinduced generation of singlet oxygen intracellularly under illumination conditions compatible with applications in vivo. Thus, these supramolecular strategies to control the excitation dynamics of multichromophoric assemblies in the intracellular environment can evolve into valuable protocols for photodynamic therapy.An amphiphilic

  12. Enhanced intracellular delivery and antibacterial efficacy of enrofloxacin-loaded docosanoic acid solid lipid nanoparticles against intracellular Salmonella

    PubMed Central

    Xie, Shuyu; Yang, Fei; Tao, Yanfei; Chen, Dongmei; Qu, Wei; Huang, Lingli; Liu, Zhenli; Pan, Yuanhu; Yuan, Zonghui

    2017-01-01

    Enrofloxacin-loaded docosanoic acid solid lipid nanoparticles (SLNs) with different physicochemical properties were developed to enhance activity against intracellular Salmonella. Their cellular uptake, intracellular elimination and antibacterial activity were studied in RAW 264.7 cells. During the experimental period, SLN-encapsulated enrofloxacin accumulated in the cells approximately 27.06–37.71 times more efficiently than free drugs at the same extracellular concentration. After incubation for 0.5 h, the intracellular enrofloxacin was enhanced from 0.336 to 1.147 μg/mg of protein as the sizes of nanoparticles were increased from 150 to 605 nm, and from 0.960 to 1.147 μg/mg of protein when the charge was improved from −8.1 to −24.9 mv. The cellular uptake was more significantly influenced by the size than it was by the charge, and was not affected by whether the charge was positive or negative. The elimination of optimal SLN-encapsulated enrofloxacin from the cells was significantly slower than that of free enrofloxacin after removing extracellular drug. The inhibition effect against intracellular Salmonella CVCC541 of 0.24 and 0.06 μg/mL encapsulated enrofloxacin was stronger than 0.6 μg/mL free drug after all of the incubation periods and at 48 h, respectively. Docosanoic acid SLNs are thus considered as a promising carrier for intracellular bacterial treatment. PMID:28112240

  13. Increase in intracellular Zn2+ concentration by thimerosal in rat thymocytes: intracellular Zn2+ release induced by oxidative stress.

    PubMed

    Hashimoto, Erika; Oyama, Toshihisa B; Oyama, Keisuke; Nishimura, Yumiko; Oyama, Tomohiro M; Ueha-Ishibashi, Toshiko; Okano, Yoshiro; Oyama, Yasuo

    2009-09-01

    Thimerosal (TMR), an ethylmercury-containing preservative in pharmaceutical products, was recently reported to increase intracellular Zn(2+) concentration. Therefore, some health concerns about the toxicity of TMR remain because of physiological and pathological roles of Zn(2+). To reveal the property of TMR-induced increase in intracellular Zn(2+) concentration, the effect of TMR on FluoZin-3 fluorescence, an indicator of intracellular Zn(2+), of rat thymocytes was examined. TMR at concentrations ranging from 0.3 microM to 10 microM increased the intensity of FluoZin-3 fluorescence in a concentration-dependent manner under external Ca(2+)- and Zn(2+)-free condition. The threshold concentration was 0.3-1 microM. The increase in the intensity was significant when TMR concentration was 1 microM or more. N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a chelator for intracellular Zn(2+), completely attenuated the TMR-induced augmentation of FluoZin-3 fluorescence. Hydrogen peroxide (H(2)O(2)) and N-ethylmaleimide, reducing cellular thiol content, significantly increased FluoZin-3 fluorescence intensity and decreased 5-chloromethylfluorescein (5-CMF) fluorescence intensity, an indicator for cellular thiol. The correlation coefficient between TMR-induced augmentation of FluoZin-3 fluorescence and attenuation of 5-CMF fluorescence was -0.882. TMR also attenuated the 5-CMF fluorescence in the presence of TPEN. Simultaneous application of H(2)O(2) and TMR synergistically augmented the FluoZin-3 fluorescence. It is suggested that TMR increases intracellular Zn(2+) concentration via decreasing cellular thiol content.

  14. Intracellular calcium levels as screening tool for nanoparticle toxicity

    PubMed Central

    Meindl, Claudia; Kueznik, Tatjana; Bösch, Martina; Roblegg, Eva; Fröhlich, Eleonore

    2015-01-01

    The use of engineered nano-sized materials led to revolutionary developments in many industrial applications and in the medical field. These materials, however, also may cause cytotoxicity. In addition to size, surface properties and shape were identified as relevant parameters for cell damage. Cell damage may occur as disruption of membrane integrity, induction of apoptosis and by organelle damage. Generation of oxidative stress may serve as an indicator for cytotoxicity. Effects occurring upon short contact of particles with cells, for instance in the systemic blood circulation, could be identified according to increases of intracellular [Ca2+] levels, which are caused by variety of toxic stimuli. Negatively charged, neutral and positively charged polystyrene particles of different sizes were used to study the role of size and surface properties on viability, membrane disruption, apoptosis, lysosome function, intracellular [Ca2+] levels and generation of oxidative stress. Silica particles served to test this hypothesis. Twenty nm polystyrene particles as well as 12 nm and 40 nm silica particles caused membrane damage and apoptosis with no preference of the surface charge. Only 20 nm plain and amine functionalized polystyrene particles cause oxidative stress and only the plain particles lysosomal damage. A potential role of surface charge was identified for 200 nm polystyrene particles, where only the amidine particles caused lysosomal damage. Increases in intracellular [Ca2+] levels and cytotoxicity after 24 h was often linked but determination of intracellular [Ca2+] levels could serve to characterize further the type of membrane damage. © 2015 The Authors. Journal of Applied Toxicology Published by John Wiley & Sons Ltd. Nano-sized materials may cause cytotoxicity. Negatively charged, neutral and positively charged polystyrene particles of different sizes and silica nanoparticles were used to study the role of size and surface properties on viability, membrane

  15. Analysing intracellular deformation of polymer capsules using structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Xi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Yan, Yan; Noi, Ka Fung; Ping, Yuan; Caruso, Frank

    2016-06-01

    Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties.Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces

  16. Amino-terminal cysteine residues differentially influence RGS4 protein plasma membrane targeting, intracellular trafficking, and function.

    PubMed

    Bastin, Guillaume; Singh, Kevin; Dissanayake, Kaveesh; Mighiu, Alexandra S; Nurmohamed, Aliya; Heximer, Scott P

    2012-08-17

    Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

  17. Ahcyl2 upregulates NBCe1-B via multiple serine residues of the PEST domain-mediated association.

    PubMed

    Park, Pil Whan; Ahn, Jeong Yeal; Yang, Dongki

    2016-07-01

    Inositol-1,4,5-triphosphate [IP3] receptors binding protein released with IP3 (IRBIT) was previously reported as an activator of NBCe1-B. Recent studies have characterized IRBIT homologue S-Adenosylhomocysteine hydrolase-like 2 (AHCYL2). AHCYL2 is highly homologous to IRBIT (88%) and heteromerizes with IRBIT. The two important domains in the N-terminus of AHCYL2 are a PEST domain and a coiled-coil domain which are highly comparable to those in IRBIT. Therefore, in this study, we tried to identify the role of those domains in mouse AHCYL2 (Ahcyl2), and we succeeded in identifying PEST domain of Ahcyl2 as a regulation region for NBCe1-B activity. Site directed mutagenesis and coimmunoprecipitation assay showed that NBCe1-B binds to the N-terminal Ahcyl2-PEST domain, and its binding is determined by the phosphorylation of 4 critical serine residues (Ser151, Ser154, Ser157, and Ser160) in Ahcyl2 PEST domain. Also we revealed that 4 critical serine residues in Ahcyl2 PEST domain are indispensable for the activation of NBCe1-B using measurement of intracellular pH experiment. Thus, these results suggested that the NBCe1-B is interacted with 4 critical serine residues in Ahcyl2 PEST domain, which play an important role in intracellular pH regulation through NBCe1-B.

  18. Frequency domain nonlinear optics

    NASA Astrophysics Data System (ADS)

    Legare, Francois

    2016-05-01

    The universal dilemma of gain narrowing occurring in fs amplifiers prevents ultra-high power lasers from delivering few-cycle pulses. This problem is overcome by a new amplification concept: Frequency domain Optical Parametric Amplification - FOPA. It enables simultaneous up-scaling of peak power and amplified spectral bandwidth and can be performed at any wavelength range of conventional amplification schemes, however, with the capability to amplify single cycles of light. The key idea for amplification of octave-spanning spectra without loss of spectral bandwidth is to amplify the broad spectrum ``slice by slice'' in the frequency domain, i.e. in the Fourier plane of a 4f-setup. The striking advantages of this scheme, are its capability to amplify (more than) one octave of bandwidth without shorting the corresponding pulse duration. This is because ultrabroadband phase matching is not defined by the properties of the nonlinear crystal employed but the number of crystals employed. In the same manner, to increase the output energy one simply has to increase the spectral extension in the Fourier plane and to add one more crystal. Thus, increasing pulse energy and shortening its duration accompany each other. A proof of principle experiment was carried out at ALLS on the sub-two cycle IR beam line and yielded record breaking performance in the field of few-cycle IR lasers. 100 μJ two-cycle pulses from a hollow core fibre compression setup were amplified to 1.43mJ without distorting spatial or temporal properties. Pulse duration at the input of FOPA and after FOPA remains the same. Recently, we have started upgrading this system to be pumped by 250 mJ to reach 40 mJ two-cycle IR few-cycle pulses and latest results will be presented at the conference. Furthermore, the extension of the concept of FOPA to other nonlinear optical processes will be discussed. Frequency domain nonlinear optics.

  19. Field Evolution of Antiferromagnetic Domains and Domain Walls

    NASA Astrophysics Data System (ADS)

    Fullerton, Eric E.; Hellwig, Olav; Berger, Andreas K.

    2003-03-01

    We have used magnetron sputtered [Co(4Å)Pt(7Å)]X Co(4Å)Ru(9Å)N multiplayer films to create artificially layered antiferromagnets. In contrast to atomic antiferromagnets our model system has an antiferromagnetic (AF) exchange energy comparable to the Zeemann energy in moderate fields and allows to fine tune the relative magnitude of the different magnetic energy terms by varying the parameters X and N. With increasing X and N we observe a transition from traditionally observed sharp AF domain walls towards AF domain walls with a finite width which consist of ferromagnetic stripes, i.e. the AF domains have zero net moment whereas the domain walls carry a finite magnetic moment. Such AF domain walls have not been observed before and are a direct consequence of balancing out exchange and Zeeman energy. We also show that such domain walls are expected from theoretical energy calculations. In this contribution we study the nature and field evolution of the AF stripe domain walls by Magnetic Force Microscopy (MFM). The surface sensitivity of MFM and the finite moment of the AF domain walls allow us to image AF domains as well as domain walls. We are showing first experiments to study the AF domain wall evolution in real space while applying an external field. O.H. was supported by the Deutsche Forschungsgemeinschaft via a Forschungsstipendium under the contract number HE 3286/1-1.

  20. Space Domain Awareness

    DTIC Science & Technology

    2012-09-01

    information required to characterize a space object. Another key parameter to be considered is the frequency of observation. This sampling rate varies...useful to define the values of these parameters that approximate the current and future state of the space domain. The current catalog and network... Parameters used in estimating data needs for SDA Current Threshold Objective βmo , βimg 0.1, 10Kb 0.1, 10Kb 0.1, 10Kb Number of Objects (Na , Np

  1. Pico gauges for minimally invasive intracellular hydrostatic pressure measurements.

    PubMed

    Knoblauch, Jan; Mullendore, Daniel L; Jensen, Kaare H; Knoblauch, Michael

    2014-11-01

    Intracellular pressure has a multitude of functions in cells surrounded by a cell wall or similar matrix in all kingdoms of life. The functions include cell growth, nastic movements, and penetration of tissue by parasites. The precise measurement of intracellular pressure in the majority of cells, however, remains difficult or impossible due to their small size and/or sensitivity to manipulation. Here, we report on a method that allows precise measurements in basically any cell type over all ranges of pressure. It is based on the compression of nanoliter and picoliter volumes of oil entrapped in the tip of microcapillaries, which we call pico gauges. The production of pico gauges can be accomplished with standard laboratory equipment, and measurements are comparably easy to conduct. Example pressure measurements are performed on cells that are difficult or impossible to measure with other methods.

  2. Evolution of the Calcium-Based Intracellular Signaling System

    PubMed Central

    Marchadier, Elodie; Oates, Matt E.; Fang, Hai; Donoghue, Philip C.J.; Hetherington, Alistair M.; Gough, Julian

    2016-01-01

    To progress our understanding of molecular evolution from a collection of well-studied genes toward the level of the cell, we must consider whole systems. Here, we reveal the evolution of an important intracellular signaling system. The calcium-signaling toolkit is made up of different multidomain proteins that have undergone duplication, recombination, sequence divergence, and selection. The picture of evolution, considering the repertoire of proteins in the toolkit of both extant organisms and ancestors, is radically different from that of other systems. In eukaryotes, the repertoire increased in both abundance and diversity at a far greater rate than general genomic expansion. We describe how calcium-based intracellular signaling evolution differs not only in rate but in nature, and how this correlates with the disparity of plants and animals. PMID:27358427

  3. Characterization of a Mycobacterium intracellulare Variant Strain by Molecular Techniques

    PubMed Central

    Menendez, M. C.; Palenque, E.; Navarro, M. C.; Nuñez, M. C.; Rebollo, M. J.; Garcia, M. J.

    2001-01-01

    This paper describes a Mycobacterium intracellulare variant strain causing an unusual infection. Several isolates obtained from an immunocompromised patient were identified as members of the Mycobacterium avium complex (MAC) by the commercial AccuProbe system and biochemical standard identification. Further molecular approaches were undertaken for a more accurate characterization of the bacteria. Up to seven different genomic sequences were analyzed, ranging from conserved mycobacterial genes such as 16S ribosomal DNA to MAC-specific genes such as mig (macrophage-induced gene). The results obtained identify the isolates as a variant of M. intracellulare, an example of the internal variability described for members of the MAC, particularly within that species. The application of other molecular approaches is recommended for more accurate identification of bacteria described as MAC members. PMID:11724827

  4. Electrochemical Visualization of Intracellular Hydrogen Peroxide at Single Cells.

    PubMed

    He, Ruiqin; Tang, Huifen; Jiang, Dechen; Chen, Hong-yuan

    2016-02-16

    In this Letter, the electrochemical visualization of hydrogen peroxide inside one cell was achieved first using a comprehensive Au-luminol-microelectrode and electrochemiluminescence. The capillary with a tip opening of 1-2 μm was filled with the mixture of chitosan and luminol, which was coated with the thin layers of polyvinyl chloride/nitrophenyloctyl ether (PVC/NPOE) and gold as the microelectrode. Upon contact with the aqueous hydrogen peroxide, hydrogen peroxide and luminol in contact with the gold layer were oxidized under the positive potential resulting in luminescence for the imaging. Due to the small diameter of the electrode, the microelectrode tip was inserted into one cell and the bright luminescence observed at the tip confirmed the visualization of intracellular hydrogen peroxide. The further coupling of oxidase on the electrode surface could open the field in the electrochemical imaging of intracellular biomolecules at single cells, which benefited the single cell electrochemical detection.

  5. Practical aspects of measuring intracellular calcium signals with fluorescent indicators.

    PubMed

    Kao, Joseph P Y; Li, Gong; Auston, Darryl A

    2010-01-01

    The use of fluorescent indicators for monitoring calcium (Ca(2+)) signals and for measuring Ca(2+) concentration ([Ca(2+)]) in living cells is described. The following topics are covered in detail: (1) ratiometric and nonratiometric fluorescent indicators and the principles underlying their use, (2) techniques for loading Ca(2+) indicators and Ca(2+) buffers into living cells, (3) calibration of indicator fluorescence intensity measurements to yield values of intracellular [Ca(2+)], (4) analysis of nonratiometric fluorescence intensity data and caveats relating to their interpretation, (5) techniques for manipulating intracellular and extracellular [Ca(2+)], and (6) the use of fluorescent indicators to monitor Ca(2+) signals in mitochondria. The chapter aims to present these fundamental topics in a manner that is practically useful and intuitively accessible. The origins of key mathematical equations used in the article are outlined in two appendices.

  6. Zinc Chelation Mediates the Lysosomal Disruption without Intracellular ROS Generation

    PubMed Central

    Matias, Andreza Cândido; Manieri, Tânia Maria; Cerchiaro, Giselle

    2016-01-01

    We report the molecular mechanism for zinc depletion caused by TPEN (N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine) in neuroblastoma cells. The activation of p38 MAP kinase and subsequently caspase 3 is not due to or followed by redox imbalance or ROS generation, though these are commonly observed in literature. We found that TPEN is not responsible for ROS generation and the mechanism involves essentially lysosomal disruption caused by intracellular zinc depletion. We also observed a modest activation of Bax and no changes in the Bcl-2 proteins. As a result, we suggest that TPEN causes intracellular zinc depletion which can influence the breakdown of lysosomes and cell death without ROS generation. PMID:27123155

  7. Intracellular localization of titanium dioxide-biomolecule nanocomposites.

    SciTech Connect

    Paunesku, T.; Stojicevic, N.; Vogt, S.; Maser, J.; Lai, B.; Rajh, T.; Thurnauer, M.; Woloschak, G.

    2002-10-30

    Emerging areas of nanotechnology hold the promise of overcoming the limitations of existing technology for intracellular manipulation. These new developments include the creation of nanocomposites that can be introduced into the cells, targeted to specific subcellular sites, and subsequently used as platforms for initiation of intracellular processes dependent on or aided by locally high concentrations of specific molecules delivered as components of the nanocomposites. Nanocomposites that combine functional properties of biomolecules with the functional properties of inorganic components could provide new tools for biology, medicine, chemistry and material sciences. Here we describe how we introduced TiO{sub 2}-DNA nanocomposites into cells, and localized titanium in the cells by mapping the Ti K{alpha} X-ray fluorescence induced at the 2-ID-E microprobe of the SRI-CAT at the Advanced Photon Source at Argonne National Laboratory.

  8. Loligomers: design of de novo peptide-based intracellular vehicles.

    PubMed Central

    Sheldon, K; Liu, D; Ferguson, J; Gariépy, J

    1995-01-01

    Defined branched peptides (loligomers) incorporating cytoplasmic translocation signals, nuclear localization sequences, and fluorescent probes were designed and synthesized to demonstrate the feasibility and simplicity of creating novel classes of intracellular vehicles. Loligomers containing all the above signals were rapidly internalized by Chinese hamster ovary (CHO) cells and accumulated in their nucleus. At 4 degrees C, the interaction of peptide constructs with CHO cells was limited to membrane association. Loligomers entered cells at higher temperatures by adsorptive endocytosis. Inhibitors of ATP synthesis affected cytoplasmic import only weakly but abolished nuclear uptake. The peptide signals guided both cytoplasmic and nuclear localization events. The properties exhibited by loligomers suggest a strategy for the facile design of "guided" classes of intracellular agents. Images Fig. 3 PMID:7892224

  9. Monitoring the intracellular calcium response to a dynamic hypertonic environment

    NASA Astrophysics Data System (ADS)

    Huang, Xiaowen; Yue, Wanqing; Liu, Dandan; Yue, Jianbo; Li, Jiaqian; Sun, Dong; Yang, Mengsu; Wang, Zuankai

    2016-03-01

    The profiling of physiological response of cells to external stimuli at the single cell level is of importance. Traditional approaches to study cell responses are often limited by ensemble measurement, which is challenging to reveal the complex single cell behaviors under a dynamic environment. Here we report the development of a simple microfluidic device to investigate intracellular calcium response to dynamic hypertonic conditions at the single cell level in real-time. Interestingly, a dramatic elevation in the intracellular calcium signaling is found in both suspension cells (human leukemic cell line, HL-60) and adherent cells (lung cancer cell line, A549), which is ascribed to the exposure of cells to the hydrodynamic stress. We also demonstrate that the calcium response exhibits distinct single cell heterogeneity as well as cell-type-dependent responses to the same stimuli. Our study opens up a new tool for tracking cellular activity at the single cell level in real time for high throughput drug screening.

  10. Modulation of Host miRNAs by Intracellular Bacterial Pathogens

    PubMed Central

    Das, Kishore; Garnica, Omar; Dhandayuthapani, Subramanian

    2016-01-01

    MicroRNAs (miRNAs) are short non-coding RNAs that regulate the expression of protein coding genes of viruses and eukaryotes at the post-transcriptional level. The eukaryotic genes regulated by miRNAs include those whose products are critical for biological processes such as cell proliferation, metabolic pathways, immune response, and development. It is now increasingly recognized that modulation of miRNAs associated with biological processes is one of the strategies adopted by bacterial pathogens to survive inside host cells. In this review, we present an overview of the recent findings on alterations of miRNAs in the host cells by facultative intracellular bacterial pathogens. In addition, we discuss how the altered miRNAs help in the survival of these pathogens in the intracellular environment. PMID:27536558

  11. Regulatory role of intracellular sodium ions in neurotransmitter secretion.

    PubMed

    Melinek, R; Lev-Tov, A; Meiri, H; Erulkar, S D; Rahamimoff, R

    1982-01-01

    Calcium ions are the main inducer of quantal transmitter release of the frog neuromuscular junction; but even in their virtual absence from the extracellular medium, nerve stimulation causes a prolonged augmentation of transmitter release. These facts led to the hypothesis that an accumulation of intracellular sodium can serve as a slow secondary regulator of neurosecretion. Three lines of evidence presented in this article substantiate this hypothesis: firstly, veratridine, which is known to increase sodium fluxes through the voltage-dependent sodium channels, increases transmitter release after nerve stimulation. Secondly, monensin, which was shown to induce sodium transport through nerve membranes, increases evoked transmitter release, tetanic potentiation and posttetanic potentiation. Thirdly, sodium-filled phosphatidylcholine liposomes increase transmitter release. These effects of sodium are probably not due to a direct effect on the transmitter release mechanism, but are caused by sodium-induced calcium translocation from intracellular stores.

  12. Extra- and intracellular innate immune recognition in endothelial cells.

    PubMed

    Opitz, Bastian; Hippenstiel, Stefan; Eitel, Julia; Suttorp, Norbert

    2007-08-01

    The innate immune system represents the principal sensor of infections in multicellular organisms and might also mediate responses to some endogenous molecules. In this context, endothelial cells are among the first cells coming into contact with microbial or endogenous (danger-associated) molecules or whole pathogens entering the bloodstream. Since many bacteria and viruses invade the endothelium, endothelial cells are equipped with both extracellular and cytosolic surveillance systems capable of sensing microbial components, and endogenous danger-associated molecules. The receptor molecules, called pattern recognition receptors (PRRs), are classified as transmembrane or cytosolic molecules. While the transmembrane PRRs recognize extracellular and membrane-enclosed foreign organisms, the cytosolic PRRs appear to sense intracellular infections. Here we focus on both PRR classes in general, and outline the current knowledge of extra- and intracellular pattern recognition in endothelial cells and its potential role in vascular diseases and sepsis.

  13. An evolutionary strategy for a stealthy intracellular Brucella pathogen.

    PubMed

    Martirosyan, Anna; Moreno, Edgardo; Gorvel, Jean-Pierre

    2011-03-01

    Brucella is an intracellular bacterial pathogen that causes abortion and infertility in mammals and leads to a debilitating febrile illness that can progress into a long lasting disease with severe complications in humans. Its virulence depends on survival and replication properties in host cells. In this review, we describe the stealthy strategy used by Brucella to escape recognition of the innate immunity and the means by which this bacterium evades intracellular destruction. We also discuss the development of adaptive immunity and its modulation during brucellosis that in course leads to chronic infections. Brucella has developed specific strategies to influence antigen presentation mediated by cells. There is increasing evidence that Brucella also modulates signaling events during host adaptive immune responses.

  14. Non-contact intracellular binding of chloroplasts in vivo

    NASA Astrophysics Data System (ADS)

    Li, Yuchao; Xin, Hongbao; Liu, Xiaoshuai; Li, Baojun

    2015-06-01

    Non-contact intracellular binding and controllable manipulation of chloroplasts in vivo was demonstrated using an optical fiber probe. Launching a 980-nm laser beam into a fiber, which was placed about 3 μm above the surface of a living plant (Hydrilla verticillata) leaf, enabled stable binding of different numbers of chloroplasts, as well as their arrangement into one-dimensional chains and two-dimensional arrays inside the leaf without damaging the chloroplasts. Additionally, the formed chloroplast chains were controllably transported inside the living cells. The optical force exerted on the chloroplasts was calculated to explain the experimental results. This method provides a flexible method for studying intracellular organelle interaction with highly organized organelle-organelle contact in vivo in a non-contact manner.

  15. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins.

    PubMed

    Enz, Ralf

    2012-01-01

    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.

  16. Supramolecular nanoreactors for intracellular singlet-oxygen sensitization.

    PubMed

    Swaminathan, Subramani; Fowley, Colin; Thapaliya, Ek Raj; McCaughan, Bridgeen; Tang, Sicheng; Fraix, Aurore; Captain, Burjor; Sortino, Salvatore; Callan, John F; Raymo, Françisco M

    2015-09-07

    An amphiphilic polymer with multiple decyl and oligo(ethylene glycol) chains attached to a common poly(methacrylate) backbone assembles into nanoscaled particles in aqueous environments. Hydrophobic anthracene and borondipyrromethene (BODIPY) chromophores can be co-encapsulated within the self-assembling nanoparticles and transported across hydrophilic media. The reversible character of the noncovalent bonds, holding the supramolecular containers together, permits the exchange of their components with fast kinetics in aqueous solution. Incubation of cervical cancer (HeLA) cells with a mixture of two sets of nanoparticles, pre-loaded independently with anthracene or BODIPY chromophores, results in guest scrambling first and then transport of co-entrapped species to the intracellular space. Alternatively, incubation of cells with the two sets of nanocarriers in consecutive steps permits the sequential transport of the anthracene and BODIPY chromophores across the plasma membrane and only then allows their co-encapsulation within the same supramolecular containers. Both mechanisms position the two sets of chromophores with complementary spectral overlap in close proximity to enable the efficient transfer of energy intracellularly from the anthracene donors to the BODIPY acceptors. In the presence of iodine substituents on the BODIPY platform, intersystem crossing follows energy transfer. The resulting triplet state can transfer energy further to molecular oxygen with the concomitant production of singlet oxygen to induce cell mortality. Furthermore, the donor can be excited with two near-infrared photons simultaneously to permit the photoinduced generation of singlet oxygen intracellularly under illumination conditions compatible with applications in vivo. Thus, these supramolecular strategies to control the excitation dynamics of multichromophoric assemblies in the intracellular environment can evolve into valuable protocols for photodynamic therapy.

  17. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus

    PubMed Central

    Sharma, Onkar; O’Seaghdha, Maghnus; Velarde, Jorge J.; Wessels, Michael R.

    2016-01-01

    A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS) has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase). When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO), and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase) that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells. PMID:26938870

  18. Mycobacterium intracellulare infection in a capybara (Hydrochoerus hydrochaeris).

    PubMed

    Pezzone, Natalia; Eberhardt, Ayelen T; Fernández, Analia; Garbaccio, Sergio; Zumárraga, Martín; Gioffré, Andrea; Magni, Carolina; Beldomenico, Pablo M; Marini, M Rocío; Canal, Ana M

    2013-12-01

    This report describes the first case of Mycobacterium intracellulare infection with typical granulomatous lesions of mycobacteriosis in a capybara (Hydrochoerus hydrochaeris). The individual was a captive-bred young female, part of the control group of an experimental study on stress. Multiple granulomatous lesions were detected in a mesenteric lymph node of this young female. Mycobacterial infection was confirmed by bacteriologic culture and molecular identification methods. Clinical lesions were characterized by histopathology.

  19. Mycobacterium avium-intracellulare: a rare cause of subacromial bursitis.

    PubMed

    Sinha, Raj; Tuckett, John; Hide, Geoff; Dildey, Petra; Karsandas, Alvin

    2015-01-01

    Septic subacromial bursitis is an uncommon disorder with only a few reported cases in the literature. The most common causative organism is Staphylococcus aureus. We report the case of a 61-year-old female with a septic subacromial bursitis where the causative organism was found to be Mycobacterium avium-intracellulare (MAI). The diagnosis was only made following a biopsy, and we use this case to highlight the importance of recognising the need to consider a biopsy and aspiration in atypical situations.

  20. Evaluation of two novel methods for assessing intracellular oxygen

    NASA Astrophysics Data System (ADS)

    Williams, Catrin F.; Kombrabail, M.; Vijayalakshmi, K.; White, Nick; Krishnamoorthy, G.; Lloyd, David

    2012-08-01

    The ability to resolve the spatio-temporal complexity of intracellular O2 distribution is the ‘Holy Grail’ of cellular physiology. In an effort to obtain a non-invasive approach of mapping intracellular O2 tensions, two methods of phosphorescent lifetime imaging microscopy were examined in the current study. These were picosecond time-resolved epiphosphorescence microscopy (single 0.5 µm focused spot) and two-photon confocal laser scanning microscopy with pinhole shifting. Both methods utilized nanoparticle-embedded Ru complex (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of Ti-sapphire Tsunami lasers (710-1050 nm). The former method used a 1 ps pulse width excitation beam with vertical polarization via a dichroic mirror (610 nm, XF43) and a 20× objective (NA 0.55, Nikon). Transmitted luminescence (1-2 × 104 counts s-1) was collected and time-correlated single photon counted decay times measured. Alternatively, an unmodified Zeiss LSM510 Confocal NLO microscope with 40× objective (NA 1.3) used successively shifted pinhole positions to collect image data from the lagging trail of the raster scan. Images obtained from two-photon excitation of a yeast (Schizosaccharomyces pombe) and a flagellate fish parasite (Spironucleus vortens), electroporated with Ru complex, indicated the intracellular location and magnitude of O2 gradients, thus confirming the feasibility of optical mapping under different external O2 concentrations. Both methods gave similar lifetimes for Ru complex phosphorescence under aerobic and anaerobic gas phases. Estimation of O2 tensions within individual fibroblasts (human dermal fibroblast (HDF)) and mammary adenocarcinoma (MCF-7) cells was possible using epiphosphorescence microscopy. MCF-7 cells showed lower intracellular O2 concentrations than HDF cells, possibly due to higher metabolic rates in the former. Future work should involve construction of higher resolution 3D maps of Ru coordinate complex lifetime

  1. Bafilomycin A1 and intracellular multiplication of Legionella pneumophila.

    PubMed Central

    Cattani, L; Goldoni, P; Pastoris, M C; Sinibaldi, L; Orsi, N

    1997-01-01

    Multiplication of Legionella pneumophila in HeLa cells was found to be inhibited by noncytotoxic concentrations of bafilomycin A1, with blockage of bacterial growth at a concentration 15.6 nM. The inhibiting action was evident only when the antibiotic was present during the initial phase of intracellular multiplication, i.e., during the formation of the phagosome, whereas the addition of the drug did not affect microorganisms already actively multiplying within the phagosome. PMID:8980784

  2. The druggability of intracellular nucleotide-degrading enzymes.

    PubMed

    Rampazzo, Chiara; Tozzi, Maria Grazia; Dumontet, Charles; Jordheim, Lars Petter

    2016-05-01

    Nucleotide metabolism is the target of a large number of anticancer drugs including antimetabolites and specific enzyme inhibitors. We review scientific findings that over the last 10-15 years have allowed the identification of several intracellular nucleotide-degrading enzymes as cancer drug targets, and discuss further potential therapeutic applications for Rcl, SAMHD1, MTH1 and cN-II. We believe that enzymes involved in nucleotide metabolism represent potent alternatives to conventional cancer chemotherapy targets.

  3. Intracellular accumulation of boceprevir according to plasma concentrations and pharmacogenetics.

    PubMed

    Cusato, Jessica; Allegra, Sarah; De Nicolò, Amedeo; Boglione, Lucio; Fatiguso, Giovanna; Abdi, Adnan Mohamed; Cariti, Giuseppe; Di Perri, Giovanni; D'Avolio, Antonio

    2015-06-01

    Boceprevir (BOC) is a directly-acting antiviral agent for the treatment of hepatitis C virus genotype 1 (HCV-1) infection. It is a mixture of two stereoisomers, the inactive R and the active S isomers. No data have previously been published on BOC intracellular accumulation. In this study, BOC isomer concentrations in peripheral blood mononuclear cells (PBMCs) and plasma were determined. The influence of various single nucleotide polymorphisms (SNPs) on plasma and intracellular drug exposure at Week 4 of triple therapy were also evaluated. Plasma and intracellular BOC concentrations were determined at the end of the dosing interval (C(trough)) using a UPLC-MS/MS validated method. Allelic discrimination was performed through real-time PCR. Median plasma concentrations were 65.97 ng/mL for the S isomer and 36.31 ng/mL for the R isomer; the median S/R plasma concentration ratio was 1.66. The median PBMC concentration was 2285.88 ng/mL for the S isomer; the R isomer was undetectable within PBMCs. The median S isomer PBMC/plasma concentration ratio was 28.59. A significant positive correlation was found between plasma and PBMC S isomer concentrations. ABCB1 1236, SLC28A2 124 and IL28B rs12979860 SNPs were associated with the S isomer PBMC/plasma concentration ratio. In regression models, S isomer plasma levels and FokI polymorphism were able to predict S isomer intracellular exposure, whereas SNPs in AKR1, BCRP1 and SLC28A2 predicted the S isomer PBMC/plasma concentration ratio. No similar data regarding BOC pharmacogenetics and pharmacokinetics have been published previously. This study adds a novel and useful overview of the pharmacological properties of this drug.

  4. Role of intracellular carbon metabolism pathways in Shigella flexneri virulence.

    PubMed

    Waligora, E A; Fisher, C R; Hanovice, N J; Rodou, A; Wyckoff, E E; Payne, S M

    2014-07-01

    Shigella flexneri, which replicates in the cytoplasm of intestinal epithelial cells, can use the Embden-Meyerhof-Parnas, Entner-Doudoroff, or pentose phosphate pathway for glycolytic carbon metabolism. To determine which of these pathways is used by intracellular S. flexneri, mutants were constructed and tested in a plaque assay for the ability to invade, replicate intracellularly, and spread to adjacent epithelial cells. Mutants blocked in the Embden-Meyerhof-Parnas pathway (pfkAB and pykAF mutants) invaded the cells but formed very small plaques. Loss of the Entner-Doudoroff pathway gene eda resulted in small plaques, but the double eda edd mutant formed normal-size plaques. This suggested that the plaque defect of the eda mutant was due to buildup of the toxic intermediate 2-keto-3-deoxy-6-phosphogluconic acid rather than a specific requirement for this pathway. Loss of the pentose phosphate pathway had no effect on plaque formation, indicating that it is not critical for intracellular S. flexneri. Supplementation of the epithelial cell culture medium with pyruvate allowed the glycolysis mutants to form larger plaques than those observed with unsupplemented medium, consistent with data from phenotypic microarrays (Biolog) indicating that pyruvate metabolism was not disrupted in these mutants. Interestingly, the wild-type S. flexneri also formed larger plaques in the presence of supplemental pyruvate or glucose, with pyruvate yielding the largest plaques. Analysis of the metabolites in the cultured cells showed increased intracellular levels of the added compound. Pyruvate increased the growth rate of S. flexneri in vitro, suggesting that it may be a preferred carbon source inside host cells.

  5. Biochemical properties of an intracellular serpin from Echinococcus multilocularis.

    PubMed

    Merckelbach, Armin; Ruppel, Andreas

    2007-11-01

    A serpin of the intracellular type from the tapeworm Echinococcus multilocularis was expressed in Escherichia coli, purified by ion exchange chromatography and tested for inhibitory activity against several proteinases. The recombinant protein, which after transcriptional induction, represents about 20 % of total cellular protein, is biochemically active and inhibits trypsin and the trypsin-like plasmin as well as pig pancreatic and human neutrophil elastase. Implications regarding its biochemistry and biological function are discussed.

  6. Novel intracellular proteins associated with cellular vitamin D action.

    PubMed

    Angelo, Giana; Wood, Richard J; Mayer, Jean

    2002-07-01

    Work with vitamin D-resistant New World primates has revealed novel cellular proteins involved in vitamin D action. An "intracellular vitamin D-binding protein" functions to bind vitamin D metabolites in the cell and enhances vitamin D action. By contrast, a "vitamin D response element-binding protein" inhibits vitamin D receptor binding to the DNA and is responsible for vitamin D resistance in New World primates.

  7. Diffusive spatio-temporal noise in a first-passage time model for intracellular calcium release

    NASA Astrophysics Data System (ADS)

    Flegg, Mark B.; Rüdiger, Sten; Erban, Radek

    2013-04-01

    The intracellular release of calcium from the endoplasmic reticulum is controlled by ion channels. The resulting calcium signals exhibit a rich spatio-temporal signature, which originates at least partly from microscopic fluctuations. While stochasticity in the gating transition of ion channels has been incorporated into many models, the distribution of calcium is usually described by deterministic reaction-diffusion equations. Here we test the validity of the latter modeling approach by using two different models to calculate the frequency of localized calcium signals (calcium puffs) from clustered IP3 receptor channels. The complexity of the full calcium system is here limited to the basic opening mechanism of the ion channels and, in the mathematical reduction simplifies to the calculation of a first passage time. Two models are then studied: (i) a hybrid model, where channel gating is treated stochastically, while calcium concentration is deterministic and (ii) a fully stochastic model with noisy channel gating and Brownian calcium ion motion. The second model utilises the recently developed two-regime method [M. B. Flegg, S. J. Chapman, and R. Erban, "The two-regime method for optimizing stochastic reaction-diffusion simulations," J. R. Soc., Interface 9, 859-868 (2012)], 10.1098/rsif.2011.0574 in order to simulate a large domain with precision required only near the Ca2+ absorbing channels. The expected time for a first channel opening that results in a calcium puff event is calculated. It is found that for a large diffusion constant, predictions of the interpuff time are significantly overestimated using the model (i) with a deterministic non-spatial calcium variable. It is thus demonstrated that the presence of diffusive noise in local concentrations of intracellular Ca2+ ions can substantially influence the occurrence of calcium signals. The presented approach and results may also be relevant for other cell-physiological first-passage time problems with

  8. Role of Diatoms in the Spatial-Temporal Distribution of Intracellular Nitrate in Intertidal Sediment

    PubMed Central

    Stief, Peter; Kamp, Anja; de Beer, Dirk

    2013-01-01

    Intracellular nitrate storage allows microorganisms to survive fluctuating nutrient availability and anoxic conditions in aquatic ecosystems. Here we show that diatoms, ubiquitous and highly abundant microalgae, represent major cellular reservoirs of nitrate in an intertidal flat of the German Wadden Sea and are potentially involved in anaerobic nitrate respiration. Intracellular nitrate (ICNO3) was present year-round in the sediment and was spatially and temporally correlated with fucoxanthin, the marker photopigment of diatoms. Pyrosequencing of SSU rRNA genes of all domains of life confirmed that ICNO3 storage was most likely due to diatoms rather than other known nitrate-storing microorganisms (i.e., large sulfur bacteria and the eukaryotic foraminifers and gromiids). Sedimentary ICNO3 concentrations reached up to 22.3 µmol dm-3 at the sediment surface and decreased with sediment depth to negligible concentrations below 5 cm. Similarly, the ICNO3/fucoxanthin ratio and porewater nitrate (PWNO3) concentrations decreased with sediment depth, suggesting that ICNO3 of diatoms is in equilibrium with PWNO3, but is enriched relative to PWNO3 by 2-3 orders of magnitude. Cell-volume-specific ICNO3 concentrations in a diatom mat covering the sediment surface during spring were estimated at 9.3-46.7 mmol L-1. Retrieval of 18S rRNA gene sequences related to known nitrate-storing and nitrate-ammonifying diatom species suggested that diatoms in dark and anoxic sediment layers might be involved in anaerobic nitrate respiration. Due to the widespread dominance of diatoms in microphytobenthos, the total nitrate pool in coastal marine sediments may generally be at least two times larger than derived from porewater measurements and partially be recycled to ammonium. PMID:24023845

  9. Identification of iron-responsive proteins expressed by Chlamydia trachomatis reticulate bodies during intracellular growth.

    PubMed

    Dill, Brian D; Dessus-Babus, Sophie; Raulston, Jane E

    2009-01-01

    The obligate intracellular bacterium Chlamydia trachomatis serovar E is the most prevalent cause of bacterial sexually transmitted disease. With an established requirement for iron, the developmental cycle arrests at the intracellular reticulate body stage during iron restriction, resulting in a phenomenon termed persistence. Persistence has implications in natural infections for altered expression of virulence factors and antigens, in addition to a potential role in producing chronic infection. In this study, chlamydial proteins in iron-restricted, infected HEC-1B cells were radiolabelled during mid-developmental cycle growth, harvested, and separated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Of approximately 250 radiolabelled protein species visualized, densitometric analysis revealed 25 proteins that increased in expression under iron restriction compared to iron-sufficient control samples; ten protein species identified by mass spectrometry are involved in the oxidative damage response (alkyl hydroperoxide reductase, 6-phosphogluconolactonase and acyl carrier protein synthase), transcription (RNA polymerase subunit alpha and transcription anti-termination factors NusA and NusG), protein modification (peptide deformylase and trigger factor), and virulence (Chlamydia protein associating with death domains, CADD). Transcript-level expression patterns of ahpC, devB, cadd, fabF and ct538 were measured by quantitative RT-PCR throughout the developmental cycle, and each gene examined demonstrated a significant but small mid-cycle increase in transcript level in iron-restricted cultures compared to iron-replete controls. Taken together, these data suggest that the primary response of chlamydiae to reduced iron availability is to increase expression of proteins involved in protection against oxidative damage via iron-catalysed generation of reactive oxygen species and adaptation to stress by increasing expression of transcriptional machinery

  10. Role of diatoms in the spatial-temporal distribution of intracellular nitrate in intertidal sediment.

    PubMed

    Stief, Peter; Kamp, Anja; de Beer, Dirk

    2013-01-01

    Intracellular nitrate storage allows microorganisms to survive fluctuating nutrient availability and anoxic conditions in aquatic ecosystems. Here we show that diatoms, ubiquitous and highly abundant microalgae, represent major cellular reservoirs of nitrate in an intertidal flat of the German Wadden Sea and are potentially involved in anaerobic nitrate respiration. Intracellular nitrate (ICNO3) was present year-round in the sediment and was spatially and temporally correlated with fucoxanthin, the marker photopigment of diatoms. Pyrosequencing of SSU rRNA genes of all domains of life confirmed that ICNO3 storage was most likely due to diatoms rather than other known nitrate-storing microorganisms (i.e., large sulfur bacteria and the eukaryotic foraminifers and gromiids). Sedimentary ICNO3 concentrations reached up to 22.3 µmol dm(-3) at the sediment surface and decreased with sediment depth to negligible concentrations below 5 cm. Similarly, the ICNO3/fucoxanthin ratio and porewater nitrate (PWNO3) concentrations decreased with sediment depth, suggesting that ICNO3 of diatoms is in equilibrium with PWNO3, but is enriched relative to PWNO3 by 2-3 orders of magnitude. Cell-volume-specific ICNO3 concentrations in a diatom mat covering the sediment surface during spring were estimated at 9.3-46.7 mmol L(-1). Retrieval of 18S rRNA gene sequences related to known nitrate-storing and nitrate-ammonifying diatom species suggested that diatoms in dark and anoxic sediment layers might be involved in anaerobic nitrate respiration. Due to the widespread dominance of diatoms in microphytobenthos, the total nitrate pool in coastal marine sediments may generally be at least two times larger than derived from porewater measurements and partially be recycled to ammonium.

  11. Nanoscale intracellular organization and functional architecture mediating cellular behavior.

    PubMed

    LeDuc, Philip P; LeDuc, Philip R; Bellin, Robert R; Bellin, Robert M

    2006-01-01

    Cells function based on a complex set of interactions that control pathways resulting in ultimate cell fates including proliferation, differentiation, and apoptosis. The inter-workings of this immensely dense network of intracellular molecules are influenced by more than random protein and nucleic acid distribution where their interactions culminate in distinct cellular function. By probing the design of these biological systems from an engineering perspective, researchers can gain great insight that will aid in building and utilizing systems that are on this size scale where traditional large-scale rules may fail to apply. The organized interaction and gradient distribution in intracellular space imply a structural architecture that modulates cellular processes by influencing biochemical interactions including transport and binding-reactions. One significant structure that plays a role in this modulation is the cell cytoskeleton. Here, we discuss the cytoskeleton as a central and integrating functional structure in influencing cell processes and we describe technology useful for probing this structure. We explain the nanometer scale science of cytoskeletal structure with respect to intracellular organization, mechanotransduction, cytoskeletal-associated proteins, and motor molecules, as well as nano- and microtechnologies that are applicable for experimental studies of the cytoskeleton. This biological architecture of the cytoskeleton influences molecular, cellular, and physiological processes through structured multimodular and hierarchical principles centered on these functional filaments. Through investigating these organic systems that have evolved over billions of years, understanding in biology, engineering, and nanometer-scaled science will be advanced.

  12. Intracellular glutathione determines bortezomib cytotoxicity in multiple myeloma cells

    PubMed Central

    Starheim, K K; Holien, T; Misund, K; Johansson, I; Baranowska, K A; Sponaas, A-M; Hella, H; Buene, G; Waage, A; Sundan, A; Bjørkøy, G

    2016-01-01

    Multiple myeloma (myeloma in short) is an incurable cancer of antibody-producing plasma cells that comprise 13% of all hematological malignancies. The proteasome inhibitor bortezomib has improved treatment significantly, but inherent and acquired resistance to the drug remains a problem. We here show that bortezomib-induced cytotoxicity was completely dampened when cells were supplemented with cysteine or its derivative, glutathione (GSH) in ANBL-6 and INA-6 myeloma cell lines. GSH is a major component of the antioxidative defense in eukaryotic cells. Increasing intracellular GSH levels fully abolished bortezomib-induced cytotoxicity and transcriptional changes. Elevated intracellular GSH levels blocked bortezomib-induced nuclear factor erythroid 2-related factor 2 (NFE2L2, NRF2)-associated stress responses, including upregulation of the xCT subunit of the Xc- cystine-glutamate antiporter. INA-6 cells conditioned to increasing bortezomib doses displayed reduced bortezomib sensitivity and elevated xCT levels. Inhibiting Xc- activity potentiated bortezomib-induced cytotoxicity in myeloma cell lines and primary cells, and re-established sensitivity to bortezomib in bortezomib-conditioned cells. We propose that intracellular GSH level is the main determinant of bortezomib-induced cytotoxicity in a subset of myeloma cells, and that combined targeting of the proteasome and the Xc- cystine-glutamate antiporter can circumvent bortezomib resistance. PMID:27421095

  13. Intracellular calcium buffering declines in aging adrenergic nerves.

    PubMed

    Tsai, H; Hewitt, C W; Buchholz, J N; Duckles, S P

    1997-01-01

    Stimulation-evoked norepinephrine release from rat tail artery adrenergic nerves increased with advancing age in the Fischer-344 rat when function of norepinephrine uptake mechanisms and prejunctional alpha-2 adrenoceptors were blocked. When calcium channels were bypassed with the ionophore, ionomycin (4 microM), norepinephrine release from aged nerves (20 months) was still elevated as compared to 6-month-old nerves. Norepinephrine release stimulated by high K+ was also higher in 20-month nerves. The intracellular calcium chelator, 1,2 bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetomethylester (BAPTA/AM), was used to determine whether age-related increases in norepinephrine release could be reversed with the addition of an artificial intracellular calcium buffer. Exposure to BAPTA/AM decreased stimulation-evoked norepinephrine release in both old and young tail arteries; however, the effect was significantly greater in older arteries. When mitochondrial calcium uptake was compromised using the uncoupler of mitochondrial oxidative phosphorylation, dinitrophenol, BAPTA caused a further decrease in stimulation-evoked norepinephrine release in 20-month tail arteries with much less effect in 6-month-old nerves. These results suggest that intracellular calcium buffering is less efficient in older nerves.

  14. Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy

    PubMed Central

    Zhang, Jinxie; Zhang, Xudong; Liu, Gan; Chang, Danfeng; Liang, Xin; Zhu, Xianbing; Tao, Wei; Mei, Lin

    2016-01-01

    The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy. However, the details of the intracellular trafficking pathway of nanoparticles in cells have been poorly investigated. Here, we investigate in detail the intracellular trafficking pathway of protein nanocapsules using more than 30 Rab proteins as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy. We observed that FITC-labeled protein nanoparticles were internalized by the cells mainly through Arf6-dependent endocytosis and Rab34-mediated micropinocytosis. In addition to this classic pathway: early endosome (EEs)/late endosome (LEs) to lysosome, we identified two novel transport pathways: micropinocytosis (Rab34 positive)-LEs (Rab7 positive)-lysosome pathway and EEs-liposome (Rab18 positive)-lysosome pathway. Moreover, the cells use slow endocytosis recycling pathway (Rab11 and Rab35 positive vesicles) and GLUT4 exocytosis vesicles (Rab8 and Rab10 positive) transport the protein nanocapsules out of the cells. In addition, protein nanoparticles are observed in autophagosomes, which receive protein nanocapsules through multiple endocytosis vesicles. Using autophagy inhibitor to block these transport pathways could prevent the degradation of nanoparticles through lysosomes. Using Rab proteins as vesicle markers to investigation the detail intracellular trafficking of the protein nanocapsules, will provide new targets to interfere the cellular behaver of the nanoparticles, and improve the therapeutic effect of nanomedicine. PMID:27698943

  15. Quantification of intracellular payload release from polymersome nanoparticles

    PubMed Central

    Scarpa, Edoardo; Bailey, Joanne L.; Janeczek, Agnieszka A.; Stumpf, Patrick S.; Johnston, Alexander H.; Oreffo, Richard O. C.; Woo, Yin L.; Cheong, Ying C.; Evans, Nicholas D.; Newman, Tracey A.

    2016-01-01

    Polymersome nanoparticles (PMs) are attractive candidates for spatio-temporal controlled delivery of therapeutic agents. Although many studies have addressed cellular uptake of solid nanoparticles, there is very little data available on intracellular release of molecules encapsulated in membranous carriers, such as polymersomes. Here, we addressed this by developing a quantitative assay based on the hydrophilic dye, fluorescein. Fluorescein was encapsulated stably in PMs of mean diameter 85 nm, with minimal leakage after sustained dialysis. No fluorescence was detectable from fluorescein PMs, indicating quenching. Following incubation of L929 cells with fluorescein PMs, there was a gradual increase in intracellular fluorescence, indicating PM disruption and cytosolic release of fluorescein. By combining absorbance measurements with flow cytometry, we quantified the real-time intracellular release of a fluorescein at a single-cell resolution. We found that 173 ± 38 polymersomes released their payload per cell, with significant heterogeneity in uptake, despite controlled synchronisation of cell cycle. This novel method for quantification of the release of compounds from nanoparticles provides fundamental information on cellular uptake of nanoparticle-encapsulated compounds. It also illustrates the stochastic nature of population distribution in homogeneous cell populations, a factor that must be taken into account in clinical use of this technology. PMID:27404770

  16. Calpeptin Attenuated Apoptosis and Intracellular Inflammatory Changes in Muscle Cells

    PubMed Central

    Nozaki, Kenkichi; Das, Arabinda; Ray, Swapan K.; Banik, Naren L.

    2011-01-01

    In idiopathic inflammatory myopathies (IIMs), extracellular inflammatory stimulation is considered to induce secondary intracellular inflammatory changes including expression of major histocompatibility complex class-I (MHC-I) and to produce self-sustaining loop of inflammation. We hypothesize that activation of calpain, a Ca2+-sensitive protease, bridges between these extracellular inflammatory stress and intracellular secondary inflammatory changes in muscle cells. In this study, we demonstrated that treatment of rat L6 myoblast cells with interferon-gamma (IFN-γ) caused expression of MHC-I and inflammation related transcription factors (phosphorylated-extracellular signal-regulated kinase 1/2 and nuclear factor-kappa B). We also demonstrated that treatment with tumor necrosis factor-alpha (TNF-α) induced apoptotic changes and activation of calpain and cyclooxygenase-2. Further, we found that post-treatment with calpeptin attenuated the intracellular changes induced by IFN-γ or TNF-α. Our results indicate that calpain inhibition attenuates apoptosis and secondary inflammatory changes induced by extracellular inflammatory stimulation in the muscle cells. These results suggest calpain as a potential therapeutic target for treatment of IIMs. PMID:21290412

  17. Gamma Band Activity in the RAS-intracellular mechanisms

    PubMed Central

    Garcia-Rill, E.; Kezunovic, N.; D’Onofrio, S.; Luster, B.; Hyde, J.; Bisagno, V.; Urbano, F.J.

    2014-01-01

    Gamma band activity participates in sensory perception, problem solving, and memory. This review considers recent evidence showing that cells in the reticular activating system (RAS) exhibit gamma band activity, and describes the intrinsic membrane properties behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN), intralaminar parafascicular nucleus (Pf), and pontine Subcoeruleus nucleus dorsalis (SubCD) all fire in the gamma band range when maximally activated, but no higher. The mechanisms involve high threshold, voltage-dependent P/Q-type calcium channels or sodium-dependent subthreshold oscillations. Rather than participating in the temporal binding of sensory events as in the cortex, gamma band activity in the RAS may participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. We address three necessary next steps resulting from these discoveries, an intracellular mechanism responsible for maintaining gamma band activity based on persistent G-protein activation, separate intracellular pathways that differentiate between gamma band activity during waking vs during REM sleep, and an intracellular mechanism responsible for the dysregulation in gamma band activity in schizophrenia. These findings open several promising research avenues that have not been thoroughly explored. What are the effects of sleep or REM sleep deprivation on these RAS mechanisms? Are these mechanisms involved in memory processing during waking and/or during REM sleep? Does gamma band processing differ during waking vs REM sleep after sleep or REM sleep deprivation? PMID:24309750

  18. Twenty years of fluorescence imaging of intracellular chloride

    PubMed Central

    Arosio, Daniele; Ratto, Gian Michele

    2014-01-01

    Chloride homeostasis has a pivotal role in controlling neuronal excitability in the adult brain and during development. The intracellular concentration of chloride is regulated by the dynamic equilibrium between passive fluxes through membrane conductances and the active transport mediated by importers and exporters. In cortical neurons, chloride fluxes are coupled to network activity by the opening of the ionotropic GABAA receptors that provides a direct link between the activity of interneurons and chloride fluxes. These molecular mechanisms are not evenly distributed and regulated over the neuron surface and this fact can lead to a compartmentalized control of the intracellular concentration of chloride. The inhibitory drive provided by the activity of the GABAA receptors depends on the direction and strength of the associated currents, which are ultimately dictated by the gradient of chloride, the main charge carrier flowing through the GABAA channel. Thus, the intracellular distribution of chloride determines the local strength of ionotropic inhibition and influences the interaction between converging excitation and inhibition. The importance of chloride regulation is also underlined by its involvement in several brain pathologies, including epilepsy and disorders of the autistic spectra. The full comprehension of the physiological meaning of GABAergic activity on neurons requires the measurement of the spatiotemporal dynamics of chloride fluxes across the membrane. Nowadays, there are several available tools for the task, and both synthetic and genetically encoded indicators have been successfully used for chloride imaging. Here, we will review the available sensors analyzing their properties and outlining desirable future developments. PMID:25221475

  19. Probing cytoskeleton dynamics by intracellular particle transport analysis

    NASA Astrophysics Data System (ADS)

    Götz, M.; Hodeck, K. F.; Witzel, P.; Nandi, A.; Lindner, B.; Heinrich, D.

    2015-07-01

    All cellular functions arise from the transport of molecules through a heterogeneous, highly dynamic cell interior for intracellular signaling. Here, the impact of intracellular architecture and cytoskeleton dynamics on transport processes is revealed by high-resolution single particle tracking within living cells, in combination with time-resolved local mean squared displacement (I-MSD) analysis. We apply the I-MSD analysis to trajectories of 200 nm silica particles within living cells of Dictyostelium discoideum obtained by high resolution spinning disc confocal microscopy with a frame rate of 100 fps and imaging in one fixed focal plane. We investigate phases of motor-driven active transport and subdiffusion, normal diffusion, as well as superdiffusion with high spatial and temporal resolution. Active directed intracellular motion is attributed to microtubule associated molecular motor driven transport with average absolute velocities of 2.8 μm s-1 for 200 nm diameter particles. Diffusion processes of these particles within wild-type cells are found to exhibit diffusion constants ranging across two orders of magnitude from subdiffusive to superdiffusive behavior. This type of analysis might prove of ample importance for medical applications, like targeted drug treatment of cells by nano-sized carriers or innovative diagnostic assays.

  20. Quantification of intracellular payload release from polymersome nanoparticles

    NASA Astrophysics Data System (ADS)

    Scarpa, Edoardo; Bailey, Joanne L.; Janeczek, Agnieszka A.; Stumpf, Patrick S.; Johnston, Alexander H.; Oreffo, Richard O. C.; Woo, Yin L.; Cheong, Ying C.; Evans, Nicholas D.; Newman, Tracey A.

    2016-07-01

    Polymersome nanoparticles (PMs) are attractive candidates for spatio-temporal controlled delivery of therapeutic agents. Although many studies have addressed cellular uptake of solid nanoparticles, there is very little data available on intracellular release of molecules encapsulated in membranous carriers, such as polymersomes. Here, we addressed this by developing a quantitative assay based on the hydrophilic dye, fluorescein. Fluorescein was encapsulated stably in PMs of mean diameter 85 nm, with minimal leakage after sustained dialysis. No fluorescence was detectable from fluorescein PMs, indicating quenching. Following incubation of L929 cells with fluorescein PMs, there was a gradual increase in intracellular fluorescence, indicating PM disruption and cytosolic release of fluorescein. By combining absorbance measurements with flow cytometry, we quantified the real-time intracellular release of a fluorescein at a single-cell resolution. We found that 173 ± 38 polymersomes released their payload per cell, with significant heterogeneity in uptake, despite controlled synchronisation of cell cycle. This novel method for quantification of the release of compounds from nanoparticles provides fundamental information on cellular uptake of nanoparticle-encapsulated compounds. It also illustrates the stochastic nature of population distribution in homogeneous cell populations, a factor that must be taken into account in clinical use of this technology.

  1. Mechanisms of Borrelia burgdorferi internalization and intracellular innate immune signaling.

    PubMed

    Petnicki-Ocwieja, Tanja; Kern, Aurelie

    2014-01-01

    Lyme disease is a long-term infection whose most severe pathology is characterized by inflammatory arthritis of the lower bearing joints, carditis, and neuropathy. The inflammatory cascades are initiated through the early recognition of invading Borrelia burgdorferi spirochetes by cells of the innate immune response, such as neutrophils and macrophage. B. burgdorferi does not have an intracellular niche and thus much research has focused on immune pathways activated by pathogen recognition molecules at the cell surface, such as the Toll-like receptors (TLRs). However, in recent years, studies have shown that internalization of the bacterium by host cells is an important component of the defense machinery in response to B. burgdorferi. Upon internalization, B. burgdorferi is trafficked through an endo/lysosomal pathway resulting in the activation of a number of intracellular pathogen recognition receptors including TLRs and Nod-like receptors (NLRs). Here we will review the innate immune molecules that participate in both cell surface and intracellular immune activation by B. burgdorferi.

  2. Potent Antibacterial Nanoparticles against Biofilm and Intracellular Bacteria

    NASA Astrophysics Data System (ADS)

    Mu, Haibo; Tang, Jiangjiang; Liu, Qianjin; Sun, Chunli; Wang, Tingting; Duan, Jinyou

    2016-01-01

    The chronic infections related to biofilm and intracellular bacteria are always hard to be cured because of their inherent resistance to both antimicrobial agents and host defenses. Herein we develop a facile approach to overcome the above conundrum through phosphatidylcholine-decorated Au nanoparticles loaded with gentamicin (GPA NPs). The nanoparticles were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS) and ultraviolet‑visible (UV‑vis) absorption spectra which demonstrated that GPA NPs with a diameter of approximately 180 nm were uniform. The loading manner and release behaviors were also investigated. The generated GPA NPs maintained their antibiotic activities against planktonic bacteria, but more effective to damage established biofilms and inhibited biofilm formation of pathogens including Gram-positive and Gram-negative bacteria. In addition, GPA NPs were observed to be nontoxic to RAW 264.7 cells and readily engulfed by the macrophages, which facilitated the killing of intracellular bacteria in infected macrophages. These results suggested GPA NPs might be a promising antibacterial agent for effective treatment of chronic infections due to microbial biofilm and intracellular bacteria.

  3. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

    PubMed Central

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  4. Ca2+ signaling and intracellular Ca2+ binding proteins.

    PubMed

    Niki, I; Yokokura, H; Sudo, T; Kato, M; Hidaka, H

    1996-10-01

    Changes in cytosolic Ca2+ concentrations evoke a wide range of cellular responses and intracellular Ca(2+)-binding proteins are the key molecules to transduce Ca2+ signaling via enzymatic reactions or modulation of protein/protein interations (Fig.1). The EF hand proteins, like calmodulin and S100 proteins, are considered to exert Ca(2+)-dependent actions in the nucleus or the cytoplasm. The Ca2+/phospholipid binding proteins are classified into two groups, the annexins and the C2 region proteins. These proteins, distributed mainly in the cytoplasm, translocate to the plasma membrane in response to an increase in cytosolic Ca2+ and function in the vicinity of the membrane. Ca2+ storage proteins in the endoplasmic or sarcoplasmic reticulum provide the high Ca2+ capacity of the Ca2+ store sites, which regulate intracellular Ca2+ distribution. The variety and complexity of Ca2+ signaling result from the cooperative actions of specific Ca(2+)-binding proteins. This review describes biochemical properties of intracellular Ca(2+)-binding proteins and their proposed roles in mediating Ca2+ signaling.

  5. KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

    PubMed Central

    Goitre, Luca; Balzac, Fiorella; Degani, Simona; Degan, Paolo; Marchi, Saverio; Pinton, Paolo; Retta, Saverio Francesco

    2010-01-01

    KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1−/− cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1−/− cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45α, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the

  6. KRIT1 regulates the homeostasis of intracellular reactive oxygen species.

    PubMed

    Goitre, Luca; Balzac, Fiorella; Degani, Simona; Degan, Paolo; Marchi, Saverio; Pinton, Paolo; Retta, Saverio Francesco

    2010-07-26

    KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1(-/-) cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1(-/-) cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45alpha, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the cell

  7. The C-terminal domain controls the mobility of Crumbs 3 isoforms.

    PubMed

    Djuric, Ivona; Siebrasse, Jan Peter; Schulze, Ulf; Granado, Daniel; Schlüter, Marc A; Kubitscheck, Ulrich; Pavenstädt, Hermann; Weide, Thomas

    2016-06-01

    The physiological function of epithelia depends on an asymmetric distribution of their membrane domains. Polarity proteins play a crucial role for distribution processes, however, little is known about their mobility in epithelial cells. In this study, we analyzed the intracellular and plasma-membrane-associated mobility of fluorescence-labeled Crb3A and Crb3B. Both variants belong to the Crumbs protein family, which control size and identity of apical membranes in epithelial cells. Fluorescence recovery after photo-bleaching measurements revealed different mobilities for the two Crb3 variants. They also differentially affected mobility and localization of the Pals1/Mpp5 protein, which binds to Crb3A but not to Crb3B. In addition, tracking of intracellular vesicles indicated that Crb3A containing vesicles are slightly more immobile than Crb3B ones. Taken together, our data revealed different intracellular mobility patterns for Crb3A and Crb3B.

  8. Calcium-dependent stoichiometries of the KCa2.2 (SK) intracellular domain/calmodulin complex in solution.

    PubMed

    Halling, D Brent; Kenrick, Sophia A; Riggs, Austen F; Aldrich, Richard W

    2014-02-01

    Ca(2+) activates SK Ca(2+)-activated K(+) channels through the protein Ca(2+) sensor, calmodulin (CaM). To understand how SK channels operate, it is necessary to determine how Ca(2+) regulates CaM binding to its target on SK. Tagless, recombinant SK peptide (SKp), was purified for binding studies with CaM at low and high Ca(2+) concentrations. Composition gradient multi-angle light scattering accurately measures the molar mass, stoichiometry, and affinity of protein complexes. In 2 mM Ca(2+), SKp and CaM bind with three different stoichiometries that depend on the molar ratio of SKp:CaM in solution. These complexes include 28 kD 1SKp/1CaM, 39 kD 2SKp/1CaM, and 44 kD 1SKp/2CaM. A 2SKp/2CaM complex, observed in prior crystallographic studies, is absent. At <5 nM Ca(2+), 1SKp/1CaM and 2SKp/1CaM were observed; however, 1SKp/2CaM was absent. Analytical ultracentrifugation was used to characterize the physical properties of the three SKp/CaM stoichiometries. In high Ca(2+), the sedimentation coefficient is smaller for a 1SKp:1CaM solution than it is for either 2SKp:1CaM or 1SKp:2CaM. At low Ca(2+) and at >100 µM protein concentrations, a molar excess of SKp over CaM causes aggregation. Aggregation is not observed in Ca(2+) or with CaM in molar excess. In low Ca(2+) both 1SKp:1CaM and 1SKp:2CaM solutions have similar sedimentation coefficients, which is consistent with the absence of a 1SKp/2CaM complex in low Ca(2+). These results suggest that complexes with stoichiometries other than 2SKp/2CaM are important in gating.

  9. A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE.

    PubMed

    Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

    2016-03-18

    Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N cyt/C exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders.

  10. Modulation of the Activity of Mycobacterium tuberculosis LipY by Its PE Domain.

    PubMed

    Garrett, Christopher K; Broadwell, Lindsey J; Hayne, Cassandra K; Neher, Saskia B

    2015-01-01

    Mycobacterium tuberculosis harbors over 160 genes encoding PE/PPE proteins, several of which have roles in the pathogen's virulence. A number of PE/PPE proteins are secreted via Type VII secretion systems known as the ESX secretion systems. One PE protein, LipY, has a triglyceride lipase domain in addition to its PE domain. LipY can regulate intracellular triglyceride levels and is also exported to the cell wall by one of the ESX family members, ESX-5. Upon export, LipY's PE domain is removed by proteolytic cleavage. Studies using cells and crude extracts suggest that LipY's PE domain not only directs its secretion by ESX-5, but also functions to inhibit its enzymatic activity. Here, we attempt to further elucidate the role of LipY's PE domain in the regulation of its enzymatic activity. First, we established an improved purification method for several LipY variants using detergent micelles. We then used enzymatic assays to confirm that the PE domain down-regulates LipY activity. The PE domain must be attached to LipY in order to effectively inhibit it. Finally, we determined that full length LipY and the mature lipase lacking the PE domain (LipYΔPE) have similar melting temperatures. Based on our improved purification strategy and activity-based approach, we concluded that LipY's PE domain down-regulates its enzymatic activity but does not impact the thermal stability of the enzyme.

  11. Swarming in bounded domains

    NASA Astrophysics Data System (ADS)

    Armbruster, Dieter; Motsch, Sébastien; Thatcher, Andrea

    2017-04-01

    The Vicsek model is a prototype for the emergence of collective motion. In free space, it is characterized by a swarm of particles all moving in the same direction. Since this dynamic does not include attraction among particles, the swarm, while aligning in velocity space, has no spatial coherence. Adding specular reflection at the boundaries generates global spatial coherence of the swarms while maintaining its velocity alignment. We investigate numerically how the geometry of the domain influences the Vicsek model using three type of geometry: a channel, a disk and a rectangle. Varying the parameters of the Vicsek model (e.g. noise levels and influence horizons), we discuss the mechanisms that generate spatial coherence and show how they create new dynamical solutions of the swarming motions in these geometries. Several observables are introduced to characterize the simulated patterns (e.g. mass profile, center of mass, connectivity of the swarm).

  12. Beyond the Number Domain

    PubMed Central

    Cantlon, Jessica F.; Platt, Michael L.; Brannon, Elizabeth M.

    2009-01-01

    In a world without numbers, we would be unable to build a skyscraper, hold a national election, plan a wedding, or pay for a chicken at the market. The numerical symbols used in all these behaviors build on the approximate number system (ANS) which represents the number of discrete objects or events as a continuous mental magnitude. In this review, we first discuss evidence that the ANS bears a set of behavioral and brain signatures that are universally displayed across animal species, human cultures, and development. We then turn to the question of whether the ANS constitutes a specialized cognitive and neural domain--a question central to understanding how this system works, the nature of its evolutionary and developmental trajectory, and its physical instantiation in the brain. PMID:19131268

  13. Gating of two pore domain potassium channels.

    PubMed

    Mathie, Alistair; Al-Moubarak, Ehab; Veale, Emma L

    2010-09-01

    Two-pore-domain potassium (K2P) channels are responsible for background leak currents which regulate the membrane potential and excitability of many cell types. Their activity is modulated by a variety of chemical and physical stimuli which act to increase or decrease the open probability of individual K2P channels. Crystallographic data and homology modelling suggest that all K(+) channels possess a highly conserved structure for ion selectivity and gating mechanisms. Like other K(+) channels, K2P channels are thought to have two primary conserved gating mechanisms: an inactivation (or C-type) gate at the selectivity filter close to the extracellular side of the channel and an activation gate at the intracellular entrance to the channel involving key, identified, hinge glycine residues. Zinc and hydrogen ions regulate Drosophila KCNK0 and mammalian TASK channels, respectively, by interacting with the inactivation gate of these channels. In contrast, the voltage dependence of TASK3 channels is mediated through its activation gate. For KCNK0 it has been shown that the gates display positive cooperativity. It is of much interest to determine whether other K2P regulatory compounds interact with either the activation gate or the inactivation gate to alter channel activity or, indeed, whether additional regulatory gating pathways exist.

  14. Gating of two pore domain potassium channels

    PubMed Central

    Mathie, Alistair; Al-Moubarak, Ehab; Veale, Emma L

    2010-01-01

    Two-pore-domain potassium (K2P) channels are responsible for background leak currents which regulate the membrane potential and excitability of many cell types. Their activity is modulated by a variety of chemical and physical stimuli which act to increase or decrease the open probability of individual K2P channels. Crystallographic data and homology modelling suggest that all K+ channels possess a highly conserved structure for ion selectivity and gating mechanisms. Like other K+ channels, K2P channels are thought to have two primary conserved gating mechanisms: an inactivation (or C-type) gate at the selectivity filter close to the extracellular side of the channel and an activation gate at the intracellular entrance to the channel involving key, identified, hinge glycine residues. Zinc and hydrogen ions regulate Drosophila KCNK0 and mammalian TASK channels, respectively, by interacting with the inactivation gate of these channels. In contrast, the voltage dependence of TASK3 channels is mediated through its activation gate. For KCNK0 it has been shown that the gates display positive cooperativity. It is of much interest to determine whether other K2P regulatory compounds interact with either the activation gate or the inactivation gate to alter channel activity or, indeed, whether additional regulatory gating pathways exist. PMID:20566661

  15. Structure of sorting nexin 11 (SNX11) reveals a novel extended phox homology (PX) domain critical for inhibition of SNX10-induced vacuolation.

    PubMed

    Xu, Jinxin; Xu, Tingting; Wu, Bin; Ye, Yinghua; You, Xiaojuan; Shu, Xiaodong; Pei, Duanqing; Liu, Jinsong

    2013-06-07

    Sorting nexins are phox homology (PX) domain-containing proteins involved in diverse intracellular endosomal trafficking pathways. The PX domain binds to certain phosphatidylinositols and is recruited to vesicles rich in these lipids. The structure of the PX domain is highly conserved, containing a three-stranded β-sheet, followed by three α-helices. Here, we report the crystal structures of truncated human SNX11 (sorting nexin 11). The structures reveal that SNX11 contains a novel PX domain, hereby named the extended PX (PXe) domain, with two additional α-helices at the C terminus. We demonstrate that these α-helices are indispensible for the in vitro functions of SNX11. We propose that this PXe domain is present in SNX10 and is responsible for the vacuolation activity of SNX10. Thus, this novel PXe domain constitutes a structurally and functionally important PX domain subfamily.

  16. Effects of eliminating a disulfide bridge within domain II of Pseudomonas aeruginosa exotoxin A.

    PubMed Central

    Madshus, I H; Collier, R J

    1989-01-01

    Cysteines 265 and 287 of Pseudomonas aeruginosa exotoxin A (ETA) were substituted by serine, thereby eliminating a disulfide bridge within domain II, the putative membrane insertion-translocation domain. Purified mutant toxin was 80-fold less toxic for mouse L cells than was wild-type ETA while retaining the same specific activity in the ADP-ribosyltransferase reaction as did wild-type toxin. Binding of the nonionic detergent Triton X-114 by mutant ETA occurred at a slightly higher pH than did binding by wild-type ETA, suggesting that the mutant protein more readily undergoes a conformational change exposing hydrophobic regions. Data are presented supporting the notion that the mutant and wild-type toxins enter from the same intracellular compartment. The lower cytotoxicity of the mutant protein could be due to accelerated intracellular degradation or abortive, premature membrane insertion. Images PMID:2499539

  17. Cholesterol-rich Fluid Membranes Solubilize Ceramide Domains

    PubMed Central

    Castro, Bruno M.; Silva, Liana C.; Fedorov, Alexander; de Almeida, Rodrigo F. M.; Prieto, Manuel

    2009-01-01

    A uniquely sensitive method for ceramide domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide concentrations, ranging from low or no cholesterol (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high cholesterol (similar to mammalian plasma membrane). Diverse fluorescence spectroscopy and microscopy experiments were conducted showing that for low cholesterol amounts ceramide segregates into gel domains that disappear upon increasing cholesterol levels. This was observed in different raft (sphingomyelin/cholesterol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Cholesterol-ceramide interactions have been described mainly as raft sphingomyelin-dependent. Here sphingomyelin independence is demonstrated. In addition, ceramide-rich domains re-appear when either cholesterol is converted by cholesterol oxidase to cholestenone or the temperature is decreased. Ceramide is more soluble in cholesterol-rich fluid membranes than in cholesterol-poor ones, thereby increasing the chemical potential of cholesterol. Ceramide solubility depends on the average gel-fluid transition temperature of the remaining membrane lipids. The inability of cholestenone-rich membranes to dissolve ceramide gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. We also show that the solubility of cholesterol in ceramide domains is low. The results are rationalized by a ternary phospholipid/ceramide/cholesterol phase diagram, providing the framework for the better understanding of biochemical phenomena modulated by cholesterol-ceramide interactions such as cholesterol oxidase activity, lipoprotein metabolism, and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed unless a

  18. Surface functionalized mesoporous silica nanoparticles for intracellular drug delivery

    NASA Astrophysics Data System (ADS)

    Vivero-Escoto, Juan Luis

    Mesoporous silica nanoparticles (MSNs) are a highly promising platform for intracellular controlled release of drugs and biomolecules. Despite that the application of MSNs in the field of intracellular drug delivery is still at its infancy very exciting breakthroughs have been achieved in the last years. A general review of the most recent progress in this area of research is presented, including a description of the latest findings on the pathways of entry into live mammalian cells together with the intracellular trafficking, a summary on the contribution of MSNs to the development of site-specific drug delivery systems, a report on the biocompatibility of this material in vitro andin vivo, and a discussion on the most recent breakthroughs in the synthesis and application of stimuli-responsive mesoporous silica-based delivery vehicles. A gold nanoparticles (AuNPs)-capped MSNs-based intracellular photoinduced drug delivery system (PR-AuNPs-MSNs) for the controlled release of anticancer drug inside of human fibroblast and liver cells was synthesized and characterized. We found that the mesoporous channels of MSNs could be efficiently capped by the photoresponsive AuNPs without leaking the toxic drug, paclitaxel, inside of human cells. Furthermore, we demonstrated that the cargo-release property of this PR-AuNPs-MSNs system could be easily photo-controlled under mild and biocompatible conditions in vitro. In collaboration with Renato Mortera (a visiting student from Italy), a MSNs based intracellular delivery system for controlled release of cell membrane impermeable cysteine was developed. A large amount of cysteine molecules were covalently attached to the silica surface of MSNs through cleavable disulfide linkers. These cysteine-containing nanoparticles were efficiently endocytosed by human cervical cancer cells HeLa. These materials exhibit 450 times higher cell growth inhibition capability than that of the conventional N-acetylcysteine prodrug. The ability to

  19. Ligand binding by PDZ domains.

    PubMed

    Chi, Celestine N; Bach, Anders; Strømgaard, Kristian; Gianni, Stefano; Jemth, Per

    2012-01-01

    The postsynaptic density protein-95/disks large/zonula occludens-1 (PDZ) protein domain family is one of the most common protein-protein interaction modules in mammalian cells, with paralogs present in several hundred human proteins. PDZ domains are found in most cell types, but neuronal proteins, for example, are particularly rich in these domains. The general function of PDZ domains is to bring proteins together within the appropriate cellular compartment, thereby facilitating scaffolding, signaling, and trafficking events. The many functions of PDZ domains under normal physiological as well as pathological conditions have been reviewed recently. In this review, we focus on the molecular details of how PDZ domains bind their protein ligands and their potential as drug targets in this context.

  20. Multifunctionalities driven by ferroic domains

    SciTech Connect

    Yang, J. C.; Huang, Y. L.; Chu, Y. H.; He, Q.

    2014-08-14

    Considerable attention has been paid to ferroic systems in pursuit of advanced applications in past decades. Most recently, the emergence and development of multiferroics, which exhibit the coexistence of different ferroic natures, has offered a new route to create functionalities in the system. In this manuscript, we step from domain engineering to explore a roadmap for discovering intriguing phenomena and multifunctionalities driven by periodic domain patters. As-grown periodic domains, offering exotic order parameters, periodic local perturbations and the capability of tailoring local spin, charge, orbital and lattice degrees of freedom, are introduced as modeling templates for fundamental studies and novel applications. We discuss related significant findings on ferroic domain, nanoscopic domain walls, and conjunct heterostructures based on the well-organized domain patterns, and end with future prospects and challenges in the field.

  1. AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons.

    PubMed

    Rathje, Mette; Fang, Huaqiang; Bachman, Julia L; Anggono, Victor; Gether, Ulrik; Huganir, Richard L; Madsen, Kenneth L

    2013-08-27

    NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling. Here, we demonstrate that in somatic and dendritic regions of hippocampal neurons a large fraction of the fluorescent signal originates from intracellular pH-GluA2, and that the decline in fluorescence in response to NMDA and AMPA primarily describes an intracellular acidification, which quenches the pHluorin signal from intracellular receptor pools. Neurons expressing an endoplasmic reticulum-retained mutant of GluA2 (pH-GluA2 ΔC49) displayed a larger response to NMDA than neurons expressing wild-type pH-GluA2. A similar NMDA-elicited decline in pHluorin signal was observed by expressing cytosolic pHluorin alone without fusion to GluA2 (cyto-pHluorin). Intracellular acidification in response to NMDA was further confirmed by using the ratiometric pH indicator carboxy-SNARF-1. The NMDA-induced decline was followed by rapid recovery of the fluorescent signal from both cyto-pHluorin and pH-GluA2. The recovery was sodium-dependent and sensitive to Na(+)/H(+)-exchanger (NHE) inhibitors. Moreover, recovery was more rapid after shRNA-mediated knockdown of the GluA2 binding PDZ domain-containing protein interacting with C kinase 1 (PICK1). Interestingly, the accelerating effect of PICK1 knockdown on the fluorescence recovery was eliminated in the presence of the NHE1 inhibitor zoniporide. Our results indicate that the pH-GluA2 recycling assay is an unreliable assay for studying AMPA receptor trafficking and also suggest a role for PICK1 in regulating intracellular pH via modulation of NHE activity.

  2. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling

    PubMed Central

    Stephen, Terri-Leigh; Higgs, Nathalie F.; Sheehan, David F.; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I. Lorena

    2015-01-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca2+. Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca2+-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca2+ in astrocytic processes. Thus, the regulation of intracellular Ca2+ signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca2+ wave propagation, gliotransmission, and ultimately neuronal function. SIGNIFICANCE STATEMENT Mitochondria are key cellular organelles that play important roles in providing cellular energy and buffering intracellular calcium ions. The mechanisms that control mitochondrial distribution within the processes of glial cells called astrocytes and the impact this may have on calcium signaling remains unclear. We show that activation of glutamate receptors or increased neuronal

  3. Systematic investigation on the intracellular trafficking network of polymeric nanoparticles.

    PubMed

    Zhang, Jinxie; Chang, Danfeng; Yang, Yao; Zhang, Xudong; Tao, Wei; Jiang, Lijuan; Liang, Xin; Tsai, Hsiangi; Huang, Laiqiang; Mei, Lin

    2017-02-22

    Polymeric nanoparticles such as PLGA-based nanoparticles are emerging as promising carriers for controlled drug delivery. However, little is known about the intracellular trafficking network of polymeric nanoparticles. Here, more than 30 Rab proteins were used as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy to investigate in detail the intracellular trafficking pathways of PLGA nanoparticles. We observed that coumarin-6-loaded PLGA nanoparticles were internalized by the cells mainly through caveolin and clathrin-dependent endocytosis and Rab34-mediated macropinocytosis. Then the PLGA nanoparticles were transported to early endosomes (EEs), late endosomes (LEs), and finally to lysosomes. Two novel transport pathways were identified in our research: the macropinocytosis (Rab34 positive)-LE (Rab7 positive)-lysosome pathway and the EE-liposome (Rab18)-lysosome pathway. Moreover, the slow (Rab11 and Rab35 positive), fast (Rab4 positive) and apical (Rab20 and Rab25 positive) endocytic recycling endosome pathways could transport the PLGA nanoparticles to lysosomes. The PLGA nanoparticles were transported out of the cells by GLUT4 transport vesicles (Rab8, Rab10 positive), classic secretory vesicles (Rab3, Rab27 positive vesicles) and melanosomes (Rab32, Rab38 positive vesicles). Besides, the PLGA nanoparticles were observed in autophagosomes (LC3 positive), which means that the nanoparticles can be delivered by the autophagy pathway. Multiple cross-talk pathways were identified connecting autophagy and endocytosis or exocytosis by screening the co-localization of the Rab proteins with the LC3 protein. Degradation of nanoparticles through lysosomes can be blocked by autophagy inhibitors (3 MA and CQ). A better understanding of intracellular trafficking mechanisms involved in polymeric nanoparticle-based drug delivery is a prerequisite to clinical application.

  4. Intracellular Neutralization of Virus by Immunoglobulin A Antibodies

    NASA Astrophysics Data System (ADS)

    Mazanec, Mary B.; Kaetzel, Charlotte S.; Lamm, Michael E.; Fletcher, David; Nedrud, John G.

    1992-08-01

    IgA is thought to neutralize viruses at the epithelial surface of mucous membranes by preventing their attachment. Since IgA, a polymeric immunoglobulin, is transported through the lining of epithelial cells by the polymeric-immunoglobulin receptor and since viruses are obligate intracellular parasites, we hypothesized that IgA antibodies may also interfere with viral replication by binding to newly synthesized viral proteins within infected cells. Polarized monolayers of Madin-Darby canine kidney epithelial cells expressing the polymeric-immunoglobulin receptor were infected on the apical surface with Sendai virus. Anti-Sendai virus IgA monoclonal antibody delivered from the basolateral surface colocalized with viral protein within the cell, as documented by immunofluorescence. More importantly, anti-viral IgA reduced virus titers >1000-fold (P < 0.0001) in apical supernatants and >10-fold (P < 0.0001) in cell lysates from monolayers treated with anti-viral IgA compared with those treated with either anti-viral IgG or an irrelevant IgA monoclonal antibody. We believe that the differences in viral titers between cell layers treated with specific IgA, which enters the epithelial cell by binding to the polymeric-immunoglobulin receptor, and those treated with specific IgG, which does not enter the cells, or irrelevant IgA indicate that specific intracellular IgA antibodies can inhibit viral replication. Thus, in addition to the classical role of humoral antibodies in extracellular defense, IgA antibody may be able to neutralize microbial pathogens intracellularly, giving IgA a role in host defense that has traditionally been reserved for cell-mediated immunity.

  5. Transient fluctuations of intracellular zinc ions in cell proliferation

    SciTech Connect

    Li, Yuan; Maret, Wolfgang

    2009-08-15

    Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn{sup 2+}). By employing a fluorescent Zn{sup 2+} probe, FluoZin-3 acetoxymethyl ester, intracellular Zn{sup 2+} concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn{sup 2+} concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn{sup 2+} concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn{sup 2+} concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn{sup 2+} concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn{sup 2+} concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn{sup 2+} concentrations, suggest a role for Zn{sup 2+} in the control of the cell cycle. Interventions targeted at these picomolar Zn{sup 2+} fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.

  6. Lysophosphatidic acids. Influence on platelet aggregation and intracellular calcium flux.

    PubMed Central

    Gerrard, J. M.; Kindom, S. E.; Peterson, D. A.; Peller, J.; Krantz, K. E.; White, J. G.

    1979-01-01

    Decanoyl-, palmitoyl-, and oleoyl-lysophosphatidic acid (LPA) were studied for their effects on platelet aggregation and intracellular calcium flux. Palmitoyl-LPA and oleoyl-LPA both caused a concentration-dependent aggregation of human blood platelets at concentrations of 12--300 microM. Aggregation by adenosine diphosphate (ADP) was enhanced at slightly lower concentrations. First-wave aggregation induced by these LPAs was not blocked by aspirin, indomethacin, or heparin, suggesting similarities to ADP aggregation. However, in washed platelets with a high calcium concentration, no serotonin secretion was observed, even though full aggregation occurred, suggesting that aggregation was not due to released ADP. This concept was supported by studies of platelets deficient in the storage pool of ADP and serotonin, which had a normal first-wave aggregation response to palmitoyl-LPA. Aggregation induced by palmitoyl LPA was inhibited by prostaglandin E1 (PGE1), theophylline, and ethylenediaminotetraacetate (EDTA), though in the presence of EDTA shape change occurred. Aggregation stimulated by palmitoyl-LPA or oleoyl-LPA was characterized by changes in the shape of the platelets with development of pseudopods and centralization of granules closely surrounded by contractile microfilaments and supporting microtubules. The addition of palmitoyl-LPA and oleoyl-LPA, but not decanoyl-LPA, caused the release of calcium from a platelet membrane fraction that contains elements of the intracellular calcium storage system and actively concentrates this cation in the presence of adenosine triphosphate (ATP) and magnesium. It is suggested that LPAs cause aggregation by stimulating the release of calcium intracellularly. Images Figure 1 Figure 2 Figure 3 Figure 4 Text-Figure 6 PMID:112871

  7. Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth ▿

    PubMed Central

    Nasrallah, Gheyath K.; Riveroll, Angela L.; Chong, Audrey; Murray, Lois E.; Lewis, P. Jeffrey; Garduño, Rafael A.

    2011-01-01

    The Gram-negative intracellular pathogen Legionella pneumophilareplicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophilaexpressing a translocation reporter consisting of the Bordetella pertussisadenylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophilamay require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophilareplication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophilaproliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila. PMID:21742865

  8. Copper transporter 2 regulates intracellular copper and sensitivity to cisplatin.

    PubMed

    Huang, Carlos P; Fofana, Mariama; Chan, Jefferson; Chang, Christopher J; Howell, Stephen B

    2014-03-01

    Mammalian cells express two copper (Cu) influx transporters, CTR1 and CTR2. CTR1 serves as an influx transporter for both Cu and cisplatin (cDDP). In mouse embryo fibroblasts, reduction of CTR1 expression renders cells resistant to cDDP whereas reduction of CTR2 makes them hypersensitive both in vitro and in vivo. To investigate the role of CTR2 on intracellular Cu and cDDP sensitivity its expression was molecularly altered in the human epithelial 2008 cancer cell model. Intracellular exchangeable Cu(+) was measured with the fluorescent probe Coppersensor-3 (CS3). The ability of CS3 to report on changes in intracellular Cu(+) was validated by showing that Cu chelators reduced its signal, and that changes in signal accompanied alterations in expression of the major Cu influx transporter CTR1 and the two Cu efflux transporters, ATP7A and ATP7B. Constitutive knock down of CTR2 mRNA by ∼50% reduced steady-state exchangeable Cu by 22-23% and increased the sensitivity of 2008 cells by a factor of 2.6-2.9 in two separate clones. Over-expression of CTR2 increased exchangeable Cu(+) by 150% and rendered the 2008 cells 2.5-fold resistant to cDDP. The results provide evidence that CS3 can quantitatively assess changes in exchangeable Cu(+), and that CTR2 regulates both the level of exchangeable Cu(+) and sensitivity to cDDP in a model of human epithelial cancer. This study introduces CS3 and related sensors as novel tools for probing and assaying Cu-dependent sensitivity to anticancer therapeutics.

  9. Intracellular Mono-ADP-Ribosylation in Signaling and Disease

    PubMed Central

    Bütepage, Mareike; Eckei, Laura; Verheugd, Patricia; Lüscher, Bernhard

    2015-01-01

    A key process in the regulation of protein activities and thus cellular signaling pathways is the modification of proteins by post-translational mechanisms. Knowledge about the enzymes (writers and erasers) that attach and remove post-translational modifications, the targets that are modified and the functional consequences elicited by specific modifications, is crucial for understanding cell biological processes. Moreover detailed knowledge about these mechanisms and pathways helps to elucidate the molecular causes of various diseases and in defining potential targets for therapeutic approaches. Intracellular adenosine diphosphate (ADP)-ribosylation refers to the nicotinamide adenine dinucleotide (NAD+)-dependent modification of proteins with ADP-ribose and is catalyzed by enzymes of the ARTD (ADP-ribosyltransferase diphtheria toxin like, also known as PARP) family as well as some members of the Sirtuin family. Poly-ADP-ribosylation is relatively well understood with inhibitors being used as anti-cancer agents. However, the majority of ARTD enzymes and the ADP-ribosylating Sirtuins are restricted to catalyzing mono-ADP-ribosylation. Although writers, readers and erasers of intracellular mono-ADP-ribosylation have been identified only recently, it is becoming more and more evident that this reversible post-translational modification is capable of modulating key intracellular processes and signaling pathways. These include signal transduction mechanisms, stress pathways associated with the endoplasmic reticulum and stress granules, and chromatin-associated processes such as transcription and DNA repair. We hypothesize that mono-ADP-ribosylation controls, through these different pathways, the development of cancer and infectious diseases. PMID:26426055

  10. Intracellular pathogen detection by RIG-I-like receptors

    PubMed Central

    Dixit, Evelyn; Kagan, Jonathan C.

    2014-01-01

    The RIG-I-like receptors (RLR) RIG-I, MDA5 and LGP2 trigger innate immune responses against viral infections that serve to limit virus replication and to stimulate adaptive immunity. RLRs are cytosolic sensors for virus-derived RNA and thus responsible for intracellular immune surveillance against infection. RLR signaling requires the adapter protein MAVS to induce type I interferon, interferon-stimulated genes and proinflammatory cytokines. This review focusses on the molecular and cell biological requirements for RLR signal transduction. PMID:23611287

  11. Intracellular Assessment of ATP Levels in Caenorhabditis elegans

    PubMed Central

    Palikaras, Konstantinos; Tavernarakis, Nektarios

    2017-01-01

    Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP levels can serve as a metabolic gauge for cellular homeostasis and survival (Artal-Sanz and Tavernarakis, 2009; Gomes et al., 2011; Palikaras et al., 2015). In this protocol, we describe a method for the determination of intracellular ATP levels using a bioluminescence approach in the nematode Caenorhabditis elegans. PMID:28194429

  12. Intracellular survival of Burkholderia cepacia complex in phagocytic cells.

    PubMed

    Valvano, Miguel A

    2015-09-01

    Burkholderia cepacia complex (Bcc) species are a group of Gram-negative opportunistic pathogens that infect the airways of cystic fibrosis patients, and occasionally they infect other immunocompromised patients. Bcc bacteria display high-level multidrug resistance and chronically persist in the infected host while eliciting robust inflammatory responses. Studies using macrophages, neutrophils, and dendritic cells, combined with advances in the genetic manipulation of these bacteria, have increased our understanding of the molecular mechanisms of virulence in these pathogens and the molecular details of cell-host responses triggering inflammation. This article discusses our current view of the intracellular survival of Burkholderia cenocepacia within macrophages.

  13. Intracellular coagulation inhibits the extraction of proteins from Prochloron

    NASA Technical Reports Server (NTRS)

    Fall, R.; Lewin, R. A.; Fall, L. R.

    1983-01-01

    Protein extraction from the prokaryotic alga Prochloron LP (isolated from the ascidian host Lissoclinum patella) was complicated by an irreversible loss of cell fragility in the isolated algae. Accompanying this phenomenon, which is termed intracellular coagulation, was a redistribution of thylakoids around the cell periphery, a loss of photosynthetic O2 production, and a drastic decrease in the extractability of cell proteins. Procedures are described for the successful preparation and transport of cell extracts yielding the enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase as well as other soluble proteins.

  14. Dependence of cerebral arterial contractions on intracellularly stored Ca++.

    PubMed

    Sasaki, T; Kassell, N F; Zuccarello, M

    1986-01-01

    The purpose of the present study was to evaluate the dependence of the arterial contractions induced by different vasoactive agents upon intracellularly stored calcium in canine versus monkey cerebral arteries. The potency for inducing contractions in Ca++-free media was in the order of 9,11-epithio-11,12-metano-thromboxane A2 (STXA2) greater than prostaglandin F2 alpha (PGF2 alpha) much greater than serotonin greater than K+ in canine basilar arteries, and STXA2 greater than PGF2 alpha much greater than serotonin = K+ in monkey basilar arteries.

  15. The mystery of intracellular developmental programmes and timers.

    PubMed

    Raff, M

    2006-11-01

    There has been a revolution in understanding animal development in the last 25 years or so, but there is at least one area of development that has been relatively neglected and therefore remains largely mysterious. This is the intracellular programmes and timers that run in developing precursor cells and change the cells over time. The molecular mechanisms underlying these programmes are largely unknown. My colleagues and I have studied such programmes in two types of rodent neural precursor cells: those that give rise to oligodendrocytes, which make myelin in the CNS (central nervous system), and those that give rise to the various cell types in the retina.

  16. Imaging intracellular RNA distribution and dynamics in living cells.

    PubMed

    Tyagi, Sanjay

    2009-05-01

    Powerful methods now allow the imaging of specific mRNAs in living cells. These methods enlist fluorescent proteins to illuminate mRNAs, use labeled oligonucleotide probes and exploit aptamers that render organic dyes fluorescent. The intracellular dynamics of mRNA synthesis, transport and localization can be analyzed at higher temporal resolution with these methods than has been possible with traditional fixed-cell or biochemical approaches. These methods have also been adopted to visualize and track single mRNA molecules in real time. This review explores the promises and limitations of these methods.

  17. Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

    NASA Astrophysics Data System (ADS)

    Appert-Rolland, C.; Ebbinghaus, M.; Santen, L.

    2015-09-01

    Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized. The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach. It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer. We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the

  18. Voltage-dependent gating rearrangements in the intracellular T1-T1 interface of a K+ channel.

    PubMed

    Wang, Guangyu; Covarrubias, Manuel

    2006-04-01

    The intracellular tetramerization domain (T1) of most eukaryotic voltage-gated potassium channels (Kv channels) exists as a "hanging gondola" below the transmembrane regions that directly control activation gating via the electromechanical coupling between the S4 voltage sensor and the main S6 gate. However, much less is known about the putative contribution of the T1 domain to Kv channel gating. This possibility is mechanistically intriguing because the T1-S1 linker connects the T1 domain to the voltage-sensing domain. Previously, we demonstrated that thiol-specific reagents inhibit Kv4.1 channels by reacting in a state-dependent manner with native Zn(2+) site thiolate groups in the T1-T1 interface; therefore, we concluded that the T1-T1 interface is functionally active and not protected by Zn(2+) (Wang, G., M. Shahidullah, C.A. Rocha, C. Strang, P.J. Pfaffinger, and M. Covarrubias. 2005. J. Gen. Physiol. 126:55-69). Here, we co-expressed Kv4.1 channels and auxiliary subunits (KChIP-1 and DPPX-S) to investigate the state and voltage dependence of the accessibility of MTSET to the three interfacial cysteines in the T1 domain. The results showed that the average MTSET modification rate constant (k(MTSET)) is dramatically enhanced in the activated state relative to the resting and inactivated states (approximately 260- and approximately 47-fold, respectively). Crucially, under three separate conditions that produce distinct activation profiles, k(MTSET) is steeply voltage dependent in a manner that is precisely correlated with the peak conductance-voltage relations. These observations strongly suggest that Kv4 channel gating is tightly coupled to voltage-dependent accessibility changes of native T1 cysteines in the intersubunit Zn(2+) site. Furthermore, cross-linking of cysteine pairs across the T1-T1 interface induced substantial inhibition of the channel, which supports the functionally dynamic role of T1 in channel gating. Therefore, we conclude that the complex

  19. Structural dynamics of soluble chloride intracellular channel protein CLIC1 examined by amide hydrogen-deuterium exchange mass spectrometry.

    PubMed

    Stoychev, Stoyan H; Nathaniel, Christos; Fanucchi, Sylvia; Brock, Melissa; Li, Sheng; Asmus, Kyle; Woods, Virgil L; Dirr, Heini W

    2009-09-08

    Chloride intracellular channel protein 1 (CLIC1) functions as an anion channel in plasma and nuclear membranes when its soluble monomeric form converts to an integral-membrane form. The transmembrane region of CLIC1 is located in its thioredoxin-like domain 1, but the mechanism whereby the protein converts to its membrane conformation has yet to be determined. Since channel formation in membranes is enhanced at low pH (5 to 5.5), a condition that is found at the surface of membranes, the structural dynamics of soluble CLIC1 was studied at pH 7 and at pH 5.5 in the absence of membranes by amide hydrogen-deuterium exchange mass spectrometry (DXMS). Rapid hydrogen exchange data indicate that CLIC1 displays a similar core structure at these pH values. Domain 1 is less stable than the all-helical domain 2, and, while the structure of domain 1 remains intact, its conformational flexibility is further increased in an acidic environment (pH 5.5). In the absence of membrane, an acidic environment appears to prime the solution structure of CLIC1 by destabilizing domain 1 in order to lower the activation energy barrier for its conversion to the membrane-insertion conformation. The significantly enhanced H/D-exchange rates at pH 5.5 displayed by two segments (peptides 11-31 and 68-82) could be due to the protonation of acidic residues in salt bridges. One of these segments (peptide 11-31) includes part of the transmembrane region which, in the solution structure, consists of helix alpha1. This helix is intrinsically stable and is most likely retained in the membrane conformation. Strand beta2, another element of the transmembrane region, displays a propensity to form a helical structure and has putative N- and C-capping motifs, suggesting that it too most likely forms a helix in a lipid bilayer.

  20. Protein-protein interactions in intracellular Ca2+-release channel function.

    PubMed Central

    MacKrill, J J

    1999-01-01

    Release of Ca2+ ions from intracellular stores can occur via two classes of Ca2+-release channel (CRC) protein, the inositol 1,4, 5-trisphosphate receptors (InsP3Rs) and the ryanodine receptors (RyRs). Multiple isoforms and subtypes of each CRC class display distinct but overlapping distributions within mammalian tissues. InsP3Rs and RyRs interact with a plethora of accessory proteins which modulate the activity of their intrinsic channels. Although many aspects of CRC structure and function have been reviewed in recent years, the properties of proteins with which they interact has not been comprehensively surveyed, despite extensive current research on the roles of these modulators. The aim of this article is to review the regulation of CRC activity by accessory proteins and, wherever possible, to outline the structural details of such interactions. The CRCs are large transmembrane proteins, with the bulk of their structure located cytoplasmically. Intra- and inter-complex protein-protein interactions between these cytoplasmic domains also regulate CRC function. Some accessory proteins modulate channel activity of all CRC subtypes characterized, whereas other have class- or even isoform-specific effects. Certain accessory proteins exert both direct and indirect forms of regulation on CRCs, occasionally with opposing effects. Others are themselves modulated by changes in Ca2+ concentration, thereby participating in feedback mechanisms acting on InsP3R and RyR activity. CRCs are therefore capable of integrating numerous signalling events within a cell by virtue of such protein-protein interactions. Consequently, the functional properties of InsP3Rs and RyRs within particular cells and subcellular domains are 'customized' by the accessory proteins present. PMID:9895277

  1. Numerical simulation of water transport and intracellular ice formation for freezing of endothelial cells.

    PubMed

    Zhao, G; Xu, Y; Ding, W P; Hu, M B

    2013-01-01

    Endothelial cell detachment may cause failure of blood vessel and corneal cryopreservation, and thus successful cryopreservation of endothelial cells is regarded to be the first step to optimize cryopreservation of endothelial cells containing tissues. In this study, the pre-determined biophysical parameters were incorporated into the model for intracellular ice formation (IIF) and the growth of intracellular ice crystals (ICG) to calculate cell water loss, supercooling of intracellular solution, intracellular ice formation and the growth of intracellular ice crystals. The optimal protocols were determined according to the combination effect of both solution injury and IIF injury.

  2. Structural similarities and functional diversity of eukaryotic discoidin-like domains.

    PubMed

    Kiedzierska, A; Smietana, K; Czepczynska, H; Otlewski, J

    2007-09-01

    The discoidin domain is a approximately 150 amino acid motif common in both eukaryotic and prokaryotic proteins. It is found in a variety of extracellular, intracellular and transmembrane multidomain proteins characterized by a considerable functional diversity, mostly involved in developmental processes. The biological role of the domain depends on its interactions with different molecules, including growth factors, phospholipids and lipids, galactose or its derivatives, and collagen. The conservation of the motif, as well as the serious physiological consequences of discoidin domain disorders underscore the importance of the fold, while the ability to accommodate such an extraordinarily broad range of ligand molecules makes it a fascinating research target. In present review we characterize the distinctive features of discoidin domains and briefly outline the biological role of this module in various eukaryotic proteins.

  3. Mapping the Moral Domain

    PubMed Central

    Graham, Jesse; Nosek, Brian A.; Haidt, Jonathan; Iyer, Ravi; Koleva, Spassena; Ditto, Peter H.

    2010-01-01

    The moral domain is broader than the empathy and justice concerns assessed by existing measures of moral competence, and it is not just a subset of the values assessed by value inventories. To fill the need for reliable and theoretically-grounded measurement of the full range of moral concerns, we developed the Moral Foundations Questionnaire (MFQ) based on a theoretical model of five universally available (but variably developed) sets of moral intuitions: Harm/care, Fairness/reciprocity, Ingroup/loyalty, Authority/respect, and Purity/sanctity. We present evidence for the internal and external validity of the scale and the model, and in doing so present new findings about morality: 1. Comparative model fitting of confirmatory factor analyses provides empirical justification for a five-factor structure of moral concerns. 2. Convergent/discriminant validity evidence suggests that moral concerns predict personality features and social group attitudes not previously considered morally relevant. 3. We establish pragmatic validity of the measure in providing new knowledge and research opportunities concerning demographic and cultural differences in moral intuitions. These analyses provide evidence for the usefulness of Moral Foundations Theory in simultaneously increasing the scope and sharpening the resolution of psychological views of morality. PMID:21244182

  4. Discoidin Domain Receptor 1

    PubMed Central

    Song, Sunmi; Shackel, Nicholas A.; Wang, Xin M.; Ajami, Katerina; McCaughan, Geoffrey W.; Gorrell, Mark D.

    2011-01-01

    Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and is activated by collagens. Transcriptional profiling of cirrhosis in human liver using a DNA array and quantitative PCR detected elevated mRNA expression of DDR1 compared with that in nondiseased liver. The present study characterized DDR1 expression in cirrhotic and nondiseased human liver and examined the cellular effects of DDR1 expression. mRNA expression of all five isoforms of DDR1 was detected in human liver, whereas DDR1a demonstrated differential expression in liver with hepatitis C virus and primary biliary cirrhosis compared with nondiseased liver. In addition, immunoblot analysis detected shed fragments of DDR1 more readily in cirrhotic liver than in nondiseased liver. Inasmuch as DDR1 is subject to protease-mediated cleavage after prolonged interaction with collagen, this differential expression may indicate more intense activation of DDR1 protein in cirrhotic compared with nondiseased liver. In situ hybridization and immunofluorescence localized intense DDR1 mRNA and protein expression to epithelial cells including hepatocytes at the portal-parenchymal interface and the luminal aspect of the biliary epithelium. Overexpression of DDR1a altered hepatocyte behavior including increased adhesion and less migration on extracelular matrix substrates. DDR1a regulated extracellular expression of matrix metalloproteinases 1 and 2. These data elucidate DDR1 function pertinent to cirrhosis and indicate the importance of epithelial cell–collagen interactions in chronic liver injury. PMID:21356365

  5. Antibiotic uptake by cultured Atlantic cod leucocytes and effect on intracellular Francisella noatunensis subsp. noatunensis replication.

    PubMed

    Kaldestad, Marte; Haugland, Gyri T; Rønneseth, Anita; Wergeland, Heidrun I; Samuelsen, Ole Bent

    2014-02-04

    The granuloma disease caused by Francisella noatunensis subsp. noatunensis in farmed Atlantic cod has not been successfully treated by use of antibacterials, even when antibacterial resistance testing indicates a sufficient effect. The reason for this treatment failure may be the intracellular existence of the bacteria within immune cells, mainly macrophages. To investigate the effect of antibacterials on intracellular Francisella replication, we established a protocol for the detection of drugs within Atlantic cod immune cells using high-performance liquid chromatography (HPLC). When the uptake and intracellular concentrations of oxolinic acid and flumequine were analysed in isolated adherent head kidney leucocytes (HKLs) by HPLC, we found that uptake was rapid and the intracellular concentrations reflected the extracellular exposure concentrations. To investigate the effect of the antibacterial compounds on intracellular bacterial replication, adherent HKLs experimentally infected with the bacteria were analysed using flow cytometry and intracellular labelling of bacteria by specific antibodies. We found that flumequine did not inhibit intracellular bacterial replication. Unexpectedly, the results indicated that the intracellularly effiacy of the drug was reduced. The HPLC method used proved to be highly applicable for accurate determination of intracellular drug concentrations. When combined with sensitive and specific flow cytometry analyses for identification and measurement of intracellular bacterial replication, we suggest that this approach can be very valuable for the design of antibacterial treatments of intracellular pathogens.

  6. Domain-dependent effects of insulin and IGF-1 receptors on signalling and gene expression

    PubMed Central

    Cai, Weikang; Sakaguchi, Masaji; Kleinridders, Andre; Gonzalez-Del Pino, Gonzalo; Dreyfuss, Jonathan M.; O'Neill, Brian T.; Ramirez, Alfred K.; Pan, Hui; Winnay, Jonathon N.; Boucher, Jeremie; Eck, Michael J.; Kahn, C. Ronald

    2017-01-01

    Despite a high degree of homology, insulin receptor (IR) and IGF-1 receptor (IGF1R) mediate distinct cellular and physiological functions. Here, we demonstrate how domain differences between IR and IGF1R contribute to the distinct functions of these receptors using chimeric and site-mutated receptors. Receptors with the intracellular domain of IGF1R show increased activation of Shc and Gab-1 and more potent regulation of genes involved in proliferation, corresponding to their higher mitogenic activity. Conversely, receptors with the intracellular domain of IR display higher IRS-1 phosphorylation, stronger regulation of genes in metabolic pathways and more dramatic glycolytic responses to hormonal stimulation. Strikingly, replacement of leucine973 in the juxtamembrane region of IR to phenylalanine, which is present in IGF1R, mimics many of these signalling and gene expression responses. Overall, we show that the distinct activities of the closely related IR and IGF1R are mediated by their intracellular juxtamembrane region and substrate binding to this region. PMID:28345670

  7. Functional receptors and intracellular signal pathways of midkine (MK) and pleiotrophin (PTN).

    PubMed

    Xu, Chuanying; Zhu, Shunying; Wu, Mingyuan; Han, Wei; Yu, Yan

    2014-01-01

    Midkine (MK) and pleiotrophin (PTN) belong to the subfamily of heparin binding growth factors. They have ca. 50% structural homology, with similar C- and N-domains as well as comparable binding affinity to heparin, glycoproteins and proteoglycans. Both MK and PTN have diverse functions, such as mitogenicity, inflammation, angiogenesis, oncogenesis and stem cell self-renewal. The high expression of MK and PTN in many kinds of cancers makes them excellent as cancer biomarkers and targets for anticancer drug development. In addition, the important roles of MK and PTN in the regeneration of tissues, such as myocardium, cartilage, neuron, muscle, and bone, make them attractive candidates for the treatment of degenerative diseases such as myocardiac and cerebral infarction, Alzheimer's disease, Parkinson's disease and skeletal muscle injury. As a result, there has been a growing interest in the mechanisms of MK and PTN function, including the diverse receptors on the cell membrane and complex signal pathways in the cytoplasm. This work reviews the structures of MK and PTN, as well as the receptors and the intracellular signal pathways involving MK and PTN which will pave the way for future development of MK and PTN therapeutics.

  8. The genome of obligately intracellular Ehrlichia canis revealsthemes of complex membrane structure and immune evasion strategies

    SciTech Connect

    Mavromatis, K.; Kuyler Doyle, C.; Lykidis, A.; Ivanova, N.; Francino, P.; Chain, P.; Shin, M.; Malfatti, S.; Larimer, F.; Copeland,A.; Detter, J.C.; Land, M.; Richardson, P.M.; Yu, X.J.; Walker, D.H.; McBride, J.W.; Kyrpides, N.C.

    2005-09-01

    Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, a-proteobacterium is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, and 17 putative pseudogenes, and a substantial proportion of non-coding sequence (27 percent). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences, and a unique serine-threonine bias associated with the potential for O-glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly G:C tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Proteins associated with pathogen-host interactions were identified including a small group of proteins (12) with tandem repeats and another with eukaryotic-like ankyrin domains (7).

  9. Functional dissection of SseF, a membrane-integral effector protein of intracellular Salmonella enterica.

    PubMed

    Müller, Petra; Chikkaballi, Deepak; Hensel, Michael

    2012-01-01

    During intracellular life, the bacterial pathogen Salmonella enterica translocates a complex cocktail of effector proteins by means of the SPI2-encoded type III secretions system. The effectors jointly modify the endosomal system and vesicular transport in host cells. SseF and SseG are two effectors encoded by genes within Salmonella Pathogenicity Island 2 and both effector associate with endosomal membranes and microtubules and are involved in the formation of Salmonella-induced filaments. Our previous deletional analyses identified protein domains of SseF required for the effector function. Here we present a detailed mutational analysis that identifies a short hydrophobic motif as functionally essential. We demonstrate that SseF and SseG are still functional if translocated as a single fusion protein, but also mediate effector function if translocated in cells co-infected with sseF and sseG strains. SseF has characteristics of an integral membrane protein after translocation into host cells.

  10. Sustained accurate recording of intracellular acidification in living tissues with a photo-controllable bioluminescent protein.

    PubMed

    Hattori, Mitsuru; Haga, Sanae; Takakura, Hideo; Ozaki, Michitaka; Ozawa, Takeaki

    2013-06-04

    Regulation of an intracellular acidic environment plays a pivotal role in biological processes and functions. However, spatiotemporal analysis of the acidification in complex tissues of living subjects persists as an important challenge. We developed a photo-inactivatable bioluminescent indicator, based on a combination of luciferase-fragment complementation and a photoreaction of a light, oxygen, and voltage domain from Avena sativa Phototropin1 (LOV2), to visualize temporally dynamic acidification in living tissue samples. Bioluminescence of the indicator diminished upon light irradiation and it recovered gradually in the dark state thereafter. The recovery rate was remarkably sensitive to pH changes but unsusceptible to fluctuation of luciferin or ATP concentrations. Bioluminescence imaging, taken as an index of the recovery rates, enabled long-time recording of acidification in apoptotic and autophagous processes in a cell population and an ischemic condition in living mice. This technology using the indicator is widely applicable to sense organelle-specific acidic changes in target biological tissues.

  11. Biochemical characterization of extra- and intracellular endoxylanse from thermophilic bacterium Caldicellulosiruptor kronotskyensis

    PubMed Central

    Jia, Xiaojing; Qiao, Weibo; Tian, Wenli; Peng, Xiaowei; Mi, Shuofu; Su, Hong; Han, Yejun

    2016-01-01

    Caldicellulosiruptor kronotskyensis grows on lignocellulosic biomass by the catalysis of intrinsic glycoside hydrolase, and has potential application for consolidated bioprocessing. In current study, two predicted extra- (Xyn10A) and intracellular (Xyn10B) xylanase from C. kronotskyensis were comparatively characterized. Xyn10A and Xyn10B share GH10 catalytic domain with similarity of 41%, while the former contains two tandem N-terminus CBM22s. Xyn10A showed higher hydrolytic capability than Xyn10B on both beechwood xylan (BWX) and oat spelt xylan (OSX). Truncation mutation experiments revealed the importance of CBMs for hydrolytic activity, substrate binding and thermostability of Xyn10A.While the quantity of CBM was not directly related to bind and thermostability. Although CBM was considered to be crucial for substrate binding, Xyn10B and Xyn10A as well as truncations performed similar binding affinity to insoluble substrate OSX. Analysis of point mutation revealed similar key residues, Glu493, Glu601 and Trp658 for Xyn10A and Glu139, Glu247 and Trp305 for Xyn10B. Both Xyn10A and Xyn10B exhibited hydrolytic activity on the mechanical pretreated corncob. After pre-digested by Xyn10A or Xyn10B, the micropores inthe the mechanical pretreated corncob were observed, which enhanced the accessibility for cellulase. Compared with corncob hydrolyzed with cellulase alone, enhanced hydrolytic performance of was observed after pre-digestion by Xyn10A or Xyn10B. PMID:26899227

  12. Sustained accurate recording of intracellular acidification in living tissues with a photo-controllable bioluminescent protein

    PubMed Central

    Hattori, Mitsuru; Haga, Sanae; Takakura, Hideo; Ozaki, Michitaka; Ozawa, Takeaki

    2013-01-01

    Regulation of an intracellular acidic environment plays a pivotal role in biological processes and functions. However, spatiotemporal analysis of the acidification in complex tissues of living subjects persists as an important challenge. We developed a photo-inactivatable bioluminescent indicator, based on a combination of luciferase-fragment complementation and a photoreaction of a light, oxygen, and voltage domain from Avena sativa Phototropin1 (LOV2), to visualize temporally dynamic acidification in living tissue samples. Bioluminescence of the indicator diminished upon light irradiation and it recovered gradually in the dark state thereafter. The recovery rate was remarkably sensitive to pH changes but unsusceptible to fluctuation of luciferin or ATP concentrations. Bioluminescence imaging, taken as an index of the recovery rates, enabled long-time recording of acidification in apoptotic and autophagous processes in a cell population and an ischemic condition in living mice. This technology using the indicator is widely applicable to sense organelle-specific acidic changes in target biological tissues. PMID:23690604

  13. Engineered antibody therapies to counteract mutant huntingtin and related toxic intracellular proteins

    PubMed Central

    Butler, David C.; McLear, Julie A.; Messer, Anne

    2013-01-01

    The engineered antibody approach to Huntington's disease (HD) therapeutics is based on the premise that significantly lowering the levels of the primary misfolded mutant protein will reduce abnormal protein interactions and direct toxic effects of the misfolded huntingtin (HTT). This will in turn reduce the pathologic stress on cells, and normalize intrinsic proteostasis. Intracellular antibodies (intrabodies) are single-chain (scFv) and single-domain (dAb; nanobody) variable fragments that can retain the affinity and specificity of full-length antibodies, but can be selected and engineered as genes. Functionally, they represent a protein-based approach to the problem of aberrant mutant protein folding, post-translational modifications, protein-protein interactions, and aggregation. Several intrabodies that bind on either side of the expanded polyglutamine tract of mutant HTT have been reported to improve the mutant phenotype in cell and organotypic cultures, fruit flies, and mice. Further refinements to the difficult challenges of intraneuronal delivery, cytoplasmic folding, and long-term efficacy are in progress. This review covers published studies and emerging approaches on the choice of targets, selection and engineering methods, gene and protein delivery options, and testing of candidates in cell and animal models. The resultant antibody fragments can be used as direct therapeutics and as target validation/drug discovery tools for HD, while the technology is also applicable to a wide range of neurodegenerative and other diseases that are triggered by toxic proteins. PMID:22120646

  14. Hydrophobic Compounds Reshape Membrane Domains

    PubMed Central

    Barnoud, Jonathan; Rossi, Giulia; Marrink, Siewert J.; Monticelli, Luca

    2014-01-01

    Cell membranes have a complex lateral organization featuring domains with distinct composition, also known as rafts, which play an essential role in cellular processes such as signal transduction and protein trafficking. In vivo, perturbations of membrane domains (e.g., by drugs or lipophilic compounds) have major effects on the activity of raft-associated proteins and on signaling pathways, but they are difficult to characterize because of the small size of the domains, typically below optical resolution. Model membranes, instead, can show macroscopic phase separation between liquid-ordered and liquid-disordered domains, and they are often used to investigate the driving forces of membrane lateral organization. Studies in model membranes have shown that some lipophilic compounds perturb membrane domains, but it is not clear which chemical and physical properties determine domain perturbation. The mechanisms of domain stabilization and destabilization are also unknown. Here we describe the effect of six simple hydrophobic compounds on the lateral organization of phase-separated model membranes consisting of saturated and unsaturated phospholipids and cholesterol. Using molecular simulations, we identify two groups of molecules with distinct behavior: aliphatic compounds promote lipid mixing by distributing at the interface between liquid-ordered and liquid-disordered domains; aromatic compounds, instead, stabilize phase separation by partitioning into liquid-disordered domains and excluding cholesterol from the disordered domains. We predict that relatively small concentrations of hydrophobic species can have a broad impact on domain stability in model systems, which suggests possible mechanisms of action for hydrophobic compounds in vivo. PMID:25299598

  15. Downregulation of transferrin receptor surface expression by intracellular antibody

    SciTech Connect

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin . E-mail: guanxin_shen@yahoo.com.cn

    2007-03-23

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 {+-} 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.

  16. Optochemokine Tandem for Light-Control of Intracellular Ca2+

    PubMed Central

    Weissbecker, Juliane; Sauer, Frank; Wood, Phillip G.; Bamberg, Ernst

    2016-01-01

    An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light. PMID:27768773

  17. Elevated intracellular Na(+) concentrations in developing spinal neurons.

    PubMed

    Lindsly, Casie; Gonzalez-Islas, Carlos; Wenner, Peter

    2017-03-01

    Over 25 years ago it was first reported that intracellular chloride levels (Cl(-)in ) were higher in developing neurons than in maturity. This finding has had significant implications for understanding the excitability of developing networks and recognizing the underlying causes of hyperexcitability associated with disease and neural injury. While there is some evidence that intracellular sodium levels (Na(+)in ) change during the development of non-neural cells, it has largely been assumed that Na(+)in is the same in developing and mature neurons. Here, using the sodium indicator SBFI, we test this idea and find that Na(+)in is significantly higher in embryonic spinal motoneurons and interneurons than in maturity. We find that Na(+)in reaches ~ 60 mM in mid-embryonic development and is then reduced to ~ 30 mM in late embryonic development. By retrogradely labeling motoneurons with SBFI we can reliably follow Na(+)in levels in vitro for hours. Bursts of spiking activity, and blocking voltage-gated sodium channels did not influence observed motoneuron sodium levels. On the other hand, Na(+)in was reduced by blocking the Na(+) -K(+) -2Cl(-) cotransporter NKCC1, and was highly sensitive to changes in external Na(+) and a blocker of the Na(+) /K(+) ATPase. Our findings suggest that the Na(+) gradient is weaker in embryonic neuronal development and strengthens in maturity in a manner similar to that of Cl(-) .

  18. Transient light-induced intracellular oxidation revealed by redox biosensor

    SciTech Connect

    Kolossov, Vladimir L.; Beaudoin, Jessica N.; Hanafin, William P.; DiLiberto, Stephen J.; Kenis, Paul J.A.; Rex Gaskins, H.

    2013-10-04

    Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.

  19. Mechanisms of Obligatory Intracellular Infection with Anaplasma phagocytophilum

    PubMed Central

    Rikihisa, Yasuko

    2011-01-01

    Summary: Anaplasma phagocytophilum persists in nature by cycling between mammals and ticks. Human infection by the bite of an infected tick leads to a potentially fatal emerging disease called human granulocytic anaplasmosis. A. phagocytophilum is an obligatory intracellular bacterium that replicates inside mammalian granulocytes and the salivary gland and midgut cells of ticks. A. phagocytophilum evolved the remarkable ability to hijack the regulatory system of host cells. A. phagocytophilum alters vesicular traffic to create an intracellular membrane-bound compartment that allows replication in seclusion from lysosomes. The bacterium downregulates or actively inhibits a number of innate immune responses of mammalian host cells, and it upregulates cellular cholesterol uptake to acquire cholesterol for survival. It also upregulates several genes critical for the infection of ticks, and it prolongs tick survival at freezing temperatures. Several host factors that exacerbate infection have been identified, including interleukin-8 (IL-8) and cholesterol. Host factors that overcome infection include IL-12 and gamma interferon (IFN-γ). Two bacterial type IV secretion effectors and several bacterial proteins that associate with inclusion membranes have been identified. An understanding of the molecular mechanisms underlying A. phagocytophilum infection will foster the development of creative ideas to prevent or treat this emerging tick-borne disease. PMID:21734244

  20. Origins of intracellular calcium mobilization evoked by infrared laser stimulation

    NASA Astrophysics Data System (ADS)

    Olsovsky, Cory A.; Tolstykh, Gleb P.; Ibey, Bennett L.; Beier, Hope T.

    2015-03-01

    Cellular delivery of pulsed IR laser energy has been shown to stimulate action potentials in neurons. The mechanism for this stimulation is not completely understood. Certain hypotheses suggest the rise in temperature from IR exposure could activate temperature- or pressure-sensitive channels, or create pores in the cellular outer membrane. Studies using intensity-based Ca2+-responsive dyes show changes in Ca2+ levels after various IR stimulation parameters; however, determination of the origin of this signal proved difficult. An influx of larger, typically plasma-membrane-impermeant ions has been demonstrated, which suggests that Ca2+ may originate from the external solution. However, activation of intracellular signaling pathways, possibly indicating a more complex role of increasing Ca2+ concentration, has also been shown. By usingCa2+ sensitive dye Fura-2 and a high-speed ratiometric imaging system that rapidly alternates the excitation wavelengths, we have quantified the Ca2+ mobilization in terms of influx from the external solution and efflux from intracellular organelles. CHO-K1 cells, which lack voltage-gated Ca2+ channels, and NG-108 neuroblastoma cells, which do not produce action potentials in an early undifferentiated state, are used to determine the origin of the Ca2+ signals and investigate the role these mechanisms may play in IR neural stimulation.

  1. Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

    PubMed Central

    Umezaki, Mariko; Tashiro, Ryo; Oda, Masataka; Kobayashi, Keiko; Shibutani, Masahiro; Takagishi, Teruhisa; Ishidoh, Kazumi; Fukuda, Mitsunori; Sakurai, Jun

    2012-01-01

    Clostrid